用CTAB-PVP法提取棉花各组织总RNA的研究

Extraction of total RNA from cotton(Gossypium hirsutum L.) tissues with CTAB-PVP method

针对棉花组织总RNA难以提取、不同组织的提取方法难以统一的问题，借鉴Jaakola等的越橘果实总RNA提取方法（即CTAB-PVP法），对棉花组织总RNA的提取效果进行了分析。实验结果表明：得到的棉花纤维、胚珠、叶片、胚根、花瓣、下胚轴和花药各组织的RNA完整性好，其D260／D280比值为1．7～2．0，产率30～180pg／g（鲜质量）；用开花后15d纤维的RNA为材料进行反转录，再利用RT-PCR和3’RACE技术进行克隆，各获得1个大小为542和712bp的cDNA克隆，经序列分析鉴定，这2个片段分别编码棉花微管结合蛋白和微丝结合蛋白。

A new improved CTAB （Hexadecetyltrimethyl ammonium bromide） method, the CTAB-PVP method, was developed in order to isolate high-quality RNA from the most tissues of cotton （Gossypium hirsutum L. ）. The total RNAs were extracted from fiber, ovule, leaf, radicle, petal, hypocotyls and anther using CTAB-PVP. The extracted total RNA was of high integrality and the D260/D280 ratio of each sample was between 1.7 and 2.0. The average yield of RNA was ranged from 30 to 180μg/gfw （gram fresh weight）. The total RNA extracted with this method can be suitably used for most RNA-based analyses, including reverse transcription and RT-PCR, etc.