| Bombyx mori bidensovirus(Bm BDV)is an important virus,which brings huge economic losses to the sericulture industry every year.mi RNAs(micro RNAs)are a class of endogenous non-coding small RNA molecules,consisting of 18 to 24 nucleotides,and regulating the life activities of organisms.In this study,the 3rd instar larva infected with Bm BDV virus for 36 hours and the control group were sequenced by high-throughput sequencing technology,the sequencing data were analyzed by bioinformatics,and then screening out 24 different expression mi RNAs.The specific stem-loop primers of mi RNAs were synthesized,then using RT-PCR and q RT-PCR methods to verify 24 differentially expressed mi RNAs.Three significantly differentially expressed mi RNAs were selected and analyzed for tissue expression profile.We choose newly discovered novel-mir-16 for further study,validate four target genes of the novel-mir-16 through double luciferase reporter system,and select MAN1A1(mannosyl-oligosaccharide alpha-1,2-mannosidase IA)to do the preliminary research.The specific experimental results were as follows:1.Analysis the high-throughput sequencing results by bioinformaticsThe 3rd instar larva of silkworm Jingsong×Haoyue strain was fed with Bm BDV virus,and after 36 h the silkworm of the infected and the control group were collected respectively,then small RNA libraries were established,which was used to revealing changes in mi RNAs expression profiles by high-throughput sequencing.Analyzing sequencing results: the control and the infected group obtained 8 084 812 and 8 465 313 clean sequences respectively,and then the clean sequences were compared to the genome,with 16.9% in the control group and 15.36% in the infected group.In addition,the clean sequences were classified and annotated,as well as predicted the new mi RNAs,totally 273 mi RNAs and 24 novel mi RNAs were obtained,which including 24 differentially expressed mi RNAs,among of that the up-and down-regulated were 12.Bioinformatics software were used to predict the target genes of mi RNA,and GO analysis showed that these target genes were mainly involved in cellular processes,catalysis,binding and metabolic processes,while only a small part was involved in apoptosis,stress response and immune system processes.2.Differentially expressed mi RNAs were validated by q RT-PCRThe stem-loop RT-PCR and q RT-PCR methods were used to identify and verify the expression levels of 24 differentially expressed mi RNAs in the 3rd instar silkworm infected with Bm BDV 36 h and midgut of 5th instar silkworm after infected 48 h,so as to select out mi R-1923 which was significant up-regulated and mi R-13b-3p was down-regulated in the silkworm,as well as novel-mir-16 was significantly up-regulated in midgut tissue.Analysing the expression levels of three mi RNAs in various tissues,among them,mi R-1923 was the highest expressed in fat body,mi R-13b-3p and novel-mir-16 were both highly expressed in skin.Online software was used to predict the target genes of mi R-1923,mi R-13b-3p and novel-mir-16,then speculating the potential role of mi RNAs,and providing a theoretical basis for screening mi RNA for further research.3.Validation target genes of novel-mir-16novel-mir-16 was significantly up-regulated in midgut after infection with Bm BDV,so we used RNAhybrid online software to predict the binding site of novel-mir-16 with target genes,then verified the expression level of 9 target genes in midgut by q RT-PCR method.Initially,selecting out four down-regulated target genes among them,including: mannosyl-oligosaccharide alpha-1,2-mannosidase IA?apoptosis inducing factor?inhibit of growth protein5 and phosphoserine aminotransferase.We use the dual luciferase reporter system to verify at the cellular level,the results showed that: novel-mir-16 bound to AIF,MAN1A1 and Psat1 at the site of coding region and the target genes expression was down-regulated;which also has a binding site with the 3'UTR of ING5 and negatively regulates target gene expression.4.A preliminary study on the function of MAN1A1 geneWe choos MAN1A1 for further study and explored the function of novel-mir-16 initially.First,we analysis the tissue differential expression of MAN1A1 and the results showed that it was significantly higher in fat body;Then,the protein was predicted by biological software and the analysis showed that the protein was a stable,hydrophilic protein without signal peptide but with a transmembrane domain.Overexpression mimics inhibits the expression of MAN1A1 gene,which indirectly affects the down-regulation of some immune-related genes;and after overexpression MAN1A1,it may promote up-regulation of immune-related genes,indicating that changes in the MAN1A1 gene have an impact on the immune process of cells.However,after silkworm infected with Bm BDV,novel-mir-16 was up-regulated in the midgut tissue,and the expression of MAN1A1 gene was suppressed,so we speculate that novel-mir-16 is involved in the innate immune response of silkworm,and the specific regulatory mechanism needs further experiments to verify.As a result of the above study,it is more helpful to study the regulatory role of differentially expressed mi RNAs in the process of silkworm infection,providing a new theoretical basis for further study of the interaction mechanism between silkworm and Bm BDV,and also supporting a new idea for developing the biological control of Bm BDV. |
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