家蚕肽聚糖识别蛋白基因（BmPGRP-L1）参与Imd信号通路

发布时间：2018-05-16 07:02

本文选题：家蚕 + 先天免疫　；参考：《江苏科技大学》2016年硕士论文

【摘要】：肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)作为一个主要的模式识别受体,在家蚕Bombyx mori的天然免疫中具有重要的作用。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。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-L1dsRNA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、Moricin、葛佬素)的表达情况。本文从家蚕的组织中克隆了BmPGRP-L1基因,通过序列分析发现BmPGRP-L1与果蝇的DmPGRP-LC的相似度较高,而果蝇的DmPGRP-LC在被革兰氏阴性菌感染后从而激活Imd途径,那么家蚕BmPGRP-L1是否参与Imd信号通路?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BmPGRP-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BmPGRP-L1转录水平没有变化;利用RNAi技术成功敲低BmPGRP-L1表达。对5龄敲低的家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,相应地抗菌肽表达也有不同程度的下调。微生物诱导实验结果显示BmPGRP-L1基因参与家蚕对革兰氏阴性菌大肠杆菌的免疫响应;RNAi实验结果显示BmPGRP-L1基因在家蚕的体壁和头中参与Imd信号通路。这些结果说明BmPGRP-L1基因在家蚕的先天性免疫中扮演着重要的角色。
[Abstract]:Peptidoglycan recognition proteinsm (PGRPs), as a major pattern recognition receptor, plays an important role in the innate immunity of silkworm Bombyx mori. The purpose of this study was to investigate the function of PGRP-L1 gene and its immune signaling pathway in Bombyx mori (Bombyx mori). The PGRP-L1 gene of Bombyx mori was amplified by RT-PCR. E. coli Escherichia from 5th instar silkworm, Saccharomyces cerevisiae Saccharomyces cerevisiae, Bacillus subtilis Bacillus subtilis and Bacillus subtilis were extracted from body wall and head of silkworm for 12 h, then the expression level of BmPGRP-L1 gene was determined by RT-PCR and gel electrophoresis. Three kinds of inactivated microbes or PBSs were injected into the 5th instar silkworm (Bombyx mori) from the 5th instar to PGRP-L1dsRNA or PBS5 for 6 h. Then the immune related transcription factors of silkworm body wall and head, Relishtus and several antimicrobial peptides of Bombyx mori (Bombyx mori) were detected. The expression of Moricin. BmPGRP-L1 gene was cloned from Bombyx mori tissues. Sequence analysis showed that the similarity between BmPGRP-L1 and Drosophila's DmPGRP-LC was high, while the DmPGRP-LC of Drosophila was activated Imd pathway after being infected by Gram-negative bacteria, so whether the BmPGRP-L1 of Bombyx mori participated in Imd signaling pathway? Microbial induction test showed that the BmPGRP-L1 gene transcription level of the experimental group injected with Escherichia coli was significantly up-regulated than that of the control group injected with PBS, but the BmPGRP-L1 transcription level of the experimental group injected with Saccharomyces cerevisiae and Bacillus subtilis did not change. RNAi technique was used to successfully knock down the expression of BmPGRP-L1. The expression of relish factor in the experiment group was lower than that in the control group, and the expression of antimicrobial peptide was down-regulated in different degree. The results of microbial induction experiment showed that BmPGRP-L1 gene was involved in the immune response of Bombyx mori to gram-negative bacteria Escherichia coli. The results showed that BmPGRP-L1 gene was involved in the Imd signaling pathway in the body wall and head of Bombyx mori. These results suggest that BmPGRP-L1 gene plays an important role in the innate immunity of Bombyx mori.
【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S881.2

【相似文献】