Trizol试剂法快速高效提取3种作物不同组织总RNA

Fast and effective total RNA extraction from different tissues in 3 crops through the Trizol reagent method

【目的】建立适用于禾本科作物不同组织总RNA的快速、高效提取方法,为进行后续的分子生物学研究奠定基础。【方法】采用Trizol试剂法,通过改变抽提上清液吸取量、沉淀方法及沉淀漂洗次数,对甘蔗、玉米和水稻的叶片、茎尖和根尖的总RNA提取方法进行优化。【结果】与吸取0.4mL上清液相比,吸取0.2mL上清液的总RNA OD260/OD280、OD260/OD230值分别在1.95～2.00和2.11～2.44,所获得的总RNA纯度较高。沉淀漂洗两次的OD260/OD230值(2.11～2.47)明显高于沉淀漂洗1次的总RNA OD260/OD230值(1.01～1.61),总RNA纯度较高。经异丙醇沉淀所得的各材料的总RNA28S、18S和5S谱带清晰,无拖尾现象,总RNA完整性较好;LiCl沉淀法的RNA条带较模糊粘连,5S条带明显弥散,总RNA产率相对较低。以Trizol-异丙醇法提取的甘蔗总RNA为模板进行GAPDH基因片段扩增,所获甘蔗的叶片、茎尖和根尖的GAPDH基因表达丰度很强,无特异扩增,目的片段长度为153bp。【结论】改良Trizol-异丙醇法提取甘蔗、玉米和水稻不同组织的总RNA带型清晰、完整性好、纯度和产率高,适用于快速、高效提取禾本科植物不同组织总RNA。

[Objective]The present study was conducted to establish a fast and effectively method for isolating the to- tal RNA in gramineous crops in order to provide foundation for subsequent molecular biology researches. [Method]Using the Trizol reagent method, the supernatant amount changed. Then, by the precipitation method and precipitation rinse times, the total RNA extraction from the leaves, stem tips, and roots of sugarcane, maize, and rice was optimized. [Result] Compared with the 0.4 mL supernatant absorbent, the 0.2 mL supernatant absorbent＇s total RNA OD260/OD280 and OD260/OD230 values were between 1.95-2.00 and 2.l 1-2.44 respectively, in which the total RNA obtained was highly puri- fied. The total RNA OD260/OD230 value （2.11-2.47） that underwent precipitation rinse two times was significantly higher than the total RNA OD260/OD230 value （1.01-1.61） that underwent precipitation rinse 1 time, so the total RNA of the higher OD^0/OD~ value was purer. The spectra of total RNA 28S, 185, and 5S resulted from the isopropanol precipitation in various materials were clear and without trailing; therefore the total RNA integrality was better. However, from the LiC1 precipitation method, the resulting RNA band was adhesively fuzzy and the 5S band was apparently diffused, so the total yield of RNA was relatively low. Utilizing the Trizol-isopropanol extraction method, the total RNA from sugarcane was ex-tracted as template for GAPDH gene fragmentation, and the resuhing GAPDH gene expressions obtained from sugarcane leaves, stalk tips, and root tips were very strong with no specific amplifications. Hence, the objective-to-fragment length was 153 bp. [Conclusion]The total RNA tissues extracted from sugarcane, maize, and rice through the modified Trizol- isopropanol extraction method showed clear bands, good integrity, high purity, and high yield. Therefore, this extraction method is suitable for the fast and efficient extraction of total RNA from different tissues of gramineous plants.