Functional Analysis Of MiRNAs Encoded By Bombyx Morinuclear Polyhedrosis Virus

Sericulture production is an important economic industry. Bombyx mori serves as a Lepidopteran model system for genetics and molecular studies. The B. mori nucleopolyhedrosis virus(BmNPV) is a natural pathogen of B. mori; it causes a very high mortality that in turn leads to a significant loss in silk cocoons and therefore a major economic damage to the silk industry. Varieties which have a strong resistance to the virus and which can eliminate the source of infection have been bred to control BmNPV damages; however the affected individuals cannot be cured once they are infected(Singh et al., 2010). Micro RNAs(miRNAs) are non-coding RNA molecules of 20-23 nt in length. These molecules regulate the expression of genes by guiding the RNA-induced silencing complex(RISC)to a target sequence, which is usually located at the 3’ UTR of m RNAs. Increasing evidence suggests that miRNAs have indispensable roles in host-virus interactions. In this article, 14 virus encoded miRNAs was found from silkworm haemolymph post infected BmNPV through high-throughput sequencing. We verified 7 of them by experimental methods, we also predicted the target genes of 2 of the miRNAs and do some research on regulation.The main results were as follows:1. We analyzed the miRNAs from silkworm haemolymph post infected Bombyx mori.nuclear polyhedrosis virus(BmNPV) through high-throughput sequencing, and found 14 virus encoded miRNAs, we verified seven of them by stem-loop PCR, and submitted them to NCBI database, we focus on 2 of the highest expression abundance one(BmNPV-miR-364, BmNPV-miR-415), and made a further research. We analyzed BmNPV-miR-364, BmNPV-miR-415 and 3 ’UTRs of silkworm and BmNPV by RNA22 and RNA hybrid software, and found 6 target genes of BmNPV-miR-364 and 9 target genes of BmNPV-miR-415.2. Recombinant expression vectors of BmNPV-miR-364 precursor, BmNPV-miR-415 precursor and 15 3 ’UTR of target genes were constructed, we transfected the miRNA and its corresponding target gene into Bm N cells one by one. Through the dual luciferase detection system(DLR) analysis we found that BmNPV-miR-364 targeting silkworm gene Prx5037、att A and viral gene GTA, BmNPV-miR-415 targeting viral gene DNA polymerase. We made BmNPV-miR-364 and BmNPV-miR-415 excessive and lack of expression by miRNA mimic and miRNA inhibitor, and found that BmNPV-miR-364 and BmNPV-miR-415 can inhibit the expression of their target genes respectively.3. BmNPV-miR-415 was transfected into silkworm Bm N cells, by using two-dimensional electrophoresis technology analysis we found that the group transfected with BmNPV-miR-415 have one more protein than the control group. We analyzed the differential protein by mass spectrometry and found that this protein is not each one of the predicted target genes before, but the target gene of rapamycin 2(TOR2). The proteins encoded by target genes of BmNPV-miR-415 may be too low expression or covered by high abundance ratios protein.4. We analyzed BmNPV-miR-415 and the sequence of TOR2 gene and found that TOR2 is not the target gene of BmNPV-miR-415, but the target gene of bmn-miR-5738 which was also obtained through high-throughput sequencing. The recombinant expression vectors of bmn-miR-5738 and TOR2 were constructed and transfected into silkworm Bm N cells. Measured by DLR, we found BmNPV-miR-415 and bmn-miR-5738 can all raise expression level of TOR2. We analyzed the expression of bmn-miR-5738 and TOR2 of silkworm haemolymph infected BmNPV by q PCR and found that the expression trend of bmn-miR-5738 and BmNPV-miR-415 is consistent in the first 24 h after viral infection, after that, BmNPV-miR-415 expression level has been rising, while the expression of bmn-miR-5738 and TOR2 down regulate. We injected BmNPV-miR-415 mimic and BmNPV-miR-415 inhibitor into the day 1 fifth silkworm larvae and found that in the experimental group injected BmNPV-miR-415 mimic, expression level of bmn-miR-5738 and TOR2 increased significantly(P < 0.05), and in the experimental group injected BmNPV-miR-415 inhibitor, expression level of bmn-miR-5738 and TOR2 significantly reduced(P < 0.05). According to the result of the above, we speculated that BmNPV-miR-415 can induce the generation of bmn-miR-5738, which in turn increases the expression TOR2.