Screening And Identification Of Key Genes Of Replication And Proliferation In Bombyx Mori Nuclearpolyhedrosis Virus(BmNPV)

Bombyx mori is a model insect of Lepidopteran, which has important economic values. It plays an important role in the cultural transmission, economic and society development of world. However, the annual loss caused by silkworm disease up to25%proportion of the total silk production. Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of the most serious hazards and the most common type of pathogenic microorganisms. With the completion of the BmNPV genome sequencing in1999, the study of the molecular mechanisms of BmNPV infection and proliferation has been gradually deepened.In this study, using silkworm endogenous microRNA backbone by RNAi technology, screening and identification of key genes of proliferation and replication in BmNPV. The main results are as follows:1. Screening and cloning of the genes related to proliferation and replication of BmNPVAccording to the literature of baculovirus gene function analysis,34genes which releated proliferation and replication of BmNPV were preliminary screened and cloned in BmNPV. There are nine genes involving in viral DNA replication,28genes involving in viral BV generation,12genes associating with the nucleocapsid producing, eight genes affecting late gene expression, and seven genes affecting the very late gene expression.2. The key genes identification of proliferation and replication in BmNPVThe eight candidate genes were screened from the34screening genes for the further experiments study. They were dbp, lef2, lef3, lef7, lef10, lef11, p143, vp1054, respectively. The eight genes were constructed to the correspond interference vector and luciferase vector, which transfected to BmN-SWU1cells, respectively. Luciferase activity was measured at48h post transfection. The results reveal that the inhibitory effects of lef2and lefll reached more than90%, dbp, lef3, p143, vp1054inhibitory effect were more than80%, while the inhibitory effect of lef10was only12.4%,lef7has no inhibitory effect analyed by luciferase activity. According to the statistical analysis of the virus infection rate, the viral infection rate of cells which transfected pIZ-DsRed-milef11was only9.16%, pIZ-DsRed-mileflO was26.2%, while pIZ-DsRed-midbp, pIZ-DsRed-milef3were58.4%and54.6%, respectively pIZ-DsRed-mip143and pIZ-DsRed-mivp1054were71.1%and67.3%, respectively, pIZ-DsRcd-milcf2and pIZ-DsRed-milef7were100%. The result indicates that lefll was the key gene which in screened eight candidate genes of proliferation and replication in BmNPV.3. Effect on viral proliferation and replication by different promoters driving the same interference vectorSeven different activity promoters were selected. They were A4, IE1, IE1-295, IE2, IE2-339, hr3A4, hsp70promoters, respectively. pIZ-DsRed-milef11promoter was replaced with one of the seven promoters and constructed the correspond interference vector. The seven correspond interference vector and correspond luciferase vector were co-transfected to BmN-SWU1cells, respectively. The inhibitory effects of lefll examined using luciferase activity at48h post transfection. The results indicate that the inhibitory effects of A4, IE1, IE1-295, IE2, IE2-339, hr3A4, hsp70were92.6%,97.9%,96.9%,94.8%,95.3%,96.8%,97.9%, respectively.According to the statistical analysis of the virus infection rate, the viral infection rate of cells which transfected A4, IE1, IE1-295, IE2, IE2-339, hr3A4, hsp70were18.1%,8.34%,8.41%,11.1%,11.9%,12.1%and11.8%, respectively. By the analysis from the interference rates and the effect on the proliferation of virus replication, the results showed that IE1promoter was a best promoter in the screening promoters which drives pIZ-DsRed-milef11interference vector and pIZ-IE1-DsRed-milefll vector which drived by IE1promoter was also an optimized interference vector in this study.