Cloning, Sequencing And Detecting By PCR-SSCP Of TTF-1 In Sheep

Thyroid transcription factor-1 (TTF-1) is a 38-kDa nuclear protein which is expressed in the embryonic forebrain , mature thyroid gland and lung epithelial cells.It plays an important role in activating TG, TPO and TSHR gene transcription. Mammalian reproductive ability also can be influenced potentially by TTF-1.Firstly,Sheep DNA was extracted from blood cell in this study, three pair of primers, SP1/SP2 ,SP3/SP4 and SP5 /SP6, were designed according to the TTF-1 gene sequences reported previously in other species for amplifying and synthesizing aim gene.Result showed that three DNA fragments ,T1 ,T2 and T3 ,were amplified by PCR.The plasmid DNA library was constructed with T4 polymerase by ligating the DNA fragments into the SacⅡ和SpeⅠsite of the cloning plasmid pGEM?-T Vector. And then, by performed the following steps: isolating from DNA library, transfecting the competent E.coli-DH5α, extracting the plasmid DNA from transfected cells, identifying restrictly the recombinant plasmids and sequence analysing, we obtained the sheep TTF-1 coding sequences T1(ACCESSION NO:DQ823383)and T2(ACCESSION NO:DQ010920)and T3. T1 has 375 nucleotides ,and T2&T3 has 457 nucleotide (T2and T3 were merged because some nucleotides were superimposed). T1 and T2 &T3 and encodes a polypeptide containing 106 and 126 amino acids ,respectively. Sequence Analysis indedicate that the T1 fragment contains a 74-amino acids N-terminal transcription active domain; T2 & T3 fragment contains 60 amino that is highly conserved in the NKX2 gene family .Homogeneous analysis demonstrated that nucleotide and amino acids sequences of sheep TTF-1 gene between different species mammal are comparatively conservative.In addition,data showed that nucleotide sequences of TTF-1 gene were not polymorphic among three sheep breeds by PCR-SSCP .