大花蕙兰基因组DNA提取及RAPD反应条件探索  

Genomic DNA Extraction and RAPD Protocols for Cymbidium hybridium

作　　者：李冬梅[1] 朱根发[2] 叶庆生[1] 

机构地区：[1]华南师范大学生命科学学院广东省植物发育生物工程重点实验室,广州510631 [2]广东省农业科学院花卉研究所,广州510640

出　　处：《热带亚热带植物学报》2006年第1期25-30,共6页Journal of Tropical and Subtropical Botany

基　　金：广东省科技攻关重大专项(2003A201040);广州市农业局招标项目(GK0302105)资助

摘　　要：用SDS、CTAB和改良CTAB法分别提取大花蕙兰叶片基因组DNA,发现以改良CTAB法提取的DNA纯度高,产率也较高。以5’-GGTGCTCCGT-3(’BA440)为随机引物,并以大花蕙兰品‘种金杯(’Cymbidiumhybridiumcv.HiroshimaGoldenCu“pSunnyMoon”)的DNA为模板,对RAPD(RandomAmplifiedPolymorphicDNA)反应体系进行了优化研究,结果表明,25μl反应体系中,Mg2+、TaqDNA聚合酶、引物、模板DNA和dNTP5种主要成分的适宜浓度或用量分别是:2.0mmol/L、1.0U、0.20μmol/L、25ng和0.20mmol/L。扩增程序优化为:94℃预变性4min;94℃变性0.5min,37℃退火1min,72℃延伸1min,40个循环;最后72℃延伸7min。SDS, CTAB and modified CTAB methods were used to extract DNA from the leaves of Cymbidium hybridium cv. Hiroshima Golden Cup ＂Sunny Moon＂ and cv. Fortissimo ＂Pianist＂. The results showed that modified CTAB method was best for DNA quality and production. The optimal reaction of RAPD in C. hybridium was studied with primer 5＇-GGTGCTCCGT-3 ＇ for cv. Hiroshima Golden Cup ＂Sunny Moon＂. The optimum concentration of five important components such as Mg^2＋, Taq DNA polymerase, primer, template DNA, and dNTP in 25 μl RAPD reaction system were 2.0 mmol/L, 1.0 U, 0.20 μmol/L, 25 ng, and 0.20 mmol/L, respectively. Modified thermal profile consisted of an initial denaturation step at 94℃ for 4 min, followed by 40 cycles of 94℃ for 30 s, 37℃ for 1 min and 72℃ for 1 min and a final exposure to 72℃ for 7 min.

关 键 词：大花蕙兰 基因组DNA提取 CTAB RAPD 

分 类 号：S682.31[农业科学—观赏园艺]