Cloning And Preliminary Study On Function Of The Key Flowering Gene FT/TFL1 From Rong’an Kumquat

Flowering Locus T (FT)/Terminal Flower Like 1 (TFL1) gene family is an important integration factor of plant flowering regulation pathway. It is the key for the regulation of plant flowering and plays a key role in the flower development. At present, FT/TFL1 homologous genes in apple, grape, pecan, orange, longan, mango and many kinds of fruit trees were cloned and the part of their function were studied. The flowering habit of Kumquat is different with other citrus. The molecular mechanism of its flowering research is relatively rare. To prove the effect of FT/TFL1 genes of Kumquat flowering regulation preliminary, Rong’an Kumquat is used as experimental materials for this paper and we do the study on the homologous gene cloning, bioinformatics analysis, characterization and transgenic. The main results are as follows:1. Three CDS sequences of FT/TFL1 homologous genes were isolated from Rong’an Kumquat and they were named FcFT1 (531 bp), FcFT2 (516 bp), FcTFL1 (519 bp). According to the analysis of the bioinformatics, it showed:FcFT1 encoded 177 amino acids, total protein atoms is 2791, the molecular formula:C888H1377N255O266S5; the weight of theoretical protein molecular is 20.0 kDa, isoelectric point:8.97; FcFT2 encoded 172 amino acids, total protein atoms is 2653, the molecular formula:C845H1328N230O240S10; the theoretical protein molecular weight is 18.87kDa, isoelectric point is 7.81; FcTFL1 encoded 173 amino acids, there are 2733 protein atoms at total. The molecular formula is C869H1359N249O250S6; the weight of theoretical protein molecular is 19.49kDa, isoelectric point is 8.89.The three protein all with the phosphatidylethanolamine-binding protein (PEBP) domain.2. Homologous analysis on the amino acid sequence indicated that the three sequence had high identity to homologous protein of other fifteen species. The homologous percentage is 77.48%. Phylogenetic analysis revealed that FcFT1 was closelyrelated to the CiFT and FT homologous gene of Citrus trifoliate; FcFT2 was gathered together with clementina and longan FT homologous genes; FcTFL1 was also related to Citrus clementina and Citrus trifoliate.3. Using real-time fluorescence quantitative analyze the FcFTl, FcFT2, FcTFLl expression quantity of Rong’an Kumquat’different organizations and different flower organs, as well as, analyzing the day cycle expression quantity of the flower, stem and leaf.(1) In the result tree, FcFTl, FcFT2, FcTFLl expression quantity of stem and leaf tissues were higher than sapling. FcFT1 was more highly expressed in the mature leaves than in the mature stems and flower bud, and that FcFT2, FcTFLl expression quantity was slightly lower in the mature leaves than in the mature stems.(2) In the different tissues of flower organs, the expression patterns of three genes displayed distinct difference. The average expression quantity of FcFTl: anther>torus>filament>petal>ovary, the average expression quantity of FcFT2: ovary>filament>anther>torust>petal, the average expression quantity of FcTFLl:filament> anther>torus>petal>ovary.(3) In the daily cycle, day expression quantity of FcFTl, FcFT2, FcTFLl were higher than the night expression quantity. The expression all of the three genes in flowers have peaked at midday. FcFT1, FcFT2 of the stem got high level expression at the same time. But in stem and leaf tissue, the highest expression quantity of FcTFLl than it of FcFT1, FcFT2 was delayed for two hours.(4) In the flower, stem and leaf, the average expression quantity of three genes shown that:FcTFL1>FcFT2>FcFTl, the average expression level of three tissues was: stem>flower>leaf. FcFTl, FcFT2 and FcTFLl can be expressed in lots of tissues during the growth and development period of Kumquat.4. The eukaryotic expression vector pBI121 was successfully constructed of Rong’an Kumquat FcFTl, FcTFL1 and the eukaryotic expression vector Cam35S-gfp was successfully constructed of Rong’an Kumquat FcFT2. Through the floral dip method, the expression vector was introduced into Arabidopsis thaliana by Agrobacterium-tumefaciens. We harvested TO and T1 generation seeds. In the phenotypic analysis of T1 generation transformational plants, we found that the transgenic plants of FcFT2 appear early flowering.