Identification And Function Analysis Of GhWRKY11, A WRKY Gene From Cotton

Living plant tissues are host to various stresses from environment, including drought aswell as salt stresses, pathogen attack and wounding. To cope with these threats, plants developa wide array of mechanisms in a highly extensive and temporal manner to protect themselvesfrom attacks. In the complicated signaling pathways, the activation of defence-related genes isachieved by a network of various transcription factors. By now, many plant transcriptionfactors have been shown to contribute to this regulation, such as EREBP, bZIP, MYB proteinsand WRKY transcription factors.WRKY proteins are key zinc finger transcription factors in plants. Since the first WRKYgene (SPF1) was identified from sweet potato, an increasing number of reports about WRKYproteins have been found. Recently, many studies indicated that WRKY is involved in plantdevelopment process, biotic as well as abiotic stresses. However, exploring its function is stillgoing on because of its largely family members and complicated mechanisms. In this study,we isolated a WRKY transcription factor gene, termed as GhWRKY11, from cotton. And aseries of experiments have been conducted on sequence analysis, expression patterns andfunctions. The main results as follows:(1) Based on the homology-based cloning, we isolated a WRKY gene from cotton, which isnamed as GhWRKY11(GenBank number: HQ828074). Sequence analysis showed that thefull-length cDNA consisting of1306nucleotides with a5′untranslated region (UTR) of111bp,a3′UTR of142bp and a1053bp open reading frame (ORF). The ORF encodes a373aminoacid protein with a predicted molecular weight of38.90kDa and isoelectric point of8.99.Real-time PCR showed that GhWRKY11existes as a single copy in cotton genome. Aminoacid sequence alignment and the phylogenetic tree revealed that GhWRKY11is a member ofgroup IId WRKY protein. Subcellular localization experiment indicated that GhWRKY11protein is localized to the nucleus, which reveals that GhWRKY11transcription factor functions in vitro.(2) With the help of I-PCR and RACE-PCR, we obtained a1025bp fragment ofGhWRKY115′flanking region. The PlantCARE and PLACE databases reveal various putativecis-acting elements involved in defense responses and development processes in the promoterregion of GhWRKY11. Thus the possibility that GhWRKY11may be a critical transcriptionalfactor in regulating various aspects in cotton should be considered.(3) Semi-quantitative RT-PCR analysis reveals that the level of GhWRKKY11transcriptincreased gradually inoculated with C. gossypii and wounding. In addition, there are stronginductions of GhWRKY11by SA, JA, ET and H2O2. Above all, we speculated thatGhWRKY11may be involved in plant defense responses.(4) N.benthamiana was transformed with pBI121-GhWRKY11construct using A.tumefaciens-mediated transformation method. Two independent lines (OE1and OE2) wereused for functional analysis. Compared with wild-type plants, overexpression lines showedmore resistance to viral, fungal and bacterial infections.(5) Semi-quantitative RT-PCR analysis and H2O2staining assay showed that GhWRKY11may play important roles in regulating plant defense responses through SA-andROS-mediated pathways.