东京四照花盐胁迫响应下实时荧光定量PCR内参基因的筛选与验证  

Screening and Verification of Reference Genes for Real-time Fluorescence Quantitative PCR in Cornus hongkongensis subsp.tonkinensis Under Salt Stress

作　　者：孙大伟 袁佳秋 蔡梅 洑香香 SUN Da-Wei;YUAN Jia-Qiu;CAI Mei;FU Xiang-Xiang(College of Forestry,Nanjing Forestry University,Nanjing 210037,China;Co-Innovation Center for Sustainable Forestry in Southern China,Nanjing Forestry University,Nanjing 210037,China)

机构地区：[1]南京林业大学林学院,南京210037 [2]南京林业大学南方现代林业协同创新中心,南京210037

出　　处：《农业生物技术学报》2023年第5期1088-1099,共12页Journal of Agricultural Biotechnology

基　　金：江苏省林业科技创新与推广项目(LYKJ[2018]06)。

摘　　要：利用实时荧光定量PCR技术(real-time fluorescence quantitative PCR,qRT-PCR)分析基因的表达时,内参基因的选择对保证研究结果的准确性至关重要。本研究以东京四照花(Cornus hongkongensis subsp.tonkinensis)经过不同盐胁迫时间的根与叶为材料,基于转录组数据,选择9个候选内参基因:18S核糖体RNA(18S ribosome RNA,18S rRNA)、翻译起始因子2(initiation factor 2,IF2)、转录延伸因子1(elongation factor-1α1,EF-1-α1)、EF-1-α2、腺嘌呤磷酸核糖转移酶(adenine phosphoribosyltransferase,APT)、亲环蛋白1(cycloidea1,cyc1)、3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、GAPDH2和β微管蛋白(β-tubulin,TUB)基因进行qRT-PCR扩增。运用GeNorm、NormFinder和BestKeeper 3种软件对9个候选内参基因进行表达稳定性分析,最后用RefFinder程序综合分析、得出最适的内参基因,并利用目的基因超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)基因进行验证。实验结果表明,18S rRNA和GAPDH为盐胁迫下东京四照花各组织中表达最稳定的基因,EF-1-α2是最不稳定的基因。但18S rRNA的Ct值较高,意味着18S rRNA在各个组织中的表达量比GAPDH低。验证结果也表明以GAPDH为内参基因时,目的基因相对表达量与转录组测序结果较为一致。因此认为GAPDH为盐胁迫下东京四照花荧光定量PCR基因分析的最适内参基因。本研究可为后期研究东京四照花相关耐盐基因表达及功能提供参考。For analyzing the expression of genes accurately by quantitative real-time PCR(qRT-PCR),screening of stabilized reference genes is of great important.Based on transcriptome data,9 candidate genes including 18S ribosome RNA(18S rRNA),initiation factor 2(IF2),elongation factor-1α1(EF-1-α1),EF-1-α2,adenine phosphoribosyltransferase(APT),cycloidea1(cyc1),glyceraldehyde-3-phosphatedehydrogenase(GAPDH),GAPDH2 andβ-tubulin(TUB)were amplified by qRT-PCR in leaves and roots of Cornus hongkongensis subsp.tonkinensis suffering from different salt stress time.The expression stability of 9 candidate genes were measured by GeNorm,NormFinder and BestKeeper.Suitable reference gene was finally screened based on comprehensive evaluation of expression stability with RefFinder.Then the selected reference genes were verified by target genes superoxide dismutase(SOD)and catalase(CAT).The results showed that 18S rRNA and GAPDH were the optimal candidate reference genes in various organs under salt stress,whereas EF-1-α2 was the least stable gene.However,the Ct value of 18S rRNA was higher,indicated that the expression of 18S rRNA was lower than that of GAPDH in various organs.The results also showed that the relative expression levels of target genes were consistent with the transcriptome sequencing results when using GAPDH as reference gene.This study determined that the most suitable reference gene was GAPDH for qRT-PCR,which could provide the reference for further study on the expression of related gene in Cornus hongkongensis subsp.tonkinensis under salt stress.

关 键 词：东京四照花 实时荧光定量PCR 内参基因 

分 类 号：S722[农业科学—林木遗传育种]