大豆总rna 提取方法比较及其在基因克隆和表达分析中的应用 .pdf

分子植物育种，2013 年，第11 卷，第2 期，第255-261 页 Molecular Plant Breeding, 2013, Vol.11, No.2, 255-261 技术主题 Technology Feature 大豆总RNA 提取方法比较及其在基因克隆和表达分析中的应用 1,2 1 1,2 1 1 2* 郭彬 侯思宇 黄可盛 路阳 韩渊怀 王玉国 1 山西农业大学生物工程研究所, 太谷, 030801; 2 山西农业大学农学院, 太谷, 030801 * 通讯作者, tgwygn@126.com 摘 要 为确立大豆叶、花组织提取总RNA 的最佳方法，比较了植物总RNA 提取试盒剂法、改良的 CTAB-LiCL 法和改良的热硼酸法的提取效果。通过RNA 产量、纯度、凝胶电泳及后续的基因克隆和荧光定 量PCR 等分析，结果表明：热硼酸法所提取的叶、花总RNA 含量约为其他两种方法的6 倍；总RNA OD260/OD280 值介于 1.98~2.1；电泳条带完整清晰；应用获得的总RNA 所克隆ZAT9-like 基因，经测序获得 885 bp 的核酸序列与ZAT9-like (登录号: XM_003517371.1)基因同源率达99% ；荧光定量分析Histone H3 基因的扩增曲线与融解曲线峰型良好。说明该方法能够满足一般分子生物学下游实验的要求，是一种理想的 针对大豆叶、花总RNA 的的提取方法。 关键词 大豆, 总RNA, 热硼酸法 Different Methods for Extracting Total RNA and Their Application in Gene Cloning and Gene Expression Analysis in Soybean (Glycine max) Guo Bin 1,2 Hou Siyu 1 Huang Kesheng 1,2 Lu Yang 1 Han Yuanhuai 1 Wang Yuguo 2* 1 Institute of Agricultural Bioengineerings, Taigu, 030801; 2 School of Agriculture, Shanxi Agricultural University, Taigu, 030801 * Corresponding author, tgwygn@126.com DOI: 10.3969/mpb.011.000255 Abstract In order to find out the optimal method for extracting total RNA from leaves and flowers in soybean (Glycine max), extracting efficiency was compared among three methods of RNA extraction, RNApure plant kit, modified CTAB and hot borate. The yield and purity of total RNA were determined by nucleic acid and protein detector; integrity and quality of total RNA were analyzed by agrose gel electrophoresis, gene cloning and real-time PCR. The results showed that the yield of total RNA by using hot borate was six times that by the other methods (RNApure plant kit and modified CTAB). The value of OD260/OD280 was between 1.98 to 2.01; The electr- ophoresis result of RNA showed satisfactory bands with sharpness and integrity. ZAT9-like gene was obtained by cloning from the total RNA, sequenced to the nucleic acid sequence of 885 bp, showing