| 刘小飞，于 波，黄丽丽，孙映波.杜鹃红山茶实时定量PCR 内参基因筛选及验证[J].广东农业科学,2020,47(12):203-211 |
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杜鹃红山茶实时定量PCR 内参基因筛选及验证 |
| Screening and Validation of Reference Genes of Camellia azalea by Quantitative Real-time PCR |
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| DOI：10.16768/j.issn.1004-874X.2020.12.021 |
| 中文关键词: 实时荧光定量PCR 杜鹃红山茶 内参基因 GeNorm NormFinder |
| 英文关键词: qRT-PCR Camellia azalea reference gene GeNorm NormFinder |
| 基金项目:广东省重点领域研发计划项目（2018B020202002）；广东省科技计划项目（2018A050506054）；广州市科技计划项目（201604020031，201807010016） |
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| 中文摘要: |
| 【目的】实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）以高灵敏度和特异性等优点，成为基因表达分析的主要工具，而选择适合不同条件下工作的内参是qRT-PCR分析的前提。筛选得到适用于杜鹃红山茶不同器官、不同时期花瓣的qRT-PCR分析的内参基因，为后续相关基因功能研究提供可用的内参基因。【方法】选择转录延伸因子编码基因（EF1α）、α-tubulin（TUA）、β-tubulin（TUB）、Ubiquitin（UBQ）、肌动蛋白（Actin）和甘油醛-3-磷酸脱氢酶（Glyceraldehyde-3-phosphate dehydrogenase，GAPDH）6个看家基因，使用2种不同的计算程序GeNorm和NormFinder评估了6个基因表达的稳定性。进一步通过CaGASA3基因的表达模式验证了所选内参基因的适用性。【结果】在不同器官中通过GeNorm和NormFinder筛选出稳定性最好的基因，排名前2位的均为TUA和GAPDH，GAPDH基因在两种计算程序评估中稳定性均最佳。而在不同时期花瓣中，通过2种程序得出排名前2位的内参基因均为TUB和UBQ；UBQ基因在GeNorm程序评估中稳定性最好，TUB基因在NormFinder程序的评估中稳定性最佳。【结论】杜鹃红山茶不同器官中最佳内参基因为TUA和GAPDH，不同时期花瓣中最适内参基因为TUB和UBQ。 |
| 英文摘要: |
| 【Objective】Quantitative real-time PCR（qRT-PCR）has become an main tool for gene expression analysis due to its high sensitivity and specificity, and selecting reference genes suitable for different conditions is the prerequisite for qRT-PCR analysis. The reference genes suitable for qRT-PCR analysis of different organs and petals in different periods of Camellia azalea were screened to provide usable reference genes for subsequent related gene function researches.【Method】In this study, six housekeeping genes were selected, which were the coding genes of transcription elongation factor（EF1α）, α-tubulin（TUA）, β-tubulin（TUB）, Ubiquitin（UBQ）, Actin and glyceraldehyde- 3-phosphate dehydrogenase（GAPDH）, and two different calculation programs GeNorm and NormFinder were used to evaluate the expression stability of 6 genes. The applicability of the selected reference genes were further verified by the expression mode of CaGASA3.【Result】In different organs, GeNorm and NormFinder were used to screen out the most stable genes. The top two were TUA and GAPDH. GAPDH had the best stability in the evaluation of the two calculation programs. In petals of different periods, the top 2 reference genes obtained by the two programs were TUB and UBQ. UBQ had the best stability in the evaluation of GeNorm program, and TUB had the best stability in evaluation of NormFinder program.【Conclusion】The optimal reference genes in different organs of C. azalea were TUA and GAPDH, and the most suitable reference genes in petals of different periods were TUB and UBQ. |
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