High-throughput identification of heavy metal binding proteins from the byssus of chinese green mussel (Perna viridis) by combination of transcriptome and proteome sequencing

Abstract

The Byssus, which is derived from the foot gland of mussels, has been proved to bind heavy metals effectively, but few studies have focused on the molecular mechanisms behind the accumulation of heavy metals by the byssus. In this study, we integrated high-throughput transcriptome and proteome sequencing to construct a comprehensive protein database for the byssus of Chinese green mussel ( Perna viridis ), aiming at providing novel insights into the molecular mechanisms by which the byssus binds to heavy metals. Illumina transcriptome sequencing generated a total of 55,670,668 reads. After filtration, we obtained 53,047,718 clean reads and subjected them to de novo assembly using Trinity software. Finally, we annotated 73,264 unigenes and predicted a total of 34,298 protein coding sequences. Moreover, byssal samples were analyzed by proteome sequencing, with the translated protein database from the foot transcriptome as the reference for further prediction of byssal proteins. We eventually determined 187 protein sequences in the byssus, of which 181 proteins are reported for the first time. Interestingly, we observed that many of these byssal proteins are rich in histidine or cysteine residues, which may contribute to the byssal accumulation of heavy metals. Finally, we picked one representative protein, Pvfp-5-1, for recombinant protein synthesis and experimental verification of its efficient binding to cadmium (Cd 2+ ) ions.

Genomics and transcriptomics of the green mussel explain the durability of its byssus

“…Thus, Pvfp-3 is likely to exist as a multiple copy gene, as suggested in M. galloprovincialis fp-3 39 , and the copies were generated through tandem duplications. Pvfp-4, reported by Zhang et al 23 (GGRR01022802) almost matched the protein encoded by pvir_s00068g44 (Supplementary Fig. S9).…”

“…Three major pre-collagen genes reported by Zhang et al 23 were discovered among the genome scaffolds. Col-D and Col-NG genes were identified close together on the same scaffold (pvir_s00637g8 and pvir_s00637g10), suggesting generation of the two genes through tandem duplication (Supplementary Figs.…”

“…Interestingly, pvir_s00637g9, the gene between the two collagen genes encodes a highly glycine-rich protein, but it is not a collagen because the glycine residues are not arranged in a characteristic G-X-Y repeat. Col-P 23 corresponded to pvir_s00107g4, although there are some differences in predicted N- and C-terminal sequences (Supplementary Fig. S4).…”

“…Genes encoding major fps reported in the green mussel, i.e., those for Pvfp-1 through -6 were searched in the green mussel genome scaffolds using previously reported sequences 23 , 24 as queries (Table 4 ). Known sequences of Pvfp-1 were aligned to different parts of the protein encoded by a single gene, s00107g6, identified in this study (Supplementary Fig.…”

“…Using genome sequence information with the transcriptomic data, we identified genes involved in byssus formation. Although transcriptomic and proteomic studies have been reported in this species previously 23 , 24 , the combination of genomic and foot-specific transcriptomic analyses were expected to yield information specific to characteristics of the byssus. After confirming that our data contained genes encoding known fps and collagens, we listed genes exclusively expressed in the foot, the site of byssus production, to find more components involved in byssus formation.…”