蔡丽平, 梅辉坚, 陈奶莲, 苗作云, 吴鹏飞, 邹显花, 马祥庆. 濒危植物沉水樟ISSR-PCR反应体系的建立与优化[J]. 西南林业大学学报, 2013, 33(5): 40-45. DOI: 10.3969/j.issn.2095-1914.2013.05.007
引用本文: 蔡丽平, 梅辉坚, 陈奶莲, 苗作云, 吴鹏飞, 邹显花, 马祥庆. 濒危植物沉水樟ISSR-PCR反应体系的建立与优化[J]. 西南林业大学学报, 2013, 33(5): 40-45. DOI: 10.3969/j.issn.2095-1914.2013.05.007
CAI Liping1, MEI Huijian2, CHEN Nailian3, MIAO Zuoyun4, WU Pengfei1, ZOU Xianhua1, MA Xiangqing1. Establishment and Optimization of ISSR-PCR System for Endangered Plant Species Cinnamomun micranthum[J]. Journal of Southwest Forestry University, 2013, 33(5): 40-45. DOI: 10.3969/j.issn.2095-1914.2013.05.007
Citation: CAI Liping1, MEI Huijian2, CHEN Nailian3, MIAO Zuoyun4, WU Pengfei1, ZOU Xianhua1, MA Xiangqing1. Establishment and Optimization of ISSR-PCR System for Endangered Plant Species Cinnamomun micranthum[J]. Journal of Southwest Forestry University, 2013, 33(5): 40-45. DOI: 10.3969/j.issn.2095-1914.2013.05.007

濒危植物沉水樟ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR System for Endangered Plant Species Cinnamomun micranthum

  • 摘要: 为从分子水平上探讨濒危物种沉水樟种质资源的保护机制,以其基因组DNA为模版,开展ISSR-PCR反应体系的研究,对各反应因子水平、引物退火温度和循环参数进行优化,确定适于沉水樟ISSR-PCR反应的最佳体系:20μL反应体系中各反应物的最适含量为模板75ng、引物04μmol/L、dNTP 200μmol/L、Taq酶1.25U、Mg2+ 1.5mmol/L;扩增程序为:94℃预变性7min;94 ℃变性30s,530~580 ℃退火45s,72 ℃延伸90s,45个循环;72℃延伸7min,4 ℃保存。

     

    Abstract: In order to explore the germplasm resource conservation mechanism for the endangered species Cinnamomun micranthum from the molecular level, the most appropriate ISSR-PCR reaction system with its genomic DNA was experimented by optimizing the level of each reacting factors, the primer annealing temperature and cycle parameters. The results showed that the suitable dosage of each reacting factor for C. micranthum ISSR-PCR reaction system was as follows: 20μL reaction system, including 75ng template, 0.4 μmol/L primer, 200 μmol/L dNTP, 1.25 U Taq enzyme, and 1.5 mmol/L Mg2+. The best amplification program was as: denaturation for 7 min at 94 ℃ in advance, then denaturation at 94 ℃ for 30s, annealing for 45s at 530-580 ℃, extension for 90s at 72 ℃, after 45 cycles, extension at 72 ℃ for 7 min, preservation at 4 ℃.

     

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