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.
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)
关键词
湿加松
MSAP
DNA甲基化
引物筛选
Pinus elliottii × P. caribaea
MSAP
DNA methylation
primers screening
