南京林业大学学报(自然科学版) ›› 2022, Vol. 46 ›› Issue (6): 279-287.doi: 10.12302/j.issn.1000-2006.202109036

所属专题: 南京林业大学120周年校庆特刊

• 研究论文 • 上一篇    下一篇

薄荷MhWRKY57基因克隆及响应茉莉酸信号的表达分析

张婷1(), 柏杨(), 亓希武1, 于盱1, 房海灵1, 李莉1, 刘冬梅1, 梁呈元1,*(), 李维林2   

  1. 1.江苏省中国科学院植物研究所,江苏 南京 210014
    2.南京林业大学林学院,南方现代林业协同创新中心,江苏 南京 210037
  • 收稿日期:2021-09-22 修回日期:2022-01-21 出版日期:2022-11-30 发布日期:2022-11-24
  • 通讯作者: 梁呈元
  • 基金资助:
    国家自然科学基金项目(31970353);江苏省植物资源研究与利用重点实验室开放基金项目(JSPKLB202029)

Cloning and expression analyses of MhWRKY57 response to jasmonic acid in Mentha canadensis

ZHANG Ting1(), BAI Yang(), QI Xiwu1, YU Xu1, FANG Hailing1, LI Li1, LIU Dongmei1, LIANG Chengyuan1,*(), LI Weilin2   

  1. 1. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
    2. Co-Innovation Center for Sustainable Forstry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
  • Received:2021-09-22 Revised:2022-01-21 Online:2022-11-30 Published:2022-11-24
  • Contact: LIANG Chengyuan

摘要:

【目的】分析薄荷转录因子MhWRKY57的基因和氨基酸特性、亚细胞定位、转录活性、组织表达及茉莉酸甲酯处理下的基因表达水平,为揭示MhWRKY57响应茉莉酸信号调控薄荷精油合成的分子机制提供理论依据。【方法】 利用薄荷转录组数据,采用RT-PCR方法获得MhWRKY57基因编码序列,利用生物信息学分析其基因和氨基酸特性,烟草叶片瞬时表达系统进行亚细胞定位,酵母双杂交系统分析转录激活活性,实时荧光定量 PCR (qRT-PCR)技术分析MhWRKY57基因在各组织及茉莉酸甲酯诱导下的表达模式。【结果】 获得薄荷MhWRKY57基因的编码序列(CDS),编码276个氨基酸,具有典型的WRKY结构域,含34个磷酸化位点,属于非跨膜亲水蛋白,定位于细胞核且具有转录激活活性。系统进化分析表明,薄荷MhWRKY57与大豆Glyma.01G056800.2.p亲缘关系最近。qRT-PCR分析结果表明,MhWRKY57基因在幼叶、成熟叶、花、茎、根中均表达,在根和叶中均明显受到茉莉酸甲酯诱导表达。【结论】 MhWRKY57是一个响应茉莉酸信号的转录因子,可能参与茉莉酸信号介导调控的薄荷精油合成过程。

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

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