Cloning, Methylation Status Of Full-length CDNA Of A Thermal Stress Gene From Japanese Scallop Mizuhopecten Yessoensis

Japanese Scallop(Mizuhopecten yessoensis) since the introduction of Chinese twentieth Century at the beginning of the 80’s after the rapid development of aquaculture research and practice. In recent ten years, Japanese Scallop appeared slow growth, low yield and large scale problems of seedling death, aquaculture suffered huge economic losses, seriously restricted the development of China’s shellfish industry. So the exploration on the immune system of shellfish can help us to effectively control the disease development of shellfish aquaculture Scallop, thereby better. This paper obtained 2 ESTs sequences from the constructed c DNA Library of Japanese Scallop(Mizuhopecten yessoensis), specific primers design and RACE technology, combined with the cloned complete c DNA sequence of the 2 stress gene, and the full-length sequence was obtained from the structural analysis and function prediction. The main research contents of this thesis are to do for these 2 stress genes are as follows:1. Cloning, sequence analysis, characterization and expression of full-length c DNA of a Tyrosinase gene from Japanese scallop Mizuhopecten yessoensisTyrosinase is a copper-containing metal enzymes, widely distributed inmicroorganisms, plants, animals and humans. It plays an important role in the innate immune defense. In this study, we report the cloning and characterization of a Tyrosinase(TYR)gene from Japanese scallop, Mizuhopecten yessoensis. Quantitative Real time PCR(q R T-PCR) was employed to measure TYR expression in different tissues and after bacte-ria(Vibrioanguillarum) injection. The full-length 1850 bp TYR m RNA contained a 37 bp 5’ untranslated region, a 1470 open reading frame encoding 489 amino acids and a343 bp 3’ UTR. As the q RT-PCR result showed, among the hepatopancreas, mantle, kidney, adductor, gill and hemocytes, the m RNA expression level is the highest in the mantle. 6 hours after the injection of Vibrioanguillarum, TYR gene expression was significantly lower than that of the control group, while the 12-36 hours after injection, the expression level of TYR gene was significantly higher than that of the control gr-oup, the data indicate that the gene may play an important role in antimicrobial immunity.2. Cloning, sequence analysis, expression and methylation status of full-length c DNA of a heat shock protein 60 gene from Japanese scallop Mizuhopecten yessoensisThe HSP60(heat shock protein 60) as a member of the highly conserved heat shock protein family, not only in the condition of stress to help other protein folding correctly and restore the natural conformation, can also be used as a danger signal to the natural immune system. Specific transcriptome sequence primer design based on HSP60, with the molecular cloning technique, we report the heat shock protein 60(HSP60) gene sequence from Japanese scallop Mizuhopecten yessoensis. The c DNA sequence was 3 368 bp in length, including 5 ’ UTR 68 bp, 1 569 bp 3’ UTR and 1 731 bp open reading frame encoding 576 amino acids. The use of gene walking amplification of the promoter region of My HSP60 gene sequences obtained and sequenced, the promoter region of My HSP60 gene sequences of the 1 236 bp, the area contains 2 TATA boxes, 4 CCAAT boxes, 3 heat shock element. Comparison of 10 Scallop in promoter sequence, did not find the putative SNP sites. The phylogenetic tree based on amino acid sequence and traditional species trees based on basic anastomosis. As the real-time PCR result shows, HSP60 in different tissues of Mizuhopecten yessoensis are expressed, the tissue expression increased after heating, in which expression of kidney significantly higher, while the expression of hepatopancreas, mantle, hemolymph and muscle increased significantly(P < 0.05), indicating that the gene plays a role in heat stress process. Detect and analyz the scallop DNA promoter region and the methylation degree of coding rigion by BSP method. The experimental results show that the Scallop DNA methylation occurs within the gene promoter region, which were not methylated sites, whereas in the coding region of DNA methylation.