一种改良的云杉针叶总RNA提取方法  

An Improved Method for the Extraction of Total RNA from Spruce Needles

作　　者：刘洁[1] 江静怡 徐吉臣[1] 

机构地区：[1]北京林业大学林木育种国家工程实验室,北京100083

出　　处：《分子植物育种》2014年第3期554-561,共8页Molecular Plant Breeding

基　　金：林业公益性行业科研专项(201004009)资助

摘　　要：云杉(Picea)是一种重要的绿化和用材树种。根据云杉的特点,本研究提出了改良的云杉RNA提取方法即SDS酚-LiCl法,以不同种云杉(青杄,白杄和红皮)的针叶为试验材料,采用TRIzol法、CTAB法、改进的SDS酚法和SDS酚-LiCl法提取RNA,并进行了质量验证。结果表明:TRIzol法不能提取云杉针叶总RNA;CTAB法和改进的SDS酚法提取的RNA质量少,并存在不同程度的多糖、多酚和DNA污染。SDS酚-LiCl法适当增加了提取液中SDS、PVP以及β-巯基乙醇的浓度,结合酸性条件、高盐溶液(3 mol/L NaAc)和LiCl等减少DNA、多糖污染的步骤,最终提取的RNA质量好、得率高。进一步反转录后,利用特异引物进行扩增验证,发现扩增的片段条带清晰,得率高,片段大小符合预期。研究结果表明,SDS酚-LiCl法可以较好的提取云杉RNA并进行后续的基因表达及转录组研究,为推动云杉在转录水平上的研究奠定了基础。Spruce （Picea） is one of the important virescence and timber species. According to the characteristics of spruce, we improved a new RNA extraction method called SDS phenol-LiC1. Needles of three spruce species Picea wilsonii, Picea meyeri and Picea koraiensis were used as experiment materials for RNA extraction. The RNA extracting effects of SDS phenol-LiC1 method and the previous TRIzol, CTAB, improved SDS-phenol methods were analyzed and compared. The results showed that TRIzol method failed for RNA extraction from spruce. CTAB and improved SDS-phenol methods could be used to extract spruce RNA but with less amount and contamination of polysaccharides, polyphenol and DNA. With increased addition of SDS, PVP and β-mercaptoethanol, and combined use of acidic condition, high concentration of salt （3 mol/L NaAc） and LiC1 to decrease the DNA and polysaccharides contamination, the spruce RNA extracted by SDS phenol-LiC1 method was in high quality and yield. The reverse transcribed 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. It confirmed that the total RNA can be successfully extracted by SDS phenol-LiC1 method from spruce, and further used in gene expression to extend the transcriptome study in spruce.

关 键 词：云杉 RNA 多糖 多酚 DNA SDS酚-LiCl法 

分 类 号：S791.18[农业科学—林木遗传育种]