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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (12): 3387-3398.doi: 10.3724/SP.J.1006.2023.23081

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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 Online:2023-12-12 Published: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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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

Fig. 1

Protein domain of ZmMYC2 and phylogenetic tree of MYC family in plants A: protein domain of ZmMYC2, drawing with IBS software. B: phylogenetic tree of MYC family system, drawing with MEGA 6.0 by adjacency method."

Fig. 2

Relative expression pattern of ZmMYC2 genes A: the relative expression pattern of ZmMYC2 genes in different tissues. Red, yellow, and white indicate high, medium, and low levels of gene expression, respectively. B: Tissue-specific expression patterns of the ZmMYC2 genes, R: root; S: stem; L: leaf; T: tassel; SK: corn silk; CB: corncob; ST: stegophyll; EM: embryo; EN: endosperm."

Fig. 3

Subcellular localization of ZmMYC2 and screening of interacting proteins Nuclear localization of pMDC43-35S-GFP and ZmMYC2-GFP. Scale bar: 20 μm."

Fig. 4

Relative expression pattern of ZmMYC2 genes under various hormones treatments"

Fig. 5

Sensitivity of ZmMYC2 overexpression, backfeeding, and mutant strains to JA A: wild type, back fed strains, mutant materials, and over-expressed materials treated with 50 μmol L-1 JA for 7 days; B: root length after JA treatment for 7 days. Data are means ± SDs. * and ** denote significant differences in WT at the 0.05 and 0.01 probability levels according to Student’s tests, respectively."

Fig. 6

Resistance of ZmMYC2 overexpressed strain to Pst DC3000 A, B: leaf phenotype after 7 days of treatment with Pst DC3000; C, D: DAB and Evans blue staining; E, F, I, J: the proportion of lesion area of leaves; G, H: Bacterial content in leaves. Data are means ± SD. * and ** denote significant differences from WT at the 0.05 and 0.01 probability levels according to Student’s tests, respectively."

Fig. 7

Phenotype of Arabidopsis overexpressed strain after JA and Pst DC3000 treatment A: after spraying JA 1 d, Pst DC3000 was treated for 7 days for leaf phenotype; B: DAB and Evans blue staining; C, D: bacterial content in leaves; E: the proportion of lesion area of leaves. Data are means ± SD. * and ** denote significant differences in WT at the 0.05 and 0.01 probability levels according to Student’s tests, respectively."

Fig. 8

Expression analysis of stress-related genes in pathogenic bacteria A, B, C, D: the relative overexpression gene; F, G, H, I, J: the relative expression of mutant and backfed lines. Data are means ± SD. * and ** denote significant differences in WT at the 0.05 and 0.01 probability levels according to Student’s tests, respectively."

Fig. 9

Interaction between ZmMYC2 and JAZ protein DDO: synthetic dropout without Leu-Trp, QDO: synthetic dropout without Leu-Trp-His-Ade."

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