The Response Of Metallothionein To Heavy Metals And Its Transcriptional Mechanism Of Hyriopsis Schlegelii

Metallothioneins?MTs?are a family of cysteine-rich,low-molecular weight?6–7kDa?,inducible muti-functional proteins in which numerous?about 30%?cysteine residues.MTs are ubiquitous found in organisms including microorganism,plants and animals,and play important roles in detoxify non-essential heavy metals,balancing the concentrations of essential heavy metals and anti-oxidation.Hyriopsis schlegelii,which was introduced into China from the Biwa Lake of Japan in 1997,is a commercially important aquaculture species of freshwater mussel used for pearl cultivation.Due to their filter feeding habits and slow-moving,bivalves are directly and unavoidably exposed in aquatic environments containing heavy metals.To date,however,limited papers about using MTs of freshwater pearl mussels as bio-markers for environments polluted by heavy metals and the regulatory mechanisms of MT genes,are found.The contents,results and conclusions of this paper are as follows:1.In this study,Cu,Zn,Pb and Cd levels under normal conditions were determined in different tissues of different ages?from 1 to 3 years old?of H.schlegelii.The results indicated that the gill tissues contained the highest proportion of metals?about35%-60%?.The Cd concentrations in different tissues from mussels generally varied as follows:gills>digestive gland>mantle>visceral mass>foot.The Zn concentrations in different tissues from mussels generally varied as follows:gills>mantle?foot>digestive gland?visceral mass.However,there is no tissue-specific relationship of Cu and Pb concentrations.For example,the gills in one-year old mussels showed the highest Cu accumulation percent?60.06%?among the five tissues,however,this percent dropped to 20.17%in three year-old mussels.As a result,the highest Cu accumulation percent?about 35.12%?were found in visceral mass or digestive gland in three year-old mussels.As for the Pb content,the gills showed the highest accumulation rate among the parts tested,but not tissue-specific in other four tissues.In sum,the high percentage of heavy metals could be accumulated in H.schlegelii and its gill is the target tissue of heavy metals accumulation.Furthermore,one-year old mussels were exposed to 0,0.005,0.05 and 0.5 mg/L Cd²⁺for 1,3,5,and 7 days,to determine the relationship between Cd accumulation and MT levels in different tissues.Elevated Cd accumulation and MT concentrations are mainly observed in organisms in 0.05 and 0.5 mg/L Cd exposure.Both MT protein levels and Cd bioaccumulation appeared to be dose-responsive following Cd treatment for 1,3,5and 7 days,respectively;however,no time-dependent responses were found.Correlation analysis revealed that MT seem to be relevant biomarkers of Cd exposure due to its protein levels are highly correlated to Cd accumulation in tissues.Taken together,MT contents in tissues of H.schlegelii could be used as biomarkers to monitor the concentrations of Cd.2.Two metallothionein genes?HsMT1 and HsMT2?were first identified and described from H.schlegelii by molecular biology means.Thefull-length cDNAs of HsMT1 and HsMT2 were of 589 and 667 bp.The open reading frame?ORF?sequences of HsMT1 has 216 bp in length,encoding a hypothetical protein of 71amino acids with a predicted molecular mass about 7.14 kDa andisoelectric point?pI?of 7.24.The deduced Hs MT1 amino acid sequences contained 21 Cys residues which were calcuLated as 29.6%of the total amino acids,and lacked histidines and aromatic residues?phenylalanine and tyrosine?.It contained nine characteristic Cys-X-Cys,one Cys-X-X-Cys,six Cys-X-X-X-Cys and one Cys-Cys motif arrangementsandaconservedstructuralpattern Cys-x-Cys-x?3?-Cys-Thr-Gly-Cys-x?3?-Cys-x-Cys-x?3?-Cys-x-Cys-Argatthe C-terminus.Thefull length cDNA of HsMT2 consisted of an ORF 222 bp encoding a protein of 73 amino acids,which had a calculated molecular weight of 7.99 kDa and the theoretical pI of 4.58.The sequence of HsMT2 contained the characteristic eight Cys-X-Cys and seven Cys-X-X-X-Cys motifs,with cysteine contents?27.4%?,but lacked Cys-Cys motifs and aromatic acids?phenylalanine and tyrosine?.The structure of C-terminus was Cys-x-Cys-x?3?-Cys-Lys-Gly-x?3?-Cys-x-Cys-x?3?-Cys-x-Cys-His.Both complete sequences of HsMT1 and HsMT2 were submitted to GenBank under accession Nos.KJ019820 and KJ019821.The deduced amino acid sequences of HsMT1 and HsMT2 revealed strong homologies on positions of the abundant cysteine residues,though they only shared an identity of 46.5%.The phylogenetic tree showed HsMT1 shared a high similarity with that of other molluscs,but HsMT2 was split into a distinct group with zebrafish separated from known freshwater and marine molluscan MTs.HsMT1 exhibited constitutive expression in all examined tissues and the highest expression occurred in hepatopancreas,however,nearly all HsMT2 was just detected in gonad.After 5ppm Cd exposure,their m RNA levels presented similiar expression patterns with trends of“decrease-recovery-decrease”.The transgenic bacteria of HsMT1 showed higher tolerance than HsMT2 in Cd environment.It was implied that HsMT1 and HsMT2 were members of metallothionein family and involved in metal response but HsMT2 might have other physiological functions.3.To further investigate the transcription regulation mechanisms of metallothionein genes?HsMTs?of H.schlegelii,two different PCR strategies including Genome walking and Genome walker methods were employed.As a result,we obtained two fragments of 1,121 bp and 1,270 bp in size of the promoter regions of HsMT1 and HsMT2,respectively.Their nucleotide sequences have been submitted to the Gen Bank database under Accession Numbers KX279831 and KX279832,respectively.The two promoters shared partially conserved features and possessed distinct characteristics such as the number or position of metal response elements?MREs?.Analysis of the two regions revealed that both promoters had high A+T content?61.5%for HsMT1 and 60.5%for HsMT2,respectively?and contained a canonical TATA sequence?TATAAA?.One copy of metal response element?MRE,TGCGCAC?in the HsMT1 promoter was identified at-588 to-594 bp upstream of the start codon.On the other hand,two MREs?MREa and MREb?in HsMT2 promoter were identified.The proximal?MREa?and distal MRE?MREb?were located from-135 to-141 bp?TGCACAC?and from-540 to-546 bp?TGCGCAC?upstream of the start codon,respectively.Further analysis of the HsMT1 and HsMT2 promoters was performed by the reporter assay using the luciferase gene.Both promoters were activated by various metals,and presented different levels of metal ions inducibility in human hepatoblastoma cells?HepG2?.Deletion mutant assays demonstrated that both the longest promoter regions achieved the maximum inducibility,and the metal inducibility was dependent on the presence of the MRE in HsMT1 and the distal MRE in HsMT2.However,when the MRE in HsMT1 or the distal MRE in HsMT2 was absent,the metal inducibility was disappeared.4.In addition,we cloned a putative metal responsive transcription factor?hereby designated as HsMTF-like?and studied its effect on HsMTs expression in human HepG2 cells.The HsMTF-like cDNA showed a length of 2,033 bp and contained an open reading frame of 1,551 bp that encodes a putative polypeptide of 516 amino acids,with an estimated molecular weight of 57.32 kDa.Further analysis revealed that the Hs MTF-like possessed six putative zinc fingers regions that shared moderate similarity to that of human?39.44%?,mouse?39.44%?,pufferfish?38.89%?,and zebrafish?38.89%?.The nucleotide sequence of HsMTF was submitted to GenBank as Accession Number KY211621.To explore the possible effect of HsMTF-like on the expression of HsMT1 and HsMT2 in HepG2 cells,we constructed the eukaryotic expression vectors pcDNA-HsMTF-like and promoter-luciferase reporter vectors with mutated MREs,and then cotransfected into HepG2 cells.An in vivo assay demonstrated that Hs MTF-like activates basal HsMT1 or HsMT2 transcription level,and the MRE in the Hs MT1 or HsMT2 promoters mediates this activation process.Moreover,this basal transcription level can be further boosted by zinc treatment.Meanwhile,the two MREs in HsMT2 show different activity and contributions for the activation of HsMT2 gene.Combined the previous reports on the transcriptional regulation mechanisms of MT in vertebrate and Pacific oyster,we inferred that the regulation mechanisms for MT activation in H.schlegelii should be similar and evolutionarily conserved.