钩藤实时荧光定量PCR分析中内参基因的筛选及稳定性评价  

Screening and Stability Evaluation of Reference Genes in Uncaria rhynchophylla qRT-PCR Analysis

作　　者：于晓松 王晓红[1] 李雪[1] 贾娜 上官黎阳 强玮 张明生[1] YU Xiao-Song;WANG Xiao-Hong;LI Xue;JIA Na;SHANGGUAN Li-Yang;QIANG Wei;ZHANG Ming-Sheng(Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education)/College of Life Sciences,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学生命科学学院/山地植物资源保护与种质创新教育部重点实验室,贵阳550025

出　　处：《农业生物技术学报》2021年第3期599-609,共11页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划(2016YFC0502604);贵州省科技计划重大专项(黔科合平台人才[2017]5411-06;黔科合平台人才[2017]5788);贵州省高层次创新型人才培养计划(黔科合人才[2015]4031);国家喀斯特石漠化防治工程技术研究中心建设项目(2012FU125X13);贵州省中药材现代产业技术体系建设项目(GZCYTX-02);贵州省生物学一流学科建设项目(GNYL[2017]009FX1KT02)。

摘　　要：钩藤(Uncaria rhynchophylla)的药用成分钩藤碱和异钩藤碱受各种酶促反应基因调控,筛选钩藤qRT-PCR实验体系中的最佳内参基因,对影响钩藤药用成分基因的表达研究具有重要意义。本研究旨在筛选钩藤不同组织、不同遮光处理和不同乙烯处理条件下稳定表达的内参基因。以钩藤为材料,应用qRT-PCR分析β-肌动蛋白(β-actin,β-act)、α-微管蛋白(α-tubulin,α-tub)、18S核糖体RNA (18S ribosomal RNA, 18S rRNA)、翻译延伸因子(translation elongation factor, ef-1)、甘油醛三磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, gapdh)、β-微管蛋白(β-tubulin,β-tub)、核糖体蛋白(ribosomal protein, rpl)和泛素连接酶(ubiquitin-ligase enzyme, ubc)共计8个内参基因在不同组织和不同处理下的表达变化,借助GeNorm程序确定内参基因数目,再结合NormFinder和BestKeeper程序对候选内参基因稳定性进行评价,随后通过几何平均值法对不同软件所得结果进行整合并明确综合排名。结果显示,在不同组织和处理中,皆可选用单一内参基因;在不同组织中,应选择ef-1作为内参基因;在遮光处理下,应选择gapdh作为内参基因;在乙烯处理下,ubc和β-act皆可作为内参基因;如果多种表达研究固定采用一个稳定性相对较好的内参基因,应当选择ubc。本研究为钩藤在不同实验体系中的相关基因表达分析提供了校正和标准化基因,有助于提高实验的准确性和可靠性。The medicinal components of Uncaria rhynchophylla, rhynchophylline and isorhynchophylline, are regulated by various enzymatic reaction genes. Screening the best internal reference genes in the qRT-PCR system of U. rhynchophylla is of great significance for the study of gene expression of medicinal components in U. rhynchophylla. The present study aimed to select reference genes stably expressed in different tissues,different shading treatments and different ethylene treatments in U. rhynchophylla. U. rhynchophylla was selected as the material, and the expression of 8 reference genes of β-actin(β-act), α-tubulin(α-tub), 18 S ribosomal RNA(18 S rRNA), translation elongation factor(ef-1), glyceraldehyde-3-phosphate dehydrogenase(gapdh), β-tubulin(β-tub), ribosomal protein(rpl) and ubiquitin-ligase enzyme(ubc) in different tissues and different treatments were analyzed by qRT-PCR. The GeNorm program was used to determine the number of reference genes, and then NormFinder and BestKeeper programs were combined to evaluate the stability of the reference genes. Finally, the geometric mean method was used to integrate the results obtained from different software and obtained comprehensive ranking. The results showed that single reference gene could be used in different tissues and treatments. In different tissues, ef-1 should be selected as the reference gene;under shading treatments, gapdh should be selected as the reference gene;under ethylene treatments, both ubc and β-act could be used as the reference gene. If a relatively stable reference gene is used in multiple expression studies, ubc should be selected. This study provides correction and standardization genes for the expression analysis of related genes in different experimental systems of U. rhynchophylla, and is helpful to improve the accuracy and reliability of the experiments.

关 键 词：钩藤 内参基因 实时荧光定量PCR 稳定性 

分 类 号：S-3[农业科学] Q94-336[生物学—植物学]