Studies On The Molecular Mechanism Of Porcine Myostatin For Muscle Growth

rLean 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 amimals 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 to probe the relation between myostatin and high lean meat rate and plump-hipped trait,we sythesized the C'80 Amino Acids Coding Sequence of Porcine Myostatin and costructed the cloning and expressing vector of it. The C' 80 amino acids coding sequence of MSTN optimized according to common codons ofE.coli was divided twelve single segments, FI> p2> FS> R$> R^ R^ p4> FS> F6^ RS^ R2and RI.These segments were synthesized by chemical means.Each pair of adjancent segment overlaps with 20bp.The length of Fiand Riis 38bp and 36bp.The length of other segments is 40bp.The end of Fjand RI segment are designed with recognition site sequence of restriction enzyme^co I and Xho I .Using the overlaped segment as primer,We obtained 254bp segment by PCR. After electrophorised on 1% agarose gel, the PCR production was purified with Agarose Gel DNA Extraction kit.The segment was ligated with vector pMD18-T and then was tranformed into the competent cell of DH5 a .A construction MSTND-pMD18T was generated by inserting the sequence of 254bp into pMD18-T vector and selecting the sense clones.Positive clone was identified by three ways:endonuclease digestion,PCR and sequencing.The result showed that the cloned sequence coincides with the designed sequence.This construction was digested with Nco I and Xho I and ligated the pET28a(+) vector digested with the same enzymes using DNA Ligation Kit.The production of ligation reaction was transformed into the competent cell of BL21(DE3).After 12-16 hours of culture,several colnes appeared on the plate.Some positive clones were selected toextract their plasmid.These plasmids were digested by Nco I and Xho I and indentified by PCR.A contraction, MSTND- pET28a was generated.The result showed that the cloned sequence coincides with the designed sequence. The sense clone was induced with IPTG.The expression of MSTND peptide was observed on SDS-PAGE.The results lay a good foundation in the application for molecular biological technique in animal nutrition.