Flower Research Institute of Yunnan Academy of Agricultural Sciences, Kunming 650205, China.
Fujian Forestry Science and Technology Experimental Center, Zhangzhou 363600, China.
Genes (Basel). 2023 May 19;14(5):1112. doi: 10.3390/genes14051112.
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 , 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 (), polyubiquitin (), glyceraldehyde-3-phosphate dehydrogenase (), elongation factor 1-α (), β-tubulin (), α-tubulin (), 60S ribosomal protein L13-1 (), aquaporin PIP1-2 (), Intima protein () and Cyclin () 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, exhibited the highest stability and was selected as the most reliable. The reliability and accuracy of were confirmed through qRT-PCR analysis of fifteen chlorophyll pathway-related genes. The expression patterns of these genes via normalization were consistent with the results by RNA-Seq. 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 .
叶色突变体（LCMs）是研究多种代谢过程的重要资源，如叶绿体发生和分化、色素的生物合成和积累以及光合作用。然而，在 ，由于缺乏用于 qRT-PCR（定量实时逆转录 PCR）归一化的可靠 RG（参考基因），LCM 尚未得到充分研究和利用。因此，本研究利用先前发布的转录组数据，选择并评估了十个候选 RG 的适用性，包括肌动蛋白（）、多聚泛素（）、甘油醛-3-磷酸脱氢酶（）、延伸因子 1-α（）、β-微管蛋白（）、α-微管蛋白（）、60S 核糖体蛋白 L13-1（）、水通道蛋白 PIP1-2（）、内膜蛋白（）和细胞周期蛋白（），通过 qRT-PCR 来归一化与叶色相关基因的表达水平。通过常用软件 Best-Keeper、GeNorm 和 NormFinder 进行稳定性排名分析表明，这十个基因均符合 RG 的要求。其中，表现出最高的稳定性，被选为最可靠的 RG。通过对 15 个叶绿素途径相关基因的 qRT-PCR 分析，验证了 的可靠性和准确性。通过 归一化的这些基因的表达模式与 RNA-Seq 的结果一致。我们的结果为与叶色相关基因的功能特征提供了关键的遗传资源，并为 在分子水平上解析叶色突变铺平了道路。
