CN1256712A - 植物病害控制 - Google Patents
植物病害控制 Download PDFInfo
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- CN1256712A CN1256712A CN98805198A CN98805198A CN1256712A CN 1256712 A CN1256712 A CN 1256712A CN 98805198 A CN98805198 A CN 98805198A CN 98805198 A CN98805198 A CN 98805198A CN 1256712 A CN1256712 A CN 1256712A
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Abstract
本发明涉及蛋白新种类及它们的受体。本发明提供控制植物害虫的步骤、测定和方法。
Description
本发明涉及控制植物病害的蛋白新种类。
植物病害是导致世界重要商业农作物损失的主要因素,导致世界主要区域的农民损失和当地人口的营养贫乏。广谱化学杀虫剂已广泛用于控制或根除具有重要农业意义的害虫。但仍有发展有效的可选择的杀虫剂的需要。
通过使用生物分子控制各种害虫仅在有限情况下可行。已知生物分子的最好例子是来自革兰氏阳性形成孢子的微生物苏云芽孢杆菌(Bacillus Thruingiensis,Bt)的δ-内毒素。已知各种Bt产生超过25种不同的相关δ-内毒素。Bt菌株在形成孢子期间产生δ-内毒素,它们的应用有限,因为它们仅对很少的一些昆虫害虫有活性。
Bt内毒素的限制特异性至少部分取决于昆虫肠中毒素的活化(Haider,M.Z.等,1986,Eur.J.Biochem.156:531-540)和其与呈现在昆虫肠上皮细胞上的特异受体结合的能力(Hofmann,C.P.等,1988,PNAS 85:7844-7848)。因此,目前使用δ-内毒素控制特定昆虫害虫的能力依赖于找到具有所需活性范围的适当δ-内毒素的能力。在很多情况下还未知这样的δ-内毒素,并且甚至不能肯定存在这样一种δ-内毒素。
植物也常常受真菌和细菌侵染,并且许多微生物种类进化为利用正在生长的植物提供的小环境。除了真菌和细菌侵染,一些植物疾病由线虫引起,它们是土生的并且侵染根部,当一些农作物品种在相同地区土壤连续栽培几年时,通常引起严重损失。
病害破坏过程的严重性取决于植物病原体的进攻性和宿主的反应。大多数植物育种计划的一个目标是提高宿主植物对病害的抗性。由于植物病原性克服抗性基因的快速进化,开发用于病害抗性的新基因来源及组合通常在一些农作物-病原体系统中只有有限的成功应用期间。
因此,很明显,科学家必须不断地寻找保护农作物免受植物病害侵袭的新方法。本发明中已发现可用于控制植物病害的新蛋白种类。
编程性细胞死亡是发育或环境刺激物激活导致细胞死亡的遗传程序的过程(Jacobson,M.D.等,1977,细胞88:347-354)。这种遗传潜力存在于大多数,如果不是全部,多细胞生物中。对于无脊椎动物,编程性细胞死亡看来通过作为昆虫发育过程的内在部分和针对尤其病毒性质的侵袭的应答机制的内在部分起双重作用(Clem,R.J.等,1991,科学254:1388-1390)。编程性细胞死亡看来以几种不同方式进行,导致凋亡、萎缩或分化。凋亡是编程性细胞死亡的最好典型类型,包含细胞学变化包括膜结合的凋亡体(apoptotic body)和胞质空泡形成以及分子变化如以寡心核长度片段产生为特征的核内溶解(VauX,D.L和Strasser,A.,1996,PNAS 93:2239-2244)。尽管在不同生物中确实观察到一般的凋亡现象,但是值得指出,对于昆虫细胞,胞质空泡形成和增大看来是与凋亡过程有关的细胞学特征(Bowen,I.D.等,1996,Micros.Res.Techniq.34:202-217)。本发明中公开的新蛋白种类表现出诱导编程性细胞死亡并发挥杀昆虫效应。
本发明涉及VIP3A(C)蛋白及其同系物。本发明也提供VIP3类蛋白的结构域,包括毒性结构域和稳定结构域。本发明优选的实施方案是VIP3A(a)蛋白及其同系物的毒性结构域。另一优选的实施方案是VIP3类蛋白的抗体,但优选地VIP3A(C)蛋白的抗性。
本发明也提供含有VIP3类蛋白的毒性结构域的杂交毒素。在优选的实施方案中,杂交毒素是带有与异源结构域可操作连接的毒性核心结构域的嵌合蛋白。在一另优选实施方案中,杂交毒素包含抗体或其免疫活性片段,它们与来自其它蛋白的毒性结构域可操作连接识别VIP3受体,其中来自各种细胞毒性蛋白的毒素结构域包括但不限于芽孢杆菌毒素,包括内毒素和植物性杀昆虫蛋白。
本发明也包括含有编码VIP3类蛋白,但优选地VIP3A(C)蛋白的DNA序列的植物。优选的实施方案包括选自玉米、高粱、小麦、向日葵、蕃茄、农作物、棉花、水稻、大豆、甜菜、甘蕉、烟草、大麦和油籽油菜的植物。在特别优选的实施方案中,植物是玉米植物。
本发明也提供含有编码VIP3类蛋白、但优选地VIP3A(C)蛋白的DNA序列的微生物。在优选的实施方案中,微生物选自细菌、杆状病毒、藻类和真菌。在另一优选实施方案中,微生物选自芽孢杆菌、假单胞菌、棍状杆菌和根瘤菌。本发明进一步包括杀昆虫组合物,包含含有编码VIP3类蛋白、但优选地VIP3A(C)蛋白的DNA序列的微生物。
本发明进一步涉及进一步包含编码第二种杀昆虫蛋白的第二种DNA序列的植物和微生物。特别优选的是编码δ-内毒素、VIP3类蛋白或VIP1或VIP2类蛋白的第二种DNA序列。在更优选的实施方案中,δ-内毒素对选自鳞翅目和鞘翅目的昆虫有活性。在更优选的实施方案中,δ-内毒素对玉米螟(Ostrinia)或叶甲(Diabrotica)有活性。另外优选的是编码选自Cry1、Cry3、Cry5和Cry9的δ内毒素蛋白的第二种DNA序列。在更优选的实施方案中,δ内毒素选自Cry1Aa、Cry1Ab、Cry1B、Cry1C、Cry1D、Cry1Ea、Cry1Fa、Cry3A、Cry9A、Cry9C和Cry9B。最优选的是选自Cry1Ab、Cry1Ba和Cy9C蛋白的δ-内毒素。
本发明进一步提供控制昆虫的方法,用杀昆虫量的VIP3类蛋白、但优选地VIP3A(C)或者杀昆虫量的VIP3类蛋白的受体的化学配体作用于昆虫。在一个优选的实施方案中,昆虫与含有表达VIP3类蛋白、但优选地VIP3a(C)蛋白的DNA序列的转基因植物接触。在另一优选实施方案中,用含有VIP3类蛋白、但优选的VIP3A(C)蛋白或者含有表达VIP3类蛋白、但优选地的VIP3A(c)蛋白的DNA序列的杀昆虫组合物作用于昆虫。在另一优选实施方案中,转基因植物包含表达VIP3A(a)蛋白的DNA序列。在另一优选实施方案中,昆虫选自鞘翅目、双翅目、膜翅目、鳞翅目、食毛目、同翅目、半翅目、直翅目、缨翅目、革翅目、等翅目和蜱螨目。在特别优选的实施方案中昆虫是鞘翅目或膜翅目昆虫。在另一优选实施方案中,昆虫选自Agrotis ipsilon(小地老虎)、Helicoverpazea(棉铃虫)、Spodoptera frugiperda(草地夜蛾)、Diatraea grandiosella(西南玉米杆草螟)、Diatraea saccharalis(小蔗杆草螟蛾)、S.ornithogalli(黄条粘虫夜蛾)、S.exigua(非洲夜蛾)、Sesamia nonagroides(地中海玉米螟)。Trichoplusia ni(粉蚊夜蛾)、Anticarsia gemmatalis(大豆夜蛾)、Plutella xylostella(菜蛾)和Heliothis virescens(烟芽夜蛾)。
本发明也提供控制昆虫的方法,其中转基因植物或微生物进一步包含编码第二种杀昆虫蛋白如此前所述的那些蛋白的第二种DNA序列。
本发明进一步提供编码VIP3A(c)蛋白及其同系物的重组DNA序列。在另一优选实施方案中,DNA序列是已改变用于植物中最佳表达的合成序列,特别是其中DNA序列已优化用于玉米植物中表达。优选的是包含合成部分和天然部分的DNA序列。在特别优选的实施方案中,编码VIP3A(c)蛋白的DNA序列已优化用于玉米植物中表达。另一优选实施方案是与编码VIP3A(c)蛋白的DNA序列同源的DNA序列。特别优选的是在中等严格条件下与vip3A(c)编码序列杂交的DNA序列。本发明另一实施方案是重组DNA序列,它在异源启动子的控制下表达VIP3类蛋白、优选地VIP3A(c)蛋白,或者其中编码区域整合入其天然不表达生物的基因组或者以比天然存在更高的水平表达。
本发明进一步涉及鉴定和分离VIP3A(c)蛋白的同系物或编码所述蛋白和DNA序列的方法。
本发明也提供含有与启动子可操作连接编码VIP3类蛋白、但优选地VIP3A(c)蛋白的DNA序列的表达盒。在一个优选实施方案中,启动子选自用于植物中表达的组成型、组织偏爱的和组织特异的启动子。在特别优选的实施方案中,启动子选自遍在蛋白、PEP羧化酶、LPT和MTL启动子。在另一优选实施方案中,启动子在微生物中有活性。
本发明进一步提供VIP3类蛋白的受体及其DNA序列。在本发明另一实施方案中,受体包含致死结构域和重复的EGF基序。本发明更优选的实施方案包括VIP3A(a)的受体。特别优选的实施方案是SEQ IDNO:9中所示的受体蛋白序列及其同系物。本发明也包括编码这些受体蛋白的DNA序列,例如SEQ ID NO:8中所示的DNA序列及其同系物。VIP3的受体的cDNA包含在质粒pCIB7113中,该质粒已于1997年3月29日在布达佩斯条约下保藏于NRRL[农业研究机构保藏中心(NRRL),北部区域研究中心,1815 North University Street,Peoria,Illinois 61604,USA]并获得保藏号B-21676。本发明也包括VIP3类蛋白的受体。
本发明也提供鉴定作为VIP3受体的化学配体并具有杀昆虫活性的化合物的方法,包括使细胞、优选地昆虫细胞与受检化合物接触,并分析所述细胞的凋亡活性,在本发明另一实施方案中,方法包括测定VIP3受体和受检化合物之间的特异结合。优选的实施方案是通过此方面鉴定的VIP3受体的配体。定义
“植物病害”是指任何已知与植物有关并且作为该关系的结果引起对植物健康和活力有害影响的生物。植物病害包括但不限真菌、细菌、昆虫和线虫。如此处所用的,术语植物包括完整植物和植物部分如根、茎、叶和种子以及植物或植物部分内的细胞和组织。
“VIP3类蛋白”包括VIP3A(a)、VIP3A(b)和VIP3A(c)及它们的同系物。“同系物”全文用于指所指的蛋白或多肽具有与VIP3类蛋白其它成员的特定关系。此特定关系包括但不限于1)在序列水平上与另一VIP3类蛋白成员有至少30%、更优选地80%并且最优选地90%相同性,同时保留杀昆虫活性的蛋白,2)与免疫识别另一VIP3类蛋白成员的抗体交叉反应的蛋白,3)与另一VIP3类蛋白成员的受体交叉反应并保留诱导编程性细胞死亡的能力的蛋白,和4)在序列水平上与另一VIP3类蛋白成员的毒性核心区域有至少70%、更优选地80%并且最优选地90%相同性,同时也保留杀昆虫活性的蛋白。
“杂交毒素”用于指具有可操作连接的结构域的遗传融合,翻译时形成具有线个雨特征的功能性嵌合蛋白。“结构域”用于指赋予对蛋白总体功能有作用的可辩认的结构或功能的蛋白区域或部分。可以认为此定义也包括编码蛋白结构域的DNA序列。
“异源的”用于指相对于其目前宿主有不同天然来源的蛋白、多肽或核苷酸序列。例如,如果将来自苏云金芽孢杆菌的Vip3A(a)基因遗传转化入植物细胞,就将此基因描述为相对于其目前宿植物细胞是异源的。而且,如果将来自苏云金芽孢杆菌的vip3基因遗传转化入假单胞菌属细菌,那么也将基因描述为相对于假单胞菌为异源的。“异源的”也用于指嵌合蛋白、多肽或核苷酸序列中存在的一个或多个结构域相对于其它存在的结构域的天然来源不同。例如,如果将来自VIP3A(a)蛋白的毒性结构域与来自VIP1A(a)蛋白的结合结构域融合制备功能性杀昆虫蛋白,那么嵌合融合体就具有互相异源的结构域。另外,含有来自VIP3A(a)蛋白的毒性结构域和来自VIP1A(a)蛋白的结合结构域的融合体的异源嵌合蛋白或多肽在植物中表达时也会认为相对于植物宿主异源。
术语“嵌合基因”用于指由一些结构域组成的蛋白、多肽或核苷酸序列,其中至少一个结构域具有相对于其它存在的结构域的异源来源,这些嵌合蛋白或多肽由已融合或连接在一起的核苷酸序列编码,导致天然不存在的编码序列。这样的嵌合构建体也可称为“重组”。
“表达盒”此处用于指能指导植物中基因表达、包含与启动子和终止子可操作连接的氨基酸编码序列的DNA序列。基因可以是嵌合的,即基因的至少一个成分相对于基因的至少一个其它成分是异源的。基因也可以是天然存在的,但已经以重组形式得到用于植物或微生物的遗传转化。
附图
图1:从cDNA翻译得来的VIP3A(a)的受体的氨基酸序列。表示了蛋白的几个特征:虚线-信号肽(氨基酸13-35);斜体-跨越公认致死结构域的区域(氨基酸81-205);双下划线-与保守致死结构域中存在的序列有强同源性的序列;黑体-跨越EGF基序的CKC基序重复六次;下划线-EGF基序内重复的序列。
节肢动物
为本发明目的,昆虫包括选鞘翅目、双翅目、膜翅目、鳞翅目、食毛目、同翅目、半翅目、直翅目、缨翅目、革翅目、等翅目和蜱螨目的昆虫,尤其是鞘翅目和膜翅目昆虫。
例如,WO96/10083的3-20页的表1-10中提供与主要农作物相关的昆虫目录,此处引用作为参考。另外在下面表1-8中提供其它害虫。这些害虫包括在本发明范围内。表1:鳞翅目(蝶和蛾)玉米 十字花科植物(嫩茎花椰菜、甘蓝、花椰
菜、羽衣甘蓝)
Sesamia nonagroides(地中海玉米螟) Artogeia rapae(菜粉蝶)
Ostrinia fumacalis(亚洲玉米螟) Pieris brassicae(大菜粉蝶)
棉花 Trichoplusia ni(粉蚊夜蛾)
Helicoverpa armigera(棉铃虫) Plutella xylostella(小菜粉蝶)
水稻 Spodoptera exigua(非洲夜蛾)
Chilo suppressalis(二化螟) Agrotis lpsilon(小地老虎)
Scirpohaga属种类 Agrotis segetrm(黄地老虎)
Mamestra configura(披肩粘虫)
蕃茄 葡萄
Helicoverpa zea(美洲棉铃虫) Endopiza viteana(葡萄小食心虫)
Spodoptera exigua(非洲夜蛾) 脱叶水果和坚果
Spodoptera frugiperda(草地夜蛾) Cydia pomonella(苹果蠹蛾)
Spodoptera omithogalli(黄条粘虫夜蛾) Platynota idaeusalis(tufted apple bud
Spodoptera praefica(西部黄条粘虫夜蛾) moth)
Spodoptera eridania(亚热带粘虫夜蛾)
Agrotis ipsilon(小地老虎) 胡椒
Peridroma saucia(豆杂角夜蛾) Ostrinia nubilalis(欧洲玉米螟)
Papaipema nebris(普通蛀茎夜蛾) Spodoptera exigua(非洲夜蛾)
Trichoplusia ni(粉纹夜蛾) Spodoptera eridania(南方夜蛾)
Keiferia lycopersicella(番茄蠹蛾) 马铃薯
Manduca sexta(烟草夜蛾) Ostrinie nubilalis(欧洲玉米螟)
Manduca quinquemaculate(番茄夜蛾) Phthorimaea operculella(马铃薯块茎蛾)
Plutella xylostella(小菜蛾)
甘蔗
Diatraea saccharalis(蔗螟)表2:鞘翅目(甲虫)水稻
Oulema oryzea(稻负泥虫)番茄
Leptinotarsa decemlineate(马铃薯甲虫)
Epitrix hirtipennis烟草跳甲十字花科植物(嫩茎花椰菜、甘蓝、花椰菜、羽衣甘蓝)
Phyllotreta cruciferae(芜菁黄条跳甲)
Phyllotreta pusilla(柔弱黑跳甲)胡椒
Anthonomus eugenii(胡椒跳甲)马铃薯
Leptinotarsa decemlineata(马铃薯甲虫)
Hemicrepidus memnonius(马铃薯甲虫)
Hemicrepidus memnonius(捻转血矛线虫)
Melanpotus属种类(捻转血矛线虫)Canola
torhycyus assimilis(甘蓝荚象甲)
yllotreta cruciferae(芜菁黄条跳甲)表3:同翅目(粉虱、蚜虫等)水稻
Nilaparvata lugens(稻褐飞虱)
Sogatella furcifera(白背飞虱)
Laodelphaax striatellus(稻灰虱)蕃茄
Myzus persicae(桃蚜)
Macrosiphum euphorbiae(大戟长管蚜)
Trileurodes vaporariorum(温室白粉虱)
Bemisia argentifolii(silverleaf whitefly)十字花科植物(嫩茎花椰菜、甘蓝、花椰菜、羽衣甘蓝)
Brevicoryne brassicae(甘蓝蚜)
Myzus persicae(桃蚜)胡椒
Myzus persicae(桃蚜)马铃薯
Empoasca fabae(蚕虫微叶蝉)
Myzus persicae(桃蚜)
Macrosiphum euphorbiae(马铃薯蚜)
Paratrioza cockerelli(potato psyllid)瓜
Bemisia argentifolii(silverleaf whitefly)
Bemisia tabaci(烟粉虱)胡萝卜
Cavariella aegopodii(伞形花二尾蚜)Canola
Brevicoryne brassicae(甘蓝蚜)蔬菜
Aphis fabae(蚕豆蚜)甜菜
Pemphigus popullivenae(甜菜多脉瘿绵蚜)脱叶水果和核果
Dysaphis plantaginea(苹粉红劣蚜)甘蔗
Saccharosydne saccharivora(长稻虱)canefly
Sipha flava(蔗黄伪毛蚜)表4:半翅目(臭虫)番茄
长蝽
Acrostemum hilare(春绿蝽)
Euschistus servus(褐臭蝽)表5:直翅目(蝗虫、蟋蟀和蟑螂)小麦
Melanoplus sanguinipes(迁徒蚱蜢)表6:双翅目(蝇和蚊)蕃茄
Liriomyza trifolii(潜叶虫)
Liriomyza sativae(蔬菜潜叶虫)
Scrobipalpula absoluta(蕃茄潜叶虫)十字花科植物(嫩茎花椰菜、甘蓝、花椰菜、羽衣甘蓝)
Delia brassicae(甘蓝种蝇)
Delia radicum(甘蓝地种蝇)胡萝卜
Psilia rosae(胡萝卜茎潜蝇)甜菜
Tetanops myopaeformis(甜菜根斑蝇)蔬菜
Liviomyza sativae(菜潜叶蝇)表7:缨翅目(蓟马)蕃茄
Frankliniella occidentakis(苜蓿蓟马)
Frankliniella fusca烟草褐蓟马
Thrips tabaci(烟蓟马)十字花科植物(嫩茎花椰菜、甘蓝、花椰菜、羽衣甘蓝)
Thrips tabaci(烟蓟马)胡椒
Thrips palmi(瓜蓟马)马铃薯
Thrips palmi(瓜蓟马)表8:蜱螨目(螨和蜱)马铃薯
Tetranychus urticae(棉红蜘蛛)
Aculops lycopersici(蕃茄叶刺皮瘿螨)
Steneotarsonemus pallidus(樱草狭趺线螨)柑桔
Panonychus citri(桔全爪螨)
Brevipalpus lewisi(桔短须螨)
Phyllocoptrutra oleivora(桔锈螨)脱叶水果和坚果
Panonychus ulmi(苹果红蜘蛛)
Tetranchus属种类(红叶螨)
对于本发明的目的,病害也包括植物的植物真菌病原体。在表9中提供与主要农作物植株相关的真菌病害目录。这样的病害包含在本发明范围之内。表9:植物真菌病穗霉病
赤霉菌穗霉病 玉蜀黍赤霉菌
G.saubinetii
曲霉腐病 黄曲霉
寄生曲霉
色二孢 玉蜀黍色二孢
大孢色二孢
镰孢穗霉病 串珠镰孢
亚粘团串珠镰孢
柄腐病
腐霉柄腐病 瓜果腐霉
炭疽病柄腐病 禾生刺盘孢
C.tucumanensis
Glomerella graminicola色二孢柄腐病 Diplodia maydis(玉蜀黍色二孢)
D.zeae-maydis(玉蜀黍色二孢)
Stenocarpella maydis
Macrodiplodia zeae
Sphaeria maydis
S.zeae
大孢色二孢镰孢柄腐病 串珠镰孢赤霉菌柄腐病 玉蜀黍赤霉菌
G.saubinettiStewart’s萎蔫&叶枯萎 Erwinia stewartii叶疾病南方玉米叶枯萎 Exserohilum turcicum北方玉米叶枯萎 Bipolaris maydis灰叶斑 Cercospora zeae-maydis(玉蜀黍尾孢)
C.sorghi var.maydis(高梁尾孢玉蜀黍变
种)炭疽病叶枯萎 禾生刺盘孢普通锈病 高粱柄锈菌
P.maydis南方锈病 多堆柄锈菌
Dicaeoma polysorum丝黑穗病 丝轴黑粉菌普通黑穗病 玉蜀黍黑粉菌Carbonum叶斑病 炭色长蠕孢眼点 玉蜀黍球梗孢霜霉病高梁霜霉病 Peronoslerospora sorghi褐斑霜霉病 Sclerophthora rayssiae甘蔗霜霉病 Peronosclerospora sacchari菲律宾霜霉病 Peronscler philippinensis爪哇霜霉病 Peronosclerospora maydisSpontaneum霜霉病 Peronosclerospora spontaneaRajasthan霜霉病 Peronosclerospora heteropogoni禾生霜霉病 禾生指梗霉锈病 小麦禾柄锈菌
小麦隐匿柄锈菌
条形柄锈菌黑粉病 小麦腥黑粉菌
小麦矮腥黑粉菌
印度腥黑粉菌
小麦散黑粉菌
小麦条黑粉菌根腐烂、底部腐烂和枯萎 禾顶囊孢
腐霉属种类
大刀镰孢
禾本科镰孢
燕麦镰孢
Drechslere tritici-repentis
丝核菌属种类
Colletotrichum graminicola
长蠕孢属
Microdochium nivale
Pseudocercosporella herpotrichoides霉腐 小麦禾白粉菌
大孢子指疫霉各种真菌病 小麦壳针孢
颖枯壳针孢
芽孢杆菌属种类在营养生长阶段将VIP3类蛋白分泌到培养基中。VIP3A(a)是新发现一类的表现杀昆虫活性的蛋白的成员,表现对广谱鳞翅目昆虫包括小地老虎(Agrotis ipsilon)、草地夜蛾(Spodopterafrugiperda)、非洲夜蛾(S.exigua)、黄条粘虫夜蛾(S.ornithogalli)、西南玉米杆草螟(Diatraea grandiosella)、小蔗杆草螟(D.saccharalis)、棉铃虫(Helicovepa zea)、地中海玉米螟(Sesamia nonagrodies)、粉蚊夜蛾(Trichoplusia ni)、大豆夜蛾(Anticarsia gemmatalis)、菜蛾(Pluteelaxylostella)、烟草芽蛾(Heliothis virescens)的杀昆虫活性。已证明这其中的一些昆虫对其它杀昆虫蛋白如δ-内毒素有很强抗性。例如,所报道的针对小地老虎最有效的δ-内毒素Cry1A(C)的LC50大于6000ng/cm2[maclntosh等,无脊椎动物病理学杂志56:258-266(1990)]。与之相比,只需少260倍的VIP3A(a)蛋白来杀死50%的小地老虎幼虫。因此,VIP3A(a)蛋白表现独特的杀昆虫活性范围。
本发明提供VIP3类蛋白的新成员,即从菌株AB51分离的VIP3A(c)蛋白[于1997年3月28日以保藏号NRRL B-21675保藏,所有保藏按照布达佩斯条约通过交给农业研究机构保藏中心(NRRL),北部研究中心,1815 North University Stree,Peoria,Illinois 61604,USA]公开于SEQ ID NO:5-6。
编码VIP3A(c)蛋白的DNA序列通过使用PCR技术鉴定和分离。具体地,可以制备识别编码的保守或可变区域的引物,然后将其用于筛选从已知或未知菌株得到的DNA样品。
目前公认有很多方法鉴定和分离VIP3类蛋白的同系物和编码它们的DNA序列,这些方法为本领域技术人员熟知。
VIP 3A(a)和VIP3A(c)蛋白的DNA和蛋白序列在表12中比较。
表12:VIP3A(a)(上行)与VIP 3A(c)(下行)的比较1 MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDE 50 SEQ ID NO:2
|||||.||||||||||||||||||||||||||||||||||||||||.|||1 MNKNNAKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLALDE 50 SEQ ID NO:651 ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL 100
||.|||||||||||||||||||||||||||||||||||||||||||||||51 ILENQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL 100101 NDVNNKLDAINTMLRVYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEIS 150
||||||||||||||||||||||||||||||||||||||||||||||||||101 NDVNNKLDAINTMLRVYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEIS 150
151 DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG 200
||||||||||||||||||||||||||||||||||||||||||||||||||
151 DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG 200
201 SPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALK 250
||||| |||.||||||||||.|||||||||||||||||||||||||||||
201 SPADIRDELSELTELAKSVTQNDVDGFEFYLNTFHDVMVGNNLFGRSALK 250
251 TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLAD 300
||||||||||||||||||||||||||||||||||||||||.|||||||||
251 TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTPCRKLLGLAD 300
301 IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVE 350
||||||||||||||||||||||||||||||||||||||||||||||||||
301 IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVE 350
351 AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL 400
||||||||||||||||||||||||||||||||||||||||||||||||||
351 AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL 400
401 CPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI 450
|||||:||||||||||||||||||||||||||||||||||||||||||||
401 CPDQSGQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI 450
451 DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS 500
||||||||||||||||||||||||||||||||||||||||||||||||||
451 DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS 500
501 RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLE 550
||||||||||||||||||||||||||||||||||||||||||||||||||
501 RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLE 550
551 PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT 600
||||||||||||||||||||||||||||||||||||||||||||||||||
551 PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT 600
601 VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILK 650
||||||||||||||||||||||||||||||||||||||||||||||||||
601 VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILK 650
651 SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLY 700
||||||||||||||||||||||||||||||||||||||||||||||||||
651 SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLY 700
701 QGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYM 746
|||||||||||||||||||||||||||||||||||||||||||: :
701 QGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKKDI 746VIP3类蛋白的多肽结构域
已表明VIP3A(a)蛋白与昆虫幼虫的肠液混合时受到蛋白水解切割。当将来自小地老虎的肠液与纯化的VIP3A(a)混合时,可以鉴定出来自VIP3A(a)的四种主要蛋白水解产物,具有约65、45、33和22kDa的分子量。22kDa条带含有VIP3A(a)蛋白的N端从SEQ ID NO:2的氨基酸1至氨基酸198。66kDa条带含有VIP3A(a)蛋白的剩余部分从SEQ IDNO:2的氨基酸200至氨基酸789。45和33kDa条带通过蛋白水解来自66kDa条带并分别具有SEQ ID NO:2的氨基酸412-氨基酸789和氨基酸200-氨基酸455。33kDa是VIP3A(a)蛋白的主要组成成分,在温育超过两小时的时间后仍然保留。此VIP3A(a)蛋白的33kDa“毒性核心”结构域(SEQ ID NO:2的氨基酸200-氨基酸455)保持针对广谱鳞翅目昆虫的完全杀昆虫活性。将VIP3A(a)与从对VIP3A(a)敏感的另一昆虫草地夜蛾分离的肠液一起温育时,得到相同结果。
除了毒性核心结构域,VIP3A(a)蛋白也含有C端的稳定结构域。使用VIP3A(a)蛋白和VIP3A(c)蛋白的突变体发现稳定结构域的作用,当它们被已知对VIP3A(a)敏感的昆虫时摄入时都不表现杀昆虫活性。当用VIP3A(a)突变体、具体地用含有位于羧基端结构域的三个点突变[氨基酸742(E→D);氨基酸770(S→P)和氨基酸784(Y→H)]的VIP3A(a)蛋白突变体针对小地老虎体液中稳定性进行相似研究时,发现蛋白完全水解。对于从AB51分离的VIP 3A(c)(SEQ ID NO:6)蛋白[与VIP3A(a)蛋白有96%的总相同性但缺少VIP3A(a)羧基端]得到相似结果。然而,突变体和VIP3A(a)蛋白都对昆虫细胞系Sf-9有活性。这些结果表明VIP3类蛋白的羧基端结构域提供蛋白在敏感昆虫胃环境中的稳定性。含有VIP3区域和异源区域的杂交毒素
通过制备框架内遗传融合体,可以将毒素、酶、转录因子、抗体、细胞结合组分或其它蛋白结构域与本发明的新型蛋白可操作连接,融合体由核糖体翻译时产生具有VIP和用于融合的其它成分的结合特征的融合蛋白。而且,与VIP融合的蛋白结构域有对另一蛋白、核酸、糖类、脂类或其它化学品或因子的亲和性,然后可以形成三组成成分的复合物。此复合物将具有其所有组成成分的特征。相似原理可用于制备四或更多组成成分的复合物。这些复合物可用作杀昆虫毒素、药品、实验室试剂和诊断试剂等。常用的这样的复合物的例子是用于潜在癌症治疗的融合毒素、ELISA分析中的试剂和免疫印迹分析。
本发明的杂交毒素包括含有与稳定结构域异源的毒性核心结构域的嵌合蛋白。也可通过将免疫识别VIP3受体的抗体或其免疫活性片段与来自其它蛋白的毒性结构域结合来制备杂交毒素。毒性结构域从许多细胞毒性蛋白得到。这些蛋白包括但不限于芽孢杆菌毒素,包括内毒素和植物性杀昆虫蛋白。见例如1993年3月25日递交的美国申请序列号08/037,057和1992年9月25日递交的美国申请序列号07/951,715,此处引用作为参考。其它毒素包括催化性核糖体灭活剂如gelonin、假单胞菌内毒素A或商陆素[假单胞菌内毒素的结构已在Chaudhary等,生物化学杂志265:16303-16310(1990)]中得到很好鉴定;细胞代谢干扰剂如核糖核酸酶[见例如Mariani等,自然347:737-741](1990)];芽孢杆菌RNA酶毒素(或PE-Bar),从假单胞菌内毒素A和核糖核酸酶衍生的嵌合毒素[见Prior等,细胞64:1023(1991)];在膜中产生孔隙的亲水性肽(见Frohlich和Wells,Int.J.Peptide Protein Res.37:2-6(1991))。VIP3A(a)的作用模式
已表明VIP3A(a)蛋白对广谱植物病害有活性。例如组织病理学观察结果表面敏感昆虫如小地老虎(Agrotis ipsilon)和草地夜蛾(Spodopterafrugiperda)以4ng/cm2低浓度摄入VIP3A(a)引起肠麻痹,在大于40ng/cm2浓度时肠上皮细胞的完全溶解导致幼虫死亡。更不敏感的昆虫如欧洲玉米螟(Ostrinia nubilalis)摄入VIP3A(a)时不发展任何病症。而从易感和非敏感昆虫得到的中肠液对VIP3A(a)蛋白的蛋白水解切割是相同的,体内免疫定位研究表明VIP3A(a)结合局限于敏感昆虫的肠细胞。因此,VIP3A(a)的昆虫宿主范围看来取决于其结合肠细胞的能力。组织病理学观察结果表明敏感昆虫的中肠上皮细胞是VIP3A(a)杀昆虫蛋白的首要目标,并且它们随后的溶解是致死时的主要机制。
编程性细胞死亡是自身破坏的活性过程,看来对多细胞生物的发育和维持很重要[Clem,R.J.等,科学254:1388-1390(1991)]。细胞经历凋亡(编程性细胞死亡的一种形式)产生膜结合的凋亡体和活性内源核酸酶,将染色质切成不连续的片段。来自草地夜蛾的SF-9昆虫细胞与VIP3A(a)蛋白接触经历一系列细胞学变化和分子变化包括膜突出、大量空泡形成和凋亡类型编程性细胞死亡的特征核内溶解。组织学研究已证明VIP3A(a)蛋白靶向敏感昆虫的中肠上皮细胞,在细胞溶解和幼虫死亡前启动一系列细胞学变化包括大量空泡形成和膨胀。如DNA片段化的原位检测所揭示的,这些中肠细胞与VIP3A(a)蛋白接触时也经历核内溶解过程。这些结果表明VIP3A(a)通过激发凋亡类型的编程性细胞死亡对敏感昆虫发挥其杀昆虫特性。VIP3A(a)的受体已得到分离
上面提供的免疫组织化学结果表明VIP3A(a)具有与中肠上皮细胞的顶端膜结合的能力,并且这种结合激发最终以细胞溶解结束的进程。这表明存在位于顶端膜的一种或多种蛋白作用为受体识别并与VIP3A(a)结合。此受体具有与VIP3A(a)的相互作用的信号并激发凋亡过程。因此,受体将介导昆虫细胞对VIP3A(a)的反应。
为分离此受体,对从来自小地老虎中肠组织的mRNA制备的cDNA文库进行筛选。筛选的目的是在两元杂交系统中鉴定和分离编码将与VIP3A(a)相互作用的蛋白的cDNA序列(见Fields,S.和Song,O.-K自然340:245-246(1989))。此方法得到其编码蛋白与VIP3A(a)蛋白强烈相互作用的一种cDNA的鉴定和分离。此1.75kb长的cDNA(SEQ ID NO:8)编码约48kDa的蛋白(396个氨基酸,见SEQ ID NO:9)。克隆的cDNA与由Northern分析的编码cDNA的mRNA大小相似。编码头5-20个氨基酸的DNA序列部分可能缺失。在cDNA编码蛋白中可以鉴定到以下特征(见图1):1)它包含信号肽;2)它包含与所谓的致死结构域(Feinstein,E.等,Trends in Biochem.20:342-344(1995))同源的结构域和3)它包含EGF样基序或重复(Fantl,W.J.等,Annu.Rev.Biochem.62:453-481(1993))。使用VIP3A(a)的受体的蛋白数据研究表明其与称为肌腱蛋白(Pearson,C.A.等,EMBO J.7:2677-2681(1988)或Hexabrachion(Nies,D.E.等,J.Biol.Chem.266:2818-2823(1991))的外糖蛋白家族的同源性。此蛋白家族含有EGF样重复、与多个配体结合并在细胞粘着和/或信号转导中起作用。在VIP3受体中观察到的致死结构域和重复的EGF基序在编程性细胞死亡受体中是独特。
另外,VIP3A(a)受体的一部分与所谓的“致死结构域”同源。致死结构域是参与蛋白与蛋白相互作用的60-70氨基酸长的基序,并且存在于具有不同细胞功能的蛋白中(Feinstein,E.等,Trends in Biochem.20:342-344(1995))。已知一些含有致死结构域基序的蛋白成员包括受体与凋亡过程有关。一些例子包括Fas受体(Brakebush,C.等,EMBO J.11:943-950(1992))和肿瘤坏死因子(TNF)(Tartaglia,L.A.等,细胞74:845-853(1993))。
VIP3A(a)受体的同系物可以通过各种方法,例如通过核酸杂交得到鉴定和分离。可以对已经过限制性酶切、琼脂糖电泳并印迹到硝酸纤维素和/或尼龙膜上的来自昆虫或真菌细胞的DNA样品进行Southern印迹分析。在低度严格杂交和洗涤条件下,用编码VIP3A(a)蛋白受体的全长或部分核酸探测Southern印迹。基因可容易地从cDNA或基因组文库克隆并测序。也可以得到所选大小的基因组文库以有助于目的基因的克隆。进行上述实验的技术方案易于得到(见例如分子克隆,实验室手册,第二版,1-3卷,Sambrook等(编辑)冷泉港实验室出版社,冷泉港,NY(1989)和其中引用的参考文献)。VIP 3A(a)及其受体的抗体
本发明提供针对VIP3蛋白或其受体的多克隆和单克隆抗体,包括免疫识别任一上述蛋白部分的其片段。通过利用VIP3蛋白或其受体作为抗原可以制备本发明的抗体和单克隆抗体。
本发明的抗体包括单克隆和多克隆抗体以及保持其与VIP3蛋白或其受体结合能力的其片段。如果抗体、单克隆抗体或其片段能够与分子特异反应由此使分子与抗体、单克隆抗体或其片段结合,那么就说它能结合分子。术语“抗体”(Ab)或“单克隆抗体”(MAb)是指包括完整分子并能结合半抗原的其片段或结合区域或其结构域(如Fab和F(ab)2片段)。这样的片段通常通过蛋白水解切割如木瓜蛋白酶和胃蛋白酶产生。选择性地,半抗体结合片段可以通过重组DNA技术或合成化学技术的应用得到制备。
用于制备本发明的抗体的方法在本领域内公知。例如见抗体,实验室手册,Ed Harlow和Derid Lane(编辑)冷泉港实验室,NY(1988)以及其中引用的参考文献。说明免疫学基本原理的标准参考文献包括Klein免疫学杂志,细胞-非细胞辨别的科学,John Willey&Sons,NY(1982);Dennett,R.等,单克隆抗体,杂交瘤:A New Dimension in BiologicalAnalyses,Plenum Press,NY(1980)和Campbell,A.“单克隆抗体技术”,于生物化学和分子生物学方面实验室技术,13卷,Burdon等(编辑),Elsevier,Amsterdam(1984)。也见美国专利号4,609,893;4,713,325;4,714,681;4,716,117和4,720,459。
可以认为按照此处所述的方法,对特定VIP3蛋白或其受体特异的抗体可以得到制备。具有所需结合特异性的MAb系的亚群可以用作用于克隆特定单克隆抗体cDNA的信使RNA来源。
然后克隆的DNA可以通过本领域已知的方法测序。见例如Sambrook等,分子克隆:实验室手册,第二版,冷泉港实验室出版社,NY(1989)1-3卷以及其中引用的参考文献)。从核酸序列可以推出所选MAb结合区域的蛋白序列。
本发明的抗体和单克隆抗体的一个用途包括但不限于杂交毒素的制备。即连接时单克隆抗体或抗体片段保持其结合特性而毒素组分保持其细胞毒性的特性。
已知各种得到抗体基因的方法,一个方法是在噬菌体中克隆抗体基因的随机文库并筛选文库结合VIP3蛋白或其受体结合的能力。另一可用的方法是制备与VIP3蛋白或其受体结合的单克隆抗体,然后从这些抗体克隆抗体基因。对于本实施例,使用第二种方法。可以使用恒定区和可变区的构架区内保守DNA序列引物从杂交瘤细胞克隆抗体基因。通常见Mullis等,酶学方法155:335-350(1987);Erlich(编辑),PCR技术,Stockton Press(New York 1989)。由Kabat等,US Dept Health andHuman Services,US Government Printing Offices(1991)收集的小鼠重链和轻链序列的数据已用于制备克隆抗体基因的同种型特异的引物和简并的引物(Jones等,Bio/technology 9:88-89(1991))。另外用于克隆具有原始抗体结合特征的抗体更小片段(Fab)的技术众所周知。完整抗体是大分子(150kDa),但已证明小得多的Fab和Fv抗原结合片段(12kDa-50kDa)保持了完全的结合亲和性。其中Vh和VI结构域由亲水和可弯曲肽连接的单链Fv片段(scFv)已成功地用于将酶和毒素靶向特异细胞(Bird,科学423:423-426(1988);Huston,PNAS 85:5879-5883(1988)。称为最小识别单位(m.r.u)的单独Vh结构域(Dabs)和单独的小到20个氨基酸的互补决定区也已用于抗原结合[Ward,自然341:544-546(1989);Taub,J.Biol.Chem 264:259-265(1989);Williams,PNAS 86:5537-5541(1989)]因此,有可能将VIP3或其受体特异的结合结构域减小到非常小的长度。
聚合酶链反应技术和特异寡核苷酸引物用于克隆免疫球蛋白基因或免疫球蛋白基因区域。对IgM和三种IgG同种型的轻链和重链特异的PCR引物从上述的Kabat数据库选出。设计编码NH2-末端和成熟可变区的区域的引物在第一个构架区起始并制备带有一些简并性的引物以使它们用作通用引物。从轻链和重链的第一个恒定区(CH1)和保守序列设计用于可变区特异PCR扩增的3’引物。不同的3’引物用于免疫球蛋白同种型IgG1、IgG3和IgM。IgG2A和IgG2B可以使用用于IgG1的相同引物扩增。将抗体可变区分别克隆入有内质网信号肽和IgG1轻链和重链恒定区的轻链和重链表达载体。用于小鼠免疫球蛋白轻链和重链可变区的PCR克隆的引物序列在文献中公开(Coloma等,Bio/Techniques 11:152-156(1991);Jones等,Bio/Technilogy 9:88-89(1991))。寡核苷酸用如下述标准方法在Applied Biosystems DNA合成仪380B(Applied Biosystems,Foster City,CA)上制备。PCR引物掺入了限制性位点,并且在扩增和消化后可以将其克隆入表达载体中植物可表达启动的控制下。选择已知在测序的抗体基因中不存在的限制性位点。
本发明的单克隆和/或多克隆位点的另一用途包括用抗体靶向Vip3A的受体刺激凋亡。参与控制细胞生长、针对细胞表面定位蛋白产生的抗体相互作用通过防止所述受体与其天然配体结合导致凋亡的诱导。例如,抗APO-1的抗体完全阻断带有APO-1蛋白的白血病细胞的增殖并激发这些细胞中的凋亡(Trauth,B.C.等,科学245:301-305(1989))。而且,通过用针对受体产生的抗体取代配体模拟由特定受体和配体之间相互作用所导致的活性。例如,向在其细胞表面带有Fas受体的细胞加入某些抗Fas的抗体将以与加入Fas受体的配体时相似的模式介导凋亡[Itoh,N.等,细胞66:233-243(1991)]。
从小地老虎分离的Vip3A(a)受体表现与已知为肌腱蛋白、具体地肌腱蛋白X的细胞外糖蛋白的同源性(Bristow,J.等,J.Cell Biol.122:265-278(1993))。已知肌健蛋白X参与细胞与细胞粘着和信号转导。通过突变或通过除去基因使肌健蛋白-X的功能缺失将导致死亡。因此,针对Vip3A(a)的受体产生的抗体有效地阻断受体与其配体的结合或模拟VIP3A(a)蛋白的相互作用激发凋亡。此方法可推延到具有相似生物功能的不同受体。在这个意义上,针对昆虫细胞产生的抗体参与决定性的细胞生长和导致凋亡的诱导的相互作用过程并用作控制昆虫的策略。筛选其作用方式为凋亡的新型杀昆虫活性
在本发明中所述的物质用于筛选具有杀昆虫特征、激发凋亡反应的化学配体。化学配体包括小有机分子、肽和蛋白。在本发明的一个实施方案中,昆虫细胞系用作模式昆虫有机体以筛选由于其诱导凋亡的能力而能杀昆虫的化合物。这些细胞系以大规模筛选模式处理,其中细胞在多孔培养板中生长并与多种化合物接触。酵母也用作模式生物。使用本领域已知或此处所述的方法,测定化合物是否由于诱导凋亡而杀昆虫。
通过与已知受体相互作用鉴定激发凋亡反应的化合物的一种方法是采用参与在凋亡昆虫细胞系中激发的信号转导通路的鉴定的受体。将这些受体转入异源细胞系产生纯合系,其中之一含有用于特定受体表达的基因而另一个不含有或表达这样的基因。这些细胞系以大规模筛选方式处理,由此表达受体的转化细胞通过它们与该受体的特异反应而具有针对激发凋亡的化合物的不同反应。
本发明也包括特异性地鉴定经历凋亡的昆虫细胞系的生物化学和/分子标记的特征鉴定。例如,有可能分离在特定昆虫细胞系内凋亡过程中诱导的特异cDNA。尽管致死核心通路看来是系统发育上保守的[Nagata,S.,细胞88:355-365(1997)],但是从受体到致死核心通路的信号转导通路在生物间有变异。在经历凋亡的昆虫细胞中差异表达的信使RNA通过许多容易使用的技术如差异显示[Bauer,D.等,核酸研究21:4272-4280(1993)]或减法文库[Sommer,H.等,EMBOJ.9:605-613(1990)]来鉴定。差异表达的cDNA编码的蛋白用作特异昆虫细胞系中凋亡的标记。含有编码VIP3类蛋白的DNA序列的转基因植物
表达至少一个本发明序列的宿主植物已加强了对植物病害侵袭的抗性,因此更好地避免了与这样的侵袭相关的农作物损失。对于植物,是指可用通过本领域已知方法遗传转化的任何植物品种。本领域已知的用于植物转化的方法在下面讨论。宿主植物包括但不限于那些先前作为靶农作物列出的物种。植物表达盒
构建植物表达盒以及将外源DNA导入植物的方法学在本领域内得到描述。这样的表达盒可以包含与启动子、终止子、增强子、前导序列、内含子和其它调控序列可操作连接的杀昆虫蛋白编码序列。可以进一步认为VIP3基因的启动子或终止子可用于表达盒。
来自微生物的毒素基因也可能与植物基因不同。植物基因与微生物中发现的基因的区别在于它们的转录RNA不具有确定的与起甲硫氨酸相邻的核糖体结合位点。因此,微生物基因表达可通过在ATG处包含真核生物共有的翻译起始子得到加强[kozak,细胞44:283-292(1986)]。Clontech(1993/1994目录,第210页)已提出了作为保守翻译起始子的序列GTCGACCATG TTC(SEQ ID NO:…)用于在植物中表达大肠杆菌uidA基因。另外,Joshi[核酸研究15:6643-6653(1987)]比较了多种植物与ATG相邻的序列并提出保守序列TAAACAATGGCT(SEQID NO:…)。当微生物ORF在植物中表达遇到困难的情况下,在起始ATG处包含一个这样的序列可以增强翻译。在这样的情况下,由于它们第二个AA残基的修饰,保守序列的最后三个核苷酸可能不适于包含在修饰序列中。优选的与起始甲硫氨酸相邻的序列在不同植物物种之间可以不同。通过研究GenBank EMBL数据库中存在的玉米基因序列,可以区分哪些与ATG相邻的核苷酸应该得到修饰以加强导入玉米的毒素基因的翻译。
另外,已证明去除不合理剪切位点可以加强导入基因的表达和稳定性。从非植物来源克隆的并且未优化以在植物中表达的基因可能包含在植物中可识别为5’或3’剪接位点的基序,因此,转录过程可能非成熟地终止产生截短或缺失的mRNA。可以使用多种技术加工毒素基因以去掉这些不合适的剪切位点。首先潜在剪切位点可以通过DNA序列的计算机分析得到鉴定。与剪切位点有关的保守序列在本领域内已知。见例如Goodall,G.J.和Filipowicz,W.,EMBO J.10,2635-2644(1991)和Brown,J.W.S.,核酸研究14,9549-9559(1986)。选择性地,可以通过将来自基因的cDNA的PCR分析与实际基因产物进行比较来鉴定植物实际的剪切位点。比推测产物更小就是剪切的标志。然后克隆这样的更小产物并对其进行序列测定,确定剪切的确切定位。也认为可以使用计算机搜索和PCR分析的结合。
本发明的新型毒素基因作为其天然序列或作为如上所述优化的合成序列可与植物中表达的启动子包括组成型的、可诱导的、时序调控的、发育调控的、化学调控、组织偏爱和/或组织特异性启动子可操作地融合以制备重组DNA分子即嵌合基因。优选的组成型启动子包括CaMV355和19S启动子(1994年10月4日授予的Fraley等,美国专利5,352,605)。另一优选的启动子来自任一已知在大多数细胞类型中表达的肌动蛋白基因,McElroy等[Mol.Gen.Genet.231:150-160(1991)]描述的启动子。启动子表达盒可以容易地得到修饰以表达新型毒素基因并特别地在单子叶植物宿主中使用。
另一优选的组成型启动子来自另一已知其产物在多种细胞类型中积累的遍在蛋白基因。已从几个物种克隆了遍在蛋白启动子以用于转基因植物(如向日葵-Binet等,植物科学79:87-94(1991);玉米-Christensen等,植物分子生物学12:619-632(1989))。单子叶植物转基因系统中已开发了玉米遍在蛋白启动子,其序列和构建用于单子叶植物转化的载体公开于EPO 342 926。遍在蛋白启动子适于转基因植物中,特别是单子叶植物中新型毒素基因的表达。
用于植物中新型毒素基因表达的启动子是例如组织特异性或组织偏受性启动子如在WO93/07278中公开的启动子、在EP-A 0332 104中公开的化学诱导型启动子,此处全文引用作为参考。
除了启动子,也可得到多种转录终止子用于采用本发明新型毒素基因的嵌合基因构建。转录终止子负责在转基因外终止转录及其正确的多聚腺苷酸化。合适的转录终止子及已知在植物中起作用的那些转录终止子包括CaMV35S终止子、tml终止子、胭脂碱合酶终止子、豌豆rbcS E9终止子及其它本领域已知的终止子。这些终止子可用单子叶和双子叶植物。
已知来自病毒的许多非翻译前导序列如在例如WO 96/10083中报告的那些可增强表达,并且在双子叶植物细胞中尤其有效。具体地,已证明来自烟草花叶病毒(TWV,“Ω-序列”)、玉米褪绿条纹病毒(MCMV)和苜蓿花叶病毒(AMV)的前导序列对增强表达有效[例如Gallie等,核酸研究,15:8693-8711(1987);Skuzeshi等,植物分子生物学15:65-78(1990)]。已证明多种内含子序列在加到5’调控区时增强表达,尤其是在单子叶细胞中。例如,已发现玉米Adhl基因的内含子导入玉米细胞时在其同源启动子作用下显著增强野生型基因的表达(Callis等,基因发育1:1183-1200(1987))。优化vip3基因用于植物表达
本发明的杀昆虫基因可以得到优化以加强植物中的表达。见例如EPA0359472;EPA0385962;WO91/16432和Perlak等,美国国家科学院院报88:3324-3328(1991)。以此方式,优化用于植物表达的编码序列可以得到合成。
在本发明的一个实施方案,根据美国序列号07/951,715中公开的方法制备vip3A(a),此处引用作为参考。在此方法中,使用玉米偏爱的密码子即最频繁编码玉米中氨基酸的单个密码子。编码特定氨基酸的玉米偏爱的密码子可以例如从已知的玉米基因序列推出。来自玉米植物的28种基因的玉米密码子用法见于Murray等,核酸研究17:477-498(1989),其公开内容在此引用作为参考。用玉米优先的密码子制备的合成序列的例子在SEQ ID NO:7(VIP3A(a)),SEQ ID NO:19(VIP3A(b))和SEQ ID NO:20(VIP3A(c))中列出。
以此方式,核苷酸序列可以得到优化以在任何植物中表达。可以认为基因序列的全部或任何部分可以得到优化或合成。即也可以使用合成或部分优化的序列。植物转化
重组DNA分子可以用很多本领域公认的方法导入植物细胞。本领域技术人员将同意,方法的选择可能依赖于要转化的靶植物类型,即单子叶或双子叶植物。转化植物细胞的适当方法例如分别在WO96/10083和WO97/46105中得到描述,包括微注射、电穿孔、土壤土壤杆菌介导的转化、直接基因转化和使用可从Agracetus,Inc.,Madison,Wisconsin和Dupont,Inc.,Wilmington,Delaware得到的仪器的弹粒加速。
优选的实施方案是如欧洲专利申请EP0 292 435和美国专利号5,350,689中公开的玉米原生质体转化方法,此处全文引用作为参考。用于通过微粒轰击将本发明的表达元件盒导入玉米的一套特别优选的实施方案可以在美国专利号5,610,042中找到,此处全文引用作为参考。
植物转化可以用一种DNA分子或多种DNA分子(即共转化)进行。这两种技术都适合用于本发明的表达元件盒。很多转化载体适于植物转化,本发明的表达元件盒可与任何这样的载体结合使用。载体的优选依赖于优选的转化技术和转化的目的品种。
可得到很多用于根瘤土壤杆菌转化的载体。它们通常具有至少一个T-DNA边界序列,并且包括诸如pBIN19的载体[Bevan,核酸研究(1984)]。在一个优选实施方案中,可以将本发明的新型毒素基因插入用于土壤杆菌的双元载体pCIB200和pCIB2001之一,其构建例如公开于WO95/33818(实施例35)(也见EP0 332 104,实施例19)中。
用于土壤杆菌介导的转化的另一载体是双元载体pCIB10,它包含编码用于植物中选择的卡那霉素抗性基因、T-DNA左右边界序列,并且掺入了来自广谱宿主范围质粒pRK252的序列使它在大肠杆菌和土壤杆菌中都能复制。它的构建由Rothstein等[基因53:153-161(1987)]描述。已构建了多种pCIB10的衍生物,它们掺入了由Gritz等[基因25:179-188(1983)]所述的编码码潮霉素B磷酸转移酶的基因。这些衍生物使得可以只用潮霉素(pCIB743)或用潮霉素和卡地霉素(pCIB715,pCIB717)选择转基因植物。
使用直接基因转化或土壤杆菌介导的转化形式的方法常常但不必须采用可以提供对抗生素(如卡那霉素、潮霉素或氨甲喋呤)或除草剂抗性的选择标记进行。然而,用于植物转化的选择标记的选择对本发明并不关键。
对于某些植物品种,优选的是不同的抗生素或除草剂选择标记。转化中常用的选择标记包括赋予对卡那霉素和相关抗生素抗性的nptII基因(messing&Vierra,基因19:259-268(1982);Vevan等,自然304:184-187(1983)),赋予对除草剂膦丝菌素抗性的bar基因(White等,核酸研究18:1062(1990),Spencer等,Theor Appl.Genet.79:625-632(1990)),赋予对抗生素潮霉素抗性的hph基因(Blochinger&Diggelmann,分子细胞生物学4:2929-2931)和赋予对氨甲喋呤抗性的dhfr基因(Bourouis等,EMBO J.2:1099-1104(1983)),允许在甘露糖作为碳源上选择的甘露糖磷酸异构酶基因(EP530 129,WO94/20627)。
用于直接基因转移技术并与用除草剂Basta(或膦丝菌素)选择结合的载体是pCIB3064。此载体基于质粒pCIB246,它包含与大肠杆菌GUS基因可操作融合的CaMV35S启动子和CaMV35S转录终止子并在PCT公开申请WO93/07298中有所描述,此处引用作为参考。另一有用的选择标记通过连接遍在蛋白的启动子、合成的PAT基因和nos终止子得到。含有此标记的载体的一个例子是pCIB9804。
另一转化载体是pSOG35,它利用赋予氨甲喋呤抗性的大肠杆菌基因二氢叶酸还原酶(DHFR)作为选择标记,其构建例如在WO95/33818(实施例35)中描述。
另一转化载体是载体pGL2[Shimamoto等,自然338,274-276(1989)],它包含与35S启动子和35S终止子序列可操作连接的吸水链霉素磷酸转移酶基因(hpt)。
转基因植物也可通过使用可评级标记得到鉴定。用于本发明的可评级标记的例子是β-葡糖醛酸糖苷酶、绿色荧光蛋白以及玉米花色素苷途径的C1和B-peru调控基因。另外表达VIP3蛋白的转基因植物可以在不需可评级或可选择标记的情况下通过筛选它们的杀昆虫活性得到鉴定。
用编码根据上述任一方法所述的VIP3类蛋白、但优选地VIP3A(c)蛋白的DNA序列转化玉米可以容易地通过未成熟合子胚或可连续繁殖的I型胚发生愈伤组织的微粒轰击实现。
对于使用未成熟合子的转化。将未成熟合子胚立即转移到相同但含12%蔗糖的培养基上。4小时后,使用来自BioRad公司的PDS-1000/HE装置用质粒轰击未成熟合子胚。质粒包含可选择标记如赋予对膦丝菌素抗体的基因或可评级标记如绿色荧光蛋白以及根据上面描述制备的用于转移入植物并在植物中表达的编码VIP3类蛋白的基因。基本上按照BioRad公开的方法将质粒沉淀在1μm的金颗粒上。使用1550psi爆发压力(burst pressure)的氦气转移颗粒。每一靶平板用质粒和金颗粒制品轰击两次。既然在本发明的一个实施方案中,质粒含有编码对膦丝菌素抗性的嵌合基因,那么此物质优选地用于体外选择转化的细胞。如果使用,选择试剂在基因转移那天以10mg/L使用,约1个月后增加到40mg/L。如果使用选择标记,这样得到的胚发生愈伤组织可以在选择试剂膦丝菌素存在的情况下再生。从所选的胚发生愈伤组织得到植株。分析再生植物对敏感昆虫的抗性。如通过在植物中使用ELISA分析测定VIP3蛋白所证实的,所有对昆虫有抗体的植物也表达导入的编码VIP3类蛋白的嵌合基因。对昆虫有抗体并表达VIP3蛋白的植物得到了转化。
为了用I型胚发生愈伤组织转化玉米,用标准培养技术从未成熟合子胚获得愈伤。为基因转移,通过用手术刀片切割或通过在基因转移前传代培养3-5天制备约300mg I型愈伤组织。基因转移前,将制备的愈伤组织放在含12%蔗糖的半固体培养基上。约4小时后,用BioRad公司的PDS-1000/He Biolistic装置轰击组织。质粒包含可选择标记如赋予对膦丝菌素抗体的基因或可评级标记如绿色荧光蛋白以及根据上面描述制备的用于转移入植物并在植物中表达的编码VIP3类蛋白的基因。基本上用BioRad标准方法将质粒沉淀在1μm金颗粒上。基因转移后约16个小时,将愈伤组织转移至含2%蔗糖以及如果使用1mg/L可选择标记膦丝菌素的标准培养基中。将愈伤组织选择传代培养8周,然后将存活和生长的愈伤组织转移至标准再生培养基上以产生植株。分析再生植物对敏感昆虫的抗性。如通过在植物中使用ELISA分析测定VIP3蛋白所证实的,所有对昆虫有抗体的植物也表达导入的编码VIP3类蛋白的嵌合基因。对昆虫有抗体并表达VIP3蛋白的植物得到了转化。补充昆虫控制原理
本发明的杀昆虫蛋白可与Btδ-内毒素或其它杀昆虫蛋白联合使用以增加靶昆虫范围。而且本发明的VIP、但优选地VIP3A(c)蛋白与Btδ-内毒素或不同性质的其它杀昆虫原理的联合使用具有防止和/或控制昆虫抗性的特别用处。
根据它们的活性范围和序列相似性,已将来自苏云金芽孢杆菌的不同杀昆虫晶体蛋白分类。按Hfte和Whiteley,微生物学综述53:242-255(1989)提出的分类将已知的杀昆虫晶体蛋白分成四大类。一般地,大类按它们的活性范围来分,Cry1蛋白对鳞翅目有活性,Cry2蛋白对鳞翅目和双翅目都有活性,Cry3蛋白对鞘翅目有活性,Cry4蛋白对双翅目有活性。
在每一大类中,根据序列相似性将δ-内毒素分组。Cry1蛋白典型地以130-140KDa前毒素蛋白合成,并经蛋白酶剪切产生约60-70KDa的活性毒素蛋白。δ-内毒素的活性部分位于全长分子的氨基端部分。Hfte和Whiteley,同上将已知的Cry1蛋白分成六组1Aa,1Ab,1Ac,1B,IC和1D。从那以后,分类命名为Cry1Ea,Cry1Fa,Cry9A,Cry9C和Cry9B的蛋白也得到鉴定。
来自苏云金芽孢杆菌的每个δ-内毒素的杀昆虫活性范围趋于十分狭窄,所给的δ-内毒素只对一些昆虫有效。特异性是由于产生活性毒素蛋白涉及的各个步骤的效率和其随后与昆虫消化道上皮细胞相互作用的能力。
在一个优选的实施方案中,VIP在转基因植物中的表达伴随着一种或多种Btδ-内毒素的表达。特别优选的Btδ-内毒素是在美国申请序列号07/951,715中那些,此处引用作为参考。
众所周知一些来自苏云金芽孢杆菌的δ-内毒素蛋白确实以前毒素表达。这些前毒素在昆虫肠的碱性环境中溶解并被蛋白酶蛋白水解转化成毒性核心片段(Hfte和Whiteley,Microbiol.Rev.53:242-255(1989))。对于Cry1类的δ-内毒素蛋白,毒性核心片段位于蛋白的N端一半。可以用于植物转化载体以赋予宿主植物杀昆虫特性的编码新型毒素蛋白的全长前毒素形式或截短的毒性核心片段的基因在本发明的范围之内。
其它杀昆虫原因包括蛋白酶抑制剂(丝氨酸和半脱氨酸类型)、凝集素、α-淀粉酶、过氧化物酶和胆固醇氧化酶。如美国序列号08/463,483中公开的其它VIP基因如vip1A(a)和vip2A(a)也可用于本发明,此处引用作为参考。
多于一种杀昆虫原理在相同转基因植物中的共表达可以通过遗传操作植物以包含和表达所有必需基因来得到实现。选择性地,一个植物,亲本1可以受到遗传操作以表达VIP。第二植株,亲本2可以受到遗传操作以表达补充的昆虫控制原理。通过亲体1和亲本2的杂交,得到表达所有导入亲本1和2的基因的子代植株。含有VIP3类蛋白和基因的重组微生物
可以认为,可以将本发明的编码VIP3类蛋白,但优选地VIP3A(c)蛋白的分离基因转入任何微生物宿主并赋予宿主杀昆虫特性。可以选择用于本发明的新型基因的可选择宿主以适于克隆目的、适于鉴定基因或编码蛋白的形式和功能的目的、用作提高毒素蛋白产量的发酵宿主、适于更有效地将至少一种毒素蛋白转移入靶昆虫的目的或适于将新型毒素蛋白导入昆虫病原体如杆状病毒(核型多角体病毒,例如苜蓿银纹夜蛾)以增强它们的效力。
可以设想,应将所述可选择宿主施用到环境或植物或动物或宿主中以控制昆虫。可以选择已知占据一种或多种目的农作物的“植物环境”(叶表面、叶环境、根环境和/或根表面)的微生物宿主。选择这些微生物以便能成功地在特定环境中与野生型微生物竞争;提供表达多肽杀昆虫剂的基因的稳定保持和表达、并且更受欢迎地提供更好的杀昆虫剂保护以避免环境降解和失活。
这样的微生物包括细菌、藻类和真菌,特别感兴趣的是以下微生物:例如细菌如芽孢杆菌、柄杆菌、Agmenellum、假单胞菌、欧文氏菌、沙雷氏菌、克雷伯氏菌、黄单胞菌、链霉菌、根瘤菌、红假单胞菌、Methylius、土壤杆菌、醋杆菌、乳酸杆菌、节杆菌、固氮杆菌、明串珠菌、产碱菌;真菌尤其是酵母如酵母属、隐球菌属和短梗霉属。特别感兴趣的是以下植物环境细菌如芽孢杆菌属种类、丁香假单孢菌、荧光假单胞菌、粘质沙雷氏菌、木醋杆菌、土壤杆菌、类球红假单孢菌、野油菜黄单胞菌、苜蓿根瘤菌、真养产碱菌、木棍状杆菌、棕色固氮菌;以及植物环境酵母种类如深红酵母、红酵母、海滨红酵母、橙黄酵母、浅白隐球酵母、流散酵母、掷孢酵母、香气掷孢酵母、佛地酵母和出芽短梗霉。特别感兴趣的是形成色素的微生物。
当含杀昆虫剂的细胞将得到处理以延长细胞中毒素的活性然后将处理的细胞施用于靶病害的环境时,适当宿主细胞可以包括真核生物或原核生物,一般限制于不产生对高等生物如哺乳动物有毒的物质的那些细胞。然而,当毒素是不稳定的或者施用水平足够低以避免对哺乳动物宿主的任何毒性时,可以使用产生对高等生物有毒的物质的生物作为宿主,特别感兴趣的将是原核生物和低等真核生物如真菌。举例性的原核生物(革兰氏阳性和阴性)包括肠杆菌科如欧文氏菌、埃希氏菌、志贺氏菌、沙氏菌和变形杆菌;杆菌科;根瘤菌科如根瘤菌;螺菌科如发光菌、发酵单胞菌、沙雷氏菌、气单胞菌、弧菌、去磺弧菌、螺旋菌;乳酸杆菌科;假单胞菌科如假单胞菌和醋杆菌;固氮菌科和硝杆菌科(Nitrobacteraceae),在真核生物中是真菌如藻状菌纲和子囊菌纲包括酵母如酵母属和裂殖酵母属和担子菌纲酵母如红酵母属、短梗霉属、掷孢酵母属等。
在选择用于生产目的的宿主细胞时特别感兴趣的特征包括将蛋白基因导入宿主的可行性、表达系统的可行性、宿主中蛋白的稳定性和辅助遗传能力的存在。用作杀昆虫剂微胶囊的感兴趣特征包括杀昆虫剂的保护性品质如厚细胞壁、色素形成和细胞内包装或内含体的形式;叶亲和性;无哺乳动物毒性;吸引昆虫摄入的吸引力;不损伤毒素的情况下杀死和固定的可行性。其它考虑因素包括配制和处理的可行性、经济、贮存能力等。
特别感兴趣的宿主包括酵母如红酵母属种类、短梗霉属种类、酵母属种类和掷孢酵母属种类,叶表面生物如埃希氏属种类、乳酸杆菌属种类、芽孢杆菌属种类等。具体微生物包括铜绿短梗霉、荧光假单胞菌、酿酒酵母、苏云金芽孢杆菌、大肠杆菌、枯草杆菌等。
许多方法可用于在基因稳定保持和表达的条件下将表达杀昆虫蛋白的基因导入微生物。例如,可以构建包含与用于DNA构建体表达的转录和翻译调控信号可操作连接的目的DNA构建体和与宿主生物中序列同源的DNA序列的表达盒,因此将发生整合;和/或包含在宿主中有功能的复制系统的表达盒,由此将发生整合或稳定维持。
转录和翻译调控信号包括但不限于启动子、转录起始开始位点、操纵子、激活物、增强子、其它调控元件、核糖体结合位点、起始密码子、终止信号等,见例如美国专利5,039,523;美国专利号4,853,331;EPO0480762A2;Sambrook等,同上,分子克隆,实验室手册,第二版,Maniatis等(编辑)冷泉港实验室出版社,冷泉港,NY(1989)以及其中引用的参考文献)。
可以使用本领域公知的方法将新型基因或其重组形式转化入这样的可选择宿主中。一个这样的优选方法是微生物细胞的电穿孔,按照例如Dower方法所述的(美国专利号5,186,800)。另一优选的方法是Schurter等的方法[Mol.Gen.Genet.218:177-181(1989)],也公开于美国序列号07/353,565中,此处全文引用作为参考。
可以将编码VIP3类蛋白的基因导入在植物上(附生寄生物)或植物中(植物内寄生物)繁殖的微生物中以将VIP3类蛋白传递给潜在靶害虫。许多细菌物种能在植物维管组织中存活。大多数这样的植物内寄生物和附生寄生物看来对植物生长和繁殖的生理学影响极小。
例如根部集群细菌可以通过本领域已知方法从目的植物分离。具体地,可以从植物根分离在根部集群的蜡状芽孢杆菌(例如见J.Handelsman,S.Raffel,E.Mester,L.Wunderlich和C.Grau,应用环境植物学56:713-718(1990))。也可以通过本领域已知的方法将Vip3基因导入根部集群蜡状芽孢杆菌。具体地,可以使用标准方法通过接合,将来自菌株AB88的编码VIP3类蛋白的基因导入根部集群蜡状芽孢杆菌[J.Gonzalez,B.Brown和B.Carlton,美国国家科学院院报79:6951-6955(1982)]。
而且,可以通过电转化将本发明的新型基因导入根部集群芽孢杆菌。例如,可以将vip3A(a)克隆入穿梭载体如pHT3101[D.lereclus等,FEMS Microbiol.letts.,60:211-218(1989)]。然后可以通过电穿孔方法将含有编码序列的穿梭载体pHT3101转化入根部集群芽孢杆菌[D.leveclus等,FEMS Microbiol.letts.,60:211-218(1989)]。也可以使用棉花集群megaterium杆菌。
由植物内寄生菌木棍状菌提供另一例子,来自已知包含引起植物矮化的植物病原细菌的属/种。这种细菌可以在植物的维管系统中生长到非常高的水平。将δ-内毒素导入此植物内寄生菌,当它进入植物时,提供玉米螟的良好控制。其它植物内寄生物也已知。
可以设计表达系统以使VIP3蛋白分泌到革兰氏阴性细菌例如大肠杆菌的细胞质外。使VIP3蛋白分泌的优点是1)它可以提高表达的VIP3蛋白水平,以及2)可以有助于VIP3蛋白的纯化。
可以通过将适当的大肠杆菌信号肽与VIP3信号肽的氨基末端融合或者用大肠杆菌信号肽替换VIP3信号肽来制备例如在大肠杆菌中分泌的VIP3蛋白。被大肠杆菌识别的信号肽可以在已知在大肠杆菌中分泌的蛋白如OmpA中发现[J.Ghrayeb.H,Kimura,M.Takahara,H.Hsiung Y.Masui和M.Inouye,EMMO J.,3.2437-2442(1984)]。OmpA是大肠杆菌外膜的主要蛋白,因此认为其信号肽在转运过程中有效。而且OmpA信号肽不需要象其它信号肽如脂蛋白信号肽可能的那样在操作前进行修饰[G.Duffuad,P.March和M.Inouge酶学方法,153:492(1989)]。
具体地,可以使用本领域已知的标准方法将单一BamHI限制性位点导入VIP编码序列的氨基和羧基末端。可以将这些BamHI片段在框架内克隆入载体pIN-III-ompA1、A2或A3中[J.Ghrayeb.H,Kimura,M.Takahara,H.Hsiung Y.Masui和M.Inouye,EMMO J.,3.2437-2442(1984)],由此产生分泌到壁膜间隙内的ompA:VIP融合基因。在pIN-III-ompA多接头中的另一限制性位点可以用本领域已知的标准方法去除以便VIP3氨基端氨基酸编码序列直接在ompA信号肽切割位点之后。于是,在大肠杆菌中分泌的VIP3序列与天然VIP3序列相同。
当不需要VIP3天然信号肽用于成熟蛋白的适当折叠时,可以除去这样的信号序列并用ompA信号序列代替。可以在前蛋白编码序列的氨基端直接在VIP3信号肽编码序列之后和VIP3编码序列的羧基末端处导入单一BamHI限制性位点。然后可以将这些BamHI片段导入如上所述的pIN-III-ompA载体。
在杀昆虫控制或在基因操作作为杀昆虫试剂的其它生物时使用本发明菌株的基本方法在本领域内已知。见例如美国专利号5,039,523和EP0480762A2。
VIP3可以在细菌宿主中发酵产生,并且以与苏云金芽孢杆菌用作杀昆虫喷酒剂相同的方式对所得细菌进行加工并用作微生物喷洒剂。以从芽孢杆菌分泌的VIP3为例,使用本领域已知的方法除去分泌信号或进行突变。这样的突变或删除防止在发酵过程中VPI3蛋白分泌到生长培养基中。VIP3蛋白可以保留在细胞中,然后对细胞进行加工以产生胶囊化的VIP3蛋白。任何适当的微生物都可用于此目的。假单胞菌已用于表达作为胶囊化蛋白的苏云金芽孢杆菌内毒素,并且对所得的细胞进行加工并作为杀昆虫剂喷洒(H.Gaertner等,1993,于AdvancedEngineered Pesticides,L.Rim编)。
以此方式使用各种苏云金芽孢杆菌菌株。这样的Bt菌株产生内毒素蛋白和VIP3。选择性地,这样的菌株可以仅产生VIP3。已证明枯草杆菌的孢子形成缺陷菌株产生高水平的苏云金芽孢杆菌Cry 3A内毒素(Agaisse,H.和Lereclus,D.,“在枯草杆菌中表达苏云金芽孢杆菌Cry 3A毒素蛋白不依赖孢子形成特异的δ因子并且在spoOA突变体中增加”,细菌学杂志176:4743-4741(1994))。相似的spoOA突变体也可以在苏云金芽孢杆菌中制备并用于产生不分泌到培养基中、只保留在细胞内的胶囊化的VIP3。
本发明要保护的靶作物包括例如下列植物物种:谷物(小麦、大麦、黑麦、燕麦、水稻、高粱及相关作物),甜菜(甜菜和饲料甜菜),饲料草(果园草、羊茅等),梨果,核果和软果(苹果、梨、李、桃、杏、樱桃、草莓、木莓和黑莓),豆科植物(菜豆、小扁豆、豌豆、大豆),油料植物(油菜、芥子、罂栗属植物、橄榄、向日葵、椰子、蓖麻、可可豆、落花生),瓜类植物(南瓜、黄瓜、甜瓜),纤维植物(棉花、亚麻、大麻、黄麻),柑橘属植物(橘子、柠檬、葡萄柚、柑橘),蔬菜(菠菜、莴苣、芦笋、卷心菜及其它芸苔属蔬菜、洋葱、蕃茄、土豆、辣椒),月桂属植物(鳄梨、胡萝卜、樟属植物、樟树),落叶树和针叶树(如菩提树、紫松树、橡树、赤杨、白杨、桦树、冷杉、落叶松、杉树)或诸如玉米、烟草、坚果、咖啡、甘蔗、茶、蛇麻草、香蕉和天然橡胶的植物以及观赏植物(包括复合植物)。
已经过遗传修饰含有杀昆虫基因和蛋白的微生物可以用于保护农业作物和产品免受病害。在本发明的一个方面,将细胞施用给靶害虫的环境时,用延长细胞中产生的毒素活性的试剂处理产生毒素的生物的完整即未裂解的细胞。
选择性地,通过将异源基因导入细胞宿主生产杀昆虫剂。异源基因的表达直接或间接地导致杀昆虫剂的细胞内合成和保持。然后在将细胞施用给靶害虫的环境时,在延长细胞中产生的毒素活性的条件下处理这些细胞。所得产物保持毒素的毒性。然后可以按照用于向生活着靶害虫的环境如土壤、水和叶表面施用的常规技术对这些天然胶囊化的杀昆虫剂进行配制。见例如EPA0192319,此处引用作为参考。
本发明的活性成分一般以组合物形式施用并可以与其它化合物同时或连续施用给要处理的农作物区域或植株。这些化合物可以是肥料或微量营养物供体或其他影响植物生长的制品。它们也可以是选择的除草剂、杀昆虫剂、杀真菌剂、杀细菌剂、杀线虫剂、杀软体动物剂或几种这样制品的混合物,如果需要,可进一步与在配制技术中常规使用的农学上可接受的载体、表面活性剂或施用促进佐剂结合。适当的载体和佐剂可以是固体或液体,并符合配制技术中一般使用的物质,例如天然的或再生的矿物质、溶剂、分散剂、润湿剂、粘着剂、粘合剂或肥料。
施用本发明的活性成分或本发明的含有至少一种由本发明的细菌菌株产生的杀昆虫蛋白的农业化学组合物的优选的方法是叶面施用、种子包裹和土壤施用。施用方法和比率取决于相应病害侵染的密度。含有苏云金芽孢杆菌菌株的杀昆虫组合物
本发明进一步提供含有重组苏云金芽孢杆菌菌株或其衍生物或突变体的杀昆虫组合物,苏云金芽孢杆菌菌株以重组形式含有至少一种新型毒素基因和农学上的佐剂如载体、表面活性剂或施用促进佐剂。组合物也可以包含进一步的选自肥料、微量营养物供体、其他影响植物生长的制品、除草剂、杀昆虫剂、杀真菌剂、杀细菌剂、杀线虫剂、杀软体动物剂或其混合物的生物活性化合物。按重量比,组合物可以包含0.1-99%的以重组形式含有至少一种新型基因的重组苏云金芽孢杆菌菌株或其衍生物或突变体、1-99.9%的固体或液体佐剂和0-25%的表面活性剂。以重组形式含有至少一种新型基因的重组苏云金芽孢杆菌菌株或含有其的组合物可以与某一其它杀昆虫剂或化学品(1993农作物保护化学品参考手册,化学品和药品出版社,加拿大)结合施用给要保护植物或农作物而不失去效力。其可以与大多数其它通用的农业喷洒物质相容但不应在强碱性喷洒溶液中使用。其可以以粉末、悬浮液、可湿性粉末或任何对农业应用适当的其它物质形式施用。
以重组形式含有至少一种新型基因的重组苏云金芽孢杆菌菌株以组合物的形式施用,也可与其它生物活性化合物同时或连续施用给要处理的农作物区域或植株。这些化合物可以是肥料或微量营养物供体或其他影响植物生长的制品。它们也可以是选择的除草剂、杀昆虫剂、杀真菌剂、杀细菌剂、杀线虫剂、杀软体动物剂或几种这样制品的混合物,如果需要,可进一步与在配制技术中常规使用的载体、表面活性剂或施用促进佐剂结合。适当的载体和佐剂可以是固体或液体,并符合配制技术中一般使用的物质,例如天然的或再生的矿物质、溶剂、分散剂、润湿剂、粘着剂或肥料。以重组形式含有至少一种新型基因的重组苏云金芽孢杆菌菌株作为活性成分的配制物,即杀昆虫组合物、制品或其与其它活性成分和适当的固体或液体佐剂的混合物,可按已知的方法制备,例如通过将活性成分与填料如溶剂、固体载体和某些情况下的表面活性化合物(表面活性剂)均匀混合和/或研磨来配制。
在配制技术中常规使用并可在本发明内适当使用的溶剂、载体、表面活性剂、表面活性化合物公开于例如WO96/10083中。
本发明的杀昆虫组合物另一特别优选的特征是当施用于植物和土壤时活性成分的持久性。引起活性丧失的可能原因包括由于紫外光、热、叶分泌物和PH的失活。例如,在高PH,尤其存在还原剂时,δ-内毒素晶体溶解,从而变得更容易受到蛋白水解失活。叶的高PH值也可能是重要的,特别是在叶表面PH可以在8-10的范围内的地方。本发明的杀昆虫组合物配方可以通过加入添加剂帮助防止活性成分的丧失或将物质胶囊化,以此方法保护活性成分免于失活来解决这些问题。胶囊化可以用化学方法(McGuire和Shasha,1992)或生物方法(Barnes和Cunmings,1986)进行。化学胶囊化涉及用聚合物包裹活性成分的步骤,而生物胶囊化涉及在微生物中表达δ-内毒素基因,对于生物胶囊化,在配方中使用包含毒素蛋白的完整微生物作为活性成分。加紫外线防护剂可以有效地减少辐射损伤,由于热引起的失活也可以通过加入合适的添加剂控制。
杀昆虫组合物通常含有0.1%-99%、优选地0.1-95%的以重组形式含有至少一种新型基因的苏云金芽孢杆菌菌株或与其它其活性成分的混合物;1-99.9%固体或液体佐剂和0-25%、优选地0.1-25%的表面活性剂。同时商业产品优选地配制成浓缩物,而最终使用者一般将使用实际上更低浓度的稀释配制物。杀昆虫组合物也可能包含进一步成分,比如稳定剂、消泡剂、粘度调节剂、粘合剂、粘着剂、肥料或其它活性成分以得到特殊的效果。控制昆虫的方法
鉴于本发明的上述描述,很明显有几种单独或与补充昆虫控制原理如δ-内毒素与VIP类蛋白结合使用作为杀昆虫原理控制昆虫的方法。任何传递VIP3蛋白给敏感昆虫摄入的方法将导致该昆虫的控制。
在本发明的一个实施方案中,根据要控制的昆虫性质,用编码VIP3类蛋白的基因转化植物。蛋白的表达可以在植物生长发育的任何时间出现。例如,根据本发明,VIP3类蛋白可以在根、茎、叶、种子、花粉等器官中表达。这提供了优势,即蛋白仅在靶昆虫食用的那些细胞或组织中表达。使敏感昆虫摄入表达VIP3蛋白的植物细胞或组织将导致此昆虫的控制。在本发明的一个实施方案中,VIP3蛋白在植物的茎或柄中表达以控制小地老虎。植物可以田间或温室条件下生长。含有VIP3蛋白的种子在贮存期间可以得到针对昆虫破坏的保护。
实施例
WO 96/10083的73~82页的实施例16~18描述了苏云金芽孢杆菌菌株AB88和AB424的分离和生物鉴定、VIP3A(a)蛋白的纯化和鉴定、vip3A(a)和vip3A(b)基因的克隆以及通过杂交对新vip基因的鉴定。上述诸实施例在此全文引用作为参考。下面实施例进一步描述用于实施本发明的材料和方法以及随后的结果。这些实施例以举例说明的方式提供,不应将其引用视为对本发明的保护范围的限定。实施例1:芽孢杆菌分离物中vip3样基因和VIP3样蛋白的存在
当对采用的培养物上清进行测试时,芽孢杆菌(而不是AB88)表现出针对鳞翅目幼虫的杀昆虫活性。分析一些对小地老虎有活性的分离物中vip3样基因的存在和VIP3样蛋白的产生。
采用标准PCR分析以确定对小地老虎有活性的芽孢杆菌分离物是否含有vip3样基因。采用PCR引物对GW 110(5’-CGA TTA ATG TTGGCC TC-3’;SEQ ID NO:17)和GW111(5’-CAT TAG CAT CTC CGGACA CAG-3’;SEQ ID NO:18)确定了所有对小地老虎有活性的分离物都产生728bp的vip3基因产物,与菌株类型AB88所产生的大小相等。一个对小地老虎无活性的芽孢杆菌分离物AB51产生相同大小的vip3产物。而其它对小地老虎无活性的芽孢杆菌分离物均未产生vip3 PCR产物。
用标准Western印迹方法进行VIP3蛋白产物的分析。用上述实施例中描述的针对VIP 3A(a)蛋白产生的抗体来检测免疫反应蛋白。将每份来自生成孢子的培养物的无细胞培养物上清用标准方法在SDS-PAGE凝胶中电泳。然后,进行标准Western印迹操作以确定是否存在VIP3样蛋白。所有具有728bp PCR产物并对小地老虎有抗性的芽孢杆菌分离物都产生了对VIP 3A(a)抗体有免疫反应的80kDa蛋白。具有正确大小的vip3 PCR产物但对小地老虎无活性的AB51分离物产生了截短的免疫反应蛋白,表明这一点可能就是观察到对小地老虎无生物活性的原因。实施例2:包含vip3样基因的苏云金芽孢杆菌株AB51的鉴定
称为AB51的苏云金芽孢杆菌株,用兔多克隆抗Vip3A(a)的抗体进行Western分析表明其包含VIP3类的蛋白。将vip3样基因克隆到pKS中产生pCLB7112。该基因命名为vip3A(c)。vip3A(c)的DNA序列在SEQID NO:5中公开,其编码蛋白序列在SEQ ID NO:6中公开。VIP3A(c)蛋白长746个氨基酸,比其VIP3A(a)和VIP3A(b)同系物短43个氨基酸。实施例3:针对VIP3A(a)蛋白的抗体的诱发
在兔和山羊体内制备针对纯化的Vip3A(a)杀昆虫蛋白的抗血清。对于兔,将硝酸纤维素结合的蛋白(50μg)溶于DMSO中,用弗氏完全佐剂(Difo)乳化,并在三个内每月皮下注射两次。对于山羊,三个月内每月肌肉内注射两次活性可溶纯Vip3A蛋白(300μg)。在第二次和第三次注射后10天对它们抽血,并从血样中回收血清(Harlow,E.和Lane,D.抗体:实验室手册,冷泉港实验室出版社,NY,1988)。然后,用葡萄球菌蛋白A通过亲和层析对抗血清进行分级分离,并将得到的IgG组分通过含有固定的大肠杆菌裂解物的柱过滤来进一步纯化(Yu,C.G.等,应用环境微生物学63:532-536(1997))。
通过Western印迹分析Vip3A(a)蛋白来鉴定兔和山羊抗血清。蛋白通过SDS/PAGE分离并转移到硝酸纤维素上。硝酸纤维印迹在20mMTris-HCl,PH7.5/0.15M NaCl/0.02%NaN3/5%脱脂奶粉中封闭。分别使用200ng/ml或100ng/ml浓度的兔产生的或山羊产生的抗Vip3A(a)的抗体呈现印迹。碱性磷酸酶偶联的羊抗兔IgG或兔抗羊血清以1μg/ml浓度用作二抗(Kirkegaard & Perry Labratories,Inc.)。溴氯吲哚磷酸盐和硝基蓝四唑物用作碱性磷酸酶反应的底物。兔和山羊产生的抗Vip3A(a)的抗体是多克隆的。从山羊得到的抗Vip3A(a)的抗体比从兔得到的抗体效价更高。在实验过程中,来自兔的抗Vip3A(a)的抗体应以原始血清的1/500稀释(200ng/ml)使用。作为对比,来自山羊的抗Vip3A(a)的抗体可从原始血清稀释不大于1/2000(100ng/ml)。兔产生的抗体仅仅识别Vip3A(a)蛋白的N末端部分,而来自山羊的抗体与Vip3A(a)蛋白全长上存在的表位反应。实施例4:植物表达盒的构建
植物表达盒包括可以组成型或以组织特异性方式驱动编码序列的表达的启动子、要表达的编码序列和允许mRNA多腺苷酸化以及其正确翻译的终止序列。
本发明的DNA构建体中选择的启动子包括:组成型启动子如来自玉米遍在蛋白基因的启动子(Christensen等,植物分子生物学12:619-632,1989)(pCIB8029,图4;pCIB8055,图5;pCIB9806,图6)以及组织特异性启动子如提供根优先表达来自玉米金属硫蛋白样基因的启动子(de Framond,A.FEBS 290:103-106,1991)(pCIB8030,图7;pCIB8056,pCIB9805)、提供绿色组织特异表达来自玉米PEPC基因的启动子(Hudspeth,R.L.和Grula,J.W.,植物分子生物学12:579-589,1989)(pCIB5535,图8;pCIB9807)和提供茎优先表达来自大麦非特异性脂转移蛋白LTP4的启动子(pCIB9819,图9)(Molina,A.和Garcia-Olmedo,F.植物杂志,4:983-991,1993)。为增强基因表达的目的,本发明中采用的所有构建体都包含由35S CaMV衍生的终止序列和从玉米PEPC基因衍生的内含子9。质粒pCIB8029、pCIB8055和pCIB9806包含位于玉米遍在启动子和vip3A(a)基因之间的玉米遍在基因的内含子#1。以BamHI-EcoRI双酶切将包含vip3A(a)基因的编码序列、内含子9和35S终止序列的构建体加工到具有不同启动子的受体质粒中。
将植物表达盒本身用于植物转化试验或使用在质粒骨架的AmpR基因中切割的限制性酶将它们切割成线形。在一些试验中,通过限制性酶切和片段纯化将包含启动子、目的基因、内含子和终止子的片段从质粒骨架的其它部分分离出来。在这些情况下,片段纯化如下操作:用适当酶对500μg的DNA进行酶切,并在0.8%琼脂糖凝胶上分离。鉴定目的片段,从凝胶上切下并用Durapore Millipone滤膜(0.45μm)纯化。包含片段的滤液用乙酸钠和乙醇沉淀。片段重新悬浮于TE中并用于转化试验。实施例5:表达VIP3A(a)的玉米植株的杀昆虫活性
用下述方式测试了表达VIP3A(a)蛋白的玉米植株对下表列出的昆虫种类的杀昆虫效果。从转基因和对照玉米植株的叶子上切下1~4cm小块。将每个叶块置于50×9mm培养皿中的湿润滤纸圆片上。每个叶片上放五只测试物种的新生虫,每一种植物共测试5~20只幼虫。培养皿放在黑暗中于30℃培养。48~72小时后评估死亡率。结果示于表16。表16
实施例6:玉米植株中的vip3A(a)表达
测试的昆虫种类 | 死亡率百分率 | |
VIP3A(a) | 对照 | |
玉米害虫小地老虎(Agrotis ipsilon)草地夜蛾(Spodoptera frugiperda)小蔗杆草螟(Diatrea saccharalis)西南玉米杆草螟(Diatraea grandiosella)棉铃虫(Helicoverpa zea)地中海玉米螟(Sesamia nonagroides) | 100100100100100100 | 00001015 |
其它鳞翅目害虫非洲夜蛾(S.Exigua)黄条粘虫夜蛾(S.Omithogalli)粉纹夜蛾(Trichoplusia ni) | 100100100 | 0020 |
采用I型愈伤组织的颗粒轰击实现了具有Vip3基因的玉米良种Ciba近交系CG00526和2154的转化。对于使用I型胚发生愈伤组织的转化,用标准培养技术从合子胚得到愈伤组织并在轰击前传代培养1~2天。将~20、直径3~5mm块以环形排布置于含有12%蔗糖的培养基中,制备用于轰击的愈伤组织。在轰击前将愈伤组织置于此培养基中四小时。用于玉米愈伤组织转化的DNA是含有在不同植物启动子控制下的Vip3基因的圆形质粒DNA、线形质粒DNA或纯化的DNA片段。在采用可选择试剂的试验中,基因允许对膦丝菌素的抗性或允许在有甘露糖的情况下生长。根据来自Biorad实验室,Hercules,CA的已发表的方法,将通过过滤分离的质粒或DNA片段沉淀在0.3μm金颗粒上。金颗粒用650psi爆发压力的氦转移。每个靶板用DNA包裹的颗粒轰击两次。轰击后16~20个小时,将CG00526愈伤组织转移到标准保持培养基上。轰击后7天将组织转移到含有100mg/L浓度选择试剂Basta的培养基上。Basta是Hoechst制造的glufosinate ammonium的商业制品。轰击后1-7天将2154的愈伤组织保持在12%蔗糖中1~7天,并在第7天转移到含20~30mg/L Basta的标准培养基中。愈伤组织2154和CG00526分别在含30或100mg/L Basta的情况下传代培养八周。将存活选择的组织在更低水平的Basta(5-40mg/L)上传代培养约5-8周时间以使组织长大,然后转移到无选择压力的标准再生培养基上以产生植株。一般地,轰击制备的转化愈伤组织中有12%的愈伤块在Basta选择下存活。每个转化愈伤组织通常再生产生20-30株植株。
从没有使用选择的实验得到一些结果。在这些实验中,转移至再生培养基之前,愈伤组织在保持培养基上生长9-10周。结果1337是通过筛选植物杀昆虫活性从没有可选择的或可评级的标记的转化实验得到的转化的VIP3结果的例子。
转化的愈伤组织也从使用甘露糖选择的实验得到制备。在这些转化中,在pCIB9818的玉米遍在蛋白启动子控制下的磷酸甘露糖异构酶与Vip3基因一起轰击。在保持培养基中包含0.5-1.5%的甘露糖,时间为12周,在再生培养基中不包含甘露糖。
通过昆虫生物测定和ELISA分析评估转基因植物VIP3A(a)蛋白的表达。从2-4叶阶段的植物取叶块用于使用小地老虎和草地夜蛾生物分析的评估。使用放在装有叶块的平板中的新孵出的幼虫进行生物测定。同时使用标准方法通过ELISA对来自转基因植物的组织进行不同植物组织中Vip3蛋白水平的定量。对植物组织进行抽提,表17提供了得到的代表性结果和它们相应的昆虫生物分析结果。
用含有在各种启动子如玉米PEP羧化酶启动子(PEPC)、玉米遍在蛋白启动子(Vbi)和玉米金属硫蛋白样启动子(MTL)的控制下的Vip3基因的各种质粒转化转基因的玉米植株。选择标记基因是在pUBIAC中的玉米遍在蛋白启动子控制下的PAT基因。表17中所列的代表性结果表明用不同质粒或来自质粒的DNA片段得到的结果。用限制性酶消化并在0.8%琼脂糖凝胶中通过电泳进行大小分离来制备DNA片段。从凝胶上切下DNA片段、破碎并通过经过0.45微米DuraporeMillipore滤膜的过滤然后乙醇沉淀来纯化。用含有在玉米PEPC启动子控制下的Vip3基因的pCIB5535的环状质粒DNA得到转化的玉米结果。也用含有在玉米遍在蛋白启动子控制下的Vip3基因的pCIB5535和pCIB8029的线形质粒DNA转化。其它结果通过轰击只含有Vip3基因和启动子的纯化DNA限制性酶切片段得到。对应于Vip3基因的片段包括:来自pCIB5535带有玉米PEPC启动子的4906bp EcoRI/HindIII片段;来自pCIB8030带有MTL启动子的5142bp kpnI/HindIII片段;带有玉米遍在蛋白启动子的pCIB8029的4597bp kpnI/HindIII片段;带有玉米遍在蛋白启动子的pCIB8055的4818bp HindIII片段;带有MTL启动子的pCIB8056的5364bp HindIII片段;带有MTL启动子的pCIB9805的5964 Ascl片段;带有玉米遍在蛋白启动子的pCIB9806的5418bp Ascl片段和带有玉米PEPC启动的pCIB9807的5727bp Ascl片段。表17
实施例7:含有Vip3和Bt的δ-内毒素的玉米植株的杀昆虫活性
结果数 | 所用质粒 | 嵌合基因 | 死亡率 | |
草地夜蛾 | 小地老虎 | |||
891 | pCIB5535 | PEPC:vip3A(a) | 100 | 100 |
906 | pCIB5535和pCIB8029 | PEPC:vip3A(a)和Ubi:vip3A(a) | 100 | 100 |
946 | pCIB5535和pCIB8030 | PEPC:vip3A(a)和MTL:vip3A(a) | 100 | 100 |
VIP3A(a)对欧洲玉米螟(ECB)只有极小的活性。为制备具有广谱鳞翅目控制能力的植物,使含有vip3A(a)基因的玉米植株与含有对ECB有活性的cry1B的玉米植株杂交。如实施例1所述对杂交子代进行针对ECB和草地夜蛾(FAW)的生物侧定。结果在表18中表示。约34%的子代对两个物种都没有活性,15.4%仅对ECB有活性,23.1%仅对FAW有活性,并且27.9%对两个物种都有活性。使用其它的Btδ-内毒素、具体地Cry1Ab或Cry9C得到相同结果。表18
实施例8 VIP3A(a)溶解敏感昆虫的中肠上皮细胞喂养和中肠排空研究
杂交 | %ECB活性 | %FAW活性 | %ECB&FAW活性 | %无活性 |
VIP3A(a)×Cry1B | 15.4 | 23.1 | 27.9 | 34.6 |
从起始施予到幼虫死亡,对敏感昆虫小地老虎(BCW)二龄幼虫摄入含VIP3A(a)的食物后的症状时间顺序进行记录。与对照食物接触的幼虫表现活跃进食并保持未受影响的肠平衡。相反,食物中VIP3A(a)蛋白的加入对进食行为有显著影响。当以4ng/cm2低浓度加入时,在10-20分钟时期内断断续续地进食。昆虫中肠内蓝颜色的存在表明进食,但通过粪便数量判断中肠内容物的排空受到剧烈影响。随着每cm2 4ngVIP3A(a)加入食物,48小时培养后幼虫发育受到明显损害,但没有观察到死亡率。以每cm2 40ng Vip3A(a)浓度时,幼虫摄入分钟量食物即患中肠麻痹,并且没有观察到粪便,表明几乎完全缺乏中肠排空。在此条件下,48小时后记录到50%死亡率。当使用高于每cm2 40ng VIP3A(a)浓度时,幼虫仅吃几口后就变得垂死,没有粪便并且死亡率达100%。当用另一敏感昆虫草地夜蛾进行相似实验时,观察到相似行为模式。相反,在以每cm2 400ng VIP3A(a)蛋白的高浓度向食物加入VIP3A(a)蛋白时,欧洲玉米螟不改变其进食行为。Vip3A(a)蛋白影响的组织学观察。
对已摄入含VIP3A(a)的食物的二龄和三龄幼虫进行VIP3A(a)蛋白对BCW影响的组织病理学观察,BCW肠横切片的分析表明了对中肠上皮的严重影响,证明中肠组织是蛋白五作用的原始位置。在前肠和后肠没有观察到损伤。来自未处理幼虫的中肠上皮细胞互相紧密结合,没有显示出损伤的证据。来自已用含有Vip3A(a)的食物喂养24小时的幼虫的切片表明柱状上皮细胞的顶部已变得膨胀和隆起。尽管杯状细胞表现一些形状学变化,但它们在此阶段未表现损伤症状。柱状上皮细胞持续这样衰退,摄入含Vip3A(a)食物48小时后,内腔充满了破碎的细胞碎片。杯状细胞48小时后也表现损伤症状,但两种类型的细胞仍粘在基膜上。小地老虎幼虫在72小时时死亡,上皮层完全脱落。对于草地夜蛾,观察到相似组织病理学结果,欧洲玉米螟在相似实验条件下不表现任何组织损伤。Vip3A(a)蛋白的体内免疫定位
在加有每cm2100-200ng VIP3A(a)的人工食物上喂养的三龄小地老虎和欧洲玉米螟用于VIP3A(a)与中肠结合切片的免疫细胞化学鉴定。用事先已通过蛋白A Sepharose和大肠杆菌固定柱纯化的兔抗VIP3A(a)抗体使结合的VIP3A(a)显现出来(Yu,C.G.等,应用环境微生物学63:532-536,1997)。在中肠上皮检测到VIP3A(a)结合,而没有表现与欧洲玉米螟中肠的结合。来自用对照食物喂养的小地老虎的中肠切片没有表现VIP3A(a)结合。VIP3A(a)结合看来与顶端微绒毛特异相关,并且与柱状细胞最相关,在杯状细胞中没有可检测到的信号。实施例9昆虫细胞中VIP3A(a)和VIP3A(b)诱导凋亡
通过VIP3A(a)和VIP3A(b)对来自草地夜蛾(对VIP3A(a)敏感的昆虫)的昆虫细胞系(Sf-9)的杀昆虫效应的特征鉴定,表明VIP3A(a)和VIP3A(b)是凋亡诱导蛋白。当VIP3A(a)在整个实验过程中保持存在时,它表现对昆虫细胞的杀昆虫活性,当SF-9昆虫细胞与VIP3A(a)和VIP3A(b)短暂接触时,它们的细胞存活力甚至在短到5分钟接触时间的情况下也显著降低。通过显微镜监测与VIP3A(a)短暂接触的SF-9细胞中出现的细胞学变化。在处理细胞与VIP3A(a)蛋白接触后10-15分钟之间的某一时间,小突起出现在处理细胞的表面。在此阶段,用在具有活性膜潜力的线粒体中积累的染料罗丹明123染色揭示,细胞线粒体保持功能完整(Johnson,L.V.等,美国国家科学院院报77:990-994,1980)。这些突起最后消失,细胞进入持续另外30-60分钟的大量空泡形成阶段。在此最后阶段,观察到细胞在瓦解前膨胀。对于每个细胞,整个过程需要1-2小时。尤其考虑到某些昆虫变形过程中编程性细胞死亡伴随着细胞空泡形成和膨胀,所有这些细胞事件与以往对经历凋亡的细胞的研究一致(Schwartz,L.M.等,美国国家科学院院报90:980-984(1993))。
最近研究表明在经历凋亡的动物细胞早期阶段,质膜中的磷脂分布受到影响(Martin,S.J.等,实验医学杂志182:1545-1556,1996),特别是磷脂酰丝氨酸(PS)的表面化。此过程可以通过使用Annexin V(对磷脂酰丝氨酸有高亲和性的抗凝血蛋白)显现出来。当VIP3A(a)处理的SF-9细胞与Annexin V一起培养时,在早至与VIP3A(a)接触5-10分钟后,就在昆虫细胞膜中发现PS的表面化,表明凋亡的开始。
关键分子事件之一即凋亡的特点是核内溶解,导致双链DNA断裂释放200碱基对寡核小体大小的片段并成倍增加。我们使用原位测定方法并通过琼脂糖凝胶电泳分析DNA,从而检测用VIP3A(a)处理的SF-9细胞中核内溶解的存在。采用因DNA片段化产生的DNA末端、基于Klenow酶掺入修饰的核苷酸的能力,SF-9昆虫细胞在早至与VIP3A(a)蛋白接触30分钟时就表现核内溶解迹象。此阶段与显微镜观察中显现的膜结合亚细胞凋亡体的出现相符。这些核内溶解活性的早期指示特征通过在琼脂糖凝胶中检测DNA片段得到证实,染色质梯度的特征在此过程中稍后出现。这些结果证实了从细胞学观察获得的迹象,即SF-9一旦与VIP3A(a)蛋白接触就启动凋亡型编程性细胞死亡。
基于它们针对一些鳞翅目的杀昆虫特征,发现了VIP3A(a)和VIP3A(b)蛋白。因此我们有兴趣知道VIP3A(a)蛋白是否会在摄入其的敏感昆虫的肠细胞中诱导凋亡通路,并因此将通过激发细胞死亡的活性过程发挥其杀昆虫特征。组织学和组织化学研究已表明VIP3A(a)蛋白特异地靶向敏感昆虫中肠上皮柱状细胞,激起特征在于膜突起和大量空泡形成的细胞变化,导致细胞死亡。这些由昆虫肠细胞中VIP3A(a)诱导的细胞学变化类似于上述对于SF-9细胞所述的那些。然后我们通过DNA片段化的原位测定[Cuvillier等,自然381:800-803(1986)]检测是否敏感昆虫的上皮细胞一经摄入含有VIP3A(a)的食物就经历凋亡。当中肠组织切片来自用含有VIP3A(a)的食物或对照食物喂养的小地老虎幼虫时,DNA片段的核染色指示只能在与VIP3A(a)蛋白接触的中肠柱状细胞中检测到。此结果表明蛋白在中肠上皮细胞中诱导与前述细胞变化同时发生的核内溶解过程。我们的结论是VIP3A(a)蛋白很可能通过激活敏感昆虫中肠上皮细胞的凋亡型编程性细胞死亡来发挥其杀昆虫特性。实施例10:从小地老虎分离VIP3A(a)的受体
小地老虎对VIP3A(a)敏感,因此将此昆虫用于分离VIP3A(a)受体。通过解剖收集三龄小地老虎幼虫的中肠并立即在液氮中冷冻。通过遵循Clontech(1997)提供的二元杂交cDNA文库构建试剂盒中所述的方法,用1g中肠组织分离mRNA。10μg polyA+RNA用作起始材料。在第一链合成中,用MML反转录酶并在分开的合成中使用随机和锚定的oligo(dT)25d(A/C/G)引物。第二链cDNA合成用在第二链酶组合中适当比例的DNA聚合酶和RNaseH活性实现。然后将新合成的双链cDNA与EcoR1-NotI-SalI接头连接。将cDNA连入提供活化结构域的pGAD10(Vijaychander,S.等,CLONTECHnigues IX-3:8-10,1994)。将vip3A(a)基因导入质粒pGBT9的多接头位点,在含有GAL4-DNA结合结构域的框架内(Bartel,P.L.等,发育中的细胞相互作用:实用方法,第153-179页Oxford University Press,1993)。通过电穿孔(Estruch,J.J.等,生物技术16:610-612,1994)将重组pGBT9-vip3A(a)转化入酵母菌株GGYl:171C(Gill,G.和Ptashne,M.,细胞51:121-126,1987)。在没有色氨酸的基本培养基中选择转化的酵母(Bartel,P.L.等,发育中细胞相互作用:实用方法,第153-179页,Oxford University Press,1993)。VIP3A(a)蛋白在重组酵母中的表达通过Western分析证实。用pGAD10中所示的小地老虎cDNA文库转化酵母菌株GGYl:171-VIP3A(a)。GGYl:171具有GAL4识别位点控制下的HIS3标记。HIS3基因允许用2个相互作用杂合构建体转化的克隆的阴性生长选择。用多于200,000的重组克隆涂板后,只有一个能在没有组氨酸的基本培养基中生长。通过酵母裂解缓冲液方法[Kaiser,P.和Auer,B.,生物技术14:552(1993)]分离阳性酵母克隆的质粒DNA并将其电击入大肠杆菌。将含有cDNA的插入亚克隆入pBluescript(stratagere)的EcoRI位点并用Sanger等,美国国家科学院院报74:5463-5467(1997)的双脱氧终止法、使用PRISM备好反应染料双脱氧终止循环测序试剂盒和PRISM测序酶终止双链DNA测序试剂盒进行测序,并在ABI373自动测序仪上分析。
鉴定编码与Vip3A相互作用的蛋白的克隆的另一方法包括涂布用pGAD10中所示的小地老虎cDNA文库转化的酵母。在将所述转化的酵母种群转移到硝酸纤维素膜上后,用生物素标记的Vip3A蛋白对它们进行筛选,然后与含有与碱性磷酸酶(AP)偶联的抗生素蛋白链霉素的溶液一起温育。充分洗涤后,表达具有结合Vip3A能力的蛋白的克隆通过用底物5-溴-4-氯-3-吲哚磷酸(BCIP)结合硝基蓝四唑物(NBT)来显现。AP催化不溶性沉淀的形成,沉淀在硝酸纤维素滤膜上显色为黑色。实验过程在Sambrook等,分子克隆:实验手册,第12.21-12.24页,冷泉港实验室,1989中详细描述。在重新过夜培养酵母平板后,将滤膜中显色的点与原始平板中的酵母克隆比较。培养这些克隆,分离它们的质粒并如上述鉴定它们的插入片段。实施例11用受体基因转化的昆虫细胞在与VIP3A(a)蛋白接触时表现凋亡
将小地老虎中肠细胞中的VIP3A(a)蛋白的受体克隆入Smart2粘粒载体的XhoI-BamHI中[Speek,M等,基因64:173-177(1988)],用磷酸钙共沉淀法使用重组载体转化Schneider 2(S2)果蝇细胞[Clem,R.J.和Miller,L.K.Mol.Cel.Biol.14:521 2-5222(1994)]。Smart 2带有用于细菌转化的标记tet(四环素)和用于果蝇细胞转化的neo(新霉素)。neo可选择标记在果蝇hsp70启动子的控制下表达。转化的S2细胞在加有10%胎血清白蛋白和G418(1mg/ml)的S2果蝇培养基中于30℃选择(见GIBCO目录,1997)。在上述培养基中选择45天之后,建立稳定转化的S2细胞系。通过向含有转化的S2细胞的培养基加入VIP3A(a)蛋白(以每ml 1.7mg的终浓度)来测试转化的S2细胞对VIP3A(a)的敏感性,培养基中所含的转化的S2细胞已事先在42℃热击30分钟。转化的S2细胞中凋亡的诱导通过显微镜观察并通过TACS试剂盒以及在原位凋亡检测试剂盒中证实(详细描述见Trevigen目录1996)。实施例12:从其它昆虫分离受体同系物
小地老虎中肠上皮细胞有由VIP3A(a)蛋白识别的受体。通过在Southern分析中鉴定DNA序列从已知对VIP3A(a)敏感的其它昆虫分离受体。制备DNA、酶切、在琼脂糖凝胶中电泳并印迹到硝酸纤维素和/或尼龙滤膜上。使用低严格杂交和洗涤条件,用编码来自小地老虎的受体的cDNA探测滤膜。与小地老虎VIP3A(a)受体有低于50%相似性的基因得到鉴定。以分离含有相似结构域的基因为目的,Southern分析也可以探测编码特异结构域如致死结构域或EGF样基序的cDNA部分序列,尽管它们功能与VIP3A(a)的小地老虎受体不同。
通过Fields,S.和Song,O.-K.自然340:245-246(1989)中描述的二元杂交系统实现VIP3A(a)小地老虎受体同系物的分离。从目的生物获得分离的mRNA,合成cDNA并将它们克隆入pGAD10或相应质粒。用带有vip3A(a)基因(或此基因同系物)的pGB9共转化cDNA文库并通过活化基于VIP3A(a)蛋白(或同系物)和推测受体间蛋白-蛋白相互作用的标记搜索酵母中推测受体。
通过从目的生物分离cDNA文库,将其克隆入适当表达载体并转化入已知不具有与VIP3A(a)结合的能力和/或对VIP3A(a)敏感的宿主细胞如酵母或昆虫细胞中。基于它们与VIP3A(a)一起温育时得到的结合VIP3A(a)或经历凋亡反应的特性筛选转化细胞。在此情况下,蛋白VIP3A(a)用作探针,其结合通过针对VIP3A(a)的抗体或通过标鉴如与VIP3A(a)结合的抗生素来监测。实施例13筛选诱导昆虫细胞中凋亡的新型化合物
将不同目的昆虫的模式细胞系(一些例子包括鳞翅目的Sf-9细胞、鞘翅目的马铃薯甲虫、双翅目来自果蝇的S2)用于筛选作用方式为凋亡诱导的新型化合物。细胞在多孔培养板上生长用于筛选上千种化合物(大分子量和小分子量)的大量分析。将作为单一或成分或作为混合物的化合物加入。诱导凋亡的化合物如下鉴定:1)细胞膜中可见膜突起,2)通过使用与可见标记连接的对磷脂酰丝氨酸有高亲和性的特异蛋白如Annexin-V可检测到含有磷脂酰丝氨酸的膜脂的重建,3)细胞质中可见空泡形成,4)通过使用在线粒体中积累的生命染料如罗丹明口3可见活性线粒体,5)通过琼脂糖凝胶中DNA分析、通过核小体释放的ELISA检测或通过DNA缺口的体内检测可检测到DNA的片段化。所有这些细胞学和分子特征是凋亡的指示特征。
将小地老虎VIP3A(a)的受体转化入S2细胞系。因此可得到含有和不含有该受体的同源S2细胞系。将这些细胞系用于筛选由于所述受体的存在提供不同反应的化合物。一经与某些化合物接触就经历凋亡的转化S2细胞如上述得到鉴定。转化与非转化的不同反应是化合物作用由克隆的受体介导的指示特征。对用小地老虎VIP3A(a)的受体的受体同系物转化的昆虫细胞进行相似操作。
本说明书提及的所有公开文献和专利申请代表本发明涉及的领域中普通技术人员的技术水平。所有公开文献和专利申请在此相同程度地引用作为参考,即每个单独公开文献或专利申请都具体和单独地指定引用的参考。
尽管为了清楚理解的目的,前述本发明已通过举例说明和实施例的方法得到详细描述,但是很明显某些变化和修改的实际应用在所附的权利要求书范围之内。
序列表(1)基本信息
(i)申请人:NOVARTIS AG
(A)街道:Schwarzwaldallee 215
(B)城市:Basel
(C)国家:瑞士
(D)邮政编号:4058
(E)电话:+41 61 324 1111
(F)传真:+41 61 322 75 32
(ii)发明名称:植物病害控制
(iii)序列数:20
(iv)计算机可读形式
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release#1.0,版本#1.30(2)关于SEQ ID NO:1的信息:
(i)序列特征
(A)长度:2378个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线形
(ii)分子类型:DNA(基因组)
(iii)假设:否
(ix)特征:
(A)名称/关键:CDS
(B)位置:9..2375
(B)其它信息:/备注=“pCIB7104中所包含的来自AB88的编码VIP3A(a)蛋白的天然DNA序列”
(xi)序列描述:SEQ ID NO:1:AGATGAAC ATG AAC AAG AAT AAT ACT AAA TTA AGC ACA AGA GCC TTA CCA 50
Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro
1 5 10AGT TTT ATT GAT TAT TTT AAT GGC ATT TAT GGA TTT GCC ACT GGT ATC 98Ser Phe Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile15 20 25 30AAA GAC ATT ATG AAC ATG ATT TTT AAA ACG GAT ACA GGT GGT GAT CTA 146Lys Asp Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu
35 40 45ACC CTA GAC GAA ATT TTA AAG AAT CAG CAG TTA CTA AAT GAT ATT TCT 194Thr Leu Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser
50 55 60GGT AAA TTG GAT GGG GTG AAT GGA AGC TTA AAT GAT CTT ATC GCA CAG 242Gly Lys Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln
65 70 75GGA AAC TTA AAT ACA GAA TTA TCT AAG GAA ATA TTA AAA ATT GCA AAT 290Gly Asn Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn
80 85 90GAA CAA AAT CAA GTT TTA AAT GAT GTT AAT AAC AAA CTC GAT GCG ATA 338Glu Gln Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile95 100 105 110AAT ACG ATG CTT CGG GTA TAT CTA CCT AAA ATT ACC TCT ATG TTG AGT 386Asn Thr Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser
115 120 125GAT GTA ATG AAA CAA AAT TAT GCG CTA AGT CTG CAA ATA GAA TAC TTA 434Asp Val Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu
130 135 140AGT AAA CAA TTG CAA GAG ATT TCT GAT AAG TTG GAT ATT ATT AAT GTA 482Ser Lys Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val
145 150 155AAT GTA CTT ATT AAC TCT ACA CTT ACT GAA ATT ACA CCT GCG TAT CAA 530Asn Val Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln
160 165 170AGG ATT AAA TAT GTG AAC GAA AAA TTT GAG GAA TTA ACT TTT GCT ACA 578Arg Ile Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr175 180 185 190GAA ACT AGT TCA AAA GTA AAA AAG GAT GGC TCT CCT GCA GAT ATT CTT 626Glu Thr Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu
195 200 205GAT GAG TTA ACT GAG TTA ACT GAA CTA GCG AAA AGT GTA ACA AAA AAT 674Asp Glu Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn
210 215 220GAT GTG GAT GGT TTT GAA TTT TAC CTT AAT ACA TTC CAC GAT GTA ATG 722Asp Val Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met
225 230 235GTA GGA AAT AAT TTA TTC GGG CGT TCA GCT TTA AAA ACT GCA TCG GAA 770Val Gly Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu
240 245 250TTA ATT ACT AAA GAA AAT GTG AAA ACA AGT GGC AGT GAG GTC GGA AAT 818Leu Ile Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn255 260 265 270GTT TAT AAC TTC TTA ATT GTA TTA ACA GCT CTG CAA GCC CAA GCT TTT 866Val Tyr Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Gln Ala Phe
275 280 285CTT ACT TTA ACA ACA TGC CGA AAA TTA TTA GGC TTA GCA GAT ATT GAT 914Leu Thr Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp
290 295 300TAT ACT TCT ATT ATG AAT GAA CAT TTA AAT AAG GAA AAA GAG GAA TTT 962Tyr Thr Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe
305 310 315AGA GTA AAC ATC CTC CCT ACA CTT TCT AAT ACT TTT TCT AAT CCT AAT 1010Arg Val Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn
320 325 330TAT GCA AAA GTT AAA GGA AGT GAT GAA GAT GCA AAG ATG ATT GTG GAA 1058Tyr Ala Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu335 340 345 350GCT AAA CCA GGA CAT GCA TTG ATT GGG TTT GAA ATT AGT AAT GAT TCA 1106Ala Lys Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser
355 360 365ATT ACA GTA TTA AAA GTA TAT GAG GCT AAG CTA AAA CAA AAT TAT CAA 1154Ile Thr Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln
370 375 380GTC GAT AAG GAT TCC TTA TCG GAA GTT ATT TAT GGT GAT ATG GAT AAA 1202Val Asp Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys
385 390 395TTA TTG TGC CCA GAT CAA TCT GAA CAA ATC TAT TAT ACA AAT AAC ATA 1250Leu Leu Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile
400 405 410GTA TTT CCA AAT GAA TAT GTA ATT ACT AAA ATT GAT TTC ACT AAA AAA 1298Val Phe Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys415 420 425 430ATG AAA ACT TTA AGA TAT GAG GTA ACA GCG AAT TTT TAT GAT TCT TCT 1346Met Lys Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser
435 440 445ACA GGA GAA ATT GAC TTA AAT AAG AAA AAA GTA GAA TCA AGT GAA GCG 1394Thr Gly Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala
450 455 460GAG TAT AGA ACG TTA AGT GCT AAT GAT GAT GGG GTG TAT ATG CCG TTA 1442Glu Tyr Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu
465 470 475GGT GTC ATC AGT GAA ACA TTT TTG ACT CCG ATT AAT GGG TTT GGC CTC 1490Gly Val Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu
480 485 490CAA GCT GAT GAA AAT TCA AGA TTA ATT ACT TTA ACA TGT AAA TCA TAT 1538Gln Ala Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr495 500 505 510TTA AGA GAA CTA CTG CTA GCA ACA GAC TTA AGC AAT AAA GAA ACT AAA 1586Leu Arg Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys
515 520 525TTG ATC GTC CCG CCA AGT GGT TTT ATT AGC AAT ATT GTA GAG AAC GGG 1634Leu Ile Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly
530 535 540TCC ATA GAA GAG GAC AAT TTA GAG CCG TGG AAA GCA AAT AAT AAG AAT 1682Ser Ile Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn
545 550 555GCG TAT GTA GAT CAT ACA GGC GGA GTG AAT GGA ACT AAA GCT TTA TAT 1730Ala Tyr Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr
560 565 570GTT CAT AAG GAC GGA GGA ATT TCA CAA TTT ATT GGA GAT AAG TTA AAA 1778Val His Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys575 580 585 590CCG AAA ACT GAG TAT GTA ATC CAA TAT ACT GTT AAA GGA AAA CCT TCT 1826Pro Lys Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser
595 600 605ATT CAT TTA AAA GAT GAA AAT ACT GGA TAT ATT CAT TAT GAA GAT ACA 1874Ile His Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr
610 615 620AAT AAT AAT TTA GAA GAT TAT CAA ACT ATT AAT AAA CGT TTT ACT ACA 1922Asn Asn Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr
625 630 635GGA ACT GAT TTA AAG GGA GTG TAT TTA ATT TTA AAA AGT CAA AAT GGA 1970Gly Thr Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly
640 645 650GAT GAA GCT TGG GGA GAT AAC TTT ATT ATT TTG GAA ATT AGT CCT TCT 2018Asp Glu Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser655 660 665 670GAA AAG TTA TTA AGT CCA GAA TTA ATT AAT ACA AAT AAT TGG ACG AGT 2066Glu Lys Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser
675 680 685ACG GGA TCA ACT AAT ATT AGC GGT AAT ACA CTC ACT CTT TAT CAG GGA 2114Thr Gly Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly
690 695 700GGA CGA GGG ATT CTA AAA CAA AAC CTT CAA TTA GAT AGT TTT TCA ACT 2162Gly Arg Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr
705 710 715TAT AGA GTG TAT TTT TCT GTG TCC GGA GAT GCT AAT GTA AGG ATT AGA 2210Tyr Arg Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg
720 725 730AAT TCT AGG GAA GTG TTA TTT GAA AAA AGA TAT ATG AGC GGT GCT AAA 2258Asn Ser Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys735 740 745 750GAT GTT TCT GAA ATG TTC ACT ACA AAA TTT GAG AAA GAT AAC TTT TAT 2306Asp Val Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr
755 760 765ATA GAG CTT TCT CAA GGG AAT AAT TTA TAT GGT GGT CCT ATT GTA CAT 2354Ile Glu Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His
770 775 780TTT TAC GAT GTC TCT ATT AAG TAA 2378Phe Tyr Asp Val Ser Ile Lys
785(2)关于SEQ ID NO:2的信息:
(i)序列特征
(A)长度:789个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线型
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:2:Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe1 5 10 15Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp
20 25 30Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu
35 40 45Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys
50 55 60Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn65 70 75 80Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln
85 90 95Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr
100 105 110Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val
115 120 125Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys
130 135 140Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val145 150 155 160Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile
165 170 175Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr
180 185 190Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu
195 200 205Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val
210 215 220Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly225 230 235 240Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile
245 250 255Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr
260 265 270Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Gln Ala Phe Leu Thr
275 280 285Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr
290 295 300Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val305 310 315 320Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala
325 330 335Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys
340 345 350Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr
355 360 365Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp
370 375 380Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu385 390 395 400Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe
405 410 415Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys
420 425 430Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly
435 440 445Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr
450 455 460Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val465 470 475 480Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala
485 490 495Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg
500 505 510Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile
515 520 525Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile
530 535 540Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr545 550 555 560Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His
565 570 575Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys
580 585 590Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His
595 600 605Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn
610 615 620Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr625 630 635 640Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu
645 650 655Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys
660 665 670Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly
675 680 685Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg
690 695 700Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg705 710 715 720Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser
725 730 735Arg Glu Val Leu Phe Giu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val
740 745 750Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu
755 760 765Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr
770 775 780Asp Val Ser Ile Lys785(2)关于SEQ ID NO:3的信息:
(i)序列特征
(A)长度:2612个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线形
(ii)分子类型:DNA(基因组)
(iii)假设:否
(ix)特征:
(A)名称/关键:CDS
(B)位置:118..2484
(B)其它信息:/备注=“来自AB424的编码VIP3A(b)的天然DNA序列”
(xi)序列描述:SEQ ID NO:3:ATTGAAATTG ATAAAAAGTT ATGAGTGTTT AATAATCAGT AATTACCAAT AAAGAATTAA 60GAATACAAGT TTACAAGAAA TAAGTGTTAC AAAAAATAGC TGAAAAGGAA GATGAAC 117ATG AAC AAG AAT AAT ACT AAA TTA AGC ACA AGA GCC TTA CCA AGT TTT 165Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe790 795 800 805ATT GAT TAT TTC AAT GGC ATT TAT GGA TTT GCC ACT GGT ATC AAA GAC 213Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp
810 815 820ATT ATG AAC ATG ATT TTT AAA ACG GAT ACA GGT GGT GAT CTA ACC CTA 261Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu
825 830 835GAC GAA ATT TTA AAG AAT CAG CAG CTA CTA AAT GAT ATT TCT GGT AAA 309Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys
840 845 850TTG GAT GGG GTG AAT GGA AGC TTA AAT GAT CTT ATC GCA CAG GGA AAC 357Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn
855 860 865TTA AAT ACA GAA TTA TCT AAG GAA ATA TTA AAA ATT GCA AAT GAA CAA 405Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln870 875 880 885AAT CAA GTT TTA AAT GAT GTT AAT AAC AAA CTC GAT GCG ATA AAT ACG 453Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr
890 895 900ATG CTT CGG GTA TAT CTA CCT AAA ATT ACC TCT ATG TTG AGT GAT GTA 501Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val
905 910 915ATG AAA CAA AAT TAT GCG CTA AGT CTG CAA ATA GAA TAC TTA AGT AAA 549Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys
920 925 930CAA TTG CAA GAG ATT TCT GAT AAG TTG GAT ATT ATT AAT GTA AAT GTA 597Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val
935 940 945CTT ATT AAC TCT ACA CTT ACT GAA ATT ACA CCT GCG TAT CAA AGG ATT 645Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile950 955 960 965AAA TAT GTG AAC GAA AAA TTT GAG GAA TTA ACT TTT GCT ACA GAA ACT 693Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr
970 975 980AGT TCA AAA GTA AAA AAG GAT GGC TCT CCT GCA GAT ATT CGT GAT GAG 741Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Arg Asp Glu
985 990 995TTA ACT GAG TTA ACT GAA CTA GCG AAA AGT GTA ACA AAA AAT GAT GTG 789Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val
1000 1005 1010GAT GGT TTT GAA TTT TAC CTT AAT ACA TTC CAC GAT GTA ATG GTA GGA 837Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly
1015 1020 1025AAT AAT TTA TTC GGG CGT TCA GCT TTA AAA ACT GCA TCG GAA TTA ATT 885Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile1030 1035 1040 1045ACT AAA GAA AAT GTG AAA ACA AGT GGC AGT GAG GTC GGA AAT GTT TAT 933Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr
1050 1055 1060AAC TTC CTA ATT GTA TTA ACA GCT CTG CAA GCA AAA GCT TTT CTT ACT 981Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe Leu Thr
1065 1070 1075TTA ACA CCA TGC CGA AAA TTA TTA GGC TTA GCA GAT ATT GAT TAT ACT 1029Leu Thr Pro Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr
1080 1085 1090TCT ATT ATG AAT GAA CAT TTA AAT AAG GAA AAA GAG GAA TTT AGA GTA 1077Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val
1095 1100 1105AAC ATC CTC CCT ACA CTT TCT AAT ACT TTT TCT AAT CCT AAT TAT GCA 1125Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala1110 1115 1120 1125AAA GTT AAA GGA AGT GAT GAA GAT GCA AAG ATG ATT GTG GAA GCT AAA 1173Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys
1130 1135 1140CCA GGA CAT GCA TTG ATT GGG TTT GAA ATT AGT AAT GAT TCA ATT ACA 1221Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr
1145 1150 1155GTA TTA AAA GTA TAT GAG GCT AAG CTA AAA CAA AAT TAT CAA GTC GAT 1269Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp
1160 1165 1170AAG GAT TCC TTA TCG GAA GTT ATT TAT GGC GAT ATG GAT AAA TTA TTG 1317Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu
1175 1180 1185TGC CCA GAT CAA TCT GGA CAA ATC TAT TAT ACA AAT AAC ATA GTA TTT 1365Cys Pro Asp Gln Ser Gly Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe1190 1195 1200 1205CCA AAT GAA TAT GTA ATT ACT AAA ATT GAT TTC ACT AAA AAA ATG AAA 1413Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys
1210 1215 1220ACT TTA AGA TAT GAG GTA ACA GCG AAT TTT TAT GAT TCT TCT ACA GGA 1461Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly
1225 1230 1235GAA ATT GAC TTA AAT AAG AAA AAA GTA GAA TCA AGT GAA GCG GAG TAT 1509Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr
1240 1245 1250AGA ACG TTA AGT GCT AAT GAT GAT GGG GTG TAT ATG CCG TTA GGT GTC 1557Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val
1255 1260 1265ATC AGT GAA ACA TTT TTG ACT CCG ATT AAT GGG TTT GGC CTC CAA GCT 1605Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala1270 1275 1280 1285GAT GAA AAT TCA AGA TTA ATT ACT TTA ACA TGT AAA TCA TAT TTA AGA 1653Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg
1290 1295 1300GAA CTA CTG CTA GCA ACA GAC TTA AGC AAT AAA GAA ACT AAA TTG ATC 1701Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile
1305 1310 1315GTC CCG CCA AGT GGT TTT ATT AGC AAT ATT GTA GAG AAC GGG TCC ATA 1749Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile
1320 1325 1330GAA GAG GAC AAT TTA GAG CCG TGG AAA GCA AAT AAT AAG AAT GCG TAT 1797Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr
1335 1340 1345GTA GAT CAT ACA GGC GGA GTG AAT GGA ACT AAA GCT TTA TAT GTT CAT 1845Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His1350 1355 1360 1365AAG GAC GGA GGA ATT TCA CAA TTT ATT GGA GAT AAG TTA AAA CCG AAA 1893Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys
1370 1375 1380ACT GAG TAT GTA ATC CAA TAT ACT GTT AAA GGA AAA CCT TCT ATT CAT 1941Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His
1385 1390 1395TTA AAA GAT GAA AAT ACT GGA TAT ATT CAT TAT GAA GAT ACA AAT AAT 1989Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn
1400 1405 1410AAT TTA GAA GAT TAT CAA ACT ATT AAT AAA CGT TTT ACT ACA GGA ACT 2037Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr
1415 1420 1425GAT TTA AAG GGA GTG TAT TTA ATT TTA AAA AGT CAA AAT GGA GAT GAA 2085Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu1430 1435 1440 1445GCT TGG GGA GAT AAC TTT ATT ATT TTG GAA ATT AGT CCT TCT GAA AAG 2133Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys
1450 1455 1460TTA TTA AGT CCA GAA TTA ATT AAT ACA AAT AAT TGG ACG AGT ACG GGA 2181Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly
1465 1470 1475TCA ACT AAT ATT AGC GGT AAT ACA CTC ACT CTT TAT CAG GGA GGA CGA 2229Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg
1480 1485 1490GGG ATT CTA AAA CAA AAC CTT CAA TTA GAT AGT TTT TCA ACT TAT AGA 2277Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg
1495 1500 1505GTG TAT TTC TCT GTG TCC GGA GAT GCT AAT GTA AGG ATT AGA AAT TCT 2325Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser1510 1515 1520 1525AGG GAA GTG TTA TTT GAA AAA AGA TAT ATG AGC GGT GCT AAA GAT GTT 2373Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val
1530 1535 1540TCT GAA ATG TTC ACT ACA AAA TTT GAG AAA GAT AAC TTC TAT ATA GAG 2421Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu
1545 1550 1555CTT TCT CAA GGG AAT AAT TTA TAT GGT GGT CCT ATT GTA CAT TTT TAC 2469Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr
1560 1565 1570GAT GTC TCT ATT AAG TAAGATCGGG ATCTAATATT AACAGTTTTT AGAAGCTAAT 2524Asp Val Ser Ile Lys
1575TCTTGTATAA TGTCCTTGAT TATGGAAAAA CACAATTTTG TTTGCTAAGA TGTATATATA 2584GCTCACTCAT TAAAAGGCAA TCAAGCTT 2612(2)关于SEQ ID NO:4的信息:
(i)序列特征
(A)长度:789个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线型
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:4:Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe1 5 10 15Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp
20 25 30Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu
35 40 45Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys
50 55 60Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn
65 70 75 80Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln
85 90 95Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr
100 105 110Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val
115 120 125Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys
130 135 140Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val145 150 155 160Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile
165 170 175Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr
180 185 190Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Arg Asp Glu
195 200 205Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val
210 215 220Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly225 230 235 240Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile
245 250 255Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr
260 265 270Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Lys Ala Phe Leu Thr
275 280 285Leu Thr Pro Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr
290 295 300Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val305 310 315 320Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala
325 330 335Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys
340 345 350Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr
355 360 365Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp
370 375 380Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu385 390 395 400Cys Pro Asp Gln Ser Gly Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe
405 410 415Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys
420 425 430Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly
435 440 445Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr
450 455 460Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val465 470 475 480Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala
485 490 495Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg
500 505 510Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile
515 520 525Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile
530 535 540Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr545 550 555 560Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His
565 570 575Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys
580 585 590Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His
595 600 605Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn
610 615 620Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr625 630 635 640Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu
645 650 655Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys
660 665 670Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly
675 680 685Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg
690 695 700Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg705 710 715 720Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser
725 730 735Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val
740 745 750Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu
755 760 765Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr
770 775 780
Asp Val Ser Ile Lys785(2)关于SEQ ID NO:5的信息:
(i)序列特征
(A)长度:2364个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线形
(ii)分子类型:DNA(基因组)
(iii)假设:否
(ix)特征:
(A)名称/关键:CDS
(B)位置:56..2295
(B)其它信息:/产物=“VIP3A(c)蛋白的”
(xi)序列描述:SEQ ID NO:5:AATACAATTT ACGAGGGATA AGTGTTACAA AGAATAGCTG AGGAGGGAGA TGAAC ATG 58
Met
1AAC AAG AAT AAT GCT AAA TTA AGC ACA AGA GCC TTA CCA AGT TTT ATT 106Asn Lys Asn Asn Ala Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe Ile
5 10 15GAT TAT TTC AAT GGC ATT TAT GGA TTT GCC ACT GGT ATC AAA GAC ATT 154Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile
20 25 30ATG AAC ATG ATT TTT AAA ACG GAT ACA GGT GGT GAT CTA GCC CTA GAC 202Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Ala Leu Asp
35 40 45GAA ATT TTA GAG AAT CAG CAG CTA CTA AAT GAT ATT TCT GGT AAA TTG 250Glu Ile Leu Glu Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys Leu50 55 60 65GAT GGG GTG AAT GGA AGC TTA AAT GAT CTT ATC GCA CAG GGA AAC TTA 298Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn Leu
70 75 80AAT ACA GAA TTA TCT AAG GAA ATA TTA AAA ATT GCA AAT GAA CAA AAT 346Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln Asn
85 90 95CAA GTT TTA AAT GAT GTT AAT AAC AAA CTC GAT GCG ATA AAT ACG ATG 394Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr Met
100 105 110CTT CGG GTA TAT CTA CCT AAA ATT ACC TCT ATG TTG AGT GAT GTA ATG 442Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val Met
115 120 125AAA CAA AAT TAT GCG CTA AGT CTG CAA ATA GAA TAC TTA AGT AAA CAA 490Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys Gln130 135 140 145TTG CAA GAG ATT TCT GAT AAG TTG GAT ATT ATT AAT GTA AAT GTA CTT 538Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val Leu
150 155 160ATT AAC TCT ACA CTT ACT GAA ATT ACA CCT GCG TAT CAA AGG ATT AAA 586Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile Lys
165 170 175TAT GTG AAC GAA AAA TTT GAG GAA TTA ACT TTT GCT ACA GAA ACT AGT 634Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr Ser
180 185 190TCA AAA GTA AAA AAG GAT GGC TCT CCT GCA GAT ATT CGT GAT GAG TTA 682Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Arg Asp Glu Leu
195 200 205AGT GAG TTA ACT GAA CTA GCG AAA AGT GTA ACA CAA AAT GAT GTG GAT 730Ser Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Gln Asn Asp Val Asp210 215 220 225GGT TTT GAA TTT TAC CTT AAT ACA TTC CAC GAT GTA ATG GTA GGA AAT 778Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly Asn
230 235 240AAT TTA TTC GGG CGT TCA GCT TTA AAA ACT GCA TCG GAA TTA ATT ACT 826Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile Thr
245 250 255AAA GAA AAT GTG AAA ACA AGT GGC AGT GAG GTC GGA AAT GTT TAT AAC 874Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr Asn
260 265 270TTC CTA ATT GTA TTA ACA GCT CTG CAA GCA CAA GCT TTT CTT ACT TTA 922Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Gln Ala Phe Leu Thr Leu
275 280 285ACA CCA TGC CGA AAA TTA TTA GGC TTA GCA GAT ATT GAT TAT ACT TCT 970Thr Pro Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr Ser290 295 300 305ATT ATG AAT GAA CAT TTA AAT AAG GAA AAA GAG GAA TTT AGA GTA AAC 1018Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val Asn
310 315 320ATC CTC CCT ACA CTT TCT AAT ACT TTT TCT AAT CCT AAT TAT GCA AAA 1066Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala Lys
325 330 335GTT AAA GGA AGT GAT GAA GAT GCA AAG ATG ATT GTG GAA GCT AAA CCA 1114Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys Pro
340 345 350GGA CAT GCA TTG ATT GGG TTT GAA ATT AGT AAT GAT TCA ATT ACA GTA 1162Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr Val
355 360 365TTA AAA GTA TAT GAG GCT AAG CTA AAA CAA AAT TAT CAA GTC GAT AAG 1210Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp Lys370 375 380 385GAT TCC TTA TCG GAA GTT ATT TAT GGC GAT ATG GAT AAA TTA TTG TGC 1258Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu Cys
390 395 400CCA GAT CAA TCT GGA CAA ATC TAT TAT ACA AAT AAC ATA GTA TTT CCA 1306Pro Asp Gln Ser Gly Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe Pro
405 410 415AAT GAA TAT GTA ATT ACT AAA ATT GAT TTC ACT AAA AAA ATG AAA ACT 1354Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys Thr
420 425 430TTA AGA TAT GAG GTA ACA GCG AAT TTT TAT GAT TCT TCT ACA GGA GAA 1402Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly Glu
435 440 445ATT GAC TTA AAT AAG AAA AAA GTA GAA TCA AGT GAA GCG GAG TAT AGA 1450Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr Arg450 455 460 465ACG TTA AGT GCT AAT GAT GAT GGG GTG TAT ATG CCG TTA GGT GTC ATC 1498Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val Ile
470 475 480AGT GAA ACA TTT TTG ACT CCG ATT AAT GGG TTT GGC CTC CAA GCT GAT 1546Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala Asp
485 490 495GAA AAT TCA AGA TTA ATT ACT TTA ACA TGT AAA TCA TAT TTA AGA GAA 1594Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg Glu
500 505 510CTA CTG CTA GCA ACA GAC TTA AGC AAT AAA GAA ACT AAA TTG ATC GTC 1642Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile Val
515 520 525CCG CCA AGT GGT TTT ATT AGC AAT ATT GTA GAG AAC GGG TCC ATA GAA 1690Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile Glu530 535 540 545GAG GAC AAT TTA GAG CCG TGG AAA GCA AAT AAT AAG AAT GCG TAT GTA 1738Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr Val
550 555 560GAT CAT ACA GGC GGA GTG AAT GGA ACT AAA GCT TTA TAT GTT CAT AAG 1786Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His Lys
565 570 575GAC GGA GGA ATT TCA CAA TTT ATT GGA GAT AAG TTA AAA CCG AAA ACT 1834Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys Thr
580 585 590GAG TAT GTA ATC CAA TAT ACT GTT AAA GGA AAA CCT TCT ATT CAT TTA 1882Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His Leu
595 600 605AAA GAT GAA AAT ACT GGA TAT ATT CAT TAT GAA GAT ACA AAT AAT AAT 1930Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn Asn610 615 620 625TTA GAA GAT TAT CAA ACT ATT AAT AAA CGT TTT ACT ACA GGA ACT GAT 1978Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr Asp
630 635 640TTA AAG GGA GTG TAT TTA ATT TTA AAA AGT CAA AAT GGA GAT GAA GCT 2026Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu Ala
645 650 655TGG GGA GAT AAC TTT ATT ATT TTG GAA ATT AGT CCT TCT GAA AAG TTA 2074Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys Leu
660 665 670TTA AGT CCA GAA TTA ATT AAT ACA AAT AAT TGG ACG AGT ACG GGA TCA 2122Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly Ser
675 680 685ACT AAT ATT AGC GGT AAT ACA CTC ACT CTT TAT CAG GGA GGA CGA GGG 2170Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg Gly690 695 700 705ATT CTA AAA CAA AAC CTT CAA TTA GAT AGT TTT TCA ACT TAT AGA GTG 2218Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg Val
710 715 720TAT TTC TCT GTG TCC GGA GAT GCT AAT GTA AGG ATT AGA AAT TCT AGG 2266Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser Arg
725 730 735GAA GTG TTA TTT GAA AAA AAG GAT ATA TGA GC GGCGCTAAAG ATGTTTCTGA 2318Glu Val Leu Phe Glu Lys Lys Asp Ile
740 745AATGTTCACT ACAAAATTGA AAGATAACTT CTATATAGAG CTTTCT 2364(2)关于SEQ ID NO:6的信息:
(i)序列特征
(A)长度:747个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线型
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:6:Met Asn Lys Asn Asn Ala Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe1 5 10 15Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp
20 25 30Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Ala Leu
35 40 45Asp Glu Ile Leu Glu Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys
50 55 60Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn65 70 75 80Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln
85 90 95Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr
100 105 110Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val
115 120 125Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys
130 135 140Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val145 150 155 160Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile
165 170 175Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr
180 185 190Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Arg Asp Glu
195 200 205Leu Ser Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Gln Asn Asp Val
210 215 220Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly225 230 235 240Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile
245 250 255Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr
260 265 270Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Gln Ala Phe Leu Thr
275 280 285Leu Thr Pro Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr
290 295 300Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val305 310 315 320Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala
325 330 335Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys
340 345 350Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr
355 360 365Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp
370 375 380Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu385 390 395 400Cys Pro Asp Gln Ser Gly Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe
405 410 415Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys
420 425 430Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly
435 440 445Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr
450 455 460Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val465 470 475 480Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala
485 490 495Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg
500 505 510Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile
515 520 525Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile
530 535 540Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr545 550 555 560Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His
565 570 575Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys
580 585 590Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His
595 600 605Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn
610 615 620Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr625 630 635 640Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu
645 650 655Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys
660 665 670Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly
675 680 685Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg
690 695 700Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg705 710 715 720Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser
725 730 735Arg Glu Val Leu Phe Glu Lys Lys Asp Ile
740 745(2)关于SEQ ID NO:7的信息:
(i)序列特征
(A)长度:2403个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线形
(ii)分子类型:其它核酸
(A)描述:/desc=“合成DNA’
(iii)假设:否
(ix)特征:
(A)名称/关键:misc特征
(B)位置:11..2389
(B)其它信息:/备注=“编码VIP3A(a)蛋白的玉米优化DNA序列”
(xi)序列描述:SEQ ID NO:7:GGATCCACCA ATGAACATGA ACAAGAACAA CACCAAGCTG AGCACCCGCG CCCTGCCGAG 60CTTCATCGAC TACTTCAACG GCATCTACGG CTTCGCCACC GGCATCAAGG ACATCATGAA 120CATGATCTTC AAGACCGACA CCGGCGGCGA CCTGACCCTG GACGAGATCC TGAAGAACCA 180GCAGCTGCTG AACGACATCA GCGGCAAGCT GGACGGCGTG AACGGCAGCC TGAACGACCT 240GATCGCCCAG GGCAACCTGA ACACCGAGCT GAGCAAGGAG ATCCTTAAGA TCGCCAACGA 300GCAGAACCAG GTGCTGAACG ACGTGAACAA CAAGCTGGAC GCCATCAACA CCATGCTGCG 360CGTGTACCTG CCGAAGATCA CCAGCATGCT GAGCGACGTG ATGAAGCAGA ACTACGCCCT 420GAGCCTGCAG ATCGAGTACC TGAGCAAGCA GCTGCAGGAG ATCAGCGACA AGCTGGACAT 480CATCAACGTG AACGTCCTGA TCAACAGCAC CCTGACCGAG ATCACCCCGG CCTACCAGCG 540CATCAAGTAC GTGAACGAGA AGTTCGAAGA GCTGACCTTC GCCACCGAGA CCAGCAGCAA 600GGTGAAGAAG GACGGCAGCC CGGCCGACAT CCTGGACGAG CTGACCGAGC TGACCGAGCT 660GGCCAAGAGC GTGACCAAGA ACGACGTGGA CGGCTTCGAG TTCTACCTGA ACACCTTCCA 720CGACGTGATG GTGGGCAACA ACCTGTTCGG CCGCAGCGCC CTGAAGACCG CCAGCGAGCT 780GATCACCAAG GAGAACGTGA AGACCAGCGG CAGCGAGGTG GGCAACGTGT ACAACTTCCT 840GATCGTGCTG ACCGCCCTGC AGGCCCAGGC CTTCCTGACC CTGACCACCT GTCGCAAGCT 900GCTGGGCCTG GCCGACATCG ACTACACCAG CATCATGAAC GAGCACTTGA ACAAGGAGAA 960GGAGGAGTTC CGCGTGAACA TCCTGCCGAC CCTGAGCAAC ACCTTCAGCA ACCCGAACTA 1020CGCCAAGGTG AAGGGCAGCG ACGAGGACGC CAAGATGATC GTGGAGGCTA AGCCGGGCCA 1080CGCGTTGATC GGCTTCGAGA TCAGCAACGA CAGCATCACC GTGCTGAAGG TGTACGAGGC 1140CAAGCTGAAG CAGAACTACC AGGTGGACAA GGACAGCTTG AGCGAGGTGA TCTACGGCGA 1200CATGGACAAG CTGCTGTGTC CGGACCAGAG CGAGCAAATC TACTACACCA ACAACATCGT 1260GTTCCCGAAC GAGTACGTGA TCACCAAGAT CGACTTCACC AAGAAGATGA AGACCCTGCG 1320CTACGAGGTG ACCGCCAACT TCTACGACAG CAGCACCGGC GAGATCGACC TGAACAAGAA 1380GAAGGTGGAG AGCAGCGAGG CCGAGTACCG CACCCTGAGC GCGAACGACG ACGGCGTCTA 1440CATGCCACTG GGCGTGATCA GCGAGACCTT CCTGACCCCG ATCAACGGCT TTGGCCTGCA 1500GGCCGACGAG AACAGCCGCC TGATCACCCT GACCTGTAAG AGCTACCTGC GCGAGCTGCT 1560GCTAGCCACC GACCTGAGCA ACAAGGAGAC CAAGCTGATC GTGCCACCGA GCGGCTTCAT 1620CAGCAACATC GTGGAGAACG GCAGCATCGA GGAGGACAAC CTGGAGCCGT GGAAGGCCAA 1680CAACAAGAAC GCCTACGTGG ACCACACCGG CGGCGTGAAC GGCACCAAGG CCCTGTACGT 1740GCACAAGGAC GGCGGCATCA GCCAGTTCAT CGGCGACAAG CTGAAGCCGA AGACCGAGTA 1800CGTGATCCAG TACACCGTGA AGGGCAAGCC ATCGATTCAC CTGAAGGACG AGAACACCGG 1860CTACATCCAC TACGAGGACA CCAACAACAA CCTGGAGGAC TACCAGACCA TCAACAAGCG 1920CTTCACCACC GGCACCGACC TGAAGGGCGT GTACCTGATC CTGAAGAGCC AGAACGGCGA 1980CGAGGCCTGG GGCGACAACT TCATCATCCT GGAGATCAGC CCGAGCGAGA AGCTGCTGAG 2040CCCGGAGCTG ATCAACACCA ACAACTGGAC CAGCACCGGC AGCACCAACA TCAGCGGCAA 2100CACCCTGACC CTGTACCAGG GCGGCCGCGG CATCCTGAAG CAGAACCTGC AGCTGGACAG 2160CTTCAGCACC TACCGCGTGT ACTTCAGCGT GAGCGGCGAC GCCAACGTGC GCATCCGCAA 2220CAGCCGCGAG GTGCTGTTCG AGAAGAGGTA CATGAGCGGC GCCAAGGACG TGAGCGAGAT 2280GTTCACCACC AAGTTCGAGA AGGACAACTT CTACATCGAG CTGAGCCAGG GCAACAACCT 2340GTACGGCGGC CCGATCGTGC ACTTCTACGA CGTGAGCATC AAGTTAACGT AGAGCTCAGA 2400TCT 2403(2)关于SEQ ID NO:8的信息:
(i)序列特征
(A)长度:1638个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线形
(ii)分子类型:cDNA
(iii)假设:否
(ix)特征:
(A)名称/关键:CDS
(B)位置:44..1191
(B)其它信息:/产物=“编码VIP3A(a)受体的cDNA的翻译”
(xi)序列描述:SEQ ID NO:8:T AGT GGA TCC CCC GGG CTG CAG GAA TTC GCG GCC GCG TCG ACC ATG 46
Met
1 5 10 15TAC TCT AGA ATA TTT TTC CTC CTT GTG ATA GTG TGT GCT GTT AAG GCT 94Tyr Ser Arg Ile Phe Phe Leu Leu Val Ile Val Cys Ala Val Lys Ala
20 25 30TCT CTG TTT ACT GTA AAT GTG TAT GAT GAT AAC CCC GAA ACT GAA ATT 142Ser Leu Phe Thr Val Asn Val Tyr Asp Asp Asn Pro Glu Thr Glu Ile
35 40 45GCG AGT AGT CTA AAA GGC TGT AAC CCC CAA GAG TGT GAC CAG CGG TGT 190Ala Ser Ser Leu Lys Gly Cys Asn Pro Gln Glu Cys Asp Gln Arg Cys
50 55 60CGT AGA CTG AAG TTT CCC GGT GGC GCC TGT GTC AAT GGT CGC TGC AAG 238Arg Arg Leu Lys Phe Pro Gly Gly Ala Cys Val Asn Gly Arg Cys Lys
65 70 75TGT GAC AAC TTC CTC AGT GTA AAA GAT GAC GTG TCT GTT GAA GAG CCT 286Cys Asp Asn Phe Leu Ser Val Lys Asp Asp Val Ser Val Glu Glu Pro80 85 90 95GCG ATT CTC AAA GAT TTG GTG TCA TTA GAA GCT GAA CAG GCA GCG AAA 334Ala Ile Leu Lys Asp Leu Val Ser Leu Glu Ala Glu Gln Ala Ala Lys
100 105 110AGT AGA TGC AGA AAC AGA GTG TGT GAC GCG GTG TGC CGT GCC CTA CAC 382Ser Arg Cys Arg Asn Arg Val Cys Asp Ala Val Cys Arg Ala Leu His
115 120 125AAC ACC AGT GGT GCC TGT GTT GAT GGA CAA TGC AAG TGT ACT AAT AAG 430Asn Thr Ser Gly Ala Cys Val Asp Gly Gln Cys Lys Cys Thr Asn Lys
130 135 140ATC AGT GCA GGA GAT ATT GTG TCT GAT CCT GCT GAA TCG CTA CGC ACT 478Ile Ser Ala Gly Asp Ile Val Ser Asp Pro Ala Glu Ser Leu Arg Thr
145 150 155TGT AAC CCT ATA AGG TGT GAC GAA CAA TGT AGA AGA AAT GGC CAT GAA 526Cys Asn Pro Ile Arg Cys Asp Glu Gln Cys Arg Arg Asn Gly His Glu160 165 170 175TTT GGT GTT TGC TTC AAA GGA CAA TGC AAG TGT GAT TAC TTC CTC AAG 574Phe Gly Val Cys Phe Lys Gly Gln Cys Lys Cys Asp Tyr Phe Leu Lys
180 185 190GAA GAA GTC GAT GAA CCT GAA GTT ACA AGC CTT CCA AAA AAC TGC AAC 622Glu Glu Val Asp Glu Pro Glu Val Thr Ser Leu Pro Lys Asn Cys Asn
195 200 205CCC CAA GAG TGT GAC CAG CGT TGT CGT AGA CTG AAG TTC CCC GGT GGC 670Pro Gln Glu Cys Asp Gln Arg Cys Arg Arg Leu Lys Phe Pro Gly Gly
210 215 220GCC TGT GTC AAC GGG CGC TGC AAG TGT GAC AAC TTC TTC AGT GCA GGA 718Ala Cys Val Asn Gly Arg Cys Lys Cys Asp Asn Phe Phe Ser Ala Gly
225 230 235GAT ATT GTG TCT GAT CCT GCC GAA TCG CTA CGC TCT TGT AAC CCT ATA 766Asp Ile Val Ser Asp Pro Ala Glu Ser Leu Arg Ser Cys Asn Pro Ile240 245 250 255AGG TGT GAC GAA CAA TGT AGA AGA AAT GGC CAT GAA TTT GGT GTT TGC 814Arg Cys Asp Glu Gln Cys Arg Arg Asn Gly His Glu Phe Gly Val Cys
260 265 270TTC AAA GGA CAA TGC AAG TGT GAT TAC TTC CTC AAC TCA GAA GTA GAC 862Phe Lys Gly Gln Cys Lys Cys Asp Tyr Phe Leu Asn Ser Glu Val Asp
275 280 285GCT GTT AAT GAG TTT CCT CAA GCG GGC TCA AAA CGC TAC TGC AAC TTA 910Ala Val Asn Glu Phe Pro Gln Ala Gly Ser Lys Arg Tyr Cys Asn Leu
290 295 300ACG CAA TGC AAC CAG ACG TGC GCC AAT CGT TTC TAT GAT AGT GCT AGA 958Thr Gln Cys Asn Gln Thr Cys Ala Asn Arg Phe Tyr Asp Ser Ala Arg
305 310 315GTG ATC CAC GGC TGG TGC AAA TGC TAC AGT AAG ATG GAA AGA CAG GAT 1006Val Ile His Gly Trp Cys Lys Cys Tyr Ser Lys Met Glu Arg Gln Asp320 325 330 335GCA TCT CCA TTA AAC GAT GTG ACT GAG GAT GAA AAT GAA GTT TCT AAC 1054Ala Ser Pro Leu Asn Asp Val Thr Glu Asp Glu Asn Glu Val Ser Asn
340 345 350GAT ATC CTG AGG ACT GTT GCA GAG GAG CTG TCT GAT GTG TCA CCT AGG 1102Asp Ile Leu Arg Thr Val Ala Glu Glu Leu Ser Asp Val Ser Pro Arg
355 360 365GCC TGC AAA TCA GCG AGC TGC AAT CAA GCA TGT CGC GCC TTC TAC TTT 1150Ala Cys Lys Ser Ala Ser Cys Asn Gln Ala Cys Arg Ala Phe Tyr Phe
370 375 380AAA GGA GGG TGG TGT CGC TTT GGA CGA TGC CAA TGC TTC TA 1191Lys Gly Gly Trp Cys Arg Phe Gly Arg Cys Gln Cys Phe
385 390 395AAATTAGTAT GATATATGAA TTTTGTATTA TTCGGTTAAT TGTGTTATGT TTAAAAAACA 1251TAATGTCTTC ATTTTAGAAA AAAGTACCTT CACTAAAGCG CAACAATTAA CTAGTAGTTA 1311ATTATTAACT AGTAGTTAAA TTATTGATGA TTATGATTAT CTTAGTAGTA GTTAATTATA 1371ATCATCAACT ATTAACTAGT AGTTAATTAT TAACTAGTAG TTAAATTATT GATGATTATG 1431ATTATCTTAG TAGTAGTTAA TTATTGTTTC TTATAATAAT CTAGTATGTT GGTAGGTACT 1491TAATAATAAC GCTTCTGACA AAAAATTTAA AATTAAATAA TTCTATCAAA CATAAATAAT 1551AACTGAAATA AAAATTTATA AGAGAAAAAA AAAAAGTCGA CGCGGCCGCG AATTCGATAT 1611CAAGCTTATC GATACCGTCG ACCTCGA 1638(2)关于SEQ ID NO:9的信息:
(i)序列特征
(A)长度:396个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线型
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:9:Ser Gly Ser Pro Gly Leu Gln Glu Phe Ala Ala Ala Ser Thr Met Tyr1 5 10 15Ser Arg Ile Phe Phe Leu Leu Val Ile Val Cys Ala Val Lys Ala Ser
20 25 30Leu Phe Thr Val Asn Val Tyr Asp Asp Asn Pro Glu Thr Glu Ile Ala
35 40 45Ser Ser Leu Lys Gly Cys Asn Pro Gln Glu Cys Asp Gln Arg Cys Arg
50 55 60Arg Leu Lys Phe Pro Gly Gly Ala Cys Val Asn Gly Arg Cys Lys Cys65 70 75 80Asp Asn Phe Leu Ser Val Lys Asp Asp Val Ser Val Glu Glu Pro Ala
85 90 95Ile Leu Lys Asp Leu Val Ser Leu Glu Ala Glu Gln Ala Ala Lys Ser
100 105 110Arg Cys Arg Asn Arg Val Cys Asp Ala Val Cys Arg Ala Leu His Asn
115 120 125Thr Ser Gly Ala Cys Val Asp Gly Gln Cys Lys Cys Thr Asn Lys Ile
130 135 140Ser Ala Gly Asp Ile Val Ser Asp Pro Ala Glu Ser Leu Arg Thr Cys145 150 155 160Asn Pro Ile Arg Cys Asp Glu Gln Cys Arg Arg Asn Gly His Glu Phe
165 170 175Gly Val Cys Phe Lys Gly Gln Cys Lys Cys Asp Tyr Phe Leu Lys Glu
180 185 190Glu Val Asp Glu Pro Glu Val Thr Ser Leu Pro Lys Asn Cys Asn Pro
195 200 205Gln Glu Cys Asp Gln Arg Cys Arg Arg Leu Lys Phe Pro Gly Gly Ala210 215 220Cys Val Asn Gly Arg Cys Lys Cys Asp Asn Phe Phe Ser Ala Gly Asp225 230 235 240Ile Val Ser Asp Pro Ala Glu Ser Leu Arg Ser Cys Asn Pro Ile Arg
245 250 255Cys Asp Glu Gln Cys Arg Arg Asn Gly His Glu Phe Gly Val Cys Phe
260 265 270Lys Gly Gln Cys Lys Cys Asp Tyr Phe Leu Asn Ser Glu Val Asp Ala
275 280 285Val Asn Glu Phe Pro Gln Ala Gly Ser Lys Arg Tyr Cys Asn Leu Thr
290 295 300Gln Cys Asn Gln Thr Cys Ala Asn Arg Phe Tyr Asp Ser Ala Arg Val305 310 315 320Ile His Gly Trp Cys Lys Cys Tyr Ser Lys Met Glu Arg Gln Asp Ala
325 330 335Ser Pro Leu Asn Asp Val Thr Glu Asp Glu Asn Glu Val Ser Asn Asp
340 345 350Ile Leu Arg Thr Val Ala Glu Glu Leu Ser Asp Val Ser Pro Arg Ala
355 360 365Cys Lys Ser Ala Ser Cys Asn Gln Ala Cys Arg Ala Phe Tyr Phe Lys
370 375 380Gly Gly Trp Cys Arg Phe Gly Arg Cys Gln Cys Phe385 390 395(2)关于SEQ ID NO:10的信息:
(i)序列特征
(A)长度:14个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线型
(ii)分子类型:肽
(iii)假设:否
(v)片段类型:N-端
(vi)原始来源:
(A)生物:苏云金芽孢杆菌
(B)菌株:AB88
(ix)特征:
(A)名称/关键:肽
(B)位置:1..14
(D)其它信息:/备注=“已知为阴离子交换组分23(更小)的蛋白N-端氨基酸序列”
(xi)序列描述:SEQ ID NO:10:Xaa Glu Pro Phe Val Ser Ala Xaa Xaa Xaa Gln Xaa Xaa Xaa1 5 10(2)关于SEQ ID NO:11的信息:
(i)序列特征
(A)长度:13个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线型
(vi)原始来源:
(A)生物:苏云金芽孢杆菌
(xi)序列描述:SEQ ID NO:11:Xaa Glu Tyr Glu Asn Val Glu Pro Phe Val Ser Ala Xaa1 5 10(2)关于SEQ ID NO:12的信息:
(i)序列特征
(A)长度:14个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线型
(vi)原始来源:
(A)生物:苏云金芽孢杆菌
(xi)序列描述:SEQ ID NO:12:Met Asn Lys Asn Asn Thr Lys Leu Pro Thr Arg Ala Leu Pro1 5 10(2)关于SEQ ID NO:13的信息:
(i)序列特征
(A)长度:15个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线型
(ii)分子类型:肽
(iii)假设:否
(v)片段类型:N-端
(vi)原始来源:
(C)生物:苏云金芽孢杆菌
(D)菌株:AB88
(ix)特征:
(B)位置:1..15
(D)其它信息:/备注=“对小地老虎有活性的35kDa VIP的N-端氨基酸序列”
(xi)序列描述:SEQ ID NO:13:Ala Leu Ser Glu Asn Thr Gly Lys Asp Gly Gly Tyr Ile Val Pro1 5 10 15(2)关于SEQ ID NO:14的信息:
(i)序列特征
(A)长度:9个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线型
(vi)原始来源:
(A)生物:苏云金芽孢杆菌
(xi)序列描述:SEQ ID NO:14:Met Asp Asn Asn Pro Asn Ile Asn Glu1 5(2)关于SEQ ID NO:15的信息:
(i)序列特征
(A)长度:9个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线型
(ii)分子类型:肽
(iii)假设:否
(v)片段类型:N-端
(ix)特征:
(A)名称/关键:肽
(B)位置:1..9
(D)其它信息:/备注=“80kDaδ-内毒素的N-端序列
(xi)序列描述:SEQ ID NO:15:Met Asp Asn Asn Pro AsnIle Asn Glu1 5(2)关于SEQ ID NO:16的信息:
(i)序列特征
(A)长度:11个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线型
(ii)分子类型:肽
(iii)假设:否
(v)片段类型:N-端
(vi)原始来源:
(A)生物:苏云金芽孢杆菌
(ix)特征:
(A)名称/关键:肽
(B)位置:1..11
(D)其它信息:/备注=“60kDa δ-内毒素的N-端序列”
(xi)序列描述:SEQ ID NO:16:Met Asn Val Leu Asn Ser Gly Arg Thr Thr Ile1 5 10(2)关于SEQ ID NO:17的信息:
(i)序列特征
(A)长度:17个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线形
(ii)分子类型:其它核酸
(A)描述:/desc=“引物序列”
(iii)假设:否
(xi)序列描述:SEQ ID NO:17:CGATTAATGT TGGCCTC 17(2)关于SEQ ID NO:18的信息:
(i)序列特征
(A)长度:21个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线形
(ii)分子类型:其它核酸
(A)描述:/desc=“引物序列”
(iii)假设:否
(xi)序列描述:SEQ ID NO:18:CATTAGCATC TCCGGACACA G 21(2)关于SEQ ID NO:19的信息:
(i)序列特征
(A)长度:2370个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线形
(ii)分子类型:其它核酸
(A)描述:/desc=“编码VIP3A(a)的合成序列”
(iii)假设:否
(xi)序列描述:SEQ ID NO:19:ATGAACAAGA ACAACACCAA GCTGAGCACC CGCGCCCTGC CGAGCTTCAT CGACTACTTC 60AACGGCATCT ACGGCTTCGC CACCGGCATC AAGGACATCA TGAACATGAT CTTCAAGACC 120GACACCGGCG GCGACCTGAC CCTGGACGAG ATCCTGAAGA ACCAGCAGCT GCTGAACGAC 180ATCAGCGGCA AGCTGGACGG CGTGAACGGC AGCCTGAACG ACCTGATCGC CCAGGGCAAC 240CTGAACACCG AGCTGAGCAA GGAGATCCTT AAGATCGCCA ACGAGCAGAA CCAGGTGCTG 300AACGACGTGA ACAACAAGCT GGACGCCATC AACACCATGC TGCGCGTGTA CCTGCCGAAG 360ATCACCAGCA TGCTGAGCGA CGTGATGAAG CAGAACTACG CCCTGAGCCT GCAGATCGAG 420TACCTGAGCA AGCAGCTGCA GGAGATCAGC GACAAGCTGG ACATCATCAA CGTGAACGTC 480CTGATCAACA GCACCCTGAC CGAGATCACC CCGGCCTACC AGCGCATCAA GTACGTGAAC 540GAGAAGTTCG AAGAGCTGAC CTTCGCCACC GAGACCAGCA GCAAGGTGAA GAAGGACGGC 600AGCCCGGCCG ACATCCTGGA CGAGCTGACC GAGCTGACCG AGCTGGCGAA GAGCGTGACC 660AAGAACGACG TGGACGGCTT CGAGTTCTAC CTGAACACCT TCCACGACGT GATGGTGGGC 720AACAACCTGT TCGGCCGCAG CGCCCTGAAG ACCGCCAGCG AGCTGATCAC CAAGGAGAAC 780GTGAAGACCA GCGGCAGCGA GGTGGGCAAC GTGTACAACT TCCTGATCGT GCTGACCGCC 840CTGCAGGCCA AGGCCTTCCT GACCCTGACC CCCTGTCGCA AGCTGCTGGG CCTGGCCGAC 900ATCGACTACA CCAGCATCAT GAACGAGCAC TTGAACAAGG AGAAGGAGGA GTTCCGCGTG 960AACATCCTGC CGACCCTGAG CAACACCTTC AGCAACCCGA ACTACGCCAA GGTGAAGGGC 1020AGCGACGAGG ACGCCAAGAT GATCGTGGAG GCTAAGCCGG GCCACGCGTT GATCGGCTTC 1080GAGATCAGCA ACGACAGCAT CACCGTGCTG AAGGTGTACG AGGCCAAGCT GAAGCAGAAC 1140TACCAGGTGG ACAAGGACAG CTTGAGCGAG GTGATCTACG GCGACATGGA CAAGCTGCTG 1200TGTCCGGACC AGAGCGGGCA AATCTACTAC ACCAACAACA TCGTGTTCCC GAACGAGTAC 1260GTGATCACCA AGATCGACTT CACCAAGAAG ATGAAGACCC TGCGCTACGA GGTGACCGCC 1320AACTTCTACG ACAGCAGCAC CGGCGAGATC GACCTGAACA AGAAGAAGGT GGAGAGCAGC 1380GAGGCCGAGT ACCGCACCCT GAGCGCGAAC GACGACGGCG TCTACATGCC ACTGGGCGTG 1440ATCAGCGAGA CCTTCCTGAC CCCGATCAAC GGCTTTGGCC TGCAGGCCGA CGAGAACACC 1500CGCCTGATCA CCCTGACCTG TAAGAGCTAC CTGCGCGAGC TGCTGCTAGC CACCGACCTG 1560AGCAACAAGG AGACCAAGCT GATCGTGCCA CCGAGCGGCT TCATCAGCAA CATCGTGGAG 1620AACGGCAGCA TCGAGGAGGA CAACCTGGAG CCGTGGAAGG CCAACAACAA GAACGCCTAC 1680GTGGACCACA CCGGCGGCGT GAACGGCACC AAGGCCCTGT ACGTGCACAA GGACGGCGGC 1740ATCAGCCAGT TCATCGGCGA CAAGCTGAAG CCGAAGACCG AGTACGTGAT CCAGTACACC 1800GTGAAGGGCA AGCCATCGAT TCACCTGAAG GACGAGAACA CCGGCTACAT CCACTACGAG 1860GACACCAACA ACAACCTGGA GGACTACCAG ACCATCAACA AGCGCTTCAC CACCGGCACC 1920GACCTGAAGG GCGTGTACCT GATCCTGAAG AGCCAGAACG GCGACGAGGC CTGGGGCGAC 1980AACTTCATCA TCCTGGAGAT CAGCCCGAGC GAGAAGCTGC TGAGCCCGGA GCTGATCAAC 2040ACCAACAACT GGACCAGCAC CGGCAGCACC AACATCAGCG GCAACACCCT GACCCTGTAC 2100CAGGGCGGCC GCGGCATCCT GAAGCAGAAC CTGCAGCTGG ACAGCTTCAG CACCTACCGC 2160GTGTACTTCA GCGTGAGCGG CGACGCCAAC GTGCGCATCC GCAACTCCCG CGAGGTGCTG 2220TTCAAGAAGA GGTACATGAG CGGCGCCAAG GACGTGAGCG AGATGTTCAC CACCAAGTTC 2280GAGAAGGACA ACTTCTACAT CGAGCTGAGC CAGGGCAACA ACCTGTACGG CGGCCCGATC 2340GTGCACTTCT ACGACGTGAG CATCAAGTAG 2370(2)关于SEQ ID NO:20的信息:
(i)序列特征
(A)长度:2241个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线形
(ii)分子类型:其它核酸
(A)描述:/desc=“编码VIP3A(c)的合成序列”
(iii)假设:否
(xi)序列描述:SEQ ID NO:20:
ATGAACAAGA ACAACGCCAA GCTGAGCACC CGCGCCCTGC CGAGCTTCAT CGACTACTTC 60
AACGGCATCT ACGGCTTCGC CACCGGCATC AAGGACATCA TGAACATGAT CTTCAAGACC 120
GACACCGGCG GCGACCTGGC CCTGGACGAG ATCCTGGAGA ACCAGCAGCT GCTGAACGAC 180
ATCAGCGGCA AGCTGGACGG CGTGAACGGC AGCCTGAACG ACCTGATCGC CCAGGGCAAC 240CTGAACACCG AGCTGAGCAA GGAGATCCTT AAGATCGCCA ACGAGCAGAA CCAGGTGCTG 300AACGACGTGA ACAACAAGCT GGACGCCATC AACACCATGC TGCGCGTGTA CCTGCCGAAG 360ATCACCAGCA TGCTGAGCGA CGTGATGAAG CAGAACTACG CCCTGAGCCT GCAGATCGAG 420TACCTGAGCA AGCAGCTGCA GGAGATCAGC GACAAGCTGG ACATCATCAA CGTGAACGTC 480CTGATCAACA GCACCCTGAC CGAGATCACC CCGGCCTACC AGCGCATCAA GTACGTGAAC 540GAGAAGTTCG AAGAGCTGAC CTTCGCCACC GAGACCAGCA GCAAGGTGAA GAAGGACGGC 600AGCCCGGCCG ACATCCGGGA CGAGCTGAGC GAGCTGACCG AGCTGGCGAA GAGCGTGACC 660CAGAACGACG TGGACGGCTT CGAGTTCTAC CTGAACACCT TCCACGACGT GATGGTGGGC 720AACAACCTGT TCGGCCGCAG CGCCCTGAAG ACCGCCAGCG AGCTGATCAC CAAGGAGAAC 780GTGAAGACCA GCGGCAGCGA GGTGGGCAAC GTGTACAACT TCCTGATCGT GCTGACCGCC 840CTGCAGGCCC AGGCCTTCCT GACCCTGACC CCCTGTCGCA AGCTGCTGGG CCTGGCCGAC 900ATCGACTACA CCAGCATCAT GAACGAGCAC TTGAACAAGG AGAAGGAGGA GTTCCGCGTG 960AACATCCTGC CGACCCTGAG CAACACCTTC AGCAACCCGA ACTACGCCAA GGTGAAGGGC 1020AGCGACGAGG ACGCCAAGAT GATCGTGGAG GCTAAGCCGG GCCACGCGTT GATCGGCTTC 1080GAGATCAGCA ACGACAGCAT CACCGTGCTG AAGGTGTACG AGGCCAAGCT GAAGCAGAAC 1140TACCAGGTGG ACAAGGACAG CTTGAGCGAG GTGATCTACG GCGACATGGA CAAGCTGCTG 1200TGTCCGGACC AGAGCGGGCA AATCTACTAC ACCAACAACA TCGTGTTCCC GAACGAGTAC 1260GTGATCACCA AGATCGACTT CACCAAGAAG ATGAAGACCC TGCGCTACGA GGTGACCGCC 1320AACTTCTACG ACAGCAGCAC CGGCGAGATC GACCTGAACA AGAAGAAGGT GGAGAGCAGC 1380GAGGCCGAGT ACCGCACCCT GAGCGCGAAC GACGACGGCG TCTACATGCC ACTGGGCGTG 1440ATCAGCGAGA CCTTCCTGAC CCCGATCAAC GGCTTTGGCC TGCAGGCCGA CGAGAACAGC 1500CGCCTGATCA CCCTGACCTG TAAGAGCTAC CTGCGCGAGC TGCTGCTAGC CACCGACCTG 1560AGCAACAAGG AGACCAAGCT GATCGTGCCA CCGAGCGGCT TCATCAGCAA CATCGTGGAG 1620AACGGCAGCA TCGAGGAGGA CAACCTGGAG CCGTGGAAGG CCAACAACAA GAACGCCTAC 1680GTGGACCACA CCGGCGGCGT GAACGGCACC AAGGCCCTGT ACGTGCACAA GGACGGCGGC 1740ATCAGCCAGT TCATCGGCGA CAAGCTGAAG CCGAAGACCG AGTACGTGAT CCAGTACACC 1800GTGAAGGGCA AGCCATCGAT TCACCTGAAG GACGAGAACA CCGGCTACAT CCACTACGAG 1860GACACCAACA ACAACCTGGA GGACTACCAG ACCATCAACA AGCGCTTCAC CACCGGCACC 1920GACCTGAAGG GCGTGTACCT GATCCTGAAG AGCCAGAACG GCGACGAGGC CTGGGGCGAC 1980AACTTCATCA TCCTGGAGAT CAGCCCGAGC GAGAAGCTGC TGAGCCCGGA GCTGATCAAC 2040ACCAACAACT GGACCAGCAC CGGCAGCACC AACATCAGCG GCAACACCCT GACCCTGTAC 2100CAGGGCGGCC GCGGCATCCT GAAGCAGAAC CTGCAGCTGG ACAGCTTCAG CACCTACCGC 2160GTGTACTTCA GCGTGAGCGG CGACGCCAAC GTGCGCATCC GCAACTCCCG CGAGGTGCTG 2220TTCGAGAAGA AGGACAAGTA G 2241
Claims (38)
1.一种VIP3类的蛋白,是VIP3A(c)及其同系物。
2.一种蛋白,其中氨基酸序列包含VIP3类蛋白的毒性结构域。
3.权利要求2的蛋白,其中毒性结构域是VIP3A(a)蛋白的毒性结构域。
4.一种转基因植物,包含编码VIP3类蛋白的DNA序列。
5.权利要求4的转基因植物,其中DNA序列编码VIP3A(a)蛋白。
6.权利要求6的转基因植物,其中DNA序列编码VIP3A(c)蛋白。
7.根据权利要求4-6中的任意一项所述的转基因植物,其中所述植物选自玉米、高粱、小麦、向日葵、番茄、农作物、棉花、水稻、大豆、甜菜、甘蔗、烟草、大麦和油籽油菜。
8.权利要求7的转基因植物,其中所述植物是玉米植物。
9.根据权利要求4-8中的任意一项所述的转基因植物,进一步包含编码第二种杀昆虫蛋白的第二种DNA序列。
10.权利要求9的转基因植物,其中所述第二种DNA序列编码δ-内毒素、VIP3类的另一种蛋白、VIP1类的蛋白或VIP2类的蛋白。
11.权利要求10的转基因植物,其中所述第二种DNA序列是δ-内毒素。
12.一种包含异源DNA序列的微生物,其中DNA序列编码VIP3A(a)蛋白。
13.权利要求12的微生物,其中DNA序列编码VIP3A(c)蛋白。
14.权利要求12或13的微生物,其中所述的微生物选自细菌、杆状病毒、藻类和真菌。
15.权利要求14的微生物,其中所述的微生物选自芽孢杆菌、假单胞菌、棍状杆菌和根瘤菌。
16.根据权利要求12-14中的任意一项所述的微生物,进一步包含编码第二种杀昆虫蛋白的第二种DNA序列。
17.权利要求16的微生物,其中所述第二种DNA序列编码δ-内毒素、VIP3类的另一种蛋白、VIP1类的蛋白的或VIP2类的蛋白。
18.权利要求17的微生物,其中所述第二种DNA序列是δ-内毒素。
19.一种杀昆虫组合物,包含根据权利要求12-14中的任意一项所述的微生物。
20.一种控制昆虫的方法,其中用杀昆虫量的VIP3类蛋白的受体的化学配体或VIP3类蛋白的受体的抗体作用于昆虫。
21.权利要求20的方法,其中昆虫与含有表达VIP3类蛋白、优选地VIP3A(c)蛋白的DNA序列的转基因植物接触。
22.权利要求20的方法,其中用杀昆虫组合物作用于昆虫,杀昆虫组合物包含含有能表达VIP3类蛋白、优选地VIP3A(c)蛋白的异源DNA序列的微生物。
23.一种重组DNA序列,编码VIP3A(c)蛋白及其同系物。
24.权利要求23的重组DNA序列,其中DNA序列是设计用于植物中最佳表达的合成序列。
25.权利要求24的重组DNA序列,其中植物是玉米植物。
26.一种表达盒,包含与异源启动子可操作连接的编码VIP3A(c)蛋白的DNA序列。
27.权利要求26的表达盒,其中在植物起作用的所述启动子选自可诱导的、组成型的、组织偏爱的和组织特异的启动子。
28.权利要求27的表达盒,其中启动子选自遍在蛋白、PEP羧化酶、LPT和MTL启动子。
29.VIP类蛋白的受体。
30.一种DNA序列,编码VIP3类的受体。
31.权利要求29的受体,包含致死结构域和重复EGF基序。
32.权利要求31的受体,具有SEQ ID NO:9中所列的序列。
33.根据权利要求30所述的DNA序列,如SEQ ID NO:9中所示。
34.VIP3类蛋白的受体的抗体。
35.一种鉴定和分离VIP3类蛋白的受体的同系物或编码VIP3类蛋白的受体的DNA序列的方法,包括得到
(a)编码VIP3类蛋白的受体的DNA序列,将该DNA序列与来自受检微生物的DNA杂交,检测与来自所述微生物的DNA的杂交并从该微生物分离所述同系物;
(b)来自微生物的DNA,使用编码VIP3类蛋白的受体的DNA序列的引物,得到反应产物,然后从该微生物分离编码VIP3类蛋白的受体的DNA序列;
(c)来自受检微生物的蛋白样品,得到VIP3类蛋白的受体的抗体,使该抗体与所述蛋白样品反应,通过免疫反应的存在检测和分离同系物。
36.一种鉴定作为VIP3受体的化学配体并具有杀昆虫活性的化合物的方法,包括使VIP3受体与受检化合物接触,并分析受体和受检化合物之间的相互作用。
37.根据权利要求36所述的方法,其中VIP3受体在细胞内表达,而所分析的相互作用是编程性细胞死亡。
38.根据权利要求36所述的方法,其中分析的相互作用是VIP3受体与受检化合物之间的特异结合。
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US83226397A | 1997-04-03 | 1997-04-03 | |
US83226597A | 1997-04-03 | 1997-04-03 | |
US08/832,265 | 1997-04-03 | ||
US08/838,219 US5877012A (en) | 1993-03-25 | 1997-04-03 | Class of proteins for the control of plant pests |
US08/838,219 | 1997-04-03 | ||
US08/832,263 | 1997-04-03 |
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EP (1) | EP0972062A2 (zh) |
JP (1) | JP2001524817A (zh) |
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CN (1) | CN1256712A (zh) |
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AU (1) | AU727218B2 (zh) |
BR (1) | BR9808483A (zh) |
CA (1) | CA2286284A1 (zh) |
IL (1) | IL132039A0 (zh) |
IN (1) | IN1998CH00710A (zh) |
MX (1) | MX219628B (zh) |
PL (1) | PL336081A1 (zh) |
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- 1998-04-02 AU AU68325/98A patent/AU727218B2/en not_active Expired
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- 1998-04-02 CA CA002286284A patent/CA2286284A1/en not_active Abandoned
- 1998-04-02 WO PCT/EP1998/001952 patent/WO1998044137A2/en not_active Application Discontinuation
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- 1998-04-02 EP EP98913751A patent/EP0972062A2/en not_active Withdrawn
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CN100439507C (zh) * | 2002-10-29 | 2008-12-03 | 辛根塔参与股份公司 | Cot102杀虫棉花 |
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US20020078473A1 (en) | 2002-06-20 |
AR059470A2 (es) | 2008-04-09 |
RU2222597C2 (ru) | 2004-01-27 |
CA2286284A1 (en) | 1998-10-08 |
IL132039A0 (en) | 2001-03-19 |
MX9909043A (en) | 1999-12-31 |
BR9808483A (pt) | 2000-05-23 |
MX219628B (en) | 2004-03-30 |
WO1998044137A2 (en) | 1998-10-08 |
US6291156B1 (en) | 2001-09-18 |
TR199902426T2 (xx) | 2000-06-21 |
WO1998044137A3 (en) | 1998-12-17 |
AU6832598A (en) | 1998-10-22 |
IN1998CH00710A (en) | 2005-03-04 |
AU727218B2 (en) | 2000-12-07 |
KR20010006015A (ko) | 2001-01-15 |
PL336081A1 (en) | 2000-06-05 |
US6429360B1 (en) | 2002-08-06 |
EP0972062A2 (en) | 2000-01-19 |
JP2001524817A (ja) | 2001-12-04 |
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