Molecular Cloning And Functional Analysis Of The FLOWERING LOCUS T(FT) Homolog GhFT1 From Gossypium Hirsutum L.

Flowering is the critical event in flowering plants for the plant successful reproduction. FLOWERING LOCUS T(FT) encodes a member of the phosphatidylethanolamine-binding protein(PEBP) family that functions as the mobile floral signal, playing an important role in regulating the transition from vegetative to reproductive growth. The FT gene encodes a small globular protein that is able to translocate from the leaves to the shoot apex meristem(SAM) through the phloem. At the SAM, FT interacts with the b ZIP transcription factor FD to formed complex, enabling the activation of expression of floral meristem identity genes such as APETALA1(AP1), LEAFY(LFY), SUPPERSSOR OF OVEREXPRESSION OF CONSTANS1(SOC1), FRUITFULL(FUL), and then induced early flowering of plants.Object: This research aim to further analyse the function of the Gh FT1 gene in cotton, expression pattern of Gh FT1 as well as its polymorphism, which will play a foundation for the further research on the function of Gh FT1 gene.Methods: Gh FT1 gene was isolated from cotton through RT-PCR and RACE, and the full length of Gh FT1 gene was cloned via PCR. The expression pattern of Gh FT1 was performed by semi-quantitative RT-PCR and quantitative Real-time PCR(q RT-PCR), respectively. The overexpression vector 35S::Gh FT1 was constracted, and then transformed into Col-0 and ft-10 mutants by floral dipping method. The 35S::Gh FT1-GFP was constracted and transformed into Col-0, and after the root cells of the transgenic plants that were plasmolyzed by sucrose treatment and the subcellular location of Gh FT1-GFP was determined by confocal laser scanning microscope.Results:(1) We re-cloned the Gh FTL1 gene from Gossypium hirsutum and named this gene as Gh FT1 in this research. The 5’ non-coding region was amplified, and the full-length of the Gh FT1 c DNA was 818 bp, and encoded 174 amino acids. Genomic structural analysis revealed that the genomic DNA of FT gene in cotton was 3151 bp and consists of four exons and three introns. Bioinformatics analysis indicated that Gh FT1 has conservative motifs in PEBP family and contains two critical residues, Gh FT1 also has a conserved floral promoters domains of ‘segment B’. Phylogenetic analysis revealed that Gh FT1 had a closer relationship with Tc PEBP, both of them belonged to FT-like subfamily.(2) Gh FT1 showed a broad expression pattern in different tissues, predominantly expressed in flower organs as well as in stems and leaves. Our results showed that Gh FT1 transcript level was strongly upregulated in fiber cells during the period of fiber initiation and early elongation. Gh FT1 expression level was strongly upregulated during the developmet of cotton. Gh FT1 transcript also has a robust diurnal rhythm in both Lond-day(LD) and Short-day(SD) conditions.(3)The FT homolog in allotetraploid cotton Gossypium hirsutum, diploid cotton Gossypium raimondii and Gossypium arboretum both contain four exons and three introns, respectively. However, the its relative copy numbers were different, the relative copy number of FT gene in allotetraploid cotton was 4 and 2 in diaploid cotton.(4) The prokaryotic expression vector of Gh FT1 was successfully constructed and the recombinant protein of Gh FT1 was efficiently expressed in BL21. Gh FT1 fusion protein existed mainly in inclusion bodies as well as in soluble proteins. The ELISA assay showed that the recombinant protein had a good immune response with antibody sera.(5) The transgenic plants carrying the 35S::Gh FT1 construct flowered earlier and had fewer rosette leaves at the time of flowering than the control plant in LD and SD. q RT-PCR analysis showed that multiple flower meristem identity genes, for example, At AP1, At SOC1, At LFY, and At FUL, and were upregulated in the transgenic plants. Ectopic overexpression of Gh FT1 partially restored ft-10 mutant phenotypes in Arabidopsis.(6) Our study indicated that Gh FT1 is a cytoplasm and nucleus localization protein. Introduction of 35S::Gh FT1-GFP into wild-type plants caused these plants to flower much earlier than the wild-type plants.Conclusion: Gh FT1 is an FT orthologous gene in Gossypium hirsutum, and Gh FT1 genome consists of four exons and three introns. The open reading frame of Gh FT1 was 525 bp, encoding 174 amino acids. The relative copy number of FT gene in Gossypium hirsutum was 4, but in Gossypium raimodii and Gossypium arboreum was 2. Gh FT1 showed a broad expression pattern in different tissues and showed robust diurnal rhythm under light photoperiod. Gh FT1 is a cytoplasm and nucleus localization protein. Overexpression of Gh FT1 in Arabidopsis showed extreme early flowering. Flower meristem identity genes were upregulated in the 35S::Gh FT1 transgenic plants. Ectopic overexpression of Gh FT1 partially restored ft-10 mutant phenotypes in Arabidopsis.