两面针ISSR-PCR反应体系的建立及优化

【目的】建立适合两面针简单重复序列—聚合酶链反应(ISSR-PCR)体系和扩增程序。【方法】采用改良的高盐低pH法提取两面针基因组DNA,通过单因素试验和正交试验相结合对ISSR反应体系中的主要成分(Mg2+、Taq DNA聚合酶、引物和模板)进行筛选和优化,并考察退火温度对扩增结果的影响。【结果】最佳的PCR反应体系为:总体积为25μL,含1.50 mmol/L Mg2+、150μmol/L dNTPs、0.04 U/μL Taq DNA聚合酶、0.60μmol/L引物、2.40 ng/μL DNA模板。扩增程序为:94℃预变性5 min;94℃变性30 s,51.4℃退火45 s,72℃延伸2 min;35个循环;72℃再延伸10 min。【结论】优化了两面针ISSR-PCR反应体系和扩增程序,为两面针ISSR遗传多样性分析打下基础。

Establishment and Optimization of ISSR-PCR System for Zanthoxylum nitidum (Roxb.) DC.

Objective To establish an optimal inter-simple sequence repeat-polymerase chain reaction(ISSR-PCR) system and amplification sequence for Zanthoxylum nitidum(Roxb.) DC..Methods Modified high-salt low-pH method was used for the extraction of genomic DNA from Zanthoxylum nitidum.Single-factor analysis and orthogonal experiment method were used to optimize the main reaction system elements(Mg2+,Taq DNA polymerase,primer and template DNA) and annealing temperature for ISSR-PCR system.Results A reaction system and amplified procedure suitable for Zanthoxylum nitidum(Roxb.) DC.were established.The total volume of 25μl reaction system contained 1.50 mmol/L Mg2+,150 μmol/L dNTPs,0.04 U/μL Taq DNA polymerase,0.60 μmol/L primer,and 2.40 ng/μL template DNA.The optimal amplified procedure was predenaturing for 5 min at 94℃,and then 35 cycles of denaturing for 30s at 94℃,annealing for 45s at 51.4℃,and extension for 120 s at 72℃,with a final extension step for 10 min at 72℃.Conclusion The optimized ISSR-PCR system will lay a foundation for the analysis of genetic diversity of Zanthoxylum nitidum(Roxb.)DC..