Construction Of The RNAi Vectors With Inducible Promoter To Against BmNPV Infection

Nuclear polyhedrosis is caused by the Bombyx mori nuclearpolyhedrosisvirus (BmNPV) infection, which causes serious economic loss. The most economical and effective way to prevent the disease is breeding the antiviral silkworm strain. RNA interference (RNAi)-mediated gene-specific silencing can be used in viral inhibition. Nowadays. RNA interference combining transgenesis technology provides a new approach to elicit antiviral infection in silkworm larvae. Using a strong constitutive promoter could increase the efficiency of RNAi, but the constitutive expression of the foreign gene may affect the development of transgenic silkworm. Therefore, selecting a suitable promoter and constructing the stable vector to express dsRNA would eliminate the influence of RNAi and to development the antivirus ability of silkworm simultaneously.In this study, we uesd lucifer's reporter gene to detect the inducible promoters plef3 and p39k from BmNPV. Then we constructed the RNAi vectors with inducible promoter to against BmNPV infection. The results were described as following:1 Promoter selection and cloningAccording to knowledge on the baculovirus coordinately regulated cascade fashion, we selected promoters of lef3 and 39k gene in BmNPV. We cloned the 39k and lef3 promoter region from BmNPV genome. The sequencing results showed that the cloned promoters contain transcriptional core promoter region which are the obvious characteristics of baculovirus promoter, TATA box, cap site, early gene transcription start site of the conserved sequence (CAGT), translation start codon ATG, which are coincident with the characteristics of eukaryotic promoters.2 Promoter activity assayTo investigate the activity of promoter, we constructed pGL3-Basic-39k and pGL3-Basic-lef3 vectors. respectively. The transient transfection results showed that the activity of 39k promoter in Sf9 and BmE-SWUl cells was increased significantly when the transgenic insect cells infected with AcMNPV or BmNPV. In Sf9 cell, the activity of p39k is lower than in the cell infected BmNPV (relative activity increased from the 0.411 to 64.9268, increased about 158 times). In the host cell BmE-SWU1 the constitutive activity is also lower than in the cell infected AcMNPV (relative activity increase from the 6.0151 to 12.6497. increased about 2.103 times). The constitutive activity in Sf9 cells is much lower than that BmE-SWUl cells. But the activity of lef3 promoter in the case of whether the virus infected was far below the 39k promoter (P＜0.01). Further, we determined 39k promoter activity at various time points after transfection in BmE-SWU1 cells, found that,39k promoter expression increased with time, the activity of p39k is lower than in the cell infected BmNPV (P＜0.01)3 Construction of transgenic RNAi vector and detection of antiviral efficiency in vitroWe identified the structure of RNAi vector and constructed the transgenic RNAi vector which can produce stable double-stranded hairpin RNA (hpRNA) in silkworm cells. We used antibiotic G418 to screen the stable transgenic RNAi silkworm cell line.4 months later, the stable transgenic cell line was established. We analyzed the proliferation of the virus in transgenic cells infected with Bm-BacPAK. When using a higher titer of virus for infection (1.014×1010PFU/mL-1.014×l08PFU/mL),a significant antiviral effect (P＜0.01 or P＜0.05) in transgenic cell lines appeared during the virus infection early phase (48-84h). When using a lower titer(1.014×107PFU/mL-1.014×106PFU/mL) for infection, the significant anti-viral effect (P＜0.01 or P＜0.05) in transgenic cell lines occurred during late phase (84-108h).The results indicated that the interfere efficiency was closely related to the virus titer and virus proliferation time.