高原鼠兔ISSR引物反应体系的优化与筛选  

Optimization and Screening of ISSR Primer Reaction System for Ochotona curzoniae

作　　者：葛艳丽[1,2] 慈海鑫[1,2] 唐利洲[1,2] 林恭华[1,2] 苏建平[1] 

机构地区：[1]中国科学院高原生物适应与进化重点实验室中国科学院西北高原生物研究所,青海西宁810001 [2]中国科学院研究生院,北京100039

出　　处：《安徽农业科学》2008年第33期14422-14424,14430,共4页Journal of Anhui Agricultural Sciences

基　　金：中国科学院知识创新工程领域前沿项目(CXLY-2002-3)

摘　　要：[目的]筛选和优化高原鼠兔(Ochotona curzoniae)适宜的ISSR反应体系,以在对高原鼠兔进行ISSR分析时获得清晰和多态性好的扩增结果。[方法]以高原鼠兔基因组DNA为模板,通过单因素试验,对体系中的模板浓度、Mg2+浓度、dNTPs浓度、Taq酶用量、引物用量、退火温度进行探讨。[结果]结果表明,高原鼠兔ISSR-PCR扩增的最佳条件为:25μl PCR反应体系,其中4lμDNA模板,1.5 mmol/LMgCl2,0.2 mmol/L dNTPs,1.25 UTaq聚合酶,1.5μmol/L引物,复性温度4560℃(退火温度随引物不同而确定)。用11条引物进行了PCR扩增,筛选出效果较好的6条引物。[结论]该反应体系的建立为鼠兔遗传多样性和分子系统学研究提供技术支持。[ Objective ] The research aimed to study the suitable ISSR reaction system for Ochotona curzoniae, to obtain high resolution and multiple polymorphie bands. [ Method] Based on the geuotnic DNA extracted from Ochotona curzoniae, the effects of the main reaction system elements such as Mg2＋ , dNTPs, primer, template DNA, Taq DNA polymerase and annealing temperature were presented by single factor experimeut. [ Result ] The resuhs showed that the optimum conditions of ISSR-PCR amplification were as follow：The total volume of the reactiou system was 25 μl,consisted of 4μl DNA template, 1.5mmol/L MgCl2 ,0.2 mm/L dNTPs, 1.25 U Taq DNA polymerase, 1.5μmol/L primer and annealing temperature of 45- 60 ℃. The 11 ISSR primers were used to screen the suitable primers, of which 6 ISSR primers were serecued. [ Conclusiou] This optimal system offered a technical system of ISSR analysis suitable to Octvotona curzoniae.

关 键 词：高原鼠兔 ISSR 优化FCR 

分 类 号：S813.3[农业科学—畜牧学]