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| 药用植物苦参SSR-PCR体系的优化与验证 |
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段永红1, 渠云芳2, 王长彪3, 毕红园3, 王玉庆1, 孙毅3,4, 杨武德1
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| (1.山西农业大学 农学院, 山西 太谷 030801;2.1. 山西农业大学 农学院, 山西 太谷 030801;3.山西省农业科学院 生物技术研究中心, 太原 030031;4.农业部黄土高原作物基因资源与种质创制重点实验室, 太原 030031) |
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| 摘要: |
| 为有效利用SSR-PCR技术对药用植物苦参进行遗传多样性分析，采用正交设计L16(45)对影响苦参SSR-PCR体系的5个因素(Taq 酶、Mg2+、模板DNA、dNTP、引物)在4个水平上进行优化，PCR 结果用DPS 数据处理软件分析，筛选出各因素的最佳水平，建立适宜苦参SSR-PCR 的反应体系和扩增程序，并选用内蒙古苦参对该体系进行稳定性验证。结果表明：在10 μL 的苦参SSR-PCR体系中，模板DNA 的用量为20.0 ng，Taq DNA 聚合酶的用量为0.2~0.6 U，Mg2+ 的浓度为2.5 mmol/L，dNTPs 浓度为0.1 mmol/L，引物的浓度为0.6 μmol/L。扩增程序为：94 ℃预变性3 min；94 ℃变性1 min，56 ℃退火45 s，72 ℃ 1 min，35个循环；72 ℃延伸10 min。4 ℃保存。选用12份苦参DNA对该体系进行稳定性验证，该体系具有较高的扩增稳定性，可用于药用植物苦参SSR 标记的研究。 |
| 关键词: 苦参 SSR 优化 正交设计 |
| DOI：10.11841/j.issn.1007-4333.2014.05.13 |
| 投稿时间：2013-11-10 |
| 基金项目:山西省科技攻关项目（20130311004-3）；山西省财政支农项目（2011NYGX-05）；中国博士后基金项目（112118）；山西农业大学博士科研启动经费（xb2011011） |
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| Optimization and testing for the SSR-PCR system of Sophora flavescens medicinal plant |
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DUAN Yong-hong1, QU Yun-fang2, WANG Chang-biao3, BI Hong-yuan3, WANG Yu-qing1, SUN Yi3,4, YANG Wu-de1
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| (1.College of Agronomy, Shanxi Agricultural University, Taigu 030801, China;2.1. College of Agronomy, Shanxi Agricultural University, Taigu 030801, China;3.Biotechnology Research Center, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, China;4.Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau of Ministry of Agriculture, Taiyuan 030031, China) |
| Abstract: |
| The SSR molecular markers were used for analysing genetic diversity of Sophora flavescens medicinal plant, which was helpful for comprehensive evaluating germplasm resources of Sophora flavescens.The orthogonal design L16(45)on Sophora flavescens SSR-PCR system of 5 elements(Taq enzyme, Mg2+, template DNA, dNTP, primers) in the four levels was optimized, The result of PCR was analyzed by software DPS, filtering out the best level of response factors established for the SSR-PCR Pinus reaction.An optimal 10 μL volumes SSR-PCR system consisted of template DNA was 20.0 ng, Taq DNA polymerase was 0.2-0.6 U, Mg2+ was 2.5 mmol/L, dNTPs was 0.1 mmol/L and primer was 0.6 μmol/L.PCR procedures was pre-denaturation for 3 min at 94 ℃, followed by 35 cycles of denaturation for 1 min at 94 ℃, anneal for 45 s at 56 ℃, extension for 1 min at 72 ℃, and a final extension at 72 ℃ for 10 min, then stored at 4 ℃.Total DNA of 12 Sophora from different origin were tested to amplify by SSR markers, which verified the robustness of the system.The results showed that the system had high amplification stability, it can be used to study the Sophora medicinal plant SSR markers. |
| Key words: Sophora flavescens SSR optimization orthogonal design |
