小麦TaPAT1-2D基因的克隆与表达分析

doi:10.3969/j.issn.1004-1524.2023.01.03

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (1): 23-32.DOI: 10.3969/j.issn.1004-1524.2023.01.03

小麦TaPAT1-2D基因的克隆与表达分析

董飞燕¹^,²(), 宋婧含¹^,³, 张华东¹^,², 吴昊天², 李雅倩¹^,², 刘孟伟², 高春保¹^,², 方正武¹^,^*(), 刘易科²^,^*()

1.长江大学农学院,农业农村部长江中游作物绿色高效生产重点实验室(部省共建),湖北荆州 434025
2.湖北省农业科学院粮食作物研究所,粮食作物种质创新与遗传改良湖北省重点实验室,农业农村部华中地区小麦病害生物学科学观测实验站,湖北武汉 430064
3.浙江大学农业与生物技术学院,浙江杭州 310058

收稿日期:2022-04-06 出版日期:2023-01-25 发布日期:2023-02-21
通讯作者: *方正武,E-mail:fangzhengwu88@163.com;刘易科,E-mail:hbliuyk@foxmail.com
作者简介:董飞燕(1997—),女,河南驻马店人,硕士研究生,研究方向为小麦育种栽培。E-mail: 2064276160@qq.com
基金资助:
湖北省自然科学基金(2021CFB393);湖北省中央引导地方科技发展专项(2020ZYYD011);国家小麦产业技术体系建设专项(CARS-03)

Clonging and expression analysis of TaPAT1-2D gene in wheat

DONG Feiyan¹^,²(), SONG Jinghan¹^,³, ZHANG Huadong¹^,², WU Haotian², LI Yaqian¹^,², LIU Mengwei², GAO Chunbao¹^,², FANG Zhengwu¹^,^*(), LIU Yike²^,^*()

1. MARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtza River (Co-Construction by Ministry and Province), College of Agronomy, Yangtze University, Jingzhou 434025, Hubei, China
2. Hubei Provincial Key Laboratory of Germplasm Innovation and Genetic Improvement of Food Crops, Wheat Disease Biology Research Station on Central China, Ministry of Agriculture and Rural Affairs, Food Crops Institute, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
3. College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China

Received:2022-04-06 Online:2023-01-25 Published:2023-02-21

摘要/Abstract

摘要：

GRAS(GIBBERELLIN-INSENSITIVE, repressor of ga1-3 and SCARECROW)基因家族作为重要的植物转录因子在调控植物生长发育、抵抗逆境胁迫的各种信号转导途径中发挥重要作用。为进一步挖掘该家族小麦抗赤霉病相关基因,从禾谷镰刀菌诱导的小麦转录组测序数据筛选出差异表达基因TaPAT1-2D(TraesCS2D02G198200.1),克隆该基因的全长序列,并对其进行生物信息学和表达模式分析,以及亚细胞定位和酵母转录激活活性研究。生物信息学分析结果表明:TaPAT1-2D序列全长1 668 bp,编码555个氨基酸,分子量约为61.34 ku;TaPAT1-2D蛋白含有典型GRAS功能结构域,在进化关系上与水稻OsCIGR2(LOC_Os07g39470.1)关系较近;TaPAT1-2D启动子区包含茉莉酸甲酯、脱落酸、生长素等植物激素响应元件与光应答元件等。实时荧光定量PCR结果显示,接种禾谷镰刀菌孢子液72 h后,TaPAT1-2D基因在4个不同赤霉病抗性小麦品种中的相对表达水平明显上调,表明该基因参与赤霉病的响应过程。农杆菌介导的烟草中瞬时表达试验结果表明,TaPAT1-2D蛋白定位于细胞核和细胞膜中。酵母转录激活活性实验表明,TaPAT1-2D蛋白具有转录自激活能力。研究结果为深入研究TaPAT1-2D基因的功能奠定了基础。

关键词: 小麦, GRAS转录因子, 亚细胞定位, 表达分析, 自激活

Abstract:

As a plant-specific transcription factor, the GRAS (GIBBERELLIN-INSENSITIVE, repressor of ga1-3 and SCARECROW) gene family plays a vital role in plant growth and stress tolerance. To further explore the gene involved in response to Fusarium head blight of wheat in this family, the research screened out the differentially expressed gene TaPAT1-2D(TraesCS2D02G198200.1) from the Fusarium graminearum spore-induced wheat transcriptome sequencing data, and conducted its full-length cloning, bioinformatics analysis, expression pattern analysis, subcellular localization, and transcription activation studies. The results of bioinformatics analysis showed that the exon sequence of TaPAT1-2D was 1 668 bp, encoded 555 amino acids, and had a molecular weight of around 61.34 ku. Its protein sequence comprised a typical GRAS functional domain and was phylogenetically related to rice OsCIGR2 (LOC_Os07g39470.1). In the promoter region, TaPAT1-2D contained some plant hormone response elements and light-responsive elements including methyl jasmonate response elements, auxin response elements, abscisic acid response elements, Etc. Quantitative real-time PCR results revealed that expression level of TaPAT1-2D gene was up-regulated at 72 h after inoculating with F. graminearum spore liquid in four distinct resistants, indicating that this gene was involved in response to Fusarium head blight. Agrobacterium tumefaciens-mediated transient expression was carried out in tobacco and the result showed that TaPAT1-2D protein was localized in the nucleus and cell membrane. The yeast transcription activation experiments verified that the protein could self-activate transcription. This study laid the foundation for studing TaPAT1-2D gene’s function.

Key words: wheat, GRAS transcription factor, subcellular localization, expression analysis, self-activation

中图分类号:

S512

董飞燕, 宋婧含, 张华东, 吴昊天, 李雅倩, 刘孟伟, 高春保, 方正武, 刘易科. 小麦TaPAT1-2D基因的克隆与表达分析[J]. 浙江农业学报, 2023, 35(1): 23-32.

DONG Feiyan, SONG Jinghan, ZHANG Huadong, WU Haotian, LI Yaqian, LIU Mengwei, GAO Chunbao, FANG Zhengwu, LIU Yike. Clonging and expression analysis of TaPAT1-2D gene in wheat[J]. Acta Agriculturae Zhejiangensis, 2023, 35(1): 23-32.

图/表 11

表2 本研究涉及的引物

Table 2 Primers used in the study

引物名称Primer name	序列Sequence(5'→3')
pCAMBIA2300-TaPAT1-2D-GFP-F	CTCGGTACCCGGGGATCCTCTAGAATGGCCGACACTCCAACTTCCCGG
pCAMBIA2300-TaPAT1-2D-GFP-R	GCCCTTGCTCACCATGGTGTCGACGTGCCATGCGGAGGAGACGACGAGG
qPCR-TaPAT1-2D-F	CTACAAGGCCTTGAGGTGCA
qPCR-TaPAT1-2D-R	TGGAGGAGGGAGATCCACTG
Ta2291-F	GCTCTCCAACAACATTGCCAAC
Ta2291-R	GCTTCTGCCTGTCACATACGC
pGBKT7-TaPAT1-2D-F	CATATGGCCATGGAGGCCGAATTCATGGCCGACACTCCAACTTCCCGG
pGBKT7-TaPAT1-2D-R	CGGCCGCTGCAGGTCGACGGATCCTCAGTGCCATGCGGAGGAGACGACGAGG

表1 生物信息学分析工具与数据库

Table 1 Tools and databases used in bioinformatics analysis

名称Name	描述Description	网址Website
SMART	功能结构域Functional domain	http://smart.embl-hei-delberg.de/
GSDS	基因结构Structure of gene	http://gsds.gao-lab.org/
ProtParam	理化性质Physical and chemical properties	https://web.expasy.org/protparam/
NetPhos	磷酸化位点Phosphorylation site	http://www.cbs.dtu.dk/services/ NetPhos/
ProtScale	亲疏水性Hydrophilicity	https://web.expasy.org/protscale/
SOPMA	二级结构Secondary structure	https://npsa-prabi.ibcp. fr/cgi-bin/secpred_sopma.pl
PlantCARE	顺式元件Cis-acting element	http://bioinformatics.psb.ugent.be/webtools/plantcare/html/
SignalP	信号肽Signal peptide	https://services.healthtech.dtu.dk/service.php?SignalP-5.0
TMHMM	跨膜区Transmembrane area	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0
Softberry	亚细胞定位Subcellular localization	http://www.softberry.com/
Ensembl	基因组Genome	https://asia.ensembl.org/index.html

图1 PCR扩增结果 M,DL5 000 marker;1~3,PCR扩增产物。

Fig.1 PCR amplification result M, DL5 000 marker; 1-3, PCR amplification product.

图2 TaPAT1-2D的基因结构和蛋白结构域

Fig.2 Gene structure and protein domains of TaPAT1-2D

图3 TaPAT1-2D蛋白质的生物信息学分析 A,亲水性/疏水性分析。B,磷酸化位点分析。C,二级结构预测;蓝色,α螺旋;橙色,无规则卷曲;红色,延伸链;绿色,β转角。

Fig.3 Bioinformatics analysis of TaPAT1-2D protein A, Analysis of hydrophilic and hydrophobic amino acid. B, Phosphorylation sites analysis. C, Prediction of secondary structure; Blue, Alpha helix; Orange, Random coil; Red, Extended strand; Green, β turn.

图4 TaPAT1-2D与拟南芥、水稻GRAS蛋白系统发育分析 ★,TaPAT1-2D蛋白质所在位置。

Fig.4 Phylogenetic analysis of TaPAT1-2D and GRAS proteins of Arabidopsis thaliana and Oryza sativa ★, Location of the TaPAT1-2D protein.

图5 TaPAT1-2D与拟南芥、水稻GRAS蛋白的氨基酸序列比对

Fig.5 Sequence alignment of TaPAT1-2D and GRAS proteins of Arabidopsis thaliana and Oryza sativa

表3 TaPAT1-2D基因启动子区顺式作用元件

Table 3 Cis-acting element of TaPAT1-2D gene in the promoter regions

顺式作用元件名称 Name of cis-acting element	功能 Function
TGA-element	生长素反应元件Auxin-responsive element
TATA-box	转录起始-30区核心启动子元件Core promoter element around-30 of transcription start
TGACG-motif	参与茉莉酸甲酯响应的顺式作用调控元件Cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif	参与茉莉酸甲酯响应的顺式作用调控元件Cis-acting regulatory element involved in the MeJA-responsiveness
AuxRR-core	参与生长素反应响应的顺式作用调控元件Cis-acting regulatory element involved in auxin responsiveness
CCAAT-box	MYBHv1结合位点MYBHv1 binding site
ARE	对厌氧诱导必需的顺式作用元件Cis-acting regulatory element essential for the anaerobic induction
CAAT-box	启动子和增强子区的一般顺式作用元件Common cis-acting element in promoter and enhancer regions
ABRE	参与脱落酸反应的顺式作用元件Cis-acting element involved in the abscisic acid responsiveness
Box 4	部分光响应保守元件Part of a conserved DNA module involved in light responsiveness
G-box	参与光反应的顺式作用调节元件Cis-acting regulatory element involved in light responsiveness
MRE	参与光响应的MYB结合位点MYB binding site involved in light responsiveness

图6 TaPAT1-2D基因的qRT-PCR结果同一品种不同处理时间数据上没有相同小写字母表示差异显著(P<0.05)。

Fig.6 qRT-PCR results of TaPAT1-2D gene The bars with different letters show the significant difference at the level of 0.05.

图7 小麦TaPAT1-2D的亚细胞定位 A,pCAMBIA2300-GFP空载体;B,pCAMBIA2300-TaPAT1-2D-GFP融合载体。

Fig.7 Subcellular localization of TaPAT1-2D in wheat A, pCAMBIA2300-GFP empty vector; B, pCAMBIA2300-TaPAT1-2D-GFP fusion vector.

图8 小麦TaPAT1-2D转录激活活性分析 10-1、10-2、10-3、10-4为酵母菌液的4个梯度稀释;pGBKT7-53+pGADT7-T为阳性对照组,pGBKT7-Lam+pGADT7-T为阴性对照组,pGBKT7-EV+pGADT7-EV为空载体组,pGBKT7-TaPAT1-2D+pGADT7-EV为试验组;SD-T-L为SD/-Trp-Leu营养缺陷培养基,SD-T-L-H-A为SD/-Trp-Leu-His-Ade营养缺陷培养基。

Fig.8 Transactivation activity of TaPAT1-2D in wheat 10-1, 10-2, 10-3 and 10-4 were four different dilution concentration of yeast solution; pGBKT7-53+pGADT7-T was positive control group; pGBKT7-Lam+pGADT7-T was negative control group; pGBKT7-EV+pGADT7-EV was empty carrier group and pGBKT7-TaPAT1-2D+pGADT7-EV was the experimental group; SD-T-L was SD/-Trp-Leu nutrient deficiency medium and SD-T-L-H-A was SD/-Trp-Leu-His-Ade nutrient deficiency medium.

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小麦TaPAT1-2D基因的克隆与表达分析

Clonging and expression analysis of TaPAT1-2D gene in wheat

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