Screening And Functional Analysis Of WRKY Related To Disease Resistance Gene Of Soybean Downy Mildew

Soybean downy mildew is a fungal disease caused by Peronospora manshurica(Naoun.)Sydow,which is a serious threat to soybean production and occurs in the world’s soybean producing areas.In the northeast and north China,the occurrence is especially prevalent,which can caused the reduction by 30%to 50%.In the previous study,we found that the expression of soybean serine/alanine-glyoxylic acid transaminase(GmSAGT1)gene was closely related to the resistance of soybean downy mildew.And the average expression level of GmSAGT1 in high resistance cultivars was 13 times higher than that in high resistance cultivars.GmSAGTl was involved in the resistance of soybean downy mildew at the transcriptional level,and the coding function of GmSAGTl in the resistant cultivars was different.The GmSAGTl promoter sequence analysis revealed that there were two transcription factors,WRKY’s binding site.It is known that WRKYs are an important transcription factor of plants and play an important role in the biological processes such as stress resistance and disease resistance of many plants.So far,WRKYs transcription factor and soybean downy mildew resistance research have not been reported in the literature.Therefore,this study on soybean downy mildew resistance related transcription factor WRKY were screened and studied,the main reasults as followed:1.The expression of WRKYs was analyzed before and after inoculation,and seven WRKYs were detected by transcriptome sequencing.The results showed that.the expression of these seven genes(WRKY5,WRKYY56,WRKY81,WRKY106,WRKY109,WRKY139 and WRKY161)in the resistant cultivars was higher than that of the susceptible cultivars after inoculation,suggesting that they were involved in the regulation of GmSAGT1 gene expression and soybean downy mildew resistance2.Real-time quantitative PCR was used to verify the sequencing results of the transcriptome.The results showed that the resistant expression of seven WRKYs genes in the susceptible cultivars was consistent with that of the transcriptome.The results of the transcriptase sequencing were reliable.3.In order to identify the activity of GmSAGTl promoter by using the transient expression technique of plant,three plant expression vectors were constructed:1)The plant expression vector pG958∷GFP expressing GFP was driven with 958bp GmSAGT1 full-length promoter G958;2)GFP-expressing plant expression vector pG355::GFP was amplified by G355 at the 3’-end 355bp core region of the GmSAGTl promoter;3)The GFP-expressing plant expression vector pG181 ∷ GFP was driven into the Arabidopsis thaliana protoplasts by G181 at the 3 ’end of the GmSAGT1 promoter.G958 ∷ GFP,G355 ∷GFP and G181::GFP showed green fluorescence in Arabidopsis thaliana protoplast cells,G958 ∷ GFP was the strongest,G355 ∷ GFP and G181 ∷ GFP Arabidopsis thaliana protoplast cells in the green fluorescence of the same brightness.The resuts showed that all three promoters had the ability to initiate gene expression and could be used for the construction of bait vector in yeast single hybridization.4.WRKY5,WRKYY56,WRKY106,WRKY109.WRKY139 and WRKY161 were not found to be associated with two WRKY bingding sites at-235 and-272 at the beginning of the GmSAGTl transcription initiation site(W-box),WRKY81 can binds to W-boxl(TGACGTAC)upstream of GmSAGTl transcription initiation site and also bind to W-box2(GACTGAC),which indicating that WRKY81 can regulate GmSAGT1 expression at transcription level and participate in soybean Resistance to downy mildew.In this paper,we found that WRKY were closely related to the resistance to downy mildew of soybean,and WRKY81 regulated the downy mildew resistance of soybean by binding to two W-boxes on GmSAGT1 promoter.It was suggested that the expression of WRKY81 was up-regulated by the pathogen-stimulated soybean,and the expression of GmSAGTl gene was activated by binding to the GmSAGT1 promoter to provide the active oxygen content in the host plant leaves.Then the occurrence of hypersensitivity was induced,and the resistance of soybean to downy mildew was improved.The Plant disease resistance is an extremely complex reaction,it involves multiple networks and a variety of mechanisms,This study with great significance revealed the molecular mechanism of resistance to soybean downy mildew,and we hope it can provide a good start.