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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 their isoforms associated with Apis mellifera ligustica humoral immune pathways |
投稿时间:2024-09-08 修订日期:2024-11-07 |
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 full-length transcript humoral immune pathway-related genes 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(2022J01131334);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: |
【目的】 利用已获得的纳米孔(Nanopore)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和全长转录本,为深入开展相关基因和剪接体的功能研究提供资源与基础。【方法】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预测和分析体液免疫通路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(motif)。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。【结果】 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,分别延伸了6个基因的5’端和14个基因的3’端,同时延长了13个基因的5’端和7个基因的3’端。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。【结论】 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: |
【Aim】To identify and analyze the genes and full-length transcripts related to the humoral immune pathway in Apis mellifera ligustica by using the obtained Nanopore long read sequencing data, and to provide resources and basis for further functional studies of related genes and isoforms.【Methods】The previously identified all the full-length transcripts of A. m. ligustica were aligned to the Nr database by Blast tool, a total of genes and their full-length transcripts relevant to the humoral immune pathway were identified. Structures of annotated genes were optimized by using gffcompare software to compare endocytosis-associated full-length transcripts with annotated transcripts in the A. mellifera reference genome (Amel_HAv3.1).Types of AS events in genes related to the humoral immune pathway were identified using Astalavista software. Visualization of isoforms’structure was performed with an IGV browser. The authenticity of AS events was verified using PCR. Prediction and investigation of APA sites were done using a TAPIS pipeline, followed by identification of motifs upstream of APA sites with MEME software. Multiple motifs were identified in upstream of APA sites, and the consistent sequence was:A total number of n AS events of genes related to humoral immune pathway were identified, including 2 alternative 3’ splice site (A3SS), 17 intron retention (IR), 14 alternative 5’ splice site (A5SS) and 36 exon skipping (ES).RT-PCR showed that the sizes of amplified fragments were consistent with expected sizes, confirming that the authenticity of the1 randomly selected AS event and the APA sites of the two genes.【Results】26 genes and 41 splices related to Toll signaling pathway, 9 genes and 11 splices related to Imd signaling pathway, 18 genes and 21 splices related to JNK-MAPK-p38 signaling pathway, 9 genes and 13 splices related to JAK-STAT signaling pathway were identified respectively. A total of 20 genes annotated to the humoral immune pathway in the A.mellifera reference genome were structurally optimized, extending the 5’ end of 6 genes and the 3’ end of 14 genes, respectively, while extending the 5’ end of 13 genes and the 3’ end of 7 genes. A total of 69 AS events of genes related to humoral immune pathway were identified, including 2 alternative 3’ splice site (A3SS), 17 intron retention (IR), 14 alternative 5’ splice site (A5SS) and 36 exon skipping (ES).A total of 40 genes related to humoral immune pathway were identified with one or more APA sites. Multiple motifs were identified upstream of the APA site, and the consensus sequence was : GGWRRWRTHAARHTWKSYGAYTTTGGTRTWTCNGSDHRMHTDRYHGMWWS.The authenticity of the APA sites of the two genes was confirmed by 3’RACE.【Conclusion】62 genes and 86 splices related to the humoral immune pathway of A. m. ligustica were identified, and 20 gene structures related to the humoral immune pathway of A. m. ligustica were optimized. Sixty-nine AS events and 106 APA sites of genes related to the humoral immune pathway were discovered, which confirmed the authenticity of AS events and APA sites of related genes. |
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