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温郁金SCOT-PCR反应体系优化及遗传多样性分析摘要本实验的目的在于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，及分析温郁金的遗传多样性为分子鉴定等研究提供技术支持。在方差分析基础上，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。通过实验建立了温郁金SCoT-PCR的最佳反应体系（20 μL）：引物0.8 μmol·L-1，dNTPs 0.4 mmol·L-1，Mg2+ 1.5 mmol·L-1，Taq酶0.5 U，模板DNA 10 ng，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。从36个SCoT 引物中的筛选出9个引物，对20个郁金进行扩增，得到68 条DNA 条带,占所观察到的总扩增带数的76.47% 。遗传相似系数在0.62到0.94之间。这表明20个郁金个体有较高遗传多样性。47586
In order to provide technical support for the research of the molecular identification in Curcuma wenyujin Y.H.Chen et C.Ling, we established and optimized SCoT–PCR (start codon targeted polymorphism-polymerase chain reaction) and system genetic persity in this study. In this research, the L25(56) 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. The results of optimized system by intuitive analysis and variance analysis are as follows: a total volume of 20 μL system including 1.0 μmol·L-1 primer, 0.8 mmol·L-1 dNTPs, 1.5 mmol·L-1 Mg2 + , 1.0 U Taq DNA polymerase and 10 ng templates DNA. The effective degree order of the factors on the SCoT-PCR is as below: dNTPs >Taq DNA polymerase > primer > Mg2+> template DNA. The key factor affecting the SCoT-PCR system is the concentration of dNTPs. Furthermore, the optimal reaction system was used to verify with multiple varieties Curcuma wenyujin Y.H.Chen et C.Ling samples, which showed good stability，repeatability and abundent polymorphism. Nine informative primers were selected from 36 SCoT primers to amplification twenty Curcuma wenyujin .A total of 68 bands were produced,76.47% of them were polymorphic.The genetic similarity coefficient between 0.62 to 0.94. It means the twenty Curcuma wenyujin have highly variable.
毕业论文关键词：温郁金； SCoT-PCR； 体系优化； 正交设计； 遗传多样性
Key words: Curcuma wenyujin; Start codon targeted polymorphism - polymerase chain reaction; System optimization; Orthogonal design ; genetic persity
目 录
1材料和方法 5
1.1材料及主要试剂 5
1.2方法 5
1.2.1基因组 DNA的提取 5
1.2.2 SCoT-PCR反应体系正交设计 6
1.2.3 SCoT-PCR 扩增及检测 7
1.3数据统计与处理 7
1.4 SCoT最佳反应体系的稳定性和通用性检测 7
1.5 遗传多样性分析 7
2结果与分析 7
2.1温郁金DNA电泳结果 7
2.2正交设计电泳结果评分 8
2.2.1 PCR正交设计直观分析 8
2.2.2 PCR正交设计方差分析