拟南芥开花抑制因子TFL1与GRFs蛋白的相互作用

doi:10.3864/j.issn.0578-1752.2017.10.003

摘要/Abstract

摘要： 【目的】研究拟南芥开花抑制因子TFL1与2个GRFs家族成员GRF4和GRF7之间的互作关系，为进一步解析TFL1抑制植物开花的分子机制奠定基础。【方法】以拟南芥cDNA作为模板，利用基因特异性引物，克隆TFL1、GRF4和GRF7，分别连接入门载体pCR8，经菌落PCR扩增和测序鉴定分别获得这3个基因的入门载体TFL1-pCR8、GRF4-pCR8和GRF7-pCR8。利用LR重组的方法将上述3个入门载体分别与目标载体pGADT7和pGBKT7重组获得酵母双杂交试验载体TFL1-BD、GRF4-AD和GRF7-AD。将TFL1-BD载体分别与GRF4-AD或GRF7-AD载体共同转化酵母感受态细胞，于双缺（-Leu/-Trp）培养基上30℃培养2—3 d直至长出酵母克隆，选取合适大小的酵母菌落转移到双缺（-Leu/-Trp）和四缺（-Leu/-Trp/-His/-Ade）缺陷培养基上，通过观察酵母菌落的生长情况判断TFL1与GRFs之间的互作关系。利用LR重组的方法将上述3个入门载体分别与目标载体px-nYFP和px-cYFP重组获得TFL1-nYFP、TFL1-cYFP、GRFs-nYFP、GRFs-cYFP双分子荧光互补试验载体，并分别转化农杆菌感受态细胞。将转化TFL1-nYFP或TFL1-cYFP载体的农杆菌分别与转化GRFs-nYFP或GRFs-cYFP载体的农杆菌共注射烟草叶片，培养48 h后在荧光共聚焦显微镜下观察烟草细胞中YFP荧光的表达情况。通过YFP荧光信号的有无来判断TFL1与GRFs之间的互作关系。【结果】成功克隆到拟南芥中的3个基因，分别是534 bp的TFL1、888 bp的GRF4和798 bp的GRF7，并分别获得其入门载体（TFL1-pCR8、GRF4-pCR8和GRF7-pCR8）、酵母双杂交试验载体（TFL1-BD、GRF4-AD和GRF7-AD）和双分子荧光互补试验载体（TFL1-nYFP、TFL1-cYFP、GRFs-nYFP和GRFs-cYFP）。在酵母双杂交试验中，相较于阴性对照组，共同转化TFL1-BD与GRFs载体的酵母菌落在双缺（-Leu/-Trp）和四缺（-Leu/-Trp/-His/-Ade）培养基上都生长较好，结果表明TFL1与GRF4、GRF7在酵母中直接相互作用。在双分子荧光互补试验中，相较于阴性对照组，将转化TFL1-cYFP载体的农杆菌与转化GRFs-nYFP载体的农杆菌共注射烟草细胞之后均在烟草细胞核内产生较强的YFP荧光信号；与此同时，将转化TFL1-nYFP载体的农杆菌与转化GRFs-cYFP载体的农杆菌共注射烟草细胞之后同样在烟草细胞核内产生较强的YFP荧光信号。结果表明，TFL1与GRF4、GRF7在烟草中直接相互作用。【结论】拟南芥开花抑制因子TFL1与GRFs家族的2个成员GRF4和GRF7均直接相互作用。

关键词: 拟南芥, TFL1, GRFs, 酵母双杂交, 双分子荧光互补

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

袁敏，邢继红，王莉，葛伟娜，郭棣，张岚. 拟南芥开花抑制因子TFL1与GRFs蛋白的相互作用[J]. 中国农业科学, 2017, 50(10): 1772-1780.

YUAN Min, XING JiHong, WANG Li, GE WeiNa, GUO Di, ZHANG Lan. Interaction Between TFL1 and GRFs in Arabidopsis thaliana[J]. Scientia Agricultura Sinica, 2017, 50(10): 1772-1780.

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