基于乙烯响应筛选调控番茄成熟且影响呼吸的基因及其功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2026-0003

摘要/Abstract

摘要：

目的筛选番茄果实中响应乙烯、调控成熟且与呼吸代谢相关的关键因子，揭示其对果实成熟的调控作用，为解析乙烯介导的果实成熟分子网络提供理论依据。方法以番茄栽培种为材料，通过乙烯/1-MCP处理绿熟期果实鉴定乙烯响应基因；对果实成熟及呼吸跃变过程5个关键阶段果实进行转录组测序，筛选表达趋势与呼吸峰同步的正相关基因集；整合2组数据后经GO富集分析筛选候选基因，采用VIGS技术进行功能验证。结果鉴定出1 025个乙烯响应基因，与2 356个呼吸跃变正相关基因取交集获得220个核心基因，这些基因显著富集于呼吸代谢通路；筛选出AOX1a、MPC1、NDB2、PCO2、HIGD3 五个候选基因，VIGS沉默后均导致果实成熟障碍。结论 AOX1a、MPC1、NDB2、PCO2、HIGD3 是响应乙烯信号的关键因子，其核心功能为调控番茄果实成熟，且与呼吸代谢相关，可能间接影响果实呼吸相关生理过程。

关键词: 番茄, 果实成熟, 呼吸跃变, 乙烯, 转录组, VIGS

Abstract:

Objective This study is aimed to screen key factors responding to ethylene, regulating fruit ripening and associated with respiratory metabolism in tomato (Solanum lycopersicum) fruits. Their regulatory roles were clarified, which may provide a theoretical basis for deciphering the ethylene-mediated molecular network of tomato fruit ripening. Method Tomato cultivars were used as materials. Ethylene-responsive genes were identified by treating mature green fruits with ethylene/1-methylcyclopropene (1-MCP). Transcriptome sequencing was performed on the samples at five key stages of fruit ripening and respiratory climacteric. It was used to screen gene set with expression trends positively correlated with the respiratory peak. After integrating the two datasets, candidate genes were selected via Gene Ontology (GO) enrichment analysis and virus-induced gene silencing (VIGS) technology was used for functional verification. Result A total of 1 025 ethylene-responsive genes were identified. The intersection with 2 356 genes positively correlated with respiratory climacteric yielded 220 core genes, which were significantly enriched in respiratory metabolism pathways. Five candidate genes AOX1a, MPC1, NDB2, PCO2, and HIGD3 were selected. The VIGS silencing of each gene led to fruit ripening defects. Conclusion AOX1a, MPC1, NDB2, PCO2, and HIGD3 are key factors responding to ethylene signals, whose core function is to regulate tomato fruit ripening. They are associated with respiratory metabolism and may indirectly affect fruit respiration-related physiological processes.

Key words: tomato, fruit ripening, climacteric respiration, ethylene, transcriptome, VIGS

王潇奕, 李金焱, 邢醒, 朱鸿亮. 基于乙烯响应筛选调控番茄成熟且影响呼吸的基因及其功能分析[J]. 生物技术通报, 2026, 42(3): 275-282.

WANG Xiao-yi, LI Jin-yan, XING Xing, ZHU Hong-liang. Screening and Functional Analysis of Ethylene-responsive Genes Regulating Tomato Fruit Ripening and Respiration[J]. Biotechnology Bulletin, 2026, 42(3): 275-282.

图/表 5

图1 番茄果实呼吸速率及乙烯释放量动态变化A：不同时期番茄果实呼吸速率及乙烯释放量图；B：番茄果实成熟进程图；MG代表绿熟，Br代表破色

Fig. 1 Dynamic changes of respiration rate and ethylene release amount in tomato fruitsA: Respiration rate and ethylene release of tomato fruit in different periods. B: Tomato fruit ripening process diagram.MG means mature green, Br means breaker

图2 乙烯及1-MCP处理差异表达基因韦恩图

Fig. 2 Venn diagram of differentially expressed genes treated with ethylene and 1-MCP

图3 番茄果实转录组样本相关性分析及差异表达基因聚类分析A：样本相关性热图；B：差异表达基因聚类图

Fig. 3 Transcriptome sample correlation analysis and differentially expressed gene clustering analysis of tomato (S. lycopersicum cultivar Ailsa Craig) fruitA: Sample correlation heat map. B: Cluster diagram of differentially expressed genes

图4 调控番茄果实成熟且影响果实呼吸的乙烯响应关键基因筛选A：乙烯响应基因与呼吸跃变正相关基因交集韦恩图；B：交集基因GO富集分析；C：交集基因KEGG富集分析；D：候选基因在果实成熟各阶段的表达趋势

Fig. 4 Screening of key ethylene-responsive genes regulating tomato fruit ripening and affecting fruit respirationA: Venn diagram of the intersection of ethylene-responsive genes and respiratory jump positive correlation genes. B: GO enrichment analysis of intersection genes. C: KEGG enrichment analysis of intersection genes. D: The expression trend of candidate genes in different stages of fruit ripening

图5 候选基因VIGS沉默表型及表达量验证A：VIGS处理后果实表型（Bar=1 cm）；B：候选基因在果实红黄区域的相对表达量（不同小写字母表示P<0.05水平显著差异）

Fig. 5 VIGS silencing phenotype and expression verification of candidate genesA: Fruit phenotype after VIGS treatment (Bar = 1 cm) . B: The relative expression of candidate genes in the red and yellow regions of fruit (different lowercase letters indicate a significant difference at P<0.05 level)

参考文献 33

[1]	Alexander L, Grierson D. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening [J]. J Exp Bot, 2002, 53(377): 2039-2055.
[2]	Giovannoni JJ. Genetic regulation of fruit development and ripening [J]. Plant Cell, 2004, 16(): S170-S180.
[3]	Bapat VA, Trivedi PK, Ghosh A, et al. Ripening of fleshy fruit: Molecular insight and the role of ethylene [J]. Biotechnol Adv, 2010, 28(1): 94-107.
[4]	Karakas E, Fernie AR. What metabolomics has taught us about tomato fruit ripening and quality [J]. J Exp Bot, 2025, 76(21): 6245-6258.
[5]	Osorio S, Alba R, Damasceno CMB, et al. Systems biology of tomato fruit development: combined transcript, protein, and metabolite analysis of tomato transcription factor (nor, rin) and ethylene receptor (Nr) mutants reveals novel regulatory interactions [J]. Plant Physiol, 2011, 157(1): 405-425.
[6]	Wu SB, Wu D, Song J, et al. Metabolomic and transcriptomic analyses reveal new insights into the role of abscisic acid in modulating mango fruit ripening [J]. Hortic Res, 2022, 9: uhac102.
[7]	Yang YF, Liu XY, Wang KR, et al. Molecular and functional diversity of organelle RNA editing mediated by RNA recognition motif-containing protein ORRM4 in tomato [J]. New Phytol, 2020, 228(2): 570-585.
[8]	Xu F, Yuan S, Zhang DW, et al. The role of alternative oxidase in tomato fruit ripening and its regulatory interaction with ethylene [J]. J Exp Bot, 2012, 63(15): 5705-5716.
[9]	Iglesias-Sanchez A, Del-Saz NF, Ezquerro M, et al. Activation of alternative oxidase ensures carbon supply for ethylene and carotenoid biosynthesis during tomato fruit ripening [J]. Plant Physiol, 2025, 199(3): kiaf516.
[10]	De Paepe A, Van Der Straeten D. Ethylene biosynthesis and signaling: An overview [J]. Vitam Horm, 2005, 72: 399-430.
[11]	Yadav P, Ansari MW, Kaula BC, et al. Regulation of ethylene metabolism in tomato under salinity stress involving linkages with important physiological signaling pathways [J]. Plant Sci, 2023, 334: 111736.
[12]	Lanahan MB, Yen HC, Giovannoni JJ, et al. The never ripe mutation blocks ethylene perception in tomato [J]. Plant Cell, 1994, 6(4): 521-530.
[13]	Li S, Zhu BZ, Pirrello J, et al. Roles of RIN and ethylene in tomato fruit ripening and ripening-associated traits [J]. New Phytol, 2020, 226(2): 460-475.
[14]	Wang XY, Wen HY, Suprun A, et al. Ethylene signaling in regulating plant growth, development, and stress responses [J]. Plants, 2025, 14(3): 309.
[15]	Wang RF, Lammers M, Tikunov Y, et al. The rin, nor and Cnr spontaneous mutations inhibit tomato fruit ripening in additive and epistatic manners [J]. Plant Sci, 2020, 294: 110436.
[16]	Fu DQ, Zhu BZ, Zhu HL, et al. Virus-induced gene silencing in tomato fruit [J]. Plant J, 2005, 43(2): 299-308.
[17]	Brumos J. Gene regulation in climacteric fruit ripening [J]. Curr Opin Plant Biol, 2021, 63: 102042.
[18]	Ming YC, Jiang LB, Ji DC. Epigenetic regulation in tomato fruit ripening [J]. Front Plant Sci, 2023, 14: 1269090.
[19]	Kou XH, Zhou JQ, Wu CE, et al. The interplay between ABA/ethylene and NAC TFs in tomato fruit ripening: a review [J]. Plant Mol Biol, 2021, 106(3): 223-238.
[20]	Huang W, Hu N, Xiao ZN, et al. A molecular framework of ethylene-mediated fruit growth and ripening processes in tomato [J]. Plant Cell, 2022, 34(9): 3280-3300.
[21]	Karlova R, Chapman N, David K, et al. Transcriptional control of fleshy fruit development and ripening [J]. J Exp Bot, 2014, 65(16): 4527-4541.
[22]	Vanlerberghe GC, Martyn GD, Dahal K. Alternative oxidase: a respiratory electron transport chain pathway essential for maintaining photosynthetic performance during drought stress [J]. Physiol Plant, 2016, 157(3): 322-337.
[23]	Weaver RJ, McDonald AE. Mitochondrial alternative oxidase across the tree of life: Presence, absence, and putative cases of lateral gene transfer [J]. Biochim Biophys Acta Bioenerg, 2023, 1864(4): 149003.
[24]	Møller IM, Rasmusson AG, Siedow JJN, et al. The product of the alternative oxidase is still H₂O [J]. Arch Biochem Biophys, 2010, 495(1): 93-94.
[25]	He CM, Hartmann A, Li MX, et al. Functional characterisation of alternative oxidase protein isoproteins in Arabidopsis thaliana [J]. Physiol Plant, 2025, 177(5): e70468.
[26]	Yuan C, He LL, Chen DH, et al. TSJT1 and glutamate is required for aluminum tolerance associated with mitochondrial pyruvate carrier 1 in Arabidopsis [J]. Plant Signal Behav, 2025, 20: 2526765.
[27]	Schell JC, Olson KA, Jiang L, et al. A role for the mitochondrial pyruvate carrier as a repressor of the Warburg effect and colon cancer cell growth [J]. Mol Cell, 2014, 56(3): 400-413.
[28]	Sweetman C, Waterman CD, Wong DCJ, et al. Altering the balance between AOX1A and NDB2 expression affects a common set of transcripts in Arabidopsis [J]. Front Plant Sci, 2022, 13: 876843.
[29]	Wanniarachchi V, Dametto L, Sweetman C, et al. Alternative respiratory pathway component genes (AOX and ND) in rice and barley and their response to stress [J]. Int J Mol Sci, 2018, 19(3): 915.
[30]	Weits DA, Giuntoli B, Kosmacz M, et al. Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway [J]. Nat Commun, 2014, 5: 3425.
[31]	White MD, Kamps JJAG, East S, et al. The plant cysteine oxidases from Arabidopsis thaliana are kinetically tailored to act as oxygen sensors [J]. J Biol Chem, 2018, 293(30): 11786-11795.
[32]	Vercellino I, Sazanov LA. The assembly, regulation and function of the mitochondrial respiratory chain [J]. Nat Rev Mol Cell Biol, 2022, 23(2): 141-161.
[33]	Fernando W, Rupasinghe HP, Hoskin D. Regulation of hypoxia-inducible factor-1α and vascular endothelial growth factor signaling by plant flavonoids [J]. Mini Rev Med Chem, 2015, 15(6): 479-489.