Cloning and characterization of a novel CONSTANS-like gene from Phalaenopsis hybrida

The CONSTANS (CO) or CONSTANS-like (COL) gene plays a key role in the photoperiodic flowering pathway. A novel cDNA encoding COL gene was isolated from Phalaenopsis hybrida (cv. Wedding Promenade) and designated as PhalCOL. The full cDNA sequence was 1,202 bp in length with an open reading frame (ORF) of 987 bp, encoding 329 amino acids with a molecular weight of 34.87 kDa and a theoretical isoelectric point of 5.60. The genomic clone contained two exons and one 106 bp intron at the conserved site. The analysis based on amino acid sequence alignment showed that PhalCOL shared a high identity with COL (49%) from Hordeum vulgare, Hd1 (45%) from Oryza sativa and AtCOL4 (46%) from Arabidopsis thaliana, respectively. PhalCOL had two B-box zinc finger motifs and a CCT domain. A transient expression assay using PhalCOL/GFP fusion protein showed that PhalCOL was nucleus-localized protein. Semi-quantitative RT–PCR analysis showed that PhalCOL was expressed in all organs throughout development. Its transcripts accumulated at higher levels in the stem during the transition from vegetative to reproductive growth. Over-expression of PhalCOL in tobacco caused an early-flowering phenotype. These results indicated PhalCOL was a COL homologue and may play an important role in initiating buds and promoting flowering.