安徽农业大学学报2011，Vol.38Issue(5)：736-741,6.DOI:34-1162/s.20110829.1430.013
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
摘要
Abstract
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 pbarGPEl digested by the same restriction enzyme to construct a recombinant plasmid pbarGPEl-vip3Aa. The plasmid expression vector linearized by Sea I was transformed into Bbl3 strain of Beauveria bassiana, one of the most important entomopathogenic fungus, by blastospore-based transformation system. Reverse Transcription-PCR detection of Vip3Aa transcription in Bbl3V suggested that Vip3Aa gene was consistently expressed in the transformant Bbl3V. To compare the virulence of Bbl3 and Bbl3V 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 Bbl3V and spray for Bbl3. Conidial suspension concentrations were treated at 1×105,l × 106,l ×107,l ×108 and 2×108 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 Bbl3V treated with the 3 methods increased by over 30% for the suspensions of 1 ×105, 1×106, 1×107, and 1×108 conidia·mL-1 and virulence by spray was increased by 33.6~62.1 folds; mortality caused by ingestion was higher than by spray for all concentrations. Under the concentration of 1×108, mortality 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Key words
Beauveria bassiana/ Bacillus thuringiensis/ Vip3Aa gene/ vector construction/ blastospore transformation/ gene engineering分类
农业科技引用本文复制引用
任小芳,刘振邦,朱虹,黄勃,李增智..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[J].安徽农业大学学报,2011,38(5):736-741,6.基金项目
国家”863”计划(2006AA 10A212)和国家自然科学基金(30972368)共同资助. （2006AA 10A212）
