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作物杂志,2024, 第2期: 30–39 doi: 10.16035/j.issn.1001-7283.2024.02.005

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

玉米ZmMAPKKK21基因的克隆和生物信息学分析

张倩1,2(), 任雯2, 赵冰兵2, 周秒依2, 李韩帅2, 刘亚2(), 杜何为1()   

  1. 1长江大学生命科学学院，434025，湖北荆州
    2北京市农林科学院玉米研究所/玉米DNA指纹及分子育种北京重点实验室，100097，北京
  • 收稿日期:2023-02-12 修回日期:2023-05-11 出版日期:2024-04-15 发布日期:2024-04-15
  • 通讯作者: 杜何为，研究方向为玉米遗传育种，E-mail：200457@yangtzeu.edu.cn；刘亚，研究方向为玉米遗传育种，E-mail：srlyyd@gmail.com
  • 作者简介:张倩，研究方向为玉米分子生物学，E-mail：zg0502q@163.com
  • 基金资助:
    北京市农林科学院科技创新能力建设专项(KJCX20230203)

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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摘要：

丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）在植物应对生物和非生物胁迫过程中发挥着重要作用。在前期研究中我们挖掘到ZmMAPKKK21这一潜在的重要抗旱基因，本研究从玉米抗旱自交系J24中克隆获得ZmMAPKKK21基因，并对其进行生物信息学分析。结果表明，玉米ZmMAPKKK21基因开放阅读框长1419 bp，编码472个氨基酸，是不具有信号肽的亲水性蛋白，且其启动子序列含有多个与逆境胁迫和激素有关的顺式作用元件；玉米中的MAPKKK21蛋白预测定位在细胞核上，蛋白互作预测结果显示，ZmMAPKKK21蛋白与参与植物逆境胁迫相关的MAPKK3蛋白和ZIM家族蛋白发生互作，其结构与高粱（Sorghum bicolor L.）和谷子（Setaria italica L.）等高抗旱禾本科作物相比，不但在STKc_MAPKKK结构域上高度保守，且具有更加相似的二级结构和三级结构；qRT-PCR分析发现，ZmMAPKKK21基因在玉米根系中表达水平较高，干旱胁迫后该基因在根和叶中表达上调，进一步验证了ZmMAPKKK21基因的表达与干旱胁迫响应的相关性。

关键词: 玉米, MAPKKK, 生物信息学分析, 表达分析

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

表1

引物序列及用途

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

表2

生物信息学分析网址

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/

图1

PCR扩增电泳图 (a) 基因组DNA电泳图，(b) ZmMAPKKK21基因全长扩增电泳图，(c) ZmMAPKKK21基因启动子扩增电泳图，M：1 kb DNA分子量标准。

表3

ZmMAPKKK21基因启动子顺式作用元件分析

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

图2

ZmMAPKKK21启动子顺式作用元件分析（a）和CpG岛分析（b） (b) 蓝色区域为CpG岛。

表4

ZmMAPKKK21蛋白一级结构预测结果

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

图3

ZmMAPKKK21蛋白理化性质分析 (a) 亲疏水性分析，(b) 信号肽预测，(c) 跨膜结构预测，(d) 磷酸化位点预测。

图4

ZmMAPKKK21蛋白与其他植物MAPKKK基因编码的氨基酸序列的系统进化树

图5

ZmMAPKKK21蛋白及其同源蛋白的保守结构域分析

图6

ZmMAPKKK21蛋白及其同源蛋白三级结构预测

图7

亚细胞定位预测（a）和蛋白互作网络预测（b）

图8

ZmMAPKKK21基因在各器官的相对表达量 R：根，L：叶，S：茎。

图9

干旱胁迫下ZmMAPKKK21基因在根、叶组织中的的表达 “**”代表P < 0.01水平上的显著性差异。

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