川牛膝MSAP反应体系的优化及引物筛选

Optimization of MSAP Reaction System for Cyathula officinalis Kuan and Primers Screening

目的:首次对川产道地药材川牛膝表观遗传多样性进行研究,建立并优化川牛膝甲基化敏感扩增多态性技术(MSAP)最佳反应体系。方法:以川牛膝苗期叶片为材料,利用正交试验设计对川牛膝MSAP反应体系中预扩增和选择性扩增中关键影响因素进行考察,建立川牛膝MSAP反应最佳体系。结果:川牛膝MSAP最佳反应体系为:酶切体系(20μL),300 ng DNA、1.5μL Eco RI、1.5μL Hpa II或1.5μL Msp I(Hpa II);连接体系(25μL):15μL酶切产物、1μL Eco RI adaptor、1μL Hpa II/Msp I adaptor、0.2μL T4连接酶;预扩增体系(25μL):连接产物1μL、r Taq酶1 U、引物分别1.5μL、d NTP 2μL;选择性扩增体系(25μL):预扩产物稀释50倍、r Taq酶1 U、引物1.5μL、d NTP 3μL,并运用优化后的体系,最终从川牛膝MSAP的256对引物中筛选出有效引物6对。结论:优化后的体系保证了稳定的图谱、清晰的条带,筛选出的6对引物组合特异性好,能够用于后续川牛膝DNA甲基化相关实验研究,同时,为其他药用植物MSAP分析提供了参考。

This study was aimed to establish and optimize the reaction system of MSAP for analysis on DNA methylation in Cyathula officinalis Kuan of Sichuan medicinal. The tender leaves of C. officinalis Kuan were used as materials. Orthogonal method was used in the study of main factors which affected the system quality of MSAP. The results showed that the optimal reaction systems of MSAP included： enzyme digestion（20 μL）, 300 ng DNA, 1.5 μL Eco RI, 1.5 μL Hpa II or 1.5 μL Msp I（Hpa II）; ligation（25 μL）： 15 μL digestion products, 1 μL Eco R I adaptor, 1 μL Hpa II/Msp I adaptor, 0.2 μL T4 ligase; pre-amplication mixture（25 μL）： ligation products 1 μL, r Taq polymerase 1 U, each primer 1.5 μL, d NTP 2 μL; selective amplification mixture（25 μL）： pre-amplification product was diluted 50 times, r Taq polymerase 1 U, each primer 1.5 μL, d NTP 3 μL. The optimal MSAP reaction system was used to screen for 6 pairs of effective primers from 256 pairs of primers of MSAP. It was concluded that the optimized system ensured the stable and clear bands and the screened 6 pairs of primers were with good specificity. It provided useful references for further studies of epigenetic on C. officinalis Kuan DNA methylation and MSAP analysis for other medicinal plants.