Study On Metallothionein (MT)-Ⅲ/-Ⅳ Gene Characteristics Of Yak And Expression Of MT-Ⅲ

The yak species(Bos grunniens) represents a unique bovine species adapted to the Tibetan plateau of China at altitudes of 3,000m above sea level with intense ultraviolet radiation.Consequently,yak adapted to this environment likely have special physiological adaptive mechanisms to protect their central nervous systems against hypoxic and oxidative injury.The metallothionein(MT) family is a class of low molecular, intracellular,and cysteines-rich proteins with a high affinity for metals.Accumulating studies have demonstrated MTs fulfill unique biological roles in scavenger of free radicals,metal detoxication and homeostasis,and homeostasis of the central nervous system and in the etiology of neuropathological disorders. The MTs of yak maybe important for protecting yak from hypoxic and oxidative injury and it is essential to study MTs gene of yak.However,there were no detailed reports concerning MT-â…¢and MT-â…£gene in Yak. In this study,MT-â…¢and MT-â…£gene were cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and 5'-RACE methods firstly.To analysis the gene structure and the protein characteristics of MT-â…¢and MT-â…£by bioinformatics methods,and established the expression system of MT-â…¢proteins in E. coli..The main results were got as following:1.The translated region sequence of MT-â…¢gene in yak was gotten,the total length was 1484bp,the length of 5' UTR,exonl,intronl,exon2,intron2,exon3 and 3'UTR was 56bp,31bp,66bp,817bp,110bp and 172bp respectively.The sequences had been deposited in the GenBank data base(Accession No.DQ492300 and EU653285 ).2.The coding regions of Yak MT-â…¢were composed by 207 nucleotides,encoded a 68-amino acid protein, and contained 19 cysteine residues,no aromatic AA.Compared to 61 amino acids for MT-â… and MT-â…¡isoforms,MT-â…¢contained 2 inserts,a single threonine in the N terminal region and a glutamic acid rich hexapeptide(GEGAEA) in the C-terminal region.The molecular weight of Yak MT-â…¢is 6.94 KDa,analysis of hydrophobicity,transmembrane region,and signal peptides suggested that MT-â…¢of the yak are nonsecretory proteins.3.The corresponding conservative Cys³⁰ of MT-â…¢in human and mouse was replaced by Ser³⁰,due to a mutation at the 88th nucleotide acid where T mutated into A,it may implied some structural/or dynamic and physiological function differences.4.The sequence length of MT-â…£gene in yak is 2099bp,including 3 exons and 2 introns,the length of 3 exons were respectively 31bp,66bp and 92bp,the length of 2 introns were respectively 1392bp and 518bp,the sequences had been deposited in the GenBank data base(Accession No.EU665491).The coding regions were composed by 189 nucleotides,encode a 62-amino acid protein,and contain 20 cysteine residues,no aromatic AA.Compared to MT-â… and MT-â…¡isoforms,MT-â…£contained a single glutamic inserts in the N terminal region.The molecular weight of Yak MT-â…¢was 6.25 KDa.,Analysis of hydrophobicity,transmembrane region,and signal peptides suggested that MT-â…£of the yak are nonsecretory proteins.5.The coding region of MT-â…£was cloned by RT-PCR from liver,spleen,brain and pituitary in yak,yellow cattle,sheep and goat,in which there are no cornified stratified squamous epithelium,it applied the expression of MT-â…£not only limited in stratified squamous epithelium.6.The coding regions and amino acid sequence of MTs among mammalian are highly identical,and highly conserved in evolution.But the identity of the noncoding regions sequence are very low,these sequence is special for species likely.7.Modelling on three-dimensional solution structure of mouse MT-â…¢,the three-dimensional solution structure ofα-domain of MT-â…¢in yak was predict,and lainα-helix from the Glu^43rd to Ala^46th.Modelling on three-dimensional solution structure of rabbit MT-â…¡,the three-dimensional solution structure of MT-â…£in yak was predict,different from other MT isforms,the conserved hinge region between two domain in other MT isoforms were replaced by RKS in MT-â…£.8.Establishing pET-42a(+)-MT-â…¢expression system in E.Coli BL21(DE3)plysS with IPTG.