The Cloning And Vectors Construction Of Vip3A Gene, EF1α Promoter And Transformation In Tobacco

Vegetative insecticidal proteins (Vips), an insecticidal protein, are secreted during vegetative stage of Bacillus thuringiensis. The Vips are known as a novel high virulence protein, plays a significant role in constructing broad spectrum and high toxicity genetically engineered microorganism, transgenic insect resistant plants, and controlling the resistance development of pests. Vips genes are regarded as the second generation insect resistant genes, and Vip3A protein gene has been studied most clearly.In this study, Vip3A gene was cloned by PCR. Sequence analysis showed that full-length of the gene is 2370 bp, predicting protein of the gene encoded 789 amino acids, which shared more than 99% homology with other deduced Vip3A protein, its molecular weight was 88 KDa, and belonged to weak acid protein. The bioinformatics prediction showed that the front of Vip3A protein was a signal peptide, which was a transmembrane signal and an extracellular secreting protein, The C-terminal of Vip3A protein had a stable functional region, the prediction of its secondary structure showed that the protein has obvious alpha helixes, beta sheets and coils. The specific structures imply potential function.Because of low level expression of insect-resistant genes in transgenic plants, in order to study the applications of Vip3A gene in transgenic insect-resistant plants, EF1αpromoter of tobacco also was cloned by PCR in the experiment, the promoter is a flower-specific promoter. Sequence analysis showed that full-length of the promoter was 1115 bp; and the promoter contained the initial 63 bp encoding sequence of EF1αgene, and it contains typical TATA-box and CAAT box consensus and cis-acting elements,it also contains other cis-acting elements,such as efficient transcription element 5 UTR Py-rich stretch, antioxidant element ARE, light responsive elements (BoxI, Sp1); ethylene responsive element ERE; heat shock element HSE; MYB protein binding sites MBS.The pBI121 used as a vector, three plants'expression vectors have been constructed successfully with the cloned Vip3A gene and EF1αpromoter: pBIEF vector, in which GUS gene was drove by EF1αpromoter; pBIVip3A vector, in which Vip3A gene was drove by constitutive CaMV35S promoter; pBIEFVip3A vector, in which Vip3A gene was drove by flower-specific promoter EF1α. The three vectors were transferred into tobaccos via Agrobacterium mediated method. Transgenic plants were indentified by PCR detection. The positive transgenic plants containing GUS gene have been detected by GUS histochemical assay and the transgenic lines containing Vip3A gene have been analyzed by RT-PCR expression analysis again. The results showed that some transgenic lines have obtained.