Analysis Of MiRNA Expressional Variation In Tachypleus Tridentatus After Vibrio Parahaemolyticus Injection And MiRNAs Computational Mining

Chinese horseshoe crab(Tachypleus tridentatus)is a kind of rare and endangered aquatic arthropods.When Chinese horseshoe crabs were infected by bacteria,especially Vibrio parahaemolyticus,their normal physiological activity will be severely affected,even leading to death.Many studies showed that MicroRNAs(miRNAs)are a class of small non-coding RNA in organism,which involved in the regulation of immune system development and function.However,the miRNAs related to immune system of T.tridentatus have not been reported.Therefore,the study of miRNA is conducive to reveal the mechanisms of immune response of T.tridentatus.In this study,we constructed two small RNA libraries using hemocytes from bacteria-injected and control(without injected)adults of T.tridentatus.The bacteria used in this study were V.parahaemolyticus.The expressed miRNAs of these two libraries were obtained using Solexa technology.The miRNA expression spectrum and differential expression were analyzed through bioinformatics methods.537 miRNAs were obtained including 292 conserved miRNAs and 245 newly discovered miRNAs.89 differentially expressed miRNAs were found between infected and control groups(P?0.05).The differentially expressed miRNAs were identified by Realtime quantitative PCR.The results showed that the expression level of miR-745,miR-87 and miR-305 was upregulated after infection,meanwhile that of miR-10 was downregulated.The results provide an important theoretical basis to study miRNA regulation in innate immune of T.tridentatus.The miRNAs and miRNA precursors of T.tridentatus were identified through sequence alignment.The potential 63 miRNA precursors were screened out with 46~102 nt in length.These precursors contained 49 mature miRNAs,with the length between 19~23nt.The conservation of miRNAs between T.tridentatus and other animals such as zebra fish,mice,humans,drosophila melanogaster were analyzed.The results showed that these four families of miR-10,miR-219,miR-1 andmiR-190 families between T.tridentatus and the above bilaterian,and their precursor sequences were highly conservative.The target genes of ttr-miR-34-5p,ttr-miR-2b and ttr-miR-8-3p were filtered out though miRanda and PITA prediction softwares.We found that the target gene function of ttr-miR-34-5p mainly involved in intracellular solidification inhibitor?,serine proteolytic enzyme,blood cells transglutaminase;ttr-miR-2b mainly involved in 11 kinds of related proteins such as HDID stents,arginine phosphokinase,ribosomal protein and so on;ttr-miR-8-3p regulating target genes function is relatively wide,involving cell components,molecular functions and biological processes.In this study we identified the first miRNA transcriptome profile of T.tridentatus,which further expanded the number of Chinese horseshoe crabs miRNAs by bioinformatics analysis.The miRNAs obtained in this study will lay the foundation of further research on regulatory mechanism of various physiological activities in T.tridentatus.