Abstract: 【Objective】The objective of this study is to identify whether the flowering repressor TFL1 interacts with the two GRFs family members GRF4 and GRF7, and to provide a basis for illustrating the mechanism of TFL1 repressing flowering.【Method】The TFL1, GRF4 and GRF7 genes were cloned by specific primers using the Arabidopsis cDNA as the templates. These three genes were linked into pCR8 vector to get the entry vectors. The correct entry vectors TFL1-pCR8, GRF4-pCR8 and GRF7-pCR8 were obtained by colony PCR screening and sequencing. The yeast two hybrid assay vectors, including TFL1-BD, GRF4-AD and GRF7-AD, were obtained by LR reaction between these three entry vectors and the destination vectors pGADT7 or pGBKT7. The yeast competent cells which were co-transformed with TFL1-BD plus GRF4-AD or GRF7-AD vectors were incubated on –Leu/-Trp growth medium under 30℃ for 2-3 days until the yeast colonies show up. The yeast colonies in proper size were chosen and transferred to both –Leu/-Trp and -Leu/-Trp/-His/-Ade growth medium. The interaction between TFL1 and GRFs was determined through observing the growth conditions of those yeast colonies on -Leu/-Trp/-His/-Ade growth medium. The BiFC assay vectors, including TFL1-nYFP, TFL1-cYFP, GRFs-nYFP and GRFs-cYFP, were also obtained by LR reaction between these three entry vectors and the destination vectors px-nYFP or px-cYFP, and were transformed into Agrobacterium competent cells. The tobaccos, which were co-transformed by the Agrobacterium harboring TFL1-nYFP or TFL1-cYFP vector and the Agrobacterium harboring GRFs-nYFP or GRFs-cYFP vector, were grown for more 48 hours before observing YFP fluorescence signals under confocal microscopy. The interaction between TFL1 and GRFs was determined if the fluorescence signals in tobacco cells were observed under confocal microscopy.【Result】The three genes, including 534 bp TFL1, 888 bp GRF4 and 798 bp GRF7, were cloned successfully. The entry vectors (TFL1-pCR8, GRF4-pCR8 and GRF7-pCR8), yeast two hybrid assay vectors (TFL1-BD, GRF4-AD and GRF7-AD), and BiFC assay vectors (TFL1-nYFP, TFL1-cYFP, GRFs-nYFP and GRFs-cYFP) of the three genes were obtained successfully. Compared with the negative controls, the yeast colonies which were co-transformed with TFL1-BD plus GRFs-AD vectors grew well in both -Leu/-Trp and -Leu/-Trp/-His/-Ade media in yeast two hybrid assay. Compared with the negative controls, the obvious nuclear YFP fluorescence signals were observed in the tobacco cells which were co-transformed with the Agrobacterium harboring TFL1-cYFP vector or GRFs-nYFP vector. Meanwhile, the obvious nuclear YFP fluorescence signals were also observed in the tobacco cells which were co-transformed with the Agrobacterium harboring TFL1-nYFP vector or GRFs-cYFP vector.【Conclusion】The flowering repressor TFL1 directly interacts with the two GRFs family members GRF4 and GRF7 in Arabidopsis.

Key words: Arabidopsis thaliana, TFL1, GRFs, yeast two hybrid, BiFC