【作者】 苏胜彦；

【导师】 谢庄；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 从分子水平上筛选影响鹅肥肝性状的主效基因,并在早期选择有较大产肝潜力的朗德鹅是迅速提高鹅产肝性能和朗德鹅产业经济效益的方法之一。然而朗德鹅肥肝性状受众多基因和信号途径的调控,难以确定控制鹅肥肝性状的主效基因,因此从信号通路和基因表达调控网络的角度进行分析将是揭示鹅肥肝形成的分子机制的有效途径。针对朗德鹅肥肝性状的特点,文章通过图论的方法从整体上把握肝脏脂平衡的调控网络,然后寻找网络中具有整体调节作用的信号通路,并从试验的角度分析填饲造成网络中重要节点和脂代谢相关基因的表达变化。而对于图论发现的调控通路也必须通过试验的方法验证和分析。根据原始的研究文献、综述及分子生物学数据库,将分子实体作为顶点,分子实体名称对应标记分子网络中的分子实体,边对应于分子实体间的关系,对分子调控网络资料进行收集,并构建对应的邻接矩阵。然后将原始各个邻接矩阵综合为分子调控网络综合邻接矩阵。根据邻接矩阵A,通过计算R=A+A2+A3+…+An,得到可达矩阵。然后根据可达矩阵所研究分子所在的行和列的数据资料来判断分子间的调节与被调节关系。将分子调控网络的邻接矩阵Spath进行k次幂来判断肝脏脂代谢核心相关分子调控网络中的分子的上游与下游信号分子间的调控关系、路径长度及路径数。依据综合邻接矩阵,通过Matlab软件绘制了以核心基因为基础的肝脏脂代谢调控图。填饲后朗德鹅的肝重、腹脂重、肝重指数和体重显著增加。通过CT分析发现对照组肝脾比值大于1,而填饲组肝脾比值呈负数,与人的脂肪肝结果分析类似。在血清生化指标方面,填饲组朗德鹅血清谷丙转氨酶、甘油三酯和H-胆固醇与对照组相比差异极显著；胆碱脂酶显著高于对照组。通过组织切片分析发现：填饲处理后,朗德鹅肝脏细胞胀大,并且细胞质内充满大小不等的脂泡。通过气相色谱法测定了朗德鹅肝脏的脂肪酸组分发现：填饲可以降低朗德鹅肝脏中饱和脂肪酸、C18:2、C20:4、PUFA、SFA/UFA的含量、PUFA/UFA和PUFA/MUFA的值；提高MUFA、C18:1、UFA的含量和MUFA/UFA的值(P<0.01)。用荧光实时定量PCR法检测了填饲对脂生成相关基因和相关分子mRNA表达的影响。结果显示：与对照组相比,填饲可以显著提高朗德鹅肝脏中ACCα、PPARα、aP2、LXRα、PPARγ、Spot14α、Spot14β、C/EBPα、C/EBPβ、GH基因mRNA的表达水平。肝脏组织中ACCα、LXRα基因的表达量与血清TG含量呈极显著正相关。而肝脏组织中aP2、LXRα、PPARγ基因的表达量与肝重指数呈极显著相关(P<0.01)。填饲可以降低SREBP1c基因mRNA的表达量(P<0.05)。肝脏组织中SREBP1c基因的表达量与肝重指数呈极显著相关(P<0.01),与血清TG成负相关(P<0.05)。T3处理可以显著提高体重、肝重(P<0.01),添加甜菜碱后可以逆转这种变化。与对照组和填饲组相比,T3处理可以显著提高血清Che, HDL和LDL的浓度,但是添加甜菜碱不能够逆转这种变化。T3处理组空泡数量明显减少,细胞质内脂滴增加,肝细胞膨胀。添加甜菜碱后,空泡数量进一步减少,肝细胞内脂滴比较均匀,细胞结构的变化得到有效的逆转。相对于填饲组,T3处理组显著提高肝脏中不饱和脂肪酸的含量(P<0.05),并且添加甜菜碱后可以逆转这种变化。相对于填饲组,T3处理可以显著提高ACCα基因的表达量,添加甜菜碱后可逆转T3处理对ACCα基因表达量的影响。相对于填饲组,T3处理可以显著降低PPARγ、PPARα、C/EBPβ、LXRα和GH基因mRNA的表达量(P<0.01),显著提高aP2基因mRNA的表达水平,添加甜菜碱后可逆转T3处理对GH和aP2基因表达量的影响(P<0.05)。肝脏组织中ACCα, Spot14α基因的表达量与血清TG呈极显著相关。肝脏中SREBP1c基因mRNA表达量与肝重指数呈极显著负相关,与血清TG呈显著负相关(P<0.05)。相对于填饲组,添加甜菜碱后肝重和肝重指数显著增加,腹脂重降低,血清中Che,HDL, ALT浓度显著增加(P<0.01),而TG和LDL浓度没有受到甜菜碱的影响。甜菜碱处理后,空泡数量明显减少,脂滴沉积分布均匀,数量较多。然而添加甜菜碱对脂肪酸组分没有影响。相对于填饲组,甜菜碱处理可降低C/EBPβ、PPARα、Spot14β、PPARγ和LXRαmRNA的表达水平(P<0.01)。而甜菜碱处理后可以显著提高Spot14α基因mRNA的表达。Spot14αcDNA全序列长792bp,与鸭比有87%的同源性,并已提交到基因库中,序列号为EU710582,编码128个氨基酸,与鸭比有84%的同源性。序列中有2个CpG岛,一个位于61～375区,另一个位于433～645区。移码突变是由于399～400位之间插入一个C或T引起的,其可导致亮氨酸拉链结构的丢失,从而引起Spot14α同源二聚体的形成。对Spot14α基因转录起始位点(-8～+374)区域的33个CpG位点进行分析发现：对照组该区域69.6%的位点甲基化,而经过填饲处理后,有87.9%的甲基化；T3处理后有94.0%的位点甲基化。与填饲组相比,T3处理后甲基化程度增加,并达到显著水平。甜菜碱处理后有82.6%的位点甲基化。与填饲组相比,甜菜碱处理甲基化程度降低,但是没有达到显著水平。本文试验结果显示：T3和甜菜碱处理均能提高朗德鹅的产肝性能,而且T3的适当剂量和合适的添加时间不会在肥肝中残留。这对鹅肥肝的生产有实践意义。更多还原

【Abstract】 An experiment was conducted research the candidate gene and early breeding projects of the fattty liver of Landes goose which had big markets. Performance of Landes geese fatty livers were controlled by lots of genes and regulation pathways. So the signal pathway and gene regulation network were the effect one to analysis the mechanism of geese fatty livers.The graphic model was established in order to grasp the regulation network of hepatic lipid homeostasis and discover the useful and important signal transduction because of complication of the fatty livers. The mRNA expression level of lipogenic genes involved the key signal pathway were detected by realtime PCR. Meanwhile, histological examination and analysis of fatty acids of the fatty livers were done used H & E stain and gas chromatography respectively. The signal transduction was proved by the trials and further study by the epigenetic method.Collect the data of molecular interaction from original research articles, reviews and molecular biological databank, and define the molecular entity as vertex, the relation between molecules as the edge in the graph, and mark the signaling direction with arrows, and then express the results with corresponding adjacency matrixes and obtain integrated adjacency matrix.By calculating the R=A+A2+A3+…+An, the accessible matrix was got. Then, we can induce the regulation relation between any molecules by accessible matrix. Through power the integrated adjacency matrix, the regulation relation between molecules upstream and downstream the molecules, as well as the crosstalk between signaling pathways were achieved. Based on the integrated adjacency matrix, the graph of regulation of molecular network related with lipid metabolism of Landes geese was produced using Matlab 6.0.Histological examination and analysis of fatty acids of the fatty liver were done used H & E stain and gas chromatography respectively. CT was used to investigate the fat deposition of living Landes goose. Compared to control group, overfeeding can increase the liver weight, liver index, serum ALT, TG, Che, HDL levels and change the positive ratio of liver and spleen to negative. Swelling of hepatocyte were observed within the cytoplasm of liver cells in goose treated with overfeeding compared to control. The values for the SFA, C18:2, C20:4, PUFA, SFA/UFA,、PUFA/UFA and PUFA/MUFA were significantly higher in control group than in overfeeding group. Higher values for MUFA, C18:1, UFA and MUFA/UFA in livers were found for goose in overfeeding group.The mRNA expression level of lipogenic genes were detected by realtime PCR. Compared to the control group, the mRNA expression level of ACCα, PPARα, aP2, LXRα, PPARγ, Spot14α, Spot14β, C/EBPα, C/EBPβand GH were increased in overfeeding group. The positive correlation between the transcriptional level of ACC a, LXR a and serum TG was observed (P<0.01). Significant positive correlations were also found between aP2, LXRα, PPARγin liver and liver/body weight.SREBP1c mRNA expression level in liver was decreased in overfeeding group compared to that of control. The significant negative correlations were observed between the expression of SREBP1c in liver and liver/body weight, serum TGThe overfed supplemented with T3 markedly increased the liver weight, liver/body weight, compared with control and overfed diet. Plasma levels of Che, HDL and LDL were higher in control group than in the overfed supplemented with T3 geese. T3 significantly increased the number of fatty droplets and exhibited bigger lipid droplets. Geese fed diets supplemented with T3 had higher concentration of UFA in liver, compared to control and overfeeding group. The effect of T3 on the UFA content can be reversed by betaine.The effect of T3 on ACC a and aP2 gene expression were also determined in Landes geese livers using real time PCR. ACC a and aP2 mRNA expression were significantly increased in T3-fed geese compared with control and overfed group. The transcriptional level of ACC a and aP2 gene were reversed by betaine significantly. Compared to the overfeeding group, the mRNA expression level of PPARγ、PPARα、C/EBPβ、LXRαand GH were decreased in T3 treatment group. The significant negative correlations were observed between the expression of SREBPlc in liver and liver/body weight, serum TG However, the significant correlations were observed between the expression of ACC a and Spot14αin liver and serum TG content.The geese fed the betaine diet showed increased liver weight and decreased abdominal adipose weight compared with the control diet and overfeeding group. Betaine treatment also increased body weighy (BW) (P<0.01) and increased serum triglyceride (TG), Che and low density lipoproteins (LDL) compared with the control group and no increase in these parameters relative to the overfeeding group. Diffuse yellow lesions and swelling of hepatocytes were observed in the betaine-treated group, which also had significantly decreased macrovesicular or microvesicular steatosis. Geese treated with betaine had no effect on the fatty acids profile in the livers compared with the control diet.C/EBPβ、PPARα、Spot14β、PPARγand LXRαmRNA expression were significantly decreased in betaine-fed geese compared with overfeeding group. The transcriptional level of Spot14αwas increased by the diets supplemented with betaine.We cloned the cDNA sequence of goose Spot14α(EU710582). The gene was predicted to encode a peptide of 128 amino acids, which has 87% and 84% sequence identities at the cDNA and amino acid levels, respectively, with the duck counterparts. High percentage of G and C nucleotides were found in exon and 3’untranslated region of the goose Spot 14 a cDNA.A novel frameshift mutation that leads to a damaged leucine zipper motif was observed at nucleotide position 399～400. This can influence the homodimerization of Spot14α, probably resulting in dysfunction in the Spot 14 family in vivo.We performed sodium bisulfite sequencing of individual alleles of this region (between+374 and -8 base pairs relative to the transcription start site), which contains 33 CpG dinucleotides of Spot14α. In the overfed group and T3 treatment group, the average methylation at the 33 CpGs sites were 87.9% and 94% in contrast to 69.6% in control group respectively. The significant differences were obsereved among the three groups. Compared to the overfeeding group, betaine treatment (82.6%) of the average methylation at these CpGs sites can not achieve the statistic significant change. But the level of methylation in this region was decreased.The result of this article showed that T3 and betaine treatment can make the liver weight of Landes geese increased. Meanwhile, no T3 was found in the fatty liver in the condition of adaptive time and suitable dose. The result might be significitant for the production of Landes geese fatty liver.更多还原