怀地黄ISSR扩增条件优化的研究

用CTAB法提取怀地黄嫩叶DNA,进行简单重复间序列标记(ISSR)分析.通过单因子实验分别研究了退火温度、Taq酶单位、Mg2+浓度、dNTP浓度、引物浓度和模板DNA浓度对ISSR-PCR反应的影响,找出各自的合适条件,而且每一个合适条件确定以后都被作为后续研究的一个条件.通过各个因子的组合研究建立了适宜于怀地黄ISSR分析的扩增体系25μL PCR反应体积,1×Taq DNA酶缓冲液(10 mmol/L Tris-HCl,50 mmol/L KCl,0.1% TrionX-100,pH9.0 ),2.5 mmol/L MgCl2,1.5～1.0 U Taq酶,60 ng模板DNA,0.4 μmmol/L引物,各0.4mmol/L的dATP、dGTP、dCTP和dTTP.合适的退火温度为53～55℃.为用ISSR技术分析鉴定怀地黄种质资源奠定了良好的基础.

Optimization of ISSR-PCR amplification in Huai Rehmannia glutinosa

Huai Rehmannia glutinosa Libosch. genomic DNA,extracted from its fresh young leaves by CTAB method,was used as ISSR-PCR amplification template.The effects of main elements,i.e.temperature,Taq polymerase dosage,dNTP concentration,Mg2+ concentration,primer concentration and template DNA concentration on ISSR-PCR amplication were tested by single factor experiment,respectively,to determine its optimal levels and establish the following optimal reaction system for ISSR analysis in Huai Rehmannia glutinosa Libosch.:PCR reaction volume of 25 μL,1.5～1.0 U Taq DNA polymerase,3.0 mmol/L Mg2+,1 × Taq DNA polymerase buffer (10 mmol/L Tris-HCl,50 mmol/L KCl,0.1% Trion X-100,pH9.0 ),60 ng template DNA,0.4 μmmol/L primer,0.4 mmol/L each of dATP,dGTP,dCTP and dTTP.Proper annealing temperature was 53～55℃.This has laid the good foundation for ISSR analysis under way in Huai Rehmannia glutinosa Libosch..