| 宋宇轩，张荣华，董舒楠，王梦怡，范小雪，李婧娴，宓诗雨，王美仪，邱剑丰，陈大福，严提珍，郭 睿,2025,意大利蜜蜂体液免疫通路相关基因及其剪接分析[J].环境昆虫学报,(4):1226-1237 |
| 意大利蜜蜂体液免疫通路相关基因及其剪接分析 |
| Identification of genes and isoforms associated with humoral immune pathways in Apis mellifera ligustica |
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| DOI： |
| 中文关键词: 西方蜜蜂 意大利蜜蜂 纳米孔测序 体液免疫 可变剪接 可变多聚腺苷酸化 |
| 英文关键词:Apis mellifera Apis mellifera ligustica Nanopore sequencing humoral immune pathway alternative splicing alternative polyadenylation |
| 基金项目:国家自然科学基金面上项目（32372943）；国家现代农业产业技术体系专项资金（CARS-44-KXJ7）；福建省自然科学基金面上项目（2022J01133）；福建农林大学科技创新专项基金项目（KFb22060XA）；福建省大学生创新创业训练计划项目（S202410389057，202410389180） |
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| 中文摘要: |
| 本研究旨在利用已获得的纳米孔长读段测序数据鉴定和分析意大利蜜蜂Apis mellifera ligustica体液免疫通路相关基因及全长转录本，为深入开展功能研究提供资源与基础。使用Blast工具将鉴定到的所有全长转录本比对Nr数据库以筛选出体液免疫通路相关基因和剪接体。采用gffcompare软件将全长转录本与西方蜜蜂Apis mellifera参考基因组（Amel_HAv3.1）上注释的转录本进行比较来优化已注释基因的结构。利用TAPIS pipeline预测和分析相关基因的可变多聚腺苷酸化（Alternative polyadenylation，APA）位点，再通过TBtools软件鉴定APA位点上游的基序。使用Astalavista软件鉴定可变剪接（Alternative splicing，AS）事件，进而通过IGV浏览器进行结构可视化。通过RT-PCR验证AS事件的真实性。分别鉴定到Toll信号通路相关的26个基因和41条剪接体，Imd信号通路相关的9个基因和11条剪接体，JNK-MAPK-p38信号通路相关的18个基因和21条剪接体，JAK-STAT信号通路相关的9个基因和13条剪接体。共对西方蜜蜂参考基因组上注释到体液免疫通路的20个基因进行了结构优化，正链和负链结构优化的基因分别有5个和15个，5'端和3'端延长的基因有13个和7个，其中有两个基因(LOC724728; LOC411861)的5'端和3'端都有延长。共鉴定到体液免疫通路相关基因的69次AS事件，包括2次可变3'端剪接（Alternative 3' splice site，A3SS）、17次内含子保留（Intron retention，IR）、14次可变5'端剪接（Alternative 5' splice site，A5SS）和36次外显子跳跃（Exon skipping，ES）。RT-PCR结果显示目的片段大小符合预期，证实了随机选择的6次AS事件的真实性。共鉴定到体液免疫通路相关的40个基因含1个及以上APA位点。在APA位点上游鉴定到多个基序，一致性序列为：GGWRRWRTHAARHTWKSYGAYTTTGGTRTWTCNGSDHRMHTDRYHGMWWS。通过3' RACE证实了2个基因的APA位点真实性。鉴定到意大利蜜蜂体液免疫通路相关的62个基因和86条剪接体，优化了西方蜜蜂体液免疫通路相关的20个基因结构，发掘出体液免疫通路相关基因的69次AS事件与106个APA位点，证实了相关基因的AS事件和APA位点的真实性。 |
| 英文摘要: |
| This study aims to identify and analyze the genes and full-length transcripts related to humoral immune pathways in Apis mellifera ligustica using the obtained Nanopore long-read sequencing data, providing resource and basis for further functional study. All of the identified full-length transcripts in A. m. ligustica were aligned to the Nr database via BLAST tool, screening genes and isoforms related to humoral immune pathways. Structures of annotated genes were optimized by comparing full-length transcripts with annotated transcripts in the A. mellifera reference genome (Amel_HAv3.1) using gffcompare software. The TAPIS pipeline was applied to predict and analyze alternative polyadenylation (APA) sites of relevant genes, followed by identification of motifs upstream of APA sites by MEME software. Astalavista software was used to identify alternative splicing (AS) events, while visualization of isoforms' structures was performed by IGV browser. The authenticity of AS events was verified using RT-PCR. Specifically, 26 genes and 41 isoforms associated with Toll signaling pathway, 9 genes and 11 isoforms related to Imd signaling pathway, 18 genes and 21 isoforms relative to JNK-MAPK-p38 signaling pathway, 9 genes and 13 splices linked to JAK-STAT signaling pathway were identified, respectively. A total of 20 genes annotated to humoral immune pathways in the reference genome of A. mellifera were structurally optimized, including: 5 and 15 genes with positive and negative chain structure optimization, respectively; 13 genes with 5'-end extensions and 7 with 3'-end extensions, including two genes (LOC724728 and LOC411861) extended at both ends. Additionally: 69 AS events of humoral immune pathway-associated genes were identified, including: 2 alternative 3' splice sites (A3SS), 17 intron retention (IR), 14 alternative 5' splice sites (A5SS) and 36 exon skipping (ES). RT-qPCR results were indicative of the expected sizes of target fragments, confirming the authenticity of randomly selected 6 AS events. 40 genes related to humoral immune pathway were discovered to have one or more APA sites. Multiple motifs were identified upstream of the APA sites, and the consensus sequence was GGWRRWRTHAARHTWKSYGAYTTTGGTRTWTCNGSDHRMHTDRY HGMWWS. APA sites of two genes were validated by 3' RACE. We identified 62 genes and 86 splices relative to the humoral immune pathways of A. m. ligustica, and optimized structures of 20 related genes in A. mellifera, discovered 69 AS events and 106 APA sites and confirmed the authenticity of their AS events and APA sites. |
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