[关键词]
[摘要]
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。
[Key word]
[Abstract]
Objective To establish and optimize the MSAP reaction system for analysis on epigenetic diversity in Akebia trifoliata.Methods The leaves of A. trifoliata were as materials, using orthogonal L 16 (4 5 ) method to establish pre-amplification and selectiveamplification optimum reaction system of the MSAP. Results The optimal MSAP reaction systems include: 10 U HpaII/MspI andEcoR I in the enzyme digestion; in the 20 μL pre-amplication mixture contained 2 μL of ligation products, 125 ng of each pre-selectiveprimers, E00 and HM0; 0.312 5 mmol/L of dNTPs, 1.875 mmol/L of Mg 2+ , 2 U of Taq DNA polymerase; in the 20 μL selectiveamplification mixture contained 5 μL of 1∶200 diluted pre-amplification products, 50 ng of each selective primer, 0.250 mmol/L ofdNTPs, 1.250 mmol/L of Mg 2+ , and 1 U of Taq DNA polymerase. Using the optimal MSAP reaction system to screen for six pairs ofeffective primers from 80 pairs of primers of MSAP. Conclusion Those results provide fundamental reference for further epigeneticstudies on A. trifoliata DNA methylation.
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[基金项目]
陕西省科技攻关项目(2010K17-05-02);2010年国家级大学生创新性实验计划资助项目(101071825)