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作物学报 ›› 2024, Vol. 50 ›› Issue (8): 1920-1933.doi: 10.3724/SP.J.1006.2024.33070

• 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ZmEREB180调控根系生长发育及耐逆的功能研究

刘宸铭1,2(), 赵克勇2(), 悦曼芳2, 赵延明1,*(), 吴忠义2,*(), 张春2,*()   

  1. 1青岛农业大学农学院, 山东青岛 266109
    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:2023-11-24 接受日期:2024-04-01 出版日期:2024-08-12 网络出版日期:2024-04-19
  • 通讯作者: * 张春, E-mail: zhangchun@babrc.ac.cn;吴忠义, E-mail: zwu22@126.com;赵延明, E-mail: zhaoym796@sina.com
  • 作者简介:刘宸铭, E-mail: 237588660@qq.com;
    赵克勇, E-mail: zhaokeyong@baafs.net.cn
    ** 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(32372053)

Functional study on the regulation of root growth and development and stress tolerance by maize transcription factor ZmEREB180

LIU Chen-Ming1,2(), ZHAO Ke-Yong2(), YUE Man-Fang2, ZHAO Yan-Ming1,*(), WU Zhong-Yi2,*(), ZHANG Chun2,*()   

  1. 1College of Agriculture, Qingdao Agricultural University, Qingdao 266109, Shandong, China
    2Institute of Biotechnology, Beijing Academy of Agriculture and Forestry Sciences / Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing 100097, China
  • Received:2023-11-24 Accepted:2024-04-01 Published:2024-08-12 Published online:2024-04-19
  • Contact: * E-mail: zhangchun@babrc.ac.cn;E-mail: zwu22@126.com;E-mail: zhaoym796@sina.com
  • About author:** Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(32001430);National Natural Science Foundation of China(32372053)

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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、响应逆境胁迫、调节激素信号转导和物质代谢等多种生物学过程中发挥着重要作用。目前AP2/ERF超家族基因在许多植物物种中的生物学功能已经得到验证, 但对玉米(Zea mays L.)中AP2/ERF基因的结构和功能的研究报道较少。前期工作中我们发现, ZmEREB180 (ethylene-responsive element binding)转录因子, 在鉴定的玉米六叶期(V6)、十二叶期(V12)和抽雄期(VT)等关键发育时期, 根系中的表达量均存在显著差异; 通过组织表达分析发现该基因主要在玉米根系中表达, 且在幼根中的表达量显著高于成熟根, 推测该基因可能参与玉米根系生长发育调控。本研究克隆了ZmEREB180 (Gene ID: 100192457)基因, 结合生物信息学分析、实时荧光定量PCR (RT-qPCR)、亚细胞定位和转基因拟南芥(Arabidopsis thaliana L.)株系的耐逆表型鉴定等生物学手段, 初步分析了该基因的表达模式和生物学功能。该基因包含2个外显子, 编码序列全长1023 bp, 编码340个氨基酸, 具有AP2/ERF家族所特有的保守结构域; 该基因在玉米根系中表达量最高, 且在高盐、干旱、高氮和低氮等胁迫处理条件下的玉米根部皆有不同程度的诱导表达, 其中, 低氮处理较高氮处理具有更高的表达量和更快的响应速率; 在含0.10、0.15 mol L-1 NaCl以及0.15、0.20和0.30 mol L-1甘露醇(mannitol)的1/2 MS固体培养基上, 转ZmEREB180基因拟南芥的主根长度均显著长于野生型; 土壤环境中, 高盐和干旱胁迫条件下的转基因植株比野生型拟南芥具有更健康的生长状态、更高的绿叶率、更低的丙二醛含量和更高的过氧化物酶活性。转录因子ZmEREB180可能在调控玉米根系生长发育方面具有积极的促进作用, 并且能增强玉米植株对高盐、干旱、渗透、低氮等逆境胁迫的耐受性。本研究为下一步鉴定转录因子ZmEREB180在玉米中的生物学功能和分子机制奠定了良好的基础。

关键词: 玉米, ZmEREB180, 转录因子, 根系, 非生物胁迫, 氮利用效率

Abstract:

The AP2/ERF (APETALA2/ethylene-responsive factor) family is one of the largest families of transcription factors in plants, and plays an important role in regulating a variety of biological processes such as plant growth and development, respond-ing to adversity stress, and regulating hormone signaling and substance metabolism. The biological functions of AP2/ERF family genes in many plant species have been validated, but fewer studies have been reported in maize (Zea mays L.). In the previous work, there was significant difference in the relative expression level of the ZmEREB180 transcription factor in root system between the identified critical developmental stages of maize at the six-leaf (V6), the twelve-leaf (V12), and tasseling (VT) stages, the tissue expression analysis revealed that this gene was mainly expressed in maize root system and was significantly higher in young roots than in mature roots, and it was hypothesized that this gene might be involved in the regulation of maize root growth and development. In this study, we cloned the ZmEREB180 (Gene ID: 100192457) transcription factor gene, and preliminarily analyzed the relative expression pattern and biological functions of ZmEREB180 by bioinformatics, RT-qPCR, subcellular localization, and stress-resistant phenotype identification of transgenic Arabidopsis (Arabidopsis thaliana L.) lines. This gene contained two exons and the full-length cDNA was 1023 bp, encoding 340 amino acids. The gene had a conserved domain unique to the AP2/ERF family, which expressed most highly in root system of maize; and the gene had different degrees of induced expression under high salt, drought, high nitrogen, and low nitrogen treatment conditions, with a more rapid and higher expression in response to low nitrogen than high nitrogen; root length of ZmEREB180 transgenic Arabidopsis lines were all significantly longer than the wild type (WT) on 1/2 MS medium containing 0.10 mol L-1, 0.15 mol L-1 NaCl, and 0.15 mol L-1, 0.20 mol L-1, 0.30 mol L-1 mannitol (MNT). Under high salt and drought stress conditions in soil environments, transgenic Arabidopsis lines had healthier growth status, higher green leaf percentage, lower malondialdehyde (MDA) content, and higher peroxidase (POD) activity than WT. The transcription factor ZmEREB180 may play a positive and promotional role in regulating the growth and development of maize root system and enhance the tolerance of maize plants under high salt, drought, osmosis, low nitrogen, and other adversity stresses. This study lays a good foundation for further identification of the biological function and molecular mechanism of transcription factor ZmEREB180 in maize.

Key words: maize (Zea mays L.), ZmEREB180, transcription factor, root system, abiotic stress, nitrogen use efficiency

表1

本实验中所用的引物及序列"

引物名称Primer name 引物序列Primer sequence (5'-3')
pZmEREB180 OE-F CTGAAATCACCAGTCGGTACCATGTGCGGAGGCGCCATCC (Kpn I)
pZmEREB180 OE-R gcccttgctcaccatgGTACCTCAGAAAACAGAACCGTCG (Kpn I)
pZmEREB180RT-F CTGACGAGCTGGCGTTC
pZmEREB180RT-R TCAGAAAACAGAACCGTCG
pGAPDHRT-F CCCTTCATCACCACGGACTAC
pGAPDHRT-R AACCTTCTTGGCACCACCCT
pActinRT-F CCGTGAAGCCAGAAGCTACG
pActinRT-R AACTTGTGGCCGTTTACGTCG
pZmEREB180-F CTGACGAGCTGGCGTTC
pZmEREB180-R TCAGAAAACAGAACCGTCG

图1

ZmEREB180基因及其蛋白的生物信息学分析 A: 蛋白空间结构域分析; B: 蛋白跨膜分析; C: 蛋白三级结构预测; D: 启动子区序列分析。"

图2

ZmEREB180在玉米不同组织中的相对表达量 不同小写字母表示显著差异(P < 0.05)。"

图3

ZmEREB180在不同非生物胁迫下的相对表达量 A~D: ZmEREB180分别在脱水、高盐、渗透、低温处理后的表达量分析; *: P < 0.05; **: P < 0.01。"

图4

ZmEREB180在玉米高氮和低氮处理下的相对表达量 A~B: ZmEREB180分别在高氮、低氮处理后的表达量分析; *: P < 0.05; **: P < 0.01。"

图5

ZmEREB180在玉米不同激素处理下的相对表达量 A~E: ZmEREB180分别在JA、ABA、SA、2,4-D和GA处理后的表达量分析。*: P < 0.05; **: P < 0.01。"

图6

ZmEREB180蛋白在烟草叶片中的亚细胞定位 GFP: 绿色荧光蛋白图; Merged: 叠加图; Bright field: 明场; CK: 转入空载体的烟草叶片; ZmEREB180-EGFP: 转入目的基因载体的烟草叶片; 标尺为20 μm。"

图7

转基因拟南芥(T3)PCR检测和RT-qPCR检测 A: 转基因拟南芥(T3代)PCR检测; B: 转基因拟南芥(T3代)RT-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: 转基因拟南芥(T3代)株系; **: P < 0.01; ***: P < 0.001。"

图8

不同盐浓度下转基因拟南芥株系的表型分析 A~D: 转基因拟南芥植株和WT植株分别在0、0.10、0.15和0.18 mol L-1 NaCl处理的1/2 MS培养基上的生长情况; E: 1/2 MS培养基上植株初生根长的平均数据; WT: 野生型拟南芥; L-3、L-5、L-6: 转ZmEREB180拟南芥株系; 标尺为1.5 cm; *: P < 0.05; **: P < 0.01。"

图9

不同甘露醇浓度下转基因拟南芥株系的表型分析 A~D: 转基因拟南芥植株和WT植株分别在0、0.15、0.20和0.30 mol L-1 甘露醇处理的1/2 MS培养基上的生长情况; E: 1/2 MS培养基上植株初生根长的平均数据。缩写同图8; 标尺为1.5 cm; *: P < 0.05; **: P < 0.01。"

图10

土壤中干旱胁迫处理下拟南芥株系表型分析 A: 转基因及野生型 拟南芥植株的表型; B: 干旱处理后绿叶率统计分析结果; C: MDA含量; D: POD活性; E: 复水处理后绿叶率统计分析结果。缩写同图8; *: P < 0.05;**: P < 0.01。"

图11

土壤中高盐胁迫处理下拟南芥株系表型分析 A: 转基因及野生型拟南芥植株的表型; B: 高盐处理后绿叶率统计分析; C: MDA含量; D: POD活性。缩写同图8; *: P < 0.05;**: P < 0.01。"

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