长牡蛎钙调蛋白基因克隆及多态性分析

Cloning and polymorphism analysis of calmodulin(CaM)gene obtained from Crassostrea gigas

【目的】从钙调蛋白(CaM)基因克隆和序列分析入手,对长牡蛎CaM基因的功能进行深入研究,为生产实践提供参考依据。【方法】利用SMARTRACE和EPIC-PCR技术克隆长牡蛎CaM基因,然后采用PCR-SSCP技术和DNA测序方法分析长牡蛎CaM基因编码区多态性。【结果】扩增获得长牡蛎CaM基因cDNA全长序列为917bp,包括开放阅读框(ORF)全长450bp,编码149个氨基酸;5'非编码区含222bp,3'非编码区含245bp;ORF内有3段内含子序列,分别为In-tron-1:110bp、Intron-2:137bp和Intron-3:143bp。PCR-SSCP分析获得C5引物野生型TT型、突变型GG型和杂合型GT型3种基因型。基因型和等位基因频率统计结果表明,GG型和G等位基因频率明显高于TT型和T等位基因;而最小二乘分析结果表明,CaM基因位于ORF内第158位T→G的SNPs位点,对长牡蛎的壳重有显著性影响(P<0.05),但对壳长、壳高、壳宽、体总重及肉重无显著影响。【结论】CaM基因对长牡蛎贝壳形成具有一定的影响作用。

[ Objective ]The aim of this study was to conduct in-depth analysis of calmodulin （CAM） gene functions in Crassostrea gigas using molecular approaches in order to provide with references for commercial production. [ Method ] CaM gene was cloned by SMART RACE and ERIC-PCR, and polymorphism in coding sequence of CaM gene was analyzed by PCR-SSCP and DNA sequencing. [Result]Amplified cDNA of CaM gene had a length of 917 bp, including an ORF of 450 bp encoding a polypeptide of 149 amino acids, 5＇non-coding region of 222 bp, and 3＇non-coding region of 245 bp. Three intron sequences in ORF of CaM gene were as follows： Intron-1,110 bp; Intron-2,137 bp; and Intron-3,143 bp. Through PCR-SSCP analysis, three genotypes were obtained from C5： TT （wild）, GG （mutant） and GT（heterozygous）. According to statistics from genotypes, allele frequency of GG and G was evidently higher than that of TT and T. Least square analysis showed that the 158th T→G SNP site of CaM gene in ORF was significantly related to shell weight （P〈0.05）, while it had no significantly effects on shell length, shell height, shell width, gross weight and meat weight. [ Conclusion ] CaM gene affected the formation of the oyster shells in some extent.