Abstract:

Gymnocypris eckloni is an important cold-water indigenous fish from the upper reaches of the Yellow River.Because of its slow growth and development and late sexual maturity,its population has been declining for many years.MSTN(Muscle Growth Inhibitor) is a factor that negatively regulates muscle growth.This study cloned the MSTN-1 gene of G.eckloni and examined its expression characteristics to provide basic information on the molecular regulation mechanism of growth retardation in G.eckloni.The full-length cDNA sequence of MSTN-1 gene was obtained by PCR,5'-RACE and 3'-RACE.qPCR was used to detect the expression characteristics of MSTN-1 gene in different tissues of G.eckloni and in muscle tissues of different Cyprinidae fishes.The full length of the cDNA sequence of MSTN-1 gene was 2 190 bp,with an open reading frame of 1 128 bp,a 5'UTR of 96 bp,and a 3'UTR of 966 bp,encoding a total of 375 amino acids.MSTN-1 was an unstable hydrophilic secretory protein containing a signal peptide and no transmembrane structure,which was mainly distributed in the nucleus,mitochondria and cytoplasm.The secondary structure of MSTN-1 protein was dominated by random curls,and exists in two TGF-β structural domains:namely,the TGF-β prepeptide region (37—268 aa) and the TGF-β functional region (281—375 aa).MSTN-1 protein contained one the proteolytic site RIRR and nine conserved cysteinee in the TGF-β functional region.Amino acid sequence homology analysis showed that the amino acid sequence of MSTN-1 from G.eckloni had high similarity with other Cyprinidae fishes MSTN-1s and low similarity with mammalian and avian MSTN-1s.Phylogenetic analyses showed that the MSTN-1 of the G.eckloni clustered in the same evolutionary branch with other Cyprinidae fishes.The results of Real-time Fluorescence Quantitative PCR showed that MSTN-1 was expressed in brain,muscle,gill,heart,liver and kidney tissues of the G.eckloni,but the expression was higher in brain and muscle tissues.The gene was expressed in the muscle tissues of different Cyprinidae fishes,with the highest expression in the muscle tissues of the G.przewalskii,followed by the higher expression in the muscle tissues of the G.eckloni and the Cyprinuscaurpio.The full-length cDNA sequence of the MSTN-1 gene of G.eckloni was obtained by cloning,and bioinformatics analysis and qPCR assay were performed.The characteristic differences in the expression of MSTN-1 in the muscle tissues of these indigenous fishes can help to further understand the molecular mechanism of growth retardation in highland fishes.

Key words: Gymnocypris eckloni, Muscle growth inhibitor (MSTN), Gene cloning, RACE, Gene expression