| 王鹤潼,何蕾,宋杰,孙梨宗,崔伟娜,台培东,贾春云,刘宛.改进MSAP-PCR技术应用于Cd胁迫下拟南芥DNA甲基化分析[J].农业环境科学学报,2015,34(8):1618-1624. |
| 改进MSAP-PCR技术应用于Cd胁迫下拟南芥DNA甲基化分析 |
| Assay of DNA Methylation in Arabidopsis Under Cd Stress Using Improved MSAP-PCR Technique |
| 投稿时间：2015-03-16 |
| DOI：10.11654/jaes.2015.08.027 |
| 中文关键词: 甲基化 MSAP-PCR 拟南芥 生物标记物 表观遗传损伤 |
| 英文关键词: methylation MSAP-PCR Arabidopsis biomarker epigenetic damage |
| 基金项目:国家自然基金项目(21347007);国家自然科学基金项目(2107-7113,40930739,20977095);辽宁省自然科学基金项目(201202224);国家科技重大专项(2012ZX7505-001);沈阳大学区域污染环境生态修复教育部重点实验室基金 |
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| 摘要点击次数: 3260 |
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| 中文摘要: |
| 全基因组DNA甲基化分析是植物胁迫表观遗传损伤研究的主要方向之一,目前可用手段虽多,然而多数较繁琐,不利于表观遗传损伤的快速诊断。为此应用MSAP-PCR法研究Cd胁迫下拟南芥基因组甲基化变化,并通过优化实验条件、筛选引物(共5条,包括通用引物MLG2和本实验室设计引物AP-1、AP-2、AP-3、AP-4)以及检验多态性和敏感性,综合评估该方法在植物甲基化研究中的应用前景。研究结果发现:该方法模板量选择在150~250 ng并使用4%聚丙烯酰胺凝胶电泳(含50%尿素)分辨PCR产物多态性最佳;该方法对镉敏感性高,在0.2 mg·L-1 Cd2+水平就可检测约30个位点的甲基化多态性;引物AP-4对CpG位点甲基化变化最敏感,而AP-3对CHG位点甲基化变化敏感性最高。该方法操作简便、成本低廉、结果准确且敏感性高,可作为植物全基因组甲基化研究的理想方法,以及植物抗逆研究和环境污染早期诊断的生物胁迫标记物。 |
| 英文摘要: |
| Global genomic DNA methylation assay is a hot research topic in plant epigenetic damages induced by stress. However, there is no method used for a rapid diagnosis of epigenetic damages. In this study, Arabidopsis was used as the test plant to examine the applicability of the methylation-sensitive arbitrarily primed PCR(MSAP-PCR) in plant methylation assay. The experimental condition optimization, primer screening, and polymorphism and sensitivity analysis were performed. A higher methylation polymorphism of MSAP-PCR products could be obtained with the optimum template DNA of 150~250 ng and 4% PAGE gel with 50% urea. MSAP-PCR had higher sensitivity to Cd stresses, with 30 sites of methylation polymorphism detected under 0.2 mg·L-1 Cd2+. The primer AP-4 was sensitive to methylation in CpG sites whereas the primer AP-3 was sensitive in CHG sites. In summary, MSAP-PCR was a method with simple procedure, low cost, high accuracy, and superior sensitivity. It could be an ideal method for studying the plant global genomic methylation, the plant stress resistance, and the early diagnosis of environmental contamination. |
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