转Vip3Aa基因的球孢白僵菌工程菌的构建及其对马尾松毛虫毒力的增效作用  

Generation of transgenic Beauveria bassiana strain with Vip3Aa gene and its enhanced virulence on Dendrolimus punctatus

作　　者：任小芳[1] 刘振邦[1] 朱虹[1] 黄勃[1] 李增智[1] 

机构地区：[1]安徽农业大学微生物防治省重点实验室,合肥230036

出　　处：《安徽农业大学学报》2011年第5期736-741,共6页Journal of Anhui Agricultural University

基　　金：国家"863"计划(2006AA10A212);国家自然科学基金(30972368)共同资助

摘　　要：根据苏云金杆菌营养期杀虫蛋白基因Vip3Aa序列设计全基因扩增引物,并在引物两端添加合适的酶切位点进行PCR扩增,纯化的PCR产物和载体pbarGPE1分别经Xho I和EcoR I双酶切、连接,构建真菌表达载体pbarGPE1-vip3Aa。将构建好的质粒经Sca I酶切线性化后,利用芽生孢子转化法转入昆虫病原真菌球孢白僵菌Bb13菌株内,得到白僵菌工程菌株Bb13V。RT-PCR结果证明Vip3Aa在工程菌株中已得到成功转录。在室内恒温25℃条件下,用喂食、喷雾以及两者相结合的方法,分别测定1×10^5、1×10^6、1×10^7、1×10^8和2×10^8孢子.mL^-1 5个不同孢子浓度下原始菌株和工程菌株对二龄马尾松毛虫的毒力。结果表明,在前4种不同浓度的处理条件下,采取3种不同的处理方式,工程菌株均比喷雾处理的原始菌株致死率提高30%以上;喷雾处理的毒力提高33.6-62.1倍;喂食处理的致死率均高于喷雾处理。在1×108孢子.mL^-1浓度下,喂食处理的致死率显著高于喷雾处理,致死中时缩短6.89 d。因此,Vip3Aa基因在转入白僵菌后得到了表达,从而赋予白僵菌可观的胃毒作用,使得工程菌对松毛虫的毒力明显增强。Based on the sequence of Vip3Aa gene for a kind of vegetative insecticidal proteins of Bacillus thuringiensis,primers were designed with appropriate restriction sites,to amplify the full-length of Vip3Aa gene.The PCR products digested by Xho I and EcoR I were ligated with the vector pbarGPE1 digested by the same re-striction enzyme to construct a recombinant plasmid pbarGPE1-vip3Aa.The plasmid expression vector linearized by Sca I was transformed into Bb13 strain of Beauveria bassiana,one of the most important entomopathogenic fungus,by blastospore-based transformation system.Reverse Transcription-PCR detection of Vip3Aa transcription in Bb13V suggested that Vip3Aa gene was consistently expressed in the transformant Bb13V.To compare the virulence of Bb13 and Bb13V on the third instar larvae of the Masson’s pine caterpillar,Dendrolimus punctatus,a bioassay was conducted by treatments of spray,ingestion of conidial suspension and both for Bb13V and spray for Bb13.Conidial suspension concentrations were treated at 1×10^5,1×10^6,1×10^7,1×10^8 and 2×10^8 conidia·mL^-1,with 0.05% Tween 80 as control.The results showed that as compared to the wild-type strain Bb13,the corrected cumulative mortality caused by Bb13V treated with the 3 methods increased by over 30% for the suspensions of 1×10^5,1×10^6,1×10^7,and 1×10^8 conidia·mL^-1 and virulence by spray was increased by 33.6-62.1folds;mortal-ity caused by ingestion was higher than by spray for all concentrations.Under the concentration of 1×10^8,mor-tality by spray was significantly higher than by spray and LT50 shortened by 6.89 days.Consequently,Vip3Aa gene was expressed in the transgenic strain so as to present B.bassiana with stomach toxicity,which infect hosts mainly through cuticle,and its virulence on the pine caterpillars was raised substantially as a result.

关 键 词：球孢白僵菌 苏云金杆菌 Vip3Aa基因 载体构建 芽生孢子转化 基因工程 

分 类 号：S476.12[农业科学—农业昆虫与害虫防治]