| Pteria penguin is one of the marine pearl oyster species with black shell which can cultivate large high quality sea pearls.In this paper,the full-length c DNA of Tyr and Bcl2 genes were cloned,and the expression patterns of different tissues in Pteria penguin were analyzed.The arbutin(a melanin inhibitor)with different concentrations was used to treat Pteria penguin for different times.Then the expression levels of Tyr,Creb and Bcl2 genes were compared after treatment of arbutin.The melanin in mantle was extracted to determine the content of melanin after arbutin treatment.The results were as follows:1.The full-length c DNA of Tyr gene was characterized from Pteria penguin by RACE-PCR.The full-length c DNA of Tyr was 1728 bp,including an open reading frame of 951 bp which encoded a deduced protein of 474 amino acids and a 5’UTR of11 bp,a 3’UTR of 295 bp.The analysis of the biological information software showed that,the predicted molecular weight was 52.78 k D,isoelectric point was 6.23;the transmembrane domain was from 5 to 27 amino acids;the signal peptide was from 1to 18 amino acids and the tyrosinase domain was from 131 to 324 amino acids.There were two Cu-binding regions,named Cu(A)/ Cu(B)in the tyrosinase domain.The amino acid sequence alignment showed that Tyr in Pteria penguin shared 50.4%sequence identity with Tyr in Pinctada margaritifera.However there was only 12.4%sequence identify with that of Octopus bimaculoides.The realtime-PCR showed that Tyr was constitutively expressed in different tissues.There was a highest level in mantle(P<0.05),and there was no significant difference between other tissues expression(P>0.05).The Tyr gene may be involved in the formation of shell color in Pteria penguin.After treated Pteria penguin with different concentrations of 10 m M,50 m M and 250 m M melanin inhibitor arbutin with 9 hours,18 hours and 36 hours,compared with the control,the expression of Tyr gene was not change in 10 m M and50 m M concentration for short time(9h),but reduced significant in 50 m M after 18 hours and was reduce with the increase of arbutin concentration and action time.The expression of Creb gene was not reduced with the low concentration arbutin,but was reduced in 250 m M concentration for 9h,18 h and 36 h.The results indicated Tyr and Creb gene played an important role in melanin synthesis of Pteria penguin.2.The full-length c DNA of Bcl2 was 1482 bp,including a 5’UTR of 355 bp,a3’UTR of 566 bp and an open reading frame of 561 bp,which encoded a deduced protein of 187 amino acids.The analysis of the biologcal information software showed that,the predicted molecular weight was 21.21 k D,isoelectric point was 4.77.The predicted Bcl2 protein comprised of a BCL domain and four BH domains including BH1,BH2,BH3 and BH4.The sequence alignment showed that Bcl2 in Pteria penguin shared 67.4% and 52.7% sequence identity with Bcl2 in Crassostrea gigas and Mytilus galloprovincialis,respectively.However there was only 18.0%sequence identify with that of Biomphalaria glabrata.The realtime-PCR showed that Bcl2 was constitutively expressed in different tissues.There was a highest level in testis,and foot in second,a lowest level in adductor muscle(P<0.05).After treated Pteria penguin with different concentrations of melanin inhibitor arbutin with 9h,18 h and 36 h,the expression of Bcl2 gene increased compared with the control.But there was no significant difference between treatment groups.The results indicated that arbutin didn’t induce apotosis of cells,and was relatively safe to Pteria penguin.3.After treatment of melanin inhibitor arbutin with different concentrations at10 m M,50 m M and 250 m M for 24 h in Pteria penguin,the melanin content in mantle was determined by ultraviolet spectrophotometer.The result showed that the melanin content reduced with the increase of arbutin concentration,which was consistent to the expression levels of Tyr,Creb and Bcl2. |
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