Cloning and Fuctional Analysis of CaUBC38 Gene in Pepper

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

Abstract

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

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.

Figures/Tables 11

Fig. 1 Fruits of peppers at 4 stages

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

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

Fig. 3 Comparative analysis of protein sequences

Fig. 4 Conserved domain of CaUBC38 protein

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

Fig. 6 Phylogenetic tree analysis of CaUBC38

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

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)

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

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

References 32

[1]	Nagels Durand A, Pauwels L, Goossens A. The ubiquitin system and jasmonate signaling [J]. Plants, 2016, 5(1): 6.
[2]	Liu XT, Zhou YY, Du MS, et al. The calcium-dependent protein kinase CPK28 is targeted by the ubiquitin ligases ATL31 and ATL6 for proteasome-mediated degradation to fine-tune immune signaling in Arabidopsis [J]. Plant Cell, 2022, 34(1): 679-697.
[3]	Gil KE, Kim WY, Lee HJ, et al. ZEITLUPE contributes to a thermoresponsive protein quality control system in Arabidopsis [J]. Plant Cell, 2017, 29(11): 2882-2894.
[4]	Kim SH, Park JS, Lee MH, et al. The N-degron pathway governs autophagy to promote thermotolerance in Arabidopsis [J]. Nat Commun, 2025, 16(1): 5889.
[5]	Liu WG, Tang X, Fu X, et al. Functional characterization of potato UBC13-UEV1s genes required for ubiquitin Lys63 chain to polyubiquitination [J]. Int J Mol Sci, 2023, 24(3): 2412.
[6]	Cheng MC, Kuo WC, Wang YM, et al. UBC18 mediates ERF1 degradation under light-dark cycles [J]. New Phytol, 2017, 213(3): 1156-1167.
[7]	Pickart CM. Mechanisms underlying ubiquitination [J]. Annu Rev Biochem, 2001, 70: 503-533.
[8]	Ye YH, Rape M. Building ubiquitin chains: E2 enzymes at work [J]. Nat Rev Mol Cell Biol, 2009, 10(11): 755-764.
[9]	Wang PC, Guo K, Su Q, et al. Histone ubiquitination controls organ size in cotton (Gossypium hirsutum) [J]. Plant J, 2022, 110(4): 1005-1020.
[10]	董发才, 宋纯鹏. 植物细胞中的泛素及其生理功能 [J]. 植物生理学通讯, 1999, 35(1): 54-59.
	Dong FC, Song CP. Ubiquitin in plant cells and its physiological functions [J]. Plant Physiol Commun, 1999, 35(1): 54-59.
[11]	Dong C, Hu HG, Jue DW, et al. The banana E2 gene family: Genomic identification, characterization, expression profiling analysis [J]. Plant Sci, 2016, 245: 11-24.
[12]	Jue DW, Sang XL, Shu B, et al. Characterization and expression analysis of genes encoding ubiquitin conjugating domain-containing enzymes in Carica papaya [J]. PLoS One, 2017, 12(2): e0171357.
[13]	Kim S, Park M, Yeom SI, et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species [J]. Nat Genet, 2014, 46(3): 270-278.
[14]	李广存, 庄飞云, 王立浩, 等. 蔬菜生物育种发展概述 [J]. 中国基础科学, 2022, 24(4): 29-36.
	Li GC, Zhuang FY, Wang LH, et al. Development of vegetable bio-breeding [J]. China Basic Sci, 2022, 24(4): 29-36.
[15]	邹学校, 马艳青, 戴雄泽, 等. 辣椒在中国的传播与产业发展 [J]. 园艺学报, 2020, 47(9): 1715-1726.
	Zou XX, Ma YQ, Dai XZ, et al. Spread and industry development of pepper in China [J]. Acta Hortic Sin, 2020, 47(9): 1715-1726.
[16]	邹学校, 朱凡. 辣椒的起源、进化与栽培历史 [J]. 园艺学报, 2022, 49(6): 1371-1381.
	Zou XX, Zhu F. Origin, evolution and cultivation history of the pepper [J]. Acta Hortic Sin, 2022, 49(6): 1371-1381.
[17]	胡悦. 辣椒中辣椒红素抗氧化活性理论与实验研究 [D]. 包头: 内蒙古科技大学, 2024.
	Hu Y. Theoretical and experimental study on antioxidant activity of capsanthin in pepper [D]. Baotou: Inner Mongolia University of Science & Technology, 2024.
[18]	邹学校, 杨莎, 戴雄泽, 等. 中国辣椒产业快速发展40年回顾与展望 [J]. 园艺学报, 2025, 52(1): 247-258.
	Zou XX, Yang S, Dai XZ, et al. The rapid development of China’s chili pepper industry over the past 40 years [J]. Acta Hortic Sin, 2025, 52(1): 247-258.
[19]	高成安. 发育阶段和高温、干旱对辣椒果实辣椒素代谢的影响 [D]. 杭州: 浙江农林大学, 2025.
	Gao CA. Effects of development stage, high temperature and drought on capsaicin metabolism in pepper fruit [D]. Hangzhou: Zhejiang A & F University, 2025.
[20]	梁宝萍, 段莹, 姜俊, 等. 高温胁迫对辣椒果实活性氧代谢的影响 [J]. 陕西农业科学, 2022, 68(8): 93-96, 102.
	Liang BP, Duan Y, Jiang J, et al. Effect of high-temperature stress on active oxygen metabolism in Capsicum annuum L fruit [J]. Shaanxi J Agric Sci, 2022, 68(8): 93-96, 102.
[21]	Lin TH, Lin SW, Wang YW, et al. Growing environment and heat treatment effects on intra- and interspecific pollination in Chile pepper (Capsicum spp.) [J]. Agronomy, 2021, 11(7): 1275.
[22]	李连英, 谢培菡, 曾广明, 等. 赣南地区蔬菜产业基本情况调研报告 [J]. 北方园艺, 2021(7): 135-142.
	Li LY, Xie PH, Zeng GM, et al. Investigation report on the basic situation of vegetable industry in southern Jiangxi [J]. North Hortic, 2021(7): 135-142.
[23]	常雪瑞, 王田田, 王静. 辣椒E2基因家族的鉴定及分析 [J]. 生物技术通报, 2024, 40(6): 238-250.
	Chang XR, Wang TT, Wang J. Identification and analysis of E2 gene family in pepper (Capsicum annuum L.) [J]. Biotechnol Bull, 2024, 40(6): 238-250.
[24]	马艳青, 戴雄泽, 李雪峰, 等. 辣椒骨干亲本6421及其衍生系的创制与利用 [J]. 中国蔬菜, 2015(6): 11-16.
	Ma YQ, Dai XZ, Li XF, et al. Creation and utilization of pepper backbone parent 6421 and its derivated lines [J]. China Veg, 2015(6): 11-16.
[25]	田士林. 辣椒果实中辣椒红素合成的分子机理及其调控研究 [D]. 杨凌: 西北农林科技大学, 2014.
	Tian SL. Study on molecular mechanism and regulation of capsaicin synthesis in pepper fruit [D]. Yangling: Northwest A & F University, 2014.
[26]	Liu WG, Tang X, Qi XH, et al. The ubiquitin conjugating enzyme: an important ubiquitin transfer platform in ubiquitin-proteasome system [J]. Int J Mol Sci, 2020, 21(8): 2894.
[27]	Gao YY, Wang Y, Xin HP, et al. Involvement of ubiquitin-conjugating enzyme (E2 gene family) in ripening process and response to cold and heat stress of Vitis vinifera [J]. Sci Rep, 2017, 7: 13290.
[28]	罗传英. 草莓泛素结合酶基因家族鉴定和调控果实成熟的功能初探 [D]. 雅安: 四川农业大学, 2022.
	Luo CY. Identification of ubiquitin-binding enzyme gene family in strawberry and preliminary study on its function of regulating fruit ripening [D]. Ya’an: Sichuan Agricultural University, 2022.
[29]	张明先, 张芮豪, 李平平, 等. 辣椒CMB1基因克隆与表达分析 [J]. 广东农业科学, 2023, 50(11): 50-58.
	Zhang MX, Zhang RH, Li PP, et al. Cloning and expression analysis of CMB1 gene in pepper [J]. Guangdong Agric Sci, 2023, 50(11): 50-58.
[30]	董晨, 李金枝, 郑雪文, 等. 基于转录组的荔枝泛素结合酶基因家族的鉴定及表达分析 [J]. 热带作物学报, 2022, 43(5): 882-892.
	Dong C, Li JZ, Zheng XW, et al. Transcriptome-wide identification and analysis of UBC gene family in Litchi chinensis sonn [J]. Chin J Trop Crops, 2022, 43(5): 882-892.
[31]	Wang YY, Wang WH, Cai JH, et al. Tomato nuclear proteome reveals the involvement of specific E2 ubiquitin-conjugating enzymes in fruit ripening [J]. Genome Biol, 2014, 15(12): 548.
[32]	高维东, 胡城祯, 张龙, 等. 小麦泛素结合酶TaUBC16基因的克隆与功能分析 [J]. 作物学报, 2024, 50(8): 1971-1988.
	Gao WD, Hu CZ, Zhang L, et al. Cloning and functional analysis of ubiquitin-conjugating enzymes TaUBC16 gene in wheat [J]. Acta Agron Sin, 2024, 50(8): 1971-1988.