家蚕肽聚糖识别蛋白L1参与Imd信号通路

Peptidoglycan recognition protein L1 is involved in the Imd pathway in the silkworm,Bombyx mori

【目的】肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRP)作为一个重要的模式识别受体,在家蚕Bombyx mori的先天免疫中发挥重要的作用。本研究主要探讨家蚕PGRP-L1基因的功能及其所参与的免疫信号通路。【方法】本实验通过RT-PCR扩增获得家蚕PGRP-L1基因。利用微生物诱导实验对5龄起蚕分别注射大肠杆菌Escherichia coli、酿酒酵母Saccharomyces cerevisiae、枯草芽孢杆菌Bacillus subtilis和PBS,12 h后取体壁和头部提取RNA,然后采用RT-PCR和凝胶电泳技术测定BmPGRP-L1基因的表达水平;利用RNA干涉实验向5龄起蚕注射PGRP-L1 dsRNA或PBS,6 h后再分别注射3种灭活的微生物或PBS,然后检测家蚕体壁及头部的免疫相关转录因子(Rel,Cactus和Relish)以及家蚕多个抗菌肽(antimicrobial peptides,AMPs)基因(攻击素基因Attacin,Moricin和葛佬素基因Gloverin)的表达情况。【结果】微生物诱导实验显示,注射大肠杆菌的实验组比注射PBS的对照组Bm PGRP-L1基因转录水平显著上调,而注射酿酒酵母和枯草芽孢杆菌的实验组Bm PGRP-L1转录水平没有变化。利用RNAi技术成功敲低Bm PGRP-1表达,对Bm PGRP-L1敲低的5龄起蚕注射灭活的微生物,敲低实验组relish因子表达低于正常对照组,相应地抗菌肽基因的表达也有不同程度的下调。【结论】结果提示,BmPGRP-L1基因参与家蚕对革兰氏阴性菌大肠杆菌的免疫响应;Bm PGRP-L1基因在家蚕的体壁和头中参与Imd信号通路。

【Aim 】 As an important pattern recognition receptor, peptidoglycan recognition protein（ PGRP） plays an important role in the innate immunity of the silkworm,Bombyx mori. The study aims to identify and characterize a new PGRP gene PGRP-L1 from Bombyx mori,analyze its immune function and the immune signaling pathway that the gene is involved in. 【Methods】We successfully cloned the gene BmPGRP-L1 from Bombyx mori by RT-PCR. Newly-exuviated 5th instar larvae of B. mori were injected with Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis and phosphate buffered saline,respectively. After 12 h,the integument and head were collected and used for RNA extraction. RT-PCR and gel electrophoresis were then performed to measure the transcriptional level of Bm PGRP-L1 gene.Newly-exuviated 5th instar larvae were injected with dsRNA specific for Bm PGRP-L1 and PBS,respectively,then 6 h later injected with the inactivated three microorganisms and phosphate buffered saline,and another 6 h later,tissues were collected and used to detect the transcription levels of related transcription factors（ Rel,Cactus and Relish） and antimicrobial peptides（ AMPs） genes（ Attacin,Moricin and Gloverin）. 【Results】Microbial induction experiments showed that Bm PGRP-L1 mRNA level was upregulated in the integument and head of B. mori 12 h after injection of E. coli but presented no change after injection of S. cerevisiae or B. subtilis. RNA interference experiments showed that the expression level of BmPGRP-L1 was successfully knocked down,and the expression level of relish in the newly-exuviated 5th instar larvae subjected to Bm PGRP-L1 RNAi was significantly lower than the normal control post E. coli challenge,and the corresponding expression levels of the relevant antimicrobial peptide genes were downregulated to different degrees. 【Conclusion】The results suggest that Bm PGRPL1 gene participates in the immune response of B. mori against gram negative bacteria E. coli,and Bm PGRP-L1 participates in the Imd signal transduction pathway in integument and head of B. mori.