Isolation, Expression Analysis And Genetic Transformation Of The TaWRKY1Gene In Wheat(Triticum Aestivum L.)

The WRKY family is one of the biggest groups of plant-specific transcriptional regulators. The defining feature of WRKY transcription factors is their DNA binding domain.WRKY, Which binds specifically to the W-box elements in the promoter regions of the target genes. WRKY genes are considerd to play an regulatory functions role in response to biotic and abiotic stresses. However, no systematic identification, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated one wheat WRKY gene with complete open reading frame encoding a putative WRKY protein. Bioinformatic analysis and expression patterns in different organs of wheat were carried out. To futher elucidate the biological function of this WRKY gene, we constructed overexpression vector and transform it into wheat. The main results were as follows.(1) cDNA cloning:Wheat ESTs homologous to WRKY were retrieved from TIGR, on the basis of the almost invariant WRKY amino acid sequences. The overlapped ESTs were assembled into contigs with putative open reading frames(ORFs). Finally we got the cDNA of one WRKY gene by RT-PCT. Sequence alignment showeed that this gene shared82%homology in the region of81%at the amino acid level with OsWRKY1,and thus designated TaWRKY1. The cDNA sequence of TaWRKY1contained a entire ORFs in length of1113bp, and was predicted to encode a polypeptide of371amino acid residues consisting of two WRKY domains, each with a zinc finger motif of C2H2belongs to the WRKY subgroup I.(2) Expression patterns, analysis:Semi-quantitative RT-PCR was performed to examine its expression pattern in different tissues. The result showed that TaWRKY1is constitutive expressed in the leaves, steams and roots and the leaves had highest leave. To analyse the expression of TaWRKY1under various abiotic stress, the wheat seedlings were treated with Nacl(100mM), ABA (100μM), PEG-6000(20%) and cold(4℃), respectively and RT-PCR analysis was conducted; and the results indicated that the expression of TaWRKY1was markedly upregulated after treatment with ABA at1h maintained till6h after treatment, and finally recovered to a normal leavels at12h after treatment. While the expression of TaWRKY1shows no obvious changes to the other three stress treatments.(3) Transgenic research:To further analyse the roles of TaWKRY1under stress, The cDNA of TaWKRY1gene was subcloned into expression vector PBI121.The resulting vector thus named PBI121-TaWKRY1, harbors the TaWKRY1under the control of the CaMV35S promoter and also contains a gus gene as a selectable marker. This PBI121-TaWKRY1plasmid was transformed into immature embryos of zhengmai9023by particle bombardment. After tissue culture, regenerated plants were obtained. Finally, two positive TaWRKY1transgenic plants were confirmed by PCR technology.Results of present study might provide usuful information for studying the biological function and regulatory mechanisms of TaWRKY1in the future.