适用于转录组测序的毛葡萄叶片总RNA提取方法筛选  

Screening of Total RNA Extraction Method of Vitis quinquangularis Leaves Suitable for Transcriptome Sequencing

作　　者：张树伟[1,3] 丁峰 黄羽[2] 彭宏祥[3] 卢江[1] Zhang Shuwei;Ding Feng;Huang Yu;Peng Hongxiang;Lu Jiang(Guangxi Crop Genetic Improvement and Biotechnology Laboratory,Guangxi Academy of Agricultural Sciences,Nanning,530007;Grape and wine research istitute Guangxi Academy of Agriculural Sciences,Nanning.530007;Horicultural Research Istitute,Guangxi Academy of Agricultural Sciences,Nanning 530007)

机构地区：[1]广西农业科学院,广西作物遗传改良生物技术重点开放实验室,南宁530007 [2]广西农业科学院葡萄与葡萄酒研究所,南宁530007 [3]广西农业科学院园艺研究所,南宁530007

出　　处：《分子植物育种》2020年第11期3593-3597,共5页Molecular Plant Breeding

基　　金：广西农业科学院博士后基金项目(桂农科博2014022);广西自然科学基金项目(2015GXNSFBA139099);中国博士后科学基金项目(2015M582745XB);广西作物遗传改良生物技术重点开放实验室建设项目开放课题(15-140-33-1);国家科技支撑计划课题(2014BAD16B05);广西农科院园艺所科技基金(001001026)共同资助。

摘　　要：毛葡萄叶片中含有大量多糖、多酚等多种次生代谢物,严重影响RNA提取质量,筛选从毛葡萄叶片中提取高质量RNA的方法是开展相关分子研究的基础。本研究采用改良CTAB法、改良SDS/酚法和三种试剂盒法,筛选适合转录组测序的提取毛葡萄叶片总RNA的方法。结果显示,五种方法均可以获得毛葡萄叶片总RNA,但在纯度和完整性上差别较大。改良CTAB和改良SDS/酚法提取的总RNA OD260/OD230比值小于1.8,试剂盒A法提取的总RNA在纯度和完整性上均可以满足转录组测序要求,试剂盒B法和试剂盒C法提取的总RNA降解严重。相比其他四种方法,试剂盒A法提取的毛葡萄叶总RNA质量更好,在纯度和完整性上均能满足转录组测序及其他分子生物学实验的要求。Leaves of Vitis quinquangularis were rich in polysaccharides,polyphenols and other secondary metabolites,which influenced the quality of total RNA seriously.It was the basic of molecular research to screen extraction methods for high-quality RNA from leaves of Vitis quinquangularis.In this study,modified cetyltrimethylammonium bromide(CTAB),modified SDS/phenol and three commercial kits extraction methods were screened to isolate total RNA for transcriptome sequencing.The results showed that these five methods all could obtain total RNA from leaves of Vitis quinquangularis,but purity and integrity were quite different.OD260/OD230 ratio for RNA samples prepared by improved CTAB and SDS/phenol methods was less than 1.80.The total RNA extracted by kit A method can meet the requirements of transcriptome sequencing in terms of purity and integrity.Total RNA extracted with Kit B and Kit C was seriously degraded.Compared with other four protocols,Kit A method could generate high-quality total RNA which could meet the request of transcriptome sequencing and other molecular biological experiments.

关 键 词：毛葡萄(Vitis quinquangularis) 叶片 RNA提取方法 转录组测序 

分 类 号：S663.1[农业科学—果树学]