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CN1657620A - 用双链rna抑制鉴定基因功能 - Google Patents

用双链rna抑制鉴定基因功能 Download PDF

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CN1657620A
CN1657620A CN200510052742XA CN200510052742A CN1657620A CN 1657620 A CN1657620 A CN 1657620A CN 200510052742X A CN200510052742X A CN 200510052742XA CN 200510052742 A CN200510052742 A CN 200510052742A CN 1657620 A CN1657620 A CN 1657620A
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G·普莱廷克
C·普拉托伊夫
K·莫蒂尔
T·波格尔特
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Abstract

提供负责在细胞中赋予特定表型的DNA的鉴定方法，所述方法包括：a)以相应于启动子、当合适的转录因子结合于所述启动子时能够启动从所述cDNA或DNA转录出双链(ds)RNA的方向，在合适的载体中构建所述细胞DNA的cDNA文库或基因组文库，b)将所述文库引入包含所述转录因子的一种或多种所述细胞中，且c)鉴定和分离包含所述文库的特定表型的所述细胞，并鉴定所述文库中负责赋予所述表型的DNA或cDNA片段。采用该技术，可以将功能确定到已知DNA序列，方法为：a)鉴定细胞中所述DNA序列的同源物，b)从所述细胞中分离出相关的DNA同源物或其片段，c)将所述同源物或其片段以相应于合适启动子、当合适的转录因子结合于所述启动子时能够启动从所述DNA同源物或其片段转化dsRNA的方向，克隆到合适的载体中，d)将所述载体引入得自步骤a)的包含所述转录因子的所述细胞中。

Description

用双链RNA抑制鉴定基因功能
本发明为分案申请，原申请的申请号为99810196.6，申请日为1999年7月2日，发明名称为“用双链RNA抑制鉴定基因功能”。
本发明涉及采用双链RNA抑制(dsRNAi)来特征鉴定或鉴定基因的功能，涉及鉴定负责在细胞中诱导特定表型的DNA的方法以及将功能确定到已知基因序列的方法。
最近，在Nature第391卷，第806-811页(1998年2月)中已经描述了：将双链RNA引入到细胞中，导致有效且特异性地干扰细胞中内源基因的表达，并且这种干扰比在反义技术中提出的单独提供任一RNA链要有效得多。也发现当将所述RNA引入到线虫Caenorhabditis elegans(C.celegans)蠕虫基因组或体腔中时，在这种蠕虫中存在该基因活性的这种特异性降低。
本发明人已经利用了这种技术，并应用其进一步设计新的发明方法，以将功能确定至已经在各种计划诸如人类基因组计划中测序、并且还给予了一种特定功能的基因或DNA片段上，并且将其用于鉴定负责赋予特定表型的DNA。
因此，按照本发明的第一个方面，提供一种鉴定负责在细胞中赋予表型的DNA的方法，所述方法包括：a)以相应于启动子的方向，构建所述细胞DNA的cNDA文库或基因组文库，使得在合适的转录因子结合于所述启动子时能够促进从所述cDNA或DNA转录出双链(ds)RNA，b)将所述文库引入到包含所述转录因子的一种或多种所述细胞中，且c)鉴定并分离包含所述文库的所需表型的所述细胞，并鉴定所述文库中负责赋予所述表型的DNA或cDNA片段。
在本发明的一个优选实施方案中，在步骤b)之前，可以将所述文库组成为如在所提供的实施例中更详细描述的等级库，以致包括例如基因家族。
按照本发明的再一方面，也提供一种将功能确定到已知DNA序列的方法，所述方法包括a)鉴定细胞中所述DNA的同源物，b)从所述细胞中分离出相关的DNA同源物或其片段，c)以相应于合适的转录因子结合于启动子时能够促进dsRNA转录的启动子的方向，将所述同源物或片段克隆到合适的载体中，d)将所述载体引入到得自步骤a)的包含所述转录因子的所述细胞中，以及e)鉴定与野生型相比所述细胞的表型。
在本发明的每一方面，均可以以相应于具有以上所定义特性的单个启动子的有义和反义方向提供所述核苷酸序列或DNA序列，或者可选择地，可以在两个相同启动子之间提供所述核苷酸序列或DNA序列。在这两个实施方案中，在启动子结合其合适的转录因子后，由所述启动子启动的转录提供dsRNA。
按照本发明的细胞可以得自一种生物或包含于一种生物中。在所述细胞包含于一种生物中的场合下，可以改变所述生物以表达合适的转录因子。所述生物可以为植物、动物、真菌或酵母中的任何一种，但最好可以是线虫蠕虫C.elegans，它可以是C.elegans野生型、nuc-1或pha-ts突变体中的任何一种或所述突变的组合。在一个替代实施方案中，可以便利地将所述DNA或cDNA文库或所述DNA同源物或其片段转染或转化到可以饲喂给所述生物的微生物中，诸如细菌或酵母细胞，所述生物最好是线虫蠕虫C.elegans。在本发明的该实施方案中，可以将所述微生物改变以表达所述合适的转录因子。所述微生物最好是大肠杆菌。
在本发明的每个方面，可以在包含编码所述转录因子的核苷酸序列的合适的DNA载体中，构建所述DNA文库、DNA同源物或DNA片段。或者，可以由另一载体编码所述转录因子。在再一替代方法中，所述细胞或生物可以表达所述转录因子，或者可以将其改变以表达所述转录因子。最好是，用于按照本发明的方法中的任一所述载体包含一种选择标记，可以例如为编码sup-35或其片段的核苷酸序列。可以将所述核苷酸序列相应于启动子定向，以致转录因子结合至所述启动子而启动从所述DNA转录出双链RNA。图10图解了使得在C.elegans中能够产生双链RNA的载体和DNA序列的方向。因此，在一个实施方案中，所述DNA位于载体上的两个启动子之间，当合适的转录因子结合于所述启动子时，所述载体能够表达dsRNA。或者，所述载体包含两个拷贝的所述DNA序列，所述两个拷贝以相应于所述启动子的有义和反义方向组织，而且当所述标记包含于pha-1突变C.elegans中时，所述标记为可选择的。所述启动子最好是T7、T3或SP6启动子中的任一种，所述转录因子包括所述合适的聚合酶。
所述选择标记最好包含能够在所述细胞中抑制或防止基因表达的核苷酸序列，而且所述基因负责赋予已知的表型。该核苷酸序列可以是赋予所述表型的所述基因的一部分，或与所述基因相同，而所述核苷酸序列本身的定向与在合适的转录因子结合于所述启动子时能够启动双链RNA转录的合适启动子的定向相应。或者，所述核苷酸序列可以是赋予所述表型的所述基因序列的一部分，或与所述基因序列相同，而所述核苷酸序列是这样的序列，以致允许所述合适的载体或另一载体通过同源重组整合到所述细胞的基因组中，并且在所述整合后，所述核苷酸序列能够抑制赋予所述表型的所述基因序列的表达。在该实施方案中，所述核苷酸序列包含足以防止所述核苷酸序列在其整合到所述基因组中之后进行翻译的终止密码子。
有利地是，在所述方法中，可以将化合物加到所述细胞上或生物中，以便筛选所需表型，诸如当与野生型相比时，具有对该化合物的抗性或敏感性。所述启动子最好是诱导型的。在某些实施方案中，所述转录因子可以得自噬菌体，诸如为由噬菌体启动子驱动的T7聚合酶。然而，当利用C.elegan时，可以使用蠕虫特异性或组织特异性启动子，例如Let858、SERCA、UL6、myo-2或myo-3。所述大肠杆菌菌株最好是RNA酶III阴性菌株，甚至更优选为RNA酶阴性菌株。
本发明的再一方面提供一种产生包含一种外源转录因子和一种转基因的非人类转基因生物的方法，其中所述转基因包含一个有效连接至DNA片段的启动子，当所述转录因子结合于所述启动子时表达所述DNA片段；所述方法包括：a)提供包含第一构成物的第一转基因生物和包含第二构成物的第二转基因生物，所述第一构成物掺入了编码外源转录因子的DNA，而所述第二构成物包括至少一个有效连接至所需DNA序列的启动子，在所述第一转基因生物的转录因子结合于所述启动子时，表达所述所需DNA序列，b)将所述第一转基因生物与所述第二转基因生物杂交，并选择表达所述所需DNA序列的后代。在一个实施方案中，通过将所述第一和第二构成物转化到相应的微生物中，随后用所述微生物饲喂所述相应的生物，产生所述第一和第二转基因生物。最好是，所述第二构成物包含所述所需DNA序列，所述所需DNA序列相应于所述启动子的方向，使得当所述转录因子结合于所述启动子时，能够启动从所述DNA转录出dsRNA。在该实施方案中，所述第二构成物包含两个邻接所述所需DNA序列的启动子，所述启动子在所述转录因子与其结合时，可以启动从所述DNA序列转录出dsRNA。或者，以相应于所述启动子的有义和反义的方向提供所述DNA序列，使得可以在所述转录因子结合于所述启动子时产生dsRNA。在这些实施方案中的每一个中，可能最好为所述第一构成物和/或第二构成物提供一个有效连接至启动子的报道基因，所述启动子在所述转录因子与其结合时能够启动所述报道基因的转录。最好是，所述报道基因编码荧光素酶、绿荧光蛋白、β半乳糖苷酶或β-内酰胺酶中的任一个。
本发明也包括在酵母双杂种载体实验中所鉴定的克隆的验证方法，所述实验是本领域技术人员众所周知的，并且所述实验由Chien等(1991)首先提出，用于探测蛋白-蛋白相互作用。按照本发明的方法包括：提供一种构成物，所述构成物包含在双杂种载体实验中所鉴定的蛋白的编码DNA，所述构成物使得以相应于一个或多个启动子的方向提供所述DNA，所述启动子在合适的转录因子结合于所述启动子时，能够促进从所述DNA转录出双链RNA；用所述构成物转化诸如细菌细胞的细胞，或者转化包含所述转录因子的生物；并且与野生型相比，鉴定在所述细胞或生物中的表型变化，所述生物可以是C.elegan或诸如此类的生物。最好是，所述转录因子在所述细胞或生物中为可诱导的。如上所述，可以再一次将所述DNA序列定位于两种启动子之间，或以相对于单一启动子的有义和反义两个方向定位。最好是，所述启动子为噬菌体聚合酶启动子，而所述转录因子为RNA聚合酶，最好是T7聚合酶。本发明的范围也包括用于转化所述细胞或生物的载体以及所述细胞或生物本身。
在本发明的再一方面，提供减轻植物害虫侵染的方法，所述方法包括：a)从所述害虫中鉴定出或者对于其生存、生长、增殖或者对于其生殖重要的DNA序列，b)在合适的载体中，以相对于一个或多个启动子的方向克隆得自步骤a)的所述序列或其片段，所述启动子在合适的转录因子与其结合时能够从所述序列转录出RNA或dsRNA，并且c)将所述载体引入到所述植物中。
因此，有利的是，按照本发明的方法提供一种特别具有选择性的机制，用于减轻害虫侵染，而且在某些情况下用于减轻植物寄生虫侵染，使得当所述害虫在所述植物上摄食时，它将摄入植物中所表达的dsRNA，因此抑制对于害虫生长、生存、增殖或生殖重要的DNA在害虫中的表达。在一个优选实施方案中，所述害虫可以为小垫刃线虫属物种(Tylenchulus ssp.)、穿孔线虫属物种(Radopholus ssp.)、细杆滑刃线虫属物种(Rhadinaphelenchus ssp.)、异皮线虫属物种(Heteroderassp.)、小盘旋线虫属物种(Rotylenchulus，ssp.)、短体线虫属物种(Pratylenchus ssp.)、刺线虫属物种(Belonolaimus ssp.)、Canjanus ssp.、根结线虫属物种(Meloidogyne ssp.)、球异皮线虫属物种(Globoderassp.)、科布线虫属物种(Nacobbus ssp.)、茎线虫属物种(Ditylenchusssp.)、滑刃线虫属物种(Aphelenchoides ssp.)、潜根线虫属物种(Hirschmenniella ssp.)、鳗线虫属物种(Anguina ssp.)、纽带线虫属物种(Hoplolaimus ssp.)、螺旋线虫属物种(Heliotylenchus ssp.)、小环线虫属物种(Criconemella ssp.)、剑线虫属物种(Xiphinema ssp.)、长针线虫属物种(Longidorus ssp.)、毛刺线虫属物种(Trichodorus ssp.)、副毛刺线虫属物种(Paratrichodorus ssp.)、真滑刃线虫属物种(Aphelenchs ssp.)中的任何一种。可以将按照本发明该方面的DNA序列或其片段克隆在两个组织特异性启动子之间，诸如在两个根特异性启动子之间。
本发明的再一方面涉及用于本发明每个方法中用于构建所述文库的载体，所述载体包含两个相同的启动子，所述启动子的定向使得在合适的转录因子结合于所述启动子时，所述启动子能够启动从位于所述启动子之间的DNA序列转录出dsRNA。所述DNA序列可以例如包括一个多克隆位点。最好是，所述表达载体包含一段编码选择标记的核苷酸序列。在一个实施方案中，编码所述选择标记的所述核苷酸序列位于两个相同的启动子之间，所述启动子的定向使得在合适的转录因子结合于所述启动子时，所述启动子能够启动从位于所述启动子之间的DNA转录出双链RNA。为了引入到具有pha-1突变的C.elegans中，所述选择标记最好包含一段编码sup-35的核苷酸序列。
最好是，所述转录因子或者包括一个结合于其相应启动子的噬菌体聚合酶，或者包括一个C.elegans特异性启动子，甚至更优选T7聚合酶。最好是，所述载体包含一个在所述相同的启动子之间的多克隆位点。
在本发明的再一方面，提供一种表达载体，用于表达供按照本发明方法使用的合适的转录因子，所述载体包含一段有效连接至合适的表达控制序列的、编码所述转录因子的核苷酸序列。所述表达控制序列最好包括启动子，所述启动子为诱导型启动子、组成型启动子、普遍性启动子或组织特异性启动子或它们的组合。最好是所述转录因子包括噬菌体聚合酶，最好为T7、T3或SP6的RNA聚合酶。
本发明的再一方面提供一个选择系统，用于鉴定用按照本发明的载体对细胞或生物的转化，所述系统包括一个按照本发明的载体，其中所述选择标记包括一段在所述细胞或生物中能够抑制或防止负责赋予已知表型的基因表达的核苷酸序列。所述核苷酸序列最好对应于赋予所述已知表型的所述基因的一部分，或与所述基因相同，而且所述核苷酸序列本身位于两个相同启动子之间，所述启动子在合适的转录因子与其结合时，能够启动双链RNA的转录。或者，所述核苷酸序列包含的一段核苷酸序列是赋予所述细胞或生物已知表型的所述基因序列的一部分，或与所述基因相同，并且使得在所述载体通过同源重组而整合到所述细胞或生物的染色体中之后，所述序列抑制赋予所述已知表型的所述基因序列的表达。最好是，按照该实施方案，所述核苷酸序列包含足以防止所述核苷酸序列在整合到所述染色体之后进行翻译的终止密码子。所述已知基因序列最好包含可选择的sup-35基因或其片段，可通过鉴定将其引入到pha-1 et123ts突变型C.elegans蠕虫中之后于高于25℃的温度下生长的后代，可选择出该基因或片段。
在本发明的再一方面，提供所述已知基因的序列，所述序列包括通过鉴定将所述载体引入到pha-1 et123ts突变型C.elegans蠕虫中之后后代在高于25℃的温度下生长可选择出的sup-35基因或其片段。又一个方面包括将功能确定至多细胞生物的DNA序列的方法，所述方法包括：a)在能够从所述启动子启动子转录的多细胞生物中，提供i)构成物，所述构成物包含在所述多细胞生物中能够促进转录的两个启动子之间克隆的所述DNA片段；b)与野生型相比，鉴定所述多细胞生物的表型。
参考附图，通过以下仅为示例性的实施例，可以更清楚地理解本发明，在附图中：
图1是按照本发明的质粒PGN1的核苷酸序列。
图2是按照本发明的质粒PGN100的核苷酸序列。
图3是按照本发明的方法中使用的质粒和转化方案的示意图。
图4是按照本发明使用的表达载体的说明。
图5是用于转化C.elegans的T7 RNA聚合酶表达载体的示意图。
图6是质粒PGN1的图解。
图7是用于dsRBA抑制、编码sup-35 dsRNA的增强载体的图示说明。
图8是用于整合到C.elegans基因组中的载体的说明。
图9是相对于从所述DNA序列启动dsRNA表达的合适启动子的DNA序列位置的说明。
图10是质粒pGN108的说明。
图11是质粒pGN105的说明。
图12是质粒pGN400的说明。
图13是质粒pGN401的说明。
图14是质粒pGN110的说明。
图15是具有正向和反向T7/T3/SP6启动子的质粒pAS2的说明。
图16是具有正向和反向T7/T3/SP6启动子的质粒pGAD424的说明。
图17是质粒pAS2-cyh2-HA+both T7-final的说明。
图18为质粒pGAD424-without-FULL-ICE-BOTH-T7的说明。
图19(a)为质粒pGN205的说明，而(b)为质粒pGN207的说明。
实施例A：有序等级汇集的cDNA文库的构建及其应用
随机有序和汇集的文库：
所述载体为一种大肠杆菌载体，带有两个T7启动子，在所述两个启动子之间有一个多克隆位点(MCS)。所述两个启动子彼此相对地定向并朝向所述MCS定向。在存在在大肠杆菌、C.elegans或任何其它生物中表达的T7 RNA聚合酶的情况下，将从所述两个T7启动子开始，产生RNA。由于这些启动子以相反的方向定向，因此将从插入(或克隆)到位于所述两个启动子之间的MCS中的DNA，产生两条RNA链，当T7 RNA聚合酶与启动子结合时，导致产生双链RNA(dsRNA)。
采用标准分子生物学技术，在所述MCS中构建了一个C.eleganscDNA文库。将所述文库转化到大肠杆菌中，让所产生的大肠杆菌在在培养基中生长并贮存在96孔多孔板中。在该阶段，可以分离质粒DNA并将其对应于大肠杆菌菌落的96多孔板贮存在96孔多孔板中。记录了大约100,000个菌落。以这种方式，该文库将含有约5倍于总的表达的C.elegans cDNA变异的cDNA变异，这为低表达序列出现在该文库中提供了机会。这将产生约1041块96孔板。必要时将所述板进行等级汇集。目前，将所述克隆的汇集范围安排在10-100个。如果按每8个或每12个进行等级汇集(因为96孔板具有8排12列，则这些数字更为方便)，则这产生大约87块多孔板，大约8352个孔。如果按96孔即整个板进行等级汇集，则这产生约11块板，大约1041个孔。在等级汇集的任一阶段，均可以分离质粒DNA，当使用较少的板时，工作量可能较少，但将导致复杂度的降低，尽管在按12列汇集的条件下不应该是这种情况。也可以用所述原始DNA进行所述DNA的汇集。
以下实验描述了应该如何对所述DNA文库以及对大肠杆菌文库进行等级汇集。
用于RNAi技术的、含有C.elegans基因组中每个基因的有序文库及其应用
由于基因组测序计划即将进入尾声，因而可以将这种信息用于T7RNA抑制技术的应用中。采用PCR技术，可以克隆C.elegans基因组的每个基因。优选克隆最小长度为500bp的外显子。如果外显子太小，则用PCR分离较小的片段，或甚至用PCR技术分离多个内含子的部分和相邻的外显子，使得至少克隆足够部分的所述基因的翻译区。为此，需要进行至少17000个PCR反应。将这种PCR产物的收集物克隆在所述T7载体(两个T7启动子彼此相对地定向，在其之间具有一个多克隆位点)中。独立地克隆每种PCR产物，或可以用全部PCR产物产生类似于所述cDNA文库的随机文库。如果单独克隆每种PCR产物，则可以以不同方式汇集所产生的细菌和质粒DNA。首先，可以如在随机文库中所述，将这种分别在T7 RNAi载体中克隆的PCR产物的收集物随机地汇集。也可以以更合理的方式进行这种汇集。例如，可以采用生物信息工具(载于silico biology)分析C.elegans基因组的基因。该基因组的不同基因将属于一个基因家族，或在该基因组中具有同源物。汇集该基因家族的这些成员，或将为同源物的成员汇集。以这种方式，将约17000个克隆的总数降至更合用的量。在按照本发明的方法中可以用该文库筛选表型。所产生的表型给出了对C.elegans基因组的所述基因或基因家族或基因同源物的功能描述。由于该文库由该基因组中每个基因的一部分构成，因此该方法使得能够用功能-表型术语描述整个基因组。为此，需要将双链RNA(dsRNA)引入到所述蠕虫中。可以以所述的几种方式，完成这种单独的克隆、或者随机汇集或合理汇集的汇集克隆的引入。
用于表达双链RNAi的载体的实例
可以使用含有T7启动子的任何载体，且所述载体含有一个多克隆位点(有许多市售的载体)。设计具有合适末端的含有T7启动子的引物和具有合适末端的反向互补链的引物。可以将这些引物杂交，并且如果很好地设计，可以将其克隆在选择的载体中。作为T7启动子的最小序列为TAATACGACTCACTATAGGGCGA。尽管可以使用任何载体构建一个T7表达载体，但以下是用载体pGEM-3zf(-)如何完成这一过程的实例。
-用HindIII和SalI消化载体pGEM-3zf(+)(PROMEGA)。
-将引物oGN1和oGN2以1μg/30μl的终浓度混合在一起，将其煮沸并缓慢冷却至室温。
-采用标准连接程序将所述引物连接到该载体中。所产生的载体为pGN1(示于图1)，它含有彼此相对地定向的两个T7启动子，两个启动子之间含有一个多克隆位点。
oGN1和oGN2的序列为：
-oGN1：AGC TGT AAT ACG ACT CAC TAT AGG GCG AGA AGC TT
-oGN2：TCG AAA GCT TCT CGC ATA ATA GTG AGT CGT ATT AC
                  文库构建实施例
从对RNAi敏感的每种生物中分离RNA。一般接着将分离的RNA拷贝为双链cDNA，随后在合适的载体中制备用于克隆。存在几种方法，分子生物学试剂盒可以购自不同公司，包括promega，clontech，boehringer Mannheim、BRL等，所述试剂盒使得能够：
-分离RNA，
-最终可以分离polyA RNA(可利用几种技术和试剂盒)
-用AMV逆转录酶、随机六聚引物和/或寡聚(dT)引物合成第一条链
-用RNA酶H、DNA聚合酶I合成第二条链，
-用T4 DNA聚合酶填平末端
-用T4 DNA连接酶加入一个连接物
-最后用T4多核苷酸激酶处理
-将所述cDNA克隆到所述载体中。
所产生的连接混合物可以被认为是cDNA文库。连接翻译物含有连接到目的载体中的所有该步骤的cDNA。为了安排文库，需要将该连接反应物转化到大肠杆菌菌株中。
           该大肠杆菌文库或DNA文库的应用
产生T7 RNA的菌株：
-一种标准的菌株为BL21(DE3)：F-ompT[lon]hsds(r-m-)；和大肠杆菌B菌株)λ(DE3)。最后可使用BL21(DE3)的变异体，尽管使用BL21(DE3)pLysS。
-需要构建可以利用的、产生T7 RNA聚合酶的任何其它大肠杆菌菌株。可以用市售的噬菌体容易地产生这种菌株，在这种情况下，使用λCE6载体(由Promega提供)。可以用该噬菌体转染几乎每种大肠杆菌，并将产生T7 RNA聚合酶。
-RNA酶III突变大肠杆菌：
原则上可利用各种菌株，我们选择在第一个实验中使用菌株AB301-105：rna-19，suc-11，bio-3，gdhA2，his95，rnc-105，relA1，spoT1，metB1。(kinder等，1973 Mol.Gen.Genet 126：53)，但其它菌株可能更适合。用λDE6感染该菌株，由此构建生产T7的变异体。
可以在细菌库(bacteria pool)上培养野生型C.elegans蠕虫。所述细菌正在表达T7 RNA聚合酶。这在C.elegans肠中产生大量的dsRNA，dsRNA将在这种生物中扩散，并导致抑制表达。现在可以用该文库筛选几种表型。这种技术的优点是：与已知的C.elegans技术相比，它探测某些途径中的相关基因要快得多。此外，如果发现一种感兴趣的表型，则可以容易地克隆负责的基因。
采用等级汇集，人们可以容易地在第二次筛选中发现该库的相关克隆。然后可以对该克隆的插入DNA进行测序。该实验以一个步骤产生遗传和生化的数据。
可以将野生型C.elegans株系与化合物混合，以根据表型、药物抗性和或药物敏感性进行筛选。所述C.elegans株系可以是突变型株系，根据增强的表型、减弱的表型或新表型筛选。所述C.elegans株系可以是一种突变株系，并且可以将所述文库的筛选与化合物相结合。因此，人们可以筛选药物抗性、药物敏感性、增强的表型、减弱的表型或新表型。所述大肠杆菌菌株可以是表达T7 RNA聚合酶的任何菌株，例如像BL21(DE3)，但通过采用RNA酶III阴性的特殊大肠杆菌菌株，可以增强双链RNA的形成。最后，可以使用非RNA酶III的RNA酶缺失的大肠杆菌，或可以使用一种或多种RNA酶缺失的大肠杆菌。T7 RNA聚合酶在大多数已知的大肠杆菌菌株和可用来产生生产T7 RNA聚合酶的大肠杆菌菌株的构成物中的表达，一般包括一个诱导型启动子。以这种方式，调节T7 RNA聚合酶的产生，并由此产生所述dsRNA。最好是，可以用这一特征来“脉冲式”饲喂特定生长阶段的C.elegans蠕虫。让所述蠕虫生长在非诱导的大肠杆菌菌株上。当所述蠕虫长到目的阶段时，在所述细菌中诱导产生T7 RNA。这使得可以在所述动物生活周期中的任何点研究任一基因的功能。筛选文库中推定的目的人类基因的同源物并将功能确定至这些基因
已经在各种计划(为基因组计划、差异表达排列、杂交研究等)中分离出数百个基因。所描述的cDNA文库可以提供一种以快速有效的方式验证功能和或将功能确定至这些基因的方法。首先，需要通过生物信息工具(载于silico biology)鉴定出该蠕虫的一种或多种同源物或所述基因。研制出PCR引物，并用PCR技术分离所述cDNA片段。可以在等级库上进行PCR。将含带有合适cDNA的细菌的阳性库或阳性孔的所述细菌饲喂C.elegans并记录表型。
可以在从C.elegans中分离的cDNA上进行PCR。可将产生的DNA克隆到T7载体中，并转化到所述生产dsRNA的大肠杆菌中，然后用该大肠杆菌饲喂C.elegans蠕虫。需要根据哪种方式更快和更可靠而作出选择。
如果该基因属于一个基因家族，则可能需要用细菌混合物饲喂所述蠕虫。细菌混合物中的每种细菌均含有该基因家族成员的一部分。可以如上所述使用大肠杆菌菌株、生长条件及进行与化合物的混合。
如果使用其中以组织和结构方式克隆C.elegans的所有基因的该文库的合理形式，则可以用silico biology容易地在该文库中追踪回该基因家族的C.elegans同源物，并最终追踪回该基因家族的其它同源物、orthologues和成员。在该步骤中不涉及PCR，可以分离用于养所述蠕虫的细菌和或DNA。
实施例
所述系列实验的：既测试所述RNAi载体和也测试构建的各种大肠杆菌菌株。
1)试验质粒的构建
可以使用当被失效或用于RNAi实验中时在蠕虫中产生清楚表型的任何cDNA。已知unc-22是一个良好的候选者，但可能其它基因也是可以的。我们选择可以用于较晚阶段的一个敏感型系统。用在pha-1背景中的sup-35测试该系统。通过PCR分离sup-35的外显子5，并将其克隆到T7启动子载体pGN1中。产生的载体被命名为pGN2。pha-1(e2123)突变型蠕虫不能在高于25℃的温度下产生后代。这是由于胚胎发生中的发育问题而引起的。当在该株系中sup-35被失效或抑制时，后代可以在该温度下生长。pha-1突变型蠕虫和sup-35 RNAi的组合是一个验证各种可选择之物的良好系统。
2)用产生dsRNA的大肠杆菌菌株测试所述RNAi
-将pGN2引入大肠杆菌菌株BL21(DE3)中，并用IPTG诱导T7RNA聚合酶。在该细菌上接种C.elegans蠕虫(pha-1(e2123))，让其在25℃限制温度下生长。因为该突变体在该温度下是胚胎突变体，所以将观察不到后代。如果大肠杆菌中存在的sup-35 dsRNA有效抑制sup-35基因，则将观察到后代。
-将pGN2引入大肠杆菌AB301-105(DE3)中，并用IPTG诱导T7RNA聚合酶。在该细菌上接种C.elegans蠕虫(pha-1(e2123))，让其在25℃限制温度下生长。因为该突变体在该温度下是胚胎突变体，所以观察不到后代。如果大肠杆菌中存在的sup-35 dsRNA有效抑制sup-35基因，则将观察到后代。
3)改良蠕虫株系以更好地摄入dsRNA
在将pha-1 C.elegans接种到产生双链sup-35 RNA的大肠杆菌菌株平板上之前，用EMS(甲磺酸乙酯)诱变该蠕虫。然后将诱变蠕虫的后代接种到所述细菌平板上。摄食该细菌的蠕虫产生较大的后代，所述后代具有导致dsRNA摄入改善的突变，可以将其用于进一步的实验。
所述产生dsRNA的载体稳定地整合到产生T7 RNA聚合酶的蠕虫的基因组中
构建具有以下特征的大肠杆菌载体：彼此相对的两个T7启动子，在所述两个启动子之间具有一个限制位点或一个多克隆位点。此外，该载体可以含有C.elegans sup-35基因组DNA，对该载体的工程改造方式使得其在不同的间隔含有几个终止密码子，以致从sup35基因组DNA片段不可能表达出全长蛋白，如图8所示。在所述两个T7启动子之间的多克隆位点中可以克隆任何cDNA或cDNA片段。如果将该载体引入到表达T7 RNA聚合酶的C.elegans株系中，则在所述两个T7启动子之间克隆的cDNA或cDNA片段将转录，并从所克隆的片段产生dsRNA。
设计该载体以用于表达T7 RNA聚合酶的pha-1(e2123)突变型蠕虫中。T7 RNA聚合酶的表达可以是组成型的，或可以被调节，可以是普遍性的或是组织特异性的。这些pha-1(e2123)蠕虫在高于25℃的温度下不能产生后代，这是由于胚胎发生中的发育问题引起的。当在该株系中sup-35被失效或抑制时，后代可以在该温度下生长。
如果将该载体引入到所述蠕虫中，则该载体可以通过同源重组而整合(Campbell样整合)。已经表明，在C.elegans中发生同源重组，尽管其频率低(Plasterk和Groenen，EMBO J.11：287-290，1992)。与sup-35基因的同源重组将导致该基因的失效，因为所产生的两个sup-35基因将含有终止密码子。如果这种重组发生在卵中，则所产生的蠕虫及其后代将具有一个拷贝的整合到基因组中的所述载体。可以将其选择出来，因为仅有sup-35已经被失效的蠕虫在高于25℃的温度下具有后代。此外，所产生的蠕虫将稳定地从所述两个T7启动子之间的克隆的DNA片段产生双链RNA。这种蠕虫现在可以被认为是基因功能减低的稳定的转基因蠕虫株系，而所述基因是所述两个T7启动子之间所克隆片段所属的基因。
可以通过几种技术将所述DNA传递至蠕虫，所述技术包括注射、弹道转化(ballistic transformation)、浸泡在所述DNA溶液中、饲喂所述细菌。可以考虑提高转化率的新方法和其它方法。
另外，C.elegans靶株系可以具有除pha-1(e2123)突变以外的其它突变，并且可以表达除T7 RNA聚合酶以外的其它基因。
实施例B：酵母双杂种-RNAi载体
可以构建含有所述两个T7启动子的酵母双杂种载体。所述载体可被设计为既在酵母中复制，又在大肠杆菌中复制。一般而言，可以在Gal4或LexA载体中制备适合于所述酵母双杂种系统的cDNA文库。在具有这些基因中的一个基因的激活域的载体中构建该文库。可以构建一种载体，该载体在酵母双杂种筛选中仍可以执行功能，但它也含有两个彼此相对定向的T7启动子，并且在所述两个启动子之间具有一个克隆位点。在该质粒中所述序列的顺序则将为“质粒骨架、(GAL4-T7)、MCS、T7、骨架”。在实验中在该载体中构建的C.elegans cDNA文库可以用作标准酵母双杂种文库，以分离与给定蛋白相互作用的蛋白。一旦分离出克隆，则可以将该质粒引入到表达T7RNA聚合酶的大肠杆菌菌株中，并因此将产生所克隆片段的dsRNA。可以将产生该dsRNA的细菌饲喂所述蠕虫，并可记录表型。如先前的实施例中，这种对于新分离的酵母双杂种克隆的验证方法明显比标准方法省时，标准方法需要PCR步骤和/或克隆步骤、RNA实验和/或失效实验。在大多数情况下，首先对分离出的克隆测序，在该序列的基础上，作出继续进行进一步的实验的决定。在本发明中，可以容易地将每个分离出的克隆引入到合适的大肠杆菌中并将其饲喂所述蠕虫。然后通过表型分析进行验证。
为了应用该方法，用一个已知基因作饵，用新构建的文库作为靶，使用酵母双杂种。在所述靶中，由所述克隆编码的与饵蛋白相互作用的蛋白，将产生表达报道分子的阳性酵母克隆，所述报道分子诸如可以通过用X-gal的LacZ染色观察到的报道分子。直接从该酵母菌株中分离出编码靶蛋白的质粒，并将其引入到大肠杆菌中。该大肠杆菌为产生T7 RNA聚合酶的大肠杆菌。在这种情况下，从克隆在该载体的多克隆位点中的DNA产生双链RNA。如果用前述方法将该dsRNA饲喂所述蠕虫，则所述基因在蠕虫中被抑制，产生特定表型。
-这种酵母双杂种载体可以有利地用来构建一个先前实施例中所述的有序且等级汇集的文库。
-也可以构建一种有条件地产生T7 RNA聚合酶的酵母菌株。在酵母双杂种实验后，可以诱导所述T7聚合酶的表达，导致在酵母细胞中产生dsRNA。因此，可以将该酵母饲喂蠕虫。可得到的证明表明，C.elegans蠕虫可以摄食酵母。
产生T7 RNA聚合酶的菌株的构建及其应用
可以构建表达T7 RNA聚合酶的C.elegans株系。表达可以是普遍的和组成型的，但也可以在一个组织特异性启动子、诱导型启动子或瞬时启动子或含有这些特征之一或特征组合的启动子之下而被调节。可以将DNA引入到该C.elegans株系中。这种引入或者通过注射、用粒子射击、电穿孔进行，或者如上所述通过摄食进行。如果所述DNA为前述实施例中描述的质粒，即在两个侧翼T7启动子之间具有所克隆cDNA片段或PCR片段的质粒，则在所述细胞或整个生物中形成该cDNA或PCR片段的dsRNA，导致向下调节相应的基因。所引入的DNA可以具有有效的瞬时向下调节作用。所引入的DNA可以形成染色体外布置(array)，所述布置可能导致更具催化性的功能表型的剔除或降低。该质粒也可能整合到所述生物的基因组中，导致同样催化性的功能表型的剔除或降低，但这可被稳定地传递。
-如实施例A)和B)中所述，可以通过标准技术，将在两个T7启动子之间具有一个克隆的C.elegans cDNA或cDNA部分或EST或PCR片段的质粒引入到T7 RNA聚合酶蠕虫中。可以分析表型-可以通过标准技术(注射、射击)，将如同实施例A)中的、得自有序汇集文库的DNA引入到T7 RNA聚合酶蠕虫中。可以分离表型。用所述等级库，可以容易地发现原始克隆。
-可以用表达T7 RNA聚合酶的突变蠕虫完成同样的步骤。筛选增强的、降低的或新的表型。
-可以使用能够筛选化合物的方法。或者用野生型株系或者用突变株系筛选增强的表型或新表型。
-可以用新方法将所述DNA引入到蠕虫中。其中一个方法是用大肠杆菌传递DNA。在这种情况下，用所述等级汇集文库饲喂所述动物。为了防止所述大肠杆菌DNA在线虫肠中被消化，优先使用DNA酶缺陷型C.elegans，例如nuc-1(e1392)。这种方法将的最令人感兴趣的方法，因为它与其它技术的转化效率无关，并且一般更快且工作量更少。
2)该方法推定的增强作用
-设计一种载体，使其含有sup-35 cDNA或该cDNA的一部分，将其克隆在两个T7启动子之间。该载体的其余部分如实施例A)和B)中所述。可以将该载体引入到pha-lts突变C.elegans中。在这种情况下存在一个温度选择系统，只有已经摄入所述DNA并表达双链sup-35RNA的那些蠕虫将在限制温度下存活。可以通过上述任何方法传递所述等级汇集文库。
-可以用该载体构建一个文库，将所述载体引入到表达T7 RNA聚合酶的大肠杆菌中。在这种情况下，我们已经如部分A)中所述，类似地进行了筛选，并在sup-35的dsRNA有活性的场合下，对蠕虫进行额外的筛选。
-可以在不同质粒上传递sup-35的DNA和或dsRNA。对于饲喂，即对于DNA饲喂(实施例C)和dsRNA饲喂实施例A)和B)，这意味着所述两种质粒可以存在于一种细菌中，或意味着用细菌混合物饲喂蠕虫，其中一种细菌含有sup-35构成物。
构建产生T7 RNA的C.elegans的实施例
为了在所述蠕虫中产生T7 RNA聚合酶，有几种可能性。所述T7聚合酶可以在不同启动子下表达，即在诱导型启动子、组成型启动子、普遍性启动子和组织(细胞)特异性启动子或它们的组合下表达。这些启动子的实例是热激启动子hsp-6、肠启动子ges 1、得自cet858的启动子，而且也有dpy 7的启动子和启动子元件GATA1。在该实施例中，在pPD49.78载体中可利用的hsp-16启动子控制下表达所述T7 RNA聚合酶。用GN3和GN4引物，分离作为PCR产物的T7 RNA聚合酶。
用NheI和NcoI消化所产生的PCR产物，同样消化我们想用于克隆的载体，即Fire载体pPD49.78。所产生的载体为图2中说明的pGN100，包括oGN3：CAT GGC AGG ATG AAC ACG ATT AAC ATCGC oGN4：ATG GCC CCA TGG TTA CGG GAA CGC GAA GTC CGpGN100。
用标准技术例如微注射，将该载体引入到蠕虫中。
然后构建以下株系：
-野生型(pGN100)
-nuc-1(e1392)(pGN100)
-pha-1(e2123)(pGN100)
-pha-1；nuc-1(pGN100)
当温度诱导时，或者用金属诸如应用重金属镉或汞时，所有这些株系均能够产生T7 RNA聚合酶。温度诱导方法是将动物转移至30-33℃的温度下达至少1小时，然后可将所述动物转移回到标准温度(15-25℃)下。
可以用产生T7 RNA聚合酶的野生型株系在所述蠕虫中产生任何RNA。更具体地讲，可以将得自所述文库的质粒引入到这些蠕虫中，并可记录表型。
用nuc-1突变蠕虫，通过喂给蠕虫的细菌来引入DNA。因为nuc-1蠕虫不消化所述DNA，所以所述质粒DNA可以穿过肠壁。如果所述DNA被产生T7 RNA聚合酶的细胞吸收，则将产生dsRNA，由此抑制转录出所述RNA的基因。
产生T7 RNA聚合酶的pha-1突变株系可以用来增强上述方法。可以通过射击、微注射或摄食引入DNA。更具体地讲，对于从sup-35和从目的基因产生dsRNA的载体，可以使用该株系，所述目的基因可以是PCR产物、cDNA或所述的文库。
产生T7 RNA聚合酶的pha-1、nuc-1突变体可以用于所述DNA的细菌传递。DNA将优选为从sup-35和目的基因产生dsRNA的质粒。该蠕虫株系将优先在肠中产生T7 RNA聚合酶。优选通过用带有该质粒的细菌饲喂蠕虫而发生传递。
RNAi技术在植物中的应用
线虫是使植物受到损害的一个很大原因，对于农业产业中使用的植物更尤其如此。可以将按照本发明的RNAi方法用于植物，以阻止这些寄生线虫摄食更长时间。第一步，从寄生于植物的线虫中分离出对于所述动物生存或生长或摄食或增殖重要的cDNA片段。必需表达的任何基因均适用于此目的。
克隆该基因、外显子或cDNA的一部分。可以将该DNA片段克隆置于组织特异性启动子的影响之下，所述启动子优选根特异性启动子，甚至更优选将该DNA片段克隆在两个根特异性启动子之间。可以用植物转基因技术，将在根特异性启动子控制下的所克隆基因的DNA引入目的植物中。对于每种寄生线虫，可能需要不同片段的DNA，同样，对于每个植物品种，将需要不同的启动子。
当利用根特异性启动子时，根将从引入的DNA片段产生RNA或dsRNA。因为线虫在植物上摄食，故所述线虫将消耗或摄入所述RNA和/或dsRNA。所述RNA和/或dsRNA可以进入线虫的细胞，并对所述靶DNA发挥其抑制作用。根据所克隆的蠕虫的DNA片段的性质，所述线虫将不能存活、进食、增殖等，在所有情况下均阻止该动物在所述植物上更长久地摄食，并因此保护植物。
产生T7 RNA聚合酶的C.elegans的构建
为了在动物中、更具体地讲在线虫中，更尤其在C.elegans中产生T7 RNA聚合酶或其它RNA聚合酶，可以设想几种可能性。可以在各种启动子下表达T7 RNA聚合酶。这些启动子可以是诱导型启动子、组成型启动子、普遍性启动子、组织特异性启动子或它们的组合。
实施例1：
构建用于在C.elegans中表达T7聚合酶的基本载体
用引物oGN26(ATGGAATTCTTACGCGAACGCGAAGTCCG)和oGN46(CTCACCGGTAATGAACACGATTAACATCGC)，采用标准方法(PCR，A practical A practical approach，1993，J.McPherson等编辑，IRL Press)，从λCE6(Novagen，Madison，USA)经PCR扩增T7聚合酶的编码序列。用AgeI和EcoRI消化所得的编码T7 RNA聚合酶的DNA片段，并插入用AgeI和EcoRI消化的Fire载体pPD97.82中。所得的构成物编码一个与SV40大T抗原核定位信号(NLS)融合的T7 RNA聚合酶的可读框，其中NLS的氨基酸序列为MTAPKKKRKVPV。将T7RNA聚合酶从胞质转运至细胞核需要这种核定位信号序列，在细胞核中T7 RNA聚合酶能够结合于名为T7启动子的其特异性启动子上。T7聚合酶融合蛋白编码序列的上游为最小启动子(myo-2)，在最小启动子之前为一个多克隆位点(MCS)，在其中可以插入几个C.elegans启动子。该质粒(示于图11的pGN105)为基本T7 RNA聚合酶质粒，该质粒能够在C.elegans中表达T7聚合酶。其中将启动子克隆到该质粒多克隆位点中的该质粒衍生物，在C.elegans中可提供T7 RNA聚合酶的诱导型、组成型、普遍型和组织特异性表达，因为表达将受到在多克隆位点中所克隆的启动子的调节。
尽管不限于这些实施例，但对于以下启动子，已知它们在以下组织中诱导表达：let-858(遍在表达)、myo-2(咽表达)、myo-3(体壁肌)、egl-15(阴户肌)、unc-119(全身神经元)。
实施例2：
构建用于在C.elegans肌肉组织中表达T7 RNA聚合酶的载体
用引物oGN43
(GCCACCGGTGCGAGCTCATGAACACGATTAACATCGC)和oGN44(CACTAGTGGGCCCTTACGCGAACGCGAAGTCCG)，从λCE6经PCR扩增T7 RNA聚合酶的编码序列，用AgeI/SpeI消化，并插入用AgeI/SpeI消化的pGK13载体中。(该载体含有在咽、阴户肌、尾部和体壁肌中驱动表达的强SERCA启动子)。通过将两个重叠寡核苷酸oGN45(CCGGATGACTGCTCCAAAGAAGAAGCGTAAGCT)和oGN46(CTCACCGGTAATGAACACGATTAACATCGC)插入SacI/AgeI限制位点中，将SV40大T抗原的核定位信号(NLS)插入T7聚合酶编码序列之前。将所得的构成物称为pGN108，如图10所示。将该质粒引入到C.elegans中，导致T7 RNA聚合酶在咽、阴户肌、尾部和体壁肌中表达。
为了测试T7 RNA聚合酶在C.elegans中处于SERCA启动子调节下的表达和功能性，将编码在SERCA启动子控制下的T7 RNA聚合酶的pGN108注射到C.elegans中。共同注射一种试验载体。该试验载体编码在T7启动子控制下的GFP(图13中的pGN401)。通过将两个重叠的寡核苷酸oGN41(CCCGGGATTAATACGACTCACTATA)和oGN42(CCGGTATAGTGAGTCGTATTAATCCCGGGAGCT)插入SacI/AgeI打开的Fire载体pPD97.82中，产生一个T7启动子，来构建质粒pGN401。此外，共注射一种选择标记以选择转化体(rol6，pRF4)。后一选择载体pRF4是本领域技术人员众所周知的。可以容易地分离出转基因F1，因为它们表现出rol6表型。这些转基因C.elegans均在咽、阴户肌、尾部和体壁肌中表达GFP。该数据清楚地表明，T7 RNA聚合酶在SERCA启动子调节下进行功能性表达，并且所表达的T7RNA聚合酶结合于pGN401中存在的T7启动子，从而启动GFP基因的转录，然后功能性地表达GFP，在SERCA正在诱导表达T7 RNA聚合酶的肌肉组织中产生荧光。
实施例3：
构建用于在C.elegans中遍在表达T7聚合酶的载体
用XmaI/Bsp1201从pGN108中分离NLS-T7 RNA聚合酶融合基因，将其克隆到用XmaI/Bsp120I消化的Fire载体pPD103.05中。这产生一种载体，其中将T7 RNA聚合酶克隆在let858启动子调节之下。这种特异性启动子使得能够在所有组织中表达T7 RNA聚合酶。将所得的质粒命名为pGN110(图14)。
实施例4：
构建一种载体，用于经T7 RNA聚合酶介导在T7启动子控制下 表达DNA片段、基因和cDNA
用SacI/AgeI消化Fire载体pPD97.82，通过将两个重叠寡核苷酸oGN41(CCCGGGATTAATACGACTCACTATA)和oGN41(CCGGTATAGTGAGTCGTATTAATCCCGGGAGCT)插入SacI/AgeI限制性内切核酸酶位点中，产生一个T7启动子序列。该构成物(图12中的pGN400)含有一个克隆在SacI和EcoRI限制性内切核酸酶位点之间的、在T7启动子调节之下的GFP可读框。通过缺失作为AgeI/SacI片段的GFP基因，并将目的DNA片段插入该载体中，可以在该载体中克隆任何基因、cDNA或DNA片段。优选可以通过PCR扩增获得目的DNA片段，将其插入引物的SacI/AfeI位点中。将PCR扩增后所得的DNA片段消化，用扩增的DNA片段取代pGN400中的GFP基因。对于在C.elegans中由T7 RNA聚合酶诱导的表达而言，可以使用含有T7启动子的每种载体，所述载体诸如市售的pGEM载体和pBluescript载体。通过pGN401载体清楚地表明了这一点：pGN401在表达T7 RNA聚合酶的转基因C.elegans中，在T7启动子调节下表达GFP。
应用pGN400的优点是：该载体包括得自unc-54的3’UTR片段，该片段增强所述RNA的转录或稳定性。
永久性、组织特异性“假失效”RNAi C.elegans系的产生
目前，在随机大规模诱变和PCR碱基同胞选择后，获得C.elegans中的基因失效。该方法工作量大、非常耗时并且烦琐。已经描述了，将双链RNA引入到细胞中导致内源基因表达的有效而特异性的干扰。在C.elegans中，通过将RNA注射到该蠕虫的体腔中、将蠕虫浸泡在含dsRNA的溶液中，或饲喂表达对应于目的基因的dsRNA的大肠杆菌，可以向下调节基因的表达。C.elegans细胞具有从其胞外环境中摄入dsRNA的能力。已经报道：mRNA是该ds RNA介导的遗传干扰的靶(Montgomery和Fire 1998)。也提示了，在可以发生翻译之前，所述靶RNA在细胞核中被降解。虽然RNAi介导的基因表达的降低可以传递至下一代，但可遗传性差，在更远的后代期间该效应迅速丧失。这可能是由于dsRNA库的持续减少。我们在这里提出一种方法，来构建具有永久性的、可遗传的RNAi表型的C.elegans系。该方法包括：通过将含有义方向和反义方向靶基因cDNA片段的质粒引入并置于蠕虫启动子的控制之下，或通过从单一构成物转录反向重复的所述cDNA，产生转基因C.elegans系。或者，可以在表达T7聚合酶的C.elegans株系中，从含有邻接两个T7启动子的cDNA的载体转录dsRNA。结果是具有可遗传的稳定的“假失效”表型的转基因蠕虫。所述cDNA或T7聚合酶的表达可以是普遍型的和组成型的，但也可以在组织特异性启动子下而被调节。与通过外来ds RNAi(注射的、浸泡的或饲喂的)诱导的RNAi对比，该方法使人们能够获得基因表达的有条件的、组织特异性抑制。
通过RNA干扰抑制unc-22表达导致“痉挛(twicthing)”表型
将unc 22 cDNA(外显子22)以有义和反义方向克隆到pPD103.05中。(A.Fire nr L2865)含有能够在所有组织中表达RNA序列的let 858启动子。将所得的质粒命名为pGN205(图19a)和pGN207(图19b)。将这些构成物与一种选择标记(rol-6；GFP)一起引入到C.elwgans中。转基因F1个体(表达rol-6或GFP)表现出“痉挛”表型，表明RNAi可以通过从转基因DNA内源转录的RNA来介导。RNAi表型与所述选择标记共分离到更远的后代。这导致具有永久性RNAi表型的C.elegans系的产生。
稳定系T7 RNA聚合酶系的产生和双转基因蠕虫的产生
在C.elegans中基于外源RNA聚合酶的一种表达系统要求两种质粒。一种质粒编码在特异性启动子控制之下的RNA聚合酶，而另一种质粒编码在T7启动子调节下表达的DNA片段。对于也称为假稳定失效的半稳定RNAi而言，将目的DNA克隆到两个T7启动子之间，使得可以产生dsRNA。
因为已知T7 RNA聚合酶表达系统是一种高效表达系统，所以这将导致产生双转基因动物的问题。如果要在C.elegans线虫中表达的基因有毒性，则这将导致致死效应，并因此导致在构建出的C.elegans中没有目的基因的高度调节的稳定表达。如果目的基因对于所述生物的生存是必不可少的，则具有得自该基因的DNA片段的RNAi也将导致致死效应，使得不可能假稳定失效。
为了解决这一问题，本发明人已经设计出一种由两种转基因动物组成的系统。第一种动物为T7 RNA聚合酶的转基因动物，正如前述实施例中所表明的，这种T7 RNA聚合酶可以在所有细胞或特定细胞或组织中表达。第二种转基因动物是目的DNA片段的转基因动物。这可以是连接于T7启动子的基因或cDNA，或者如果人们想要进行RNAi，则它可以是克隆在两个T7启动子之间的这样的基因的DNA片段。
这两种转基因动物均是活的，并且不表现出任何异常表型。这是因为在第一种转基因生物中表达的T7 RNA聚合酶即使以相对高水平表达，也对该生物无毒性。在第二种转基因生物中，不表达目的基因，或者不产生所述dsRNA，因为这些转基因动物不含有T7 RNA聚合酶。
通过让这两种转基因动物交配，可以获得目的基因或目的cDNA的表达、或具有DNA片段的RNAi。这些转基因动物的后代是双转基因动物，并且表达目的基因，或表达目的DNA片段的dsRNA。为了在这种交配中产生足够的雄性动物，其中一种所述转基因雄性动物可以是具有适于产生雄性的表型的C.elegans突变体。这样一种突变体的实例是him-5。将优选用这样一种突变体来产生T7 RNA聚合酶的转基因C.elegans，而雌雄同体动物含有在T7启动子调节下的所述DNA片段。
为了有效选择所述双转基因后代，可以将一种第二转基因引入到所述第二种转基因动物中。这种转基因含有一个在T7启动子调节下的报道基因。所述报道基因可以是GFP、荧光素酶、β半乳糖苷酶或β-内酰胺酶。这种转基因的实例是载体pGB400和pGN401。
为了在C.elegans中获得转基因的可诱导的组织特异性表达，我们可以产生这样的雄性原种(即him-5)：所述雄性原种(即him-5)携带使得能够进行例如组织特异性表达的不同C.elegans启动子控制下的T7聚合酶构成物。可以使这种雄性动物与携带在T7启动子控制下的目的基因的雌雄同体交杂。
此外，可以将所述转基因整合到动物的基因组中。已经描述了导致质粒稳定地整合到动物基因组中的方法(Methods in cell biology，第48卷，1995，epstein和Shakes编辑，Academic Press)，所述方法涉及对所述动物辐照。可以对两种动物辐照，但优选对表达T7 RNA聚合酶的动物进行这种处理。这产生一群在不同启动子控制下稳定表达T7RNA聚合酶的C.elegans线虫，这样的启动子的实例是myo-2(咽表达)、myo-3(体壁肌)、dgl-15(阴户肌)、unc-119(全身神经元)、SERCA(肌肉)、let858(所有细胞)、ges-1(肠)。
RNAi T7启动子酵母双杂种载体的构建
具有正向和反向T7T3和或Sp6的pGAD424
在大多数双杂种实验中，用已经进行工程改造的、在多接头中具有额外限制位点(诸如Ncol位点)的质粒pGAD424(图16)(Clontech)，克隆一个cDNA文库。该文库便于在酵母双杂种实验中筛选结合蛋白。我们构建了一种新的酵母双杂种载体，该载体具有完成酵母双杂种的同样的可能性，但它含有两个额外的T7启动子，使得该载体可以用于T7 RNA聚合酶诱导的假稳定失效。为此，通过用一个T7接头(由以下引物aattcttaatacgactcactatagggcc和catgggccctatagtgagtcgtattaag构成)，我们将一个正向T7插入pGAD424的EcoRI-NcoI位点中。我们将所得的载体命名为pGAD424-without-FULL-ICE-both-T7。小心地去除终止密码子并使用最大多接头可匹配的氨基酸。对于反向T7(由两种引物gatccgtcgacagatctccctatagtgagtcgtattactgca和gtaatacgactcactatagggagatctgtcgacg构成)，我们采用同一策略并使用BamH1和Pat1。为了避免丧失SalI，我们在所述引物中包括了这一位点。
所述SalI位点是重要的，因为大多数文库均在该位点克隆，连接物也买得到。这使得新构建的载体与现有的载体相容。
具有正向和反向T7/T3和或Sp6的pAS2
基于pAS2(Clontech)构建了一种类似的酵母双杂种载体。通过用EcoRV部分消化，我们能够去除cyh2基因相当大的部分。可以分离出正确的构成物，并通过用BglII限制消化进行检查，该位点存在于被去除的PAS2的EcoRV片段中。这消除了略有毒性并涉及生长延迟的cyh2基因。该基因对于进行RNAi和酵母双杂种实验不是必不可少的。在消除EcoR片段后，位于编码GAL4DB和HA(表位)的DNA序列之间的EcoRI限制位点成为该载体唯一的EcoRI限制位点，可以用来用含接头的T7启动子取代HA。这确保所有的限制位点存留，允许既符合读框地克隆，也与先前的载体和pAD424相容。我们使用了以下接头(引物：aattcttaatacgactcactatagggca和tatgccctatagtgagtcgtattaag)，使用EcoRI和NdeI克隆位点。我们采用同一策略用于反向T7(引物：gatccgtcgacagatctccctatagtgagtcgtattactgcacatgggccctatagtgagtcgtattaag和gtaatacgactcactatagggagatctgtcgacg)，使用BamH1和Pst1。为了避免丧失Sal1，我们在所述引物中包括了该位点。将所得的载体命名为pAS2-cyh2-HA+both T7-final。
在PGAD424中具有T7启动子(或者T3，或SP6启动子)，使得可以很快地从相互作用蛋白转变至RNAi，并将功能确定至所分离的DNA片段。另一优点是具有这样一种能力，即能够通过与体外翻译偶联的体外转录(有一个ATG或者GAL4DB或者GAL4AD的读框符合)，制备可以用于体外控制(例如摧毁(pull down)测定)实际蛋白-蛋白相互作用的标记蛋白。
在以下提供的序列表中确定了所产生的质粒序列和SP6和T3聚合酶的序列。
SP6 DNA依赖性RNA聚合酶：                              SEQENCE ID NO.1
swissprot登录号P06221
蛋白序列：
  1 mqdlhaiqlq leeemfnggi rrfeadqqrq iaagsesdta wnrrllseli apmaegiqay
 61 keeyegkkgr apralaflqc venevaayit mkvvmdmlnt datlqaiams vaeriedqvr
121 fskleghaak yfekvkkslk asrtksyrha hnvavvaeks vaekdadfdr weawpketql
181 qigttlleil egsvfyngep vfmramrtyg gktiyylqts esvgqwisaf kehvaqlspa
241 yapcvipprp wrtpfnggfh tekvasrirl vkgnrehvrk ltqkqmpkvy kainalqntq
301 wqinkdvlav ieevirldlg ygvpsfkpli dkenkpanpv pvefqhlrgr elkemlspeq
361 wqqfinwkge carlytaetk rgsksaavvr mvgqarkysa fesiyfvyam dsrsrvyvqs
421 stlspqsndl gkallrfteg rpvngvealk wfcinganlw gwdkktfdvr vsnvldeefq
481 dmcrdiaadp ltftqwakad apyeflawcf eyaqyldlvd egradefrth lpvhqdgscs
541 giqhysamlr devgakavnl kpsdapqdiy gavaqvvikk nalymdadda ttftsgsvtl
601 sgtelramas awdsigitrs ltkkpvmtlp ygstrltcre svidyivdle ekeaqkavae
661 grtankvhpf eddrqdyltp gaaynymtal iwpsisevvk apivamkmir qlarfaakrn
721 eglmytlptg fileqkimat emlrvrtclm gdikmslqve tdivdeaamm gaaapnfvhg
781 hdashliltv celvdkgvts iavihdsfgt hadntltlrv alkgqmvamy idgnalqkll
841 eehevrwmvd tgievpeqge fdlneimdse yvfa
T3DNA依赖性RNA聚合酶：                                SEQENCE ID NO.2
swissprot登录号P07659
蛋白序列：
  1 mniieniekn dfseielaai pfntladhyg salakeqlal ehesyelger rflkmlerqa
 61 kageiadnaa akpllatllp klttrivewl eeyaskkgrk psayaplqll kpeasafitl
121 kvilasltst nmttiqaaag mlgkaiedea rfgrirdlea khfkkhveeq lnkrhgqvyk
181 kafmqvvead migrgllgge awsswdkett mhvgirliem liestglvel qrhnagnags
241 dhealqlaqe yvdvlakrag alagispmfq pcvvppkpwv aitgggywan grrplalvrt
301 hskkglmrye dvympevyka vnlaqntawk inkkvlavvn eivnwkncpv adipslerqe
361 lppkpddidt neaalkewkk aaagiyrldk arvsrrisle fmleqankfa skkaiwfpyn
421 mdwrgrvyav pmfnpqgndm tkglltlakg kpigeegfyw lkihgancag vdkvpfperi
481 afiekhvddi lacakdpinn twwaeqdspf cflafcfeya gvthhglsyn cslplafdgs
541 csgiqhfsam lrdevggrav nllpsetvqd iygivaqkvn eilkqdaing tpnemitvtd
601 kdtgeisekl klgtstlaqq wlaygvtrsv tkrsvmtlay gskefgfrqq vlddtiqpai
661 dsgkglmftq pnqaagymak liwdavsvtv vaaveamnwl ksaakllaae vkdkktkeil
721 rhrcavhwtt pdgfpvwqey rkplqkrldm iflgqfrlqp tintlkdsgi dahkqesgia
781 pnfvhsqdgs hlrmtvvyah ekygiesfal ihdsfgtipa dagklfkavr etmvityenn
841 dvladfysqf adqlhetqld kmpplpkkgn lnlqdilksd fafa
pGN108：                                                          SEQUENCE ID NO.3
gttgtcgtaaagagatgtttttattttactttacaccgggtcctctctctctgccagcacagctcagtgttggctgtgtgctcgggctcctgccaccggcgg
cctcatcttcttcttcttcttctctcctgctctcgcttatcacttcttcattcattcttattccttttcatcatcaaactagcatttcttactttatttatttttttcaattttca
attttcagataaaaccaaactacttgggttacagccgtcaacagatccccgggattggccaaaggacccaaaggtatgtttcgaatgatactaacataa
catagaacattttcaggaggacccttgcttggagggtaccggatgactgctccaaagaagaagcgtaagctcatgaacacgattaacatcgctaagaa
cgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgag
tcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcact
accctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaag
aaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgc
aatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaag
cgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcncgtggca
taaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtagg
tcaagactctgagactatcgaactcgacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttg
cgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagc
actgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcg
gtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgac
atgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaagacaaggctcgcaagtctcgccgtatcagccttgagttcatg
cttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggttcgtgtttacgctgtgtcaatgttcaacccgc
aaggtaacgatatgaccaaaggacgtcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactg
tgcgggtgtcgataaggtttcgtttcctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacac
ttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgc
tggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgt
tcaggacatctacgggattgttgctaagaaagtcaacgagattctgcaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgat
gagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagc
gttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtc
tgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatccgtgagcgtgacggtggtagctgcggttgaagcaatgaac
tggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactgagagattcttcgcaagcgttgcgctgtgcattgggtaactcct
gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacacca
acaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtg
tgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgc
gaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagc
acttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaagggcccactagtcggccgtacgggccctttcgtct
cgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagc
ccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgt
gaaataccgcacagatgcgtaaggagaaaataccgcatcaggcggccttaagggcctcgtgatacgcctatttttggttaatgtcatgataataat
ggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaat
aaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttt
tgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc
cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagca
actcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatg
cagtgctgccataaccatgagtggataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgg
gggatcatgtaactcgccttgatcgttggggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaa
caacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttc
tgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggt
aagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaa
gcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctca
tgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctg
ctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagc
gcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctcatacctcgctctgctaatcctgttac
cagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggg
gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaaggg
agaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtc
ctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttac
ggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctc
gccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccg
attcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccca
ggctttacactttatgcttccggcccgtatgttgtgtggaattgtgagcggataacaacttcacacaggaaacagctatgaccatgattacgccaagctgt
aagtttaaacatgatcttactaactaactattctcatttaaattttcagagcttaaaaatggctgaaaccactcacaacgatggatacgctaacaacttggaa
atgaaataagcttgcatgcctgcagagcaaaaaaatactgcttttccttgcaaaattcggtgctttcttcaaagagaaacttttgaagtcggcgcgagcat
ttccttctttgacttctctctttccgccaaaaagcctagcatttttattgataatttgattacacacactcagagttcttcgacatgataaagtgtttcattggcac
tcgccctaacagtacatgacaagggcggattattatcgatcgatattgaagacaaactccaaatgtgtgctcattttggagccccgtgtggggcagctg
ctctcaatatattactagggagacgaggagggggaccttatcgaacgtcgcatgagccattctttcttctttatgcactctcttcactctctcacacattaat
cgattcatagactcccatattccttgatgaaggtgtgggtttttagctttttttcccgatttgtaaaaggaagaggctgacgatgttaggaaaaagagaacg
gagccgaaaaaacatccgtagtaagtcttccttttaagccgacactttttagacagcattcgccgctagttttgaagtttaaattttaaaaaataaaaattag
tttcaattttttttaattactaaataggcaaaagttttttcaagaactctagaaaaactagcttaattcatgggtactagaaaaattcttgttttaaatttaatattta
tcttaagatgtaattacgagaagcttttttgaaaattctcaattaaaagaatttgccgatttagaataaaagtcttcagaaatgagtaaaagctcaaattaga
agtttgtttttaaaggaaaaacacgaaaaaagaacactatttatcttttcctccccgcgtaaaattagttgttgtgataatagtgatccgctgtctatttgcact
cggctcttcacaccgtgcttcctctcacttgacccaacaggaaaaaaaaacatcacgtctgagacggtgaattgccttatcaagagcgtcgtctctttca
cccagtaacaaaaaaaatttggtttctttactttatatttatgtaggtcacaaaaaaaaagtgatgcagttttgtgggtcggttgtctccacaccacctccgc
ctccagcagcacacaatcatcttcgtgtgttctcgacgattccttgtatgccgcggtcgtgaatgcaccacattcgacgcgcaactacacaccacactc
actttcggtggtattactacacgtcatcgttgttcgtagtctcccgctctttcgtccccactcactcctcattattccccttggtgtattgattttttttaaatggta
caccactcctgacgtttctaccttcttgttttccgtccatttagattttatctggaaatttttttaaaattttaggccagagagttctagttcttgttctaaaagtcta
ggtagacatacattttctatttctcatcaaaaaaaaagttgataaagaaaactggttattcagaaagagtgtgtctcgttgaaattgattcaaaaaaaaatt
cccacccctcgcttgtttctcaaaatatgagatcaacggattttttccttctcgattcaattttttgctgcgctctgtctgccaaagtgtgtgtgtccgagcaaa
agatgagagaatttacaaacagaaatgaaaaaaagttggccaaataatgaagttttatccgagattgatgggaaagatattaatgttctttacggtttgga
ggggagagagagatagattttcgcatcaaactccgccttttacatgtcttttagaatctaaaatagatttttctcatcatttttaatagaaaatcgagaaatta
cagtaatttcgcaattttcttgccaaaaatacacgaaatttgtgggtctcgccacgatctcggtcttagtggttcatttggtttaaaagtttataaaatttcaaa
ttctagtgtttaatttccgcataattggacctaaaatgggtttttgtcatcattttcaacaagaaatcgtgaaaatcctgttgtttcgcaattttcttttcaaaaata
cacgaaatatatggtaatttcccgaaatattgagggtctcgccacgatttcagtcacagtggccaggatttatcacgaaaaaagttcgcctagtctcacat
ttccggaaaaccgaatctaaattagttttttgtcatcattttgaacaaaaaatcgagacatccctatagtttcgcaattttcgtcgcttttctctccaaaaatga
cagtctagaattaaaattcgctggaactgggaccatgatatcttttctccccgtttttcattttattttttattacactggattgactaaaggtcaccaccaccg
ccagtgtgtgccatatcacacacacacacacacacaatgtcgagattttatgtgttatccctgcttgatttcgttccgttgtctctctctctctattcatcttttg
agccgagaagctccagagaatggagcacacaggatcccggcgcgcgatgtcgtcgggagatggcgccgcctgggaagccgccgagagatatca
gggaagatcggtctgatttctcctcggatgccacctcatctctcgagtttctccgcctgttactccctgccgaacctgatatttccc
pGN105：                                                         SEQUENCE ID NO.4
aagcttgcatgcctgcaggccttggtcgactctagacacttttcagctacctagatacatggatatccccgcctcccaatccacccacccagggaaaaaa
gaagggctcgccgaaaaatcaaagttatctccaggctcgcgcatcccaccgagcggttgacttctctccaccacttttcattttaaccctcggggtacg
ggattggccaaaggacccaaaggtatgtttcgaatgatactaacataacatagaacattttcaggaggacccttgcttggagggtaccgagctcagaa
aaaatgactgctccaaagaagaagcgtaaggtaccggtaatgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatccc
gttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaa
gatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaa
cgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatca
ccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcg
cttcggtcggtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcattta
tgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgct
gcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacct
gaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcatt
actggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcct
gaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcat
tgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaa
cgtgctgccgctgctgtgtaccgcaaggacagggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccat
aaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgccgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgctt
acgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctg
agcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttct
gcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatc
cagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgagaccgttcaggacatctacgggattgttgctaag
aaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaa
gtcaagctgggcactaaggcactggctggtcaatggctggctcacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtc
caaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtccgatgttcactcagccgaatcaggctg
ctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagtcgctgg
ctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaataca
agaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacaca
aacaggagtctggtatcgctcctaactttgtacacagccaagacggttagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcga
atcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtctt
gtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacct
ccgtgacatcttagagtcggacttcgcgttcgcgtaagaattccaactgagcgccggtcgctaccattaccaacttgtctggtgtcaaaaataataggg
gccgctgtcatcagagtaagtttaaactgagttctactaactaacgagtaatatttaaattttcagcatctcgcgcccgtgcctctgacttctaggtccaatt
actcttcaacatccctacatgctctttctccctgtgctcccaccccctatttttgttattatcaaaaaaacttcttcttaatttctttgttttttagcttcttttaagtca
cctctaacaatgaaattgtgtagattcaaaaatagaattaattcgtaataaaaagtcgaaaaaaattgtgctccctccccccattaataataattctatccca
aaatctacacaatgttctgtgtacacttcttatgttttttttacttctgataaattttttttgaaacatcatagaaaaaaccgcacacaaaataccttatcatatgtt
acgtttcagtttatgaccgcaatttttatttcttcgcacgtctgggcctctcatgacgtcaaatcatgctcatcgtgaaaaagttttggagtatttttggaattttt
caatcaagtgaaagtttatgaaattaattttcctgcttttgctttttgggggtttcccctattgtttgtcaagagttcgaggacggcgtttttcttgctaaaatca
caagtattgatgagcacgatgcaagaaagatcggaagaaggtttgggtttgaggctcagtggaaggtgagtagaagttgataatttgaaagtggagta
gtgtctatggggtttttgccttaaatgacagaatacattcccaatataccaaacataactgtttcctactagtcggccgtacgggccctttcgtctcgcgcg
tttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtca
gggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaata
ccgcacagatgcgtaaggagaaaataccgcatcaggcggccttaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttctt
agacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct
gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctca
cccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgag
agttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccaggcaagagcaactcg
gtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtg
ctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggat
catgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacg
ttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgct
cggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccc
tcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattg
gtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgacca
aaatcccttaacgtgagtttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgctt
gcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcaga
taccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtg
gctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcg
tgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaa
aggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtc
gggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagccttggaaaaacgccagcaacgcggcctttttacggttc
ctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccg
cagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattca
ttaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctt
tacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctgtaagttt
aaacatgatcttactaactaactattctcatttaaattttcagagcttaaaaatggctgaaatcactcacaacgatggatacgctaacaacttggaaatgaa
at
pGN400：                                                             SEQUENCE ID NO.5
aagcttgcatgcctgcaggccttggtcgactctagacacttttcagctacctagatacatggatatccccgcctcccaatccacccacccagggaaaaa
gaagggctcgccgaaaaatcaaagttatctccaggctcgcgcatcccaccgagcggtttgacttctctccaccacttttcattttaaccctcggggtacg
ggattggccaaaggacccaaaggtatgtttcgaatgatactaacataacatagaacattttcaggaggacccttgcttggagggtaccgagctcccgg
gattaatacgactcactataccggtagaaaaaatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatg
ggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatgg
gtaagtttaaacatatatatactaactaaccctgattatttaaattttcagccaacacttgtcactactttctgttatggtgttcaatgcttctcgagatacccag
atcatatgaaacggcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacac
gtaagtttaaacagttcggtactaactaaccatacatatttaaattttcaggtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaa
aggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaat
ggaatcaaagttgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagaacttcaaaattagacacaacattgaagatggaagcgttca
actagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagtacaaccattacctgtccacacaatctgccctttcgaaagatc
ccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaatagcattcgtagaattc
caactgagcgccggtcgctaccattaccaacttgtctggtgtcaaaaataataggggccgctgtcatcagagtaagtttaaactgagttctactaactaa
cgagtaatatttaaattttcagcatctcgcgcccgtgcctctgacttctaagtccaattactcttcaacatccctacatgctctttctccctgtgctcccaccc
cctatttttgttattatcaaaaaaacttcttcttaatttctttgttttttagcttcttttaagtcacctctaacaatgaaattgttgtagattcaaaaatagaattaatttcg
taataaaaagtcgaaaaaaattgtgctccctccccccattaataataattctatcccaaaatctacacaatgttctgtgtacacttcttatgttttttttacttctg
ataaattttttttgaaacatcatagaaaaaaccgcacacaaaataccttatcatatgttacgtttcagtttatgaccgcaatttttatttcttcgcacgtctgggc
ctctcatgacgtcaaatcatgctcatcgtgaaaaagttttggagtatttttggaatttttcaatcaagtgaaagtttatgaaattaattttcctgcttttgctttttg
ggggtttcccctattgtttgtcaagagtttcgaggacggcgtttttcttgctaaaatcacaagtattgatgagcacgatgcaagaaagatcggaagaagg
tttgggtttgaggctcagtggaaggtgagtagaagttgataatttgaaagtggagtagtgtctatggggtttttgccttaaatgacagaatacattcccaat
ataccaaacataactgtttcctactagtcggccgtacgggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccg
gagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggctta
actatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcggc
cttaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcgggggaaatgtgcgcggaacc
cctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtatt
caacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagtt
gggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaa
gttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcac
cagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctga
caacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagc
cataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggc
aacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccg
gtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactat
ggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaa
acttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccg
tagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg
atcaagagctaccaactcttttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttagggccaccactt
caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtgggcgataagtcgtgtcttaccgggttggactca
agacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactga
gatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggag
agcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtc
aggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatccc
ctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaag
cggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggc
agtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcgg
ataacaatttcacacaggaaacagctatgaccatgattacgccaagctgtaagtttaaacatgatcttactaactaactattctcatttaaattttcagagctt
aaaaatggctgaaatcactcacaacgatggatacgctaacaacttggaaatgaaat
pGN401                                                           SEQUENCE ID NO.6
gatcccggcgcgcgatgtcgtcgggagatggcgccgcctgggaagccgccgagagatatcagggaagatcgtctgatttctcctcggatgccacct
catctctcgagtttctccgcctgttactccctgccgaacctgatatttcccgttgtcgtaaagagatgtttttattttactttacaccgggtcctctctctctgcc
agcacagctcagtgttggctgtgtgctcgggctcctgccaccggcggcctcatcttcttcttcttcttctctcctgctctcgcttatcacttcttcatcattctt
attccttttcatcatcaaactagcatttcttactttatttatttttttcaattttcaattttcagataaaaccaaactacttgggttacagccgtcaacagatccccg
ggattggccaaaggacccaaaggtatgtttcgaatgatactaacataacatagaacattttcaggaggacccttgcttggagggtaccggtagaaaaa
atgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtga
aggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatgggtaagtttaaacatatatatactaactaaccct
gattatttaaattttcagccaacacttgtcactactttctgttatggtgttcaatgcttctcgagatacccagatcatatgaaacggcatgactttttcaagagt
gccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtaagtttaaacagttcggtactaactaaccat
acatatttaaattttcaggtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattctt
ggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttgtaagtttaaacttggacttac
taactaacggattatatttaaattttcagaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaa
ttggcgatggccctgtccttggaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttct
tgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaatagcattcgtagaattccaactgagcgccggtcgctaccattaccaac
ttgtctggtgtcaaaaataataggggccgctgtcatcagagtaagtttaaactgagttctactaactaacgagtaatatttaaattttcagcatctcgcgccc
gtgcctctgacttctaagtccaattactcttcaacatccctacatgctcttctccctgtgctcccaccccctatttttgttattatcaaaaaaacttcttcttaatt
tctttgttttttagcttcttttaagtcacctctaacaatgaaattgtgtagattcaaaaatagaattaattcgtaataaaaagtcgaaaaaaattgtgctccctcc
ccccattaataataattctatcccaaaatctacacaatgttctgtgtacacttcttatgttttttttacttctgataaattttttttgaaacatcatagaaaaaaccg
cacacaaaataccttatcatatgttacgtttcagtttatgaccgcaatttttatttcttcgcacgtctgggcctctcatgacgtcaaatcatgctcatcgtgaaa
aagttttggagtatttttggaatttttcaatcaagtgaaagtttatgaaattaattttcctgcttttgctttttgggggtttcccctattgtttgtcaagagtttcgag
gacggcgtttttcttgctaaaatcacaagtatttgatgagcacgatgcaagaaagatcggaagaaggtttgggtttgaggctcagtggaaggtgagtag
aagttgataatttgaaagtggagtagtgtctatggggtttttgccttaaatgacagaatacattcccaatataccaaacataactgtttcctactagtcggcc
gtacgggcccggtacccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgct
cacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgc
ccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcc
tcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggata
acgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccct
gacgagcatcaacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtg
cgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcag
ttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaa
cccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtg
gtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccgg
caaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacg
gggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaag
ttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccat
agttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccg
gctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgc
cgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttggttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttc
attcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagt
aagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtacctcaa
ccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtg
ctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttc
agcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaata
ctcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttc
cgcgcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggc
cgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtgga
ctccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaa
gcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaa
aggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtc
ccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaag
gcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattg
gagctccaccgcggtggcggccgctctagaactagtg
pGN110：                                                      SEQUENCE ID NO.7
gatcctccaaaatcgtcttccgctctgaaaaacgaaagtggacctttgacatccgaaaaaatgggcgaaaaaatgaaattgagctttttgggtcgaaaa
aaatgtttttagaatgctgagaacacgttaaacacgaagatcatatttattttgagacccggatgctctgaaaatgtctgacatagatttaaaaaagcatat
atatattttttcattttcaacgtgaaagttttgtgcaactttatagaatctcctattggcacattgttttttatttaactgaggcagtttttgaacacctttttgaaactt
tgaatctctttgaagtatactgtcgaaaagactgacttgagcgttcgaaatgccagaagaaaactatatttgaatctcgcgctaaattgagaaatgcaacc
gcgctccactggacaattggaaaaaaaatttattcggaggcgacaacggtattttcgaaattgattttctgtgtattttctcattttttataaattcttctttgattt
atcgttcgtttgtgagaaatttaattgtattcaaacttttttatagtaagataccggtggtaccgctagccgtacgaacccgggattggccaaaggaccca
aaggtatgtttcgaatgatactaacataacatagaacattttcaggaggacccttgcttggagggtaccggatgactgctccaaagaagaagcgttaagc
tcatgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagct
cgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcgga
taacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaag
cgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgaca
atacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaa
gaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtcta
ctcggtggcgaggcgtggtcttcgtggccataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttag
cttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcg
ctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcatttactggtggtggctattgggctaacggtcgtcgtcctct
ggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacacc
gcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaa
gaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaagacaaggctc
gcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcg
gttcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggacgtcttacgctggcgaaaggtaaaccaatcggtaaggaagg
ttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggtttcgtttcctgagcgcatcaagttcattgaggaaaaccacgagaacat
catggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagc
accacggcctgagctataactgctcccttccgctttcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtg
gtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctgcaagcagacgcaatcaatg
ggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggct
ggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagatac
cattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaacccgtgagcgt
gacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttc
gcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaactgatgttcctcgg
tcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagcca
agacggtagcccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggc
tgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccag
ttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaaggg
ccctcgttcgagtcggtcacaatcacctgaaactccaaaggcagccagtgaggaacgtgaagaagaagaaaaagagtcatctgaacaggtttgatttt
ctttctggtcaaaaagatgaaattattgattttcagccagatactcccaaaactagcagcgagaagtctgcaagtcgttcacagtcgcccagagaatcgc
gggaagtgagccaagaggtatgtttttcaaaaatcaataactgatcataatttttattgtttggtgaatttaagaaaataatattcgaaaattcctctgaattat
caagattgcagtattaatttcgagaaaaattgagatattcatagagctattgtaaattttcttgatttcagactgaaacttcggaaaatcaagagaaaatcaa
agaaaaggatgacggggatgatcagcctggcacaccgaacagctatagaagccgggaaacttcaccagctccaaaaaggtccaaggagaccag
gtttgtcaaaagcttcctgcgattaattctcatttcaatttttcagagaatcagagtctcctgaaaaatccccggttcgttcaagatctcccagaaggtcttca
gcacgttccccgtcacgatctcctagacggcgccgagaaagaagctcagaaagaaagcaatccgaagagccagcaccgctaccagagaaaaaga
agaaagagccgctggatattctacgaacaagaaccggaggagcatatattccacccgccaaacttcgacttatgcaacaacagattggtgataagca
aagtgaacagtatcagagaatgaattgggaaagaatgaagaaaaagattcacggattggttaacagagtcaacgcgaagaatcttgttcaaattgtca
gagaacttcttcaagagaatgtgattcgttcaaagtgagtgagaaaatcgaaggaaaaggaaagaattaatttaaatttttcaggggacttctctgccgtg
acattattcaagctcaggctttctcaccaggattctctaacgtctatgcagctttggcggcagttatcaactcgaaattccctcatgtcggtgaacttcttctc
cgtcgtctgattgtacagttcaaaagaagtttccgtagaaatgacagaggcgtcacggtgaacgtgatcaaattcatcgcacatttgattaatcaacaag
ttgctcacgaagttcttgcgctggaaatcatgattctgatgcttgaagaaccaactgatgattcagttgaagtcgccattgcgttcctgaaagagtgtgga
gcaaagcttctggagattgctccagcagctcttaacagtgtctacgaccgtcttcgtgcaattctcatggaaactgaaagatcggaaaatgcactggatc
gacgtattcagtatatgattgagactgcaatgcagattcgaaaggacaaatttgcggtaaggtagaatatataaatagtttattagaaaaaaataaattag
aataatttaaattcctactagccaatcaggcgacctttttgcgcatagttctattattgaaaaatttggagaatttctcatattctcgctcggaaatctggaatt
cgacgagatcttctggcttctgtgcagctgcatcgctttgtgctccctttctcgcttgtcttctgtgtacaccaagaaccttgttgagttcatcaactgaatct
gtgactggcttgttgctcactggatgcactagacgactgattctcgagaaatcagattgagttgcgattagggtgacctagaaattgggaataatacgaa
cttttgaaaatattcaggaggattaaaaaaattattctcgacaatcctacaaatttacttattgcaccatgttgctccaacatttttcattaaaagttaatgaaaa
aatgtagaaaatcggaaattggcaattttcagaccatttttaagcattttcaaaaaaaaattgcagctgaaataaatgtcattttcagataaatcgagcgatt
ttctgttgtctgacactagtttttagttttaaaaaatgttggaagaacatggtgcaataggtaatttcatagaatttccatgtgttttttttcaattaaccaattatc
caaatcttccaaactcacattttgcggagctgggctatcaagaatctgctgcagttttataagacgagcatctctgatatcactgaaaattaatttttaatca
aaacttgaatatcaactaaacccacttattaactttctcgatcttctgtcgttcggtacgatgacggtgaagaagccaattgtagtagttgatttggttcaagc
cctttcggtgttgtacgtcagtgtcctgcaatgctatttagttataacttaggcctaagattcaatttaatgaagtgattaaatttgttctctgaacctcttaaga
tgatcttttggattagaaacatataagacaggtttacctatctattaaaaaacagatcaaaatagatacgaccaaatcggataatccatgcctacctggcat
ctaggaacgtgttcttagaagatttcttacgtaatcgtatgaagaaataacaatttgatcgttggccagcaaaaatagggttttaagtgggatagtgtttttat
tagctaaccggaaaattttatagtttttttttgcaagaaaccactgaaaaccccctaattgtatacattttttggagcagcttctggtctttttgagcaataaaat
tcgataaaacagaatttaagtgtaaattgttcacatttagtttctattttatcaaattttgttgctcaaaaacattcgaagctgctctaaaaaaatgcattaaaaa
aggggttttcagtggtttttcacattaaaaaagctaattttaactaaaaatccatcatatttccaactttgtcacaacaataaaatgctggtcaaaatgtgttcg
aaaaaatgtttttttttttaatttttataatttaaaaatagttttctttcgctgggacacatacatttttgggcgtaaattttcagttcaaatttccatttttacaaccat
aatcataaagctacgtctgatctctctcgcacttacctgcgcctgattcgaaagaacaaccgtagccaaaagaacaagaagaacaagcacgtagttgt
ggtagtggacgttcatcacgcaatactgaccaatggtcgtggggtctcactttccgtactattgagagaggggagactgaagatggcaattgaggaca
gtgtcttcgacgcacgcatgcatccataagcataatccaggagggatggagagaaaaatcttgtttctaagcccctccctttgtaatacatacacatatct
aataccgaagaatggctaattgaatggacgtcagctgttgctgtagttgccaaggcatcatcgatgaaataactgaaagaaagaattaaataattattgc
aggcgtatccggcggtcattgaagacttggacttgattgaggaggaggatcagatcatccatacacttaatttggaggatgcggttgatccggaaaatg
ggcttagtaagtgactgaccacacgcggggggcattaatttaataaattgaattccatttcagatgtgttcaaactagatccagaattcgaaaagaacga
ggaggtttatgaggagatccgtaaggaaatcattggaaacgccgatatttcggatgaggatggtggcgacgagttggatgatgaagaagagggtagt
gatgtggaagaggctccgaagaagactacagagattattgataatactgatcagaattgactgctttcagaaggtattcattttgagttttgggccggcaa
atctgtaagttgccggttgccgaaaatttgctgaatttgccggaaaaaaaaattccggaatttatttaaaaactttttgtaaaaattaaattaaatttgcaactt
ttcagagaagtctacctgacaatgcaatcatctttggactaccaagaagctgctcacaaattgctgaaaatgaagattccagacagcatgcaggtcagc
gatgttgcaaagaaaaattttcgaccaaaaaaaccaaccaatcataaaatttaaaaaaaaactccgtttttttctttttttttatacgagaaaaaccaaaaaa
atgtatttttgccaaattctaaaatactatccccgaaattttcaatattttctctttcagaacgaactctgcgcgatgcttgtcgattgttgtgctcaacagcgta
cctacgagcgattctacggaatgctcatcgaacgtttctgccgacttcgcctcgaataccagcaatactttgaaaagctctgccaggacacgtattccac
gattcaccgaattgacatcacaaaactgcggaatttggctcgccttattgctcatttgctctcgacggatgctattgactggaagattttggccgatatgaa
aatgaccgaagaggacacaacttcttctggcagaatctatattaaatatatatttaatgaacttgtggaggcgatgggaatggttaacttcattcgagag
ttactgatccgtgagtttcctagagagagttgttttcgtattcaattttccctattttcagaactttggctcattgctttgttggattattcccacgaactaatccg
aacagcgcacgattttcgatcaacttcttcacaatgattggattgggtggtttgacgttggaacttcgtgaatggctggcaaagggtctcaagaagaaga
agggaatgctggatcagttgaaggccgaatcaagctcagattcatcgtcgtcttcggattcgtcagactcgtctgattcttcggattctgacgattcatcc
gactcgtcttcagattcctcatcttcttcagaatcagagccagaaccaccgaagaaaaagaagaagaagaacagtgaagagagttccaaaaagaag
gaaaaagagaatattggtcgacgggatcgtggatacaagagagctgaacgtcatcgtgatcaaagtgtggagaacaaggacaaggatcgtcgacg
tcgccaggattctgacgaaaatcgtcggccagaacgaggagatgaccgcaaggatcggagtaaagatcgtcgtcgtcaagactcggatgatgagg
atcggaaaggtcgtgaacgtcgggaagattcaggggaaagacgtcgcggagatcgggatcgacgtgatcgaaacaaggatcaggaggatcaccg
tgaagatcgccgtgaccgaagcaaggatcgtgaggatcgacgtgatcgccgtcgtcatgactctgatgatgatcgtaaaactcgtcgggatagaagt
gaagagcgaggaggacgtcgtcgtgaagtggaatcggatgatcgacgccgacgtcgttgaattttcaaattttaaatactgaatatttgttttttttcctatt
atttatttattctctttgtgttttttttcttgctttctaaaaattaattcaatccaaatctaaacatgagcggttttttttctctttccgtctcccaattcgtattccgct
cctctcatctgaacacaatgtgcaagtttatttatcttctcgctttcatttcattaggacgtggggggaattggtggaagggggaaacacacaaaaggatg
atggaaatgaaataaggacacacaatatgcaacaacattcaattcagaaatatggaggaaggtttaaaagaaaacataaaaatatatagaggaggaa
ggaaaactagtaaaaaataagcaaagaaattaggcgaacgatgagaattgtcctcgcttggcaaatgcgaatccgtatggagaggcacgtttggcga
aggcaaatgttcggtatggagatctgtaaaaatttttaagttgaaatttggtgttgctcttttacaaaattttccgattttcgcttgaaattacggtgccaggtct
cgacacgtcttccaatttttcaaattcaaaagagcctttaatgggctgtagttgctaatttctcgtttttgaaaatttttcttccgtttaatcgaaatttgatgtattt
tatttatgattttcaataaatttcaaagaaactggtgaaaactcggaaaattgtgaactacagtaatccaatccttaaaggcgcacaccttttaaatgtccgc
cccaatacgatatttttttaagattcgctagagcggccgccaccgcggtggagctccaattcgccctatagtgagtcgtattacaattcactggccgtcgt
tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccccttcgccagctggcgtaatagcgaagaggccc
gcaccgatcgcccttcccaacagttgcgtagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgc
gcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctcta
aatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccct
gatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattctttt
gatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttca
ggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgc
ttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaac
gctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgcc
ccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcat
acactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataag
catgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctttttttcacaacatgggggatcatgtaactcg
ccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaact
attaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttcc
ggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgt
agttatctacacgacgggcagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcag
accaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaa
cgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaa
aaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaaactggcttcagcagagcgcagataccaaatact
gtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccag
tggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcc
cagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacagg
tatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggggaacgccttggtatctttatagtcctgtcgggtttcgccac
ctctgacttgagcgtcgatttttgtgatgctcgtcaggggggccgagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgct
ggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacga
ccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctg
gcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttacctcactcattaggcaccccaggctttacactttatgct
tccggctcctatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctcggaattaaccctcactaa
agggaacaaaagctgggggg
pAS2-cyh2-HA+bothT7-final                                             SEQUENCE ID NO.8
gatccgtcgacagatctccctatagtgagtcgtattactgcagccaagctaattccgggcgaatttcttatgatttatgatttttattattaaataagttataaaaaaa
ataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttgttcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggt
cgctcttattgaccacacctctaccggcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatac
gagccggaagcataaagtgtaaagcctggggtgcctaatgagtgaggtaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgt
cgtgccagctggattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcg
ttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcc
agcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagag
gtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtc
cgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaac
cccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaaca
ggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactaacggctacactagaaggacagtatttggtatctgcgctctgct
gaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgc
agaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattacaaa
aaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggc
acctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgc
aatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatcc
gcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtca
cgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcct
ccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgac
tggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaac
tttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactg
atcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaat
actcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgc
gcacatttccccgaaaagtgccacctgaacgaagcatctgtgcttcattttgtagaacaaaaatgcaacgcgagagcgctaatttttcaaacaaagaatctga
gctgcatttttacagaacagaaatgcaacgcgaaagcgctattttaccaacgaagaatctgtgcttcatttttgtaaacaaaaatgcaacgcgagagcgctaa
tttttcaaacaaagaatctgagctgcatttttacagaacagaaatgcaacgcgagagcgctattttaccaacaaagaatctatacttcttttttgttctacaaaaatg
catcccgagagcgctatttttctaacaaagcatcttagattacttttttctcctttgtgcgctctataatgcagtctcttgataactttttgcactgtaggtccgttaag
gttagaagaaggctactttggtgtctattttctcttccataaaaaaagcctgactccacttcccgcgtttactgattactagcgaagctgcgggtgcattttttcaag
ataaaggcatccccgattatattctataccgatgtggattgcgcatactttgtgaacagaaagtgatagcgttgatgattcttcattggtcagaaaattatgaacg
gtttcttctattttgtctctatatactacgtataggaaatgtttacattttcgtattgttttcgattcactctatgaatagttcttactacaatttttttgtctaaagagtaatac
tagagataaacataaaaaatgtagaggtcgagtttagatgcaagttcaaggagcgaaaggtggatgggtaggttatatagggatatagcacagagatatata
gcaaagagatacttttgagcaatgtttgtggaagcggtattcgcaatattttagtagctcgttacagtccggtgcgtttttggttttttgaaagtgcgtcttcagagc
gcttttggttttcaaaagcgctctgaagttcctatactttctagagaataggaacttcggaataggaacttcaaagcgtttccgaaaacgagcgcttccgaaaat
gcaacgcgagctgcgcacatacagctcactgttcacgtcgcacctatatctgcgtgttgcctgtatatatatatacatgagaagaacggcatagtgcgtgtttat
gcttaaatgcgtacttatatgcgtctatttatgtaggatgaaaggtagtctagtacctcctgtgatattatcccattccatgcggggtatcgtatgcttccttcagca
ctaccctttagctgttctatatgctgccactcctcaattggattagtctcatccttcaatgctatcatttcctttgatattggatcatattaagaaaccattattatcatga
cattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca
cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcag
agcagattgtactgagagtgcaccatagatcaacgacattactatatatataatataggaagcatttaatagacagcatcgtaatatatgtgtactttgcagttatg
acgccagatggcagtagtggaagatattctttattgaaaaatagcttgtcaccttacgtacaatcttgatccggagcttttctttttttgccgattaagaattaattcg
gtcgaaaaaagaaaaggagagggccaagagggagggcattggtgactattgagcacgtgagtatacgtgattaagcacacaaaggcagcttggagtatg
tctgttattaatttcacaggtagttctggtccattggtgaaagtttgcggcttgcagagcacagaggccgcagaatgtgctctagattccgatgctgacttgctg
ggtattatatgtgtgcccaatagaaagagaacaattgacccggttattgcaaggaaaatttcaagtcttgtaaaagcatataaaaatagttcaggcactccgaa
atacttggttggcgtgtttcgtaatcaacctaaggaggatgttttggctctggtcaatgattacggcattgatatcgtccaactgcatggagatgagtcgtggca
agaataccaagagttcctcggtttgccagttattaaaagactcgtatttccaaaagactgcaacatactactcagtgcagcttcacagaaacctcattcgtttatt
cccttgtttgattcagaagcaggtgggacaggtgaacttttggattggaactcgatttctgactgggttggaaggcaagagagccccgaaagcttacattttat
gttagctggtggactgacgccagaaaatgttggtgatgcgcttagattaaatggcgttattggtgttgatgtaagcggaggtgtggagacaaatggtgtaaaa
gactctaacaaaatagcaaatttcgtcaaaaatgctaagaaataggttattactgagtagtatttatttaagtattgtttgtgcacttgccgatctatgcggtgtgaa
ataccgcacagatgcgtaaggagaaaataccgcatcaggaaattgtaaacgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaacc
aataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgt
ggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaag
cactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggag
cgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcgcgccattcg
ccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctatacgccagctggcgaaagggggatgtgctgcaaggcgattaagtt
gggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtcgtccaagctttcgcgagctcgagatcccgagctttgcaaattaaagccttc
gagcgtcccaaaaccttctcaagcaaggttttcagtataatgttacatgcgtacacgcgtctgtacagaaaaaaaagaaaaatttgaaatataaataacgttctt
aatactaacataactataaaaaaataaatagggacctagacttcaggttgtctaactccttccttttcggttagagcggatgtggggggagggcgtgaatgtaa
gcgtgacataactaattacatgatatccttttgttgtttccgggtgtacaatatggacttcctcttttctggcaaccaaacccatacatcgggattcctataatacctt
cgttggtctccctaacatgtaggtggcggaggggagatatacaatagaacagataccagacaagacataatgggctaaacaagactacaccaattacactg
cctcattgatggtggtacataacgaactaatactgtagccctagacttgatagccatcatcatatcgaagtttcactaccctttttccatttgccatctattgaagta
ataataggcgcatgcaacttcttttctttttttttcttttctctctcccccgttgttgtctcaccatatccgcaatgacaaaaaaaatgatggaagacactaaaggaaa
aaattaacgacaaagacagcaccaacagatgtcgttgttccagagctgatgaggggtatcttcgaacacacgaaactttttccttccttcattcacgcacacta
ctctctaatgagcaacggtatacggccttccttccagttacttgaatttgaaataaaaaaagtttgccgctttgctatcaagtataaatagacctgcaattattaatc
ttttgtttcctcgtcattgttctcgttccctttcttccttgtttctttttctgcacaatatttcaagctataccaagcatacaatcaactccaagcttgaagcaagcctcct
gaaagatgaagctactgtcttctacgaacaagcatgcgatatttgccgacttaaaaagctcaagtgctccaaagaaaaaccgaagtgcgccaagtgtctga
agaacaactgggagtgtcgctactctcccaaaaccaaaaggtctccgctgactagggcacatctgacagaagtggaatcaaggctagaaagactggaaca
gctatttctactgatttttcctcgagaagaccttgacatgattttgaaaatggattctttacaggatataaaagcattgttaacaggattatttgtacaagataatgtg
aataaagatgccgtcacagatagattggcttcagtggagactgatatgcctctaacattgagacagcatagaataagtgcgacatcatcatcggaagagagt
agtaacaaaggtcaaagacagttgactgtatcgccggaattcttaatacgactcactatagggcatatggccatggaggccccggg
pGAD424-without-FULL-ICE-BOTH-T7                                       SEQUENCE ID NO.9
gatccgtcgacagatctccctatagtgagtcgtattactgcagagatctatgaatcgtagatactgaaaaaccccgcaagttcacttcaactgtgcatcgtgca
ccatctcaatttctttcatttatacatcgttttgccttcttttatgtaactatactcctctaagtttcaatcttggccatgtaacctctgatctatagaattttttaaatgacta
gaattaatgcccatcttttttttggacctaaattcttcatgaaaatatatacgagggcttattcagaagctttggacttcttcgccagaggtttggtcaagtctccaa
tcaaggttgtcggcttgtctaccttgccagaaatttacgaaaagatggaaaagggtcaaatcgttggtagatacgttgttgacacttctaaataagcgaatttctt
atgatttatgatttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttgttcttgagtaactctttcc
tgtaggtcaggttgctttctcaggtatagcatgaggtcgctcttattgaccacacctctaccggcatgcccgaaattcccctaccctatgaacatattccattttgt
aatttcgtgtcgtttctattatgaatttcatttataatgtttatgtacaaatatcataaaaaaagagaatctttttaagcaaggattttcttaacttcttcggcgacagca
tcaccgacttcggtggtactgttggaaccacctaaatcaccagttctgatacctgcatccaaaacctttttaactgcatcttcaatggccttaccttcttcaggcaa
gttcaatgacaatttcaacatcattgcagcagacaagatagtggcgatagggtcaaccttattctttggcaaatctggagcagaaccgtggcatggttcgtaca
aaccaaatgcggtgttcttgtctggcaaagaggccaaggacgcagatggcaacaaacccaaggaacctgggataacggaggcttcatcggagatgatatc
accaaacatgttgctggtgattataataccatttaggtgggttgggttcttaactaggatcatggcggcagaatcaatcaattgatgttgaaccttcaatgtagga
aattcgttcttgatggtttcctccacagtttttctccataatcttgaagaggccaaaacattagctttatccaaggaccaaataggcaatggtggctcatgttgtag
ggccatgaaagcggccattcttgtgattctttgcacttctggaacggtgtattgttcactatcccaagcgacaccatcaccatcgtcttcctttctcttaccaaagt
aaatacctcccactaattctctgacaacaacgaagtcagtacctttagcaaattgtggcttgattggagataagtctaaaagagagtcggatgcaaagttacat
ggtcttaagttggcgtacaattgaagttctttacggatttttagtaaaccttgttcaggtctaacactacctgtaccccatttaggaccacccacagcacctaacaa
aacggcatcaaccttcttggaggcttccagcgcctcatctggaagtgggacacctgtagcatcgatagcagcaccaccaattaaatgattttcgaaatcgaac
ttgacattggaacgaacatcagaaatagctttaagaaccttaatggcttcggctgtgatttcttgaccaacgtggtcacctggcaaaacgacgatcttcttaggg
gcagacattaagaatggtatatccttgaaatatatatatatattgctgaaatgtaaaaggtaagaaaagttagaaagtaagacgattggctaaccacctattggaaa
aaacaataggtccttaaataatattgtcaacttcaagtattgtgatgcaagcatttagtcatgaacgcttctctattctatatgaaaagccggttccggcctctcac
ctttcctttttctcccaatttttcagttgaaaaaggtatatgcgtcaggcgacctctgaaattaacaaaaaatttccagtcatcgaatttgattctgtgcgatagcgc
ccctgtgtgttctcgttatgttgaggaaaaaaataatggttgctaagagattcgaactcttgcatcttacgatacctgagtattcccacagttggggatctcgactc
tagctagaggatcaattcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaa
agcctggggtgcctaatgagtgaggtaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctggattaatgaatc
ggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatc
agctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgta
aaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggact
ataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcg
tggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctg
cgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt
aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaa
agagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaa
gatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcctttt
aaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatt
tcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacg
ctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgt
tgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttca
ttcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagtt
ggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcat
tctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaa
acgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcacc
agcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatatt
attgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcc
acctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacct
ctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggt
gtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccataacgcatttaagcataaacacgcactatgccgttcttctcatgtatat
atatatacaggcaacacgcagatataggtgcgacgtgaacagtgagctgtatgtgcgcagctcgcgttgcattttcggaagcgctcgttttcggaaacgcttt
gaagttcctattccgaagttcctattctctagctagaaagtataggaacttcagagcgcttttgaaaaccaaaagcgctctgaagacgcactttcaaaaaacca
aaaacgcaccggactgtaacgagctactaaaatattgcgaataccgcttccacaaacattgctcaaaagtatctctttgctatatatctctgtgctatatccctata
taacctacccatccacctttcgctccttgaacttgcatctaaactcgacctctacattttttatgtttatctctagtattactctttagacaaaaaaattgtagtaagaac
tattcatagagtgaatcgaaaacaatacgaaaatgtaaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttctgaccaatgaa
gaatcatcaacgctatcactttctgttcacaaagtatgcgcaatccacatcggtatagaatataatcggggatgcctttatcttgaaaaaatgcacccgcagctt
cgctagtaatcagtaaacgcgggaagtggagtcaggctttttttatggaagagaaaatagacaccaaagtagccttcttctaaccttaacggacctacagtgc
aaaaagttatcaagagactgcattatagagcgcacaaaggagaaaaaaagtaatctaagatgctttgttagaaaaatagcgctctcgggatgcatttttgtaga
acaaaaaagaagtatagattctttgttggtaaaatagcgctctcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcg
cgttgcatttttgttttacaaaaatgaagcacagattcttcgttggtaaaatagcgctttcgcgttgcatttctgttctgtaaaaaatgcagctcagattctttgtttgaa
aaattagcgctctcgcgttgcatttttgttctacaaaatgaagcacagatgcttcgttgcttgcatgaacttcttttcttttttttttcttttctctctcccccgttgttgtct
caccatatccgcaatgacaaaaaaaatgatggaagacactaaggaaaaaattaacgacaaagacagcaccaacagatgtcgttgttccagagctgatga
ggggtatcttgaaacacgaaactttttccttccttcattcacgcacactactctctaatgagcaacggtatacggccttccttccagttacttgaatttgaaataa
aaaaagtttgccgctttgctatcaagtataaatagacctgcaattattaatcttttgtttcctcgtcattgttctcgttccctttcttccttgtttctttttctgcacaatattt
caagctataccaagcatacaatcaactccaagctttgcaaagatggataaagcggaattaattcccgagcctccaaaaaagaagagaaaggtcgaattggg
taccgccgccaattttaatcaaagtgggaatattgctgatagctcattgtccttcactttcactaacagtagcaacggtccgaacctcataacaactcaaacaaa
ttctcaaagcgctttcacaaccaattgcctcctctaacgttcatgataacttcatgaataatgaaatcacggctagtaaaattgatgatggtaataattcaaaaccac
tgtcacctggttggacggaccaaactgcgtataacgcgtttggaatcactacagggatgtttaataccactacaatggatgatgtatataactatctattcgatga
tgaagataccccaccaaacccaaaaaaagagatcgaattcttaatacgactcactatagggcccatggacgaagaatccagttcattcttatgtacctatgctg
agaatcgtgccaataagaagccaatacttccttagatgatgcaataaatattaaaataaaacaaaacagaaggctg
pGN205：                                                            SEQUENCE ID NO.10
ccggtggtaccgggccccccctcgaggtcgacggtatcgataagctttcgtcattgaaaagaaggataagaatggacgatgggaagaagctctcgtt
gttccaggagatcagaaaacagcaactgttccaaatcttaaggagggagaagaatatcaattcagaatttctgctcgtaacaaggctggcaacttggaga
tccttctgatccttctgatcgtgttgttgcgaagccaagaaaccttgctccaagaattcatcgtgaagatctttctgatacaactgtcaaggtcggagccac
tctcaagttcattgttcatattgatggtgagccagcaccagatgtaacatggtcattcaatggaaaaggaatcggagagagcaaggctcaaattgaaaa
tgagccatacatctcgagatttgctttgccaaaggcacttcgtaagcaaagtggaaaatataccatcactgcaaccaacattaatggaactgacagtgtc
actatcaatatcaaggtaaaaagcaagccaacgaaaccaaagggaccaatcgaggtaactgatgtcttcgaagatcgtgcaactcttgactggaaac
caccagaggatgacggaggagagccaattgagttctatgaaattgaaaagatgaacaccaaggacggaatctgggttccatgtggacgtagtggag
atacccacttcacagtcgattcactcaacaagggagatcattacaagttccgtgtcaaggctgtcaacagcgaaggaccttctgatccattggaaactg
aaaccgatattttggctaaaaatccatttgatcgtccagatagaccaggtcgtccagagccaactgattgggattctgatcatgttgatctaaagtgggat
ccactagttctagaagcgctgctaagggggccctcgtcgagtcggtcacaatcacctgaaactccaaaggcagccagtgaggaacgtgaagaaga
agaaaaagagtcatctgaacaggtttgattttctttctggtcaaaaagatgaaattattgattttcagccagatactcccaaaactagcagcgagaagtct
gcaagtcgttcacagtcgcccagagaatggcgggaagtgagccaagaggtatgtttttcaaaaatcaataactgatcataatttttattgtttggtgaattt
aagaaaataatattcgaaaattcctctgaattatcaagattgcagtattaatttcgagaaaaattgagatattcatagagctattgtaaattttcttgatttcag
actgaaacttcggaaaatcagagaaaatcaaagaaaaggatgacggggatgatcagcctggcacaccgaacagctatagaagccgggaaacttc
accagctccaaaaaggtccaaggagaccaggtttgtcaaaagcttcctgcgattaattctcatttcaatttttcagagaatcagagtctcctgaaaaatcc
ccggttcgttcaagatctcccagaaggtcttcagcacgttccccgtcacgatctcctagacggcgccgagaaagaagctcagaaagaaagcaatccg
aagagccagcaccgctaccagagaaaaagaagaaagagccgctggatattctacgaacaagaaccggaggagcatatattccacccgccaaactt
cgacttatgcaacaacagattagtgataagcaaagtgaacagtatcagagattgaattgggaaagaatgaagaaaaagattcacggattggttaacag
agtcaacgcgaagaatcttgttcaaattgtcagagaacttcttcaagagaatgtgattcgttcaaagtgagtgagaaaatcgaaggaaaaggaaagaat
taatttaatttttcaggggacttctctgccgtgacattattcaagctcaggctttctcaccaggattctctaacgtctatgcagctttggcggcagttatcaac
tcgaaattccctcatgtcggtgaacttcttctccgtcgtctgattgtacagttcaaaagaagtttccgtagaaatgacagaggcgtcacggtgaacgtgat
caaattcatcgcacatttgattaatcaacaagttgctaacgaagttcttgcgctggaaatcatgattctgatgcttgaagaaccaactgatgattcagttga
agtcgccattgcgttcctgaaagagtgtggagcaaagcttctggagattgctccagcagctcttaacagtgtctacgaccgtcttcgtgcaattctcatg
gaaactgaaagatcggaaaatgcactggatcgacgtattcagtatatgattgtgactgcaatgcaagattcgaaaggacaaatttgcggtaaggtagaa
tatataaatagtttattagaaaaaaataaattagaataatttaaattcctactagccaatcaggcgacctttttgcgcatagttctattattgaaaaatttggag
aatttctcatattctcgctcggaaatctggaattcgacgagatcttctggcttctgtgcagctgcatcgctttgtgctccctttctcgcttgtcttctgtgtaca
ccaagaaccttgttgagttcatcaactgaatctgtgactggcttgttgctcactggatgcactagacgactgattctcgagaaatcagattgagttgcgatt
agggtgacctagaaattgggaataatacgaacttttgaaaatattcaggaggtttaaaaaaattattctcgacaatcctacaaatttacttattgcaccatgt
tgctccaacatttttcattaaaagttaatgaaaaaatgtagaaaatcggaaattggcaattttcagaccattttaagcattttcaaaaaaaaattgcagctga
aataaatgtcattttcagataaatcgagcgattttctgttgtctgacactagtttttagttttaaaaaatgttggaagaacatggtgcaataggtaatttcatag
aatttccatgtgttttttttcaattaaccaattatccaaatcttccaaactcacattttgcggagctgggctatcaagaatctgctgcagttttataagacgagc
atctctgatatcactgaaaattaatttttaatcaaaacttgaatatcaactaaacccacttattaactttctcgatcttctgtcgttcggtacgatgacggtgaa
gaagccaattgtagtagttgatttggttcaagtcctttcggtgttgtacgtcagtgtcctgcaatgctatttagttattaacttaggcctaagattcaatttaatg
aagtgattaaatttgttctctgaacctcttaggatgatcttttggattagaaacatataagacaggtttacctatctattaaaaaacagatcaaaatagatacg
accaaatcggataatccatgcctacctggcatctaggaacgtgttcttagaagatttcttacgtaatcgtatgaagaaataacaatttgatcgttggccag
caaaaatagggttttaagtgggatagtgtttttattagctaaccggaaaattttatagtttttttttgcaagaaaccactgaaaaccccctaattgtatacatttt
ttggagcagcttctggtctttttgagcaataaaattcgataaaacagaatttaagtgtaaattgttcacatttagtttctattttatcaaattttgttgctcaaaaa
cattcgaagctgctctaaaaaaatgcattaaaaaaggggttttcagtggtttttcacattaaaaaagctaattttaactaaaaatccatcatatttccaactttg
tcacaacaataaaatgctggtcaaaatgtgttcgaaaaaatgtttttttttttaatttttataatttaaaaatagttttctttcgctgggacacatacatttttgggc
gtaaattttcagttcaaatttccatttttacaaccataatcataaagctacgtctgatctctctcgcacttacctgcgcctgattcgaaagaacaaccgtagc
caaaagaacaagaagaacaagcacgtagttgtggtagtggacgttcatcacgcaatactgaccaatggtcgtggggtctcactttccgtactattgaga
gaggggagacgaagatggcaattgaggacagtgtcttcgacgcacgcatgcatccataagcataatccaggagggatggagagaaaaatcttgttt
ctaagcccctccctttgtaatacatacacatatctaataccgaagaatggctaattgaatggacgtcagctgttgctgtagttgccaaggcatcatcgatg
aaataactgaaagaaagaattaaataattattgcaggcgtatccggcggtcattgaagacttggacttgattgaggaggaggatcagatcatccataca
cttaatttggaggatgcggttgatccggaaaatgggcttagtaagtgactgaccacacgcggggggcattaatttaataaattgaattccatttcagatgt
gttcaaactagatccagaattcgaaaagaacgaggaggtttatgaggagatccgtaaggaaatcattggaaacgccgatatttcggatgaggatggtg
gcgacgagttggatgatgaagaagagggtagtgatgttggaagaggctccgaagaagactacagagattattgataatactgatcagaattgactgctt
tcagaaggtattcattttgagttttgggccggcaaatctgtaagttgccggttgccgaaaatttgctgaatttgccggaaaaaaaaattccggaatttattta
aaaactttttgtaaaaattaaattaaatttgcaacttttcagagaagtctacctgacaatgcaatcatctttggactaccaagaagctgctcacaaattgctg
aaaatgaagattccagacagcatgcaggtcagcgatgttgcaaagaaaaattttcgaccaaaaaaaccaaccaatcataaaatttaaaaaaaaactcc
gtttttttctttttttttatacgagaaaaaccaaaaaaatgtatttttgccaaattctaaaatactatccccgaaattttcaatattttctctttcagaacgaactctg
cgcgatgcttgtcgattgttgtgctcaacagcgtacctacgagcgattctacggaatgctcatcgaacgtttctgccgacttcgcctcgaataccagcaa
tactttgaaaagctctgccaggacacgtattccacgattcaccgaattgacatcacaaaactgcggaatttggctcgccttattgctcatttgctctcgacg
gatgctattgactggaagattttggccgatatgaaaatgaccgaagaggacacaacttcttctggcagaatctatattaaatatatatttaatgaacttgtg
gaggcgatgggaatggttaaacttcattcgagagttactgatccgtgagtttcctagagagagttgttttcgtattcaattttccctattttcagaactttggct
cattgctttgttggattattcccacgaactaatccgaacagcgcacgattttcgatcaacttcttcacaatgattggattgggtggtttgacgttggaacttc
gtgaatggctggcaaagggtctcaagaagaagaagggaatgctggatcagttgaaggccgaatcaagctcagattcatcgtcgtcttcggattcgtca
gactcgtctgattcttcggattctgacgattcatccgactcgtcttcagattcctcatcttcttcagaatcagagccagaaccaccgaagaaaaagaagaa
gaagaacagtgaagagagttccaaaaagaaggaaaaagagaatattggtcgacgggatcgtggagacaagagagctgaacgtcatcgtgatcaaa
gtgtggagaacaaggacaaggatcgtcgacgtcgccaggattctgacgaaaatcgtcggccagaacgaggagatgaccgcaaggatcggagtaa
agatcgtcgtcgtcaagactcggatgatgaggatcggaaaggtcgtgaacgtcgggaagattcaggggaaagacgtcgcggagatcgggatcgac
gtgatcgaaacaaggatcaggaggatcaccgtgaagatcgccgtgaccgaagcaaggatcgtgaggatcgacgtgatcgccgtcgtcatgactctg
atgatgatcgtaaaactcgtcgggatagaagtgaagagcgaggaggacgtcgtcgtgaagtggaatcggatgatcgacgccgacgtcgttgaatttt
caaattttaaatactgaatatttgttttttttcctattatttatttattctctttgtgttttttttcttgctttctaaaaaattaattcaatccaaatctaaacatgagcggtt
ttttttctctttccgtctcccaattcgtattccgctcctctcatctgaacacaatgtgcaagtttatttatcttctcgctttcatttcattaggacgtggggggaatt
ggtggaagggggaaacacacaaaaggatgatggaaatgaaataaggacacacaatatgcaacaacattcaattcagaaatatggaggaaggtttaa
aagaaaacataaaaatatatagaggaggaaggaaaactagtaaaaaataagcaaagaaattaggcgaacgatgagaattgtcctcgcttggcaaatg
cgaatccgtatggagaggcacgtttggcgaaggcaaatgttcggtatggagatctgtaaaaatttttaagttgaaatttggtgttgctcttttacaaaatttt
ccgattttcgcttgaaattacggtgccaggtctcgacacgtcttccaatttttcaaattcaaaagagcctttaatgggctgtagttgctaatttctcgtttttga
aaatttttcttccgtttaatcgaaatttgatgtattttatttatgattttcaataaatttcaaagaaactggtgaaaactcggaaaattgtgaactacagtaatcca
atccttaaaggcgcacaccttttaaatgtccgccccaatacgatatttttttaagattcgctagagcggccgccaccgcggtggagctccaattcgccct
atagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccc
cttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgtagcctgaatggcgaatgggacgcgccctgtagcg
gcgcattaagcgcggcgggtgtggtggttacgcgcagcaggaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttct
cgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgat
tagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaact
ggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaa
cgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatat
gtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttg
cggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgga
tctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtatt
gacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcat
gacagtaagagaattatgcagtgctgccataagcatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaacc
gctttttttcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgat
gcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttattactctagcttcccggcaacaattaatagactggatggaggcggata
aagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcag
cactggggccagatggtaagccctcccgtatcgtagttatctacacgacgggcagtcaggcaactatggatgaacgaaatagacagatcgctgagat
aggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaa
gatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcc
tttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggt
aactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacct
cgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagc
ggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaag
cgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggg
aacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgggatgctcgtcaggggggccgagcctatggaaaaacg
ccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcct
ttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgc
ctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagtta
cctcactcattaggcaccccaggctttacactttatgcttccggctcctatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatg
accatgattacgccaagctcggaattaaccctcactaaagggaacaaaagctgggggggatcctccaaaatcgtcttccgctctgaaaaacgaaagt
ggacctttgacatccgaaaaaatgggcgaaaaaatgaaattgagctttttgggtcgaaaaaaatgtttttagaatgctgagaacacgttaaacacgaag
atcatatttattttgagacccggatgctctgaaaatgtctgacatagatttaaaaaagcatatatatatttttcattttcaacgtgaaagttttgtgcaactttata
gaatctcctattggcacattgttttttatttaactgaggcagtttttgaacacctttttgaaactttgaatctctttgaagtatactgtcgaaaagactgacttga
gcgttcgaaatgccagaagaaaactatatttgaatctcgcgctaaattgagaaatgcaaccgcgctccactggacaattggaaaaaaaatttattcgga
ggcgacaacggtattttcgaaagattttctgtgtattttctcattttttataaattcttctttgatttatcgttcgtttgtgagaaatttaattgtattcaaactttttt
atagtaagata
pGN207：                                                    SEQUENCE ID NO.11
ccggtggtaccgctagccgtacgaacccgggttctagaactagtggatcccacttgagatcaacatgatcagaatcccaatcagttggctctggacga
cctggtctatctggacgatcaaatggatttttagccaaaatatcggtttcagtttccaatggatcagaaggtccttcgctgttgacagccttgacacggaa
cttgtaatgatctcccttgttgagtgaatcgactgtgaagtgggtatctccactacgtccacatggaacccagattccgtccttggtgttcatcttttcaattt
catagaactcaattggctctcctccgtcatcctctggtggtttccagtcaagagttgcacgatcttcgaagacatcagttacctcgattggtccctttggttt
cgttggcttgctttttaccttgatattgatagtgacactgtcagttccattaatgttggttgcagtgatggtatattttccactttgcttacgaagtgcctttggca
aagcaaatctcgagatgtatggctcattttcaatttgagccttgctctctccgattccttttccattgaatgaccatgttacatctggtgctggctcaccatca
atatgaacaatgaacttgagagtggctccgaccttgacagttgtatcagaaagatcttcacgatgaattcttggagcaaggtttcttggcttcgcaacaac
acgatcagaaggatcagaaggtactccagttccagccttgttacgagcagaaattctgaattgatattcttctccctccttaagatttggaacagttgctgt
tttctgatctcctggaacaacgagagcttcttcccatcgtccattcttatccttcttttcaatgacgaaagcttatcgataccgtcgacctagagggggggc
cctcgtcgagtcggtcacaatcacctgaaactccaaaggcagccagtgaggaacgtgaagaagaagaaaaagagtcatctgaacaggtttgattttct
ttctggtcaaaaagatgaaattattgattttcagccagatactcccaaaactagcagcgagaagtctgcaagtcgttcacagtcgcccagagaatcgcg
ggaagtgagccaagaggtatgtttttcaaaaatcaataactgatcataatttttattgtttggtgaatttaagaaaataatattccgaaaattcctctgaattatc
aagattgcagtattaatttcgagaaaaattgagatattcatagagctattgtaaattttcttgatttcagactgaaacttcggaaaatcaagagaaaatcaaa
gaaaaggatgacggggatgatcagcctggcacaccgaacagctatagaagccgggaaacttcaccagctccaaaaaggtccaaggagaccaggt
ttgtcaaaagcttcctgcgattaattctcatttcaatttttcagagaatcagagtctcctgaaaaatccccggttcgttcaagatctcccagaaggtcttcag
cacgttccccgtcacgatctcctagacggcgccgagaaagaagctcagaaagaaagcaatccgaagagccagcaccgctaccagagaaaaagaa
gaaagagccgctggatattctacgaacaagaaccggaggagcatatattccacccgccaaacttcgacttatgcaacaacagattagtgataagcaa
agtgaacagtatcagagaatgaattgggaaagaatgaagaaaaagattcacggattggttaacagagtcaacgcgaagaatcttgttcaaattgtcag
agaacttcttcaagagaatgtgattcgttcaaagtgagtgagaaaatcgaaggaaaaggaaagaattaatttaatttttcaggggacttctctgccgtgac
attattcaagctcaggctttctcaccaggattctctaacgtctatgcagctttggcggcagttatcaactcgaaattccctcatgtcggtgaacttcttctcc
gtcgtctgattgtacagttcaaaagaagtttccgtagaaatgacagaggcgtcacggtgaacgtgatcaaattcatcgcacatttgattaatcaacaagtt
gctcacgaagttcttgcgctggaaatcatgattctgatgcttgaagaaccaactgatgattcagttgaagtcgccattgcgttcctgaaagagtgtggag
caaagcttctggagattgctccagcagctcttaacagtgtctacgaccgtcttcgtgcaattctcatggaaactgaaagatcggaaaatgcactggatcg
acgtattcagtatatgattgagactgcaatgcagattcgaaaggacaaatttgcggtaaggtagaatatataaatagtttattagaaaaaaataaattaga
ataatttaaattcctactagccaatcaggcgacctttttgcgcatagttctattattgaaaaatttggagaatttctcatattctcgctcggaaatctggaattc
gacgagatcttctggcttctgtgcagctgcatcgctttgtgctccctttctcgcttgtcttctgtgtacaccaagaaccttgttgagttcatcaactgaatctgt
gactggcttgttgctcactggatgcactagacgactgattctcgagaaatcagattgagttgcgattagggtgacctagaaattgggaataatacgaact
tttgaaaatattcaggaggattaaaaaaattattctcgacaatcctacaaatttacttattgcaccatgttgctccaacatttttcattaaaagttaatgaaaaa
atgtagaaaatcggaaaaggcaattttcagaccatttttaagcattttcaaaaaaaaattgcagctgaaataaatgtcattttcagataaatcgagcgatttt
ctgttgtctgacactagtttttagttttaaaaaatgttggaagaacatggtgcaataggtaatttcatagaatttccatgtgttttttttcaattaaccaattatcc
aaatcttccaaactcacattttgcggagctgggctatcaagaatctgctgcagttttataagacgagcatctctgatatcactgaaaattaatttttaatca
aacttgaatatcaactaaacccacttattaactttctcgatcttctgtcgttcggtacgatgacggtgaagaagccaattgtagtagttgatttggttcaagtc
ctttcggtgttgtacgtcagtgtcctgcaatgctatttagttataacttaggcctaagattcaatttaatgaagtgattaaatttgttctctgaacctcttaagat
gatcttttggattagaaacatataagacaggtttacctatctattaaaaaacagatcaaaatagatacgaccaaatcggataatccatgcctacctggcat
ctaggaacgtgttcttagaagatttcttacgtaatcgtatgaagaaataacaatttgatcgttggccagcaaaaatagggttttaagtgggatagtgtttttat
tagctaaccggaaaattttatagtttttttttgcaagaaaccactgaaaaccccctaattgtatacattttttggagcagcttctggtctttttgagcaataaaat
tcgataaaacagaatttaagtgtaaattgttcacatttagtttctattttatcaaattttgttgctcaaaaacattcgaagctgctctaaaaaaatgcattaaaaa
aggggttttcagtggtttttcacattaaaaaagctaattttaactaaaaatccatcatatttccaactttgtcacaacaataaaatgctggtcaaaatgtgttcg
aaaaaatgtttttttttttaatttttataatttaaaaatagttttctttcgctgggacacatacatttttgggcgtaaattttcagttcaaatttccatttttacaaccatt
aatcataaagctacgtctgatctctctcgcacttacctgcgcctgattcgaaagaacaaccgtagccaaaagaacaagaagaacaagcacgtagttgt
ggtagtggacgttcatcacgcaatactgaccaatggtcgtggggtctcactttccgtactattgagagaggggagactgaagatggcaattgaggaca
gtgtcttcgacgcacgcatgcatccataagcataatccaggagggatggagagaaaaatcttgtttctaagcccctccctttgtaatacatacacatatct
aataccgaagaatggctaattgaatggacgtcagctgttgctgtagttgccaaggcatcatcgatgaaataactgaaagaaagaattaaataattattgc
aggcgtatatccggcggtcattgaagacttggacttgattgaggaggaggatcagatcatccatacacttaatttggaggatgcggttgatccggaaaatg
ggcttagtaagtgactgaccacacgcggggggcattaatttaataaattgaattccatttcagatgtgttcaaactagatccagaattcgaaaagaacga
ggaggtttatgaggagatccgtaaggaaatcattggaaacgccgatatttcggatgaggatggtggcgacgagttggatgatgaagaagagggtagt
gatgtggaagaggctccgaagaagactacagagattattgataatactgatcagaattgactgctttcagaaggtattcattttgagttttgggccggcaa
atctgtaagttgccggttgccgaaaatttgctgaatttgccggaaaaaaaaattccggaatttatttaaaaactttttgtaaaaattaaattaaatttgcaactt
ttcagagaagtctacctgacaatgcaatcatctttggactaccaagaagctgctcacaaattgctgaaaatgaagattccagacagcatgcaggtcagc
gatgttgcaaagaaaaattttcgaccaaaaaaaccaaccaatcataaaatttaaaaaaaaactccgtttttttctttttttttatacgagaaaaaccaaaaaa
atgtatttttgccaaattctaaaatactatccccgaaattttcaatattttctctttcagaacgaactctgcgcgatgcttgtcgattgttgtgctcaacagcgta
cctacgagcgattctacggaatgctcatcgaacgtttctgccgacttcgcctcgaataccagcaatactttgaaaagctctgccaggacacgtattccac
gattcaccgaattgacatcacaaaactgcggaatttggctcgccttattgctcatttgctctcgacggatgctattgactggaagattttggccgatatgaa
aatgaccgaagaggacacaacttcttctggcagaatctatattaaatatatatttaatgaacttgtggaggcgatgggaatggttaaacttcattcgagag
ttactgatccgtgagtttcctagagagagttgttttcgtattcaattttccctattttcagaactttggctcattgctttgttggattattcccacgaactaatccg
aacagcgcacgattttcgatcaacttcttcacaatgattggattgggtggtttgacgttggaacttcgtgaatggctggcaaagggtctcaagaagaaga
agggaatgctggatcagttgaaggccgaatcaagctcagattcatcgtcgtcttcggattcgtcagactcgtctgattcttcggattctgacgattcatcc
gactcgtcttcagattcctcatcttcttcagaatcagagccagaaccaccgaagaaaaagaagaagaagaacagtgaagagagttccaaaaagaag
gaaaaagagaatattggtcgacgggatcgtggagacaagagagctgaacgtcatcgtgatcaaagtgtggagaacaaggacaaggatcgtcgacg
tcgccaggattctgacgaaaatcgtcggccagaacgaggagatgaccgcaaggatcggagtaaagatcgtcgtcgtcaagactcggatgatgagg
atcggaaaggtcgtgaacgtcgggaagattcaggggaaggacgtcgcggagatcgggatcgacgtgatcgaaacaaggatcaggaggatcaccg
tgaagatcgccgtgaccgaagcaaggatcgtgaggatcgacgtgatcgccgtcgtcatgactctgatgatgatcgtaaaactcgtcgggatagaagt
gaagagcgaggaggacgtcgtcgtgaagtggaatcggatgatcgacgccgacgtcgttgaattttcaaattttaaatactgaatatttgttttttttcctatt
atttatttattctctttgtgttttttttcttgctttctaaaaaattaattcaatccaaatctaaacatgagcggttttttttctctttccgtctcccaattcgtattccgct
cctctcatctgaacacaatgtgcaagtttatttatcttctcgctttcatttcattaggacgtggggggaattggtggaagggggaaacacacaaaaggatg
atggaaatgaaataaggacacacaatatgcaacaacattcaattcagaaatatggaggaaggtttaaaagaaaacataaaaatatatagaggaggaa
ggaaaactagtaaaaaataagcaaagaaattaggcgaacgatgagaattgtcctcgcttggcaaatgcgaatccgtatggagaggcacgtttggcga
aggcaaatgttcggtatggagatctgtaaaaattttaagttgaaatttggtgttgctcttttacaaaattttccgattttcgcttgaaattacggtgccaggtct
cgacacgtcttccaatttttcaaattcaaaagagcctttaatgggctgtagttgctaatttctcgtttttgaaaatttttcttccgtttaatcgaaatttgatgtattt
tatttatgattttcaataaatttcaaagaaactggtgaaaactcggaaaattgtgaactacagtaatccaatccttaaaggcgcacaccttttaaatgtccgc
cccaatacgatatttttttaagattcgctagagcggccgccaccgcggtggagctccaattcgccctatagtgagtcgtattacaattcactggccgtcgt
tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccccttcgccagctggcgtaatagcgaagaggccc
gcaccgatcgcccttcccaacagttgcgtagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgc
gcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttccttctcgccacgttcgccggctttccccgtcaagctcta
aatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccct
gatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattctttt
gatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttca
ggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgc
ttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaac
gctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgcc
ccgaagaacgttttccaatgatgagcactttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcat
acactattctcagaatgacttggttgagtactcacagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataag
catgagtgataaacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctttttttcacaacatgggggatcatgtaactcg
ccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaact
attaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttcc
ggctggctggtttattgctgataaatcttggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgt
agttatctacacgacgggcagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcag
accaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaa
cgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaa
aaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatact
gtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccag
tggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcc
cagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacagg
tatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggggaacgcctggtatctttatagtcctgtcgggtttcgccac
ctctgacttgagcgtcgatttttgtgatgctcgtcaggggggccgagcctatgaaaaacgccagcaacgcggcctttttacggttcctggccttttgct
ggccttttgctcacatgttctttcctgcgttatctcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacga
ccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctg
gcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttacctcactcattaggcaccccaggctttacactttatgct
tccggctcctatgttgtgtggaattgtgagcggataacaatttcacacaggaaacggctatgaccatgattacgccaagctcggaattaaccctcactaa
agggaacaaaagctgggggggatcctccaaaatcgtcttccgctctgaaaaacgaaagtggacctttgacatccgaaaaaatgggcgaaaaaatga
aattgagctttttgggtcgaaaaaaatgtttttagaatgctgagaacacgttaaacacgaagatcatatttattttgagacccggatgctctgaaaatgtctg
acatagatttaaaaaagcatatatatatttttcattttcaacgtgaaagttttgtgcaactttatagaatctcctattggcacattgttttttatttaactgaggcag
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gctaattgagaaatgcaaccgcgctccactggacaattggaaaaaaaatttattcggaggcgacaacggtattttcgaaattgattttctgtgtattttct
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Claims (9)

1.减轻植物害虫侵染的方法，所述方法包括：
a)从所述害虫中鉴定出对其生存、生长、增殖重要的DNA序列，
b)在合适的载体中，以相对于启动子的方向克隆得自步骤a)的所述序列或其片段，使所述启动子在合适的转录因子与所述启动子结合时能够启动从所述DNA序列转录出RNA或dsRNA，并且
c)将所述载体引入所述植物中。
2.根据权利要求1的方法，其中所述植物害虫是在所述植物上取食的植物害虫。
3.根据任一前述权利要求的方法，其中所述害虫为线虫蠕虫。
4.根据权利要求3的方法，其中所述线虫包括小垫刃线虫属物种(Tylenchulus ssp.)、穿孔线虫属物种(Radopholus ssp.)、细杆滑刃线虫属物种(Rhadinaphelenchus ssp.)、异皮线虫属物种(Heterodera ssp.)、小盘旋线虫属物种(Rotylenchulus，ssp.)、短体线虫属物种(Pratylenchusssp.)、刺线虫属物种(Belonolaimus ssp.)、Canjanus ssp.、根结线虫属物种(Meloidogyne ssp.)、球异皮线虫属物种(Globodera ssp.)、科布线虫属物种(Nacobbus ssp.)、茎线虫属物种(Ditylenchus ssp.)、滑刃线虫属物种(Aphelenchoides ssp.)、潜根线虫属物种(Hirschmenniella ssp.)、鳗线虫属物种(Anguina ssp.)、纽带线虫属物种(Hoplolaimus ssp.)、螺旋线虫属物种(Heliotylenchus ssp.)、小环线虫属物种(Criconemellassp.)、剑线虫属物种(Xiphinema ssp.)、长针线虫属物种(Longidorusssp.)、毛刺线虫属物种(Trichodorus ssp.)、副毛刺线虫属物种(Paratrichodorus ssp.)、真滑刃线虫属物种(Aphelenchs ssp.)中的任一种。
5.根据权利要求1或2的方法，其中所述植物害虫为昆虫。
6.根据任一前述权利要求的方法，其中所述DNA序列在两个启动子之间提供，使所述转录因子与所述启动子的结合导致从所述DNA转录出dsRNA。
7.根据任一前述权利要求的方法，其中所述DNA序列以相对所述启动子为有义和反义的方向提供，使所述转录因子与所述启动子的结合导致从所述DNA转录出dsRNA。
8.根据任一前述权利要求的方法，其中所述DNA序列或其片段克隆在两个组织特异性启动子之间，最好是克隆在两个根特异性启动子之间。
9.根据任一前述权利要求的方法，用于减轻植物的寄生虫侵染。
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