Porcine Myostatin Gene Expression And Its Location In Different Tissues

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 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 tissues distribution of myostatin. we devised three trial: C-terminal 88 amino acid Chemical Synthesis and Prokaryotic expression of porcine myostatin gene; C-terminal 88 amino acid antibody preparation and MSTN'tissue distribution of porcine myostatin gene;Eukaryotic expression and its checking of porcine myostatin gene.The C' 88 amino acids coding sequence of MSTN optimized according to common codons of E.coli was divided twelve single segments.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 are 40bp.Both end of F1and R1 segment are connected with recognition site sequence of restriction enzyme,Nco I and Xho I .Using the overlaped segment as primer,We obtained 254bp segment by PCR. The PCR product was purified with Agarose Gel DNA Extraction kit.The segment was ligated with pMD18-T and was tranformed into the competent cell of DH5.A construction MSTND-pMD18T was generated by inserting the sequence of 254bp into pMD18-T vector and selecting the sense clones.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.Theproduction of ligation reaction was transformed into the competent cell of BL21(DE3).After culture,several colnes appeared on the plate.These plasmids were digested by Nco I and xho I and indentified by PCR.A contruction, 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.In order to study tissue distribution of porcine MSNT.Induced the expression of MSTND protein with IPTG at final concentration of ImM.Expression of MSTND was detected on SDS-PAGE.The protein expressed by the plasmid of pET28a(+) MSTND in E.coli was purified and ligated by EDCI,then the ligation was injected in the adult rabbits three times in two mouths. Antibody against MSTN which tite was 1:6400 was obtained by determination of ELISA and Western-blotting.At the same time,Study tissue distribution of porcine MSTN through immunohistochemistry and Western-blotting. The results of immunohistochemistry showed: Heaviness stain were on lung, liver, cerebellum, heart, pancreas, adipocyte, little stain were on muscles, kindey, metra-cells; The results of western-blot showe: lots of distribution on lung, liver, cerebellum, heart, pancreas, adipocyte. Inferior distribution on muscle, on kindey, metra-cells have no fund.In order to express the coding sequence of porcine myostatin in eukaryotic system.two special primers(CCD8951wf-l, CCD8951wf-2) were designed according to the myostatin cDNA gene including two restriction endonucleases(EcoR LSal I) sites.Using these primers,PCR was proceeded to amplify the coding sequence of myostatin.The PCR products were purified in 1% agarose gel electrophoresis and recovered by Agrose gel DNA Extraction Kit,and then inserted into the pMD18-T vector between the sites of EcoR 1 and Sal l.The ligated products were transformed to the competence E.coli DH5 a .The recombinant positive clones were screened by complementation,identified by restriction endonucleases digetion,PCR and sequencing.The cloning plasmid and pef-dhfrla vector were digested by EcoR I and Sal 1 simultaneously.The eukaryotic expression plasmid pef-dhfrla-MSTN was constructedby inserting the coding sequence of...