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作物学报 ›› 2024, Vol. 50 ›› Issue (1): 76-88.doi: 10.3724/SP.J.1006.2024.33007

• 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ZmMYB12提高植物抗旱性和低磷耐受性的功能鉴定

王丽平(), 王晓钰, 傅竞也, 王强*()   

  1. 四川农业大学农学院 / 西南作物基因资源发掘与利用国家重点实验室, 四川成都 611130
  • 收稿日期:2023-02-12 接受日期:2023-06-29 出版日期:2024-01-12 网络出版日期:2023-07-20
  • 通讯作者: *王强, E-mail: qwang@sicau.edu.cn
  • 作者简介:E-mail: wlp2019@sina.com
  • 基金资助:
    国家自然科学基金项目(31971825)

Functional identification of maize transcription factor ZmMYB12 to enhance drought resistance and low phosphorus tolerance in plants

WANG Li-Ping(), WANG Xiao-Yu, FU Jing-Ye, WANG Qiang*()   

  1. State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China / College of Agronomy, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China
  • Received:2023-02-12 Accepted:2023-06-29 Published:2024-01-12 Published online:2023-07-20
  • Contact: *E-mail: qwang@sicau.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31971825)

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摘要：

玉米生长发育过程中会遭受干旱、高温、高盐及营养元素匮乏等多种非生物胁迫, 导致其产量和品质下降, 造成严重的农业减产。MYB类转录因子在植物中广泛分布, 参与植物生长发育和环境响应, 筛选及鉴定出具有抗逆功能的MYB类转录因子能为玉米抗逆遗传改良提供理论依据。本研究从玉米干旱处理的材料中克隆了一个R2R3-MYB家族转录因子基因ZmMYB12, 其响应自然干旱、ABA及PEG处理, 基因表达被诱导上调。病毒诱导沉默ZmMYB12后的玉米植株对干旱更加敏感, 活性氧积累更多, 根系更小; 复水后ZmMYB12沉默植株存活率更低, 表明ZmMYB12是干旱正调控因子。进一步构建ZmMYB12稳定过表达拟南芥, 干旱处理后ZmMYB12过表达株系活性氧积累更少, 侧根更多, 抗旱能力增强。同时, 发现ZmMYB12过表达拟南芥在低磷胁迫下侧根数量更多, 根系酸化程度增加, 叶绿素及花青素含量更多, 体内无机磷含量高于野生型, 表明ZmMYB12参与到低磷胁迫过程中, 并且提高了植株对磷元素的吸收及利用率。研究表明ZmMYB12调控干旱抗性并响应低磷胁迫, 为作物抗旱及耐低磷育种提供了一个良好的基因资源。

关键词: 玉米, MYB转录因子, 干旱胁迫, 低磷胁迫, 根系

Abstract:

Maize usually suffers from various abiotic stresses such as drought, high temperature, high salt, and deficiency of nutrient elements during growth and development period, which will eventually lead to the decline of yield and quality, resulting in serious agricultural yield reduction. MYB transcription factors are widely distributed in plants and involved in the whole process of plant growth and development and environmental response. Therefore, the screening and identification of MYB transcription factors conferring stress resistance can provide the theoretical basis and technical support for genetic improvement in maize. In this study, a R2R3-MYB family transcription factor gene, ZmMYB12, was cloned from maize materials under drought treatment. This gene showed inducible expression in response to drought, ABA, and PEG treatments. Maize plants with virus induced gene silencing (VIGS) of ZmMYB12 had higher sensitivity to drought and accumulated more reactive oxygen species (ROS), as well as smaller roots. The survival rate of ZmMYB12 silencing plants was lower after re-watering than the wild type plants (WT), indicating that ZmMYB12 was a positive regulator of drought response. Overexpression of ZmMYB12 in Arabidopsis thaliana resulted in less ROS accumulation and more lateral roots than WT, thus elevating drought resistance. After low phosphorus stress, ZmMYB12 overexpression Arabidopsis had more lateral roots and stronger root system, as well as higher contents of chlorophyll and anthocyanin. The content of inorganic phosphorus in ZmMYB12 overexpression Arabidopsis was also higher than WT. In conclusion, this study indicates that ZmMYB12 positively regulates drought response and increases the uptake and utilization of phosphorus in plants, providing an elite gene for maize breeding to increase abiotic resistance.

Key words: maize, MYB, drought stress, low phosphorus stress, root

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 引物用途
Primer usage
ZmMYB12-F ATGGGGAGAGCTCCGTGCTG 基因克隆
ZmMYB12-R CTAATTCATCCCAAGCTTTC Gene cloning
MYB12-VIGS-F TGTCCGAGTCTGAGGTACCAGTCGCGCAACAAGG 病毒诱导基因沉默
MYB12-VIGS-R GAAGGGGAGGTTCTAGACAGCTCCTCCCAGCT Virus induced gene silencing (VIGS)
p3301-ZmMYB12-F AAACCATGG ATGGGGAGAGCTCCGTGCTG 植物表达载体构建
p3301-ZmMYB12-R AAAAGATCT CTAATTCATCCCAAGCTTTC Plant expression vector construction
ZmMYB12-qPCR-F CTGCGACAAGGCTACTGTGA qRT-PCR检测
ZmMYB12-qPCR-R GTTTGGCCGGAGGTAGTTGA qRT-PCR detection
ZmEf1a-qPCR-F TGGTGTCATCAAGCCTGGTA 内参引物
ZmEf1a-qPCR-R AACATTGTCACCCGGAAGAG Control primer

图1

ZmMYB12的表达模式分析和进化分析 A: PEG、ABA和自然干旱处理下ZmMYB12的表达模式分析; 以WT为1计算相对上调倍数, 每个处理设置3次生物学重复, 利用 SPSS 软件进行显著性分析(***: P < 0.001)。B: ZmMYB12的进化树分析。登录号来自MaizeGDB和NCBI。ZmMYB12登录号为Zm00001d013164, ZmMYB111登录号为Zm00001d021296, ZmMYB148登录号为Zm00001d026203, ZmMYB167登录号为Zm00001d032032, ZmMYB48登录号为Zm00001d030678, ZmMYB94登录号为Zm00001d022227, ZmMYB134登录号为Zm00001d024726, ZmMYB31登录号为Zm00001d006236, ZmMYB3R登录号为Zm00001d042910, ZmMYB119登录号为Zm00001d037994, OsMYB48登录号为LOC_Os01g 74410, OsMYBS1登录号为LOC_Os01g34060, OsMYB91登录号为LOC_Os12g38400。"

图2

玉米ZmMYB12-VIGS沉默植株的干旱处理表型 A: ZmMYB12 基因沉默效率检测; B: 沉默植株干旱处理图; C: 复水后存活率统计; D: 干旱处理后 DAB 染色; E: 正常生长GFP及沉默植株根系形态; F: 干旱处理后GFP及基因沉默植株根系形态, 以GFP作为对照, 每个处理设置3次生物学重复, 利用SPSS软件进行显著性分析(***: P < 0.001)。"

图3

ZmMYB12通过减少活性氧积累增强拟南芥抗旱 A: 阳性植株ZmMYB12-OE基因表达量检测; B: 抗旱表型鉴定图; C: 复水后存活率统计。D: 20% PEG模拟干旱处理离体叶片不同时间点活性氧积累情况, 每个处理设置3次生物学重复; E: 正常及干旱胁迫下MDA含量及酶活性测定。利用SPSS软件进行显著性分析每个处理设置3次生物学重复, 利用 SPSS 软件进行显著性分析(***: P < 0.001, 不同小写字母表示P < 0.05水平有显著差异)。"

图4

ZmMYB12通过调控侧根发育来响应低磷胁迫 A: 低磷处理7 d和21 d根系形态发育; B: 低磷处理21 d的拟南芥幼苗; C: 低磷处理21 d侧根密度统计; D: 正常和低磷处理下侧根形态的观察; E: 正常和低磷处理下侧根长度统计; F: 正常和低磷处理下根毛形态的观察; G: 正常和低磷处理下根毛长度统计; H: 正常和低磷处理下根毛密度统计。每处理设置3次生物学重复, 利用SPSS软件进行显著性分析(不同小写字母表示P < 0.05水平下有显著差异)。"

图5

过表达ZmMYB12增强拟南芥对磷的吸收 A: 正常及低磷处理下野生型(WT)和过表达植株根系溴甲酚紫原位显色; B: 正常及低磷处理野生型(WT)和过表达植株的根系酸化定量; C: 正常及低磷处理野生型(WT)和过表达植株叶片无机磷含量; D: 正常及低磷处理野生型(WT)和过表达植株根系无机磷含量; E: 正常及低磷处理下叶绿素a含量; F: 正常及低磷处理下叶绿素b含量; G: 正常及低磷处理下花青素含量测定。每个处理设置3次生物学重复, 利用SPSS软件进行显著性分析(*: P < 0.05, 不同小写字母表示在P < 0.05水平差异显著)。"

图6

ZmMYB12过表达影响拟南芥种子萌发 A: 正常MS平板生长1周野生型和过表达种子萌发情况; B: 正常生长萌发率统计; C: 正常生长子叶绿化率统计; D: MS+GA处理后种子萌发情况; E: MS+GA处理后萌发率统计; F: MS+GA处理后子叶绿化率统计; G: MS+ABA抑制剂处理后种子萌发情况; H: MS+ABA抑制剂处理后萌发率统计。每个处理设置3次生物学重复, 利用SPSS软件进行显著性分析(*: P < 0.05, ***: P < 0.001, 不同小写字母表示P < 0.05水平差异显著)。"

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