辣椒CaUBC38基因的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0363

摘要/Abstract

摘要：

目的泛素结合酶E2（ubiquitin conjugating enzyme, UBC）是底物泛素化的关键酶，在植物生长发育中具有调控作用。探究CaUBC38基因在辣椒果实成熟和高温胁迫响应过程中的作用，为辣椒的分子育种奠定基础。方法以辣椒（Capsicum annuum）骨干亲本‘6421’为材料，从辣椒中克隆CaUBC38，并对其蛋白序列、蛋白结构、亚细胞定位、表达模式进行详细分析；通过RT-qPCR技术分析CaUBC38在辣椒根、茎、叶、花、果实不同发育时期的表达模式，构建CaUBC38病毒诱导的基因沉默（virus induced gene silencing, VIGS）载体，以离体辣椒果实为材料，探索CaUBC38基因的功能；同时构建CaUBC38过表达载体，得到转基因株系。结果辣椒CaUBC38编码区393 bp，共编码130个氨基酸；包含典型的UBCc超家族保守结构域，属于UBC基因家族；蛋白的分子质量为14.80 kD，推测为酸性的不稳定蛋白，无跨膜结构域和信号肽；含有9个丝氨酸磷酸化位点；CaUBC38的二级、三级结构预测结果表明，其主要由无规卷曲和α-螺旋组成。系统进化分析表明，CaUBC38与茄科其他物种的同源性较高。亚细胞定位结果显示CaUBC38蛋白主要定位于细胞质膜、细胞核上。RT-qPCR检测发现，CaUBC38在辣椒果实中的表达量高于根、茎、叶、花，且果实转色期的表达量高于红熟期、绿熟期、未熟期。通过VIGS技术沉默CaUBC38后发现，辣椒果实转色延缓。同时构建CaUBC38过表达载体，得到转基因拟南芥株系，CaUBC38过表达拟南芥植株耐热性降低。结论沉默CaUBC38延缓辣椒果实成熟，过表达CaUBC38拟南芥株系耐热性降低。

关键词: 辣椒, CaUBC38, 泛素结合酶E2, 理化性质分析, 果实, 表达模式分析, 高温, 过表达载体

Abstract:

Objective Ubiquitin-conjugating enzyme (UBC) is a pivotal enzyme in substrate ubiquitination, playing a crucial regulatory role in plant growth and development. Elucidating the function of the CaUBC38 gene in regulating pepper (Capsicum annuum) fruit ripening and thermotolerance responses to advance molecular breeding strategies. Method We cloned CaUBC38 from the pepper backbone parent ‘6421’ and conducted a comprehensive analysis of its protein sequence, structure, subcellular localization, and expression patterns. In addition, RT-qPCR was used to analyze the expression patterns of CaUBC38 in pepper roots, stems, leaves, flowers and fruits at different developmental stages, and a virus-induced gene silencing (VIGS) vector of CaUBC38 virus was constructed to explore the function of CaUBC38 gene in vitro pepper fruits. At the same time, the CaUBC38-overexpressed vector was constructed to obtain the transgenic line. Result The coding region of CaUBC38 spans 393 bp, encoding a protein of 130 amino acids. This protein harbors a typical conserved domain of the UBCc superfamily, classifying it within the UBC gene family. The molecular mass of CaUBC38 is 14.80 kD, and it is predicted to be an acidic, unstable protein lacking transmembrane domains and signal peptides. It contains 9 serine phosphorylation sites. Secondary and tertiary structure predictions indicated that CaUBC38 is predominantly composed of random coils and α-helices. Phylogenetic analysis demonstrated that CaUBC38 has high homology with other species of Solanaceae. The subcellular localization results showed that the CaUBC38 protein was mainly localized on the plasma membrane and nucleus. RT-qPCR detection revealed that the expression of CaUBC38 in pepper fruits was higher than that in the roots, stems, leaves, and flowers. Moreover, the expression during the fruit color-changing stage was higher than that during the red-ripe stage, green-ripe stage, and unripe stage. Silencing CaUBC38 viaVIGS technology, delayed pepper fruit maturation. At the same time, the CaUBC38-overexpressing vector was constructed to obtain a transgenic Arabidopsis line, and the tolerance of CaUBC38 overexpressing Arabidopsis plants to heat was reduced. Conclusion Silencing of CaUBC38 delays pepper fruit ripening, while its overexpression impairs thermotolerance in transgenic Arabidopsis.

Key words: CaUBC38, ubiquitin conjugating enzyme, analysis of physicochemical properties, fruit, analysis of expression pattern, high temperature, overexpression vectors Capsicum annuum

王静, 常雪瑞, 贾旭, 黄嘉欣, 王田田, 梁燕平. 辣椒CaUBC38基因的克隆及功能分析[J]. 生物技术通报, 2025, 41(10): 242-252.

WANG Jing, CHANG Xue-rui, JIA Xu, HUANG Jia-xin, WANG Tian-tian, LIANG Yan-ping. Cloning and Fuctional Analysis of CaUBC38 Gene in Pepper[J]. Biotechnology Bulletin, 2025, 41(10): 242-252.

图/表 11

图1 辣椒4个时期的果实

Fig. 1 Fruits of peppers at 4 stages

表１试验所用引物名称及序列

Table 1 Names and sequences of the primers used in the experiment

引物名称 Primer name	引物序列 Primer sequence （5′‒3′）
CaUBC38-F	GCGGAAAGTCCTTATCATGG
CaUBC38-R	GATCAACAGGACCTGCCATT
qCaUBC38-F	GCGGAAAGTCCTTATCATGG
qCaUBC38-R	GATCAACAGGACCTGCCATT
qβ-Actin-F qβ-Actin-R	CCACCTCTTCACTCTCTGCTCT ACTAGGAAAAACAGCCCTTGGT
CaUBC38-pART-CAM-EGFP-F CaUBC38-pART-CAM-EGFP-R	CATTTGGAGAGGACACGCATGTCTTCTCCGAGCAAACG TCGCCCTTGCTCACCATGAATGGATCAACAGGACCTGC
CaUBC38-VIGS-F	GAAGGCCTCCATGGGGATCCATGACTACAATATTTGCAAAAC
CaUBC38-VIGS -R	GCCTCGAGACGCGTGAGCTCATAAAATACATCGTTTAGAG
PTRV2-SEQS	AACAAAGTCCGTTCCCCTAT
CaUBC38-OE-F	GAGCTTTCGCGAGCTCGGTACCATGTCTTCTCCGAGCAAACG
CaUBC38-OE-R	GCAGGTCGACTCTAGAGGATCCCTATGGATCAACAGGACCTGC

图2 CaUBC38基因PCR扩增（A）和CaUBC38全长核苷酸序列及预测氨基酸序列（B）M：DNA marker；1：PCR产物

Fig. 2 PCR amplification of CaUBC38 gene (A) and full-length nucleotide sequence of CaUBC38 and predicting amino acid sequence (B)M: DNA marker; 1: PCR amplified product

图3 蛋白序列对比分析

Fig. 3 Comparative analysis of protein sequences

图4 CaUBC38蛋白的保守结构域

Fig. 4 Conserved domain of CaUBC38 protein

图5 辣椒CaUBC38蛋白的基本信息A：亲疏水性预测；B：蛋白磷酸化位点预测；C：二级结构；D：三级结构；E：跨膜结构；F：信号肽

Fig. 5 Basic information on CaUBC38 protein in pepperA: Hydrophilic and hydrophobic prediction. B: Protein phosphorylation site prediction. C: Secondary structure. D: Tertiary structure. E: Transmembrane structure. F: Signal peptide

图6 CaUBC38的系统进化树分析

Fig. 6 Phylogenetic tree analysis of CaUBC38

图7 CaUBC38蛋白的亚细胞定位从左到右分别为GFP绿色荧光蛋白、PM-mcherry通道、叶绿体荧光通道、明场和叠加图。比例尺=25.0 μm

Fig. 7 Subcellular localization of CaUBC38From left to right, GFP green fluorescent protein, PM-mcherry channel, chloroplast fluorescence channel, bright, and merge. Scale bar = 25.0 μm

图8 CaUBC38基因在辣椒不同组织（A）及果实不同发育时期（B）的表达模式不同小写字母表示差异显著（P<0.05）

Fig. 8 Expression patterns of CaUBC38 gene in different tissues (A) and fruit development stages (B) of pepperDifferent lower letters indicate significant differences (P<0.05)

图9 VIGS载体鉴定（A）、沉默效率（B）和CaUBC38基因沉默果实表型（C、D）M：DNA marker 2000；1‒3：VIGS载体鉴定；pTRV2：基因沉默阴性对照；pTRV2：CaUBC38： CaUBC38基因沉默果实

Fig. 9 Identification of VIGS vector (A), silencing efficiency (B), and CaUBC38 genes silenced fruit phenotype (C, D)M: DNA marker 2000; 1‒3: identification of VIGS vector; pTRV2: negative control; pTRV2: CaUBC38: CaUBC38-silenced fruit

图10 过表达载体鉴定（A）、CaUBC38基因过表达拟南芥表型（B）及死细胞含量测定（C）M：DNA marker 2000；1：过表达载体鉴定

Fig. 10 Identification of overexpression vectors (A), phenotypes of Arabidopsis thaliana overexpressing the CaUBC38 gene (B), and determination of dead cells content (C)M: DNA marker 2000; 1: identification of over-expression vector

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