Cloning and Expression Analysis of AP2/ERF Transcription Factor NtESR2 in Nicotiana tabacum

doi:10.13560/j.cnki.biotech.bull.1985.2024-0894

Abstract

Abstract:

Objective ESR2 gene in the AP2/ERF (APETALA2/ethylene-responsive factor) transcription factor family plays an important role in the regulation of plant leaf bud development. To clone the tobacco NtESR2 gene and analyze its function, we aimed to provide a target gene for the regulation of tobacco leaf bud development. Method NtESR2 gene was cloned from cultivated tobacco (Nicotiana tabacum L.) by homologous cloning, and its bioinformatics, gene expression, hormone response, and subcellular localization analysis were performed. Meanwhile, CRISPR/Cas9 editing technology was used to create the NtESR2a and NtESR2b double mutant ntesr2, and the phenotype of the ntesr2 mutant was observed. Result NtESR2 has two copies in cultivated tobacco, NtESR2a and NtESR2b, with CDS lengths of 1 188 bp and 1 200 bp, encoding 395 and 399 amino acids, respectively. NtESR2a protein is located in the nucleus, while NtESR2b protein is located in the nucleus and cell membrane. Both proteins have only one AP2 domain and belong to the ERF subfamily, of which NtESR2a is from Nicotiana sylvestris and NtESR2b is from Nicotiana tomentosiformis. NtESR2 gene is expressed in the roots, stems, leaves, terminal buds, axillary buds and flowers of tobacco, among which the expression in terminal buds and axillary buds is higher, indicating that NtESR2 gene may have an important regulatory role in these tissues. Cis-acting element analysis and exogenous hormone treatment showed that the gene is regulated by a variety of plant hormones. The ntesr2 mutant showed the characteristics of cotyledon fusion and meristem loss, and the expression of NtCUC2, NtLAS, NtREV, NtYUCCA and NtPIN1 genes was significantly downregulated. Conclusion Two members of the ERF transcription factor NtESR2 are cloned from tobacco. They are highly expressed in apical buds and axillary buds and are regulated by a variety of plant hormones such as auxin. NtESR2 regulates the transport of auxin to affect the morphological establishment process of tobacco leaf buds. The research results provide a theoretical basis for regulating the development of tobacco leaf buds.

Key words: tobacco, NtESR2, CRISPR/Cas9, cotyledon fusion, meristem, bioinformatics analysis, subcellular localization

LU Yong-jie, XIA Hai-qian, LI Yong-ling, ZHANG Wen-jian, YU Jing, ZHAO Hui-na, WANG Bing, XU Ben-bo, LEI Bo. Cloning and Expression Analysis of AP2/ERF Transcription Factor NtESR2 in Nicotiana tabacum[J]. Biotechnology Bulletin, 2025, 41(4): 266-277.

Figures/Tables 12

References 31

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引物Primer	序列Sequence （5′‒3′）	用途Usage
NtESR2a-F	ATGGAAGATGCCATGAG	NtESR2a基因克隆 NtESR2a amplification
NtESR2a-R	TTAAGAATTCTGCAGCTTAG	NtESR2a基因克隆 NtESR2a amplification
NtESR2b-F	ATGCGCGGTGTTAAAGC	NtESR2b基因克隆 NtESR2b amplification
NtESR2b-R	TCAAGAATTCTGCAGCTTAG	NtESR2b基因克隆 NtESR2b amplification
Actin-Q-F	CGGAATCCACGAGACTACATA	RT-qPCR内标基因检测 RT-qPCR internal standard gene detection
Actin-Q-R	GGGAAGCCAAGATAGAGC	RT-qPCR内标基因检测 RT-qPCR internal standard gene detection
ESR2a-Q-F	CCAACTTCCACTAACGATGGGT	RT-qPCR检测NtESR2a基因表达量 RT-qPCR detection of NtESR2a gene expression
ESR2a-Q-R	GTGTCTGAAGAAAGGGAACTGGT
ESR2b-Q-F	GTGAACTTCTTAGTTCGAACCCCT	RT-qPCR检测NtESR2b基因表达量 RT-qPCR detection of NtESR2b gene expression
ESR2b-Q-R	GACTTGATGGGCTTGGGCT
NtESR2a-CRISPR test-F	CATCTGTTGGGGCGGTGAGGTAT	检测NtESR2a基因突变
NtESR2a-CRISPR test-R	CTGGTAGTATTGGAGGAATTG	Detection of NtESR2a gene mutation
NtESR2b-CRISPR test-F	CATCTGTCGGGGCGGTGAGGTAC	检测NtESR2b基因突变
NtESR2b-CRISPR test-R	CAAAGAATCATTGCTTTTCTGA	Detection of NtESR2b gene mutation
CUC2-Q-F	TCCTCTGTCGGTGCCAATTC	RT-qPCR检测NtCUC2基因表达量
CUC2-Q-R	TTGGGTCGAAACTGTGCGTA	RT-qPCR detection of NtCUC2 gene expression
LAS-Q-F	ACAGCTGCAGTAAAGGTGCC	RT-qPCR检测NtLAS基因表达量
LAS-Q-R	GCGAGGATACTCTCCCAGCA	RT-qPCR detection of NtLAS gene expression
REV-Q-F	CCACTGGGCCACACAATAGAA	RT-qPCR检测NtREV基因表达量
REV-Q-R	ACAGATCGTGCACTGTAGCC	RT-qPCR detection of NtREV gene expression
YUCCA-Q-F	TGCTAAGTACGACGAAGCTTGT	RT-qPCR检测NtYUCCA基因表达量
YUCCA-Q-R	ATGCCAGAATTTCCACAGCCTA	RT-qPCR detection of NtYUCCA gene expression
PIN1-Q-F	TCGAACCAAGTGAGGGCATC	RT-qPCR检测NtPIN1基因表达量
PIN1-Q-R	AGCTGCAATCAGAGAAGCGA	RT-qPCR detection of NtPIN1 gene expression

引物Primer	序列Sequence （5′‒3′）	用途Usage
NtESR2a-F	ATGGAAGATGCCATGAG	NtESR2a基因克隆 NtESR2a amplification
NtESR2a-R	TTAAGAATTCTGCAGCTTAG	NtESR2a基因克隆 NtESR2a amplification
NtESR2b-F	ATGCGCGGTGTTAAAGC	NtESR2b基因克隆 NtESR2b amplification
NtESR2b-R	TCAAGAATTCTGCAGCTTAG	NtESR2b基因克隆 NtESR2b amplification
Actin-Q-F	CGGAATCCACGAGACTACATA	RT-qPCR内标基因检测 RT-qPCR internal standard gene detection
Actin-Q-R	GGGAAGCCAAGATAGAGC	RT-qPCR内标基因检测 RT-qPCR internal standard gene detection
ESR2a-Q-F	CCAACTTCCACTAACGATGGGT	RT-qPCR检测NtESR2a基因表达量 RT-qPCR detection of NtESR2a gene expression
ESR2a-Q-R	GTGTCTGAAGAAAGGGAACTGGT
ESR2b-Q-F	GTGAACTTCTTAGTTCGAACCCCT	RT-qPCR检测NtESR2b基因表达量 RT-qPCR detection of NtESR2b gene expression
ESR2b-Q-R	GACTTGATGGGCTTGGGCT
NtESR2a-CRISPR test-F	CATCTGTTGGGGCGGTGAGGTAT	检测NtESR2a基因突变
NtESR2a-CRISPR test-R	CTGGTAGTATTGGAGGAATTG	Detection of NtESR2a gene mutation
NtESR2b-CRISPR test-F	CATCTGTCGGGGCGGTGAGGTAC	检测NtESR2b基因突变
NtESR2b-CRISPR test-R	CAAAGAATCATTGCTTTTCTGA	Detection of NtESR2b gene mutation
CUC2-Q-F	TCCTCTGTCGGTGCCAATTC	RT-qPCR检测NtCUC2基因表达量
CUC2-Q-R	TTGGGTCGAAACTGTGCGTA	RT-qPCR detection of NtCUC2 gene expression
LAS-Q-F	ACAGCTGCAGTAAAGGTGCC	RT-qPCR检测NtLAS基因表达量
LAS-Q-R	GCGAGGATACTCTCCCAGCA	RT-qPCR detection of NtLAS gene expression
REV-Q-F	CCACTGGGCCACACAATAGAA	RT-qPCR检测NtREV基因表达量
REV-Q-R	ACAGATCGTGCACTGTAGCC	RT-qPCR detection of NtREV gene expression
YUCCA-Q-F	TGCTAAGTACGACGAAGCTTGT	RT-qPCR检测NtYUCCA基因表达量
YUCCA-Q-R	ATGCCAGAATTTCCACAGCCTA	RT-qPCR detection of NtYUCCA gene expression
PIN1-Q-F	TCGAACCAAGTGAGGGCATC	RT-qPCR检测NtPIN1基因表达量
PIN1-Q-R	AGCTGCAATCAGAGAAGCGA	RT-qPCR detection of NtPIN1 gene expression

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