Isolation And Expression Analysis Of A Novel Gene, Ghwrky3, In Cotton (gossypium Hirsutum L.)

WRKY transcription superfamily mainly exists in the plant. It had been extensive studied since the first WRKY transcription factor named SPF1 identified from sweet potato by Ishiguro and Nakamura. Now, many WRKY genes had been identified from various plants and their functions had been elucidated using genetic and molecular approaches. The increasing data proved that WRKY transcription factors are involved in various biological processes, especially in disease defense responses. In the present study, we isolated a novel WRKY gene, GhWRKY3, from cotton, and then we analyzed its expression patterns and functions. The main results as follows:1. A novel WRKY gene, named GhWRKY3, was isolated from cotton (Gossypium hirsutum L). The deduced full-length cDNA sequence of WRKY gene consisted of 1,705 nucleotides, containing a 72 bp 5′untranslated region (UTR) and a 109 bp 3′UTR. The GhWRKY3 cDNA contains a 1,524 bp ORF encoding a protein of 507 amino acid residues. The deduced GhWRKY3 was found to possess two WRKY domains and two putative zinc finger motifs(C-X₄-C-X_22–23-H-X₁-H). In addition, the deduced GhWRKY3 protein sequence contains a putative nuclear localization signal (NLS), PKRR. Comparison of the genomic sequence and cDNA sequence of GhWRKY3 gene showed that there were three introns and four extrons.2. Using I-PCR and nested PCR, a 720bp fragment of 5′-flanking region of GhWRKY3 gene was isolated from cotton genomic DNA. Then, several putative cis-acting elements involved in ABA-responsiveness, light responsiveness, defense and stress responsiveness and endosperm expression were predicted in 5′-flanking region using the PlantCARE databases. The basic cis-acting elements TATA-box and CAAT-box also existed in this region. The results suggested that GhWRKY3 may be involved in plant defense responses and development processes.3. Semi-quantitative RT-PCR showed that the transcripts of GhWRKY3 could be up-regulated by SA, ABA, MeJA, ET, GA₃ and H₂O₂. Furthermore, the expression of GhWRKY3 was also induced by wounding and infection with three fungal pathogens including Rhizoctonia solani, Colletotrichum gossypii, and Fusarium oxysporum f. sp. vasinfectum, but not induced by 6-BA, NAA, drought, NaCl, and cold.4. A sense expression vector pBI121-GhWRKY3 was constructed, and was transformed into tobacco. Using PCR and semi-quantitative RT-PCR methods, the identification of the transgenic plants were verified, and the results showed that GhWRKY3 had been expressed successfully in transgenic plant.5. Several T₁ transgenic plants were obtained by self-reproduction of T0 transgenic plant seeds. Based on the results of molecular identification, the separation ratio of T₁ transgenic plants is 3:1. And the result above is consistent with the law of segregation. Seveal seeds of T₁ transgenic plants were selected for analyzing the germination and the growth situations of the seedlings, the results showed that the transgentic plant germinated earlier than wild plant. Analysis of germination and the growth situations of plant on MS with different concentration of ABA or GA3, the results suggested that GhWRKY3 may be involved in regulating plant growth and development processes mediated by ABA and GA.