Acetyl-CoA carboxylase α (ACC-alpha) is the key regulatory enzyme in fatty acid biosynthesis. This biotin-dependent enzyme is regulated by the Acetyl- CoA carboxylase α gene (ACC-alpha) and can catalyze irreversible carboxylation of Acetyl-coA for producing malonyl-CoA (Badaoui et al. 2007; García-Fernández et al. 2010a). Malonyl-CoA is considered as substrate for the synthesis of long chain fatty acids (García-Fernández et al. 2010b). Lipogenic tissues including adipose tissues, liver and lactating mammary gland are the target tissues where the ACC-alpha is predominantly expressed (Ponce-Castañeda et al. 1991; Zhang et al. 2010). The complete sequence of the bovine ACC-alpha, located in chromosome 19, was first reported by Mao et al. (2001). Bovine ACC-alpha is regulated in a tissue-specific pattern depending on four various promoters (Barber et al. 2005). The ACC-alpha gene in sheep is under the control of a single gene located in chromosome 11 (Barber and Travers, 1995). This gene involves a number of 58 exons which encodes a protein including 2346 amino acids (Barber and Travers, 1995). By sequencing a large fragment of caprine ACC-alpha gene, Badaoui et al. (2007) reported the existence of a great amino acid identity among goat and sheep. Genetic polymorphisms of the ACC-alpha gene have been reported in various species including cattle, sheep, goat and fish (Badaoui et al. 2007; Signorelli et al. 2009; Zhang et al. 2010; Cheng et al. 2011; Crepaldi et al. 2013). Several studies have also reported related association effects between ACC-alpha gene polymorphisms and milk and meat traits in farm animals (Crepaldi et al. 2012; Crepaldi et al. 2013; Badaoui et al. 2007; Milanesi et al. 2010). ACC-alpha gene has been proposed as a candidate marker for meat quality traits in Hanwoo beef steers (Shin et al. 2011). Significant association has also been reported among polymorphic conformations of promoter I of the ACC-alpha gene and back fat thickness, triacylglycerol content and fatty acid composition of longissimus dorsi muscle (Zhang et al. 2010). Badaoui et al. (2007) identified the association among a silent SNP existing in the exon 45 region of the ACC-alpha gene and milk fat, lactose and somatic cell count in lactating goats. Signorelli et al. (2009) reported three SNPs in promoter III of the ACC-alpha gene that was associated with milk fat yield in goat. Possible existing polymorphism in the exon 1 region of the ACC-alpha gene has not been reported in any goat breeds, so far. In this study we aimed to find polymorphism in this region, in Mahabadi goats of Iran using two different methods (including PCR-RFLP and PCR-SSCP).
MATERIALS AND METHODS
Random blood samples were collected from a total of 150 Mahabadi goats belonging to the experimental farm of the TehranUniversity, Alborz, Iran. Samples were collected from does at different ages. For each individual approximately 5 mL of vein blood was gathered in EDTA tubes and was transferred to -20 ˚C freezer.
DNA extraction and polymerase chain reaction (PCR)
Genomic DNA was extracted from blood samples using modified salting-out protocol on whole blood (Miller et al. 1988). A 390 bp fragment of the exon 1 region of ACC-alpha gene was amplified through PCR technique. Primer pairs were designed utilizing Vector NTI (Vector NTI®, Infor Max Inc, North Bethesda, MD) and based on the DNA sequence of the ovine ACC-alpha gene )Gene Bank access number AJ292285.1). Arrangement of forward and reverse primers (synthesized by the Metabion company) is presented in Table 1. The PCR reaction was carried out in a 25 µL final volume containing 1X reaction buffer, 0.5 ppm of each primer, 0.2 mM of each dNTP, 2 mM MgCl2 , 1 unit of Taq DNA polymerase and 150 ng of goat genomic DNA as template. Thermal cycling condition was set at 95 ˚C for 5 min for denaturation, followed by 33 cycles of 95 ˚C for1 min, 60 ˚C for 45 sec. for annealing, DNA extension at 72˚C for 45 sec. and a final elongation of 10 min at 72 ˚C. About 10 µL of the PCR product was mixed with 8 µL of gel loading solution containing 95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol. The volume of the solution was adjusted using distilled H2O.
Genotyping of each animal was done using RFLP technique. Aliquots of 10 µL of the amplified DNA were digested with 5 U of Hinf1 endonuclease at 37 ˚C for 10 hours following the supplier’s guidelines for buffer condition. The digested products were then visualized by loading on 14% PAGE in 0.5x Tris-Borate-EDTA (TBE) buffer and constant voltage of 100 V for 5 h.
SNP genotyping using PCR-SSCP
Two µL PCR product was admixed with 18 µL SSCP DYE (1960 µL formamid, 60 µL 0.5 M EDTA, 1 µL zinol sianid and 1 µL bromophenol blue). The mixture was denatured at 96˚C for 10 min, cooled on ice for 10 min and was loaded on nondenaturing 12% polyacrylamide gels (37.5:1 acrylamide to bis-acrylamide). Electerophoresis was performed in 0.5x TBE buffer at 250 V for 15 hours at -4˚C. After electrophoresis, the DNA fragments in the gel were detected by silver staining.
The PCR product of each SSCP pattern of the exon 1 region of ACC-alpha gene was purified and sequenced by Biotech Company in both directions (Seoul, south Korea) in order to identify the existing SNPs in different genotypic patterns.
RESULTS AND DISCUSSIONACC-alpha gene has been reported as a polymorphic marker for selection in milk or meat fat in farm animals (Badaoui et al. 2007; Crisà et al. 2010; Signorelli et al. 2009; Crepaldi et al. 2013). Therefore, the effect of the ACC-alpha genetic polymorphism is important due to its potential impact on milk and meat quality traits. As expected, the amplified goat ACC-alpha gene resulted in a 390 bp DNA fragment covering the exon 1 region. HinfI recognizes 5'G▼ANTC3' cutting site. No polymorphism was observed after digestion of the PCR products using Hinf1 restriction enzyme. Under PCR-SCCP two different conformations were detected after electrophoresis on gel (Figure 1).
a According to IUPAC nucleotide codes, R refers to A or G.
*** (P<0.001).NS: non significant.