Analysis Of Interacting Protein And Downstream Gene Related To GmNFYB1 Drought Tolerant Gene Of Soybean

Water is one of the necessary and important element during the growth and development process of soybean.Water deficit and drought can affect soybean growth process, even more, severe drought and other abiotic stress environment would cause abnormal growth and development of soybean and cause yield and quality decline. Useing modern biotechnology to explore soybean drought mechanism and breed new varieties with drought-resistant is critical for substantial soybean production in China.Plant transcription factor NFY plays important roles in regulation of growth and development, photoperiod, flowering, drought and salt stress tolerance response and other aspects.Transcription factor NFY regulates the expression of downstream genes by binding to CCAAT-box elements with the composite which consiste of A,B,C three subunits.Many NFYA,NFYB and NFYC genes involved in plant defense reactions have been cloned in Arabid opsis, rice, soybeans and other plants. The previous results in our lab have proved drought and salt tolerance in Arabidopsis and in soybean by overexpressing GmNFYB1 gene.The results showed that Gm NFYB1 gene closely related to soybean drought and may play an important role in the drought-resistant in soybean.To further study Gm NFYB1 regulation mechanisms, Gm NFYB1 and genes from NFYA and NFYC family of soybean were amplified,and then the protein interactions between soybean Gm NFYB1 and NFYA as well NFYC were analyzed by yeast two-hybrid system. Eighteen up-regulated genes and thirteen down-regulated genes involved in plant drought and regulated by Gm NFYB1 were obtained by q RT-PCR. These genes were selected from the transcription sequencing results with transgenic Gm NFYB1 Arabidopsis.Subsequently, EMSA analysis bewteen Gm NFYB1 protein and the downstream gene probe regulated by Gm NFYB1 was conducted.The main results are as follows:(1) By analyzing the bioinformatics and evolution tree of the 21 genes from Gm NFYA family and 15 genes from GmNFYC family,Gm NFYC4,Gm NFYC6,Gm NFYC9,Gm NFYA5,Gm NFYA16 and Gm NFYA19 were selected for further analyses.We found these six genes all participated in plant drought reaction according to the analysis of expression and function of their h omologous genes in Arabidopsis in data BAR.(2) Full-length sequence of Gm NFYB1,Gm NFYC4,Gm NFYC6,Gm NFYC9,Gm NFYA5, Gm NFYA16 and GmNFYA19 was isolated from soybean genome.And then the bait vector p GBKT7-Gm NFYB1 and vectors p GADT7-Gm NFYC4,pGADT7-GmNFYC6,p GADT7-Gm NFY, p GADT7-Gm NFYA5,p GADT7-Gm NFYA16 and p GADT7-Gm NFYA19 were built.(3) Self-activating experiment proved that there was no self-activating in Gm NFYB1.We identified that Gm NFYB1 protein had interaction relationship wtih Gm NFYC4,Gm NFYC6 and Gm NFYC9 proteins by the yeast two-hybrid experiments and result proved that NFYB subunit can interact with NFYC subunit in soybean.(4) Thirteen up-regulated and seven down-regulated genes involved in drought reaction regulated by Gm NFYB1 were identified by using q RT-PCR from eighteen up-regulated genes and thirteen down-regulated genes based on RNA-seq results(5) The protein inducing vector PET-29b(+)-Gm NFYB1 was built and the protein of Gm NFYB1 was successfully purified.The binding reaction between GmNFYB1 protein and probes was analyzed by EMSA experiment. The results showed that the pathway and mechanism of Gm NFYB1 regulation downstream genes is not binded to the CCAAT-box directly by Gm NFYB1 protein.