Regulation Mechanism On Grass Carp Intestinal Flora And Biological Characteristics Of Bacillus Subtilis H2

Functional ingredients such as Bacillus subtilis are used in aquaculture to improve fish condition,modulating microbiota and promoting a healthy intestinal system.However,the underlying mechanisms of grass crap intestine treated with B.subtilis are not fully characterized.An isolated strain was identified from sewage in this study as Bacillus subtilis and named as B.subtilis H2.The biological characteristics of the strain were investigated by physiological and biochemical tests,tolerance experiments,and whole genome sequencing.To explore its application in aquaculture,the grass crap was administered intragastrically with the B.subtilis H2then challenged with the pathogen Aeromonas hydrophila to analyse the regulatory mechanism of B.subtilis H2 on the intestinal microorganisms and immune system of grass carp using histopathology,16S intestinal community diversity,transcriptome sequencing and other methed.The main results obtained were:1.To obtain the microbes efficiently degrading pollutants,a new bacterial strain was successfully isolated that has the ability to degrade chemical oxygen demand（COD）from sewage efficiently.According to physiological,biochemical characteristics,and 16S rDNA sequencing phylogenetic analysis,the isolated strain is identified as Bacillus subtilis and named as B.subtilis H2.The isolate H2 was found to be highly tolerant to a wide range of temperature,pH,and salinity conditions,however,the optimal growth conditions were at 42℃,6.5,and 1,respectively.From the growth curve,it can be analyzed that the H2 grew rapidly and entered the logarithmic growth phase in 2 hours.2.To further explore the mechanism of action of this strain,the whole genome of B.subtilis H2 was determined in this study.The whole-genome sequencing of the B.subtilis H2 isolate resulted in 4 111 243 bp with GC content of 43.46%,encoding4 213 genes accounted for 87.67%of the gene coding region,including 31 rRNA and 87 tRNA genes.Comparative genomics analysis revealed,there were 3 551 core genes shared by this strain with B.subtilis 168.3.Grass carp were treated with B.subtilis H2 and normal saline.After 7 days,some intestinal microbial samples of grass carp were collected.The remaining grass carp were challeged with A.hydrophila respectively,and intestinal microbial samples were collected 3 days later.The composition of intestinal microorganisms in the above four groups of samples were analyzed by 16S sequencing.The 16S rDNA of the intestinal flora was found that he dominant bacterial phyla identified in all samples were,in descending order,Proteobacteria,Actinobacteria,Fusobacteria,Bacteroidetes and Acidobacteria.The proportion of the five dominant phylum bacteria in the infected group and the control group grass carp intestinal flora was over 90%.In addition,Aeromonas and Shewanella of A.hydrophila group accounted for more than the other three groups.A.hydrophila reduced the microbial diversity of intestinal tissues,and B.subtilis H2 had a significant regulatory protective effect on intestinal tissue.4.Grass carp were treated with B.subtilis H2（treatment group）and normal saline（control group）.After 7 days,intestinal RNA was extracted from grass carp.Sequential transcriptome sequencing of two samples with Illumina HiSeq^TM 2500.The grass carp after treated of B.subtilis H2,we found that the expression of the intestinal tissue gene was to be significantly,and comparative genome analysis showed 824 differentially expressed genes（DEGs）,including 365 up-regulated and459 down-regulated genes.Six DEGs were randomly selected for further validation by quantitative real-time RT-PCR and the results showed that were consistent with the RNA-seq data.Compared with the control group,many different genes related to immune response were found in the intestinal tissue,so this study also screened and verified eigth immune-related genes（IL-4,IL-11,IFN-α,CSF,FOSB,MAPK12b,IGHV3-11,IGHV3-21）and which were significantly up-regulated by B.subtilis H2effect.In addition,According to KEGG fat digestion and absorption pathway,we selected out eleven significantly up-regulated genes to analyze its fat digestion and absorption gene expression.The result show that the expression levels of almost all genes increase,especially Apob-48,ABCG8 and DGAT.In conclusion,B.subtilis H2 has a strong ability to degrade COD,improve the intestinal tract of grass carp and resist A.hydrophila.The protective effect to intestine is related to the fact that the bacteria can improve the ability of fat metabolism in the intestinal tract of grass carp.The results of this study provide a reference for the development of highly efficient microbial preparation of B.subtilis.