Computational identification and characterization of microRNAs and their targets in Penaeus monodon

摘要

Study on shrimp miRNAs was limited and just 7 mature miRNA sequences of Marsupenaeus japonicus are deposited in mirBase database.In this study,miRNAs and their target gene candidates were computationally identified from shrimp Penaeus monodon and then experimentally validated.Using 39 908 expressed sequence tags (ESTs) and 21 124 genome survey sequences (GSSs) of P.monodon (pmo) as reference dataset,a comprehensive approach based on inter-species homolog search was employed to investigate the candidate miRNAs (i.e.pmo-miRNA).A total of eight miRNAs belonging to 7 families were computationally identified and five out of them were subsequently validated by PCR and sequencing.Of these,pmo-miR-4961a,pmo-miR-4961b,pmo-miR-4979 and pmo-miR-3819 were first identified from shrimps.Both the mature pmo-miRNAs and the corresponding precursors were conserved among different species.Based on perfect or near-perfect match to the target region,the target gene candidates of pmomiRNAs were predicted from 10 331 mRNA sequences ofP.monodon.A total of 20 genes were predicted as the targets of pmo-miR-4961a,pmo-miR-4961b,pmo-miR-4979 and pmo-miR-6492.Experimental validation by dual luciferase reporter assay confirmed the targeting between 3 pmo-miRNAs and one or two of their target genes,especially the pmo-miR-4979 which could significantly down-regulate the expression of target gene (JR226772).This study updates the miRNAs and their targets in P.monodon and lays a solid foundation for future RNAi study.