山莨菪ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Anisodus tanguticus

以山莨菪为实验材料,通过正交设计与单因素两种试验方法对山莨菪ISSR-PCR反应体系中的5种主要因素(模板DNA浓度,Mg2+,Taq酶,d NTPs及引物)进行4水平优化与筛选。经过PCR扩增结果的比较与分析,确定各因素的最适浓度,建立山莨菪的最佳ISSR-PCR反应体系。结果表明,25μL最佳反应体系为:3.0 mmol/L Mg2+、0.2 mol/L d NTPs、0.4μmol/L引物、0.5 U Taq DNA酶、40 ng DNA、2.5μL 10×Buffer。在最佳优化体系条件下,从100条引物中筛选出13条ISSR扩增引物,并确定各自最佳退火温度。建立的山莨菪ISSR-PCR反应体系,经过11份山莨菪样品检测,证明该体系稳定,可用于山莨菪遗传差异性分析。

In this research the ISSR-PCR amplification system in Anisodus tanguticus was optimized by using the orthogonal experimental design combined with signal factor analysis to screen five main factors（DNA template,Mg2＋, Taq DNA polymerase, d NTPs and primers） of PCR reaction in four levels. Through the deep analyzed of PCR results, a suitable ISSR-PCR reaction system was established. Results showed that the optimal 25 μL reaction mixture contained 40 ng DNA template, Mg2＋3.0 mmol/L, d NTP 0.2 mol/L, primer 0.4 μmol/L, Taq polymerase0.5 U, 2.5 μL 10 ×Buffer. Under the above optimized reaction system, 13 amplified primers were screened from100 ISSR primers, and the optimal annealing temperature for each primer was determined. A. tanguticus of 11 samples with the optimized ISSR system confirmed that the established ISSR-PCR was steady, thus the system provided the basis for the genetic analysis of A. Tanguticus.