锥栗总RNA提取方法比较及LFY基因克隆

Method of Total RNA Extraction and Cloning of LFY Gene in Castanea henryi

锥栗总RNA的提取质量是后期锥栗空棚分子机理研究的关键。以锥栗花序为材料,采用2种植物RNA快速提取试剂盒、改良Trizol法和改良CTAB法提取锥栗总RNA,并采用多种方法检测提取出的总RNA的质量。结果表明:采用改良CTAB法获得的总RNA可明显看到28S和18S条带,且亮度比约为2∶1,DNA基本无残留,点样孔无杂质,OD260/OD280均接近2,而OD260/OD230均超过2,经逆转录可作为模板,获得LFY基因。改良后CTAB法最合适,所得的总RNA产量高、比较纯净、完整,可用于后续的逆转录并进行基因片段的扩增。

The quality of extracted total RNA is the key to study the molecular mechanism of chinquapin later.In this study,chinquapin inflorescences were taken as the material.The study used two types of plant RNA Rapid Extraction kit,a modified trizol method and a improved CTAB method to extract the chinquapin total RNA,and used some methods to test the quality of the RNA.The total RNA obtained by improved CTAB method could significantly observe 28S and 18S bands and the brightness ratio was about 2：1,DNA hardly leave,spotting holes without impurities,OD260/OD280 were close to 2,while OD260/OD230＞2.The RNA can be used as a template after reverse transcription to obtain LFY gene.The improved CTAB method was most appropriate,the total RNA had high yield and relatively pure,complete and could be used for subsequent reverse transcription reaction to clone.