Isolation And Expression Analysis Of The Promoter Of CaWRKY5 In Tobacco Transient Expression System

Proteins in WRKY superfamily include many plant-specific transcription factors, which play important roles in the regulation of plant growth, development and stresses responses. CaWRKY5 is a member of the pepper (Capsicum annuum L.) WRKY family. Our previous study found that the expression of CaWRKY5 was induced by heat, high salinity and pathogen infection, indicating the possible involvement of CaWRKY5 in plant defense reaction against biotic and abiotic stresses. However the molecular mechanism of the transcription expression of CaWRKY5 against different stresses remains unclear. In this study, to gain insight into the molecular basis and possible signaling pathway of the induction of CaWRKY5 response to environment stress, we isolated the promoter of CaWRKY5. Deletion analysis of the CaWRKY5 promoter against different stresses as well as exogenous hormones was carried out via an Agrobacterium-mediated transient expression assay in tobacco leaves. The results are as followings:(1) The 936bp upstream promoter sequence of the CaWRKY5 was isolated from the genomic DNA of pepper by LA-PCR. Bioinformatics analysis showed that the TATA-box of the promoter locates in -140bp to -136bp, and several cis-acting elements including W-box, S-box, SA-responsive element, ABRE responsive to different stresses or hormones were found in the promoter.(2)The -936bp region upstream of translation start codon in CaWRKY5 seemed to be an intact promoter. The fragment from translation start codon to -936bp and from translation start codon to -886bp in the promoter were enough for GUS expression against pathogen infection and MeJA induction, However the fragment from translation start codon to -489bp abolished the response of GUS to pathogen infection and MeJA induction. These results suggested that within region from -886bp to -489bp in the promoter of CaWRKY5 there may exist the pathogen and MeJA responsive cis-element. The result also showed that in promoter of CaWRKY5, the fragment from translation start codon to -336bp were sufficient for GUS expression in response to mechanical wounding and SA treatment, suggesting that wounding and SA responsive cis-element may exist in this region. The deletion analysis of CaWRKY5 promoter against exogenous ethylene treatment suggested the ethylene responsive cis-element may locate within the region from translation start codon to -336bp region and from -936bp to -886bp of the promoter. Whereas, the promoter region from -886bp to -336bp were showed to inhibit the expression of GUS, indicating that some ethylene-related negative cis-element appeared to be located within the region from -489bp to -336bp of the promoter.