油茶SSR-PCR反应体系的优化研究

摘要

[目的]优化油茶(Camellia oleifera) SSR-PCR反应体系.[方法]应用改良CTAB法提取油茶基因组DNA,采用L16(45)正交设计试验,对影响油茶SSR-PCR反应体系的5个因素(Taq聚合酶浓度、模板浓度、dNTPs浓度、引物浓度和Mg2+浓度)在4水平上进行筛选.PCR结果经统计分析软件DPS分析,并对退火温度进行了摸索,确立了油茶SSR-PCR的优化体系.[结果]油茶SSR-PCR的优化体系总体积为20μl,包括Taq聚合酶量为1.0 U/20μ1,模板浓度75 ng/20 μl,dNTPs浓度为0.15 mmol/L,引物浓度为0.40μmoL/L,Mg2+浓度为1.50 mmol/L,退火温度为50℃.[结论]该研究确定的优化体系可以为油茶资源遗传多样性分析奠定技术基础.%[Objective]The research aimed to optimize the reaction system of C. Oleifera. [Method]The genomic DNA extracted from leaves of C. Oleifera with an improved method of CTAB was applied to optimize the SSR-PCR system. The 4 levels of 5 factors( Taq DNA polymerase, DNA, dNTPs concentration, primers concentration, and Mg2+ concentration) in SSR-PCR system were selected by L16(45) orthogonal design. The electrophoresis profiles of SSR-PCR were analyzed by software DPS. The annealing temperaturp of SSR-PCR reaction was also explored. [Result]A suitable SSR-PCR system (20 μl) was established, including 1.0 U/20 μl Taq DNA polymerase, 75 ng/20 μl DNA, 0. 15 mmol/L dNTPs, 0.40 μmol/L primers, 1.50 mmol/L Mg2+, and annealing temperature was 50 ℃. [Conclusion]The optimization SSR-PCR system can provide some technical foundations for the study of the genetic diversity of C. Oleifera resources.