苏云金杆菌以色列亚种杀蚊蛋白Cyt1Aa在离中不粘柄菌中的表达  

Expression of mosquitocidal Cyt1 Aa toxin from Bacillus thuringiensis subsp. israelensis in Asticcacaulis excentricus

作　　者：郑大胜[1] Neil Crickmore 蔡亚君[1] 闫建平[1] 袁志明[1] 

机构地区：[1]中国科学院武汉病毒研究所 [2]University of Sussex,Brighton,BN19QG,United Kingdom

出　　处：《微生物学报》2007年第2期217-220,共4页Acta Microbiologica Sinica

基　　金：中国科学院提供赴外研修资助;英国Wellcome Trust提供项目资助

摘　　要：在蚊幼虫生活水域里的离中不粘柄菌(Asticcacaulis excentricus,Ae)中已成功表达苏云金芽孢杆菌以色列亚种(Bacillus thuringiensis subsp.israelensis,Bti)杀蚊蛋白基因cry11Aa的基础上,将另一Bti杀蚊蛋白基因cyt1Aa转化入Ae中表达。构建并转化了分别单独含有cyt1Aa基因、及同时含有cry11Aa基因的表达质粒pSODCyt20和pSODCryCyt20,蛋白免疫杂交检测相应的Ae重组子分别表达产生了Cyt1Aa和Cry11Aa蛋白。为了探究Ae(pSODCryCyt20)重组子不能表达cyt1Aa的原因,提取了重组子总RNA、并与同是革兰氏染色阴性的大肠杆菌的总RNA比较,结果显示两者RNA系统显著不同,推测Ae中多个外源基因的表达,可能要求每个基因必需一个启动子。Asticcacaulis excentricus, who lives in upper-layer waters providing food resource to the mosquito larvae and has been proven to be a successful host to produce the mosquitocidal binary toxins or CryllAa toxin from Bacilli （Liu et al., 1996, Nat Biotech 14： 343; Armengol, et al., 2005, Curr Microbiol 51 ： 430）, was developed to express cytlAa gene from Bacillus thuringieusis subsp, israeleusis （ Bti ）. Two A. excentricus transformants were constructed with the attempt of producing Cytl Aa alone and alongside with Cryl 1Aa, repectively. Detection of expressed Cryl 1Aa and Cytl Aa proteins by immunoblot in the recombinant A. excentricus clones showed that either cryllAa or cytlAa was expressed well solely but not simultaneously although both restriction analyses of plasmid DNA and DNA sequencing showed that the transformed plasmid was identical to scheme. To investigate the reason why the recombinant A. excentricus harboring both genes and their ribosome binding site （RBS） sequences expressed only Cryl 1Aa, the total RNA of A. excentricus cell was extracted and revealed three-band pattern in which all RNA molecule weights are not greater than 16S RNA of Escherichia coli by formamide agarose gel electrophoresis, indicating that different RNA systems within these two Gramnegative strains required distinguishingly organised constructs to express multiple foreign genes. It is hypothesized that an extra promoter upstream of RBS sequence is required to express cytlAa in the cryllAa-cytlAa tandom plasmid.

关 键 词：离中不粘柄菌 苏云金杆菌以色列亚种 cyt1Aa cryllAa 多外源基因表达 总RNA 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]