雌牙鲆CYP19A基因启动子“CpG”岛区的单核苷酸多态性及与繁殖性能的关联分析

Polymorphisms in CpG Island Region of CYP19A Gene Promoter Associated with Reproductive Performance in Japanese Flounder(Paralichthys olivaceus)

芳香化酶基因(CYP19)编码的芳香化酶是雄激素转化为雌激素的关键酶,在鱼类性腺发育及繁殖内分泌过程中起着重要的作用。本研究采用聚合酶链式反应-单链构象多态(PCR-SSCP)和放射性免疫方法(RAI)对61尾雌牙鲆进行了CYP19A基因启动子CpG岛区多态性检测,并将其与繁殖性能进行了关联分析。CpG岛区存在3种类型的基因型(pattern1、pattern2、pattern3),经测序发现CpG岛区有以下突变:T-1927插入或缺失、C-1899插入或缺失、T-1772C、C-1766T、TGTCAAC-1752^-1744插入或缺失(11个SNPs连锁突变)。方差分析结果表明:此位点对雌二醇(E2)水平的效应达到显著(P<0.05)水平,经多重比较分析,具有pattern1基因型的个体的雌激素(E2)水平显著低于pattern2和pattern3基因型个体的雌激素水平(P<0.05),同时pattern1基因型的个体的mRNA表达量显著低于pattern2和pattern3基因型个体的mRNA表达量(P<0.05)。以上结果表明,CYP19A基因调控区甲基化位点突变可能导致芳香化酶的表达量,进而影响牙鲆繁殖内分泌过程;同时也阐明了这些与牙鲆繁殖性状相关的SNPs可以用于生产实践中进行标记辅助选择具有优良繁殖性能的牙鲆。

Cytochrome P450arom, encoded by the CYP19 gene, is an important component of the enzymatic aromatase complex converting androgens to estrogens, which has been shown to play key role in ovarian development and reproductive endocrinology in fish. In this study, single nucleotide polymorphisms （SNPs） within CpG island region of the CYP19A gene promoter were tested and the associations between their genotypes and four reproductive traits were analyzed in 61 Japanese flounder individuals with polymerase chain reaction and single-stranded conformational polymorphism （PCR-SSCP）. Results indicated that three patterns in the promoter of CYP19A gene were identified, which were T--1927 insertion or deletion, C-- 1899 insertion or deletion, T-- 1772C, C-- 1766T, TGTCAAC-- 1752-- 1744 inser- tion or deletion（ll SNPs linkaged） by sequencing. Statistic results showed that the pair of primer was significantly associated with 17β-estradiol （E2） （P〈0.05）. Individuals with genotype pattern1 had significantly lower serum E2 levels （P〈0.05） than those of genotype pattern2 or pattern3. In addition, mRNA expression of CYP19A gene in pattern1 was significantly lower than those of patternt and pattern3. The evidence of the associations between genetic variants with serum E2 level may help explain that mutation in methylation site of CYP19A gene could change aromatase expression and further affect reproductive endocrinology of Japanese flounder. At the same time, these results implied that those SNPs might be used as molecular marker selection for Japanese flounder with super reproductive performance.