珍稀濒危植物大果木莲ISSR-PCR反应体系的建立  

Establishment of ISSR-PCR Reaction System for the Rare and Endangered Plant Manglietia grandis Hu et Cheng

作　　者：毛云玲[1] 陈少瑜[1] 韩燕[2] 吴涛[1] 司马永康[1] 郝佳波[1] 付玉嫔[1] 

机构地区：[1]云南省林业科学院国家林业局云南珍稀濒特森林植物保护和繁育重点实验室云南省森林植物培育与开发利用重点实验室,云南昆明650204 [2]西南林学院资源学院,云南昆明650224

出　　处：《安徽农业科学》2009年第31期15163-15166,共4页Journal of Anhui Agricultural Sciences

基　　金：云南省自然科学基金项目(2006C0066M)

摘　　要：[目的]建立高效稳定的大果木莲ISSR-PCR反应体系。[方法]以珍稀濒危植物大果木莲新鲜嫩叶为材料,在高盐沉淀法提取高质量基因组DNA的基础上,通过ISSR-PCR反应体系中5个主要因素的单因子梯度试验,优化并最终建立了大果木莲稳定而高效的ISSR-PCR反应体系和反应程序。[结果]试验建立的大果木莲的ISSR-PCR反应体系为:20.0μl反应体系中含10×buffer2.0μl、Mg2+2.0 mmol/L、Taq酶0.5 U、DNA模板40 ng、引物0.8μmol/L、dNTPs0.2 mmol/L和ddH2O13.3μl,其反应程序为:94℃预变性5 min;94℃变性1 min,50～60℃退火45 s,72℃延伸2 min,40个循环;72℃完全延伸7 min。[结论]该研究为进一步揭示大果木莲群体的遗传多样性和遗传结构状况以及制定科学的保护措施提供了理论依据。[Objective] The study aimed to establish the effective and stable ISSR-PCR reaction system for Manglietia grandis Hu et Cheng. Method ] With the leaves of rare and endangered plant M. grandis as tested material,based on high quality DNA extracted by high salt precipitation method,the single factor grade experinaent with 5 factors in ISSR-PCR reaction system was taken to optimize and finally establish the effective and slable ISSR-PCR reaction system through single-factor gradient test. [ Result] The effective and stable ISSR-PCR reaction system for M. grandis was established, which contained 10 × buffer 2.0 μl, Mg2 ＋ 2.0 mmoL/L, Tax/enzyme O. 5 U, DNA template 40 ng, primer 0.8 μmol/L, dNTPs 0.2 mmol/L,ddH,0 13.3 μl in total 20.0 μl system. The PCR amplification program was that the initial denaturation was for 5 min at 94℃ ,followed by 40 cycles of denaturing at 94 ℃ for 1 min,annealing at 50 -60 ℃ for 45 s,extension at 72 ℃ for 2 min,and a final extension was at 72 %~ for 7 rain. [ Conclusion] The study provided the theoretical basis for further opening out the genetic diversity and structure and making out the scientific protection measures.

关 键 词：珍稀濒危植物 大果木莲 基因组DNA ISSR—PCR反应体系 

分 类 号：S188[农业科学—农业基础科学]