Isolation And Function Analysis Of GhWRKY25 From Cotton

Land plants, being unable to move, are constantly challenged with various types of stresses. In order to survive these challenges, plants have evolved elaborate and intricate mechanisms to perceive external signals and achieve optimal adaption to these unfavorable stresses. Among these mechanisms, transcriptional modulation and stress signaling transduction are very important parts. The WRKY transcription factor is one of the largest families of transcriptional regulators in plants. Since the first WRKY cDNA, SPF1, cloned from sweet potato, more and more WRKY transcriptional factors have been identified from different species. WRKY proteins bind the cognate cis-acting W box elements(TTGACC/T),which are present in the promoter region of target genes associated with defence, thus regulated the defense process to abiotic and biotic stresses. WRKY transcription factors have also been reported to be involved in plant development and metabolism process. Most studies about WRKY transcription factors have been performed on model plants, Arabidopsis or rice,but little known about cotton, one of the most important fibre and oil crops. In the present study, upland cotton,(Gossypium hirsutum L. cv. lumian 22) was selcted as the experimental material, and a group I WRKY gene named GhWRKY25 was isolated using the homologous cloning. The multiple sequences alignment and phylogenetic analysis was performed, also did the biological function analysis. The main results are as follows:(1) Sequence analysis revealed that the full-length cDNA consisted of 1798 nucleotides,containing a 1551-bp open reading frame(ORF), a 75-bp 5’-untranslated region(5’-UTR) and a 172-bp 3’-UTR. This ORF was predicted to encode a protein of 516 amino acid residues with a putative molecular weight of 56.27 kDa and an isoelectric point of 8.47. Multiple alignments and phylogenetic analysis revealed that GhWRKY25 had high similarities with CsWRKY4, JcWRKY6, GhWRKY3 and TcWRKY3, which were group I WRKY transcription factors. A comparison between the genomic DNA sequence and cDNA sequenceshowed that there were three introns, and the insert position of the first intron was the conserved character of group I WRKY family. All of these results indicate that GhWRKY25 is a member of WRKY group I.(2) Inverse PCR was performed to isolate a 792-bp fragment of the GhWRKY25 promoter. Predictions using the PlantCARE databases revealed that some putative cis-acting elements related to low temperature, drought, gibberellin and endosperm expression existed in this region, suggesting the multiple functions of GhWRKY25.(3) The expression patterns analysis showed that the expression level of GhWRKY25 was induced by salt and low temperature, but deduced by PEG and wounding. Signaling molecules, such as GA3, 6-BA, ABA and SA, can strongly increase the transcripts level of GhWRKY25, but ET and MeJA have no significantly influence on the expression level. All these results revealed that GhWRKY25 might be involved in multiple signaling pathways and play an important role in development and defense processes.(4) Recombinant expression vector about GhWRKY25 and the report gene, GFP, was constructed and were transiently transformed into onion epidermal cells using particle bombardment. The resulting green fluorescence was visualized using fluorescence microscopy. The result showed that the GhWRKY25-GFP fusion protein emitted green fluorescence predominantly in the nucleus whereas the fluorescence of free GFP was detected almost around all cell. These results reveal that the GhWRKY25 protein is localized in the nucleus.(5) The GhWRKY25 cDNA was inserted into the binary vector pBI121 for the construction of the recombinant vector pBI121-GhWRKY25, and then the recombinant vector was transformed to the Nicotiana benthamiana using A. tumefaciens-mediated leaf disk method. Result of RT-PCR analysis showed that GhWRKY25 was successfully expressed in transgenic plants. Comparison with the wild type plant, overexpression of GhWRKY25 in Nicotiana benthamiana deduced its tolerance to drought stress, as indicted by the lower survival rate, lower relative water content and higher water loss rate. All of these results suggested GhWRKY25 might act as a negative regulator in regulating the drought stress.(6) Comparison with the wild type plant, the ROS level in transgenic plants was much higher after the drought treatment. The expression level of the genes related to the ROSproduction was higher in transgenic plants, and the expression level of the genes related to the ROS eliminating was lower in transgenic plants. The activities of antioxidases were also lower in transgenic plants after drought treatment. All of these results suggested that GhWRKY25 might participate in the ROS signaling pathway.(7) Overexpression of GhWRKY25 in transgenic plants enhanced salt tolerance. This result revealed that GhWRKY25 might act as a positive regulator in regulating the salt stress.(8) Comparison with the wild type plant, GhWRKY25-overexpressing plants enhanced the susceptibility to the infection of Botrytis cinerea. After infection, the expression level of the genes related to the SA signaling pathway was lower in transgenic plants, and the expression level of the genes related to the JA/ET signaling pathway was higher in transgenic plants. All of the results revealed that the cross talk between SA- and JA/ET-mediated signaling pathway might contribute to the enhanced pathogen susceptibility of the GhWRKY25-overexpressing plants.In summary, overexpression of GhWRKY25 enhanced salt tolerance, drought sensitivity and pathogen susceptibility. This study will broaden our knowledge about the function of the group I WRKY transcription factors, and provide a basis for the construction of the transgenic cotton with the high stress and pathogen resistance.