| There are many microbial communities in the intestinal cavity of metazoan.There are bacteria that are beneficial to the host,as well as harmful bacteria that do damage to the metabolism of the host,destroy the host's intestinal microenvironment and even get the host sick in these communities.For example,gas-producing bacteria cause enteritis,Vibrio causes red-legged disease and Vibrio cholerae causes bad eye disease in shrimp.In addition,there are a large number of neutral bacteria in the host gut.How the host tolerates so many bacteria and maintain the homeostasis of their gastrointestinal microbial community has not been fully revealed.Previous studies have shown that the DUOX system plays an important role in the homeostasis of the Drosophila intestinal microflora in addition to antimicrobial peptides.In intestinal cells,the DUOX system can produce reactive oxygen species(ROS)which can directly destroy the DNA,RNA,proteins and lipids of microorganism and cause the degradation of these substances to kill these microorganisms.The DUOX system is regulated by multiple pathways including G protein-coupled receptor(GPCR)pathway.GPCR is a type of 7 transmembrane receptor that is widely distributed in a variety of animals and plays very important roles,such as vision,smell,taste and immunity.In the DUOX expression pathway regulated by GPCR,after the activation of GPCR by ligand,the activity and expression level of DUOX can be separately regulated via a rapid Ca2+ pathway and a slow expression regulation pathway,thereby increasing the level of ROS produced by DUOX and directly killing intestinal bacteria.However,it is not clear whether the system exists in shrimp and what are the receptors and ligands of the system.In this paper,we obtained 12 GPCRs by intestine transcriptome sequencing of Marsupenaeus japonicus.We then screened these GPCRs by analyzing hydrogen peroxide levels,ROS levels and the expression level of DUOXs after knockdown of the MjGPCRs.Two GPCRs,MjGPCR7 and MjGPCR10,which can regulate the intestinal ROS level and MjDUOXs expression in shrimp,were identified.We then conducted an in-depth study of these two GPCRs.We analyzed the tissue distribution of MjGPCR 7 and MjGPCR10 in M.japonicus and the expression pattern of these two GPCRs after oral infected with Vibrio anguillarum.It was found that the two GPCRs were highly expressed in heart,stomach and intestine.After oral feeding with V.anguillarum,the expression levels of these two GPCRs were increased.In order to confirm that the GPCRs we selected can really regulate the expression of DUOX and thus affect the production of ROS,we perforemed an RNA interference assay.When the expression of MjGPCR7 or MjGPCR10 was disturbed,the Ca2+ level in the cytoplasm of M.japonicus intestinal cells was significantly decreased after oral delivery with V.anguillarum.In addition,when the GPCR downstream protein MjGaq was disturbed,the expression levels of MjDUOXl and MjDUOX2 in the intestinal cells were decreased and the level of ROS was also decreased.Finally,in order to find out the ligands of MjGPCR7 and MjGPCR10,we did a preliminary exploration experiment.The CLECT domain at the N-terminus of MjGPCR7,can bind to different bacteria.One h after oral feeding of adenine,guanine and cytosine,the relative levels of ROS in the intestinal tract of M.japonicus did not change significantly.However,the relative levels of ROS increased significantly 1 h after oral feeding of V.anguillarum,thymine and uracil.All these results suggested that the two MjGPCRs(MjGPCR7 and MjGPCR10)we screened can regulate the expression of MjDUOXs through MjGaq and Ca2+ pathway,up-regulate the level of ROS in the intestinal tract of M.japonicus and thus affect the homeostasis of shrimp intestinal microbiota.In addition,we also found that thymine and uracil can induce the production of ROS in the intestinal tract of M.japonicus.and these micro-moleculars may be ligands for MjGPCR7 and MjGPCR10. |
PDF Full Text Request