Cloning And Expression Of TαWRKYⅢ-A37 And TαWRKYⅡc-D2 Genes And Identification Of ABC1 Gene Family In Triticum Aestivum L.

As a unique family of transcription factors(TFs)in higher plants,WRKY TFs are involved in the physiological process and stress response mechanism of plants.There are many WRKY genes in wheat,but the molecular regulation mechanism of drought resistance genes is still unclear.Predecessors have reported that the Tα WRKYI II-A3 7 and TαWRKYⅡc-D2 genes in the WRKY family respond the most to drought stress.Therefore,we cloned two TαWRKY genes from wheat,and performed bioinformatics analysis and expression patterns under different stresses.In addition,the ABC1 family is an atypical protein kinase family.Studies showed that the ABC1 gene family played a role in a variety of plant metabolism and stress response processes,while the wheat ABC1 family had less research on stress.In this study,the wheat ABC1 gene family was identified at the genome-wide level,and the phylogenetic relationships,gene structure,protein conservation motifs and protein interaction regulatory networks of its family members were comprehensively studied.Meanwhile,10 ABC1 genes with obvious response to drought stress were selected to analyze their expression patterns in different tissues and under different stresses,so as to provide basis for further study on the stress resistance function of TαABCl gene.The main findings are as following:1.Two WRKY genes were cloned from wheat and named TαWRKYⅢ-A37 and TαWRKYIIc-D2.Boinformatics analysis showed that they are both hydrophilic and unstable non-secreted nuclear proteins;Cis-acting element predicted that the promoters of two TαWRKY genes contained ABA and MeJA stress related elements;Phylogenetic analysis showed that TαWRKYⅢ-A37 protein has the closest relationship with its two homologous copies,while TαWRKYⅡc-D2 protein has the closest relationship with Aegilops tauschii.2.qRT-PCR results showed that the two TαWRKY genes were expressed in wheat roots,stems and leaves,but their expression levels were different.TαWRKYⅢ-A37 and TαWRKYⅡc-D2 had the highest expression levels in wheat roots and leaves,respectively,while had low expression levels in stems.Two TαWRKY genes can respond to the stress of PEG,NaCl,H2O2 and ABA in the seedling stage and the filling stage of wheat.3.The overexpression vector and subcellular localization vector of TαWRKYⅢ-A37 and TαWRKYⅡc-D2 genes were successfully constructed,which laid a foundation for further study of gene function.4.A totle of 43 ABC1 family members were identified from the wheat protein database,and they were renamed according to the distribution of genes on the chromosome and gene homology.The prediction of physicochemical properties showed that the number of ABC1 amino acids in wheat varied greatly,and most of TαABC1 proteins belonged to hydrophilic and unstable chloroplast proteins;Chromosome mapping results showed that TαABC1 genes arerandomly distributed on 20 chromosomes,except for chromosome 1D.Phylogenetic analysis showed that 73 ABC1 members of wheat,rice and Arabidopsis thaliana were divided into 3 categories and 9 subcategories.Analysis of gene structure and conserved motifs showed that the number of exons in TαABC1 family members ranged from 5 to 21;all TαABC1 family members contained motif 1,2,4,7,and 8,indicating that the motif of TαABC1 protein is relatively conservative.5.Homology analysis showed that wheat TαABC1 had higher homology with barley.The analysis of gene duplication events showed that there were 39 pairs of segmental duplication genes in the ABC1 gene family,which did not contain tandem duplication genes,indicating that segmental duplication events were the main driving force for the evolution of the TαABC1 family.In addition,the Ka/Ks of 39 pairs of segmental duplication genes were all less than 1,indicating that this family had carried out purification selection during the evolution process.6.Analysis of cis-acting elements showed that the promoter region of TαABC1 genes contain 1137 cis-regulatory elements which related to hormones and hypoxia-induced stress,and all members contained elements that related to light,abscisic acid and hypoxia stress.Protein interaction network analysis showed that TαABC1-A2,TαABC 1-D5,TαABC1-A11,TαABC1-B3,TαABC1-B12 and TαABC1-B4 interacted with other proteins.7.qRT-PCR results showed that the expression of TαABC1 genes were tissue-specific,with the highest expression levels in wheat leaves;and the family members were subjected to ABA,H2O2,PEG,heat stress and cold stress.The expression is up-regulated or down-regulated after stress,indicating that the wheat ABC1 family is involved in abiotic stresses.