Molecular Cloning And Charateristic Analysis Of The Myosin Heavy China(MyHC) From The Fast Skeletal Muscle Of The Madarin Fish, Siniperca Chuatsi

Myosin heavy chain (MyHC) is one of the major structural and contracting proteins of muscle. The white skeletal muscle of siniperca chuatsi was selected as experimental material in the experiment. The total RNA was isolated from the muscle tissue and the single-stranded cDNA was cloned using reverse transcription-PCR. Based on the sequences of teleost MyHC cDNA published in the GenBank, three pairs of special primers were designed for cloning the coding sequence of MyHC in the siniperca chuatsi. After PCR amplification, three target fragments were obtained and assembled into the full-length open reading frame of the MyHC cDNA.The ORF of the MyHC gene is5814bp long, encoding1937amino acid residues. The molecular weight of the MyHC is about220kDa and its isoelectric point is5.61. The MyHC contains606hydrophobic amino acids and451hydrophilic amino acids. The hydrophilic amino acids were concentrated in the first half of the protein from100-800position,and hydrophobic amino acids were concentrated in the Myosin-tail₁conserved region. According to the proportion of hydrophobic and hydrophilic amino acids, the MyHC is the hydrophobic protein. The309strongly basic amino acids and355strongly acidic amino acid are in the protein. The C and T content is relatively low,20.9%and20.31%respectively, in the MyHC base composition. The base content in the various sites of the MyHC gene is also different. The content of the A base is high in the first and second base of the MyHC gene, but the G base is high in the first and third base.The myosin heavy chain of Siniperca chuatsi is mainly composed of three parts: subfragment-1(S1), subfragment-2(S2) and the light meromyosin(LMM) regions. The S1region has three ATP binding sites, three actin binding sites and two Loop s rings. The putative sequences of the S1, S2and LMM regions showed89.9%-99.5%homology to the corresponding regions of other fish MyHCs.The phylogenetic tree constructed by using software CLUSTALX1.83and MEGA4.0showed that the MyHC of siniperca chuatsi had closer phylogenetic relationship with that of siniperca kneri. Our work on the MyHC gene from the siniperca chuatsi added useful information for fish molecular biology and fish genomics.