泥鳅MSAP技术反应体系的建立及优化

摘要

A reaction system of MSAP was established by orthogonal experiment in order to obtain genomic DNA methylation level and pattern of different ploidy of loach Misgurnus anguillicaudatus and used for MSAP analysis of diploid loach. The results showed that the best double digestion systems for MSAP including 10 U EcoR I, 10 U Hpa II, and 10 U Msp I were observed for 8 h at 37 ℃;the best pre-selective amplification mixture contained 0. 2 mmol/L dNTPs, 1. 5 mmol/L Mg2+, Taq 1 U and selective primers 0. 8 μL in total 20 μL;the best selective am-plification mixture contained 0. 375 mmol/L dNTPs, 1. 5 mmol/L Mg2+, Taq 1 U and selective primers 1. 5 μL in total 20 μL. The atlas showed that full methylation rate of three samples were 24. 1%, 20. 8% and 22. 3%, re-spectively;hemi-methylation rates were 41 . 4%, 20 . 8% and 33 . 3%, respectively;and total methylation rate was 65. 5%, 41. 6% and 55. 6%, respectively. The findings indicated that this MSAP reaction system was stable, reli-able and reproducible, providing the foundation for the MSAP technology with loach-related research.%为获得不同倍性泥鳅基因组DNA甲基化水平及模式,以泥鳅Misgurnus anguillicaudatus为研究对象,利用正交试验建立了甲基化敏感扩增多态性( MSAP)方法的反应体系,并利用该方法对二倍体泥鳅鳍基因组DNA进行了MSAP分析。结果表明：最佳双酶切反应体系为800 ng的泥鳅基因组DNA,用EcoR I、 Hpa II和Msp I各10 U,在37℃下反应8 h后即可酶切；最佳预扩增反应体系为模板4μL、预扩增引物0·8μL、0·2 mmol/L dNTPs、1·5 mmol/L Mg2+和 Taq 1 U；最佳选择性扩增反应体系为预扩增产物稀释20倍的模板2μL、引物1·5μL、0·375 mmol/L dNTPs、1·5 mmol/L Mg2+和 Taq 1 U；二倍体泥鳅全甲基化率分别为24·1%、20·8%、22·3%,半甲基化率分别41·4%、20·8%、33·3%,总甲基化率分别为65·5%、41·6%、55·6%。研究表明,该反应体系稳定、可靠、重复性好,为MSAP技术在多倍体泥鳅相关研究中的应用奠定了基础。