【作者】 束婧婷；

【导师】 陈国宏；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 肌苷酸(Inosine monophosphate, IMP)是重要的风味物质,对肌肉的鲜味程度起着巨大作用,目前世界上已有不少国家以肌苷酸的含量作为肉类新鲜程度的指标之一。肌苷酸(IMP)在体内的合成代谢过程十分复杂,从头合成涉及十种关键酶。本研究以鸡肌苷酸合成代谢相关酶中的谷氨酰胺磷酸核糖焦磷酸酰胺转移酶(GPAT)基因、5-氨基咪唑核苷酸羧化酶/5-氨基咪唑-4-(N-琥珀酰胺)核苷酸合成酶(AIRC)基因和氨基咪唑氨甲酰核苷酸转甲酰基酶/次黄嘌呤核苷酸环水解酶(PURH)基因为候选基因,采用PCR-SSCP和测序相结合的方法,在丝羽乌骨鸡、萧山鸡、白耳鸡、藏鸡等4个地方鸡种以及1个外来鸡种-隐性白羽肉鸡群体中对鸡GPAT、AIRC和PURH基因进行单核苷酸多态性(SNPs)扫描并分析其与肌肉肌苷酸含量之间的关系;利用相对定量RT-PCR法检测了ADSL、GARS-AIRS-GART、GPAT、AIRC和PURH等5个候选基因在白耳鸡不同组织中的时空表达规律;同时结合本实验室已有的对ADSL、GARS-AIRS-GART基因多态性研究结果,在白耳鸡群体中对ADSL、GARS-AIRS-GART、GPAT、AIRC和PURH基因进行多基因聚合分析,探讨不同基因组合之间合并基因型对肌肉肌苷酸含量的效应大小。主要研究结果如下:1.以鸡IMP合成代谢过程中紧密连锁的谷氨酰胺磷酸核糖焦磷酸酰胺转移酶(GPAT)基因、5-氨基咪唑核苷酸羧化酶/5-氨基咪唑-4-(N-琥珀酰胺)核苷酸合成酶(AIRC)基因为候选基因,对该两个基因的启动子区域、所有外显子和部分内含子区域进行单核苷酸多态性(SNPs)扫描。共检测出6个多态位点,分别为GPAT基因外显子2中29777处的碱基A缺失突变、内含子4中G34993A突变、内含子5中T35188A突变、AIRC基因外显子3中T1993C突变、外显子8中C5406G突变以及启动子区G-158C突变,将核苷酸序列翻译成相应的氨基酸序列后发现,位于编码区的突变均为沉默突变。2.在GPAT基因中,仅在外显子2的多态位点上存在显著的基因型效应,AA型个体胸肌IMP含量显著的高于AB型与BB型个体,AB型个体IMP含量稍高于BB型,但二者之间差异不显著。AIRC基因的两个多态位点在5个鸡种中均存在显著的基因型效应,在外显子3中CC型个体胸肌IMP含量显著高于TC型和TT型个体,TC型个体IMP含量均值稍高于TT型个体,但两者之间的差异不显著;而在外显子8中,CG型个体的胸肌IMP含量最高,并显著高于CC型和GG型个体,GG型个体IMP含量也显著地高于CC型个体。两基因启动子区的多态位点也与胸肌IMP含量之间存在着显著相关,GG型个体胸肌IMP含量显著高于GC型和CC型个体,GC型个体IMP含量稍高于CC型个体,但两者之间的差异不显著。推测GPAT/AIRC基因应是肌肉肌苷酸含量的主效QTL或与其主效QTL紧密连锁,有望用于标记辅助选择。3.以鸡IMP合成过程中的氨基咪唑氨甲酰核苷酸转甲酰基酶/次黄嘌呤核苷酸环水解酶(PURH)基因为候选基因,对该基因的所有16个外显子、外显子/内含子的结合区域以及3’侧翼部分序列进行SNPs扫描。共检测到两个多态位点,分别为外显子9中的A8023T突变和外显子16中的T17446C突变(从转录起始位点开始计起所得到的核苷酸位置),但这两个突变均为沉默突变,并没有导致对应的氨基酸序列发生变异。两个SNPs位点在不同鸡种中的基因型分布差异显著,进一步的关联分析显示,仅外显子16中的多态位点与肌肉IMP含量之间存在着显著的关联,其TT型个体胸肌IMP含量显著高于CT型与CC型个体,CT型个体IMP含量稍高于CC型,但二者之间差异不显著,初步认为该位点可以作为肌肉IMP含量的辅助选择标记。4.在白耳鸡群体中对肌苷酸合成相关酶基因(ADSL、GARS-AIRS-GART、GPAT、AIRC和PURH基因)在不同组织和肌肉中的表达规律进行了研究。结果在白耳鸡的不同生长阶段(3,6,9,12和15周龄)不同组织中均检测到了5个基因的mRNA,在某些时期的表达量上存在强弱之分,但没有发现有和无的差别,说明IMP合成过程中的5个基因是通过作用于一系列不同的组织器官而发挥其生理功能;并且ADSL、GARS-AIRS-GART、GPAT和PURH基因在所有组织中表达量的变化趋势基本一致,呈明显的时序性变化:12周龄前随着周龄的增加,表达量不断增加,在12周龄时达到最高,而后开始下降;而AIRC基因在不同组织中的表达随着周龄的增加并没有一定的规律,可能是由于其在不同组织中的表达存在特殊的调节机制。位于同一染色体上的基因(ADSL与GARS-AIRS-GART,GPAT与AIRC) mRNA表达量之间均存在显著或极显著的正相关,ADSL与AIRC、PURH与GPAT/AIRC基因mRNA表达量之间也存在显著的正相关,这些结果说明IMP合成涉及的关键酶的调控存在关联,它们之间存在着协同作用,并最终影响着肌苷酸的含量。5.在较大规模的白耳鸡群体中对IMP合成过程中的5个相关酶基因进行多基因聚合效应分析,分别分析了单基因、两基因、三基因、四基因以及五基因聚合后对胸肌IMP含量的影响效应大小,结果表现出五基因合并效应>四基因合并效应>三基因合并效应>两基因合并效应>单基因效应,合并基因型的效应不是各自基因型效应的简单相加,但却要高于最好的单个基因型效应,这提示我们在分析多基因控制的数量性状时,需要同时对多个位点组成的合并基因型(单倍型)进行分析,这样才能得到基因与性状之间的真实相关性,这个结论对于利用DNA标记进行辅助选择特别有意义,表明评估品种或种群的遗传改良必须以合并基因型的影响效应为准。更多还原

【Abstract】 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.更多还原