【作者】 赵阿勇；

【导师】 何瑞国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 超饲养（Overfeeding）引起鹅肝脏代谢发生改变，使得大量脂肪沉积在肝脏中形成肥肝（fatty liver）。不同品种鹅对超饲养导致的肝脏代谢改变的敏感程度不同，所以肥肝性能差异很大。但目前鹅肥肝形成的分子机理仍然不清楚。本研究以朗德鹅和溆浦鹅为试验材料，利用mRNA差异显示技术和荧光定量PCR技术，对两个品种之间、肥肝和普通肝脏之间差异表达基因进行了分离和鉴定，并通过生物信息学方法从蛋白质和核酸两个水平上进行了结构与功能预测，取得了如下研究结果：实验使用了90对差异显示引物来研究两个品种鹅肥肝和普通肝脏中mRNA表达水平对超饲养的反应情况，并用荧光定量PCR进行了验证。6个基因被证实在肥肝和普通肝脏之间差异表达，其中3个基因表达水平被上调，2个基因被下调，1个基因在两个品种试验鹅中表现了相反的调控状态。基因8216的表达水平在肥肝中被上调了，并且朗德鹅肝脏（LL和LFL）中的表达水平高于溆浦鹅（XpL和XpFL）。这个基因的全长cDNA是1797bp，GenBank登陆号：EF488993，经过比对，这个基因的全长cDNA序列与鸡硒结合蛋白1（selenium binding protein 1，SELENBP1；XM 423397.2）基因有92％的同源性。序列分析发现，这个cDNA序列含有一个1413bp的开放读码框（ORF），编码一个471个氨基酸的蛋白质。这个蛋白质中含有一个56 kDa的硒结合蛋白（SBP56）保守结构域。聚类分析表明，该蛋白质与鸡、大鼠、人、猴、狗、牛、小鼠的SBP56蛋白分别具有95％、86％、86％、86％、86％、86％、84％的同源性。组织表达分析显示，鹅基因8216在肝脏和肾脏中表达丰富，在脾脏中中等表达，在卵巢、子宫、肌胃和腹脂中表达量较低。基因9105的表达水平在肥肝中被上调了。经过cDNA克隆，得到一个包含全长CDS的1248bp的cDNA片段，GenBank登陆号：EF541127。比对的结果表明，该片段与鸡的转化生长因子β-2 mRNA（transforming growth factor beta 2，TGFB2；NM₀01031045.1）有94％的同源性，与其它物种的TGFB2也有很高的同源性。序列分析发现，这个cDNA序列含有一个1239bp的开放读码框架（ORF），编码一个有412个氨基酸的蛋白质。BlastP分析表明，该蛋白质序列存在着2个保守的功能域，一个是引导序列（TGFb前肽），另一个是功能结构域（TGFβ）。聚类分析表明，该蛋白质与鸡、非洲爪蛙、大鼠、人、短尾猊、牛、小鼠、猪、绵羊的TGFb2蛋白分别具有99％、96％、87％、90％、91％、89％、88％、90％、89％的同源性。组织表达分析显示，鹅基因9105在肌胃中表达很丰富，在肝脏、脾脏和卵巢中表达也较丰富，在子宫、肺和肌肉中中等表达，而在肾脏和腹脂中表达量较低。基因7501的表达水平在肥肝中被上调了，肥肝中（LFL和XpFL）高于普通肝脏（LL和XpL），朗德鹅（LL和LFL）高于溆浦鹅（XpL和XpFL）。经过cDNA克隆，得到一个1446bp的cDNA片段。经过比对，这个基因的部分cDNA序列与鸡的细胞质NADP（+）-依赖型苹果酸酶1mRNA（malic enzyme 1，NADP（+）-dependent，cytosolic，ME1；NM₂04303.1）有93％的同源性，与其它物种的ME1也有很高的同源性。组织表达分析显示，鹅基因7501在肝脏中表达很丰富，在脾脏、卵巢、子宫、肺、肾脏、肌胃和肌肉中中等表达，而在腹脂中表达量最低。基因8407的表达水平在肥肝中被下调了。经过cDNA克隆，得到一个包含全长CDS的1269bp的cDNA片段，GenBank登陆号：EF541128。比对的结果表明，该片段与鸡的细胞质异柠檬酸脱氢酶mRNA（cytosolic NADP-dependent isocitrate dehydrogenase，IDH1；XM₄21965）有95％的同源性，与其它物种的IDH1也有很高的同源性。序列分析发现，这个cDNA序列含有一个1248bp的开放读码框架（ORF），编码一个有415个氨基酸的蛋白质。BlastP分析表明，该蛋白质序列存在着一个保守的结构域，这个结构域与已知的真核蛋白Icd具有较高的同源性。聚类分析表明，该蛋白质与鸡、大鼠、人、猿、牛、小鼠的Icd蛋白分别具有99％、89％、90％、90％、88％、88％的同源性。组织表达分析显示，鹅基因8407在肝脏中表达丰富，在脾脏、肾脏和肌胃中中等表达，在卵巢、子宫、肺、肌肉和腹脂中表达量较低。基因9302的表达水平在肥肝中被下调了。经过cDNA克隆，得到一个943bp的cDNA片段。经过比对，这个基因的部分cDNA序列与鸡的胆固醇-7α羟化酶基因（Cytochrome P450，family 7，subfamily A，polypeptide 1，CYP7A1；XM₄19217）有93％的同源性。组织表达分析显示，鹅基因9302在肝脏中表达很丰富，在其它组织中不表达。基因7102的表达水平在两种鹅中表现了相反的调控状态，朗德鹅肥肝中基因7102的表达水平被上调了，而在溆浦鹅肥肝中被下调了。经过cDNA克隆，得到一个923bp的cDNA片段。经过比对，这个基因的部分cDNA序列与鸡的NAD依赖型苹果酸脱氢酶基因（malate dehydrogenase 1，NAD（soluble），mRNA，MDH1；NM₀01006395.1）有95％的同源性，与其它物种的MDH1基因也有很高的同源性。组织表达分析显示，鹅基因7102在肝脏中表达很丰富，在其它组织中表达量较低。鹅通过短期内超饲养（Overfeeding）大量高能饲料可以形成肥肝。在肥肝发生过程中，肝脏的氧化应激增强，造成自由基在肝脏中大量产生，对肝细胞和肥肝品质产生了影响。本试验研究采用单因子梯度设计，研究日粮中添加不同水平的茶多酚对超饲养朗德鹅脂肪代谢和肝脏抗氧化性能的影响。试验鹅被分为5组，以第1组为对照组，其余4组为试验组，每组饲喂含不同水平茶多酚的日粮，4个试验组超饲养日粮中茶多酚的浓度分别为：40、80、160、320mg／Kg。通过对屠宰性能、血清生化指标和肝脏抗氧化指标的分析，取得了如下的研究结果：经过21天的超饲养，每只鹅的平均超饲养量大致相同（13．54-0．23Kg）。屠宰后对各项屠宰指标的分析结果表明，各组之间屠宰率、半净膛率、全净膛率、胸肌重和腿肌重等没有显著性差异（p>0．05），体重和体增重呈现了显著的下降趋势（p<0．01）：腹脂重与超饲养后体重的比值和肥肝重都表现了先上升然后下降的趋势，但总体来说变化不显著。这些结果说明，茶多酚对肝脏中脂肪的合成与沉积，以及合成后脂类在机体中的分布都没有显著的影响。血清中生化指标的分析结果表明，甘油三酯（TG）水平的变化与肥肝重的变化基本上是一致的，总胆固醇（TC）水平总体来说是下降的，但试验组之间没有差异（p>0．05），而与对照组有极显著的差异（p<0．01），高密度脂蛋白胆固醇（HDL-C）水平相对平稳，总体上各组之间差异不显著（p>0．05），低密度脂蛋白胆固醇（LDL-C）水平有显著的下降（p<0．01），球蛋白与清蛋白的比例、谷丙转氨酶（ALT）和谷草转氨酶（AST）都没有显著性的变化（p>0．05）。这些结果说明，茶多酚可能影响了肝脏与外周组织之间脂类转运的状态，但对机体的免疫系统功能没有产生影响。肝脏中抗氧化指标的分析结果表明，日粮中不同茶多酚水平对肝脏总超氧化物歧化酶（T-SOD）和谷胱甘肽过氧化物酶（GSH-Px）活性的影响表现出了相同的趋势，但总体来说，在本试验条件下，肝脏中总超氧化物歧化酶（T-SOD）和谷胱甘肽过氧化物酶（GSH-Px）的活性在各组之间基本稳定，但肝脏中丙二醛水平随着日粮中茶多酚添加水平的上升显著下降的趋势（p<O．01）。这些结果说明，茶多酚被吸收进入机体或细胞后，抑制了机体组织或细胞中的脂质过氧化反应，但其所产生的保护作用可能是通过茶多酚本身的某些成分和一些代谢产物而独立发挥作用的，茶多酚在机体中的抗氧化功能可能不是通过与机体内其它抗氧化体系相互作用而产生。更多还原

【Abstract】 The liver metabolize of goose was dramatically changed induced by overfeeding,and causing large fats deposited in liver. The sensibility of liver is different among goosebreeds in responses to overfeeding, which resulting in differential fatty liver performance.Up till now, the molecular mechanism of fatty liver forming is still remaining un-clear. Inthis study, mRNA differential display and real-time quantitative PCR were applied toisolate and identify the differentially expressed gene between fatty liver and normal liverof Landes and Xupu goose. The bio-informatics technique was also used in predicting thearchitecture and functions on protein and nucleic acid levels, and the main results asfollows:Ninety primer pairs were used in this study to obtain an understanding of the mRNAlevels in response to overfeeding in the two breeds. The identified genes were confirmedby using real-time quantitative PCR technique. Six genes were confirmed as up regulated,two were confirmed as down regulated, and one gene was confirmed as up regulated inLandes, but down regulated in Xupu goose.The expression level of gene 8216 was confirmed as up regulated in both fatty livers,and its expression level is higher in Landes than in Xupu goose. The complete cDNAsequence of this gene was 1797 bp, and the GenBank accession number was EF488993.By blast in NCBI server, this sequence shows 92％identity with chicken seleniumbinding protein 1 (SELENBP1) mRNA (XM-423397.2). The sequence analysis revealedthat its open reading frame (ORF) of 1413 bp encodes a protein of 471 amino acids,which contains a putative conserved domain of 56 kDa selenium binding protein (SBP56)and has high homology with its homologues-chicken 95％, rat 86％, mouse 84％, human86％, monkey 86％, dog 86％, and cattle 86％. The tissue expression analysis indicatedthat goose SELENBP1 mRNA is higher expressed in liver and kidney, but moderate inspleen, and lowest in ovary, uterus, muscular stomach, and abdominal fat.The expression level of gene 9105 was confirmed as up regulated in both fatty livers.A 1248 bp cDNA fragments which contains the complete CDS was obtained thoughcDNA cloning. The accession number of GenBank is EF541127. By blast in NCBI server,this sequence shows 94％identity with chicken transforming growth factor beta 2(TGFB2) mRNA (NM-001031045.1), and also has high identities with TGFB2 of otherspecies. The sequence analysis revealed that its open reading frame (ORF) of 1239 bpencodes a protein of 412 amino acids, which contains two putative conserved domains ofTGFb-propeptide and TGFβ, and has high homology with its homologues-chicken 99％, Xenopus laevis 96％, rat 87％, human 90％, Monodelphis domestica 91％, cattle 89％,mouse 88％, pig 90％, Ovis aries 89％. The tissue expression analysis indicated that goosegene 9105 is higher expressed in muscular stomach, but moderate in liver, spleen, andovary, and lowest in uterus, abdominal fat, and kidney.The expression level of gene 7501 was confirmed as up regulated in both fatty livers,and its expression level is higher in Landes than in Xupu goose. A cDNA fragment of1446 bp was obtained by cDNA cloning. By blast in NCBI server, this sequence shows93％identity with chicken cytosolic NADP (+)-dependent malic enzyme (ME1) mRNA(NM-204303.1), and also has high identities with ME1 of other species. The tissueexpression analysis indicated that goose gene 7501 is higher expressed in liver, butmoderate in muscular stomach, spleen, ovary, uterus, and kidney, and lowest in abdominalfat.The expression level of gene 8407 was confirmed as down regulated in both fattylivers. A 1269 bp cDNA fragments which contains the complete CDS was obtainedthough cDNA cloning. The accession number of GenBank is EF541128. By blast in NCBIserver, this sequence shows 95％identity with chicken cytosolic NADP-dependentisoeitrate dehydrogenase (IDH1) mRNA (NM-421965), and also has high identities withIDH1 of other species. The sequence analysis revealed that its open reading frame (ORF)of 1248 bp encodes a protein of 415 amino acids, which contains a putative domains ofIcd, which conserved among species, and has high homology with itshomologues-chicken 99％, rat 89％, human 90％, monkey 90％, cattle 88％, mouse 88％.The tissue expression analysis indicated that goose gene 8407 is higher expressed in liver,but moderate in muscular stomach, liver, and spleen, and lowest in ovary, uterus,abdominal fat, lung, and muscle.The expression level of gene 9302 was confirmed as down regulated in both fattylivers. A cDNA fragment of 943 bp was obtained by cDNA cloning. By blast in NCBIserver, this sequence shows 93％identity with chicken cytochrome P450, family 7,subfamily A, polypeptide 1 (CYP7A1) mRNA (XM-419217). The tissue expressionanalysis indicated that goose gene 9302 is highly expressed in liver, but non-expressed inother tested tissues.The expression level of gene 7102 was contrary between these two breeds. It was upregulated in Landes, but down regulated in Xupu goose. A cDNA fragment of 923 bp wasobtained by cDNA cloning. By blast in NCBI server, this sequence shows 95％identitywith chicken malate dehydrogenase 1, NAD (soluble) mRNA (MDH1)(NM-001006395.1), and also has high identities with MDH1 of other species. The tissue expression analysis indicated that goose gene 9302 is highly expressed in liver, but lowerexpressed in other tested tissues. Goose can develops fatty liver though overfeeding with feed which contains high energy concentrations in short-term. In the process of fatty liver developments, the oxidative stress of liver increased correspondingly and the free radical accumulated in liver followed by liver cell damaged and the quality of fatty liver decline. A single factor trial based on the Tea Polyphenols (TP) concentration was designed to study the effects of TP in diets on fat metabolism and the capability of antioxidation in overfeeding goose in this study. Geese were assigned to five groups and treated with feeds which contained different levels of TP using the first group as control. The levels of TP in the rest feed were: 40, 80, 160, and 320 mg/Kg. the performance of slaughter, bio-indexes of serum, and the capability of liver antioxdation were analyzed, and the main results are as follows:After 21d of overfeeding, the average intake was approximately same (13.5±0.23Kg). The analysis results revealed that the dressing percentage, semi-eviscerated carcass percentage, eviscerated carcass percentage, breast muscle weight and leg muscle weight had no significant differences among groups (p>0.05), the weight after overfeeding and weight gain presented a significant down trend (p<0.01); The ratio of abdominal weight to the weight after overfeeding showed an up and then down trend, but the changes did not significant. These results indicated that TP had no effects on hepatic lipogenesis and fat depositing, and also had no effects on the assignments of fat in body. The analysis results about serum bio-index among groups revealed that the alteration of triglyceride (TG) level coincided with fatty liver weight, and the total cholesterol (TC) level in serum showed down in total, but had no significant difference among trial groups (p>0.05) and had significant difference compared with control (p<0.01). The High density lipoprotein cholesterol (HDL-C) level were relatively steady, and had no significant difference among groups (p>0.05). The Low density lipoprotein cholesterol (LDL-C) level in serum was significant down (p<0.01). Others had no significant differences ((p>0.05). These results revealed that the migration of fat between liver and other tissues might affected by TP which were absorbed from diets, but the TP involved in diets had no effects on immune system. The analysis results about antioxidation index of liver among groups revealed that TP which from diets had no effects on the activity of Total superoxide dismutase (T-SOD) and Glutathione peroxidase (GSH-Px) in liver, but the Malonaldehyde (MDA) level were significantly decreased according to the TP level in diets (p<0.01). These results indicated that TP could be absorbed into tissues from diets and finally into cell. The intracellular TP and it metabolites suppressed the oxides reaction of fat, but it protecting function was exerted through some signaling pathway by itself and its metabolites, other than via interacted with some antioxidant system.更多还原