Effects of Ethylene Signal Treansduction Factor CmEIL01 Gene on Volatile Biosynthesis of Melon Fruit

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

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (4): 1202-1214.doi: 10.16420/j.issn.0513-353x.2025-0057

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Previous Articles Next Articles

Effects of Ethylene Signal Treansduction Factor CmEIL01 Gene on Volatile Biosynthesis of Melon Fruit

GUO Xinqi, CHEN Qiang, ZHANG Ying, ZHANG Chong^*()

College of Life Science，Shenyang Normal University，Shenyang 110000，China

Received:2025-12-04 Revised:2026-02-12 Online:2026-04-25 Published:2026-04-20
Contact: ZHANG Chong

Abstract

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

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.

Figures/Tables 6

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

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

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	_

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

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

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

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Effects of Ethylene Signal Treansduction Factor CmEIL01 Gene on Volatile Biosynthesis of Melon Fruit

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