Study Of Silkworm BmSMYD4-X1 Gene Resistance To Bombyx Mori Nuclearpolyhedrosisvirus

The Bombyx mori Nuclear Polyhedrosis Virus(Bm NPV)is the causative agent of silkworm hemato-purulent disease and one of the main pathogens that harm the sericulture industry.Once silkworms are infected with Bm NPV,a large number of deaths will occur,affecting the stable development of silk production.In order to effectively control Bm NPV infection,from breeding new varieties of disease-resistant silkworms to screening relevant resistance genes,sericulture scholars have conducted a large number of studies to try to reveal the interaction mechanism between silkworms and Bm NPV,but so far,the mechanism is still unknown.SMYD4 is a lysine methyltransferase gene containing SET And MYND Domain Containing 4,which is involved in gene expression and development regulation.Analysis of tissue expression in the middle intestine of silkworms found significant differences in the expression of Bm SMYD4-X1 gene between the resistant strain AN of nucleotype polyhedravirus(Bm NPV)and the sensibility strain C108.In this thesis,the differences in Bm SMYD4-X1 gene expression between the Bm SMYD4-X1 gene after the silkworm karyotype polyhedravirus Bm NPV infected with silkworm cells were studied by transcriptomics,molecular biology,bioinformatics and other analytical methods.The main results are as follows.1.Transcriptome sequencing results analysis and verificationIn the early stage,the research group used the silkworm perceptual strain C108 and the resistant strain AN to repeatedly cross C108 and combined with the progressive Bm NPV attack selection to construct a near-isogenic gene line C108＿AN;C108 and C108＿AN were used to inject Bm NPV polyhedra 1×10~8/m L suspension impregnated mulberry leaves from the 5th instar,and the middle intestine tissue of 12 h,24 h,36 h and 48 h after infection was used for transcriptome sequencing.The difference in the expression of Bm SMYD4-X1 gene in the midgut tissue between the antagonistic cultivar AN and the perceptual strain C108 in the transcriptome analysis reached 2.014-3.682,equivalent to 4.04-12.83 times,and the effect of Bm NPV attack on the expression of the Bm SMYD4-X1 gene was not obvious by quantitative detection of the fluorescence of the midgut tissue at different time points after the antagonistic strain AN and the perceptual strain C108 Bm NPV attack.There was no virus infection induced expression,and the expression patterns of Bm SMYD4-X1 gene between 0 h and 48 h after silkworming in infancy 5 years were similar,but the gene expression of the strains was significantly higher than that of C108 at most time points.2.Silkworm Bm SMYD4-X1 gene cloning and molecular characteristics analysisAccording to the silkworm genome database silk DB3.0 and silk base,the nucleotide sequence of Bm SMYD4-X1 gene was obtained,and the CDS full-length sequence of Bm SMYD4-X1 gene was successfully cloned from the PRIMER 5.0 software primer and the C108 strain of silkworm AN strain.Homologous comparison analysis of Bm SMYD4-X1amino acid sequences showed that Bm SMYD4-X1 had evolutionary specificity in the process of domestication.3.Analysis of the effect of Bm N cell overexpression of Bm SMYD4-X1 gene on Bm NPV replicationAfter the Bm SMYD4-X1 gene was expressed,the re-infection of Bm NPV virus was subjected to fluorescence quantitative PCR detection,and the quantitative results showed that after transfection of the recombinant vector,the expression of Bm SMYD4-X1 gene was significantly increased,and the gene expression was upregulated by about 25-40 times after6 h to 48 h after virus infection.The red fluorescence and green fluorescence intensities of the images at each time point were calculated,and the Bm SMYD4-X1 gene overexpression correction(green fluorescence value/red fluorescence value)was performed as a benchmark to eliminate the error caused by differences in plasmid transfection levels.The corrected green fluorescence intensity ratio(control group/overexpression group)after 6 h,12 h,24 h and 48 h of virus infestation was 3.54,3.98,6.62,2.10,and this result showed that the significant inhibitory effect of Bm NPV replication and proliferation continued to be shown at 6 h-48 h after overexpression of Bm SMYD4-X14.Effect of Bm SMYD4-X1 gene on transcription level and DNA replication of Bm NPV gene by Bm N cellsThe effect of overexpression of Bm SMYD4-X1 gene on the transcriptional level of representative genes of Bm NPV in different periods was analyzed by q RT-PCR detection,and the results showed that under the condition of overexpression of Bm SMYD4-X1 genes,lef-3,vp39 and p10 appeared expression inhibition from 6 h of viral infection,and all showed the strongest expression inhibition at 24 h of viral infection,and the maximum inhibition multiples of lef-3,vp39 and p10 reached 10.13,respectively.556.41 and 29.45times.At the same time,the effect of Bm SMYD4-X1 gene expression on DNA replication of Bm NPV virus was found that the DNA quantification results of the three viral genome loci were basically the same,and the Bm NPV genomic DNA decreased by 1.99-3.25 times at 6 h and 12 h after infection,the Bm NPV genomic DNA decreased by 8.17-12.73 times after infection 24 h,and the inhibition effect basically disappeared 48 h after infection.5.Effect of Bm N cells interfering with Bm SMYD4-X1 gene on transcriptional level of Bm NPV gene.At the cellular level,after RNA interference with the Bm SMYD4-X1 gene in Bm N cells,the quantitative results were shown at different time points,and the interference efficiency of the experimental group reached 70%compared with the control group.After RNA interference with Bm SMYD4-X1 in Bm N cells,BV-EGFP infected Bm N cells,and the expression of genes at different time points of the virus was detected at different time points,and the quantitative results showed that compared with the control group,there was no difference in the expression of lef-3,vp39 and p10 of Bm NPV at 6 h,and the expression of vp39 gene of Bm NPV in the interference group increased to 3.182 times that of the control group at 12 h(P<0.01),other gene expression differences were not significant.At 24 h and48 h of Bm NPV infestation,the expression of lef-3,vp39 and p10 genes in the interference group was slightly reduced by about 0.4-1.0 times(P<0.05).The results showed that RNA interference with Bm SMYD4-X1 had no significant effect on the proliferation efficiency of Bm NPV virus.In summary,the results of this study suggest that the high expression of Bm SMYD4-X1gene may be one of the molecular mechanisms of the resistance of host silkworms to Bm NPV,and the Bm SMYD4-X1 gene may be a new functional gene source for molecular breeding of Bm NPV resistance.