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李义良,赵奋成,陈铭甄,李宪政,钟岁英,邓乐平,李福明,张应中
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美高梅林业研究所,美高梅林业研究所,广东药学院生命科学与生物制药学院,美高梅林业研究所,广东省台山红岭种子园,广东省台山红岭种子园,广东省台山红岭种子园,美高梅林业研究所
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摘要: |
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为建立适合湿加松的MSAP（甲基化敏感扩增多态性）反应体系，以湿加松幼嫩针叶为材料，建立了湿加松MSAP反应体系，即：400ng基因组DNA用EcoRⅠ，HapⅡ或MspⅠ各3U，37℃保温24h。预扩增体系为：酶切-连接产物1μL、上下游引物各1μL、2×PCR mastermix10μL和ddH2O2 7μL。20μL选择性扩增体系中，含有10倍稀释的模板DNA2μL、上下游引物各1μL、2×PCR mastermix10μL和ddH2O2 6μL。利用反应体系，筛选出了13对适宜于湿加松MSAP分析的引物，建立的MSAP反应体系可用于湿加松基因组DNA甲基化差异分析。
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关键词:
湿加松，MSAP，DNA甲基化，引物筛选
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DOI：
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分类号：
S791.24
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基金项目：林木遗传育种国家重点实验室（中国林业科学研究院）开放课题（TGB2013005)，广东省科技创新专项（2014KJCX013），国家林业局林业公益性行业科研专项（20110414）。 |
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Establishment of MSAP reaction systems for Pinus elliottii × P.caribaea and primers screening |
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liyiliang,zhaofencheng,,,,, and
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Guangdong Academy of Forestry,,,,,,
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Abstract: |
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In order to establish suitable MSAP (Methylation Sensitive Amplification Polymorphism) reaction system for Pinus elliottii × P.caribaea, Young coniferous of Pinus elliottii × P.caribaea was used as material to screen the key factors in the system. It was found that 400 ng genomic DNA could be fully digested by EcoRⅠ, HapⅡor MspⅠ, 3 U respectively, at 37℃for 24h. The20μL pre-amplification reaction mixture contained the follow factors, DNA template 1μL, 2× PCR master mix10μL, ddH2O2 7μL, primers E/HM 1μL, respectively. The amplification products were diluted 10 times for the selective amplification. The 20μL selective pre-amplification reaction mixture contained DNA template 2μL, 2×PCR master mix10μL, ddH2O2 6μL, primer E-ACC/HM-CAC 1μL respectively. Finally，by using the system，13 pairs of primers were chosen for the MSAP analysis of Pinus elliottii × P.caribaea plants. Those results provide fundamental reference for further epi美高梅 studies on Pinus elliottii × P.caribaea DNA methylation.
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Key words:
Pinus elliottii × P.caribaea; MSAP; DNA methylation; primers screening
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