蘇云金芽孢桿菌營養(yǎng)期殺蟲蛋白基因Vip3Aa在蟲生真菌孢白僵菌中的表達.pdf

1、安徽農(nóng)業(yè)大學碩士學位論文蘇云金芽孢桿菌營養(yǎng)期殺蟲蛋白基因Vip3Aa在蟲生真菌孢白僵菌中的表達姓名：任小芳申請學位級別：碩士專業(yè)：微生物學指導教師：李增智;黃勃2011-06Abstract Beauveria bassiana is one of the most common entomogenous fungi, with wide host range and great convenience for massproducti

2、on, and in its natural medium, sometimes it possibly causes epidemic in between the host population which is beneficial to represses pests breeding rapidly or abundantly. However, like other entomogenous fungi, it always

3、 kills insects more slowly than chemical insecticides and this disadvantage discourages the use of it in insect pest control. Therefore, improving its virulence using genetic engineering technology to increase mycoinsect

4、icide products in market is of significant application value. In this study, foreign gene Vip3Aa from Bacillus thuringiensis was transformed into two initial strains Bb13 and Bb1944, resulting in construction of two mono

5、copic chitinase-overexpressive transgenic engineered strains. Based on the sequence of Vip3Aa, gene for a kind of vegetative insecticidal proteins of Bacillus thuringiensis, primers were designed with appropriate restric

6、tion sites, to amplify the full-length Vip3Aa gene. The PCR products digested by XhoI and EcoRI were ligated with the vector pbarGPE1 digested by the same restriction enzyme to construct a recombinant plasmid pbarGPE1-vi

7、p3Aa. The plasmid expression vector linearized by ScaI was transformed into Bb13 and Bb1944 strains of Beauveria bassiana, one of the most important entomopathogenic fungus, by blastospore-based transformation system. Re

8、verse Transcription-PCR detection of Vip3Aa transcription in Bb13V and Bb1944V suggested that Vip3Aa gene was consistently expressed in the transformants. To compare the virulence of Bb13 and Bb13V on the third instar l

9、arvae 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 concentration wa

10、s settled at 1×105、1×106、1×107、1×108 and 2×108 conidia/ml, with 0.05% Tween 80 as control. The results showed that as compared to the wild-type strain Bb13, the corrected cumulative mortality cau

11、sed by Bb13V treated with the 3 methods increased by over 30% for the suspensions of 1×105、1×106、1×107、and 1×108 conidia/ml and virulence by spray was increased by 33.6~62.1-folds; mortality caused by

12、 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. To compare the virulence of