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目的研究三叶木通表观遗传多样性,建立并优化三叶木通甲基化敏感扩增多态性(methylation sensitive amplifiedpolymorphism,MSAP)反应体系。方法以三叶木通叶片为材料,利用正交试验建立三叶木通MSAP的预扩增和选择性扩增的最佳反应体系。结果建立的MSAP最佳反应体系为酶切反应:HpaII/MspI 10 U,EcoR I 10 U;预扩增反应:总体积20μL,连接产物2μL,E00、HM00各125 ng,dNTPs 0.312 5 mmol/L,Mg2+1.875 mmol/L,Taq DNA聚合酶2 U;选择性扩增反应:总体积20μL,稀释200倍的预扩增产物5μL,引物E-ACT、HM-CAT各50 ng,dNTPs 0.250 mmol/L,Mg2+1.250mmol/L,Taq DNA酶1 U。利用优化的体系,最终从MSAP的80对引物中筛选出有效引物6对。结论建立的MSAP反应体系可用于后续三叶木通DNA甲基化的相关研究。
Objective To study the epigenetic diversity of Cloverwood and establish and optimize methylation sensitive amplified polymorphism (MSAP) reaction system. Methods Cloverwood leaves as the material, the orthogonal test was used to establish the optimum reaction system for preamplification and selective amplification of MSAP. Results The optimal reaction system for MSAP was HpaII / MspI 10 U, EcoR I 10 U; preamplification reaction: total volume 20 μL, 2 μL ligation product, 125 ng E00 and HM00, 0.312 5 mmol / L dNTPs , Mg2 + 1.875 mmol / L, Taq DNA Polymerase 2 U; selective amplification reaction: total volume 20μL, diluted 200 times pre-amplification products 5μL, primers E-ACT, HM-CAT each 50 ng, dNTPs 0.250 mmol /L,Mg2+1.250mmol/L, Thaq DNase 1U. With the optimized system, six pairs of effective primers were finally screened from 80 pairs of primers of MSAP. Conclusion The established MSAP reaction system can be used for the subsequent DNA methylation research.