Bombyx Mori Nuclear Polyhedrosis Virus-encoded Mirna And Its Preliminary Functional Analysis

MicroRNAs (miRNAs) are 21-25nt non-coding RNAs. They regulate the expression of related genes and play crucial roles in various physiological processes, including growth, development, metabolism and many other important cellular processes. The first miRNA was detected in Caenorhabits elegan. Recently miRNAs have been detected in many viruses too.In order to study the function of miRNAs encoded by Bombyx mori nuclear polyhedrosis virus (BmNPV), we used the method of bioinformatics and high-throughput sequencing to identifiy BmNPV-encoded miRNAs, and utilized the online software to predict the possible viral target genes of these miRNAs; and preliminary analysed the function of two known BmNPV miRNAs m1 and m2 by experiments. The results are as follows.(1) Prediction and RT-PCR verification of BmNPV miRNA precursors. To predict BmNPV ecoded miRNA precursors, the complete BmNPV genome was screened with vMir software, and the predicted sequences were scored and their hairpin structures were analyzed. Thus 7 candidate BmNPV encoded miRNA precursors were obtained after removing the sequences located in protein coding regions. The hybrid silkworm S16·S17×A1·A16, Bombyx mori was used as experimental materials, and fifth instar larvae were infected orally with BmNPV suspension of 5×107 PIBs/mL. The hemolymph was harvested from the BmNPV-infected larvae 72 h post infection for RNA extraction. And 5 sequences were amplified by reverse transcription PCR (RT-PCR), which are the possible miRNA precursor sequences. After analysis of hairpin structure of above 5 precursors, their mature miRNA sequences were verified by RT-PCR and 6 mature miRNA sequences, which are consistent with the virus genome and locate between ORFs, were amplified. In the 6 sequences, 2 were the positive sequences and 4 were reverse sequences. Therefore, we deduce that these 6 mature miRNA sequences are encoded by BmNPV. Moreover, real time-PCR analysis showed that the expression of above 5 precursors and 6 miRNAs increased as time goes on.(2) High-throughput sequencing of BmNPV miRNA sequences. The high-throughput sequencing method was used to detect miRNA sequences encoded by BmNPV. And 14 sequences were obtained inform the BmNPV genome. But only 7 sequences locating in different ORFs could be identified by RT-PCR, and 3 of them were the positive sequences and the other 4 were reverse ones.(3) Prediction of target genes of BmNPV miRNAs in the virus genome. By using online RNA target gene prediction programs RNAhybrid combined with RNA 22 software, viral target genes of the above 13 BmNPV miRNAs were analysed and 28 potential viral target genes were predicted. In which 4 are envelope protein coding genes, 3 are the capsid protein coding genes, 5 of them are lef-5, lef-6, SOD, Polyhedrin and GP37 coding genes, and the other 16 are unknown function genes. Therefore we consider that BmNPV miRNAs may be involved in the process of host infection.(4) Analysis of function of BmNPV miRNAs m1 and m2. By applying online RNA target gene prediction programs RNAhybrid, target genes of BmNPV miRNAs m1 and m2 were predicted. Then the precursor sequences of m1, m2 and the 3'-UTR sequences of predicted target genes were amplified by PCR from the genome of BmNPV. The pcDNA recombinant plasmid expressing m1 and m2 miRNAs and PGL recombinant plasmids expressing the 3'-UTR of the target genes were constructed respectively and co-transfected to BmN cells. The analysis of dual luciferase reporter gene assay system showed that overexpression of m1 down regulated expression of PGL-38k 3'-UTR by 14%, overexpression of m2 own regulated expression of PGL-Polyhedrin3-UTR and PGL-ODV-EC27 3-UTR by 9.8% and 9.2%, respectively. It suggests that 38K and Polyhedrin, ODV-EC27 are the possible target genes of m1 and m2 separately.