Identification, Cloning Of Wheat WRKY Transcription Factors Families And Functional Analysis Of TaWRKY10Gene In Wheat (Triticum Aestivum L.)

Environmental stresses, including drought, salinity and low temperature, are the primary causes of declines in crop yield and quality worldwide. To combat these challenges, plants have evolved sophisticated molecular networks resulting in adaptive responses through physiological and morphological changes. The adaptive responses commonly use transcriptional activation or repression of genes upon signal perception and transduction of the external stimuli. Plant adaptability is mainly operated by the regulation of various transcription factors. Significant progress has been made in understanding the genes response to various stresses, and numerous transcription factors and cis-regulatory sequences in plants have been identified. Among them, the plant WRKY transcription factors, comprising a large family of regulatory proteins, are shown to play an important role in response to various stresses. However, the mechanisms for its action remain unclear and less research on WRKY in wheat has been reported because of its complicated hexaploid nature.Wheat is one of the the staple crop for human food and livestock feed. Current and future concerns include improving wheat yield and quality under hostile environments. Considering the diverse roles of WRKY transcription factors under complex environmental conditions, identification of WRKY transcription factor gene family and clarifying the functions of each WRKY members in wheat remains a challenge.The perpose of present study is to investigate whether wheat WRKY genes can confer abiotic stress tolerances. Firstly, after searching for WRKY domains, ten WRKY transcription factors were identified from wheat database. We analysed the relationship between WRKY transcription factor family genes and abiotic stress in model wheat variety Chinese Spring. Furthermore, we characterized the function of TaWRKY10in abiotic stresses by using genetic, physiological and biochemical and molecular approaches. The main results are as follows.After searching for WRKY domains and eliminating repeats in the DFCI database and the wheat genome database, full-length cDNAs of ten WRKY transcription factors were successfully cloned byRT-PCR using mRNA isolated from wheat leaves. They were designated TaWRKY1-TaWRKY10, respectively. Domain prediction of the full-length deduced proteins of the TaWRKYs clearly showed that these proteins contained the conserved WRKY DNA-binding domain and zinc finger region. These WRKYs were further divided into three subgroups:TaWRKY4,8, and9belong to group Ⅰ; TaWRKY1,2,3,6, and10belong to subgroup II; and TaWRKY5and7belong to group III.The TaWRKY10cDNA is791bp in length (GenBank accession no. HQ700327), including a complete ORF of672bp encoding a putative protein of223amino acids. The alignment results revealed that the TaWRKY10protein contains a conserved DNA-binding domain (WRKY domain) of60amino acids and a zinc finger region (C-X4-C-X23-H-X-H). TaWRKY10is very similar to other WRKY proteins, such as WRKYs Hordeum vulgare and Zea mays.TaWRKY10was detected to have varying degrees of expression levels in root, stem and leaves of10-day-old seedlings. Further characterization of TaWRKY10showed that the TaWRKY10gene was induced by PEG6000, NaCl, cold and H2O2treatment. Southern blot assay of genomic DNA was performed. The result revealed that TaWRKY10was existed as three copies in the genomes of hexaploid wheat. The TaWRKY10-GFP fusion construct was bombarded into onion epidermal cells. The nucleus location of TaWRKY10-GFP was confirmed by GFP and DAPI merging images showing a complete match. Yeast two hybrid experiment result showed that the His and LacZ reporter genes were activated by full-length TaWRKY10protein and N-terminal domain, indicating that TaWRKY10is functional transcription factor and its transcriptional active domain is located at its N-terminus.To further investigate the role of TaWRKY10in drought/osmotic stress tolerance, transgenic tobacco plants over-expressing TaWRKY10under the control of CaMV35S promoter were generated. Over-expression of TaWRKY10in tobacco resulted in increased drought/osmotic tolerance, which was demonstrated that transgenic lines had higher seed germination rate and longer root length.After drought or salt treatment for three weeks, compared with transgenic plants, the wild type plants were smaller and more withered. The transgenic lines exhibited lower rates of leaf yellowing and higher survival rates than wild type lines under drought and salt treatments. Compared with the wild type plants, the transgenic lines showed remarkably higher levels of RWC, proline and soluble sugar, but lower levels of MDA under drought and salt conditions. After exposure to drought or salt treatments, the ROS level of wild type plants accumulated greater than transgenic seedlings. The expression of three genes in overexpressed seedlings was obviously higher in transgenic lines than in control plants. The expression of NtERD10C, NtSPSA and NtGPX was significant higher than wild type under normal condition, suggesting that TaWRKY10constitutively induced the expression of osmotic stress and oxidative stress genes in tobacco plants.To study the function of TaWRKY10in wheat, we constructed pAHC25-TaWRKY10over-expression vector, and bombarded it into wheat (Zhengmai9023). The PCR and Western Blot results of T1generation wheat showed that the TaWRKY10gene inherit stably. The transgenic wheat offspring breeding work is presently in operation.In conclusion, the TaWRKY10conferred drought, osmotic and oxdidative stress tolerance through regulating the osmotic balance, ROS scavenging and transcription of stress related genes thus preventing plants from oxidative damage.