Zhou Bujin, Chen Peng, Khan Aziz, Zhao Yanhong, Chen Lihong, Liu Dongmei, Liao Xiaofang, Kong Xiangjun, Zhou Ruiyang
Key Laboratory of Plant Genetics and Breeding, College of Agriculture, Guangxi UniversityNanning, China.
Cash Crop Institute of Guangxi Academy of Agricultural SciencesNanning, China.
Front Plant Sci. 2017 Sep 1;8:1520. doi: 10.3389/fpls.2017.01520. eCollection 2017.
Cytoplasmic male sterility (CMS) is a maternally inherited trait that results in the production of dysfunctional pollen. Based on reliable reference gene-normalized real-time quantitative PCR (RT-qPCR) data, examining gene expression profile can provide valuable information on the molecular mechanism of kenaf CMS. However, studies have not been conducted regarding selection of reference genes for normalizing RT-qPCR data in the CMS and maintainer lines of kenaf crop. Therefore, we studied 10 candidate reference genes (, and ) to assess their expression stability at three stages of pollen development in CMS line 722A and maintainer line 722B of kenaf. Five computational statistical approaches (GeNorm, NormFinder, ΔCt, BestKeeper, and RefFinder) were used to evaluate the expression stability levels of these genes. According to RefFinder and GeNorm, the combination of , and was identified as an internal control for the accurate normalization across all sample set, which was further confirmed by validating the expression of . Furthermore, the combination of , and was used to differentiate the expression pattern of five mitochondria FF-ATPase subunit genes (, and ) by RT-qPCR during pollen development in CMS line 722A and maintainer line 722B. We found that , and exhibited significantly different expression patterns during pollen development in line 722A compared with line 722B. This is the first systematic study of reference genes selection for CMS and will provide useful information for future research on the gene expressions and molecular mechanisms underlying CMS in kenaf.
细胞质雄性不育（CMS）是一种母系遗传性状，会导致产生功能失调的花粉。基于可靠的参考基因标准化实时定量PCR（RT-qPCR）数据，检测基因表达谱可为红麻CMS的分子机制提供有价值的信息。然而，尚未针对红麻作物CMS系和保持系中用于标准化RT-qPCR数据的参考基因选择进行研究。因此，我们研究了10个候选参考基因（……），以评估它们在红麻CMS系722A和保持系722B花粉发育三个阶段的表达稳定性。使用了五种计算统计方法（GeNorm、NormFinder、ΔCt、BestKeeper和RefFinder）来评估这些基因的表达稳定性水平。根据RefFinder和GeNorm，……的组合被确定为所有样本集准确定量的内参，通过验证……的表达进一步证实了这一点。此外，……的组合用于通过RT-qPCR区分CMS系722A和保持系722B花粉发育过程中五个线粒体FF-ATPase亚基基因（……）的表达模式。我们发现，……在722A系与722B系花粉发育过程中表现出明显不同的表达模式。这是首次对CMS参考基因选择进行系统研究，将为今后红麻CMS基因表达及分子机制的研究提供有用信息。
