基于转录组与蛋白质组联合分析揭示羔羊断母乳应激调控机制研究

发布时间：2018-02-24 19:28

本文关键词： 湖羊断奶羔羊断奶应激肠道 RNA-seq iTRAQ　出处：《中国农业科学院》2016年博士后论文　论文类型：学位论文

【摘要】：湖羊是我国著名的多胎品种,平均产羔率达256%,高繁殖率选育群可达300%以上。在实际生产过程中,如母羊多胎或母体体质弱、母乳不足、羔羊规模化育肥和工厂化生产、优良品种母羊一年两产等情况,需要羔羊早期或超早期断奶。先前的研究主要围绕羔羊早期断奶的方法和缓解断奶应激的技术措施展开。对于断奶过程中的分子调节机制的研究相对较少。本研究选用初生双胞胎湖羊作为研究对象,随母哺乳至3日龄,采用配对试验设计,对照组随母哺乳,试验组饲喂代乳品至15日龄屠宰,取羔羊空肠组织。对断母乳羔羊肠道组织进行切片分析,同时采用RNA-Seq技术和iTRAQ技术分别对断母乳湖羊羔羊肠道差异表达基因和差异表达蛋白进行分析。结果如下：1.组织形态学分析表明断母乳羔羊肠道组织形态发生明显变化,主要表现为肠绒毛长度缩短,隐窝深度增加。2.采用RNA-seq技术分析断母乳对羔羊肠道组织基因mRNA水平的影响。RNA-seq结果显示,肠道组织样品表达转录本注释得到的基因有24592个,对所有能够比对上基因组的基因按padj 0.05的条件筛选,共有482个差异表达基因。其中,断母乳组表达上调的基因有235个,表达下调的基因有247个。差异表达基因GO和KEGG分析结果显示,差异表达基因主要参与调节机体营养素的代谢,消化系统、免疫系统和内分泌系统。通过RNA-seq数据分析,我们发现4个免疫相关的新基因,分别为ENSOARG00000010572, ENSOARG00000015002, ENSOARG00000009042和ENSOARG00000014493。3.采用iTRAQ技术对断母乳羔羊肠道组织进行蛋白质组学分析,共鉴定得到蛋白5338个,根据筛选标准FC1.2或0.83,且经统计检验其p-value0.05的蛋白作为差异蛋白,断母乳组羔羊共得到差异蛋白389个,其中表达上调蛋白143个,表达下调蛋白246个。差异表达蛋白主要参与生物学过程包括免疫系统、物质代谢、生物黏附和细胞迁移等过程。本研究采用联合组学技术,从mRNA水平和蛋白质水平探讨断母乳羔羊肠道基因表达模式和代谢通路,为后续羔羊揭示羔羊断奶应激的分子调控机制提供依据。
[Abstract]:Hu is a famous prolific species, average lambing rate of 256%, the high reproduction rate of breeding groups can reach more than 300%. In the actual production process, such as multiple births or ewe maternal physical weakness, lack of breast milk, lamb scale breeding and factory production, fine varieties of ewe two a year production situation, need Lamb the early or super early weaning. Previous studies mainly focus on the methods of lamb early weaning and technical measures to alleviate weaning stress. The molecular regulation mechanism of the weaning process is relatively small. This research selects the newborn twins sheep as the research object, the mother breast-feeding to 3 days of age, using a paired experimental design, the control group with lactation the test group, fed milk replacer to 15 days of age were slaughtered lamb. Lamb of the jejunum tissue off the breast intestinal tissue slice analysis, using RNA-Seq technology and iTRAQ technology on broken milk sheep Differential expression of gene and protein expression of intestinal lamb difference was analyzed. The results are as follows: 1. the morphological analysis showed that the broken milk lamb intestinal morphology changed significantly, mainly for intestinal villus length shortened, crypt depth increased.2. RNA-seq technology was used to analyze the fault breast effect on gene level of mRNA lamb intestinal tissue.RNA-seq results showed that the intestinal tissue the sample expressed transcripts annotated from 24592 genes, all of which can be aligned on the genome Padj gene according to the condition of 0.05 screening, a total of 482 differentially expressed genes. Among them, broken breast milk group was up-regulated and 235 genes down regulated genes, 247 differentially expressed genes. GO and KEGG analysis results showed that the differentially expressed genes were mainly involved in the regulation of metabolism, body nutrients digestive system, immune system and endocrine system. Through the analysis of RNA-seq data, I They found 4 immune related genes were ENSOARG00000010572, ENSOARG00000015002, ENSOARG00000009042 and ENSOARG00000014493.3. by iTRAQ analysis of broken breast lamb intestinal tissue proteome, 5338 proteins were identified, according to the screening criteria of FC1.2 or 0.83, and the statistical test of p-value0.05 protein as a protein, milk broken group lamb there are 389 different proteins, including 143 up-regulated proteins and 246 down regulated proteins. The differentially expressed proteins are mainly involved in biological processes including immune system, metabolism, biological adhesion and cell migration process. This research adopts combined group technology, to explore the gene expression pattern of intestinal lamb weaning and metabolic pathways from the mRNA level and the protein level, provide the basis for further revealing the molecular mechanism of lamb lamb weaning stress.

【学位授予单位】：中国农业科学院
【学位级别】：博士后
【学位授予年份】：2016
【分类号】：S826

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