半夏块茎高质量总RNA提取的一种有效方法  

Efficient method for extraction of high quality RNA from microtubers of Pinellia ternata in vitro

作　　者：黄月琴[1] 徐有明[2] 薛建平[1] 

机构地区：[1]淮北煤炭师范学院生命科学学院,安徽淮北235000 [2]华中农业大学园艺林学学院,湖北武汉430070

出　　处：《中国中药杂志》2008年第15期1810-1813,共4页China Journal of Chinese Materia Medica

基　　金：国家农业成果转化资金重点项目(05EFN213400124);淮北市重点项目(06085)

摘　　要：目的：探讨一种有效提取半夏块茎总RNA方法。方法：以半夏试管苗的叶片、叶柄和块茎为试验材料，将常规的异硫氰酸胍提取植物总RNA的方法做了改进，在RNA提取缓冲液中加入高浓度的β-巯基乙醇以除去酚类物质，用酸酚／氯仿抽提去除蛋白质污染，以及利用异丙醇和醋酸钠联合沉淀多糖等手段，建立了一种简单实用的从富含多酚类和多糖等物质的植物材料中提取总RNA的方法。结果：该方法所提RNA的A260/A230均大于2．0，A260/A230的值为1．7～2．0，电泳条带清晰，RNA完整性好。结论：利用本方法提取的半夏试管小块茎总RNA具有很高的质量和纯度，且得率很高，完全满足后续的DDRT-PCR和反向Northern blotting等分子生物学研究，而且简单经济、实验结果稳定，重复性好，适合富含多酚类和多糖等物质的药用植物组织总RNA的提取。Objective： To extract RNA from Pinellia ternata and lay a foundation for studying the formation mechanism of P. ternata. Method： By modifying the method recommended by Guanidinium for extracting total RNA from plant tissues rich in phenolic and polysaccharidic compounds, a simple and convenient method for extraction of total RNA from the tubers, stems and leaves of P. ternate containing abundant polyphenols and polysaccharides was established. High concentrated β-mercaptoethanol was added in the RNA extracted buffer to remove polyphenols, phenol and chloroform were used to eliminate proteins, and isopropanol and sodium acetate were used to precipitate polysaccharides. Result： The A260/A230 value of RNA extracted with improved method were all over 2.0 and the values of A260/A230 were between 1.7 and 2.0. The electrophoresis bands were cleared on agarosegel and integrity of RNA was good. Conclusion： The results showed that RNA obtained from the tubers, stems and leaves of P. ternate with this method had high purity and quality and could be used in molecular biological research, as DDRT-PCR and reverse Northern blotting analysis directly. This method is simple, economic, stable performance, and has a good repeatability as well as is suitable for extracting total RNA of medicinal plants with high concentrations of phenolics and polysaccharides.

关 键 词：半夏 试管块茎 多酚类物质 多糖 总RNA 提取 

分 类 号：R284[医药卫生—中药学]