拟南芥NiNJA基因酵母双杂诱饵载体构建及互作蛋白的筛选

Construction of Bait Vector for NiNJA Gene in Arabidopsis and Screening the Interacted Proteins Using Yeast Two-hybrid

JAZ蛋白是植物茉莉酸信号途径的重要负调控因子,JAZ与NiNJA形成蛋白复合体抑制茉莉酸下游转录因子的转录活性,NiNJA蛋白是联系JAZ蛋白与下游转录因子的重要因子。为了研究NiNJA调控的下游基因,首先构建了NiNJA基因的诱饵载体pGBKT7-NiNJA,然后转化酵母Y2HGold感受态,通过自转录激活实验,发现诱饵载体pGBKT7-NiNJA没有自转录激活活性。在此基础上,从拟南芥"Mate&PlateTM"Library进行酵母双杂筛选,获得若干个与NiNJA互作的蛋白,为下一步鉴定NiNJA的互作蛋白及茉莉酸的信号调控途径打下基础。

JAZ proteins are important negative factors for jasmonic acid （ JA） signaling pathways in Arabidopsis thaliana. NiNJA interacted with JAZ proteins to inhibit the transcription activity of JA-related transcription factors. NiNJA is a key factor that connected the JAZs and JA-related transcription factors. In order to screen NiNJA interacted proteins,NiNJA full-length cDNA sequence was PCR amplified inserted into pGBKT7 vector to construct a bait vector for yeast two-hybrid system. Furthermore,the vector was transformed into Y2H Gold strain. The result of autoactivation showed that pGBKT7-NiNJA vector had no autoactivation. Finally,the bait vector was used to screen the interacted proteins in ＂Mate PlateTM ＂Library and several potential interacted proteins were obtained. This work laid a foundation for further identification of NiNJA interacted proteins and related signaling pathway.