中国芒属植物ISSR-PCR扩增反应体系的优化

›› 2013, Vol. 21 ›› Issue (1): 167-173.

中国芒属植物ISSR-PCR扩增反应体系的优化

卢玉飞, 蒋建雄, 易自力

湖南农业大学生物科学技术学院, 湖南长沙 410128

收稿日期:2012-08-31 修回日期:2012-11-06 出版日期:2013-02-15 发布日期:2013-02-27
通讯作者: 易自力
作者简介:卢玉飞(1976-),男,广西贵港人,讲师,博士研究生,研究方向为植物遗传学,E-mail:lyff1002@126.com;
基金资助:
国家自然科学基金项目(30971832);国家高技术研究发展计划(863计划)项目(2011AA10020903)资助

Optimization the ISSR-PCR Amplification Reaction System of Miscanthus from China

LU Yu-fei, JIANG Jian-xiong, YI Zi-li

College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan Province 410128, China

Received:2012-08-31 Revised:2012-11-06 Online:2013-02-15 Published:2013-02-27

摘要/Abstract

摘要： 芒属植物(Miscanthus)被认为适合作为新一代能源作物开发利用而成为当前国内外研究热点之一。为给后续相关研究奠定基础,本研究从中国芒属植物的芒(Miscanthus sinensis)、五节芒(M. floridulus)、荻(M. sacchariflorus)、南荻(M. lutarioriparius)、尼泊尔芒(M. nepalensis)、双药芒(M. nudipes)和红山茅(M. paniculatus)等类群中挑取部分材料,以其基因组DNA为模板,采用同一试验考察多个因素及水平的筛选方式对ISSR-PCR扩增体系中的Mg²⁺、dNTP、引物、模板的浓度以及循环数进行优化,建立适用于中国芒属植物的最佳ISSR-PCR反应体系。该体系为20 μL,含Mg²⁺2.5 mmol·L^-1,dNTP 0.25 mmol·L^-1,引物0.5 μ mol·L^-1,DNA 2 μL及1 U Taq Plus DNA聚合酶和1×PCR buffer。结果将为进一步开展中国芒属植物的相关遗传分析研究提供技术参考。

关键词: 芒属, ISSR, PCR反应体系, 优化

Abstract: Miscanthus andresson (Poaceae) has recently become a research focus because it has been defined as a second-generation non-food energy crop. In this study, genomic DNAs of Miscanthus species from China including Miscanthus sinensis, M. floridulus, M. sacchariflorus, M. lutarioriparius, M. nepalensis, M. nudipes, and M. paniculatus were chosen as specimen samples for subsequent assay. The concentration of Mg²⁺, dNTP, primer, DNA template and the number of amplification cycles suitable for ISSR-PCR amplification reaction of Miscanthus species were optimized using the multifactor and multilevel experiments. Finally, an optimal ISSR-PCR amplification reaction system for the Miscanthus of China was established successfully. The 20 μL volumes of reaction system contained 1×PCR buffer, Mg²⁺ 2.5 mmol·L^-1, 0.25 mmol·L^-1 of each dNTP, primer 0.5 μmol·L^-1, 2 μL DNA, and 1 unit of plus Taq DNA polymerase, which undoubtedly supplied the technical reference for further related genetic analysis of Miscanthus in China.

Key words: Miscanthus, ISSR, PCR reaction system, Optimization

中图分类号:

Q943.1

卢玉飞, 蒋建雄, 易自力. 中国芒属植物ISSR-PCR扩增反应体系的优化[J]. , 2013, 21(1): 167-173.

LU Yu-fei, JIANG Jian-xiong, YI Zi-li. Optimization the ISSR-PCR Amplification Reaction System of Miscanthus from China[J]. , 2013, 21(1): 167-173.

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