The Research Of Metallothionein Gene Of Cadmium Tolerance Colpoda

One of the toxic effects of cadmium to the biological effect is to induce expression of Metallothionein,MTS.Metallothionein is a wide range of heavy metalloid binding proteins in the biological boundaries,with metalion balance and detoxification,but there is less research on protozoa,sulfur proteins,and the related gene information is insufficient,and the study of the gene may be cadmium biological monitoring provides targets.The protozoa is more sensitive to the toxicity of toxic substances in the water environment.It is an ideal indication species that deteriorates the deterioration of the water ecosystem in the early forecast.This paper is screened from three protozoa of Colpoda with a high tolerance of cadmium,which is clearly related to cadmium resistance to metallothionein.And cloned the nematode metallothionein gene,analyzing its sequence characteristics and response to gene expression on cadmium stress.Provide a basis for further exploration of the relationship between the metallothionein of Colpoda with its cadmium tolerance and molecular mechanism.The specific results are as follows:1.Screening high cadmium tolerance Colpoda.The three types of Colpoda were purified and cultured.According to morphological identification and phylogenetic tree construction based on the ribosomal small subunit gene sequences,the species were identified as Colpoda cf.grandis,Colpoda inflata and Colpoda sp.respectively.The growth curves,population growth rates and generation times of three Colpoda species under different concentrations of Cd stress were analyzed for 168 h.A Colpoda that could tolerate Cd concentration up to 10mg/L was obtained and identified as Colpoda inflata Harbin population.The metallothionein content of Colpoda inflata was measured at four consecutive time points within 96 h.It was found that the metallothionein content of this Colpoda increased significantly under the induction of cadmium.2.Cloning and analysis of metallothionein gene of Colpoda inflata.There is currently no this metallothionein associated genes of Colpoda related to cadmium stress to cadmium stress.According to the nucleotide sequence of the Tetrahymena of the gene,the inverted c DNA is expanded to increase the 241 bp length fragment.The GC content of this fragment accounts for 44.98%,and the maximum open reading box is 159 bp.It is predicted that the DNA molecular weight is about77.44 k Da,the average isoelectric point is 4.96,theoretical isotropic point is 5.33,it is predicted as non-secreted protein.This sequence is up to 30.90% of Tetrahymena americais of metallothionein gene in the native animal.The homology of Drosophila melanogaster of metallothionein gene is up to 42.20% in high animals.And metallothionein family sequence-specific conservative sequence Cys-Cys,X-Cys-x,which belongs to the 7a isoform of metallothionein gene.3.Analyze the relationship between expression of metallothionein and cadmium stress.At 60 h,84 h and 106 h,6 concentrations of cadmium,the expression quantity of the metallothionein gene was detected by q RT-PCR.The results showed that the expression of metallothionein was induced by high concentration cadmium,but the induction of high concentration cadmium on metallothionein decreases as the treatment time was prolonged.