專利名稱：：δ-內(nèi)毒素基因及其使用方法5-內(nèi)毒素基因及其使用方法本申請是申請日為2004年02月20日、申請?zhí)枮?00480007563.8、發(fā)明名稱為"5-內(nèi)毒素基因及其使用方法"的發(fā)明專利申請的分案申請。發(fā)明領(lǐng)域本發(fā)明涉及分子生物學(xué)領(lǐng)域。所提供的是編碼殺蟲蛋白質(zhì)的新基因。這些蛋白質(zhì)及編碼它們的核酸序列可用于制備殺蟲制劑以及生產(chǎn)轉(zhuǎn)基因的抗害蟲植物。發(fā)明背景蘇云金芽孢桿菌是形成孢子的革蘭氏陽性土壤細菌，特征在于它能產(chǎn)生對某些目和種的昆蟲有特異性毒性、但對植物和其它非靶向生物體無害的晶狀內(nèi)含物。因此，含蘇云金芽孢桿菌菌林或它們的殺蟲蛋白質(zhì)的組合物可用作環(huán)境可接受的殺蟲劑，以抑制農(nóng)業(yè)有害昆蟲或針對多種人或動物疾病的昆蟲載體。來自蘇云金芽孢桿菌的晶體(Cry)蛋白質(zhì)(5-內(nèi)毒素)具有主要針對鱗翅類、雙翅類和鞘翅類幼蟲的有效殺蟲活性。這些蛋白質(zhì)還顯示出抗膜翅目、同翅目、虱、食毛目和螨等有害目以及諸如線蟲、扁形動物(Platyhelminthes)和Sarcomastigorphora等其它無脊推動物目的活性(Feitelson(1993)蘇云金芽孢桿菌家族樹，高級基因工程殺蟲劑中(TheBacillusThuringiensisfamilytree,AdvancedEngineeredPesticides)MarcelDekker，Inc.,NewYork,N.Y.)。這些蛋白質(zhì)主要基于其殺蟲活性而最初被劃分為Cryl至CryV。主要的種類是鱗翅類特異的(I)、鱗翅類和雙翅類特異的(II)、鞘翅類特異的(III)、雙翅類特異的(IV)以及線蟲類特異的(V)和(VI)。所述蛋白質(zhì)進一步被劃分為亞家族，各個家族內(nèi)更密切相關(guān)的蛋白質(zhì)被指派上分類字母，諸如CrylA、CrylB、CrylC等。在各亞類內(nèi)更緊密相關(guān)的蛋白質(zhì)則命名為諸如CrylCl、CrylC2等。最近出現(xiàn)了一種針對Cry基因的新命名法，其基于的是氨基酸序列同源性而非昆蟲靼特異性(Crickmore爭(1998)#/cro6/o/.Wo厶We7.62:807-813)。在該新的分類法中，每種毒素被分配了一個獨特的名稱，其中合并了第一等級(阿拉伯?dāng)?shù)字)、第二等級(大寫字母)、第三等級(小寫字母)和第四等級(另一阿拉伯?dāng)?shù)字)。在新的分類中，羅馬數(shù)字被轉(zhuǎn)換成第一等級的阿拉伯?dāng)?shù)字。序列同一性小于45%的蛋白質(zhì)具有不同的第一等級，而對于第二和第三等級而言這一標準分別為78%和95%。晶體蛋白質(zhì)在被昆蟲中腸攝取和溶解前不呈現(xiàn)殺蟲活性。被攝取的毒素原在昆蟲消化道內(nèi)被蛋白酶水解成活性的有毒分子。(貼fte和Whiteley(1989)飾ro6/o人紐53:242-255)。此毒素與乾目標幼蟲中腸內(nèi)的頂端刷狀邊緣受體結(jié)合并插入頂端膜中，產(chǎn)生離子通道或孔，導(dǎo)致幼蟲死亡。5-內(nèi)毒素通常具有5個保守的序列結(jié)構(gòu)域和三個保守的結(jié)構(gòu)性結(jié)構(gòu)域(參閱，例ft口，deMaagd爭(2001)rre/7dsCe/7ef/cs17:193-199)。第一個保守的結(jié)構(gòu)性結(jié)構(gòu)域由7個ot螺旋組成，并涉及膜插入和孔形成。結(jié)構(gòu)域II由排列成希臘語鑰匙構(gòu)形的三個P折疊片組成，而結(jié)構(gòu)域III由"果凍-巻"形式的兩個反向平行卩折疊組成(deMaagd爭(2001)同上文)。結(jié)構(gòu)域II和III涉及受體識別和結(jié)合，并因此被認為是毒素特異性的決定區(qū)。因為昆蟲可造成的破壞，所以一直需要發(fā)現(xiàn)新形式的蘇云金芽孢桿菌5-內(nèi)毒素。發(fā)明簡述本發(fā)明提供了賦予細菌、植物、植物細胞、組織和種子以殺蟲劑抗性的組合物和方法。組合物包括編碼5-內(nèi)毒素和5-內(nèi)毒素相關(guān)多肽序列的分離核酸分子、包含這些核酸分子的載體和包含這些載體的宿主細胞。組合物還包括內(nèi)毒素的分離或重組多肽序列、包含這些多肽的組合物以及針對那些多肽的抗體。所述核酸序列可用于DNA構(gòu)建體或表達盒中，用于生物體中的轉(zhuǎn)化和表達，包括微生物和植物。所述核苷酸或氨基酸序列可以是已被設(shè)計成最適于在生物體中表達的合成序列，包括但不局限于，微生物或植物。組合物還包括被轉(zhuǎn)化的細菌、植物、植物細胞、組織和種子。具體而言，本發(fā)明提供了含有編碼SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29中所示氨基酸序列的核苷酸序列或SEQIDN0:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28中所給出的核苷酸序列的分離核酸分子以及它們的變體序列也包括在內(nèi)。還提供了可用于產(chǎn)生本發(fā)明的多肽的方法，以及利用那些多肽來控制或殺死鱗翅類或鞘翅類害蟲的方法。其是細菌和植物。這些生物體及其來源的組合物是農(nóng)業(yè)用途所需要的。素或5-內(nèi)毒素相關(guān)蛋白質(zhì)，或用于檢測產(chǎn)品或生物體中5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)或者核酸的存在。附圖描述圖1顯示了AXMI-004(SEQIDNO:3)與crylAc(SEQIDNO:31)、crylCa卿IDNO:32)、cry2Aa(SEQIDNO:34)、cry3Aal(SEQIDNO:35)、crylla(SEQIDNO:33)和cry7Aa(SEQIDN0:41)的序列比對。具有C-末端非毒性結(jié)構(gòu)域的毒素如圖所示被人為截短。該序列比對顯示了以黑色標示的最高度保守氨基酸殘基和以灰色標示的高度保守氨基酸殘基。發(fā)現(xiàn)保守組l是從SEQIDN0:3的大約第174位至大約第196位氨基酸殘基。發(fā)現(xiàn)保守組2是從SEQIDNO:3的大約第250位至大約笫292位氨基酸殘基。發(fā)現(xiàn)保守組3是從SEQIDNO:3的大約第476位至大約第521位氨基酸殘基。發(fā)現(xiàn)保守組4是從SEQIDNO:3的大約第542位至大約552位氨基酸殘基。發(fā)現(xiàn)保守組5是從SEQIDNO:3的大約第618位至大約第628位氨基酸殘基。圖2A、B和C顯示了AXMI-006(SEQIDNO:7)與crylAa(SEQIDNO:30)、crylAc(SEQIDNO:31)、crylla(SEQIDNO:33)、cry3Aal(SEQIDNO:35)、cry3Ba(SEQIDNO:36)、cry4Aa(SEQIDNO:38)、cry6Aa(SEQIDNO:40)、cry7Aa(SEQIDNO:41)、cry8Aa(SEQIDNO:42)、crylOAa(SEQIDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)和cry24Aa(SEQIDNO:47)的序列比對。具有C-末端非毒性結(jié)構(gòu)域的毒素如圖所示被人為截短。該序列比對顯示了以黑色標示的最高度保守氨基酸殘基和以灰色標示的高度保守氨基酸殘基。發(fā)現(xiàn)保守組1是從SEQIDNO:7的大約第218位至大約第239位氨基酸殘基。發(fā)現(xiàn)保守組2是從SEQIDNO:7的大約第300位至大約第350位氨基酸殘基。發(fā)現(xiàn)保守組3是從SEQIDNO:7的大約第547位至大約第592位氨基酸殘基。發(fā)現(xiàn)保守組4是從SEQIDN0:7的大約笫611位至大約621位氨基酸殘基。發(fā)現(xiàn)保守組5是從SEQIDNO:7的大約第694位至大約第704位氨基酸殘基。圖3A、B和C顯示了AXMI-007(SEQIDNO:9)與crylAa(SEQIDNO:30)、crylAc(SEQIDNO:31)、crylla(SEQIDNO:33)、cry3Aal(SEQIDNO:35)、cry3Ba(SEQIDNO:36)、cry4Aa(SEQIDNO:38)、cry6Aa(SEQIDNO:40)、cry7Aa(SEQIDNO:41)、cry8Aa(SEQIDNO:42)、crylOAa卿IDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)和cry24Aa(SEQIDNO:47)的序列比對。具有C-末端非毒性結(jié)構(gòu)域的毒素如圖所示被人為截短。該序列比對顯示了以黑色標示的最高度保守氨基酸殘基和以灰色標示的高度保守氨基酸殘基。發(fā)現(xiàn)保守組l是從SEQIDN0:9的大約第217位至大約第238位氨基酸殘基。發(fā)現(xiàn)保守組2是從SEQIDNO:9的大約第299位至大約笫347位氨基酸殘基。發(fā)現(xiàn)保守組3是從SEQIDNO:9的大約第445位至大約笫590位氨基酸殘基。發(fā)現(xiàn)保守組4是從SEQIDN0:9的大約笫609位至大約619位氨基酸殘基。發(fā)現(xiàn)保守組5是從SEQIDNO:9的大約第692位至大約第702位氨基酸殘基。圖4A、B和C顯示了AXMI-008(SEQIDNO:14)與crylAa(SEQIDNO:30)、crylAc(SEQIDNO:31)、crylla(SEQIDNO:33)、cry2Aa(SEQIDNO:34)、cry3Aal(SEQIDNO:35)、cry3Bb(SEQIDNO:37)、cry4Aa(SEQIDNO:38)、cry4Ba(SEQIDNO:39)、cry6Aa(SEQIDNO:40)、cry7Aa(SEQIDNO:41)、cry8Aa(SEQIDNO:42)、crylOAa(SEQIDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)、cry24Aa(SEQIDNO:47)、cry25Aa(SEQIDNO:48)、cry39Aal(SEQIDN0:49)和cry楊al(SEQIDN0:51)的序列比對。具有C-末端非毒性結(jié)構(gòu)域的毒素如圖所示被人為截短。該序列比對顯示了以黑色標示的最高度保守氨基酸殘基和以灰色標示的高度保守氨基酸殘基。發(fā)現(xiàn)保守組1是從SEQIDNO:14的大約第185位至大約第206位氨基酸殘基。發(fā)現(xiàn)保守組2是從SEQIDNO:14的大約第276位至大約第318位氨基酸殘基。發(fā)現(xiàn)保守組3是從SEQIDN0:14的大約第497位至大約第547位氨基酸殘基。發(fā)現(xiàn)保守組4是從SEQIDNO:14的大約第576位至大約586位氨基酸殘基。發(fā)現(xiàn)保守組5是從SEQIDNO:14的大約第657位至大約第667位氨基酸殘基。圖5A和B顯示了AXMI-008orf2(SEQIDNO:18)與cryl9Aa-orf2(SEQIDNO:46)、crybun2-orf2卿IDNO:50)、crybun3-orf2(SEQIDNO:52)、cry4Aa(SEQIDNO:38)和cry4Ba(SEQIDNO:39)的序列比對。該序列比對顯示了以黑色標示的最高度保守氨基酸殘基和以灰色標示的高度保守氨基酸殘基。圖6A、B和C顯示了AXMI-009(SEQIDNO:20)與crylAa(SEQIDNO:30)、crylAc(SEQIDNO:31)、crylCa(SEQIDNO:32)、crylla(SEQIDNO:33)、cry3Aal(SEQIDNO:35)、cry3Ba(SEQIDNO:36)、cry3Bb(SEQIDNO:37)、cry4Aa(SEQIDNO:38)、cry6Aa(SEQIDNO:40)、cry7Aa卿IDNO:41)、cry8Aa卿IDNO:42)、crylOAa(SEQIDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)、cry24Aa(SEQIDNO:47)、cry25Aa(SEQIDNO:48)和cry40Aal(SEQIDNO:51)的序列比對。具有C-末端非毒性結(jié)構(gòu)域的毒素如圖所示被人為截短。該序列比對顯示了以黑色標示的最高度保守氨基酸殘基和以灰色標示的高度保守氨基酸殘基。發(fā)現(xiàn)保守組1是從SEQIDNO:20的大約笫196位至大約第217位氨基酸殘基。發(fā)現(xiàn)保守組2是從SEQIDNO:20的大約第269位至大約第311位氨基酸殘基。發(fā)現(xiàn)保守組3是從SEQIDNO:20的大約第514位至大約第556位氨基酸殘基。發(fā)現(xiàn)保守組4是從SEQIDN0:20的大約第574位至大約584位氨基酸殘基。發(fā)現(xiàn)保守組5是從SEQIDNO:20的大約第651位至大約第661位氨基酸殘基。圖7A、B和C顯示了AXMI-014(SEQIDNO:27)與crylAa(SEQIDNO:30)、crylAc卿IDNO:31)、crylla(SEQIDNO:33)、cry2Aa(SEQIDNO:34)、cry3Aal(SEQIDNO:35)、cry3Bb(SEQIDNO:37)、cry4Aa(SEQIDNO:38)、cry4Ba(SEQIDNO:39)、cry6Aa(SEQIDNO:40)、cry7Aa(SEQIDNO:41)、cry8Aa(SEQIDNO:42)、crylOAa(SEQIDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)、cry24Aa(SEQIDNO:47)、cry25Aa(SEQIDNO:48)、cry39Aal(SEQIDN0:49)和cry錫al(SEQIDN0:51)的序列比對。具有C-末端非毒性結(jié)構(gòu)域的毒素如圖所示被人為截短。該序列比對顯示了以黑色標示的最高度保守氨基酸殘基和以灰色標示的高度保守氨基酸殘基。發(fā)現(xiàn)保守組1是從SEQIDN0:27的大約第177位至大約第188位氨基酸殘基。發(fā)現(xiàn)保守組2是從SEQIDN0:27的大約第251位至大約第293位氨基酸殘基。發(fā)現(xiàn)保守組3是從SEQIDNO:27的大約第483位至大約第533位氨基酸殘基。發(fā)現(xiàn)保守組4是從SEQIDNO:27的大約第552位至大約562位氨基酸殘基。圖8顯示了4-20%梯度SDS丙烯酰胺凝膠的照片。泳道1-4包括表達69kDAXMI-004蛋白質(zhì)的各種濃度的形成孢子的芽孢桿菌細胞培養(yǎng)物。泳道5-8包括各種濃度的BSA。泳道9包含分子量標記物。箭頭指示69kD的帶。發(fā)明詳述本發(fā)明針對的是用于調(diào)控生物體、尤其是植物或植物細胞中害蟲抗性的組合物和方法。所述方法包括用編碼本發(fā)明5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的核苷酸序列轉(zhuǎn)化生物體。具體而言，本發(fā)明的核苷酸序列可用于制備具有殺蟲活性的植物和微生物。因而提供了已轉(zhuǎn)化的細菌、植物、植物細胞、植物組織和種子。組合物是蘇云金芽孢桿菌的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)核酸和蛋白質(zhì)。所述序列可用于構(gòu)建表達載體用于隨后轉(zhuǎn)化入目的生物體內(nèi)，作為探針用于分離其它的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)基因，以及用本領(lǐng)域已知方法產(chǎn)生改變的殺蟲蛋白質(zhì)，諸如結(jié)構(gòu)域交換或DM改組。所述蛋白質(zhì)可用于抑制或殺死鱗翅類或鞘翅類有害種群以及用于產(chǎn)生具有殺蟲活性的組合物。定義"5-內(nèi)毒素"指來自蘇云金芽孢桿菌且對一種或多種害蟲具有毒性的毒素，所述害蟲包括、但不局限于鱗翅目、雙翅目和鞘翅目等的種類。在某些情況下，5-內(nèi)毒素蛋白質(zhì)已從其它生物體分離出來了，包括雙酶梭狀芽孢桿菌(C/os"/d/"/z76/尸e/7Z7e/^a/zs)和？類芽孢桿菌(戶ae"^ac27/^/o/7//7/ae)。5—內(nèi)毒素蛋白質(zhì)包含由本文所公布的全長核苷酸序列推導(dǎo)出來的氨基酸序列，以及由于使用備選的下游起始位點或者由于產(chǎn)生具有殺蟲活性的較短蛋白質(zhì)的加工所造成的比全長序列更短的氨基酸序列。加工可發(fā)生在所述蛋白質(zhì)所表達的生物體內(nèi)，或攝取所述蛋白質(zhì)后在害蟲體內(nèi)。5-內(nèi)毒素包括被確認為cryl至cry43、cytl和cyt2的蛋白質(zhì)以及Cyt-類毒素。目前有超過250種具有廣泛特異性和毒性的已知5-內(nèi)毒素。更全面的名單參閱Crickmore爭(1998)，#/cro6/o/,#。人齡.62:807-813，而定期的更新資料參閱畫.biols.susx.ac.uk/Home/Neil-Crickmore/Bt/index上的Crickmore爭(20(J3)"蘇云金芽孢桿菌毒素命名(丑ac/7/usf力uri/^/e/7s/stoxinnomenclature)"。細菌基因，諸如本發(fā)明的AXMI基因，通常具有多個甲硫氨酸起始密碼子接近開發(fā)閱讀框架的起始點。經(jīng)常地，開始于一個或多個這些起始密碼子的翻譯會導(dǎo)致產(chǎn)生功能性蛋白質(zhì)。這些起始密碼子可包括ATG密碼子。不過，諸如芽孢桿菌等的細菌還將密碼子GTG識別為起始密碼子，而翻譯起始于GTG密碼子的蛋白質(zhì)在笫一個氨基酸處含有曱硫氨酸。此外，并非常?？梢誀幭却_定這些密碼子中的哪一個在細菌中天然使用。因此，可以理解，利用這些變通性曱疏氨酸密碼子之一還可導(dǎo)致產(chǎn)生編碼殺蟲活性的5-內(nèi)毒素蛋白質(zhì)。例如，本發(fā)明AXMI-0045-內(nèi)毒素的一個備選起始位點是SEQIDNO:1第385位堿基對處。從此備選起始位點開始的翻譯產(chǎn)生了SEQIDNO:5中所顯示的氨基酸序列。本發(fā)明AXMI-0075-內(nèi)毒素蛋白質(zhì)的一個備選起始位點可以是SEQIDN0:8第151位堿基對處。從此備選起始位點開始的翻譯產(chǎn)生了SEQIDNO:11中所顯示的氨基酸序列。本發(fā)明的AXMI-0085-內(nèi)毒素蛋白質(zhì)的一個備選起始位點可以是SEQIDNO:12的第177位核苷酸處。從此備選起始位點開始的翻譯產(chǎn)生了SEQIDN0:16中所示的氨基酸序列。本發(fā)明的AXMI-0095-內(nèi)毒素蛋白質(zhì)的一個備選起始位點可以是SEQIDN0:19的第34位核苷酸處。從此備選起始位點開始的翻譯產(chǎn)生了SEQIDNO:22中所示的氨基酸序列。本發(fā)明的AXMI-0095-內(nèi)毒素蛋白質(zhì)的另一個備選起始位點可以是SEQIDNO:1的第64位核苷酸處。從此備選起始位點開始的翻譯產(chǎn)生了SEQIDNO:24中所示的氨基酸序列。本發(fā)明的AXMI-0145-內(nèi)毒素蛋白質(zhì)的一個備選起始位點可以是SEQIDN0:25的第136位核苷酸處。從此備選起始位點開始的翻譯產(chǎn)生了SEQIDNO:29中所示的氨基酸序列。這些5-內(nèi)毒素蛋白質(zhì)都包括在本發(fā)明的范圍內(nèi)，可用于本發(fā)明的方法中。此外，在所公布的編碼一個或多個5-內(nèi)毒素相關(guān)蛋白質(zhì)的核普酸序列中可以有一個或多個另外的開發(fā)閱讀框架。"5-內(nèi)毒素相關(guān)蛋白質(zhì)"指用不同于本發(fā)明5-內(nèi)毒素所用，使用另一開發(fā)閱讀框架由此處所公布的核苷酸序列編碼的蛋白質(zhì)。諸如此類的蛋白質(zhì)在本領(lǐng)域中已知可作為輔助蛋白質(zhì)、穩(wěn)定序列或5-內(nèi)毒素相關(guān)蛋白質(zhì)。這些5-內(nèi)毒素相關(guān)蛋白質(zhì)可具有殺蟲活性，或可在促進5-內(nèi)毒素蛋白質(zhì)表達中起重要作用。用于檢測殺蟲活性以及用于測定5-內(nèi)毒素相關(guān)蛋白質(zhì)對5-內(nèi)毒素蛋白質(zhì)表達和晶體形成的影響的方法是本領(lǐng)域已知的(參閱，例如，Park爭(1999)尸i^51V/c/"o6/。/.181:319-327;Ge爭(1998)層S165:35-41;Rosso和Delecluse(1997)j/7/7/.i"/2"'ro/7.63:4449—4455)。這些5—內(nèi)毒素相關(guān)蛋白質(zhì)包括在本發(fā)明范圍內(nèi)，并可單獨或與已知的5-內(nèi)毒素蛋白質(zhì)聯(lián)合用于本文所公布的方法中。在某一實施方案中，5-內(nèi)毒素相關(guān)蛋白質(zhì)具有存在于SEQIDNO:18中的氨基酸序列，由SEQIDNO:17的核苷酸序列編碼。"植物細胞"指所有已知形式的植物，包括未分化的組織(如愈傷組織)、懸浮培養(yǎng)細胞、原生質(zhì)體、葉細胞、根細胞、韌皮部細胞、植物種子、花粉、繁殖體、胚等等。"植物表達盒"指能導(dǎo)致蛋白質(zhì)在植物細胞中從開放閱讀框架表達的DNA構(gòu)建體。一般其中包含啟動子和編碼序列。經(jīng)常地，這樣的構(gòu)建體還包含3，非翻譯區(qū)。所述構(gòu)建體可包含"信號序列"或"前導(dǎo)序列"，以促進肽在翻譯的同時或翻譯后轉(zhuǎn)運至某些細胞內(nèi)結(jié)構(gòu)，諸如葉綠體(或其它質(zhì)體)、內(nèi)質(zhì)網(wǎng)或高爾基體。"信號序列"指已知或猜測會導(dǎo)致翻譯時或翻譯后的跨細胞膜肽轉(zhuǎn)運的序列。在真核細胞中，這通常包括分泌入高爾基體內(nèi)，具有某些由此產(chǎn)生的糖基化作用。"前導(dǎo)序列"指在翻譯時產(chǎn)生的氨基酸序列足以引起肽鏈共翻譯轉(zhuǎn)運至亞細胞器的任何序列。因此，這包括通過進入內(nèi)質(zhì)網(wǎng)、穿越液泡、包括葉綠體、線粒體等在內(nèi)的質(zhì)體而耙向轉(zhuǎn)運和/或糖基化作用的前導(dǎo)序列。"植物轉(zhuǎn)化栽體"指植物細胞有效轉(zhuǎn)化所必需的DM分子。這樣的分子可由一個或多個植物表達盒組成，且可組裝入一個以上的"載體"DNA分子中。例如，二元載體是利用兩個非毗連的DNA載體編碼(Hellens和Mullineaux(2000)7ye/7ffs//尸7fl/2f5We/7ce5:446-451)。"栽體"指設(shè)計成在不同宿主細胞之間轉(zhuǎn)移的核酸構(gòu)建體."表達載體"指能在外來細胞中摻入、整合并表達異源DNA序列或片段的載體。"轉(zhuǎn)基因植物"或"已轉(zhuǎn)化的植物"或"穩(wěn)定轉(zhuǎn)化的植物或細胞組織"指在植物細胞中已摻入或整合了外源核酸序列或DNA片段的植酸序列，以及可能是內(nèi)源的或存在于未轉(zhuǎn)化的植物細胞內(nèi)的那些核酸序列。"異源的"通常指對于它們所存在的細胞或部分天然基因組而言不是內(nèi)源性的，且已通過感染、轉(zhuǎn)染、顯微注射、電穿孔、顯微投景^(microprojection)等方式力口入細胞內(nèi)的核酸序歹'J。"啟動子"指起到指導(dǎo)下游編碼序列轉(zhuǎn)錄的功能的核酸序列。啟動子與其它轉(zhuǎn)錄和翻譯調(diào)節(jié)核酸序列(也稱"調(diào)控序列")一起是目的DM序列表達所必需的。本文提供了賦予殺蟲活性的新的分離核苷酸序列。還提供了5-內(nèi)毒素和5-內(nèi)毒素相關(guān)蛋白質(zhì)的氨基酸序列。由此基因翻譯所產(chǎn)生的蛋白質(zhì)可使細胞抑制或殺死攝取它的害蟲。"分離的"或"純化的"核酸分子或蛋白質(zhì)或者其生物學(xué)活性部分指的是，在用重組技術(shù)生產(chǎn)時基本上無其它細胞物質(zhì)或培養(yǎng)基，或者在化學(xué)合成時基本上無化學(xué)前體或其它化學(xué)物質(zhì)。優(yōu)選的，"分離核酸兩側(cè)(即，位于所述核酸5，和3，末端的序列)的序列(優(yōu)選蛋白質(zhì)編碼序列)。就本發(fā)明的目的而言，"分離的"用于描述核酸分子時，并不包括分離的染色體。例如，在各種實施方案中，分離的編碼5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白的核酸分子可包含天然存在于所述核酸來源的細胞的基因組DNA中該核酸分子兩側(cè)的小于大約5kb、4kb、3kb、2kb、1kb、0.5kb或0.1kb的核苷酸序列。基本上無細胞物質(zhì)的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)包括具有少于大約30%、20%、10%或5%(千重)的非5-內(nèi)毒素或非5-內(nèi)毒素相關(guān)蛋白質(zhì)(在此也稱為"污染蛋白質(zhì)")的蛋白質(zhì)制品。本發(fā)明的各個方面在以下部分有更進一步的詳述。分離的核酸分子及其變體和片段本發(fā)明的一方面是關(guān)于含有編碼5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)和多肽或其生物學(xué)活性部分的核苷酸序列的分離核酸分子，以及足以用作雜交探針來鑒定編碼5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的核酸分子。用于此處時，術(shù)語"核酸分子"意在包括DNA分子(如，cDNA或基因組DNA)和RNA分子(如，mRNA)和用核苷酸類似物產(chǎn)生的所述DNA或RNA的類似物。所述核酸分子可以是單鏈或雙鏈的，但優(yōu)選是雙鏈DNA。編碼本發(fā)明蛋白質(zhì)的核普酸序列包括SEQIDN0S:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26和28中所提出的序列及其互補序列。"互補序列"意指足以與給定核苷酸序列互補、從而可與該給定核苷酸序列雜交形成穩(wěn)定的雙聯(lián)體的核普酸序列。由這些核苷酸序列編碼的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的相應(yīng)氨基酸序列如SEQIDN0S:3、5、7、9、11、14、16、18、20、22、24、27和29中所示。作為這些5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)編碼核苷酸序列的片段的核酸分子也包括在本發(fā)明內(nèi)。"片段"指編碼5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的核苷酸序列的一部分。核苷酸序列的片段可編碼5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的生物學(xué)活性部分，或可能是可以用下文所述方法作為雜交探針或PCR引物的片段。作為5-內(nèi)毒素或5-內(nèi)毒素相關(guān)核苷酸序列的片段的核酸分子包含至少約15、20、50、75、100、200、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2100、2200、2300、2400、2500、3000、3500、4000、4500、5000、5500個核苷酸，或者多達本文所公布的全長5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)編碼核苷酸序列的核苷酸數(shù)目(例如，對于SEQIDNO:1而言是2190個核苷酸，對于SEQIDNO:2而言是1890個核苷酸，等等)，這將取決于目的用途。本發(fā)明的核苷酸序列的片段將編碼保留5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的生物學(xué)活性、從而分別保持了殺蟲活性或5-內(nèi)毒素相關(guān)蛋白質(zhì)活性的蛋白質(zhì)片段。"5-內(nèi)毒素活性"指殺蟲活性。"5-內(nèi)毒素相關(guān)蛋白質(zhì)活性"指所述蛋白質(zhì)具有殺蟲活性，或所述蛋白質(zhì)可提高5-內(nèi)毒素蛋白質(zhì)的表達。這種蛋白質(zhì)表達的提高可以以任何機制發(fā)生。"保持活性"指所述片段具有5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的活性的至少約30%、優(yōu)選至少約50%、更優(yōu)選至少約70%、還更優(yōu)選至少約80%。用于測定5-內(nèi)毒素相關(guān)蛋白質(zhì)對5-內(nèi)毒素蛋白質(zhì)表達和晶體形成的影響的方法是本領(lǐng)域所已知的(參閱，例如，Park爭(1999)尸^/y181:319-327;Ge爭(1998)#/cro6/o/.丄e〃.165:35-41;Rosso和Delecluse(1997)銣/.63:4449-4455)。用于測定殺蟲活性的方法在本領(lǐng)域內(nèi)是眾所周知的。參閱，例如，Czapla和Lang(1990)/.fco/.射畫/.83(6):2480-2485;Andrews爭(1988)腸c力e瓜/.252:199-206;Marrone爭(1985)/.o/*Aco/70/z/c^^o/z7o/o^f78:290-293;和美國專利5,743,477，所有這些都全文收編于此作為參考。編碼本發(fā)明蛋白質(zhì)生物學(xué)活性部分的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)編碼核苷酸序列片段將編碼本發(fā)明全長5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)中的至少約15、25、30、50、75、100、125、150、175、200、250、300、350、400、450、500、550、600或65。個連續(xù)的氨基酸，或最多可達其中所存在的總氨基酸數(shù)目(例如，對于SEQIDNO:3而言是629個氨基酸，對于SEQIDNO:5而言是601個氨基酸，等等)。優(yōu)選的本發(fā)明5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)是由與SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29中的核苷酸序列足夠一致的核苷酸序列編碼的。"足夠一致的"指用標準參數(shù)，通過本文所述序列比對程序之一與參考序列比較而言具有至少約60%或65%序列同一性、優(yōu)選至少約70%或75%序列同一性、更優(yōu)選至少約稀或85%序列同一性、最優(yōu)選至少約90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的氨基酸或核苷酸序列。本領(lǐng)域技術(shù)人員應(yīng)認識到，這些數(shù)值可適當(dāng)調(diào)整，以確定由于密碼子簡并性、氨基酸相似性、閱讀框架定位等原因而由兩種核苷酸序列編碼的蛋白質(zhì)的相應(yīng)同一性。為了確定兩種氨基酸序列或兩種核酸的同一性百分比，將這些序列進行比對以達到最佳的比較效果。兩個序列之間的同一性百分比是所述序列共有的相同位置數(shù)目的函數(shù)(即，同一性百分比=相同位置的數(shù)目/總位置數(shù)目(如重疊位置)x100)。在某一實施方案中，所述的兩個序列長度相同。兩個序列之間的同一性百分比可以用類似于下文所述方法的技術(shù)確定，允許或不允許有缺口。在計算同一性百分比時，通常嚴格匹配的才被計算在內(nèi)。兩個序列之間同一性百分比的確定可用數(shù)學(xué)運算法則完成。用于比較兩個序列的數(shù)學(xué)運算法則的非局限性例子是文獻Karlin和Altschul(1990)尸roc.#<2〃.87:2264中提出的運算法則，根據(jù)文獻Karlin和Altschul(1993)尸roc.Ms〃.Jcat/.5W.90:5873-5877進行修正。這樣的運算法則被合并入文獻Altschul爭(1990)/.215:403的BLASTN和BLASTX程序中。BLAST核苷酸搜索可用BLASTN軟件進行(得分=100，詞長=12)，以獲得與本發(fā)明5-內(nèi)毒素或5-內(nèi)毒素相關(guān)核酸分子同源的核苷酸序列。BLAST蛋白質(zhì)搜索可用BLASTN軟件進行(得分=50，詞長=3)，以獲得與本發(fā)明5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)分子同源的氨基酸序列。為了獲得帶缺口的序刊比對以進行比較，可如文獻Altschul爭(1997)腸/e/cJci&組25:3389中所述利用GappedBLAST?；蛘撸捎肞SI-Blast進行檢測兩個分子之間疏遠關(guān)聯(lián)的反復(fù)搜索。參閱，Altschul爭(1997),同上文。在應(yīng)用BLAST、GappedBLAST和PSI-Blast程序時，可使用各個程序(如，BLASTX和BLASTN)默認的參數(shù)。見麗.ncbi.nlm.nih.gov,另一用于比較序列的數(shù)學(xué)運算法則的非限制性例子是ClustalW運算法則(Higgins爭(1994)腸/e/"c油ies.22:4673-4680)。ClustalW可比較序列，并將氨基酸或DNA序列全部比對，因此可提供有關(guān)整個氨基酸序列的序列保守性的資料.ClustalW運算法則被用于數(shù)個商品化的DNA/氨基酸分析軟件包中，諸如栽體NTiProgramSuite(Informax，Inc)的ALIGNX才莫塊。用ClustalW進行氨基酸序列的比對后，可評估氨基酸同一性百分比?？捎糜贑lustalW序列比對分析的軟件程序的非局限性例子是GeneDocTM。GenedocTM(KarlNicholas)可評估多個蛋白質(zhì)之間的氨基酸(或DNA)相似性和同一性。用于序列比較的數(shù)學(xué)運算法則的另一非限制性例子是文獻Myers和Miller(1988)""i7^4:11-17中的運算法則。這樣的運算法則被引入ALIGN程序(版本2.0)中，它是GCG序列比對軟件包的一部分(可獲自Accelrys，Inc.,9865Sc訓(xùn)tonRd.，SanDiego,California,USA)。當(dāng)應(yīng)用ALIGN程序來比較氨基酸序列時，可使用PAM120加權(quán)殘基表、缺口長度罰分12和缺口罰分4。本發(fā)明還包括變體核酸分子。5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)編碼核苷酸序列的"變體"包括編碼本文所公布的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)、但由于遺傳密碼簡并性而有保守性差別的序列，以及如上所述足夠相似的那些序列。天然存在的等位基因變體可用眾所周知的分子生物學(xué)技術(shù)鑒定，諸如聚合酶鏈式反應(yīng)(PCR)和下文概述的雜交技術(shù)。變體核苷酸序列還包括合成來源的核普酸序列，例如如下所述通過定位誘變產(chǎn)生、但仍然編碼本發(fā)明公布的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的序列。包含在本發(fā)明范圍內(nèi)的變體蛋白質(zhì)是有生物學(xué)活性的，也就是說，它們繼續(xù)具有天然蛋白質(zhì)的目的生物學(xué)活性，即保持殺蟲活性。"保持活性"是指變體具有5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的活性的至少約30%、優(yōu)選至少約50%、更優(yōu)選至少約70%、還更優(yōu)選至少約80%。用于測量殺蟲活性的方法在本領(lǐng)域中是眾所周知的。參閱，例如，Czapla和Lang(1990)/.fco/7.f/^咖o/.83(6):2480-2485;Andrews爭(1988)"/oc/e瓜/252:199-206;Marrone爭(1985)/.fco/7咖/cf/^咖o/og/78:290-293;以及美國專利5，743，477，所有這些均全部收編于此作為參考。本發(fā)明還包括變體核酸分子。&-內(nèi)毒素或5-內(nèi)毒素相關(guān)編碼核苷酸序列的"變體"包括那些編碼本文所公布的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)、但由于遺傳密碼簡并性而有保守性差異的序列以及如上所述充分相同的那些序列。天然存在的等位基因變體可用眾所周知的分子生物學(xué)技術(shù)鑒定，諸如聚合酶鏈式反應(yīng)(PCR)和下述雜交技術(shù)。變體核苷酸序列還包括合成來源的核苷酸序列，例如如下所述通過定位誘變產(chǎn)生、但仍然編碼本發(fā)明公布的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的序列。技術(shù)熟練人員應(yīng)認識到，可通過突變在本發(fā)明的核苷酸序列中引入變異，從而導(dǎo)致所編碼的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的氨基酸序列中的變化，但不改變所述蛋白質(zhì)的生物學(xué)活性。因此，可通過在本^#會布的相應(yīng)核苷酸序列中引入一個或多個核苷酸取代、添加或缺失而制備變異的分離核酸分子，從而在所編碼的蛋白質(zhì)中引入一個或多個氨基酸取代、添加或缺失。突變可通過標準技術(shù)引入，諸如定位誘變和PCR介導(dǎo)誘變。這樣的變異核苷酸序列也包括在本發(fā)明的范圍內(nèi)。例如，優(yōu)選的，可以在一個或多個預(yù)知的、優(yōu)選非必需氨基酸殘基處進行保守氨基酸取代。"非必需的"氨基酸殘基是可以在5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)序列的野生型序列中發(fā)生改變、而不改變生物學(xué)活性的殘基，而"必需的"氨基酸殘基是生物學(xué)活性所必需的。"保守的氨基酸取代"是用具有相似側(cè)鏈的氨基酸殘基替換某一氨基酸殘基。具有相似側(cè)鏈的氨基酸殘基家族在本領(lǐng)域中已有定義。這些家族包括具有堿性側(cè)鏈的氨基酸(如，賴氨酸、精氨酸、組氨酸)、具有酸性側(cè)鏈的氨基酸(如，天冬氨酸、谷氨酸)、具有不帶電荷的極性側(cè)鏈的氨基酸(如，甘氨酸、天冬酰胺、谷氨酰胺、絲氨酸、蘇氨酸、酪氨酸、半胱氨酸)、具有非極性側(cè)鏈的氨基酸(如，丙氨酸、纈氨酸、亮氨酸、異亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有P-分支側(cè)鏈的氨基酸(如，蘇氨酸、纈氨酸、異亮氨酸)和具有芳香族側(cè)鏈的氨基酸(如，酪氨酸、苯丙氨酸、色氨酸、組氨酸)。在5-內(nèi)毒素蛋白質(zhì)之間通常有5個高度保守的區(qū)域，主要集中在結(jié)構(gòu)域的中心或結(jié)構(gòu)域之間的連接處(Rajamohan爭(1998)戶/"^A7/c/e/cJc/VM/.S/o/.60:1-23)。多種5-內(nèi)毒素的保守氨基酸塊以及共有序列可參閱以下文獻Schnepf爭(1998)Vo/.》io/.TeK.62:775-806和Lereclus爭(1989)，編碼蘇云金芽孢桿菌類芽孢5-內(nèi)毒素的基因的作用、結(jié)構(gòu)和分子組成(Role，Structure,andMolecularOrganizationoftheGenesCodingfortheParasporald-endotoxinsof^3c///t/stA￡yr//^7e/2s/s)。在"原核發(fā)育的調(diào)控(RegulationofProcaryoticDevelopment)"一書中，IssarSmit，Slepecky，R.A.，Setlow，P.美國孩i生物學(xué)會，Washington,D.C.20006。已提出具有這些保守區(qū)的5-內(nèi)毒素可共享相似的結(jié)構(gòu)，由三個結(jié)構(gòu)域組成(Li爭(199DA^^re353:815-821)。結(jié)構(gòu)域I在5-內(nèi)毒素之間具有最高的相似性(Bravo(1997)/.5sc"n'o/.179:2793—2801)?？稍诜潜Ｊ貐^(qū)內(nèi)進行保持活性的氨基酸取代。一般而言，不對保守氨基酸殘基、或定位在保守基元內(nèi)的氨基酸殘基進行這樣的取代，其中所述的殘基對于蛋白質(zhì)活性是必需的。保守的、并且可能為蛋白質(zhì)活性所必需的殘基例子包括，例如，在所提供的序列比對中含有的所有蛋白質(zhì)之間均相同的殘基。保守的、但可能允許發(fā)生保守性氨基酸取代而仍保持活性的殘基的例子包括例如在所提供的序列比對中所包括的所有蛋白質(zhì)之間僅有保守性取代的殘基。不過，本領(lǐng)域技術(shù)人員應(yīng)理解，功能性變體可在保守殘基中具有微小的保守或非保守變異?；蛘?，可通過諸如飽和誘變等方法沿著所有或部分編碼序列隨機引入突變而制備變異核苷酸序列，并可對所產(chǎn)生的突變體篩選賦予5-內(nèi)毒素或5-內(nèi)毒素相關(guān)活性的能力，以鑒別保持活性的突變體。誘變后，所編碼的蛋白質(zhì)可重組表達，而蛋白質(zhì)的活性可用標準檢測技術(shù)測定。用諸如PCR、雜交等方法可鑒定相應(yīng)的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)序列，這樣的序列與本發(fā)明的序列基本上相同。參閱，例如，SambrookJ.和Russell,D.W.(2001)分子克隆實驗室手冊M/"f/"rC7o/2i/^:JZa6orator7Va/zi/aA(冷，良港實驗室出版;土，冷，良港，)PCR流程方法和應(yīng)用指南戶^尸ro&coh;爿Cu2efo#<^力0&a/7t/Jp/7//c<3〃o"51(AcademicPress,NY)。在雜交方法中，可用全部或部分5-內(nèi)毒素或5-內(nèi)毒素相關(guān)核苷酸序列篩選cDNA或基因組文庫。所述cDM和基因組文庫的構(gòu)建方法是本領(lǐng)域普遍已知的，并被描述在Sambrook和Russell，2001中。所謂的雜交探針可以是基因組DNA片段、cDNA片段、RNA片段或其它寡核苷酸，它們可用諸如"P等可檢測基團或諸如其它放射性同位素、焚光化合物、酶或酶輔因子等任何其它可檢測標記物進行標記?？赏ㄟ^標記基于本文所公布的已知5-內(nèi)毒素或5-內(nèi)毒素相關(guān)編碼核苷酸序列的合成寡核苷酸制備用于雜交的探針。此外，還可使用基于核苷酸序列或所編碼的氨基酸序列中的保守核苷酸或氨基酸殘基設(shè)計的簡并引物。所述探針通常包含在嚴謹條件下與本發(fā)明5-內(nèi)毒素或5-內(nèi)毒素相關(guān)編碼核苷酸序列或其片段或變體的至少約12個、優(yōu)選約25個、更優(yōu)選至少約50、75、100、125、150、175、200、250、300、350或400個連續(xù)核苷酸雜交的核苷酸序列區(qū)。用于雜交的探針的制備是本領(lǐng)域所普遍已知的，并且公開在此處收編作為參考的文獻Sambrook和Russell，2001中。在雜交技術(shù)中，已知的核苷酸序列的全部或部分被用作與來自選定生物體的克隆基因組DNA片段或cDNA片段群(即，基因組或cDNA文庫)中存在的其它相應(yīng)核苷酸序列選擇性雜交的探針。雜交探針可以是基因組DM片段、cDNA片段、RM片段或其它寡核苷酸，且可用諸如"P等可檢測基團或任何其它可檢測標記物標記。因此，例如，用于雜交的探針可通過標記基于本發(fā)明的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)序列的合成寡核苷酸而制備。雜交探針的制備方法以及cDNA和基因組文庫的構(gòu)建方法是本領(lǐng)域通常已知的，并公開在Sambrook爭(1989)分子克隆實驗室手冊腺ecw/arC7o/7一"a6or"or/#2細/(第二版，冷泉港實驗室出版社，Plainview，紐約)中。例如，本文所公布的整個5-內(nèi)毒素或5-內(nèi)毒素相關(guān)序列或其一個或多個部分可用作能特異性地與相應(yīng)5-內(nèi)毒素或5-內(nèi)毒素相關(guān)樣序探針。為了實現(xiàn)各種條件下的特異雜交，所述探針包含獨特的序列，并且優(yōu)選長度為至少約IO個核苷酸、最優(yōu)選至少約20個核苷酸。這樣的探針可用于通過PCR從選定的生物體中擴增相應(yīng)的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)序列。這一技術(shù)可用于從目的生物體中分離額外的編碼序列，或作為診斷檢測法用于確定生物體中編碼序列的存在與否。雜交技術(shù)包括鋪板的DNA文庫(或者噬斑或者菌落，參閱，例如，Sarabrook爭(1989)分子克隆實驗室手冊(第二版，冷泉港實驗室出版社，Plainview，紐約))的雜交篩選。這些序列的雜交可在嚴謹條件下進行。"嚴謹條件"或"嚴謹?shù)碾s交條件"指，在此條件下，探針與其靶序列的雜交程度可檢測性地高于其它序列(例如，至少高于背景2倍)。嚴謹條件是隨序列而定的，在不同的環(huán)境下有所不同。通過控制雜交和/或漂洗條件的嚴謹性，可鑒別與探針100%互補的靶序列(同源探測)?；蛘?，可將嚴謹條件調(diào)整成允許序列中有一些錯配，從而檢測較低程度的相似性(異源探測)。通常，探針的長度小于約1000個核苷酸，優(yōu)選長度小于500個核苷酸。通常，嚴謹條件是這樣的條件，其中在pH7.0至8.3時鹽濃度小于約1.5MNa離子、通常約0.01至1.0MNa離子濃度(或其它鹽)，并且對于短探針(如10-50個核苷酸)而言溫度至少約30°C，而對于長探針(例如，大于50個核苷酸)而言至少約60°C。還可通過添加諸如甲酰胺等去穩(wěn)定劑來達到嚴謹條件。示范性的低嚴謹條件包括用30-35%甲酰胺、1MNaCl、1%SDS(十二烷基硫酸鈉)的緩沖液在37。C進行雜交，在IX至2XSSC(20XSSC=3.0MNaC1/0.3M檸檬酸三鈉)在50-55。C漂洗。示范性的中等嚴謹條件包括在40-45%甲酰胺、1.0MNaCl、1%SDS中于37°C雜交，并在0.5X至IXSSC于55-60。C漂洗。示范性的高嚴謹條件包括在50%甲酰胺、1MNaCl、1%SDS中于37°C進行雜交，并在0.IXSSC中于60-65°C漂洗。任選的，漂洗緩沖液中可包含約0.1%至約1°/。SDS。雜交的持續(xù)時間一般少于大約24小時，通常約為4至12小時。一般而言，特異性是雜交后漂洗條件的函數(shù)，關(guān)鍵因素是最終漂洗溶液的離子強度和溫度。對于DNA-DNA雜交，L可用Meinkoth等Wahl(1984)細/.扁c力e邁.138:267-284中的公式估計:Tra=81.50C+16.6(logM)+0.41(%GC)—0.61(%form)-500/L;其中的M是單價陽離子的摩爾濃度，％GC是DNA中鳥噤呤和胞嘧啶核苷酸的百分比，°/。form是雜交溶液中曱酰胺的百分比，而L是堿基對中雜交體的長度。L是互補靶序列的50%與完全匹配的探針雜交的溫度(在確定的離子強度和pH下)。對于每1%的錯配，L下降約1。C;因此，為了與所期望的同一性的序列雜交，可以調(diào)節(jié)乙、雜交和/或漂洗條件。例如，如果搜尋具有^90%同一性的序列，乙可下降10。C。通常，將嚴謹條件選擇成比該具體序列與其互補序列在確定的離子強度和pH的熱融點(L)低約5°C。不過，嚴格的嚴謹條件可利用比熱融點(Tm)低l、2、3或4°C的條件進行雜交和/或漂洗；中等的嚴謹條件可利用比熱融點(TJ低6、7、8、9或10°C的條件進行雜交和/或漂洗；低嚴謹條件可利用可利用比熱融點(TJ低11、12、13、14、15或20"C的條件進行雜交和/或漂洗。利用所述公式、雜交和漂洗組合物以及預(yù)期的乙，普通技術(shù)人員可理解雜交和/或漂洗溶液的嚴謹性的變化已得到了本質(zhì)的描述。如果所需程度的錯配導(dǎo)致L小于45°C(水溶液)或32。C(曱酰胺溶液)，則優(yōu)選提高SSC濃度，以利用較高的溫度。有關(guān)核酸雜交的詳盡指南可參閱文獻Tijssen(1993)生化和分子生物學(xué)實驗技術(shù)-用核酸探針進行雜交(Ls6or"or/7ecA/2/《"es//Jc/dZ^o6e",第I部分，第二章(Elsevier,紐約)；和Ausubel爭薦禪(1995)分子生物學(xué)通用方法(a/rreW尸r^oco/s//7#o/ecu/ar)，第二章(GreenePublishingandWiley-Interscience，NewYork)。參閱Sambrook爭(1989)分子克隆實驗室手冊(淑ecw/ar"a6or"c/r#雄")(第二版，冷泉港實驗室出版社，Plainview，NewYork)。分離蛋白質(zhì)及其變體和片段5-內(nèi)毒素和5-內(nèi)毒素相關(guān)蛋白質(zhì)也包含在本發(fā)明的范圍內(nèi)。"5-內(nèi)毒素蛋白質(zhì)"指具有SEQIDNO:3、5、7、9、11、14、16、20、22、24、27或29中所示氨基酸序列的蛋白質(zhì)。"5-內(nèi)毒素相關(guān)蛋白質(zhì)"指具有SEQIDNO:18中所示氨基酸序列的蛋白質(zhì)。同時還提供了它們的片段、生物學(xué)活性部分和變體，它們可用于實踐本發(fā)明的方法。"片段"或"生物學(xué)活性部分"包括含有編碼如SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29中所示5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的氨基酸序列的一部分且保持5-內(nèi)毒素活性或5一內(nèi)毒素相關(guān)活性的多肽片段。5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的生物學(xué)活性部分可以是例如10、25、50、IOO或更多個氨基酸長的多肽。這樣的生物學(xué)活性部分可通過重組技術(shù)制備，并評估其5-內(nèi)毒素或5-內(nèi)毒素相關(guān)活性。檢測殺蟲活性的方法是本領(lǐng)域眾所周知的。參閱，例如，Czapla和Lang(1990)/.腸/7.艇畫/.83(6):2480-2485;Andrews爭(1988)飾c力e瓜/.252:199-206;Marrone爭(1985)/.o,fco;30iZ7/cA/2&邊o/o^F78:290-293;以及美國專利5，743，477，所有這些均全部收編于此作為參考。用于此處時，片段含有SEQIDN0:3、5、7、9、11、14、16、18、20、22、24、27或29中的至少8個毗連氨基酸。不過，本發(fā)明還包含其它的片段，諸如該蛋白質(zhì)中的任何大于約10、20、30、50、100、150、200、250、300、350、400、450、500、550、600和650個氨基酸的片段。"變體"指具有與SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列至少約60°/。、65°/。、優(yōu)選至少約70%、75%、更優(yōu)選至少約80°/。、85%、最優(yōu)選至少約90%、91%、92%、93%、94%、95%、96%、97%、98°/4或99%相同的氨基酸序列的蛋白質(zhì)或多肽。變體還包括在嚴謹條件下與SEQIDNO:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28的核酸分子或其互補分子雜交的核酸分子編碼的多肽。這樣的變體通常保持5-內(nèi)毒素或5-內(nèi)毒素相關(guān)活性。變體包括由于誘變而氨基酸序列有所區(qū)別的多肽。本發(fā)明所包含的變體蛋白質(zhì)是有生物學(xué)活性的，即，它們繼續(xù)具有天然蛋白質(zhì)的所需生物學(xué)活性，即保持殺蟲活性。檢測殺蟲活性的方法是本領(lǐng)域眾所周知的。參閱，例如，Czapla和Lang(1990)/.fco幾iW咖o/.83(6):2480-2485;Andrews爭(1988)扁c力e邁./.252:199-206;Marrone爭(1985)/.o尸fcw2c邁/cj^M邁o7(^/78:290-293;以及美國專利5,743,477,所有這些均全部收編于此作為參考。改變或改良的變體已認識到可用各種方法改變5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的DNA序列，并且這些改變可能產(chǎn)生所編碼的蛋白質(zhì)氨基酸序列不同于本發(fā)明5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)氨基酸序列的DNA序列。這一蛋白質(zhì)可以多種方式改變，包括氨基酸取代、缺失、截短和插入。這些操作方法是本領(lǐng)域通常已知的。例如，5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的氨基酸序列變體可通過DNA中的突變來制備。這還可以通過數(shù)種誘變形式之一和/或定向進化來完成。在某些方面，氨基酸序列中編碼的改變基本上不會影響蛋白質(zhì)的功能。這樣的變體將具有所需的殺蟲活性。不過，應(yīng)理解5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)賦予殺蟲活性的能力可基于本發(fā)明組合物通過使用所述技術(shù)得以提高。例如，可以在于DNA復(fù)制期間呈現(xiàn)出高比率堿基錯誤摻入的宿主細胞中表達5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)，諸如XL-1Red(Stratagene)。在所述林系中增殖后，可分離5-內(nèi)毒素或5-內(nèi)毒素相關(guān)DM(例如通過制備質(zhì)粒，或通過PCR擴增并將產(chǎn)生的PCR片段克隆入載體中)，在非誘變性菌林中培養(yǎng)5-內(nèi)毒素或5-內(nèi)毒素相關(guān)突變，鑒定具有殺蟲活性的突變的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)基因，例如進行檢測來測試殺蟲活性。通常，將所述蛋白質(zhì)混和并用于喂服試驗中。參閱，例如，Marrone爭(1985)/.o尸fco/20邁yc^7^/zzo/c^/78:290-293。這樣的試驗可包括使植物接觸一種或多種害蟲并測定植物存活的能力和/或引起害蟲死亡的能力。導(dǎo)致毒性增強的突變的例子參閱Schnepf爭(1998)#/cro6/o/."/'o人yer.62:775-806。或者，可在氛基或羧基末端對許多蛋白質(zhì)的序列進行改變而基本上不影響其活性。這可包括利用現(xiàn)代分子方法引入的插入、刪除或改變，諸如PCR,包括由于在PCR擴增所用的寡核苷酸中含有氨基酸編碼序列而改變或延長蛋白質(zhì)編碼序列的PCR擴增?；蛘?，所加入的蛋白質(zhì)序列可包括完整的蛋白質(zhì)編碼序列，諸如那些在本領(lǐng)域中通常用于產(chǎn)生蛋白質(zhì)融合體的序列。這樣的融合蛋白質(zhì)常常用于(1)增加目的蛋白質(zhì)的表達，(2)引入結(jié)合結(jié)構(gòu)域、酶促活性或表位以方便蛋白質(zhì)純化、蛋白質(zhì)檢測或本領(lǐng)域已知的其它實驗性應(yīng)用，(3)使蛋白質(zhì)的分泌或翻譯定向于亞細胞性細胞器，諸如革蘭氏陰性細菌的周質(zhì)空間，或真核細胞的內(nèi)質(zhì)網(wǎng)，后者常常導(dǎo)致蛋白質(zhì)的糖基化。本發(fā)明的變體核苷酸和氨基酸序列還包括來自諸如DNA改組等誘變和重組方法的序列。通過這樣的步驟，可利用一個或多個不同的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)編碼區(qū)構(gòu)建具有所需特性的新的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)。以此方式，由含有具有充分的序列同一性且可體外或體內(nèi)同源重組的序列區(qū)的相關(guān)序列多核苷酸群產(chǎn)生重組多核苷酸文庫。例如，用此方法，可在本發(fā)明的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)基因與其它已知5-內(nèi)毒素或5-內(nèi)毒素相關(guān)基因之間對編碼目的結(jié)構(gòu)域的序列基元進行改組，以獲得編碼具有改良目的特性的蛋白質(zhì)的新基因，諸如提高殺蟲活性。所述的DM改組策略是本領(lǐng)域所已知的。參閱，例如，Stemmer(1994)戶roc.Jcad.Sc/.^S^91:10747-10751;Ste咖er(1994)Y"i/re370:389-391;Crameri爭(1997)5/由c//.15:436-438;Moore爭(1997)/.Wo/.272:336-347;Zhapg夢(7W7J戶roc.^caA^SVi94:4504-4509;Crameri爭(1998)iV"ure391:288-291;以及美國專利5，605，793和5，837，458。結(jié)構(gòu)域交換或改組是產(chǎn)生改變的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的另一機制。結(jié)構(gòu)域II和III可在5-內(nèi)毒素蛋白質(zhì)之間進行交換，產(chǎn)生具有改良的殺蟲活性或目標譜的雜合或嵌合毒素。產(chǎn)生重組蛋白質(zhì)并檢測其殺蟲活性的方法在本領(lǐng)域中是眾所周知的(參閱，例如，Naimov爭(200D銣/.紐/r肌Wcro6/o入67:5328-5330;deMaagd爭(1996)f脂'層.飾roWo厶62:1537-1543;Ge爭(1991)/.C力e瓜266:17954-17958;Schnepf爭(1990j/.A/o人C力e邁.265:20923-20930;Rang爭91999)^2"'r0".#/cr/oWo/.65:2918-2925)。植物轉(zhuǎn)化可通過本領(lǐng)域已知的數(shù)種技術(shù)之一完成植物細胞的轉(zhuǎn)化。首先，以允許其在植物細胞中表達的方式改造5-內(nèi)毒素或5-內(nèi)毒素相關(guān)基因。通常表達這種蛋白質(zhì)的構(gòu)建體應(yīng)含有驅(qū)使基因轉(zhuǎn)錄的啟動子以及允許轉(zhuǎn)錄終止和聚腺苷酸化的3，非翻譯區(qū)。這樣的構(gòu)建體的組成在本領(lǐng)域中是眾所周知的。在某些情況下，可有效改造基因，使得產(chǎn)生的肽被分泌出來，或定向于植物細胞中。例如，可改造基因，使其含有信號肽，以方便所述的肽轉(zhuǎn)移至內(nèi)質(zhì)網(wǎng)。還可能優(yōu)選的是，改造植物表達盒使其含有內(nèi)含子，從而使內(nèi)含子的mRNA加工為表達所必需。通常這一"植物表達盒"將被插入"植物轉(zhuǎn)化載體"中。該植物轉(zhuǎn)化栽體可由完成植物轉(zhuǎn)化所需的一個或多個DNA載體組成。例如，本領(lǐng)域的通用慣例是利用由一個以上毗連DNA區(qū)段組成的植物轉(zhuǎn)化載體。這些載體在本領(lǐng)域中常常稱為"二元載體"。二元栽體以及具有輔助質(zhì)粒的載體是最常用于農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化中的，其中完成有效轉(zhuǎn)化所需的DNA區(qū)段的大小和復(fù)雜性是相當(dāng)大的，并且將功能分攤到分開的DNA分子上比較有利。二元載體通常包含含有T-DM轉(zhuǎn)移所必需的順式作用序列(諸如左邊界和右邊界)、經(jīng)改造能在植物細胞中表達的可選擇標記物以及"目的基因"(經(jīng)改造能在期望產(chǎn)生轉(zhuǎn)基因植物的植物細胞中表達的基因)的質(zhì)粒載體。還存在于此質(zhì)粒載體中的是細菌復(fù)制所必需的序列。順式作用序列以可有效轉(zhuǎn)移入植物細胞并在其中表達的形式安排。例如，可選擇標記基因和目的基因定位于左和右邊界之間。常常有另一個質(zhì)粒載體包含介導(dǎo)T-DM從農(nóng)桿菌轉(zhuǎn)移至植物細胞的的反式作用因子。按照本領(lǐng)域中所理解的，此質(zhì)粒常常具有使植物細胞可被農(nóng)桿菌感染并通過在邊界序列處的斷裂以及病毒介導(dǎo)的DNA轉(zhuǎn)移來轉(zhuǎn)移DNA的致病作用(Vir基因)(Hellens和Mullineaux(2000)7>e/](/s/刀尸/a力tSc/e刀ce，5:446—451)。數(shù)種類型的農(nóng)桿菌菌林(如LBA4404、GV3101、EHAIOI、EHA105等)可用于植物轉(zhuǎn)化。該第二種質(zhì)粒載體不是用諸如顯微投影、顯微注射、電穿孔、聚乙二醇等其它方法轉(zhuǎn)化植物所必需的。一般而言，植物轉(zhuǎn)化方法包括將異源DNA轉(zhuǎn)移入靶植物細胞(如，不成熟或成熟的胚、懸浮培養(yǎng)物、未分化的愈傷組織、原生質(zhì)體，等等)中，隨后進行最大極限水平的適當(dāng)選擇(取決于可選擇標記基因)，以從未轉(zhuǎn)化細胞集合組中回收轉(zhuǎn)化的植物細胞。通常將外植體轉(zhuǎn)移至新鮮置換的同種培養(yǎng)基中并進行常規(guī)培養(yǎng)。隨后，在放置于補充了最后將所述的芽轉(zhuǎn)移到選擇性i根4養(yǎng)l中以回收生根的芽或苗。然i將轉(zhuǎn)基因苗培養(yǎng)成成熟的植物并產(chǎn)生能繁殖的種子(如Hiei爭(1994)T7e7Vai3//o"r加/6:271-282;Ishida爭(1996)^"wre"/Wec/wo7o^r14:745-750)。通常將外植體轉(zhuǎn)移至新鮮補充的同種培養(yǎng)基中并進行常規(guī)培養(yǎng)。產(chǎn)生轉(zhuǎn)基因幼苗的有關(guān)技術(shù)和方法的全面描述參閱文獻Ayres和Park，1994(C"7/ca//e"譜//7尸/fl;2fSc/e腦13:219-239)以及Bommineni和Jauhar，19970Va7力'ca42:107-120)。由于被轉(zhuǎn)化的物質(zhì)包含許多細胞，轉(zhuǎn)化和未轉(zhuǎn)化的細胞都存在于任何受試靶愈傷片或組織或細胞組中。殺死未轉(zhuǎn)化細胞并允許轉(zhuǎn)化細胞增殖的能力使被轉(zhuǎn)化的植物培養(yǎng)物得以生長。通常，去除未轉(zhuǎn)化細胞的能力是對快速回收被轉(zhuǎn)化植物細胞并成功產(chǎn)生轉(zhuǎn)基因植物的限制。轉(zhuǎn)基因植物的產(chǎn)生可用數(shù)種方法之一完成，包括但不局限于通過農(nóng)桿菌將異源DNA引入植物細胞中(農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化)、用粘附于顆粒上的外源性外來DNA轟擊植物細胞以及各種其它非粒子定向介導(dǎo)的方法(如Hiei爭(1994)r力e戶/a^/owr/za/6:271-282;Ishida爭(1996)A"f/reAi"ec力/Jauhar(1997)Va/cTica42:107-120)轉(zhuǎn)移DNA。轉(zhuǎn)化方法以及將核苷酸序列引入植物的方法可能隨著待轉(zhuǎn)化的植物或植物細胞的類型(即單子葉或雙子葉植物)而變化。將核苷酸(Crossway爭(1986)S/Wec/w/^/es4:320—334)、電穿孔(Riggs爭(1986)尸roc.」cad.Sc厶83:5602-5606)、農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化(美國專利5,563,055;美國專利5,981,840)、直接基因轉(zhuǎn)移(Paszkowski爭(1984)灘O/,3:2717-2722)和彈道顆粒加速(參閱,例如，美國專利4，945，050;美國專利5，879，918;美國專利5，886，244;美國專利5,932,782;Tomes爭(1995)"通過顯微投射轟擊將DNA定向轉(zhuǎn)移入完整的植物細胞中(Direct腿TransferintoIntactPlantCellsviaMicroprojectileBombardment)"，在"植物細胞、組織和器官培養(yǎng)基本方法(尸/a/7fCe//，77^we,Orga/iOz〃d/re;尸w3^2/pe/2fa7Vef力od^)"一"^'哞，Gamborg和Phillips編禪(Springer—Verlag，Berlin);McCabe爭(1988)A/ofec力/2o7og/6:923-926)、氣溶膠束轉(zhuǎn)化(美國已出版專利申請20010026941;美國專利4,945,050;國際專利申請WO91/00915;美國已出版專利申請2002015066)以及Lecl轉(zhuǎn)化(WO00/28058)。也可參閱Weissinger爭(1988)細.紐6^/7"22:421-477;Sanford爭(1987)*戎j(戶a/^/ci;/afeSciencerec/wo/og/)5:27—37;Christou爭(1988)尸/aWM/s/oA87:671-674;McCabe爭(1988)萬/o/Tec//7o/0g/6:923—926;Finer和McMullen(1991)ZnP7troCe//Per."/o人27P:175-182;Singh爭(1998)r力eor.Jp;/.96:319-324(大豆)；Datta爭(1990)"/otec力/70/0^78:736-740;Klein爭(1988)尸roc.iVa〃.Jcacf.Sc/.〃W85:4305-4309;美國專利5,240,855;美國專利5，322，783和5，324，646;Tomes等柳""通過微粒轟擊將DNA定向轉(zhuǎn)移入完整的植物細胞中(DirectDNATransferintoIntactPlantCellsviaMicroprojec出eBombardment)"，在"植物細胞、組織和器官培養(yǎng)基本方法(/Va射*，薦襌Gamborg和Phillips(Springer-Verlag，Berlin);Klein爭(1988)戶/a"f尸//"'o厶91:440-444;Hooykaas—VanSlogteren爭(1984)^""re"o/^oW311:763-764;美國專利5，736,369;ByteMer爭(1987)細c.咖84:5345-5349(Liliaceae);DeWet爭(1985)在"胚珠組織的實驗操作(7TeAjr77er/邁e77"/#fl/7//7i//a〃0"o尸6Vw/e77ss"es)"—書中，編輯Chapman爭(Longman,NewYork),笫197-209頁；Kaeppler爭(1990)植物細胞報告戶/a/^Ce//9:415-418和Kaeppler爭(1992)7力eor.場/.84:560-566;D'Halluin爭(1992)4:1495-1505;Li爭(1993)戶/aWCe/77e/7or〃12:250-255以及Christou和Ford(1995)^■/so尸淑<3//75:407-413;0sjoda爭(1996)艇i/reA/ofec/wo/o^F14:745-750，所有這些均收編于此作為參考。在將外源性外來DNA整合入植物細胞中后，在培養(yǎng)基中進行最大極限水平的適當(dāng)選擇，以殺死未轉(zhuǎn)化的細胞，并通過定期轉(zhuǎn)移至新鮮培養(yǎng)基中而分離和增殖在此選擇處理中存活的據(jù)推定已轉(zhuǎn)化的細胞。通過連續(xù)傳代并進行適當(dāng)?shù)倪x擇，可識別并增殖被所述質(zhì)粒載體轉(zhuǎn)化的細胞。然后用分子和生化方法證實轉(zhuǎn)基因植物基因組中被整合的異源目的基因的存在。已4皮轉(zhuǎn)化的細胞可以傳統(tǒng)方式生長成植物。參閱，例如McCormick爭(1986)P7a;^Ce/7;e;or〃5:81-84。然后可培養(yǎng)這些植物，用同樣的被轉(zhuǎn)化抹系或不同的林系授粉，并鑒定具有所需表型特征的組成型表達的所得雜合體?？膳囵B(yǎng)兩代或多代，以保證所需表型特征的表達穩(wěn)定維持和遺傳，然后收獲種子以保證已實現(xiàn)了所需表型特征的表達。以此方式，本發(fā)明提供了在其基因組中穩(wěn)定摻入了本發(fā)明的核苷酸構(gòu)建體、例如本發(fā)明的表達盒的已轉(zhuǎn)化種子(也稱為"轉(zhuǎn)基因種子")。本發(fā)明的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)序列可存在于用于在目的植物中表達的表達盒中。所述的盒將包括可操作性的與本發(fā)明序列連接的5'和3'調(diào)控序列。"可操作性的連接"指在啟動子和第二序列之間的功能性聯(lián)接，其中的啟動子序列起始并介導(dǎo)了與第二序列相應(yīng)的DNA序列的轉(zhuǎn)錄。通常，可操作性連接意味著被連接的核酸序列是毗鄰的，并且，在需連接兩個蛋白質(zhì)編碼區(qū)時，則是毗鄰且處于同一閱讀框架中。所述的盒可另外含有至少一個其它基因以共轉(zhuǎn)化入生物體中?；蛘?，所述的其它基因可存在于多個表達盒中。向所述的表達盒中提供多個限制性位點，以供插入5-內(nèi)毒素或5-內(nèi)毒素相關(guān)序列，處于調(diào)控區(qū)的轉(zhuǎn)錄調(diào)控下。所述的表達盒沿5'-3'轉(zhuǎn)錄方向?qū)谥参镏邪l(fā)揮作用的轉(zhuǎn)錄和翻譯起始區(qū)(即，啟動子)、本發(fā)明的DNA序列以及轉(zhuǎn)錄和翻譯終止區(qū)(即終止區(qū))。啟動子對于植物宿主和/或本發(fā)明的DNA序列而言可能是天然的或類似的、或者外源的或異源的。此外，啟動子可以是天然序列或可以是合成序列。啟動子對于植物宿主而言是"天然的"或"同源的"，指的是啟動子在該啟動子將被引入的天然植物中存在。啟動子對于本發(fā)明DNA序列而言是"外源的"或"異源的"，指的是啟動子對于可操作性連接的本發(fā)明DNA序列而言不是天然或自然存在的啟動子。終止區(qū)可以是對于轉(zhuǎn)錄起始區(qū)而言是天然的，可以是就可操作性連接的目的DNA序列而言是天然的，可以是就植物宿主而言是天然的，或者可以來自其它來源(即，對于啟動子、目的DNA序列、植物宿主或它們的任何組合而言是外源的或異源的)。方便的終止區(qū)可從#癌農(nóng)桿菌的Ti-質(zhì)粒獲得，諸如章魚堿合酶和胭脂堿合酶終止區(qū)。還可參閱Guerineau爭(1991)Ge/.(e刀".262:141-144;Proudfoot(1991)6W/64:671-674;Sanfacon爭(1991)Ce総紐5:141-149;Mogen爭(1990)尸/aflfCe//2:1261-1272;Munroe爭(1990)Ge/e91:151-158;Ballas爭(1989)^c/e/cJc油紐17:7891-7903;以及Joshi爭(1987)腸/e/.cJc/"es.15:9627-9639。在適當(dāng)?shù)那闆r下，可將基因最優(yōu)化以提高其在被轉(zhuǎn)化宿主細胞中的表達。即，所述基因可以用宿主細胞偏好的密碼子合成以促進表達，或者可以宿主偏好的密碼子使用頻率用密碼子合成。例如，有關(guān)宿主偏好的密碼子使用的討論參閱Campbell和Gowri(1990)尸/a;^尸力7^'o7.92:1-11。合成植物偏好基因的方法是本領(lǐng)域所已知的。參閱，例如，美國專利6，320，100、6，075，185、5，380，831和5，436，391，美國已出版專利申請20040005600和20010003849，以及Murray爭(1989)A^/c/e/c^c/"/e義17:477-498，在此收編作為參考。在一個實施方案中，目的核酸被定向于葉綠體中表達。以此方式，當(dāng)目的核酸不是直接插入葉綠體的情況下，表達盒將另外含有編碼轉(zhuǎn)運肽的核酸以指導(dǎo)目的基因產(chǎn)物進入葉綠體。這樣的轉(zhuǎn)運肽在本領(lǐng)域中是已知的。參閱，例如，VonHeijne爭(199D/Vai^Vo/.飾/.雖9:104-126;Clark爭(1989)/.W/'o/.C力e瓜264:17544-17550;Della-Cioppa爭(1987)戶/aW腳Wo/.84:965-968;Romer爭(1993)A/oc力e邁."/o/7力/義Co/zww/7.196:1414—1421;k乂及Shah爭(1986)5We/ce233:478-481。用于葉綠體轉(zhuǎn)化的方法在本領(lǐng)域中是已知的。參閱，例如，Svab爭(1990)戶roc.射/.JcacT.5W.〃W87:8526-8530;Svab和Maliga(1993)尸roc.艇/.JcacT.<SW.蹈90:913-917;Svab和Maliga(1993)W仿O/.12:601-606。所述方法依賴于含可選擇標記的DNA的粒子槍投遞以及通過同源重組將DNA定向于質(zhì)體基因組。此外，質(zhì)體轉(zhuǎn)化可通過用核編碼及質(zhì)體指導(dǎo)的RNA聚合酶的組織優(yōu)選表達而反式激活沉默的質(zhì)體攜帶的轉(zhuǎn)基因而完成。這樣的體系已被報道于文獻McBride爭(1994)iVis".91:7301-7305中?？蓛?yōu)化定向于葉綠體的目的核酸以表達于葉綠體中，從而解決植物細胞核和此細胞器之間的密碼子使用中的差異。以此方式，目的核酸可以用葉綠體偏好的密碼子合成。參閱，例如，美國專利5，380,831,在此收編作為參考。植物轉(zhuǎn)化的評估將異源性的外來DNA引入植物細胞中后，用各種方法證實植物基因組中異源基因的轉(zhuǎn)化或整合，諸如與被整合基因相關(guān)的核酸、蛋白質(zhì)和代謝物的分析。PCR分析PCR分析是在移植至土壤中之前的較早階段篩選已轉(zhuǎn)化的細胞、組織或芽是否存在被摻入基因的快速方法(Sambrook和Russell，2001)。PCR是用特異于目的基因或農(nóng)桿菌栽體背景等的寡核苷酸引物進行的。DNA雜交分析植物轉(zhuǎn)化通過基因組DNA的DNA印跡分析證實(Sambrook和Russell,2001)。通常，從轉(zhuǎn)化林中提取總DNA，用適當(dāng)?shù)南拗泼赶?，在瓊脂糖凝膠中進行分級分離并轉(zhuǎn)移至硝酸纖維素或尼龍膜上。然后依照標準技術(shù)，用例如放射性標記了32P的耙DNA片段探測所述的膜或"印跡"，以證實被導(dǎo)入基因在植物基因組中的整合(Sambrook和Russell,2001.分子克隆實驗室手冊#o/eci//arC7o72//7^vJL260rafor/Va/7i/a/.冷7良港實驗室出版^土，冷7良港，紐約)。RNA雜交分析依照本領(lǐng)域中常規(guī)使用的標準方法，從轉(zhuǎn)化林的特異組織中分離RNA，在甲醛瓊脂糖凝膠中分級分離，印跡至尼龍濾膜上(Sambrook，J.和Russell,D.W.2001.分子克隆實驗室手冊#o/ecw/ar6V0/z/i2^vjL360rafor/#a/7i/<37.冷7良港實驗室出版it，冷泉港，紐約)。然后用本領(lǐng)域已知方法(Sambrook和Russell,2001),通過將所述濾膜與來自5-內(nèi)毒素或S-內(nèi)毒素相關(guān)蛋白質(zhì)的放射性探針雜交而測試5-內(nèi)毒素或與5-內(nèi)毒素相關(guān)基因編碼的RNA的表達。蛋白質(zhì)印跡和生化檢測可通過標準步驟(Sambrook,J.和Russell,D.W.2001.分子克隆實驗室手冊Vo/ecu/drC7oj7/;^:船/n/a/.冷泉港實驗室出版社，冷泉港，紐約)，利用結(jié)合5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)上存在的一個或多個表位的抗體對轉(zhuǎn)基因植物進行蛋白質(zhì)印跡和生化檢測及類似測定，以證實由5-內(nèi)毒素或5-內(nèi)毒素相關(guān)基因編碼的蛋白質(zhì)的存在。植物中的殺蟲活性在本發(fā)明的另一方面，可產(chǎn)生表達具有殺蟲活性的5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的轉(zhuǎn)基因植物。可利用上述舉例說明的方法產(chǎn)生轉(zhuǎn)基因植物，但產(chǎn)生轉(zhuǎn)基因植物細胞的方式對本發(fā)明而言不是關(guān)鍵的?？衫弥T如農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化、氣溶膠束(aerosolbeam)、biolisUc轉(zhuǎn)化及非粒子介導(dǎo)方法等本領(lǐng)域中已知或描述的方法來進行，具體所用方法依實驗者而定?？捎帽绢I(lǐng)域中描述的通用方法分離表達5-內(nèi)毒素或5-內(nèi)毒素相關(guān)蛋白質(zhì)的植物，例如，通過轉(zhuǎn)化愈傷組織、篩選被轉(zhuǎn)化的愈傷組織以及從所述的轉(zhuǎn)基因愈傷組織再生出可育植物。在所述過程中，可利用任何基因作為可選擇標記，只要通過它在植物細胞中的表達能識別或選擇已被轉(zhuǎn)化的細胞即可。已開發(fā)了許多與植物細胞一起使用的標記物，諸如對氯霉素、氨基糖苷類G418、潮霉素等等的抗性。編碼葉綠體代謝中所涉及的產(chǎn)物的其它基因也可用作選擇標記。例如，能提供對諸如草甘膦、溴苯腈(bromoxynil)或口米峻淋酉同(imidazolinone)等植物除草劑的抗'f生的基因可能有具體用途。這樣的基因已有報道(Stalker爭(1985)/.萬/o人C力e瓜263:6310-6314(溴苯腈抗性腈水解酶基因)；以及Sathasivan爭(199(J)^uc/.」c/'^ies.18:2188(AHAS咪唑啉酮抗性基因)。可對表達5-內(nèi)毒素或S-內(nèi)毒素相關(guān)蛋白質(zhì)的可育植物測試殺蟲活性，并選擇顯示出最佳活性的植物進行進一步的培育。本領(lǐng)域中有可供利用的方法能夠用于檢測對害蟲的活性。通常，所述蛋白質(zhì)被混合并用于喂服試驗中。參閱，例如，Marrone爭(1985)/.o尸fco/70邁/c78:290—293。殺蟲控制中的應(yīng)用般方法是本領(lǐng)域所已知的。參閱，例如，美國專利5,039,523和EP0480762A2。本發(fā)明的芽孢桿菌林系或經(jīng)遺傳改造而含有殺蟲基因和蛋白質(zhì)的微生物可用于保護農(nóng)作品和產(chǎn)品免受害蟲的侵襲。在本發(fā)明的一個方面，當(dāng)將產(chǎn)生毒素的細胞施用于有目標害蟲的環(huán)境中時，用延長所述細胞中產(chǎn)生的毒素的活性的試劑處理生產(chǎn)毒素(殺蟲劑)的生物體的完整(即未裂解)細胞?；蛘?，通過將異源基因引入細胞宿主而產(chǎn)生殺蟲劑。異源基因的表達直接或間接的導(dǎo)致了殺蟲劑在細胞內(nèi)的產(chǎn)生和保持。在本發(fā)明的一個方面，當(dāng)將所述細胞施用于有目標害蟲的環(huán)境中時，在延長該細胞所產(chǎn)生的毒素的活性的條件下處理這些細胞。所產(chǎn)生的產(chǎn)物保持了毒素的毒性。然后可依照常規(guī)技術(shù)配制這些自然封裝的殺蟲劑，以便施用于含有目標害蟲的環(huán)境中，如，土壤、水和植物的葉子。參閱，例如EPA0192319以及其中引用的文獻。或者，可對表達本發(fā)明基因的細胞進行配制，以便可將所產(chǎn)生的物質(zhì)作為殺蟲劑施用。本發(fā)明的活性成分通常以組合物的形式施用，并可與其它化合物同時或接連地施用于待處理的農(nóng)作物地區(qū)或植物。這些化合物可以是肥料、除草劑、防凍劑、表面活性劑、去垢劑、殺蟲皂、休眠油、聚合體和/或在單次施用制劑后可長期對目標區(qū)域進行給藥的隨時間釋放型或可生物降解型載體制劑。它們也可以是選擇性除草劑、化學(xué)殺蟲劑、殺病毒劑、殺微生物劑、殺變形蟲劑、殺蟲劑、殺真菌劑、殺菌劑、殺線蟲劑、殺軟體動物劑或數(shù)種這些制劑的混合物，如果需要，可進一步與農(nóng)業(yè)可接受載體、表面活性劑或制備領(lǐng)域中常規(guī)采用的促進應(yīng)用的佐劑一起施用。合適的載體和佐劑可以是固體或液體，并與制備工藝中通常應(yīng)用的物質(zhì)相對應(yīng)，如，天然或再生的礦物、溶劑、分散劑、增濕劑、粘附劑、粘合劑或肥料。同樣的，所述制劑可制備成可食用的"餌"或作成害蟲"陷阱"，使得可被殺蟲制劑的目標害蟲所飼用或攝取。應(yīng)用本發(fā)明活性成分或含本發(fā)明細菌菌林所產(chǎn)生的殺蟲性蛋白質(zhì)中至少一種的本發(fā)明農(nóng)用化學(xué)品組合物的優(yōu)選方法是葉片施用、種子包裹和土壤施用。施用次數(shù)和施用頻率取決于相應(yīng)害蟲的侵襲強度。所述組合物可制備成粉劑、塵劑、丸劑、顆粒劑、噴霧劑、乳劑、膠體、溶液等等，且可用諸如含所述多肽的細胞培養(yǎng)物的干燥、凍干、勻漿、提取、過濾、離心、沉降和濃縮等常規(guī)方式制備。在所有含至少一種所述殺蟲多肽的所述組合物中，所述多肽可以重量約1%-約99%的濃度存在。利用本發(fā)明方法可殺死鱗翅類或鞘翅類害蟲，或者減少其在特定區(qū)域內(nèi)的數(shù)目，或者可預(yù)防性的施用于周圍區(qū)域以防止易感害蟲的蔓延。優(yōu)選的，所述害蟲攝取或接觸殺蟲有效量的所述多肽。"殺蟲有效量"指能導(dǎo)致至少一種害蟲死亡或能顯著降低害蟲的生長、伺用或正常生理發(fā)育的殺蟲劑用量。這個量將取決于各種因素而變化，諸如待控制的具體目標害蟲；待處理的特異環(huán)境、位置、植物、農(nóng)作物或農(nóng)業(yè)場所、環(huán)境條件，以及該殺蟲有效性多肽組合物施用的方法、速率、濃度、穩(wěn)定性和用量。制劑還可隨氣候條件、環(huán)境因素和/或施用頻率和/或害蟲侵襲的嚴重程度等而發(fā)生變化。所述的殺蟲劑組合物可通過制備細菌細胞、晶體和/或孢子懸浮液或者與所需的農(nóng)業(yè)可接受栽體有組合的分離蛋白質(zhì)組分制成。所述組合物可在施用前配制成適當(dāng)?shù)男问?，諸如凍千、冷凍干燥、脫水或在水性載體、培養(yǎng)基或諸如鹽水或其它緩沖液等適當(dāng)?shù)娜軇┲?。制備好的組合物可以是塵土或顆粒物形式，或者油(植物油或礦物油)中的懸浮液，或者水或油/水乳劑，或作為可濕粉末，或者與適于農(nóng)業(yè)應(yīng)用的任何其它載體物質(zhì)相組合。適當(dāng)?shù)霓r(nóng)業(yè)載體可以是固體或液體，在本領(lǐng)域中是眾所周知的。術(shù)語"農(nóng)業(yè)可接受載體"涵蓋了通常應(yīng)用于殺蟲劑制備工藝中的所有佐劑、惰性成分、分散劑、表面活性劑、粘著劑、粘合劑等，這些是殺蟲劑制備業(yè)中的技術(shù)人員眾所周知的。所述制劑可與一種或多種固體或液體佐劑混合，并通過各種方式制備，例如，用常規(guī)的制備工藝將殺蟲組合物與適當(dāng)?shù)淖魟┚鶆蚧旌稀⒒旌秃?或碾磨。合適的制劑和應(yīng)用方法參閱美國專利6,468,523，該文件在此收編作為參考。"害蟲"包括但不局限于，昆蟲、真菌、細菌、線蟲、螨、蜱等等。昆蟲類的害蟲包括選自以下目的昆蟲鞘翅目、雙翅目、膜翅目、鱗翅目、食毛目、同翅目、半翅目、直翅目、纓翅目、革翅目、等翅目、虱目、蚤目、毛翅目等，尤其是鞘翅目、鱗翅目和雙翅目。昆蟲類害蟲包括選自以下目的昆蟲鞘翅目、雙翅目、膜翅目、鱗翅目、食毛目、同翅目、半翅目、直翅目、纓翅目、革翅目、等翅目、虱目、蚤目、毛翅目等，尤其是鞘翅目和鱗翅目。針對主要農(nóng)作物的本發(fā)明昆蟲類害蟲包括玉米&"http://7/3/2"6//a//s，歐洲玉米螟；y4^ro〃s2'/7s/7o/，小地虎；〃e/2'coFer/7flzea，谷實夜蛾；S/7odo/^e/^/Vt/^7/7e油，草地夜蛾；^ra""/^"",巨座玉米螟(西南玉米桿草螟)；fy^/z7，//^705^//ws，南美玉米苗斑螟(小玉米螟)；"/"raeasaccAara//51，小蔗桿草螟；Z/a67"o〃car/r^7/"ers，玉米(幼芽)根葉曱；"/fl6ro〃ca/an^7cori7/s6ar6er/，長角葉甲；"/<a6r<9〃caW2f/ec/'邁/7w/i""flAowrd/，黃瓜十一星葉曱；spp.，才危爪口卩頭蟲；Cyc/ocep力s/a6orea//s，；i匕方圓頭厚金龜(圓頭獨角仙)；C/c/oce;力a/a/邊邁acw7a"，南方圓頭犀金龜(圓頭無斑獨角仙)；尸o/^7/is力;7o/j/ca，弧麗鲅(日本麗金龜)；C力aefoci7e邁ar/a，玉米(銅色)跳曱；5^力e/30/7力o/"s邁a2/，玉米隱咮象(玉米谷象)；^opa7os/;7力wz7鵬yf/2、玉米坊(玉米縊管辨)；J/i/ra/7力/s邁aWrad/c2、玉米根辨；"http://^i/s/ewco/^erf/s/ewco/7feri/s，麥長蝽(美洲谷長蝽)；#e/fl/zo/7/f/s尸e邁wrrw6r咖，赤脛黑蝗(赤腿鲊蜢)；#e/a/op/M^/2^///2//es，血黑蝗(遷徙蚱蜢)；外/e邁"/7/"wrs，(玉米)種錄'，^ro邁yzfl/7ar7/cor/7/s，玉米斑潛錄；J/a/7力o^/7;^o6sci7/ri/s，玉米黃呆薊馬(美;川維尾薊馬)；iSb/e/70/7s/s/p//e"a，竊蚊；7Wr<3/zyc/wsi/r〃cae，二斑葉螨(普通紅葉螨)；高粱C///o/w"e/風(fēng)玉米禾螟；5^oc^era/Vwg/per^s,草地夜蛾；〃e//core_rpazea，谷實夜蛾；A/as邁o/a/;7f/51//^720se/7z^，南美玉米苗斑螟(小玉米螟)；尸e/〃aSi/We"a/7ea，粒膚地虎；尸/7//0/7力<3^2cr/"27a，蜂螬(金龜幼蟲)；^7eocTes,寬胸口卩甲(C畫cTe價)和細/mspp.，金針蟲(叩曱幼蟲)；邁e/a/o;us，黑角負泥蟲(橙足負泥蟲)；C/aefoc/e邁apw/2'car2'a，玉米(銅色)刃t曱；5^Ae/o;力or:^邁fl/cT2、玉米隱味象(玉米谷象)；i力o/7a/os/;7力w7/邁a/d/s，玉米坊(玉米纟益管辨)；57/力a/Vara,美甘蔑偽毛辨；"7/'"i/s7eyco/^erws7ewco/^e/^，麥長棒(美洲谷長蝽)；Co/7&r/i3/aso/^力/co/a，高粱癭蚊；re"a/2/c//^c/w/zahar/zzys,紅葉蝶(朱/^,葉竭)，refra/7/c/u^ur^2.cae，葉竭(普通紅葉螨)；小麥sewc^s7e〃ai/"/，""a，粘蟲；5"/7odo/^era/Vw^7/ercfa，草地夜蛾；^fi7as/z/o/7a/;7ws//g/70se/7iAy，南美玉米苗斑螟(小玉米螟)；^ro〃VoH力^w7/a，西方灰地老虎；f/a57Z70/a/;7W5"http://^■/o<ye//w<y，南美玉米苗斑螟；Ou/e則邁e/a/70/ws，黑角負泥蟲(橙足負泥蟲)；^rperaj3mc""，三葉草葉象(車軸草葉象)；Z/a6ro〃cafy/7￡/ec//z7pi//7""a力(？附rd/，黃瓜十一星葉甲；俄國麥長管坊；i"c力/zap力/s^Ta邁/;3w邁，麥二盆坊；Macros//7力m7are加e，麥長管蚜；〃e/a/3o;7/f^/e邁i/rrz/6r"邁，赤脛黑蟲皇(赤腿蚱蛭)；Ve7a/7o/7/w(///Tere/2〃a/2:，異黑蟲皇(殊種蚱蛭)；^e/a/o//i^■sa/zgu/zij'pes,血,繁、蟲皇(遷徙坤樣)；//^yet/o/acfestrz;cfor，小麥瘦墳；57fod/;7/os/s邁ose/7a/7a，麥紅吸裝蟲；^/isrc>/z7_rzaa辺er/ca/3a，美洲桿蠅(美洲麥桿黃潛蠅)；i^7e/z^acoarc""，麥癭種蠅(冬作種蠅)；Fra/i7//7/e//fl/7^cfl，煙褐花薊馬(煙草褐薊馬)；Ce/力^c/'/z"i/s，麥莖蜂；Jcer/a^//;7ae，麥癃螨(郁金香瘤螨)；向曰葵5^/e/邊f(xié)l力e/i'fl/2fAa/za，向日葵芽ii蟲；〃o邁oeoso/na向日葵(同)斑螟；^F^0gra邁iz7ae;rc/a邁a〃on/s，向日葵葉甲；g/66o薦，胡蘿卜利犀金龜(胡蘿卜金龜)；腸/as/一era/nwrf/Wi/〃fl/7fl，向日葵籽癀蚊；棉花〃e7/c^力hF/resce/2s，煙芽夜蛾；〃e7/ccyei77azea，棉鈴蟲；5^odo/^eraei/^/a,甜菜夜蛾；尸ect/z70/7力oragoss/;2'e/7a，紅鈴麥域(棉紅鈴蟲)；爿/f力o/2o邁wsgr2/3c^,墨西哥棉鈴象(甲)；爿;力/sgos，//，棉坊；尸se:/f/afo邁osce7/sser/aff/s，棉序盲蜂(棉逸匕盲蜂)；rr/a/ei/rodes由〃/畫a，苘麻粉虱(結(jié)翅粉IU;Z/戸/2'/2eo/ar/、(美國)牧草盲蜂；Ve/a/20//M/e/z7i/rrz/6r"邁，赤脛黑嫂(赤腿蚱蛭)；^/a卿/Md/〃erey7〃a/2、異黑蟲皇(殊種蚱蛭)；7Tr/;7S"6aci'，煙薊馬(蔥薊馬)；/^2/1///^2'6//3/""a，煙褐花薊馬(煙草褐薊馬)；refra刀/c力w51c/z7/s6arj'/7ws，紅葉竭(朱^1^、葉竭)；ref/"a/7/c力w51z;r〃cae，二斑葉螨(普通紅葉螨)；稻"/"raeasaccAara/M小蔑軒草螺；5^0do;7fera/Vug/pe/Y/a,草地夜域；〃e//coye_r/7a么ea，沖帛鈴蟲；Co/as//s6rwinea，葡萄肖葉甲；Z/ssor力o/^rwsor7zop力/7w51，稻才艮象；57^0/7力//"<or/zae，米象；Ae;7力c^eft/jri/^ro/7/cff/s，,、尾葉蜂；"http://mws/ewco;^erws/ei/co/7fe/^，麥長蝽(美洲谷長蝽)；Jcro"ei7咖力27are，擬緣椿(喜綠棒)；大豆/^ewdo/7/i/Wa//7c/i/de/^，大豆(尺)夜蛾；J/7〃csr".age鵬afa//51，梨豆夜蛾；尸/af力7Pe/7asca6ra，苜著綠夜域；6^t_r//7/a/7i/627a/2，歐夕川玉米螟；J^ro〃s0/2，小地虎；Spoffo/^eraeu'gf;a，甜菜夜蛾；y/i"esce/s，煙芽夜蛾；〃e//coFer/>azea，棉鈴蟲'，^/7//ac力/M7ar/resf/s，墨、西哥(大)豆孤蟲；/7ers/cae，書匕對；fzz7/oa"a尸a6ae，蠶豆微葉蟬；Jcro"e/72咖力/7are，擬緣蜂(喜綠蝽)；#e/a/2op/i/s/e/z/wrri;6r叫赤脛黑蟲皇(赤腿蚱蛭)；t^e7a/o/7wsf/i//ere/7〃a7i、異黑蝗(殊種蛑蜢)；//fl"ra，(玉米)種繩；Ayer2'cof/r//^rar2's6///s，大豆牧草蟲；7!ftr//751fa6acy，煙薊馬(蔥薊馬)；re"s;/c/"s"rires"i7/，土耳其葉螨(草莓葉螨)；r"ra/z/c力^wr〃cae，二斑葉螨(普通紅葉螨)；大麥0s"/zz/a/7u6//a//s，歐、,川玉米螺；J^rof/s/;7s//an，'J、地虎；ScA/zap力/s^Ta邊/i3"/zz，麥二盆坊；A/2'^w51/ewco/^erws/ewco/7ferus，麥長棒(美洲谷長蝽)；Jcro"eiV2咖A/7are，擬緣捲(喜綠棒)；^;sc力/"i/51ser^/51，煙草漆(褐臭蝽)；/e//a/77"i/ra，(灰地)種蠅；艙7e〃0/af/e"ru"or，小麥癭蚊；戶e"o6/a/"e/s，麥巖螨；油籽油菜Are"'cor//7e6ras"'cae，甘藍椅(菜奸)；"http:///c^refa"f;c/尸erae，十字花跳菜曱；船zz/es"ac^7/7^y/""a,蓓帶夜蛾(披肩夜蛾)；尸/"e7/s^x/o"e"a，菜蛾；Ze〃assp.，地種竭(花錄)。線蟲類包括寄生性線蟲，例如根節(jié)、胚嚢和損害線蟲，包括異皮線蟲(#e"refersspp.)、根結(jié)線蟲(^e/o油，espp.)和球異皮線蟲(W^w/eraspp.)，具體而言是胚嚢線蟲類，包括但不局限于，ero^/erag/jc/aes(大豆異皮線蟲)、Vefero(/erasc力ac力〃/(甜菜胞嚢線蟲)、射erc^era<s,ae(燕麥胞嚢線蟲)和67o耐eraro"oc力/e脂、(馬鈴薯金線蟲)以及蒼白球異皮線蟲(67由dera/7a/7/da)。4員害線蟲包括短軒線蟲(Praf/Ze^c/"-spp.)。以下實施例僅供例證說明而非起限制作用。實施例實施例1:質(zhì)粒DNA的抽提以如下方式由菌株ATX13026或ATX13002制備質(zhì)粒DNA。在大量的豐富培養(yǎng)基中培養(yǎng)菌抹ATX13026或ATX13002的純培養(yǎng)物。將培養(yǎng)液離心以收獲細胞沉淀塊。然后以本領(lǐng)域已知方法用SDS處理細胞沉淀塊，導(dǎo)致細胞壁破損且DNA釋放。然后通過高鹽濃度沉淀蛋白質(zhì)和大基因組DNA。通過標準乙醇沉淀來沉淀質(zhì)粒DM。通過在氯化銫梯度中的高速離心將質(zhì)粒DNA與任何剩余的染色體DM分開。使用UV光顯現(xiàn)梯度中的DNA，并使用注射器吸取較低密度條帶(即較低條帶)。此條帶含有來自菌林(或是ATX13026或是ATX13002)的質(zhì)粒DM。利用本領(lǐng)域已知方法通過在瓊脂糖凝膠上的顯影來檢查DNA的質(zhì)量。實施例2:基因的克隆正如本領(lǐng)域已知的，將純化的質(zhì)粒DNA剪切成5-10kb大小的片段，并且在存在所有四種dNTP的條件下使用T4DNA聚合酶和Klenow片段修補5'和3'單鏈突出。然后，如本領(lǐng)域已知的，通過T4多核普酸激酶處理而在5'末端添加磷酸根。如本領(lǐng)域已知的，將修復(fù)的DNA片段過夜連接到適當(dāng)制備以接受插入片段(例如通過限制酶消化而生成平端)的標準高拷貝載體(即pBluescriptSK+)中。通過用已知在克隆位點側(cè)翼具有切割位點的限制酶消化克隆亞群來分析文庫的質(zhì)量。確定了高百分比的克隆含有插入片段，平均長度5-6kb。實施例3:文庫平板的高通量測序?qū)⑼ㄟ^上述方法制備的文庫涂布在含有適當(dāng)抗生素的豐富培養(yǎng)基上以維持質(zhì)?？寺?，并將菌落單獨挑到裝有2ml含有適當(dāng)抗生素的培養(yǎng)基的96孔板中。將這些板于37。C、以350rpm搖速培養(yǎng)過夜。將板離心而將細胞收集到板底。通過標準的堿裂解法以高通量方式由這些培養(yǎng)物分離質(zhì)粒DNA。以如下方式對來自每塊板的大量克隆測定來自該文庫的克隆的末端序列。4吏用熒光染料終止物測序技術(shù)(AppliedBiosystems)和位于克隆位點每一側(cè)側(cè)翼的標準引物測定選擇用于分析的每個克隆的DNA序列。一旦反應(yīng)在熱循環(huán)儀中完成，則4吏用標準乙醇沉淀法來沉淀DNA。將DNA重懸于水，并加載到毛細管測序儀上。由克隆位點的任一側(cè)測定每個文庫平板的DM,每個平板的每個插入片段產(chǎn)生兩個閱讀結(jié)果。實施例4:測序數(shù)據(jù)的裝配和篩選將得到的DNA序列匯編成裝配方案，并排列在一起以形成毗連序列群。這可以使用電腦程序有效進行，諸如VectorNTi,或者使用DNA比對和分析程序的Pred/Phrap套餐。將這些毗連序列群連同未能添加到毗連序列群中的任何個別閱讀序列與所有類型的已知殺蟲劑基因的匯編數(shù)據(jù)庫進行比較。進一步分析鑒定為與已知內(nèi)毒素或殺蟲劑基因具有同一性的毗連序列群或個別閱讀序列。在得到的序列中，克隆pAX004、pAX006、pAX007、pAX008、pAX009和pAX014含有鑒定為與已知內(nèi)毒素基因具有同源性的DNA。因此，選擇這些克隆用于進一步測序。實施例5:5-內(nèi)毒素基因的測序和鑒定設(shè)計與內(nèi)毒素基因具有同源性的序列退火的引物，使得由這些引物產(chǎn)生的DNA序列將與克隆中的現(xiàn)有DNA序列交疊。這一稱為"寡聚物行走"的方法在本領(lǐng)域是眾所周知的。該方法被用于測定顯示與已知內(nèi)毒素基因有同源性的區(qū)域的完整DNA序列。在上文所述克隆的情況中，該方法被用于測定整個克隆的DNA序列，從而得到每個基因的單一核苷酸序列。然后將完整DNA序列放回最初的大裝配結(jié)果中以進一步確認。這能夠?qū)⒏嗟腄NA序列閱讀結(jié)果插入毗連序列群，產(chǎn)生覆蓋整個區(qū)域的多個閱讀結(jié)果。對與已知內(nèi)毒素基因同源的每個區(qū)域進行的DNA序列分析鑒定得到了與已知5-內(nèi)毒素基因同源的開放讀碼框。將由pAX004鑒定得到的開放讀碼框命名為AXMI-004。將由pAX006鑒定得到的開放讀碼框命名為AXMI-006。將由pAX007鑒定得到的開放讀碼框命名為AXMI-007。將由pAX008鑒定得到的開放讀碼框命名為AXMI-008。將由pAX009鑒定得到的開放讀碼框命名為AXMI-009。將由pAX014鑒定得到的開放讀碼框命名為AXMI-014。SEQIDNOS:1和2提供了AXMI-004的DM序列，而SEQIDN0:3提供了預(yù)測的AMXI-004蛋白質(zhì)的氨基酸序列。位于SEQIDN0:1笫385位核苷酸處的AXMI-004備選起始位點產(chǎn)生了SEQIDNO:5中提供的氨基酸序列。SEQIDNOS:6提供了AXMI-006的DNA序列，而SEQIDNO:7提供了預(yù)測的AMXI-006蛋白質(zhì)的氨基酸序列，SEQIDN0:8提供了AXMI-007的DM序列，而SEQIDNO:9提供了預(yù)測的AMXI-007蛋白質(zhì)的氨基酸序列。位于SEQIDNO:8第151位核苷酸處的AXMI-007備選起始位點產(chǎn)生了SEQIDNO:11中提供的氨基酸序列。SEQIDNOS:12和13提供了AXMI-008的DNA序列，而SEQIDNO:M提供了預(yù)測的AMXI-008蛋白質(zhì)的氨基酸序列。位于SEQIDNO:12笫177位核苷酸處的AXMI-008備選起始位點產(chǎn)生了SEQIDN0:16提供中的氨基酸序列。進一步的分析鑒定得到了緊臨AXMI-008開放讀碼框3'末端的開放讀碼框。SEQIDNO:18中提供了在本文中稱為AXMI-008orf2的該開放讀碼框的這一預(yù)測的氨基酸序列。SEQIDNOS:19中提供了AXMI-009的DNA序列，而SEQIDNO:20中提供了預(yù)測的AMXI-009蛋白質(zhì)的氨基酸序列。位于SEQIDNO:19笫34位核苷酸處的AXMI-009備選起始位點產(chǎn)生了SEQIDNO:22中提供的氨基酸序列。位于SEQIDNO:19第64位核苷酸處的另一個AXMI-009備選起始位點產(chǎn)生了SEQIDNO:24中提供的氨基酸序列。SEQIDNOS:25和26中提供了AXMI-014的DM序列，而SEQIDNO:27提供了預(yù)測的AMXI-008蛋白質(zhì)的氨基酸序列。位于SEQIDNO:25第136位核普酸處的AXMI-014備選起始位點產(chǎn)生了SEQIDNO:29中提供的氨基酸序列。實施例6:分離基因與已知內(nèi)毒素基因的同源性用本發(fā)明的DNA序列和氨基酸序列搜索DM和蛋白質(zhì)數(shù)據(jù)庫揭示出了這些序列與已知內(nèi)毒素同源。圖1顯示了AXMI-004與幾種內(nèi)毒素的序列比對。Blast搜索確定了crylCa具有最強的同源區(qū)，在毒性結(jié)構(gòu)域的整體序列同一性為43%(見表1)。表1.AXMI-004與示范性內(nèi)毒素類型的氨基酸同一性<table>tableseeoriginaldocumentpage159</column></row><table>AXMI-006圖2顯示了AXMI-006與幾種內(nèi)毒素的序列比對。Blast搜索確定了cry4Aa具有最強的同源區(qū)，盡管AMXI-006蛋白質(zhì)(SEQIDNO:7)與一大組內(nèi)毒素蛋白質(zhì)的序列比對顯示最同源的蛋白質(zhì)是crylOAa。crylOAa與AXMI-006的整體氨基酸同一性是25%(見表2)。對AXMI-006氨基酸序列的檢查提示它不含幾種內(nèi)毒素家族中存在的C-末端非毒性結(jié)構(gòu)域。由序列比對中除去毒素的這個C-末端蛋白質(zhì)后，序列比對反映了氨基酸同一性只存在于毒素結(jié)構(gòu)域中(見表2，第3列)。圖2顯示了這一"修剪過的"序列比對。表2.AXMI-006與示范性內(nèi)毒素類型的氨基酸同一性<table>tableseeoriginaldocumentpage46</column></row><table>AXMI-007Blast搜索確定了c/r^a具有與AXMI-007最強的同源區(qū)，盡管AMXI-007蛋白質(zhì)(SEQIDNO:9)與一大組內(nèi)毒素蛋白質(zhì)的序列比對顯示最同源的蛋白質(zhì)是c/r7Wa。cir7Wa與AXMI-007的整體氨基酸同一性是25%(見表3)。對AXMI-007氨基酸序列的檢查提示它不含幾種內(nèi)毒素家族中存在的C-末端非毒性結(jié)構(gòu)域。由序列比對中除去毒素的這個C-末端蛋白質(zhì)后，序列比對反映了氨基酸同一性只存在于毒素結(jié)構(gòu)域中(見表3，第3列)。圖3顯示了這一"修剪過的"序列比對。表3.AXMI-007與示范性內(nèi)毒素類型的氨基酸同一性<table>tableseeoriginaldocumentpage47</column></row><table>Blast搜索確定了CiyW^具有與AXMI-008最強的同源區(qū)，而且AMXI-008蛋白質(zhì)(SEQIDNO:14)與一大組內(nèi)毒素蛋白質(zhì)的序列比對顯示最同源的蛋白質(zhì)是cry40Aa。crj^Wa與AXMI-008的整體氨基酸同一性是66%(見表4)。對AXMI-008氨基酸序列的檢查提示它不含幾種內(nèi)毒素家族中存在的C-末端非毒性結(jié)構(gòu)域。由序列比對中除去毒素的這個C-末端蛋白質(zhì)后，序列比對反映了氨基酸同一性只存在于毒素結(jié)構(gòu)域中(見表4,第3列)。圖4顯示了這一"修剪過的"序列比對。表4.AXMI-008與示范性內(nèi)毒素類型的氨基酸同一性<table>tableseeoriginaldocumentpage48</column></row><table>緊臨AXMI-008編碼區(qū)下游(3')的開放讀碼框與已知內(nèi)毒素相關(guān)蛋白質(zhì)具有同源性。Blast搜索確定了c/y6w^r/7(crj^^a的下游開放讀碼框)具有最強的同源區(qū)。數(shù)據(jù)庫中存在幾種其它orf-2樣蛋白質(zhì)，而且圖5顯示了AMXI-008-orf2蛋白質(zhì)(SEQIDNO:18)與這些蛋白質(zhì)中的一組的序列比對。這些蛋白質(zhì)還與cir^a和cr/MaC-末端非毒性結(jié)構(gòu)域享有同源性。AXMI-8-orf2與CTr^^aor/7的整體氨基酸同一性是86%(見表5)。表5.AXMI-008-orf2與相關(guān)蛋白質(zhì)的氨基酸同一性<table>tableseeoriginaldocumentpage49</column></row><table>Blast搜索確定了cryBAa具有與AXMI-009最強的同源區(qū)，而且AMXI-009蛋白質(zhì)(SEQIDN0:20)與一大組內(nèi)毒素蛋白質(zhì)的序列比對顯示最同源的蛋白質(zhì)是cry3Ba和cryl6Aa。cry3Ba和cryl6Aa與AXMI-009的整體氨基酸同一性是26%(見表6)。對AXMI-009氨基酸序列的檢查提示它不含幾種內(nèi)毒素家族中存在的C-末端非毒性結(jié)構(gòu)域。由序列比對中除去毒素的這個C-末端蛋白質(zhì)后，序列比對反映了氨基酸同一性只存在于毒素結(jié)構(gòu)域中(見表6，笫3列)。圖6顯示了這一"修剪過的"序列比對。表6.AXMI-009與示范性內(nèi)毒素類型的氨基酸同一性<table>tableseeoriginaldocumentpage50</column></row><table>Blast搜索確定了cr7^Ma具有與AXMI-014最強的同源區(qū)，而且AMXI-0014蛋白質(zhì)(SEQIDNO:27)與一大組內(nèi)毒素蛋白質(zhì)的序列比對顯示最同源的蛋白質(zhì)是cry40Aa。crr^4a與AXMI-014的整體氨基酸同一性是55%(見表7)。對AXMI-014氨基酸序列的檢查提示它不含幾種內(nèi)毒素家族中存在的C-末端非毒性結(jié)構(gòu)域。由序列比對中除去毒素的這個C-末端蛋白質(zhì)后，序列比對反映了氨基酸同一性只存在于毒素結(jié)構(gòu)域中(見表7，第3列)。圖7顯示了這一"修剪過的"序列比對。表7.AXMI-014與示范性內(nèi)毒素類型的氨基酸同一性<table>tableseeoriginaldocumentpage51</column></row><table>實施例7:AXMI-006與AXMI-007之間的同源性對氨基酸序列AXMI-007與AXMI-006的比較顯示了這兩種毒素共享顯著的氨基酸同源性。氨基酸序列AXMI-006(SEQIDNO:7)和AXMI-007(SEQIDNO:9)的比對顯示了這兩種蛋白質(zhì)在氨基酸水平上85%相同。因此，AXMI-006和AXMI-007組成一類新的相關(guān)內(nèi)毒素。實施例8:殺蟲活性的測定法常常以多種方式評估殺蟲劑蛋白質(zhì)作為殺蟲劑作用于害蟲的能力。本領(lǐng)域眾所周知的一種方法是進行喂食測定法。在這種喂食測定法中，將害蟲暴露于含有待測化合物的樣品或?qū)φ諛悠?。通常，這是通過將待測物質(zhì)或其合適稀釋物置于害蟲將要攝食的物質(zhì)諸如人工詞料上來進行的。待測物質(zhì)可以由液體、固體或漿液構(gòu)成?？梢詫⒋郎y物質(zhì)置于表面之上，然后使其干燥。或者，可以將待測物質(zhì)與熔化的人工飼料混和，然后分裝到測定槽中。測定槽可以是例如杯、盤或微量滴定板的孔。針對吸食害蟲(例如坊蟲)的測定法可以包括使用隔離物將測試物質(zhì)與昆蟲分開，理想的是吸食昆蟲的吸食口器能夠刺穿的物體，從而能夠攝食測試物質(zhì)。通常，將測試物質(zhì)與喂食刺激物諸如蔗糖混和以促進測試化合物的攝食。其它類型的測定法可以包括將測試物質(zhì)顯微注射到害蟲的口或腸中，以及開發(fā)轉(zhuǎn)基因植物，隨后測試害蟲以轉(zhuǎn)基因植物為食的能力。植物測試可以包括分離通常取食的植物部分，例如將小籠附于葉片上，或者在裝有昆蟲的籠中分離整個植抹。本領(lǐng)域還知道測定害蟲的其它方法和途徑，可以在如下文獻中找到Robertson,J.L.和H.LPreisler，1992，《4吏用節(jié)肢動物的殺蟲齊寸生物領(lǐng)1定法》(尸e"j'6/oassa/sw'MartAro/70cfs),CRC，伯克萊屯，佛羅里達州?；蛘?，測定法一般描述于如下期刊中"ArthropodManagementTests"和"JournalofEconomicEntomology"，或者是與美國昆蟲學(xué)學(xué)會(EntomologicalSocietyofAmerica,ESA)成員的討論。實施例9:克隆AXMI-006用于蛋白質(zhì)表達如下將AXMI-006克隆到用于大腸桿菌表達的載體中。pAX480含有用于選擇轉(zhuǎn)化子的卡那霉素抗性基因，以及可通過IPTG誘導(dǎo)來調(diào)控蛋白質(zhì)表達的"c啟動子。通過將編碼"His標簽區(qū)的DNA片段緊臨著插入插入片段克隆區(qū)的上游來修飾pAX480，使得得到的克隆在所表達的蛋白質(zhì)的N端含有6xHis標簽。本領(lǐng)域眾所周知表達含有6xHis-使用尸尸"^"raTM高保真DNA聚合酶(Stratagene)PCR擴增AXMI-006的編碼序列。設(shè)計寡核苷酸引物，使得得到的PCR產(chǎn)物在兩個末端附近含有期望的限制性位點，以便于克隆。用合適的限制酶消化所得到的PCR產(chǎn)物(約2.2kb),并亞克隆到經(jīng)過修飾的pAX480中。通過限制性分析鑒定含有插入片段的克隆。得到的克隆pAX906含有與6xHis標簽融合的AXMI-006開放讀碼框，其轉(zhuǎn)錄和翻譯導(dǎo)致生成具有一段六個組氨酸的"融合蛋白"。通過DNA序列分析確認了pAX906的DNA序列，隨后如制造商(Stratagene，拉霍亞，加利福尼亞州)所述轉(zhuǎn)化到化學(xué)感受態(tài)大腸桿菌BL21中。將pAX906在BL21中的單菌落接種到添加了卡那霉素的LB培養(yǎng)基中，并于37。C以劇烈振搖培養(yǎng)幾個小時。將這些培養(yǎng)物培養(yǎng)至0D6。。為0.6-0.8，然后通過添加G.1mMIPTG來誘導(dǎo)蛋白質(zhì)生成。將培養(yǎng)物在i秀導(dǎo)條件下培養(yǎng)3小時，然后通過離心沉淀細胞并重懸于PBS。采用IO秒超聲間隔，使用MisonixSonicator3000超聲處理細胞，十30秒，并在冰上保溫l分鐘。"、4實施例10:AXMI-006對煙芽夜蛾和草地夜蛾的活性的生物測定法4吏用人工飼料(多物種飼料，SouthlandProducts,LakeVillage,阿肯色州)進行經(jīng)過超聲處理的pAX906培養(yǎng)物的生物測定法。通過將經(jīng)過超聲處理的pAX906細胞或作為對照的大腸桿菌菌林的細胞施加到飼料表面并使飼料表面干燥來進行生物測定法。在24孔組織培養(yǎng)板中進行生物測定法。生物測定法在黑暗中、25。C和65%相對濕度下進行。用Breathe簡易密封帶(DiversifiedBiotech,波士頓，馬薩諸塞州)密封盤子。笫5天記錄結(jié)果。表8.AXMI-006對草地夜蛾的生物測定法<table>tableseeoriginaldocumentpage54</column></row><table>阻礙發(fā)育被定義為昆蟲體形變小，而且一些幼蟲攝食減少。發(fā)育受阻礙的昆蟲還可能表現(xiàn)出相對于未處理飼料而言回避經(jīng)過處理的飼料。實施例11:AXMI-008、AXMI-009和AXMI-014對粉紋夜蛾的殺蟲活性將含有pAX008、pAX009或pAX-014的大腸桿菌菌林，以及未轉(zhuǎn)化的大腸桿菌的培養(yǎng)物在2mlLB培養(yǎng)基(Luria-Bertani肉湯，BectonDickinson&Company,斯帕克斯，馬里蘭州)中于37。C以250rpm振搖培養(yǎng)24小時。將含有質(zhì)粒的菌抹在含有適當(dāng)抗生素的LB培養(yǎng)基中培養(yǎng)，以選擇質(zhì)粒在大腸桿菌中的維持。使用人工飼料(多物種飼料，SouthlandProducts,LakeVillage,阿肯色州)在24孔組織培養(yǎng)板中進行生物測定法。通過將含有pAX-014的大腸桿菌培養(yǎng)物施加到飼料表面并使飼料表面干燥來進行生物測定法。將菌林的整個培養(yǎng)物以每孔40^1培養(yǎng)物的濃度施加于伺料。生物測定法在黑暗中、25°C和65°/。相對濕度下進行。用Breathe簡易密封帶(DiversifiedBiotech,波士頓，馬薩諸塞州)密封盤子。笫5天記錄結(jié)果。表IO.對粉發(fā)在喊的殺蟲活性<table>tableseeoriginaldocumentpage55</column></row><table>實施例12:5-內(nèi)毒素基因在芽孢桿菌中的表達通過PCR由來自實施例4的克隆擴增AXMI-004、AXMI-006、AXMI-007、AXMI-008和AXMI-009,并通過本領(lǐng)域眾所周知的方法克隆到芽孢桿菌表達栽體pAX916中。對于AXMI-004，將得到的克隆命名為pAX920。對于AXMI-006，將得到的克隆命名為pAX921。對于AXMI-007，將得到的克隆命名為pAX919。對于AXMI-008,將得到的克隆命名為pAX922。對于AXMI-009，將得到的克隆命名為pAX917。所得到的克隆在轉(zhuǎn)化到c/y(S)蘇云金芽孢桿菌菌林的細胞中后表達相應(yīng)蛋白質(zhì)?？梢栽诙喾N常規(guī)培養(yǎng)基中培養(yǎng)含有5-內(nèi)毒素基因且表達5-內(nèi)毒素蛋白質(zhì)的芽孢桿菌菌林。在CYS培養(yǎng)基(10g/1Bacto-casitone;3g/1酵母提取物；6g/1KH2P04;14g/1K2HPO"0.5mMMgS04;0.05mMMnCl2;0.05mMFeS04)中培養(yǎng)含有期望基因的芽孢桿菌菌林，直至通過顯微鏡檢查觀察到孢子形成。制備樣品，并在針對重要昆蟲害蟲的生物測定法中對5-內(nèi)毒素蛋白質(zhì)測試殺蟲活性。才法配制CYS培養(yǎng)基10g/1Bacto-casitone;3g/1酵母提取物；6g/1KH2P04;14g/1L線；0.5mMMgS(V'0.05mMMnCl2;0.05mMFeS04。CYS混合物應(yīng)當(dāng)是pH7，如果需要就進行調(diào)節(jié)。優(yōu)選用NaOH或HC1。然后將培養(yǎng)基高壓滅菌，并在高壓滅菌后添加100mlIOX經(jīng)過濾的葡萄糖。如果得到的溶液渾濁，可以在室溫攪動至清澈。實施例13:在芽孢桿菌中表達的AXMI-004的N端氨基酸序列對于在芽孢桿菌中表達的AXMI-004的分析說明在這些培養(yǎng)物中檢測到的蛋白質(zhì)產(chǎn)物在長度上相對于全長AXMI-004蛋白質(zhì)而言可能是縮短的。因為許多內(nèi)毒素蛋白質(zhì)在芽孢桿菌中表達后在N端受到切割，我們測定了由芽孢桿菌表達產(chǎn)生的AXMI-004蛋白質(zhì)的N末端。在PAGE凝膠上分離來自AXMI-004的蛋白質(zhì)樣品，并且通過本領(lǐng)域眾所周知的方法將蛋白質(zhì)轉(zhuǎn)移到PVDF膜上。切下與AXMI-004對應(yīng)的蛋白質(zhì)條帶。通過本領(lǐng)域已知的連續(xù)Edman降解法測定該蛋白質(zhì)的N端氨基酸序列。得到的序列如下ERFDK畫LE該氨基酸序列與全長AXMI-004序列(SEQIDNO:3)的比較證明該氨基酸序列來自AXMI-004在芽孢桿菌中表達后的內(nèi)在切割，導(dǎo)致N末端與SEQIDNO:3第28位氨基酸相對應(yīng)的蛋白質(zhì)(公開于SEQIDNO:5)。實施例14:AXMI-004對昆蟲害蟲的生物測定法利用公認的生物測定法流程，使用形成孢子的表達AXMI-004的芽孢桿菌細胞培養(yǎng)物裂解物確定AXMI-004的殺蟲活性。在50mlCYS培養(yǎng)基中培養(yǎng)芽孢桿菌培養(yǎng)物，以用于標準生物測定法和LCs。生物測定法。然后將培養(yǎng)物于30。C、以250rpm培養(yǎng)2-3天，直至細胞形成孢子。通過顯微鏡檢查孢子的存在來確定孢子形成。通過將形成孢子的培養(yǎng)物以12，000xg離心10分鐘來制備AXMI-004蛋白質(zhì)樣品。收集沉淀并重懸于4ml20mMTris-HCl，pH8.0。將管置于水上的同時將懸浮液超聲處理20秒(大功率，使用微型探針)。通過在SDS4-20y。梯度丙烯酰胺凝膠上與已知量的牛血清清蛋白(BSA)—起電泳來測定樣品的蛋白質(zhì)濃度(圖8)。AXMI-004的濃度經(jīng)測定是0.4pg/iil。使用按照本領(lǐng)域所知制備的人工飼料(多物種飼料，SouthlandProducts,LakeVillage,阿肯色州)的表面處理生物測定法來測試AXMI-004的殺蟲活性。通過將表達AXMI-004的芽孢桿菌培養(yǎng)物施加到飼料表面并使表面通風(fēng)干燥來進行生物測定法。在標準生物測定法中，每個孔使用5個蟲卵，而LCs。生物測定法每個孔使用IO條新生昆蟲幼蟲。^使用細尖毛刷施加蟲卵或幼蟲。標準表面生物測定法在24孔組織培養(yǎng)板中進行。將40ul每種樣品施加到每個孔中。因為每個孔的表面積是2cm2(板的來源)，40pi細胞裂解物樣品含有大約0.4ug/ulAXMI-O(H。測定LCs。的生物測定法在48孔組織培養(yǎng)板中進行，每個孔展示的表面積是1cm2(來源)，每個孔使用大約20ul0.4pg/ulAXMI-004。每個生物測定法中AXMI-004蛋白質(zhì)的最終量是大約8pg/cm2。用Breathe簡易密封帶(DiversifiedBiotech,波士頓，馬薩諸塞州)密封生物測定法的板。對照樣品包括只含培養(yǎng)基的樣品，以及未經(jīng)樣品處理的孔。然后將生物測定法在黑暗中于25。C和65%相對濕度下放置5天，并記錄結(jié)果。表11.AXMI-004的殺蟲活性<table>tableseeoriginaldocumentpage57</column></row><table>AXMI-004顯示針對歐洲玉米螟和煙芽夜蛾有強殺蟲活性(100%致死)。AXMI-004還顯示針對紅鈴麥蛾有50-75%致死的殺蟲活性。濃度為43jag/cn^的AXMI-004對紅鈴麥蛾產(chǎn)生70%死亡率。AXMI-004嚴重阻礙小地虎、棉鈴蟲和草地夜蛾的發(fā)育。實施例15:AXMI-004針對煙芽夜蛾和歐洲玉米螟的殺蟲活性的量化通過在昆蟲生物測定法中測試一定范圍的AXMI-004蛋白質(zhì)濃度并將這些蛋白質(zhì)樣品施加到昆蟲飼料表面來測定AXMI-004蛋白質(zhì)對歐洲玉米螟和煙芽夜蛾幼蟲的LC5。。記錄每個蛋白質(zhì)濃度的死亡率，并使用Probit分析軟件進行分析。結(jié)果在95%置信空間是顯著的。因為測定是通過表面污染來進行的，在假設(shè)全部蛋白質(zhì)樣品在測定過程中都保留在表面上而在昆蟲的攝食水平下很少有擴散的前提下測定LC5。。因此，測定的數(shù)值可能有些低估AXMI-004蛋白質(zhì)對測試昆蟲的毒性。表12.AXMI-004對歐洲玉米螟的LC<table>tableseeoriginaldocumentpage58</column></row><table>表13.AXMI-004對煙芽夜蛾的LC<table>tableseeoriginaldocumentpage59</column></row><table>LC50=2874ng/cm2;95%CI=2189-3933實施例16:AXMI-004、AXMI-006、AXMI-007和AXMI-009針對牧草盲蝽(Lyguslineolaris)的殺蟲活性的量化如下制備細菌裂解物切下芽孢桿菌在50mlCYS培養(yǎng)基中培養(yǎng)60小時。然后將芽孢桿菌培養(yǎng)物以12，000rpm離心IO分鐘，并丟棄上清液。將沉淀重懸于5ml20mMTrisHC1pH8。如下進行生物測定法切下Eppendorf管的底尖和蓋子，形成喂食槽。將殺蟲劑蛋白質(zhì)或?qū)φ找匀芤旱谷牍苊敝胁⒏采w昆蟲能夠刺穿從而攝食的石蠟?zāi)?PechineyPlasticPackaging,芝加哥，伊利諾斯州)從而呈現(xiàn)給昆蟲。將Eppendorf管倒置管帽朝下置于黑暗中，并用細尖毛刷將一齡或二齡Ar^/s/7//z7//^置于Eppendorf槽中。用石蠟?zāi)っ芊馇邢碌腅ppendorf管尖制成測定槽。將得到的測定槽帽端朝下于環(huán)境溫度保溫。在15°/。葡萄糖溶液中以濃度6.6ug/ml測試殺蟲劑蛋白質(zhì)。<table>tableseeoriginaldocumentpage159</column></row><table>實施例17:AXMI-008對黃粉蟲(Tenebriomolitor)的生物測定法制備表達AXMI-008的芽孢桿菌培養(yǎng)物的樣品，并測試對黃粉蟲的殺蟲活性。當(dāng)pAX922制備成pH4.0的不溶組分時，它顯示對通常稱為大黃粉蟲的昆蟲有活性。由含有pAX922的芽孢桿菌菌林的培養(yǎng)物制備AXMI-008的樣品。如下制備生物測定法樣品將培養(yǎng)物在CYS培養(yǎng)基中培養(yǎng)4天，直至孢子形成。將樣品以10，000rpm離心10分鐘，丟棄上清液。用20mMTrispH8清洗沉淀，并以10,000rpm離心10分鐘。丟棄上清液，并將沉淀重懸于3ml50mM檸檬酸鈉和25mM氯化鈉及2mMDTTpH4。將樣品于37。C保溫1小時。保溫后，將樣品以13,000rpm離心10分鐘，丟棄上清液。將沉淀重懸于50mM檸檬酸鈉和25mM氯化鈉pH4。使用人工飼料(SouthernCornRootwormDiet,Bioserv，F(xiàn)renchtown,新澤西州，#F9757B)在24孔組織培養(yǎng)板中進行樣品對黃粉蟲的生物測定法。通過表面處理以Axmi008濃度8ug/cm'施加樣品，并使之空氣干燥。使用細尖毛刷施加昆蟲。用Breathe簡易密封帶(DiversifiedBiotech,波士頓，馬薩諸塞州)密封生物測定盤，并在無光條件下于65°/。相對濕度和25。C保溫7天并記錄結(jié)果。表15.AXMI-008對黃粉蟲的殺蟲活性<table>tableseeoriginaldocumentpage61</column></row><table>*其余黃粉蟲發(fā)育受到阻礙。發(fā)育受阻即觀察到幼蟲體形較小、生長較慢，而且攝食劇烈減少或僅有最低限度。昆蟲還可能表現(xiàn)出相對于未處理飼料而言回避經(jīng)過處理的飼料。實施例18:AXMI-009蛋白質(zhì)對鞘翅類害蟲的生物測定法通過將40pl不溶組分施加到2cm2飼料表面上、達到最終總蛋白質(zhì)濃度為8pg/cm2來進行AXMI-009蛋白質(zhì)制劑的生物測定法。使用南方玉米食蟲祠料(Bioserv，F(xiàn)renchtown，新澤西州，井F9757B)測試玉米根葉甲(Diabroticavirgiferavirgifera)和黃瓜十一星葉曱(Diabroticaundecimpunctata)。通過將表達AXMI-009的芽孢桿菌培養(yǎng)物施加到伺料表面并使飼料表面干燥來進行生物測定法。生物測定法在24孔組織培養(yǎng)板中進行。標準生物測定法每個孔使用25個蟲卵。將蟲卵施加到含有0.1%瓊脂和30ug/ml制霉菌素的溶液中。用Breathe簡易密封帶(DiversifiedBiotech,波士頓，馬薩諸塞州)密封盤子并將蓋子蓋回盤上。將生物測定法避光于65%相對濕度(RH)和25。C保溫7天。對于玉米根葉甲和黃瓜十一星葉甲都看到了不溶組分的活性。對照是培養(yǎng)基、緩沖液1mMTrispH10.5、和芽孢桿菌表達載體pAX916。表16.玉米根葉曱<table>tableseeoriginaldocumentpage61</column></row><table>表17.黃瓜十一星葉曱<table>tableseeoriginaldocumentpage62</column></row><table>實施例19:用于植物表達的栽體將5-內(nèi)毒素編碼區(qū)DNA可操作連接用于在植物中表達的合適啟動子和終止子序列。這些序列在本領(lǐng)域是眾所周知的，而且可以包括用于在單子葉植物中表達的水稻肌動蛋白啟動子或玉米泛素啟動子、用于在雙子葉植物中表達的擬南芥UBQ3啟動子或CaMV35S啟動子、以及nos或PinII終止子。用于構(gòu)建和鑒定啟動子-基因-終止子構(gòu)建體的技術(shù)在本領(lǐng)域同樣是眾所周知的。將上文所述植物表達盒與合適的植物選擇標記結(jié)合使用，以便于選擇經(jīng)轉(zhuǎn)化的細胞和組織，并連接到植物轉(zhuǎn)化載體中。這些載體可以包括用于由農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化的二元載體或用于氣霧劑或biolistic轉(zhuǎn)化的單一質(zhì)粒載體。實施例20:玉米細胞的轉(zhuǎn)化授粉后8-12天采集玉米穗。由穗分離胚，并將大小為0.8-1.5mm的那些胚用于轉(zhuǎn)化。將胚盾片朝上置于合適的培養(yǎng)基上，諸如DN62A5S培養(yǎng)基(3.98g/LN6鹽；1mL/L(1000x儲液)N6維生素；800mg/LL-天冬酰胺；100mg/L肌醇；1.4g/LL-脯氨酸；100mg/LCasaminoacids;50g/L蔗糖；1mL/L(1mg/mL儲液)2，4-D)，并于25。C在黑暗中保溫過夜，將由此產(chǎn)生的外植體轉(zhuǎn)移到篩網(wǎng)上(每塊板30-40粒)，轉(zhuǎn)移到滲透培養(yǎng)基上達30-45分鐘，然后轉(zhuǎn)移到光照板上(見例如PCT公開號WO/0138514和美國專利號5,240,842)。采用基本上如PCT公開號WO/0138514中描述的條件(aerosolbeamaccelerator),使用氣溶膠氣流加速器，將設(shè)計在植物細胞中表達5-內(nèi)毒素的DNA構(gòu)建體加速進入植物組織。光照后，將胚在滲透培養(yǎng)基上保溫30分鐘，然后置于保溫培養(yǎng)基上在黑暗中于25。C保溫過夜。為了避免過度損傷經(jīng)過光照的外植體，在轉(zhuǎn)移至恢復(fù)培養(yǎng)基前將它們至少保溫24小時。再將胚散布到恢復(fù)期培養(yǎng)基上，在黑暗中于25。C保溫5天，再轉(zhuǎn)移至選擇培養(yǎng)基上。將外植體在選擇培養(yǎng)基中保溫可長達8周，這取決于所用具體選擇方法的本質(zhì)和特征。選擇期后，將由此產(chǎn)生的愈傷組織轉(zhuǎn)移至胚成熟培養(yǎng)基，直至觀察到成熟體細胞胚的形成。然后將由此產(chǎn)生的成熟體細胞胚置于低光照下，并通過本領(lǐng)域已知方法啟動再生程序。使由此產(chǎn)生的芽在生根培養(yǎng)基上生根，并將由此產(chǎn)生的植抹轉(zhuǎn)移至苗圃罐中，繁殖成轉(zhuǎn)基因植物。辦DN62A5S培養(yǎng)基成分絲來源Chu'SN6M鹽^給物(Prod.No.C416)3.98g/LPhytotechnologyLabsChu，sN6維生素溶液(Prod.No.C149)1mL/L(lOOOx儲紀PhytotechnologyLabsL-天冬纖800mg/LPhytotechnologyLabs肌醇100mg/LSigmaL-脯氨酸1.4g/LPhytotechnologyLabsCasaminoacids100mg/LFisherScientific庶^^50g/LPhytotechnologyLabs2，4-D(Prod.No.D~7299)1mL/L(1mg/mL#^USigma用1NK0H/1NKC1將溶液的pH調(diào)至pH5，8，添加Gelrite(Sigma)至3g/L，并高壓滅菌。冷卻至50°C后，添加2ml/L硝酸銀(PhytotechnologyLabs)的5mg/ml儲液。it酉己方帝J備大纟勺20塊平板。實施例21:通過由農(nóng)桿菌介導(dǎo)的轉(zhuǎn)化轉(zhuǎn)化到植物細胞中授粉后8-12天采集穗。由穗分離胚，并將大小為0.8-1.5mm的那些胚用于轉(zhuǎn)化。將胚盾片朝上置于合適的培養(yǎng)基上，并在黑暗中于25。C保溫過夜。但是，本質(zhì)上并不必將胚保溫過夜。使胚接觸含有用于Ti質(zhì)粒介導(dǎo)的轉(zhuǎn)移的合適載體的農(nóng)桿菌菌林達5-10分鐘，然后鋪板在共培養(yǎng)培養(yǎng)基上，在黑暗中于25。C保溫3天。共培養(yǎng)后，將外植體轉(zhuǎn)移至恢復(fù)期培養(yǎng)基，在黑暗中于25。C保溫5天。將外植體在選擇培養(yǎng)基中保溫可長達8周，這取決于所用具體選擇方法的本質(zhì)和特征。選擇期后，將由此產(chǎn)生的愈傷組織轉(zhuǎn)移至胚成熟培養(yǎng)基，直至觀察到成熟體細胞胚的形成。然后將由此產(chǎn)生的成熟體細胞胚置于低光照下，并通過本領(lǐng)域已知方法啟動再生程序。使由此產(chǎn)生的芽在生根培養(yǎng)基上生根，并將由此產(chǎn)生的植林轉(zhuǎn)移至苗圃罐中，繁殖成轉(zhuǎn)基因植物。結(jié)論正如上文實施例中所示，本發(fā)明的5-內(nèi)毒素蛋白質(zhì)針對多種害蟲具有活性。AXMI-004對包括歐洲玉米螟、小地虎、棉鈴蟲、草地夜蛾、煙芽夜蛾、紅鈴麥蛾、煙草天蛾、粉紋夜蛾、和牧草盲蝽在內(nèi)的害蟲有殺蟲活性。AXMI-006對包括煙芽夜蛾、草地夜蛾、和牧草盲蝽在內(nèi)的害蟲有殺蟲活性。AXMI-007對包括牧草盲蝽在內(nèi)的害蟲有殺蟲活性。AXMI-008對包括黃粉蟲和粉紋夜蛾在內(nèi)的害蟲有殺蟲活性。AXMI-009對包括牧草盲蝽、粉紋夜蛾、玉米根葉甲、和黃瓜十一星葉甲在內(nèi)的害蟲有殺蟲活性。AXMI-014對包括粉紋夜蛾在內(nèi)的害蟲有殺蟲活性。說明書中提到的所有發(fā)表物和專利申請指示了本發(fā)明所屬領(lǐng)域熟練技術(shù)人員的技術(shù)水平。本文收錄所有發(fā)表物和專利申請作為參考，其程度與專門和個別指出收錄每一項發(fā)表物或?qū)＠暾堊鳛閰⒖枷嗤?。雖然出于清楚理解的目的通過例示和實施例已經(jīng)較為詳細的描述了上述發(fā)明，顯然可以在所附權(quán)利要求的范圍內(nèi)實踐某些改變和修飾。序列表<110>Caxozzi,NadineHargiss,TracyKoziel,MichaelG.Duck,NicholasB.Carr,Brian<i2o>6-內(nèi)毒素基因及其使用方法<130>045600/274379<150>60/448,632<151>2003-02-20<150>60/448,633<151>2003-02-20<150>60/448,797<131>2003-02-20<:150>60/448,806<151>2003-02-20<150>60/448,810<151>2003-02-20<150>60/448,812<:151>2003-02-<160>S2<170>FastSEQforWindowsVersion4.0<210>1<211>2190<212>DNA<2工3>蘇云金芽孢桿菌<400>1tatgatgataELCtttg3caacagccgcatcccaagcttattttgtacaaattacactttagcatataagaaaattttttcatatataagttaggataaactttacttgtaaacacaagtaaaaatatttacgaatccta±ctataaaaatatgagtgaattgaaggggaaatttaagaaaataataaatataggaggaagaggtatgaataatgatttaagtgatcgccaatattaatatgattggtatgtccattgtatctgaacttatttttgtgtttaatcaattgtggtctcgtttacatgt的agg幼ttaattgatactaaaatagaattagcaggtatacaaagaaaccttgaaaatgatattg幼幼ctcaaa的ctcaaggtcaggcggttgaaaggagtstgcctc祖tttactgtctatgtgcaagctgctaatcttcatggaaagtgttggggatggtcggagcagaaatatacccatg站tacacaaatcattgtgtaaaaaataaaggttcttcttatcaagattggactcttactgttttagatatcgttgctttaataacaaccgttgetcagctaacaagggaacctaaattacattctgtgtctcaattacctagaactccacatctgatggaatttttaagagtgggaagaaactattattggggaggacataatataagatcacctctatat的tagagagttttatggacccgtttttaagacgttatcacataacgtactttttcaatattaattgtat60ttgtatgcataatagtttcttattgattga120gaaaatatttttgcttttcttttttttatt180aatagtttattagagatagagcctcatttt240ctaattttaaaattattccttttaaatgta300agtactaatcgaacttgttgtttgctaaaa360tcaaaggasicatgattatctaaaagtttgt420訴acggtttgataagaatgatgcactggaa480ggtatgattccaggcggaacagctttgcaaS40ggtgattctggatggaatgcgttcatggaa600gaagggtatgcaaaaaat站agccttatctacatatatacaattacgt貼tgaatgg訴a720aaggtagctaattactat訴幼gtcttgag780gcagtggagaattttgaagtaccactttta840ttattattattaa訴gatgtttc的tttat900attaaaatttattatgat貼acagattaagS60站ttggtataataaag訴cttgaga訴tta1020tac站ttataatcgtttccgta訴g站at:g1080ttcccgcactELt訴tgtacaLaacttatcca1140gtttatacggatcctttacttaattttaat1200agttttagtgacatgga站atgcaacaatt1260atgctaacaatttatacagattggtatagt1320cgcgtgacgtcttaccatgtaggaggagag1380gcaaatcaagaggttcct的的atttttat"40aagccgactcta的accattacagcagcct1500gcaccagctcggtagtttta33tcce1gttgcgtaaatctgt3tcaaattgtctcasagatagtattaatgsctttsggttgcacaasgcagataaaattgctccttttaatgtatcgtgaggttacctcsggaccccttaccaatacaatggtcaggcaccaggtcctggatcgtgtaagggaccaccatctgagggctatatttagatt&atttattcctttacgtagcaagaggatcgtaaggtatactgaatcaaattactgtaaggaacatttggagattcgttattaat3ttggttagaacagtaaggtatacaatgttgaatagttagagggsggtagattcttagtcaccgttggtgccatctattattacgcgatatgagaagcttctacgaaacttcccgatcgtttagtagcattttctgt￡iga3ttcc3ttaatgagtttatgtcatgctttcttggacgattcacaggtaaatattaacagatttacaGtccatttaggagttcsagacactcctacagccgccttttaacgtttgttacatcgtagtagtsaaagcaaggggatatatgcaccattaatttgtcacgtttctcaaatagtttatgtg15601620:U8Q174018001860192019802040210021602190<210>2<211>1890<212>DNAdlh蘇云金芽孢桿菌<220><221>CDS<222>(1)…UB90)<400>23tgagtgaaMetSerGluttgaaggggaaatttaagaaaagtactaatcgaacttgtLeuLysGlyLysPheLysLysSerThrAsnArgThrCys510IS48tgtttgetaaaaataataaatataggaggaagaggtatgaatteaaagCysLeuLeuLyslielieAsnlieGlyGlyArgGlyMetAsnSerLys20253096gaacatgattatetaaaagtttgtaatgatttaagtgacgecaatattG3LuHisAspTyrLeuLysValCysAsiiAspLeuSerAspAlaAsnlie3S4045144aatatggaaeggtttgataagaatgatgcactggaaattggtatgtecAsnMetGluArgPheAspLysAsnAspAlaLeuGlulieGly.MetSer505560192attgtatctgaacttattggtatgattccaggcggaacagetttgcaalieValSerGluLeulieGlyMetlieProGlyGlyThrAlaLeuGin65707580240tttgtgtttaatcaattgt的tctcgtttaggtgattctggatggaatPheValPheAsnGinLeuTrpSerArgLeuGlyAspSerGlyTrpAsn859095288gcgttcatggaacatgtggaggaattaattgatactaaaatsigaag的AlaPheMetGluHisValGluGluLeu工leAspThrLys工leGluGly100105110336tstgcaaaaaataaagecttatctgaattagcaggtatacaaagaaacTyrAlaIiysAsnLysAlaLeuSerGlulieuAlaGlylieGinArgAsn115120125384cttgaaacatatatsicaattacgtaatgaat的gaaaatgatattgaaLeuGluThrTyrlieGinLeuArgAsnGluTrpGluAsnAsplieGlu13013S140432站CtC2L抑3AsnSerLys145getcaaggtaagAlaGinGlyLysISOgtagetaattactatgaaagtcttgagValAlaAsnTyrTyrGluSerLeuGlu155160480caggcggttgaaaggagtatgcctcaatttgcagtggagaattttgaaGinAlaValGluArgSerMetProGinPheAlaValGluAsnPheGlu165170175S28gtaccacttttaactgtctatgtgcaagetgetaatcttcatttatta57SValProLeuLeuThrValTyrValGinAlaAlaAsnLeuHisLeuLeu180185190ttattaagagatgttteagtttatggaaagtgttggggatggteggagLeuLeuArgAspValSerValTyrGlyLysCysTrpGlyTrpSerGlu19520020S624Gin3332103ttlieLys3ttlietatTyrtatTyr215gatAspS33LiysGin3ttlie220tatTyr3CCThrC3tHisgaaGlu672t3CTyr225￡L￡LtestHistgtCys230￡L3tJ\ST\TrptatTyrs的235ggaGlycttLeugagGlu3g3ArgLeu240720As119gtGlytct245tcttatTyrGinAsptggTrp250t3CTyrsatAsnt3tTyr33tAsncgtArg255ttcPhe768cgtArg3g3ArggaaGluatgMet2603Ctct匕l(fā)ieusetThrgttValttaLeu265ga匕AspateliegttValgetAlatt3lieu270ttcPheccgPro816C5LCHisTyrgatAsp275ValC33GinactThrtatCC3Pro280lieThr3CCThrgttValgetAla28SGinCt3Ieu864頓Argg貼Glu290tatTyrThrAspcctPro295ttacttLeuttt300cctProttalieucatHis912tct30S9tgValtctGinttaLeucctPro310SertttPheagtSerMet315gasGluAsngcaAlaThrattlie320ArgsetThrCCELProcatHisctgLeu325atgMetg站GlutttPhett3LeuagaArg330atgMetetaLeuThr8ttlietatTyr335Thr1008gattggtatagtgtgggaagaaactattattggggaggacatcgcgtgAspTrpTyr*SerValGlyArgAsnTyrTyrGlyGlyHisArgVal3403453501056acgtcttaccatgtaggaggagagaatataagateacctetatatggtThrSerTyrHisValGlyGlyGluAsnlieArgSerPi:oLeuTyrGly3553601104agagaggcaaatcaagaggttcctagagatttttatttttatggacccArgGluAlaAsnGinGluValProArgAspPheTyrPheTyrGlyPro3703753801152gttVal385tttPhe的gacgThrLeutedSer3S0站gccgProactThretaLeu的aArg39SccaProLeuGinGincctPro4001200gfcaccagetcctccttttaatttacgtagettagagggagtagaattcAlaProAlaProProPheAsnLeuArgSerLeuGluGlyValGluPhe40S4104151248cacactccfcacaggtagttttatgtatcgtgaaagaggateggtagatHisThrProThrGlySerPheMetTyrArgGluArgGlySerValAsp42042S4301296tctSetttt站tAsn435弼GluttglieuccgProcctProtttPhe440aatAsnProgttVal卿GlyttaLeu445cctProestHis站g1344gtatacagtca<=cgtttatgtcatgcaacgtttgttcgtaaatctggg1392LeuCysHisAlaThrPheValArgLysSerGly4504554603CCThr465cctProtatTyrttaThra_C3470ggtGlygccAlaatelietttPhetctSer475tggTrpThrC3tHiscgtArgSer4801440getAlagaaGluGlu3CCThr33tAsn485ThrattliegaaGluteaSera_3tAsn4903ttlie3tt工leacgThrGin3tClie495ccgPro1488LeugtaVal335Ala500ta匕TyrGin3tt工legggGlytC3Ser505GlyactThractThrgtaValArg510333Gly1536ProGlyttcPhe515Thr卿GlygggGlygatAsp3t3lie520cttArgArg抓Gly525cctPro卿GlyThr1584t匕tPheggaGly530gatAspatgMet3ga工lesatAsia535attlieAsnAlaCC3Prott3Leu540tctSerC33GinArgtatTyr1S32cgtArg545gtaValArgliecgtArgtatTyr5S0getAlatctSeraegThrThrgatAsp5S5ttaLeuc貼GintttPhegtcValacgThr5601680agtSer3ttlie站tAsng的Gly■accThr5653CCThrattlie€L6ttAsnattlieGly57033CAsnttcPheccgProsetThr5753ttlie1728aataatetaaatactttacggttctgagggctatagaacagtategtttAsnAsnLeuAsnThrLeuGlySerGl\iGlyTyrArgThrValSerPheS805855901776agtSer3CtThrCC3Pro595tttPheSerttcPhetC3Ser￡131600Alac貼GinageSersita_工letttPhe605ttaLieuGly18243t3lieC33GinG10gcaAlatttPhetctGly615C33Ging33GlugttValt3tTyrgtgVal620gatAsp333attliegaaGlu1872ttt.Phe625attliecctProgttValGlutag1890<210>3<211>629<212>PRT<2">蘇云金芽孢桿菌<400>3MetSerGlulieuGlyLys1SCyslieuLeuLyslielieAsnlie20GluHisAspTyrlieuLysValCys3S40AsnMetGluArgPheAspLysAsnSOSSlieValSerGlulieulieGlyMetPheValPheAsnGinLeuTrpSer85Ala昨sMetGluBisValGluGlu100Ly曰LysSerThrAsiaArgThrCys1015GlyGlyArgGlyMetAsnSerLys2S30AsnAspLeuSerAspAlaAsnlie4SAspAlalieuGlulieGlyMetSer60lieProGlyGlyThrAlalieuGin75B0ArglieuGlyAspSerGlyTrpAsnSOLeulieAspThrLyslieGluGly<table>tableseeoriginaldocumentpage70</column></row><table><210><211><212><213><220〉<221><222>41806DNA蘇云金芽孢桿菌CDS(1).(180S)<400>4atgaatMetAsnteaSer站gljysgaaGlucatHisgatAsptatTyrCt3Leu333Ijys10gttValtgtCys33tAsngatAspLeu15a_gtSer48AspAlaAsialie20AsnMetGlueggArgtttPhe25gatAspLysAsngatAspAla30ctgLieuGlu96attlieggtGlyMet35tecSerattliegt3ValtctSergaaGlu40cttLeu3ttlieGlyMetlie45ProggcGlyGly144ThrgetAla50ttgLieucaia_GintttPhegtgValtttPhe55aatAsnC33GinttgLieutggTrptctSerGOcgtArgttaLeuggtGlyga±Asp192tctSerGlyt的TrpaatAlattcPhe70atgMetgaacatHisVal弼Glu75Glutt3LeuattliegatAspsetThr80240liegaaGlu鵬tatTyr85Ala6L6L3LAsn祖eiAla90ttalieutctSergaaGluIjeugcaAlaGly2B83t3lieGillagaArgAsn100CttlieugaaGluThrtatTyratalie105GinttalieucgtsatAsng的Glu110tggTrpgasGlu33633tgatAspattlie11593aGluteaSeraaggetAla120Gin93tGlyLyeValgetAla125aattacTyrtatTyr384g犯GluagtSer130CttlieugagGluGinAlagttVal13SgaaGluaggArgSerMetcct140c站GintttEheAla9t9Val432gagGlu145AsntttPheg幼GluValPro150cttLeuttaIjeuactgtcThrValtatTyr1559tgValGingetAlagetAlaAsn160480cttcatttattattattaagagatgttteagtttatggaaagtgttggLeuHisLeuLeuLeuLeuArgAspValSerValTyrGlyLysCysTrp16S170175S2BggatggtcggagcagaaaattaaaatttattatgataaacagattaagGlyTrpSerGluGinLyslieLyslieTyrTyrAspLysGinlieLys180185190S76tatacccatgaatacacaaatcattgtgtaaattggtataataaag訴TyrThrHisGluTyrThrAsnHisCysValAsnTrpTyrAsnLysGly195200205cttgagagattaaaaaataaaggttcttcttatcaagattggtacaatLeuGluArgLeuLysAsnLysGlySerSerTyrGinAspTrpTyrAsn210215220€72tataatcgtttccgtagagaaatgactcttactgttttagatategttTyrAsnArgPheArgArgGluMetThrLeuThrValLeuAsplieVal230235240getAlattaLieuttcPheccgProescHis245tatTyrgatAspgtaValGinactThr250tatTyrcca_ProatalieThr3CCThr255gttValgetAlacagGina_c3Thr2603ggArgGlugttValtatTyra_cgThr265gatAspcctPrott￡LLeucttLeu3attAsn270tttPheAsn816cctProIjysttaIjeu275catHistctgtgValtctSerca_3Gin280tt3LeucctProagtSertttPheagtSer285AspatgMetgaaGlu86433tAsngcaAla290ThrattlieagaArg3CtThrCC3Pro295catHisctgIjeuMetGlutttPhe300Leuaga_Arg3tgMetetaLeuacaatttatacagattggtatagtgtgggaagaaactattattggggaThrlieTyrThrAspTrpTyrSerValGlyArgAsnTyrTyrTrpGly305310315320960GlyC3tHiscgcArggtgVal3egThr325tctSert3CTyrestHisgtaValggaGly330GlyGlu33tAsn3t3lieArg335tC3Ser1008cctetatatggtagagaggcaaatcaagaggttcctagagatttttatProLeuTyrGlyArgGluAlaAsnGinGluValProArgAspPheTyx3403453S010S6ttttatTyrGly355cccPro3ttValtttPhesagLysacgThr360tt3LieuteaSerLyaProactThr365etaIjeuagaArgCC3Pro1工04ttacagGin370GincctProgcaAlaCC3ProgetAla375cctProcctProtttPhe33tAsnttalieu380cgtageSerlieugagGlu1152Gly385gtaValGluttcescHis3CtThr390cctProThrggtGly3gtSerttt395atgMetTyrcgtArgGluArg4001200ggateggtagattcttttaatgagttgccgccttttaatccagttgggGlySerValAspSerPheAsnGluLeuProProPheAsnProValGly4054104151248ttacctcataaggtatacagtcaccgtttatgtcatgcaacgtttgttLeuProHisLysValTyrSerHisArgLeuCysHisAlaThrPheVal4204254301296cgtArgtct435的gGlyThrcctProTyrttsi440ThrThr的tGlygecAlaatelie445ttttctSertggTrp1344acacatcgtagtgetgaagaaaccaatacaattgaateaaatattattThrHisArgSerAlaGluGluThrAsnThrlieGluSerAsnlielie4504S54601392acgcaaateccgttagtaaaagcatatcaaattgggteaggcactactThrGinlieProlieuValLysAlaTyrGinlieGlySerGlyThrThr4654704754801440gtaaggaaaggaccaggattcacaggaggggatatacttcgaagaacaValArgLysGlyProGlyPteThrGlyGlyAsplieLeuArgArgThr4854904SS1488ggtcctggaacattt的agatatgagaataaatatt站tgcaccattaGlyProGlyThrPheGlyAspMetArglieAsnlieAsnAlaProLeu153€510tctcaaagatatcgtgtaaggattcgttatgettctacgacagatttaSerGinArgTyrArgValArglieArgTyrAlaSerThrThrAspLeu5155205251584caatttgtcacgagtattaatgggaccaccattaatattggtaacttcGinPheValThrSerlieAsnGlyThrThrlieAsnlieGlyAsnPhe5305355401632ccgaaaactattaataatetaaatactttaggttctgagggctatagaProLysThrlieAsnAsnLeuAsnThrLeuGlySerGluGlyTyxArg5455505555601680acagtategtttagtactccatttagtttcteaaatgcacaaageataThrValSerPheSerThrProPheSerPheSerAsnAlaGinSerlie5655705751728tttagattaggtatacaagcattttctggagttcaagaagtttatgtgPheArgLeuGlylieGinAlaPheSerGlyValGinGluValTyrVal5805855901776gataaaattgaatttattcctgttgaatagAspLyslieGluPhelieProValGlu*5956001806<210>5<211>601<212>PRT<"3>蘇云金芽孢桿菌<400>MetAsnSerLysGluHisAspTyrlieuLysValCysAsnAspLeuSer1510ISAspAlaAsnlie20AsnMetGluArgPhe25AspUysAsnAspAla30LeuGlulieGlyMet35Ser工leValSerGlu40IjeulieGlyMetlie45ProGlyGlyThrAla50IjeuGinPheValPhe55AsilGinLeuTrpSer60ArgLeuGlyAspSerGlyTrpAsnAlaPheMetGluHisValGluGlulieu工leAspThr65'707580LysneGluGlyTyr85AlaLysAsnLiysAla90lieuSerGluLeuAla95GlylieGluArgAsn100LeuGluThrTyrlie105GinlieuArgAsnGlu110TrpGluAsilAsplie115GluAsnSerLysAla120GinGlyLysValAla125AsnTyrTyrGluSer130lieuGluGinAlaVal135GluArgSerMetPro140GinPheAlaValGluAsnGluValProLeulieuThrvalTyrValGinAlaAlaAsn145ISO155160lieuHisLeulieuIjeuLeuArgAspValSerValTyrGlyLysCysTrp1653L70GlyTrpSerGluGinLyslieLyslieTyrTyrAspLysGilllieLys180185TyrThrHisGluTyrThrAsnHiaCysValAsnTrpTyrAsnLysGly195200205IjeuGluArglieuLysAsnLysGlySerSerTyrGinAspTrpiyrAsa210215220TyrAsnArgPheArgArgGluMetThrLeuThrValLeuAsplieVal225230235240AlaLeuPheProHis245TyrAspValGinThr2S0TyrProlieThrThr2SSValAlaGinUeuThr260ArgGluValTyrThr265AspProlieulieuAsn270PheAsnProLyslieuHisSerValSerGinLeuProSerPheAspMetGlu275280285AsnAlaThrlieArgThrProHisLeuMetGluPheLeuArgMetLeu290295300ThrlieTyrThrAspTrpTyrSerValGlyArgAsnTyrTyrTrpGly305310315320GlyHisArgValThrSerTyrHisValGlyGlyGluAsnlieArgSer325330335ProLeuTyrGlyArgGluAlaAsnGinGluValProArgAspPheTyr340345350PheTyrGlyProValPheLysThrIjeuSerLysProThrIjeuArgPro355360365IjeuGluGinProAlaProAlaProProPheAsnIjeuArgSerLeuGlu370375380GlyValGluPheHisThrProThrGlySerPheMetTyrArgGluArg385390395400GlySerValAspSerPheAsnGluIj已UProProPheAsnProValGly405410UeuProHisLysValTyxSerHisArgLeuCysHisAlaThrPheVal420425430ArgLysSerGlyThrProTyrLeuThrThrGlyAlaliePheSerTrp435440445ThrHisArgSerAlaGluGluThrAsnThr工leGluSerAsnlielie450455460ThrGin工leProIjeuValIjysAlaTyrGinlieGlySerGlyThrThr465470475480ValArgLysGlyProGlyPheThrGlyGlyAsplielieuArgArgThr485490495GlyProGlyThrPheGlyAspMetA巧lieAsn工leAs:nAlaProIjeu500505510SerGinArgTyrArgValArglieArgTyrAlaSerThrThrAspLeu515520525GinPheValThrSerlieAsnGlyThrThrlieAsnlieGlyAsnPhe530S35S40ProLysThrlieAsnAsnlieuAsnThrUeuGlySer*GluGlyTyrArg545550555560ThrValSerPheSerThrProPheSerPheSerAsnAlaGinSerlie56S570575PheArglieuGlylieGinAlaPheSerGlyValGinGluValTyrVal580585590AspLyslieGluPhelieProValGlu595<210>6<211>2208<212>DNA<2工3>蘇云金芽孢桿菌<220><221>CDS<222>(1)-.，(2208)<400>6atgcaaaat幼cgataat站cgaatatgaaELttattgattegcatMetAsnGinAsnAsnA6pAsnAsnGluTyrGlulielieAspSerHis151015ccttattttccgagaaacagtaatgattctagataccctThrSerProTyxProAanArgAsnSerAsnAspSerArgTyrPro202S30t3CaatSLSlt站tcaattacaa站c站ttsc站￡1弼TyrThrAsnAsnProAsnGinProLeuGinAsaThrAsnTyrLysGlu354045tggetcaatatgtgtc站gggcaatatggtgataatttc訴gTrpLeuMetCysGinGlyAsnTharGinTyrGlyAspAsnPheGlu505560acatttgetagtgetgatacaattgetgcagtt的tgcaggtactattThrPheAlaSerAlaAspThrlieAlaAlaValSerAlaGlyThrlie65707580"0gtatecValSerggtactctgGlyThrLeuB5ttalieugecAlaggtGly3t3lie抓Gly90gggGlyetcLieuactThrtctSer3ta_工le95tecSer28BggaccgGlyProataggaatalieGlylie100ggtGlygetAla3teilie105lieatalietctSertttPheGly110a_ccThrcta3363tC3CtlieThrgtcttttggValPheTrpliscccProAlaGly120GluGingacAsp3333C3Thr12SgtaValtggTrpThr384catstttGinPhe130attaaastg工leLysMetGlyGlu135attlietttPhegttValgatAsp3C3Thr140ccgPrott3LeuThrgaaGlu432ageataSerlie145aaacagctaLysGinLeusagLys150ttalieuGinsetThrttaUeu9站Glu155的3Glyttt3g3ArgGinlie160480ttacaaLeuGinagetataatSerTyxAsn165ThrgcaAlaLteu93tAspgatAsp170tggTrp3g3Arg3站LysttaLeu333Lys175Arg528Ct3C33IjeuGingetcctggaAlaProGly180tt3IjeuCC3ProCC3ProtC3Ser185tC3SerAlattaLeuC33GinGin190getAlaAla576ttgactLeuThrcttaaaatalieuLyslie19Scga_ArgtttgagGlu200站tgttC3CHis站tgatAsp20stttPheattliecgaArg€24g33a_ta>Glulie210cctggtttcProGlyPheGincttIjeu215GluactThrt&tTyracgThr220LeuIjeuCt3cctPro672atttatlieTyr225gcgcastgetAlaGinAlagetAla230sattttPhecatHisttaUeuaatAsn235ttaLeutt3Ginc貼GinGly240720getgaattggetgatgaatggaatgcagatatacatcctteacaaattAlaGluLeuAlaAspGluTrpAsnAlaAsplieHisProSerGinlie245250255768gaacctaatgetggaacateagatgactattataaacttttaaaagaaGluProAsnAlaGlyThrSerAspAspTyxTyrLysLeuLeuLysGlu2602S5270816aatatacctaaatatagtaactattgtgcaaatacctatagaacaggaAsnlieProLysTyrSerAsnTyrCysAlaAsnThrTyrArgThrGly27S2802858"ctaaaaaatcttagagacgaaccaaatatgaaatggagtatatttaatLeuLysAsnLeuArgAspGluProAsnMetLysTrpSer工lePheAsn29029S300312訴ctatcgaagatatatgaccattactgtatta訴taccatetctcaaAspTyrArgArgTyrMetTfarlieThrVallieuAspThrlieSerGin30S310315320960ttttctttatatgatataaaaagatatagagatteaataggaggaataPheSerLeuTyrAsplieLysArgTyrArgAspSerlieGlyGlylie3253303351008gaagtaaaaggcattaagaatgaaetcacaagagaa_atttatacaactGluValLysGly工leLysAsnGlulieuThrArgGlulieTyrThrThr340345350GlulieaatAsn355tttgatcgtcttPheAspArgLeucctPro360caa_ctta_gaGinJjeuArggttcaaValGin365cccPro33tAsnctalieu1104getacgatggaatataatttaacacgtgcaagttttaaattatttteaAlaThrMetGluTyrAsnLeuThrArgAlaSerPheLysLeuPheSer3703753801152tttttagaacaa匕ttattttttatacagaaaatacaaatttcgggaatPheLeuGluGinPheliePheTyrThrGluAsnThrAsnPheGlyAsn3853903954001200cgtttagttggtatttctaa匕cgtgatgcacctacttatageaatactArgLeuValGlylieSerAsnArgAspAlaProThrTyrSerAsnThr4054104151248ata工leactThrgaaGluactttatatggagaaThrLeuTyrGlyGlu420agaacaggtArgThr*Gly425tca_cccSerPro5LCELThr4306LC8LThrEL貼1296aca^taagaccatttgaatcttataaagttteaattgtaactgatagaThrlieArgProPheGluSerTyrLysValSerlieValThrAspArg4354404451344caateacctcctgttteccctattcaaccacactttataattaatcaaGinSerProProValSerPro工leGinProHisPhelielieAsnGin450455"01392attgaactttatttaaatggcteatctaacaacacaetcaaatattealieGlulieuTyrlieuAsnGlySerSerAsnAsnThrLeuLysTyrSer4654704754B01440gcaAlag9a999tctttatctaattatcaaaacacaactttttttcaatttGlyGlySerLeuSerAsnTyrGinAsnThrThrPhePheGinPhe4854904951488cctagaaaaaaagactgcaatctagttattgatccaggttgtteaccaProArgLysLysAspCysAsnLeuVallieAspProGlyCysSerPro5005055101536aactttaataactatagtcatattttateccattttteattatttactAsnPheAsnAsnTyrSerHislieLeuSerHisPheSerLeuPheThrSISS20S251584tattectatgtgattggattacagctacaaatattagatacaggtgtaTyxSerTyxVallieGlyLeuGinLeuGinlieLeuAspThrGlyVal53053S5401632ttaggatggacacacagtagtgttgatagatataafcgcaatateagatLeuGlyTrpThrHisSerSerValAspArgTyrAsnAlalieSerAsp5455505555601680aaaataattacaatgateccagcaateaaaggtaacaatcttgatacaLyslielieThrMetlieProAlalieLysGlyAsnAsnlieuAspThr5655705751728aactctaaggtaatt訴aggacctggtcatacaggaggaaacttggttAsnSerLysVallieGluGlyProGlyHisThrGlyGlyAsnLeuVal58058SS901776tatttacaaagtcaagggcgtttagaaattacatgtgaaactcctaatTyrLeuGinSerGinGlyArgLeuGlulieThrCysGluThrProAsnS356006051824tcttctttcattagaicttcgaitatgetgetSerThrGinSerTyrPhelieArgLeuTyrAlaThrAsnGlyAla610615cttcctaatatatctctta_taccaigta的3GlyAsnThrLeuPro工leSerLeuThrlieProGlyVallieGly625630635640CC3cctcgaetcactttttctggttat站tlieProProGinArgLeuAsnAshThrPheSerGlyAsnTyrAsn645650655ttatacgattttgggtatttctt匕CC3gtalieuGinTyrGlyAspPheGlyTyrPheGinPheProSerThrVal"5670ttacctttsaatCC3ttttttcgtgcagatThrLieuProIjeuAsnArgAsnlieProPheliePheAsnArgAlaA印675680685gtateatceiattttsatesttgatattgaatttataccaiValSerAsnlieLeulielieAsplieGluPhelieProlie690695700acttectctatgC3Caatagas站c站ttagaaacta±cThrSerSerMetHisGinAsnArgGluLysGinLysIjeuGluThrlie705710715720tttttcCSLtcttGinThrLyslieAsnPhePheThrHisThrThrLeu725730735<=210>7<211>735<212>PRT<213>蘇云金芽孢桿菌<400>7MetAsnGinAsnAsnAspAsnAsnGluTyrGlulie工leAspSerHis151015ThrSerProTyrPheProAsnArgAsnSerAsnAspSerArgTyrPro202530TyrThrAsnProGinProLeuGinAsnThrAsnTyrLysGlu354045TrpLeuAsnMetCysGinGlyAsnThrGillTyrGlyAspAsnPheGlu505560ThrPheAlaSerAlaAspThrlieAlaAlaValSerAlaGlyThrlie707580ValSerGlyThrUeuLeuAlaGlylieGlyGlyLeuThrSerlieSer85GlyProlieGlylielieGlyAlalielielieSerPheGlyThrLeu100105110lieThrValPheTrpProAlaGlyGluGinAspLysThrValTrpThr115120125GinPhelieLysMetGlyGluliePheValAspThrProlieuThrGlu13013SSerlieLysGinLeuLysLeuGinThrlieuGluGlyPheArgGinlie145150155lieuGinSerTyrAsnThrAlaLeuAepAspTrpArgLysLeuLysArg165170175lieuGinAlaProGlyLeuProProSerSerAlaLeuGlxiGinAlaAla180185190LeuThrLeuLyslieArgPheGluAsnValHisAsnAspPhelieArg19520020SGlulieProGlyGinlieuGluThrTyrLysThrlieulieulieuPro187219201968201620642112216022&8210215220lieTyrAlaGinAlaAlaAsnPheHisLeuAsnIjeuLeuGluGillGly225230235240AlaGluLeuAlaAspGluTrpAsnAlaAsplieHisProSerGinlie250255GluProAsnAlaCJlyThrSerAspAspTyrTyrLysLeuLeuLysGlu2S5270AsnlieProLiysTyrSerAsnTyrCysAlaAsnThrTyrArgThrGly27528028SUeuIiysAsnIjeuArgAspGluProAsnMetLysTrpSerliePheAsn290295300AspTyrArg■ArgTyrMetThrlieThrValLeuAspThrlieSerGin305310315320PheSerIjeuTyrAsplieIiysArgTyrArgAspSerlieGlyGlylie325330335GluValLjysGlylieLysAsnGluJjeuThrArgGlu工leTyrThrThr340350GlulieAsnPheAspArgLeuProGinLeuArgValGinProAsnIjeu355360AlaThrMetGluTyrAsnLeuThrArgAlaSerPheIiysLeuPheSer370375380PheIjeuGluGinPheliePheTyrThrGluAsnThrAsnPheGlyAsn385390395400ArgLieuValGly工leSerAsnArgAspAlaProThr"TyrSerAsnThr405410415lieThrGluThrIjeuTyrGlyGluArgThrGlySerProThrThrLys420425430ThrlieArgProPheGluSerTyrLysValSer工leValThrAspArg435440445GinSerProProValSerProlieGinProHisPhelielieAsnGin450455460lieGluLeuTyrIjeuAsnGlySerSerAsnAsnThrLysTyrSer"547047S480AlaGlyGlySerlieuSerAsnTyrGinAsnThrThrPhePheGinPhe48549049SProArgLysLysJVspCyslieuVal工leAspProGlyCysSerPro500S05510AsnPheAsnAsnTyrSerHislieleuSerHisPheSerlieuPheThr515520525■TyrSerTyrVallieGlyIjeuGinljetiGinlielieuAspThrGlyVal530535540lieuGlyTrpThrHisSerValAspArgTyrAsnAlalieSerAsp54S550555560LyslielieThrMetlieProAlalieLysGlyAsnAsnLeuAspThr570575AsnSerLysVallieGluGlyProGlyHisThrGlyGlyAsnIjeuVal580585TyrLeuGinSerGinGlyArgLeuGlulieThrCysGluThrProAsn595600605SerThrGinSerTyrPhelieArgLeuArgTyrAlaThrAsnGlyAla610615GlyAsnThrlieuProAsnlieSerLeuThrlieProGlyVallieGly625。0635640lieProProGinArgLeuAsnAsnThrPheSerGlyThrAsnTyrAsn645AsnLeuGluTyrGlyPheGlyTyr<31nPheProSerThrVal660665670ThrUeuProUeuAsnArgAsnlieProPheliePheAsnArgAlaAsp67S68068SValSerAsnSerlieLeulieUeAspIyslieGluPhelieProlie690700ThrSerSerMetHisGinAsnArgGluLysGinLysLeuGluThrlie705710715720GinThrLyslieAsnThrPhePheThrAsaHisThrLysThrLeu72573073S<210>8<211>2235<212>DNA<213>蘇云金芽孢桿菌<220:<221=CDS<400>8gtgaatMetAsnC33GinaatAsn33tAsnaatsatAsnAsngaatatgagGluTyrGlu10attatelieliegatteaa_agAspSerLys15Asn48tt3tctUeuSertatTyrcctPro20tctSerAsnArga^atattgatAsnlieAsp25cattctHisSeragsLta_ccctArgTyrPro30tELCTyx96ThrAsn33tAsn35Pro33tAsnCC3GinProttacaiasacLeuGinAsn40ThrAsntacaaagagTyrLysGlu45Trp144etceiatlieuAsn50a_tgMettgtCysGingggaatGlyAsn55acacaatatThrGinTyrggtgstGlyAsp60aa_tttcgagAsnPheGluThi:192tttgetagtgetgatacaattgetgcagttagtgcaggtactattgtaPheAlaSerAlaAspThrlieAlaAlaValSerAlaGlyThrlieVal65707580240tecggtactctgttagecggtataggtgggetcacttctatatecggaSerGlyThrLeuLeuAlaGlylieGlyGlyLeuThrSerlieSerGly859095288ccgatag訴ataataggtgetataataatatcttttggtaccetaateProlieGlylielieGlyAlalielielieSerPheGlyThrLeulie100105110336actgtcttttggcccgcgggagaacaagacaaaacagtatggacacaaThrVelIPheTrpProAlaGlyGluGinAspLysThrValTrpThrGin115120125384tttattPhelie130333MetggaGlygaa3ttGlulie135tttgttgatPheValAspacaccgThrPro140tta_eicagas3gcUeuThrGluSer4323teiaaacagcteiaagttaicastactttagaaggatttagacslbl&tatt&lieLysGinlieuLysLeuGinThrLeuGluGlyPheArgGinlieLeu145ISO1551604B0caaagetataatacagcattagatgattggagaaaattaaaaagaetaGinSerTyrAsnThrAlaLeuAspAspTrpArgLysLeuLysArgLeu16517017S528caagetcctggattaccaccateateagcattacaacaagetgecttgGinAlaProGlyLeuProProSerSerAlaLeuGinGI31AlaAlaLeu180185190S7SactcttaaaatacgatttgagaatgttcacaatgattttattcgagaaThrLeuLyslieArgPheGluAsnValHisAsnAspPhelieArgGlu195200205624atacctggtttccaacttgaaacttataaaacgetattaetacctattlieProGlyPheGinLeuGluThrTyrLysThrLeulieuLeuProlie210215220672tatgcgcaagetgetaattttcatttaaatttattacaacaaggtgetTyrAlaGinAlaAlaAsnPheHisLeuAsnLeuLeuGinGinGlyAla22S230235240720gaattggetgatgaatggaatgcagatatacatcctteacaaattgaaGluLeuAlaAspGluTrpAsnAlaAsplieHisProSerGinlieGlu2452502S5768cctaatgetggaacateagatgactattataaacttttaaaagaaaatProAsnAlaGlyThrSerAspAspTyxTyrLysLeuLeuLysGluAsn2S0265270816atacctaaatatagtaactattgtgcaaatacctatagagaaggaetalieProLysTyrSerAsnTyrCysAlaAsnThrTyrArgGluGlyLeu27S280285864aataaacttcgaaacgaacctaatatgagatggagtatatttaatgatAsnLysLeuArgAsnGluProAsnMetArgTrpSer*liePheAsnAsp290295300912tatcgaagatatatgactattactgtattagatactategetcaatttTyrArgArgTyrMetThrlieThrValLeuAspThrlieAlaGinPhe3053103153209S0tctttttatgatataaagagatacaaagatteaataggaagaataggtSerPheTyrAsplieLysArgTyrLysAspSerlieGlyArglieGly3253303351008ggcattaaaactgaacttacaagagaaatttatacaactgaaataaatGlylieLysThrGluLeuThrArgGlulieTyrThrThrGlulieAsn3403453501056tttgsiccgtcttacttaccttgaasttcsiaccceieitetcgetatasttgPheAspArgLeuThrTyrLeuGlulieGinProAsnLeuAlalieMet3553603651104gaatataatttaacacgtteagggcttagattatttteatttttagatGluTyrAsnLeuThrArgSerGlyLeuArglieuPheSerPheLeuAsp3703753801152gaacttatattttatacaaaaaatgaaacgtacgggaatcgtttagttGluLeuliePheTyrThrLysAsnGluThrTyrGlyAsnArgLeuVal3853903954001200ggtattgcgaatcgtaatagatctacttatgetacgacaggaactgaaGlylieAlaAsnArgAsnArgSet:ThrTyrAlaThrThrGlyThrGlu4054104151248attatatatggagaaagaacaggtccacccacaacaaaaactttaatalielieTyrGlyGluArgThrGlyProProThrThrLysThrlieulie420425430ccatttgaatectataaagttteaattgtaactgatagacaagtaactProPheGluSerTyrLysValSerlieValThrAspArgGinValThr4354404451344cctacttecccttttProThrSerProPhe450cctaacatatactttacaattaatcaaattgaaProAsnlieTyrPheThrlieAsnGinlieGlu45S4601392ctttatttaaataatteacctagtaataaattaacatatteaget的glieuTyrLeuAsnAsnSer*ProSerAsnLyslieuThrTyrSerAlaGly4右54704754801440gggaatttatctaatgataaaaaaacaactgattttcaatttcctgtaGlyAsnLeuSerAsnAspLysLysThrThrAspPheGinPheProVal48S4304351488幼aaaagactgtaaaccaattattaatccaaattgtttaccaagetatLysLysAepCysLysProlielieAsnProAsnCyeLeuProSerTyrS0050SS1015363at3gttatAsnSerTyr515agtcatSerHisattlieLeuteccagSerGinS20tttPhetctSerttaLeutttPhe525AsntatTyrtecSer1584tst3a_aattTy;rLyslie530ggattaGlyLeugcgAlaCt3Leu53533tstaAsnlieLeutatTyrThr540GlygcaAlattaLeuGlytggacacacTrpThrHis545agtagtSerSergttVal550tagaaatArgAsn33tAsngcaAla5553t3lietC6LSerAsp333Lys3t3工le560attacaatglieThrMet3tcccaliePro565gcaAlalieasaggtLysGly33CAsxi570agtSercttgatA印ThrAsn575tctSer1728aaggtaattLysValliegasggaGluGly580cctProGlyC3t3C3HisThr585GlyGlyAsnttgLeugtt590tatTyrttaLieu1776caasgtcaaGinSerGin595gggcgtGlyArgLeu弼Gluattaca工leThr600tgtCysArgactThrcctPro605AsntctSerThr1824C33tCtt3tGinSerTyx610tscattTyrlieArgcttLeu615cgatacArgTyrgetAlaThrsatAsn620ggtGlygetAlaggaGlyAsn1872setcttcctThrUeuPro625aatataAsn工letctSercttThrlieProGly635gtaValatalie3t3liePro6401920cctcaacgaProGinArgCtc33CIjSuAsn"5站cAsnactThrttttctPheSerggtGly650Thr站tTyr33taatAsn655lieuc站tacggaGinTyxGlygattttAspPhe"0鵬GlytatTyxttccaia_PheGinSS5tttPheCC3ProSerThrVal670ttaLeu2016ccttteLaatProIjeuAsn675cgsa^cArgAsn3t3lieProtttataPhelie680tttPhe的tAsncgtAla68593tAspgtaValtC3Seraatteaa_ttAsnSer工le690ttaateLeulieattliegatAsp695aeiaattUyslieGlutttPheata工le700Prolie3CtThrtecSer2112tctgtacgcSerValArg705Caa8L￡Lt3諷GinAsnArg710Glu3站LysGin￡1站Lystta715gaaGluactThratelieC站3C3GinThr720aaaataaatacatttttcacaaaitcatacaaaaaatactttaaatataLyslieAsnThrPhePheThrAsnHisThrLysAsnThrLeuAsnlie7257307352208gaagecacaaactat訴tattgattaaGluAlaThrAeuTyrAsplieAsp*7402235<210>9<211>744<212>PRT<2">蘇云金芽a菌<400>9<table>tableseeoriginaldocumentpage82</column></row><table>TrpThrHisSerSerValAsnArgAsnAsnAlalieSerAspLyslieS45550S55S60lieThrMetlieProAlalieLysGlyAsnSerlieuAspThrAfinSer565570575LysVallieGluGlyProGlyHisThrGlyGlyAsnLeuValTyr!Leu580585590GinSerGinGlyArgLeuGlulieThrCysArgThrProAsnSerThr595600GinSerTyrTyrlieAxgLeuArgTyrAlaThrAsnGlyAlaGlyAsn610620TtirLeuProAsnlieSerIjeuThrlieProGlyVallieGly工lePro625630635640ProGinArgLeuAsnThrPheSerGlyThrAsnTyrAsnAsnIjeu"565SGinTyrGlyAspPheGlyTyrPheGinPheProSerThrValThrLeuProLeuAsnArgAsnlieProPheliePheAsnArgAlaAspValSer675685AsnSerlielieu工lelieAspLyslieGluPhelieProlieThrSer690695700SerValArgGinAsnArgGluLysGinliysLeuGluThrlieGinThr705710715720LyslieAsnThrPhePheThrAsnHisThrLysAsnThrLieuAsnlie72573073SGluAlaThrAsnTyrAsplieAsp740<210><211><212><213>102085DNA蘇云金芽孢桿菌<220><221>CDS<222>(1).<2085)<400>10atgtgtcaaMetCysGingggaatacacaatatGlyAsnThrGinTyrggtgataatttcgagacatttget48GlyAspAsnPheGVuThrPheAla1015agt9ct9atacaatt9ct9cagttagtgcaggtactatt'gtatecggtSerAlaAspThrlieAlaAlaValSerAlaGlyThrlieValSerGly20253096actctgttagec的tataggtgggetcacttctatatecggaccgataThrLeuLeuAlaGlylieGlyGlyLeuThrSerlieSer(31yProlie354045144ggastaataggtgetataataatatcttttggtaccetaateactgtcGlylielieGlyAlalielielieSerPheGlyThrLeulieThrVal50S560192ttttggcccgcgggagaacaagacaaaacagtatggacacaatttattPlieTrpProAlaGlyGluGinAspLysThrValTrpThrGinPhelie65707580240aaaatgggagaaatttttgttgatacaccgttaacagaaageataaaa288LysMetGlyGluliePheValAspThrProLeuThrGluSerlieLys85909Scagetaaagttac站actttagaag訴tttagacaastattacaaageGinLeuLys'LeuGinThrI*euGluGlyPlieArgGinlieLeuGinser100105110tat站tacagcattagat訴ttggaga站atta站aagaetacaagetTyrAsnThrAlaLeuAspAspTrpArgLysLeuLysArglieuGinAla384115120125cctggaProGly130ttaProccateateagcattacaacaagetgccttgactcttProSerSerAlaLeuGinGinAlaAlaLeuThrLeu135140432aaaatacgatttgagaatgttcacaatgattttattcgagaaatacctLyslieArgPheGluAsnValHisAsnAspPhelieArgGluliePro1451501551G0480ggtttccaacttgaaacttataaaacgetattaetacctatttatgcgGlyPheGinLeuGluThrTyrLysThrLeuLeuLeuProlieTyxAla165170175S28caagetgetaattttcatttaaatttattacaacaaggtgetgaattgGinAlaAlaAsnPheHisLeuAsnLeuLeuGinGinGlyAlaGluLeu180185190576getgatgaatggaatgcagatatacatcctteacaaattgaacctaatAlaAspGluTrpAsnAlaAsplieHisProSerGinlieGluProAsn1S5200205624getggsacateagatgactattataaacttttaga_aaa_tstacctAlaGlyThrSerAspAspTyrTyxLysLeuLeuLysGluAsnliePro21021S220672aaatatagtaactattgtgcaaatacctatagagaaggaetaaataaaLysTyxSerAsnTyrCysAlaAsnThrTyxArgGluGlyLeuAsntiys225230235240720cttcgaaacgaacctaatatgagatggagtatatttaatgattatcgaLeuArgAsnGluProAsnMetArgTrpSerliePheAsnAspTyrArg2452S0255768agatatatgactattactgtattagatactategetcaattttcttttArgTyrMetThrlieThrValLeuAspThrlieAlaGinPheSerPlie2602S5270816tatgettataaagagataca_aagatteaataggaagaataggtggcattTyrAsplieLysArgTyrLysAspSerlieGlyArglieGly*Glylie2752802858S4aaaactgaaLysThrGlu290cttacaagagaaattlieuThrArgGlulie295tatacaactgaaataaattttgacTyrThrThrGlulieAsnPheAsp300912cgtcttacttaccttgaaattcaaArgIjeuThrTyrlieuGlulieGin305310cccaatetcgetataatggaatatProAsnLeu^lalieMetGluTyx315320960aatttaacacgtteagggcttagattaAsnLeuThrArgSerGlyljeuArg:Leu325tttteatttttagatgaacttPheSerPhelieuAspGluIjeu3303351008atattttatacaaaaaatgaaacgtacg的aatcgtttagttggtattliePheTyrThrLysAsnGluThrTyrGlyAsnArgLeuValGlylie34034S3501056gcgaatcgtaatagatctacttatgetacgaca的aactgaaattataAlaAsnArgAsnArgSerThrTyrAlaThrThrGlyThrGlulielie3S53603651104tstgga訴a的aacaggtccacccacaacaaaasetttaatacceitttTyrGlyGluArgThrGlyProProThrThrLysThrlieulieProPhe37037538011S2gaatectataaagttteaattgtaact訴tagacaagtaactcctactGluSerTyrLysValSerlieValThrAapArg<31nValThrProThr*1200385390395400tecccttttcctaacatatactttacaattaatcaaattgaactttatSerProPheProAsnlieTyrPheThrlieAsnGinlieGluLeuTyr40541041S1248ttaaataatteacctagtaataaattaacatatteagetggggggaatUeuAsnAsnSerProSerAsnLysLeuThrTyrSerAlaGlyGlyAsn4204254301296ttatctaatgataaaaaaacaactgattttcaatttcctgtaaaaaaaLieuSerAsnAspLysLysThrThrAspPheGinPheProValLysLys4354404451344gactgtaaaAspCysLys450CC3Pro3tt3tt工le工leaatccaaattgtAsnProAsnCys455tt5LCC3LeuPro460ageSertatTyr3a±Asn1392tatsgtestTyrSerHis465liettatec:LeuSer470cagttttctttaGinPheSerLeutttaatPheAsn475TyrtecSerTyr4B01440attggattagcgetaaatatattatatacaggtgcattaggatggacalieGlyLeuAlalieuAsnlieTbeuTyrThrGlyAlaIjeuGlyTrpThr4854904S51488ca_cagtagtHisSerSergttVal500aatagaaataatgcaataAsnAsnAlalieS05teagatSerAspa祖Lys3tslieS103tt工le3C3ThratgateccaMetliePro515Alaateaa>3lieLysggtaacagtcttGlyAsnSerLeu520gatacaAspThrAsn525tctSersagVal1584attgaaggalieGluGly530cctProggtcatGlyHisseaggagg3aacThrGlyGlyAsn535ttggttLeuVal540t3tttalieuGinSei:1632csagggcgtGinGlyArg545gagattGlulie550acatgtagaactThrCysArgThrcctaieitProAsn555tctSera_caGintctSer5601680teittacattTyrTyrlieArgcttcgaUeuArg565tacgetacaaatTyrAlaThrAsn570ggtgetGlyAla99333tactThr575cttlieu1728cctsatataProAsnlietctcttacaSerlieuThr5803t3ccaggagtalieProGlyVal585atagga>lieGly3t3lieCC3Pro590cctProGin1776cgaetcaacaacactttttctggtacaaattataataatttacaatacArgLeuAenAsnThrPheSearGlyThrAsnTyrAsnAsnLeu<31nTyr59560060$1824ggagattttgggtatttccaatttccaagtacagt汰acattacctttaGlyAepPhe'GlyTyrPheGinPheProSerTlirValThrLeuPro3Jeu€10615€201872aatc9a幼cataccatttatatttaatcgtgcagatgtateaaatteaAsnArgAsnlieProPheliePheAsnArgAlaAspValSerA8iiSer625630635640attttaateattgataaaattgaatttataccaattacttectctgbalielieulielieAspLyslieGluPhelieProlieThrSerSer*Val"S650cgccaaaat的agaaaaacaaaaatta.gaaactatecaaacaa—aataArgGinAenArgGluLysGinLysLeuGluThrlie61nThrLyslie:2016"06S5670aatacatttttcacaaatcatacaaaaaatactttaaatatagaagccAsnThrPhePheThrAsnHisThrLysAsnThrLeuAsnlieGluAla67568068520643caaactatThrAsnTyr690gatattgattaaAsplieAsp*2085<210>11<211>694<212>PRT<2">蘇云金芽孢桿菌<400>11MetCysGinGlyThrGinGlyAspAsnPheGlqTh;rPheAla151015SerAlaAspThr20lieAlaAlaVal25GlyThrlieVal30SerGlyThrLeuAlaGlylieGlyGlyLeuThrSerlieSerGlyProlie35404SGlylie50lieGlyAlalielie55lieSerPheGlyThrIjeu60lieThrValiPheTrpProAlaGlyGluGinAspLysThrValTrpThrGinPhelie65707580LysMetGlyGlulie85PheValAspThrPro90LeuThrGluSerlie9SLysGinlieuLysLeu100GinThrlieuGluGlyPhe105ArgGinlieLeu110GinSerTyrAsnThrAlalieuAspAspTrpArgLysLeuLysArgIjeuGinAla115120125ProGlyProProSerSerAlaIjeuGinGluAlSAl2LLieuTlirlieu130135140ljysliePheGluAsnValHisAsnAspPhelieArgGluliePro145150155160GlyPheGinlieuGlu165ThrTyrLysThrIjeu170LeuUeuProlieTyr175AlaGinAlaAlaAsn180PheHisIjeuAsnLeuLieu185GinGinGlyAla190GluUeuAlaAspGlu195TrpAsnAlaAsplie200HisProSerGinlie205GluProAsnAlaGly210ThrSerAspAspTyr215TyrLysIjeuLeuLysGlu220AsnlieProLysTyrSerAsnTyrCysAlaAsnThrTyrArgGluGlyIjeuAsnLys225230235240IjeuArgAsnGluPro245AsnMetArgTrpSer250liePheAsnAspTyr255ArgArgTyrMetThrlieThrValLeuAspThr*lieAlaGinPheSerPhe260265270TyrAsplieLysArgTyrLysAspSerlieGlyArglieGlyGlylie275280285LysThrGluIjeuThrArgGlulieTyrThrThrGlulieAsnPheAsb235300ArgLeuThrTyrLeuGlulieGinProAsnlieuAlalieMetGluTyr30531031S320AsnlieuThrArgSer32SGlyLeuArgLeuPhe330SerPhelieuAspGlu335lieuliePheTyrThi1LysAsnGluThrTyrGlyAsnArgLeuValGlylie34034S350AlaAspArgAsnArgSerThrTyrAlaThrThrGlyThrGlulielie355365TyrGlyGluArgThrGlyProProThrThrLysThrLeulieProPhe37037S380GluSerTyrValSerlieValThrAspArgGinValThrProThr385330400SerProPheProAsnUeTyrThrlieAsnGinlieGluLeuTyr40S410415IjeuAsnAsnSerProSerAsnLysLeuThrTyrSerAlaGlyGlyAsn420425430LeuSerAsnAspLys:LysThrThrAspPheGinPheProValLysLys435440AspCysLysProlielieAsnProAsnCysIjeuProSerTyrAsnSer450455460TyrSerHislieLeuSerGinPheSerLeuPheAsnTyrSerTyrIiys470475480lieGlyIjeuAlaIjeuAsnlieLeuTyrThrGlyAlaIjeuGlyTrpThr485490495HisSerSerValAsnArgAsnAsnAlalieSerAsplielieThr500505510Met工leProAla工leLysGlyLeuAspThrAsnSerLysVal515520S25lieGluGlyProGlyHisThrGlyGlyAsnLeuValTyi:LeuGinSer530535540GinGlyArgLieuGVulieThrCysArgThrProAsnSerThrGinSer545550555TyrTyrlieArgLeuArgTyrAlaThrAsnGlyAlaGlyAsnThrLeu565570575ProAsnlieSerLeuThrlieProGlyVallieGly工leProProGin580585ArgLeuAsnAsnThrPheSerGlyThrAsnTyrAsnAsnIjeuGinTyr5S5600605GlyAspPheGlyTyrPheGinPheProSerThrValTharLeuProLeu615AsnArgAsnlieProPheliePheAsnArgAlaAsjpValSerAsnSer62S635"0lielieulielieAspIiyslieGluPhelieProlieThrSerSerVal65SArgGinAsnArgGluLysGinLysLeuGluThrlieGinThrLyslie665670AsnThrPhePheThrAsnHisThrLysAsnThrLeuAsnlieGluAla675680685ThrAsnTyrAsplieAsp690<210>12<211>5，<212>DNA。lh蘇云金芽孢桿菌<400>12tacatgcaataceitaaagagaaggtttaaatatttgtagaaacattgttacaggaatacact站caLtatatttaLttaggtgtcttaaaaagtccatatcaaaataaaaatgaatatgaaacgccaaacagatatccttttgcaa汰taatcgtcagggaatctcat的gatgaaatttgggtaaatcttat3gagtttgtgatagaacctataatagga^aactaAttccgactaatcgtctattatctataattcgftaaagaggtagccgttgacggtaaattga站aattatagagagtaagacggtaaaccacttcaa站gacaaatattaaactttcagaaagagatttcaatgaaactcaagtattgttattacctacttttgcacgggettgcagttcaatataaagcac站t的tcg訴att站tatcacctaaceigtggttgtg站tgcaa幼cggcagagtataccaattattagat站aacagg的的gacaiggtgctgacaaaatgacgttggcggtattggatattatcgatccattgccaacacatgtagagtt訴ctecatcgggaacttatagttggttacggaattatggaacacggg3acct33tttagttacttatgtttcgagatattttgccggctg93taggtaacggtatttttcaacgtatgtctggakaaatcaatacctcaccaaaaataatgggtt60ttggggtactacgaatatatagaaagstcac120taaggactaitataaggagtgaaaaa訴tgsi180tattggaatcctcatcgaataacacaaata240gggatatgtctactatgtcttggaatgatt300aatcagtcgaaacgataacaagtattggga360gtttgggtggaaittaat3C3ctattatc貼420aaactgtgtcagcactttctatatgtgatt480atagtgttttaagtgatgcgattgcagattS40attatctttcttatcttggggcttggctta600attctgatatcggacaattsigtttattatt660ttctagga的gtcattgtcsLagaaacaatg720aagctgcaaatgtgcagttattactattaa780tcccatttttgagtgcagagaatgtaagat840attttacc的tgattactatgagc9attasL300gtttatattggtatcaggtaggtttaaatccttggtcgaaattt站taaatttcgtagag1020ctat汰tttccaacttatgattttgagaaat1080gggaaatttatac的atgcagtgggatatt1140ggcctaatactttt站tgggttagaggcta1200ggcttagcaaaata訴tatatataatgagt1260g站ctc9tcattatgaagactacacaaagg1320ctacgagtaatgatctacgtaatattgatt1380atttaaattacacttgtattgtgttgtatagtgaaaatatttcaa^SLtts3ttt33tccgttagattgatgttttgtatcttaaattcgctcccaagtaaacgcggtgaagctgtttaactacgttasaatatgtcagatgacattcggtEicagatgcgtaatagatgagtagtaaagattgttccasatatcatcgtcttatgtatgcaatttgattttcttattaeiaaacaacacstgtgtagatgcatceiaLaeLcgcttgctccaggtta3tttatatatggcscgcaatcaaattgggttacgtgtgagtagaggtaattttatatagcscacgggttgtgtattacaaattatacttggctgtacatgcga站站ttaacggacatccccaatatttaaatgttaagtgaatgcaattttgttcgt的tacataaccgggttaaatgttttcccagaattcacaaataattcttgattcgtttgactcttgcaggtttcgstcat3cttgccatttgagctttttttttgaattaagataatcttggtttcttttcacggtctgtacgatgt3tataccagagtattgatgcaggtgcactctaattggaaactccaattgaagcctcttgctaatttttttaagaagctgctttcgcttgaaaaatgaaattatcgaatagttttcgatctgtttaaataagcgaatagaaacgagaacaaaatcagtcagtctcaatgatattaggagcacgagtctaaattaagatttggcctcagtatgtgatgcccgcaaacatattgeicaatttctaatcctaacaggtggagaaaaaacttattttacaagatcgactggatgaactatggatgcacgaaacaggaaatgagcgcgggggattgccaaaa一caggei站cacgatagcatcgaatatgtatgcaagggtataaacaatgatgtgtaatcaagaaaascctacgattaatgaacssttcctgattttggtatttgatatgagcaacagtcggcctagttcatctcccttgtttcttgatatcctgtgttcttggccsttttsa訴gattcaattttcatcatattcgttgtatttgctgaagattgtggatacc幼ccattctBtattt訴atascccgctgaccaatcttcattctgtaaaasaaattacttcgtgtttcaaaataatgggcggtgttagagagagttaacggcctstacctacaaaatcaga_aaactgcagtttagtgtattcttactctgacacctccatcaatcagatgttagaatatgsccaattaaaaaaaggttgtcagattatgagtaatctacttcataggatcacatatatggaaccatatactggtaacacgatatgcagccgccaagggtaatatag&tagccgg3g站gtcagatggataaagcattggc9ttgg9貼acatgaacaagatacagtcgatatgtatatcaatgtcataacccgcggtELCstatctc33a3ttaagttcacgtgatcgtgtagttgctttgagcaaagttacaasc5t3Ct3acctgcattcgatataaccgtcac幼幼tcaggtctccttaatactcctttccagaag3tstgatggaccaataatatgataaaattaattaaagcctcttccaagtaatattcgttgtactctcgaaatatggtctatacataatgagatctgtttcgtgtcacttgtttcattctctacctgttaaacggtaaatatta幼atctcttatgttctcgccattagctataggcagattttaagcaggatgaccctctcatatatggttgatgtaataaagacaaagttaatctttatccgasattatgatacctagttcagggagaacggagtcctatgaatagatcaaaatastgttaccaaaatggaCS3t33tCCtBactct3tttacgttatcgattacgggaaa_taLaitccttC3LtCCt3C3CC3aaagcatgtgagatacaaatcgctacagta3catgtg站agttacactattccctatgtaagagttaaacaaatggtgaccttgcatgctttcttgcgaaacggattgaatatgcaaggataatggaaatcggttgcsLcattaacgattctattgcat5Lt站agcatttcaaacggtcca站ttctaagtaataggagctaaatcacaggtctaELt的acgccttc3貼caaa的attttttcgctttctatatagcttaatgaacccctaatctattaagagtctgcaaacagtaagtgaagcttttcttgaaa站tctcccatagaatcttctgcggtcatgaacctatcgtagggtagacaeLttgaagacacatacaggcggtgaagaggcsctcatagtttatgcaccgtattcgtcgatttaaaaaaacattcagtctcga_cateLa_ggacctagaattactagaattttacaagtagcggccattttgggatgaaagacttttctgatttttaaaggtaagagggtgaaattgatgaagtgtcacgacaaatgtaggggaatctagtatgatccagcatattacttteLCcatasittgcacgtatcatttacacaagggagcttcsigcaagatcatctatgtaacgagaaggatataataggagaaatctagtaatatgtaaccaagta貼taagaacataacataataaggagatttttaggctgag的gcatgcagtaaacgataccaatgcagattgaagtaacccgctccttgcttcaatttgctactaaaitcct站tgctgtgtttcccttcgaatttgttctgattaagttgcagaaattgaatgcagt訴ttctaaagttaacaggtggctatgatatcgtagctgctgtttcatccttaagaataacgacggcctactcgtaggagagatctacgttccaatgcggcatagtttaaaacagttattaccacsggtggggtttcgttatgggttcaaatctatagtgatt3tggatgtggattgaattccaggtggtatttaaaactggcggaatgattggaaaatttcttctgtcaaaattgtgggaagctatcatgaaaitcgctgtgaatgctcccgaatcgtcatccgtattgatgtaagtcattgasg站atggaacaaaacaggctagatcatatggttaccg站tgcaaggttagattacagggtaittagttctatggatatgttgatggattgatactagcgatgtataatcaggcataactctcaacatcattttttcgtttttggtgttttgctactttttactttcacctsgtaagccggtatgacacttcttttgsaatcttcttgatgagcta站tgt3幼站3tcgcactccattcatgctacttttttctttggtttttacaggcgggacaaagctgtatggttttagttcgtgagaatgagtattacaacgcgtggtgtgcatat祖tt諷tccc1440150015601620168017401800I860192019802040210021602220228023402400246025202S8026402700276028202880294030003060312031803240330033G0342034803S40360036S037203*7803840390039604020408041404200426043204380"4045004560468047404800486049204980504051005160522052805340S4005460attcttttttaatgataatcacttgttttaatttctccatatttatttaccgttgttgtgggtaatggtttcaatgcagccttatcatcatcataatgtaggcgtttctgstcttctattgaaaa_3ctctccata_a_tcggagcggttgtacgttcaacgttccccttttcatgcccgctataatgcatttgaaattgtaaaaatgcatccgtgactacagcggcagataagttatcaatttgaaataatcgttttaagccttctaaaaagcgtaaaacaaaacgttcttgatccactgtg5520tccattgagaagaccgttaggtcttccaaa5S80ttccaaaggtcttcaatcatgacttccttcS640gcttgtacttctaaccattgtgcgagttga5700aaccaattgtttcgtgtataactgactttc5760taaggattacaaggttgcacttcaaagtta5820gtataagttcgattatcaccttttcctact5880cgaagatgcgttggtacaccaccagcttga5940ccttctgtat5980<210>13<211>2082<212>DNAdlh蘇云金芽孢桿菌<220><221>CDS(1)-.(2082)<400>13gtgaaaaagLysUysMet;atgagtMetSer5CC3Prot3tc站GinsateL33Lys10331AsngaaGlutatTyargasGlu3t3lie15ttgLeu48gaaGlutecteaSerSerteg站t20AsnTtiraicgThr2Scca_ProtatTyrcctPro30tttPheAla96aataategggatatgtctactatgtcttggaatgattgtcagggaateAsnAsnArgAspMetSerThrMetSerTrpAsnAspCysGin(Slylie354045144teatgggatgaaatttgggaateagtcgaaacgataacaagtattgggSerTrpAspGlulieTrpGluSerValGluThrlieThrSerlieGly50S560"1S2lie65aatcttAsnlieuatagaglieGlutttPhe70ValataliegaaGlucctProagtSer75ttgLeuggtGlyGlyattlie貼t80240ThretattaIjeulieutC3Serlie85lieGly333cta_Leuattlie卯ccgProactThraatcgtArgc幼Gin95actThr288gtgteagcactttctatatgtgatttattettctataattcgtaaageigValSerAlalieuSerlieCysAsplieuLeuSerlielieArgLysGlu10010511033GgtagecgatagtgttttaagtgatgcgattgcagattttgacggtaaaValAlaAspSerValLeuSerAspAlalieAlaAspPheAspGlyLys115120125384ttgaaaaattatagagagtattatctttcttatcttggggettggcttLeuLysAsnTyrArgGluTyrTyrLeuSerTyrlieuGlyAlaTrpLeu130135140432SLSL5Lgateggtaaaccacttcaaaagaca幼taattctgatateg訴caaLysAspGlyLysProLeuGinLysThrAsnAsnSerAsplieGlyGin145ISO155160480ttagtttattatttta站cttteagaaagagatttcaatgaaattetaLeuValTyrTyrPheLy曰LeuSerGluArgAspPheAsnGlulieLeu17017552899a999teattgteaa訴aacaatgetcaagtattgttattacctact<31yGlySerLeuSerArgAsnAsnAlaGinVallieulieuIeuProThr57618018S190tttPhegca^c的AlaGin195getAlagcaaatgtgAlaAsnVaicagtta_GinLeu200ttalieuCt3ljeutt3lieu鄉(xiāng)Arg205gatAspgcaAlagttVal624GintataaaTyrIjys210gcaAlacmtggttcGinTrpPhe215ccatttProPhettgLeuagtSerAla220gagGlu貼tAsngtaValArg672tegSer225gaattaGluLeulieteacctaacSerProAsn230a9tggtSerGlytgtCysgatAsp235tttPheThrggtGlygatAspt3CTyr240720tatTyrgagcgaGluArgttaLeu333tgC333IiysCysLys245ThrAlaGlu2S0t3tTyrThr33tAsntatTyrtgtCys2S5tta7"匕attggta匕caggtaggtttaaatcaga_taaaacagggggggeicaggtTyrTrpTyrGinValGlyLeuAsnGin工leLysGinGlyGlyThrGly260265270getAlagacactAspThr275tggTrptegaaatttSerIjysPhe站tAsnLys280tttPhecgtArg3g3Arg933Glu28Sa±gMet3cgThrttg8"9c3AlagtattgVallieu290gatAspattategetlielieAla295atatttliePheCC3ProsetThrTyr300AsptttPhe9&933a>912tstTyr30SccattgProLeuCC3ProacacatgtaThxHisVal310g3gttgGlulieuactThr鄉(xiāng)Arg315Sf貼GluattlieTyrThrgatA印320%0gcagtgggatatteategggaacttatagttggttaeggaattggcctAlaValGlyTyrSerSerGlyThrTyrSerTrpLeuArgAsnTrpPro3253303351008站tacttttThrPhe站tAsn340gggttagagGlyIjeuGlugetaatAI3Asn345Gly3C3ThreggArg9g3Glycc仁Pro350ggtGlyttaLeu1056gttacttggcttageaaaataggtatatataatgagtatgtttegagaValThrTrpUeuSerLyslieGlylieTyrAsnGluTyrValSerArg35S3603651104tattttgccggctgggtaggaactcgtcattatgaagactacacaaagTyrPheAlaGlyTrpValGlyThrArgHisTyrGluAspTyrThrLys3703753801152ggtaacggtatttttcaacgtatgtctggaactacgagtaatgatetaGlyAsnGlyliePheGinArgMetSerGlyThrThrSerAsnAspLeu3853903954001200cgtaatattgattttcagaatgccgatgtatataaaattactteattaArgAsnlieAspPheGinAsnAlaAspValLyslieThrSerLeu405"041S1248getateatgaacetagtagga訴gactaceget的accagagtatcgtAlalieMetAsnlieuValGlyGluThrThrAlaArgProGluTyrArg4204254301236gttteaaaggca諷ttttcgtagggtagggggacctgatttaaattatValSerIiysAlaAspPheArgArgValGlyGlyProAspIeuAsnTyr435440"513"gatgcaggtaataatg的etaageaggatgacaattgaatctacgttcAspAlaGlyAsnAsnGly*LeuSerArgMetThrlie虹uSerThrPhe4S0455460CC3cttProLeu465gtaValttgcacIjeuHistctSet470aatAsnggtGlygttagaggaValArgGly475ccctctcatagattaProSerHisArgLeu4801440teaaatSe:rAs:nAlagcatgtAlsCys485gttValgtatatTyrggaaactecGlyAsnSer490agagttaacgtatatArgValAsnValTyr4951488ggttggGlyTrpThrC3t3C3HisThr500agtSertt3Leu333IjyscgtgaaaatArgGluAsnS05ataattgaagecaatlielieGluAlaAsn5101536caaattGinlieThr515GinlieccgProAlaVal520aagagttatLysSerTyrtaccttcaa33ttstTyrLeuGinAsnTyr5251584cttgetLeuAla530AsngectatAla_Tyr3CCThrt3tTyr535gtaValataaaaggclieLysGlyactcatacaggtgggThrHisThrGlyGly5401632gatttaAspLeu5453tCliecgttttArgPhettaLeu550agaArgThraaateagagIjysSerGlu555tataacgcagtttatTyrAsnAlaValTyr5601680gca_ggtAlaGlyggcGlyggaattGlylieS65agattglieuattlieattaataaclieAsnAsnS70aaaactgcaggacaaLysThrAlaGlyGin5751728agttaccgtattcgttttcgttatgetgcagataaagetgetttctttSerTyrArglieArgPheArgTyrAlaAlaAspLysAlaAlaPhePhe5S0S855901776agtgtatatctttatccagga^gttggggtteaaatcgttttgtategSerValTyrLeuTyrProGlyGlyTrpGlySerAsnArgPheValSer595GOO6051824cttgaalieuGlu610333IjystcttactctSerGly615astAsnt3tgsicgatTyrAspAsptta站atatagtgatLeuIiysTyrSerAsp6201872ttt3EiaPheIiys625ttcPhegetgaaAlaGluattlie6303tClieThrcctccattaProProIjeu635cctagtteaaacattProSerSerAsnlie6401920cag3tgGinMetgatAspgtggagValGlu"5atgMetC33GinAlaaatagttttAsnSerPhe650caateagatgtaaacGinSerAspValAsn1968gtggttetcgacaaaattgaattcetcccaagtaatacaacaactttaValValLeuAspLyslieGluPheLeuProSerAsnThrThrThrLeu"06656702016gaatatgagggagaac的gacetagaaaaaacaaagaacgcggtgaacGluTyrGluGlyGluArgAspI*euGluLyeThrLysAsnAlaValAsn67S68068SgatctgtttaccaattaaAspIeuPheThrAsn*6902082<210>14<211>633<212>PRT<213>蘇云金芽孢桿菌<400>14MetLysMetSerProTyrGinAsnAsnGluTyrGlulieLeu151015GluSerSerSerAsnAsixThrAsnThrProAsnArgTyrProPheAla202530AsnAsnArgAspMetSerThrMetSerTrpAsnAspCysGinGlylie354045SerTrpAspGlulieTrpGluSerValGluThrlieThrSerlieGly505560lieAsnLeulieGluPheVallieGluProSerLieuGlyGlylieAsn707580ThrIjeuLeuSerlielieGlyLyslieulieProThrAsnArgGinThr859095ValSerAlaL>euSerlieCysAspLieuLieuSer工le工leArgGlu100105110ValAlaAspSerValIjeuSerAspAla工leAlaAspPheAspGlyIjys115120125LeuLysTyrArgGluTyrTyrLeuSerTyrLeuGlyAlaTrpL/eu130135140LysAspGlyLysProLeuGinLysThrAsnAsnSerAsplieGlyGin145150155160UeuValTyrTyrPheLyslieuSerGluArgA叩PheAsnGlulielieu165170175GlyGlySerUeuSerArgAsnAsnAlaGinValUeuLeuUeuProThr18018S190PheAlaGinAlaAlaAsnValGinLieuLeuLeuUeuArgAspAlaVal195200205GluTyr!>ysAlaGinTrpPheProPheSerAlaGluAsnValArg210215220SerGluUeulieSerProAsnSerGlyCysAspPheThrGlyAspTyr225230235240TyrGluArglieuijysCysLysThrAlaGluTyrThrAsnTyrCysIjeu245250255TyrTrpTyrGinValGlyLeuAsnGlulieLysGinGlyGlyThrGly265270AlaAspThrTrpSerLysPheAsnIjysPheArgArgGluMetThrIjeu275280285AlaVallieuAsplielieAlaliePheProThrTyrAspPheGluLys290295300TyrProLeuProThrHisValGluIjeuThrArgGlulieTyrThrAsp305310315320AlaValGlyTyxSerSerGlyThrTyrSerTrpIjeuArgAsnTrpPro32533033SAsnThrPheAsnGlyIjeuGluAlaAsnGlyThrArgGlyProGlyUeu34034S350ValThrTrpIjeulieGlylieTyrGluTyrValSerArg3553603G5AlaGlyTrpValGlyThrArgHisiyrGluAspTyrThrLys37037S380GlyAsnGlyliePheGinArgMetSerGlyThrThrSerAsnAspLeu385390395400ArgAsnlieAspPheGillAsnAlaA8pValTyrLyslieThrSerlieu405410415AlalieMetAsnlieuValGlyGluThrThrAlaArgProGluTyrArg420425430ValSerLysAlaAspPheArgArgValGlyGlyProAsplieuAsnTyr435440"5AspAla<31yAsnAsnGlylieuSerArgMetThrlieGluSerThrPhe45045S460ProLeuVallieuHisSerAsnGlyValArgGlyProSerHisArgIjeu465470475480Sea:AsnAlaAlaCysVal"ValTyrGlyAsnSerArgValAsnValTyr485<31yTrpThrHisThrSerlieuLysArg<31uAsnlielieGluAlaAsnSOOS05GinlieThrGinlieProAlaValIiysSerTyrTyrlieuGinAsaTyrS15520525IjeuAlaAsnAlaTyrThrTyrVallieLys<31yThrHisThrGlyGlyS3053SS40AsplieulieArgPheIjeuArgThrLysSerGluTyrAsnAlaValTyr545550555560AlaGlyGlyGlylie565ArgLeulielieAsn570AsnIiysThrAlaGinSerTyrArglie580ArgPheArgTyrAla585AlaAspliysAlaAla590PhePheSerValTyr595lieuTyrProGlyGlyGOOTrpGlySerAsnArg605PheValSerLeuGlu610ljysSerTyrSerGly615AsnTyrAspAspLeu620LysTyrSerAspPheLysPheAlaGlulielieThrProProlieuProSerSerAsnlie625630635640GinMetAspValGlu645MetGinAlaAsnSer650PheGinSerAspVal655AsnValValLieuAspliyslieGluPheIjeuProSerAsnThrThr670ThrUeuGluTyrGlu675GlyGluArgAspLeu680GluIjysThrLysAsn685AlaValAsnAspLeuPheThrAsn690<210>15<211>2073<212>DNA<213>蘇云金芽孢桿菌<220><221>CDS<222><1)…(2073)<400>15atgagtccaMetSerProtatcaaaataaaaatgaatatgaaatattggaatecteaTyrGinAsnLysAsnGluTyrGlulielieuGluSerSer5101548tegaataacacaaatacgccaaacagatatccttttgcaaataateggSerAsnAsnThxAsnThrProAsnArgTyrProPheAlaAsnAsnArg20253096gatatgtctactatgtcttggaatgattgtcagggaateteatgggatAspMetSerThrMetSerTrpAsnAspCysGinGlylieSerTrpAsp354045144gaaatttgggaateagtcgaaacgataacaagtattgggataaatcttGlulieTrpGluSerValGluThrlieThrSerlieGlylieAsnLeu505560192atagagtttgtgatagaacctagtttgggtggaattaatacaetattalieGluPheVallieGluProSerLeuGlyGlylieAsnThrLeuLeu65707580240teaataataggaaaaetaattccgactaatcgtcaaactgtgteagcaSerlielieGiyLysLeulieProThrAsnArgGinThrVaiSerAla8S9095288ctttctatatgtgatttattatctataattcgtaaagaggtagecgatUeuSerlieCysAspLeulieuSerlielieArgLysGluValMaAsp10010S110336agtgttttaagtgatgcgattgcagattttgacggtaaattgaaaaatSerVallieuSerAspAlalieAlaAspPheAspGlyliysLeuItysAsn11512012S384tataga訴gtattatctttcttatcttg的gettggcttaaa訴cggtTyrArgGluTyrTyrLeuSerTyrLeuGlyAlaTrpLeuLysAspGly130135140432acaaataattctgatateLysProLeuGinLysThrAsnAsnSerAsplie145150155ggacaattagttGlyGinLeuValTyr160480tattttaaacttteagaaagagatttcaatgaaTyrPheLysLeuSerGluArgAspPheAsnGlu165170attetaggagggtealieDeuGlyGlySer175528ttgteaagaaacaatgetcaagtattgttattaLeuSerArgAsnAsnAlaGinValLeuLeuLeu180185cctacttttgcacaaProThrPheAlaGin190576getgcaaatgtgcagttattaetattaagggatAlaAlaAsnValGinLeuLeuLeuLeuArgAsp195200gcagttcaatataaaAlaValGinTyrLys205624gcacaatggttcccatttttgagtgcagagaatAlaGinTrpPheProPheLeuSerAlaGluAsn210215gtaagateggaattaValArgSerGluLeu220672atateacctaacagtggttgtgattttaccggtlieSerProAsnSerGlyCysAspPheTht:Gly225230235gattactatgagAspTyrTyrGlucgaArg240720ttaaaatgcaaaacggcagagtataccaattatLeuLysCysLysThrAlaGluTyrThrAsnTyr2452S0tgtttatattggtatCyslieuTyrTrpTyr255768caggtaggtttaaatcagataaaacagggggggseaggtgetgacactGltiValGlyLeuAsnGinlieLysGinGlyGlyThrGlyAlaAspThr2602S5270816t的tegaaatttaataaatttcgtagagaaatgacgttggcggtattgTrpSerLysPheAsnLysPheArgArgGluMetThrlieuAlaVallieu27S280285gatattettcgetatatttccaacttatgattttgagaaatatccattgAsplielieAlaliePheProThrTyrAspPheGluLysTyrProLeu2902953008S4912ccaacac&tgtagagttgactagggaaatttatProThrHisValGluLeuThrArgGlulieTyr305310315acagatgcagtgThrA叩A(chǔ)laVal993Gly320960tatteategggaacttatagttggttaeggaattggcctaatacttttTyrSerSerGiyThrTyrSerTrpLeuArgAsnTrpProAsnThrPhe32S3303351008aatgggttagaggetaatggaacac的ggacctggtttagttacttggAsnGlyLeuGluAlaAanGlyThrArgGlyProGlyLeuValThrTrp3403453501056cttageaaaata的tatatataatgagtatgtttegagatattttgecLeuSerLyslieGiylieTyrAsnGluTyrValSerArgTyrPheAla35S3603651104ggctgggtaggaactcgtcattat訴agactacacaaagggtaacggtGlyTrpValGlyThrArgHisTyrGluAspTyrThrliysGlyAsnGly37037530011S2atttttcaacgtatgtctggaactacgagtaatgatetacgtaatattliePheGinArgMetSerGlyThrThrSerAsnAspLeuArgAsnlie3853903954001200gattttcag站tgecgatgtatataaaattactteattagetateatgAepPheGinAsnAlaAspValTyrLyslieTbrSerlieuAlalieMet40S4104151248aacctagtaggagagactaccgetagaccagagtatcgtgttteaaagAsnLeuValGlyGluThrThrAlaArgProGluTyrArgValSerLys4204254301296gesigattttcgtagggtagggggacctgatttaaattatgatgcaggtAlaAspPheArgArgValGlyGlyProAspLeuAsnTyrAspAlaGly4354404451344aettaatgggetaageaggatgacaattgaatctacgttcccacttgtaAsnAsnGlyLeuSesrArgMetThrlieGluSerThrPheProLeuVal4504554601392ttgcactctaatggtgttagaggaccctctcatagattateaaatgcgUeuHisSerAsnGlyValArgGlyProSerHisArgLeuSer*AsnAla4654704754801440gcatgtgttgtatatggaaactecagagttaacgtatatggttggacaAlaCysValValTyrGlyAsnSerArgValAsnValTyrGlyTrpThr4854904951488catacaagtttaaaacgtgaaaatataattgaagecaatcaaattacaHisThrSerLeuLysArgGluAsnlielieGluAlaAsnGinlieThr5005055101536caaataccggcggtgaagagttattaccttcaaaattatcttgetaatGinlieProAlaValLysSerTyrTyrLeuGinAsnTyrLeuAlaAsn515S205251584gectatacctatgtaataaaaggcactcatacaggtggggatttaateAlaTyrThrTyrVallieIysGlyThrHisThrGlyGlyAspUeulie530535540cgttttttaagaacaaaateagagtsttaacgcagtttatgcaggtggcArgPheLeuArgThrLysSerGluTyrAsnAlaValTyrAlaGlyGly5455505555"ggaattagattgattattaataacaaaactgcaGiylieArgLeulielieAsnAsnLysThrAla565S70ggacaaagttaccgtGlyGinSerTyxArg5751728attcgttttcgttatgetgcagataaagetgetttctttagtgtatatlieArgPheArgTyrAlaAlaAspLysAlaAlaPhePheSerValTyr580S8S5901776ctttatccaggaggttggggtteaaatcgttttgtategcttgaaaaaIjeuTyrProGlyGlyTrpGlySea:AsnArgPheValperLeuGluIiys59560060S1824tcttactctggaaattatgacgatttaaaatatagtgattttaaattcSerTyrSerGlyAsnTyrAspAspLeuLysTyrSerAspPheLysPhe"06201872getgaaattateacacctccattacctAlaGlulielieThrProProLeuPro625630agtteaaacattcagatggatSerSerAsnlieGinMetAsp63S64013203tggagatgcaagcgaatagttttcaateagatgtaaacgtggttetcValGluMetGinAlaAsnSerPheGinSerAspValAsnValVallieu"S65065S1968gacaaaattgaattcetcccaagtaatacaacaactttagaatatgagAspLyslieGluKieLeuProSerAsnThrThrThrLeuGluTyrGlu€606656702016ggagaaegggaceta訴aaaaacaaagaacgcggtgaac訴tctgtttGlyGluArgAspLeuGluLysThrLysAsnAlaValAsnAspLeu跑e67S68068520"accaatta_a20*73ThrAsn*690<210>16<211>690<212>PRT<2lh蘇云金芽孢桿菌<400>16MetSerProTyrGinAsnLysAsn15SerAsnAsnThrAsnThrProAsn20AspMetSerThrMetSerTrpAsn3540GlulieTrpGluSerValGluThr5055lieGluPheVallieGluProSer6570SerlielieGlyLyslieuliePro85LieuSerlieCysAspIjeuLeuSer100SerValLeuSerAspAlalieAla115120TyrArgGluTyrTyrIjeuSerTyr130135LysProIjeuGinThrAsnAsn145150TyrPheLysIjeuSerGluArgAspLeuSerArgAsnAsnAlaGinVal180AlaAlaAsnValGinLeuLeuLeu195200AlaGinTrpPheProPheLeuSer210215lieSerProAsnSerGlyCysAsp225230IjeuLysCysLysThrAlaGluTyr245GinValGlyLeuAsnGinlieLysTrpSerLysPheAsnLysPheArg275280AsplielieAlaliePheProThr2902S5ProThrHisValGlulieuThrArg305310TyrSerSerGlyThrTyrSerTrp325AsnGlylieuGluAlaAsnGlyThr340lieuSerLyslieGlylieTyrAsn355360GlyTrpValGlyThrArgHisTyr370375liePheGinArgMetSerGlyThr385390AspPheGinAsnAlaAspValTyr405AsnLeuValGlyGluThrThrAla420AlaAspPheArgArgValGlyGly43S440GluTyrGlulie!LeuGluSerSer1015ArgTyrProPheAlaAsnAsnArg2530AspCysGinGlylieSerTrpAsp45lieThrSer工leGlylieAsnLeu60LeuGlyGlylieAsnTharLeuLieu7580ThrAsnArgGinThrValSerAla9095lielieArgLysGluValAlaAsp105110AspPheAspGlyLysIjeuUysAsn125IjeuGlyAlaTrpLeuIjysAspGly140SerAsplieGlyGinUeuValTyr155160PheAsnGlulieLeuGlyGlySer170175LeuLeuUeuProThrPheAlaGin185190IjeuArgAspAlaValGinTyrLys205AlaGluAsnValArgSerGluLeu220PheThrGlyAspTyrTyrGluArg235240ThrAsnTyrCysIjeuTyrTrpTyr2S0255GinGlyGlyThrGlyAlaAspThr265270ArgGluMetThrLeuAlaVallieu285TyrAspPheGluLysTyrProLieu300GlulieTyrThrAspAlaValGly315320LeuArgAsn.TrpProAsnThrPhe330335ArgGlyProGlylieuValThrTrp345350GluTyrValSerArgPheAla3S5GluAapTyrThrLysGlyAsnGly380ThrSerAsnAspLeuArgAsnlie335400LyslieThrSerLeuAlalieMet"0415ArgProGluTyrArgValSerLys42S430ProAspLeuAsnTyrAspAlaGly"5AsnAsnGlyLeuSerArgMetThrlieGluSerThrPheProUeuVal450455460LeuHisSerAsnGlyValArgGlyProSerHisArgLeuSerAsnAla465470475480AlaCysValValTyr485GlyAsnSerArgVal490AsnValTyrGlyTrp495ThrHisThrSerLeu500LysArgGluAsnlie505lieGluAlaAsnGin510lieThrGinliePro515AlaValLysSerTyr520TyrLieuGinAsnTyr525LieuAlaAsnAlaTyrThrTyrVallieLysGlyThrHisThrGlyGlyAspLeulie530535540ArgPheLeuArgThrIjysSerGluTyrAsnAlaValTyrAlaGlyGly545550555GlylieArgLeulielieAsnAsnLysThrAlaGlyGinSerTyrArg565570575lieArgPheArg580TyrAidAlaAspLys585AlaAlaPhePheSer590ValTyrLeuTyrPro595GlyGlyTrpGlySer600AsnArgPheValSer605LeuGluLysSerTyrSerGlyAsnTyrAspAspIjeuLysTyrSerA印PheLysPhe620AlaGlulielieThrProPro!LeuProSerAsnlieGinMetAsp625630ValGluMetGinAlaAsnSerPheGinSerAspValAsnValValLeu645650SS5AspLyslieGluPheLeuProSerAsnThrThrThrlieuGluTyrGlu660665670GlyGluArgG75AspIjeuGluLysThr680LysAsnAlaValAsn685AsplieuPhe690<210>17<211>1686<212>DNA<213>蘇云金芽孢桿菌<220><221>CDS<222>(1)…(1686)<400>17gtgagtcctatgtttacaagtagtacgaaaaatacgttaaaaatagaa48MetSerProMetPheThrSerSerThrLysAsnThrLeuLyslieGlu151015atcgacagattsitgaaatagatcaagcggccattThrThrAspTyrGlulieAspGinAlaAlalie2025tcteitELgaatgtatgSerlieGluCysMet3096teagatgaacaaaatcctcaggaaaaaataatgttatgggatgaaataSerAspGluGinAsnProGinGluLyslieMetlieuTrpAspGlulie3S4045144ctgLeuSOgcaAlaa站c站GincttagtcaglieuSerGin55tctcgtaatSerArgAsnetaetccaaaatggaLeuIjeuGinAsnGly60192gacttttctgggaatgattggacattcggtaatgatattateatagga240AspPheSerGlyAsnAspTrpThrPhe<3lyAsnAsplielielieGly6S707580tecaataatcctatttttaaag訴aaatttetacagatgcgt訴agcaSerAsnAsnProliePheLysGlyLysPheLeuGinMetArgGlyAla859095288<table>tableseeoriginaldocumentpage98</column></row><table>ttacagggatggcacgcaacaggaaatgccgcggtacaacaaatggatLeuGinGlyTrpHisAlaThrGlyAsnAlaAlaValGinGinMetAsp3703753801152Gly385getAlatC3SergtattagttValLeuVal390etateaaattggageLeuSerAsnTrpSer39SAlagggGlygtaValtctSerGin4001200aacttgcatgetcaagatcatcatggatatgtgttacgtgtgattAsnLeuHisAlaGinAspHisHisGlyTyrValLeuArgVal工le405410415gccAla1248aaaaaagaaggacctggaaaagggta_tgtaacgatgatggattgtaatLysLysGluGlyProGlyIjysGlyTyrValThrMetMetAspCysAsn4204254301296ggaaagcaggaaacacttaagttcacttcttgcgaagaaggatatatgGlyLysGinGluThrLeuLysPheThrSerCysGluGluGlyTyrMet435"04451344seaaaaacagtagaggtattcccagaaagtgatcgtgtaeggattThrLysThrValGluValPheProGluSerAspArgValArglie4504554S0Glu13923taggagaaeiccgaaggtacattttatatagatageategagttgcttlieGlyGluThrGluGlyThfPheTyrlieAspSerlieGluLeuLeu46547047S4801440tgtatgcaaggatatgataacaata_ataacctgcacacgggtaatatgCysMetGinGlyTyrAspAsnAsnAsnAsnLeuHisThrGlyAsnMet4854S04951488tatgagcaaagttataatggaaattataatcaaaatactagegatgtgTyrGluGinSerTyrAanGlyAsnTyrAsnGinAsnThrSerAspVal500S055101536tattacca_a_gggtata_ca_etacaactataaccaagactctagtaatatgTyrTyrGinGlyTyxThrAsnAsnTyrAsnGinA印SerSerAsnMetS155205251584tataatcaaaattatactaacaatgatgacctgcattecggttgcacaTyrAsnGinAsnTyxT)irAsnAsnAspAspLeuHisSerGlyCysThr53053S5401632tgtaaccaagggcataactctggctgtacatgtaatcaaggatataacCysAsnGinGlyHisAsnSerGlyCysThrCysAsnGinGlyTyrAsn54S550S555S01680cgttaaiArg*<210>18<211>561<212>PRT<S13>蘇云金芽孢桿菌<40()>18MetSerProMetPheThrSerSer15ThrThrAspTyrGlulieAspGin20SerAspGluGinAsnProGinGlu3540LysUeuAlaLysGltiLeuSerGin50S5AspPileSetGlyAsnAfipTrpThrThrLyeAsnThrLeuI*yslieGlu1015AlaAlalieSerlieGluCysMet2530liyslieMetLeuTrpA8pGlulie45SerArgAsnlieulieuGinAsnGly60PheGlyAsnAsplielielieGly65707580SerAsnAsnProliePheGlyLyaPheLeuGinMetArgGlyAla8595ArgAsplieTyrGlyThrLeuPheProThrTyrlieCysGinlie100105110AspGluSertjysProTyrThrArgTyrArgValArgGlyPhe115120125ValGlySerSerLysAspLeuliysLeuMetValThrArgTyrGlyLys130135140GlulieAspAlalieMetAsnValProAsnAspLeuAlaTyrMetGin1451501S5ISOProAsnProSerCysGlyAspTyrArgCysGluSerSerGinTyr165170175ValSerGinGlyTyrProThrProThrAspGlyTyrAlaProAspMet180185190TyrAlaCysProGinAsnlieAspArgLysHisValIjysCysHisAsp195200205ArgHisProPheAspPheHislieAspThxGlyGluValAspThrAsn210215220ThrAsnValGlylieAspValLeuLeuLyslieSerAsnProAspGly22S230235240TyrAlaThrValGlyAsnLeuGluVallieGluGluGlyProLeuThr24S25025SGlyGluAlaLeuAlaHisValIjysGinIjysGluLysLysTrpLysGin265270HisMetGluUysUysArgTrpGluThrGinGinAlaTyrAspProAla275280285LysGinAlaValAspAlaIjeuPheThrAsnGluGinGlulieuHisTyr290295300HislieThrLeuAspHislieGinAsnAlaAspArgIeuValGinSer30531031S320lieProTyrValTyrHisAsnTrpLeuProAsnAlaProGlyMetAsn325330335TyrAspValTyrGinGlulieuAsnAlaArglieMetGinGlyTyrAsn340345350LeuTyxAspAlaArgAsnVallieThrAsnGlyAspPheThrGinGly35536SLeuGinGlyTrpHisAlaThrGlyAsnAlaAlaValGinGinMetAsp370375380GlyAlaSerLeuValLieuSerAsnTrpSerAlaGlyValSerGin385390400asx1IiSUHisAlaGinAspHisHisGlyTyrValLeuArgVallieAla405410415GluGlyProGlyUysGlyTyrValThrMetMetAspCysAsn420425430GlyLysGinGluThrLeuLysPheThrSerCysGluGluGlyTyrMet435"0"5ThrLysThrValGluValPheProGluSerAspArgValArglieGlu4S0455460lieGlyGluThrGluGlyThrPheTyrlieAspSerlieGluLeuUeu470475480CysMetGinGlyTyrAspAsnAsnAsxiAsnlieuHisThrGlyAsnMet48543043STyrGlu<3ltlSerTyrAsnGlyAsnTyrAsnGinAbxiThrSerAspVal500S05S10TyrTyrGinGlyTyrThrAsnAsnTyrAsnGinAspSerSerAsnMet515520S25TyrAsnGinAsnTyrThrAsnAsnAspAspLeuHisSerGlyCysThr530S3S540CysAsnGin<31yHisAsnSerGlyCysThrCysAsnGluGlyTyrAsn545S50555S€0Arg<210>IS<211>2049<212>mA<2lh蘇云金芽孢桿菌<220><221>CDS<222>(1)…(2049)<400>19atgaatteatata_a_a_a^tMetAsnSerTyrLysAsn15aaaaatgaatatgaaatgttggatgetttaLysAsnGluTyrGluMetLeuAspAlaLeu101548cgaateaactctaatatgArglieAsnSerAsnMet20tctaattgttatccaaggtatccaetaSerAsnCysTyxProArgTyxProLeu2530gcaAla96aaagatccacaaa匕gactIjysAspProGinMetThr35atgcgaaacacgaacta匕aaagaatggetaMetArgAsnThrAsnTyrLysGluTrpLeu404S144aatatgAsnMet50tgtgatteaaatCysAspSerAsnacacaatttattggtgatataageacgtatThrGinPhelieGlyAsplieSerThrTyr5560192tctagecctgaagetgetttaagtgtacgagatgetgt匕ttaacgggtSerSerProGluAlaAlaLeuSerValArgAflpAlaValLeuThrGly707580240attaacagtgtagggactatactttegaatttaggggtccctttglieAsnSerValGlyThrlieLeuSerAsnLeuGl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agtaaaaacggaattgactagggaaAspProArgArgTyrProLeuAlaValLysThrGluLeuThrArgGlu275280285864gtttatacagatccagtagggtttactggggtattagaaagtggaggtValTyrThrAspProValGlyPheThrGlyValLeuGluSerGlyGly2S029S300912aggacttacccttggtataatcctaataatacaacctttactgetatgArgThrTyrProTrpTyrAsnProAsnAsnThrThrPheThrAlaMet305310315320960gaaaataacgcaagacgacgtccttcttataccacttggcttaatcgtGluAsnAsnAlaArgArgArgProSerTyrThrThrTrpLeuAsnArg3253303351008atttttgtatatacaaggactetaggtaatatgtctgatgtgagaaatliePheValTyrThrArgThrLeuGlyAsnMetSerAspValArgAsn3403453501056atttggggagggcatacattagttgaaaatggaaatgatggttctgaalieTrpGlyGlyHisThrLeuValGluAsnGlyAsnABpGlySerGlu3553603651104ataacccataactttggtaaaactgattctattactcctattcaatatlieThrHisAsnPheGlyLysThrAspSerlieThrProlieGinTyr3703753801152tttaatttcgcgaacctttctgttttcagtattgagteacttgetcgtPheAsnPheAlaAsnLeuSerValPheSerlieGluSerLeuAlaArg3853903954001200atatatttaggaggaacagaggetaataattatattactagtcagtatlieTyrlieuGlyGlyThrGluAlaAenAsnTyrlieThrSerGinTyx40S4104151248ggagtctegagagttatttttaatacateaaatataaataatgtacctG^yValSerArgValliePheAsnThrSerAsnlieAsnAsnValPro4204254301296ggatctttaagatacgaagtgcctgetaatcttccateccaaactataGlySerLeuArgTyrGluValProAlaAsnLeuProSerGinThrlie43544044S1344ttateagaattaccaggaaaggataagccaagaccaaacgcaggagatLeuSet*GluLeuProGl女LysAspLysProArgProAsnAlaGlyAsp4504554601392ttcagecatagattatcttatatatea貼ttttgatgcac的cgaagtPheSerHisArgI*euSerTyrlieSerAsnPheAspAlaArgArgSer46547047S4801440agtteaggcggtattgttagtcttttaacgtttggttgggcacataccSerSer<31yGlylieValSerLeuLeuThrPheGlyTxpAlaHisThr4854904951488agtatggatcgtaataatcgtcttgaaccagataaaattactcaaataSerMetAspArgAsnAsnArgLeuGluProAspLyslieThrGinlie5005055101536gatgcagttaaaggttggggggggaatategggtttgtcateccaggaAspAlaValLysGlyTrpGlyGlyAsnlieGlyPheVallieProGly51552052S1S84CCtProactThr530鵬GlygggGly貼tttgAsnIjsugtaVal535gtcValSergatagtAspSer540TrpC3tHisteaSercttlieu1632Lys545gttValc站GingcaAlaCC3ProGin550ArgGinseaThrSertatcgtTyrArg555liecgtArgttgLeucgt5601680tatTyrgetAlatgtttaLeugttaccValThr565catHisgggGlygatAspgetAla570attttt工lePheValgasGluHis575age1728GlySeragtSercatHis580BitsgttlieValteaSertttPhetttPhe585gatAsptgcteaCysSsraattea590tC3ggtGly1776cgtArgccaProteaSer595AsnactcttThrlieueta弼Glu600SergatAsptttcgcPheArgTyr605attliegatAspgttVal1824CC3Pro的tGly610attlietttPheThrProteaSei:615lie33tAstlCCCProttaataLeulie620tatTyraga1872C3ELGill625agetttPheggtGly￡LCCC3CThrHis630Ala3t3liegacAsp貼3tttgaaPheGlu635tttPhe.attlieCC3ProcttLeu64019203CtThrtttPheccgProAsnGin€45SerttaLeug貼Glu貼s650的sgaaArgGluGing33GlugtaVal65533tAsnAspLeutttPheatelie660aattaa1986'<210>24<211:>661<212>ERT<213>蘇云金芽孢桿菌<400>24MetSerAsnCysTyrProArgTyrProLeuAlaLysAspProGinMet151015ThrMet;ArgAsnThrAsnTyrLysGluTrplieuAsnMetCysAspSer202S30AsnThrGinPhelieGlyAsplieSerThrTyrSerSerProGluAla35404SAlalieuSerValArgAspAlaValIeuThrGlylieAsnSerValGly505560ThrlielieuSerAsnlieuGlyValProLeuAlaSerGinSetPheGly65707S80lieSerArglieulieGlylieLeuTrpAlaGlyProAspProPhe859095GluAlaUeuMetValIi的ValGluGluIjeulieLysLysSerlieAsp10010S110GinArgValArgGluAsnAlaLeuArgGluIjeuGluGlylieuGinGly115120125lieMetArgLeuTyrGinThrArglieuGinAlaTrplieuValAsn130135AsilAspAspAsnArgArgAlaLeu14S150AsnPhePheGluAsnMetProlieIjeuLeulieuProValTyrAla180LeuIjeuArgAspAlaA印TyrPhe19S200AspGlulieArgAspAsnTyrUe210215TyrLysAspHisCyslieThrPhe225230AsnArgSerAsnAlaGinAspTrp245AspMetThrLeuThrValLieuAsp260AspProArgArgTyrProLeuAla275280ValTyrThrAspProValGlyPhe290295ArgThrTyrProTrpTyrAsnPro305310GluAsnAsrxAlaArgArgArgPro325liePheValTyrThrArgThrIjeu340lieTrpGlyGlyHisThrUeuVal355lieThrHisAsnPheGlyLysThr370375PheAsnAlaAsnLeuSerVal385lieTyrLeuGlyGlyThrGluAlaGlyValSerArgValliePheAsnGlySerLeuArgTyrGluValPro435440IjeuSerGlulieuProGlyUysAsp450455PheSerHisArgIjeuSerTyrlie465470SerSerGlyGlylieValSerLeu485SerMetAspArgAsnAsnArgLeu500AspAlaValLysGlyTrpGlyGlySIS520ProThrGlyGlyAsnLeuValLys530535LysValGinAlaProGinArgGinS45550TyrAlaCysheuValThrHis<31y565GlySerSerHislieValSer*Phe580ArgProSerAsnThrLeulieuGluS95600ProGlylie處eThrProSeirlieGinSerPheGlyThrHisAlaUeAsnThrPheProAsnGinSerlieu"5AspUeulieAsn660140ValThrGinTyrAlalieValAsp155160LysPheLysGluArgAsnpheGlu170175GinAlaAlaAsnLeuHisLeulie185190GlyAlaGinTrpGinLeuGlyAsp205ArglieuGinGlyIjeulieArgGlu220TyrAsnGinGlyLeuAsnGinPhe235ValSerPheAsnArgPheArgThr250255LeuAlalieLeuPheProAsnTyr265270ValLysThrGluLeuThrArgGlu285ThrGlyValLeuGluSerGlyGly300AsnAsnThrThrPheThrAlaMet315320SerTyrThrThrTrpLeuAsnArg33033SGlyAsnMetSerAspValArgAsn345350GluAsnGlyAsnAspGlySerGluAspSer工leThxPi:olieGinTyr380PheSerlieGluSerLeuAlaArg395400AsnAsnTyrlieThrSerGinTyr41041SThrSerAsnlieAsnAsnValPro425430AlaAsnLeuProSerGinThrlie445LysProArgProAsnAlaGlyAspSerAsnPheAspAlaArgArgSer475480LeuThrPheGlyTrpJUaHisThrGluProAspLyslieThrGinlie505510AsnlieGlyPheVallieProGly525ValSerAspSerTrpHisSerLeu540ThrSerTyrArglieArgLeuArg555AspMaliePheValGluHisSerS70S7SPheAspCysSerAsnSerSerGly585SerAspPheArgTyrlieAspVal605AsnProLeulieArgTyrArgThrAspLysPheGluPhelieProLeuLysArgGluGltiGluValAsn650655<table>tableseeoriginaldocumentpage113</column></row><table>5055603C3Thr65TyrcttLieu3t3lieC33GinttcPhe70ttgLeuctaLeugaaGlucccProggtGly753t3l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LeuGlyTyrLeuSerGlyTrpGlyGlyThrArgHisTyrGluAspPhe3553603G5ThrLjysGlyAsnGlyAlaPheGinArgMetSerGlyThrThrSerAsn370375380AsnProArgAsn工leliePheGlyAstiThrAspliePheIiyslielie3853S0395400SerLeuAlaArgTyr405AlaMetGinProPhe410ValGlyTyrSerlie415ProArgHisLeuValSerArgAlaGluPhePheProThrThrLeuAsnThr420425430PheLieuTyr435GluValAsnSerSer440GlyTyrSerGinThr445UeGluSerValLieu450ProGlylieAsnUys45SAspLeuProProSer460ArgThrAsnTyrSerHisArgUeuSerAsnAlaAlaCysValGinAsnGluThrSerArg470475480ValAsnValPheGly485TrpThrH丄sThrSer490MetljysljysAspAsn495ArglieTyrProAsp500LyslieThrGinlie505ProAlaVallaysAla510PheAlaLieuProAlaGlyThrGlyTyrAlaGlyGlyTyrValThrAlaGlyPro515520525GlyTyrThrGlyGlyAspValValThrLeuProTyrGinAlaSerLeu530535540UyslieArgLeuThrSerAlaProThrAsnLysTyrArgValArg545SSO555560lieuArgTyrAlaSerGlyGlyProGlyProPheArgValGluArgTrpS6S570575SerProSerSerValSerAsnAlaAsnPheSerArgProAlaThrGly580585590GlyTyrSerSerPheAspTyrVal600AspThrLeuValThr605ThrPheAsnGinSer610GlyValGlulielielieGinAsnLeuSer620GlyTyrHisIjeulieValAspLysValGluPhelieProlieAsplieGin工leGluUys625630635CysThrljysCysGinPheGluGlyAsplieCysArgCysGluGlyValS45650655GinSerIjeuGlu660ThrLysGlulie"5ValAsnSerlieuPhe670lieAsn<210><211><212><213>282010DNA蘇云金芽孢桿菌<220><221>CDS<222>(1)(2010)<400>28atgaattctMetAsnSertatTyrCS3GluAsnThraatgaaAsnGlutatTyr10gaaatt(31uliectglieugatAspggttecGlySerIS48ccg站tProAsa站cAbuThr2033tAsnatgteaMetSeraacag這AsnArg25tatTyrccttttProPhegcaAlaaagLys30訴tccaAspPro96站tatatttcctattaacctggacgettgtcagggaaggccat的caaAsnliePheProlieAsnLeuAspAlaCysGinGlyArgProTrpGin354045gat.AspacgThrSOTrpgaaGlutCSLSergtcValtcggatSerAsp553t3liegt￡iVal3Ct5LttThrlie60gggGlyThrTyrcttLeu192atalie6SGinttcPhettgLeuetaIjeug貼Glu70cccggtProGlyatalieggtGlyggaa_ttGlylie75cctProgta_VallietttPhe80240tC3SerlielieAsn3a_3Uys85etcLeuattccglieProtctSertct90ggtcaaGlyGintctSerValgcaAla95Ala288ctttctatatgtgatttagtatctataattcgtaaagaggtagacgagLeuSerlieCysAspLeuValSerlielieArgLysGluValAspGlu100105110336agegtgttaagtgacggggttgcagattttgagggtgaaatgactgetSerValLeuSerAspGlyValAlaAspPheGluGlyGluMetThrAla115120125384tatcaagattattatcttcattatcttgaggattggcttacagataaaTyrGinAspTyrTyrLeuHisTyxLeuGluAspTrpLeuThrAspLys130135140432Ser145貼tAsncctProLyscttLeu150getgacgtagttAlaAspValVal155cagGinttcPheGingcaAlaeggArg160480gaagaagatttcactaaacttttagcagggteattateaagacagaaaGluGluAspPheThrLysLeuLeuAlaGlySerLeuSerArgGinLys16517017S528getgaaatattattattgcctacgtatgtgcaagetgcaaatgtgcatAlaGlulieLeuLeuLeuProThrTyrValGinAlaAlaAsnValHis180185190576ttattaetattaagggacgcagttaaatataaaaaagaatggggaetaIjeuLeuLeuLeuArgAspAlaValLysTyrLysLysGluTrpGlyLeu"5200205624gt'gtgtccaccgttgtatccagggteagggagaactgattgtaacgagValCysProProLeuTyrProGlySerGlyArgThrAspCysAsnGlu21021S220eggArg225ttaIjeu3站LysAla333Lys3t3lie230aaagagtatactLysGluTyrThr235tatTyrtgtCysgtaValgggGly240720tataacaagggtttagatcagEttaagacaggcgggtacaagtgetgaaTyrAsnLysGlyLeuAspGinlieArgGinAlaGlyThrSerAlaGlu24S250255gtttggtcgaaatttaat幼atttcgtagagaaatgacgttggcggtaValTrpSerLysPheAsnLysPheArgArgGluMetThrlieuAlaVal2S5270816ttggatattattgetatatttccaacttatgattttgaaaaatatccaLeuAsplielieAlaliePheProThrTyrAspPheGluLysTyrPro27S280285864ttagcaacaagtgtagagttaactagggaaatttatacagatccagtgLeuAlaThrSerValGlulieuThrArgGlulieTyrThrAspProVal290235300912ggatatteagggggaaattatggttgggaaeggttttttagetttaatGlyTyrSerGlyGlyAsnTyrGlyTrpGluArgPhePheSerPheAsn30S310315320360tcggtagaagcaaatggaacseggggacctggtttagttacttggcttSerValGluAlaAsnGlyThrArgGlyProGlyLeuValThrTrpLeu3253303351008caagetatagatatatatagcattctattaatcttcagcttggttatGinAlalieAsplieTyrSerHisSerlieAsnLeuGinLeuGlyTyr3403453501056cttagtggctgggggggaactcgtcattatgaagacttcacasagggtLeuSerGlyTrpGlyGlyThrArgHisTyrGluAspPheThrLysGly3553603651104aacggtgettttcaacgtatgtctggaactacgagtaeitaatccacgtAsnGiyAlaPheGinArgMetSerGlyThrThrSerAsnAsnProArg3703753801152aatattatttttggcaataccgatatatttaaaattattteattagetAsnlieliePtieGlyAsnThrAspliePheLyslie工leSerLeuAla3853903954001200agatatArgTyrAla3tgC33MetGin405ccgtttgttgggProPheValGlytatTyr410teaSeratelieccaeggProArgcatHis415cttLeu1248gttteacgtgcagaattttttccgacaacaetaaatactttcctgtatValSerArgAlaGluPhePheProThrThrLeuAsnThrPheLeuTyr4204254301296gaggtaaacagttctgggtacteacagacaattgaatctgtgttaccaGluVSlAsnSerSerGlyTyrSerGinThrlieGluSerValLeuPro4354404451344ggtattaataaggatetaccacctagtcgtacaaattactctcatagaGlylieAsnLysAspLeuProProSerArgThrAsnTyrSerHisArg4S04554601392ttateaaatgcggcatgtgttcaaaatgaaacctecagagttaacgtaLeuSerAsnAlaAlaCysValGinAsnGluThrSerArgValAanVal4654704754801440tttggttggacacatacaagtatgaaaaaagataatcgaatttatccaPheGlyTrpThrHisThrSerMetLysLysAspAsnArglieTyrPro4854904951488gataaaattacgcaaattcctgcagtaaaagettttgecetaccaAspLyslieThrGinlieProAlaValLysAlaPheAlaLeuPro500505510AlaggtacaggatatgcaggaggttacgtcacagetgggcctggttatacaGlyThrGlyTyrAlaGlyGlyTyrValThrAlaGlyProGlyTyrThr515520S2S158499aggagatgtagtaacgttaccttatcaagcaagtttaaaaatacgtGlyGlyAspValValThrLeuProTyrGinAlaSerLeuLyslieArg53053SS401632ttaacttctgcacccacgtaaaaattaccgtgttagacttcgctacLeuThrSerAlaProThrAsnLysAsiaTyrArgValArgLeuArgTyr5455S0SS5S€01680gcgagtggaggacctggtccgttccgagtagaaagatggtegccaagtAlaSerGlyGlyProGlyProPheArgValGluArgT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spLeuThrTyr580585590GlySerPheGlyTyrlieGluTyrSerThrThrlieGinPheProAsp605GluHisProLyslieThrIjeuHisIjeuSerAspIjeuSerAsnAsnSer610620SerPheTyxValAspSerlieGluPhelieProValAspValAsnTyr630635AlaGluLysGluLysLeuGluLysAlaGinLysAlaValAsnThr650655PheThrGluGlyArgAsnAlaIjeuGinLysAspValThrAspTyrLysValAspGinValSerlieIjeuValAspCyslieSerGlyAspLeuTyr675680ProAsnGluLysArgGluLeuGinAsnLeuValLysTyrAlaLysArg690700LieuSerTyxSerArgAsnLeuLeuLeuAspProThrPheAspSerlie705710715720AsnSerSerGluGluAsnGlyTrpTyrGlySerAsnGlylieVallie725730735GlyAsnGlyAspPheValPheLysGlyAsnTyrLeuliePheSerGly740745750ThrAsnAspThrGinTyrProThrTyrIjeuTyrGinLyslieAspGlu755760765SerLyslieuLy，GluTyrThrArgTyrIjysLeuLysGlyPhelieGlu770775780SerSerGinAsplieuGluAlaTyrVallieArgTyrAspAlaLysHis785790795800ArgThrLieuAspValSerAspAsnLeuIjeuProAsplielieuProGlu805810815AsnThrCysGlyGluProAsnArgCysAlaAlaGinGinT^xLeuAsp820825830GluAsnProSerProGluCysSerSerMetGinAspGlylieLeuSer835840845AspSerHisSerPheSerIjeuAsnlieAspThrGlySerlieAsnHis850855860AsnGluAsnUeuGlylieTrpValLeuPheLyslieSerThrLeuGlu.865870875880GlyTyrAlaIiysPheGlyAsnLeuGluVallieGluAspGlyProVal885890lieGlyGluAlaLeuAlaArgValLysArgGinGluThrLysTrpArg900905910AsnLysIjeuAlaGinUeuThrThrGluThrGinAlalieTyrThrArg91SS25AlaIysGinAlaLeuAspAsnIjeuPheAlaAsnAlaGinAspSerHis935LeuLysArgAspValThrPheAlaGlulieAlaAlaAlaArgLyslie945350355S60ValGinSerlieArgGluAlaTyrMetSerTrpIjeuSerValValPro965370GlyValAsnHisProliePheThrGluLeuSerGlyArgValGinArg980S8S990AlaPheGinUeuTyrAspValArgAsnValValArgAsnGlyArgPhe99510001005lieuAsnGlyLeuSerAspTrplievalThrSerAspValLysValGin101010151020GluGluAsnGlyAsnAenVallieuVallieuA8nAsnTrpAspAlaGin10251030103S1040ValLeuGinAsnValLyslieuTyrGinAspArgGlyTyrlieLeuHis104510501055ValThrAlaArgLyslieGlylieGlyGluGlyTyrlieThrlieThr10S010651070AspGluGluGlyHisThrAspGinLeuArgPheThrAlaCysGluGlu107510801085lieAspAlaSerAsnAlaPhelieSerGlyTyrlieThrLysGluLeu10901100GluPhePheProAspThrGluLysValHislieGlulieGlyGluThr1105111011151120GluGlyliePheLeuValGluSerlieGluLeuLeuMetGluGlu112511301135LieuCys<210>42<211>1157<212>PRT<213>蘇云金芽孢桿菌<400>42MetSerProAsnAsnGluAsnGluTyrGlulielieAspAlaThrPro1510SerThrSerValSerSerAspSerAsiaArgTyrProPheAlaAsnGlu202530ProThrAspAlaLeuGinAsnMetAsnTyrLysAspTyrLeuLysMet354045SerGlyGlyGluAsnProGlu:LeuPheGlyAsnProGluThrPhelie5055GOSerSerSerThrlieGinThrGly工leGlylieValGlyArglieLeu65707580GlyAlalieuGlyValProPheAlaSerGinlieAlaSerPheTyrSer8590PhelieValGlyGinLeuTrpProSerLysSerValAsplieTrpGly100105110Glu工leMetGluArgValGluGluLeuValAspGinLyslieGluLys115120125Tyrval]jysAspLysAlaLeuAlaGluLeuLysGlyLeuGlyAsnAla130135140LeuAspValTyrGinGinSerLeuGluAspTrpUeuGluAsnArgAsn14515015S160AepAlaArgThrArgSerValValSerAsnGinPhelieAlaLeuAsp1G5170175IjeuAstlPheValSerSerlieProSerPheAlaValSerGlyHisGlu180185ValLeuLeuLeuAlaValTyrAl3GinAlaValAsnLeuHislieuLeu200205'LieuLeuArgAspAlaSerliePheGlyGluGluTrpGlyPheThrPro210215220GlyGlulieSerArgPheTyrAsnArgGinValGinLeuThrAlaGlu225230235240TyrSerAspTyrCysValLiysTrpTyrLyslieGlylieuAspIiysLeu245250255LysGlyThrThrSerLysSerTrpLieuAsnTyrHisGinPheArgArg260265270GluMetThrIjeuValIjeuAsp3JeuValAlaLeuPheProAsnTyr275280285AspThrHisMetTyrProlieGluThrThrAlaGinLeuThrArgAsp290300ValTyrThrAspProlieAlaPheAsnlieValThrSerThrGlyPhe305310315320CysAsnProTrpSerThrHisSerGlylieLeuPheTyrGluValGlu325330335AsnAsnVallieArgProProHisLeuPheAsplielieuSerSerVal34034S350GlulieAsnThrSerArgGlyGlylieThrlieuAsnAsnAspAlaTyx355365lieAsnTyrTrpSerGlyHisThrlieuLysTyrArgArgThrAlaAsp370375380SerThrValThrTyrThrAlaAsnTyrGlyArglieThrSerGluLys385390395400AsnSerPheAlalieuGluAspArgAspliePheGlulieAstiSerThr405410ValAlaAsnlieuAlaAsnTyrTyrGinLysAlaTyrC51yValProGly42042S430SerTrpPheHisMetValLysArgGlyThrSerSerThrThrAlaTyr43S"5lieuTyrSerLysThrHisThrAlaLeuGinGlyCysThr*GinValTyr455460GluSerSerAspGlulieProLeuAspArgThrValProValAlaGlu470475480SerTyrSerHisArgLeuSerHis工leThrSerHisSerPheSerLys485490495AsnGlySerAlaTyrTyrGlySerPheProValPheValTrpThrHis500505S10ThrSerAlaAspLeuAsnAsnThrlieTyrSerAspLyslieThrGin515520525工leProAlaValLysGlyAspMetLeuTyrlieuGlyGlySerValVal530535540GinGlyProGlyPheThrGlyGlyAsplieIjeuLysArgThrAsnPro545550555560SerlieLeuGlyThrPheAlaValThi:V汪lAsnGlySerLeuSerGin570575ArgTyrArgValArglieArgTyrAlaSerThrThrAspPheGluPhe580585590ThrLeuTyrLeuGlyAspThrlieGluLysAsnArgPheAsnLysThr595600605MetAspAsnGlyAlaSerLeuThrTyrGluThrPheLysPheAlaSer620PhelieThrAspPheGinPheArgGluThrGinAspLyslieIjeuLieu630。5640SerMetGlyA印PheSerSerGlyGinGluValTyrlieAspArglie€50655GluPhelieProValAspGluThrTyrGluAlaGluGinGlu670AlaAlaLysLysAlaValAsnAlaLeuPheThrAsnThrLysAspGly6806B5LeuArgProGlyValThrAspTyrGluValAsnGinAlaAlaAsnLeu。0700ValGluCysUeuSerAspAspUeuTyrProAsnGluLysArglieu705710715720PheAspAlaValArgGluAlaLysArgLeuSerGlyAlaArgAsnLeu725730735LeaGinAspProAspPheGinGlu工leAsnGlyGluAsnGlyTrpAla740745750AlaSerThrGlylieGlulieValGluGlyAspAlaValPheIiysGly755765ArgTyrLeuArgUeuProGlyAlaArgGlulieAspThrGluThrTyr770775780ProThrTyrUeuTyrGliiIiysValGluGluGlyValUeuIiysProTyr785790795800Thi:ArgTyrArgLeuArgGlyPheValGlySerSerGinGlyLeuGlu80581081S工leTyrThrlieArgHisGinThrAsnArglieValLysAsnValPro820825830AspAsplieuJjeuProAspValSerProValAsnSerAspGlySerlie835840845AsnArgCysSerGluGinLysTyrValAsnSerArgIjeuGluGlyGlu850855AsnArgSerGlyAspAlaHisGluPheSerProlieAsplieGly865870875880GluIj€UAspTyrAsnGluAsnAlaGlylieTrpVeQGlyPheLyslie885895ThrAspProG工uGlyTyrAlaThrI>euGlyAsnlieuGluUeuValGlu900905910GluGlyProLeuSerGlyAspAlalieuGluArglieuGinArgGluGlu920S2SGinGinTrpLyslieGlaMetThrArgArgArgGluGluThrAspArg930335ArgTyrMetAlaSerLysGinAlaValAspArgLeuTyrAlaAspTyr94S950SS5GinAspGinGinI/euAsnProAspValGlulieThrA印LeuThrAla$70AlaGinAeplieulieGinSerlieProTyrValTyrAsnGluMetPhe380S85ProGlulieProGlyMetAsnTyrThrLysThrGluLeuThrAsp99510001005ArgLeuGinGinAlaTrpAsnLeuTyrAspGinArgAsnAlaliePro101010151020AsnGlyAspPheArgAsnGlyLeuSerAsnTrpAsnAlaThrProGly10251030103S1040ValGluValGinGinlieAsnHisThrSerValLeuVallieProAsn104510501055TrpAspGluGinValSerGinGinPheThrGinProAsnGinArg106010651070TyrVal:LeiiArgValThrAlaArgLysGluGlyValGlyAsrtGlyTyr107510801085ValSerlieArgAspGlyGlyAsnGinSerGluThrUeuThrPheSer109010951100AlaSerAspTyrAspThrAsnGlyValTyrAsnAspGinThrGlyTyr1105111011151120工leThrIjysThrValThrPhelieProTyrThrAspGinMetTrplie112511301135GluUeSerGluThrGluGlyThrPheTyrlieGluSerValGlulieu114011451150工leValAspValGlu1155<210>43<211>675<212>PRT蘇云金芽孢桿菌<400>43MetAsnProTyrGluAsnIjysAsnGluTyrGluliePheAsnAlaPro151015SerAsiiGlyPheSerLysSerAsnAsnTyrSerArgTyrProlieuAla202530AsnLysProAsnGinProLeuLysAsnThrAsnTyrLysAspTrpUeu354045AsnValCysGinAspAsnGinGinTyrGlyAsnAsnAlaGlyAsnPhe5055AlaSerSerGluThrlieValGlyValS6rAIelGlylielieValVal65707580GlyThrMetLeuGlyAlaPheAlaAlaProValLeuAlaAlaGlylie859095lieSerPheGlyThrLeuLeuProliePheTrpGinGlySerAspPro100105110AlaAsnValTrpGinAspUeuAsn工leGlyGlyArgProlieGin115120125GlulieAspAsnlielieAsnValIjSUThrSerlieValThrP:ro130135140UeLysAsnGinlieuAspLysTyrGlxiGluPhePheAspLysTrpGlu145150155ProAlaArgThrHisAlaAsnAlaLysAlaValHisAspLeuPheThr165170175TlirLeuGluProHelieAspLysAspIjeuAspMetLeuLy日AsnAsn180185ISOAlaSerTyrArglieProThrLeuProAl3TyrAlaGinlieAlaThr200205TrpHisAen￡jeuLeuIiysHisAlaAlsThrTyrTyrAsnlieTrp210215220lieuGinAsn<31nGlylieAenProSerThrPheAsnSerSerAsnTyr225230235240TyrGinGlyTyrLeuLysArgLyslieGluGluTyrThrAspTyrCys24S2S02SSUeGinThrTyrAsnAlaGlyLeuThrMetlieArgThrAsnThrAsn260265270AlaThrTrpMetTyrAsnThrTyrArgLeuGluMetThrI*euThr27528028SValLeuAsplieulieAlaliePheProAbjiTyrAspProGluLysI*yr29S300ProlieValSerGlulieulieArgGluValTyrThrAsnVal30S310AsnSerAspThrPheArgThrlie325ArgProThr340LieuPheThrTrpArgAsnSer355ArgAsplieIjeuGlyAsnGinMetAlaPheThrHis370375TrpGlyAlaValHisGlyAsnlie385390PheProPheTyrArgAsnLysPro405HisArgGluTyr420SerAsp工lelieSerSerGlu435PheArgTyxSer440TyrLysArgThrAspSerTyrMet450455GluGluTyrGlyHisThrLieuSer470PheSerValValArgGluArgArg485ThrSerValAspsooPheGinAsnThrlieHisAla515IjeuLysAlaLeuLys520LysGlyProGlyHisThrGlyGly530535MetAspPheArgValPheLeuS45550ValArgUeArgTyrAlaThrAsn565ThrGlylieAsp580ThrlieSerValGinAsnProAsnAlaThrAspLeuGOOThrPheProArgThrValPro615AsnLeuLeuMetThrLeuTyrGlyThr625630lieAspLyslieGluPhelieProThrGluLysGin660lieGluLysValAsn675<210>44<211>648<212>PRT<213>蘇云金芽孢桿菌<400>44MetHisTyrTyrGlyA6txArgAsn15SerAsnAspSerAsnMetSerAsn20AsnProGinGluAspLeuMetGlu3540AsnValCysGluGlyTyrHislieSO5SArgAlaGlyLeuGlyLysGlylieu6570PhePheGlyGlySerlielieLeu85lieSerGluLeuTrpProGlu315320ThrGluLeu<31uAsnGlyThr33033S工leAsnGinGlyArgPheTyrThr345350ProTyrAspliePteSerPheThrThrAsnA印AspArgAsnlielie380工leSerGinAspThrSerLysVal395400lieASpLysValGlulieValArg410415Tyr'GluMetliePhePheSerAsn425430SerAsnSerThrlieGluAsnAsn445工leProLysGinThrTrpLysAsnTyrlieLysThrAspAsnTyr工le475480ArgValAlaPheSerTrpThrHis49049S工leAspLeuAspAsnlieThrGin505S10ValSerSerAspSerLyslieVal525AspLeuVallieUeuLysAspSer540Ly日AsnValSerArgGinTyrGin555560AlaProLysThrThrValPheIjeu570575GluLeuProSerThxThrSearArg5855S0ThrTyrAlaAspPheGlyTyrValSOSLysThrPheGluGlyGluAspThr620ProAsnHisSerTyrAsnlieTyr635lieThrGinSerValUeuAspTyrThrGinLyslieValAsnAsplieuMSGluTyrAsplieLeuAsnAlaSer10ThrTyrProArgTyrProlieuAla2530AsaThrAsnTyxLysAspTrpLeu45GluAsnProArgGluAlaSerVal60GlylieValSerThrlieVal<31y7580AspThrlieGlyLeuPheTyrGin9095AspAspThrGin<31nTyxThrTrp100GinAsplieMet115GluVallieArg130LysValAspAsp145LiysSerThrGlyAspSerAspPhe180ProGlyTyrGlu195IjeuHisLeuLeu210TrpSerAlaArg225GluLysThrLyBGlyljeuAsnAsp260TyrArgArgGlu275Pro工leTyrAsp290ThrArgGlulie305MetThrProTyrAlaProH丄slieuSerlieSerTyr355TyrThrAsnAsn370ThrAspTyrGly385TyrValTyrAsnGlyAspProVal420AsnSerSerLys435V3lVaiArgThr450lieuAlaLiysTyr465AsnGlyGluThrThrHisSerSer500ThrGin工lePro51SlieGlulysGly530AlaAspSerGly54SLysTyrArgVallieI*euArgLys580AsnlieSerPro595lieThrThrThr610ProTyrGlyArg625LyslieGluPheH丄sValGlu120GlyAsnAla工le135TyrAsnAsnTrpISOAsnLeuSerThrAsnGlyAla工leLeuLeuLeuLeu200lieuLieuArgAsp215AlaAsnAlaArg230GluTyrThrGlu245PheArgThrAlaMetThrUeuThr280AlaArgLeuTyr295TyrSerAspVal310PheSerPheGlu325PheThrTrpJjeuTrpThrPhelieuSerSerlieAsn37SGlyThrSerSerLeuTrpThrGlu405AsnlieThrijysGluLeulieTyr440AspPheAspPhe455AsnAspTyrAsn470PheGlyGinLys485ValAspProAsnValValLysAla520ProGlyPheThr535LeuThrMetArgS50ArglieArgTyrTrpLysGlyGluThrTyrGlyAla600GluAsnliePhe615GinPheValGlu630LeuProThrAsn105AsplieAspArgThrLeuAla140LeuLysLysTrp155LeuValThr170ArgThrValAsn185ProValTyrAlaAla(Unlieoyr220AspAsnTyrTyr235TyrCyslieAsn250GlyGinTrpVal265VallieuAsplieProThrGluVal300lieAsnGlyGlu315LysAlaGlu330■LysGlyPheArg345SerGlyGlyGinGluGlySerPhe380ThrlieAsnlie395AsnTyrGluTyrMetAsnPheSer425GlyAlaHisArgLeuThrAsn460HislieLeuSer475ArgHisGlyTyr4S0AsnThrlieAlaSOSSerSerlieAsnplyGlyAspLeu540PheLysAlaGluS55LysCysAenTyrS70GlyTVrlieGin58SPheSerTyrLeuAspLeuThrMetASplieProSer635110ArglieThr125AspLeuGinGlyLysAspAspProPileThi:AlaIjeu175AsnGinGlySerGinlieAlaAsn205GlyAepLysTrpGinlieGinLeu240TrpTyrAsnLys255AsnPheAsnArg270lieSerMetPhe285LysThrGluLieulieTyrGlylieu320lieu*TyrThrArg335PheValThrAsn350AsixLysTyrSer365ArgGlyGinAspProSerAsnSer400lieTyrProTrp415ValThrAspAsn430ThrAsnLysPro445GluGlyThrGluTyrMetLeulie480SerPheAlaPheAlaAsnLyslie510GlySerlieSer525ValLysMetArgLeulieuAspLysSerSerLysLeuS75饑nGinlieHis5S0GluSerPheThr605GluValThrTyrlieulielieuAsp"5<210>45<211>682<212>PRT<2">蘇云金芽孢桿菌<400>45MetAsnSerTyrGinAsnLysAsnGluTyrg:lulieLeuAspAlaLys151015ArgAsnThrCysHisMetSerAsnCysTyrProLysTyrProIjeuAla202530AsnAspProGinMetTyrIjeuArgAsnThrHisTyxIiysAspTrplie354045AsnMetCysGluGluAlaSerTyrAlaSerSerGlyProSerGinLeu505560PheValGlyGlySerlieValAlaLyslielieuGlyMetliePro65707580GluValGlyProLieuLieuSerTrpMetSerlieuPheTrpProThr859095lieGluGluAsnThrValTrpGluAspMet工leLysTyrV3lAla100105110AsnLeuljeuLysGinGluLeuThrAsnAspThxlieuAsnArgAlaThr115120125SerAsnUeuSerGlylieuAsnGluSerlieuAsnlieTyrAsnArgAla130135140LeuAlaAlaTrpLtysGinAsnLyaAsnAsnPheAlaSerGlyGluIjeu14S150155160lieArgSerTyr工leAsnAspIjeuHislieLeuJPheThrArgAsplie170175GinSerAspPheSerLeuGlyGlyTyrGluThrValIjeuIjeuProSer180185190TyrAlaSerAlaAlaAsnlieuHisIjeuLeuLeuLeuArgAspValAla195200205lieTyrGlyLysGlulieuGlyTyrProSerThrAspValGluPheTyr210215220TyrAsnGluGinLysTyrTyrThrGluJjysTyrSerAsnTyrCysVal225230235240AsnThrTyrLysSerGlyLeuGluSerLysLysGin工leGlyTrpSer245250A印PheAsnArgTyrArgArgGluMetThrLeuSerValLieuAsplie270ValAlaLeuPheProLeuTyrAspThrGlyLeuTyrProSerLysAsp275280285GlyLyslieHisValIysAlaGluLieuThrArgGlulieTyrSerAsp290295300VallieAsnAspHisValTyrGlyLeuMetValProTyrlieSerPhe305310315320GluHisAlaGluSerLeuTyrThrArgArgProH丄sAlaPheThrTrp325330335LeuIiysGlyPheArgPheValThrAsnSerlieAsnSerTrpThrPhe340345350LeuSerGlyGlyGluAsnArgTyrPheIjeuThrHisGlyGluGlyThr355lieTyrAsnGlyProPheLeuGlyGinAspThrGluTyrGlyGlyThr370375380SerSerTyrlieAsplieSerAsnAsnSerSerlieTyrAsnLeuTrp38S3S0395400ThrLysASHTyrGluTrplieTyrProTrpThrAspProValAsnlie405410415ThrLyslieAsnPheSerlieThrAspAsnSerAsnSerSerGluSer42042S430lieTyrGlyAlaGluArgMetAsnLysProThrValArgThrAspPhe435445AsnPheLeuLeuAsnArgAlaGlyAsnGlyProThrThrTyrAsnAsp4S0TyrAsnHislieLeuSerTyrMetlieulieAsn<3lyGluThrPheGly465470475480GinLysArgHisGlyTyrSerPheAlaPheThrHisSerSerValAsp485490495ArgTyrAsnThrlieValProAspIiyslieValGinlieProAlaVal500505510LysThrAsnLeuValGlyAlaAsn工lelieLysGlyProGlyHisThr515520525GlyGlyAspLeuIjeuLyslieuGluTyrGluArgPHeLeuSerLeuArg530535540lieLysLeulieAlaSerMetThrPheArglieArglieArgTyrAla545550555SerAsnlieSerGlyGinMetMet工leAsnlieGlyTyrGinAsnPro565570575ThrTyrPheAsnlielieProThrThrSerArgAspTyrThrGlulieu580585590laysPheGluAspPheGinLeuValAspThrSerTyr工leTyrSerGly595600605GlyProSerlieSerSerAsnThrUeuTrpUeuAspAsnPheSerAsn610615GlyProVallielieAspLyslieGluPhe工leProGlylieThr625630635640LeuAsnGinAlaGinGlyTyrAspThrTyrAspGinAsnAlaAsnGly655MetTyrHisGinAsnTyrSerAsnSerGlyTyrAsnTyrAsnGinGlu665670TyrAsnThrTyrTyrGinSerTyrAsnAsn680<210>46<211>529<212>PRT蘇云金芽孢桿菌<400>46AsnPheMetlieuThrSerGlyAlaAsnMetLeuLysIeuGlu151015ThrThrAspTyrGlulieAspGinMetAlaAsnAlalieGluAsnMet202530SerGlyGluGluTyrSerGinGluLysMetMetGinTrpHisAsplie354045LysTyrAlaLysGinLeuSerGinAlaArgAstiLeuLeuGinAsnGly5055AspPheGluAspIseuPheSerGlyTrpThrThrSerAsnGinMetSer707580lieGinAlaAspAsnAlaThrPheLysGlyAsnTyrLeuHisMetSer8595GlyAlaArgAsplieTyrGlyThrliePheProThrTyrlieTyrGin100105110LyslieAspGluSerLyslieuLysProTyrThrArgTyrlieuValArg115120125GlyPheValGlySerSsrLysAspIjeuGluLeuMetValMetArgTyr130135140GlyLysGlulieAspThrValMetAsnValProAsnAsplieProTyr150155ValProSerMetValCysAsnGluIjeuTyrAspGlyGinGinPro165170175TyrProAsnArgHisValGlyTyrTyrAsnProMetProValSerGin180185ISOProSerTyrThrSerAspThrCysGinCysThrProGlyLysLysHis20020SValValCysHisAspSerHisGinPheLysPheHislieAspThrGly210220GluValAspTyrAsnThrAsnLeu<33LylieTrpValLeuPheLyslie22523023S240SerSerProAspGlyTyrAlaTfarLeuAspAsnlieuGluVallieGlu2452S02S5GluGlyProValArgGlyGluAlaValThrHisValLysGinLysGlu260265270LysTrp275AsnGinGinMetGlu280LysLysArgMetGlu285ThrLysArgValTyr290AspArgAlaIjysGinAlaValAspAlaIjeu300PheThrGlyGluGluLieuAsnTyrAspValThrLeuSerHislieIjysAsnAlaAspAsp305310315320lieuValGinSerHe325ProTyrValHisAsn330GluTrpIjeuProAsp335PheProGlyMetAsn340TyrAsplieTyrGin345GluLeuAsnAlaArg350lieMetGinAlaArg355TyrLeuTyrAspAla360ArgAsnVal工leThrAsnGlyAspPheAlaGinGlyLeuGinGlyTrpHisAlaGluGlyIjysValGluVal370375380GinGinMetAsnGlyThrSerValLeuValLeuSerAsnTrpSerSer385390395400GlyValSerGinAsn405]jeuHisValGinHis410ProHisGlyTyrLeu415lieuArgValSerAlaijysIjysGluGlySerGlyLiysGlyTyrValThrArg420425430MetSerCys435AsnGlyLysGinGlu440ThrtieuThrPheThr445SerCysAspGlyGly450TyrMetThrIjysThr4S5ValGluValPheProGluSerAspArgValArglieGlulieGlyGluThrGluGlySerPheTyrlieGluSer470475480lieGluLeu工leCys485MetAsnGlyTyrThr490SerAsnAsnAsnGlu495AsnMetSer■AsnMetTyrAspGinSerTyrGlyAsnTyrSerGinAsn500S05510ThrSerAspMetTyrAspGinGlyGlySerValAlaLysPheGluIjys51S520525Glu<210>47<211>674<212>PRT<2">蘇云金芽孢桿菌<400>47MetAsnGinTyrGinAbuLys15GinAsnAsnMet20AsnMetProAsnAsnAlaVal35MetLysAsnGlyAsn40GluGlySOSerAsnlieSerPro55SerlieValSerlieVallieulaysThr6570LeuAlaAspSerlie85LysSerSerGluAsnAsnVal100SerGinValSerAsnArgArg115lieGinGluThrlie120AsnGly130LeuValAlalieTyr135AsnAlaTrpAsnAsnAsnAsn150GluAlaPtieLysThrValGluArglieTyrArgThr180SerSerSerGinGinAlaAlaAsnIi€UHislieuSerGluTyr10ArgTyr2STyrLysProAl3LeuAlaLeuGly90MetVal105lieuAspArgAsplieAsnGlulie185Met170ThrLeuGlu工leProPheAspTrpAlaAl360LysAlei7SlieSerGinVallieuGlyTyrlieuTyrGin1S5PheThrlieulieuArgAspLeuAlaVal45lievalLysHisGlu125GlyThrLysProAlaGluAsp30AsnThrAlaThrGin110SerAlaAsnLeuThr190ValSerSer15AspProGluCysSerSer80lieThrlielieSerLeulieuGluValAla160LysGly175PheThrMetTyr195200205GinGluGlyTrpAsnlieuGinSerHislieAsnTyrSerLysGluIjeu210215220AspAspAlaLeuGluAspTyrThrAsnTyrCysValGluValTyrThr22S230235240I/ysGlylieuAsnAlaLeuArgGlyThrAlalieAspTrpLeuGlu24525025SPheAsnSerPheArgArgAspMetThrIjeuMetValLeuAspIjeuVal260265270AlaliePheProAsnTyrAsnProValArgTyrProLeuSerThrIiys275280285lieSerLieuSerArglieTyrThrAspProValGlyArgThrAsp29029S300SerProSerPheGlyAspTrpThrAsnThrGlyArgThrLeuAlaAsn305310315320PheAsnAspIjeuGluArgGluValThrAspSerProSerIjeuValIjys325330335TrpLeuGlyAspMetThrlieTyrThrGlyAlalieAspSerTyrArg340345350ProThrSerProGlyAspArglieGlyValTrpTyrGlyAsn工leAsn355360365AlaPheTyrHisThrGlyArgThrAspValValMetPheArgGinThr370375380GlyAspThrAlaTyrGluAspProSerThrPhelieSerAsnlieLeu385390395TyrAspAsplieTyrLysLeuAspLeuArgAlaAlaAlaValSerThr405410415lieGinGlyAlaMetA印ThrThrPheGlyValSerSerSerArgPhe420425PheAsplieArgGlyArgAsnGin'LieuTyrGluSerAsnLysProTyr43S440445ProSerLeuProlieThrlieThrPheProGlyGluGluSerSerGlu4S045SGlyAsnAlaAsnAspTyrSerHisIj6ULeuCysAspValLyslieLeu465470475.480GinGluAspSerSerASlllieCysGluGlyArgSerSerIjeuLeuSer485490495HisAlaTrpThrHisAlaSerLeuAspArgAsnAsnThrlieLeuPro500505510AspGlulieThrGinlieProAlaValThrAlaTyrGluIjeuArgGly515520525AsnSerSerValValAlaGlyProGlySerThrGlyGlyAspLeuVal530535540LysMetSerTyrHisSerValTrpSerPheLysValTyrCysSerGlu545550555560LeuLysAsnTyrArgValArg工leArgTyrAlaSerHisGlyAsnCys565570S75GinPheLeuMetLysArgTrpProSerThrGlyValAlaProArgGin580585590TrpAlaHisAsnValGinGlyThrPheSerA掃nSerMetArgTyr595600GluAlaPheljysTyrlieuAspliePheThrlieThrProGluGluAsn€10615620AsnPheAlaPheThrlieAspLeuGluSerGlyGlyAspLeuPhelie€2563063SAspLyslieGluPhelieProValSerGlyPheGlutTyrGlu"5GlyLysGinAsnlieGluLysThrGinLysAlaValAsnAspLeuPhe660665670lieAsn<210>48<211>。5<212>PRT<2">蘇云金芽孢桿菌MetAsnProTyxGinAsnLysSerGluCysGlulieIeuAsnAlaPro151015LeuAsnAsnlieAsnMetProAsnArgTyi:ProPheAlaAsnAspPro202530AlaValMetLysAsnGlyAsnTyi:LiysAspTrpLeuAsnGluCys354045AspGly工leThrProSer工lePheGlyThrIjeuGlyValLeuAlaSer505560lieVallieSerThrlieAsnLieuAlaThrSerProSerlieGlyAsp707580AlaPheAlaLeuValSerSerlieGlyGluTyrTrpProGluThrLys859095ThrSerPheProIjeuSerValAlaAspValAsnArgLeulieArgGlu100105110AlaLeuAspGinAsnAlalieAsnArgAlaThrGlyLysPheAsnGly115120125IjeuMetAspThrTyrAsnThrValTyrLeuLysAsnLeuGinAspTrp130135140TyrAspThrArg工leProAlaAsnProGinGlyAspSerGinIjeuArg145150155160GluAlaAlaArgArgSerLeuGluGlu工leGluArgAspPheArgLys170175AlaLeuAlaGlyGluPheAlaGluAlaGlySerGinlieValIjeulieu180185190ProlieTyrAlaGinAlaAlaAsnlieHisIjeulieulieUeuLysAsp200205AlaMetGinPheArgThrAspGlyUeulieArgProValGlyVal210215220Pxo工leThrThrSerAlaGluAspProPheGluSerGluPheIjeuIjeu225230235240ArglieLysLysTyrThrAspHisCyslieSerTyrTyrAspAspGly245250255lieuAlaLyslieArgSerArgGlySerAspGlyGluThrTrpTrpGlu2602S5270PheAsntiysPheArgArgGluMetThrLeuThrValLeuAspI/euVal275280285AlaLeuTyrProThrHisAsnlieLysLeuTyrProlieProThrGin290295300ThrGluLeuSerArgValValTyr*ThrAspProValGlyCysPheGly305310315320AsnArgLysSerAspliePheSerArgLieuAsnPheAspTyrlieuGlu325330335AsnArglieuThrArgProArgGluProPheAsnTyrIjeuAsnSerVal3403453S0GinI/euPheAlaSerThrValSerAsnSerAsnAsnGlyGluValLeu35536036SArgGlyAsnI^euAsiiLyslieMetPheGluGlyGlyTrpThrAlaSer370375380ArgSerGlyAspGlyValThrThrGlyThrProPheSerThrMetAsp385390395400TrpSerTyrGlyTrpGlyTyrProArgLysHisTyrAlaGlulieThr405410415SerArgSerGinAlalieuProGlylieuAsnAsnSerUeHisVallie420425430ValGlylieAspSerPheArgAlalieGlyProGlyGlyGinGlyAsp435440"5HisThrPheSerlieuProGlyGlyAspMetTyrAspCysGlyLysVal4S04SS460GinlieAsaProLeuGluAspTyrArgAsnSerAspHisTrplieSer465470475480AspMetMetThrlieAsnGinSerValGinlieuAlaSerAsnProThr4&S490GinThrPheAlaPheSerAlaLeuSerlieuGlyTrpHisHisSerSerSOOS05S10AlaGlyAsnArgAsnValTyrValTyrAspLyslieThrGinliePro515520S2SAlaThrIiysThrValArgGluHisProMetlieLysGlyPro530535540ThrGlyGlyAspLeuAlaAspLeuSerSerAsnSerAsplieLeuGinS45S50555TyrAsplieuArgSerAspTyrAspAspArgLeuThrGluAspValPro565570575PheArglieArglieArgCysAlaSerlieGlyValSerThrlieSer580585590ValAspAsnTrpGlySerSerSerProGinValThrValAlaSerThr595605AlaAlaSerAspThrLeuLysTyxGluSerPheGinTyrValSer620lieProGlyAsnTyrTyrPheAspSerAlaProArglieArgLeuLeu630640ArgGinProGlyArglieuLeuValAspArglieGlulielieProVal"5655AsnPhePheProSerGluGinGluAsnLysSerValAspSerLeu670PhelieAsn675<210>49<211>6S9<212>PRT<213>蘇云金芽孢桿菌<400>49AsnSerTyrGluAsnLysAsnGluTyrGlulieLeuAsnAspSerLys151015LysSerAsnMetSerAsnProTyrlieuArgTyrProLeuAlaAsnAspAla2025、，30SerLeuSerMetGinAsnThrAsnTyrLysAspTrpLeuThrMet3S4045CysAspArgThrAspThrAspValLeuSerSerArgGlyAlaValSer5055GOThrGlyValGlyMetLeuSerThrlielieuSerUeuPlieGlyliePro65707580LeulieGlyGluGlylieAspLeuLeuUeuGlyAlaAlaAspPheLeu8S9095TrpProGluSerAspGlyGlyHisGinTyrThrTrpGluAspUeuMet100105110AsnHislieGluGluIjeuMetAspGluArgLeuGluThrGluIiysArg115120125ThrThxAlalieuAspAspLeuArgGlyLeuLysAlaLeuIjeuGlyIjeu130135140PheArgAspAlaPheA印SerTrpGluLysAsnGinAsnAspProlieISO155160AlaLysAsnArgValGlyGlyTyrPheGluAspValHisThrHisPhe170175ValLysAspMetAlaSerliePheSerAlaThrAsnTyrGluVallieu180185ISOUeuIjeuProValTyrAlaGinAIelAlaAsnUeuHisLeuLeulieuLeu19520020SArgGluGlyVallieTyrGlySerArgTrpGlylieAlaProAlaAla210215220AspPheTyrHisAspGinLeuLeuLysTyrThrAlalieTyrAlaAsn225230235240HisCysValThrTrpTyrAsnAsnGlyLeuAlaGinGluLysGlulieu2452502S5PheAlaIiysSerProAsaTrpAsnArgPheAsnAlsiTyrArgArgAsp270MetThrlieThrValLeuAsplielieAlaLeuPheProThrTyrAsp275280285AlaArglieuTyrThrLysProlieLysThrGluLeuThrArg<31ulie2卯295300TyrSerAspVallieuAsnLeuAspValTyrGlyValGinGinThrAsp30531031S320LeuAsnLysAsnGluAl3AlaPheThrArgSerProHisLeuValThr325330335ArgLeuArgGlyPheAspPheTyrThrArgThrLysTyrAlaTyrTrp340345350ArgTyrLeuAlaGlyHisThrAsnTyrPheSerPheThrGlyAsnGly355360Thr工leTyrSerSerSerPheAsnAsnTrpTyrAspThrAspMetThr370375380LysSerThrlieAsnlieProAspTyrAlaAsnlieTyrLysLeuTrp385395400ThrLysSerTyrThrAsnlieSerProTyrThrAspPi:oValGlylie405410415SerGinMetGinPheSerLeuThrAsnAsnGinGinLeuThrTyrThrf420425430GlyThrSerAlaProLysTyrProVal■ArgGluThrPhePheGlulie435440445ProProThrAspGluLysProIjeuThrTyrGluAsnTyrSerHislie4504S5460IjeuSerTyrMetThrSerAlaGinH丄sPheGlyAspLysLyslieGly465470475480TyirThrPheAlaTrpMetHisGluSerValAspPheAspAsn485435AspProAspLyslieThrGinlieProAlaValLysGlyAspTyrIjeu500505510GinTyrGlyTyrValGinGlyProGlyHisThrGlyGlyAspLeu515520525ValSerMetlieArgThrAspArgLeuGlylieAsiiValTyrPhePro530535540GinProLieuAspTyrArglieArglieArgTyrSerThrSerSerAsn545550555560GlyTyrUeuTyr工JLeTyrSerProAsnThrLyslieValTyrIjeuPro570575ProThrThrLeuValAspGlyGinProThrPheAspProMetAspPhe58058SS90SerAlaPheArgValValGluValProAlaSerPheArgAlaSerVal595600605AlaGlyTyrThrAsnPheThrlieGluAlaGlyPheGlyProValTyr€15工leAsplieGluPhelieProAspAsnThrThrThrlieuGluTyr625630635640GluGlyGlyArgAspLeuGluUysThrLysAsnAlaValAsnAsplieu650655PheThrAsn<210>50<211>55B<212>PRT<213>蘇云金芽孢桿菌<400>50MetPhelieSerAsnlieLysAsnThr3beuLyslieGluThrThrAsp1S1015GlulieAspGinAlaAlalieSerlieGluCysMetSerAsnGlu202530HisSerSerI*ysGluGluMetMetLeuTrpAspGluGinAla354045LysGinIjeuSerTrpSerArgAsnIjeuLeuTyrAsnGlyAspPheGlu505560AspValSerAsnGlyTrpLysThrSerAsnThrlieGlulieArgGlu65707580AsnSearProValPheLysGlyHisTyrlieuHisMetPheGlyAlaArg8530AsplieAspGlyThrLeuPheProThrTyrlieTyr<3lnliyslieGlu10S110GluSerLysLeuLysProTyrThrArgTyrArgValArg<31yPheValUS12012SGlySerSerLysAspLeuLysLeuMetValThrArgTyr(SlyGlu<table>tableseeoriginaldocumentpage155</column></row><table>SOS560GluPheLieuMetGluProSerLeuGlyGlylieAsnThrLeuPheSer65707580lielieGlyLyslieulieProThrAsnHisGinSerValSerAlaLeu859095SerlieCysAspLeuLeuSerlielieArgLiysGluValAlaAspSer100105110ValLeuSerAspAlalieCysArgPhelieuAspGlyLysUeuLysAsn115120125TyrArgGluTyrTyrLieuProTyrL;euGluAlaTrpLeuIjysAspGly130135140LysProGinliysThrAsnAsnSerAsplieGlyGinLieuValLiys145150155160TyrPheGluLeuSerGluArgAspPheAsnGlu工leLeuGlyGlySer165170175LieuAlaArgAsnAsnAlaGinlieLeuUeuIjeuProTyrPheCysAla180IBS190SerCysLysCysGinLeuIjeuLeuArgAspAlaValGinTyrGlu195200205GluGinTrpPheProPheLeuSerAlaGluAsnValArgSerGluLeu210215220lieSerProAsnSerGlyCysAspPheThrGlyAspTyrTyrGluArg225230235240LeuLysCysLyslieAlaGluTyrTtirAspTyrCysGluTyrTrpTyr245250255GinAlaGlyLeuAsnGilllieLysGinAlaGlyThrGlyAlaAspThr260265270TrpAlaIjysPheAsnLysPheArgArgGluMetThrLeuThrValUeu27528028SAsplielieAla工leGinThrTyrAspPheLysLysTyrProIjeu290295300Pro丁hrHisValGluLeuThrArgGlu工leTyrThrAspProValGly305310315320TyrSerSerGlyThrTyrSerTrpLieuLiysTyrTrpThrGlyAlaPhe325330335AsnThrLeuGluAlaAsnGlyThrArgGlyProGlyIjeuValThrTrp34034S350IjeuArgSerlieGlylieTyrAsnGluTyrValSerArgTyrPheSer355360365GlyTrpValGlyThrArgHisTyrGluAspTyrThrThrGlyAsnGly370375380AsnPheGinArgMetSerGlyThrThrSerAsnAspLeuArgAsplie38539039S400Sei:PheProAsnSerAspliePheLyslieGluSerLysAlalieMet405410415AsnLeuValGlyGlulieAsnAlaArgProGluTyrArgValSerArg420425430AlaGluPheSerGluSerThrAlaPhelieTyrLeuTyrAspAlaGly435440445AsnSerGlyLeuSerSerMetThrlieThrSerLysLeuProGlylie450455"0LysAsnProGluProSerTyrArgAspTyrSerHisArgLeuSerAsn470475480AlaAlaCysValGlyAlaGlyAsnSerArglieAsnValTyrGlyTrp4854904S5ThrHisThrSerMetSerIiysTyrAsnLeulieTyrProAspLyslie500505510Thr<51nneProAlaValLysAlaPheAsplieSerAspThrPro51SS20525GlyGinVallieAlaGlyProGlyHisThrGlyGlyAfinValValSerS30S35540lieuProTyrTyrSerArglieuUyslieArglieulieProAlaSerThr54555055S560AsnLysAsnTyrIjeuValArgValArgT^rThrSerThrSerAeaGlyS6557057SArgIjeuLeuValGluArgTrpSerProSerSerlielieAsnSerTyr580S8SS90PhePheLeuProSerThrGlyProAspPheGlyTyrValAsp59S600605ThrLeuVaiThrThrPheAsnGinProGlyValGlulielielieGin610615620AsnLeuAspThrProlieAsnValAspUysValGluPhelieProVal625630635640AsnSerThrAlaLieuGluTyrGluGlyLrysGinSerLeuGluLysAlaGinAspValValAsnAspIteuPheValLys660665<210;>52<211>558<212>PRT<21>蘇云金芽孢桿菌<400>52MetPheThrAsnGlyThrLysAsnThrLeuIiyslieGluThrThrAsp151015GlulieAspGinAla'AlalieSer工leGluCysMetSerAspGlu202530HisSerProIiysGluLysMetMetIjeuTrpAspGluValLysArgAla354045LysIjeuIjeuSerGinSerArgAsnLieuLeuGinAsnGlyAspPheGly50S560AspPheTyrGlyAsnAspTrpLysPheGlyAanAsnlielielieGly65707580SerAanAsnSerliePheLysGlyAsnPhelieuGinMetSerGlyAla859095ArgAspUeTyrGlyThrliePheProThrTyrlieTyrGinLys工le100105110AspGluSerLysUeuLysProTyrThrArgTyrArgValGlyPhe11S120125ValSerSerIjysAspLeuArgUeuMetValThrArgTyrGlyLys130135140GlulieAspAlaMetMetAsnValProAsnAsplieuAlaTyrMetGin145150155160ProAsnProSerCysGlyAspSerArgCysGluSerSerSerGinTyr165170175"ValSerGinGlyTyrProThrProThrAspGlyTyrAlaProAspArg180185190TyrAlaCysProSerSerSerAspLysLysHisValMetCysHisAsp200205ArgHisProPheAspPheHislieAspThrGlyGluLeuAspThrAsn210215220ThrAsnValGlylieAspValLeuPheLyslieSerAsnProAspGly22S230235240TyrAlaThrLeuGlyAsnLeuGluVallieGluGluGlyProLeuThr2452502S5GlyGluAlaIjeuThrHisValLys(31nLysGluLysTrpLysGin260270HisMetGluLiysLysArgTrpGluThrGinGinAlaTyrAspProAla27S280285IiysGinAlaValAspThrPheThrAsnGluGinGluLeuHisTyr295300HislieThr乙euAspTyrlieGltiThrLeulieAspTrpTyrSerArg305310315320PheProlieTyrThrMetThrGlyTyrArgAspAlaProGlyMetAsn32S33033STyrAsp<31yTyrGin<31yLeuAsnAlaArglieMetGinAlaTyrAsn34034S350LeuTyrAspAlaArgAsnVallieThrAenGlyAspPheThrLys<3iy3SS360365LeuGinGlyTrpHisAlaAlaGlyLysAlaAlaValGinGlulieAsp370375380GlyAlaSerVallieuValIjeuSerAsnTrpSerAlaGlyValSerGin38S330400AsnlieuHisAlaGinAspHisHisGlyTyrMetlieuArgVallieAla405LysGluGlyProGlyLysGly420GlyAsnGinGluThrLeuLysPhe435440ThrliysThrValGluValPhePi:o4S0455lieGlyGluThrGluGlyThrPhe465470CysMetGinGlyTyrAlaSerAsn485TyrGlyGinSerTyrAsnGlyAsn500TyrHisGinGlyTyrThrAsnAsn515520TyrAsnGinAsnTyrThrHisAsn530535CysAsnGinGlyHisAsnSerGly545550410415TyrValThrMetMetAspCysAsn425430ThrSerCysGluGlu445GlyTyrMetGluSerAspArgValAxglieGluTyrValAsp475SerlieGluLeutjeu480AsnAsnProHisThrGly495MetTyrAsnGinAsnTh;rSerAspVal505510TyrAsnGinAsnSer525SerAsnMetAspAspLeuHis540SerGlyCysThrCysThrCysSerGinGly55權(quán)利要求1.選自下組的分離核酸分子a)含有SEQIDNO1、2、4、6、8、10、12、13、15、17、19、21、23、25、26、或28所示核苷酸序列的核酸分子；b)含有與SEQIDNO1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列具有至少95％序列同一性的核苷酸序列的核酸分子，其中所述核苷酸序列編碼具有殺蟲活性的多肽；c)編碼含有SEQIDNO3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列的多肽的核酸分子；d)含有編碼具有與SEQIDNO3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列有至少95％氨基酸序列同一性的多肽的核苷酸序列的核酸分子，其中所述多肽具有殺蟲活性；和e)a)-d)中任一項的互補物。2.權(quán)利要求1的分離核酸分子，其中所述核苷酸序列是設(shè)計成在植物中表達的合成序列。3.權(quán)利要求2的核酸分子，其中所述合成序列具有升高的GC含量。4.含有權(quán)利要求1的核酸分子的載體。5.權(quán)利要求4的載體，其中還含有編碼異源多肽的核酸分子。6.含有權(quán)利要求4的載體的宿主細胞。7.權(quán)利要求6的宿主細胞，它是細菌宿主細胞。8.權(quán)利要求6的宿主細胞，它是植物細胞。9.含有權(quán)利要求8的宿主細胞的轉(zhuǎn)基因植物。10.權(quán)利要求9的轉(zhuǎn)基因植物，其中所述植物選自下組玉米、高粱、小麥、向日葵、番茄、十字花科植物、胡椒屬植物、馬鈴薯、棉花、稻、大豆、甜菜、甘蔗、煙草、大麥、和油籽油菜。,11.權(quán)利要求9的植物的轉(zhuǎn)基因種子。12.選自下組的分離多肽a)含有SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列的多肽；b)由SEQIDNO:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列編碼的多肽，其中所述多肽具有殺蟲活性；c)含有與SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列具有至少95%序列同一性的氨基酸序列的多肽，其中所述多肽具有殺蟲活性；和d)由與SEQIDNO:l、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列至少95%相同的核苷酸序列編碼的多肽。13.權(quán)利要求12的多肽，其中還含有異源氨基酸序列。14.選擇性結(jié)合權(quán)利要求12的多肽的抗體。15.含有權(quán)利要求12的多肽的組合物。16.權(quán)利要求15的組合物，其中所述組合物選自下組粉劑、塵劑、丸劑、顆粒劑、噴霧劑、乳液、膠體和溶液。17.權(quán)利要求15的組合物，其中所述組合物是通過對蘇云金芽孢桿菌細胞進行干燥、凍干、勻漿、抽提、過濾、離心、沉降、或濃縮而制備的。18.權(quán)利要求15的組合物，其中含有約1重量％至約99重量％的所述多肽。19，用于生產(chǎn)具有殺蟲活性的多肽的方法，其中包括在編碼所述多肽的核酸分子得到表達的條件下培養(yǎng)權(quán)利要求6的宿主細胞，所述多肽選自下組a)含有SEQIDN0:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列的多肽；b)由SEQIDN0:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列編碼的多肽，其中所述多肽具有殺蟲活性；c)含有與SEQIDN0:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列具有至少95%序列同一性的氨基酸序列的多肽，其中所述多肽具有殺蟲活性；和d)由與SEQIDN0:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列至少95%相同的核苷酸序列編碼的多肽。20.用于控制鱗翅類或鞘翅類害蟲種群的方法，其中包括使所述種群接觸殺蟲有效量的權(quán)利要求12的多肽。21.用于殺死鱗翅類或鞘翅類害蟲的方法，其中包括使所述害蟲接觸或給其喂食殺蟲有效量的權(quán)利要求12的多肽。22.在其基因組中穩(wěn)定摻入了DNA構(gòu)建體的植物，其中所述DNA構(gòu)建體含有編碼具有殺蟲活性的蛋白質(zhì)的核苷酸序列，該核苷酸序列選自下組a)SEQIDN0:1、2、4、6、8、10、12、13、15、19、21、23、25、26、或28所示核苷酸序列；b)與SEQIDN0:1、2、4、6、8、10、12、13、15、19、21、23、25、26或28所示核苷酸序列具有至少95%序列同一性的核苷酸序列，其中所述核苷酸序列編碼具有殺蟲活性的多肽；c)編碼含有SEQIDN0:3、5、7、9、11、14、16、20、22、24、27或29所示氨基酸序列的多肽的核苷酸序列；和d)編碼與SEQIDNO:3、5、7、9、11、14、16、20、22、24、27或29所示氨基酸序列具有至少95%氨基酸序列同一性的多肽的核苷酸序列，其中所述多肽具有殺蟲活性；表達的啟動子。23,權(quán)利要求22的植物，其中所述植物還含有SEQIDNO:17所示核苷酸序列。24.在其基因組中穩(wěn)定摻入了DNA構(gòu)建體的植物細胞，其中所述DNA構(gòu)建體含有編碼具有殺蟲活性的蛋白質(zhì)的核苷酸序列，該核苷酸序列選自下組a)SEQIDN0:1、2、4、6、8、10、12、13、15、19、21、23、25、26、或28所示核苷酸序列；b)與SEQIDNO:l、2、4、6、8、10、12、13、15、19、21、23、25、26或28所示核苷酸序列具有至少95%序列同一性的核苷酸序列，其中所述核苷酸序列編碼具有殺蟲活性的多肽；c)編碼含有SEQIDNO:3、5、7、9、11、14、16、20、22、24、27或29所示氨基酸序列的多肽的核苷酸序列；和d)編碼與SEQIDNO:3、5、7、9、11、14、16、20、22、24、27或29所示氨基酸序列具有至少95%氨基酸序列同一性的多肽的核苷酸序列，其中所述多肽具有殺蟲活性；中表達的啟動4。"''—一25.權(quán)利要求24的植物細胞，其中所述植物細胞還含有SEQIDNO:17所示核苷酸序列。全文摘要本發(fā)明提供了賦予細菌、植物、植物細胞、組織和種子以殺蟲活性的組合物和方法。本發(fā)明提供了含有δ-內(nèi)毒素和δ-內(nèi)毒素相關(guān)多肽的編碼序列的組合物。所述編碼序列可用于DNA構(gòu)建體或表達盒中，以便用于植物和細菌中的轉(zhuǎn)化和表達。組合物還包括被轉(zhuǎn)化的細菌、植物、植物細胞、組織和種子。具體而言，本發(fā)明提供了分離的δ-內(nèi)毒素和δ-內(nèi)毒素相關(guān)核酸分子。另外，本發(fā)明還涵蓋與多核苷酸對應(yīng)的氨基酸序列。具體而言，本發(fā)明提供了含有編碼SEQIDNO3、5、7、9、11、14、16、18、20、22、24、27和29中所示氨基酸序列的核苷酸序列和SEQIDNO1、2、4、6、8、10、12、13、15、17、19、21、23、25、26和28中所示核苷酸序列的分離的核酸分子及其變體和片段。文檔編號C07K16/12GK101328484SQ20081013609公開日2008年12月24日申請日期2004年2月20日優(yōu)先權(quán)日2003年2月20日發(fā)明者B·卡爾,M·G·科奇埃爾,N·B·達克,N·卡羅奇,T·哈吉斯申請人:阿則耐克斯公司