摘要
为建立适合二月兰的SSR-PCR反应体系,采用正交试验设计方法对影响二月兰SSR反应体系的5个因素(DNA模板、Mg^(2+)、d NTPs、引物和Taq聚合酶)进行优化试验,筛选出每个因素的最佳水平。结果表明,10μL反应体系中,Mg^(2+)浓度2.0 mmol/L,d NTPs浓度0.4 mmol/L,引物浓度0.6μmol/L,DNA模板量20 ng,Taq聚合酶量0.6 U。以3份二月兰基因组DNA为模板,利用优化后的SSR-PCR反应体系进行引物筛选,从50对SSR引物中成功筛选出扩增条带清晰、具有多态性的引物有12对,证明该反应体系具有较好的稳定性和通用性。研究建立和优化的二月兰SSR-PCR反应体系,为进一步利用SSR标记技术开展二月兰分子生物学研究提供了理论依据和技术参考。
In order to establish and optimize the SSR-PCR reaction system of Orychophragmus violaceus, theL25(56) orthogonal design was applied to optimize the five main factors(DNA template, Mg^(2+), d NTPs, primersand Taq enzyme) at five levels of SSR-PCR reaction system in this study. The PCR result was analyzed toselect the best levels of the five factors, and a suitable SSR reaction system for Orychophragmus violaceus wasestablished. The result showed that the optimal SSR-PCR system of 10 μL volumes consisted of 2.0 mmol/LMg^(2+), 0.4 mmol/L d NTPs, 0.6 μmol/L primer, 20 ng DNA template, and 0.6 U Taq polymerase. The optimizedSSR-PCR reaction system was used to verify with three Orychophragmus violaceus germplasms, 12 primerpairs were selected with clear banding patterns and abundant polymorphism bands from 50 primer pairs, andthe result showed that the optimal reaction system had excellent stability and generality. The establishment ofSSR-PCR reaction system provides a theoretical basis and technical references for molecular biology researchon Orychophragmus violaceus.
出处
《中国农学通报》
2017年第5期40-46,共7页
Chinese Agricultural Science Bulletin
基金
国家科技支撑项目"重要花卉种质资源发掘与创新利用"(2013BAD01B)
关键词
二月兰
SSR-PCR
反应体系
引物筛选
Orychophragmus violaceus
SSR-PCR
reaction system
primer selection
