Screening Of Aeromonas DNA Barcode And Analysis Of Cyprinus Carpio Koi Antibacterial Immune Response

Aeromonas is a kind of pathogenic bacteria widely existing in aquatic environment,which is a common disease of human,livestock and aquatic animals.DNA barcoding can effectively and accurately identify Aeromonas bacteria and provide scientific and accurate basis for rational selection of anti-infective drugs.The frequent outbreak of Aeromonas infection disease seriously endangers human health and limits the development of aquaculture industry.Understanding the genes and pathways that play an important role in host immune response and exploring the sequence information and tissue distribution of key genes in immune response after Aeromonas veronii infection can provide the basis for the discovery of drug targets and the development of antibacterial drugs and provide a new idea for the comprehensive prevention and treatment of pathogenic bacteria.In this study,33 strains of Aeromonas were used as test materials,and the ppsA,recA and cpn60 genes of different strains were amplified by PCR with the designed universal primers,and the sequencing information of the sequence was obtained and analyzed.Using PCR amplification success rate and sequencing success rate,genetic distance,DNA barcoding gap and phylogenetic tree as evaluation criteria,it aims to screen barcode genes with good versatility and high accuracy.We found that the variation sites of cpn60 gene accounted for27.68% of the total bases,which could provide more species identification information;the intraspecific genetic distance of 97.9% sequences of cpn60 gene was less than 0.04,while the interspecific genetic distance of 98.1%sequences was greater than 0.04,and the interspecific genetic distance of cpn60 was 13.3%,which was significantly greater than 1.7%.The bar code interval of cpn60 is obvious,which can effectively distinguish different strains in the same genus.The average interspecific genetic distance of recA gene is 0.049,which is larger than the average intraspecific genetic distance of 0.023,but the recA gene has a large degree of genetic distance overlap,and there is no obvious barcode gap.The success rate of ppsA gene amplification and sequencing is too low and its versatility is poor,so it cannot be used for the identification of Aeromonas.In summary,the cpn60 gene can be used as a DNA barcode for Aeromonas to identify different species.In order to better understand the immune response of koi carp to pathogenic stimuli,we performed transcriptome sequencing on the spleen of koi carp artificially infected with Aeromonas veronii for 24 hours.Differential expression analysis screened a total of 373 differentially expressed genes(DEGs),of which 240 genes were up-regulated and 133 genes were down-regulated.The expression levels of NLRP3,CD11 b,CD11c and IL-1β,which are closely related to inflammasomes,were significantly up-regulated.Further annotation and enrichment analysis showed that DEGs were mainly enriched in phagosomes,cell adhesion molecules,IL-17 signaling pathway,and antigen processing and presentation.The above-mentioned molecules and signal pathways may play an important role in the elimination of invading pathogens.To verify the accuracy of the RNA-seq data,10 immune-related genes were selected for RT-qPCR experiments,and the results showed that their expression was highly consistent with the RNA-seq data.Additionally,RT-qPCR also confirmed that caspase-1,caspase-3,caspase-9,CARD and ASC genes were up-regulated.These genes were considered to be related to the assembly and activation of NLRP3 inflammasome,indicating that Aeromonas veronii challenge significantly induced the assembly and maturation of NLRP3 inflammasome in the host spleen.TLR4(Toll-like receptor 4)is the main receptor of lipopolysaccharide.After being stimulated,it can cause the release of a series of inflammatory factors and activate the immune response.We cloned the koi carp TLR4 gene sequence.The full-length c DNA sequence is 1237 bp,the open reading frame is 432 bp,and it encodes 143 amino acids.The bioinformatics software predicted that the molecular weight of the amino acid sequence of koi carp TLR4 was 16114.2,and the theoretical isoelectric point was 5.87.In comparison with other species,the amino acid sequence similarity of koi carp TLR4 and common carp TLR4 was the highest(98.43%),which indicated that koi carp had the closest genetic relationship with common carp.Tissue expression analysis showed that TLR4 gene expression in spleen and intestine was 789 times and 466 times higher than that in muscle,respectively.