The Cloning And Function Analysis Of A Novel WRKY Transcription Factors From Pepper

Pepper(Capsicum annuum L.) is an important vegetable worldwide and a diseases sentitive Solanaceae. Frequent diseases always cause heavy loss in yield and quality. Development and extention of disease resistant cultivars is the most feasible and efficient technological strategy to overcome the problem of diseases. The results of plant diseases resistant mechanism in the past decades indicated that defense raction could be triggered in plants when exposed to biotic or abiotic stresses, which is largely transcriptionally mediated by various transcription factors,such as WRKY, ERF, MYB and NAC. Isolation and functional identification of different stresses responding transcription factors is a feasible approach for the elucidation of plant diseases resistance mechanism. So, in this paper, a full cDNA of a novel WRKY of pepper, named CaWRKY, was isolated from a normalized cDNA library. The structure, subcullar localization and its binding to W-box and its expression pattern were analyzed. The main results were as followings:1. According to bioinformatics analysis results, CaWRKY encodes a 37_KDa protein and contains typical WRKYGQK domain and C2HC zinc finger domain which belong to WRKY transcription factors. Basing on characters of its conserved domains, it was classified into the third group of WRKY gene family.2. The CaWRKY-GFP fusion expression vector was transformed into onion cells utilizing partical gun and CaWRKY was observed to locate in nuclei.3. The effector plasmid containing CaWRKY and the reporter plasmid including w-box were contransformated into onion cells. And then transient expression of CaWRKY showed that CaWRKY can bind to w-box.4. Transactivation activity assays showed the C-terminal region of CaWRKY protein contains a transcriptional activation domain.5. The relative expression levels of CaWRKY were investigated in different tissues of young seedlings as well as mature pepper plants. Results showed that CaWRKY was detectable in all tissues, and mature leaf possesses the highest transcript levels which suggested CaWRKY plays an critical role in regulating development and leaf. The expression patterns of CaWRKY in response to different biotic and abiotic stresses were studied via real-time PCR. The results showed that CaWRKY was up-regulated under hot, cold, wound or Ralstonia solanacearum treatment. Similar results were obtained when pepper plants were treated by SA, MeJA, NAA or Ethephon. However, the expression of CaWRKY was changed slightly under ABA , H2O2 or NaCl treatment.6. The over-expression vector of CaWRKY was constructed and transformed into tobacco plant through Agrobacterium-mediated way. 30 lines of T0 generation of transgenic tobacco were obtained, and among them 22 lines were proved to be postive transgenic line with PCR.