Cloning And Characterization Of Transcription Factor TaWRKY35 In Wheat

Owing to climate change, drought, high salinity and extreme temperatures are major limitations to wheat production worldwide. Wheat is one of the most important food crops in the world; however, its yield fluctuation severely impacts on global food security. Mining and utilization of stress responsive genes to enhance the tolerances to abiotic stresse is a promising way to protect food security, as well as benefiting our understanding of the molecular mechanism for abiotic stress resistance, and facilitating the improvement in crop production. Transcription factors play an important role in the regulation of plant growth and development, physiological and biochemical responses to external environmental stimuli. Here, our aim is to decipher the roles of WRKY transcription factors in abiotic stress signaling and facilitates the utilization WRKY genes in the improvement of abiotic stress tolerance in wheat.A wheat WRKY gene designated TaWRKY35 was cloned via the wheat full-length cDNA libraries. By characterizing gene sequence, expression patterns, subcellular localization, chromosomal origination, and phenotypic traits of transgenics, we initially discovered TaWRKY35's expression patterns, function and potential regulation of signaling network under abiotic stress. Our results are mainly as follows:1. The cDNA of Ta WRYK35 contains an 1134 bp open reading frame, encoding a 377-amino acid protein. The molecular weight of TaWRKY35 is about 40 k Da, and isoelectric point is 6.50. Ta WRYK35 possesses a typical WRKY domain in the N-terminal and a C2 HC type zinc finger domain in the C-terminal, belonging to group ? of WRKY superfamily. Phynogenetic analysis indicates TaWRKY35 has 79%?60%, 50% and 45% similarity to the counterparts from barly, Brachipodium, rice and Arabidopsis.2. The dynamic expression of TaWRKY35 was identified in different tissues at various developmental stages, and the highest expression occurred in the root base of seedlings, while the lowest expression was observed in seedling leaf. Furthermore, its transcript was inducible by ABA, PEG, Na Cl and low temperature treatments, yet the expression patterns to difference stress varied significantly.3. Bioinformatics prediction results showed TaWRKY35 may be located in the nucleus. Subcellular localization demonstrated that TaWRKY35 specifically located in the nucleus.4. Yeast one-hybrid experiment showed that both the N-terminal and C-terminal flanking the WRKY domain possessed self-activation ability, yet the WRKY domain has no self-activation capability. With the construct harbing the WRKY domain, four candidate proteins probably interacting with TaWRKY35 were identified via screening the yeast library constructed with the full-length cDNA of wheat.5. Transgenic results show there is no difference between WT and TaWRKY35 transgenics under normal growing conditions, however, the rates of whiten cotyledon for transgenic plants was significantly lower than the control, the survival rate and cell membrane stability of transgenic plants were significantly higher than WT under salt stress conditions, suggesting overexpression of TaWRKY35 results in enhanced tolerance to high salinity in Arabidopsis. Additionally, there is no difference between transgenic plants and WT under low temperature, ABA and osmotic stress conditions on MS medium.6. A pair of gene-specific primers was introuduced to amplify the genomic sequence of TaWRKY35. The results showed the target band could only be amplified in wheat species containing A genome, suggesting TaWRKY35 may be originate from A genome. The genomic fragement of TaWRKY35 is 2.55 kb containing three exons and two introns. To probe the nucleotide polymorphism of TaWRKY35, 32 wheat accessions with high genetic diversity were used to detect single nucleotide polymorphism(SNP), and no SNP was identified. The high conservative property of TaWRKY35 reveals that it might play crucial roles in plant growth and development.To sum up, our experimental results show that TaWRKY35 can significantly enhance salt tolerance without negative effects in transgenic plants, thus it can be used as a candidate gene to improve the tolerance to salt stress in crops.