【作者】 朱丽慧；

【导师】 龚道清； 孟和；

【作者基本信息】 扬州大学 ， 动物营养与饲料科学， 2010， 硕士

【摘要】 本研究以朗德鹅为试验材料,运用抑制性消减杂交(SSH)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,对筛选出的差异表达基因进行功能聚类分析和代谢途径分析(KEGG),推测筛选的基因的功能,并绘制肥肝形成过程中脂类代谢调控示意图。选择12个与脂类代谢或糖代谢相关的基因进行荧光定量PCR(qRT-PCR)验证,并检测其中4个基因的时空表达规律,另外还测定了朗德鹅填肥前、填肥10天和填肥20天的血清生化指标和组织营养成分等。结果如下:(1)填肥鹅血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、甘油三酯(TG)、总胆固醇和高密度脂蛋白(HDL)水平显著增加,γ-谷氨酰转肽酶(GGT)在填肥过程中呈现出先升高后降低;脂肪主要在肝脏沉积,其他组织无明显差异,蛋白质在肌肉和肝脏中相对增加;填饲朗德鹅肝脏细胞胀大,肝细胞胞浆中充满大量大小不等的脂肪滴。表明填肥使鹅的肝脏脂肪代谢和血清发生改变。(2)采用SSH技术在填肥组和对照组间共检测到107条差异表达片段,其中未知基因18个,假设性基因26个,与鹅肥肝相关上调表达基因83个,下调表达基因24个。GO聚类分析显示这些差异表达基因主要参与细胞生化过程、信号转导、脂肪合成代谢等生物学过程。(3)荧光实时定量PCR技术检测结果证明,部分代谢相关差异表达基因的表达规律与消减杂交的结果一致。表明SSH技术是检测鹅肥肝形成过程中差异表达基因的有效方法。(4)差异表达基因代谢途径分析(KEGG)表明,在强饲的高能饲料诱导下,脂肪酸合成途径和TG合成途径加强,而糖酵解途径受阻,其中SCD、Elovl-6、ACSL1、NADP-ME等基因参与的不饱和脂肪酸合成途径在肥肝形成过程中发挥极其重要的作用。更多还原

【Abstract】 In present study, suppression subtractive hybridization (SSH) was used to detect differential expression of genes in the livers of overfeeding and normal feeding Landes geese. These differential expression bands were cloned, sequenced and aligned homology with known genes from the GenBank.And then, they were classified into functional categories and analyzed by Kyoto Encyclopaedia of Genes and Genomes (KEGG) automatic annotation server for ortholog assignment and pathway mapping, and the possible functions and schematic representation of the roles of the genes were predicted and drawn. Twelve genes that related to metabolism processes and cellular processes were confirmed using quantitative real time PCR (qRT-PCR). Four genes were selected and detected in different tissues and period of overfeeding. The serum biochemical parameters were measured during the period of overfeeding and tissue nutrient composition and histological changes in liver were also measured, results are as follows:(1) The concentration of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total cholesterol (TC) and high density lipoprotein (HDL) in overfeeding Landes geese serum were significantly increased. Theγ-glutamyl transpeptidase (GGT) level was increased and then decreased during the overfeeding period. The tissue nutrient component analysis showed that fat was mainly deposited in liver, and the content of protein was increased in muscle and liver after overfeeding. Histology observation shown that liver cytoplasm was full of fat in overfeeding group. The results indicated that overfeeding has changed the liver lipid metabolism and serum component of geese. (2) One hundred and seven differential expression genes containing 16 unknown sequences and 26 hypothetical genes in the livers of overfeeding geese and normal feeding geese were detected by SSH, of which 83 were up-regulated and 24 were down-regulated. The functional categories showed that these genes were maily related to lipids metabolism process, ceculler process and signal transduction.(3) Tweleve different expression genes selected from SSH results confirmed by qRT-PCR, which indicated that SSH was an efficient way to screening different expression genes related to goose fatty liver.(4) KEGG analysis showed that most genes were associated with pathways related to metabolism or biosynthesis pathways, especially for the biosynthesis of fatty acids. The fatty acids and TG synthesis were enhanced and glycolysis was was reduced in response to overfeeding. Several genes such as stearoyl-CoA desaturase (delta-9-desaturase) (SCD), ELOVL family member 6 (Elovl-6), NADP+ dependent malic enzyme (NADP-ME) and long-chain acyl-CoA synthetase 1 (ACSL1) are involved in the biosynthesis of unsaturated fatty acids which play an important role in the formation of goose fatty liver.更多还原