Functional Analysis Of WRKY50 In Sweet Sorghum Under Salt Stress

The WRKY transcription factor family is involved in plant responses to stress.Members of this family contain a conserved WRKY domain,which can regulate plant physiological and biochemical responses by binding to the W-box of the downstream target gene promoter region.In this experiment,we screened a sweet sorghum SbWRKY50?Sb09G005700?gene from the transcriptome and overexpressed this gene in wild-type Arabidopsis to obtain overexpressed lines W50-12,W50-13.We analyzed the bioinformatics information of the gene,construction of a GFP-tagged vector for analysis of SbWRKY50 location,and constructed a SbWRKY50-pGBKT7 vector for transcriptional activity analysis.The downstream target genes of wild type?WT?,over-expressed physiological indexes at germination and seedling stage under salt treatment were determined and analyzed.The downstream target genes regulated by SbWRKY50 were screened by qPCR,yeast one hybrid and luciferase complementary experiments,and the mechanism of the gene regulating plant salt resistance was further clarified.The main conclusions of this study are as follows:1.The CDS sequence of SbWRKY50 has a total length of 621 bp and encodes207 amino acids.It has a conserved WRKY domain at the N-terminal and a zinc finger structure of C₂H₂ at the C-terminal.It belongs to a typical class II WRKY family protein.The molecular formula of C₉₆₉H₁₄₅₃N₂₇₅O₃₀₅S₈ is 22 kDa.It is a hydrophobic protein without transmembrane helix.2.Analysis of the location of SbWRKY50 by constructing a GFP-tagged vector showed that the gene was localized in the nucleus.The results showed that the gene was located in the nucleus.SbWRKY50-pGBKT7 vector was constructed and its transcriptional activity was analyzed.The results of yeast transformation showed that the gene had transcriptional activation activity.3.RT-PCR analysis showed that the expression of SbWRKY50 was higher in W50-12 and W50-13 lines than in wild type,which indicated that SbWRKY50 had been successfully overexpressed in Arabidopsis thaliana.4.Statistics of germination rate and root length of wild type and overexpressing plants showed that the germination rate and root length of overexpressing plants were significantly lower than that of wild type plants.Determination of physiological indexes of plants at seedling stage showed that compared with the wild type,the biomass and potassium content of overexpressing lines were significantly reduced under salt stress,while the contents of hydrogen peroxide,superoxide anion and sodium ion were significantly increased.5.Under salt stress,the expression of some genes responding to osmotic stress?SOD and APX?,oxidative stress?RD29B and p5CS1?,and ion stress?CLC-C,SOS1and HKT1?in the over expressed lines was detected by real-time PCR.The results of yeast one hybrid and luciferase complementary imaging showed that SbWRKY50could directly bind to the upstream promoters of SOS1 gene in Arabidopsis thaliana,SOS1 and HKT1 genes in sweet sorghum.These results indicate that the new WRKY transcription factor SbWRKY50 participates in plant salt reaction by controlling ion balance.However,there are different regulatory mechanisms in sweet sorghum and Arabidopsis,which may explain why they have different salt tolerance.These data provide basis for salt tolerance transformation of different crop varieties.