Expression And Function Study Of CaWRKY3 Transcription Factor In Pepper

Previous studies have suggested that WRKY genes play key roles in modulating plant defense against different biotic and abiotic stresses. Isolation and functional identification of different members of plant WRKY superfamily is an important way to elucidate the molecular mechanism of plants stress resistance. The previous work in our lab isolated full length cDNA of named CaWRKY3 from cDNA library of pepper leaves challenged with UV, which is 1137 bps in length and harboring open reading frame of 378 aa. The gene belongs to ClassificationⅠof the WRKY superfamily according to its sequence and the two conserved WRKY domains. The previous study in our lab also found that CaWRKY3 gene is the nuclear localizating protein and could bind to W-box in transient expression analysis in onion epidermal cells. In this study, the expression of CaWRKY3 was detected by real time PCR against different stresses treatment, the effect of CaWRKY3 overexpression in T2 transgenic tobacco lines on growth, response to exogenous phytohormones treatment, as well as resistance to biotic and abiotic stresses was carried out, and proteins of CaWRKY3 was also acquired by its prokaryotic expression. The main results are as followings:1.By real time PCR analysis of the expression of CaWRKY3 against different stresses as well exogenous phytohormones application, it was found that the expression of CaWRKY3 was reduced by pathogen infection, as well as the applicaton of SA and ethylene, and was enhanced by NaCl, low temperature and wounding treatment.2. By using the overexpression transgenic tobacco T2 lines of CaWRKY3, the effect of CaWRKY oversexpression on resistance against biotic and abiotic stresses was studied. The results indicated that, the overexpression of CaWRKY3 increased the susceptivity of tobacco to Pseudomonas solanacearum infection, as well as exogenous ethylene and SA application, and the overexpression of CaWRKY3 also increased the resistance to NaCl treatment to some degress. These results suggested that CaWRKY3 could act as a negetive regulator to disease resistance and a positive regulator to salt stress resistance.3.The prokaryotic expression vector pET32a-CaWRKY3 was constructed by cloning the full length cDNA of CaWRKY3 in frame to the cloning sites of pET32, which was transformed into E.coli BL21 and cultured in LB media for 20 hour on large scale and the fused protein was induced by 1.0mM IPTG in 20℃. The extract product of the harvested E.coli BL21 was subjected to SDS-PAGE, and new band of about 62 KDa was found in transformed E.coli BL21, which was consistent with the anticipating molecular weight of 6×his- CaWRKY3 protein.The N-terminal amino-acid sequencing showed that the expressed protein was the targeted protein. The fused 6×his-CaWRKY3 protein was purified by Ni2 + affinity chromatography. The purified fused protein acquired in this study laid the foundation to prepare the antibody of CaWRKY3 for its use in further study.