昆虫病原真菌降解寄主体壁酶基因的克隆及球孢白僵菌高毒力重组菌株的获得
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摘要
随着大面积使用化学农药所造成的环境污染和农药残留等问题日益突出，以生物防治为主的害虫防治策略正受到广泛重视。在众多的杀虫微生物中，由于昆虫病原真菌具有主动感染寄主、能形成循环侵染和寄主难以产生抗性等优点，因此目前正得到越来越多的关注。但是，昆虫病原真菌存在击倒害虫时间长和防效不稳定等缺点，这限制了它们的进一步应用。为了改变这一局面，有必要加强昆虫病原真菌致病分子机理的研究，为利用基因工程技术改良菌株奠定理论基础和提供目的基因。穿透体壁是在昆虫病原真菌感染寄主的过程中重要的步骤之一。昆虫体壁主要由蛋白质和几丁质组成，昆虫病原真菌则通过机械压力和蛋白酶、几丁质酶等水解酶的作用降解昆虫体壁。已有研究表明，类枯草杆菌蛋白酶在金龟子绿僵菌致病寄主的过程中起重要作用，而几丁质酶所起作用较小。本文首先研究类枯草杆菌蛋白酶在球孢白僵菌致病过程中的作用，然后从蛋白酶和几丁质酶相互作用的角度来研究几丁质酶在球孢白僵菌感染昆虫过程中的作用，并在此基础上获得球孢白僵菌高毒力菌株。主要研究结果如下：
1．球孢白僵菌类枯草杆菌蛋白酶基因CDEP-1及其上游调控序列的克隆与分析
蛋白质是昆虫体壁的主要组成成份，降解昆虫体壁蛋白的类枯草杆菌蛋白酶Pr1被证明是金龟子绿僵菌的重要致病因子，为了研究Pr1类蛋白酶在球孢白僵菌感染寄主过程中的作用，我们首先从该病原真菌中克隆Pr1类蛋白酶基因。
从在蝉蜕诱导培养基内培养的球孢白僵菌芽管状物中提取总RNA，并用于构建诱导cDNA文库。完整cDNA文库的滴度为3.1×10~4 pfu／μl，外源片段的长度集中在0.7kb到1.6kb之间。以Pr1类蛋白酶基因的部分DNA序列BbP为探针从该cDNA文库中筛选到球孢白僵菌类枯草杆菌蛋白酶基因CDEP-1的全长cDNA。该cDNA长为1557bp，含有1134bp的开放阅读框，编码一377个氨基酸的蛋白酶前体，其氮端18个氨基酸为信号肽序列。CDEP-1对应的基因组序列为gCDEP-1(Genebank登录号为AY040532)，其中有3个内含子。Southern杂交分析表明，gCDEP-1在球孢白僵菌基因组上为单拷贝。CEDP-1推导的氨基酸序列与金龟子绿僵菌的MaPr1、白色念珠菌的蛋白酶K和球孢白僵菌的BbPr1的相似性分别为54.7％、57.9％和83.3％。CDEP-1推导的氨基酸序列氮端327个氨基酸和BbPr1的几乎完全相同，其后CDEP-1有50个氨基酸，BbPr1则只有33个，而且二者差异很大。因此，CDEP-1为Pr1类蛋白酶基因的新成员。
在蝉蜕诱导培养基中，加入葡萄糖能阻遏CDEP-1的表达，加入丙氨酸则只能部分阻遏CDEP-1的表达。为了进一步研究CDEP-1的表达规律，利用YADE
中 文摘要
技术克隆它的上游调控序列。YADE法扩增出了 CDEP＊上游长为 1705hp的调
控序列CDEPP。序列分析发现，CDEPP中含有多个葡萄糖阻遏于的结合位点（共
同序列为 5卜（g／C）YggRg＜3‘人和氮阻遏子的结合位点（相隔很近的DNA序列
“ GATA”）。
L球抱白僵菌内切几了质酶＊bCMd的纯化及其基因的克隆与分析
几丁质占昆虫体壁17－50％，在抵御外来做生物入侵时起重要作用。在感染
昆虫的过程中，昆虫病原真菌分泌多种几丁质酶，但是有关几了质酶在病原真菌
致病寄主过程中所起作用的研究很少。到目前为止，还未有从球抱白僵菌中克隆
几了质酶基因的报道。为研究几丁质酶在球抱白僵菌感染寄主过程中的作用，我
们从该病原真菌中纯化几丁质酶，并克隆其相应的基因。
2．二 内切几丁质酶＊bCMd的纯化及氮端序列测定
从球抱白僵菌在胶体几丁质诱导培养基内培养了168h的诱导液中，利用硫
酸氨沉淀、凝胶过滤层析和阴离子交换层析纯化出了内切几了质酶 BbChit。
Bbchitl的分子量为33kDa，等电点为5石万 氮端22个氨基酸为Ala七ly1hr－
Cys－Ala－Thl－Lys－GlyATg－Pro－Ala－Gly－Lys－ValLeu－Gin－G卜Tyr－Tp－Gin－Asn－Tp。
2．二球抱自僵菌内切几丁质酶基因BbChitl的克隆
根据内切几丁质酶Bbchitl的氮端氨基酸序列设计简并引物，采用3‘RACE
和 YADE技术获得了 BbChitl的基因和该基因的部分上游调控序列，该基因命名
为Bbchitl（Genbank登录号为AY145440）。Bbchitl含有长为1047hp 的开放阅
读框架，编码一348个氨基酸的几丁质酶前体，其中N端28个氨基酸为信号肽
序列。Bbchitl 的基因组序列不含内含子，在球施白僵菌的基因组上为单拷贝。
在BbChitl 的上游调控序列中有7个葡萄糖阻遏子结合位点（共同序列为
5”叶g／C）YggRg《”人这与BbChitl的表达受葡萄糖阻遏的结果一致。在推导的氨
基酸序列中含有几丁质酶典型的保守区域 SXGG和 DGIDXDXE。有趣的是，
Bbchitl推导的氨基酸序列与其它来自昆虫病原真菌的几丁质酶基因的相似性很
低。BbChitl与金龟子绿僵菌的几了质酶基因CHITZ、CHIZ、CHI］j和黄绿绿
僵菌的几丁质酶基因 CHH的相似性分别为 20％，ZI％上3％和 25％。表明，BbChitl
是从昆虫病原真菌中克隆的一新几了质酶基因。
3．球抱白僵菌几丁质酶基因BbChitZ的克隆与分析
在昆虫病原真菌感染寄主的过程?
Introduction
Insect pest is one of major factors that cause yield reduction of crop, therefore, pest control is conducted through the process of agricultural practice. At the onset of 1950s, the 'green revolution' and the slogan 'better living through chemistry' shifted the pest control focus from a biocontrol approach to a chemical approach. However, the widely application of chemical pesticide has left the agriculture with pesticide resistance, agro-ecosystem collapse, environmental contamination and human health concerns. Under such circumstance, the biological strategy for pest control is receiving much attention.
Compared with other microbial pathogens of insect, entomopathogenic fungi can invade host actively, and can infect pest circularly. Moreover, host can rarely develop resistance to entomopathogneic fungi. Therefore, mycoinsecticides are being considered as alternative and supplement to chemical pesticides. However, the acceptance of fungal products for pest control is very limited. What make limited acceptance of fungal insecticides is that they are not as fast acting as chemical products, sensitive to adverse environment, lose their effectiveness more rapidly. Therefore, entomoapathogenic fungal strain improvement is necessary. With the elucidation of molecular basis of fungal entomopathogenicity, gene engineering might
Summary
overcome these drawbacks and could lead to an ideal biocontrol microorganism.
Six integrated steps are involved in fungal pathogenesis in insect, and they are as following: 1) spore adhering to the cuticle of insect, 2) spore germination and infection structure formation, 3) penetration of host cuticle, 4) growth in host's hemocoel, 5) saprophytic feeding and host death and 6) hypha reemergence from inside host and sporulation on host cuticle. Penetrating host's cuticle is one of the key steps in infection. In common with phytopathogenic fungi, entry of insect fungi into the host also involves both mechanical pressure and enzymatic degradation. The complex refractory nature of insect cuticle suggests that penetration would require the synergistic action of several different enzymes including chitinase, protease and lipase. Among theses enzymes, subtilisin-like protease had been proved to play important role in the cuticle degradation in Metarhizium anisopliae. The constitutive expression of Prla was shown to enhance virulence of M. anisoplia greatly. Subtilisin-like protease gene had
been found in several other entomopathogenic fungi, however, its role in the pathogenicity of these fungi remains to be established.
Chitin accounts for 17%-50% of insect cuticle, and chitin synthesis-inhibited larvae of Maduca Sexta by Dimilin was more easily attacked by M. anisopliae, demonstrating that chitin was one of barriers that could protect insect from being invaded by foreign microorganisms. Therefore, It can be deduced that chitinases play important role in fungal pathogenesis in insect. However, overexpressing a chitinase gene CHII did not alter the virulence of M. anisopliae to the larvae of Maduca sexta, sggesting that this chitinase was not involved in cuticle degrading. Nevertheless, entomopahtogenic fungi, for example M. anisopliae, produced different kinds of chitinases in the process of penetrating host's cuticle, and the roles of these chitinases may be of difference, therefore, the general role of chitinases in fungal pathogenesis in insect remains to be further studied.
In the medium using insect cuticle as sole carbon and nitrogen resource, chitinases were proved to act synergistically with proteolytic enzymes to degrade insect cuticle. As mentioned above, chitin-synthesis-inhibited larvae were easily attacked by M anisopliae. These two results made us think that proteases and chitinases may also act synergistically when entomopathogenic fungi penetrated insect cuticle. Therefore, we should investigate the role of chitinases in fungal pathogenesis in insect from the point of their interaction with proteases
Beauveria bassiana is a widely employed entomopa
1．球孢白僵菌类枯草杆菌蛋白酶基因CDEP-1及其上游调控序列的克隆与分析
蛋白质是昆虫体壁的主要组成成份，降解昆虫体壁蛋白的类枯草杆菌蛋白酶Pr1被证明是金龟子绿僵菌的重要致病因子，为了研究Pr1类蛋白酶在球孢白僵菌感染寄主过程中的作用，我们首先从该病原真菌中克隆Pr1类蛋白酶基因。
从在蝉蜕诱导培养基内培养的球孢白僵菌芽管状物中提取总RNA，并用于构建诱导cDNA文库。完整cDNA文库的滴度为3.1×10~4 pfu／μl，外源片段的长度集中在0.7kb到1.6kb之间。以Pr1类蛋白酶基因的部分DNA序列BbP为探针从该cDNA文库中筛选到球孢白僵菌类枯草杆菌蛋白酶基因CDEP-1的全长cDNA。该cDNA长为1557bp，含有1134bp的开放阅读框，编码一377个氨基酸的蛋白酶前体，其氮端18个氨基酸为信号肽序列。CDEP-1对应的基因组序列为gCDEP-1(Genebank登录号为AY040532)，其中有3个内含子。Southern杂交分析表明，gCDEP-1在球孢白僵菌基因组上为单拷贝。CEDP-1推导的氨基酸序列与金龟子绿僵菌的MaPr1、白色念珠菌的蛋白酶K和球孢白僵菌的BbPr1的相似性分别为54.7％、57.9％和83.3％。CDEP-1推导的氨基酸序列氮端327个氨基酸和BbPr1的几乎完全相同，其后CDEP-1有50个氨基酸，BbPr1则只有33个，而且二者差异很大。因此，CDEP-1为Pr1类蛋白酶基因的新成员。
在蝉蜕诱导培养基中，加入葡萄糖能阻遏CDEP-1的表达，加入丙氨酸则只能部分阻遏CDEP-1的表达。为了进一步研究CDEP-1的表达规律，利用YADE
中 文摘要
技术克隆它的上游调控序列。YADE法扩增出了 CDEP＊上游长为 1705hp的调
控序列CDEPP。序列分析发现，CDEPP中含有多个葡萄糖阻遏于的结合位点（共
同序列为 5卜（g／C）YggRg＜3‘人和氮阻遏子的结合位点（相隔很近的DNA序列
“ GATA”）。
L球抱白僵菌内切几了质酶＊bCMd的纯化及其基因的克隆与分析
几丁质占昆虫体壁17－50％，在抵御外来做生物入侵时起重要作用。在感染
昆虫的过程中，昆虫病原真菌分泌多种几丁质酶，但是有关几了质酶在病原真菌
致病寄主过程中所起作用的研究很少。到目前为止，还未有从球抱白僵菌中克隆
几了质酶基因的报道。为研究几丁质酶在球抱白僵菌感染寄主过程中的作用，我
们从该病原真菌中纯化几丁质酶，并克隆其相应的基因。
2．二 内切几丁质酶＊bCMd的纯化及氮端序列测定
从球抱白僵菌在胶体几丁质诱导培养基内培养了168h的诱导液中，利用硫
酸氨沉淀、凝胶过滤层析和阴离子交换层析纯化出了内切几了质酶 BbChit。
Bbchitl的分子量为33kDa，等电点为5石万 氮端22个氨基酸为Ala七ly1hr－
Cys－Ala－Thl－Lys－GlyATg－Pro－Ala－Gly－Lys－ValLeu－Gin－G卜Tyr－Tp－Gin－Asn－Tp。
2．二球抱自僵菌内切几丁质酶基因BbChitl的克隆
根据内切几丁质酶Bbchitl的氮端氨基酸序列设计简并引物，采用3‘RACE
和 YADE技术获得了 BbChitl的基因和该基因的部分上游调控序列，该基因命名
为Bbchitl（Genbank登录号为AY145440）。Bbchitl含有长为1047hp 的开放阅
读框架，编码一348个氨基酸的几丁质酶前体，其中N端28个氨基酸为信号肽
序列。Bbchitl 的基因组序列不含内含子，在球施白僵菌的基因组上为单拷贝。
在BbChitl 的上游调控序列中有7个葡萄糖阻遏子结合位点（共同序列为
5”叶g／C）YggRg《”人这与BbChitl的表达受葡萄糖阻遏的结果一致。在推导的氨
基酸序列中含有几丁质酶典型的保守区域 SXGG和 DGIDXDXE。有趣的是，
Bbchitl推导的氨基酸序列与其它来自昆虫病原真菌的几丁质酶基因的相似性很
低。BbChitl与金龟子绿僵菌的几了质酶基因CHITZ、CHIZ、CHI］j和黄绿绿
僵菌的几丁质酶基因 CHH的相似性分别为 20％，ZI％上3％和 25％。表明，BbChitl
是从昆虫病原真菌中克隆的一新几了质酶基因。
3．球抱白僵菌几丁质酶基因BbChitZ的克隆与分析
在昆虫病原真菌感染寄主的过程?
Introduction
Insect pest is one of major factors that cause yield reduction of crop, therefore, pest control is conducted through the process of agricultural practice. At the onset of 1950s, the 'green revolution' and the slogan 'better living through chemistry' shifted the pest control focus from a biocontrol approach to a chemical approach. However, the widely application of chemical pesticide has left the agriculture with pesticide resistance, agro-ecosystem collapse, environmental contamination and human health concerns. Under such circumstance, the biological strategy for pest control is receiving much attention.
Compared with other microbial pathogens of insect, entomopathogenic fungi can invade host actively, and can infect pest circularly. Moreover, host can rarely develop resistance to entomopathogneic fungi. Therefore, mycoinsecticides are being considered as alternative and supplement to chemical pesticides. However, the acceptance of fungal products for pest control is very limited. What make limited acceptance of fungal insecticides is that they are not as fast acting as chemical products, sensitive to adverse environment, lose their effectiveness more rapidly. Therefore, entomoapathogenic fungal strain improvement is necessary. With the elucidation of molecular basis of fungal entomopathogenicity, gene engineering might
Summary
overcome these drawbacks and could lead to an ideal biocontrol microorganism.
Six integrated steps are involved in fungal pathogenesis in insect, and they are as following: 1) spore adhering to the cuticle of insect, 2) spore germination and infection structure formation, 3) penetration of host cuticle, 4) growth in host's hemocoel, 5) saprophytic feeding and host death and 6) hypha reemergence from inside host and sporulation on host cuticle. Penetrating host's cuticle is one of the key steps in infection. In common with phytopathogenic fungi, entry of insect fungi into the host also involves both mechanical pressure and enzymatic degradation. The complex refractory nature of insect cuticle suggests that penetration would require the synergistic action of several different enzymes including chitinase, protease and lipase. Among theses enzymes, subtilisin-like protease had been proved to play important role in the cuticle degradation in Metarhizium anisopliae. The constitutive expression of Prla was shown to enhance virulence of M. anisoplia greatly. Subtilisin-like protease gene had
been found in several other entomopathogenic fungi, however, its role in the pathogenicity of these fungi remains to be established.
Chitin accounts for 17%-50% of insect cuticle, and chitin synthesis-inhibited larvae of Maduca Sexta by Dimilin was more easily attacked by M. anisopliae, demonstrating that chitin was one of barriers that could protect insect from being invaded by foreign microorganisms. Therefore, It can be deduced that chitinases play important role in fungal pathogenesis in insect. However, overexpressing a chitinase gene CHII did not alter the virulence of M. anisopliae to the larvae of Maduca sexta, sggesting that this chitinase was not involved in cuticle degrading. Nevertheless, entomopahtogenic fungi, for example M. anisopliae, produced different kinds of chitinases in the process of penetrating host's cuticle, and the roles of these chitinases may be of difference, therefore, the general role of chitinases in fungal pathogenesis in insect remains to be further studied.
In the medium using insect cuticle as sole carbon and nitrogen resource, chitinases were proved to act synergistically with proteolytic enzymes to degrade insect cuticle. As mentioned above, chitin-synthesis-inhibited larvae were easily attacked by M anisopliae. These two results made us think that proteases and chitinases may also act synergistically when entomopathogenic fungi penetrated insect cuticle. Therefore, we should investigate the role of chitinases in fungal pathogenesis in insect from the point of their interaction with proteases
Beauveria bassiana is a widely employed entomopa
引文
1． 樊美珍，李增智．1994．营养物和培养条件对虫生真菌附着胞形成的影响．安徽农业大学学报，21(2)：123-130．
2． 樊美珍，黄勃，王建林．1999．几种虫生真菌附着胞的荧光电镜及扫描电镜观察．菌物系统，18(3)：249-253．
3． 方卫国，张永军，杨星勇，王中康，裴炎．2002a．球孢白僵菌降解昆虫体壁蛋白酶基因CDEP-1的克隆与序列分析．遗传学报，29(3)：278-282．
4． 方卫国，张永军，杨星勇，裴炎．2002b．根癌农杆菌介导真菌遗传转化的研究进展．中国生物工程杂志，22(5)：41-45．
5． 方卫国，杨星勇，张永军，裴炎．2002c．真菌核酸的一种快速提取方法．应用与环境生物学报，8(3)：305～307．
6． 方卫国，张永军，马金成，肖月华，杨星勇，裴炎．2003．用YADE法克隆球孢白僵菌类枯草杆菌蛋白酶基因CDEP-1的启动子及启动子序列分析．菌物系统，(待印刷)．
7． 方卫国，张永军，肖月华，马金成，杨星勇，裴炎．2003．金龟子绿僵菌羧基转运蛋白基因MaJEN1及其启动子的克隆与分析．遗传学报，30(3)：283-288．
8． 冯明光．1998．生物杀虫剂与新的农业科技革命．植物保护21世纪展望暨第三届全国青年植物保护科技工作者学术研讨会论文集，北京：中国科学技术出版社．
9． 冯明光．1998．开发真菌杀虫剂防治刺吸式口器害虫．’98海峡两岸害虫生物防治学术研讨会摘要，pp8-11．
10．黄大昉，林敏．2001，农业微生物基因工程的安全性．农业微生物基因工程，北京：科学出版社，pp457-493．
11．根井 外喜男编，金连缘译．1982．微生物保存法．上海：上海科学技术出版社，pp6．
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