Cloning ,Expression Of Swine Myostatin CDNA And Its Tissue Expression Pattern

Abstract: Myostatin (MSTN),a member of the transforming growth factor-beta(TGF-beta) superfamily of secreted growth and differentiation factors that is expressed in vertebrate skeletal muscle, has been shown to be a negative regulator of myogenesis. Targeted disruption of the MSTN gene in mice and a mutation in the third exon of MSTN gene in double-muscled Belgian Blue and Piedmontese cattle breeds respit in double muscle phenotype that resulted from muscle fiber hypertrophy and hyperplasia. Myostatin is expressed early in gestation and then maintained to adulthood in certain muscles. Mystatin expression in bovine muscle is highest during gestation when muscle fibers are forming and some of the myogenic regulatory factors have elevated expression over the same period as myostatin. In this experiment, total RNAs were isolated from skeletal muscle, cardiac muscle. liver, kidney, lung, spleen, pancreas, and fat cells of a new pig breed JunMu-l using Trizol reagent. Using a sensitive reverse transcription-polymerase chain reaction (RT-PCR) and a nested PCR, it was found that the expression of mRNAs were 86 detected in skeletal muscle, cardiac muscle and fat, and not detected in the other tissues. The result was coincident with the published literatures. The eDNA fragment of MSTN was amplified by RT-PCR and a nested PCR, using total RNA of skeletal muscle as templet and primers spanning the coding sequence of MSTN (OYHSOOI,OYHSOO5 and OYHSOO4).The resjdted cDNA fragment with the expected size was cloned into vector pMD I 8-T and subsequently subjected to restriction enzyme analysis and sequencing .The resi.dt 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 causing only one amino acid changed, which indicated that the coding sequence of MST7N was conserved. A pair of primers was designed spanning the complete coding sequence of MSTN but without the coding sequence of its signal peptide (OYI-1S029 and OYHSOO4). A PCR was performed using this pair of primers and the total mRNA of skeletal muscle. And a eDNA fragment of 1225 bp was obtained which include the complete coding sequence of mature MSTN protein. Then a MSTN coding region cDNA construct pMD I 8-T-MSTN was generated by inserting the eDNA fragment into pMD I 8-T vector and selecting the sense clones by PCR with primers OYHSO29 and OYHSOO4 and restriction enzyme analysis. This construct was digested with Sal I and Barn H I and ligated the pET23(+) victor digested with the same enzymes using T4 DNA ligase. And a construct pET28(?-MSTN was generated by transforming the competent cell of BL2 I (DE3) of E.coli. The sense clones were obtained by PCR and restriction enzyme analysis. The plasmid of the construct pET28(-t)-MSTN was 87 sequenced and the sequence was confirmed to be right. The expression of MSTN mRNA was detected by RT-PCR and Northern blot. Incubate the genetic engineering bacteria in LB medium (7fQ~4r) and induce the expression of MSTN protein with IPTG at a final concentration of I mM. Unfortunately, no expression of MSTN protein was observed on SDS-PAGE. which maybe resulted from the rare codons existed in the coding s...