紫云英ISSR引物的筛选及PCR反应体系的优化

Optimization of ISSR′s Primer and PCR Reaction System for Astragalus sinicus L.

以紫云英为研究材料,用哥伦比亚大学(UBC)公布的100条ISSR引物和11种株系紫云英品种的DNA为模板进行PCR扩增,筛选出33条扩增条带较好的ISSR引物,对其中的ISSR引物进行梯度PCR,筛选出最佳的退火温度。再采用正交试验和单因素试验相结合的方法对紫云英ISSR-PCR反应体系的5种因素(模板、Mg2+、TaqDNA聚合酶、dNTP及引物)进行优化浓度。确立了适合紫云英的ISSR分析的反应体系。在25μl反应体系中,其反应浓度为:DNA模版50.00ng,Mg2+2.00mol/L,Taq聚合酶1.0 U,dNTP 0.25mmol/L,引物0.20μmol/L,2.5μl 10×buffer。本试验为以后利用ISSR技术进行紫云英遗传多样性分析和物种保护奠定了技术基础。

The factors that affecting the ISSR（inter-simple sequence repeat）result of Astragalus sinicus L.were researched.We studied and selected 33 ISSR primers which can amplify bands from the DNA of Astragalus sinicus L.and the 33 ISSR primers all from the ISSR primers published by Columbia University.By setting the temperature gradient,the available primers′best annealing temperature were explored.The effect of the five main reaction elements（Mg2＋,TaqDNA polymerase,dNTP、template and primer）on ISSR-PCR were all optimized by combining single factor experiments and orthogonal test mathed.Then we established the best ISSR-PCR reaction system.The best ISSR-PCR reaction system of Astragalus sinicus L.is 25μl reaction system concention：the DNA template 50.00ng,Mg2＋concentration 2.00mmol/L,Taq enzyme dosages 1.0U,dNTP 0.25mmol/L,primers 0.20μmol/L,2.5μl 10×buffer.The selected ISSR primers and annealing temperature were widespread and representative in Astragalus sinicus L.ISSR-PCR marker research and the result could provide the basis for the analysis of diversity,protection,and exploitation of SSR primers of Astragalus sinicus L..