CN1307639A - 杀虫毒素和编码这些毒素的核苷酸序列 - Google Patents
杀虫毒素和编码这些毒素的核苷酸序列 Download PDFInfo
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Abstract
此处公开和要求保护的是新的苏云金芽孢杆菌的分离物、杀虫毒素、基因和用于鉴定编码对抗害虫有活性的毒素的基因的核苷酸探针及引物。这些引物可以用于通过PCR技术产生编码这些毒素的基因的特征性基因片段。本发明提供了可以由芽孢杆茵培养物上清液得到的全新的毒素家族。
Description
发明背景
昆虫和其它害虫,使农民由于产量降低和用于抑制这些害虫的花费,每年损失数十亿美元。昆虫害虫在农业生产环境中引起的损失,包括农作物产量降低、农作物质量下降、和收获费用上升。
刺激不定根系生长的耕作方法,诸如轮作和高水平氮的应用,已经部分提出了农业害虫引起的难题。利用农田的经济需要限制了使用轮作。此外,有些昆虫的越冬性状正在破坏一些地区的轮作。因此严重依赖化学杀虫剂来实现期望的抑制水平,导致了杀虫剂结合或掺入到土壤中。
使用化学杀虫剂有几个缺点。连续使用杀虫剂导致抗性昆虫的进化。诸如极高密度幼虫、大雨、和杀虫剂应用设备的不适当标准等情形,可能导致抑制效果差。使用杀虫剂常常唤起环境关注,诸如土壤污染和地表及地下水源的污染。公众也已经开始关注可能会在食品上发现的人工合成化学药品的残留量。操作杀虫剂还可能对使用它们的人造成危险。因此,人们正日益观察人工合成化学杀虫剂的潜在有毒环境后果,这样做是正确的。广泛使用的人工合成化学杀虫剂的实例,包括有机氯,例如DDT、灭蚁灵(mirex)、十氯酮(kepone)、林丹(lindane)、艾氏剂(aldrin)、氯丹(chlordane)、涕灭威(aldicarb)、和狄氏剂(dieldrin);有机磷杀虫剂,例如氯螨硫磷(chlorpyrifos)、柏拉息昂(parathion)、马拉硫磷(malathion)、和二嗪农(diazinon);和氨基甲酸酯。对使用杀虫剂的新的严谨限制和从市场上清除一些有效的杀虫剂,可能限制了控制有破坏性的和代价昂贵的害虫的经济和有效的选择。
由于与使用有机合成化学杀虫剂有关的难题,显然存在着限制使用这些试剂的需要和鉴定替换控制剂的需要。用生物学杀虫剂取代人工合成化学杀虫剂或这些试剂的组合,可以降低有毒化学药品在环境中的水平。
声望渐增的一种生物学杀虫剂是土壤微生物苏云金芽孢杆菌(Bacillus thuringiensis,B.t.)。土壤微生物苏云金芽孢杆菌是一种形成芽孢的革兰氏阳性细菌。B.t.的大多数菌株不具有杀虫活性。一些B.t.菌株产生伴孢晶体蛋白质内含物,并以此为特征。这些内含物通常在显微镜下表现为独特形状的晶体。一些B.t.蛋白质对害虫(诸如昆虫)有高度毒性,并且它们的毒性活性是特异的。某些杀虫B.t.蛋白质与内含物有关。这些δ-内毒素与具有非特异性宿主范围的外毒素不同。芽孢杆菌的其它物种也产生杀虫蛋白质。
已经分离得到某些芽孢杆菌毒素的基因并测序,而且已经生产了基于重组DNA的产品并批准使用。此外,通过使用基因工程技术,正在发展将这些毒素投递到农业环境中的方法,包括用毒素基因将植物经基因工程改造成具有昆虫抗性的应用,和将稳定的完整微生物细胞作为毒素投递载体的应用。由此,分离的芽孢杆菌毒素基因日益具有商业价值。
直到最近十五年,商业性使用的B.t.杀虫剂才被严谨限制到针对小范围的鳞翅目(毛虫)害虫。苏云金芽孢杆菌库尔斯塔克亚种(B.thuringiensis subsp.kurstaki)的芽孢和晶体,作为针对鳞翅目害虫的商业性杀虫剂,已经使用了许多年。例如,苏云金芽孢杆菌库尔斯塔克HD-1变种(B.thuringiensis var.kurstaki HD-1)产生对许多鳞翅目昆虫的幼虫有毒的结晶状δ-内毒素。
然而,在最近几年中,调查人员已经发现对更广范围的害虫具有特异性的B.t.杀虫剂。例如其它B.t.物种即以色列亚种和莫氏亚种(a.k.a.tenebrionis,a.k.a.B.t.M-7,a.k.a.B.t.san diego),已经分别商业性应用于控制双翅目和鞘翅目昆虫。已经有报道,苏云金芽孢杆菌粉虫变种(Bacillus thuringiensis var.tenebrionis)对鞘翅目的两种甲虫(Colorado potato beetle,马铃薯叶甲(Lepinotarsa decemlineata)和蓝毛臀萤叶甲(Agelastica alni))有活性。
更近的,已经鉴定了B.t.新亚种,而且已经分离得到了负责有活性的δ-内毒素蛋白质的基因。H_fte和Whiteley将结晶状蛋白质基因分类成四个主要种类(H.H_fte,H.R.Whiteley[1989]Microbiological Reviews 52(2):242-255):Cry I(鳞翅目特异的)、CryII(鳞翅目和双翅目特异的)、CryIII(鞘翅目特异的)、和CryIV(双翅目特异的)。已经报道了对其它害虫有特异毒性的株系。例如,已经提出用CryV和CryVI命名对线虫特异的一类毒素基因。
H_fte和Whiteley对结晶状蛋白质的1989年命名和分类方案,是基于推导的氨基酸序列和毒素的宿主范围。那种系统覆盖了14种不同类型的毒素,分成五个主要种类。经测序的苏云金芽孢杆菌结晶状蛋白质基因的数目目前超过50种。已经有人提出了仅仅基于氨基酸同一性的经修正的命名方案(Crickmore等人[1996]无脊椎动物病理学学会,第29次年会,第三次苏云金芽孢杆菌讨论会,University ofClrdoba,Spain,September 1-6,1996,abstract)。除了仍然保留为单独类型的cytA和cytB外,新方案保留了所有毒素基因中的“cry”。将第一级中的罗马数字改为阿拉伯数字,并除去了第三级中的圆括号。虽然许多种类进行了重新分类,但是保留了许多最初的名称,除了注明的例外。
现在已经鉴定了许多其它B.t.基因。WO 94/21795、WO 96/10083、WO 98/44137、和J.J.Estruch等人(1996)PNAS 93:5389-5394描述了由芽孢杆菌微生物得到的Vip1A(a)、Vip1A(b)、Vip2A(a)、Vip2A(b)、Vip3A(a)、和Vip3A(b)毒素。据报道,这些毒素产生于营养细胞生长期,并因此命名为营养杀虫蛋白质(vegetativeinsecticidal protein,VIP)。据报道,这些毒素对某些鳞翅目和鞘翅目害虫有活性。WO 98/18932公开了杀虫毒素的新类型。
芽孢杆菌毒素成功应用于农业的障碍包括昆虫发展对B.t.的抗性。此外,某些昆虫可能是不受芽孢杆菌毒素作用的。后者包括诸如棉铃象甲(boll weevil)和小地虎(black cutworm)等昆虫,以及迄今已经证明对B.t.δ-内毒素没有明显重大敏感性的大多数物种的成年昆虫。当B.t.转基因植物技术中的抗性处理策略已经成为兴趣焦点时,仍然很需要发展能够在植物中表达以有效控制多种昆虫的其它基因。
除那些描述于WO 98/18932中的之外,本申请提供了毒素和基因的新类型,并与WO 94/21795、WO 96/10083、WO 98/44137、和Estruch等人公开的那些截然不同。
发明的简要概述
本发明涉及对控制非哺乳动物害虫(特别是植物害虫)有用的物质和方法。在一个实施方案中,本发明提供了针对非哺乳动物害虫具有有利活性的新的芽孢杆菌分离物。在另一个实施方案中,本发明提供了对控制非哺乳动物害虫有用的新毒素。在优选的实施方案中,这些害虫是鳞翅目和/或鞘翅目昆虫。本发明的毒素包括δ-内毒素,以及可以由芽孢杆菌培养物上清液得到的可溶性毒素。
本发明还提供了编码本发明毒素的核苷酸序列。本发明还提供了对鉴定和分析杀虫毒素的编码基因有用的核苷酸序列和方法。
在一个实施方案中,本发明涉及作为杂交探针和/或PCR技术中的引物有用的独特核苷酸序列。这些引物产生可以用于鉴定、分析、和/或分离特异毒素基因的特征性基因片段。本发明的核苷酸序列编码的毒素与先前描述的毒素截然不同。
在特殊的实施方案中,本发明提供了具有有利杀虫活性的毒素新类别。这些毒素类别可以由多核苷酸序列编码,所述多核苷酸序列特征为能够与某些例示序列杂交,和/或能够使用某些例示引物由PCR扩增。
本发明的一方面是关于具有有利杀虫特性的芽孢杆菌毒素的全新家族的鉴定和分析。本发明包括此处称为MIS-7和MIS-8的基因和毒素新类型。本发明还公开了新的WAR和SUP类基因和毒素。此处还进一步分析了某些MIS-1和MIS-2毒素和基因。
这些毒素家族和它们的编码基因,可以由为例如毒素或基因的大小、DNA或氨基酸序列、杀虫活性、和/或抗体反应性鉴别。关于本发明毒素新家族的编码基因,当前公开内容提供了独特杂交探针和PCR引物,可以用于鉴定和分析每种例示家族的DNA。
在本发明的一个实施方案中,可以在导致微生物高度增殖的条件下培养芽孢杆菌分离物。在对微生物进行处理以提供单链基因组核酸之后,可以将DNA与本发明的引物接触并用于PCR扩增。毒素编码基因的特征性片段将通过此步骤得到扩增,由此鉴定毒素编码基因的存在。
本发明的另一方面是将公开的核苷酸序列作为探针使用,以检测对抗害虫有活性的芽孢杆菌毒素的编码基因。
本发明的另一方面包括使用此处公开的方法和核苷酸序列鉴定的基因和分离物。由此鉴定的基因编码对抗害虫有活性的毒素。相似的,分离物具有对抗这些害虫的活性。在优选的实施方案中,这些害虫是鳞翅目或鞘翅目害虫。
在优选的实施方案中,本发明涉及用至少一种本发明的多核苷酸序列转化的植物细胞,从而经转化的植物细胞在目的害虫取食的组织中表达杀虫毒素。正如此处描述的,根据本发明有用的毒素可以是通过将多种毒素部分结合产生的嵌合毒素。此外,可以根据本发明使用毒素的混合物和/或组合物。
用此处公开的基因构建物转化植物,可以使用本领域技术人员熟知的技术实现,通常涉及对基因进行修饰以优化毒素在植物中的表达。
或者,可以将本发明的芽孢杆菌分离物、或表达此处描述的毒素的重组微生物用于控制害虫。在这点上,本发明包括对基本上完整的芽孢杆菌细胞、和/或表达本发明毒素的重组细胞进行处理,使当基本上完整的细胞被应用于目的害虫的环境时可延长杀虫活性。经处理的细胞作为杀虫毒素的保护性覆料。毒素经目的害虫消化后变得有活性。
序列的简要描述
SEQ ID NO.1是编码来自B.t.菌株Javelin 1990的毒素的核苷酸序列。
SEQ ID NO.2是Javelin 1990毒素的氨基酸序列。
SEQ ID NO.3是根据本发明使用的正向引物。
SEQ ID NO.4是根据本发明使用的反向引物。
SEQ ID NO.5是来自B.t.菌株PS66D3的毒素基因的核苷酸序列。
SEQ ID NO.6是来自66D3毒素的氨基酸序列。
SEQ ID NO.7是来自B.t.菌株PS177C8的MIS毒素基因的核苷酸序列。
SEQ ID NO.8是来自177C8-MIS毒素的氨基酸序列。
SEQ ID NO.9是来自B.t.菌株PS177I8的毒素基因的核苷酸序列。
SEQ ID NO.10是177I8毒素的氨基酸序列。
SEQ ID NO.11是编码来自B.t.菌株PS177C8的177C8-WAR毒素基因的核苷酸序列。
SEQ ID NO.12是来自B.t.菌株PS177C8的177C8-WAR毒素的氨基酸序列。
SEQ ID NO.13-21是根据本发明使用的引物。
SEQ ID NO.22是SEQ ID NO.14引物的反向互补链。
SEQ ID NO.23是SEQ ID NO.15引物的反向互补链。
SEQ ID NO.24是SEQ ID NO.17引物的反向互补链。
SEQ ID NO.25是SEQ ID NO.18引物的反向互补链。
SEQ ID NO.26是SEQ ID NO.19引物的反向互补链。
SEQ ID NO.27是SEQ ID NO.20引物的反向互补链。
SEQ ID NO.28是SEQ ID NO.21引物的反向互补链。
SEQ ID NO.29是MIS-7正向引物。
SEQ ID NO.30是MIS-7反向引物。
SEQ ID NO.31是MIS-8正向引物。
SEQ ID NO.32是MIS-8反向引物。
SEQ ID NO.33是来自B.t.菌株PS157C1、命名为157C1-A的MIS-7毒素基因的核苷酸序列。
SEQ ID NO.34是来自B.t.菌株PS157C1、命名为157C1-A的MIS-7毒素的氨基酸序列。
SEQ ID NO.35是来自B.t.菌株PS201Z的MIS-7毒素基因的核苷酸序列。
SEQ ID NO.36是来自B.t.菌株PS31F2的MIS-8毒素基因的核苷酸序列。
SEQ ID NO.37是来自B.t.菌株PS185Y2的MIS-8毒素基因的核苷酸序列。
SEQ ID NO.38是来自B.t.菌株PS33F1的MIS-1毒素基因的核苷酸序列。
SEQ ID NO.39是根据本发明使用的MIS引物。
SEQ ID NO.40是根据本发明使用的MIS引物。
SEQ ID NO.41是根据本发明使用的WAR引物。
SEQ ID NO.42是根据本发明使用的WAR引物。
SEQ ID NO.43是来自PS205C的MIS-7基因的部分核苷酸序列。
SEQ ID NO.44是来自PS205C的MIS-7毒素的部分氨基酸序列。
SEQ ID NO.45是来自PS205C的WAR基因的部分核苷酸序列。
SEQ ID NO.46是来自PS205C的WAR毒素的部分氨基酸序列。
SEQ ID NO.47是来自PS31F2的MIS-8基因的核苷酸序列。
SEQ ID NO.48是来自PS31F2的MIS-8毒素的氨基酸序列。
SEQ ID NO.49是来自PS31F2的WAR基因的核苷酸序列。
SEQ ID NO.50是来自PS31F2的WAR毒素的氨基酸序列。
SEQ ID NO.51是根据本发明使用的SUP引物。
SEQ ID NO.52是根据本发明使用的SUP引物。
SEQ ID NO.53是来自KB59A4-6的SUP基因的核苷酸序列。
SEQ ID NO.54是来自KB59A4-6的SUP毒素的氨基酸序列。
发明的详细公开
本发明涉及控制非哺乳动物害虫的物质和方法。在特殊的实施方案中,本发明涉及新的苏云金芽孢杆菌分离物,和对抗鳞翅目和/或鞘翅目昆虫具有活性的毒素。本发明还涉及编码杀虫毒素的新基因,和鉴定及分析编码具有有用特性的毒素的芽孢杆菌基因的方法。本发明不仅涉及编码这些毒素的多核苷酸序列,而且还涉及使用这些多核苷酸序列,以产生表达毒素的重组宿主。本发明的蛋白质与先前从苏云金芽孢杆菌中分离得到的蛋白质毒素截然不同。
根据本发明有用的B.t.分离物已经保存于农业研究机构专利培养物收藏中心(Agricultural Research Service Patent CultureCollection)(NRRL),北方地区研究中心(Northern RegionalResearch Center),北方大学街1815号(1815 North UniversityStreet),Peoria,伊利诺斯(Illinois)61604美国。B.t.菌株培养物保藏编号如下:
表1. | |||
培养物 | 保藏号 | 存放日期 | 专利号 |
B.t.PS157C1(MT104) | NRRL B-18240 | July 17,1987 | 5,262,159 |
B.t.PS31F2 | NRRL B-21876 | October 24,1997 | |
B.t.PS66D3 | NRRL B-21858 | October 24,1997 | |
B.t.PS177C8a | NRRL B-21867 | October 24,1997 | |
B.t.PS177I8 | NRRL B-21868 | October 24,1997 | |
KB53A49-4 | NRRL B-21879 | October 24,1997 | |
KB68B46-2 | NRRL B-21877 | October 24,1997 | |
KB68B51-2 | NRRL B-21880 | October 24,1997 | |
KB68B55-2 | NRRL B-21878 | October 24,1997 | |
PS33F1 | NRRL B-21977 | April 24,1998 | |
PS71G4 | NRRL B-21978 | April 24,1998 | |
PS86D1 | NRRL B-21979 | April 24,1998 | |
PS185V2 | NRRL B-21980 | April 24,1998 | |
PS191A21 | NRRL B-21981 | April 24,1998 | |
PS201Z | NRRL B-21982 | April 24,1998 | |
PS205A3 | NRRL B-21983 | April 24,1998 | |
PS205C | NRRL B-21984 | April 24,1998 | |
PS234E1 | NRRL B-21985 | April 24,1998 | |
PS248N10 | NRRL B-21986 | April 24,1998 | |
KB63B19-13 | NRRL B-21990 | April 29,1998 | |
KB63B19-7 | NRRL B-21989 | April 29,1998 | |
KB68B62-7 | NRRL B-21991 | April 29,1998 | |
KB68B63-2 | NRRL B-21992 | April 29,1998 | |
KB69A125-1 | NRRL B-21993 | April 29,1998 | |
KB69A125-3 | NRRL B-21994 | April 29,1998 | |
KB69A125-5 | NRRL B-21995 | April 29,1998 | |
KB69A127-7 | NRRL B-21996 | April 29,1998 | |
KB69A132-1 | NRRL B-21997 | April 29,1998 | |
KB69B2-1 | NRRL B-21998 | April 29,1998 | |
KB70B5-3 | NRRL B-21999 | April 29,1998 | |
KB71A125-15 | NRRL B-30001 | April 29,1998 | |
KB71A35-6 | NRRL B-30000 | April 29,1998 | |
KB71A72-1 | NRRL B-21987 | April 29,1998 | |
KB71A134-2 | NRRL B-21988 | April 29,1998 | |
PS185Y2 | NRRL B-30121 | May 4,1999 | |
KB59A4-6 | NRRLB- | ||
MR992 | NRRL B-30124 | May 4,1999 | |
MR983 | NRRL B-30123 | May 4,1999 | |
MR993 | NRRL B-30125 | May 4,1999 | |
MR951 | NRRL B-30122 | May 4,1999 |
所述培养物已进行了保藏,在该专利申请的待审期内,保证根据37 CFR 1.14和35 U.S.C.122.授权的专利和商标委员会所确定的人能获取所述培养物。在提交了该申请的副本或其后续申请的国家中,可以按照该国专利法的要求提供所述保藏物。但是,应当明白保藏物的可获得性并不构成对以侵犯由政府行为授予的专利权来实施本发明的许可。
此外,应按照微生物保藏的布达佩斯条约款项来保存和对公众发放所述保藏培养物,即应小心储存以保证其在最近一次要求重新提供保藏物样品后至少5年内,以及任何情况中在保藏日后至少30年内或公开该培养物的专利授权后的有效期内存活并不被污染。由于保藏物状况使得无法由保藏物提供样品时,保藏者承担替换保藏物的责任。公众获取所述培养物的全部限制在公开该培养物的专利被授权时去除。
通过公开这些菌株的专利的发行效力,通过在公共收藏中心的保藏,或通过它们在商业中的销售,可以容易的获得根据本发明有用的许多菌株。例如,用于商业生产的B.t.菌株Javelin和HD分离物都可以公开得到。
此处提及的分离物的突变体可以通过本领域熟知的步骤产生。例如,通过甲烷磺酸乙酯(EMS)诱变分离物,可以得到不产孢子的突变体。使用紫外线和亚硝基胍,通过本领域熟知的步骤也可以产生突变体。
在一个实施方案中,本发明涉及包括用于分离、分析、和鉴定芽孢杆菌基因(编码对抗非哺乳动物害虫有活性的蛋白质毒素)的核苷酸引物和探针的物质和方法。此处描述的核苷酸序列还可以用于鉴定新的杀虫芽孢杆菌分离物。本发明还涉及使用此处描述的方法和物质鉴定到的基因、分离物、和毒素。
此处提供的新的毒素和多核苷酸序列由多个参数详细说明。此处描述的毒素的一个特征是杀虫活性。在特殊的实施方案中,这些毒素具有针对鞘翅目和/或鳞翅目害虫的活性。本发明的毒素和基因还可以由它们的氨基酸和核苷酸序列详细说明。分子的序列可以由与某些例示序列的同源性,以及与某些例示探针杂交、或被某些例示引物扩增的能力详细说明。此处提供的毒素还可以根据它们与某些抗体的免疫反应性鉴定。
本发明的一个重要方面是芽孢杆菌毒素新家族和编码这些毒素的基因的鉴定和分析。这些家族已经命名为MIS-7和MIS-8。此处还公开了新的WAR和SUP类毒素家族。这些家族的毒素,以及编码这些家族内的毒素的基因,可以容易的如此处所述,通过例如大小、氨基酸或DNA序列、和抗体活性等鉴定。氨基酸和DNA序列特征包括与例示序列的同源性、与DNA探针杂交的能力、和用特殊引物扩增的能力。
此处还进一步分析了MIS-1家族的一种基因和毒素(可从PS33F1得到)和MIS-2家族的一种基因和毒素(可从PS66D3得到)。
此处鉴定的毒素新家族是MIS-7家族。这个家族包括可以由B.t.分离物PS157C1、PS205C、和PS201Z得到的毒素。本发明进一步提供了用于鉴定MIS-7基因和毒素的探针和引物。
此处鉴定的另一个毒素新家族是MIS-8家族。这个家族包括可以由B.t.分离物PS31F2和PS185Y2得到的毒素。本发明进一步提供了用于鉴定MIS-8基因和毒素的探针和引物。
在优选的实施方案中,MIS家族基因编码分子量为大约70-大约100kDa的毒素,最优选大小为大约80kDa的毒素。这些毒素通常是可溶性的,而且可以由此处所述的芽孢杆菌培养物的上清液得到。这些毒素具有针对非哺乳动物害虫的毒性。在优选的实施方案中,这些毒素具有针对鞘翅目害虫的活性。MIS蛋白质由于它们在细胞中形成孔的能力而进一步有用。这些蛋白质可以与第二种实体,例如其它蛋白质一起使用。当与第二种实体一起使用时,MIS蛋白质将促进第二种试剂进入靶细胞。在优选的实施方案中,MIS蛋白质与靶细胞中的MIS受体相互作用,并引起靶细胞中孔的形成。第二种实体可以是毒素或需要进入细胞的其它分子。
本发明还涉及命名为WAR类毒素的毒素家族。WAR毒素通常大小为大约30-50kDa,而且最常见的大小为大约40kDa。这些毒素通常是可溶性的,而且可以由此处所述的芽孢杆菌培养物的上清液得到。WAR毒素可以用此处所述的引物以及抗体鉴定。
根据本发明提供的其它毒素家族是命名为SUP类的毒素。这些毒素通常是可溶性的,而且可以由此处所述的芽孢杆菌培养物的上清液得到。在优选的实施方案中,SUP毒素对抗鳞翅目害虫有活性。SUP毒素通常大小为大约70-100kDa,优选大约80kDa。SUP家族此处由来自分离物KB59A4-6的毒素例示。本发明提供了对鉴定SUP家族毒素和基因有用的探针和引物。
本发明还提供其它的芽孢杆菌毒素和基因,包括其它的MIS、WAR、和SUP毒素和基因。
MIS、WAR、和SUP家族的毒素都是可溶性的,而且可以如此处所述由芽孢杆菌培养物的上清液得到。这些毒素可以单独或与其它毒素组合使用来控制害虫。例如,MIS家族的毒素可以与WAR类毒素结合使用以实现控制害虫,特别是鞘翅目害虫。这些毒素可以,例如,与由芽孢杆菌分离物得到的δ-内毒素一起使用。
表2提供了本发明的毒素和基因新家族的概要。某些MIS家族在此处通过可以由表2所示的特殊B.t.分离物得到的毒素具体例示。编码这些家族的毒素的基因可以通过多种高度特异参数鉴定,包括与表2列出的特殊探针杂交的能力。与表2列出的探针超过大约80%相同的序列,也可以用来鉴定多种家族的基因。表2还例示了可以用来扩增本发明的基因的特殊引物对。用至少一对列举的引物,通常可以扩增所指家族内的基因的一部分。在优选的实施方案中,被扩增的部分将大约是所指的片段大小。表2所示引物由编码此处所附序列表中所示的肽的多核苷酸序列组成。本领域的那些技术人员可以容易的构建其它引物和探针,从而可以利用编码相同氨基酸序列的其它多核苷酸序列鉴定和/或分析编码杀虫毒素的其它基因。在优选的实施方案中,可以由芽孢杆菌分离物得到这些其它毒素和它们的基因。
表2.家族 分离物 探针 引物对 片段大小
(SEQ ID NO.) (SEQ ID NO.) (nt)MIS-1 PS33F1 37 13和22 69
13和23 506
14和23 458MIS-2 PS66D3 5 16和24 160
16和25 239
16和26 400
16和27 509
16和28 703
17和25 102
17和26 263
17和27 372
17和28 566
18和26 191
18和27 300
18和28 494
19和27 131
19和28 325
20和28 213MIS-7 PS205C、PS157C1(157C1-A)、33,35 29和30 598
PS201ZMIS-8 PS31F2、PS185Y2 36,37 31和32 585SUP KB59A4-6 1 51和52
此外,根据本发明可以使用嵌合毒素。已经发展了通过B.t.蛋白质各部分的组合产生有用嵌合毒素的方法。组合的各部分自身不需要是杀虫的,只要组合各部分产生的嵌合蛋白质是杀虫的即可。这可以使用限制酶实现,例如欧洲专利0 228 838;A.Z.Ge,N.L.Shivarova,D.H.Dean(1989)美国国家科学院进展(Proc.Natl.Acad.Sci.USA)86:4037-4041;A.Z.Ge,D.Rivers,R.Milne,D.H.Dean(1991)生物化学杂志266:17954-17958;H.E.Schnepf,K.Tomczak,J.P.Ortega,H.R.Whiteley(1990)生物化学杂志265:20923-20930;G.Honee,D.Convents,J.Van Rie,S.Jansens,M.Peferoen,B.Visser(1991)分子微生物学5:2799-2806中所述。或者,利用细胞重组机制的重组可以实现相似的结果。见,例如,T.Caramori,A.M.Albertini,A.Galizzi(1991)基因98:37-44;W.R.Widner,H.R.Whiteley (1990)细菌学杂志172:2826-2832;D.Bosch,B.Schipper,H.van der Kliej,R.A.de Maagd,W.J.Stickema(1994)生物技术(Biotechnology)12:915-918。本领域已有许多其它方法可以产生这些嵌合DNA。本发明意欲包括利用本申请鉴定的新序列的嵌合蛋白质。
用此处提供的教学,本领域的技术人员可以容易的产生和使用此处所述的多种毒素和多核苷酸序列。
基因和毒素。根据本发明有用的基因和毒素不仅包括全长序列,还有这些序列的片段、变体、突变体、和融合蛋白质(保留了此处特别例示毒素的特征性杀虫活性)。根据本发明的教学,也可以利用通过组合一种以上芽孢杆菌毒素或基因的部分产生的嵌合基因和毒素。如此处所用,术语基因的“变体”或“突变体”指编码相同毒素或编码具有杀虫活性的相当毒素的核苷酸序列。如此处所用,术语“相当毒素”指具有与例示毒素相同或本质上相同的对目的害虫的生物学活性的毒素。例如,美国专利号5,605,793描述了通过重新组装经随机片段化的DNA产生的多种其它分子的方法。
对本领域技术人员是明显的,可以通过多种方法鉴定和得到编码活性毒素的基因。可以从保存于上述培养物保藏单位的分离物得到此处例示的特殊基因。这些基因,或者它们的部分或变体,也可以通过人工合成构建,例如,使用基因合成仪。使用产生点突变的常规技术,可以容易的构建基因的变体。还有,根据常规步骤,使用商业性可获得的核酸外切酶或核酸内切酶,可以产生这些基因的片段。例如,可以用酶诸如Bal31或定点诱变来从这些基因的末端系统切除核苷酸。还可以使用多种限制酶得到编码活性片段的基因。可以使用蛋白酶直接得到这些毒素的活性片段。
使用此处提供的教学,可以由芽孢杆菌分离物和/或DNA文库得到相当毒素和/或编码这些相当毒素的基因。有许多方法可以得到本发明的杀虫毒素。例如,可以使用此处公开和要求保护的杀虫毒素的抗体,从蛋白质混合物中鉴定和分离毒素。具体地说,可以制得针对毒素中最保守和与其它芽孢杆菌毒素相比最独特的部分的抗体。然后通过免疫沉淀、酶联免疫吸附反应(enzyme linked immunosorbent assay,ELISA)、或Western印渍,可以将这些抗体用于特别鉴定具有特征性活性的相当毒素。使用本领域的常规步骤,可以容易的制备针对此处公开的毒素、相当毒素、或这些毒素的片段的抗体。然后可以由微生物得到编码这些毒素的基因。
保留了例示毒素的杀虫活性的片段和相当物属于本发明的范围之内。还有,因为遗传密码的冗余,多种不同DNA序列可以编码此处公开的氨基酸序列。产生编码相同或本质上相同的毒素的这些替换DNA序列,是属于本领域训练的工作人员的技术范围内的。这些变体DNA序列属于本发明的范围之内。如此处所用,提及的“本质上相同的”序列指具有本质上不影响杀虫活性的氨基酸取代、删除、增加、或插入的序列。这个定义还包括保留了杀虫活性的片段。
鉴定本发明毒素和基因的另一种方法是使用寡核苷酸探针。这些探针是可检测的核苷酸序列。探针提供了鉴定本发明毒素编码基因的快速方法。可以使用DNA合成仪和常规步骤,人工合成根据本发明作为探针使用的核苷酸片段。
此处已经特别例示了本发明的某些毒素。既然这些毒素仅仅是本发明毒素的范本,显然本发明应当包含具有与例示毒素相同或相似杀虫活性的变体或相当毒素(和编码相当毒素的核苷酸序列)。相当毒素与例示毒素具有氨基酸同源性。这种氨基酸同一性通常超过60%,优选超过75%,更优选超过80%,更优选超过90%,而且可以超过95%。这些同一性是使用常规排序技术测定的。氨基酸同源性在确定生物学活性或涉及决定最终负责生物学活性的三维构型的毒素关键区将是最高的。在这一点上,某些氨基酸取代是可接受的而且可预计的,如果这些取代位于对活性不重要的区域内或是不影响分子三维构型的保守氨基酸取代。例如,氨基酸可以归为下列种类:非极性氨基酸、不带电氨基酸、碱性氨基酸、和酸性氨基酸。只要取代本质上不改变化合物的生物学活性,用一种氨基酸取代其它同类氨基酸的保守取代属于本发明的范围之内。表3提供了属于每一类的氨基酸实例的列表。
表3.氨基酸类型 氨基酸实例非极性 Ala、Val、Leu、Ile、Pro、Met、Phe、Trp不带电、极性 Gly、Ser、Thr、Cys、Tyr、Asn、Gln酸性 Asp、Glu碱性 Lys、Arg、His
在某些情况中,还可以进行非保守取代。关键因素是这些取代不能显著影响毒素的生物学活性。
本发明的δ-内毒素的特征可以用如上所述的毒素的形状和位置表述。
如此处所用,提及的“分离的”多核苷酸和/或“经纯化的”毒素指这些分子与在自然条件下共存的其它分子分开。因此,提及“分离和经纯化的”表示涉及此处所述的“人为处理”。嵌合毒素和基因也涉及“人为处理”。
重组宿主。可以将本发明的毒素编码基因导入广泛的微生物或植物宿主。毒素基因的表达直接的或间接的导致杀虫剂的产生和维持。对于合适的微生物宿主,例如假单胞菌(Pseudomonas),可以应用于害虫地点,它们将增殖并被摄取。结果是控制了害虫。或者,在延长毒素活性和稳定细胞的条件下,可以杀死和处理含有毒素基因的微生物。然后可以将保留了毒性活性的、经处理的细胞应用于目的害虫的环境。
通过合适的载体将芽孢杆菌毒素基因导入微生物宿主,并将该宿主以活的状态应用于环境时,重要的是使用特定的宿主微生物。微生物宿主选自已知占据一种或多种感兴趣农作物的“植物群落”(叶表、叶圈、根际和/或根表)。选择这些微生物是为了能够成功的在特殊环境中(农作物和其它昆虫栖息地)与野生型微生物竞争,提供表达多肽杀虫剂的基因的稳定维持和表达,并理想地使杀虫剂免于环境降解和灭活。
已知大量的微生物栖息于广泛的重要农作物的叶面(植物叶片表面)和/或根际(植物根部周围的土壤)。这些微生物包括细菌、藻类、和真菌。特别感兴趣的是微生物,诸如细菌,例如,假单胞菌属(Pseudomonas)、欧文氏菌属(Erwinia)、沙雷氏菌属(Serratia)、克雷白氏菌属(Klebsiella)、黄单胞菌属(Xanthomonas)、链霉菌属(Streptomyces)、根瘤菌属(Rhizobium)、红假单胞菌属(Rhodopseudomonas)、嗜甲基菌属(Methylophilius)、土壤杆菌属(Agrobacterium)、醋杆菌属(Acetobacter)、乳杆菌属(Lactobacillus)、节杆菌属(Arthrobacter)、固氮菌属(Azotobacter)、明串珠菌属(Leuconostoc)、和产碱菌属(Alcaligenes);真菌,特别是酵母,例如,酵母属(Saccharomyces)、隐球酵母属(Cryptococcus)、克鲁维酵母属(Kluyveromyces)、掷孢酵母属(Sporobolomyces)、红酵母属(Rhodotorula)、和短梗霉属(Aureobasidium)。特别感兴趣的是这些植物群落细菌物种,如丁香假单胞菌(Pseudomonas syringae)、荧光假单胞菌(Pseudomonasfluorescens)、粘质沙雷氏菌(Serratia marcescens)、木醋杆菌(Acetobacter xylinum)、根瘤土壤杆菌(Agrobacteriumtumefaciens)、球形红假单胞菌(Rhodopseudomonas spheroides)、野油菜黄单胞菌(Xanthomonas campestris)、苜蓿根瘤菌(Rhizobiummelioti)、真养产碱菌(Alcaligenes entrophus)、和维涅兰德固氮菌(Azotobacter vinlandii);和植物群落酵母物种,诸如深红酵母(Rhodotorula rubra)、胶粘红酵母(R.glutinis)、海滨红酵母(R.marina)、橙黄色红酵母(R.aurantiaca)、浅白隐球酵母(Cryptococcus albidus)、流散隐球酵母(C.diffluens)、罗伦隐球酵母(C.laurentii)、罗氏酵母(Saccharomyces rosei)、普地酵母(S.pretoriensis)、啤酒酵母(S.cerevisiae)、红色掷孢酵母(Sporobolomyces roseus)、香气掷孢酵母(S.odorus)、佛地克鲁维酵母(Kluyveromyces veronae)、和Aureobasidium pollulans。特别感兴趣的是有色微生物。
有许多方法可以在允许基因稳定维持和表达的条件下将编码毒素的芽孢杆菌基因导入微生物宿主。这些方法对于本领域的技术人员是众所周知的,并描述于,例如,美国专利号5,135,867,此处引用作为参考。
还可以将与本发明毒素功能相当的人工合成基因用于转化宿主。可以在,例如,美国专利号5,380,831中找到产生人工合成基因的方法。
细胞的处理。正如上文提到的,可以对表达芽孢杆菌毒素的芽孢杆菌或重组细胞进行处理,以延长毒素活性和稳定细胞。形成的杀虫微囊在细胞结构中包含芽孢杆菌毒素,该细胞结构经稳定处理并在微囊应用于目的害虫环境时保护毒素。合适的宿主细胞包括原核细胞或真核细胞。作为宿主,特别感兴趣的是原核细胞和低级真核细胞,诸如真菌。细胞通常是完整的,并在处理时基本上处于繁殖形式而非孢子形式。
微生物细胞,例如含有芽孢杆菌毒素基因的微生物的处理,可以通过化学的或物理的方法,或者通过化学和/或物理方法的组合,只要该技术不损害毒素的特性,并且不减小细胞保护毒素的能力即可。处理微生物细胞的方法公开于美国专利号4,695,455和4,695,462,此处引用作为参考。
用于控制害虫的方法和制剂。可以通过本领域技术人员知道的多种方法,使用本发明的分离物、毒素、和基因来实现控制害虫。这些方法包括,例如,将芽孢杆菌分离物应用于害虫(或它们的所在地)、将重组微生物应用于害虫(或它们的所在地)、和用编码本发明的杀虫毒素的基因转化植物。转化可以由本领域技术人员使用常规技术进行。此处公开了用于这些转化的必要物质,或者熟练的技术人员可以通过其它方法容易的获得。
可以将配制的含有引诱剂和芽孢杆菌分离物毒素、或包含可由此处公开的芽孢杆菌分离物得到的基因的重组微生物的诱饵颗粒应用于土壤。还可以将配制的产品作为种子覆料或根部处理或作物生长周期晚期的完整植株处理应用。植物和土壤的芽孢杆菌细胞处理可以以可润湿粉末、颗粒或粉尘,通过与多种惰性物质混合,诸如无机矿物质(phyllosilicates、碳酸盐、硫酸盐、磷酸盐、等等)或植物材料(玉米穗轴粉、稻壳、胡桃壳、等等)混合使用。制剂可以包括扩散-增稠佐剂、稳定剂、其它杀虫添加剂、或表面活性剂。液体制剂可以是基于水的或非水的,并以泡沫、凝胶、悬浮液、可乳化浓缩物等等形式使用。成分可以包括流变剂、表面活性剂、乳化剂、分散剂、或聚合物。
本领域技术人员可以理解,杀虫剂浓度将由于特殊制剂的本性广泛变化,特别是可作为浓缩物或直接使用。杀虫剂将以至少1%(重量计)存在,而且可能是100%(重量计)。干燥制剂通常有大约1-95%(重量计)的杀虫剂,而液体制剂将通常是液相中固体重量大约1-60%。含有细胞的制剂将通常含有大约102-大约104个细胞/mg。这些制剂将以每公顷大约50mg(液体的或干的)-1kg或更多的量使用。
通过喷、撒、洒、等等,可以将制剂应用于害虫环境,例如土壤和植物。
多核苷酸探针。众所周知的,DNA具有叫做碱基互补性的基本特性。自然界中,DNA通常以成对的反向平行链的形式存在,每条链上的碱基从该链上凸出并指向相对链。一条链上的腺嘌啉(adenine,A)碱基总是对应另一条链上的胸腺嘧啶(thymine,T)碱基,而鸟嘌啉(guanine,G)碱基对应胞嘧啶(eytosine,C)碱基。由于碱基能够以这种特殊方式形成氢键,它们保持并置。虽然每一个单独的键是相对微弱的,但是许多邻近碱基的氢键结合的的净作用,与碱基堆叠作用一起,形成两条互补链的稳定连接。这些键可以通过处理诸如高pH或高温打断,而且这些条件导致两条链的解离,或“变性”。然后如果将DNA置于热动力学上有利于碱基氢键形成的条件中,那么DNA链将退火,或“杂交”,并重新形成最初的双链DNA。如果在适当的条件下进行,这种杂交可以是高度特异的。即只有具有高度碱基互补的链能够形成稳定的双链结构。杂交特异性与反应条件的关系是众所周知的。由此,杂交可以用于测试两段DNA的碱基序列是否互补。正是这种杂交机制促进了使用本发明的探针来容易的检测和分析感兴趣的DNA序列。
探针可以是RNA、DNA、或PNA(peptide nucleic acid,肽核酸)。探针将通常具有至少大约10个碱基,更通常至少大约17个碱基,而且可以具有多至大约100个碱基或更多。可以容易的利用较长的探针,而且这种探针可以是例如长度为几千个碱基。探针序列被设计成至少与编码感兴趣的毒素的基因的一部分基本互补。探针不需要与它要杂交的序列完全互补。可以使用本领域技术人员熟知的技术,对探针进行标记。
使用本发明作为探针的尝试,首先需要通过Southern印渍分析,鉴定与公开的核苷酸序列同源的芽孢杆菌分离物的所有DNA片段基因库。由此,仍可能在没有生物学分析帮助的情况下,预先知道许多新的芽孢杆菌分离物、和给定的芽孢杆菌分离物表达的个别基因产物的大概活性。这种探针分析提供了在各种B.t.亚种中快速鉴定具有潜在商业价值的杀虫毒素基因的方法。
根据本发明有用的一种杂交操作,通常包括分离感兴趣的DNA样品并化学纯化之的起始步骤。可以使用经裂解的细菌或从细菌分离得到并经片段化的所有核酸。可以使用已知技术对细胞进行处理,以释放它们的DNA(和/或RNA)。使用适当的限制酶可以将DNA样品切成片段。通过凝胶(通常是琼脂糖或丙烯酰胺)电泳可以将片段根据大小分开。可以将感兴趣的片段转移到固定化膜上。
特别的杂交技术对本发明并不重要。由于杂交技术中做了改进,所以它们可以容易的应用。
然后可以将探针和样品在杂交缓冲液中混合,并在适当的温度中保持到发生退火。此后,洗膜至无无关物质,留下样品和结合的探针分子,通常通过自显影和/或液闪计数检测和定量。正如本领域众所周知的,如果探针分子和核酸样品通过形成两分子间强非共价键杂交,那么可以合理的推测探针和样品基本相同。探针的可检测标记提供了以已知方式测定杂交是否发生的方法。
将核苷酸片段作为探针使用时,用本领域技术人员知道的任何合适的标记,包括放射性和非放射性标记,对特别的探针进行标记。典型的放射性标记包括32P、35S等等。非放射性标记包括,例如,配基诸如生物素或甲状腺素,以及酶诸如水解酶或perixodase,或各种化学发光剂诸如萤光素、或荧光化合物如荧光素及其衍生物。可以使探针固有的发荧光,如国际申请号WO 93/16094中描述的。
可以应用各种程度的严谨条件。越严谨的条件,形成双螺旋要求的互补程度越高。可以通过温度、探针浓度、探针长度、离子强度、时间等等控制严谨程度。优选的,通过本领域众所周知的技术在中度-高度严谨条件下进行杂交,例如,G.H.Keller,M.M.Manak(1987)DNA探针,Stockton出版社,New York,NY.,pp.169-170所述。
如此处使用的杂交的“中度-高度严谨”条件指,达到与本申请人应用的条件的杂交相同或大致相同程度特异性的条件。此处提供了中度和高度严谨条件的实例。具体地说,使用常规方法(Maniatis等人)进行Southern印渍上固定的DNA与32P标记的基因特异探针的杂交。通常,在中度-高度严谨条件下进行杂交和随后的清洗,这种严谨条件下能够检测到与例示毒素基因同源的目的序列。对于双链DNA基因探针,杂交于低于DNA杂合体熔解温度[melting temperature,Tm])20-25℃下在6X SSPE、5X Denhardt氏溶液、0.1%SDS、0.1mg/ml变性DNA中进行过夜。熔解温度由下面的公式描述(G.A.Beltz,K.A.Jacobs,T.H.Eickbush,P.T.Cherbas,和F.C.Kafatos[1983]酶学方法,R.Wu,L.Grossman和K.Moldave[编]Academic Press,NewYork 100:266-285)。
Tm=81.5℃+16.6 Log[Na+]+0.41(%G+C)-0.61(%甲酰胺)-600/双螺旋长度(单位:碱基对)。
清洗通常如下进行:
(1)于室温在1X SSPE、0.1%SDS中两次15分钟(低度严谨条件清洗)。
(2)于Tm-20℃在0.2X SSPE、0.1%SDS中一次15分钟(中度严谨条件清洗)。
对于寡核苷酸探针,杂交于低于杂合体的熔解温度Tm 10-20℃下在6X SSPE、5X Denhardt氏溶液、0.1%SDS、0.1mg/ml变性DNA中进行过夜。寡核苷酸探针的Tm由下面的公式描述:
Tm(℃)=2(T/A碱基对数目)+4(G/C碱基对数目)(S.V.Suggs,T.Miyake,E.H.Kawashime,M.J.Johnson,K.Itakura,和R.B.Wallace[1981]ICN-UCLA symp.Dev.Biol.Using PurifiedGenes,D.D.Brown[编],学术出版社,纽约,23:683-693)。
清洗通常如下进行:
(1)于室温在1X SSPE、0.1%SDS中两次15分钟(低度严谨条件清洗)。
(2)于杂交温度在1X SSPE、0.1%SDS中一次15分钟(中度严谨条件清洗)。
通常,可以改变盐和/或温度来改变严谨条件。对于长度>70碱基左右的、经标记的DNA片段,可以使用下面的条件:
低度:1或2X SSPE,室温
低度:1或2X SSPE,42℃
中度:0.2X或1X SSPE,65℃
高度:0.1X SSPE,65℃
双螺旋的形成和稳定性取决于杂合体两条链之间实质上的互补性,并且,如上所述,可以承受一定程度的错配。因此,本发明的探针序列包括所述序列的(单个和多个)突变、删除、插入、及其组合,其中所述突变、插入和删除可以允许与感兴趣的目的多核苷酸形成稳定的杂合体。可以用许多方法在给定的多核苷酸序列中产生突变、插入、和删除,而且普通熟练工作人员知道这些方法。将来可以有其它一些方法。
由此,通过那些本领域技术人员熟知的方法,可以容易的制备公开的核苷酸序列的突变的、插入的、和删除的变体。这些变体可以以与例示引物序列相同的方式使用,只要变体与原始序列具有实质上的序列同源性即可。如此处所用的,实质上的序列同源性指足够使得变体探针以与原始探针相同能力发挥功能的同源性。优选的,此同源性超过50%;更优选的,此同源性超过75%;最优选的,此同源性超过90%。变体以预计能力发挥功能需要的同源性程度,将取决于序列预计用途。产生设计用来改进序列的功能或提供方法学优点的突变、插入、和删除突变的技术,属于本领域训练的技术人员的技术范围之内。
PCR技术。聚合酶链反应(PCR)是一种反复的、酶催化的、引导性的核酸序列合成。该过程是公知并被本领域技术人员广泛使用的(参见,Mullis.美国专利4,683,195、4,683,202和4,800,159;Saiki.Randall K.,Stephen Scharf,Fred Faloona.Kary B.Mullis,GlennT.Horn,Henry A.Erlich,Norman Artiheim[1985]″诊断镰形细胞贫血病的β-珠蛋白基因组序列的酶法扩增以及限制位点分析″科学230:1350-1354.)。PCR基于对侧翼为两个与目的序列相对链可杂交的寡核苷酸引物的目的DNA片段进行酶法扩增。该引物的方向是3’端相对。反复循环的模板热变性、引物与其互补序列退火以及DNA聚合酶引导的退火引物的延伸使由PCR引物5’末端界定的片段得以扩增。由于每个引物的延伸产物可以作为另一引物的模板,每次循环基本能使前一循环产生的DNA片段的量加倍。这就导致特异靶片段的指数累积,在几个小时内达到几百万倍。通过使用热稳定的DNA聚合酶,比如Taq聚合酶(其分离自嗜热杆菌水生栖热菌),扩增过程可以完全自动化。其他可以使用的酶是本领域技术人员已知的。
本发明的DNA序列可以作为PCR扩增的引物使用。在进行PCR扩增中,可以承受引物和模板之间一定程度的错配。因此,例示引物的突变、删除、和插入(尤其在5’末端加入核苷酸)属于本发明的范围。通过普通熟练技工知道的方法,可以在给定的引物中产生突变、插入和删除。
此处引用的所有文献由此作为参考引用。
下列实施例举例说明了实行本发明的步骤。不应当把这些实施例解释为限制。除非特别注明,所有百分比是指重量百分比,而所有溶剂混合物的比例是指体积比。实施例1-根据本发明有用的芽孢杆菌分离物的培养
含有芽孢杆菌杀虫基因的细胞宿主可以在任何方便的营养培养基中培养。然后可以用常规方法收获这些细胞。或者,可以在收获之前对细胞进行处理。
本发明的芽孢杆菌细胞可以使用常规技术培养基和发酵技术培养。在发酵循环中,可以使用本领域众所周知的方法,首先从发酵液中分离芽孢杆菌营养细胞、芽孢、晶体、和裂解细胞碎片,从而收获细菌。使用众所周知的技术,可以回收形成的任何芽孢杆菌芽孢或δ-内毒素晶体,并作为传统的B.t.δ-内毒素制备物使用。发酵工艺中的上清中含有本发明的毒素。使用众所周知的技术分离并纯化毒素。
芽孢杆菌分离物的传代培养物或它们的突变体,可以用来接种下面叫做TB的培养基:
胰蛋白胨 12 g/l
酵母提取物 24 g/l
甘油 4 g/l
KH2PO4 2.1 g/l
K2HPO4 14.7 g/l
pH 7.4
经高压灭菌的培养液冷却后加入磷酸钾。将烧瓶于30℃在旋转摇床上以250rpm培养24-36小时。
上述步骤可以通过本领域众所周知的步骤容易的放大至大发酵罐。
可以通过本领域众所周知的步骤,分离在上述发酵中得到的芽孢杆菌。经常使用的步骤是对收获的发酵液进行分离技术,如离心。在特殊的实施方案中,可以从上清液中得到根据本发明有用的芽孢杆菌蛋白质。含有活性蛋白质的培养物上清液可以用于生物实验。
或者,芽孢杆菌分离物的传代培养物或它们的突变体,可以用来接种下面的蛋白胨、葡萄糖、盐培养基:
细菌用蛋白胨 7.5 g/l
葡萄糖 1.0 g/l
KH2PO4 3.4 g/l
K2HPO4 4.35 g/l
盐溶液 5.0 ml/l
CaCl2 5.0 ml/l
pH 7.2盐溶液(100ml)
MgSO4·7H2O 2.46g
MnSO4·H2O 0.04g
ZnSO4·7H2O 0.28g
FeSO4·7H2O 0.40gCaCl2溶液(100ml)
CaCl2·2H2O 3.66g
将盐溶液和CaCl2溶液过滤除菌,并在接种时加到经高温灭菌的培养液中。烧瓶于30℃在旋转摇床上以200rpm培养64小时。
上述步骤可以通过本领域众所周知的步骤容易的放大至大发酵罐。
可以通过本领域众所周知的步骤,分离在上述发酵中得到的芽孢杆菌芽孢和/或晶体。经常使用的步骤是对收获的发酵液进行分离技术,如离心。实施例2-用于PCR的细胞DNA的分离和制备
可以从在Spizizen氏琼脂或其它本领域技术人员众所周知的基本或丰富琼脂上培养了大约16小时的细胞制备DNA。Spizizen氏酪蛋白氨基酸琼脂含有23.2g/l Spizizen氏基本盐[(NH4)2SO4,120g;K2HPO4,840g;KH2PO4,360g;柠檬酸钠,60g;MgSO4·7H2O,12g。合计:1392g]、1.0g/l不含维生素的酪蛋白氨基酸、15.0g/l Difco琼脂。在制备琼脂过程中,将混合物高压灭菌30分钟,然后加入无菌的50%葡萄糖溶液至终浓度0.5%(1/100体积)。一旦细胞培养了大约16小时,从琼脂上刮取大约1cm2菌苔,加到300μl 10mMTris-HCl(pH8.0)-1mM EDTA中。加入蛋白酶K至50μg/ml,并于55℃温育15分钟。也可以使用其它适当的无核酸酶活性的蛋白酶。然后将样品置于沸水浴中15分钟,使蛋白酶失活并使DNA变性。这还使不想要的成分沉淀。然后将样品于14,000g在Eppendorf微量离心机中于室温离心5分钟,除去细胞碎片。将含有粗制DNA的上清液转移到新管中,并冻存于-20℃直至用于PCR反应。
或者,可以使用购自Qiagen(Santa Clarita,CA)的QIAamp TissueKit,依照制造商的说明书,从平板培养的细胞制备总细胞DNA。实施例3-对分析和/或鉴定毒素基因有用的引物
下面的PCR引物对可以用于鉴定和/或分析编码杀虫毒素的本发明的基因:
GGRTTAMTTGGRTAYTATTT(SEQ ID NO.3)
ATATCKWAYATTKGCATTTA(SEQ ID NO.4)
本发明公开的序列中的冗余核苷酸密码子与IUPAC协定一致,并且包括:
R=A或G
M=A或C
Y=C或T
K=G或T
W=A或T实施例4-编码来自芽孢杆菌菌株的新可溶性蛋白质毒素的基因的鉴 定和测序
在从广泛的B.t.菌株分离得到的总细胞基因组DNA上,使用引物SEQ ID NO.3和SEQ ID NO.4进行PCR。选择那些产生大约1kb条带的样品,通过DNA测序进行分析。首先,如供应商(Invitrogen,SanDiego,CA)所述,将扩增的DNA片段克隆到PCR DNA TA-克隆质粒载体pCR2.1中。从重组克隆中分离质粒,并使用质粒载体引物T3和T7通过PCR测试大约1kbp插入片段的存在。
下列菌株产生期望的大约1000bp条带,由此说明MIS类毒素基因的存在:PS66D3、PS177C8、PS177I8、PS33F1、PS157C1(PS157C1-A)、PS201Z、PS31F2、和PS185Y2。
然后使用QIAGEN(Santa Clarita,CA)微量制备试剂盒,如供应商所述分离质粒作为测序模板使用。使用购自PE AppliedBiosystems的Dye Terminator Cycle Sequencing Ready ReactionKit,在ABI PRISM 377 Automated Sequencer上,进行测序反应。使用购自PE ABI的ABI PRISM 377 Collection、Factura、和AutoAssembler软件收集、编辑、并装配序列数据。
测定了来自下列分离物的新毒素基因部分的DNA序列:PS66D3、PS177C8、PS177I8、PS33F1、PS157C1(157C1-A)、PS201Z、PS31F2、和PS185Y2。这些核苷酸序列分别显示于SEQ ID NO.5、7、9、38、33、35、36、和37。推导了编码的、来自下列分离物的新可溶性毒素部分的多肽序列:PS66D3、PS177C8、PS177I8、和PS157C1(毒素157C1-A)。这些核苷酸序列分别显示于SEQ ID NO.6、8、10、和34。实施例5-来自苏云金芽孢杆菌菌株的毒素的限制性片段长度多态性 (Restriction Fragment Length Polymorphism,RFLP)
从培养至600nm可见光处的光密度为0.5-0.8的各种苏云金芽孢杆菌(B.t.)菌株制备总细胞DNA。使用Qiagen Genomic-tip 500/G kit和Genomic DNA Buffer Set,根据用于革兰氏阳性细菌的方案(QiagenInc.,Valencia,CA),提取DNA。
在总基因组DNA制备物中,用常规Southern杂交(使用32P标记的探针)鉴定和分析新的毒素基因。将制备的总基因组DNA用各种限制酶消化、在1%琼脂糖凝胶上电泳、并使用常规方法固定到支持尼龙膜上(Maniatis等人)。
将PCR扩增的长度为1.0-1.1 kb的DNA片段凝胶纯化,作为探针使用。使用DNA聚合酶I Klenow片段(New England Biolabs)、随机六核苷酸引物(Boehringer Mannheim)和32P dCTP,每种DNA片段大约25ng作为模板,用来引发DNA合成。
每种32P标记的片段充当针对相应基因组DNA印渍的特异探针。经固定的DNA与随机标记的32P探针的杂交,在由5X SSPE、5X Denhardt’s溶液、0.5%SDS、0.1mg/ml组成的常规含水缓冲液中,于65℃进行过夜。在中等严谨条件下,在0.2X SSC、0.1%SDS中于65℃清洗印渍,并对胶片曝光。得到每种菌株的显示特异杂交条带(包含所有或部分感兴趣的新基因)的RFLP数据。
表3 | ||
(菌株)/ 探针的 RFLP数据(大致条带大小)基因名 序列编号 | ||
(PS)66D3 | 24 | BamH I:4.5kbp,Hind III:>23kbp,Kpn I:23kbp,Pst I:15kbp,Xba I:>23kbp |
(PS)177I8 | 33 | BamH I:>23kbp,EcoR I:10kbp,Hind III:2kbp,Sal I:>23kbp,Xba I:3.5kbp |
在单独的实验中,通过32P自显影或通过使用DIG核酸标记和检测系统(Boehringer Mannheim,Indianapolis,IN)的非放射性方法,分别使用MIS和WAR基因的探针,在基因组DNA的Southern印渍上检测新的毒素基因。从质粒pMYC2450中,使用与相应基因5’和3’末端同源的引物,PCR扩增长度大约为2.6kbp(PS177C8 MIS毒素基因;SEQ ID NO.7)和1.3kbp(PS177C8 WAR毒素基因;SEQ ID NO.11)的DNA片段。pMYC2450是在pHTBlue II(包含pBluescriptS/K[Stratagene,La Jolla,CA]和来自B.t.常住质粒的复制起点的大肠杆菌/苏云金芽孢杆菌穿梭载体[D.Lereclus等人1989,FEMS微生物学通讯60:211-218])含有大约14kbp Cla I片段上的PS177C8MIS和WAR基因的重组质粒。将这些DNA片段作为MIS RFLP类型A-N和WAR RFLP类型A-L的探针使用。表4中类型O的RFLP数据是使用引物S1-633F(CACTCAAAAAATGAAAAGGGAAA;SEQ ID NO.39)和S1-2269R(CCGGTTTTATTGATGCTAC;SEQ ID NO.40)扩增的、大约1636bp的MIS片段产生的。表5中类型M的RFLP数据是使用引物S2-501F(AGAACAATTTTTAGATAGGG;SEQ ID NO.41)和S2-995R(TCCCTAAAGCATCAGAAATA;SEQ ID NO.42)扩增的、大约495bp的WAR片段产生的。
将片段凝胶纯化,每种DNA片段取大约25ng用32P随机标记用于放射性检测,或者取300ng用DIG High Prime kit随机标记用于非放射性检测。经固定的DNA与随机标记的32P探针的杂交,在常规甲酰胺条件:50%甲酰胺、5X SSPE、5X Denhardt’s溶液、2%SDS、0.1mg/ml经超声波降解的精子DNA中,于42℃进行过夜。在低严谨条件下,在2X SSC、0.1%SDS中于42℃清洗印渍,并对胶片曝光。得到每种菌株的显示DNA条带(包含所有或部分感兴趣的新基因)的RFLP数据。如上所述使用MIS探针的RFLP数据如下:
表4 | ||
RFLP类型 | 菌株名 | RFLP数据(大致条带大小,单位:碱基对) |
A | 177C8,74H3,66D3 | Hind III:2,454;1,645Xba I:14,820;9,612;8,138;5,642;1,440 |
B | 177I8 | Hind III:2,454Xba I:3,500(很弱7,000) |
C | 66D3 | Hind III:2,454(弱20,000)Xba I:3,500(弱7,000) |
D | 28M,31F2,71G5,71G7,71I1,71N1,146F,185Y2,201JJ7,KB73,KB68B46-2,KB71A35-4,KB71A116-1 | Hind III:11,738;7,614Xba I:10,622;6,030 |
D1 | 70B2,71C2 | Hind III:11,738;8,698;7,614Xba I:11,354;10,622;6,030 |
E | KB68B51-2,KB68B55-2 | Hind III:6,975;2,527Xba I:10,000;6,144 |
F | KB53A49-4 | Hind III:5,766Xba I:6,757 |
G | 86D1 | Hind III:4,920Xba I:11,961 |
H | HD573B,33F1,67B3 | Hind III:6,558;1,978Xba I:7,815;6,558 |
I | 205C,40C1 | Hind III:6,752Xba I:4,618 |
J | 130A3,143A2,157C1 | Hind III:9,639;3,943,1,954;1,210Xba I:7,005;6,165;4,480;3,699 |
K | 201Z | Hind III:9,639;4,339Xba I:7,232;6,365 |
L | 71G4 | Hind III:7,005Xba I:9.639 |
M | KB42A33-8,KB71A72-1,KB71A133-11 | Hind III:3,721Xba I:3,274 |
N | KB71A134-2 | Hind III:7,523Xba I:10,360;3,490 |
O | KB69A125-3,KB69A127-7,KB69A136-2,KB71A20-4 | Hind III:6,360;3,726;1,874;1,098Xba I:6,360;5,893;5,058;3,726 |
如上所述使用WAR探针的RFLP数据如下:
实施例6-WAR毒素的分析和/或鉴定
表5 | ||
RFLP类型 | 菌株名 | RFLP数据(大致条带大小,单位:碱基对) |
A | 177C8,74H3 | Hind III:3,659,2,454,606Xba I:5,457,4,469,1,440,966 |
B | 177I8,66D3 | 数据难以获得 |
C | 28M,31F2,71G5,71G7,71I1,71N1,146F,185Y2,201JJ7,KB73,KB68B46-2,KB71A35-4,KB71A116-1 | Hind III:7,614Xba I:10,982,6,235 |
C1 | 70B2,71C2 | Hind III:8,698,7,614Xba I:11,354,6,235 |
D | KB68B51-2,KB68B55-2 | Hind III:7,200Xba1:6,342(51-2有11,225)(55-2有9,888) |
E | KB53A49-4 | Hind III:5,766Xba I:6,757 |
F | HD573B,33F1,67B3 | Hind III:3,348,2,037(只有HD573B有6,558)Xba I:6,953(只有HD573B有7,815;6,185) |
G | 205C,40C1 | Hind III:3,158Xba I:6,558,2,809 |
H | 130A3,143A2,157C1 | Hind III:4,339,3,361,1,954,660,349Xba I:9.043,4,203,3,583,2,958,581,464 |
I | 201Z | Hind III:4,480,3,819,703Xba I:9,336,3,256,495 |
J | 71G4 | Hind III:7,005Xba I:9,639 |
K | KB42A33-8,KB71A72-1,KB71A133-11 | 没有杂交信号 |
L | KB71A134-2 | Hind III:7,523Xba I:10.360 |
M | KB69A125-3,KB69A127-7,KB69A136-2,KB71A20-4 | Hind III:5,058;3,726;3,198;2,745;257Xba I:5,255;4,341;3,452;1,490;474 |
在本发明的另一个实施方案中,可以通过杀虫毒素与此处例示的杀虫毒素抗体的反应水平来进行分析和/或鉴定。在特殊的实施方案中,可以制备针对WAR毒素(诸如可从PS177C8a得到的毒素)的抗体。然后其它WAR毒素可以通过与这些抗体的反应来进行鉴定和/或分析。在优选的实施方案中,抗体是多克隆抗体。在这个实施例中,与177C8a-WAR毒素具有最大相似性的毒素,应该与多克隆抗体有最大反应性。具有较大差异的WAR毒素与177C8a多克隆抗体发生反应,但是程度较弱。可以从例如命名为PS177C8a,PS17718,PS66D3,KB68B55-2,PS185Y2,KB53A49-4,KB68B51-2,PS31F2,PS74H3,PS28M,PS71G6,PS71G7,PS71I1,PS71N1,PS201JJ7,KB73,KB68B46-2,KB71A35-4,KB71A116-1,PS70B2,PS71C2,PS86D1,HD573B,PS33F1,PS67B3,PS205C,PS40C1,PS130A3,PS143A2,PS157C1,PS201Z,PS71G4,KB42A33-8,KB71A72-1,KB71A133-11,KB71A134-2,KB69A125-3,KB69A127-7,KB69A136-2,和KB71A20-4的分离物,得到与针对177C8a WAR毒素的多克隆抗体发生免疫反应的毒素。分离物PS31F2和KB68B46-2显示非常微弱的抗体反应,暗示有差异。实施例7-来自苏云金芽孢杆菌菌株PS205C的可溶性杀虫蛋白质(MIS 和WAR)基因的分子克隆和DNA序列分析
从在Luria Bertani(LB)培养液中培养至600nm可见光处的光密度为0.5-0.8的苏云金芽孢杆菌菌株PS205C制备总细胞DNA。使用Qiagen Genomic-tip 500/G kit和Genomic DNA Buffer Set,根据用于革兰氏阳性细菌的方案(Qiagen Inc.,Valencia,CA)提取DNA。使用PS205C总细胞DNA经Nde II部分消化的插入片段,在SuperCos载体(Stratagene)中构建了PS205C粘粒文库。将经包装的粘粒转染XL1-Blue细胞(Stratagene),得到抗羧苄青霉素和卡那霉素的克隆。576个粘粒菌落在96孔板中、1ml LB+羧苄青霉素(100μg/ml)+卡那霉素(50μg/ml)中于37℃培养18小时,并将平板复制到尼龙滤膜上用于杂交筛选。
如实施例4所述,使用引物SEQ ID NO.3和SEQ ID NO.4,从PS205基因组DNA扩增得到包含大约1000bp PS205C MIS基因的PCR扩增子。使用Qiaex II extraction(Qiagen),将DNA片段凝胶纯化。使用Prime-It II kit(Stratagene),用32P-dCTP对探针进行放射性标记,并与菌落复制滤膜在含水杂交溶液(6X SSPE、5X Denhardt氏溶液、0.1%SDS、0.1mg/ml变性DNA)于65℃杂交16小时。将菌落复制滤膜用2XSSC/0.1%SDS于室温简单洗一次,随后再在0.5XSSC/0.1%SDS中洗10分钟两次。然后用滤膜使X光胶片曝光5.5小时。选择与探针强杂交的一个粘粒克隆,进行进一步的分析。通过使用引物SEQ IDNO.3和SEQ ID NO.4的PCR扩增,确认了这个粘粒克隆包含MIS基因。这个粘粒克隆命名为pMYC3105;包含pMYC3105的重组大肠杆菌XL-Blue MR细胞命名为MR992。
MR992的传代培养物于1999年5月4日保存于专利培养物收藏中心(NRRL),Regional Research Center,1815 North UniversityStreet,Peoria,Illinois,61604美国。编号是NRRL B-30124。PS205C经截短的质粒克隆也于1999年5月4日收存。编号是NRRL B-30122。
为了对PS205C MIS和WAR基因进行测序,使用GPS-1 GenomePriming System和方案(New England Biolabs)产生了pMYC3105的随机转座子插入。选择编码氯霉素抗性的GPS2转座载体用于选择包含插入的粘粒。通过转化和在含有氨苄青霉素、卡那霉素和氯霉素的培养基上选择大肠杆菌XL1-Blue细胞,鉴定获得了转座子的pMYC3105粘粒。使用Multiscreen 96-well plasmid prep(Millipore),从单个菌落制备粘粒模板,作为测序模板使用。使用ABI377自动测序系统和相关软件,用GPS2引物对pMYC3105编码的MIS和WAR毒素基因进行测序。发现MIS和WAR基因在一个表观转录操纵子中彼此相邻。其核苷酸和推导的多肽序列命名为SEQ ID NO.43-46。实施例8-来自苏云金芽孢杆菌菌株PS31F2的可溶性杀虫蛋白质(MIS 和WAR)基因的分子克隆和DNA序列分析
a.基因组DNA的制备和克隆
从在Luria Bertani(LB)培养液中培养至600nm可见光的光学密度为0.5-0.8的苏云金芽孢杆菌菌株PS31F2制备总细胞DNA。使用Qiagen Genomic-tip 500/G kit或Genomic-tip 20/G和GenomicDNA Buffer Set(Qiagen Inc.,Valencia,CA),根据用于革兰氏阳性细菌的方案,提取DNA。
由经Nde II部分消化的DNA制备包含来自苏云金芽孢杆菌菌株PS31F2的总基因组DNA的λ文库。在0.7%琼脂糖凝胶上进行部分NdeII限制性消化产物的电泳,从凝胶上切下包含DNA片段大小范围为9-20kbp的区域。在0.1XTAE缓冲液中,于大约30V进行一小时,将DNA从凝胶块上电洗脱下来,并使用Elutip-d层析柱(Schleicher和Schuell;Keene,NH)纯化。
将经纯化、片段化的DNA连接到经BamH I消化的λ-GEM-11臂(Promega Corp.,Madison,WI)中。然后使用Gigapack III Goldpackaging extract(Stratagene Corp.,La Jolla,CA)将经连接的DNA包装成λ噬菌体。用重组噬菌体感染大肠杆菌菌株KW251,并在LB顶层琼脂中铺到LB平板上。将噬斑复制到硝酸纤维素滤膜上,并准备用于使用常规方法(Maniatis等人)的杂交。从质粒pMYC2450中PCR扩增长度大约为1.1kb(PS177C8 MIS)或700bp(PS177C8 WAR)的DNA片段,作为探针使用。将片段凝胶纯化,并且每种DNA片段取大约25ng,用32P-dCTP进行随机标记。经固定的DNA与随机32p标记的PS177C8探针的杂交在常规甲酰胺条件:50%甲酰胺、5X SSPE、5XDenhardt氏溶液、2%SDS、0.1mg/ml中,于42℃进行过夜。在低严谨条件下,在2X SSC、0.1%SDS中于42℃清洗印渍,并使对胶曝光。从平板上分离杂交噬斑,并重悬于SM缓冲液。使用LambdaSorb phageadsorbent(Promega,Madison,WI)制备噬菌体DNA。使用寡核苷酸引物SEQ ID NO.3和SEQ ID NO.4,噬菌体DNA模板,进行PCR以检验目的基因的存在。两份DNA样品的PCR反应都产生期望的1kb条带,确认了那些噬菌体克隆含有感兴趣的基因。为进行亚克隆,将噬菌体DNA用各种酶消化、在1%琼脂糖凝胶上分离、并转膜用于Southern分析。如上所述进行Southern分析。鉴定了大小大约为8kb的HindIII片段含有PS31F2毒素基因。将这个片段凝胶纯化,并克隆到pBluescript II(SK+)的Hind III位点;这个质粒克隆命名为pMYC2610。重组大肠杆菌XL10 Gold[pMYC2610]菌株命名为MR983。
MR983的传代培养物于1999年5月4日保存于专利培养物收藏中心(NRRL),Regional Research Center,1815 North UniversityStreet,Peoria,Illinois,61604美国。编号是NRRL B-30123。
b.DNA测序
通过限制性消化、在0.7%琼脂糖凝胶上分离、并使用Qiaex IIkit(Qiagen Inc.;Valencia,CA)从凝胶基质上纯化,分离了含有PS31F2毒素基因的pMYC2610 Hind III片段。然后将经凝胶纯化的插入DNA,分别用限制酶Alu I、Mse I、或Rsa I消化,并在1%琼脂糖凝胶上分离。从凝胶上切下0.5-1.5kb之间的DNA片段,并使用QiaexII kit纯化。将回收的片段连接到经EcoRV消化的pBluescript II中,并转化到大肠杆菌XL10 Gold细胞中。从随机选择的转化体制备质粒DNA,用Not I和Apa I消化以检验插入片段的大小,并作为测序模板和与质粒载体序列同源的引物一起使用。由引物步行法(primerwalking)来完成序列。使用dRhodamine或BigDye Sequencing kit(ABI Prism/Perkin Elmer Applied Biosystems),并在ABI 373或377自动测序仪上进行测序反应。使用Factura、Autoassembler(ABIPrism)和Gentics Computer Group(Madison,WI)程序分析数据。发现MIS和WAR基因在一个表观转录操纵子中彼此相邻。WAR基因位于MIS基因的5’,并且两个基因被4个核苷酸碱基分开。
来自PS31F2的新的MIS和WAR基因的核苷酸序列和推导的肽序列报导于新的SEQ ID NO.47-50。
c.苏云金芽孢杆菌的亚克隆和转化
为了在天然的芽孢杆菌启动子后表达,通过8kb的Hind III片段,将PS31F2毒素基因从pMYC2610亚克隆到大肠杆菌/苏云金芽孢杆菌穿梭载体pHT370(O.Arantes和D.Lereclus,1991,基因108:115-119)中。得到的质粒构建物命名为pMYC2615。从重组大肠杆菌XL10Gold制备pMYC2415质粒DNA,通过电穿孔转化无晶体(Cry-)苏云金芽孢杆菌宿主CryB(A.Aronson,Purdue University,West Lafayette,IN)。重组CryB[pMYC2615]菌株命名为MR558。实施例9-来自苏云金芽孢杆菌菌株KB59A4-6的新的SUP毒素基因的 分子克隆和DNA序列分析
从在Luria Bertani(LB)培养液中培养至600nm可见处的光学密度为0.5-0.8的苏云金芽孢杆菌菌株KB59A4-6制备总细胞DNA。使用QiagenGenomic-tip 500/G kit和Genomic DNA Buffer Set,根据用于革兰氏阳性细菌的方案(Qiagen Inc.;Valencia,CA)提取DNA。将DNA用HindIII消化,并进行0.7%琼脂糖凝胶电泳,用于常规方法(Maniatis等人)的Southern印渍分析。使用寡核苷酸“3A-atg”(GCTCTAGAAGGAGGTAACTTATGAACAAGAATAATACTAAATTAAGC)(SEQ ID NO.51)和“3A-taa”(GGGGTACCTTACTTAATAGAGACATCG)(SEQ ID NO.52),得到含有来自Javelin-90基因组DNA的SUP类基因(SEQ ID NO.1)的PCR扩增子。将这个DNA片段凝胶纯化,并使用Prime-It II Random PrimerLabeling Kit(Stratagene)用放射性的32P-dCTP进行标记,作为探针使用。Southern印渍滤膜的杂交,在溶液6X SSPE、5X Denhardt氏溶液、0.1%SDS、0.1mg/ml变性DNA中,在振摇水浴中于42℃进行过夜。随后将滤膜在1X SSPE和0.1%SDS中于25℃清洗一次,随后于37℃清洗两次。然后用经杂交的滤膜对X光胶片于-80U曝光。鉴定了KB59A4-6基因组DNA大约1kbp的Hind III片段与Javelin 90 SUP探针杂交。
如下构建了KB59A4-6基因组DNA的λ文库。将DNA用Sau3A部分消化,并在琼脂糖凝胶上进行大小分离。切下含有9.0-23kbp之间片段的凝胶区域,并通过在0.1XTAE缓冲液中电洗脱来分离DNA,随后使用Elutip-d层析柱(Schleicher和Schuell,Keene,NH)纯化。将经大小分离的DNA插入片段连接到经BamH I消化的λ-Gem 11(Promega)中,并使用Gigapack III XL Packing Extract(Stratagene)包装重组噬菌体。将噬菌体铺在大肠杆菌VCS257细胞上,用于杂交筛选。将噬斑转移到尼龙滤膜上,并在真空中于80℃干燥。然后如上所述与Javelin 90 Sup基因探针进行杂交。选择给出阳性信号的一个噬斑,使用巴斯德移液管得到一个栓子。将栓子在1mL SM缓冲液+10μLCHCl3中于室温浸泡过夜。从用这种噬菌体感染的大肠杆菌KW251裂解液中得到大量噬菌体DNA的制备物(Maniatis等人)。
通过经Southern杂交鉴定的大约5.5kbp Sac I/Xba I片段,将KB59A4-6毒素基因亚克隆到大肠杆菌/苏云金芽孢杆菌穿梭载体pHT370(0.Arantes和D.Lereclus,1991,Gene基因108:115-119)中。这个质粒亚克隆命名为pMYC2473。含有这种构建物的重组大肠杆菌XL10-Gold细胞(Stratagene)命名为MR993。使用pMYC2473质粒和PCR扩增子作为DNA模板,通过引物前行法对杀虫毒素基因测序。使用购自PE Applied Biosystems的Dye Terminator CycleSequencing Ready Reaction Kit,在ABI PRISM 377 AutomatedSequencer上进行测序反应。使用PE ABI PRISM 377 Collection、Factura、和AutoAssembler软件分析序列数据。KB59A4-6毒素的DNA序列和推导的肽序列分别报导于新的SEQ ID NO.53和SEQ ID NO.54。
MR993的传代培养物于1999年5月4日保存于专利培养物收藏中心(NRRL),Regional Research Center,1815 North UniversityStreet,Peoria,Illinois,61604美国。编号是NRRL B-30125。实施例10-测试针对鳞翅目昆虫和鞘翅目昆虫的活性的生物实验
使用常规生物实验步骤,可以确认本发明的分离物和毒素的生物学活性。一种这样的实验是烟芽夜蛾-棉铃虫(Heliothisvirescens[Fabricius]和Helicoverpa zea[Boddie])实验。通过施用到人工昆虫食物表面,或食物中掺入样品,进行鳞翅目生物实验。测试了所有鳞翅目昆虫,从初生阶段到二龄。用烤大豆粉人工食物或小地虎人工食物(BioServ,Frenchtown,NJ)进行所有实验。
可以通过将样品与人工食物以6mL悬浮液+54mL食物的比例混和进行食物掺入。漩涡混匀后,将这种混合物倒入划分成3ml孔的塑料盘(Nutrend Container Corporation,Jacksonville,FL)。不含B.t.的水空白作为对照使用。将一龄幼虫(USDA-ARS,Stoneville,MS)置于食物混合物上。然后用Mylar薄膜(ClearLam Packaging,IL)和烙铁将孔封闭,并在每个孔上戳几个针孔以提供气体交换。将幼虫在14∶10(白天∶黑夜)饲养间中于25℃放置6天。六天后记录死亡率和发育迟缓率。
通过表面施药方法的生物实验,使用如上所列相同的样品和食物制备物。将样品施用到昆虫食物表面。在特殊的实施方案中,表面面积范围为0.3-大约0.8cm2(取决于盘子的大小),除了上面列出的形式,还使用96孔组织培养板。施药后,在昆虫食用前,将样品在空气中干燥。不含B.t.的水空白可以作为对照使用。将虫卵置于每个经处理的孔中,然后用Mylar薄膜(ClearLam Packaging,IL)和烙铁将孔封闭,并在每个孔上戳几个针孔以提供气体交换。生物实验在14∶10(白天∶黑夜)饲养间中于25℃进行7天或于28℃进行4天。在每个生物实验的最后记录死亡率和昆虫发育迟缓现象。
根据本发明有用的另一个实验是玉米幼芽根叶甲(western cornrootworm)实验。通过将样品以160ml/cm2的比例施用到基于琼脂的人工食物表面上,可以进行样品对抗初生的玉米幼芽根叶甲幼虫(Diabrotica virgifera virgifera)的生物实验。人工食物可以分发到48孔组织培养板或相似板的0.78cm2孔中,并使其变硬。食物固化后,将样品用移液管分发到食物表面。然后从表面蒸发掉过量的液体,再用骆驼毛刷向每个孔的食物表面上转移大约三个初生的幼虫。为了防止昆虫在气体交换时逃跑,孔用2-mil打孔的聚酯薄膜和27HT粘合剂(Oliver Product Company,Grand Rapids,Michigan)热封。生物实验在黑暗中于25℃进行,并在四天后记录死亡率。
可以由那些本领域的技术人员进行类似的生物实验,以评定对其它害虫,诸如小地虎(Agrotis ipsilon)的活性。
结果显示于表6。
表6.筛选鳞翅类和鞘翅类活性的经浓缩的B.t.上清液的功能和遗传学 | ||||||||
菌株 | 大约339bpPCR片段 | 总蛋白质(μg/cm2) | 约80-100kDa蛋白质(μg/cm2) | 烟芽夜蛾H.virescens | 棉铃虫H.zen | Diabrotica%死亡率 | ||
%死亡率 | 发育迟缓 | %死亡率 | 发育迟缓 | |||||
PS157C1(#1) | - | 24 | 2 | 43 | 是 | 13 | 是 | - |
PS157C1(#2) | - | 93 | 8 | - | - | - | - | 40 |
PS157C1(#3) | - | 35 | 3 | - | - | - | - | 18 |
Javelin 1990 | ++ | 43.2 | 3.6 | 100 | 是 | 96 | 是 | NT |
水 | 0-8 | - | 0-4 | - | 12 |
实施例11-玉米幼芽根叶甲生物实验的结果和毒素的进一步分析
测试根据本发明得到的经浓缩的液体上清液溶液对抗玉米幼芽根叶甲(WCRW)的活性。发现来自下列分离物的上清液引起WCRW死亡:PS31F2、PS66D3、PS177I8、KB53A49-4、KB68B46-2、KB68B51-2、KB68B55-2、和PS177C8。
还发现来自下列分离物的上清液引起WCRW死亡:PS205A3、PS185V2、PS234E1、PS71G4、PS248N10、PS191A21、KB63B19-13、KB63B19-7、KB68B62-7、KB68B63-2、KB69A125-1、KB69A125-3、KB69A125-5、KB69A127-7、KB69A132-1、KB69B2-1、KB70B5-3、KB71A125-15、和KB71A35-6;确认了这种活性是热不稳定的。而且,确定了下列分离物的上清液与WAR抗体(见实施例12)不发生反应(产生阴性测试结果),并且与MIS(SEQ ID NO.31)和WAR(SEQ ID NO.51)探针不发生反应:PS205A3、PS185V2、PS234E1、PS71G4、PS248N10、PS191A21、KB63B19-13、KB63B19-7、KB68B62-7、KB68B63-2、KB69A125-1、KB69A125-5、KB69A132-1、KB69B2-1、KB70B5-3、KB71A125-15、和KB71A35-6;分离物KB69A125-3和KB69A127-7的上清液产生阳性测试结果。实施例12-31F2克隆的培养和31F2毒素对玉米幼芽根叶甲(wCRW) 的生物实验
在装有50ml DIFCO Terrific Broth培养基的250ml底部带挡板烧瓶中,培养大肠杆菌MR983和阴性对照菌株MR948(大肠杆菌XL1-Blue[pSupercos];载体对照)。培养物在New Brunswick摇床中,以250rpm振摇,于30℃温育~23小时。温育23小时后,无菌操作吸取样品,在显微镜下检查培养物,以检查污染物的存在。将30ml培养物分装到50ml离心管中,并在Sorvall离心机中于15,000rpm离心20分钟。保存1X上清液,并用于对抗wCRW的生物实验。将沉淀5×重悬于10mM Tris缓冲液中,并在用于对抗wCRW的生物实验之前超声波处理。
以相同方式培养(除了从Terrific Broth培养基中省去甘油)B.t.菌株MR558和阴性对照MR539(B.t.cryB[pHT BlueII];载体对照)。在超声波破碎之前,将B.t.细胞沉淀重悬于水而非缓冲液中。
使用与实施例10中用于玉米幼芽根叶甲的相同的实验设计(除了将上清液样品以~160μl/cm2的剂量施用到食物表面上),进行含有31F2毒素基因的大肠杆菌克隆MR983和苏云金芽孢杆菌克隆MR558的实验。对于两个克隆,将5X浓度的B.t.细胞沉淀样品以~150和~75μl/cm2的剂量施用到食物表面上,MR558苏云金芽孢杆菌克隆以~35μl/cm2的剂量施用(两个克隆的有活性的毒素量未知)。将样品蒸发之后,立即将大约6-8个幼虫转移到食物上。用Mylar薄膜和烙铁将生物实验板封闭,并在每个孔上戳针孔以提供气体交换。与毒素阴性克隆MR948和MR539相比,MR983和MR558克隆都证明了对抗玉米幼芽根叶甲的生物活性程度(较大的死亡率)。
表7列出了显示克隆的PS31F2毒素对玉米幼芽根叶甲的生物活性的结果。
表7
实施例13-目的害虫
wCRW死亡百分比 | ||||||
上清液 | 沉淀5X | 沉淀5X | 沉淀5X | |||
菌株 | 毒素基因 | 比率_ | 160ul/cm2 | 150ul/cm2 | 75ul/cm2 | 35ul/cm2 |
MR983 | 31F2 | 7%(4/56) | 19%(5/27) | - | - | |
MR948 | 无 | 4%(1/24) | 26%(6/23) | - | - | |
MR983 | 31F2 | 3%(5/147) | - | 20%(49/245) | - | |
MR948 | 无 | 27%(19/70) | - | 51%(79/154) | - | |
MR983 | 31F2 | 13%(32/243) | - | 33%(85/259) | - | |
MR948 | 无 | 9%(14/155) | - | 20%(55/273) | - | |
MR558 | 31F2 | 35%(41/118) | 88%(43/49) | 9%(9/100) | 13%(13/97) | |
MR539 | 无 | 10%(14/134) | 14%(3/21) | 15%(17/111) | 17%(19/111) | |
MR558 | 31F2 | 3%(1/29) | 35%(17/48) | 29%(15/52) | 13%(7/55) | |
MR539 | 无 | 19%(5/27) | 20%(9/46) | 31%(18/57) | 18%(9/49) | |
MR558 | 31F2 | 13%(9/69) | 38%(19/50) | 18%(15/85) | 15%(10/65) | |
MR539 | 无 | 29%(16/55) | 24%(14/58) | 14%(13/91) | 28%(18/64) | |
MR558 | 31F2 | 7%(5/74) | 14%(9/66) | 17%(14/83) | 11%(6/57) | |
MR539 | 无 | 11%(9/79) | 32%(19/59) | 9%(7/78) | 15%(10/67) |
本发明的毒素可以单独或与其它毒素组合使用来控制一种或多种非哺乳动物害虫。这些害虫可能是例如那些列于表8中的。使用此处提供的生物实验、这些实验的改进、和/或本领域技术人员熟知的其它生物实验,可以容易的确定活性。
表8.目的害虫种类目/俗名(英) 拉丁名鳞翅目(LEPIDOPTERA)欧洲玉米螟(European Corn Borer) Ostrinia nubilalis对Cry1A类毒素有抗性的欧洲玉米螟 Ostrinia nubilalis小地虎(Black Cutworm) Agrotis ipsilon秋粘虫(Fall Armyworm) Spodoptera
frugiperda巨座玉米螟(Southwestern Corn Borer) Diatraea
grandiosella谷实夜蛾/棉铃虫(Corn Earworm/Bollworm) Helicoverpa zea烟芽夜蛾(Tobacco Budworm) Heliothis virescens对Cry1A类毒素有抗性的烟芽夜蛾 Heliothis virescens向日葵同斑螟(Sunflower Head Moth) Homeosoma
ellectellum向日葵细卷叶蛾(Banded Sunflower Moth) Cochylis hospes阿根廷尺蠖(Argentine Looper) Rachiplusia nu灯蛾(Spilosoma) Spilosoma virginica披肩粘虫(Bertha Armyworm) Mamestra configurata小菜蛾(Diamondback Moth) Plutella xylostells对Cry1A类毒素有抗性的小菜蛾 Plutella xylostells鞘翅目(COLEOPTERA)向日葵籽象甲(Red Sunflower Seed Weevil) Smicronyx fulvus向日葵枝象甲(Sunflower Stem Weevil) Cylindrocopturus
adspersus向日葵叶甲(Sunflowe Beetle) Zygoramma
exclama tionis芜菁菜跳甲(Canola Flea Beetle) Phyllotreta
cruciferae玉米幼芽根叶甲(Western Corn Rootworm) Diabrotica virgifera
virgifera双翅目(DIPTERA)小麦瘿蚊(Hessian Fly) Mayetiola destructor同翅目HOMOPTERA麦二岔蚜(Greenbug) Schizaphis graminum半翅目(HEMIPTERA)豆荚草盲蝽(Lygus Bug) Lygus lineolaris线虫(Nematoda) Heterodera glycines实施例14-将毒素基因插入植物
本发明的一个方面是用本发明的杀虫毒素的编码基因转化植物。经转化的植物具有针对目的害虫攻击的抵抗力。
使用本领域众所周知的技术,可以将杀虫毒素的编码基因,如此处公开的,插入到植物细胞里。例如,可获得大量的包含大肠杆菌中的复制系统和可用于进行转化细胞选择的标记的克隆载体,用于制备插入到高等植物中的外源基因。这些载体包括,例如,pBR322、pUC系列、M13mp系列、pACYC184等等。相应的,可以将芽孢杆菌毒素的编码序列插入到载体的合适限制性位点。将得到的载体用于转化大肠杆菌。将大肠杆菌细胞在合适的营养培养基中培养,然后收获并裂解。回收质粒。通常进行序列分析、限制性分析、电泳、和其它生化-分子生物学方法作为分析方法。每步操作后,可以将使用的DNA序列切下并与下一个DNA序列连接。每种质粒序列可以克隆到相同或其它质粒中。取决于将想要的基因插入到植物中的方法,其它DNA序列可能是必需的。如果,例如,使用Ti或Ri质粒转化植物细胞,那么必需连接至少Ti或Ri质粒T-DNA的右边界序列,但是经常同时连接右和左边界序列,作为将要插入的基因的侧翼区域。
使用T-DNA转化植物细胞,已经深入研究并充分描述于EP 120516;Hoekema(1985):The Binary Plant Vector System,Offset-durkkerij Kanters B.V.,Alblasserdam,第5章;Fraley等人,Crit.Rev.Plant Sci.4:1-46;和An等人(1985)EMBO J4:277-287。
一旦插入的DNA已经整合到基因组中,它在那儿相对稳定,并且原则上不再出来。它通常含有选择性标记,赋予经转化的植物细胞以对杀生物剂(biocide)或抗生素(诸如卡那霉素、G418、博来霉素、潮霉素、或氯霉素及其它)的抗性。单独应用的标记应当相应的允许选择经转化的细胞,而非不含插入DNA的细胞。
可以获得大量的技术用于将DNA插入到植物宿主细胞中。那些技术包括使用根瘤土壤杆菌(Agrobacterium tumefaciens)或发根土壤杆菌(Agrobacterium rhizogenes)作为转化剂用T-DNA转化、嵌合、注射、biolistics(微粒轰击)、或电穿孔以及其它可能的方法。如果使用土壤杆菌转化,要插入的DNA必需克隆到特殊质粒,即中间载体或二元载体中。由于有与T-DNA序列同源的序列,中间载体可通过同源重组整合到Ti或Ri质粒中。Ti和Ri质粒还包含T-DNA转移必需的vir区。中间载体自身在土壤杆菌中不能复制。通过辅助质粒的方法(接合),中间载体可以转移到根瘤土壤杆菌中。二元载体自身可以在大肠杆菌中和在土壤杆菌中复制。它们包含侧翼为T-DNA右和左边界区的选择性标记基因和接头或多克隆位点。它们可以直接转化到土壤杆菌中(Holsters等人[1978]Mol.Gen.Genet.163:181-187)。作为宿主细胞使用的土壤杆菌要包含携带vir区的质粒。vir区对将T-DNA转移到植物细胞中是必需的。可以含有另外的T-DNA。这样转化的细菌被用于转化植物细胞。植物外植体可以方便的与根瘤土壤杆菌或发根土壤杆菌一起培养以将DNA转移到植物细胞中。然后由经感染的植物材料(例如,叶片、茎段、根,还有原生质体或悬浮培养的细胞),可以在合适的培养基(可以含有抗生素或杀生物剂用于选择)中再生成完整植株。然后可以测试这样得到的植物中插入DNA的存在。注射和电穿孔对质粒没有特殊要求。可以使用普通质粒,诸如pUC衍生物。在biolistic转化中,可以使用质粒DNA或线性DNA。
以常规方式,将经转化的细胞再生成形态正常的植物。如果转化事件涉及种系细胞,那么插入的DNA和相应的表型特征将遗传给后代植物。这样的植物可以以常规方式培养,并可以与具有相同的转化遗传因子或其它遗传因子的植物杂交。得到的杂合体个体具有相应的表型特征。
在本发明优选的实施方案中,用为植物优化了密码子用法的基因转化植物。见,例如,U.S.专利号5,380,831。还有,将方便的使用编码截短毒素的植物。截短毒素通常将编码全长毒素的大约55%-大约80%。产生用于植物的人工合成的芽孢杆菌基因的方法在本领域是知道的。
应当这样理解,此处描述的实施例和实施方案只是为了举例说明的目的,并且本领域的技术人员根据本发明可以领会各种更改和变化,这些将被认为是包括在本申请及所附权利要求书的精神和范围之内的。
序列表(1)一般资料(i)申请人:
申请人: MYCOGEN CORPORATION
街道地址: 5501 Oberlin Drive
城市: San Diego
州/省: Caiifornia
国家: US
邮政编码/ZIP:92121
电话号码: (800)745-7475
传真号码: (619)453-0142(ii)发明题目:新的杀虫毒素和编码这些毒素的核苷酸序列(iii)序列数:54(iv)通信地址:
(A)收件人:Saliwanchik,Lloyd和Saliwanchik(公司)
(B)街道: 2421N.W.41st Street,Suite A-1
(C)城市: Gainesville
(D)州: FL
(E)国家: US
(F)ZIP: 32606-6669(v)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,Version#1.30(vi)现在的申请资料:
(A)申请号:
(B)申请日期:
(C)分类: (vii)在先申请资料:
(A)申请号: US 09/073,898
(B)申请日期:1998年5月5日(viii)代理人资料:
(A)姓名: Jay M.Sanders
(B)注册号: 39,355
(C)参考/文档号:MA-708C2(ix)电讯资料:
(A)电话:352-375-8100
(B)电传:352-372-5800(2)SEQ ID NO.1的资料:(i)序列特征
(A)长度:2375个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(vi)最初来源:
(C)单独的分离物:Jav90(xi)序列描述:SEQ ID NO.1ATGAACAAGA ATAATACTAA ATTAAGCACA AGAGCCTTAC CAAGTTTTAT TGATTATTTT 60AATGGCATTT ATGGATTTGC CACTGGTATC AAAGACATTA TGAACATGAT TTTTAAAACG 120GATACAGGTG GTGATCTAAC CCTAGACGAA ATTTTAAAGA ATCAGCAGTT ACTAAATGAT 180ATTTCTGGTA AATTGGATGG GGTGAATGGA AGCTTAAATG ATCTTATCGC ACAGGGAAAC 240TTAAATACAG AATTATCTAA GGAAATATTA AAAATTGCAA ATGAACAAAA TCAAGTTTTA 300AATGATGTTA ATAACAAACT CGATGCGATA AATACGATGC TTCGGGTATA TCTACCTAAA 360ATTACCTCTA TGTTGAGTGA TGTAATGAAA CAAAATTATG CGCTAAGTCT GCAAATAGAA 420TACTTAAGTA AACAATTGCA AGAGATTTCT GATAAGTTGG ATATTATTAA TGTAAATGTA 480CTTATTAACT CTACACTTAC TGAAATTACA CCTGCGTATC AAAGGATTAA ATATGTGAAC 540GAAAAATTTG AGGAATTAAC TTTTGCTACA GAAACTAGTT CAAAAGTAAA AAAGGATGGC 600TCTCCTGCAG ATATTCTTGA TGAGTTAACT GAGTTAACTG AACTAGCGAA AAGTGTAACA 660AAAAATGATG TGGATGGTTT TGAATTTTAC CTTAATACAT TCCACGATGT AATGGTAGGA 720AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCATCGG AATTAATTAC TAAAGAAAAT 780GTGAAAACAA GTGGCAGTGA GGTCGGAAAT GTTTATAACT TCTTAATTGT ATTAACAGCT 840CTGCAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGG CTTAGCAGAT 900ATTGATTATA CTTCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGA ATTTAGAGTA 960AACATCCTCC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATGCAAA AGTTAAAGGA 1020AGTGATGAAG ATGCAAAGAT GATTGTGGAA GCTAAACCAG GACATGCATT GATTGGGTTT 1080GAAATTAGTA ATGATTCAAT TACAGTATTA AAAGTATATG AGGCTAAGCT AAAACAAAAT 1140TATCAAGTCG ATAAGGATTC CTTATCGGAA GTTATTTATG GTGATATGGA TAAATTATTG 1200TGCCCAGATC AATCTGAACA AATCTATTAT ACAAATAACA TAGTATTTCC AAATGAATAT 1260GTAATTACTA AAATTGATTT CACTAAAAAA ATGAAAACTT TAAGATATGA GGTAACAGCG 1320AATTTTTATG ATTCTTCTAC AGGAGAAATT GACTTAAATA AGAAAAAAGT AGAATCAAGT 1380GAAGCGGAGT ATAGAACGTT AAGTGCTAAT GATGATGGGG TGTATATGCC GTTAGGTGTC 1440ATCAGTGAAA CATTTTTGAC TCCGATTAAT GGGTTTGGCC TCCAAGCTGA TGAAAATTCA 1500AGATTAATTA CTTTAACATG TAAATCATAT TTAAGAGAAC TACTGCTAGC AACAGACTTA 1560AGCAATAAAG AAACTAAATT GATYGTCCCG CCAAGTGGTT TTATTAGCAA TATTGTAGAG 1620AACGGGTCCA TAGAAGAGGA CAATTTAGAG CCGTGGAAAG CAAATAATAA GAATGCGTAT 1680GTAGATCATA CAGGCGGAGT GAATGGAACT AAAGCTTTAT ATGTTCATAA GGACGGAGGA 1740ATTTCACAAT TTATTGGAGA TAAGTTAAAA CCGAAAACTG AGTATGTAAT CCAATATACT 1800GTTAAAGGAA AACCTTCTAT TCATTTAAAA GATGAAAATA CTGGATATAT TCATTATGAA 1860GATACAAATA ATAATTTAGA AGATTATCAA ACTATTAATA AACGTTTTAC TACAGGAACT 1920GATTTAAAGG GAGTGTATTT AATTTTAAAA AGTCAAAATG GAGATGAAGC TTGGGGAGAT 1980AACTTTATTA TTTTGGAAAT TAGTCCTTCT GAAAAGTTAT TAAGTCCAGA ATTAATTAAT 2040ACAAATAATT GGACGAGTAC GGGATCAACT AATATTAGCG GTAATACACT CACTCTTTAT 2100CAGGGAGGAC GAGGGATTCT AAAACAAAAC CTTCAATTAG ATAGTTTTTC AACTTATAGA 2160GTGTATTTTT CTGTGTCCGG AGATGCTAAT GTAAGGATTA GAAATTCTAG GGAAGTGTTA 2220TTTGAAAAAA GATATATGAG CGGTGCTAAA GATGTTTCTG AAATGTTCAC TACAAAATTT 2280GAGAAAGATA ACTTTTATAT AGAGCTTTCT CAAGGGAATA ATTTATATGG TGGTCCTATT 2340GTACATTTTT ACGATGTCTC TATTAAGTAA CCCAA 2375(2)SEQ ID NO.2的资料:(i)序列特征
(A)长度:790个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:蛋白质(vi)最初来源:
(C)单独的分离物:Jav90(xi)序列描述:SEQ ID NO.2Met 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 50 155 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 Lys 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 GIy 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 780Asp Val Ser Ile Lys Pro785 790(2)SEQ ID NO.3的资料:(i)序列特征
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.3GGRTTAMTTG GRTAYTATTT 20(2)SEQ ID NO.4的资料:(i)序列特征
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.4ATATCKWAYA TTKGCATTTA 20(2)SEQ ID NO.5的资料:(i)序列特征 (A)长度:1042个碱基对(B)类型:核酸(C)链型:单链(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(vi)最初来源:(C)单独的分离物:66D3(xi)序列描述:SEQ ID NO.5TTAATTGGGT ACTATTTTAA AGGAAAAGAT TTTAATAATC TTACTATATT TGCTCCAACA 60CGTGAGAATA CTCTTATTTA TGATTTAGAA ACAGCGAATT CTTTATTAGA TAAGCAACAA 120CAAACCTATC AATCTATTCG TTGGATCGGT TTAATAAAAA GCAAAAAAGC TGGAGATTTT 180ACCTTTCAAT TATCGGATGA TGAGCATGCT ATTATAGAAA TCGATGGGAA AGTTATTTCG 240CAAAAAGGCC AAAAGAAACA AGTTGTTCAT TTAGAAAAAG ATAAATTAGT TCCCATCAAA 300ATTGAATATC AATCTGATAA AGCGTTAAAC CCAGATAGTC AAATGTTTAA AGAATTGAAA 360TTATTTAAAA TAAATAGTCA AAAACAATCT CAGCAAGTGC AACAAGACGA ATTGAGAAAT 420CCTGAATTTG GTAAAGAAAA AACTCAAACA TATTTAAAGA AAGCATCGAA AAGCAGCCTG 480TTTAGCAATA AAAGTAAACG AGATATAGAT GAAGATATAG ATGAGGATAC AGATACAGAT 540GGAGATGCCA TTCCTGATGT ATGGGAAGAA AATGGGTATA CCATCAAAGG AAGAGTAGCT 600GTTAAATGGG ACGAAGGATT AGCTGATAAG GGATATAAAA AGTTTGTTTC CAATCCTTTT 660AGACAGCACA CTGCTGGTGA CCCCTATAGT GACTATGAAA AGGCATCAAA AGATTTGGAT 720TTATCTAATG CAAAAGAAAC ATTTAATCCA TTGGTGGCTG CTTTTCCAAG TGTCAATGTT 780AGCTTGGAAA ATGTCACCAT ATCAAAAGAT GAAAATAAAA CTGCTGAAAT TGCGTCTACT 840TCATCGAATA ATTGGTCCTA TACAAATACA GAGGGGGCAT CTATTGAAGC TGGAATTGGA 900CCAGAAGGTT TGTTGTCTTT TGGAGTAAGT GCCAATTATC AACATTCTGA AACAGTGGCC 960AAAGAGTGGG GTACAACTAA GGGAGACGCA ACACAATATA ATACAGCTTC AGCAGGATAT 1020CTAAATGCCA ATGTACGATA TA 1042(2)SEQ ID NO.6的资料:(i)序列特征
(A)长度:347个氨基酸 (B)类型:氨基酸(C)链型:单链(D)拓扑结构:线性(ii)分子类型:肽(vi)最初来源:(C)单独的分离物:66D3(xi)序列描述:SEQ ID NO.6Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Thr Ile1 5 10 15Phe Ala Pro Thr Arg Glu Asn Thr Leu Ile Tyr Asp Leu Glu Thr Ala
20 25 30Asn Ser Leu Leu Asp Lys Gln Gln Gln Thr Tyr Gln Ser Ile Arg Trp
35 40 45Ile Gly Leu Ile Lys Ser Lys Lys Ala Gly Asp Phe Thr Phe Gln Leu
50 55 60Ser Asp Asp Glu His Ala Ile Ile Glu Ile Asp Gly Lys Val Ile Ser65 70 75 80Gln Lys Gly Gln Lys Lys Gln Val Val His Leu Glu Lys Asp Lys Leu
85 90 95Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Lys Ala Leu Asn Pro Asp
100 105 110Ser Gln Met Phe Lys Glu Leu Lys Leu Phe Lys Ile Asn Ser Gln Lys
115 120 125Gln Ser Gln Gln Val Gln Gln Asp Glu Leu Arg Asn Pro Glu Phe Gly
130 135 140Lys Glu Lys Thr Gln Thr Tyr Leu Lys Lys Ala Ser Lys Ser Ser Leu145 150 155 160Phe Ser Asn Lys Ser Lys Arg Asp Ile Asp Glu Asp Ile Asp Glu Asp
165 170 175Thr Asp Thr Asp Gly Asp Ala Ile Pro Asp Val Trp Glu Glu Asn Gly
180 185 190Tyr Thr Ile Lys Gly Arg Val Ala Val Lys Trp Asp Glu Gly Leu Ala
195 200 205Asp Lys Gly Tyr Lys Lys Phe Val Ser Asn Pro Phe Arg Gln His Thr
210 215 220Ala Gly Asp Pro Tyr Ser Asp Tyr Glu Lys Ala Ser Lys Asp Leu Asp225 230 235 240Leu Ser Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro
245 250 255Ser Val Asn Val Ser Leu Glu Asn Val Thr Ile Ser Lys Asp Glu Ash
260 265 270Lys Thr Ala Glu Ile Ala Ser Thr Ser Ser Asn Asn Trp Ser Tyr Thr
275 280 285Asn Thr Glu Gly Ala Ser Ile Glu Ala Gly Ile Gly Pro Glu Gly Leu
290 295 300Leu Ser Phe Gly Val Ser Ala Asn Tyr Gln His Ser Glu Thr Val Ala305 310 315 320Lys Glu Trp Gly Thr Thr Lys Gly Asp Ala Thr Gln Tyr Asn Thr Ala
325 330 335Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr
340 345(2)SEQ ID NO.7的资料:(i)序列特征
(A)长度:2645个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(vi)最初来源:
(C)单独的分离物:PS177C8a(xi)序列描述:SEQ ID NO.7ATGAAGAAGA AGTTAGCAAG TGTTGTAACG TGTACGTTAT TAGCTCCTAT GTTTTTGAAT 60GGAAATGTGA ATGCTGTTTA CGCAGACAGC AAAACAAATC AAATTTCTAC AACACAGAAA 120AATCAACAGA AAGAGATGGA CCGAAAAGGA TTACTTGGGT ATTATTTCAA AGGAAAAGAT 180TTTAGTAATC TTACTATGTT TGCACCGACA CGTGATAGTA CTCTTATTTA TGATCAACAA 240ACAGCAAATA AACTATTAGA TAAAAAACAA CAAGAATATC AGTCTATTCG TTGGATTGGT 300TTGATTCAGA GTAAAGAAAC GGGAGATTTC ACATTTAACT TATCTGAGGA TGAACAGGCA 360ATTATAGAAA TCAATGGGAA AATTATTTCT AATAAAGGGA AAGAAAAGCA AGTTGTCCAT 420TTAGAAAAAG GAAAATTAGT TCCAATCAAA ATAGAGTATC AATCAGATAC AAAATTTAAT 480ATTGACAGTA AAACATTTAA AGAACTTAAA TTATTTAAAA TAGATAGTCA AAACCAACCC 540CAGCAAGTCC AGCAAGATGA ACTGAGAAAT CCTGAATTTA ACAAGAAAGA ATCACAGGAA 600TTCTTAGCGA AACCATCGAA AATAAATCTT TTCACTCAAA AAATGAAAAG GGAAATTGAT 660GAAGACACGG ATACGGATGG GGACTCTATT CCTGACCTTT GGGAAGAAAA TGGGTATACG 720ATTCAAAATA GAATCGCTGT AAAGTGGGAC GATTCTYTAG CAAGTAAAGG GTATACGAAA 780TTTGTTTCAA ATCCGCTAGA AAGTCACACA GTTGGTGATC CTTATACAGA TTATGAAAAG 840GCAGCAAGAG ACCTAGATTT GTCAAATGCA AAGGAAACGT TTAACCCATT GGTAGCTGCT 900TTTCCAAGTG TGAATGTTAG TATGGAAAAG GTGATATTAT CACCAAATGA AAATTTATCC 960AATAGTGTAG AGTCTCATTC ATCCACGAAT TGGTCTTATA CAAATACAGA AGGTGCTTCT 1020GTTGAAGCGG GGATTGGACC AAAAGGTATT TCGTTCGGAG TTAGCGTAAA CTATCAACAC 1080TCTGAAACAG TTGCACAAGA ATGGGGAACA TCTACAGGAA ATACTTCGCA ATTCAATACG 1140GCTTCAGCGG GATATTTAAA TGCAAATGTT CGATATAACA ATGTAGGAAC TGGTGCCATC 1200TACGATGTAA AACCTACAAC AAGTTTTGTA TTAAATAACG ATACTATCGC AACTATTACG 1260GCGAAATCTA ATTCTACAGC CTTAAATATA TCTCCTGGAG AAAGTTACCC GAAAAAAGGA 1320CAAAATGGAA TCGCAATAAC ATCAATGGAT GATTTTAATT CCCATCCGAT TACATTAAAT 1380AAAAAACAAG TAGATAATCT GCTAAATAAT AAACCTATGA TGTTGGAAAC AAACCAAACA 1440GATGGTGTTT ATAAGATAAA AGATACACAT GGAAATATAG TAACTGGCGG AGAATGGAAT 1500GGTGTCATAC AACAAATCAA GGCTAAAACA GCGTCTATTA TTGTGGATGA TGGGGAACGT 1560GTAGCAGAAA AACGTGTAGC GGCAAAAGAT TATGAAAATC CAGAAGATAA AACACCGTCT 1620TTAACTTTAA AAGATGCCCT GAAGCTTTCA TATCCAGATG AAATAAAAGA AATAGAGGGA 1680TTATTATATT ATAAAAACAA ACCGATATAC GAATCGAGCG TTATGACTTA CTTAGATGAA 1740AATACAGCAA AAGAAGTGAC CAAACAATTA AATGATACCA CTGGGAAATT TAAAGATGTA 1800AGTCATTTAT ATGATGTAAA ACTGACTCCA AAAATGAATG TTACAATCAA ATTGTCTATA 1860CTTTATGATA ATGCTGAGTC TAATGATAAC TCAATTGGTA AATGGACAAA CACAAATATT 1920GTTTCAGGTG GAAATAACGG AAAAAAACAA TATTCTTCTA ATAATCCGGA TGCTAATTTG 1980ACATTAAATA CAGATGCTCA AGAAAAATTA AATAAAAATC GTACTATTAT ATAAGTTTAT 2040ATATGAAGTC AGAAAAAAAC ACACAATGTG AGATTACTAT AGATGGGGAG ATTTATCCGA 2100TCACTACAAA AACAGTGAAT GTGAATAAAG ACAATTACAA AAGATTAGAT ATTATAGCTC 2160ATAATATAAA AAGTAATCCA ATTTCTTCAA TTCATATTAA AACGAATGAT GAAATAACTT 2220TATTTTGGGA TGATATTTCT ATAACAGATG TAGCATCAAT AAAACCGGAA AATTTAACAG 2280ATTCAGAAAT TAAACAGATT TATAGTAGGT ATGGTATTAA GTTAGAAGAT GGAATCCTTA 2340TTGATAAAAA AGGTGGGATT CATTATGGTG AATTTATTAA TGAAGCTAGT TTTAATATTG 2400AACCATTGCA AAATTATGTG ACAAAATATA AAGTTACTTA TAGTAGTGAG TTAGGACAAA 2460ACGTGAGTGA CACACTTGAA AGTGATAAAA TTTACAAGGA TGGGACAATT AAATTTGATT 2520TTACAAAATA TAGTRAAAAT GAACAAGGAT TATTTTATGA CAGTGGATTA AATTGGGACT 2580TTAAAATTAA TGCTATTACT TATGATGGTA AAGAGATGAA TGTTTTTCAT AGATATAATA 2640AATAG 2645(2)SEQ ID NO.8的资料:(i)序列特征
(A)长度:881个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:肽(vi)最初来源:
(C)单独的分离物:PS177C8a(xi)序列描述:SEQ ID NO.8Met Lys Lys Lys Leu Ala Ser Val Val Thr Cys Thr Leu Leu Ala Pro1 5 10 15Met Phe Leu Asn Gly Asn Val Asn Ala Val Tyr Ala Asp Ser Lys Thr
20 25 30Asn Gln Ile Ser Thr Thr Gln Lys Asn Gln Gln Lys Glu Met Asp Arg
35 40 45Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Ser Asn Leu
50 55 60Thr Met Phe Ala Pro Thr Arg Asp Ser Thr Leu Ile Tyr Asp Gln Gln65 70 75 80Thr Ala Asn Lys Leu Leu Asp Lys Lys Gln Gln Glu Tyr Gln Ser Ile
85 90 95Arg Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe
100 105 110Asn Leu Ser Glu Asp Glu Gln Ala Ile Ile Glu Ile Asn Gly Lys Ile
115 120 125Ile Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Gly
130 135 140Lys Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Thr Lys Phe Asn145 150 155 160Ile Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys Ile Asp Ser
165 170 175Gln Asn Gln Pro Gln Gln Val Gln Gln Asp Glu Leu Arg Asn Pro Glu
180 185 190Phe Asn Lys Lys Glu Ser Gln Glu Phe Leu Ala Lys Pro Ser Lys Ile
195 200 205Asn Leu Phe Thr Gln Lys Met Lys Arg Glu Ile Asp Glu Asp Thr Asp
210 215 220Thr Asp Gly Asp Ser Ile Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr225 230 235 240Ile Gln Asn Arg Ile Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys
245 250 255Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu Ser His Thr Val Gly
260 265 270Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser
275 280 285Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Val
290 295 300Asn Val Ser Met Glu Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Ser305 310 315 320Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr
325 330 335Glu Gly Ala Ser Val Glu Ala Gly Ile Gly Pro Lys Gly Ile Ser Phe
340 345 350Gly Val Ser Val ASn Tyr Gln His Ser Glu Thr Val Ala Gln Glu Trp
355 360 365Gly Thr Ser Thr Gly Asn Thr Ser Gln Phe Asn Thr Ala Ser Ala Gly
370 375 380Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val Gly Thr Gly Ala Ile385 390 395 400Tyr Asp Val Lys Pro Thr Thr Ser Phe Val Leu Asn Asn Asp Thr Ile
405 410 415Ala Thr Ile Thr Ala Lys Ser Asn Ser Thr Ala Leu Asn Ile Ser Pro
420 425 430Gly Glu Ser Tyr Pro Lys Lys Gly Gln Asn Gly Ile Ala Ile Thr Ser
435 440 445Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu Asn Lys Lys Gln Val
450 455 460Asp Asn Leu Leu Asn Asn Lys Pro Met Met Leu Glu Thr Asn Gln Thr465 470 475 480Asp Gly Val Tyr Lys Ile Lys Asp Thr His Gly Asn Ile Val Thr Gly
485 490 495Gly Glu Trp Asn Gly Val Ile Gln Gln Ile Lys Ala Lys Thr Ala Ser
500 505 510Ile Ile Val Asp Asp Gly Glu Arg Val Ala Glu Lys Arg Val Ala Ala
515 520 525Lys Asp Tyr Glu Asn Pro Glu Asp Lys Thr Pro Ser Leu Thr Leu Lys
530 535 540Asp Ala Leu Lys Leu Ser Tyr Pro Asp Glu Ile Lys Glu Ile Glu Gly545 550 555 560Leu Leu Tyr Tyr Lys Asn Lys Pro Ile Tyr Glu Ser Ser Val Met Thr
565 570 575Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Thr Lys Gln Leu Asn Asp
580 585 590Thr Thr Gly Lys Phe Lys Asp Val Ser His Leu Tyr Asp Val Lys Leu
595 600 605Thr Pro Lys Met Asn Val Thr Ile Lys Leu Ser Ile Leu Tyr Asp Asn
610 615 620Ala Glu Ser Asn Asp Asn Ser Ile Gly Lys Trp Thr Asn Thr Asn Ile625 630 635 640Val Ser Gly Gly Asn Asn Gly Lys Lys Gln Tyr Ser Ser Asn Asn Pro
645 650 655Asp Ala Asn Leu Thr Leu Asn Thr Asp Ala Gln Glu Lys Leu Asn Lys
660 665 670Asn Arg Asp Tyr Tyr Ile Ser Leu Tyr Met Lys Ser Glu Lys Asn Thr
675 680 685Gln Cys Glu Ile Thr Ile Asp Gly Glu Ile Tyr Pro Ile Thr Thr Lys
690 695 700Thr Val Asn Val Asn Lys Asp Asn Tyr Lys Arg Leu Asp Ile Ile Ala705 710 715 720His Asn Ile Lys Ser Asn Pro Ile Ser Ser Ile His Ile Lys Thr Asn
725 730 735Asp Glu Ile Thr Leu Phe Trp Asp Asp Ile Ser Ile Thr Asp Val Ala
740 745 750Ser Ile Lys Pro Glu Asn Leu Thr Asp Ser Glu Ile Lys Gln Ile Tyr
755 760 765Ser Arg Tyr Gly Ile Lys Leu Glu Asp Gly Ile Leu Ile Asp Lys Lys
770 775 780Gly Gly Ile His Tyr Gly Glu Phe Ile Asn Glu Ala ser Phe Asn Ile785 790 795 800Glu Pro Leu Gln Asn Tyr Val Thr Lys Tyr Lys Val Thr Tyr Ser Ser
805 810 815Glu Leu Gly Gln Asn Val Ser Asp Thr Leu Glu Ser Asp Lys Ile Tyr
820 825 830Lys Asp Gly Thr Ile Lys Phe Asp Phe Thr Lys Tyr Ser Xaa Asn Glu
835 840 845Gln Gly Leu Phe Tyr Asp Ser Gly Leu Asn Trp Asp Phe Lys Ile Asn
850 855 860Ala Ile Thr Tyr Asp Gly Lys Glu Met Asn Val Phe His Arg Tyr Asn865 870 875 880Lys(2)SEQ ID NO.9的资料:(i)序列特征
(A)长度:1022个碱基对
(B)类型:核酸 (C)链型:单链(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(vi)最初来源:(C)单独的分离物:177I8(xi)序列描述:SEQ ID NO.9TGGATTAATT GGGTATTATT TCAAAGGAAA AGATTTTAAT AATCTTACTA TGTTTGCACC 60GACACGTGAT AATACCCTTA TGTATGACCA ACAAACAGCG AATGCATTAT TAGATAAAAA 120ACAACAAGAA TATCAGTCCA TTCGTTGGAT TGGTTTGATT CAGAGTAAAG AAACGGGCGA 180TTTCACATTT AACTTATCAA AGGATGAACA GGCAATTATA GAAATCGATG GGAAAATCAT 240TTCTAATAAA GGGAAAGAAA AGCAAGTTGT CCATTTAGAA AAAGAAAAAT TAGTTCCAAT 300CAAAATAGAG TATCAATCAG ATACGAAATT TAATATTGAT AGTAAAACAT TTAAAGAACT 360TAAATTATTT AAAATAGATA GTCAAAACCA ATCTCAACAA GTTCAACTGA GAAACCCTGA 420ATTTAACAAA AAAGAATCAC AGGAATTTTT AGCAAAAGCA TCAAAAACAA ACCTTTTTAA 480GCAAAAAATG AAAAGAGATA TTGATGAAGA TACGGATACA GATGGAGACT CCATTCCTGA 540TCTTTGGGAA GAAAATGGGT ACACGATTCA AAATAAAGTT GCTGTCAAAT GGGATGATTC 600GCTAGCAAGT AAGGGATATA CAAAATTTGT TTCGAATCCA TTAGACAGCC ACACAGTTGG 660CGATCCCTAT ACTGATTATG AAAAGGCCGC AAGGGATTTA GATTTATCAA ATGCAAAGGA 720AACGTTCAAC CCATTGGTAG CTGCTTTYCC AAGTGTGAAT GTTAGTATGG AAAAGGTGAT 780ATTATCACCA AATGAAAATT TATCCAATAG TGTAGAGTCT CATTCATCCA CGAATTGGTC 840TTATACGAAT ACAGAAGGAG CTTCCATTGA AGCTGGTGGC GGTCCATTAG GCCTTTCTTT 900TGGAGTGAGT GTTAATTATC AACACTCTGA AACAGTTGCA CAAGAATGGG GAACATCTAC 960AGGAAATACT TCACAATTCA ATACGGCTTC AGCGGGATAT TTAAATGCCA ATATACGATA 1020TA 1022(2)SEQ ID NO.10的资料:(i)序列特征 (A)长度:340个氨基酸(B)类型:氨基酸(C)链型:单链(D)拓扑结构:线性(ii)分子类型:肽(vi)最初来源:(C)单独的分离物:177I8(xi)序列描述:SEQ ID NO.10Gly Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Thr1 5 10 15Met Phe Ala Pro Thr Arg Asp Asn Thr Leu Met Tyr Asp Gln Gln Thr
20 25 30Ala Asn Ala Leu Leu Asp Lys Lys Gln Gln Glu Tyr Gln Ser Ile Arg
35 40 45Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe Asn
50 55 60Leu Ser Lys Asp Glu Gln Ala Ile Ile Glu Ile Asp Gly Lys Ile Ile65 70 75 80Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Glu Lys
85 90 95Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Thr Lys Phe Asn Ile
100 105 110Asp Ser Lys Thr Phe Lys Glu Leu Lys Leu Phe Lys Ile Asp Ser Gln
115 120 125Asn Gln Ser Gln Gln Val Gln Leu Arg Asn Pro Glu Phe Asn Lys Lys
130 135 140Glu Ser Gln Glu Phe Leu Ala Lys Ala Ser Lys Thr Asn Leu Phe Lys145 150 155 160Gln Lys Met Lys Arg Asp Ile Asp Glu Asp Thr Asp Thr Asp Gly Asp
165 170 175Ser Ile Pro Asp Leu Trp Glu Glu Asn Gly Tyr Thr Ile Gln Asn Lys
180 185 190Val Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys Gly Tyr Thr Lys
195 200 205Phe Val Ser Asn Pro Leu Asp Ser His Thr Val Gly Asp Pro Tyr Thr
210 215 220Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser Asn Ala Lys Glu225 230 235 240Thr Phe Asn Pro Leu Val Ala Ala Xaa Pro Ser Val Asn Val Ser Met
245 250 255Glu Lys Val Ile Leu Ser Pro Asn Glu Asn Leu Ser Asn Ser Val Glu
260 265 270Ser His Ser Ser Thr Asn Trp Ser Tyr Thr Asn Thr Glu Gly Ala Ser
275 280 285Ile Glu Ala Gly Gly Gly Pro Leu Gly Leu Ser Phe Gly Val Ser Val
290 295 300Asn Tyr Gln His Ser Glu Thr Val Ala Gln Glu Trp Gly Thr Ser Thr305 310 315 320G1y Asn Thr Ser Gln Phe Asn Thr Ala Ser Ala Gly Tyr Leu Asn Ala
325 330 335Asn Ile Arg Tyr
340(2)SEQ ID NO.11的资料:(i)序列特征
(A)长度:1341个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(vi)最初来源:
(C)单独的分离物:PS177C8a(xi)序列描述:SEQ ID NO.11ATGTTTATGG TTTCTAAAAA ATTACAAGTA GTTACTAAAA CTGTATTGCT TAGTACAGTT 60TTCTCTATAT CTTTATTAAA TAATGAAGTG ATAAAAGCTG AACAATTAAA TATAAATTCT 120CAAAGTAAAT ATACTAACTT GCAAAATCTA AAAATCACTG ACAAGGTAGA GGATTTTAAA 180GAAGATAAGG AAAAAGCGAA AGAATGGGGG AAAGAAAAAG AAAAAGAGTG GAAACTAACT 240GCTACTGAAA AAGGAAAAAT GAATAATTTT TTAGATAATA AAAATGATAT AAAGACAAAT 300TATAAAGAAA TTACTTTTTC TATGGCAGGC TCATTTGAAG ATGAAATAAA AGATTTAAAA 360GAAATTGATA AGATGTTTGA TAAAACCAAT CTATCAAATT CTATTATCAC CTATAAAAAT 420GTGGAACCGA CAACAATTGG ATTTAATAAA TCTTTAACAG AAGGTAATAC GATTAATTCT 480GATGCAATGG CACAGTTTAA AGAACAATTT TTAGATAGGG ATATTAAGTT TGATAGTTAT 540CTAGATACGC ATTTAACTGC TCAACAAGTT TCCAGTAAAG AAAGAGTTAT TTTGAAGGTT 600ACGGTTCCGA GTGGGAAAGG TTCTACTACT CCAACAAAAG CAGGTGTCAT TTTAAATAAT 660AGTGAATACA AAATGCTCAT TGATAATGGG TATATGGTCC ATGTAGATAA GGTATCAAAA 720GTGGTGAAAA AAGGGGTGGA GTGCTTACAA ATTGAAGGGA CTTTAAAAAA GAGTCTTGAC 780TTTAAAAATG ATATAAATGC TGAAGCGCAT AGCTGGGGTA TGAAGAATTA TGAAGAGTGG 840GCTAAAGATT TAACCGATTC GCAAAGGGAA GCTTTAGATG GGTATGCTAG GCAAGATTAT 900AAAGAAATCA ATAATTATTT AAGAAATCAA GGCGGAAGTG GAAATGAAAA ACTAGATGCT 960CAAATAAAAA ATATTTCTGA TGCTTTAGGG AAGAAACCAA TACCGGAAAA TATTACTGTG 1020TATAGATGGT GTGGCATGCC GGAATTTGGT TATCAAATTA GTGATCCGTT ACCTTCTTTA 1080AAAGATTTTG AAGAACAATT TTTAAATACA ATCAAAGAAG ACAAAGGATA TATGAGTACA 1140AGCTTATCGA GTGAACGTCT TGCAGCTTTT GGATCTAGAA AAATTATATT ACGATTACAA 1200GTTCCGAAAG GAAGTACGGG TGCGTATTTA AGTGCCATTG GTGGATTTGC AAGTGAAAAA 1260GAGATCCTAC TTGATAAAGA TAGTAAATAT CATATTGATA AAGTAACAGA GGTAATTATT 1320AAGGTGTTAA GCGATATGTA G 1341(2)SEQ ID NO.12的资料:(i)序列特征
(A)长度:446个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:肽(vi)最初来源:
(C)单独的分离物:PS177C8a(xi)序列描述:SEQ ID NO.12Met Phe Met Val Ser Lys Lys Leu Gln Val Val Thr Lys Thr Val Leu1 5 10 15Leu Ser Thr Val Phe Ser Ile Ser Leu Leu Asn Asn Glu Val Ile Lys
20 25 30Ala Glu Gln Leu Asn Ile Asn Ser Gln Ser Lys Tyr Thr Asn Leu Gln
35 40 45Asn Leu Lys Ile Thr Asp Lys Val Glu Asp Phe Lys Glu Asp Lys Glu
50 55 60Lys Ala Lys Glu Trp Gly Lys Glu Lys Glu Lys Glu Trp Lys Leu Thr65 70 75 80Ala Thr Glu Lys Gly Lys Met Asn Asn Phe Leu Asp Asn Lys Asn Asp
85 90 95Ile Lys Thr Asn Tyr Lys Glu Ile Thr Phe Ser Met Ala Gly Ser Phe
100 105 110Glu Asp Glu Ile Lys Asp Leu Lys Glu Ile Asp Lys Met Phe Asp Lys
115 120 125Thr Asn Leu Ser Asn Ser Ile Ile Thr Tyr Lys Asn Val Glu Pro Thr
130 135 140Thr Ile Gly Phe Asn Lys Ser Leu Thr Glu Gly Asn Thr Ile Asn Ser145 150 155 160Asp Ala Met Ala Gln Phe Lys Glu Gln Phe Leu Asp Arg Asp Ile Lys
165 170 175Phe Asp Ser Tyr Leu Asp Thr His Leu Thr Ala Gln Gln Val Ser Ser
180 185 190Lys Glu Arg Val Ile Leu Lys Val Thr Val Pro Ser Gly Lys Gly Ser
195 200 205Thr Thr Pro Thr Lys Ala Gly Val Ile Leu Asn Asn Ser Glu Tyr Lys
210 215 220Met Leu Ile Asp Asn Gly Tyr Met Val His Val Asp Lys Val Ser Lys225 230 235 240Val Val Lys Lys Gly Val Glu Cys Leu Gln Ile Glu Gly Thr Leu Lys
245 250 255Lys Ser Leu Asp Phe Lys Asn Asp Ile Asn Ala Glu Ala His Ser Trp
260 265 270Gly Met Lys Asn Tyr Glu Glu Trp Ala Lys Asp Leu Thr Asp Ser Gln
275 280 285Arg Glu Ala Leu Asp Gly Tyr Ala Arg Gln Asp Tyr Lys Glu Ile Asn
290 295 300Asn Tyr Leu Arg Asn Gln Gly Gly Ser Gly Asn Glu Lys Leu Asp Ala305 310 315 320Gln Ile Lys Asn Ile Ser Asp Ala Leu Gly Lys Lys Pro Ile Pro Glu
325 330 335Asn Ile Thr Val Tyr Arg Trp Cys Gly Met Pro Glu Phe Gly Tyr Gln
340 345 350Ile Ser Asp Pro Leu Pro Ser Leu Lys Asp Phe Glu Glu Gln Phe Leu
355 360 365Asn Thr Ile Lys Glu Asp Lys Gly Tyr Met Ser Thr Ser Leu Ser Ser
370 375 380Glu Arg Leu Ala Ala Phe Gly Ser Arg Lys Ile Ile Leu Arg Leu Gln385 390 395 400Val Pro Lys Gly Ser Thr Gly Ala Tyr Leu Ser Ala Ile Gly Gly Phe
405 410 415Ala Ser Glu Lys Glu Ile Leu Leu Asp Lys Asp Ser Lys Tyr His Ile
420 425 430Asp Lys Val Thr Glu Val Ile Ile Lys Val Leu Ser Asp Met
435 440 445(2)SEQ ID NO.13的资料:(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:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.13GCTGATGAAC CATTTAATGC C 21(2)SEQ ID NO.14的资料: (i)序列特征
(A)长度:22个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.14CTCTTTAAAG TAGATACTAA GC 22(2)SEQ ID NO.15的资料:(i)序列特征
(A)长度:24个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.15GATGAGAACT TATCAAATAG TATC 24(2)SEQ ID NO.16的资料:(i)序列特征
(A)长度:33个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.16CGAATTCTTT ATTAGATAAG CAACAACAAA CCT 33(2)SEQ ID NO.17的资料:(i)序列特征
(A)长度:24个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.17GTTATTTCGC AAAAAGGCCA AAAG 24(2)SEQ ID NO.18的资料:(i)序列特征
(A)长度:31个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.18GAATATCAAT CTGATAAAGC GTTAAACCCA G 31(2)SEQ ID NO.19的资料:(i)序列特征
(A)长度:23个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.19GCAGCYTGTT TAGCAATAAA AGT 23(2)SEQ ID NO.20的资料:
(i)序列特征
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO.20CAAAGGAAGA GTAGCTGTTA 20(2)SEQ ID NO.21的资料:
(i)序列特征
(A)长度:25个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO.21CAATGTTAGC TTGGAAAATG TCACC 25(2)SEQ ID NO.22的资料:
(i)序列特征
(A)长度:22个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组)
(xi)序列描述:SEQ ID NO.22GCTTAGTATC TACTTTAAAG AG 22(2)SEQ ID NO.23的资料:(i)序列特征
(A)长度:24个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.23GATACTATTT GATAAGTTCT CATC 24(2)SEQ ID NO.24的资料:(i)序列特征
(A)长度:24个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.24CTTTTGGCCT TTTTGCGAAA TAAC 24(2)SEQ ID NO.25的资料:(i)序列特征
(A)长度:31个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组) (xi)序列描述:SEQ ID NO.25CTGGGTTTAA CGCTTTATCA GATTGATATT C 31(2)SEQ ID NO.26的资料:(i)序列特征
(A)长度:23个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.26ACTTTTATTG CTAAACARGC TGC 23(2)SEQ ID NO.27的资料:(i)序列特征
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.27TAACAGCTAC TCTTCCTTTG 20(2)SEQ ID NO.28的资料:(i)序列特征
(A)长度:25个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性 (ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.28GGTGACATTT TCCAAGCTAA CATTG 25(2)SEQ ID NO.29的资料:(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:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.29CCAGTCCAAT GAACCTCTTA C 21(2)SEQ ID NO.30的资料:(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:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.30AGGGAACAAA CCTTCCCAAC C 21(2)SEQ ID NO.31的资料:(i)序列特征
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.31CARMTAKTAA MTAGGGATAG 20(2)SEQ ID NO.32的资料:(i)序列特征
(A)长度:22个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.32AGYTTCTATC GAAGCTGGGR ST 22(2)SEQ ID NO.33的资料:(i)序列特征
(A)长度:1035个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.33GGGTTAATTG GGTATTATTT TAAAGGGAAA GATTTTAATA ATCTGACTAT GTTTGCACCA 60ACCATAAATA ATACGCTTAT TTATGATCGG CAAACAGCAG ATACACTATT AAATAAGCAG 120CAACAAGAGT TCAATTCTAT TCGATGGATT GGTTTAATAC AAAGTAAAGA AACAGGTGAC 180TTTACATTCC AATTATCAGA TGATAAAAAT GCCATCATTG AAATAGATGG AAAAGTTGTT 240TCTCGTAGAG GAGAAGATAA ACAAACTATC CATTTAGAAA AAGGAAAGAT GGTTCCAATC 300AAAATTGAGT ACCAGTCCAA TGAACCTCTT ACTGTAGATA GTAAAGTATT TAACGATCTT 360AAACTATTTA AAATAGATGG TCATAATCAA TCGCATCAAA TACAGCAAGA TGATTTGAAA 420ATCCTGAATT TAATAAAAAG GAAACGAAAG AGCTTTTATC AAAAACAGCA AAAAGAACCT 480TTTCTCTTCA AAACGGGGTT GAGAAGCGAT GAGGATGATG ATCTAGGATA CAGATGGTGA 540TAGCATTCCT GGATAATTGG GAAATGAATG GATATACCAT TCAAACGAAA AATGGCAGTC 600AAATGGGATG ATTCATTTGC AGAAAAAGGA TATACAAAAT TTGTTTCGAA TCCATATGAA 660GCCCATACAG CAGGAGATCC TTATACCGAT TATGAAAAAG CAGCAAAAGA TATTCCTTTA 720TCGAACGCAA AAGAAGCCTT TAATCCTCTT GTAGCTGCTT TTCCATCTGT CAATGTAGGA 780TTAGAAAAAG TAGTAATTTC TAAAAATGAG GATATGAGTC AGGGTGTATC ATCCAGCACT 840TCGAATAGTG CCTCTAATAC AAATTCAATT GGTGTTACCG TAGATGCTGG TTGGGAAGGT 900TTGTTCCCTA AATTTGGTAT TTCAACTAAT TATCAAAACA CATGGACCAC TGCACAAGAA 960TGGGGCTCTT CTAAAGAAGA TTCTACCCAT ATAAATGGAG CACAATCAGC CTTTTTAAAT 1020GCAAATGTAC GATAT 1035(2)SEQ ID NO.34的资料:(i)序列特征
(A)长度:345个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:蛋白质(xi)序列描述:SEQ ID NO.34Gly Leu Ile Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asn Leu Thr1 5 10 15Met Phe Ala Pro Thr Ile Asn Ash Thr Leu Ile Tyr Asp Arg Gln Thr
20 25 30Ala Asp Thr Leu Leu Asn Lys Gln Gln Gln Glu Phe Asn Ser Ile Arg
35 40 45Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp Phe Thr Phe Gln
50 55 60Leu Ser Asp Asp Lys Asn Ala Ile Ile Glu Ile Asp Gly Lys Val Val65 70 75 80Ser Arg Arg Gly Glu Asp Lys Gln Thr Ile His Leu Glu Lys Gly Lys
85 90 95Met Val Pro Ile Lys Ile Glu Tyr Gln Ser Asn Glu Pro Leu Thr Val
100 105 110Asp Ser Lys Val Phe Asn Asp Leu Lys Leu Phe Lys Ile Asp Gly His
115 120 125Asn Gln Ser His Gln Ile Gln Gln Asp Asp Leu Lys Ile Leu Asn Leu
130 135 140Ile Lys Arg Lys Arg Lys Ser Phe Tyr Gln Lys Gln Gln Lys Glu Pro145 150 155 160Phe Leu Phe Lys Thr Gly Leu Arg Ser Asp Glu Asp Asp Asp Leu Gly
165 170 175Tyr Arg Trp Xaa Xaa His Ser Trp Ile Ile Gly Lys Xaa Met Asp Ile
180 185 190Pro Phe Lys Arg Lys Met Ala Val Lys Trp Asp Asp Ser Phe Ala Glu
195 200 205Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Tyr Glu Ala His Thr Ala
210 215 220Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Lys Asp Ile Pro Leu225 230 235 240Ser Asn Ala Lys Glu Ala Phe Asn Pro Leu Val Ala Ala Phe Pro Ser
245 250 255Val Asn Val Gly Leu Glu Lys Val Val Ile Ser Lys Asn Glu Asp Met
260 265 270Ser Gln Gly Val Ser Ser Ser Thr Ser Asn Ser Ala Ser Asn Thr Asn
275 280 285Ser Ile Gly Val Thr Val Asp Ala Gly Trp Glu Gly Leu Phe Pro Lys
290 295 300Phe Gly Ile Ser Thr Asn Tyr Gln Asn Thr Trp Thr Thr Ala Gln Glu305 310 315 320Trp Gly Ser Ser Lys Glu Asp Ser Thr His Ile Asn Gly Ala Gln Ser
325 330 335Ala Phe Leu Asn Ala Asn Val Arg Tyr
340 345(2)SEQ ID NO.35的资料:(i)序列特征(A)长度:1037个碱基对(B)类型:核酸(C)链型:单链(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.35GGGTTAATTG GGTATTATTT TAAAGGGAAA GATTTTAATA ATCTGACTAT GTTTGCACCA 60ACCATAAATA ATACGCTTAT TTATGATCGG CAAACAGCAG ATACACTATT AAATAAGCAG 120CAACAAGAGT TCAATTCTAT TCGATGGATT GGTTTAATAC AAAGTAAAGA AACAGGTGAC 180TTTACATTCC AATTATCAGA TGATAAAAAT GCCATCATTG AAATAGATGG AAAAGTTGTT 240TCTCGTAGAG GAGAAGATAA ACAAACTATC CATTTAGAAA AAGGAAAGAT GGTTCCAATC 300AAAATTGAGT ACCAGTCCAA TGAACCTCTT ACTGTAGATA GTAAAGTATT TAACGATCTT 360AAACTATTTA AAATAGATGG TCATAATCAA TCGCATCAAA TACAGCAAGA TGATTTGAAA 420AATCCTGAAT TTAATAAAAA AGAAACGAAA GAGCTTTTAT CAAAAACAGC AAAAAGRAAC 480CTTTTCTCTT CAAACGRRGT KGAGAAGCGA TGAGGATGAT RATCYTAGAT ACAGGTGGKG 540ATAGCATTCC YKGATAATTG GGGAAATGAA WGGRTATACC ATTCAACSGA AAAATGGSAG 600TCAAATGGGA TGATTCATTT GCGGAAAAAG GATATACAAA ATTTGTTTCG AATCCATATG 660AAGCCCATAC AGCAGGAGAT CCTTATACCG ATTATGAAAA AGCAGCAAAA GATATTCCTT 720TATCGAACGC AAAAGAAGCC TTTAATCCTC TTGTAGCTGC TTTTCCATCT GTCAATGTAG 780GATTAGAAAA AGTAGTAATT TCTAAAAATG AGGATATGAG TCAGGGTGTA TCATCCAGCA 840CTTCGAATAG TGCCTCTAAT ACAAATTCAA TTGGTGTTAC CGTAGATGCT GGTTGGGAAG 900GTTTGTTCCC TAAATTTGGT ATTTCAACTA ATTATCAAAA CACATGGACC ACTGCACAAG 960AATGGGGCTC TTCTAAAGAA GATTCTACCC ATATAAATGG AGCACAATCA GCCTTTTTAA 1020ATGCAAATGT ACGATAT 1037(2)SEQ ID NO.36的资料:(i)序列特征 (A)长度:1048个碱基对(B)类型:核酸(C)链型:单链(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.36TGGGTTAATT GGGTATTATT TTAAAGGGCA AGAGTTTAAT CATCTTACTT TGTTCGCACC 60AACACGTGAT AATACCCTTA TTTATGATCA ACAAACAGCG AATTCCTTAT TAGATACCAA 120GCAACAAGAA TATCAATCTA TTCGCTGGAT TGGTTTAATT CAAAGTAAAG AAACGGGTGA 180TTTCACATTT AACTTATCAG ATGATCAACA TGCAATTATA GAAATCGATG GCAAAATCAT 240TTCGCATAAA GGACAGAATA AACAAGTTGT TCACTTAGAA AAAGGAAAGT TAGTCCCGAT 300AAAAATTGAG TATCAATCAG ATCAACTATT AAATAGGGAT AGTAACATCT TTAAAGAGTT 360TAAATTATTC AAAGTAGATA GTCAGCAACA CGCTCACCAA GTTCAACTAG ACGAATTAAG 420AAACCCTGCG TTTAATAAAA AGGAAACACA ACAATCTTAA GAAAAAGCAT CCAAAAACAA 480TCTTTTTACA CCAGGGACAT TAAAAGGAAG ATACTGATGA TGATGATAAG GATAACAGGA 540TGGGAGATTC TATTCCTGGA CCTTTTGGGG GAAGAAAATG GGTATACCAA TCCCAAAATA 600AAATAGCTGG TCCAAGTGGG ATGTTCATTC GCCGCGAAAG GGTATACAAA TTTGTTTCTT 660AATCCACTTG ATAGTCATAC AGTTGGAGAT CCCTATACGG ATTATGAAAA AGCAGCAAGA 720GATTTAGACT TGGCCCAATG CAAAAGAAAC ATTTAACCCA TTAGTAGCTG CTTTTCCAAG 780TGTGAATGTG AATTTGGAAA AAGTCATTTT ATCTAAAGAT GAAAATCTAT CCAATAGTGT 840AGAGTCACAT TCCTCCACCA ACTGGTCTTA TACGAATACA GAAGGAGCTT CTATCGAAGC 900TGGGGCTAAA CCAGAGGGTC CTACTTTTGG AGTGAGTGCT ACTTATCAAC ACTCTGAAAC 960AGTTGCAAAA GAATGGGGAA CATCTACAGG AAATACCTCG CAATTTAATA CAGCTTCAGC 1020AGGATATTTA AATGCAAATG TACGATAT 1048(2)SEQ ID NO.37的资料:(i)序列特征
(A)长度:1175个碱基对 (B)类型:核酸(C)链型:单链(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.37ACCTCTAGAT GCANGCTCGA GCGGCCGCCA GTGTGATGGA TATCTGCAGA ATTCGGATTA 60CTTGGGTATT ATTTTAAAGG GAAAGAGTTT AATCATCTTA CTTTGTTCGC ACCAACACGT 120GATAATACCC TTATTTATGA TCAACAAACA GCGAATTCCT TATTAGATAC CAAACAACAA 180GAATATCAAT CTATTCGCTG GATTGGTTTG ATTCAAAGTA AAGAAACAGG TGATTTCACG 240TTTAACTTAT CTGATGATCA AAATGCAATT ATAGAAATAG ATGGCAAAAT CATTTCGCAT 300AAAGGACAGA ATAAACAAGT TGTTCACTTA GAAAAAGGAA AGTTAGTCCC GATAAAAATT 360GAGTATCAAT CAGATCAGAT ATTAACTAGG GATAGTAACA TCTTTAAAGA GTTCAATTAT 420TCAAAGTAGA TAGTCAAGCA ACACTCTCAC CAAAGTTCAA CTTAGGNCNG AATTAAGNAA 480CCCTNGGATT TTAANTTNAA AAAAAGGAAC CCNCANCATT CTTTAGGAAA AAGCAGCAAN 540AACCAAATCC TTTTTTACCA CAGGATATTG AAAAGGAGAT ACGGGNTNGA TGATGGATTG 600ATACCGGGAT ACCAGTTGGG GNTTCTANTC CCTGACCTTT GGGGAAAGAA AATNGGTATA 660CCNATCCCAA AANTTAAGCC AGCTGTCCAG GTGGGATGAT TCAATTCGCC CGCGAAAGGG 720TATACCAAAA TTTGTTTCTT AATCCACTTG AGAGTCATAC AGTTGGAGAT CCCTATACGG 780ATTATGAAAA AGCAGCAAGA GATTTAGACT TGGCCAATGC AAAAGAAACA TTTAACCCAT 840TAGTAGCTGC TTTTCCAAGT GTGAATGTGA ATTTGGAAAA AGTAATATTA TCCCCAGATG 900AGAATTTATC TAACAGTGTA GAATCTCATT CGTCTACAAA TTGGTCTTAT ACGAATACTG 960AAGGAGCTTC TATCGAAGCT GGGGGTGGTC CATTAGGTAT TTCATTTGGA GTGAGTGCTA 1020ATTATCAACA CTCTGAAACA GTTGCAAAAG AATGGGGAAC ATCTACAGGA AATACCTCGC 1080AATTTAATAC AGCTTCAGCA GGATATTTAA ATGCCAATGG TCGATNTAAG CCGAATNCCA 1140NCACACTGNC GGCCGTTAGT AGTGGCACCG AGCCC 1175(2)SEQ ID NO.38的资料:(i)序列特征 (A)长度:1030个碱基对(B)类型:核酸(C)链型:单链(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.38GGRTTAMTTG GGTATTATTT TAAAGGGAAA GATTTTAATG ATCTTACTGT ATTTGCACCA 60ACGCGTGGGA ATACTCTTGT ATATGATCAA CAAACAGCAA ATACATTACT AAATCAAAAA 120CAACAAGACT TTCAGTCTAT TCGTTGGGTT GGTTTAATTC AAAGTAAAGA AGCAGGCGAT 180TTTACATTTA ACTTATCAGA TGATGAACAT ACGATGATAG AAATCGATGG GAAAGTTATT 240TCTAATAAAG GGAAAGAAAA ACAAGTTGTC CATTTAGAAA AAGGACAGTT CGTTTCTATC 300AAAATAGAAT ATCAAGCTGA TGAACCATTT AATGCGGATA GTCAAACCTT TAAAAATTTG 360AAACTCYTTA AAGTAGATAC TAAGCAACAG TCCCAGCAAA TTCAACTAGA TGAATTAAGA 420AACCCTGRAA TTTAATAAAA AAGAAACACA AGAATTTCTA ACAAAAGCAA CAAAAACAAA 480CCTTATTACT CAAAAAGTGA AGAGTACTAG GGATGAAGAC ACGGATACAG ATGGAGATTC 540TATTCCAGAC ATTTGGGAAG AAAATGGGTA TACCATCCAA AATAAGATTG CCGTCAAATG 600GGATGATTCA TTAGCAAGTA AAGGATATAC GAAATTTGTT TCAAACCCAC TAGATACTCA 660CACGGTTGGA GATCCTTATA CAGATTATGA AAAAGCAGCA AGGGATTTAG ATTTGTCAAA 720TGCAAAAGAA ACATTTAACC CATTAGTTGC GGCTTTTCCA AGTGTGAATG TGAGTATGGA 780AAAAGTGATA TTGTCTCCAG ATGAGAACTT ATCAAATAGT ATCGAGTCTC ATTCATCTAC 840GAATTGGTCG TATACGAATA CAGAAGGGGC TTCTATTGAA GCTGGTGGGG GAGCATTAGG 900CCTATCTTTT GGTGTAAGTG CAAACTATCA ACATTCTGAA ACAGTTGGGT ATGAATGGGG 960AACATCTACG GGAAATACTT CGCAATTTAA TACAGCTTCA GCGGGGTATT TAAATGCCAA 1020TRTAMGATAT 1030(2)SEQ ID NO.39的资料:(i)序列特征
(A)长度:23个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.39CACTCAAAAA ATGAAAAGGG AAA 23(2)SEQ ID NO.40的资料:(i)序列特征
(A)长度:19个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.40CCGGTTTTAT TGATGCTAC 19(2)SEQ ID NO.41的资料:(i)序列特征
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.41AGAACAATTT TTAGATAGGG 20(2)SEQ ID NO.42的资料:(i)序列特征
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.42TCCCTAAAGC ATCAGAAATA 20(2)SEQ ID NO.43的资料:(i)序列特征
(A)长度:1170个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.43ATGAAGAAAC AAATAGCAAG CGTTGTAACT TGTACGCTAT TAGCCCCTAT GCTTTTTAAT 60GGAGATATGA ACGCTGCTTA CGCAGCTAGT CAAACAAAAC AAACACCTGC AGCTCAGGTA 120AACCAAGAGA AAGAAGTAGA TCGAAAAGGA TTACTTGGCT ATTACTTTAA AGGGAAAGAT 180TTTAATGATC TTACTGTATT TGCACCAACG CGTGGGAATA CTCTTGTATA TGATCAACAA 240ACAGCAAATA CATTACTAAA TCAAAAACAA CAAGACTTTC AGTCTATTCG TTGGGTTGGT 300TTAATTCAAA GTAAAGAAGC AGGCGATTTT ACATTTAACT TATCAGATGA TGAACATACG 360ATGATAGAAA TCGATGGGAA AGTTATTTCT AATAAAGGGA AAGAAAAACA AGTTGTCCAT 420TTAGAAAAAG GACAGTTCGT TTCTATAAAA TGATTCAGCT GATGAACCAT TTAATGCGGT 480AGTAAACCTT TAAAAATTTG AAACTCTTTA AAGTAGATAC TAAGCAACAG TCCCAGCAAA 540TTCAACTAGA TGAATTAAGA AACCCTGAAT TTAATAAAAA AGAAACACAA GAATTTCTAA 600CAAAAGCAAC AAAAACAAAC CTTATTACTC AAAAAGTGAA GAGTACTAGG GATGAAGACA 660CGGATACAGA TGGAGATTCT ATTCCAGACA TTTGGGAAGA AAATGGGTAT ACCATCCAAA 720ATAAATTGCC GTCAAATGGG ATGATTCATT AGCAAGTAAA GGATATACGA AATTTGTTTC 780AAACCCACTA GATACTCACA CGGTTGGAGA TCCTTATACA GATTATGAAA AAGCAGCAAG 840GGATTTAGAT TTGTCAAATG CAAAAGAAAC ATTTAACCCA TTAGTTGCGG CTTTTCCAAG 900TGTAATTGAG TATGGAAAAA GGATTTGTTC CAGATGAGAA CTTATCAAAT AGTATCGAGT 960TCATTCATTC CTACAATTGG TCGATACGAA TACAGAAGGG GCTTCTATTG AAGCTGGTGG 1020GGGAGCATTA GGCCTATCTT TTGGTGTAAG TGCAAACTAT CAACATTCTG AAACAGTTGG 1080GTATGAATGG GGAACATCTA CGGGAAATAC TTCGCAATTT AATACAGCTT CAGCGGGGTA 1140TTTAAATGCG AATGTTGCTA CAATAACGTG 1170(2)SEQ ID NO.44的资料:(i)序列特征
(A)长度:348个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:蛋白质(xi)序列描述:SEQ ID NO.44Met Lys Lys Gln Ile Ala Ser Val Val Thr Cys Thr Leu Leu Ala Pro1 5 10 15Met Leu Phe Asn Gly Asp Met Asn Ala Ala Tyr Ala Ala Ser Gln Thr
20 25 30Lys Gln Thr Pro Ala Ala Gln Val Asn Gln Glu Lys Glu Val Asp Arg
35 40 45Lys Gly Leu Leu Gly Tyr Tyr Phe Lys Gly Lys Asp Phe Asn Asp Leu
50 55 60Thr Val Phe Ala Pro Thr Arg Gly Asn Thr Leu Val Tyr Asp Gln Gln65 70 75 80Thr Ala Asn Thr Leu Leu Asn Gln Lys Gln Gln Asp Phe Gln Ser Ile
85 90 95Arg Trp Val Gly Leu Ile Gln Ser Lys Glu Ala Gly Asp Phe Thr Phe
100 105 110Asn Leu Ser Asp Asp Glu His Thr Met Ile Glu Ile Asp Gly Lys Val
115 120 125Ile Ser Asn Lys Gly Lys Glu Lys Gln Val Val His Leu Glu Lys Gly
130 135 140Gln Phe Val Ser Xaa Lys Xaa Xaa Xaa Xaa Ala Asp Glu Pro Phe Asn145 150 155 160Ala Xaa Ser Xaa Thr Phe Lys Asn Leu Lys Leu Phe Lys Val Asp Thr
165 170 175Lys Gln Gln Ser Gln Gln Ile Gln Leu Asp Glu Leu Arg Asn Pro Glu
180 185 190Phe Asn Lys Lys Glu Thr Gln Glu Phe Leu Thr Lys Ala Thr Lys Thr
195 200 205Asn Leu Ile Thr Gln Lys Val Lys Ser Thr Arg Asp Glu Asp Thr Asp
210 215 220Thr Asp Gly Asp Ser Ile Pro Asp Ile Trp Glu Glu Asn Gly Tyr Thr225 230 235 240Ile Gln Asn Xaa Ile Ala Val Lys Trp Asp Asp Ser Leu Ala Ser Lys
245 250 255Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Asp Thr His Thr Val Gly
260 265 270Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg Asp Leu Asp Leu Ser
275 280 285Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala Ala Phe Pro Ser Val
290 295 300Asn Xaa Ser Met Glu Lys Xaa Ile Leu Xaa Pro Asp Glu Asn Leu Ser305 310 315 320Asn Ser Ile Glu Xaa His Ser Phe Leu Xaa Ile Gly Arg Ile Arg Ile
325 330 335Gln Lys Gly Leu Leu Leu Lys Leu Val Gly Glu His
340 345(2)SEQ ID NO.45的资料:(i)序列特征
(A)长度:3个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性 (ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.45ATG 3(2)SEQ ID NO.46的资料:(i)序列特征
(A)长度:1个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:蛋白质(xi)序列描述:SEQ ID NO.46Met1(2)SEQ ID NO.47的资料:(i)序列特征
(A)长度:2583个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.47ATGACATATA TGAAAAAAAA GTTAGTTAGT GTTGTAACTT GCACGTTATT GGCTCCGATA 60TTTTTGACTG GAAATGTACA TCCTGTTAAT GCAGACAGTA AAAAAAGTCA GCCTTCTACA 120GCGCAGGAAA AACAAGAAAA GCCGGTTGAT CGAAAAGGGT TACTCGGCTA TTTTTTTAAA 180GGGAAAGAGT TTAATCATCT TACTTTGTTC GCACCAACAC GTGATAATAC CCTTATTTAT 240GATCAACAAA CAGCGAATTC CTTATTAGAT ACCAAACAAC AAGAATATCA ATCTATTCGC 300TGGATTGGTT TGATTCAAAG TAAAGAAACA GGTGATTTCA CGTTTAACTT ATCTGATGAT 360CAAAATGCAA TTATAGAAAT AGATGGCAAA ATCATTTCGC ATAAAGGACA GAATAAACAA 420GTTGTTCACT TAGAAAAAGG AAAGTTAGTC CCGATAAAAA TTGAGTATCA ATCAGATCAG 480ATATTAACTA GGGATAGTAA CATCTTTAAA GAGTTTCAAT TATTCAAAGT AGATAGTCAG 540CAACACTCTC ACCAAGTTCA ACTAGACGAA TTAAGAAACC CTGATTTTAA TAAAAAAGAA 600ACACAACAAT TCTTAGAAAA AGCAGCAAAA ACAAATCTTT TTACACAGAA TATGAAAAGA 660GATACGGATG ATGATGATGA TACGGATACA GATGGAGATT CTATTCCTGA CCTTTGGGAA 720GAAAATGGGT ATACCATCCA AAATAAAGTA GCTGTCAAGT GGGATGATTC ATTCGCCGCG 780AAAGGGTATA CAAAATTTGT TTCTAATCCA CTTGAGAGTC ATACAGTTGG AGATCCCTAT 840ACGGATTATG AAAAAGCAGC AAGAGATTTA GACTTGGCCA ATGCAAAAGA AACATTTAAC 900CCATTAGTAG CTGCTTTTCC AAGTGTGAAT GTGAATTTGG AAAAAGTAAT ATTATCCCCA 960GATGAGAATT TATCTAACAG TGTAGAATCT CATTCGTCTA CAAATTGGTC TTATACGAAT 1020ACTGAAGGAG CTTCTATCGA AGCTGGGGGT GGTCCATTAG GTATTTCATT TGGAGTGAGT 1080GCTAATTATC AACACTCTGA AACAGTTGCA AAAGAATGGG GAACATCTAC AGGAAATACC 1140TCGCAATTTA ATACAGCTTC AGCAGGATAT TTGAATGCGA ATGTTCGATA CAATAATGTG 1200GGAACAGGTG CGATTTATGA GGTGAAACCT ACAACAAGTT TTGTATTAGA TAAAGATACT 1260GTAGCAACAA TTACCGCAAA ATCGAATTCG ACAGCTTTAA GTATATCTCC AGGAGAAAGT 1320TATCCCAAAA AAGGACAAAA TGGAATTGCA ATTAATACAA TGGATGATTT TAATTCCCAT 1380CCGATTACAT TAAATAAACA ACAATTAGAT CAACTATTAA ATAATAAACC TCTTATGTTA 1440GAAACAAATC AGGCAGATGG TGTTTATAAA ATAAAGGATA CAAGCGGTAA TATTGTGACT 1500GGTGGAGAAT GGAACGGTGT TATCCAACAA ATTCAAGCAA AAACAGCCTC TATTATCGTT 1560GATACGGGAG AAAGTGTTTC AGAAAAGCGT GTCGCAGCAA AAGATTATGA TAATCCTGAG 1620GATAAAACAC CTTCTTTATC TTTAAAAGAG GCACTTAAAC TTGGATATCC AGAAGAAATT 1680AAAGAAAAAG ATGGATTGTT GTACTATAAG GACAAGCCAA TTTACGAATC TAGTGTTATG 1740ACTTATCTAG ATGAGAATAC AGCCAAGGAA GTGGAAAAAC AATTACAGGA TACAACCGGA 1800ATATATAAAG ATATCAATCA TTTATATGAT GTGAAATTAA CACCTACAAT GAATTTTACG 1860ATTAAATTAG CTTCCTTATA TGATGGAGCT GAAAATAATG ATGTGAAGAA TGGTCCTATA 1920GGACATTGGT ATTATACCTA TAATACAGGG GGAGGAAATA CTGGAAAACA CCAATATAGG 1980TCTGCTAATC CCAGTGCAAA TGTAGTTTTA TCTTCTGAAG CGAAAAGTAA GTTAGATAAA 2040AATACAAATT ACTACCTTAG TATGTATATG AAAGCTGAGT CTGATACAGA GCCTACAATA 2100GAAGTAAGTG GTGAGAATTC TACGATAACG AGTAAAAAGG TAAAACTAAA CAGTGAGGGC 2160TATCAAAGAG TAGATATTTT AGTGCCGAAT TCTGAAAGAA ATCCAATAAA TCAAATATAT 2220GTAAGAGGAA ATAATACAAC AAATGTATAC TGGGATGATG TTTCAATTAC AAATATTTCA 2280GCTATAAACC CAAAAACTTT AACAGATGAA GAAATTAAAG AAATATATAA AGATTTTAGT 2340GAGTCTAAAG ACTGGCCTTG GTTCAATGAT GTTACGTTTA AAAATATTAA ACCATTAGAG 2400AATTATGTAA AACAATATAG AGTTGATTTC TGGAATACTA ATAGTGATAG ATCATTTAAT 2460AGGATTAAGG ACAGTTACCC AGTTAATGAA GATGGAAGTG TTAAAGTCAA CATGACAGAA 2520TATAATGAAG GATATCCACT TAGAATTGAA TCCGCCTACC ATTTAAATAT TTCAGATCTA 2580TAA 2583(2)SEQ ID NO.48的资料:(i)序列特征
(A)长度:860个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:蛋白质(xi)序列描述:SEQ ID NO.48Met Thr Tyr Met Lys Lys Lys Leu Val Ser Val Val Thr Cys Thr Leul 5 10 15Leu Ala Pro Ile Phe Leu Thr Gly Asn Val His Pro Val Asn Ala Asp
20 25 30Ser Lys Lys Ser Gln Pro Ser Thr Ala Gln Glu Lys Gln Glu Lys Pro
35 40 45val Asp Arg Lys Gly Leu Leu Gly Tyr Phe Phe Lys Gly Lys Glu Phe
50 55 60Asn His Leu Thr Leu Phe Ala Pro Thr Arg Asp Asn Thr Leu Ile Tyr65 70 75 80Asp Gln Gln Thr Ala Asn Ser Leu Leu Asp Thr Lys Gln Gln Glu Tyr
85 90 95Gln Ser Ile Arg Trp Ile Gly Leu Ile Gln Ser Lys Glu Thr Gly Asp
100 105 110Phe Thr Phe Asn Leu Ser Asp Asp Gln Asn Ala Ile Ile Glu Ile Asp
115 120 125Gly Lys Ile Ile Ser His Lys Gly Gln Asn Lys Gln Val Val His Leu
130 135 140Glu Lys Gly Lys Leu Val Pro Ile Lys Ile Glu Tyr Gln Ser Asp Gln145 150 155 160Ile Leu Thr Arg Asp Ser Asn Ile Phe Lys Glu Phe Gln Leu Phe Lys
165 170 175Val Asp Ser Gln Gln His Ser His Gln Val Gln Leu Asp Glu Leu Arg
180 185 190Asn Pro Asp Phe Asn Lys Lys Glu Thr Gln Gln Phe Leu Glu Lys Ala
195 200 205Ala Lys Thr Asn Leu Phe Thr Gln Asn Met Lys Arg Asp Thr Asp Asp
210 215 220Asp Asp Asp Thr Asp Thr Asp Gly Asp Ser Ile Pro Asp Leu Trp Glu225 230 235 240Glu Asn Gly Tyr Thr Ile Gln Asn Lys Val Ala Val Lys Trp Asp Asp
245 250 255Ser Phe Ala Ala Lys Gly Tyr Thr Lys Phe Val Ser Asn Pro Leu Glu
260 265 270Ser His Thr Val Gly Asp Pro Tyr Thr Asp Tyr Glu Lys Ala Ala Arg
275 280 285Asp Leu Asp Leu Ala Asn Ala Lys Glu Thr Phe Asn Pro Leu Val Ala
290 295 300Ala Phe Pro Ser Val Asn Val Asn Leu Glu Lys Val Ile Leu Ser Pro305 310 315 320Asp Glu Asn Leu Ser Asn Ser Val Glu Ser His Ser Ser Thr Asn Trp
325 330 335Ser Tyr Thr Asn Thr Glu Gly Ala Ser Ile Glu Ala Gly Gly Gly ProLeu Gly Ile Ser Phe Gly Val Ser Ala Asn Tyr Gln His Ser Glu Thr
355 360 365Val Ala Lys Glu Trp Gly Thr Ser Thr Gly Asn Thr Ser Gln Phe Asn
370 375 380Thr Ala Ser Ala Gly Tyr Leu Asn Ala Asn Val Arg Tyr Asn Asn Val385 390 395 400Gly Thr Gly Ala Ile Tyr Glu Val Lys Pro Thr Thr Ser Phe Val Leu
405 410 415Asp Lys Asp Thr Val Ala Thr Ile Thr Ala Lys Ser Asn Ser Thr Ala
420 425 430Leu Ser Ile Ser Pro Gly Glu Ser Tyr Pro Lys Lys Gly Gln Asn Gly
435 440 445Ile Ala Ile Asn Thr Met Asp Asp Phe Asn Ser His Pro Ile Thr Leu
450 455 460Asn Lys Gln Gln Leu Asp Gln Leu Leu Asn Asn Lys Pro Leu Met Leu465 470 475 480Glu Thr Asn Gln Ala Asp Gly Val Tyr Lys Ile Lys Asp Thr Ser Gly
485 490 495Asn Ile Val Thr Gly Gly Glu Trp Asn Gly Val Ile Gln Gln Ile Gln
500 505 510Ala Lys Thr Ala Ser Ile Ile Val Asp Thr Gly Glu Ser Val Ser Glu
515 520 525Lys Arg Val Ala Ala Lys Asp Tyr Asp Asn Pro Glu Asp Lys Thr Pro
530 535 540Ser Leu Ser Leu Lys Glu Ala Leu Lys Leu Gly Tyr Pro Glu Glu Ile545 550 555 560Lys Glu Lys Asp Gly Leu Leu Tyr Tyr Lys Asp Lys Pro Ile Tyr Glu
565 570 575Ser Ser Val Met Thr Tyr Leu Asp Glu Asn Thr Ala Lys Glu Val Glu
580 585 590Lys Gln Leu Gln Asp Thr Thr Gly Ile Tyr Lys Asp Ile Asn His Leu
595 600 605Tyr Asp Val Lys Leu Thr Pro Thr Met Asn Phe Thr Ile Lys Leu Ala
610 615 620Ser Leu Tyr Asp Gly Ala Glu Asn Asn Asp Val Lys Asn Gly Pro Ile625 630 635 640Gly His Trp Tyr Tyr Thr Tyr Asn Thr Gly Gly Gly Asn Thr Gly Lys
645 650 655His Gln Tyr Arg Ser Ala Asn Pro Ser Ala Asn Val Val Leu Ser Ser
660 665 670Glu Ala Lys Ser Lys Leu Asp Lys Asn Thr Asn Tyr Tyr Leu Ser Met
675 680 685Tyr Met Lys Ala Glu Ser Asp Thr Glu Pro Thr Ile Glu Val Ser Gly
690 695 700Glu Asn Ser Thr Ile Thr Ser Lys Lys Val Lys Leu Asn Ser Glu Gly705 710 715 720Tyr Gln Arg Val Asp Ile Leu Val Pro Asn Ser Glu Arg Asn Pro Ile
725 730 735Asn Gln Ile Tyr Val Arg Gly Asn Asn Thr Thr Asn Val Tyr Trp Asp
740 745 750Asp Val Ser Ile Thr Asn Ile ser Ala Ile Asn Pro Lys Thr Leu Thr
755 760 765Asp Glu Glu Ile Lys Glu Ile Tyr Lys Asp Phe Ser Glu Ser Lys Asp
770 775 780Trp Pro Trp Phe Asn Asp Val Thr Phe Lys Asn Ile Lys Pro Leu Glu785 790 795 800Asn Tyr Val Lys Gln Tyr Arg Val Asp Phe Trp Asn Thr Asn Ser Asp
805 810 815Arg Ser Phe Asn Arg Ile Lys Asp Ser Tyr Pro Val Asn Glu Asp Gly
820 825 830Ser Val Lys val Asn Met Thr Glu Tyr Asn Glu Gly Tyr Pro Leu Arg
835 840 845Ile Glu Ser Ala Tyr His Leu Asn Ile Ser Asp Leu
850 855 860(2)SEQ ID NO.49的资料:(i)序列特征
(A)长度:1356个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.49ATGGTATCCA AAAAGTTACA ATTAGTCACA AAAACTTTAG TGTTTAGTAC AGTTTTGTCA 60ATACCGTTAT TAAATAATAG TGAGATAAAA GCGGAACAAT TAAATATGAA TTCTCAAATT 120AAATATCCTA ACTTCCAAAA TATAAATATC GCTGATAAGC CAGTAGATTT TAAAGAGGAT 180AAAGAAAAAG CACGAGAATG GGGAAAAGAA AAAGAAAAAG AGTGGAAACT AACTGCTACT 240GAAAAAGGGA AAATTAATGA TTTTTTAGAT GATAAAGATG GATTAAAAAC AAAATACAAA 300GAAATTAATT TTTCTAAGAA TTTTGAATAT GAAACAGAGT TAAAACAGCT TGAAAAAATT 360AATAGCATGC TAGATAAAGC AAATCTAACA AATTCAATTG TCACGTATAA AAACGTTGAG 420CCTACAACAA TAGGATTCAA TCACTCTTTG ACTGATGGGA ATCAAATTAA TTCCGAAGCT 480CAACAGAAGT TCAAGGAACA GTTTTTAGGA AATGATATTA AATTTGATAG TTATTTGGAT 540ATGCACTTAA CTGAACAAAA TGTTTCCGGT AAAGAAAGGG TTATTTTAAA AGTTACAGTA 600CTTAGTGGGA AAGGTTCTAC TCCAACAAAA GCAGGTGTTG TTTTAAATAA TAAAGAATAC 660AAAATGTTGA TTGATAATGG ATATATACTA CATGTAGAAA ACATAACGAA AGTTGTAAAA 720AAAGGACAGG AATGTTTACA AGTTGAAGGA ACGTTAAAAA AGAGCTTGGA CTTTAAAAAT 780GATAGTGACG GTAAGGGAGA TTCCTGGGGA AAGAAAAATT ACAAGGAATG GTCTGATTCT 840TTAACAAATG ATCAGAGAAA AGACTTAAAT GATTATGGTG CGCGAGGTTA TACCGAAATA 900AATAAATATT TACGTGAAGG GGGTACCGGA AATACAGAGT TGGAGGAAAA AATTAAAAAT 960ATTTCTGACG CACTAGAAAA GAATCCTATC CCTGAAAACA TTACTGTTTA TAGATATTGC 1020GGAATGGCGG AATTTGGTTA TCCAATTCAA CCCGAGGCTC CCTCCGTACA AGATTTTGAA 1080GAGAAATTTT TGGATAAAAT TAAGGAAGAA AAAGGATATA TGAGTACGAG CTTATCAAGT 1140GATGCGACTT CTTTTGGCGC AAGAAAAATT ATCTTAAGAT TGCAGATACC AAAAGGAAGT 1200TCAGGAGCAT ATGTAGCTGG TTTAGATGGA TTTAAACCAG CAGAGAAGGA GATTCTTATT 1260GATAAGGGAA GCAAGTATCA TATTGATAAA GTAACAGAAG TAGTTGTGAA AGGTATTAGA 1320AAACTCGTAG TAGATGCGAC ATTATTATTA AAATAA 1356(2)SEQ ID NO.50的资料:(i)序列特征
(A)长度:451个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.50Met Val Ser Lys Lys Leu Gln Leu Val Thr Lys Thr Leu Val Phe Ser1 5 10 15Thr Val Leu Ser Ile Pro Leu Leu Asn Asn Ser Glu Ile Lys Ala Glu
20 25 30Gln Leu Asn Met Asn Ser Gln Ile Lys Tyr Pro Asn Phe Gln Asn Ile
35 40 45Asn Ile Ala Asp Lys Pro Val Asp Phe Lys Glu Asp Lys Glu Lys Ala
50 55 60Arg Glu Trp Gly Lys Glu Lys Glu Lys Glu Trp Lys Leu Thr Ala THr65 70 75 80Glu Lys Gly Lys Ile Asn Asp Phe Leu Asp Asp Lys Asp Gly Leu Lys
85 90 95Thr Lys Tyr Lys Glu Ile Asn Phe Ser Lys Asn Phe Glu Tyr Glu Thr
100 105 110Glu Leu Lys Gln Leu Glu Lys Ile Asn Ser Met Leu Asp Lys Ala Asn
115 120 125Leu Thr Asn Ser Ile Val Thr Tyr Lys Asn Val Glu Pro Thr Thr Ile
130 135 140Gly Phe Asn His Ser Leu Thr Asp Gly Asn Gln Ile Asn Ser Glu Ala145 150 155 160Gln Gln Lys Phe Lys Glu Gln Phe Leu Gly Asn Asp Ile Lys Phe Asp
165 170 175Ser Tyr Leu Asp Met His Leu Thr Glu Gln Asn Val Ser Gly Lys Glu
180 185 190Arg Val Ile Leu Lys Val Thr Val Leu Ser Gly Lys Gly Ser Thr Pro
195 200 205Thr Lys Ala Gly Val Val Leu Asn Asn Lys Glu Tyr Lys Met Leu Ile
210 215 220Asp Asn Gly Tyr Ile Leu His Val Glu Asn Ile Thr Lys val Val Lys225 230 235 240Lys Gly Gln Glu Cys Leu Gln Val Glu Gly Thr Leu Lys Lys Ser Leu
245 250 255Asp Phe Lys Asn Asp Ser Asp Gly Lys Gly Asp Ser Trp Gly Lys Lys
260 265 270Asn Tyr Lys Glu Trp Ser Asp Ser Leu Thr Asn Asp Gln Arg Lys Asp
275 280 285Leu Asn Asp Tyr Gly Ala Arg Gly Tyr Thr Glu Ile Asn Lys Tyr Leu
290 295 300Arg Glu Gly Gly Thr Gly Asn Thr Glu Leu Glu Glu Lys Ile Lys Asn305 310 315 320Ile Ser Asp Ala Leu Glu Lys Asn Pro Ile Pro Glu Asn Ile Thr Val
325 330 335Tyr Arg Tyr Cys Gly Met Ala Glu Phe Gly Tyr Pro Ile Gln Pro Glu
340 345 350Ala Pro Ser Val Gln Asp Phe Glu Glu Lys Phe Leu Asp Lys Ile Lys
355 360 365Glu Glu Lys Gly Tyr Met Ser Thr Ser Leu Ser Ser Asp Ala Thr Ser
370 375 380Phe Gly Ala Arg Lys Ile Ile Leu Arg Leu Gln Ile Pro Lys Gly Ser385 390 395 400Ser Gly Ala Tyr Val Ala Gly Leu Asp Gly Phe Lys Pro Ala Glu Lys
405 410 415Glu Ile Leu Ile Asp Lys Gly Ser Lys Tyr His Ile Asp Lys Val Thr
420 425 430Glu Val Val Val Lys Gly Ile Arg Lys Leu Val Val Asp Ala Thr Leu
435 440 445Leu Leu Lys
450(2)SEQ ID NO.的资料:51(i)序列特征
(A)长度:47个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.51GCTCTAGAAG GAGGTAACTT ATGAACAAGA ATAATACTAA ATTAAGC 47(2)SEQ ID NO.52的资料: (i)序列特征
(A)长度:27个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.52GGGGTACCTT ACTTAATAGA GACATCG 27(2)SEQ ID NO.53的资料:(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:线性(ii)分子类型:DNA(基因组)(xi)序列描述:SEQ ID NO.53ATGAATATGA ATAATACTAA ATTAAACGCA AGGGCCCTAC CGAGTTTTAT TGATTATTTT 60AATGGCATTT ATGGATTTGC CACTGGTATC AAAGACATTA TGAATATGAT TTTTAAAACG 120GATACAGGTG GTAATCTAAC CTTAGACGAA ATCCTAAAGA ATCAGCAGTT ACTAAATGAG 180ATTTCTGGTA AATTGGATGG GGTAAATGGG AGCTTAAATG ATCTTATCGC ACAGGGAAAC 240TTAAATACAG AATTATCTAA GGAAATCTTA AAAATTGCAA ATGAACAGAA TCAAGTCTTA 300AATGATGTTA ATAACAAACT CGATGCGATA AATACGATGC TTCATATATA TCTACCTAAA 360ATCACATCTA TGTTAAGTGA TGTAATGAAG CAAAATTATG CGCTAAGTCT GCAAGTAGAA 420TACTTAAGTA AACAATTGAA AGAAATTTCT GATAAATTAG ATGTTATTAA CGTAAATGTT 480CTTATTAACT CTACACTTAC TGAAATTACA CCTGCATATC AACGGATTAA ATATGTAAAT 540GAAAAATTTG AAGAATTAAC TTTTGCTACA GAAACCACTT TAAAAGTAAA AAAGGATAGC 600TCGCCTGCTG ATATTCTTGA CGAGTTAACT GAATTAACTG AACTAGCGAA AAGTGTTACA 660AAAAATGACG TGGATGGTTT TGAATTTTAC CTTAATACAT TCCACGATGT AATGGTAGGA 720AATAATTTAT TCGGGCGTTC AGCTTTAAAA ACTGCTTCAG AATTAATTGC TAAAGAAAAT 780GTGAAAACAA GTGGCAGTGA AGTAGGAAAT GTTTATAATT TCTTAATTGT ATTAACAGCT 840CTACAAGCAA AAGCTTTTCT TACTTTAACA ACATGCCGAA AATTATTAGG CTTAGCAGAT 900ATTGATTATA CATCTATTAT GAATGAACAT TTAAATAAGG AAAAAGAGGA ATTTAGAGTA 960AACATCCTTC CTACACTTTC TAATACTTTT TCTAATCCTA ATTATGCAAA AGTTAAAGGA 1020AGTGATGAAG ATGCAAAGAT GATTGTGGAA GCTAAACCAG GACATGCATT GGTTGGGTTT 1080GAAATTAGTA ATGATTCAAT GACAGTATTA AAAGTATATG AAGCTAAGCT AAAACAAAAT 1140TACCAAGTTG ATAAGGATTC CTTATCGGAA GTCATTTATA GTGATATGGA TAAATTATTG 1200TGCCCAGATC AATCTGAACA AATTTATTAT ACAAATAATA TAGTATTTCC AAATGAATAT 1260GTAATTACTA AAATTGATTT TACTAAGAAA ATGAAAACTT TAAGATATGA GGTAACAGCT 1320AATTCTTACG ATTCTTCTAC AGGAGAAATT GACTTAAATA AGAAGAAAGT AGAATCAAGT 1380GAAGCGGAGT ATAGGACGTT AAGTGCTAAT AATGATGGAG TATATATGCC GTTAGGTGTC 1440ATCAGTGAAA CATTTTTGAC TCCAATTAAT GGATTTGGCC TCCAAGCTGA TGAAAATTCA 1500AGATTAATTA CTTTAACATG TAAATCATAT TTAAGGGAAC TACTACTAGC GACAGACTTA 1560AGCAATAAAG AAACTAAATT GATTGTCCCG CCTATTAGTT TTATTAGTAA TATTGTAGAA 1620AATGGGAACT TAGAGGGAGA AAACTTAGAG CCGTGGATAG CAAATAACAA AAATGCGTAT 1680GTAGATCATA CAGGTGGTAT AAATGGAACT AAAGTTTTAT ATGTTCATAA GGATGGTGAG 1740TTTTCACAAT TTGTTGGAGG TAAGTTAAAA TCGAAAACAG AATATGTAAT TCAATATATT 1800GTAAAGGGAA AAGCTTCTAT TTATTTAAAA GATAAAAAAA ATGAGAATTC CATTTATGAA 1860GAAATAAATA ATGATTTAGA AGGTTTTCAA ACTGTTACTA AACGTTTTAT TACAGGAACG 1920GATTCTTCAG GGATTCATTT AATTTTTACC AGTCAAAATG GCGAGGGAGC ATTTGGAGGA 1980AACTTTATTA TCTCAGAAAT TAGGACATCC GAAGAGTTAT TAAGTCCAGA ATTGATTATG 2040TCGGATGCTT GGGTTGGATC CCAGGGAACT TGGATCTCAG GAAATTCTCT CACTATTAAT 2100AGTAATGTAA ATGGAACCTT TCGACAAAAT CTTCCGTTAG AAAGTTATTC AACCTATAGT 2160ATGAACTTTA CTGTGAATGG ATTTGGCAAG GTGACAGTAA GAAATTCTCG TGAAGTATTA 2220TTTGAAAAAA GTTATCCGCA GCTTTCACCT AAAGATATTT CTGAAAAATT TACAACTGCA 2280GCCAATAATA CCGGATTATA TGTAGAGCTT TCTCGCTCAA CGTCGGGTGG TGCAATAAAT 2340TTCCGAGATT TTTCAATTAA GTAA 2364(2)SEQ ID NO.54的资料:(i)序列特征
(A)长度:787个氨基酸
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性(ii)分子类型:蛋白质(xi)序列描述:SEQ ID NO.54Met Asn Met Asn Asn Thr Lys Leu Asn Ala 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 Asn Leu Thr Leu
35 40 45Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Glu 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 His Ile Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val
115 120 125Met Lys Gln Asn Tyr Ala Leu Ser Leu Gln Val Glu Tyr Leu Ser Lys
130 135 140Gln Leu Lys Glu Ile Ser Asp Lys Leu Asp Val 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 190Thr Leu Lys Val Lys Lys Asp Ser 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 A a Leu Lys Thr Ala Ser Glu Leu Ile
245 250 255Ala 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 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 Val Gly Phe Glu Ile Ser Asn Asp Ser Met 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 Ser 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 Ser 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 Asn 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 Ile Ser Phe Ile Ser Asn Ile Val Glu Asn Gly Asn Leu
530 535 540Glu Gly Glu Asn Leu Glu Pro Trp Ile Ala Asn Asn Lys Asn Ala Tyr545 550 555 560Val Asp His Thr Gly Gly Ile Asn Gly Thr Lys Val Leu Tyr Val His
565 570 575Lys Asp Gly Glu Phe Ser Gln Phe Val Gly Gly Lys Leu Lys Ser Lys
580 585 590Thr Glu Tyr Val Ile Gln Tyr Ile Val Lys Gly Lys Ala Ser Ile Tyr
595 600 605Leu Lys Asp Lys Lys Asn Glu Asn Ser Ile Tyr Glu Glu Ile Asn Asn
610 615 620Asp Leu Glu Gly Phe Gln Thr Val Thr Lys Arg Phe Ile Thr Gly Thr625 630 635 640Asp Ser Ser Gly Ile His Leu Ile Phe Thr Ser Gln Ash Gly Glu Gly
645 650 655Ala Phe Gly Gly Asn Phe Ile Ile Ser Glu Ile Arg Thr Ser Glu Glu
660 665 670Leu Leu Ser Pro Glu Leu Ile Met Ser Asp Ala Trp Val Gly Ser Gln
675 680 685Gly Thr Trp Ile Ser Gly Asn Ser Leu Thr Ile Asn Ser Asn Val Asn
690 695 700Gly Thr Phe Arg Gln Asn Leu Pro Leu Glu Ser Tyr Ser Thr Tyr Ser705 710 715 720Met Asn Phe Thr Val Asn Gly Phe Gly Lys Val Thr Val Arg Asn Ser
725 730 735Arg Glu Val Leu Phe Glu Lys Ser Tyr Pro Gln Leu Ser Pro Lys Asp
740 745 750Ile Ser Glu Lys Phe Thr Thr Ala Ala Asn Asn Thr Gly Leu Tyr Val
755 760 765Glu Leu Ser Arg Ser Thr Ser Gly Gly Ala Ile Asn Phe Arg Asp Phe
770 775 780Ser Ile Lys785
Claims (48)
1.一种分离的、编码有杀虫活性的蛋白质的多核苷酸,其中选自下组的核苷酸序列可以与编码所述蛋白质或者与编码该蛋白质的核苷酸序列互补的核苷酸序列在严谨条件下杂交:SEQ ID NO.29,SEQID NO.30,SEQ ID NO.31,SEQ ID NO.32,SEQ ID NO.33,SEQ ID NO.35,SEQ ID NO.36,SEQ ID NO.37,SEQ ID NO.38,SEQ ID NO.39,SEQ ID NO.40,SEQ ID NO.41,SEQ ID NO.42,SEQ ID NO.43,SEQ ID NO.45,SEQ IDNO.47,SEQ ID NO.49,SEQ ID NO.51,SEQ ID NO.52,SEQ ID NO.53,和SEQ ID NO.54。
2.一种分离的多核苷酸,其至少编码选自SEQ ID NO.34、SEQ IDNO.44、SEQ ID NO.46、SEQ ID NO.48、SEQ ID NO.50、和SEQ ID NO.54的氨基酸序列的杀虫活性部分。
3.一种分离的多核苷酸,其至少编码选自B.t.分离物PS33F1产生的MIS-1蛋白质、MIS-7蛋白质、MIS-8蛋白质和KB59A4-6产生的SUP蛋白质的蛋白质的杀虫活性部分。
4.一种分离的多核苷酸,其编码由选自下组的分离物产生的杀虫活性蛋白质:PS33F1,PS71G4,PS86D1,PS185V2,PS191A21,PS201Z,PS205A3,PS205C,PS234E1,PS248N10,KB63B19-13,KB63B19-7,KB68B62-7,KB68B63-2,KB69A125-1,KB69A125-3,KB69A125-5,KB69A127-7,KB69A132-1,KB69B2-1,KB70B5-3,KB71A125-15,KB71A35-6,KB71A72-1,KB71A134-2,PS185Y2,和KB59A4-6.
5.权利要求4的多核苷酸,其中所述蛋白质是由选自下组的分离物产生的MIS蛋白质:PS177CSa,PS66D3,PS177I8,PS31F2,PS185Y2,KB68B46-2,KB68B51-2,KB68B55-2,KB53A49-4,PS86D1,HD573B,PS33F1,PS205C,PS157C1,PS201Z,PS71G4,KB71A72-1,KB71A134-2,KB69A125-3,和KB69A127-7.
6.权利要求4的多核苷酸,其中所述蛋白质是由选自下组的分离物产生的WAR蛋白质:PS177C8a,PS66D3,PS177I8,PS31F2,PS185Y2,KB68B46-2,KB68B51-2,KB68B55-2,KB53A49-4,HD573B,PS33F1,PS205C,PS157C1,PS201Z,PS71G4,KB71A72-1,KB71A134-2,KB69A125-3,和KB69A127-7.
7.权利要求4的多核苷酸,其中所述蛋白质是由选自下组的分离物产生的WAR蛋白质:KB68B46-2,PS86D1,HD573B,PS33F1,PS205C,PS157C1,PS201Z,PS71G4,KB71A72-1,KB71A134-2,KB69A125-3,KB69A127-7,PS31F2,和KB68B46-2.
8.权利要求4的多核苷酸,其中所述蛋白质对抗玉米幼芽根叶甲有活性,并且所述蛋白质是由选自下组的分离物产生的:PS205A3,PS185V2,PS234E1,PS71G4,PS248N10,PS191A21,KB63B19-13,KB63B19-7,KB68B62-7,KB68B63-2,KB69A125-1,KB69A125-3,KB69A125-5,KB69A127-7,KB69A132-1,KB69B2-1,KB70B5-3,KB71A125-15,和KB71A35-6.
9.权利要求3的多核苷酸,其中所述蛋白质是B.t.分离物PS157C1一A产生的MIS-7蛋白质。
10.权利要求3的多核苷酸,其中所述蛋白质至少包含SEQ IDNO.34所示氨基酸序列的杀虫部分。
11.权利要求3的多核苷酸,其中所述多核苷酸至少包含SEQ IDNO.33所示核苷酸序列中足以编码杀虫活性蛋白质的一部分。
12.权利要求3的多核苷酸,其中所述蛋白质是B.t.分离物PS201Z产生的MIS-7蛋白质。
13.权利要求3的多核苷酸,其中所述多核苷酸至少包含SEQ IDNO.35所示核苷酸序列中足以编码杀虫活性蛋白质的一部分。
14.权利要求3的多核苷酸,其中所述多核苷酸包含SEQ ID NO.35所示核苷酸序列。
15.权利要求3的多核苷酸,其中所述蛋白质是B.t.分离物PS205C产生的MIS-7蛋白质。
16.权利要求3的多核苷酸,其中所述蛋白质至少包含SEQ IDNO.44所示氨基酸序列的杀虫部分。
17.权利要求3的多核苷酸,其中所述多核苷酸至少包含SEQ IDNO.43所示核苷酸序列中足以编码杀虫活性蛋白质的一部分。
18.权利要求3的多核苷酸,其中所述蛋白质是B.t.分离物PS31F2产生的MIS-8蛋白质。
19.权利要求3的多核苷酸,其中所述蛋白质至少包含SEQ IDNO.48所示氨基酸序列的杀虫部分。
20.权利要求3的多核苷酸,其中所述多核苷酸至少包含SEQ IDNO.47所示核苷酸序列中足以编码杀虫活性蛋白质的一部分。
21.权利要求3的多核苷酸,其中所述蛋白质是B.t.分离物PS185Y2产生的MIS-8蛋白质。
22.权利要求3的多核苷酸,其中所述多核苷酸包含SEQ ID NO.37所示核苷酸序列。
23.权利要求3的多核苷酸,其中所述多核苷酸包含SEQ ID NO.38所示核苷酸序列。
24.权利要求1的多核苷酸,其中所述多核苷酸编码至少包含SEQID NO.46所示氨基酸序列的杀虫部分的蛋白质。
25.权利要求1的多核苷酸,其中所述多核苷酸至少包含SEQ IDNO.45所示核苷酸序列中足以编码杀虫活性蛋白质的一部分。
26.权利要求1的多核苷酸,其中所述多核苷酸编码至少包含SEQID NO.50所示氨基酸序列的杀虫部分的蛋白质。
27.权利要求1的多核苷酸,其中所述多核苷酸至少包含SEQ IDNO.49所示核苷酸序列中足以编码杀虫活性蛋白质的一部分。
28.权利要求1的多核苷酸,其中所述多核苷酸编码至少包含SEQID NO.54所示氨基酸序列的杀虫部分的蛋白质。
29.权利要求1的多核苷酸,其中所述多核苷酸至少包含编码SEQID NO.53所示核苷酸序列中足以编码杀虫活性蛋白质的一部分。
30.包含权利要求1的至少一种多核苷酸的重组宿主。
31.权利要求30的重组宿主,其中所述宿主是植物细胞。
32.权利要求30的重组宿主,其中所述宿主是植物。
33.包含权利要求2的至少一种多核苷酸的重组宿主。
34.包含权利要求3的至少一种多核苷酸的重组宿主。
35.包含权利要求4的至少一种多核苷酸的重组宿主。
36.权利要求1的多核苷酸编码的杀虫活性蛋白质。
37.权利要求2的多核苷酸编码的杀虫活性蛋白质。
38.权利要求3的多核苷酸编码的杀虫活性蛋白质。
39.权利要求4的多核苷酸编码的杀虫活性蛋白质。
40.一种控制非哺乳动物害虫的方法,包括使所述害虫与权利要求1的多核苷酸编码的至少一种杀虫活性蛋白质接触。
41.一种控制非哺乳动物害虫的方法,包括使所述害虫与权利要求2的多核苷酸编码的至少一种杀虫活性蛋白质接触。
42.一种控制非哺乳动物害虫的方法,包括使所述害虫与权利要求3的多核苷酸编码的至少一种杀虫活性蛋白质接触。
43.一种控制非哺乳动物害虫的方法,包括使所述害虫与权利要求4的多核苷酸编码的至少一种杀虫活性蛋白质接触。
44.一种控制玉米根虫(corn rootworm)的方法,其中所述方法包含将所述玉米根虫与权利要求1的多核苷酸编码的至少一种杀虫活性蛋白质接触,所述蛋白质是由选自下组的分离物产生的:PS205A3、PS185V2,PS234E1,PS71G4,PS248N10,PS191A21,KB63B19-13,KB63B19-7,KB68B62-7,KB68B63-2,KB69A125-1,KB69A125-3,KB69A125-5,KB69A127-7,KB69A132-1,KB69B2-1,KB70B5-3,KB71A125-15,和KB71A35-6.
45.权利要求48的方法,其中所述玉米根虫是玉米幼芽根叶甲。
46.控制玉米根虫的方法,其中所述方法包含将所述玉米根虫与权利要求1的多核苷酸编码的至少一种杀虫活性蛋白质接触,其中所述蛋白质是由B.t.分离物PS31F2产生的。
47.产生权利要求1的多核苷酸编码的杀虫活性蛋白质的B.t.分离物的生物学纯培养物,其中所述分离物选自下组:PS33F1,PS71G4,PS86D1,PS185V2,PS191A21,PS201Z,PS205A3,PS205C,PS234E1,PS248N10,KB63B19-13,KB63B19-7,KB68B62-7,KB68B63-2,KB69A125-1,KB69A125-3,KB69A125-5,KB69A127-7,KB69A132-1,KB69B2-1,KB70B5-3,KB71A125-15,KB71A35-6,KB71A72-1.KB71A134-2,PS185Y2,和KB59A4-6.
48.作为探针或引物使用、能够与权利要求1的多核苷酸杂交的诊断多核苷酸,其中所述诊断多核苷酸包含选自下组的核苷酸序列:SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ IDNO.33、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ IDNO.43、SEQ ID NO.45、SEQ ID NO.47、SEQ ID NO.49、SEQ ID NO.51、SEQ ID NO.52、SEQ ID NO.53和SEQ ID NO.54。
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Publication number | Priority date | Publication date | Assignee | Title |
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CN100420752C (zh) * | 2006-03-10 | 2008-09-24 | 浙江大学 | 一种杀虫基因及其用途 |
CN110903361A (zh) * | 2019-12-24 | 2020-03-24 | 隆平生物技术(海南)有限公司 | 一种植物抗虫基因mVip3Aa及其载体和应用 |
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US6242669B1 (en) | 2001-06-05 |
WO1999057282A3 (en) | 2000-06-22 |
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HUP0101991A3 (en) | 2003-03-28 |
EP1075522A2 (en) | 2001-02-14 |
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