桔小实蝇肽聚糖识别蛋白基因BdPGRP-SB1的克隆及功能鉴定

【目的】为探究肽聚糖识别蛋白(PGRP)基因BdPGRP-SB1在桔小实蝇Bactrocera dorsalis免疫中的作用。【方法】本研究利用PCR克隆桔小实蝇BdPGRP-SB1全长cDNA序列；利用生物信息学软件对该基因核苷酸序列及其编码的氨基酸序列特征进行分析。采用RT-qPCR分析BdPGRP-SB1在桔小实蝇不同发育阶段(卵、幼虫、蛹、成虫)及5日龄成虫不同组织(中肠、马氏管、后肠、脂肪体、卵巢和精巢)中的表达模式；对桔小实蝇5日龄雌成虫分别注射大肠杆菌Escherichia coli 0111:B4肽聚糖(PGN-EB)和金黄色葡萄球菌Staphylococcus aureus肽聚糖(PGN-SA)后检测BdPGRP-SB1表达水平变化。利用RNAi沉默BdPGRP-SB1的表达，测定大肠杆菌和金黄色葡萄球菌诱导后桔小实蝇雌成虫的死亡率及大肠杆菌诱导后抗菌肽(AMP)基因attacin-A, defensin和diptercin表达变化情况。【结果】克隆获得桔小实蝇BdPGRP-SB1的全长cDNA序列(GenBank登录号: MN892482)，开放阅读框长558 bp，编码185个氨基酸，其编码蛋白预测分子量为21.45 kD，等电点为8.57。序列分析表明，BdPGRP-SB1无跨膜结构域，具有PGRP保守结构域，前端具有信号肽，为分泌型蛋白；具有Zn²⁺依赖性酰胺酶活性和DAP型肽聚糖识别位点。系统进化分析发现，BdPGRP-SB1与辣椒实蝇B. latifrons的PGRP-SB1亲缘关系最近，氨基酸序列一致性达96%。发育表达模式表明，BdPGRP-SB1在桔小实蝇3日龄幼虫和成虫期高表达；组织表达谱结果显示BdPGRP-SB1在5日龄成虫各组织中均有表达，在脂肪体内表达量最高。PGN-EB和PGN-SA均能诱导桔小实蝇雌成虫体内BdPGRP-SB1表达水平变化。通过RNAi抑制BdPGRP-SB1表达后，注射大肠杆菌导致桔小实蝇雌成虫死亡率显著升高，以及attacin-A, defensin和diptercin表达量显著上调。【结论】结果说明桔小实蝇BdPGRP-SB1参与识别革兰氏阴性细菌，并可能参与桔小实蝇Imd途径调控其免疫反应。

Cloning and functional characterization of the peptidoglycan recognition protein geneBdPGRP-SB1 inBactrocera dorsalis(Diptera: Tephritidae)

【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. Thenucleotide and amino acid sequence characteristics of this gene were analyzed usingbioinformatics software. The relative expression levels of BdPGRP-SB1 in differentdevelopmental 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 analyzedby 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-SAfrom Staphylococcus aureus, respectively, were detected by RT-qPCR. After the expressionof BdPGRP-SB1 was suppressed by RNAi, the mortality of female adults of B. dorsalis postinjection 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. dorsalispost infection ofiE. colwere assayed. 【Results】 The full-length cDNA sequence ofBdPGRP-SB1 (GenBank accession no.: MN892482) was successfully cloned, and its ORF is 558bp in length, encoding a protein of 185 amino acid residues with a predicted molecularweight of 21.45 kD and a theoretical pI of 8.57. Sequence analysis indicated thatBdPGRP-SB1 is a secreted protein with a signal peptide and a conserved PGRP domain butwithout transmembrane domain, and has the Zn²⁺-dependent amidase activity and therecognition 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 acidsequence identity. The developmental expression profile revealed that BdPGRP-SB1 washighly expressed in the 3-day-old larva and adult of B. dorsalis, and the tissueexpression profile showed that it was expressed in various tissues of the 5-day-oldadults, with the highest expression level in fat body. Both PGN-EB and PGN-SA induced theexpression 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 andsignificant up-regulation of attacin-A, defensin and diptercin in female adults of B.dorsalis. 【Conclusion】 The results suggest that BdPGRP-SB1 is involved in therecognition of gram-negative bacteria and may participate in Imd pathway to regulateimmune response in B. dorsalis.