| Cotton is an important economic crop in the world.Many factors,such as biotic stress,abiotic stress and premature senescence have seriously affected the yield and quality of cotton.According to previous studies,WRKY transcription factor is one of the largest transcription factor families in plants,which consists of one or two conserved WRKY domains.The N-terminal is a highly conserved sequence,(WRKYGQK),and the C-terminal is a zinc finger structure(CX4-5CX22-23HX1H or CX7CX23HX1C).According to the different characteristics of the number of conserved WRKY domains and zinc finger sequences,WRKY proteins can be divided into three groups.Group I has two WRKY domains,the zinc finger structure type is C2H2,the second group and the third group contain a WRKY domain,and the zinc finger structure types are C2H2 and C2HC,respectively.According to the evolutionary relationship of WRKY domain and some amino acid motifs,the second group can be divided into five subgroups:Ⅱ a,Ⅱ b,Ⅱ c,Ⅱ d,Ⅱ e.WRKY transcription factors are involved in plant defense responses in a variety of plants,including high temperature,low temperature,drought,saline-alkali stress,as well as plant senescence.However,little is known about WRKY transcription factors involved in leaf senescence in cotton.Through the whole genome identification and analysis of cotton WRKY transcription factor,combined with a transcriptome database of leaf senescence,the up-regulated GhWRKY33 gene expression under cotton leaf senescence and abiotic stress was obtained.In this study,GhWRKY33 gene was cloned from cotton,and its function was analyzed,and the results were as follows:1.GhWRKY33 was cloned from upland cotton CCRI10.The ORF was 1533 bp and encoded 510 amino acid residues.GhWRKY33 contained two WRKY conserved domains and possessed two C2H2-type Znic finger structure.The gene belonged to the family of type I WRKY transcription factors.2.The 35S::GhWRKY33-GFP fusion expression vector was constructed and injected into tobacco leaves mediated by Agrobacterium tumefaciens.The results showed that the gene was located in the nucleus,indicating that the gene plays a role in the nucleus.The fluorescence quantitative results of tissue-specific expression showed that the expression of GhWRKY33 was the highest in buds,the second in stamens,pistils and leaves,and the lowest in roots and stems.The gene expression was up-regulated by PEG,ethylene,jasmonic acid and abscisic acid.3.The 2000-bp length promoter sequence of GhWRKY33was cloned and analyzed by PlantCARE software.It was found that there were several cis-acting elements,such as the element involved in abscisic acid regulation response,the element ABER,involved in light response,the MYB binding site involved in drought stress response,the element CAT-box related to plant tissue meristem expression,and so on.pBI121 vector,in which CaMV35S was replaced by the GhWRKY33promoter,was transformed into pre-cultured onion epidermal cells by Agrobacterium tumefaciensmediated method,and GUS staining test was carried out.The results showed that this fragment could promote the expression of GUS and had the function of promoter.4.The overexpression vector of GhWRKY33 gene was constructed and transformed into Arabidopsisthaliana by Agrobacterium tumefaciens.The results showed that the natural senescence of transgenic Arabidopsis thaliana was delayed,and the expression of senescence-related genes AtSA G12 and AtSAG13 was down-regulated.PEG6000 was used to simulate drought treatment.The results showed that the drought resistance of transgenic Arabidopsis thaliana was enhanced,and the expression of drought stress-related genes AtRD29A,AtCOR1 5A and AtP5CS was up-regulated.In summary,the GhWRKY33 gene was cloned from upland cotton,which belonged to the typical class I WRKY transcription factor.It had the highest expression level in the apical bud and obvious tissue expression specificity.This gene responded to drought,ethylene,jasmonic acid and abscisic acid induction.GhWRKY33was located in the nucleus by subcellular localization.Gh WRKY33 was used as a positive regulator to regulate drought resistance in Arabidopsis thalianaand as a negative regulator to regulate Arabidopsis thaliana senescence.. |
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