Molecular Cloning And Expression Nanlysis Of Hemocyanin Gene From Sepiella Maindroni

Hemocyanin were copper-containing respiratory protein in the hemolymph of arthropods and mollusks.It has the function of carrying oxygen, storage protein, immunology and regulation of molting, and also hasa variety of enzymatic activity. In the present study, S.maindroni hemocyanin(designated SmHC) gene wascloned from S. maindroni de Rochebrune. specific primers were designed based on the sequenceinformation of related genes getted by EST sequencing methods. A series of molecular biology techniquessuch as RNA extraction, reverse transcription, PCR were used to clone the SmHC gene and analyse thegene’s sequence. Real-time fluorescence quantitative PCR was employed to assess the mRNA expressionof SmHC in various tssues, hypoxia stress, bacteria challenge and hydrogen peroxide challenge.The full-length cDNA of SmHC gene was1502bp, containing a5’ untranslated region (UTR) of51bp, a3’ UTR of160bp with a poly (A) tail, and an open reading frame (ORF) of10032bp encoding apolypeptide of3343. The predicted molecular weight of mature peptide is381kDa, and the theoreticalisoelectric point is5.75, containing8tyrosinase domains and metalloprotease theme of HExxxH. SMARTanalysis revealed that SmHC has100%homology respectively with Sepia officinalis(GeneBank accessionNo. ABD47515.1),99%with Sepia officinalis(GeneBank accession No. ABD47516.1) and99%homologyrespectively with Enteroctopus dofleini (GeneBank accession No. AAK28276.2), Haliotis tuberculata(GeneBank accession No. CAC20588.1), Nucula nucleus (GeneBank accession No. CAH10286.1),Aplysia californica (GeneBank accession No. CAD88977.1), Haliotis diversicolor (GeneBank accessionNo. ACU00134.1), Nautilus pompilius (GeneBank accession No. CAF03590.1), Megathura crenulata(GeneBank accession No. CAG28309.1).The mRNA transcript of SmHC could be detected in all the examined tissues with highest expressionlevel in gills. The expression of SmHC gene was decreased, then increased to the maximum value, and thendecreased again, during hypoxia stress, bacteria challenge and hydrogen peroxide challenge. These resultsshowed that SmHC was involved in respiration and had the function of carrying oxygen, and presumed thatSmHC could be involved in immune function and could possess catalase CAT and tyrosinase activity,providing an important basis for the variety of enzymatic activity of hemocyanin.