INTRODUCTION

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 3'.

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 genotype number.

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 locus:

y_ijl = μ+s_i+g_j+e_ijl

where, y_ijl is an observation on the slaughter traits, μ is the overall mean, s_i is the effect of sex, g_j is the effect of genotype and e_ijl is the random residual. The data were analyzed by GLM procedure.

RESULTS

PCR-SSCP
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.

Fig. 1:	Agarose gel electrophoresis of PCR product of goose PPAR gene

Fig. 2:	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:

Fig. 3:	Mutation site of PPAR gene partial sequence

Table 1:	Table of gene frequency and genotype frequency
χ20.05(2) = 5.99

Table 2:	Relation of genotype and performance in goose of different gender
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 1.

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).