目的为研究三叶木通种质资源遗传多样性,建立并优化三叶木通AFLP反应体系。方法以三叶木通叶片为材料,对影响AFLP反应体系中连接、预扩增和选择性扩增的各因素进行分析,建立优化的AFLP反应体系。结果 建立三叶木通AFLP反应优化体系:连接体系加入T4连接酶2 U,MseI接头100 pmol,EcoR I接头10 pmol;预扩增反应总体积20μL,加入连接产物2.0μL,E-0、M-0各100 ng,dNTPs 0.250 mmol/L,Mg²⁺1.875mmol/L,TaqDNA聚合酶2 U;选择性扩增反应总体积20μL,加入5μL稀释100倍的预扩增产物,E3、M3各50 ng,dNTPs 0.250 mmol/L,Mg²⁺1.875 mmol/L,TaqDNA酶1.5 U。结论 建立适用于三叶木通种质资源遗传多样性研究的AFLP反应优化体系。

Objective To establish and optimize the AFLP reaction system for genetic diversity analysis on Akebia trifoliata.Methods The leaves of A.trifoliata were used to analyze the influencing factors of the ligation,the pre-amplification,and the selective amplification of AFLP,so that the optimal AFLP reaction system could be built up.Results The optimal AFLP reaction system has been set up: added 10 pmol of EcoR I adapter,100 pmol of Mse I adapter,and 2 U of T4 DNA ligase in the ligation mixture;The pre-amplification mixture(20 μL) contained 2.0 μL of ligation products,100 ng of each pre-selective primer,E-0 and M-0,0.250 mmol/L of dNTPs,1.875 mmol/L of Mg²⁺,2 U of Taq DNA polymerase;the selective amplification mixture(20 μL) contained 5 μL of 1:100 diluted pre-amplification products,50 ng of each selective primer,E3,M3,0.250 mmol/L of dNTPs,1.875 mmol/L of Mg²⁺,1.5 U of Taq DNA polymerase.Conclusion The study on A.trifoliata could establish and optimize the AFLP reaction system which can be used in the research of genetic diversity of the species in future.

陕西省科技攻关项目(2010K17-05)