Cloning And Expression Analysis Of Genes Involved In The Nacre Color Of Shell In Hyriopsis Cumingii

The freshwater mussel Hyriopsis cumingii is a native pearl mussel widely distributed in China and is the most commercially important mussel species exploited for freshwater pearl production. Color is an important character for pearl quality. The nacre color of shell(inner shell color) of the donor pearl mussel or oyster has a significant effect on the color of pearls. The formation mechanism of pearl color is still under discussion, primarily focused on metal ion coloring, organic pigment coloring and structural coloring. Based on these three essential factors affecting the color of pearls, and comparative analysis of the transcriptome in tissues secreting purple and white nacre in the H cumingii, four genes related to nacre color of shell were identified using purple selected mussels(mussels with purple inner shell color) and white selected mussels(mussels with white inner shell color).1. Cloning and expression analysis of Hc GIFLP genes from H. cumingiiTwo gastric intrinsic factor-like protein genes, Hc GIFLP1 and Hc GIFLP2 were cloned and characterized from H. cumingii. The full length of Hc GIFLP1 c DNA is 619 bp which encodes 140 aa and the full length of Hc GIFLP2 c DNA is 821 bp which encodes 148 aa. Gen Bank accession number are KR822703 and KR822704 separately. Both proteins contain a putative signal peptide with 23 residues in the N-terminal, and without transmembrane structure. The similarity of c DNA sequences and amino acid sequences between Hc GIFLP1 and Hc GIFLP2 are both very high. Real-time Q-PCR revealed that Hc GIFLP1 gene was ubiquitously expressed in all tissues with the highest expressin level in hepatopancreas. The expression level of the hepatopancreas in purple mussels was significantly higher than that of white mussels(P<0.01). Hc GIFLP2 gene was ubiquitously expressed in all tissues with the highested expressin level in mantle, and the expression level of the posterior mantle pallial in purple mussels was significantly higher than that of white mussels(P<0.01). The result of in situ hybridization showed the same expression site of the two genes in the outer epithelial cells at the mantle pallial and middle fold of mantle edge. The result of prokaryotic expression showed that the recombinant proteins of two genes existed in the form of inclusion body. Western-Blot analysis showed that the antibody of Hc GIFLP1 was highly specific. The antibody of Hc GIFLP1 can only detect recombinant proteins of its own, but the antibody of Hc GIFLP2 could not detect recombinant proteins of its own and Hc GIFLP1.2. Cloning and expression analysis of Hc CUBDC gene from H.cumingiiA CUB domain containing protein gene, Hc CUBDC was cloned and characterized from H. cumingii. The full length of Hc CUBDC c DNA is 5158 bp which encodes 639 aa. Gen Bank accession number is KP067952. Hc CUBDC has 4 CUB domains and belongs to CUB superfamily. It contains a putative signal peptide with 19 residues in the N-terminal, and without transmembrane structure. Real-time Q-PCR revealed that Hc CUBDC gene was ubiquitously expressed in all tissues with the lowest expressin level in hepatopancreas. In the purple mussels, the expression level of posterior mantle pallial(p MP) was significantly higher compared with anterior mantle pallial(a MP)(P<0.01). In the white mussels, there was no difference between a MP and p MP. Hc CUBDC expression level of the p MP in purple mussels was significantly higher than that of white mussels(P<0.01). There was no difference about the m RNA levels of a MP between the purple and white mussels. In situ hybridization showed that Hc CUBDC gene was mainly expressed in outer epithelial cells at the mantle pallial.3. Cloning and expression analysis of Hc CA2 gene from H. cumingiiA carbonic anhydrase gene, Hc CA2 was cloned and characterized from H. cumingii. The full length of Hc CA2 c DNA is 1770 bp which encodes 332 aa. Gen Bank accession number is KR822703. Hc CA2 has 1 CA domain and belongs to α-CA superfamily. It contains a putative signal peptide with 18 residues in the N-terminal, and without transmembrane structure. Real-time Q-PCR revealed that Hc CA2 gene was mainly expressed mantle tissues with extremely low expressin level in other tissues. In the purple mussels, the expression level of p MP was significantly higher than a MP(P<0.01). In the white mussels, there was no difference between a MP and p MP. Hc CA2 expression level of the p MP in purple mussels was significantly higher than that of white mussels(P<0.01). There was no difference about the m RNA levels of a MP between the purple and white mussels. The Hc CA2 expression level of pearl sac was extremely low before day 19 after implantation, and was significantly higher and steady after day 19. The Hc CA2 expression level of control varied during shell regeneration. The Hc CA2 expression level of treatment group was significantly lower than that of control at previous 7 time points, and Hc CA2 was scarcely expressed at post 7 time points. In situ hybridization showed that Hc CA2 gene was expressed both in the outer and inner epithelial cells. The hybridization signal showed mainly in mantle pallial of outer epithelial cells, and the signal at mantle pallial of inner epithelial cells was stronger than that of mantle edge.