Isolation and functional analysis of CYC2d orthologous genes from several plants of the tribe Anthemideae

As the important gene of regulating flower symmetry, cyc-like proteins have been shown to mainly regulate the identity and development of ray floret (bilaterally symmetrical) in Asteraceae. The presence or absence of ray floret in Asteraceae and its molecular regulation mechanism as well as the evolutionary process have been highly concerned. Orthologous genes of CYC2d from Ajania potaninii, Brachanthemum titovii and Chrysanthemum indicum var. aromaticum were obtained by homology-based cloning. Their sequence alignment and conserved motif analysis were performed with the amino sequence of CmCYC2d, respectively. The results showed that their homology was more than 90% and all these proteins contained the conserved TCP and R domains. Furthermore, according to the result of semi-quantitative RT-PCR assay, CYC2d was strongly expressed in the young inflorescence of the groundcover chrysanthemum 'Mao xiangyu', while slightly expressed in that of A.potaninii and B.titovii. Therefore, the transcription levels of CmCYC2d were examined in ray and disc florets of 'Mao xiangyu' at six developing stages using quantitative real-time PCR. The results indicated that it was weakly expressed in disc florets of all stages, while highly expressed in ray florets of the corresponding stages. Moreover, in three F1 progenies with various whorls of ray florets, the CmCYC2d was expressed at much higher levels in ray florets of different whorls than in disc florets. The recombinant plasmid pSUPER1300-CmCYC2d-GFP was transiently expressed into the epidermal cells of Nicotiana benthamiana by agrobacterium-mediated transformation, and subcellular localization analysis revealed that the CmCYC2d protein mainly localized into the nucleus of epidermal cells. Furthermore, CmCYC2d was overexpressed in wild type Arabidopsis and the TCP1 mutant used the floral-dip method. The results showed that the vegetative growth and the flowering time of the positive transgenic lines were repressed and postponed. Moreover, the size and arrangement of the petals seemed to be changed, making the petal arrangement showed bilateral symmetry from original radial symmetry. These results indicate that the transcription factor CmCYC2d is essential in regulating ray floret identity in chrysanthemum. Our study lays a foundation for the research of molecular mechanisms for the evolutionary process of ray floret in Asteraceae.