山茱萸RAPD反应体系的正交优化研究  

The Orthogonal Optimizaiton of RAPD Reaction System in Cornus officinalis

作　　者：叶凡[1] 李开言[1] 王亚芹[1] 鲁阳芳[1] 麻光磊 刘青蓝 任雪玲[1] 

机构地区：[1]郑州大学药学院,河南郑州450001

出　　处：《河南农业科学》2012年第4期113-116,共4页Journal of Henan Agricultural Sciences

基　　金：国家级大学生创新性实验计划项目(101045902)

摘　　要：为了优化山茱萸RAPD反应条件,采用改良CTAB法提取山茱萸基因组DNA,并采用三因素三水平正交试验对RAPD反应体系中Mg2+、dNTP、引物浓度进行初步优化,在此基础上,对温度条件进一步优化。结果表明:在25μL的RAPD反应体系中各因素的最佳浓度分别为1.925mmol/L Mg2+、0.275mmol/L dNTPs、0.55μmol/L引物、DNA模板40ng和1UTaq DNA聚合酶;引物Tm为36.9℃时,最佳退火温度为35℃;引物Tm为41.0℃时,最佳退火温度为38℃。在最优反应条件下,对100种随机引物进行RAPD扩增,其中93种随机引物均扩增出条带,建立了可靠的反应体系和反应程序,为山茱萸的DNA指纹图谱鉴定建立了基础。To optimize the amplification system of RAPD used to characterization Cornus officinalis,the genomic DNA was extracted by modified CTAB from Cornus officinalis and used as template for PCR assay.The orthogonal experiments of three factors（concentration of Mg2＋,dNTP and primers in PCR reaction mixture） and three levels were used to determine the optimum system of RAPD assay.Then,the single factor,annealing temperature,was also optimized.The results showed that the optimal RAPD amplification system contained 1.925 mmol/L Mg2＋,0.275 mmol/L dNTPs,0.55 μmol/L random primer,40 ng DNA template,and 1U Taq enzyme in a total volume of 25 μL.Tm of primer（36.9 ℃） corresponded to the optimal annealing temperature of 35 ℃,while the optimal annealing temperature of 38 ℃ corresponded to Tm of primer of 41 ℃.There were 100 random primers used for RAPD,of which 93 could amplify polymorphic bands.The optimal reaction parameters of RAPD-PCR were determined,which laid a foundation for construction and analysis of DNA fingerprinting of Cornus officinalis.

关 键 词：山茱萸 正交优化 随机引物扩增多态性DNA DNA指纹图谱 

分 类 号：S567.239[农业科学—中草药栽培]