Isolation And Functional Analyses Of GhWRKY39in Cotton

Environmental stresses (e.g. pathogen infection and high salinity) cause serious damage on plant growth, and plants have developed a series of mechanisms to regulate their own growth and development, defense biotic and abiotic stresses in the long evolutionary process. Among the adaptive mechanisms, gene expression in the transcription regulation plays an important role in the response of plants to environmental signals stimulation. Transcription factors are the most important regulatory genes, and WRKY transcription factor, being plant-specific, has been received a wide attention in recent years. WRKY proteins specifically bind to the W-box in target gene promoters to participate in plant defense response and regulate plant development. A large body evidence have shown that WRKY transcription factor acts as an important regulator in the response to biotic and abiotic stresses. However, previous studies concerning WRKYs have primarily focused on the model plants (e.g. Arabidopsis and rice), and fairly limited research has been reported in cotton.In the present study, the economically important species cotton (Gossypium hirsutum L. cv. lumian22) was selected as the experiment material, and a WRKY family gene named GhWRKY39was isolated by homologous cloning, and biological function analyses were performed. The main results are as follows:(1) Sequence analysis indicated that the full-length cDNA of GhWRKY39consisted of1348nucleotides that contained a972bp open reading frame encoding a324amino acid polypeptide with a predicted molecular mass of36.225kDa. GhWRKY39contained a typical WRKY domain and zinc finger motif as well as the distinct characteristic of the WRKY Group IId, C-region. Phylogenetic analysis indicated that GhWRKY39was highly similar to Group IId WRKY family members. For the putative WRKY clone from cotton was highly homologous to WRKY39of Arabidopsis thaliana and Brassica napus, so it was designated as GhWRKY39. Quantitative PCR was performed to examine the copy number of GhWRKY39and the result indicated that it is likely a single-copy gene in cotton. A transient expression analysis in onion epidermal cells indicated that GhWRKY39was located in the nucleus. (2) A1377bp fragment of the GHWRKY39promoter was obtained using high-efficiency TAIL-PCR (hiTAIL-PCR). Predictions using the PlantCARE databases revealed that different putative cis-acting elements existed in this region, including development-related, biotic and abiotic stresses responsive elements.(3) Glucuronidase (GUS) activity assays indicated that GhWRKY39could be induced by pathogen infection and high salt stress; Quantitative PCR (qPCR) anaslysis further indicated that the expression of GhWRKY39was induced by biotic and abiotic stresses.(4) The constitutive overexpression of GHWRKY39in Nicotiana benthamiana conferred more resistance to bacterial and fungal pathogen infections; The transgenic plants also exhibited elevated mRNA accumulation of several pathogenesis-related (PR) genes and non-expression of PR1gene (NPR1).(5) Following the treatment of bacterial and fungal pathogen, transgenic plants exhibited much less H2O2accumulation and higher activities of antioxidant enzymes (SOD, POD and CAT) compared with the wild type plants.(6) GhWRKY39-oxerexpressing plants displayed enhanced tolerance to salt stress with a higher germination rate at the germination stage, a higher root length at the seeding stage and more tolerance to high salt stress at the vegetable-growth stage.(7) GhWRKY39-overexpressing plants exhibited less accumulation of ROS and less oxidative damage compared with WT plants under high salt stress, higher transcription and activities of antioxidant enzymes were displayed in transgenic plants than WT plants.