入侵植物牛膝菊ISSR-PCR反应体系的建立与优化

Establishment and optimization of ISSR-PCR reaction system of Galinsoga parviflora

通过L16(45)正交试验,研究了Mg2+、引物、dNTPs、Taq DNA聚合酶、模板DNA浓度5个因素在4个水平上对ISSR-PCR的影响,PCR结果应用DPS数据处理软件分析,建立了适合于牛膝菊ISSR-PCR的反应体系。优化体系(25μL)为:Mg2+浓度2.0 mmol/L,dNTP浓度0.5 mmol/L,Taq DNA聚合酶浓度0.08 U/μL,引物浓度0.2μmol/L,DNA浓度1.5 ng/μL。筛选出扩增稳定、多态性高、扩增条带清晰的ISSR引物8条,并确定了8条引物各自的最佳退火温度。

The effects of various factors on ISSR-PCR reaction, including concentration of Mg2＋, dNTPs and primers, DNA template and Taq DNA polymerase, were investigated to optimize the ISSR-PCR reaction system of Galinsoga parviflora through orthogonal tests. The result of PCR was analyzed by software DPS, and the most suitable ISSR-PCR system for G. parviflora was established, the 25 μL reaction system contained 2.0 mmol/L Mg2＋, 0.5 mmol/L dNTPs, 0.08 U/ μL Taq DNA polymerase, 0.2μmol/L primer, DNA template 1.5 ng/μL. Based on the above conditions, 8 primers with high polymorphism, clear amplified bands and good repeatability were selected. The optimal annealing temperatures of 8 primers were proposed by gradient PCR.