改进亚硫酸盐测序技术研究拟南芥DNA甲基化水平  

An Improved Bisulfite Sequencing Technique for Analysis of DNA Methylation Level in Arabidopsis thaliana

作　　者：马珊珊[1,2] 孙晓霞[3] 李照令[1,2] 贾春云[1] 刘宛[1] 台培东[1] 赵晓辉[4] 李培军[1] 

机构地区：[1]中国科学院沈阳应用生态研究所污染生态与环境工程重点实验室,沈阳110016 [2]中国科学院大学,北京100049 [3]辽宁大学,沈阳110036 [4]沈阳农业大学,沈阳110866

出　　处：《农业环境科学学报》2013年第5期889-894,共6页Journal of Agro-Environment Science

基　　金：国家自然科学基金项目(20977095;40930739);辽宁省自然科学基金项目(201202224);国家科技重大专项(2012ZX07505-001);沈阳大学区域污染环境生态修复教育部重点实验室基金资助

摘　　要：DNA胞嘧啶甲基化是一种重要的表观遗传修饰形式,在细胞增殖、分化、发育、基因组印迹、基因表达调控等过程中起着重要作用。随着对DNA甲基化研究的深入,各种DNA甲基化检测方法被开发出来满足不同类型的研究需求。通过实验建立的一种改进亚硫酸盐测序技术,将拟南芥基因组DNA经酶切纯化后,用亚硫酸盐修饰液修饰,将PCR产物克隆到pEASY-T1载体上,每个样品随机挑取15个阳性克隆,测序分析,研究拟南芥错配修复基因MutL-homologue 1(MLH1)启动子区域的甲基化水平。结果表明:与传统亚硫酸盐测序法相比,改进方法有利于DNA完全修饰,既减少了修饰时间,又提高了PCR目的片段的特异性、稳定性和重复性;MLH1基因启动子区域甲基化比率为27.3%,而不是45.6%,避免了非甲基化位点的错认。为植物DNA甲基化分析提供了一种更为理想的研究手段。DNA cytosine methylation is a central epigenetic modification that has essential roles in the control of many critically important biological processes,including cell proliferation,differentiation,development,genomic imprinting and regulation of gene expression.With progresses in study on DNA methylation,detection techniques of DNA methylation have been developed to meet requirements of various researches.This article has established an improved bisulfite sequencing technique for analysis of MutL-homologue 1(MLH1) promoter methylation level of mismatch repair gene in Arabidopsis thaliana.After the genomic DNA in Arabidopsis thaliana was digested,purified,and then modified with bisulfite liquid,the PCR products were cloned into the pEASY-T1 vector.15 positive clones were randomly selected from each sample and then sequenced.The results showed that compared with the traditional method of methylation analysis,the improved method modified the digested DNA completely,reduced the modification time greatly,and improved the specificity,stability and repeatability of the PCR products.MLH1 promoter methylation level was 27.3% instead of 45.6%.The improved bisulfite sequencing technique avoided misidentification at non-methylated sites,and would provide a better means of research for analysis of DNA methylation in plant.

关 键 词：亚硫酸盐测序 DNA甲基化 MLH1 

分 类 号：Q2-33[生物学—细胞生物学]