硅胶干燥罗布麻叶片DNA提取及RAPD反应体系建立  

Extraction of Apocynum Genomic DNA and Establishment of RAPD System

作　　者：苏前[1] 吕杰[1] 任曼丽[1] 冉启洋[1] 张雪妮[1] 吕光辉[1] 

机构地区：[1]新疆大学资源与环境科学学院/绿洲生态教育部重点实验室,乌鲁木齐830046

出　　处：《新疆农业科学》2013年第3期524-529,共6页Xinjiang Agricultural Sciences

基　　金：国家自然科学重点基金(41130531)

摘　　要：【目的】得到适用于罗布麻RAPD的最优体系。【方法】以伊犁地区新源县罗布麻硅胶干燥叶片为材料,采用TIANGEN试剂盒提取罗布麻基因组DNA,得到满足RAPD(Random amplified polymorphic DNA)分析的罗布麻基因组DNA。【结果】通过单因素优化实验,得到罗布麻RAPD分析的最佳反应体系为:Mg2+(2.0mmol/L)、dNTPs(0.5 mmol/L)、primer(0.4 mmol/L)、DNA(1μL)、Taq酶取1μL、10×buffer取1μL,总反应体系为10μL;扩增程序为:在94℃下预变性,然后进行42个循环(94℃变性30 s、38℃退火30 s、72℃延伸2min),最后在72℃延伸7 min。【结论】提取的罗布麻总DNA均有较高的质量,适合RAPD分析;Tiangen植物基因组DNA提取试剂盒对罗布麻基因组DNA有着良好的提取效果;对罗布麻RAPD-PCR中Mg2+、dNTPs、DNA聚合酶和引物浓度进行优化,较为理想的各因子用量和扩增程序为:Mg2+(2.0 mmol/L)、dNTPs(0.5mmol/L)、primer(0.4 mmol/L)、Taq酶(0.5 U/μL)、DNA(40 ng),总反应体系为10μL;扩增程序为:在94℃下预变性5 min,然后进行42个循环(94℃变性30 s、38℃退火30 s、72℃延伸2 min),最后在72℃延伸7 min。[ Objective] The purpose of this project is to obtain the optimal RAPD reaction system that might apply to apocynum. [ Method ] Apocynum in Xinyuan County is used as experimental materials and the genomic DNA was extracted from young leaves of Apocynum by means of Tiangen Kit until sufficient RAPD （ Random amplified polymorphic DNA） is got to carry out analysis of Apocynum. [ Result ] Through single factor experiments, The best RAPD reaction system optimized was： Mg^2＋ （2.0 mmol/L）, dNTPs（0.5 mmol/ L）, primer（0.4 mmol/L） ,DNA（0.3 U）, Taq DNA polymerase 1 L, 10 × buffer 1 L,10 L PCR solution;The RAPD PCR was programmed by a cycle for 5 min at 94%, followed by 42 cycles for 30s at 94℃, 30s at 38% and 2 min at 72℃, and finally by a cycle for 2 min at 72℃. [ Conclusion ] The obtained DNA is of high quality, which applies to RAPD analysis.

关 键 词：罗布麻 RAPD体系 DNA提取 

分 类 号：S567.23[农业科学—中草药栽培] S188[农业科学—作物学]