Analysis Of Antimicrobial Immune Response Mechanism Of Largemouth Bass Based On Transcriptome

Largemouth bass(Micropterus salmoides),belonging to Centrarchiformes,Centrarchidae and Micropterus,is an important freshwater breeding species in China.In recent years,with the expansion of breeding scale and the increase of intensification,bacterial and viral diseases occur frequently in the growth process of largemouth bass,and the abuse of antibiotics is becoming more and more common.In addition to the economic loss,it also caused pollution to the water environment and seriously restricted the development of largemouth bass culture industry.In depth study on immune-relevant genes and immune response molecular mechanism of largemouth bass,using immunological methods to prevent and control diseases is of great significance for the healthy and efficient cultivation of largemouth bass,and the development of an environmentally friendly antibacterial drug instead of traditional antibiotics is an effective measure to solve the bacterial resistance and water environmental pollution.In this study,Lipopolysaccharide(LPS)and polyinosinic-polycytidylic acid(poly I:C)were used to simulate bacterial and viral stimulation of largemouth bass,respectively Spleen was selected for RNA-Seq and transcriptome analysis.The transcriptome data were screened and mined to obtain a large number of key genes of immune-relevant signaling pathways,such as Toll-like receptors(TLRs)TLR1,TLR2,TLR3,TLR5 M,TLR5S,TLR7,TLR8,TLR9,TLR13,TLR14,TLR20,TLR21 and TLR22,Interleukins(ILs)IL-1β,IL-2,IL-6,IL-7,IL-8,IL-12,IL-15,IL-16,IL18,IL-27,IL-34.Also,a variety of antimicrobial peptide genes,including Piscidin,Defensin,Hepcidin,Leap-2 and NK-Lysin.Based on the partial gene sequence obtained from transcriptome mining,8 TLRs(Ms TLR1,Ms TLR2,Ms TLR3,Ms TLR5 S,Ms TLR8,Ms TLR9,Ms TLR21 and Ms TLR22),3 ILs(MSIL-1β,MSIL-15 and MSIL-16)and an antimicrobial peptide(Ms Piscidin-3)full-length c DNA were cloned by RT-PCR(Reverse Transcription Polymerase Chain Reaction)and RACE(Rapid Amplification of c DNA Ends)Each c DNA sequence contained two in-frame stop codons,with one upstream of the start codon and one downstream of the stop codon,indicating that complete open reading frames(ORFs)had been obtained.Sequence analysis and multi-sequence alignment revealed that all Ms TLRs except Ms TLR5 S had typical TLR family structural characteristics,including several tandem leucine-rich repeats(LRRs)in extracellular domain,transmembrane domain and three conserved box motifs in Toll/IL-1 receptor(TIR)domain in intracellular domain.Ms ILs all have conserved domain,with N-terminal prodomain,IL-1 superfamily and IL-1 family signature in Ms IL-1β,IL-15 superfamily domain in Ms IL-15 and two PDZ domains in Ms IL-16.Ms Piscidin-3 has the antibacterial12 domain,which is essential for its antibacterial activity.Homology and phylogenetic analysis showed that Ms TLRs and Ms ILs had high homology with other teleosts of Perciformes,which was consistent with the classification status of largemouth bass.Based on sequence alignment and phylogenetic tree,the antimicrobial peptide gene obtained was classified as Group I piscidin-3,named Ms Piscidin-3.Specific primers were designed according to the obtained c DNA.The expression of Ms TLRs,Ms ILs,and Ms Piscidin-3 in normal tissues(brain,gill,head kidney,intestine,liver,muscle,epidermis,and spleen)of largemouth bass and the expression changes in spleen after LPS and poly I:C stimulation were detected by q PCR(Quantitative Real-Time PCR).The results showed that Ms TLRs,Ms ILs,and Ms Piscidin-3 were constitutive expression in all tested normal tissues,but there were differences in expression levels.Ms TLR1,Ms TLR2,Ms TLR8,Ms TLR9,Ms TLR21 and Ms TLR22 had the highest expression in spleen,Ms TLR3 had the highest expression in intestine,Ms TLR5 S had the highest expression in liver,and Ms TLR1 also had a high expression level in intestine.Ms TLR21 was also highly expressed in muscle.The expression levels of Ms IL-1 β,Ms IL-15 and Ms IL-16 were the highest in skin,muscle and head kidney,respectively.The expression levels of Ms IL-1β were also higher in gills,Ms IL-15 in gills and intestines,and Ms IL-16 in spleen.The expression of Mspiscidin-3 was highest in gills,and also higher in intestine and skin.It is speculated that all Ms TLRs,Ms ILs and Ms Piscidin-3are closely related to the immune response of largemouth bass.Ms TLR1,Ms TLR3,Ms IL-1β,Ms IL-15 and Ms Piscidin-3 may play an important role in the mucosal immunity of largemouth bass.Ms TLR5 S may play a role as a bridge between metabolism and immune response of largemouth bass,and Ms TLR21 and Ms IL-15 may also be involved in physiological activities of largemouth bass.After LPS and poly I:C stimulation,the expression levels of all Ms TLRs,Ms ILs and Ms Piscidin-3 were upregulated to varying degrees,confirming that all of them were involved in antibacterial and antiviral immune responses of largemouth bass.Based on the sequence of Ms Piscidin-3,a derivative peptide with short length and high stability was designed through amino acid site analysis and domain prediction.The above peptide was synthesized by solid-phase synthesis,and the purity was over 95%after purification.The molecular weight was 2245.40 Da detected by mass spectrometry,which was consistent with the theoretical molecular weight,indicating that the target derived peptide was successfully synthesized.The antimicrobial activity of the synthetic peptide was detected by double dilution method.It had been showed that the synthetic peptide had a strong inhibitory effect on Aeromonas hydrophila,a certain inhibitory effect on Vibrio splendidus,and a general inhibitory effect on Staphylococcus aureus and Edwardsiella tarda.However,there was little inhibition against Escherichia coli and Aeromonas veronii.These results indicate that Ms Piscidin-3 derived peptides can inhibit and kill some pathogenic bacteria in aquaculture,which has the potential to be developed as antimicrobial drugs in aquaculture,and also provide a new idea for the development of drugs to replace antibiotics.