成熟油菜次生休眠种子RNA的快速高效提取方法  

A fast and efficient method for extraction of RNA from secondary dormant seeds of mature rapeseed

作　　者：赵祥祥[1,2] 刘福霞[1] 唐瑭[1] 赵祥强[3] 王兴龙[3] 卢长明[2] 

机构地区：[1]淮阴师范学院江苏省环洪泽湖生态农业生物技术重点实验室,江苏淮安223300 [2]中国农业科学院油料作物研究所/基因工程与转基因安全研究室,湖北武汉430062 [3]南通大学生命科学学院,江苏南通226019

出　　处：《扬州大学学报（农业与生命科学版）》2013年第1期64-67,共4页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(30900217);江苏省自然科学基金资助项目(BK2011410);江苏省"333高层次人才培养工程"培养对象;江苏省"青蓝工程"中青年学术带头人培养对象资助项目

摘　　要：针对成熟油菜次生休眠种子富含多糖和多酚、老健组织部分富含RNA酶、生理活性低等特点,通过选用RNAplant plus reagent植物总RNA提取试剂,添加β-巯基乙醇抑制RNA酶活性、防止酚类物质氧化,应用醋酸钠去除多糖类物质对RNA的影响,以及缩短抽提时间等措施,建立成熟油菜次生休眠种子RNA的快速高效提取方法。结果表明:采用本方法提取的RNA D260nm/D280nm为1.92～2.05,28SrRNA和18SrRNA条带清晰而完整,且无明显降解,可以应用于后续的mRNA分离、建库和高通量转录组测序。Isolation of high quality RNA is an essential prerequisite for gene expression analysis on rape seed develop- ment mechanism. The secondary dormant seeds of mature rapeseed were rich in polysaccharids and polyphenols, while the old tissues had high Rnase activity and low physiological activity. According to these characteristics, we developed a rapid and simple method for the isolation of total RNA through choosing RNA plant plus reagent DP437 （TIANGEN） and tak- ing some measures such as adding β-mercaptoethanol, NaAc and reducing the extraction times to inhibit RNA enzyme ac- tivity, preventing oxidation of phenolic compounds and removing the effects of polysaccharide substance respectively. Two clear bands of 28S rRNA and 18S rRNA isolated from rapeseeds could be detected by agarose gel electrophoresis. RNA extracted by this method was intact without degradation. The D260 nm/D280 nm absorbance ratio was 1. 92 -- 2.05. These results showed that the RNA obtained from rapeseed with this method had high quality and were suitable for the subsequent molecular studies, including mRNA separation, standard mRNA library constructing and high-throughput RNA-seg etc. At the same time this method would provide technical guarantee for exploring the molecular mechanism of secondary dormancy in rapeseed, and also could be used for isolation of total RNA from mature dormant seeds rich in pol- ysaccharides and polyphenols of other plants.

关 键 词：油菜 种子 次生休眠 RNA提取 

分 类 号：S634.3[农业科学—蔬菜学]