何首乌ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Polygonum multiflorum Thunb.

目的建立和优化ISSR-PCR反应体系。方法通过单因素实验研究ISSR反应体系中主要成分(模板、TaqDNA聚合酶、Mg2+、引物、dNTPs)以及退火温度对扩增结果的影响。结果建立了适合何首乌ISSR分析的反应体系和扩增程序,即在25μl反应体系中,内含1×Taq酶配套缓冲液、100 ng模板、1.5 UTaqDNA聚合酶、1.5 mmol/L Mg2+、0.6μmol/L引物、0.2 mmol/L dNTPs。扩增程序为94℃预变性5 min;然后是94℃变性45 s,(据不同引物的退火温度)复性1 min,72℃延伸1.5 min,共35个循环;72℃延伸7 min,4℃保存。结论这一优化体系的建立为今后利用ISSR标记技术进行何首乌鉴定及种质资源遗传多样性分析提供了一个标准化程序。

Objective To establish and optimize ISSR - PCR reaction system for Polygonum multiflorum Thunb. Methods Single factor experiment method was used to present the effect of the main reaction system elements （Taq DNA polymerase, template DNA, Mg^2＋ , primer, dNTPs） and annealing temperature on ISSR -PCR. Results A reaction system and amplified procedure suitable for Polygonum multiflorum Thunb. were established, that was, 25 μl reaction system containing 1 ×PCR buffer, 100ng template DNA, 1.5 U Taq DNA polymerase, 1.5 mmol/L Mg^2＋ ,0.6 μmol/L primer,0.2 mmol/L dNTPs. The optimal amplified procedure was as follows ： after a pre - denaturing of 5 min at 94℃ , 35 cycles were performed with denaturing of 45 s at 94℃ , annealing of 1 rain according to denaturing temperature of different primer, extension of 1.5 min at 72℃ , a final extension step of 7min at 72℃ and hold at 4℃. Conclusion This optimal system lay the standardization program for the identification of Polygohum multiflorum Thunb. and the genetic diversity analysis of its germplasm resource.