Identification Of BmCPV Encoded MicroRNAs And DsRNA Interference On BmCPV RDRP Gene

Bombyx mori cytoplasmic polyhedron virus BmCPV is a type of silkworm gut virus that poses great dangers in sericulture. Research on the polyhedron virus-encoding microRNA(miRNA) and RNA interference(RNAi) can help clarify the relationship between virus and its host to identify targets for viral miRNA control; on the other hand, it can also help explore the feasibility of using RNAi to disrupt viral replication that may have theoretical and practical significance in viral disease molecular treatment and resistant silkworm strain selection. In this study, we predicted BmCPV-encoding miRNAs, two of which underwent bioinformatic analysis, identification, and expression feature detection to explore preliminarily their impact on viral gene expression; we also investigated the interference of BmCPV-RDRP gene expression by double-stranded RNA(dsRNA) and constructed dsRNA-expressing transgenic vector. This study provides important reference value for further exploration of the relationship between miRNAs and silkworm genome,effects of miRNAs on the expression of host genes, and future detailed studies on the interaction between miRNAs and the genes on sub-sections of BmCPV virus; at the same time, it also provides a technical basis for the breeding of BmCPV-resistant silkworm varieties. The findings of this study are reported as follows.1. BmCPV encodes miRNAs. Total small RNAs were extracted from BmCPV-infected silkworm midgut, and small RNA library was established by deep sequencing to predict BmCPV-encoded miRNAs. Bioinformatic analysis, verification and expression pattern detection were done for two of the predicted miRNAs. The two miRNAs were miR-3,encoded by the 3rd fragment of BmCPV genome(478 ~ 497 bp), and miR-5, encoded by the fifth fragment of BmCPV genome(2481 ~ 2500 bp), respectively; both were 20 bp and the encoding positions were both in the ORF region; their secondary and 3-D structures were deduced through online analysis; Stem-loop RT-qPCR and Northern blot both confirmed the existence of the two miRNAs; gene expression analysis revealed that miR-3’s expression level was relatively higher than miR-5’s, and miRNA expression could be detected 5 h after BmCPV infection that started to increase at 24 h. The results indicated that BmCPV is capable of encoding miRNAs, which laid the foundation for further investigations on BmCPV-encoded miRNAs and their functions.2. BmCPV-encoded miRNAs regulate viral gene expression. After BmCPV infection,high-dose miR and inhibition-miR were injected into silkworms via cavity. High-dose miR led to significantly enhanced expression of some viral genes, as revealed by RT-qPCR detection.3. dsRNA interferes with the gene expression of BmCPV-RDRP. Three dsRNA sequences were designed based on the gene BmCPV-RDRP encoding RNA-dependent RNA polymerase of Bombyx mori cytoplasmic polyhedrosis virus. The dsRNAs were injected into body cavity after BmCPV infection and RT-PCR was used to measure the expression level of targeted gene. Each five-instar silkworm was injected with 4 ~ 6 ng/mg dsRNA to effectively interfere with the gene expression of BmCPV-RDRP; the interference rate could reach 93% in the treatment zone when dsRNA was injected before the infection, while the rate became as high as 99.9% when the infection was before the dsRNA injection; at the same time, the expression levels of BmCPV structural gene fragments, S1 and S5, were reduced along with decreasing RDRP expression levels. These results indicated that dsRNA interference of BmCPV-RDRP expression affects BmCPV viral amplification.4. Construction of dsRNA-expression transgenic vector. Silkworm midgut-specific promoter P2 was cloned to construct intermediate expression vector PBM-P2-dsRNA-eGFP.After transient transfection of the vector, P2 was able to promote the expression of dsRNA and formation of shRNA(small hairpin RNA); using P2-dsRNA sequences as target,dsRNA-expression transgenic vector, pig-P2-dsRNA-ploy(A)-eGFP-neo, was constructed based on piggyBac transposon. Transient transfection of the vector led to expressed dsRNA as revealed by qRT-PCR.