Identification And Function Analysis Of GhWRKY44, A WRKY Transcription Factor From Cotton

Plants constantly encounter various unfavorable environmental factors that affect growth and metabolism of plants. Plants have evolved intricate mechanisms that increase tolerance in order to adapt to a variety of biotic and abiotic stresses. Transcriptional modulation plays a pivotal role in plant defense responses. The WRKY is a superfamily of transcriptional factors in plants. WRKY proteins specifically bind to the W-box cis-elements to change the expression of related genes and to regulate the physiological and biochemical responses. Many reports indicated that WRKY transcription factors play essential roles in response to abiotic and biotic stresses, such as pathogens infection, salinity and drought. So far, many researches about WRKY have been reported, but studies of WRKY in cotton are limited. Cotton (Gossypium hirsutum L.) is the important fiber and oil crop in the world. Its growth and yield are severely inhibited by various adverse environmental factors. In this study, we isolated a WRKY transcription factor, GhWRKY44, from cotton. The sequence analysis, expression patterns and functions identification of GhWRKY44have been conducted. The main results are as follows:(1) The full-length cDNA sequence of WRKY44gene was successfully obtained by the homology cloning. The deduced full-length sequence of cDNA fragment consisted of1,716nucleotides, including188bp5’-untranslated region (UTR),127bp3’-UTR, and1,401bp open reading frame (ORF). This cDNA encodes a polypeptide of467amino acids with a putative molecular weight of51.39kDa and an isoelectric point of8.76. Alignment analysis and phylogenetic tree analysis revealed that GhWRKY44belonged to group I WRKY family. Subcellular localization indicated that GhWRKY44is a nuclear protein, which might function in the nucleus.(2) A1418bp fragment of GhWRKY44promoter was also determined from cotton genomic DNA by hiTAIL-PCR method. PlantCARE databases predicted various cis-elements associated with defense responses. These data suggest that GhWRKY44maybe in function multiple signal transduction pathways.(3) To identify the expression patterns of GhWRKY44gene, semi-quantitative RT-PCR was performed. The expression patterns showed that GhWRKY44can be induced by biotic stimuli, such as the bacterial pathogen R. Solanacearum and fungal pathogen R. solani; diverse signalling molecules, such as jasmonate (JA), methyl jasmonate (MeJA), ethylene (ET), and Hydrogen Peroxide (H2O2); abiotic stimuli, such as salt and drought. Overall, these results indicated that GhWRKY44may be involved in responses to multiple abiotic and biotic stresses.(4) To better understand GhWRKY44function, a sense expression vector pBI-GhWRKY44was constructed and transformed into Nicotiana benthamiana with Agrobacterium tumefaciens-mediated leaf disc method. Three independent lines were used for functional analysis. Overepression of GhWRKY44enhances plant resistance to R. Solanacearum and R. solani compared with wild-type plants. These results indicated that GhWRKY44may be the positive regulator in disease response.(5) The DAB and NBT staining were used to detect the cellular levels accumulation of H2O2and O2-in transgenic plants after inoculation with R. solanacearum and R. solani. These results showed that GhWRKY44-overexpressing plants decreased the levels of H2O2and O2-accumulation and reduced the ROS-associated oxidative injury.(6) The overepression of GhWRKY44exhibited increased RNA expression of SA and JA synthesis-related genes in transgenic plants, such as NPR1, PR1, PR2, PR4and PR5. These results showed that GhWRKY44may be involved in regulating plant defense responses through SA-and JA-mediated pathways.