光滑鳖甲肽聚糖识别蛋白ApPGRP的表达与功能分析

Expression and functional analysis of the peptidoglycan recognition protein ApPGRP from Anatolica polita(Coleoptera: Tenebrionidae)

【目的】旨在研究光滑鳖甲Anatolica polita肽聚糖识别蛋白(peptidoglycan recognition proteins, PGRPs)基因ApPGRP表达模式及其对下游免疫相关基因的表达调控,以及重组蛋白ApPGRP与细菌的结合能力。【方法】构建原核表达载体pET-28a-ApPGRP,转化大肠杆菌Escherichia coli BL21(DE3),诱导表达并纯化重组蛋白His-ApPGRP,分别测定其与金黄色葡萄球菌Staphylococcus aureus和肽聚糖(peptidoglycan, PGN)的结合能力。金黄色葡萄球菌S.aureus刺激后,利用qRT-PCR检测光滑鳖甲6龄幼虫体内ApPGRP、抗菌肽基因ApAttacin2和ApAttacin1、防御素基因ApDefensin、丝氨酸蛋白酶基因ApSP和丝氨酸蛋白酶抑制剂基因ApSerpin等免疫相关基因的表达水平;采用RNAi技术沉默光滑鳖甲6龄幼虫体内ApPGRP基因的表达,并利用qRT-PCR分别检测RNAi处理以及RNAi后再用S.aureus刺激时幼虫体内上述基因的表达水平变化。【结果】通过原核表达获得了重组蛋白His-ApPGRP,其具有结合金黄色葡萄球菌和肽聚糖的能力。注射金黄色葡萄球菌后,检测的光滑鳖甲6龄幼虫免疫相关基因(ApSP除外)的表达都显著上调;RNAi沉默光滑鳖甲6龄幼虫ApPGRP后,其他免疫相关基因表达量均显著降低,其响应金黄色葡萄球菌刺激后的表达量也显著低于对照组。【结论】这些结果表明,ApPGRP在光滑鳖甲免疫防御中起着识别外源微生物,激活信号通路并调控抗菌肽表达的作用。

【Aim】 This study aims to investigate the expression patterns of the peptidoglycan recognition protein gene ApPGRP in the desert beetle Anatolica polita, its regulation of the downstream immune-related genes in vivo and the binding capability of the recombinant protein ApPGRP with bacteria in vitro.【Methods】 The expression vector pET-28 a-ApPGRP was constructed and transformed into Escherichia coli BL21(DE3). The recombinant protein His-ApPGRP was expressed and purified, and its binding capabilities with Staphylococcus aureus and peptidoglycan(PGN) were detected, respectively. The expression levels of immune-related genes including ApPGRP, antimicrobial peptide genes ApAttacin2 and ApAttacin1, defensin gene ApDefensin, serine protease gene ApSP and serine protease inhibitor gene ApSerpin in the 6 th instar larva of A. polita infected by S. aureus were determined by qRT-PCR. The expression levels of the immune-related genes in the 6 th instar larvae of A. polita after RNAi of ApPGRP and S. aureus stimulation following RNAi of ApPGRP were also detected by qRT-PCR, respectively.【Results】 The purified recombinant protein His-ApPGRP had the binding capability with both S. aureus and PGN. Except for ApSP, the expression of the other tested immune-related genes in the 6 th instar larva of A. polita was significantly up-regulated after S. aureus injection. When ApPGRP was knocked down by RNAi, the expression of all the other tested immune-related genes in the 6 th instar larva of A. polita was significantly down-regulated and the expression levels of these genes in response to subsequent S. aureus challenge in the RNAi-treated larva were also significantly lower than those of the control group.【Conclusion】 These results show that ApPGRP plays an important role in recognizing exogenous microorganisms, activating signaling pathways and regulating the expression of antimicrobial peptides in the immune defense of A. polita.