CN114616334A - 杀虫基因和使用方法 - Google Patents

杀虫基因和使用方法 Download PDF

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CN114616334A
CN114616334A CN202080034147.6A CN202080034147A CN114616334A CN 114616334 A CN114616334 A CN 114616334A CN 202080034147 A CN202080034147 A CN 202080034147A CN 114616334 A CN114616334 A CN 114616334A
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C.邦宗-奥迪夫林
W.阿乔瓦克
M.巴拉卡特
P.奥泰特
D.瓦莱内特
T.霍伊林
C.萨劳德
M.博西奥
V.盖恩
W.保罗
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Pu Keshi
Sai Mei
Centre National de la Recherche Scientifique CNRS
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Genective SA
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National Interprofessional Association For Seeds And Plants GNIS
Proteus SA
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Genective SA
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Abstract

本发明涉及分子生物学领域，特别是编码可用于防治害虫，特别是植物害虫的杀虫蛋白的新基因。这些蛋白质和编码它们的核酸序列可用于制备杀虫组合物和生产转基因抗害虫植物。

Description

杀虫基因和使用方法
技术领域
本发明涉及分子生物学领域，特别是编码可用于防治害虫，特别是植物害虫的杀虫蛋白的新基因。这些蛋白以及编码它们的核酸序列可用于制备杀虫组合物以及用于生产转基因害虫抗性植物。
背景技术
在世界范围内，作物受到多种威胁，例如害虫、植物病害、杂草。由于害虫和病害造成的损失极大地威胁着全球粮食供应，因此有必要开发解决方案以避免培育物部分或完全破坏。主要解决方案是化学品、生物防治或GMO。
目前的GMO策略使用表达毒素的基因来产生转基因作物。毒素是毒力决定因子，其在微生物致病性中发挥重要作用。这些毒素通常衍生自苏云金芽孢杆菌(Bacillusthuringiensis)，其为一种革兰氏阳性孢子形成土壤细菌。它们称为Cry(结晶蛋白)或VIP(植物杀虫蛋白)。表达杀虫蛋白毒素的转基因作物用于对抗昆虫对作物的损害。
农民广泛采用芽孢杆菌毒素来防治田间昆虫，导致世界许多地方的一些目标害虫对苏云金芽孢杆菌(Bt)毒素产生抗性。解决该问题的一种方法是在转基因植物中堆叠具有不同作用模式的杀虫基因。为了找到具有新作用模式的新毒素，该策略在于发现来自除苏云金芽孢杆菌以外的其他来源的新毒素。这些新毒素可用作衍生自苏云金芽孢杆菌的毒素的替代物，用于部署于昆虫和害虫抗性转基因植物。因此，需要新的毒素蛋白。
发明内容
本发明涉及一种分离的核酸序列，其编码选自由以下组成的组的蛋白质：
a)与SEQ ID NO:1(GDI005)具有至少70％同一性的第1组蛋白质，和
b)与SEQ ID NO:2(GDI006)具有至少65％同一性的第2组蛋白质。
因此，根据本发明，如SEQ ID NO:1所示的GDI0005蛋白和与SEQ ID NO:1具有至少70％同一性的蛋白形成第1组蛋白质。类似地，如SEQ ID NO:2所示的GDI0006蛋白和与SEQID NO:2具有至少65％同一性的蛋白形成第2组蛋白质。
如SEQ ID NO:3、5、28、29、30、31、32、33或34所示的分离的核酸序列编码优选的第1组蛋白质。如SEQ ID NO:4、6、35、36、37、38、39、40或41所示的分离的核酸序列编码优选的第2组蛋白质。
由上述核酸序列编码的分离的多肽也是本发明的目的。如SEQ ID NO:1、11、12、13、14、15、16或17所示的多肽是优选的第1组蛋白质。如SEQ ID NO:2、18、19、20、21、22、23或24所示的多肽是优选的第2组蛋白质。
本发明的一个目的是昆虫害虫(包括西方玉米根虫(Diabrotica virgiferavirgifera)幼虫)的二元毒素。当包含选自与SEQ ID NO:1(GDI0005A)具有至少70％同一性的第1组蛋白质和选自与SEQ ID NO:2(GDI0006A)具有至少65％同一性的第2组蛋白质的蛋白质时，本发明的二元毒素是有效的。本发明的一个目的还是本发明的二元毒素的蛋白质组分，其选自如上所述的第1组或第2组蛋白质。
本发明的另一个目的是包含至少一种上述核酸序列的载体。这种载体因此包含编码本发明的二元毒素的第1组或第2组的蛋白质之一的至少一种核酸序列，或包含各自编码本发明的二元毒素的蛋白质之一的核酸两者，即编码第1组蛋白质和第2组蛋白质两者。
优选的载体包含两种核酸序列，其中：
-第一种如SEQ ID NO:1所示，或编码与SEQ ID NO:1具有至少70％同一性的蛋白质，或如SEQ ID NO:3、5、28、29、30、31、32、33或34所示，以及
-第二种如SEQ ID NO:2所示，或编码与SEQ ID NO:2具有至少65％同一性的蛋白质，或如SEQ ID NO:4、6、35、36、37、38、39、40或41所示。
更优选地，该载体包含两种核酸序列，其中第一种如SEQ ID NO:3、5、28、29、30、31、32、33或34所示，第二种如SEQ ID NO:4、6、35、36、37、38、39、40或41所示。
优选地，包含在本发明的载体中的核酸序列与启动子可操作地连接。
另一方面，本发明涉及一种包含上述本发明载体的宿主细胞。特别地，该宿主细胞包含
至少一种核酸序列，其编码本发明的第1组或第2组的二元毒素的蛋白质中的一种，或包含各自编码本发明的第1组和第2组的二元毒素的蛋白质中的一种的核酸序列两者。
优选地，该宿主细胞是可以包含一种或两种如上所述的核酸序列的植物细胞。
仍然，本发明的另一个目的是一种包含至少一种如上所述的核酸序列的转基因植物。特别地，所述转基因植物表达本发明的二元毒素的第1组或第2组蛋白质中的至少一种，或本发明的二元毒素的两种蛋白质。因此，这种转基因植物是害虫耐受性植物。
有利地，本发明的转基因植物为玉米植物。此外，来自转基因植物，优选玉米植物的转基因种子构成本发明的另一个目的。
本发明的另一个目的是一种生产害虫耐受性转基因植物的方法，其中所述方法包含用如上所述的本发明的载体转化植物细胞，选择包含本发明的核酸序列的转化的植物细胞，以及从所述转化的植物细胞再生表达至少一种如上所述的本发明的多肽的害虫耐受性转基因植物。
在另一方面，本发明涉及一种用于防治田间害虫群体的方法，其包含在所述田间种植如上所述的转基因植物或其种子。
本发明还涉及一种用于鉴定具有改善的害虫耐受性的植物的方法，其包含如下的步骤：在植物群体中鉴定表达本发明的二元毒素的第1组或第2组蛋白质中的至少一种或本发明的二元毒素的两种蛋白质的植物，尤其是包含如上所定义的载体或包含其的宿主细胞的那些。
在另一方面，本发明还涉及一种用于在生物样品中检测如上所定义的本发明的核酸序列或多肽的存在或缺乏的方法，其包含以下步骤：获得生物样品，从该生物样品中提取DNA或RNA或蛋白质；以及检测如上所定义的本发明的核酸序列或多肽的存在或缺乏。
本发明的生物样品可以定义为细胞裂解物。细胞可以是属于古细菌(Archaea)或细菌的原核生物、真核生物诸如真菌或植物细胞。根据需要，可以使用澄清的裂解物或粗裂解物。
用于在这种方法中进行上述检测步骤的工具(means)也是本发明的一个目的，其中所述工具选自由以下组成的组：
-至少两种用于扩增本发明的核酸序列的引物，
-至少一种与本发明的核酸序列杂交的标志物，以及
-至少一种识别本发明的多肽的抗体。
本发明的一个目的是如上定义的本发明的分离的核酸序列用于筛选具有杀虫活性的同源物序列的用途。
本发明的另一个目的是一种针对昆虫害虫的杀虫组合物，其包含有效量的如本文所述的第1组或第2组的至少一种蛋白质，或包含来自如本文所述的第1组和第2组两者的蛋白质。
本发明的一个目的是一种生产所述杀虫组合物的方法，其包含培养细胞，该细胞包含编码第1组或第2组蛋白质，或优选地编码第1组和第2组蛋白质两者的至少一种核酸序列，以及回收第1组或第2组的至少一种蛋白质，或第1组和第2组蛋白质两者。该细胞可以是野生型细胞或宿主细胞。宿主细胞可以是原核生物、真核生物诸如真菌或植物细胞。
本发明的另一个目的是一种处理植物免于害虫侵染或处理受害虫侵染的植物的方法，其包含向该植物或其部分施用本发明的二元毒素的第1组或第2组中的至少一种蛋白质，或来自本发明的二元毒素的第1组和第2组的每种的蛋白质两者。
附图说明
图1：包括GDI0005和GDI0006基因和相应序列的推定操纵子。
图2：GDI0005和7个同源物序列之间的同一性百分比。
图3：GDI0006和7个同源物序列之间的同一性百分比。
图4：用于植物转化的GDI0005盒。
图5：用于植物转化的GDI0006盒。
图6：在96孔板(每次处理n＝48孔，2018)的人工饮食中，在包含GDI0005和/或GDI0006的细菌裂解物处理中或阴性对照(缓冲液中的空菌株)中，在处理后3天和5天，新生西方玉米根虫幼虫的发育迟缓率(stunting)(A)和死亡率(B)。
图7：在96孔板(每次处理n＝48孔，2018)的人工饮食中，在GDI0005A同源物+GDI0006A同源物细菌裂解物处理中或在阴性对照(缓冲液中的空菌株)中，新生西方玉米根虫幼虫在处理后5天的死亡率(A)和在处理后3天的发育迟缓率(亚致死效应)(B)。
图8：在包含GDI0005和GDI0006蛋白的细菌裂解物处理中或在阴性对照(缓冲液中的空裂解物，Tris缓冲液和UTC)或阳性对照(Cry34/35裂解物)，无稀释后，处理后5天，易感WCRW幼虫(A)、Cry3Bb1抗性WCRW幼虫(B)和Cry34/35抗性WCRW幼虫(C)的幼虫重量(mg)。
图9：蛋白质印迹显示转化烟草叶的总蛋白提取物。用GDI0005A(SEQ ID NO:1)或其同源物GDI0175A(SEQ ID NO:11)、GDI0177A(SEQ ID NO:12)、GDI0179A(SEQ ID NO:13)、GDI0181A(SEQ ID NO:14)、GDI0183A(SEQ ID NO:15)、GDI0185A(SEQ ID NO:16)、GDI0187A(SEQ ID NO:17)中的一种转化烟草植物。
图10：蛋白质印迹显示转化烟草叶的总蛋白提取物。用GDI0006A(SEQ ID NO:2)或其同源物GDI0176A(SEQ ID NO:18)、GDI0178A(SEQ ID NO:19)、GDI0180A(SEQ ID NO:20)、GDI0182A(SEQ ID NO:21)、GDI0184A(SEQ ID NO:22)、GDI0186A(SEQ ID NO:23)中的一种转化烟草植物。
图11：在96孔板的人工饮食中，用包含GDI0005A(SEQ ID NO:1)和GDI0006A(SEQID NO:2)或GDI0005A(SEQ ID NO:1)和GDI0176A(SEQ ID NO:18)或GDI0005A(SEQ ID NO:1)和GDI0178A(SEQ ID NO:19)或GDI0005A(SEQ ID NO:1)和GDI0184A(SEQ ID NO:22)或GDI0005A(SEQ ID NO:1)和GDI0186A(SEQ ID NO:23)或GDI0005A(SEQ ID NO:1)和GDI0188A(SEQ ID NO:24)的细菌裂解物或阴性对照(缓冲液中的空菌株)或阳性对照(Cry34/Cry35)处理后3天，新生西方玉米根虫幼虫的发育迟缓率。
具体实施方式
蛋白质
根据本发明，本发明的蛋白质或多肽或毒素是杀虫的。杀虫是指该蛋白质/多肽/毒素能够诱导昆虫害虫的发育迟缓(亚致死效应)和/或杀死(致死效应)。
第1组蛋白质和第2组蛋白质具有杀虫活性。
启动子
根据本发明，启动子可以选自能够驱动基因在细菌、真菌、酵母或植物细胞中表达的启动子。
启动子可以是天然启动子或异源启动子。异源启动子为不是位于下游的基因的天然启动子的启动子。
在一个优选的实施方案中，能够驱动基因在细菌细胞中表达的启动子能够驱动在大肠杆菌(E.coli)中表达。在另一个优选的实施方案中，这种启动子能够驱动基因在酵母细胞中的表达，例如在解脂耶氏酵母(Yarrowia lipolytica)中的表达。
在另一个优选的实施方案中，能够驱动与其可操作连接的基因在植物细胞中表达的“在植物中具有功能性”的启动子能够驱动基因在玉米细胞中表达。
为了得到表达，编码如上定义的要过表达的蛋白质，优选如SEQ ID NO:1或SEQ IDNO:2所示的蛋白质的序列可以在组成型、组织特异性、发育调控、诱导型或减数分裂启动子的控制下存在。此类启动子通常是本领域熟知的。可以使用其它合适的启动子。这可以是组织特异性启动子，诸如叶特异性启动子、种子特异性启动子、BETL特异性启动子等。在文献中描述了许多组织特异性启动子，并且可以使用它们中的任何一种，例如US 20130024998中公开的启动子。
其它有用的启动子是在种子发育期间受调控的启动子，诸如小麦的HMWG启动子(高分子量麦谷蛋白)(Anderson&Greene,1989；Robert et al.,1989)，玉米的蜡质(waxy)、玉米醇溶蛋白或bronze启动子，或US 20150007360、US 20120011621、US 20100306876、US20090307795或US 20070028327中公开的启动子。
尽管一些启动子在不同物种中可具有相同的调控模式，但在植物中通常优选在单子叶植物中使用单子叶启动子，而在双子叶植物中使用双子叶启动子。
在一个优选的实施方案中，启动子是pCsVMV+OsActin+内含子，如SEQ ID NO:7所示。
同源物序列的定义
本发明的同源物序列可以从公共或私人保藏物中分离得到，也可以基于本发明中呈现的肽序列的全部或部分，或者使用其编码核苷酸序列的全部或部分，通过各种常规方法制备，诸如随机诱变、定点诱变、基因合成或基因改组。此类同源物包含例如蛋白质的氨基酸序列中一个或多个残基的缺失、插入或取代，或其组合。本发明还涉及本文公开的序列GDI0005A和GDI0006A的任何同源物，条件是这些同源物单独(GDI0005型或GDI0006型)或以二元形式(GDI0005型与GDI0006型)保留杀虫活性。
根据本发明，GDI0005A同源物是与SEQ ID NO:1具有至少70％序列同一性，优选至少73％序列同一性，优选至少75％序列同一性，优选至少80％序列同一性，优选至少85％序列同一性，优选至少90％序列同一性，优选至少90％序列同一性，优选至少95％序列同一性，优选至少98％序列同一性，优选至少99％序列同一性，优选至少99.2％序列同一性，优选至少99.5％序列同一性，优选至少99.8％序列同一性，优选至少99.9％序列同一性的蛋白质。
在一个优选的实施方案中，GDI0005A同源物选自序列SEQ ID NO:11至SEQ ID NO:17。
根据本发明，GDI0006A同源物是与SEQ ID NO:2具有至少65％序列同一性，优选至少68％序列同一性，优选至少70％序列同一性，优选至少75％序列同一性，优选至少80％序列同一性，优选至少85％序列同一性，优选至少90％序列同一性，优选至少95％序列同一性，优选至少98％序列同一性，优选至少99％序列同一性，优选至少99.2％序列同一性，优选至少99.5％序列同一性，优选至少99.8％序列同一性，优选至少99.9％序列同一性的蛋白质。
在一个优选的实施方案中，GDI0006A同源物选自序列SEQ ID NO:18至SEQ ID NO:24。
同源物序列表现出序列的50％或更多的序列对查询覆盖(GDI0005A或GDI0006A)长度和此比对区域中的同一性>30％。该同一性百分比优选通过使用BLASTP算法获得。
“序列同一性百分比”可通过在比较窗上比较两个最佳比对的序列来确定，其中与参考序列(其不包含添加或缺失)相比，比较窗中的多核苷酸或多肽序列的部分可包含添加或缺失(即，缺口)以用于两个序列的最佳比对。通过确定在两个序列中出现相同的核酸碱基或氨基酸残基的位置的数目以得到匹配的位置的数目，将匹配的位置的数目除以比较窗中的位置的总数并将结果乘以100以得到序列同一性的百分比来计算百分比。优选地，如本发明上下文中定义的序列同一性百分比是通过在其全长上比较的序列的全局比对确定的。
通过应用BLASTP程序(Altschul et al.(1997),Nucleic Acids Res.25:3389-3402；Altschul et al.(2005)FEBS J.272:5101-5109)至SEQ ID NO:1或SEQ ID NO:2，使用默认算法参数，可以鉴定同源物蛋白。
二元毒素
杀虫二元毒素由两种组分构成，一种来自第1组蛋白质，另一种来自第2组蛋白质，并且当将这两种组分组合施用至昆虫害虫时诱导杀灭或发育迟缓。当单独施用时，毒素蛋白中的一些可以具有致死或亚致死特性。然而，当两种组分蛋白一起施用时，杀虫二元毒素蛋白的毒性要大得多。
GDI0005A蛋白及其同源物，例如SEQ ID NO:11所示的GDI0175A、SEQ ID NO:12所示的GDI0177A、SEQ ID NO:13所示的GDI0179A、SEQ ID NO:14所示的GDI0181A、SEQ ID NO:15所示的GDI0183A、SEQ ID NO:16所示的GDI0185A和SEQ ID NO:17所示的GDI0187A属于第1组蛋白质。
GDI0006蛋白及其同源物，例如SEQ ID NO:18所示的GDI0176A、SEQ ID NO:19所示的GDI0178A、SEQ ID NO:20所示的GDI0180A、SEQ ID NO:21所示的GDI0182A、SEQ ID NO:22所示的GDI0184A、SEQ ID NO:23所示的GDI0186A和SEQ ID NO:24所示的GDI0188A属于第2组蛋白质。
在本发明的一个实施方案中，杀虫二元毒素可以用选自第1组的任何蛋白质与选自第2组的任何蛋白质组合形成，以最大化对昆虫幼虫的致死或亚致死效应。
在具体的实施方案中，GDI0005A和GDI0006A形成第一二元毒素，GDI0175A和GDI0176A形成第二二元毒素，GDI0177A和GDI0178A形成第三二元毒素，GDI0179A和GDI0180A形成第四二元毒素，GDI0181A和GDI0182A形成第五二元毒素，GDI0183A和GDI0184A形成第六二元毒素，GDI0185A和GDI0186A形成第七二元毒素，GDI0187A和GDI0188A形成第八二元毒素。
在二元毒素的其它具体实施方案中，GDI0005A可以与任何GDI0006A同源物组合，诸如与SEQ ID NO:18所示的GDI0176A，SEQ ID NO:19所示的GDI0178A，SEQ ID NO:20所示的GDI0180A，SEQ ID NO:21所示的GDI0182A，SEQ ID NO:22所示的GDI0184A，SEQ ID NO:23所示的GDI0186A或SEQ ID NO:24所示的GDI0188A组合。类似地，GDI0006A可与任何GDI0005A同源物组合，诸如与SEQ ID NO:11所示的GDI0175A，SEQ ID NO:12所示的GDI0177A，SEQ ID NO:13所示的GDI0179A，SEQ ID NO:14所示的GDI0181A，SEQ ID NO:15所示的GDI0183A，SEQ ID NO:16所示的GDI0185A或SEQ ID NO:17所示的GDI0187A组合。
盒与载体
在一个实施方案中，编码蛋白GDI0005A或其同源物中的一种和GDI0006A或其同源物中的一种的核酸序列在盒中的单个启动子的控制下。将克隆在单个载体中的单个盒插入宿主细胞，诸如选自微藻、植物、细菌、古细菌、酵母或真菌的细胞，如下文举例说明的。转化这些细胞类型所需的载体是本领域熟知的。转化的细胞能够表达GDI0005A(或其同源物中的一种)和GDI0006A(或其同源物)的融合体。
在一个具体的实施方案中，两种蛋白可以表达为一种蛋白，在二元毒素的两种组分之间具有蛋白接头。
该接头可为长度为4-21个氨基酸残基的序列。该接头可为天然序列或设计的序列。接头可包含蛋白水解切割位点以分离两种融合蛋白。接头可以是长且柔性的，以保持两种蛋白融合并允许两种蛋白的正确折叠。接头示例于Chen X,Zaro JL,Shen WC.Fusionprotein linkers:property,design and functionality.Adv Drug Deliv Rev.2012；65(10):1357-69。
在第二个实施方案中，这两种蛋白由克隆在同一载体中的两个不同的盒编码。用所述载体转化宿主细胞。该宿主细胞能够表达这两种蛋白。
在第三个实施方案中，这两种蛋白由克隆在两个单独的载体中的两个不同的盒编码。
在特定实施方案中，用这两种载体转化宿主细胞。该宿主细胞能够表达这两种蛋白。
根据本发明，载体还可以携带选择标志物。术语“选择标志物”意图指一种如下的基因，其表达赋予含有该基因的宿主细胞以使得可以选择如此转化的所述宿主细胞(阳性选择)的特征。例如，其为赋予抗生素抗性或热稳定性抗生素抗性的基因，或编码能够互补营养缺陷型(天然的，诸如克服必需氨基酸的缺乏，或工程化的，诸如URA3缺陷型突变体，其中URA3是尿嘧啶生物合成所必需)的酶的基因。选择标志物基因包括赋予抗生素抗性的基因，诸如卡那霉素(nptII基因)、潮霉素(aph IV)、壮观霉素(aadA)和庆大霉素(aac3和aacC4)，或赋予除草剂抗性的基因，诸如草铵膦(glufosinate)(bar或pat)、麦草畏(dicamba)(DMO)和草甘膦(glyphosate)(aroA或EPSPS)。也可以使用允许直接视觉鉴定转化体的选择标志物，例如，表达有色或荧光蛋白诸如荧萤光素酶或绿色荧光蛋白(GFP)的基因，或表达β-葡糖醛酸糖苷酶或uidA基因(GUS)的基因，对于这些基因的各种显色底物是已知的。它们可具有宿主特异性。选择标志物是本领域熟知的。
在另一个实施方案中，用至少一种编码二元毒素的第一蛋白的载体转化宿主细胞，并用至少一种编码二元毒素的第二蛋白的另一种载体转化另一种宿主细胞。当宿主细胞是植物细胞时，然后将由其衍生的两种生长的植物杂交。在后代中选择表达这两种蛋白的植物。当宿主细胞是微生物细胞时，单独培养表达二元毒素的蛋白质中的一种的每种宿主细胞，以表达二元毒素的蛋白质组分中的一种。在另一个实施方案中，当宿主细胞是微生物细胞时，二元毒素的两种蛋白在同一宿主细胞中表达。
在一个优选的实施方案中，这种载体包含启动子pCsVMV+OsActin+内含子，如SEQID NO:7所示。
本领域技术人员可以通过熟知的常规手段将至少一个拷贝的上述核酸或至少一个拷贝的载体引入宿主细胞。例如，可以采用氯化钙处理、电穿孔、使用基因枪、病毒介导的转化、通过显微注射的转化、土壤杆菌(Agrobacterium)介导的植物转化(Ishida et al.,Nat.Biotechnol.,14:745-750,1996)。
根据一个实施方案，本领域技术人员可以通过常规方法将几个拷贝的上述核酸或载体引入宿主细胞。
转化步骤之后可以是选择步骤和再生步骤。专用于每种物种的选择和再生方法是本领域技术人员熟知的。
信号肽/靶向
根据本发明，通过将信号肽核酸序列与编码GDI0005和GDI0006的核酸序列融合，可以将二元毒素的两种组分靶向于特定的细胞区室中。
信号肽可以将本发明的蛋白靶向不同的细胞器，例如细胞核、液泡、ER、线粒体、叶绿体。在革兰氏阴性菌中，它们将本发明的蛋白质靶向周质。在革兰氏阳性菌中，它们将本发明的蛋白质靶向分泌到细胞外(培养基中)。
本发明的信号肽可以为叶绿体靶向信号，诸如玉米RUBISCO活化酶(RCA)(SEQ IDNO:42)，玉米酮醇酸还原异构酶KARI2(SEQ ID NO:43)，玉米乙酰乳酸合酶1(AHAS)(SEQ IDNO:44)，US 8426204 B2中描述的莱茵衣藻(C.reinhardtii)5-烯醇丙酮基莽草酸-3-磷酸合酶(EPSPS)，Sallaud et al.(2009)中描述的番茄Z,Z-法呢基焦磷酸合酶，番茄檀香烯和香柠檬烯(bergamotene)合酶(Sallaud et al.(2009))，稻RUBISCO SSU(Jang et al.(2002))，拟南芥RUBISCO SSU(Wong et al.1992)。
其它叶绿体靶向信号已在文献(例如Von Heijne et al.(1991))中确定，并且可用于靶向本发明的蛋白质。
本发明的信号肽可以为线粒体靶向信号，诸如Huang et al.(2009)中描述的OsPPR_02g02020和Os01g49190，或质外体分泌信号，诸如大麦α-淀粉酶信号序列(Rogers1985)和烟草发病相关蛋白，PR1a(Pen et al.1992)。
本发明的信号肽可以在具有ER保留信号(诸如C末端KDEL基序)的情况下将蛋白质靶向到内质网(ER)中，在具有大麦aleuraine信号肽(Holwerda et al.(1992))或烟草壳多糖酶A信号肽(Neuhaus et al.(1994))的情况下将蛋白质靶向到液泡中。
本发明的其它信号肽为过氧化物酶体靶向性C末端PTS1信号、SKL、SRL和变体(Hayashi et al.(1996),Kragler et al.(1998))以及核靶向信号(NLS)诸如猿猴病毒SV40(Kalderon at al.(1984))。Raikhel N.(1992)还描述了植物NLS序列。
利用例如Torrent et al.(2009)描述的玉米γ玉米醇溶蛋白信号肽，可以将本发明的蛋白质靶向到蛋白质体中，以改善细胞中的蛋白积累。
此外，玉米细胞基因组学数据库(http://maize.jcvi.org/cellgenomics/index.php)中描述了定位于不同植物区室并因此具有靶向信号的玉米蛋白质。植物区室包括：细胞壁，叶绿体，造粉体，高尔基体，线粒体，细胞核，过氧化物酶体，质膜，胞间连丝，蛋白质体，糙面内质网，光滑内质网，液泡膜，液泡，囊泡，核周间隙。
在第一个实施方案中，本发明的第1组和第2组蛋白质均靶向于相同的细胞区室中。
在第二个实施方案中，本发明的第1组和第2组蛋白质靶向于不同的细胞区室中。
宿主细胞
宿主细胞可以是属于古细菌或细菌的原核生物、真核生物诸如真菌或植物细胞。
根据本发明，如上所述的宿主细胞可为微生物细胞，诸如木霉属(Trichoderma)，曲霉属(Aspergillus)，脉孢菌属(Neurospora)，腐质霉属(Humicola)，青霉属(Penicillium)，镰刀菌属(Fusarium)，热单孢菌属(Thermomonospora)，芽孢杆菌属(Bacillus)，假单胞菌属(Pseudomonas)，埃希氏菌属(Escherichia)，梭菌属(Clostridium)，纤维单胞菌属(Cellulomonas)，链霉菌属(Streptomyces)，耶氏酵母属(Yarrowia)，毕赤酵母属(Pichia)或酵母属(Saccharomyces)。
例如，根据一个实施方案，如上所述的微生物宿主细胞可选自里氏木霉(Trichoderma reesei)，绿色木霉(Trichoderma viridae)，康宁木霉(Trichodermakoningii)，黑曲霉(Aspergillus niger)，构巢曲霉(Aspergillus nidulans)，文氏曲霉(Aspergillus wentii)，米曲霉(Aspergillus oryzae)，海要曲霉(Aspergillusphoenicis)，粗糙脉孢菌(Neurospora crassa)，灰腐质霉(Humicola grisae)，嗜松青霉(Penicillium pinophilum)，草酸青霉菌(Penicillium oxalicum)，大肠杆菌(Escherichia coli)，丙酮丁醇梭菌(Clostridium acetobutylicum)，解糖梭菌(Clostridium saccharolyticum)，Clostridium bejerinckii，丁酸梭菌(Clostridiumbutylicum)，毕赤酵母(Pichia pastoris)，荧光假单胞菌(Pseudomonas fluorescens)和解脂耶氏酵母(Yarrowia lipolytica)。
根据本发明，如上所述的宿主细胞可为诸如属于蓝细菌物种的微藻细胞。
例如，根据一个实施方案，如上所述的微藻宿主细胞可为聚球藻属种(Synechococcus sp.)或集胞藻属种(Synechocystis sp.)。
根据本发明，如上所述的宿主细胞是选自双子叶植物和单子叶植物的植物细胞。
例如，根据一个实施方案，如上所述的植物宿主细胞选自双子叶植物诸如烟草、棉、大豆、向日葵、油菜籽和单子叶植物诸如小麦、玉米、稻、大麦、高粱，并且优选玉米。
本发明涉及包含如上所述的本发明的载体的宿主细胞。特别是，宿主细胞包含至少一种编码本发明的第1组或第2组蛋白质的二元毒素的蛋白质之一的核酸序列，或包含各自编码本发明的二元毒素的第1组和第2组蛋白质之一的核酸序列两者。
优选地，宿主细胞为可以包含一种或两种如上所述的核酸序列的植物细胞。
转基因植物
转基因植物可选自双子叶植物和单子叶植物。
根据一个实施方案，转基因植物选自双子叶植物诸如烟草、棉、大豆、向日葵、油菜籽和单子叶植物诸如小麦、玉米、稻、大麦、高粱，并且优选玉米。
防治害虫群体/耐受性
根据本发明，防治害虫群体意味着限制害虫的发育、停止其发育或杀死害虫。这包括诱导害虫(成虫或幼虫)的生理或行为变化，诸如生长发育迟缓，死亡率增加，繁殖能力降低，摄食行为或运动的减少或停止，或变态阶段发育的减少或停止。
害虫生存力的降低诱导植物对害虫侵染的耐受性增强。
在一个优选的实施方案中，本发明实现了昆虫耐受性，更优选鞘翅目耐受性，更优选对西方玉米根虫(WCRW)Diabrotica virgifera virgifera的耐受性。
在一个实施方案中，本发明涉及一种用于防治田间害虫群体的方法，其包含在所述田间种植本发明的转基因植物或来自其的转基因种子。
在一个优选的实施方案中，本发明涉及一种防治田间昆虫群体，更优选鞘翅目昆虫群体，更优选西方玉米根虫(WCRW)Diabrotica virgifera virgifera群体的方法。
制备方法
根据本发明，二元毒素的蛋白质组分可以在宿主细胞中一起产生，或者分别在单独的宿主细胞中产生，该单独的宿主细胞可以是相同种类或不同的，然后以期望的剂量组合在单一组合物中，以提供其有效量，用于应用于植物或其环境以及应用于害虫。
本发明的二元毒素的蛋白质可通过培养宿主细胞单独产生或组合产生以用于组合物中。将目的基因克隆入表达载体，然后插入宿主细胞中。转化的宿主细胞在需要时在诱导下在表达条件下培养。
或者，本发明的二元毒素的每种蛋白质组分可以通过培养野生型微生物(细菌、古细菌、真菌和原核和真核微藻)来产生。野生型微生物的生长条件得到修饰并且适合于改善生长或二元毒素的生成，或者都利用这些微生物在选择压力下的适应性进化能力。
在一个实施方案中，包含表达本发明的二元毒素的第1组或第2组蛋白质中的至少一种，或来自本发明的二元毒素的第1组或第2组中的每一个的蛋白质两者的野生型金黄杆菌属的组合物是本发明的一个目的。这种组合物可以特别用于通过将该组合物施用于植物或其部分来处理植物以防昆虫害虫侵染或处理受害虫侵染的植物。
野生型金黄杆菌属是可以在自然界中发现的金黄杆菌属细菌菌株。这种天然存在的菌株未经修饰、转化或突变。
例如在下面列出可根据本发明使用的这种金黄杆菌菌株。
Chryseobacterium arthrosphaerae CC-VM-7(Jeong et al.,2016.GenomeAnnounc 4(5):e01168-16.doi:10.1128/genomeA.01168-16)，一种从球马陆(pillmillipede)Arthrosphaera magna Attems的粪便分离的菌株(Kampfer etal.2010International Journal of Systematic and Evolutionary Microbiology,60,1765–1769)。该菌株表达SEQ ID NO:1和SEQ ID NO:2。
Chryseobacterium carnipullorum菌株25581是一种革兰氏阴性、棒状、非孢子形成的、非运动性细菌，其从南非布隆方丹的家禽加工厂的生鸡中分离得到(Charimba etal.2013)。该菌株可通过莱布尼茨研究所DSMZ-德国微生物和细胞培养物保藏中心(Leibniz Institute DSMZ-German Collection of Microorganisms and CellCultures)获得。该菌株表达GDI0175A(SEQ ID NO:11)和GDI0176A(SEQ ID NO:18)。
Chryseobacterium shigense菌株DSM17126(也称为BAMY 1001和GUM-kaji)是一种革兰氏阴性、严格需氧、棒状、非运动性细菌，其从日本的乳酸饮料中分离(Shimomura etal.2005)。该菌株可通过莱布尼茨研究所DSMZ-德国微生物和细胞培养物保藏中心获得。该菌株表达GDI0177A(SEQ ID NO:12)和GDI0178A(SEQ ID NO:19)。
Chryseobacterium kwangjuense菌株KJ1R5(也称为KACC 13029(T)和JCM 15904(T))是一种革兰氏阴性棒状细菌，其从韩国光州的辣椒植物番椒(Capsicum annuum)的根部分离得到(Sang et al.2013)。该菌株可通过JCM Riken获得。该菌株表达GDI0183A(SEQID NO:15)和GDI0184A(SEQ ID NO:22)。
金黄杆菌OV705表达GDI0185A(SEQ ID NO:16)和GDI0186A(SEQ ID NO:23)。
产吲哚金黄杆菌(Chryseobacterium indologenes)表达GDI0187A(SEQ ID NO:17)和GDI0188A(SEQ ID NO:24)。
无论蛋白质是由野生型微生物或重组微生物产生的，蛋白质都可以在之前有或没有细胞破碎的情况下从细胞沉淀中回收，或者可以从细胞沉淀、培养物上清液或整个培养物中提取、纯化和浓缩它们。
组合物
本发明的杀虫组合物含有一种或多种活性剂(包括本发明的二元毒素的第1组和第2组蛋白质中的一种或两种)与一种或多种其它组分(诸如例如载体，佐剂，表面活性剂，乳化剂，包封剂，释放剂，渗透剂，可检测剂以及本领域已知赋予活性剂或组合物本身以下特性中的一种或多种的其它组分：诸如活性剂的稳定性、乳化、悬浮或增溶，活性剂在环境中的持久性或植物渗透性，活性剂进入植物的渗透性，害虫的吸收)的组合。
本发明的二元毒素的蛋白质的市售制剂可用于生产稳定且易于使用的农业和园艺产品。市售制剂包括可湿性粉剂、悬浮浓缩物、水分散颗粒、油可溶混悬浮剂、胶囊悬浮剂和颗粒剂。
本发明的组合物还可含有其他活性剂，诸如除草剂、杀真菌剂、杀细菌剂、杀线虫剂、杀软体动物剂或其他杀虫剂。该组合物还可含有其他有利于植物生长的活性剂，诸如肥料、营养物、植物生长促进根瘤菌(PGPR)或微量营养物供体。
用法
根据本发明，可以通过将二元毒素或制备二元毒素的两种蛋白质组分中的任一种施用于昆虫害虫来处理植物免于昆虫害虫的侵染或处理受害虫侵染的植物。因此，该处理可以是预防性的，即在昆虫害虫侵染之前，或者是治愈性的，即一旦害虫侵染存在。可以通过培养表达有效量的本发明的二元毒素的一种或两种蛋白质组分的转基因植物来施用毒素或蛋白质。或者，可通过将包含有效量的本发明二元毒素的一种或两种蛋白质组分的组合物应用于害虫的环境来处理植物。所述组合物可应用于任何植物部分，直接应用至其叶子，或应用于其根部，或应用于两者，或应用于种子，通常应用于植物的叶子上，或通过常规方法诸如喷雾施用于作物上。其它应用方法包括但不限于，撒粉，喷洒，充气，土壤浸泡，土壤注射，种子包衣，幼苗包衣，叶面喷雾，喷雾，熏蒸，雾化以及本领域技术人员熟知的任何其它应用方法。此外，可以将包含有效量的本发明的二元毒素的至少一种蛋白质组分的组合物应用于表达其他蛋白质组分的转基因植物。对于包含本发明的二元毒素的单一组分的组合物，这种组合物可以单独应用，或者应用这些组合物中的两种，每种组合物具有二元毒素的一种蛋白质组分；它们可以同时或顺序施用。
“有效量”是足以在幼虫阶段或成年阶段逆转、减缓、阻碍、延迟或停止昆虫害虫的生长，或导致幼虫或成年昆虫害虫死亡的量。可以在一种或多种治疗应用中或经由如本文所公开的一种或多种方法施用有效量。
如本领域技术人员将理解的，有效量将随具体制剂的性质而广泛地变化，特别是其是在使用前稀释的浓缩物还是直接使用的浓缩物。制剂可含有约1重量％至约100重量％的本发明的二元毒素。干制剂可具有约1-95重量％的二元毒素，而液体制剂通常可具有液相中约1-60％重量的固体。表达本发明的二元毒素的全细胞组合物通常可具有约102至约104个细胞/mg。这些组合物可以每公顷约50mg(液体或干)至1kg或更多使用。
根据本发明，可以通过种植表达二元毒素的两种蛋白中的至少一种的转基因植物来控制害虫群体，然后可以将二元毒素的另一种蛋白以如上所述的处理的形式应用于植物。害虫群体还可通过种植经修饰以表达二元毒素的两种蛋白的植物来防治。
本发明的组合物可配制成含有引诱剂和本发明的二元毒素或表达它们或本文所述的其蛋白质组分之一的全细胞的诱饵颗粒。可将诱饵颗粒应用于土壤。本发明的组合物还可以在作物周期的后期阶段作为种子包衣或根处理或全植物处理应用。对于植物和土壤处理，表达如本文所述的本发明二元毒素或其组分蛋白质之一的细胞可通过与各种惰性材料(诸如无机矿物(层状硅酸盐(phyllosilicate)、碳酸盐、硫酸盐、磷酸盐等)或植物材料(玉米芯粉、稻壳、胡桃壳等))混合而用作可湿性粉剂、颗粒剂或粉剂。制剂可包括涂抹剂-粘着剂佐剂、稳定剂、其它杀虫添加剂或表面活性剂。液体制剂可为泡沫、凝胶、悬浮液、可乳化浓缩物等形式的水性或非水性制剂。成分可包括流变剂、表面活性剂、乳化剂、分散剂或聚合物。
在本文公开的方法和组合物的另一个实施方案中，表达本发明的二元毒素的宿主细胞培养物可在液氮中快速冷冻，然后在最终冷冻温度下储存。该培养物也可以更渐进的方式冷冻，或本领域已知的用于冷冻宿主细胞培养物的任何其它方法冷冻。
实施例
实施例1：基因组测序和DNA分析
通过Picogreen测定法定量从来自含有金黄杆菌属物种的私人保藏中心的细菌中分离的DNA样品，并用琼脂糖凝胶电泳分析检验质量。对DNA样品进行标准化，之后采用可适应聚焦声学(Adaptative Focused Acoustics)技术片段化。从每个初始DNA样品产生Illumina相容的无PCR文库。在此过程期间，每个文库用独特的双索引策略单独进行条形码化。然后通过qPCR测量对每个文库进行定量，之后合并。将96个文库合并在一起并使用配对末端2x100个碱基策略进行测序。每个合并物产生大约50Mb。
来自每个样品的读段(read)序列使用工具cutadapt 1.8.3版(https://pypi.org/project/cutadapt/)进行衔接头(adapter)和质量修整。修整后的读段进一步用de novo assembler SPADES 3.5.0版(http://cab.spbu.ru/software/spades/)进行组装，并且蛋白质编码基因用工具Prodigal 2.6.3版(https://github.com/hyattpd/Prodigal)进行预测。
在公共数据库中检索同源序列：使用BLASTP将测序基因组中编码区的预测蛋白序列与公共数据库中发现的具有证实的或推定的杀虫活性的蛋白进行比对。在表现出50％序列或更多的序列相对于查询覆盖长度和该比对区中同一性>30％的新序列为潜在候选物。
实施例2：二元毒素基因候选物和具有同源物序列的7种相应二元毒素的鉴定
发现从实施例1鉴定的蛋白序列GDI0005A(SEQ ID NO:1)和GDI0006A(SEQ ID NO:2)与专利申请WO 2016/114973中描述的PIP45蛋白具有非常低的同一性。GDI0005A与PIP45-Ga1具有39％的同一性，GDI0006A与PIP45-Ga2具有32％的同一性。然而，从纤维弧菌(Cellvibrio japonicus)分离的PIP45-Ga1加PIP45-Ga2描述为具有检测不到的西方玉米根虫(WCRW)杀虫活性，而来自假单胞菌属的相关蛋白(诸如PIP74Aa1加PIP74Aa2，其分别与GDI0005A和GDI0006A具有36％和30％的同一性)具有WCRW活性。所有这些蛋白质均为二元蛋白毒素。
GDI0005A和GDI0006A一起位于推定的操纵子中，从而增强了它们可以是具有杀虫活性的二元毒素的可能性(图1；SEQ ID NO:45)。
含有GDI0005A和GDI0006A的菌株的基因组序列与菌株Chryseobacteriumarthrosphaerae CC-VM-7(Jeong et al.,2016.Genome Announc 4(5):e01168-16.doi:10.1128/genomeA.01168-16)的公开序列非常密切相关(99％同一性)，Chryseobacteriumarthrosphaerae CC-VM-7是一种从球马陆Arthrosphaera magna Attems的粪便分离的菌株(Kampfer et al.2010International Journal of Systematic and EvolutionaryMicrobiology,60,1765–1769)。GDI0005A与Chryseobacterium arthrosphaerae蛋白A0A1B8Z9L3(GN＝BBI00_22205)具有99％的同一性，GDI0006A与A0A1B8Z9X7(GN＝BBI00_22200)具有100％的同一性。然而，这两个基因都被注释为“未表征的蛋白质”。
通过UniProt数据库中的BLASTP分析鉴定的与GDI0005A同源的蛋白由SEQ ID NO:11至SEQ ID NO:17表示。序列之间的同一性百分比在图2中呈现。所有蛋白质均见于金黄杆菌属的各物种的基因组，Chryseobacterium carnipullorum(GDI0175A；SEQ ID NO:11)，Chryseobacterium shigense(GDI0177A；SEQ ID NO:12)，Chryseobacterium kwangjuense(GDI0183A；SEQ ID NO:15)，OV705(GDI0185A；SEQ ID NO:16)，产吲哚金黄杆菌(GDI0187A；SEQ ID NO:17)。它们均在GDI0005和GDI0006等推定的操纵子中构造。
通过UniProt数据库中的BLASTP分析鉴定的与GDI0006A同源的蛋白质由SEQ IDNO:18至SEQ ID NO:24表示。序列之间的同一性百分比在图3中呈现。所有蛋白质均见于金黄杆菌的各物种的基因组，C.carnipullorum(GDI0176A；SEQ ID NO:18)，C.shigense(GDI0178A；SEQ ID NO:19)，C.kwangjuense(GDI0184A；SEQ ID NO:22)，OV705(GDI0186A；SEQ ID NO:23)，产吲哚金黄杆菌(GDI0188A；SEQ ID NO:24)。它们均在GDI0005和GDI0006等推定的操纵子中构造。
Chryseobacterium carnipullorum菌株25581是一种革兰氏阴性、棒状、非孢子形成的、非运动性细菌，其从南非布隆方丹的家禽加工厂的生鸡中分离得到(Charimba etal.2013)。该菌株可通过莱布尼茨研究所DSMZ-德国微生物和细胞培养物保藏中心获得。
Chryseobacterium shigense菌株DSM17126(也称为BAMY 1001和GUM-kaji)是一种革兰氏阴性、严格需氧、棒状、非运动性细菌，其从日本的乳酸饮料中分离(Shimomura etal.2005)。该菌株可通过莱布尼茨研究所DSMZ-德国微生物和细胞培养物保藏中心获得。
Chryseobacterium kwangjuense菌株KJ1R5(也称为KACC 13029(T)和JCM 15904(T))是一种革兰氏阴性棒状细菌，其从韩国光州的辣椒植物番椒(Capsicum annuum)的根部分离得到(Sang et al.2013)。该菌株可通过JCM Riken获得。
实施例3：GDI0005A和GDI0006A在大肠杆菌中的克隆和表达
为了在大肠杆菌中表达GDI0005A和GDI0006A，将缺少编码N末端Met和C端密码子终止的序列的DNA基因编码序列优化用于在大肠杆菌中表达(SEQ ID NO:26和SEQ ID NO:27)。将这些序列克隆到Electra克隆载体pD441-HMBP(ATUM(ex DNA2.0))中，从而将N末端His+MBP TAG编码序列与每个基因融合。将克隆转化到大肠杆菌菌株BL21(DE3)中，并在50mL体积的自诱导培养基(
Figure BDA0003336155620000191
Switch培养基/Delphi Genetics/GE-AIME-04)中生长。将培养物离心并重悬于12mL具有6μL溶菌酶(50mg/mL)和6μL DNaseI(2mg/ml)的化学细胞裂解缓冲液(NZY Bacterial Cell Lysis Buffer/NZYtech/MB17801)中。
以类似的方式，在大肠杆菌中表达SEQ ID NO:11至SEQ ID NO:17的GDI0005A同源蛋白(针对大肠杆菌优化的核酸序列，SEQ ID NO:28至SEQ ID NO:34)和SEQ ID NO:18至SEQ ID NO:24的GDI0006A同源蛋白(针对大肠杆菌优化的核酸序列，SEQ ID NO:35至SEQID NO:41)，并制备用于昆虫幼虫活性测定的细菌裂解物。
实施例4：使用GDI0005A和GDI0006A细菌裂解物进行易感鞘翅目生物测定
用裂解物进行杀虫活性生物测定以评估杀虫蛋白对鞘翅目昆虫，西方玉米根虫Diabrotica virgifera virgifera LeConte的作用。根据Pleau et al.(2002)和Moar etal.(2017Plos ONE)，使用96孔板对根虫特异性人工饲料(Frontier Scientific LtdF9800B饲料加上冻干玉米根,按照Pleau et al.2002Ent.Exp.et Appl.)进行幼虫喂养试验。将20μL不同大肠杆菌克隆的澄清液体裂解物应用于每孔190μL固体人工饲料的0.8cm2表面上。裂解物含有GDI0006A蛋白或GDI0005A蛋白，或两者的50：50混合物(均为HIS&MBPTAGs)。类似体积的澄清的空裂解物及其NZY-tech Tris缓冲液用作阴性对照。将1只西方玉米根虫新生幼虫置于每个孔中喂养5天。如果幼虫死亡率和发育迟缓率在统计学上高于阴性对照，则认为结果是阳性的。
如在本实施例和以下实施例中使用的空裂解物是在相同条件下并由相同细菌菌株(其含有与含有杀虫蛋白的裂解物相同的表达载体，唯一的例外是该表达载体不含有编码毒素的基因)产生的细菌裂解物。
三天后，与每种单一的GDI0005A和GDI0006A毒素和对照的活性相比，含有组合的GDI0006A和GDI0005A蛋白的裂解物在幼虫中表现出强烈的发育迟缓(图6A)。五天后，发现了类似的发育迟缓作用，并且在根虫幼虫中由组合的GDI0006A和GDI0005A蛋白引起显著死亡率(图6B)。
三天后，含有组合的GDI0006A同源物和GDI0005A同源物蛋白的裂解物在幼虫中表现出强烈的发育迟缓(图7B)。五天后，结果显示由组合的GDI0006A同源物和GDI0005A同源物蛋白引起的根虫幼虫的显著死亡率(图7A)。
实施例5：使用GDI0005A和GDI0006A细菌裂解物对抗性和易感鞘翅目进行生物测定以确定模式作用
杀虫活性：
用如Huynh MP,et al.(PLoS ONE 12(11):e0187997)所述生产的饮食，称为WCRMO-1饮食，利用人工饮食生物测定法测定GDI005(His_MBP_GDI0005A)和GDI006(His_MBP_GDI0006A)细菌裂解物对WCRW(西方玉米根虫，Diabrotica virgifera virgiferaLeConte)的杀虫活性。针对3种WCRW新生幼虫株，WCRW易感、WCRW Cry3Bb1抗性和WCRWCry34/35抗性，进行了四种测定(每种株系一个)。WCRW Cry34/35抗性群体呈现对Cry34/35毒素的不完全抗性，如Ludwick et al.(2017)和Gassmann et al.(2018)所报道的。这种不完全抗性对于新发现的田间进化抗性是典型的。
每个测定法由一系列3个96孔测定板(Costar 3370,Corning Inc.,KennebunkME)组成，其中每个板在每个测试中含有重复的样品。与UTC、空裂解物、Tris缓冲液和阳性对照相比，分别针对易感、Cry3Bb1抗性和Cry34/35抗性WCRW，测定评估了未稀释(1X)时GDI005和GDI006等比例的二元混合物的WCRW活性，所述阳性对照是天然细菌裂解物Cry34/35的1：1混合物。
数据评价：
每个测试有3个重复，每个重复包含每种毒素、阴性对照和阳性对照以及UTC的1列，每个测试每个处理总共24个数据点。包括测试毒素(GDI005/GDI006)、空裂解物、Tris缓冲液和POS的每个处理孔通过在约200μL的WCRMO-1饮食中添加20μL的测试物质来处理。UTC没有覆盖测试物质，因为它仅仅是饮食。在覆盖测试物质后，将托盘在层流通风橱下干燥约30分钟。接下来，使用细尖水彩刷将一只新生WCRW幼虫侵染到96孔测定板的每个处理孔中。幼虫侵染后，将各个板用密封膜(Excel Scientific,Inc.,Thermalseal RTSTM,TSS-RTQ-100)覆盖，并置于25℃黑暗的生长室中5天。在第5天，取出各个板，并评估每个孔中的幼虫的死亡率(活的或死的)。在死亡率评估后，使用以mg为单位的分析天平，将幼虫称重作为每种处理的每个列每重复96孔测定板的合并组。评估了死亡率和幼虫重量后，对数据进行分析。数据示于图8A-8B-8C。如果与UTC相比，当暴露于毒素5天时，结果显示重量减少至少50％，则认为毒素是有效的。以空裂解物、Tris缓冲液和POS作为阳性对照和阴性对照，以评估每个测试的质量和可靠性。此外，POS用于测量毒素与已在商业上使用的已知毒素类别相比的有效性。
结果：
试验显示二元毒素对Cry34/35和Cry3B1抗性株的毒性。Cry34/35和Cry3Bb1抗性集落的结果显示，本发明的二元毒素呈现与Cry34/35和Cry3Bb1毒素中的一种不同的模式作用。
实施例6：LC50
GDI005和GDI006的纯化蛋白用于测定二元混合物的LC50值。LC50方法的第一步是通过范围探索体外生物测定(range finding in vitro bioassay)确定在3次重复的测定中杀死约50％和100％的昆虫的二元混合物的浓度。这些测定将标准蛋白质覆盖在饮食测定之上，其中将20μL测试物质移液到约200μL的WCRMO-1饮食上。在覆盖测试物质后，将托盘在层流通风橱下干燥约30分钟。接下来，使用细尖水彩刷将一个新生WCRW幼虫侵染到96孔测定板的每个处理孔中。幼虫侵染后，将各个板用密封膜(Excel Scientific,Inc.,Thermalseal RTSTM,TSS-RTQ-100)覆盖，并置于25℃黑暗的生长室中5天。在第5天，取出各个板，并评估每个孔中的幼虫的死亡率(活的或死的)。在死亡率评估后，使用以mg为单位的分析天平，将幼虫称重作为每种处理的每个列每重复96孔测定板的合并组。评估死亡率和幼虫重量后，对数据进行分析。每个浓度最初产生至少24个数据点，如果需要，添加更多的数据点。在易感的WCRW上进行初始范围探索生物测定，以确定纯化蛋白质二元混合物的基线死亡率。
范围探索测定完成后，在来自范围探索测定的全强度浓度和50％浓度开始制备几种浓度的二元纯化蛋白质混合物，并使用低熔融琼脂掺入WCRMO-1饮食中。首先评估易感WCRW幼虫以测定LC50。由于饮食掺入的复杂性质，使用了48孔板。每个孔用7-10只新生幼虫侵染，并在5天后评估死亡率。此外，通过重复和通过浓缩在合并的基础上评估幼虫重量。在易感WCRW群体上建立LC50后，评估Cry3Bb1和Cry34/35抗性群体并测定LC50。
实施例7：烟草瞬时测定
将编码GDI0005A的合成玉米密码子优化序列加N末端组氨酸TAG(SEQ ID NO:5)克隆在与存在于植物二元载体中的稻肌动蛋白5'UTR(SEQ ID NO:7)连接的强组成型CsVMV启动子(Verdaguer et al.(1996))和高粱HSP多聚腺苷酸化序列(SEQ ID NO:8)之间。GDI0005基因盒(SEQ ID NO:9)在图4中呈现。类似地，将编码GDI0006A加上N末端组氨酸TAG的合成玉米密码子优化序列(SEQ ID NO:6)克隆到另一种植物载体中。GDI0006基因盒(SEQID NO:10)在图5中呈现。另外，将上述GDI0005A和GDI0006A植物表达盒一起克隆到同一植物二元载体中。
根据Komari et al.(1996)，将得到的二元质粒转移到土壤杆菌菌株LBA4404(pSB1)中，提供菌株T11467(含有GDI0005)和T11466(含有GDI0006)和T11522(含有GDI0005A和GDI0006A)。使用标准烟草(本氏烟草(Nicotiana benthamiana))土壤杆菌渗入(agroinfiltration)方案(基本上如https://bio-protocol.org/bio101/e95中所述)，用单一菌株或单一基因株两者的共渗入瞬时转化叶扇区(leaf sector)。以类似的方式，将GDI0005A同源物和GDI0006A同源物瞬时转化到烟草中。
从转化的烟草叶中提取总蛋白，并分别使用对SEQ ID NO:1具有特异性并能够识别GDI0005同源物的多克隆抗体或对SEQ ID NO:2具有特异性并能够识别GDI0006同源物的多克隆抗体，通过蛋白质印迹检测目的蛋白的表达。
图9的蛋白质印迹显示蛋白质GDI0005A(SEQ ID NO:1)及其同源物GDI0175A(SEQID NO:11)、GDI0177A(SEQ ID NO:12)、GDI0179A(SEQ ID NO:13)、GDI0181A(SEQ ID NO:14)、GDI0183A(SEQ ID NO:15)、GDI0185A(SEQ ID NO:16)、GDI0187A(SEQ ID NO:17)在转化烟草植物中表达。被特异性抗体识别的蛋白以53kDa的预期大小累积。
图10的蛋白质印迹显示蛋白质GDI0006A(SEQ ID NO:2)及其同源物GDI0176A(SEQID NO:18)、GDI0178A(SEQ ID NO:19)、GDI0180A(SEQ ID NO:20)、GDI0182A(SEQ ID NO:21)、GDI0184A(SEQ ID NO:22)、GDI0186A(SEQ ID NO:23)在转化烟草植物中表达。被特异性抗体识别的蛋白以53kDa的预期大小累积，并通过纯化的GDI0006的大小确认。
基本上如实施例4所述，将粗蛋白提取物也用于昆虫幼虫毒素活性测定。在处理后3天和5天评估幼虫的死亡率和发育迟缓率。
实施例8：玉米植株转化
基本上如Ishida et al.(1996)所述，将实施例7中描述的植物二元构建体菌株T11467、T11466和T11522转化到玉米自交系A188中。还将菌株T11467和T11466共转化到玉米自交系A188中。每个构建体产生最少10个个体，单拷贝转化体或具有完整T-DNA的共转化体。对T0叶材料进行QRT-PCR和蛋白质印迹分析。基本上如实施例4所述，将粗蛋白提取物用于昆虫幼虫毒素活性测定。
在处理后3天和5天评估幼虫的死亡率和发育迟缓率。
以类似的方式，在玉米中转化GDI0005同源物和GDI0006同源物。基本上如实施例4所述，将粗蛋白提取物也用于昆虫幼虫毒素活性测定。在处理后3天和5天评估幼虫的死亡率和发育迟缓率。
实施例9：GDI0005A和GDI0006A在荧光假单胞菌(Pseudomonas fluorescens)中的克隆和表达
为了在荧光假单胞菌中表达GDI0005A和GDI0006A，将缺少编码N末端Met和C末端密码子终止的序列的DNA基因编码序列优化用于在荧光假单胞菌中表达(分别为SEQ IDNO:46和SEQ ID NO:47)。将这些序列克隆到pCOM10克隆载体中，从而将N末端His TAG编码序列与每个基因融合。将克隆转化到荧光假单胞菌中并在50mL体积的LB培养基中生长。加入二环丙基酮诱导蛋白表达。将培养物离心并重悬于1mL重悬浮缓冲液(Tris 20mM pH8，NaCl 150mM，DTT 1mM)中。用二氧化硅和玻璃珠机械裂解细胞。
以类似的方式，将编码SEQ ID NO:11至SEQ ID NO:17的GDI0005A同源蛋白的核酸序列和编码SEQ ID NO:18至SEQ ID NO:24的GDI0006A同源蛋白的核酸序列进行优化以在假单胞菌中表达，并在荧光假单胞菌中表达。
制备细菌裂解物用于昆虫幼虫活性测定。
实施例10：从野生型菌株生产GDI0005A和GDI0006A蛋白
为了从野生型菌株产生GDI0005A和GDI0006A蛋白或GDI0005A和GDI0006A同源蛋白，在每种菌株的最佳温度下，使实施例2的金黄杆菌属物种在200mL体积的丰富矿物质培养基中生长，进行或不进行通过与幼虫或昆虫的直接接触的诱导。在对数生长期和稳定生长期收获培养物并离心。用二氧化硅和玻璃珠机械裂解细胞并重悬于10mL新鲜培养基中。
用培养物上清液(毒素分泌物)和裂解物进行杀虫活性生物测定，以评估杀虫蛋白对鞘翅目物种幼虫的影响。
实施例11：用GDI0005A和GDI0006A或GDI0006同源物细菌裂解物进行易感鞘翅目生物测定
将如实施例3所述获得的转化大肠杆菌制备成裂解物，并如实施例4所述进行测试。
表达SEQ ID NO:1的裂解物的活性结合其自身配偶体SEQ ID NO:2以及结合表达GDI0176A(SEQ ID NO:18)、GDI0178A(SEQ ID NO:19)、GDI0184A(SEQ ID NO:22)、GDI0186A(SEQ ID NO:23)或GDI0188A(SEQ ID NO:24)的裂解物进行测试。在3天后针对西方玉米根虫，与阴性对照的7％和8％的基础死亡率相比，这6种组合显示出相当的活性，发育迟缓率范围为88.9％至97.2％。
该实验表明，二元毒素可以由选自第1组的一种蛋白和选自第2组的另一种蛋白形成，并且即使这两种蛋白不是源自金黄杆菌属的相同物种也是有效的。
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Shimomura,K；Kaji,S；Hiraishi,A(September 2005)."Chryseobacteriumshigense sp.nov.,a yellow-pigmented,aerobic bacterium isolated from a lacticacid beverage".International Journal of Systematic and EvolutionaryMicrobiology.55(Pt 5):1903–6.doi:10.1099/ijs.0.63690-0.PMID 16166686.
Torrent M,Llompart B,Lasserre-Ramassamy S,Llop-Tous I,Bastida M,Marzabal P,Westerholm-Parvinen A,Saloheimo M,Heifetz PB,Ludevid MD.(2009)Eukaryotic protein production in designed storage organelles.BMC Biol.7:5doi:10.1186/1741-7007-7-5.
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von Heijne G,Hirai T,
Figure BDA0003336155620000271
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序列表
<110> GROUPEMENT NATIONAL INTERPROFESSIONNEL DES SEMENCES,
GRAINES ET PLANTS (G.N.I.S.)
吉奈克蒂夫公司
普鲁泰斯公司
原子能和代替能源委员会
ALTERNATIVES
<120> 杀虫基因和使用方法
<130> B377601PCT D39230
<150> EP19305304.8
<151> 2019-03-13
<160> 47
<170> PatentIn version 3.5
<210> 1
<211> 579
<212> PRT
<213> Chryseobacterium arthrosphaerae
<400> 1
Met Lys Lys Phe Asn Thr Pro Ala Tyr Gln Ala Glu Lys Asp Phe Lys
1 5 10 15
Gln Tyr Pro His Leu Gly Glu Gln Leu Glu Asn Ala Trp Ser Asn Tyr
20 25 30
Val Lys Tyr Cys Thr Ile Asn Ser Ile Met Gly Asn Pro Trp Ser Ser
35 40 45
Thr Tyr Asp His Pro Arg Ser Trp Tyr Tyr Asn Pro Leu Val Asp Asp
50 55 60
Val Val Pro Thr Glu Gln Asn Thr Val Pro Ile Gln Trp Asn Ala Phe
65 70 75 80
Pro Asn Arg Ile Asn His Tyr Phe Thr Gly Leu Phe Thr Lys Gln Phe
85 90 95
Gly Ser Ala Glu Tyr Glu Asp Lys Leu His Glu Leu Ala Asp Ile Gly
100 105 110
Pro Ala Ala Phe Gly Lys Lys Tyr Asn Met Asp Leu Thr Val Pro Lys
115 120 125
Asn Pro Cys Asp Pro Ser Asp Thr Arg Thr Lys Pro Phe Gly Pro Ser
130 135 140
Gly Pro Arg Gly Trp Gln Asp Glu Tyr Cys Glu Trp Ala Val Thr Arg
145 150 155 160
Asp Glu Asn Gly Asp Ile Thr Ala Val Asp Phe Thr His Glu Asn Pro
165 170 175
Glu Tyr Trp Phe His Met Trp Lys Ile Ser Pro Asp Ile Val Val Ala
180 185 190
Leu Tyr Gln Glu Ile Leu Asn Asn Lys Asn Val Lys Lys Glu Asp Leu
195 200 205
Tyr Leu Leu Asp Ser Thr Gly Asn Pro Val Ile Val Arg Glu Thr Gly
210 215 220
Glu Pro Ala Tyr Asn Pro Ile Asn Lys Trp Asn Asn Gly Pro Glu Ala
225 230 235 240
Thr Pro Glu Gly Gly Gly Ala Val His Leu Thr Ser Pro Pro Asn Ser
245 250 255
Leu Gly Ala Glu Ile Tyr Leu Gly Ala Ala Ala Thr Ile Leu Arg Val
260 265 270
Lys Asn Asn Gln Val Ile Thr Asp Ala Asn Ala Leu Ile Cys Ala Ala
275 280 285
Gln Tyr Gly Gln Ile Tyr Arg Asn Ser Asp Pro Arg Ile Gly Gln Asn
290 295 300
Val Asn Ser Leu Val Tyr Asn His Lys Gln Gln Ile Thr Leu Thr Asn
305 310 315 320
Pro Ile Ala Leu Tyr Gly Gln Val Pro Asp Phe Asp Gln Phe Glu Met
325 330 335
Pro Ser Thr Ala Gly Ser Tyr Lys Ile Gln Asp Cys Tyr Thr Val Val
340 345 350
Arg Gly Glu Glu Arg Asn Lys Gly Ile Thr Phe Tyr Pro Phe Asn Met
355 360 365
Leu Leu His Thr Arg Phe Ser Val Pro Lys Gly Ala Asn Phe Lys Leu
370 375 380
Ser Glu Ile Lys Val Lys Gly Lys Leu Leu Lys Trp Gly Ser Gln Ile
385 390 395 400
Ala Asp Thr Phe Phe Val Gln Leu Ala Gly Thr Gly Lys Ser Pro Gly
405 410 415
Ala Gly Glu Gln Pro Glu Lys Phe Pro Pro Val Gly Asp Pro Ala Thr
420 425 430
Thr Leu Pro Asn Val Gln Tyr Leu Leu Asp Asn Asn Leu Leu Gln Ala
435 440 445
Ser Leu Tyr Asn Lys Leu Asn Thr Phe Ser Asn Leu Thr Ser Cys Ile
450 455 460
Thr Gln Ile Glu Ala Gly Thr Ser Thr Glu Gly Ile Ala Val Leu Thr
465 470 475 480
Asn Ala Ala Thr Lys Glu Thr Gln Phe Asp Phe Gly Pro Gly Ile Ser
485 490 495
Val Met Val Thr Asp Phe Gln Asn Ile Asp Glu Asp Thr Gln Leu Phe
500 505 510
Leu Ile Thr Ile Thr Ala Asp Ala Asp Thr Ser Leu Gly Glu Lys Pro
515 520 525
Leu Ser Leu Tyr Asn Asn Ala Ser Asp Pro Lys Tyr Ala Leu Ser Gly
530 535 540
Val Leu Glu Val Val Pro Asn Gly Ser Leu Pro Lys Ile Asn Thr Thr
545 550 555 560
Pro Asn Leu Ala Leu Leu Ser Gly Gln Gln Val Glu Gln Val Lys Lys
565 570 575
Ile Leu Lys
<210> 2
<211> 466
<212> PRT
<213> Chryseobacterium arthrosphaerae
<400> 2
Met Lys Thr Leu Val Phe Ser Leu Phe Ile Leu Ser Phe Ile Ser Cys
1 5 10 15
Glu Ser Gly Lys Lys Asp Gln Arg Leu Asn Ser Ala Thr Gly Phe Ser
20 25 30
Lys Val Ala Asp Ser Glu Ser Ile Tyr Cys Gly Ala Phe Cys Asp Pro
35 40 45
Pro Ser Ile Thr Tyr Lys Leu Ala Gly Asp Ser Thr Cys Ala His Ser
50 55 60
Ser Gln Asp Val Leu Asn Cys Phe Ala Trp Lys Asn Phe Ile Ala Leu
65 70 75 80
Asn Trp Ala Ala Ser Ala Gln Arg Gly Val Pro Asp Thr Thr Ala Thr
85 90 95
Ala Ala Asn Tyr Gly Met Pro Gly Asp Tyr Ser Pro Thr Val Trp Glu
100 105 110
Ser Phe Ala Ser Asn Asp Glu Val Phe Ala Pro Lys Asn Leu Leu Thr
115 120 125
Trp Asn Leu Lys Ser Lys Asn Gly Tyr Val Lys Gln Ile Asn Glu Ile
130 135 140
Asn Lys Phe Thr Asp Ile Asn Ile Ser Ile Pro Lys Ala Thr Leu Arg
145 150 155 160
Ala Ala Val Gly Asn Ser Asn Val Asp Glu Ile Leu Gln Ala Glu Gly
165 170 175
Ser Trp Leu Thr Asp Gln Ser Gly Asn Ile Val Trp Tyr Glu Ile Lys
180 185 190
Ile Asn Asn Ile Glu Ser Asp Phe Ile Arg Arg Asn Lys Leu Tyr Asp
195 200 205
Tyr Asn Ser Leu Lys Glu Tyr Gly Thr Ala Asn Asn Gly Val Trp Leu
210 215 220
Pro Met Glu Ser Ile Glu Leu Lys Ala Ala Trp Arg Val Ile Pro Glu
225 230 235 240
Asp Lys Leu Asp Ser Leu Lys Asn Tyr Tyr Lys Ile Ser Lys Ala Met
245 250 255
Val Pro Glu Ile Lys Gly Phe Lys Asp Lys Lys Pro Val Phe Gly Lys
260 265 270
Ser Thr Gln Lys Tyr Leu Gly Leu Val Gly Leu His Ile Ile Arg Lys
275 280 285
Thr Pro Gln Ser Pro Gln Phe Asn Trp Met Thr Phe Glu His Ile His
290 295 300
Asn Ala Pro Asn Glu Gly Gln Ala Asp Pro Ser Val Arg Tyr Cys Phe
305 310 315 320
Tyr Asn Pro Lys Ser Thr Lys Thr Pro Asn Ile Ala Pro Val Ile Gly
325 330 335
Lys Asp Ser Leu Asn Thr Pro Val Gln Val Val Arg Val Asn Lys Ile
340 345 350
Lys Thr Lys Leu Gln Lys Leu Asn Thr Gln Met Gln Gln Leu Ile Arg
355 360 365
Ala Ser Asn Pro Lys Ser Val Trp Gln Tyr Tyr Gln Leu Val Asn Ile
370 375 380
Gln Trp Pro Glu Asn Pro Ile Gln Asp Asn Gly Asn Asn Lys Ser Ala
385 390 395 400
Pro Leu Met Glu Gly Gly Ile Thr Pro Ser Asp Ile Ser Asn Thr Thr
405 410 415
Met Glu Thr Tyr Ala Gln Gln Lys Gln Cys Met Asp Cys His Lys Tyr
420 425 430
Ala Ser Val Val Gly Ser Gly Met Pro Pro Thr Asp Tyr Ser Phe Ile
435 440 445
Phe Leu Lys Val Lys Pro Glu Lys Gln Ile Pro Lys Gly Lys Thr Pro
450 455 460
Val Lys
465
<210> 3
<211> 1740
<212> DNA
<213> Chryseobacterium arthrosphaerae
<400> 3
atgaaaaaat ttaacacccc tgcctatcag gccgagaagg attttaaaca gtatccgcat 60
ttaggagagc agcttgaaaa tgcctggagc aattatgtaa aatactgtac catcaattca 120
atcatgggaa atccctggtc aagtacgtat gatcatccca gaagctggta ttataatcct 180
ctggtggatg atgtcgtacc tacagaacag aataccgttc ctatacagtg gaatgccttt 240
cccaacagga tcaatcatta ttttaccggg cttttcacca aacaatttgg cagcgcggaa 300
tatgaagata aactgcatga gctggctgat ataggtccgg ctgcttttgg aaaaaaatat 360
aatatggatc ttacagtccc taaaaacccg tgtgatcctt ctgatacccg tacgaaacca 420
tttggcccgt caggacctcg cgggtggcag gatgaatatt gtgaatgggc tgttacaagg 480
gatgaaaacg gggatatcac agctgttgat tttacccatg agaatccgga gtattggttt 540
catatgtgga aaatttctcc ggatatagtg gtcgcacttt atcaggaaat cctcaataat 600
aagaacgtta aaaaagaaga cctgtacctg ctggacagca ctggaaatcc agttatcgta 660
agagaaacag gagagcctgc ctacaacccg attaataaat ggaataatgg cccggaggca 720
actcctgaag gcggaggagc cgtacatctt acaagtcctc caaattcatt gggtgctgaa 780
atctaccttg gagcggcagc cactatctta cgggtaaaaa acaatcaggt gattacagac 840
gccaatgctt tgatctgcgc tgcccagtac ggacaaattt atagaaacag tgatccccgc 900
ataggacaga atgttaattc attggtatac aaccataagc aacaaataac tttaaccaat 960
cctattgcac tctatgggca ggttccggat tttgatcaat ttgaaatgcc ttctaccgcc 1020
ggcagttaca aaattcagga ttgctatact gttgtacgag gggaagaacg gaataaagga 1080
attactttct atccttttaa tatgcttttg cacacaagat tttctgttcc gaaaggcgcc 1140
aattttaagc tgagtgaaat aaaagtaaaa ggaaagctct taaaatgggg ctcacagatt 1200
gctgacacct ttttcgtaca gctggcaggt accggaaaaa gtcccggggc cggagagcag 1260
cctgaaaaat ttcctccggt aggagatccg gcaacaactc ttcctaatgt acaatatctt 1320
ttggataaca atttgctgca ggcaagtctt tacaataaac tgaatacttt ttctaatctg 1380
acatcctgca ttacacagat tgaagcagga acgagtactg aaggtattgc tgttctgacg 1440
aatgcggcca cgaaggaaac ccagtttgat ttcggtcccg gaatcagtgt tatggtcact 1500
gatttccaga atatcgatga ggatacacaa ctgtttctga ttacgattac agcagatgct 1560
gatacaagcc tgggagaaaa gccattaagc ctgtacaata atgcttcaga tcctaagtat 1620
gcactatccg gagtattgga ggtagtaccc aatggctctt tacctaaaat aaatacaact 1680
cctaatcttg cattgctttc ggggcagcaa gttgaacaag ttaaaaaaat attgaaatga 1740
<210> 4
<211> 1401
<212> DNA
<213> Chryseobacterium arthrosphaerae
<400> 4
atgaaaactt tagtattttc gctttttata ttgagcttca tctcatgtga atccggtaag 60
aaagaccagc gactgaattc agcaaccggt ttttcaaaag tagcagacag tgaatcgatc 120
tattgcggtg cgttttgtga tcctccttct attacttata aacttgccgg ggattccacc 180
tgtgcgcaca gttcgcagga tgtgctgaat tgttttgcat ggaagaattt tattgcacta 240
aactgggctg cttctgctca aagaggtgtt ccggatacca ctgcaacagc agccaattac 300
ggtatgcccg gggattacag ccctacagtc tgggagagct ttgcaagcaa tgacgaagtt 360
tttgccccca aaaatctttt aacatggaac ctgaaaagta agaatgggta tgtaaaacaa 420
attaatgaga ttaataaatt tacagatatt aatatcagta ttcccaaagc tactctcaga 480
gctgcagtag gaaatagtaa tgttgatgaa atattacagg ctgaaggttc ctggcttact 540
gatcagagcg gaaatattgt ttggtatgaa attaaaatca acaatattga aagtgatttt 600
atccgccgga ataagctgta tgattataat agtctgaaag agtatggtac tgcaaataac 660
ggcgtgtggc tgcccatgga gtctatagaa cttaaagctg cctggcgtgt cattcctgaa 720
gataaacttg actctttgaa aaattattat aaaatttcaa aagccatggt tccggaaatt 780
aaaggtttta aagataaaaa gcctgtcttt ggaaaatcca cacagaaata tttgggactg 840
gtgggcctcc atattatcag gaaaactcca cagtcacctc agtttaactg gatgacattt 900
gagcatatac ataatgctcc taatgaagga caggctgatc cctctgtaag gtattgcttc 960
tacaatccta aaagtacaaa gactcccaat atcgctccgg taatagggaa ggacagtctg 1020
aatactcctg ttcaggtcgt acgggtgaac aaaatcaaaa caaagcttca aaagctgaac 1080
acccagatgc agcagcttat cagggcaagt aaccctaaat cagtatggca gtattaccag 1140
cttgtgaata tccagtggcc ggaaaatccg atacaggata atggaaataa taagtctgct 1200
ccacttatgg aaggagggat taccccttcg gatatttcga atacaacgat ggaaacctat 1260
gctcaacaaa aacagtgcat ggactgtcat aagtatgctt cagtagtggg gagcggtatg 1320
ccgcctaccg attacagttt tatattttta aaggtaaaac cggaaaagca gattccaaaa 1380
ggaaaaactc ctgtaaaata a 1401
<210> 5
<211> 1767
<212> DNA
<213> 人工序列
<220>
<223> His-GDI005核优化植物
<400> 5
atgcaccacc accatcacca catcgatatg aagaagttca acacccctgc ttaccaggcc 60
gaaaaagact tcaaacagta cccccacctg ggcgagcaac tcgagaacgc gtggagcaat 120
tacgtgaagt actgtacaat caactccatt atggggaatc cgtggtcctc aacctatgac 180
cacccgcgct cgtggtacta caatccactc gttgatgacg tggtgcctac tgaacagaat 240
accgtgccaa ttcagtggaa tgcattccca aaccgcatca accactactt cactgggctg 300
tttaccaagc agttcggttc ggctgagtac gaggataagc tgcacgagct tgccgatatc 360
ggccctgcgg cattcgggaa gaagtataac atggacctta cagtgccgaa gaacccatgt 420
gacccgagcg atacacggac caaaccattt gggccttcag gtccacgcgg ttggcaagac 480
gaatattgcg agtgggcggt cacacgggac gagaacgggg acattacagc ggtcgacttt 540
acccacgaaa accctgagta ctggttccat atgtggaaga tatcaccaga catcgtggtg 600
gccttgtatc aggagatcct gaacaacaag aatgtgaaga aggaagacct ctacttgctc 660
gactcgaccg gtaaccccgt catcgttcgg gagacagggg aaccggcata caaccccatc 720
aacaagtgga ataacggacc agaggccaca ccagagggtg ggggtgcagt tcatctcacc 780
tcaccaccga actccctggg tgcagagatc tacctgggcg cagccgcgac catactccgg 840
gttaagaata accaggtcat caccgacgct aacgccctca tctgcgcggc ccagtacggc 900
cagatctacc ggaacagcga cccacggatc gggcagaatg tcaactcact cgtctacaat 960
cataagcagc agatcacact aaccaacccc atcgcgctgt atggacaagt cccagacttt 1020
gatcagttcg agatgccaag caccgcgggg tcgtataaga ttcaggactg ctacacggtc 1080
gtgagaggag aggaaaggaa caagggtatc accttctacc cctttaacat gctcctccac 1140
acaaggtttt cggttcccaa gggggcgaat ttcaagctca gcgagatcaa ggtcaagggc 1200
aagctcctga agtgggggag ccaaatcgcg gacaccttct tcgtgcagct ggcgggaacc 1260
ggcaagagtc ccggtgccgg ggaacagccg gaaaagtttc ctccagtggg tgacccggcg 1320
accacactgc ccaacgtgca gtacctgctc gataataacc tgctgcaggc ctccctctac 1380
aacaaactga acaccttcag caatctgacc tcgtgcatca cgcaaatcga ggccggcaca 1440
tcgactgaag gtatcgcagt gttgacaaac gccgctacca aggaaactca gttcgacttc 1500
ggtccgggca tcagcgtgat ggtgacagac ttccagaaca tcgatgaaga cacgcaactg 1560
tttcttatca ccatcacagc agacgcggat acatcgctgg gagagaagcc actttccctc 1620
tacaacaatg caagcgatcc aaagtacgcc ctgtccggcg tgctggaagt ggtccccaac 1680
ggtagcctgc cgaagattaa cactacaccc aatctggcgc tcttgtccgg acaacaggtg 1740
gagcaagtca agaaaatcct taagtag 1767
<210> 6
<211> 1428
<212> DNA
<213> 人工序列
<220>
<223> His-GDI0006核优化植物
<400> 6
atgcaccacc accatcacca catcgatatg aagacgctgg tctttagcct gttcatcctc 60
tcattcatct cgtgtgaatc agggaaaaag gaccaacggc ttaatagcgc aaccggtttc 120
tcgaaggtgg ctgactccga gagcatctac tgtggagcgt tttgcgatcc tccatcaatc 180
acatacaaac tggcaggaga cagcacctgc gcacacagct cgcaggacgt tctcaactgc 240
ttcgcgtgga agaacttcat cgcactgaat tgggcggcct ccgcgcagag aggtgtccca 300
gacaccacag caacagcagc gaactatggc atgccagggg actattcgcc gacagtctgg 360
gagtccttcg cttccaacga cgaggtgttc gcacccaaga acctcctgac atggaatctg 420
aagagtaaga acggttacgt gaaacagatc aacgagatta acaagttcac cgatatcaac 480
atcagcatac ccaaggcaac cctcagagca gctgtcggca acagcaatgt ggatgaaatc 540
ctgcaagcgg agggttcgtg gcttactgac cagtcgggca acatcgtctg gtacgagatt 600
aagatcaata acatcgaatc ggacttcatc agacggaata agctctacga ctacaatagc 660
ctcaaggagt acgggaccgc aaacaatggg gtgtggctcc ccatggagag catagaactg 720
aaagcggcat ggagagtgat cccagaggac aaactggata gcctgaaaaa ctactataag 780
atctccaaag ccatggtccc agagatcaag gggttcaagg ataagaaacc cgtgttcgga 840
aagtccaccc agaagtacct gggtctcgtg gggctgcaca ttatccggaa gaccccacaa 900
agcccacaat tcaactggat gacattcgag catatccaca acgccccaaa tgaggggcag 960
gcggatccta gcgtccggta ctgcttctac aaccccaaga gcacaaaaac accaaatatc 1020
gcgcctgtca tcggaaagga ttcactgaac acaccggtgc aagtcgttcg ggtcaataag 1080
attaaaacca aactgcagaa gctgaacacc caaatgcagc agctgattcg ggcgagcaat 1140
cccaagagcg tgtggcagta ttaccagctg gtgaacatcc agtggccaga gaatccgatc 1200
caggacaacg gtaacaataa gagtgcccca ctcatggagg gagggattac ccccagcgat 1260
atctccaata ccacgatgga gacctacgcc caacagaagc agtgtatgga ctgtcacaag 1320
tacgcatcgg ttgttggtag cgggatgcca cctaccgact atagcttcat cttcctgaag 1380
gtgaagccag aaaaacagat tccaaagggg aaaaccccag tgaagtag 1428
<210> 7
<211> 1018
<212> DNA
<213> 人工序列
<220>
<223> pCsVMV+OsActin+内含子
<400> 7
aaggtaatta tccaagatgt agcatcaaga atccaatgtt tacgggaaaa actatggaag 60
tattatgtga gctcagcaag aagcagatca atatgcggca catatgcaac ctatgttcaa 120
aaatgaagaa tgtacagata caagatccta tactgccaga atacgaagaa gaatacgtag 180
aaattgaaaa agaagaacca ggcgaagaaa agaatcttga agacgtaagc actgacgaca 240
acaatgaaaa gaagaagata aggtcggtga ttgtgaaaga gacatagagg acacatgtaa 300
ggtggaaaat gtaagggcgg aaagtaacct tatcacaaag gaatcttatc ccccactact 360
tatcctttta tatttttccg tgtcattttt gcccttgagt tttcctatat aaggaaccaa 420
gttcggcatt tgtgaaaaca agaaaaaatt tggtgtaagc tattttcttt gaagtactga 480
ggatacaact tcagagaaat ttgtccctcc cccctccccc tccgccgccg ccgcgccggt 540
aaccaccccg cccctctcct ctttctttct ccgttttttt tttccgtctc ggtctcgatc 600
tttggccttg gtagtttggg tgggcgagag gcggcttcgt gcgcgcccag atcggtgcgc 660
gggaggggcg ggatctcgcg gctggggctc tcgccggcgt ggatccggcc cggatctcgc 720
ggggaatggg gctctcggat gtagatctgc gatccgccgt tgttggggga gatgatgggg 780
ggtttaaaat ttccgccatg ctaaacaaga tcaggaagag gggaaaaggg cactatggtt 840
tatattttta tatatttctg ctgcttcgtc aggcttagat gtgctagatc tttctttctt 900
ctttttgtgg gtagaatttg aatccctcag cattgttcat cggtagtttt tcttttcatg 960
atttgtgaca aatgcagcct cgtgcggagc ttttttgtag gtagacgata tctccacc 1018
<210> 8
<211> 450
<212> DNA
<213> 人工序列
<220>
<223> terSbHSP
<400> 8
gcatccatgg acgtggattg aattgaaggt gtactactgc tgtgctggtc cgtggatcgt 60
ggctgtcatg catggtttgc tgtgtcttct acgatatgta cttccctttg ttccgtatat 120
gtacatcttc ctcgtttggt tcatgtattt tcctttgaat aataataaat aaatcgggct 180
ttccatatcg gatgctttta tatctgtgtg tatggagatt gtggtatatg gtttcatctc 240
aagttgttta cgtcaagaac taaagatatt tcctcaaaaa aaaagaacta aagatataat 300
caatgtcatt aacataactc atttccatga ggagaggacg aaggacgaag tcataataag 360
tagattggtt gatattttat aatcattcaa aactgcaggg gttataagat cttcattttg 420
tagaagtttt agatcttccg aggggttctc 450
<210> 9
<211> 3238
<212> DNA
<213> 人工序列
<220>
<223> 构建体GDI005
<400> 9
aaggtaatta tccaagatgt agcatcaaga atccaatgtt tacgggaaaa actatggaag 60
tattatgtga gctcagcaag aagcagatca atatgcggca catatgcaac ctatgttcaa 120
aaatgaagaa tgtacagata caagatccta tactgccaga atacgaagaa gaatacgtag 180
aaattgaaaa agaagaacca ggcgaagaaa agaatcttga agacgtaagc actgacgaca 240
acaatgaaaa gaagaagata aggtcggtga ttgtgaaaga gacatagagg acacatgtaa 300
ggtggaaaat gtaagggcgg aaagtaacct tatcacaaag gaatcttatc ccccactact 360
tatcctttta tatttttccg tgtcattttt gcccttgagt tttcctatat aaggaaccaa 420
gttcggcatt tgtgaaaaca agaaaaaatt tggtgtaagc tattttcttt gaagtactga 480
ggatacaact tcagagaaat ttgtccctcc cccctccccc tccgccgccg ccgcgccggt 540
aaccaccccg cccctctcct ctttctttct ccgttttttt tttccgtctc ggtctcgatc 600
tttggccttg gtagtttggg tgggcgagag gcggcttcgt gcgcgcccag atcggtgcgc 660
gggaggggcg ggatctcgcg gctggggctc tcgccggcgt ggatccggcc cggatctcgc 720
ggggaatggg gctctcggat gtagatctgc gatccgccgt tgttggggga gatgatgggg 780
ggtttaaaat ttccgccatg ctaaacaaga tcaggaagag gggaaaaggg cactatggtt 840
tatattttta tatatttctg ctgcttcgtc aggcttagat gtgctagatc tttctttctt 900
ctttttgtgg gtagaatttg aatccctcag cattgttcat cggtagtttt tcttttcatg 960
atttgtgaca aatgcagcct cgtgcggagc ttttttgtag gtagacgata tctccaccat 1020
gcaccaccac catcaccaca tcgatatgaa gaagttcaac acccctgctt accaggccga 1080
aaaagacttc aaacagtacc cccacctggg cgagcaactc gagaacgcgt ggagcaatta 1140
cgtgaagtac tgtacaatca actccattat ggggaatccg tggtcctcaa cctatgacca 1200
cccgcgctcg tggtactaca atccactcgt tgatgacgtg gtgcctactg aacagaatac 1260
cgtgccaatt cagtggaatg cattcccaaa ccgcatcaac cactacttca ctgggctgtt 1320
taccaagcag ttcggttcgg ctgagtacga ggataagctg cacgagcttg ccgatatcgg 1380
ccctgcggca ttcgggaaga agtataacat ggaccttaca gtgccgaaga acccatgtga 1440
cccgagcgat acacggacca aaccatttgg gccttcaggt ccacgcggtt ggcaagacga 1500
atattgcgag tgggcggtca cacgggacga gaacggggac attacagcgg tcgactttac 1560
ccacgaaaac cctgagtact ggttccatat gtggaagata tcaccagaca tcgtggtggc 1620
cttgtatcag gagatcctga acaacaagaa tgtgaagaag gaagacctct acttgctcga 1680
ctcgaccggt aaccccgtca tcgttcggga gacaggggaa ccggcataca accccatcaa 1740
caagtggaat aacggaccag aggccacacc agagggtggg ggtgcagttc atctcacctc 1800
accaccgaac tccctgggtg cagagatcta cctgggcgca gccgcgacca tactccgggt 1860
taagaataac caggtcatca ccgacgctaa cgccctcatc tgcgcggccc agtacggcca 1920
gatctaccgg aacagcgacc cacggatcgg gcagaatgtc aactcactcg tctacaatca 1980
taagcagcag atcacactaa ccaaccccat cgcgctgtat ggacaagtcc cagactttga 2040
tcagttcgag atgccaagca ccgcggggtc gtataagatt caggactgct acacggtcgt 2100
gagaggagag gaaaggaaca agggtatcac cttctacccc tttaacatgc tcctccacac 2160
aaggttttcg gttcccaagg gggcgaattt caagctcagc gagatcaagg tcaagggcaa 2220
gctcctgaag tgggggagcc aaatcgcgga caccttcttc gtgcagctgg cgggaaccgg 2280
caagagtccc ggtgccgggg aacagccgga aaagtttcct ccagtgggtg acccggcgac 2340
cacactgccc aacgtgcagt acctgctcga taataacctg ctgcaggcct ccctctacaa 2400
caaactgaac accttcagca atctgacctc gtgcatcacg caaatcgagg ccggcacatc 2460
gactgaaggt atcgcagtgt tgacaaacgc cgctaccaag gaaactcagt tcgacttcgg 2520
tccgggcatc agcgtgatgg tgacagactt ccagaacatc gatgaagaca cgcaactgtt 2580
tcttatcacc atcacagcag acgcggatac atcgctggga gagaagccac tttccctcta 2640
caacaatgca agcgatccaa agtacgccct gtccggcgtg ctggaagtgg tccccaacgg 2700
tagcctgccg aagattaaca ctacacccaa tctggcgctc ttgtccggac aacaggtgga 2760
gcaagtcaag aaaatcctta agtagaacgc atccatggac gtggattgaa ttgaaggtgt 2820
actactgctg tgctggtccg tggatcgtgg ctgtcatgca tggtttgctg tgtcttctac 2880
gatatgtact tccctttgtt ccgtatatgt acatcttcct cgtttggttc atgtattttc 2940
ctttgaataa taataaataa atcgggcttt ccatatcgga tgcttttata tctgtgtgta 3000
tggagattgt ggtatatggt ttcatctcaa gttgtttacg tcaagaacta aagatatttc 3060
ctcaaaaaaa aagaactaaa gatataatca atgtcattaa cataactcat ttccatgagg 3120
agaggacgaa ggacgaagtc ataataagta gattggttga tattttataa tcattcaaaa 3180
ctgcaggggt tataagatct tcattttgta gaagttttag atcttccgag gggttctc 3238
<210> 10
<211> 2899
<212> DNA
<213> 人工序列
<220>
<223> 构建体GDI006
<400> 10
aaggtaatta tccaagatgt agcatcaaga atccaatgtt tacgggaaaa actatggaag 60
tattatgtga gctcagcaag aagcagatca atatgcggca catatgcaac ctatgttcaa 120
aaatgaagaa tgtacagata caagatccta tactgccaga atacgaagaa gaatacgtag 180
aaattgaaaa agaagaacca ggcgaagaaa agaatcttga agacgtaagc actgacgaca 240
acaatgaaaa gaagaagata aggtcggtga ttgtgaaaga gacatagagg acacatgtaa 300
ggtggaaaat gtaagggcgg aaagtaacct tatcacaaag gaatcttatc ccccactact 360
tatcctttta tatttttccg tgtcattttt gcccttgagt tttcctatat aaggaaccaa 420
gttcggcatt tgtgaaaaca agaaaaaatt tggtgtaagc tattttcttt gaagtactga 480
ggatacaact tcagagaaat ttgtccctcc cccctccccc tccgccgccg ccgcgccggt 540
aaccaccccg cccctctcct ctttctttct ccgttttttt tttccgtctc ggtctcgatc 600
tttggccttg gtagtttggg tgggcgagag gcggcttcgt gcgcgcccag atcggtgcgc 660
gggaggggcg ggatctcgcg gctggggctc tcgccggcgt ggatccggcc cggatctcgc 720
ggggaatggg gctctcggat gtagatctgc gatccgccgt tgttggggga gatgatgggg 780
ggtttaaaat ttccgccatg ctaaacaaga tcaggaagag gggaaaaggg cactatggtt 840
tatattttta tatatttctg ctgcttcgtc aggcttagat gtgctagatc tttctttctt 900
ctttttgtgg gtagaatttg aatccctcag cattgttcat cggtagtttt tcttttcatg 960
atttgtgaca aatgcagcct cgtgcggagc ttttttgtag gtagacgata tctccaccat 1020
gcaccaccac catcaccaca tcgatatgaa gacgctggtc tttagcctgt tcatcctctc 1080
attcatctcg tgtgaatcag ggaaaaagga ccaacggctt aatagcgcaa ccggtttctc 1140
gaaggtggct gactccgaga gcatctactg tggagcgttt tgcgatcctc catcaatcac 1200
atacaaactg gcaggagaca gcacctgcgc acacagctcg caggacgttc tcaactgctt 1260
cgcgtggaag aacttcatcg cactgaattg ggcggcctcc gcgcagagag gtgtcccaga 1320
caccacagca acagcagcga actatggcat gccaggggac tattcgccga cagtctggga 1380
gtccttcgct tccaacgacg aggtgttcgc acccaagaac ctcctgacat ggaatctgaa 1440
gagtaagaac ggttacgtga aacagatcaa cgagattaac aagttcaccg atatcaacat 1500
cagcataccc aaggcaaccc tcagagcagc tgtcggcaac agcaatgtgg atgaaatcct 1560
gcaagcggag ggttcgtggc ttactgacca gtcgggcaac atcgtctggt acgagattaa 1620
gatcaataac atcgaatcgg acttcatcag acggaataag ctctacgact acaatagcct 1680
caaggagtac gggaccgcaa acaatggggt gtggctcccc atggagagca tagaactgaa 1740
agcggcatgg agagtgatcc cagaggacaa actggatagc ctgaaaaact actataagat 1800
ctccaaagcc atggtcccag agatcaaggg gttcaaggat aagaaacccg tgttcggaaa 1860
gtccacccag aagtacctgg gtctcgtggg gctgcacatt atccggaaga ccccacaaag 1920
cccacaattc aactggatga cattcgagca tatccacaac gccccaaatg aggggcaggc 1980
ggatcctagc gtccggtact gcttctacaa ccccaagagc acaaaaacac caaatatcgc 2040
gcctgtcatc ggaaaggatt cactgaacac accggtgcaa gtcgttcggg tcaataagat 2100
taaaaccaaa ctgcagaagc tgaacaccca aatgcagcag ctgattcggg cgagcaatcc 2160
caagagcgtg tggcagtatt accagctggt gaacatccag tggccagaga atccgatcca 2220
ggacaacggt aacaataaga gtgccccact catggaggga gggattaccc ccagcgatat 2280
ctccaatacc acgatggaga cctacgccca acagaagcag tgtatggact gtcacaagta 2340
cgcatcggtt gttggtagcg ggatgccacc taccgactat agcttcatct tcctgaaggt 2400
gaagccagaa aaacagattc caaaggggaa aaccccagtg aagtagaacg catccatgga 2460
cgtggattga attgaaggtg tactactgct gtgctggtcc gtggatcgtg gctgtcatgc 2520
atggtttgct gtgtcttcta cgatatgtac ttccctttgt tccgtatatg tacatcttcc 2580
tcgtttggtt catgtatttt cctttgaata ataataaata aatcgggctt tccatatcgg 2640
atgcttttat atctgtgtgt atggagattg tggtatatgg tttcatctca agttgtttac 2700
gtcaagaact aaagatattt cctcaaaaaa aaagaactaa agatataatc aatgtcatta 2760
acataactca tttccatgag gagaggacga aggacgaagt cataataagt agattggttg 2820
atattttata atcattcaaa actgcagggg ttataagatc ttcattttgt agaagtttta 2880
gatcttccga ggggttctc 2899
<210> 11
<211> 578
<212> PRT
<213> Chryseobacterium carnipullorum
<400> 11
Met Lys Lys Phe Asp Thr Pro Ala Tyr Gln Ala Glu Lys Asp Phe Lys
1 5 10 15
Asp Gln Pro Asp Leu Lys Ile Gln Ile Glu Asn Ala Trp Ser Asp Tyr
20 25 30
Val Lys Tyr Cys Thr Ile Asn Ser Gln Met Gly Asn Pro Trp Ser Ser
35 40 45
Ser Tyr Asp His Pro Arg Ser Trp Tyr Tyr Asn Pro Leu Val Thr Pro
50 55 60
Val Thr Pro Ile Lys Asn Asn Thr Val Pro Ile Gln Trp Gly Ala Phe
65 70 75 80
Pro Asn Arg Ile Asn His Tyr Phe Thr Asp Leu Phe Thr Gln Lys Phe
85 90 95
Gly Lys Ala Asp Ala Thr Asp Lys Leu His Glu Leu Ala Asp Ile Gly
100 105 110
Pro Asp Ala Phe Ser Lys Lys Tyr Asp Ile Thr Leu Thr Val Thr Lys
115 120 125
Asn Pro Cys Asp Pro Asn Asn Thr Ala Thr Lys Pro Phe Gly Pro Ser
130 135 140
Gly Pro Arg Gly Trp Gln Asp Glu Tyr Cys Glu Trp Ala Val Thr Arg
145 150 155 160
Asp Thr Asn Gly Asp Ile Ile Ala Val Asp Phe Thr His Glu Asn Pro
165 170 175
Glu Tyr Trp Phe His Met Trp Lys Val Ser Pro Asp Met Val Val Ser
180 185 190
Leu Tyr Gln Glu Ile Leu Asn Asn Ser Asn Val Lys Lys Glu Asp Leu
195 200 205
Tyr Leu Leu Asp Glu Gln Asn Asn Pro Val Ile Val Arg Glu Thr Gly
210 215 220
Leu Pro Ala Tyr Asn Pro Ile Asn Lys Trp Asn Asn Gly Ser Ser Ala
225 230 235 240
Thr Thr Ala Gly Gly Gly Ala Val His Leu Thr Ser Pro Pro Asn Ser
245 250 255
Leu Gly Ala Glu Ile Tyr Leu Gly Ala Ala Ala Thr Ile Leu Arg Ala
260 265 270
Val Asn Gly Lys Val Ile Thr Asp Ala Asn Ala Leu Ile Cys Ala Ala
275 280 285
Gln Tyr Gly Gln Ile Tyr Arg Asn Ser Asp Pro Arg Ile Gly Gln Asn
290 295 300
Val Asn Ser Leu Val Tyr His Asn Lys Val Lys Ile Ser Leu Thr Asn
305 310 315 320
Pro Ile Ala Leu Tyr Gly Gln Leu Pro Asp Phe Thr Gln Phe Thr Met
325 330 335
Pro Ala Ser Ala Glu Gly Tyr Lys Ile Glu Asp Cys Tyr Lys Ile Ile
340 345 350
Arg Gly Arg Asp Ile Asn Pro Gly Thr Thr Phe Tyr Pro Asn Asn Met
355 360 365
Val Leu His Ser Arg Phe Glu Val Pro Ala Gly Ala Asn Phe Lys Leu
370 375 380
Ser Glu Ile Leu Val Gln Asn Gln Pro Leu Lys Trp Gly Ser Gln Ile
385 390 395 400
Ala Asp Val Phe Lys Val Gln Leu Ala Gly Thr Gly Ile Pro Gly Gly
405 410 415
Gly Asp Lys Pro Gln Glu Tyr Pro Pro Val Gly Asp Pro Asp Val Ala
420 425 430
Leu Pro Ser Val Gln Tyr Leu Leu Asp Asn Arg Leu Leu Gln Ala Ser
435 440 445
Leu Tyr Asn Lys Leu Asn Thr Phe Ser Asn Leu Thr Ser Cys Ile Thr
450 455 460
Gln Ile Glu Ala Gly Thr Thr Thr Lys Gly Ile Ala Val Leu Ala Ser
465 470 475 480
Asp Ala Asn Gln Lys Thr Gly Phe Asp Phe Gly Ala Gly Ile Ile Ala
485 490 495
Ser Ile Thr Asp Phe Gln Asp Leu Gly Asn Asp Asn Gln Leu Phe Ile
500 505 510
Ile Asp Ile Thr Val Gln Ser Asp Ala Val Leu Gly Glu Lys Pro Leu
515 520 525
Ala Leu Tyr Asn Ser Ser Ser Asp Pro Lys Tyr Thr Ile Ser Gly Val
530 535 540
Leu Glu Val Val Ala Ala Gly Ser Leu Pro Lys Leu Asn Met Met Pro
545 550 555 560
Asn His Thr Leu Leu Ser Asp Gln Gln Ile Gln Gln Val Gln Lys Ile
565 570 575
Leu Lys
<210> 12
<211> 578
<212> PRT
<213> Chryseobacterium shigense
<400> 12
Met Lys Lys Phe Asp Thr Pro Ala Tyr Gln Ala Glu Lys Asp Phe Lys
1 5 10 15
Asp Gln Pro Asn Leu Lys Ile Gln Ile Glu Asn Ala Trp Ser Asn Tyr
20 25 30
Val Lys Tyr Cys Thr Ile Asn Ser Gln Met Gly Asn Pro Trp Ser Ser
35 40 45
Ser Tyr Asp His Pro Arg Ser Trp Tyr Tyr Asn Pro Leu Val Thr Pro
50 55 60
Val Thr Pro Ile Lys Asn Asn Thr Val Pro Ile Gln Trp Gly Ala Phe
65 70 75 80
Pro Asn Arg Ile Asn His Tyr Phe Thr Asp Leu Phe Thr Gln Lys Phe
85 90 95
Gly Lys Ala Asp Ala Thr Asp Lys Leu His Glu Leu Ala Asp Ile Gly
100 105 110
Pro Asp Ala Phe Ser Lys Lys Tyr Asp Ile Thr Leu Thr Val Thr Lys
115 120 125
Asn Pro Cys Asp Pro Asp Asn Thr Ala Thr Lys Pro Phe Gly Pro Ser
130 135 140
Gly Pro Arg Gly Trp Gln Asp Glu Tyr Cys Glu Trp Ala Val Thr Arg
145 150 155 160
Asp Thr Asn Gly Gly Ile Ile Ala Val Asp Phe Thr His Glu Asn Pro
165 170 175
Glu Tyr Trp Phe His Met Trp Lys Val Ser Gln Asp Met Val Val Ser
180 185 190
Leu Tyr Gln Gln Ile Leu Asn Asn Ser Asn Val Lys Lys Glu Asp Leu
195 200 205
Tyr Leu Leu Asp Glu His His Asn Pro Val Ile Val Arg Glu Thr Gly
210 215 220
Leu Pro Ala Tyr Asn Pro Ile Asn Lys Trp Asn Lys Gly Ser Ser Ala
225 230 235 240
Thr Ala Glu Gly Gly Gly Ala Val His Leu Thr Ser Pro Pro Asn Ser
245 250 255
Leu Gly Ala Glu Ile Tyr Leu Gly Ala Ala Ala Thr Ile Leu Arg Val
260 265 270
Val Asn Gly Lys Val Ile Thr Asp Ala Asn Thr Leu Ile Cys Ala Ala
275 280 285
Gln Tyr Gly Gln Ile Tyr Arg Asn Ser Asp Pro Arg Ile Gly Gln Asn
290 295 300
Val Asn Ser Leu Val Tyr Asn Asn Lys Leu Lys Ile Ser Leu Thr Asn
305 310 315 320
Pro Ile Ala Leu Tyr Gly Gln Leu Pro Asp Phe Thr Gln Phe Thr Met
325 330 335
Pro Ala Ser Ala Glu Gly Tyr Lys Ile Glu Asp Cys Tyr Lys Ile Ile
340 345 350
Arg Gly Thr Asp Ile Asn Pro Gly Thr Thr Phe Tyr Pro Ser Asn Met
355 360 365
Ile Leu His Ser Arg Phe Glu Val Pro Ala Gly Ala Asn Phe Lys Leu
370 375 380
Ser Glu Ile Leu Val Gln Asn Gln Pro Leu Lys Trp Gly Ser Gln Ile
385 390 395 400
Ala Asp Val Phe Lys Val Gln Leu Ala Gly Thr Gly Ile Pro Thr Ser
405 410 415
Gly Glu Lys Pro Gln Glu Tyr Pro Pro Val Gly Asp Pro Asp Val Thr
420 425 430
Leu Pro Ser Val Gln Tyr Leu Leu Asp Asn Gly Leu Leu Gln Ala Ser
435 440 445
Leu Tyr Asn Lys Leu Asn Thr Phe Ser Asn Leu Thr Ser Cys Ile Thr
450 455 460
Gln Ile Glu Ala Gly Thr Thr Thr Arg Gly Ile Ala Val Leu Ala Ser
465 470 475 480
Asp Ala Asn Gln Lys Thr Gly Phe Asp Phe Gly Ala Gly Ile Asn Ala
485 490 495
Ser Val Thr Asp Phe Gln Asp Leu Gly Asn Asp Asn Gln Leu Phe Ile
500 505 510
Ile Asp Ile Thr Ala Glu Ala Asp Ala Val Leu Gly Glu Lys Pro Leu
515 520 525
Ala Leu Tyr Asn Asn Ala Ser Asp Ala Arg Tyr Thr Leu Ser Gly Val
530 535 540
Leu Glu Val Val Ala Pro Gly Ser Leu Pro Lys Leu Asn Ile Ala Ala
545 550 555 560
Asn His Thr Leu Leu Ser Asp Gln Gln Ile Gln Gln Val Gln Lys Ile
565 570 575
Leu Lys
<210> 13
<211> 577
<212> PRT
<213> 金黄杆菌属种
<400> 13
Met Lys Gln Phe Asp Thr Pro Ala Tyr Gln Ala Asp Lys Asp Phe Lys
1 5 10 15
Asp Gln Pro Asp Leu Arg Asn Gln Ile Glu Asn Ala Trp Ser Asn Tyr
20 25 30
Val Lys Tyr Cys Thr Ile Asn Ser Gln Met Gly Asn Pro Trp Ser Ser
35 40 45
Ser Tyr Asp His Pro Arg Ser Trp Tyr Tyr Asn Pro Leu Val Thr Pro
50 55 60
Val Thr Pro Asn Lys Asn Asn Thr Val Pro Ile Gln Trp Gly Ala Phe
65 70 75 80
Pro Asn Arg Ile Asn His Tyr Phe Thr Ser Leu Phe Thr Lys Val Phe
85 90 95
Pro Asn Glu Ala Gln Asp Lys Leu His Glu Leu Ala Asp Ile Gly Pro
100 105 110
Lys Ala Phe Thr Glu Lys Tyr Gly Thr Gln Leu Val Val Pro Lys Asn
115 120 125
Pro Cys Asp Pro Thr Asn Thr Asp Thr Lys Ala Phe Gly Pro Ser Gly
130 135 140
Pro Arg Gly Trp Gln Asp Glu Tyr Cys Glu Trp Ser Val Thr Arg Asp
145 150 155 160
Thr Asn Gly Asp Ile Ile Ala Val Asn Phe Thr His Glu Asn Pro Glu
165 170 175
Tyr Trp Phe His Met Trp Lys Val Ser Gln Asp Met Val Val Ser Leu
180 185 190
Tyr Gln Glu Ile Leu Asn Asn Pro Asn Val Gln Lys Glu Asp Leu Tyr
195 200 205
Leu Leu Asp Glu Asn Gly Asn Pro Val Ile Val Arg Glu Thr Gly Leu
210 215 220
Pro Ala Tyr Asn Pro Ile Asn Lys Trp Asn Asn Gly Ser Ser Ala Thr
225 230 235 240
Ala Glu Gly Gly Gly Ala Val His Leu Thr Ser Pro Pro Asn Ser Leu
245 250 255
Gly Ala Glu Ile Tyr Leu Gly Ala Ala Ala Thr Ile Leu Arg Val Val
260 265 270
Gly Gly Lys Val Ile Thr Asp Ala Asn Thr Leu Ile Cys Ala Ala Gln
275 280 285
Tyr Gly Gln Ile Tyr Arg Asn Ser Asp Pro Arg Ile Gly Gln Asn Val
290 295 300
Asn Ala Leu Val Tyr Asn Lys Lys Leu Lys Ile Ser Leu Thr Asn Pro
305 310 315 320
Ile Ala Leu Tyr Gly Gln Met Pro Asp Phe Thr Gln Phe Ala Met Pro
325 330 335
Asp Ser Ala Glu Gly Tyr Thr Ile Glu Asp Cys Tyr Lys Ile Ile Arg
340 345 350
Gly Thr Ala Thr Asn Pro Gly Thr Asp Phe Tyr Pro Phe Asn Met Ile
355 360 365
Leu His Ser Arg Phe Glu Val Pro Ala Gly Ala Gln Phe Lys Leu Ser
370 375 380
Asp Ile Lys Val Gln Gly Gln Pro Leu Lys Trp Gly Ser Gln Ile Ala
385 390 395 400
Asp Val Phe Lys Val Gln Leu Ala Gly Thr Gly Ile Pro Gly Gly Ser
405 410 415
Asp Lys Pro Gln Glu Tyr Pro Pro Val Gly Asp Pro Asp Val Thr Leu
420 425 430
Pro Ser Val Gln Tyr Leu Leu Asp Asn Asn Leu Leu Gln Ala Ser Leu
435 440 445
Tyr Asn Lys Leu Asn Thr Phe Ser Asn Leu Thr Ser Cys Ile Thr Gln
450 455 460
Ile Glu Ala Gly Thr Thr Thr Ser Gly Ile Ala Val Leu Ala Ser Asp
465 470 475 480
Ala Asn Lys Glu Thr Gly Phe Asp Phe Gly Pro Gly Ile Ser Val Ser
485 490 495
Val Thr Asp Tyr Gln Asp Leu Gly Asn Asp Asn Gln Leu Phe Met Ile
500 505 510
Asp Ile Thr Val His Ala Ala Ala Ser Leu Gly Glu Lys Pro Leu Ala
515 520 525
Leu Tyr Asn Asn Thr Ser Asp Pro Lys Tyr Thr Val Ser Gly Val Leu
530 535 540
Glu Val Val Ala Pro Gly Ser Leu Pro Lys Leu Asn Ile Thr Pro Asn
545 550 555 560
Gln Thr Leu Leu Ser Asp Gln Gln Ile Lys Gln Val Gln Lys Ile Leu
565 570 575
Lys
<210> 14
<211> 578
<212> PRT
<213> 金黄杆菌属种
<400> 14
Met Asn Lys Phe Asn Thr Pro Ala Tyr Gln Ala Glu Lys Asp Phe Lys
1 5 10 15
Asp Gln Pro Asp Leu Lys Asn Lys Ile Glu Asn Ala Trp Ser Asn Tyr
20 25 30
Val Lys Tyr Cys Thr Ile Asn Ser Gln Met Gly Asn Pro Trp Ser Ser
35 40 45
Ser Tyr Asp His Pro Arg Ser Trp Tyr Tyr Asn Pro Leu Val Thr Pro
50 55 60
Ala Ile Pro Ala Lys Asn Asn Thr Val Pro Ile Gln Trp Gly Ala Phe
65 70 75 80
Pro Asn Arg Ile Asn His Tyr Phe Ser Asp Leu Phe Ala Gln Lys Phe
85 90 95
Gly Lys Gly Glu Ala Gln Asp Lys Leu Tyr Glu Leu Ala Asp Ile Gly
100 105 110
Ser Glu Ala Phe Ser Lys Lys Tyr Ala Ile Glu Leu Thr Val Pro Lys
115 120 125
Asn Pro Cys Asp Pro Asn Asn Thr Ala Lys Lys Pro Phe Gly Pro Ala
130 135 140
Gly Pro Arg Gly Trp Gln Asp Glu Tyr Cys Glu Trp Ala Val Thr Arg
145 150 155 160
Asp Ala Ser Gly Asp Ile Ile Ala Val Asn Phe Thr His Glu Asn Pro
165 170 175
Glu Tyr Trp Phe His Met Trp Lys Phe Ser Pro Asp Thr Val Val Ser
180 185 190
Leu Tyr Gln Gln Ile Leu Asn Asn Pro Asn Val Lys Lys Glu Asp Leu
195 200 205
Tyr Leu Leu Asp Ser Ser Asn Asn Pro Val Ile Val Arg Glu Thr Gly
210 215 220
Leu Pro Ala Tyr Asn Pro Ile Asn Lys Trp Asn Arg Gly Ser Ser Ala
225 230 235 240
Thr Glu Thr Glu Gly Gly Ala Val His Leu Thr Ser Pro Pro Asn Ser
245 250 255
Leu Gly Ala Glu Ile Tyr Leu Gly Ala Ala Ala Thr Ile Leu Arg Val
260 265 270
Val Asp Gly Asn Val Ile Thr Asp Ala Asn Thr Leu Val Cys Ala Ala
275 280 285
Gln Tyr Gly Gln Ile Tyr Arg Asn Ser Asp Pro Arg Ile Gly Gln Asn
290 295 300
Val Asn Ser Leu Val Tyr Ile Asn Lys Leu Lys Val Ser Leu Thr Asn
305 310 315 320
Pro Ile Ala Leu Tyr Gly Gln Leu Pro Asp Phe Thr Gln Phe Gln Met
325 330 335
Pro Asp Ser Ala Glu Gly Tyr Thr Ile Glu Asp Cys Tyr Lys Ile Ile
340 345 350
Arg Gly Thr Asp Ile Asn Pro Gly Thr Thr Phe Tyr Pro Asn Asn Met
355 360 365
Ile Leu His Ser Arg Phe Glu Ala Pro Ala Gly Ala Arg Phe Lys Leu
370 375 380
Ser Asp Ile Leu Val Gln Gly Gln Pro Leu Lys Trp Gly Ser Gln Ile
385 390 395 400
Ala Asn Val Phe Asn Val Gln Leu Ala Gly Thr Gly Ile Pro Gly Gly
405 410 415
Gly Asp Arg Pro Gln Glu Tyr Pro Pro Val Gly Asn Pro Ala Val Thr
420 425 430
Leu Pro Ser Ile Gln Tyr Val Leu Asp His Asn Leu Leu Leu Ala Ser
435 440 445
Leu His Asn Lys Leu Asn Thr Leu Ser Asn Leu Thr Ser Cys Thr Thr
450 455 460
Gln Val Glu Ala Gly Thr Thr Thr Lys Gly Ile Ala Val Leu Ala Ser
465 470 475 480
Asp Ala Asn Arg Glu Thr Gly Phe Asp Phe Gly Ala Gly Ile Ser Val
485 490 495
Ser Val Lys Asp Phe Gln Asp Leu Gly Asn Asp Asn Gln Leu Phe Ile
500 505 510
Ile Asp Ile Thr Val Asp Ala Ala Ala Leu Leu Gly Glu Lys Pro Leu
515 520 525
Ala Leu Tyr Asn Asn Thr Ser Asp Pro Lys Tyr Asn Val Ser Gly Val
530 535 540
Leu Glu Val Val Ala Thr Gly Ser Leu Pro Gln Leu Asn Ser Leu Pro
545 550 555 560
Asn Gln Thr Leu Leu Ser Asp Gln Gln Ile Thr Gln Val Gln Lys Leu
565 570 575
Leu Lys
<210> 15
<211> 579
<212> PRT
<213> Chryseobacterium kwangjuense
<400> 15
Met Thr Lys Phe Asp Thr Pro Ala Tyr Gln Ala Glu Lys Asp Phe Lys
1 5 10 15
Asn Asp Pro Asp Leu Lys Ser Gln Ile Glu Asn Ala Trp Ser Asn Tyr
20 25 30
Val Lys Tyr Cys Thr Ile Asn Ser Gln Met Gly Asn Pro Trp Ser Ser
35 40 45
Ser Tyr Asp His Pro Arg Ser Trp Tyr Tyr Asn Pro Leu Val Thr Pro
50 55 60
Ala Glu Pro Ala Lys Asn Asn Thr Val Ala Val Gln Trp Gly Ala Phe
65 70 75 80
Pro Asn Arg Ile Asn His Tyr Phe Thr Asn Leu Phe Val Glu Lys Phe
85 90 95
Gly Lys Thr Asp Ala Pro Asp Lys Leu His Glu Leu Ala Asp Ile Gly
100 105 110
Pro Asp Ala Phe Ser Lys Lys Tyr Asn Ile Thr Leu Val Val Ser Lys
115 120 125
Asn Pro Cys Asp Pro Ser Asn Thr Glu Thr Lys Pro Phe Gly Pro Ser
130 135 140
Gly Pro Arg Gly Trp Gln Asp Glu Tyr Cys Glu Trp Ala Val Thr Arg
145 150 155 160
Asp Ala Ser Gly Asp Ile Ile Ala Val Asp Phe Thr His Glu Asn Pro
165 170 175
Glu Tyr Trp Phe His Met Trp Lys Val Ser Pro Asp Met Val Val Ser
180 185 190
Leu Tyr Gln Gln Ile Leu Asn Asn Pro Asn Val Lys Lys Glu Asp Leu
195 200 205
Tyr Leu Leu Asp Glu Lys Gly Asn Tyr Val Ile Val Arg Glu Thr Gly
210 215 220
Leu Pro Ala Tyr Asn Pro Ile Asn Lys Trp Asn Arg Gly Ser Ser Ala
225 230 235 240
Thr Ala Gly Gly Gly Gly Thr Val His Leu Thr Ser Pro Pro Asn Ser
245 250 255
Leu Gly Ala Glu Ile Tyr Leu Gly Ala Ala Ala Thr Ile Leu Arg Val
260 265 270
Asn Ser Gln Gly Arg Val Ile Thr Asp Ala Asn Glu Leu Ile Cys Ala
275 280 285
Ala Gln Tyr Gly Gln Ile Tyr Arg Asn Ser Asp Pro Arg Ile Gly Gln
290 295 300
Asn Val Asn Ser Leu Val Tyr Asn Lys Lys Leu Lys Ile Ser Leu Thr
305 310 315 320
Asn Pro Ile Ala Leu Tyr Gly Gln Gln Pro Asp Phe Thr Gln Phe Thr
325 330 335
Met Pro Asp Ser Ala Lys Gly Tyr Lys Ile Glu Asp Cys Tyr Lys Val
340 345 350
Ile Arg Gly Ser Glu Ser Asn Pro Gly Thr Thr Phe Tyr Pro Phe Asn
355 360 365
Met Ile Leu His Ser Arg Phe Glu Ala Pro Ala Gly Ala Lys Phe Lys
370 375 380
Leu Ser Asp Ile Met Val Lys Gly Ser Lys Ile Lys Trp Gly Ser Gln
385 390 395 400
Ile Ala Asp Val Phe Lys Val Gln Leu Ala Gly Thr Gly Ile Pro Gly
405 410 415
Gly Gly Asp Gln Pro Gln Gln Tyr Pro Ala Val Gly Asp Pro Ala Val
420 425 430
Thr Leu Pro Ser Val Gln Tyr Val Leu Asp Asn Asn Leu Leu Gln Ala
435 440 445
Ser Leu Tyr Asn Lys Leu Asn Thr Phe Ser Asn Leu Thr Ser Cys Ile
450 455 460
Thr Gln Val Glu Ala Gly Thr Ile Thr Ser Gly Ile Ala Ile Leu Ala
465 470 475 480
Ser Asp Ala Asn Lys Gly Thr Gly Phe Asp Phe Gly Pro Gly Ile Ser
485 490 495
Val Ala Val Thr Asp Phe Gln Asp Leu Gly Asn Asp Asn Gln Leu Phe
500 505 510
Ile Val Asp Ile Asn Val His Ser Ala Ala Leu Leu Gly Glu Lys Pro
515 520 525
Leu Ala Leu Phe Asn Asn Thr Thr Asp Pro Lys Tyr Thr Ile Ala Gly
530 535 540
Val Leu Glu Val Val Ala Pro Gly Ser Leu Pro Lys Leu Asp Val Val
545 550 555 560
Pro Asn His Ser Leu Leu Ser Asp Gln Gln Val Ser Gln Val Gln Lys
565 570 575
Met Leu Lys
<210> 16
<211> 578
<212> PRT
<213> 金黄杆菌属种OV705
<400> 16
Met Lys Thr Phe Asp Thr Pro Ala Tyr Gln Ala Glu Lys Asp Phe Lys
1 5 10 15
Asp Asn Pro Ala Leu Arg Glu Lys Leu His Asn Ala Trp Ser Asn Tyr
20 25 30
Val Lys Tyr Cys Thr Val Asn Ser Ile Met Gly Asn Pro Trp Ser Ser
35 40 45
Thr Tyr Asp His Pro Arg Ser Trp Tyr Tyr Asn Pro Leu Val Thr Pro
50 55 60
Ser Ile Pro Asn Glu Asn Asn Thr Val Pro Ile Gln Trp Asn Ala Phe
65 70 75 80
Pro Asn Arg Ile Asn His Tyr Phe Thr Thr Leu Phe Thr Asp Lys Phe
85 90 95
Gly Lys Gln Asp Tyr Glu Asp Lys Leu His Glu Leu Ala Asp Ile Gly
100 105 110
Pro Ile Ala Phe Gly Gln Lys Tyr Asn Met Lys Leu Thr Val Pro Arg
115 120 125
Asn Pro Cys Asp Pro Thr Asp Thr Gly Thr Lys Ala Phe Gly Pro Ser
130 135 140
Gly Pro Arg Gly Trp Gln Asp Glu Tyr Cys Glu Trp Ser Val Thr Arg
145 150 155 160
Asp Glu Ser Gly Asp Ile Ile Ala Val Asn Phe Thr His Glu Asn Pro
165 170 175
Glu Tyr Trp Phe His Met Trp Lys Ile Ser Pro Asp Thr Val Val Ser
180 185 190
Leu Tyr Gln Glu Ile Leu Asn Asn Glu Asn Val Gln Lys Glu Asp Leu
195 200 205
Tyr Leu Leu Asp Ser His Gly Asn Pro Val Ile Val Arg Glu Thr Gly
210 215 220
Leu Pro Ala Tyr Asn Pro Ile Asn Lys Trp Asn Asn Gly Pro Asp Ala
225 230 235 240
Thr Ser Ser Gly Gly Gly Ala Val His Leu Thr Ser Pro Pro Asn Ser
245 250 255
Leu Gly Ala Glu Ile Tyr Leu Gly Ala Ala Ala Thr Ile Leu Arg Val
260 265 270
Val Asn Gly Lys Val Ile Thr Asp Ala Asn Thr Leu Ile Cys Ala Ala
275 280 285
Gln Tyr Gly Gln Ile Tyr Arg Asn Ser Asp Pro Arg Ile Gly Gln Asn
290 295 300
Val Asn Ser Leu Val Tyr Asn His Asn Val Gln Val Ser Leu Thr Asn
305 310 315 320
Pro Ile Ala Leu Tyr Gly Gln Ile Pro His Phe Asp Gln Phe Glu Met
325 330 335
Pro Ala Thr Ala Asn Tyr Lys Ile Glu Asp Cys Tyr Thr Val Val Arg
340 345 350
Gly Ala Leu Lys Asn Lys Gly Ile Thr Tyr Tyr Pro Asn Asn Met Leu
355 360 365
Leu His Thr Arg Phe Ser Val Pro Ala Asp Ala Asn Phe Lys Leu Ser
370 375 380
Asp Ile Leu Val Asn Lys Lys Pro Leu Lys Trp Gly Ser Gln Ile Ala
385 390 395 400
Asp Thr Phe Phe Val Gln Leu Ala Gly Thr Gly Leu Ser Pro Ala Gln
405 410 415
Gly Gln Gln Ser Glu Lys Phe Pro Pro Val Gly Ile Pro Ala Thr Thr
420 425 430
Leu Pro Ser Val Gln Tyr Leu Leu Asp Asn Asn Leu Leu Gln Ala Ser
435 440 445
Leu Tyr Asn Lys Leu Asn Thr Phe Ser Asn Leu Thr Ser Cys Ile Thr
450 455 460
Gln Val Glu Ala Gly Thr Thr Thr Glu Gly Ile Ala Val Leu Ala Asn
465 470 475 480
Gly Ala Ile Gln Gln Thr Gly Phe Asp Phe Gly Pro Gly Val Thr Val
485 490 495
Ala Val Thr Asp Phe Gln Asn Leu Asp Glu Asp Thr Gln Leu Phe Leu
500 505 510
Ile Ser Ile Thr Thr Asp Gly Gly Val Ala Leu Gly Glu Lys Pro Leu
515 520 525
Thr Leu Tyr Asn Asn Ala Ser Asp Pro Gly Phe Ala Leu Ser Gly Val
530 535 540
Leu Glu Val Val Ala Ala Gly Ser Leu Pro Lys Thr Asp Ser Thr Pro
545 550 555 560
Asn Arg Thr Leu Leu Ser Ser Gln Gln Ile Glu Gln Val Lys Lys Ile
565 570 575
Leu Lys
<210> 17
<211> 579
<212> PRT
<213> 产吲哚金黄杆菌
<400> 17
Met Lys Lys Phe Asn Thr Pro Ala Tyr Gln Ala Glu Lys Asp Phe Lys
1 5 10 15
His Glu Pro His Leu Gly Ala Gln Leu Glu Asn Ala Trp Ser Asn Tyr
20 25 30
Val Gln Tyr Cys Thr Ile Asn Ser Ile Met Gly Asn Pro Trp Ser Ser
35 40 45
Thr Tyr Asp His Pro Arg Ser Trp Tyr Tyr Asn Pro Leu Val Asp Pro
50 55 60
Val Ile Pro Thr Glu Lys Asn Thr Val Ala Ile Gln Trp Asn Ala Phe
65 70 75 80
Pro Asn Arg Ile Asn His Tyr Phe Thr Asn Leu Phe Thr Gln Lys Phe
85 90 95
Gly Lys Asp Gln Phe Asp Asp Lys Leu His Glu Leu Ala Asp Ile Gly
100 105 110
Pro Ala Ala Phe Gly Lys Lys Tyr Asp Met Val Leu Thr Val Pro Lys
115 120 125
Asn Pro Cys Asp Pro Ser Asp Thr Gly Thr Lys Pro Phe Gly Pro Ser
130 135 140
Gly Pro Arg Gly Trp Gln Asp Glu Tyr Cys Glu Trp Ala Val Thr Arg
145 150 155 160
Asp Glu Asn Gly Asp Ile Ile Ala Val Asp Phe Thr His Glu Asn Pro
165 170 175
Glu Tyr Trp Phe His Met Trp Lys Val Ser Pro Asp Ile Val Val Ser
180 185 190
Leu Tyr Gln Glu Ile Leu Asn Asn Lys Asn Val Lys Lys Glu Asp Leu
195 200 205
Tyr Leu Leu Asp Ser Asn Gly Asn Pro Val Ile Val Arg Glu Thr Gly
210 215 220
Glu Pro Ala Tyr Asn Pro Ile Asn Lys Trp Asn Arg Gly Pro Val Ala
225 230 235 240
Thr Pro Glu Gly Gly Gly Ala Val His Leu Thr Ser Pro Pro Asn Ser
245 250 255
Leu Gly Ala Glu Ile Tyr Leu Gly Ala Ala Ala Thr Ile Leu Arg Val
260 265 270
Lys Asp Asn Gln Val Ile Thr Asp Ala Asn Ala Leu Ile Cys Ala Ala
275 280 285
Gln Tyr Gly Gln Ile Tyr Arg Asn Ser Asp Pro Arg Ile Gly Gln Asn
290 295 300
Val Asn Ser Leu Val Tyr Asn Tyr Asn Gln Lys Ile Thr Leu Thr Asn
305 310 315 320
Pro Ile Ala Leu Tyr Gly Gln Val Pro Asp Phe Asp Gln Phe Asp Met
325 330 335
Pro Ser Thr Ala Gly Asn Tyr Thr Ile Glu Asp Cys Tyr Thr Val Val
340 345 350
Arg Gly Glu Glu Arg Asn Asn Gly Ile Thr Phe Tyr Pro Phe Asn Met
355 360 365
Leu Leu His Thr Arg Phe Ser Val Pro Glu Gly Ala Asn Phe Lys Leu
370 375 380
Ser Asp Ile Lys Val Lys Gly Lys Leu Leu Lys Trp Gly Ser Gln Ile
385 390 395 400
Ala Asp Thr Phe Phe Val Gln Leu Ala Gly Thr Gly Lys Asp Pro Ala
405 410 415
Ala Gly Glu Gln Pro Glu Lys Phe Pro Pro Val Gly Asp Pro Ala Thr
420 425 430
Ile Leu Pro Asn Val Gln Tyr Leu Leu Asp Asn Asn Leu Leu Gln Ala
435 440 445
Ser Leu Tyr Asn Lys Leu Asn Thr Phe Ser Asn Leu Thr Ser Cys Ile
450 455 460
Thr Gln Ile Glu Ala Gly Thr Ala Thr Glu Gly Ile Ala Val Leu Thr
465 470 475 480
Asn Gly Ala Val Arg Asp Thr His Phe Asp Phe Gly Pro Gly Ile Ser
485 490 495
Val Thr Val Thr Asp Phe Gln Asn Val Asp Glu Asp Thr Gln Leu Phe
500 505 510
Leu Ile Thr Ile Glu Thr Asp Ala Asn Val Ser Leu Gly Glu Lys Pro
515 520 525
Leu Ser Leu Tyr Asn Asn Ala Ser Asp Pro Lys Tyr Ala Leu Ser Gly
530 535 540
Val Leu Glu Val Val Ala Ser Gly Ser Leu Pro Lys Val Asn Ile Thr
545 550 555 560
Pro Asn Arg Thr Leu Leu Ser Gly Gln Gln Val Glu Gln Val Gln Lys
565 570 575
Ile Leu Lys
<210> 18
<211> 457
<212> PRT
<213> Chryseobacterium carnipullorum
<400> 18
Met Arg Thr Leu Leu Phe Ser Phe Phe Leu Leu Ser Leu Ile Ser Ile
1 5 10 15
Ser Cys Glu Ser Gly Lys Lys Asp Lys Lys Leu Asn Ser Arg Leu Gly
20 25 30
Phe Ser Lys Glu Met Ala Asp Ile Asp Cys Gly Ala Phe Cys Asp Pro
35 40 45
Pro Thr Val Gly Tyr Glu Leu Pro Ala Asp Ser Thr Cys Gly His Ser
50 55 60
Ser Gln Ser Val Leu Asn Cys Phe Ala Trp Lys Asn Phe Leu Ala Leu
65 70 75 80
Asn Trp Arg Ala Ser Asp Glu Arg Gly Leu Pro Asp Thr Thr Ala Val
85 90 95
Ala Ala Asp Tyr Gly Met Pro Gly Asp Tyr Ser Pro Thr Val Trp Glu
100 105 110
Ser Tyr Leu Ser Ala Asp Asp Val Phe Ala Ala Lys Gln Pro Glu Gln
115 120 125
Trp Asn Leu Lys Ser Lys Asn Gly Tyr Ile Lys Tyr Ile Asn Glu Ile
130 135 140
Asn Lys Phe Thr Asp Ile Asn Ala Ser Leu Pro Lys Pro Lys Leu Arg
145 150 155 160
Ala Met Leu Gly Gly Asn Val Asp Glu Ile Met Gln Ala Lys Gly Ala
165 170 175
Trp Leu Thr Asp Gln Ser Gly Asn Ile Val Trp Tyr Glu Ile Arg Met
180 185 190
Asn Ser Ile Glu Ser Asp Phe Ile Arg Lys Asn Lys Leu Tyr Ser Ser
195 200 205
Glu Asn Leu Asn Ala Phe Ala Ala Lys Asn Gln Gly Val Trp Leu Pro
210 215 220
Met Glu Ser Ile Glu Ile Lys Ala Ala Trp Arg Val Ile Pro Glu Asn
225 230 235 240
Gln Leu Glu Ser Leu Lys Asn Phe Tyr Lys Ile Ser Lys Ala Met Val
245 250 255
Pro Glu Ile Lys Gly Phe Asp Lys Asn Asn Gln Pro Ile Tyr Gly Lys
260 265 270
Tyr Thr Gln Lys Tyr Leu Gly Leu Val Gly Leu His Ile Ile Arg Lys
275 280 285
Thr Asn Gln Ser Pro Gln Phe Thr Trp Met Thr Phe Glu His Val Asn
290 295 300
Asn Ala Pro Thr Glu Gly Gln Val Asp Pro Ser Ile Lys Tyr Cys Phe
305 310 315 320
Tyr Asn Pro Lys Ser Thr Asp Lys Pro Asn Gln Ser Pro Val Pro Gly
325 330 335
Lys Asp Ser Leu Ser Lys Pro Val Gln Val Ile Arg Ile Ala Asn Asn
340 345 350
Ala Ile Thr Pro Glu Ile Gln Asn Leu Asn Lys Gln Ile Arg Asp Met
355 360 365
Ile Lys Ala Ser Asn Pro Lys Ser Val Trp Gln Tyr Tyr Gln Leu Val
370 375 380
Asn Val Gln Trp Pro Glu Asn Pro Ile Gln Asp Gly Asn Asn Asn Lys
385 390 395 400
Thr Ala Pro Leu Met Asp Gly Gly Ile Thr Pro Asn Asn Ile Ala Asn
405 410 415
Val Thr Met Glu Thr Tyr Ile Gln Glu Lys Gln Cys Met Asp Cys His
420 425 430
Lys Asn Ala Ser Val Gly Ala Gln Lys Tyr Pro Thr Asp Tyr Ser Phe
435 440 445
Ile Phe Leu Lys Val Lys Gln Ala Lys
450 455
<210> 19
<211> 457
<212> PRT
<213> Chryseobacterium shigense
<400> 19
Met Arg Thr Leu Ile Phe Ser Phe Phe Leu Leu Ser Leu Ile Ser Ile
1 5 10 15
Ser Cys Glu Ser Gly Lys Lys Asp Lys Lys Leu Asn Ser Arg Leu Gly
20 25 30
Phe Ser Lys Glu Met Ala Asp Met Asp Cys Gly Ala Phe Cys Asp Pro
35 40 45
Pro Thr Val Gly Tyr Gln Leu Pro Ala Asp Ser Ile Cys Gly His Ser
50 55 60
Ser Gln Ser Val Leu Asn Cys Phe Ala Trp Lys Asn Phe Leu Ala Leu
65 70 75 80
Asn Trp Lys Ala Ser Asp Glu Arg Gly Leu Pro Asp Thr Thr Ala Val
85 90 95
Ala Ala Asp Tyr Gly Met Pro Gly Asp Tyr Ser Pro Thr Val Trp Glu
100 105 110
Ser Tyr Leu Ser Ala Asp Asp Val Phe Ala Ala Lys Gln Pro Glu Gln
115 120 125
Trp Asn Leu Lys Ser Lys Asn Gly Tyr Ile Lys Tyr Ile Asn Glu Ile
130 135 140
Asn Lys Phe Thr Asp Val Asn Ala Ser Leu Pro Lys Pro Lys Leu Arg
145 150 155 160
Ala Met Leu Gly Gly Asn Val Asp Glu Ile Met Gln Ala Lys Gly Ala
165 170 175
Trp Leu Thr Asp Gln Ser Gly Asn Ile Val Trp Tyr Glu Ile Arg Met
180 185 190
Asn Ser Ile Glu Ser Asp Phe Ile Arg Lys Asn Lys Leu Tyr Ser Ser
195 200 205
Glu Asn Leu Asn Ala Phe Ala Ala Lys Asn Gln Gly Val Trp Leu Pro
210 215 220
Met Glu Ser Ile Glu Ile Lys Ala Ala Trp Arg Ile Ile Pro Glu Asp
225 230 235 240
Gln Leu Glu Ser Leu Lys Asn Phe Tyr Lys Ile Ser Lys Ala Met Val
245 250 255
Pro Glu Ile Lys Gly Phe Asp Lys Asn Asn Gln Pro Ile Tyr Gly Lys
260 265 270
Tyr Thr Gln Lys Tyr Leu Gly Leu Val Gly Leu His Ile Ile Arg Lys
275 280 285
Thr Asn Gln Ser Pro Gln Phe Thr Trp Met Thr Phe Glu His Val Asn
290 295 300
Asn Ala Pro Thr Glu Gly Gln Val Asp Pro Ser Val Lys Tyr Cys Phe
305 310 315 320
Tyr Asn Pro Lys Ser Thr Asp Gln Pro Asn Gln Ser Pro Val Pro Gly
325 330 335
Lys Asp Ser Leu Ser Lys Pro Val Gln Val Met Arg Ile Ala Asn Asn
340 345 350
Ala Ile Thr Pro Glu Ile Gln Asn Leu Asn Lys Gln Ile Arg Asp Met
355 360 365
Ile Lys Ala Ser Asn Pro Lys Ser Val Trp Gln Tyr Tyr Gln Leu Val
370 375 380
Asn Val Gln Trp Pro Glu Asn Pro Ile Gln Asp Gly Asn Asn Asn Lys
385 390 395 400
Thr Ala Pro Leu Met Asp Gly Gly Ile Thr Pro Asn Asn Ile Ala Asn
405 410 415
Val Thr Met Glu Thr Tyr Ile Gln Glu Lys Gln Cys Met Asp Cys His
420 425 430
Lys Asn Ala Ser Val Gly Ala Gln Lys Tyr Pro Thr Asp Tyr Ser Phe
435 440 445
Ile Phe Leu Lys Val Lys Gln Ala Lys
450 455
<210> 20
<211> 457
<212> PRT
<213> 金黄杆菌属种
<400> 20
Met Arg Thr Leu Leu Phe Ser Phe Phe Leu Leu Ser Leu Ile Ser Ile
1 5 10 15
Ser Cys Glu Ser Gly Lys Lys Asp Lys Lys Leu Asn Ser Arg Val Gly
20 25 30
Phe Ser Gln Lys Met Ala Asp Phe Asp Cys Gly Ala Phe Cys Asp Pro
35 40 45
Pro Ser Val Thr Tyr Gln Leu Pro Ala Asp Ser Ser Cys Val Asn Ser
50 55 60
Ser Gln Asn Val Met Asn Cys Phe Ala Trp Lys Asn Phe Leu Ala Leu
65 70 75 80
Asn Trp Leu Ala Ser Asp Gln Arg Gly Val Pro Asp Thr Ala Ala Val
85 90 95
Ala Ala Asp Tyr Gly Met Pro Gly Asp Tyr Lys Pro Thr Val Trp Glu
100 105 110
Ser Tyr Leu Ser Ile Asp Asp Val Phe Ala Ala Lys Pro Pro Ala Gln
115 120 125
Trp Asn Leu Arg Ser Lys Asn Gly Tyr Met Lys Tyr Ile Asn Glu Ile
130 135 140
Asn Lys Phe Thr Asp Ile Asn Ala Ser Leu Pro Lys Pro Lys Leu Arg
145 150 155 160
Ala Met Leu Gly Gly Asn Val Asp Glu Ile Met Gln Ala Lys Gly Ala
165 170 175
Trp Leu Thr Asp Gln Ser Gly Asn Ile Val Trp Tyr Glu Ile Arg Met
180 185 190
Asn Thr Ile Glu Ser Asp Phe Val Arg Asp Asn Lys Leu Tyr Asn Tyr
195 200 205
Gly Ser Leu Ser Ala Phe Ala Ala Gln Asn Gln Gly Val Trp Phe Pro
210 215 220
Met Glu Ser Ile Glu Ile Lys Ala Ala Trp Arg Ile Ile Pro Glu Asp
225 230 235 240
Gln Leu Glu Ser Leu Lys Asn Phe Tyr Lys Ile Ser Met Ala Met Val
245 250 255
Pro Glu Ile Lys Gly Phe Asp Lys Asn Asn Lys Pro Ile Tyr Gly Lys
260 265 270
Tyr Leu Gln Lys Tyr Leu Gly Leu Val Gly Leu His Ile Ile Arg Lys
275 280 285
Thr Asn Gln Ser Pro Gln Phe Thr Trp Met Thr Phe Glu His Val Asn
290 295 300
Asn Ala Pro Thr Asp Gly Gln Ile Asp Pro Ser Val Lys Tyr Cys Phe
305 310 315 320
Tyr Asp Pro Lys Ser Lys Asp Lys Pro Asn Gln Ser Pro Val Pro Gly
325 330 335
Lys Asp Ser Leu Asn Lys Pro Val Gln Val Val Arg Ile Ala Asp Asn
340 345 350
Ala Ile Ser Pro Glu Ile Gln Gln Leu Asn Lys Gln Ile Gln Asn Met
355 360 365
Ile Lys Ala Ser Asn Pro Lys Ser Val Trp Gln Tyr Tyr Gln Leu Val
370 375 380
Asn Val Gln Trp Pro Glu Asn Pro Val Lys Asp Lys Asp Asn Asn Lys
385 390 395 400
Lys Ala Pro Leu Met Thr Gly Gly Ile Thr Pro Lys Asn Ile Ala Asn
405 410 415
Val Thr Met Glu Thr Tyr Ile Gln Glu Lys Gln Cys Met Asp Cys His
420 425 430
Lys Asn Ala Ser Val Gly Asp Gln Lys Tyr Pro Thr Asp Tyr Ser Phe
435 440 445
Ile Phe Leu Lys Val Lys Pro Gly Asn
450 455
<210> 21
<211> 455
<212> PRT
<213> 金黄杆菌属种
<400> 21
Met Arg Thr Leu Leu Phe Ser Phe Phe Leu Leu Ser Leu Ile Ser Ile
1 5 10 15
Ser Cys Glu Ser Gly Lys Lys Asp Lys Lys Leu Asn Ser Arg Ile Gly
20 25 30
Phe Ser Lys Glu Leu Asp Asp Tyr Gly Cys Gly Ala Phe Cys Asp Pro
35 40 45
Pro Ser Val Gly Tyr Gln Leu Thr Asp Asp Asn Cys Leu His Ser Asp
50 55 60
Gln Asn Ser Met Asn Cys Phe Ala Trp Lys Asn Phe Leu Ala Leu Asn
65 70 75 80
Trp Ile Ala Ser Asp Gln Arg Gly Ile Pro Asp Thr Thr Ala Leu Ala
85 90 95
Ser Asp Tyr Gly Met Pro Gly Asp Tyr Lys Pro Thr Val Trp Glu Ser
100 105 110
Tyr Leu Ser Ile Asn Asp Val Phe Thr Ala Gln Gln Pro Ala Gln Trp
115 120 125
Ser Leu Lys Ser Lys Ser Gly Tyr Ile Lys Tyr Ile Asn Glu Ile Asn
130 135 140
Lys Phe Thr Asp Ile Asn Val Asn Ile Pro Lys Pro Lys Leu Arg Ala
145 150 155 160
Met Leu Gly Gly Asn Val Asp Glu Ile Met Gln Ala Lys Gly Ala Trp
165 170 175
Leu Thr Asp Gln Ser Gly Asn Ile Val Trp Tyr Glu Ile Arg Met Asn
180 185 190
Asn Ile Glu Ser Asp Phe Val Arg Asn Asn Lys Leu Tyr Asn Ser Glu
195 200 205
Asn Leu Asn Ala Phe Ala Ala Lys Asn Gln Gly Val Trp Leu Pro Met
210 215 220
Glu Ser Ile Glu Ile Lys Ala Ala Trp Arg Val Ile Pro Glu Asn Gln
225 230 235 240
Leu Glu Ser Leu Lys Asp Phe Tyr Lys Ile Ser Met Ala Met Val Pro
245 250 255
Glu Ile Lys Gly Phe Asp Lys Asn Asn Gln Pro Ile Tyr Gly Lys Tyr
260 265 270
Thr Gln Lys Tyr Leu Gly Leu Val Gly Leu His Ile Ile Arg Lys Thr
275 280 285
Ser Gln Ser Pro Gln Phe Thr Trp Met Thr Phe Glu His Ile Asn Asn
290 295 300
Ala Pro Thr Glu Gly Gln Val Asp Pro Ser Val Asn Tyr Cys Phe Tyr
305 310 315 320
Asn Pro Lys Ser Lys Asp Lys Pro Asn Gln Ser Pro Val Pro Gly Lys
325 330 335
Asp Ser Leu Asn Lys Pro Val Gln Val Val Arg Ile Ala Asn Asn Ala
340 345 350
Ile Thr Pro Glu Ile Gln Gln Leu Asn Lys Gln Ile Gln Ser Met Ile
355 360 365
Arg Ala Ser Asn Pro Lys Ser Val Trp Gln Tyr Tyr Gln Leu Val Asn
370 375 380
Val Gln Trp Pro Glu Asn Pro Val Gln Asp Lys Asp Asn Lys Asn Thr
385 390 395 400
Pro Pro Leu Arg Asp Gly Gly Ile Lys Pro Lys Asn Ile Ala Asn Val
405 410 415
Thr Met Glu Thr Tyr Ile Gln Asp Lys Gln Cys Met Asp Cys His Lys
420 425 430
Asn Ala Ser Thr Val Ala Thr Lys Tyr Pro Thr Asp Tyr Ser Phe Ile
435 440 445
Phe Leu Lys Val Lys Pro Lys
450 455
<210> 22
<211> 457
<212> PRT
<213> Chryseobacterium kwangjuense
<400> 22
Met Arg Thr Leu Leu Ile Ser Phe Phe Leu Leu Ser Leu Ile Ala Ile
1 5 10 15
Ser Cys Glu Ser Gly Lys Lys Asp Lys Lys Leu Asn Ser Arg Val Gly
20 25 30
Phe Ser Gln Glu Leu Ala Asp Phe Asp Cys Gly Ala Phe Cys Asp Pro
35 40 45
Pro Ser Val Ser Tyr Gln Leu Pro Val Asp Ser Thr Cys Gly His Ser
50 55 60
Ser Gln Asn Val Leu Asn Cys Phe Ala Trp Lys Asn Phe Leu Ala Leu
65 70 75 80
Asn Trp Lys Ala Ser Asp Glu Arg Gly Leu Pro Asp Thr Thr Ala Val
85 90 95
Ala Ala Asp Tyr Gly Met Pro Gly Asp Tyr Ser Pro Thr Val Trp Glu
100 105 110
Ser Tyr Leu Ser Ile Glu Asp Val Phe Ser Ala Arg Gln Pro Gln Thr
115 120 125
Trp Asn Leu Lys Ser Lys Asn Gly Tyr Ile Lys Tyr Ile Asn Glu Ile
130 135 140
Asn Lys Phe Thr Asp Ile Asn Ala Ala Leu Pro Lys Pro Lys Leu Arg
145 150 155 160
Ala Met Leu Gly Gly Asn Val Asp Glu Ile Met Gln Ala Lys Gly Ala
165 170 175
Trp Leu Thr Asp Gln Ser Gly Asn Ile Val Trp Tyr Glu Ile Arg Met
180 185 190
Asn Asn Ile Glu Ser Asp Phe Val Arg Gln Asn Lys Leu Tyr Asn Ser
195 200 205
Asp Asn Leu Asn Ala Phe Ala Ala Lys Asn Gln Gly Val Trp Leu Pro
210 215 220
Met Glu Ser Ile Glu Ile Lys Ala Ala Trp Arg Ile Ile Pro Asp Ser
225 230 235 240
Gln Leu Glu Ser Leu Lys Asn Leu Tyr Lys Ile Ser Arg Ala Met Val
245 250 255
Pro Glu Ile Lys Gly Phe Asp Lys Asn Asn Gln Pro Ile Tyr Gly Lys
260 265 270
Tyr Ser Pro Lys Tyr Leu Gly Leu Val Gly Leu His Ile Ile Arg Lys
275 280 285
Thr Asn Gln Ser Pro Gln Phe Thr Trp Met Thr Phe Glu His Val Asn
290 295 300
Asn Ala Pro Thr Glu Gly Gln Val Asp Pro Ser Val Lys Tyr Cys Phe
305 310 315 320
Tyr Asn Pro Lys Ser Lys Asp Lys Pro Asn Gln Ser Pro Val Pro Gly
325 330 335
Gln Asp Ser Leu Asn Thr Pro Val Gln Val Val Arg Ile Ala Asp Asn
340 345 350
Ala Ile Ser Ala Asp Ile Gln Gln Leu Asn Lys Gln Ile Gln Ala Met
355 360 365
Ile Lys Lys Ser Asn Pro Lys Ser Val Trp Gln Tyr Tyr Gln Leu Val
370 375 380
Asn Val Gln Trp Pro Glu Asn Pro Ile Gln Asp Thr Asn Asn Asn Lys
385 390 395 400
Thr Ala Pro Leu Met Asp Gly Gly Ile Thr Pro Ser Asn Ile Ala Asn
405 410 415
Val Thr Met Glu Thr Tyr Ile Gln Glu Lys Gln Cys Met Asp Cys His
420 425 430
Lys Asn Ala Ser Val Gly Ala Gln Lys Tyr Pro Thr Asp Tyr Ser Phe
435 440 445
Ile Phe Leu Lys Ala Lys Pro Gly Lys
450 455
<210> 23
<211> 457
<212> PRT
<213> 金黄杆菌属种 OV705
<400> 23
Met Lys Ala Leu Ile Leu Ile Trp Ser Leu Ala Ile Val Ser Ile Val
1 5 10 15
Ser Cys Glu Ser Ser Lys Lys Asp Lys Lys Leu Asn Ser Ala Thr Gly
20 25 30
Phe Ser Lys Lys Val Thr Gly Glu Thr Ile Phe Cys Gly Ala Tyr Cys
35 40 45
Asn Pro Pro Ala Ile Ser Tyr Glu Leu Ala Ala Asp Ser Thr Cys Ala
50 55 60
His Ser Ser Gln Glu Val Leu Asn Cys Phe Ala Trp Lys Asn Phe Ile
65 70 75 80
Ala Leu Asn Trp Ile Ala Ser Ala Gln Arg Gly Ile Pro Asp Thr Thr
85 90 95
Ala Thr Ala Ala Asn Tyr Gly Met Pro Gly Asp Tyr Ser Pro Thr Val
100 105 110
Trp Glu Ser Phe Leu Ser Ile Asp Asp Val Phe Ala Pro Lys Pro Pro
115 120 125
Leu Ala Trp Asn Leu Lys Ser Lys Thr Gly Tyr Ile Lys Arg Ile Asn
130 135 140
Glu Ile Asn Lys Phe Thr Asp Ile Ile Ser Ser Leu Pro Lys Ala Thr
145 150 155 160
Leu Arg Ala Ala Val Gly Ser Ser Asn Val Asp Glu Ile Met Gln Ala
165 170 175
Glu Gly Ala Trp Leu Thr Asp Gln Asn Gly Asn Ile Val Trp Tyr Glu
180 185 190
Ile Arg Ile Asn Asn Leu Glu Ser Asp Phe Ile Arg Gln Asn Lys Leu
195 200 205
Tyr Asp Tyr Asp Asn Leu Lys Ala Phe Gly Thr Lys Asn Asn Gly Val
210 215 220
Trp Leu Pro Asn Glu Ser Ile Glu Leu Lys Ala Ala Trp Arg Val Ile
225 230 235 240
Pro Asp Asp Gln Leu Asp Ser Leu Lys Asn Tyr Tyr Lys Ile Ser Lys
245 250 255
Ala Met Val Pro Glu Ile Lys Gly Phe Asn Gly Lys Lys Pro Ile Tyr
260 265 270
Gly Lys Tyr Thr Gln Lys Tyr Leu Gly Leu Val Gly Leu His Ile Ile
275 280 285
Arg Lys Thr Pro Gln Ser Pro Gln Leu Asn Trp Met Thr Phe Glu His
290 295 300
Val Asn Asn Ala Pro Gly Pro Gly Pro Ala Asp Pro Ser Val Lys Tyr
305 310 315 320
Ser Phe Tyr Asn Pro Asn Ser Lys Asp Pro Ala Asn Gln Ser Pro Val
325 330 335
Pro Gly Lys Asp Ser Leu Asn Lys Pro Val Gln Val Val Arg Val Asn
340 345 350
Arg Ile Ser Gln Ser Val Gln Lys Leu Asn Ala Gln Met Gln Gln Leu
355 360 365
Ile Arg Ala Ser Asn Pro Lys Ser Val Trp Gln Tyr Tyr Gln Leu Val
370 375 380
Asn Ile Gln Trp Pro Glu Asn Pro Val Ala Asp Lys Asp Asn Asn Thr
385 390 395 400
Gln Thr Pro Leu Met Glu Gly Gly Val Lys Pro Asn Gln Ile Ser Asn
405 410 415
Thr Thr Met Glu Thr Tyr Ala Gln Ser Lys Gln Cys Met Asp Cys His
420 425 430
Lys Asn Ala Pro Val Ile Gly Thr Ser Ile Pro Thr Asp Tyr Ser Phe
435 440 445
Ile Phe Leu Lys Val Lys Pro Lys Lys
450 455
<210> 24
<211> 466
<212> PRT
<213> 产吲哚金黄杆菌
<400> 24
Met Lys Ala Leu Ile Cys Ser Leu Phe Ile Leu Gly Leu Ile Ser Cys
1 5 10 15
Glu Ser Gly Lys Lys Asp Lys Lys Leu Asn Ser Ala Thr Gly Phe Ser
20 25 30
Lys Glu Val Ala Gly Glu Ser Ile Tyr Cys Gly Ala Tyr Cys Asp Pro
35 40 45
Pro Ser Ile Thr Tyr Lys Leu Ala Gly Asp Ser Thr Cys Ala His Ser
50 55 60
Ser Gln Asp Val Leu Asn Cys Phe Ala Trp Lys Asn Phe Ile Ala Leu
65 70 75 80
Asn Trp Ile Ala Ser Ala Gln Arg Gly Val Pro Asp Thr Thr Ala Thr
85 90 95
Ala Ala Asn Tyr Gly Met Pro Gly Asp Tyr Ser Pro Thr Val Trp Glu
100 105 110
Ser Phe Ala Ser Asn Asp Glu Val Phe Ala Ala Lys Asn Pro Leu Ala
115 120 125
Trp Asn Leu Lys Ser Arg Asn Thr Tyr Val Lys Gln Ile Asn Glu Ile
130 135 140
Asn Lys Phe Thr Asp Ile Asn Ile Ser Ile Pro Lys Ala Thr Leu Arg
145 150 155 160
Ala Ala Val Gly Ser Asn Asn Val Asp Glu Ile Leu Gln Ala Glu Gly
165 170 175
Ser Trp Leu Thr Asp Gln Ser Gly Asn Ile Val Trp Tyr Glu Ile Lys
180 185 190
Ile Asn Asn Ile Glu Ser Asp Phe Ile Arg Lys Asn Lys Leu Tyr Asp
195 200 205
Tyr Asn Ser Leu Lys Asp Tyr Gly Thr Ala Asn Lys Gly Val Trp Leu
210 215 220
Pro Met Glu Ser Ile Glu Leu Lys Ala Ala Trp Arg Ile Ile Pro Glu
225 230 235 240
Asp Lys Leu Asp Ser Leu Lys Asn Tyr Tyr Lys Ile Ser Lys Ala Met
245 250 255
Val Pro Glu Ile Ile Gly Phe Lys Asp Lys Lys Pro Ile Phe Gly Lys
260 265 270
Ser Thr Gln Lys Tyr Leu Gly Leu Val Gly Leu His Ile Ile Arg Lys
275 280 285
Thr Pro Gln Ser Pro Gln Phe Asn Trp Met Thr Phe Glu His Val Asn
290 295 300
Asn Ala Pro Asn Glu Gly Gln Ala Asp Pro Ala Val Lys Tyr Cys Phe
305 310 315 320
Tyr Asn Pro Lys Ser Lys Asp Thr Pro Asn Ile Ala Pro Lys Ile Gly
325 330 335
Tyr Asp Ser Leu Asn Lys Pro Val Gln Val Val Arg Val Asn Lys Ile
340 345 350
Lys Thr Lys Leu Gln Lys Leu Asn Ala Gln Met Gln Gln Leu Ile Arg
355 360 365
Ala Ser Asn Pro Lys Ser Val Trp Gln Tyr Tyr Gln Leu Val Asn Ile
370 375 380
Gln Trp Pro Glu Asn Pro Ile Gln Asp Asn Gly Asn Asn Ser Ala Ala
385 390 395 400
Pro Leu Met Glu Gly Gly Ile Thr Pro Ser Asp Ile Ser Asn Thr Thr
405 410 415
Met Glu Thr Tyr Ala Gln Thr Lys Gln Cys Met Asp Cys His Lys Tyr
420 425 430
Ala Ser Val Val Gly Ser Gly Met Pro Pro Thr Asp Tyr Ser Phe Ile
435 440 445
Phe Leu Lys Val Lys Pro Val Lys Gln Leu Pro Lys Lys Thr Ala Pro
450 455 460
Val Lys
465
<210> 25
<211> 6182
<212> DNA
<213> 人工序列
<220>
<223> 构建体GDI005-GDI006
<400> 25
aaggtaatta tccaagatgt agcatcaaga atccaatgtt tacgggaaaa actatggaag 60
tattatgtga gctcagcaag aagcagatca atatgcggca catatgcaac ctatgttcaa 120
aaatgaagaa tgtacagata caagatccta tactgccaga atacgaagaa gaatacgtag 180
aaattgaaaa agaagaacca ggcgaagaaa agaatcttga agacgtaagc actgacgaca 240
acaatgaaaa gaagaagata aggtcggtga ttgtgaaaga gacatagagg acacatgtaa 300
ggtggaaaat gtaagggcgg aaagtaacct tatcacaaag gaatcttatc ccccactact 360
tatcctttta tatttttccg tgtcattttt gcccttgagt tttcctatat aaggaaccaa 420
gttcggcatt tgtgaaaaca agaaaaaatt tggtgtaagc tattttcttt gaagtactga 480
ggatacaact tcagagaaat ttgtccctcc cccctccccc tccgccgccg ccgcgccggt 540
aaccaccccg cccctctcct ctttctttct ccgttttttt tttccgtctc ggtctcgatc 600
tttggccttg gtagtttggg tgggcgagag gcggcttcgt gcgcgcccag atcggtgcgc 660
gggaggggcg ggatctcgcg gctggggctc tcgccggcgt ggatccggcc cggatctcgc 720
ggggaatggg gctctcggat gtagatctgc gatccgccgt tgttggggga gatgatgggg 780
ggtttaaaat ttccgccatg ctaaacaaga tcaggaagag gggaaaaggg cactatggtt 840
tatattttta tatatttctg ctgcttcgtc aggcttagat gtgctagatc tttctttctt 900
ctttttgtgg gtagaatttg aatccctcag cattgttcat cggtagtttt tcttttcatg 960
atttgtgaca aatgcagcct cgtgcggagc ttttttgtag gtagacgata tctccaccat 1020
gcaccaccac catcaccaca tcgatatgaa gaagttcaac acccctgctt accaggccga 1080
aaaagacttc aaacagtacc cccacctggg cgagcaactc gagaacgcgt ggagcaatta 1140
cgtgaagtac tgtacaatca actccattat ggggaatccg tggtcctcaa cctatgacca 1200
cccgcgctcg tggtactaca atccactcgt tgatgacgtg gtgcctactg aacagaatac 1260
cgtgccaatt cagtggaatg cattcccaaa ccgcatcaac cactacttca ctgggctgtt 1320
taccaagcag ttcggttcgg ctgagtacga ggataagctg cacgagcttg ccgatatcgg 1380
ccctgcggca ttcgggaaga agtataacat ggaccttaca gtgccgaaga acccatgtga 1440
cccgagcgat acacggacca aaccatttgg gccttcaggt ccacgcggtt ggcaagacga 1500
atattgcgag tgggcggtca cacgggacga gaacggggac attacagcgg tcgactttac 1560
ccacgaaaac cctgagtact ggttccatat gtggaagata tcaccagaca tcgtggtggc 1620
cttgtatcag gagatcctga acaacaagaa tgtgaagaag gaagacctct acttgctcga 1680
ctcgaccggt aaccccgtca tcgttcggga gacaggggaa ccggcataca accccatcaa 1740
caagtggaat aacggaccag aggccacacc agagggtggg ggtgcagttc atctcacctc 1800
accaccgaac tccctgggtg cagagatcta cctgggcgca gccgcgacca tactccgggt 1860
taagaataac caggtcatca ccgacgctaa cgccctcatc tgcgcggccc agtacggcca 1920
gatctaccgg aacagcgacc cacggatcgg gcagaatgtc aactcactcg tctacaatca 1980
taagcagcag atcacactaa ccaaccccat cgcgctgtat ggacaagtcc cagactttga 2040
tcagttcgag atgccaagca ccgcggggtc gtataagatt caggactgct acacggtcgt 2100
gagaggagag gaaaggaaca agggtatcac cttctacccc tttaacatgc tcctccacac 2160
aaggttttcg gttcccaagg gggcgaattt caagctcagc gagatcaagg tcaagggcaa 2220
gctcctgaag tgggggagcc aaatcgcgga caccttcttc gtgcagctgg cgggaaccgg 2280
caagagtccc ggtgccgggg aacagccgga aaagtttcct ccagtgggtg acccggcgac 2340
cacactgccc aacgtgcagt acctgctcga taataacctg ctgcaggcct ccctctacaa 2400
caaactgaac accttcagca atctgacctc gtgcatcacg caaatcgagg ccggcacatc 2460
gactgaaggt atcgcagtgt tgacaaacgc cgctaccaag gaaactcagt tcgacttcgg 2520
tccgggcatc agcgtgatgg tgacagactt ccagaacatc gatgaagaca cgcaactgtt 2580
tcttatcacc atcacagcag acgcggatac atcgctggga gagaagccac tttccctcta 2640
caacaatgca agcgatccaa agtacgccct gtccggcgtg ctggaagtgg tccccaacgg 2700
tagcctgccg aagattaaca ctacacccaa tctggcgctc ttgtccggac aacaggtgga 2760
gcaagtcaag aaaatcctta agtagaacgc atccatggac gtggattgaa ttgaaggtgt 2820
actactgctg tgctggtccg tggatcgtgg ctgtcatgca tggtttgctg tgtcttctac 2880
gatatgtact tccctttgtt ccgtatatgt acatcttcct cgtttggttc atgtattttc 2940
ctttgaataa taataaataa atcgggcttt ccatatcgga tgcttttata tctgtgtgta 3000
tggagattgt ggtatatggt ttcatctcaa gttgtttacg tcaagaacta aagatatttc 3060
ctcaaaaaaa aagaactaaa gatataatca atgtcattaa cataactcat ttccatgagg 3120
agaggacgaa ggacgaagtc ataataagta gattggttga tattttataa tcattcaaaa 3180
ctgcaggggt tataagatct tcattttgta gaagttttag atcttccgag gggttctcgt 3240
gatctatgtc gggtgcggag aaagaggtaa tgaaatggca ggaaaggtaa ttatccaaga 3300
tgtagcatca agaatccaat gtttacggga aaaactatgg aagtattatg tgagctcagc 3360
aagaagcaga tcaatatgcg gcacatatgc aacctatgtt caaaaatgaa gaatgtacag 3420
atacaagatc ctatactgcc agaatacgaa gaagaatacg tagaaattga aaaagaagaa 3480
ccaggcgaag aaaagaatct tgaagacgta agcactgacg acaacaatga aaagaagaag 3540
ataaggtcgg tgattgtgaa agagacatag aggacacatg taaggtggaa aatgtaaggg 3600
cggaaagtaa ccttatcaca aaggaatctt atcccccact acttatcctt ttatattttt 3660
ccgtgtcatt tttgcccttg agttttccta tataaggaac caagttcggc atttgtgaaa 3720
acaagaaaaa atttggtgta agctattttc tttgaagtac tgaggataca acttcagaga 3780
aatttgtccc tcccccctcc ccctccgccg ccgccgcgcc ggtaaccacc ccgcccctct 3840
cctctttctt tctccgtttt ttttttccgt ctcggtctcg atctttggcc ttggtagttt 3900
gggtgggcga gaggcggctt cgtgcgcgcc cagatcggtg cgcgggaggg gcgggatctc 3960
gcggctgggg ctctcgccgg cgtggatccg gcccggatct cgcggggaat ggggctctcg 4020
gatgtagatc tgcgatccgc cgttgttggg ggagatgatg gggggtttaa aatttccgcc 4080
atgctaaaca agatcaggaa gaggggaaaa gggcactatg gtttatattt ttatatattt 4140
ctgctgcttc gtcaggctta gatgtgctag atctttcttt cttctttttg tgggtagaat 4200
ttgaatccct cagcattgtt catcggtagt ttttcttttc atgatttgtg acaaatgcag 4260
cctcgtgcgg agcttttttg taggtagacg atatctccac catgcaccac caccatcacc 4320
acatcgatat gaagacgctg gtctttagcc tgttcatcct ctcattcatc tcgtgtgaat 4380
cagggaaaaa ggaccaacgg cttaatagcg caaccggttt ctcgaaggtg gctgactccg 4440
agagcatcta ctgtggagcg ttttgcgatc ctccatcaat cacatacaaa ctggcaggag 4500
acagcacctg cgcacacagc tcgcaggacg ttctcaactg cttcgcgtgg aagaacttca 4560
tcgcactgaa ttgggcggcc tccgcgcaga gaggtgtccc agacaccaca gcaacagcag 4620
cgaactatgg catgccaggg gactattcgc cgacagtctg ggagtccttc gcttccaacg 4680
acgaggtgtt cgcacccaag aacctcctga catggaatct gaagagtaag aacggttacg 4740
tgaaacagat caacgagatt aacaagttca ccgatatcaa catcagcata cccaaggcaa 4800
ccctcagagc agctgtcggc aacagcaatg tggatgaaat cctgcaagcg gagggttcgt 4860
ggcttactga ccagtcgggc aacatcgtct ggtacgagat taagatcaat aacatcgaat 4920
cggacttcat cagacggaat aagctctacg actacaatag cctcaaggag tacgggaccg 4980
caaacaatgg ggtgtggctc cccatggaga gcatagaact gaaagcggca tggagagtga 5040
tcccagagga caaactggat agcctgaaaa actactataa gatctccaaa gccatggtcc 5100
cagagatcaa ggggttcaag gataagaaac ccgtgttcgg aaagtccacc cagaagtacc 5160
tgggtctcgt ggggctgcac attatccgga agaccccaca aagcccacaa ttcaactgga 5220
tgacattcga gcatatccac aacgccccaa atgaggggca ggcggatcct agcgtccggt 5280
actgcttcta caaccccaag agcacaaaaa caccaaatat cgcgcctgtc atcggaaagg 5340
attcactgaa cacaccggtg caagtcgttc gggtcaataa gattaaaacc aaactgcaga 5400
agctgaacac ccaaatgcag cagctgattc gggcgagcaa tcccaagagc gtgtggcagt 5460
attaccagct ggtgaacatc cagtggccag agaatccgat ccaggacaac ggtaacaata 5520
agagtgcccc actcatggag ggagggatta cccccagcga tatctccaat accacgatgg 5580
agacctacgc ccaacagaag cagtgtatgg actgtcacaa gtacgcatcg gttgttggta 5640
gcgggatgcc acctaccgac tatagcttca tcttcctgaa ggtgaagcca gaaaaacaga 5700
ttccaaaggg gaaaacccca gtgaagtaga acgcatccat ggacgtggat tgaattgaag 5760
gtgtactact gctgtgctgg tccgtggatc gtggctgtca tgcatggttt gctgtgtctt 5820
ctacgatatg tacttccctt tgttccgtat atgtacatct tcctcgtttg gttcatgtat 5880
tttcctttga ataataataa ataaatcggg ctttccatat cggatgcttt tatatctgtg 5940
tgtatggaga ttgtggtata tggtttcatc tcaagttgtt tacgtcaaga actaaagata 6000
tttcctcaaa aaaaaagaac taaagatata atcaatgtca ttaacataac tcatttccat 6060
gaggagagga cgaaggacga agtcataata agtagattgg ttgatatttt ataatcattc 6120
aaaactgcag gggttataag atcttcattt tgtagaagtt ttagatcttc cgaggggttc 6180
tc 6182
<210> 26
<211> 1734
<212> DNA
<213> 人工序列
<220>
<223> GDI005核优化大肠杆菌
<400> 26
aagaaattta acaccccggc gtaccaggcg gagaaagact tcaagcaata tccgcacctg 60
ggcgagcagc tggaaaacgc gtggagcaac tacgtgaagt attgcaccat caacagcatt 120
atgggcaacc cgtggagcag cacctacgat cacccgcgta gctggtacta taacccgctg 180
gttgacgatg tggttccgac cgagcagaac accgttccga tccaatggaa cgcgttcccg 240
aaccgtatta accactactt caccggtctg tttaccaaac agttcggcag cgcggagtat 300
gaagacaagc tgcacgaact ggcggatatc ggtccggcgg cgtttggcaa gaaatacaac 360
atggacctga ccgttccgaa aaacccgtgc gacccgagcg atacccgtac caaaccgttt 420
ggtccgagcg gtccgcgtgg ctggcaagat gagtactgcg aatgggcggt gacccgtgac 480
gagaacggtg atatcaccgc ggttgacttt acccacgaga acccggaata ttggttccac 540
atgtggaaaa tcagcccgga tattgtggtt gcgctgtacc aggagattct gaacaacaag 600
aacgtgaaga aagaagacct gtatctgctg gatagcaccg gtaacccggt gatcgttcgt 660
gaaaccggcg aaccggcgta caacccgatt aacaaatgga acaacggtcc ggaggcgacc 720
ccggaaggtg gcggtgcggt tcatctgacc agcccgccga acagcctggg tgcggaaatc 780
tatctgggtg cggcggcgac cattctgcgt gtgaagaaca accaagttat caccgatgcg 840
aacgcgctga tttgcgcggc gcagtacggt caaatctatc gtaacagcga tccgcgtatt 900
ggccagaacg tgaacagcct ggtttacaac cacaaacagc aaatcaccct gaccaacccg 960
attgcgctgt atggtcaggt gccggacttc gatcaatttg agatgccgag caccgcgggc 1020
agctacaaaa tccaagactg ctataccgtg gttcgtggtg aggaacgtaa caagggcatt 1080
accttctacc cgtttaacat gctgctgcac acccgtttta gcgtgccgaa aggtgcgaac 1140
ttcaagctga gcgaaatcaa ggttaaaggc aagctgctga aatggggcag ccaaattgcg 1200
gataccttct ttgttcagct ggcgggtacc ggtaaaagcc cgggtgcggg cgagcagccg 1260
gaaaagtttc cgccggtggg cgacccggcg accaccctgc cgaacgttca atacctgctg 1320
gataacaacc tgctgcaggc gagcctgtat aacaaactga acaccttcag caacctgacc 1380
agctgcatca cccaaattga ggcgggtacc agcaccgaag gtattgcggt gctgaccaac 1440
gcggcgacca aggaaaccca gttcgacttt ggcccgggta tcagcgtgat ggttaccgat 1500
tttcaaaaca ttgacgagga tacccagctg ttcctgatca ccattaccgc ggacgcggat 1560
accagcctgg gtgaaaaacc gctgagcctg tacaacaacg cgagcgaccc gaagtatgcg 1620
ctgagcggtg ttctggaagt ggttccgaac ggcagcctgc cgaaaatcaa caccaccccg 1680
aacctggcgc tgctgagcgg tcagcaagtg gaacaggtta agaaaattct gaag 1734
<210> 27
<211> 1395
<212> DNA
<213> 人工序列
<220>
<223> GDI006核优化大肠杆菌
<400> 27
aagaccctgg ttttcagcct gtttattctg agcttcatca gctgcgagag cggtaagaaa 60
gatcagcgtc tgaacagcgc gaccggcttc agcaaagtgg cggacagcga aagcatttac 120
tgcggtgcgt tttgcgatcc gccgagcatc acctataaac tggcgggtga cagcacctgc 180
gcgcacagca gccaggatgt tctgaactgc ttcgcgtgga aaaactttat tgcgctgaac 240
tgggcggcga gcgcgcaacg tggtgtgccg gacaccaccg cgaccgcggc gaactatggt 300
atgccgggcg attatagccc gaccgtgtgg gagagcttcg cgagcaacga cgaagttttt 360
gcgccgaaga acctgctgac ctggaacctg aaaagcaaaa acggttacgt taaacagatt 420
aacgagatca acaagttcac cgatatcaac attagcattc cgaaagcgac cctgcgtgcg 480
gcggtgggta acagcaacgt tgacgagatt ctgcaggcgg aaggtagctg gctgaccgat 540
caaagcggca acatcgtgtg gtacgagatt aagatcaaca acattgaaag cgactttatc 600
cgtcgtaaca aactgtacga ttataacagc ctgaaggaat atggtaccgc gaacaacggc 660
gtttggctgc cgatggagag cattgaactg aaagcggcgt ggcgtgtgat cccggaggac 720
aaactggata gcctgaagaa ctactataaa attagcaagg cgatggtgcc ggaaatcaaa 780
ggtttcaagg acaagaaacc ggtttttggc aaaagcaccc agaagtatct gggtctggtg 840
ggcctgcaca tcattcgtaa gaccccgcag agcccgcaat tcaactggat gacctttgag 900
cacatccaca acgcgccgaa cgagggtcag gcggatccga gcgttcgtta ctgcttttat 960
aacccgaaaa gcaccaagac cccgaacatt gcgccggtga tcggcaaaga cagcctgaac 1020
accccggtgc aagtggttcg tgttaacaag attaaaacca agctgcagaa actgaacacc 1080
caaatgcagc aactgatccg tgcgagcaac ccgaaaagcg tgtggcagta ctatcaactg 1140
gttaacattc agtggccgga gaacccgatc caagataacg gtaacaacaa aagcgcgccg 1200
ctgatggagg gtggcattac cccgagcgac atcagcaaca ccaccatgga aacctacgcg 1260
cagcaaaaac agtgcatgga ttgccacaag tatgcgagcg tggttggtag cggcatgccg 1320
ccaaccgact acagcttcat ttttctgaaa gttaagccgg aaaaacaaat cccgaaaggc 1380
aagaccccgg tgaag 1395
<210> 28
<211> 1731
<212> DNA
<213> 人工序列
<220>
<223> GDI0175A_GDI0005A_like_Binary优化大肠杆菌
<400> 28
aagaaatttg acaccccggc gtaccaggcg gagaaggact tcaaagatca gccggacctg 60
aagatccaaa ttgaaaacgc gtggagcgat tacgttaaat attgcaccat taacagccaa 120
atgggcaacc cgtggagcag cagctatgac cacccgcgta gctggtacta taacccgctg 180
gtgaccccgg ttaccccgat caagaacaac accgtgccga ttcagtgggg tgcgtttccg 240
aaccgtatca accactactt caccgacctg ttcacccaaa agttcggcaa agcggatgcg 300
accgacaaac tgcacgagct ggcggatatc ggtccggacg cgtttagcaa gaaatatgat 360
attaccctga ccgttaccaa gaacccgtgc gacccgaaca acaccgcgac caagccgttt 420
ggtccgagcg gtccgcgtgg ttggcaggac gagtactgcg aatgggcggt tacccgtgat 480
accaacggtg acatcattgc ggtggatttt acccacgaga acccggaata ttggttccac 540
atgtggaagg ttagcccgga catggtggtt agcctgtacc aggagatcct gaacaacagc 600
aacgtgaaga aagaggatct gtatctgctg gacgaacaaa acaacccggt gatcgttcgt 660
gaaaccggcc tgccggcgta caacccgatt aacaaatgga acaacggtag cagcgcgacc 720
accgcgggtg gcggtgcggt tcatctgacc agcccgccga acagcctggg tgcggaaatc 780
tatctgggtg cggcggcgac cattctgcgt gcggtgaacg gtaaagttat caccgatgcg 840
aacgcgctga tttgcgcggc gcagtacggc caaatctatc gtaacagcga cccgcgtatt 900
ggtcagaacg tgaacagcct ggtttaccac aacaaggtga aaatcagcct gaccaacccg 960
attgcgctgt atggccagct gccggatttc acccaattta ccatgccggc gagcgcggag 1020
ggttacaaga tcgaagattg ctacaagatc attcgtggcc gtgacattaa cccgggtacc 1080
accttttacc cgaacaacat ggttctgcat agccgttttg aggtgccggc gggtgcgaac 1140
ttcaagctga gcgaaatcct ggttcagaac caaccgctga agtggggtag ccagattgcg 1200
gatgtgttca aagttcaact ggcgggtacc ggtatcccgg gcggtggcga caaaccgcag 1260
gagtacccgc cggtgggcga cccggatgtt gcgctgccga gcgtgcagta cctgctggac 1320
aaccgtctgc tgcaagcgag cctgtataac aaactgaaca ccttcagcaa cctgaccagc 1380
tgcatcaccc agattgaagc gggcaccacc accaagggta tcgcggtgct ggcgagcgat 1440
gcgaaccaaa aaaccggctt cgactttggt gcgggcatca ttgcgagcat taccgatttt 1500
caggacctgg gtaacgataa ccaactgttc atcattgata tcaccgttca gagcgatgcg 1560
gtgctgggcg agaagccgct ggcgctgtac aacagcagca gcgacccgaa atataccatc 1620
agcggcgttc tggaagtggt tgcggcgggt agcctgccga aactgaacat gatgccgaac 1680
cacaccctgc tgagcgacca gcaaatccag caagtgcaaa agattctgaa a 1731
<210> 29
<211> 1731
<212> DNA
<213> 人工序列
<220>
<223> GDI0177A_GDI0005A_like_Binary优化大肠杆菌
<400> 29
aagaaatttg acaccccggc gtaccaggcg gagaaggact tcaaagatca gccgaacctg 60
aagatccaaa ttgaaaacgc gtggagcaac tacgttaaat attgcaccat caacagccaa 120
atgggtaacc cgtggagcag cagctatgac cacccgcgta gctggtacta taacccgctg 180
gtgaccccgg ttaccccgat caaaaacaac accgtgccga ttcagtgggg cgcgtttccg 240
aaccgtatca accactactt caccgacctg ttcacccaaa agttcggtaa agcggacgcg 300
accgataaac tgcatgagct ggcggacatt ggtccggatg cgtttagcaa gaaatatgac 360
attaccctga ccgttaccaa gaacccgtgc gacccggata acaccgcgac caaaccgttt 420
ggtccgagcg gtccgcgtgg ctggcaggat gagtactgcg aatgggcggt tacccgtgac 480
accaacggtg gcatcattgc ggtggatttt acccacgaga acccggaata ttggttccac 540
atgtggaaag ttagccaaga catggtggtt agcctgtacc agcaaatcct gaacaacagc 600
aacgtgaaga aagaggacct gtatctgctg gatgaacacc acaacccggt gatcgttcgt 660
gaaaccggtc tgccggcgta caacccgatt aacaagtgga acaaaggcag cagcgcgacc 720
gcggagggtg gcggtgcggt tcatctgacc agcccgccga acagcctggg tgcggaaatc 780
tatctgggtg cggcggcgac cattctgcgt gtggttaacg gcaaggtgat caccgacgcg 840
aacaccctga tttgcgcggc gcagtacggt caaatctatc gtaacagcga tccgcgtatt 900
ggccagaacg tgaacagcct ggtttacaac aacaagctga aaatcagcct gaccaacccg 960
attgcgctgt atggtcagct gccggatttc acccaattta ccatgccggc gagcgcggag 1020
ggctacaaga tcgaagactg ctataaaatc attcgtggta ccgatattaa cccgggcacc 1080
accttttacc cgagcaacat gatcctgcac agccgttttg aggttccggc gggtgcgaac 1140
ttcaaactga gcgaaattct ggtgcagaac caaccgctga agtggggcag ccagatcgcg 1200
gacgtgttca aagttcaact ggcgggcacc ggtattccga ccagcggcga gaaaccgcag 1260
gaatacccgc cggtgggcga cccggatgtt accctgccga gcgtgcagta cctgctggat 1320
aacggtctgc tgcaagcgag cctgtataac aaactgaaca ccttcagcaa cctgaccagc 1380
tgcatcaccc agattgaagc gggtaccacc acccgtggta ttgcggttct ggcgagcgac 1440
gcgaaccaaa agaccggttt cgattttggc gcgggtatta acgcgagcgt gaccgacttt 1500
caggatctgg gcaacgacaa ccaactgttc atcattgaca tcaccgcgga ggcggatgcg 1560
gttctgggtg aaaagccgct ggcgctgtac aacaacgcga gcgatgcgcg ttataccctg 1620
agcggtgtgc tggaagtggt tgcgccgggc agcctgccga aactgaacat tgcggcgaac 1680
cacaccctgc tgagcgacca gcaaatccag caagtgcaga agattctgaa a 1731
<210> 30
<211> 1728
<212> DNA
<213> 人工序列
<220>
<223> GDI0179A_GDI0005A_like_Binary优化大肠杆菌
<400> 30
aagcagtttg ataccccggc gtaccaagcg gacaaggatt tcaaagacca gccggatctg 60
cgtaaccaaa tcgagaacgc gtggagcaac tacgttaaat attgcaccat taacagccag 120
atgggtaacc cgtggagcag cagctatgac cacccgcgta gctggtacta taacccgctg 180
gtgaccccgg ttaccccgaa caagaacaac accgttccga tccaatgggg cgcgtttccg 240
aaccgtatta accactactt caccagcctg tttaccaagg tgttcccgaa cgaggcgcag 300
gacaaactgc acgaactggc ggatattggt ccgaaggcgt ttaccgaaaa atatggcacc 360
caactggtgg ttccgaagaa cccgtgcgac ccgaccaaca ccgataccaa agcgtttggt 420
ccgagcggtc cgcgtggctg gcaggatgag tactgcgaat ggagcgttac ccgtgacacc 480
aacggtgata tcattgcggt gaactttacc cacgagaacc cggaatattg gttccacatg 540
tggaaggtta gccaggacat ggtggttagc ctgtaccaag agatcctgaa caacccgaac 600
gtgcaaaaag aggacctgta tctgctggat gaaaacggta acccggtgat cgttcgtgaa 660
accggcctgc cggcgtacaa cccgattaac aagtggaaca acggtagcag cgcgaccgcg 720
gagggtggcg gtgcggttca tctgaccagc ccgccgaaca gcctgggtgc ggaaatctat 780
ctgggtgcgg cggcgaccat tctgcgtgtg gttggcggta aagtgatcac cgatgcgaac 840
accctgattt gcgcggcgca gtacggtcaa atctatcgta acagcgaccc gcgtattggc 900
cagaacgtga acgcgctggt ttacaacaag aaactgaaga tcagcctgac caacccgatt 960
gcgctgtatg gtcagatgcc ggacttcacc caatttgcga tgccggatag cgcggagggc 1020
tacaccattg aagactgcta taaaatcatt cgtggtaccg cgaccaaccc gggcaccgat 1080
ttctacccgt ttaacatgat cctgcacagc cgttttgagg ttccggcggg tgcgcagttc 1140
aagctgagcg acatcaaagt gcagggtcaa ccgctgaagt ggggcagcca gattgcggat 1200
gtgtttaaag tgcagctggc gggtaccggt atcccgggcg gtagcgacaa accgcaggaa 1260
tacccgccgg tgggcgaccc ggatgttacc ctgccgagcg tgcagtacct gctggacaac 1320
aacctgctgc aagcgagcct gtataacaag ctgaacacct tcagcaacct gaccagctgc 1380
atcacccaga ttgaggcggg taccaccacc agcggcattg cggttctggc gagcgacgcg 1440
aacaaagaaa ccggtttcga ttttggcccg ggtatcagcg tgagcgttac cgactaccag 1500
gatctgggca acgacaacca actgttcatg atcgatatta ccgtgcatgc ggcggcgagc 1560
ctgggcgaga agccgctggc gctgtacaac aacaccagcg atccgaaata taccgtgagc 1620
ggtgttctgg aagtggttgc gccgggcagc ctgccgaagc tgaacatcac cccgaaccag 1680
accctgctga gcgaccagca aatcaaacag gtgcaaaaga ttctgaaa 1728
<210> 31
<211> 1731
<212> DNA
<213> 人工序列
<220>
<223> GDI0181A_GDI0005A_like_Binary优化大肠杆菌
<400> 31
aacaagttta acaccccggc gtaccaggcg gaaaaggact tcaaagatca accggacctg 60
aagaacaaaa tcgagaacgc gtggagcaac tacgtgaaat attgcaccat taacagccag 120
atgggtaacc cgtggagcag cagctacgat cacccgcgta gctggtacta taacccgctg 180
gtgaccccgg cgatcccggc gaagaacaac accgttccga ttcagtgggg tgcgtttccg 240
aaccgtatca accactattt cagcgacctg tttgcgcaga agttcggcaa aggcgaggcg 300
caagataagc tgtacgaact ggcggacatt ggcagcgagg cgtttagcaa gaaatatgcg 360
atcgaactga ccgttccgaa aaacccgtgc gatccgaaca acaccgcgaa gaaaccgttt 420
ggtccggcgg gtccgcgtgg ctggcaggac gagtactgcg aatgggcggt gacccgtgat 480
gcgagcggtg acatcattgc ggttaacttc acccacgaga acccggaata ttggtttcac 540
atgtggaagt tcagcccgga taccgtggtt agcctgtacc agcaaattct gaacaacccg 600
aacgtgaaga aagaagatct gtatctgctg gacagcagca acaacccggt gattgttcgt 660
gaaaccggtc tgccggcgta caacccgatc aacaaatgga accgtggcag cagcgcgacc 720
gaaaccgaag gtggcgcggt tcatctgacc agcccgccga acagcctggg tgcggagatc 780
tatctgggtg cggcggcgac cattctgcgt gtggttgatg gcaacgtgat caccgatgcg 840
aacaccctgg tttgcgcggc gcagtacggt caaatctatc gtaacagcga tccgcgtatt 900
ggccagaacg tgaacagcct ggtttacatt aacaagctga aagtgagcct gaccaacccg 960
atcgcgctgt atggtcaact gccggatttc acccagtttc aaatgccgga cagcgcggag 1020
ggctacacca ttgaagattg ctataagatc attcgtggta ccgacatcaa cccgggcacc 1080
accttttacc cgaacaacat gattctgcat agccgttttg aagcgccggc gggtgcgcgt 1140
ttcaagctga gcgacatcct ggtgcagggt caaccgctga aatggggcag ccagattgcg 1200
aacgtgttca acgttcagct ggcgggtacc ggtatcccgg gtggcggtga tcgtccgcaa 1260
gagtacccgc cggtgggtaa cccggcggtt accctgccga gcatccaata tgttctggac 1320
cacaacctgc tgctggcgag cctgcacaac aagctgaaca ccctgagcaa cctgaccagc 1380
tgcaccaccc aggtggaagc gggtaccacc accaaaggca ttgcggttct ggcgagcgat 1440
gcgaaccgtg aaaccggttt cgactttggc gcgggtatca gcgtgagcgt taaagatttt 1500
caggacctgg gcaacgataa ccaactgttc atcattgata ttaccgttga tgcggcggcg 1560
ctgctgggtg aaaagccgct ggcgctgtac aacaacacca gcgacccgaa atataacgtg 1620
agcggtgttc tggaagtggt tgcgaccggc agcctgccgc agctgaacag cctgccgaac 1680
caaaccctgc tgagcgacca gcaaatcacc caggttcaaa agctgctgaa a 1731
<210> 32
<211> 1734
<212> DNA
<213> 人工序列
<220>
<223> GDI0183A_GDI0005A_like_Binary优化大肠杆菌
<400> 32
accaagtttg acaccccggc gtatcaggcg gagaaggatt tcaaaaacga cccggatctg 60
aaaagccaaa tcgaaaacgc gtggagcaac tacgtgaaat attgcaccat taacagccag 120
atgggtaacc cgtggagcag cagctatgac cacccgcgta gctggtacta taacccgctg 180
gttaccccgg cggagccggc gaagaacaac accgtggcgg ttcaatgggg cgcgtttccg 240
aaccgtatca accactactt caccaacctg tttgtggaga agttcggtaa aaccgacgcg 300
ccggataaac tgcacgaact ggcggacatc ggcccggatg cgtttagcaa gaaatataac 360
attaccctgg tggttagcaa gaacccgtgc gacccgagca acaccgaaac caaaccgttt 420
ggtccgagcg gtccgcgtgg ctggcaggat gagtactgcg aatgggcggt gacccgtgat 480
gcgagcggtg atatcattgc ggttgacttt acccacgaga acccggaata ctggttccac 540
atgtggaaag tgagcccgga tatggtggtt agcctgtatc agcaaatcct gaacaacccg 600
aacgttaaga aagaggacct gtacctgctg gatgaaaagg gtaactatgt gatcgttcgt 660
gaaaccggcc tgccggcgta taacccgatt aacaaatgga accgtggtag cagcgcgacc 720
gcgggtggcg gtggcaccgt tcatctgacc agcccgccga acagcctggg tgcggagatt 780
tatctgggtg cggcggcgac cattctgcgt gtgaacagcc agggccgtgt tatcaccgac 840
gcgaacgaac tgatttgcgc ggcgcagtac ggtcaaatct atcgtaacag cgatccgcgt 900
attggccaaa acgtgaacag cctggtttac aacaagaaac tgaagatcag cctgaccaac 960
ccgattgcgc tgtatggtca gcaaccggac ttcacccagt ttaccatgcc ggatagcgcg 1020
aagggctaca aaatcgagga ctgctataaa gtgattcgtg gtagcgaaag caacccgggc 1080
accaccttct atccgtttaa catgattctg catagccgtt ttgaggcgcc ggcgggtgcg 1140
aagttcaaac tgagcgacat tatggttaag ggtagcaaga tcaaatgggg cagccagatt 1200
gcggatgtgt ttaaagttca gctggcgggt accggtatcc cgggtggcgg tgaccagccg 1260
cagcaatatc cggcggtggg tgatccggcg gttaccctgc cgagcgtgca gtacgttctg 1320
gataacaacc tgctgcaagc gagcctgtat aacaagctga acaccttcag caacctgacc 1380
agctgcatca cccaagtgga agcgggtacc attaccagcg gcatcgcgat tctggcgagc 1440
gacgcgaaca aaggcaccgg tttcgatttt ggcccgggta tcagcgtggc ggttaccgac 1500
tttcaggatc tgggtaacga caaccaactg ttcatcgtgg atattaacgt tcacagcgcg 1560
gcgctgctgg gtgaaaagcc gctggcgctg ttcaacaaca ccaccgaccc gaaatacacc 1620
attgcgggtg ttctggaagt ggttgcgccg ggcagcctgc cgaagctgga tgtggttccg 1680
aaccacagcc tgctgagcga tcagcaagtg agccaggttc aaaagatgct gaaa 1734
<210> 33
<211> 1731
<212> DNA
<213> 人工序列
<220>
<223> GDI0185A_GDI0005A_like_Binary优化大肠杆菌
<400> 33
aagacctttg acaccccggc gtaccaggcg gaaaaggact tcaaagataa cccggcgctg 60
cgtgagaagc tgcacaacgc gtggagcaac tacgttaaat attgcaccgt gaacagcatc 120
atgggtaacc cgtggagcag cacctatgat cacccgcgta gctggtacta taacccgctg 180
gttaccccga gcatcccgaa cgaaaacaac accgtgccga ttcaatggaa cgcgtttccg 240
aaccgtatca accactactt caccaccctg tttaccgaca agttcggcaa acaggactat 300
gaggataagc tgcacgaact ggcggatatc ggtccgattg cgtttggcca aaagtacaac 360
atgaaactga ccgttccgcg taacccgtgc gacccgaccg ataccggtac caaagcgttt 420
ggtccgagcg gtccgcgtgg ctggcaggac gagtactgcg aatggagcgt tacccgtgac 480
gagagcggcg atatcattgc ggtgaacttt acccacgaga acccggaata ttggttccac 540
atgtggaaga ttagcccgga taccgtggtt agcctgtacc aggagatcct gaacaacgaa 600
aacgtgcaaa aagaggacct gtatctgctg gatagccacg gtaacccggt gattgttcgt 660
gaaaccggcc tgccggcgta caacccgatc aacaagtgga acaacggtcc ggatgcgacc 720
agcagcggtg gcggtgcggt tcatctgacc agcccgccga acagcctggg tgcggagatc 780
tatctgggtg cggcggcgac cattctgcgt gtggttaacg gcaaagtgat caccgacgcg 840
aacaccctga tttgcgcggc gcagtacggt caaatctatc gtaacagcga tccgcgtatt 900
ggccagaacg ttaacagcct ggtgtacaac cacaacgtgc aagttagcct gaccaacccg 960
atcgcgctgt atggtcagat tccgcacttc gaccaatttg aaatgccggc gaccgcgaac 1020
tacaagattg aggattgcta taccgtggtt cgtggtgcgc tgaagaacaa aggcatcacc 1080
tactatccga acaacatgct gctgcacacc cgttttagcg ttccggcgga cgcgaacttc 1140
aagctgagcg atattctggt gaacaagaaa ccgctgaaat ggggtagcca gatcgcggac 1200
accttctttg ttcaactggc gggtaccggt ctgagcccgg cgcagggtca gcaaagcgaa 1260
aagtttccgc cggttggtat cccggcgacc accctgccga gcgtgcagta cctgctggat 1320
aacaacctgc tgcaagcgag cctgtataac aaactgaaca ccttcagcaa cctgaccagc 1380
tgcatcaccc aagttgaagc gggtaccacc accgagggta ttgcggtgct ggcgaacggt 1440
gcgattcagc aaaccggctt cgactttggc ccgggtgtga ccgttgcggt gaccgatttt 1500
cagaacctgg acgaagatac ccaactgttc ctgatcagca ttaccaccga cggcggtgtt 1560
gcgctgggcg agaaaccgct gaccctgtac aacaacgcga gcgatccggg ttttgcgctg 1620
agcggcgtgc tggaagtggt tgcggcgggc agcctgccga agaccgacag caccccgaac 1680
cgtaccctgc tgagcagcca gcaaatcgag caggtgaaga aaattctgaa a 1731
<210> 34
<211> 1734
<212> DNA
<213> 人工序列
<220>
<223> GDI0187A_GDI0005A_like_Binary优化大肠杆菌
<400> 34
aagaaattta acaccccggc gtaccaggcg gagaaagact tcaagcacga accgcatctg 60
ggtgcgcagc tggagaacgc gtggagcaac tacgtgcaat attgcaccat caacagcatt 120
atgggcaacc cgtggagcag cacctacgat cacccgcgta gctggtacta taacccgctg 180
gttgacccgg ttatcccgac cgagaaaaac accgttgcga ttcagtggaa cgcgtttccg 240
aaccgtatca accactactt caccaacctg tttacccaga aattcggcaa ggaccaattt 300
gacgataagc tgcacgaact ggcggatatt ggtccggcgg cgtttggcaa gaaatatgac 360
atggtgctga ccgttccgaa aaacccgtgc gacccgagcg ataccggtac caaaccgttt 420
ggtccgagcg gtccgcgtgg ctggcaggac gagtactgcg aatgggcggt gacccgtgac 480
gaaaacggcg atatcattgc ggttgatttt acccacgaga acccggaata ttggttccac 540
atgtggaaag tgagcccgga catcgtggtt agcctgtacc aagagattct gaacaacaag 600
aacgttaaga aagaagacct gtatctgctg gatagcaacg gtaacccggt gattgttcgt 660
gaaaccggcg aaccggcgta caacccgatc aacaaatgga accgtggtcc ggtggcgacc 720
ccggagggtg gcggtgcggt tcatctgacc agcccgccga acagcctggg tgcggaaatc 780
tatctgggtg cggcggcgac cattctgcgt gtgaaggaca accaggttat caccgatgcg 840
aacgcgctga tttgcgcggc gcagtacggt caaatctatc gtaacagcga cccgcgtatt 900
ggccagaacg tgaacagcct ggtttacaac tataaccaaa aaatcaccct gaccaacccg 960
attgcgctgt acggtcaggt gccggacttc gatcaatttg atatgccgag caccgcgggc 1020
aactacacca ttgaggactg ctataccgtg gttcgtggtg aggaacgtaa caacggcatc 1080
accttctatc cgtttaacat gctgctgcac acccgtttta gcgtgccgga aggtgcgaac 1140
ttcaaactga gcgatattaa ggttaaaggc aagctgctga agtggggcag ccagatcgcg 1200
gataccttct ttgttcaact ggcgggtacc ggtaaagatc cggcggcggg cgagcagccg 1260
gaaaagtttc cgccggtggg cgatccggcg accatcctgc cgaacgttca gtacctgctg 1320
gacaacaacc tgctgcaagc gagcctgtat aacaaactga acaccttcag caacctgacc 1380
agctgcatca cccaaattga ggcgggtacc gcgaccgaag gcattgcggt gctgaccaac 1440
ggtgcggttc gtgacaccca cttcgatttt ggcccgggta tcagcgtgac cgttaccgat 1500
tttcagaacg tggacgagga tacccaactg ttcctgatca ccattgaaac cgacgcgaac 1560
gttagcctgg gcgaaaaacc gctgagcctg tacaacaacg cgagcgatcc gaagtatgcg 1620
ctgagcggtg tgctggaagt ggttgcgagc ggcagcctgc cgaaggttaa cattaccccg 1680
aaccgtaccc tgctgagcgg tcagcaagtg gaacaggttc aaaaaatcct gaag 1734
<210> 35
<211> 1368
<212> DNA
<213> 人工序列
<220>
<223> GDI0176A_GDI0006A_like_Binary优化大肠杆菌
<400> 35
cgtaccctgc tgttcagctt ctttctgctg agcctgatca gcattagctg cgagagcggt 60
aagaaagaca agaaactgaa cagccgtctg ggcttcagca aggaaatggc ggacatcgat 120
tgcggtgcgt tttgcgaccc gccgaccgtg ggttatgagc tgccggcgga tagcacctgc 180
ggtcacagca gccagagcgt tctgaactgc ttcgcgtgga aaaactttct ggcgctgaac 240
tggcgtgcga gcgatgaacg tggcctgccg gataccaccg cggtggcggc ggactatggt 300
atgccgggcg attatagccc gaccgtgtgg gagagctatc tgagcgcgga cgatgttttc 360
gcggcgaagc agccggaaca atggaacctg aaaagcaaga acggttacat caagtacatc 420
aacgagatca acaagttcac cgacattaac gcgagcctgc cgaagccgaa actgcgtgcg 480
atgctgggtg gcaacgtgga cgaaatcatg caggcgaagg gtgcgtggct gaccgatcaa 540
agcggcaaca ttgtttggta cgagatccgt atgaacagca ttgaaagcga tttcatccgt 600
aagaacaaac tgtatagcag cgagaacctg aacgcgtttg cggcgaagaa ccagggtgtg 660
tggctgccga tggagagcat cgaaattaaa gcggcgtggc gtgttattcc ggagaaccaa 720
ctggaaagcc tgaagaactt ctacaagatt agcaaagcga tggtgccgga aatcaagggt 780
tttgacaaaa acaaccagcc gatctacggc aagtataccc aaaaatatct gggtctggtt 840
ggcctgcaca tcattcgtaa gaccaaccag agcccgcaat tcacctggat gacctttgag 900
cacgtgaaca acgcgccgac cgagggtcag gttgatccga gcattaaata ctgcttttat 960
aacccgaaaa gcaccgacaa accgaaccag agcccggttc cgggcaagga tagcctgagc 1020
aaaccggtgc aagttatccg tattgcgaac aacgcgatta ccccggagat ccagaacctg 1080
aacaagcaaa ttcgtgatat gatcaaggcg agcaacccga aaagcgtgtg gcagtactat 1140
caactggtga acgttcagtg gccggaaaac ccgatccaag acggtaacaa caacaaaacc 1200
gcgccgctga tggatggtgg cattaccccg aacaacatcg cgaacgttac catggaaacc 1260
tacattcagg aaaagcaatg catggactgc cacaaaaacg cgagcgtggg tgcgcagaag 1320
tacccgaccg attatagctt catctttctg aaggttaaac aagcgaaa 1368
<210> 36
<211> 1368
<212> DNA
<213> 人工序列
<220>
<223> GDI0178A_GDI0006A_like_Binary优化大肠杆菌
<400> 36
cgtaccctga ttttcagctt ctttctgctg agcctgatca gcattagctg cgagagcggt 60
aagaaagaca agaaactgaa cagccgtctg ggcttcagca aggaaatggc ggacatggat 120
tgcggtgcgt tttgcgaccc gccgaccgtg ggttaccagc tgccggcgga tagcatctgc 180
ggtcacagca gccaaagcgt tctgaactgc ttcgcgtgga agaactttct ggcgctgaac 240
tggaaggcga gcgatgagcg tggcctgccg gataccaccg cggttgcggc ggactatggt 300
atgccgggcg attatagccc gaccgtgtgg gagagctatc tgagcgcgga cgatgttttc 360
gcggcgaagc agccggaaca atggaacctg aaaagcaaga acggttacat caagtacatc 420
aacgagatca acaagttcac cgacgtgaac gcgagcctgc cgaagccgaa actgcgtgcg 480
atgctgggtg gcaacgttga cgaaatcatg caggcgaagg gtgcgtggct gaccgatcaa 540
agcggcaaca ttgtgtggta cgagatccgt atgaacagca ttgaaagcga tttcatccgt 600
aagaacaaac tgtatagcag cgagaacctg aacgcgtttg cggcgaagaa ccagggtgtt 660
tggctgccga tggagagcat cgaaattaaa gcggcgtggc gtatcattcc ggaggaccaa 720
ctggaaagcc tgaagaactt ctacaagatt agcaaagcga tggtgccgga aatcaagggt 780
tttgataaaa acaaccagcc gatctacggc aagtataccc aaaaatatct gggtctggtg 840
ggcctgcaca tcattcgtaa aaccaaccag agcccgcaat tcacctggat gacctttgag 900
cacgttaaca acgcgccgac cgagggtcag gtggacccga gcgttaagta ctgcttttat 960
aacccgaaaa gcaccgacca gccgaaccaa agcccggtgc cgggcaagga tagcctgagc 1020
aaaccggtgc aggttatgcg tattgcgaac aacgcgatta ccccggagat ccagaacctg 1080
aacaagcaaa ttcgtgatat gatcaaggcg agcaacccga aaagcgtttg gcagtactat 1140
caactggtga acgttcagtg gccggaaaac ccgatccaag acggtaacaa caacaaaacc 1200
gcgccgctga tggatggtgg cattaccccg aacaacatcg cgaacgtgac catggaaacc 1260
tacattcagg aaaagcaatg catggactgc cacaaaaacg cgagcgtggg tgcgcagaag 1320
tacccgaccg attatagctt catctttctg aaggttaaac aagcgaaa 1368
<210> 37
<211> 1368
<212> DNA
<213> 人工序列
<220>
<223> GDI0180A_GDI0006A_like_Binary优化大肠杆菌
<400> 37
cgtaccctgc tgttcagctt ctttctgctg agcctgatca gcattagctg cgagagcggt 60
aagaaagaca agaaactgaa cagccgtgtg ggctttagcc agaaaatggc ggacttcgat 120
tgcggtgcgt tttgcgaccc gccgagcgtt acctaccagc tgccggcgga tagcagctgc 180
gtgaacagca gccaaaacgt tatgaactgc ttcgcgtgga agaactttct ggcgctgaac 240
tggctggcga gcgaccagcg tggcgtgccg gataccgcgg cggttgcggc ggactatggt 300
atgccgggcg attataaacc gaccgtgtgg gaaagctatc tgagcatcga cgatgttttt 360
gcggcgaagc cgccggcgca atggaacctg cgtagcaaaa acggttacat gaagtacatc 420
aacgagatca acaagttcac cgatattaac gcgagcctgc cgaagccgaa actgcgtgcg 480
atgctgggtg gcaacgtgga cgaaatcatg caggcgaagg gtgcgtggct gaccgatcaa 540
agcggcaaca tcgtgtggta cgagattcgt atgaacacca tcgaaagcga cttcgttcgt 600
gataacaaac tgtacaacta tggcagcctg agcgcgtttg cggcgcagaa ccaaggtgtt 660
tggtttccga tggagagcat cgaaattaag gcggcgtggc gtatcattcc ggaggaccag 720
ctggaaagcc tgaaaaactt ctacaagatt agcatggcga tggtgccgga gatcaaaggt 780
tttgataaaa acaacaagcc gatctacggc aaatatctgc aaaagtatct gggtctggtt 840
ggcctgcaca tcattcgtaa gaccaaccag agcccgcaat tcacctggat gacctttgaa 900
cacgtgaaca acgcgccgac cgacggccag attgatccga gcgttaaata ctgcttctat 960
gacccgaaaa gcaaagataa gccgaaccag agcccggtgc cgggtaaaga cagcctgaac 1020
aagccggttc aagtggttcg tatcgcggat aacgcgatca gcccggagat tcagcaactg 1080
aacaagcaga tccaaaacat gattaaagcg agcaacccga aaagcgtgtg gcagtactat 1140
caactggtga acgttcagtg gccggaaaac ccggttaaag acaaggataa caacaagaaa 1200
gcgccgctga tgaccggtgg cattaccccg aaaaacatcg cgaacgtgac catggaaacc 1260
tacattcagg aaaaacaatg catggactgc cacaagaacg cgagcgtggg cgaccaaaag 1320
tacccgaccg attatagctt catctttctg aaagttaagc cgggtaac 1368
<210> 38
<211> 1362
<212> DNA
<213> 人工序列
<220>
<223> GDI0182A_GDI0006A_like_Binary优化大肠杆菌
<400> 38
cgtaccctgc tgttcagctt ctttctgctg agcctgatca gcattagctg cgagagcggt 60
aagaaagaca agaaactgaa cagccgtatc ggcttcagca aagaactgga cgattatggt 120
tgcggtgcgt tttgcgaccc gccgagcgtg ggttatcagc tgaccgacga taactgcctg 180
cacagcgatc aaaacagcat gaactgcttc gcgtggaaga actttctggc gctgaactgg 240
atcgcgagcg accagcgtgg cattccggat accaccgcgc tggcgagcga ctatggtatg 300
ccgggcgatt ataaaccgac cgtgtgggag agctacctga gcatcaacga cgtttttacc 360
gcgcagcaac cggcgcagtg gagcctgaaa agcaagagcg gttacatcaa gtacatcaac 420
gagatcaaca agttcaccga tattaacgtg aacatcccga agccgaaact gcgtgcgatg 480
ctgggtggca acgttgacga aatcatgcag gcgaagggtg cgtggctgac cgatcaaagc 540
ggcaacattg tgtggtacga gatccgtatg aacaacattg aaagcgactt cgttcgtaac 600
aacaagctgt ataacagcga gaacctgaac gcgtttgcgg cgaaaaacca gggtgtgtgg 660
ctgccgatgg agagcatcga aattaaagcg gcgtggcgtg ttatcccgga gaaccaactg 720
gaaagcctga aggacttcta caaaatcagc atggcgatgg tgccggaaat taagggtttt 780
gataaaaaca accagccgat ttacggcaag tatacccaaa aatatctggg tctggttggc 840
ctgcacatca ttcgtaagac cagccagagc ccgcaattca cctggatgac ctttgagcac 900
atcaacaacg cgccgaccga gggtcaggtg gatccgagcg ttaactactg cttttataac 960
ccgaaaagca aagataaacc gaaccagagc ccggtgccgg gcaaggatag cctgaacaaa 1020
ccggttcaag tggttcgtat tgcgaacaac gcgattaccc cggaaatcca gcaactgaac 1080
aagcagattc aaagcatgat ccgtgcgagc aacccgaaaa gcgtgtggca gtactatcaa 1140
ctggtgaacg ttcagtggcc ggagaacccg gttcaagaca aggataacaa aaacaccccg 1200
ccgctgcgtg acggtggcat caaaccgaag aacattgcga acgttaccat ggaaacctac 1260
atccaggaca agcaatgcat ggattgccac aaaaacgcga gcaccgtggc gaccaagtac 1320
ccgaccgatt acagcttcat ttttctgaag gttaagccga aa 1362
<210> 39
<211> 1368
<212> DNA
<213> 人工序列
<220>
<223> GDI0184A_GDI0006A_like_Binary优化大肠杆菌
<400> 39
cgtaccctgc tgatcagctt ctttctgctg agcctgatcg cgattagctg cgagagcggt 60
aagaaagaca agaaactgaa cagccgtgtg ggcttcagcc aggaactggc ggacttcgat 120
tgcggtgcgt tttgcgaccc gccgagcgtg agctaccagc tgccggttga tagcacctgc 180
ggccacagca gccaaaacgt gctgaactgc ttcgcgtgga aaaactttct ggcgctgaac 240
tggaaagcga gcgatgagcg tggtctgccg gataccaccg cggttgcggc ggactatggt 300
atgccgggcg attatagccc gaccgtgtgg gagagctatc tgagcatcga agacgttttc 360
agcgcgcgtc agccgcaaac ctggaacctg aaaagcaaga acggctacat caagtacatc 420
aacgagatca acaagttcac cgatatcaac gcggcgctgc cgaagccgaa actgcgtgcg 480
atgctgggtg gcaacgttga cgaaattatg caggcgaagg gtgcgtggct gaccgatcaa 540
agcggcaaca ttgtgtggta cgagatccgt atgaacaaca ttgaaagcga cttcgttcgt 600
cagaacaaac tgtataacag cgataacctg aacgcgtttg cggcgaagaa ccaaggtgtg 660
tggctgccga tggagagcat cgaaattaag gcggcgtggc gtatcattcc ggacagccag 720
ctggagagcc tgaaaaacct gtacaagatc agccgtgcga tggttccgga aattaaaggt 780
ttcgataaga acaaccaacc gatctacggc aaatatagcc cgaagtatct gggtctggtg 840
ggcctgcaca tcattcgtaa aaccaaccag agcccgcaat tcacctggat gacctttgag 900
cacgttaaca acgcgccgac cgagggtcag gtggacccga gcgttaagta ctgcttttat 960
aacccgaaaa gcaaagacaa gccgaaccag agcccggtgc cgggtcaaga tagcctgaac 1020
accccggttc aagtggttcg tatcgcggac aacgcgatta gcgcggatat ccagcaactg 1080
aacaaacaga ttcaagcgat gatcaagaaa agcaacccga aaagcgtgtg gcagtactat 1140
caactggtga acgttcagtg gccggaaaac ccgattcaag acaccaacaa caacaaaacc 1200
gcgccgctga tggatggtgg catcaccccg agcaacattg cgaacgtgac catggaaacc 1260
tacatccagg aaaaacaatg catggactgc cacaagaacg cgagcgttgg cgcgcagaaa 1320
tacccgaccg attatagctt catttttctg aaagcgaagc cgggtaag 1368
<210> 40
<211> 1367
<212> DNA
<213> 人工序列
<220>
<223> GDI0186A_GDI0006A_like_Binary优化大肠杆菌
<400> 40
aaggcgctga tcctgatttg gagcctggcg atcgtgagca ttgttagctg cgagagcagc 60
aagaaagaca agaaactgaa cagcgcgacc ggtttcagca agaaagtgac cggtgaaacc 120
atcttctgcg gtgcgtactg caacccgccg gcgatcagct atgaactggc ggcggatagc 180
acctgcgcgc acagcagcca ggaagttctg aactgcttcg cgtggaaaaa ctttattgcg 240
ctgaactgga ttgcgagcgc gcaacgtggt attccggaca ccaccgcgac cgcggcgaac 300
tatggtatgc cgggcgatta tagcccgacc gtgtgggaga gcttcctgag cattgacgat 360
gtttttgcgc cgaagccgcc gctggcgtgg aacctgaaaa gcaaaaccgg ttacatcaag 420
cgtattaacg aaatcaacaa attcaccgac atcattagca gcctgccgaa agcgaccctg 480
cgtgcggcgg tgggcagcag caacgttgac gagattatgc aggcggaggg tgcgtggctg 540
accgatcaaa acggcaacat cgtgtggtat gagatccgta ttaacaacct ggaaagcgac 600
ttcattcgtc agaacaagct gtacgactat gataacctga aggcgtttgg taccaaaaac 660
aacggcgtgt ggctgccgaa cgagagcatt gaactgaaag cggcgtggcg tgttatcccg 720
gacgatcagc tggatagcct gaagaactac tataagatta gcaaagcgat ggtgccggag 780
atcaaaggtt ttaacggcaa gaaaccgatc tacggcaagt atacccagaa atacctgggt 840
ctggttggcc tgcacatcat tcgtaagacc ccgcagagcc cgcaactgaa ctggatgacc 900
ttcgaacacg tgaacaacgc gccgggtccg ggtccggcgg atccgagcgt taagtacagc 960
ttttataacc cgaacagcaa agatccggcg aaccagagcc cggtgccggg caaggatagc 1020
ctgaacaaac cggtgcaagt ggttcgtgtt aaccgtatca gccagagcgt tcaaaagctg 1080
aacgcgcaga tgcagcaact gattcgtgcg agcaacccga aaagcgtgtg gcagtactat 1140
caactggtta acatccaatg gccggagaac ccggtggcgg acaaggataa caacacccag 1200
accccgctga tggagggtgg cgttaaaccg aaccaaatta gcaacaccac catggaaacc 1260
tacgcgcaga gcaagcaatg catggactgc cacaaaaacg cgccggtgat tggtaccagc 1320
atcccgaccg attatagctt catctttctg aaggttaaac cgaagaa 1367
<210> 41
<211> 1395
<212> DNA
<213> 人工序列
<220>
<223> GDI0188A_GDI0006A_like_Binary优化大肠杆菌
<400> 41
aaagcgctga tctgcagcct gttcattctg ggtctgatca gctgcgagag cggcaagaaa 60
gataagaaac tgaacagcgc gaccggtttt agcaaagagg tggcgggtga aagcatttac 120
tgcggcgcgt attgcgaccc gccgagcatc acctacaaac tggcgggtga cagcacctgc 180
gcgcacagca gccaggatgt gctgaactgc ttcgcgtgga agaactttat tgcgctgaac 240
tggatcgcga gcgcgcaacg tggtgttccg gacaccaccg cgaccgcggc gaactatggt 300
atgccgggcg attatagccc gaccgtgtgg gagagcttcg cgagcaacga cgaagttttt 360
gcggcgaaaa acccgctggc gtggaacctg aaaagccgta acacctacgt taagcagatc 420
aacgaaatca acaagttcac cgatatcaac atcagcattc cgaaggcgac cctgcgtgcg 480
gcggtgggta gcaacaacgt tgacgagatt ctgcaggcgg aaggtagctg gctgaccgat 540
caaagcggca acatcgtgtg gtacgagatc aagatcaaca acatcgaaag cgacttcatc 600
cgtaagaaca aactgtacga ctataacagc ctgaaggatt atggtaccgc gaacaaaggc 660
gtttggctgc cgatggagag catcgaactg aaggcggcgt ggcgtatcat tccggaggac 720
aaactggata gcctgaagaa ctactataaa attagcaagg cgatggtgcc ggaaatcatt 780
ggtttcaaag ataagaaacc gatctttggc aaaagcaccc aaaagtacct gggtctggtt 840
ggcctgcaca tcattcgtaa gaccccgcag agcccgcaat tcaactggat gacctttgag 900
cacgtgaaca acgcgccgaa cgagggtcag gcggacccgg cggttaagta ctgcttctat 960
aacccgaaaa gcaaagacac cccgaacatt gcgccgaaga tcggctatga tagcctgaac 1020
aaaccggtgc aagtggttcg tgttaacaag attaaaacca agctgcagaa gctgaacgcg 1080
caaatgcagc aactgatccg tgcgagcaac ccgaaaagcg tgtggcagta ctatcaactg 1140
gttaacattc agtggccgga gaacccgatc caagataacg gtaacaacag cgcggcgccg 1200
ctgatggagg gtggcattac cccgagcgac atcagcaaca ccaccatgga aacctacgcg 1260
cagaccaagc aatgcatgga ttgccacaaa tatgcgagcg tggttggtag cggcatgccg 1320
ccaaccgact acagcttcat ctttctgaaa gtgaagccgg ttaagcagct gccgaagaaa 1380
accgcgccgg tgaaa 1395
<210> 42
<211> 53
<212> PRT
<213> 玉米
<400> 42
Met Ala Ala Ala Phe Ser Ser Thr Val Gly Ala Pro Ala Ser Thr Pro
1 5 10 15
Thr Arg Ser Ser Phe Leu Gly Lys Lys Leu Asn Lys Pro Gln Val Ser
20 25 30
Ala Ala Val Thr Tyr His Gly Lys Ser Ser Ser Ser Asn Ser Arg Phe
35 40 45
Lys Ala Met Ala Ala
50
<210> 43
<211> 70
<212> PRT
<213> 玉米
<400> 43
Met Ala Ala Ala Thr Ser Ser Ser Thr Tyr Leu Ala Ile Gly Arg Lys
1 5 10 15
Thr Leu Asn Pro Ala Pro Ser Val Ala Thr Ala Ala Thr Ser Val Ser
20 25 30
Phe Pro Ala Thr Gln His Pro Cys Leu Val Ala Ala Ser Ala Asp Arg
35 40 45
Arg Arg Ala Val Ala Ala Lys Val Ser Ser Pro Ser Val Ile Gly Thr
50 55 60
Ala Met Pro Ser Leu Asp
65 70
<210> 44
<211> 66
<212> PRT
<213> 玉米
<400> 44
Met Ala Thr Ala Ala Ala Ala Ser Thr Ala Leu Thr Gly Ala Thr Thr
1 5 10 15
Ala Ala Pro Lys Ala Arg Arg Arg Ala His Leu Leu Ala Thr Arg Arg
20 25 30
Ala Leu Ala Ala Pro Ile Arg Cys Ser Ala Ala Ser Pro Ala Met Pro
35 40 45
Met Ala Pro Pro Ala Thr Pro Leu Arg Pro Trp Gly Pro Thr Asp Pro
50 55 60
Arg Lys
65
<210> 45
<211> 1398
<212> DNA
<213> Chryseobacterium arthrosphaerae
<400> 45
atgaaaactt tagtattttc gctttttata ttgagcttca tctcatgtga atccggtaag 60
aaagaccagc gactgaattc agcaaccggt ttttcaaaag tagcagacag tgaatcgatc 120
tattgcggtg cgttttgtga tcctccttct attacttata aacttgccgg ggattccacc 180
tgtgcgcaca gttcgcagga tgtgctgaat tgttttgcat ggaagaattt tattgcacta 240
aactgggctg cttctgctca aagaggtgtt ccggatacca ctgcaacagc agccaattac 300
ggtatgcccg gggattacag ccctacagtc tgggagagct ttgcaagcaa tgacgaagtt 360
tttgccccca aaaatctttt aacatggaac ctgaaaagta agaatgggta tgtaaaacaa 420
attaatgaga ttaataaatt tacagatatt aatatcagta ttcccaaagc tactctcaga 480
gctgcagtag gaaatagtaa tgttgatgaa atattacagg ctgaaggttc ctggcttact 540
gatcagagcg gaaatattgt ttggtatgaa attaaaatca acaatattga aagtgatttt 600
atccgccgga ataagctgta tgattataat agtctgaaag agtatggtac tgcaaataac 660
ggcgtgtggc tgcccatgga gtctatagaa cttaaagctg cctggcgtgt cattcctgaa 720
gataaacttg actctttgaa aaattattat aaaatttcaa aagccatggt tccggaaatt 780
aaaggtttta aagataaaaa gcctgtcttt ggaaaatcca cacagaaata tttgggactg 840
gtgggcctcc atattatcag gaaaactcca cagtcacctc agtttaactg gatgacattt 900
gagcatatac ataatgctcc taatgaagga caggctgatc cctctgtaag gtattgcttc 960
tacaatccta aaagtacaaa gactcccaat atcgctccgg taatagggaa ggacagtctg 1020
aatactcctg ttcaggtcgt acgggtgaac aaaatcaaaa caaagcttca aaagctgaac 1080
acccagatgc agcagcttat cagggcaagt aaccctaaat cagtatggca gtattaccag 1140
cttgtgaata tccagtggcc ggaaaatccg atacaggata atggaaataa taagtctgct 1200
ccacttatgg aaggagggat taccccttcg gatatttcga atacaacgat ggaaacctat 1260
gctcaacaaa aacagtgcat ggactgtcat aagtatgctt cagtagtggg gagcggtatg 1320
ccgcctaccg attacagttt tatattttta aaggtaaaac cggaaaagca gattccaaaa 1380
ggaaaaactc ctgtaaaa 1398
<210> 46
<211> 1767
<212> DNA
<213> 人工序列
<220>
<223> GDI0005核优化假单胞菌
<400> 46
catatgcacc accaccacca ccacaagaag ttcaacaccc cggcctacca ggccgaaaag 60
gacttcaagc agtacccgca cctgggcgaa cagctggaga acgcctggag caactacgtg 120
aagtactgca ccatcaacag catcatgggc aacccgtgga gcagcaccta cgaccacccg 180
cgcagctggt actacaaccc gctggtcgac gacgtggtgc cgaccgaaca gaacaccgtg 240
ccgatccagt ggaacgcctt cccgaaccgc atcaaccact acttcaccgg cctgttcacc 300
aagcagttcg gcagcgccga atacgaggac aagctgcacg agctggccga catcggcccg 360
gccgccttcg gcaagaagta caacatggac ctgaccgtgc cgaagaaccc gtgcgacccg 420
agcgacaccc gcaccaagcc gttcggcccg agcggcccgc gcggctggca ggacgaatac 480
tgcgagtggg ccgtgacccg cgacgaaaac ggcgacatca ccgccgtgga cttcacccac 540
gaaaacccgg agtactggtt ccacatgtgg aagatcagcc cggacatcgt ggtggccctg 600
taccaggaaa tcctgaacaa caagaacgtg aagaaggaag acctgtacct gctggacagc 660
accggcaacc cggtgatcgt gcgcgaaacc ggcgagccgg cctacaaccc gatcaacaag 720
tggaacaacg gcccggaagc caccccggaa ggcggcggcg ccgtgcacct gaccagcccg 780
ccgaacagcc tgggcgccga gatctacctg ggcgccgccg caaccatcct gcgcgtgaag 840
aacaaccagg tgatcaccga cgccaacgcc ctgatctgcg ccgcccagta cggccagatc 900
taccgcaaca gcgacccgcg catcggccag aacgtgaaca gcctggtgta caaccacaag 960
cagcagatca ccctgaccaa cccgatcgcc ctgtacggcc aggtgccgga cttcgaccag 1020
ttcgaaatgc cgagcaccgc cggcagctac aagatccagg actgctacac cgtggtgcgc 1080
ggcgaagaac gcaacaaggg catcaccttc tacccgttca acatgctgct gcacacccgc 1140
ttcagcgtgc cgaagggcgc caacttcaag ctgagcgaga tcaaggtgaa gggcaagctg 1200
ctgaagtggg gcagccagat cgccgacacc ttcttcgtgc agctggccgg caccggcaag 1260
agcccgggcg ccggcgaaca gccggagaag ttcccgccgg tgggcgaccc ggccaccacc 1320
ctgccgaacg tgcagtacct gctggacaac aacctgctgc aggccagcct gtacaacaag 1380
ctgaacacct tcagcaacct gaccagctgc atcacccaga tcgaagccgg caccagcacc 1440
gaaggcatcg ccgtgctgac caacgccgcc accaaggaga cccagttcga cttcggcccg 1500
ggcatcagcg tgatggtgac cgacttccag aacatcgatg aagacaccca gctgttcctg 1560
atcaccatca ccgccgacgc cgacaccagc ctgggcgaga agccgctgag cctgtacaac 1620
aacgccagcg acccgaagta cgccctgagc ggcgtgctgg aagtggtgcc gaacggcagc 1680
ctgccgaaga tcaacaccac cccgaacctg gccctgctga gcggccagca ggtggagcag 1740
gtgaagaaga tcctgaagtg aggatcc 1767
<210> 47
<211> 1428
<212> DNA
<213> 人工序列
<220>
<223> GDI0006核优化假单胞菌
<400> 47
catatgcacc accaccacca ccacaagacc ctggtgttca gcctgttcat cctgagcttc 60
atcagctgcg aaagcggcaa gaaggaccag cgcctgaaca gcgccaccgg cttcagcaag 120
gtggccgaca gcgagagcat ctactgcggc gccttctgcg acccgccgag catcacctac 180
aagctggccg gcgacagcac ctgcgcccac agcagccagg acgtgctgaa ctgcttcgcc 240
tggaagaact tcatcgccct gaactgggcc gccagcgccc agcgcggcgt gccggacacc 300
accgccaccg ccgccaacta cggcatgccg ggcgactaca gcccgaccgt gtgggaaagc 360
ttcgccagca acgacgaggt gttcgccccg aagaacctgc tgacctggaa cctgaagtcg 420
aagaacggct acgtgaagca gatcaacgaa atcaacaagt tcaccgacat caacatcagc 480
atcccgaagg ccaccctgcg cgccgccgtg ggcaacagca acgtggacga aatcctgcag 540
gccgagggca gctggctgac cgaccagagc ggcaacatcg tgtggtacga aatcaagatc 600
aacaacatcg agagcgactt catccgtcgc aacaagctgt acgattacaa cagcctgaag 660
gagtacggca ccgccaacaa cggcgtgtgg ctgccgatgg aaagcatcga gctgaaggcc 720
gcctggcgcg tgatcccgga agacaagctg gacagcctga agaactacta caagatcagc 780
aaggccatgg tgccggagat caagggcttc aaggacaaga agccggtgtt cggcaagagc 840
acccagaagt acctgggcct ggtgggcctg cacatcatcc gcaagacccc gcagagcccg 900
cagttcaact ggatgacctt cgaacacatc cacaacgccc cgaacgaggg ccaggccgac 960
ccgagcgtgc gctactgctt ctacaacccg aagtcgacca agaccccgaa catcgccccg 1020
gtgatcggca aggacagcct gaacaccccg gtgcaggtgg tgcgcgtgaa caagatcaag 1080
accaagctgc agaagctgaa cacccagatg cagcagctga tccgcgccag caacccgaag 1140
tcggtgtggc agtactacca gctggtgaac atccagtggc cggaaaaccc gatccaggac 1200
aacggcaaca acaagagcgc cccgctgatg gaaggcggca tcaccccgag cgacatcagc 1260
aacaccacca tggagaccta cgcccagcag aagcagtgca tggactgcca caagtacgcc 1320
agcgtggtgg gcagcggcat gcctccgacc gactacagct tcatcttcct gaaggtgaag 1380
ccggagaagc agatcccgaa gggcaagacc ccggtgaagt gaggatcc 1428

Claims (24)

1.一种分离的核酸序列，其编码选自由以下组成的组的蛋白质：
a)与SEQ ID NO:1(GDI0005A)具有至少70％同一性的第1组蛋白质，以及
b)与SEQ ID NO:2(GDI0006A)具有至少65％同一性的第2组蛋白质。
2.根据权利要求1所述的分离的核酸序列，其具有选自由以下组成的组的序列：
a)编码所述第1组蛋白质的SEQ ID NO:3、5、28、29、30、31、32、33或34，以及
b)编码所述第2组蛋白质的SEQ ID NO:4、6、35、36、37、38、39、40或41。
3.一种由权利要求1或2所述的核酸序列编码的分离的多肽。
4.根据权利要求3所述的分离的多肽，其具有选自由以下组成的组的序列：
a)对于第1组蛋白质，SEQ ID NO:1、11、12、13、14、15、16或17，以及
b)对于第2组蛋白质，SEQ ID NO:2、18、19、20、21、22、23或24。
5.一种包含至少一种权利要求1或2所述的核酸序列的载体。
6.根据权利要求5所述的载体，其包含两种核酸序列，其中一种核酸序列是如权利要求1a)或权利要求2a)中定义的序列的，而另一种核酸序列是如权利要求1b)或权利要求2b)中定义的序列的。
7.根据权利要求5或6所述的载体，其中所述核酸序列与启动子可操作地连接。
8.一种包含权利要求5至7中任一项所述的载体的宿主细胞。
9.一种转基因植物，其包含至少一种权利要求1或2所述的核酸序列。
10.根据权利要求9所述的转基因植物，其中所述植物为玉米植物。
11.一种来自权利要求9或10中任一项所述的转基因植物的转基因种子。
12.一种用于防治田间害虫群体的方法，其包括在所述田间种植权利要求9或10中任一项所述的植物或权利要求11所述的种子。
13.一种生产害虫耐受性转基因植物的方法，其中所述方法包括：
a)用权利要求5至7中任一项所述的载体转化植物细胞；
b)选择包含权利要求1或2所述的核酸的转化的植物细胞；以及
c)从所述转化的植物细胞再生表达至少一种权利要求3或4所述的多肽的害虫耐受性转基因植物。
14.一种鉴定具有改善的害虫耐受性的植物的方法，其包括在植物群体中鉴定权利要求9至10中任一项所述的植物的步骤。
15.一种在生物样品中检测根据权利要求1或2所述的核酸序列或根据权利要求3或4所述的多肽的存在或缺乏的方法，其包括以下步骤：
a)获得生物样品；
b)从所述生物样品中提取DNA或RNA或蛋白质；以及
c)检测权利要求1或2所述的核酸序列或权利要求3或4所述的多肽的存在或缺乏。
16.用于进行权利要求15所述的方法的工具(means)，其中使用选自由以下组成的组的工具之一来进行步骤c)：
a)至少两种用于扩增权利要求1或2的核酸序列的引物，
b)至少一种与权利要求1或2的核酸序列杂交的标志物，以及
c)至少一种识别权利要求3和4所述的多肽的抗体。
17.权利要求1或2的分离的核酸序列用于筛选具有杀虫活性的同源物序列的用途。
18.一种生产杀虫组合物的方法，其中所述方法包括：
a)培养细胞，所述细胞包含至少一种编码选自由以下组成的组的蛋白质的核酸序列：
i)与SEQ ID NO:1(GDI0005A)具有至少70％同一性的第1组蛋白质，以及
ii)与SEQ ID NO:2(GDI0006A)具有至少65％同一性的第2组蛋白质；以及
b)回收所述蛋白质的至少一种。
19.根据权利要求18所述的方法，其中所述细胞选自野生型细胞和权利要求8的宿主细胞。
20.一种对昆虫害虫具有杀虫活性的组合物，其包含有效量的至少一种选自以下的蛋白质：
a)与SEQ ID NO:1(GDI0005A)具有至少70％同一性的第1组蛋白质，以及
b)与SEQ ID NO:2(GDI0006A)具有至少65％同一性的第2组蛋白质。
21.根据权利要求20所述的组合物，其包含第1组的蛋白质和第2组的蛋白质。
22.根据权利要求20至21中任一项所述的组合物，其包含表达至少一种选自由以下组成的组的蛋白质的天然金黄杆菌：
a)与SEQ ID NO:1(GDI0005A)具有至少70％同一性的第1组蛋白质，以及
b)与SEQ ID NO:2(GDI0006A)具有至少65％同一性的第2组蛋白质。
23.一种处理植物免于昆虫害虫侵染或处理受害虫侵染的植物的方法，其包括向所述植物或其一部分施用至少一种选自由以下组成的组的蛋白质：
a)与SEQ ID NO:1(GDI0005A)具有至少70％同一性的第1组蛋白质，以及
b)与SEQ ID NO:2(GDI0006A)具有至少65％同一性的第2组蛋白质。
24.根据权利要求23所述的处理植物的方法，其中通过应用权利要求20至22中任一项所述的组合物或通过培养权利要求9或10所述的转基因植物或种植权利要求11所述的种子来施用所述至少一种蛋白质。
CN202080034147.6A 2019-03-13 2020-03-13 杀虫基因和使用方法 Pending CN114616334A (zh)

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