SOX9和INSL3基因在克隆猪隐睾中的表达及甲基化模式

SOX9 and INSL3 Gene Expression and Methylation Patterns in Cryptorchidism of Cloned Pigs

为查明本课题组所获得的体细胞克隆猪隐睾发生的可能原因,本研究采用相对荧光定量PCR技术和亚硫酸氢盐测序法分析SOX9和INSL3基因在克隆猪隐睾中的表达量及其启动子区CpG位点甲基化状态。结果表明:除了SOX9基因在克隆猪C2中的表达量显著高于对照组N(1.82 vs 1,P<0.05)外,克隆猪SOX9和INSL3基因的表达量都显著低于对照组N(P<0.05);亚硫酸氢盐测序结果显示SOX9基因启动子区在克隆猪C1和C2中的甲基化程度没有显著变化,而INSL3基因启动子区在克隆猪C1和C2中的甲基化程度显著高于对照猪N(86.25%、78.75%vs 57.50%,P<0.05)。结果说明,INSL3基因在克隆猪隐睾中DNA甲基化重编程异常,而且这种异常影响其正常表达,由此推测这可能是导致克隆猪发生隐睾的重要因素之一。

In order to identify the possible causes of the cryptorchidism in cloned pigs which producted by our research group,The relative fluorescence quantitative PCR techniques and bisulfite salt sequencing analysis be used to analysis the expression and promoter methylation status of the promoter CpG sites of SOX9 and INSL3 genes in the cloned pig's cryptorchidism.The results showed that,the expression of SOX9 gene in cloned pig C2 was significantly higher than that of control group N(1.82 vs 1,P<0.05),expression of SOX9 and INSL3 genes in cloned pigs were all significantly decreased(P<0.05).Bisulfite sequencing PCR results showed that there are no significant changes of SOX9 gene methylation in cloned pig C1 and C2;and the high level of INSL3 gene methylation in cloned pig C1 and in C2 were significantly higher than that in N(86.25%,78.75% vs 57.50%,P<0.05).Results indicated that the abnormal DNA methylation reprogramming of INSL3 gene occurred in cloned porcine cryptorchidism,which affected normal expression levels of INSL3 gene,suggesting that itmay be an important factor in the occurrence of cryptorchidism cloned pigs.