Isolation And Function Study Of WRKY Transcription Factors In Pepper (Capsicum Annuum)

Pepper (Capsicum annuum) and tobacco (Nicolin tobacco) are the Solanaceae crops susceptible of diseases and other abiotic stresses, which usually result in lower yield and poor quality. Pesticides commonly used in pepper diseases protection are not effective enough but bring environment pollution. Development and useage of disease and other stresses resistant crop cultivars are the basic measures to solve the problem. Transcription factors play important roles in the plant response and resistance to stresses and the engineering of transcription factor has been proved to be a prospective way of genetic improvement of plant stresses resistance in the past decade. Therefore, a better understanding of the function of stress-inducible transcription factors is not only important in dissecting the molecular basis of interactions between plants and environment, but also important for genetic improvement of plant stresses resistance. In this paper, the full length cDNAs of 4 putative WRKY transcription factors were isolated from a pepper cDNA library, and their functions were studied by real time PCR, transient expression, subcellular localization and overpression in transgenic tobacco, the main results are as followings:1. Four putative WRKY transcription factors were isolated from pepper cDNA library. The results of sequence functionally annotated by Blast in NCBI indicate they contained one or two conserved WRKY domains with zin-finger motif of C₂H₂, which maybe involved in diffenent signal transduction pathways.2. Four putative WRKY transcription factors were localized in nuclei, which was consistent with software prediction. So they had the basic feacture of transcription factors.3. Transient expression verified that four putative WRKY transcription factors could bind specially to sequences harboring double W box cis-elements and promote the following GUS gene expression.4. Real-time PCR technique was applied to clarify the express pattens response to various biotic and abiotic stresses about 4 WRKY transcription factors. CaWRKY was induced by JA and wound, and had lower express level response to other elicitors; CaWRKY2 had abundance expression induced by wound, SA, JA and UV-B; CaWRKY3 had obvious increased expression level response to Phytophthora capsici and UV-B; CaWRKY3 were expressed at diffenent levels in various elicitors with the highest abundance by JA and wound, next in high salt, declined in SA. CaWRKY5 express decreaced by Phytophthora capsici, UV-B and low temperature. 5. Four overexpression vectors for four genes were construced and transformed into tobacco by agrobacterium-mediated method. 40 TO independent lines carrying overexpression vectors were generated. CaWRKY-overexpression plants exhibited more or less drawf. T1 lines of the other three genes showed different resistance with abiotic stesses. CaWRKY2-overexpression plants were higher altitude and less axillary than wild-type, increased resistence to heavy metal; GaWRKY3-overexpression plants were much sensitive to high temperature; CaWRKY4-overexpression plants showed obvirous resistance to low temperature.