一种改良的红豆杉成熟针叶总RNA提取方法

A Modified Method for Total Ribonucleic Acids Extraction from Taxus Chinensis Needles

红豆杉(Taxus chinensis(Pilger)Rehd.)是一种重要的药源植物,根据红豆杉针叶特点,本研究提出改良RNA提取方法,即十六烷基三甲基溴化铵-氯化锂(CTAB-Li Cl)联用法,以红豆杉的幼嫩、成熟针叶为材料,采用TRIzol法提取RNA,结果表明,TRIzol法提取嫩针叶中的RNA质量完整,成熟针叶中的RNA质量不完整;针对成熟针叶采用CTAB法、SDS-酚法和改良的CTAB-Li Cl法提取RNA,并进行了质量验证比对显示,CTAB法和SDS-酚法提取的RNA质量差,并存在不同程度的多糖、多酚和DNA污染,改良CTAB-Li Cl联用法提取红豆杉成熟针叶的RNA质量完整,A_(260)/A_(280)比值在1.9~2.0之间,RNA产率最高,分别为CTAB法和SDS-酚法提取的RNA产率的1.37和1.24倍。进一步反转录后,利用特异性引物进行扩增验证,发现扩增的片段条带清晰,片段大小符合预期。成熟针叶中高质量的RNA更有利于红豆杉分子生物学方面的深入研究。

Taxus chinensis(Pilger)Rehd.is one of the most important medicinal plants.According to the characteristics of the needles,a new RNA extraction method,CTAB-LiCl,was developed.RNA was extracted from young and mature needles of Taxus Chinensis by TRIzol method.The result showed that the quality of RNA extracted from young needles was complete,and that of RNA from mature needles was incomplete.As a comparison,cetyl trimethyl ammonium bromide(CTAB)method,SDS-phenol method and improved CTAB-LiCl method were respectively used for extraction of RNA from mature needles.The comparative result showed that the quality of RNA extracted by CTAB method and SDS-phenol method was incomplete,accompanied with different degrees of contamination of polysaccharides,polyphenols and DNA.The improved CTAB-LiCl method could better extract RNA from mature needles.The ratio of A 260/A 280 was in the range of 1.9-2.0,and the yield of RNA was the highest,which was 1.37 and 1.24 times compared with that by CTAB and SDS phenol extraction,respectively.The reverse transcription cDNA was further tested through PCR with the specific primers.The amplified fragments displayed in clear and bright bands in accordance with the expected size.High quality RNA from mature needles has more advantages in molecular biology study of Taxus chinensis.