关键词: 陆地棉, GhWRKY33, 胁迫, 抗旱分析

Abstract:

【Objective】In this study, GhWRKY33 gene in upland cotton was cloned, and its function was analyzed in the drought resistance, which will lay a foundation for the mechanism dissection of drought resistance and molecular breeding in cotton.【Method】The ORF of GhWRKY33 was cloned from Upland cotton CCRI10 by homologous cloning method. Bioinformatics analysis was carried out to analyze the secondary structure and hydrophobicity, and to predict the phosphorylation sites and cis-acting elements in the promoter region. In the NCBI website, the protein sequences with high homology were searched by BLASTP for sequence alignment, and the phylogenetic tree was constructed. The expression vector of 35S::GhWRKY33-GFP fusion protein was constructed and injected into tobacco leaves mediated by Agrobacterium tumefaciens to observe the fluorescence signal. qRT-PCR was conducted to detect the tissue-specific expression of the gene and the expression pattern under drought, ABA, JA and ET treatments. Plant overexpression vector was constructed and transformed into Arabidopsis thaliana. T₃ transgenic lines were performed to observe the phenotype after drought treatment and to determine the physiological and biochemical indexes such as the content of proline and malondialdehyde. The expression levels of drought response genes AtP5CS, AtRD29A and AtCOR15A were identified in the wild type and transgenic lines before and after PEG treatment. 【Result】The open reading frame (ORF) of GhWRKY33 gene was 1533 bp, which encoded 510 amino acid residues. GhWRKY33 protein included two WRKY conserved domains and two C₂H₂ zinc finger structures, and belonged to the family of type I WRKY transcription factors. Secondary structure prediction showed that GhWRKY33 was mainly composed of random curl and contained 26 threonine phosphorylation sites, which was closely related to phosphorylation. The hydrophobicity prediction showed that the protein belonged to a hydrophilic protein. Phylogenetic tree analysis displayed that the gene had the highest homology with GrWRKY33. The GhWRKY33 protein was located in the nucleus. qRT-PCR showed that the expression of the gene possessed tissue specificity and was the highest in the apical bud tissue, and significantly increased after drought, JA and ET treatments. Compared to the wild type, the transgenic lines displayed stronger drought resistance, and the wilting degree of the plants was weaker. Under drought treatment, the content of proline was significantly higher than that of wild type, while the content of MDA was lower than that of wild type. The expression levels of GhWRKY33 and drought response genes AtP5CS, AtRD29A and AtCOR15A were significantly increased. It was speculated that PEG could induce the expression of GhWRKY33, and then regulated the up-regulation expression of the drought response genes, thus, transgenic Arabidopsis thaliana showed higher resistance to drought stress. 【Conclusion】GhWRKY33 responded to drought stress and improved the drought resistance of transgenic Arabidopsis thaliana.

Key words: upland cotton, GhWRKY33, stress, drought resistance analysis