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作物学报 ›› 2025, Vol. 51 ›› Issue (9): 2318-2329.doi: 10.3724/SP.J.1006.2025.53011

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米低温响应基因ZmNTL1和ZmNTL5的鉴定及功能分析

高源**(), 王宇琦**(), 姜佳宁, 赵健雄, 王雪贺缘, 王浩宇, 张芮嘉, 徐晶宇, 贺琳*()   

  1. 黑龙江八一农垦大学, 黑龙江大庆 163319
  • 收稿日期:2025-02-19 接受日期:2025-06-01 出版日期:2025-09-12 网络出版日期:2025-06-10
  • 通讯作者: *贺琳, E-mail: linlinhe65@sina.com
  • 作者简介:高源, E-mail: 644909333@qq.com;王宇琦, E-mail: 3496668542@qq.com
    **同等贡献
  • 基金资助:
    本研究由国家自然科学基金项目(31701328);黑龙江省博士后科研启动金项目(LBH-Q21160)

Identification and functional analysis of low temperature responsive genes ZmNTL1 and ZmNTL5 in maize

GAO Yuan**(), WANG Yu-Qi**(), JIANG Jia-Ning, ZHAO Jian-Xiong, WANG Xue-He-Yuan, WANG Hao-Yu, ZHANG Rui-Jia, XU Jing-Yu, HE Lin*()   

  1. Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang, China
  • Received:2025-02-19 Accepted:2025-06-01 Published:2025-09-12 Published online:2025-06-10
  • Contact: *E-mail: linlinhe65@sina.com
  • About author:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(31701328);Postdoctoral Research Foundation of Heilongjiang Province(LBH-Q21160)

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摘要：

低温是限制东北地区玉米产量的主要逆境之一。膜NAC转录因子在植物抵御逆境过程中发挥着重要作用, 探究其功能将为玉米耐低温育种提供一定的理论依据。本研究通过生物信息技术分析了9条玉米NTL基因的分子特性, 利用已发表的玉米低温RNA-seq数据, 发现6个成员在根和叶中同时受到低温诱导显著上调表达。选取蛋白同源性达到80.04%的ZmNTL1与ZmNTL5基因, 采用基因敲除技术构建了该2条基因的双敲材料zmntl1zmntl5。在低温胁迫条件下, zmntl1zmntl5株系的叶片离子渗透率显著高于B104株系; 而其地上部干重和鲜重、地下部干重和鲜重以及叶绿素含量均显著低于B104株系; zmntl1zmntl5株系的O2-和H2O2含量显著高于B104对照株系, 而GST、SOD、POD和CAT活性均显著低于B104株系。此外, 低温胁迫下ZmGST24、ZmNCED3、ZmDREB1.6和ZmDREB2A基因在zmntl1zmntl5株系中的表达量较B104株系显著降低。综上所述, ZmNTL1与ZmNTL5的缺失降低了植物对低温的耐受性, 表明二者是玉米低温耐受性的正调控因子。本研究为耐冷玉米品种改良和新品种培育提供了重要的理论基础和宝贵的遗传资源。

关键词: 玉米, 低温, RNA-seq, NTL, 基因编辑

Abstract:

Low temperature is one of the major abiotic stresses limiting maize yield in Northeast China. Membrane-bound NAC transcription factors play important roles in plant stress responses. Investigating their function provides a theoretical basis for breeding maize with enhanced cold tolerance. In this study, the molecular characteristics of nine maize NTL genes were analyzed using bioinformatics approaches. Analysis of publicly available RNA-seq data revealed that six of these genes were significantly upregulated by low temperature stress in both roots and leaves. Among them, ZmNTL1 and ZmNTL5, which share 80.04% sequence homology, were selected for further study. A double knockout mutant (zmntl1zmntl5) was generated using gene-editing techniques. Under low temperature stress, the ion leakage rate in the leaves of zmntl1zmntl5 was significantly higher than in the wild-type B104. The fresh and dry weights of both aboveground and belowground tissues, as well as chlorophyll content, were significantly lower in zmntl1zmntl5 compared to B104. In contrast, zmntl1zmntl5 exhibited significantly higher levels of O2- and H2O2, while the activities of antioxidant enzymes GST, SOD, POD, and CAT were significantly reduced. Furthermore, the expression levels of ZmGST24, ZmNCED3, ZmDREB1.6, and ZmDREB2A were markedly decreased in zmntl1zmntl5 compared to B104 under cold stress. In summary, the absence of ZmNTL1 and ZmNTL5 compromises cold tolerance in maize, suggesting that both genes function as positive regulators in the cold stress response. This study provides a valuable theoretical foundation and genetic resources for the improvement of cold-tolerant maize varieties and the development of new cultivars.

Key words: maize, low temperature, RNA-seq, NTL, gene editing

表1

本研究所用引物"

引物名称
Primer name		 序列
Sequence (5′-3′)		 作用
Function
pCPB-Cas9-F GACGGCCAGTGCCAAGCTTGCTGTTTTTGTTAGCCCCATCG 载体构建 Vector construction
pCPB-Cas9-R CTAGCTCTAAAACAATCCTGGTGGCAGGTGCATAATTCGGTGCTTGC 载体构建 Vector construction
ZmNTL1-F CTTCTGCATTTCTAGGCATGATTG 测序 Sequencing
ZmNTL1-R CCGTCTATCTGCCTATGATTCTCAA 测序 Sequencing
ZmNTL5-F CTCGGGTGCTGTGTTTCTGTTTG 测序 Sequencing
ZmNTL5-R GAGGATACAGATTGACAGGCATCACTT 测序 Sequencing
ZmUbi-F TGGTTGTGGCTTCGTTGGTT 实时荧光定量PCR qRT-PCR
ZmUbi-R GCTGCAGAAGAGTTTTGGGTACA 实时荧光定量PCR qRT-PCR
ZmGST24-F CCGGAGTGCACCTGATAGC 实时荧光定量PCR qRT-PCR
ZmGST24-R TGACCCACGACTTGTACCTC 实时荧光定量PCR qRT-PCR
ZmNCED3-F CCGAGGAAAGCGGACGG 实时荧光定量PCR qRT-PCR
ZmNCED3-R CGTAGACCCCGTCGATGAAG 实时荧光定量PCR qRT-PCR
ZmDREB1.6-F TTGAGGTGGATGTGTTCGGC 实时荧光定量PCR qRT-PCR
ZmDREB1.6-R CCACAGCTCCATCTCACCAG 实时荧光定量PCR qRT-PCR
ZmDREB2A-F ATCACTCGGACATCGCATCC 实时荧光定量PCR qRT-PCR
ZmDREB2A-R AGATACCAGGGCTGTCCCAT 实时荧光定量PCR qRT-PCR

表2

ZmNTLs基因理化性质信息统计"

基因名称
Gene name		 基因ID
Gene ID		 染色体位置
Chromosomal
location		 蛋白质长度
Protein length (aa)		 分子量
Molecular weight
(kD)		 理论等电点
Theoretical isoelectric point
ZmNTL1 GRMZM2G064541 3 517 57.50 5.49
ZmNTL2 GRMZM2G125777 4 665 71.82 4.53
ZmNTL3 GRMZM2G113950 4 657 71.98 4.62
ZmNTL4 GRMZM2G004531 7 709 77.98 4.67
ZmNTL5 GRMZM2G167492 8 518 57.33 5.52
ZmNTL6 GRMZM2G163914 9 612 66.81 5.58
ZmNTL7 GRMZM2G003715 10 664 73.61 4.81
ZmNTL8 GRMZM2G158204 8 313 34.83 9.62
ZmNTL9 AC196475.3_FG005 4 633 70.66 4.90
基因名称
Gene name		 不稳定指数
Instability index		 脂肪酸指数
Fatty acid index		 平均亲疏水性
Grand average of hydropathicity		 低温叶log2FC
log2FC of leaves under low temperature		 低温根log2FC
log2FC of roots under low temperature
ZmNTL1 49.37 70.60 -0.641 5.19 4.83
ZmNTL2 36.98 74.56 -0.365 -1.00 0.79
ZmNTL3 43.32 67.32 -0.530 2.19 1.47
ZmNTL4 46.98 68.27 -0.508 2.25 2.06
ZmNTL5 49.92 68.40 -0.607 3.32 2.56
ZmNTL6 49.88 79.07 -0.408 -1.21 0.62
ZmNTL7 47.62 67.12 -0.602 4.05 3.50
ZmNTL8 27.62 99.36 0.257 - -
ZmNTL9 56.17 65.94 -0.772 2.93 3.35

图1

ZmNTLs基因的分子特性和低温下表达模式分析 A: ZmNTLs的基因结构。B: ZmNTLs的保守基序。C: ZmNTLs蛋白跨膜域预测。D: ZmNTLs在低温胁迫下的表达模式。E: NTLs的进化关系分析。绿圈代表7个拟南芥NTLs, 蓝圈代表5个水稻NTLs, 黄圈代表9个玉米NTLs, 红圈代表1个小麦NTL。"

图2

zmntl1zmntl5突变体材料的创制 A: ZmNTL1与ZmNTL5蛋白序列比对。B: pCPB-Cas9重组载体示意图。C: ZmNTL1与ZmNTL5的共有靶点设计。D, E: 基因敲除材料中突变等位基因序列的比对。"

图3

ZmNTL1和ZmNTL5的敲除对玉米低温耐受性的影响 A: 低温胁迫下zmntl1zmntl5突变体的表型。B~E: 低温胁迫下zmntl1zmntl5突变体的地上部鲜重/干重、地下部鲜重/干重变化。F, G: 低温胁迫下zmntl1zmntl5突变体的离子渗透率和叶绿素含量变化。标尺为10 cm。**, P < 0.01; ***, P < 0.001。"

图4

ZmNTL1和ZmNTL5的敲除对低温下玉米抗氧化防御系统的影响 A~D: 低温胁迫下zmntl1zmntl5突变体的GST、CAT、POD和SOD酶活变化。E, F: zmntl1zmntl5突变体的DAB和NBT染色。G, H: 低温胁迫下zmntl1zmntl5突变体的O2-和H2O2含量变化。*, P < 0.05; **, P < 0.01。"

图5

ZmNTL1和ZmNTL5敲除对低温胁迫下逆境相关基因表达的影响 A~D: 低温胁迫下zmntl1zmntl5突变体中ZmGST24、ZmNCED3、ZmDREB1.6和ZmDREB2A的相对表达水平变化。**, P < 0.01; ***, P < 0.001。"

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