Identification Of The Silk Protein Of Emerald Mussel And Its Mechanism Of Mercury, Cadmium Resistance And Enrichment Behavior

Mussel,a common marine biovalves living in the wave of the stone reefs,belongs to phylum Mollusca.Due to the excellent adhension of byssal protein,mussels proved to be capable of binding heavy metals.It is estimated that there are more than 35 kinds of byssal proteins,but only a few of them have been discovered and identified.In the present study,transcriptome and proteome analysis of byssal protein?Perna viridis?was carried out in an attempt to explore the mechanism of heavy metal binding.Via Illumina transcriptome sequencing,73,571 unigenes were obtained,among them 37.7% unigenes being annotated.A total number of 34,298 protein coding sequences were predicted based on the alignment of known protein database and ESTscan software.Meanwhile,byssal proteins were analyzed by LC-MS/MS proteome sequencing with the protein database of P.viridis foot transcriptome as the reference for protein prediction.Consequently,we obtained 252 protein sequences.Finally,by means of homologous alignment of Mytilus edulis mussel protein,six and three proteins were identified in the transcriptome and protein database,respectively.Pvfp6 and Antistasin like protein were selected for further analysis due to their high content of cysteine,histidine and tyrosine,which may contribute to metal binding.In addition,Pvfp6 has elastase-specific inhibitor and serine protease inhibitor domains,which may protect other proteins from degrading by protease or peptidase.For Antistasin like protein,its high content of cysteine domain is similar to cys-x-cys in metallothionein,which has been confirmed to be able to enrich heavy metals.Genetic engineering technology was applied to study whether Antistasin like protein and Pvfp6 are heavy metal tolerant proteins.After two coding genes were respectively cloned into pET-30 a and then transformed into E.coli BL21?DE3?,the recombinant strains AL1 and P6 were constructed successfully.Then the mercury and cadmium resistance and quantity of equilibrium accumulation of each recombinant strain were evaluated.The results showed that both AL1 and P6 were able to grow in the presence of 5 mg/L Hg²⁺,or 120 mg/L and 160 mg/L Cd²⁺,respectively.Hg²⁺ equilibrium accumulation capacities of AL1,P6 and negative control E.coli BL21 reached 121.95 mg/g,109.89 mg/g and 93.46 mg/g respectively.Furthermore,there is no obvious difference in Cd²⁺ accumulation among three kinds of bacteria?20.92 mg/g,19.27 mg/g and 16.50 mg/g respectively?.