| Based on the mantle transcriptome library from Pinctada martensii completed in the previous study. Four with the comparative analysis of MC and MO transcriptome database, were screened out from the specifically-expressed genes in the MC. The full-length cDNA sequence of four candidate genes were obtained by the RACE technology, and the amino acid sequences and structural characteristics of the candidate genes were analyzed. The real-time fluorescence quantitative PCR(qRT-PCR) was performed to test the tissues distribution, and the RNAi induced silencing to explore the function of the candidate genes to shell mineralization. The results summarized as follows:1. Screening of specifically-expressed genes in the MC: Based on the mantle transcriptome library of Pinctada martensii, differentially expressed genes in the mantle were filtered. 2298 genes with significantly higher expression in MC were possessed,among which 228 genes were specifically-expressed in MC. 197 of 228 genes were annotated by the NR database, included the tyrosinase, nacre proteins, calcium binding proteins, tissue inhibitor of metalloproteinase TIMP, thioester-containing protein-F,MRNP, ribosomal proteins as well as some hypothetical proteins, such as prestin-like,tyrosine-protein kinase, solute carrier family etc. The specificitly high expressive genes in MC were carried on characteristic sequence analysis in this paper, such as the secondary structure and the domains. Finally, four genes including chitin binding protein, alpha-2macroglobulin-like, MRNP(methionine-rich nacre protein) and a novel gene 5915 were selected for the further study in this study.2. The cDNA sequence of chitin binding protein was cloned by RACE. The full-length sequence was 2126 bp, 5’UTR 143 bp, 3’ UTR 492 bp, the open reading frame(ORF) was1491 bp, encoding a protein of 496 amino acids. Chitin binding protein was predicted with Chitin-bind-3 domain by SMART. qRT-PCR results showed that chitin binding protein gene was significantly high expressed(p<0.05) in pearl sac, the mantle pallial region and the mantle central region in Pinctada martensii. The expression of chitin binding protein mRNA in the mantle were significantly inhibited after the injection ofds RNA of chitin binding protein, which leaded to abnormal phenomena such as the incompletion of nacre crystal morphology and fragmentation of layered plate even cave porosities in alignment of crystal in the inner surface nacrous layer of shells. And the nacrous layer of normal crystal was regular arranged, the plate arranged closely and clear crystal. Therefore chitin binding protein may be involved in the formation of nacrous layer in the process of shell mineralization.3. The α2M-like full-length was 3127 bp, with the 5’UTR of 390 bp, 3’ UTR of 322 bp,the ORF was 2415 bp, encoding 804 amino acids. α2M-like protein contained α-2macroglobulin domain predicted by SMART. Three conservative domains of typical α2M were the internal thioester region, receptor binding region and bait region in the C-terminal end of α2M-like, and the typical thioester bond sequences were298-GCGEQNM-304. But the N-terminal end missed partial sequences compared with typical α2M. At the same time, consistented with α2M-like in the shell protein of Crassostrea gigas, this indicated that α2M-like may have specialized in bivalve.qRT-PCR showed that α2M-like gene significantly higher expressed in the mantle than in other tissues(p<0.05). Compared with the control group, the mRNA expression ofα2M-like was significantly inhibited(p<0.05) in the mantle edge and central region by injecting α2M-like-dsRNA. However the expression in the mantle pallial was no declined significantly. Scanning electron microscopy showed that the α2M-like gene silencing resulted in the ultrastructural changes in the nacreous layer and prismatic layer of shells.The crystals were shaped irregularly, arranged loosely, even destroyed. Therefore, theα2M-like probably participate in the prismatic and nacreous layer of shell biomineralization in Pinctada martensii.4. The full-length of methionine-rich nacre protein(Pm-MRNP) from Pinctada martensii was 1226 bp, 5’UTR 99 bp, 3’ UTR 446 bp, the ORF was 681 bp, encoding 226 amino acids. The characteristics analysises of Pm-MRNP sequences showed that MRNP belonged to the alkalic insoluble matrix protein, contained 8 conserved Cys residues,which formed potential 4 pairs of disulfide bonds. MG-repeats domains were rich in Pm-MRNP. Multiple sequences alignment of the amino acid showed that the consistency of MRNP in Pinctada martensii, Pinctada margaritifera and Pinctada maxima was up to85%, which indicated that MRNP was highly conserved in the Pearl oyster. qRT-PCR results showed that Pm-MRNP gene was significantly highly expressed(p<0.05) in pearl sac, the mantle pallial and the mantle central tissues in Pinctada martensii. The RANi experimental results showed that compared with the control group, the relative expression of Pm-MRNP mRNA was significantly declined(p<0.05) in the mantle in theexperimental group of injection Pm-MRNP-dsRNA. The ultrastructural changes in the nacreous layer of the internal surface from the experimental group shells were observed by SEM, which showed that the new nacreous crystals morphology were incomplete and irregular, the plate crystal was small. Thus, Pm-MRNP could be involved in the formation of the nacreous layer in shell mineralizative process.5. The full-length cDNA sequence of an unknown function gene 82.8 kDa protein gene was 3083 bp, 5’UTR 199 bp, 3’ UTR 592 bp, the ORF was 2292 bp, encoding 763 amino acids. 65 glycosylative sites and 26 phosphorylative sites were predicted by softwares,which indicated may undergo complex glycosylative and phosphorylative modifications after translation. The 82.8 kDa protein was alkalic insoluble protein predicted by ProtParam software. The 82.8 kDa protein was intrinsically unstructured/disordered proteins(IUPs or IDPs) with Repeated low complexity domains(RLCDs) structures predicted by IUPred and SMART online softwares. α-helices, β-sheet and random coil were composed in the secondary structure. qRT-PCR results showed that the gene was significantly highly expressed(p<0.05) in pearl sac, the mantle pallial and the mantle central tissues in Pinctada martensii. The mRNA relative expression of 82.8 kDa protein gene was significantly declined(p<0.05) in the mantle by injecting α2M-like-dsRNA.SEM detected that in the nacreous layer of inner surface in shells from the experimental group, the new nacreous crystals morphology were incomplete, slice of layered plates were finely, crystals existed interspaces in alignment. Thus 82.8 kDa protein probably involve in the formation of nacreous layer of shell mineralizative process. |
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