【作者】 徐国庆；

【导师】 龚道清； 孟和；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 为了探索鹅肥肝形成的分子机理,本研究以12只朗德鹅为试验材料,分填肥组和对照组,每组6只,运用mRNA差异显示(DDRT-PCR)技术,检测填肥组和对照组之间的差异表达片段,将所获得的差异片段克隆测序后与GenBank中已知基因进行序列比对,寻找差异片段的同源基因或序列,并推测差异表达基因的功能。另外,由于鹅肥肝的形成与脂肪代谢密切相关,本研究还选择了7个脂肪性状的候选基因:脂肪细胞型脂肪酸结合蛋白(Adipocyte fatty acid binding protein, A-FABP)、肝脏型脂肪酸结合蛋白(liver fatty acid binding protein, L-FABP)、过氧化物酶体增殖物受体α(proxisome proliferators-activated receptor alpha, PPARα)和γ(proxisome proliferators-activated receptor gmma, PPARγ)、脂蛋白酯酶(lipoprotein lipase, LPL)、肝X受体-α(liver X receptor-α, LXRα)及脂联素(aidponectin, AdipoQ),利用半定量RT-PCR技术检测候选基因和新获得的差异表达基因在鹅肥肝中的表达水平,探讨它们在肥肝形成中的作用机理。结果如下:(1)用90对引物组合在填肥组和对照组间检测到40条差异表达片段,经片段回收、二次PCR及克隆测序后共成功获得了22条差异片段序列。经序列比对,在22条差异表达片段中,发现了8条与已知功能基因高度同源;6条与已知EST序列或cDNA序列同源,但其功能未知;8条在GenBank中没有找到相似序列,归为新EST序列。(2) 8条已知功能的差异表达基因为:纤维蛋白原α(fibrinogen alpha chain , FGA)基因、纤维蛋白素原γ(fibrinogen gamma chain, FGG)基因、色素框同源物6(Chromobox homolog 6, CBX6)基因、RNA结合基序7(RNA binding motif protein7, RBM7)基因、跨膜蛋白53(transmembrane protein 53, TMEM53)基因、基质金属蛋白酶11(matrix metallopeptidase-11, MMP-11 )基因、C18orf1基因和锌指蛋白469(zinc finger protein 469, ZNF469)基因。(3) DDRT-PCR结合半定量RT-PCR检测发现,在鹅肥肝中表达上调的基因有FGA、CBX6、TMEM53、MMP-11、ZNF469、A-FABP、L-FABP和PPARγ基因。而呈现下调趋势的有5条基因,它们是编码FGG、RBM 7、C18orf1、PPARα和LPL的基因。另外,LXRα基因在对照组和填肥组肝脏之间没有显著的表达差异,AdipoQ基因在两组间均不表达。(4)上述基因与脂肪代谢、肿瘤的转移、RNA修饰及调控有密切的关系,在强饲的高能饲料诱导下,这些基因在肝脏中的特异性表达变化可能是促进鹅肝脏中脂肪沉积的重要因素,可以将它们作为鹅肥肝形成的候选基因。更多还原

【Abstract】 In order to search for the molecular mechanism of the goose fatty liver, 12 Landes geese were used in this study. These geese were divided into two groups and 6 geese in each group. The differential expression bands between overfeeding group and control group were detected with technique of mRNA differential display. These differential expression bands were cloned and sequenced and found homologous genes through comparison with known genes from the GenBank, and the possible functions should be predicted. In addition, because of the closely correlation between fat metabolism and development of goose fatty liver, 7 candidate genes of fatty trait were involved in the present study, such as: Adipocyte fatty acid binding protein (A-FABP) gene, liver fatty acid binding protein (L-FABP) gene, proxisome proliferators-activated receptor alpha and gmma gene (PPARαand PPARγ) gene, lipoprotein lipase(LPL)gene, liver X receptor(LXRs) gene and adiponectin(AdipoQ) gene. The mRNA level of these candidate genes and new differential expression genes in goose fatty liver were detected with technique of semi-quantitative RT-PCR, and their effect on the development of fatty liver were explored. The results are as follows:(1) 40 differential expression bands were found between overfeeding group and control group using 90 pairs primer combination, and 22 differential expression sequences were obtained successfully through recovering bands, twice PCR, cloning and sequencing. From the 22 differential expression bands, 8 bands that have homologization highly with known functional genes were found; 6 bands were similar with known EST or cDNA, but their functions were unknown; other 8 bands were new EST, their homologies were not found in GenBank.(2) 8 differential expression genes whose functions are known, are fibrinogen alpha chain(FAG) gene, fibrinogen gamma chain(FGG) gene, Chromobox homolog 6(CBX6) gene, RNA binding motif protein 7(RBM7), transmembrane protein 53(TMEM 53), matrix metallopeptidase-11 (MMP-11 ) gene, C18orf1 gene and zinc finger protein 469(ZNF469 ) gene.(3) With the combination of the DDRT-PCR and semi-quantitative RT-PCR analysis, the following genes were significantly up-regulated in goose fatty liver: FGA, CBX 6, TMEM 53, MMP-11, ZNF469, A-FABP, L-FABP and PPARγ, while 5 genes including FGG, RBM7, C18orf1, PPARαand LPL gene were down-regulated in goose fatty liver. Additionally, the difference of LXRαexpressed between overfeeding group and control group was not significant, and AdipoQ was not expressed in the two groups.(4) According to the previous studies, these genes were involved in the fat metabolism, tumorous transfer, RNA modify and regulation. Induced by the high-energy feeds, the special changes of the expression of these genes in goose fatty liver may be important factors in promoting the fatty accumulation in goose fatty liver. So these genes may be the candidate genes of the development of goose fatty liver.更多还原