五节芒ISSR-PCR反应体系的优化  

Optimization of ISSR-PCR Reaction System for Miscanthus floridulus

作　　者：金琳[1] 葛刚[1] 陈少风[1] 杨赛钢[1] 章泉[1] 

机构地区：[1]南昌大学生命科学与食品工程学院,江西南昌330031

出　　处：《安徽农业科学》2009年第29期14041-14043,14077,共4页Journal of Anhui Agricultural Sciences

基　　金：国家"973"项目子项目

摘　　要：［目的］保护五节芒的种质资源并为其开发利用奠定基础。[方法]以五节芒DNA为材料，分析DNA浓度、Mg^2＋浓度、dNTP浓度、Taq DNA聚合酶的含量以及退火温度对ISSR-PCR扩增结果的影响。并通过单因子试验对ISSR-PCR反应体系进行优化。［结果］建立了五节芒ISSR-PCR的最佳体系：25 μl反应体系中10×PCR Buffer 2.5 μl、0.2 mmol/L 4×dNTP、1.5 mmol/L Mg^2＋、0.75 U Taq DNA聚合酶。PCR反应程序为94 ℃预变性5 min；94 ℃变性30s；51-53 ℃退火30 s，72 ℃延伸50 s，40个循环；72 ℃再延伸7 min。利用优化反应体系从100个ISSR通用引物中筛选出了11个稳定性高、重复性好的引物。［结论］这一优化体系的建立为今后利用ISSR标记技术研究五节芒遗传多样性奠定了基础。[ Objective] The research aimed to protect the germplasm resources of Miscanthus floridulus and lay the foundation for its developmcnt and utilization, [Method] With DNA of M. floridulus as materials, the effects of DNA concentration, Mg^2＋ concentration, dNTP concentration and Taq DNA polymerase content, and annealing temperature on the results of amplification ISSR-PCR results were analyzed. By using single factor test, ISSR-PCR reaction system was optimized. [ Result] The optimum system of ISSR-PCR for M. floridulus was as follows： 25 μl system containing 0.2 mmoL/L 4 × dNTP, 1.5 mmoL/L Mg^2＋ ,0.75 U Taq DNA polymerase. PCR reaction procedure was as follows： pre-denaturizing at 94℃ for 5 min, denaturizing at 94 ℃ for 30 s, annealing at 51 -53 ℃ for 30 s, extension at 72 ℃ for 50 s, 40 cycles; re-extension at 72 ℃ for 7 rain. 1 1 primers with high stability and good repeatability were screened from 100 ISSR common primers by using the optimized reaction system. [ Conclusion ] This optimal system laid the foundation for the research on the genetic diversity of M. floridulus by using ISSR technology.

关 键 词：五节芒 基因组DNA ISSR-PCR 优化 

分 类 号：S188[农业科学—农业基础科学]