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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 study the methylation of Pinus elliottii × P. caribaea, we established the MSAP (methylation-sensitive amplification polymorphism) reaction system of softwood pine needles, in which : Digestion-ligation system: 400 ng of genomic DNA was double digested with 3 U each of Eco RⅠ + HapⅡ or Eco RⅠ + MspI, incubated at 37 ° C for 24 h and ligated overnight at 16 ° C with T4 DNA ligase. The best pre-amplification system: digestion-ligation products 1μL, upstream and downstream primers 1μL, 2 × PCR mastermix 10μL and ddH 2O2 7μL. In a 20 μL selective amplification system, 2 μL of 10-fold diluted template DNA, 1 μL each of upstream and downstream primers, 10 μL of 2 × PCR mastermix, and 6 μL of ddH202 were prepared. Using the reaction system, 13 pairs of primers suitable for MSAP analysis of P. xylostella were screened out. The established MSAP reaction system can be used for methylation difference analysis of genomic DNA of P. vulgaris.