PfmPif97-like regulated by Pfm-miR-9b-5p participates in shell formation in Pinctada fucata martensii.

Xinwei Xiong,Zhe Zheng,Yu Jiao,Bingyi Xie,Xiaodong Du,Yuewen Deng,Thiyagarajan Vengatesen

doi:10.1371/journal.pone.0226367

Abstract

Mollusk shell matrix proteins are important for the formation of organic frameworks, crystal nucleation, and crystal growth in Pinctada fucata martensii (P. f. martensii). MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in many biological processes, including shell formation. In this study, we obtained the full-length sequence of Pif97-like gene in P. f. martensii (PfmPif97-like). PfmPif97-like was mainly distributed in mantle pallial and mantle edge. Correlation analysis indicated that the average shell thickness and weight showed a positive correlation with PfmPif97-like expression (P < 0.05). The inner surface of the nacreous layer and prismatic layer showed atypical growth when we knocked down the expression of PfmPif97-like by RNA interference (RNAi). We used a luciferase reporter assay to identify that miR-9b-5p of P. f. martensii (Pfm-miR-9b-5p) downregulated the expression of PfmPif97-like by interacting with the 3′-untranslated region (UTR) while we obtained the same result by injecting the Pfm-miR-9b-5p mimics in vivo. After injecting the mimics, we also observed abnormal growth in nacre layer and prismatic layer which is consistent with the result of RNAi. We proposed that PfmPif97-like regulated by Pfm-miR-9b-5p participates in shell formation of P. f. martensii. These findings provide important clues about the molecular mechanisms that regulate biomineralization in P. f. martensii.

Highlights

Biomineralization is a special biological process that achieves precise regulation through organic matrix [1]
Total RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions with some modification and cDNA was synthesized by M-MLV reverse transcriptase (Promega, USA). miRNAs were extracted by using SanPrep Column microRNA Extraction Kit (Sangon Biotech) and miRNA First-Strand was synthesized by using Mir-X miRNA First-Strand Synthesis Kit (TaKaRa)
The full-length cDNA of PfmPif97-like (GenBank accession numbers: MK962312) was 2356 bp, containing an 1821 bp open reading frame (ORF) encoding a putative protein of 606 aa, 50UTR of 47 bp, and 30UTR of 458 bp (Fig 1)

Summary

Introduction

Biomineralization is a special biological process that achieves precise regulation through organic matrix [1]. Mollusk shell is a stable organic mineral product consisting of calcium carbonate and organic matrices, including proteins, polysaccharides, and lipids [2, 3]. Shell matrix protein (SMP) is involved in nucleation, polymorphism, orientation, morphology, and organization of calcium carbonate crystallites in the shell [4]. Nacrein [5], MSI60 [6], N19 [7], N16 [8], Pif80 [9], Pif97 [9], and P10 [10] participate in nacre layer formation. MSI31 [6], MSI7 [11], aspein [12], prisilkin-39 [13], prismalin-14 [14], KRMP family [15], and prismin family [16] have key effects in prismatic layer.

Methods

Results

Discussion

Conclusion