| 中麻黄ISSR—PCR反应体系的建立和优化 |
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| 引用本文： | 朱田田,晋玲,杜弢,崔治家,林丽,高天鹏,张裴斯.中麻黄ISSR—PCR反应体系的建立和优化[J].中国药房,2013(43):4033-4037. |
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| 作者姓名： | 朱田田 晋玲 杜弢 崔治家 林丽 高天鹏 张裴斯 |
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| 作者单位： | [1]甘肃中医学院,兰州730000 [2]甘肃省高校中（藏）药化学与质量研究省级重点实验室,兰州730000 [3]甘肃中医学院药用植物遗传育种研究所,兰州730000 [4]兰州城市学院城市生态与环境生物技术中心,兰州730070 |
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| 基金项目： | 基金项目：国家自然科学基金资助项目（No.31160118）;甘肃省教育厅科研项目（No.1106B-07）;甘肃中医学院中青年科研基金资助项目（No.ZQ2011-12） |
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| 摘 要： | 目的：建立适用于中麻黄的简单重复序列区间．聚合酶链反应（ISSR—PCR）最佳反应体系。方法：以凝胶成像系统下特异谱带特征为评价指标，以Mg2＋、dNTPs、TaqDNA聚合酶、引物和DNA模板为考察因素，采用L16（4^5）正交试验优选条件；通过单因素试验对反应体系中各种影响因素进行筛选。结果：中麻黄ISSR-PCR最佳反应体系（20p1）为30ngDNA模板、0．25mmol／LdNTPs、2．0mmol／LMg2＋（10xPCRBuffer）、0．4μmol／LISSR引物、1．5UTaqDNA聚合酶；并筛选出了12条ISSR-PCR引物的最佳退火温度。结论：所建中麻黄ISSR—PCR反应体系经过10份中麻黄材料检验证明该体系稳定可靠，可用于中麻黄遗传分析。
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| 关 键 词： | 中麻黄 ISSR反应体系 建立 优化 |
Establishment and Optimization of ISSR-PCR Reaction System for Ephedra intermedia |
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| ZHU Tian-tian,JIN Ling,DU Tao,CUI Zhi-jia,LIN Li,GAO Tian-peng,ZHANG Pei-si.Establishment and Optimization of ISSR-PCR Reaction System for Ephedra intermedia[J].China Pharmacy,2013(43):4033-4037. |
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| Authors: | ZHU Tian-tian JIN Ling DU Tao CUI Zhi-jia LIN Li GAO Tian-peng ZHANG Pei-si |
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| Institution: | 1.Gansu Col- lege of TCM, Lanzhou 730000, China; 2. Key Lab of Chemistry and Quality for Traditional Chinese (Tibetan) Medicines of the College of Gansu Province, Lanzhou 730000, China; 3. Institute of Medicinal Plant Genetic Breeding, Gansu College of TCM, Lanzhou 730000, China; 4. Centre of Urban Ecology and Environmental Bio- technology, Lanzhou City University, Lanzhou 730070, China) |
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| Abstract: | OBJECTIVE： To establish optimal ISSR-PCR reaction system for Ephedra intermedia. METHODS： The reaction system was optimized by L18（4^5） orthogonal test with specific bands under gel imaging system as index using Mg2＋, dNTPs, Taq DNA polymerase and primer, DNA template as factors. The influential factors of reaction system were screened by single factor test. RESULTS： The optimal ISSR-PCR reaction system of E. intermedia（20 μl） contained 30 ng DNA template, 0.25 mmol/L dNTPs, 2.0 mmol/L Mg2＋（10×PCR Buffer）, 0.4 μmol/L ISSR primer and 1.5 U Taq DNA polymerase. The suitable annealing tem- perature of 12 primers was determined. CONCLUSIONS： It is proved that the established ISSR-PCR reaction system of E. interme- dia would be stable and credible by the materials testing result of 10 samples of E. intermedia. This would provide the basis for the genetic analysis of E. intermedia. |
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| Keywords: | Ephedra intermedia ISSR reaction system Establishment Optimization |
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