Probing the Single Nucleotide Polymorphism (Snp) of Swine PPAR Delta Gene
Recently, it was reported that PPAR delta (PPARδ, alias
PPARβ) serves as a key nuclear transcription factor regulating the expression
of myriad genes in placent nutrient absorption and energy metabolism (lipid
and glucose metabolism) which suggested it might be vital in placental development
and pregnancy. In order to investigate and identify potential genetic markers
in swine breeding, we designed two pairs of primers for Single Strand Conformation
Polymorphism (SSCP) analysis based on the complete mRNA coding sequences of
PPARδ gene and its 5- flanking region. The sequencing result showed the
amplification product of a pair of primers was polymorphic and one mutation
C→T was identified in the second intron of PPARδ gene. Before the study,
genetic polymorphisms of swine PPARδ gene were rarely reported. The present
study provided useful information for future research in relevant medicinal
genetics and animal breeding.
Received: November 15, 2013;
Accepted: November 23, 2013;
Published: February 03, 2014
PPARs (Peroxisome proliferator-activated receptors) are a kind of transcription
factors belonging to the Nuclear Receptors (NRs) super-family. When PPARs are
activated, they will affect and/or regulate the transcription of many genes
controlling vital physiological processes. Whole body metabolism related tissues,
such as skeletal muscle, adipose tissue, heart and liver, are prone to get inflammation
in metabolic disturbance. It was pointed out that PPARs had diverse functions
and wide distributions serving as important links between lipids, metabolites
and innate immunity (Hutter et al., 2013; Kruger
et al., 2010). Herein, PPARs were considered as effective targets
of drug remedy blocking obesity, diabetes and other metabolic disease.
PPARs play key roles in the metabolic syndrome and overall health of organisms
including regeneration of tissues, adipocyte differentiation, lipid and glucose
metabolism and immune response (Nagy et al., 2012).
From a nutritional viewpoint, the PPARs are of importance because of their ability
to be activated by long chain fatty acids and their metabolites (Kruger
et al., 2010). In addition, several evidences showed the important
role of PPARs in reproductive organs (Martinez et al.,
2011; Shalom-Barak et al., 2004). Therefore,
PPARs are recognized as candidates in order to improve metabolism and health
and pregnancy through suitable diet.Up to date, there are three well-known PPARs
subtypes, i.e., PPARa, PPARα (alias PPARβ) and PPARγ (classified
as three isoforms in human, i.e., PPARγ1, PPARγ2, PPARγ3) (Asami-Miyagishi
et al., 2004; Hutter et al., 2013;
Matsuda et al., 2013; Schoonjans
et al., 1996). Each subtype of PPARs is a product of a separate gene
with a distinct tissue specific distribution and distinct functions. Among these
subtypes, PPARα is predominantly expressed in the heart, kidney and liver,
etc., mainly involving in fatty acid oxidation; PPARγ is mainly associated
with adipose tissue,in which it controls adipocyte differentiation and insulin
sensitivity; PPARδ is the only subtype currently untapped as candidate
genes in metabolism and health and pregnancy research (Barak
et al., 1999; Lehrke and Lazar, 2005; Lockyer
et al., 2010; Martinez et al., 2011;
Reilly and Lee, 2008; Robinson
and Grieve, 2009; Wagner and Wagner, 2010). PPARδ
is regarded as a member of nuclear hormone receptors super-family intimately
regulating the expression of myriad genes involving in cell differentiation,
apoptosis, energy metabolism and inflammation; It was found expressed in most
metabolically active tissues, such as adipose and skeletal muscle tissues (Castillero
et al., 2013; Kruger et al., 2010;
Reilly and Lee, 2008; Wang
et al., 2003). However, its fuction has not been clearly defined yet.
On the other hand, improvement in reproductive traits (e.g., litter size), are
of interest to swine producers and breeders (Johnson
et al., 1999; Rothschild et al., 1996;
Wang et al., 2013a). However it is frequently
difficult to make genetic selection to improve the quality and quantity of animal
reproductive traits due to low heritability and/or sex-linked inheritance pattern
(Johnson et al., 1999; Rothschild
et al., 1996). PPARs may play key roles in linking lipid and glucose
metabolism and reproduction systems. Previous researches revealed that PPARγ
is associated with body conditions, reproduction hormones and their receptor
expression. The PPARγ/RXRα signaling was proved important in placenta,
cytotrophoblast and cell fusions (Asami-Miyagishi et
al., 2004; Batista et al., 2012; Hutter
et al., 2013; Matsuda et al., 2013;
Wang et al., 2013b). It is now clear that PPARs
are important in the control of placental development. Nevertheless, unlike
PPARα and PPARγ, little is known about the detailed roles of PPARδ
gene in placental development and pregnancy nutrient regulation. In view of
PPARδ as an important nuclear transcription factor implicated in adipocyte
and myocyte differentiation, lipid and glucose metabolism, skeletal muscle wasting
remedy and pregnancy in domesticated animals, we had designed animal experiments
to investigate the function of PPARδ gene. In this study, a Single Nucleotide
Polymorphism (SNP) corresponding to C→T substitution was detected and reported
in the second intron of swine PPARδ gene locus in two swine strains, Yarkshire
MATERIALS AND METHODS
Sampling and PCR-SSCP amplification: In total, 30 sows and 30 boars
of Yarkshire strains, 22 sows and 20 boars of Landrace strains, were used in
the study. Pieces of ear tissues were sampled and genomic DNA was extracted
according to the manufacturers
protocol. PCR primers were designed to detect SNP of porcine PPARδ gene
locus (Table 1). The following SSCP analysis was employed
based on the complete coding region and the reported 5'-regulator region of
two published porcine PPARδ mRNA sequences (GenBank accession No. DQ437886,
PCR amplifications were carried out in a eppendorf tube with a designed reaction
system. The reaction system was a mixture being composed of multiple components
(100-500 ng of genomic DNA, 2.5 μL of 10xPCR buffer, 200 μM of each
dNTP, 10 pM of each primers, 2U of Taq DNA polymerase and sterile double-distilled
water) with a total volume of 25 μL reaction mixture. The 10xPCR buffer
contains 100 mM Tris-HCl (pH = 8.0), 500 mM KCl, 10 mM of MgCl2 and
0.1% glutin. Following an initial denaturation at 95°C for 5 min, 35 cycles
of 1min denature at 94°C, 30 sec annealing at annealing temperature, 30
sec synthesis at 72°C, with a final cycle of 7 min extension at 72°C
(Table 1). The amplified mixture was denatured 10 min at 98°C
and then cooled on ice for 5 min with 2 μL of the PCR product and 8 μL
of the loading buffer. Genetic polymorphism was detected by Single Strand Conformation
Polymorphism (SSCP) in agarose gel electrophoresis and polyacrylamide gel electrophoresis,
respectively. Final DNA band patterns were detected by silver staining.
Polyacrylamide gel electrophoresis: A total volume of 1.5 μL PCR
product was transferred in the eppendorf tube, mixed with 6 μL gel loading
solution containing 98% formamide, 0.025% bromophenol blue, 0.025% xylene cyanol,
20 mmol L-1 EDTA (pH = 8.0) and 10% glycerol. The reaction mixture
was centrifuged and denatured at 98°C for 10 min, followed with a chill
on ice for 5 min and loaded on 10-12% neutral polyacrylamide gels (acrylamide:
bisacrylamide = 29:1). Polyacrylamide gel electrophoresis was performed in 1xTris
borate-EDTA buffer (pH = 8.3) at 9-15 V cm-1 for 14-16 h at 4°C.
Finally, polyacrylamide gels were stained with silver nitrate to identify SNP
Cloning and sequencing: After accomplishing the runs of polyacrylamide
gel electrophoresis, PCR amplifications of different homozygous genotypes were
separated on 0.7-1.0% agarose gels and recovered using geneclean II kit (Promega).
Each DNA fragment was ligated into the pGEM-T easy vector (Promega) according
to the manufacturers protocol
at 4°C overnight. The ligation reactions were carried out in a 5 μL
reaction mixture containing 1.5 μL of PCR product, 0.5 μL of pGEM-T
vector (50 ng μL), 0.5 μL of T4 ligase (3 U μL-1 and
2.5 μL of 2xligation buffer, according to the protocol's instruction. Then
recombinant plasmids were transformed into Escherichia coli DH5α
competing cells. Positive clones of transformed cells were identified by restriction
enzyme digestion. Final target clones of each homozygous genotype were sequenced
from both directions by Shanghai Invitrogen Biotechnology Co. Ltd.
|| Information of primer sequences
RESULTS AND DISCUSSION
Genomic DNA extracted: Genomic DNA extracted from ear tissues was dissolved
at room temperature for 24 h. Before carrying out PCR-SSCP analysis, the quality
of genomic DNA should be checked with 1 μL sample on the 0.7-1.0% agarose
gel electrophoresis. Figure 1 showed that the majority of
genomic DNA had clear bands and was suitable for PCR-SSCP amplification.
SNP mutation detected in porcine PPARδ gene: Two pairs of primers
were designed for PCR-based SSCP analysis of PPARδ gene. There was a mutation
C→T at 107 bp of the PCR amplified fragment of PPARδ gene 5'-regulator
region (GenBank accession No: AY188501.1). Allele gene A corresponded to base
C; allele gene B corresponded
to base T.
The representative SNP sequencing output for homozygote AA or BB, heterozygote
AB individuals is shown in Fig. 2b.
After reading and distinguishing heterozygotes from homozygotes of PPARδ
genotypes in the PCR-SSCP analysis it was found that there was little polymorphism
in the PCR-SSCP product of primer pair P1 but primer pair P2. A mutation C→T
was identified in the second intron of PPARδ gene with primer pair P2.
Therefore, all the following analysis proceeded with the result of primer pair
P2. According to international research reports and their naming rules (Komatsu
et al., 2010; Msalya et al., 2009)
the SSCP patterns in primer pair P2 were read as AA, AB and BB genotypes respectively
(Fig. 2). Every individual was tested in the sampled populations
of Yarkshire strains and Landrace strains and we found both heterozygotes and
homozygotes with PCR-SSCP analysis. Though the distribution of genotype frequencies
was not strictly following the rules of Hardy-Weinberg equilibrium, we counted
the numbers of genotypes and calculated corresponding gene frequency. There
were 8 individuals genotyped as BB and 7 individuals genotyped as AB in Yarkshire
strains while there were 5 individuals genotyped as BB and 9 individuals genotyped
as AB in Landrace strains. The gene frequency of B was calculated as 0.1333)
in Yarkshire strains and 0.3450 in Landrace strains, whereas the gene frequency
of A was calculated as 0.8667 in Yarkshire strains and 0.6550 in Landrace strains
according Hardy-Weinberg equilibrium.
The genetic polymorphisms of swine PPARδ gene and its Untranslated Region
(UTR) were rarely reported. On the contrary, polymorphisms at the PPARα
gene loci have been frequently identified (Bener et
al., 2013; Domenici et al., 2013; Maciejewska-Karlowska
et al., 2013; Motavallian et al., 2013;
Sahmani et al., 2013; Wang
et al., 2011, 2013a, b;
Yang et al., 2013; Zhang
et al., 2013).
|| Genomic DNA extracted
||PCR-SSCP analysis for the swine PPARδ gene (a) Agarose
gel electrophoresis for primers P1 and P2 and (b), Polyacrylamide gel electrophoresis
for primers P2 (i.e., genotypes of the SSCP products for primers P2)
||Sequencing result of SNP (single nucleotide polymorphism)
in PPARδ gene (a) Sequencing for genotype AA and (b) Sequencing for
Among these reports, Wang et al. (2011) found
two SNP mutations in swine PPARβ gene were polymorphic and significantly
correlated with litter sizes (Wang et al., 2011)
(Fig. 3). They also performed a few experiments to test whether
other candidate genes were correlated with swine litter sizes (personal communications).
In the present study, for the first time, a SNP mutation was identified in the
second intron of PPARδ gene and no mutation in the coding region was detected.
The deficiency in the present study was mainly the lack of the correlation analysis
of PPARδ gene mutation genotype frequencies associated with the corresponding
data of swine reproductive traits. As the PPARδ gene was considered as
a potential genetic marker associated with growth and reproductive traits in
domesticated animals, further enlarged experiments are required for the investigation
on the specific SNP mutation of PPARδ gene and its association with specific
It was revealed that the role of PPARs serving as vital metabolic sensors controlling
cell functions including many essential cellular and physiological processes
in organisms ranging from yeast to mammals. PPARδ is the only subtype of
PPARs that is currently not a candidate or target gene in metabolism and health
and pregnancy research. In the study, we designed two pairs of primers for Single
Strand Conformation Polymorphism (SSCP) analysis based on complete coding region
and the 5- flanking region of PPARδ gene and found a SNP mutation
C→T in the second intron. This mutation could provide a potential genetic
marker for swine breeding. The SNP mutation and polymorphic information from
this experiment would be useful for further research in medicinal genetics and
animal breeding of PPARδ gene. Future research (both in vivo and
in vitro) should be designed to identify novel markers and dissect the
functions of PPARδ gene for growth and reproductive traits, as well as
the progression of lipid and glucose metabolism and metabolic disorders. In
our next experiment it will be designed and carried out to detect whether positive
associations of PPARδ polymorphisms are associated with lipid and/or glucose
content and relevant genetic data. According to the present study and previous
reports, PPARδ could be considered as a candidate or target gene for swine
We are grateful to the anonymous reviewers for the suggestions. The partial
financial support of the following programs is also greatly acknowledged. This
study is supported by National Natural Science Foundations of China (No. 31301965),
Anhui Provincial Natural Science Foundations (No. 1308085QC63), Programs of
Anhui Provincial Educational Commission Natural Science Foundation (No. KJ2012A216,
No. KJ2013B198), National Statistical Scientific Research Project (No. 2013LY051)
and the Fuyang Normal College Educational Project (No. 2012JYXM71).
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