Cloning, Sequencing, Detecting Of The Expression Position Of TTF-1 In Sheep

There are nearly 700 varieties in the world's sheep breeds library, with the exception of a small number of sheep having high lambing rate, precocious sexual maturity and perennial oestrus. The majority of them are one-child production capacity of a year and single lamb of every fetal. The world's major sheep producing countries attach great importance to studying the prolificacy of sheep. TTF-1(Thyroid transcription factor-1) is a homeodomain transcription factor, a member of the NKX2 gene families, which are specifically expressed in thyroid, lung and forebrain. It regulates the tissue-specific gene expressions of thyroglobulin. Mammalian reproductive ability also can be influenced potentially by TTF-1. Studying of molecular biology methods used to control sheep lamb traits, is one of the effective ways to improve the reproductive rate of sheep.Firstly, DNA was extracted from blood cell of sheep in this study. Three pairs of primers, SP1/SP2, SP3/SP4 and SP5 /SP6 were designed according to other species having been reported. The target DNA sequence was amplified through using the primers. After transfecting the competent DH5α, extracting the plasmid DNA, restriction identification of recombinant plasmids and sequencing analysis, the sequence was submitted to Gen Bank and accession number: FJ177515 was obtained. The sequence length is 2972bp, including the full coding region, intone and 5'-, 3'- end partial sequence. Homogeneous analysis demonstrated that nucleotide and amino acids sequences of sheep TTF-1 gene among different mammal species are comparatively conservative. After a pair of primer SP7/SP8 was designed, the target DNA sequence was amplified and cloned by RT-PCR. A lot of experiments have testified that the TTF-1 genes not only exists in thyroid, lung and forebrain, but also in liver and kidney, but can't be found in spleen, ovarian and uterine. There is only a matter of the expression quantity. Lastly, the polymorphisms of TTF-1 gene partial sequence were predicted. Amplifying using the primer SP3/SP4, the result suggested that it had no mutation in the first exon and prior to 166bp department of the second exon of the coding region, but it had four sites mutation within the region of the intron: G/A at 111, A/G at 525, A/G at 540, G/A at 737.