| At present, in aquaculture, the wide use of antibiotics results in the development of resistance ofpathogen to antibiotics. The antibiotic residues and enrichment in issues can bring the risk to the qualitysafety of aquatic products and human health. To find an efficient, safe and new broad-spectrumantimicrobial agent is very important to instead the traditional antibiotics. Antimicrobial peptides with aremostly the short cationic peptides, high temperature resistant and broad-spectrum antimicrobial activity, aswell as the appearance of the drug resistance, which can be used as the potential antibacterial agent tosubstitute the antibiotics. Antimicrobial peptides have been used in medicine, animal husbandry and foodpreservation. So far, the antimicrobial peptides have been reported the more than the1000kinds, mostlywith the amphiphilic cationic α-helix structure of antimicrobial peptides. However, in fish, the studies onantimicrobial peptides are still relatively few.In this study, based on the conservative sequences of H2A genes from GenBank, the primers weredesigned using Primer5.0to amplify the gene H2A in Carassius aurutus in Qihe River. The extracted totalRNA in fish muscle was used as the template to amplify the coding sequence of H2A by RT-PCR Thetarget sequences were connected to the PMDTM-19vector, transferred into JM-109competent Escherichiacoli, then cloned and sequenced. At last, the364bp coding sequence of histone H2A was successfulobtained. The same procedure was used to clone the histone H2A gene sequences in Mylopharyngodonpiceus, Ctenopharyngodon idellus, Hypophthalmichthys molitrix and Aristichthys mobilis means. It wasfound that proteins are unstableby online software analysis, with the average hydrophilic range of0.179~0.264and PI value range of10.48~10.48. The homology of the histone H2A genes was analyzedusing software DNAman, with the homology of93%for coding sequences, and the homology of99%foramino acid sequences. Compared with Sinocyclocheilus angustiporus, the homology is92%, and withDanio rerio is83%. Compared with the S. angustiporus for amino acid, the homology is98%, with D. rerio,is91%. But, compared with the other species, the homology was lower.The H2A N-terminal2~52(except the first methionine) from C. aurutus in Qihe River, M. piceus,C. idellus, H. molitrix and A. mobilis were corresponding with the antibacterial peptide hipposin with51amino acids from Hippoglossus Hippoglossus, the antimicrobial peptide hipposin is without the acidicamino acid, and with15positivecharges. However, the cysteine in the first position of hipposin was replaced by the serine in C. aurutus in Qihe River, M. piceus, C. idellus, H. molitrix and A. mobilis and thehistidine in Site41of hipposin was replaced by the glutamate, with the14cations. The predicted spatialstructure model is with alpha helix structure. The histone H2A in C. aurutus in Qihe River was predictedusing the cutting tools ExPaSy, to be hydrolyzed in Site52into the short peptides by chymotrypsin, pepsin,proteinase K and thermophilic bacteria protease. The methionine in the first position can be hydrolyzed bypancreatic curd protease and removed by cyanogen bromide. The antibacterial peptide sequence with51amino acids in C. aurutus was corresponding with the hipposin in H. Hippoglossus, named Ca-L-hipposinantimicrobial peptides. The like-hipposin was formed in other fish (e.g., M. piceus, C. idellus, H. molitrixand A. mobilis) by the corresponding hydrolysis proteases. It was indicated that the like-hipposinantimicrobial peptide was derived from the N-terminal sequence of the histone H2A.The histone H2A ORF sequence in C. aurutus is387bp, encoding128amino acids. The3’-UTRcontains466bp, with AATAAA poly including a termination signal loci, the5’-UTR contains55bp, thelength of the histone H2A complete cDNA was908bp. The histone H2A in C. aurutus belongs to cationicα-helix antibacterial peptide. The spatial structure of histone H2A was similar with Salmo salar,Oncorhynchus mykiss and D.rerio with96%. The histone H2A and the derived like-hipposin antibacterialpeptide in C. aurutus wrer researched to provide the basis for the further studies.The mRNA expression levels of histone H2A in the various organs and tissues (liver, kidney,brain, spleen, gill, heart, muscle and intestinal) in C. aurutus in Qihe River was detected using real-timefluorescence quantitative RT-PCR technology. The results indicated that the histone H2A mRNA expressionlevel was the highest in kidney, the second was spleen, then was gill, brainliver, intestinal and heart in order,and the least in muscle.The C. aurutus in Qihe River was infected by by intraperitoneal injection of Aeromonashydrophila. The real-time fluorescence quantitative RT-PCR and the2-ΔΔctmethod were used to detectedthe responses of expression levels of histone H2A and antimicrobial peptides Ca-L-hipposin geneafterchallenged with the A. hydrophila. The histone H2A and Ca-L-hipposin expressed significantly in liver,spleen and kidney (P<0.01**). The expression data of H2A and Ca-L-hipposin at different times have beenmade a scatterplot, and the curve fitting analysis was performed indicating the positive correlation. Theresponse of H2A and Ca-L-hipposin to A. hydrophila infection present in the similar pattern. It was demonstrated that the Ca-L-hipposin and H2A mRNA were not expressed independently respectively, and itwas speculated that the antibacterial peptide Ca-L-hipposin was produced by enzymatic hydrolysis ofhistone H2A. In addition, the sensitive responses of Ca-L-hipposin to bacterial infection indicated that theCa-L-hipposin plays an important role in the fish immunity. Antimicrobial peptide produced by the generecombined expression, as a potential antimicrobial regent, will be used promisingly in aquaculture. |
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