專利名稱：發(fā)酵產(chǎn)生含硫精細(xì)化學(xué)品的方法(metA)的制作方法
描述本發(fā)明涉及通過(guò)使用表達(dá)編碼高絲氨酸O-乙酰轉(zhuǎn)移酶(metA)基因的核苷酸序列的細(xì)菌，發(fā)酵產(chǎn)生含硫精細(xì)化學(xué)品，尤其是L-甲硫氨酸的方法。
現(xiàn)有技術(shù)含硫精細(xì)化學(xué)品如甲硫氨酸、高半胱氨酸、S-腺苷甲硫氨酸、谷胱甘肽、半胱氨酸、生物素、硫胺素、硫辛酸通過(guò)天然代謝過(guò)程在細(xì)胞中產(chǎn)生并且用于許多工業(yè)領(lǐng)域，包括食品、動(dòng)物飼料、化妝品和制藥工業(yè)。這些統(tǒng)稱為“含硫精細(xì)化學(xué)品”的物質(zhì)包括有機(jī)酸、蛋白原性(proteinogenic)和非蛋白原性氨基酸、維生素和輔因子。通過(guò)培養(yǎng)細(xì)菌可以極其方便地大規(guī)模生產(chǎn)這些物質(zhì)，已經(jīng)開(kāi)發(fā)這些細(xì)菌以產(chǎn)生并大量分泌在每種情況下所希望的物質(zhì)。尤其適于該目的的生物是棒狀細(xì)菌，它們是革蘭氏陽(yáng)性非病原性細(xì)菌。
公知通過(guò)棒狀細(xì)菌，尤其是谷氨酸棒桿菌(Corynebacteriumglutamicum)的發(fā)酵生產(chǎn)氨基酸。由于非常重要，所以生產(chǎn)方法不斷改進(jìn)。方法改進(jìn)可涉及測(cè)定相關(guān)的發(fā)酵技術(shù)方面如攪拌和氧供給，或者涉及營(yíng)養(yǎng)培養(yǎng)基組分如發(fā)酵過(guò)程中的糖濃度，或者涉及得到產(chǎn)物的操作(work-up)，例如通過(guò)離子交換層析，或者涉及微生物自身的內(nèi)在性能特性。
通過(guò)菌株選擇已經(jīng)開(kāi)發(fā)了從含硫精細(xì)化學(xué)品產(chǎn)生各種所希望的化合物的許多突變菌株。通過(guò)應(yīng)用誘變、選擇和突變選擇的方法，在特定分子的產(chǎn)生方面所述微生物的性能特性得到提高。然而，這是一種費(fèi)時(shí)而且困難的方法。以這種方式獲得了例如對(duì)下述抗代謝物具有抗性或者對(duì)于調(diào)節(jié)重要的代謝物為營(yíng)養(yǎng)缺陷型的并產(chǎn)生含硫精細(xì)化學(xué)品如L-甲硫氨酸的菌株，所述抗代謝物如甲硫氨酸類(lèi)似物α-甲基甲硫氨酸、乙硫氨酸、正亮氨酸、n-乙?；涟彼?、S-三氟甲基高半胱氨酸、2-氨基-5-heprenoitic acid、硒代蛋氨酸、甲硫氨酸亞砜胺(methioninesulfoximine)、methoxine、1-氨基環(huán)戊烷羧酸。
重組DNA技術(shù)的方法通過(guò)擴(kuò)增單個(gè)氨基酸生物合成基因并研究其對(duì)氨基酸產(chǎn)生的影響數(shù)年來(lái)也已經(jīng)被用于改良產(chǎn)生L-氨基酸的棒桿菌菌株。
發(fā)明簡(jiǎn)述本發(fā)明的一個(gè)目的是提供含硫精細(xì)化學(xué)品尤其是L-甲硫氨酸的改良的發(fā)酵生產(chǎn)的新方法。
我們已經(jīng)發(fā)現(xiàn)通過(guò)提供一種含硫精細(xì)化學(xué)品的發(fā)酵生產(chǎn)方法實(shí)現(xiàn)了該目的，該方法包括在棒狀細(xì)菌中表達(dá)編碼具有metA活性的蛋白質(zhì)的異源核苷酸序列。
本發(fā)明首先涉及用于發(fā)酵產(chǎn)生至少一種含硫精細(xì)化學(xué)品的方法，其包括下面的步驟a)發(fā)酵產(chǎn)生目的含硫精細(xì)化學(xué)品的棒狀細(xì)菌培養(yǎng)物，該棒狀細(xì)菌表達(dá)至少一種這樣的異源核苷酸序列，該序列編碼具有高絲氨酸O-乙酰轉(zhuǎn)移酶(metA)活性的蛋白質(zhì)；b)濃縮培養(yǎng)基或細(xì)菌細(xì)胞中的含硫精細(xì)化學(xué)品；和c)分離含硫精細(xì)化學(xué)品，其優(yōu)選含有L-甲硫氨酸。
上面的異源編碼metA的核苷酸序列與谷氨酸棒桿菌ATCC 13032的編碼metA的序列優(yōu)選具有100％以下且優(yōu)選70％以上的同源性。編碼metA的序列優(yōu)選來(lái)自下面表I生物中的任一種。
表I
ATCC美國(guó)典型培養(yǎng)物保藏中心，位于美國(guó)Rockville，MD。
本發(fā)明使用的metA-編碼序列優(yōu)選含有根據(jù)SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43和45的編碼序列或者編碼具有metA活性蛋白質(zhì)的與它們同源的核苷酸序列。
此外，本發(fā)明使用的metA-編碼序列優(yōu)選編碼具有metA活性的蛋白質(zhì)，所述蛋白質(zhì)含有根據(jù)SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44和46的氨基酸序列或代表具有metA活性的蛋白質(zhì)的與它們同源的氨基酸序列。
編碼metA的序列優(yōu)選為可以在棒狀細(xì)菌中復(fù)制或者被穩(wěn)定整合到染色體中的DNA或RNA。
根據(jù)一個(gè)優(yōu)選的實(shí)施方案，本發(fā)明的方法通過(guò)下面的步驟實(shí)施a)使用質(zhì)粒載體轉(zhuǎn)化的細(xì)菌菌株，該質(zhì)粒載體攜帶處于調(diào)節(jié)序列控制下的至少一份編碼metA序列的拷貝，或者b)使用這樣的菌株，該菌株中編碼metA的序列已經(jīng)被整合到細(xì)菌染色體中。
此外，發(fā)酵優(yōu)選過(guò)量表達(dá)編碼metA的序列。
還希望發(fā)酵這樣的細(xì)菌，其中目的含硫精細(xì)化學(xué)品的生物合成途徑的至少另一基因已經(jīng)被擴(kuò)增；和/或其中至少一條代謝途徑已經(jīng)至少部分被關(guān)閉，其中所述該代謝途徑降低目的含硫精細(xì)化學(xué)品的產(chǎn)生。
還希望發(fā)酵這樣的細(xì)菌，其中額外地目的含硫精細(xì)化學(xué)品的生物合成途徑的至少另一基因不被代謝的代謝物不利地影響。
因此，根據(jù)本發(fā)明方法的另一實(shí)施方案，發(fā)酵這樣的棒狀細(xì)菌，其中同時(shí)存在選自a)基因lysC，其編碼天冬氨酸激酶，b)基因asd，其編碼天冬氨酸-半醛脫氫酶，c)甘油醛-3-磷酸脫氫酶編碼基因gap，d)3-磷酸甘油酸激酶編碼基因pgk，e)丙酮酸羧化酶編碼基因pyc，f)磷酸丙糖異構(gòu)酶編碼基因tpi，g)甲硫氨酸合酶編碼基因metH，h)γ胱硫醚合酶編碼基因metB，
i)γ胱硫醚裂合酶編碼基因metC，j)絲氨酸羥甲基轉(zhuǎn)移酶編碼基因glyA，k)O-乙酰高絲氨酸硫化氫解酶編碼基因metY，l)亞甲基四氫葉酸還原酶編碼基因metF，m)磷酸絲氨酸氨基轉(zhuǎn)移酶編碼基因serC，n)磷酸絲氨酸磷酸酶編碼基因serb，o)絲氨酸乙酰轉(zhuǎn)移酶編碼基因cysE，p)高絲氨酸脫氫酶編碼基因hom的至少一種基因被過(guò)量表達(dá)。
根據(jù)本發(fā)明方法的另一實(shí)施方案，發(fā)酵這樣的棒桿菌，其中同時(shí)有選自上面的組a)到p)的基因的至少一種基因以某種方式突變使得相應(yīng)蛋白質(zhì)的活性與未突變蛋白質(zhì)相比，被所代謝的代謝物影響程度較小(如果有)，并且尤其是精細(xì)化學(xué)品的發(fā)明性生產(chǎn)不被不利地影響。
根據(jù)本發(fā)明的方法的另一實(shí)施方案，發(fā)酵這樣的棒桿菌，其中同時(shí)存在選自q)高絲氨酸激酶編碼基因thrB，r)蘇氨酸脫水酶編碼基因ilvA，s)蘇氨酸合酶編碼基因thrC，t)內(nèi)消旋-二氨基庚二酸D-脫氫酶編碼基因ddh，u)磷酸烯醇丙酮酸羧激酶編碼基因pck，v)葡萄糖-6-磷酸6-異構(gòu)酶編碼基因pgi，w)丙酮酸氧化酶編碼基因poxB，x)二氫吡啶二羧酸合酶編碼基因dapA，y)二氫吡啶二羧酸還原酶編碼基因dapB；或z)二氨基吡啶甲酸脫羧酶編碼基因lysA的至少一種基因被弱化，尤其通過(guò)降低相應(yīng)基因的表達(dá)速率而被弱化。
根據(jù)本發(fā)明的另一實(shí)施方案，發(fā)酵這樣的棒桿菌，其中同時(shí)存在至少一種選自上面組q)到z)的基因以某種方式突變使得相應(yīng)蛋白質(zhì)的酶活性被部分或完全降低。
在本發(fā)明的方法中，優(yōu)選谷氨酸棒桿菌種的微生物。
本發(fā)明還涉及從發(fā)酵液產(chǎn)生含L-甲硫氨酸的動(dòng)物飼料添加劑的方法，該方法包括下面的步驟a)在發(fā)酵培養(yǎng)基中培養(yǎng)和發(fā)酵產(chǎn)生L-甲硫氨酸的微生物；b)從含L-甲硫氨酸的發(fā)酵液除去水；c)除去發(fā)酵過(guò)程中形成的生物量重量的0到100％；和d)干燥根據(jù)b)和/或c)所得發(fā)酵液，以得到所希望的粉劑或粒劑形式的動(dòng)物飼料添加劑。
本發(fā)明同樣涉及第一次從上面的微生物分離的編碼metA的序列，涉及由其編碼的高絲氨酸O-乙酰轉(zhuǎn)移酶，還分別涉及這些多核苷酸和蛋白質(zhì)的功能同系物。
發(fā)明詳述a)一般術(shù)語(yǔ)具有高絲氨酸O-乙酰轉(zhuǎn)移酶活性的蛋白質(zhì)，也稱作metA(EC2.3.1.31)，被描述為能夠?qū)⒏呓z氨酸和乙酰輔酶A轉(zhuǎn)化成O-乙?；呓z氨酸的蛋白質(zhì)。技術(shù)人員能夠區(qū)分高絲氨酸O-乙酰轉(zhuǎn)移酶的活性和高絲氨酸O-琥珀酰轉(zhuǎn)移酶的活性，但是高絲氨酸O-琥珀酰轉(zhuǎn)移酶在文獻(xiàn)中也被稱為metA。對(duì)于后一種酶，琥珀酰輔酶A而不是乙酰輔酶A作為反應(yīng)的底物。技術(shù)人員可以通過(guò)酶測(cè)定法檢測(cè)高絲氨酸O-乙酰轉(zhuǎn)移酶的酶活性，該酶測(cè)定法的方案可以是Park SD.Lee JY.Kim Y.Kim JH.Lee HS.Molecules& Cells.8(3)286-94，1998。
在本發(fā)明的范圍中，術(shù)語(yǔ)“含硫精細(xì)化學(xué)品”包括含有至少一個(gè)共價(jià)結(jié)合的硫原子并且可通過(guò)本發(fā)明的發(fā)酵方法得到的化合物。它們的非限制性實(shí)例為甲硫氨酸、高半胱氨酸、S-腺苷甲硫氨酸，尤其是甲硫氨酸和S-腺苷甲硫氨酸。
在本發(fā)明的范圍內(nèi)，術(shù)語(yǔ)“L-甲硫氨酸”、“甲硫氨酸”、高半胱氨酸和S-腺苷甲硫氨酸還包括相應(yīng)的鹽如甲硫氨酸鹽酸鹽或甲硫氨酸硫酸鹽。
“多核苷酸”通常指多核糖核苷酸(RNA)和多脫氧核糖核苷酸(DNA)，其可以分別是未修飾的RNA和DNA，或者分別是修飾的RNA和DNA。
根據(jù)本發(fā)明，“多肽”指含有通過(guò)肽鍵連接的兩個(gè)或多個(gè)氨基酸的肽或蛋白質(zhì)。
術(shù)語(yǔ)“代謝的代謝物”指在生物體的代謝中發(fā)生的作為中間產(chǎn)物或者作為終產(chǎn)物并且，它們除了作為化學(xué)結(jié)構(gòu)單元的性質(zhì)，還可以對(duì)酶和對(duì)它們的催化活性具有調(diào)節(jié)作用的化合物。從文獻(xiàn)中已知這些代謝的代謝物可以以抑制和刺激的方式作用于酶活性(Biochemistry，Stryer，Lubert，1995W.H.Freeman & Company，New York，紐約)。在文獻(xiàn)中還描述了可能在生物體中產(chǎn)生酶，其中代謝的代謝物的影響已經(jīng)被一些措施改變，這些措施為例如通過(guò)紫外輻射、電離輻射或誘變而突變基因組DNA，隨后選擇特定表型(Sahm H.，Eggeling L.，de Graaf AA.，Biological Chemistry381(9-10)899-910，2000；Eikmanns BJ.，Eggeling L.，Sahm H.，Antonie vanLeeuwenhoek.，64145-63，1993-94)。這些改變的特性也可以通過(guò)特定測(cè)量實(shí)現(xiàn)。技術(shù)人員公知還可能以如此方法特異修飾編碼蛋白質(zhì)的DNA的酶基因中特定核苷酸從而由表達(dá)的DNA序列得到的蛋白質(zhì)具有某些新的性質(zhì)，例如，代謝的代謝物對(duì)未修飾的蛋白質(zhì)的調(diào)節(jié)作用被改變。
術(shù)語(yǔ)“表達(dá)”和“擴(kuò)增”或“過(guò)量表達(dá)”在本發(fā)明的上下文中描述了微生物中相應(yīng)DNA編碼的一種或多種酶的產(chǎn)生或細(xì)胞內(nèi)活性增加。為此，例如，可將基因?qū)肷矬w以通過(guò)另一基因替換現(xiàn)有基因，增加該一種基因或幾種基因的拷貝數(shù)，使用強(qiáng)啟動(dòng)子或使用編碼具有高活性的相應(yīng)酶的基因，并且適當(dāng)時(shí)可以組合這些措施。
b)本發(fā)明的metA蛋白質(zhì)本發(fā)明同樣包括上面的表I中具體公開(kāi)的生物metA酶的“功能等同物”。
在本發(fā)明范圍內(nèi)，具體公開(kāi)的多肽的“功能等同物”或類(lèi)似物是與其不同的多肽，而且其具有目的生物學(xué)活性如底物特異性。
根據(jù)本發(fā)明，“功能等同物”指特定突變體，其在上面提到的序列位置的至少一個(gè)位置具有不同于特定提到的氨基酸的氨基酸，但是仍然具有上面提到的生物學(xué)活性之一。從而“功能等同物”還包括通過(guò)一個(gè)或多個(gè)氨基酸添加、替換、缺失和/或倒位可以得到的突變，所述修飾可能在該序列的任何位置發(fā)生，只要它們導(dǎo)致具有本發(fā)明特性的突變體。尤其當(dāng)突變體和未修飾多肽的反應(yīng)模式定性地匹配，即，例如相同的底物以不同速率被轉(zhuǎn)化時(shí)，則存在功能等同物。
“功能等同物”自然也包括從其他生物可以得到的多肽，和天然存在的變體。例如，通過(guò)序列比較可以發(fā)現(xiàn)同源序列區(qū)，按照本發(fā)明的特定指導(dǎo)方針可以建立等同酶。
“功能等同物”同樣包括本發(fā)明多肽的片段、優(yōu)選單個(gè)結(jié)構(gòu)域或序列基序，它們具有例如目的生物學(xué)功能。
“功能等同物”還包括融合蛋白質(zhì)，其具有上面提到的多肽序列之一或者衍生自該序列的功能等同物以及在N-或C-末端功能性連接的與該序列功能不同的至少一種其他異源序列(即，融合蛋白部分的功能的可忽略功能損失)。這些異源序列的非限制性實(shí)例為，例如，信號(hào)肽、酶、免疫球蛋白、表面抗原、受體或受體配體。
根據(jù)本發(fā)明，“功能等同物”包括具體公開(kāi)的蛋白質(zhì)的同系物。這些同系物與具體公開(kāi)的序列之一具有至少20％，或者約30％、40％、50％，優(yōu)選至少約60％、65％、70％，或75％，尤其至少85％，如90％、95％或99％的同源性，該同源性通過(guò)Pearson和Lipman(Proc.Natl.Acad.，Sci.(USA)85(8)，1988，2444-2448)的算法計(jì)算。
通過(guò)誘變，例如通過(guò)蛋白質(zhì)的點(diǎn)突變或截短可以產(chǎn)生本發(fā)明的蛋白質(zhì)或多肽的同系物。如此處所用的術(shù)語(yǔ)“同系物”涉及蛋白質(zhì)的變體形式，其作為蛋白質(zhì)活性的激動(dòng)劑或拮抗劑。
通過(guò)篩選突變體組合文庫(kù)如截短突變體組合文庫(kù)，可以鑒定本發(fā)明蛋白質(zhì)的同系物?？衫缤ㄟ^(guò)核酸水平的組合誘變，例如，通過(guò)合成的寡核苷酸混合物的酶促連接產(chǎn)生蛋白質(zhì)變體的多樣化文庫(kù)。有多種方法可用于從簡(jiǎn)并寡核苷酸序列制備潛在同系物的文庫(kù)。簡(jiǎn)并基因序列的化學(xué)合成可以在自動(dòng)DNA合成儀中進(jìn)行，然后合成的基因可以被連接到適宜的表達(dá)載體中。一組簡(jiǎn)并基因的使用使得可能在一種混合物中提供編碼一組目的潛在蛋白質(zhì)序列的全部序列。合成簡(jiǎn)并寡核苷酸的方法是技術(shù)人員公知的(例如，Narang，S.A.，(1983)Tetrahedron 393；Itakura等，(1984)Annu.Rev.Biochem.53323；Itakura等，(1984)Science 1981056；Ike等，(1983)Nucleic Acids Res.11477)。
此外，蛋白質(zhì)密碼子片段的文庫(kù)可用于產(chǎn)生蛋白質(zhì)片段的多樣化群體，該群體用于篩選和隨后選擇本發(fā)明蛋白質(zhì)的同系物。在一個(gè)實(shí)施方案中，可以如下產(chǎn)生編碼序列片段的文庫(kù)，這可通過(guò)用核酸酶在一定條件下處理編碼序列的雙鏈PCR片段，在該條件下切開(kāi)發(fā)生僅僅約為每個(gè)分子一次，變性雙鏈DNA，復(fù)性該DNA形成雙鏈DNA，其可含有不同切口產(chǎn)物的有意/反義對(duì)，通過(guò)S1核酸酶處理重新形成的雙鏈體除去單鏈部分并將所得片段文庫(kù)連接到表達(dá)載體而實(shí)現(xiàn)?？赏ㄟ^(guò)該方法設(shè)計(jì)編碼本發(fā)明蛋白質(zhì)的N-末端、C-末端和內(nèi)部片段的表達(dá)文庫(kù)，這些片段具有不同大小。
在現(xiàn)有技術(shù)中公知一些技術(shù)用于從已經(jīng)通過(guò)點(diǎn)突變或截短產(chǎn)生的組合文庫(kù)篩選基因產(chǎn)物和篩選DNA文庫(kù)以得到具有所選擇特性的基因產(chǎn)物。這些技術(shù)可適于快速篩選通過(guò)本發(fā)明的同系物的組合誘變所產(chǎn)生的基因文庫(kù)。用于篩選經(jīng)歷高通量分析的大基因文庫(kù)的最經(jīng)常使用的技術(shù)包括將基因文庫(kù)克隆到可復(fù)制的表達(dá)載體中，用所得載體文庫(kù)轉(zhuǎn)化適宜的細(xì)胞并在一定條件下表達(dá)組合基因，其中在該條件下對(duì)目的活性的檢測(cè)方便了這樣的載體的分離，該載體的基因編碼的產(chǎn)物已經(jīng)被檢測(cè)。遞歸整體誘變(Recursive ensemble mutagenesis，REM)—增加文庫(kù)中功能突變體頻率的一種技術(shù)—可以與篩選試驗(yàn)組合使用以鑒定同系物(Arkin und Yourvan(1992)PNAS 897811-7815；Delgrave等(1993)，Protein Engineering 6(3)327-331。
c)本發(fā)明的多核苷酸本發(fā)明還涉及編碼上面的metA酶之一的核酸序列(單鏈和雙鏈DNA和RNA如cDNA和mRNA)及其功能等同物，其也可以通過(guò)例如使用人工核苷酸類(lèi)似物得到。
本發(fā)明涉及分離的核酸分子，其編碼本發(fā)明的多肽或蛋白質(zhì)或者其生物學(xué)活性部分，還涉及這樣的核酸片段，該片段可用作例如用于鑒定或擴(kuò)增本發(fā)明的編碼核酸的雜交探針或引物。
此外，本發(fā)明的核酸分子可以含有基因編碼區(qū)的3’和/或5’端的非翻譯序列。
“分離的”核酸分子分離自存在于該核酸的天然來(lái)源的其他核酸分子并且還可以基本上無(wú)其他細(xì)胞物質(zhì)或培養(yǎng)基(如果其通過(guò)重組技術(shù)制備)，或者無(wú)化學(xué)前體或其他化學(xué)品(如果其通過(guò)化學(xué)合成)。
本發(fā)明還包括與具體描述的核苷酸序列或其部分互補(bǔ)的核酸分子。
本發(fā)明的核苷酸序列使得可產(chǎn)生可用于鑒定和/或克隆其他細(xì)胞型或生物中的同源序列的探針和引物。這些探針和引物通常組成這樣的核苷酸序列區(qū)，其在嚴(yán)格條件下與本發(fā)明核酸序列的有意鏈或者相應(yīng)的反義鏈的至少約12、優(yōu)選至少約25，如40、50或75個(gè)連續(xù)核苷酸雜交。
本發(fā)明的其他核酸序列來(lái)自SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43或45并且通過(guò)添加、替換、插入或缺失一個(gè)或多個(gè)核苷酸而與它們不同，但是仍然編碼具有目的特性的多肽。這些可以是在至少約50％、55％、60％、65％、70％、80％或90％，優(yōu)選在至少約95％、96％、97％、98％或99％的序列位置中與上面的序列相同的多核苷酸。
本發(fā)明還包括通過(guò)與特定提到的序列比較，按照特定來(lái)源或宿主生物的密碼子使用，含有“沉默”突變或被修飾的那些核酸序列，以及天然存在的變體如剪接變體或等位基因變體。本發(fā)明還涉及通過(guò)保守核苷酸替換(即，相關(guān)氨基酸被具有相同電荷、大小、極性和/或溶解性的氨基酸替換)可以得到的序列。
本發(fā)明還涉及通過(guò)序列多態(tài)性從具體公開(kāi)的核酸衍生的分子。這些遺傳多態(tài)性可以由于群體內(nèi)個(gè)體間的天然變異而存在。這些天然變異通常導(dǎo)致基因的核苷酸序列中1到5％的變化。
本發(fā)明還包括與上面提到的編碼序列雜交或者與它們互補(bǔ)的核酸序列。這些多核苷酸可以在篩選基因組或cDNA文庫(kù)時(shí)發(fā)現(xiàn)，并且適宜時(shí)，通過(guò)PCR使用適宜的引物從它們擴(kuò)增，然后，例如，用適宜的探針?lè)蛛x。另一可能性是用本發(fā)明的多核苷酸或載體轉(zhuǎn)化適宜的微生物，繁殖該微生物從而增殖該多核苷酸，然后分離這些多核苷酸。另一可能性是通過(guò)化學(xué)途徑合成本發(fā)明的多核苷酸。
能夠“雜交”多核苷酸的性質(zhì)指多核苷酸或寡核苷酸能夠在嚴(yán)格條件下結(jié)合幾乎互補(bǔ)的序列，而非互補(bǔ)序列在這些條件下沒(méi)有非特異結(jié)合。為此，序列應(yīng)該70-100％，優(yōu)選90-100％互補(bǔ)，互補(bǔ)序列能夠特異地相互結(jié)合的性質(zhì)被例如用于RNA印跡技術(shù)或DNA印跡技術(shù)或者PCR或者RT-PCR(對(duì)于引物結(jié)合的情況)中。具有長(zhǎng)為30個(gè)堿基對(duì)或更多堿基對(duì)的寡核苷酸通常用于該目的。嚴(yán)格條件指，例如，在RNA印跡技術(shù)中，使用50-70℃，優(yōu)選60-65℃的洗滌溶液，例如，含有0.1％SDS的0.1×SSC緩沖液(20×SSC；3M NaCl，0.3M檸檬酸鈉，pH 7.0)用于洗脫非特異雜交的cDNA探針或寡核苷酸。在該情況下，如上面提到的，僅僅具有高度互補(bǔ)性的核酸保持相互結(jié)合。嚴(yán)格條件的設(shè)置是技術(shù)人員公知的并且在例如Ausubel等，Current Protocols in Molecular Biology，John Wiley & Sons，N.Y.(1989)，6.3.1-6.3.6中描述。
d)編碼metA基因的分離可以以本身公知的方法從上面表I的生物分離編碼高絲氨酸O-乙酰轉(zhuǎn)移酶的metA基因。
為了分離上面表I的生物的metA基因或其他基因，首先在大腸桿菌(E.coli)中產(chǎn)生該生物的基因文庫(kù)?；蛭膸?kù)的產(chǎn)生在一般已知的教科書(shū)和手冊(cè)中詳細(xì)描述?？梢蕴峒暗膶?shí)例是WinnackerGene und Klone，EineEinführung in die Gentechnologie(Verlag Chemie，Weinheim，德國(guó)，1990)的教科書(shū)，和Sambrook等分子克隆實(shí)驗(yàn)指南(冷泉港，1989)。一種非常熟知的基因文庫(kù)是大腸桿菌K-12菌株W3110的基因文庫(kù)，其由Kohara等人(Cell50，495-508(1980))在λ載體中產(chǎn)生。
為了在大腸桿菌中產(chǎn)生來(lái)自表I中生物的基因文庫(kù)，可以使用粘粒如粘粒載體SuperCos I(Wahl等人，1987，Proceedings of the NationalAcademy of Sciences USA，842160-2164)或者質(zhì)粒如pBR322(BoliVal；Life Sciences，25，807-818(1979))或pUC9(Vieira等人，1982，Gene，19259-268)。適宜的宿主尤其是限制性和重組缺陷的大腸桿菌菌株。該菌株的一個(gè)實(shí)例是菌株DH5αmcr，其已經(jīng)由Grant等人(Proceedings of theNational Academy of Sciences USA，87(1990)4645-4649)描述。用粘粒克隆的長(zhǎng)DNA片段然后又可以亞克隆到適于測(cè)序的通用載體中并隨后被測(cè)序，如在Sanger等人(proceedings of the National Academy of Sciences of theUnited States of America，745463-5467，1977)中所描述的。
然后所得DNA序列可以使用公知的算法或序列分析程序研究，這些算法或序列分析程序?yàn)槿鏢taden(Nucleic Acids Research 14，217-232(1986))的算法、Marck(Nucleic Acids Research 16，1829-1836(1988))的算法或者Butler(Methods of Biochemical Analysis 39，74-97(1998))的GCG程序。
發(fā)現(xiàn)了來(lái)自上面表I生物的編碼metA的DNA序列。具體地，發(fā)現(xiàn)了根據(jù)SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43和45的DNA序列。此外，使用上述方法，從存在的所述DNA序列得到了相應(yīng)蛋白質(zhì)的氨基酸序列。SEQ IDNO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44和46描述了metA基因產(chǎn)物的所得氨基酸序列。
由于遺傳密碼的簡(jiǎn)并性從根據(jù)SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43和45的序列得到的編碼DNA序列也是本發(fā)明的主題。同樣，本發(fā)明涉及與所述序列或從它們衍生的序列的部分雜交的DNA序列。
通過(guò)雜交鑒定DNA序列的教導(dǎo)可以由技術(shù)人員在例如來(lái)自Boehringer Mannheim GmbH的手冊(cè)《濾膜雜交的DIG系統(tǒng)用戶指南》(Mannheim，德國(guó)，1993)和在Liebl等人(International Journal ofSystematic Bacteriology(1991)41255-260)中發(fā)現(xiàn)。利用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增DNA序列的教導(dǎo)尤其可以由技術(shù)人員在Gait編著的手冊(cè)O(shè)ligonucleotide synthesisA Practical Approach(IRL Press，Oxford，UK，1984)以及Newton和GrahamPCR(Spektrum Akademischer Verlag，Heidelberg，德國(guó)，1994)中找到。
還公知蛋白質(zhì)的N-和/或C-末端的改變不實(shí)質(zhì)性地?fù)p害其功能或者甚至可穩(wěn)定所述功能。關(guān)于此的信息可以由技術(shù)人員尤其在Ben-Bassat等人(Journal of Bacteriology 169751-757(1987))、O′Regan等人(Gene 77237-251(1989)、Sahin-Toth等人(Protein Seiences 3240-247(1994))、Hochuli等人(Biotechnology 61321-1325(1988))以及在遺傳學(xué)和分子生物學(xué)的公知教科書(shū)中找到。
因此從SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44和46獲得的氨基酸序列同樣是本發(fā)明的部分。
e)本發(fā)明使用的宿主細(xì)胞本發(fā)明還涉及作為宿主細(xì)胞的微生物，尤其是棒細(xì)菌，該微生物含有攜帶本發(fā)明定義的至少一種metA基因的載體，尤其是穿梭載體或質(zhì)粒載體，或者其中本發(fā)明的metA基因被表達(dá)或擴(kuò)增。
這些微生物可以從葡萄糖、蔗糖、乳糖、果糖、麥芽糖、糖蜜、淀粉、纖維素或從甘油和乙醇產(chǎn)生含硫精細(xì)化學(xué)品，尤其是L-甲硫氨酸。所述微生物優(yōu)選為棒狀細(xì)菌，尤其是棒桿菌屬的細(xì)菌。對(duì)于棒桿菌屬，必須提及的是谷氨酸棒桿菌，在文獻(xiàn)中已知其能夠產(chǎn)生L-氨基酸。
可以提及的棒狀細(xì)菌的適宜菌株的實(shí)例是棒桿菌屬的菌株，尤其是谷氨酸棒桿菌(C.glutamicum)種的菌株，如谷氨酸棒桿菌ATCC 13032、醋谷氨酸棒桿菌(Corynebacterium acetoglutamicum)ATCC 15806、
嗜乙酰乙酸棒桿菌(Corynebacterium acetoacidophilum)ATCC13870、熱產(chǎn)氨棒桿菌(Corynebacterium thermoaminogenes)FERMBP-1539、Corynebacterium melassecola ATCC 17965或者短桿菌屬(Brevibacterium)的菌株，如黃色短桿菌(Brevibacterium flavum)ATCC 14067、乳發(fā)酵短桿菌(Brevibacterium lactofermentum)ATCC 13869和叉開(kāi)短桿菌(Brevibacterium divaricatum)ATCC 14020；后者從中衍生的菌株，如谷氨酸棒桿菌KFCC10065、谷氨酸棒桿菌ATCC21608，其同樣產(chǎn)生目的精細(xì)化學(xué)品或者其前體。
縮寫(xiě)KFCC指韓國(guó)培養(yǎng)物保藏聯(lián)合會(huì)(Korean Federation of CultureCollection)，縮寫(xiě)ATCC指美國(guó)典型菌株培養(yǎng)物保藏中心，縮寫(xiě)FERM BP指日本工業(yè)科學(xué)和技術(shù)局的國(guó)立生命科學(xué)和人體技術(shù)研究所的保藏中心。
f)實(shí)施本發(fā)明的發(fā)酵根據(jù)本發(fā)明，發(fā)現(xiàn)棒狀細(xì)菌過(guò)量表達(dá)來(lái)自表I生物的metA基因后，以有利的方式產(chǎn)生含硫精細(xì)化學(xué)品，尤其是L-甲硫氨酸。
為了實(shí)現(xiàn)過(guò)量表達(dá)，技術(shù)人員可以采用單獨(dú)的或聯(lián)合的不同措施。從而可能增加適宜基因的拷貝數(shù)或者突變啟動(dòng)子和調(diào)節(jié)區(qū)或者位于結(jié)構(gòu)基因上游的核糖體結(jié)合位點(diǎn)。摻入結(jié)構(gòu)基因上游的表達(dá)盒以同樣的方式作用。可誘導(dǎo)的啟動(dòng)子使得還可能在發(fā)酵性L-甲硫氨酸產(chǎn)生過(guò)程中增加表達(dá)。通過(guò)延長(zhǎng)mRNA壽命的措施也可以提高表達(dá)。此外，通過(guò)防止酶蛋白質(zhì)的降解也可以增強(qiáng)酶活性?；蚧蚧驑?gòu)建體可以或者以不同的拷貝數(shù)存在于質(zhì)粒中或者被整合到染色體并在染色體中擴(kuò)增。另一可能的備選方案是通過(guò)改變培養(yǎng)基組分和操縱培養(yǎng)實(shí)現(xiàn)相關(guān)基因的過(guò)量表達(dá)。
過(guò)量表達(dá)的教導(dǎo)可以由技術(shù)人員在Martin等人(Biontechnology 5，137-146(1987))、Guerrero等人(Gene 138，35-41(1994))、Tsuchiya和Morinaga(Bio/Technology 6，428-430(1988))、Eikmanns等人(Gene 102，93-98(1991))、歐洲專利0472869、美國(guó)專利4,601,893、Schwarzer和Pühler(Biotechnology 9，84-87(1991)、Remscheid等人(Applied andEnvironmental Microbiology 60，126-132(1994)、LaBarre等人(Journal ofBacteriology 175，1001-1007(1993))、專利申請(qǐng)WO 96/15246、Malumbres等人(Gene 134，15-24(1993))、日本公開(kāi)的說(shuō)明書(shū)JP-A-10-229891、Jensen和Hammer(Biotechnology and Bioengineering 58，191-195(1998))、Makrides(Microbiological Reviews 60512-538(1996)以及遺傳學(xué)和分子生物學(xué)的公知教科書(shū)中找到。
本發(fā)明因此還涉及含有處于調(diào)節(jié)性核酸序列的遺傳控制下的編碼本發(fā)明多肽的核酸序列的表達(dá)構(gòu)建體，還涉及含有至少一種那所述表達(dá)構(gòu)建體的載體。本發(fā)明的這類(lèi)構(gòu)建體優(yōu)選包括特定編碼序列5’上游的啟動(dòng)子和3’下游的終止子序列和適當(dāng)時(shí)包括其他調(diào)節(jié)元件，在每種情況下它們均可操作地連接到編碼序列。“可操作地連接”指啟動(dòng)子、編碼序列、終止子序列和適宜時(shí)其他調(diào)節(jié)元件的順序排列從而每種調(diào)節(jié)元件可以在編碼序列的表達(dá)中正確發(fā)揮其功能?？刹僮鞯剡B接的序列的實(shí)例為活化序列和增強(qiáng)子等。其他調(diào)節(jié)元件包括可選擇標(biāo)記、擴(kuò)增信號(hào)、復(fù)制起點(diǎn)等。適宜的調(diào)節(jié)序列在例如Goeddel，基因表達(dá)技術(shù)酶學(xué)方法185，Academic Press，SanDiego，CA(1990)中描述。
除了人工調(diào)節(jié)序列外，天然調(diào)節(jié)序列仍然可以存在于實(shí)際的結(jié)構(gòu)基因的上游。遺傳修飾可以在適宜時(shí)關(guān)閉該天然調(diào)節(jié)并且增加或減少該基因的表達(dá)。基因構(gòu)建體也可以具有更簡(jiǎn)單的設(shè)計(jì)，即沒(méi)有額外的調(diào)節(jié)信號(hào)被插入結(jié)構(gòu)基因的上游并且天然啟動(dòng)子與其調(diào)節(jié)沒(méi)有被除去。取而代之的是，天然調(diào)節(jié)序列被突變從而調(diào)節(jié)不再發(fā)生并且基因表達(dá)被增強(qiáng)或減弱?；驑?gòu)建體可以含有核酸序列的一份或多份拷貝。
有用的啟動(dòng)子的實(shí)例為來(lái)自谷氨酸棒桿菌啟動(dòng)子的ddh、amy、lysC、dapA、lysA，以及革蘭氏陽(yáng)性啟動(dòng)子SPO2，如在《枯草芽孢桿菌及其最接近的菌株》，Sonenshein，Abraham L.，Hoch，James A.，Losick，Richard；ASM Press，華盛頓哥倫比亞特區(qū)以及Patek M.Eikmanns BJ.，Patek J.，Sahm H.，Microbiology.142 1297-309，1996中所描述的，或者優(yōu)選有利地用于革蘭氏陰性細(xì)菌中的cos、tac、trp、tet、trp-tet、lpp、lac、lpp-lac、lacIq、T7、T5、T3、gal、trc、ara、SP6、λ-PR和λ-PL啟動(dòng)子。還優(yōu)選使用可誘導(dǎo)的啟動(dòng)子如光可誘導(dǎo)的啟動(dòng)子，尤其是溫度可誘導(dǎo)的啟動(dòng)子如PrPL啟動(dòng)子。原則上可以使用具有調(diào)節(jié)序列的所有天然啟動(dòng)子。此外，還可以有利地使用合成的啟動(dòng)子。
所提及的調(diào)節(jié)序列旨在使得核酸序列的特異表達(dá)成為可能。根據(jù)宿主生物，這可以指例如基因僅僅在誘導(dǎo)后被表達(dá)或過(guò)量表達(dá)，或者其被立即表達(dá)和/或過(guò)量表達(dá)。
關(guān)于這一點(diǎn)，調(diào)節(jié)序列和因子可以優(yōu)選對(duì)表達(dá)具有有益影響，并能從而增加或減少表達(dá)。從而，可能并有利地通過(guò)使用強(qiáng)轉(zhuǎn)錄信號(hào)如啟動(dòng)子和/或增強(qiáng)子增強(qiáng)轉(zhuǎn)錄水平上的調(diào)節(jié)元件。然而，除了這之外還可通過(guò)例如提高mRNA的穩(wěn)定性增強(qiáng)翻譯。
通過(guò)將適宜的啟動(dòng)子、適宜的SD序列融合到metA核苷酸序列和適宜的終止信號(hào)制備表達(dá)盒。為此，使用常規(guī)重組和克隆技術(shù)，如在CurrentProtocols in Molecular Biology，1993，John Wiley & Sons，Incorporated，New York，紐約；PCR Methods，Gelfand，David H.，Innis，Michael A.，Sninsky，John J.，1999，Academic Press，Incorporated，California，SanDiego；PCR Cloning Protocols，Methods in Molecular Biology Ser.，192卷，第二版，Humana Press，New Jersey；Totowa.T.Maniatis，E.F.Fritsch和J.Sambrook，分子克隆實(shí)驗(yàn)指南，冷泉港實(shí)驗(yàn)室，冷泉港，NY(1989)；以及T.J.Silhavy，M.L.Berman和L.W.Enquist，Experiments with GeneFusions，Cold Spring Harbor Laboratory，Cold Spring Harbor，NY(1984)；以及Ausubel，F(xiàn).M.等人，Current Protocols in Molecular Biology，Greene Publishing Assoc.and Wiley Interscience(1987)中描述的那些技術(shù)。
通過(guò)將重組核酸構(gòu)建體或基因構(gòu)建體有利地插入宿主特異的載體而實(shí)現(xiàn)在適宜的宿主生物中表達(dá)所述重組核酸構(gòu)建體或基因構(gòu)建體，其中所述載體使得可能在宿主中最優(yōu)表達(dá)這些基因。載體是本領(lǐng)域技術(shù)人員熟知的并且可以在例如，“Cloning Vectors”(Pouwels P.H.等人，Hrsg，Elsevier，Amsterdam-New York-Oxford，1985)中找到。術(shù)語(yǔ)“載體”除了質(zhì)粒，還指技術(shù)人員公知的所有其他載體，如噬菌體、轉(zhuǎn)座子、IS元件、質(zhì)粒、粘粒和線性或環(huán)狀DNA。這些載體可以在宿主生物中自主復(fù)制或者隨染色體復(fù)制。
通過(guò)例如利用游離型質(zhì)粒過(guò)量表達(dá)本發(fā)明的metA基因而擴(kuò)增這些基因。適宜的質(zhì)粒為在棒狀細(xì)菌中復(fù)制的那些質(zhì)粒。許多公知的質(zhì)粒載體如pZ1(Menkel等人，Applied and Environmental Microbiology(1989)64549-554)、pEKEx1(Eikmanns等人，Gene 10293-98(1991))或pHS2-1(Sonnen等人，Gene 10769-74(1991))是基于隱性質(zhì)粒(cryptic plasmid)pHM1519、pBL1或pGA1。其他質(zhì)粒載體如pCLiK5MCS或者基于pCG4(US-A 4,489,160)或pNG2(Serwold-Davis等人，F(xiàn)EMS MicrobiologyLetters 66，119-124(1990))或pAG1(US-A 5,158,891)的那些質(zhì)?？梢砸韵嗤绞绞褂谩?br> 適宜的質(zhì)粒載體還包括通過(guò)它們可以應(yīng)用通過(guò)整合到染色體擴(kuò)增基因的方法的那些質(zhì)粒載體，如Remscheid等人(Applied and EnvironmentalMicrobiology 60，126-132(1994))已經(jīng)描述的用于復(fù)制和擴(kuò)增hom-thrB操縱子的那些質(zhì)粒載體。在該方法中，完整基因被克隆到質(zhì)粒載體中，該質(zhì)粒載體可以在宿主(一般為大腸桿菌)但是不能在谷氨酸棒桿菌中復(fù)制。適宜的載體為例如pSUP301(Sirnon等人，Bio/Technology 1，784-791(1983))、pK18mob或pK19mob(Sch_fer等人，Gene 145，69-73(1994))，Bernard等人，Journal of Molecular Biology，234534-541(1993))、pEM1(Schrumpf等人，1991，Journal of Bacteriology 1734510-4516)或pBGS8(Spratt等人，1986，Gene 41337-342)。含有待擴(kuò)增基因的質(zhì)粒載體然后通過(guò)轉(zhuǎn)化被轉(zhuǎn)移到目的谷氨酸棒桿菌菌株中。轉(zhuǎn)化方法在例如Thierbach等人(Applied Microbiology and Biotechnology 29，356-362(1988))、Dunican和Shivnan(Biotechnology 7，1067-1070(1989))和Tauch等人(FEMSMicrobiological Letters 123，343-347(1994))中描述。
酶的活性可以被相應(yīng)基因中的突變影響從而使得酶反應(yīng)的速率被部分或完全降低。這些突變的實(shí)例是技術(shù)人員公知的(Motoyama H.，Yano H.，Terasaki Y.，Anazawa H.，Applied & Environmental Microbiology.673064-70，2001，Eikmanns BJ.，Eggeling L.，Sahm H.，Antonie vanLeeuwenhoek.64145-63，1993-94)。
此外，對(duì)于含硫精細(xì)化學(xué)品尤其是L-甲硫氨酸的產(chǎn)生有利的是，除了表達(dá)和擴(kuò)增本發(fā)明的metA基因，還擴(kuò)增各自生物合成途徑、半胱氨酸途徑、天冬氨酸-半醛合成、糖酵解、回補(bǔ)、磷酸戊糖代謝、檸檬酸循環(huán)或者氨基酸輸出的一種或多種酶。
從而，可以擴(kuò)增一種或多種下面的基因以產(chǎn)生含硫精細(xì)化學(xué)品，尤其是L-甲硫氨酸-基因lysC，其編碼天冬氨酸激酶(EP 1 108 790 A2；DNA-SEQ NO.281)，-基因asd，其編碼天冬氨酸半醛脫氫酶(EP 1 108 790 A2；DNA-SEQNO.282)，-甘油醛-3-磷酸脫氫酶編碼基因gap(Eikmanns(1992)，Journal ofBacteriology 1746076-6086)，-3-磷酸甘油酸激酶編碼基因pgk(Eikmanns(1992)，Journal ofBacteriology 1746076-6086)，-丙酮酸羧化酶編碼基因pyc(Eikmanns(1992)，Journal ofBacteriology 1746076-6086)，-磷酸丙糖異構(gòu)酶編碼基因tpi(Eikmanns(1992)，Journal ofBacteriology 1746076-6086)，-甲硫氨酸合酶編碼基因metH(EP 1 108 790 A2)，-γ胱硫醚合酶編碼基因metB(EP 1 108 790 A2；DNA-SEQ NO.3491)，-γ胱硫醚裂合酶編碼基因metC(EP 1 108 790 A2；DNA-SEQ NO.3061)，-絲氨酸羥甲基轉(zhuǎn)移酶編碼基因glyA(EP 1 108 790 A2；DNA-SEQNO.1110)，-O-乙酰基高絲氨酸硫化氫解酶編碼基因metY(EP 1 108 790 A2；DNA-SEQ NO.726)，-亞甲基四氫葉酸還原酶編碼基因metF(EP 1 108 790 A2；DNA-SEQNO.2379)，-磷酸絲氨酸氨基轉(zhuǎn)移酶編碼基因serC(EP 1 108 790 A2；DNA-SEQNO.928)，-磷酸絲氨酸磷酸酶編碼基因serB(EP 1 108 790 A2；DNA-SEQ NO.334，DNA-SEQ NO.467，DNA-SEQ NO.2767)，-絲氨酸乙酰轉(zhuǎn)移酶編碼基因cysE(EP 1 108 790 A2；DNA-SEQ NO.2818)，-基因hom，其編碼高絲氨酸脫氫酶(EP 1 108 790 A2；DNA-SEQ NO.1306)從而，對(duì)在棒狀細(xì)菌中產(chǎn)生含硫精細(xì)化學(xué)品尤其是L-甲硫氨酸，有利的是同時(shí)突變至少一種下面的基因，從而相應(yīng)蛋白質(zhì)的活性與未突變的蛋白質(zhì)的相比，受代謝的代謝物影響程度較小或不受影響-基因lysC，其編碼天冬氨酸激酶(EP 1 108 790 A2；DNA-SEQ NO.281)，-丙酮酸羧化酶編碼基因pyc(Eikmanns(1992)，Journal ofBacteriology 1746076-6086)，-甲硫氨酸合酶編碼基因metH(EP 1 108 790 A2)，-γ胱硫醚合酶編碼基因metB(EP 1 108 790 A2；DNA-SEQ NO.3491)，-γ胱硫醚裂合酶編碼基因metC(EP 1 108 790 A2；DNA-SEQ NO.3061)，-絲氨酸羥甲基轉(zhuǎn)移酶編碼基因glyA(EP 1 108 790 A2；DNA-SEQNO.1110)，-O-乙?；呓z氨酸硫化氫解酶編碼基因metY(EP 1 108 790 A2；DNA-SEQ NO.726)，-亞甲基四氫葉酸還原酶編碼基因metF(EP 1 108 790 A2；DNA-SEQNO.2379)，-磷酸絲氨酸氨基轉(zhuǎn)移酶編碼基因serC(EP 1 108 790 A2；DNA-SEQNO.928)，-磷酸絲氨酸磷酸酶編碼基因serB(EP 1 108 790 A2；DNA-SEQ NO.334，DNA-SEQ NO.467，DNA-SEQ NO.2767)，-絲氨酸乙酰轉(zhuǎn)移酶編碼基因cysE(EP 1 108 790 A2；DNA-SEQ NO.2818)，-基因hom，其編碼高絲氨酸脫氫酶(EP 1 108 790 A2；DNA-SEQ NO.1306)另外對(duì)產(chǎn)生含硫精細(xì)化學(xué)品尤其是L-甲硫氨酸有利的是，除了表達(dá)和擴(kuò)增本發(fā)明的metA基因之一外，還弱化一種或多種下面的基因，尤其是減少它們的表達(dá)，或者將它們關(guān)閉-高絲氨酸激酶編碼基因thrB(EP 1 108 790 A2；DNA-SEQ NO.3453)，-蘇氨酸脫水酶編碼基因ilvA(EP 1 108 790 A2；DNA-SEQ NO.2328)，-蘇氨酸合酶編碼基因thrC(EP 1 108 790 A2；DNA-SEQ NO.3486)，-內(nèi)消旋二氨基庚二酸D-脫氫酶編碼基因ddh(EP 1 108 790 A2；DNA-SEQ NO.3494)，-磷酸烯醇丙酮酸羧激酶編碼基因pck(EP 1 108 790 A2；DNA-SEQNO.3157)，-葡萄糖-6-磷酸6-異構(gòu)酶編碼基因pgi(EP 1 108 790 A2；DNA-SEQNO.950)，-丙酮酸氧化酶編碼基因poxB(EP 1 108 790 A2；DNA-SEQ NO.2873)，-二氫吡啶二羧酸合酶編碼基因dapA(EP 1 108 790 A2；DNA-SEQNO.3476)，-二氫吡啶二羧酸還原酶編碼基因dapB(EP 1 108 790 A2；DNA-SEQNO.3477)-二氨基吡啶甲酸脫羧酶編碼基因lysA(EP 1 108 790 A2；DNA-SEQNO.3451)。
另外對(duì)含硫精細(xì)化學(xué)品尤其是L-甲硫氨酸的產(chǎn)生有利的是，除了在棒狀細(xì)菌中表達(dá)和擴(kuò)增本發(fā)明的metA基因之一外，同時(shí)突變至少一種下面的基因使得相應(yīng)蛋白質(zhì)的酶活性部分或完全降低-高絲氨酸激酶編碼基因thrB(EP 1 108 790 A2；DNA-SEQ NO.3453)，-蘇氨酸脫水酶編碼基因ilvA(EP 1 108 790 A2；DNA-SEQ NO.2328)，-蘇氨酸合酶編碼基因thrC(EP 1 108 790 A2；DNA-SEQ NO.3486)，-內(nèi)消旋二氨基庚二酸D-脫氫酶編碼基因ddh(EP 1 108 790 A2；DNA-SEQ NO.3494)，-磷酸烯醇丙酮酸羧激酶編碼基因pck(EP 1 108 790 A2；DNA-SEQNO.3157)，-葡萄糖-6-磷酸6-異構(gòu)酶編碼基因pgi(EP 1 108 790 A2；DNA-SEQNO.950)，-丙酮酸氧化酶編碼基因poxB(EP 1 108 790 A2；DNA-SEQ NO.2873)，-二氫吡啶二羧酸 合酶編碼基因dapA(EP 1 108 790 A2；DNA-SEQNO.3476)，-二氫吡啶二羧酸還原酶編碼基因dapB(EP 1 108 790 A2；DNA-SEQNO.3477)-二氨基吡啶甲酸脫羧酶編碼基因lysA(EP 1 108 790 A2；DNA-SEQNO.3451)。
另外對(duì)含硫精細(xì)化學(xué)品尤其是L-甲硫氨酸的產(chǎn)生有利的是，除了表達(dá)和擴(kuò)增本發(fā)明的一種metA基因，還消除不需要的副反應(yīng)(Overproductionof Microbial Products(Krumphanzl，Sikyta，Vanek(編輯)，Academic Press，英國(guó)倫敦，1982)一書(shū)中Nakayama產(chǎn)氨基酸微生物的培養(yǎng))。
根據(jù)本發(fā)明產(chǎn)生的微生物可以連續(xù)地或者分批地或者補(bǔ)料分批或者反復(fù)補(bǔ)料分批方法培養(yǎng)以產(chǎn)生含硫精細(xì)化學(xué)品，尤其是L-甲硫氨酸。公知的培養(yǎng)方法的概述可以在Chmiel的教科書(shū)(Bioprozeβtechnik 1.Einführungin die Bioverfahrenstechnik(Gustav Fischer Verlag，Stuttgart，1991))或者Storhas的教科書(shū)(Bioreaktoren und periphere Einrichtungen(ViewegVerlag，Braunschweig/Wiesbaden，1994))中找到。
所用的培養(yǎng)基必須以適當(dāng)?shù)姆绞綕M足特定菌株的要求。美國(guó)細(xì)菌學(xué)協(xié)會(huì)(the American Society for Bacteriology)的教科書(shū)″Manual of Methodsfür General Bacteriology″包含各種微生物培養(yǎng)基的描述。
可以根據(jù)本發(fā)明使用的所述培養(yǎng)基通常含有一種或多種碳源、氮源、無(wú)機(jī)鹽、維生素和/或微量元素。
優(yōu)選的碳源為糖如單糖、二糖或多糖。非常好的碳源的實(shí)例為葡萄糖、果糖、甘露糖、半乳糖、核糖、山梨糖、核酮糖、乳糖、麥芽糖、蔗糖、棉子糖、淀粉和纖維素。也可通過(guò)復(fù)雜化合物如糖蜜或其他糖精煉的副產(chǎn)物將糖加入培養(yǎng)基。還有利的是加入不同碳源的混合物。其他可能的碳源為油和脂肪如大豆油、向日葵油、花生油和椰子脂，脂肪酸如棕櫚酸、硬脂酸和亞油酸，醇如甘油、甲醇和乙醇以及有機(jī)酸如乙酸和乳酸。
氮源通常為有機(jī)或無(wú)機(jī)氮化合物或含有所述化合物的物質(zhì)。氮源的實(shí)例包括氨氣或銨鹽如硫酸銨、氯化銨、磷酸銨、碳酸銨和硝酸銨、硝酸鹽、尿素、氨基酸和復(fù)雜氮源如玉米漿、大豆粉、大豆蛋白、酵母提取物、肉膏等。氮源可以單獨(dú)地或者作為混合物使用。
可以包括在培養(yǎng)基的無(wú)機(jī)鹽化合物包括鈣、鎂、鈉、鈷、鉬、鉀、錳、鋅、銅和鐵的氯化物、磷酸鹽或硫酸鹽。
無(wú)機(jī)含硫化合物如硫酸鹽、亞硫酸鹽、連二亞硫酸鹽、連四硫酸鹽、硫代硫酸鹽、硫化物或者有機(jī)含硫化合物如硫醇類(lèi)也可用作產(chǎn)生含硫精細(xì)化學(xué)品尤其是甲硫氨酸的硫源。
磷酸、磷酸二氫鉀和磷酸氫二鉀或相應(yīng)的含鈉鹽可用作磷源。
可向培養(yǎng)基中加入螯合劑以保持溶液中的金屬離子。尤其適宜的螯合劑包括二羥基酚類(lèi)如兒茶酚或原兒茶酸以及有機(jī)酸如檸檬酸。
根據(jù)本發(fā)明使用的發(fā)酵培養(yǎng)基通常還含有其他生長(zhǎng)因子如維生素或生長(zhǎng)促進(jìn)劑，其包括例如生物素、核黃素、硫胺素、葉酸、煙酸、泛酸和吡哆醇。生長(zhǎng)因子和鹽經(jīng)常來(lái)自復(fù)雜的培養(yǎng)基組分如酵母提取物、糖蜜、玉米漿等。還可向培養(yǎng)基中加入適宜的前體。培養(yǎng)基的精確組分很大程度上取決于特定實(shí)驗(yàn)并且對(duì)于每種特定情況單獨(dú)決定。優(yōu)化培養(yǎng)基的信息可以在教科書(shū)″Applied Microbiol.Physiology，A Practical Approach″(編者P.M.Rhodes，P.F.Stanbury，IRL Press(1997)53-73頁(yè)，ISBN 0 199 635773)中發(fā)現(xiàn)。還可以從供應(yīng)商得到生長(zhǎng)培養(yǎng)基，例如Standard 1(Merck)或BHI(腦心浸液，DIFCO)等。
所有培養(yǎng)基組分通過(guò)熱(1.5巴下20分鐘，121℃)或通過(guò)無(wú)菌過(guò)濾除菌。各組分可以一起或者，如果需要，分開(kāi)滅菌。所有培養(yǎng)基組分可以在培養(yǎng)開(kāi)始時(shí)存在或者根據(jù)需要連續(xù)或者分批加入。
培養(yǎng)溫度通常為15℃到45℃，優(yōu)選25℃到40℃，并且可以保持恒定或者在實(shí)驗(yàn)過(guò)程中改變。培養(yǎng)基的pH應(yīng)該為5到8.5，優(yōu)選約7.0。培養(yǎng)的pH可以在培養(yǎng)中通過(guò)加入堿性化合物如氫氧化鈉、氫氧化鉀、氨和氨水或者酸性化合物如磷酸或硫酸控制。通過(guò)使用防沫劑如脂肪酸聚乙二醇酯控制起泡沫。為了保持質(zhì)粒的穩(wěn)定，可向培養(yǎng)基加入具有選擇作用的適宜物質(zhì)，例如抗生素。通過(guò)將氧氣或者含氧的氣體混合物如空氣導(dǎo)入培養(yǎng)基可以保持需氧條件。培養(yǎng)溫度通常為20℃到45℃。連續(xù)培養(yǎng)直到目的產(chǎn)物達(dá)到最大量。該目標(biāo)通常在10到160小時(shí)內(nèi)實(shí)現(xiàn)。
以這種方法得到的發(fā)酵液，尤其是含有L-甲硫氨酸的培養(yǎng)基，通常含有按重量計(jì)7.5到25％的干生物量。
另一額外的益處是至少在末尾，但是優(yōu)選在至少30％的發(fā)酵期間實(shí)施限糖發(fā)酵。這表示在該時(shí)間內(nèi)發(fā)酵培養(yǎng)基中可利用糖的濃度保持在或者減小到≥0到3g/l。
然后進(jìn)一步處理發(fā)酵液。生物量可以根據(jù)需要通過(guò)分離方法如離心、過(guò)濾、倒出或這些方法的組合從發(fā)酵液完全或部分除去或者完全保留在所述發(fā)酵液中。
隨后，使用公知的方法如利用旋轉(zhuǎn)蒸發(fā)器、薄膜蒸發(fā)器、降膜蒸發(fā)器、反向滲透或者通過(guò)納過(guò)濾(nanofiltration)增稠或者濃縮發(fā)酵液。該濃縮的發(fā)酵液然后可以通過(guò)冷凍干燥、噴霧干燥、噴霧?；蚱渌椒ㄌ幚怼?br> 然而，還可進(jìn)一步純化含硫精細(xì)化學(xué)品，尤其是L-甲硫氨酸。為此，含產(chǎn)物的發(fā)酵液，在除去生物量后，使用適宜的樹(shù)脂進(jìn)行層析，目的產(chǎn)物或者雜質(zhì)完全或部分保留在層析樹(shù)脂上。如果需要，可以使用相同的或者不同的層析樹(shù)脂重復(fù)這些層析步驟。技術(shù)人員熟悉適宜的層析樹(shù)脂的選擇和它們最有效的應(yīng)用。純化的產(chǎn)物可以通過(guò)過(guò)濾或者超濾濃縮并保持在某一溫度下，在該溫度下產(chǎn)物的穩(wěn)定性最大。
通過(guò)本領(lǐng)域技術(shù)可以確定所分離的一種或幾種化合物的身份和純度。這些技術(shù)包括高效液相層析(HPLC)、光譜方法、染色方法、薄層層析、NIRS、酶測(cè)定法或微生物學(xué)測(cè)定法。這些分析方法在Patek等人(1994)Appl.Environ.Microbiol.60133-140；Malakhova等人(1996)Biotekhnologiya 11 27-32；和Schmidt等人(1998)Bioprocess Engineer.1967-70.Ulmann′s Encyclopedia of Industrial Chemistry(1996)Bd.A27，VCHWeinheim，89-90頁(yè)，521-540頁(yè)，540-547頁(yè)，559-566頁(yè)，575-581和581-587頁(yè)；Michal，G.，(1999)Biochemical PathwaysAn Atlas ofBiochemistry and Molecular Biology，John Wiley and Sons；Fallon，A.等人(1987)，在Laboratory Techniques in Biochemistry and Molecular Biology，17卷的HPLC在生物化學(xué)中的應(yīng)用中概述。
下面的非限制性實(shí)施例和附圖更詳細(xì)時(shí)描述本發(fā)明

圖1顯示了質(zhì)粒pClysC的質(zhì)粒圖；圖2顯示了質(zhì)粒pCISlysCthr311ile的質(zhì)粒圖；圖3顯示了質(zhì)粒pC_metA_Cd的質(zhì)粒圖；限制性切割位點(diǎn)及它們各自的位置(在括號(hào)中)在質(zhì)粒圖中顯示。必需的序列片段以粗體印刷。KanR指卡那霉素抗性基因；ask指天冬氨酸激酶基因。
實(shí)施例1pCLiK5MCS的構(gòu)建首先，使用寡核苷酸p1.3(SEQ ID NO47)和p2.3(SEQ ID NO48)，利用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增載體pBR322的氨芐青霉素抗性和復(fù)制起點(diǎn)。
p1.3(SEQ ID NO47)5‘-CCCGGGATCCGCTAGCGGCGCGCCGGCCGGCCCGGTGTGAAATACCGCACAG-3‘p2.3(SEQ ID NO48)5‘-TCTAGACTCGAGCGGCCGCGGCCGGCCTTTAAATTGAAGACGAAAGGGCCTCG-3‘寡核苷酸p1.3(SEQ ID NO47)除了含有與pBR322互補(bǔ)的序列外，還含有5’-3’方向限制性核酸酶SmaI、BamHI、NheI和AscI的切割位點(diǎn)，寡核苷酸p2.3(SEQ ID NO48)含有5’-3’方向限制性核酸內(nèi)切酶XbaI、XhoI、NotI和DraI的切割位點(diǎn)。根據(jù)標(biāo)準(zhǔn)方法如Innis等(PCR Protocols.A Guide to Methods and Applications，Academic Press(1990))使用PfuTurbo聚合酶(Stratagene，La Jolla，USA)實(shí)施PCR反應(yīng)。得到的大小約2.1kb的DNA片段用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明純化。使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據(jù)生產(chǎn)商的使用說(shuō)明將DNA片段的鈍端相互連接并根據(jù)標(biāo)準(zhǔn)方法，如Sambrook等人(分子克隆實(shí)驗(yàn)指南，冷泉港實(shí)驗(yàn)室，(1989))中描述方法將連接混合物轉(zhuǎn)化到感受態(tài)大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中。通過(guò)將細(xì)胞涂在含有氨芐青霉素(50μg/ml)的LB瓊脂(Lennox，1955，Virology，1190)板選擇攜帶質(zhì)粒的細(xì)胞。
使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離單個(gè)克隆的質(zhì)粒DNA并將它們通過(guò)限制性消化檢查。以這種方法得到的質(zhì)粒稱為pCLiK1。
以質(zhì)粒pwLT1(Liebl等，1992)作為PCR反應(yīng)的模板開(kāi)始，使用寡核苷酸neol(SEQ ID NO49)和neo2(SEQ ID NO50)擴(kuò)增卡那霉素抗性盒。
neo1(SEQ ID NO49)5‘-GAGATCTAGACCCGGGGATCCGCTAGCGGGCTGCTAAAGGAAGCGGA-3‘neo2(SEQ ID NO50)5‘-GAGAGGCGCGCCGCTAGCGTGGGCGAAGAACTCCAGCA-3‘寡核苷酸neol除了含有與pWLT1互補(bǔ)的序列外，還含有5’-3’方向限制性內(nèi)切酶XbaI、SmaI、BamHI、NheI的切割位點(diǎn)，寡核苷酸neo2(SEQID NO50)含有5’-3’方向限制性內(nèi)切酶AscI和NheI的切割位點(diǎn)。使用PfuTurbo聚合酶(Stratagene，La Jolla，USA)根據(jù)標(biāo)準(zhǔn)方法如Innis等(PCR Protocols.A Guide to Methods and Applications，Academic Press(1990))的方法實(shí)施PCR反應(yīng)。得到的約1.3kb大小的DNA片段用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)純化。DNA片段用限制性內(nèi)切酶XbaI和AscI(New England Biolabs，Beverly，USA)切割并且，之后，再次用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)純化。載體pCLiK1也用限制性內(nèi)切酶XbaI和AscI切割并使用堿性磷酸酶(Roche Diagnostics，Mannheim)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)去磷酸。在0.8％強(qiáng)度的瓊脂糖凝膠中電泳后，線性化載體(約2.1kb)使用GFXTMPCR、DNA和凝膠帶純化試劑盒(AmershamPharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離。該載體片段使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據(jù)生產(chǎn)商的使用說(shuō)明用切割的PCR片段連接并將根據(jù)標(biāo)準(zhǔn)方法，如Sambrook等(分子克隆實(shí)驗(yàn)指南，冷泉港，(1989))中描述的方法將連接混合物轉(zhuǎn)化到感受態(tài)大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中。通過(guò)涂在含有氨芐青霉素(50μg/ml)和卡那霉素(20μg/ml)的LB瓊脂(Lennox，1955，Virology，1190)板上選擇攜帶質(zhì)粒的細(xì)胞。
使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離單個(gè)克隆的質(zhì)粒DNA并將它們通過(guò)限制性消化檢查。以這種方法得到的質(zhì)粒稱為pCLiK2。
載體pCLiK2用限制性內(nèi)切酶DraI(New England Biolabs，Beverly，USA)切割。在0.8％強(qiáng)度的瓊脂糖凝膠中電泳后，使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離約2.3kb載體片段。該載體片段使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據(jù)生產(chǎn)商的使用說(shuō)明重新連接并將根據(jù)標(biāo)準(zhǔn)方法，如Sambrook等(分子克隆實(shí)驗(yàn)指南，冷泉港，(1989))中描述的方法將連接混合物轉(zhuǎn)化到感受態(tài)大腸桿菌XL-1Blue(Stratagene，La JoHa，USA)中。通過(guò)涂在含有卡那霉素(20μg/ml)的LB瓊脂(Lennox，1955，Virology，1190)板上選擇攜帶質(zhì)粒的細(xì)胞。
使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離單個(gè)克隆的質(zhì)粒DNA并將它們通過(guò)限制性消化檢查。以這種方法得到的質(zhì)粒稱為pCLiK3。
以質(zhì)粒pWLQ2(Liebl等，1992)作為PCR反應(yīng)的模板開(kāi)始，使用寡核苷酸cg1(SEQ ID NO51)和cg2(SEQ ID NO52)擴(kuò)增復(fù)制起點(diǎn)pHM1519。
cg1(SEQ ID NO51)5‘-GAGAGGGCGGCCGCGCAAAGTCCCGCTTCGTGAA-3‘
cg2(SEQ ID NO52)5‘-GAGAGGGCGGCCGCTCAAGTCGGTCAAGCCACGC-3‘寡核苷酸cg1(SEQ ID NO51)和cg2(SEQ ID NO52)除了含有與pWLQ2互補(bǔ)的序列外，還含有限制性內(nèi)切酶NotI的切割位點(diǎn)。使用PfuTurbo聚合酶(Stratagene，La Jolla，USA)根據(jù)標(biāo)準(zhǔn)方法如Innis等(PCR Protocols.A Guide to Methods and Applications，Academic Press(1990))的方法實(shí)施PCR反應(yīng)。得到DNA片段大小約2.7kb并用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)純化。DNA片段用限制性內(nèi)切酶NotI(NewEngland Biolabs，Beverly，USA)切割，并且之后再次用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)純化。載體pCLiK3也用限制性內(nèi)切酶NotI切割并使用堿性磷酸酶(Roche Diagnostics，Mannheim)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)去磷酸。在0.8％強(qiáng)度的瓊脂糖凝膠中電泳后，線性化載體(約2.3kb)使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離。該載體片段使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據(jù)生產(chǎn)商的使用說(shuō)明用切割的PCR片段連接并根據(jù)標(biāo)準(zhǔn)方法，如Sambrook等(分子克隆實(shí)驗(yàn)指南，冷泉港，(1989))中描述的方法將連接混合物轉(zhuǎn)化到感受態(tài)大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中。通過(guò)涂在含有卡那霉素(20μg/ml)的LB瓊脂(Lennox，1955，Virology，1190)板上選擇攜帶質(zhì)粒的細(xì)胞。
使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離單個(gè)克隆的質(zhì)粒DNA并將它們通過(guò)限制性消化檢查。以這種方法得到的質(zhì)粒稱為pCLiK5。
通過(guò)組合兩種合成的基本互補(bǔ)的寡核苷酸HS445((SEQ ID NO53)和HS446(SEQ ID NO54))通過(guò)多克隆位點(diǎn)(MCS)延伸PCLik5，HS445和HS446含有限制性內(nèi)切酶SwaI、XhoI、AatI、ApaI、Asp718、MluI、NdeI、SpeI、EcoRV、SalI、ClaI、BamHI、XbaI和SmaI的切割位點(diǎn)，延伸后通過(guò)將它們一起加熱到95℃，然后緩慢冷卻得到雙鏈DNA片段。
HS445(SEQ ID NO53)5‘-TCGAATTTAAATCTCGAGAGGCCTGACGTCGGGCCCGGTACCACGCGTCATATGACTAGTTCGGACCTAGGGATATCGTCGACATCGATGCTCTTCTGCGTTAATTAACAATTGGGATCCTCTAGACCCGGGATTTAAAT-3‘HS446(SEQ ID NO54)5‘-GATCATTTAAATCCCGGGTCTAGAGGATCCCAATTGTTAATTAACGCAGAAGAGCATCGATGTCGACGATATCCCTAGGTCCGAACTAGTCATATGACGCGTGGTACCGGGCCCGACGTCAGGCCTCTCGAGATTTAAAT-3‘載體pCLiK5用限制性內(nèi)切酶XhoI和BamHI(New England Biolabs，Beverly，USA)切割并用堿性磷酸酶(I(Roche Diagnostics，Mannheim))根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)去磷酸。在0.8％強(qiáng)度的瓊脂糖凝膠中電泳后，線性化載體(約5.0kb)使用GFXTMPCR、DNA和凝膠帶純化試劑盒(AmershamPharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離。該載體片段使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據(jù)生產(chǎn)商的使用說(shuō)明與合成的雙鏈DNA片段連接，并根據(jù)如Sambrook等(分子克隆實(shí)驗(yàn)指南，冷泉港，(1989))中描述的標(biāo)準(zhǔn)方法將連接混合物轉(zhuǎn)化到感受態(tài)大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中。通過(guò)涂在含有卡那霉素(20μg/ml)的LB瓊脂(Lennox，1955，Virology，1190)板上選擇攜帶質(zhì)粒的細(xì)胞。
使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離單個(gè)克隆的質(zhì)粒DNA并將它們通過(guò)限制性消化檢查。以這種方法得到的質(zhì)粒稱為pCLiK5MCS。
根據(jù)Sanger等人(1977)所述(Proceedings of the National Academy ofSciences USA 745463-5467)實(shí)施測(cè)序反應(yīng)。分段進(jìn)行測(cè)序反應(yīng)并通過(guò)ABIPrism 377(PE Applied Biosystems，Weiterstadt)分析。
所得質(zhì)粒pCLiK5MCS如SEQ ID NO57所示。
實(shí)施例2pCLiK5MCS integrativ sacB的構(gòu)建以質(zhì)粒pK19mob(Sch_fer等人，Gene 145，69-73(1994))作為模板開(kāi)始PCR反應(yīng)，使用寡核苷酸BK1732和BK1733擴(kuò)增枯草芽孢桿菌sacB基因(編碼果聚糖蔗糖酶)。
BK1732(SEQ ID NO55)5‘-GAGAGCGGCCGCCGATCCTTTTTAACCCATCAC-3‘BK1733(SEQ ID NO56)5‘-AGGAGCGGCCGCCATCGGCATTTTCTTTTGCG-3‘寡核苷酸BK1732和BK1733除了含有與pEK19mobsac互補(bǔ)的序列外，還含有限制性內(nèi)切酶NotI的切割位點(diǎn)。使用PfuTurbo聚合酶(Stratagene，La Jolla，USA)根據(jù)標(biāo)準(zhǔn)方法如Innis等(PCR Protocols.AGuide to Methods and Applications，Academic Press(1990))的方法實(shí)施PCR反應(yīng)。得到大小約1.9kb的DNA片段用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)純化。DNA片段用限制性內(nèi)切酶NotI(New England Biolabs，Beverly，USA)切割并且，之后再次用GFXTMPCR、DNA和凝膠帶純化試劑盒(AmershamPharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)純化。
載體pCLiK5MCS(根據(jù)實(shí)施例1制備)也用限制性內(nèi)切酶NotI切割并使用堿性磷酸酶(I(Roche Diagnostics，Mannheim))根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)去磷酸。在0.8％強(qiáng)度的瓊脂糖凝膠中電泳后，使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離約2.4kb大小的載體。該載體片段使用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)根據(jù)生產(chǎn)商的使用說(shuō)明與切割的PCR片段連接并根據(jù)標(biāo)準(zhǔn)方法，如Sambrook等(分子克隆實(shí)驗(yàn)指南，冷泉港，(1989))中描述的方法將連接混合物轉(zhuǎn)化到感受態(tài)大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中。通過(guò)涂在含有卡那霉素(20μg/ml)的LB瓊脂(Lennox，1955，Virology，1190)板上選擇攜帶質(zhì)粒的細(xì)胞。
使用Qiaprep spin miniprep試劑盒(Qiagen，Hilden)根據(jù)生產(chǎn)商的使用說(shuō)明書(shū)分離單個(gè)克隆的質(zhì)粒DNA并將它們通過(guò)限制性消化檢查。以這種方法得到的質(zhì)粒稱為pCLiK5MCS integrativ sacB。
根據(jù)Sanger等人(1977)所述(Proceedings of the National Academy ofSciences USA 745463-5467)實(shí)施測(cè)序反應(yīng)。分段進(jìn)行測(cè)序反應(yīng)并通過(guò)ABIPrism 377(PE Applied Biosystems，Weiterstadt)分析。
所得質(zhì)粒pCLiK5MCS integrativ sacB如SEQ ID NO58所示。
可以類(lèi)似方式制備適于metA基因的發(fā)明性表達(dá)或過(guò)量產(chǎn)生的其他載體。
實(shí)施例3從谷氨酸棒桿菌菌株LU1479分離lysC基因菌株構(gòu)建的第一步計(jì)劃為谷氨酸棒桿菌ATCC13032(以下稱為L(zhǎng)U1479)中編碼天冬氨酸激酶的lysC野生型基因的等位基因替換。計(jì)劃在lysC基因中實(shí)施核苷酸替換從而在所得蛋白質(zhì)中，311位的氨基酸Thr改變?yōu)榘被酙le。
以來(lái)自LU1479的染色體DNA作為模板開(kāi)始PCR反應(yīng)，用寡核苷酸引物SEQ ID NO59和SEQ ID NO60 lysC，利用Pfu-Turbo PCR系統(tǒng)(Stratagene USA)按照生產(chǎn)商的使用說(shuō)明書(shū)實(shí)施擴(kuò)增。如Tauch等人(1995)Plasmid 33168-179或Eikmanns等人(1994)Microbiology 1401817-1828描述的制備谷氨酸棒桿菌ATCC 13032的染色體DNA。所擴(kuò)增的片段的5，端側(cè)翼位為SalI限制性切割，其3，端側(cè)翼位為MluI限制性切割?？寺∏埃瑪U(kuò)增的片段用這兩種限制酶消化并用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)純化。
SEQ ID NO595‘-GAGAGAGAGACGCGTCCCAGTGGCTGAGACGCATC-3‘SEQ ID NO605‘-CTCTCTCTGTCGACGAATTCAATCTTACGGCCTG-3‘所得多核苷酸通過(guò)SalI和MluI切割被克隆到pCLIK5MCS integrativSacB(此后稱為pCIS；實(shí)施例2的SEQ ID NO58)并被轉(zhuǎn)化到大腸桿菌XL-1blue中。通過(guò)涂含有卡那霉素(20μg/ml)的LB瓊脂(Lennox，1955，Virology，1190)板實(shí)現(xiàn)對(duì)攜帶質(zhì)粒的細(xì)胞的選擇。分離質(zhì)粒并通過(guò)測(cè)序驗(yàn)證預(yù)期的核苷酸序列。通過(guò)Quiagen的方法并使用來(lái)自Quiagen的材料制備質(zhì)粒DNA。如Sanger等人(1977)Proceedings of the National Academyof Sciences USA 745463-5467描述的實(shí)施測(cè)序反應(yīng)。使用ABI Prism 377(PE Applied Biosystems，Weiterstadt)分離測(cè)序反應(yīng)物并對(duì)其評(píng)定。所得質(zhì)粒pCIS lysC如SEQ ID NO61所示。相應(yīng)的質(zhì)粒圖在圖1中顯示。
序列SEQ ID NO61包括下面的必需部分-區(qū)域基因座pCIS\lysC 5860bpDNA 環(huán)狀特征 定位/定義(Qualifiers)CDS1)155..1420/vntifkey＝″4″/label＝lysCCDS 互補(bǔ)的2)(3935..5356)/vntifkey＝″4″/label＝sacB\(枯草芽孢桿菌)啟動(dòng)子 互補(bǔ)的(5357..5819)/vntifkey＝″30″/label＝啟動(dòng)子\sacBC_region 互補(bǔ)的(3913..3934)/vntifkey＝″2″/label＝sacB\下游區(qū)CDS 1974..2765/vntifkey＝″4″/label＝Kan\RCDS 互補(bǔ)的(3032..3892)/vntifkey＝″4″/label＝Ori\-EC\(pMB)1)編碼序列
2)在互補(bǔ)鏈上實(shí)施例4谷氨酸棒桿菌lysC基因的誘變使用QuickChange試劑盒(Stratagene/USA)按照生產(chǎn)商的使用說(shuō)明書(shū)實(shí)施谷氨酸棒桿菌lysC基因(實(shí)施例3)的定點(diǎn)誘變。在質(zhì)粒pCIS lysC，SEQID NO61中實(shí)施誘變。利用Quickchange方法(Stratagene)合成了下面的寡核苷酸引物，其用于將thr311變?yōu)?11ile。
SEQ ID NO625‘-CGGCACCACCGACATCATCTTCACCTGCCCTCGTTCCG-3‘SEQ ID NO635‘-CGGAACGAGGGCAGGTGAAGATGATGTCGGTGGTGCCG-3‘Quickchange反應(yīng)中這些寡核苷酸引物的使用導(dǎo)致lysC基因中932位核苷酸的替換(用T代替C)(參考SEQ ID NO64)和相應(yīng)酶中311位氨基酸的替換(Thr→Ile)(參考SEQ ID NO65)。LysC基因中所得氨基酸替換Thr311Ile通過(guò)轉(zhuǎn)化到大腸桿菌XL1-blue和質(zhì)粒制備后測(cè)序來(lái)檢驗(yàn)。該質(zhì)粒稱為pCIS lysC thr311ile并且如SEQ ID NO66所示。相應(yīng)的質(zhì)粒圖在圖2中顯示。
序列SEQ ID NO66包括下面的必需部分區(qū)域基因座 pCIS\lysC\thr311ile 5860bpDNA環(huán)狀特征定位/定義CDS1)155..1420/vntifkey＝″4″/label＝lysCCDS 互補(bǔ)的2)(3935..5356)/vntfkey＝″4″/label＝sacB\(枯草芽孢桿菌)啟動(dòng)子 互補(bǔ)的(5357..5819)
/vntifkey＝″30″/label＝啟動(dòng)子\sacBC_region 互補(bǔ)的(3913..3934)/vntifkey＝″2″/label＝sacB\下游區(qū)CDS1974..2765/vntifkey＝″4″/label＝Kan\RCDS互補(bǔ)的(3032..3892)/vntifkey＝″4″/label＝Ori\-EC\(pMB)1)編碼區(qū)2)在互補(bǔ)鏈上通過(guò)如Liebl，等人(1989)FEMS Microbiology Letters 53299-303描述的電穿孔法將質(zhì)粒pCIS lysC thr311ile轉(zhuǎn)化到谷氨酸棒桿菌LU1479中。方案的修改在DE-A-10046870中描述。使用標(biāo)準(zhǔn)方法通過(guò)如Sambrook等人((1989)，分子克隆實(shí)驗(yàn)指南，冷泉港)描述的DNA印跡和雜交檢查單個(gè)轉(zhuǎn)化體的lysC基因座的染色體排列。從而確保轉(zhuǎn)化體為具有通過(guò)同源重組整合在lysC基因座的被轉(zhuǎn)化質(zhì)粒的轉(zhuǎn)化體。這些菌落在沒(méi)有抗生素的培養(yǎng)基中生長(zhǎng)過(guò)夜后，將細(xì)胞涂布在蔗糖-CM瓊脂培養(yǎng)基(10％蔗糖)的平板上并在30℃孵育24小時(shí)。
因?yàn)榇嬖谟谳d體pCIS lysC thr311ile中的sacB基因?qū)⒄崽寝D(zhuǎn)化成毒性產(chǎn)物，所以僅僅那些通過(guò)另一同源重組步驟將野生型lysC基因和突變的基因lysC thr311ile之間的sacB基因缺失的菌落能夠生長(zhǎng)。野生型基因或者突變基因與sacB基因一起可以在同源重組過(guò)程中被缺失。當(dāng)sacB基因與野生型基因一起被除去時(shí)，產(chǎn)生突變的轉(zhuǎn)化體。
挑選生長(zhǎng)菌落并檢查卡那霉素敏感表型。缺失SacB基因的克隆必須同時(shí)表現(xiàn)出卡那霉素-敏感的生長(zhǎng)行為。在搖瓶中研究這種Kan-敏感克隆的賴氨酸產(chǎn)量(見(jiàn)實(shí)施例6)。生長(zhǎng)未處理的菌株LU1479用于比較目的。選擇賴氨酸產(chǎn)量比對(duì)照增加的克隆，得到染色體DNA，并通過(guò)PCR反應(yīng)擴(kuò)增lysC基因的相應(yīng)區(qū)域并測(cè)序。具有增加的賴氨酸合成和具有l(wèi)ysC中932位經(jīng)證實(shí)突變的這種克隆之一稱為L(zhǎng)U1479lysC 311ile。
實(shí)施例5乙硫氨酸抗性谷氨酸棒桿菌菌株的產(chǎn)生在第二個(gè)菌株構(gòu)建步驟，處理所得菌株LU1479lysC 311ile(實(shí)施例4)以誘導(dǎo)對(duì)乙硫氨酸的抗性(Kase，H.Nakayama K.Agr.Biol.Chem.39，153-106，1975)，通過(guò)谷氨酸棒桿菌的甲硫氨酸類(lèi)似物抗性突變株產(chǎn)生L-甲硫氨酸)BHI培養(yǎng)基(Difco)中的過(guò)夜培養(yǎng)物用檸檬酸鹽緩沖液(50mMpH 5.5)洗滌并在30℃用N-甲基亞硝基胍(50mM檸檬酸鹽pH 5.5中10mg/ml)處理20分鐘。用化學(xué)誘變劑N-甲基亞硝基胍處理后，洗滌(檸檬酸鹽緩沖液50mM pH 5.5)細(xì)胞并將其涂布在含有下面組分的培養(yǎng)基平板上，在500ml中含有10g(NH4)2SO4、0.5g KH2PO4、0.5g K2HPO4、0.125gMgSO4·7H2O、21g MOPS、50mg CaCl2、15mg原兒茶酸(proteocatechuate)、0.5mg生物素、1mg硫胺素、5g/l D，L-乙硫氨酸(SigmaChemicals Deutschland)，pH 7.0。此外，培養(yǎng)基含有0.5ml微量鹽溶液，其由10g/l FeSO4·7H2O、1g/l MnSO4·H2O、0.1g/l ZnSO4·7H2O、0.02g/lCuSO4、0.002g/l NiCl2·6H2O組成，所有鹽溶于0.1M HCl中。所完成的培養(yǎng)基通過(guò)過(guò)濾除菌，并且加入40ml無(wú)菌50％葡萄糖溶液后，加入液體無(wú)菌瓊脂，其終濃度為1.5％瓊脂，并將混合物倒入培養(yǎng)皿中。
將已經(jīng)經(jīng)歷誘變處理的細(xì)胞應(yīng)用于含有上述培養(yǎng)基的平板并在30℃孵育3-7天。分離所得克隆，單個(gè)克隆在選擇培養(yǎng)基上分離至少一次然后在裝有培養(yǎng)基II(見(jiàn)實(shí)施例6)的搖瓶中分析它們的甲硫氨酸產(chǎn)量。
實(shí)施例6使用菌株LU1479lysC 311ile ET-16制備甲硫氨酸實(shí)施例5中產(chǎn)生的菌株在含有CM培養(yǎng)基的瓊脂板上于30℃生長(zhǎng)2天。
CM瓊脂10.0g/l D-葡萄糖、2.5g/l NaCl、2.0g/l尿素、10.0g/l細(xì)菌培養(yǎng)用蛋白胨(Difco)、5.0g/l酵母提取物(Difco)、5.0g/l牛肉膏(Difco)、22.0g/l瓊脂(Difco)，高壓滅菌(20分鐘，121℃)細(xì)胞隨后從平板刮下并重懸在鹽水中。對(duì)于主培養(yǎng)，向100ml錐形燒瓶中的10ml培養(yǎng)基II和0.5g高壓滅菌的CaCO3(Riedel de Haen)接種細(xì)胞懸浮物至OD 600nm為1.5并在有軌搖床上以200轉(zhuǎn)/分鐘在30℃下孵育72小時(shí)。
培養(yǎng)基II40g/l蔗糖60g/l糖蜜(基于100％糖含量)10g/l(NH4)2SO40.4g/l MgSO4·7H2O0.6g/l KH2PO40.3mg/l 硫胺素·HCl1mg/l生物素(來(lái)自用NH4OH調(diào)節(jié)至pH 8.0的1mg/ml過(guò)濾除菌的母液)2mg/lFeSO42mg/lMnSO4用NH4OH建立7.8的pH并將混合物高壓滅菌(121℃，20分鐘)。此外，加入來(lái)自母液(200μg/ml，過(guò)濾除菌的)的維生素B12(羥鈷胺素，SigmaChemicals)至終濃度100μg/l。
使用Agilent氨基酸確定方法在Agilent 1100 Series LC System HPLC上確定培養(yǎng)液中形成的甲硫氨酸以及其他氨基酸。用正-鄰苯二醛(ortho-phtalaldehyde)柱前衍生使得可以確定所形成的氨基酸量。氨基酸混合物在柱上分離。氨基酸混合物在Hypersil AA柱(Agilent)上分離。
分離甲硫氨酸產(chǎn)量比原始菌株LU1479lysC 311ile的產(chǎn)量高至少2倍的克隆。一個(gè)這種克隆用于進(jìn)一步的實(shí)驗(yàn)中，并命名為L(zhǎng)U1479lysC 311ileET-16。
實(shí)施例7從白喉棒桿菌克隆metA并克隆到質(zhì)粒pC metA_Cd中白喉棒桿菌的染色體DNA得自美國(guó)典型菌株培養(yǎng)物保藏中心(ATCC，Atlanta-USA)，來(lái)自菌株ATCC 700971，目錄號(hào)700971D。
使用寡核苷酸引物SEQ ID NO67和SEQ ID NO68、作為模板的白喉棒桿菌染色體DNA和Pfu Turbo聚合酶(Stratagene)，通過(guò)聚合酶鏈?zhǔn)椒磻?yīng)(PCR)，按照標(biāo)準(zhǔn)方法如Innis等人(1990)PCR Protocols.A Guide toMethods and Applications，Academic Press擴(kuò)增了約1.4kb的DNA片段，其含有包括5’非編碼區(qū)(啟動(dòng)子區(qū))的metA基因。擴(kuò)增的片段在其5’端側(cè)翼為XhoI限制性切割位點(diǎn)，其3’末端側(cè)翼為Nde I限制性切割位點(diǎn)，這些位點(diǎn)已經(jīng)通過(guò)寡核苷酸引物導(dǎo)入。
SEQ ID NO675’-GAGACTCGAGGTTGGCTGGTCATCATAGG-3’和SEQ ID NO685’GAAGAGAGCATATGTCAGCGCTCTAGTTTGGTTC-3’所得DNA片段用GFXTMPCR、DNA和凝膠帶純化試劑盒(AmershamPharmacia，F(xiàn)reiburg)按照生產(chǎn)商的使用說(shuō)明書(shū)純化。此后，將其用限制酶XhoI和Nde I(Roche Diagnostics，Mannheim)切割并通過(guò)凝膠電泳分離。隨后使用GFXTMPCR、DNA和凝膠帶純化試劑盒(Amersham Pharmacia，F(xiàn)reiburg)從瓊脂糖分離約1.4kb DNA片段。
載體pClik5MCS SEQ ID NO57，此后稱為pC，用限制酶XhoI和Nde I(Roche Diagnostics，Mannheim)切割，并將約5kb片段通過(guò)電泳分開(kāi)然后使用GFXTMPCR、DNA和凝膠帶純化試劑盒分離。
利用快速DNA連接試劑盒(Roche Diagnostics，Mannheim)按照生產(chǎn)商的使用說(shuō)明書(shū)將載體片段與PCR片段連接，使用如Sambrook等人(分子克隆實(shí)驗(yàn)指南，冷泉港，(1989))描述的標(biāo)準(zhǔn)方法將連接反應(yīng)物轉(zhuǎn)化到感受態(tài)大腸桿菌XL-1Blue(Stratagene，La Jolla，USA)中。通過(guò)涂布在含有卡那霉素(20μg/ml)的LB瓊脂(Lennox，1955，Virology，1190)板上實(shí)現(xiàn)含有質(zhì)粒的細(xì)胞的選擇。
使用Quiagen的方法和來(lái)自Quiagen的材料制備質(zhì)粒DNA。如Sanger等人(1977)Proceedings of the National Academy of Sciences USA745463-5467描述的實(shí)施測(cè)序反應(yīng)。通過(guò)ABI Prism 377(PE AppliedBiosystems，Weiterstadt)分離和評(píng)價(jià)測(cè)序反應(yīng)物。
所得質(zhì)粒pC metA_Cd(白喉棒桿菌)如SEQ ID NO69所示。相應(yīng)的質(zhì)粒圖在圖3中顯示。
基因座 pC_metA_Cd 6472bpDNA 環(huán)狀特征 定位/定義CDS313..1416/vntifkey＝″4″/label＝metA\白喉棒桿菌CDS1838..2629/vntifkey＝″4″/label＝Kan\RCDS4910..6031/vntifkey＝″4″/label＝Rep\蛋白質(zhì)CDS3902..4576/vntifkey＝″4″/label＝ORF\1CDS互補(bǔ)的(2896..3756)/vntifkey＝″4″/label＝Ori\-EC\(pMB)實(shí)施例8用質(zhì)粒pC metA_Cd轉(zhuǎn)化菌株LU1479lysC 311ile ET-16通過(guò)上述方法(Liebl，等人(1989)FEMS Microbiology Letters53299-303)用質(zhì)粒pC metA_Cd轉(zhuǎn)化菌株LU1479lysC 311ile ET-16。將轉(zhuǎn)化混合物涂布到額外含有20mg/l卡那霉素的CM板上以得到對(duì)含有質(zhì)粒的細(xì)胞的選擇。挑選所得Kan-抗性克隆并分離單個(gè)克隆。在搖瓶實(shí)驗(yàn)中研究克隆的甲硫氨酸產(chǎn)量(見(jiàn)實(shí)施例6)。菌株LU1479lysC 311ile ET-16pCmetA_Cd與LU1479lysC 311ile ET-16相比產(chǎn)生明顯更多的甲硫氨酸。
序列表<110>巴斯福股份公司<120>發(fā)酵產(chǎn)生含硫精細(xì)化學(xué)品的方法(metA)<130>M/43127<140>
<141>
<160>58<210>1<211>1104<212>DNA<213>白喉棒桿菌(corynebacterium diphteriae)<220>
<221>CDS<222>(1)..(1101)<223>RDI00386<400>1atg ctc acc acc aca ggg acg ctc acg cac caa aaa atc gga gac ttt48Met Leu Thr Thr Thr Gly Thr Leu Thr His Gln Lys Ile Gly Asp Phe1 5 10 15tac acc gaa gcc gga gcg acg ctt cac gac gta acc atc gcc tac caa96Tyr Thr Glu Ala Gly Ala Thr Leu His Asp Val Thr Ile Ala Tyr Gln20 25 30gca tgg ggc cac tac acc ggc acc aat ctc atc gtt ctc gaa cat gcc144Ala Trp Gly His Tyr Thr Gly Thr Asn Leu Ile Val Leu Glu His Ala35 40 45ctg acc ggc gac tct aac gct att tca tgg tgg gac gga ctg att ggc192Leu Thr Gly Asp Ser Asn Ala Ile Ser Trp Trp Asp Gly Leu Ile Gly50 55 60cct ggc aaa gca ctc gac acc aac cgc tac tgc atc cta tgc acc aac240Pro Gly Lys Ala Leu Asp Thr Asn Arg Tyr Cys Ile Leu Cys Thr Asn65 70 75 80gtg ctc gga gga tgc aaa gga tcc acc gga ccg agc agt cca cac cca288Val Leu Gly Gly Cys Lys Gly Ser Thr Gly Pro Ser Ser Pro His Pro85 90 95gac gga aaa cca tgg gga tcc aga ttt cca gcc ctt tca atc cgt gac336Asp Gly Lys Pro Trp Gly Ser Arg Phe Pro Ala Leu Ser Ile Arg Asp100 105 110ctt gtc aat gcc gaa aaa caa ctt ttc gac cac ctc ggc atc aat aaa384Leu Val Asn Ala Glu Lys Gln Leu Phe Asp His Leu Gly Ile Asn Lys115 120 125att cac gca atc atc ggc gga tcc atg gga ggc gca cgc acc ctc gaa432Ile His Ala Ile Ile Gly Gly Ser Met Gly Gly Ala Arg Thr Leu Glu130 135 140tgg gct gca ctc cac cca cac atg atg acg act gga ttc gtc ata gca 480Trp Ala Ala Leu His Pro His Met Met Thr Thr Gly Phe Val Ile Ala145 150 155 160
gtc tca gca cgc gca agc gct tgg caa atc ggt att caa act gca caa528Val Ser Ala Arg Ala Ser Ala Trp Gln Ile Gly Ile Gln Thr Ala Gln165 170 175atc agc gcc ata gaa ctc gac ccc cac tgg aac ggc ggc gat tac tac576Ile Ser Ala Ile Glu Leu Asp Pro His Trp Asn Gly Gly Asp Tyr Tyr180 185 190agc ggt cac gca cca tgg gaa gga atc gcc gcc gct cgc cgg atc gcc624Ser Gly His Ala Pro Trp Glu Gly Ile Ala Ala Ala Arg Arg Ile Ala195 200 205cac ctc acc tat cgc ggc gaa cta gaa ata gac gaa cga ttc ggc act672His Leu Thr Tyr Arg Gly Glu Leu Glu Ile Asp Glu Arg Phe Gly Thr210 215 220tcc gca caa cac ggt gaa aac cca ctc ggc ccc ttc cga gat cca cat720Ser Ala Gln His Gly Glu Asn Pro Leu Gly Pro Phe Arg Asp Pro His225 230 235 240caa cgt ttt gcg gtc acg agc tac ctc caa cac caa ggc atc aaa ctc768Gln Arg Phe Ala Val Thr Ser Tyr Leu Gln His Gln Gly Ile Lys Leu245 250 255gct caa cga ttc gat gca ggt agt tac gtc gtg ctt acc gaa gcc ctc816Ala Gln Arg Phe Asp Ala Gly Ser Tyr Val Val Leu Thr Glu Ala Leu260 265 270aat cgt cat gac atc gga cgc ggc cga ggc gga ctc aac aaa gcc ctc864Asn Arg His Asp Ile Gly Arg Gly Arg Gly Gly Leu Asn Lys Ala Leu275 280 285agc gca atc aca gtc ccc atc atg att gct ggc gtt gat acc gat att912Ser Ala Ile Thr Val Pro Ile Met Ile Ala Gly Val Asp Thr Asp Ile290 295 300ctc tac ccc tat cac cag caa gaa cac cta tca cga aat cta ggc aac960Leu Tyr Pro Tyr His Gln Gln Glu His Leu Ser Arg Asn Leu Gly Asn305 310 315 320cta ctc gct atg gca aaa atc agc tca cca gta ggc cac gac gct ttc1008Leu Leu Ala Met Ala Lys Ile Ser Ser Pro Val Gly His Asp Ala Phe325 330 335ctc aca gaa ttc cga caa atg gag cga atc cta aga cat ttc atg gag1056Leu Thr Glu Phe Arg Gln Met Glu Arg Ile Leu Arg His Phe Met Glu340 345 350ctt tcg gaa gga atc gac gat tcc ttc cga acc aaa cta gag cgc1101Leu Ser Glu Gly Ile Asp Asp Ser Phe Arg Thr Lys Leu Glu Arg355 360 365tga1104<210>2<211>367<212>PRT<213>白喉棒桿菌<400>2Met Leu Thr Thr Thr Gly Thr Leu Thr His Gln Lys Ile Gly Asp Phe1 5 10 15Tyr Thr Glu Ala Gly Ala Thr Leu His Asp Val Thr Ile Ala Tyr Gln20 25 30
Ala Trp Gly His Tyr Thr Gly Thr Asn Leu Ile Val Leu Glu His Ala35 40 45Leu Thr Gly Asp Ser Asn Ala Ile Ser Trp Trp Asp Gly Leu Ile Gly50 55 60Pro Gly Lys Ala Leu Asp Thr Asn Arg Tyr Cys Ile Leu Cys Thr Asn65 70 75 80Val Leu Gly Gly Cys Lys Gly Ser Thr Gly Pro Ser Ser Pro His Pro85 90 95Asp Gly Lys Pro Trp Gly Ser Arg Phe Pro Ala Leu Ser Ile Arg Asp100 105 110Leu Val Asn Ala Glu Lys Gln Leu Phe Asp His Leu Gly Ile Asn Lys115 120 125Ile His Ala Ile Ile Gly Gly Ser Met Gly Gly Ala Arg Thr Leu Glu130 135 140Trp Ala Ala Leu His Pro His Met Met Thr Thr Gly Phe Val Ile Ala145 150 155 160Val Ser Ala Arg Ala Ser Ala Trp Gln Ile Gly Ile Gln Thr Ala Gln165 170 175Ile Ser Ala Ile Glu Leu Asp Pro His Trp Asn Gly Gly Asp Tyr Tyr180 185 190Ser Gly His Ala Pro Trp Glu Gly Ile Ala Ala Ala Arg Arg Ile Ala195 200 205His Leu Thr Tyr Arg Gly Glu Leu Glu Ile Asp Glu Arg Phe Gly Thr210 215 220Ser Ala Gln His Gly Glu Asn Pro Leu Gly Pro Phe Arg Asp Pro His225 230 235 240Gln Arg Phe Ala Val Thr Ser Tyr Leu Gln His Gln Gly Ile Lys Leu245 250 255Ala Gln Arg Phe Asp Ala Gly Ser Tyr Val Val Leu Thr Glu Ala Leu260 265 270Asn Arg His Asp Ile Gly Arg Gly Arg Gly Gly Leu Asn Lys Ala Leu275 280 285Ser Ala Ile Thr Val Pro Ile Met Ile Ala Gly Val Asp Thr Asp Ile290 295 300Leu Tyr Pro Tyr His Gln Gln Glu His Leu Ser Arg Asn Leu Gly Asn305 310 315 320Leu Leu Ala Met Ala Lys Ile Ser Ser Pro Val Gly His Asp Ala Phe325 330 335Leu Thr Glu Phe Arg Gln Met Glu Arg Ile Leu Arg His Phe Met Glu340 345 350Leu Ser Glu Gly Ile Asp Asp Ser Phe Arg Thr Lys Leu Glu Arg355 360 365<210>3
<211>1149<212>DNA<213>麻瘋分枝桿菌(Mycobacterium leprae)<220>
<221>CDS<222>(1)..(1146)<223>RML02951<220>
<221>不確定的<222>224..224<223>所出現(xiàn)的n表示任一核苷酸<400>3atg aca atc tcc aag gtc cct acc cag aag ctg ccg gcc gaa ggc gag48Met Thr Ile Ser Lys Val Pro Thr Gln Lys Leu Pro Ala Glu Gly Glu1 5 10 15gtc ggc ttg gtc gac atc ggc tca ctt acc acc gaa agc ggt gcc gtc96Val Gly Leu Val Asp Ile Gly Ser Leu Thr Thr Glu Ser Gly Ala Val20 25 30atc gac gat gtc tgc atc gcc gtt cag cgc tgg ggg gaa ttg tcg ccc144Ile Asp Asp Val Cys Ile Ala Val Gln Arg Trp Gly Glu Leu Ser Pro35 40 45acg cga gac aac gta gtg atg gta ctg cat gca ctc acc ggt gac tcg192Thr Arg Asp Asn Val Val Met Val Leu His Ala Leu Thr Gly Asp Ser50 55 60cac atc acc ggg ccc gcc gga ccg gga cat cnc aca ccc ggc tgg tgg240His Ile Thr Gly Pro Ala Gly Pro Gly His Xaa Thr Pro Gly Trp Trp65 70 75 80gac tgg ata gct gga ccg ggt gca cca atc gac acc aac cgc tgg tgc288Asp Trp Ile Ala Gly Pro Gly Ala Pro Ile Asp Thr Asn Arg Trp Cys85 90 95gcg ata gcc acc aac gtg ctg ggc ggt tgc cgt ggc tcc acc ggc cct336Ala Ile Ala Thr Asn Val Leu Gly Gly Cys Arg Gly Ser Thr Gly Pro100 105 110agt tcg ctt gcc cgc gac gga aag cct tgg ggt tca aga ttt ccg ctg384Ser Ser Leu Ala Arg Asp Gly Lys Pro Trp Gly Ser Arg Phe Pro Leu115 120 125ata tct ata cgc gac cag gta gag gca gat atc gct gca ctg gcc gcc432Ile Ser Ile Arg Asp Gln Val Glu Ala Asp Ile Ala Ala Leu Ala Ala130 135 140atg gga att aca aag gtt gcc gcc gtc gtt gga gga tct atg ggc ggg480Met Gly Ile Thr Lys Val Ala Ala Val Val Gly Gly Ser Met Gly Gly145 150 155 160gcg cgt gca ctg gaa tgg atc atc ggc cac ccg gac caa gtc cgg gcc528Ala Arg Ala Leu Glu Trp Ile Ile Gly His Pro Asp Gln Val Arg Ala165 170 175ggg ctg ttg ctg gcg gtc ggt gtg cgc gcc acc gcc gac cag atc ggc576Gly Leu Leu Leu Ala Val Gly Val Arg Ala Thr Ala Asp Gln Ile Gly180 185 190acc caa acc acc caa atc gca gcc atc aag aca gac ccg aac tgg caa624Thr Gln Thr Thr Gln Ile Ala Ala Ile Lys Thr Asp Pro Asn Trp Gln195 200 205
ggc ggt gac tac tac gag aca ggg agg gca cca gag aac ggc ttg aca 672Gly Gly Asp Tyr Tyr Glu Thr Gly Arg Ala Pro Glu Asn Gly Leu Thr210 215 220att gcc cgc cgc ttc gcc cac ctg acc tac cgc agc gag gtc gag ctc 720Ile Ala Arg Arg Phe Ala His Leu Thr Tyr Arg Ser Glu Val Glu Leu225 230 235 240gac acc cgg ttt gcc aac aac aac caa ggc aat gag gac ccg gcg acg 768Asp Thr Arg Phe Ala Asn Asn Asn Gln Gly Asn Glu Asp Pro Ala Thr245 250 255ggc ggg cgt tac gca gtg cag agt tac cta gag cac cag ggt gac aag816Gly Gly Arg Tyr Ala Val Gln Ser Tyr Leu Glu His Gln Gly Asp Lys260 265 270cta ttg gcc cgc ttt gac gca ggc agc tac gtg gtc ttg acc gaa acg864Leu Leu Ala Arg Phe Asp Ala Gly Ser Tyr Val Val Leu Thr Glu Thr275 280 285ctg aac agc cac gac gtt ggc cgg ggc cgc gga ggg atc ggt aca gcg912Leu Asn Ser His Asp Val Gly Arg Gly Arg Gly Gly Ile Gly Thr Ala290 295 300ctg cgc ggg tgc ccg gta ccg gtg gtg gtg ggt ggc att acc tcg gat960Leu Arg Gly Cys Pro Val Pro Val Val Val Gly Gly Ile Thr Ser Asp305 310 315 320cgg ctc tac cca ctg cgc ttg cag cag gag ctg gcc gag atg ctg ccg1008Arg Leu Tyr Pro Leu Arg Leu Gln Gln Glu Leu Ala Glu Met Leu Pro325 330 335ggc tgc acc ggg ctg cag gtt gta gac tcc acc tac ggg cac gac ggc1056Gly Cys Thr Gly Leu Gln Val Val Asp Ser Thr Tyr Gly His Asp Gly340 345 350ttc ctg gtg gaa tcc gag gcc gtc ggc aaa ttg atc cgt caa acc ctc1104Phe Leu Val Glu Ser Glu Ala Val Gly Lys Leu Ile Arg Gln Thr Leu355 360 365gaa ttg gcc gac gtg ggt tcc aag gaa gac gcg tgt tcg caa1146Glu Leu Ala Asp Val Gly Ser Lys Glu Asp Ala Cys Ser Gln370 375 380tga1149<210>4<211>382<212>PRT<213>麻瘋分枝桿菌<220>
<221>不確定的<222>75..75<223>所出現(xiàn)的Xaa表示任一氨基酸<400>4Met Thr Ile Ser Lys Val Pro Thr Gln Lys Leu Pro Ala Glu Gly Glu1 5 10 15Val Gly Leu Val Asp Ile Gly Ser Leu Thr Thr Glu Ser Gly Ala Val20 25 30Ile Asp Asp Val Cys Ile Ala Val Gln Arg Trp Gly Glu Leu Ser Pro
35 40 45Thr Arg Asp Asn Val Val Met Val Leu His Ala Leu Thr Gly Asp Ser50 55 60His Ile Thr Gly Pro Ala Gly Pro Gly His Xaa Thr Pro Gly Trp Trp65 70 75 80Asp Trp Ile Ala Gly Pro Gly Ala Pro Ile Asp Thr Asn Arg Trp Cys85 90 95Ala Ile Ala Thr Asn Val Leu Gly Gly Cys Arg Gly Ser Thr Gly Pro100 105 110Ser Ser Leu Ala Arg Asp Gly Lys Pro Trp Gly Ser Arg Phe Pro Leu115 120 125Ile Ser Ile Arg Asp Gln Val Glu Ala Asp Ile Ala Ala Leu Ala Ala130 135 140Met Gly Ile Thr Lys Val Ala Ala Val Val Gly Gly Ser Met Gly Gly145 150 155 160Ala Arg Ala Leu Glu Trp Ile Ile Gly His Pro Asp Gln Val Arg Ala165 170 175Gly Leu Leu Leu Ala Val Gly Val Arg Ala Thr Ala Asp Gln Ile Gly180 185 190Thr Gln Thr Thr Gln Ile Ala Ala Ile Lys Thr Asp Pro Asn Trp Gln195 200 205Gly Gly Asp Tyr Tyr Glu Thr Gly Arg Ala Pro Glu Asn Gly Leu Thr210 215 220Ile Ala Arg Arg Phe Ala His Leu Thr Tyr Arg Ser Glu Val Glu Leu225 230 235 240Asp Thr Arg Phe Ala Asn Asn Asn Gln Gly Asn Glu Asp Pro Ala Thr245 250 255Gly Gly Arg Tyr Ala Val Gln Ser Tyr Leu Glu His Gln Gly Asp Lys260 265 270Leu Leu Ala Arg Phe Asp Ala Gly Ser Tyr Val Val Leu Thr Glu Thr275 280 285Leu Asn Ser His Asp Val Gly Arg Gly Arg Gly Gly Ile Gly Thr Ala290 295 300Leu Arg Gly Cys Pro Val Pro Val Val Val Gly Gly Ile Thr Ser Asp305 310 315 320Arg Leu Tyr Pro Leu Arg Leu Gln Gln Glu Leu Ala Glu Met Leu Pro325 330 335Gly Cys Thr Gly Leu Gln Val Val Asp Ser Thr Tyr Gly His Asp Gly340 345 350Phe Leu Val Glu Ser Glu Ala Val Gly Lys Leu Ile Arg Gln Thr Leu355 360 365Glu Leu Ala Asp Val Gly Ser Lys Glu Asp Ala Cys Ser Gln370 375 380<210>5
<211>1140<212>DNA<213>結(jié)核分枝桿菌(Mycobacterium tuberculosis)<220>
<221>CDS<222>(1)..(1137)<223>RMTB03565<400>5atg acg atc tcc gat gta ccc acc cag acg ctg ccc gcc gaa ggc gaa48Met Thr Ile Ser Asp Val Pro Thr Gln Thr Leu Pro Ala Glu Gly Glu1 5 10 15atc ggc ctg ata gac gtc ggc tcg ctg caa ctg gaa agc ggg gcg gtg96Ile Gly Leu Ile Asp Val Gly Ser Leu Gln Leu Glu Ser Gly Ala Val20 25 30atc gac gat gtc tgt atc gcc gtg caa cgc tgg ggc aaa ttg tcg ccc 144Ile Asp Asp Val Cys Ile Ala Val Gln Arg Trp Gly Lys Leu Ser Pro35 40 45gca cgg gac aac gtg gtg gtg gtc ttg cac gcg ctc acc ggc gac tcg192Ala Arg Asp Asn Val Val Val Val Leu His Ala Leu Thr Gly Asp Ser50 55 60cac atc act gga ccc gcc gga ccc ggc cac ccc acc ccc ggc tgg tgg240His Ile Thr Gly Pro Ala Gly Pro Gly His Pro Thr Pro Gly Trp Trp65 70 75 80gac ggg gtg gcc ggg ccg agt gcg ccg att gac acc acc cgc tgg tgc288Asp Gly Val Ala Gly Pro Ser Ala Pro Ile Asp Thr Thr Arg Trp Cys85 90 95gcg gta gct acc aat gtg ctc ggc ggc tgc cgc ggc tcc acc ggg ccc336Ala Val Ala Thr Asn Val Leu Gly Gly Cys Arg Gly Ser Thr Gly Pro100 105 110agc tcg ctt gcc cgc gac gga aag cct tgg ggc tca aga ttt ccg ctg384Ser Ser Leu Ala Arg Asp Gly Lys Pro Trp Gly Ser Arg Phe Pro Leu115 120 125atc tcg ata cgt gac cag gtg cag gcg gac gtc gcg gcg ctg gcc gcg432Ile Ser Ile Arg Asp Gln Val Gln Ala Asp Val Ala Ala Leu Ala Ala130 135 140ctg ggc atc acc gag gtc gcc gcc gtc gtc ggc ggc tcc atg ggc ggc480Leu Gly Ile Thr Glu Val Ala Ala Val Val Gly Gly Ser Met Gly Gly145 150 155 160gcc cgg gcc ctg gaa tgg gtg gtc ggc tac ccg gat cgg gtc cga gcc528Ala Arg Ala Leu Glu Trp Val Val Gly Tyr Pro Asp Arg Val Arg Ala165 170 175gga ttg ctg ctg gcg gtc ggt gcg cgt gcc acc gca gac cag atc ggc576Gly Leu Leu Leu Ala Val Gly Ala Arg Ala Thr Ala Asp Gln Ile Gly180 185 190acg cag aca acg caa atc gcg gcc atc aaa gcc gac ccg gac tgg cag624Thr Gln Thr Thr Gln Ile Ala Ala Ile Lys Ala Asp Pro Asp Trp Gln195 200 205agc ggc gac tac cac gag acg ggg agg gca cca gac gcc ggg ctg cga672Ser Gly Asp Tyr His Glu Thr Gly Arg Ala Pro Asp Ala Gly Leu Arg210 215 220
ctc gcc cgc cgc ttc gcg cac ctc acc tac cgc ggc gag atc gag ctc 720Leu Ala Arg Arg Phe Ala His Leu Thr Tyr Arg Gly Glu Ile Glu Leu225 230 235 240gac acc cgg ttc gcc aac cac aac cag ggc aac gag gat ccg acg gcc768Asp Thr Arg Phe Ala Asn His Asn Gln Gly Asn Glu Asp Pro Thr Ala245 250 255ggc ggg cgc tac gcg gtg caa agt tat ctg gaa cac caa gga gac aaa816Gly Gly Arg Tyr Ala Val Gln Ser Tyr Leu Glu His Gln Gly Asp Lys260 265 270ctg tta tcc cgg ttc gac gcc ggc agc tac gtg att ctc acc gag gcg864Leu Leu Ser Arg Phe Asp Ala Gly Ser Tyr Val Ile Leu Thr Glu Ala275 280 285ctc aac agc cac gac gtc ggc cgc ggc cgc ggc ggg gtc tcc gcg gct912Leu Asn Ser His Asp Val Gly Arg Gly Arg Gly Gly Val Ser Ala Ala290 295 300ctg cgc gcc tgc ccg gtg ccg gtg gtg gtg ggc ggc atc acc tcc gac960Leu Arg Ala Cys Pro Val Pro Val Val Val Gly Gly Ile Thr Ser Asp305 310 315 320cgg ctc tac ccg ctg cgc ctg cag cag gag ctg gcc gac ctg ctg ccg 1008Arg Leu Tyr Pro Leu Arg Leu Gln Gln Glu Leu Ala Asp Leu Leu Pro325 330 335ggc tgc gcc ggg ctg cga gtc gtc gag tcg gtc tac gga cac gac ggc1056Gly Cys Ala Gly Leu Arg Val Val Glu Ser Val Tyr Gly His Asp Gly340 345 350ttc ctg gtg gaa acc gag gcc gtg ggc gaa ttg atc cgc cag aca ctg1104Phe Leu Val Glu Thr Glu Ala Val Gly Glu Leu Ile Arg Gln Thr Leu355 360 365gga ttg gct gat cgt gaa ggc gcg tgt cgg cgg tga1140Gly Leu Ala Asp Arg Glu Gly Ala Cys Arg Arg370 375<210>6<211>379<212>PRT<213>結(jié)核分枝桿菌<400>6Met Thr Ile Ser Asp Val Pro Thr Gln Thr Leu Pro Ala Glu Gly Glu1 5 10 15Ile Gly Leu Ile Asp Val Gly Ser Leu Gln Leu Glu Ser Gly Ala Val20 25 30Ile Asp Asp Val Cys Ile Ala Val Gln Arg Trp Gly Lys Leu Ser Pro35 40 45Ala Arg Asp Asn Val Val Val Val Leu His Ala Leu Thr Gly Asp Ser50 55 60His Ile Thr Gly Pro Ala Gly Pro Gly His Pro Thr Pro Gly Trp Trp65 70 75 80Asp Gly Val Ala Gly Pro Ser Ala Pro Ile Asp Thr Thr Arg Trp Cys85 90 95Ala Val Ala Thr Asn Val Leu Gly Gly Cys Arg Gly Ser Thr Gly Pro
100 105 110Ser Ser Leu Ala Arg Asp Gly Lys Pro Trp Gly Ser Arg Phe Pro Leu115 120 125Ile Ser lie Arg Asp Gin Val Gln Ala Asp Val Ala Ala Leu Ala Ala130 135 140Leu Gly Ile Thr Glu Val Ala Ala Val Val Gly Gly Ser Met Gly Gly145 150 155 160Ala Arg Ala Leu Glu Trp Val Val Gly Tyr Pro Asp Arg Val Arg Ala165 170 175Gly Leu Leu Leu Ala Val Gly Ala Arg Ala Thr Ala Asp Gln Ile Gly180 185 190Thr Gln Thr Thr Gln Ile Ala Ala Ile Lys Ala Asp Pro Asp Trp Gln195 200 205Ser Gly Asp Tyr His Glu Thr Gly Arg Ala Pro Asp Ala Gly Leu Arg210 215 220Leu Aia Arg Arg Phe Ala His Leu Thr Tyr Arg Gly Glu Ile Glu Leu225 230 235 240Asp Thr Arg Phe Ala Asn His Asn Gln Gly Asn Glu Asp Pro Thr Ala245 250 255Gly Gly Arg Tyr Ala Val Gln Ser Tyr Leu Glu His Gln Gly Asp Lys260 265 270Leu Leu Ser Arg Phe Asp Ala Gly Ser Tyr Val Ile Leu Thr Glu Ala275 280 285Leu Asn Ser His Asp Val Gly Arg Gly Arg Gly Gly Val Ser Ala Ala290 295 300Leu Arg Ala Cys Pro Val Pro Val Val Val Gly Gly Ile Thr Ser Asp305 310 315 320Arg Leu Tyr Pro Leu Arg Leu Gln Gln Glu Leu Ala Asp Leu Leu Pro325 330 335Gly Cys Ala Gly Leu Arg Val Val Glu Ser Val Tyr Gly His Asp Gly340 345 350Phe Leu Val Glu Thr Glu Ala Val Gly Glu Leu Ile Arg Gln Thr Leu355 360 365Gly Leu Ala Asp Arg Glu Gly Ala Cys Arg Arg370 375<210>7<211>972<212>DNA<213>微溫綠菌(chlorobium tepidum)<220>
<221>CDS<222>(1)..(969)<223>RCL01447<400>7gtg agg gtc gct tac cgt acc tgg ggt acg cta aac gca gag aaa agc48Val Arg Val Ala Tyr Arg Thr Trp Gly Thr Leu Asn Ala Glu Lys Ser
1 5 10 15aac gtg att ctg gtc tgc cac gcg ctg acc ggc aac gcc gac gcc gac96Asn Val Ile Leu Val Cys His Ala Leu Thr Gly Asn Ala Asp Ala Asp20 25 30agc tgg tgg tgc ggc atg ttc ggt gag gga cgg gcg ttc gac gag act144Ser Trp Trp Cys Gly Met Phe Gly Glu Gly Arg Ala Phe Asp Glu Thr35 40 45cgg gac ttc atc gta tgc agc aac gtg ctt gga agc tgc tac gga acg192Arg Asp Phe Ile Val Cys Ser Asn Val Leu Gly Ser Cys Tyr Gly Thr50 55 60acc ggg ccg atg tcg gtg aat ccg ctg agt ggc agg cac tac ggt ccc240Thr Gly Pro Met Ser Val Asn Pro Leu Ser Gly Arg His Tyr Gly Pro65 70 75 80gat ttt ccg cgc att acc att cgc gac atg gtg aat gtt cag cga tta288Asp Phe Pro Arg Ile Thr Ile Arg Asp Met Val Asn Val Gln Arg Leu85 90 95ttg ctt cgt tcg ctc ggc atc gac cgg atc cgg ctc atc gtt ggt gca336Leu Leu Arg Ser Leu Gly Ile Asp Arg Ile Arg Leu Ile Val Gly Ala100 105 110tcg ctt ggc ggg atg cag gtg ctc gaa tgg ggc gca atg tat ccc gaa384Ser Leu Gly Gly Met Gln Val Leu Glu Trp Gly Ala Met Tyr Pro Glu115 120 125atg gcc ggg gcg ctg atg ccg atg ggc gtt tcg ggt cgt cat tcg gcg432Met Ala Gly Ala Leu Met Pro Met Gly Val Ser Gly Arg His Ser Ala130 135 140tgg tgc atc gcg cag agc gag gcg cag cgg cag gct atc gcc gcc gat480Trp Cys Ile Ala Gln Ser Glu Ala Gln Arg Gln Ala Ile Ala Ala Asp145 150 155 160gcg gag tgg caa gat ggc tgg tat gat ccg gag gtg cag cca cgc aaa528Ala Glu Trp Gln Asp Gly Trp Tyr Asp Pro Glu Val Gln Pro Arg Lys165 170 175gga ctt gcc gcc gcg cgg atg atg gcg atg tgc acc tac cgc tgc ttc576Gly Leu Ala Ala Ala Arg Met Met Ala Met Cys Thr Tyr Arg Cys Phe180 185 190gag aac tac cag caa cgc ttt ggc cgc aag cag cgc gag gac ggc ttg624Glu ASn Tyr Gln Gln Arg Phe Gly Arg Lys Gln Arg Glu Asp Gly Leu195 200 205ttc gaa gcc gaa agc tac gtg cgt cac cag ggc gac aag ctg gtt ggg672Phe Glu Ala Glu Ser Tyr Val Arg His Gln Gly Asp Lys Leu Val Gly210 215 220cgc ttt gat gca aac acc tat atc acg ctc acc aga gcg atg gac atg720Arg Phe Asp Ala Asn Thr Tyr Ile Thr Leu Thr Arg Ala Met Asp Met225 230 235 240cac gac ctc ggg cgc gga cgc gac tcc tac gaa gcg gcg ctc gga gcg768His Asp Leu Gly Arg Gly Arg Asp Ser Tyr Glu Ala Ala Leu Gly Ala245 250 255ctg aag atg ccg gtc gag att ctc tcc atc gac tcg gac gtg ctc tat816Leu Lys Met Pro Val Glu Ile Leu Ser Ile Asp Ser Asp Val Leu Tyr260 265 270
ccc agg cag gag cag gag gaa ctt gcc cgc ctc att ccc ggc tca cgc864Pro Arg Gln Glu Gln Glu Glu Leu Ala Arg Leu Ile Pro Gly Ser Arg275 280 285ctg ctt ttc ctt gac gaa ccc tat ggc cac gac gcc ttt ctt atc gac912Leu Leu Phe Leu Asp Glu Pro Tyr Gly His Asp Ala Phe Leu Ile Asp290 295 300acc gag acc gtc agc cgc atg gtc tgc gag ttc aag agg cag ttg ata960Thr Glu Thr Val Ser Arg Met Val Cys Glu Phe Lys Arg Gln Leu Ile305 310 315 320gtt gac aat tga972Val Asp Asn<210>8<211>323<212>PRT<213>微溫綠菌<400>8Val Arg Val Ala Tyr Arg Thr Trp Gly Thr Leu Asn Ala Glu Lys Ser1 5 10 15Asn Val Ile Leu Val Cys His Ala Leu Thr Gly Asn Ala Asp Ala Asp20 25 30Ser Trp Trp Cys Gly Met Phe Gly Glu Gly Arg Ala Phe Asp Glu Thr35 40 45Arg Asp Phe Ile Val Cys Ser Asn Val Leu Gly Ser Cys Tyr Gly Thr50 55 60Thr Gly Pro Met Ser Val Asn Pro Leu Ser Gly Arg His Tyr Gly Pro65 70 75 80Asp Phe Pro Arg Ile Thr Ile Arg Asp Met Val Asn Val Gln Arg Leu85 90 95Leu Leu Arg Ser Leu Gly Ile Asp Arg Ile Arg Leu Ile Val Gly Ala100 105 110Ser Leu Gly Gly Met Gln Val Leu Glu Trp Gly Ala Met Tyr Pro Glu115 120 125Met Ala Gly Ala Leu Met Pro Met Gly Val Ser Gly Arg His Ser Ala130 135 140Trp Cys Ile Ala Gln Ser Glu Ala Gln Arg Gln Ala Ile Ala Ala Asp145 150 155 160Ala Glu Trp Gln Asp Gly Trp Tyr Asp Pro Glu Val Gin Pro Arg Lys165 170 175Gly Leu Ala Ala Ala Arg Met Met Ala Met Cys Thr Tyr Arg Cys Phe180 185 190Glu Asn Tyr Gln Gln Arg Phe Gly Arg Lys Gln Arg Glu Asp Gly Leu195 200 205Phe Glu Ala Glu Ser Tyr Val Arg His Gln Gly Asp Lys Leu Val Gly210 215 220Arg Phe Asp Ala Asn Thr Tyr Ile Thr Leu Thr Arg Ala Met Asp Met225 230 235 240
His Asp Leu Gly Arg Gly Arg Asp Ser Tyr Glu Ala Ala Leu Gly Ala245 250 255Leu Lys Met Pro Val Glu Ile Leu Ser Ile Asp Ser Asp Val Leu Tyr260 265 270Pro Arg Gln Glu Gln Glu Glu Leu Ala Arg Leu Ile Pro Gly Ser Arg275 280 285Leu Leu Phe Leu Asp Glu Pro Tyr Gly His Asp Ala Phe Leu Ile Asp290 295 300Thr Glu Thr Val Ser Arg Met Val Cys Glu Phe Lys Arg Gln Leu Ile305 310 315 320Val Asp Asn<210>9<211>1149<212>DNA<213>新月柄桿菌(caulobacter crescentus)<220>
<221>CDS<222>(1)..(1146)<223>RCO00727<400>9atg gct gcg ctc gat ccg atc acg ccc gcc ggc ggg gga acc tgg cgg48Met Ala Ala Leu Asp Pro Ile Thr Pro Ala Gly Gly Gly Thr Trp Arg1 5 10 15ttt cct gcg aat gaa cct ctg cgg ctg gac tcc gga ggc gtc atc gaa96Phe Pro Ala Asn Glu Pro Leu Arg Leu Asp Ser Gly Gly Val Ile Glu20 25 30ggt ctg gaa atc gcc tac cag acc tac ggc cag ctg aac gcg gac aag144Gly Leu Glu Ile Ala Tyr Gln Thr Tyr Gly Gln Leu Asn Ala Asp Lys35 40 45tcc aac gcc gtc ctg atc tgc cac gcc ctg acg ggc gac cag cat gtg192Ser Asn Ala Val Leu Ile Cys His Ala Leu Thr Gly Asp Gln His Val50 55 60gcc tcg ccc cac ccc acc acc ggc aag ccc ggc tgg tgg caa cgc ctt240Ala Ser Pro His Pro Thr Thr Gly Lys Pro Gly Trp Trp Gln Arg Leu65 70 75 80gtt ggt ccc ggt aag ccg ctg gat ccc gcg cgg cac ttc atc atc tgc288Val Gly Pro Gly Lys Pro Leu Asp Pro Ala Arg His Phe Ile Ile Cys85 90 95tcg aac gtg atc ggc ggc tgc atg ggc tcg acg ggc ccg gcc tcg atc336Ser Asn Val Ile Gly Gly Cys Met Gly Ser Thr Gly Pro Ala Ser Ile100 105 110aat ccg gcc acg ggc aag acc tat ggc ctg tcg ttc cca gtc atc acc384Asn Pro Ala Thr Gly Lys Thr Tyr Gly Leu Ser Phe Pro Val Ile Thr115 120 125atc gcc gat atg gtg cgg gcc cag gcc atg ctg gtc tct gcg ctc ggg432Ile Ala Asp Met Val Arg Ala Gln Ala Met Leu Val Ser Ala Leu Gly130 135 140
gtc gag acc ctg ttc gcc gtc gtc ggc ggc tcg atg ggc ggc atg cag480Val Glu Thr Leu Phe Ala Val Val Gly Gly Ser Met Gly Gly Met Gln145 150 155 160gtc cag caa tgg gcc gtg gac tat ccc gag cgg atg ttc agc gcc gtg528Val Gln Gln Trp Ala Val Asp Tyr Pro Glu Arg Met Phe Ser Ala Val165 170 175gtg ctg gcc tcg gcc tcg cgc cac tcg gcc cag aac atc gcg ttc cac576Val Leu Ala Ser Ala Ser Arg His Ser Ala Gln Asn Ile Ala Phe His180 185 190gag gtg ggc cgc cag gcg atc atg gcc gat ccc gac tgg cgc ggc ggc624Glu Val Gly Arg Gln Ala Ile Met Ala Asp Pro Asp Trp Arg Gly Gly195 200 205gcc tat gcc gag cac ggc gtg cgg ccc gag aag ggc ctg gcc gtg gcg672Ala Tyr Ala Glu His Gly Val Arg Pro Glu Lys Gly Leu Ala Val Ala210 215 220cgg atg gcc gcg cac atc acc tat ctg tcc gag ccc gcc ctg cag cgg720Arg Met Ala Ala His Ile Thr Tyr Leu Ser Glu Pro Ala Leu Gln Arg225 230 235 240aag ttc ggc cgc gag cta cag cgc gac ggc ctc tcc tgg ggc ttt gac768Lys Phe Gly Arg Glu Leu Gln Arg Asp Gly Leu Ser Trp Gly Phe Asp245 250 255gcc gac ttc cag gtc gag agc tat cta cgc cac cag ggg tcc agc ttc816Ala Asp Phe Gln Val Glu Ser Tyr Leu Arg His Gln Gly Ser Ser Phe260 265 270gtc gac cgg ttc gac gcc aac agc tat ctc tac atc acc cgg gcc atg864Val Asp Arg Phe Asp Ala Asn Ser Tyr Leu Tyr Ile Thr Arg Ala Met275 280 285gac tat ttc gac atc gcc gcc agc cat ggc ggg gtg ctg gcc aag gcg912Asp Tyr Phe Asp Ile Ala Ala Ser His Gly Gly Val Leu Ala Lys Ala290 295 300ttc acc cga gcg cgg aat gtg cgc ttc tgc gtg ctg agc ttc tcc agc960Phe Thr Arg Ala Arg Asn Val Arg Phe Cys Val Leu Ser Phe Ser Ser305 310 315 320gac tgg ctc tat ccg acc gcc gag aac cgc cac ctg gtc cgc gcc ctg1008Asp Trp Leu Tyr Pro Thr Ala Glu Asn Arg His Leu Val Arg Ala Leu325 330 335acc gcc gcc ggg gcc cgc gcg gcc ttc gcc gag atc gag agc gac aag1056Thr Ala Ala Gly Ala Arg Ala Ala Phe Ala Glu Ile Glu Ser Asp Lys340 345 350ggc cat gac gcc ttc ctg ctg gac gag ccg gtg atg gac gcc gcg ctg1104Gly His Asp Ala Phe Leu Leu Asp Glu Pro Val Met Asp Ala Ala Leu355 360 365gaa ggc ttc ctg gcc tcg gcc gaa cgc gat cgg ggg ctg gtt1146Glu Gly Phe Leu Ala Ser Ala Glu Arg Asp Arg Gly Leu Val370 375 380tga1149<210>10<211>382<212>PRT<213>新月柄桿菌
<400>10Met Ala Ala Leu Asp Pro Ile Thr Pro Ala Gly Gly Gly Thr Trp Arg1 5 10 15Phe Pro Ala Asn Glu Pro Leu Arg Leu Asp Ser Gly Gly Val Ile Glu20 25 30Gly Leu Glu Ile Ala Tyr Gln Thr Tyr Gly Gln Leu Asn Ala Asp Lys35 40 45Ser Asn Ala Val Leu Ile Cys His Ala Leu Thr Gly Asp Gln His Val50 55 60Ala Ser Pro His Pro Thr Thr Gly Lys Pro Gly Trp Trp Gln Arg Leu65 70 75 80Val Gly Pro Gly Lys Pro Leu Asp Pro Ala Arg His Phe Ile Ile Cys85 90 95Ser Asn Val Ile Gly Gly Cys Met Gly Ser Thr Gly Pro Ala Ser Ile100 105 110Asn Pro Ala Thr Gly Lys Thr Tyr Gly Leu Ser Phe Pro Val Ile Thr115 120 125Ile Ala Asp Met Val Arg Ala Gln Ala Met Leu Val Ser Ala Leu Gly130 135 140Val Glu Thr Leu Phe Ala Val Val Gly Gly Ser Met Gly Gly Met Gln145 150 155 160Val Gln Gln Trp Ala Val Asp Tyr Pro Glu Arg Met Phe Ser Ala Val165 170 175Val Leu Ala Ser Ala Ser Arg His Ser Ala Gln Asn Ile Ala Phe His180 185 190Glu Val Gly Arg Gln Ala Ile Met Ala Asp Pro Asp Trp Arg Gly Gly195 200 205Ala Tyr Ala Glu His Gly Val Arg Pro Glu Lys Gly Leu Ala Val Ala210 215 220Arg Met Ala Ala His Ile Thr Tyr Leu Ser Glu Pro Ala Leu Gln Arg225 230 235 240Lys Phe Gly Arg Glu Leu Gln Arg Asp Gly Leu Ser Trp Gly Phe Asp245 250 255Ala Asp Phe Gln Val Glu Ser Tyr Leu Arg His Gln Gly Ser Ser Phe260 265 270Val Asp Arg Phe Asp Ala Asn Ser Tyr Leu Tyr Ile Thr Arg Ala Met275 280 285Asp Tyr Phe Asp Ile Ala Ala Ser His Gly Gly Val Leu Ala Lys Ala290 295 300Phe Thr Arg Ala Arg Asn Val Arg Phe Cys Val Leu Ser Phe Ser Ser305 310 315 320Asp Trp Leu Tyr Pro Thr Ala Glu Asn Arg His Leu Val Arg Ala Leu325 330 335Thr Ala Ala Gly Ala Arg Ala Ala Phe Ala Glu Ile Glu Ser Asp Lys340 345 350
Gly His Asp Ala Phe Leu Leu Asp Glu Pro Val Met Asp Ala Ala Leu355 360 365Glu Gly Phe Leu Ala Ser Ala Glu Arg Asp Arg Gly Leu Val370 375 380<210>11<211>1140<212>DNA<213>淋病奈瑟氏球菌(Neisseria gonorrhoeae)<220>
<221>CDS<222>(1)..(1137)<223>RNG00132<400>11atg agt caa aat acc tcg gtg ggc att gta acg ccc caa aaa att ccg48Met Ser Gln Asn Thr Ser Val Gly Ile Val Thr Pro Gln Lys Ile Pro1 5 10 15ttt gaa atg ccg ctg gtt ttg gaa aac ggt aaa act ttg ccg cgt ttc96Phe Glu Met Pro Leu Val Leu Glu Asn Gly Lys Thr Leu Pro Arg Phe20 25 30gat ctg atg att gaa acc tac ggc gag ctg aat gct gaa aaa aac aat144Asp Leu Met Ile Glu Thr Tyr Gly Glu Leu Asn Ala Glu Lys Asn Asn35 40 45gcg gtt tta atc tgc cac gcg ctg tcg ggc aac cat cac gtt gcg ggc192Ala Val Leu Ile Cys His Ala Leu Ser Gly Asn His His Val Ala Gly50 55 60agg cat tcg gcg gag gat aaa tat acg ggc tgg tgg gac aat atg gtc240Arg His Ser Ala Glu Asp Lys Tyr Thr Gly Trp Trp Asp Asn Met Val65 70 75 80ggt ccc gga aaa ccg att gat acg gaa cgt ttt ttc gtg gtc ggg ttg288Gly Pro Gly Lys Pro Ile Asp Thr Glu Arg Phe Phe Val Val Gly Leu85 90 95aac aat ctg ggc ggc tgc gac ggc agc agc ggg cct ttg tcg atc aat336Asn Asn Leu Gly Gly Cys Asp Gly Ser Ser Gly Pro Leu Ser Ile Asn100 105 110cct gaa acg ggc agg gaa tac ggc gcg gat ttt ccg atg gtt acg gtg384Pro Glu Thr Gly Arg Glu Tyr Gly Ala Asp Phe Pro Met Val Thr Val115 120 125aag gac tgg gta aaa tca caa gcc gcg ctt gcc gat tat ctc ggc atc432Lys Asp Trp Val Lys Ser Gln Ala Ala Leu Ala Asp Tyr Leu Gly Ile130 135 140gaa caa tgg gcg gcg gtt gtc ggc ggc agc ttg ggc ggc atg cag gct480Glu Gln Trp Ala Ala Val Val Gly Gly Ser Leu Gly Gly Met Gln Ala145 150 155 160ttg cag tgg gcg att tcc tat ccc gaa cgt gtg cgc cac gcc ttg gtg528Leu Gln Trp Ala Ile Ser Tyr Pro Glu Arg Val Arg His Ala Leu Val165 170 175att gcg tct gcg ccg aaa ctg tcc gcg caa aat atc gcg ttt aat gat576Ile Ala Ser Ala Pro Lys Leu Ser Ala Gln Asn Ile Ala Phe Asn Asp180 185 190
gta gca cgt cag gcg att ttg acc gac ccc gat ttc aat gaa gga cat624Val Ala Arg Gln Ala Ile Leu Thr Asp Pro Asp Phe Asn Glu Gly His195 200 205tac cgc agc cac aac acc gtt ccc gcg cgc ggt ttg cgg att gcc cgt672Tyr Arg Ser His Asn Thr Val Pro Ala Arg Gly Leu Arg Ile Ala Arg210 215 220atg atg gga cac att acg tat ctt gcc gaa gac ggt ttg ggc aaa aaa720Met Met Gly His Ile Thr Tyr Leu Ala Glu Asp Gly Leu Gly Lys Lys225 230 235 240ttc gga cgc gat ttg cgt tcc aac ggc tat caa tac ggc tat agc gtt768Phe Gly Arg Asp Leu Arg Ser Asn Gly Tyr Gln Tyr Gly Tyr Ser Val245 250 255gaa ttt gaa gta gaa tcc tat ctc cgc tat caa ggc gac aaa ttc gtc816Glu Phe Glu Val Glu Ser Tyr Leu Arg Tyr Gln Gly Asp Lys Phe Val260 265 270ggg cgg ttt gat gct aat aca tat ttg ctg atg acc aaa gct ttg gac864Gly Arg Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr Lys Ala Leu Asp275 280 285tat ttc gat ccg gcg gcg gat ttc ggc aac agc ctg acc cgc gcc gtg912Tyr Phe Asp Pro Ala Ala Asp Phe Gly Asn Ser Leu Thr Arg Ala Val290 295 300cag gat gtg cag gca aaa ttc ttt gtc gcc agc ttc agc acc gac tgg960Gln Asp Val Gln Ala Lys Phe Phe Val Ala Ser Phe Ser Thr Asp Trp305 310 315 320cgt ttc gcg ccc gaa cgt tcg cac gaa ctg gtc aag gca ctg att gcc 1008Arg Phe Ala Pro Glu Arg Ser His Glu Leu Val Lys Ala Leu Ile Ala325 330 335gcc caa aaa tcc gtg cag tat atc gaa gtc aag tcc gca cac ggg cac 1056Ala Gln Lys Ser Val Gln Tyr Ile Glu Val Lys Ser Ala His Gly His340 345 350gat gcc ttt tta atg gaa gac gaa gcc tat atg cgc gcc gta acg gct 1104Asp Ala Phe Leu Met Glu Asp Glu Ala Tyr Met Arg Ala Val Thr Ala355 360 365tat atg aac aat gtt gac aag gat tgc cga tta tga 1140Tyr Met Asn Asn Val Asp Lys Asp Cys Arg Leu370 375<210>12<211>379<212>PRT<213>淋病奈瑟氏球菌<400>12Met Ser Gln Asn Thr Ser Val Gly Ile Val Thr Pro Gln Lys Ile Pro1 5 10 15Phe Glu Met Pro Leu Val Leu Glu Asn Gly Lys Thr Leu Pro Arg Phe20 25 30Asp Leu Met Ile Glu Thr Tyr Gly Glu Leu Asn Ala Glu Lys Asn Asn35 40 45Ala Val Leu Ile Cys His Ala Leu Ser Gly Asn His His Val Ala Gly50 55 60
Arg His Ser Ala Glu Asp Lys Tyr Thr Gly Trp Trp Asp Asn Met Val65 70 75 80Gly Pro Gly Lys Pro Ile Asp Thr Glu Arg Phe Phe Val Val Gly Leu85 90 95Asn Asn Leu Gly Gly Cys Asp Gly Ser Ser Gly Pro Leu Ser Ile Asn100 105 110Pro Glu Thr Gly Arg Glu Tyr Gly Ala Asp Phe Pro Met Val Thr Val115 120 125Lys Asp Trp Val Lys Ser Gln Ala Ala Leu Ala Asp Tyr Leu Gly Ile130 135 140Glu Gln Trp Ala Ala Val Val Gly Gly Ser Leu Gly Gly Met Gln Ala145 150 155 160Leu Gln Trp Ala Ile Ser Tyr Pro Glu Arg Val Arg His Ala Leu Val165 170 175Ile Ala Ser Ala Pro Lys Leu Ser Ala Gln Asn Ile Ala Phe Asn Asp180 185 190Val Ala Arg Gln Ala Ile Leu Thr Asp Pro Asp Phe Asn Glu Gly His195 200 205Tyr Arg Ser His Asn Thr Val Pro Ala Arg Gly Leu Arg Ile Ala Arg210 215 220Met Met Gly His Ile Thr Tyr Leu Ala Glu Asp Gly Leu Gly Lys Lys225 230 235 240Phe Gly Arg Asp Leu Arg Ser Asn Gly Tyr Gln Tyr Gly Tyr Ser Val245 250 255Glu Phe Glu Val Glu Ser Tyr Leu Arg Tyr Gln Gly Asp Lys Phe Val260 265 270Gly Arg Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr Lys Ala Leu Asp275 280 285Tyr Phe Asp Pro Ala Ala Asp Phe Gly Asn Ser Leu Thr Arg Ala Val290 295 300Gln Asp Val Gln Ala Lys Phe Phe Val Ala Ser Phe Ser Thr Asp Trp305 310 315 320Arg Phe Ala Pro Glu Arg Ser His Glu Leu Val Lys Ala Leu Ile Ala325 330 335Ala Gln Lys Ser Val Gln Tyr Ile Glu Val Lys Ser Ala His Gly His340 345 350Asp Ala Phe Leu Met Glu Asp Glu Ala Tyr Met Arg Ala Val Thr Ala355 360 365Tyr Met Asn Asn Val Asp Lys Asp Cys Arg Leu370 375<210>13<211>1140<212>DNA<213>腦膜炎奈瑟氏球菌(Neisseria meningitidis ser.A)
<220>
<221>CDS<222>(1)..(1137)<223>RNM00815<400>13atg agt caa aat gcc tcg gtg ggc att gta acg ccc caa aaa att ccg48Met Ser Gln Asn Ala Ser Val Gly Ile Val Thr Pro Gln Lys Ile Pro1 5 10 15ttt gaa atg ccg ctg gtt ttg gaa aac ggt aaa act ttg ccg cgt ttc96Phe Glu Met Pro Leu Val Leu Glu Asn Giy Lys Thr Leu Pro Arg Phe20 25 30gat ctg atg att gaa acc tac ggc gag ctg aat gcc gaa aaa aat aat144Asp Leu Met Ile Glu Thr Tyr Gly Glu Leu Asn Ala Glu Lys Asn Asn35 40 45gcg gtt tta atc tgt cat gcg ctg tca ggc aac cat cat gtt gcg ggc192Ala Val Leu Ile Cys His Ala Leu Ser Gly Asn His His Vai Ala Gly50 55 60agg cat tcg gcg gag gat aaa tat acg ggc tgg tgg gac aat atg gta240Arg His Ser Ala Glu Asp Lys Tyr Thr Gly Trp Trp Asp Asn Met Val65 70 75 80gga ccc ggc aaa ccg att gat aca gaa cgt ttt ttc gtg gtc ggt ttg288Gly Pro Gly Lys Pro Ile Asp Thr Glu Arg Phe Phe Val Val Gly Leu85 90 95aac aat ctg ggc ggc tgc gac ggc agc agc gga cct ttg tcg atc aat336Asn Asn Leu Gly Gly Cys Asp Gly Ser Ser Gly Pro Leu Ser Ile Asn100 105 110cct gaa acg ggc agg gaa tac ggc gcg gat ttt ccg gtg gtt acg gtg384Pro Glu Thr Gly Arg Glu Tyr Gly Ala Asp Phe Pro Val Val Thr Val115 120 125aag gac tgg gta aaa tcc caa gcc gcg ctt acc gat tat ctc ggc atc432Lys Asp Trp Val Lys Ser Gln Ala Ala Leu Thr Asp Tyr Leu Gly Ile130 135 140ggg caa tgg gcg gcg gtt gtc ggc ggc agc ttg ggc ggt atg cag gct480Gly Gln Trp Ala Ala Val Val Gly Gly Ser Leu Gly Gly Met Gln Ala145 150 155 160ttg cag tgg acg att tcc tat ccc gag cgc gtg cgc cat gcc tta gtg528Leu Gln Trp Thr Ile Ser Tyr Pro Glu Arg Val Arg His Ala Leu Val165 170 175att gcg tcc gcg ccg aaa ctg tcc acg caa aat atc gcg ttt aat gat576Ile Ala Ser Ala Pro Lys Leu Ser Thr Gln Asn Ile Ala Phe Asn Asp180 185 190gta gca cgt cag gcg att ttg acc gat ccc gat ttc aac gaa gga cat624Val Ala Arg Gln Ala Ile Leu Thr Asp Pro Asp Phe Asn Glu Gly His195 200 205tac cgc agc cgc aac acc gtt ccc gct cgg ggc ttg cgg att gcc cgc672Tyr Arg Ser Arg Asn Thr Val Pro Ala Arg Gly Leu Arg Ile Ala Arg210 215 220atg atg ggg cac atc acc tat ctt gcc gaa gac ggt ttg ggc aaa aaa720Met Met Gly His Ile Thr Tyr Leu Ala Glu Asp Gly Leu Gly Lys Lys225 230 235 240ttc gga cgc gat ttg cgt tcc aac ggc tat caa tac ggc tat ggc gtt768
Phe Gly Arg Asp Leu Arg Ser Asn Gly Tyr Gln Tyr Gly Tyr Gly Val245 250 255gaa ttt gaa gta gaa tcc tat ctg cgc tat caa ggc gat aaa ttc gtc816Glu Phe Glu Val Glu Ser Tyr Leu Arg Tyr Gln Gly Asp Lys Phe Val260 265 270ggg cgg ttt gat gcc aac acc tat ttg ctg atg acc aag gct ttg gac864Gly Arg Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr Lys Ala Leu Asp275 280 285tat ttc gat ccg gcg gcg gat ttc ggc aac agc ctg acc cgc gcc gtg912Tyr Phe Asp Pro Ala Ala Asp Phe Gly Asn Ser Leu Thr Arg Ala Val290 295 300cag gat gtt cag gca aaa ttc ttt gtc gcc agc ttc agc acc gat tgg960Gln Asp Val Gln Ala Lys Phe Phe Val Ala Ser Phe Ser Thr Asp Trp305 310 315 320cgt ttc gcg ccc gaa cgt tcg cac gaa ctg gtc aag gcc ctg att gcc1008Arg Phe Ala Pro Glu Arg Ser His Glu Leu Val Lys Ala Leu Ile Ala325 330 335gcc caa aaa tcc gtg cag tat atc gaa gtc aaa tcc gca cac ggg cac1056Ala Gln Lys Ser Val Gln Tyr Ile Glu Val Lys Ser Ala His Gly His340 345 350gat gcc ttt tta atg gaa gac gaa gcc tat atg cgt gcg gtc gcc gcc1104Asp Ala Phe Leu Met Glu Asp Glu Ala Tyr Met Arg Ala Val Ala Ala355 360 365tat atg aac aac gtt tat aag gaa tgt cag caa tga1140Tyr Met Asn Asn Val Tyr Lys Glu Cys Gln Gln370 375<210>14<211>379<212>PRT<213>腦膜炎奈瑟氏球菌<400>14Met Ser Gln Asn Ala Ser Val Gly Ile Val Thr Pro Gln Lys Ile Pro1 5 10 15Phe Glu Met Pro Leu Val Leu Glu Asn Gly Lys Thr Leu Pro Arg Phe20 25 30Asp Leu Met Ile Glu Thr Tyr Gly Glu Leu Asn Ala Glu Lys Asn Asn35 40 45Ala Val Leu Ile Cys His Ala Leu Ser Gly ASn His His Vai Ala Gly50 55 60Arg His Ser Ala Glu Asp Lys Tyr Thr Gly Trp Trp Asp Asn Met Val65 70 75 80Gly Pro Gly Lys Pro Ile Asp Thr Glu Arg Phe Phe Val Val Gly Leu85 90 95Asn Asn Leu Gly Gly Cys Asp Gly Ser Ser Gly Pro Leu Ser Ile Asn100 105 110Pro Glu Thr Gly Arg Glu Tyr Gly Ala Asp Phe Pro Val Val Thr Val115 120 125Lys Asp Trp Val Lys Ser Gln Ala Ala Leu Thr Asp Tyr Leu Gly Ile
130 135 140Gly Gln Trp Ala Ala Val Val Gly Gly Ser Leu Gly Gly Met Gln Ala145 150 155 160Leu Gln Trp Thr Ile Ser Tyr Pro Glu Arg Val Arg His Ala Leu Val165 170 175Ile Ala Ser Ala Pro Lys Leu Ser Thr Gln Asn Ile Ala Phe Asn Asp180 185 190Val Ala Arg Gln Ala Ile Leu Thr Asp Pro Asp Phe Asn Glu Gly His195 200 205Tyr Arg Ser Arg Asn Thr Val Pro Ala Arg Gly Leu Arg Ile Ala Arg210 215 220Met Met Gly His Ile Thr Tyr Leu Ala Glu Asp Gly Leu Gly Lys Lys225 230 235 240Phe Gly Arg Asp Leu Arg Ser Asn Gly Tyr Gln Tyr Gly Tyr Gly Val245 250 255Glu Phe Glu Val Glu Ser Tyr Leu Arg Tyr Gln Gly Asp Lys Phe Val260 265 270Gly Arg Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr Lys Ala Leu Asp275 280 285Tyr Phe Asp Pro Ala Ala Asp Phe Gly Asn Ser Leu Thr Arg Ala Val290 295 300Gln Asp Val Gln Ala Lys Phe Phe Val Ala Ser Phe Ser Thr Asp Trp305 310 315 320Arg Phe Ala Pro Glu Arg Ser His Glu Leu Val Lys Ala Leu Ile Ala325 330 335Ala Gln Lys Ser Val Gln Tyr Ile Glu Val Lys Ser Ala His Gly His340 345 350Asp Ala Phe Leu Met Glu Asp Glu Ala Tyr Met Arg Ala Val Ala Ala355 360 365Tyr Met Asn Asn Val Tyr Lys Glu Cys Gln Gln370 375<210>15<211>1140<212>DNA<213>熒光假單胞菌(Pseudomonas fluorescens)<220>
<221>CDS<222>(1)..(1137)<223>RPU01633<400>15atg cca gct gcc ttt ccc ccc gat tct gtt ggt ctg gtg acg ccg caa48Met Pro Ala Ala Phe Pro Pro Asp Ser Val Gly Leu Val Thr Pro Gln1 5 10 15acg gcg cac ttc agc gaa ccg ctg gcc ctg gcc tgc ggc cgt tcg ctg96Thr Ala His Phe Ser Glu Pro Leu Ala Leu Ala Cys Gly Arg Ser Leu20 25 30
gcc gat tat gac ctg atc tac gaa acc tac ggc acg ctg aac gcg caa144Ala Asp Tyr Asp Leu Ile Tyr Glu Thr Tyr Gly Thr Leu Asn Ala Gln35 40 45gcg agc aac gcc gtg ctg atc tgc cac gcc ttg tcc ggc cac cac cat192Ala Ser Asn Ala Val Leu Ile Cys His Ala Leu Ser Gly His His His50 55 60gct gcg ggt tat cac agc gtc gac gac cgc aag ccc ggt tgg tgg gac240Ala Ala Gly Tyr His Ser Val Asp Asp Arg Lys Pro Gly Trp Trp Asp65 70 75 80agc tgc atc ggc ccc ggc aaa ccg atc gac acc aac aag ttc ttc gtg288Ser Cys Ile Gly Pro Gly Lys Pro Ile Asp Thr Asn Lys Phe Phe Val85 90 95gtc agc ctg aac aac ctc ggc ggt tgc aat ggt tct acc ggc ccg agc336Val Ser Leu Asn Asn Leu Gly Gly Cys Asn Gly Ser Thr Gly Pro Ser100 105 110agc ctc aat ccg gaa acc ggc aag ccg ttc ggc gcc gac ttc ccg gtg384Ser Leu Asn Pro Glu Thr Gly Lys Pro Phe Gly Ala Asp Phe Pro Val115 120 125ctg acc gtg gaa gac tgg gtg cac agc cag gca cgc ctg gcc gac ctg432Leu Thr Val Glu Asp Trp Val His Ser Gln Ala Arg Leu Ala Asp Leu130 135 140ctc ggc atc ggc cag tgg gcg gcg gtg atc ggc ggc agc ctg ggc ggc480Leu Gly Ile Gly Gln Trp Ala Ala Val Ile Gly Gly Ser Leu Gly Gly145 150 155 160atg cag gcg ctg caa tgg acc atc acc tat ccg gat cgc gtt cgc cac528Met Gln Ala Leu Gln Trp Thr Ile Thr Tyr Pro Asp Arg Val Arg His165 170 175tgc ctg gcc atc gcc tcg gcc ccc aag ctg tcg gcg cag aac atc gcc576Cys Leu Ala Ile Ala Ser Ala Pro Lys Leu Ser Ala Gln Asn Ile Ala180 185 190ttc aac gaa gtg gcg cgc cag gcg atc ctc act gac ccg gaa ttc cac624Phe Asn Glu Vai Ala Arg Gln Ala Ile Leu Thr Asp Pro Glu Phe His195 200 205ggc ggc tcg ttc cag gaa cac ggc gtg atc ccc aag cgc ggc ctg atg672Gly Gly Ser Phe Gln Glu His Gly Val Ile Pro Lys Arg Gly Leu Met210 215 220ctg gcg cgg atg gtg ggg cac atc acc tac ctg tcc gac gac tcc atg720Leu Ala Arg Met Val Gly His Ile Thr Tyr Leu Ser Asp Asp Ser Met225 230 235 240ggt gag aaa ttc ggc cgt ggc ctg aag agc gaa aag ctc aac tac gac768Gly Glu Lys Phe Gly Arg Gly Leu Lys Ser Glu Lys Leu Asn Tyr Asp245 250 255ttc cac agc gtc gag ttc cag gtc gaa agc tac ctg cgc tat cag ggc816Phe His Ser Val Glu Phe Gln Val Glu Ser Tyr Leu Arg Tyr Gln Gly260 265 270gaa gag ttc tcc ggg cgc ttc gat gcc aac acc tat ctg ttg atg acc864Glu Glu Phe Ser Gly Arg Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr275 280 285aag gcg ctg gac tacttc gat ccg gcg gcg aac ttc aac gat aac ctg 912Lys Ala Leu Asp Tyr Phe Asp Pro Ala Ala Asn Phe Asn Asp Asn Leu290 295 300
gcg aaa acc ttc gaa ggt gca aaa gcc aag ttc tgc gtg atg tcg ttc960Ala Lys Thr Phe Glu Gly Ala Lys Ala Lys Phe Cys Val Met Ser Phe305 310 315 320acc acc gac tgg cgc ttc tcc ccg gcc cgc tcg cga gaa ctg gtg gat1008Thr Thr Asp Trp Arg Phe Ser Pro Ala Arg Ser Arg Glu Leu Val Asp325 330 335gcg ctg atg gcg gcg cgc aaa gac gtc agc tac ctg gaa atc gac gcg1056Ala Leu Met Ala Ala Arg Lys Asp Val Ser Tyr Leu Glu Ile Asp Ala340 345 350ccc cag ggc cac gac gcc ttc ctg att ccg atc ccg cgc tac ttg cag1104Pro Gln Gly His Asp Ala Phe Leu Ile Pro Ile Pro Arg Tyr Leu Gln355 360 365gcg ttc ggc aat tac atg aac cgc att acg ttg tga1140Ala Phe Gly Asn Tyr Met Asn Arg Ile Thr Leu370 375<210>16<211>379<212>PRT<213>熒光假單胞菌<400>16Met Pro Ala Ala Phe Pro Pro Asp Ser Val Gly Leu Val Thr Pro Gln1 5 10 15Thr Ala His Phe Ser Glu Pro Leu Ala Leu Ala Cys Gly Arg Ser Leu20 25 30Ala Asp Tyr Asp Leu Ile Tyr Glu Thr Tyr Gly Thr Leu Asn Ala Gln35 40 45Ala Ser Asn Ala Val Leu Ile Cys His Ala Leu Ser Gly His His His50 55 60Ala Ala Gly Tyr His Ser Val Asp Asp Arg Lys Pro Gly Trp Trp Asp65 70 75 80Ser Cys Ile Gly Pro Gly Lys Pro Ile Asp Thr Asn Lys Phe Phe Val85 90 95Val Ser Leu Asn Asn Leu Gly Gly Cys Asn Gly Ser Thr Gly Pro Ser100 105 110Ser Leu Asn Pro Glu Thr Gly Lys Pro Phe Gly Ala Asp Phe Pro Val115 120 125Leu Thr Val Glu Asp Trp Val His Ser Gln Ala Arg Leu Ala Asp Leu130 135 140Leu Gly Ile Gly Gln Trp Ala Ala Val Ile Gly Gly Ser Leu Gly Gly145 150 155 160Met Gln Ala Leu Gln Trp Thr Ile Thr Tyr Pro Asp Arg Val Arg His165170175Cys Leu Ala Ile Ala Ser Ala Pro Lys Leu Ser Ala Gln Asn Ile Ala180185190Phe Asn Glu Val Ala Arg Gln Ala Ile Leu Thr Asp Pro Glu Phe His195200205
Gly Gly Ser Phe Gln Glu His Gly Val Ile Pro Lys Arg Gly Leu Met210 215 220Leu Ala Arg Met Val Gly His Ile Thr Tyr Leu Ser Asp Asp Ser Met225 230 235 240Gly Glu Lys Phe Gly Arg Gly Leu Lys Ser Glu Lys Leu Asn Tyr Asp245 250 255Phe His Ser Val Glu Phe Gln Val Glu Ser Tyr Leu Arg Tyr Gln Gly260 265 270Glu Glu Phe Ser Gly Arg Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr275 280 285Lys Ala Leu Asp Tyr Phe Asp Pro Ala Ala Asn Phe Asn Asp Asn Leu290 295 300Ala Lys Thr Phe Glu Gly Ala Lys Ala Lys Phe Cys Val Met Ser Phe305 310 315 320Thr Thr Asp Trp Arg Phe Ser Pro Ala Arg Ser Arg Glu Leu Val Asp325 330 335Ala Leu Met Ala Ala Arg Lys Asp Val Ser Tyr Leu Glu Ile Asp Ala340 345 350Pro Gln Gly His Asp Ala Phe Leu Ile Pro Ile Pro Arg Tyr Leu Gln355 360 365Ala Phe Gly Asn Tyr Met Asn Arg Ile Thr Leu370 375<210>17<211>1140<212>DNA<213>銅綠假單胞菌(Pseudomonas aeruginosa)<220>
<221>CDS<222>(1)..(1137)<223>RPA04460<400>17atg ccc aca gtc ttc ccc gac gac tcc gtc ggt ctg gtc tcc ccc cag48Met Pro Thr Val Phe Pro Asp Asp Ser Val Gly Leu Val Ser Pro Gln1 5 10 15acg ctg cac ttc aac gaa ccg ctc gag ctg acc agc ggc aag tcc ctg96Thr Leu His Phe Asn Glu Pro Leu Glu Leu Thr Ser Gly Lys Ser Leu20 25 30gcc gag tac gac ctg gtg atc gaa acc tac ggc gag ctg aat gcc acg144Ala Glu Tyr Asp Leu Val Ile Glu Thr Tyr Gly Glu Leu Asn Ala Thr35 40 45cag agc aac gcg gtg ctg atc tgc cac gcc ctc tcc ggc cac cac cac192Gln Ser Asn Ala Val Leu Ile Cys His Ala Leu Ser Gly His His His50 55 60gcc gcc ggc tac cac agc gtc gac gag cgc aag ccg ggc tgg tgg gac240Ala Ala Gly Tyr His Ser Val Asp Glu Arg Lys Pro Gly Trp Trp Asp65 70 75 80agc tgc atc ggt ccg ggc aag ccg atc gac acc cgc aag ttc ttc gtc288Ser Cys Ile Gly Pro Gly Lys Pro Ile Asp Thr Arg Lys Phe Phe Val
85 90 95gtc gcc ctc aac aac ctc ggc ggt tgc aac gga tcc agc ggc ccc gcc336Val Ala Leu Asn Asn Leu Gly Gly Cys Asn Gly Ser Ser Gly Pro Ala100 105 110agc atc aat ccg gcg acc ggc aag gtc tac ggc gcg gac ttc ccg atg384Ser Ile Asn Pro Ala Thr Gly Lys Val Tyr Gly Ala Asp Phe Pro Met115 120 125gtt acg gtg gaa gac tgg gtg cat agc cag gcg cgc ctg gca gac cgc432Val Thr Val Glu Asp Trp Val His Ser Gln Ala Arg Leu Ala Asp Arg130 135 140ctc ggc atc cgc cag tgg gcc gcg gtg gtc ggc ggc agc ctc ggc ggc480Leu Gly Ile Arg Gln Trp Ala Ala Val Val Gly Gly Ser Leu Gly Gly145 150 155 160atg cag gcg ctg caa tgg acc atc agc tat ccc gag cgc gtc cgt cac528Met Gln Ala Leu Gln Trp Thr Ile Ser Tyr Pro Glu Arg Val Arg His165 170 175tgc ctg tgc atc gcc agc gcg ccg aag ctg tcg gcg cag aac atc gcc576Cys Leu Cys Ile Ala Ser Ala Pro Lys Leu Ser Ala Gln Asn Ile Ala180 185 190ttc aac gaa gtc gcc cgg cag gcg att ctt tcc gac cct gag ttc ctc624Phe Asn Glu Val Ala Arg Gln Ala Ile Leu Ser Asp Pro Glu Phe Leu195 200 205ggc ggc tac ttc cag gag cag ggc gtg att ccc aag cgc ggc ctc aag672Gly Gly Tyr Phe Gln Glu Gln Gly Val Ile Pro Lys Arg Gly Leu Lys210 215 220ctg gcg cgg atg gtc ggc cat atc acc tac ctg tcc gac gac gcc atg720Leu Ala Arg Met Val Gly His Ile Thr Tyr Leu Ser Asp Asp Ala Met225 230 235 240ggc gcc aag ttc ggc cgt gta ctg aag acc gag aag ctc aac tac gac768Gly Ala Lys Phe Gly Arg Val Leu Lys Thr Glu Lys Leu Asn Tyr Asp245 250 255ctg cac agc gtc gag ttc cag gtc gag agt tac ctg cgc tac cag ggc816Leu His Ser Val Glu Phe Gln Val Glu Ser Tyr Leu Arg Tyr Gln Gly260 265 270gag gag ttc tcc acc cgc ttc gac gcc aat acc tac ctg ctg atg acc864Glu Glu Phe Ser Thr Arg Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr275 280 285aag gcg ctg gac tacttc gac ccc gcc gcc gcc cac ggc gac gac ctg 912Lys Ala Leu Asp Tyr Phe Asp Pro Ala Ala Ala His Gly Asp Asp Leu290 295 300gtg cgc acc ctg gag ggc gtc gag gcg gac ttc tgc ctg atg tcc ttc960Val Arg Thr Leu Glu Gly Val Glu Ala Asp Phe Cys Leu Met Ser Phe305 310 315 320acc acc gac tgg cgt ttc tcg ccg gcc cgc tcg cgg gaa atc gtc gac1008Thr Thr Asp Trp Arg Phe Ser Pro Ala Arg Ser Arg Glu Ile Val Asp325 330 335gcc ctg atc gcg gcg aaa aag aac gtc agc tac ctg gag atc gac gcc1056Ala Leu Ile Ala Ala Lys Lys Asn Val Ser Tyr Leu Glu Ile Asp Ala340 345 350ccg caa ggc cac gac gcc ttc ctc atg ccg atc ccc cgg tac ctg caa1104
Pro Gln Gly His Asp Ala Phe Leu Met Pro Ile Pro Arg Tyr Leu Gln355 360 365gcc ttc agc ggt tac atg aac cgc atc agc gtg tga 1140Ala Phe Ser Gly Tyr Met Asn Arg Ile Ser Val370 375<210>18<211>379<212>PRT<213>銅綠假單胞菌<400>18Met Pro Thr Val Phe Pro Asp Asp Ser Val Gly Leu Val Ser Pro Gln1 5 10 15Thr Leu His Phe Asn Glu Pro Leu Glu Leu Thr Ser Gly Lys Ser Leu20 25 30Ala Glu Tyr Asp Leu Val Ile Glu Thr Tyr Gly Glu Leu Asn Ala Thr35 40 45Gln Ser Asn Ala Val Leu Ile Cys His Ala Leu Ser Gly His His His50 55 60Ala Ala Gly Tyr His Ser Val Asp Glu Arg Lys Pro Gly Trp Trp Asp65 70 75 80Ser Cys Ile Gly Pro Gly Lys Pro Ile Asp Thr Arg Lys Phe Phe Val85 90 95Val Ala Leu Asn Asn Leu Gly Gly Cys Asn Gly Ser Ser Gly Pro Ala100 105 110Ser Ile Asn Pro Ala Thr Gly Lys Val Tyr Gly Ala Asp Phe Pro Met115 120 125Val Thr Val Glu Asp Trp Val His Ser Gln Ala Arg Leu Ala Asp Arg130 135 140Leu Gly Ile Arg Gln Trp Ala Ala Val Val Gly Gly Ser Leu Gly Gly145 150 155 160Met Gln Ala Leu Gln Trp Thr Ile Ser Tyr Pro Glu Arg Val Arg His165 170 175Cys Leu Cys Ile Ala Ser Ala Pro Lys Leu Ser Ala Gln Asn Ile Ala180 185 190Phe Asn Glu Val Ala Arg Gln Ala Ile Leu Ser Asp Pro Glu Phe Leu195 200 205Gly Gly Tyr Phe Gln Glu Gln Gly Val Ile Pro Lys Arg Gly Leu Lys210 215 220Leu Ala Arg Met Val Gly His Ile Thr Tyr Leu Ser Asp Asp Ala Met225 230 235 240Gly Ala Lys Phe Gly Arg Val Leu Lys Thr Glu Lys Leu Asn Tyr Asp245 250 255Leu His Ser Val Glu Phe Gln Val Glu Ser Tyr Leu Arg Tyr Gln Gly260 265 270Glu Glu Phe Ser Thr Arg Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr275 280 285
Lys Ala Leu Asp Tyr Phe Asp Pro Ala Ala Ala His Gly Asp Asp Leu290 295 300Val Arg Thr Leu Glu Gly Val Glu Ala Asp Phe Cys Leu Met Ser Phe305 310 315 320Thr Thr Asp Trp Arg Phe Ser Pro Ala Arg Ser Arg Glu Ile Val Asp325 330 335Ala Leu Ile Ala Ala Lys Lys Asn Val Ser Tyr Leu Glu Ile Asp Ala340 345 350Pro Gln Gly His Asp Ala Phe Leu Met Pro Ile Pro Arg Tyr Leu Gln355 360 365Ala Phe Ser Gly Tyr Met Asn Arg Ile Ser Val370 375<210>19<211>1146<212>DNA<213>洋蔥伯克霍爾德氏菌(Burkholderia cepacia)<220>
<221>CDS<222>(1)..(1143)<223>RBU12675<400>19atg gaa tcg atc ggt atc gtc gct ccc caa aaa atg cat ttc acc gag48Met Glu Ser Ile Gly Ile Val Ala Pro Gln Lys Met His Phe Thr Glu1 5 10 15ccg ctg ccg ttg cag aac ggc agt tcg ctc gcc ggt tac gac ctg atg96Pro Leu Pro Leu Gln Asn Gly Ser Ser Leu Ala Gly Tyr Asp Leu Met20 25 30gtc gag acc tac ggc acg ctc aac gcc gcg cgt agc aac gcg gtg ctg144Val Glu Thr Tyr Gly Thr Leu Asn Ala Ala Arg Ser Asn Ala Val Leu35 40 45gtg tgc cac gcg ctc aac gcg tcg cac cac gtg gcg ggc gtg tat gcc192Val Cys His Ala Leu Asn Ala Ser His His Val Ala Gly Val Tyr Ala50 55 60gac aac ccc agg gac atc ggc tgg tgg gac aac atg gtc ggc ccg ggc240Asp Asn Pro Arg Asp Ile Gly Trp Trp Asp Asn Met Val Gly Pro Gly65 70 75 80aag ccg ctc gac act gac aag ttc ttc gtg atc ggc gtg aac aac ctc288Lys Pro Leu Asp Thr Asp Lys Phe Phe Val Ile Gly Val Asn Asn Leu85 90 95gga tcg tgc ttc ggc tcg act ggg ccg atg agc atc gat ccg tct acc336Gly Ser Cys Phe Gly Ser Thr Gly Pro Met Ser Ile Asp Pro Ser Thr100 105 110ggc aat ccg tac ggc gcg acg ttt ccc gtc gtg acg gtg gaa gac tgg384Gly Asn Pro Tyr Gly Ala Thr Phe Pro Val Val Thr Val Glu Asp Trp115 120 125gtc aac gcc cag gcg cgc gtc gcg gat caa ttc ggc atc acg cgc ttt 432Val Asn Ala Gln Ala Arg Val Ala Asp Gln Phe Gly Ile Thr Arg Phe130 135 140
gcg gcg gtg atg ggc ggc agc ctc ggc ggc atg cag gcg ctc gcg tgg480Ala Ala Val Met Gly Gly Ser Leu Gly Gly Met Gln Ala Leu Ala Trp145 150 155 160agc atg atg tat ccg gag cgc gtc gct cac tgc atc gtg gtc gcg tcc528Ser Met Met Tyr Pro Glu Arg Val Ala His Cys Ile Val Val Ala Ser165 170 175aca ccc aag ctg tcg gcg cag aac atc gcg ttc aac gag gtt gcg cgc576Thr Pro Lys Leu Ser Ala Gln Asn Ile Ala Phe Asn Glu Val Ala Arg180 185 190tcg gcg atc ctg tcg gac ccg gac ttc cac ggc ggc aac tac tac gcg624Ser Ala Ile Leu Ser Asp Pro Asp Phe His Gly Gly Asn Tyr Tyr Ala195 200 205cac aac gtt aag ccg aag cgc ggc ctg cgc gtc gcg cgc atg atc ggc672His Asn Val Lys Pro Lys Arg Gly Leu Arg Val Ala Arg Met Ile Gly210 215 220cac atc acg tat ctg tcg gac gac gac atg gcc gag aaa ttc ggc cgc720His Ile Thr Tyr Leu Ser Asp Asp Asp Met Ala Glu Lys Phe Gly Arg225 230 235 240tcg ctg cgg cgc gcg gaa ggc gcg ctg gac gcg tac aac ttc aac ttc768Ser Leu Arg Arg Ala Glu Gly Ala Leu Asp Ala Tyr Asn Phe Asn Phe245 250 255gac gtg gag ttc gag gtg gag tcg tac ctg cgc tac cag ggc gac aag816Asp Val Glu Phe Glu Val Glu Ser Tyr Leu Arg Tyr Gln Gly Asp Lys260 265 270ttc gcc gac tacttc gac gcg aat acg tat ctg ctg atc acc cgc gcg 864Phe Ala Asp Tyr Phe Asp Ala Asn Thr Tyr Leu Leu Ile Thr Arg Ala275 280 285ctc gac tac ttc gat ccg gcc aag gcc ttc gcc ggc gac ctg acg gcc912Leu Asp Tyr Phe Asp Pro Ala Lys Ala Phe Ala Gly Asp Leu Thr Ala290 295 300gcg gtc gcg cac acc acg gcg aaa tat ctg atc gcc agc ttc acg acc960Ala Val Ala His Thr Thr Ala Lys Tyr Leu Ile Ala Ser Phe Thr Thr305 310 315 320gac tgg cgc ttc gcg ccg gcc cgc tcg cgt gaa ctg gtg aag gcg ctg1008Asp Trp Arg Phe Ala Pro Ala Arg Ser Arg Glu Leu Val Lys Ala Leu325 330 335ctc gat cac aag cgc acg gtc acc tac gcg gaa atc gac gcg ccg cac1056Leu Asp His Lys Arg Thr Val Thr Tyr Ala Glu Ile Asp Ala Pro His340 345 350ggc cac gac gcc ttc ctg ctc gac gac gcg cgc tat cac aac ctg atg1104Gly His Asp Ala Phe Leu Leu Asp Asp Ala Arg Tyr His Asn Leu Met355 360 365cgc gct tac tac gaa cgt att gcg aac gag gtg aac gca tga1146Arg Ala Tyr Tyr Glu Arg Ile Ala Asn Glu Val Asn Ala370 375 380<210>20<211>381<212>PRT<213>洋蔥伯克霍爾德氏菌<400>20
Met Glu Ser Ile Gly Ile Val Ala Pro Gln Lys Met His Phe Thr Glu1 5 10 15Pro Leu Pro Leu Gln Asn Gly Ser Ser Leu Ala Gly Tyr Asp Leu Met20 25 30Val Glu Thr Tyr Gly Thr Leu Asn Ala Ala Arg Ser Asn Ala Val Leu35 40 45Val Cys His Ala Leu Asn Ala Ser His His Val Ala Gly Val Tyr Ala50 55 60Asp Asn Pro Arg Asp Ile Gly Trp Trp Asp Asn Met Val Gly Pro Gly65 70 75 80Lys Pro Leu Asp Thr Asp Lys Phe Phe Val Ile Gly Val Asn Asn Leu85 90 95Gly Ser Cys Phe Gly Ser Thr Gly Pro Met Ser Ile Asp Pro Ser Thr100 105 110Gly Asn Pro Tyr Gly Ala Thr Phe Pro Val Val Thr Val Glu Asp Trp115 120 125Val Asn Ala Gln Ala Arg Val Ala Asp Gln Phe Gly Ile Thr Arg Phe130 135 140Ala Ala Val Met Gly Gly Ser Leu Gly Gly Met Gln Ala Leu Ala Trp145 150 155 160Ser Met Met Tyr Pro Glu Arg Val Ala His Cys Ile Val Val Ala Ser165 170 175Thr Pro Lys Leu Ser Ala Gln Asn Ile Ala Phe Asn Glu Val Ala Arg180 185 190Ser Ala Ile Leu Ser Asp Pro Asp Phe His Gly Gly Asn Tyr Tyr Ala195 200 205His Asn Val Lys Pro Lys Arg Gly Leu Arg Val Ala Arg Met Ile Gly210 215 220His Ile Thr Tyr Leu Ser Asp Asp Asp Met Ala Glu Lys Phe Gly Arg225 230 235 240Ser Leu Arg Arg Ala Glu Gly Ala Leu Asp Ala Tyr Asn Phe Asn Phe245 250 255Asp Val Glu Phe Glu Val Glu Ser Tyr Leu Arg Tyr Gln Gly Asp Lys260 265 270Phe Ala Asp Tyr Phe Asp Ala Asn Thr Tyr Leu Leu Ile Thr Arg Ala275 280 285Leu Asp Tyr Phe Asp Pro Ala Lys Ala Phe Ala Gly Asp Leu Thr Ala290 295 300Ala Val Ala His Thr Thr Ala Lys Tyr Leu Ile Ala Ser Phe Thr Thr305 310 315 320Asp Trp Arg Phe Ala Pro Ala Arg Ser Arg Glu Leu Val Lys Ala Leu325 330 335Leu Asp His Lys Arg Thr Val Thr Tyr Ala Glu Ile Asp Ala Pro His340 345 350Gly His Asp Ala Phe Leu Leu Asp Asp Ala Arg Tyr His Asn Leu Met
355 360 365Arg Ala Tyr Tyr Glu Arg Ile Ala Asn Glu Val Asn Ala370 375 380<210>21<211>1134<212>DNA<213>歐洲亞硝化單胞菌(Nitrosomonas europaea)<220>
<221>CDS<222>(1)..(1131)<223>RNE02005<400>21atg tcc aca caa gat tct gat tcg atc ggc atc gta tcg gca cga cgc48Met Ser Thr Gln Asp Ser Asp Ser Ile Gly Ile Val Ser Ala Arg Arg1 5 10 15gcc cat ttc gac acc ccg ctc agc ctg aaa agc gga gct gta ctg gac96Ala His Phe Asp Thr Pro Leu Ser Leu Lys Ser Gly Ala Val Leu Asp20 25 30agc tac gag ctc gtc tat gaa acc tat ggg gag ctg aat gca gac cga144Ser Tyr Glu Leu Val Tyr Glu Thr Tyr Gly Glu Leu Asn Ala Asp Arg35 40 45tcc aat gca gtg ctg atc tgc cat gct tta tcc ggc aac cac cat gtt192Ser Asn Ala Val Leu Ile Cys His Ala Leu Ser Gly Asn His His Val50 55 60gcc ggt gtt tat gca gat aac ccc aag aat acc gga tgg tgg aac aac240Ala Gly Val Tyr Ala Asp Asn Pro Lys Asn Thr Gly Trp Trp Asn Asn65 70 75 80atg atc ggt ccg ggc aaa ccg gtc gat acc cga aaa ttc ttt gtc atc288Met Ile Gly Pro Gly Lys Pro Val Asp Thr Arg Lys Phe Phe Val Ile85 90 95ggt atc aat aat ctc ggg ggt tgc cat ggc tcc acc ggg ccc atc agc336Gly Ile Asn Asn Leu Gly Gly Cys His Gly Ser Thr Gly Pro Ile Ser100 105 110atc aac gac aag acc ggt aaa cgc ttc ggc ccg gat ttt ccg ctg gta384Ile Asn Asp Lys Thr Gly Lys Arg Phe Gly Pro Asp Phe Pro Leu Val115 120 125acg aca gct gac tgg gca aaa acc tat gtc cgt ttc gcc gat cag ttc432Thr Thr Ala Asp Trp Ala Lys Thr Tyr Val Arg Phe Ala Asp Gln Phe130 135 140agc atc gac tgt ttt gcc gcc gtc atc ggt ggc agt ctg ggc ggg atg480Ser Ile Asp Cys Phe Ala Ala Val Ile Gly Gly Ser Leu Gly Gly Met145 150 155 160tcg gcc atg caa ctg gcg ctc gat gca ccg gaa aga gtt cgt cat gcc528Ser Ala Met Gln Leu Ala Leu Asp Ala Pro Glu Arg Val Arg His Ala165 170 175ata gtg gtt gca gca tcg gcc agg ctg aca gca cag aac atc gct ttc576Ile Val Val Ala Ala Ser Ala Arg Leu Thr Ala Gln Asn Ile Ala Phe180 185 190aat gat gtc gcg cgt cag gcg att ctg acc gac cct gat ttt cac gac624Asn Asp Val Ala Arg Gln Ala Ile Leu Thr Asp Pro Asp Phe His Asp
195 200 205ggc gac tat tat tcc cat ggc acc cac ccg cgc aga ggt tta cgc ctt672Gly Asp Tyr Tyr Ser His Gly Thr His Pro Arg Arg Gly Leu Arg Leu210 215 220gcc cgc atg ctt ggc cac atc acc tac ctg tcg gac gac tcc atg gcc720Ala Arg Met Leu Gly His Ile Thr Tyr Leu Ser Asp Asp Ser Met Ala225 230 235 240agc aaa ttc ggc cgt gag tta cgt aac ggc tcg ctt gct ttc aat tat768Ser Lys Phe Gly Arg Glu Leu Arg Asn Gly Ser Leu Ala Phe Asn Tyr245 250 255gat gtg gaa ttc cag atc gaa tcc tat ctg cac cat cag ggc gac aaa816Asp Val Glu Phe Gln Ile Glu Ser Tyr Leu His His Gln Gly Asp Lys260 265 270ttt gcc gac ctg ttc gac gca aac act tat ctg ctg atg acg aag gcg864Phe Ala Asp Leu Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr Lys Ala275 280 285ctc gat tat ttc gat ccg gcc cag gat tac gat ggc aac ctg agt gca912Leu Asp Tyr Phe Asp Pro Ala Gln Asp Tyr Asp Gly Asn Leu Ser Ala290 295 300gcc ttt gcc cgt gca caa gcg gat ttt ctg gta ctt tcc ttt act tcc960Ala Phe Ala Arg Ala Gln Ala Asp Phe Leu Val Leu Ser Phe Thr Ser305 310 315 320gac tgg cgt ttt tcc ccg gag cgt tcg cgc gat atc gtc aag gca ctg 1008Asp Trp Arg Phe Ser Pro Glu Arg Ser Arg Asp Ile Val Lys Ala Leu325 330 335ctc gac aac aaa ctg aat gtc agt tat gcg gaa att ccc tcc tcg tac1056Leu Asp Asn Lys Leu Asn Val Ser Tyr Ala Glu Ile Pro Ser Ser Tyr340 345 350gga cat gat tcc ttt ctc atg cag gac gac tac tat cac cag ttg ata1104Gly His Asp Ser Phe Leu Met Gln Asp Asp Tyr Tyr His Gln Leu Ile355 360 365cgt gct tac atg aac aat atc gct ctc tag1134Arg Ala Tyr Met Asn Asn Ile Ala Leu370 375<210>22<211>377<212>PRT<213>歐洲亞硝化單胞菌<400>22Met Ser Thr Gin Asp Ser Asp Ser Ile Gly Ile Val Ser Ala Arg Arg1 5 10 15Ala His Phe Asp Thr Pro Leu Ser Leu Lys Ser Gly Ala Val Leu Asp20 25 30Ser Tyr Glu Leu Val Tyr Glu Thr Tyr Gly Glu Leu Asn Ala Asp Arg35 40 45Ser Asn Ala Val Leu Ile Cys His Ala Leu Ser Gly Asn His His Val50 55 60Ala Gly Val Tyr Ala Asp Asn Pro Lys Asn Thr Gly Trp Trp Asn Asn65 70 75 80
Met Ile Gly Pro Gly Lys Pro Val Asp Thr Arg Lys Phe Phe Val Ile85 90 95Gly Ile Asn Asn Leu Gly Gly Cys His Gly Ser Thr Gly Pro Ile Ser100 105 110Ile Asn Asp Lys Thr Gly Lys Arg Phe Gly Pro Asp Phe Pro Leu Val115 120 125Thr Thr Ala Asp Trp Ala Lys Thr Tyr Val Arg Phe Ala Asp Gln Phe130 135 140Ser Ile Asp Cys Phe Ala Ala Val Ile Gly Gly Ser Leu Gly Gly Met145 150 155 160Ser Ala Met Gln Leu Ala Leu Asp Ala Pro Glu Arg Val Arg His Ala165 170 175Ile Val Val Ala Ala Ser Ala Arg Leu Thr Ala Gln Asn Ile Ala Phe180 185 190Asn Asp Val Ala Arg Gln Ala Ile Leu Thr Asp Pro Asp Phe His Asp195 200 205Gly Asp Tyr Tyr Ser His Gly Thr His Pro Arg Arg Gly Leu Arg Leu210 215 220Ala Arg Met Leu Gly His Ile Thr Tyr Leu Ser Asp Asp Ser Met Ala225 230 235 240Ser Lys Phe Gly Arg Glu Leu Arg Asn Gly Ser Leu Ala Phe Asn Tyr245 250 255Asp Val Glu Phe Gln Ile Glu Ser Tyr Leu His His Gln Gly Asp Lys260 265 270Phe Ala Asp Leu Phe Asp Ala Asn Thr Tyr Leu Leu Met Thr Lys Ala275 280 285Leu Asp Tyr Phe Asp Pro Ala Gln Asp Tyr Asp Gly Asn Leu Ser Ala290 295 300Ala Phe Ala Arg Ala Gln Ala Asp Phe Leu Val Leu Ser Phe Thr Ser305 310 315 320Asp Trp Arg Phe Ser Pro Glu Arg Ser Arg Asp Ile Val Lys Ala Leu325 330 335Leu Asp Asn Lys Leu Asn Val Ser Tyr Ala Glu Ile Pro Ser Ser Tyr340 345 350Gly His Asp Ser Phe Leu Met Gln Asp Asp Tyr Tyr His Gln Leu Ile355 360 365Arg Ala Tyr Met Asn Asn Ile Ala Leu370 375<210>23<211>1077<212>DNA<213>流感嗜血桿菌(Haemophilus influenzae)<220>
<221>CDS<222>(1)..(1074)
<223>RHI02681<400>23atg tct gtg caa aat gta gtg ctt ttt gac aca cag cct tta act ctg48Met Ser Val Gln Asn Val Val Leu Phe Asp Thr Gln Pro Leu Thr Leu1 5 10 15atg ctt ggc ggc aaa ctt tcc cat att aat gtc gcg tat caa act tat96Met Leu Gly Gly Lys Leu Ser His Ile Asn Val Ala Tyr Gln Thr Tyr20 25 30ggc acg ctc aat gcc gaa aaa aat aat gcg gta tta att tgc cac gct144Gly Thr Leu Asn Ala Glu Lys Asn Asn Ala Val Leu Ile Cys His Ala35 40 45ttg act ggt gat gct gag cct tat ttc gat gat ggt cga gat ggc tgg192Leu Thr Gly Asp Ala Glu Pro Tyr Phe Asp Asp Gly Arg Asp Gly Trp50 55 60tgg cag aat ttt atg gga gca ggt tta gca ttg gat acg gat cgt tat240Trp Gln Asn Phe Met Gly Ala Gly Leu Ala Leu Asp Thr Asp Arg Tyr65 70 75 80ttt ttt att agc tcg aac gta tta ggt ggt tgc aag gga aca act ggg288Phe Phe Ile Ser Ser Asn Val Leu Gly Gly Cys Lys Gly Thr Thr Gly85 90 95cct tca tca att aat ccg caa acg ggt aaa cct tat ggc agc caa ttt336Pro Ser Ser Ile Asn Pro Gln Thr Gly Lys Pro Tyr Gly Ser Gln Phe100 105 110cct aat att gtt gtg caa gat att gtt aaa gta caa aaa gcc ttg ctt384Pro Asn Ile Val Val Gln Asp Ile Val Lys Val Gln Lys Ala Leu Leu115 120 125gat cat ctt ggt att agc cat tta aaa gcc att att ggt gga tct ttt 432Asp His Leu Gly Ile Ser His Leu Lys Ala Ile Ile Gly Gly Ser Phe130 135 140ggc ggc atg caa gcg aat caa tgg gcg att gat tat cct gat ttt atg480Gly Gly Met Gln Ala Asn Gln Trp Ala Ile Asp Tyr Pro Asp Phe Met145 150 155 160gat aat atc gtg aat ctt tgc tca tcc att tat ttt agt gct gaa gcc528Asp Asn Ile Val Asn Leu Cys Ser Ser Ile Tyr Phe Ser Ala Glu Ala165 170 175ata ggt ttt aat cac gta atg cgt caa gcg gtc att aat gat ccc aat576Ile Gly Phe Asn His Val Met Arg Gln Ala Val Ile Asn Asp Pro ASn180 185 190ttt aac ggc ggc gat tat tat gag ggt aca ccg cca gat caa ggg tta624Phe Asn Gly Gly Asp Tyr Tyr Glu Gly Thr Pro Pro Asp Gln Gly Leu195 200 205tct att gca cgt atg cta ggt atg ctg act tac cgc acc gat tta caa672Ser Ile Ala Arg Met Leu Gly Met Leu Thr Tyr Arg Thr Asp Leu Gln210 215 220ctt gcg aaa gcc ttt ggg cgt gcc aca aaa tca gat ggc agc ttt tgg720Leu Ala Lys Ala Phe Gly Arg Ala Thr Lys Ser Asp Gly Ser Phe Trp225 230 235 240ggc gat tac ttt caa gtg gaa tcc tat ctt tct tac caa ggc aaa aaa768Gly Asp Tyr Phe Gln Val Glu Ser Tyr Leu Ser Tyr Gln Gly Lys Lys245 250 255
ttc tta gaa cgt ttt gat gcc aat agt tat ttg cat ttg tta cgt gcg816Phe Leu Glu Arg Phe Asp Ala Asn Ser Tyr Leu His Leu Leu Arg Ala260 265 270ttg gat atg tat gat cca agt ttg ggg tat gac aat gtt aaa gag gca864Leu Asp Met Tyr Asp Pro Ser Leu Gly Tyr Asp Asn Val Lys Glu Ala275 280 285tta tca cgt att aaa gca cgc tat acg ttg gtt tct gtg aca acg gat912Leu Ser Arg Ile Lys Ala Arg Tyr Thr Leu Val Ser Val Thr Thr Asp290 295 300caa ctt ttt aaa ccc att gat ctt tat aaa agt aaa cag ctt tta gag960Gln Leu Phe Lys Pro Ile Asp Leu Tyr Lys Ser Lys Gln Leu Leu Glu305 310 315 320caa agt gga gtc gat cta cat ttt tat gaa ttc cca tca gat tac gga1008Gln Ser Gly Val Asp Leu His Phe Tyr Glu Phe Pro Ser Asp Tyr Gly325 330 335cac gat gcg ttt tta gtg gat tat gat cag ttt gaa aaa cga att cga1056His Asp Ala Phe Leu Val Asp Tyr Asp Gln Phe Glu Lys Arg Ile Arg340 345 350gat ggt ttg gca ggt aat taa1077Asp Gly Leu Ala Gly Asn355<210>24<211>358<212>PRT<213>流感嗜血桿菌<400>24Met Ser Val Gln Asn Val Val Leu Phe Asp Thr Gln Pro Leu Thr Leu1 5 10 15Met Leu Gly Gly Lys Leu Ser His Ile Asn Val Ala Tyr Gln Thr Tyr20 25 30Gly Thr Leu Asn Ala Glu Lys Asn Asn Ala Val Leu Ile Cys His Ala35 40 45Leu Thr Gly Asp Ala Glu Pro Tyr Phe Asp Asp Gly Arg Asp Gly Trp50 55 60Trp Gln Asn Phe Met Gly Ala Gly Leu Ala Leu Asp Thr Asp Arg Tyr65 70 75 80Phe Phe Ile Ser Ser Asn Val Leu Gly Gly Cys Lys Gly Thr Thr Gly85 90 95Pro Ser Ser Ile Asn Pro Gln Thr Gly Lys Pro Tyr Gly Ser Gln Phe100 105 110Pro Asn Ile Val Val Gln Asp Ile Val Lys Val Gln Lys Ala Leu Leu115 120 125Asp His Leu Gly Ile Ser His Leu Lys Ala Ile Ile Gly Gly Ser Phe130 135 140Gly Gly Met Gln Ala Asn Gln Trp Ala Ile Asp Tyr Pro Asp Phe Met145 150 155 160Asp Asn Ile Val Asn Leu Cys Ser Ser Ile Tyr Phe Ser Ala Glu Ala165 170 175
Ile Gly Phe Asn His Val Met Arg Gln Ala Val Ile Asn Asp Pro Asn180 185 190Phe Asn Gly Gly Asp Tyr Tyr Glu Gly Thr Pro Pro Asp Gln Gly Leu195 200 205Ser Ile Ala Arg Met Leu Gly Met Leu Thr Tyr Arg Thr Asp Leu Gln210 215 220Leu Ala Lys Ala Phe Gly Arg Ala Thr Lys Ser Asp Gly Ser Phe Trp225 230 235 240Gly Asp Tyr Phe Gln Val Glu Ser Tyr Leu Ser Tyr Gln Gly Lys Lys245 250 255Phe Leu Glu Arg Phe Asp Ala Asn Ser Tyr Leu His Leu Leu Arg Ala260 265 270Leu Asp Met Tyr Asp Pro Ser Leu Gly Tyr Asp Asn Val Lys Glu Ala275 280 285Leu Ser Arg Ile Lys Ala Arg Tyr Thr Leu Val Ser Val Thr Thr Asp290 295 300Gln Leu Phe Lys Pro Ile Asp Leu Tyr Lys Ser Lys Gln Leu Leu Glu305 310 315 320Gln Ser Gly Val Asp Leu His Phe Tyr Glu Phe Pro Ser Asp Tyr Gly325 330 335His Asp Ala Phe Leu Val Asp Tyr Asp Gln Phe Glu Lys Arg Ile Arg340 345 350Asp Gly Leu Ala Gly Asn355<210>25<211>1296<212>DNA<213>鹽桿菌屬中的種(Halobacterium sp)<220>
<221>CDS<222>(1)..(1293)<223>ETX_HALN1<400>25atg ggc cac gat cac gga ctc cac acc aac agt gta cac gcc ggc cag48Met Gly His Asp His Gly Leu His Thr Asn Ser Val His Ala Gly Gln1 5 10 15cgc gtc gac ccg gcc acg ggc gct cgc gcg ccg cca ctc tac cag acc96Arg Val Asp Pro Ala Thr Gly Ala Arg Ala Pro Pro Leu Tyr Gln Thr20 25 30acg tcg tac gcc ttc gag gac agc gcc gat gcc gcc ggc cag ttc gcc144Thr Ser Tyr Ala Phe Glu Asp Ser Ala Asp Ala Ala Gly Gln Phe Ala35 40 45ctt gag cgg gac ggc tac atc tac tcg cgg ctg atg aac ccc acc gtg192Leu Glu Arg Asp Gly Tyr Ile Tyr Ser Arg Leu Met Asn Pro Thr Val50 55 60gag acc ctc cag gac cgc ctc gcc gcc ctc gaa ggc ggc gtc ggc gcg240Glu Thr Leu Gln Asp Arg Leu Ala Ala Leu Glu Gly Gly Val Gly Ala
65 70 75 80gtc gcc acc gcg tcc gga atg gcc gcc ctg gac ctc gcg acg ttc ctg288Val Ala Thr Ala Ser Gly Met Ala Ala Leu Asp Leu Ala Thr Phe Leu85 90 95ctg gca cgc gcc ggc gac tcc gtc gtc gcc gcc agc gac ctc tac ggc336Leu Ala Arg Ala Gly Asp Ser Val Val Ala Ala Ser Asp Leu Tyr Gly100 105 110ggc acc gtg acg tac ctc acg cac agc gcc cag cgc cgc ggc gtc gac384Gly Thr Val Thr Tyr Leu Thr His Ser Ala Gln Arg Arg Gly Val Asp115 120 125acg acg ttc gtg gac gtc ctc gac tac gac gcc tac gcc gac gcc atc432Thr Thr Phe Val Asp Val Leu Asp Tyr Asp Ala Tyr Ala Asp Ala Ile130 135 140gac gcc gac acc gcc tac gtg ctc gtc gaa acc gtc ggc aac ccc agc480Asp Ala Asp Thr Ala Tyr Val Leu Val Glu Thr Val Gly Asn Pro Ser145 150 155 160ctg atc acg ccc gac ctc gaa cgc atc gcc gac atc gcc cac gac aac528Leu Ile Thr Pro Asp Leu Glu Arg Ile Ala Asp Ile Ala His Asp Asn165 170 175ggc gtt ccc ctg ctg gtg gac aac acg ttc gcg acc ccc gcg ctg gca576Gly Val Pro Leu Leu Val Asp Asn Thr Phe Ala Thr Pro Ala Leu Ala180 185 190acc ccg atc gac cac ggt gcc gac atc gtc tgg cac tcc acc acc aaa624Thr Pro Ile Asp His Gly Ala Asp Ile Val Trp His Ser Thr Thr Lys195 200 205tgg atc cac ggt gcc ggc acc acc gtc ggc ggc gcg ctc gtc gac gcc672Trp Ile His Gly Ala Gly Thr Thr Val Gly Gly Ala Leu Val Asp Ala210 215 220ggc agc ttc gac tgg gac gcc cac gcc gcc gac tac ccc gag atc gcc720Gly Ser Phe Asp Trp Asp Ala His Ala Ala Asp Tyr Pro Glu Ile Ala225 230 235 240cag gaa aac ccc gcc tac cac ggc gtg acc ttc acc gat cgc ttc ggg768Gln Glu Asn Pro Ala Tyr His Gly Val Thr Phe Thr Asp Arg Phe Gly245 250 255gac gcc gcg ttc acg tac gcc gca atc gcc cgc ggg ctg cgc gat ctg816Asp Ala Ala Phe Thr Tyr Ala Ala Ile Ala Arg Gly Leu Arg Asp Leu260 265 270ggc aac cag cag tcg ccg ttc gac gcc tgg cag acc ctc cag aag ctc864Gly Asn Gln Gln Ser Pro Phe Asp Ala Trp Gln Thr Leu Gln Lys Leu275 280 285gaa acg ctc ccg ctg cgc atg caa caa cac tgc cgg aac gcc cag ctc912Glu Thr Leu Pro Leu Arg Met Gln Gln His Cys Arg Asn Ala Gln Leu290 295 300gtc gcc gaa cac ctc cgg gac cac ccc aac gtg tcg tgg gtc aac tac960Val Ala Glu His Leu Arg Asp His Pro Asn Val Ser Trp Val Asn Tyr305 310 315 320ccc ggg ctg gcc gac cac gac acc cac gac aac gca acc acc tac ctc 1008Pro Gly Leu Ala Asp His Asp Thr His Asp Asn Ala Thr Thr Tyr Leu325 330 335gat tcg ggc tac gga ggc atg ctc acg ttc ggc gtc gag gac ggc tac 1056
Asp Ser Gly Tyr Gly Gly Met Leu Thr Phe Gly Val Glu Asp Gly Tyr340 345 350gag gcc gcc caa tcg gtc acc gag gag acc acg ctt gcc agc ctg ctg1104Glu Ala Ala Gln Ser Val Thr Glu Glu Thr Thr Leu Ala Ser Leu Leu355 360 365gcg aac gtc ggc gac gcc aaa acg ctc gtg atc cac ccc gcc tcc acc1152Ala Asn Val Gly Asp Ala Lys Thr Leu Val Ile His Pro Ala Ser Thr370 375 380acc cac cag cag ctc acc ccc gaa gcc cag cgc gcc ggc ggt gtg cgc1200Thr His Gln Gln Leu Thr Pro Glu Ala Gln Arg Ala Gly Gly Val Arg385 390 395 400ccc gag atg gtg cgg gtg tcg gtc ggc atc gag gac ccc gcc gac atc1248Pro Glu Met Val Arg Val Ser Val Gly Ile Glu Asp Pro Ala Asp Ile405 410 415gtc gcg gac ctc gaa acc gcc atc gag gcc gcg gtc ggg tcg gcg1293Val Ala Asp Leu Glu Thr Ala Ile Glu Ala Ala Val Gly Ser Ala420 425 430tag1296<210>26<211>431<212>PRT<213>鹽桿菌屬中的種<400>26Met Gly His Asp His Gly Leu His Thr Asn Ser Val His Ala Gly Gln1 5 10 15Arg Val Asp Pro Ala Thr Gly Ala Arg Ala Pro Pro Leu Tyr Gln Thr20 25 30Thr Ser Tyr Ala Phe Glu Asp Ser Ala Asp Ala Ala Gly Gln Phe Ala35 40 45Leu Glu Arg Asp Gly Tyr Ile Tyr Ser Arg Leu Met Asn Pro Thr Val50 55 60Glu Thr Leu Gln Asp Arg Leu Ala Ala Leu Glu Gly Gly Val Gly Ala65 70 75 80Val Ala Thr Ala Ser Gly Met Ala Ala Leu Asp Leu Ala Thr Phe Leu85 90 95Leu Ala Arg Ala Gly Asp Ser Val Val Ala Ala Ser Asp Leu Tyr Gly100 105 110Gly Thr Val Thr Tyr Leu Thr His Ser Ala Gln Arg Arg Gly Val Asp115 120 125Thr Thr Phe Val Asp Val Leu Asp Tyr Asp Ala Tyr Ala Asp Ala Ile130 135 140Asp Ala Asp Thr Ala Tyr Val Leu Val Glu Thr Val Gly Asn Pro Ser145 150 155 160Leu Iie Thr Pro Asp Leu Glu Arg Ile Ala Asp Ile Ala His Asp Asn165 170 175Gly Val Pro Leu Leu Val Asp Asn Thr Phe Ala Thr Pro Ala Leu Ala180 185 190
Thr Pro Ile Asp His Gly Ala Asp Ile Val Trp His Ser Thr Thr Lys195 200 205Trp Ile His Gly Ala Gly Thr Thr Val Gly Gly Ala Leu Val Asp Ala210 215 220Gly Ser Phe Asp Trp Asp Ala His Ala Ala Asp Tyr Pro Glu Ile Ala225 230 235 240Gln Glu Asn Pro Ala Tyr His Gly Val Thr Phe Thr Asp Arg Phe Gly245 250 255Asp Ala Ala Phe Thr Tyr Ala Ala Ile Ala Arg Gly Leu Arg Asp Leu260 265 270Gly Asn Gln Gln Ser Pro Phe Asp Ala Trp Gln Thr Leu Gln Lys Leu275 280 285Glu Thr Leu Pro Leu Arg Met Gln Gln His Cys Arg Asn Ala Gln Leu290 295 300Val Ala Glu His Leu Arg Asp His Pro Asn Val Ser Trp Val Asn Tyr305 310 315 320Pro Gly Leu Ala Asp His Asp Thr His Asp Asn Ala Thr Thr Tyr Leu325 330 335Asp Ser Gly Tyr Gly Gly Met Leu Thr Phe Gly Val Glu Asp Gly Tyr340 345 350Glu Ala Ala Gln Ser Val Thr Glu Glu Thr Thr Leu Ala Ser Leu Leu355 360 365Ala Asn Val Gly Asp Ala Lys Thr Leu Val Ile His Pro Ala Ser Thr370 375 380Thr His Gln Gln Leu Thr Pro Glu Ala Gln Arg Ala Gly Gly Val Arg385 390 395 400Pro Glu Met Val Arg Val Ser Val Gly Ile Glu Asp Pro Ala Asp Ile405 410 415Val Ala Asp Leu Glu Thr Ala Ile Glu Ala Ala Val Gly Ser Ala420 425 430<210>27<211>1143<212>DNA<213>嗜熱棲熱菌(Thermus thermophilus)<220>
<221>CDS<222>(1)..(1140)<223>RTT00268<400>27atg agc gag atc gcc ctc gag gcc tgg ggg gag cac gag gcc ctc ctc48Met Ser Glu Ile Ala Leu Glu Ala Trp Gly Glu His Glu Ala Leu Leu1 5 10 15ctc aag ccc ccc cgc tcc ccc ctc tcc atc ccc ccg ccc aag ccc cgc96Leu Lys Pro Pro Arg Ser Pro Leu Ser Ile Pro Pro Pro Lys Pro Arg20 25 30acc gcc gtc ctc ttc ccc agg cgg gag ggg ttc tac acg gag ctc ggg144
Thr Ala Val Leu Phe Pro Arg Arg Glu Gly Phe Tyr Thr Glu Leu Gly35 40 45ggg tac ctc ccc gag gtg cgc ctc cgc ttt gag acc tac ggg acc ctc192Gly Tyr Leu Pro Glu Val Arg Leu Arg Phe Glu Thr Tyr Gly Thr Leu50 55 60tcc cgc agg cgg gat aac gcc gtc ctc gtc ttc cac gcc ctc acg ggg240Ser Arg Arg Arg Asp Asn Ala Val Leu Val Phe His Ala LeuThr Gly65 70 75 80agc gcc cac ctg gcg ggg acc tac gac gag gaa acc ttt aga agc ctc288Ser Ala His Leu Ala Gly Thr Tyr Asp Glu Glu Thr Phe Arg Ser Leu85 90 95tcc ccc ctg gag cag gcc ttc ggc cgg gaa ggg tgg tgg gac agc ctg336Ser Pro Leu Glu Gln Ala Phe Gly Arg Glu Gly Trp Trp Asp Ser Leu100 105 110gtg ggg ccc ggg cgg atc ctg gac ccc gcc ctc tac tac gtg gtc tcc384Val Gly Pro Gly Arg Ile Leu Asp Pro Ala Leu Tyr Tyr Val Val Ser115 120 125gcc aac cac ctg gga agc tgc tac ggc tcc acc ggc ccc ctc tcc cta432Ala Asn His Leu Gly Ser Cys Tyr Gly Ser Thr Gly Pro Leu Ser Leu130 135 140gac ccc cac acg ggc cgc ccc tac ggg agg gac ttc cct ccc ctt acc480Asp Pro His Thr Gly Arg Pro Tyr Gly Arg Asp Phe Pro Pro Leu Thr145 150 155 160atc cgc gac ctg gcc cgg gcc cag gcg agg ctt ctg gac cat ctg ggg528Ile Arg Asp Leu Ala Arg Ala Gln Ala Arg Leu Leu Asp His Leu Gly165 170 175gtg gag aag gcc atc gtc atc ggg ggg agc ctc ggg ggg atg gtg gcc576Val Glu Lys Ala Ile Val Ile Gly Gly Ser Leu Gly Gly Met Val Ala180 185 190ctg gag ttc gcc ctc atg tac ccg gag agg gtg aag aag ctc gtg gtc624Leu Glu Phe Ala Leu Met Tyr Pro Glu Arg Val Lys Lys Leu Val Val195 200 205ctg gcg gcc ccc gca cgg cac ggc ccc tgg gcc cgg gcc ttc aac cac672Leu Ala Ala Pro Ala Arg His Gly Pro Trp Ala Arg Ala Phe Asn His210 215 220ctc tcc cgc cag gcc atc ctc caa gac ccc gag tac cag aag ggc aac720Leu Ser Arg Gln Ala Ile Leu Gln Asp Pro Glu Tyr Gin Lys Gly Asn225 230 235 240cct gcc ccc aag ggc atg gcc ctc gcc cgg gga atc gcc atg atg agc768Pro Ala Pro Lys Gly Met Ala Leu Ala Arg Gly Ile Ala Met Met Ser245 250 255tac cgg gcc ccc gag ggg ttt gag gcc cgc tgg ggc gcg gag ccc gag816Tyr Arg Ala Pro Glu Gly Phe Glu Ala Arg Trp Gly Ala Glu Pro Glu260 265 270ctc ggg gaa atc cac ctg gac tac cag ggg gag aag ttc ctc cgg cgc864Leu Gly Glu Ile His Leu Asp Tyr Gln Gly Glu Lys Phe Leu Arg Arg275 280 285ttc cac gcc gag agc tac ctc gtc ctc tcc cgg gcc atg gac aac cac912Phe His Ala Glu Ser Tyr Leu Val Leu Ser Arg Ala Met Asp Asn His290 295 300
gac gtg ggc cgg ggc cgg ggc ggg gtg gag gag gcc ctg aag cgc ctc 960Asp Val Gly Arg Gly Arg Gly Gly Val Glu Glu Ala Leu Lys Arg Leu305 310 315 320agg gcc atc ccc tcc ctc ttc gtg ggc att gac acc gac ctc ctc tac1008Arg Ala Ile Pro Ser Leu Phe Val Gly Ile Asp Thr Asp Leu Leu Tyr325 330 335ccc gcc tgg gag gtg agg cag gcg gcc aag gcg gcg ggg gcc cgc tac1056Pro Ala Trp Glu Val Arg Gin Ala Ala Lys Ala Ala Gly Ala Arg Tyr340 345 350cgg gag atc aaa agc ccc cac ggg cac gac gcc ttc ctc ata gag acc1104Arg Glu Ile Lys Ser Pro His Gly His Asp Ala Phe Leu Ile Glu Thr355 360 365gac cag gtg gag gag atc ctg gac gcc ttc ctc ccg tag1143Asp Gln Val Glu Glu Ile Leu Asp Ala Phe Leu Pro370 375 380<210>28<211>380<212>PRT<213>嗜熱棲熱菌<400>28Met Ser Glu Ile Ala Leu Glu Ala Trp Gly Glu His Glu Ala Leu Leu1 5 10 15Leu Lys Pro Pro Arg Ser Pro Leu Ser Ile Pro Pro Pro Lys Pro Arg20 25 30Thr Ala Val Leu Phe Pro Arg Arg Glu Gly Phe Tyr Thr Glu Leu Gly35 40 45Gly Tyr Leu Pro Glu Val Arg Leu Arg Phe Glu Thr Tyr Gly Thr Leu50 55 60Ser Arg Arg Arg Asp Asn Ala Val Leu Val Phe His Ala Leu Thr Gly65 70 75 80Ser Ala His Leu Ala Gly Thr Tyr Asp Glu Glu Thr Phe Arg Ser Leu85 90 95Ser Pro Leu Glu Gln Ala Phe Gly Arg Glu Gly Trp Trp Asp Ser Leu100 105 110Val Gly Pro Gly Arg Ile Leu Asp Pro Ala Leu Tyr Tyr Val Val Ser115 120 125Ala Asn His Leu Gly Ser Cys Tyr Gly Ser Thr Gly Pro Leu Ser Leu130 135 140Asp Pro His Thr Gly Arg Pro Tyr Gly Arg Asp Phe Pro Pro Leu Thr145 150 155 160Ile Arg Asp Leu Ala Arg Ala Gln Ala Arg Leu Leu Asp His Leu Gly165 170 175Val Glu Lys Ala Ile Val Ile Gly Gly Ser Leu Gly Gly Met Val Ala180 185 190Leu Glu Phe Ala Leu Met Tyr Pro Glu Arg Val Lys Lys Leu Val Val195 200 205Leu Ala Ala Pro Ala Arg His Gly Pro Trp Ala Arg Ala Phe Asn His
210 215 220Leu Ser Arg Gln Ala Ile Leu Gln Asp Pro Glu Tyr Gln Lys Gly Asn225 230 235 240Pro Ala Pro Lys Gly Met Ala Leu Ala Arg Gly Ile Ala Met Met Ser245 250 255Tyr Arg Ala Pro Glu Gly Phe Glu Ala Arg Trp Gly Ala GluPro Glu260 265 270Leu Gly Glu Ile His Leu Asp Tyr Gln Gly Glu Lys Phe Leu Arg Arg275 280 285Phe His Ala Glu Ser Tyr Leu Val Leu Ser Arg Ala Met Asp Asn His290 295 300Asp Val Gly Arg Gly Arg Gly Gly Val Glu Glu Ala Leu Lys Arg Leu305 310 315 320Arg Ala Ile Pro Ser Leu Phe Val Gly Ile Asp Thr Asp Leu Leu Tyr325 330 335Pro Ala Trp Glu Val Arg Gln Ala Ala Lys Ala Ala Gly Ala Arg Tyr340 345 350Arg Glu Ile Lys Ser Pro His Gly His Asp Ala Phe Leu Ile Glu Thr355 360 365Asp Gln Val Glu Glu Ile Leu Asp Ala Phe Leu Pro370 375 380<210>29<211>1005<212>DNA<213>耐輻射奇異球菌(Deinococcus radiodurans)<220>
<221>CDS<222>(1)..(1002)<223>RDR01287<400>29gtg acc gcc gtg ctc gcg ggc cac gcc tct gcc ctg ctg ctg acc gaa48Val Thr Ala Val Leu Ala Gly His Ala Ser Ala Leu Leu Leu Thr Glu1 5 10 15gaa ccc gac tgt tcg ggg ccg cag acg gtc gtt ctc ttc cgg cgt gag96Glu Pro Asp Cys Ser Gly Pro Gln Thr Val Val Leu Phe Arg Arg Glu20 25 30ccg ctg ctg ctc gac tgc gga cgg gcg ctg agc gac gtg cgg gtg gcc144Pro Leu Leu Leu Asp Cys Gly Arg Ala Leu Ser Asp Val Arg Val Ala35 40 45ttt cac acc tac ggc acg ccg cgc gcc gac gcc acg ctg gtg ctg cac192Phe His Thr Tyr Gly Thr Pro Arg Ala Asp Ala Thr Leu Val Leu His50 55 60gcc ctg acc ggc gac agc gcg gtg cac gag tgg tgg ccc gac ttt ctg240Ala Leu Thr Gly Asp Ser Ala Val His Glu Trp Trp Pro Asp Phe Leu65 70 75 80ggc gcg ggc cgg cca ctg gac ccg gca gac gac tac gtg gtg tgc gcc288Gly Ala Gly Arg Pro Leu Asp Pro Ala Asp Asp Tyr Val Val Cys Ala85 90 95
aac gtc ctc ggc ggg tgc gcc ggc acg acg agc gcc gct gaa ctc gcc336Asn Val Leu Gly Gly Cys Ala Gly Thr Thr Ser Ala Ala Glu Leu Ala100 105 110gcc acc tgt tcc gga ccg gtg ccg ctc agc ctg cgc gac atg gcc cgg384Ala Thr Cys Ser Gly Pro Val Pro Leu Ser Leu Arg Asp Met Ala Arg115 120 125gtg ggg cgc gcc ctg ctg gat tct ctc ggc gtg cga cgg gtg cgg gtc432Val Gly Arg Ala Leu Leu Asp Ser Leu Gly Val Arg Arg Val Arg Val130 135 140atc ggc gcg agc atg ggc ggg atg ctc gcc tac gcc tgg ctg ctg gag480Ile Gly Ala Ser Met Gly Gly Met Leu Ala Tyr Ala Trp Leu Leu Glu145 150 155 160tgc ccc gac ctg gtg gaa aag gcc gtg att ata gga gcc ccg gcg cgg528Cys Pro Asp Leu Val Glu Lys Ala Val Ile Ile Gly Ala Pro Ala Arg165 170 175cac tcg ccc tgg gct att gga ctg aac acg gcg gcc cgc agc gcc att576His Ser Pro Trp Ala Ile Gly Leu Asn Thr Ala Ala Arg Ser Ala Ile180 185 190gcc ctc gct ccc ggc ggc gag ggg ctg aag gtg gcg cgc cag att gcc624Ala Leu Ala Pro Gly Gly Glu Gly Leu Lys Val Ala Arg Gln Ile Ala195 200 205atg ctc agt tac cgc agc ccc gaa agc cta agc cgc acg cag gcg ggg672Met Leu Ser Tyr Arg Ser Pro Glu Ser Leu Ser Arg Thr Gln Ala Gly210 215 220cag cgc gtg ccg ggg gtg ccc gcc gtt acg tct tac ctg cac tac caa720Gln Arg Val Pro Gly Val Pro Ala Val Thr Ser Tyr Leu His Tyr Gln225 230 235 240ggc gaa aaa ctc gcc gcc cgc ttc gac gag cag acc tac tgc gcc ctc768Gly Glu Lys Leu Ala Ala Arg Phe Asp Glu Gln Thr Tyr Cys Ala Leu245 250 255acc tgg gcg atg gac gcc ttt cag ccg agc agc gcc gac ctc aaa gcg816Thr Trp Ala Met Asp Ala Phe Gln Pro Ser Ser Ala Asp Leu Lys Ala260 265 270gtg cgc gcg ccg gta ctc gtc gtc ggc atc tcc agc gat ctg ctc tac864Val Arg Ala Pro Val Leu Val Val Gly Ile Ser Ser Asp Leu Leu Tyr275 280 285ccc gcc gcc gag gtc cgc gcc tgc gcc gcc gag ctt ccc cac gcc gac912Pro Ala Ala Glu Val Arg Ala Cys Ala Ala Glu Leu Pro His Ala Asp290 295 300tac tgg gaa ctg ggc agc att cac ggc cac gac gcc ttt ttg atg gac960Tyr Trp Glu Leu Gly Ser Ile His Gly His Asp Ala Phe Leu Met Asp305 310 315 320cca cag gac ttg ccg gag cgg gtg ggg gcg ttt ctc agg agt1002Pro Gln Asp Leu Pro Glu Arg Val Gly Ala Phe Leu Arg Ser325 330tga1005<210>30<211>334<212>PRT
<213>耐輻射奇異球菌<400>30Val Thr Ala Val Leu Ala Gly His Ala Ser Ala Leu Leu Leu Thr Glu1 5 10 15Glu Pro Asp Cys Ser Gly Pro Gln Thr Val Val Leu Phe Arg Arg Glu20 25 30Pro Leu Leu Leu Asp Cys Gly Arg Ala Leu Ser Asp Val Arg Val Ala35 40 45Phe His Thr Tyr Gly Thr Pro Arg Ala Asp Ala Thr Leu Val Leu His50 55 60Ala Leu Thr Gly Asp Ser Ala Val His Glu Trp Trp Pro Asp Phe Leu65 70 75 80Gly Ala Gly Arg Pro Leu Asp Pro Ala Asp Asp Tyr Val Val Cys Ala85 90 95Asn Val Leu Gly Gly Cys Ala Gly Thr Thr Ser Ala Ala Glu Leu Ala100 105 110Ala Thr Cys Ser Gly Pro Val Pro Leu Ser Leu Arg Asp Met Ala Arg115 120 125Val Gly Arg Ala Leu Leu Asp Ser Leu Gly Val Arg Arg Val Arg Val130 135 140Ile Gly Ala Ser Met Gly Gly Met Leu Ala Tyr Ala Trp Leu Leu Glu145 150 155 160Cys Pro Asp Leu Val Glu Lys Ala Val Ile Ile Gly Ala Pro Ala Arg165 170 175His Ser Pro Trp Ala Ile Gly Leu Asn Thr Ala Ala Arg Ser Ala Ile180 185 190Ala Leu Ala Pro Gly Gly Glu Gly Leu Lys Val Ala Arg Gln Ile Ala195 200 205Met Leu Ser Tyr Arg Ser Pro Glu Ser Leu Ser Arg Thr Gln Ala Gly210 215 220Gln Arg Val Pro Gly Val Pro Ala Val Thr Ser Tyr Leu His Tyr Gln225 230 235 240Gly Glu Lys Leu Ala Ala Arg Phe Asp Glu Gln Thr Tyr Cys Ala Leu245 250 255Thr Trp Ala Met Asp Ala Phe Gln Pro Ser Ser Ala Asp Leu Lys Ala260 265 270Val Arg Ala Pro Val Leu Val Val Gly Ile Ser Ser Asp Leu Leu Tyr275 280 285Pro Ala Ala Glu Val Arg Ala Cys Ala Ala Glu Leu Pro His Ala Asp290 295 300Tyr Trp Glu Leu Gly Ser Ile His Gly His Asp Ala Phe Leu Met Asp305 310 315 320Pro Gln Asp Leu Pro Glu Arg Val Gly Ala Phe Leu Arg Ser325 330
<210>31<211>1461<212>DNA<213>釀酒酵母(saccharomyces cerevisiae)<220>
<221>CDS<222>(1)..(1458)<223>RSC08123<400>31atg tcg cat act tta aaa tcg aaa acg ctc caa gag ctg gac att gag48Met Ser His Thr Leu Lys Ser Lys Thr Leu Gln Glu Leu Asp Ile Glu1 5 10 15gag att aag gaa act aac cca ttg ctc aaa cta gtt caa ggg cag agg96Glu Ile Lys Glu Thr Asn Pro Leu Leu Lys Leu Val Gln Gly Gln Arg20 25 30att gtt caa gtt ccg gaa cta gtg ctt gag tct ggc gtg gtc ata aat144Ile Val Gln Val Pro Glu Leu Val Leu Glu Ser Gly Val Val Ile Asn35 40 45aat ttc cct att gct tat aag acg tgg ggt aca ctg aat gaa gct ggt192Asn Phe Pro Ile Ala Tyr Lys Thr Trp Gly Thr Leu Asn Glu Ala Gly50 55 60gat aat gtt ctg gta att tgt cat gcc ttg act ggg tcc gca gat gtt240Asp Asn Val Leu Val Ile Cys His Ala Leu Thr Gly Ser Ala Asp Val65 70 75 80gct gac tgg tgg ggc cct ctt ctg ggt aac gac tta gca ttc gac cca288Ala Asp Trp Trp Gly Pro Leu Leu Gly Asn Asp Leu Ala Phe Asp Pro85 90 95tca agg ttt ttt atc ata tgt tta aac tct atg ggc tct cca tat ggg336Ser Arg Phe Phe Ile Ile Cys Leu Asn Ser Met Gly Ser Pro Tyr Gly100 105 110tct ttt tcg cca tta acg ata aat gag gag acg ggc gtt aga tat gga384Ser Phe Ser Pro Leu Thr Ile Asn Glu Glu Thr Gly Val Arg Tyr Gly115 120 125ccc gaa ttc cca tta tgt act gtg cgc gat gac gtt aga gct cac aga432Pro Glu Phe Pro Leu Cys Thr Val Arg Asp Asp Val Arg Ala His Arg130 135 140att gtt ctg gat tct ctg gga gta aag tca ata gcc tgt gtt att ggt480Ile Val Leu Asp Ser Leu Gly Val Lys Ser Ile Ala Cys Val Ile Gly145 150 155 160ggc tct atg ggg ggg atg ctg agt ttg gaa tgg gct gcc atg tat ggt528Gly Ser Met Gly Gly Met Leu Ser Leu Glu Trp Ala Ala Met Tyr Gly165 170 175aag gaa tat gtg aag aat atg gtt gct ctg gcg aca tca gca aga cat576Lys Glu Tyr Val Lys Asn Met Val Ala Leu Ala Thr Ser Ala Arg His180 185 190tct gcc tgg tgc ata tcg tgg tct gag gct caa aga caa tcg att tac624Ser Ala Trp Cys Ile Ser Trp Ser Glu Ala Gln Arg Gln Ser Ile Tyr195 200 205tca gat ccc aac tac ttg gac ggg tac tat ccg gta gag gag caa cct672Ser Asp Pro Asn Tyr Leu Asp Gly Tyr Tyr Pro Val Glu Glu Gln Pro210 215 220
gtg gcc gga cta tcg gct gca cgt atg tct gca ttg ttg acg tac agg 720Val Ala Gly Leu Ser Ala Ala Arg Met Ser Ala Leu Leu Thr Tyr Arg225 230 235 240aca aga aac agt ttc gag aac aaa ttc tcc aga aga tct cct tca ata768Thr Arg Asn Ser Phe Glu Asn Lys Phe Ser Arg Arg Ser Pro Ser Ile245 250 255gca caa caa caa aaa gct caa agg gag gag aca cgc aaa ccatct act 816Ala Gln Gln Gln Lys Ala Gln Arg Glu Glu Thr Arg Lys Pro Ser Thr260 265 270gtc agc gaa cac tcc cta caa atc cac aat gat ggg tat aaa aca aaa864Val Ser Glu His Ser Leu Gln Ile His Asn Asp Gly Tyr Lys Thr Lys275 280 285gcc agc act gcc atc gct ggc att tct ggg caa aaa ggt caa agc gtg912Ala Ser Thr Ala Ile Ala Gly Ile Ser Gly Gln Lys Gly Gln Ser Val290 295 300gtg tcc acc gca tct tct tcg gat tca ttg aat tct tca aca tcg atg960Val Ser Thr Ala Ser Ser Ser Asp Ser Leu Asn Ser Ser Thr Ser Met305 310 315 320act tcg gta agt tct gta acg ggt gaa gtg aag gac ata aag cct gcg1008Thr Ser Val Ser Ser Val Thr Gly Glu Val Lys Asp Ile Lys Pro Ala325 330 335cag acg tat ttt tct gca caa agt tac ttg agg tac cag ggc aca aag1056Gln Thr Tyr Phe Ser Ala Gln Ser Tyr Leu Arg Tyr Gln Gly Thr Lys340 345 350ttc atc aat agg ttc gac gcc aat tgt tac att gcc atc aca cgt aaa1104Phe Ile Asn Arg Phe Asp Ala Asn Cys Tyr Ile Ala Ile Thr Arg Lys355 360 365ctg gat acg cac gat ttg gca aga gac aga gta gat gac atc act gag1152Leu Asp Thr His Asp Leu Ala Arg Asp Arg Val Asp Asp Ile Thr Glu370 375 380gtc ctt tct acc atc caa caa cca tcc ctg atc atc ggt atc caa tct1200Val Leu Ser Thr Ile Gln Gln Pro Ser Leu Ile Ile Gly Ile Gln Ser385 390 395 400gat gga ctg ttc aca tat tca gaa caa gaa ttt ttg gct gag cac ata1248Asp Gly Leu Phe Thr Tyr Ser Glu Gin Glu Phe Leu Ala Glu His Ile405 410 415ccg aag tcg caa tta gaa aaa att gaa tct ccc gaa ggc cac gat gcc1296Pro Lys Ser Gln Leu Glu Lys Ile Glu Ser Pro Glu Gly His Asp Ala420 425 430ttc cta ttg gag ttt aag ctg ata aac aaa ctg ata gta caa ttt tta1344Phe Leu Leu Glu Phe Lys Leu Ile Asn Lys Leu Ile Val Gln Phe Leu435 440 445aaa acc aac tgc aag gcc att acc gat gcc gct cca aga gct tgg gga1392Lys Thr Asn Cys Lys Ala Ile Thr Asp Ala Ala Pro Arg Ala Trp Gly450 455 460ggt gac gtt ggt aac gat gaa acg aag acg tct gtc ttt ggt gag gcc1440Gly Asp Val Gly Asn Asp Glu Thr Lys Thr Ser Val Phe Gly Glu Ala465 470 475 480gaa gaa gtt acc aac tgg tag1461Glu Glu Val Thr Asn Trp485
<210>32<211>486<212>PRT<213>釀酒酵母<400>32Met Ser His Thr Leu Lys Ser Lys Thr Leu Gln Glu Leu Asp Ile Glu1 5 10 15Glu Ile Lys Glu Thr Asn Pro Leu Leu Lys Leu Val Gln Gly Gln Arg20 25 30Ile Val Gln Val Pro Glu Leu Val Leu Glu Ser Gly Val Val Ile Asn35 40 45Asn Phe Pro Ile Ala Tyr Lys Thr Trp Gly Thr Leu Asn Glu Ala Gly50 55 60Asp Asn Val Leu Val Ile Cys His Ala Leu Thr Gly Ser Ala Asp Val65 70 75 80Ala Asp Trp Trp Gly Pro Leu Leu Gly Asn Asp Leu Ala Phe Asp Pro85 90 95Ser Arg Phe Phe Ile Ile Cys Leu Asn Ser Met Gly Ser Pro Tyr Gly100 105 110Ser Phe Ser Pro Leu Thr Ile Asn Glu Glu Thr Gly Val Arg Tyr Gly115 120 125Pro Glu Phe Pro Leu Cys Thr Val Arg Asp Asp Val Arg Ala His Arg130 135 140Ile Val Leu Asp Ser Leu Gly Val Lys Ser Ile Ala Cys Val Ile Gly145 150 155 160Gly Ser Met Gly Gly Met Leu Ser Leu Glu Trp Ala Ala Met Tyr Gly165 170 175Lys Glu Tyr Val Lys Asn Met Val Ala Leu Ala Thr Ser Ala Arg His180 185 190Ser Ala Trp Cys Ile Ser Trp Ser Glu Ala Gln Arg Gln Ser Ile Tyr195 200 205Ser Asp Pro Asn Tyr Leu Asp Gly Tyr Tyr Pro Val Glu Glu Gln Pro210 215 220Val Ala Gly Leu Ser Ala Ala Arg Met Ser Ala Leu Leu Thr Tyr Arg225 230 235 240Thr Arg Asn Ser Phe Glu Asn Lys Phe Ser Arg Arg Ser Pro Ser Ile245 250 255Ala Gln Gln Gln Lys Ala Gln Arg Glu Glu Thr Arg Lys Pro Ser Thr260 265 270Val Ser Glu His Ser Leu Gln Ile His Asn Asp Gly Tyr Lys Thr Lys275 280 285Ala Ser Thr Ala Ile Ala Gly Ile Ser Gly Gln Lys Gly Gln Ser Val290 295 300Val Ser Thr Ala Ser Ser Ser Asp Ser Leu Asn Ser Ser Thr Ser Met305 310 315 320
Thr Ser Val Ser Ser Val Thr Gly Glu Val Lys Asp Ile Lys Pro Ala325 330 335Gln Thr Tyr Phe Ser Ala Gln Ser Tyr Leu Arg Tyr Gln Gly Thr Lys340 345 350Phe Ile Asn Arg Phe Asp Ala Asn Cys Tyr Ile Ala Ile ThrArg Lys355 360 365Leu Asp Thr His Asp Leu Ala Arg Asp Arg Val Asp Asp Ile Thr Glu370 375 380Val Leu Ser Thr Ile Gln Gln Pro Ser Leu Ile Ile Gly Ile Gln Ser385 390 395 400Asp Gly Leu Phe Thr Tyr Ser Glu Gln Glu Phe Leu Ala Glu His Ile405 410 415Pro Lys Ser Gln Leu Glu Lys Ile Glu Ser Pro Glu Gly His Asp Ala420 425 430Phe Leu Leu Glu Phe Lys Leu Ile Asn Lys Leu Ile Val Gln Phe Leu435 440 445Lys Thr Asn Cys Lys Ala Ile Thr Asp Ala Ala Pro Arg Ala Trp Gly450 455 460Gly Asp Val Gly Asn Asp Glu Thr Lys Thr Ser Val Phe Gly Glu Ala465 470 475 480Glu Glu Val Thr Asn Trp485<210>33<211>1470<212>DNA<213>粟酒裂殖酵母(schizosaccharomyces pombe)<220>
<221>CDS<222>(1)..(1467)<223>RSO01936<400>33atg gaa tct caa tct ccg att gaa tca att gtc ttt act gac tcc tgt48Met Glu Ser Gln Ser Pro Ile Glu Ser Ile Val Phe Thr Asp Ser Cys1 5 10 15cat ccg tct cag caa gaa aat aaa ttt gtt cag ctt att tca gat caa96His Pro Ser Gln Gln Glu Asn Lys Phe Val Gln Leu Ile Ser Asp Gln20 25 30aaa att gca att gtt ccc aaa ttt acg ttg gag tgt ggc gac atc ctt144Lys Ile Ala Ile Val Pro Lys Phe Thr Leu Glu Cys Gly Asp Ile Leu35 40 45tac gat gtt ccc gtt gcc ttc aag act tgg ggt act ttg aat aaa gaa192Tyr Asp Val Pro Val Ala Phe Lys Thr Trp Gly Thr Leu Asn Lys Glu50 55 60gga aac aat tgt ctt ctt ctt tgt cat gct tta agt ggt tct gct gat240Gly Asn Asn Cys Leu Leu Leu Cys His Ala Leu Ser Gly Ser Ala Asp65 70 75 80gct gga gat tgg tgg ggt cct tta ctc ggt cct ggt cgt gcg ttt gat288
Ala Gly Asp Trp Trp Gly Pro Leu Leu Gly Pro Gly Arg Ala Phe Asp85 90 95cca tca cat ttc ttt atc gta tgc ctt aat tct ctt ggt agc cca tac336Pro Ser His Phe Phe Ile Val Cys Leu Asn Ser Leu Gly Ser Pro Tyr100 105 110gga agc gcc tct cct gtt aca tgg aac gct gag act cat agt gtt tat384Gly Ser Ala Ser Pro Val Thr Trp Asn Ala Glu Thr His Ser Val Tyr115 120 125ggg cca gaa ttt cct tta gca acc ata cgt gat gat gta aac atc cat432Gly Pro Glu Phe Pro Leu Ala Thr Ile Arg Asp Asp Val Asn Ile His130 135 140aaa ctt att tta caa aga ttg ggt gta aag caa att gct atg gca gta480Lys Leu Ile Leu Gln Arg Leu Gly Val Lys Gln Ile Ala Met Ala Val145 150 155 160ggt ggc tcc atg ggt ggt atg ctg gtt ttg gag tgg gca ttt gat aag528Gly Gly Ser Met Gly Gly Met Leu Val Leu Glu Trp Ala Phe Asp Lys165 170 175gaa ttt gtg cga tca att gtt ccc att tct acc tct ctt cgt cat tcc576Glu Phe Val Arg Ser Ile Val Pro Ile Ser Thr Ser Leu Arg His Ser180 185 190gcg tgg tgc att agc tgg tct gaa gcg caa cgc cag agt ata tat tct624Ala Trp Cys Ile Ser Trp Ser Glu Ala Gln Arg Gln Ser Ile Tyr Ser195 200 205gac cct aag ttt aat gat gga tac tac ggc ata gac gat cag cct gta672Asp Pro Lys Phe Asn Asp Gly Tyr Tyr Gly Ile Asp Asp Gln Pro Val210 215 220agt ggc ctt gga gct gct cgt atg tct gcc ttg ttg aca tat cgc tcc720Ser Gly Leu Gly Ala Ala Arg Met Ser Ala Leu Leu Thr Tyr Arg Ser225 230 235 240aaa tgt tct ttc gaa cgt cgc ttt gcc cgt act gtt cct gat gcg tct768Lys Cys Ser Phe Glu Arg Arg Phe Ala Arg Thr Val Pro Asp Ala Ser245 250 255cgt cac ccc tat cca gat cgt tta ccc act cct ctc acg ccc agt aat816Arg His Pro Tyr Pro Asp Arg Leu Pro Thr Pro Leu Thr Pro Ser Asn260 265 270gca cat tgg gtc gtt cac aac gaa gga aac cgt aat cgc cgt gaa cga864Ala His Trp Val Val His Asn Glu Gly Asn Arg Asn Arg Arg Glu Arg275 280 285cct tgt cga tcc aat gga tca tca cct act tct gaa agt gct tta aat912Pro Cys Arg Ser Asn Gly Ser Ser Pro Thr Ser Glu Ser Ala Leu Asn290 295 300tcc ccc gcc tct tct gtc tcg tct tta cct tct tta ggt gcc tct cag960Ser Pro Ala Ser Ser Val Ser Ser Leu Pro Ser Leu Gly Ala Ser Gln305 310 315 320act aca gac agt tct tcc ctt aac cag agt tcg tta tta aga cgt cct1008Thr Thr Asp Ser Ser Ser Leu Asn Gln Ser Ser Leu Leu Arg Arg Pro325 330 335gct aat act tac ttc tct gcg caa tcg tat tta cgt tac caa gcg aag1056Ala Asn Thr Tyr Phe Ser Ala Gln Ser Tyr Leu Arg Tyr Gln Ala Lys340 345 350
aag ttt gta agt cgc ttt gat gct aat tgt tac att tcg att act aaa1104Lys Phe Val Ser Arg Phe Asp Ala Asn Cys Tyr Ile Ser Ile Thr Lys355 360 365aag ttg gac acc cat gat att act cgt gga cgc ggt tca gac tct cct1152Lys Leu Asp Thr His Asp Ile Thr Arg Gly Arg Gly Ser Asp Ser Pro370 375 380aag gaa gtc atg aag gat ttg tct tta ccc gta ctc gta ctc ggt att1200Lys Glu Val Met Lys Asp Leu Set Leu Pro Val Leu Val Leu Gly Ile385 390 395 400gaa agc gat ggt ctt ttc aca ttt gac gaa caa gtt gaa att gcc aaa1248Glu Ser Asp Gly Leu Phe Thr Phe Asp Glu Gln Val Giu Ile Ala Lys405 410 415tct ttt ccc aat gct acc ttg gaa aaa att att tcg gcc gaa ggc cac1296Ser Phe Pro Asn Ala Thr Leu Glu Lys Ile Ile Ser Ala Glu Gly His420 425 430gac ggt ttt ttg ctt gag ttt act caa gta aac tca cat att caa aaa1344Asp Gly Phe Leu Leu Glu Phe Thr Gln Val Asn Ser His Ile Gln Lys435 440 445ttc caa aag gaa cat tta att gat atc atg tct caa act aat tcc ttt1392Phe Gln Lys Glu His Leu Ile Asp Ile Met Ser Gln Thr Asn Ser Phe450 455 460gag cga ctt gat tcc caa gtt aat gat acc aac cgc gaa agc gtt ttt1440Glu Arg Leu Asp Ser Gln Val Asn Asp Thr Asn Arg Glu Ser Val Phe465 470 475 480gga gaa atg gaa gac ata acc tcc tgg taa1470Gly Glu Met Glu Asp Ile Thr Ser Trp485<210>34<211>489<212>PRT<213>粟酒裂殖酵母<400>34Met Glu Ser Gln Ser Pro Ile Glu Ser Ile Val Phe Thr Asp Ser Cys1 5 10 15His Pro Ser Gln Gln Glu Asn Lys Phe Val Gin Leu Ile Ser Asp Gln20 25 30Lys Ile Ala Ile Val Pro Lys Phe Thr Leu Glu Cys Gly Asp Ile Leu35 40 45Tyr Asp Val Pro Val Ala Phe Lys Thr Trp Gly Thr Leu Asn Lys Glu50 55 60Gly Asn Asn Cys Leu Leu Leu Cys His Ala Leu Ser Gly Ser Ala Asp65 70 75 80Ala Gly Asp Trp Trp Gly Pro Leu Leu Gly Pro Gly Arg Ala Phe Asp85 90 95Pro Ser His Phe Phe Ile Val Cys Leu Asn Ser Leu Gly Ser Pro Tyr100 105 110Gly Ser Ala Ser Pro Val Thr Trp Asn Ala Glu Thr His Ser Val Tyr115 120 125
Gly Pro Glu Phe Pro Leu Ala Thr Ile Arg Asp Asp Val Asn Ile His130 135 140Lys Leu Ile Leu Gln Arg Leu Gly Val Lys Gln Ile Ala Met Ala Val145 150 155 160Gly Gly Ser Met Gly Gly Met Leu Val Leu Glu Trp Ala Phe Asp Lys165 170 175Glu Phe Val Arg Ser Ile Val Pro Ile Ser Thr Ser Leu Arg His Ser180 185 190Ala Trp Cys Ile Ser Trp Ser Glu Ala Gln Arg Gln Ser Ile Tyr Ser195 200 205Asp Pro Lys Phe Asn Asp Gly Tyr Tyr Gly Ile Asp Asp Gln Pro Val210 215 220Ser Gly Leu Gly Ala Ala Arg Met Ser Ala Leu Leu Thr Tyr Arg Ser225 230 235 240Lys Cys Ser Phe Glu Arg Arg Phe Ala Arg Thr Val Pro Asp Ala Ser245 250 255Arg His Pro Tyr Pro Asp Arg Leu Pro Thr Pro Leu Thr Pro Ser Asn260 265 270Ala His Trp Val Val His Asn Glu Gly Asn Arg Asn Arg Arg Glu Arg275 280 285Pro Cys Arg Ser Asn Gly Ser Ser Pro Thr Ser Glu Ser Ala Leu Asn290 295 300Ser Pro Ala Ser Ser Val Ser Ser Leu Pro Ser Leu Gly Ala Ser Gln305 310 315 320Thr Thr Asp Ser Ser Ser Leu Asn Gln Ser Ser Leu Leu Arg Arg Pro325 330 335Ala Asn Thr Tyr Phe Ser Ala Gln Ser Tyr Leu Arg Tyr Gln Ala Lys340 345 350Lys Phe Val Ser Arg Phe Asp Ala Asn Cys Tyr Ile Ser Ile Thr Lys355 360 365Lys Leu Asp Thr His Asp Ile Thr Arg Gly Arg Gly Ser Asp Ser Pro370 375 380Lys Glu Val Met Lys Asp Leu Ser Leu Pro Val Leu Val Leu Gly Ile385 390 395 400Glu Ser Asp Gly Leu Phe Thr Phe Asp Glu Gln Val Glu Ile Ala Lys405 410 415Ser Phe Pro Asn Ala Thr Leu Glu Lys Ile Ile Ser Ala Glu Gly His420 425 430Asp Gly Phe Leu Leu Glu Phe Thr Gln Val Asn Ser His Ile Gln Lys435 440 445Phe Gln Lys Glu His Leu Ile Asp Ile Met Ser Gln Thr Asn Ser Phe450 455 460Glu Arg Leu Asp Ser Gln Val Asn Asp Thr Asn Arg Glu Ser Val Phe465 470 475 480Gly Glu Met Glu Asp Ile Thr Ser Trp
485<210>35<211>1113<212>DNA<213>杏仁木桿菌(Xylella almond)<220>
<221>CDS<222>(1)..(1110)<223>RXFX01562<400>35atg acc gaa ttt atc cct ccg ggc agc cta ttc cat gcg ctc tcc tct48Met Thr Glu Phe Ile Pro Pro Gly Ser Leu Phe His Ala Leu Ser Ser1 5 10 15cca ttt gcg atg aag cgt ggc gga caa ctc cac cac gcc cgc atc gct96Pro Phe Ala Met Lys Arg Gly Gly Gln Leu His His Ala Arg Ile Ala20 25 30tac gaa aca tgg ggc cgc ctc aat gcc agc gcc acc aat gcc att ctg144Tyr Glu Thr Trp Gly Arg Leu Asn Ala Ser Ala Thr Asn Ala Ile Leu35 40 45atc atg cct ggc tta tca ccc aat gca cat gcc gca cac cat gac agc192Ile Met Pro Gly Leu Ser Pro Asn Ala His Ala Ala His His Asp Ser50 55 60aat gct gag cca ggc tgg tgg gag tca atg cta ggt cca ggc aaa ccc240Asn Ala Glu Pro Gly Trp Trp Glu Ser Met Leu Gly Pro Gly Lys Pro65 70 75 80atc gac aca gac cgt tgg ttc gtg atc tgt gtc aac tca ctt ggt agc288Ile Asp Thr Asp Arg Trp Phe Val Ile Cys Val Asn Ser Leu Gly Ser85 90 95tgc aaa gga tcg act ggc cct gca tcg tac aac ccc atc acg cag gcc336Cys Lys Gly Ser Thr Gly Pro Ala Ser Tyr Asn Pro Ile Thr Gln Ala100 105 110atg tat cgt ttg gac ttt cca gca ctg tca atc gaa gac ggg gcc aac384Met Tyr Arg Leu Asp Phe Pro Ala Leu Ser Ile Glu Asp Gly Ala Asn115 120 125tcc gca att gaa gtg gta cat gca ctg ggc atc aag caa ctt gcc agc432Ser Ala Ile Glu Val Val His Ala Leu Gly Ile Lys Gln Leu Ala Ser130 135 140ctg atc ggc aat tca atg ggc ggc atg acg gca ctg gcc atc ctg ctg480Leu Ile Gly Asn Ser Met Gly Gly Met Thr Ala Leu Ala Ile Leu Leu145 150 155 160tta cat cca gat ata gcc cgc agc cac atc aac atc tca ggc agc gcg528Leu His Pro Asp Ile Ala Arg Ser His Ile Asn Ile Ser Gly Ser Ala165 170 175cag gca tta ccg ttt tcc atc gcc att cgc tcg cta caa cgc gag gcg576Gln Ala Leu Pro Phe Ser Ile Ala Ile Arg Ser Leu Gln Arg Glu Ala180 185 190atc cgc ctg gac ccc cat tgg agg cag gga gac tac gac gac acc cac624Ile Arg Leu Asp Pro His Trp Arg Gln Gly Asp Tyr Asp Asp Thr His195 200 205tac ccg gaa tcg ggg cta cgc atc gca cgc aaa ctt ggg gtg atc acc672
Tyr Pro Glu Ser Gly Leu Arg Ile Ala Arg Lys Leu Gly Val Ile Thr210 215 220tac cgc tcc gcg ctg gaa tgg gac ggg cgt ttt ggc cgg gta cgc ttg720Tyr Arg Ser Ala Leu Glu Trp Asp Gly Arg Phe Gly Arg Val Arg Leu225 230 235 240gat tcg gac caa acc aac gac aca cca ttc gga ctg gaa ttc caa att768Asp Ser Asp Gln Thr Asn Asp Thr Pro Phe Gly Leu Glu Phe Gln Ile245 250 255gaa aac tac ttg gaa agc cat gca cac cgc ttc gtg cac acc ttc gac816Glu Asn Tyr Leu Glu Ser His Ala His Arg Phe Val His Thr Phe Asp260 265 270cca aac tgc tac ctg tac ctg agc cgc tcc atg gac tgg ttc gac gtg864Pro Asn Cys Tyr Leu Tyr Leu Ser Arg Ser Met Asp Trp Phe Asp Val275 280 285gcc gag tac gcc aat gga gac att ctt gcc ggg ctg gcc agg atc cga912Ala Glu Tyr Ala Asn Gly Asp Ile Leu Ala Gly Leu Ala Arg Ile Arg290 295 300atc caa cgc gca ctc gcc atc ggt agc cat acc gac atc ctc ttt cca960Ile Gln Arg Ala Leu Ala Ile Gly Ser His Thr Asp Ile Leu Phe Pro305 310 315 320ata caa cag caa caa caa att gcc gaa ggg cta cgc cgt ggc ggt aca1008Ile Gln Gln Gln Gln Gln Ile Ala Glu Gly Leu Arg Arg Gly Gly Thr325 330 335cac gcc acc ttc ctg ggc ctt gac tca ccg cag ggg cat gat gcg ttc1056His Ala Thr Phe Leu Gly Leu Asp Ser Pro Gln Gly His Asp Ala Phe340 345 350ctt gtg gat atc gca aga ttt ggc cct cca gtg aag gaa ttt ctg gac1104Leu Val Asp Ile Ala Arg Phe Gly Pro Pro Val Lys Glu Phe Leu Asp355 360 365gaa ctg tga1113Glu Leu370<210>36<211>370<212>PRT<213>杏仁木桿菌<400>36Met Thr Glu Phe Ile Pro Pro Gly Ser Leu Phe His Ala Leu Ser Ser1 5 10 15Pro Phe Ala Met Lys Arg Gly Gly Gln Leu His His Ala Arg Ile Ala20 25 30Tyr Glu Thr Trp Gly Arg Leu Asn Ala Ser Ala Thr Asn Ala Ile Leu35 40 45Ile Met Pro Gly Leu Ser Pro Asn Ala His Ala Ala His His Asp Ser50 55 60Asn Ala Glu Pro Gly Trp Trp Glu Ser Met Leu Gly Pro Gly Lys Pro65 70 75 80Ile Asp Thr Asp Arg Trp Phe Val Ile Cys Val Asn Ser Leu Gly Ser85 90 95
Cys Lys Gly Ser Thr Gly Pro Ala Ser Tyr Asn Pro Ile Thr Gln Ala100 105 110Met Tyr Arg Leu Asp Phe Pro Ala Leu Ser Ile Glu Asp Gly Ala Asn115 120 125Ser Ala Ile Glu Val Val His Ala Leu Gly Ile Lys Gln Leu Ala Ser130 135 140Leu Ile Gly Asn Ser Met Gly Gly Met Thr Ala Leu Ala Ile Leu Leu145 150 155 160Leu His Pro Asp Ile Ala Arg Ser His Ile Asn Ile Ser Gly Ser Ala165 170 175Gln Ala Leu Pro Phe Ser Ile Ala Ile Arg Ser Leu Gln Arg Glu Ala180 185 190Ile Arg Leu Asp Pro His Trp Arg Gln Gly Asp Tyr Asp Asp Thr His195 200 205Tyr Pro Glu Ser Gly Leu Arg Ile Ala Arg Lys Leu Gly Val Ile Thr210 215 220Tyr Arg Ser Ala Leu Glu Trp Asp Gly Arg Phe Gly Arg Val Arg Leu225 230 235 240Asp Ser Asp Gln Thr Asn Asp Thr Pro Phe Gly Leu Glu Phe Gln Ile245 250 255Glu Asn Tyr Leu Glu Ser His Ala His Arg Phe Val His Thr Phe Asp260 265 270Pro Asn Cys Tyr Leu Tyr Leu Ser Arg Ser Met Asp Trp Phe Asp Val275 280 285Ala Glu Tyr Ala Asn Gly Asp Ile Leu Ala Gly Leu Ala Arg Ile Arg290 295 300Ile Gln Arg Ala Leu Ala Ile Gly Ser His Thr Asp Ile Leu Phe Pro305 310 315 320Ile Gln Gln Gln Gln Gln Ile Ala Glu Gly Leu Arg Arg Gly Gly Thr325 330 335His Ala Thr Phe Leu Gly Leu Asp Ser Pro Gln Gly His Asp Ala Phe340 345 350Leu Val Asp Ile Ala Arg Phe Gly Pro Pro Val Lys Glu Phe Leu Asp355 360 365Glu Leu370<210>37<211>1113<212>DNA<213>夾竹桃木桿菌(Xylella oleander)<220>
<221>CDS<222>(1)..(1110)<223>RXFY01729<400>37
atg acc gaa ttt atc cct ccg ggc agc cta ttc cat gcg ctc tcc tct48Met Thr Glu Phe Ile Pro Pro Gly Ser Leu Phe His Ala Leu Ser Ser1 5 10 15cca ttt gcg atg aag cgt ggc gga caa ctc cac cac gcc cgc atc gct96Pro Phe Ala Met Lys Arg Gly Gly Gln Leu His His Ala Arg Ile Ala20 25 30tac gaa aca tgg ggc cgc ctc aat gcc agc gcc acc aat gcc att ctg144Tyr Glu Thr Trp Gly Arg Leu Asn Ala Ser Ala Thr Asn Ala Ile Leu35 40 45atc atg cct ggc tta tca ccc aat gca cat gcc gca cac cat gac agc192Ile Met Pro Gly Leu Ser Pro Asn Ala His Ala Ala His His Asp Ser50 55 60aat gct gag cca ggc tgg tgg gag tca atg cta ggt cca ggc aaa ccc240Asn Ala Glu Pro Gly Trp Trp Glu Ser Met Leu Gly Pro Gly Lys Pro65 70 75 80atc gac aca gac cgt tgg ttc gtg atc tgt gtc aac tca ctt ggt agc288Ile Asp Thr Asp Arg Trp Phe Val Ile Cys Val Asn Ser Leu Gly Ser85 90 95tgc aaa gga tcg act ggc cct gca tcg tac aac ccc atc acg cag gcc336Cys Lys Gly Ser Thr Gly Pro Ala Ser Tyr Asn Pro Ile Thr Gln Ala100 105 110atg tat cgt ttg gac ttt cca gca ctg tca atc gaa gac ggg gcc aac384Met Tyr Arg Leu Asp Phe Pro Ala Leu Ser Ile Glu Asp Gly Ala Asn115 120 125gcc gca att gaa gtg gta cat gca ctg ggc atc aag caa ctt gcc agc432Ala Ala Ile Glu Val Val His Ala Leu Gly Ile Lys Gln Leu Ala Ser130 135 140ctg atc ggc aat tca atg ggg ggc atg acg aca ctg gcc atc ctg ctg480Leu Ile Gly Asn Ser Met Gly Gly Met Thr Thr Leu Ala Ile Leu Leu145 150 155 160tta cat cca gat att gcc cgc agc cac atc aac atc tca ggc agc gcg528Leu His Pro Asp Ile Ala Arg Ser His Ile Asn Ile Ser Gly Ser Ala165 170 175cag gca tta ccg ttt tcc atc gcc att cgc tcg cta caa cgc gag gcg576Gln Ala Leu Pro Phe Ser Ile Ala Ile Arg Ser Leu Gln Arg Glu Ala180 185 190atc cgc ctg gac ccc cat tgg aag cag gga gac tac gac gac acc cac624Ile Arg Leu Asp Pro His Trp Lys Gln Gly Asp Tyr Asp Asp Thr His195 200 205tac ccg gaa tcg ggg cta cgc atc gca cgc aaa ctc ggg gtg atc acc672Tyr Pro Glu Ser Gly Leu Arg Ile Ala Arg Lys Leu Gly Val Ile Thr210 215 220tac cgc tcc gcg ctg gaa tgg gac ggg cgt ttt ggc cgg gta cgc ttg720Tyr Arg Ser Ala Leu Glu Trp Asp Gly Arg Phe Gly Arg Val Arg Leu225 230 235 240gat tcg gac caa acc aac gac aca cca ttc gga ctg gaa ttc caa att768Asp Ser Asp Gln Thr Asn Asp Thr Pro Phe Gly Leu Glu Phe Gln Ile245 250 255gaa aac tac ttg gaa agc cat gca cac cgc ttc gtg cac acc ttc gac816Glu Asn Tyr Leu Glu Ser His Ala His Arg Phe Val His Thr Phe Asp260 265 270
cca aac tgc tac ctg tac ctg agc cgc tcc atg gac tgg ttc gac gtg864Pro Asn Cys Tyr Leu Tyr Leu Ser Arg Ser Met Asp Trp Phe Asp Val275 280 285gcc gag tac gcc aat gga gac att ctt gcc ggg ctg gcc agg atc cga912Ala Glu Tyr Ala Asn Gly Asp Ile Leu Ala Gly Leu Ala Arg Ile Arg290 295 300atc caa cgc gca ctt gcc atc ggt agc cat acc gac atc ctc ttt cca960Ile Gln Arg Ala Leu Ala Ile Gly Ser His Thr Asp Ile Leu Phe Pro305 310 315 320ata caa cag caa caa caa att gcc gaa ggg cta cgc cgt ggc ggt aca1008Ile Gln Gln Gln Gln Gln Ile Ala Glu Gly Leu Arg Arg Gly Gly Thr325 330 335cac gcc acc ttc ctg ggc ctt gac tca ccg cag gga cat gat gcg ttc1056His Ala Thr Phe Leu Gly Leu Asp Ser Pro Gln Gly His Asp Ala Phe340 345 350ctt gtg gat atc gca gga ttt ggc cct cca gtg aag gaa ttt ctg ggc1104Leu Val Asp Ile Ala Gly Phe Gly Pro Pro Val Lys Glu Phe Leu Gly355 360 365gaa ctg tga1113Glu Leu370<210>38<211>370<212>PRT<213>夾竹桃木桿菌<400>38Met Thr Glu Phe Ile Pro Pro Gly Ser Leu Phe His Ala Leu Ser Ser1 5 10 15Pro Phe Ala Met Lys Arg Gly Gly Gln Leu His His Ala Arg Ile Ala20 25 30Tyr Glu Thr Trp Gly Arg Leu Asn Ala Ser Ala Thr Asn Ala Ile Leu35 40 45Ile Met Pro Gly Leu Ser Pro Asn Ala His Ala Ala His His Asp Ser50 55 60Asn Ala Glu Pro Gly Trp Trp Glu Ser Met Leu Gly Pro Gly Lys Pro65 70 75 80Ile Asp Thr Asp Arg Trp Phe Val Ile Cys Val Asn Ser Leu Gly Ser85 90 95Cys Lys Gly Ser Thr Gly Pro Ala Ser Tyr Asn Pro Ile Thr Gln Ala100 105 110Met Tyr Arg Leu Asp Phe Pro Ala Leu Ser Ile Glu Asp Gly Ala Asn115 120 125Ala Ala Ile Glu Val Val His Ala Leu Gly Ile Lys Gln Leu Ala Ser130 135 140Leu Ile Gly Asn Ser Met Gly Gly Met Thr Thr Leu Ala Ile Leu Leu145 150 155 160Leu His Pro Asp Ile Ala Arg Ser His Ile Asn Ile Ser Gly Ser Ala
165 170 175Gln Ala Leu Pro Phe Ser Ile Ala Ile Arg Ser Leu Gln Arg Glu Ala180 185 190Ile Arg Leu Asp Pro His Trp Lys Gln Gly Asp Tyr Asp Asp Thr His195 200 205Tyr Pro Glu Ser Gly Leu Arg Ile Ala Arg Lys Leu Gly Val Ile Thr210 215 220Tyr Arg Ser Ala Leu Glu Trp Asp Gly Arg Phe Gly Arg Val Arg Leu225 230 235 240Asp Ser Asp Gln Thr Asn Asp Thr Pro Phe Gly Leu Glu Phe Gln Ile245 250 255Glu Asn Tyr Leu Glu Ser His Ala His Arg Phe Val His Thr Phe Asp260 265 270Pro Asn Cys Tyr Leu Tyr Leu Ser Arg Ser Met Asp Trp Phe Asp Val275 280 285Ala Glu Tyr Ala Asn Gly Asp Ile Leu Ala Gly Leu Ala Arg Ile Arg290 295 300Ile Gln Arg Ala Leu Ala Ile Gly Ser His Thr Asp Ile Leu Phe Pro305 310 315 320Ile Gln Gln Gln Gln Gln Ile Ala Glu Gly Leu Arg Arg Gly Gly Thr325 330 335His Ala Thr Phe Leu Gly Leu Asp Ser Pro Gln Gly His Asp Ala Phe340 345 350Leu Val Asp Ile Ala Gly Phe Gly Pro Pro Val Lys Glu Phe Leu Gly355 360 365Glu Leu370<210>39<211>1578<212>DNA<213>構(gòu)巢裸孢殼(Emericella nidulans)<220>
<221>CDS<222>(1)..(1575)<223>REN00010<400>39atg agt ccg ctg aac ggc gtc gct cgt tcc ttt ccg cgg ccc ttc cag48Met Ser Pro Leu Asn Gly Val Ala Arg Ser Phe Pro Arg Pro Phe Gln1 5 10 15gcc gtg acc agg cgg cct ttt cga gtt gtc cag ccg gcc atc gcc tgt96Ala Val Thr Arg Arg Pro Phe Arg Val Val Gln Pro Ala Ile Ala Cys20 25 30ccg tcc aac agc cgg tcg ttt aac cat tct cga tca tta cga tca acg144Pro Ser Asn Ser Arg Ser Phe Asn His Ser Arg Ser Leu Arg Ser Thr35 40 45ggg tct cag tcc ccc gct cca tcc cca cgc gac tcc tcg aat ccc gcg192Gly Ser Gln Ser Pro Ala Pro Ser Pro Arg Asp Ser Ser Asn Pro Ala
50 55 60ctg tcc ttc cct tgc ctc gac gcc cag gag gcc aag tcc gct ctt ctt240Leu Ser Phe Pro Cys Leu Asp Ala Gln Glu Ala Lys Ser Ala Leu Leu65 70 75 80tcc gcg cga tct ctt ggt tca ggc cct gaa ccc tcc tat acc gcc ggc288Ser Ala Arg Ser Leu Gly Ser Gly Pro Glu Pro Ser Tyr Thr Ala Gly85 90 95cac cac gaa cga ttc cat tcc gac gaa ccg ctg ctc ctt gat tgg ggc336His His Glu Arg Phe His Ser Asp Glu Pro Leu Leu Leu Asp Trp Gly100 105 110ggt ttg ctt cca gaa ttt gat atc gca tat gag aca tgg ggc cag ctg384Gly Leu Leu Pro Glu Phe Asp Ile Ala Tyr Glu Thr Trp Gly Gln Leu115 120 125aac gag aag aag gat aat gtc att ctg ctg cat acc ggt ctg tct gca432Asn Glu Lys Lys Asp Asn Val Ile Leu Leu His Thr Gly Leu Ser Ala130 135 140tct agc cat gcg cac agc acc gaa gcg aac ccg aag ccc ggc tgg tgg480Ser Ser His Ala His Ser Thr Glu Ala Asn Pro Lys Pro Gly Trp Trp145 150 155 160gag aaa ttc ata ggt cct ggg aag acg cta gat acg gac aag tac ttt528Glu Lys Phe Ile Gly Pro Gly Lys Thr Leu Asp Thr Asp Lys Tyr Phe165 170 175gtg atc tgc acc aat gtc ctt gga ggg tgc tac ggt agc acg ggg ccc576Val Ile Cys Thr Asn Val Leu Gly Gly Cys Tyr Gly Ser Thr Gly Pro180 185 190tcg acg gtg gac ccg tcg gat ggg aag aag tat gct acg cgg ttt ccc624Ser Thr Val Asp Pro Ser Asp Gly Lys Lys Tyr Ala Thr Arg Phe Pro195 200 205atc ctg aca att gaa gat atg gtg cga gcg cag ttc cgc ctt ttg gac672Ile Leu Thr Ile Glu Asp Met Val Arg Ala Gln Phe Arg Leu Leu Asp210 215 220cat ctt ggg gtt cgg aaa ctc tac gcg tcc gtc ggc tcc agc atg ggt720His Leu Gly Val Arg Lys Leu Tyr Ala Ser Val Gly Ser Ser Met Gly225 230 235 240ggt atg cag agt ctt gca gcc ggt gtt ctg ttc cca gag cga gtg ggc768Gly Met Gln Ser Leu Ala Ala Gly Val Leu Phe Pro Glu Arg Val Gly245 250 255aag att gtg tcg att agc ggt tgt gct cga agc cat ccg tac agc att816Lys Ile Val Ser Ile Ser Gly Cys Ala Arg Ser His Pro Tyr Ser Ile260 265 270gct atg cgc cat acc cag cgg cag gtg ttg atg atg gat cca aat tgg864Ala Met Arg His Thr Gln Arg Gln Val Leu Met Met Asp Pro Asn Trp275 280 285gct cga ggt ttc tac tac gat tcg atc cca cct cat tca ggc atg aag912Ala Arg Gly Phe Tyr Tyr Asp Ser Ile Pro Pro His Ser Gly Met Lys290 295 300ctc gct cgc gag att gcc acc gtc acg tac cgc agc gga cca gaa tgg960Leu Ala Arg Glu Ile Ala Thr Val Thr Tyr Arg Ser Gly Pro Glu Trp305 310 315 320gag aaa cgc ttt ggt cgg aaa cgg gct gat ccg agc aaa cag cct gcg1008
Glu Lys Arg Phe Gly Arg Lys Arg Ala Asp Pro Ser Lys Gln Pro Ala325 330 335ctt tgc ccc gac ttt ctc atc gag acg tat ctc gac cac gcc ggt gaa1056Leu Cys Pro Asp Phe Leu Ile Glu Thr Tyr Leu Asp His Ala Gly Glu340 345 350aaa ttc tgc ttg gaa tac gat gcc aac agc ctg ctc tac atc tcc aag1104Lys Phe Cys Leu Glu Tyr Asp Ala Asn Ser Leu Leu Tyr Ile Ser Lys355 360 365gcg atg gat ctg ttt gac cta ggg ttg act cag caa ctc gcg acg aag1152Ala Met Asp Leu Phe Asp Leu Gly Leu Thr Gln Gln Leu Ala Thr Lys370 375 380aag cag agg gcg gag gcc cag gcg aag att agc agc gga aca aac act1200Lys Gln Arg Ala Glu Ala Gln Ala Lys Ile Ser Ser Gly Thr Asn Thr385 390 395 400gtc aat gat gcg tcg tgc agc ctt aca ctt cct gaa cag cca tac cag1248Val Asn Asp Ala Ser Cys Ser Leu Thr Leu Pro Glu Gln Pro Tyr Gln405 410 415gag cag cca tct gcc tcg aca tcc gcc gag cag tct gct tcc gct tca1296Glu Gln Pro Ser Ala Ser Thr Ser Ala Glu Gln Ser Ala Ser Ala Ser420 425 430gag acc ggg tcg gct ccg aac gat ctt gtt gcc ggg ctt gcg ccg ctg1344Glu Thr Gly Ser Ala Pro Asn Asp Leu Val Ala Gly Leu Ala Pro Leu435 440 445aaa gac cat cag gtg ctg gta atc gga gtc gca agc gac att ctc ttc1392Lys Asp His Gln Val Leu Val Ile Gly Val Ala Ser Asp Ile Leu Phe450 455 460ccg gcg tgg caa cag cgc gag atc gcg gag act ctg att caa gca ggg1440Pro Ala Trp Gln Gln Arg Glu Ile Ala Glu Thr Leu Ile Gln Ala Gly465 470 475 480aac aag acc gtg gag cat att gag ctg ggc aac gac gtg tct ctc ttt1488Asn Lys Thr Val Glu His Ile Glu Leu Gly Asn Asp Val Ser Leu Phe485 490 495ggt cat gac aca ttc ctc ctt gat gtc aga acg tcg gag gcg cag ttc1536Gly His Asp Thr Phe Leu Leu Asp Val Arg Thr Ser Glu Ala Gln Phe500 505 510gca agt tcc gta cta gtc ggc tcg cac ata att gta caa tag1578Ala Ser Ser Val Leu Val Gly Ser His Ile Ile Val Gln515 520 525<210>40<211>525<212>PRT<213>構(gòu)巢裸孢殼<400>40Met Ser Pro Leu Asn Gly Val Ala Arg Ser Phe Pro Arg Pro Phe Gln1 5 10 15Ala Val Thr Arg Arg Pro Phe Arg Val Val Gln Pro Ala Ile Ala Cys20 25 30Pro Ser Asn Ser Arg Ser Phe Asn His Ser Arg Ser Leu Arg Ser Thr35 40 45
Gly Ser Gln Ser Pro Ala Pro Ser Pro Arg Asp Ser Ser Asn Pro Ala50 55 60Leu Ser Phe Pro Cys Leu Asp Ala Gln Glu Ala Lys Ser Ala Leu Leu65 70 75 80Ser Ala Arg Ser Leu Gly Ser Gly Pro Glu Pro Ser Tyr Thr Ala Gly85 90 95His His Glu Arg Phe His Ser Asp Glu Pro Leu Leu Leu Asp Trp Gly100 105 110Gly Leu Leu Pro Glu Phe Asp Ile Ala Tyr Glu Thr Trp Gly Gln Leu115 120 125Asn Glu Lys Lys Asp Asn Val Ile Leu Leu His Thr Gly Leu Ser Ala130 135 140Ser Ser His Ala His Ser Thr Glu Ala Asn Pro Lys Pro Gly Trp Trp145 150 155 160Glu Lys Phe Ile Gly Pro Gly Lys Thr Leu Asp Thr Asp Lys Tyr Phe165 170 175Val Ile Cys Thr Asn Val Leu Gly Gly Cys Tyr Gly Ser Thr Gly Pro180 185 190Ser Thr Val Asp Pro Ser Asp Gly Lys Lys Tyr Ala Thr Arg Phe Pro195 200 205Ile Leu Thr Ile Glu Asp Met Val Arg Ala Gln Phe Arg Leu Leu Asp210 215 220His Leu Gly Val Arg Lys Leu Tyr Ala Ser Val Gly Ser Ser Met Gly225 230 235 240Gly Met Gln Ser Leu Ala Ala Gly Val Leu Phe Pro Glu Arg Val Gly245 250 255Lys Ile Val Ser Ile Ser Gly Cys Ala Arg Ser His Pro Tyr Ser Ile260 265 270Ala Met Arg His Thr Gln Arg Gln Val Leu Met Met Asp Pro Asn Trp275 280 285Ala Arg Gly Phe Tyr Tyr Asp Ser Ile Pro Pro His Ser Gly Met Lys290 295 300Leu Ala Arg Glu Ile Ala Thr Val Thr Tyr Arg Ser Gly Pro Glu Trp305 310 315 320Glu Lys Arg Phe Gly Arg Lys Arg Ala Asp Pro Ser Lys Gln Pro Ala325 330 335Leu Cys Pro Asp Phe Leu Ile Glu Thr Tyr Leu Asp His Ala Gly Glu340 345 350Lys Phe Cys Leu Glu Tyr Asp Ala Asn Ser Leu Leu Tyr Ile Ser Lys355 360 365Ala Met Asp Leu Phe Asp Leu Gly Leu Thr Gln Gln Leu Ala Thr Lys370 375 380Lys Gln Arg Ala Glu Ala Gln Ala Lys Ile Ser Ser Gly Thr Asn Thr385 390 395 400Val Asn Asp Ala Ser Cys Ser Leu Thr Leu Pro Glu Gln Pro Tyr Gln
405 410 415Glu Gln Pro Ser Ala Ser Thr Ser Ala Glu Gln Ser Ala Ser Ala Ser420 425 430Glu Thr Gly Ser Ala Pro Asn Asp Leu Val Ala Gly Leu Ala Pro Leu435 440 445Lys Asp His Gln Val Leu Val Ile Gly Val Ala Ser Asp Ile Leu Phe450 455 460Pro Ala Trp Gln Gln Arg Glu Ile Ala Glu Thr Leu Ile Gln Ala Gly465 470 475 480Asn Lys Thr Val Glu His Ile Glu Leu Gly Asn Asp Val Ser Leu Phe485 490 495Gly His Asp Thr Phe Leu Leu Asp Val Arg Thr Ser Glu Ala Gln Phe500 505 510Ala Ser Ser Val Leu Val Gly Ser His Ile Ile Val Gln515 520 525<210>41<211>1170<212>DNA<213>百脈根根瘤菌(Mesorhizobium loti)<220>
<221>CDS<222>(1)..(1167)<223>NP_104621<400>41atg gcc gct ctg cgc gca gga aag acc aac aac gag gcc gac cag ccg48Met Ala Ala Leu Arg Ala Gly Lys Thr Asn Asn Glu Ala Asp Gln Pro1 5 10 15tcg agc ccg gtg ttg cgc ttc ggg gcg gac aag ccg ctc aag ctc gac96Ser Ser Pro Val Leu Arg Phe Gly Ala Asp Lys Pro Leu Lys Leu Asp20 25 30gcc ggc acg ctt ttg tcg ccg ttc cag atc gcc tat cag acc tac ggc144Ala Gly Thr Leu Leu Ser Pro Phe Gln Ile Ala Tyr Gln Thr Tyr Gly35 40 45acg ctg aac gat gcc cgc tcc aat gcc atc ctc gtc tgc cat gcg ctg192Thr Leu Asn Asp Ala Arg Ser Asn Ala Ile Leu Val Cys His Ala Leu50 55 60acc ggc gac cag cat gtc gcc aac acc aat ccg gtg acc ggc aag ccg240Thr Gly Asp Gln His Val Ala Asn Thr Asn Pro Val Thr Gly Lys Pro65 70 75 80gga tgg tgg gaa gtg ctg atc ggc ccc ggc agg atc atc gac acc aac288Gly Trp Trp Glu Val Leu Ile Gly Pro Gly Arg Ile Ile Asp Thr Asn85 90 95cgt ttc ttc gtc atc tgc tcc aac gtc atc ggc ggt tgt ctg ggc tcc336Arg Phe Phe Val Ile Cys Ser Asn Val Ile Gly Gly Cys Leu Gly Ser100 105 110acc ggc ccg gcc tcg acc aac ccc gcc acc ggc aag ccc tac ggg ctc384Thr Gly Pro Ala Ser Thr Asn Pro Ala Thr Gly Lys Pro Tyr Gly Leu115 120 125
gac ctg ccg gtc atc acc atc cgc gat atg gtg cgc gcg cag cag atg432Asp Leu Pro Val Ile Thr Ile Arg Asp Met Val Arg Ala Gln Gln Met130 135 140ctg atc gat cat ttc ggc atc gag aaa ctg ttc tgc gtg ctc ggc ggc480Leu Ile Asp His Phe Gly Ile Glu Lys Leu Phe Cys Val Leu Gly Gly145 150 155 160tcg atg ggc gga atg cag gtg ctg gaa tgg gcg tcg agc tac ccc gag528Ser Met Gly Gly Met Gln Val Leu Glu Trp Ala Ser Ser Tyr Pro Glu165 170 175cgc gtc ttt tcg gca ctg ccg atc gcc acc ggc gcg cgc cat tcc tcg576Arg Val Phe Ser Ala Leu Pro Ile Ala Thr Gly Ala Arg His Ser Ser180 185 190cag aac atc gcc ttc cac gag gtc ggc cgg cag gct gtc atg gcc gat624Gln Asn Ile Ala Phe His Glu Val Gly Arg Gln Ala Val Met Ala Asp195 200 205ccg gac tgg cac ggc ggc aaa tat ttc gaa aac ggc aaa cgc ccg gaa672Pro Asp Trp His Gly Gly Lys Tyr Phe Glu Asn Gly Lys Arg Pro Glu210 215 220aag ggc ctg gcg gta gcg cgc atg gcc gcc cac ata acc tat ctg tcg720Lys Gly Leu Ala Val Ala Arg Met Ala Ala His Ile Thr Tyr Leu Ser225 230 235 240gaa gcc gcc ctg cac cgg aaa ttc ggc cgc aat ctg cag gat cgc gag768Glu Ala Ala Leu His Arg Lys Phe Gly Arg Asn Leu Gln Asp Arg Glu245 250 255gcg ctg acc ttc ggc ttc gac gcc gac ttc cag atc gaa agc tat ctg816Ala Leu Thr Phe Gly Phe Asp Ala Asp Phe Gln Ile Glu Ser Tyr Leu260 265 270cgc cac caa ggc atg acc ttc gtc gac cgc ttc gac gcc aat tcc tat864Arg His Gln Gly Met Thr Phe Val Asp Arg Phe Asp Ala Asn Ser Tyr275 280 285ctc tac atg acg cgg tcg atg gac tat ttc gac ctc gcc gcc gat cat912Leu Tyr Met Thr Arg Ser Met Asp Tyr Phe Asp Leu Ala Ala Asp His290 295 300ggc ggg cgg ctg gcg gat gcc ttt gcc ggc acc aaa acc cgc ttc tgc960Gly Gly Arg Leu Ala Asp Ala Phe Ala Gly Thr Lys Thr Arg Phe Cys305 310 315 320ctg gtg tcc ttc acc tcg gat tgg ttg ttt ccg acc gaa gag agc cgc1008Leu Val Ser Phe Thr Ser Asp Trp Leu Phe Pro Thr Glu Glu Ser Arg325 330 335tcg atc gtg cac gcg ctc aac gcc gcc ggc gcg tcc gtg tcc ttc gtc1056Ser Ile Val His Ala Leu Asn Ala Ala Gly Ala Ser Val Ser Phe Val340 345 350gaa atc gag acc gac cgc ggc cac gat gcc ttc ctg ctc gac gag ccg1104Glu Ile Glu Thr Asp Arg Gly His Asp Ala Phe Leu Leu Asp Glu Pro355 360 365gaa ctg ttc gcc gcc atc aac ggc ttc atc ggc tcc gcg gcg cgg gcg1152Glu Leu Phe Ala Ala Ile Asn Gly Phe Ile Gly Ser Ala Ala Arg Ala370 375 380aga ggg cta agc gca tga1170Arg Gly Leu Ser Ala385
<210>42<211>389<212>PRT<213>百脈根根瘤菌<400>42Met Ala Ala Leu Arg Ala Gly Lys Thr Asn Asn Glu Ala Asp Gln Pro1 5 10 15Ser Ser Pro Val Leu Arg Phe Gly Ala Asp Lys Pro Leu Lys Leu Asp20 25 30Ala Gly Thr Leu Leu Ser Pro Phe Gln Ile Ala Tyr Gln Thr Tyr Gly35 40 45Thr Leu Asn Asp Ala Arg Ser Asn Ala Ile Leu Val Cys His Ala Leu50 55 60Thr Gly Asp Gln His Val Ala Asn Thr Asn Pro Val Thr Gly Lys Pro65 70 75 80Gly Trp Trp Glu Val Leu Ile Gly Pro Gly Arg Ile Ile Asp Thr Asn85 90 95Arg Phe Phe Val Ile Cys Ser Asn Val Ile Gly Gly Cys Leu Gly Ser100 105 110Thr Gly Pro Ala Ser Thr Asn Pro Ala Thr Gly Lys Pro Tyr Gly Leu115 120 125Asp Leu Pro Val Ile Thr Ile Arg Asp Met Val Arg Ala Gln Gln Met130 135 140Leu Ile Asp His Phe Gly Ile Glu Lys Leu Phe Cys Val Leu Gly Gly145 150 155 160Ser Met Gly Gly Met Gln Val Leu Glu Trp Ala Ser Ser Tyr Pro Glu165 170 175Arg Val Phe Ser Ala Leu Pro Ile Ala Thr Gly Ala Arg His Ser Ser180 185 190Gln Asn Ile Ala Phe His Glu Val Gly Arg Gln Ala Val Met Ala Asp195 200 205Pro Asp Trp His Gly Gly Lys Tyr Phe Glu Asn Gly Lys Arg Pro Glu210 215 220Lys Gly Leu Ala Val Ala Arg Met Ala Ala His Ile Thr Tyr Leu Ser225 230 235 240Glu Ala Ala Leu His Arg Lys Phe Gly Arg Asn Leu Gln Asp Arg Glu245 250 255Ala Leu Thr Phe Gly Phe Asp Ala Asp Phe Gln Ile Glu Ser Tyr Leu260 265 270Arg His Gln Gly Met Thr Phe Val Asp Arg Phe Asp Ala Asn Ser Tyr275 280 285Leu Tyr Met Thr Arg Ser Met Asp Tyr Phe Asp Leu Ala Ala Asp His290 295 300Gly Gly Arg Leu Ala Asp Ala Phe Ala Gly Thr Lys Thr Arg Phe Cys305 310 315 320
Leu Val Ser Phe Thr Ser Asp Trp Leu Phe Pro Thr Glu Glu Ser Arg325 330 335Ser Ile Val His Ala Leu Asn Ala Ala Gly Ala Ser Val Ser Phe Val340 345 350Glu Ile Glu Thr Asp Arg Gly His Asp Ala Phe Leu Leu Asp Glu Pro355 360 365Glu Leu Phe Ala Ala Ile Asn Gly Phe Ile Gly Ser Ala Ala Arg Ala370 375 380Arg Gly Leu Ser Ala385<210>43<211>1155<212>DNA<213>產(chǎn)黃頂孢霉(acremonium crysogenum)<220>
<221>CDS<222>(1)..(1152)<223>P39058<400>43tgt cgc ctc aga tcg cca atc gct tcg agg ctt cgc tag atg ccc aag48Cys Arg Leu Arg Ser Pro Ile Ala Ser Arg Leu Arg Xaa Met Pro Lys1 5 10 15aca tag cca gaa tat cgc tct tca cac tgg aat ctg gcg tca tcc ttc96Thr Xaa Pro Glu Tyr Arg Ser Ser His Trp Asn Leu Ala Ser Ser Phe20 25 30gcg atg tac ccg tgg cat aca aat cgt ggg gtc gca tga atg tct caa144Ala Met Tyr Pro Trp His Thr Asn Arg Gly Val Ala Xaa Met Ser Gln35 40 45ggg ata act gcg tca tcg tct gcc aca cct tga cga gca gcg ccc atg192Gly Ile Thr Ala Ser Ser Ser Ala Thr Pro Xaa Arg Ala Ala Pro Met50 55 60tca cct cgt ggt ggc cca cac tgt ttg gcc aag gca ggg ctt tcg ata240Ser Pro Arg Gly Gly Pro His Cys Leu Ala Lys Ala Gly Leu Ser Ile65 70 75 80cct ctc gct act tca tca tct gcc taa att atc tcg gga gcc cct ttg288Pro Leu Ala Thr Ser Ser Ser Ala Xaa Ile Ile Ser Gly Ala Pro Leu85 90 95gga gtg ctg gac cat gtt cac cgg acc ccg atg cag aag gcc agc gcc336Gly Val Leu Asp His Val His Arg Thr Pro Met Gln Lys Ala Ser Ala100 105 110cgt acg ggg cca agt ttc ctc gca cga cga ttc gag atg atg ttc gta384Arg Thr Gly Pro Ser Phe Leu Ala Arg Arg Phe Glu Met Met Phe Val115 120 125ttc atc gcc agg tgc tcg aca ggt tag gcg tca ggc aaa ttg ctg ccg432Phe Ile Ala Arg Cys Ser Thr Gly Xaa Ala Ser Gly Lys Leu Leu Pro130 135 140tag tcg gcg cat cca tgg gtg gaa tgc aca ctc tgg aat ggg cct tct480Xaa Ser Ala His Pro Trp Val Glu Cys Thr Leu Trp Asn Gly Pro Ser145 150 155 160
ttg gtc ccg agt acg tgc gaa aga ttg tgc cca tcg cga cat cat gcc528Leu Val Pro Ser Thr Cys Glu Arg Leu Cys Pro Ser Arg His His Ala165 170 175gtc aga gcg gct ggt gcg cag ctt ggt tcg aga cac aga ggc agt gca576Val Arg Ala Ala Gly Ala Gln Leu Gly Ser Arg His Arg Gly Ser Ala180 185 190tct atg atg acc cca agt acc tgg acg ggg agt acg acg tag acg acc624Ser Met Met Thr Pro Ser Thr Trp Thr Gly Ser Thr Thr Xaa Thr Thr195 200 205agc ctg tcc ggg ggc tcg aaa cag cgc gca aga ttg cga atc tca cgt672Ser Leu Ser Gly Gly Ser Lys Gln Arg Ala Arg Leu Arg Ile Ser Arg210 215 220aca aga gca aac ctg cga tgg acg agc gct tcc ata tgg ctc cag gag720Thr Arg Ala Asn Leu Arg Trp Thr Ser Ala Ser Ile Trp Leu Gln Glu225 230 235 240tcc aag ccg gcc gga ata tca gca gcc agg atg cga aga agg aaa tca768Ser Lys Pro Ala Gly Ile Ser Ala Ala Arg Met Arg Arg Arg Lys Ser245 250 255acg gca cag aca gcg gca aca gcc acc gtg ctg gcc agc cca ttg aag816Thr Ala Gln Thr Ala Ala Thr Ala Thr Val Leu Ala Ser Pro Leu Lys260 265 270ccg tat ctt cct atc tcc ggt acc agg ccc aga agt ttg ccg cga gct864Pro Tyr Leu Pro Ile Ser Gly Thr Arg Pro Arg Ser Leu Pro Arg Ala275 280 285tcg acg cca act gct aca tcg cca tga cac tca agt tcg aca ccc acg912Ser Thr Pro Thr Ala Thr Ser Pro Xaa His Ser Ser Ser Thr Pro Thr290 295 300aca tca gca gag gcc ggg cag gat caa tcc cgg agg ctc tgg caa tga960Thr Ser Ala Glu Ala Gly Gln Asp Gln Ser Arg Arg Leu Trp Gln Xaa305 310 315 320tta cac aac cag cgt tga tca ttt gcg cca ggt cag acg gtc tgt act1008Leu His Asn Gln Arg Xaa Ser Phe Ala Pro Gly Gln Thr Val Cys Thr325 330 335cgt ttg acg agc acg ttg aga tgg ggc gca gta tcc caa aca gtc gtc1056Arg Leu Thr Ser Thr Leu Arg Trp Gly Ala Val Ser Gln Thr Val Val340 345 350ttt gcg tgg tgg aca cga atg agg gtc atg act tct ttg taa tgg aag1104Phe Ala Trp Trp Thr Arg Met Arg Val Met Thr Ser Leu Xaa Trp Lys355 360 365cgg aca agg tta atg atg ccg tca gag gat tcc tcg atc agt cat taa1152Arg Thr Arg Leu Met Met Pro Ser Glu Asp Ser Ser Ile Ser His Xaa370 375 380tgt1155<210>44<211>384<212>PRT<213>產(chǎn)黃頂孢霉<220>
<221>不確定的
<222>13..13<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>18..18<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>45..45<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>59..59<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>89..89<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>137..137<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>145..145<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>206..206<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>297..297<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>320..320<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>326..326<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>366..366<223>所出現(xiàn)的Xaa表示任一氨基酸<220>
<221>不確定的<222>384..384<223>所出現(xiàn)的Xaa表示任一氨基酸
<400>44Cys Arg Leu Arg Ser Pro Ile Ala Ser Arg Leu Arg Xaa Met Pro Lys1 5 10 15Thr Xaa Pro Glu Tyr Arg Ser Ser His Trp Asn Leu Ala Ser Ser Phe20 25 30Ala Met Tyr Pro Trp His Thr Asn Arg Gly Val Ala Xaa Met Ser Gln35 40 45Gly Ile Thr Ala Ser Ser Ser Ala Thr Pro Xaa Arg Ala Ala Pro Met50 55 60Ser Pro Arg Gly Gly Pro His Cys Leu Ala Lys Ala Gly Leu Ser Ile65 70 75 80Pro Leu Ala Thr Ser Ser Ser Ala Xaa Ile Ile Ser Gly Ala Pro Leu85 90 95Gly Val Leu Asp His Val His Arg Thr Pro Met Gln Lys Ala Ser Ala100 105 110Arg Thr Gly Pro Ser Phe Leu Ala Arg Arg Phe Glu Met Met Phe Val115 120 125Phe Ile Ala Arg Cys Ser Thr Gly Xaa Ala Ser Gly Lys Leu Leu Pro130 135 140Xaa Ser Ala His Pro Trp Val Glu Cys Thr Leu Trp Asn Gly Pro Ser145 150 155 160Leu Val Pro Ser Thr Cys Glu Arg Leu Cys Pro Ser Arg His His Ala165 170 175Val Arg Ala Ala Gly Ala Gln Leu Gly Ser Arg His Arg Gly Ser Ala180 185 190Ser Met Met Thr Pro Ser Thr Trp Thr Gly Ser Thr Thr Xaa Thr Thr195 200 205Ser Leu Ser Gly Gly Ser Lys Gln Arg Ala Arg Leu Arg Ile Ser Arg210 215 220Thr Arg Ala Asn Leu Arg Trp Thr Ser Ala Ser Ile Trp Leu Gln Glu225 230 235 240Ser Lys Pro Ala Gly Ile Ser Ala Ala Arg Met Arg Arg Arg Lys Ser245 250 255Thr Ala Gln Thr Ala Ala Thr Ala Thr Val Leu Ala Ser Pro Leu Lys260 265 270Pro Tyr Leu Pro Ile Ser Gly Thr Arg Pro Arg Ser Leu Pro Arg Ala275 280 285Ser Thr Pro Thr Ala Thr Ser Pro Xaa His Ser Ser Ser Thr Pro Thr290 295 300Thr Ser Ala Glu Ala Gly Gln Asp Gln Ser Arg Arg Leu Trp Gln Xaa305 310 315 320Leu His Asn Gln Arg Xaa Ser Phe Ala Pro Gly Gln Thr Val Cys Thr325 330 335Arg Leu Thr Ser Thr Leu Arg Trp Gly Ala Val Ser Gln Thr Val Val340 345 350
Phe Ala Trp Trp Thr Arg Met Arg Val Met Thr Ser Leu Xaa Trp Lys355 360 365Arg Thr Arg Leu Met Met Pro Ser Glu Asp Ser Ser Ile Ser His Xaa370 375 380<210>45<211>1077<212>DNA<213>惡臭假單胞菌(Pseudomonas putida)<220>
<221>CDS<222>(1)..(1074)<223>AAK49778<400>45atg tca act gtc ttt ccc gaa gat tcc gtc ggt ctg gta gta cgg caa48Met Ser Thr Val Phe Pro Glu Asp Ser Val Gly Leu Val Val Arg Gln1 5 10 15acc tcc cgg ttc gat gaa ccg ctg gca ctg gcc tgt ggc cgt tca ctg96Thr Ser Arg Phe Asp Glu Pro Leu Ala Leu Ala Cys Gly Arg Ser Leu20 25 30gcc agt tac gaa ctg gtc tac gag acc tat ggc acc ctg aac gcc agc144Ala Ser Tyr Glu Leu Val Tyr Glu Thr Tyr Gly Thr Leu Asn Ala Ser35 40 45gcg agc aac gcc gtg ctg atc tgc cat gcc ctg tcc ggc cac cac cat192Ala Ser Asn Ala Val Leu Ile Cys His Ala Leu Ser Gly His His His50 55 60gcc gct ggc tac cat gcc gcc acc gac cgc aag ccg ggc tgg tgg gac240Ala Ala Gly Tyr His Ala Ala Thr Asp Arg Lys Pro Gly Trp Trp Asp65 70 75 80agc tgc atc ggc ccc gga aaa ccg atc gat acc aac cgc ttc ttc gtg288Ser Cys Ile Gly Pro Gly Lys Pro Ile Asp Thr Asn Arg Phe Phe Val85 90 95gtc agc ctg aac aac ctc ggc ggc tgc aac ggc agc acc ggc ccc agc336Val Ser Leu Asn Asn Leu Gly Gly Cys Asn Gly Ser Thr Gly Pro Ser100 105 110agt gtc aac cca gcc acc ggt aaa ccc tat ggc gcc gag ttc ccg gta384Ser Val Asn Pro Ala Thr Gly Lys Pro Tyr Gly Ala Glu Phe Pro Val115 120 125ttg acc gtg gaa gac tgg gtg cac agc cag gca cgg ctg gcc gac cgc432Leu Thr Val Glu Asp Trp Val His Ser Gln Ala Arg Leu Ala Asp Arg130 135 140ctg ggc atc cag cag tgg gca gct atc gtc ggc ggt agc ctg ggt ggc480Leu Gly Ile Gln Gln Trp Ala Ala Ile Val Gly Gly Ser Leu Gly Gly145 150 155 160atg cag gcg ctg caa tgg acg atg acc tac ccc gag cgc gta cgc cac528Met Gln Ala Leu Gln Trp Thr Met Thr Tyr Pro Glu Arg Val Arg His165 170 175tgc gtc gac att gcc tcg gcc ccc aag ctg tcg gcg cag aac atc gcc576
Cys Val Asp Ile Ala Ser Ala Pro Lys Leu Ser Ala Gln Asn Ile Ala180 185 190ttc aac gag gtg gcg cgt cag gcc att ctt acc gac cct gag tac cgc 624Phe Asn Glu Val Ala Arg Gln Ala Ile Leu Thr Asp Pro Glu Tyr Arg195 200 205aga ggc tcg ttt cca gga cca ggt gtg atc ccc aag cgc ggc ctg atg672Arg Gly Ser Phe Pro Gly Pro Gly Val Ile Pro Lys Arg Gly Leu Met210 215 220ctg gca cgg atg gtc ggc cac att acc tat ctg tcc gat gat tcg atg720Leu Ala Arg Met Val Gly His Ile Thr Tyr Leu Ser Asp Asp Ser Met225 230 235 240ggt gaa aaa ttc ggc cga gag ctg aaa gcg aca agc tca act acg act768Gly Glu Lys Phe Gly Arg Glu Leu Lys Ala Thr Ser Ser Thr Thr Thr245 250 255tcc aca gcg tcg agt tcc agg tcg aaa gct acc tgc gct atc agg gcg816Ser Thr Ala Ser Ser Ser Arg Ser Lys Ala Thr Cys Ala Ile Arg Ala260 265 270agg agt ttt ccg gcc gtt tcg acg cca aca cct acc ttg atg acc aag864Arg Ser Phe Pro Ala Val Ser Thr Pro Thr Pro Thr Leu Met Thr Lys275 280 285gca ctg gac tat ttc gac ccg gcc gcc acg cac ggt ggt gat ctg gcc912Ala Leu Asp Tyr Phe Asp Pro Ala Ala Thr His Gly Gly Asp Leu Ala290 295 300gcc acc ctg gcc cac gtc acg gcg gac tac tgc atc tgt cgt tca cca960Ala Thr Leu Ala His Val Thr Ala Asp Tyr Cys Ile Cys Arg Ser Pro305 310 315 320ccg act gcg ctt ctc tcc ggc ccg ttc gcg cga gat cgt cga cgc gct1008Pro Thr Ala Leu Leu Ser Gly Pro Phe Ala Arg Asp Arg Arg Arg Ala325 330 335gat ggc cgc gcg caa gaa cgt ctg cta cct gga gat cga ttc gcc cta1056Asp Gly Arg Ala Gln Glu Arg Leu Leu Pro Gly Asp Arg Phe Ala Leu340 345 350cgg gca cga tgc att tcc tga1077Arg Ala Arg Cys Ile Ser355<210>46<211>358<212>PRT<213>惡臭假單胞菌<400>46Met Ser Thr Val Phe Pro Glu Asp Ser Val Gly Leu Val Val Arg Gln1 5 10 15Thr Ser Arg Phe Asp Glu Pro Leu Ala Leu Ala Cys Gly Arg Ser Leu20 25 30Ala Ser Tyr Glu Leu Val Tyr Glu Thr Tyr Gly Thr Leu Asn Ala Ser35 40 45Ala Ser Asn Ala Val Leu Ile Cys His Ala Leu Ser Gly His His His50 55 60Ala Ala Gly Tyr His Ala Ala Thr Asp Arg Lys Pro Gly Trp Trp Asp
65 70 75 80Ser Cys Ile Gly Pro Gly Lys Pro Ile Asp Thr Asn Arg Phe Phe Val85 90 95Val Ser Leu Asn Asn Leu Gly Gly Cys Asn Gly Ser Thr Gly Pro Ser100 105 110Ser Val Asn Pro Ala Thr Gly Lys Pro Tyr Gly Ala Glu Phe Pro Val115 120 125Leu Thr Val Glu Asp Trp Val His Ser Gln Ala Arg Leu Ala Asp Arg130 135 140Leu Gly Ile Gln Gln Trp Ala Ala Ile Val Gly Gly Ser Leu Gly Gly145 150 155 160Met Gln Ala Leu Gln Trp Thr Met Thr Tyr Pro Glu Arg Val Arg His165 170 175Cys Val Asp Ile Ala Ser Ala Pro Lys Leu Ser Ala Gln Asn Ile Ala180 185 190Phe Asn Glu Val Ala Arg Gln Ala Ile Leu Thr Asp Pro Glu Tyr Arg195 200 205Arg Gly Ser Phe Pro Gly Pro Gly Val Ile Pro Lys Arg Gly Leu Met210 215 220Leu Ala Arg Met Val Gly His Ile Thr Tyr Leu Ser Asp Asp Ser Met225 230 235 240Gly Glu Lys Phe Gly Arg Glu Leu Lys Ala Thr Ser Ser Thr Thr Thr245 250 255Ser Thr Ala Ser Ser Ser Arg Ser Lys Ala Thr Cys Ala Ile Arg Ala260 265 270Arg Ser Phe Pro Ala Val Ser Thr Pro Thr Pro Thr Leu Met Thr Lys275 280 285Ala Leu Asp Tyr Phe Asp Pro Ala Ala Thr His Gly Gly Asp Leu Ala290 295 300Ala Thr Leu Ala His Val Thr Ala Asp Tyr Cys Ile Cys Arg Ser Pro305 310 315 320Pro Thr Ala Leu Leu Ser Gly Pro Phe Ala Arg Asp Arg Arg Arg Ala325 330 335Asp Gly Arg Ala Gln Glu Arg Leu Leu Pro Gly Asp Arg Phe Ala Leu340 345 350Arg Ala Arg Cys Ile Ser355<210>47<211>52<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>47cccgggatcc gctagcggcg cgccggccgg cccggtgtga aataccgcac ag 52
<210>48<211>53<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>48tctagactcg agcggccgcg gccggccttt aaattgaaga cgaaagggcc tcg53<210>49<211>47<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>49gagatctaga cccggggatc cgctagcggg ctgctaaagg aagcgga 47<210>50<211>38<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>50gagaggcgcg ccgctagcgt gggcgaagaa ctccagca 38<210>51<211>34<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>51gagagggcgg ccgcgcaaag tcccgcttcg tgaa 34<210>52<211>34<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>52gagagggcgg ccgctcaagt cggtcaagcc acgc 34<210>53<211>140<212>DNA
<213>人工序列<220>
<223>人工序列的描述PCR引物<400>53tcgaatttaa atctcgagag gcctgacgtc gggcccggta ccacgcgtca tatgactagt 60tcggacctag ggatatcgtc gacatcgatg ctcttctgcg ttaattaaca attgggatcc 120tctagacccg ggatttaaat 140<210>54<211>140<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>54gatcatttaa atcccgggtc tagaggatcc caattgttaa ttaacgcaga agagcatcga 60tgtcgacgat atccctaggt ccgaactagt catatgacgc gtggtaccgg gcccgacgtc 120aggcctctcg agatttaaat 140<210>55<211>33<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>55gagagcggcc gccgatcctt tttaacccat cac 33<210>56<211>32<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>56aggagcggcc gccatcggca ttttcttttg cg32<210>57<211>5091<212>DNA<213>人工序列<220>
<223>人工序列的描述質(zhì)粒<400>57gccgcgactg ccttcgcgaa gccttgcccc gcggaaattt cctccaccga gttcgtgcac 60acccctatgc caagcttctt tcaccctaaa ttcgagagat tggattctta ccgtggaaat 120tcttcgcaaa aatcgtcccc tgatcgccct tgcgacgttg gcgtcggtgc cgctggttgc 180gcttggcttg accgacttga tcagcggccg ctcgatttaa atctcgagag gcctgacgtc 240gggcccggta ccacgcgtca tatgactagt tcggacctag ggatatcgtc gacatcgatg 300ctcttctgcg ttaattaaca attgggatcc tctagacccg ggatttaaat cgctagcggg 360ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag aaacggtgct gaccccggat 420
gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt 480agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga 540accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg 600gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagatctg atcaagagac 660aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc 720ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc 780cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc 840cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg 900cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt 960gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc 1020catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga 1080ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga 1140tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct 1200caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc 1260gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt 1320ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg 1380cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat 1440cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc 1500gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa 1560aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat 1620ctcatgctgg agttcttcgc ccacgctagc ggcgcgccgg ccggcccggt gtgaaatacc 1680gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga 1740ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 1800acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 1860aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 1920tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 1980aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 2040gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 2100acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 2160accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 2220ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 2280gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 2340gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 2400ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 2460gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 2520cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 2580cttcacctag atccttttaa aggccggccg cggccgcgca aagtcccgct tcgtgaaaat 2640tttcgtgccg cgtgattttc cgccaaaaac tttaacgaac gttcgttata atggtgtcat 2700gaccttcacg acgaagtact aaaattggcc cgaatcatca gctatggatc tctctgatgt 2760cgcgctggag tccgacgcgc tcgatgctgc cgtcgattta aaaacggtga tcggattttt 2820ccgagctctc gatacgacgg acgcgccagc atcacgagac tgggccagtg ccgcgagcga 2880cctagaaact ctcgtggcgg atcttgagga gctggctgac gagctgcgtg ctcggccagc 2940gccaggagga cgcacagtag tggaggatgc aatcagttgc gcctactgcg gtggcctgat 3000tcctccccgg cctgacccgc gaggacggcg cgcaaaatat tgctcagatg cgtgtcgtgc 3060cgcagccagc cgcgagcgcg ccaacaaacg ccacgccgag gagctggagg cggctaggtc 3120gcaaatggcg ctggaagtgc gtcccccgag cgaaattttg gccatggtcg tcacagagct 3180ggaagcggca gcgagaatta tcgcgatcgt ggcggtgccc gcaggcatga caaacatcgt 3240aaatgccgcg tttcgtgtgc cgtggccgcc caggacgtgt cagcgccgcc accacctgca 3300ccgaatcggc agcagcgtcg cgcgtcgaaa aagcgcacag gcggcaagaa gcgataagct 3360gcacgaatac ctgaaaaatg ttgaacgccc cgtgagcggt aactcacagg gcgtcggcta 3420acccccagtc caaacctggg agaaagcgct caaaaatgac tctagcggat tcacgagaca 3480ttgacacacc ggcctggaaa ttttccgctg atctgttcga cacccatccc gagctcgcgc 3540tgcgatcacg tggctggacg agcgaagacc gccgcgaatt cctcgctcac ctgggcagag 3600aaaatttcca gggcagcaag acccgcgact tcgccagcgc ttggatcaaa gacccggaca 3660cggagaaaca cagccgaagt tataccgagt tggttcaaaa tcgcttgccc ggtgccagta 3720tgttgctctg acgcacgcgc agcacgcagc cgtgcttgtc ctggacattg atgtgccgag 3780ccaccaggcc ggcgggaaaa tcgagcacgt aaaccccgag gtctacgcga ttttggagcg 3840ctgggcacgc ctggaaaaag cgccagcttg gatcggcgtg aatccactga gcgggaaatg 3900ccagctcatc tggctcattg atccggtgta tgccgcagca ggcatgagca gcccgaatat 3960gcgcctgctg gctgcaacga ccgaggaaat gacccgcgtt ttcggcgctg accaggcttt 4020ttcacatagg ctgagccgtg gccactgcac tctccgacga tcccagccgt accgctggca 4080tgcccagcac aatcgcgtgg atcgcctagc tgatcttatg gaggttgctc gcatgatctc 4140aggcacagaa aaacctaaaa aacgctatga gcaggagttt tctagcggac gggcacgtat 4200cgaagcggca agaaaagcca ctgcggaagc aaaagcactt gccacgcttg aagcaagcct 4260gccgagcgcc gctgaagcgt ctggagagct gatcgacggc gtccgtgtcc tctggactgc 4320tccagggcgt gccgcccgtg atgagacggc ttttcgccac gctttgactg tgggatacca 4380gttaaaagcg gctggtgagc gcctaaaaga caccaagggt catcgagcct acgagcgtgc 4440
ctacaccgtc gctcaggcgg tcggaggagg ccgtgagcct gatctgccgc cggactgtga 4500ccgccagacg gattggccgc gacgtgtgcg cggctacgtc gctaaaggcc agccagtcgt 4560ccctgctcgt cagacagaga cgcagagcca gccgaggcga aaagctctgg ccactatggg 4620aagacgtggc ggtaaaaagg ccgcagaacg ctggaaagac ccaaacagtg agtacgcccg 4680agcacagcga gaaaaactag ctaagtccag tcaacgacaa gctaggaaag ctaaaggaaa 4740tcgcttgacc attgcaggtt ggtttatgac tgttgaggga gagactggct cgtggccgac 4800aatcaatgaa gctatgtctg aatttagcgt gtcacgtcag accgtgaata gagcacttaa 4860ggtctgcggg cattgaactt ccacgaggac gccgaaagct tcccagtaaa tgtgccatct 4920cgtaggcaga aaacggttcc cccgtagggt ctctctcttg gcctcctttc taggtcgggc 4980tgattgctct tgaagctctc taggggggct cacaccatag gcagataacg ttccccaccg 5040gctcgcctcg taagcgcaca aggactgctc ccaaagatct tcaaagccac t 5091<210>58<211>4323<212>DNA<213>人工序列<220>
<223>人工序列的描述質(zhì)粒<400>58tctctcagcg tatggttgtc gcctgagctg tagttgcctt catcgatgaa ctgctgtaca 60ttttgatacg tttttccgtc accgtcaaag attgatttat aatcctctac accgttgatg 120ttcaaagagc tgtctgatgc tgatacgtta acttgtgcag ttgtcagtgt ttgtttgccg 180taatgtttac cggagaaatc agtgtagaat aaacggattt ttccgtcaga tgtaaatgtg 240gctgaacctg accattcttg tgtttggtct tttaggatag aatcatttgc atcgaatttg 300tcgctgtctt taaagacgcg gccagcgttt ttccagctgt caatagaagt ttcgccgact 360ttttgataga acatgtaaat cgatgtgtca tccgcatttt taggatctcc ggctaatgca 420aagacgatgt ggtagccgtg atagtttgcg acagtgccgt cagcgttttg taatggccag 480ctgtcccaaa cgtccaggcc ttttgcagaa gagatatttt taattgtgga cgaatcaaat 540tcagaaactt gatatttttc atttttttgc tgttcaggga tttgcagcat atcatggcgt 600gtaatatggg aaatgccgta tgtttcctta tatggctttt ggttcgtttc tttcgcaaac 660gcttgagttg cgcctcctgc cagcagtgcg gtagtaaagg ttaatactgt tgcttgtttt 720gcaaactttt tgatgttcat cgttcatgtc tcctttttta tgtactgtgt tagcggtctg 780cttcttccag ccctcctgtt tgaagatggc aagttagtta cgcacaataa aaaaagacct 840aaaatatgta aggggtgacg ccaaagtata cactttgccc tttacacatt ttaggtcttg 900cctgctttat cagtaacaaa cccgcgcgat ttacttttcg acctcattct attagactct 960cgtttggatt gcaactggtc tattttcctc ttttgtttga tagaaaatca taaaaggatt 1020tgcagactac gggcctaaag aactaaaaaa tctatctgtt tcttttcatt ctctgtattt 1080tttatagttt ctgttgcatg ggcataaagt tgccttttta atcacaattc agaaaatatc 1140ataatatctc atttcactaa ataatagtga acggcaggta tatgtgatgg gttaaaaagg 1200atcggcggcc gctcgattta aatctcgaga ggcctgacgt cgggcccggt accacgcgtc 1260atatgactag ttcggaccta gggatatcgt cgacatcgat gctcttctgc gttaattaac 1320aattgggatc ctctagaccc gggatttaaa tcgctagcgg gctgctaaag gaagcggaac 1380acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct 1440atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca 1500tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg 1560gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca 1620aggatctgat ggcgcagggg atcaagatct gatcaagaga caggatgagg atcgtttcgc 1680atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 1740ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 1800gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 1860caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 1920ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 1980gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 2040cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 2100atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 2160gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 2220ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 2280ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 2340atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 2400ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 2460gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc 2520tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg 2580ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg 2640
cccacgctag cggcgcgccg gccggcccgg tgtgaaatac cgcacagatg cgtaaggaga 2700aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 2760cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 2820ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 2880aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 2940cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 3000cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 3060gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 3120tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 3180cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 3240ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 3300gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 3360gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 3420accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 3480ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 3540tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 3600aaggccggcc gcggccgcca tcggcatttt cttttgcgtt tttatttgtt aactgttaat 3660tgtccttgtt caaggatgct gtctttgaca acagatgttt tcttgccttt gatgttcagc 3720aggaagctcg gcgcaaacgt tgattgtttg tctgcgtaga atcctctgtt tgtcatatag 3780cttgtaatca cgacattgtt tcctttcgct tgaggtacag cgaagtgtga gtaagtaaag 3840gttacatcgt taggatcaag atccattttt aacacaaggc cagttttgtt cagcggcttg 3900tatgggccag ttaaagaatt agaaacataa ccaagcatgt aaatatcgtt agacgtaatg 3960ccgtcaatcg tcatttttga tccgcgggag tcagtgaaca ggtaccattt gccgttcatt 4020ttaaagacgt tcgcgcgttc aatttcatct gttactgtgt tagatgcaat cagcggtttc 4080atcacttttt tcagtgtgta atcatcgttt agctcaatca taccgagagc gccgtttgct 4140aactcagccg tgcgtttttt atcgctttgc agaagttttt gactttcttg acggaagaat 4200gatgtgcttt tgccatagta tgctttgtta aataaagatt cttcgccttg gtagccatct 4260tcagttccag tgtttgcttc aaatactaag tatttgtggc ctttatcttc tacgtagtga 4320gga 4323<210>59<211>35<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>59gagagagaga cgcgtcccag tggctgagac gcatc 35<210>60<211>34<212>DNA<213>人工序列<220>
<223>人工序列的描述PcR引物<400>60ctctctctgt cgacgaattc aatcttacgg cctg 34<210>61<211>5860<212>DNA<213>人工序列<220>
<223>人工序列的描述質(zhì)粒<400>61cccggtacca cgcgtcccag tggctgagac gcatccgcta aagccccagg aaccctgtg 60
agaaagaaaa cactcctctg gctaggtaga cacagtttat aaaggtagag ttgagcgggt 120aactgtcagc acgtagatcg aaaggtgcac aaaggtggcc ctggtcgtac agaaatatgg 180cggttcctcg cttgagagtg cggaacgcat tagaaacgtc gctgaacgga tcgttgccac 240caagaaggct ggaaatgatg tcgtggttgt ctgctccgca atgggagaca ccacggatga 300acttctagaa cttgcagcgg cagtgaatcc cgttccgcca gctcgtgaaa tggatatgct 360cctgactgct ggtgagcgta tttctaacgc tctcgtcgcc atggctattg agtcccttgg 420cgcagaagcc caatctttca cgggctctca ggctggtgtg ctcaccaccg agcgccacgg 480aaacgcacgc attgttgatg tcactccagg tcgtgtgcgt gaagcactcg atgagggcaa 540gatctgcatt gttgctggtt tccagggtgt taataaagaa acccgcgatg tcaccacgtt 600gggtcgtggt ggttctgaca ccactgcagt tgcgttggca gctgctttga acgctgatgt 660gtgtgagatt tactcggacg ttgacggtgt gtataccgct gacccgcgca tcgttcctaa 720tgcacagaag ctggaaaagc tcagcttcga agaaatgctg gaacttgctg ctgttggctc 780caagattttg gtgctgcgca gtgttgaata cgctcgtgca ttcaatgtgc cacttcgcgt 840acgctcgtct tatagtaatg atcccggcac tttgattgcc ggctctatgg aggatattcc 900tgtggaagaa gcagtcctta ccggtgtcgc aaccgacaag tccgaagcca aagtaaccgt 960tctgggtatt tccgataagc caggcgaggc tgcgaaggtt ttccgtgcgt tggctgatgc1020agaaatcaac attgacatgg ttctgcagaa cgtctcttct gtagaagacg gcaccaccga1080catcaccttc acctgccctc gttccgacgg ccgccgcgcg atggagatct tgaagaagct1140tcaggttcag ggcaactgga ccaatgtgct ttacgacgac caggtcggca aagtctccct1200cgtgggtgct ggcatgaagt ctcacccagg tgttaccgca gagttcatgg aagctctgcg1260cgatgtcaac gtgaacatcg aattgatttc cacctctgag attcgtattt ccgtgctgat1320ccgtgaagat gatctggatg ctgctgcacg tgcattgcat gagcagttcc agctgggcgg1380cgaagacgaa gccgtcgttt atgcaggcac cggacgctaa agttttaaag gagtagtttt1440acaatgacca ccatcgcagt tgttggtgca accggccagg tcggccaggt tatgcgcacc1500cttttggaag agcgcaattt cccagctgac actgttcgtt tctttgcttc cccacgttcc1560gcaggccgta agattgaatt cgtcgacatc gatgctcttc tgcgttaatt aacaattggg1620atcctctaga cccgggattt aaatcgctag cgggctgcta aaggaagcgg aacacgtaga1680aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga1740caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat1800agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct1860ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct1920gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg1980aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg2040actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg2100
ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg2160aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg2220ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc2280tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc2340tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc2400gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc2460aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg2520atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct2580tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt2640tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc2700tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt2760tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc2820acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg2880ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccacgc2940tagcggcgcg ccggccggcc cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc3000gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc3060ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata3120acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg3180cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct3240caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa3300gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc3360tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt3420aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg3480ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg3540cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct3600tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc3660tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg3720ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc3780aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt3840aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaaggccg3900gccgcggccg ccatcggcat tttcttttgc gtttttattt gttaactgtt aattgtcctt3960gttcaaggat gctgtctttg acaacagatg ttttcttgcc tttgatgttc agcaggaagc4020tcggcgcaaa cgttgattgt ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa4080
tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta aaggttacat4140cgttaggatc aagatccatt tttaacacaa ggccagtttt gttcagcggc ttgtatgggc4200cagttaaaga attagaaaca taaccaagca tgtaaatatc gttagacgta atgccgtcaa4260tcgtcatttt tgatccgcgg gagtcagtga acaggtacca tttgccgttc attttaaaga4320cgttcgcgcg ttcaatttca tctgttactg tgttagatgc aatcagcggt ttcatcactt4380ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt gctaactcag4440ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc ttgacggaag aatgatgtgc4500ttttgccata gtatgctttg ttaaataaag attcttcgcc ttggtagcca tcttcagttc4560cagtgtttgc ttcaaatact aagtatttgt ggcctttatc ttctacgtag tgaggatctc4620tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc gatgaactgc tgtacatttt4680gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg ttgatgttca4740aagagctgtc tgatgctgat acgttaactt gtgcagttgt cagtgtttgt ttgccgtaat4800gtttaccgga gaaatcagtg tagaataaac ggatttttcc gtcagatgta aatgtggctg4860aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg aatttgtcgc4920tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat agaagtttcg ccgacttttt4980gatagaacat gtaaatcgat gtgtcatccg catttttagg atctccggct aatgcaaaga5040cgatgtggta gccgtgatag tttgcgacag tgccgtcagc gttttgtaat ggccagctgt5100cccaaacgtc caggcctttt gcagaagaga tatttttaat tgtggacgaa tcaaattcag5160aaacttgata tttttcattt ttttgctgtt cagggatttg cagcatatca tggcgtgtaa5220tatgggaaat gccgtatgtt tccttatatg gcttttggtt cgtttctttc gcaaacgctt5280gagttgcgcc tcctgccagc agtgcggtag taaaggttaa tactgttgct tgttttgcaa5340actttttgat gttcatcgtt catgtctcct tttttatgta ctgtgttagc ggtctgcttc5400ttccagccct cctgtttgaa gatggcaagt tagttacgca caataaaaaa agacctaaaa5460tatgtaaggg gtgacgccaa agtatacact ttgcccttta cacattttag gtcttgcctg5520ctttatcagt aacaaacccg cgcgatttac ttttcgacct cattctatta gactctcgtt5580tggattgcaa ctggtctatt ttcctctttt gtttgataga aaatcataaa aggatttgca5640gactacgggc ctaaagaact aaaaaatcta tctgtttctt ttcattctct gtatttttta5700tagtttctgt tgcatgggca taaagttgcc tttttaatca caattcagaa aatatcataa5760tatctcattt cactaaataa tagtgaacgg caggtatatg tgatgggtta aaaaggatcg5820gcggccgctc gatttaaatc tcgagaggcc tgacgtcggg 5860<210>62<211>38<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>62cggcaccacc gacatcatct tcacctgccc tcgttccg38<210>63<211>38<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>63cggaacgagg gcaggtgaag atgatgtcgg tggtgccg 38<210>64<211>1266<212>DNA<213>LysC突變體<220>
<221>CDS<222>(1)..(1266)<223>
<400>64gtg gcc ctg gtc gta cag aaa tat ggc ggt tcc tcg ctt gag agt gcg48Val Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala1 5 10 15gaa cgc att aga aac gtc gct gaa cgg atc gtt gcc acc aag aag gct96Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala20 25 30gga aat gat gtc gtg gtt gtc tgc tcc gca atg gga gac acc acg gat 144Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp35 40 45gaa ctt cta gaa ctt gca gcg gca gtg aat ccc gtt ccg cca gct cgt 192Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg50 55 60gaa atg gat atg ctc ctg act gct ggt gag cgt att tct aac gct ctc 240Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu65 70 75 80gtc gcc atg gct att gag tcc ctt ggc gca gaa gcc caa tct ttc acg 288Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr85 90 95ggc tct cag gct ggt gtg ctc acc acc gag cgc cac gga aac gca cgc 336Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg100 105 110att gtt gat gtc act cca ggt cgt gtg cgt gaa gca ctc gat gag ggc 384Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly115 120 125aag atc tgc att gtt gct ggt ttc cag ggt gtt aat aaa gaa acc cgc 432Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg130 135 140gat gtc acc acg ttg ggt cgt ggt ggt tct gac acc act gca gtt gcg 480Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala
145 150 155 160ttg gca gct gct ttg aac gct gat gtg tgt gag att tac tcg gac gtt 528Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val165 170 175gac ggt gtg tat acc gct gac ccg cgc atc gtt cct aat gca cag aag 576Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys180 185 190ctg gaa aag ctc agc ttc gaa gaa atg ctg gaa ctt gct gct gtt ggc 624Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly195 200 205tcc aag att ttg gtg ctg cgc agt gtt gaa tac gct cgt gca ttc aat 672Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn210 215 220gtg cca ctt cgc gta cgc tcg tct tat agt aat gat ccc ggc act ttg 720Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu225 230 235 240att gcc ggc tct atg gag gat att cct gtg gaa gaa gca gtc ctt acc 768Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr245 250 255ggt gtc gca acc gac aag tcc gaa gcc aaa gta acc gtt ctg ggt att 816Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile260 265 270tcc gat aag cca ggc gag gct gcg aag gtt ttc cgt gcg ttg gct gat 864Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp275 280 285gca gaa atc aac att gac atg gtt ctg cag aac gtc tct tct gta gaa 912Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu290 295 300gac ggc acc acc gac atc atc ttc acc tgc cct cgt tcc gac ggc cgc 960Asp Gly Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg305 310 315 320cgc gcg atg gag atc ttg aag aag ctt cag gtt cag ggc aac tgg acc 1008Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr325 330 335aat gtg ctt tac gac gac cag gtc ggc aaa gtc tcc ctc gtg ggt gct 1056Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala340 345 350ggc atg aag tct cac cca ggt gtt acc gca gag ttc atg gaa gct ctg 1104Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu355 360 365cgc gat gtc aac gtg aac atc gaa ttg att tcc acc tct gag att cgt 1152Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg370 375 380att tcc gtg ctg atc cgt gaa gat gat ctg gat gct gct gca cgt gca 1200Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala385 390 395 400ttg cat gag cag ttc cag ctg ggc ggc gaa gac gaa gcc gtc gtt tat 1248Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr405 410 415gca ggc acc gga cgc taa 1266
Ala Gly Thr Gly Arg420<210>65<211>421<212>PRT<213>LysC突變體<400>65Val Ala Leu Val Val Gln Lys Tyr Gly Gly Ser Ser Leu Glu Ser Ala1 5 10 15Glu Arg Ile Arg Asn Val Ala Glu Arg Ile Val Ala Thr Lys Lys Ala20 25 30Gly Asn Asp Val Val Val Val Cys Ser Ala Met Gly Asp Thr Thr Asp35 40 45Glu Leu Leu Glu Leu Ala Ala Ala Val Asn Pro Val Pro Pro Ala Arg50 55 60Glu Met Asp Met Leu Leu Thr Ala Gly Glu Arg Ile Ser Asn Ala Leu65 70 75 80Val Ala Met Ala Ile Glu Ser Leu Gly Ala Glu Ala Gln Ser Phe Thr85 90 95Gly Ser Gln Ala Gly Val Leu Thr Thr Glu Arg His Gly Asn Ala Arg100 105 110Ile Val Asp Val Thr Pro Gly Arg Val Arg Glu Ala Leu Asp Glu Gly115 120 125Lys Ile Cys Ile Val Ala Gly Phe Gln Gly Val Asn Lys Glu Thr Arg130 135 140Asp Val Thr Thr Leu Gly Arg Gly Gly Ser Asp Thr Thr Ala Val Ala145 150 155 160Leu Ala Ala Ala Leu Asn Ala Asp Val Cys Glu Ile Tyr Ser Asp Val165 170 175Asp Gly Val Tyr Thr Ala Asp Pro Arg Ile Val Pro Asn Ala Gln Lys180 185 190Leu Glu Lys Leu Ser Phe Glu Glu Met Leu Glu Leu Ala Ala Val Gly195 200 205Ser Lys Ile Leu Val Leu Arg Ser Val Glu Tyr Ala Arg Ala Phe Asn210 215 220
Val Pro Leu Arg Val Arg Ser Ser Tyr Ser Asn Asp Pro Gly Thr Leu225 230 235 240Ile Ala Gly Ser Met Glu Asp Ile Pro Val Glu Glu Ala Val Leu Thr245 250 255Gly Val Ala Thr Asp Lys Ser Glu Ala Lys Val Thr Val Leu Gly Ile260 265 270Ser Asp Lys Pro Gly Glu Ala Ala Lys Val Phe Arg Ala Leu Ala Asp275 280 285Ala Glu Ile Asn Ile Asp Met Val Leu Gln Asn Val Ser Ser Val Glu290 295 300Asp Gly Thr Thr Asp Ile Ile Phe Thr Cys Pro Arg Ser Asp Gly Arg305 310 315 320Arg Ala Met Glu Ile Leu Lys Lys Leu Gln Val Gln Gly Asn Trp Thr325 330 335Asn Val Leu Tyr Asp Asp Gln Val Gly Lys Val Ser Leu Val Gly Ala340 345 350Gly Met Lys Ser His Pro Gly Val Thr Ala Glu Phe Met Glu Ala Leu355 360 365Arg Asp Val Asn Val Asn Ile Glu Leu Ile Ser Thr Ser Glu Ile Arg370 375 380Ile Ser Val Leu Ile Arg Glu Asp Asp Leu Asp Ala Ala Ala Arg Ala385 390 395 400Leu His Glu Gln Phe Gln Leu Gly Gly Glu Asp Glu Ala Val Val Tyr405 410 415Ala Gly Thr Gly Arg420<210>66<211>5860<212>DNA<213>人工序列<220>
<223>人工序列的描述質(zhì)粒<400>66cccggtacca cgcgtcccag tggctgagac gcatccgcta aagccccagg aaccctgtgc 60agaaagaaaa cactcctctg gctaggtaga cacagtttat aaaggtagag ttgagcgggt120aactgtcagc acgtagatcg aaaggtgcac aaaggtggcc ctggtcgtac agaaatatgg180
cggttcctcg cttgagagtg cggaacgcat tagaaacgtc gctgaacgga tcgttgccac 240caagaaggct ggaaatgatg tcgtggttgt ctgctccgca atgggagaca ccacggatga 300acttctagaa cttgcagcgg cagtgaatcc cgttccgcca gctcgtgaaa tggatatgct 360cctgactgct ggtgagcgta tttctaacgc tctcgtcgcc atggctattg agtcccttgg 420cgcagaagcc caatctttca cgggctctca ggctggtgtg ctcaccaccg agcgccacgg 480aaacgcacgc attgttgatg tcactccagg tcgtgtgcgt gaagcactcg atgagggcaa 540gatctgcatt gttgctggtt tccagggtgt taataaagaa acccgcgatg tcaccacgtt 600gggtcgtggt ggttctgaca ccactgcagt tgcgttggca gctgctttga acgctgatgt 660gtgtgagatt tactcggacg ttgacggtgt gtataccgct gacccgcgca tcgttcctaa 720tgcacagaag ctggaaaagc tcagcttcga agaaatgctg gaacttgctg ctgttggctc 780caagattttg gtgctgcgca gtgttgaata cgctcgtgca ttcaatgtgc cacttcgcgt 840acgctcgtct tatagtaatg atcccggcac tttgattgcc ggctctatgg aggatattcc 900tgtggaagaa gcagtcctta ccggtgtcgc aaccgacaag tccgaagcca aagtaaccgt 960tctgggtatt tccgataagc caggcgaggc tgcgaaggtt ttccgtgcgt tggctgatgc1020agaaatcaac attgacatgg ttctgcagaa cgtctcttct gtagaagacg gcaccaccga1080catcatcttc acctgccctc gttccgacgg ccgccgcgcg atggagatct tgaagaagct1140tcaggttcag ggcaactgga ccaatgtgct ttacgacgac caggtcggca aagtctccct1200cgtgggtgct ggcatgaagt ctcacccagg tgttaccgca gagttcatgg aagctctgcg1260cgatgtcaac gtgaacatcg aattgatttc cacctctgag attcgtattt ccgtgctgat1320ccgtgaagat gatctggatg ctgctgcacg tgcattgcat gagcagttcc agctgggcgg1380cgaagacgaa gccgtcgttt atgcaggcac cggacgctaa agttttaaag gagtagtttt1440acaatgacca ccatcgcagt tgttggtgca accggccagg tcggccaggt tatgcgcacc1500cttttggaag agcgcaattt cccagctgac actgttcgtt tctttgcttc cccacgttcc1560gcaggccgta agattgaatt cgtcgacatc gatgctcttc tgcgttaatt aacaattggg1620atcctctaga cccgggattt aaatcgctag cgggctgcta aaggaagcgg aacacgtaga1680aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga1740caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat1800agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct1860ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct1920gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt cgcatgattg1980aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg2040actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg2100ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg2160aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg2220
ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc2280tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc2340tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc2400gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc2460aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg2520atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct2580tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt2640tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc2700tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt2760tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc2820acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg2880ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccacgc2940tagcggcgcg ccggccggcc cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc3000gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc3060ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata3120acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg3180cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct3240caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa3300gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc3360tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt3420aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg3480ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg3540cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct3600tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc3660tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg3720ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc3780aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt3840aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaaggccg3900gccgcggccg ccatcggcat tttcttttgc gtttttattt gttaactgtt aattgtcctt3960gttcaaggat gctgtctttg acaacagatg ttttcttgcc tttgatgttc agcaggaagc4020tcggcgcaaa cgttgattgt ttgtctgcgt agaatcctct gtttgtcata tagcttgtaa4080tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta aaggttacat4140cgttaggatc aagatccatt tttaacacaa ggccagtttt gttcagcggc ttgtatgggc4200
cagttaaaga attagaaaca taaccaagca tgtaaatatc gttagacgta atgccgtcaa4260tcgtcatttt tgatccgcgg gagtcagtga acaggtacca tttgccgttc attttaaaga4320cgttcgcgcg ttcaatttca tctgttactg tgttagatgc aatcagcggt ttcatcactt4380ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt gctaactcag4440ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc ttgacggaag aatgatgtgc4500ttttgccata gtatgctttg ttaaataaag attcttcgcc ttggtagcca tcttcagttc4560cagtgtttgc ttcaaatact aagtatttgt ggcctttatc ttctacgtag tgaggatctc4620tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc gatgaactgc tgtacatttt4680gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg ttgatgttca4740aagagctgtc tgatgctgat acgttaactt gtgcagttgt cagtgtttgt ttgccgtaat4800gtttaccgga gaaatcagtg tagaataaac ggatttttcc gtcagatgta aatgtggctg4860aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg aatttgtcgc4920tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat agaagtttcg ccgacttttt4980gatagaacat gtaaatcgat gtgtcatccg catttttagg atctccggct aatgcaaaga5040cgatgtggta gccgtgatag tttgcgacag tgccgtcagc gttttgtaat ggccagctgt5100cccaaacgtc caggcctttt gcagaagaga tatttttaat tgtggacgaa tcaaattcag5160aaacttgata tttttcattt ttttgctgtt cagggatttg cagcatatca tggcgtgtaa5220tatgggaaat gccgtatgtt tccttatatg gcttttggtt cgtttctttc gcaaacgctt5280gagttgcgcc tcctgccagc agtgcggtag taaaggttaa tactgttgct tgttttgcaa5340actttttgat gttcatcgtt catgtctcct tttttatgta ctgtgttagc ggtctgcttc5400ttccagccct cctgtttgaa gatggcaagt tagttacgca caataaaaaa agacctaaaa5460tatgtaaggg gtgacgccaa agtatacact ttgcccttta cacattttag gtcttgcctg5520ctttatcagt aacaaacccg cgcgatttac ttttcgacct cattctatta gactctcgtt5580tggattgcaa ctggtctatt ttcctctttt gtttgataga aaatcataaa aggatttgca5640gactacgggc ctaaagaact aaaaaatcta tctgtttctt ttcattctct gtatttttta5700tagtttctgt tgcatgggca taaagttgcc tttttaatca caattcagaa aatatcataa5760tatctcattt cactaaataa tagtgaacgg caggtatatg tgatgggtta aaaaggatcg5820gcggccgctc gatttaaatc tcgagaggcc tgacgtcggg 5860<210>67<211>29<212>DNA<213>人工序列<220>
<223>人工序列的描述PcR引物<400>67gagactcgag gttggctggt catcatagg 29
<210>68<211>34<212>DNA<213>人工序列<220>
<223>人工序列的描述PCR引物<400>68gaagagagca tatgtcagcg ctctagtttg gttc 34<210>69<211>6472<212>DNA<213>人工序列<220>
<223>人工序列的描述質(zhì)粒<400>69tcgaggttgg ctggtcatca taggaatcaa cctggccact ttatggtggg caccaccgtc 60gcaaacaaca tatcttgcag caggcgtgtc gattctttcc gccatcattg tttggtttct 120tcccggcgca cacccgctat ggaatcgccg tcgcattgct tcacgcaaac aacagtccac 180cggtagacgt cgacaagccc ccaaacgatc aagccaccct caaacggcgg aatttagcca 240acaacaatag actagacaga gctgtccatg tagcatgaac tcgattatca actgccacga 300gaggtcgggg tcatgctcac caccacaggg acgctcacgc accaaaaaat cggagacttt 360tacaccgaag ccggagcgac gcttcacgac gtaaccatcg cctaccaagc atggggccac 420tacaccggca ccaatctcat cgttctcgaa catgccctga ccggcgactc taacgctatt 480tcatggtggg acggactgat tggccctggc aaagcactcg acaccaaccg ctactgcatc 540ctatgcacca acgtgctcgg aggatgcaaa ggatccaccg gaccgagcag tccacaccca 600gacggaaaac catggggatc cagatttcca gccctttcaa tccgtgacct tgtcaatgcc 660gaaaaacaac ttttcgacca cctcggcatc aataaaattc acgcaatcat cggcggatcc 720atgggaggcg cacgcaccct cgaatgggct gcactccacc cacacatgat gacgactgga 780ttcgtcatag cagtctcagc acgcgcaagc gcttggcaaa tcggtattca aactgcacaa 840atcagcgcca tagaactcga cccccactgg aacggcggcg attactacag cggtcacgca 900ccatgggaag gaatcgccgc cgctcgccgg atcgcccacc tcacctatcg cggcgaacta 960gaaatagacg aacgattcgg cacttccgca caacacggtg aaaacccact cggccccttc1020cgagatccac atcaacgttt tgcggtcacg agctacctcc aacaccaagg catcaaactc1080gctcaacgat tcgatgcagg tagttacgtc gtgcttaccg aagccctcaa tcgtcatgac1140atcggacgcg gccgaggcgg actcaacaaa gccctcagcg caatcacagt ccccatcatg1200attgctggcg ttgataccga tattctctac ccctatcacc agcaagaaca cctatcacga1260aatctaggca acctactcgc tatggcaaaa atcagctcac cagtaggcca cgacgctttc1320
ctcacagaat tccgacaaat ggagcgaatc ctaagacatt tcatggagct ttcggaagga1380atcgacgatt ccttccgaac caaactagag cgctgacata tgactagttc ggacctaggg1440atatcgtcga catcgatgct cttctgcgtt aattaacaat tgggatcctc tagacccggg1500atttaaatcg ctagcgggct gctaaaggaa gcggaacacg tagaaagcca gtccgcagaa1560acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag1620cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt1680tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa1740gccctgcaaa gtaaactgga tggctttctt gccgccaagg atctgatggc gcaggggatc1800aagatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca1860cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac1920aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt1980tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc2040gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg2100aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc2160tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc2220ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat2280ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc2340cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca2400tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga2460ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat2520tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc2580tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact2640ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc2700accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg2760atcctccagc gcggggatct catgctggag ttcttcgccc acgctagcgg cgcgccggcc2820ggcccggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc2880cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc2940tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat3000gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt3060ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg3120aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc3180tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt3240ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa3300
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta3360tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa3420caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa3480ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt3540cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt3600ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat3660cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat3720gagattatca aaaaggatct tcacctagat ccttttaaag gccggccgcg gccgcgcaaa3780gtcccgcttc gtgaaaattt tcgtgccgcg tgattttccg ccaaaaactt taacgaacgt3840tcgttataat ggtgtcatga ccttcacgac gaagtactaa aattggcccg aatcatcagc3900tatggatctc tctgatgtcg cgctggagtc cgacgcgctc gatgctgccg tcgatttaaa3960aacggtgatc ggatttttcc gagctctcga tacgacggac gcgccagcat cacgagactg4020ggccagtgcc gcgagcgacc tagaaactct cgtggcggat cttgaggagc tggctgacga4080gctgcgtgct cggccagcgc caggaggacg cacagtagtg gaggatgcaa tcagttgcgc4140ctactgcggt ggcctgattc ctccccggcc tgacccgcga ggacggcgcg caaaatattg4200ctcagatgcg tgtcgtgccg cagccagccg cgagcgcgcc aacaaacgcc acgccgagga4260gctggaggcg gctaggtcgc aaatggcgct ggaagtgcgt cccccgagcg aaattttggc4320catggtcgtc acagagctgg aagcggcagc gagaattatc gcgatcgtgg cggtgcccgc4380aggcatgaca aacatcgtaa atgccgcgtt tcgtgtgccg tggccgccca ggacgtgtca4440gcgccgccac cacctgcacc gaatcggcag cagcgtcgcg cgtcgaaaaa gcgcacaggc4500ggcaagaagc gataagctgc acgaatacct gaaaaatgtt gaacgccccg tgagcggtaa4560ctcacagggc gtcggctaac ccccagtcca aacctgggag aaagcgctca aaaatgactc4620tagcggattc acgagacatt gacacaccgg cctggaaatt ttccgctgat ctgttcgaca4680cccatcccga gctcgcgctg cgatcacgtg gctggacgag cgaagaccgc cgcgaattcc4740tcgctcacct gggcagagaa aatttccagg gcagcaagac ccgcgacttc gccagcgctt4800ggatcaaaga cccggacacg gagaaacaca gccgaagtta taccgagttg gttcaaaatc4860gcttgcccgg tgccagtatg ttgctctgac gcacgcgcag cacgcagccg tgcttgtcct4920ggacattgat gtgccgagcc accaggccgg cgggaaaatc gagcacgtaa accccgaggt4980ctacgcgatt ttggagcgct gggcacgcct ggaaaaagcg ccagcttgga tcggcgtgaa5040tccactgagc gggaaatgcc agctcatctg gctcattgat ccggtgtatg ccgcagcagg5100catgagcagc ccgaatatgc gcctgctggc tgcaacgacc gaggaaatga cccgcgtttt5160cggcgctgac caggcttttt cacataggct gagccgtggc cactgcactc tccgacgatc5220ccagccgtac cgctggcatg cccagcacaa tcgcgtggat cgcctagctg atcttatgga5280ggttgctcgc atgatctcag gcacagaaaa acctaaaaaa cgctatgagc aggagttttc5340
tagcggacgg gcacgtatcg aagcggcaag aaaagccact gcggaagcaa aagcacttgc5400cacgcttgaa gcaagcctgc cgagcgccgc tgaagcgtct ggagagctga tcgacggcgt5460ccgtgtcctc tggactgctc cagggcgtgc cgcccgtgat gagacggctt ttcgccacgc5520tttgactgtg ggataccagt taaaagcggc tggtgagcgc ctaaaagaca ccaagggtca5580tcgagcctac gagcgtgcct acaccgtcgc tcaggcggtc ggaggaggcc gtgagcctga5640tctgccgccg gactgtgacc gccagacgga ttggccgcga cgtgtgcgcg gctacgtcgc5700taaaggccag ccagtcgtcc ctgctcgtca gacagagacg cagagccagc cgaggcgaaa5760agctctggcc actatgggaa gacgtggcgg taaaaaggcc gcagaacgct ggaaagaccc5820aaacagtgag tacgcccgag cacagcgaga aaaactagct aagtccagtc aacgacaagc5880taggaaagct aaaggaaatc gcttgaccat tgcaggttgg tttatgactg ttgagggaga5940gactggctcg tggccgacaa tcaatgaagc tatgtctgaa tttagcgtgt cacgtcagac6000cgtgaataga gcacttaagg tctgcgggca ttgaacttcc acgaggacgc cgaaagcttc6060ccagtaaatg tgccatctcg taggcagaaa acggttcccc cgtagggtct ctctcttggc6120ctcctttcta ggtcgggctg attgctcttg aagctctcta ggggggctca caccataggc6180agataacgtt ccccaccggc tcgcctcgta agcgcacaag gactgctccc aaagatcttc6240aaagccactg ccgcgactgc cttcgcgaag ccttgccccg cggaaatttc ctccaccgag6300ttcgtgcaca cccctatgcc aagcttcttt caccctaaat tcgagagatt ggattcttac6360cgtggaaatt cttcgcaaaa atcgtcccct gatcgccctt gcgacgttgg cgtcggtgcc6420gctggttgcg cttggcttga ccgacttgat cagcggccgc tcgatttaaa tc647權(quán)利要求
1.發(fā)酵產(chǎn)生至少一種含硫精細(xì)化學(xué)品的方法，該方法包括下面的步驟a)發(fā)酵產(chǎn)生目的含硫精細(xì)化學(xué)品的棒狀細(xì)菌培養(yǎng)物，其中所述棒狀細(xì)菌表達(dá)至少一種這樣的異源核苷酸序列，該序列編碼具有高絲氨酸O-乙酰轉(zhuǎn)移酶(metA)活性的蛋白質(zhì)；b)濃縮培養(yǎng)基或細(xì)菌細(xì)胞中的含硫精細(xì)化學(xué)品；和c)分離含硫精細(xì)化學(xué)品。
2.權(quán)利要求1所述的方法，其中含硫精細(xì)化學(xué)品包含L-甲硫氨酸。
3.前述權(quán)利要求任一項(xiàng)中所述的方法，其中編碼異源metA的核苷酸序列與來(lái)自谷氨酸棒桿菌(Corynebacterium glutamicum)ATCC 13032的metA編碼序列的同源性小于100％。
4.權(quán)利要求3所述的方法，其中metA編碼序列來(lái)自任一下列生物體
5.前述權(quán)利要求任一項(xiàng)中所述的方法，其中metA編碼序列包含根據(jù)SEQ ID NO1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43和45的編碼序列或者包含與它們同源的編碼具有metA活性的蛋白質(zhì)的核苷酸序列。
6.前述權(quán)利要求任一項(xiàng)中所述的方法，其中metA編碼序列編碼具有metA活性的蛋白質(zhì)，所述蛋白質(zhì)包含根據(jù)SEQ ID NO2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44和46的氨基酸序列或包含與它們同源的代表具有metA活性的蛋白質(zhì)的氨基酸序列。
7.前述權(quán)利要求任一項(xiàng)中所述的方法，其中編碼metA的序列為可以在棒狀細(xì)菌中復(fù)制或者被穩(wěn)定整合到其染色體中的DNA或RNA。
8.權(quán)利要求7所述的方法，其中a)使用經(jīng)這樣的質(zhì)粒載體轉(zhuǎn)化的細(xì)菌菌株，其中所述質(zhì)粒載體攜帶處于調(diào)節(jié)序列控制下的至少一個(gè)拷貝的metA編碼序列，或者b)使用其中metA編碼序列已經(jīng)被整合入該細(xì)菌染色體中的菌株。
9.前述權(quán)利要求任一項(xiàng)中所述的方法，其中編碼metA的序列被過(guò)量表達(dá)。
10.前述權(quán)利要求任一項(xiàng)中所述的方法，其中發(fā)酵這樣的細(xì)菌，該細(xì)菌中目的含硫精細(xì)化學(xué)品生物合成途徑的至少另一種基因額外地被擴(kuò)增或突變從而其活性不受代謝的代謝物影響。
11.前述權(quán)利要求任一項(xiàng)中所述的方法，其中發(fā)酵這樣的細(xì)菌，該細(xì)菌中降低目的含硫精細(xì)化學(xué)品產(chǎn)生的至少一條代謝途徑至少被部分關(guān)閉。
12.前述權(quán)利要求任一項(xiàng)中所述的方法，其中發(fā)酵這樣的棒狀細(xì)菌，該細(xì)菌中同時(shí)對(duì)選自a)基因lysC，其編碼天冬氨酸激酶，b)甘油醛-3-磷酸脫氫酶編碼基因gap，c)3-磷酸甘油酸激酶編碼基因pgk，d)丙酮酸羧化酶編碼基因pyc，e)磷酸丙糖異構(gòu)酶編碼基因tpi，f)亞甲基四氫葉酸還原酶編碼基因metF，g)γ胱硫醚合酶編碼基因metB，h)γ胱硫醚裂合酶編碼基因metC，i)絲氨酸羥甲基轉(zhuǎn)移酶編碼基因glyA，j)O-乙酰高絲氨酸硫化氫解酶編碼基因metY，k)維生素B12依賴的甲硫氨酸合酶編碼基因metH，l)磷酸絲氨酸氨基轉(zhuǎn)移酶編碼基因serC，m)磷酸絲氨酸磷酸酶編碼基因serB，n)絲氨酸乙酰轉(zhuǎn)移酶編碼基因cysE，和o)高絲氨酸脫氫酶編碼基因hom，的至少一種基因以某種方式過(guò)量表達(dá)或者突變，從而使得相應(yīng)蛋白質(zhì)的活性與未突變的蛋白質(zhì)相比，受代謝的代謝物影響程度如果有的話較小。
13.前述權(quán)利要求任一項(xiàng)中所述的方法，其中發(fā)酵這樣的棒狀細(xì)菌，該細(xì)菌中同時(shí)對(duì)選自a)高絲氨酸激酶編碼基因thrB，b)蘇氨酸脫水酶編碼基因ilvA，c)蘇氨酸合酶編碼基因thrC，d)內(nèi)消旋-二氨基庚二酸D-脫氫酶編碼基因ddh，e)磷酸烯醇丙酮酸羧激酶編碼基因pck，f)葡萄糖-6-磷酸6-異構(gòu)酶編碼基因pgi，g)丙酮酸氧化酶編碼基因poxB，h)二氫吡啶二羧酸合酶編碼基因dapA，i)二氫吡啶二羧酸還原酶編碼基因dapB；和j)二氨基吡啶甲酸脫羧酶編碼基因，的至少一種基因通過(guò)改變表達(dá)速率或者通過(guò)導(dǎo)入特定的突變而被弱化。
14.前述權(quán)利要求一項(xiàng)或多項(xiàng)中所述的方法，其中使用谷氨酸棒桿菌種的微生物。
15.從發(fā)酵液產(chǎn)生含L-甲硫氨酸的動(dòng)物飼料添加劑的方法，該方法包括下面的步驟a)在發(fā)酵培養(yǎng)基中培養(yǎng)并發(fā)酵產(chǎn)生L-甲硫氨酸的微生物；b)從含L-甲硫氨酸的發(fā)酵液除去水；c)除去發(fā)酵過(guò)程中形成的生物量重量的0到100％；和d)干燥根據(jù)b)和/或c)所得發(fā)酵液，以得到目的粉劑或粒劑形式的動(dòng)物飼料添加劑。
16.權(quán)利要求15所述的方法，其中使用根據(jù)權(quán)利要求1到14任一項(xiàng)中所定義的微生物。
全文摘要
本發(fā)明涉及通過(guò)使用表達(dá)編碼甲硫氨酸合酶(metA)基因的核苷酸序列的細(xì)菌，發(fā)酵產(chǎn)生含硫精細(xì)化學(xué)品尤其是L－甲硫氨酸的方法。
文檔編號(hào)C12P13/12GK1788090SQ03820574
公開(kāi)日2006年6月14日 申請(qǐng)日期2003年8月26日 優(yōu)先權(quán)日2002年8月26日
發(fā)明者B·克勒格爾, O·策爾德?tīng)? C·克洛普羅格, H·施羅德, S·哈夫納 申請(qǐng)人:巴斯福股份公司