Molecular Cloning And Characterization Analysis Of Myostatin Gene From Siniperca Scherzeri

Myostatin is the strongest negative regulatory factor of muscle growth and development as everyone knowns.The genomic DNA was isolated from the white skeletal muscle of Siniperca scherzeri by phenol-cholorform extraction method, myostatin promoter sequence was cloned by three Tail-PCR methods and two fragments of myostatin structural gene were cloned by two conventional PCR technologies.After purification recovery and sequencing of the PCR products. Sequencing result of the three target fragments were obtained into a size of3228bp sequence, which included a868bp long upstream regulatory sequence and a2360bp long structural gene. The structural gene consists of three exons and two introns, The total length of three exons is1131bp, encoding an amino acid sequence of376residues.Bioinformatics analysis of myostatin promoter and its structural gene sequences(1) myostatin promoter contains a consensus sequence of a TATAA-box, a CAAT-box and four putative E-boxes and possesses many transcription factor binding sites, such as USF、myoD、myogenin、C/EBP、NF-Y and so on.The phylogenetic tree of the myostatin gene promoters from14teleost species was constructed by using software Clustal W and MEGA3.1. The result indicated that the myostatin promoter of Siniperca scherzeri had closer phylogenetic relationship with that of Micropterus salmoides, which was consistent with general morphological taxonomy.(2) Myostatin gene of each teleost just contains two introns and the length of intron is not related to phylogenetic relationship after comparison of13teleost.Some similar promoter elements were found in two introns of Siniperca scherzeri gene, such as a TATAA-box, nine E-box and seven CAAT-box.Some studies about other genes has demonstrated that these similar promoter elements in their introns can regulate the transcription efficiency of their genes.however,the true and specific function of these similar promoter elements needs to be confirmed by further studies.(3) The C-terminal mature peptide region is exactly conservative, the precursor protein is strong conservative, and the N-terminal propeptide is weak conservative after the comparison of homology of Siniperca scherzeri myostatin deduced amino acids.sequence of myostatin deduced amino acids consists of a signal peptide of22amino acids, a RARR (264-267aa) motif which is the conservatively hydrolytic site and consistent with RXXR, and a conservative cysteine knot include9cysteines.The result of the phylogenetic tree of myostatin amino acids from14vertebrate show that Siniperca scherzeri had the closest phylogenetic relationship with that of Micropterus salmoides and had the most distant genetic relationship with that of species belonged to mammalia and aves.Tissue distribution pattern of Siniperca scherzeri myostatin geneThe total RNA was isolated from heart, liver, spleen, kidney, brain, the white skeletal muscle of Siniperca scherzeri by Trizol method of RNA isolation.Quantitative analysis of myostatin gene expression in different tissues by RT-PCR indicated that myostatin had mass expression in different tissues of Siniperca scherzeri,it was different from the specific expression in mammalian skeletal muscle.The relative expression level is Heart>liver>kidney>spleen>brain>the white skeletal muscle. Comparing heart with otner tissues revealed significant differences in their expression level of myostatin mRNA (P<0.05);Myostatin mRNA expression level in liver was significantly different from that in the white skeletal muscle (P<0.05).comparison of the white muscle, heart, spleen and kidney showed no significant differences in their expression levels and it also showed no significant differences comparing liver with heart,spleen and kidney (P>0.05). Expression analysis of myostatin gene in Siniperca scherzeri embryosThe Whole mount in situ hybridization was used to detect the spatial and temporal expression characteristics of myostatin gene in Siniperca scherzeri embryos at different embryonic development periods. The result showed that none of the myostatin transcripts were detected in these embryos during just fertilized eggs, blastula, gastrula and last neurula periods. However, the myostatin expression was first detected in early of sarcomere period with ten somites of development and the distribution of myostatin expression increased with the increase of the numer of somites with the development of embryos during sarcomere, tail Vesicle, muscular Contraction, heartbeat periods.Finally,the distribution decreased in the middle of dorsal muscle and concentrated on the tail of dorsal muscle,head, eye. chest, abdomen and back of neck in the embryos during hatching out and early larval periods. This study would contribute to the future research on the regulation of expression and functional analysis of teleost myostatin after cloning of myostatin gene and its promoter sequences and analyzing characteristics of myostatin expression in Siniperca scherzeri tissues and embryos.