Cloning And Expression Analysis Of Genes Involved In The Pearl Formation Of Hyriopsis Cumingii

The triangle pearl mussel (Hyriopsis cumingii) is an important freshwater pearlmussel in China. In this study, the first work is to compare the relative expression ofseven housekeeping genes across different tissue types and in the mantle or pearl sacduring three biomineralization processes: seasonal shell growth, shell healing and pearlsac formation in H. cumingii to help analyze the characterization of matrix protein.Three programs evaluated the expression stabilities of the seven genes: BestKeeper,geNorm and NormFinder. To investigate the potential role of matrix protein infreshwater mussel shell and pearl formation, we cloned two C-type lectin gene andBMP7gene depend on the H. cumingii transcriptome data. In the current study, wealso investigated the expression pattern of the three gene in various tissues, duringshell healing and early pearl formation. Meanwhile, we analyzed the expression mantlelayers of three gene by in situ hybridization (ISH). The main results as followed:(1) H. cumingii house keeping gene selectionTo obtain the quantitative real-time RT-PCR suitable housekeeping genes, weevaluated the expression stabilities of the seven genes across different tissue types andin the mantle or pearl sac during three biomineralization processes: seasonal shellgrowth, shell healing and pearl sac formation in H. cumingii using three programs:BestKeeper, geNorm and NormFinder. The results showed that the expressions ofUbiquitin (Ubi) and Ribosomal protein L18(Rpl18) and Elongation factor1-alpha(EF1) were more stable than the remaining four genes. Therefore, we suggest thatUbi, Rpl18and EF1are suitable reference genes.(2) The cloning and analysis of2perlucin gene in H.cumingii The full-length cDNA transcribed from the Hc-perlucin1and Hc-perlucin2genewas1460bp and1973bp long, encoding a protein of161and183amino acids,respectively. Only Hc-perlucin1have a putative signal peptide. The both Hc-perlucin1and Hc-perlucin2peptide contained six conserved cysteine residues and a carbohydraterecognition domain, similar to other members of the C-type lectin families. In addition,a―QPS‖and―WND‖motif near the C-terminal region were also found inHc-perlucin1and Hc-perlucin2, respectively. These means that Hc-perlucin1andHc-perlucin2are specific binding to galactose and mannose, respectively. The bothmRNA of Hc-perlucin1and Hc-perlucin2were constitutively expressed in all tested H.cumingii tissues, with the highest expression levels observed in the mantle, adductorand gill. However, Hc-perlucin1also high express in hemocytes, and Hc-perlucin2gene express in intestine. The significant Hc-perlucin1mRNA expression was detectedon day14post shell damage and implantation. However, the Hc-perlucin2mRNAexpression only increase during pearl formation. In situ hybridization was used todetect the presence of Hc-perlucin1and Hc-perlucin2mRNA in the mantle, and theresult showed that the both mRNA were specifically expressed in the epithelial cells ofthe dorsal mantle pallial, an area known to express genes involved in the biosynthesisof the nacreous layer of the shell.(3) The cloning and analysis of Hc-BMP7geneThe full-length cDNA transcribed from the Hc-BMP7gene was2080bp long,encoding a protein of428amino acids. Hc-BMP7have a TGF-β family signature,TGF-beta propeptide and TGFB domains. The mRNA of Hc-BMP7were constitutivelyexpressed in all tested H. cumingii tissues, with the highest expression levels observedin the mantle, kidney, gill and gonad. The expression of Hc-BMP7have not changeduring shell and pearl formation, although Hc-BMP7was specifically expressed in theepithelial cells of the dorsal mantle pallia and the inner epithelial cells of the outer foldof the mantle edge.(4) Purification and Optimization of Prokaryotic Expression of Hc-perlucin1ProteinWe amplified the open reading frame of Hc-perlucin1from the freshwater pearlmussel, H. cumingii. And then the ORF was ligated into the expression plasmidpET-28a, and the recombinant plasmid was transferred into host bacteria, Escherichia coli BL21(DE3) cells. The recombinant pET-28a-Hcperlucin protein was expressedby induction with isopropyl-β-thiogalactopyranoside (IPTG). The result showed thatthe optimal conditions for recombinant protein expression was inducing the cells at37℃in0.1mmol/L of IPTG for6h. Inclusion body was the main existence form ofthe recombinant protein. We detected the expression products showed the recombinantexpression plasmid of pET-28a-Hcperlucin expressed a21kD protein, using SDS-Pageand Western-Blot.