Identification And Functional Studies Of The MicroRNAs From The Marginal And Central Zone Of Pinctada Martensii

Mantle is the most important organ for shell formation in Pinctada martensii. According to the different functions and structures in different shell parts, from the shell’s top to the bottom, the mantle is divided into three regions: marginal, pallial and central zones. It is generally recognized that the central zone is majorate in the formation nacreous layers and the marginal zone is important for the prismatic layers formation. To explore the mechanism underlying the different functions between central and marginal zones, we have obtained the transcriptiome databases of microRNA and mRNA of the marginal and central zones in our precious research. In this study, based on the obtained database, we have screened the differential expressed microRNA and used mir-RACE to verify the sequence of the screened miRNAs. The expression level of microRNA in different tissues were determined by stem-loop qRT–PCR. Using Dual-luciferase reporter system, the miRNA-mRNA target interaction was verified. Furthermore, after overexepression of miRNAs in vivo, combined qRT–PCR and scanning electron microscope technology, the function of the miRNA and predicted potential target genes was validated. These results were summarized as follows:1. Screening and identification of mi RNA: from the transcriptome data of the central and marginal zones, we obtained the highly expressed miRNA in the marginal zone(miR-124, miR-8-3p and miR-9a-5p) and central zone(miR-10a-3p and miR-183) from 189 known miRNAs. By mir-RACE technology, the sequence of the obtained five miRNAs was validated. Redults showed the sequence of miR-124, miR-8-3p, miR-9a-5p and miR-183 were consistent with that from sequencing. While the 3’end sequence of miR-10a-3p’s was one bases shorter than that from sequencing. By stem-loop qRT-PCR, the expression patterns of the the five miRNAs in different tissues were detected. Results showed that the expression levels detected by stem-loop qRT-PCR were consistent with that from sequencing.2. Functional study of miRNAs: The SEM results showed that, the growth status of the nacre layer was disordered after over-expression of mi R-9a-5p, mi R-10a-3p or miR-124. This suggested that mi R-9a-5p, miR-10a-3p and miR-124 participated in nacre layer formation.3. Target prediction and identification of miRNA: By target gene prediction software, RNAhybrid and miRNAda, combined with transcriptome data, based on the complementarity between miRNA and target gene. 4 immunity, growth and biomineralization related-genes were chosen. The predicted targets of miR-10a-3p’s was nAChRsα, NPY and PfCHS1. The predicted targets of miR-124 was: nAChRsα, NPY and PfCHS1. The predicted targets of miR-9a-5p was: TRAF2 and PfCHS1. By qRT-PCR, we found over-expression of miR-9a-5p could down-regulated the expression of PfCHS1 gene, and over-expression of miR-10a-3p could down-regulated the expression of NPY gene, which further verified the target relationship between miR-9a-5p and PfCHS1, and between miR-10a-3p and NPY. Thus we proposed that miR-9a-5p and mi R-10a-3p participated in shell formation by targeting PfCHS1 and NPY, respectively. Furthermore we found that over-expression of miR-9a-5p could down-regulate the expression of TRAF2 gene, and over-expression of mi R-10a-3p could down-regulate the expression of nAChRsα and PfCHS1 gene, and over-expression of miR-124 could down-regulate the expression of NPY, nAChRsα and PfCHS1. These results indicated that there were indirect regulatory relationship between these genes and the related miRNAs, while the mechanism was still unknown. Using Dual-luciferase reporter gene system, miR-9a-5p and PfCHS1 were conformed to have targeted regulation relationship, and miR-10a-3p and NPY have the targeted regulation relationship. Targeted regulation does not exist between the other miRNA and predicted target genes.