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作物学报 ›› 2022, Vol. 48 ›› Issue (12): 3018-3028.doi: 10.3724/SP.J.1006.2022.13074

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

玉米苗期根汞胁迫响应中miRNA的鉴定及初步验证

秦永田1,2(), 陈黎霞3, 汤继华1, 陈建辉1, 马拴红1, 张雪海1, 丁冬1(), 付志远1()   

  1. 1河南农业大学农学院 / 省部共建小麦玉米作物学国家重点实验室 / 河南粮食作物协同创新中心, 河南郑州 450046
    2鹤壁市农业科学院, 河南鹤壁458030
    3华北水利水电大学物理与电子学院, 河南郑州450000
  • 收稿日期:2021-12-14 接受日期:2022-03-25 出版日期:2022-12-12 网络出版日期:2022-04-20
  • 通讯作者: 丁冬,付志远
  • 作者简介:秦永田, E-mail: 442869057@qq.com第一联系人：

    **同等贡献

  • 基金资助:
    河南省重点研发与推广专项(科技攻关)(212102110061);国家转基因生物新品种培育重大专项(2018ZX0800908B);省部共建作物逆境适应与改良国家重点实验室(河南大学) 2021年度开放课题(2021KF07)

Identification and validation of miRNA involved in mercury stress response in maize seedling roots

QIN Yong-Tian1,2(), CHEN Li-Xia3, TANG Ji-Hua1, CHEN Jian-Hui1, MA Shuan-Hong1, ZHANG Xue-Hai1, DING Dong1(), FU Zhi-Yuan1()   

  1. 1College of Agronomy, Henan Agricultural University / National Key Laboratory of Wheat and Maize Crop Science / Collaborative Innovation Center of Henan Grain Crops, Zhengzhou 450046, Henan, China
    2Hebi Academy of Agricultural Sciences, Hebi 458030, Henan, China
    3School of Physics and Electronics, North China University of Water Resources and Electric Power, Zhengzhou 450000, Henan, China
  • Received:2021-12-14 Accepted:2022-03-25 Published:2022-12-12 Published online:2022-04-20
  • Contact: DING Dong,FU Zhi-Yuan
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    Key Technologies Research & Development Program of Henan Province(212102110061);National Major Project for Developing New GM Crops(2018ZX0800908B);Open Project Funding of the State Key Laboratory of Crop Stress Adaptation and Improvement(2021KF07)

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摘要：

汞污染已成为威胁全球作物生产的主要重金属污染源之一。microRNA (miRNA)是调控植物生长发育和非生物胁迫响应的重要因子, 但其在单子叶植物汞胁迫响应中的作用尚不明确。为挖掘响应汞胁迫的关键miRNA, 本研究对B73和郑58 (Zheng 58, Z58)自交系幼苗在HgCl2处理后的表型和差异表达的miRNA进行了分析。结果表明, B73对汞胁迫较郑58更敏感, miRNA166l是B73和Z58中共同鉴定到的下调表达的miRNA。为进一步验证miRNA166在汞胁迫中的作用, 利用STTM (short tandem target mimic)技术获得了STTM165/166拟南芥转基因稳定株系。STTM166稳定系在HgCl2处理后的表型与玉米幼苗在汞胁迫处理后的表型类似, 叶片失绿萎蔫、根长变短。本研究证明miRNA166在玉米汞胁迫响应中有重要调控作用, 对其作用机制进行更深入的研究有重要意义。

关键词: 玉米, 汞胁迫, microRNA, 短的串联重复靶标模拟(STTM)

Abstract:

Mercury is one of the most important sources of heavy mental pollution to crop production in the worldwide. MicroRNA (miRNA) is a critical regulator in plant development and abiotic stress responses. However, its function on mercury stress response is still unknown in monocots. To identify the critical miRNA in response to mercury, we analyzed phenotype changes and differentially expressed miRNA in seedlings of two maize inbred lines B73 and Zheng 58 (Z58) under HgCl2 stress. The results showed that B73 was more sensitive to mercury than Z58 and miRNA166l was down-regulated in both B73 and Z58 under HgCl2 stress. miRNA165/166 knock-out stable lines of Arabidopsis were created using STTM technology to validate its role in mercury stress response. These lines showed wilted and etiolated leaf and shorten root, which were similar with maize seedlings after mercury treatment. This study verified that miRNA166 was important for mercury stress modulation, which promoted us to explore the molecular mechanism of miRNA166 in heavy metal response in the following experiments.

Key words: maize, mercury stress, microRNA, STTM

图1

HgCl2胁迫处理后的表型变化 A: B73对照和HgCl2处理6 d的苗与根; B: Z58对照和HgCl2处理6 d的苗与根。"

图2

HgCl2胁迫处理后B73和Z58苗期表型的差异分析"

表1

不同组织汞含量的统计分析"

组织
Tissue		 含量Content (mean±SD) P值
P-value
CK (μg L-1) Hg (μg L-1)
B73地下部分Part below the ground of B73 26.79±0.21 28.41±0.09 1.07E-08**
B73地上部分Part above the ground of B73 20.577±0.48 26.64±0.54 1.88E-06**
郑58地下部分Part below the ground of Z58 20.29±0.82 28.25±0.04 1.86E-06**
郑58地上部分Part above the ground of Z58 14.45±0.81 24.77±0.87 8.09E-06**

表2

HgCl2胁迫处理后B73和Z58中的差异表达miRNA"

B73 郑58 Zheng 58
基因序列号
Gene ID		 log2 (Fold Change) Padj-value 基因序列号
Gene ID		 log2 (Fold Change) Padj-value
zma-miRNA156b 2.1 1.65E-09 zma-miRNA156b 2.1 5.24E-05
zma-miRNA156i 2.8 3.09E-11 zma-miRNA156i -2.3 6.64E-03
zma-miRNA156l 2.4 7.91E-05 zma-miRNA156l -2.2 3.29E-04
zma-miRNA159a -3.9 3.09E-33 zma-miRNA159a 1.6 3.23E-16
zma-miRNA159b -4.0 1.27E-27 zma-miRNA159b 1.6 3.39E-16
zma-miRNA159c -1.4 8.17E-04 zma-miRNA159c 3.8 1.19E-45
zma-miRNA159d -1.4 3.35E-03 zma-miRNA159d 3.9 3.94E-44
zma-miRNA159f -3.6 2.38E-30 zma-miRNA159f 1.8 6.23E-20
zma-miRNA159j -4.2 8.07E-40 zma-miRNA159j 1.6 2.07E-16
zma-miRNA159k -4.0 2.70E-31 zma-miRNA159k 1.6 6.04E-17
zma-miRNA166j -2.5 5.90E-06 zma-miRNA166j 1.1 2.65E-02
zma-miRNA166k -3.5 3.84E-12 zma-miRNA166k 1.0 2.91E-02
zma-miRNA166l -2.8 1.07E-05 zma-miRNA166l -1.4 4.30E-05
zma-miRNA167b 3.1 1.14E-07 zma-miRNA167b -2.1 3.79E-10
zma-miRNA167c 2.4 2.38E-03 zma-miRNA167c -1.1 2.00E-02
zma-miRNA167g 1.2 1.68E-03 zma-miRNA167g -1.6 1.90E-04
zma-miRNA169g 10.8 3.70E-05 zma-miRNA169g -4.3 2.80E-02
zma-miRNA171d 8.9 4.04E-02 zma-miRNA171d -7.4 3.19E-02
zma-miRNA171g 1.9 4.27E-02 zma-miRNA171g 2.2 4.80E-02
zma-miRNA171l 8.9 4.04E-02 zma-miRNA171l -8.9 1.00E-04
zma-miRNA171n 10.3 6.35E-04 zma-miRNA171n -8.1 3.70E-03
zma-miRNA393b 12.4 4.92E-11 zma-miRNA393b -4.1 4.03E-02
zma-miRNA396c 2.7 1.73E-12 zma-miRNA396c -1.9 1.57E-14
zma-miRNA396d 2.2 1.84E-10 zma-miRNA396d -1.9 1.03E-16
zma-miRNA398a -1.9 6.31E-04 zma-miRNA398a 1.2 7.49E-04
zma-miRNA398b -1.3 1.30E-03 zma-miRNA398b 1.1 2.59E-04
zma-miRNA408a -2.0 1.01E-03 zma-miRNA408a 3.9 1.02E-80
zma-miRNA408b -2.2 6.31E-04 zma-miRNA408b 3.9 5.01E-74
zma-miRNA2118a -24.4 1.65E-06 zma-miRNA397b -4.7 2.33E-94
zma-miRNA159e -23.9 2.77E-06 zma-miRNA319d 2.8 1.86E-39
zma-miRNA399a -23.9 2.77E-06 zma-miRNA319b 2.8 1.27E-35
zma-miRNA169a -4.1 8.97E-06 zma-miRNA319a 2.2 5.98E-20
zma-miRNA166n -3.3 5.31E-09 zma-miRNA319c 2.2 1.70E-16
zma-miRNA2118b -2.5 1.72E-02 zma-miRNA390b -3.9 7.90E-14
zma-miRNA166h -2.5 1.46E-18 zma-miRNA171f -10.0 1.12E-07
zma-miRNA166m -2.4 4.24E-03 zma-miRNA169b -2.7 1.34E-06
zma-miRNA166b -2.4 1.35E-23 zma-miRNA390a -3.3 1.43E-05
zma-miRNA166g -2.3 3.18E-21 zma-miRNA1432 -3.1 1.68E-05
zma-miRNA166i -2.2 4.79E-15 zma-miRNA164b 2.5 5.24E-05
zma-miRNA166d -2.2 7.66E-14 zma-miRNA164c 2.6 5.42E-05
zma-miRNA166f -2.1 2.65E-13 zma-miRNA164g 2.6 9.17E-04
zma-miRNA166a -2.1 2.27E-13 zma-miRNA172d -8.4 1.26E-03
zma-miRNA166c -2.0 2.95E-15 zma-miRNA164a 2.3 3.02E-03
zma-miRNA166e -1.9 9.86E-14 zma-miRNA167d -1.7 3.19E-03
zma-miRNA528b -1.7 1.40E-02 zma-miRNA397a -4.9 3.70E-03
zma-miRNA528a -1.5 1.49E-03 zma-miRNA164h 2.4 2.04E-02
zma-miRNA167i 1.1 2.66E-03 zma-miRNA399h -7.4 3.13E-02
zma-miRNA167j 1.3 3.96E-02 zma-miRNA164d 2.1 3.55E-02
zma-miRNA167h 1.4 4.27E-02 zma-miRNA171m -7.4 4.00E-02
zma-miRNA827 1.5 1.63E-03
zma-miRNA171h 1.5 2.78E-02
zma-miRNA167f 1.5 8.83E-03
zma-miRNA171k 1.6 2.07E-02
zma-miRNA156d 1.7 1.36E-03
zma-miRNA168a 1.7 1.07E-05
zma-miRNA156g 1.8 1.51E-12
zma-miRNA168b 1.8 7.65E-05
zma-miRNA396f 2.0 2.41E-02
zma-miRNA162 2.0 3.46E-03
zma-miRNA156e 2.1 1.46E-18
zma-miRNA156f 2.1 3.52E-04
zma-miRNA164e 2.3 1.57E-05
zma-miRNA156a 2.3 1.55E-10
zma-miRNA396g 2.3 1.34E-02
zma-miRNA156k 2.5 4.12E-09
zma-miRNA156h 2.7 9.32E-08
zma-miRNA156h 2.7 9.32E-08
zma-miRNA169r 9.2 2.47E-02
zma-miRNA395b 9.2 2.47E-02
zma-miRNA395p 9.2 2.07E-02
zma-miRNA171b 9.4 1.11E-02
zma-miRNA171e 9.8 3.46E-03
zma-miRNA399j 10.0 1.63E-03
zma-miRNA169k 10.2 8.17E-04
zma-miRNA171i 10.6 1.20E-04
zma-miRNA169m 11.0 8.17E-06
zma-miRNA172b 22.2 1.30E-05
zma-miRNA395o 22.2 1.30E-05

图3

miRNA基因的表达验证"

图4

STTM165/166的拟南芥表型 对照Col和STTM165/166转基因稳定系经0、25、50、75、100 mg L-1的HgCl2处理3 d后的表型。"

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