珍稀药用植物竹节参SRAP-PCR反应体系的构建与优化  

Optimization by SRAP-PCR reaction system in rare medicinal herb Panax japonicus C. A. Mey

作　　者：宋佳[1] 张绍鹏[1] 孙巧[1] 李天佩 曾万勇[1] 陈平[1] 

机构地区：[1]武汉轻工大学生物与制药工程学院,湖北武汉430023

出　　处：《武汉轻工大学学报》2015年第2期14-19,共6页Journal of Wuhan Polytechnic University

基　　金：国家自然科学基金项目(81274023);湖北省教育厅科学研究青年项目(Q20141704)

摘　　要：人参属重要的药用植物竹节参(Panax japonicus C.A.Mey)野生资源已近濒危,开展其遗传多样性研究对于资源保护和可持续利用具有重要意义。研究构建并优化了竹节参DNA的SRAP-PCR扩增体系。以竹节参根茎为材料,对PCR反应体系中的d NTPs、DNA模板、引物、Taq DNA聚合酶浓度和复性第二阶段的退火温度等条件进行探索和研究,得到了能稳定扩增竹节参基因组的最佳反应体系(25μl):DNA 40ng,d NTPs 0.2 mmol/L,Primer 0.36μmol/L,10×PCR Buffer 2.5 ul,Taq DNA polymerase 2U,退火温度50℃。进一步检测了随机组合的24对引物,进行扩增,得到了2对带型清楚、重复性较好的引物Me1/Em5和Me3/Em1。以这2对引物对10个不同产地竹节参DNA样品进行SRAP-PCR扩增。产物的电泳图谱表明,竹节参不同产地的DNA谱带多态性丰富,带型清晰,重复性好。说明建立的该SRAP-PCR反应体系稳定可靠,为利用SRAP分子标记技术研究竹节参的植物遗传多样性奠定了工作基础。Panax japonicus C.A.Mey, an important medicinal herb, its wild resources are becoming endangered. Genetic diversity research of P.japonicas is significant for the conservation and sustainable utilization of these re-sources.In this study, optimum SRAP-PCR reaction system for P.japonicus DNA was constructed for the first time.The DNA of rhizome in P.japoncius was abstracted as PCR template.And, the concentration gradient of dNTPs, primer, DNA and Taq polymerase and Tm were assessed respectively to optimize SRAP-PCR reaction sys-tem.An optimal and stable SRAP-PCR system for P.japonicus（Total of 25μl） was as follows：DNA 40 ng, dNTPs 0.2 mmol/L, primer 0.36 umol/L, 10 ×PCR Buffer 2.5 μ,l Taq polymerase 2U , Tm50.Then,24 random prim-ers were chosen for further test.2 couple of primers, Me1/Em5 and Me3/Em1, which can get clear and repeatable bands, were obtained after PCR.Finally, 10 different P.japonicus DNA samples were used to test this method u-sing these 2 primers, which showed that rich polymorphism among P.japoncius plants from different area.Thus, this stable and suitable SRAP -PCR system can be used as an important method of molecular markers analysis for Panax plants genetic diversity studying.

关 键 词：竹节参 SRAP-PCR反应体系 构建 优化 

分 类 号：Q949.95[生物学—植物学] TQ460.6[化学工程—制药化工]