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作物学报 ›› 2020, Vol. 46 ›› Issue (12): 2008-2016.doi: 10.3724/SP.J.1006.2020.03022

• 研究简报 • 上一篇    

玉米ZmbHLH161基因的克隆及功能研究

杨梦婷1,2(), 张春2(), 王作平2, 邹华文1,*(), 吴忠义2,*()   

  1. 1长江大学农学院, 湖北荆州 434023
    2北京市农林科学院 / 北京农业生物技术研究中心 / 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:2020-03-28 接受日期:2020-06-02 出版日期:2020-06-22 网络出版日期:2020-06-22
  • 通讯作者: 邹华文,吴忠义
  • 基金资助:
    北京市农林科学院青年基金项目(QNJJ201724);北京市农林科学院科技创新能力建设专项(KJCX20200204);北京市农林科学院科技创新能力建设专项(KJCX20200205);北京市农林科学院科技创新能力建设专项(KJCX20200407);国家自然科学基金项目(31971839);国家自然科学基金项目(31471510)

Cloning and functional analysis of ZmbHLH161 gene in maize

Meng-Ting YANG1,2(), Chun ZHANG2(), Zuo-Ping WANG2, Hua-Wen ZOU1,*(), Zhong-Yi WU2,*()   

  1. 1College of Agriculture, Yangtze University, Jingzhou 434023, Hubei, China
    2Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences / Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing 100097, China
  • Received:2020-03-28 Accepted:2020-06-02 Published:2020-06-22 Published online:2020-06-22
  • Contact: Hua-Wen ZOU,Zhong-Yi WU
  • Supported by:
    Beijing Academy of Agricultural Youth Fund(QNJJ201724);Beijing Academy of Agricultural and Forestry Sciences(KJCX20200204);Beijing Academy of Agricultural and Forestry Sciences(KJCX20200205);Beijing Academy of Agricultural and Forestry Sciences(KJCX20200407);National Natural Science Foundation of China(31971839);National Natural Science Foundation of China(31471510)

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摘要：

bHLH转录因子是植物第二大转录因子家族, 在调节植物生长发育、信号转导和逆境胁迫响应等方面发挥着重要的作用。为了研究玉米bHLH家族基因在逆境胁迫响应中的功能, 本研究从玉米根组织中克隆了ZmbHLH161 (AC: NC_AQK75074)基因。生物信息学分析表明: 该基因包含3个外显子, cDNA全长1460 bp, 编码序列全长1059 bp, 编码352个氨基酸; 在玉米基因组中以单拷贝形式存在, 功能未知; ZmbHLH161蛋白分子量为37.1 kD, 理论等电点为6.10, 具有bHLH转录因子家族特有的保守结构域, 但不具跨膜结构, 无信号肽, 为亲水性蛋白, 蛋白二级结构无规则卷曲所占比例最大, 为42.05%。玉米原生质体瞬时表达试验表明, ZmbHLH161定位在细胞核内。实时荧光定量PCR (qPCR)分析表明, 正常生长条件下, ZmbHLH161主要在根系和幼胚中表达; 在脱水和干旱处理下, ZmbHLH161在玉米苗期叶片中上调表达。转基因异源表达ZmbHLH161拟南芥株系经不同浓度NaCl处理后, 其根长与野生型差异不显著, 而不同浓度甘露醇处理后其根长优于野生型。由此推测ZmbHLH161基因可能参与玉米对渗透胁迫应答。

关键词: 玉米, ZmbHLH161, 转录因子, 原生质体, qPCR, 渗透胁迫

Abstract:

bHLH transcription factors are the second largest family of transcription factors in plants and play an important role in regulating plant growth and development, signal transduction, and stress response. In order to study the function of maize bHLH family genes in response to stress, ZmbHLH161 (AC: NC_AQK75074) gene from maize root tissue was cloned in this study. Bioinformatic analysis showed that this gene contains 3 exons, the full-length of its cDNA is 1460 bp, coding sequence is 1059 bp in length, encoding 352 amino acids. It exists as a single copy in the maize genome and its function is unknown. The molecular weight of ZmbHLH161 protein is 37.1 kD, and its theoretical isoelectric point is 6.10, with a conserved domain unique to the bHLH transcription factor family, but does not have a transmembrane structure or signal peptide. It is a hydrophilic protein, and the secondary structure of the protein has a maximum proportion of 42.05%. Transient expression experiments in maize protoplasts showed that ZmbHLH161 was localized in the nucleus. Real-time quantitative PCR (qPCR) analysis showed that under normal growth conditions, ZmbHLH161 was mainly expressed in roots and immature embryos, while under dehydration and drought treatment, ZmbHLH161 was up-regulated in maize seedling leaves. After treated with different concentrations of NaCl, the root length of ZmbHLH161 transgenic heterologous Arabidopsis strains was not significantly different from that of wild type, and their root was longer than that of wild type after being treated with different concentrations of mannitol. It is speculated that ZmbHLH161 gene may be involved in the response of maize to osmotic stress.

Key words: maize, ZmbHLH161, transcription factor, protoplast, qPCR, osmotic stress

表1

本试验中所用的引物"

引物对编号
Number of primer pairs		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
1 pZmbHLH161-F AGCGTTAGTCACAGTTGTTGCC
pZmbHLH161-R CGTAAGCTGGTCCAATCGGTAC
2 pZmbHLH161SL-F ATGAATTCATGGCGCTCGAAG (EcoR I)
pZmbHLH161SL-R AATAAGCTTGTGGCCTGGCC (Hind III)
3 pZmbHLH161RT-F AGCATCAGCTCCTGTCCGTGTC
pZmbHLH161RT-R CTCTCCATCTCGTCCGTGTTCC
4 pZmbHLH161At-F ATGAATTCAGCGTTAGTCACAGTTGTTGCC (EcoR I)
pZmbHLH161At-R ATGGTACCTCAGTGGCCTGGCCCCCCGC (Kpn I)
5 Actin-F TACGAGATGCCTGATGGTCAGGTCA
Actin-R TGGAGTTGTACGTGGCCTCATGGAC

图1

ZmbHLH161生物信息学分析 A: 氨基酸序列比对; B: 蛋白二级结构预测; C: 跨膜结构预测。"

图2

拟南芥、水稻和玉米bHLH转录因子氨基酸序列的系统发育树"

图3

ZmbHLH161启动子序列分析"

图4

ZmbHLH161蛋白在玉米叶片原生质体中的亚细胞定位 GFP: GFP标记的ZmbHLH161在玉米原生质体的亚细胞定位; DAPI: DAPI标记的原生质体细胞核; Bright: 同一视野光镜下的原生质体; Merged: 光镜及荧光照片的叠加; CK: 转入空载体的原生质体; ZmbHLH161: 转入目的基因载体的原生质体; 标尺为3 μm。"

图5

ZmbHLH161在玉米不同组织中的相对表达量"

图6

实时荧光定量PCR分析不同处理条件下ZmbHLH161基因在叶片和根中的表达情况 A: 干旱处理; B: 脱水处理; C: 低温(4℃)处理; D: 高盐(200 mmol L-1 NaCl)处理。数据为3个生物学重复±标准差。"

图7

T1代拟南芥PCR检测 M: DL2000 marker; P: 阳性对照; W: 野生型拟南芥; N: 阴性对照; 1~3: T1代拟南芥。"

图8

不同盐浓度下转基因拟南芥根长比较 A~D分别为0、0.10、0.15和0.18 mol L-1盐处理的生长情况; E: 不同盐浓度下拟南芥平均主根长度; WT: 野生型拟南芥; OE: 转ZmbHLH161拟南芥; 标尺为1.5 cm。"

图9

不同甘露醇浓度下转基因拟南芥根长比较 A~D分别为0、0.15、0.2和0.3 mol L-1甘露醇处理的生长情况; E: 不同甘露醇浓度下拟南芥平均主根长度; WT: 野生型拟南芥; OE: 转ZmbHLH161拟南芥; 标尺为1.5 cm。*和**分别表示转基因拟南芥主根长度较野生型呈差异显著水平(P<0.05)和差异极显著水平(P<0.01)。"

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