湿加松MSAP反应体系建立及引物筛选  

Establishment of MSAP Reaction Systems for Pinus elliottii×P,caribaea and Primers Screening

作　　者：李义良[1,2] 赵奋成[1,2] 李宪政[1,2] 陈铭甄 钟岁英 邓乐平 李福明 张应中[1,2] 

机构地区：[1]广东省森林病虫害生物防治重点实验室,广东广州510520 [2]广东省林业科学研究院,广东广州510520 [3]广东药学院生命科学与生物制药学院,广东广州510006 [4]广东省台山红岭种子园,广东台山529200

出　　处：《广东林业科技》2015年第3期35-39,共5页Forestry Science and Technology of Guangdong Province

基　　金：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"湿加松及其亲本DNA甲基化水平与杂种优势关系研究"(TGB2013005);广东省科技创新专项"湿加松优良新品系评价与选育技术研究"(2014KJCX013);国家林业局林业公益性行业科研专项"国外松高世代良种培育关键技术研究"(20110414)

摘　　要：为开展湿加松(Pinus elliottii×P.caribaea)甲基化研究,以湿加松幼嫩针叶为材料,建立了湿加松MSAP(甲基化敏感扩增多态性)反应体系,其中:酶切-连接体系为:400 ng基因组DNA用Eco RⅠ+HapⅡ或Eco RⅠ+MspⅠ各3 U进行双酶切,37℃保温24 h,用T4 DNA ligase 16℃连接过夜。最佳预扩增体系为:酶切-连接产物1μL、上下游引物各1μL、2×PCR mastermix 10μL和dd H2O2 7μL。20μL选择性扩增体系中,含有10倍稀释的模板DNA 2μL、上下游引物各1μL、2×PCR mastermix10μL和dd H2O2 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。In order to establish suitable MSAP （ Methylation Sensitive Amplification Polymorphis ） reaction system for Pinus elliottii× P caribaea, young pine needles of Pinus elliottii × P caribaea were used as material to screen the key factors in the system. It was found that 400 ng genomic DNA could be fully digested by EcoR Ⅰ , Hap Ⅱ or MspⅠ , 3 U respectively, at 37℃ for 24 h. The 20 μL pre-amplification reaction mixture contained the follow factors, DNA template 1μL, 2 × PCR master mix10 μL, ddH2O2 7 μL, primers E/HM 1 μL, respectively. The amplification products were diluted 10 times for the selective amplification. The 20 μL selective pre-amplification reaction mixture contained DNA template 2 μL, 2 ×PCR master mix10 μL, ddH2O2 6 μL, primer E-ACC/HM-CAC 1 μL respectively. Finally, by using the system, 13 pairs of primers were chosen for theMSAP analysis ofPinus elliottii × P. caribaea. Those results provide fundamental reference for further epigenetic studies on Pinus elliottii × P. caribaea DNA methylation.

关 键 词：湿加松 MSAP DNA甲基化 引物筛选 

分 类 号：S791.24[农业科学—林木遗传育种]