黄瓜CsERF025L转录因子的克隆及表达分析

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

摘要/Abstract

摘要：

【目的】ERF转录因子是AP2/ERF基因家族的一个亚族，在植物生长发育和抵抗外界胁迫方面具有重要作用，克隆CsERF025-like（CsERF025L）并分析其表达模式，为黄瓜CsERF025L功能研究奠定基础。【方法】通过RT-PCR从黄瓜茎尖中克隆CsERF025L，对CsERF025L进行蛋白理化性质、亚细胞定位、互作蛋白等生物信息学分析，采用实时荧光定量PCR分析CsERF025L在黄瓜中的表达模式。【结果】CsERF025L编码序列为510 bp，编码169个氨基酸，具有一个AP2/ERF结构域，属于AP2/ERF家族的ERF亚家族。CsERF025L蛋白是一种不稳定的疏水性蛋白，位于细胞核，蛋白CsERF025L与P450基因家族成员、BEE3转录因子、DIR1蛋白和MATE家族成员存在互作。CsERF025L启动子含有光响应、激素响应、应激响应等元件。基因CsERF025L在黄瓜种子时期表达量最高，显著高于叶、茎和根。比较CsERF025L在不同叶序黄瓜中的表达，发现对生叶序黄瓜cop1茎尖中CsERF025L表达量均显著高于互生叶序黄瓜R1。【结论】CsERF025L是一个位于细胞核的ERF转录因子，可能在黄瓜对生叶序性状的形成与调控中具有潜在功能。

关键词: 黄瓜, CsERF025L转录因子, 生物信息学, 表达分析, 叶序

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

肖雅茹, 贾婷婷, 罗丹, 武喆, 李丽霞. 黄瓜CsERF025L转录因子的克隆及表达分析[J]. 生物技术通报, 2024, 40(4): 159-166.

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.

图/表 8

表1 CsERF025L转录因子实时荧光定量和克隆引物

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

图1 黄瓜CsERF025L cDNA序列PCR扩增电泳图（A）和与参考序列比对结果（B）

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

表2 黄瓜CsERF025L蛋白理化性质分析

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

图2 黄瓜CsERF025L蛋白分析 A：黄瓜CsERF025L蛋白二级结构；B：黄瓜CsERF025L蛋白三级结构；C：黄瓜CsERF025L磷酸化预测；D：黄瓜CsERF025L蛋白互作网络模型图

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

图3 黄瓜CsERF025L启动子顺式作用元件

Fig. 3 Cis-acting elements of cucumber CsERF025L promoter

图4 黄瓜CsERF025L保守基序（A）和结构域分析（B）

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

图5 黄瓜与其他物种ERF025L的系统进化树

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

图6 CsERF025L在黄瓜中的特异性表达 S：种子（浸种后1 d）；M：茎；L：叶；R：根；T1：茎尖1（浸种后7 d）；T2：茎尖2（浸种后12 d）；T3：茎尖3（浸种后17 d）；R1:互生叶序黄瓜；cop1：对生叶序黄瓜；不同小写字母表示在0.05水平差异显著

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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