| Hyriopsis cumingii is an economically important freshwater pearl mussel with high pearl quality that is endemic in China. Investigation of genes relevant to pearl quality is important for increased income of pearl producer. Based on the EST sequences of mantle cDNA library of H. cumingii, the complete cDNA of37kDa LRP and one metal binding protein gene in H. cumingii were cloned by rapid amplification of cDNA ends (RACE) in this study, then analyzed the characteristics of these two amino acids. The real-time quantitative PCR was used to study the two genes expression in eight tissues. Using the in situ hybridization studied the distribution of37kDa LRP gene, and the Prokaryotic expression techniques was used to express one metal binding protein gene in H. cumingii. The research results are as follows:1. Full-length cDNA cloning and molecular characterization of tow genes from H. cumingiiThe full-length cDNA sequence (1,133bp) of the37kDa LRP (accession No. JX441866) was obtained after amplification and found to comprise a5’UTR of71bp, a3’UTR of159bp and an ORF of903bp, encoding300amino-acid residues. The total atomic weight was4,625and the molecular weight approximately33.1kDa. The amino-acid sequence of H. cumingii37LRP exhibited the highest similarity with those of Pinctada fucata (80%), with Bos Taurus, Chlorocebus aethiops and Rattus norvegicus (73%), and with Homo sapiens and Xenopus laevis (72%).The metal binding protein full-length cDNA sequence was508bp(accession No. JQ358609) including a35bp5’-untranslated region, a119bp3’-untranslated region and an open reading frame (ORF)354bp in length, which encodes for117amino acids with the predicted molecular weight of12.7kDa and the isoelectric point of7.40. Online analytical results show that this metal binding protein contains a signal peptide of19amino acids, a transmembrane at3-22.2. Real-time quantitative analysis of tow genes from H. CumingiiQuantitative PCR analysis showed that37LRP was expressed in mantle, blood, gill, foot, liver, kidney, intestine and adductor muscle tissues. Highest expression was detected in liver, lowest in blood and similar low levels in all other tissues evaluated. During shell repair, expression of the37LRP in liver was initially increased, followed by decreased expression and a subsequent increase. Expression in the experimental group was significantly higher than that in the control group during the first and second day after shell damage and lower than that in the control group after4days Expression of the37LRP in the mantle initially increased and subsequently decreased during shell repair. Its expression in the experimental group was higher than that in the control group at each time-point, with highest expression at24h and recovery to normal levels on repair completion (Fig.5). These data indicated that the37LRP plays an important role in shell repair and mantle growth.Real-time fluorescence quantitative results show that this metal-binding protein gene expression in the gill tissue expression, followed by, in the mantle. The expression of this metal binding protein gene has the significant difference between the white and purple mantle. The result showed that there is correlation between the metal binding protein gene and shell color3. In situ hybridization of the37LRP in the mantleA strong positive signal for expression of the37LRP was observed in the external epithelial cells in the mantle, internal and external epithelial cells in the evagination and the epithelial cell layer in the peritoneum.4.The prokaryotic expression of this metal-binding proteinUnder30℃,0.2mM IPTG inducted the expression of this metal-binding protein recombinant plasmid (PET300-MBP, PET300-Metal Binding Protein) in the expression strain, the result of SDS-PAGE tested showed, one metal-binding protein from H. Cumingii existence in the tow forms of inclusion body. The largest expression at3h, it’s also the optimized conditions. |
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