Cloning And Expression Analysis Of Two Antimicrobial Peptide Genes From Hyriopsis Cumingii

According to the EST sequence in cDNA library in our lab, the full-length cDNAsequences of theromacin and big defensin gene were cloned in the triangle-shell pearlmussel Hyriopsis cumingii by the RACE-PCR，then the characteristics of the two aminoacids were analyzed.The cloned beta-actin gene as internal gene, half-quantitative PCR detected thedifferential expression of the two genes in eghit tissues, mantle, blood, liver, kidney,stomach, intestine, gill and foot respectively in Hyriopsis cumingii, and after infected byAeromonas hydrophilia, real-time quantitative PCR studied the two genes expression inmantle, blood, liver, intestine, gill and foot at different time points. By constructedexpression vector of two antimicrobial peptide genes, IPTG, as the inducer, induced therecombinant plasmids expressing in the expression strain E.coli BL21(DE3), and thenanalyzed preliminarily by SDS-PAGE. The main research results are as follows:1) cloning and molecular characteristics analysis of theromacin geneA970bp cDNA sequence contained a123bp5’-untranslation region,526bp3’-untranslation region and300bp open reading frame(ORF), which encoded99aminoacids with a signal peptides of27amino acids and a mature peptides of72amino acids,and molecular mass is10924.7u. Amino acid sequence analysis shows that thesequence exists an obvious transmembrane and a hydrophobic region. Homologyanalysis indicates that this full-length amino acid sequence showed the highestsimilarity with Aplysia californica theromacin (56%), lower similarity with suchasdefensins,mytilins,myticins and mytimycins in.mussels, so speculated that this genebelongs to theromacin gene families of antibacterial peptides.The prediction results of Jpred3software for H.cumingii theromacin proteinsecondary structure showed that the places of2-22,57-62amino acid residues exist thealpha helix, accordingly, theromacin in H.cumingii was the membrane protein containng transmembrane spiral. Hydramacin-1A chain tertiary structure of Hydramagnipapillata as a template, tertiary structure of theromacin protein was forecastthrough the ESyPred3D and the results showed, the tertiary structure of this protein wassimilar to Hydramacin-1.2) cloning and molecular characteristics analysis of big defensin geneA604bp cDNA sequence contained a canonical polyadenylation signal sequenceAATAA and a poly（A）tail，166bp5’-untranslation region,96bp3’-untranslation regionand342bp open reading frame(ORF), which encoded113amino acids with a signalpeptides of23amino acids and a mature peptides of90amino acids, and molecularmass is12.5kDa. Amino acid sequence analysis shows that the sequence exists anobvious transmembrane and a hydrophobic region. Homology analysis indicates thatthis full-length amino acid sequence showed the highest similarity with amphioxus（Branchiostoma floridae）(64%), lower similarity with such known defensins（α-、β-、θ-defensins and insect defensins）. Multiple alignment of the known big defensins,revealed that the consensus conserved pattern C-X6-C-X3-C-X13(14)-C-X4-C-C wasobserved in the mature peptide of all big defensins. The result of the constructedphylogenetic tree showed the big defensin and the known big defensins got together.The prediction results of Jpred3software for H.cumingii big defensin proteinsecondary structure showed that the places of7-35and43-66amino acids residues existthe alpha helix, while41-42,95-97and104-108amino acid residues exist the beta sheet.So big defensin in H.cumingii was the membrane protein containng transmembranespiral. Using the tertiary structure of beta-defensin from Japanese horseshoe crab as atemplate reported by Takahide Kouno(PDB id：2RNG), the tertiary structure of bigdefensin was predicted by ESyPred3D, the two protein structures were similar.3) Semi quantitative and real-time quantitative analysisAnalysis of the tissue expression pattern of the theromacin and big defensin genewas detected by RT-PCR, beta-actin as inner control gene, in healthy mantle, blood,liver, stomach, intestine, gill and foot.Theromacin gene was all expressed in8tissues, the expression in blood was highest,liver and gills trace expression, and the expression in the kidney, stomach, intestine andfoot was less.Big defensin gene was not fully expressed in8tissues, only expression in mantle,blood, liver, kidney, stomach and intestine, while the relative expression in mantle and blood were higher, lower in the liver, kidney, stomach and intestine and no expressionin the gills and foot.2h,4h,6h,12h,24h,48h and72h after Aeromonas hydrophila infection thetheromacin, big defensin expressions were detected in mantle, blood, liver, intestine,gill and foot.In the mantle, theromacin and big defensin expressed highest respectively at4h and6h, and both lowest at the12h; in the blood, theromacin and big defensin both hadhighest expression at the72h, respectively, lowest at6h and48h; in the liver,theromacin at4h expressed highest, and big defensin at48h reached peak, both at the24h had lowest expression; in the intestine, the peak and lowest expression oftheromacin, big defensin were respectively at4h and24h,72h and6h; in the gill, bothof the peak and lowest was respectively at4h and24h,2h and6h; in the foot, the tworeached peak both at4h, lowest respectively at24h and12h4) prokaryotic expressionUnder37℃,1mmol/L IPTG inducted the expression of theromacin and bigdefensin recombinant plasmid in the expression strain, then SDS-PAGE tested, showed,both protein existence in the form of inclusion body, expression increased with theinduction time extension, both the largest expression at6h. The result shows that therecombinant plasmid could have expression in the expression strain E.c oli BL21(DE3)expression under induction condition.