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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。
A total of 33 ISSR amplified bands were amplified by PCR using 100 samples of ISSR primers published by Columbia University (UBC) and 11 strains of astragalus mongholicus as template. Primer, among them ISSR primer gradient PCR, screening out the best annealing temperature. Then five factors (template, Mg2 +, Taq DNA polymerase, dNTP and primer) of ISSR-PCR reaction system of Astragalus sinicus were optimized by the combination of orthogonal test and single factor test. A suitable reaction system for ISSR analysis was established. In 25μl reaction system, the reaction concentration was 50.00ng for DNA template, 2.00mol / L for Mg2 +, 1.0 U for Taq polymerase, 0.25mmol / L for dNTP, 0.20μmol / L for primer and 2.5μl for 10 × buffer. This experiment lays the technical foundation for the future analysis of ISSR genetic diversity and species conservation.