The Polymorphisms of β2-Adrenergic Receptor Gene on two Cattle Breeds in China
The beta-adrenergic receptors (β-AR) are a class of metabotropic G protein-coupled receptors that responsible for the muscle growth, fat deposition or lactation characteristics of domestic animals. The aim of this article was to scan nucleotide mutations in partial coding region of β2-AR gene on cattle. In this study, the polymorphisms of β2-AR gene on cattle were detected by methods of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing in two cattle breeds in China, Xinjiang Brown cattle and Chinese Holstein cows. The results revealed that there were two genotypes and homozygote variant was not found in the two breeds. The genotypic frequencies at the Sma I locus were significantly different between the two breeds. The wild-type allele was ascendancy in the two breeds but its allele frequency was distinctly lower in the Chinese Holstein cows than that in the Xinjiang Brown cattle. DNA alignment results showed that three single nucleotide polymorphisms (SNPs), C11A, G53A and C129T were found in 5-coding region of β2-AR gene. The C11A mutation resulted in disappearance of Sma I cleavage site. In addition, the C11A and G53A mutations caused two amino acid residues replacement at the 4th and 18th of β2-AR. However, the C129T mutation was a synonymous one. In conclusion, the heterozygous frequency in Chinese Holstein cows was obviously higher than that in the Xinjiang Brown cattle. The Sma I locus would be a potential genetic marker for lactation performance of cattle.
Received: March 18, 2011;
Accepted: April 26, 2011;
Published: June 15, 2011
The β-adrenergic receptors (β-ARs, or adrenoceptors) are a class
of metabotropic G protein-coupled receptors that are targets of the catecholamines,
especially norepinephrine and epinephrine (Perez, 2006).
The β-ARs have been subdivided into at least three distinct groups: β1,
β2 and β3, classically identified in cardiac,
airway smooth muscle and adipose tissue, respectively (Johnson,
1998). β-ARs are present on the surface of almost every type of mammalian
cell but the distribution of subtypes and proportion of each varies between
tissues in a given species (Mersmann, 1998). The use
of exogenous β-AR agonists to promote muscle growth and limit fat deposition
has been extensively evaluated (Liang and Mills, 2001;
Mills et al., 2003; Kim
et al., 2010). In general, the most profound effects of exogenous β-AR
agonists are observed in cattle and sheep (Mersmann, 1998;
Beckett et al., 2009).
Bruckmaier et al. (1991) reported that isoproterenol
(β-AR agonists) enhanced milk flow without affecting milk ejection. Blum
et al. (1989) found that using phentolamine (α-AR antagonists)
or aleudrin benefited lactating a lot. Over the past decade, researches indicated
that stimulation of β-ARs in the bovine mammary gland affected milking
characteristics such as milk yield and peak flow rate (Inderwies
et al., 2003a). Milk yield and peak flow rate decreased during α-AR
stimulation and during the oxytocin receptor blockade and increased during β-adrenergic
stimulation (Inderwies et al., 2003b). So if
nucleotide mutation take place in β-AR gene, the lactation performance
of dairy cattle would be influenced. However, there is little information about
polymorphisms of β-AR gene in cattle at present.
In this study, two breeds of cattle (Xinjiang Brown cattle and Chinese Holstein cows) which have obviously differences in lactation performance were chosen as the samples. PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) and DNA sequencing methods were used to scan single nucleotide polymorphisms (SNPs) in the parts of the encoding region of β2-AR gene, in order to identify potential genetic markers for lactation performance.
MATERIALS AND METHODS
Blood sample source: Ninety eight all unrelated animals (Chinese Holstein cow, n = 42; Xinjiang Brown cattle, n = 56) were collected and used in this research in December of 2009 to July of 2010. The Chinese Holstein cows were from Zhongzhou cattle farm of Yili county of Xinjiang in China, the Xinjiang Brown cattle were from Tacheng Brown Cattle Breeding Centre of Xinjiang in China. Blood samples were drawn from the jugular vein into vacuum blood collection tubes containing ACD (anticoagulant citrate dextrose). The samples were stored at -20°C for latter analyze.
DNA extraction: Genomic DNA was extracted with the use of phenol/chloroform
(Sambrook and Russell, 2001) and detected with 0.8% agarose
gel electrophoresis (0.5 x Tris-borate-EDTA buffers).
Primer design: According to the DNA sequence of bovine β2-AR
(GeneBank accession number: NM_174231), one pair of PCR primers was designed
with Oligo 6.0 software package (Qiong et al., 2011).
Primer sequences used for PCR were as follows:
||Forward: 5-TGC GCT CAC CTG CCA GC-3
||Reverse: 5-TGC CAG GCC CAT GAC CAG GTC AG-3
The primers were synthesized by Invitrogen by Life Technologies (Shanghai) and used to amplify 281 bp PCR products, containing partial 5-UTR (26 bp) and coding region (255 bp) of bovine β2-AR gene.
PCR amplification: Amplification of the DNA was performed by the PCR
with the following conditions in a final volume of 20 μL: 100 ng genomic
DNA, 5 pmol of each primer, 0.2 mM of each dNTP, 4 μL colorless GoTaq reaction
buffer, 0.2 μL GoTaqTM polymerase (5 IU μL-1,
Promega, Shanghai), nuclease-free water was added to achieve the final volume
(Dhanapal et al., 2010). Samples were amplified
in a thermocycler (Mycycler, Bio-Rad, USA) using the following programs: 94°C
for 5 min followed by 10 cycles of 94°C for 1 min, 70°C for 35 sec,
72°C for 45 sec, then another 25 cycles of 94°C for 45 sec, annealing
63°C for 30 seconds, 72°C for 40 seconds and a final extension at 72°C
for 7 min. PCR products were visualized on 1% (w/v) agarose gels prior to RFLP
Deletion mutation polymorphism in the β2-AR gene: Restriction
enzymatic digestion was performed with the following conditions in a final volume
of 20 μL: 5 μL PCR products of β2-AR gene, 2 μL
0.1% BSA, 2 μL 10xT buffer, 0.5 μL Sma I (10 IU μL-1,
TaKaRa, Japan), deionized water was added to achieve the final volume. All reagents
mixed gently and incubated at 30°C for 16 h and then 65 °C for 20 min
to inactivate the endonucleases. Afterwards, 2 μL 10xloading buffer was
added to terminate the reaction. Aliquots (10 μL) of the mixture were loaded
onto 10% polyacrylamide gel and electrophoresed at a constant voltage (200 V)
for 2 h (Alashawkany et al., 2008). At the end
of electrophoresis, the gels were stained with ethidium bromide (200 ng mL-1)
for 15 min and the gel images were captured with a GelDoc XR gel-documentation
system (Bio-Rad, USA).
DNA sequencing: The PCR amplicons from different patterns in the two
breeds purified with a DNA Fragment Qiuck Purification Kit (DingGuo, Beijing,
China) and cloned in Escherichia coli DH5α by using pUCm-T vector
(Bio Basic Inc, Canada). The recombinant plasmids DNA were isolated and sequenced
by using ABI PRISM 377 automated sequencer (Perkin Elmer-Applied Biosystems
Division, USA) with primers M13 universal forward primer (5-GTT GTA AAA
CGA CGG CCA GT-3) and M13 reverse primer (5-CAG GAA ACA GCT ATG
ACC-3) by Invitrogen by Life Technologies (Shanghai). These sequences
were then compared to those available in GenBank by using Basic Local Alignment
Search Tool (BLAST) at http://blast.ncbi.nlm.nih.gov/Blast.cgi
(Fang et al., 2010).
Statistical analysis: Based on the genotypes of Sma I locus in the two
cattle breeds, the genotypic frequencies and allelic frequencies were calculated
and Hardy-Weinberg equilibriums were detected by using PopGen32 software package
(Jun et al., 2010). The distribution of these
genotypes among all cattle populations was analyzed using χ2-test
which were performed by PASW Statistics 18.0 (Norusis, 2009).
Population genetic indexes, such as He (gene heterozygosity), Ho (gene homozygosity),
Ne (effective allele numbers) and PIC (Polymorphism Information Content) were
calculated according to Nei and Roychoudhury (1974),
Nei and Li (1979), respectively.
PCR amplification: All of the PCR amplification products were detected by 1% agarose and obtained the expected fragments with the clear and specific bands. The agarose electrophoresis profiles of β2-AR gene obtained from the primers are shown in Fig. 1. The result showed that the amplification products had a good specificity and it could be used for enzymatic digestion.
RFLP typing: In this study, polymorphisms of β2-AR gene
were scanned by PCR-RFLP in the two breeds. The results revealed that there
were 2 genotypes (named as genotype CC and CA) and 2 alleles (namely C and A)
at Sma I locus (Fig. 2). The restriction fragment lengths
included 243 and 38 bp for CC genotpye and 281, 243 and 38 bp for CA genotype.
Genotypic and allele frequencies of β2-AR gene in the two breeds
were shown in Table 1. The results showed that both of CC
and CA genotype existed in the two breeds and the frequency of CC genotype was
higher than that of CA. in both of the two breeds. The frequency of CC genotype
in Chinese Holstein cows was 0.5714 which was obviously lower than that in the
Xinjiang Brown cattle. Frequencies of allele C in the analyzed populations were
0.9018 and 0.7857 for Xinjiang Brown cattle and Chinese Holstein cow. The genotypic
frequencies at the Sma I locus were significantly different between Xinjiang
Brown cattle and Chinese Holstein cows based on a χ2-test (χ2
= 6.208, df = 1, p = 0.013).
|| Genotype and allele frequencies of β2-AR
gene in different breeds
|χ2 (HWE): Hardy-Weinberg equilibrium χ2
|| The atlas of β2-AR gene PCR products. Line
1 and 2: PCR products; line M: DNA marker
||The PCR-RFLP PAGE patterns of β2-AR-Sma I
locus. Line 1 and 3 to 7: CC genotype, line 2: CA genotype. Line M: pBR322/BsuR
I DNA marker
The C allele was ascendancy in the two breeds but its allele frequency was
distinctly lower in the Chinese Holstein cows than that in the Xinjiang brown
cattle. The χ2-test showed that the genotype distributions of
Xinjiang Brown cattle and Chinese Holstein cow breeds were in consistent with
Hardy-Weinberg equilibrium (p>0.05).
The population genetic parameters (namely, homozygosity, heterozygosity, effective allele numbers (Ne) and PIC (Polymorphism Information Content)) were demonstrated in Table 2. Value of homozygosty estimate varied from 0.663 to 0.823 and Ne ranged from 1.215 to 1.508. PIC values varied from 0.161 to 0.280.
DNA sequencing results: According to the sequencing and sequence of bovine β2-AR gene (GeneBank accession No. NM_174231), CC genotype was indentified as wild-type and CA genotype as heterozygote. Alignment results showed that three SNPs, C11A, G53A and C129T were found in coding region at β2-AR gene (Fig. 3). The C11A mutation resulted in disappearance of Sma I cleavage sites (CCCGGG→CACGGG). In addition, amino acid sequence analysis showed that the C11A and G53A mutations leaded to the fouth and eighteenth amino acid residues changed from the proline to histidine and arginine changed to histidine of β2-AR. However, the C129T mutation was a synonymous one.
|| Genetic index in two cattle breeds in China
|Ho: Gene homozygosity; He: Gene heterozygosity; Ne: Effective
allele number; PIC: Polymorphic information content
|| Sequencing maps from different genotype of β2-AR
β2-AR prevails on the surface of cardiac muscle cell, adipose
cell, nervous system, ren tissue and mammary gland (Bray
and Boerwinkle, 2000; Inderwies et al., 2003c).
It is involved in the process of physiological and metabolism regulation, including
heart rate, blood pressure, remaining of renal water and milk yields (Stoffel
and Meyer, 1993; Bray and Boerwinkle, 2000). Roets
et al. (1986) reported that there were some relevance between milk
yields of cows and α2-AR and β2-AR which were
found both in mammary papilla organs and blood cells. So nucleotide mutation
of β2-AR may influence the lactation performance of cattle.
However, there have been no reports about whether nucleotide mutation of β2-AR
gene may generate some influence on the quantity of β2-AR or
on the lactation performance of cattle.
In this study, PCR-RFLP was adopted to analyze polymorphism of β2-AR
gene in two cattle breeds in China and the results showed that there were two
genotypes, namely CC and CA. DNA sequence showed that there were three mutation
sites. The C11A mutation resulted in the disappearing of the recognition site
of restriction enzyme Sma I. Amino acid sequence showed that proline was changed
into histidine at the forth amino acid residue. Due to the fact that proline
is an imino acid while histidine is a basic amino acid, meanwhile, this site
is very conservative in cattle, swine and sheep (Carron
et al., 2005; Chen et al., 2002; Zhang
et al., 2010), so the three-dimensional structure and function of
β2-AR would be affected by the C11A mutation. This mutation
whether affect milk yield needs more further study.
Xinjiang Brown cattle are an improved breed with some Brown Swiss, Alatau and
Kesiteluomu blood. They are dual-purpose, medium-sized cattle (Suttie
and Reynolds, 2003). Its milk yields situate between 1600-3000 kg (305-day
basis) (Zheng, 1984). However, Chinese Holstein cows (named
Chinese Black and White before the year of 1997) bred with abroad Holstein cows
and indigenous yellow cattle and its milk yields situate between 6500-7500 kg
(305-day basis). In the present study, the CA genotypic frequency in Chinese
Holstein cows was obviously higher than that in the Xinjiang Brown cattle. There
seems to be certain relationship between CA genotype and lactation performance.
To prove this presumption, more researches still need.
In this research, the population genetic parameters were calculated. PIC values
varied from 0.161 to 0.280. According to the classification of PIC (high polymorphism
if PIC value>0.5, median polymorphism if 0.25<PIC value<0.5 and low
polymorphism if PIC value<0.25) (Botstein et al.,
1980). Among the loci of the two populations showed low polymorphism.
In conclusion, three SNPs existed in the 5`-conding region of β2-AR gene on two cattle breeds in China. The heterozygous frequency in Chinese Holstein cows was obviously higher than that in the Xinjiang Brown cattle. The Sma I locus would be a potential genetic marker for lactation performance of cattle.
This study was supported by National Natural Science Foundation of P.R. China (Grant No. 30860195) and the project of the Ministry of Agriculture of P.R. China (Grant No. nyhyzx07-036-10).
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