Cloning and expression analysis of SsWRKY1 gene in Solanum sisymbriifolium under stress of verticillium wilt disease

【Objective】The WRKY1 gene of Solanum sisymbriifolium(SsWRKY1) was cloned and its expression pattern under the stress of verticillium wilt disease was analyzed to provide a theoretical reference for exploring the regulatory mechanism of WRKY1 gene in the resistance to verticillium wilt.【Method】SsWRKY1 gene was cloned by the genome walking technique,and its sequence was analyzed by bioinformatics software and a phylogenetic tree was constructed.The subcellular localization of WRKY1 protein was determined by its GFP fusion protein expression under its native promoter. The expression of SsWRKY1 in different tissues of S. sisymbriifolium at different times under verticillium wilt disease stress was analyzed by real-time quantitative PCR(qRT-PCR). The correlation analysis between disease index and WRKY1 gene expression of 9 wild eggplant materials inoculated with the verticillium wilt pathogen was also analyzed.【Result】The full-length open reading frame(ORF)sequence of SsWRKY1 gene(GenBank accession number:MZ846202) was 1224 bp,encoding 407 amino acids residues. Its protein molecular weight was 44.87 kD and its theoretical isoelectric point(pI)was 6.91,which indicated that SsWRKY1 was a hydrophilic and unstable protein. Its subcellular localization in the nucleus conformed to the characteristics of transcription factors. The SsWRKY1 protein contained two conserved WRKY domains and a C2H2-type zinc finger structure,indicating that SsWRKY1 was a class I WRKY transcription factor.Its secondary structure was mainly composed of α-helix(7.13%),β-bridge(4.67%),random coil(73.71%) and extended strand(14.50%). S. sisymbriifolium was closely related to S. lycopersicum,S. pennellii,S. arcanum and S. tuberosum. The relative expression of SsWRKY1 in roots and stems was significantly higher than that in leaves(P<0.05,the same below). The relative expression of SsWRKY1 at different times after the inoculation of the verticillium wilt pathogen was lower than that of the control group(sterile water inoculation),but the difference was significant at 24 h. There was a significant positive correlation between disease index and the variation in WRKY1 expression in 9 wild eggplant materials inoculated with verticillium wilt pathogen.【Conclusion】SsWRKY1 belongs to the class I of WRKY transcription factors,its sequence is highly conserved and is expressed in the nucleus. The gene expression of SsWRKY1 is tissue-specific and the expression of this gene decreased under verticillium wilt disease stress. SsWRKY1 gene may play a negative regulatory role in wild eggplant resistance to verticillium wilt.