【摘要】 为筛选出适合百合叶片RNA提取方法,以铁炮百合‘白天堂’组培苗叶片为材料,比较了改进的CTAB、SDS及传统的Trizol、CTAB、SDS方法提取RNA的效果。结果表明:不论传统及改进的SDS法和Trizol法均不能有效去除百合叶片组织中的多糖、蛋白等杂质,传统CTAB法提取RNA存在DNA污染。针对百合叶片组织富含多糖多酚的特点,改进了CTAB法,采用醋酸钠及无水乙醇去除多糖,酚/氯仿反复抽提去除蛋白,DNaseⅠ去除DNA,LiCl有效沉淀RNA。采用此方法提取的RNA条带清晰,经紫外光谱分析D260/D280比值为2.0,D260/D230为2.1,电泳28S、18S条带清晰,比值约为1.5。RT-PCR结果表明,改进的CTAB法提取的总RNA可直接用于分子克隆和基因表达分析等分子生物学实验。更多还原

【Abstract】 Five extraction methods: common Trizol,SDS/phenol extraction,CTAB and the modified methods of SDS/phenol extraction and CTAB were compared for extracting total RNA from lily leaves.The SDS and Trizol methods were not effective in removing polysaccharides,while the RNA extracted by traditional CTAB method was ineffective in removing DNA.Because lily leaves are rich in polysaccharides and secondary metabolites,the modified CTAB method was improved by adding KAc and ethanol to remove polysaccharides,phenol/ chloroform to remove protein,LiCl to precipitate RNA and DNaseⅠ to remove DNA.The modified CTAB can extract high-quality and high-integrity RNA,as determined by UV-spectroscopic analysis showing the D260/D280 at 2.0 and D260/D230 at 2.1.The electrophoresis profile showed that the lightness at 28S was 1.5 times higher than that at 18S.The modified CTAB can efficiently remove polysaccharides,protein and DNA.The results of RT-PCR indicated that this improved method satisfied the standard for gene cloning and other molecular biological experiments.更多还原