浙贝母鳞茎总RNA提取方法的研究

Total RNA Extraction from the Bulbs of Fritillaria thunbergii Miq.

建立适合功能基因分析的浙贝母Fritillaria thunbergiiMiq.鳞茎总RNA的提取方法,将为后续与药效成分相关功能基因的分析奠定良好的工作基础。本研究通过紫外分光光度法、非变性琼脂糖电泳法以及DDRT-PCR方法比较异硫氰酸胍法、改良的异硫氰酸胍法、改良的SDS法和改良CTAB法提取浙贝母鳞茎总RNA的质量。结果表明,通过改良的CTAB法提取出的总RNA的完整性好(28 s/18 s约为2),纯度高(其A260/A280和A260/A230分别为1.95和2.30),以其为模板进行DDRT-PCR分析时能获得清晰的差异条带。通过本研究建立的改良CTAB法可于浙贝母鳞茎总RNA的提取,并可为其它植物鳞茎的总RNA提取提供参考。

To develop a suitable protocol for the extraction of total RNA with higher quality to be used in the functional genes analysis from bulbs of Fritillaria thunbergii Miq.. The improved methods based on guanidine isothinocyanate, SDS, and CTAB were applied. Total RNA was extracted and their quality was analyzed by UV spectrophotometry, agarose gel electrophoresis, and DDRT-PCR amplification with silver staining. Higher quality total RNA were extracted from the bulbs of F. thunbergii by the improved CTAB method. Compared with poor results in other 3 methods, the improved CTAB method produced a rational agarose gel electrophoresis results （28S/18S≈2.0） and a good UV spectrophotometric ratio （A260/A280 = 1.95 ,A260/A230 = 2.30）. By using the improved CTAB method, the total RNA from F. thunbergii can be analyzed in differentially displayed transcripts analyzing by DDRT-PCR method. The improved CTAB method is suitable for total RNA extraction from bulbs of Fritillaria thunbergii Miq. , and can be spread to other bulb plants.