Cloning and Expression Analysis of CsERF025L Transcription Factor in Cucumber

doi:10.13560/j.cnki.biotech.bull.1985.2023-0962

Abstract

Abstract:

【Objective】ERF transcription factor is a subfamily of AP2/ERF gene family, which plays a key role in plant growth and development and resistance to external stress. Cloning CsERF025-like（CsERF025L）and analyzing its expression pattern may lay a foundation for studying the function of CsERF025L. 【Method】CsERF025L from cucumber（Cucumis sativus L.）stem tip was cloned by RT-PCR, and bioinformatics analysis of CsERF025L protein physicochemical properties, subcellular localization and interacting proteins was performed. Real-time fluorescence quantitative PCR was used to analyze the expression characteristics of CsERF025L in cucumber. 【Result】The encoding sequence of CsERF025L was 510 bp, encoding 169 amino acids, with an AP2/ERF domain, belonging to the ERF subfamily of the AP2/ERF family. CsERF025L protein was an unstable hydrophobic protein located in the nucleus. CsERF025L protein interacts with members of P450 gene family, BEE3 transcription factor, DIR1 protein and MATE family members. CsERF025L promoter contained light response, hormone response, stress response and other elements. The expressions of CsERF025L in the cucumber seeds was the highest, which was significantly higher than that in leaves, stems and roots. The expression patterns of CsERF025L in the cucumber with different phyllotaxis types were compared, and the expressions of CsERF025L in the stem tip of opposite phyllotaxis cop1 were significantly higher than those of cucumber alternate phyllotaxis R1. 【Conclusion】The findings of this study suggest that CsERF025L is a nuclear-localized ERF transcription factor, potentially involved in the formation and regulation of cucumber alternate phyllotaxis.

Key words: cucumber, CsERF025L transcription factor, bioinformatics, expression analysis, phyllotaxis

XIAO Ya-ru, JIA Ting-ting, LUO Dan, WU Zhe, LI Li-xia. Cloning and Expression Analysis of CsERF025L Transcription Factor in Cucumber[J]. Biotechnology Bulletin, 2024, 40(4): 159-166.

Figures/Tables 8

Table 1 Primers for CsERF025L transcription factor cloning and real-time fluorescence quantification

用途 Application	引物名称 Primer name	引物序列 Primer sequence（5'-3'）
实时荧光定量PCR RT-qPCR	TUA-F	AGCGTTTGTCTGTTGACTATGGA
	TUA-R	TGCAGATATCATAAATGGCTTCGT
	CsERF025L-qF	GGTTGCAATGCCGTTCTCAA
	CsERF025L-qR	ACAGCGCTTCTTCATCGACA
基因克隆 Gene cloning	CsERF025L-F	ATGGCCTCTACATCCTCCAAGAG
基因克隆 Gene cloning	CsERF025L-R	CTAGTGGTAGCTCCAAAGATTTCCAT

Fig. 1 PCR amplification electrophoresis of cucumber CsERF025L cDNA sequences (A) and the aligned results with the reference sequence (B)

Table 2 Physicochemical properties of proteins in cucumber CsERF025L

基因名称 Gene name	氨基酸数 NAA	分子质量 Mw/kD	等电点 pI	不稳定系数 II	亲水系数 GRAVY	带负电荷的残基总数 Asp+Glu	带正电荷的残基总数 Arg+Lys	脂肪族指数 AI	亚细胞定位 SL
CsERF025L	169	1.87	6.64	57.22	-0.620	17	16	58.46	细胞核

Fig. 2 Protein analysis of cucumber CsERF025L A: Secondary structure of cucumber CsERF025L. B: Tertiary structure of cucumber CsERF025L protein. C: Prediction map of cucumber CsERF025L phosphorylation. D: Protein interaction network model of cucumber CsERF025L

Fig. 3 Cis-acting elements of cucumber CsERF025L promoter

Fig. 4 CsERF025L motif（A）and domain analysis（B）of cucumber

Fig. 5 Phylogenetic tree of ERF025L in cucumber and other species

Fig. 6 Specific expression of CsERF025L in cucumber S: Seed（the first day after soaking）; M: stem; L: leaf; R: root; T1: stem tip 1（the seventh day after soaking）; T2: stem tip 2（the 12th day after soaking; T3: stem tip 3（the 17th day after soaking）; R1: alternate phyllotaxis in cucumber; cop1: opposite phyllotaxis in cucumber; Different lowercases indicate significant differences at the 0.05 level

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