| Dicer-2(Dcr2)is a ribonuclease of RNase Ⅲ family,it can specifically recognize endogenous or exogenous double stranded RNA(dsRNA)disease and gradually cut it into si RNA.Bombyx mori nuclear polyhedrosis virus(BmNPV)can cause a kind of acute infectious disease,which is the most serious infectious disease in sericulture.Breeding resistant varieties and screening resistant genes are topics that sericulture researchers have been exploring.In this study,the relationship between Dcr2 and BmNPV resistance of Bombyx mori was studied.The main results are as follows:1.The partial sequence of Dcr2 of silkworm was cloned successfully.The sequence length was 423 bp,and 141 amino acidswere encoded.The molecular weight of the predicted protein was about 15.5 k Da.The Dcr2 has no signal peptide and is not a secretory protein.Analysis domain display with DEXHc＿Dicer domain,MPH1 super family domain,two RIBOc domains,Dicer＿dimer and PAZ super family domains.Phylogenetic tree analysis showed that the closest relationship between Dcr2 of B.mori and Dcr of Bombyx mandarina.2.The constructed recombinant expression plasmid was transfected into the expression strain BL21 and induced to express the recombinant protein.The results of SDS-PAGE and Western blot showed that the recombinant protein was successfully expressed in E.coli,the recombinant protein was purified by Ni column,and the corresponding rabbit polyclonal antibody was prepared by the protein delivery company.3.The transcription and translation levels of Dcr2 in various tissues of silkworm were detected by fluorescence quantitative PCR and Western blot,respectively.The results showed that Dcr2 was expressed in all tissues and different developmental stages examined,and the highest expression was found in haemocytes and moth stage.And Dcr2 expression in adult stage was significantly higher than that in other stages.4.After injection of dsBmSPH-1,the expression of BmSPH-1 and Dcr2 were detected by RT-q PCR.The results showed that the expression of BmSPH-1 gene was down regulation at 48 h,meantime Dcr2 was significantly up-regulated.5.The larvae of silkworm were infected with BmNPV,the replication of virus in midgut and the expression of Dcr2 in midgut and haemocyts were detected.The results showed that the replication of the virus increased gradually,and the transcription level and translation level of Dcr2 in midgut and haemocyts were up-regulated.It is suggested that the expression of Dcr2 can be induced by BmNPV infection in B.mori.6.DsDcr2 or dsGFP were injected into silkworm to detect whether the interference of Dcr2 was successful.When Dcr2 was successfully interfered in silkworm,then oral feed BmNPV.The amount of replication of the virus in silkworm was detected.Compared with the other two groups,the amount of replication of the virus was significantly increased after Dcr2 was interfered.To sum up,this study found that Dcr2 is involved in the resistance to BmNPV through RNAi pathway,which is helpful to further supplement the research on the resistance mechanism of B.mori to BmNPV... |
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