CAJ | 학술논문

为确立大豆叶、花组织提取总RNA的最佳方法,比较了植物总RNA提取试盒剂法、改良的CTAB-LiCL法和改良的热硼酸法的提取效果.通过RNA产量、纯度、凝胶电泳及后续的基因克隆和荧光定量PCR等分析,结果表明:热硼酸法所提取的叶、花总RNA含量约为其他两种方法的6倍;总RNA OD260/OD280值介于1.98~2.1;电泳条带完整清晰;应用获得的总RNA所克隆ZAT9-like基因,经测序获得885 bp的核酸序列与ZAT9-like (登录号:XM_003517371.1)基因同源率达99%;荧光定量分析Histone H3基因的扩增曲线与融解曲线峰型良好.说明该方法能够满足一般分子生物学下游实验的要求,是一种理想的针对大豆叶、花总RNA的的提取方法.
위학립대두협、화조직제취총RNA적최가방법,비교료식물총RNA제취시합제법、개량적CTAB-LiCL법화개량적열붕산법적제취효과.통과RNA산량、순도、응효전영급후속적기인극륭화형광정량PCR등분석,결과표명:열붕산법소제취적협、화총RNA함량약위기타량충방법적6배;총RNA OD260/OD280치개우1.98~2.1;전영조대완정청석;응용획득적총RNA소극륭ZAT9-like기인,경측서획득885 bp적핵산서렬여ZAT9-like (등록호:XM_003517371.1)기인동원솔체99%;형광정량분석Histone H3기인적확증곡선여융해곡선봉형량호.설명해방법능구만족일반분자생물학하유실험적요구,시일충이상적침대대두협、화총RNA적적제취방법.
@@@@In order to find out the optimal method for extracting total RNA from leaves and flowers in soybean (Glycine max), extracting efficiency was compared among three methods of RNA extraction, RNApure plant kit, modified CTAB and hot borate. The yield and purity of total RNA were determined by nucleic acid and protein detector; integrity and quality of total RNA were analyzed by agrose gel electrophoresis, gene cloning and real-time PCR. The results showed that the yield of total RNA by using hot borate was six times that by the other methods (RNApure plant kit and modified CTAB). The value of OD260/OD280 was between 1.98 to 2.01;The electr-ophoresis result of RNA showed satisfactory bands with sharpness and integrity. ZAT9-like gene was obtained by cloning from the total RNA, sequenced to the nucleic acid sequence of 885 bp, showing up to 99%homology to ZAT9-like gene. In real-time PCR amplification, curve peak type and the melting curve peak type were better with RNA extracted with hot borate than the other methods. The quality of the RNA by hot borate was of high quality for gene cloning, real-time PCR and the other downstream application in molecular biology.