枸杞红瘿蚊Gephyraulus lycantha Jiao & Kolesik是为害宁夏枸杞Lycium babarum L.（以下简称枸杞）的一种致瘿害虫，是我国枸杞产区重发、频发的成灾性害虫。成虫寿命极短，防治窗口期短暂，生产中防治难度极大，长期滥用化学农药会导致抗性增强，导致枸杞子农残问题突出，花蕾危害率即为损失率。嗅觉在枸杞红瘿蚊寻找寄主植物、配偶和产卵场所等行为过程中起着重要作用，而气味结合蛋白GlycOBPs和化学感受蛋白GlycCSPs是枸杞红瘿蚊与外部环境接触和交流的重要环节，是其嗅觉识别的第一步。课题组前期通过转录组测序，在枸杞红瘿蚊触角中鉴定出了多个GlycOBPs和GlycCSPs，但对其在枸杞红瘿蚊嗅觉识别过程中的功能尚不清楚。鉴于此，本文基于枸杞红瘿蚊触角转录组的分析结果，研究GlycOBPs和GlycCSPs的组织表达特性，选择具有性别和组织特异性高表达的蛋白，通过分子对接和荧光竞争结合等方法明确重组蛋白与枸杞红瘿蚊相关信息化学物质（寄主、非寄主、瘿蚊科性信息素类似物）的结合特性，以期为设计基于嗅觉蛋白为靶标，研发枸杞红瘿蚊引诱剂和趋避剂提供参考。主要研究结果如下：

1. 明确了枸杞红瘿蚊部分OBPs和CSPs的组织特异性表达模式。

（1）通过qRT-PCR分析了枸杞红瘿蚊7 个候选内参基因（GAPDH、RPS18、RPS20、RPL12、γ-Tublin、UBC、Actin）的表达水平，根据GeNorm、Normfinder、Bestkeeper及RefFinder综合分析，明确GAPDH为雌雄成虫不同组织中表达最稳定的内参基因，可为本文后续实验提供参照。（2）转录组数据表明GlycOBP2/5/12/13/14/155/16/17/20/21/24/26在雌雄触角里差异表达；利用qPCR验证发现5 个GlycOBPs（GlycOBP2/14/15/21/26）和3 个GlycCSPs（GlycCSP1/2/3）在枸杞红瘿蚊不同性别、不同组织（触角、胸部、腹部）中的表达水平与前期转录组数据中FPKM值的分析结果基本一致，推测这些基因可能是识别寄主植物挥发物或性信息素的关键蛋白。结果显示，除GlycOBP21外，GlycOBP2/14/15/26均触角高表达，且表达差异显著。GlycOBP2在雄虫腹部，GlycOBP15/26在成虫胸部，GlycOBP21在雌虫胸腹部的表达水平也相对较高

2. 分子对接预测了触角中高表达的气味结合蛋白OBPs和化学感受蛋白CSPs与配体化合物之间的结合作用。

（1）利用Alphafold3成功构建了GlycOBP2、GlycOBP14、GlycOBP15、GlycOBP26和GlycCSP1、GlycCSP3蛋白的三维结构模型。并通过pLDDT、PAE和iPTM等置信度指标及Procheck拉式构象图，评估了这些预测模型结果是可靠和准确的。（2）在获得高质量三维蛋白结构的基础上，本研究采用分子对接技术，对筛选出的枸杞红瘿蚊35 种关键信息化学物质（包括9 种寄主植物的挥发性物质；11 种瘿蚊科性信息素类似物；15 种非寄主植物释放的挥发物）进行相互作用分析。探究不同目的蛋白与不同类别信息化学物质的结合模式和潜在调控机制。根据6个蛋白与候选出的35种化学信息物质的分子对接结果，筛选出15种对接较好的化学信息物质，为β-石竹烯、α-法呢烯、α-蒎烯、假荆芥内酯、β-蒎烯、萜品油烯、1,8-桉叶素、3-蒈烯、d-柠檬烯、莰烯、薄荷酮、对乙基苯乙酮、丁香酚、月桂烯和(2S,8Z)-2-丁酰氧基十七碳-8-烯。GlycOBP14与GlycOBP26是所选目的嗅觉蛋白中与化学物分子对接结果普遍较好的两个蛋白，因此选择这两个蛋白作为荧光竞争实验。

3. 验证了枸杞红瘿蚊关键气味结合蛋白GlycOBP14和GlycOBP26与植物挥发物和性信息素化合物之间的结合活性。

（1）通过基因克隆技术获取GlycOBP26与GlycOBP14编码基因，经测序验证表明其与已知CDS序列相同，可用于后续的蛋白纯化及原核表达分析。亚细胞定位研究显示，GlycOBP14主要在细胞质内发挥生物学功能；而GlycOBP26主要在细胞膜上执行功能。序列分析结果表明，两个蛋白的信号肽区域均位于N端，对应第1至20位氨基酸残基，该区域序列中包含有6 个保守的半胱氨酸（Cys）残基。（2）凝胶电泳（SDS-PAGE）分析结果显示，GlycOBP14和GlycOBP26主要以包涵体结构的形式存在，分离后蛋白样品在电泳图谱中呈现单一清晰条带，其大小约为16 kDa，这与理论预测值基本吻合且纯度较高，可用于开展后续荧光竞争结合实验。（3）通过荧光竞争结合发现GlycOBPs重组蛋白与15 个配体化合物，包括8 种寄主植物挥发物；6 种非寄主植物挥发物和1 种瘿蚊科性信息素类似物能进行有效结合。3 类化合物中，GlycOBP14与对乙基苯乙酮、α-法尼烯和(2S,8Z)-2-丁酰氧基十七碳-8-烯的结合力较强，结合常数分别为3.70 μmol/L、3.02 μmol/L和6.18 μmol/L；GlycOBP26与莰烯、β-石竹烯和(2S,8Z)-2-丁酰氧基十七碳-8-烯有较强的亲和力，结合常数分别为3.60 μmol/L、5.17 μmol/L和11.60 μmol/L。总体来看，GlycOBP14和GlycOBP26与配体化合物结合亲和力普遍都较好，表明这两个蛋白具有广谱性功能，在枸杞红瘿蚊识别寄主和配偶的行为过程中，均能发挥嗅觉功能。

The gall midge Gephyraulus lycantha Jiao & Kolesik is a serious gall-forming pest that causes severe damage in the wolfberry, Lycium barbarum L. in China. It is a disastrous pest with frequent and severe outbreaks in wolfberry-producing regions across the country. Adults of G. lycantha have an extremely short lifespan, resulting in a narrow control window period, which makes field management highly challenging. The long-term overuse of chemical pesticides has led to increased resistance in the pest and exacerbated pesticide residue issues in wolfberry fruits. Notably, the damage rate to flower buds directly equates to the crop loss rate.Olfactory plays crucial role in the behavioral processes of G. lycantha, such as host location, mating and oviposition. Previous researches have found that odorant-binding proteins (GlycOBPs) and chemosensory proteins (GlycCSPs) serve as critical intermediaries in the insect's interaction and communication with the external environment, representing the first step in olfactory recognition. Previous transcriptomic sequencing by our research group identified multiple GlycOBPs and GlycCSPs in the antennae of G. lycantha, but their functional roles in olfactory on remain unclear. In order to better understand the olfactory perception mechanism of G. lycantha, this paper studied the tissue-specific expression characteristics of GlycOBPs and GlycCSPs, based on the analysis results of the antennal transcriptom of G. lycantha. Proteins exhibiting high expression levels with sex and tissue-specificity were selected for further characterization. Fluorescence competitive binding assays, and molecular docking were employed to elucidate the binding properties of recombinant proteins with key semiochemicals (host/non-host plant volatiles and sex pheromone analogs) relevant to G. lycantha. The findings aim to provide a theoretical foundation for developing attractants and repellents targeting olfactory proteins for pest control. The main results are as follows:

1. Tissue-specific expression patterns of selected OBPs and CSPs in G. lycantha 

 (1) Real-time quantitative PCR (qPCR) was used to assess the expression stability of seven candidate reference genes(GAPDH, RPS18, RPS20, RPL12, γ-tubulin, UBC, and Actin)  in different adult tissues of G. lycantha. Comprehensive evaluation using GeNorm, NormFinder, BestKeeper, and RefFinder identified GAPDH as the most stable reference gene for subsequent experiments. (2) qPCR analysis revealed the expression profiles of five GlycOBPs (GlycOBP2/14/15/21/26) and three GlycCSPs (GlycCSP1/2/3) across different sexes and tissues (antennae, thorax, abdomen). Most GlycOBPs and GlycCSPs were highly expressed in the antennae, with some also detected in the thorax and abdomen. Notably, significant differences in expression levels were observed between male and female antennae.

2. Predicted binding properties of key OBPs to ligand compounds

   (1) AlphaFold3 was employed to successfully construct three-dimensional structural models of GlycOBP2, GlycOBP14, GlycOBP15, GlycOBP26, GlycCSP1, and GlycCSP3. The reliability and accuracy of these models were assessed using confidence metrics (pLDDT, PAE, iPTM) and Procheck Ramachandran plots. (2) Molecular docking was performed between the high-quality protein models and 35 key semiochemicals (including 9 host plant volatiles, 11 Cecidomyiidae sex pheromone components, and 15 non-host plant volatiles).Based on molecular docking results between 6 proteins and 35 candidate chemical information substances, 15 compounds exhibiting favorable docking performance were identified: β-Caryophyllene, α-Farnesene, α-Pinene, Nepetalactone, β-Pinene, Terpinolene, 1,8-Cineole, 3-Carene, d-Limonene, Camphene, Menthone, para-Ethylacetophenone, Eugenol, Myrcene and (2S,8Z)-2-Butyryloxyheptadec-8-ene.Based on molecular docking results, GlycOBP14 and GlycOBP26 demonstrated superior binding affinities with semiochemicals among the selected olfactory proteins. Therefore, these two proteins were chosen for fluorescence competitive binding assays.

3. Sequence analysis, protein purification and recombinant expression of GlycOBP14 and GlycOBP26.

   (1) GlycOBP26 and GlycOBP14 genes were cloned and sequenced, confirming that their coding sequences (CDS) matched known sequences, enabling subsequent protein purification and prokaryotic expression analysis. GlycOBP14 primarily functions in the cytoplasm, while GlycOBP26 operates on the cell membrane. Both proteins contain a signal peptide region spanning amino acids 1-20 at the N-terminus, with six conserved cysteine residues.(2) Gel electrophoresis revealed that GlycOBP14 and GlycOBP26 predominantly existed as inclusion bodies, displaying single, distinct bands at approximately 16 kDa, consistent with predicted molecular weights and high purity, qualifying them for fluorescence competitive binding assays.(3) Fluorescence competitive binding assays demonstrated that recombinant GlycOBPs effectively bound to 15 ligand compounds, including 8 host plant volatiles, 6 non-host plant volatiles, and 1 Cecidomyiidae sex pheromone component. Among these:GlycOBP14 exhibited strong binding to 4-ethylacetophenone,，α-farnesene, and (2S,8Z)-2-butanoyloxyheptadec-8-ene, with binding constants of 3.70 μM, 3.02 μM, and 6.18 μM, respectively.GlycOBP26 showed high affinity for camphene, β-caryophyllene, and (2S,8Z)-2-butanoyloxyheptadec-8-ene, with binding constants of 3.60 μM, 5.17 μM, and 11.60 μM, respectively.Overall, GlycOBP14 and GlycOBP26 displayed robust and broad-spectrum ligand-binding affinities, suggesting their dual roles in mediating olfactory-driven host plant recognition and mate-seeking behaviors in G. lycantha.