Cloning Of Functional Genes Involved In Formation Of Nacre Matrix In Pinctada Martensii

Based on our previous studies, on the full spectrum of matrix proteins of Pinctada martensii, six genes that may be associated with the formation of nacre were choosed on the basis of highly expressing in the transcriptome databases of pearl sac and mantle pallial. Of these six genes, four genes have no functional annotation, another two genes were annotated by serine protease inhibitors. We have cloned where as the full-length cDNA sequences of these six genes and analysed the structural features of these sequences by bioinformatic methods. Study of tissue distribution of individual genes was conducted by Real time qPCR technology, RNAi combined with real-time PCR and scanning electron microscopy(SEM) techniques were applied to analyse its function on the formation of nacre, and the localization of these fragments was studied in situ hybridization. Furtherly, the prokaryotic expression technology was applied to obtain the recombinant protein and antibody of genes for western blotting experiment, so as to confirm the correspondence between genes and proteins associated with the mineralization formation of nacre, and the study will provide the basis of revealing the molecular mechanisms study.1. According to the existing naming items, those four genes without functional annotation were named as Argnine-rich matrix protein(Pm-RRMP), Glycine-rich matrix(Pm-GRMP), Proline-rich matrix protein(Pm-PRMP), Serine-rich matrix protein(Pm-RRMP), which the full-length of them were 1063 bp, 2088 bp, 2312 bp, 3154 bp, respectively coding 273, 565, 589 and 958 amino acids All of them carried with a signal pepitide to perform the function as secretting proteins. Similarly, the method of real-time PCR was applied to to detect the expression of them in different tissues or organs. Except for the Pm-RRMP was highly expressed in the outside edge of the mantle, the other three genes showed highly expression levels in the mantle pallial and pearl sac. After RNA interference experiments, the expression of the target genes significantly decreased in mantle pallial and the shell nacre crystallized irregularly, blurred the boundaries between the sheets, and merged with each other by SEM. These results showed that four genes plays an important role in the formation of shells. In situ hybridization studies have shown that the addition of epidermal cells martensii Pm-PRMP of mRNA expression in the outer mantle outer epidermal cell.2.The two genes with functional annotation were named by PmNSPI-1, PmNSPI-2, which the full-length of them were 766 bp, 702 bp, respectively, and encoded 164,183.All of them carried with a signal peptide to perform the function of secretting proteins. Mature peptides respectively are comprised of two Kunitz-type serine protease inhibitor family domain. Based on amino acid sequence characteristics as Kunitz-type serine protease inhibitor family, we initially and cloned and identified three sequences are Kunitz-type serine protease inhibitor family in this experiment. Real-time PCR indicated that PmNSPI-1, PmNSPI-2 expressed in different tissues, exspecially that these two genes were highly expressed in the mantle pallial and the pearl sac tissue. After the RNA interference experiments, as above, we discovered that the expression of the two genes significantly decreased in mantle pallial; Surface of the nacre crystal showed roughness, malalignment, and the hexagonal structure was not obvious. In situ hybridization experiment, we found that mRNA of PmNSPI-2 gene specifically expressed in the outer pleated outer mantle epithelium. Building the recombinant expression vector of PmNSPI-1 and PmNSPI-2, the recombinant proteins were optimized in E. coli at the optimal expression conditions and concluded that the optimal expression conditions of PmNSPI-1 was 0.1 Mm, 12 h, 37℃ while the PmNSPI-2 was 0.8 mM, 8 h, 37℃; under the optimal conditions, the recombinant protein was obtained and confirmed by Western blotting the recombinant fusion protein could integrate with His-Tag monoclonal antibody, fusion proteins exhibit the same size pieces, combining with sequencing results inferred, it confirm that the recombinant fusion protein was expressed successfully.