温郁金SCoT-PCR反应体系的正交优化  

Orthogonal Optimization of Start Codon Targeted Polymorphism System in Curcuma wenyujin Y.H.Chen et C.Ling

作　　者：陈喆[1] 冯尚国[1] 王振[1] 何仁锋[1] 姜梦莹 王慧中[1] 

机构地区：[1]杭州师范大学生命与环境科学学院浙江省药用植物种质改良和质量监控重点实验室,浙江杭州310036

出　　处：《北方园艺》2015年第12期84-90,共7页Northern Horticulture

基　　金：国家自然科学基金资助项目(31070298;30870180);浙江省科技计划资助项目(2008C12081);浙江省自然科学基金资助项目(LQ13H280006);浙江省药用植物种质改良与质量控制技术重点实验室开放资助项目(201304);浙江省大学生科技创新活动计划暨新苗人才计划资助项目(ZX13005002067);浙江省大学生科技创新活动计划暨新苗人才计划资助项目(ZX1305002007)

摘　　要：以温郁金为试材,运用L25(56)正交设计在5个水平上对影响温郁金SCoT-PCR反应的模板DNA、Mg2+、dNTPs、Taq酶和引物5个因素进行优化试验,对PCR结果进行极差分析。建立并优化温郁金的目标起始密码子多态性-聚合酶链式反应(SCoT-PCR)体系,以期为温郁金的遗传多样性分析及分子鉴定等研究提供技术支持。结果表明:建立了温郁金SCoT-PCR的最佳反应体系(20μL):引物0.8μmol/L,dNTPs 0.4mmol/L,Mg2+1.5mmol/L,Taq酶0.5U,模板DNA 40ng,且确定各因素对温郁金SCoT-PCR反应效果的影响大小依次为:dNTPs>Taq酶>引物>Mg2+>模板DNA,其中dNTPs对体系影响最大。优化的温郁金SCoT-PCR反应体系在多个温郁金品种遗传多样性研究中得到了验证,结果表现出良好的稳定性、重复性和多态性丰富等特点,可用于今后温郁金品种遗传多样性分析、系统发育分析、遗传图谱构建、基因定位和分子标记辅助育种等研究。Taking Curcuma zoenyujin Y. H. Chen et C. Ling as test material,the Le5 （58） orthogonal design was applied to optimize the five factors （template DNA, Mg2＋ ,dNTPs, Taq DNA polymerase and primer concentration） of SCoT-PCR amplification system at five concentration levels, and range analysis was conducted on the PCR results. The SCoT-PCR （start codon targeted polymorphism-polymerase chain reaction） system was established and optimized,in order to provide technical support for the research of the genetic diversity and molecular identification in Curcurna zoenyujin Y. H. Chen et C. Ling. The results showed as follows, a total volume of 20 μL system included 0. 8 /~mol/L primer, 0. 4 rnmol/L dNTPs,1.5 mmol/L Mg2＋ ,0. 5 U Taq DNA polymerase and 40 ng templates DNA. The effective degree order of the factors on the SCoT-PCR was as below：dNTPs〉Taq DNA polymerase〉primer〉Mgz＋ 〉template DNA. The key factor affecting the SCoT-PCR system was the concentration of dNTPs. Furthermore, the optimal reaction system was used to verify with multiple varieties Curcuma zoenyujin Y. H. Chen et C. Ling samples, which showed good stability, repeatability and abundent polyrnorphism. It was proved to be suitable for the analysis of genetic diversity and phylogenetic,the establishment of genetic maps, gene localizations and molecular mark assisted breeding researches in Curcuma wenyujin Y. H. Chen et C. Ling in future.

关 键 词：温郁金 SCoT-PCR 体系优化 正交设计 

分 类 号：S567.239[农业科学—中草药栽培]