cry11Aa4基因的克
发布时间：2018-11-28 08:36
【摘要】：苏云金芽孢杆菌LLP29是一株具有自主知识产权的高效杀蚊菌株，为进一步了解该菌株的杀蚊毒素，本研究成功克隆分析了LLP29含有的cry11基因，结果发现该基因和已报道的cry11Aa同源性达99%以上，是一类杀虫谱较广且毒力较高的杀蚊基因。将该基因登陆NCBI，获得登陆号XQ228567，并提交Bt命名委员会，被正式命名为cry11Aa4。原核表达并纯化Cry11Aa4杀蚊蛋白，以致倦库蚊为靶标进行生物测定，，其LC50为1340ng/mL。 以Cry11Aa4为抗原免疫新西兰大白兔，获得Cry11Aa4的多克隆抗体。免疫印迹试验表明，Cry11Aa4可与致倦库蚊中肠蛋白有效结合。为了分析获得的结合蛋白，构建了生物素-亲和素系统，基于生物素-亲和素的沉降试验，成功分离到了三个Cry11Aa4的结合蛋白，其分子量分别为46、52和100KDa。经质谱分析，上述蛋白分别为ATP结合蛋白、热激蛋白和未知蛋白。其中，未知蛋白可能为一种APN。本研究为深入研究毒素与受体的相互作用、毒素的作用机制及蚊子抗性产生机理提供重要基础。
[Abstract]:Bacillus thuringiensis (LLP29) is a highly efficient mosquito killing strain with independent intellectual property rights. In order to further understand the insecticidal toxin of Bacillus thuringiensis, the cry11 gene contained in LLP29 was cloned and analyzed successfully. The results showed that the homology of this gene with the reported cry11Aa was over 99%, and it was a class of insecticidal genes with high virulence and broad insecticidal spectrum. The gene is logged in to NCBI, to obtain the landing number XQ228567, and submitted to the Bt naming committee, officially named cry11Aa4. The prokaryotic expression and purification of Cry11Aa4 insecticidal protein led to the biometric determination of Culex pipiens quinquefasciatus with a LC50 of 1340ng / mL. New Zealand white rabbits were immunized with Cry11Aa4 as antigen to obtain polyclonal antibodies against Cry11Aa4. Western blot test showed that Cry11Aa4 could effectively bind to midgut protein of Culex pipiens quinquefasciatus. In order to analyze the binding proteins, the biotin-avidin system was constructed. Three Cry11Aa4 binding proteins with molecular weight of 460.52 and 100KDawere successfully isolated by the sedimentation test based on biotin-avidin. The above proteins were identified as ATP binding proteins, heat shock proteins and unknown proteins by mass spectrometry. Among them, the unknown protein may be a kind of APN.. This study provides an important basis for the further study of the interaction between toxin and receptor, the mechanism of toxin action and the mechanism of mosquito resistance.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：S476
本文编号：2362363
[Abstract]:Bacillus thuringiensis (LLP29) is a highly efficient mosquito killing strain with independent intellectual property rights. In order to further understand the insecticidal toxin of Bacillus thuringiensis, the cry11 gene contained in LLP29 was cloned and analyzed successfully. The results showed that the homology of this gene with the reported cry11Aa was over 99%, and it was a class of insecticidal genes with high virulence and broad insecticidal spectrum. The gene is logged in to NCBI, to obtain the landing number XQ228567, and submitted to the Bt naming committee, officially named cry11Aa4. The prokaryotic expression and purification of Cry11Aa4 insecticidal protein led to the biometric determination of Culex pipiens quinquefasciatus with a LC50 of 1340ng / mL. New Zealand white rabbits were immunized with Cry11Aa4 as antigen to obtain polyclonal antibodies against Cry11Aa4. Western blot test showed that Cry11Aa4 could effectively bind to midgut protein of Culex pipiens quinquefasciatus. In order to analyze the binding proteins, the biotin-avidin system was constructed. Three Cry11Aa4 binding proteins with molecular weight of 460.52 and 100KDawere successfully isolated by the sedimentation test based on biotin-avidin. The above proteins were identified as ATP binding proteins, heat shock proteins and unknown proteins by mass spectrometry. Among them, the unknown protein may be a kind of APN.. This study provides an important basis for the further study of the interaction between toxin and receptor, the mechanism of toxin action and the mechanism of mosquito resistance.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：S476
【参考文献】
相关期刊论文 前1条
1 关雄;;苏云金芽孢杆菌研究回顾与展望[J];中国农业科技导报;2006年06期
本文编号：2362363
本文链接：https://www.wllwen.com/falvlunwen/zhishichanquanfa/2362363.html
