茶愈伤组织实时定量PCR分析中内参基因选取.PDF

安徽农业大学学报, 2014, 41(6): 905-910 Journal of Anhui Agricultural University [DOI] 10.13610/ki.1672-352x021 网络出版时间：2014-10-29 10:59:46 [URL] /kcms/doi/10.13610/ki.1672-352x021.html 茶愈伤组织实时定量 PCR 分析中内参基因的选取 史成颖，李正国，徐 乾，宛晓春* (安徽农业大学农业部、教育部茶叶生物化学和生物技术重点实验室，合肥 230036) 摘 要：为了选取合适的内参基因来分析培养基中前体物诱导及对照的茶愈伤组织中茶氨酸代谢相关基因的差 异表达，利用GenBank 上登录的茶树基因序列以及通过构建文库测序所得的基因（具有完整的阅读框）共7 个持 家基因设计引物。在分析这些引物的扩增效率和特异性后，测定了它们在茶愈伤组织生长过程中（对照和愈伤培养 基中添加含氮外源物的情况下）的表达水平。利用geNorm 和NormFinder 软件分析了这些持家基因的稳定性，确 定在该条件下合适的内参基因为β-actin 和GAPDH。 关键词：实时定量PCR；内参基因；基因表达；茶树 中图分类号：S571.1 文献标识码：A 文章编号：1672−352X (2014)06−0905−06 Reference gene selection for real-time PCR normalization in tea callus SHI Chengying, LI Zhengguo, XU Qian, WAN Xiaochun (Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Agriculture and Ministry of Education, Anhui Agricultural University, Hefei 230036) Abstract: In order to obtain suitable reference genes for analysis of the related gene expression in calli of the control and treatment groups, 7 housekeeping genes from the GenBank and ESTs of cDNA library of Camellia sinensis young roots （with complete open reading frame ）were chosen for primer design. After analysis the am- plification efficiency and primers specificity, the expression levels of the 7 housekeeping genes in tea calli of the control and treatment groups at different growth stages were determined, and their stabilities of expressions were analyzed using geNorm and NormFinder softwares. As a result, β-actin and GAPDH are suitable reference genes under this condition. Key words: real -time PCR; reference gene; gene expression; Camellia sinensis 研究植物代谢途径及其机理关键在于研究代谢 法如 Northern blot 、半定量RT-PCR 检测技术相比， 途径中的相关基因表达量与代谢产物合成的关系。