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作物学报 ›› 2023, Vol. 49 ›› Issue (10): 2717-2726.doi: 10.3724/SP.J.1006.2023.23080

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米GST31基因的克隆与表达分析

孙兰兰(), 麻荣慧, 薛飞, 杨慕菡, 徐洪乐, 苏旺苍, 鲁传涛, 吴仁海()   

  1. 河南省农业科学院植物保护研究所 / 河南省农作物病虫害防治重点实验室, 河南郑州 450002
  • 收稿日期:2022-12-12 接受日期:2023-02-21 出版日期:2023-10-12 网络出版日期:2023-03-02
  • 通讯作者: 吴仁海, E-mail: renhai.wu@163.com
  • 作者简介:E-mail: sll30711034@163.com
  • 基金资助:
    河南省自然科学基金青年科学基金项目(212300410171);河南省农业科学院自主选题项目(2022ZC36);河南省科技研发计划联合基金项目(222301420109)

Cloning and the relative expression pattern of GST31 gene in maize

SUN Lan-Lan(), MA Rong-Hui, XUE Fei, YANG Mu-Han, XU Hong-Le, SU Wang-Cang, LU Chuan-Tao, WU Ren-Hai()   

  1. Henan Key Laboratory of Crop Pest Control, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
  • Received:2022-12-12 Accepted:2023-02-21 Published:2023-10-12 Published online:2023-03-02
  • Contact: E-mail: renhai.wu@163.com
  • Supported by:
    Science Foundation of Henan Province, China(212300410171);Independent Topic Selection Project of Henan Academy of Agricultural Sciences(2022ZC36);Joint Fund for Scientific and Technological Research and Development of Henan(222301420109)

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摘要：

为探究ZmGST31基因的序列特征及除草剂与安全剂对基因表达模式的影响, 本研究以玉米品种“郑单958”为试验材料, 利用反转录PCR (RT-PCR)技术克隆该基因, 分析其核苷酸和编码蛋白序列特性, 并利用实时荧光定量PCR (qRT-PCR)技术研究其在玉米根茎叶以及除草剂烟嘧磺隆(nicosulfuron, NS)和安全剂双苯噁唑酸(isoxadifen-ethyl, IE)单施和共施24 h、48 h的表达情况。结果显示: ZmGST31编码序列(CDS)长为765 bp, 可编码一个分子式为C1276H2021N331O353S4, 氨基酸大小为254个氨基酸, 分子量为27.78 kD, 等电点为6.60的稳定疏水性蛋白。该蛋白主要定位于细胞质, 且无信号肽和跨膜结构, 含有保守的GST_C_Tau结构域和GST_N_Tau结构域, 属于Tau亚家族, 与扫帚黍(Dichanthelium oligosanthes) GSTU17蛋白具有较高的相似性。蛋白互作预测结果显示, ZmGST31与10个蛋白存在互作, 其中包括2个谷胱甘肽合成酶(glutathione synthetase)和5个谷胱甘肽过氧化物酶(glutathione peroxidase)。ZmGST31启动子区含有大量的光响应和多种激素响应相关元件。qRT-PCR结果显示ZmGST31在玉米苗期不同部位中均有表达, 其中根中表达量最高, 其次是叶片, 茎最低。除草剂烟嘧磺隆和安全剂双苯噁唑酸处理均能诱导ZmGST31基因在玉米的地上部分中表达, 其诱导作用在处理24 h时最强。本研究表明, ZmGST31的表达受除草剂烟嘧磺隆与安全剂双苯噁唑酸调控, 可能在玉米除草剂毒性降解或安全剂介导的除草剂毒性降解中发挥重要作用。

关键词: 玉米, ZmGST31, 生物信息学分析, 表达模式

Abstract:

To investigate the sequence characteristics of the ZmGST31 gene and the relative expression pattern under herbicide and safener application, ZmGST31 gene was cloned from maize variety ‘Zhengdan 958' by using Reverse Transcription PCR (RT-PCR). A series of bioinformatics analyses were performed subsequently to reveal its nucleotide and coding protein sequence characteristics. Real-time fluorescence quantitative PCR (qRT-PCR) was used to study its relative expression level in root, stem, and leaf with the application of Nicosulfuron (NS) and Isoxadifen-Ethyl (IE) for 24 hours and 48 hours. The results showed that the coding sequence (CDS) of ZmGST31 was 765 bp, which encoded an stable hydrophobic protein with molecular formula of C1276H2021N331O353S4, amino acid size of 254 aa, molecular weight of 27.78 kD, and isoelectric point of 6.60. The protein was mainly localized in the cytoplasm and had no signal peptide and transmembrane structure. Based on the conserved GST_C_Tau and GST_N_Tau structural domain, it was suggested to be a member of the Tau subfamily. ZmGST31 had higher homology with Dichanthelium oligosanthes GSTU17 protein. Protein-protein interaction prediction result showed that ZmGST31 could interact with ten proteins, including two glutathione synthetases and five Glutathione peroxidase proteins. The promoter region of ZmGST31 containd a large number of Cis-acting elements such as light response and hormone response. qRT-PCR results revealed that ZmGST31 was expressed in different parts of maize seedlings, with the highest expression in roots, followed by leaves, and stems. The relative expression of ZmGST31 gene in the aboveground part of maize could be induced by the nicosulfuron and isoxadifen-ethyl, the induction effect was the strongest at 24 hours. In conclusion, the relative expression level of ZmGST31 was regulated by herbicide Nicosulfuron and safener Isoxadifen-Ethyl and might play an important role in herbicide toxicity degradation or safener-mediated herbicide toxicity degradation in maize.

Key words: Zea mays L., ZmGST31, bioinformatics analysis, the relative expression pattern

图1

ZmGST31 cDNA扩增产物电泳检测结果 M: DL2000标记。"

图2

ZmGST31蛋白二级结构(A)与三级结构(B) 蓝色h: α-螺旋; 绿色t: β-折叠; 橙色c: 无规则卷曲; 红色e: 延伸链。"

图3

ZmGST31保守结构域"

表1

ZmGST31蛋白基序"

基序名称
Motif name		 基序序列
Motif sequence		 E值
E-value		 宽度
Width		 位点
Location		 基序名称
Motif name		 基序序列
Motif sequence		 E值
E-value		 宽度
Width		 位点
Location
Motif 1 RFWAQY
RLCAHP		 1.4e+001 6 120-125
218-223		 Motif 10 DAIPDA
DAHARA		 3.6e+003 6 227-232
112-117
Motif 2 FGGDSVGY
FVEFSVTY		 1.3e+002 8 173-180
235-242		 Motif 11 LKGTDD
LKGVEF		 4.2e+003 6 137-142
48-53
Motif 3 HHGKPIS
SFSKPIN		 1.8e+002 7 82-88
244-250		 Motif 12 RVIGLW
KIAGVT		 8.0e+003 6 29-34
197-202
Motif 4 PWAIRI
PFVIRV		 4.1e+002 6 131-136
37-42		 Motif 13 EEVVGR
SELLLR		 8.0e+003 6 57-62
64-69
Motif 5 GMEQAA
KMSEAA		 4.3e+002 6 144-149
22-27		 Motif 14 PAFLPV
TVVLPV		 9.8e+003 6 106-111
9-14
Motif 6 QYIDEVW
QLLEEAF		 1.0e+003 7 95-101
157-163		 Motif 15 GWVKAV
LLDKAK		 1.9e+004 6 190-195
203-208
Motif 7 MSRLTTT
DIALVSH		 2.1e+003 7 1-7
182-188		 Motif 16 ELSELG
ESLIIV		 2.4e+004 6 15-20
89-94
Motif 8 PNLVAW
SNPVHK		 3.8e+002 6 210-215
70-75		 Motif 17 SFSKPI
KIPVLL		 3.1e+004 6 244-249
76-81
Motif 9 AQLSQG
GQLSAA		 3.3e+003 6 164-169
150-155

图4

ZmGST31蛋白基序LOGO"

图5

ZmGST31蛋白互作网络模型"

图6

GST蛋白系统进化树"

表2

ZmGST31启动子顺式作用元件分析"

元件
Motif		 数量
Number		 功能
Function
TATA-box 77 核心启动子元件 Core promoter element
CAAT-box 14 核心启动子元件 Core promoter element
CGTCA-motif 8 茉莉酸甲酯响应 MeJA-responsiveness
CAT-box 3 分生组织表达 Meristem expression
ATCT-motif 3 光反应 Light responsiveness
TGA-element 3 生长素响应元件 Auxin-responsive element
ARE 2 厌氧诱导 Anaerobic induction
GA-motif 2 光响应元件 Light responsive element
ABRE 2 脱落酸响应 Abscisic acid responsiveness
GC-motif 1 缺氧特异性诱导 Anoxic specific inducibility
CCAAT-box 1 MYBHv1结合位点 MYBHv1 binding site
GA-motif 1 光反应元件 Light responsive element
AT-rich element 1 富含AT的DNA结合蛋白结合位点 Binding site of AT-rich DNA binding protein
ACE 1 光反应 Light responsiveness
CAT-box 1 顺式作用调控元件 Cis-acting regulatory element
GCN4_motif 1 胚乳发育 Endosperm expression
MSA-like 1 细胞周期调控 Cell cycle regulation

图7

不同组织部位与不同处理下ZmGST31相对表达量 A: 根、茎、叶中ZmGST31相对表达量。B: 对照组(CK)处理0 h、24 h和48 h ZmGST31相对表达量。C: 对照组(CK)、双苯噁唑酸(IE)、烟嘧磺隆(NS)、双苯噁唑酸+烟嘧磺隆(IE+NS)处理24 h ZmGST31相对表达量。D: 对照组(CK)、双苯噁唑酸(IE)、烟嘧磺隆(NS)、双苯噁唑酸+烟嘧磺隆(IE+NS)处理48 h ZmGST31相对表达量。所有数据显示为3个重复的平均值±标准偏差, 各栏上方不同的小写字母代表了不同处理间在P < 0.05时的差异显著。"

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