关键词: 西方蜜蜂, 东方蜜蜂微孢子虫, 肽聚糖识别蛋白, 纳米孔测序, 剪接异构体

Abstract: 【Aim】 This study aims to systematically identify and analyze the genes related to peptidoglycan recognition proteins (PGRPs) and their splice variants in Apis mellifera, to explore the physicochemical properties and molecular characteristics of PGRP gene splice variants-encoding proteins, and to determine the expression profile of PGRP gene splice variants in response of adult workers of A. mellifera to Nosema ceranae infection, so as to offer the reference and basis for functional study on splice variants of PGRP genes of A. mellifera. 【Methods】Based on the obtained nanopore sequencing data from the midguts of the 8- and 11-day-old adult workers of A. mellifera, the previously identified all full-length transcripts were aligned to the Nr and KEGG databases by using the Blast tool to screen PGRP genes of A. mellifera and their splice variants, and RT-PCR was used to validate the authenticity of the sequences of five splice variants of PGRP genes randomly selected. Gffcompare software was utilized to align the sequences of the identified PGRP genes to the reference genome of A. mellifera, thus optimizing the gene structures. Astalavista software was employed to identify the types of alternative splicing (AS) events in PGRP genes, and AS events were then verified by RT-PCR and Sanger sequencing. Related software was used to predict and analyze the physicochemical properties and molecular characteristics of the proteins encoded by the splice variants of PGRP genes. RT-qPCR was conducted to determine the relative expression levels of PGRP gene splice variants in the midguts of A. mellifera adult workers after inoculation with N. ceranae.【Results】 A total of four PGRP-associated genes (PGRP-3, PGRP-S2, PGRP-LC and PGRP-LB) and their 19 splice variants of A. mellifera were identified. RT-PCR and Sanger sequencing results confirmed the authenticity of expression and sequences of five splice variants (rna14029, ONT.6350.8, ONT.6350.10, rna24089, ONT.6350.7) of PGRP genes. The 5′ and 3′ ends of PGRP-3 (gene10434) were elongated by 8 and 1 055 bp,  respectively, while those of PGRP-S2 (gene10435) were elongated by 27 and 234 bp, respectively. The authenticity of AS events in PGRP-S2 was verified by RT-PCR. The splice variant ONT.6350.2-encoded protein had a molecular formula of C₉₆₆H₁₅₀₂N₂₇₂O₂₇₅S₇, an approximate molecular weight of 21 527.63 D, a theoretical isoelectric point (pI) of 8.94, an average hydrophilicity of -0.119, a signal peptide, a PGRP domain and no transmembrane domain. The protein encoded by the splice variant ONT.6350.6 had a molecular formula of C₈₄₁H₁₃₀₁N₂₄₃O₂₄₄S₅, an approximate molecular weight of 18 860.46 D, a theoretical pI of 9.14, an average hydrophilicity of -0.324, and no transmembrane domain and signal peptide. PGRP-S2 proteins from A. mellifera and A. cerana were clustered into a single clade on the phylogenetic tree. The expression level of the splice variant ONT.6350.2 in the midguts of adult workers inoculated with N. ceranaewas significantly upregulated at 2 and 4 d post inoculation and extremely significantly upregulated at 3 d post inoculation as compared with that in the midguts of A. mellifera adult workers uninoculated with N. ceranae in the control group. Additionally, the splice variant ONT.6350.6 had the same expression pattern as ONT.6350.2. Moreover, both ONT.6350.2 and ONT.6350.6 in the midguts of adult workers inoculated with N. ceranae exhibited an overall increased expression trend at 1－4 d post inoculation as compared with those in the midguts of A. mellifera adult workers uninoculated with N. ceranae in the control group. 【Conclusion】 This study optimized the structure of PGRP-3 and PGRP-S2 annotated on the A. mellifera reference genome. The splice variant ONT.6350.2-encoded protein is a potential secretary protein, whereas the splice variant ONT.6350.6-encoded protein is a putative intracellular protein. The homology of PGRP-S2 between A. mellifera and A. cerana is the highest. The expression of the splice variants ONT.6350.2 and ONT.6350.6 is activated in response of A. mellifera adult workers to N. ceranae infection.

Key words: Apis mellifera, Nosema ceranae, peptidoglycan recognition protein, nanopore sequencing, splice variant