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Reference Gene Selection of Real-time Quantitative PCR in Cymbidium Hybrid
Lin Rongyan1,2,3, Zhong Huaiqin1,2,3, Huang Minling1,2,3*, Fan RongHui1,2,3, Wu Jianshe1,2,3
1Crop Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; 2Flower Research Center, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; 3Fujian Engineering Research Center for Characteristic Floriculture, Fuzhou 350013, China
Abstract: To provide reference genes for the studies of gene function expression and regulation， sequence of Ch18S rRNA， Ch28S rRNA， ChACT， ChTUA， ChTUB， ChUBQ， ChEF-1a and ChrpoB genes were cloned by RTPCR using the leaf of Cymbidium Golden Elf ‘Sun-dust’ as materials， Real-time quantitative PCR (qPCR) analyses were employed to determine the primer amplification efficiency and their relative quantities for each gene in leaves of ten different Cymbidium hybrid cultivars， geNorm， NormFinder and BestKeeper software programs were used to analyze the expression stability of each candidate gene and the expression pattern of ChPDS in different parts of the flower organs was analyzed to verify the reference genes selected in this study. The RT-PCR and qPCR results showed that the length of each candidate gene fragments were 171 bp， 148 bp， 183 bp， 146 bp， 130 bp， 147 bp， 203 bp and 139 bp， while the primer efficiencies were 1.94， 2.19， 1.88， 1.99， 2.12， 2.20， 2.11 and 1.98， respectively. The evaluation results of three software programs showed that ChACT was the optimal reference genes from the different cultivars， ChACT， ChTUB， ChUBQ and ChEF-1α were the optimal reference genes from the different parts of the flower organs， but， for all the samples in the experiment， the most stable internal gene was ChTUB. The results indicated that different reference gene was required in different test conditions. Analysis results of the relative expression level for ChPDS confirmed the reliability of the reference genes selected， the relative expression of ChPDS using ChUBQ， ChEF-1α， ChTUB， ChACT， the combination of ChUBQ and ChEF-1α for normalization was petal>labellum>gynandrium.
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