Construction, Expression And Activity Assay Of Fused Gene With Vip3Aa And Cry1Ac From Bacillus Thuringiensis

Since microbial pesticides have the advantage of ample raw materials, specificprevention, less resistant among treatment objects and environment friendly, theyhave become a pillar industry of the biological control. Bacillus thuringiensisinsecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (VIPs) are themost important substances for the toxicity of Bacillus thuringiensis. Cry1AC is aninsecticidal crystal protein which formed in the stationary phase of Bacillusthuringiensis, it has high insecticidal activity against lepidopteran. Vip3Aa is avegetative insecticidal protein that formed in the vegetative growth phase and thensecreted into the extracellular, it has a broad insecticidal activity against pests. Thereis neither sequence homology nor same mechanisms of interaction between these twoproteins, so combination of them can be used as a novel toxin in pest control.Construction of fusion gene can save the cost of production, change the sectetionpositon of vip3Aa like protein， broaden the insecticidal spectrum, enhanceinsecticidal activity and delay pest resistance.In this study, two fusion expression vectors expressed fusion proteins namedVip3Aa11-GFP and Vip3Aa11-Linker16-GFP, which expressed in engineeredbacterias named HD8E-VG and HD8E-VLG, are constructed to determine the role oflinker. The results revealed that both of the fusion proteins can produce fluorescence.The fluorescence of HD8E-GFP, which only produces the protein of GFP, is strongerthan HD8E-VG or HD8E-VLG. But there was no significant difference betweenHD8E-VG and HD8E-VLG. The linker did not affect the activity of GFPs in fusionproteins.In order to reduce the consumption of energy in the progress of producingproteins and improve the yield of proteins, N-terminal region of Cry1Ac wasexpressed to determine the insecticidal activity and solubility. The results ofSDS-PAGE showed that it could express in a high level in Bt, and its solubilizationincreased in the presence of reducing agents. But the results of bioassay showed thatits insecticidal activity suffered a slight decline. The vip3Aa11-cry1Ac fusion gene under the control of the cry8E promoter wascloned into the plasmid pHT315-8E21b. In order to ensure the insecticidal activity offusion protein, some fusion expression vectors with different linkers and differentsequences are constructed in our study. The bioassay results showed that each of thefusion proteins showed a good insecticidal activity. The Vip3A-Cry1Ac fusion proteinenhanced the activity of Cry1Ac protein by3.1-fold against Plutella xylostella,Cry1Ac-Vip3A fusion protein enhanced the activity of Vip3Aa11protein by2.5-foldagainst Spodoptera exigua.This is the first time that Cry1Ac protein had been fused with Vip3Aa11proteinand expressed in Bacillus thuringiensis, it reduced the cost of fermentation andenhanced insecticidal activity. In this study we explored the role of the linker peptidein fusing genes, which provides the basis for the research of fusion protein, broadensthe insecticidal spectrum of Bt and delays pest resistance. At the same time, itprovides valuable materials for transgenic plants.