关键词: 薄荷, WRKY转录因子, 组织表达分析, 亚细胞定位, 茉莉酸信号, 转录活性

Abstract:

【Objective】Mint(Mentha canadensis) is a medicinal and edible herb of the Mentha genus in the Lamiaceae family that is rich in medicinal essential oil. WRKY family is a type of plant-specific transcription factor family, and plays an important role in regulating plant secondary metabolism. In this study, MhWRKY57 gene was cloned from mint, and its gene and amino acid characteristics, subcellular localization, transcriptional activity and tissue expression were studied. Additionally, the gene expression levels of MhWRKY57 with essential oil biosynthesis-related gene MhGPPS-s under methyl jasmonate treatment were analyzed, which provides a theoretical basis for revealing the molecular mechanism of MhWRKY57 regulating mint essential oil biosynthesis responding to jasmonic acid signal.【Method】 Based on the transcriptome database of mint treated with methyl jasmonate, a WRKY transcription factor gene significantly induced by methyl jasmonate was screened and then cloned by RT-PCR, sequenced, and named MhWRKY57, according to homology. The gene and amino acid characteristics of MhWRKY57 were analyzed by bioinformatics, and the phylogenetic tree was constructed with MEGA7 by the maximum likelihood method for a homology analysis. The subcellular localization of MhWRKY57 was demonstrated using a tobacco leaf transient expression system combined with green fluorescent protein (GFP) fusion expression system followed by laser confocal scanning microscopy (LCSM) to detect the fluorescence signal. To verify the transcriptional activity of MhWRKY57, the MhWRKY57 coding sequence was cloned into the pBD vector so that transcriptional activation could be assayed in a yeast two-hybrid system. The expression patterns of genes in various tissues and induced by methyl jasmonate were analyzed by quantitative real-time PCR (qRT-PCR). 【Result】The coding sequence (CDS) of MhWRKY57 was cloned, which contained an open reading frame with 831 bases and encoded 276 amino acids. The secondary structure of MhWRKY57 was predicted, including mainly random coil, then alpha helix, extended strand and beta turn. The amino acids of MhWRKY57 were predicted to have 34 phosphorylation sites in which the serine may be mainly phosphorylated. MhWRKY57 is a non-transmembrane protein with certain hydrophilicity and instability. The amino acid sequences of WRKY57 ortholog proteins from M. canadensis and other ten plant species were used for a cluster analysis, and the results showed that MhWRKY57 was closer to soybean Glyma.01G056800.2.p than other WRKY57 ortholog proteins in the evolutionary relationship. The domain analysis and amino acid sequence alignment showed that MhWRKY57 was a typical transcription factor that belonged to the WRKY family and contained a conserved WRKYGQK domain at the N-terminus, and an incomplete C2H2 zinc-finger structure at the C-terminus. The MhWRKY57 protein was predicted to be located in the nucleus, and transiently co-expressed in onion epidermal cells of MhWRKY57-GFP fusion protein together with the OsD53-RFP, which is a nuclear localized marker protein. The subcellular distributions of these two fusion proteins were merged further, indicating that MhWRKY57 was a nuclear-localized protein. The transcriptional activity verification of MhWRKY57 was performed on SD/-Trp and SD/-Trp-His-Ade media using Y2H Gold yeast cells. The yeast strains harboring the BD- MhWRKY57 plasmid could grow on both SD/-Trp and SD/-Trp-His-Ade media, whereas the yeast strains transformed with the pBD empty vector could only grow on SD/-Trp medium, which suggested that MhWRKY57 had transcriptional activation activity. The tissues of young and mature leaves, flowers, stems and roots were prepared individually to be used for the total RNA extraction, and then for a tissue expression analysis using qRT-PCR. The qRT-PCR analysis showed that MhWRKY57 gene was expressed in young and mature leaves, flowers, stems and roots, and the highest expression level of MhWRKY57 gene was in flowers, followed by roots, stems, mature leaves and young leaves. The mint seedlings cultured in water were treated with 0.2 mol/L MeJA to analyze the response of MhWRKY57 gene in roots and leaves to MeJA, respectively. The expression of MhWRKY57 gene in roots and leaves was induced most significantly at 12 h after treatment, while the expression was down regulated to the control level after 24 h of treatment. In addition, the expression levels of MhGPPS-s gene in leaves and roots, which plays an important role in mint essential oil biosynthesis under treatment of 0.2 mol/L MeJA, were also detected. Compared with the control, the mRNA levels of MhGPPS-s gene in leaves were evidently enhanced at 9 and 12 h after treatment, while those of MhGPPS-s gene in leaves were enhanced evidently at 2 and 4 h after treatment. These results indicated that the expression of MhGPPS-s and MhWRKY57 genes were both induced by MeJA. 【Conclusion】 MhWRKY57 is widely expressed in major tissues of mint and encodes a transcription factor, which is located in the nucleus. MhWRKY57 is a typical WRKY family protein that possesses a conserved WRKY domain and has transcriptional activation activity. Considering that WRKY family genes play important roles in secondary metabolism and MhGPPS-s gene functions in the biosynthesis of mint essential oil, both the expression of MhWRKY57 and MhGPPS-s genes in mint leaves can be significantly induced by MeJA, which implies that MhWRKY57 gene may play a role in the biosynthesis of mint essential oil by regulating the expression of MhGPPS-s gene in response to jasmonic acid signal.

Key words: minty(Mentha canadensis), WRKY transcription factor, tissue expression analysis, subcellular localization, jasmonic acid signaling, transcriptional activity