Construction Of The Yeast Two-hybrid System Muscle CDNA Library And Myostain Gene Bait Vector In Bovine

Myostatin is an important regular factor of muscle growth after being discovered, which acts to limit muscle fiber growth and differentiation. The new research shows it can inhibit the growth and differentiation of the white and brown adipocyte. If Myostatin can’t express or have on activity after expressing in the living beings, the growth of muscle will become very quickly and the pattern become much thicker than the normal animals. The fat between muscle fiber will increase but the fat under the skin will decrease. Many researchers want to get the new breeds producing more and better meat and growing fast by the biotechnology of Gene Knock-out and Transgene which can inhibit the Myostatin’s activity in the Agricultural production. During the Clinical and Agricultural production, to find the method inhibits the biology activity of Myostatin having very actual application value. But at present, people still uncertain about the regular principle of Myostatin. There are some unknown proteins interacting with Myostatin in vivo of animals when it completes the fuction. And some proteins can prevent the effects of Myostatin after binding with it because the Myostatin isn’t in the activity situation this time. When being departed from the binding protein, the Myostatin will have biology activity to interact with the receptor on the surface of myoblast, and send the negative regulate signal to the growth of muscle fiber. To research and discover the unknown protein which binding the Myostatin will provide a new way to inhibit the biology activity of Myostatin and promote the growth of muscle. In this research, I constructed a cDNA Library of Bovine Muscle which used for yeast two-hybrid method screening for proteins and the binding combine plasimid. These will lay a foundation to screen for proteins interacting with Myostatin and research the activity and function of Myostatin.The cDNA Library of Bovine Muscle that I constructed in this research are directly used for the Matchmaker GAL4-based Yeast two-hybrid system. The interactions between the bait protein and libraries of prey proteins will activate the Upstream Activating Sequence(UAS) of Gal and make the transcription of four independent reporter genes, so the positive clone got in the selective medium indicates existing interactions between the bait protein and libraries of prey proteins. Next sequencing the insert DNA of libraries of prey proteins and analyses its biological characteristic. In this paper, firstly getting the target gene of the Myostatin C-domain and combine with the procaryote vector which cloned in colibacillus and then construct the bait combine plasmid of Myostatin C-domain(pGBKT7-MSTN).Secondly,extract high-quality RNA and synthetize the2nd cDNA chain by SMART and PCR. Then transform it with linear vector to Yeast to construct the cDNA Library of Bovine Muscle which can be directly used for the Yeast two-hybrid system to screen for proteins.We got such results in this research as following, the the bait combine plasmid, pGBKT7-MSTN which having no toxicity and self-activation to the yeast and can be used for the Yeast two-hybrid system to screen for proteins interacting with Myostatin; the Yeast two-hybrid cDNA Library of Bovine Muscle.