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作物学报 ›› 2023, Vol. 49 ›› Issue (12): 3387-3398.doi: 10.3724/SP.J.1006.2023.23081

• 研究简报 • 上一篇    下一篇

玉米ZmMYC2基因的克隆及功能分析

杜明1(), 陈明超3, 方玉1,2, 武健东3,*()   

  1. 1上海中科荃银分子育种技术有限公司, 上海200030
    2安徽荃银高科种业股份有限公司, 安徽合肥230088
    3安徽农业大学 / 作物抗逆育种与减灾国家地方联合工程实验室, 安徽合肥230036
  • 收稿日期:2022-12-21 接受日期:2023-07-03 出版日期:2023-12-12 网络出版日期:2023-07-13
  • 通讯作者: * 武健东, E-mail: wujiandong@ahau.edu.cn
  • 作者简介:E-mail: dm@zkwbreeding.com
  • 基金资助:
    荃银高科国家企业技术中心创新项目(2021M002);安徽省自然科学基金项目(2008085MC70);安徽省高等学校自然科学研究项目(KJ2021A0172)

Cloning and functional analysis of maize ZmMYC2 gene

DU Ming1(), CHEN Ming-Chao3, FANG Yu1,2, WU Jian-Dong3,*()   

  1. 1Shanghai ZKW Molecular Breeding Technology Co., Ltd, Shanghai 200030, China
    2Anhui Win-all Hi-tech Seed Co., Ltd., Hefei 230088, Anhui, China
    3Anhui Agricultural University / National Engineering Laboratory of Crop Stress Resistance, Heifei 230036, Anhui, China
  • Received:2022-12-21 Accepted:2023-07-03 Published:2023-12-12 Published online:2023-07-13
  • Contact: * E-mail: wujiandong@ahau.edu.cn
  • Supported by:
    National Enterprise Technology Center Innovation Project of Anhui Win-all Hi-tech Seed Co., Ltd(2021M002);Anhui Provincial Natural Science Foundation(2008085MC70);Anhui Provincial Key Research and Development Project(KJ2021A0172)

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摘要：

植物在生长发育过程经常受到病原菌的侵害, 严重影响作物产量。MYC2属于bHLH家族转录因子, 在茉莉酸介导的信号途径中有着重要的调控作用。在前期工作中, 通过对MYC家族进化树分析, 找到了与拟南芥同源的玉米基因ZmMYC2。本研究克隆了ZmMYC2, 该基因全长2118 bp, 编码705个氨基酸残基, 其编码蛋白定位于细胞核。RT-qPCR分析表明, ZmMYC2在玉米各个组织中均有表达, 但在叶中的表达量最高。诱导表达模式分析表明, ZmMYC2可以响应茉莉酸(jasmonic acid, JA)、水杨酸(salicylic acid, SA)和乙烯(ethylene, ETH)的诱导。在50 μmol L-1茉莉酸处理的培养基上, 过表达拟南芥的根长显著短于野生型, 突变体拟南芥的根长显著长于野生型, 回补材料拟南芥根长与野生型无明显差异。对过表达ZmMYC2拟南芥材料接种丁香假单胞杆菌(psedumonas syrinage pv.tomato DC3000, Pst DC3000), 野生型、突变体和回补拟南芥相对于过表达表现出较好的状态, 推测ZmMYC2降低了拟南芥对病原菌侵害的防御能力。接种前, 外源施加茉莉酸激素, 过表达拟南芥叶片死亡面积和叶内含菌量均有所减少, 茉莉酸激素增强了ZmMYC2对病原菌侵害的抵抗能力。亚细胞定位显示, ZmMYC2为核定位蛋白。酵母双杂交表明ZmMYC2可以与JAZ蛋白中的JAZ1和JAZ3相互作用。茉莉酸、水杨酸和防御基因以及细胞程序性死亡基因的表达量分析表明, ZmMYC2负调控PDF1.2的表达, 正调控PR1、MED25、COI1以及细胞程序性死亡基因HIN1的表达。综上所述, ZmMYC2通过与JAZ1和JAZ3相互作用, 参与茉莉酸信号的转导作用, 降低了拟南芥对病原菌胁迫的抵抗能力。

关键词: 玉米, ZmMYC2, 病原菌防御, 茉莉酸

Abstract:

Plants are often harmed by pathogens during their growth and development, which seriously affect crop yield. MYC2 belongs to the bHLH family of transcription factors and plays an important role in the regulation of JA-mediated signaling pathways. In the previous work, maize gene ZmMYC2 which was homologous to Arabidopsis thaliana was detected through the analysis of the MYC family evolutionary tree. In this study, we cloned ZmMYC2, which had a total length of 2118 bp and encoded 705 amino acid residues. The encoded protein was localized in the nucleus. RT-qPCR analysis showed that ZmMYC2 was expressed in all tissues of maize, but the relative expression level was the highest in leaves. The results showed that ZmMYC2 could be induced by jasmonic acid (jasmonic acid, JA), salicylic acid (salicylic acid, SA), and ethylene (ethylene, ETH). In 50 μmol L-1 JA medium, root length of overexpressed Arabidopsis thaliana was significantly shorter than wild type, and that of mutant Arabidopsis thaliana was significantly longer than wild type. There was no significant difference in root length between Arabidopsis thaliana and wild type. Pst DC3000 (Psedumonas syrinage pv.tomato DC3000, Pst DC3000) was inoculated with ZmMYC2 Arabidopsis materials, and the wild type, mutant, and complement Arabidopsis showed better state relative to over expression. It was speculated that ZmMYC2 decreased the defense ability of Arabidopsis thaliana against pathogen. Before inoculation, the dead area of leaves and the amount of bacteria in leaves with the overexpression of Arabidopsis thaliana were reduced by exogenous JA hormone. JA hormone enhanced the resistance of ZmMYC2 to pathogens. Subcellular localization showed that ZmMYC2 was a nuclear localization protein. The yeast two-hybrid showed that ZmMYC2 could interact with JAZ1 and JAZ3 in JAZ protein. The relative expression levels of JA, SA, defense genes, and programmed cell death genes showed that ZmMYC2 negatively regulated the relative expression level of PDF1.2, and positively regulated the relative expression level of PR1, MED25, COI1, and programmed cell death gene HIN1. In conclusion, ZmMYC2 was involved in JA signal transduction by interacting with JAZ1 and JAZ3, which reduced the defense ability of Arabidopsis thaliana against pathogen stress.

Key words: maize, ZmMYC2, pathogen defense, jasmonic acid

图1

ZmMYC2的蛋白结构域分析和植物中MYC家族系统进化树 A: ZmMYC2的蛋白结构域, 采用IBS软件绘制。B: MYC家族系统进化树分析, 采用邻接法利用MEGA 6.0进行绘制。"

图2

ZmMYC2组织特异性表达模式 A: ZmMYC2在不同组织中的表达模式。红色、黄色和白色分别表示高、中和低水平的基因表达。B: ZmMYC2基因的组织特异性表达模式, R: 根; S: 茎; L: 叶; T: 雄蕊; SK: 花丝; CB: 玉米芯; ST: 苞叶; EM: 胚; EN: 胚乳。"

图3

ZmMYC2的亚细胞定位 pMDC43-35S-GFP、ZmMYC2-GFP的亚细胞定位, 标尺为20 μm。"

图4

ZmMYC2在多种激素处理下的表达模式"

图5

ZmMYC2过表达、回补和突变体株系对茉莉酸的敏感性 A: 50 μmol L-1的茉莉酸处理7 d的野生型、回补株系、突变体材料和过表达材料图片; B: 茉莉酸处理7 d后的根长统计。误差线表示3个独立试验的标准差, *表示显著性差异(0.01 < P < 0.05); **表示极显著差异(P < 0.01)。"

图6

拟南芥对Pst DC3000的抗性 A、B: Pst DC3000处理7 d后叶片表型; C、D: DAB和Evans blue染色, E、F、I、J: 叶片病变面积比例, G、H: 叶片内细菌含量。误差线表示3个独立试验的标准差, *表示显著性差异(0.01 < P < 0.05); **表示极显著差异(P < 0.01)。"

图7

茉莉酸和Pst DC3000处理后拟南芥过表达株系表型 A: 喷施JA 1 d后Pst DC3000处理7 d叶片表型; B: DAB和Evans blue染色结果; C, D: 叶片内细菌含量; E: 叶片病变面积比例。误差线表示3个独立试验的标准差, *表示显著性差异(0.01 < P < 0.05); **表示极显著差异(P < 0.01)。"

图8

病原菌胁迫相关基因表达量分析 A, B, C, D: 过表达基因表达量分析; F, G, H, I, J: 表示突变体和回补株系表达量分析。误差线表示3个独立试验的标准差, *表示显著性差异(0.01 < P < 0.05); **表示极显著差异(P < 0.01)。"

图9

ZmMYC2与JAZ蛋白的相互作用 DDO: SD/-Leu-Trp缺陷型培养基, QDO: SD/-Leu-Trp-His-Ade缺陷型培养基。"

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