【作者】 孙云子；

【导师】 陈代文；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 为了探讨哺乳期和断奶后饲喂不同日粮的仔猪小肠发育的分子机制,本研究利用猪全基因组表达谱芯片进行两个试验,分析不同生长发育阶段仔猪小肠全基因组转录谱的变化,并对差异表达基因进行不同层次的数据挖掘。结果如下:试验一哺乳仔猪小肠发育全基因组转录谱研究仔猪小肠发育对动物的生长具有重要意义。本试验利用猪表达谱芯片,检测大约克哺乳仔猪(出生0d,3d,8d,14d和21d)肠道转录谱变化,结果如下:1、全基因组转录谱芯片(PGA)分析结果:序列试验基因表达模式分析(STC)得到80个表达模式,其中模式66和13达极显著水平(p＜0.01),提示哺乳阶段仔猪小肠发育与这些模式所属基因差异表达有关。2、对6个差异表达基因进行qRT-PCR验证结果:两种实验方法检测结果均为正相关,相关系数为0.934±0.025,表明本研究结果准确可靠,检测数据能够真实反映基因的表达变化规律。3、上调模式66所属基因Gene Ontology(GO)分析结果:该簇基因涉及的显著生物学过程(BP)主要有细胞周期及促进真菌类细胞壁的形成等(p＜0.01)。下调模式13所属基因GO分析发现,该簇基因涉及的显著BP主要有维生素及离子转运等(p＜0.01)。提示这些生物学过程对新生仔猪肠道发育具有重要意义。4、通径分析结果:差异表达基因主要涉及细胞色素参与的外源物质代谢及亚麻油酸代谢等通路发生了扰动,从一定程度上揭示了肠道发育过程的分子基础。5、基因共表达网络构建和分析:调控网络的构建从一定程度上反映了肠道发育过程的复杂性,同时还鉴定出20个在网络中具有重要调控能力的基因,建议作为对肠道发育具有重要影响和研究价值的种子基因进行深入研究。试验二不同蛋白源对断奶仔猪小肠全基因组转录谱的影响断奶日粮对仔猪小肠发育的影响是显著的。为了探讨不同蛋白源日粮对仔猪小肠发育影响的差异,本试验利用猪表达谱芯片,检测不同蛋白源日粮下断奶仔猪(断奶0d,3d,8d和14d)小肠转录谱,结果如下:1、PGA结果:乳源蛋白替代30%总蛋白组(处理1)获得370个差异表达基因。STC分析得到26个显著模式中,模式5和11显著性水平最高(p＜0.01),全植物蛋白日粮处理(处理2)获得263个差异表达基因。STC分析得到26个主流模式,其中模式3和22显著水平最高(p＜0.01)。提示不同蛋白源日粮对小肠的影响和这些模式所属基因差异表达相关。2、分别对6个差异表达基因进行qRT-PCR验证结果:芯片结果和qRT-PCR结果均为正相关,相关系数分别为0.902±0.023和0.855±0.036。表明本研究结果准确可靠,检测数据能够真实反映基因的表达变化规律。3、GO分析结果:乳源蛋白组模式5所属基因涉及的显著BP包括提高细胞内钙离子浓度等,模式11所属基因涉及的显著BP有对外加营养刺激的反应等(p＜0.01)。在植物蛋白组中,模式3所属基因涉及的显著BP主要有正向调节平滑肌收缩等,模式22所属基因涉及外皮细胞的生长等分子事件(p＜0.01)。提示不同断奶日粮下,差异基因主要体现了这些生物学过程。4、通径分析结果:不同蛋白源日粮组转录谱差异表达基因参与的信号通路相似,主要显著通路有PPAR信号通路、氨基糖代谢、T细胞受体信号通路以及脂肪代谢等通路(p＜0.01)。提示这些通路对断奶后小肠发育具有重要意义。5、基因共表达网络构建和分析:基因共表达网络一定程度上揭示了不同蛋白源日粮对肠道发育影响的明显差异。同时分别鉴定出12个和9个在两个处理调控网络中具有重要影响力基因以及9个在两个处理中的网络调控地位发生改变的共表达基因。建议可作为影响不同蛋白源日粮下肠道发育关键基因进行研究。本研究筛选出了对仔猪肠道发育可能具有重要影响和研究价值的基因,初步揭示了生长发育过程中肠细胞基因表达的规律、互作关系以及不同蛋白源日粮下肠道发育差异的分子基础。更多还原

【Abstract】 To investigate the molecular mechanism of intestinal development of postnatal piglets during suckling and after weaming fed with different weanling diets in this study, two experiments were conducted by using porcine genome array to analyze the transcription profile of piglets during different growth and development stages. The differentially expressed genes were screened and these data were mined. The results were as followings:Experiment 1. Transcription profile of whole-genome for the developmental intestine of suckling pigletsThe intestinal development of postnatal piglets has an important impact on health and growth performance. The transcription profiles of nursing Yorkshire at the age of 0, 3, 8, 14 and 21 days were detected by using porcine genome array (PGA) in this study. The results were as followings:1. The results of PGA during suckling: More than 8000 differentially expressed transcripts in intestine of piglets during suckling were obtained. Series Test Cluster were analyzed for these differential expression genes and 80 profiles were established, among which profiles 66 and 13 were the most significant (P<0.01). This result indicated that the differentially expressed genes in co-expression of these trends are related to the intestinal development during suckling.2. Six differentially expressed genes were selected and quantified by qRT-PCR. Results illustrated the expression patterns of the six genes were similar to those from PGA. The two experimental methods were both positively correlated with correlation coefficient being 0.934±0.025. This result demonstrateed that the microarray technique used in this study is accurate and reproducible.3. Gene Ontology(GO)analysis of the profile 66 indicated that the co-expression genes in this profile were mainly involved in cell cycle and fungal-type cell wall organization. GO of the profile 13 showed that the co-expression genes in this profile were mainly involved in vitamin and ionic transports. Furthermore, there was a significant inflection point on d3 of age, which suggested that d3 of age was a crucial period for piglets.4. Significant pathway analysis of differentially expressed genes in profiles 66 and 13 indicated that the pathways which the differentially expressed genes participated during suckling were mainly PPAR signaling pathway, linoleic acid metabolism and so on. This result suggested that these pathways were important to the intestinal development.5. Construction and analysis of gene regulatory networks (GRNs) initially revealed that there were 20 genes which possessed important regulatory ability (p<0.01), These genes could regulate other genes in GRN.Experiment 2. Effect of different weaning diets on transcription profile of genome-wide in intestine for weanling pigletsThe weanling diets have an obvious impact on the intestinal development. To investigate the impact of different weaning diets on the intestinal development, in the present study, PGA was employed to detect the difference on transcription spectrum of intestine for weanling piglets fed with different diets with or without milk protein.1. Results of PGA: Analysis of expression profiles in genome-wide transcription in weanling piglets fed with the diets with milk protein (treatment 1) was conducted and 370 differentially expressed transcripts were screened. Among these model profiles, there were 26 significant profiles and profiles 5 and 11 were the most significant (P<0.01). Meanwhile, analysis of expression profiles in genome-wide transcription in weanling piglets fed with the vegetable protein without milk protein (treatment 2) showed that 263 differentially expressed transcripts were screened. Series Test Cluster were analyzed for these differential expression genes and 26 significant profiles were obtained, among these model profiles, profiles 3 and 22 were the most significant (P<0.01). This result indicated that the differentially expressed genes in co-expression of these trends are related to the intestinal development of weanling piglets.2. Six differentially expressed genes were selected and quantified by qRT-PCR. Results illustrated that the expression patterns of the six genes were similar to those from PGA. The two experimental methods were both positively correlated with correlation coefficient being 0.902±0.023 and 0.855±0.036. These resultes indicated that the microarray technique used in this study is accurate and reproducible. 3. The co-expression of genes in these significant profiles were assayed using GO: For the treatment 1, it was found from the results of GO that the genes in profile 5 were mainly involved in some biology process such as tachykinin signaling pathway, elevation of Cytosolic calcium ion concentration and so on, while the genes in profile 11 were mainly involved in positive regulation of viral transcription, inflammatory response, histidine metabolic process, cellular metabolic process (p<0.01) and so on. For the treatment 2, the results of GO revealed that the genes in profile 3 were mainly involved in positive regulation of smooth muscle contraction, while the genes in profile 22 were mainly involved in cell growth.4. Pathway analysis: The signal pathways that the differentially expressed genes participated in both treatments were similar, including PPAR signaling pathway, Aminosugars metabolism, T cell receptor signaling pathway and so on. These pathways changed obviously after weanling (p<0.01).5. Construction and analysis of Dynamic-GeneNet of the differentially co-expression genes in significant profiles: Based on DGN model, gene regulatory networks (GRNs) for the genes in profile 5、11、3 and 22 in treatment land 2 were constructed. The GRNs revealed that there were 12 genes and 9 genes which possessed important regulatory ability in GRN. Meanwhile, 9 genes in these two GRNs were found to have changed the regulatory site. We can choose seed genes from sub-networks that have higher rank scores as potential key genes on the intestinal development of piglets.These results highlight some possible candidate genes for developmental intestine of piglets and provide some data on which to base further study of the molecular mechanism of the developmental intestine of piglets.更多还原