杀虫基因和使用方法与流程

本发明涉及用于防治害虫，特别是植物害虫的方法和组合物。相关申请的交叉引用本申请要求于2015年6月22日提交的美国临时申请序列号62/182,855的权益，其通过引用整体并入本文。经由efs-web作为文本文件提交的序列表的提及序列表的官方拷贝作为ascii格式化序列表经由efs-web以电子方式提交，文件名为agb015pct-seqlisting-1010353.txt，于2016年6月16日创建，具有952,518字节的大小，且与说明书同时提交。包含在该ascii格式化文件中的序列表是说明书的一部分，并且通过引用整体并入本文。背景害虫、植物疾病和杂草对作物可以是严重的威胁。由于害虫和疾病的损失据估计占全世界农业生产的37％，其中由于昆虫、细菌和其他生物的占13％。毒素是在微生物致病性和/或宿主免疫应答逃避中起重要作用的毒力决定簇。来自革兰氏阳性细菌芽孢杆菌属(bacillus)，特别是苏云金芽孢杆菌(bacillusthuringensis)的毒素已被用作杀昆虫蛋白质。目前的策略使用表达这些毒素的基因来产生转基因作物。表达杀昆虫蛋白质毒素的转基因作物被用于对抗来自昆虫的作物损害。尽管使用芽孢杆菌毒素已成功地控制昆虫，但在其中这些毒素已大量使用的世界上许多地方的一些靶害虫中已发展了针对bt毒素的抗性。解决这个问题的一个方法是播种bt作物与常规非bt作物(避难所)的交替行。避免或减缓昆虫抗性发展的可替代方法是在转基因植物中堆积具有针对昆虫的不同作用方式的杀昆虫基因。使用表达杀昆虫蛋白质毒素的转基因作物的目前策略越来越重视在已经衍生自细菌苏云金芽孢杆菌的那些毒素之外发现新毒素。这些毒素可以证明可用作衍生自苏云金芽孢杆菌的那些毒素的替代物用于在昆虫和害虫抗性转基因植物中的应用。因此，需要新的毒素蛋白质。概述提供了具有杀虫活性的组合物及其使用方法。组合物包括具有杀虫活性的分离的和重组的多肽序列，编码杀虫多肽的重组和合成的核酸分子，包含核酸分子的dna构建体，包含核酸分子的载体，包含载体的宿主细胞和针对杀虫多肽的抗体。编码本文提供的多肽的核苷酸序列可以用于dna构建体或表达盒中，用于在目的生物包括微生物和植物中的转化和表达。本文提供的组合物和方法可用于生产具有增强的害虫抗性或耐受性的生物。这些生物和包含生物的组合物对于农业目的是期望的。还提供了包含编码本发明的杀虫蛋白质的核苷酸序列的转基因植物和种子。这种植物对昆虫和其他害虫具有抗性。提供了用于产生本文公开的各种多肽以及使用这些多肽控制或杀死害虫的方法。还包括用于检测样品中的本发明多肽的方法和试剂盒。发明详述现在将在下文中更全面地描述本发明。实际上，这些发明可以以许多不同的形式来体现，并且不应被解释为限制于本文阐述的实施方案；相反，提供这些实施方案使得本公开内容将满足适用的法律要求。受益于上述说明中呈现的教导，这些发明所属领域的技术人员将会想到本文阐述的本发明的许多修改和其他实施方案。因此，应理解本发明并不限于所公开的特定实施方案，并且修改和其他实施方案预期被包括在所附权利要求的范围内。虽然本文采用了特定的术语，但它们仅以一般的和描述性的含义使用，而不是用于限制的目的。i.多核苷酸和多肽提供了用于对生物体赋予杀虫活性的组合物和方法。经修饰的生物体显示出杀虫抗性或耐受性。提供了保留杀虫活性的重组杀虫蛋白质或多肽及其片段和变体，并且包括在seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223和/或224中阐述的那些。杀虫蛋白质针对害虫包括昆虫、真菌、线虫等等是生物学活性的(例如杀虫的)。编码杀虫多肽、包括例如seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223和/或224或其活性片段或变体的核苷酸，可以用于产生转基因生物，例如植物和微生物。杀虫蛋白质针对害虫包括昆虫、真菌、线虫等等是生物学活性的(例如杀虫的)。编码杀虫多肽、包括例如seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223和/或224，或其活性片段或变体的多核苷酸可以用于产生转基因生物，例如植物和微生物。经转化的生物的特征在于包含至少一个稳定掺入的dna构建体的基因组，所述dna构建体包含本文公开的杀虫蛋白质的编码序列。在一些实施方案中，编码序列可操作地连接至驱动所编码的杀虫多肽的表达的启动子。相应地，提供了经转化的微生物、植物细胞、植物组织、植物、种子和植物部分。各种多肽、其活性变体及其片段和编码其的多核苷酸在下表1中阐述。如表1所示，提供了各种形式的多肽。提供了全长杀虫多肽以及原始全长序列的修饰形式(即变体)。表1进一步指示“crybp1”序列。这样的序列(seqidno:11和93)包含可以与一些毒素基因相关的辅助多肽。在这种情况下，crybp1序列可以单独使用或与本文提供的任何杀虫多肽组合使用。表1进一步提供了split-cryc末端多肽(seqidno:106)。此类序列包含与cry类毒素基因的c末端具有同源性的下游蛋白质的序列，并且通常在并非全长且缺失预期的c末端区域的cry基因之后发现。i.杀虫蛋白质的类别本文提供的杀虫蛋白质和编码其的核苷酸序列在用于影响害虫的方法中是有用的。即，本发明的组合物和方法在农业中用于控制或杀死害虫，包括许多作物植物的害虫。本文提供的杀虫蛋白质是来自细菌的毒素蛋白质，并显示出针对某些害虫的活性。杀虫蛋白质来自几个毒素类别，包括cry、cyt、bin、mtx毒素。关于本文提供的各种seqidnos的特定蛋白质分类参见例如表1。另外，在本公开内容自始至终参考pfam数据库条目。pfam数据库是蛋白质家族的数据库，每个蛋白质家族都由多重序列比对和概况隐马尔可夫模型表示。finn等人(2014)nucl.acidres.databaseissue42:d222-d230。苏云金芽孢杆菌(bt)是革兰氏阳性细菌，在其生长的孢子形成期过程中以晶体包涵体形式产生杀昆虫蛋白质。苏云金芽孢杆菌(bt)的蛋白质包涵体被称为晶体蛋白质或δ-内毒素(或cry蛋白)，其对昆虫纲和其他无脊椎动物的成员是有毒的。类似地，cyt蛋白是来自bt的伴孢包涵体蛋白，其显示出溶血(细胞溶解)活性或与已知的cyt蛋白具有明显的序列相似性。这些毒素对其靶生物是高度特异性的，对人、脊椎动物和植物无害。cry毒素的结构显示有五个保守的氨基酸块，主要集中在结构域的中心或结构域之间的连接处。cry毒素由三个结构域组成，每个结构域具有特定的功能。结构域i是七个α-螺旋的束，其中中央螺旋被六个外部螺旋完全包围。该结构域牵涉膜中的通道形成。结构域ii显示为三个反向平行的β-折叠片的三角形柱，其与免疫球蛋白的抗原结合区相似。结构域iii包含β-夹心形式的反向平行β-链。毒素蛋白质的n端部分负责其毒性和特异性，并且含有5个保守区域。c末端部分通常是高度保守的，并且可能负责晶体形成。参见例如美国专利号8,878,007。苏云金芽孢杆菌菌株显示针对不同昆虫目(鳞翅目(lepidoptera)、双翅目(diptera)、鞘翅目(coleoptera)、膜翅目(hymenoptera)、同翅目(homoptera)、虱目(phthiraptera)例如食毛目(mallophaga)和蜱螨亚纲(acari))和其他无脊椎动物(线虫动物(nemathelminthes)、扁形动物(platyhelminthes)和sarocomastebrates)的广泛范围的特异性。基于对各种昆虫和无脊椎动物群体的毒性，cry蛋白已被分类成几个组。一般地，cryi表现出对鳞翅目的毒性，cryii表现出对鳞翅目和双翅目的毒性，cryiii表现出对鞘翅目的毒性，cryiv表现出对双翅目的毒性，并且cryv和cryvi表现出对线虫的毒性。基于氨基酸同一性，新的cry蛋白可以得到鉴定并且分配到一个cry组中。参见例如，通过引用并入本文的bravo，a.(1997)j.ofbacteriol.179:2793-2801；bravo等人(2013)microb.biotechnol.6:17-26。超过750种不同的cry基因序列已被分成73个组(cry1-cry73)，而且这个基因家族的新成员还在不断被发现(crickmore等人(2014)www.btnomenclature.info/)。cry基因家族由可以具有不同作用模式的几个系统发育上无关的蛋白质家族组成：三结构域cry毒素家族，杀蚊cry毒素家族，二元样毒素家族和cyt毒素家族(bravo等人，2005)。一些bt菌株产生另外的杀昆虫毒素，vip毒素。还参见cohen等人(2011)j.mol.biol.413:4-814；在万维网上lifesci.sussex.ac.uk/home/neil_crickmore/bt/处找到的crickmore等人(2014)苏云金芽孢杆菌毒素命名法；crickmore等人(1988)microbiol.mol.biol.rev.62:807-813；gill等人(1992)ann.rev.entomol.37:807-636；goldbert等人(1997)appl.environ.microbiol.63:2716-2712；knowles等人(1992)proc.r.soc.ser.b.248:1-7；koni等人(1994)microbiology140:1869-1880；lailak等人(2013)biochem.biophys.res.commun.435:216-221；lopez-diaz等人(2013)environ.microbiol.15:3030-3039；perez等人(2007)cell.microbiol.9:2931-2937；promdonkoy等人(2003)biochem.j.374:255-259；rigden(2009)febslett.583:1555-1560；schnepf等人(1998)microbiol.mol.biol.rev.62:775-806；soberon等人(2013)peptides41:87-93；thiery等人(1998)j.am.mosq.controlassoc.14:472-476；thomas等人(1983)febslett.154:362-368；wirth等人(1997)proc.natl.acad.sci.u.s.a.94:10536-10540；wirth等人(2005)appl.environ.microbiol.71:185-189；和zhang等人(2006)biosci.biotechnol.biochem.70:2199-2204；其各自通过引用整体并入本文。cyt指示具有细胞溶解活性的来自苏云金芽孢杆菌的伴孢晶体包涵体蛋白，或与已知cyt蛋白具有序列相似性的蛋白质。(crickmore等人(1998)microbiol.mol.biol.rev.62:807-813.)。该基因被命名为cyt。这些蛋白质与cry蛋白在结构和活性方面不同(gill等人(1992)annu.rev.entomol.37:615-636)。cyt毒素首先在苏云金芽孢杆菌以色列亚种中发现(goldberg等人(1977)mosq.news.37:355-358)。在目前的命名法中存在3个cyt毒素家族，包括11个正模式标本(holotype)毒素(在万维网上lifesci.sussex.ac.uk/home/neil_crickmore/bt/处找到的crickmore等人(2014)苏云金芽孢杆菌毒素命名法)。具有cyt基因的大多数苏云金芽孢杆菌分离物显示针对双翅目昆虫(特别是蚊子和黑蝇)的活性，但也存在靶向鳞翅目或鞘翅目昆虫的苏云金芽孢杆菌菌株中已描述的cyt基因(guerchicoff等人(1997)appl.environ.microbiol.63:2716-2721)。通过x射线晶体学解析的cyt2a的结构显示单一结构域，其中有两个α-螺旋外层缠绕在混合β-折叠周围。cyt毒素的进一步可用晶体结构支持具有两个α-螺旋发夹的保守α-β结构模型，所述α螺旋发夹侧接含有七至八条β-链的β-折叠核心。(cohen等人(2011)j.mol.biol.413:804-814.)。诱变研究将β-折叠残基鉴定为对于毒性是关键的，而螺旋结构域中的突变不影响毒性(adang等人，diversityofbacillusthuringiensiscrystaltoxinsandmechanismofaction.in:t.s.dhadialla和s.s.gill编辑，advancesininsectphysiology，vol.47，oxford:academicpress，2014，第39-87页.)。cyt毒素的代表性结构域是β-内毒素bac_thur_toxin(pfampf01338)。对于cyt毒素的作用模式存在多种建议模式，而且这仍然是活跃研究领域。一些cyt蛋白(cyt1a)已显示需要辅助蛋白的存在用于结晶。cyt1a和cyt2a原毒素在n和c末端的相同位点处被消化蛋白酶加工为稳定的毒素核心。cyt毒素然后与非饱和的膜脂质(例如磷脂酰胆碱、磷脂酰乙醇胺和鞘磷脂)相互作用。对于cyt毒素，孔形成和去污剂样膜破裂已被提议作为非排他性的机制；并且一般公认两者均可以取决于毒素浓度而发生，其中较低的浓度有利于寡聚孔，较高的浓度导致膜破裂。(butko(2003)appl.environ.microbiol.69:2415-2422)在孔形成模型中，cyt毒素与细胞膜结合，诱导膜囊泡中阳离子选择性通道的形成，导致细胞的胶体-渗透性裂解。(knowles等人(1989)febslett.244:259-262；knowles等人(1992)proc.r.soc.ser.b.248:1-7和promdonkoy等人(2003)biochem.j.374:255-259.)在去污剂模型中，在脂双层的表面上存在毒素的非特异性聚集，导致膜分解和细胞死亡。(butko(2003)同上；manceva等人(2005)biochem.44:589-597.)多个研究已显示cyt毒素与其他苏云金芽孢杆菌毒素，特别是cry、bin和mtx毒素之间的协同活性。这种协同作用甚至已显示克服昆虫对其他毒素的抗性。(wirth1997，wirth2005，thiery1998，zhang2006.)。cry毒素的cyt协同效应推测涉及cyt1a与cry毒素结构域ii在溶液中或膜平面上的结合，促进cry毒素预孔寡聚物以形成。该寡聚物的形成不依赖于cyt寡聚化、结合或插入。(lailak2013，perez2007，lopez-diaz2013.)在营养生长期间，与cry蛋白无关的许多杀虫蛋白质由苏云金芽孢杆菌和蜡状芽孢杆菌(b.cereus)的一些菌株产生(estruch等人(1996)procnatlacadsciusa93:5389–5394；warren等人(1994)wo94/21795)。这些营养型杀昆虫蛋白质或vip不形成伴孢晶体蛋白质，并且明显由细胞分泌。vip目前被排除在cry蛋白命名法之外，因为它们不是晶体形成蛋白。术语vip在含义上是一个误称，因为一些苏云金芽孢杆菌cry蛋白在营养生长期间以及在静止期和孢子形成期过程中也产生，最著名的是cry3aa。苏云金芽孢杆菌基因组中的vip基因的位置据报道位于也编码cry基因的大质粒中(mesrati等人(2005)femsmicrobiol.lett.244(2):353-358)。可在万维网上的lifesci.sussex.ac.uk处以路径“/home/neil_crickmore/bt/”和在：“btnomenclature.info/”处找到bt毒素命名法的网站。还参见schnepf等人(1998)microbiol.mol.biol.rev.62(3):775-806。这些参考文献通过引用并入本文。迄今为止已鉴定了四类vip。一些vip基因形成二元双组分蛋白复合物；“a”组分通常是“活动”部分，“b”组分通常是“结合”部分。(pfampfam.xfam.org/family/pf03495)。vip1和vip4蛋白一般含有二元毒素b蛋白结构域。vip2蛋白一般含有二元毒素a蛋白结构域。vip1和vip2蛋白是显示出针对鞘翅目的毒性的二元毒素的两个组分。vip1aa1和vip2aa1针对玉米根虫，特别是玉米根叶甲是有非常高活性的(diabroticavirgifera)和长角叶甲(diabroticalongicornis)(han等人(1999)nat.struct.biol.6:932-936；warrengw(1997)“vegetativeinsecticidalproteins:novelproteinsforcontrolofcornpests”in:carozzinb，kozielm(编辑)advancesininsectcontrol，theroleoftransgenicplants；taylor&francisltd，london，第109-121页)。膜结合的95kdavip1多聚体提供了用于52kdavip2adp-核糖酶进入靶西方玉米根虫细胞的细胞质的途径(warren(1997)同上)。nad依赖性adp-核糖基转移酶vip2可能在arg177处修饰单体肌动蛋白以阻断聚合，导致肌动蛋白细胞骨架的损失，并且由于体内肌动蛋白丝内的快速亚单位交换导致最终细胞死亡(carlierm.f.(1990)adv.biophys.26:51-73)。如同cry毒素，活化的vip3a毒素是能够在膜中制备稳定离子通道的成孔蛋白质(lee等人(2003)appl.environ.microbiol.69:4648–4657)。vip3蛋白针对几种主要的鳞翅目害虫具有活性(rang等人(2005)appl.environ.microbiol.71(10):6276-6281；bhalla等人(2005)femsmicrobiol.lett.243:467-472；estruch等人(1998)wo9844137；estruch等人(1996)procnatlacadsciusa93:5389-5394；selvapandiyan等人(2001)appl.environmicrobiol.67:5855–5858；yu等人(1997)appl.environmicrobiol.63:532–536)。vip3a针对黑切根虫(agrotisipsilon)、草地贪夜蛾(spodopterafrugiperda)、甜菜夜蛾(spodopteraexigua)、烟芽夜蛾(heliothisvirescens)和玉米穗虫(helicoverpazea)有活性(warren等人(1996)wo96/10083；estruch等人(1996)procnatlacadsciusa93:5389–5394)。如同cry毒素，vip3a蛋白质必须在特异性膜蛋白质的中肠上皮表面上识别之前被蛋白酶激活，所述特异性膜蛋白质不同于由cry毒素识别的那些。毒素蛋白质的mtx家族的特征在于保守结构域etx_mtx2(pfam03318)的存在。该家族的成员与来自球形芽孢杆菌(bacillussphaericus)的杀蚊毒素mtx2和mtx3以及来自产气荚膜梭菌(clostridiumperfringens)的ε毒素etx共享序列同源性(cole等人(2004)nat.struct.mol.biol.11:797-8；thanabalu等人(1996)gene170:85-89)。mtx样蛋白在结构上不同于三结构域cry毒素，因为它们具有延长且主要是基于β-折叠的结构。然而，类似于三结构域毒素，mtx样蛋白被认为在靶细胞的膜上形成孔(adang等人(2014)，同上)。与三结构域cry蛋白不同，mtx样蛋白的长度小得多，范围从267个氨基酸(cry23)到340个氨基酸(cry15a)。迄今为止，仅15种属于mtx样毒素家族的蛋白质被分配了cry名称，因而其与三结构域cry家族相比是相对较小的类别(crickmore等人(2014)同上；adang等人(2014)同上)。mtx样毒素家族的成员包括cry15、cry23、cry33、cry38、cry45、cry46、cry51、cry60a、cry60b和cry64。这个家族显示出一系列的杀昆虫活性，包括针对鳞翅目和鞘翅目的昆虫害虫的活性。这个家族的一些成员可以与其他蛋白质形成二元合作关系，这对于杀昆虫活性可能是需要的或也可能是不需要的。cry15是一种在苏云金芽孢杆菌血清变型thompsonihd542中鉴定的34kda蛋白质；它与大约40kda的一种无关蛋白质一起天然存在于晶体中。编码cry15及其伙伴蛋白的基因在操纵子中一起排列。cry15单独已显示具有针对鳞翅目昆虫害虫的活性，所述昆虫害虫包括烟草天蛾(manducasexta)、苹果蠹蛾(cydiapomonella)和菜粉蝶(pierisrapae)，其中所述40kda蛋白的存在已显示仅增加cry15针对苹果蠹蛾的活性(brownk.和whiteleyh.(1992)j.bacteriol.174:549-557；naimov等人(2008)appl.environ.microbiol.74:7145–7151)。需要进一步的研究来阐明cry15的伙伴蛋白质的功能。类似地，cry23是一种29kda蛋白质，其已显示与其伙伴蛋白质cry37一起具有针对鞘翅目害虫赤拟谷盗(triboliumcastaneum)和日本弧丽金龟(popilliajaponica)的活性(donovan等人(2000)美国专利号6,063,756)。mtx样家族的新成员仍在不断被鉴定出来。最近在苏云金芽孢杆菌血清变型tolworthi菌株na205-3的基因组中鉴定了etx_mtx毒素基因。发现该菌株针对鳞翅目害虫棉铃虫(helicoverpaarmigera)是有毒性的，并且它还含有cry1、cry11、vip1、vip2和vip3的同系物(palma等人(2014)genomeannounc.2(2):e00187-14.2014年3月13日在线发表于doi:10.1128/genomea.00187-14；pmcid:pmc3953196处)。因为mtx样蛋白相对于三结构域cry蛋白具有独特的结构域结构，所以它们被认为具有独特的作用模式，从而使其成为昆虫防治和抵抗昆虫抗性中有价值的工具。细菌细胞产生针对宿主和非宿主生物具有不同特异性的大量毒素。已在许多细菌家族中鉴定了二元毒素的大家族，包括具有针对昆虫害虫的活性的毒素。(poopathi和abidha(2010)j.physiol.path.1(3):22-38)。球形赖氨酸芽孢杆菌(lysinibacillussphaericus)(ls)，以前称为球形芽孢杆菌(ahmed等人(2007)int.j.syst.evol.microbiol.57:1117-1125)，是众所周知的昆虫生物防治菌株。ls产生几种杀昆虫蛋白质，包括高度有效的二元复合物bina/binb。这种二元复合物在ls细胞中形成伴孢晶体，对于双翅目昆虫、特别是蚊子具有强而特异的活性。在一些地区，已报道了对现有ls杀蚊菌株的昆虫抗性。具有不同靶特异性或克服昆虫抗性的能力的新二元毒素的发现具有重大意义。ls二元杀昆虫蛋白质复合物含有两种主要的多肽，即分别命名为bina和binb的42kda多肽和51kda多肽(ahmed等人(2007)同上)。这两种多肽协同作用以对其靶赋予毒性。作用方式涉及所述蛋白质与幼虫中肠内受体的结合。在一些情况下，所述蛋白质通过在幼虫肠道中的蛋白酶消化来修饰，产生活化形式。binb组分被认为涉及结合，而bina组分赋予毒性(nielsen-leroux等人(2001)appl.environ.microbiol.67(11):5049–5054)。当分别克隆和表达时，bina组分对蚊子幼虫是有毒性的，而binb组分则没有。然而，所述蛋白质的共同施用显著增加毒性(nielsen-leroux等人(2001)同上)。已从细菌来源中描述了少量bin蛋白同系物。priest等人(1997)appl.environ.microbiol.63(4):1195-1198描述了鉴定新的ls菌株的杂交努力，虽然他们鉴定的大部分基因编码的蛋白质与已知的bina/binb蛋白质相同。bina蛋白含有被称为毒素10超家族结构域的确定的保守结构域。该毒素结构域最初通过其在bina和binb中的存在得到确定。这两种蛋白质均具有该结构域，尽管bina和binb之间的序列相似性在这个区域是有限的(<40％)。也具有杀昆虫活性的cry49aa蛋白也具有该结构域(下文描述)。ls的cry48aa/cry49aa二元毒素具有杀死致倦库蚊(culexquinquefasciatus)幼虫的能力。这些蛋白质属于这样一个蛋白质结构类别，该类别与苏云金芽孢杆菌(bt)的cry蛋白复合物(一个众所周知的杀昆虫蛋白质家族)具有一些相似性。bt的cry34/cry35二元毒素也已知会杀死昆虫，包括西方玉米根虫(一种重要的玉米害虫)。已鉴定了几种变体的cry34是一种小型(14kda)多肽，而cry35(也由几种变体编码)是一种44kda的多肽。这些蛋白质与bina/binb蛋白质组具有一些序列同源性，并且被视为在进化上是相关的(ellis等人(2002)appl.environ.microbiol.68(3):1137-1145)。磷脂酰肌醇磷脂酶c蛋白质(pi-plc；也称为磷脂酰肌醇磷脂酶c)是更大的磷脂酶c蛋白质组的成员。这些蛋白质中的许多在信号转导中起重要作用，作为正常细胞生理学的一部分。几种重要的细菌毒素还含有与这些蛋白具有相似性的结构域(titball，r.w.(1993)microbiologicalreviews.57(2):347-366))。重要的是，这些蛋白质牵涉通过btcry蛋白在昆虫细胞中毒期间的信号放大(valaitis，a.p.(2008)insectbiochemistryandmolecularbiology.38:611-618)。pi-plc毒素类别在芽孢杆菌分离株中出现，通常与其他描述的毒素类别(例如二元毒素)的同源物共同出现。这类序列与磷脂酰肌醇磷酸二酯酶(也称为磷脂酰肌醇特异性磷脂酶c-pi-plc)具有同源性。heinz等人(heinz等人(1995)theembojournal.14(16):3855-3863)解决了蜡样芽孢杆菌pi-plc的晶体结构及其活性位点。通过等人使用定点诱变、动力学和晶体结构分析(等人(1997)biochemistry.36(42):12802-13)研究了蜡状芽孢杆菌pi-plc活性位点氨基酸残基在催化和底物结合中的作用。这些pi-plc毒素蛋白质含有plc样磷酸二酯酶、timβ/α-桶结构域(ipr017946)和/或磷脂酶c，磷脂酰肌醇特异性x结构域(ipr000909)(也称为pi-plcx-盒结构域)。我们也已看到具有这些结构域与其他典型芽孢杆菌蛋白质毒素结构域组合的蛋白质。该列表最通常包括凝集素结构域(ipr000772)，可以以一个或多个拷贝存在并且被认为结合细胞膜的糖结合结构域，以及杀昆虫晶体毒素(ipr008872)(也被称为毒素10或p42)，这是二元毒素的限定结构域。这个结构域结构的一个例子在序列apg00732中。这种pi-plc类别的毒素之前在美国专利号8,318,900b2seqidno30(dna)和79(氨基酸)，美国专利公开号20110263488a1seqidno8(dna)和9(氨基酸)，以及美国专利号8,461,421b2seqidno3(dna)和63(氨基酸)中有定义。本文提供了来自这些毒素类别的杀虫蛋白质。杀虫蛋白质根据其结构、与已知毒素的同源性和/或其杀虫特异性进行分类。ii.杀虫蛋白质和编码其的多核苷酸的变体和片段本发明的杀虫蛋白质或多肽包括在seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223和/或224中所示的那些及其片段和变体。“杀虫毒素”或“杀虫蛋白质”或“杀虫多肽”是指针对一种或多种害虫(包括昆虫、真菌、线虫等等)有活性的毒素或蛋白质或多肽，使得所述害虫被杀死或受到控制。“分离的”或“纯化的”多肽或蛋白质或其生物学活性部分基本上或实质上不含如在其天然存在的环境中发现的，通常伴随所述多肽或蛋白质或者与所述多肽或蛋白质相互作用的组分。因此，分离的或纯化的多肽或蛋白质当通过重组技术产生时基本上不含其他细胞材料或培养基，或者当化学合成时基本上不含化学前体或其他化学物质。基本上不含细胞物质的蛋白质包括具有小于约30％、20％、10％、5％或1％(按干重计)的污染蛋白质的蛋白质制剂。当重组产生本发明的蛋白质或其生物学活性部分时，理想地培养基占化学前体或非目的蛋白质的化学物质的少于约30％、20％、10％、5％或1％(按干重计)。术语“片段”指本发明的多肽序列的一部分。“片段”或“生物学活性部分”包括包含足够数目的邻接氨基酸残基以保留生物学活性(即具有杀虫活性)的多肽。杀虫蛋白质的片段包括比全长序列更短的那些，其或者是由于使用替代的下游起始位点所致，或者是由于产生具有杀虫活性的较短蛋白质的加工所致。加工可以在蛋白质在其中表达的生物体中，或者在摄入蛋白质后在害虫中发生。蛋白质片段的实例可以在表1中找到。杀虫蛋白质的生物学活性部分可以是例如下述任一序列的长度上10、25、50、100、150、200、250个或更多个氨基酸的多肽：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223和/或224。这些生物学活性部分可以通过重组技术制备并且评估杀虫活性。如本文使用的，片段包含seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223和/或224的至少8个邻接氨基酸。细菌基因(包括编码本文公开的杀虫蛋白质的那些)在开放读码框的开始附近经常具有多个甲硫氨酸起始密码子。通常，在这些起始密码子中的一个或多个处的翻译起始将导致功能蛋白质的生成。这些起始密码子可以包括atg密码子。然而，细菌例如芽孢杆菌属菌种(bacillussp.)也将密码子gtg识别为起始密码子，并且在gtg密码子处起始翻译的蛋白质在第一个氨基酸处含有甲硫氨酸。在极少数情况下，细菌系统中的翻译可以在ttg密码子处起始，尽管在这种情况下ttg编码甲硫氨酸。此外，通常不能事先确定这些密码子中的哪一个在细菌中天然使用。因此，应理解使用替代甲硫氨酸密码子之一也可以导致杀虫蛋白质的生成。这些杀虫蛋白质涵盖在本发明中，并且可以用于本文公开的方法中。应理解当在植物中表达时，有必要将可替代起始密码子改变为atg用于正确的翻译。在各种实施方案中，本文提供的杀虫蛋白质包括由全长核苷酸序列推导的氨基酸序列，以及由于使用替代的下游起始位点而比全长序列更短的氨基酸序列。因此，本发明的核苷酸序列和/或包含本发明的核苷酸序列的载体、宿主细胞和植物(以及制备和使用本发明的核苷酸序列的方法)可以包含编码替代起始位点的核苷酸序列。可认识到可以对本文提供的杀虫多肽作出修饰，产生变体蛋白质。通过应用定点诱变技术可以引入由人设计的变化。可替代地，也可以鉴定落入本发明范围内的与本文公开的序列在结构和/或功能上相关的天然、然而迄今未知的或迄今未鉴定的多核苷酸和/或多肽。保守氨基酸取代可以在不改变杀虫蛋白质功能的非保守区域中进行。可替代地，可以进行改善毒素活性的修饰。通过结构域iii交换修饰cry毒素在一些情况下已得到了一些杂合毒素，其针对某些昆虫物种具有改善的毒性。因此，结构域iii交换可以是改善cry毒素的毒性或产生针对下述害虫具有毒性的新型杂合毒素的有效策略，所述害虫对于亲本cry毒素未显示敏感性。结构域ii环序列的定点诱变可以导致具有增加的杀昆虫活性的新毒素。结构域ii环区是初始cry毒素的关键结合区，其是具有改善的杀昆虫性质的cry毒素的诱变和选择的合适靶。cry毒素的结构域i可以进行修饰，以引入蛋白酶切割位点而改善针对某些害虫的活性。用于在大量cry基因中改组这三个不同结构域的策略和高通量生物测定筛选方法可以提供具有改善的或新型的毒性的新型cry毒素。如所指出的，本文公开的多肽的片段和变体将保留杀虫活性。杀虫活性包括组合物实现减少靶害虫的发生或活性的可观察效应的能力，包括例如导致至少一个害虫的死亡，或者害虫生长、进食或正常生理发育的明显下降。数目、害虫生长、进食或正常发育的这些下降可以包含任何统计学上显著的降低，包括例如约5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、85％、90％、95％或更多的降低。认识到杀虫活性相对于天然蛋白质的活性而言可以不同或改善，或者它可以保持不变，只要保留杀虫活性即可。用于测量杀虫活性的方法是本领域众所周知的。参见例如czapla和lang(1990)j.econ.entomol.83:2480-2485；andrews等人(1988)biochem.j.252:199-206；marrone等人(1985)j.ofeconomicentomology78:290-293；和美国专利号5,743,477，所有这些文献通过引用整体并入本文。本公开内容的多肽变体包括具有与下述中任一个氨基酸序列有至少约60％、约65％、约70％、约75％、约80％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％相同、并且保留杀虫活性的氨基酸序列的多肽：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223和/或224。注意，表1提供了seqidno:1-224中每一个的变体多肽(和编码其的多核苷酸)的非限制性实例。本发明的杀虫多肽的生物学活性变体可以有少至约1-15个氨基酸残基，少至约1-10，例如约6-10、少至5、少至4、少至3个、少至2个、或少至1个氨基酸残基的差异。在具体的实施方案中，多肽可以包含n'末端或c'末端截短，其可以包括从多肽的n'或c'末端缺失至少10、15、20、25、30、35、40、45、50个氨基酸或更多个氨基酸。表2提供了基于pfam数据的在seqidno:1-224中发现的蛋白质结构域。结构域描述和在给定seqidno内的位置两者都在表2中提供。在特定的实施方案中，包含seqidno:1-224中任何一个的活性变体可以与seqidno:1-224中任何一个有至少70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性，并且进一步包含表2中所示的保守结构域中的至少一个。例如，在一个实施方案中，活性变体将与seqidno:3有至少70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且进一步包含在位置207-400处的天然氨基酸。表2.seqidno:1-224每一个中的pfam结构域的概述还提供了编码本文公开的杀虫多肽的重组或合成核酸。特别有兴趣的是已被设计用于在目的植物中表达的核酸序列。即，可以对核酸序列进行优化以增加宿主植物中的表达。本发明的杀虫蛋白质可以被反向翻译以产生包含为了在特定宿主例如农作物中表达而优化的密码子的核酸。在另一个实施方案中，可以将编码本文提供的多肽的多核苷酸进行优化，用于增加在经转化的植物中的表达。即，多核苷酸可以使用植物偏好的密码子来合成用于改善表达。关于宿主偏好的密码子使用的讨论，参见例如campbell和gowri(1990)plantphysiol.92:1-11。本领域可获得用于合成植物偏好基因的方法。参见例如通过引用并入本文的美国专利号5,380,831和5,436,391，以及murray等人(1989)nucleicacidsres.17:477-498。通过经转化的植物(例如双子叶植物或单子叶植物)表达这种编码序列将导致产生杀虫多肽，并且在植物中赋予针对害虫的增加抗性。编码本发明杀虫蛋白质的重组和合成核酸分子不包括编码该蛋白的天然存在的细菌序列。“重组多核苷酸”或“重组核酸”包含两种或更多种化学连接的核酸区段的组合，其在自然界中未发现直接连接。“直接连接”是指两个核酸区段直接相邻并且通过化学连键彼此连接。在具体的实施方案中，重组多核苷酸包含目的多核苷酸或其变体或片段，使得另外的化学连接核酸区段位于目的多核苷酸的5'、3'或内部。可替代地，重组多核苷酸的化学连接的核酸区段可以通过序列缺失而形成。另外的化学连接的核酸区段或缺失以连接所连接的核酸区段的序列可以具有任何长度，包括例如1、2、3、4、5、6、7、8、9、10、15、20个或更多个核苷酸。用于制备这种重组多核苷酸的各种方法包括化学合成或通过基因工程技术操作分离的多核苷酸区段。在具体的实施方案中，重组多核苷酸可以包含重组dna序列或重组rna序列。“重组多核苷酸或核酸的片段”包含两个或更多个化学连接的氨基酸区段的组合中的至少一种，所述氨基酸区段在自然界中未发现直接连接。多核苷酸(rna或dna)的片段可以编码保留活性的蛋白质片段。在具体的实施方案中，重组多核苷酸或重组多核苷酸构建体的片段包含所述两个或更多个化学连接的或可操作地连接的核酸区段的至少一个连接，所述核酸区段在自然界中未发现直接连接。编码保留杀虫活性的多肽的生物学活性部分的多核苷酸的片段将编码如下所述序列的全长多肽中存在的至少25、30、40、50、60、70、75、80、90、100、110、120、125、130、140、150、160、170、175、180个邻接氨基酸或高达全部数目的氨基酸：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223和/或224。在具体的实施方案中，此类多肽片段是活性片段，在另外的其他实施方案中，多肽片段包含重组多肽片段。如本文使用的，重组多肽的片段包含两个或更多个化学连接的氨基酸区段的组合中的至少一种，所述氨基酸区段在自然界中未发现直接连接。“变体”意指基本上相似的序列。对于多核苷酸，变体包含在天然多核苷酸内的一个或多个内部位点处一个或多个核苷酸的缺失和/或添加和/或在天然多核苷酸的一个或多个位点处一个或多个核苷酸的取代。如本文使用的，“天然”多核苷酸或多肽分别包含天然存在的核苷酸序列或氨基酸序列。也可以通过比较由变体多核苷酸编码的多肽与由参考多核苷酸编码的多肽之间的序列同一性百分比来评估本发明的特定多核苷酸(即参考多核苷酸)的变体。因此，例如，公开了编码与具有下述序列的多肽有给定百分比序列同一性的多肽的分离的多核苷酸seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224。任何两个多肽之间的序列同一性百分比可以使用本文其他地方所述的序列比对程序和参数来计算。当通过比较由它们编码的两种多肽共享的百分比序列同一性来评估本发明的任何给定多核苷酸对时，两种编码多肽之间的序列同一性百分比为与下述序列有至少约40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％，或者更高序列同一性：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224。在其他实施方案中，本文提供的多核苷酸的变体与天然序列的差别有至少1、2、3、4、5、6、7、8、9、10个或更多个核苷酸。变体多核苷酸和蛋白质还涵盖衍生自诱变和重组程序如dna改组的序列和蛋白质。使用这样的程序，操纵本文公开的一种或多种不同的杀虫蛋白质(seqidno:1-224)，以产生具有所需性质的新杀虫蛋白质。以这种方式，由相关序列多核苷酸群体生成重组多核苷酸文库，所述相关序列多核苷酸包含具有基本序列同一性并且可在体外或体内同源重组的序列区域。例如，使用这种方法，编码目的结构域的序列基序可以在本文提供的杀虫序列和其他已知的杀虫基因之间改组，以获得编码具有改善的目的性质的蛋白质的新基因，例如在酶的情况下有增加的km。这种dna改组的策略是本领域已知的。参见例如，stemmer(1994)proc.natl.acad.sci.usa91:10747-10751；stemmer(1994)nature370:389-391；crameri等人(1997)naturebiotech.15:436-438；moore等人(1997)j.mol.biol.272:336-347；zhang等人(1997)proc.natl.acad.sci.usa94:4504-4509；crameri等人(1998)nature391:288-291；以及美国专利号5,605,793和5,837,458。“改组”核酸是通过改组程序例如本文所述的任何改组程序产生的核酸。通过例如以人工和任选递归的方式重组(物理上或实质上)两个或更多个核酸(或字符串)来产生改组的核酸。一般地，在改组过程中使用一个或多个筛选步骤来鉴定目的核酸；这个筛选步骤可以在任何重组步骤之前或之后执行。在一些(但不是全部)改组实施方案中，理想的是在选择之前执行多轮重组，以增加待筛选的库的多样性。重组和选择的整个过程任选递归地重复。根据上下文，改组可以指重组和选择的整个过程，或可替代地，可以简单地指整个过程的重组部分。在一个实施方案中，提供了获得编码包含杀虫活性的改善的多肽的多核苷酸的方法，其中所述改善的多肽具有超过seqidno:1-224中任何一个的至少一种改善的性质。此类方法可包括(a)重组多个亲本多核苷酸，以产生编码重组杀虫多肽的重组多核苷酸文库；(b)筛选文库以鉴定编码改善的重组杀虫多肽的重组多核苷酸，所述重组多核苷酸具有比亲本多核苷酸改善的增强的性质；(c)回收编码(b)中鉴定的改善的重组杀虫多肽的重组多核苷酸；和(d)使用步骤(c)中回收的重组多核苷酸作为重复步骤(a)中的多个亲本多核苷酸之一，重复步骤(a)、(b)和(c)。iii.序列比较如本文使用的，当关于比对氨基酸序列的特定对使用时，术语“同一性”或“百分比同一性”指氨基酸序列同一性百分比，其通过计数在比对中的相同匹配数目，并且将这样的相同匹配数目除以比对序列的长度来获得。如本文使用的，当关于比对氨基酸序列的特定对使用时，术语“相似性”或“百分比相似性”指从比对中的每个氨基酸对的评分矩阵获得的评分总和除以比对序列的长度。除非另有说明，否则将通过needleman-wunsch总体比对和评分算法(needleman和wunsch(1970)j.mol.biol.48(3):443-453)计算同一性和相似性，如通过作为emboss软件包的部分分配的“针”程序执行的(rice，p.等人(2000)emboss:theeuropeanmolecularbiologyopensoftwaresuite，trendsingenetics16(6):276-277，在其他资源中，可在embnet.org/resource/emboss和emboss.sourceforge.net处从embnet获得的版本6.3.1)，使用默认缺口罚分和评分矩阵(用于蛋白质的eblosum62和用于dna的ednafull)。也可以使用等价程序。“等价程序”指任何序列比较程序，对于所讨论的任何两个序列，当与通过来自emboss6.3.1版的针所生成的相应比对比较时，其生成具有相同核苷酸残基匹配和相同序列同一性百分比的比对。另外的数学算法是本领域中已知的并且可以用于比较两个序列。参见例如karlin和altschul(1990)proc.natl.acad.sci.usa87:2264的算法，如karlin和altschul(1993)proc.natl.acad.sci.usa90:5873-5877中改动。这种算法被引入altschul等人(1990)j.mol.biol.215:403的blast程序内。可以用blastn程序(针对核苷酸序列搜索的核苷酸查询)执行blast核苷酸搜索，以获得与本发明的杀虫样核酸分子同源的核苷酸序列，或用blastx程序(针对蛋白质序列搜索的翻译核苷酸查询)，以获得与本发明的杀虫核酸分子同源的蛋白质序列。可以用blastp程序(针对蛋白质序列搜索的蛋白质查询)执行blast蛋白质搜索，以获得与本发明的杀虫蛋白质分子同源的氨基酸序列，或用tblastn程序(针对翻译的核苷酸序列搜索的蛋白质查询)，以获得与本发明的杀虫蛋白质分子同源的核苷酸序列。为了获得用于比较目的的缺口比对，gappedblast(在blast2.0中)可以如altschul等人(1997)nucleicacidsres.25:3389中所述利用。可替代地，psi-blast可以用于执行迭代搜索，其检测分子之间的遥远关系。参见altschul等人(1997)同上。当利用blast、gappedblast和psi-blast程序时，可以使用相应程序(例如blastx和blastn)的默认参数。比对也可以通过检查手动执行。当使用限定的氨基酸取代矩阵(例如，blosum62)、缺口存在罚分和缺口延伸罚分，以便获得对于该对序列可能的最高评分，对两个序列就相似性评分进行比对时，它们是“最佳比对的”。氨基酸取代矩阵及其在量化两个序列之间的相似性中的用途是本领域众所周知的，并且在例如dayhoff等人(1978)“amodelofevolutionarychangeinproteins.”in“atlasofproteinsequenceandstructure,”第5卷，suppl.3(编辑m.o.dayhoff)，第345-352页.natl.biomed.res.found.，washington，d.c.和henikoff等人(1992)proc.natl.acad.sci.usa89:10915-10919中描述。blosum62矩阵经常被用作序列比对方案中的缺省评分取代矩阵。对于在比对的序列之一中引入单个氨基酸缺口而施加缺口存在罚分，对于插入已经开放的缺口内的每个另外的空氨基酸位置施加缺口延伸罚分。由比对在每种序列上开始和结束的氨基酸位置、并且任选地通过在一个或两个序列中插入缺口或多个缺口、以便达到最高的可能评分来确定比对。尽管可以手动完成最佳比对和评分，但通过使用计算机执行的比对算法来促进该过程，所述算法例如缺口blast2.0，如altschul等人(1997)nucleicacidsres.25:3389-3402中所述的，并且在美国国家生物技术信息中心网站(nationalcenterforbiotechnologyinformationwebsite)(www.ncbi.nlm.nih.gov)上可公开获得。最佳比对(包括多重比对)可以使用例如psi-blast来制备，所述psi-blast可通过www.ncbi.nlm.nih.gov获得，其如altschul等人(1997)nucleicacidsres.25:3389-3402所述。关于与参考序列最佳比对的氨基酸序列，氨基酸残基“对应于”在比对中与残基配对的参考序列中的位置。“位置”由数目指示，该数目基于其相对于n末端的位置依次鉴定参考序列中的每个氨基酸。例如，在seqidno:1中，位置1是l，位置2是s，位置3是i等。当测试序列与seqidno:1最佳比对时，测试序列中与在位置3处的i比对的残基被说成“对应于seqidno:1的位置3”。由于在确定最佳比对时必须考虑的缺失、插入、截短、融合等，一般而言，如通过从n末端简单计数确定的测试序列中的氨基酸残基数目不一定与参考序列中其对应位置的数目相同。例如，在其中比对的测试序列中存在缺失的情况下，在缺失位点处没有氨基酸对应于参考序列中的位置。当比对的参考序列中存在插入时，该插入将不对应于参考序列中的任何氨基酸位置。在截短或融合的情况下，在参考序列或比对序列中可以存在不对应于相应序列中的任何氨基酸的氨基酸段。iv.抗体还涵盖了针对本发明多肽或其变体或片段的抗体。用于产生抗体的方法是本领域众所周知的(参见例如harlow和lane(1988)antibodies:alaboratorymanual、coldspringharborlaboratory、coldspringharbor、n.y.；和美国专利号4,196,265)。这些抗体可以用于检测和分离毒素多肽的试剂盒中。因此，本公开内容提供了包含抗体的试剂盒，所述抗体与本文所述的多肽、包括例如具有下述序列的多肽特异性结合：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133,134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212,213、214、215、216、217、218、219、220、221、222、223和/或224。ii.害虫本文提供的组合物和方法可用于对抗各种害虫。“害虫”包括但不限于昆虫、真菌、细菌、线虫、螨、原生动物病原体、动物寄生性肝吸虫等等。特别感兴趣的害虫是昆虫害虫，特别是对农业植物造成严重损害的昆虫害虫。昆虫害虫包括选自鞘翅目、双翅目、膜翅目、鳞翅目、食毛目、同翅目、半翅目、orthroptera、缨翅目(thysanoptera)、革翅目(dermaptera)、等翅目(isoptera)、虱目、蚤目(siphonaptera)、毛翅目(trichoptera)或线虫的昆虫。在非限制性实施方案中，昆虫害虫可以包括西方玉米根虫，玉米根萤叶甲(diabroticavirgiferavirgifera)；秋粘虫，草地贪夜蛾；科罗拉多马铃薯甲虫，马铃薯甲虫(leptinotarsadecemlineata)；玉米穗虫，玉米穗虫(在北美相同物种攻击棉花且被称为棉铃虫)；欧洲玉米螟，欧洲玉米螟(ostrinianubilalis)；小地老虎，黑切根虫；小菜蛾，小菜蛾(plutellaxylostella)；黍豆毛毛虫，大豆夜蛾(anticarsiagemmatalis)；西南玉米螟，西南玉米螟(diatraeagrandiosella)；棉铃虫，棉铃虫(在全世界除美国外的其他地区发现)；南绿蝽象，稻绿蝽(nezaraviridula)；绿蝽象，chinaviahalaris；brownmarmorated蝽象，褐翅蝽象(halyomorphahalys)；和褐臭蝽，褐臭蝽(euschistusservus)、大豆褐蝽(euschistusheros)(新热带棕蝽象和/或大豆蝽象)；红蝽(piezodorusguildinii)(红带蝽象)；dichelopsmelacanthus(无通用名)和/或dichelopsfurcatus(无通用名)；蚜虫，例如大豆蚜虫。在其他实施方案中，害虫包含线虫，包括但不限于：根结线虫(meloidogynehapla)(北方根结线虫)；象耳豆根结线虫(meloidogyneenterolobii)、花生根结线虫(meloidogynearenaria)(花生根结线虫)；和/或爪哇根结线虫(meloidogynejavanica)。如本文使用的，术语“害虫”指昆虫和其他类似的害虫，例如蜱螨亚纲的那些，包括但不限于螨和蜱。本发明的昆虫害虫包括但不限于鳞翅目昆虫，例如小蜡螟(achoroiagrisella)、黑头长翅卷蛾(aclerisgloverana)、aclerisvariana、苹果小卷叶蛾(adoxophyesorana)、黑切根虫、棉叶波纹夜蛾(alabamaargillacea)、秋尺蠖(alsophilapometaria)、脐橙螟蛾(amyeloistransitella)、地中海粉螟(anagastakuehniella)、桃枝麦蛾(anarsialineatella)、橙纹犀额蛾(anisotasenatoria)、柞蚕(antheraeapernyi)、大豆夜蛾、黄卷蛾属物种(archipssp.)、带卷蛾属物种(argyrotaeniasp.)、athetismindara、家蚕(bombyxmori)、棉潜蛾(bucculatrixthurberiella)、粉斑螟蛾(cadracautella)、色卷蛾属物种(choristoneurasp.)、cochyllshospes、苜蓿黄蝶(coliaseurytheme)、米蛾(corcyracephalonica)、cydialatiferreanus、苹果蠹蛾、古月桃黄蝶(datanaintegerrima)、dendrolimussibericus、desmiafeneralis、甜瓜绢野螟(diaphaniahyalinata)、黄瓜绢野螟(diaphanianitidalis)、西南玉米螟、小蔗螟(diatraeasaccharalis)、白尺蠖蛾(ennomossubsignaria)、墨西哥稻螟(eoreumaloftini)、烟草粉螟(esphestiaelutella)、erannistilaria、estigmeneacrea、euliasalubricola、eupocoelliaambiguella、女贞细卷蛾(eupoeciliaambiguella)、褐尾蠹(euproctischrysorrhoea)、暗缘地老虎(euxoamessoria)、大蜡螟(galleriamellonella)、梨小食心虫(grapholitamolesta)、黑拟蛉蛾(harrisinaamericana)、helicoverpasubflexa、玉米穗虫、烟芽夜蛾、hemileucaoliviae、向日葵斑螟(homoeosomaelectellum)、美国白蛾(hyphantiacunea)、番茄蠹蛾(keiferialycopersicella)、lambdinafiscellariafiscellaria、lambdinafiscellarialugubrosa、柳毒蛾(leucomasalicis)、葡萄花翅小卷蛾(lobesiabotrana)、草地螟(loxostegesticticalis)、舞毒蛾(lymantriadispar)、macallathyrisalis、天幕毛虫属物种(malacosomasp.)、甘蓝夜蛾(mamestrabrassicae)、蓓带夜蛾(mamestraconfigurata)、番茄天蛾(manducaquinquemaculata)、烟草天蛾(manducasexta)、豆荚野螟(marucatestulalis)、melanchrapicta、冬尺蛾(operophterabrumata)、毒蛾属物种(orgyiasp.)、欧洲玉米螟、春尺蠖(paleacritavernata)、美洲大芷凤蝶(papiliocresphontes)、红棉铃虫(pectinophoragossypiella)、加州槲蛾(phryganidiacalifornica)、斑幕潜叶蛾(phyllonorycterblancardella)、暗脉菜粉蝶(pierisnapi)、菜粉蝶(pierisrapae)、苜蓿绿夜蛾(plathypenascabra)、platynotaflouendana、荷兰石竹小卷蛾(platynotastultana)、platyptiliacarduidactyla、印度谷螟(plodiainterpunctella)、小莱蛾(plutellaxylostella)、pontiaprotodice、美洲粘虫(pseudaletiaunipuncta)、pseudoplasiaincludens、杂食尺蠖(sabulodesaegrotata)、红抚天社蛾(schizuraconcinna)、麦蛾(sitotrogacerealella)、苹果小卷叶蛾(spilontaocellana)、斜纹夜蛾属物种(spodopterasp.)、thaurnstopoeapityocampa、衣蛾(tinsolabisselliella)、粉纹夜蛾(trichoplusiahi)、温室结网野螟(udearubigalis)、xylomygescuriails和苹果巢蛾(yponomeutapadella)。昆虫害虫还包括选自双翅目、膜翅目、鳞翅目、食毛目、同翅目、半翅目、orthroptera、缨翅目、革翅目、等翅目、虱目、蚤目、毛翅目、鞘翅目的昆虫。对于主要作物的本发明的害虫包括但不限于：玉蜀黍：欧洲玉米螟，欧洲玉米螟；黑切根虫，黑切根虫；玉米穗虫，玉米穗虫；草地贪夜蛾，秋粘虫；西南玉米螟，西南玉米螟；小玉米茎蛀虫(elasmopalpuslignosellus)，小玉米茎蛀虫；小蔗螟，甘蔗螟虫；西方玉米根虫，例如玉米根萤叶甲；北方玉米根虫，例如diabroticalongicornisbarberi；南方玉米根虫，例如diabroticaundecimpunctatahowardi；梳爪叩甲属物种(melanotusspp.)，线虫；北方圆头犀金龟(cyclocephalaborealis)，北方假面金龟子(蛴螬)；南方圆头犀金龟(cyclocephalaimmaculata)，南方假面金龟子(蛴螬)；日本弧丽金龟，日本金龟子；玉米跳甲(chaetocnemapulicaria)，玉米跳甲；玉米象虫(sphenophorusmaidis)，玉米象虫；玉米蚜(rhopalosiphummaidis)，玉米蚜；玉蜀黍根蚜(anuraphismaidiradicis)，玉米根蚜；blissusleucopterusleucopterus，麦小蝽；melanoplusfemurrubrum，红腿蚱蜢；迁徒蚱蜢(melanoplussanguinipes)，迁徒蚱蜢；灰地种蝇(hylemyaplatura)，种蝇；美洲黍潜叶蝇(agromyzaparvicornis)，玉米斑潜叶蝇；玉米黄呆蓟马(anaphothripsobscrurus)，草蓟马；红火蚁(solenopsismilesta)，盗蚁；二斑叶螨(tetranychusurticae)，二斑叶螨；高粱：玉米斑点螟(chilopartellus)，高粱螟；草地贪夜蛾，秋粘虫；玉米穗虫，玉米穗虫；小玉米茎蛀虫，小玉米茎蛀虫；粒肤地老虎(feltiasubterranea)，颗粒地老虎；phyllophagacrinita，蛴螬；伪金针虫属(eleodes)，宽胸叩头虫属(conoderus)和aeolusspp.，线虫；谷叶甲虫(oulemamelanopus)，谷物叶甲；玉米跳甲，玉米跳甲；玉米象虫，玉米象虫；玉米蚜，玉米蚜；蔗黄伪毛蚜(siphaflava)，黄甘蔗蚜虫；麦小蝽，例如blissusleucopterusleucopterus；高粱瘿蚊(contariniasorghicola)，高粱瘿蚊；朱砂叶螨(tetranychuscinnabarinus)，朱砂叶螨；二斑叶螨，二斑叶螨；小麦：pseudaletiaunipunctata，粘虫；草地贪夜蛾；秋粘虫；小玉米茎蛀虫，小玉米茎蛀虫；谷叶甲虫，谷物叶甲；三叶草叶象，三叶草叶象；南方玉米根虫，例如diabroticaundecimpunctatahowardi；俄罗斯小麦蚜虫；麦二叉蚜(schizaphisgraminum)，麦二叉蚜；麦长管蚜(macrosiphumavenae)，麦长管蚜；melanoplusfemurrubrum，红腿蚱蜢；殊种蚱蜢(melanoplusdifferentialis)，殊种蚱蜢；迁徒蚱蜢，迁徒蚱蜢；小麦瘿蚊(mayetioladestructor)，小麦瘿蝇；麦红吸浆虫(sitodiplosismosellana)，小麦吸浆虫；美州麦杆蝇(meromyzaamericana)，麦杆蝇；麦种蝇(hylemyacoarctata)，麦种蝇；烟草褐花蓟马(frankliniellafusca)，烟草蓟马；麦茎蜂(cephuscinctus)，小麦茎蜂；小麦曲叶螨(aceriatulipae)，小麦卷叶螨；向日葵：向曰葵茎象甲(cylindrocupturusadspersus)，向日葵茎象甲；红色种子象(smicronyxfulus)，红色向日葵种子象鼻虫；灰色种子象(smicronyxsordidus)，灰色向日葵种子象鼻虫；向日葵食芽蛾(suleimahelianthana)，向日葵芽蛾；向日葵斑螟，向日葵螟；向日葵叶甲(zygogrammaexclamationis)，向日葵甲虫；胡萝卜金龟(bothyrusgibbosus)，胡萝卜甲虫；向日葵籽瘿蚊(neolasiopteramurtfeldtiana)，向日葵籽瘿蚊；棉花：烟芽夜蛾，烟夜蛾；玉米穗虫，玉米穗虫；甜菜夜蛾(spodopteraexigua)，甜菜夜蛾；红棉铃虫，红铃虫；棉铃象鼻虫，例如棉铃象甲(anthonomusgrandis)；棉蚜(aphisgossypii)，棉蚜；棉盲蝽(pseudatomoscelisseriatus)，棉盲蝽；纹翅粉虱(trialeurodesabutilonea)，纹翅粉虱；美国牧草盲蝽(lyguslineolaris)，牧草盲蝽；melanoplusfemurrubrum，红腿蚱蜢；殊种蚱蜢，殊种蚱蜢；烟蓟马(thripstabaci)，葱蓟马；烟草褐花蓟马，烟草蓟马；朱砂叶螨，朱砂叶螨；二斑叶螨，二斑叶螨；稻：小蔗螟，甘蔗螟虫；草地贪夜蛾，秋粘虫；玉米穗虫，玉米穗虫；葡萄肖叶甲(colaspisbrunnea)，葡萄肖叶甲；稻水象(lissorhoptrusoryzophilus)，稻水象甲；米象(sitophilusoryzae)，米象；二条黑尾叶蝉(nephotettixnigropictus)，稻叶蝉；麦小蝽，例如blissusleucopterusleucopterus；拟绿蝽(acrosternumhilare)，绿蝽；大豆：大豆夜蛾，大豆尺蠖；大豆夜蛾，黍豆毛毛虫；苜蓿绿夜蛾，绿夜蛾；欧洲玉米螟，欧洲玉米螟；黑切根虫，黑切根虫；甜菜夜蛾，甜菜夜蛾；烟芽夜蛾，烟夜蛾；玉米穗虫，玉米穗虫；墨西哥豆瓢虫(epilachnavarivestis)，墨西哥豆甲虫；桃蚜(myzuspersicae)，绿桃蚜虫；马铃薯小绿叶蝉(empoascafabae)，马铃薯叶蝉；拟绿蝽，绿蝽；melanoplusfemurrubrum，红腿蚱蜢；殊种蚱蜢，殊种蚱蜢；灰地种蝇，种蝇；大豆蓟马(sericothripsvariabilis)，大豆蓟马；烟蓟马，葱蓟马；土耳其斯坦叶螨(tetranychusturkestani)，草莓蜘蛛螨；二斑叶螨，二斑叶螨；大麦：欧洲玉米螟，欧洲玉米螟；黑切根虫，黑切根虫；麦二叉蚜，麦二叉蚜；麦小蝽，例如blissusleucopterusleucopterus；拟绿蝽，绿蝽；褐臭蝽，褐臭蝽；jylemyaplatura，种蝇；小麦瘿蚊，小麦瘿蝇；麦岩螨，棕色小麦螨；油籽油菜：甘蓝蚜(vrevicorynebrassicae)，卷心菜蚜虫；十字花科跳甲(phyllotretacruciferae)，十字花科跳甲；黄曲条跳甲(phyllotretastriolata)，黄条跳甲；大豆淡足跳甲(phyllotretanemorum)，条纹萝卜跳甲；油菜露尾甲(meligethesaeneus)，油菜甲虫；以及花粉甲虫meligethesrufimanus、meligethesnigrescens、meligethescanadianus和meligethesviridescens；马铃薯：马铃薯甲虫，科罗拉多马铃薯甲虫。本发明提供的方法和组合物可以有效抗半翅目，例如草盲蝽(lygushesperus)、美国牧草盲蝽、牧草盲蝽(lyguspratensis)、长毛草盲蝽(lygusrugulipennispopp)、lyguspabulinus、土豆盲蝽(calocorisnorvegicus)、泛奥盲蝽(orthopscompestris)、苹果盲蝽(plesiocorisrugicollis)、cyrtopeltismodestus、黑斑烟盲蝽(cyrtopeltisnotatus)、白斑盲蝽(spanagonicusalbofasciatus)、diaphnocorischlorinonis、labopidicolaallii、棉盲蝽(pseudatomoscelisseriatus)、苜蓿盲蝽(adelphocorisrapidus)、四线盲蝽(poecilocapsuslineatus)、麦长蝽(blissusleucopterus)、小长蝽(nysiusericae)、nysiusraphanus、褐臭蝽、稻绿蝽、扁盾蝽属(eurygaster)、缘蝽科(coreidae)、红蝽科(pyrrhocoridae)、tinidae、负子蝽科(blostomatidae)、猎蝽科(reduviidae)和臭虫科(cimicidae)。感兴趣的害虫还包括咖啡豆象(araecerusfasciculatus)，咖啡豆象；大豆象(acanthoscelidesobtectus)，大豆象；蚕豆象(bruchusrufmanus)，蚕豆象；豌豆象(bruchuspisorum)，豌豆象；墨西哥豆象(zabrotessubfasciatus)，墨西哥豆象；黄瓜叶甲(diabroticabalteata)，带状黄瓜甲虫；菜豆萤叶甲(cerotomatrifurcata)，豆叶甲；玉米根叶甲，墨西哥玉米根虫；黄瓜跳甲，马铃薯跳甲；甘薯跳甲(chaetocnemaconfinis)，甘薯跳甲；苜蓿叶象(hyperapostica)，苜蓿象甲；苹果花象(anthonomusquadrigibbus)，苹果象鼻虫；豆茎象(sternechuspaludatus)，豆茎象鼻虫；埃及苜蓿叶象(hyperabrunnipennis)，埃及苜蓿象甲；谷象(sitophilusgranaries)，谷象；葡萄象(craponiusinaequalis)，葡萄象鼻虫；玉米象(sitophiluszeamais)，玉米象；李象(conotrachelusnenuphar)，李象鼻虫；西印度群岛甘薯象甲，西印度甘薯象甲；紫绒鲍角金龟(maladeracastanea)，亚洲花园甲虫；欧洲金龟(rhizotrogusmajalis)，欧洲金龟子；蔷薇刺金龟(macrodactylussubspinosus)，玫瑰金龟子；杂拟谷盗(triboliumconfusum)，杂拟谷盗；黑粉虫(tenebrioobscurus)，黑粉虫；赤拟谷盗(triboliumcastaneum)，赤拟谷盗；黄粉虫(tenebriomolitor)，黄粉虫。线虫包括寄生线虫例如根节、孢囊和病变线虫，包括异皮线虫属物种(heteroderaspp.)、根结线虫属物种(meloidogynespp.)和球异皮线虫属物种(globoderaspp.)；特别是孢囊线虫的成员，包括但不限于大豆孢囊线虫(heteroderaglycines)(大豆孢囊线虫)；甜菜孢囊线虫(heteroderaschachtii)(甜菜孢囊线虫)；禾谷孢囊线虫(heteroderaavenae)(禾谷孢囊线虫)；以及马铃薯金线虫(globoderarostochiensis)和马铃薯白线虫(globoderapailida)(马铃薯孢囊线虫)。病变线虫包括短体线虫属物种(pratylenchusspp.)。可以在早期发育阶段(例如幼虫或其他未成熟形式)对昆虫害虫测试本发明组合物的杀虫活性。昆虫可以在完全黑暗中在约20℃至约30℃和约30％至约70％相对湿度下饲养。生物测定可以如czapla和lang(1990)j.econ.entomol.83(6):2480-2485中所述执行。还参见本文的实验部分。iii.表达盒可以在表达盒中提供编码本文提供的杀虫蛋白质的多核苷酸，用于在目的生物中表达。所述盒将包括与编码本文提供的杀虫多肽的多核苷酸可操作地连接的5'和3'调节序列，其允许多核苷酸的表达。所述盒可以另外含有待共转化到生物内的至少一种另外的基因或遗传元件。当包括另外的基因或元件时，各组分可操作地连接。可替代地，可以在多个表达盒上提供另外的基因或元件。这样的表达盒有多个限制性位点和/或重组位点，用于插入多核苷酸以处于调节区的转录调节之下。表达盒可以另外含有选择标记基因。表达盒将在5'-3'转录方向上包括转录和翻译起始区(即启动子)、本发明的杀虫多核苷酸、以及在目的生物即植物或细菌中起作用的转录和翻译终止区(即终止区)。本发明的启动子能够指导或驱动编码序列在宿主细胞中的表达。调节区(即启动子、转录调节区和翻译终止区)对于宿主细胞或彼此之间可以是内源的或异源的。如本文使用的，提及序列时“异源”是源自外来物种的序列，或者如果来自相同物种，则通过有意的人为干预而在组成和/或基因组基因座中从其天然形式实质上修饰而来。如本文使用的，嵌合基因包含可操作地连接至对编码序列异源的转录起始区的编码序列。方便的终止区可从根瘤土壤杆菌(a.tumefaciens)的ti质粒获得，例如章鱼碱合酶和胭脂碱合酶终止区。还参见guerineau等人(1991)mol.gen.genet.262:141-144；proudfoot(1991)cell64:671-674；sanfacon等人(1991)genesdev.5:141-149；mogen等人(1990)plantcell2:1261-1272；munroe等人(1990)gene91:151-158；ballas等人(1989)nucleicacidsres.17:7891-7903；和joshi等人(1987)nucleicacidsres.15:9627-9639。另外的调节信号包括但不限于转录开始起始位点、操纵子、激活子、增强子、其他调节元件、核糖体结合位点、起始密码子、终止信号等等。参见例如，美国专利号5,039,523和4,853,331；ep0480762a2；sambrook等人(1992)molecularcloning:alaboratorymanual，编辑maniatis等人(coldspringharborlaboratorypress，coldspringharbor，n.y.)，下文称为“sambrook11”；davis等人，编辑(1980)advancedbacterialgenetics(coldspringharborlaboratorypress)，coldspringharbor，n.y.，以及其中引用的参考文献。在制备表达盒时，可以操纵各种dna片段，以便提供以正确取向并且适当地正确读码框的dna序列。为此，衔接子或接头可以用于连接dna片段，或者可以涉及其他操纵以提供方便的限制性位点、去除多余的dna、去除限制性位点等等。为此目的，可以涉及体外诱变、引物修复、限制性、退火、再取代例如转换和颠换。在本发明的实践中可以使用许多启动子。可以基于所需结果来选择启动子。核酸可以与组成型、诱导型、组织偏好型或其他启动子组合，用于在目的生物中表达。参见例如，wo99/43838以及美国专利号8,575,425；7,790,846；8,147,856；8,586832；7,772,369；7,534,939；6,072,050；5,659,026；5,608,149；5,608,144；5,604,121；5,569,597；5,466,785；5,399,680；5,268,463；5,608,142；和6,177,611中所述的启动子；各自通过引用并入本文。为了在植物中表达，组成型启动子还包括camv35s启动子(odell等(1985)nature313:810-812)；水稻肌动蛋白(mcelroy等人(1990)plantcell2:163-171)；遍在蛋白(christensen等人(1989)plantmol.biol.12:619-632和christensen等人(1992)plantmol.biol.18:675-689)；pemu(last等人(1991)theor.appl.genet.81:581-588)；mas(velten等人(1984)emboj.3:2723-2730)。诱导型启动子包括驱动发病机制相关蛋白(pr蛋白)表达的那些启动子，所述pr蛋白在被病原体感染后被诱导。参见例如redolfi等人(1983)neth.j.plantpathol.89:245-254；uknes等人(1992)plantcell4:645-656；和vanloon(1985)plantmol.virol.4:111-116；和wo99/43819，引入本文作为参考。也可以使用在病原体感染位点处或附近局部表达的启动子(marineau等人(1987)plantmol.biol.9:335-342；matton等人(1989)molecularplant-microbeinteractions2:325-331；somsisch等人(1986)proc.natl.acad.sci.usa83:2427-2430；somsisch等人(1988)mol.gen.genet.2:93-98；和yang(1996)proc.natl.acad.sci.usa93:14972-14977；chen等人(1996)plantj.10:955-966；zhang等人(1994)proc.natl.acad.sci.usa91:2507-2511；warner等人(1993)plantj.3:191-201；siebertz等人(1989)plantcell1:961-968；cordero等人(1992)physiol.mol.plantpath.41:189-200；美国专利号5,750,386(线虫诱导型)；以及其中引用的参考文献)。创伤诱导型启动子可以用于本发明的构建体中。此类创伤诱导型启动子包括pinii启动子(ryan(1990)ann.rev.phytopath.28:425-449；duan等人(1996)naturebiotechnology14:494-498)；wun1和wun2(美国专利5,428,148)；win1和win2(stanford等人(1989)mol.gen.genet.215:200-208)；系统素(mcgurl等人(1992)science225:1570-1573)；wip1(rohmeier等人(1993)plantmol.biol.22:783-792；eckelkamp等人(1993)febsletters323:73-76)；mpi基因(corderok等人(1994)plantj.6(2):141-150)；等等，通过引用并入本文。用于本发明的组织偏好型启动子包括在下述中阐述的那些：yamamoto等人(1997)plantj.12(2):255-265；kawamata等人(1997)plantcellphysiol.38(7):792-803；hansen等人(1997)mol.gengenet.254(3):337-343；russell等人(1997)transgenicres.6(2):157-168；rinehart等人(1996)plantphysiol.112(3):1331-1341；vancamp等人(1996)plantphysiol.112(2):525-535；canevascini等人(1996)plantphysiol.112(2):513-524；yamamoto等人(1994)plantcellphysiol.35(5):773-778；lam(1994)resultsprobl.celldiffer.20:181-196；orozco等人(1993)plantmolbiol.23(6):1129-1138；matsuoka等人(1993)procnatl.acad.sci.usa90(20):9586-9590；和guevara-garcia等人(1993)plantj.4(3):495-505。叶偏好的启动子包括在下述中阐述的那些：yamamoto等人(1997)plantj.12(2):255-265；kwon等人(1994)plantphysiol.105:357-67；yamamoto等人(1994)plantcellphysiol.35(5):773-778；gotor等人(1993)plantj.3:509-18；orozco等人(1993)plantmol.biol.23(6):1129-1138；和matsuoka等人(1993)proc.natl.acad.sci.usa90(20):9586-9590。根偏好的启动子是已知的，并且包括下述中的那些：hire等人(1992)plantmol.biol.20(2):207-218(大豆根特异性谷氨酰胺合成酶基因)；keller和baumgartner(1991)plantcell3(10):1051-1061(根特异性控制元件)；sanger等人(1990)plantmol.biol.14(3):433-443(根瘤土壤杆菌的甘露碱合成酶(mas)基因；和miao等人(1991)plantcell3(1):11-22(胞质谷氨酰胺合成酶(gs))；bogusz等人(1990)plantcell2(7):633-641；leach和aoyagi(1991)plantscience(limerick)79(1):69-76(rolc和rold)；teeri等人(1989)emboj.8(2):343-350；kuster等人(1995)plantmol.biol.29(4):759-772(vfenod-grp3基因启动子)；和capana等人(1994)plantmol.biol.25(4):681-691(rolb启动子)。也参见美国专利号5,837,876；5,750,386；5,633,363；5,459,252；5,401,836；5,110,732；和5,023,179。“种子偏好型”启动子包括“种子特异性”启动子(在种子发育过程中活跃的那些启动子，例如种子贮藏蛋白的启动子)以及“种子萌发”启动子(在种子萌发过程中活跃的那些启动子)两者。参见thompson等人(1989)bioessays10:108。种子偏好型启动子包括但不限于cim1(细胞分裂素诱导的信息)；cz19b1(玉蜀黍19kda玉米醇溶蛋白)；milps(肌醇-1-磷酸合酶)(参见wo00/11177和美国专利号6,225,529)。γ-玉米醇溶蛋白是胚乳特异性启动子。球蛋白1(glb-1)是代表性的胚特异性启动子。对于双子叶植物，种子特异性启动子包括但不限于豆β-菜豆球蛋白、napin、β-伴大豆球蛋白、大豆凝集素、十字花科蛋白等等。对于单子叶植物，种子特异性启动子包括但不限于玉蜀黍15kda玉米醇溶蛋白、22kda玉米醇溶蛋白、27kda玉米醇溶蛋白、γ-玉米醇溶蛋白、蜡质、收缩蛋白(shrunken)1、收缩2、球蛋白1等。也参见wo00/12733，其中公开了来自end1和end2基因的种子偏好型启动子。为了在细菌宿主中表达，在细菌中起作用的启动子是本领域众所周知的。这样的启动子包括任何已知的晶体蛋白质基因启动子，包括本发明的任何杀虫蛋白质的启动子，以及对于苏云金芽孢杆菌σ因子特异性的启动子。可替代地，诱变的或重组的晶体蛋白质编码基因启动子可以被重组改造并且用于促进本文公开的新型基因区段的表达。表达盒还可以包含用于选择经转化的细胞的可选择标记物基因。可选择标记物基因用于选择经转化的细胞或组织。标记物基因包括编码抗生素抗性的基因，例如编码新霉素磷酸转移酶ii(neo)和潮霉素磷酸转移酶(hpt)的基因，以及赋予对除草剂化合物如草铵膦、溴苯腈、咪唑啉酮和2,4-二氯苯氧乙酸(2,4-d)的抗性的基因。另外的可选择标记物是已知的，并且均可以用于本发明的实践中。参见例如通过引用整体并入本文的于2015年12月18日提交的pct/us2015/066648，其公开了可以用作可选择标记物的草铵膦抗性序列。iv.方法、宿主细胞和植物细胞如所指出的，包含编码杀虫蛋白质或其活性变体或片段的核苷酸序列的dna构建体可以用于转化感兴趣的植物或感兴趣的其他生物。用于转化的方法涉及将核苷酸构建体引入植物内。“引入”是指将核苷酸构建体以这样的方式引入植物或其他宿主细胞，使得构建体进入植物细胞或宿主细胞的内部。本发明的方法不需要将核苷酸构建体引入植物或宿主细胞的特定方法，只要核苷酸构建体进入植物或宿主生物的至少一个细胞的内部即可。用于将核苷酸构建体引入植物和其他宿主细胞内的方法是本领域已知的，包括但不限于稳定转化方法、瞬时转化方法和病毒介导的方法。所述方法产生经转化的生物，例如植物，包括完整植物，以及植物器官(例如叶、茎、根等)、种子、植物细胞、繁殖体、胚及其后代。植物细胞可以是分化的或未分化的(例如愈伤组织、悬浮培养细胞、原生质体、叶细胞、根细胞、韧皮部细胞、花粉)。“转基因植物”或“经转化的植物”或“稳定转化的”植物或细胞或组织指已掺入或整合了编码本发明的至少一种杀虫多肽的多核苷酸的植物。已认识到其他外源或内源核酸序列或dna片段也可以掺入该植物细胞内。土壤杆菌和生物弹道介导的转化仍然是两种主要采用的方法。然而，可以通过感染、转染、显微注射、电穿孔、微粒轰击、生物弹道或粒子轰击、电穿孔、二氧化硅/碳纤维、超声介导、peg介导、磷酸钙共沉淀、聚阳离子dmso技术、deae葡聚糖程序、agro和病毒介导(caulimoriviruses、双生病毒、rna植物病毒)、脂质体介导等等来执行。转化方案以及用于将多肽或多核苷酸序列引入植物内的方案可以根据靶向用于转化的植物或植物细胞的类型(即单子叶或双子叶植物)而变化。转化方法是本领域已知的，包括美国专利号：8,575,425；7,692,068；8,802,934；7,541,517中所述的那些；所述美国专利号各自通过引用并入本文。还参见rakoczy-trojanowska，m.(2002)cellmolbiollett.7:849-858；jones等人(2005)plantmethods1:5；rivera等人(2012)physicsoflifereviews9:308-345；bartlett等人(2008)plantmethods4:1-12；bates，g.w.(1999)methodsinmolecularbiology111:359-366；binns和thomashow(1988)annualreviewsinmicrobiology42:575-606；christou，p.(1992)theplantjournal2:275-281；christou，p.(1995)euphytica85:13-27；tzfira等人(2004)trendsingenetics20:375-383；yao等人(2006)journalofexperimentalbotany57:3737-3746；zupan和zambryski(1995)plantphysiology107:1041-1047；jones等人(2005)plantmethods1:5。转化可导致核酸稳定或瞬时掺入细胞内。“稳定转化”意指引入宿主细胞内的核苷酸构建体整合到宿主细胞的基因组内，并且能够被其后代继承。“瞬时转化”意指将多核苷酸被引入宿主细胞内，并且不整合到宿主细胞的基因组内。用于叶绿体转化的方法是本领域已知的。参见例如，svab等人(1990)proc.natl.acad.sci.usa87:8526-8530；svab和maliga(1993)proc.natl.acad.sci.usa90:913-917；svab和maliga(1993)emboj.12:601-606。该方法依赖于含有可选择标记物的dna的粒子枪递送，并且通过同源重组将dna靶向质体基因组。另外，质体转化可以通过由核编码的和质体定向的rna聚合酶的组织偏好表达反式激活沉默的质体携带的转基因来实现。这种系统已在mcbride等人(1994)proc.natl.acad.sci.usa91:7301-7305中得到报道。已转化的细胞可以按照常规方式生长成植物。参见例如，mccormick等人(1986)plantcellreports5:81-84。然后可以使这些植物生长，并且用相同的转化品系或不同品系授粉，并且鉴定具有所需表型特征的组成型表达的所得到的杂种。可以生长两代或更多代以确保所需表型特征的表达得到稳定维持和继承，然后收获种子以确保已实现了所需表型特征的表达。以这种方式，本发明提供了具有稳定整合到其基因组内的本发明的核苷酸构建体(例如本发明的表达盒)的经转化的种子(也称为“转基因种子”)。在具体的实施方案中，本文提供的序列可以被靶向宿主细胞或植物细胞的基因组内的特定位点。此类方法包括但不限于针对目的植物基因组序列设计的大范围核酸酶(meganucleases)(d’halluin等人2013plantbiotechnolj)；crispr-cas9、talen和用于基因组的精确编辑的其他技术(feng等人(2013)cellresearch23:1229-1232；podevin等人(2013)trendsbiotechnology，在线公开；wei等人(2013)jgengenomics；zhang等人(2013)wo2013/026740)；cre-lox位点特异性重组(dale等人(1995)plantj7:649-659；lyznik等人(2007)transgenicplantj1:1-9；flp-frt重组(li等人(2009)plantphysiol151:1087-1095)；bxb1介导的整合(yau等人plantj(2011)701:147-166)；锌指介导的整合(wright等人(2005)plantj44:693-705)；cai等人(2009)plantmolbiol69:699-709)；和同源重组(lieberman-lazarovich和levy(2011)methodsmolbiol701:51-65)；puchta(2002)plantmolbiol48:173-182)。本文提供的序列可以用于转化任何植物物种，包括但不限于单子叶植物和双子叶植物。感兴趣的植物的实例包括但不限于玉米(玉蜀黍)、高粱、小麦、向日葵、番茄、十字花科植物、辣椒、马铃薯、棉花、稻、大豆、甜菜、甘蔗、烟草、大麦和油菜、芸苔属物种(brassicasp.)、苜蓿、黑麦、小米、红花、花生、甘薯、木薯(cassaya)、咖啡、椰子、菠萝、柑橘树、可可、茶、香蕉、鳄梨、无花果、番石榴、芒果、橄榄、木瓜、腰果、澳洲坚果、杏仁、燕麦、蔬菜、观赏植物和针叶树。蔬菜包括但不限于番茄，莴苣，绿豆，利马豆，豌豆和黄瓜属(curcumis)的成员，例如黄瓜、哈密瓜和甜瓜。观赏植物包括但不限于杜鹃花、绣球花、芙蓉、玫瑰、郁金香、水仙、矮牵牛、康乃馨、一品红和菊花。优选地，本发明的植物是作物植物(例如玉蜀黍、高粱、小麦、向日葵、番茄、十字花科植物、辣椒、马铃薯、棉花、稻、大豆、甜菜、甘蔗、烟草、大麦、油籽油菜等)。如本文使用的，术语植物包括植物细胞、植物原生质体、由其可以再生植物的植物细胞组织培养物、植物愈伤组织、植物团块和在植物或植物部分中完整的植物细胞例如胚、花粉、胚珠、种子、叶、花、枝、果实、果仁、穗、穗轴、外壳、茎、根、根尖、花药等等。谷类意指通过商业种植者为了除生长或繁殖物种外的目的而生产的成熟种子。再生植物的后代、变体和突变体也包括在本发明的范围内，条件是这些部分包含所引入的多核苷酸。进一步提供了保留了本文公开的序列的经加工的植物产物或副产物，包括例如豆粕。在另一个实施方案中，编码杀虫蛋白质的基因可以用于转化昆虫致病生物。这样的生物包括杆状病毒、真菌、原生动物、细菌和线虫。可以选择已知占据一种或多种目的作物的“植物圈”(叶面、叶际、根际和/或根面(rhizoplana))的微生物宿主。选择这些微生物以便能够在特定的环境中与野生型微生物成功竞争，提供表达杀虫蛋白质的基因的稳定维持和表达，并且期望地提供杀虫剂免于环境降解和灭活的更好保护。这样的微生物包括古细菌、细菌、藻类和真菌。特别感兴趣的是微生物，例如细菌，例如芽孢杆菌属、假单胞菌属(pseudomonas)、欧文氏菌属(erwinia)、沙雷氏菌属(serratia)、克雷伯氏菌属(klebsiella)、黄单胞菌属(xanthomonas)、链霉菌属(streptomyces)、根瘤菌属(rhizobium)、红假单胞菌属(rhodopseudomonas)、methylius、土壤杆菌属(agrobacterium)、乙杆菌属(acetobacter)、乳杆菌属(lactobacillus)、节杆菌属(arthrobacter)、固氮菌属(azotobacter)、明串珠菌属(leuconostoc)和产碱菌属(alcaligenes)。真菌包括酵母，例如酵母属(saccharomyces)、隐球酵母属(cryptococcus)、克鲁维酵母属(kluyveromyces)、掷孢酵母属(sporobolomyces)、红酵母属(rhodotorula)和短梗霉属(aureobasidium)。特别感兴趣的是这些植物圈细菌物种，如丁香假单胞菌(pseudomonassyringae)、铜绿假单胞菌(pseudomonasaeruginosa)、荧光假单胞菌(pseudomonasfluorescens)、粘质沙雷氏菌(serratiamarcescens)、木乙杆菌(acetobacterxylinum)、土壤杆菌属、球形红假单胞菌(rhodopseudomonasspheroides)、野油菜黄单胞菌(xanthomonascampestris)、苜蓿根瘤菌(rhizobiummelioti)、真养产碱杆菌(alcaligenesentrophus)、木质棒状杆菌(clavibacterxyli)和维氏固氮菌(azotobactervinlandir)，以及植物圈酵母物种例如深红酵母(rhodotorularubra)、粘红酵母(r.glutinis)、海滨红酵母(r.marina)、橙红酵母(r.aurantiaca)、浅白隐球酵母(cryptococcusalbidus)、流散假丝酵母(c.diffluens)、生防酵母(c.laurentii)、罗斯酵母(saccharomycesrosei)、s.pretoriensis、酿酒酵母(s.cerevisiae)、sporobolomycesrosues、s.odorus、kluyveromycesveronae、禾口出芽短梗霉(aureobasidiumpollulans)、苏云金芽孢杆菌、大肠杆菌(escherichiacoli)、枯草芽孢杆菌等等。示例性的原核生物(革兰氏阴性和革兰氏阳性两者)包括肠杆菌科(enterobacteriaceae)，例如埃希氏菌属(escherichia)、欧文氏菌属、志贺氏菌属(shigella)、沙门氏菌属(salmonella)和变形杆菌属(proteus)；芽孢杆菌科(bacillaceae)；根瘤菌科(rhizobiceae)，例如根瘤菌属；螺菌科(spirillaceae)，例如发光杆菌属(photobacterium)、发酵单胞菌属(zymomonas)、沙雷氏菌属、气单胞菌属(aeromonas)、弧菌属(vibrio)、脱硫弧菌属(desulfovibrio)、螺旋菌属(spirillum)；乳杆菌科(lactobacillaceae)；假单胞菌科(pseudomonadaceae)，例如假单胞菌属和乙杆菌属；固氮菌科(azotobacteraceae)和硝化杆菌科(nitrobacteraceae)。真菌包括藻菌纲(phycomycetes)和子囊菌纲(ascomycetes)，例如酵母，如酵母属和裂殖酵母属(schizosaccharomyces)；和担子菌纲(basidiomycetes)酵母，例如红酵母属、短梗霉属、掷孢酵母属等等。编码杀虫蛋白质的基因可以借助于电转化、peg诱导的转化、热休克、转导、缀合等等引入。具体地，可以将编码杀虫蛋白质的基因克隆到穿梭载体例如pht3101内(lerecius等人(1989)femsmicrobiol.letts.60:211-218)。含有特定杀虫蛋白质基因的编码序列的穿梭载体pht3101可以例如借助于电穿孔转化到根部定植的芽孢杆菌内(lerecius等人(1989)femsmicrobiol.letts.60:211-218)。可以设计表达系统，通过将适当的信号肽融合到杀虫蛋白质的氨基末端，使得杀虫蛋白质分泌到革兰氏阴性细菌的细胞质外。由大肠杆菌识别的信号肽包括ompa蛋白(ghrayeb等人(1984)emboj，3:2437-2442)。杀虫蛋白质及其活性变体可以在细菌宿主中发酵，并且可以将得到的细菌加工且以与苏云金芽孢杆菌菌株被用作杀虫喷雾的相同方式用作微生物喷雾。在杀虫蛋白质由芽孢杆菌分泌的情况下，使用本领域已知的操作去除或突变分泌信号。这种突变和/或缺失可防止杀虫蛋白质在发酵过程期间分泌到生长培养基内。杀虫蛋白质保留在细胞内，然后加工细胞以产生包封的杀虫蛋白质。可替代地，通过将异源基因引入细胞宿主内来产生杀虫蛋白质。异源基因的表达直接或间接地导致杀虫剂的细胞内生产和维持。然后将这些细胞在细胞施用于靶害虫的环境时能延长细胞中产生的毒素的活性的条件下进行处理。所得到的产物保留了毒素的毒性。然后可以根据常规技术配制这些天然包封的杀虫蛋白质，用于施用于具有靶害虫的环境例如土壤、水和植物叶子。参见例如美国专利号6,468,523和美国公开号20050138685，以及其中引用的参考文献。在本发明中，经转化的微生物(其包括完整生物、细胞、孢子、杀虫蛋白质、杀虫组分、冲击害虫的组分、突变体、活细胞或死细胞和细胞组分，包括活细胞和死细胞以及细胞组分的混合物，并且包括破碎的细胞和细胞组分)或分离的杀虫蛋白质可以用可接受的载体配制成杀虫或农业组合物，其为例如悬浮液、溶液、乳剂、撒粉剂、可分散颗粒剂、可湿性粉剂和可乳化浓缩物、气溶胶、浸渍颗粒剂、佐剂、可涂布糊剂以及包封在例如聚合物物质中。如本文公开的，农业组合物可以包含多肽、重组多肽或其变体或片段。本文公开的农业组合物可以应用于植物的环境或栽培区域，或者应用于植物、植物部分、植物细胞或种子。上述公开的这种组合物可以通过添加表面活性剂、惰性载体、防腐剂、湿润剂、进食刺激剂、引诱剂、包封剂、粘合剂、乳化剂、染料、uv保护剂、缓冲剂、流动剂或肥料、微量营养素供体或影响植物生长的其他制剂而获得。一种或多种农业化学品，包括、但不限于除草剂、杀昆虫剂、杀真菌剂、杀细菌剂、杀线虫剂、杀软体动物剂、杀螨剂、植物生长调节剂、收获助剂和肥料，可以与载体、表面活性剂或配制领域常规采用的佐剂或其他组分相组合，以促进产品处理和对于特定靶害虫的应用。合适的载体和佐剂可以是固体或液体，并且对应于制剂技术中通常采用的物质，例如天然或再生矿物质、溶剂、分散剂、润湿剂、增粘剂、粘合剂或肥料。本发明的活性成分通常以组合物的形式应用，并且可以应用于待处理的作物区域、植物或种子。例如，本发明的组合物可以应用于准备在粮仓或筒仓等贮存或者在粮仓或筒仓等贮存期间的谷类。本发明的组合物可以与其他化合物同时或相继应用。应用本发明的活性成分或含有由本发明的细菌菌株产生的至少一种杀虫蛋白质的本发明农业化学组合物的方法包括但不限于叶面施用、种子包被和土壤施用。施用次数和施用速率取决于相应害虫的侵扰强度。合适的表面活性试剂包括、但不限于阴离子化合物，例如金属的羧酸盐；长链脂肪酸的羧酸盐；n-酰基肌氨酸盐；磷酸与脂肪醇乙氧基化物的单酯或二酯或者这些酯的盐；脂肪醇硫酸盐例如十二烷基硫酸钠、十八烷基硫酸钠或十六烷基硫酸钠；乙氧基化脂肪醇硫酸盐；乙氧基化烷基酚硫酸盐；木质素磺酸盐；石油磺酸盐；烷基芳基磺酸盐例如烷基苯磺酸盐或低级烷基萘磺酸盐，例如丁基萘磺酸盐；磺化萘-甲醛缩合物的盐；磺化苯酚-甲醛缩合物的盐；更复杂的磺酸盐例如酰胺磺酸盐，例如油酸和n-甲基牛磺酸的磺化缩合产物；或二烷基磺基琥珀酸盐，例如琥珀酸二辛酯的磺酸钠。非离子型试剂包括脂肪酸酯、脂肪醇、脂肪酸酰胺或脂肪烷基或烯基取代的酚与环氧乙烷的缩合产物，多元醇醚的脂肪酸酯如脱水山梨糖醇脂肪酸酯，这些酯与环氧乙烷的缩合产物，例如聚氧乙烯脱水山梨糖醇脂肪酸酯，环氧乙烷和环氧丙烷的嵌段共聚物，炔二醇如2,4,7,9-四乙基-5-癸炔-4,7-二醇或乙氧基化炔二醇。阳离子表面活性试剂的实例包括例如脂肪族单胺、二胺或多胺如乙酸盐、萘酸盐或油酸盐；或含氧的胺如聚氧乙烯烷基胺的氧化胺；通过羧酸与二胺或多胺缩合制备的酰胺连接的胺；或季铵盐。惰性材料的实例包括但不限于无机矿物质如高岭土、层状硅酸盐、碳酸盐、硫酸盐、磷酸盐，或植物材料如软木、粉状玉米芯、花生壳、稻壳和核桃壳。本发明的组合物可以以直接施用的合适形式，或者作为主要组合物的浓缩物，其在施用前需要用合适量的水或其他稀释剂稀释。杀虫浓度将根据具体制剂的性质而变化，具体地，无论它是浓缩物还是直接使用。该组合物含有1至98％的固体或液体惰性载体，以及0至50％或0.1至50％的表面活性剂。这些组合物将以用于商业产物的标记速率施用，例如当以干燥形式时为约0.01lb-5.0lb/英亩，当以液体形式时为约0.01pts.-10pts./英亩。在一个进一步的实施方案中，本文提供的组合物以及经转化的微生物和杀虫蛋白质可以在配制前进行处理，以在施用于靶害虫的环境时延长杀虫活性，只要预处理对该杀虫活性没有害处即可。这种处理可以通过化学和/或物理手段进行，只要处理不会有害地影响组合物的性质即可。化学试剂的例子包括但不限于卤化剂；醛如甲醛和戊二醛；抗感染药如苯扎氯铵；醇如异丙醇和乙醇；和组织固定剂如bouin固定剂和helly固定剂(参见例如humason(1967)animaltissuetechniques(w.h.freemanandco.))。在一个方面，可以通过将本发明的杀虫蛋白质施加到一个给定区域而杀死该区域内的害虫或减少其中的害虫数目。可替代地，杀虫蛋白质可以预防性地应用于环境区域，以预防易感害虫的侵扰。优选地，害虫摄取或接触杀虫有效量的所述多肽。“杀虫有效量”意指能够使至少一种害虫死亡或明显降低害虫生长、进食或正常生理发育的杀虫剂的量。这个量取决于这种因素，如例如待控制的特定靶害虫、特定环境、地点、植物、作物或待处理的农业地点、环境条件，以及杀虫有效多肽组合物的施用方法、速率、浓度、稳定性和数量而变化。制剂或组合物也可以根据气候条件、环境考虑因素和/或施用频率和/或害虫侵扰的严重程度而变化。活性成分通常以组合物的形式应用，并且可以应用于待处理的作物区域、植物或种子。因此提供了用于对植物、植物细胞、种子、植物部分或栽培区域提供有效量的包含所述多肽、重组多肽或其活性变体或片段的农业组合物的方法。“有效量”是指足以杀死或控制害虫，或者导致害虫生长、进食或正常生理发育的显著下降的蛋白质或组合物的量。害虫数目、害虫生长、害虫进食或害虫正常发育的这种下降可以包括任何统计学上显著的下降，包括例如下降约5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、85％、90％、95％或更多。例如，所述组合物可以应用于准备在粮仓或筒仓等中贮存或者在粮仓或筒仓等中贮存期间的谷类。所述组合物可以与其他化合物同时或相继应用。应用包含如本文公开的多肽、重组多肽或者其变体或片段中的至少一种的活性成分或农业化学组合物的方法包括但不限于叶面施用、种子包被和土壤施用。提供了用于增加植物产量的方法。所述方法包括提供表达编码本文公开的杀虫多肽序列的多核苷酸的植物或植物细胞，以及使所述植物或其种子在侵染了所述多肽对其具有杀虫活性的害虫(或易受其侵染)的田地中生长。在一些实施方案中，所述多肽具有针对鳞翅目、鞘翅目、双翅目、半翅目或线虫害虫的杀虫活性、并且所述田地被鳞翅目、半翅目、鞘翅目、双翅目或线虫害虫侵扰。如本文定义的，植物的“产量”指由植物产生的生物质的质量和/或数量。“生物质”意指任何测量的植物产物。生物质产量中的增加是测量的植物产物的产量中的任何改善。增加植物产量具有几种商业应用。例如，增加植物叶生物量可以增加用于人或动物消费的叶用蔬菜的产量。另外，增加叶生物量可以用于增加植物衍生的药物或工业产物的生产。产量增加可以包括任何统计学上显著的增加，包括、但不限于与不表达所述杀虫序列的植物相比，产量有至少1％增加、至少3％增加、至少5％增加、至少10％增加、至少20％增加、至少30％、至少50％、至少70％、至少100％或更大增加。在具体方法中，由于表达本文公开的杀虫蛋白质的植物的害虫抗性改善，植物产量增加。杀虫蛋白质的表达导致害虫侵袭或进食的能力降低。还可以用一种或多种化学组合物(包括一种或多种除草剂、杀昆虫剂或杀真菌剂)处理植物。非限制性实施方案包括：实施方案1.具有杀虫活性的分离的多肽，其包含(a)包含选自由下述序列组成之组中的氨基酸序列的多肽：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224；或(b)包含与选自由下述序列组成之组中的氨基酸序列至少有表1所示的序列同一性百分比的氨基酸序列的多肽：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224。实施方案2.实施方案1的多肽，其中所述多肽包含seqidno.1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224中所示的氨基酸序列。实施方案3.实施方案2的多肽，其进一步包含异源氨基酸序列。实施方案4.包含实施方案1、2、3中的至少一个所述的多肽的组合物。实施方案5.编码实施方案1或2所述的多肽的重组核酸分子，其中所述重组核酸分子不是编码所述多肽的天然存在的序列。实施方案6.实施方案5的重组核酸，其中所述核酸分子是已被设计用于在植物中表达的合成序列。实施方案7.实施方案5或6的重组核酸分子，其中所述核酸分子与能够指导在植物细胞中的表达的启动子可操作地连接。实施方案8.实施方案5或6的重组核酸分子，其中所述核酸分子与能够指导在细菌中的表达的启动子可操作地连接。实施方案9.含有实施方案5-8中任何一个所述的重组核酸分子的宿主细胞。实施方案10.实施方案9的宿主细胞，其中所述宿主细胞是细菌宿主细胞。实施方案11.包含可操作地连接至重组核酸分子的启动子的dna构建体，所述启动子驱动在植物细胞中的表达，所述重组核酸分子包含：(a)编码包含下述任一氨基酸序列的多肽的核苷酸序列：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224；或(b)编码包含与选自下述序列的组中的氨基酸序列有至少表1中所示的序列同一性百分比的氨基酸序列的多肽的核苷酸序列：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224。实施方案12.实施方案11的dna构建体，其中所述核苷酸序列是已被设计用于在植物中表达的合成dna序列。实施方案13.包含实施方案11的dna构建体的载体。实施方案14.含有实施方案11或12的dna构建体或者实施方案13的载体的宿主细胞。实施方案15.实施方案14的宿主细胞，其中所述宿主细胞是植物细胞。实施方案16.包含实施方案14或15的宿主细胞的转基因植物。实施方案17.包含实施方案9、10、14或15的宿主细胞的组合物。实施方案18.实施方案17的组合物，其中所述组合物选自粉末、粉剂、丸剂、颗粒剂、喷雾剂、乳剂、胶体和溶液。实施方案19.实施方案17的组合物，其中所述组合物包含按重量计约1％至约99％的所述多肽。实施方案20.用于控制害虫群体的方法，其包括使所述群体与杀虫有效量的实施方案17-19中任何一个所述的组合物接触。实施方案21.用于杀死害虫群体的方法，其包括使所述群体与杀虫有效量的实施方案17-19中任何一个所述的组合物接触。实施方案22.用于产生具有杀虫活性的多肽的方法，其包括在其中编码所述多肽的核酸分子被表达的条件下培养实施方案9、10、14或15所述的宿主细胞。实施方案23.具有稳定整合到其基因组内的dna构建体的植物，所述dna构建体包含编码具有杀虫活性的蛋白质的核苷酸序列，其中所述核苷酸序列包含：(a)编码包含下述任一氨基酸序列的多肽的核苷酸序列：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224；或(b)编码包含与选自下述序列的组中的氨基酸序列有至少表1中所示的序列同一性百分比的氨基酸序列的多肽的核苷酸序列：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224。实施方案24.实施方案23所述的植物的转基因种子。实施方案25.用于保护植物免受昆虫害虫的方法，其包括在植物或其细胞中表达编码杀虫多肽的核苷酸序列，其中所述核苷酸序列包含：(a)编码包含下述任一氨基酸序列的多肽的核苷酸序列：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224；或(b)编码包含与选自下述序列的组中的氨基酸序列有至少表1中所示的序列同一性百分比的氨基酸序列的多肽的核苷酸序列：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224。实施方案26.实施方案25的方法，其中所述植物产生具有针对鳞翅目害虫或鞘翅目害虫中的至少一种的杀虫活性的杀虫多肽。实施方案27.用于增加植物产量的方法，其包括在田间生长具有稳定整合到其基因组内的dna构建体的植物或其种子，所述dna构建体包含可操作地连接至编码杀虫多肽的核苷酸序列的启动子，所述启动子驱动在植物中的表达，其中所述核苷酸序列包含：(a)编码包含下述任一氨基酸序列的多肽的核苷酸序列：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224；或(b)编码包含与选自下述序列的组中的氨基酸序列有至少表1中所示的序列同一性百分比的氨基酸序列的多肽的核苷酸序列：seqidno:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223或224。实施方案28.提供了获得编码包含杀虫活性的改善多肽的多核苷酸的方法，其中所述改善多肽具有超过seqidno:1-224中任何一个的至少一种改善的性质，所述方法包括：(a)将包含seqidno:1-224或者其活性变体或片段的多个亲本多核苷酸重组，以产生编码重组杀虫多肽的重组多核苷酸文库；(b)筛选所述文库以鉴定编码改善的重组杀虫多肽的重组多核苷酸，所述重组多核苷酸相比于亲本多核苷酸具有改善的更好的性质；(c)回收编码(b)中鉴定的改善的重组杀虫多肽的重组多核苷酸；和(d)使用步骤(c)中回收的重组多核苷酸作为重复步骤(a)中的多个亲本多核苷酸之一，重复步骤(a)、(b)和(c)。下述实施例以举例说明性而非限制性的方式提供。实验实验1：通过测序和dna分析发现新型基因微生物培养物在标准实验室培养基中以液体培养物生长。在dna制备之前，使培养物生长至饱和(16至24小时)。通过去污剂裂解从细菌细胞中提取dna，随后与二氧化硅基质结合并且用乙醇缓冲液洗涤。纯化的dna用弱碱性水性缓冲液从二氧化硅基质中洗脱下来。通过分光光度法测试用于测序的dna的纯度和浓度。根据制造商的方案，使用nexteraxt文库制备试剂盒制备测序文库。根据illuminahiseq2000系统用户指南协议，在hiseq2000上生成序列数据。使用clcbioassemblycell软件包将测序读数装配成基因组草图。在组装之后，通过几种方法进行基因调用，并且分析所得到的基因序列，以鉴定杀虫基因的新型同系物。新型基因通过blast、结构域组成、以及与一组靶杀虫基因的成对比对进行鉴定。这些序列的概括在表1中阐述。通过pcr从细菌dna扩增同源性搜索中鉴定的基因，并且克隆到含有框内融合的纯化标签的细菌表达载体内。克隆的基因在大肠杆菌中表达并且通过柱层析纯化。在昆虫饮食生物测定研究中评价纯化的蛋白质，以鉴定活性蛋白质。使用小麦胚芽和琼脂人造饮食执行昆虫饮食生物测定，将纯化蛋白质作为表面处理应用于所述饮食。将昆虫幼虫应用于经处理的饮食并且监测死亡率。昆虫饮食生物测定使用包含在其中添加了纯化蛋白质的膜小袋中的蔗糖液体饮食执行。允许昆虫幼虫以饮食小袋为食并且监测死亡率。在生物测定中测试的昆虫包括褐臭蝽(bsb)euschistusservus(褐臭蝽)和南绿蝽象(sgsb)nezaraviridula(稻绿蝽)。实施例2.在大肠杆菌中的异源表达将表3和表4中阐述的每个开放读码框克隆到含有麦芽糖结合蛋白(pmbp)的大肠杆菌表达载体内。将表达载体转化到bl21*ripl内。用单菌落接种补充有羧苄青霉素的lb培养物，并且使用0.5％过夜培养物在37℃下生长过夜，接种新鲜培养物并且在37℃下培养至对数期。使用250mmiptg在16℃下将培养物诱导18小时。将细胞形成团块并且重悬浮于补充有蛋白酶抑制剂的10mmtrisph7.4和150mmnacl中。通过sds-page评估蛋白质表达。实施例3.针对鞘翅目和鳞翅目的杀虫活性蛋白质表达：如实施例2中所述，将表3中阐述的每种序列在大肠杆菌中表达。接种400ml的lb并且生长至od600达到0.6。培养物在16℃下用0.25mmiptg诱导过夜。将细胞离心下来，并且将细胞团块重悬浮于5ml缓冲液中。重悬浮物在冰上超声处理2分钟。生物测定：从商业养虫室(benzonresearchinc.，carlisle，pa)购买秋粘虫(faw)、玉米穗虫(cew)、欧洲玉米螟(ecb)西南玉米螟(swcb)和小菜蛾(dbm或px)卵。将faw、cew、ecb和bcw卵孵化至羽化在测定设置的12小时内发生的点。将swcb和dbm作为新生幼虫引入测定中。在含有多物种鳞翅目饮食(southlandproductsinc.，lakevillage，ar)的24孔盘中进行测定。将超声处理的裂解产物样品应用于饮食表面(饮食覆盖层)，并且允许蒸发且浸泡到饮食内。对于cew、faw、bcw、ecb和swcb，将125μl超声处理的裂解产物加入饮食表面并干燥。对于dbm，将50μl1:2稀释的超声处理的裂解产物加入饮食表面。将生物测定板用带针孔的平板密封膜密封。将板在26℃、65％相对湿度(rh)下在percival中以16:8白天:黑夜循环温育5天。评价测定中的死亡率水平、生长抑制和进食抑制。对于西方玉米根虫生物测定法，通过在24孔板(cellstar，24孔，greinerbio)的孔中的饮食顶部表面上分配60μl体积并且允许干燥，在昆虫生物测定中评估蛋白质构建体/裂解产物。每孔含有500μl饮食(marrone等人，1985)。使用精细尖端涂料刷在每个孔中引入十五至二十个新生幼虫，并且用膜(viewseal，greinerbioone)覆盖板。将生物测定贮存于环境温度下，并且在第4天时对死亡率和/或生长/进食抑制进行评分。对于科罗拉多马铃薯甲虫(cpb)，从马铃薯叶中切下8号木塞钻孔器大小的叶盘，并且浸入蛋白质构建物/裂解产物中直至彻底湿润，置于滤盘(millipore，玻璃纤维滤纸，13mm)之上。将60μldh2o加入每个滤盘中，并且置于24孔板(cellstar，24孔，greinerbioone)的每个孔中。允许叶盘干燥，并且使用精细尖端涂料刷在每个孔中引入五至七个一龄幼虫。将板用膜(viewseal，greinerbioone)覆盖，并且在膜的每个孔中穿孔。构建体用一式四份进行评估，并且在第3天时对死亡率和叶损害进行评分。表3提供了各种序列针对鞘翅目和鳞翅目的杀虫活性的概括。表格代码：“-”指示并未看到活性；“+”指示杀虫活性；“nt”指示未经测试；“s”指示发育迟缓；“ss”指示轻微发育迟缓；“lf”指示低进食，“m”指示死亡率。表3.针对鞘翅目和鳞翅目的杀虫活性概括。实施例4.针对半翅目的杀虫活性蛋白质表达：如实施例2中所述，将表4中阐述的每种序列在大肠杆菌中表达。接种400ml的lb并且生长至od600达到0.6。培养物在16℃下用0.25mmiptg诱导过夜。将细胞离心下来，并且将细胞团块重悬浮于5ml缓冲液中。重悬浮物在冰上超声处理2分钟。二龄sgsb从商业养虫室(benzonresearchinc.，carlisle，pa)获得。以超声处理的裂解产物样品与20％蔗糖的50％v/v比用于生物测定。将拉伸的石蜡膜用作进食膜，以使sgsb暴露于饮食/样品混合物。将板在25℃：21℃的16:8白天：黑夜循环于65％rh下温育5天。对于每个样品评分死亡率。结果在表4中阐述。虚线指示未检测到死亡率。表4中列出的蛋白质显示针对南绿蝽象25％的死亡率或50％的死亡率(如所示)(4只中的1只臭虫死亡)。阴性对照(表达结合结构域的空载体和仅缓冲液)两者均未显示死亡率(4只中有0只臭虫死亡)。表4.针对半翅目的杀虫活性概括apgseqid针对sgsb测试apg00078225％实施例5.大豆的转化将包含与在植物中有活性的启动子可操作连接的seqidno:1-224中的每一个或其活性变体或片段的dna构建体克隆到转化载体内，并且引入土壤杆菌属内，如于2015年12月18日提交的pct申请pct/us2015/066702中所述，其通过引用整体并入本文。接种前四天，将几个环的土壤杆菌划线接种到补充有合适抗生素**(壮观霉素、氯霉素和卡那霉素)的yep*培养基的新鲜板上。细菌在28℃下在黑暗中生长两天。两天后，在125ml锥形瓶中将几个环的细菌转移到3ml具有抗生素的yep液体培养基内。将烧瓶置于28℃下250rpm的旋转振荡器上过夜。接种前一天，将2-3ml过夜培养物转移至500ml锥形瓶中的具有抗生素的125mlyep内。将烧瓶置于28℃下250rpm的旋转振荡器上过夜。在接种之前，在od620处检查细菌培养物的od。0.8-1.0的od指示培养物处于对数期。培养物在oakridge管中以4000rpm离心10分钟。弃去上清液，并且将团块重悬浮于大豆感染培养基(si)体积中，以达到所需的od。将培养物伴随定期混合保持直至需要接种。接种前两天或三天，使用氯气将大豆种子表面灭菌。在通风橱中，将具有种子的培养皿置于盖打开的钟形罩中。将1.75ml12nhcl缓慢加入在钟形罩内的250ml锥形瓶中的100ml漂白剂中。将盖立即放在钟形罩的顶部上。使种子灭菌14-16小时(过夜)。将顶部从钟形罩上取下，并且替换培养皿的盖子。然后将表面灭菌的培养皿在层流中打开约30分钟，以散开任何剩余的氯气。将种子用无菌di水或大豆感染培养基(si)吸胀1-2天。将20-30粒种子在100x25mm培养皿中用液体覆盖，并且在24℃下在黑暗中温育。吸胀后，弃去不发芽的种子。子叶外植体在具有无菌滤纸的无菌纸板上进行加工，其中无菌滤纸采用美国专利号7,473,822(通过引用并入本文)的方法使用si培养基弄湿。通常，每个处理接种16-20片子叶。弃去用于保持外植体的si培养基，并且用25ml土壤杆菌属培养物(od620＝0.8-20)替换。在所有的外植体都浸没后，接种进行30分钟，伴随盘的周期旋转。30分钟后，去除土壤杆菌属培养物。通过将一片无菌纸覆盖在大豆共培养基(scc)上来制备共培养板。无需印迹，将接种的子叶近轴侧面朝下地在滤纸上培养。每块板上可以培养约20个外植体。将板用parafilm密封，并且在24℃和约120μmolesm-2s-1(在percival培养箱中)培养4-5天。共培养后，将子叶在具有200mg/l头孢噻肟和特美汀的25ml大豆洗涤培养基中洗涤3次。将子叶印迹到无菌滤纸上，然后转移到大豆芽诱导培养基(ssi)。外植体的结节末端略微压入培养基内，其中远端以约45度保持高于表面。在每块板上培养不超过10个外植体。用micropore带包裹板并且在24℃和约120μmolesm-2s-1在percival中培养。14天后将外植体转移到新鲜的ssi培养基中。弃去来自茎尖和子叶节的新芽。芽诱导在相同的条件下继续另外14天。在芽诱导4周后，将子叶从结节端分离，并且在芽诱导区(芽垫)下面进行平行切割。将平行切割的区域放置在大豆芽伸长培养基(sse)上，并且将外植体在24℃和约120μmolesm-2s-1下在percival中培养。这个步骤每两周重复一次，高达8周，只要芽继续伸长。当芽达到2-3cm的长度时，将它们转移到plantcon容器中的大豆生根培养基(sr)中，并且在相同的条件下温育2周，或者直到根达到约3-4cm的长度。这之后，将植物转移到土壤。注意，对于大豆转化提到的所有培养基都可见于paz等人(2010)agrobacterium-mediatedtransformationofsoybeanandrecoveryoftransgenicsoybeanplants；planttransformationfacilityofiowastateuniversity中，所述参考文献通过引用整体并入本文。(参见agron-www.agron.iastate.edu/ptf/protocol/soybean.pdf.)实施例6.玉蜀黍的转化玉蜀黍穗最好在授粉后8-12天收集。将胚从穗中分离出来，尺寸为0.8-1.5mm的那些胚优选用于转化。将胚以盾片侧面向上的方式放置在合适的温育培养基如dn62a5s培养基(3.98g/ln6盐；1ml/l(1000x原液)n6维生素；800mg/ll-天冬酰胺；100mg/l肌醇；1.4g/ll-脯氨酸；100mg/l酪蛋白氨基酸；50g/l蔗糖；1ml/l(1mg/ml原液)2,4-d)上。然而，除dn62a5s外的培养基和盐是合适的，并且是本领域已知的。胚在黑暗中在25℃下温育过夜。然而，将胚温育过夜本身并不是必要的。将所得到的外植体转移到网格正方形(30-40个/板)，转移到渗透培养基上约30-45分钟，然后转移到光束板(参见例如pct公开号wo/0138514和美国专利号5,240,842)。设计用于在植物细胞中表达本发明的grg蛋白质的dna构建体使用气溶胶光束加速器加速到植物组织内，使用基本上如pct公开号wo/0138514中所述的条件。光束照射后，将胚在渗透培养基上温育约30分钟，并且置于温育培养基上在黑暗中在25℃下过夜。为了避免过度损伤受光束照射的外植体，在转移至恢复培养基之前将它们温育至少24小时。然后将胚铺展到恢复期培养基上约5天，在25℃下在黑暗中，然后转移到选择培养基上。外植体在选择培养基中温育高达八周，这取决于所利用的特定选择的性质和特征。选择期后，将所得到的愈伤组织转移至胚成熟培养基，直至观察到成熟体细胞胚的形成。然后将得到的成熟体细胞胚置于弱光下，并且通过本领域已知的方法启动再生过程。使所得到的芽在生根培养基上生根，并且将所得到的植物转移到育苗盆中且作为转基因植物繁殖。实施例7.针对线虫的杀虫活性大豆孢囊线虫(heteroderaglycine)的体外测定将大豆孢囊线虫分配到96孔测定板内，每孔具有100ul总体积和100个j2。如seqidno:1-224中任何一个所述的感兴趣蛋白质被分配到孔内，并且保持在室温下用于评价。最后，分析含有scnj2的96孔板的运动性。数据表示为与对照相比的％抑制。命中被定义为大于或等于70％抑制。大豆孢囊线虫(大豆孢囊线虫)的植物上测定表达seqidno:1-224中的一个或多个的大豆植物如本文其他地方所述生成。3周龄的大豆切条用5000个scn卵/植物进行接种。这种感染保持70天，然后收获用于计数已在植物上发育的scn孢囊。数据表示为与对照相比的％抑制。命中被定义为大于或等于90％抑制。南方根结线虫(meloidogyneincognita)(根结线虫)体外测定将根结线虫分配到96孔测定板内，每孔具有100ul总体积和100个j2。包含seqidno:1-398中任何一个的感兴趣蛋白质被分配到孔内，并且保持在室温下用于评价。最后，分析含有rknj2的96孔板的运动性。数据表示为与对照相比的％抑制。命中被定义为大于或等于70％抑制。南方根结线虫(根结线虫)植物上测定表达seqidno:1-224中的一个或多个的大豆植物如本文其他地方所述生成。3周龄的大豆用5000个rkn卵/植物进行接种。这种感染保持70天，然后收获用于计数已在植物中发育的rkn卵。数据表示为与对照相比的％抑制。命中被定义为大于或等于90％抑制。实施例8.关于杀虫活性的进一步测定可以以多种方式测试seqidno:1-224中所示的各种多肽充当害虫的杀虫剂。一种这样的方法是执行进食测定。在这样的进食测定中，将害虫暴露于含有待测试的化合物的样品或对照样品。通常这通过将待测试的材料或这种材料的适当稀释物放置在害虫将摄取的材料例如人造饮食上执行。待测试的材料可以由液体、固体或浆料组成。待测试的材料可以放置在表面上，然后使其干燥。可替代地，待测试的材料可以与熔化的人造饮食混合，然后分配到测定室内。测定室可以是例如杯子、盘或微量滴定板的孔。用于刺吸式害虫(例如蚜虫)的测定可能涉及通过分隔物(理想的是可被刺吸式昆虫的吸式口器刺穿的部分)使测试材料与昆虫隔开，以允许测试材料的摄取。通常将测试物质与进食刺激剂(如蔗糖)混合，以促进测试化合物的摄取。其他类型的测定可以包括将测试物质显微注射到害虫的口腔或肠内，以及转基因植物的开发，随后测试害虫以转基因植物为食的能力。植物测试可能涉及到正常消耗的植物部分的分离，例如，附着到叶上的小笼，或者在含有昆虫的笼中分离整个植物。测定害虫的其他方法和途径是本领域已知的，并且可见于例如robertson和preisler编辑(1992)pesticidebioassayswitharthropods，crc，bocaraton，fla中。可替代地，测定通常在期刊arthropodmanagementtests和journalofeconomicentomology中或通过与美国昆虫学会(entomologicalsocietyofamerica)(esa)会员的讨论进行描述。可以表达seqidno:1-224中任何一个，并且用于如本文实施例3和4中所述的测定中。说明书中提及的所有出版物和专利申请指示本发明所属领域的技术人员的技术水平。所有的出版物和专利申请都通过引用并入本文，其程度与每个个别出版物或专利申请被特别且个别地指出通过引用并入相同。尽管为清楚理解起见，前述发明已通过举例说明和实施例的方式较为详细地进行了描述，但显而易见的是，可以在所附权利要求书的范围内实施某些改变和修改。序列表<110>agbiome,inc.<120>杀虫基因和使用方法<130>9686991010353<150>us62/182,855<151>2015-06-22<160>224<170>patentinversion3.5<210>1<211>724<212>prt<213>芽孢杆菌属的种(bacillussp.)<400>1leuserileileargilearglysmetasnlyslysileserarggly151015argleucysasnmetgluleulysvalasnaspilegluilealaser202530asnasplysglytyrserhisaspserserileglytyrserasnpro354045asntrpmetasnlysmetproglythrleulysilesergluleuser505560ileproglythrhisasnsermetserargtyrglyglyglyglyleu65707580tyrproaspasptyrilelysthrglnthrmetaspvalasnthrgln859095leuasnserglyileargtyrleuaspileargleuarglysgluser100105110asnalaleuleulysalatyrhisglyservalasnglnasnalaile115120125pheserseraspileleuasplysilethralapheleuargthrasn130135140proglygluthrilephemetargvallysasnglucysasnglypro145150155160glythrglylyscysthraspalaserthrthrlysthrtrpalagln165170175ilephegluaspasntyrtyrasnasnalasertyrserasntyrphe180185190trplysglyalaserasnasnprothrleuasngluvallysglylys195200205ilevalvalmetserasppheprothraspglylysargpheglyile210215220protyrasnthrleuasnlysglnaspglnpheaspvalaspaspser225230235240alaaspalamettyralalystrpthralavalglyasnhisleulys245250255alaalaasnalaserasplysglnseriletyrleuasntyrleuser260265270glyasnglyalatrpargpheilesermetglyalaalaprotyrphe275280285valalaserglytyrargglyserglythrasnglusermetlysmet290295300ilethrgluhisargthrasplystrpproasptyrproargglytyr305310315320tyrglyglnvalphetyrglyglythrasnleuleuthrthrglntyr325330335ileglnglntrpproilearghisthrglyileilealaalaaspphe340345350proglylysglyleuileaspargvalileargleuasngluarghis355360365asnserglyaspvalargtyrilethrprolysglyserserglyleu370375380argvalglypheglyglyaspalatyrleuargasnsertyrvalval385390395400tyrargasnglythrtyrileglyhisvalaspserglyserprotyr405410415tyrsertyrtrpasplysthraspvalglyhisaspleuargpheglu420425430glyleualaleuphethrglyasplysileaspvaltyrvallysasn435440445glyglyserserth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1>258<212>prt<213>链霉菌属的种(streptomycessp.)<400>13valthrleuileseralaleualaargproproalaaspprovalgly151015aspglyglyasnglyaspaspargthrproglyhisargargglyser202530proglythrvalvalserglyproserserseralaargargserleu354045arghisthraspproserleuileasnargleuphelysgluaspser505560leualaalaserphelysthrvalilegluvalglyproalahisleu65707580aspglnalaglnalaleuglyargalapheglnaspalailealapro859095alathrvalasntyrasppheasphisileargaspalaalaalaala100105110ileproaspserserilevallysmetvalargglytrpglyleugln115120125gluhisalaprovalleuvalmetvalleuserleulysglualaval130135140argglnalaleuproprogluseralaglualaserphetrpaspthr145150155160valgluarggluleuvalaspalaphethrglyleualaalaglnglu165170175glyleuproglyleusertyrtyrglugluglyalaaspargthrarg180185190tyrtyrargaspleuphephealaleuglnaspglugluthrglygly195200205hismettyralailealaphecysileaspvalthrileglyleuasp210215220lysalaargvalglyalaleuglumetthraspalavalpropheala225230235240leuargleuasnalailevalleuargglngluleuthrargalaleu245250255thrala<210>14<211>304<212>prt<213>芽孢杆菌属的种<400>14metthraspthrlysmetthrileglutyrleuphethrprotyrtyr151015glyvalasnproasnglyserasnileasnprotyrleuleuasnala202530asnhistrpasnservalmetcystyrpheasntyrileserprotyr354045thrgluileargasnargaspthrleuglylysleuleutyrserser505560valasnglnglnglnalaphetyrargphepheaspvaltyrasnleu65707580glnaspvalpheglnproilepheaspleuhisargthrileserasn859095alaserthrtrptrpthrileglnglyhisglnglnilethrleuleu100105110valglnaspileargserphethrasnileleucysvalarghisile115120125asplysleuasnglnileilelysaspvalglnlysgluargvalser130135140thrlysthrlysaspglnphethraspilecysilegluleuglnala145150155160argthrserglytyrtyrglnargleuaspileleuaspglnserlys165170175lysphevalasnileleuleulystyrasnproalailehisthrthr180185190leuasnhisleuglulysiletyrseraspsergluphelysalaval195200205asplyshistyrphelyslysleuleuserphevalarglysglngln210215220ilephevalglntyrtyrthrtyrthrileglythrleuglnalaleu225230235240thrasnleuargglyiletrpthrglutyrglyleuasnleuglnlys245250255leuileglualacysaspasnasnleuvaltyrpheasnserleuleu260265270alaargleulysleugluasnalaileasnalatrpglnaspvallys275280285lyslysalaaspargpheleuglnthrtrpglnaspaspthrargcys290295300<210>15<211>391<212>prt<213>芽孢杆菌属的种<400>15metlysservalserlyslysvalmetthrglyleuphealaglyala151015metglyleuserilephealaproalaserglnalaalathrproglu202530glnasnglutyrtyrasnilelysleulysalaasnthrtyrlysile354045trpaspvalaspasnalaserseraspasnaspargvalileargleu505560trpglyglyserthrthrglualaasnargglnphevalphephepro65707580leuaspglyglyalatyralailevalasnlysasnserglylyspro859095valthrpheglyasnglyalatrppheserileasnglyglyserser100105110ileleualaglyalaaspileleuglnglnglnsertrpthrglyser115120125alaasnglnglntrptyrleuargaspglnglyasnasnasntyrglu130135140ilevalasnglnglyasnglylysvalalasertyralatrpglygly145150155160thrleualaglnasntrpglutyrvalaspleuaspgluserlyspro165170175tyraspserasnasnvalpheasnilealaasnserglyserserile180185190serleuproasnleuproalalysglyaspargproasnalaproasp195200205tyrasnglnthrglyargileaspglnglnleuproglnthrserglu210215220servalvalileglyalaserleuileproserilemetvallysasp225230235240asnseralaserasptyrthrlysilehisasnserprotyrtyrval245250255leuglulysgluglutyrtrpglulysvalargsergluilevalpro260265270alaglyglyserserlystyrthrvallysargglyvalserthrval275280285aspglnglnlysmetthraspthrleuasnmetlyspheglythrasp290295300leuglyphelyspheglyaspthrthralaserilelyssergluile305310315320serlysthrleuglnthrgluvalserlysthrserthraspalathr325330335glugluthrlysglulysglypheserserthrserglylysaspthr340345350glyleuthrvaltyrglnleuvalthrlystyrthrleulysargthr355360365aspglyserthrvalsersersertyrvalargaspglyaspglnthr370375380leugluargthrilealalys385390<210>16<211>325<212>prt<213>乳球菌属的种(lactococcussp.)<400>16metlyslysasnlyslystyrvalmetvalilesercysargleuleu151015ileleuserpheglymetvalprolysasnvalleualaleuasngln202530glyglyalaglyasnserglyserglytyrleuvalasnasnmetser354045glulyshisgluaspileileaspprogluaspilepheserasnmet505560leuasnalavalglylystyrlysgluhislysasppropheleuphe65707580valthrargasnserileglyserleusersertyrserasnpheasn859095alagluglnaspglyaspproileilegluasngluserleuleuphe100105110valglygluserileleuthrasnasnmetasnthrgluglnthrleu115120125lysthrasnserpheserlysthrmetserasnthrvalsersergln130135140valthrhisglyphemetleuglythrglualaargthrserphegly145150155160ileproleuileglygluthrgluleuserleulysalaglutyrasn165170175phesersersergluthrasngluileserglusertyrthrtyrgly180185190valgluproglngluileleuvalproprohisserservallysval195200205argvalaspleuasnthralaaspileserglylysvallysleumet210215220alathrprolysthrasppheglyaspileaspglytyrphehistyr225230235240glyproaspgluglnasnilealagluserileprophethrtyrasn245250255trpasnthrleuthrlysgluvalargglnleuasnleuprovalasp260265270aspileasptyrasppheasnasnglnthrprolysleuvalglyser275280285glylystyrlysalalystyrglythrthrphelysvalgluvalile290295300proileasnasnprolysalagluglytyrsertyrserileglnpro305310315320thrhisthralaasn325<210>17<211>546<212>prt<213>芽孢杆菌属的种<400>17metlysglulyslysasnileglngluargileglyaspasnvallys151015tyrasngluarglyslysalalysargasntyrlyslysalavalleu202530thralaalathralapheserileglyvalthrserileglyglyala354045thrseralaphealaalaglulysproproalaglnglnglnprogln505560serservallysservalglyglyalaaspaspvaltyrserasnglu65707580ileasnasnproprophelysilegluglyaspalaleuthrleuthr859095lysasnpheleuserglyaspleualaalathralalyslysmettrp100105110glyserserleuasnleualavalglnseralaglnglylysasnval115120125leuasnvalasnasnleualaargaspmetvalmetvalglythrser130135140alaileprotyrglyglyvalileleuserserileileglyvalleu145150155160trpprometserglythrasnglnmetglnalaleumetasplysmet165170175glyglnilemetaspglnlysileglulystyraspasnthrthrleu180185190thrserlysileasnalavalgluvalalaleulysargphegluthr195200205ileleutyrglythraspaspvaltyrseraspthrasnlysalagly210215220glualaleuargglnalathrvalileglnasnalaphethrgluleu225230235240ileasngluserlyslysgluglutyrgluthrsergluleuproala245250255tyrthrmetalaalathralahisleuleupheleuserphemetval260265270thrasnglythrserileleuglyleugluproaspthrmetlysasn275280285sertyrasnasnaspleulysasnalaleuserglutyrasnserhis290295300ileglnargileglyseraspserasptyrlysleuserglnleupro305310315320glnlysileaspalaalaargvalgluileglulysalalysalagly325330335metasnproproileglyalaalaalaglytyrtrpalagluiletyr340345350glnglnlysserglngluleuhisasnleuleugluglnglnlysgln355360365leuthraspleuvalasnserthrthrlysasnglualaphealaala370375380alaleulysseralalysthrthralaaspasnlysglyilelystyr385390395400gluglulysaspserserlysilevalaspglyglythrtyrlysile405410415ileseralaleuasnasnglyglntyrvalaspasnglnarggluthr420425430lysasnvalseriletyrhisasnthrargseraspasnglnasntrp435440445lyspheilepheaspserserlysglnalatyrlysileglnserval450455460tyraspserargvalleualathrserserseralaasntyrasnval465470475480phethrtyrasnpheaspglysergluaspglnphetrpileproglu485490495aspileglyasnglythrtyrilephelysasnlyslystyrproasp500505510argvalleuaspilephegluglylysalagluaspserthrasnile515520525thralatyrproargasnasnglyalaglyglnlysphelysvalile530535540glngln545<210>18<211>432<212>prt<213>芽孢杆菌属的种<400>18leuvalleuserphetyrilealalysvalileleupheargasnlys151015serileserasnvalglntyrasnpheilephecysasnleuvalgly202530ileleuprothrlysleuglyglyasnilemetlysserileserlys354045lysvalmetalaglyleuglyvalglyalametserleuseriletrp505560alaproserserglnalaalaalaproalaasnasnglutyrhisthr65707580ilehisleualaalaasnthrasnilealatrpaspvaltyrlysgly859095trpglnthraspaspalaglyilevalleutrpaspglyvalarggly100105110aspasngluglnphevalphepheproleuaspglyglyalatyrala115120125ilevalasnlysasnserglylysprovalglyileglyseralaile130135140ileglyseraspglyvalargasnpheprosertyrasnasnglngly145150155160leuargglnthrsertrpthrglyalaproasngluglntrptyrleu165170175argasplysglyasnasnasnphegluilevalasnglnglysergly180185190lysvalalasertrpalaglyvalglumettyrglyphelysasptyr195200205valaspleuaspgluserasnproserasplysaspargleupheasn210215220ileproalaalaargserthrpheserleuprothrleuprothrval225230235240glythrargproasnalaproasptyrasnthrserglyalaileasp245250255glnglnleuproglnthrserasnservalvalvalglyalaserleu260265270ileproserilemetvallysaspasnglyalaserasptyrthrlys275280285ilehisasnserprotyrtyrvalleuvallysgluglutyrtrpglu290295300lysvalargsergluileileproalaglyalathrserlystyrthr305310315320vallysserglyvalsergluileaspglnlyslysmetseraspthr325330335leualametserpheglyseraspleuglyleulyspheglygluser340345350serleuserilelysserservalserargthrileglnthrgluval355360365thrlysthrserthrasnalathrglugluthrgluglulysserile370375380proserileproglylysasnthrglyphethralatyrglnleuval385390395400thrlystyrthrleulysargthraspglyservalvalthrasppro405410415serilevalargaspserlysglnthrilealaargthrilealalys420425430<210>19<211>420<212>prt<213>芽孢杆菌属的种<400>19valileleupheargasnthrserileserasnvalglncysthrile151015ilephecysasnleuvalglyileleuprothrlysleuglyglyasn202530ilemetlysserileserlyslysilemetalaglyleuglyvalgly354045alametserleuseriletrpalaproserserglualaalaalapro505560alalysasnglutyrhisthrilehisleualaalaasnthrasnleu65707580alatrpaspvalaspasnglyservalaspasnaspargalaileleu859095leutrpasnglyaspargasnaspasngluglnpheleuphephepro100105110leuaspglyglysertyralailevalasnlysasnserglylyspro115120125valthrpheglyglyprothrtrpserglyserglyglyileleugly130135140asnglnasnleuasnglnargsertrpthrglyserserasnglugln145150155160trptyrleuargaspargglyasnasnasntyrgluilevalasngln165170175glyasnglylysvalalasertyralatyrglntrpalaserarghis180185190ilegluhisvalaspleuaspgluserasnproseraspasnasparg195200205leuphehisileproalaalaalaargserthrpheserleuprothr210215220leuproalavalglythrargprovalalaproglutyrthrglngly225230235240glyserileaspglnglnleuproglnthrserasnservalvalval245250255glyalaserleuvalprocysilemetvallysaspserglnalaser260265270asptyrlyslysilehisasnserprotyrtyrthrleuvallysglu275280285glutyrtrpasplysthrpheseralavalileproalaglyleuthr290295300argasntyrthrphelysthrglymetthrservalaspglnglnlys305310315320metthraspthrleusermetlysileglyalaasppheglyleulys325330335pheglyaspalaseralaserilelyssergluileserlysthrile340345350glnthrgluileserthrthrasnthrglualathrglugluthrile355360365thrserthrvalthrsergluleuglylysthrthrglyphethrglu370375380tyrglnleualathrlystyrthrleulysargalaaspglyserval385390395400valseraspprotrpvalvallysasnasnlysilethrvalalaarg405410415lysasnserile420<210>20<211>301<212>prt<213>芽孢杆菌属的种<400>20leuargglylysphemetlyslysleumetpheserleuvalalathr151015thrilesermetglyleuileleuglyseralaproalalysalaasp202530valserasnlysasnseralatyrglnaspileglygluargvallys354045lysmetalaglnseralaalatrpglyglyglnglutyrargserhis505560asnilelysaspilegluleulysglyasnleuileaspglysermet65707580valgluasnserglnvalleuthrvalserseraspvalleugluasn859095asnleuglyhisthrvalasnmetproserthrglytyrgluhisglu100105110pheglugluthrthrserthrthrasnthrserglytrplysphegly115120125tyrasntyrasnalaserpheservalleumetvalseralasergln130135140serpheservalglutyraspmetserthrseraspthrhisgluthr145150155160lysglulysarglysphethrvalproserilegluvalprovalpro165170175alaglylyslystyrlysvalglutyrvalpheglulysvallysval180185190serglylysasnlysileaspalaserleupheglyaspvalthrtyr195200205tyrtyrasnasnglnprometserproglnleuleutyrservalgln210215220glyleualathrasplysglnglyphegluglnvalileargaspser225230235240alaileglyasnaspargpheglyilelysthrthrglyileglygln245250255pheserthrglupheglythrargleuthrargthrleuthraspile260265270thraspalaargasnprovallysleugluthrlysasnileproval275280285gluphelysthrleuserileaspthrargvalilelys290295300<210>21<211>585<212>prt<213>芽孢杆菌属的种<400>21metlysprolysserglugluthrglnproproserprolystrpser151015lysserglutyrasnasntyrargasnglytyralatyrasnalaasn202530ileglytyrgluasnlysasntrpmetasnalaleuargglygluthr354045lysileserglnleuserileproglythrhisglyserthralaleu505560hisglyileproglyalaaspargtrpileileasnglnargmetser65707580ilethrthrglnleuasnserglyileargtyrleuaspileargala859095argargthrglythrserphealamethishisglyglnalatyrgln100105110lyslysmetpheglyaspileleuasngluvalthralapheleuarg115120125glnhisproglygluthrvalleumetargleulysglugluhisthr130135140progluaspglyserargserpheglugluileleulysglytyrtrp145150155160aspasnprosertyrlysasntyrphetrpglnproalaglysergly165170175asnglyserglyvalasnalaasnprogluleulysaspvalarggly180185190lysilevalleuleuglnglnphealavalserlysalatyrglyile195200205asntyrtrpasnleuglulysglnasphisaspasnvalargpheser210215220metaspglymettyralalystrpthralavallysasnhisleuile225230235240lysalaasnseraspglnasnlysiletyrleuasnhisleusergly245250255asnglyglyproserglyglyalaalaprogluphevalalaserala260265270tyrglyglyaspglygluproleumetglythrglyserprosergln275280285tyrproasppheprothrgluasntyrargleutyrpheglyglymet290295300asnilemetthrasnglntyrileproargserglyleuglntyrthr305310315320glyilevalalaalaasppheproglylysglyleuvalgluargval325330335ilelysleuasnasplyshisileaspvalseralailehishismet340345350glyleuasnalaglnlyslysvalgluileglyphethrglyasntyr355360365leuserasnasntyrvalilethrlysasnglyglutyrilealahis370375380tyrasplysglythralatyrtyralatyrleuserlysthrasptyr385390395400glyhisleuleuthrhisaspaspglytyrserphepheasnglyasp405410415lysilealavalserleugluhisserasnglyalaargthrleuleu420425430lysgluphethrvalasnileaspgluasnasnleuglugluvallys435440445ileproaspglylystyrasnilevalseralaleuasnthrserlys450455460valalaaspmetthrglyasnasnasnvalvalleutyrthrlyshis465470475480glyasplysasnglnglntrplysphevaltyraspalaasnlysgln485490495sertyrglnileargserglutrpasnthrasnvalalaleusertyr500505510valserglyserglyasnvalleuglygluserileasnthrthrlys515520525glugluglntyrtrplysleuargargalaglyasnglytyrleutyr530535540valgluasnlyslysasnglytrpvalleuaspvalgluargserser545550555560thrseraspglythrasnileileleuphevalargthrglythrthr565570575asnglnlysphelysleualaargser580585<210>22<211>348<212>prt<213>芽孢杆菌属的种<400>22metlysthrlysilelysleuproasnlyslysleumetalalysser151015serileilealaleualathrserphealaleuileserproaspleu202530valasnalaaspvalileasnleulyslysglyphegluglnglnlys354045glnilealaalaaspvalaspproileprovalproilegluasnval505560lysglyleuglnaspleutrpleuaspserlysglumetlyscyspro65707580leualaaspleuileglythralaprohisasnleuserasnleuhis859095pheasnpheleutyrtyrlysgluprosertyrservalaspvalile100105110lyslysasngluglulysproserserprovaltyrmetglythrthr115120125asnleuserhisserglyglythrsergluglnthrleutyrsergln130135140alapheserlysthrvalthrserservalthrthrthrilethrhis145150155160glyalalysvalglyalalysalaglyalalysleuglnvalproleu165170175valglygluthrservalgluleuasnalaglutyrasnphealalys180185190alaglysergluthrglythrgluasnvalthrtyrthrvalproser195200205glnprovallysleuglnproglygluseralalysilethralaasn210215220leuargmetalalysalaserglygluvalargleuargthrthrtyr225230235240thrglygluileglnserasnphephevalasnthrprotyrglypro245250255metgluaspseraspthrthrglyleuglyglutrpmetlysthrleu260265270valasppheasngluaspleulysasnglntrpthrpheserprothr275280285tyralaservalalatyrglnaspglylysglythrtyrasnalathr290295300tyrglyalavalleuaspilehisvalaspilepheglyglyserasn305310315320lysglyarglyslysargaspalaaspserthrproileargsertyr325330335sertyrgluvalthrprogluilevalgluasnasn340345<210>23<211>291<212>prt<213>芽孢杆菌属的种<400>23metalailealaaspleuaspalatyrleuilealaleuglylyslys151015ileglypheproserilethrglyvalasnthrserasnleuargval202530gluasnalaglusertyrglyphegluilelysaspserlysprogln354045glythrmetphemetglyglusergluleulysasnaspthraspgln505560thrglnthrilelysseraspserphethrlysthrilethraspser65707580valthrleuservalthrasnglyvalthrthrglyvalservalthr859095ileserglylysilepheglyileglyalaglusersermetserphe100105110gluvalserthrserthrthrasngluglnthrserglugluserval115120125alatyrthrvalproserglnasnvalleuvalproserarglysthr130135140tyrtyrvaltyrthrserleuglnargserglnleugluglythrile145150155160argleulysalaaspleuserasnhispheserphelysthrsergly165170175asnvalserproalaleumetasniletyraspphevalasnhistyr180185190glnserleuhisproleuproserglyileserleuasnhislysasp195200205lysserilehisphegluglyvalalaglutyriletyrglythrgly210215220thrlysphetyrvalthrileserasplysproserserglnglythr225230235240glngluhislysalapheaspvallysthrglyleuglythrtyrala245250255ileargleuaspglylysargpheaspleuthrleuasnaspleulys260265270asphisleugluprolysasppheglulysilemetgluleuglnasn275280285gluileval290<210>24<211>288<212>prt<213>芽孢杆菌属的种<400>24metseriletyrasnvalglualaaspleupheaspleucysargglu151015tyralailelyshistyraspalaasnaspalathrphehisglyala202530thriletyraspargservalleuasnsergluvalmetproaspgln354045valglupheasnvalileproaspleuserasnthrvalileasnthr505560ilethrasnalathrserilegluglnsermetserthrlysleuser65707580thrlysthrilegluthrthrserphethrthrthrgluglytyrlys859095valglythrserilelysasnthrleugluphelysalaglyglyleu100105110pheserileglyglyvalgluhislysphegluvalservalsergly115120125glutyrasnhissersersergluthrthrthrasnthrthrglulys130135140leutrpglutyrthralaproilelysvalproprolysthrlysval145150155160gluglylysileglniletyrserglyalaileprovalprovalthr165170175leulysserthrvalleuglyleuglyglnserproasnglylysleu180185190glnasnalaleuthrserleutrptyrlysasplysalaglylysglu195200205trpvalaspthrtyrglnthrserhisleutyrthrleuarggluglu210215220trpserglytyrlysproiletyrvalglyseraspglyasnglyval225230235240lysleuglnglylysglythrvalglyleuaspleuglyleutyrala245250255valvalthrphelysglugluproleuproglyasntyrsermetgly260265270asngluargthrtyrserlysvalilepheargaspglyargvalile275280285<210>25<211>332<212>prt<213>芽孢杆菌属的种<400>25metlysarglysasnasnargasnlysserleuleuilevalalathr151015ileserthrileglyleuthrglyileserserthrproalapheala202530alagluthrproalaileasnserlysalalysglusertyrtyrser354045glythrileaspleumetpheileasnglupheasnlysasncysleu505560serileaspasnasnasnserargthrileilelysaspleulyspro65707580alaarggluglnthrgluserileaspserphegluvalaspasnser859095asnserasnserthrargilephelysserglnglnlysthrtyrlys100105110asnthraspthrvalthrvalthrhisalalysglupheserphegly115120125glngluileseralagluleuglyalaprolysleuleugluasnleu130135140leuasnmetalalysileasnleulysalathrpheglyglnlysile145150155160thrasnserlysalaglualalysserglngluglnserthrthrtyr165170175glyglyaspasnilegluvallysproglyglnlysvallysvalglu180185190tyrglutyrilethrasniletyrserglythrleugluthrleuala195200205proleulystyrasnvallysaspprosermetsertyrvalserpro210215220lysaspileprotyrtyrlyslysthrleuprotyrvalalalysleu225230235240serglyglylyslystyrserthrasptyrgluphephelysaspval245250255hisasnthrproaspasptrpalavalleuilealatyrasplysgly260265270aspalatyrphephetyralatrpgluleulysaspthrthrthrleu275280285asnaspasnlyslysglnvaltyrserlysglyseralalyspheglu290295300alavalargglythrglyvallysvallysvalthraspleuaspthr305310315320glylysilevallysasplysvalvalsertyrphe325330<210>26<211>595<212>prt<213>芽孢杆菌属的种<400>26metlystyrlysasparglysaspalalysarglystyrlysglnala151015leuleualathrvalalathrmetthrleuglyvalserthrleugly202530serthralaseralaphealagluglulysasplysasnilealagln354045glnglyalagluglnglygluproalaglulysaspthrthralathr505560glnglnglnasnthrvalglnglnlysasnproglythrtyrtyrglu65707580aspglulysleuasnproilealaglnleuasplystrpthrglnasp859095leuglylysasnthrglyalaalaasntyrlysthrserleuserala100105110valglulysilemetproleuleuilelysaspilethrlysaspasn115120125tyrasnasnserleuargalavalthrmetpheglyalaalaleuile130135140protyrglyglyserpheileserproleuileglyleuiletrppro145150155160gluserlysaspaspalaalaleulysleulyslysglnleuaspasp165170175leuarglysgluleuseraspleumetaspglulysileaspargphe180185190aspglngluglyileglnalaalaalaasnvalileasngluasnleu195200205lyslysleugluaspglyilealaglylysthrproglyilephetyr210215220aspasnglnlyslysleugluaspglyilealaglylysserglugly225230235240serphetyraspserglnserglyphethrvalvaltyrserileasn245250255seralaphegluleuleuleualaasnalaserlysproserphelys260265270metglngluleuproleutyrthrglyleualaalaalahisleugln275280285pheleugluphevallysglnasnglylysasnprolysilealaleu290295300thrasngluasntyrasnthrphevalasnlysilealaglulysthr305310315320provaltyrthrglnhisilegluaspthrtyrasnglnpheargser325330335lysilegluglnlysmetthrproleuleuglnglyvalileglyval340345350thrseraspgluglnglnasnleuglyalaalapheasplysthrile355360365gluleuglnarglysvalvalglnilethrglyglyaspargleugly370375380ileasnserlysasplysglnlysleuglnthrpheileaspasnlys385390395400gluasntyrlyslysleumetgluthrlysserasptyrtyrasnser405410415thrvalglyleuphealavalthrglnglyileproserlysilegln420425430lysprogluthrthrprothrtrpglyproleulysthrleuglngly435440445provalalaasnglythrtyrlysilealaserlysleuaspserasn450455460metleuleuaspasnasphisasnargasnargleualathrleuasn465470475480lysaspilethrthrlysaspasnleutrpglyleuglytrpgluleu485490495argtyrasnproasplysglnalatyrlyspheileserlysleugly500505510seraspgluleualathrasnglyglnaspaspthrvalphealathr515520525thraspargasnaspasnaspleuargtyrtrpthrleugluproala530535540alagluaspglytyrphepheilelysasnlysalaserglyargval545550555560leuaspvalthrglyglyasnthralaalaglythraspilelysthr565570575asnvalglnasnthrproalavalalaalaglnleuphelysleuglu580585590thrvalgln595<210>27<211>303<212>prt<213>赖氨酸芽孢杆菌属的种(lysinibacillussp.)<400>27metargglutyrphemetlyslysleuthrleualathrleualathr151015phemetilevalglyleuilepheglyalavalproalagluvallys202530alasergluhisglylysasnasnvaltyrglnaspleuaspgluarg354045vallysargmetleuileseralaalaalaproglythrglutyrarg505560tyrhisasnileilehisalagluleuasnglyglyaspvalglugly65707580thrleuilegluasnsergluthrleuthrvalserargasnthrleu859095gluasnasnpheasphisgluilegluleuprothrserglytyrglu100105110hisgluphethrgluthrthrserthrthrasnserthrglytrpthr115120125pheglytyrasntyrasnvalgluileserileprometvalsergly130135140glyhisserpheservalasptyrasnmetserthralaasnthrval145150155160glulyssergluthrargargpheservalproservalleuilepro165170175valproalaglyarglystyrleuvalglutyrargpheglulysgln180185190thrvalserglyargasnargileasnalaaspleupheglyaspala195200205valtyrtyrpheasnasnglnprometserproglnleuleutyrser210215220alaleuargphealathraspthrglnglyphegluglnvalilearg225230235240aspseralavalglyasnaspargpheglyileargglythrglyval245250255glyglnpheargthrglupheglythrserleutyrileargthrthr260265270aspilethrasnserglngluprovalileilegluthrilethrile275280285provallysilegluthrileserglulysthrlysvalvalglu290295300<210>28<211>341<212>prt<213>芽孢杆菌属的种<400>28metargtyrlystyrlysilelyshishisasnlyslyslysglyala151015ileasnmetlyslysasnlyslyslysglnileleuserleualaile202530leuthrserileglyalasermetthrleualaserproglyserala354045seralaaspglnileserproileglnlysilethrglngluglngln505560asnvalalaileserasptrpargglupropheglnlysthrtyrlys65707580seralalysleuargaspproasnargtyrpheserasnglualalys859095ileaspleutrpglyvaltyrglnserservalglualaaspglyser100105110prothrilethrasnserlysserleuphevalglyargthrileleu115120125thrasnhisthrasnglnalaglnthrleuthrthrasnglupheser130135140lysserpheglyasnservalthrasnserthrthrasnglypheasn145150155160leuglyvalserthrseralaserpheglyileproleuileglyglu165170175thrglyilegluileserthrglutyrasnpheserasnthralaglu180185190lysthrlysthrglusertyrmettyrthralaserproglnasnile195200205leuvalproalaasnserservalgluvalilevalasnleuasnthr210215220alalysleuasnglyasnvallysleuleuserhisvalasnglygln225230235240leumettrpasnthrserlysthrglyglnlysileserthrserphe245250255lysglypheleuglulysalaalaargproasngluilealaserasn260265270vallysalahisproargasnglyalatyrleuileglylysglylys275280285tyrseralaglutyrglythrglupheservalthrvalargproval290295300glnlysproserthrvalalaalalysserlyssermetvalserglu305310315320hisserlysthrthrserglusertyrthrtyrthrvallysproglu325330335vallyslysglugln340<210>29<211>380<212>prt<213>芽孢杆菌属的种<400>29metleuglnasnlysasnlysleuphephethrglyileseralaleu151015thrilemetthrserglyleuthrileserthrprothralaserala202530asnthrglngluserthrproservaltyrproileglnglythrglu354045pheseraspgluasnglyglnvalileserproglyalaphealathr505560leuserprotyrtrpleuileasnaspserasnaspglnleuleuasn65707580phetyrasnglnvallysglnargleuleuasplysglnasnthrglu859095glnasnmetleulysleuaspglutyrleuasnaspleutrppheval100105110glyalavalsergluleutyralathrprothrasptyrasnlysarg115120125leuglualaserglnleuvalargtrptyrlysasnasnasnalaala130135140argprothrphegluileleugluserlysleugluasnvalglupro145150155160ileleuaspilethrgluproileglytyrphealathrgluserglu165170175tyrthrasnasnserprohisglnglyglutyrleuileproglytrp180185190serasnthrthrserthrthrtyrasnthrthrthrthrlysglyphe195200205serileglyalaasnthrglyilegluileproleuprophemetasp210215220lysleuglyleuthrglysertyrasnpheserlysalagluasnval225230235240serthrthraspserthrthrleuthrileprovalglnthrilegln245250255valserpropheserhisserlysthrserphetyrpheglnthrhis260265270glntyrargalalyspheaspvalthrserlysphelysalaaspval275280285glyargaspalaglutyrthrlyslysargseralaphetyrleuleu290295300alalystyrasnglutyrleuprolysglyilevalvalgluasngly305310315320glylysleuthrleugluglylysglygluileservalproasnhis325330335ileasnglyphelysleuvallysgluthrlysgluthrproleuleu340345350glyglygluglylysthrthrlysgluiletyrthrlysaspproasn355360365thrasnlysleuileproaspalaserserlysser370375380<210>30<211>257<212>prt<213>芽孢杆菌属的种<400>30metaspleuserhisleuasnleusergluvalsergluglyilelys151015lysleuglualaseraspphetyrmetasnglntyrleugluleutyr202530aspargproalahisleuvalileileserglnasnvalsergluser354045ileaspproasntyrglnsermetsertyrleuproalaasnglyala505560aspaspthralapheaspleuleulyslyspheaspproalavalval65707580thrserproalaproproglyglyasnserglyleumetmetaspmet859095alavalalaglnlysilealalysserleulystyrglyvalileglu100105110glyvalaspilelysserlysglnserasnservallysilealaasp115120125hisilethralavaltyrasnmetileserserleuvalthrthrval130135140alaseraspservalserglyphethrserileleulysalaglyile145150155160thrasnleuphethraspileglylysgluvalaspasnglyalagly165170175glyilelyslysargtrpalavalvalthrtyrasplysserasnglu180185190leusertyrthrtyrasnglnmetphealatyrproasnthrasngly195200205lystyrileglymetmetleumetserthrglnthrlysilethrser210215220asnserleuaspleuleuileleulysprotyraspilealathrval225230235240asnserthrthrasnileileargleuvalhislysileglyproval245250255glu<210>31<211>947<212>prt<213>芽孢杆菌属的种<400>31metilemetglnmetvalthrlysalametasnprolysleuglyala151015leuproasppheileaspasppheasnglyilepheglyphemetgly202530asnilethraspvalmetserthrilepheglyvalaspthrglyasp354045serserileaspaspvalleuasnasnglugluleuleulysglnmet505560metgluglnmetaspthrmetglnaspserleugluaspileleulys65707580asnglngluileserlysasnileglngluglnileleualaleuthr859095thraspleualathrserileasnlysgluleuvallysileglugly100105110ileleuthrthrtyrleuasnalai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ngluglyargprogly225230235240leuserphetyrglugluserglyaspargthrargphetyrargasp245250255leuphephealaleuglnaspglugluthrglyasphismettyrala260265270leualaphecysvalaspvalthrvalgluleuprolysalagluthr275280285leualaleuaspleuthraspvalvalserphealavalargleuasn290295300alailevalvalargglnalaleualavalserval305310315<210>108<211>336<212>prt<213>芽孢杆菌属的种<400>108metleuilevalcystyrleuargsertyrilepheasnglyglyasn151015lysmetthrasnthrglnmetaspasnlystyrleupheargproser202530tyrglualalysproaspglythrthrvalasnprotyrtyrileile354045ileasnaspleulysargtrpprocystyrvalthraspasnargasn505560pheileserserserglugluleuglnthrargvalalaprosertyr65707580aspprothrglylystyraspglythrproseralaasppheasnarg859095valtyrargasptyralaleuaspproalapheargproleutyrala100105110ileasnalaglnileleuprolystyrasntyrtrphisglnglyasn115120125trpglntyrasngluleuglyargvalserglnleuglyglnaspile130135140glnasnphealaasnleuilecysvallystyrileasngluleuasn145150155160glnileileaspaspthrglnasnasplysleuserglnlysaspile165170175aspthrphelysasnilecysseraspleuglnserglnileglypro180185190cysglnargargserargasniletyrasnglutyrglnasnalaile195200205asnileleuleuserglnserhishisaspalaalaasntyralaphe210215220thrilevalaspasnalatyrserthralalysserglyaspglnasn225230235240alaleuglyglyileargsertyralaserservalleuasntyrala245250255asnalatyraspglnglyvalilethralasermetasnleuglngly260265270mettrpmetphepheglyaspasnleuglngluleuileserasnleu275280285lysaspproilevalasphisasnalaleuilealaserleuglnleu290295300gluilealailegluglutrpglnasnvalserthrasnalaalaglu305310315320pheilelysglutrpalaglytyrglyalaserleuglytyraspcys325330335<210>109<211>307<212>prt<213>芽孢杆菌属的种<400>109leuseriletyrasntyrargilethrsertrppheglyaspargser151015ileleuproserasngluthrleuglnglnglnsertrpthrglyglu202530alalysglnlystrpasnleuargaspleuglyasptyrserpheglu354045ilevalasnglnalaasnglylysvalprothrtyrglntrpalagly505560serleuvalaspasnvalglutyrleulysleuasptyrserasnpro65707580seraspproasplysvalpheglyileileasnargserpheleuphe859095aspglythrasntyralailehisalagluaspleuvallysilegln100105110lysleuproaspvalglythrargproaspalaproasntyrthrgly115120125glyvalaspglnglnleuproglnvalseraspservalvalvalgly130135140alaserleuileprocysilemetvalasnaspasnglnalaserasp145150155160tyrthrlysilehisasnserprotyrtyrthrleuvallysgluglu165170175tyrtrpasplysthrtyrseralavalileproalaglyvalthrhis180185190lystyrserphethrthrglymetserservalaspglnglnlysmet195200205thraspthrleusermetserileglyalaasppheglyleuserphe210215220glyalaalaseralaserleulysthrgluileserargthrilegln225230235240thrgluileserserthrasnthrasnalaserglugluasnvalthr245250255argaspvalglnsergluproglylysthrthrglyphethrglutyr260265270glnleualathrargtyrthrleulysargalaaspglyserileval275280285seraspprotrpvalvallysasnasnlysilethrilethrarglys290295300asnalagln305<210>110<211>328<212>prt<213>赖氨酸芽孢杆菌属的种<400>110metarglysasnlyslysilevalglnlysthrleuthrilealaala151015ilealaserleuglythrthrleualaileserserproserleuala202530seralaaspglnileasnthrsergluasnasnleuglnarggluval354045lysalahisthrileleuasptrplysaspproilepheasnalathr505560gluilevalglylysasnleuleuvalproglyiletyrleuphethr65707580asnserthrtyrhishismetglytyrtyrglyvallystyrglugln859095pheservalglualaaspglyserproserhisthrasnserthrser100105110ilephevalglylysthrthrleuthrasnilethraspglnglugln115120125thrleuserthrasnserpheserlysmetileserasnservalser130135140asnserthrthrhisglyphelyspheglythrlysalaseralalys145150155160pheglnileprophevalglygluthrglyilegluleuseralaglu165170175tyrasnpheseraspthrserserthrthrasnthrglusertyrthr180185190tyrilealaserproglnasnilelysvalproalahisserthrval195200205gluvalleuvalasnleuasnthrvallysalalysglyasnvallys210215220leuleuthrlysvalserglyargaspglnglyvalphevalpheasp225230235240proglnthrglyaspileserlyssertyrtyrtyraspleuhisphe245250255asnglnilevalglulysalaserlyspheglulysleuhisasnile260265270serserasnseraspglyasnthrvalasnileileglyserglylys275280285tyrglualaglutyrglythrglupheservalthrvalargproval290295300aspasnlysglyargserileasnglyglytyrthrpheasnvallys305310315320proglulysileilelysvalglu325<210>111<211>338<212>prt<213>类芽孢杆菌属的种(paenibacillussp.)<400>111metarglyshisvalleulyshisglyleuvalileglyleuserala151015leumetleuglyseralaseralaproproglnvaltyralagluala202530serilealaglnglulysserileglyilevalaspvalasngluval354045leulyslysvalglythrtyrtyrtyrlysasnhisleualaasnthr505560valalaasnprogluserasnglypheargtyrthrleuargalaasn65707580proaspservalthrprovalileaspleuserileglualaargthr859095gluasnilehistyraspserthrileproilepheileglygluasn100105110thrpheproasnasnleuaspglugluglnsertyrasnthrleulys115120125tyrserlysthrtyrthrgluserthrserthrserilesersergly130135140phelysvalglyglyalaglyaspleuglyasnlyspheileleupro145150155160leuileleugluglyalaglylysileasnleuglutyrasnthrgly165170175serthrgluthrasnthrthrserglusertyrthrleugluvalpro180185190proglnprovallysvalproprohislysthrtyrlysthrvalval195200205glupheserglnargthrtyrargglyaspvalvalphethrglyarg210215220thrgluserthrmetalaasnargserileileasnalathrglyile225230235240tyrthrglytrpmetglymetglnaspserlyslysphethrtyrasp245250255asntyrleuglyvalhistyralaglyleuthralaserglnglngln260265270gluilelysasnalaglyvalglnilethrthrserglytyrglyala275280285alaalaasnargpheserserilegluvalthrglylysglyserphe29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25330335asppheargvalasnalaaspvalglyphevalvalgluglntyrthr340345350ala<210>128<211>654<212>prt<213>未知<220><223>来自环境样品的生物体<400>128valhisleuvalpheleuileleuiletyrthrglnilephelysglu151015gluphetyrmetasnasnvalleuasnserglyargthrthrilecys202530aspglyhisasnvalmetalaglnasppropheasnpheglnhislys354045serleuaspthrileglnlysglutrpthrglutrplysargaspasp505560hisserleutyrvalalaprovalvalglythrvalalavalpheleu65707580leulyslysvalglyserphealaalalysargileleuasnglyleu859095trpaspleuvalpheproasnaspasnthrlysleumetglnaspile100105110leuarggluthrglulyspheleuasnglnargleuasnalaaspthr115120125leuserargvalasnalagluleugluglyleuglnlysasnvalala130135140glupheasnargglnvalaspasnpheleuasnproasnargasnpro145150155160thrleuleuserilethrserservalasnthrmetglnglnleuphe165170175leuasnargleuthrglnpheglnleuargglytyrgluleuleuleu180185190leuproleuphealaglnalaalaasnleuhisleuserpheilearg195200205aspvalileleuasnalagluglutrpglyileseralaalathrleu210215220argthrtyrargasptyrleulysasptyrthrlysglutyrserasn225230235240tyrcysileasnthrtyrglnthralaphelyslysleuasnthrpro245250255leuhisglumetleuglupheargthrtyrmetpheleuasnvalphe260265270glutyrvalseriletrpserleuphelystyrglnserleuleuval275280285serserglyalaasnleutyralaserglyserglyproglnglnlys290295300glnserpheseralaglnasntrppropheleutyrserleuphegln305310315320valasnserasntyrvalleuthrglypheserglyserargserthr325330335vallyspheproglyglyglyglyleuleuproproglyserthrthr340345350thrtyralaleuhisseralaargalaasntyrlysgluglyvalser355360365serglyileileglyglythrasnpheasnhisasnphelyscysarg370375380thrileleuproproleuleuthrprophevalargsertrpleuasp385390395400serglythrasparggluglyvalthrthrphethrasntrpglnthr405410415glualaphegluthrthrpheglyileargglyglyalapheglypro420425430argglyasnalaasntyrpheproasptyrpheileargasnileser435440445glyleuproleuvalvalarglysgluaspleuthrlysproleuhis450455460tyrasngluilelysasnilegluserproserglythrproglygly465470475480alaargalatyrmetvalservalhisasnarglysasnasniletyr485490495aspalahisgluasnglythrmetilehisleualaproaspaspasp500505510thrglyphethrileserproilehisalathrglnvalasnasngln515520525ileargthrpheileserglulyspheglyasnglnglyaspserleu530535540argphegluglnthrasnthralaalaargtyrthrpheargglyasn545550555560glyasnasntyrthrleutyrleuargvalserserglnglyasnser565570575thrileargvalasnileasnglyargvalpheasnvalproasnval580585590asnthrthrthrasnasnaspglyvalileaspasnglyalaargphe595600605seraspileargileglyasnilevalalaserasnasnthrasnile610615620proleuaspileasnvalthrleuasnserglythrglnphegluleu625630635640metasnilemetphevalproasnasnleuproproleutyr645650<210>129<211>648<212>prt<213>人工序列<220><223>天然序列的合成变体<400>129valhisleuvalpheleuileleuiletyrthrglnilephelysglu151015gluphetyrmetasnasnvalleuasnserglyargthrthrilecys202530aspglyhisasnvalmetalaglnasppropheasnpheglnhislys354045serleuaspthrileglnlysglutrpthrglutrplysargaspasp505560hisserleutyrvalalaprovalvalglythrvalalavalpheleu65707580leulyslysvalglyserphealaalalysargileleuasnglyleu859095trpaspleuvalpheproasnaspasnthrlysleumetglnaspile100105110leuarggluthrglulyspheleuasnglnargleuasnalaaspthr115120125leuserargvalasnalagluleugluglyleuglnlysasnvalala130135140glupheasnargglnvalaspasnpheleuasnproasnargasnpro145150155160thrleuleuserilethrserservalasnthrmetglnglnleuphe165170175leuasnargleuthrglnpheglnleuargglytyrgluleuleuleu180185190leuproleuphealaglnalaalaasnleuhisleuserpheilearg195200205aspvalileleuasnalagluglutrpglyileseralaalathrleu210215220argthrtyrargasptyrleulysasptyrthrlysglutyrserasn225230235240tyrcysileasnthrtyrglnthralaphelyslysleuasnthrpro245250255leuhisglumetleuglupheargthrtyrmetpheleuasnvalphe260265270glutyrvalseriletrpserleuphelystyrglnserleuleuval275280285serserglyalaasnleutyralaserglyserglyproglnglnlys290295300glnserpheseralaglnasntrppropheleutyrserleuphegln305310315320valasnserasntyrvalleuthrglypheserglyserargserthr325330335vallyspheproglyglyglyglyleuleuproproglyserthrthr340345350thrtyralaleuhisseralaargalaasntyrlysgluglyvalser355360365serglyileileglyglythrasnpheasnhisasnphelyscysarg370375380thrileleuproproleuleuthrprophevalargsertrpleuasp385390395400serglythrasparggluglyvalthrthrphethrasntrpglnthr405410415glualaphegluthrthrpheglyileargglyglyalapheglypro420425430argglyasnalaasntyrpheproasptyrpheileargasnileser435440445glyleuproleuvalvalarglysgluaspleuthrlysproleuhis450455460tyrasngluilelysasnilegluserproserglythrproglygly465470475480alaargalatyrmetvalservalhisasnarglysasnasniletyr485490495aspalahisgluasnglythrmetilehisleualaproaspaspasp500505510thrglyphethrileserproilehisalathrglnvalasnasngln515520525ileargthrpheileserglulyspheglyasnglnglyaspserleu530535540argphegluglnthrasnthralaalaargtyrthrpheargglyasn545550555560glyasnasntyrthrleutyrleuargvalserserglnglyasnser565570575thrileargvalasnileasnglyargvalpheasnvalproasnval580585590asnthrthrthrasnasnaspglyvalileaspasnglyalaargphe595600605seraspileargileglyasnilevalalaserasnasnthrasnile610615620proleuaspileasnvalthrleuasnserglythrglnphegluleu625630635640metasnilemetphevalproasn645<210>130<211>394<212>prt<213>芽孢杆菌属的种<400>130metlysileileileglyglyserlysmetlyslysmetleuilethr151015glyvalleuleuthralavalalathrasntyrleuileprovalasn202530alaphealaalagluasnhisleugluvalglnglnileasnsergln354045glngluargalaseraspthrleuserleuserleuargargvalgly505560serasnseralaleuleuglnglytyrglyleuvalileleuglngln65707580serasnilethrleuproalametglyserleuthrthrhisglnlys859095ilealalysthrasnvallysglutrpleuaspgluhisasnprolys100105110leuilealaleuasnglnaspilelyslyspheserthrlyspheasn115120125glytyrtyrglnthrleutyraspleualaglyglnvalasngluasn130135140gluglnglulysglnasnpheglulysglyleuserthrleulysasp145150155160glnglyglnasnthrlysserthrmetasnaspthrleuserlysphe165170175lysaspphelysthrleuleuaspaspaspvalglulyspheserthr180185190alaalaseraspalaileglnservalglnglyproglyglyaspala195200205ileglnleuarggluasnilelyslysleuglnglnaspileglnthr210215220gluleuthrasnileleuasnargprosergluileileasnglyser225230235240ileasnpheglylyslysvaltyrthrileglymetserseralaglu245250255serglnthrileaspleuvalserileglnserleuthrglyaspleu260265270leuaspleuserasphislysilelysgluseralaasnlysileasp275280285alaileasnasngluleuvalleuleuthrlysasnleuseraspmet290295300leuileglnvalthrglyilethrpheilegluaspglnvalthrgly305310315320phealaglymetileaspargglnilethrthrleuglupheleumet325330335lysasptrpaspvalileasnglulysileasnglnileglnserasn340345350leuaspserglyalaasnserglyleuleulysaspglnleuvalglu355360365leulyslysleuasnaspglumetgluasnglnserlysglnpheglu370375380asppheleuthrvalgluilethrglnile385390<210>131<211>525<212>prt<213>芽孢杆菌属的种<400>131leuleuglyvalmetglygluleuglnasnileleutyrlysargser151015glylysmetasnthrhisglylysasngluglutrpasnglnhishis202530aspglythrcysglycysglnglnasnlysglnasptyrglytyrglu354045glnileprogluhisasplysglnglntyralaserhisglutyrlys505560asnglnglutyrthrtyrlysaspsercysglygluileglythrgly65707580tyrhisasngluasnasnglyasppheglncysaspthrileargtyr859095thrthrproasnleuprovalgluserthrargpheglnthrilethr100105110asnileasnthrasnaspasnglyhisargvalmetaspalametasn115120125ilealavalglythrglyleugluglnargvalglyglycysprotrp130135140glnileaspvalgluasnthrvalvalasngluglnvalargpheasn145150155160asnglnserglnleuphevalphetyrglnthraspserglyglnphe165170175valilealaasnargglyasnglyargvalleuglumetvalgluphe180185190proasnseraspproglntrphisglnleuleuileserserphetyr195200205gluasnargargasnglnleupheaspargargileileasnasnthr210215220asppheleuleuthrthrilethrgluthrglyservalpheglyval225230235240glnasncysasnasnasnproasnalatrpglniletyrthrthrval245250255asngluasngluservalglypheargasnasnthrtyrargphearg260265270thrgluasnasnleuproileasnileproproleuargserglyglu275280285proleuglyproleuproalaleuargtyrleuglnaspserglyleu290295300serproalaglualaproargalavalileglyseralaleuleupro305310315320alailephevalgluaspvalleuserleuproalaargmetalagln325330335serprotyrtyrvalleuglutyrargglntyrtrphisargleutrp340345350seraspileileproalaasnglyglnsertrppheasngluthrthr355360365glymetleuglyglyalaproglnglnglyasnmetargasnthrile370375380sermetserilealaproaspleuglyileargpheglythrglnser385390395400metpropheserilelysilealagluglyleuglyargglnargser405410415asnalaasnileaspileglygluthrserglnasnileargtyrasp420425430asnproglnasnglnprothrargphealaargphealaleualahis435440445glutyralaleuleuargmetaspglyargvalileasnargtrpthr450455460valvalaspargsersermettyrleuleuserpheproglnasnala465470475480leuleuthrmetglncysasplysileileargthraspasnsertyr485490495aspleuservaltrplysthrprometglnilelysserglyglnval500505510ilethrlysasnglulysasnserlysprotyrasnala515520525<210>132<211>463<212>prt<213>芽孢杆菌属的种<400>132valargserglyileasnmetaspleuserhisalaasnhisaspthr151015aspasnglniletyrlysthrsercysglucysasnglnlysthrglu202530trpprometserlyslysglngluglnthrhisthraspthrhistyr354045lysalathrglnpheasnserproiletrpasnleuproilegluser505560argargpheglnthrilethraspvalasnserserglnglyarggly65707580leuasnalaleuargthrtyrproglyglnleuleuglyglyaspleu859095glyalaphecysargthrvalthrleuglnasnglnvalvalasnasp100105110leuthrargglyasnilealaargpheleuphetyrasnleuaspasn115120125glyglyphevalilealaasnalaasnvalglygluvalleuglnglu130135140metasnalaasnasnserserleuilethrasnthrtyrthrglygln145150155160thrasnglnphephearglysserproilethrglnasphisphearg165170175leuleuthrasngluasnmetilevalglyprocysglyasnasnala180185190serasnsertrpthrargilethrleualaleupheserasnglnasn195200205pheargtyrarghisgluprosergluasnglnproileasnleupro210215220glnalaalaalathrsergluleualaproleuproalaleuthrser225230235240leuasnaspproglyproserprovalvalalaproargvalthrile245250255glyseralaleuileprocysilephevalasnaspvalileproleu260265270glnhisargileargglnserprotyrtyrvalleuglucysarggln275280285tyrtrphisargiletrpseraspvalleuthralaglyseraspala290295300phetyrglugluargthrglyileseraspglyalaglnileaspile305310315320argasnthrmetaspileserileglyserasptrptyrleuargphe325330335glyserleuserserpropheargglyglnileserserglyleuarg340345350thrglnproserpheserasnileaspleualagluthrvalmetile355360365asnarghisileasnproasnserphethrthrargpheserargphe370375380thrlysalaileglutyrargleuleuargmetaspglythrvalval385390395400argsertrpvalalavalaspasnargsermettyrtrplysthrphe405410415proasnasnalaglnserthrmetglnserglnlysileileglnser420425430aspthrsertyraspleuservaltrplysthrprometvalilearg435440445aspglygluileileglulysasnasphisasnserlysthrgln450455460<210>133<211>952<212>prt<213>芽孢杆菌属的种<400>133metilemetglnmetvalthrlysalametthrprolysleuglyala151015leuproasppheileaspaspphe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