Identification of genes and isoforms associated with humoral immune pathways in Apis mellifera ligustica

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.