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作物学报 ›› 2024, Vol. 50 ›› Issue (8): 2001-2013.doi: 10.3724/SP.J.1006.2024.33059

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米N-乙酰转移酶ZmNAT1基因响应非生物胁迫的功能分析

郭思语1,2(), 赵克勇2(), 代正罡1,2, 邹华文1,*(), 吴忠义2,*(), 张春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:2023-10-17 接受日期:2024-01-31 出版日期:2024-08-12 网络出版日期:2024-02-28
  • 通讯作者: * 张春, E-mail: spring2007318@163.com;邹华文, E-mail: zouhuawen@yangtzeu.edu.cn;吴忠义, E-mail: zwu22@126.com
  • 作者简介:郭思语, E-mail: 1030997671@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(32171952);国家自然科学基金项目(31971839)

Functional analysis of maize N-acetyltransferase ZmNAT1 gene in response to abiotic stress

GUO Si-Yu1,2(), ZHAO Ke-Yong2(), DAI Zheng-Gang1,2, ZOU Hua-Wen1,*(), WU Zhong-Yi2,*(), ZHANG Chun2,*()   

  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:2023-10-17 Accepted:2024-01-31 Published:2024-08-12 Published online:2024-02-28
  • Contact: * E-mail: spring2007318@163.com;E-mail: zouhuawen@yangtzeu.edu.cn;E-mail: zwu22@126.com
  • About author:** 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)

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摘要：

GNAT (Gcn5-related N-acetyltransferase)家族蛋白在调控植物生长发育和响应逆境胁迫等过程中发挥着重要作用。目前GNAT家族基因在多个物种中的生物学功能已有报道, 但在玉米(Zea mays L.)中的功能验证研究却很少。探究玉米GNAT家族基因的功能, 不仅能丰富我国的玉米育种基因资源, 同时也可为玉米的新种质资源创制提供重要依据。本研究克隆了ZmNAT1基因(Gene ID: 541936, GRMZM2G123159), 通过生物信息学分析发现, 该基因CDS全长为519 bp, 编码172个氨基酸, 具有GNAT家族特有的保守结构域。通过对ZmNAT1基因在玉米不同时期不同组织中的表达量和不同逆境胁迫下表达模式分析发现: ZmNAT1在成熟根中的表达量最高, 在不同非生物逆境胁迫处理下, ZmNAT1基因均有不同程度的诱导表达。通过异源表达获得了3株独立的表达量较高的转基因拟南芥(Arabidopsis thaliana L.)纯合株系, 对其进行了不同逆境胁迫处理下表型鉴定实验, 结果表明, 转基因拟南芥相对于野生型拟南芥有更好的表型, 在盐胁迫、渗透胁迫和干旱条件下的转基因株系的根显著长于野生型, 且植株较野生型植株的绿叶率和叶绿素含量均升高、丙二醛含量降低, 差异均达到显著水平。由此推测, ZmNAT1基因可能参与玉米对干旱、盐等非生物逆境胁迫的应答。本研究为进一步解析ZmNAT1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, ZmNAT1, 根系, 生长发育, 逆境胁迫

Abstract:

The GNAT (Gcn5-related N-acetyltransferase) family proteins play a crucial role in regulating plant growth and development, and responding to stress. At present, the biological functions of GNAT family genes have been reported in many species, but there are few studies on its function validation in maize (Zea mays L.). Exploring the functions of maize GNAT family genes not only enriches the genetic resources for maize breeding in China, but also provides an important basis for the creation of new germplasm resources of maize. In this study, the ZmNAT1 gene (Gene ID: 541936, GRMZM2G123159) was cloned. Bioinformatics analysis showed that the CDS of this gene was 519 bp, encoding 172 amino acids. ZmNAT1 contained a conserved domain unique to GNAT family. The relative expression level of ZmNAT1 gene in different tissues of maize at different stages under different stress conditions showed that the expression level of ZmNAT1 was the highest in mature roots, and the relative expression of ZmNAT1 gene could be induced in different degrees under different abiotic adversity stress conditions. Three independent transgenic Arabidopsis (Arabidopsis thaliana L.) pure lines with higher expression were obtained by heterologous expression, and phenotypic characterisation experiments under different adversity stress treatments were carried out on them, the results showed that transgenic Arabidopsis had a better phenotype relative to wild-type Arabidopsis, and the roots of transgenic plants under salt stress, osmotic stress, and drought were significantly longer than those of the wild-type,and the plants showed higher green leaf rate and chlorophyll content, and lower malondialdehyde content than the wild-type plants, which were significant difference. We speculated that ZmNAT1 gene may be involved in response to abiotic stress such as drought and salt in maize. This study provides an important reference for further analysis of the biological functions of ZmNAT1 in maize.

Key words: maize (Zea mays L.), ZmNAT1, root system, growth and development, adversity stress

表1

引物信息"

引物名称Primer name 引物序列Primer sequence (5'-3')
pZmNAT1-F CCCGGGCTGCAGAATTCATGACGACAATCCGTCGGTTCTG
pZmNAT1-R CCATGGTACCCTCGAGAAGCTTGTTCAAAAATAAACTAGTGG
pZmNAT1RT-F AAGGGTGCTCCGGTACTACT
pZmNAT1RT-R AGTGGACGACAAATATTCGAGA
pGAPDHRT-F CCCTTCATCACCACGGACTAC
pGAPDHRT-R AACCTTCTTGGCACCACCCT
pActinRT-F GGTAACATTGTGCTCAGTGGTGG
pActinRT-R AACGACCTTAATCTTCATGCTGC

图1

ZmNAT1生物信息学和进化分析 A: 保守结构域分析; B: 跨膜结构预测; C: ZmNAT1启动子序列分析; D: 拟南芥、水稻和玉米HAT家族蛋白的系统发育树。"

图2

ZmNAT1在玉米中不同发育时期不同组织中的相对表达量的分析 不同小写字母表示差异显著(P < 0.05)。"

图3

不同非生物胁迫下玉米地上部和根部中ZmNAT1的相对表达量 A: 脱水处理; B: 高盐处理; C : 渗透处理; D: 低温处理。*: P < 0.05; **: P < 0.01。"

图4

T3代ZmNAT1转基因拟南芥阳性鉴定及相对表达量分析 A: T3代转基因拟南芥ZmNAT1基因PCR鉴定; B: T3代转基因拟南芥ZmNAT1基因RT-qPCR鉴定。M: DL2000 marker; P: 阳性对照; N: 阴性对照; W: 水对照; WT: 野生型拟南芥; L-1~L-6: T3代ZmNAT1转基因拟南芥; **: P < 0.01。"

图5

ZmNAT1转基因拟南芥在不同盐浓度处理下的生长发育情况和根长统计 A~E: ZmNAT1转基因拟南芥和野生型拟南芥在0、0.10、0.12、0.15和0.18 mol L-1盐处理下的生长发育情况; F: 转基因拟南芥和野生型拟南芥在不同浓度盐处理下的平均主根长统计和比较; WT: 野生型拟南芥; L-2, L-5, L-6: 转ZmNAT1基因拟南芥; 标尺为1.5 cm。 *: P < 0.05; **: P < 0.01。"

图6

ZmNAT1转基因拟南芥在不同甘露醇浓度处理下的生长发育情况和根长统计 缩写同图5。A~D: ZmNAT1转基因拟南芥和野生型拟南芥在0、0.15、0.20和0.30 mol L-1甘露醇处理下的生长发育情况; E: 转基因拟南芥和野生型拟南芥在不同浓度甘露醇处理下的平均主根长统计和比较。标尺为1.5 cm。*: P < 0.05; **: P < 0.01。"

图7

ZmNAT1转基因拟南芥在干旱处理下的生长发育情况和生理指标分析 A: 分别是正常条件、干旱处理、复水后野生型拟南芥和转基因拟南芥在土壤中的生长情况; B: 对照组和实验组WT和转基因拟南芥MDA含量; C: 对照组和实验组WT和转基因拟南芥总叶绿素含量; D: 干旱处理后WT和转基因拟南芥绿叶率统计; E: 复水处理2 d后WT和转基因拟南芥绿叶率统计。缩写同图5。*: P < 0.05; **: P < 0.01。"

图8

ZmNAT1转基因拟南芥在盐处理下的生长发育情况和生理指标分析 A: 分别是正常条件、盐处理后野生型拟南芥和转基因拟南芥在土壤中的生长情况; B: 对照组和实验组WT和转基因拟南芥MDA含量; C: 对照组和实验组WT和转基因拟南芥总叶绿素含量; D: 盐处理后WT和转基因拟南芥绿叶率统计。缩写同图5。*: P < 0.05; **: P < 0.01。"

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