Expression And Regulation Of Duck Myostatin Gene And Its Alternative Splicesomes

MSTN (Myostatin) is a member of transforming growth fator-β superfamily (TGF-β) and plays a negative regulatory role in skeletal muscle growth and development. The inactive MSTN can give rise to diameter and number of muscle fiber increasing significantly, and called "double muscling" in mammals. The MSTN gene was found in vertebrates, and has different genome characters:single copy gene in mammals and birds and four MSTN genes in fish causing by gene duplication. Tissue-specific and developing expression profiles showed different expression patterns between fish and mammals, suggesting functional diverged in MSTN genes. Recently, alternative splicing isoform of MSTN genes were found in bird, however, there were no further reports about the numbers, splicing sites and expression profiles of alternative splicing isoform of MSTN in duck. Based on ESTs of duck MSTN, which were obtained by preliminary tests, we employed Peking duck as materials to amplify the mRNA and DNA sequence of duck MSTN sequences, and to analyze the alternative splicing sites, tissue-specific and developing expression profiles, and then alternative splicing isoform transfection assay was performed to detect the regulation roles to downstream gene expression. The main results of this study are as follows:(1) RT-PCR and RACE assays were performed to obtain the full-mRNA and DNA sequences of alternative splicing isoform of duck MSTN gene. Finally, we have got a DNA sequences with length6,082bp, and four splicing isoforms of duck MSTN gene and were named MSTN-a, MSTN-b, MSTN-c and MSTN-d. Based on the comparison between each mRNA variant and the DNA sequence, we found that all the intron splicing of duck MSTN were consistent with GU-AG rules with8splicing sites, and within two alternative splicing methods:exon skipping and3’SSs selective using. The structure and function were changed by alternative splicing:Because of premature stop codon and the deletion of RSRR site and mature domain, the MSTN-b and MSTN-c retained only the N-terminal LAP domains. However, MSTN-d was not missing these domains, in contrast to MSTN-a.(2) The qRT-PCR assay was performed to analysis the expression profiles in30d Peking duck with13tissues and found that the alternative splicing isoform of duck MSTN has different expression profiles between tissues and sex.(3) The real-time PCR results showed that MSTN-a was expressed in breast muscle, leg muscle and heart with higher level than MSTN-b, MSTN-c and MSTN-d. There was no significant difference between breast muscle and leg muscle in MSTN-a mRNA expression, and MSTN-a was found to have essential roles during heart development at late stage. MSTN-b was thought to play roles during leg muscle development with higher expression level than which in breast muscle. MSTN-c has significant lower expression levels in breast muscle and leg muscle, and could not have function in regulating muscle development. During duck embryo muscle development, MSTN-d showed the different expression patterns between leg muscle and breast muscle, suggesting that it should relate to muscle fiber type.(4) The transfection assay showed that, MyoD was down-regulated by MSTN-a, and Akirin2and MyoG were down-regulated by MSTN-b, showing that MSTN-a probably have major roles in myoblast proliferation, and MSTN-b in differentiation.