CN107267514A - 用于根结线虫防治的方法和组合物 - Google Patents
用于根结线虫防治的方法和组合物 Download PDFInfo
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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Abstract
本发明公开了用于遗传防治由根结线虫属的线虫(根结线虫)引起的植物疾病的基因靶标、构建体和方法。本发明涉及通过确认目标编码序列和使用用于转录后阻抑或抑制植物寄生线虫细胞中的目标编码序列的表达的重组DNA技术获得植物保护效应。所公开的基因靶标在不同根结线虫种的直系同源基因之间在核苷酸水平上表现出显著的保守性，从而利于RNA干扰的属范围的靶标选择。
Description
本申请是申请日为2009年04月10日和发明名称为“用于根结线虫防治的方法和组合物”的200980115310.5号发明专利申请的分案申请。
相关申请的交叉引用
本申请要求2008年4月10日提交的美国临时申请61/044,015和2008年7月28日提交的美国临时申请61/084,205的优先权，其公开内容通过引用并入本申请中。
通过引用并入计算机可读形式的序列表
作为本公开的一部分的序列表包括2009年创建的名称为“MNDI004WO_ST25.txt”计算机可读形式的113KB的文件，其包含本发明的核苷酸序列。序列表的主题通过引用全部并入本申请中。
发明领域
本发明一般涉及由植物寄生的线虫引起的疾病的遗传防治。更具体地，本发明涉及目标编码序列的确认，并涉及使用重组DNA技术在植物寄生线虫的细胞中进行转录后阻抑或抑制目标编码序列的表达，从而提供植物保护效果。
发明技术
植物受到多种潜在的引起疾病的物质的影响，包括植物寄生的线虫，其为生活在潮湿表面上或液体环境(包括土壤内的水膜和其它生物体内的湿润组织)中的活性的、柔软的、细长的生物体。有许多的植物寄生性线虫物种，包括各种根结线虫(例如根结线虫属(Meloidogyne)的种)、根腐线虫(lesion nematodes)(如短体线虫属(Pratylenchus)的种)、胞囊线虫(cyst nematodes)(例如异皮线虫属(Heterodera)的种)、剑线虫(daggernematodes)(例如剑线虫属(Xiphinema)和茎线虫(stem and bulb nematodes)(例如茎线虫属(Ditylenchus)的种))及其它。垫刃线虫(Tylenchid nematodes)(垫刃目的成员)(包括异皮科(Heteroderidae)、根结线虫科(Meloidogynidae)和短体科(Pratylenchidae))是最大和最具经济重要性的一组植物寄生的线虫。线虫类通过一系列的生命周期阶段和蜕皮期生长。典型地，具有五个阶段和四个蜕皮期：卵阶段、J1(即第一幼虫阶段)、M1(即第一蜕皮期)、J2(第二幼虫阶段，有时由卵孵化)、M2、J3、M3、J4、M4、A(成虫)。幼虫(“J”)阶段有时也称为蚴(“L”)阶段。基因表达可能对于一个或多个生命周期阶段是特异性的。
植物特异性和动物特异性的线虫种类已进化成为非常成功的寄生虫，并在农业和畜牧业方面造成重大的经济损失和造成人的疾病和死亡。植物的线虫寄生虫可以寄居在植物的所有部分，包括根、发育的花蕾、叶和茎。植物寄生虫根据其摄食习惯分成宽范围的迁移性外寄生虫(migratory ectoparasites)、迁移性内寄生虫(migratory endoparasites)和定居性内寄生虫(sedentary endoparasites)。定居性内寄生虫(包括根结线虫种类(Meloidogyne spp.)和胞囊线虫(球形胞囊线虫属(Globodera)和异皮线虫)诱生摄食位点(feeding site)(在根结线虫的情况中是“巨细胞”和在胞囊线虫的情况中是“合胞体”)并在根中形成长期的感染，这通常对作物造成非常大的危害。据估计，按所有主要作物每年估计平均12％的损失，寄生的线虫在全世界每年对园艺业和农业造成超过780亿美元的损失。例如，据估计线虫每年引起全世界大约32亿美元的大豆损失(Barker等,1994)。
已经提供了几种形式的用于防治线虫感染的组合物、方法和药物。在许多情况下尝试了生物和耕作防治方法(包括植物检疫)。在某些作物中，已确认了提供线虫抗性或耐受性的植物抗性基因。化学组合物(如杀线虫剂)通常应用于其中存在植物寄生性线虫的土壤中。但是，存在着对安全和高效的线虫防治的急迫需求。与当前防治策略的劣势相关的因素包括对于农业可持续性的更高的关切和可能禁止或严厉限制许多可用的农业化学驱虫剂的使用的新政府法规。
化学剂通常是非选择性的并对非目标生物体产生作用，从而在施用该化学剂后的一段时间内严重地破坏了有益微生物的种群。化学剂可能持久地存在于环境中且仅缓慢地代谢。杀线虫土壤薰剂(如三氯硝基甲烷和甲基溴及相关化合物)是高毒性的。甲基溴已被确认为消耗臭氧的化合物，且其在美国使用的注册已经被取消。这些药剂也可能累积在地下水或食物链中，并累积在较高营养层次的物种中。这些药剂也可能具有突变剂和/或致癌剂的作用以引起不可逆的和有害的遗传改变。因此，线虫防治的替代方法(如遗传方法)越来越多地受到研究。
RNA干扰(“RNAi”)是利用内源细胞途径的方法，从而双链RNA(dsRNA)特异性的目标基因导致目标mRNA的降解。RNAi通过内源途径发挥作用，包括能够从原dsRNA产生大约21个核苷酸的小干扰RNA(siRNA)的Dicer蛋白复合体和使用siRNA引导(siRNA guides)识别并降低或阻断相应mRNA的翻译的RNA-诱导沉默复合体(RISC)。仅与该siRNA互补的转录物受到影响，并因此mRNA表达的降低通常是序列特异性的。RNAi的基因沉默效应持续数天且在试验条件下可导致目标转录物丰度的90％或更高的下降，随之导致相应蛋白质水平的下降。
附图简要说明
图1显示了用于选择组合化学品(BASTA)和荧光(DsRed)以产生具有杀线虫目标基因(GOI)的均匀表达的毛根(Hairy root)的毛根表达构建体的示意性实例。GOI可编码杀线虫核苷酸，如靶向于关键线虫基因的双链RNA(dsRNA)。卡那霉素抗性用于细菌宿主内的质粒扩增(plasmid propagation)。BASTA(草铵膦铵盐:草胺膦)耐受性通过在甘露碱(mannopine)合成酶启动子和终止子的控制下的BAR基因(草胺膦乙酰转移酶)表达赋予。GOI可受到源自花椰菜花叶病毒35S启动子(35S)或玄参花叶病毒(FMV)的强组成型启动子的驱动，或者可以使用多种植物启动子(如泛素3启动子)和终止子(如E6、E9或章鱼碱合成酶(OCS)终止子)。红色荧光蛋白DsRed可由强组成型病毒启动子或其它启动子(如肌动蛋白7植物启动子)驱动，并使用终止子如E6、E9或OCS。
图2显示了实施例2中使用的线虫评定量表(1-4)的例子。
发明概述
在一个方面中，本发明包括选自以下的多核苷酸：(a)SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ IDNO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47的任何核酸序列的至少21个连续核苷酸的片段，其中植物寄生的线虫摄取包含至少一个与所述片段互补的链的双链核糖核苷酸序列将抑制该线虫的生长；和(b)(a)中的序列的互补序列。在特定的实施方式中，该多核苷酸定义为与异源启动子可操作地连接。“异源的”意思是未发现天然地与所述多核苷酸结合的任何序列(如启动子)，包括例如，来自相同植物的未发现天然地结合在一起的核酸序列的组合。在特定的实施方式中，该多核苷酸包含在植物转化载体上。在本发明的进一步的实施方式中，这里提供的多核苷酸序列可以定义为分离的多核苷酸序列。
本发明的另一方面是由这种多核苷酸的表达产生的双链核糖核苷酸序列，其中植物寄生的线虫摄取该核糖核苷酸序列将抑制该线虫的生长。在特定的实施方式中，该双链核糖核苷酸序列进一步定义为通过制备包含第一、第二和第三多核苷酸序列的重组多核苷酸序列产生，其中所述第一多核苷酸序列包含选自以下的多核苷酸：(a)SEQ ID NO:1、SEQID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ IDNO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47的任何核酸序列的至少21个连续核苷酸的片段，其中植物寄生的线虫摄取包含至少一个与所述片段互补的链的双链核糖核苷酸序列将抑制该线虫的生长；和(b)(a)中的序列的互补序列；且其中所述第三多核苷酸序列通过所述第二多核苷酸序列与第一多核苷酸连接，且其中第三多核苷酸序列基本上为第一多核苷酸序列的反向互补序列，从而第一和第三多核苷酸序列在转录成核糖核酸时杂交以形成由所连接的第二多核苷酸序列稳定的双链核糖核苷酸分子。在特别的实施方式中，当该多核苷酸序列被植物寄生的线虫摄取时，该双链核糖核苷酸序列抑制基本上与该多核苷酸序列互补的核苷酸序列的表达。
本发明的另一方面是包含选自以下的核苷酸序列的植物转化载体：(a)SEQ IDNO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ IDNO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ IDNO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47的任何核酸序列的至少21个连续核苷酸的片段，其中植物寄生的线虫摄取包含至少一个与所述片段互补的链的双链核糖核苷酸序列将抑制该线虫的生长；和(b)(a)中的序列的互补序列，其中该DNA序列与在植物细胞中起作用的异源启动子可操作地连接。本发明的进一步的实施方式是用这种多核苷酸转化的细胞。在特定的实施方式中，该细胞定义为原核细胞或真核细胞。在特别的实施方式中，该细胞定义为植物细胞。
本发明的另一实施方式涉及用选自以下的多核苷酸转化的植物：(a)SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ IDNO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47的任何核酸序列的至少21个连续核苷酸的片段，其中植物寄生的线虫摄取包含至少一个与所述片段互补的链的双链核糖核苷酸序列将抑制线虫的生长；和(b)(a)中的序列的互补序列。在特定的实施方式中，该植物进一步定义为选自农作物，其选自玉米、小麦、大麦、黑麦、稻、马铃薯、西红柿、黄瓜、辣椒、苜蓿、豆科植物、大豆、豌豆、紫花苜蓿、甘蔗、甜菜、烟草、胡萝卜、棉花、油菜籽(加拿大油菜)、向日葵、红花、高粱、草莓、香蕉、草皮和观赏植物。这种植物的种子(其中该种子包含所述多核苷酸)是本发明的另一实施方式。在一些实施方式中，所述多核苷酸在植物或植物细胞(如根细胞)中表达为双链核糖核苷酸序列。在其它实施方式中，植物寄生的线虫是根结线虫种类。在特别的实施方式中，植物寄生的线虫是南方根结线虫(Meloidogyne incognita)。在再其它的实施方式中，植物寄生的线虫摄取抑制量的双链核糖核苷酸序列将抑制线虫的生长和繁殖。
本发明的另一方面是由包含选自以下的多核苷酸的植物生产的商品：(a)SEQ IDNO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ IDNO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ IDNO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47的任何核酸序列的至少21个连续核苷酸的片段，其中植物寄生的线虫摄取包含至少一个与所述片段互补的链的双链核糖核苷酸序列将抑制线虫的生长；和(b)(a)中的序列的互补序列，其中该商品包含可检测量的所述多核苷酸或由其表达的核糖核苷酸。
本发明的另一方面是用于控制植物寄生线虫的群体数量的方法，包括提供包含在由线虫摄取时发挥作用的双链核糖核苷酸序列的药剂，以抑制该线虫体内的生物学功能，其中该药剂包含选自SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ IDNO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQID NO:47及其互补序列的核苷酸序列。
本发明的再另一方面是用于控制植物寄生线虫群体数量的方法，包括提供包含在由植物寄生的线虫摄取时发挥作用的第一多核苷酸序列的药剂，以抑制该线虫体内的生物学功能，其中该多核苷酸序列沿着至少大约19-大约25个连续核苷酸表现出与源自线虫的编码序列的大约95％-大约100％核苷酸序列同一性，并与第二多核苷酸序列杂交，该第二多核苷酸序列与第一多核苷酸序列互补，且其中所述源自线虫的编码序列选自SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ IDNO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47及其互补序列。在特定的实施方式中，线虫是根结线虫种类。在特别的实施方式中，线虫是南方根结线虫。
本发明的另一实施方式是用于控制植物寄生线虫群体数量的方法，包括在植物寄生线虫的宿主植物中提供表达选自以下的多核苷酸序列的转化植物细胞：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47、这些多核苷酸序列的任一个的至少21个连续核苷酸的片段及其互补序列，其中该多核苷酸被表达来产生在由植物寄生的线虫摄取时发挥作用的双链核糖核酸，从而抑制线虫体内目标序列的表达并导致线虫或线虫群的生长或繁殖相对于缺乏所述转化植物细胞的宿主上的生长或繁殖降低。在特定的实施方式中，该线虫在感染宿主植物后表现出生长降低。构成本发明一部分的该方法的特别实施方式中，目标序列编码其预期功能如下的蛋白质：DNA复制、细胞周期控制、转录、RNA加工、翻译、核糖体功能、tRNA合成、tRNA功能、蛋白质运输(protein trafficking)、分泌、蛋白质修饰、蛋白质稳定、蛋白质降解、能量产生、线粒体功能、中间代谢、细胞结构、信号转导、内吞作用、离子调节、卵产生、繁殖和运输。在特别的实施方式中，线虫选自根结线虫种类。在更特别的实施方式中，线虫是南方根结线虫。在一些实施方式中，该多核苷酸在由植物寄生的线虫摄取时发挥作用以抑制其执行的功能对于线虫的存活、繁殖或生长关键的基因的表达，所述功能选自DNA复制、细胞周期控制、转录、RNA加工、翻译、核糖体功能、tRNA合成、tRNA功能、蛋白质运输、分泌、蛋白质修饰、蛋白质稳定、蛋白质降解、能量产生、线粒体功能、中间代谢、细胞结构、信号转导、内吞作用、离子调节、卵产生和运输。
本发明的另一方面是用于减少宿主植物的根组织中形成的根结线虫(RKN)摄食位点的数目的方法，包括在根结线虫的宿主植物中提供表达选自以下的多核苷酸序列的转化植物细胞：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ IDNO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ IDNO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47、这些多核苷酸的任一个的至少21个连续核苷酸的片段及其互补序列，其中该多核苷酸被表达以产生在由根结线虫摄取时发挥作用的双链核糖核酸，从而抑制所述线虫体内目标序列的表达并导致所形成的摄食位点数目相对于缺乏所述转化植物细胞的宿主上形成的摄食位点数目降低。
本发明的另一实施方式是防治植物中的植物线虫虫害的方法，包括在植物线虫害虫的食物中提供dsRNA，其包含a)有义核苷酸序列；和b)与有义核苷酸序列互补的反义核苷酸序列，其中有义核苷酸序列包含选自以下的核苷酸序列或与选自以下的核苷酸序列互补：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ IDNO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47及其互补序列。在一个实施方式中，所述食物包含经转化以表达所述有义和反义核苷酸序列的植物细胞。
本发明还涉及用于提高由遭受植物寄生线虫感染的作物植物产生的作物产量的方法，所述方法包括以下步骤：a)将这里描述的多核苷酸引入所述作物植物中；和b)种植该作物植物以使得所述多核苷酸表达，其中该多核苷酸的表达抑制植物寄生线虫感染、生长、繁殖或由于植物寄生线虫感染导致的产量损失。在特定实施方式中，该作物植物选自玉米、小麦、大麦、黑麦、稻、马铃薯、西红柿、黄瓜、辣椒、苜蓿、豆科植物、大豆、豌豆、紫花苜蓿、甘蔗、甜菜、烟草、胡萝卜、棉花、油菜籽(加拿大油菜)、向日葵、红花、高粱、草莓、香蕉、草皮和观赏植物。在一些实施方式中，所述多核苷酸的表达产生抑制已经与所述作物植物的一部分接触的植物寄生线虫中的至少第一目标基因的RNA分子，其中该目标基因执行至少一种选自以下的关键功能：DNA复制、细胞周期控制、转录、RNA加工、翻译、核糖体功能、tRNA合成、tRNA功能、蛋白质运输、分泌、蛋白质修饰、蛋白质稳定、蛋白质降解、能量产生、线粒体功能、中间代谢、细胞结构、信号转导、内吞作用、离子调节、卵产生、繁殖和运输。在特定的实施方式中，植物寄生的线虫是垫刃线虫。在特别的实施方式中，植物寄生的线虫是根结线虫种类。在更特别的实施方式中，植物寄生的线虫是南方根结线虫。
本发明的另一方面是用于提高遭受植物寄生线虫感染的作物植物的渗透胁迫(osmotic stress)耐受性的方法，所述方法包括以下步骤：a)将权利要求1的多核苷酸引入所述作物植物；b)种植该作物植物以允许所述多核苷酸表达，其中所述多核苷酸的表达提高作物植物的渗透胁迫耐受性。在一些实施方式中，渗透胁迫耐受性定义为耐旱性。
本发明的再另一方面是产生商品的方法，包括获得包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ IDNO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47任一核酸序列的至少21个连续核苷酸，其中植物寄生的线虫摄取包含至少一个与所述片段互补的链的双链核糖核苷酸序列将抑制该线虫的生长；和(b)(a)中的序列的互补序列的植物或其部分；和从该植物或其部分制备商品。本发明还涉及生产食物或饲料的方法，包括获得这种植物或其部分和从所述植物或其部分制备食物或饲料。在特定的实施方式中，食物或饲料定义为油、粗磨粉、蛋白质、淀粉、面粉或青贮饲料。
本发明的再另一方面是调节植物寄生线虫细胞中目标基因的表达的方法，该方法包括：(a)用包含选自以下的核酸序列转化植物细胞：SEQ ID NO:1、SEQ ID NO:3、SEQ IDNO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ IDNO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ IDNO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ IDNO:44、SEQ ID NO:46、SEQ ID NO:47及这些序列中任一的至少21个连续核苷酸的片段，其中该核酸序列编码dsRNA并可操作地与启动子和转录终止序列连接；(b)在足以允许包含多个转化的植物细胞的植物细胞培养物发育的条件下培养转化的植物细胞；(c)选择已经将所述核酸序列整合到其基因组中的转化植物细胞；(d)筛选表达由所述核酸序列编码的dsRNA的转化植物细胞；和(e)选择表达该dsRNA的植物细胞。本发明的另一实施方式是进一步包括从表达所述dsRNA的植物细胞再生植物，从而植物中基因的表达足以调节与转化的植物或植物细胞接触的植物寄生线虫细胞中目标基因的表达。
特别地，本发明涉及以下各项：
1.一种多核苷酸，包含：
(a)SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ IDNO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ IDNO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47的任一核酸序列的至少21个连续核苷酸的片段；和
(b)(a)中片段的反向互补序列。
2.第1项的多核苷酸，其中植物寄生的线虫摄取包含所述至少21个连续核苷酸的核糖核苷酸将抑制该线虫的生长。
3.第1项的多核苷酸，定义为可操作地与外源启动子连接。
4.第1项的多核苷酸，在所述片段和该片段的反向互补序列之间包含间隔多核苷酸序列。
5.包含第1项的多核苷酸的转化载体。
6.第5项的转化载体，其中所述多核苷酸可操作地与在植物细胞中发挥功能的外源启动子连接。
7.从第1项的多核苷酸表达产生的双链核糖核苷酸分子，其中植物寄生的线虫摄取所述核糖核苷酸分子将抑制该线虫的生长。
8.第7项的双链核糖核苷酸分子，其中所述核糖核苷酸分子在植物寄生的线虫中抑制基本上与所述多核苷酸分子互补的核苷酸序列的表达。
9.用第1项的多核苷酸转化的细胞。
10.第9项的细胞，定义为原核细胞。
11.第9项的细胞，定义为真核细胞。
12.第9项的细胞，定义为植物细胞。
13.用第1项的多核苷酸转化的植物。
14.第13项的植物，进一步定义为选自玉米、小麦、大麦、黑麦、稻、马铃薯、西红柿、黄瓜、辣椒、苜蓿、豆科植物、大豆、豌豆、紫花苜蓿、甘蔗、甜菜、烟草、胡萝卜、棉花、油菜籽(加拿大油菜)、向日葵、红花、高粱、草莓、香蕉、草皮和观赏植物的作物。
15.第13项的植物的种子，其中所述种子包含所述多核苷酸。
16.第13项的植物，其中所述多核苷酸在所述植物中表达为双链核糖核苷酸分子。
17.第16项的植物，其中植物寄生的线虫摄取包含所述双链核糖核苷酸分子的植物的组织将抑制该线虫的生长。
18.第17项的植物，其中所述植物寄生的线虫是根结线虫种类(Meloidogynespp)。
19.第17项的植物，其中所述植物寄生的线虫是南方根结线虫(Meloidogyneincognita)。
20.由第13项的植物产生的商品，其中所述商品包含可检测量的所述多核苷酸或由其表达的核糖核苷酸。
21.一种用于控制植物寄生线虫群体数量的方法，包括提供包含双链核糖核苷酸分子的药剂，该双链核糖核苷酸分子在被线虫摄取时发挥抑制线虫内的生物学功能的作用，其中所述药剂包含选自以下的核苷酸序列：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ IDNO:44、SEQ ID NO:46和SEQ ID NO:47，及其互补序列。
22.一种用于控制植物寄生线虫群体数量的方法，包括提供包含在由植物寄生的线虫摄取时发挥作用以抑制线虫体内的生物学功能的第一多核苷酸序列的药剂，其中该多核苷酸序列沿至少大约19-大约25个连续核苷酸表现出与源自线虫的转录序列的大约95％-大约100％的核苷酸序列同一性，并与第二多核苷酸序列杂交，该第二多核苷酸序列与第一多核苷酸序列互补，且其中所述源自线虫的转录序列选自SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ IDNO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47及其互补序列。
23.第22项的方法，其中所述线虫是根结线虫种类。
24.第22项的方法，其中所述线虫是南方根结线虫。
25.一种用于控制植物寄生线虫的群体数量的方法，包括在植物寄生线虫的宿主植物中提供表达第1项的多核苷酸分子的转化植物细胞，其中该多核苷酸被表达以产生在由植物寄生的线虫摄取时发挥作用的双链核糖核酸，从而抑制所述线虫体内目标序列的表达并导致线虫或线虫群体的生长或繁殖相对于在缺乏所述转化植物细胞的宿主上的生长或繁殖降低。
26.第25项的方法，其中所述线虫在感染宿主植物后表现出生长降低。
27.第25项的方法，其中所述目标序列编码蛋白质，该蛋白质具有选自如下的预期功能：DNA复制、细胞周期控制、转录、RNA加工、翻译、核糖体功能、tRNA合成、tRNA功能、蛋白质运输、分泌、蛋白质修饰、蛋白质稳定、蛋白质降解、能量产生、线粒体功能、中间代谢、细胞结构、信号转导、内吞作用、离子调节、卵产生、繁殖和运输。
28.第25项的方法，其中所述线虫选自根结线虫种类。
29.第25项的方法，其中所述线虫是南方根结线虫。
30.第25项的方法，其中所述多核苷酸在由植物寄生的线虫摄取时发挥作用以抑制其执行的功能对于线虫的存活、繁殖或生长关键的基因的表达，所述功能选自DNA复制、细胞周期控制、转录、RNA加工、翻译、核糖体功能、tRNA合成、tRNA功能、蛋白质运输、分泌、蛋白质修饰、蛋白质稳定、蛋白质降解、能量产生、线粒体功能、中间代谢、细胞结构、信号转导、内吞作用、离子调节、卵产生和运输。
31.一种用于减少宿主植物的根组织中形成的RKN摄食位点的数目的方法，包括在根结线虫的宿主植物中提供表达第1项的多核苷酸的转化植物细胞，其中所述多核苷酸被表达以产生在由根结线虫摄取时发挥作用的双链核糖核酸，从而抑制所述线虫体内目标序列的表达并导致所形成的摄食位点数目相对于在缺乏所述转化植物细胞的宿主上形成的摄食位点数目降少。
32.一种用于防治植物中的植物线虫虫害的方法，包括在植物线虫害虫的食物中提供dsRNA，其包含
a)有义核苷酸序列；和
b)与所述有义核苷酸序列互补的反义核苷酸序列，其中所述有义核苷酸序列包含第1项的核苷酸序列或与其互补。
33.第32项的方法，其中所述食物包含经转化以表达所述有义和所述反义核苷酸序列的植物细胞。
34.一种用于提高由遭受植物寄生线虫感染的作物植物产生的作物产量的方法，所述方法包括以下步骤：
a)将第1项的多核苷酸引入所述作物植物中；和
b)种植该作物植物以使得所述多核苷酸表达，其中该多核苷酸的表达抑制植物寄生线虫感染、生长、繁殖或由于植物寄生线虫感染导致的产量损失。
35.第34项的方法，其中所述作物植物选自玉米、小麦、大麦、黑麦、稻、马铃薯、西红柿、黄瓜、辣椒、苜蓿、豆科植物、大豆、豌豆、紫花苜蓿、甘蔗、甜菜、烟草、胡萝卜、棉花、油菜籽(加拿大油菜)、向日葵、红花、高粱、草莓、香蕉、草皮和观赏植物。
36.第34项的方法，其中所述多核苷酸的表达产生抑制已经与所述作物植物的一部分接触的植物寄生线虫中的至少第一目标基因的RNA分子，其中所述目标基因执行至少一种选自以下的关键功能：DNA复制、细胞周期控制、转录、RNA加工、翻译、核糖体功能、tRNA合成、tRNA功能、蛋白质运输、分泌、蛋白质修饰、蛋白质稳定、蛋白质降解、能量产生、线粒体功能、中间代谢、细胞结构、信号转导、内吞作用、离子调节、卵产生、繁殖和运输。
37.第36项的方法，其中所述植物寄生的线虫是垫刃线虫。
38.第37项的方法，其中所述植物寄生的线虫是根结线虫种类。
39.第37项的方法，其中所述植物寄生的线虫是南方根结线虫。
40.一种用于提高遭受植物寄生线虫感染的作物植物的渗透胁迫耐受性的方法，所述方法包括以下步骤：
a)将第1项的多核苷酸引入所述作物植物；
b)种植该作物植物以允许所述多核苷酸表达，其中所述多核苷酸的表达提高作物植物的渗透胁迫耐受性。
41.第40项的方法，其中所述渗透胁迫耐受性定义为耐旱性。
42.一种用于生产商品的方法，包括获得第13项的植物或其部分，和从该植物或其部分制备商品。
43.一种用于生产食品或饲料的方法，包括获得第13项的植物或其部分和从所述植物或其部分制备食品或饲料。
44.第43项的方法，其中所述食品或饲料定义为油、粗磨粉、蛋白质、淀粉、面粉或青贮饲料。
45.一种用于调节植物寄生线虫细胞中目标基因的表达的方法，该方法包括：
(a)用包含编码dsRNA的核酸序列的载体转化植物细胞，所述核酸序列选自SEQ IDNO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ IDNO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ IDNO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:47及其至少21个连续核苷酸的片段，其中所述核酸序列编码dsRNA并可操作地与启动子和转录终止序列连接；
(b)在足以允许包含多个转化的植物细胞的植物细胞培养物发育的条件下培养转化的植物细胞；
(c)选择已经将所述核酸序列整合到其基因组中的转化植物细胞；
(d)筛选表达由所述核酸序列编码的dsRNA的转化植物细胞；和
(e)选择表达该dsRNA的植物细胞。
46.第45项的方法，进一步包括从表达所述dsRNA的植物细胞再生植物，从而植物中核酸序列的表达足以调节与转化的植物或植物细胞接触的植物寄生线虫细胞中目标基因的表达。
47.一种多核苷酸，包含：
(a)SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ IDNO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ IDNO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47的任一核酸序列的连续核苷酸的片段；和
(b)(a)中片段的反向互补序列，
其中在植物寄生的线虫的食物中提供表达足以调节所述线虫中目标基因表达的多核苷酸的植物细胞。
发明详述
下面是用于帮助本领域技术人员实施本发明的本发明详细说明。本领域技术人员可以在这里描述的实施方式中进行改进和变化而不脱离本发明的精神或范围。
本发明提供了用于遗传防治植物寄生线虫虫害，特别是用于遗传防治植物的根结线虫属(根癌)线虫虫害的方法和组合物。本发明还提供了根结线虫属植物寄生线虫的生活周期中关键的基因的确认和其用作线虫种群的dsRNA介导的防治的靶标的方法。编码dsRNA分子的DNA质粒载体设计为抑制对于生长、发育、摄食或繁殖关键的线虫基因。例如，本发明提供了用于在植物寄生线虫中的转录后阻抑或抑制目标编码序列的表达的方法和重组DNA技术，以通过允许植物寄生的线虫食用由所有或一部分目标编码序列转录的一种或多种双链或小干扰核糖核酸(RNA)分子而提供保护效应，从而防治感染。
本发明公开了根癌植物线虫害虫(根结线虫种类)的来自保守的和对于植物寄生线虫的存活力关键的基因靶标的核苷酸和氨基酸序列。本发明进一步描述了这些序列用于通过在根结线虫的食物中提供包含本发明的一种或多种多核苷酸分子的一部分、或全部或基本上全部的dsRNA改变至少根结线虫细胞中一种或多种目标多核苷酸或蛋白质分子的表达。这些目标根结线虫序列的高核苷酸保守性有利于以少数的dsRNA构建体同时靶向于多种根结线虫种，同时提供对相似或同源的人和植物序列的选择性，并还提供对存在于其它非目标生物体(如有益的昆虫，例如蜜蜂或蝴蝶)中的相似或同源序列的选择性。
用于防治根结线虫的环境良好但高效的替代方法是使用对关键线虫基因进行RNA干扰以控制植物的线虫虫害。这是通过植物中与目标线虫基因互补的双链RNA(dsRNA)的转基因表达实现。该dsRNA与目标基因的互补性可能在选为靶标的序列中是完全的(即100％)，或者dsRNA的序列可能沿选为靶标的序列基本上互补(例如90％或95％以上)。
因此，本发明涉及使用双链RNA(dsRNA)，包括小的干扰RNA(siRNA)，序列特异性地抑制编码序列的表达，从获得预期水平的根结线虫防治。
本发明还提供了抑制根结线虫(根结线虫种类)内的目标基因功能的方法，其可以通过RNA干扰完成，从而导致病原体生活周期的破坏。用于破坏的最佳目标基因包括生命周期关键基因，该破坏引起寄生虫种群的高渗透性死亡或通过以对植物的最小摄食损伤阻止繁殖、减少所形成的摄食位点的数目、使所产生的卵的数目最小化、使卵的存活力最小化和使达到下一世代的存活逃脱蠕虫的数目最小化而导致“遗传性死亡”。在特别的实施方式中，可以通过将由施用本发明的方法和组合物的根结线虫属的线虫产生的卵的数目与在类似条件下生长但未施用这些方法和组合物的根结线虫属的线虫产生的卵的数目进行比较测定效率。本发明的另一方面提供预计对根结线虫种类生长和/或发育(如摄食或卵的产生)关键的目标基因的核酸。用于预测这类目标的特征包括与具有强的和可再现的RNA干扰表型的已知秀丽隐杆线虫(C.elegans)基因的直系同源性、与大豆胞囊线虫(H.glycines)(大豆的胞囊线虫)中RNAi验证的基因和根结线虫种类中的表达模式的直系同源性。
在本发明的再另一实施方式中，提供了如SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27至SEQ IDNO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQID NO:47或者其互补序列的一系列分离的和纯化的核苷酸序列。本发明还提供了用于表达一种或多种RNA以抑制植物寄生的线虫中由这些序列及其片段表达的目标基因的表达的稳定dsRNA分子。稳定的dsRNA(包括dsRNA或siRNA分子)可以包含至少两个转录序列，如沿相对于至少一个启动子的有义和反义方向排列的编码序列，其中包含有义链和反义链的核苷酸序列通过至少大约1至大约1000个核苷酸的间隔序列连接或接合，其中该有义链和反义链可能长度不同，且其中两个转录的序列各与以下所示的任何一个或多个核苷酸序列共有至少80％的序列同一性，至少90％、至少95％、至少98％、至少99％或100％的序列同一性：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47或其互补序列。
在再另一方面中，本发明提供了包含编码这里描述的dsRNA分子的核酸分子的重组DNA构建体。dsRNA可以通过从与选自以下的核苷酸序列至少大约80％至大约100％相同的核苷酸序列转录该dsRNA分子的一个链而形成：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27至SEQ IDNO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQID NO:47及其互补序列的至少21个连续核苷酸至全长的片段。这些重组DNA构建体可以定义为产生能够在摄食时抑制植物寄生线虫细胞中内源目标序列的表达的dsRNA分子。该构建体可以包含可操作地与在宿主细胞(如植物细胞)中发挥作用的启动子序列连接的本发明的核苷酸序列。这种启动子可以是组织特异性的且可以是例如对作为植物寄生线虫攻击对象的组织类型特异性的。在例如分别为根或叶病原体的情况中，可能希望使用分别提供根或叶优先表达的启动子。
根据本发明的核苷酸序列的核酸构建体可以包含至少一个非天然存在的核苷酸序列，该非天然存在的核苷酸序列可以转录成能够通过分子间或分子内杂交在体内形成dsRNA分子的单链RNA。这些dsRNA序列自组装并可以在植物寄生线虫的营养源中提供以获得希望的抑制作用。
重组DNA构建体可以包含一个或多个不同的非天然存在的序列，该序列在体内表达为dsRNA序列并在植物寄生线虫的宿主植物的组织中提供时，抑制植物寄生线虫中至少两个不同目标基因的表达。在特定的实施方式中，至少2、3、4、5、6、8或10个或更多的不同dsRNA在具有线虫抑制效应的细胞或包含该细胞的植物中产生。dsRNA可以从在不同转化事件中引入或可能引入到单一核酸分子上的多个构建体表达。dsRNA可以使用单个启动子或多个启动子表达。在本发明的一个实施方式中，产生包含与植物寄生线虫体内的多个基因座同源的核酸的单一dsRNA。
在再另一方面中，本发明提供了在其基因组中具有至少一个重组DNA序列的重组宿主细胞，该重组DNA序列被转录以产生至少一个在被植物寄生的线虫摄食时发挥功能的dsRNA分子，从而抑制目标基因在线虫中的表达。dsRNA分子可以由这里描述并如序列表中所示的任何核酸序列编码。本发明还提供了在其基因组中具有至少一个这里描述的重组DNA序列的转化植物细胞。还提供了包含这种转化植物细胞的转基因植物，包括任何世代的后代植物、种子和植物产物，各包含该重组DNA。本发明的dsRNA分子可以在转基因植物细胞中发现，例如在细胞质中。它们也可以在非原质体空间中发现。
本发明进一步提供选自SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47及其互补序列的核酸序列的片段。该片段可以定义为当表达为dsRNA并被线虫摄取时引起死亡、生长抑制、繁殖降低或中止根结线虫属线虫的虫害或摄食。该片段可以例如包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47或其互补序列中任何一个或多个序列的至少大约19、21、23、25、40、60、80、100、125、200、250、300、400或更多的连续核苷酸或其全长。用于本发明中的有益DNA片段的一个例子是至少大约19至大约23、或大约23至大约100个核苷酸，但少于大约2000个核苷酸的长度。特别有用的是与包括19、21、23、25、40、60、80、100、125、200、250、300、400个或更多连续核苷酸的线虫目标序列同源的包括大约23至大约300个核苷酸的dsRNA序列。本发明还提供从任何这些序列表达的核糖核酸(包括dsRNA)。选择用于表达基因抑制剂的序列可以由源自一个或多个目标植物寄生线虫种类并意图用于表达RNA的单个序列构建，该RNA起到在一种或多种目标病原体中抑制单个基因或基因家族的作用，或该DNA序列可以构建为多个DNA序列的嵌合体。
在另一实施方式中，本发明提供了用于调节线虫细胞(如根结线虫种类的细胞)中目标基因的表达的方法，该方法包括：(a)用包含与启动子和转录终止序列可操作地连接的核酸序列的载体转化植物细胞，其中所述核酸序列编码dsRNA；(b)在足以允许包含多个转化的植物细胞的植物细胞培养物发育的条件下培养转化的植物细胞；(c)选择已经将该载体整合到其基因组中的转化植物细胞；(d)筛选表达由该载体编码的dsRNA的转化植物细胞；(e)选择表达该dsRNA的植物细胞；(f)任选地从表达该dsRNA的植物细胞再生植物，从而植物中核酸序列的表达足以调节与转化的植物或植物细胞接触的植物寄生线虫的细胞中目标基因的表达。基因表达的调节可以包括这种表达的部分或完全抑制。
在再另一方面中，本发明提供了用于抑制植物寄生的线虫中的基因表达的方法，包括在线虫宿主的组织中提供基因抑制量的由这里描述的核苷酸序列转录的至少一种dsRNA分子，其至少一个片段与植物寄生线虫细胞中的mRNA序列互补。该方法可以进一步包括观察植物寄生线虫的死亡、生长抑制或繁殖降低及宿主症状的程度。在一个实施方式中，被按照本发明的病原微生物摄食的dsRNA分子(包括其修饰形式如siRNA分子)可以是与由选自以下的核苷酸序列的全部或一部分转录的RNA分子至少大约80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或大约100％相同：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47。
因此，提供了用于转录本发明的dsRNA分子的分离的和基本纯化的核酸分子，包括但不限于非天然存在的核苷酸序列和重组DNA构建体，其在引入植物寄生线虫中时阻止或抑制内源编码序列或目标编码序列在植物寄生线虫体内的表达。本发明还包括转基因植物，其(a)包含编码所述分离的和基本纯化的核酸分子的核苷酸序列和用于转录防治植物寄生线虫感染的dsRNA分子的非天然存在的重组DNA构建体和(b)显示出对感染的抗性和/或增强的耐受性。本发明包括用于局部施用到植物上或动物上或动物的环境中以实现植物寄生线虫感染的消除或降低的包含本发明的dsRNA核苷酸序列的组合物。
编码对于生存关键的蛋白质或蛋白质部分的cDNA序列(如参与各种代谢或降解代谢途径、细胞分裂、繁殖、能量代谢、消化等的氨基酸序列)可以选择用于制备在植物寄生线虫的宿主植物中提供的双链RNA分子。如这里描述的，目标生物体摄食包含一个或多个dsRNA(其至少一个片段对应于目标病原体的细胞中产生的RNA的至少基本上相同的片段)可导致目标生物体的死亡或其它抑制。这些结果表明源自于植物寄生线虫的核苷酸序列(DNA或RNA)可用于构建对线虫摄食具有抗性的植物细胞。例如，线虫的宿主植物可转化为包含一个或多个源自这里提供的线虫的核苷酸序列。转化到宿主中的核苷酸序列可以编码形成为转化的宿主内的细胞或生物液体中的dsRNA序列的一个或多个RNA，因此如果/当植物寄生线虫形成与转基因宿主的营养关系时使得该dsRNA可为植物寄生线虫得到。这可以导致植物植物寄生的根结线虫类线虫的细胞中一个或多个基因的表达的抑制及其最终死亡或其生长、发育或繁殖的抑制。
本发明一般涉及宿主生物体中植物寄生的线虫的遗传防治。更特别地，本发明包括递送线虫防治药剂至植物寄生的线虫的方法。这类防治药剂引起植物寄生线虫摄食、生长或在目标宿主中另外引起疾病的能力的直接或间接损害。本发明在一个实施方式中提供了包括向植物寄生线虫递送作为抑制植物寄生线虫中的目标基因的手段的稳定的dsRNA分子，因此在线虫宿主中获得预期的植物疾病的防治。
在实现前述方法时，本发明提供了抑制植物寄生线虫中目标基因的表达的方法，从而导致生长、发育、繁殖和/或摄食的损害，并最终可能导致植物寄生线虫的死亡。在一个实施方式中，该方法包括将部分或完全稳定的双链RNA(dsRNA)核苷酸分子(包括其修饰形式如小的干扰RNA(siRNA)序列)引入植物寄生线虫的营养组合物中，和使得该营养组合物或食物源为植物寄生线虫所用。摄食包含双链或siRNA分子的营养组合物导致线虫细胞摄取分子，从而导致线虫细胞中至少一个目标基因表达的抑制。目标基因的抑制发挥对线虫的有害效应。该方法和相关的组合物可用于通过在线虫的宿主中提供一种或多种包含这里描述的dsRNA分子的组合物在线虫存在的任何宿主组织或环境中或其上限制或消除线虫对植物或植物细胞的感染或寄生。
在特定的实施方式中，本发明提供的dsRNA分子包含与以下任一所示的序列互补的核苷酸序列：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQID NO:23、SEQ ID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47，其在植物寄生的线虫中的抑制导致对线虫的生长和发育、繁殖或其它生物功能关键的蛋白质或核苷酸序列物质的减少或消除。所选择的核苷酸序列可以表现出与如下所示的一个核苷酸序列的大约80％至大约100％的同一性：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQID NO:23、SEQ ID NO:25、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47，或者其至少21个连续核苷酸的片段直至该序列的全长，包括其互补序列。在特定的其它实施方式中，能够编码有效的dsRNA分子的DNA序列选自于SEQ ID NO:27至SEQID NO:30、SEQ ID NO:32、SEQ ID NO:46和SEQ ID NO:47或其互补序列。这种抑制可描述为特异性的，因为来自目标基因一部分的核苷酸序列选自抑制性dsRNA或siRNA由其转录的序列。该方法有效地抑制至少一个目标基因的表达并可用于抑制植物寄生线虫中的许多不同类型的目标基因。
确认为具有线虫防治效果的序列可以通过建立适宜的表达构建体轻易地表达为dsRNA分子。例如，这些序列可以通过以下方式表达为发夹及茎和环结构：取得对应于选自SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47或其片段的第一片段；将这一序列连接到不与第一片段同源或互补的第二片段间隔区；并将其连接到转录RNA的第三片段，其中该第三片段的至少一部分与第一片段基本互补。这种构建体通过第一片段与第三片段的杂交形成茎和环结构且形成包含第二片段的环结构(WO94/01550、WO98/05770、US 2002/0048814A1和US 2003/0018993A1)。dsRNA可以例如以双链结构(如茎环结构，例如发夹)的形式产生，从而靶向于线虫序列的siRNA的产生通过共表达导致siRNA产生增加的靶向基因的片段(例如在另外的植物可表达盒上)而增强，或减少甲基化以阻止dsRNA发夹启动子的转录基因沉默(例如，WO05/019408)。
本发明包括的根结线虫属的典型物种包括花生根结线虫(M.arenaria)、奇特伍德根结线虫(M.chitwoodi)、不列颠根结线虫(M.artiellia)、伪哥伦比亚根结线虫(M.fallax)、北方根结线虫(M.hapla)、爪哇根结线虫(M.javanica)、南方根结线虫、小结根结线虫(M.microtyla)、M.partityla、番禺根结线虫(M.panyuensis)和M.paranaensis。可能与根结线虫一起发现的其它植物寄生线虫包括球形胞囊属、短体线虫属、拟毛刺线虫属(Paratrichodorus)、穿孔线虫属(Radopholus)、纽带线虫属(Hoplolaimus)、茎线虫属、锥线虫属(Dolichodorus)、螺旋线虫属(Helicotylenchus)、潜根线虫属(Hirschmanniella)、剑线虫属、肾形线虫属(Rotylenchulus)、毛刺线虫属(Trichodorus)、矮化线虫属(Tylenchorhynchus)、刺线虫属(Belonolaimus)和长针线虫属(Longidorus)。
本发明的方法和组合物可应用于任何单子叶或双子叶植物，取决于所希望的病原体(例如线虫)防治。本发明保护的典型植物以及与它们相关的根结和其它植物寄生的线虫物种包括，但不限于苜蓿：北方根结线虫、南方根结线虫、爪哇根结线虫、鳞球茎茎线虫(Ditylenchus dipsaci)、短体线虫类、针线虫类(Paratylenchus spp.)、剑线虫类；香蕉：南方根结线虫、花生根结线虫、爪哇根结线虫、香蕉穿孔线虫(Radopholus similis)、多带螺旋线虫(Helicotylenchus multicinctus)、咖啡短体线虫(Pratylenchus coffeae)、肾形线虫(Rotylenchulus reniformis)；蚕豆和豌豆：根结线虫类、异皮线虫类、刺线虫类、螺旋线虫类、肾形线虫、银链花拟毛刺线虫(Paratrichodorus anemones)、毛刺线虫类；木薯：根结线虫类、肾形线虫；谷类：纳西根结线虫(Meloidogyne naasi(大麦、小麦、黑麦)；鹰嘴豆：根结线虫类、木豆胞囊线虫(Heterodera cajani)、肾形线虫、塞氏纽带线虫(Hoplolaimus seinhorsti)、短体线虫类；柑橘：根结线虫类、柑橘半穿刺线虫(Tylenchulus semipenetrans)、香蕉穿孔线虫、柑橘穿孔线虫(Radopholuscitrophilus)、Hemicycliophora arenaria、短体线虫类、长尾刺线虫(Bolonolaimuslongicaudatus)、毛刺线虫类、拟毛刺线虫类、剑线虫类；三叶草：根结线虫类、三叶草胞囊线虫(Heterodera trifolii)；玉米：南方根结线虫、短体线虫类、较小拟毛刺线虫(Paratrichodorus minor)、长针线虫类、哥伦比亚纽带线虫(Hoplolaimus columbus)；棉花：南方根结线虫、长尾刺线虫(Belonolaimus longicaudatus)、肾形线虫、盔状纽带线虫(Hoplolaimus galeatus)、短体线虫类、矮化线虫类、较小拟毛刺线虫；葡萄：根结线虫类、剑线虫类、伤残短体线虫(Pratylenchus vulnus)、柑橘半穿刺线虫、肾形线虫；草类：短体线虫类、长针线虫类、Paratrichodorus christiei、剑线虫类、茎线虫类；花生：北方根结线虫、花生根结线虫、短体线虫类、小环线虫类(Criconemella spp.)、长尾刺线虫；木豆：根结线虫类、木豆胞囊线虫、肾形线虫、塞氏纽带线虫、短体线虫类；马铃薯：根结线虫类、马铃薯金线虫(Globodera rostochiensis)、马铃薯白线虫(Globodera pallida)、短体线虫类、原始毛刺线虫(Trichodorus primitivus)、茎线虫类、拟毛刺线虫类、异常珍珠线虫(Nacobbus aberrans)；稻：根结线虫类、水稻干尖线虫(Aphelenchiodes besseyi)、水稻茎线虫(Ditylenchus angustus)、潜根线虫类(Hirchmanniella spp.)、水稻胞囊线虫(Heterodera oryzae)；小果：根结线虫类、短体线虫类、剑线虫类、长针线虫类、Paratrichodorus christiei,滑刃线虫类(Aphelenchoides spp.)；大豆：南方根结线虫、爪哇根结线虫、大豆胞囊线虫、刺线虫类、哥伦比亚纽带线虫；甜菜：根结线虫类、甜菜胞囊线虫(Heterodera schachtii)、鳞球茎茎线虫、异常珍珠线虫、毛刺线虫类、长针线虫类、拟毛刺线虫类；甘蔗：根结线虫类、短体线虫类、穿孔线虫类、异皮线虫类、纽带线虫类、螺旋线虫类、盾线虫类(Scutellonema spp.)、刺线虫类、矮化线虫类、剑线虫类、长针线虫类、拟毛刺线虫类；烟草：根结线虫类、短体线虫类、烟草矮化线虫(Tylenchorhynchus claytoni)、烟草胞囊线虫(Globodera tabacum)、毛刺线虫类、美洲剑线虫(Xiphinema americanum)、鳞球茎茎线虫、拟毛刺线虫类；和西红柿：根结线虫类、短体线虫类。
本发明的各个方面特别可用于具有线虫抗性的转基因植物，包括但不限于玉米、谷类(包括小麦、大麦、黑麦和水稻)、马铃薯、西红柿、黄瓜、辣椒、三叶草、豆类(包括大豆(Glycine sp.)、蚕豆和苜蓿)、甘蔗、甜菜、烟草、胡萝卜、棉花(Gossypium sp.)、油菜籽(加拿大油菜)、向日葵、红花、高粱、草莓、香蕉、草皮和观赏植物等。
本发明还提供用于防治植物寄生线虫的感染的方法和组合物。一种方法提供了用于保护植物免受植物寄生线虫侵害的如本文中所述的dsRNA方法以及表现出与dsRNA方法和组合物不同的特征且可以干扰线虫生长、发育或繁殖的一种或多种化学剂。
A.核酸组合物和构建体
本发明提供了用于获得特定宿主靶标的稳定转化的重组DNA构建体。转化的宿主靶标可以由该重组DNA构建体表达有效水平的优选dsRNA或siRNA。分离的和纯化的核苷酸序列的对可以由cDNA文库和/或基因组文库信息提供。核苷酸序列对可以源于用作热扩增引物的任何线虫以产生本发明的dsRNA和siRNA分子。
如本文中所用的，术语“核酸”指从5’至3’端显示的脱氧核糖核苷酸或核糖核苷酸碱基的单链或双链聚合物。“核酸”也可以任选地包含允许通过聚合物正确地通读且不减少该核酸编码的多肽的表达的非天然存在的或改变的核苷酸碱基。术语“核苷酸序列”或“核酸序列”指作为单独的单链或双链体的核酸有义和反义链。术语“核糖核酸”(RNA)包括RNAi(抑制RNA)、dsRNA(双链RNA)、siRNA(小干扰RNA)、shRNA(小发夹RNA)、mRNA(信使RNA)、miRNA(微RNA)、tRNA(转移RNA，不论是否带有相应的酰化氨基酸)和cRNA(互补RNA)且术语“脱氧核糖核酸”(DNA)包括cDNA和基因组DNA及DNA-RNA杂交体。词语“核酸片段”、“核苷酸序列片段”或更一般地说“片段”，本领域技术人员理解为包括基因组序列、核糖体RNA序列、转移RNA序列、信使RNA序列、操纵子序列和表达或可适用于表达蛋白质、多肽或肽的较小的工程核苷酸序列的功能术语。
本发明提供了核苷酸序列，其表达产生基本上与植物寄生线虫中靶向基因的RNA分子的整个或部分同源的RNA序列，该靶向基因的RNA分子包含线虫基因组中核苷酸序列编码的RNA序列。因此，在消化稳定的RNA序列后，可以获得植物寄生线虫细胞中目标基因的核苷酸序列的下调，从而导致对线虫生长、生存力、增殖或繁殖的有害效应。
如本文中所使用的，用于核酸序列的术语“基本上同源”或“基本同源性”包括在严格条件下与以下任一的编码序列杂交的核苷酸序列：如序列表中所示的SEQ ID NO:1、SEQID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ IDNO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQID NO:46或SEQ ID NO:47或其互补序列。在严格条件下与如序列表中所示的SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47或其互补序列杂交的序列是使得在两个序列之间发生反平行排列的那些序列，且然后该两个序列能够在严格条件下与相对链上的相应碱基形成氢键，从而形成可使用本领域中公知的方法检测的在适当的严格性(包括高严格性)条件下充分稳定的双链体分子。基本同源的序列通常与序列表中的SEQ ID NO:1、SEQ ID NO:3、SEQID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ IDNO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27至SEQID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47任一所示的所指核苷酸序列或其互补序列具有大约70％至大约80％的序列同一性，或更特别地大约80％至大约85％的序列同一性或更特别地大约90％至大约95％的序列同一性，至大约99％的序列同一性。
如本文中所用的，术语“直系同源基因(ortholog)”指两个或多个物种中由共同的祖先核苷酸序列进化的且可以在该两个或多个物种保持相同功能的基因。
如本文中所用的，术语“序列同一性”、“序列相似性”或“同源性”用于描述两个或多个核苷酸序列之间的序列关系。两个序列之间“序列同一性”的百分比通过在比较窗口内比较两个最佳对齐序列确定，其中比较窗口中的序列部分与用于两个序列的最佳对齐的基准序列(其不包含添加或缺失)相比可以包含添加或缺失(即空隙)。该百分比通过确定两个序列中存在的相同核酸碱基或氨基酸残基的位置数目以获得匹配位置的数目，将匹配位置的数目除以比较窗口中总的位置数目并将其结果乘以100以产生序列同一性的百分比而计算。与基准序列相比在每一位置相同的序列可以说与基准序列相同，反之亦然。如果第一核苷酸序列表现出与第二或基准序列的完全互补性，则沿5’-3’方向观察的第一核苷酸序列可以说是沿3’-5’方向观察的第二或基准核苷酸序列的“互补序列”或与其互补。如本文中所用的，当沿5’-3’阅读的序列之一的每一核苷酸与沿3’-5’阅读的其它序列的每一核苷酸互补，则可以说核酸序列分子表现出“完全互补性”。与基准核苷酸序列互补的核苷酸序列表现出与基准核苷酸序列的反向互补序列相同的序列。这些术语和说明在本领域中很好地定义并易于被本领域普通技术人员理解。
如本文中所用的，“比较窗口”指至少6个连续位置，通常大约50-大约100，更通常大约100-大约150个连续位置的概念片段(conceptual segment)，其中两个序列最佳对齐后某一序列与相同数目连续位置的基准序列比较。比较窗口与用于两个序列的最佳对齐的基准序列(其不包含添加或缺失)相比可以包含大约20％或更低的添加或缺失(即空隙)。本领域技术人员可以在Wisconsin Genetics Software Package Release 7.0(GeneticsComputer Group,575Science Drive Madison,Wis.,USA)中找到用于序列比对的详细方法或从Ausubel等(1998)找到序列分析的详细说明。
本发明提供了能够在细胞或微生物中表达为RNA转录物以抑制植物寄生线虫的细胞、组织或器官中目标基因表达的DNA。该序列包含编码一种或多种不同核苷酸序列的DNA分子，其中各不同核苷酸序列包含有义核苷酸序列和反义核苷酸序列。该序列可以由编码本发明的dsRNA分子的一部分间隔序列连接。间隔序列可以构成有义核苷酸序列或反义核苷酸序列的部分或者不相关核苷酸序列，并且在该dsRNA分子中有义和反义序列之间形成。有义核苷酸序列或反义核苷酸序列基本与目标基因或其衍生物的核苷酸序列或其互补序列相同。dsRNA分子可以可操作地置于在表达该dsRNA的细胞、组织或器官中具有功能的启动子序列的控制下以产生dsRNA分子。在某些实施方式中，DNA序列可以源自如序列表中的SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47所示的核苷酸序列或其互补序列。
本发明还提供用于在植物细胞中表达的DNA序列，该植物在将该DNA表达为RNA并被植物寄生的线虫摄食时获得抑制植物寄生线虫的细胞、组织或器官中的目标基因的效果。dsRNA可以包含一个或多个结构基因序列，其中各结构基因序列包含可以通过在互补和反义序列中形成环的间隔序列连接的有义核苷酸序列和反义核苷酸序列。有义核苷酸序列或反义核苷酸序列基本与目标基因的核苷酸序列、其衍生序列或其互补序列相同。一个或多个结构基因序列可以可操作地置于一个或多个启动子序列的控制下，至少一个启动子序列在原核或真核生物体(特别是植物细胞)中是可操作的。在植物中表达基因抑制分子的方法是已知的(例如美国公开2006/0200878A1)，且可以用于表达本发明的核苷酸序列。
根据本发明的用于植物寄生线虫防治的基因序列或片段可以克隆到可在转基因植物中操作的两个组织特异性启动子(如两个根特异性启动子)之间并且在其中表达以在转基因植物细胞中产生向其中形成dsRNA分子的mRNA。根特异性启动子的实例是本领域中已知的(例如线虫诱导的RB7启动子；美国专利5,459,252；Opperman等，1994)。植物组织中包含的dsRNA分子被植物寄生的线虫摄食以获得预期的目标基因表达的抑制。
花椰菜花叶病毒35S启动子(转基因植物中通用的原型强启动子)或相关启动子如E35S或FMV启动子可以用于驱动线虫抗性基因，特别是对于根结线虫(参见实施例8)。启动子也已确认为指导在植物中线虫诱导的觅食结构中的基因表达(例如Gheysen和Fenoll,2002)。因此，可以利用在觅食位点中可再生表达的基因中确认的启动子。在线虫形成的觅食位点中上调的基因的实例包括在根和芽顶端分生组织中表达的At10.1(Mazarei等，2003)、Hs1pro-1(Thurau等，2003)、通常在伴细胞中表达的AtSUC2(Juergensen等，2003)、在维管组织和根尖中表达的At17.1(Mazarei等，2004)、FGAM合成酶(磷酸核糖甲酰甘氨脒合成酶(phosphoribosylformyl-glycinamidine synthase))(Vaghchhipawala等，2004)和ABI3(De Meutter等，2005)等。根结巨细胞具有传递细胞(transfer cell)特有的细胞壁内生形态。因此，韧皮部中的基因表达也可能适于输送效应分子到觅食位点中。
本发明提供的核苷酸序列可以包含由“间隔序列”分隔的反向重复序列。间隔序列可以是包含有助于在各重复序列之间形成二级结构(这是需要的)的任何核苷酸序列的区域。在本发明的一个实施方式中，间隔序列是mRNA的有义或反义编码序列的部分。或者间隔序列可以包含能够与核酸分子共价连接的核苷酸或其同系物的任何组合。间隔序列可以包含例如至少大约10-100核苷酸长度，或大约100-200核苷酸长度，至少大约200-400核苷酸长度或至少大约400-500核苷酸长度的核苷酸序列。
该核酸分子或核酸分子的片段或序列表中的其它核酸分子能够在某些情况下特异性地与其它核酸分子杂交。如本文中所用的，如果两个分子能够形成反平行、双链核酸结构，则可以说两个核酸分子能够彼此特异性地杂交。如果它们表现出完全互补性，则可以说某一核酸分子是另一核酸分子的互补序列。如果两个分子可以以足够的稳定性彼此杂交以允许它们在至少常规“低严格性”条件下保持彼此退火，则可以说它们是“最小互补”的。类似地，如果两个核酸分子可以以足够的稳定性彼此杂交以允许它们在常规“高严格性”条件下保持彼此退火，则可以说该分子互补。常规严格性条件由Sambrook等，(1989)和Haymes等，(1985)描述。
因此，偏离完全互补性是可允许的，只要这种偏离不完全排除分子形成双链结构的能力。因此，为了核酸分子或核酸分子的片段用作引物或探针，它只需要能够在所采用的特定溶剂和盐浓度下形成稳定双链结构的足够互补性。
促进DNA杂交的适宜严格性条件例如可以以相对低的盐和/或高温条件(如通过大约0.02M至大约0.15M NaCl和大约50℃至大约70℃提供的条件)用于需要高选择性的应用。例如，高严格性条件是用高严格性洗涤缓冲液(0.2x SSC或1x SSC,0.1％SDS,65℃)洗涤杂交过滤器至少两次。其它条件(如大约45℃下的6.0x氯化钠/柠檬酸钠(SSC)，随后在50℃下进行2.0x SSC的洗涤)也是本领域技术人员已知的且可以在Current Protocols inMolecular Biology(1989)中找到。例如，洗涤步骤中的盐深度可以选自从50℃下大约2.0xSSC的低严格性至50℃下大约0.2x SSC的高严格性。另外，洗涤步骤中的温度可以从室温(大约22℃)的低严格性条件到大约65℃的高严格性条件。温度和盐都可以变化，或者温度或盐浓度中任一可以保持恒定而另一变量发生改变。用于本发明中的核酸可以在这些条件下特异性地与来自线虫的一种或多种核酸分子或其互补序列杂交。在特定的实施方式中，用于本发明中的核酸与序列表中SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47所示的一个或多个核酸分子或其互补序列表现出至少大约80％或至少大约90％或至少大约95％或至少大约99％或甚至大约100％的序列同一性。
本发明的核酸也可以通过本领域中已知的方法完全或部分地合成，特别是在其中希望提供植物优先序列的情况中。因此，本发明的核酸的全部或一部分可以使用所选择的宿主优先的密码子合成。物种优先密码子可以例如选自在特定宿主物种中表达的蛋白质中最常用的密码子。核苷酸序列的其它修饰可以产生具有轻微改变的活性的突变体。
dsRNA或siRNA核苷酸序列包含聚合核糖核苷酸的双链且可以包括对磷酸糖骨架或核苷的修饰。RNA结构的修饰可以进行调整以允许特定的遗传抑制。在一个实施方式中，dsRNA分子可以通过酶学过程修饰以使得可以产生siRNA分子。siRNA可以在一些病原体中有效地介导一些目标基因的下调效应。这一酶学过程可以通过利用在昆虫、脊椎动物、真菌或植物细胞中存在的真核RNAi途径的RNAse III酶或DICER酶完成(Elbashir等，2001；Hamilton和Baulcombe,1999)。这一过程也可以利用通过本领域技术人员很受容易理解的重组DNA技术引入目标昆虫细胞中的重组DICER或RNAse III。DICER酶和RNAse III(天然存在于病原体中或通过重组DNA技术制备)将较大的dsRNA链切割成较小的寡核苷酸。DICER酶特异性地将dsRNA分子切割成各大约19-25核苷酸长度的siRNA段而RNAse III酶通常将dsRNA分子切割成12-15碱基对的siRNA。由任一酶产生的siRNA分子具有2-3个核苷酸的3’悬端及5’磷酸和3’羟基末端。通过RNAse III酶产生的siRNA分子与通过真核RNAi途径中的Dicer酶产生的相同，并因此随后在其后续解绕、分离成单链RNA和与目标基因转录的RNA序列杂交后成为固有的细胞RNA-降解机制的靶标并降解。这一过程导致有效降解或消除由病原体中目标基因的核苷酸序列编码的RNA序列。结果是病原体中特别靶向的核苷酸序列的沉默。酶学过程的详细说明可以在Hannon(2002)中找到。
本发明的核苷酸序列可以记录在计算机可读介质上。如本文中所用的，“计算机可读介质”指可以直接被计算机阅读和访问的任何有形的表达介质。这类介质包括，但不限于：磁性储存介质如软盘、硬盘、存储介质和磁带；光存储介质如CD-ROM；电存储介质如RAM和ROM；光学字符识别格式的计算机文件及这些类别的混合如磁/光存储介质。熟练技术人员可以很容易理解，任何目前已知的计算机可读介质可用于产生包含已在其上记录本发明的核苷酸序列的计算机可读介质的制品。
如本文中所用的，“记录”指将信息存储在计算机可读介质上的过程。熟练技术人员可以很容易地采用目前已知的用于在计算机可读介质上记录信息的任何方法以产生包含本发明的核苷酸序列信息的介质。多种数据存储结构可被熟练技术人员用于产生其上记录有本发明的核苷酸序列的计算机可读介质。数据存储结构的选择一般基于选择用于访问存储的信息的手段。另外，多种数据处理程序和格式可用于在计算机存储介质上存储本发明的核苷酸序列信息。该序列信息可以表示在字处理文本文件中，在商用软件如WordPerfect和Microsoft Word中编排，或者以ASCII文本文件的形式表示，存储在数据库应用(如DB2、Sybase、Oracle等)中。熟练技术人员可以很容易地采用多种数据处理结构化格式(例如文本文件或数据库)以获得其上存储有本发明的核苷酸序列信息的计算机可读介质。
可公开获得允许熟练技术人员访问计算机可读介质中提供的序列信息的计算机软件。在Sybase系统上执行BLAST(Altschul等，1990)和BLAZE(Brutlag等，1993)检索算法的软件可用于识别序列中的开放阅读框(ORFs)，如本文中提供的和包含与来自其它生物体的ORF或蛋白质的同源序列的Unigenes和EST。这类ORF是本发明的序列中的蛋白质编码片段且可用于产生具有商业重要性和蛋白质如用于氨基酸生物合成、代谢、转录、翻译、RNA加工、核酸和蛋白质降解、蛋白质修饰及DNA复制、限制、修饰、重组和修复的酶。
本发明进一步提供包含本文中描述的序列信息的系统，特别是基于计算机的系统。这类系统设计为识别本发明的核酸分子的具有商业重要性的片段。如本文中所用的，“基于计算机的系统”指用于分析本发明的核苷酸序列信息的硬件装置、软件装置和数据存储装置。本发明的基于计算机的系统的最小硬件装置包含中央处理器(CPU)、输入单元、输出单元和数据存储单元。熟练技术人员可很容易理解，任何当前可得的基于计算机的系统适用于本发明。
如本文中所用的，“靶结构基序”或“靶基序”指任何适当选择的序列或序列的组合，其中该一个或多个序列基于在靶基序折叠时形成的三维构型进行选择。存在本领域中已知的多种靶基序。蛋白质靶基序包括，但不限于酶活性位点和信号序列。核酸靶基序包括，但不限于启动子序列、顺式元件、发夹结构和诱导型表达元件(蛋白质结合序列)。
B.重组载体和宿主细胞转化
例如，重组DNA载体可以是线性的或封闭的环状质粒。载体系统可以是单一载体或质粒或者同时包含待引入细菌宿主的基因组中的总DNA的两个或多个载体或质粒。另外，细菌载体可以是表达载体。例如，如SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47所示的核酸分子或其互补序列或片段可适当地插入在一种或多种微生物宿主中发挥功能以驱动所连接的编码序列或其它DNA序列的表达的适当启动子控制下的载体中。许多载体可用于这一目的，且适宜载体的选择主要取决于待插入载体中的核酸的大小和将用该载体转化的特定宿主细胞。各载体包含取决于其功能(DNA的扩增或DNA的表达)和与其相容的特定宿主细胞的各种成分。用于细菌转化的载体成分一般包括，但不限于以下的一种或多种：信号序列、复制起点、一个或多个选择标记基因和允许外源DNA表达的诱导型启动子。
表达和克隆载体一般包含选择基因，也称为选择标记。这一基因编码在选择培养基中生长的转化宿主细胞存活或生长必需的蛋白质。典型的选择基因编码以下蛋白质：(a)赋予对抗生素或其它毒素(例如氨苄青霉素、新霉素、甲氨喋呤或四环素)的抗性，(b)补充营养缺陷型缺陷或(c)供应复杂培养基不提供的关键营养物(例如编码杆菌的D-丙氨酸消旋酶的基因)。成功地用异源蛋白质或其片段转化的那些细胞产生赋予药物抗性的蛋白质并因此在选择配方中存活。
用于产生mRNA的表达载体也可以包含被宿主细菌生物体识别并与核酸可操作地连接的诱导型启动子。适用于细菌宿主的诱导型启动子包括β-内酰胺酶启动子、大肠杆菌λ噬菌体PL和PR启动子及大肠杆菌半乳糖启动子、阿拉伯糖启动子、碱性磷酸酶启动子、色氨酸(trp)启动子和乳糖操纵子启动子及其变异体，以及杂合启动子如tac启动子。但是，其它已知的细菌诱导型启动子是合适的。
用于调控序列和结构核苷酸序列的术语“可操作地连接”意思是调控序列引起所连接的结构核苷酸序列的经调控的表达。“调控序列”或“控制元件”指位于结构核苷酸序列上游(5’非编码序列)、之中或下游(3’非翻译序列)的核苷酸序列，且其影响相关的结构核苷酸序列的转录、RNA加工或稳定、或翻译的时机和水平或量。调控序列可以包括启动子、翻译前导序列、内含子、增强子、茎-环结构、阻遏物结合序列和多腺苷酸化识别序列等。
包含一个或多个上列成分的合适载体的构建采用标准重组DNA技术。分离的质粒或DNA片段被切割、剪裁和重接合成产生所需要的质粒希望的形式。可用的细菌表达载体的实例包括，但不限于多功能的大肠杆菌克隆和表达载体如BluescriptTM(Stratagene,LaJolla,CA)(其中例如核酸或其片段可以接合到载体中与氨基末端Met和后续的β-半乳糖苷酶的7个残基的序列同框从而产生杂合蛋白质)、pIN载体(Van Heeke和Schuster,1989)等。
本发明还包括本发明的核苷酸序列转化到植物中以实现一种或多种dsRNA分子的线虫抑制性的表达水平。转化载体可以很容易地使用本领域中可得的方法制备。转化载体包含能够转录为RNA分子且基本上与目标线虫基因组编码的一种或多种核苷酸序列同源和/或互补的一个或多个核苷酸序列，从而由该一个或多个核苷酸序列转录的RNA被目标植物寄生线虫摄取时，线虫基因组的至少一个相应的核苷酸序列的表达下调。
转化载体可以称为dsDNA构建体且也可以定义为重组分子、疾病控制剂、遗传分子或嵌合遗传构建体。本发明的嵌合遗传构建体可以包含例如编码一个或多个反义转录物、一个或多个有义转录物、一个或多个各前述的转录物的核苷酸序列，其中由此获得的转录物的全部或部分与包含病原体基因组内的核苷酸序列编码的RNA序列的全部或部分同源。
在一个实施方式中，植物转化载体包含分离和纯化的DNA分子，其包含与本发明的一个或多个核苷酸序列可操作地连接的异源启动子。该核苷酸序列选自如序列表中所示的SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47及其互补序列。该核苷酸序列包括编码目标线虫RNA转录物中存在的RNA的全部或部分的片段且可以包含目标线虫RNA的全部或部分的反向重复序列。包含表达载体的DNA分子还可以包含位于编码序列上游或甚至编码序列中的功能内含子序列，且还可以包含位于启动子和翻译起始点之间的五一撇(5’)未翻译前导序列(即，UTR或5’-UTR)。
植物转化载体可以包含来自多于一个基因的序列，因此允许产生多于一种用于在目标线虫细胞中抑制两个或多个基因的表达的dsRNA。本领域技术人员很容易理解，其序列对应于不同基因中存在的序列的DNA的序列可组合成用于在转基因植物中表达的单个复合DNA片段。或者，已包含至少一个DNA片段的本发明的质粒可以通过在增强子和启动子或终止子序列之间顺序插入另外的DNA片段进行修饰。在设计用于抑制多个基因的本发明的疾病控制剂中，待抑制的基因可以由相同的植物寄生线虫物种获得以增强控制剂的效果。在某些实施方式中，基因可源自不同的植物寄生线虫以扩展该控制剂有效对抗的线虫的范围。当以多个基因作为抑制或表达和抑制的组合的目标时，多顺反子DNA元件可以如美国公开2004-0029283号中所表明和公开的制备。
在不同植物物种中发挥功能的启动子也是本领域中公知的。用于在植物中表达多肽的启动子包括如Odell等，(1985)所述的诱导型、病毒、合成或组成型启动子，和/或时间调节的、空间调节的和时间空间调节的启动子。可以用于本发明中的启动子包括增强的CaMV35S启动子和FMV35S启动子。也可以有利地使用表现出根特异性的CaMV35S启动子的片段。出于本发明的目的，可能希望在植物的根组织中获得这些基因的最高表达水平。多种根特异性启动子已经得到确认并且是本领域中已知的(例如美国专利5,110,732、5,837,848、5,459,252、Hirel等，1992)。
本发明的重组DNA载体或构建体可以包含赋予植物细胞选择表型的选择标记。选择标记也可用于选择包含编码本发明的多肽或蛋白质的外源核酸的植物或植物细胞。该标记可编码杀生剂抗性、抗生素抗性(例如，卡那霉素、G418博来霉素、潮霉素等)或灭草剂抗性(例如，草甘膦等)。选择标记的实例包括，但不限于编码卡那霉素抗性且可以使用卡那霉素、G418等选择的neo基因、编码双丙氨膦(bialaphos)抗性的bar基因、编码草甘膦抗性的突变EPSP合成酶基因、赋予对溴苯腈的抗性的腈水解酶基因、赋予咪唑啉酮或磺酰脲抗性的突变乙酰乳酸合成酶基因(ALS)和甲氨喋呤抗性DHFR基因。可获得赋予对氨苄青霉素、博来霉素、氯霉素、庆大霉素、潮霉素、卡那霉素、林可霉素、甲氨喋呤、草胺膦、嘌呤霉素、壮观霉素、利福平和四环素等的抗性的多重选择标记。这类选择标记的实例在美国专利5,550,318、5,633,435、5,780,708和6,118,047中说明。
本发明的重组载体或构建体还可以包括筛选标记。筛选标记可用于监测表达。典型的筛选标记包括β-葡糖苷酸酶或编码其各种发色底物已知的酶的uidA基因(GUS)(Jefferson等，1987)；各种荧光蛋白质(FP)基因中的一种或多种如绿色荧光蛋白(GFP)、红色荧光蛋白(RFP)或通常以特征性波长发荧光的一大族蛋白质中的任一种；R-基因座(其编码调节植物组织中花青素色素(红色)的产生的产物)(Dellaporta等，1988)；β-内酰胺酶基因(Sutcliffe等，1978)，编码其各种发色底物已知的酶的基因(例如，PADAC，显色头孢菌素)；荧光素酶基因(Ow等，1986)；编码可转化显色儿茶酚的儿茶酚加双氧酶的xylE基因(Zukowski等，1983)；α-淀粉酶基因(Ikatu等，1990)；编码能够将酪氨酸氧化为DOPA和多巴醌(dopaquinone)的酶的酪氨酸酶基因(Katz等，1983)，多巴醌随之缩合成黑色素；α-半乳糖苷酶，其催化显色α-半乳糖底物。
植物转化载体的实例包括源自根癌土壤杆菌(Agrobacterium tumefaciens)的Ti质粒的那些载体(例如，美国专利4,536,475、4,693,977、4,886,937、5,501,967和EP 0 122791)。毛根土壤杆菌(Agrobacterium rhizogenes)质粒(或“Ri”)也是可用的且是本领域中已知的。其它植物转化载体包括例如Herrera-Estrella(1983)、Bevan(1983)、Klee(1985)和EP 0 120 516公开的那些。
一般在，可能希望将非特异性位置的功能性重组DNA引入植物基因组中。在特殊的情况中，通过位点特异性整合插入重组DNA构建体可能是有用的。已知在植物中发挥功能的现有的几种位点特异性重组系统包括美国专利4,959,317中公开的cre-lox和美国专利5,527,695中公开的FLP-FRT。
据相信，用于本发明的合适的宿主细胞转化方法实际上包括可以将DNA引入细胞中的任何方法(参见，例如Miki等，1993)，如通过原生质体的转化(美国专利No.5,508,184；Omirulleh等，1993)、通过脱水/抑制介导的DNA摄取(Potrykus等，1985)、通过电穿孔(美国专利5,384,253)、通过用碳化硅纤维搅拌(Kaeppler等，1990；美国专利5,302,523和美国专利No.5,464,765)、通过土壤杆菌介导的转化(美国专利5,563,055；5,591,616、5,693,512、5,824,877、5,981,840、6,384,301)和通过DNA涂覆颗粒的加速(美国专利5,015,580、5,550,318、5,538,880、6,160,208、6,399,861、6,403,865；Padgette等，1995)等。通过应用如这些的技术，实际上任何物种的细胞可以稳定地转化。在多细胞物种的情况中，转基因细胞可以再生为转基因生物体。
最广泛使用的将表达载体引入植物中的方法是基于土壤杆菌的天然转化系统(例如，Horsch等，1985)。根癌土壤杆菌和毛根土壤杆菌是遗传转化植物细胞的植物病原性土壤细菌。根癌土壤杆菌和毛根土壤杆菌的Ti和Ri质粒分别携带负责植物的遗传转化的基因。用于土壤杆菌介导的基因转移的土壤杆菌载体系统和方法的由多种参考文献提供，包括Gruber等，1993、Miki等，1993、Moloney等，1989和美国专利4,940,838和5,464,763。其它天然地与植物相互作用的细菌如中华根瘤菌(Sinorhizobium)、根瘤菌(Rhizobium)和中生根瘤菌(Mesorhizobium)可以修饰以介导对许多不同的植物的基因转移。这些植物相关的共生细菌可使得通过获得去武装的Ti质粒和适宜的双运载体(binary vector)而处于基因转移的感受态(Broothaerts等，2005)。
用于产生转基因植物和特别是在植物中表达异源核酸的方法是已知的且可以用于本文提供的核酸以制备表现出对目标线虫摄食的较低易感性。例如，植物转化载体可以通过将本文公开的产生dsRNA的核酸插入植物转化载体中并将其引入植物中制备。一种已知的载体系统已通过改良根癌土壤杆菌的天然基因转移系统得到。该天然系统包含含有称为T-DNA的大片段的大Ti(肿瘤诱导)-质粒，其被转移到转化的植物。Ti质粒的另一片段，vir区域，负责T-DNA转移。T-DNA区域以末端重复序列为边界。在改良的双运载体中，肿瘤诱导的基因已被删除且vir区域的功能用于转移以T-DNA边界序列为边界的外源DNA。T-区域也可以包含用于有效回收转基因植物和细胞的选择标记及用于插入转移序列(如编码dsRNA的核酸)的多克隆位点。
使用土壤杆菌转化方法形成的转基因植物通常可能包含插入一个染色体中的单一的简单重组DNA序列，称为转基因事件。这类转基因植物可称为对于插入的外源序列为杂合的。对于转基因纯合的转基因植物可以通过使包含单一外源基因序列的独立隔离转基因植物(例如F0植物)与其自身有性交配(自交)以产生F1种子而获得。所产生的F1种子的四分之一将是对于转基因纯合的。萌发F1种子产生可以测试杂合性的植物，通亮使用SNP分析或使得能够区分杂合子和纯合子的热扩增分析(即，接合性分析)。将杂合植物与其自身或另一杂合植物杂交产生杂合后代以及纯合转基因和纯合空白后代。
C.核酸表达和目标基因抑制
举例来说，本发明提供了病原性目标生物体的转化的宿主植物、转化的植物细胞及它们的后代。转化的植物细胞和转化的植物可以进行生物工程以表达本文中所述的一种或多种在异源启动子控制下的dsRNA或siRNA序列，从而提供病原体防治效果。这些序列可以用于病原体中的基因抑制，从而在所保护的转化宿主生物体上降低病原体引起的疾病的水平和发病率。如本文中所用的，词语“基因抑制”意图指任何公知的用于降低由于基因转录为mRNA和随后mRNA的翻译而产生的蛋白质的水平的方法。
基因抑制也意指降低基因或编码序列的蛋白质表达，包括转录后基因抑制和转录抑制。转录后基因抑制是通过由作为抑制的基因或编码序列转录的mRNA的全部或部分和用于抑制的相应的双链RNA之间的同源性介导的，并且指实质和可测量地减少用于核糖体结合的细胞中可得的可利用mRNA量或阻止核糖体的翻译。转录的RNA可以是沿有义方向产生dsRNA以实现所谓的共抑制，沿反义方向产生dsRNA以实现所谓的反义抑制或沿两个方面产生dsRNA以实现所谓的RNA干扰(RNAi)。
转录抑制是通过在细胞中存在表现出与启动子DNA序列或其互补序列的基本序列同一性的dsRNA基因抑制剂以实现所谓的启动子反式抑制而介导的。基因抑制可以有效对抗与性状相关的原始植物基因以例如产生具有降低的该原始基因编码的蛋白质的水平或具有增高的或降低的感染代谢物的水平的植物。基因抑制也可以有效对抗摄食或接触含有基因抑制剂的植物材料的植物寄生线虫中的目标基因，该基因抑制剂特别设计为阻止或抑制线虫细胞中一种或多种同源或互补序列的表达。通过反义或有义定向的RNA的转录后基因抑制调节植物细胞中的基因表达公开于美国专利5,107,065、5,759,829、5,283,184和5,231,020中。dsRNA用于抑制植物中的基因的用途公开于WO 99/53050、WO 99/49029、美国公开2003/017596、美国专利申请公开2004/0029283中。
对植物寄生线虫的转录后基因抑制的有益方法采用有义定向和反义定向的，转录的RNA其是例如作为发夹或者茎和环结构稳定的。用于实现转录后基因抑制的DNA构建体的实例是其中第一片段编码表现出与作为抑制目标的基因的片段的基本同一性的、表现为反义定向的RNA，该第一片段连接到编码表现出与第一片段的基本互补性的RNA的第二片段上。这种构建体通过第一片段与第二片段的杂交形成茎和环结构及由连接该两个片段的核苷酸序列形成环结构(参见WO94/01550、WO98/05770、US 2002/0048814和US 2003/0018993)。也可以采用与另外的目标基因片段的共表达，如上所述(例如，WO05/019408)。
按照本发明的一个实施方式，提供了其体外表达导致转录与植物寄生线虫中目标基因的RNA分子基本同源的稳定RNA序列的核苷酸序列，该目标基因的RNA分子包含线虫基因组中的核苷酸序列编码的RNA序列。因此，在植物寄生的线虫摄食该稳定的RNA序列后，实现了目标线虫细胞中对应于目标基因的核苷酸序列的下调。
在本发明的特定实施方式中，可以利用SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ IDNO:47中任一核酸序列或其互补序列的至少21个连续核苷酸的片段的表达，包括最多21、36、60、100、550或1000个连续核苷酸的片段或表现出与这些序列的90-100％的同一性的序列或其互补序列的表达。在特定的实施方式中，本发明提供的核苷酸可以包含选自表4中所述的序列，包括如表4中所述的序列跨越核苷酸(sequence spanning nucleotide)上的位置。在再另外的实施方式中，本发明提供的核苷酸可以描述为包含SEQ ID NO:1、SEQ IDNO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ IDNO:27至SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQID NO:46或SEQ ID NO:47中一个或多个的核苷酸1-21、22-50、51-100、101-150、151-200、201-250、251-300、301-350、351-400、401-450、451-500、501-550、551-600、601-650、651-700、701-750、751-800、801-850、851-900、901-950、951-1000、1001-1050、1051-1100、1101-1150、1151-1200、1201-1250、1251-1300、1301-1350、1351-1400、1401-1450、1451-1500、1501-1550、1551-1600、1601-1650、1651-1700、1701-1750、1751-1800、1801-1850、1851-1900、1901-1950、1951-2000、2001-2050、2051-2100、23-75、76-125、126-175、176-225、226-275、276-325、326-375、376-425、426-475、476-525、526-575、576-625、626-675、676-725、726-775、776-825、826-875、876-925、926-975、976-1025、1026-1075、1076-1125、1126-1175、1176-1225、1226-1275、1276-1325、1326-1375、1376-1425、1426-1475、1476-1525、1526-1575、1576-1625、1626-1675、1676-1725、1726-1775、1776-1825、1826-1875、1876-1925、1926-1975、1976-2025、2026-2075、2076-2125、1-550、200-750、300-850、400-950、500-1050、600-1150、700-1250、800-1350、900-1450、1000-1550、1100-1650、1200-1750、1300-1850、1400-1950、1500-2050直至该序列的全长中一个或多个。用于选择特定的亚序列作为siRNA介导的基因抑制的靶标的方法是本领域中已知的(例如，Reynolds等，2004)。
使用本发明的稳定dsRNA技术抑制目标基因是序列特异性的，因为对应于RNA的双链区域的核苷酸序列作为基因抑制的靶标。包含与目标基因转录物的一部分相同的核苷酸序列的RNA在某些实施方式中可能对于抑制是有益的。具有相对于目标序列的插入、缺失和单点突变的RNA序列也已发现对于抑制是有效的。在进行本发明的特定实施方式中，抑制性dsRNA和目标基因的部分可以共有至少大约80％的序列同一性，或大约90％的序列同一性，或大约95％的序列同一性，或大约99％的序列同一性，或甚至大约100％的序列同一性。或者，RNA的双链区域可以功能性地定义为能够与目标基因转录物的一部分杂交的核苷酸序列。表现出更高同源性的不到全长的序列补偿了较长的低同源性序列。相同核苷酸序列的长度可以是至少大约25、50、100、200、300、400、500或至少大约1000碱基。正常地，长于20-100核苷酸的序列可能是希望的，尽管取决于目标基因的大小，长于大约200-300核苷酸的序列和长于大约500-1000核苷酸的序列可能特别地提供某些益处。在一个实施方式中，本发明具有能够耐受可预计由遗传突变、株多态性或进化趋异产生的序列变异的优势。引入的核酸分子可能不需要与目标序列绝对同源，且可能不需要相对于目标基因的初级转录产物或完全加工的mRNA的全长。因此，本领域技术人员可以理解，如本文中所公开的，实施本发明不需要RNA和目标基因之间的100％序列同一性。
目标基因表达的抑制可以通过测量内源目标RNA或由目标RNA的翻译产生的蛋白质定量，且抑制的结果可以通过检验细胞或生物体的外向特性确认。定量RNA和蛋白质的技术是本领域技术人员公知的。
在某些实施方式中，基因表达抑制至少10％，至少33％，至少50％或至少80％。在本发明的进一步的实施方式中，基因表达在病原体的细胞内抑制至少80％，至少90％，至少95％或至少99％，从而发生显著的抑制。显著抑制意思是指产生可检测表型(例如，生长中止、觅食、发育、繁殖降低、死亡等)的充分抑制或对应于所抑制的目标基因的RNA和/或蛋白质的可检测的减少。虽然在本发明的特定实施方式中，抑制在植物寄生线虫的基本上所有细胞中发生，在其它实施方式中，抑制仅在表达该基因的细胞亚群中发生。
dsRNA分子可以在体内或体外合成。dsRNA可以通过单个自身互补RNA链或由两个互补RNA链形成。细胞的内源RNA聚合酶可以介导体内转录或克隆的RNA聚合酶可用于体内或体外转录。抑制可以靶向于器官、组织或细胞类型中的特异性转录、环境条件(例如，感染、应激、温度、化学诱导剂)的刺激和/或发育阶段或时期的工程转录。RNA链可以进行或不进行多腺苷酸化，RNA链能够或不能够被细胞的翻译器官翻译成多肽。
本发明的RNA、dsRNA、siRNA或miRNA可以由本领域技术人员通过人工或自动反应化学地或酶学地产生或者在另一生物体中体内产生。RNA也可以通过部分或完全有机合成产生；任何修饰的核糖核苷酸可以通过体外酶学或有机合成引入。RNA可以通过细胞RNA聚合酶或噬菌体RNA聚合酶(例如，T3、T7、SP6)合成。表达构建体的用途和产生是本领域已知的(参见，例如，WO 97/32016、美国专利5,593,874、5,698,425、5,712,135、5,789,214和5,804,693)。如果化学合成或通过体外酶学合成，RNA可以在引入细胞之前纯化。例如，RNA可以通过溶剂或树脂的提取、沉淀、电泳、色谱或其组合由混合物纯化。或者，RNA可以无纯化或最小纯化使用以避免由于样品处理导致的损失。RNA可以干燥用于存储或溶解于水溶液中。溶液可以包含缓冲液或盐以促进退火和/或双链的稳定。
对于体内转基因或表达构建体的转录，调控区域(例如，启动子、增强子、沉默子(silencer)和多腺苷酸化)可用于转录RNA链(或多条链)。因此，在一个实施方式中，用于产生RNA分子的核苷酸序列可以可操作地连接在微生物、真菌或植物宿主细胞中发挥功能的一个或多个启动子。理想地，核苷酸序列置于通常存在于宿主基因组中的内源启动子的控制下。在可操作地连接的启动子序列的控制下的本发明的核苷酸序列可以进一步侧邻有利地影响其转录和/或所获得的转录物的稳定性的其它序列。这类序列一般位于可操作地连接的启动子的上游和/或表达构建体3’末端的下游，且可以同时存在于启动子的上游和表达构建体3’末端的下游，虽然也包括仅这种上游序列。
如本文中所用的，术语“疾病控制剂”或“基因抑制剂”指由第三RNA片段连接的第一RNA片段和第二RNA序列组成的特定RNA分子。第一和第二RNA片段存在于RNA分子的长度内且为彼此的基本反向重复序列和通过第三片段连接到一起。第一和第二RNA片段之间的互补性产生该两个片段在体内和体外杂交以形成在各第一和第二片段的一个末端由第三片段(其形成环)连接在一起的双链分子(即茎)的能力，从而整个结构形成茎和环结构，或甚至更紧密杂交的结构可以形成茎-环的节结构。第一和第二片段总是(但不是必然分别地)对应于与在意图通过摄取dsRNA分子抑制的目标病原体中由目标基因转录的目标RNA同源的有义和反义序列。控制剂也可以是基本纯化(或分离)的核酸分子且更特别地来自于基因组DNA(gDNA)或cDNA文库的核酸分子或其核酸片段分子。或者，片段可以包含具有大约15-大约250个核苷酸残基和包括大约15-大约30个核苷酸残基的较小的寡核苷酸。
如本文中所用的，术语“基因组”在用于植物寄生线虫的细胞或宿主时不仅包括细胞核中发现的染色体DNA还包括在细胞的亚细胞成分中发现的细胞器DNA。因此引入植物细胞中的本发明的DNA可以进行染色体整合或定位于细胞器中。术语“基因组”在应用于细菌时包括细菌宿主细胞内的染色体和质粒。因此引入细菌宿主细胞中的本发明的DNA可以进行染色体整合或定位于细胞器中。
如本文中所用的，术语“植物寄生线虫”指可以在经转化以表达双链基因抑制剂或涂覆双链基因抑制剂的宿主植物材料上感染、移殖、寄生或引起疾病的那些线虫。如本文中所用的，“线虫抗性”性状是引起转基因植物、转基因动物或其它转基因宿主抵抗通常能够对宿主造成损害或损失的线虫攻击的宿主特性。这种抗性可由天然突变或更典型地由引入赋予植物寄生线虫抗性的重组DNA产生。为向转基因植物施加线虫抗性，重组DNA例如可以转录成在重组植物的组织或流体内形成dsRNA分子的RNA分子。dsRNA分子部分地由与植物寄生线虫内的DNA序列编码的相应RNA片段相同的RNA的片段构成，该植物寄生线虫可引起宿主植物的疾病。目标植物寄生线虫内基因的表达受到dsRNA的抑制，且目标植物寄生线虫内基因表达的抑制产生对线虫具有抗性的植物。
dsRNA的调节效应可应用于在植物寄生线虫中表达的多种基因，包括例如负责细胞代谢或细胞转化的内源基因，包括管家基因、转录因子、蜕皮相关基因和编码参与细胞代谢或正常生长和发育的多肽的其它基因。
如本文中所用的，短语“基因表达的抑制”或“抑制植物寄生线虫细胞中目标基因的表达”指源自目标基因的蛋白质和/或mRNA产物的不存在(或其水平的可观察的降低)。特异性指抑制目标基因而没有对产生该dsRNA分子的细胞的其它基因的直接效应和没有对细胞内的任何基因的任何直接效应的能力。目标植物寄生线虫中目标内基因的基因表达的抑制可以在线虫中产生新的表型性状。
本发明部分地提供用于通过摄食宿主细胞或细胞的内容物递送线虫控制剂的递送系统。按照另一实施方式，本发明包括产生包含转录本发明的稳定dsRNA分子的重组DNA构建体的植物细胞或植物。如本文中所用的，“摄取”指试剂与目标生物体(如线虫)的细胞形成接触的过程。这可以例如通过线虫觅食、通过吮吸或通过注入发生。如本文中所用的，短语“产生转基因植物细胞或植物”指采用本领域中很容易得到的重组DNA技术(例如，Sambrook等，1989)构建转录本发明的稳定dsRNA分子的植物转化载体的方法，从而转化植物细胞或植物和产生包含转录的、稳定的dsRNA分子的转基因植物细胞或转基因植物。
可预想的是，本发明的组合物可作为整合到植物细胞基因组中的重组基因的表达产物或通过并入在播种前涂覆到种子上的涂层或种子处理剂中而并入植物物种的种子中。包含重组基因的植物细胞在本文中被认为是转基因事件。
本发明部分地提供用于向植物寄生的线虫递送疾病控制剂的递送系统。本发明的稳定dsRNA或siRNA分子可以直接引入植物寄生线虫的细胞中。用于引入的方法可以包括RNA与用于植物寄生线虫的宿主组织直接混合以及其中作为宿主的物种经遗传工程以表达dsRNA或siRNA的遗传工程途径。在一个实施方式中，RNA可以喷射到植物表面上。在再另一实施方式中，dsRNA或siRNA可以由微生物表达且微生物可以应用到植物表面上或通过物理手段(如注射)引入根、茎中。在再另一实施方式中，植物可以进行遗传工程以表达足以杀灭已知侵染植物的植物寄生线虫的量的dsRNA或siRNA。
也可以预想，通过化学或酶学合成产生的dsRNA可以以与常见农业实践协调的方式配制且用作用于防治植物疾病的喷涂产品。该制剂可以包括有效叶覆盖所需的适当的粘着剂(sticker)和湿润剂以及保护dsRNA免受UV损伤的UV保护剂。这类添加剂是生物杀虫剂工业中通常使用的且是本领域技术人员公知的。这些应用可以与其它喷涂杀虫剂应用(基于生物或不基于生物)以增强植物寄生线虫的植物保护。
本发明还涉及用于在微生物中表达的重组DNA构建体。外源核酸(目标RNA由其转录)可以使用本领域已知的方法引入微生物宿主细胞中，如细菌细胞或真菌细胞。
本发明的核苷酸序列可以引入很多种的原核和真核微生物宿主中以产生稳定的dsRNA或siRNA分子。术语“微生物”包括原核和真核物种如细胞和真菌以及线虫。真菌包括酵母和丝状真菌等。例证的原核生物(革兰氏阴性的和革兰氏阳性的)包括肠杆菌科(Enterobacteriaceae)如埃希氏菌属(Escherichia)、欧文氏菌属(Erwinia)和沙雷氏菌属(Serratia)；芽胞杆菌科(Bacillaceae)；根瘤菌科(Rhizobiaceae)如根瘤菌属(Rhizobium)；螺菌科(Spirillaceae)如发光菌属(photobacterium)、酵单胞菌属(Zymomonas)；乳杆菌科(Lactobacillaceae)；假单胞菌科(Pseudomonadaceae)如假单胞菌属(Pseudomonas)和醋酸杆菌属(Acetobacter)；固氮菌科(Azotobacteraceae)，放线菌目(Actinomycetales)和硝化杆菌科(Nitrobacteraceae)。真核生物中包括真菌如藻状菌纲(Phycomycetes)和子囊菌纲(Ascomycetes)，包括酵母属(Saccharomyces)和裂殖酵母属(Schizosaccharomyces)，和担子菌纲(Basidiomycetes)如红酵母属(Rhodotorula)、短梗霉属(Aureobasidium)等。
D.转基因植物和细胞
毛根特征在于快速生长、频繁分枝、斜生现象。毛根用作全根的强模型(strongmodel)，因为它们保持类似于完整植物的根合成化合物的能力(David等，1984)。已很好地建立了用于转移和整合位于毛根土壤杆菌的根诱导质粒Ri上的基因到植物基因组中及其用于在其中的表达的方法(White和Nester,1980)。这些类型的根在不含激素的培养基在体外继续生长且还表现出高水平的遗传稳定性(Aird等，1988)。土壤细菌毛根土壤杆菌将基因转化到宿主植物基因组中的天然能力导致根在感染位点形成，且用于产生毛根培养物。用毛根土壤杆菌菌株R-1000感染植物导致T-DNA整合到植物基因组中和表达，这引起毛根的发育。毛根培养物快速生长，显示斜生的根生长且在无激素的培养基上高度分枝。由毛根土壤杆菌诱导的转基因毛根在体外支持根结线虫(“RKN”；根结线虫类)的完整生活周期，并允许大规模的组织快速生长，这可用于鉴别和分离目标基因。毛根如下面所述且按照本领域中的知识由大豆或西红柿植物开始。
本发明提供具有一个或多个转基因事件的种子和植物。事件的组合称作“堆叠的(stacked)”转基因事件。这些堆叠的转基因事件可以是针对相同目标生物体的事件，或者它们可以针对不同目标病原体或害虫。在一个实施方式中，具有表达本文提供的核酸的能力的种子也具有表达至少一种其它物质的能力，包括但不限于其序列源自于目标病原体(如线虫)中表达的RNA序列且在种子或由该种子生长的植物细胞中表达时形成双链RNA结构的RNA分子，其中一个或多个植物细胞被目标摄食导致目标的细胞中RNA表达的抑制。
在某些实施方式中，具有表达其序列源自目标植物寄生线虫的dsRNA的能力也具有提供除草剂耐受性的“堆叠的”转基因事件。除草剂耐受性基因的一个有益的实例提供对草甘膦(N-(膦酰基甲基)氨基乙酸)的抗性，包括这种除草剂的异丙胺盐形式。
本发明提供的益处可以包括，但不限于：将dsRNA引入植物寄生线虫细胞中的方便性、可使用的dsRNA的低浓度、dsRNA的稳定性和抑制的有效性。使用低浓度的稳定dsRNA的能力避免了反义干扰的几种缺陷。本发明不限于体外用途或特定的序列组成，不限于特定系列的目标基因、目标基因的核苷酸序列的特定部分或特定转基因，或不限于特定的递送方法，这与某些本领域中已知的可用技术(如反义和共抑制)相反。此外，在不是经典遗传模型的生物体中进行遗传操作是可能的。
为了实现希望防治的植物寄生线虫物种内的目标基因的物性抑制，目标基因应通常表现出与植物或脊椎动物中的相应基因的低水平的序列同一性。在特定的实施方式中，序列同一性的水平低于大约80％，包括低于大约70％和低于大约60％。
除了用重组DNA构建体直接转化植物外，转基因植物可通过使具有重组DNA构建体的第一植物与缺乏该构建体的第二植物杂交。例如，用于基因抑制的重组DNA可引入需转化的第一植物家系以产生可与第二植物家系杂交的转基因植物，从而将用于基因抑制的重组DNA渗入第二植物家系中。
实际上，本发明可以与植物中的其它疾病防治性状组合以获得希望的用于增强植物疾病防治的性状。采用完全不同的作用模式的组合疾病防治性状可以提供具有优于携带单一防治性状的植物的保护转基因植物的耐久性，因为其在田间发展出抗性的可能性降低。
本发明还涉及包含本发明的一种或多种序列的商品，且特别地包括包含本发明的一种或多种核苷酸序列的重组植物或种子产生的商品作为本发明的实施方式。包含本发明的一种或多种序列的商品意图包括，但不限于包含本发明的一种或多种序列的重组植物或种子的粗磨粉、油或磨碎的或完整的谷粒。在本文中包括的一种或多种货物或商品中本发明的一种或多种序列的检测是该货物或商品由设计为为了使用dsRNA介导的基因抑制方法防治植物疾病的目的而表达本发明的一种或多种核苷酸序列转基因植物组成。
E.获得核酸
本发明提供了用于获得包含产生dsRNA或siRNA的核苷酸序列的核酸的方法。在一个实施方式中，这种方法包括：(a)分析在线虫中的dsRNA介导的基因抑制时一种或多种目标基因的表达、功能和表型；(b)用包含来自目标线虫的核苷酸序列或其同源序列的全部或部分的杂交探针探测cDNA或gDNA文库，该目标线虫在dsRNA介导的抑制分析中表现出改变的(例如，降低的)线虫生长、发育或繁殖表型；(c)鉴别与该杂交探针杂交的DNA克隆；(d)分离在步骤(b)中鉴别的DNA克隆；和(e)测序包含步骤(d)中分离的克隆的cDNA或gDNA片段，其中测序的核酸分子转录RNA核苷酸序列或其同源序列的全部或主要部分。
在另一实施方式中，本发明的用于获得包含产生dsRNA或siRNA的主要部分的核苷酸序列的核酸序列的方法包括：(a)合成对应于来自目标病原体的一种核苷酸序列的一部分的第一和第二寡核苷酸引物；和(b)使用步骤(a)的第一和第二寡核苷酸引物扩增存在于克隆载体中的cDNA或gDNA插入序列，其中扩增的核酸分子转录本发明的dsRNA或siRNA的主要部分。
在实施本发明时，目标基因可以源自南方根结线虫或另一线虫。可预想的是，可采用几种标准选择目标基因。根结线虫类基因可以是具有秀丽隐杆线虫同源基因的基因，在表达的RNAi敲落时具有强表型(如P0表型)的高可能性。这些目标通常是参与核心细胞过程(如DNA复制、细胞周期、转录、RNA加工、翻译、蛋白质运输、分泌、蛋白质修饰、蛋白质稳定和降解、能量产生、中间代谢、细胞结构、信号转导、通道和转动蛋白及内吞作用)蛋白质产物的那些目标基因。在某些实施方式中，有利的是选择其表达水平小的下降会导致对病原体的不利影响的基因。特别感兴趣的是具有其它垫刃线虫(例如，大豆胞囊线虫)中的RNAi验证直系同源基因的根结线虫基因。
如本文中所用的，术语“源自”指可以从特别指定的来源或物种获得的指定的核苷酸序列，尽管不是必然直接得自该指定的来源或物种。
在一个实施方式中，选择实质上参与植物寄生线虫的生长、发育或繁殖的基因。用于本发明中的其它目标基因可以包括例如，在线虫存活、生长、发育、感染和建立觅食位点中发挥重要作用的那些基因。这些目标基因可以是管家基因、转录基因等之一。另外，用于本发明中的核苷酸序列也可以源自植物、病毒、细菌或昆虫基因的同源基因(包括直系同源基因)，其功能已经由文献确定和其核苷酸序列与目标线虫基因组的目标基因共有实质的相似性。按照本发明的用于线虫防治的一个方面，目标序列可以基本源自目标植物寄生线虫。一些克隆自线虫的典型目标序列(其编码作为已知蛋白质的同源物或直系同源物的蛋白质或其片段)可以在序列表中找到，例如在SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQID NO:19、SEQ ID NO:21、SEQ ID NO:23和SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47中。
出于本发明的目的，dsRNA或siRNA分子可以通过源自gDNA或cDNA文库或其部分的目标基因序列的聚合酶链(PCR)扩增获得。DNA文库可以使用本领域技术人员已知的方法制备，且可以提取DNA/RNA。由目标生物体产生的基因组DNA或cDNA文库可以用于产生dsRNA或siRNA的PCR扩增。
然后，目标基因序列可以进行PCR扩增并使用本领域中容易获得的方法测序。本领域技术人员也许能够改变PCR条件以确保最佳的PCR产物形成。确认的PCR产物可用作体外转录的模板以利用包括的最小启动子产生有义和反义RNA。在一个实施方式中，本发明包含可用作植物寄生线虫防治剂的分离和纯化的核苷酸序列。分离和纯化的核苷酸序列可以包含如序列表中所示的那些核苷酸序列。
如本文中所用的，短语“编码序列”、“结构核苷酸序列”或“结构核酸分子”指当置于适当的调控序列控制下翻译成多肽(通常通过mRNA)的核苷酸序列。编码序列的边界通过5’末端的翻译起始密码子和3’末端的翻译终止密码子确定。编码序列可以包括，但不限于基因组DNA、cDNA、EST和重组核苷酸序列。
术语“重组DNA”或“重组核苷酸序列”指包含通过经由诱变、限制性酶等的操作的遗传工程修饰的DNA。
对于作为本发明防治的潜在靶标的许多植物寄生的线虫，可能存在有关大多数基因的序列或由特定基因的突变产生的表型的有限信息。因此，可以预想，选择用于本发明中的适当基因可以通过使用由模型生物体(如秀丽隐杆线虫)中的相应基因的研究提供的信息完成，其中该基因已经按照实施例1-8所述的分析方法鉴定。在某些情况中，有可能通过利用基因的名称或来自例如该基因由其克隆的线虫的序列检索数据库(如GenBank)而获得来自目标线虫的相应基因的序列。一旦获得了序列，PCR可以用于扩增用于本发明的目标线虫中基因的适当选择的片段。PCR引物可以基于在该基因由其克隆的另一生物体中发现的序列进行设计。该引物设计为扩增用于本发明中的足够长的DNA片段。DNA(基因组DNA或cDNA)从目标植物寄生线虫制备，且PCR引物用于扩增该DNA片段。选择扩增条件以使得扩增甚至在引物未精确匹配目标序列的情况下发生。或者，基因(或其部分)可以使用已知基因作为探针从由植物寄生线虫物种制备的gDNA或cDNA文库克隆。用于进行PCR和由文库进行克隆的技术是已知的。在实施例中提供了来自目标植物寄生线虫的DNA片段可以基于预先由其它物种克隆的基因序列分离的方法的进一步细节。本领域技术人员可认识到，多种技术可用于分离与预先由其它物种分离的基因对应的来自目标植物寄生线虫的基因片段。
实施例
本发明人在此确定了用于通过向植物寄生的线虫提供双链核糖核酸分子防治植物寄生线虫的方法和选择编码这些双链核糖核酸分子的序列的方法。在摄食时发挥作用以抑制线虫中的生物功能的双链核糖核酸分子可以例如导致以下的一种或多种特性：线虫生长的下降、线虫发育的抑制或者生存力或卵产生的下降。这些特性的任一种或任意组合可导致有效抑制植物感染或移殖，且在植物病原线虫的情况中，导致植物疾病的抑制和/或疾病症状严重性的降低。
实施例1
毛根产生方案
对于大豆A3244(易感的)或PI88788(抗性的)毛根，毛根土壤杆菌菌株K599(NCPPB2659；NCPPB,Sand Hutton,York,UK)在LB(Luria Bertani)或酵母提取物和蛋白胨(YEP)培养基上生长和维持。酵母提取物是自溶的酵母的水溶性部分。自溶小心地进行控制以保持天然存在的B复合维生素。酵母提取物通常通过在富含碳水化合物的植物培养基中生长面包酵母(baker’s yeast)(酵母属的种)制备。酵母在水中收获、洗涤和重悬浮，酵母在其中发生自溶，即利用酵母的酶自体消化。酵母提取物是这一自溶作用的全部可溶部分。自溶活性通过加热步骤终止。所获得的酵母提取物过滤透明并通过喷雾干燥干燥成粉末。使用毛根土壤杆菌菌株D1产生转基因西红柿Mountain Spring(易感的)或Fresh Mountain Plus(抗性的)毛根培养物的方法是相似的，除了使用包含酵母提取物、NaCl、胰蛋白胨、L-谷氨酸、磷酸钾、硫酸镁和生物素的MgL培养基外。大豆种子通过在受控条件下接触氯气12-16小时进行表面消毒，接着在清洁空气罩中通气至少30分钟。种子在含有1/4MS(Murashige&Skoog,1962)的皮氏培养皿中发芽。6天龄幼苗的胚轴或子叶使用手术刀切割，且切割的子叶随后浸入新生长的含目标DNA构建体的毛根土壤杆菌培养物中并真空渗透。子叶在用于种子发芽的相似条件下培养，除了抗生素头孢噻肟加入到1/4MS琼脂培养板中以防止毛根土壤杆菌随后生长过度。不定根与用毛根土壤杆菌接种的胚轴或子叶切开。假定转化的根在包含3％蔗糖+3g/l BASTA和头孢噻肟的Gamborg's B-5琼脂(Gamborg等，1976)上培养。通过选择存活的根转移到新鲜培养基中并保持在无光、大约24-30℃的温度下在培养箱中的Gamborg's B-5琼脂上。通常每2-4周切割一段根尖并转移到新鲜培养基中。
实施例2
毛根材料上的线虫生物分析
在选择毛根系后，用于植物线虫生物分析的根转移到含Gamborg's B-5培养基的新鲜培养基板上并使其生长大约两周以在用根结线虫(RKN)的第二阶段幼虫接种之前提供用于线虫感染的充足组织。单个的毛根尖置于感染板上。通常，大约20个板用于转化的根与SCN或RKN反应的病原性测试。各板包含来自单独整合的转化根。包含转化的(空载体)SCN-易感或RKN-易感的对照的另外20个板和包含转化的SCN-抗性或RKN-抗性对照的另外20个板包括在测试中。所采用的方案主要是Narayanan等，1999的方案，具有小的改进。板用大约450灭菌南方根结线虫J2s接种并在26-28℃孵育。在用南方根结线虫接种后大约两周，感染的大豆毛根从琼脂板移除并计数虫瘿的数目。虫瘿评分参照如下的评定标准基于对大小的评估进行评定：最小的虫瘿得分为1，且随着虫瘿面积变得更大，虫瘿评分提高。虫瘿评定标准的实例如图2中所示。然后1-4的标度用于评定试验中各板上的各虫瘿。
也在42天针对西红柿毛根中的RKN感染对卵数目评分(例如，参见表4)。在感染后42天，板进行微波处理并过筛以收集根。然后根在10％漂白溶液中混合并倾到一系列筛上以去除根碎片并收集卵。从各板去除卵并计数，且根进行称重。
制备灭菌的SCN和RKN幼虫以用于毛根培养系统。灭菌SCN J2如下产生：收集清洁的大豆胞囊线虫卵(即除去土壤和其它碎片的卵)并置于包含30ml的10％漂白溶液的50ml离心管中。漂白溶液温和搅拌并然后使其沉降2-3分钟。该管再次温和搅拌以重悬浮卵并然后以1000rpm离心1分钟。在无菌罩下，漂白溶液移入容器中，且25ml的无菌水加入卵管中。该管在无菌罩下重新加盖，温和搅拌以重悬浮卵和以1000rpm离心1分钟。在无菌罩下，将这一液体倾出且再将25ml的无菌水置于管中。该管在无菌罩下重新加盖，并重复搅拌和离心过程。用无菌水洗涤卵的这一过程重复大约4次以将漂白剂从卵彻底漂洗掉。在无菌罩下最后漂洗之后，除去液体以遗留大约1-2ml的卵浓缩物。灭菌卵通过在静置在5mM硫酸锌溶液中深度恰覆盖滤纸表面的潮湿滤纸上孵育卵而进行孵化。在2-3天后，J2幼虫收集在滤纸下的溶液中。J2幼虫进行离心并进一步使用洗必泰灭菌(Atkinson等，1996)。
灭菌RKN幼虫通过将切碎的RKN感染的根置于具有足够量的10％漂白溶液的搅拌机中收集卵而制备。搅拌机以5秒间隔开关。这一过程重复5-8次。然后根浆通过一系列筛，其中卵和小碎片收集在500微米筛中。将任何残留的漂白溶液从这一卵/碎片彻底漂洗掉。20毫升的卵/碎片加入50ml锥形管中且20ml的40％蔗糖溶液加入管底，达到40毫升的总体积。这一溶液然后以3750rpm离心5分钟以将卵与碎片分离。在离心后，将卵移出并彻底漂洗以除去任何残留的蔗糖溶液。然后将卵置于包含用恰好足够的加气自来水润湿的滤纸以覆盖卵的孵化盘中。1-2天后，J2幼虫收集在滤纸下的溶液中。J2幼虫进行离心并进一步使用洗必泰灭菌(Atkinson等，1996)。
实施例3
适合通过RNA干扰广谱防治根结线虫物种的根结线虫目标基因
表1描述了具有高核苷酸保守性的关键线虫基因核苷酸序列(预测的或编码的多肽列于表2中)，其有助于根结线虫类的基于RNAi的广谱防治。但是这些序列与非目标生物体(如人类、宿主植物和有益昆虫)中的关键基因具有足够的核苷酸差异以在暴露于线虫dsRNA时降低对这些另外的生物体产生毒性的可能性。
为确认特征秀丽隐杆线虫基因的南方根结线虫直系同源基因，使用BLAST程序套装(Altschul等，1990)进行同源性检索。使用秀丽隐杆线虫基因(Wormpep)的预测蛋白质序列，TBLASTN用于检索大豆胞囊线虫成簇(clustered)EST和其它由Genbank获得的序列以及最近产生的专有基因组勘测序列(genome survey sequences)。追踪Bitscore、e-值和％同一性。对于作为靶标，秀丽隐杆线虫基因必须以至少e-10或更高的e-值具有与南方根结线虫中的直系同源基因或同源序列的TBLASTN匹配。具有SCN RNAi正向直系同源基因的RKN基因赋予高优先级。
表1：核苷酸根结线虫目标序列
表1中所列的序列与各种根结线虫中的类似(例如，直系同源的或同源的)EST和其它序列对比如下：
SEQ ID NO:1对比：根结线虫EST,爪哇根结线虫gb|BI324357.1|(98％同一性,1630-2138),花生根结线虫gb|BI747188.1|(99％同一性,1687-2157),北方根结线虫gb|BM884829.1|(92％同一性,947-1461),南方根结线虫gb|BM882920.1|(98％同一性,1880-2131),奇特伍德根结线虫gb|CB933476.1|(87％,1707–2060；94％,2166-2239)。
SEQ ID NO:3对比：根结线虫EST,南方根结线虫gb|CF980444.1|(99％同一性,1-662),南方根结线虫gb|CF980487.1|(98％同一性,24-654),南方根结线虫gb|CK984584.1|(94％同一性,1-662),南方根结线虫gb|CK984580.1|(94％同一性,1-692),南方根结线虫gb|CK984571.1|(94％同一性,1-691),南方根结线虫gb|CK984607.1|(95％同一性,1-633),南方根结线虫gb|CF980817.1|(94％同一性,1-633),南方根结线虫gb|CK983672.1|(95％同一性,44-626),南方根结线虫gb|CN443018.1|(95％同一性,33-611),爪哇根结线虫gb|BG736305.1|(97％同一性,222-725),北方根结线虫gb|CF803679.1|(88％同一性,1-645),北方根结线虫gb|CF803689.1|(88％同一性,1-644),北方根结线虫gb|CF803678.1|(88％同一性,1-645),南方根结线虫gb|CN443180.1|(90％同一性,1-519),爪哇根结线虫gb|BI745534.1|(95％同一性,277-477；92％同一性,481-692)。
SEQ ID NO:5对比：根结线虫EST,花生根结线虫gb|CF357444.1|(99％同一性,1-552),北方根结线虫gb|CF357444.1|(92％同一性,892-1425),北方根结线虫gb|CN577502.1|(91％同一性,879-1425),北方根结线虫gb|BU094011.1|(94％同一性,1-438),花生根结线虫gb|BI747707.1|(100％同一性,1208-1425),南方根结线虫gb|AW829176.1|(92％同一性,662-879)。
SEQ ID NO:7对比：根结线虫EST,花生根结线虫gb|CF358371.1|(100％同一性,912-1520),花生根结线虫gb|BI501616.1|(99％同一性,1324-1846),花生根结线虫gb|BI746542.1|(96％同一性,1288-1848),南方根结线虫gb|AW829423.1|(96％同一性,1390-1851),南方根结线虫gb|AW871188.1|(96％同一性,1461-1851),南方根结线虫gb|CN443525.1|(98％同一性,1-256),南方根结线虫gb|CN443511.1|(98％同一性,1-256),南方根结线虫gb|CK984263.1|(98％同一性,1-256),南方根结线虫gb|CK984287.1|(98％同一性,1-256),南方根结线虫gb|CK984492.1|(96％同一性,1-256),南方根结线虫gb|CK984445.1|(96％同一性,1-256),南方根结线虫gb|CK984390.1|(96％同一性,1-256),南方根结线虫gb|CK984366.1|(96％同一性,1-256),南方根结线虫gb|BI703863.1|(96％同一性,1745-1851),南方根结线虫gb|BE239094.1|(96％同一性,1783-18549)。
SEQ ID NO:9对比：根结线虫EST,南方根结线虫gb|CF099482.1|(99％同一性,1-678),花生根结线虫gb|BI745992.1|(100％同一性,1-503),花生根结线虫gb|BI863117.1|(99％同一性,1-452),南方根结线虫gb|BE239183.1|(99％同一性,351-741),南方根结线虫gb|BM880799.1|(99％同一性,147-504)。
SEQ ID NO:11对比：根结线虫EST,同一性98-100％的3’-区域678-1122中多于100南方根结线虫、爪哇根结线虫和花生根结线虫EST的集合。
SEQ ID NO:13对比：无检测的根结线虫EST匹配。
SEQ ID NO:15对比：根结线虫EST,花生根结线虫gb|BI745894.1|(97％同一性,2611-3101),北方根结线虫gb|BM883184.1|(91％同一性,2079-2556),奇特伍德根结线虫gb|CB933531.1|(85％同一性,2472-2990),北方根结线虫gb|CN574157.1|(88％同一性,2827-3225)。
SEQ ID NO:17对比：无检测的根结线虫EST匹配。
SEQ ID NO:19对比：根结线虫EST,M.paranaensis gb|CK426467.1|(99％同一性,61-714),南方根结线虫gb|BM880509.1|(99％同一性,248-789),爪哇根结线虫gb|BG736965.1|(96％同一性,243-746),南方根结线虫gb|BM881612.1|(99％同一性,370-827；100％同一性,266-325),爪哇根结线虫gb|BG736194.1|(96％同一性,246-721),爪哇根结线虫gb|BG737132.1|(96％同一性,243-663),南方根结线虫gb|BM880760.1|(99％同一性,393-732；100％同一性,259-392),爪哇根结线虫gb|BG736127.1|(96％同一性,243-600)。
SEQ ID NO:21对比：根结线虫EST,爪哇根结线虫gb|BI744615.1|(98％同一性,300-700),南方根结线虫gb|CN443444.1|(100％同一性,1-237)。
SEQ ID NO:23对比：根结线虫EST,爪哇根结线虫gb|BE578613.1|(97％同一性,1-606),花生根结线虫gb|BI747271.1|(98％同一性,1-504),南方根结线虫gb|CD749365.1|(97％同一性,27-600),爪哇根结线虫gb|BI143067.1|(99％同一性,1-437),北方根结线虫gb|CF370648.1|(91％同一性,1-599),北方根结线虫gb|BM902290.1|(91％同一性,73-621),北方根结线虫gb|CN572891.1|(85％同一性,259-667)。
SEQ ID NO:25对比：根结线虫EST,南方根结线虫gb|CD749648.1|(100％同一性,1-690),南方根结线虫gb|CF099489.1|(99％同一性,15-676),花生根结线虫gb|BI746205.1|(97％同一性,203-644)。
SEQ ID NO:40对比：根结线虫EST,M.paranaensis gb|CK426467.1|(98％同一性,130-783),南方根结线虫gb|BM880509.1|(99％同一性,317-858),爪哇根结线虫gb|BG736965.1|(96％同一性,312-815),南方根结线虫gb|BM881612.1|(90％同一性,325-896；100％同一性),爪哇根结线虫gb|BG736194.1|(96％同一性,315-790),爪哇根结线虫gb|BG737132.1|(96％同一性,312–732),南方根结线虫gb|BM880760.1|(90％同一性,328-801),爪哇根结线虫gb|BG736127.1|(95％同一性,312-669)。
SEQ ID NO:42对比：根结线虫EST,南方根结线虫gb|CD749648.1|(99％同一性,1-675),南方根结线虫gb|CF099489.1|(99％同一性,1-661),花生根结线虫gb|BI746205.1|(96％同一性,185-629)。
SEQ ID NO:44对比：根结线虫EST,爪哇根结线虫gb|BG735889.1|(98％,1-255),爪哇根结线虫gb|BG736205.1|(93％,103-201)。
表2列出了由表1的核苷酸序列编码的预测氨基酸序列。
表2：RKN氨基酸序列
表2的氨基酸序列与各种线虫物种中的假定的直系同源的或同源的氨基酸序列对比如下：
SEQ ID NO:2与其线虫直系同源基因具有以下氨基酸同一性，71％马来丝虫(B.malayi),65％秀丽隐杆线虫,66％C.briggsae。
SEQ ID NO:4与其线虫直系同源基因具有以下氨基酸同一性,60％大豆胞囊线虫,47％秀丽隐杆线虫,48％C.briggsae,44％马来丝虫。
SEQ ID NO:6与其线虫直系同源基因具有以下氨基酸同一性,94％马来丝虫,93％秀丽隐杆线虫,92％C.briggsae。
SEQ ID NO:8与其线虫直系同源基因具有以下氨基酸同一性,62％马来丝虫,67％秀丽隐杆线虫,66％C.briggsae。
SEQ ID NO:10与其线虫直系同源基因具有以下氨基酸同一性,68％马来丝虫,69％秀丽隐杆线虫,69％C.briggsae。
SEQ ID NO:12与其线虫直系同源基因具有以下氨基酸同一性,78％O.volvulus,78％马来丝虫,71％秀丽隐杆线虫,75％C.briggsae。
SEQ ID NO:14与其线虫直系同源基因具有以下氨基酸同一性,82％马来丝虫,83％秀丽隐杆线虫,83％C.briggsae。
SEQ ID NO:16与其线虫直系同源基因具有以下氨基酸同一性,58％马来丝虫,53％秀丽隐杆线虫,55％C.briggsae。
SEQ ID NO:18与其线虫直系同源基因具有以下氨基酸同一性,83％马来丝虫,86％秀丽隐杆线虫,86％C.briggsae。
SEQ ID NO:20与其线虫直系同源基因具有以下氨基酸同一性,22％马来丝虫,26％秀丽隐杆线虫,22％C.briggsae。
SEQ ID NO:22与其线虫直系同源基因具有以下氨基酸同一性,51％马来丝虫,49％秀丽隐杆线虫,49％C.briggsae。
SEQ ID NO:24与其线虫直系同源基因具有以下氨基酸同一性,54％马来丝虫,55％秀丽隐杆线虫,55％C.briggsae。
SEQ ID NO:26与其线虫直系同源基因具有以下氨基酸同一性,66％马来丝虫,66％秀丽隐杆线虫,68％C.briggsae。
SEQ ID NO:41与其线虫直系同源基因具有以下氨基酸同一性:28％马来丝虫；30％秀丽隐杆线虫；27％C.briggsae。
SEQ ID NO:43与其线虫直系同源基因具有以下氨基酸同一性:70％马来丝虫；69％秀丽隐杆线虫；69％C.briggsae。
SEQ ID NO:45与其线虫直系同源基因具有以下氨基酸同一性:77％秀丽隐杆线虫；77％C.briggsae。
实施例4:
dsRNA序列和用于基因表达的启动子
图1显示了用于选择组合的化学物质和红色荧光(DsRed标记；例如，Clontech,Mountain View,CA)的毛根表达构建体以产生具有靶向于目标线虫的存活、适合度或病原性的目标基因(GOI)的均匀表达的毛根的典型示意图。GOI可以是杀线虫的核苷酸、蛋白质或能够产生杀线虫剂(例如，代谢物)的蛋白质，且通常是靶向于关键线虫基因的双链RNA(dsRNA)。卡那霉素抗性用于细菌宿主内的质粒扩增。(草铵膦:草胺膦)耐受性通过在甘露碱合成酶启动子和终止子控制下的BAR基因的表达(草胺膦乙酰基转移酶)赋予。GOI可以通过由花椰菜花叶病毒35S启动子(35S)或玄参花叶病毒(FMV)驱动的强组成型启动子(例如SEQ ID NO:36-39)或通过多种植物启动子之一(如泛素3启动子)驱动，且可以使用终止子如E6、E9或章鱼碱合成酶(OCS)终止子。红色荧光蛋白DsRed(Matz等，1999)通常利用终止子如E6、E9或OCS终止子通过强组成型病毒启动子或其它启动子如拟南芥磷酸甘油酸变位酶启动子(SEQ ID NO:35；Mazarei等，2003)表达。
表3:RKN dsRNA核苷酸序列和启动子序列
包含选择表达盒和在细菌细胞中维持质粒必需的元件的作物转化基本载体用于装配在与提供本发明的功能性的DNA片段可操作地连接时提供调控活性的DNA片段(启动子、前导序列、内含子、3’UTR)。这些DNA片段的装配可以使用重组DNA技术领域中公知的方法完成。能够编码有效dsRNA分子的DNA序列的实例是表3中的SEQ ID NO:27-30、SEQ IDNO:32和SEQ ID NO:47。
实施例5:
西红柿毛根通过转基因RNAi抗南方根结线虫的效力
培养板用大约450个灭菌南方根结线虫J2接种并在26-28℃孵育。在感染后42天，板进行微波处理并过筛以收集根。然后根在10％漂白溶液中混合并倾倒在一系列筛上以除去根碎片和收集卵。从各板除去卵并计数，且对根称重。制备表达编码例如kin-2、T26G10.1、cgh1、CplA或Vha19的基因片段(例如，SEQ ID NO:27-30、SEQ ID NO:32或SEQ IDNO:46)的转基因毛根培养物，且这些根测试RKN抗性。结果显示于表4中，证明了减少RKN繁殖的效力。
表4:典型dsRNA构建体的西红柿毛根效力
*使用T检验以0.1α具有相对于易感对照的统计显著性降低。
卵减少百分比是相对于空载体易感对照。利用分别具有或没有矮牵牛前导序列35S(E35S或E35Sp)SEQ ID NO:37和SEQ ID NO:38的增强的花椰菜花叶病毒35S启动子、玄参花叶病毒(FMV)启动子SEQ ID NO:36、D10.1(Div10)启动子SEQ ID NO:35和具有烟草花叶病毒ω翻译增强子的花椰菜花叶病毒35S启动子SEQ ID NO:39。
表4中的数据表明，启动子和目标基因以及精确的基因片段影响抗RKN效力的水平。重要的是有可能使用以对抗本文公开的基因靶标为目标的转基因RNAi途径获得抗RKN的高效力水平。
实施例6:
转基因烟草全植物产生和南方根结线虫(RKN)的分析
表5：培养基成分
制备用于预培养的外植体，例如通过在具有表面活性剂的10％Clorox中对来自一月龄烟草植物的叶表面灭菌15分钟，进行3x无菌DiH2O洗涤。将叶切成0.5cm的小片并将60-70个叶片倒置在具有2ml 4C005K液体培养基的MS104培养板上+2个灭菌滤板。这一预培养进行大约1-2天。然后外植体使用已用4C005K调节到1.2 x 109bac/ml(例如～1/5稀释)的过夜的土壤杆菌培养物接种。2ml调节的土壤杆菌培养物直接加入预培养外植体的各板中，其然后孵育10分钟。吸除土壤杆菌从使外植体尽可能干。培养板用灭菌滤板吸干并使其在24℃共培养2-3天。通过从滤纸上移除外植体并将它们均匀地隔置到选择板上(10-12外植体/板)而将外植体转移到用于选择相的MS104+草铵膦5mg/L+羧苄青霉素500μg/ml上。
在4-6周，将芽从愈伤组织切除(由各愈伤组织丛切除一个以确保独立事件)并置于MS0+草铵膦+羧苄青霉素500μg/ml生根培养基上。还未发芽的愈伤组织可以切割成独立愈伤组织块并放回到新鲜MS104选择培养板上以促进发芽。根通常在7-10天内在假定转化体上形成。此时，可以采取小的叶片并在MS104选择培养基板上重生愈伤组织以确认卡那霉素抗性(如果需要)。从生根的幼苗的根冲洗掉琼脂，且植物盆栽在小罐中、良好灌溉和置于繁殖罩下2-3天。然后繁殖罩的盖可以逐渐裂开以使幼苗受冷(harden off)而适应环境条件。这称作R0植物。
将盆栽置于温室中10周后，准备从R0植物收获R1种子。收集来自各R0植物的种子，并使其生长一周。然后每天用0.04％(草铵膦；Bayer CropScience)喷撒植物一周。按3:1的方式分离的植物被认为具有单拷贝的插入且保持用于进一步的分析。
种植和选择R1种子后12周，R2种子准备从R1植物收获。收集、种植来自R1植物的种子并使其生长一周。然后每天用0.04％(草铵膦)喷撒植物一周。正确分离(在Finale暴露后100％存活)的植物被认为是纯合的且保持用于效力测试和种子采集。对于各R2整合(integration)，植物亚群用线虫感染以测定效力且使亚群生长以产生R3种子。R2植物的草铵膦选择后三周，植物亚群每盆RKN J2用5000蠕虫形卵接种并评价8周后的虫瘿减少。
R2植物的草铵膦选择后12周，收获植物亚群从而获得R3种子。可以以类似的方式产生后续世代如R4、R5等。
表6:示例dsRNA构建体的烟草全植株效力。
| P-GOI | 5’-内含子 | DsRNA | P-DsRED | 株系 | 虫瘿减少(％) |
| E35Sp | Act7 | Kin2A | FMV | 组合 | 44* |
| E35Sp | Act7 | Kin2A | FMV | 1 | 34 |
| E35Sp | Act7 | Kin2A | FMV | 2 | 73* |
| E35Sp | Act7 | Kin2A | FMV | 3 | 38* |
| E35Sp | Act7 | Kin2A | FMV | 4 | 58* |
| E35Sp | Act7 | Kin2A | FMV | 5 | 16 |
*使用T检验以0.1α具有相对于易感对照的统计显著性减少。
表6中的数据表明，可以利用以本文中公开的基因靶标为目标的转基因RNAi途径使用另外的宿主植物物种(在该情况中即为烟草)在温室中获得抗RKN的高效力水平。
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本文中公开和要求保护的所有组合物和方法可以根据本公开制备和执行而不需要过度的试验。尽管本发明的组合物和方法按照前述的示范性实施方式进行说明，但本领域技术人员很清楚，可以对组合物、方法和本文描述的方法的步骤或步骤顺序进行变化、更换、改进和修改而不脱离本发明的实质概念、精神和范围。更具体地，很清楚，化学和生理相关的某些物质可以替代本文中描述的物质而获得相同或相似的结果。对于本领域技术人员显而易见的所有这些类似替换和改进被认为在所附权利要求限定的精神、范围和概念内。
参考文献
下列参考文献通过引用并入本文中：
美国专利4,536,475；美国专利4,693,977；美国专利4,886,937；美国专利4,940,838；美国专利4,959,317；美国专利5,015,580；美国专利5,107,065；美国专利5,110,732；美国专利5,231,020；美国专利5,283,184；美国专利5,302,523；美国专利5,384,253；美国专利5,464,763；美国专利5,464,765；美国专利5,501,967；美国专利5,508,184；美国专利5,527,695；美国专利5,538,880；美国专利5,459,252；美国专利5,550,318；美国专利5,563,055；美国专利5,591,616；美国专利5,593,874；美国专利5,633,435；美国专利5,693,512；美国专利5,698,425；美国专利5,712,135；美国专利5,759,829；美国专利5,780,708；美国专利5,789,214；美国专利5,804,693；美国专利5,824,877；美国专利5,837,848；美国专利5,981,840；美国专利6,118,047；美国专利6,160,208；美国专利6,384,301；美国专利6,399,861；美国专利6,403,865。
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序列表
<110> Bradley, John D.
Chiapelli, Brandi
Williams, Jeremy
Gao, Bingli
McCarter, James P. Gao
Verbsky, Michelle L.
<120> 用于根结线虫防治的方法和组合物
<130> MNDI:004WO
<140> 未知
<141> 2009-04-10
<150> US 61/044,015
<151> 2008-04-10
<150> US 61/084,205
<151> 2008-07-28
<160> 47
<170> PatentIn version 3.5
<210> 1
<211> 2241
<212> DNA
<213> 南方根结线虫
<400> 1
atggcaacgg agactgaacc aaacaatgct gtggcacaac aaaatggaaa agaaactgca 60
atgaattccg agtcccaaca agaattgtca aattctgttc ctgttgaaaa tggggttggt 120
gaaactaatg gagatgaact aaaaaatgac tcttctgcca aaaatgaaga tttggatgat 180
attccatggt ctcaacgaat tagggaaaaa caacaaaatt cttcaacttc taaatctgtt 240
aaaagacctg tgacacctga agaaagtggg ggagaagaag cccaacctgt tgtaaaaaaa 300
tctgccgata aaaagcggaa gacgagacaa gcatcgcctg aaagtgaaga agaatatact 360
ccaaagaaga aatcaaaatc ttccaaaacc aaacctgaaa atccaacgcc aaagtcatct 420
gctaaaaaag agacaaaagc tgaagaagat tcggattacg aagatgacaa acccgcaaaa 480
atgaaaaaga aatcaactga aaataatcat gctaaacaag agacatcttc atctgcaagc 540
cctacaaaaa agaaaaagaa ggctgaagaa gaagaggagg tttggaagtg gtgggaagaa 600
caatcacctg aacaatcaga tggaacaatt aaatggagaa ctttagagca taaagggcca 660
atgtttgcgc cagaatatgt tccgttacct aaaaatatta aattcaaata taatggaaag 720
ccaatggatc tatccccgtc tgctgaagaa gttgccactt tttatgctaa aatgcttgat 780
catgattata cttcaaagcc tatttttaat caaaactttt ttaaagattg gagaaagaca 840
atgacctctg cagaacgttc aacaataact gatttaaaaa aatgtgattt tcgtgatata 900
aatgcctatt ttctttctga aactgaaaaa agaaaggctc gaagtaaaga agagaaaaaa 960
gcagaaaaag aaattaaaga taaagaagcg gctgaatatg gttttgcaat gctggatggt 1020
tataaacaaa aaattggaaa ttttcgaatt gaaccaccgg gactttttag aggacgagga 1080
gaacacccaa aaatgggttg tttaaagcta cgagttcaac cagaagatgt tataataaat 1140
tgctctaaag gtgttacaat tcctcctccc aagggtcata aatggaaaga agttcggcat 1200
gacaatatgg tcacttggct ttgttcttgg aatgaaaatg ttttgtttag taacaaatat 1260
ataatgttaa atccttcttc aaagcttaaa ggccaaaagg attgggaaaa gttcgaaaaa 1320
gcacgaaagc ttaaaggctg tgttgggcca attcgagatc agtatttatt agactttaaa 1380
agcaaagaga tgcgaatacg acaacgtgca gttgctctct attttattga taaatttgcg 1440
cttagagctg gaaatgaaaa ggatactgat gaagctgcag atactgttgg ttgttgctct 1500
cttcgttgtg aacatataaa attacacgaa gaattggata accagaaata tgttgtagaa 1560
tttgattttc ttgggaaaga ttcaattcgt tattacaatc gtgtacctgt tgaaaaggct 1620
gtttttaaaa atttgaaaat ttttgtggaa ggaaaggaac ctggagatga tctctttgac 1680
agacttgata caagttctct aaatgcttat ttaaaagaat taatggatgg attaactgcc 1740
aaagtatttc gtacatataa tgcttcaatt actttacaag accaattgga taaattaaca 1800
aatcctgatg atacaatcca tgcaaagcta ctttcatata atcgtgcaaa tcgacaagtt 1860
gcaattcttt gtaatcacca acgtgcagtc cctaaaactc atgataaggc tatggaaaca 1920
ctacaaaaca agattaatga aaagaagaag gaatataaag aaatcaaggc tgaacttaag 1980
aaaaataaaa atgatgagaa attgcagaag aaatatactc gagtaaaaga acaacttgta 2040
aaattaaaaa cacagcatac cgacaaggat gaaaataaac aaattgcatt gggtacatcc 2100
aaacttaatt atttggatcc aagaatatca gttgcttggt gcaaaaaata tgacatccct 2160
atagaaaaga tttactcaaa aactcaacgt gacaaatttc gatgggctat agatatgaca 2220
aaagaggatt ttattttttg a 2241
<210> 2
<211> 746
<212> PRT
<213> 南方根结线虫
<400> 2
Met Ala Thr Glu Thr Glu Pro Asn Asn Ala Val Ala Gln Gln Asn Gly
1 5 10 15
Lys Glu Thr Ala Met Asn Ser Glu Ser Gln Gln Glu Leu Ser Asn Ser
20 25 30
Val Pro Val Glu Asn Gly Val Gly Glu Thr Asn Gly Asp Glu Leu Lys
35 40 45
Asn Asp Ser Ser Ala Lys Asn Glu Asp Leu Asp Asp Ile Pro Trp Ser
50 55 60
Gln Arg Ile Arg Glu Lys Gln Gln Asn Ser Ser Thr Ser Lys Ser Val
65 70 75 80
Lys Arg Pro Val Thr Pro Glu Glu Ser Gly Gly Glu Glu Ala Gln Pro
85 90 95
Val Val Lys Lys Ser Ala Asp Lys Lys Arg Lys Thr Arg Gln Ala Ser
100 105 110
Pro Glu Ser Glu Glu Glu Tyr Thr Pro Lys Lys Lys Ser Lys Ser Ser
115 120 125
Lys Thr Lys Pro Glu Asn Pro Thr Pro Lys Ser Ser Ala Lys Lys Glu
130 135 140
Thr Lys Ala Glu Glu Asp Ser Asp Tyr Glu Asp Asp Lys Pro Ala Lys
145 150 155 160
Met Lys Lys Lys Ser Thr Glu Asn Asn His Ala Lys Gln Glu Thr Ser
165 170 175
Ser Ser Ala Ser Pro Thr Lys Lys Lys Lys Lys Ala Glu Glu Glu Glu
180 185 190
Glu Val Trp Lys Trp Trp Glu Glu Gln Ser Pro Glu Gln Ser Asp Gly
195 200 205
Thr Ile Lys Trp Arg Thr Leu Glu His Lys Gly Pro Met Phe Ala Pro
210 215 220
Glu Tyr Val Pro Leu Pro Lys Asn Ile Lys Phe Lys Tyr Asn Gly Lys
225 230 235 240
Pro Met Asp Leu Ser Pro Ser Ala Glu Glu Val Ala Thr Phe Tyr Ala
245 250 255
Lys Met Leu Asp His Asp Tyr Thr Ser Lys Pro Ile Phe Asn Gln Asn
260 265 270
Phe Phe Lys Asp Trp Arg Lys Thr Met Thr Ser Ala Glu Arg Ser Thr
275 280 285
Ile Thr Asp Leu Lys Lys Cys Asp Phe Arg Asp Ile Asn Ala Tyr Phe
290 295 300
Leu Ser Glu Thr Glu Lys Arg Lys Ala Arg Ser Lys Glu Glu Lys Lys
305 310 315 320
Ala Glu Lys Glu Ile Lys Asp Lys Glu Ala Ala Glu Tyr Gly Phe Ala
325 330 335
Met Leu Asp Gly Tyr Lys Gln Lys Ile Gly Asn Phe Arg Ile Glu Pro
340 345 350
Pro Gly Leu Phe Arg Gly Arg Gly Glu His Pro Lys Met Gly Cys Leu
355 360 365
Lys Leu Arg Val Gln Pro Glu Asp Val Ile Ile Asn Cys Ser Lys Gly
370 375 380
Val Thr Ile Pro Pro Pro Lys Gly His Lys Trp Lys Glu Val Arg His
385 390 395 400
Asp Asn Met Val Thr Trp Leu Cys Ser Trp Asn Glu Asn Val Leu Phe
405 410 415
Ser Asn Lys Tyr Ile Met Leu Asn Pro Ser Ser Lys Leu Lys Gly Gln
420 425 430
Lys Asp Trp Glu Lys Phe Glu Lys Ala Arg Lys Leu Lys Gly Cys Val
435 440 445
Gly Pro Ile Arg Asp Gln Tyr Leu Leu Asp Phe Lys Ser Lys Glu Met
450 455 460
Arg Ile Arg Gln Arg Ala Val Ala Leu Tyr Phe Ile Asp Lys Phe Ala
465 470 475 480
Leu Arg Ala Gly Asn Glu Lys Asp Thr Asp Glu Ala Ala Asp Thr Val
485 490 495
Gly Cys Cys Ser Leu Arg Cys Glu His Ile Lys Leu His Glu Glu Leu
500 505 510
Asp Asn Gln Lys Tyr Val Val Glu Phe Asp Phe Leu Gly Lys Asp Ser
515 520 525
Ile Arg Tyr Tyr Asn Arg Val Pro Val Glu Lys Ala Val Phe Lys Asn
530 535 540
Leu Lys Ile Phe Val Glu Gly Lys Glu Pro Gly Asp Asp Leu Phe Asp
545 550 555 560
Arg Leu Asp Thr Ser Ser Leu Asn Ala Tyr Leu Lys Glu Leu Met Asp
565 570 575
Gly Leu Thr Ala Lys Val Phe Arg Thr Tyr Asn Ala Ser Ile Thr Leu
580 585 590
Gln Asp Gln Leu Asp Lys Leu Thr Asn Pro Asp Asp Thr Ile His Ala
595 600 605
Lys Leu Leu Ser Tyr Asn Arg Ala Asn Arg Gln Val Ala Ile Leu Cys
610 615 620
Asn His Gln Arg Ala Val Pro Lys Thr His Asp Lys Ala Met Glu Thr
625 630 635 640
Leu Gln Asn Lys Ile Asn Glu Lys Lys Lys Glu Tyr Lys Glu Ile Lys
645 650 655
Ala Glu Leu Lys Lys Asn Lys Asn Asp Glu Lys Leu Gln Lys Lys Tyr
660 665 670
Thr Arg Val Lys Glu Gln Leu Val Lys Leu Lys Thr Gln His Thr Asp
675 680 685
Lys Asp Glu Asn Lys Gln Ile Ala Leu Gly Thr Ser Lys Leu Asn Tyr
690 695 700
Leu Asp Pro Arg Ile Ser Val Ala Trp Cys Lys Lys Tyr Asp Ile Pro
705 710 715 720
Ile Glu Lys Ile Tyr Ser Lys Thr Gln Arg Asp Lys Phe Arg Trp Ala
725 730 735
Ile Asp Met Thr Lys Glu Asp Phe Ile Phe
740 745
<210> 3
<211> 957
<212> DNA
<213> 南方根结线虫
<220>
<221> 杂项特征
<222> (663)..(663)
<223> n是a, c, g或t
<220>
<221> 杂项特征
<222> (912)..(912)
<223> n是a, c, g或t
<400> 3
atggtttgcc ccttcttttt gacaattatt gcttacacag ctatcgctta ttttgttaaa 60
cgcattttga cttcgattta caacattgtt ttcccgtatt tatatgccgt cccacagaat 120
ttgcaagttt tggctggagc aaaatgggca gtaataactg gtggaactga cggtattgga 180
aaggcatacg cttatgaatt ggccaaaaaa caatttaata taattataat ttcgcgtaca 240
caatcaaaat tggatgatgt ggctaaagaa attaaagaag aatttaagga cattgaagtg 300
agaacgattg cttttgactt tacaaatcca aaattagaag attatgagag acagatattt 360
gccaaaattg atgatgttga cattggagtt ttggttaaca atgttggaat gagctatgaa 420
tttcctgaac gctttgaacg tgttcatggt ggtattaaac gtgttgctga catgacttta 480
atcaacactt ttccgacaac tgttctttct catcatattc ttcaacaaat ggctaaacgc 540
aatagaggtg tagtggttaa tgtggcttct tcagctgcca attttgattg gttttatttg 600
gctgtttata gtgctattaa aaaatatgtc actggtttgt tttcattttt acgtaaagaa 660
tangttgaca caaatattta tttccaacca gtttttccaa tgatggttgc cacaaaaagg 720
gtaaaaatta gacgttcttc attttttatt ccttcacctg aaaaatttgt tagtgaaact 780
gttcgcagtt ttggattaac tgatgagaat acggggagtt tggcacatca aattcagggt 840
gaaattatgt ttggctacat cccagaatat tttttcaaca aagttgttcg ggacaatttt 900
ttgtcaactt gnaagcgggc ctggaggaaa agggggaatt ttgcgaaggc ggaataa 957
<210> 4
<211> 318
<212> PRT
<213> 南方根结线虫
<220>
<221> 杂项特征
<222> (221)..(221)
<223> Xaa可以是任何天然存在的氨基酸
<220>
<221> 杂项特征
<222> (304)..(304)
<223> Xaa可以是任何天然存在的氨基酸
<400> 4
Met Val Cys Pro Phe Phe Leu Thr Ile Ile Ala Tyr Thr Ala Ile Ala
1 5 10 15
Tyr Phe Val Lys Arg Ile Leu Thr Ser Ile Tyr Asn Ile Val Phe Pro
20 25 30
Tyr Leu Tyr Ala Val Pro Gln Asn Leu Gln Val Leu Ala Gly Ala Lys
35 40 45
Trp Ala Val Ile Thr Gly Gly Thr Asp Gly Ile Gly Lys Ala Tyr Ala
50 55 60
Tyr Glu Leu Ala Lys Lys Gln Phe Asn Ile Ile Ile Ile Ser Arg Thr
65 70 75 80
Gln Ser Lys Leu Asp Asp Val Ala Lys Glu Ile Lys Glu Glu Phe Lys
85 90 95
Asp Ile Glu Val Arg Thr Ile Ala Phe Asp Phe Thr Asn Pro Lys Leu
100 105 110
Glu Asp Tyr Glu Arg Gln Ile Phe Ala Lys Ile Asp Asp Val Asp Ile
115 120 125
Gly Val Leu Val Asn Asn Val Gly Met Ser Tyr Glu Phe Pro Glu Arg
130 135 140
Phe Glu Arg Val His Gly Gly Ile Lys Arg Val Ala Asp Met Thr Leu
145 150 155 160
Ile Asn Thr Phe Pro Thr Thr Val Leu Ser His His Ile Leu Gln Gln
165 170 175
Met Ala Lys Arg Asn Arg Gly Val Val Val Asn Val Ala Ser Ser Ala
180 185 190
Ala Asn Phe Asp Trp Phe Tyr Leu Ala Val Tyr Ser Ala Ile Lys Lys
195 200 205
Tyr Val Thr Gly Leu Phe Ser Phe Leu Arg Lys Glu Xaa Val Asp Thr
210 215 220
Asn Ile Tyr Phe Gln Pro Val Phe Pro Met Met Val Ala Thr Lys Arg
225 230 235 240
Val Lys Ile Arg Arg Ser Ser Phe Phe Ile Pro Ser Pro Glu Lys Phe
245 250 255
Val Ser Glu Thr Val Arg Ser Phe Gly Leu Thr Asp Glu Asn Thr Gly
260 265 270
Ser Leu Ala His Gln Ile Gln Gly Glu Ile Met Phe Gly Tyr Ile Pro
275 280 285
Glu Tyr Phe Phe Asn Lys Val Val Arg Asp Asn Phe Leu Ser Thr Xaa
290 295 300
Lys Arg Ala Trp Arg Lys Arg Gly Asn Phe Ala Lys Ala Glu
305 310 315
<210> 5
<211> 1425
<212> DNA
<213> 南方根结线虫
<400> 5
atgggaatta aattcctgga atttgttaaa cctttttgtg gttttgtgcc agaggtttct 60
aaacctgaaa gaaaaattca attcagggaa aagatgcttt ggactgcgat aactcttttt 120
gtttttcttg tttgctgtca gattccactt tttggtatta tgtccactga cagtgctgat 180
cctctttatt ggcttcgagt tattatggca tctaatagag gaacgcttat ggaattggga 240
atctctccaa ttgtaacttc tggtcttatt atgcaacttt tggctggtgc aaaaattatt 300
gaagttgggg atactccaaa ggaaagagct ctttttaatg gtgcccagaa attgttcgga 360
atggtcataa ctatcggtca agctattgtt tatgttgcct ctggaatgta tggaaatcct 420
catgaaattg gtgcaggaat ttgtctttta atcgttattc aattagttgt tgctggatta 480
attgttcttt tacttgacga attacttcag aaaggctatg gacttggttc aggaatttcc 540
ctctttattg ctacaaacat ttgcgaaact attgtttgga aagcattttc acctgcgaca 600
atgaatacag gaagaggaac cgaatttgaa ggtgcaataa ttgctttatt tcatctgttg 660
tcaactcgta atgacaaagt ccgtgcactc agagaagctt tttaccgtca aaatttgcca 720
aatttgatga acttattggc tactgtgctc gtttttgctg ttgttatata ctttcagggc 780
ttccgtgttg atttgccaat taaaagttct cgttatcgtg gccaacatag ttcatatcca 840
atcaaattgt tttatacaag taatatacca ataattcttc aatctgcttt ggtctccaat 900
ttatacgtta tctctcaaat gttggcagct aaatttggag gaaatattct tgttaatata 960
cttggtacat ggtctgactc ttcaggcact tataggagtt atccaacagg aggtatttgc 1020
tactacctat caccaccaga atcattgtca catgtagttg aagacccaat tcattgtttt 1080
acctacattg tctttatgct tggttcatgt gcatttttct caaaaacctg gattgatgtt 1140
agcggttcaa gtgctaaaga tgttgcgaaa caattaaaag agcaacaaat gataatgaga 1200
ggacataggg aaaaatcaat gattcacgaa ttaaatcgtt atattcctac tgctgctgct 1260
tttggtggtt tatgtattgg agcactttct gttactgctg actttatggg agctattgga 1320
agtggaacgg gaattttact tgcggtcacc attatttatc agtatttcga aatatttgtg 1380
aaagaacagc aagaaatggg tggcgtttct ggtttattct tttga 1425
<210> 6
<211> 474
<212> PRT
<213> 南方根结线虫
<400> 6
Met Gly Ile Lys Phe Leu Glu Phe Val Lys Pro Phe Cys Gly Phe Val
1 5 10 15
Pro Glu Val Ser Lys Pro Glu Arg Lys Ile Gln Phe Arg Glu Lys Met
20 25 30
Leu Trp Thr Ala Ile Thr Leu Phe Val Phe Leu Val Cys Cys Gln Ile
35 40 45
Pro Leu Phe Gly Ile Met Ser Thr Asp Ser Ala Asp Pro Leu Tyr Trp
50 55 60
Leu Arg Val Ile Met Ala Ser Asn Arg Gly Thr Leu Met Glu Leu Gly
65 70 75 80
Ile Ser Pro Ile Val Thr Ser Gly Leu Ile Met Gln Leu Leu Ala Gly
85 90 95
Ala Lys Ile Ile Glu Val Gly Asp Thr Pro Lys Glu Arg Ala Leu Phe
100 105 110
Asn Gly Ala Gln Lys Leu Phe Gly Met Val Ile Thr Ile Gly Gln Ala
115 120 125
Ile Val Tyr Val Ala Ser Gly Met Tyr Gly Asn Pro His Glu Ile Gly
130 135 140
Ala Gly Ile Cys Leu Leu Ile Val Ile Gln Leu Val Val Ala Gly Leu
145 150 155 160
Ile Val Leu Leu Leu Asp Glu Leu Leu Gln Lys Gly Tyr Gly Leu Gly
165 170 175
Ser Gly Ile Ser Leu Phe Ile Ala Thr Asn Ile Cys Glu Thr Ile Val
180 185 190
Trp Lys Ala Phe Ser Pro Ala Thr Met Asn Thr Gly Arg Gly Thr Glu
195 200 205
Phe Glu Gly Ala Ile Ile Ala Leu Phe His Leu Leu Ser Thr Arg Asn
210 215 220
Asp Lys Val Arg Ala Leu Arg Glu Ala Phe Tyr Arg Gln Asn Leu Pro
225 230 235 240
Asn Leu Met Asn Leu Leu Ala Thr Val Leu Val Phe Ala Val Val Ile
245 250 255
Tyr Phe Gln Gly Phe Arg Val Asp Leu Pro Ile Lys Ser Ser Arg Tyr
260 265 270
Arg Gly Gln His Ser Ser Tyr Pro Ile Lys Leu Phe Tyr Thr Ser Asn
275 280 285
Ile Pro Ile Ile Leu Gln Ser Ala Leu Val Ser Asn Leu Tyr Val Ile
290 295 300
Ser Gln Met Leu Ala Ala Lys Phe Gly Gly Asn Ile Leu Val Asn Ile
305 310 315 320
Leu Gly Thr Trp Ser Asp Ser Ser Gly Thr Tyr Arg Ser Tyr Pro Thr
325 330 335
Gly Gly Ile Cys Tyr Tyr Leu Ser Pro Pro Glu Ser Leu Ser His Val
340 345 350
Val Glu Asp Pro Ile His Cys Phe Thr Tyr Ile Val Phe Met Leu Gly
355 360 365
Ser Cys Ala Phe Phe Ser Lys Thr Trp Ile Asp Val Ser Gly Ser Ser
370 375 380
Ala Lys Asp Val Ala Lys Gln Leu Lys Glu Gln Gln Met Ile Met Arg
385 390 395 400
Gly His Arg Glu Lys Ser Met Ile His Glu Leu Asn Arg Tyr Ile Pro
405 410 415
Thr Ala Ala Ala Phe Gly Gly Leu Cys Ile Gly Ala Leu Ser Val Thr
420 425 430
Ala Asp Phe Met Gly Ala Ile Gly Ser Gly Thr Gly Ile Leu Leu Ala
435 440 445
Val Thr Ile Ile Tyr Gln Tyr Phe Glu Ile Phe Val Lys Glu Gln Gln
450 455 460
Glu Met Gly Gly Val Ser Gly Leu Phe Phe
465 470
<210> 7
<211> 1851
<212> DNA
<213> 南方根结线虫
<400> 7
atggaagacc aaatttcaaa aatttctgat gctgccaatc gaatgcgtat ttcttcaatt 60
gaacagacta ctaaagcaaa ttctggacat ccaacaagta gttgtagtgc tgctgaaatt 120
gttgcgacac atttcttttc tgaaatgcgt tattcggtta aggaacctag acatgcttct 180
gctgatcgtt ttgttttgtc taagggacat gcctgtccga ttctctatgc tgcttgggag 240
gaagcaggcc tgttaactcg agaacaagtt ttgactctgc gcaaattaaa ttctgatatt 300
gaaggacatc caactccgag gctttcattt attgatgttg ctacaggctc tctcggacaa 360
ggactttctt gtgctgctgg gatggcttat actggaaaat atattgacaa cgcttcatat 420
cgagtctatt gtcttttggg cgatagtgaa agtgcagaag gatctgtttg ggaggctgca 480
gcattcgcaa gttattataa attggataat ctcgttacaa ttgttgatgt taatcgactt 540
ggacaatctc gtgaaactat gattggccat gatttgttta cttatgctaa gcggtttgaa 600
gcttttgggt ttaatagtat tattgttgat ggacacgatg ttgctgattt attgaaagct 660
tttgatgaag caagaaaagt aacaggccaa ccaacagcaa ttattgcacg tacattaaaa 720
gggaaaggaa ttgaaggagt tgaagataaa gataattgtc atggaaagcc agtaactttg 780
gacaaagcag aaattattgc ttcaaaattg ggaaataaaa cagcaaaaaa tttgtgggaa 840
ttagaaaatt taattgatga tgctcctgaa gttgattttg aaattgggaa aattaaaatg 900
agttcgccgc caaattatca aattggagag aaggtggcta ctcgtttagc ttatggaaat 960
gctcttgtta aattggctga tacaagcaaa aggattattg ctttggatgg tgacgtgtca 1020
aattccacat tttctgataa agttctcaat aaatatcctc aacaatttgt ccaatgcttt 1080
attgctgaac aaaacatggt tggcgttgct gtaggaatgt cttgtcgtgg gcgaactatt 1140
ccacatgcga gtacttttgc tgttttcttt acccgtgctg ctgaccaaat tcgaatgggg 1200
gcaatatctt ttgctaatgt taaatttgct ggttcacatg ctggtgtctc tattggagag 1260
gatgggccaa gccaaatggg acttgaagac ctcgcacttt tccgagcagt tcctaatagt 1320
atcgttcttt atccatctga tgcggtttct acagaatatg cgactgaatt ggctgctaat 1380
tataagggaa ttacttttac tcgtactgga cgtcctaata ctccagttat ttatccaaat 1440
gatgagaaat ttgagattgg aaaatgcaag gttatccgtc aaacaaatga agacaagtat 1500
ttattaatcg gcgcgggtgt aacactttat gaatgtatta aagctcatga tattttgagt 1560
tctgaaggaa ttcaagttgc tgttattgac cttttctctg tcaagccttt ggacaatcaa 1620
acacttattg agcaagctaa gcgtgttgga gggaaagttt tgactgtaga ggatcattat 1680
cagactggag gtattggtga agctgtatct ttggcgttag gtgatgttcc taatgttcgt 1740
gttcgcagtc tttgtgttaa agaaattcca cgttctggta cacctgatga gcttatggat 1800
ttgtatggaa tttctgcaaa gaaaattata gctgcagtga agaattttta a 1851
<210> 8
<211> 616
<212> PRT
<213> 南方根结线虫
<400> 8
Met Glu Asp Gln Ile Ser Lys Ile Ser Asp Ala Ala Asn Arg Met Arg
1 5 10 15
Ile Ser Ser Ile Glu Gln Thr Thr Lys Ala Asn Ser Gly His Pro Thr
20 25 30
Ser Ser Cys Ser Ala Ala Glu Ile Val Ala Thr His Phe Phe Ser Glu
35 40 45
Met Arg Tyr Ser Val Lys Glu Pro Arg His Ala Ser Ala Asp Arg Phe
50 55 60
Val Leu Ser Lys Gly His Ala Cys Pro Ile Leu Tyr Ala Ala Trp Glu
65 70 75 80
Glu Ala Gly Leu Leu Thr Arg Glu Gln Val Leu Thr Leu Arg Lys Leu
85 90 95
Asn Ser Asp Ile Glu Gly His Pro Thr Pro Arg Leu Ser Phe Ile Asp
100 105 110
Val Ala Thr Gly Ser Leu Gly Gln Gly Leu Ser Cys Ala Ala Gly Met
115 120 125
Ala Tyr Thr Gly Lys Tyr Ile Asp Asn Ala Ser Tyr Arg Val Tyr Cys
130 135 140
Leu Leu Gly Asp Ser Glu Ser Ala Glu Gly Ser Val Trp Glu Ala Ala
145 150 155 160
Ala Phe Ala Ser Tyr Tyr Lys Leu Asp Asn Leu Val Thr Ile Val Asp
165 170 175
Val Asn Arg Leu Gly Gln Ser Arg Glu Thr Met Ile Gly His Asp Leu
180 185 190
Phe Thr Tyr Ala Lys Arg Phe Glu Ala Phe Gly Phe Asn Ser Ile Ile
195 200 205
Val Asp Gly His Asp Val Ala Asp Leu Leu Lys Ala Phe Asp Glu Ala
210 215 220
Arg Lys Val Thr Gly Gln Pro Thr Ala Ile Ile Ala Arg Thr Leu Lys
225 230 235 240
Gly Lys Gly Ile Glu Gly Val Glu Asp Lys Asp Asn Cys His Gly Lys
245 250 255
Pro Val Thr Leu Asp Lys Ala Glu Ile Ile Ala Ser Lys Leu Gly Asn
260 265 270
Lys Thr Ala Lys Asn Leu Trp Glu Leu Glu Asn Leu Ile Asp Asp Ala
275 280 285
Pro Glu Val Asp Phe Glu Ile Gly Lys Ile Lys Met Ser Ser Pro Pro
290 295 300
Asn Tyr Gln Ile Gly Glu Lys Val Ala Thr Arg Leu Ala Tyr Gly Asn
305 310 315 320
Ala Leu Val Lys Leu Ala Asp Thr Ser Lys Arg Ile Ile Ala Leu Asp
325 330 335
Gly Asp Val Ser Asn Ser Thr Phe Ser Asp Lys Val Leu Asn Lys Tyr
340 345 350
Pro Gln Gln Phe Val Gln Cys Phe Ile Ala Glu Gln Asn Met Val Gly
355 360 365
Val Ala Val Gly Met Ser Cys Arg Gly Arg Thr Ile Pro His Ala Ser
370 375 380
Thr Phe Ala Val Phe Phe Thr Arg Ala Ala Asp Gln Ile Arg Met Gly
385 390 395 400
Ala Ile Ser Phe Ala Asn Val Lys Phe Ala Gly Ser His Ala Gly Val
405 410 415
Ser Ile Gly Glu Asp Gly Pro Ser Gln Met Gly Leu Glu Asp Leu Ala
420 425 430
Leu Phe Arg Ala Val Pro Asn Ser Ile Val Leu Tyr Pro Ser Asp Ala
435 440 445
Val Ser Thr Glu Tyr Ala Thr Glu Leu Ala Ala Asn Tyr Lys Gly Ile
450 455 460
Thr Phe Thr Arg Thr Gly Arg Pro Asn Thr Pro Val Ile Tyr Pro Asn
465 470 475 480
Asp Glu Lys Phe Glu Ile Gly Lys Cys Lys Val Ile Arg Gln Thr Asn
485 490 495
Glu Asp Lys Tyr Leu Leu Ile Gly Ala Gly Val Thr Leu Tyr Glu Cys
500 505 510
Ile Lys Ala His Asp Ile Leu Ser Ser Glu Gly Ile Gln Val Ala Val
515 520 525
Ile Asp Leu Phe Ser Val Lys Pro Leu Asp Asn Gln Thr Leu Ile Glu
530 535 540
Gln Ala Lys Arg Val Gly Gly Lys Val Leu Thr Val Glu Asp His Tyr
545 550 555 560
Gln Thr Gly Gly Ile Gly Glu Ala Val Ser Leu Ala Leu Gly Asp Val
565 570 575
Pro Asn Val Arg Val Arg Ser Leu Cys Val Lys Glu Ile Pro Arg Ser
580 585 590
Gly Thr Pro Asp Glu Leu Met Asp Leu Tyr Gly Ile Ser Ala Lys Lys
595 600 605
Ile Ile Ala Ala Val Lys Asn Phe
610 615
<210> 9
<211> 741
<212> DNA
<213> 南方根结线虫
<400> 9
atggcttctt cttatgatcg agcaattact attttctcgc cagatggtcg tctctttcaa 60
gttgattatg cgcaggaagc tgttaaaaaa ggctcaactg ctgtgggcgt taaagggaag 120
gattgcattg tgattggcgt tgagaagaaa tctgttcctg ttcttcaaga tgaccgcaca 180
attcgaaaga tacacaaatt ggatgatcac gtaatggctg cttttgctgg tttgagtgca 240
gatgctcgtg ttttgattga ccgtgccaga gttgcttgtg aaaattacaa attgactttg 300
gaggatcccg tcacacttga gcacatctcc cgcactattg cggacttgaa acatgaattt 360
acgcaaacta caggtcgtcg tccttttggt gtctctttac ttgttggcgg ttttgatccc 420
gatggaactc ctcatctttt tattactgaa ccatctggtg tgtattacga attacttgct 480
ggctcaatcg gccggaatga gaaggttgtc aaagaatatt tggaggagaa ttataatgat 540
ggcaatgttt caaacgaaaa cttggtcttg aagttggtgg tcaaagctct tgtcccagtt 600
gtgcaaactg gagcgcgtaa tatggagata tcggtgttga agcgtgataa tgaggggaat 660
attgttcaac gtgttctact cactgaggag ttggaggaaa ttttacgtca aattgaagct 720
gaaactatgg agactgttta g 741
<210> 10
<211> 246
<212> PRT
<213> 南方根结线虫
<400> 10
Met Ala Ser Ser Tyr Asp Arg Ala Ile Thr Ile Phe Ser Pro Asp Gly
1 5 10 15
Arg Leu Phe Gln Val Asp Tyr Ala Gln Glu Ala Val Lys Lys Gly Ser
20 25 30
Thr Ala Val Gly Val Lys Gly Lys Asp Cys Ile Val Ile Gly Val Glu
35 40 45
Lys Lys Ser Val Pro Val Leu Gln Asp Asp Arg Thr Ile Arg Lys Ile
50 55 60
His Lys Leu Asp Asp His Val Met Ala Ala Phe Ala Gly Leu Ser Ala
65 70 75 80
Asp Ala Arg Val Leu Ile Asp Arg Ala Arg Val Ala Cys Glu Asn Tyr
85 90 95
Lys Leu Thr Leu Glu Asp Pro Val Thr Leu Glu His Ile Ser Arg Thr
100 105 110
Ile Ala Asp Leu Lys His Glu Phe Thr Gln Thr Thr Gly Arg Arg Pro
115 120 125
Phe Gly Val Ser Leu Leu Val Gly Gly Phe Asp Pro Asp Gly Thr Pro
130 135 140
His Leu Phe Ile Thr Glu Pro Ser Gly Val Tyr Tyr Glu Leu Leu Ala
145 150 155 160
Gly Ser Ile Gly Arg Asn Glu Lys Val Val Lys Glu Tyr Leu Glu Glu
165 170 175
Asn Tyr Asn Asp Gly Asn Val Ser Asn Glu Asn Leu Val Leu Lys Leu
180 185 190
Val Val Lys Ala Leu Val Pro Val Val Gln Thr Gly Ala Arg Asn Met
195 200 205
Glu Ile Ser Val Leu Lys Arg Asp Asn Glu Gly Asn Ile Val Gln Arg
210 215 220
Val Leu Leu Thr Glu Glu Leu Glu Glu Ile Leu Arg Gln Ile Glu Ala
225 230 235 240
Glu Thr Met Glu Thr Val
245
<210> 11
<211> 1122
<212> DNA
<213> 南方根结线虫
<400> 11
atgtctggac atagcgtaga tgaggcacaa cttgcttctt gccaggctta tgtgcgtcaa 60
cacaatattc aacaacttgt taaagatgcc atagtttctt tatgcatcca taaacctgaa 120
aatccggtga ttttcttgcg tgaccacttt gatcagttgt cagccgatca acaaaacttc 180
tcacaggaaa cagtccattc agttcccgac gaagacgaca ccctggaaga acctccacgt 240
ctcccacaag gcaatcgccg ccgattgggc gtttctgccg aagttccaga tgaaaacgaa 300
gccgcgaatt accaacgagt agttattcca aaagatgatg atacacaaaa agctttgaga 360
caggcaatgt gccgaaatgt tttgtttgca catttggatg ttgatgaaca aaaagcaatt 420
ttcgatgcaa tgtttccagt agagaagaaa aagggagaaa ttattattga acagggagaa 480
gaaggagata atttttatgt aatcgaaagt ggagaagttg attgtttcgt taatggggaa 540
tttgttctct cagttaagga aggaggttct tttggtgaac ttgccttaat ttatggaact 600
ccacgtgctg ctactgttgt agctaagacc ccaacagtca aactttgggc tattgaccga 660
ctcacctata gagctatttt aatgggatct acaatgcgca aaagaaaaat gtatgacgaa 720
tttttgtcga aagtttcaat tcttgccgat cttgacaagt gggaacgtgc aaatgttgct 780
gatgctttgg aacaatgcca atttgaacct ggaactagaa ttgttgaaca gggccaaccc 840
ggagatgaat tctttataat tgttgaggga gaagccgagg tgctacaacg gccgaatgat 900
gatgctcctt atgaagttgt tggcaaattg ggtccttctg attattttgg cgaaattgct 960
cttttattgg atcatccacg tgcagcaacc gttgtggcta aagggcccct taagtgtgta 1020
aagttggata gagctcgatt cgagcgcgtt atgggcccag tgcgtgaaat tctcaaacgt 1080
gatgttagcc attacaattc ttatgtgaag ttgatgacct aa 1122
<210> 12
<211> 373
<212> PRT
<213> 南方根结线虫
<400> 12
Met Ser Gly His Ser Val Asp Glu Ala Gln Leu Ala Ser Cys Gln Ala
1 5 10 15
Tyr Val Arg Gln His Asn Ile Gln Gln Leu Val Lys Asp Ala Ile Val
20 25 30
Ser Leu Cys Ile His Lys Pro Glu Asn Pro Val Ile Phe Leu Arg Asp
35 40 45
His Phe Asp Gln Leu Ser Ala Asp Gln Gln Asn Phe Ser Gln Glu Thr
50 55 60
Val His Ser Val Pro Asp Glu Asp Asp Thr Leu Glu Glu Pro Pro Arg
65 70 75 80
Leu Pro Gln Gly Asn Arg Arg Arg Leu Gly Val Ser Ala Glu Val Pro
85 90 95
Asp Glu Asn Glu Ala Ala Asn Tyr Gln Arg Val Val Ile Pro Lys Asp
100 105 110
Asp Asp Thr Gln Lys Ala Leu Arg Gln Ala Met Cys Arg Asn Val Leu
115 120 125
Phe Ala His Leu Asp Val Asp Glu Gln Lys Ala Ile Phe Asp Ala Met
130 135 140
Phe Pro Val Glu Lys Lys Lys Gly Glu Ile Ile Ile Glu Gln Gly Glu
145 150 155 160
Glu Gly Asp Asn Phe Tyr Val Ile Glu Ser Gly Glu Val Asp Cys Phe
165 170 175
Val Asn Gly Glu Phe Val Leu Ser Val Lys Glu Gly Gly Ser Phe Gly
180 185 190
Glu Leu Ala Leu Ile Tyr Gly Thr Pro Arg Ala Ala Thr Val Val Ala
195 200 205
Lys Thr Pro Thr Val Lys Leu Trp Ala Ile Asp Arg Leu Thr Tyr Arg
210 215 220
Ala Ile Leu Met Gly Ser Thr Met Arg Lys Arg Lys Met Tyr Asp Glu
225 230 235 240
Phe Leu Ser Lys Val Ser Ile Leu Ala Asp Leu Asp Lys Trp Glu Arg
245 250 255
Ala Asn Val Ala Asp Ala Leu Glu Gln Cys Gln Phe Glu Pro Gly Thr
260 265 270
Arg Ile Val Glu Gln Gly Gln Pro Gly Asp Glu Phe Phe Ile Ile Val
275 280 285
Glu Gly Glu Ala Glu Val Leu Gln Arg Pro Asn Asp Asp Ala Pro Tyr
290 295 300
Glu Val Val Gly Lys Leu Gly Pro Ser Asp Tyr Phe Gly Glu Ile Ala
305 310 315 320
Leu Leu Leu Asp His Pro Arg Ala Ala Thr Val Val Ala Lys Gly Pro
325 330 335
Leu Lys Cys Val Lys Leu Asp Arg Ala Arg Phe Glu Arg Val Met Gly
340 345 350
Pro Val Arg Glu Ile Leu Lys Arg Asp Val Ser His Tyr Asn Ser Tyr
355 360 365
Val Lys Leu Met Thr
370
<210> 13
<211> 993
<212> DNA
<213> 南方根结线虫
<400> 13
aagccaagcc ctgtgcaaga agctgctatt ggacccgctc tttcgggaca agatgtgctc 60
gctcgtgcta agaatggaac aggaaagacg ggcgccttcg caatcccagt gatcgagaag 120
atcgacgtga acatcaagaa agtgcaagca atcattatgg tgccaactcg tgagctcgct 180
cttcaaacat cgcaaatctg cgtggagctt ggaaagcata tgaatgtcaa ggtgatggtt 240
acaactggcg gtacggattt gcgtgacgac attatgcgtc tcaatggaac cgttcatctg 300
attgtggcaa ctcccggacg aatcttggat ctcatcgaga agactgttgc tgatgtgagc 360
ctctgcaaga tgcttgtctt ggatgaagcc gacaaactac tttcgatcga ttttcaaggt 420
gctctcgata agatgatcag ctacttacca agagagcgcc aaattatgct tttctcggca 480
acgtttccat tggcggtggc caacttcatg caaaagtaca tgaagaaacc gtacgaaatc 540
aatctgatgg aggaactcac tcttcatggc gtcacccaat actacgctta tgtccaagag 600
aagcaaaagg ttcactgcct caatacgctt ttccggaagc tgcaagtcaa ccaatcaatc 660
atcttctgca attcaacaca gcgtgtggaa cttttggcaa agaagatcac tgaaatcggt 720
tactcctgct actacattca ttcgcgaatg atgcaacaag ataggaaccg cgtcttccac 780
gatttccgtc aaggaaactg ccgcaacttg gtctgttcgg atcttctcac tagaggcatt 840
gacatccaag ctgtgaatgt tgtgattaat tttgattttc ctcgcaactc tgagacttat 900
cttcacagaa ttggacgttc tggccgattt ggacatctcg gaatcgcaat taacctcatt 960
acttatgagg atcgttacaa tctcaagcgc att 993
<210> 14
<211> 332
<212> PRT
<213> 南方根结线虫
<400> 14
Glu Lys Pro Ser Pro Val Gln Glu Ala Ala Ile Gly Pro Ala Leu Ser
1 5 10 15
Gly Gln Asp Val Leu Ala Arg Ala Lys Asn Gly Thr Gly Lys Thr Gly
20 25 30
Ala Phe Ala Ile Pro Val Ile Glu Lys Ile Asp Val Asn Ile Lys Lys
35 40 45
Val Gln Ala Ile Ile Met Val Pro Thr Arg Glu Leu Ala Leu Gln Thr
50 55 60
Ser Gln Ile Cys Val Glu Leu Gly Lys His Met Asn Val Lys Val Met
65 70 75 80
Val Thr Thr Gly Gly Thr Asp Leu Arg Asp Asp Ile Met Arg Leu Asn
85 90 95
Gly Thr Val His Leu Ile Val Ala Thr Pro Gly Arg Ile Leu Asp Leu
100 105 110
Ile Glu Lys Thr Val Ala Asp Val Ser Leu Cys Lys Met Leu Val Leu
115 120 125
Asp Glu Ala Asp Lys Leu Leu Ser Ile Asp Phe Gln Gly Ala Leu Asp
130 135 140
Lys Met Ile Ser Tyr Leu Pro Arg Glu Arg Gln Ile Met Leu Phe Ser
145 150 155 160
Ala Thr Phe Pro Leu Ala Val Ala Asn Phe Met Gln Lys Tyr Met Lys
165 170 175
Lys Pro Tyr Glu Ile Asn Leu Met Glu Glu Leu Thr Leu His Gly Val
180 185 190
Thr Gln Tyr Tyr Ala Tyr Val Gln Glu Lys Gln Lys Val His Cys Leu
195 200 205
Asn Thr Leu Phe Arg Lys Leu Gln Val Asn Gln Ser Ile Ile Phe Cys
210 215 220
Asn Ser Thr Gln Arg Val Glu Leu Leu Ala Lys Lys Ile Thr Glu Ile
225 230 235 240
Gly Tyr Ser Cys Tyr Tyr Ile His Ser Arg Met Met Gln Gln Asp Arg
245 250 255
Asn Arg Val Phe His Asp Phe Arg Gln Gly Asn Cys Arg Asn Leu Val
260 265 270
Cys Ser Asp Leu Leu Thr Arg Gly Ile Asp Ile Gln Ala Val Asn Val
275 280 285
Val Ile Asn Phe Asp Phe Pro Arg Asn Ser Glu Thr Tyr Leu His Arg
290 295 300
Ile Gly Arg Ser Gly Arg Phe Gly His Leu Gly Ile Ala Ile Asn Leu
305 310 315 320
Ile Thr Tyr Glu Asp Arg Tyr Asn Leu Lys Arg Ile
325 330
<210> 15
<211> 3225
<212> DNA
<213> 南方根结线虫
<400> 15
atgaagcata caagacctat atttgacgaa gaaccagcaa taaaaattac tcgttgtgga 60
aattcttcta tttgttctca aagaaacgga aaaataatgc cggcatctga aaccaatcaa 120
aataattcaa atcaaacagg aggaaatgca aatggtccaa actctgccga ccattctgtt 180
gacacaaatc tttattcacg acaaatttat gcattgggcg aatctgccat gactcattta 240
cgcaaggctt cggttttgat tagtggtatt ggatccgttg ggctcgaaat cgccaagaat 300
gttatccttg gtggcgttcg tcacgtaact ctacatgacc aaaaactttt gacttttgga 360
gatttatcag cgtgttttta tgctgacgag tcaatggtgg gtgagaatcg tgccaaaatc 420
tcttttacaa agttggccga attgaatgac accgtcactt gcactcttca cacagaagca 480
ttgaatgagg aatttgtgaa acagtttgat ttagttatcg ttactgattc accttgggaa 540
gagcaaatta aaattaacaa ctggatgcgg aaacatggca agctgttcat ttctgccgat 600
gctagaggtc ttttttcata tgcttttgtt gatcttggtg agaaatttgc tgtgcacgat 660
gttgatggtg aaacttataa tgaagtatta ctcgagcatg ttaattgtga gactggtgaa 720
atatttactc ttgacaaagc ttatcatgga ttggaagatg gagatcatat tgtttttgag 780
gattttgaga ttgcagaact taataaattg acaccaaccc cagtaaaaac aactgcaaaa 840
ggtcatattg tcaatgttgg caaagctctt tctagcttct cttcagtcat tgatggccca 900
atttctcgag gaagggctcg taaagttaaa atgccgaaat ttattgagtt taaatcactt 960
gctgaagcat taaagtcccc agaatttatg atttgggatt ttgctaaatt tgatggttcg 1020
gaacatctcc accgcctctg gcaagcactc tacaaattcg agaaaaagca caaacgttca 1080
ccaaaacctc gttccaaaaa ggatgccgaa ttgtttaaag ctgaacttga agggactgga 1140
acagcagaga ttcctgaaca attgctgctc aatttttctt atcaggccac tggaaatctt 1200
caacctgtcg cttctgttat tggtggattt gttgctcaag aggcatttaa agctgtcact 1260
catcacacga cacctttgaa acaattttta tatacggact cgtgtgaagc acttcctggt 1320
gattactcta gctttgatgc tggtaaattg actgaaaagg attgtgaacc gcgccaaact 1380
cgttatgacg gtcaagcagc tgtttttggc tggtccttcc aagcagcatt atctaaacaa 1440
aattggttta ttgttggcgc aggtgccatt ggttgtgaac tttttaaaaa tatggttatg 1500
atgggattgg cagcgggaaa tggtggtcta cttaaagtta cagatccaga tactattgaa 1560
gtttctaact tgaatagaca attcctcttt cgtcgacctg atgttgggaa aaagaaatct 1620
gaagttgctg ctcgttctat gaagcaattc aacccagcag ttaatgtcca agctctttct 1680
gatattgttt ctgaggcaac agagagtgtt ttcaacgatg actttttcaa tcaattaaat 1740
ggtgtttgta atgctttgga taatgttgaa gctcgtcgct atgttgatcg acgttgtgtt 1800
tattatcagc tgccattgct tgaatctgga acaatgggag caaaaggaaa tactcaagtg 1860
gttttcccac acttaactga atcatacggt tcgacttctg atccgcccga atcagaaaca 1920
cctgtttgta cactcaagaa tttcccatat caaattcagc atacgattca atgggctcga 1980
tcgaaatttg ctgatcattt tacctcggtt gcagaaacag ccaatcaata tttggatgat 2040
gtaaatggtt ttcttggccg tcttcaacaa atgaccgttt ctcaaaagat cgaattactg 2100
cgtgcattaa ataaagcttt aattgatgaa tgcccaagca gtgctgaaga ttgtgtcaaa 2160
tgggcgcgtg atttgtttga ctcgctctat agaaatgaga ttatgcaact gttgcacaat 2220
tttcctcctg atcaggttac ttctcaagga caaaagtttt ggtcaggaac taagcgttgt 2280
cctcatccat tgaattttga cgttgacaat cccgaacatt tggaatttgt ttattcagcg 2340
gcttttctac gagcacaact ttataatatt gaaccaatta gtgaccctat caaagttgct 2400
catcttgctc gcgccatcat atcaccagaa ttcaaaccga gagaaggcat taagattgca 2460
gttactgatg cagaggctgc tgctaacgat gaagctggcc ctgaaggaga tgacagtgaa 2520
aagcttcttg atactttaaa tatgaatttg gctcgtttaa aaattgataa tattcgccgt 2580
ttaactccta tcgattttga aaaggatgat gacacaaatc accatattga ctttattact 2640
gcagcttcaa atttgcgtgc agaaaattat acgatagaga aggctgatag aatgaagacc 2700
aaacaaattg ctggtaaaat tattcctgca ttggctacaa cgacttcagt tgttgctgga 2760
cttgtttgta ttgaacttta taagactatt gaggttgaag gccaacgttc gaaggcacct 2820
attgaacgtt ttaaaaacgc ttttcttaac ctggcaactc cttttattgc tttcagtgaa 2880
cctggaaaag cgtcgaaaaa gaagtatcgt gatattgaat ttactctttg ggatagattg 2940
gaagtaaatg gtcctaaaac acttggacag tttattgaat ggattgagac acaaactgga 3000
ttaactgtct caatgatgtc ttctggtgtt tcgcttcttt atgctttctt ccagccacct 3060
agtaaagttg ctgaacgtaa aaccagagat gttattcaag ttgtggagga agtgtctcgt 3120
aataaagttc ctccatttcg tcgttcactt gtatttgagg caattactca aaatgataag 3180
gacgaggatg ttgagattcc ttatgtcaaa tataatttcc gttaa 3225
<210> 16
<211> 1074
<212> PRT
<213> 南方根结线虫
<400> 16
Met Lys His Thr Arg Pro Ile Phe Asp Glu Glu Pro Ala Ile Lys Ile
1 5 10 15
Thr Arg Cys Gly Asn Ser Ser Ile Cys Ser Gln Arg Asn Gly Lys Ile
20 25 30
Met Pro Ala Ser Glu Thr Asn Gln Asn Asn Ser Asn Gln Thr Gly Gly
35 40 45
Asn Ala Asn Gly Pro Asn Ser Ala Asp His Ser Val Asp Thr Asn Leu
50 55 60
Tyr Ser Arg Gln Ile Tyr Ala Leu Gly Glu Ser Ala Met Thr His Leu
65 70 75 80
Arg Lys Ala Ser Val Leu Ile Ser Gly Ile Gly Ser Val Gly Leu Glu
85 90 95
Ile Ala Lys Asn Val Ile Leu Gly Gly Val Arg His Val Thr Leu His
100 105 110
Asp Gln Lys Leu Leu Thr Phe Gly Asp Leu Ser Ala Cys Phe Tyr Ala
115 120 125
Asp Glu Ser Met Val Gly Glu Asn Arg Ala Lys Ile Ser Phe Thr Lys
130 135 140
Leu Ala Glu Leu Asn Asp Thr Val Thr Cys Thr Leu His Thr Glu Ala
145 150 155 160
Leu Asn Glu Glu Phe Val Lys Gln Phe Asp Leu Val Ile Val Thr Asp
165 170 175
Ser Pro Trp Glu Glu Gln Ile Lys Ile Asn Asn Trp Met Arg Lys His
180 185 190
Gly Lys Leu Phe Ile Ser Ala Asp Ala Arg Gly Leu Phe Ser Tyr Ala
195 200 205
Phe Val Asp Leu Gly Glu Lys Phe Ala Val His Asp Val Asp Gly Glu
210 215 220
Thr Tyr Asn Glu Val Leu Leu Glu His Val Asn Cys Glu Thr Gly Glu
225 230 235 240
Ile Phe Thr Leu Asp Lys Ala Tyr His Gly Leu Glu Asp Gly Asp His
245 250 255
Ile Val Phe Glu Asp Phe Glu Ile Ala Glu Leu Asn Lys Leu Thr Pro
260 265 270
Thr Pro Val Lys Thr Thr Ala Lys Gly His Ile Val Asn Val Gly Lys
275 280 285
Ala Leu Ser Ser Phe Ser Ser Val Ile Asp Gly Pro Ile Ser Arg Gly
290 295 300
Arg Ala Arg Lys Val Lys Met Pro Lys Phe Ile Glu Phe Lys Ser Leu
305 310 315 320
Ala Glu Ala Leu Lys Ser Pro Glu Phe Met Ile Trp Asp Phe Ala Lys
325 330 335
Phe Asp Gly Ser Glu His Leu His Arg Leu Trp Gln Ala Leu Tyr Lys
340 345 350
Phe Glu Lys Lys His Lys Arg Ser Pro Lys Pro Arg Ser Lys Lys Asp
355 360 365
Ala Glu Leu Phe Lys Ala Glu Leu Glu Gly Thr Gly Thr Ala Glu Ile
370 375 380
Pro Glu Gln Leu Leu Leu Asn Phe Ser Tyr Gln Ala Thr Gly Asn Leu
385 390 395 400
Gln Pro Val Ala Ser Val Ile Gly Gly Phe Val Ala Gln Glu Ala Phe
405 410 415
Lys Ala Val Thr His His Thr Thr Pro Leu Lys Gln Phe Leu Tyr Thr
420 425 430
Asp Ser Cys Glu Ala Leu Pro Gly Asp Tyr Ser Ser Phe Asp Ala Gly
435 440 445
Lys Leu Thr Glu Lys Asp Cys Glu Pro Arg Gln Thr Arg Tyr Asp Gly
450 455 460
Gln Ala Ala Val Phe Gly Trp Ser Phe Gln Ala Ala Leu Ser Lys Gln
465 470 475 480
Asn Trp Phe Ile Val Gly Ala Gly Ala Ile Gly Cys Glu Leu Phe Lys
485 490 495
Asn Met Val Met Met Gly Leu Ala Ala Gly Asn Gly Gly Leu Leu Lys
500 505 510
Val Thr Asp Pro Asp Thr Ile Glu Val Ser Asn Leu Asn Arg Gln Phe
515 520 525
Leu Phe Arg Arg Pro Asp Val Gly Lys Lys Lys Ser Glu Val Ala Ala
530 535 540
Arg Ser Met Lys Gln Phe Asn Pro Ala Val Asn Val Gln Ala Leu Ser
545 550 555 560
Asp Ile Val Ser Glu Ala Thr Glu Ser Val Phe Asn Asp Asp Phe Phe
565 570 575
Asn Gln Leu Asn Gly Val Cys Asn Ala Leu Asp Asn Val Glu Ala Arg
580 585 590
Arg Tyr Val Asp Arg Arg Cys Val Tyr Tyr Gln Leu Pro Leu Leu Glu
595 600 605
Ser Gly Thr Met Gly Ala Lys Gly Asn Thr Gln Val Val Phe Pro His
610 615 620
Leu Thr Glu Ser Tyr Gly Ser Thr Ser Asp Pro Pro Glu Ser Glu Thr
625 630 635 640
Pro Val Cys Thr Leu Lys Asn Phe Pro Tyr Gln Ile Gln His Thr Ile
645 650 655
Gln Trp Ala Arg Ser Lys Phe Ala Asp His Phe Thr Ser Val Ala Glu
660 665 670
Thr Ala Asn Gln Tyr Leu Asp Asp Val Asn Gly Phe Leu Gly Arg Leu
675 680 685
Gln Gln Met Thr Val Ser Gln Lys Ile Glu Leu Leu Arg Ala Leu Asn
690 695 700
Lys Ala Leu Ile Asp Glu Cys Pro Ser Ser Ala Glu Asp Cys Val Lys
705 710 715 720
Trp Ala Arg Asp Leu Phe Asp Ser Leu Tyr Arg Asn Glu Ile Met Gln
725 730 735
Leu Leu His Asn Phe Pro Pro Asp Gln Val Thr Ser Gln Gly Gln Lys
740 745 750
Phe Trp Ser Gly Thr Lys Arg Cys Pro His Pro Leu Asn Phe Asp Val
755 760 765
Asp Asn Pro Glu His Leu Glu Phe Val Tyr Ser Ala Ala Phe Leu Arg
770 775 780
Ala Gln Leu Tyr Asn Ile Glu Pro Ile Ser Asp Pro Ile Lys Val Ala
785 790 795 800
His Leu Ala Arg Ala Ile Ile Ser Pro Glu Phe Lys Pro Arg Glu Gly
805 810 815
Ile Lys Ile Ala Val Thr Asp Ala Glu Ala Ala Ala Asn Asp Glu Ala
820 825 830
Gly Pro Glu Gly Asp Asp Ser Glu Lys Leu Leu Asp Thr Leu Asn Met
835 840 845
Asn Leu Ala Arg Leu Lys Ile Asp Asn Ile Arg Arg Leu Thr Pro Ile
850 855 860
Asp Phe Glu Lys Asp Asp Asp Thr Asn His His Ile Asp Phe Ile Thr
865 870 875 880
Ala Ala Ser Asn Leu Arg Ala Glu Asn Tyr Thr Ile Glu Lys Ala Asp
885 890 895
Arg Met Lys Thr Lys Gln Ile Ala Gly Lys Ile Ile Pro Ala Leu Ala
900 905 910
Thr Thr Thr Ser Val Val Ala Gly Leu Val Cys Ile Glu Leu Tyr Lys
915 920 925
Thr Ile Glu Val Glu Gly Gln Arg Ser Lys Ala Pro Ile Glu Arg Phe
930 935 940
Lys Asn Ala Phe Leu Asn Leu Ala Thr Pro Phe Ile Ala Phe Ser Glu
945 950 955 960
Pro Gly Lys Ala Ser Lys Lys Lys Tyr Arg Asp Ile Glu Phe Thr Leu
965 970 975
Trp Asp Arg Leu Glu Val Asn Gly Pro Lys Thr Leu Gly Gln Phe Ile
980 985 990
Glu Trp Ile Glu Thr Gln Thr Gly Leu Thr Val Ser Met Met Ser Ser
995 1000 1005
Gly Val Ser Leu Leu Tyr Ala Phe Phe Gln Pro Pro Ser Lys Val
1010 1015 1020
Ala Glu Arg Lys Thr Arg Asp Val Ile Gln Val Val Glu Glu Val
1025 1030 1035
Ser Arg Asn Lys Val Pro Pro Phe Arg Arg Ser Leu Val Phe Glu
1040 1045 1050
Ala Ile Thr Gln Asn Asp Lys Asp Glu Asp Val Glu Ile Pro Tyr
1055 1060 1065
Val Lys Tyr Asn Phe Arg
1070
<210> 17
<211> 1716
<212> DNA
<213> 南方根结线虫
<400> 17
atgcctccat ctactcgtga agttaaagaa gcccaatatg gatatgttta tggtgtttct 60
gggcctgtcg taacggccga aaagatgtct ggtgctgcca tgtacgaatt agtccgtgtt 120
ggacataatg aacttgttgg tgaaatcatt cgtttggaag gggattatgc aactatacaa 180
gtctacgagg aaacatctgc tgtaacaatt ggagatccag ttttacgtac cggaaagcca 240
ctttcggtag aattaggtcc aggcattatg ggctgtatct ttgacggaat tcaacgtcct 300
ctacacgata ttgcacagat gacccaatct atttatatac ccaaaggaat caatacaaac 360
gccctcagtc gaacaaaaca atgggaattt attccaaaac aaagtcttcg tccaggggat 420
catgttactg gtggtgatat tgtggggata gtctatgaga gtgctttggt taaacactca 480
attatggtgc ctccaaatgc ttgtggaacg ttgaagtttt tggcacctaa agggagctat 540
aatgttacgg aaaaaatcat cgaaatcgaa ttttctggag atgttcaaaa atattctatg 600
cttcaagtct ggcctgtaag acagcctcgc ccagtcgccg aaaaattggc tgccaattat 660
ccacttttgt gtggtcaaag agttttggat gctctattcc cttgtgtaca agggggaacg 720
actgctatac ctggagcttt tggttgtgga aaaacagtaa tttcccaatc attgtcgaaa 780
tacagcaatt ctgacgcaat tatttatgtt ggctgtggag aacgtggcaa cgagatgtcc 840
gaggtattac gtgatttccc agaattaaca atggaagtag atggaaaaac aacctcaatt 900
atggaaagaa cagctttagt agctaatact tctaatatgc ctgtagctgc tagagaagct 960
agtatttata caggaattac tttggctgaa tattttcgtg atatgggttt aaatgttgca 1020
atgatggcag attcaacttc tcgttgggca gaagctttaa gagaaatttc tggacgtttg 1080
ggagaaatgc ctgctgattc tggttatcct gcctatttag ctgctagatt agcttcgttt 1140
tatgaaaggg ctgggaaagt taaatgtcgt ggatcgccgg atagagatgg atctgttact 1200
attgttgggg ctgtttctcc tcccggaggt gattttgctg atccagtaac tacagcaaca 1260
ttaggaattg ttcaagtatt ttggggatta gacaaaaaat tggctcaacg aaaacatttt 1320
ccttcaatta attggcttat atcttacagt aattatatga gagcattaga tgattattat 1380
gaaaagaatt tccctgaatt cgtccctctt cgtacaaaat acaaagaaat tttacaggag 1440
gaagaagatc tttctgagat tgtccaactt gtaggcaaag cttcattagc tgaatctgac 1500
aaaattactc ttgaagttgc aaaaattatt aaagatgatt tcctccaaca aaatggttat 1560
gcaccttatg atcgtttttg tccgttttac aaaactgttg gaatgatgaa aaatatgatt 1620
gcattttatg actttgcccg tcatgcagtt gaatctactg cacaatctga caacaaaatt 1680
acttggaacg tgcacaacgt gattcgcaag gattga 1716
<210> 18
<211> 571
<212> PRT
<213> 南方根结线虫
<400> 18
Met Pro Pro Ser Thr Arg Glu Val Lys Glu Ala Gln Tyr Gly Tyr Val
1 5 10 15
Tyr Gly Val Ser Gly Pro Val Val Thr Ala Glu Lys Met Ser Gly Ala
20 25 30
Ala Met Tyr Glu Leu Val Arg Val Gly His Asn Glu Leu Val Gly Glu
35 40 45
Ile Ile Arg Leu Glu Gly Asp Tyr Ala Thr Ile Gln Val Tyr Glu Glu
50 55 60
Thr Ser Ala Val Thr Ile Gly Asp Pro Val Leu Arg Thr Gly Lys Pro
65 70 75 80
Leu Ser Val Glu Leu Gly Pro Gly Ile Met Gly Cys Ile Phe Asp Gly
85 90 95
Ile Gln Arg Pro Leu His Asp Ile Ala Gln Met Thr Gln Ser Ile Tyr
100 105 110
Ile Pro Lys Gly Ile Asn Thr Asn Ala Leu Ser Arg Thr Lys Gln Trp
115 120 125
Glu Phe Ile Pro Lys Gln Ser Leu Arg Pro Gly Asp His Val Thr Gly
130 135 140
Gly Asp Ile Val Gly Ile Val Tyr Glu Ser Ala Leu Val Lys His Ser
145 150 155 160
Ile Met Val Pro Pro Asn Ala Cys Gly Thr Leu Lys Phe Leu Ala Pro
165 170 175
Lys Gly Ser Tyr Asn Val Thr Glu Lys Ile Ile Glu Ile Glu Phe Ser
180 185 190
Gly Asp Val Gln Lys Tyr Ser Met Leu Gln Val Trp Pro Val Arg Gln
195 200 205
Pro Arg Pro Val Ala Glu Lys Leu Ala Ala Asn Tyr Pro Leu Leu Cys
210 215 220
Gly Gln Arg Val Leu Asp Ala Leu Phe Pro Cys Val Gln Gly Gly Thr
225 230 235 240
Thr Ala Ile Pro Gly Ala Phe Gly Cys Gly Lys Thr Val Ile Ser Gln
245 250 255
Ser Leu Ser Lys Tyr Ser Asn Ser Asp Ala Ile Ile Tyr Val Gly Cys
260 265 270
Gly Glu Arg Gly Asn Glu Met Ser Glu Val Leu Arg Asp Phe Pro Glu
275 280 285
Leu Thr Met Glu Val Asp Gly Lys Thr Thr Ser Ile Met Glu Arg Thr
290 295 300
Ala Leu Val Ala Asn Thr Ser Asn Met Pro Val Ala Ala Arg Glu Ala
305 310 315 320
Ser Ile Tyr Thr Gly Ile Thr Leu Ala Glu Tyr Phe Arg Asp Met Gly
325 330 335
Leu Asn Val Ala Met Met Ala Asp Ser Thr Ser Arg Trp Ala Glu Ala
340 345 350
Leu Arg Glu Ile Ser Gly Arg Leu Gly Glu Met Pro Ala Asp Ser Gly
355 360 365
Tyr Pro Ala Tyr Leu Ala Ala Arg Leu Ala Ser Phe Tyr Glu Arg Ala
370 375 380
Gly Lys Val Lys Cys Arg Gly Ser Pro Asp Arg Asp Gly Ser Val Thr
385 390 395 400
Ile Val Gly Ala Val Ser Pro Pro Gly Gly Asp Phe Ala Asp Pro Val
405 410 415
Thr Thr Ala Thr Leu Gly Ile Val Gln Val Phe Trp Gly Leu Asp Lys
420 425 430
Lys Leu Ala Gln Arg Lys His Phe Pro Ser Ile Asn Trp Leu Ile Ser
435 440 445
Tyr Ser Asn Tyr Met Arg Ala Leu Asp Asp Tyr Tyr Glu Lys Asn Phe
450 455 460
Pro Glu Phe Val Pro Leu Arg Thr Lys Tyr Lys Glu Ile Leu Gln Glu
465 470 475 480
Glu Glu Asp Leu Ser Glu Ile Val Gln Leu Val Gly Lys Ala Ser Leu
485 490 495
Ala Glu Ser Asp Lys Ile Thr Leu Glu Val Ala Lys Ile Ile Lys Asp
500 505 510
Asp Phe Leu Gln Gln Asn Gly Tyr Ala Pro Tyr Asp Arg Phe Cys Pro
515 520 525
Phe Tyr Lys Thr Val Gly Met Met Lys Asn Met Ile Ala Phe Tyr Asp
530 535 540
Phe Ala Arg His Ala Val Glu Ser Thr Ala Gln Ser Asp Asn Lys Ile
545 550 555 560
Thr Trp Asn Val His Asn Val Ile Arg Lys Asp
565 570
<210> 19
<211> 827
<212> DNA
<213> 南方根结线虫
<400> 19
ctttgctgcg gccgcgggga gcatgtctct caaacaggag cctctgcatc tgcttccggt 60
tcatttccaa ttgttttacc acataatgaa ctaaagcagg acggtggcaa attttatttg 120
gaaggagacg aaaagaatac ctgccttcta tatattgaat cgatttctgt tgtgttatat 180
aacgtaaaac aaaaaatgtc tgcttgggcg accattaatg caacttctaa gggagcaaca 240
tatagtggta aaattgagtg tccagcaact cataatacag gacaggagtt taaaatttca 300
ttgcaagcaa ttgctggcgg tgaaggtgtt tcaggctatt atattggtga taaaaagaaa 360
gtcattttcc ggttaagcaa agtcgatgtt gatttaatat ttaactttac ttcatcagct 420
tattgggagt tggtgaatgc tgatgttagg acaataagtt tggatggcgc agaagaaggt 480
ggagttccga gtgtcgataa cacctggcga cttgtttctg gtataagcag caacaataaa 540
ggatggattt tcacagataa tcaatctccg gcctggaata ttaatgggtt taaagactac 600
gccttctctt gtggtcgaac tagtccaatt atttgggcaa aacatgatac ggatgaaaaa 660
agcctggaac atgcagttgg tttggctttt cataatttac agtttcaatt gaattcaact 720
tatatttctc cggataaaaa aagaatggca aagtttggct ggcgtgttaa tgattgtgca 780
cctctattaa gtattggtac ttggatgatt cttgtggttg caattat 827
<210> 20
<211> 275
<212> PRT
<213> 南方根结线虫
<400> 20
Leu Cys Cys Gly Arg Gly Glu His Val Ser Gln Thr Gly Ala Ser Ala
1 5 10 15
Ser Ala Ser Gly Ser Phe Pro Ile Val Leu Pro His Asn Glu Leu Lys
20 25 30
Gln Asp Gly Gly Lys Phe Tyr Leu Glu Gly Asp Glu Lys Asn Thr Cys
35 40 45
Leu Leu Tyr Ile Glu Ser Ile Ser Val Val Leu Tyr Asn Val Lys Gln
50 55 60
Lys Met Ser Ala Trp Ala Thr Ile Asn Ala Thr Ser Lys Gly Ala Thr
65 70 75 80
Tyr Ser Gly Lys Ile Glu Cys Pro Ala Thr His Asn Thr Gly Gln Glu
85 90 95
Phe Lys Ile Ser Leu Gln Ala Ile Ala Gly Gly Glu Gly Val Ser Gly
100 105 110
Tyr Tyr Ile Gly Asp Lys Lys Lys Val Ile Phe Arg Leu Ser Lys Val
115 120 125
Asp Val Asp Leu Ile Phe Asn Phe Thr Ser Ser Ala Tyr Trp Glu Leu
130 135 140
Val Asn Ala Asp Val Arg Thr Ile Ser Leu Asp Gly Ala Glu Glu Gly
145 150 155 160
Gly Val Pro Ser Val Asp Asn Thr Trp Arg Leu Val Ser Gly Ile Ser
165 170 175
Ser Asn Asn Lys Gly Trp Ile Phe Thr Asp Asn Gln Ser Pro Ala Trp
180 185 190
Asn Ile Asn Gly Phe Lys Asp Tyr Ala Phe Ser Cys Gly Arg Thr Ser
195 200 205
Pro Ile Ile Trp Ala Lys His Asp Thr Asp Glu Lys Ser Leu Glu His
210 215 220
Ala Val Gly Leu Ala Phe His Asn Leu Gln Phe Gln Leu Asn Ser Thr
225 230 235 240
Tyr Ile Ser Pro Asp Lys Lys Arg Met Ala Lys Phe Gly Trp Arg Val
245 250 255
Asn Asp Cys Ala Pro Leu Leu Ser Ile Gly Thr Trp Met Ile Leu Val
260 265 270
Val Ala Ile
275
<210> 21
<211> 3510
<212> DNA
<213> 南方根结线虫
<400> 21
atggttacaa aaattccaac ttttcccctc ctttttattt tcccattatt atttacattt 60
ttaaccacaa aatgtcaggc ttattctata ccattaatat cagaatgtaa ttcggaagaa 120
gccccagttt ttcttttgca acggaatgtt tcttctatcg ccggaactga gcctttaaga 180
actgttcctg ttactggggg atttttggaa tgtgcggaac tttgctcagc agcaaataat 240
tgtgttgccg ttaaattctc tattgaaaaa caatgccaat tattggggaa aacaacaacg 300
acaacaacaa ctttatcttt acaagacatt aatttaacac aagctagatt agctactaaa 360
agttgtgtga agagcaaaaa aatttgttct tcccccttcc attttgatgt tcacgaacaa 420
aaaatacttg ttggttttgc tagagaagtt gtatcagcag aatctataca tcaatgttta 480
actgcttgtt tagatgctgt tgatactttt ggctttgaat gcgagtcagt aatgtattat 540
ccattggatg ccgaatgtat tttaaataca gaagacagac ttgaccgtcc agatttgttt 600
gttgatgaga aggaggatac tgttgtttat ttggataata attgtgctgg atcccaatgt 660
catgcctctt atgtaaccca atatgtagct gttgaaggaa aacaattagc cgaggaattg 720
gatcataatt ttgatggaat ggaattgaca gaatgtgaac aactttgtaa tcaaagattg 780
agtgtttctg caaatgactt caattgcaaa gcatttatgt acaataacca aacaagatct 840
tgtattcttt ctgatgaacg ttcaagacct ttgggtagag ctaatttgac agatgctaaa 900
ggatggactt atcacgagaa aaaatgtttt gcctctcctc gaacttgccg aaatgttcct 960
tcatttaccc gcgtccctca aatgttatta gttggatttg cctcttttgt aatggaaaat 1020
gtcccttcag taactatgtg tttggatcaa tgtacaaatc ctccaccaga aactggacaa 1080
agttttgttt gtaaatctgt catgtattat tataatgagc aagaatgtat tttaaatgct 1140
gaatcacgtc attccaagcc agatttattt attcccgaag aagacgattt tgttgtagat 1200
tattttgata taaattgccg tctagaacaa gaacaatgta tcgatggaag aacaccccaa 1260
ttagttagaa caattaattc tgcacttcca gaaggggagg ggtctataca tgttttggaa 1320
acaattaagg gaggagttca gcaatgtgct aaaaagtgtt ttgaacacgc cccagacaaa 1380
tgtcgttctt tcaattttga taaacaagct ggtaattgta atttacttta tttggatgga 1440
caagggtctt tacgaccaga gcaaaagaca caatttgatt tatacgatgt tcactgtttg 1500
agtgggacat ctcaactttt aggagaaaat tctaaacatt ctccctctgc ttgtgttgac 1560
ccagaagggg ctatttttag tcgtttcctc tacactcgtt gggtagcaaa ttctcccaat 1620
cgtgaaattt caagtttatc actttccaaa tgtttaaatc tttgttcggt tggaggagaa 1680
caatgtgaag gtgttaatta caatcgtcga aatggttctt gtcaattatt tacttccctt 1740
ctattaaatt cttctccaaa ttctcaacaa gacaaagacg aacatgttga tttttacaga 1800
aatatttgta gagttaagga atcgaaaagt gatagtgggg ctgctaatgt acccaaaaca 1860
caacaagcaa cggctgcacc tcccccttct gttcaattaa ctactaaacc tccacaaatt 1920
cgtgatttaa acaacaacaa taaaacaaca cacaaagaac caaatattaa acttccacca 1980
caatcagcaa aacctataaa tggaaaaact ggaaaggaac aacttcctgt agggtcaaaa 2040
tcttttgggg ttactaatac gcgtgatgat ggggagaatt caataactgg aactgctcct 2100
cctcctgtag atggcaaatt aattattaaa ccttcaccac aagtttctat tccctcccct 2160
gtacttattc cggcacaaga agtacatact atttgtaatt atgaaggaat tagtgttcaa 2220
attaaacatt cttctccatt ctctggcgtt gtttttgttc gaaataaata tgatacttgc 2280
cgtgtgaagt tgaaggaaag gacagcgttg ttttggtttt ggggcttcca gcaaattttg 2340
gaaatgaagc caattgcttt aattaattca caaaaacatg gaaaagggaa taaaacacac 2400
ggagatactt tactttctat tgaaggttcc aaaaaacaaa ttgaaggggg ttcttcaact 2460
gaagatattc aattaataaa ttctcaaaaa gaccttaaac gttcaagaag acaattacaa 2520
agagattgtg gattacaaga tatggacaat ggaacttaca aaactgttat tgttgtccaa 2580
acaaataatt tgggaattcc gggacttgtt acttctatgg accaacttta tgagatttcc 2640
tgtaactatt caagtatgtt gggaggcaaa gtccagacag cagctgcatt acgtgttcac 2700
ggtccccaac cttcactaat ccagcctcgc ggcaaaatag aattgggaaa tcctgttttg 2760
atgcaaatgg ggcctgtacg tagtgaaagg caaagtgggg aagggccttt aattcaagct 2820
aaattgggag atattcttga attaaaatgg gaaattatgg caatggatga agaattggac 2880
tttttagttc gtgattgttt tgcagagccg ggaacttctg gaaatcaagg ggaaagactt 2940
cctttaattg agaatggttg tccaacacca gcagtagcac aaaaattaat tccaaatcca 3000
ataaaagcaa ttaattctgc agttaaatta acttatttac aagcattcag atttgacagt 3060
tctccagcta ttagaataac ttgtcattta gaattatgta aagaaaattg taaatcggtt 3120
aattgtaaat ttaatgatgg aattaaagaa tcgtggggca gaaaacgccg ttttgctatt 3180
gacaataaca ttaataggaa aaatgaagtt aaagaattcg aaactcgccg ttttgtcgtt 3240
ccccgttttg cccaagcaac aacttcttta gttattgtag accctttaca acaacaaaat 3300
tctgttataa aaacagaaca acaacaacaa ccatttattt cacattcctc aatatctaaa 3360
caaatatttg aaaataataa aaatataaca aaaacagcta aaaaatcctc ttctcttttt 3420
gaagctttta ctgaggctgc tggtggaagg aaaattaatt tagaattaac aacaacaaat 3480
tcagaacaac aacaactttt gtttacataa 3510
<210> 22
<211> 1169
<212> PRT
<213> 南方根结线虫
<400> 22
Met Val Thr Lys Ile Pro Thr Phe Pro Leu Leu Phe Ile Phe Pro Leu
1 5 10 15
Leu Phe Thr Phe Leu Thr Thr Lys Cys Gln Ala Tyr Ser Ile Pro Leu
20 25 30
Ile Ser Glu Cys Asn Ser Glu Glu Ala Pro Val Phe Leu Leu Gln Arg
35 40 45
Asn Val Ser Ser Ile Ala Gly Thr Glu Pro Leu Arg Thr Val Pro Val
50 55 60
Thr Gly Gly Phe Leu Glu Cys Ala Glu Leu Cys Ser Ala Ala Asn Asn
65 70 75 80
Cys Val Ala Val Lys Phe Ser Ile Glu Lys Gln Cys Gln Leu Leu Gly
85 90 95
Lys Thr Thr Thr Thr Thr Thr Thr Leu Ser Leu Gln Asp Ile Asn Leu
100 105 110
Thr Gln Ala Arg Leu Ala Thr Lys Ser Cys Val Lys Ser Lys Lys Ile
115 120 125
Cys Ser Ser Pro Phe His Phe Asp Val His Glu Gln Lys Ile Leu Val
130 135 140
Gly Phe Ala Arg Glu Val Val Ser Ala Glu Ser Ile His Gln Cys Leu
145 150 155 160
Thr Ala Cys Leu Asp Ala Val Asp Thr Phe Gly Phe Glu Cys Glu Ser
165 170 175
Val Met Tyr Tyr Pro Leu Asp Ala Glu Cys Ile Leu Asn Thr Glu Asp
180 185 190
Arg Leu Asp Arg Pro Asp Leu Phe Val Asp Glu Lys Glu Asp Thr Val
195 200 205
Val Tyr Leu Asp Asn Asn Cys Ala Gly Ser Gln Cys His Ala Ser Tyr
210 215 220
Val Thr Gln Tyr Val Ala Val Glu Gly Lys Gln Leu Ala Glu Glu Leu
225 230 235 240
Asp His Asn Phe Asp Gly Met Glu Leu Thr Glu Cys Glu Gln Leu Cys
245 250 255
Asn Gln Arg Leu Ser Val Ser Ala Asn Asp Phe Asn Cys Lys Ala Phe
260 265 270
Met Tyr Asn Asn Gln Thr Arg Ser Cys Ile Leu Ser Asp Glu Arg Ser
275 280 285
Arg Pro Leu Gly Arg Ala Asn Leu Thr Asp Ala Lys Gly Trp Thr Tyr
290 295 300
His Glu Lys Lys Cys Phe Ala Ser Pro Arg Thr Cys Arg Asn Val Pro
305 310 315 320
Ser Phe Thr Arg Val Pro Gln Met Leu Leu Val Gly Phe Ala Ser Phe
325 330 335
Val Met Glu Asn Val Pro Ser Val Thr Met Cys Leu Asp Gln Cys Thr
340 345 350
Asn Pro Pro Pro Glu Thr Gly Gln Ser Phe Val Cys Lys Ser Val Met
355 360 365
Tyr Tyr Tyr Asn Glu Gln Glu Cys Ile Leu Asn Ala Glu Ser Arg His
370 375 380
Ser Lys Pro Asp Leu Phe Ile Pro Glu Glu Asp Asp Phe Val Val Asp
385 390 395 400
Tyr Phe Asp Ile Asn Cys Arg Leu Glu Gln Glu Gln Cys Ile Asp Gly
405 410 415
Arg Thr Pro Gln Leu Val Arg Thr Ile Asn Ser Ala Leu Pro Glu Gly
420 425 430
Glu Gly Ser Ile His Val Leu Glu Thr Ile Lys Gly Gly Val Gln Gln
435 440 445
Cys Ala Lys Lys Cys Phe Glu His Ala Pro Asp Lys Cys Arg Ser Phe
450 455 460
Asn Phe Asp Lys Gln Ala Gly Asn Cys Asn Leu Leu Tyr Leu Asp Gly
465 470 475 480
Gln Gly Ser Leu Arg Pro Glu Gln Lys Thr Gln Phe Asp Leu Tyr Asp
485 490 495
Val His Cys Leu Ser Gly Thr Ser Gln Leu Leu Gly Glu Asn Ser Lys
500 505 510
His Ser Pro Ser Ala Cys Val Asp Pro Glu Gly Ala Ile Phe Ser Arg
515 520 525
Phe Leu Tyr Thr Arg Trp Val Ala Asn Ser Pro Asn Arg Glu Ile Ser
530 535 540
Ser Leu Ser Leu Ser Lys Cys Leu Asn Leu Cys Ser Val Gly Gly Glu
545 550 555 560
Gln Cys Glu Gly Val Asn Tyr Asn Arg Arg Asn Gly Ser Cys Gln Leu
565 570 575
Phe Thr Ser Leu Leu Leu Asn Ser Ser Pro Asn Ser Gln Gln Asp Lys
580 585 590
Asp Glu His Val Asp Phe Tyr Arg Asn Ile Cys Arg Val Lys Glu Ser
595 600 605
Lys Ser Asp Ser Gly Ala Ala Asn Val Pro Lys Thr Gln Gln Ala Thr
610 615 620
Ala Ala Pro Pro Pro Ser Val Gln Leu Thr Thr Lys Pro Pro Gln Ile
625 630 635 640
Arg Asp Leu Asn Asn Asn Asn Lys Thr Thr His Lys Glu Pro Asn Ile
645 650 655
Lys Leu Pro Pro Gln Ser Ala Lys Pro Ile Asn Gly Lys Thr Gly Lys
660 665 670
Glu Gln Leu Pro Val Gly Ser Lys Ser Phe Gly Val Thr Asn Thr Arg
675 680 685
Asp Asp Gly Glu Asn Ser Ile Thr Gly Thr Ala Pro Pro Pro Val Asp
690 695 700
Gly Lys Leu Ile Ile Lys Pro Ser Pro Gln Val Ser Ile Pro Ser Pro
705 710 715 720
Val Leu Ile Pro Ala Gln Glu Val His Thr Ile Cys Asn Tyr Glu Gly
725 730 735
Ile Ser Val Gln Ile Lys His Ser Ser Pro Phe Ser Gly Val Val Phe
740 745 750
Val Arg Asn Lys Tyr Asp Thr Cys Arg Val Lys Leu Lys Glu Arg Thr
755 760 765
Ala Leu Phe Trp Phe Trp Gly Phe Gln Gln Ile Leu Glu Met Lys Pro
770 775 780
Ile Ala Leu Ile Asn Ser Gln Lys His Gly Lys Gly Asn Lys Thr His
785 790 795 800
Gly Asp Thr Leu Leu Ser Ile Glu Gly Ser Lys Lys Gln Ile Glu Gly
805 810 815
Gly Ser Ser Thr Glu Asp Ile Gln Leu Ile Asn Ser Gln Lys Asp Leu
820 825 830
Lys Arg Ser Arg Arg Gln Leu Gln Arg Asp Cys Gly Leu Gln Asp Met
835 840 845
Asp Asn Gly Thr Tyr Lys Thr Val Ile Val Val Gln Thr Asn Asn Leu
850 855 860
Gly Ile Pro Gly Leu Val Thr Ser Met Asp Gln Leu Tyr Glu Ile Ser
865 870 875 880
Cys Asn Tyr Ser Ser Met Leu Gly Gly Lys Val Gln Thr Ala Ala Ala
885 890 895
Leu Arg Val His Gly Pro Gln Pro Ser Leu Ile Gln Pro Arg Gly Lys
900 905 910
Ile Glu Leu Gly Asn Pro Val Leu Met Gln Met Gly Pro Val Arg Ser
915 920 925
Glu Arg Gln Ser Gly Glu Gly Pro Leu Ile Gln Ala Lys Leu Gly Asp
930 935 940
Ile Leu Glu Leu Lys Trp Glu Ile Met Ala Met Asp Glu Glu Leu Asp
945 950 955 960
Phe Leu Val Arg Asp Cys Phe Ala Glu Pro Gly Thr Ser Gly Asn Gln
965 970 975
Gly Glu Arg Leu Pro Leu Ile Glu Asn Gly Cys Pro Thr Pro Ala Val
980 985 990
Ala Gln Lys Leu Ile Pro Asn Pro Ile Lys Ala Ile Asn Ser Ala Val
995 1000 1005
Lys Leu Thr Tyr Leu Gln Ala Phe Arg Phe Asp Ser Ser Pro Ala
1010 1015 1020
Ile Arg Ile Thr Cys His Leu Glu Leu Cys Lys Glu Asn Cys Lys
1025 1030 1035
Ser Val Asn Cys Lys Phe Asn Asp Gly Ile Lys Glu Ser Trp Gly
1040 1045 1050
Arg Lys Arg Arg Phe Ala Ile Asp Asn Asn Ile Asn Arg Lys Asn
1055 1060 1065
Glu Val Lys Glu Phe Glu Thr Arg Arg Phe Val Val Pro Arg Phe
1070 1075 1080
Ala Gln Ala Thr Thr Ser Leu Val Ile Val Asp Pro Leu Gln Gln
1085 1090 1095
Gln Asn Ser Val Ile Lys Thr Glu Gln Gln Gln Gln Pro Phe Ile
1100 1105 1110
Ser His Ser Ser Ile Ser Lys Gln Ile Phe Glu Asn Asn Lys Asn
1115 1120 1125
Ile Thr Lys Thr Ala Lys Lys Ser Ser Ser Leu Phe Glu Ala Phe
1130 1135 1140
Thr Glu Ala Ala Gly Gly Arg Lys Ile Asn Leu Glu Leu Thr Thr
1145 1150 1155
Thr Asn Ser Glu Gln Gln Gln Leu Leu Phe Thr
1160 1165
<210> 23
<211> 815
<212> DNA
<213> 南方根结线虫
<400> 23
atgtttcgaa atcagtatga tagtgatgtg actgttttta gtcctcaggg acgtttacat 60
caaaacgatt atgcagttga agcaatgcgt caaggcagtg ctactgttgc tttgcatgga 120
aaagatcatg ctgttgttgt tgctcttatg cgttctcaaa gcgaattatc ctcttatcaa 180
tcaaagatat ttgaattgga tggtcatgtg ggtctttcaa tgtctggtct tttagcggat 240
ggcagaattt tagcccgatt tattcaaaat gaatgtgcta ctttcttttc ggaattgcaa 300
acaccattgc cgatggagaa tttgcgtaca aaactaactt tgagaatgca agagaatatt 360
caggtttatg ggaaacggcc atttggagtt ggattgttaa ttattggata tgatgactct 420
ggtcctcatg ttcttaatgc tgatccatct gctaatgtta caatggttaa agctgcttca 480
attggggctc gttctcaatc tgctcgtact tatttggaaa aacattttga cgaatatgca 540
aatagtactg accaaaaaaa tgttattcgt catgctcttc ttgctcttaa agaaactctt 600
cagacaaata ctaaattgga tgagaataat actgcaattg caattgtggg taaacgatgc 660
aagtttggtt gccttccatc agagcaagtt aaagaaatta ttgcaagtct taacaacaat 720
caagctccag agccaatgca gctttgaaaa ttgtgctgac ttgcgcggat atttgtctaa 780
ataatttttt tttgattttc aacaatcttg tttga 815
<210> 24
<211> 248
<212> PRT
<213> 南方根结线虫
<400> 24
Met Phe Arg Asn Gln Tyr Asp Ser Asp Val Thr Val Phe Ser Pro Gln
1 5 10 15
Gly Arg Leu His Gln Asn Asp Tyr Ala Val Glu Ala Met Arg Gln Gly
20 25 30
Ser Ala Thr Val Ala Leu His Gly Lys Asp His Ala Val Val Val Ala
35 40 45
Leu Met Arg Ser Gln Ser Glu Leu Ser Ser Tyr Gln Ser Lys Ile Phe
50 55 60
Glu Leu Asp Gly His Val Gly Leu Ser Met Ser Gly Leu Leu Ala Asp
65 70 75 80
Gly Arg Ile Leu Ala Arg Phe Ile Gln Asn Glu Cys Ala Thr Phe Phe
85 90 95
Ser Glu Leu Gln Thr Pro Leu Pro Met Glu Asn Leu Arg Thr Lys Leu
100 105 110
Thr Leu Arg Met Gln Glu Asn Ile Gln Val Tyr Gly Lys Arg Pro Phe
115 120 125
Gly Val Gly Leu Leu Ile Ile Gly Tyr Asp Asp Ser Gly Pro His Val
130 135 140
Leu Asn Ala Asp Pro Ser Ala Asn Val Thr Met Val Lys Ala Ala Ser
145 150 155 160
Ile Gly Ala Arg Ser Gln Ser Ala Arg Thr Tyr Leu Glu Lys His Phe
165 170 175
Asp Glu Tyr Ala Asn Ser Thr Asp Gln Lys Asn Val Ile Arg His Ala
180 185 190
Leu Leu Ala Leu Lys Glu Thr Leu Gln Thr Asn Thr Lys Leu Asp Glu
195 200 205
Asn Asn Thr Ala Ile Ala Ile Val Gly Lys Arg Cys Lys Phe Gly Cys
210 215 220
Leu Pro Ser Glu Gln Val Lys Glu Ile Ile Ala Ser Leu Asn Asn Asn
225 230 235 240
Gln Ala Pro Glu Pro Met Gln Leu
245
<210> 25
<211> 690
<212> DNA
<213> 南方根结线虫
<400> 25
aattaaatgc aagaactgca agaagaaact gttgaaaata gcaacaataa ggaggatatt 60
gagactttca aagaattggg aataaatgaa gaattgtgca gtgcttgtga acgtattggt 120
tggaaaaaac ctatgcctat acaacaaaaa gttattccga ttgccttgaa gggaagagat 180
gtgattggat tggcagaaac tggttctggc aaaactgcag catttgctct tccaatactt 240
caatccttaa tgtccaatcc acagcgtctc tttgccgttg tattagctcc tacgagagaa 300
ttggcatttc aaatttcgga tcagtttgtg gctttaggtg caactatcgg cctccaagtt 360
tctacaattg tcggtggtat tgatatgtca acacagacat tgtctctttc taaaaggcct 420
catgttattg ttgcaactcc tggtcggctt gttgaccacc tcgaaaacac caaaggtttt 480
gatcttcgat cagtcaaata tttggttttg gacgaagcgg atagaatact taatatggac 540
tttgaattgg aattggataa aattcttaaa gttattccaa atacaaggca tacattttta 600
ttttcggcaa ctatgacgca caaggtttca aaattagaaa gggcccattt aaagaagcct 660
gttcgtgttg aattatctac caaatatcaa 690
<210> 26
<211> 228
<212> PRT
<213> 南方根结线虫
<400> 26
Met Gln Glu Leu Gln Glu Glu Thr Val Glu Asn Ser Asn Asn Lys Glu
1 5 10 15
Asp Ile Glu Thr Phe Lys Glu Leu Gly Ile Asn Glu Glu Leu Cys Ser
20 25 30
Ala Cys Glu Arg Ile Gly Trp Lys Lys Pro Met Pro Ile Gln Gln Lys
35 40 45
Val Ile Pro Ile Ala Leu Lys Gly Arg Asp Val Ile Gly Leu Ala Glu
50 55 60
Thr Gly Ser Gly Lys Thr Ala Ala Phe Ala Leu Pro Ile Leu Gln Ser
65 70 75 80
Leu Met Ser Asn Pro Gln Arg Leu Phe Ala Val Val Leu Ala Pro Thr
85 90 95
Arg Glu Leu Ala Phe Gln Ile Ser Asp Gln Phe Val Ala Leu Gly Ala
100 105 110
Thr Ile Gly Leu Gln Val Ser Thr Ile Val Gly Gly Ile Asp Met Ser
115 120 125
Thr Gln Thr Leu Ser Leu Ser Lys Arg Pro His Val Ile Val Ala Thr
130 135 140
Pro Gly Arg Leu Val Asp His Leu Glu Asn Thr Lys Gly Phe Asp Leu
145 150 155 160
Arg Ser Val Lys Tyr Leu Val Leu Asp Glu Ala Asp Arg Ile Leu Asn
165 170 175
Met Asp Phe Glu Leu Glu Leu Asp Lys Ile Leu Lys Val Ile Pro Asn
180 185 190
Thr Arg His Thr Phe Leu Phe Ser Ala Thr Met Thr His Lys Val Ser
195 200 205
Lys Leu Glu Arg Ala His Leu Lys Lys Pro Val Arg Val Glu Leu Ser
210 215 220
Thr Lys Tyr Gln
225
<210> 27
<211> 799
<212> DNA
<213> 南方根结线虫
<400> 27
ggcgcgcctc agcagtcgct gtcgttaccc ctgttcaaca attctagttc caggttcaaa 60
ttggcattgt tccaaagcat cagcaacatt tgcacgttcc cacttgtcaa gatcggcaag 120
aattgaaact ttcgacaaaa attcgtcata catttttctt ttgcgcattg tagatcccat 180
taaaatagct ctataggtga gtcggtcaat agcccaaagt ttgactgttg gggtcttagc 240
tacaacagta gcagcacgtg gagttccata aattaaggca agttcaccaa aagaaccccc 300
ttccttaact gagagaacaa attcccaagt actccgatcg cgttaacgct ttatcacgat 360
accttctacc acatatcact aacaacatca acactcatca ctctcgacga catccactcg 420
atcactactc tcacacgacc gattaactcc tcatccacgc ggccgcctgc gggaatttgt 480
tctctcagtt aaggaagggg gttcttttgg tgaacttgcc ttaatttatg gaactccacg 540
tgctgctact gttgtagcta agaccccaac agtcaaactt tgggctattg accgactcac 600
ctatagagct attttaatgg gatctacaat gcgcaaaaga aaaatgtatg acgaattttt 660
gtcgaaagtt tcaattcttg ccgatcttga caagtgggaa cgtgcaaatg ttgctgatgc 720
tttggaacaa tgccaatttg aacctggaac tagaattgtt gaacaggtag aacccagcgg 780
tactcgctga ggcgatcgc 799
<210> 28
<211> 832
<212> DNA
<213> 南方根结线虫
<400> 28
ggcgcgcctc agcagtcgct gtcgttacct tcacgcactg ggcccataac gcgctcgaat 60
cgagctctat ccaactttac acacttaagg ggccctttaa ccacaacggt tgctgcacgt 120
ggatgatcca ataaaagagc aatttcgcca aaataatcag aaggacccaa tttgccaaca 180
acttcataag gagcatcatc attcggccgt tgtagcacct cggcctctcc ctcaacaatt 240
ataaagaatt catctccggg ttggccctgt tcaacaattc tagttccagg ttcaaattgg 300
cattgttcca aagcatcagc aacatttgca cgttcccact aagtactccg atcgcgttaa 360
cgctttatca cgataccttc taccacatat cactaacaac atcaacactc atcactctcg 420
acgacatcca ctcgatcact actctcacac gaccgattaa ctcctcatcc acgcggccgc 480
ctgcaggagc agtgggaacg tgcaaatgtt gctgatgctt tggaacaatg ccaatttgaa 540
cctggaacta gaattgttga acagggccaa cccggagatg aattctttat aattgttgag 600
ggagaggccg aggtgctaca acggccgaat gatgatgctc cttatgaagt tgttggcaaa 660
ttgggtcctt ctgattattt tggcgaaatt gctcttttat tggatcatcc acgtgcagca 720
accgttgtgg ttaaagggcc ccttaagtgt gtaaagttgg atagagctcg attcgagcgc 780
gttatgggcc agtgcgtgaa tagaacccag cggtactcgc tgaggcgatc gc 832
<210> 29
<211> 781
<212> DNA
<213> 南方根结线虫
<400> 29
ggcgcgcctc agcagtcgct gtcgttaccg tgcgtcatag ttgccgaaaa taaaaatgta 60
tgccttgtat ttggaataac tttaagaatt ttatccaatt ccaattcaaa gtccatatta 120
agtattctat ccgcttcgtc caaaaccaaa tatttgactg atcgaagatc aaaacctttg 180
gtgttttcga ggtggtcaac aagccgacca ggagttgcaa caataacatg aggcctttta 240
gaaagagaca atgtctgtgt tgacatatca ataccaccga caattgtaga aacttggagg 300
ccgatagttg caccaagtac tccgatcgcg ttaacgcttt atcacgatac cttctaccac 360
atatcactaa caacatcaac actcatcact ctcgacgaca tccactcgat cactactctc 420
acacgaccga ttaactcctc atccacgcgg ccgcctgcag gagcggtgca actatcggcc 480
tccaagtttc tacaattgtc ggtggtattg atatgtcaac acagacattg tctctttcta 540
aaaggcctca tgttattgtt gcaactcctg gtcggcttgt tgaccacctc gaaaacacca 600
aaggttttga tcttcgatca gtcaaatatt tggttttgga cgaagcggat agaatactta 660
atatggactt tgaattggaa ttggataaaa ttcttaaagt tattccaaat acaaggcata 720
catttttatt ttcggcaact atgacgcact agaacccagc ggtactcgct gaggcgatcg 780
c 781
<210> 30
<211> 775
<212> DNA
<213> 南方根结线虫
<400> 30
ggcgcgcctc agcagtcgct gtcgttaccc gccaatggaa acgttgccga gaaaagcata 60
atttggcgct ctcttggtaa gtagctgatc atcttatcga gagcaccttg aaaatcgatc 120
gaaagtagtt tgtcggcttc atccaagaca agcatcttgc agaggctcac atcagcaaca 180
gtcttctcga tgagatccaa gattcgtccg ggagttgcca caatcagatg aacggttcca 240
ttgagacgca taatgtcgtc acgcaaatcc gtaccgccag ttgtaaccat caccttgaca 300
ttcatatgct ttccaagtac tccgatcgcg ttaacgcttt atcacgatac cttctaccac 360
atatcactaa caacatcaac actcatcact ctcgacgaca tccactcgat cactactctc 420
acacgaccga ttaactcctc atccacgcgg ccgcctgcgg aaagcatatg aatgtcaagg 480
tgatggttac aactggcggt acggatttgc gtgacgacat tatgcgtctc aatggaaccg 540
ttcatctgat tgtggcaact cccggacgaa tcttggatct catcgagaag actgttgctg 600
atgtgagcct ctgcaagatg cttgtcttgg atgaagccga caaactactt tcgatcgatt 660
ttcaaggtgc tctcgataag atgatcagct acttaccaag agagcgccaa attatgcttt 720
tctcggcaac gtttccattg gcgtagaacc cagcggtact cgctgaggcg atcgc 775
<210> 31
<211> 731
<212> DNA
<213> 南方根结线虫
<400> 31
ggcgcgcctc agcagtcgct gtcgttacct tacatgcagg gcagaggtct ttgcctttca 60
cagccatttt caaagcatca ttttgttcat caatttgatt aactatcata ataaaattat 120
ttacagactt atctactttt tttaaacaat ctttggaggg aagtttttgt attttaacta 180
gcaattgcag catttctgta tttatttgat caattgctgg gcgtaagtct gttgttaaat 240
ttcgaacttt aataagtggt atgcctgatt gattccataa attaaccaaa taaagtactc 300
cgatcgcgtt aacgctttat cacgatacct tctaccacat atcactaaca acatcaacac 360
tcatcactct cgacgacatc cactcgatca ctactctcac acgaccgatt aactcctcat 420
ccacgcggcc gcctgctatt tggttaattt atggaatcaa tcaggcatac cacttattaa 480
agttcgaaat ttaacaacag acttacgccc agcaattgat caaataaata cagaaatgct 540
gcaattgcta gttaaaatac aaaaacttcc ctccaaagat tgtttaaaaa aagtagataa 600
gtctgtaaat aattttatta tgatagttaa tcaaattgat gaacaaaatg atgctttgaa 660
aatggctgtg aaaggcaaag acctctgccc tgcatgtaat agaacccagc ggtactcgct 720
gaggcgatcg c 731
<210> 32
<211> 727
<212> DNA
<213> 南方根结线虫
<400> 32
ggcgcgcctc agcagtcgct gtcgttaccc tttagaagca atcccgcact gattgtcttt 60
gtttcgagac attcgaatat acccattttc tccccaatgt tccccccaac tatttttaac 120
aagccaataa tctccgtgga tgtcatctgt gccgtatcca acaactaaga caccatgatc 180
gagattatcg ggactgcaag cctcctcatc ataaactccg tgagtataaa gttgaaaact 240
gcgatggcca gcatcaattg ctacagaaat tgggccttgt gtggcaacag aagtactccg 300
atcgcgttaa cgctttatca cgataccttc taccacatat cactaacaac atcaacactc 360
atcactctcg acgacatcca ctcgatcact actctcacac gaccgattaa ctcctcatcc 420
acgcggccgc ctgcctgttg ccacacaagg cccaatttct gtagcaattg atgctggcca 480
tcgcagtttt caactttata ctcacggagt ttatgatgag gaggcttgca gtcccgataa 540
tctcgatcat ggtgtcttag ttgttggata cggcacagat gacatccacg gagattattg 600
gcttgttaaa aatagttggg gggaacattg gggagaaaat gggtatattc gaatgtctcg 660
aaacaaagac aatcaatgcg ggattgcttc taaagtagaa cccagcggta ctcgctgagg 720
cgatcgc 727
<210> 33
<211> 531
<212> DNA
<213> 南方根结线虫
<400> 33
ggcgcgcctc agcagtcgct gtcgttacct cttcaattat ttcctccagg atttggccca 60
ctaggctttt tgcctgcatc cacaaatgag acagacaaaa gcattaaagt aaccattaaa 120
ggcattaaat aaataattaa atttttaatt gaattagtaa acattttttt gattgaagaa 180
taattaactt taaagtactc cgatcgcgtt aacgctttat cacgatacct tctaccacat 240
atcactaaca acatcaacac tcatcactct cgacgacatc cactcgatca ctactctcac 300
acgaccgatt aactcctcat ccacgcggcc gcctgctaaa gttaattatt cttcaatcaa 360
aaaaatgttt actaattcaa ttaaaaattt aattatttat ttaatgcctt taatggttac 420
tttaatgctt ttgtctgtct catttgtgga tgcaggcaaa aagcctagtg ggccaaatcc 480
tggaggaaat aattgaagat agaacccagc ggtactcgct gaggcgatcg c 531
<210> 34
<211> 531
<212> DNA
<213> 拟南芥
<400> 34
ggcgcgcctc agcagtcgct gtcgttacct cttcaattat ttcctccagg atttggccca 60
ctaggctttt tgcctgcatc cacaaatgag acagacaaaa gcattaaagt aaccattaaa 120
ggcattaaat aaataattaa atttttaatt gaattagtaa acattttttt gattgaagaa 180
taattaactt taaagtactc cgatcgcgtt aacgctttat cacgatacct tctaccacat 240
atcactaaca acatcaacac tcatcactct cgacgacatc cactcgatca ctactctcac 300
acgaccgatt aactcctcat ccacgcggcc gcctgctaaa gttaattatt cttcaatcaa 360
aaaaatgttt actaattcaa ttaaaaattt aattatttat ttaatgcctt taatggttac 420
tttaatgctt ttgtctgtct catttgtgga tgcaggcaaa aagcctagtg ggccaaatcc 480
tggaggaaat aattgaagat agaacccagc ggtactcgct gaggcgatcg c 531
<210> 35
<211> 1144
<212> DNA
<213> 拟南芥
<400> 35
gaattcatca acaataaaac caggaactca aaacatacgg tcatataaga ttctcttgca 60
agtagatacg aggcgttgtg tagcgattag aaggagcttt ttgatatttt cgtttcatgc 120
gcaaatgcat ttgtcttgaa cccctgcctg aaattggatt gtaagtaaag tgctaatgat 180
ggatattgct gtaaacagaa acaatgatct ttaactgatt gatgtcaaag gaatgagtct 240
ggttttatct ttggtatgct ctgaacaatt tttgtatgag aagagaagac tgagatttgc 300
tgtttttaga ttgttcaata gcttccaagt actttggttt taggctttta gctaattagt 360
ttgtgccgtc ttcatatttt ccccttcttc tttgcaattt tgctatttta tggaagctga 420
ttatgaccaa gcatcaatga aggaatctct ccactataac ttatcaggtt cacataactt 480
tcttaaaagt aaaacagtga agaaccattc accttccata tctccagagc aggttcattg 540
catcttagtt agggtttaac cttcaaactt gtctttacac tatcctagga ttctattgtt 600
ttcttgtctt ctttgtattg ctgcatttga ttgacttacc actactctct tgagattggc 660
caatttttgc tgattaacag ttcatttttg ctctaagttt catcagtttt ttttcttggc 720
atggatgttt tgctgaatta tcattcgcct aatgagttac acaaacaaaa ttgtagattt 780
tcagagaaaa caaaacaaaa catatgaata aaaaagttat gcatatgata cgcgttaaga 840
aaaacaaatg attaacgtac gtcggctgtg aagtgttgtt tgatacaatt gggggaagtg 900
acggcacaat gggctggttt cacggcccct ttctaagtca tacacgtctc ctttttttat 960
tttcgttttt ttcaaaataa taaaaataat atagagagtg gcatatccga cggtttagga 1020
tctctgcctt ttctcaactt atagtatata ccaaacactt tctaaggtaa catgagatat 1080
ttcacatcct tttctctctt attcgtttct gagattccct tcttgttgtt gtagccggcg 1140
cgcc 1144
<210> 36
<211> 1052
<212> DNA
<213> 玄参花叶病毒
<400> 36
caatcccaca aaaatctgag cttaacagca cagttgctcc tctcagagca gaatcgggta 60
ttcaacaccc tcatatcaac tactacgttg tgtataacgg tccacatgcc ggtatatacg 120
atgactgggg ttgtacaaag gcggcaacaa acggcgttcc cggagttgca cacaagaaat 180
ttgccactat tacagaggca agagcagcag ctgacgcgta cacaacaagt cagcaaacag 240
acaggttgaa cttcatcccc aaaggagaag ctcaactcaa gcccaagagc tttgctaagg 300
ccctaacaag cccaccaaag caaaaagccc actggctcac gctaggaacc aaaaggccca 360
gcagtgatcc agccccaaaa gagatccgca ttccagattg ggttcaatca acaaggtacg 420
agccatatca ctttattcaa attggtatcg ccaaaaccaa gaaggaactc ccatcctcaa 480
aggtttgtaa ggaagaattc tcagtccaaa gcctcaacaa ggtcagggta cagagtctcc 540
aaaccattag ccaaaagcta caggagatca atgaagaatc ttcaatcaaa gtaaactact 600
gttccagcac atgcatcatg gtcagtaagt ttcagaaaaa gacatccacc gaagacttaa 660
agttagtggg catctttgaa agtaatcttg tcaacatcga gcagctggct tgtggggacc 720
agacaaaaaa ggaatggtgc agaattgtta ggcgcaccta ccaaaagcat ctttgccttt 780
attgcaaaga taaagcagat tcctctagta caagtgggga acaaaataac gtggaaaaga 840
gctgtcctga cagcccactc actaatgcgt atgacgaacg cagtgacgac cacaaaagaa 900
ttccctctat ataagaaggc attcattccc atttgaagga cacagaaaaa tttgctacat 960
tgtttcacaa acttcaaata ttattcattt atttgtcagc tttcaaactc tttgtttctt 1020
gtttgttgat tgagaatagg taccggcgcg cc 1052
<210> 37
<211> 1064
<212> DNA
<213> 人工序列
<220>
<223> 增强的35S花椰菜花叶病毒(E35S)启动子
<400> 37
caatcccaca aaaatctgag cttaacagca cagttgctcc tctcagagca gaatcgggta 60
ttcaacaccc tcatatcaac tactacgttg tgtataacgg tccacatgcc ggtatatacg 120
atgactgggg ttgtacaaag gcggcaacaa acggcgttcc cggagttgca cacaagaaat 180
ttgccactat tacagaggca agagcagcag ctgacgcgta cacaacaagt cagcaaacag 240
acaggttgaa cttcatcccc aaaggagaag ctcaactcaa gcccaagagc tttgctaagg 300
ccctaacaag cccaccaaag caaaaagccc actggctcac gctaggaacc aaaaggccca 360
gcagtgatcc agccccaaaa gagatcccac gtgcggaccg cctgcaggcc gcgttatcaa 420
gctaactgca ggtccgatgt gagacttttc aacaaagggt aatatccgga aacctcctcg 480
gattccattg cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct 540
cctacaaatg ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca 600
gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 660
ccacgtcttc aaagcaagtg gattgatgtg atggtccgat tgagactttt caacaaaggg 720
taatatccgg aaacctcctc ggattccatt gcccagctat ctgtcacttt attgtgaaga 780
tagtggaaaa ggaaggtggc tcctacaaat gccatcattg cgataaagga aaggccatcg 840
ttgaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 900
tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 960
ctgacgtaag ggatgacgca caatcccact atccttcgca agacccttcc tctatataag 1020
gaagttcatt tcatttggag aggaccaggt ggtaccggcg cgcc 1064
<210> 38
<211> 1156
<212> DNA
<213> 人工序列
<220>
<223> 具有矮牵牛前导序列的增强的35S花椰菜花叶病毒(E35S)启动子
<400> 38
caatcccaca aaaatctgag cttaacagca cagttgctcc tctcagagca gaatcgggta 60
ttcaacaccc tcatatcaac tactacgttg tgtataacgg tccacatgcc ggtatatacg 120
atgactgggg ttgtacaaag gcggcaacaa acggcgttcc cggagttgca cacaagaaat 180
ttgccactat tacagaggca agagcagcag ctgacgcgta cacaacaagt cagcaaacag 240
acaggttgaa cttcatcccc aaaggagaag ctcaactcaa gcccaagagc tttgctaagg 300
ccctaacaag cccaccaaag caaaaagccc actggctcac gctaggaacc aaaaggccca 360
gcagtgatcc agccccaaaa gagatcccac gtgcggaccg cctgcaggcc gcgttatcaa 420
gctaactgca ggtccgatgt gagacttttc aacaaagggt aatatccgga aacctcctcg 480
gattccattg cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct 540
cctacaaatg ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca 600
gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 660
ccacgtcttc aaagcaagtg gattgatgtg atggtccgat tgagactttt caacaaaggg 720
taatatccgg aaacctcctc ggattccatt gcccagctat ctgtcacttt attgtgaaga 780
tagtggaaaa ggaaggtggc tcctacaaat gccatcattg cgataaagga aaggccatcg 840
ttgaagatgc ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg 900
tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 960
ctgacgtaag ggatgacgca caatcccact atccttcgca agacccttcc tctatataag 1020
gaagttcatt tcatttggag aggacacaga aaaatttgct acattgtttc acaaacttca 1080
aatattattc atttatttgt cagctttcaa actctttgtt tcttgtttgt tgattgagaa 1140
taggtaccgg cgcgcc 1156
<210> 39
<211> 1462
<212> DNA
<213> 人工序列
<220>
<223> 具有烟草花叶病毒翻译前导序列的35S花椰菜花叶病毒启动子
<400> 39
atagcgacat ctatgataga gcgccacaat aacaaacaat tccaatccca caaaaatctg 60
agcttaacag cacagttgct cctctcagag cagaatcggg tattcaacac cctcatatca 120
actactacgt tgtgtataac ggtccacatg ccggtatata cgatgactgg ggttgtacaa 180
aggcggcaac aaacggcgtt cccggagttg cacacaagaa atttgccact attacagagg 240
caagagcagc agctgacgcg tacacaacaa gtcagcaaac agacaggttg aacttcatcc 300
ccaaaggaga agctcaactc aagcccaaga gctttgctaa ggccctaaca agcccaccaa 360
agcaaaaagc ccactggctc acgctaggaa ccaaaaggcc cagcagtgat ccagccccaa 420
aagagatctc ctttgccccg gagattacaa tggacgattt cctctatctt tacgatctag 480
gaaggaagtt cgaaggtgaa ggtgacgaca ctatgttcac cactgataat gagaaggtta 540
gcctcttcaa tttcagaaag aatgctgacc cacagatggt tagagaggcc tacgcagcag 600
gtctcatcaa gacgatctac ccgagtaaca atctccagga gatcaaatac cttcccaaga 660
aggttaaaga tgcagtcaaa agattcagga ctaattgcat caagaacaca gagaaagaca 720
tatttctcaa gatcagaagt actattccag tatggacgat tcaaggcttg cttcataaac 780
caaggcaagt aatagagatt ggagtctcta aaaaggtagt tcctactgaa tctaaggcca 840
tgcatggagt ctaagattca aatcgaggat ctaacagaac tcgccgtgaa gactggcgaa 900
cagttcatac agagtctttt acgactcaat gacaagaaga aaatcttcgt caacatggtg 960
gagcacgaca ctctggtcta ctccaaaaat gtcaaagata cagtctcaga agaccaaagg 1020
gctattgaga cttttcaaca aaggataatt tcgggaaacc tcctcggatt ccattgccca 1080
gctatctgtc acttcatcga aaggacagta gaaaaggaag gtggctccta caaatgccat 1140
cattgcgata aaggaaaggc tatcattcaa gatctctctg ccgacagtgg tcccaaagat 1200
ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag 1260
caagtggatt gatgtgacat ctccactgac gtaagggatg acgcacaatc ccactatcct 1320
tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac acgctcgagt 1380
ataagagctc tatttttaca acaattacca acaacaacaa acaacaaaca acattacaat 1440
tacatttaca attaccgacg tc 1462
<210> 40
<211> 1008
<212> DNA
<213> 南方根结线虫
<400> 40
atgatgaaga gatttggaat ttatacttct ttcctcgaat ttttgttaat aattttaacg 60
atttcaacag tcagaggtcc gaaaattgag catgactctc aaacaggagc ttctgcatct 120
gcttcgggtt catttccaat tgttttacca cataatgaac taaagcagga cggtggcaaa 180
ttttatttgg aaggagacga aaagaatgcc tgccttctat atattgaatc gatttctgtt 240
gtgttatata acgtaaaaca aaaaatgtct gcttgggcga ccattaatgc aacttctaag 300
ggagcaacat atagtggtaa aattgagtgt ccagcaactc ataatacagg acaggagttt 360
aaaatttcat tgcaagcaat tgctggcggt gaaggtgttt caggctatta tattggtgat 420
aaaaagaaag tcattttccg gttaagcaaa gtcgatgttg atttaatatt taactttact 480
tcatcagctt attgggagtt ggtgaatgct gatgttagga caataagttt ggatggcgca 540
gaagaaggtg gagttccgag tgtcgataac acctggcgac ttgtttctgg tataagcagc 600
aacaataaag gatggatttt cacagataat caatctccgg cctggaatat taatgggttt 660
aaagactacg ccttctcttg tggtcgaact agtccaatta tttgggcaaa acatgatacg 720
gatgaaaaaa gcctggaaca tgcagttggt ttggcttttc ataatttaca gtttcaattg 780
aattcaactt atatttctcc ggataaaaaa agaatggcaa agtttggctg gcgtgttaat 840
gattgtgcac ctctattaag tattggtact tggatgattc ttgtggttgc aattattttt 900
attggagttc tctcttttgg atttttaatg cttaattctg tccaaacaat gagccgattt 960
gacgacccaa aacaaaaaca gataattatt agttctaaag aaaattag 1008
<210> 41
<211> 335
<212> PRT
<213> 南方根结线虫
<400> 41
Met Met Lys Arg Phe Gly Ile Tyr Thr Ser Phe Leu Glu Phe Leu Leu
1 5 10 15
Ile Ile Leu Thr Ile Ser Thr Val Arg Gly Pro Lys Ile Glu His Asp
20 25 30
Ser Gln Thr Gly Ala Ser Ala Ser Ala Ser Gly Ser Phe Pro Ile Val
35 40 45
Leu Pro His Asn Glu Leu Lys Gln Asp Gly Gly Lys Phe Tyr Leu Glu
50 55 60
Gly Asp Glu Lys Asn Ala Cys Leu Leu Tyr Ile Glu Ser Ile Ser Val
65 70 75 80
Val Leu Tyr Asn Val Lys Gln Lys Met Ser Ala Trp Ala Thr Ile Asn
85 90 95
Ala Thr Ser Lys Gly Ala Thr Tyr Ser Gly Lys Ile Glu Cys Pro Ala
100 105 110
Thr His Asn Thr Gly Gln Glu Phe Lys Ile Ser Leu Gln Ala Ile Ala
115 120 125
Gly Gly Glu Gly Val Ser Gly Tyr Tyr Ile Gly Asp Lys Lys Lys Val
130 135 140
Ile Phe Arg Leu Ser Lys Val Asp Val Asp Leu Ile Phe Asn Phe Thr
145 150 155 160
Ser Ser Ala Tyr Trp Glu Leu Val Asn Ala Asp Val Arg Thr Ile Ser
165 170 175
Leu Asp Gly Ala Glu Glu Gly Gly Val Pro Ser Val Asp Asn Thr Trp
180 185 190
Arg Leu Val Ser Gly Ile Ser Ser Asn Asn Lys Gly Trp Ile Phe Thr
195 200 205
Asp Asn Gln Ser Pro Ala Trp Asn Ile Asn Gly Phe Lys Asp Tyr Ala
210 215 220
Phe Ser Cys Gly Arg Thr Ser Pro Ile Ile Trp Ala Lys His Asp Thr
225 230 235 240
Asp Glu Lys Ser Leu Glu His Ala Val Gly Leu Ala Phe His Asn Leu
245 250 255
Gln Phe Gln Leu Asn Ser Thr Tyr Ile Ser Pro Asp Lys Lys Arg Met
260 265 270
Ala Lys Phe Gly Trp Arg Val Asn Asp Cys Ala Pro Leu Leu Ser Ile
275 280 285
Gly Thr Trp Met Ile Leu Val Val Ala Ile Ile Phe Ile Gly Val Leu
290 295 300
Ser Phe Gly Phe Leu Met Leu Asn Ser Val Gln Thr Met Ser Arg Phe
305 310 315 320
Asp Asp Pro Lys Gln Lys Gln Ile Ile Ile Ser Ser Lys Glu Asn
325 330 335
<210> 42
<211> 1102
<212> DNA
<213> 南方根结线虫
<400> 42
ctgcaagaag aaactgttga ttaccgcaac aataaggagg atattgagac tttcaaagaa 60
ttgggaataa atgaagaatt gtgcagtgct tgtgaacgta ttggttggaa aaaacctatg 120
cctatacaac aaaaagttat tccgattgcc ttgaagggaa gagatgtgat tggattggca 180
gaaactggtt ctggcaaaac tgcagcattt gctcttccaa tacttcaatc cttaatgtcc 240
aatccacagc gtctctttgc cgttgtatta gctcctacga gagaattggc atttcaaatt 300
tcggatcagt ttgtggcttt aggtgcaact atcggcctcc aagtttctac aattgtcggt 360
ggtattgata tgtcaacaca gacattgtct ctttctaaaa ggcctcatgt tattgttgca 420
actcctggtc ggcttgttga ccacctcgaa aacaccaaag gttttgatct tcgatcagtc 480
aaatatttgg ttttggacga agcggataga atacttaata tggactttga attggaattg 540
gataaaattc ttaaagttat tccaaataca aggcatacat ttttattttc ggcaactatg 600
acgcacaagg tttcaaaatt agaaagggcc catttaaaga agcctgttcg tgttgaatta 660
tctaccaaat atcaaacagt tagcacattg attcaaaata tgctttttat tcccttcaag 720
tataaggagg cttatctagt ccacgttttg aacgaaaaag caggcaatac agcaatagtt 780
ttttgttcaa catgtgcaag ttcagtaaaa atagctctaa tgttaaggca attaagtttt 840
ggagcaatag ctttacatgg acaaatgtca cagccaaaac gtttgggagc tttaaataag 900
ttcaaaaaga aggatcggcc aattttggtt tgtactgatg ttgcttctcg aggtcttgat 960
attcctcatg ttgatatggt tttaaattat gacgtcccaa cgcattcaaa agattatgtt 1020
catcgtgtag gaaggactgc tagggctggg aagtcaggaa tttcaatcac aatagtcacc 1080
caatacgatg ttgaaattta tc 1102
<210> 43
<211> 367
<212> PRT
<213> 南方根结线虫
<400> 43
Leu Gln Glu Glu Thr Val Asp Tyr Arg Asn Asn Lys Glu Asp Ile Glu
1 5 10 15
Thr Phe Lys Glu Leu Gly Ile Asn Glu Glu Leu Cys Ser Ala Cys Glu
20 25 30
Arg Ile Gly Trp Lys Lys Pro Met Pro Ile Gln Gln Lys Val Ile Pro
35 40 45
Ile Ala Leu Lys Gly Arg Asp Val Ile Gly Leu Ala Glu Thr Gly Ser
50 55 60
Gly Lys Thr Ala Ala Phe Ala Leu Pro Ile Leu Gln Ser Leu Met Ser
65 70 75 80
Asn Pro Gln Arg Leu Phe Ala Val Val Leu Ala Pro Thr Arg Glu Leu
85 90 95
Ala Phe Gln Ile Ser Asp Gln Phe Val Ala Leu Gly Ala Thr Ile Gly
100 105 110
Leu Gln Val Ser Thr Ile Val Gly Gly Ile Asp Met Ser Thr Gln Thr
115 120 125
Leu Ser Leu Ser Lys Arg Pro His Val Ile Val Ala Thr Pro Gly Arg
130 135 140
Leu Val Asp His Leu Glu Asn Thr Lys Gly Phe Asp Leu Arg Ser Val
145 150 155 160
Lys Tyr Leu Val Leu Asp Glu Ala Asp Arg Ile Leu Asn Met Asp Phe
165 170 175
Glu Leu Glu Leu Asp Lys Ile Leu Lys Val Ile Pro Asn Thr Arg His
180 185 190
Thr Phe Leu Phe Ser Ala Thr Met Thr His Lys Val Ser Lys Leu Glu
195 200 205
Arg Ala His Leu Lys Lys Pro Val Arg Val Glu Leu Ser Thr Lys Tyr
210 215 220
Gln Thr Val Ser Thr Leu Ile Gln Asn Met Leu Phe Ile Pro Phe Lys
225 230 235 240
Tyr Lys Glu Ala Tyr Leu Val His Val Leu Asn Glu Lys Ala Gly Asn
245 250 255
Thr Ala Ile Val Phe Cys Ser Thr Cys Ala Ser Ser Val Lys Ile Ala
260 265 270
Leu Met Leu Arg Gln Leu Ser Phe Gly Ala Ile Ala Leu His Gly Gln
275 280 285
Met Ser Gln Pro Lys Arg Leu Gly Ala Leu Asn Lys Phe Lys Lys Lys
290 295 300
Asp Arg Pro Ile Leu Val Cys Thr Asp Val Ala Ser Arg Gly Leu Asp
305 310 315 320
Ile Pro His Val Asp Met Val Leu Asn Tyr Asp Val Pro Thr His Ser
325 330 335
Lys Asp Tyr Val His Arg Val Gly Arg Thr Ala Arg Ala Gly Lys Ser
340 345 350
Gly Ile Ser Ile Thr Ile Val Thr Gln Tyr Asp Val Glu Ile Tyr
355 360 365
<210> 44
<211> 261
<212> DNA
<213> 南方根结线虫
<400> 44
tcagaaaaat tttggcgaga aaacgctcag agatttaatg aaaagaattt tgaattaata 60
aaaatattaa ttcgattact tgattctagt gatgttttgg tactttgtgt tgctgctcat 120
gatgttggag aatatgttag acattttcca cgtggaaaag atattgtcga gcaatatcaa 180
ggcaaacaag cggtaatgaa attgttaagt gctgaagatc caaatgttag atatcatgca 240
cttttagcaa ttcaaaaatt a 261
<210> 45
<211> 87
<212> PRT
<213> 南方根结线虫
<400> 45
Ser Glu Lys Phe Trp Arg Glu Asn Ala Gln Arg Phe Asn Glu Lys Asn
1 5 10 15
Phe Glu Leu Ile Lys Ile Leu Ile Arg Leu Leu Asp Ser Ser Asp Val
20 25 30
Leu Val Leu Cys Val Ala Ala His Asp Val Gly Glu Tyr Val Arg His
35 40 45
Phe Pro Arg Gly Lys Asp Ile Val Glu Gln Tyr Gln Gly Lys Gln Ala
50 55 60
Val Met Lys Leu Leu Ser Ala Glu Asp Pro Asn Val Arg Tyr His Ala
65 70 75 80
Leu Leu Ala Ile Gln Lys Leu
85
<210> 46
<211> 705
<212> DNA
<213> 南方根结线虫
<400> 46
ggcgcgcctc agcagtcgct gtcgttacca tgactttctt tttatcacca atataatagc 60
ctgaaacacc ttcaccgcca gcaattgctt gcaatgaaat tttaaactcc tgtcctgtat 120
tatgagttgc tggacactca attttaccac tatatgttgc tcccttagaa gttgcattaa 180
tggtcgccca agcagacatt ttttgtttta cgttatataa cacaacagaa atcgattcaa 240
tatatagaag gcaggcattc ttttcgtctc cttccaaata agtactccga tcgcgttaac 300
gctttatcac gataccttct accacatatc actaacaaca tcaacactca tcactctcga 360
cgacatccac tcgatcacta ctctcacacg accgattaac tcctcatcca cgcggccgcc 420
tgcatttgga aggagacgaa aagaatgcct gccttctata tattgaatcg atttctgttg 480
tgttatataa cgtaaaacaa aaaatgtctg cttgggcgac cattaatgca acttctaagg 540
gagcaacata tagtggtaaa attgagtgtc cagcaactca taatacagga caggagttta 600
aaatttcatt gcaagcaatt gctggcggtg aaggtgtttc aggctattat attggtgata 660
aaaagaaagt cattagaacc cagcggtact cgctgaggcg atcgc 705
<210> 47
<211> 250
<212> DNA
<213> 南方根结线虫
<400> 47
atttggaagg agacgaaaag aatgcctgcc ttctatatat tgaatcgatt tctgttgtgt 60
tatataacgt aaaacaaaaa atgtctgctt gggcgaccat taatgcaact tctaagggag 120
caacatatag tggtaaaatt gagtgtccag caactcataa tacaggacag gagtttaaaa 180
tttcattgca agcaattgct ggcggtgaag gtgtttcagg ctattatatt ggtgataaaa 240
agaaagtcat 250
Claims (47)
1.一种多核苷酸，包含：
(a)SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ IDNO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47的任一核酸序列的至少21个连续核苷酸的片段；和
(b)(a)中片段的反向互补序列。
2.权利要求1的多核苷酸，其中植物寄生的线虫摄取包含所述至少21个连续核苷酸的核糖核苷酸将抑制该线虫的生长。
3.权利要求1的多核苷酸，定义为可操作地与外源启动子连接。
4.权利要求1的多核苷酸，在所述片段和该片段的反向互补序列之间包含间隔多核苷酸序列。
5.包含权利要求1的多核苷酸的转化载体。
6.权利要求5的转化载体，其中所述多核苷酸可操作地与在植物细胞中发挥功能的外源启动子连接。
7.从权利要求1的多核苷酸表达产生的双链核糖核苷酸分子，其中植物寄生的线虫摄取所述核糖核苷酸分子将抑制该线虫的生长。
8.权利要求7的双链核糖核苷酸分子，其中所述核糖核苷酸分子在植物寄生的线虫中抑制基本上与所述多核苷酸分子互补的核苷酸序列的表达。
9.用权利要求1的多核苷酸转化的细胞。
10.权利要求9的细胞，定义为原核细胞。
11.权利要求9的细胞，定义为真核细胞。
12.权利要求9的细胞，定义为植物细胞。
13.用权利要求1的多核苷酸转化的植物。
14.权利要求13的植物，进一步定义为选自玉米、小麦、大麦、黑麦、稻、马铃薯、西红柿、黄瓜、辣椒、苜蓿、豆科植物、大豆、豌豆、紫花苜蓿、甘蔗、甜菜、烟草、胡萝卜、棉花、油菜籽(加拿大油菜)、向日葵、红花、高粱、草莓、香蕉、草皮和观赏植物的作物。
15.权利要求13的植物的种子，其中所述种子包含所述多核苷酸。
16.权利要求13的植物，其中所述多核苷酸在所述植物中表达为双链核糖核苷酸分子。
17.权利要求16的植物，其中植物寄生的线虫摄取包含所述双链核糖核苷酸分子的植物的组织将抑制该线虫的生长。
18.权利要求17的植物，其中所述植物寄生的线虫是根结线虫种类(Meloidogynespp)。
19.权利要求17的植物，其中所述植物寄生的线虫是南方根结线虫(Meloidogyneincognita)。
20.由权利要求13的植物产生的商品，其中所述商品包含可检测量的所述多核苷酸或由其表达的核糖核苷酸。
21.一种用于控制植物寄生线虫群体数量的方法，包括提供包含双链核糖核苷酸分子的药剂，该双链核糖核苷酸分子在被线虫摄取时发挥抑制线虫内的生物学功能的作用，其中所述药剂包含选自以下的核苷酸序列：SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ IDNO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ IDNO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:29、SEQ ID NO:30、SEQ IDNO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQ ID NO:47，及其互补序列。
22.一种用于控制植物寄生线虫群体数量的方法，包括提供包含在由植物寄生的线虫摄取时发挥作用以抑制线虫体内的生物学功能的第一多核苷酸序列的药剂，其中该多核苷酸序列沿至少大约19-大约25个连续核苷酸表现出与源自线虫的转录序列的大约95％-大约100％的核苷酸序列同一性，并与第二多核苷酸序列杂交，该第二多核苷酸序列与第一多核苷酸序列互补，且其中所述源自线虫的转录序列选自SEQ ID NO:1、SEQ ID NO:3、SEQ IDNO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ IDNO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:29、SEQ IDNO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46和SEQID NO:47及其互补序列。
23.权利要求22的方法，其中所述线虫是根结线虫种类。
24.权利要求22的方法，其中所述线虫是南方根结线虫。
25.一种用于控制植物寄生线虫的群体数量的方法，包括在植物寄生线虫的宿主植物中提供表达权利要求1的多核苷酸分子的转化植物细胞，其中该多核苷酸被表达以产生在由植物寄生的线虫摄取时发挥作用的双链核糖核酸，从而抑制所述线虫体内目标序列的表达并导致线虫或线虫群体的生长或繁殖相对于在缺乏所述转化植物细胞的宿主上的生长或繁殖降低。
26.权利要求25的方法，其中所述线虫在感染宿主植物后表现出生长降低。
27.权利要求25的方法，其中所述目标序列编码蛋白质，该蛋白质具有选自如下的预期功能：DNA复制、细胞周期控制、转录、RNA加工、翻译、核糖体功能、tRNA合成、tRNA功能、蛋白质运输、分泌、蛋白质修饰、蛋白质稳定、蛋白质降解、能量产生、线粒体功能、中间代谢、细胞结构、信号转导、内吞作用、离子调节、卵产生、繁殖和运输。
28.权利要求25的方法，其中所述线虫选自根结线虫种类。
29.权利要求25的方法，其中所述线虫是南方根结线虫。
30.权利要求25的方法，其中所述多核苷酸在由植物寄生的线虫摄取时发挥作用以抑制其执行的功能对于线虫的存活、繁殖或生长关键的基因的表达，所述功能选自DNA复制、细胞周期控制、转录、RNA加工、翻译、核糖体功能、tRNA合成、tRNA功能、蛋白质运输、分泌、蛋白质修饰、蛋白质稳定、蛋白质降解、能量产生、线粒体功能、中间代谢、细胞结构、信号转导、内吞作用、离子调节、卵产生和运输。
31.一种用于减少宿主植物的根组织中形成的RKN摄食位点的数目的方法，包括在根结线虫的宿主植物中提供表达权利要求1的多核苷酸的转化植物细胞，其中所述多核苷酸被表达以产生在由根结线虫摄取时发挥作用的双链核糖核酸，从而抑制所述线虫体内目标序列的表达并导致所形成的摄食位点数目相对于在缺乏所述转化植物细胞的宿主上形成的摄食位点数目降少。
32.一种用于防治植物中的植物线虫虫害的方法，包括在植物线虫害虫的食物中提供dsRNA，其包含
a)有义核苷酸序列；和
b)与所述有义核苷酸序列互补的反义核苷酸序列，其中所述有义核苷酸序列包含权利要求1的核苷酸序列或与其互补。
33.权利要求32的方法，其中所述食物包含经转化以表达所述有义和所述反义核苷酸序列的植物细胞。
34.一种用于提高由遭受植物寄生线虫感染的作物植物产生的作物产量的方法，所述方法包括以下步骤：
a)将权利要求1的多核苷酸引入所述作物植物中；和
b)种植该作物植物以使得所述多核苷酸表达，其中该多核苷酸的表达抑制植物寄生线虫感染、生长、繁殖或由于植物寄生线虫感染导致的产量损失。
35.权利要求34的方法，其中所述作物植物选自玉米、小麦、大麦、黑麦、稻、马铃薯、西红柿、黄瓜、辣椒、苜蓿、豆科植物、大豆、豌豆、紫花苜蓿、甘蔗、甜菜、烟草、胡萝卜、棉花、油菜籽(加拿大油菜)、向日葵、红花、高粱、草莓、香蕉、草皮和观赏植物。
36.权利要求34的方法，其中所述多核苷酸的表达产生抑制已经与所述作物植物的一部分接触的植物寄生线虫中的至少第一目标基因的RNA分子，其中所述目标基因执行至少一种选自以下的关键功能：DNA复制、细胞周期控制、转录、RNA加工、翻译、核糖体功能、tRNA合成、tRNA功能、蛋白质运输、分泌、蛋白质修饰、蛋白质稳定、蛋白质降解、能量产生、线粒体功能、中间代谢、细胞结构、信号转导、内吞作用、离子调节、卵产生、繁殖和运输。
37.权利要求36的方法，其中所述植物寄生的线虫是垫刃线虫。
38.权利要求37的方法，其中所述植物寄生的线虫是根结线虫种类。
39.权利要求37的方法，其中所述植物寄生的线虫是南方根结线虫。
40.一种用于提高遭受植物寄生线虫感染的作物植物的渗透胁迫耐受性的方法，所述方法包括以下步骤：
a)将权利要求1的多核苷酸引入所述作物植物；
b)种植该作物植物以允许所述多核苷酸表达，其中所述多核苷酸的表达提高作物植物的渗透胁迫耐受性。
41.权利要求40的方法，其中所述渗透胁迫耐受性定义为耐旱性。
42.一种用于生产商品的方法，包括获得权利要求13的植物或其部分，和从该植物或其部分制备商品。
43.一种用于生产食品或饲料的方法，包括获得权利要求13的植物或其部分和从所述植物或其部分制备食品或饲料。
44.权利要求43的方法，其中所述食品或饲料定义为油、粗磨粉、蛋白质、淀粉、面粉或青贮饲料。
45.一种用于调节植物寄生线虫细胞中目标基因的表达的方法，该方法包括：
(a)用包含编码dsRNA的核酸序列的载体转化植物细胞，所述核酸序列选自SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ ID NO:42、SEQ IDNO:44、SEQ ID NO:46、SEQ ID NO:47及其至少21个连续核苷酸的片段，其中所述核酸序列编码dsRNA并可操作地与启动子和转录终止序列连接；
(b)在足以允许包含多个转化的植物细胞的植物细胞培养物发育的条件下培养转化的植物细胞；
(c)选择已经将所述核酸序列整合到其基因组中的转化植物细胞；
(d)筛选表达由所述核酸序列编码的dsRNA的转化植物细胞；和
(e)选择表达该dsRNA的植物细胞。
46.权利要求45的方法，进一步包括从表达所述dsRNA的植物细胞再生植物，从而植物中核酸序列的表达足以调节与转化的植物或植物细胞接触的植物寄生线虫细胞中目标基因的表达。
47.一种多核苷酸，包含：
(a)SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:40、SEQ IDNO:42、SEQ ID NO:44、SEQ ID NO:46或SEQ ID NO:47的任一核酸序列的连续核苷酸的片段；和
(b)(a)中片段的反向互补序列，
其中在植物寄生的线虫的食物中提供表达足以调节所述线虫中目标基因表达的多核苷酸的植物细胞。
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| WO2014091466A2 (en) | 2012-12-14 | 2014-06-19 | Indian Agricultural Research Institute | A method for the control of nematodes in plants |
| CN103053480B (zh) * | 2013-01-25 | 2014-04-16 | 中国农业科学院柑桔研究所 | 一种柑橘全爪螨若螨的rna干扰方法 |
| CA3046081A1 (en) * | 2016-12-06 | 2018-06-14 | Pebble Labs, Inc. | System and methods for the biocontrol of plant pathogens |
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