Isolation And Functional Characterization Of PheWRKY1 Gene Related To Disease Resistance In Moso Bamboo

The defense of plants to pathogens'infection has led to the evolution of complex adaptive responses involving in protective physical barriers and production of a diverse array of antimicrobial genes. The WRKY transcription factors, only present in plants, constitute a superfamily of transcription factors. Members of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. To study the function of WRKY transcription factors in moso bamboo (Phyllostachys edulis), a PheWRKY1 gene was cloned. Its amino acids sequence and expression patterns were analyzed. The gene had been introduced into Arabidopsis thaliana and the disease resistance of transgenic lines were analyzed.A PheWRKY1 gene (GenBank accession number: GU944762) was cloned by RT-PCR, full length of 1080 bp, with an open reading frame (ORF) of 579 bp encoding a putative protein of 193 amino acids with a theoretical isoelectric point of 5.990 and a calculated molecular mass of 20.8 KDa. The results of sequence analysis showed that PheWRKY1 encodes a peptide harboring only one WRKY domain and one zinc finger, falling intoⅡsubgroup of WRKY proteins. And the conserved sequences of the protein encoded by PheWRKY1 were highly homologous to the proteins of Saccharum sinense, Oryza sativa, Triticum aestivum and Lolium perenne with 86% identity and homologous to Arabidopsis thaliana AtWRKY51 with 47% identity. It also showed that the protein localizes in the nucleus. The tertiary structure were predicted and similar to Lolium perenne. The putative functions predicted might have transcription regulation and signal transducer activity.Total RNA samples were isolated from the leaf tissues after several different treatments, including spraying defense signaling molecules salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) on leaves, drought treatment and UV-B radiation. Fluorescence real-time quantification PCR (qRT-PCR) analysis indicated that the expression of PheWRKY1 could be rapidly activated by JA and SA, which appeared to activated more significantly. UV-B radiation could also induce the gene expression. However, ABA and drought treatments had no evident effects on its expression. The expression of PheWRKY1 of moso bamboo infected naturally was also significantly enhanced. So we proposed that PheWRKY1 might be associated with plant defense through salicylate- and jasmonate- dependent signaling but not be involved in responses to osmotic stress.In this study, the PheWRKY1 was cloned into pET-28a vector to construct recombinant prokaryotic expression vector pET28a-PheWRKY1, which was transformed to E. coli strain of BL21. After induced by IPTG, a recombinant protein was expressed.Overexpression vector for the gene was constructed and transformed into Aribidopsis thaliana by agrobacterium-mediated method. The transgenic lines were selected using MS medium containing kanamycin and RT-PCR. The transgenic lines displayed distinct morphological traits; they were larger in size, longer root and smaller pod than wide-type plants with, stronger growth vigor and advanced in flowering. So the developmental programs might be affected overexpression PheWRKY1.To determine the contribution of PheWRKY1 gene to disease resistance in A. thaliana, we examined the resistance of the transgenic lines to Pseudomonas syringae PV Tomato DC3000 (DC3000 for short). Wild-type plants appeared to sensitive to DC3000 infection, leading to more rapid spreading of disease, maceration and dehydration of leaves. By contrast, most PheWRKY1 overexpression plants did not exhibit spreading maceration to systemic leaves and displayed clearly enhanced survival of DC3000 infection. The chlorophyll fluorescence parameters of A. thaliana after infection were measured by IMAGING-PAM chlorophyll fluorometer. The results showed that the maximal photochemical efficiency of photosystemⅡ(F_v/F_m) and quantum yield of photosystemⅡelectron transport (ΦPSⅡ) of transgenic lines were higher than that of wild-type plants. The increase of Y(NO) upon DC3000 infection was largely paralleled by the decreases of Y(II) and Y(NPQ), which are complementary with the photochemical quantum yield, i.e. Y(II) + (NPQ) + Y(NO)=1, in wild-type plants than that of transgenic lines, all of which indicated that transgenic lines infected by DC3000 had a better photosynthetic capacity. Furthermore, the expression of different pathogenesis-related genes were analysed by real-time PCR. The results showed that PheWRKY1-overexpressing plants accumulated significantly more PR1, PR2, PR5 and NPR1 transcripts.In summary, transgenic A. thaliana with overexpressing PheWRKY1 gene improved resistance to bacteria infection, which provides candidate gene for improving moso bamboo stress tolerance in theory. The research provides a base for further studying on the stress-tolerant molecular mechanism and breeding of moso bamboo.