广西甜茶ISSR-PCR反应条件的建立与优化  

Establishment and Optimization of ISSR-PCR Reaction Conditions in Rubus suavissimus S. Lee

作　　者：黄夕洋[1] 唐辉[1] 孔德鑫[1] 王满莲[1] 史艳财[1] 邹蓉[1] 

机构地区：[1]广西壮族自治区中国科学院广西植物研究所,桂林541006

出　　处：《基因组学与应用生物学》2013年第4期526-532,共7页Genomics and Applied Biology

基　　金：广西自然科学基金(桂科自0991228);桂林市科技攻关项目(20110107-3);广西植物研究所基本业务费项目(桂植业09005)共同资助

摘　　要：为了探讨广西甜茶ISSR实验中的多种因素对实验结果的影响,采用单因素法对PCR反应体系中的5个潜在因素(Mg2+,dNTP,引物,Taq酶和模板DNA)在5水平上进行优化实验,并进一步优化了反应程序中的退火温度和循环次数。结果表明:25μL的最佳反应体系为,1×PCR Buffer、MgCl22.0mmol/L、dNTP0.2mmol/L、引物0.8μmol/L、Taq DNA聚合酶0.75U、模板DNA 80ng;最佳反应程序为:在94℃下进行3min预变性;随后循环扩增35次(包括94℃变性1min,54℃退火1min,72℃延伸1min);最后在72℃下延伸10min。本研究建立的广西甜茶的最佳ISSR-PCR反应条件为进一步进行遗传多样性研究奠定了基础。In order to study the influence of several potential factors on ISSR system for Rubus suavissimus S. Lee.,five factors （Mg2＋, dNTP, primer, Taq DNA polymerase and DNA template） were optimized by multiple single factor experiments at 5 concentration levels, annealing temperatures and amplification cycle numbers were also optimized. The results showed that the optimum 25 puL reaction system comprises lxPCR buffer, MgC12 2.0 retool/L, dNTP 0.2 mmol/L, primer 0.8 ixmol/L, Taq DNA polymerase 0.75 U and template DNA 80 ng; and the optimum PCR procedure is as an initial pre-denaturing at 94℃ for 3 rain, which precedes 35 cycles of denaturing at 94℃ for 1 rain, annealing at 54℃ for 1 min, and extension at 72℃ for 1 rain, with a final extension at 72℃ for 10 rain. Establish- ment of the optimum ISSR-PCR systems and conditions would serve as a base for further study on the genetic diver- sity ofRubus suavissimus S. Lee.

关 键 词：广西甜茶 ISSR PCR反应条件 单因素优化 

分 类 号：S571[农业科学—作物学]