Isolation And Functional Analysis Of GhWRKY23,A Transcription Factor Gene From Cotton

During growth and development,cotton constantly suffer various stresses,including biotic stresses(such as Verticillium dahliae,Fusarium wilt of cotton,cotton rhizoctonia and phytophagous insects bite)and abiotic stresses(such as drought and salt stresses).And these stresses bring some adverse effects on the quality and yield of cotton.So,breeding new varieties with stress-tolerance is an effective approach to solve cotton diseases problems.While,these approaches depend on the knowledge of molecular mechanisms underlying disease resistance and stress tolerance.The gene expression regulation,on signal transduction and transcription levels,play vital roles in plant defense responses.Transcription factors such as DREB,NAC,WRKY and MYB play important roles in the regulation of defense response induction.Isolating and identifying the function of transcription factors involved in the stress response provide an important way to uncover the molecular mechanism of plant stress response.Therefore,in our present study,we choose Gossypium hirsutum L.as the experiment material,and cloned a group II WRKY gene.A series of studies have been carried out on its sequence analysis,expression pattern analysis and function identification,the main results are as follows:(1)Sequence analysis revealed that the full-length cDNA of GhWRKY23 was 1194 bp long,containing a 121 bp 5'UTR,a 125 bp 3'UTR and a 948 bp open reading frame that endodes a 315 amino acid protein with the predicted molecular weight of 35.6kDa and an isoelectric point of 5.94.The genomic structure analysis,homologous sequence alignments and phylogenetic analysis revealed that the gene GhWRKY23 belongs to group IIc WRKY family.(2)The subcelluar localization of GhWRKY23 was detected in nucleus,suggesting that it may function in nucleus.(3)The transcriptional patterns of GhWRKY23 were detected by qRT-PCR,and the result revealed that drought,wounding and oxidative stresses could induce the expression level of GhWRKY23.High salinity,R.solanacearum and R.solani could suppress the transcription ofGhWRKY23.Moreover,defense-related signaling molecules including SA,MeJA,ABA and ET could induce GhWRKY23 expression.These results suggested that GhWRKY23 could respond to various biotic and abiotic stresses,and might be regulated by several signaling molecules.We speculate that GhWRKY23 play important roles in defense response.(4)A sense expression vector pBI-GhWRKY23 was constructed and transformed into Nicotiana benthamiana with Agrobacterium tumefaciens-mediated leaf disc method.The seed germination rate and root length of overexpression lines indicated that GhWRKY23 was sensitive to SA and ET,but resistant to ABA.We predict that GhWRKY23 is involved in the signaling pathways of SA,ABA and ET.(5)Detached leaves of GhWRKY23 overexpressing plants presented more severe symptoms after disease infection.Histochemical staining procedures(DAB staining and trypan blue staining)further confirmed that the overexpressing plants accumulated more ROS and formed more necrotic cells.MDA content detection also revealed a heavier oxidative damage on cell membranes in overexpressing lines.Furthermore,the accumulation of H2O2 in leaves after MV treatment indicated that transgenic lines are much easier to suffer oxidative stress.So we predict GhWRKY23 is a negative regulator in disease resistance and may be involved in the response to oxidative stress.(6)Transcriptional analysis of defense related genes in SA,ABA and ET signaling pathways before and after disease infection revealed that GhWRKY23 might play negative roles in defense response by suppress the expression of some relevant genes in SA,ABA and ET signaling pathways.(7)The down-regulation of ROS generation related gene and antioxidant related genes after disease infection revealed that GhWRKY23 might negatively regulate antioxidant system.