Transcriptome Analysis Of BmNPV-infected Silkworm Midgut Tissue And Functional Identification Of BmTret1-like Gene

Nuclear polyhedrosis caused by Bombyx mori Nucleopolyhedrovirus(BmNPV)is an acute infectious disease,once infected which causes huge losses for sericulture.Culturing resistant varieties and screening resistance genes is a topic that sericulture researchers are constantly discussing.In this study,cultivar with high resistance to BmNPV and conventional breed were used to analyze the transcriptome data of midgut,which can get more about the profile of the resistance mechanism and some correlated genes.We screened the gene Bombyx mori facilitated trehalose transporter Tret1-like(BmTret1-like)and analyzed the gene cloning and gene expression.RNAi technique was used to discuss the relationship between BmTret1-like gene and resistance mechanism.The main results were listed as follows:1 Transcriptome analysis of midgut tissue in resistant varieties and conventional varieties of SilkwormIn the experiment,we constructed two RNA libraries and obtained 12.82 Gb Clean Date(6.40 Gb for BY and 6.42 Gb for BYN).The sample Q30 base percentage is more than 86.03%.The clean Date were compared with the reference genomic sequence to obtain the position information and the unique sequence information.In the identification of single mismatches and potential single nucleotide polymorphisms(SNPs),BY were compared with 57 789 SNP and BYN were compared with 58725 SNP.More than 60% of SNP in the two libraries was in the gene region.Compared Mapped Reads with original genomic annotation information,we unearthed a total of 788 new genes.There were too many genes involved in the regulation of disease resistance in silkworm.We found 496 differentially expressed genes,of which 244 genes were upregulated,252 genes were downregulated and 486 genes were annotated.The analysis of GO classification of unigenes shows that the cell and cell membrane in the cell line are the main part.The metabolic process,the single tissue process,the cell process and the biological regulation contain many genes in the biological process.It belongs to the molecular function which has the catalytic activity and binding function.KEGG signal pathway analysis shows that 142 genes are annotated.In the COG database,it was found that the differentially expressed genes were mainly related to the metabolism and transport of amino acids,carbohydrates and inorganic ions.Silkworm infected BmNPV arise complex gene expression regulation,there are a number of pathways involved in the regulation of antiviral mechanisms.Most of the genes were mapped on a single pathway with the reference gene,and the characteristics of these genes and their effects on disease resistance needed further study.2.Cloning and expression analysis of BmTret1-like gene in silkwormBmTret1-like gene was cloned from the midgut tissue in resistant variety BYN and the conventional variety BY and about 1272 bp gene sequences were obtained.Compared with the homologous sequences in the genome of silkworm,it can be seen that the distribution in chromosomes of homologous genes is not uniform,mainly in the 27 linkage groups.The phylogenetic tree showed that the gene of BmTret1-like was closely related to Aedes albopictus,Solenopsis invicta,Takifugu rubripes and Apis mellifera,and was far from Drosophila melanogster and Caenorhabditis elegans.BmTret1-like gene was expressed in all tissues of silkworm,and had no tissue specificity.The gene expression was downregulated in the midgut tissue and the trend in conventional variety was more obvious.In the fat body tissue it shown different adiustment trend in resistant variety and conventional variety,conventional variety showing upregulated trend in addition to 12 h time points and resistant variety showing a downregulated trend.3?RNA interference of BmTret1-like geneBased on known fragments,dsRNA was designed to synthesize BmTret1-like gene,and BV virus punctured resistant varieties and conventional varieties with fluorescent groups.One group of resistant varieties was injected with dsRNA and the other group was directly puncture virus.The results showed that the fluorescent effect was observed in the resistant varieties of RNAi,and there was no fluorescence effect in the resistant varieties of the puncture virus,and the fluorescent effect was obvious in the conventional cultivars.This indicated that the antiviral mechanism in silkworm was affected,indicating that BmTret1-like gene was involved in the antiviral mechanism of silkworm.