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作物学报 ›› 2023, Vol. 49 ›› Issue (7): 1747-1757.doi: 10.3724/SP.J.1006.2023.23054

所属专题： 玉米：遗传育种·种质资源·分子遗传学

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

热诱导转录因子ZmNF-YC13调控热胁迫应答基因提高玉米耐热性

梅秀鹏1,2(), 赵子堃1,2(), 贾欣瑶1,2, 白洋1,2, 李梅3, 甘宇玲1,2, 杨秋悦1,2, 蔡一林1,2,*()   

  1. 1西南大学农学与生物科技学院, 重庆 400715
    2南方山地农业教育部工程研究中心, 重庆 400715
    3广西农业职业技术大学农学与园艺系, 广西南宁 530007
  • 收稿日期:2022-08-03 接受日期:2022-11-25 出版日期:2023-07-12 网络出版日期:2022-12-05
  • 通讯作者: *蔡一林, E-mail: caiyilin1789@163.com
  • 作者简介:梅秀鹏, E-mail: swumxp2009@163.com
    赵子堃, E-mail: 404683331@qq.com第一联系人：**同等贡献
  • 基金资助:
    本研究由国家自然科学基金项目(31901555);中国博士后科学基金项目(2019M663879XB);西南大学大学生创新创业训练计划项目(S202210635315)

Heat-inducible transcription factor ZmNF-YC13 regulates heat stress response genes to improve heat tolerance in maize

MEI Xiu-Peng1,2(), ZHAO Zi-Kun1,2(), JIA Xin-Yao1,2, BAI Yang1,2, LI Mei3, GAN Yu-Ling1,2, YANG Qiu-Yue1,2, CAI Yi-Lin1,2,*()   

  1. 1College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
    3Department of Agriculture and Horticulture, Guangxi Agricultural Vocational University, Nanning 530007, Guangxi, China
  • Received:2022-08-03 Accepted:2022-11-25 Published:2023-07-12 Published online:2022-12-05
  • Contact: *E-mail: caiyilin1789@163.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    The National Natural Science Foundation of China(31901555);The China Postdoctoral Science Foundation(2019M663879XB);The Innovation and Entrepreneurship Training Program for College Students of Southwest University(S202210635315)

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摘要：

热胁迫是影响玉米生长发育和产量形成的重要因素, 相关抗性基因的挖掘和机制解析是进行玉米耐热品种培育的重要分子基础, 而目前这方面的研究仍是缺乏。本研究鉴定到一个与热胁迫应答相关的核因子ZmNF-YC13, 该基因受高温和渗透胁迫快速诱导表达。用拟南芥热胁迫诱导表达基因AtHSP70的启动子驱动ZmNF-YC13基因, 转化玉米筛选出了热诱导表达的转基因材料HSP21Pro:ZmNF-YC13-myc。高温处理后的表型鉴定表明, 叶长、叶宽、地上部粗、地上部分和地下部分的鲜重和干重均显著高于野生型。表达分析表明, ZmNF-YC13能增强下游热胁迫应答基因响应热胁迫的程度。荧光素酶报告基因实验和ChIP-qPCR实验表明, ZmNF-YC13可调控热应激转录因子ZmHsfA2c的表达。这些结果初步证实了ZmNF-YC13可通过调控下游热胁迫应答基因来提高玉米的耐热能力, 可为利用该位点的多态性进行分子标记辅助选择和种质资源鉴定提供理论依据。

关键词: 玉米, 热胁迫, 热应激转录因子, NF-Y

Abstract:

Heat stress is an essential factor affecting maize growth and yield formation. The exploration and mechanism analysis of related tolerance genes is an important field for breeding maize heat stress tolerant varieties. However, there is few study in this aspect. In this study, we identified a nuclear factor ZmNF-YC13 associated with heat stress response, and its encoding gene expression was rapidly induced by high temperature and osmotic stress. The promoter of Arabidopsis thaliana heat stress-inducible expression gene AtHSP70 was used to drive ZmNF-YC13 and the heat-inducible expression maize material of ZmNF-YC13 (HSP21Pro:ZmNF-YC13-myc) was successfully screened. Phenotypic analysis after high temperature treatment demonstrated that leaf length, leaf width, shoot thickness, fresh and dry weight of shoot and root were significantly higher than wild type. The relative expression level showed that ZmNF-YC13 could enhance the inducible level of downstream heat stress response genes in response to heat stress. Luciferase reporter assay and ChIP-qPCR assay also revealed that ZmNF-YC13 could regulate the expression of heat stress transcription factor ZmHsfA2c. These results confirmed preliminarily that ZmNF-YC13 could improve the heat tolerance of maize by regulating downstream heat stress response genes, which could provide a theoretical basis for marker-assisted selection and germplasm identification using the polymorphism of this locus.

Key words: maize, heat stress, heat stress transcription factor, NF-Y

附表1

本研究中所用引物"

引物名称 Primer 序列Sequence (5′-3′)
ZmNF-YC13RT-F TAGGGCTGGTCTGCCACCCAT
ZmNF-YC13RT-R GGCTCCTGCCACAAATAAGTCACT
ZmDnaJ2RT-1F TCAAGTTCTGCGTTTGGGGTGTTA
ZmDnaJ2RT-1R GAAGCTAGCAAACGTATTCGAGCG
ZmDnaJ1RT-1F TACACCGCGCACGTGACGACGC
ZmDnaJ1RT-1R ATCGGCATGCCCTCCCCTCGCA
ZmHSP70RT-1F GTCGACTAAATGAGGAAATTCTGATA
ZmHSP70RT-1R AATAAGATCCACTTTGTAATTGACGC
AtHSP21Pro-F CAGCTATGACATGATTACGAATTCCTTACCAAGCTTCTGAGCATCTCC
AtHSP21Pro-R CTGAGGGGATGGTTCCATGGATCCTTGTTTCGAGTATGAGCCAAAAAT
ZmHsfA2cPro-1F AGCCTACCTTTTATGTGATACTCC
ZmHsfA2cPro-1R CAGTAGCCAAGTGTGAATCATTGT
ZmHsfA2cPro-2F TCTTGTCTCTCCTCTCCAGAACCT
ZmHsfA2cPro-2R GTCGAGACACGTCGCGAGGCTT
ZmHsfA2cPro-3F ACAGAAATATCCTAAGCGCTGAC
ZmHsfA2cPro-3R AACACGGGGCAAGGACTCGATT
ZmHsfA2cPro-4F TTCTCTTGGTCAGGGCTTGCTAAT
ZmHsfA2cPro-4R TATTGCAGAGGATTCGGATGCTCT
ZmHsfA2cPro-F GAGGTCGACGGTATCGATAAGCTTTAGTGAAGATCCAAAAGATAATA
ZmHsfA2cPro-R GGCCGCTCTAGAACTAGTGGATCCGGCCTCCGCCCAGGCCCAAGAACC
18S rRNA-F ACCTTACCAGCCCTTGACATATG
18S rRNA-R GACTTGACCAAACATCTCACGAC

图1

ZmNF-YC13在不同条件和材料中的表达分析 A: 高温和渗透胁迫处理条件下ZmNF-YC13基因叶片中的表达特点分析; B: 正常生长条件下耐热型和热敏感型玉米自交系植株叶片中ZmNF-YC13基因表达量分析; C: 高温胁迫处理后耐热型和热敏感型玉米自交系植株中ZmNF-YC13基因表达量分析; 每组试验均采用3次生物学重复, 数据显著性分析采用Student’s t测验(双尾), *: P < 0.05, **: P < 0.01; D: HSP21Pro:ZmNF-YC13-myc植株在高温处理下ZmNF-YC13-myc融合蛋白检测。"

图2

野生型与ZmNF-YC13热诱导表达材料在高温处理后的表型分析 A~D: 野生型和ZmNF-YC13诱导材料在正常条件和高温处理后的植株长势形态, D图中箭头指示为第5叶, 比例尺为5 cm; E: 高温处理后野生型和ZmNF-YC13热诱导材料叶长、叶宽、地上部粗、地上部分和地下部分的鲜重和干重统计; 显著性分析采用Student’s t测验(双尾), n > 20, *: P < 0.05, **: P < 0.01。"

图3

高温胁迫应答基因在野生型材料和ZmNF-YC13热诱导材料中的表达模式分析"

图4

ZmNF-YC13调控ZmHsfA2c基因表达 A: ZmHsfA2c基因启动子和ZmNF-YC13蛋白之间的反式激活效应分析。-ZmNF-YC13表示不含有ZmNF-YC13蛋白(对照), +ZmNF-YC13表示含有ZmNF-YC13蛋白。数据为3次独立实验的平均值。**: P < 0.01。B: ZmNF-YC13基因的启动子结合位点ChIP-qPCR分析。数据为3次独立实验的平均值。C: 用于ChIP-qPCR分析的ZmHsfA2c启动子片段示意图。"

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