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作物学报 ›› 2023, Vol. 49 ›› Issue (9): 2433-2445.doi: 10.3724/SP.J.1006.2023.23071

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米转录因子ZmEREB211对非生物逆境胁迫的应答

艾蓉1,2(), 张春2(), 悦曼芳2, 邹华文1,*(), 吴忠义2,*()   

  1. 1长江大学农学院, 湖北荆州434025
    2北京市农林科学院 / 生物技术研究所 / 农业基因资源与生物技术北京市重点实验室, 北京100097
  • 收稿日期:2022-10-21 接受日期:2023-02-21 出版日期:2023-09-12 网络出版日期:2023-03-01
  • 通讯作者: *邹华文, E-mail: zouhuawen@yangtzeu.edu.cn; 吴忠义, E-mail: zwu22@126.com
  • 作者简介:艾蓉, E-mail: airong202203@163.com
    张春, E-mail: spring2007318@163.com第一联系人：**同等贡献
  • 基金资助:
    国家自然科学基金项目(32001430);国家自然科学基金项目(32171952);国家自然科学基金项目(31971839);北京市农林科学院科技创新能力建设专项(KJCX20220402)

Response of maize transcriptional factor ZmEREB211 to abiotic stress

AI Rong1,2(), ZHANG Chun2(), YUE Man-Fang2, ZOU Hua-Wen1,*(), WU Zhong-Yi2,*()   

  1. 1College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China
    2Institute of Biotechnology, Beijing Academy of Agriculture and Forestry Sciences, Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing 100097, China
  • Received:2022-10-21 Accepted:2023-02-21 Published:2023-09-12 Published online:2023-03-01
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(32001430);National Natural Science Foundation of China(32171952);National Natural Science Foundation of China(31971839);Beijing Academy of Agricultural and Forestry Sciences(KJCX20220402)

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摘要：

AP2/ERF (APETALA2/ethylene-responsive factor)转录因子是植物中最大的转录因子家族之一, 在调控植物生长发育和响应逆境胁迫等方面起重要作用。探究玉米(Zea mays L.) AP2/ERF家族基因功能将为玉米新种质创制提供重要的基因资源。本研究克隆获得了ZmEREB211 (Gene ID: 103647485)基因, 利用生物信息学分析、实时荧光定量PCR等技术对该基因的基本特性、组织表达特性及响应逆境胁迫表达模式等进行了分析; 对转基因拟南芥株系进行了相应逆境胁迫处理和表型鉴定。结果显示: 该基因只包含1个外显子, cDNA全长为792 bp, 编码263个氨基酸; ZmEREB211蛋白分子量为27.9 kD, 理论等电点为6.01, 具有AP2家族所特有的保守结构域; ZmEREB211基因在玉米根系中的表达量最高, 且在幼根中的表达量高于成熟根中的表达量; 同时该基因在脱水、高盐、干旱和低温等处理条件下均有不同程度的诱导表达。在分别含有不同浓度梯度的NaCl、甘露醇(mannitol)和茉莉酸(jasmonic acid, JA)的1/2 MS培养基上, 转ZmEREB211基因拟南芥株系的根长显著长于野生型。在干旱和高盐处理下, 盆栽转基因拟南芥株系较野生型株系表现出更强的耐受性, 且苗期的绿叶数显著多于野生型, 过氧化物酶(POD)活性和叶绿素含量均显著高于野生型。研究表明ZmEREB211可能参与调控玉米根系生长发育, 对高盐、干旱、渗透等逆境胁迫及JA激素处理均能起到正向的调控作用。本研究为进一步解析ZmEREB211在玉米中的生物学功能提供了重要的参考依据。

关键词: 玉米, ZmEREB211, 转录因子, 异源表达, 根系, 逆境胁迫

Abstract:

AP2/ERF (APETALA2/ethylene-responsive factor) transcription factor is one of the largest transcription factor families in plants, which plays an important role in regulating plant growth and development and responding to various stresses. Exploring the function of AP2/ERF family genes in maize (Zea mays L.) will provide the important genetic resources for the creation of new maize germplasm. In this study, ZmEREB211 (Gene ID: 103647485) gene was cloned and its basic characteristics, tissue expression characteristics, and the relative expression patterns in response to stress were analyzed by bioinformatics and qRT-PCR. The transgenic Arabidopsis lines were subjected to corresponding stress treatment and phenotypic identification. The results showed that the gene contained only one exon and the full-length cDNA was 792 bp which encoding 263 amino acids. The ZmEREB211 protein had a molecular weight of 27.9 kD and a theoretical isoelectric point of 6.01. It had a conserved domain unique to the AP2/ERF family. The relative expression level of ZmEREB211 gene was the highest in maize root system and the relative expression level in young roots was higher than mature roots. At the same time, the gene had different degrees of induced expression under dehydration, high salt, drought, and low temperature treatment conditions. On 1/2 MS medium containing different concentrations of NaCl, mannitol, and jasmonic acid (JA), root length of ZmEREB211 transgenic Arabidopsis lines was significantly longer than wild type. Under drought and high salt treatments, transgenic Arabidopsis lines had stronger tolerance than wild type, and the number of green leaves at seedling stage was significantly higher than wild type and the peroxidase (POD) activity and chlorophyll content were significantly higher than wild type. ZmEREB211 may be involved in the regulation of root growth and development in maize, and can play a positive regulatory role in high salt, drought, osmotic stress, and JA hormone treatments. This study provides an important reference for further analysis of the biological function of ZmEREB211 in maize.

Key words: maize, ZmEREB211, transcription factor, heterologous expression, root system, adversity stress

表1

本实验中所用的引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')
pZmEREB211-F CTGAAATCACCAGTCGGTACCATGACCAAGAAGCTCATCT (Kpn I)
pZmEREB211-R GCCCTTGCTCACCATGGTACCTCAAAAACTAGACGCGGCG (Kpn I)
pZmEREB211RT-F AGGAGGACGCCATCATCGT
pZmEREB211RT-R GGTACATGTTCCTGTTCCTACG
Actin-F
Actin-R		 TACGAGATGCCTGATGGTCAGGTCA
TGGAGTTGTACGTGGCCTCATGGAC
pGAPDHRT-F
pGAPDHRT-R		 CCCTTCATCACCACGGACTAC
AACCTTCTTGGCACCACCCT

图1

ZmEREB211生物信息学分析 A: 保守结构域分析; B: 跨膜结构预测; C: 蛋白空间结构预测; D: 启动子区顺式作用元件分析。"

图2

ZmEREB211在玉米不同组织中的相对表达量 不同小写字母表示差异显著(P < 0.05)。"

图3

ZmEREB211在不同非生物胁迫下的相对表达量 A~D: ZmEREB211分别在脱水、高盐、渗透、低温处理后的表达量。"

图4

ZmEREB211在玉米不同激素处理中的相对表达量"

图5

T4代拟南芥PCR检测和qPCR检测 A: T4代转基因拟南芥PCR检测; B: T4代转基因拟南芥qPCR检测。M: DL2000 marker; N: 阴性对照; W: 水对照; WT: 野生型拟南芥; L-1~L-6: T4代转基因拟南芥株系; *: P < 0.05; **: P < 0.01。"

图6

不同盐浓度下转基因拟南芥株系根长比较 A~D 分别为0、0.1、0.15和0.18 mol L-1盐处理的生长情况; E: 不同盐浓度下拟南芥株系平均主根长度; WT: 野生型拟南芥; L-3、L-4、L-5: 转ZmEREB211拟南芥株系; 标尺为1.5 cm; *: P < 0.05; **: P < 0.01。"

图7

不同甘露醇浓度下转基因拟南芥株系根长比较 A~D 分别为0、0.15、0.2和0.3 mol L-1甘露醇处理的生长情况; E: 不同甘露醇浓度下拟南芥株系平均主根长度。缩写同图6; 标尺为1.5 cm; *: P < 0.05; **: P < 0.01。"

图8

不同JA和ABA浓度下转基因拟南芥株系根长比较 A: 0、50、75和100 μmol L-1 JA处理下的生长情况; B: 不同JA浓度下拟南芥株系平均主根长度; C: 0、10、25和50 μmol L-1 ABA处理下的生长情况; D: 不同ABA浓度下拟南芥株系平均主根长度。缩写同图6; 标尺为1.5 cm; *: P < 0.05; **: P < 0.01。"

图9

土壤中干旱处理下拟南芥株系表型分析 A: 拟南芥表型; B: 干旱处理后绿叶率; C: 复水处理后绿叶率; D: POD活性的测定; E: 叶绿素含量测定。缩写同图6; *: P < 0.05; **: P < 0.01。"

图10

土壤中高盐处理下拟南芥株系表型分析 A: 拟南芥表型; B: 高盐处理后绿叶率; C: POD活性的测定; D: 叶绿素含量的测定。缩写同图6; *: P < 0.05; **: P < 0.01。"

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