两种铁皮石斛总RNA提取方法的比较研究  

Comparison on Two Modified Total RNA Isolating Methods from Dendrobium officinale

作　　者：容天聚[1] 文国松[1] 钱雄[1] 查应洪 徐绍忠[1] 

机构地区：[1]云南农业大学云南省中药材规范化种植技术指导中心,云南昆明650201 [2]红河群鑫石斛种植有限公司,云南屏边661200

出　　处：《云南农业大学学报（自然科学版）》2012年第5期703-707,共5页Journal of Yunnan Agricultural University:Natural Science

基　　金：云南省重大产业项目资助项目[云发改高技(2007)1718号];国家支撑计划资助项目(2011BAI13B02-5)

摘　　要：铁皮石斛是我国重点保护药材,因其富含多糖及次生代谢物,用普通的方法难以从叶中提取到能满足构建全长cDNA文库等分子生物学实验对高质量RNA需要的总RNA。分别通过对异硫氰酸胍法和CTAB法进行改良。异硫氰酸胍法:往裂解液中加入PVP,通过PVP去除多酚;用56℃热水水浴5min加速组织细胞裂解;抽提时间由原来15 min缩短为5min;根据材料多糖多酚的多寡情况适当增加裂解液。CTAB法:根据材料多糖多酚的多寡情况适当增加CTAB裂解液;用LiCl对RNA进行选择性过夜沉淀;灵活处理,适当增大LiCl浓度。结果表明用改良后的2种方法均可提取到总RNA,经对比发现,改良CTAB法提取效果更好,可重复性强,抽取到的RNA更适合于后续的分子生物学实验。Dendrobium officinale Kimura et Migor is an important Chinese medicinal material, in whichpolysaccharide and polyphenol is rich, however, it is difficult to isolate RNA from its leaf. We modified both the GTC method and CTAB method. Guanidine thiocyanate method： PVP was added to theGTC buffer to remove polyphenol; the plant tissue was decomposed quickly with 56℃ heated water; the extracting time was shortened from 15min to 5min to decrease the loss of RNA ; regular doze of GTCbuffer was wisely added according to the the amount of polysaccharide and polyphenol. CTAB method ： adequate doze of CTAB buffer was added according to the amount of polysaccharide and polyphenol ofthe extracting material; the RNA was precipitated overnight with LiC1, which RNA but not DNA could be precipitated, and a certain doze of LiC1 was added wisely would be better. The result showed thatRNA could be extracted by the two modified GTC method and CTAB method. Moreover, it is evident that the latter is more suitable for biomolecular research, of which RNA is purer and intact.

关 键 词：铁皮石斛 多糖 总RNA提取 异硫氰酸胍法 CTAB法 

分 类 号：S567.23[农业科学—中草药栽培]