雷公藤ISSR反应体系的建立与优化  

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

作　　者：瞿印权 徐雯[1] 李欣欣[1] 胡迪科 何天友[2] 荣俊冬[1] 陈礼光[1] 郑郁善[1] QU Yin-quan;XU Wen;LI Xin-xin;HU Di-ke;HE Tian-you;RONG Jun-dong;CHEN Li-guang;ZHENG Yu-shan(College of Forestry,Fujian Agriculture and Forestry University,Fuzhou 350002,China;College of Art and Landscape Architecture,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建农林大学林学院,福州350002 [2]福建农林大学艺术学院园林学院,福州350002

出　　处：《南方农业学报》2017年第10期1755-1760,共6页Journal of Southern Agriculture

基　　金：国家科技支撑计划项目(2009BAI73B01);福建省科技创新平台建设项目(2008Y2001);中央财政林业科技推广项目(闽[2015]TG03号)

摘　　要：【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。【Objective】This research was conducted to establish a ISSR-PCR reaction system for the paternity indentification 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 the32T.wilfordii samples,including Mg2+,dNTPs,Taq DNA polymerase,primer concentration and template DNA content.And the32samples 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 reaction mixture contained2.00mmol/L Mg2+,0.30mmol/L dNTPs,1.50U Taq DNA polymerase,0.40mol/L primer,20.00ng template DNA and2.00μL10×Buffer.The optimal annealing temperature was54.7℃.【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.

关 键 词：雷公藤 ISSR 反应体系 优化 种质资源鉴定 

分 类 号：S567.1[农业科学—中草药栽培]