Study Of The Expression Position Of MT-IV Gene Of Ruminants And Its Cloning And Sequencing Analysis

Metallothioneins (MTs) are a kind of metal binding protein of low molecular weight , cysteine-rich, which extensively exists in bacterium, fungi, plants, invertebrate and vertebrate animals. Due to its unique functions, researches about various aspects of MTs receive more and more attentions in the recent years. It have the effect of cytoprotection, increase the ability to adapt to bad conditions, metal ion homeostasis, detoxification etc. MTs family divided into four kinds of subtypes MT-Ⅰ,MT-Ⅱ,MT-Ⅲand MT-Ⅳ.This research was on the basis of predecessor's study to the MT-Ⅰ/Ⅱ, primer MT-IVSP1,MT-IVSP2 and MT-IVSP3,MT-IVSP4 were designed. The target DNA sequence was amplified and cloned by RT-PCR using the primer YMT-ⅢSP1 and YMT-ⅢSP2 from the total RNA. The plasmid library was constructed with T4 polymerase by ligating the DNA fragments into the SacⅡand SpeⅠsite of the cloning plasmid pGEM?-T Vector. Then, by performed following steps: isolation of library DNA, transfection of competent DH5αextraction of plasmid DNA from transfected cells, restriction identification of recombinant plasmids and sequencing analysis, we obtained the coding sequence of various GenBank species: (yak)MT-IV(Accession No.: EF141034) , (goat ) MT-IV(Accession No.: EF470251)and MT-IV(sheep)(Accession No.: EF624067),and the sequence length which began from initiation codon ATG to termination codon TGA was 189bp, so it was yak MT-ⅢcDNA coding full length sequence. The sequences were subjected to Blastn searching in CBI and the results showed that the nucleotide sequences MT-IV exists in different animals, there are lots of conserved tripetide, such as C-X-C, C-C-X-C-C,C-X-X-C, etc. this proves that they are comparatively conservative in their evolution.The second phase of this experiment make an experiment about the hypothesis proposed by predecessors that the MT-IV only exist the double layer squamous epithelium. A lot of experiments have testified that basically the MT-IV has no tissue specificity; it's only a matter of the expression quantity.