Relative Expression Of Metallothionein Gene Induced By Water Coper In Cyprinus Carpio

In this study,Cyprinus carpio was taken as the experimental subject,based on cloning of MT(Metallotnionein)gene CDS of Cyprinus carpio by RT-PCR merthod and analysis of MT expression in different tissue,set up a RT-q PCR systerm to minitor the change regular of MT gene expression induced by different concentration water copper.This study aimed at find a new symbol for minitoring the heavy metal pollution from the molecolur.The main findings are as follows:1.The coding sequence(CDS)of Cyprinus carpio MT gene was cloned by RT-PCR successfully,which is 183 bp and encoding 60 amino acids that neither with aromatic amino acid nor with histidine.What's more,the vertebrate MT typical conserved sequence of Cys-X-Cys?Cys-X-X-Cys?Cys-X-Cys-Cys was possessed by Cyprinus carpio,in which 20 cysteine(Cys)were concentrated at the amino terminus(Nterminal)and carboxy-terminal(C-terminal)of the peptide chain.The predicted molecular weight was 6013 D and the theoretical isoelectric point was 8.38.With the multiple sequence alignment,the nucleotide sequence of Cyprinus carpio was 94% highest homology with Carassius cuvieri and the amino acid sequence was 95% highest homology with Gobiocypris rarus.2.Two pairs of specific primers were designed with the cloned sequence of cyprinus carpio MT and efficient amplification was obtained by optimizing annealing temperature and the construction of standard curve,after which a successful method to monitor the RT-q PCR of cyprinus carpio MT was developed.Taking Actin-? as an reference gene,the results suggested that the expression of cyprinus carpio MT has obvious differences in different organizations,including the hepatopancreas MT is highest expressed,followed by the kidney,brain,gills and that is minimum in muscle.Regarding the relative expression of brain tissue is unit 1,the expression quantity of MT in hepatopancreas was 56 and that were 16.6,7.9 and 3.4 in kidney,brain,gills respectively.3.RT-q PCR was adopted to analysis the MT gene expression at different time points(6h,12 h,24h,48 h,96h,192 h,12d)in hepatopancreas,kidney,brain,gill and muscle with tenements of 0,0.1,0.3,0.6,1.0,1.5mg/LCu2+.The result showed that the MT gene in Cyprinus carpio was induced upregulation of copper and has a significant organizational differences: the presence of different concentrations of a dose-response relationship,there is a certain amount of time in pre-induction(0-96h)effect;throughout induce in addition to liver and pancreas of MT gene expression level increased,the other four organizations MT as a whole showed a decline after the first increase in the development of the state,but induced peak of each organization and reaches its peak induced induction time varies;hepatopancreas the main focus of inducing kidney,gills,brain and muscle MT were: 0-48 h,0-48 h,0-24 h,0-48 h and 0-12h;between different organizations in different MT gene regulated amount,which hepatopancreas was highest,followed by kidney,gill and brain,muscle raised the least.The result showed that the MT gene sequence in Cyprinus carpio is highly conserved in vertebrates,MT gene in hepatopancreas kept rising,so the hepatopancreas MT in Cyprinus carpio could be used as a real-time minitor biomarker for heavy metal in aquaculture water environment pollution.