茶树不同器官组织总RNA提取方法的研究

A Simple and Effective Method for Extracting Total RNA from Different Organs of Tea Plant[Camellia sinensis(L.) O.Kuntze]

从茶树组织中提取高质量的总RNA,是开展茶树基因组学、功能基因组学研究的重要前提,而RNase、多酚类物质严重干扰茶树总RNA的分离提取。鉴于茶树组织总RNA提取过程难易不一、总RNA提取质量良莠不齐的现状,现对材料用量、提取液、DNA和蛋白质抽提液、RNA沉淀试剂、多酚氧化抑制剂等进行了比较研究,建立了一种适合茶树各器官组织总RNA提取的简单高效的方法(简易CTAB-LiCl法),并与实验室常用的改良Tri-Reagent法、改良CTAB法进行了比较。核酸定量和琼脂糖凝胶电泳检测结果显示,简易CTAB-LiCl法从茶树各器官组织中提取到的总RNA质量高、得率高。总RNA的得率是改良CTAB法的1.6-5倍。因此,简易CTAB-LiCl法具有效率高、适用范围广,且操作简单、实验成本低的特点。RT-PCR和cDNA-AFLP实验表明,提取的总RNA能够用于后续的分子生物学研究。

Obtaining high quality total RNA is an important prerequisite for studying genomies and functional genomies of tea plant. It is hard to extract high quality total RNA from tea due to the interference of RNase, and polyphenols. Aiming at the drawbacks in extracting method for total RNA of tea plant,a comparative study was carried out to determine material dosage, extracting buffer, protein extracting solution,precipitating reagent for extracting total RNA, in this paper. The simple CTAB-LiCl method was established, which was a simple and effective method for extracting total RNA from different organs of tea plaut. The differcnces among the improved Tri-Reagent method, the improved CTAB method and the simple CTAB-LiCl method were comparably investigated. The results of spectrophotometry testing and agarose gel electrophoresis showed that the simple CTAB-LiCl method was suitable for extracting high yield and high quality total RNA from tea organs. The intensity ratio of 28 S and 18 S rRNA bands was 2：1, the absorbance ratios of A260 to A280 were about 2. 0, the absorbance ratios of A260 to A230 were up to 2. 0, and the yield of total RNA ranged from 290.61 μg/g to 532.44 μg/g fresh weight. This method is good for extracting total RNA from different tea organs with its simple, efficient, high extracting rate and low cost. The results of RT-PCR and eDNA-AFLP analysis also revealed that the simple CTAB-15C1 method could obtain high quality total RNA from different tea organs.