Study On The Interaction Of F-box Protein FKF1 And Transcription Factor FUL In Regulating Flowering In Arabidopsis

Plant Flowering is a critical stage in the growth and development process of higher plants.The appropriate timing of flowering is extremely important for the survival and successful reproduction of plants.The process is coordinately regulated by various endogenous and exogenous factors.Numerous molecular and genetic studies,based on the research on the regulation of the flowering time of Arabidopsis,revealed that there were six major flowering pathways in Arabidopsis,including photoperiod,vernalization,temperature,gibberellin(GA)biosynthesis,aging,and autonomous pathways.These pathways are independent and interconnected,ultimately forming a complex regulatory network to achieve precise control of flowering time.F-box protein FKF1(FLAVINBINDING KELCH REPEAT F-BOX1)plays an important roles in regulating photoperiodic flowering in Arabidopsis.Studies have shown that the fkf1 mutants exhibits late flowering under long-day conditions.In order to further explore the molecular mechanism of FKF1 regulating flowering,this thesis obtained several flowering factors predicted to interact with FKF1 through bioinformatics analysis.Next,we used the yeast two-hybrid assay to identify its potential interaction partners and the results show that that FKF1 interacted with FUL(FRUITFULL).The interaction between them was further verified by other biochemical experiments.We analyzed the effect of FKF1 on the transcription and protein levels of FUL;The genetic relationship of FKF1 and FUL were analyzed.Detailed research results are as follows:(1)To investigate whether FKF1 interact with FUL protein,yeast two-hybrid assay,BIFC assay,and Co-IP assay suggested that FKF1 proteins physically interact with FUL in vitro and in vivo.(2)We found that FKF1 does not affect the protein level of FUL via semi-in vivo protein stability analysis and tobacco system protein stability analysis.However,real time quantitative polymerase chain reaction(q RT-PCR)was used to compare and analyze the expression of FUL in fkf1 mutants and the expression of FKF1 in ful mutants.We found that FKF1 can up-regulate the transcription level of FUL.Results indicated that FKF1 regulate the FUL expression in transcriptional level.(3)We observed the days to bolting and the rosette leaf number at bolting of35S-FUL-Flag plants and ful-8 mutants under long-day conditions.We found that FUL over-expressing plants showed early flowering,while ful-8 mutants showed late flowering.We examined FUL expression in developing seedlings under LD conditions and found that FUL expression levels gradually increased during floral transition.These findings indicated that FUL is a positive regulator of flowering.(4)To further investigate the relationship beween FKF1 and FUL protein in flowering,genitic analysis showed the days to bolting of 35S-FKF1-Myc / ful-8 plants delayed and the rosette leaf number at bolting increased in long-day conditions,compared with 35S-FKF1-Myc plants,which was similar to the flowering phenotype of ful-8 mutants.These showed that the early-flowering phenotype of 35S-FKF1-Myc plants was rescued by the ful-8 mutations.The transcriptional level of the flowering time integrator gene FT(FLOWERING LOCUS T)was up-regulated in 35S-FKF1-Myc plants and down-regulated in ful mutants.And the transcription level of FT in FKF1-Myc/ful-8 plants was similar to ful-8 mutants.Results demonstrate that FKF1 and FUL are on the same pathway to regulate flowering via positively regulating FT expression in Arabidopsis.