地毯草ISSR反应体系的建立与优化

Establishment and Optimization of ISSR Reaction System for Carpetgrass

在利用ISSR技术对地毯Axonopuscompessus(Sw )Beauv 种质资源的遗传多样性进行研究的实验过程中,对影响PCR扩增效果的一些因素诸如DNA的提取、模板DNA质量浓度、Taq酶的选择及其用量、Mg2+浓度、dNTP的用量以及退火温度等指标进行筛选和优化,筛选优化出可用于地毯草ISSR-PCR分析最适宜的PCR反应条件:10μLPCR反应体积中,10×Taq酶配套缓冲液(10mmol LTris＿HCl,pH9 0,50mmol LKCl,0 1%TritonX＿100),0 75UTaqDNA聚合酶(上海申能博彩),0 375μmol L引物,180μmol LdNTP,1 5～2 0mmol LMgCl2,10ng模板DNA。

Factors which affect the ISSR analysis in the study of the genetic diversity of carpetgrass, such as genomic DNA extraction, concentration of template DNA, Taq DNA polymerase, Mg^(2+) concentration, dNTP and annealing temperature, were studied for optimizing conditions of the ISSR-PCR. The results showed that the conditions being suitable for ISSR-PCR of carpetgrass were as follows: 1×Taq buffer (10 mmol/L Tris-HCl, pH 9\^0, 50 mmol/L KCl, 0.1% Triton X-100),0.75 U Taq DNA (produced by Shenneng company), 0.375 μmol/L primer, 180 μmol/L dNTP, 1\^5～2.0 mmol/L MgCl\-2, and 10 ng template DNA.