Identification Of Resistance Of Silkworm BmTret1-X1 Gene To Bombyx Mori Nucleopolyhedrovirus(BmNPV)

Sericulture industry is China’s traditional advantageous industry,China’s raw silk production accounts for about 80% of the world,occupying an absolute dominant position in the world,sericulture industry in the current China’s rural poverty alleviation and rural revitalization plays an important role.Silkworm hemato-hemorrhagic purulema caused by bombyx mori nucleopolyhedrovirus(BmNPV)infection often causes serious harm to the sericulture industry and produces huge economic losses.The resistance of silkworms to BmNPV is mainly controlled by genetic factors,and genetic studies and resistance analysis show that the resistance of silkworms to BmNPV infestation is controlled by the main effect gene and the micro-effect gene.Sericulture researchers have been working to screen resources of resistant silkworm varieties and discover resistance genes,elucidating the molecular mechanism of silkworm resistance to BmNPV.Our team has previously positioned the resistance genes of the AN strains derived from the Huakang series resistance varieties in the 27 th chain group of silkworms,but the nature of the main effect genes of silkworms has not yet been clarified,and the molecular mechanism of BmNPV infection is not yet clear.Through transcriptome sequencing and genome localization,the group found that the Silkworm Trehalose Transporter 1 Isomer X1 gene(Bombyx Mori facilitated trehalose transporter Tret1-like isoform X1,BmTret1-X1)showed significant expression differences in silkworm strains with different levels of BmNPV resistance.It is speculated that BmTret1-X1 may be involved in the silkworm anti-BmNPV infection process,but further research evidence is lacking.In this study,the role of BmTret1-X1 gene in BmTret1-X1 resistance in silkworms was studied by overexpression of BmTret1-X1 in silkworm cells and reduction of expression of BmTret1-X1 in BmN cells by si RNA transfection,and the following research results were obtained.1.Analysis and validation of transcriptome results of midgut tissue in different BmNPV resistant strains of silkwormsFirstly,the transcriptome sequencing was analyzed in combination with the results of fine localization of the previous gene,and the trehalose transporter gene BmTret1-X1 gene between the BmNPV resistance near-alle line C108＿AN the sample of the inductive variety C108 had significant upregulation expression at 12 h,24 h,36 h,48 h,and 60 h after the attack,and log2(fc)was 4.246,3.309,5.36,3.712 and 4.643,respectively.BmTret1-X1 expression was analyzed on the midgut tissues of AN,C108,C108＿AN silkworms in the BmNPV oral attack group and the conventional feeding control group by RT-q PCR,and the expression of BmTret1-X1 genes C108＿AN by resistant strains AN and near isogene lines was similar between 5 instars and 48 h,and both were much higher than the expression of C108 of the sensibility cultivar C108,and the difference was significant(P <0.01);The expression of BmTret1-X1 gene was related to the genetic basis of silkworm cultivars,and there was no induction of BmNPV infection.Comprehensive analysis of the above results suggests that the difference in BmTret1-X1 expression levels between silkworm cultivars may be related to the difference in resistance of silkworms to BmNPV.2.Silkworm BmTret1-X1 cloning and molecular characterizationBioinformatics analysis of the BmTret1-X1 sequence showed that the BmTret1-X1 nucleotide sequence contained a 654 bp 5’ terminal UTR,a 147 bp 3’ terminal UTR,and a1476 bp long open reading frame(ORF),encoding 491 amino acids,containing a signal peptide and 12 transmembrane domains,and forming 2 relatively large extramembrane functional regions.The results of protein homology comparison analysis showed that BmTret1-X1 was less homologous to Tret1 with other Lepidoptera insects.There were four amino acid coding differences in the 71 st,90th,168 th and 480 th positions of the BmTret1-X1 gene between the resistant cultivar AN and the inductive cultivar C108,and the AN strains were I,V,Y and E,and the C108 strains became V,I,C and V.3.Identification of the inhibitory effect of BmTret1-X1 gene expression on the proliferation of BmNPV virusTransfection expression vector p IZT/V5-His-m Cherry-BmTret1-X1(red fluorescence)of BmTret1-X1 gene in BmN cells followed by addition of BmNPV recombinant virus BV-EGFP(green fluorescence).The effect of BmTret1-X1 gene expression on the replication and proliferation of BmNPV virus was detected by fluorescence microscopy and quantification of fluorescence intensity,the green mean fluorescence intensity ratios of 24 h,48 h and 72 h after BmNPV infection and the BmTret1-X1 overexpression group were 2.02,3.21 and 1.52,respectively,indicating that the viral proliferation of the BmTret1-X1 overexpression group was inhibited at 24 h and 48 h of infection,and the inhibition effect was weakened at 72 h.4.Effects of overexpression of BmTret1-X1 gene on BmNPV gene expression and genome replicationThe expression of BmTret1-X1 in BmN cells in the overexpressed group increased by about 70-250 times(P<0.01);the expression of BmNPV infection was reduced by 5-45times(P<0.01),especially the vp39 gene encoding nucleocapsid protein by up to 1089 times(P <0.01)。 It is shown that the expression of BmTret1-X1 gene has a clear inhibitory effect on the expression of BmNPV viral genes.At 24 h,48 h and 72 h after virus infestation,the viral genome content of the BmTret1-X1 overexpression group was lower than that in the control group,and the inhibition effect was most significant at 24 h of virus infestation(P<0.01),indicating that overexpression of BmTret1-X1 inhibited the genome replication of BmNPV virus.5.Correlation between BmTret1-X1 anti-BmNPV virus capability and SNP siteSNP/INDEL analysis and gene cloning sequencing results showed that there were some differences in the predicted protein tertiary structure due to the amino acid sequence changes caused by four SNP sites between the silkworm resistant strain AN and the sensibility strain C108.To explore whether the polymorphism of this gene is related to the anti-BmNPV virus ability of the AN strain,we analyzed whether there was a difference in the inhibition effect of BmNPV by infecting BV-EFGP after overexpression of the AN-BmTret1-X1 and C108-BmTret1-X1 genes in BmN cells.When the transfection efficiency of the two plasmids was similar,the inhibition effect on BmNPV proliferation was basically the same.The quantitative PCR technology detected the transcription level and genome replication changes of BmNPV virus genes,and found that there was no significant difference in the inhibition of BmNPV virus gene transcription level and genome replication when the overexpression multiples of AN-BmTret1-X1 and C108-BmTret1-X1 were similar.This showed that the change of 4 amino acid loci between AN and C108 varieties had no direct effect on BmNPV resistance.6.Effects of BmN cell RNA interference on BmTret1-X1 on BmNPV gene expression and virus proliferationThe transcriptional level of BmTret1-X1 in the RNA interference group decreased by about 50%,and the expression of lef-3,vp39,p10 and gp64 of BmNPV increased at a certain level after 6 h,12 h,24 h,and 48 h of virus infection,it shows that the silencing of the BmTret1-X1 gene in host cells can significantly promote the expression of the BmNPV virus gene.Fluorescence observation showed that RNA interference with the BmTret1-X1 gene of BmN cells significantly promoted the proliferation of the virus.