Study On The Molecular Mechanism Of MdCoL And MdNF-YB Coodinately Regulated MdFT1Transcription During Floral Induction In Apple

FLOWERING LOCUS T(FT) acts as a key role in floral induction. CONSTANS(CO)protein could interact with a variety of Nuclear Factor Y(NF-Y) family proteins to regulate At FT in Arabidopsis. In order to figure out the mechanism of how CONSTANS-Like(MdCOL)and Nuclear Factor Y(NF-Y) subunit Md NF-YB family proteins regulated Md FT1 in apple(Malus domestica), we chose ‘Nagafu No.2’ and ‘Qinguan’ apple as experimental materials, and used experimental methods such as Yeast One-Hybrid System,Yeast Two-Hybrid System. Main research results were summarized as follows:1 Identification and transcript analyses of MdCOL, Md NF-YB family and Md FT1We compared the amino acid sequences of MdCOL family with the sequences of At CO,At COL1 to At COL5 and At COL9 in Arabidopsis. The amino acid sequences of Md NF-YB family and At NF-YB2 �'� At NF-YB3 were also compared for homologous analysis. Meanwhile,considering the transcriptome database obtained by our lab, we screened 7 MdCOL family genes and 1 Md NF-YB family member for transcript analysis.According to q-PCR results, Md FT1 and 6 members of MdCOL family(MdCOL2 a,MdCOL2 b, MdCOL4 a, MdCOL4 b, MdCOL5 and MdCOL12), together with Md NF-YB3 family followed expected diurnal rhythm expression pattern. It is noteworthy that Md FT1 transcript levels were significantly higher in ‘Qinguan’ than in ‘Nagafu No.2’ during the floral induction period in buds. Additionally, Md NF-YB3 was expressed coordinately with Md FT1 no matter in buds or leaves.The 6 MdCOL proteins, except MdCOL12, were divided into three domains, which were also found in the At CO homolog: the two B-Box domains and one CCT domain. The identification of Md NF-YB3 was validated by sequence comparisons with At NF-YB3 protein.They both had the histone fold domain(HFD).2 Identifying whether MdCOL, Md NF-YB family proteins could bind Md FT1 promoterDifferent regions of Md FT1 promoter(p Md FT1) were isolated and recombined to p Ab Ai vector, respectively. The linearized vectors were integrated into Y1 HGold, which together composed bait yeast strains. The 6 MdCOL and 1 Md NF-YB proteins were recombined to p GADT7 vector and transformed to the bait yeast strains, seperately. Differing with the classic CO regulating FT module, no MdCOL proteins were found to interact with p Md FT1, while only the Md NF-YB protein Md NF-YB3 showed the ability.3 Identifying whether MdCOL、Md NF-YB family proteins could interactSince no identified MdCOL proteins were found to directly bind p Md FT1, we studied the possibility that the MdCOL proteins and other Md NF-YB proteins interacted with Md NF-YB3 through Y2H assays. Coding region of Md NF-YB3 was recombined to p GBKT7 vector.MdCOL6, MdCOL9 a, MdCOL9 b and MDP0000272838, MDP0000818967, MDP0000230429,MDP0000127596 of Md NF-YB family genes, together with the 6 MdCOL genes cloned in the Yeast One-Hybrid assay, were all recombined to prey vectors and transformed to bait yeast strain respectively. As a result, no Md NF-YB proteins were found to form complex with Md NF-YB3. In contrast, MdCOL2 a, MdCOL2 b, MdCOL4 a, MdCOL4 b, MdCOL5 and MdCOL12 proteins were able to interact with it.Since the Md NF-YB3 could bind Md FT1 promoter directly, but not the 6 MdCOL proteins.Therefore, the 6 MdCOL proteins might regulate Md FT1 transcription activity by forming complex with Md NF-YB3.