IN-SILICO GENETIC ANALYSIS OF THE SEQUENCES OF MYOSTATIN GENE IN BOVIDS (CATTLE, SHEEP AND GOAT)

ABSTRACT

Double muscling is a heritable trait. It has been revealed that myostatin (MSTN) or growth differentiation factor 8(GDF8) is the genetic agent of this trait. The gene is a myokine, a protein that inhibits myogenesis (muscle cell growth and differentiation). In-Silico genetic analysis was done to analyze the sequences of Myostatin gene in cattle, sheep and goat. A total of thirty seven (37) nucleotides with their corresponding amino acid sequences comprising of (26 for goat, 9 for cattle and 2 for sheep) were retrieved from the Genebank. The genetic polymorphism with three variants (M180L, S276N, S279K), five (K178N, V151L, E247D, Q329L, G355I) and six (R98H, I119T, S125M, G133S, T165N, H328T) for goat, sheep and cattle respectively, appeared not to impair the gene function while three variants (K153F, T240K, L270Q), six (S191P, W203L, S205C,  N222Q,  D231C,  R303G),  seven (D110L,  I158A,  R175V,  K193V,  S205G,  P301L, F353N) were deleterious. The results revealed beneficial amino acid variants which can be used as possible markers for growth and development in goats, sheep and cattle. Although Capra and Ovis family had more propinquity and organized branch in the phylogenetic tree, the Neigbour- joining showed that sequences of the three species are similar. Also, goats and sheep appeared more similar in their amino acid contents compared to cattle. However, the distribution pattern was the same for the three species in respect of (aspartate 6.1, cysteine 3.5, glutamate 6.7, methionine 2.1, phenylalanine 3.7, proline 6.4 and tyrosine 3.2 with leucine 9.9, as the highest). The results also showed that this gene has a high degree of conservation during evolution of various species, which implies that MSTN, is an essential factor in biological muscle control. Physicochemical properties also showed extinction coefficient =51630 for sheep and cattle, half life=30hours for goats, sheep and cattle and aliphatic index =84.45 for goats and sheep, other parameters varied from one species to another. The secondary protein structure prediction in the bovine myostatin protein showed highest alpha helix (23.20%) and random coil (44.00%) with caprine and ovine at 22.67% and 43.20%. However, the extended strand (25.87%) and beta turn (8.27%) predictions were higher in both caprine and ovine species with bovine at 25.33% and

7.47%. Tertiary protein structure prediction of goat and sheep are the same while that of cattle

differed. Furthermore, the results showed that capra and ovis family are much similar in function compared to cattle. Finally, the comparative inferences of myostatin gene sequences of the species studied conferred similarity in goat and sheep than cattle.

1.0                  CHAPTER ONE:     GENERAL INTRODUCTION

1.1      INTRODUCTION

Improving agricultural production and human food supply are the major human concern all over the world especially in developing countries. Meat is a very important source of food to human as it supplies protein and energy (Aleriza et al., 2014). The first step in animal breeding was selection of the  best animals by ranchers, but today scientists consider recognizing genetic aspects of major genes affecting meat production. In recent years, the tools of new molecular techniques developed and caused the discovery of new growth factors that are involved in the regulation of muscle mass (Diel et al., 2010). It has already been pointed out that one of the interesting aspects of hypertrophy or double muscle is the dramatically increased muscle as a result of a combination of muscle fibre hyperplasia and hypertrophy (Mcpheron and Lee, 1997). The  gene  encoding  myostatin  was  discovered  by  geneticists  Se-Jin  Lee  and  Mcpherron Alexander who also produced a strain of mutant mice that lack the gene. These Myostatin “Knockout” mice have approximately twice as much as normal mice (Mc Pherron et al., 1997) which were subsequently named “mighty mice”. The term myostatin also known as (growth differentiation factor (GDF8) or MSTN gene is a myokine, a protein produced by muscle cells that acts on muscle cells (autocrine function) to inhibit myogenesis; muscle cell growth and differentiation. Myostatin is a secreted growth differentiation factor (GDF8) that is a member of the Transforming growth factor (TGF) beta protein family (Joulia-Ekaza and Cabello, 2007). Animal with mutant genotypes in GDF8 gene not only produce more meat, the quality of the meat in their products is also different.

In these animals, the posterior limbs are round and prominent, the muscle are in protrusion mood and clear lines under the skin are visible. Prominent examples of mammals are Belgium blue and pre-montese, which show significant characteristics of double-muscle (Kambadur et al., 1997).

Myostatin referred to as growth differentiation factor-8 (GDF8) is a member of the mammalian growth transforming family (TGF-B Super family), which is expressed specifically in developing an  adult  skeletal  muscle  (Gonzalez-Cadavid  and  Bhasin,  2004).  Mice  that  lack  myostatin indicated a widespread increase in skeletal muscle mass due to an increase in both myofiber size (hypertrophy)  and  myofiber  number  (hyperplasia):  (Mcpherron  et  al.,  1997).  Muscular

hypertrophy (mh) also called double muscling has been intensified during a study in cattle as a heritable physiological character and is found in Austriana de loss valles, Belgian blue and Redmontese breeds of cattle (Smith et al., 1997). Use of double muscle beef breeds has been encouraged as a result of their high meat yield and superior meat quality associated with high proportion of white, glycolytic muscle fibres (Shah et al., 2006). Double-muscled cattle also deposit much less fat than other breeds (Potts et al., 2003).

MSTN is synthesized as a biologically inactive precursor molecule (Full length MSTN protein) comprising 3 domains, the signal peptide, the propeptide (N-Terminus), and the C- Terminal domain. MSTN is composed of 375 amino acid precursors, and has same C-terminal fragments of about 109 amino acid residue in mice, rats, human, swine, fowl and turkey and only 3 amino acid residue in C- terminal region thereof are not the same in monkeys, cows, and sheep, The C- terminal regions are expected to include physiologically active portions of MSTN (Thomas et al., 2000). Major effect of a single gene on processing yields opened a potential channel for improving processing yields of animals using knockout technology (Arif et al., 2002). Therefore, in present study, in-silicon genetic analysis of sequence of myostatin gene in mammalian species is important in understanding the evolution, differentiation and the effects of polymorphism on the myostatin gene (MSTN ) and how they are related within and among the mammalian species under study.

1.2      Objectives of the Study

The general objective of the study was to analyze the myostatin gene in selected bovids (cattle, sheep and goat) with a view to providing relevant genetic information for breeding and selection programmes in the studied species in Nigeria.

The Specific objectives were to;

1.      Examine the attendant effects of various amino acids substitutions of the myostatin gene in the selected species.

2.      Examine the genetic diversity of myostatin in silico on their evolution and differentiation within and among species.

3.      Study the various physicochemical properties of myostatin gene and;

4.      Predict the secondary and tertiary structures of the myostatin gene in the selected species.

1.3       Justification

Myostatin genetic polymorphisms have evoked considerable research interest in recent years because of its  possible  association with growth. The  major  challenge that  faces  molecular geneticists is to identify markers for genes that control the phenotypic variation in the target traits. Recent advances in high-through put technologies have generated massive amounts of genome sequence and genotype data for a number of species. The method to identify functional SNPs from a pool, containing both functional and neutral SNPs is challenging by experimental protocols. Therefore, computational predictions have become indispensable for evaluating the impact of nonsynonymous single-nucleotide variants discovered in exome sequencing. A good knowledge of the sequences of myostatin gene will help in identifying the variants responsible for various factors attributed to the gene. This will help breeders to plan breeding programmes more easily and in turn reduce the problem of low supply of high quality protein facing the increasing populations especially in a developing economy such as Nigeria

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