Cloning, Expression And Association Analysis Of Carcass And Adipose Candidate Genes In Meat Duck

Fat deposition is controlled by several genes. However, the fat deposition is also can be changed by mutation of one gene. The more fat deposition of meat duck decreased the feed utilization ratio and carcass quality. With improvement of people's living conditions, various diseases which caused by excess fat intake have wildly concerned by people. So, the development of meat duck industry was restricted. Investigated the adipose distribution and the differences of growth and development, improving intramuscular fat content, controlling fat deposition, improving the feed conversion ratio and muscle quality in meat duck are important problems to solve in current.In this study, we collected the liver samples of Peking duck to clone the adipose candidate genes (ADRP, PPARα, PPARγand IGF-Ⅰ), further predict their structure and function by bioinformatics, analyze their expression pattern in different tissues of 8 weeks old Peking duck by RT-PCR and real-time fluorescent quantitative PCR, construction and expression of prokaryotic expression vector for PPARα, PPARγand IGF-Ⅰgene in E. coli, detect the genetic diversity of different exons of PPARα,PPARγand IGF-Ⅰgene using SSCP and sequencing assay technologies, and analyze the associations between the polymorphisms and carcass and adipose traits in Peking duck Z2, Z4, hybrid line and Cherry Valley duck populations using general linear model with SPSS 13.0. The main results were showed as following:1. Cloning and bioinformatics analysis of the adipose candidate genes in Peking duck The ADRP, PPARα, PPARγand IGF-Ⅰgene were cloned by RT-PCR. They all contained complete CDS sequences and the length were 1311bp, 1430bp, 1650bp and 620bp. We submitted them to GenBank and obtained the accession number of these sequences (EU303280, EF534215, EF546801 and EU031044, separately). Aligned the ADRP, PPARα, PPARγand IGF-Ⅰnucleotides and amino acids of duck with the sequences of people and chicken, the homology of them were all above 75%. The structure and function of ADRP, PPARα, PPARγand IGF-Ⅰwere predicted using bioinformatics software. There was no complex spatial structure in ADRP. A ZnF-C4 domain and a HOLI domain were contained in PPARαand PPARγprotein. A signal peptide cleavage site between amino acid 29 to 30 in PPARγwas found. In IGF-Ⅰ, two strong transmembrane helices, a signal peptide domain (located at 1~48 amino acids and the cleavage site between 48th and 49th) and aⅡGF domain were found.2. Expression pattern of the adipose candidate genes in Peking duckExpression pattern of ADRP, PPARα, PPARγand IGF-Ⅰgenes in breast muscle, abdominal fat, liver, kidney, heart, small intestine, lung, muscle stomach and spleen of 8 weeks old Peking ducks were studied. By RT-PCR, the results showed that the expression of ADRP was the highest in kidney in male and female ducks, and then in abdominal fat, lung, heart, small intestine, liver and breast muscle, in spleen and muscle stomach were the lowest. And the expressions of ADRP in female were higher than male in most tissues. For PPARαgene, the expression of lung was the highest, and then spleen, abdominal fat, heart, kidney, muscle stomach, breast muscle and liver, of small intestine was the lowest in male ducks; in female ducks, of the kidney was the highest, and then the heart, liver, small intestine, muscle stomach, abdominal fat, lung and spleen, of breast muscle was the lowest. And the expressions of PPARαin female were higher than male in most tissues. For PPARγgene, the expression of lung was the highest, and then spleen, abdominal fat, heart, muscle stomach, breast muscle, liver and kidney, of small intestine was the lowest in male; in female ducks, of kidney was the highest, and then small stomach, muscle stomach, liver, abdominal fat, heart, spleen and lung, of breast muscle was the lowest. And the expressions of PPARγin male were higher than female in most tissues. For IGF-Ⅰgene, the expression of heart was the highest, and then small intestine, muscle stomach, kidney, lung, liver, spleen and breast muscle, of abdominal was the lowest in male; in female, of lung was the highest, and then spleen, heart, kidney, muscle stomach, small intestine, abdominal fat and liver, of breast muscle was the lowest. And the expressions of IGF-Ⅰin male were higher than female in most tissues.By real-time fluorescent quantitative PCR, the results showed: for ADRP gene, the expressions of abdominal fat and breast muscle were higher than other tissues, of abdominal fat was the highest, and then breast muscle, liver, spleen, muscle stomach, small intestine, kidney and heart were low, of lung was the lowest in male and female ducks; and the expressions of ADRP in female was higher than male in most tissues. For PPARαgene, the expression of liver was the highest, and then abdominal fat, breast muscle, lung, spleen, heart, muscle stomach and small intestine, of kidney was the lowest in male and female ducks; and the expressions of PPARαin female was higher than male in most tissues. For PPARγgene, the expression of abdominal fat was the highest, and then lung, breast muscle, liver, muscle stomach, heart, spleen and kidney, small intestine was the lowest in male and female ducks; and the expressions of PPARγin male was higher than female in most tissues. For IGF-Ⅰgene, the expression of muscle stomach was the highest, and then abdominal fat, liver, heart, small intestine, kidney, breast muscle and spleen, of lung was the lowest in male and female ducks; and the expressions of IGF-Ⅰin female was higher than male in most tissues. The results of two methods were different. Compared the results with other species, we found that the results of real-time fluorescent quantitative PCR were more accurate than RT-PCR.3. The prokaryotic expression of IGF-Ⅰand PPARThe coding region of the PPARα, PPARγand IGF-Ⅰwere constructed into pET32a (+) vector for proeukaryotic expression. The results showed that only 34.66 kD recombinant protein with histidine tags of IGF-Ⅰwas expressed successfully in BL21 E.coli and detected by Western Blotting. The expression condition of IGF-Ⅰrecombinant protein was optimized at 28℃which was induced for 5h by IPTG (0.3 mmol/L). The recombinant protein pET32a (+)-PPARαand pET32a (+)-PPARγwere not expressed in BL21 E.coli.4. Effects of polymorphisms of PPAR and IGF-Ⅰgene on adipose traits of ducks The genetic diversities of PPARα, PPARγand IGF-Ⅰdifferent exons were analyzed using SSCP and sequencing assay technologies, and the association between the polymophisms and carcass and adipose traits were analyzed. The results showed that there were no SNP in PPARα, IGF-Ⅰand exon 1, 3, 4 and 6 of PPARγgenes. At PPARγexon 2 and exon 5 loci, an A→C mutation (at exon 2) and a G→T (at exon 5) were detected. They were synonymous mutation and not caused the change of amino acids. The two loci were all medium polymorphism.4.1 Analyzed the polymorphism and association with the carcass and adipose traits of PPARγexon 2Considered the genotype, sex and the interactions between genotype and sex, we analyzed the polymorphism and association with carcass and adipose traits in four duck populations. The results showed that the interactions have no significant effects on the four populations; there were no significant differences in different sex in Z2 population; sex has significant effects on carcass weight, leg muscle weight, eviscerated weight and percentage of abdominal fat weight in Peking duck Z4, on carcass weight, eviscerated weight, percentage of abdominal fat and IMF in Cherry Valley ducks and on carcass weight, leg muscle weight, eviscerated weight, SFW, abdominal fat weight and IMF in hybrid population (P<0.05).The results of least square analysis showed that different genotypes have significant effects on eviscerated weight, AFW, SFW, percentages of AFW and SFW and IMF content in Peking duck Z2 (P<0.05), and individuals with genotype CA were significant higher than genotype AA (P<0.05); in Peking duck Z4, different genotypes have significant effect on carcass weight, leg muscle weight, SFW, AFW, percentage of SFW and AFW and IMF content (P<0.05), and individuals with genotype AA were significant higher than genotype CA (P<0.05); in Cherry Valley ducks, different genotypes have significant effect on carcass weight, eviscerated weight, SFW, AFW, percentage of SFW and AFW (P<0.05), and individuals with genotype AA were significant higher than genotype CC (P<0.05); in hybrid ducks, different genotypes have significant effect on carcass weight, breast muscle weight, leg muscle weight, eviscerated weight and IMF content (P<0.05), and individuals with genotype CC were significant higher than genotype AA (P<0.05). There were no significant differences in other traits in different populations. It implied that exon 2 locus had significant effect on adipose traits.4.2 Analyzed the polymorphism and association with the carcass and adipose traits of PPARγexon 5The results showed that the interactions have no significant effect in the four populations; sex has significant effect on carcass weight and eviscerated weight in Peking duck Z2, on carcass weight, leg muscle weight, eviscerated weight, percentage of SFW and AFW in Peking duck Z4, on carcass weight, breast muscle weight, eviscerated weight, AFW and percentage of AFW in Cherry Valley ducks, and on carcass weight, leg muscle weight, eviscerated weight, SFW, AFW and IMF content in hybrid ducks (P<0.05). The results of least square analysis showed that different genotypes have significant effect on carcass weight, breast muscle weight, SFW, AFW, percentage of SFW and AFW in Peking duck Z2 (P<0.05), and individuals with genotype GT were significant higher than genotype GG (P<0.05); in Peking duck Z4, different genotypes have significant effect on carcass weight, SFW, AFW, percentage of SFW and AFW (P<0.05), and individuals with genotype TT were significant higher than genotype GG (P<0.05); in Cherry Valley ducks, different genotypes have significant effect on SFW, AFW and percentage of SFW and AFW (P<0.05), and individuals with genotype TT were significant higher than genotype GT (P<0.05); in hybrid ducks, different genotypes have significant effect on eviscerated weight, SFW, AFW, percentage of SFW and AFW (P<0.05), and individuals with genotype TT were significant higher than genotype GG (P<0.05). There were no significant differences in other traits in different populations. It implied that exon 5 locus had significant effect on adipose traits.