Cloning Of U6 Promoter From Mythimna Separata And Research On Silencing Of V-ATPase Subunit B

RNA interference(RNAi)is a technology which not only can be used to study gene function, but also can be used for pest control in the field. The V-ATPase widely exists in prokaryotes and eukaryotes. The main function of V-ATPase is hydrolysis ATP and proton transport. It is very important for maintain normal physiological characteristics of the organism. Therefore, any interference of the insect V-ATPase would dramatically effect growth and development, and even lead insect to death.Previous study showed that the relative expression of V-ATPase subunit B from Oriental armyworm(Mythimna separata, Walker) which injected by the dsRNA was suppressed and insect survival rates were decreased. In order to obtain siRNA continuously which can lead to gene silencing of V-ATPase, the technology of chromosome walking was used to clone the U6 promoter of Mythimna separata. And the function of the U6 promoter were analyzed and verified by bioinformatics software and transfected 293 T cells. The full-length gene of U6 promoter was 1 426 bp, which contained polymerase Ⅲ core promoter elements SPH, OCT, DSE, PSE and TATA box, and shared high homology with other insects such as Bombyx mori.Then we predicted and obtained shRNA which can result gene silencing of V-ATPase subunit B. The U6 promoter and shRNA were connected into insect baculovirus transfer vector, and obtained the recombinant baculovirus through transfected sf21 cell line. PCR and sequencing results indicated that the recombinant baculovirus had good genetic stability. Then the recombinant baculovirus was fed into 3rd instar larvae of Mythimna separata, and the gene silencing effect was analyzed by qRT-PCR. The results indicated that the relative expression level of V-ATPase subunit B gene of Mythimna separata was decreased significantly compared to the control.In this study, we cloned U6 promoter of Mythimna separata, and constructed the baculovirus shRNA expression vector which drived by U6 promoter successfully. And the recombinant baculovirus which can result V-ATPase subunit B gene silencing was obtained. Meanwhile, its genetic stability was analyzed. This study laid a foundation for using RNAi technology to control Mythimna separata and other pests.