[目的]建立并优化黄芪甲基化敏感扩增多态性(MSAP)技术扩增反应体系,对中药材黄芪甲基化水平进行研究.[方法]磁珠法提取黄芪DNA后,采用Hpa II/Msp I 2种内切酶进行酶切,将酶切产物加上连接接头序列,经预扩增和选择性扩增反应,最终从黄芪MSAP的256对引物中筛选出最佳分辨引物4对.应用筛选到的4对引物,分析12种不同产地黄芪的甲基化模式,并对甲基化水平聚类分析.[结果]经MSAP检测12个黄芪样本半甲基化率在12.5%~26.7%,全甲基化率在25.6%~51.1%,总甲基化率在48.7%~73.5%,总体上黄芪样本的全甲基化率大于半甲基化率.采用NTSYSpc软件对黄芪样品Hpa II和Msp I多态性数据进行分析,获得UP-GMA聚类图和主成分分析PCA图.[结论]MSAP技术能够很好地用于黄芪的甲基化分析,结合NTSYSpc软件能对不同产地黄芪之间甲基化水平进行聚类分析,且初步分析结果表明从UPGMA和PCA图上来看,地区相近的单株聚类分析表现出了一定的地域性.%[Objective] To establish and optimize the amplification system of methylation sensitive polymorphism ( MSAP) to study the methyl-ation level of Radix astragali.[Method] DNA of Radix astragali was extracted by magnetic bead method and digested with HpaII/MspI endo-nuclease.The digested products were connected with the linker sequence, and then pre-amplification and selectively amplification were carried out.Four pairs of primers were screened out from the 256 pairs of primers of MSAP, and were used to analyze the methylation levels for 12 dif-ferent habitat samples of R.astragali.[Result] For 12 samples, the rate of half methylation and full methylation were 12.5%-26.7% and 25.6%-51.1%, respectively.The total methylation rate was 48.7%-73.5%.The UPGMA clustering and principal component analyses (PCA) were obtained by analyzing the HpaII and MspI polymorphism data with NTSYSpc software.[Conclusion] MSAP technology can be used for the methylation analysis of R.astragali.The preliminary analysis of cluster analysis showed some regional characteristics of R.astra-gali from the UPGMA and PCA plots.
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