Construction Of Instect Baculovirus With Gene Silencing Functions Of Mythimna Separata V-ATPase

RNAi technology is a high specificity of conservative tactics. It is a new pest control strategies. V-ATPase plays a very important role in insect body, and it has a close connection with the insect body’s life activities. The experiment designs synthesis from the known sequence and selected a moderating effect of siRNA sequences, builds the baculovirus having the gene silencing effect. This is a kind of siRNA and V-ATPase combined with insect baculovirus, methods for the prevention and control of Mythimna separata. Specific as follows:1. According to the B V-ATPase subunit gene sequence, I designed and chemical synthesis of the three siRNA, by injections will chip are injected into the Mythimna separata. Using q-PCR detection technology to show that the interference effect of si2(relative expression quantity of 42.53%) is better, and choose as a carrier to build, si2 sequence;2. Transfection, mice U6 with shRNA construct promoter to insect baculovirus expression vector pBacPAK9, screening of the Mythimna separata have silence function of recombinant virus;3. The virus continues to cultivate in sf21 cells and to extend to 15 generations. By PCR technique and sequencing to detect recombinant gene, the result shows that the recombinant baculovirus expression vector with good genetic stability;4. Three age Mythimna separata feed on recombinant baculovirus, q-PCR detection starts from the third day, the results show that B subunit gene expression of relative quantity decreases obviously, this fully shows that the virus were used in insect breeding and express in the body.The experiment can successfully built a stable expression of siRNA insect baculovirus, implements the gene silence(37.60%); Got to be able to stable genetic recombinant baculovirus, which provides a new means for the biological control.