辣椒CaERF70的表达特征和转录自激活活性分析

doi:10.3969/j.issn.1004-1524.20231318

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (10): 2247-2256.DOI: 10.3969/j.issn.1004-1524.20231318

辣椒CaERF70的表达特征和转录自激活活性分析

张余¹^,²^,³(), 金明伟⁴, 任丽¹^,², 章毅颖¹^,², 赵洪¹^,², 刘昆¹^,², 邓姗¹^,², 褚云霞¹^,²^,³, 李寿国¹^,², 张靖立¹^,², 黄静艳¹^,², 陈海荣¹^,²^,³^,^*()

1.上海市农业科学院农产品质量标准与检测技术研究所,上海 201403
2.农业农村部植物新品种测试(上海)分中心,上海 201415
3.上海市设施园艺技术重点实验室·上海市农业科学院园艺研究所,上海 201403
4.安顺学院农学院,贵州安顺 561000

收稿日期:2023-11-21 出版日期:2024-10-25 发布日期:2024-10-30
作者简介:张余(1988—),男,陕西周至人,博士,助理研究员,研究方向为辣椒抗逆相关基因的克隆与功能分析。E-mail:xinongxiaoyu@163.com
通讯作者: ^*陈海荣,E-mail:sh57460009@163.com
基金资助:
上海市农业科学院农业科技创新支撑领域研究专项(JCYJ231101)

Expression patterns and transcriptional autoactivation analysis of CaERF70 in chili pepper

ZHANG Yu¹^,²^,³(), JIN Mingwei⁴, REN Li¹^,², ZHANG Yiying¹^,², ZHAO Hong¹^,², LIU Kun¹^,², DENG Shan¹^,², CHU Yunxia¹^,²^,³, LI Shouguo¹^,², ZHANG Jingli¹^,², HUANG Jingyan¹^,², CHEN Hairong¹^,²^,³^,^*()

1. Institute for Agri-Food Standards and Testing Technology, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
2. Shanghai Sub-Center for New Plant Variety Tests, Ministry of Agriculture and Rural Affairs, Shanghai 201415, China
3. Shanghai Key Lab of Protected Horticultural Technology, Horticultural Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China
4. Agricultural College, AnShun University, AnShun 561000, Guizhou, China

Received:2023-11-21 Online:2024-10-25 Published:2024-10-30

摘要/Abstract

摘要：

AP2/ERF转录因子家族广泛参与植物生长发育以及逆境应答等生物学过程,对于AP2/ERF转录因子的研究有助于提高作物抗逆性。该研究以辣椒耐盐品种SHA2022119为材料,克隆得到CaERF70,该基因cDNA全长为819 bp,编码272个氨基酸,分子量约为30.59 ku,等电点pI为7.67。生物信息学分析表明,该蛋白带负电荷,属于不稳定蛋白,存在34个潜在发生磷酸化修饰的位点,为非跨膜蛋白且在细胞核中表达。系统发育树结果显示,辣椒CaERF70与番茄、烟草、马铃薯的ERF聚为一类,暗示着CaERF70在茄科进化过程中非常保守。定量PCR结果显示,CaERF70基因受低温诱导、高温抑制表达。原核表达实验表明,CaERF70编码蛋白大小约为30 ku,与预测大小一致。转录激活活性分析表明,CaERF70具有转录激活活性。综合以上结果表明,CaERF70为AP2/ERF2转录因子,属于ERF亚组第Ⅶ亚组成员,具有转录激活活性,高温或者低温胁迫导致CaERF70表达发生显著变化,暗示着CaERF70可能在辣椒应对高温或低温胁迫过程中发挥作用。

关键词: 辣椒, 基因克隆, 转录活性, 原核表达, 非生物胁迫

Abstract:

The AP2/ERF transcription factor family is widely involved in biological processes such as plant growth and development and stress response, so it is of great significance for the study of AP2/ERF transcription factors. In this study, we cloned a ERF gene, CaERF70 from salt-tolerant variety SHA2022119 of pepper, which contains 819 bp, encoding 272 amino acids, molecular weight of about 30.59 ku, and isoelectric point pI of 7.67. Bioinformatics analysis showed that the protein had a character with negatively charged, unstable, 34 potential phosphorylation sites, non-transmembrane and expressed in the nucleus. The phylogenetic tree results indicated that CaERF70 was clustered with the ERFs of tomato, tobacco and potato, suggesting that CaERF70 was very conserved in the evolution of Solanaceae. qRT-PCR showed that the expression of CaERF70 gene was induced by low temperature and suppressed by high temperature. Prokaryotic expression experiments showed that the size of the protein encoded by CaERF70 was about 30 ku consistent with the predicted size. Transcriptional activation activity analysis showed that CaERF70 had transcriptional activation activity. Taken together, the above results indicated that CaERF70 was an AP2/ERF2 transcription factor, belonging to the ERF Ⅶ group, with transcriptional activating activity, and the expression of CaERF70 was significantly changed due to high or low temperature stress, suggesting that CaERF70 was involved in the response of pepper to high or low temperature stress.

Key words: pepper, gene cloning, transcriptional activity, prokaryotic expression, abiotic stress

中图分类号:

S641.3

张余, 金明伟, 任丽, 章毅颖, 赵洪, 刘昆, 邓姗, 褚云霞, 李寿国, 张靖立, 黄静艳, 陈海荣. 辣椒CaERF70的表达特征和转录自激活活性分析[J]. 浙江农业学报, 2024, 36(10): 2247-2256.

ZHANG Yu, JIN Mingwei, REN Li, ZHANG Yiying, ZHAO Hong, LIU Kun, DENG Shan, CHU Yunxia, LI Shouguo, ZHANG Jingli, HUANG Jingyan, CHEN Hairong. Expression patterns and transcriptional autoactivation analysis of CaERF70 in chili pepper[J]. Acta Agriculturae Zhejiangensis, 2024, 36(10): 2247-2256.

图/表 12

表1 引物列表

Table 1 Primers list

用途Usage	引物名称Primer name	引物序列Primer sequence
基因克隆Gene clone	CaERF70F	ATGGGTTCCCCACAAGAGAATTGCTC
	CaERF70R	TTATATCATGACAAGCTGAGAAT
表达量分析Expression analysis	RT-CaERF70F	GCCCCGAAAATACCCTTTATTC
	RT-CaERF70R	CGTTCGTTGAAACTCCTCTTTT
	RT-ActinF	AGGGATGGGTCAAAAGGATGC
	RT-ActinR	GAGACAACACCGCCTGAATAGC
原核表达Prokaryotic expression	GST-CaERF70F	TTCCAGGGGCCCCTGGGATCCATGGGTTCCCCACAAGAGAATTGCTC
	GST-CaERF70R	CTCGAGTCGACCCGGGAATTCTTATATCATGACAAGCTGAGAAT
自激活分析Self-activation analysis	BD-CaERF70F	ATGGCCATGGAGGCCGAATTCATGGGTTCCCCACAAGAGAATTGCTC
	BD-CaERF70R	CCGCTGCAGGTCGACGGATCCTTATATCATGACAAGCTGAGAAT

图1 CaERF70基因克隆

Fig.1 Cloning of CaERF70 gene

图2 CaERF70蛋白的结构域分析

Fig.2 Domain analysis of CaERF70 protein

图3 核苷酸和氨基酸对比图黑色下划线为预测NLS核定位信号,红色氨基酸为AP2保守结构域,绿色矩形为AP2结构域内第14和19位氨基酸。

Fig.3 Comparison of nucleotides and amino acids The black underline represents the predicted NLS nuclear localization signal, the red amino acid represents the conserved AP2 domain, and the green rectangle represents the 14th and 19th amino acids within the AP2 domain.

图4 CaERF70蛋白的跨膜结构域(A)、信号肽(B)、磷酸化位点(C)

Fig.4 Transmembrane structure (A), signal peptide (B), phosphorylation site of CaERF70 protein(C)

图5 CaERF70蛋白三级结构预测

Fig.5 Prediction of tertiary structure of CaERF70 protein

表2 CaERF70蛋白亚细胞定位预测

Table 2 Prediction of CaERF70 subcellular localization

位置权重Location weight	LocDB	PotLocDB	Neural Nets	Pentamers	Integral
细胞核Nucleus	10.0	3.0	0	0.19	8.98
质膜Plasma membrane	0	0	0.96	0	0.68
细胞外Extracellular	0	0	0.96	0.19	0
细胞质Cytoplasm	0	0	0	2.42	0
线粒体Mitochondrion	0	0	0	1.88	0
内质网Endoplasmic reticulum	0	0	0	0.36	0
过氧化物酶体Peroxisome	0	0	0.96	0	0.06
高尔基体Golgi body	0	0	0.11	0.34	0
叶绿体Chloroplast	0	0	0	0.06	0.02
液泡Vacuole	0	0	0	0	0.26

图6 CaERF70进化树分析甜橙(Citrus sinensis),XP_006490016.2;棉花(Gossypium hirsutum),XP_016737467.1;拟南芥(Arabidopsis thaliana),AAM64362.1;花生(Arachis hypogaea),XP_025632953.1;大豆(Glycine max),XP_003524068.1;苹果(Malus domestica),XP_008369419.2;黄瓜(Cucumis sativus),XP_004143677.2;葡萄(Vitis vinifera),XP_002281954.1;猕猴桃(Actinidia chinensis),PSR93298.1;烟草(Nicotiana tabacum),NP_001312428.1;番茄(Solanum lycopersicum),XP_004235185.1;辣椒(Capsicum annuum),NP_001311615.2;马铃薯(Solanum tuberosum),XP_006358219.1;小麦(Triticum aestivum),XP_044328607.1;水稻(Oryza sativa),XP_015614870.1。

Fig.6 Phylogenetic tree of CaERF70 proteins

图7 辣椒CaERF70 与茄科植物烟草, 番茄和马铃薯同源蛋白的多序列比对烟草(Nicotiana tabacum),NP_001312428.1;番茄(Solanum lycopersicum),XP_004235185.1;辣椒(Capsicum annuum),NP_001311615.2;马铃薯(Solanum tuberosum),XP_006358219.1。蓝色方框代表AP2/ERF DNA结合结构域。

Fig.7 Multi-sequence alignment of CaERF70 with homologous proteins of tobacco, tomato and potato The blue box represents the AP2/ERF DNA binding domain.

图8 CaERF70在非生物胁迫下的表达模式

Fig.8 Expression pattern of CaERF70 under abiotic stress

图9 CaERF70转录激活活性分析

Fig.9 CaERF70 transcriptional activation validation

图10 CaERF70原核表达分析

Fig.10 Prokaryotic expression analysis of CaERF70

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辣椒CaERF70的表达特征和转录自激活活性分析

Expression patterns and transcriptional autoactivation analysis of CaERF70 in chili pepper

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