枇杷日灼响应因子EjWRKY15互作蛋白的筛选及其调控网络解析

doi:10.3864/j.issn.0578-1752.2026.12.013

Abstract

Abstract:

【Objective】Loquat (Eriobotrya japonica), a significant economic fruit tree in southern China, is highly susceptible to sunburn injury during its ripening stage, which typically coincides with periods of high temperature and intense light. Sunburn manifests as fruit peel browning, tissue necrosis, and flavor deterioration, severely undermining both the marketability and economic value of the fruit. WRKY transcription factors, as plant-specific regulators of stress responses, play pivotal roles in abiotic stresses such as high temperature and drought. This study aims to analyze the function and regulatory mechanism of EjWRKY15 under sunburn stress, providing a theoretical foundation and candidate gene resources for elucidating the sunburn response pathways in loquat and developing sunburn-resistant germplasms.【Method】Centering on the transcription factor EjWRKY15, its interacting proteins were screened and validated to systematically characterize its expression profiles and molecular interaction networks under sunburn stress. A sunburn -induced yeast two-hybrid (Y2H) cDNA library of loquat fruit was constructed using SMART technology. The library capacity, titer, and recombination rate were evaluated through plating selection and sequence analysis. Using EjWRKY15 as the bait protein, potential interactors were screened from the library via the Y2H system. Functional annotation of the screened proteins was performed using NCBI, Swiss-Prot, and other databases, focusing on factors related to heat and stress responses. The physical interaction between EjWRKY15 and the candidate protein EjLHP1.2 (Like Heterochromatin Protein 1) was validated both in vitro and in vivo through one-on-one Y2H assays and bimolecular fluorescence complementation (BiFC). Based on transcriptome data and correlation analysis, a molecular interaction network for loquat sunburn response was constructed with EjWRKY15 as the core node.【Result】A high-quality cDNA library of loquat fruit with a large capacity and high recombination rate was successfully constructed. After excluding background interference via bait self-activation assays, 12 candidate proteins interacting with EjWRKY15 were obtained, with functions involving epigenetic regulation, stress signaling, and substance metabolism. Notably, EjLHP1.2, which is closely associated with sunburn response, was identified as a key interactor. One-on-one Y2H and BiFC assays confirmed that EjWRKY15 and EjLHP1.2 interact physically both in vivo and in vitro. Expression analysis revealed that EjWRKY15 and its 12 candidate interactor genes exhibited significant differential expression patterns under sunburn stress. Based on the interaction data and expression correlations, an EjWRKY15-mediated sunburn response network was preliminarily established. The network suggests that EjWRKY15 may precisely regulate downstream heat-responsive genes by recruiting EjLHP1.2 and other factors.【Conclusion】This study identified the interacting proteins of EjWRKY15 and revealed its expression patterns and molecular interaction network in the sunburn response of loquat. The results elucidate the molecular mechanism by which the core transcription factor EjWRKY15 mediates the sunburn response of loquat fruit through its protein interaction with the epigenetic regulator EjLHP1.2 and the precise modulation of the associated interaction network. These findings reveal a novel stress-regulatory pathway involving the synergetic mode of transcription factor-chromatin remodeling protein in loquat under high temperature and strong light, providing critical theoretical support and gene resources for the molecular breeding of sunburn-resistant loquat varieties.

Key words: loquat, sunburn, EjWRKY15, EjLHP1.2, yeast two-hybrid, BiFC

LI XiaoYing, CHEN XinPeng, GE Hang, CHEN JunWei, WANG Chen, XU HongXia. Screening of Interacting Proteins for the Loquat Sunburn-Responsive Factor EjWRKY15 and Analysis of Its Regulatory Network[J].Scientia Agricultura Sinica, 2026, 59(12): 2712-2725.

Figures/Tables 9

Table 1

Fig. 1

Fig. 2

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Fig. 4

Table 2

Functional annotation of EjWRKY15 interacting proteins"

基因ID Gene ID	基因长度 Gene length (bp)	基因功能注释 Gene annotation	生物过程注释 Biological process annotation
LOC103951827	4418	预测：假定蛋白LOC103951827（白梨） PREDICTED: Uncharacterized protein LOC103951827 (Pyrus bretschneideri)	GO:0045859（蛋白激酶活性的调控）；GO:0004860（蛋白激酶抑制剂活性）；GO:0005515（蛋白结合） GO:0045859 (Regulation of protein kinase activity); GO:0004860 (Protein kinase inhibitor activity); GO:0005515 (Protein binding)
EjCOP9	1748	预测：COP9信号体复合体第5a亚基类似物（苹果） PREDICTED: COP9 signalosome complex subunit 5a-like (Malus domestica)	GO:0005515（蛋白结合） GO:0005515 (Protein binding)
EjLHP1.1	2043	预测：顶端花2蛋白（苹果） PREDICTED: Terminal flower 2 protein (Malus domestica)	GO:0008017（微管结合）；GO:0045298（微管蛋白复合体）；GO:0005634（细胞核） GO:0008017 (Microtubule binding); GO:0045298 (Tubulin complex); GO:0005634 (Nucleus)
EjTFIID	2757	预测：转录起始因子TFIID 15b亚基类似物变体X2（白梨） PREDICTED: Transcription initiation factor TFIID subunit 15b-like isoform X2 (Pyrus bretschneideri)	GO:0003676（核酸结合）；GO:0008270（锌离子结合） GO:0003676 (Nucleic acid binding); GO:0008270 (Zinc ion binding)
EjR-B1	888	预测：铁氧还蛋白根部R-B1型 PREDICTED: Ferredoxin, root R-B1 (Malus domestica)	GO:0006118（电子传递）；GO:0009055（电子载体活性）；GO:0051536（铁硫簇结合）GO:0006118 (Electron transport, obsolete); GO:0009055 (Electron carrier activity); GO:0051536 (Iron-sulfur cluster binding)
LOC103439518	6025	预测：未表征蛋白 LOC103926653（白梨） PREDICTED: Uncharacterized protein LOC103926653 (Pyrus bretschneideri)	GO:0003676（核酸结合） GO:0003676 (Nucleic acid binding)
LOC103962645	1745	预测：未表征蛋白 LOC103962645（白梨） PREDICTED: Uncharacterized protein LOC103962645 (Pyrus bretschneideri)	GO:0009405（致病机制）；GO:0019865（免疫球蛋白结合）；GO:0019814（免疫球蛋白复合体） GO:0009405 (Pathogenesis); GO:0019865 (Immunoglobulin binding); GO:0019814 (Immunoglobulin complex)
EjLHP1.2	263	预测：染色质结合蛋白/类异染色质蛋白1（LHP1），PRC1复合物核心组分，参与表观遗传调控、发育阶段转变和热胁迫（苹果）PREDICTED: Chromatin-binding protein/LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), core component of PRC1 complex, involved in epigenetic regulation, developmental phase transition, and heat stress (Malus domestica)	GO:0005634（细胞核）；GO:0009408（热响应）；GO:0006355（转录调控，DNA模板依赖型） GO:0005634 (Nucleus); GO:0009408 (Response to heat); GO:0006355 (Regulation of transcription, DNA-templated)
LOC103928535	1976	预测：未表征蛋白 LOC103928535（白梨） PREDICTED: Uncharacterized protein LOC103928535 (Pyrus bretschneideri)	GO:0005975（碳水化合物代谢过程）；GO:0007155（细胞黏附）；GO:0004553（水解酶活性，水解O-糖基化合物）；GO:0030248（纤维素结合）；GO:0005576（细胞外区域）GO:0005975 (Carbohydrate metabolic process); GO:0007155 (Cell adhesion); GO:0004553 (Hydrolase activity, hydrolyzing O-glycosyl compounds); GO:0030248 (Cellulose binding); GO:0005576 (Extracellular region)
LOC103454870	903	预测：未表征蛋白 LOC103962645（白梨） PREDICTED: Uncharacterized protein LOC103962645 (Pyrus bretschneideri)	—
EjTTL	1439	预测：尿酸降解双功能蛋白TTL 变体X1（苹果） PREDICTED: uric acid degradation bifunctional protein TTL isoform X1 (Malus domestica)	GO:0005634（细胞核） GO:0005634 (Nucleus)
EjPTRG	1609	预测蛋白：PTRG（苹果） PREDICTED: Pyrenophora tritici-repentis Pt-1C-BFP conserved hypothetical protein, mRNA (Malus domestica)	—
EjWRKY15	1303	预测：WRKY转录因子15（苹果） PREDICTED: Probable WRKY transcription factor 15 (Malus domestica)	GO:0006355（转录调控，DNA模板依赖型）；GO:0003700（DNA结合转录因子活性）；GO:0043565（序列特异性DNA结合）；GO:0005667（转录调控复合体） GO:0006355 (regulation of transcription, DNA-templated); GO:0003700 (DNA-binding transcription factor activity); GO:0043565 (sequence-specific DNA binding); GO:0005667 (transcription regulator complex)

Table 2

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基因 Gene	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	用途 Application
EjWRKY15	GCATATGGCCATGGAGGCCGAATTC ATGGAGAACTGGGATTTGCAAGCTG	GCGGCCGCTGCAGGTCGACGGATCC GCAAGCACCGTTTACAGTATGAGAG	酵母双杂交载体构建 Yeast two-hybrid vector construction
EjLHP1	TATGGCCATGGAGGCCAGTGAATTC ATGAGAACCAAGGTGGGAAGAAGGG	TCTGCAGCTCGAGCTCGATGGATCC AAATGTAGGATTGTATCGGAGATGT	酵母双杂交载体构建 Yeast two-hybrid vector construction
pGADT7	TAATACGACTCACTATAGGGC	AGATGGTGCACGATGCACAG	AD载体引物 AD vector primers
pGBKT7	TAATACGACTCACTATAGGGC	CCCGGAATTAGCTTGGCTGCAAGC	BD载体引物 BD vector primers
EjWRKY15	GAGAACACGGGGGACTCTAGA ATGGAGAACTGGGATTTGCAAGCTG (XbaⅠ)	GACAGTACTATCGATGGATCC GCAAGCACCGTTTACAGTATGAGAG (BamHⅠ)	BiFC载体构建 BiFC vector construction
EjLHP1.2	GAGAACACGGGGGACTCTAGA ATGAGAACCAAGGTGGGAAGAAGGG (XbaⅠ)	GACAGTACTATCGATGGATCC AAATGTAGGATTGTATCGGAGATGT (BamHⅠ)	BiFC载体构建 BiFC vector construction
pSPYCE	GACGTAAGGGATGACGCAC	GATGCCGTTCTTCTGCTTGT	BiFC载体引物 BiFC vector primers
PSPYNE	GACGTAAGGGATGACGCAC	CGCCCTTGCTCACCATAA	BiFC载体引物 BiFC vector primers

Screening of Interacting Proteins for the Loquat Sunburn-Responsive Factor EjWRKY15 and Analysis of Its Regulatory Network

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