胡椒ISSR反应体系的建立与优化

旨在构建胡椒(Piper nigrum L.)基因组DNA的ISSR-PCR反应体系,以便为海南胡椒属植物的遗传多样性分析研究打下基础。利用单因素随机试验设计,对胡椒ISSR-PCR反应体系中各组分(TaqDNA聚合酶、dNTPs、模板DNA、引物和Mg2+)的浓度进行优化,同时筛选ISSR-PCR反应的循环数和最适退火温度。确定了最优的ISSR-PCR反应体系为:总体积20μL,其中Taq DNA聚合酶1.0 U,dNTPs 0.8 mmol/L,引物0.2 mmol/L,Mg2+1.8 mmol/L,模板DNA 50 ng。同时通过梯度PCR试验,确定引物最佳退火温度及最佳循环数。胡椒ISSR-PCR最佳反应程序为:95℃预变性5 min;94℃变性30 s,54.9℃(引物834)退火30 s,72℃延伸45 s,共计35个循环,循环结束后72℃延伸7 min,4℃保存。这一反应体系可成功地应用于海南胡椒属植物的遗传多样性分析。

Establishment and Optimization of Black Pepper ISSR Reaction System

The conditions of ISSR-PCR reaction were optimized using the method of single-factor experiment respectively.The optimal ISSR-PCR amplification was established as follows: in a 20 μl reaction mixtures containing 1.0 U Taq DNA polymerase,0.8 mM of each dNTP,0.2 mM primer,1.8 mM Mg2+,and 50 ng template DNA for 35 thermal cycles.Amplifications were performed in an Biometra TGradient Thermal Cycler using the following conditions: in 20 μl reaction volumes for each primer,initial denaturation at 95℃ for 5min;denaturation at 94℃ for 30 s;annealing at the optimum anneal temperature selected for 30 s;extension at 72℃ for 45 s;35 cycles;the last cycle followed by 7 min extension at 72℃.This reaction system can be successfully applied to the analysis of genetic diversity of Piper plants in Hainan Island.