茶树花发育MADS-box转录因子CsGLO1、CsGLO2与CsAG之间的互作关系研究

Interactions of MADS-box transcription factors CsGLO1,CsGLO2 and CsAG in Camellia sinensis flower development

利用酵母双杂交方法和双分子荧光互补技术(BiFC)研究了茶树(Camellia sinensis(L.)O.Kuntze)花发育MADS-box的B类转录因子Cs GLO1、Cs GLO2与C类转录因子CsAG间的互作关系及其可能发生互作的亚细胞定位。通过构建5个酵母表达载体,利用酵母单杂交实验检测了3个蛋白的转录激活活性,并通过酵母双杂交实验分析了蛋白间的互作关系。结果显示,3个蛋白均无转录激活活性,且两两之间可发生相互作用。进一步构建6个BiFC表达载体,采用压力注射法瞬时浸染烟草(Nicotiana benthamiana L.)叶表皮细胞,并利用激光共聚焦显微镜观察荧光信号,结果表明茶树B类CsGLO与C类CsAG蛋白可在植物活细胞内形成同源和异源二聚体,并具有在细胞质中发生互作的特定模式。本研究可为利用分子生物学技术抑制茶树"花果同现"现象提供理论依据。

In this study,the interactions between MADS-box B transcription factors GLO1 and Cs GLO2, and C transcription factor CsAG in Camellia sinensis L. in relation to flower development as well as their possible subcellular localizations were investigated using the yeast two- hybrid method and bimolecular fluorescence complementation（ BiFC）. In this study,five yeast expression vectors were constructed and the transcriptional activation activities of the three proteins were tested by yeast one-hybridization. The interactions among the three proteins were further analyzed by yeast two-hybrid assay. The results showed that the three proteins had no transcriptional activation activity,but interactions did occur among them. Six Bi FC expression vectors were constructed and transformed into the leaf epidermal cells of tobacco（ Nicotiana benthamiana L.） by pressure injection. The fluorescence signals were then observed by confocal laser scanning microscopy. The results showed that the three proteins could form homo- and heterodimers,and had specific patterns of interaction in the cytoplasm. This study could provide a theoretical basis for the inhibition of C. sinensis ‘flower and fruit present＇ using molecular biology techniques.