Cloning And Function Research Of CONSTANS-like Genes In Carrot

Carrot(Daucus carota L.) cultivar is a biennial vegetable with very important nutritional and economic values. Low temperature is a necessary condition for carrot bolting and flowering, but long day(LD) can promote flowering early. CONSTANS(CO) is a key transcription factor of the photoperiodic pathway. The accumulation of CO protein could activate the expression of downstream floral integrator genes FLOWERING LOCUS T(FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1(SOC1), and thus stimulate flowering. Based on carrot transcriptome and genome analysis, there are 24 photoperiodic pathway homologous genes in carrot, including DcGI, DcCOLs, DcFTL and so on. In this study, the ‘SongZi’wild species(Ws) and orange inbred line ‘Amsterdam’(Af) were used to analysis the functions of DcCOLs and its related genes, such as DcGI, DcFTL, DcSOC1 s.1. DcGI, DcCOL2, DcCOL4, DcCOL5 a, DcCOL5 b, DcCOL13, DcCOL15, DcFTL, DcSOC1-1 and DcSOC1-2 were cloned. Their open reading frame(ORF) were 3498 bp, 1182 bp, 1170 bp, 1041 bp, 1101 bp, 1146 bp, 1182 bp, 528 bp, 654 bp and 636 bp respectively, encoding 1166, 393, 389, 346, 366, 381, 465, 175, 217 and 211 amino acid(aa) residues. Protein sequence alignment revealed that DcGI, DcCOLs, DcFTL and DcSOC1 s respectively shared height identity with the corresponding proteins in other species. As for DcCOL2, DcCOL4, DcCOL5 a and DcCOL15, there are amino acid variations between Ws and Af.2. The circadian rhythm analysis showed that: the circadian rhythm of DcCOL5 a was similar to that of DcGI, and they were significantly positively correlated with each other; the expression of DcCOL2 was also controled by the circadian clock,and it had significantly higher expression under SD than under LD; DcFTL in Ws had high expression level under LD; DcSOC1-1 in Ws had high expression level under both SD and LD. The qPCR analysis of the expression in different growth stages showed that: the expression of DcCOL5 a and DcSOC1-1 in Ws was significantly higher than that in Af; DcCOL2 had higher expression level in Af than in Ws; DcFTL in Ws and Af both expressed at a very low level. These results made us to speculate that DcCOL2, DcCOL5 a, DcFTL and DcSOC1-1 may be played very important roles in carrot photoperiodic flowering pathway.3. The transgenic experiment showed that: over expression of DcCOL2-Ws caused late bolting and flowering under LD conditions relative to wild-type arabidopsis plants; while, over expression of DcCOL5a-Ws and DcCOL5a-Af promoted bolting and flowering and they had the same effect on promoting arabidopsis flowering. Thus, for DcCOL5 a, the two amino acid variation in Ws and Af had no effects on its function of regulating flowering time. Meanwhile, it also further confirmed that DcCOL2 and DcCOL5 a had a inhibiting and promoting effect on flowering time regulation, respectively.