LI Jia,ZHANG Na,LAI Xiaohui,et al.Establishment and Optimization of cDNA-SRAP Reaction System by Orthogonal Design for Two Species of Lepidium L.[J].Northern Horticulture,2016,40(24):99-103.[doi:10.11937/bfyy.201624025]
两种独行菜种子cDNA-SRAP反应体系的建立及优化
- Title:
- Establishment and Optimization of cDNA-SRAP Reaction System by Orthogonal Design for Two Species of Lepidium L.
- 文献标志码:
- A
- 摘要:
- 以2种独行菜种子为试材，采用cDNA-SRAP技术，研究了dNTPs、Mg2+、上下游引物、cDNA模板、Taq DNA聚合酶浓度等5个因素对独行菜cDNA-SRAP反应体系扩增结果的影响，以期得到适合2种独行菜种子的cDNA-SRAP反应体系，并且对cDNA-SRAP扩增条件进行了优化。结果表明：优化的最佳cDNA-SRAP反应体系（20 μL）为10×PCR buffer，cDNA模板用量100 ng，Mg2+浓度1.75 mmol?L-1，dNTPs浓度0.275 mmol?L-1，引物浓度1.0 μmol?L-1，Taq DNA聚合酶用量0.5 U；利用优化的cDNA-SRAP反应体系能够扩增获得清晰、数目多的条带，可用于后续深入研究2种独行菜不同处理下差异表达基因。
- Abstract:
- A cDNA sequence-related amplified polymorphism (SRAP) reaction system of two species Lepidium L.were established and optimiazed by orthogonal experimental design and single factor analysis of different concentration of dNTPs，Mg2+，primer，Taq DNA polymerase and DNA template was conducted.The results showed that the optimal reaction system for the two species of Lepidium L.was followed as 0.275 mmol?L-1 dNTPs，0.5 U Taq DNA polymerase，1.0 μmol?L-1 primer，100 ng template cDNA in total 20 μL of reaction solution.Using the optimal system of cDNA-SRAP,clear stable and repeatable bands could be amplified from two species of Lepidium plants for analyzing gene differential expression.
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备注/Memo
第一作者简介：李佳（1992-），女，硕士研究生，研究方向为植物学。E-mail：1589847368@qq.com.责任作者：李群（1971-），女，博士，副教授，现主要从事植物逆境生理与分子机制等研究工作。E-mail：liqun_007@126.com.基金项目：新疆维吾尔自治区重大科技专项科技支疆资助项目（200840102-40）；国家自然科学基金资助项目（31160051/C020407）。