Functional characteristic and differential expression of myostatin in Chlamys nobilis

ABSTRACT Myostatin (MSTN) was a conserved negative regulatory protein of muscle growth and development in numerous species. In this study, the MSTN gene was cloned and characterized from a noble scallop, Chlamys nobilis, and it was named CN-MSTN. To investigate the molecular characterization of MSTN and its gene expression profile of spatio-temporal, we isolated the MSTN cDNA sequence in C. nobilis and analysed expression patterns using quantitative real-time PCR. CN-MSTN cDNA contained a 1,374 bp open reading frame that encoded a 458 amino acids. Furthermore, the result of 3D model indicated that CN-MSTN mature peptide was similar to that of Chlamys farreri. Additionally, based on both the nucleotide and amino acid datasets, phylogenetic trees indicated that C. nobilis has the highest similarity with homologues of C. farreri. The expression patterns of different stages showed that MSTN expression was markedly higher in the gastrulae period. Additionally, remarkable expression of MSTN was observed in adductor muscle than with other tissues. Our data would provide expression and phylogenetic information of this economical important sea food, noble scallop.


Introduction
Myostatin (MSTN), which negatively regulated muscle development, was a member of the transforming growth factor beta (TGF-β) superfamily in a variety of species (McPherron et al. 1997; Thomas et al. 2000). Moreover, increasing skeletal muscle quality in mice, cattle and humans was associated with the lack of its functions, especially to the 'double muscle' feature observed in breeding cattle.
The scallop Chlamys nobilis (Pectinidae, Pterioida) is a vital and commercial marine mollusk mostly cultured in the southern sea of China (Zheng et al. 2010). In the present study, the cDNA sequences of MSTN gene were cloned and characterized. Moreover, C. nobilis MSTN of spatio-temporal expression and 3D model was structured. Additionally, the phylogenetic analysis based on nucleotide and amino acid datasets of 16 species was established. This research could provide useful MSTN evolution and function.

Samples collection
Healthy adult C. nobilis (body weight 26.11 ± 2.52 g; shell length 5.30 ± 0.13 cm; shell width 5.01 ± 0.19 cm) used in this study were collected from Lingshui in Hainan Province, China. Those scallops were retained (50 shell/tank) in 300 L tanks with circulating seawater (temperature 21 ± 0.5°C) for two weeks before experiment.
To research the distribution of CN-MSTN mRNA expression, eight tissues (including the mantle, pleopod, digestive gland, heart, gill, adductor muscle, female gonad and male gonad) were collected from five scallops. Furthermore, embryos and larvae (at least 20 each stage) at different developmental stages were collected for analysis of temporal expression. All samples were stored at 80°C after addition of 1 ml Trizol reagent (Invitrogen) for subsequent RNA extraction.

Total RNA isolation and reverse transcription
We used a Trizol kit (Promega, Madison, WI, USA) to extract total RNA from various tissues. Moreover, RNA quantity and quality (concentration) were determined with NanoDrop 2000 spectrophotometer (ThermoScientific, Waltham, USA), and to confirm integrity, it was visualized in 1.2% sepharose gel with 1× TBE buffer (Tris-Sodium acetate-EDTA). To synthesize cDNA, A Pri-meScript™ RT reagent Kit was accompanied by gDNA Eraser (TaKaRa, Japan). Total RNA (2 µg) and 0.5 µg of Oligod (T) 16 was reacted for 5 min at 70°C. After incubation for 2 min on ice, the mixture was reversely transcribed depending on the manufacturer's instructions and stored at -20°C until further use.

Bioinformatic analysis
BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) is used to calculate amino acid and nucleotide sequence similarity searches. The coding sequence of MSTN was forecasted by ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/). Moreover, to obtain theoretical isoelectric point, molecular weight and features of the predicted proteins and signal peptide, ExPASy analysis (http://us.expasy.org/tools) was utilized and SignaIP 4.1 Server was used for prediction (http://www.cbs. dtu.dk/services/). A homology model of MSTN from C. nobilis was constructed by the SWISS-MODEL Protein Modelling Server (Kopp and Schwede 2004;Arnold et al. 2006). Multiple sequence alignments and phylogenetic trees of the deduced nucleotide and amino acid sequences were performed using MEGA 6.0 program with Maximum parsimony (MP), Neighbor-Joining (NJ) and Bayesian inference (BI) methods (Lartillot et al. 2009;Tamura et al. 2013).

Gene expression analysis
Quantitative real-time PCR (qRT-PCR) was performed to analyse the mRNA levels of MSTN (Bustin et al. 2009). Specific primer pairs for MSTN (F: GACAGCAAAGCGATTACC; R: TTCCGATGT-CATTTCA-GC) and reference gene b-actin-F/R were obtained (Lu et al. 2016). The system of qRT-PCR was referred to Zhu et al. (2016). Furthermore, the relative expression was determined using the 2 -ΔΔCT method (Livak and Schmittgen 2001).

Statistical analysis
MSTN transcripts were expressed as mean ± SE. The results were performed by one-way Analysis of Variance (ANOVA), and Duncan's test was used to establish the difference between treatments. Differences were considered at a significant level of p < .05 and p < .01.

Cloning and sequence analysis of CN-MSTN
Fragments of MSTN were amplified by PCR. After identification of the fragments, it revealed that CN-MSTN [GenBank accession no. KY197473] consisted of a 1423 bp fragment which contained a 1374 bp ORF encoding a polypeptide of 458 amino acids. The predicted CN-MSTN protein had a molecular mass and an isoelectric point of 53 kDa and 5.35, respectively. Moreover, the grand average of hydropathicity of predicted CN-MSTN protein was −0.592. The result of SignalP indicated that a putative signal peptide of 19 amino acids which was estimated to be an extracellular targeting sequence was contained in the deduced amino acid sequence.
Like additional TGF-β superfamily members, a potential proteolytic processing site RXXR (amino acids 327-330) was in the CN-MSTN (Figure 1). Moreover, a propeptide and a mature peptide were produced by this site. Nine conserved cysteine residues were detected in the carboxy-terminus portion. Both the full amino acid sequences and carboxy-terminal showed higher homologies with those species in Table 1

3D structure of CN-MSTN
A 3D model of CN-MSTN mature peptide was constructed by the SWISS-MODEL Protein Modelling Server. To compare three closely species, the model of C. farreri and Mus musculus were also established. The result indicated that a 3D model of CN-MSTN mature peptide was resemblance among C. farreri and M. musculus MSTN which revealed the classics TGF-β family hand-shaped frame ( Figure 2) (Lin et al. 2006;Guo et al. 2012). Moreover, it suggested that MSTN of both scallops had a longer α-helix region than that of M. musculus (Lin et al. 2006).

Phylogeny
To know the phylogenetic relationships among molluscs, a dataset of nucleotide and amino acid sequences of 16 marine mollusk MSTN was generated (Figure 3). Based on NJ, MP and BI analyses of the nucleic acid and amino acid sequences, respectively, the results showed consistency in the topologies' structure, with a close relationship between C. nobilis and C. farreri.

Spatio-temporal expression analysis of MSTN
Spatio-temporal expression patterns indicated that MSTN expression in the gastrulae period was markedly higher than at other development stages (p < .01). After this period, the expression gradually decreased (Figure 4(A)). Additionally, the highest MSTN expression was shown in adductor muscle compared with the other tissues, with the second-best expression noted in mantle (Figure 4(B)).

Discussion
In this study, we cloned and characterized the MSTN gene (which contained 1374 bp ORF encoding 458 amino acids) from C. nobilis. Moreover, the structure of CN-MSTN was similar to other scallop MSTN, which contained 3 exons and 2 introns (Kim et al. 2004;Guo et al. 2012). Therein, the first exon contained N-terminal signal for secretion, whereas exon 3 consisted of the conserved C-terminal mature peptide, common to the A. irradians MSTN proteins (Guo et al. 2012). Furthermore, nine conserved cysteine residues and a RSKR proteolytic processing site were in the C-terminal (Guo et al. 2012).
Nucleotide and protein alignments with other molluscs prominently showed that the C. nobilis MSTN shared the highest homology with C. farreri, consistent with the fact that both C. nobilis and C. farreri were members of the Pectinidae superfamily. (Figure 3). Therefore, five adjacent species' MSTN were chosen for alignment; the result indicated that C-terminal region was highly conserved in MSTN ( Table 1. Comparison of the 3D structure of MSTN among the C. nobilis, C. farreri and M. musculus suggested that both scallops' MSTN has a longer α-helix region than that of M. musculus (Guo et al. 2012). Therefore, CN-MSTN may contain more components for effecting on other DNA sequences or amino acids, which may not be an important function in M. musculus (Cash et al. 2009).
Tissue-specific expression profile indicated that MSTN mRNA levels were found to be highest in adductor muscle, which was in keeping with the result of C. farreri, A. purpuratus, A. irradians, N. subnodosus and mammalian MSTN (Hu et al. 2010;Guo et al. 2012;Morales-Collio et al. 2014;Morelos et al. 2015). It was suggested that a highly conservation of role in muscle development. Unlike C. farreri MSTN first detected in 2-4 cells period (Hu et al. 2010), in C. nobilis, the MSTN expression was detected at the fertilized eggs period.

Conclusion
In conclusion, a close relationship between C. nobilis and C. farreri was supported by phylogenetic analysis based on both the nucleotide and amino acid sequences. Furthermore, the 3D model of CN-MSTN mature peptide was similar to that of C. farreri.
Additionally, spatio-temporal expression patterns indicated that MSTN expression was found to be remarkable in the gastrulae period and adductor muscle than in other stages and tissues, respectively.

Disclosure statement
No potential conflict of interest was reported by the authors.