RAPD技术在药用植物绞股蓝鉴别中的初步研究  

Initial Research on Authentication of Medicinal Plant Gynostemma pentaphyllum by RAPD Method

作　　者：李雄英[1] 罗育[1] 吴耀生[1] 蒋军富[1] 赵瑞强[1] 蓝秀万[1] 

机构地区：[1]广西医科大学生物化学与分子生物学教研室,南宁530021

出　　处：《武汉植物学研究》2008年第3期251-254,共4页Journal of Wuhan Botanical Research

基　　金：广西科学基金(桂科基0575065;桂科基0731065);广西区教育厅硕士研究生科研创新项目(2006105981001M02);广西大型仪器协作共用网大型仪器测试补助(484-2007-063)

摘　　要：运用RAPD技术对绞股蓝(Gynostemma pentaphyllum)及其伪品进行DNA指纹图谱的鉴别研究。采用改进的CTAB法提取七叶绞股蓝、五叶绞股蓝、乌蔹莓(Cayratia japonica)3种植物的总DNA,主要以七叶绞股蓝DNA为模板,采用随机引物WGS001进行PCR扩增,对反应体系包括模板、Mg2+、Taq酶、牛血清白蛋白(BSA)、退火温度进行优化。优化的反应体系总体积25μL,含MgCl22 mmol/L、dNTP 0.2 mmol/L、引物0.4μmol/L、模板60 ng、Taq酶1 U、BSA2μg/μL,退火温度58℃。用10条含20个碱基的随机引物对以上3种植物总DNA作PCR扩增,进行引物筛选。筛选得到的两条随机引物(WGS001、WGS004)可扩增得到识别这些物种基因组DNA的多态性片段。这些片段可以有效地鉴别绞股蓝和乌蔹莓。The random amplified polymorphic DNA （RAPD） analysis was applied to identify the resources of medicinal plant Gynostemma pentaphyllum and its spurious breed with DNA fingerprints. The modified CTAB method was used to extract the genomic DNA of several plants, including seven leaves Jiaogulan, five leaves Jiaogulan, Cayratia japonica. With the DNA extracted from seven leaves Jiaogulan as template,the random primer WGS001 for PCR has been done meanwhile RAPD reaction system has been optimized so that it could be determined for the suitable annealing temperature and concentrations of tem-plate,Mg^2＋ ,primer, Taq DNA polymerase, BSA. In the optimized system of 25μL, MgC12 2 mmol/L, primer 0.4μmol/L,template 60 ng, Taq DNA polymerase 1 U, BSA 2μg/μL and annealing temperature 58℃ were used. Based on the optimized system,ten primers of 20 bp were applied to do random amplifi-cation with total DNAs of the above mentioned three plants, respectively. The polymorphic fragments of DNA fingerprints of the above mentioned three plants were obtained with two screened random primers （WGS001, WGS004）. These polymorphic fragments can be used to distinguish G. pentaphyllum with C. japonica obviously.

关 键 词：绞股蓝 基因组DNA RAPD 鉴别 

分 类 号：Q75[生物学—分子生物学] S567.237[农业科学—中草药栽培]