Screening and Verification of Reference Genes for Real-time Fluorescence Quantitative PCR in Cornus hongkongensis subsp. tonkinensis Under Salt Stress
SUN Da-Wei1,2, YUAN Jia-Qiu1,2, CAI Mei1,2, FU Xiang-Xiang1,2*
1 College of Forestry, Nanjing Forestry University, Nanjing 210037, China; 2 Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
Abstract：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-1α 1 (EF-1-α1), EF-1-α2, adenine phosphoribosyltransferase (APT), cycloidea1 (cyc1), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), GAPDH2 and β -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.
孙大伟, 袁佳秋, 蔡梅, 洑香香. 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[J]. 农业生物技术学报, 2023, 31(5): 1088-1099.
SUN Da-Wei, YUAN Jia-Qiu, CAI Mei, FU Xiang-Xiang. Screening and Verification of Reference Genes for Real-time Fluorescence Quantitative PCR in Cornus hongkongensis subsp. tonkinensis Under Salt Stress. 农业生物技术学报, 2023, 31(5): 1088-1099.
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