The Functional Research Of Ras3 In BmNPV Infection And Relication,Bombyx Mori

Bombyx mori Ras3(BmRas3)is a small molecule protein in the GTPase superfamily,which has the activity of binding guanine nucleotidesand GTPase.It acts as a molecular switch by coupling extracellular signals to different cellular responses through the transition between Ras-GTP conformation and Ras-GDP conformation,thereby regulating and controlling cell growth,migration,adhesion,cytoskeleton integrity,survival and differentiation,etc.signal path.On the basis of sequence analysis and inducible expression analysis,this topic verified the anti-BmNPV effect of BmRas3 in vivo and in vitro by means of cell transfection and RNA interference(RNAi)technology.Real-time quantitative PCR(q RT-PCR)was used to detect the expression of key genes in the cellular pathways that BmRas3 may be involved in at the transcriptional level.It was preliminarily speculated that BmRas3 was involved in the cellular immunity of silkworm against nuclear polyhedrosis virus(BmNPV)infection pathway.The main findings are as follows:First,the full-length cloning and bioinformatics analysis of the BmRas3 gene.A new Ras GTPase superfamily gene,BmRas3(MW804669),was cloned from the silkworm p50 midgut tissue with the aid of RACE terminal rapid amplification technology.Nucleic acid sequence analysis showed that the full-length c DNA of the gene consisted of 987 bp,including 153 bp 5’UTR,279 bp 3’ UTR,and a 555 bp complete ORF encoding 184 amino acid residues.sub-composition.The amino acid sequence of BmRas3 contains three GTP/GDP binding domains(GSGGVGKS,DTAGT,NKTD),one effector binding domain(YDPTIEDSY)and one allylated CAAX motif(CIIL).It was proved that BmRas3 possesses the typical characteristics of GTPase superfamily proteins.Multiple alignment and phylogenetic analysis found that BmRas3 was most closely related to Rap1 of the genus Hyposmoko Equus,Rap-1b of Spodoptera litura and Rap1 of Helicobacter hawkmoth.Second,the expression profile analysis of BmRas3 in different developmental stages and tissues of silkworms.By detecting the transcription level of BmRas3 in each developmental stage of silkworms,it was found that the transcription level of BmRas3 m RNA in the mulberry-eating stage of each instar was significantly higher than that in the dormant stage.It is speculated that the expression of BmRas3 may be regulated by hormones.By detecting the changes of BmRas3 transcription level in each tissue after silkworms infected with BmNPV,it was found that BmRas3 was expressed in all tissues of silkworms,and the BmRas3 transcription level in the midgut and fat body of immune tissues was increased to varying degrees after inducted with BmNPV.It is indicated that BmRas3 may be an immune activation protein,which is involved in the immune response of silkworms against BmNPV invasion.Third,In vitro validation of BmRas3 inhibition of BmNPV replication.Bombyx mori ovary cells(BmN)were transfected by constructing a recombinant vector for overexpression and in vitro synthesized si RNA,overexpressing or interfering with the expression of BmRas3,observing and detecting the viral genome DNA copy of BmNPV and the expression level of late viral 39 K protein(VP39)in the cells..The results showed that the virus-infected cells,VP39 protein expression level,and viral DNA copy number in the cells overexpressing BmRas3 were significantly lower than those in the control group at each time point after BmNPV infection;The expression level of VP39 and the number of viral DNA copies were significantly higher than those in the control group at each time point after infection with BmNPV.That is,the overexpression of BmRas3 inhibited the proliferation of BmNPV,and the interference of BmRas3 promoted the proliferation of BmNPV.Fourth,the functional validation of BmRas3 againsting BmNPV in vivo.The synthesized si RNA was injected into the larvae of the third and fifth instar silkworms 24 hours after feeding,and BmNPV was orally infected to detect the survival rate and cocooning mortality of the third instar silkworm larvae after BmRas3 knockdown.The results showed that the survival rate of silkworm larvae after injection of si RNA was significantly lower than that of the control group,and the dead cage rate after cocoon formation was significantly higher than that of the control group.This indicated that knockdown of BmRas3 significantly reduced the anti-BmNPV ability of silkworms,further proving that BmRas3 played an active role in silkworms resisting BmNPV infection.Fifth,BmRas3 participates in the research on the cellular immune pathway of silkworms.q RT-PCR was used to detect the key factors involved in the signaling pathway of Ras protein in BmRas3-overexpressing cells and control cells,as well as the m RNA transcription levels of genes related to phagocytosis,apoptosis,synthesis of reactive oxygen species(ROSs),and viral DNA replication in cellular immunity.The results showed that the transcription level of Bombyx mori mitogen-activated kinase kinase 6(BmMapkk6)in the experimental group was significantly higher than that in the control group.It was speculated that the increase in the expression level of BmRas3 protein promoted the ERK tertiary phosphorylation cascade it participated in,thereby improving its own cellular immunity.In conclusion,this study identified a novel GTPase gene in Bombyx mori,BmRas3.It was proved that BmRas3 could inhibit the replication and proliferation of BmNPV when BmNPV infected silkworm.It is speculated that silkworm regulates the phosphorylation cascade of kinases in the MAPK pathway through the activation of BmRas3,which improves its own cellular immunity.The results of this research have laid a foundation for the study of the function of BmRas3 and its mechanism of resisting BmNPV infection;it also provides a breakthrough for the construction of transgenic silkworm strains resistant to BmNPV and improving the ability of silkworms to resist BmNPV.