肽聚糖识别蛋白AaPGRP-LC参与埃及伊蚊的抗细菌免疫反应  

Peptidoglycan recognition protein AaPGRP-LC is involved in immune responses against bacteria in Aedes aegypti(Diptera:Culicidae)

作　　者：杨青泰 赵莉蔺[1,2] 邹振[2] 王燕红 马瑞燕[1] 

机构地区：[1]山西农业大学农学院,山西太谷030801 [2]中国科学院动物研究所,农业虫害鼠害综合治理研究国家重点实验室,北京100101

出　　处：《昆虫学报》2018年第1期25-35,共11页Acta Entomologica Sinica

基　　金：国家自然科学基金青年基金项目(31402013);国家自然科学基金项目(31672291)

摘　　要：【目的】阐明在响应细菌感染的过程中,埃及伊蚊Aedes aegypti体内肽聚糖识别蛋白PGRPLC(Aa PGRP-LC)的功能。【方法】使用实时荧光定量PCR(q PCR)技术分析埃及伊蚊感染阴沟肠杆菌Enterobacter cloacae后不同时间抗菌肽(antimicrobial peptides,AMPs)基因的表达情况。结合RNA干扰技术和q PCR技术检测干扰Aa PGRP-LC后免疫相关基因转录模式的变化。使用原核表达系统表达Aa PGRP-LC,并通过Ni^(2+)-NTA柱纯化,通过免疫印迹分析检测所纯化蛋白的质量。【结果】阴沟肠杆菌感染埃及伊蚊6 h后埃及伊蚊体内抗菌肽基因的转录水平显著升高。干扰Aa PGRP-LC并用阴沟肠杆菌感染后,埃及伊蚊体内重要抗菌肽基因的转录水平显著降低,同时埃及伊蚊免疫缺陷(immune-deficiency,IMD)通路和Toll信号(Toll-like receptors)通路的免疫相关基因的表达量也显著降低。纯化得到条带单一的Aa PGRP-LC蛋白。【结论】Aa PGRP-LC参与调控埃及伊蚊重要抗菌肽基因的转录,在埃及伊蚊响应细菌感染的过程中起到了重要的调控作用。Aa PGRP-LC在参与调控IMD通路的同时,也可能参与了Toll信号通路的调控过程。从原核表达系统中获得的Aa PGRP-LC重组蛋白可用于下一步的功能研究。【Aim】 The objective of this study is to elucidate the roles of peptidoglycan recognition protein LC （PGRP-LC） in response to bacterial infection in Aedes aegypti. 【Methods】 The mRNA abundance of genes of antimicrobial peptides （AMPs） in mosquitoes at different time after infection of Enterobacter cloacae was measured by quantitative real-time PCR （qPCR）. After interference of PGRP-LC by RNA interference （RNAi）, the expression profiles of immune-related genes were detected by qPCR. The recombinant protein AaPGRP-LC was expressed by prokaryotic expression system and purified through a Ni2＋-NTA agarose column. The quality of the purified recombinant protein was tested by Western blotting. 【Results】 Gram-negative E. cloacae bacteria could activate the expression of genes of AMPs in Ae. aegypti at 6 h post infection. After depletion of AaPGRP-LC, the transcription levels of genes of AMPs were significantly decreased during bacterial infection. The expression levels of immune-related genes involved in Toll and IMD pathways were down-regulated after interference of AaPGRP-LC. Western blotting of the purified recombinant protein AaPGRP-LC showed a clear and unique target band. 【Conclusion】 AaPGRP-LC modulates the transcriptional expression of genes of many essential AMPs and plays an important regulatory role in response to bacterial infection. It is involved in the regulation of IMD pathway and may also regulate the Toll pathway. The obtained recombinant AaPGRP-LC can be used in the follow-up study.

关 键 词：埃及伊蚊 天然免疫 细菌 肽聚糖识别蛋白 抗菌肽 

分 类 号：Q966[生物学—昆虫学]