辣椒U-Box蛋白基因CaPUB18的耐热功能分析

doi:10.16420/j.issn.0513-353x.2024-0897

摘要/Abstract

摘要：

U-Box型E3泛素连接酶（U-box E3 ubiquitin ligase，PUB）在植物抗逆过程中发挥着重要作用，但CaPUB18基因在辣椒耐热中的功能尚不清楚。辣椒CaPUB18包含1个U-Box结构域和4个ARM结构域，与马铃薯、番茄同源蛋白的亲缘关系较近。CaPUB18定位于细胞膜，并在辣椒各个组织中均有表达，其表达量在热胁迫过程中呈先上升后下降趋势。CaPUB18基因沉默和异源过表达分别降低了辣椒和拟南芥的耐热性，均表现为叶片萎蔫加重、电导率和丙二醛含量升高、死细胞和H₂O₂积累增多、总叶绿素含量降低，且耐热标记基因上调表达受抑制。以上结果表明，CaPUB18基因沉默表达和过量表达对辣椒的耐热性均不利。研究揭示了保持U-box蛋白基因CaPUB18表达水平稳定对辣椒耐热性形成的重要性。

关键词: 辣椒, U-box蛋白, E3泛素连接酶, CaPUB18, 耐热性

Abstract:

U-box E3 ubiquitin ligase（PUB）plays important roles in plant stress resistance，while the function of CaPUB18 gene in pepper heat tolerance is still unclear. Pepper CaPUB18 protein contains one U-box domain and four ARM domains，and presents closer phylogenetic relationship to its homologues from potato and tomato. CaPUB18 is localized in the cell membrane，and expressed in various tissues. During heat stress，its expression level increases initially，and then decreases. The silencing and heterologous overexpression of CaPUB18 compromised the heat tolerance of pepper and Arabidopsis，respectively，as evidenced by the aggravated leaf wilting，increased relative conductivity and malondialdehyde（MDA）content，accumulation of dead cells and H₂O₂，reduced total chlorophyll content，and inhibited induction of heat-tolerance marker genes. These results indicate that both silencing and overexpression of CaPUB18 are detrimental to the heat tolerance of pepper. This study highlights the importance of maintaining the stability of U-box protein gene CaPUB18 expression level to the formationof heat tolerance of pepper.

Key words: pepper, U-box containing protein, E3 ubiquitin ligase, CaPUB18, heat tolerance

李莹, 郭宇玲, 赵悦, 杨巧敏, 陈涛, 安雨畅, 毛张亮, 逯明辉. 辣椒U-Box蛋白基因CaPUB18的耐热功能分析[J]. 园艺学报, 2026, 53(4): 1175-1188.

LI Ying, GUO Yuling, ZHAO Yue, YANG Qiaomin, CHEN Tao, AN Yuchang, MAO Zhangliang, LU Minghui. Functional Analysis of Pepper U-Box Domain-Containing Protein Gene CaPUB18 in Heat Tolerance[J]. Acta Horticulturae Sinica, 2026, 53(4): 1175-1188.

图/表 10

表1 本研究中所用引物及序列

Table 1 Primers and sequence used in this study

引物名称 Primer name	序列（5′-3′） Sequence	用途 Purpose
pART27-GFP-CaPUB18-F	GATGAACTATACAAAGAATTCATGATCAACGACAAATCTAACGA	基因CDS区全长扩增 Amplification of entire CDS（Coding DNA sequence）
pART27-GFP-CaPUB18-R	CAGGACTCTAGATTAGGTACCTTACCACACATTAATGAATTGATCC	基因CDS区全长扩增 Amplification of entire CDS（Coding DNA sequence）
pTRV2- CaPUB18-F	GCTCTAGATCGAAACTCGATACGTTTGATTG	基因沉默特异片段扩增 Construction of gene-silencing vector
pTRV2- CaPUB18-R	CGGGATCCTCCACCACTTCTTTAACTTCCAC	基因沉默特异片段扩增 Construction of gene-silencing vector
qCaPUB18-F	GCTTTTGGGCACACTTGAGG	基因表达分析 Analysis of gene expression
qCaPUB18-R	TGTGTGATTCTCGGGTGGTG
qCaUbi3-F	TGTCCATCTGCTCTCTGTTG
qCaUbi3-R	CACCCCAAGCACAATAAGAC
qCaHSP70.1-F	CAGGTGTGCTAGTTCAGGTGT
qCaHSP70.1-R	TGACCTGAGGCACTCCTCTT
qCaHsfA2-F	GTAGCATCAGTAGCCACAGC
qCaHsfA2-R	CAAGCAACTCTTCCCAAATA
AtActin2-F	CGCTCTTTCTTTCCAAGCTCAT
AtActin2-R	CAAATCCAGCCTTCACCAT

图1 CaPUB18的序列特征分析 A：保守结构域分析；B：系统进化分析

Fig. 1 Sequence characterization of CaPUB18 A：Analysis of conservative domains；B：Analysis of phylogenetic relationship

表2 CaPUB18启动子顺式作用元件预测

Table 2 Prediction cis-acting elements in CaPUB18 promoter

元件 Component	序列 Sequence	数量 Quantity	功能 Function
ABRE	ACGTG	2	响应脱落酸信号 Involved in the ABA-responsiveness
CGTCA-motif	CGTCA	1	响应茉莉酸甲酯信号 Involved in the MeJA-responsiveness
G-box	CACGTC	2	响应光信号 Involved in the light responsiveness
GT1-motif	GGTTAA	1	响应光信号 Involved in the light responsiveness
GT1-motif	GGTTAAT	1	响应光信号 Involved in the light responsiveness
MBS	CAACTG	1	响应干旱信号 Involved in the drought responsiveness
MYB	TAACCA	4	响应干旱信号 Involved in the drought responsiveness
TGACG-motif	TGACG	1	响应茉莉酸甲酯信号 Involved in the MeJA-responsiveness

图2 CaPUB18基因在辣椒不同组织中（A）和热胁迫下（B）的表达特性分析不同小写字母表示在P < 0.05水平下的差异显著性（n = 3）。下同

Fig. 2 Expression characteristics of CaPUB18 gene in various tissues（A）and under heat stress（B）in pepper Different lowercase letters mean the significant difference at P < 0.05 level（n = 3）. The same below

图3 CaPUB18的亚细胞定位 A：CaPUB18亚细胞定位；B：CaPUB18在常温和高温下与细胞膜marker PIP2A共定位；C：CaPUB18在高温下与PIP2A共定位的量化分析。

Fig. 3 Subcellular localization of CaPUB18 A：Subcellular localization of CaPUB18；B：Colocalization of CaPUB18 with PIP2A，the marker protein of plasma membrane，under normal and high temperature respectively；C：Quantification of colocalization of CaPUB18with PIP2A under high temperatures

图4 辣椒CaPUB18基因沉默植株创制 A：基因沉默辣椒植株表型；B：CaPUB18基因沉默效率检测，*** P < 0.001。TRV2:CaPUB18：CaPUB18沉默植株；TRV2:CaPDS：阳性对照；TRV2:00：阴性对照。下同

Fig. 4 Generation of CaPUB18-silenced pepper plants A：Phenotype of gene-silenced pepper plants；B：Test of silencing efficiency of CaPUB18，*** P < 0.001. TRV2:CaPUB18：CaPUB18-silenced plants；TRV2:CaPDS：Positive control；TRV2:00：Negative control. The same below

图5 CaPUB18基因沉默对辣椒耐热性的影响 A：热胁迫处理前后辣椒植株表型；B ~ E：热胁迫处理前后辣椒叶片相对电导率、叶片丙二醛（MDA）含量、H2O2积累和死细胞积累；F ~ G：热胁迫处理前后辣椒离体叶圆片总叶绿素含量；H、I：热胁迫处理前后辣椒叶片耐热标记基因CaHSP70.1和CaHsfA2的相对表达量

Fig. 5 Effect of CaPUB18-silenced on heat tolerance of pepper A：Phenotypes of pepper plants before and after heat-stress treatment；B-E：Relative electrical conductivity，malondialdehyde content，H2O2 accumulation and dead cells accumulation in pepper leaves before and after heat-stress treatment；F-G：Content of total chlorophyll in pepper detached leaf-discs before and after heat-stress treatment；H，I：Relative expression of heat-tolerance marker genes CaHSP70.1 and CaHsfA2 in pepper leaves before and after heat-stress treatment

图6 CaPUB18过表达拟南芥株系鉴定和幼苗存活率 A、B：拟南芥CaPUB18-OE株系的半定量PCR鉴定和定量PCR鉴定；C、D：热胁迫下（45 ℃处理50 min/22 ℃恢复3 d）5 d苗龄拟南芥幼苗的表型和存活率

Fig. 6 Identification and seedling survival rate of Arabidopsis thaliana lines CaPUB18-OE A and B：Identification of Arabidopsis CaPUB18-OE lines with semi-RT-PCR and qRT-PCR，respectively；C and D：Phenotypes and survival rate of 5-day-old Arabidopsis seedlings after heat-stress treatment with 45 ℃ for 50 min and then recovery with 22 ℃ for 3 d

图7 CaPUB18过表达拟南芥表型、相对电导率和MDA含量 A：热胁迫处理前后21 d苗龄拟南芥植株表型；B、C：热胁迫处理前后拟南芥叶片相对电导率、叶片丙二醛（MDA）含量。Col-0：拟南芥野生型；OE-2、OE-3：拟南芥CaPUB18过表达株系。

Fig. 7 Phenotypes，relative electrical conductivity and MDA of Arabidopsis thaliana lines CaPUB18-OE A：Phenotypes of 21-day-old Arabidopsis plants after heat-stress treatment；B and C：Relative electrical conductivity，malondialdehyde content in Arabidopsis leaves before and after heat-stress treatment； Col-0：Arabidopsis wild type；OE-2，and OE-3：Arabidopsis CaPUB18-overexpression lines in Col-0

图8 CaPUB18过表达对拟南芥耐热性的影响 A、D：热胁迫处理前后拟南芥离体叶圆片总叶绿素含量；B、E：热胁迫处理前后拟南芥叶片H2O2（DAB 染色）及平均灰度；C、F：热胁迫处理前后拟南芥叶片死细胞（台盼蓝染色）积累及平均灰度。Col-0：拟南芥野生型；OE-1、OE-2、OE-3：拟南芥CaPUB18过表达株系

Fig. 8 Effects of CaPUB18-overexpression on heat tolerance of Arabidopsis A and D：Content of total chlorophyll in Arabidopsis detached leaf-discs before and after heat-stress treatment；B and E：Accumulation of H2O2（DAB staining）and mean gray values of 21-day-old Arabidopsis plants after heat-stress treatment；C and F：Dead cells（Trypan blue staining）and mean gray values of 21-day-old Arabidopsis plants after heat-stress treatment. Col-0：Arabidopsis wild type；OE-1，OE-2，and OE-3：Arabidopsis CaPUB18-overexpression lines in Col-0

参考文献 40

[1]	Aleem S, Sharif I, Amin E, Tahir M, Parveen N, Aslam R, Najeebullah M,Hussain Shahid M T. 2020. Heat tolerance in vegetables in the current genomic era:an overview. Plant Growth Regulation, 92 (3):497-516. doi: 10.1007/s10725-020-00658-5
[2]	Ali S, Rizwan M, Arif M S, Ahmad R, Hasanuzzaman M, Ali B, Hussain A. 2020. Approaches in enhancing thermotolerance in plants:an updated review. Journal of Plant Growth Regulation, 39 (1):456-480. doi: 10.1007/s00344-019-09994-x
[3]	Al-Saharin R, Hellmann H, Mooney S. 2022. Plant E 3 ligases and their role in abiotic stress response. Cells, 11 (5):890. doi: 10.3390/cells11050890 URL
[4]	Bae Y, Cho J, Choi J, Lim C W, Lee S C. 2024. Pepper U-Box E 3 ubiquitin ligase 24,CaPUB24,negatively regulates drought stress response. Physiologia Plantarum, 176 (2):e14240. doi: 10.1111/ppl.v176.2 URL
[5]	Bae Y, Lim C W, Lee S C. 2023. Pepper stress-associated protein 14 is a substrate of CaSnRK2.6 that positively modulates abscisic acid-dependent osmotic stress responses. The Plant Journal, 113 (2):357-374. doi: 10.1111/tpj.v113.2 URL
[6]	Bulgari R, Franzoni G, Ferrante A. 2019. Biostimulants application in horticultural crops under abiotic stress conditions. Agronomy, 9 (6):306. doi: 10.3390/agronomy9060306 URL
[7]	Cho S K, Chung H S, Ryu M Y, Park M J, Lee M M, Bahk Y Y, Kim J, Pai H S, Kim W T. 2006. Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-Box E 3 ubiquitin ligase homolog. Plant Physiology, 142 (4):1664-1682. doi: 10.1104/pp.106.087965 URL
[8]	Clough S J, Bent A F. 1998. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal, 16 (6):735-743. doi: 10.1046/j.1365-313x.1998.00343.x pmid: 10069079
[9]	Costa G, Nascimento D, Silva B, Lopes Â C D A, Carvalho L C B, Gomes R L F. 2019. Selection of pepper accessions with ornamental potential. Revista Caatinga, 32 (2):566-574. doi: 10.1590/1983-21252019v32n230rc
[10]	Cyr D M, Hohfeld J, Patterson C. 2002. Protein quality control: U-box-containing E 3 ubiquitin ligases join the fold. Trends in Biochemical Sciences, 27 (7):368-375. doi: 10.1016/S0968-0004(02)02125-4 URL
[11]	Deng Shuqin, Gao Yingrui, Li Yutong, Wang Ying, Gong Chunmei, Bai Juan. 2025. Response of ubiquitin-ligase gene CsPUB21 to different abiotic stress in Camellia sinensis. Acta Horticulturae Sinica, 52 (3):655-670. (in Chinese) doi: 10.16420/j.issn.0513-353x.2024-0378
	邓淑琴, 高莹瑞, 李雨桐, 王瑛, 龚春梅, 白娟. 2025. 茶树泛素连接酶基因CsPUB21对非生物胁迫的响应. 园艺学报, 52 (3):655-670. doi: 10.16420/j.issn.0513-353x.2024-0378
[12]	Farooq M S, Uzair M, Raza A, Habib M, Xu Y L, Yousuf M, Yang S H, Khan M R. 2022. Uncovering the research gaps to alleviate the negative impacts of climate change on food security:a review. Frontiers in Plant Science,13:927535.
[13]	Fragkostefanakis S, Röeth S, Schleiff E, Scharf K D. 2015. Prospects of engineering thermotolerance in crops through modulation of heat stress transcription factor and heat shock protein networks. Plant,Cell & Environment, 38 (9):1881-1895. doi: 10.1111/pce.2015.38.issue-9 URL
[14]	Giordano M, Petropoulos S A, Rouphael Y. 2021. Response and defence mechanisms of vegetable crops against drought,heat and salinity stress. Agriculture, 11 (5):463. doi: 10.3390/agriculture11050463 URL
[15]	Guo M, Zhai Y F, Lu J P, Chai L, Chai W G, Gong Z H, Lu M H. 2014. Characterization of CaHsp70-1,a pepper heat-shock protein gene in response to heat stress and some regulation exogenous substances in Capsicum annuum L. International Journal of Molecular Sciences, 15 (11):19741-19759. doi: 10.3390/ijms151119741 URL
[16]	Haider S, Raza A, Iqbal J, Shaukat M, Mahmood T. 2022. Analyzing the regulatory role of heat shock transcription factors in plant heat stress tolerance:a brief appraisal. Molecular Biology Reports, 49 (6):5771-5785. doi: 10.1007/s11033-022-07190-x
[17]	Harris C J, Slootweg E J, Goverse A, Baulcombe D C. 2013. Stepwise artificial evolution of a plant disease resistance gene. Proceedings of the National Academy of Sciences, 110 (52):21189-21194.
[18]	Hassan M U, Chattha M U, Khan I, Chattha M B, Barbanti L, Aamer M, Aslam M T. 2021. Heat stress in cultivated plants:nature,impact,mechanisms,and mitigation strategies-a review. Plant Biosystems, 155 (2):211-234. doi: 10.1080/11263504.2020.1727987 URL
[19]	Koegl M, Hoppe T, Schlenker S, Ulrich H D, Mayer T U, Jentsch S. 1999. A novel ubiquitination factor,E4,is involved in multiubiquitin chain assembly. Cell, 96 (5):635-644. doi: 10.1016/s0092-8674(00)80574-7 pmid: 10089879
[20]	Kumar L S, Manisha S, Barkha R, Soma G, Sonam P, Nidhi S, Pandey G K. 2023. Overexpression of ARM repeat/U-box containing E 3 ligase,PUB 2 positively regulates growth and oxidative stress response in Arabidopsis. The Biochemical Journal, 480 (9):555-571.
[21]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT Method. Methods, 25 (4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[22]	Li Z P, Li S Q, Jin D J, Yang Y P, Pu Z Y, Han X, Hu Y R, Jiang Y J. 2023. U-box E 3 ubiquitin ligase PUB8 Attenuates abscisic acid responses during early seedling growth. Plant Physiology, 191 (4):2519-2533. doi: 10.1093/plphys/kiad044 URL
[23]	Lu J P, Guo M, Zhai Y F, Gong Z H, Lu M H. 2017. Differential responses to the combined stress of heat and Phytophthora capsici infection between resistant and susceptible germplasms of pepper(Capsicum annuum L.). Journal of Plant Growth Regulation, 36 (1):161-173. doi: 10.1007/s00344-016-9627-9 URL
[24]	Luo D, Hou X, Zhang Y, Meng Y, Zhang H, Liu S, Wang X, Chen R. 2019. CaDHN5,a dehydrin gene from pepper,plays an important role in salt and osmotic stress responses. International Journal of Molecular Sciences, 20 (8):1989. doi: 10.3390/ijms20081989 URL
[25]	Luo Y, Xie Y Y, Li W Q, Wei M H, Dai T, Li Z, Wang B Z. 2021. Physiological and transcriptomic analyses reveal exogenous trehalose is involved in the responses of wheat roots to high temperature stress. Plants, 10 (12):2644. doi: 10.3390/plants10122644 URL
[26]	Mao X G, Yu C M, Li L, Wang M, Yang L, Zhang Y, Zhang Y F, Wang J Y, Li C N, Reynolds M P, Jing R. 2022. How many faces does the plant u-box E 3 ligase have? International Journal of Molecular Sciences, 23 (4):2285. doi: 10.3390/ijms23042285 URL
[27]	Medina E, Kim S H, Yun M, Choi W G. 2021. Recapitulation of the function and role of ROS generated in response to heat stress in plants. Plants, 10 (2):371. doi: 10.3390/plants10020371 URL
[28]	Min H J, Jung Y J, Kang B G,Kim Woo Taek. 2016. CaPUB1,a hot pepper u-box E 3 ubiquitin ligase,confers enhanced cold stress tolerance and decreased drought stress tolerance in transgenic rice(Oryza sativa L.). Molecules and Cells, 39 (3):250-257. doi: 10.14348/molcells.2016.2290 URL
[29]	Oyebamiji Y O, Shamsudin N A A, Ikmal A M, Rafii M. 2023. Heat stress in vegetables:impacts and management strategies-a review. Sains Malaysiana, 52 (7):1925-1938. doi: 10.17576/jsm URL
[30]	Peng L, Wan X, Huang K, Pei L S, Xiong J, Li X Y, Wang J M. 2019. AtPUB 48 E3 ligase plays a crucial role in the thermotolerance of Arabidopsis. Biochemical and Biophysical Research Communications, 509 (1):281-286. doi: S0006-291X(18)32776-1 pmid: 30591216
[31]	Raza A. 2022. Metabolomics:a systems biology approach for enhancing heat stress tolerance in plants. Plant Cell Reports, 41 (3):741-763. doi: 10.1007/s00299-020-02635-8
[32]	Rykaczewska K. 2015. The effect of high temperature occurring in subsequent stages of plant development on potato yield and tuber physiological defects. American Journal of Potato Research, 92 (3):339-349. doi: 10.1007/s12230-015-9436-x URL
[33]	Tang B, Li X, Zhang X, Yin Q, Xie L, Zou X, Liu F, Dai X. 2022. Transcriptome data reveal gene clusters and key genes in pepper response to heat shock. Frontiers in Plant Science,13:946475.
[34]	Vierstra R D. 2009. The ubiquitin-26S proteasome system at the nexus of plant biology. Nature Reviews Molecular Cell Biology, 10 (6):385-397. doi: 10.1038/nrm2688 pmid: 19424292
[35]	Wang H, Niu H H, Liang M M, Zhai Y F, Huang W, Ding Q. 2019. A wall-associated kinase gene CaWAKL20 from pepper negatively modulates plant thermotolerance by reducing the expression of ABA-responsive genes. Frontiers in Plant Science,10:591.
[36]	Yan Bentao, Jiao Kexin, Zhao Yue, Yang Qiaomin, Chen Tao. 2023. Analysis of heat-tolerant function of pepper vesicle formation related gene CaSec16. Acta Horticulturae Sinica, 50 (11):2387-2400. (in Chinese) doi: 10.16420/j.issn.0513-353x.2022-0996
	阎本涛, 焦可心, 赵悦, 杨巧敏, 陈涛. 2023. 辣椒囊泡形成相关基因CaSec16耐热功能分析. 园艺学报, 50 (11):2387-2400. doi: 10.16420/j.issn.0513-353x.2022-0996
[37]	Zhai Y F, Wang H, Liang M M, Lu M H. 2017. Both silencing-and over-expression of pepper CaATG8c gene compromise plant tolerance to heat and salt stress. Environmental and Experimental Botany,141:10-18.
[38]	Zhou D D, Wang X H, Wang X F, Mao T L. 2023. PHYTOCHROME INTERACTING FACTOR 4 regulates microtubule organization to mediate high temperature-induced hypocotyl elongation in Arabidopsis. The Plant Cell, 35 (6):2044-2061. doi: 10.1093/plcell/koad042 URL
[39]	Zou Xuexiao, Yang Sha, Zhu Fan, Yuan Fang. 2024. Progress and trends in development of pepper industry:high quality,good taste of fresh pepper. Acta Horticulturae Sinica, 51 (1):27-38. (in Chinese) doi: 10.16420/j.issn.0513-353x.2023-0352
	邹学校, 杨莎, 朱凡, 远方. 2024. 中国高口感品质鲜食辣椒产业发展与未来趋势. 园艺学报, 51 (1):27-38. doi: 10.16420/j.issn.0513-353x.2023-0352
[40]	Zou Xuexiao, Zhu Fan. 2022. Origin,evolution and cultivation history of the pepper. Acta Horticulturae Sinica, 49 (6):1371-1381. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0853
	邹学校, 朱凡. 2022. 辣椒的起源、进化与栽培历史. 园艺学报, 49 (6):1371-1381. doi: 10.16420/j.issn.0513-353x.2021-0853