| Based on the transcriptome database of pearl sac from P. martensii obtained in ourprevious research, we predicted the secreted proteins using bioinformatical method, andselected two potential nacre formation-related genes from the predicted secreted proteins.Then, the full-length cloning, expression pattern and function in the nacre formation of thetwo potential nacre formation-related genes were analyzed. The results of this paper couldbe summarized as follows:1. The secreted proteins of pearl sac are308. According to the annotation information,there are38biomineralization related genes,15immune related genes. And255geneshave not been classified in detail, such as enzymes and cytokines. Bone morphogeneticprotein7(BMP7) and tissue inhibitors of metalloproteinase (TIMP) were selected as thetwo potential nacre formation-related genes.2. BMP7, also known as osteogenetic protein-1(OP-1), belongs to a member of TGF-βsuperfamily. In mammals, BMP7promotes the differentiation of osteoblasts and inducesectopic ossification. In this research, the full-length of BMP7from P. martensii (Pm-BMP7)was2972bp, including an open reading fragment (ORF) of1290bp encoding429aminoacids. The Pm-BMP7was a secreted protein that contained a signal peptide (1-35aa),pro-domain (36-294aa) and mature peptide (295-429aa); mature peptide was consisted of135amino acids with an estimated molecular mass of15.60kDa and a theoreticalisoelectric point of9.51, and included a TGF-β family domain with6cysteine residues.The sequence of Pm-BMP7showed66%identity to Crassostrea gigas,61%to Tegillarcagranosa,40%to Xenopus laevis,39%to Homo sapiens,39%to Danio rerio. Weperformed qRT-PCR to analyze the expression pattern of Pm-BMP7in eight tissues ofadult P. martensii. Results demonstrated Pm-BMP7was expressed in all detected tissueswith the predominant level in the mantle and gill, the lowest in haemocytes. Afterinhibiting Pm-BMP7expression using RNA interference (RNAi) method, Pm-BMP7mRNA was down-regulated significantly (P <0.05) in the mantle center (which forms thenacreous layer) and mantle edge (which forms the prismatic layer); meanwhile, by SEM,we observed the nacreous layer of the shell showed a disordered growth status and theprismatic layer appeared obvious holes. These results suggested that Pm-BMP7played acrucial role in the formation of the nacre and prismatic layer. 3. TIMP are widely identified as multifunctional proteins that play a vital role in theregulation of extracellular matrix proteins. The cDNA of TIMP gene in Pinctada martensii(Pm-TIMP) was901bp long, containing an open reading fragment (ORF) of681bpencoding a polypeptide of226amino acids with an estimated molecular mass of23.37kDaand a theoretical isoelectric point of5.42, a signal sequence with a cleavage site between20and21, and13cysteine residues. Amino acid multiple alignment showed the Pm-TIMPhad the highest (41%) identity to the TIMP from Crassostrea gigas. Tissue expressionanalysis indicated that Pm-TIMP was expressed in all the studied tissues of pearl oyster P.martensii, with the highest in the haemocytes, then was in the pearl sac, mantle and gill.After the inhibiton of Pm-TIMP gene by RNAi, we observed that Pm-TIMP mRNA wasdown-regulated significantly (P <0.05) in the mantle center. Combined with the SEMobservation, we found the nacreous layer showed a random growth, resulting in coveredwith a layer of irregular crystals in aragonitic line. These results indicated that Pm-TIMPinvolved in nacre formation. |
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