Functional Research Of GbWRKY1 Gene From Gossypium Barbadense In Verticillium Wilt Resistance

Cotton is an important economic crop in China.Its yield and fiber quality are seriously affected by Verticillium wilt.It is of great significance to improve the resistance of cotton to Verticillium wilt for achieving the goal of high yield and high quality.In this study,we have cloned the transcription factor GbWRKY1 gene in response to Verticillium dahliae,and preliminarily studied its function and mechanism of cotton resistance to Verticillium wilt,and the main results were as follows:1.The full-length coding region of WRKY1 gene was cloned from the genomic DNA of resistant variety Gossypium barbadense Pima90-53 and susceptible variety Gossypium hirsutum CRI8,respectively.The full length of GbWRKY1 and GhWRKY1genes were 1938bp and 1890 bp,respectively,which contained three introns and four exons.Sequence alignment analysis showed that GhWRKY1 was 13 bases less than GbWRKY1 in exon 4,leading to the early termination of GhWRKY1translation,resulting in 15 amino acid residues less in the predicted protein of GhWRKY1 than that of GbWRKY1.Both proteins contain two WRKY conserved domains and two C₂H₂(C-X_4-5-C-X_22-23-H-X₁-H)zinc finger structures,belonging to subgroupⅠof WRKY transcription factor family.2.The WRKY1 promoter sequences of resistant and susceptible cotton varieties were predicted and analyzed.It was found that both of them contained plant hormone regulatory elements,biotic/abiotic stress response elements,plant growth and development response elements and light response cis acting elements.It was found that the ethylene element contained in GbWRKY1 was absent in the promoter sequence of GhWRKY1.3.The expression patterns of WRKY1 gene in Pima90-53 and CRI8 were the same when the cotton seedlings were treated with Salicylic acid（SA）or Methyl jasmonate（Me JA）,and the expression levels of WRKY1 gene in Pima90-53 and CRI8 were not significantly different.The expression patterns of WRKY1 gene in Pima90-53 and CRI8 were different after Verticillium dahliae stress or ACC hormone treatment,and the expression level of WRKY1 gene in Pima90-53 was significantly higher than that in CRI8.Therefore,the expression patterns of WRKY1 gene in resistant and susceptible cotton varieties were different after Verticillium dahliae stress,which may be mainly related to Ethylene signaling pathway.4.The endogenous GbWRKY1 of Pima90-53 was silenced by VIGS technology.Compared with the control plants,the gene silencing plants showed significant increase in disease index and increased incidence rate,and the degree of vascular Brown browning was serious.The expression levels of NPR1 and PR1 in SA signaling pathway in GbWRKY1gene silenced plants were significantly higher than those in control plants,while the expression levels of MYC2,AOS,JAZ1,PR4 and ACSgenes in JA and ET signaling pathways were significantly lower than those in control plants.These results indicate that GbWRKY1 gene is involved in the resistance of cotton to Verticillium wilt through SA,JA and ET signaling pathways.5.The root growth of transgenic Arabidopsis thaliana lines with GbWRKY1 gene was more vigorous than that of WT.The relative shortening of inflorescence,relative reduction of fresh weight,disease index and disease rate of transgenic Arabidopsis thaliana were lower than those of WT.The expression of PR4 gene in transgenic plants was significantly higher than that in WT at 48 hpi and 72 hpi,the expression of PR5 gene in transgenic Arabidopsis thaliana was significantly lower than that in WT at 48 hpi and 72 hpi,and the expression of ACS2 gene in transgenic Arabidopsis thaliana was significantly higher than that in WT at 12 hpi and 24 hpi.In addition,the POD activity and lignin content of transgenic Arabidopsis thaliana plants infected with Verticillium dahliae were significantly higher than those of WT.The results showed that GbWRKY1 could improve the resistance of plants to Verticillium wilt mainly by participating in ET and other signaling pathways to regulate plant growth and development,affecting the activities of cell protective enzymes and the transcription of genes encoding resistance proteins.