Molecular Cloning, Expression And Phylogenetic Analysis Of The Tyrosinase Gene Family In The Pacific Oyster (crassostrea Gigas)

The Pacific oyster （Crassostrea gigas）, a commercially important shellfish inChina, is widely farmed. In this study, we cloned a tyrosinase gene and analyzed itsexpression in different tissues. The phylogenetic and classification of the tyrosinasegene family in the Pacific oyster is also analyzed. The results are summarized asfollows:1Molecular cloning and characterization of tyrosinase gene CgTyr2from thePacifc oysterThe cDNA of CgTyr2from the Pacific oyster was cloned by RACE techniques.The full-length cDNA of CgTyr2was2563bp, including an open reading frame （ORF）of2100encoding a mature peptide699amino acids with predicted molecular weightof77.4KDa and theoretical isoelectric point of-3.3. BLAST analysis revealedCgTyr2shared a relative high similarity with other known tyrosinase in the shellfish.Our comprehensive phylogenetic reconstructions supported that hemocyaninspossible evolved from a tyrosinase by an ancient gene duplication followed byfunctional differentiation, and the current large number of tyrosinase isoforms in C.gigas and other mollusks were originated from multiple gene duplication events thattook places before and after mollusk species were established. We also showed thatCgTyr2gene was differentially expressed with the highest level of expression inmantle edges, suggesting its potential role in the formation ofperiostracum/pigmentation.2The cloning of the four other tyrosinases partial genes from the Pacific oysterBy using BLAST in the oyster genome database, tyrosinase geneOYG₁0017214, OYG₁0011916, OYG₁0021075and OYG₁0026226wasobtained corresponding to14TYR,16TYR,75TYR and26TYR. The sequences from the oyster genome were used for designing the primers. By PCR and sequencing,14TYR,16TYR,75TYR and26TYR sequences were got. By tblastx from NCBI,14TYR and16TYR are both most similar to Pinctada martensi tyrosinase-like proteintyr-2mRNAgb （Accession number: KC912764.1）;75TYR and26TYR are both mostsimilar to partial cds Pinctada martensi tyrosinase-like protein tyr-1mRNA（KC870906.1）.3Phylogenetic and classification analysis of tyrosinase gene family in the PacificoysterThe deduced amino acid sequence characteristics, classification and phylogenyof tyrosinase gene family in the Pacific oyster were analyzed using bioinformaticsmethods. The results showed that gene duplication was the major cause of tyrosinasegene expansion in the Pacific oyster. The tyrosinase gene family in the Pacific oystercan be further classified into three types: secreted form （Type A）, cytosolic form（Type B） and membrane-bound form （Type C）. Further analysis suggested that TypeA tyrosinases of C. gigas clustered with those from cephalopods and then withnematodes and cnidarians. Type B tyrosinases were generally clustered with the sametype of tyrosinases from molluscas and nematodes, and then with those fromplatyhelminths, cnidarians and chordates. Type A tyrosinases in the Pacific oyster andthe Pearl oyster expanded independently and were divergent from membrane-boundform of tyrosinases in chordata, platyhelminthes and annelida. These observationssuggested that Type C tyrosinases in the shellfish had a distinct evolution direction.