With the development of living standard, produce and consume low-fat, high
quality meat has become mainstream. Lipid metabolism is a very complicated biochemical
response process and it cooperated with many regulation factors. PPAR
gene is known to us that it has important effect to fat deposit and differentiation.
Some researches (Kerenzvi et al., 1992; Schoonjans
et al., 1996; Lemberger et al., 1996;
Xie et al., 2005; Ma et
al., 2011; Meng et al., 2002) have showed
that activation of PPAR gene could induce redistribution of animal external
fat and lean tissue, regulate many gene that participated in lipid metabolism.
Especially some important enzymes of β-oxidation process, they charge of
fat absorption, transportation, formation, decomposition and so on.
Dongting goose is a fine two-line Hybrid Breed. It is breeding and selection
by SiChuan white goose and XuPu goose, which are famous goose breeds in China.
Dongting goose mainly used its meat, they have favorable growth rate, however,
they also have relative strong ability of deposition external fat. So, if we
can select low-fat goose line, it would bring considerable economic and social
benefits, fulfill the need of produce and consume.
Now, seldom report about PPAR in goose. We screened the SNP of PPAR
by PCR-SSCP, researched the polymorphism and its relate to production performance.
Hope to find the relation of SNP and performance of goose. It will helpful to
breeding of low-fat goose line and correlation researches in the future.
MATERIALS AND METHODS
Animals and samples preparation: Selected 282 test samples of 75 days
Dongting goose. Each collected 1 mL blood sample from wing vein. Then add into
4% EDTA to prevent the blood from concreting. Adopted saturated phenol-chloroform
method extracting genome DNA (Sambrook and Russell, 2001),
stored in -20°C.
Primer design: According to the sequence of PPARα mRNA in
GenBank, designed the prime by Prime Primer 5.0, covered partial coded sequence.
Sequence of prime are: F: 5 AATCACCCAGTGGAGCAG 3', R: 5' CAGACCTTGGCATTCGTC
PCR-SSCP: Used the prime started to PCR and SSCP analysis. The PCR reaction
system is: 10xbuffer (Mg2+) 1 μL, F, R primer (20 μmol
L-1) each 0.15 μL, dNTPs (10 μmol L-1) 0.2 μL,
Taq DNA polymerase 1U, DNA (100 ng) 0.7 μL and supplement ddH2O
to 10 μL totally volume.
Take 5 μL PCR production, mix with 10 μL denaturant, denaturalization
10 min at 98°C and put in ice 5 min at once. Then take 10 μL from them,
keep voltage at 80~120V to 15% PAGE (He et al.,
2006). When electrophoresis indicator reach bottom of the gel, terminate
it and dyeing by silver staining method, record stripline and statistic every
After electrophoresis, we PCR the DNA samples of homozygote and give them sequencing
to ShangHai Invitrogen Company.
Statistical analysis: SAS program (8.2 version) was used to statistic
and analysis all the test data (Xue et al., 2004).
Allele and genotypes frequencies were calculated from the genotypes of the 282
goose, respectively. Hardy-Weinberg equilibrium in the studied population was
tested by comparing expected and observed genotype frequencies using chi-square
test. A linear model was established to analyze the genotypic effects of the
yijl = μ+si+gj+eijl
where, yijl is an observation on the slaughter traits, μ is
the overall mean, si is the effect of sex, gj is the effect
of genotype and eijl is the random residual. The data were analyzed
by GLM procedure.
Result of agarose gel electrophoresis of PCR: We amplified partial sequence
of goose PPAR. From the result of agarose gel electrophoresis Fig.
1, the band of PCR ranged of 242~331 bp. The result showed that the PCR
condition was suitable. The size of PCR fragment and aimed gene fragment is
consistent and has nice specificity.
|| Agarose gel electrophoresis of PCR product of goose PPAR
|| PCR-SSCP electrophoresis of PPAR gene partial sequence
Genotypes 3 had come forth via SSCP: 2 homozygotic type and 1 heterozygotic
type. According to the band location of electrophoresis, 2 homozygotic type
were defined AA and BB genotype and 1 heterozygotic type was defined AB genotype
(Fig. 2). Because the existed of SNP between AA and BB, space
conformation of the two homozygotic type were different. This led the different
result of electrophoresis. It showed that mutation would change its space conformation
and electrophoresis could reflect the change.
Sequence analysis: After sequencing, the sequence of AA and BB gene
are as follows:
|| Mutation site of PPAR gene partial sequence
|| Table of gene frequency and genotype frequency
|χ20.05(2) = 5.99
|| Relation of genotype and performance in goose of different
|In the same column, the different capital letters mean significant
difference of ♂ in different genotype (p<0.05), the different small
letters mean significant difference of ♀ in different genotype (p<0.05)
and *mean significant difference of means in different genotype
Selected PCR product of AA and BB, send them to sequencing. From the figure
of above, we can deduce that the mutation of C→T in two gene sequence at
215 bp Fig. 3.
Allele frequency and genotype frequency: Upon chi-square test, the difference
of our research group was not significant (p>0.05). It showed that the population
was fitting hardy-weinberg law, belonged to balance population. Gene frequency
of A was 0.392 and B was 0.608. the specific results were listed in Table
By way of ANOVA and multiple comparisons, the results were shown in Table
2, the difference of abdominal fat weight, abdominal fat ratio and liver
weight ratio of gander, goose and their means in different genotype were significant
(p<0.05). It indicated that the Dongting goose has different fat deposit
performance in different genotype. From this point, we could deduce that SNP
may interrelate with the performance in a certain extent. It can affect the
lipid metabolism. Furthermore, the semi-eviscerated percentage of means and
eviscerated percentage of goose had significant difference in genotype of AA
and BB (p<0.05).
SSCP is a rapid, simple and sensitive mutation detection method. In order to
achieve the best results of SSCP, we should pay attention to electrophoresis
voltage and temperature. For maintain a stable conformation of single-strand
DNA, SSCP should be carried out under low temperature (4~15°C). In addition
electrophoresis process temperature, high voltage caused a rise in temperature
is the main reason. With about 100 V voltage electrophoresis, which was mainly
due to the beginning of high voltage can separate the different conformation
of DNA single strand and the gel will not increase the temperature and then
the low-voltage electrophoresis can make it further separation (He
et al., 2006). How many voltage electrophoresis we can used? It should
be based on specific test conditions to determine.
It found in the test that short-chain DNA mutation detection rate higher than
the long-chain of SSCP. This may be due to molecules of long-chain DNA, changed
single nucleotide plays a small role in maintenance of the conformation. Generally,
the midpoint mutation of 300 bp DNA detection rate is over 90%.
Screen SNP of PPAR gene in Dongting goose by PCR-SSCP. It found that
there is a G→A mutation in the two allele gene sequence of 215 bp. That
showed goose PPAR gene fragments of DNA has single-nucleotide polymorphism
and the heterozygous genotype frequencies of test samples were higher, which
could related on the breeding of Hybrid Breed System of Dongting goose.
Research has shown that PPAR gene polymorphism related with abdominal
fat weight and abdominal fat ratio. The results of this study was similar (Luo
et al., 2010). We can see the PPAR gene mutation consisting
of different genotypes of goose have some regulation with fat metabolism and
deposition. Whether it could be use as a candidate gene remains to be further
studied. If there is obviously related, PPAR gene will improve as the
candidate genes to goose meat quality trait and apply for marker-assisted selection,
provide new ways and means for the scientific breeding of goose.
In our detection, the PPARα gene frequency of A, B were 0.393,
0.607, respectively and the genotype frequency of AA, AB, BB were 0.126, 0.533,
0.341, respectively the abdominal fat weight, abdominal fat ratio and liver
weight ratio showed a tendency of BB>AA. So the genetic marker can be used
as one of the indexes of high, low fat goose strain selection.
This study was supported by National Natural Science Foundation of China (No.
31101695) and Scientific Project of Hunan Province (2012NK4015).