核桃芽总RNA提取及RT-PCR

摘要

为了从核桃芽体内提取高质量的RNA,以便进行核桃成花基因克隆研究,针对核桃芽体内富含多酚等次生代谢物的特点,在总结他人研究基础上对SDS法和CTAB法及试剂盒提取法进行了改良和比较。凝胶电泳检测表明：Spectrum Plant Total RNA试剂盒提取法及以pH 9.0硼砂为缓冲液的改良CTAB法和SDS法所提取RNA的28SrRNA和18SrRNA条带清晰,且28SrRNA的亮度约是18SrRNA的2倍,说明所提RNA完整性较好;紫外光谱分析显示,3种方法的OD260/OD280分别为1.99、1.96、1.77,OD260/OD230均〉2.0,RNA产率依次为360、180、75μg/（g.FW）。综合检测结果认为：改良CTAB法和改良SDS法所提RNA的完整性和纯度均能满足基因克隆等的研究,是一种适合核桃芽体内RNA提取的经济而有效的方法。%The aim was to extract high-quality RNA from walnut bud so that the cloning study of walnut flower gene could be carried on.In this experiment the SDS method,CTAB method and isolation kit method were compared and improved based on the conclusion of other research according to the character that polyphenol secondary metabolites were rich in walnut bud.It was the gel electrophoresis that indicated the band of 28SrRNA and 18SrRNA in the RNA extracted by Spectrum Plant Total RNA isolation kit method and CTAB method as well as SDS method improved with borate buffer solution is clear.Also the brightness of 28SrRNA was about twice than the 18SrRNA which indicated that the integrity of the extracted RNA was good.The ultraviolet spectrum analysis showed that the OD260/OD280 of these three methods were respectively 1.99,1.96,1.77,while the OD260/OD230 were all greater than 2.0 and the yield of RNA was in sequence of 360,180 and 75 μg/（g.FW）.According to the comprehensive test results,RT-PCR indicated that the integrity of the RNA extracted by improved CTAB method and improved SDS method could satisfy the studies such as gene cloning.Thus,these two methods were economic and effective methods to extract the RNA from walnut bud.