成熟葡萄果实高质量总RNA提取方法的建立  

Establishment of Extraction Methods for High Quality RNA from Mature Grape Berry

作　　者：闫冬梅 薛美昭 仪慧兰[1] 郝丽宏 乔嘉欣 YAN Dongmei;XUE Meizhao;YI Huilan;HAO Lihong;QIAO Jiaxin(College of Life Science,Shanxi University,Taiyuan 030006,China;College of Environmental and Resource Sciences,Shanxi University,Taiyuan 030006,China;College of Traditional Chinese Medicine and Food Engineering,Shanxi University of Chinese Medicine,Jinzhong 030619,China)

机构地区：[1]山西大学生命科学学院,山西太原030006 [2]山西大学环境与资源学院,山西太原030006 [3]山西中医药大学中药与食品工程学院,山西晋中030619

出　　处：《山西农业科学》2022年第1期9-14,共6页Journal of Shanxi Agricultural Sciences

基　　金：国家自然科学基金面上项目(31972132,31371868);山西中医药大学博士科研启动基金项目(2020BK06);山西省应用基础研究计划青年项目(20210302124151)。

摘　　要：成熟葡萄果实中富含多酚多糖类物质,对提取组织RNA造成了很大的干扰。研究分别采用3种试剂盒法和改良的十六烷基三甲基溴化铵(CTAB)法,以成熟的葡萄果实为材料,提取总RNA并检验其产量和质量,其中,改良CTAB法是在提取缓冲液中加入了聚乙烯吡咯烷酮、亚精胺、β-巯基乙醇及高浓度的氯化钠。对各组RNA产物进行质量检测,发现3种试剂盒法均没有得到可用的RNA,只有改良的CTAB法能够从新鲜采摘和贮藏的葡萄果实组织中提取出高质量的总RNA,基本无降解和污染,且得率可达到85μg/g以上。进一步研究表明,改良的CTAB法提取得到的RNA可用于后续的RT-PCR、qRT-PCR和CDS扩增试验。改良CTAB法适用于成熟葡萄果实RNA的提取,所得RNA质量良好,满足后续分子试验的需求。Mature grape berries are rich in polyphenols and polysaccharides that interfere with RNA extraction. In this study,we tested the efficiency of three different commercial kits and an in-house extraction protocol using improved cetyltrimethylammonium bromide(CTAB)method to extract total RNA from mature grape berries and test the yield and quality of RNA. The improved CTAB method was carried out by adding certain amounts of polyvinylpyrrolidone, spermidine, β-mercaptoethanol and high-dose of sodium chloride into the extraction buffer, and adjusting the related process. The experimental results showed that the improved CTAB method could extract high quality total RNA from fresh picked and stored grape berry without degradation and contamination, and the RNA yield could reach 85 μg/g. However, the three commercial kits were not efficient to obtain enough higher quality total RNA. In addition, it was clarified in the further studies that the total RNA extracted by the improved CTAB method was suitable for the following RT-PCR, qRT-PCR and CDS amplification experiments. The improved CTAB method is suitable for RNA extraction from mature grape berry, and the quality of RNA can meet the needs of subsequent molecular experiments.

关 键 词：成熟葡萄 改良CTAB法 多糖 多酚 RNA提取 

分 类 号：S663.1[农业科学—果树学]