Brachyury Gene Cloning And Prokaryotic Expression Of Sheep And Hy-Line Variety Brown

Brachyury gene T-box were first discovered member of the family, the main functions of the T-box family genes is as a transcription factor to regulate the growth and development of embryos. Brachyury gene mutation causes the transcription translation carboxyl terminal amino acids to reduce seriously affected the mouse embryonic mesoderm and notochord development and lead to vertebrate short tail and abnormal bone. This paper is divided into two parts, the main part of study of sheep Brachyury gene DNA sequence; The other part is the prokaryotic expression of the genes Brachyury chicken embryos.1. In order to study of sheep Brachyury gene DNA sequence, test mainly use PCR technology to WuZhu MuQin sheep, sheep barr tiger, hulun buir short-tailed sheep Brachyury gene sequence amplification, for the first time the WuZhu MuQin sheep, sheep barr tiger, hulun buir short-tailed sheep Brachyury gene sequences, and find out the differences between varieties of three sheep Brachyury gene sequences bases; Sequence from GenBank database login cows, people, rats, mice, goat, monkey and salamanders Brachyury molecular sequences in 10 different species are analyzed in comparison. Results show that the sheep Brachyury amino acid sequence and cattle Brachyury amino acid sequence homology, the highest is 96.30%; The lowest is derived from the salamander Brachyury amino acid sequence, is only 58.45%.2. In order to express Brachyury protein genes, based on the reported in GenBank jungle fowl Brachyury sequence design a pair of specific primers, tail bud to highland brown chicken embryo tissue extraction of total RNA as a template, using rt-pcr amplification sea orchid Brachyury cDNA, brown chicken embryo and cloning to the PMD-19T carrier, by colony PCR and DNA sequencing, confirmed the cDNA cloning Brachyury sequence for highland brown chicken embryos. Connect the segment sequence to expression vector PET32A, recombinant expression plasmid into a host bacterium BL21 (DE3), by IPTG induction, efficient integration expressed Brachyury protein. By changing the IPTG concentration and inducing time, determine the terms of the best induced protein expression. Western blot analysis showed that the recombinant protein could react with 6* HIS tag antibody specificity. Successful expresses the Brachyury protein genes.