Screening and Validation of Internal Reference Genes for Quantitative Real-Time PCR Analysis of Leaf Color Mutants in Dendrobium officinale

文献类型： 外文期刊

第一作者： Cao, Hua

作者： Cao, Hua;Li, Han;Lu, Lin;Ma, Lulin;Ji, Yulu;Li, Shenchong

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关键词： Dendrobium officinale; leaf color; qRT-PCR; reference gene; leaf color mutant

期刊名称：GENES （ 影响因子：3.5； 五年影响因子：3.9 ）

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年卷期： 2023 年 14 卷 5 期

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收录情况： SCI

摘要： Leaf color mutants (LCMs) are important resources for studying diverse metabolic processes such as chloroplast biogenesis and differentiation, pigments' biosynthesis and accumulation, and photosynthesis. However, in Dendrobium officinale, LCMs are yet to be fully studied and exploited due to the unavailability of reliable RGs (reference genes) for qRT-PCR (quantitative real-time reverse transcription PCR) normalization. Hence, this study took advantage of previously released transcriptome data to select and evaluate the suitability of ten candidate RGs, including Actin (Actin), polyubiquitin (UBQ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha(EF1 alpha), beta-tubulin (beta-TUB), alpha-tubulin (alpha-TUB), 60S ribosomal protein L13-1 (RPL13AD), aquaporin PIP1-2 (PIP1-2), Intima protein (ALB3) and Cyclin (CYCB1-2) for normalizing leaf color-related genes' expression levels via qRT-PCR. Stability rankings analysis via common software Best-Keeper, GeNorm, and NormFinder disclosed that all ten genes met the requirements of RGs. Of them, EF1 alpha exhibited the highest stability and was selected as the most reliable. The reliability and accuracy of EF1 alpha were confirmed through qRT-PCR analysis of fifteen chlorophyll pathway-related genes. The expression patterns of these genes via EF1 alpha normalization were consistent with the results by RNASeq. Our results offer key genetic resources for the functional characterization of leaf color-related genes and will pave the way for molecular dissection of leaf color mutations in D. officinale.

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