PPARγ2启动子的克隆及甲基化与基因表达的相关性分析

Promoter of PPARγ2 Cloning and Correlation Analysis of Methylation and Gene Expression

目的:克隆脂肪组织特异表达且表达受甲基化调控的基因启动子,并构建启动绿色荧光蛋白表达的载体模型。方法:本研究采用甲基化分析技术和荧光定量PCR技术分析PPARγ2基因调控区甲基化水平和基因表达水平的相关性,克隆其启动子,并与CMV缺失的pEGFP-N1连接构建重组质粒载体,之后转染脂肪细胞检测重组载体模型的有效性。结果:PPARγ2基因启动子的甲基化比例与mRNA表达量呈负相关,相关系数r=-0.740230314,获得PPARγ2基因启动子片段PPARγ2-P,构建的重组质粒pPPARγ2-P-EGFP在脂肪细胞中特异表达。结论:PPARγ2基因表达受启动子甲基化调控,试验成功克隆了PPARγ2基因启动子,且得到启动绿色荧光蛋白表达的载体,为后期建立甲基化依赖的启动子模型提供方法和材料,为转基因动物的制备提供新思路。

Objective ：To clone the adipose tissue -specific expression promoter regulated by the methylation, and construct a vector that can initiate the expression of green fluorescent protein. Method ： The correlation between gene expression and the methylation level in regu- latory region of PPARγ/2 gene was analyaed by Methylation and quantitative PCR. The promoter of PPARγ/2 gene was cloned and connected to EGFP that CMV was deleted to construct a recombinant plasmid, and its expression specificity was then tested by transfecting adipose cells. Result：The analysis of correlation results showed that methylation ratio of PPARγ/2 promoter is negatively related to the amount of mRNA expression （r = -0. 730230314）. The fragment of PPARγ/2 - P, the promoter of PPARγ/2 was obtained successfully, and the recom- binant plasmid pPPARγ2 - P - EGFP was efficiently expressed in adipose cells. Conclusion：The studys showed that the expression of PPARγ/2 is regulated by the promoter methylation status, and PPARγ/2 - P was successfully cloned, and the recombinant plasmid was ex- pressed in adipose cells. Our study would provide methods and materials for setting up methylation - dependent promoter model, and also provide a new idea for manufacturing of transgenic animals.