四数獐牙菜ISSR-PCR反应体系的正交优化  

Optimization for ISSR-PCR Reaction System in Swertia tetraptera Using Orthogonal Design

作　　者：杨路存[1,2] 陈桂琛[1] 周国英[1] 宋文珠[1] 李春丽[1,2] 许璟瑛[1,2] 

机构地区：[1]中国科学院西北高原生物研究所,西宁810001 [2]中国科学院研究生院,北京100039

出　　处：《生物技术通报》2010年第9期123-128,共6页Biotechnology Bulletin

基　　金：国家中西部专项项目(2001BA901A47)

摘　　要：采用正交试验与单因素设计相结合的方法,对四数獐牙菜ISSR-PCR反应体系中的4种主要因素(Mg2+、TaqDNA聚合酶、dNTP及引物)进行优化筛选,PCR结果用统计软件SPSS16.0分析。结果显示,Mg2+、TaqDNA聚合酶、dNTP这3因素的不同水平对PCR反应结果都有显著影响,其中Mg2+的浓度影响最大。筛选出各反应因素的最佳水平,建立四数獐牙菜ISSR-PCR反应的最佳体系(25μL)为3.0 mmol/L Mg2+,250μmol/L dNTP,0.6μmol/L引物,1UTaqDNA聚合酶,40 ngDNA,2.5μL10×buffer。这一体系的建立为今后利用ISSR技术进行四数獐牙菜遗传多样性分析以及物种保护奠定了技术基础。An orthogonal design combined with single factor experiment was applied to optimize ISSR（inter simple sequence repeat）-PCR（polymerase chain reaction）amplification system of Swertia tetraptera.The effect of the four main reaction system elements（Mg2 ＋,Taq DNA polymerase,dNTP and primer）on ISSR-PCR were tested.The result of PCR analyzed by software SPSS16.0,showed that the different levels of Mg2＋,Taq DNA polymerase,dNTP had an significant effect on the PCR reaction result and the concentration of Mg2＋ was the most effective factor to the result of PCR.A most suitable ISSR-PCR system for S.tetraptera containing 3.0 mmol/L Mg2 ＋,250 μmol/L dNTP,0.6 μmol/L primer,1 U Taq DNA polymerase,40 ng DNA template and 2.5 μL 10 ×buffer in the total volume of 25 μL was established.The result could provide the basis for the analysis of diversity and protection of S.tetraptera.

关 键 词：四数獐牙菜 ISSR-PCR 正交设计 体系优化 

分 类 号：R284[医药卫生—中药学]