关键词: 桔小实蝇, 肽聚糖识别蛋白, 基因克隆, 免疫, 革兰氏阴性细菌

Abstract: 【Aim】 To explore the role of a peptidoglycan recognition protein (PGRP) gene, BdPGRP-SB1, in the immunity of the oriental fruit fly, Bactrocera dorsalis. 【Methods】 The full-length cDNA sequence of BdPGRP-SB1 of B. dorsalis was cloned by PCR. The nucleotide and amino acid sequence characteristics of this gene were analyzed using bioinformatics software. The relative expression levels of BdPGRP-SB1 in different developmental stages (egg, larva, pupa and adult) and tissues (midgut, Malpighian tubules, hindgut, fat body, ovary and testis) of the 5-day-old adult of B. dorsalis were analyzed by RT-qPCR. The expression levels of BdPGRP-SB1 in the 5-day-old female adults of B. dorsalis injected with the peptidoglycan PGN-EB from Escherichia coli 0111:B4 and PGN-SA from Staphylococcus aureus, respectively, were detected by RT-qPCR. After the expression of BdPGRP-SB1 was suppressed by RNAi, the mortality of female adults of B. dorsalis post injection of E. coli and S. aureus and the expression levels of three antimicrobial peptide (AMP) genes including attacin-A, defensin and diptercin in female adults of B. dorsalis post infection ofi  E. colwere assayed. 【Results】 The full-length cDNA sequence of BdPGRP-SB1 (GenBank accession no.: MN892482) was successfully cloned, and its ORF is 558 bp in length, encoding a protein of 185 amino acid residues with a predicted molecular weight of 21.45 kD and a theoretical pI of 8.57. Sequence analysis indicated that BdPGRP-SB1 is a secreted protein with a signal peptide and a conserved PGRP domain but without transmembrane domain, and has the Zn²⁺-dependent amidase activity and the recognition sites of DAP-type peptidoglycan. Phylogenetic analysis indicated that BdPGRP-SB1 is the most closely related to PGRP-SB1 of B. latifrons, sharing 96% amino acid sequence identity. The developmental expression profile revealed that BdPGRP-SB1 was highly expressed in the 3-day-old larva and adult of B. dorsalis, and the tissue expression profile showed that it was expressed in various tissues of the 5-day-old adults, with the highest expression level in fat body. Both PGN-EB and PGN-SA induced the expression of BdPGRP-SB1 in female adults of B. dorsalis. After the suppression of BdPGRP-SB1 expression by RNAi, E. coli infection resulted in significantly higher mortality and significant up-regulation of attacin-A, defensin and diptercin in female adults of B. dorsalis. 【Conclusion】 The results suggest that BdPGRP-SB1 is involved in the recognition of gram-negative bacteria and may participate in Imd pathway to regulate immune response in B. dorsalis.

Key words: Bactrocera dorsalis, peptidoglycan recognition protein, gene cloning, immunity; gram-negative bacteria