银杏ISSR-PCR扩增反应体系的建立与优化

目的:为了对银杏进行分子鉴定和遗传关系的分析,建立银杏ISSR-PCR的最佳扩增反应体系。方法:采用正交设计和单因素梯度实验,对影响ISSR-PCR反应体系的5个主要因素(Mg2+、dNTP、引物、模板DNA及Taq DNA聚合酶)进行筛选及优化。结果:银杏25μL ISSR最佳扩增反应体系包含10×Taq反应缓冲液、2.5 mmol/L MgCl2、0.45 mmol/L dNTP、1.2μmol/L引物(UBC861)、10 ng模板DNA及0.9 U Taq DNA聚合酶,使用此ISSR扩增反应体系,获得了10株不同性别银杏DNA的清晰条带,验证了该体系的稳定性。结论:优化的反应体系为采用ISSR分子标记技术对银杏进行遗传多样性分析、遗传育种和转基因等研究奠定了一定的理论基础。

Establishment and Optimization of the ISSR-PCR System for Gingko biloba

Objective： In order to analyze the molecular markers and genetic relationship,the optimal inter simple sequence repeat（ISSR） amplification system for Gingko biloba was set up.Methods： The orthogonal design and the gradient of the single-factor experiment were conducted.The main factors influencing the ISSR-PCR system,Mg2＋,dNTP,primer,template DNA and Taq DNA polymerase were detected and optimized.Results： The results illustrated that the optimal 25 μL ISSR amplification system for G.biloba contained 10×Taq reaction buffer,2.5 mmol/L MgCl2,0.45 mmol/L dNTP,1.2 μmol/L primer（UBC861）,10 ng template DNA and 0.9 U Taq DNA polymerase,respectively.With this optimal ISSR amplification system,high quality patterns were produced on 10 G.biloba plants of different genders,which indicated the stability of the current system.Conclusion： The present study lays certain theoretical foundation for the analysis of genetic diversity,genetic breeding and transgenetic study of G.biloba using ISSR molecular marker technique.