雷公藤ISSR反应体系的建立与优化

【目的】建立适合雷公藤的ISSR-PCR反应体系,为雷公藤种质资源的亲缘关系鉴定和遗传多样性分析提供技术支持。【方法】采用单因素试验设计优化影响雷公藤ISSR-PCR扩增效果的Taq DNA聚合酶、d NTPs、Mg~(2+)、引物浓度和DNA模板含量及退火温度,并以32份雷公藤样品对优化的ISSR-PCR反应体系进行验证。【结果】优化的雷公藤种质资源ISSR-PCR反应体系为:20.00μL反应液中含1.50 U Taq DNA聚合酶、0.30 mmol/L d NTPs、2.00 mmol/L Mg~(2+)、0.40μmol/L引物、20 ng DNA模板和2.00μL 10×Buffer。ISSR-PCR扩增程序中最佳退火温度为54.7℃。【结论】建立优化的雷公藤ISSR-PCR反应体系具有较高的稳定性、重现性和适用性,可应用于雷公藤不同地理种源间的遗传多样性鉴定。

Establishment and optimization of ISSR reaction system for Tripterygium wilfordii Hook. f.

Objective]This research was conducted to establish a ISSR-PCR reaction system for the paternity indenti-fication and genetic diversity analysis of Tripterygium wilfordii Hook. f.Method]Single-factor analysis method was applied to optimize and select the main components and annealing temperature of five PCR reaction system having effects on PCR amplification of the 32 T. wilfordii samples,including Mg2+,dNTPs,Taq DNA polymerase,primer concentration and template DNA content. And the 32 samples were used to testify the optimized ISSR-PCR reaction system.Result]An ISSR-PCR reaction system applicable for indentifying germplasm resources of T. wilfordii was established:20.00μL reac-tion mixture contained 2.00 mmol/L Mg2+,0.30 mmol/L dNTPs,1.50 U Taq DNA polymerase,0.40 mol/L primer,20.00 ng template DNA and 2.00μL 10 × Buffer. The optimal annealing temperature was 54.7℃.Conclusion]The T. wilfordii ISSR-PCR reaction system established is of high stability,repeatability and applicability,indicating this reaction system is suitable for the genetic diversity analysis of T. wilfordii from different geographical provenances.