一种大量提取红豆杉中总RNA的方法研究  

A Method for Extracting Total RNA of Taxus chinensis at Large-scale

作　　者：陈小凤 曾奇峰 邓旭 曾光尧 周应军 

机构地区：[1]中南大学湘雅药学院,长沙410013

出　　处：《生物技术通报》2017年第11期60-66,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(81573314)

摘　　要：旨在确定高质量红豆杉中总RNA的提取和纯化工艺条件。以湖南长沙红豆杉叶为材料,以Trizol和CATB两种植物RNA通用提取方法为基础,以RNA的浓度、OD_(260)/OD_(280)及OD_(260)/OD_(230)的比值为标准,考察提取方法、提取试剂、提取时间等因素对RNA提取率的影响,优化了红豆杉叶中总RNA的提取方法;通过对比PVP法、β-巯基乙醇法、高盐沉淀法、低浓度乙醇沉淀法、凝胶柱层析法五种不同纯化方法来探讨对红豆杉RNA的影响。考虑到成本的问题,选择使用Trizol法提取红豆杉总RNA,确定了较优的提取条件:红豆杉叶与Trizol提取液的作用体积确定为3∶40;裂解时间确定为5 min;氯仿与水液的作用体积确定为1∶5。实验表明凝胶柱层析法能显著的提高红豆杉总RNA的纯度并能得到富含mi RNA的小分子RNA。最终可得到浓度约为1 000μg/m L,OD_(260/)OD_(280)在2.00-2.20之间及OD_(260/)OD_(230)大于1.7的高质量RNA样品。与现有的提取方法相比,本方法提取的红豆杉总RNA具有很高的质量和纯度,且大大降低了提取成本,并首次实现了植物总RNA的大量提取。This study was directed to develop the optimal method for the extraction and purification of total RNA in Taxus chinensis R.First,we investigated the effects of extraction reagent,time,and method on the yield of total RNA from T.chinensis R.using Trizol and CATB extraction methods in respect of RNA concentration and the ratios of OD_（260）/OD_（280） and OD_（260）/OD_（230）.Furthermore,we compared the effects of PVP,β-mercaptoethanol,high salt precipitation,low concentration ethanol precipitation and gel column chromatography on the purification of total RNA.The optimal extraction method was determined as follows：treatment of taxus leaves with Trizol by the action volume of 3：40 in5 minutes using chloroform and water（1：5）as solvent.It’s also found that column chromatography significantly improved the purity of total RNA of taxus and obtained small molecular RNA with rich mi RNA.Finally,the high-quality RNA in concentration of 1000μg/m L with 2.00-2.20of OD_（260）/OD_（280） and 1.7 of OD_（260）/OD_（230 ）was acquired.Compared with previous methods,this method provides taxus RNA in large-scale with high quality and purity as well as with low cost,which guarantees sufficient materials.

关 键 词：红豆杉 总RNA提取 TRIZOL法 CATB法 凝胶柱层析法 

分 类 号：Q943.2[生物学—植物学]