Selection Of Differential Expression Genes And Study Of Molecular Biomarkers Of Clam Venerupis Philippinarum Exposed To Benzo (a) Pyrene

Polycyclic aromatic hydrocarbons （PAHs） are ubiquitous environmental contaminantsand the environmental PAHs concentrations have been increasing particularly in aquaticenvironment. In present study, the toxic effect of benzo（a）pyrene （BaP） on clam Venerupisphilippinarum was studied and the molecular biomarkers were selected for PAHs pollutionassessment. The key genes of aryl hydrocarbon receptor （AhR）-mediated detoxificationmechanism, including AhR, aryl hydrocarbon receptor nuclear translocator （ARNT） and heatshock protein90（HSP90） were cloned and the effects of BaP on gene expression was studied inorder to explore the mechanism of detoxification of the clam. A subtracted cDNA library ofVenerupis philippinarum exposed to BaP was constructed by suppression subtractivehybridisation （SSH） for identification of differential expression genes. The molecular biomarkersof clam were selected and confirmed by analysis of gene expression. The resulting data mayprovide basic genomic information of the bivalve and the useful technology of biomonitoring ofPAHs by bivalves was supplied for PAHs pollution in aquatic environment.1Cloning and expression of the key genes of aryl hydrocarbon receptor （AhR）-mediateddetoxication mechanism of Venerupis philippinarum exposed to benzo（a）pyreneThe full-length VpAhR cDNA is2870bp, containing an open reading frame （ORF） of2391bp which encoded a polypeptide of796amino acids. The predicted amino acidsequences contain regions characteristic of AhRs from other species including basichelix-loop-helix （bHLH） and Per-ARNT-Sim （PAS） domains. Two nuclear translocationsequences can be identifed in the VpAhR sequence. Blast analysis showed that the deducedamino acid sequence of VpAhR shared high identity with other AhRs. The clam AhR mRNAexpression was detected in all the adult tissues tested （gill, digestive gland, adductor muscle andmantle） and highest transcription level was observed in gill compared to other tissues.The partial length VpARNT cDNA is485bp, containing an ORF of2391bp. The ORF encode a peptide of161amino acids. The predicted amino acid sequences contain a PAS domainwhich has a high identity with other species’ PAS domains. VpARNT mRNA expression wasdetected in all the adult tissues and the highest transcription level was observed in digestive gland.HSP90was an important member of molecular chaperone family. The cDNA partial length ofVpHSP90is687bp which is coding a peptide of299amino acids. An HSP90family conservedsignal region （GVVDSEDLPLNISRE） was detected in VpHSP90and it has a high identity withother species’ HSP90, such as C. farreri and A. irradians. The highest mRNA expression ofVpHSP90was detected in gill.Clams were exposed to different concentrations of BaP （0.01and0.2μg/L） for10d. Thegene expression of AhR, ARNT, HSP90and GST were increased with the enhancement ofBaP concentration and prolong of the experimental period, while there was no significantdifference of CYP4gene expression between control and BaP treatments. The potentialmolecular biomarkers were selected for further study.2Construction and analysis of subtractive cDNA library of Venerupis philippinarumexposed to benzo（a）pyreneForward and reverse subtracted cDNA library of Venerupis philippinarum exposed to BaPwere constructed. One hundred and ninety-four and ninety-three recombinants were collectedseparately from forward and reverse subtracted cDNA library. Thirty-one unigenes and twelvehypothetical proteins were identified from forward cDNA library and there were ten unigenesand four hypothetical proteins in reverse cDNA library. The cDNA library accession number ofthe clam is LIBEST₀27169. Analysis of multiple alignment and annotation showed that most ofthe SSH-selected genes were related to drug metabolic process, heterocycle metabolic process,cellular catabolic process, amine metabolic process, cellular nitrogen compound metabolicprocess and response to antibiotic.3Screening and confirmation of molecular biomarkers of Venerupis philippinarumexposed to benzo（a）pyreneA BaP （0.025,1and4μg/L） exposure for10d was conducted and the gene expression ofAhR related genes, SSH-selected genes and some functional genes of clam were tested by realtime quantitative PCR. Results showed that mRNA expression of twelve genes includingDefensin、Serine protease、Thioester protein、GST C、Mn-SOD、ARNT et al., wereup-regulated by BaP exposure and there was a significant linear relationship between BaP concentration and the gene expression, which indicated that these genes were suitablemolecular biomarkers of clam for PAHs assessment.Two sampling sites were selected according to the different PAHs pollution situation of thesea areas in Jiaozhou Bay and Dragon Bay. The PAHs contents of surface seawater and tissue ofclam and gene expression of biomarkers of clam sampled from different sites were analyzed.Results showed that the contents of total PAHs and BaP were higher in S1than S2. The geneexpression level of clams collected from S1was higher than S2which is similar to the results oflaboratory expreiment. It is indicated that the twelve genes were suitable molecular biomarkersfor PAHs pollution assessment.