【作者】 路伏增；

【导师】 刘红林；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 本试验用实时荧光定量PCR的方法研究了生长较慢的蛋鸡狼山鸡(LS)和生长相对较快的肉鸡AA鸡胚胎时期和出生早期生长轴、甲状腺轴和肾上腺轴有关基因的表达变化规律。所检测的组织包括下丘脑、垂体、肝脏、胸肌和骺软骨。营养物质可以直接或间接地作用于生长调控因子来调节生长,因此在我们的试验设计中,用营养成分含量较高的AA鸡的标准饲料饲喂AA鸡(组别以AA来表示,并以此组别作为对照组),而对狼山鸡则分别饲喂两种饲料,即营养成分含量较低的狼山鸡标准饲料(组别以LL来表示)和AA鸡标准饲料(组别以LA表示)。为了评估饲料对LL,LA和AA三个组别有关基因在出生后整体水平上mRNA表达的品种差异的影响,我们把组别和时期作为固定因素,用一般线性模型(General Linear Models,GLM)进行了数据分析。本研究首次系统分析了三条轴上五种组织中有关基因在鸡胚胎和出生后在转录水平上的变化规律,并且发现了某些基因的表达在特定的时期是相关的,狼山鸡和AA鸡在相同的AA鸡标准饲料喂养条件下某些基因的表达差异消失了。本研究结果为研究鸡某些基因在特定时期的相互调控机理在分子水平上提供了依据,同时发现从营养和某些基因型的互作效应上对鸡进行分子育种将会大大的提高鸡的选育效果,这将在鸡的生产应用中起到重大作用。1.狼山鸡和AA鸡胚胎时期和出生早期生长轴、甲状腺轴、肾上腺轴有关基因的表达本研究揭示了垂体中的生长激素(GH)和肝脏中的生长激素受体(GHR)mRNA的表达趋势在出生后第28天(P28)到P42期间是相反的,而肝脏中的胰岛素样生长因子Ⅰ(IGF-Ⅰ)和IGF-Ⅰ的受体(IGF-IR)mRNA的表达在P0-P28期间是相反的。肝脏中IGF-Ⅰ在P0-28期间对GH/IGF调控轴起着重要的作用。垂体中GH释放激素(GHRH)受体(GHRHR)在P0-28期间起着自分泌或旁分泌的功能,下丘脑中的生长激素促分泌物质受体的内源性配体ghrelin可能在胚胎时期第8天(E8)到P28期间通过刺激肝脏中的IGF-Ⅰ来影响生长。下丘脑中的ghrelin可能在P0-28期间与其中的促肾上腺皮质激素释放激素CRH相互作用。肝脏中IGF结合蛋白(IGFBP)2在P0-42期间通过调节IGF-Ⅱ的生物活性来调节生长。肝脏中的IGFBP-5是一个重要的IGF介导物。我们发现下丘脑中的GHRH(分别在E10-16和P0-42之间),生长抑素(SS,P0-28),促甲状腺激素释放激素(TRH,E10-16和P0-28),ghrelin(P0-42)和垂体中的GH在特定时期是共表达的,而下丘脑中的SS(P0-28),CRH(P0-42),TRH(E10-E18和P0-42)和促甲状腺激素β亚基(TSHβ)在特定的时期是共表达的。GLM分析显示LL组别垂体GH,下丘脑SS,Ghrelin和肝脏IGF-IR的mRNA水平比AA组别的高,而在体重,肝脏中IGF-Ⅰ和GHR的mRNA水平上的比较则正好相反。通过比较狼山鸡和AA鸡在饲喂同一种含营养成分高的AA鸡饲料时各基因表达,我们发现垂体中的GH,下丘脑中的SS,grelin,肝脏中的IGF-Ⅰ,GHR在两品种中的表达差异消失了,而体重和肝脏中的IGF-IR mRNA水平在两品种中依然存在显著差异。本研究揭示了在生长轴、甲状腺轴和肾上腺轴中某些基因在特定时期在转录水平上的表达是相关的,并且发现下丘脑、垂体和肝脏中某些基因在品种间的表达差异是受营养因素调控影响的。2.狼山鸡和AA鸡胚胎时期和出生早期胸肌中胰岛素类生长因子系统基因的表达本研究分析了营养因素对鸡胸肌中胰岛素类生长因子系统基因的影响。结果显示IGF-Ⅱ(E16-P42),IGF-IR(E18-P42),IGFBP-2(E18-P42),-5(E16-P14),-7(E12-P0),-3(E12-P0)与IGF-Ⅰ在特定时期的mRNA表达成正相关,而IGFBP-3与IGF-Ⅰ在P0-28时期成负相关。IGF-IR(E18-P42),IGFBP-2(E18-P42),-5(E14-P0),-3(E16-P0)与IGF-Ⅱ在特定时期成正相关,而IGF-IR(E10-E16),IGFBP-3(P0-P28)与IGF-Ⅱ在特定时期成负相关。另外,IGFBP-2(E16-P42),-7(E10-E16),-3(E10-E16)与IGF-IR在特定时期成正相关,而IGFBP-3与IGF-IR在P0-P28时期成负相关。最后,IGFBP-7与IGFBP-3在E12-P0时期成正相关,而IGFBP-2(P0-P28),-7(P0-P42)与IGFBP-3在相应时期成负相关。总体上,两种鸡在各自饲喂各自标准饲料的情况下,AA组别的鸡IGF-Ⅰ,IGF-IR和IGFBP-2 mRNA的表达水平比LL组别的高,而IGFBP-7的表达水平在两组间则正好相反。两种鸡在同一种饲料饲喂下,LA和AA组别IGF-Ⅰ,IGF-IR和IGFBP-7的表达差异消失了,而IGFBP-2的表达差异依然显著。狼山鸡在两种不同饲料饲喂情况下,LA组别的鸡在IGFBP-2 mRNA的表达上比LL组别的鸡高,而IGFBP-7的表达在两组比较上则正好相反。本研究首次在胸肌上发现某些胰岛素样生长因子在特定时期的mRNA表达是相关的,并且某些基因在品种表达的差异上是由于受营养因素控制的。3.狼山鸡和AA鸡胚胎时期和出生早期骺软骨中胰岛素类生长因子系统基因的表达本试验首次在狼山鸡和AA鸡胚胎时期和出生早期骺软骨中胰岛素类生长因子系统基因进行了研究。研究结果发现IGFBP-2与IGF-Ⅰ(E14-P0),IGF-Ⅱ(E14-P14)在相应时期mRNA表达成正相关,而与IGF-Ⅰ(P0-P42),IGF-Ⅱ(P14-P42)在相应时期成负相关。IGFBP-5与IGF-Ⅰ在E14-P0时期mRNA表达成正相关,而在P0-28之间成负相关。IGFBP-7和IGF-Ⅱ在P0-P42期间表达模式相反。我们发现IGFBP-3(E14-E18,P14-P42)和IGF-IR,IGFBP-3(E14-P0,P14-P42)和IGF-Ⅰ在相应时期mRNA表达成平行表达模式。内分泌调控因子与营养相互作用可以调控动物的生长。GLM分析显示AA组别IGF-Ⅰ,IGFBP-2和IGFBP-3 mRNA的水平比LL组别的高,而在IGF-Ⅱ和IGFBP-7 mRNA表达水平上的比较则正好相反。狼山鸡和AA鸡在饲喂同一种AA鸡标准饲料的情况下,在IGF-Ⅰ,IGFBP-2,IGFBP-7和IGFBP-3的转录水平的表达上差异变得不显著了,而在IGF-Ⅱ的表达上差异依然显著。我们的研究可以帮助提高鸡的分子育种。更多还原

【Abstract】 We investigated changes in mRNA expression of the somatotropic,thyrotropic,and corticotropic axes from slower growing Langshan(LS) layer and more rapidly growing Arbor Acres(AA) broiler chickens during embryonic and postnatal development.Real time RT-PCR was used to quantify the level of transcription in the hypothalamus,pituitary,liver, breast muscle and epiphyseal cartilage.Nutrient availability may control body growth directly and indirectly through its influence on regulatory factors.Moreover,AA chickens were fed a nutrient-rich AA diet(AA,as a control group) and LS chickens were fed either a less-nutritious LS diet(LL) or the AA diet(LA).For evaluating the effects of the diet on gene expression in general at the postnatal day 14(P14),P28 and P42 of LL,LA and AA groups chickens,the General Linear Models(GLM) procedure(with developmental age and strain as fixed factors) of SPSS software was used to analyze the effects of nutrient availability on the strain difference between LL,LA and AA.Our data show the first evidence that certain genes may be correlated during specific developmental periods and that strain differences in the expression of those genes in LS and AA chickens are due to differential responses to the same AA diet.These findings may help improve the molecular breeding of chickens.1.Expression of genes involved in the somatotropie,thyrotropic,and corticotropic axes during development of Langshan and Arbor Acres ChickensWe found an inverse expression profile between pituitary growth hormone(GH) and hepatic GH receptor(GHR) mRNA(postnatal day 28[P28]-42),insulin-like growth factor (IGF)-Ⅰand IGF-IR(P0-42),respectively.Hepatic IGF-Ⅰwas a major point of control in the GH/IGF axis during P0-P28.Pituitary GH-releasing hormone(GHRH) receptor(GHRHR) may serve an autocrine/paracrine function during P0-28,and hypothalamic ghrelin may affect growth by stimulating the release of hepatic IGF-Ⅰduring embryonic day 8(E8)-P28. Hypothalamic ghrelin might interact with ccorticotrophin-releasing hormone(CRH) during P0-P28.Hepatic IGF binding protein(IGFBP)-2 regulated growth by regulating hepatic IGF-Ⅱbioavailability during P0-P42.Hepatic IGFBP-5 was an important IGF mediator.A co-expression profile was found between hypothalamic GHRH(E10-E16 and P0-P42), somatostatin(SS;P0-P28),thyrotropin-releasing hormone(TRH;E10-E16 and P0-P28), ghrelin(P0-P42),and pituitary GH mRNA,hypothalamic SS(P0-P28),CRH(P0-P42), TRH(E10-E18 and P0-P42),and TSHβmRNA,respectively.GLM analysis revealed that LL chickens expressed significantly higher levels of pituitary GH,hypothalamic SS, Ghrelin,and hepatic IGF-IR mRNA than AA chicken while the opposite was true for BW, hepatic IGF-Ⅰand GHR mRNA,although strain differences on some age group were not statistically significant When comparing LS and AA chickens under the same AA diet containing higher nutrient compositions,strain differences in pituitary GH,hypothalamic SS,Ghrelin,hepatic IGF-Ⅰand GHR mRNA disappeared,while strain differences in body weight(BW) and hepatic IGF-IR mRNA still existed,which provide the new nutrient data for the application of chickens molecular breeding.Our data indicates the selected genes may be parallel expression in certain periods and strain differences in genes expression respond to nutrition differently in the hypothalamus,pituitary and liver from LS and AA chickens.2.Investigation of the insulin-like growth factor system in breast muscle during development in Langshan and Arbor Acres chickensWe analyzed the effects of nutrient availability on the breast muscle insulin-like growth factor system.According to our results,IGF-Ⅱ(E16-P42),IGF-IR(E18-P42),and IGFBP-2 (E18-P42),-5(E16-P14),-7(E12-P0),and-3(E12-P0) were positively correlated with IGF-Ⅰ,while IGFBP-3(P0-P28) was negatively correlated with IGF-Ⅰ.In comparison, IGF-IR(E18-P42),IGFBP-2(E18-P42),-5(E14-P0),and -3(E16-P0) were positively correlated with IGF-Ⅱ,while IGF-IR(E10-E16) and IGFBP-3(P0-P28) were negatively correlated with IGF-Ⅱ.Moreover,IGFBP-2(E16-P42),-7(E10-E16),and -3(E10-E16) were positively correlated with IGF-IR,while IGFBP-3(P0-P28) was negatively correlated with IGF-IR.Finally,IGFBP-7(E12-P0) was positively correlated with IGFBP-3,while IGFBP-2(P0-P28) and -7(P0-P42) were negatively correlated with IGFBP-3.Overall,the AA chickens exhibited higher levels of IGF-Ⅰ,IGF-IR,and IGFBP-2 mRNA expression than the LL chickens,while the opposite was true for IGFBP-7.No strain differences in IGF-Ⅰ,IGF-IR,and IGFBP-7 mRNA expression were detected any more between LA and AA chickens;however,a strain difference was still observed for IGFBP-2.LA chickens exhibited higher levels of IGFBP-2 than LL chickens,while the opposite was true for IGFBP-7.Our data show the first evidence that certain IGF system genes may be correlated during specific developmental periods and that strain differences in the expression of those genes in LS and AA chickens are due to differential responses to the same diet in the breast muscle.3.Role of the insulin-like growth factor system in epiphyseal cartilage on the development of Langshan and Arbor Acres chickens Gallus domesticusWe measured the mRNA transcript expression patterns for members of the insulin-like growth factor(IGF) system during embryonic and postnatal development in epiphyseal cartilage from LS and AA chickens Gallus domesticus.IGFBP-2 expression was positively correlated with expression of IGF-Ⅰfrom E14 to P0 and with IGF-Ⅱfrom E14-F14 but negatively correlated with IGF-Ⅰfrom P0-P42 and IGF-Ⅱfrom P14-P42.Expression of IGFBP-5 correlated positively with the expression of IGF-Ⅰfrom E14-P0 but negatively from P0-P28.The results suggest that these IGF axis genes are regulated in a coordinated fashion during development.An inverse expression profile was found between IGFBP-7 and IGF-Ⅱduring P0-P42.A co-expression profile was found between IGFBP-3(E14-E18, P14-P42) and IGF-IR,and between IGFBP-3(E14-P0,P 14-P42) and IGF-Ⅰ.The endocrine factors can be integrated with nutrition to regulate animal growth.In our study,AA chickens were fed a nutrient-rich AA diet,and LS chickens were fed either an AA diet(LA) or a less-rich diet(LL).GLM analysis revealed that IGF-Ⅰ,IGFBP-2,and IGFBP-3 mRNA expression was higher in AA chickens than in LL chickens,while the opposite was true for IGF-Ⅱand IGFBP-7,although the strain differences in some age groups were not significant.LS and AA chickens fed the same AA diet showed no differences any more in IGF-Ⅰ,IGFBP-2,IGFBP-7,and IGFBP-3 transcription,but did still show differences in IGF-ⅡmRNA expression.These findings may help to improve the molecular breeding of chickens.更多还原