Indentifications And Expression Analyses Of Three Antimicrobial Genes In Megalobrama Amblycephala

NK-lysin are small cationic antimicrobial peptides produced by cytotoxic T lymphocytes and natural killer cells,homologous with human granulysin.They show strong antibacterial activity against a variety of targets including bacteria,fugi,virus as well as cancer cells.Histone H2 A plays a key role in nucleosome structure formation and transcription regulation in eukaryotic cells.The derived peptides of H2 A are also known to act against various microbial pathogens.Antimicrobial peptides,the important components of innate immune systems,have attracted scientist's attention because they can be served as novel antibiotics.Megalobrama amblycephala is an economically important freshwater fish cultured in China.But diseases break out frequently because of the intensive cultivation density and the deterioration of the breeding environment.A serious disease,bacterial septicemia caused by Aeromonas hydrophila,is a big threat to M.amblycephala,which might lead to a high mortality.In this study,two NK-lysins(nkla and nklb)and one H2 A gene were obtained from previous transcriptome sequencing data of blunt snout bream.The sequences were amplified and verified by PCR.We dectected their expressions during embryonic development,in different tissues and post infection of A.hydrophila to predict its possible function in immune response.In addition,the recombinant prokaryotic expression vectors of 2 NK-lysin genes was transformed into Escherichia coli and recombined to express,which laid foundation for further study of the function of NK-lysin.Overall,the main results are as follows:1.Gene indentifications and sequence analyses of 3 antimicrobial peptide genes(nkla,nklb and H2A)from M.amblycephala.The full-length opening reading frame(ORF)of the NK-lysin genes were confirmed by PCR and sequencing,which consisted of 369(nkla)and 411(nklb)nucleotides,encoding 122 and 136 amino acids,respectively.A signal peptide and the saposin B(SapB)domain were identified in both NK-lysins,which mean they belong to the saposin-like protein family.The corresponding molecular weights and isoelectric points(p I)of the mature proteins were 14 kDa/6.71(NKLA)and 15.6 kDa/6.72(NKLB).Both NK-lysins contain 6 cysteine residues and 3 disulfide bonds were predicted to be formed in each case,which is essential for their function.Phylogenetic tree analysis showed that the deduced amino acid sequence of NK-lysin of M.amblycephala showed high identity with zabrafish,followed by tongue sole and catfish.H2 A gene had an ORF region of 387 bp,enconding 128 amino acids,with isoelectric points(pI)of 10.92 and molecular weights of 16.2 kDa.The amino acid sequence of H2 A was conservative and no transmembrane region or signal peptide was found.2.The expression patterns of the 3 M.amblycephala antimicrobial peptide genes during embryogenesis and in healthy tissues.The real-time PCR results showed that the nkla and nklb mRNA were ubiquitously expressed in all early developmental stages and all detected tissues.During early developmental stages,the nkla and nklb mRNA were lowest expressed at the beginning,then showed a sharp rise after hatching.The expression of nkla and nklb peaked at eyeball pigment stage and 1 dph,respectively.H2 A mRNA was also widely expressed at all early developmental stages,with the highest expression apeared at 7 d.In different healthy fish tissues,nkla was highly expressed in the spleen,lowly in the muscle and blood and also dectected in the liver,brain,kidney,head kidney,intestine,gill and heart.While,nklb showed different expression pattern,with highest expression in the intestine and relatively low expression in the head kidney.The different expression pattern indicated their function diversity.H2 A was dectected in all the tissues,highest in the blood and lowest in the heart.3.The expression patterns of the 3 antimicrobial peptide genes after infected with A.hydrophila in mian immune tissues.After A.hydrophila infection,the M.amblycephala nkla and nklb shared the same expression trend.In the head kidney,the mRNA level decreased remarkablely at 4 h.In the liver,the expression increased to the peak at 4 h and then fell down beyond the normal level.While,in the spleen and intestine,the expression fell to the bottom at 12 h and then reached the peak at 72 h.These results suggest that NK-lysin might play a vital role in the M.amblycephala defense against A.hydrophlia infection.For H2 A gene,its expression showed an uptrend in the liver and intestine.While in the spleen and kidney,the expression firstly decreased,and then increased to the peak.4.Prokaryotic expressions of the M.amblycephala nkla and nklb.Three different prokaryotic expression vectors of nkla and nklb were successfully constructed and then transformed into BL21(DE3).Then,we tested the effects of different IPTG concentration,induced temperature,and induced time on the expressions of fusion protein and try to get the best induced condition.Fusion protein were dectected by SDS-PAGE gel.As for pET32a-NKLA and pET32a-NKLB,fusion protein pET32a-NKLA was only dectected in the participate.While,pET32a-NKLB was in both of the participate and solutision.But the expression system was unstable.For pET28a-nkla and pET28a-nklb expression system,the former was not found and the latter was slightly expressed in inclusion bodies.The third expression vector pGEX-NKLA and pGEX-NKLB showed the best results,because they were stable and objective protein was the highest in the total protein ratio.Fusion protein was mainly in the form of inclusion bodies.There was no significant difference in the expression level of the fusion protein at the different concentration of IPTG and each temperature.When the induced temperature was 37 ? and the IPTG concentration was 0.05 mmol/L,pGEX-NKLA showed the highest protein ratio of 33.8%.For pGEX-NKLB,the highest ratio was 48.8% at temperature of 37 ? and IPTG concentrations of 0.02 mmol/L.These studies provide information for NK-lysin's further functional research.