| Myostatin is a negative regulator of skeletal muscle growth. It plays an important role during myogenic differentiation.The mature region DNAs for Myostatin of Jinhua pig, Xiaoshan chicken and Fan duck were cloned and Myostatin proteins of Jinhua pig and Xiaoshan chicken were expressed in P.pastoris in this project. The aim of the present project is to provide the material preparation for utilizing Myostatin gene and producing Myostatin vaccine that can increase the muscle mass through blocking the physiological function of Myostatin. At the same time, this project can also provide technical support for the research of expression of heterologous gene in yeast.The genomic DNAs were extracted from the bloods of Jinhua pig, Xiaoshan chicken and Fan duck respectively, their mature region DNAs for Myostatin were cloned using the pUCm-T as cloning vector and their respective sequence was obtained. The gained sequence were compared with the corresponding DNAs of other species reported in GenBank using DNAMAN software, It was found no base mutation in Jinhua pig and two base mutations (T218 C218, C219 T219) in Xiaoshan chicken. The Myostatin sequence of duck has not been reported in GenBank. The homology of the Myostatin sequences was analyzed among species. The results showed that the mature region DNA for Myostatin of Jinhua pig has the similarity of 100% % 97%, 95%, 95%, 93%, 93%, 93%, 89%, 90%, 89%, 87%, 87%, 86%, 86%, 86%, 85% and 76% compared with that of pig(Sus scrofa), horse, human, monkey, cow, hare, goat, sheep, house mouse, rat, goose, turkey, chicken(Gallus gallus), pigeon, Xiaoshan chicken, Fan duck and zebrafish respectively. The mature region DNA for Myostatin of Xiaoshan chicken has the identity of 99%, 97%, 93%, 92%, 92%, 87%, 86%, 86%, 86%, 86%, 85%, 85%, 85%, 84%, 84%, 83% and 74% compared with that of chicken(Gallus gallus), turkey, pigeon, goose. Fan duck, monkey, human, horse, pig(Sus scrofa), Jinhua pig, house mouse, rat, sheep, hare, goat, cow and zebrafish respectively. The mature region DNA for Myostatin of Fan duck has the homology of 91%, 92%, 92%, 90%, 95%, 84%, 84%, 85%, 85%, 85%, 83%, 81%, 81%, 83%, 81%, 82% and 73% compared with that of chicken(Gallus gallus), Xiaoshan chicken, turkey, pigeon, goose, monkey, human, horse, pig(Sus scrofa), Jinhua pig, house mouse, rat, sheep, hare, goat, cow and zebrafish respectively. The results indicate that Myostatin gene is highly conserved among various species. This may attribute to the important physiological function of Myostatin on much extent. Moreover, The phylogenetic tree of all species was also constructed and it revealed that the evolutionary relationship between all species is basically identical with the one made by traditional classification. But there still had two doubts: Firstly, the genetic distance between cow and hare was closer than that between cow and sheep, goat. Secondly, the genetic distance between horse and pig is quite close.The positive clones of Myostatin were digested with EcoR I and Not I, then linked with the expression vector pPIC9K cut by the same enzymes, and Myostatin-pPIC9K expression vectors of Jinhua pig and Xiaoshan chicken were constructed, then the positive recombinants were screened and transformed into P.pastoris. In order to improve recombination efficiency, the recombinants were linearized before they were transformed. Finally, the protein expression of the positive transformants was induced with methanol and the targeted proteins were identified by SDS-PAGE electrophoresis. The great advantage of P.pastoris expression system is that the expressed protein can be processed and modified after translation and its biological activity can be kept. Especially,the target protein can be secreted into the liquid culture medium, which makes the later purification more convenient and efficient.
|
PDF Full Text Request