甜瓜乙烯信号因子基因CmEIL01对果实香气物质合成的影响

doi:10.16420/j.issn.0513-353x.2025-0057

摘要/Abstract

摘要：

EIN3-like（EIL）蛋白是乙烯信号转导途径中重要的核转录因子，可启动一系列调节乙烯目标基因表达的转录级联反应。通过GST pull-down联合液相色谱串联质谱（LC-MS/MS）技术在成熟甜瓜果实中筛选与CmEIL01互作的蛋白，并进一步验证CmEIL01与候选蛋白之间的互作关系。为明确乙烯信号因子基因CmEIL01在薄皮甜瓜果实香气合成中的调控功能，筛选出9个可能与CmEIL01互作的蛋白，其中果实酯类合成关键基因CmAAT1预测的可信值最高；酵母双杂交技术（Y2H）和Pull-down分析也验证了CmEIL01与CmAAT1蛋白在体外存在相互作用；烟草双荧光素酶报告试验表明，CmEIL01显著激活了CmAAT1基因的启动子活性。通过薄皮甜瓜果实的瞬时表达发现，注射6 d后，CmEIL01基因表达水平最高，同时CmEIL02和CmAAT1的表达水平也显著提高，乙酸乙酯、乙酸己酯和己醇的含量均显著高于未注射果实。结果表明，CmEIL01可能通过调控直链酯类物质合成关键基因CmAAT1的表达，进而参与薄皮甜瓜果实芳香物质的合成。

关键词: 甜瓜, 乙烯信号转导, 果实成熟, CmEIL01, GST pull-down, 瞬时过表达, 香气物质

Abstract:

EIN3-like（EIL）proteins are crucial nuclear transcription factors in the ethylene signaling pathway，initiating a series of transcriptional cascades that regulate the expression of ethylene target genes. Using GST pull-down combined with liquid chromatography-tandem mass spectrometry（LC-MS/MS）technology，proteins interacting with CmEIL01 were screened in mature melon fruits，and the interaction between CmEIL01 and candidate proteins was further validated. To elucidate the regulatory function of the ethylene signaling factor gene CmEIL01 in the biosynthesis of aroma compounds in oriental melon，nine proteins potentially interacting with CmEIL01 were identified，among which the key gene for fruit ester synthesis，CmAAT1，had the highest predicted confidence value. Yeast two-hybrid（Y2H）and Pull-down analyses also confirmed the interaction between CmEIL01 and CmAAT1 proteins in vitro. Tobacco dual-luciferase reporter assays showed that CmEIL01 significantly activated the promoter activity of the CmAAT1 gene. Transient expression in oriental melon melon fruits revealed that the expression level of CmEIL01 peaked on the sixth day after injection，significantly increasing the expression levels of the homologous gene CmEIL02 and CmAAT1，leading to a notable rise in ethyl acetate content and the contents of hexyl acetate and hexanol were also significantly higher than those in the non-injected fruits. The results suggest that CmEIL01 may participate in the synthesis of aromatic compounds in oriental melon fruits by regulating the expression of the key gene CmAAT1 involved in straight-chain ester synthesis.

Key words: melon, ethylene signal transduction, fruit ripening, CmEIL01, GST pull-down, transient overexpression, volatile synthesis

郭欣琪, 陈强, 张颖, 张冲. 甜瓜乙烯信号因子基因CmEIL01对果实香气物质合成的影响[J]. 园艺学报, 2026, 53(4): 1202-1214.

GUO Xinqi, CHEN Qiang, ZHANG Ying, ZHANG Chong. Effects of Ethylene Signal Treansduction Factor CmEIL01 Gene on Volatile Biosynthesis of Melon Fruit[J]. Acta Horticulturae Sinica, 2026, 53(4): 1202-1214.

图/表 6

表1 用于基因表达分析的qRT-PCR引物序列

Table 1 Primer sequences for qRT-PCR analysis of gene expression

基因名称 Gene name	序号 Accession number	片段大小/bp Size of amplified	引物序列（5′-3′） Primer sequence
CmEIL01	MELO3C015210	188	F：GAACACTGGCTCCCTCTCAG R：AGCCGGTACTTCTGACTGGA
CmEIL02	MELO3C017592	214	F：GCATTTGCAGAGGAGAGAGG R：GGACTCGCATTCAGATCCAT
CmEIL03	MELO3C019931	170	F：GCACCCTGATTTTTCTTCCA R：CTTGTTTGGGCAATTGTGTG
CmEIL04	MELO3C015633	205	F：GTTCCAGAAGATGGCCAAAA R：TCGAGCCATCGAAGAAGTTT
CmAAT1	MELO3C024771	134	F：AAAACGTGGACTTTGGATGG R：CAACGAGGCATGAACATTTG

图1 CmEIL01重组蛋白的表达、纯化及互作蛋白的GST Pull-down鉴定 A：重组CmEIL01蛋白表达的SDS-PAGE检测；B：纯化后的重组CmEIL01蛋白的SDS-PAGE检测；C：重组CmEIL01蛋白的Western Blot检测；D：GST pull-down实验的SDS-PAGE结果。P1和P2为仅在CmEIL01-GST处理组中被特异性富集的互作蛋白条带；P3为GST标签蛋白或CmEIL01-GST自身条带，作为上样对照

Fig. 1 Expression，purification and identification of interacting proteins of CmEIL01 recombinant protein by GST Pull-down assay A：SDS-PAGE analysis of recombinant CmEIL01 protein expression；B：SDS-PAGE analysis of purified recombinant CmEIL01 protein；C：Western blot analysis of recombinant CmEIL01 protein；D：SDS-PAGE results of GST Pull-down assay. P1 and P2 are specifically enriched interacting protein bands only in the CmEIL01-GST treatment group；P3 represents the GST-tagged protein or the CmEIL01-GST band itself，serving as a loading control

表2 通过GST pull-down鉴定的CmEIL01结合蛋白及其启动子顺式元件

Table 2 CmEIL01-binding proteins identified by GST pull-down and promoter cis-elements of CmAAT1

基因ID Gene ID	匹配肽段数 Number of peptide matches	功能预测 Predicted protein function	等电点 pI	分子量/kD Molecular weight	启动子ECBS区预测 ECBS prediction of protomers
MELO3C024771	21	醇酰基转移酶 Alcohol acyl-transferases	51.61	8.28	AAGAAACCT（+884） AAGTAACCT（+123）
MELO3C006322.2	15	谷胱甘肽还原酶 Glutathione reductase	53.90	5.96	AAGTAACT（+564）
MELO3C014437.2	13	1-氨基环丙烷-1-羧酸氧化酶 1-Aminocyclopropane-1-carboxylate oxidase	36.07	5.34	AAGTAACCT（+123） ACGTACCT（+564） AAGGACCT（+1235）
MELO3C011043.2	9	醇脱氢酶 Alcohol dehydrogenase	42.50	5.60	AAGTAAAT（+269）
MELO3C010017.2	7	聚半乳糖醛酸酶 Polygalacturonase	45.45	6.95	_
MELO3C009647.2	5	转录因子HBP-1b Transcription factor HBP-1b-like isoform	52.21	6.33	AAGAAACT（-456）
MELO3C003106.2	4	RAS相关蛋白 Ras-related protein RABE1a	23.92	7.65	_
MELO3C025976.2	2	热激蛋白 Heat shock protein 90-2	66.48	6.21	_
MELO3C012292.2	2	ATP合成酶 ATP synthase subunit beta	59.74	5.70	_

图2 CmEIL01与CmAAT1相互作用的体外、体内及转录激活验证 A：CmAAT1重组蛋白表达的SDS-PAGE检测；B：纯化后CmAAT1重组蛋白的SDS-PAGE检测；C：CmAAT1重组蛋白的Western blot检测；D：GST pull-down实验的Western blot结果；E：CmEIL01与CmAAT1的预测互作三维结构模型；F：酵母双杂交实验；G：双荧光素酶报告实验的相对荧光素酶活性。*表明P < 0.05水平存在显著差异，**表明在P < 0.01水平存在极显著差异。ns：不显著。下同

Fig. 2 Verification of the interaction between CmEIL01 and CmAAT1 in vitro，in vivo，and by transcriptional activation assay A：SDS-PAGE analysis of CmAAT1 recombinant protein expression；B：SDS-PAGE of purified CmAAT1 recombinant protein；C：Western Blot analysis of CmAAT1 recombinant protein；D：Western Blot verification of GST pull-down results；E：Predicted three-dimensional model of CmEIL01-CmAAT1 interaction；F：Yeast two-hybrid（Y2H）assay of CmEIL01and CmAAT1 interaction；G：Relative luciferase activity in dual-luciferase report assay. *indicates differences that are statistically significant on the P < 0.05 level；**indicates differences that are extremely statistically significant on the P < 0.01 level. ns：No significant. The same below

图3 甜瓜果实瞬时超表达CmEIL01的LUC荧光成像、EIL家族基因表达及乙烯释放 A：农杆菌注射后不同时间点的果实荧光成像；B：CmEIL01 ~ 04基因的相对表达水平；C：CmEIL01过表达果实与对照果实的内源乙烯浓度水平

Fig. 3 LUC fluorescence imaging of transient overexpression of CmEIL01 in melon fruit，and analysis of EIL family gene expression and ethylene release A：Fruit fluorescence imaging at different time points after Agrobacterium-mediated transient transformation；B：Relative expression levels of CmEIL01 to CmEIL04 genes；C：Endogenous ethylene levels in CmEIL01-overexpressing fruit versus control fruit

图4 甜瓜果实中瞬时超表达CmEIL01的CmAAT1基因表达以及主要酯类和C6醇类物质的含量

Fig. 4 CmAAT1 gene expression and content of major esters and C6 in melon fruit transiently overexpressing CmEIL01

参考文献 38

[1]	Alonso J M, Stepanova A N, Solano R, Wisman E, Ferrari S, Ausubel F M, Ecker J R. 2003. Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis. Proc Natl Acad Sci, 100 (5):2992-2997. doi: 10.1073/pnas.0438070100 URL
[2]	Chao Q M, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker J R. 1997. Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell, 89 (7):1133-1144. doi: 10.1016/s0092-8674(00)80300-1 pmid: 9215635
[3]	Chen C, Zhang M, Zhang M Y, Yang M M, Dai S S, Meng Q W, Lv W, Zhuang K Y. 2023. ETHYLENE-INSENSITIVE 3-LIKE 2 regulates β-carotene and ascorbic acid accumulation in tomatoes during ripening. Plant Physiology, 192 (3):2067-2080. doi: 10.1093/plphys/kiad151 pmid: 36891812
[4]	Chen G P, Alexander L, Grierson D. 2004. Constitutive expression of EIL like transcription factor partially restores ripening in the ethylene insensitive Nr tomato mutant. Journal of Experimental Botany, 55 (402):1491-1497. doi: 10.1093/jxb/erh168 URL
[5]	Deng L, Yang T X, Li Q, Chang Z Q, Sun C L, Jiang H L, Meng X W, Huang T T, Li C B, Zhong S L, Li C Y. 2023. Tomato MED 25 regulates fruit ripening by interacting with EIN3-like transcription factors. The Plant Cell, 35 (3):1038-1057. doi: 10.1093/plcell/koac349 URL
[6]	Dudareva N, Klempien A, Muhlemann J K, Kaplan I. 2013. Biosynthesis,function and metabolic engineering of plant volatile organic compounds. New Phytologist, 198 (1):16-32. doi: 10.1111/nph.12145 pmid: 23383981
[7]	El-Sharkawy I, Manríquez D, Flores F B, Regad F, Bouzayen M, Latché A, Pech J C. 2005. Functional characterization of a melon alcoholacyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity. Plant Molecular Biology, 59 (2):345-362. pmid: 16247561
[8]	Feng J, Wang M Y, Chen X Y, Tomes S, Tao J M, Atkinson R, Nieuwenhuizen N J. 2025. EIL(ethylene-insensitive 3-like)transcription factors in appleaffect both ethylene-and cold response-dependentfruit ripening. The Plant Journal, 121 (5):e70059. doi: 10.1111/tpj.v121.5 URL
[9]	Gao G, Yang F, Wang C, Duan X Y, Li M, Ma Y, Wang F, Qi H Y. 2023. The transcription factor CmERFI-2 represses CmMYB 44 expression to increase sucrose levels in oriental melon fruit. Plant Physiology, 192 (2):1378-1395. doi: 10.1093/plphys/kiad155 pmid: 36938625
[10]	Giribaldi M, Perugini I, Sauvage F X, Schubert A. 2007. Analysis of proteins changes during grape berry ripening by 2-DE and MALDI-TOF. Proteomics, 7 (7):3154-3170. doi: 10.1002/pmic.v7:17 URL
[11]	Huang J Y, Zhao X B, Bürger M, Wang Y R, Chory J. 2021. Two interacting ethylene response factors regulate heat stress response. The Plant Cell, 33 (2):338-357. doi: 10.1093/plcell/koaa026 URL
[12]	Huang S Z, Sawaki T, Takahashi A, Mizuno S J, Takezawa K, Matsumura A, Yokotsuka M, Hirasawa Y K, Sonoda M, Nakagawa H, Sato T. 2010. Melon EIN3-like transcription factors(CmEIL1 and CmEIL2)are positive regulators of an ethylene-and ripening-induced 1-amino-cyclopropane-1-carboxylic acid oxidase gene(CM-ACO1). Plant Sci,178:251-257.
[13]	Jin Y Z, Zang C, Liu W W, Tang Y F, Qi H Y, Chen H, Cao S X. 2016. The alcohol dehydrogenase gene family in melon(Cucumis melo l.):bioinformatic analysis and expression patterns. PLoS ONE, 9 (7):670.
[14]	Kosugi S, Ohashi Y. 2000. Cloning and DNA-binding properties of a tobacco Ethylene-Insensitive3(EIN3) homolog. Nucleic Acids Res,28:960-967.
[15]	Li Y, Qi H Y, Guan X C. 2011. Effects of ethephon and 1-methycyclopropene on fruit ripening and the biosynthesis of volatiles in oriental sweet melons(Cucumis melo var makuwa Makino). J. Hortic. Sci.Biotech,86:517-526.
[16]	Liu C, Ma T, Yuan D Y, Zhou Y, Long Y, Li Z W, Dong Z Y, Duan M J, Yu D, Jing Y Z, Bai X Y, Wang Y B, Hou Q C, Liu S S, Zhang J S, Chen S Y, Li D Y, Liu X, Li Z K, Wang W S, Li J P, Wei X, Ma B, Wan X Y. 2022. The OsEIL1-OsERF115-target gene regulatory module controls grain size and weight in rice. Plant Biotechnology Journal,20:1470-1486.
[17]	Liu S, Gao P, Zhu Q L, Zhu Z C, Liu H Y, Wang X Z, Weng Y Q, Gao M L, Luan F S. 2020. Resequencing of 297 melon accessions reveals the genomic history of improvement and loci related to fruit traits in melon. Plant Biotechnology Journal,18:2545-2558.
[18]	Liu W W, Qi H Y, Xu B H, Li Y, Tian X B, Jiang Y Y, Xu X F, Lv D Q. 2012. Ethanol treatment inhibits internal ethylene concentrations and enhances ethyl ester production during storage of oriental sweet melons(Cucumis melo var. makuwa Makino). Postharvest Biology and Technology,67:75-83.
[19]	Mao C Z, Wang S M, Jia Q J, Wu P. 2006. OsEIL1,a rice homolog of the Arabidopsis EIN3 regulates the ethylene response as a positive component. Plant Molecular Biology,61:141-152.
[20]	Merchante C, Alonso J M, Stepanova A N. 2013. Ethylene signaling:simple ligand,complex regulation. Curr Opin Plant Biol,16:554-560.
[21]	Nieuwenhuizen N J, Chen X Y, Wang M Y, Matich A J, Perez R L, Allan A C, Green S A, Atkinson R G. 2015. Natural variation in monoterpene synthesis in kiwifruit:transcriptional regulation of terpene synthases by NAC and ETHYLENE-INSENSITIVE3-Like transcription factors. Plant Physiol,167:1243-1258.
[22]	Qi Hongyan, Guan Xiaochuang, Li Jinran, Qiu Liyan, Hao Jing. 2011. Analysis of ripening characteristics,aroma components and aroma-related enzymes in oriental sweet melons(Cucumis melo var. makuwa Makino). Scientia Agricultura Sinica, 44 (4):771-780. (in Chinese)
	齐红岩, 李岩, 关小川, 李金燃, 邱丽妍, 郝晶. 2011. 两个不同类型薄皮甜瓜品种成熟特性、香气成分及其相关酶活性分析. 中国农业科学, 44 (4):771-780.
[23]	Solano R, Stepanova A, Chao Q M, Ecker J R. 1998. Nuclear events in ethylene signaling:a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes & Development,12:3703-3714.
[24]	Tang Lingli, He Yuhua, Wang Qingtao, An Lulu, Xu Yongyang, Zhang Jian, Kong Weihu, Hu Keyun, Zhao Guangwei. 2025. Hormonal and transcriptome analysis of ripe fruits of non-climacteric and climacteric melon. Acta Horticulturae Sinica, 52 (4):883-896. (in Chinese) doi: 10.16420/j.issn.0513-353x.2024-0371
	唐伶俐, 贺玉花, 王庆涛, 安璐璐, 徐永阳, 张健, 孔维虎, 户克云, 赵光伟. 2025. 非呼吸跃变和呼吸跃变型甜瓜成熟果实的激素与转录组分析. 园艺学报, 52 (4):883-896. doi: 10.16420/j.issn.0513-353x.2024-0371
[25]	Tieman D M, Ciardi J A, Taylor M G, Klee H J. 2001. Members of the tomato LeEIL(EIN3-like)gene family are functionally redundant and regulate ethylene responses throughout plant development. The Plant Journal,26:47-58.
[26]	Wang F F, Cui X K, Sun Y, Dong C H. 2013. Ethylene signaling and regulation in plant growth and stress responses. Plant Cell Report,32:1099-1109.
[27]	Xiao S, Yang D D, Li F J, Tian X L, Li Z H. 2024. The EIN3/EIL-ERF9-HAK 5 transcriptional cascade positively regulates high-affinity K⁺ uptake in Gossypium hirsutum. New Phytologist, 241 (5):2090-2107. doi: 10.1111/nph.v241.5 URL
[28]	Xi Wanpeng, Yu Songlin, Zhou Zhiqin. 2013. Advances in aroma compounds biosynthesis of peach fruit. Acta Horticulturae Sinica, 40 (9):1679-1690. (in Chinese)
	席万鹏, 郁松林, 周志钦. 2013. 桃果实香气物质生物合成研究进展. 园艺学报, 40 (9):1679-1690.
[29]	Xu Qian, Yin Xueren, Cen Kunsong. 2014. EIN3/EIL and AP2/ERF are involved in transcriptional regulation on fruit quality. Acta Horticulturae Sinica, 41 (9):1913-1923. (in Chinese)
	徐倩, 殷学仁, 陈昆松. 2014. 基于乙烯受体下游转录因子的果实品质调控机制研究进展. 园艺学报, 41 (9):1913-1923.
[30]	Yanagisawa S, Yoo S D, Sheen J. 2003. Differential regulation of EIN3 stability by glucose and ethylene signalling in plants. Nature,425:521-525.
[31]	Yang C, Lu X, Ma B, Chen S Y, Zhang J S. 2015. Ethylene signaling in rice and Arabidopsis:conserved and diverged aspects. Molecular Plant, 8 (4):495-505. doi: 10.1016/j.molp.2015.01.003 URL
[32]	Yin X R, Allan A C, Chen K S, Ferguson I B. 2010. Kiwifruit EIL and ERF genes involved in regulating fruit ripening. Plant Physiol, 153 (3):1280-1292. doi: 10.1104/pp.110.157081 URL
[33]	Yokotani N, Tamura S, Nakano R, Inaba A, Kubo Y. 2003. Characterization of a novel tomato EIN3-like gene(LeEIL4). Journal of Experimental Botany, 54 (393):2775-2776. doi: 10.1093/jxb/erg308 pmid: 14623944
[34]	Yuan Guibo, Mo Shuangrong, Qian Ying, Zhang Dongnan, Yang Fan, Jiang Hongliang, Wu Yuan, Ding Haidong. 2020. Screening of interacting protein of tomato SIVQ 6 by GST pull-down. Scientia Agricultura Sinica, 53 (15):3146-3157. (in Chinese)
	原贵波, 莫双榕, 钱莹, 臧栋楠, 杨帆, 蒋红亮, 武媛, 丁海东. 2020. 应用GST pull-down技术筛选番茄SIVQ6互作蛋白. 中国农业科学, 53 (15):3146-3157. doi: 10.3864/j.issn.0578-1752.2020.15.014
[35]	Zhang Qijing, Dong Wenxuan. 2018. Mechanisms of NAC transcription factor functions during fruit development and ripening. Acta Horticulturae Sinica, 45 (10):2052-2062. (in Chinese) doi: 10.16420/j.issn.0513-353x.2018-0340
	张琪静, 董文轩. 2018. NAC转录因子在果实发育成熟过程中的作用研究进展. 园艺学报, 45 (10):2052-2062.
[36]	Zhao G W, Lian Q, Zhang Z H, Fu Q S, He Y H, Ma S W, Ruggieri V, Monforte A J, Wang P Y, Julca I, Wang H S, Liu J P, Xu Y, Wang R Z, Ji J B, Xu Z H, KongW H, Zhong Y, Shang J L, Pereira L, Argyris J, Zhang J, Mayobre C, Pujol M, Huang S W. 2019. A comprehensive genome variation map of melon identifies multiple domestication events and loci influencing agronomic traits. Nature Genetics, 51 (11):1607-1615. doi: 10.1038/s41588-019-0522-8 pmid: 31676864
[37]	Zhao W L, Zheng Z H, Aweya J J, Wang F, Li S K, Tuan T N, Yao D, Zhang Y L. 2021. Litopenaeus vannamei Notch interacts with COP 9 signalosome complex subunit 1( CNS1)to negatively regulate the NF-kB pathway. Journal of Proteomics,232:104074.
[38]	Zhu L S, Chen L, Wu C J, Shan W, Cai D L, Lin Z X, Wei W, Chen J Y, Lu W J, Kuang J F. 2023. Methionine oxidation and reduction of the ethylene signaling component MaEIL 9 are involved in banana fruit ripening. Journal of Integrative Plant Biology, 65 (1):150-166. doi: 10.1111/jipb.v65.1 URL