油茶总RNA的提取与内参基因的初选

摘要

为得到高质量的油茶总RNA及稳定的内参基因，以油茶良种‘华硕’为试验材料，采用ambion试剂盒结合CTAB法进行总RNA提取。获得的总RNA完整性好，纯度高；从前期构建的油茶转录组数据中选取ACT（肌动蛋白基因）等14个基因作为候选内参基因，共设计22对引物，通过RT-PCR反应体系的优化及琼脂糖凝胶电泳检测，快速筛选出了适合油茶不同组织（器官）的内参基因。ALB（清蛋白基因），EF1α（延伸因子基因），ETIF3H（启动因子基因），UBC2（泛素结合酶基因）可以作为油茶果实初选内参基因，EF1α可以作为油茶根、茎、花及果实内参基因，ETIF3H可以作为油茶根、茎、叶、花瓣的内参基因。%In order to obtain high quality total RNA and stable reference genes in Camellia oleifera, taking ifne cultivar‘Huashuo’ as materials, total RNA was extracted by a combination of ambion kit and CTAB method. The obtained total RNA has good integrity and high purity. A total of 14 genes were selected as candidates of reference genes based on the previously constructed transcriptome data in C. oleifera, and 22 primers were designed for the 14 genes. The reference genes suitable for different tissues or organs were rapidly screened out by RT-PCR reaction system optimization and agarose gel electrophoresis detection. ALB (albumin gene), EF1α (elongation factor gene), ETIF3H (eukaryotic translation initiation gene), UBC2 (ubiquitin-conjugating enzyme gene) can be used as the reference genes for fruits, EF1αcan be used as a reference gene for root, stem, lfower and fruit, and ETIF3H can be used as a reliable reference gene for root, stem, leaf and fruit in C. oleifera.