Molecular Mechanism Of Tamarix Hispida ThPHD3 Gene Regulating Salt Stress Response

In the process of adapting to the environment,plants can regulate the expression of certain specific transcription factors to produce specific products to enhance the adaptability of plants to adversity conditions.People have paied more and more attention to the research on the function of transcription factors in the process of coping with abiotic stress.Plant homeodomain(PHD)transcription factors,which is a zinc finger protein,play an important role in plant growth and development and particated in various biological stress response signal pathways.In this study,the ThPHD3 gene was cloned from the salt-tolerant transcriptome of T.hispida,and its salt-tolerant function and regulation mechanism were preliminary studied.The main research results obtained are as follows:1.The ThPHD3 protein is located in the nucleus.The expression of ThPHD3 gene is induced by two abiotic stress treatments(high salt and drought)and two hormonal signals(ABA and GA3.Especially under salt stress,the expression of ThPHD3 gene in T.hispida roots was significantly up-regulated,indicating it may play a role in the salt-tolerant response off T.hispida.2.The germination rate,root length and fresh weight of the overexpression ThPHD3 gene Arabidopsis thaliana seeds are higher than those of the wild type under salt stress.The cell damage degree and the reactive oxygen species content of the transgenic plants are significantly lower than that of the wild type,but the protective enzymes content in cells is higher than that of wild type.In order to verify the heterologous expression results in Arabidopsis thaliana,the ThPHD3 gene was overexpressed and suppressed,and the empty vector(control)strain were transiently transformed into T.hispida.It was found that the overexpression plants enhanced the salt tolerance of plants by increasing the content of protective enzymes and reducing the accumulation of H2O2 and MDA.It showed that ThPHD3 gene may play an important role in plant resistance to salt stress.3.RNA-Seq analysis was performed on the three transgenic T.hispida.Compared with the control group,the numbers of up-regulated and down-regulated genes in the overexpression lines were 317 and 81,respectively,while the numbers of up-regulated and down-regulated genes in in RNAi lines were 146 and 228,respectively.GO function classification results showed that most differentially expressed genes were enriched in plant hormone signal transduction,phenylpropane biosynthesis and plant pathogen interaction pathways.There were 54 differentially expressed genes were shared by the three groups,including aminopeptidase M1,zinc finger protein ZAT8,plant U-box,phosphate reaction 1 family protein,mitogen-activated protein kinase kinase A,protein phosphatase 2C 25,ethylene response transcription factor ERF017,calcium binding protein CML39 and many other types of genes.4.Through the trans yeast one-hybrid analysis,15 elements that may interact with ThPHD3 gene were screened from the random library.Yeast one-hybrid and EMSA experiment results proved that the core sequence of the AATCTCCAAT element that may bind to ThPHD3 transcription factor was TCTC.The TCTC element was constructed into the pCAMBIA1301 vector and co-transformed tobacco,GUS staining and GUS enzyme activity assay confirmed that ThPHD3 can specifically recognize TCTC elements in plants.The core element TCTC was subjected to mutation treatment and through yeast one-hybrid analysis obtained new elements CC/ATC and T/CACC that may interact with ThPHD3 gene.5.The results of ChIP experiments showed that ThPHD3 transcription factor can combined with target genes ThZAT12 and ThE3PUB23 promoter fragments which contained the cis-acting element TCTC,and this result was verified by EMSA and co-transformed tobacco in vivo.Further the salt-tolerant response of ThZAT12 and ThE3PUB23 genes in transiently transformed T.hispida were analysed and showed that the overexpression lines showed obvious salt-tolerant response.6.Transcription activation experiment found that the ThPHD3 transcription factor does not have transcription activation activity.Yeast two-hybrid and BiFC experiments found that ThPHD3 protein can interact with proteins ThPHD5,ThPHD9 or ThWRKY2 to form homodimers.Overexpression ThWRKY2 gene T.hispida improved the ROS scavenging ability,protect cells from damage under salt stress so to improve the salt tolerance of plants.Comprehensive analysis of the above results,the salt tolerance function of ThPHD3 gene was initially clarified.ThPHD3 gene interacts with ThPHD5,ThPHD9 and ThWRKY2 proteins,and binds to the TCTC elements on the promoters of downstream salt tolerance-related target genes ThZAT12 and ThE3PUB23,thereby enhancing the ROS scavenging ability and reducing cell damage of plants under salt stress.This study lays a foundation for further improving the salt tolerance mechanism of T.hispida ThPHD3 gene,and is of great significance for the depth understanding of the salt tolerance mechanism of woody plants and the cultivation of woody plants with excellent salt tolerance.