Crops ›› 2024, Vol. 40 ›› Issue (2): 30-39.doi: 10.16035/j.issn.1001-7283.2024.02.005

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Cloning and Bioinformatics Analysis of ZmMAPKKK21 Gene in Maize

Zhang Qian1,2(), Ren Wen2, Zhao Bingbing2, Zhou Miaoyi2, Li Hanshuai2, Liu Ya2(), Du Hewei1()   

  1. 1College of Life Science, Yangtze University, Jingzhou 434025, Hubei, China
    2Maize Research Institute, Beijing Academy of Agriculture & Forestry Sciences / Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing 100097, China
  • Received:2023-02-12 Revised:2023-05-11 Online:2024-04-15 Published:2024-04-15

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Abstract:

The mitogen-activated protein kinase (MAPK) plays an important role in the process of plants responsing to both biological and non-biological stresses. In the previous study, we excavated the potentially important drought- resistant gene ZmMAPKKK21. In this study, the ZmMAPKKK21 gene was cloned from the drought-resistant maize inbred line J24 and bioinformatics analysis was performed on the gene. The findings indicated that the ZmMAPKKK21 gene's open reading frame measured 1419 bp and encoded 472 amino acids. The protein was hydrophilic and devoid of a signal peptide. Its promoter sequence featured several cis-acting regions associated with hormones and stress. It is anticipated that the maize MAPKKK21 protein was found on the nucleus. The outcome of the protein interaction prediction indicated that ZmMAPKKK21 interacted with the plant stress- related proteins MAPKK3 and ZIM family. In addition to being extremely conservative on the STKc_MAPKKK domain, its secondary and tertiary structures are more akin to those of highly drought-resistant gramineous crops, such as sorghum (Sorghum bicolor L.) and foxtail millet (Setaria italica L.). The association between the expression of the ZmMAPKKK21 gene and the response to drought stress was further confirmed by qRT-PCR analysis, which revealed that the gene's expression level was high in maize roots and that it was up-regulated in roots and leaves following drought stress.

Key words: Maize, MAPKKK, Bioinformatics analysis, Expression analysis

Table 1

Sequences of primers and their usage"

引物名称
Primer name		 引物序列（5'-3'）
Primer sequence (5'-3')		 用途
Usage
ZmMAPKKK21-F AACGATGGAAACGGACCGAA 基因克隆
ZmMAPKKK21-R TGGGAATCTTGGCGTTGACA
PZmMAPKKK21-F GTCCAACTCTGACCCTAAGCG 启动子克隆
PZmMAPKKK21-R CTACCAGTTCCAGTGTCTGC
QZmMAPKKK21-F GATGCAGAGGTGGAGCAACT 实时荧光
定量PCR
QZmMAPKKK21-R GTGGACGCCTGAATGCATAG
ZmGPN1-F TGACCAAGGTGAAGAGCACTGT
ZmGPN1-R CAAATCTCACGTGGCTATGAAAC

Table 2

Bioinformatics analysis websites"

功能Function 网址Website
启动子分析Promoter analysis https://bioinformatics.psb.ugent.be/webtools/plantcare/html/
保守结构域预测Prediction of conserved domain https://www.ncbi.nlm.nih.gov/cdd/
磷酸化位点预测Phosphorylation site prediction https://services.healthtech.dtu.dk/service.php?NetPhos-3.1
亲疏水性预测Hydrophilicity prediction https://web.expasy.org/protscale/
信号肽预测Signal peptide prediction https://services.healthtech.dtu.dk/service.php?SignalP-6.0
跨膜结构预测Transmembrane structure prediction https://services.healthtech.dtu.dk/service.php?TMHMM-2.0
理化性质预测Physical and chemical properties prediction https://web.expasy.org/protparam/
二级结构预测Secondary structure prediction https://npsapbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
三级结构预测Three-level structure prediction https://swissmodel.expasy.org/
亚细胞定位Subcellular localization http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/
蛋白互作预测Protein interaction prediction https://cn.string-db.org/

Fig.1

Electrophoresis of PCR amplification (a) Electrophoresis of genomic DNA, (b) Electrophoresis for amplification of full-length ZmMAPKKK21, (c) Electrophoresis for amplification of ZmMAPKKK21 promoter, M: 1 kb DNA marker."

Table 3

Cis-acting element analysis of promoter of ZmMAPKKK21 gene"

顺式作用元件Cis-acting element 序列Sequence 功能Function 数量Number
ABRE，ABRE3a ACGTG, CACGTGTACGTG 脱落酸响应元件 5
CGTCA基序，TGACG基序CGTCA-motif, TGACG-motif CGTCA, TGACG 茉莉酸响应元件 5
G盒G-box TACGTG 光响应元件 4
GARE基序GARE-motif TCTGTTG 赤霉素响应元件 1
富含TC重复序列TC-rich repeats GTTTTCTTAC 参与防御和应激反应元件 1
TGA元件TGA-element AACGAC 生长素响应元件 1
MRE AACCTAA 参与光响应的MYB结合位点 1
AAGAA基序AAGAA-motif gGTAAAGAAA 发育相关基序 1
CCAAT盒CCAAT-box CAACGG MYBHv1结合位点 1
CCGTCC盒CCGTCC-box CCGTCC 分生组织特异性激活响应元件 1
STRE AGGGG 热诱导响应元件 4
MYB CAACCA 干旱响应元件 1
MYC CATGTG, CATTTG 干旱响应元件 2

Fig.2

Analysis of ZmMAPKKK21 gene promoter cis-acting elements (a) and CpG island (b) (b) The blue region represents CpG island."

Table 4

Prediction results of ZmMAPKKK21 protein primary structure"

一级结构特征
Characteristic of primary structure		 预测结果
Prediction result
氨基酸数量Number of amino acids 472
等电点Isoelectric point (pI) 6.41
分子量Molecular weight (kD) 49 516.58
分子式Molecular formula C2149H3399N663O657S15
正电荷残基Arg+Lys 53
负电荷残基Asp+Glu 57
平均疏水性Average hydrophobicity -0.23
脂肪系数Aliphatic index (AI) 76.12
不稳定系数(Ⅱ) Instability coefficient (Ⅱ) 42.45
半衰期Estimated half-life (h) 30.00

Fig.3

Analysis of physical and chemical properties of ZmMAPKKK21 protein (a) Analysis of hydrophilicity and hydrophobicity, (b) Signal peptide prediction, (c) Prediction of transmembrane structure, (d) Prediction of phosphorylation sites."

Fig.4

Phylogenetic tree of amino acid sequences encoded by ZmMAPKKK21 and MAPKKK from other plants"

Fig.5

Conserved domains analysis of ZmMAPKKK21 protein and homologous proteins"

Fig.6

The prediction of tertiary structural model of ZmMAPKKK21 protein and homologous proteins"

Fig.7

Subcellular localization prediction (a) and protein interaction prediction (b)"

Fig.8

Relative expression of ZmMAPKKK21 gene in various organs R: root, L: leaf, S: stem."

Fig.9

Expression of ZmMAPKKK21 gene in roots and leaves under drought stress “**”represents significant differences at the level of P < 0.01."

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