Cloning And Expression Analysis Of Two Antimicrobial Peptide Genes From Anodonta Woodiana

According to the cDNA sequence of theromacin gene in the Hyriopsis cumingii, thefull-length cDNA sequences of theromacin、mytimacin and β-actin gene were cloned inAnodonta woodiana by the RACE-PCR，then the characteristics of the three amino acids wereanalyzed. The cloned beta-actin gene as internal gene, RealTime-PCR detected the differentialexpression of the two genes in six tissues, blood, liver, mantle, kidney, gill, foot and intestinerespectively in Anodonta woodiana, and after infected by Aeromonas hydrophilia, real-timequantitative PCR studied the two genes expressions in blood, liver, mantle, kidney, gill, footand intestine at different time points. The main research results are as follows:1. cloning and molecular characteristics analysis of theromacin geneA870bp cDNA sequence contained a104bp5’-untranslation region,460bp3’-untranslation region and306bp open reading frame(ORF), which encoded101amino acidswith a signal peptides of27amino acids and a mature peptides of74amino acids, andmolecular mass is11166.9U. Amino acid sequence analysis shows that the sequence exists anobvious transmembrane, but a hydrophobic region. Homology analysis indicates that thisfull-length amino acid sequence showed the highest similarity with Hyriopsis cumingiitheromacin (73%), lower similarity with such as defensins, mytilins, myticins and mytimycinsin mussels. So speculated that this gene may not belongs to the same type as the antimicrobialpeptides found in mussels.Analysis of the tissue expression pattern of the theromacin gene was detected byRealTime-PCR, beta-actin as inner control gene, in healthy blood, liver, mantle, gill, foot, andintestine.Testing results showed that, theromacin gene was all expressed in6tissues, theexpression in mantle was highest, and the expression in the blood, liver and gill were less.After challenged with A.hydrophila, the expression of theromacin gene increasedsignificantly in6tissues at2h,4h,6h,12h,24h,48h and72h time. Testing results showedthat, theromacin gene was all expressed in6tissues，and the expression levels increasedsignificantly in early infected and then down-regulated. 2. cloning and molecular characteristics analysis of mytimacin geneA466bp cDNA sequence contained a261bp open reading frame(ORF), which encoded86amino acids with a signal peptides of24amino acids and a mature peptides of62amino acids,and molecular mass is9550.0U. Amino acid sequence analysis shows that the sequenceexists an obvious transmembrane and a hydrophobic region. Select the amino acid sequenceof the respective genes of different species by MEGA5.0sofeware phylogenetic tree wasconstructed using the neighbor joining methord and found that the mytimacin gene belongs tothe Macin family.Analysis of the tissue expression pattern of the mytimacin gene was detected byRealTime-PCR, beta-actin as inner control gene, in healthy blood, liver, mantle, gill, foot, andintestine.Testing results showed that, mytimacin gene was all expressed in6tissues, theexpression in mantle was highest, and the expression in the blood, liver and gill were less.After challenged with A.hydrophila, the expression of mytimacin gene increasedsignificantly in6tissues at2h,4h,6h,12h,24h,48h and72h time. Testing results showedthat, mytimacin gene was all expressed in6tissues，and the expression levels increasedsignificantly in early infected and then down-regulated.3. cloning and molecular characteristics analysis of β-actin geneA1392bp cDNA sequence contained a66bp5’-untranslation region,195bp3’-untranslation region and1131bp open reading frame(ORF), which encoded376aminoacids with a signal peptides of29amino acids and a mature peptides of347amino acids, andmolecular mass is41791.7U. Amino acid sequence analysis shows that the sequence existsan obvious transmembrane, but a hydrophobic region；and there have a high amino acidsequence similarity with other species.Analysis of the tissue expression pattern of the β-actin gene was detected by RT-PCR, inhealthy blood, liver, mantle, gill, foot, and intestine.Also analysis of the tissue expressionpattern of the β-actin gene was detected by RT-PCR, in6tissues of A.woodiana that afterinfected by A.hydrophila.Testing results showed that, β-actin gene was all expressed in12tissues, and the expressions were no significant differences.Analysis of the tissue expression pattern of the β-actin gene was detected byRealTime-PCR, in healthy blood, liver, mantle, gill, foot, and intestine.Testing results showedthat the expressions were also no significant differences.