RNA Interference Of The Myostatin Gene In Schizopygopsis Pylzovi

Myostatin(MSTN) is one member of transforming growth factor-beta(TGF-β) superfamily that acts as a negative regulator of skeletal muscle growth. Downregulation of the MSTN m RNA level could increase the transcription level of muscle creatine kinase, thus promote the growth of animal muscle. The two recombinant adenovirus vectors of 1P3(DSP MSTN 273+250+1737) and 1P2(DSP MSTN 195+ 1670) were constructed in this study. The efficiencies of RNA interference of MSTN gene were detected in vitro and in vivo by transfection of the vectors into fathead monnow cell line(FHM) and injection of the vectors into muscle of Schizopygopsis pylzovi. The full-length c DNA of the creatine kinase(M-CK), and the complete coding sequences of myogenic determining factor(Myo D), myogenic regulatory factor 4(MRF4), myogenic factor 5(Myf5) and Myogenin(Myo G) genes were obtain by RT-PCR and RACE methods. The transcriptional regulation of M-CK, Myo D and MRF4 genes were investigated under RNA interference using Real-time PCR. The results showed as follows:1. The recombinant adenovirus vectors were constructed successfully and then transfected into FHM cells. The efficiencies of RNA interference of MSTN gene in FHM cells were not significant.2. In order to detect the effects of RNA interference of MSTN gene in vivo, the recombinant adenovirus vectors were injected into muscle tissue of Schizopygopsis pylzovi. The Real-time PCR and western blotting results indicated that there was no significant interference effect of 1P2 vector on MSTN, compared with the HK group(virus general negative control group) and N team(blank control group). While 1P3 vector showed significant interference effect on the expression of MSTN gene(P<0.05).3. The full-length c DNA of M-CK gene was 1 599 bp, including a 1143 bp complete open reading frame(ORF)encoding 380 amino acid peptide. The 5’-unstranslate region(UTR) was 142 bp in legth. The 3’-UTR was 314 bp including a polyadenlation(AATAAA) and poly(A) tail. The Initiation codon was ATG, termination codon was TAA.4. The M-CK transcript was highly expressed in muscle, intestine, eye and heart, while weakly expressed in hepatopancreas and brain in Schizopygopsis pylzovi.5. The complete coding sequences of MyoD, Myo G, Myf5 and MRF4 genes of Schizopygopsis pylzovi were 825 bp, 762 bp, 723 bp and 720 bp, respectively. The coding sequences of Myo D, Myo G, Myf5 and MRF4 genes were identical in length to that of the corresponding gene of other species from Cyprinidae.6. With the RNAi-mediated silencing of MSTN gene by using 1P3 vector, the expression of M-CK m RNA increased significantly, while that of Myo D and MRF4 m RNA significantly decreased.