Studies On Antimicrobial Function Of Recombinant RNase1 In Blunt Snout Bream

Megalobrama amblycephala commonly known as blunt snout bream,is one of the important freshwater aquaculture fishes in China.M.amblycephala is susceptible to various pathogens,especially Aeromonas hydrophila,which led to severe economic losses.Pancreatic ribonuclease(RNase1)is a nucleic acid hydrolase secreted by the pancreas and has digestive function in phytophagous mammals.Our previous studies revealed that the recombinant RNase1 of M.amblycephala showed both antimicrobial and digestive activity.In this study,M.amblycephala was used as the target species to study the protective and immunity effect of recombinant RNase1 in fish.The NPN and ONPG fluorescent probes,scanning and transmission electron microscopic observation,A.hydrophila infection test,gene expression,enzyme content determination,proinflammatory factor concentration detection,RNase1 protein expression and 16 S rRNA sequencing were used to discover the antimicrobial function of the recombinant RNase1 in term of protective effect and immunity regulation of M.amblycephala against pathogenic bacteria infection.The main results are as belows:1.Gram-negative and Gram-positive bacteria were co-incubated with RNase1 protein as treatment groups.The fluorescence intensity of NPN fluorescent probe in treatment groups was increased gradually with time due to the destroyed of outer membrane and led to the increased of the permeability of outer membrane.The absorption value of ONPG fluorescent probe increased,the yellow colour of cell suspension deepened due to the damage of cell membrane and resulting in increased permeability of the inner membranes.24 hours after co-incubation of Gram-negative and Gram-positive bacteria with RNase1,by using scanning electron microscopy observation,the surface of Gram-negative and Gram-positive bacteria were shrivelled and irregular in shape,which resulted in the incomplete structure of bacterial cell membrane.From transmission electron microscopy observation,Gram-negative and Gram-positive bacteria were breaking up,cell deformed,cell wall boundary became blurred,and cytoplasm overflowed,RNase1 alter the permeability and integrity of bacterial cell membranes.2.After A.hydrophila infection,obvious clinical symptoms such as hemorrhage in the base of pectoral fins,caudal fins,gill,ascites,and hepatosplenomegaly were observed.The symptoms were not found in the control group(PBS injection)and the treatment group of recombinant RNase1 added 24 hours before infection.Six antioxidant genes(CAT,Cu/Zn-SOD,GCLC,GPx,GR and Nrf2)were significantly expressed in the liver and blood of healthy M.amblycephala.The Six antioxidant genes expression levels in the intestinal and blood of the treatment group was increased significantly after 12 hours infection.GSH content increased significantly in the treatment group,while GSSG content decreased significantly.Besides that,the activities of CAT and SOD increased in liver and spleen,while the contents of MDA and lysozyme decreased.The concentration of pro-inflammatory factors TNF-alpha and IL-6 in the treatment group was significantly increased in liver,blood,and kidney.After 12 hours of infection,the expression of RNase1 protein in liver,spleen,kidney and blood was higher in the treatment group.After12 hours of infection,the expression of RNase1 protein in liver,spleen,kidney and blood was higher in the treatment group.After pre-treatment with RNase1 protein for a period of time,it could alleviate oxidative stress induced by A.hydrophila infection.3.Based on 16 S rRNA V4 high-throughput sequencing,the relationship between intestinal microflora composition and abundance in control group(C),infection group(S)and treatment group(RS)was studied.A total of 1,523,379 high-quality sequences and1,068 OTUs were obtained from 30 sequencing libraries,including 15 phyla,30 classes,68 orders,135 families and 213 genera.PCoA analysis showed that the three experimental groups were distinctly differentiated due to different treatment conditions.At the phylum level,five dominant bacteria in the three experimental groups were Proteobacteria,Actinobacteria,Firmicutes,Fusobacteria,and Chlamydiae.RS group had the highest relative abundance in Proteobacteria.The relative abundance of Actinobacteria and Firmicutes were highest in C group and S group respectively.At the genus level,the five dominant bacteria were Cetobacterium,Bacillus,MNG7_norank,Gemmobacter,and Vibrio.In genus level: Bacillus was highest in S and C group.MNG7_norank and Vibrio were the highest in RS group.Predicting intestinal microbial function by PICRUSt showed that there were significant differences in the functions of intestinal microflora at level 1 and 2,The changes of microflora composition and function of intestinal microorganisms in fish were significantly affected by A.hydrophila infection,which may be related to recombinant RNase1 protein’s participation in immune response to pathogen invasion.