Identification Of Eight ERF Transcription Factors In Maize For Waterlogging Tolerance

Waterlogging disaster is one of the main factors limiting the increase of corn yield.Preliminary studies have shown that ethylene response factors（ERFs）,especially the group VII,play an important role in the regulation of plant waterlogging stress.In the preliminary research,it is found that group VII ethylene response factors（ERFVIIs）are significantly related to waterlogging resistance through the candidate gene association analysis.In addition,the group V ethylene response factor ZmEREB46 is also involved in the response to waterlogging stress.It is identified by QTL mapping of waterlogging tolerance combined with transcriptome data analysis.In this study,multiple ERFs of maize are analyzed through bioinformatics and expression patterns,and overexpressed in Arabidopsis,in order to preliminarily explore the function of key ERFs through the waterlogging phenotype identification of transformed progeny.Maize mutant materials of ERFs are constructed successfully by using CRISPR/Cas9 gene editing technology,laying a foundation for studying the function and genetic mechanism of candidate genes.The main results are as follows:1.Bioinformatics analysis and expression analysis of ZmERFVIIs.All 19ZmERFVIIs have AP2/ERF domain,13 ZmERFVIIs have N-terminal conserved motif MCGGAI（I/L）.ERFVIIs have genetic and evolutionary differences among different species.The promoter of ZmERFVIIs contains abiotic stress response elements such as anaerobic,drought,and low temperature,as well as hormone response elements such as abscisic acid,gibberellin,and auxin.The transcriptome analysis of B73 root tips at different stages of waterlogging at the seedling stage show that ZmEREB167,ZmEREB210 and ZmEREB139 are not expressed in the root tips,and 16 ZmERFVIIs are up-regulated by waterlogging stress,but the expression patterns are different.2.Bioinformatics analysis and expression analysis of ZmEREB46.The resequencing results of ZmEREB46 in A3237 and A3239 show that there is a SNP difference in the coding region that causes Ala（A3237）to change to Thr（A3239）,and there is a 911 bp fragment insertion in the A3237 promoter region.Analyzing the expression pattern of ZmEREB46 in A3237 and A3239 by q RT-PCR show that ZmEREB46 is up-regulated by waterlogging stress.The expression level of ZmEREB46 in A3237 is twice that of A3239after 8 h waterlogging.The results of the bimolecular luciferase experiment indicate that the promoter activity of ZmEREB46^A3237 is 2.5 times that of ZmEREB46^A3239.Subcellular localization results show that ZmEREB46 expressed protein is in the nucleus.3.8 ERFs ectopic overexpression materials of Arabidopsis can significantly improve the submergence tolerance of seedlings.Ectopic expression materials of 8 ERFs are successfully created by the Agrobacterium-infected Arabidopsis inflorescence method.4or more positive events are obtained for each gene and confirm by semi-quantitative detection.The flooded phenotype of WT and OE is identified by using DAB staining,MDA content,survival rate,and dry weight.The results show that the ectopic expression of 8 ERFs can improve the flood tolerance of Arabidopsis to varying degrees.4.Using CRISPR/Cas9 gene editing technology to create ERF mutants.17 maize ERFs are edited and knocked out in batches under the background of B104 by constructing CRISPR knockout vectors.The genetically transformed offspring of T₀ and T₁ have been tested for editing type.Currently,7 genes have been obtained with CRISPR editing transformation events,each gene has more than 2 editing types.The positive progeny that has been obtained provide good materials for subsequent functional studies of ERFs.5.Analysis of the binding ability of 8 ERF transcription factors and cis-acting elements.The binding ability of ERF with GCC-Box（GCCGCC）and DRE-Box（A/GCCGAC）has been tested with bi-molecule luciferase experiment,and it is found that ZmEREB116 cannot bind to two cis-elements;ZmEREB160 can bind to GCC-Box,but not bind to DRE-Box;other transcription factors can bind to two cis-acting elements of GCC-Box and DRE-Box.The experimental results indicate that ERFs may regulate the expression of downstream waterlogging stress-related genes by combining GCC-Box and DRE-Box,and affect the waterlogging tolerance of maize seedlings.