Cloning And Expression Of A Metallothionein Gene In The Sea Cucumber Apostichopus Japonicus

This study has cloned the sea cucumber(Apostichopus japonicus)metallothionein cDNA,analysised the amino acid sequence and explored MT mRNA expression pattern in different tissues which exposed to heavy metal by the means of molecular biology and bioinformatics.This article can be split into two chapter for its research framework.Firstly we obtained the sea cucumber metallothionein cDNA full-length sequence through RACE,and use kinds of bioinformatics software to analysis the information we get.Results showed:? The full-length cDNA sequence of sea cucumber metallothionein is 1351 bp,including a 5' untranslated region(UTR)of 126bp,a 3'UTR of 1069bp and an open reading frame(ORF)of 156bp encoding a polypeptide of 51 amino acids.? The sea cucumber metallothionein has isoelectric point 4.14 in theory,each ends are hydrophobic but middle area is hydrophilic,the average GRAVY value of this protein is-0.145,the entity protein is hydrophilic protein,transmembrane detection reveals that sea cucumber metallothionein is non-transmembrane protein,among all component Cys is highest content,has reached 31.37%,the protein entity 3D structure like a dumbbell.? The conservation of sea cucumber metallothionein isn't very high,phylogenetic analysis show sea cucumber is firstly together with other echinoderm in the same cluster,then in the same cluster with vertebrate species.After earned the full-length sequence of sea cucumber metallothionein cDNA,we can design primers for preparing real-time quantitative PCR according to it.Compared with the sea cucumber tissues expose to different heavy metal environment that we discovered:? After expose to Cd2+ and Zn2+ the expression level of sea cucumber metallothionein has obviously up-regulation,but the effect of heavy metals are difference,tissues exposed to Zn2+ have significant higher metallothionein expression level than expose to Cd2+,it indicates that the induction ability of sea cucumber metallothionein is Zn2+>Cd2+,and for sea cucumber,the Cd2+ has higher toxicity compared with Zn2+.? The tissues have different sensitivity to metal treating:when alimentary canal exposed to Zn2+,Cd2+ and Zn2+/Cd2+ mixture it expressed highest metallothionein among all three tissues,it indicates that the alimentary canal is the most sensitivity part to heavy metal treating,and because its great amount of expression maybe suggest that the alimentary canal is the main position for sea cucumber relieve toxicity of heavy metal.The expression of metallothionein in body wall of sea cucumber is higher thal respiratory tree when exposed to Zn2+ and Cd2+,it indicates that tissue in integument is more sensitive to heavy metal stress than respiratory tree.The heavy metal is known for its high toxicity to organism,the reduction of metallothionein expression level maybe result of it.This study also discover that the metallothionein expression peak of body wall and respiratory tree are all later when exposed to Zn2+ than Cd2+,this suggest that body wall and respiratory tree may have more tolerance to Zn2+,and respiratory tree tissues show insensitivity to heavy metal stress.? The expression of metallothionein exposed to Zn2+/Cd2+ mixture is higher than exposed to single metal treatment,but each tissue has its specific expression pattern.The metallothionein expression in alimentary canal exposed to Zn2+/Cd2+mixture is higher than single metal induction,and the time reached peak value is earlier than single metal induction.Respiratory tree has the similar expression pattern,but it's later to reach the peak value than single metal induction.Integument tissues are insensitivity to metal mixture,this maybe related to the specific structure of sea cucumber body wall.Sea cucumber metallothionein has important role in the phyletic evolution of invertebrate metallothionein,this study about sea cucumber metallothionein contribute to supplement the Holothuroidea metallothionein data.Besides,the further research of metallothionein may enhances the economic value of sea cucumber.Sea cucumber is a kind of benthic organism live in shallow sea,the metallothionein of sea cucumber could be a new potential biomarker for the heavy metal pollution of shallow sea area.