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

doi:10.3969/j.issn.1004-1524.2023.01.03

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (1): 23-32.DOI: 10.3969/j.issn.1004-1524.2023.01.03

• Crop Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S512

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.

Figures/Tables 11

References 28

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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

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

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

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

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

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

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