Isolation And Functional Analysis Of PtWRKY89in Populus Trichocarpa

WRKY transcription factors are one of the largest groups in plant transcription factor families. All of WRKY proteins contain the highly conserved amino acid sequence of WRKYGQK in N-terminus. The WRKY domain can bind to the W-box of promotors of the downstream genes and regulate expression of these genes. Based on the previous studies published, WRKY TFs play a role in regμlating the physiological functions including biotic stress, abiotic stress, seeds development, seeds dormancy and germination, morphogenesis, senescence and so on.It is demonstrated well that AtWRKY10in Arabidopsis thaliana modμlated many important plant physiology processes, especially for plant defense response. Compared with the wild type Arabidopsis, transgenic plants overexpressed AtWRKY70showed an increased resistance to fungal diseases such as Hyaloperonospora parasitica, Pseudomonas syringae, but a more susceptible symptom to bacterial diseases like Erwinia amylovora. In this study, a putative WRKY gene, PtWRKY89, which is similar to AtWRKY70in amino acid sequence, was found from Popμlus tricocarpa Torr. However, the function of PtWRKY89is still poorly understood so far. Here we characterized the expression profiles of PtWRKY89and identified its nucleus localization signal (NLS). Furthermore, the plant expression vector was constructed for genetic transformation in P. tricocarpa Carr. The role of PtWRKY89in biotic defense has been investigated in transgenic poplar plants.The detailed resμlts as follows:1. The expression pattern of PtWRKY89Tissue expression profiles of indicates PtWRKY89is expressed in various tissues of P. tricocarpa and the highest expression was observed in leaves. Different signaling molecμlar including salicylic acid, jasmonic acid, Marssonina brunnea and wounding, cold, salt stress have been applied for induction of PtWRKY89in Popμlus tricocarpa Torr. Extract the total RNA of the treated poplar and reverse transcript it into cDNA and detected the transcript level of PtWRKY89by fluorescence quantitative PCR. PtWRKY89is seen to be only response to salicylic acid treatment. The leaves of poplar were treated with H2O and salicylic acid after2h,5h,8h and24h respectively. The total RNA was extracted and reverse transcript to cDNA, finally we have observed the transcript level of PtWRKY89via semi-quantitative PCR analysis. Our resμlt indicated that the transcript level behaves as a parabolic among24h after salicylic acid treatment. PtWRKY89expression was highest at5-8h interval and started to diminish after24h of treatment.2. Isolation and characterization of PtWRKY89We designed the primers for fμll length fragment amplification of PtWRKY89based on the nucleotides sequence provided from the public database on www.phytozome.com. We extracted the total RNA from poplar and reverse transcript to cDNA. The1108bp fragment of PtWRKY89was generated. Sequence analysis showed PtWRKY89contains one WRKY domain and one CH2C which indicates PtWRKY89belongs to the group III of the WRKY superfamily.3. Subcellμlar localizationThe subcellμlar localization vector35S:GFP and35S:GFP:PtWRKY89were constructed and transformed into the onion epidermal cells, observed it via fluorescence microscope after dark cμlture for24h. Green fluorescence protein was distributed in both cell membrane and nuclear membrane in the control group. Instead, the fusion PtWRKY89protein appeared to be specific combined with the cell nuclear membrane. Our resμlt has proved that the PtWRKY89is localized to the cell nucleus.4. Construction of binary vector pCXSN with PtWRKY89.The1108bp fragment of PtWRKY89in fμll length was amplified and ligated with the PCXSN vector and introduced into the Escherichia coli strain DH5a. The single positive colony was isolated on the Luria-Bertani medium containing kanamycin. The fragment was digested with BamH1enzyme to know the insert direction. Further detection was confirmed by BGI company via sequencing. Finally the recombinant vector was introduced into the Agrobacterium tumefaciensstrain GV3101.5. Genetic transformation of Popμlus tricocarpa Carr via. Agrobacterium tumefaciens.The leaf discs of Popμlus tricocarpa Carr were transformed with transcription factor PtWRKY89via Agrobacterium strain GV3101. The plants were screened with the WPM medium containing hygromycin as selectable marker.10transgenic plants lines were obtained and demonstrated to be positive through Real Time PCR. The heterologous gene transformation was confirmed. According to the semi-quantitative PCR analysis, PtWRKY89was highly elevated in transgenic plants.6. The PtWRKY89enhances fungal pathogen resistance.The wild type poplar and PtWRKY89overexpression plants were infected with Marssonina brunnea.Disease spot appeared to mμltiply rapidly in leaves of wild type poplar, however, few sign of Marssonina brunneahas been observed in transgenic poplar. It shows that PtWRKY89enhance the plant resistance to fungal pathogen.