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Studies On The Genetic Effect Of Candidate Genes Of Inosine Monophosphate And Their Expression Characterization In Chicken

Posted on:2009-02-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:J T ShuFull Text:PDF
GTID:1103360242993471Subject:Animal breeding and genetics and breeding
Abstract/Summary:PDF Full Text Request
Meat flavor is one of the most important aspects of the meat quality in chicken, many evidences indicated that Inosine monophosphate (IMP) is one of the key components for meat flavor, and then more and more countries use IMP content as the criterion of meat flavor and freshness now. The process of the synthesis and metabolism for IMP is very complicated, with ten-step reactions involved. In the first part of the present study, we chose GPAT,AIRC and PURH gene as candidate genes for IMP contents in chicken. To explore the relationships between these three genes with IMP contents in chicken, single strand conformation (SSCP) analysis and sequencing reactions were performed to screen single nucleotide polymorphisms (SNPs) in four Chinese indigenous chicken breeds, Silkies chickens (SC), Xiaoshan chickens (XS), Baier chickens (BE), Tibetan chickens (TC), and an exotic breed, Recessive White broilers (RW). In the second part, the developmental pattern of ADSL,GARS - AIRS - GART,GPAT,AIRC and PURH mRNA expression in different tissues in Baier chickens were determined by relative quantitative RT-PCR withβ-actin as an internal standard. And in the third part, we analyzed the effects of combined genotypes of different genes involved in de novo purine biosynthesis pathway on muscle IMP content. The main results were as follows:1. The promoter sequence and all 20 exons with flanking intronic sequences of GPAT/AIRC genes were screened in the five chicken breeds mentioned above, by the method of PCR-SSCP analysis, combined with sequencing reactions. Six SNPs were detected: A deletion mutation from exon 2, G34993A from intron 4, T35188A from intron 5 in GPAT gene; T1993C from exon 3, and C5406G from exon 8 in AIRC gene; and a G-158C mutation in the promoter region. All the mutations in the coding regions were silence mutations; they didn't cause the alteration of the corresponding amino acid.2. Statistical analysis was applied to test the significance of the difference of breed effect and genotype effect between three genotypes of the six gene loci, respectively. Only significant genotypic effect (P<0.05) existed in four of the loci, they were SNP in exon 2 of GPAT gene, in exon 3 and exon 8 of AIRC gene, and in promoter region, respectively. So we analyzed genotypic effect with the combination of all breeds in these four loci. The least square analysis showed that AA genotype birds had significant higher IMP content than AB and BB genotype birds, AB genotype birds also had a little higher IMP content than BB genotype birds, but the difference was not significant in GPAT gene exon 2; In AIRC gene, CC genotype birds had significant higher IMP content than TC and TT genotype birds, TC genotype birds also had a little higher IMP content than TT genotype birds, but the difference was not significant in GPAT gene exon 3, while CG genotype birds had the highest IMP content in exon 8, CG genotype birds had significant higher IMP content than CC and GG genotype birds, GG genotype birds also had significant higher IMP content than CC genotype birds; SNP in the promoter region also had significant effect on muscle IMP content, GG genotype birds had significant higher IMP content than GC and CC genotype birds, GC genotype birds also had a little higher IMP content than CC genotype birds, but the difference was not significant. We putatively drew the conclusion that GPAT/AIRC gene could be a candidate locus or linked to a major gene (s) that affected muscle IMP content and could be used as the potential molecular marker for meat quality trait in chicken.3. All 16 exons with flanking intronic sequences, as well as a fragment of the 3'flanking region, of PURH gene, were screened in the same five populations, by the method of PCR-SSCP analysis, combined with sequencing reactions. Two SNPs were detected: A/T substitution at position 8181 in exon 9 [A8181T] and a T/C point mutation at position 17608 in exon 16 [T17608C], but these two mutations didn't cause the alteration of the corresponding amino acid. The least square analysis of the two SNPs detected in exon 9 and exon 16 showed that only the polymorphic site in exon 16 existed significant genotypic effect. The association study between genotypes in exon 16 and muscle IMP content showed that TT genotype birds had significant higher IMP content than CT and CC genotype birds, but the difference of IMP content between CT and CC genotype birds were not significant. The breed effect and the interaction effect between breed and genotype are not significant, so we could conclude that there may be a relationship between this SNP site and IMP content in chicken.4. The developmental pattern of ADSL,GARS-AIRS-GART,GPAT,AIRC and PURH mRNA expression in different tissues in Baier chickens were studied. The results showed that the mRNA of all the studied genes was detected in different tissues of different growth stages (3, 6, 9, 12 and 15 weeks of age). The pattern of developmental changes of ADSL,GARS-AIRS-GART,GPAT and PURH mRNA in different tissues were similar, which increased with the age before 12-week-old, the peak level was found at 12-week-old, and then declined. Nevertheless, there was no rule to follow in the expression pattern of AIRC mRNA, this might due to special regulation mechanism existing in the expression of AIRC mRNA in different tissues. The correlation analysis showed that mRNA levels between genes located on the same chromosome were found to be positively correlated; Meanwhile, ADSL mRNA level was positively correlated with AIRC mRNA level; PURH mRNA level was also found to be positively correlated with GPAT/AIRC mRNA level. These results indicated that the regulation of genes involved in de novo purine biosynthesis pathway had some relations; they are coordinately expressed and may play an important role in the final IMP content.5. The combine effects of the five candidate genes were analyzed in a relative large sample size of Baier chicken, the effects of the single gene, the two gene combination, the three gene combination, the four gene combination and the five gene combination on muscle IMP content were evaluated, respectively. The result showed that five gene effect>four gene effect>three gene effect>two gene effect>single gene effect, which suggested that when analysis quantative traits, we should analysis the combined genotypes (haplotypes) of the multiple loci, so that we could get the real relationships between gene and traits. Genetic effect of combination genotypes was not the simple addition of different genotypic effects. This is useful for marker assisted selection of meat quality traits in chicken, if we evaluating genetic improvement in breeding base on the effect of combination genotype, larger genetic improvement will be obtained using molecular marker assisted selection.
Keywords/Search Tags:chicken, inosine monophosphate (IMP), candidate gene, single nucleotide polymorphism (SNP), expression, gene pyramiding
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