检索
E-mail
RSS
高级检索 图表检索

当期目录 我要投稿 过刊浏览
高级检索 图表检索

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

RichHTML

3

PDF (PC)

14

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。

[1] 周秒依, 任雯, 赵冰兵, 等. 植物MAPK级联途径应答的非生物胁迫研究进展. 中国农业科技导报, 2020, 22(2):22-29.
doi: 10.13304/j.nykjdb.2019.0737
[2] Zhang M M, Zhang S Q. Mitogen-activated protein kinase cascades in plant signaling. Journal of Integrative Plant Biology, 2022, 64(2):301-341.
doi: 10.1111/jipb.13215
[3] Xu J, Zhang S Q. Mitogen-activated protein kinase cascades in signaling plant growth and development. Trends in Plant Science, 2015, 20(1):56-64.
doi: 10.1016/j.tplants.2014.10.001 pmid: 25457109
[4] Zhang M M, Su J B, Zhang Y, et al. Conveying endogenous and exogenous signals: MAPK cascades in plant growth and defense. Current Opinion in Plant Biology, 2018, 45:1-10.
doi: S1369-5266(17)30213-3 pmid: 29753266
[5] Liu D D, Zhu M, Hao L L, et al. GhMAPKKK49, a novel cotton (Gossypium hirsutum L.) MAPKKK gene, is involved in diverse stress responses. Acta Physiologiae Plantarum, 2016, 38(1):13.
doi: 10.1007/s11738-015-2029-y
[6] Jonak C, Okresz L, Bogre L, et al. Complexity,cross talk and integration of plant MAP kinase signalling. Current Opinion in Plant Biology, 2002, 5(5):415-424.
doi: 10.1016/S1369-5266(02)00285-6
[7] Rao K P, Richa T, Kumar K, et al. In silico analysis reveals 75 members of mitogen-activated protein kinase kinase kinase gene family in rice. DNA Research, 2010, 17(3):139-153.
doi: 10.1093/dnares/dsq011 pmid: 20395279
[8] 刘晨, 曹小汉, 殷丹丹, 等. MAPK信号通路调控植物响应非生物胁迫的研究进展. 安徽农业科学, 2022, 50(18):9-16.
[9] Moustafa K. Improving plant stress tolerance: potential applications of engineered MAPK cascades. Trends in Biotechnology, 2014, 32(8):389-390.
doi: 10.1016/j.tibtech.2014.06.005 pmid: 24986255
[10] Zhen W, Song Y, Ren W C, et al. Genome-wide identification of MAPK, MAPKK, and MAPKKK gene families in Fagopyrum tataricum and analysis of their expression patterns under abiotic stress. Frontiers in Genetics, 2022, 13:894048.
doi: 10.3389/fgene.2022.894048
[11] Wang N, Liu Y S, Dong C H, et al. MdMAPKKK1 regulates apple resistance to Botryosphaeria dothidea by interacting with MdBSK1. International Journal of Molecular Sciences, 2022, 23(8):4415.
doi: 10.3390/ijms23084415
[12] Li Y Y, Cai H X, Liu P, et al. Arabidopsis MAPKKK18 positively regulates drought stress resistance via downstream MAPKK3. Biochemical and Biophysical Research Communications, 2017, 484(2):292-297.
doi: 10.1016/j.bbrc.2017.01.104
[13] Zhou M Y, Zhao B B, Han S L, et al. Comprehensive analysis of MAPK cascade genes in sorghum (Sorghum bicolor L.) reveals SbMPK14 as a potential target for drought sensitivity regulation. Genomics, 2022, 114(2):110311.
doi: 10.1016/j.ygeno.2022.110311
[14] Daryanto S, Wang L, Jacinthe P A. Global synthesis of drought effects on maize and wheat production. PLoS ONE, 2017, 11(5):e0156362.
doi: 10.1371/journal.pone.0156362
[15] Rai M K, Kalia R K, Singh R, et al. Developing stress tolerant plants through in vitro selection—An overview of the recent progress. Environmental and Experimental Botany, 2011, 71(1):89-98.
doi: 10.1016/j.envexpbot.2010.10.021
[16] Liu Y, Zhou M Y, Gao Z X, et al. RNA-Seq analysis reveals MAPKKK family members related to drought tolerance in maize. PLoS ONE, 2015, 10(11):e0143128.
doi: 10.1371/journal.pone.0143128
[17] Zhang M Y, Pan J W, Kong X D, et al. ZmMKK3, a novel maize group B mitogen-activated protein kinase kinase gene, mediates osmotic stress and ABA signal responses. Journal of Plant Physiology, 2012, 169(15):1501-1510.
doi: 10.1016/j.jplph.2012.06.008 pmid: 22835533
[18] Zhang Z B, Li X L, Yu R, et al. Isolation,structural analysis, and expression characteristics of the maize TIFY gene family. Molecular Genetics and Genomics, 2015, 290(5):1849-1858.
doi: 10.1007/s00438-015-1042-6
[19] Lin L, Wu J, Jiang M, et al. Plant mitogen-activated protein kinase cascades in environmental stresses. International Journal of Molecular Sciences, 2021, 22(4):1543.
doi: 10.3390/ijms22041543
[20] Komis G, Šamajov O, Ovečka M, et al. Cell and developmental biology of plant mitogen-activated protein kinases. Annual Review of Plant Biology, 2018, 69(1):237-265.
doi: 10.1146/arplant.2018.69.issue-1
[21] Zi S, Zhao B B, Wei S, et al. Genome-wide identification and characterization of the MAPKKK, MKK, and MPK families in Chinese elite maize inbred line Huangzaosi. The Plant Genome, 2022, 15(3):e20216.
doi: 10.1002/tpg2.v15.3
[22] 卢峰, 张飞, 段有厚. 干旱胁迫对高粱苗期物质生产及生理特性的影响. 作物杂志, 2015(2):149-153.
[23] 于国红, 刘朋程, 郝洪波, 等. 不同基因型谷子对干旱胁迫的调控机制. 植物营养与肥料学报, 2022, 28(1):157-167.
[24] Wang M, Yue H, Feng K W, et al. Genome-wide identification, phylogeny and expressional profiles of mitogen activated protein kinase kinase kinase (MAPKKK) gene family in bread wheat (Triticum aestivum L.). BMC Genomics, 2016, 17(1):668.
doi: 10.1186/s12864-016-2993-7
[25] Wu J, Wang J, Pan C T, et al. Genome-wide identification of MAPKK and MAPKKK gene families in tomato and transcriptional profiling analysis during development and stress response. PLoS ONE, 2014, 9(7):e103032.
doi: 10.1371/journal.pone.0103032
[26] 李媛媛. AtMAPKKK18调节干旱胁迫抗性的分子机理研究. 泰安: 山东农业大学, 2016.
[27] 王芳, 彭云玲, 方永丰, 等. 花后干旱胁迫对不同持绿型玉米叶片衰老的影响. 水土保持通报, 2018, 38(4):60-66.
[28] 陆兰姣, 王治红. 探讨玉米自交系苗期耐旱性差异分析. 农业与技术, 2016, 36(2):7,29.
[29] 许璐璐, 王涵, 高盼盼, 等. 环境胁迫对植物根系形态的影响. 安徽农业科学, 2020, 48(14):16-19.
[1] 王怀苹, 杨明达, 张素瑜, 李帅, 关小康, 王同朝. 不同节水灌溉方式对夏玉米生长、产量及水分利用的影响[J]. 作物杂志, 2024, (2): 206–212
[2] 张俊, 蔡苏云, 徐子豪, 侯蕾, 贺润丽, 尹桂芳, 王莉花, 王艳青, 卢文洁, 孙道旺. 苦荞FtERF基因克隆、生物信息学及其表达分析[J]. 作物杂志, 2024, (2): 23–29
[3] 张玉, 杨文静, 刘璇, 聂峰杰, 张丽, 石磊, 张国辉, 郭志乾, 巩檑. 马铃薯细胞壁蔗糖转化酶基因StCWIN1启动子克隆与表达及其在干旱胁迫下的作用分析[J]. 作物杂志, 2024, (2): 54–61
[4] 胡昊驰, 王富贵, 朱孔艳, 胡树平, 王猛, 王志刚, 孙继颖, 于晓芳, 包海柱, 高聚林. 秸秆还田年限及施磷水平对玉米根系生长和产量的影响[J]. 作物杂志, 2024, (2): 80–88
[5] 冯勇, 侯旭光, 薛春雷, 张来厚, 宋国栋, 苏敏莉, 傅晓华, 孙宇燕. 内蒙古玉米品种适宜生态区划分[J]. 作物杂志, 2024, (1): 23–30
[6] 王海涛, 任春梅, 董岩, 李硕, 程兆榜, 季英华. 江苏淮安市高粱上玉米黄花叶病毒的分子检测与鉴定[J]. 作物杂志, 2024, (1): 233–238
[7] 马娟, 黄璐, 宇婷, 郭国俊, 朱卫红, 刘京宝. 玉米穗粗一般配合力多位点全基因组关联分析和基因组预测[J]. 作物杂志, 2024, (1): 31–39
[8] 吕宝莲, 杨宇昕, 崔立操, 史峰, 马亮, 孔秀英, 张立超, 倪志勇. 小麦bHLH家族转录因子的鉴定及其在盐胁迫条件下的表达分析[J]. 作物杂志, 2024, (1): 65–72
[9] 毋莹, 胡蝶, 李婷, 段乾元, 韦宁宁, 张兴华, 徐淑兔, 薛吉全. 玉米WRKY转录因子IIc亚家族分析及其在干旱胁迫下的表达分析[J]. 作物杂志, 2024, (1): 80–89
[10] 金玉, 郭新宇, 张颖, 李大壮, 王璟璐. 玉米叶片气孔表型鉴定及研究进展[J]. 作物杂志, 2023, (6): 1–10
[11] 吴琦, 明博, 高尚, 杨宏业, 张川, 初振东, 李少昆. 东北冷凉区玉米籽粒脱水模型构建策略研究[J]. 作物杂志, 2023, (6): 108–113
[12] 梁忠宇, 薛军, 张国强, 明博, 沈东萍, 方梁, 周林立, 张玉芹, 杨恒山, 王克如, 李少昆. 水肥一体化施磷量对玉米抗倒伏能力的影响[J]. 作物杂志, 2023, (6): 190–194
[13] 曹庆军, 李刚, 杨浩, 娄玉勇, 杨粉团, 孔凡丽, 李辛琲, 赵新凯, 姜晓莉. 不同耕作方式对春玉米种床质量的影响及其与幼苗群体建成和产量的关系[J]. 作物杂志, 2023, (5): 249–254
[14] 于乐, 李林, 黄红娟, 黄兆峰, 朱文达, 魏守辉. 湖北省玉米田杂草种类组成及群落特征[J]. 作物杂志, 2023, (5): 272–279
[15] 杨宗莹, 肖贵, 张红伟. 利用玉米F1群体进行玉米全株鲜重的全基因组预测分析[J]. 作物杂志, 2023, (5): 43–48
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!