The Influence of Site-directed Mutation in Bt Vip3Aa11 Protein on Insecticidal Activity Against Spodoptera exigua and Helicoverpa armigera
LIU Ming1,2, SUN Hai-Yan1, LI Hai-Tao2, GAO Ji-Guo2*
1 Testing Centre, Heilongjiang Bayi Agricultural University, Daqing 163319, China; 2 College of Life Science, Northeast Agricultural University, Harbin 150030, China
Abstract Compared with the insecticidal crystal proteins, vegetative insecticidal proteins (Vips) have no homology of amino acid sequences and have no competitive relationship with the sites of insecticidal action. As the protein structure of Vips has not yet been elucidated, the mechanistic study on insecticidal action has lagged behind. In order to identify the key amino acids affecting insecticidal activity of Bacillus thuringiensis (Bt) Vip3Aa11 protein, site-directed mutagenesis was performed on 3 amino acid sites in Vip3Aa11, and 3 mutant proteins G200S, F442S and S726T were successfully constructed, and their insecticidal activities against Spodoptera exigua and Helicoverpa armigera were determined. The results showed that the insecticidal activity of mutant protein S726T against S. exigua was 4 times of that of Vip3Aa11, while the mutant proteins G200S and F442S showed no significant change in insecticidal activity against S. exigua and H. Armigera. To find out the reason of activity change in mutants, 3 mutants and the wild type Vip3Aa11 were treated with trypsin. The results indicated that both variants and wild type Vip3Aa11 could yield a 62~66 kD fragment by trypsin digestion in vitro and the effects of the 3 mutants on trypsin sensitivity were basically consistent. Through secondary structure prediction, it was found that the conformation of mutant protein S726T was shifted backward on α-helix which indicated that the increase of insecticidal activity caused by S726T mutant might be related to the slight change of protein structure space. In this study, the difference in insecticidal activity between Vip3Aa11 protein and each mutant protein for different insects was compared, and the reason for the difference was preliminarily analyzed, the results may provide a guideline for further study on the structure and function of Vip3Aa proteins.
LIU Ming,SUN Hai-Yan,LI Hai-Tao, et al. The Influence of Site-directed Mutation in Bt Vip3Aa11 Protein on Insecticidal Activity Against Spodoptera exigua and Helicoverpa armigera[J]. 农业生物技术学报, 2019, 27(7): 1259-1265.
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