CDNA Cloning And Induced Expression Of Metallothionein MRNA In Bay Scallop Argopecten Irradians

Bay scallop, A. irradians , was introduced to China from America in 1982 and was reintroduced in 1991. It has become one of main cultured species in the north of China. In the past years, scallop culture in China has been experiencing from a mortality problem, and suffering from a great economic loss. Although it is still unclear about the causes for the scallop mortality, deterioration water quality and deterioration of the stock are suspected to be the most important causes. With the improvement and control of the marine environment, enhancing the stress and disease resistance of scallop is commonly believed to be the resolution to the these problems. The cloning and expression of the genes involved in defense mechanisms are now considered to be a basic solution in the disease control because of their potential use in the development of therapeutic agents, study of defense mechanism and genetic improvement to increase the resistance to disease.Metallothioneins (MTs) are low molecular weight, cysteine-rich, inducible metal-binding proteins found in a variety of organisms. MTs play important biological functions such as protective role against excess reactive heavy metal ions, free radical scavengers and reservoir of essential metals that can be donated to other metalloproteins. Metallothioneins were suggested to be involved in detoxification processes triggered by stress. The cloning of the full lenth cDNA of A. irradians metallothionein was a base to study the evolution process of MTs and the diversity of mollusca MTs, as well as to understand the relationship between the structure and the function of MT. The analyses of the expression after stimulations were helpful for well understanding of the expression patterns of the metallothionein genes and the mechanism of scallop defense system, and further for the genetic selection of stress resistance.A partial sequence of the bay scallop Argopecten irradians metallothionein (AIMT) gene was identified from A. irradians cDNA library by EST (Expressed sequence tagging) method. A pair of gene-specific primers was designed according to this sequence and the full-length of AIMT cDNA was obtained using RACE (Rapid amplification of cDNA end) technique. The full-length cDNA of AIMT was of 664 bp including a 5' untranslated region (UTR) of 41 bp, a 3' UTR of 290 bp and an open reading frame (ORF) of 333 bp encoding a polypeptide of 110 amino acids. The predicted protein from the AiMT gene sequence contained 40 cysteine residues organized in Cys-X(n)-Cys(n=1-3) motifs as classically described for MTs. The contents of cysteine, glycine and alanine in the AIMT amino acids sequence were 25.5%, 10.9% and 10.9% respectively, while no aromatic amino acids or histidine residue was found. The CKCXXXCXCX motif, a conserved feature for invertebrate was found in two regions of the AIMT. A specific pattern of molluscan MT C-x-C-x(3)-C-T-G-x(3)-C-x-C-x(3)-C-x-C-K was located at the C-terminal, but with C-t-x(3) instead of C-T-G-x(3) and R instead of K. The result of sequence analysis indicated that AiMT from bay scallop was the member of metallothionein family. It can be classified as Family 2(mollusc MTs) and subfamily mo(other mollusc MTs) according to the metallothionein classification system.Semi-quantitive RT-PCR method was used to analyze the expression of metallothionein after exposure to different concentrations of cadmium. mRNA expression of metallothionein in bay scallop was detected in the controls and was up regulated significantly after stimulation of Cd. There was a positive correlation between the mRNA expression and the concentration of Cd. The results indicated that this scallop metallothionein was a constitutive and inducible acute-phase protein that may be involved in essential-metal regulation and cadmium detoxification. A potential use of AIMT as an indicator of cadmium pollution was also implied.Real time quantitive RT-PCR method was used to analyze the temporal expression of metallothionein genes in Vibrio anguillarum challenged C. farreri scallop. Theresults showed that the bacteria stimulus could induce the expression of this scallop metallothionein, and there was a continuous net increase in mRNA expression within 32 hours after injection. It is indicated that the metallothionein play a critical role in scallop-pathogen interaction.