Construction Of Cloning And Expresing Vectors Of Mature Protein Coding Sequence Of Porcine Myostatin

Advisor: ZHANG Guangskeng OUYang Hongsheng Abstract: Lean meat percentage is one of the most important economic traints in pig breeding programs. Myostatin is a negative regulator of skeletal muscle growth. Null or low activity of myostatin, individual muscle of mutant animals would show a large and widespread increase in skeletal mass. Myostatin null animals have significantly larger diameter or more quantity of fiber skeletal muscle. The phenotype was termed double muscling. In order probe the relation between myostatin and high lean meat rate and plump-lipped trait, we magnified and construct cloning and expresing vector of maturity protein coding sequence of porcine myostatin. Firstly, the cDNA fragment of MSTN maturity protein coding the sequence of porcine myostatin was amplified by RT-PCR followed by a nested PCR. A 1225bp MSTN porcine myostatin fragment was prepared and amplified using pri mers(OYHSO29, OYH S005) designed from the coding sequence of porcine myostatin. The fragment includes the coding sequence of porcine myostatin gene without the coding sequence of its signal peptide. Secondly, the cDNA fragment with the expected size was ligated with pMD- 18 T vector and then was transformed into E.Coli JM1O9. Positive clone was identified successfully by three ways endonuclease digestion, PCR and sequencing . The clone in which the cDNA fragment was ligated with the plasmid of pMD-1 8 T in cis direction was identified by endonuclease digestion. The plasmid MSTN梡MD 1 8-T-029-005 was extracted. The result showed that the cloned MSTN gene fragment is 1277 bp comprising the complete coding sequence of MSTN and 99.5% homogenous to published data with replacements of 4 nucleotides but causing no amino acid changed, which indicated that the coding sequence of MSTN was conserved.. Thirdly The plasmid MSTN梡MD 1 8桾-029-005 was amplified with the primers (OYHS 051, OYHSO52), then both the purified 1.2kb fragment and the 49 Ai~L~z~C pET28a(? plasmid were cleavaged with BamH I and Sal I, and the 1.2kb fragment that digested was cloned into the pET28a(+) that with the same digested . then the recombinant was transformed into JMIO9, and transformed into BL2I (DE3) with the plasmid of JM1O9. Postive clone was identified successfully by PCR , restriction enzyme digesting and sequencing analysis, the gene encoding Myostatin protein polypeptide was cloned into a expression vector, pET-28a(+). successfully. The MSTN expression vector can be used for expression pig maturity protein coding sequence. And the results lay a good foundation in khe application for molecular biological technique in animal nutrition.