Structure of the MSTN gene
As a member of Transforming Growth Factor-β (TGF-β) family, MSTN (
myostatin), also known as growth differentiation factor 8, is a member of the transforming growth factor-β (TGF-β) family; this gene is a type of secreted polypeptide and consists of three exons and two introns (Jeanplong et al., 2001; Hinck et al., 2016).
gene of goat differs from that of pig, macaques and horses by 16, 19 and 20 amino acids, respectively. Only one amino acid difference is found between goat and sheep (Jin, 2011)
. In particular, the MSTN
C end sequence of mice, humans, pigs, chicks and turkey are highly conserved and their amino acid sequences are 100% identical. Only one to three amino acids are found different among the MSTN
of baboon, cattle and sheep (McPherron and Lee, 1997a)
The amino acid sequence of TGF-β superfamily members has low homology but exhibits similar function because they have the same functional domain that consists of one highly conservative signal peptide, one TGF-β functional peptide and one “RARR” (Daopin et al., 1992; Liu et al., 2016).
Nine immutable cysteine residues are the foundation of the tertiary protein structure of TGF super family members (Liu et al., 2016).
Eight of these residues linked via four intrachain disulphide bonds form amino acids in different positions of the polypeptide chain in the core region to the formation of a hydrogen-bonding network, which is a complementation for this region. The remaining cysteine residue and two hydrophobic surfaces form an intrachain disulphide bond, which constitutes a mature dimer structure (Bloise et al., 2019; Solinas-Toldo et al., 1995; Huang et al., 2016).
Biological function and expression of the MSTN gene
A host of research on the function of the MSTN
gene has been carried out. In 1997, McPherron was the first to verify the function of the MSTN
gene through gene editing technology and reported that mutant mice (knocked out MSTN
gene) had evident hyperplasia and hypertrophy of muscle cells and weighed two or three times higher than wild-type mice (McPherron et al., 1997b).
As an advanced biotechnology tool, gene editing technology is widely used to alter genes in breeding. For instance, the MSTN
gene of cattle (Grobet et al., 1997; Kambadur et al., 1997),
dog (Mosher et al., 2007),
pig (Bi et al., 2016),
goat (He et al., 2018),
sheep (Wang et al., 2016),
chicken (Kim et al., 2020),
rabbit (Lv et al., 2016)
and fish (Chisad et al., 2011; Dong et al., 2014)
has similar traits of natural mutation. The MSTN
gene is only specifically expressed in muscles and is closely related to muscle development in animals. However, recent studies reveal that the MSTN
gene is also expressed in other tissues and organs, implying its diverse function. In particular, the MSTN
gene impedes mouse embryonic fibroblast differentiation of preadipocytes, thereby prohibiting the formation of fat. The expression of the MSTN
gene has been to be correlated with illnesses, such as cardiac hypertrophy of human and animals (Qi et al., 2020),
diabetes mellitus (Li et al., 2020),
chronic metabolic diseases (Watanabe et al., 2019; Kuzyarova et al., 2019),
muscular atrophy (Hong et al., 2019; Liu et al., 2019)
and ischemic heart failure (Castillero et al., 2020).
Genetic diversity of the MSTN gene in domesticated animals
Although the MSTN gene is highly conserved with regard to structure, its function varies among different domestic animals when mutation occurs. The MSTN
gene of Chinese bovine remains affluent in terms of genetic diversity. For example, the MSTN
gene in Mongolian cattle, Leiqiong cattle, Dulong cattle and Bayingolin yak contains single-nucleotide polymorphisms (SNPs); however, missense mutation occurs only in one site (aa235, His®Arg) (Yi et al., 2008).
Seventeen to 29 polymorphic loci are discovered in Boomerino sheep herd from New Zealand and Stavropol merino sheep herd from Russia. A large number of SNPs are ascertained in foreign pig breeds (Landrace, Large white pig, Duroc and Pietrain pigs) and Chinese pig breeds (Meishan pig, Tongcheng pig, Laiwu pig and Wuzhishan pig) (Stinckens et al., 2008; Liu et al., 2013).
In six SNPs in Sansui duck, missense mutation occurs only in one site (g.106G>A) (Zhao et al., 2016).
In summary, poultry, whether a local or commercial variety, possesses abundant genetic diversity.
Genetic variations in the MSTN gene in domesticated animals
Studies have focused on the relationship between the MSTN
gene and skeletal muscle development. Most SNPs of MSTN
are associated with muscle development because this gene suppresses skeletal cell differentiation and myotube generation (Lee et al., 2020).
The mutation of the MSTN
gene leads to excessive proliferation of skeletal muscle cells in the animal embryonic period (Deries et al., 2016; Lehka et al., 2020);
this phenomenon results in the higher primary weight of the mutant individual than the wild type. Moreover, skeletal muscle cell hypertrophy primarily occurs after birth (Manceau et al., 2005; Gros et al., 2005).
Therefore, the performance of variations in the MSTN
gene, at a large extent, tends to increase birth weight and month age weight and amplify calving difficulty (Han et al., 2012; Casas, 1999)
. Furthermore, variations in the MSTN
gene, which is involved in differentiation of fibrocytes and anterior adipocytes, affect back-fat thickness, muscle fibre diameter and carcass quality (Li et al., 2016).
This study reports that variation in the MSTN
gene multi-allele dramatically transforms the production traits of bovine. The mutant hybrid and homozygote in Marchigiana bovine engender double-muscled cattle (Sarti et al., 2014).
Among 10 types of European breed cattle, the mutation of five of 10 polymorphic loci of the MSTN
coding sequence stimulate the formation of double muscles (Grobet et al., 1998).
A high correlation is found between MSTN
and animal birth weight and mutant individuals generally have higher birth weight than wild-type individuals. In Piedmont cattle, Angus cattle, Hayford cattle and their hybrid offsprings, G/A mutations in the exon 3 of the MSTN
gene significantly affect growth performance, such as birth weight, corrected weaning weight and different age weight and increase calving difficulty (Casas, 1999)
. The F94L mutant in the MSTN
gene is found in Limousin cattle, Angus cattle and first filial generation between Limousin cattle and Angus cattle; the birth weight of the mutant individuals increased by 2.7%, 2.2% and 3.2% relative to that of wild-type individuals (Lee et al., 2019).
The F94L site imposes marble score, eye muscle area and fat thickness. In Southern Devon cattle, 11 bp nucleotide deletion and the local beef cattle promoter g.371T> A mutation are found. These mutation sites not only yield double-muscled pigs but are also involved in fat deposition, back-fat thickness and birth weight (Wiener et al., 2002; Han et al., 2012).
The 3'UTR region g.+6723G>A in the MSTN
gene of ovine has been studied, revealing the differentiation in disparate ovine type and the function of the ovine group. In Belgian Texel sheep, the MSTN
gene mutation site g.+6723G>A generates a target site of microRNA, which prohibits the expression of the gene and leads to excessive muscular hypertrophy of Texel sheep (Clop et al., 2006).
The mutual traits of the MSTN
gene in New Zealand Texel sheep are muscle increase and fat reduction (Johnson et al., 2009).
gene mutation site g.+6723G>A in Texel sheep from Austria and white suffolk sheep leads to the same results, except for the decrease in the feed intake (Kijas et al., 2007).
The mutation site (g.+6723G>A) of the MSTN
gene also exists in multiple ovine hybrids (Dorset sheep, White Suffolk, Merino) and severely affects protein expression, increased weight of mutual tissue organ and incremental amount of muscle fibre (Haynes et al., 2013).
In Hu sheep, the MSTN
gene promoter (A®G) and exon 2 polymorphic sites (A®G) are correlated with weaning weight and June age weight. The polymorphic loci (G®T) at the 32 UTR are significantly related to weaning weight (Wang, 2010)
. Birth weight, tail weight, weaning weight and carcass traits are correlated with polymorphic loci at two sites of the MSTN
gene (c.-2449 G/C and c. -2379 T/C) in New Zealand Romney (Wang et al., 2016).
Intron 1 (c.1232 G/A) SNP in Poland Merino affects the development of the waist, front legs and rear legs (Grochowska et al., 2019).
In 2006, Liu claimed the existence of eight SNP in the 52 UTR, exon 1 and exon 2 sequences, which are associated with birth weight, end weight and weaning weight amongst diverse ovine breeds (Liu, 2006)
The occurrence of SNP in the MSTN
gene in the 52 UTR (5 bp TTTTA insertion and deletion) of Boer goat, Matou goat, Haimen goat and Nubian goat promotes growth traits (Zhang et al., 2012).
The insertion/deletion mutation of 5 bp TTTTA in the 52 non-coding region of the MSTN
gene is also significantly associated with the growth traits of Inner Mongolia White Cashmere goats (Bi et al., 2020a)
and Shaanbei White Cashmere Goat (Bi et al., 2020b),
particularly in terms of chest depth (p=0.003), height (p<0.05) and chest circumference (p < 0.05). Moreover, the SNP g.345A>T detected in the MSTN
genes of Boer goats and Anhui white goats is correlated with weight, body length and height at 12 months of age (Zhang et al., 2013).
The T/C mutation at the 3783rd in the intron 2 region of the MSTN
gene in Haimen goat dramatically affects the initial birth weight of goats (p<0.010) (Zhang, 2009)
Research on domestic pigs reveals that economic traits, such as double muscles and carcass quality, are related to the mutation of the MSTN
gene. Two polymorphic loci (g.435G> A and g.447A> G) are found in the promoter region and are correlated with pig carcass quality and average daily gain in Duro, Landrace, Duro x Lu pig and Duro x Yorkshire x Landrace. In addition, the SNP (T/A) in the 52 UTR of the MSTN
gene of the F2 generation of Jinhua pig x Pitland pig is related to porcine muscle fibre diameter, muscle percentage, back-fat thickness, muscular colour brightness and average daily weight gain at 4 months (Wu, 2009)
. In local and commercial Chinese pig breeds, the mutation of the MSTN
gene is correlated with birth weight and weaning weight (Guan, 2006, Wang, 2014)
Function and physiological pathway of MSTN mutants and proteomics
Analysis of the amino acid composition shows that the MSTN
gene has nine fairly conserved cysteine residues. When the mutation alters the position and quantity of cysteine, the biological activity of the gene will change. Activin A and MSTN
are members of the TGF-β family (Thissen et al., 2013)
and have similar protein structure and strictly conserved cysteine residues. When the mutant of activin A (cys44 and cys80) combines with the receptor, the biological activity is approximately 2% of that of wild-type activin A; when the mutant of activin A (cys4 and cys12) combines with the receptor, the biological activity of binding between the monomer and receptor decreases by two or three times (Mason et al., 1994).
A typical example is missense mutation in the exon 3 of the MSTN
gene in Piedmont cattle; this mutation induces tyrosine to replace the invariant cysteine in the mature region, resulting in complete or almost complete loss of function of the MSTN
protein (McPherron et al., 1997a)
and dysregulation of muscle development with double-muscle traits.
The transformation of function is accompanied by modification in the core structure of the protein caused by mutations in the MSTN
gene. The exon 3 of mouse MSTN
gene 175-180 nt is the core sequence that affects the function of the MSTN
protein. When a mutation occurs in this region, the two amino acids at the Y (309) and C (310) positions are deleted in the MSTN
protein structure, which destroys the disulphide bond of the mature peptide, leading to the prolongation of the β extension chain and change in the protein structure. The deficiency of a protein binding site in the MSTN
mutant protein affects its biological activity of binding to the receptor (Chen et al., 2019). MSTN
in exon 3 includes an 11 bp nucleotide deletion that results in frame shift. A similar mutation is found in mice. Targeted mutation of the MSTN
gene in mice eliminates the MSTN
mature active region and leads to muscle hypertrophy phenotype after mutation (McPherron et al., 1997a; McPherron et al., 1997b).
In Texel sheep, the G to A mutation in the 32 UTR of the MSTN
gene creates target sites for mir1 and mir206, which are highly expressed in skeletal muscles, causing translational inhibition of the gene and muscle hypertrophy (Clop et al., 2006; Ge et al., 2020).
Smad, MAPK, p38 and c-Jun N-terminal kinase and other signalling pathways participate in the physiological process of the MSTN
gene and play an irreplaceable role. For instance, combining the active dimer of mature MSTN
with activin receptors (ACVR2B) stimulates the activation of ALK4 and ALK5. Smad2 and Smad3 are phosphorylated and transferred to the nucleus, preventing Akt/TORC1/p70S6K signalling and avoiding myoblast differentiation and myotube size (Lee et al., 2020; Trendelenburg et al., 2009; Lessard et al., 2018).
Smad2, Smad3 and Smad4 induce the transcription of the MSTN
gene, which passes SBE (Smad7 binding element). Combining with increased transcription of Smad7, Smad7 regulates the expression of the MSTN
gene through a negative feedback mechanism (Zhu et al., 2004).
MyoD binds and activates the promoter of Smad7; as such, Smad7 directly interacts with MyoD and enhances MyoD transcriptional activity (Kollias et al., 2006). MSTN
can obstruct MyoD activity and expression through Smad 3 and preclude muscle cell differentiation and myotube formation (Langley et al., 2002).
When the expression of MSTN
is inhibited, Smad3 up-regulates the expression of MyoD, Myf5 and MyoG to promote the differentiation and proliferation of muscle cells (Du et al., 2016; Zeng et al., 2014; Horbelt et al., 2015).
For instance, the deletion mutation of the MSTN
gene of mouse exon 3 occurred at 175-180 nt; the expression of MSTN
gene mRNA in muscle tissues as well as MSTN
receptor activin type II receptor gene decreases, but the expression of MyoD, Myf5 and MyoG markedly increases. Mice tend to exhibit muscle hypertrophy phenotype. When the lack of functional MSTN
and lack of the expression of functional MSTN
decline, myoblast proliferation and differentiation are out of control (Chen et al., 2019).
Thus, muscle hyperplasia and hypertrophy appear during myogenic differentiation of the MSTN
With the advancement of people’s living standards, the need for high-quality meat products increases. Natural mutation and genetic engineering destroy the MSTN
gene to ameliorate animal muscle quality, fat deposition and carcass quality. This work provides new research insights into cultivating new meat types of domestic livestock and poultry and effectively enhancing the meat production performance of livestock and poultry without affecting the reproductive capacity and health status of the animals. In addition, the MSTN
gene plays a vital role in the occurrence of various metabolic diseases in humans and animals. Hence, the study of the mechanism of the MSTN
gene has certain clinical significance for diagnosis and treatment of such diseases.