Cloning Of MT-Ⅲ CDNA Gene And Sequencing Of Its Nucleotide/Protein In Yak

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. MTs have important biological functions, such as metal ion homeostasis, detoxificat- -ion, cytoprotection and tumour prevention of disease and treat. MTs family divided into four kinds of subtypes MT-â… ,MT-â…¡,MT-â…¢and MT-â…£.MT-â…¢is also named"growth inhibito- -ry factor"(GIF), mainly expresses on central nervous system and reproductive system,etc. It is a new member of the MTs family, differing from other MT subtypes, which regard as growth inhibitory factor separated out from the cultivation of the nerve cell initially. Because it has similar structure to named as MT-â…¢with MT-â… /â…¡. HMT-â…¢is composed of 68 amino acids , which has 7 amino acids more than HMT-â… /â…¡. MT-â…¢is different from known MT-â… /â…¡, it has a clear physiological effect, this kind of functional difference may come from its own unique structure.This research was on the basis of predecessor's study to the yak(B. Grunniens) MT-â… /â…¡, Two universal primers YMT -â…¢SP1 and YMT -â…¢SP2 for MT-â…¢in Yak were designed according to MT gene coding high homology. The target DNA sequence was amplified and cloned by RT-PCR using the primer YMT-â…¢SP1 and YMT-â…¢SP2 from the total RNA of yak brain tissue. The fragments of target DNA were selected by electrophoresis. The plasmid library was constructed with T4 polymerase by ligating the DNA fragments into the Sacâ…¡and Speâ… site of the cloning plasmid pGEM?-T Vector. Then, by performed following steps: isolation of library DNA, transfection of competent E.Coli.JM109, extraction of plasmid DNA from transfected cells, restriction identification of recombinant plasmids and sequencing analysis, we obtained the yak MT-â…¢coding sequence (Genbank Accession No:DQ323545), and the sequence length was 207bp. Then, the yak MT-â…¢cDNA specific primer YMT-â…¢SP3 was designed according to coding sequence. The target DNA sequence was amplified and cloned by 3'-RACE using the primer YMT-â…¢SP3 and M13 Primer4. Through the same procedure of cloning above(It was pMD18-T Vector that was used to clone the DNA target ), we obtained the yak MT-â…¢cDNA 3'-end full length sequence(Genbank Accession No:...