山羊胚胎附植阶段子宫转录组测序及附植相关基因的筛选

发布时间：2018-01-15 11:35

本文关键词：山羊胚胎附植阶段子宫转录组测序及附植相关基因的筛选　出处：《东北农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：胚胎附植是指胚胎与母体子宫内膜接触并建立结构上的联系,从而实现物质交流的过程,在哺乳动物生殖过程中起到十分关键的作用。山羊(Capra hircus)属反刍动物,反刍动物胚胎为表面附植,即胚胎不侵入到子宫基质,而是与子宫内膜上皮细胞融合并形成子叶胎盘,通过胎盘实现胎儿与母体间的交流及物质交换。胚胎附植主要涉及胚胎滋养外胚层细胞和子宫内膜上皮细胞之间的相互作用,由于其表面附植的特点,使得山羊成为研究胚胎附植阶段母胎界面相互作用的有利模型。此外山羊作为世界上最古老的物种之一,是我国草食畜牧业的重要组成部分,在乳业、肉业、纤维等产业中具有不可忽视的地位。现代胚胎工程技术在山羊优良品种的繁育中已得到广泛应用,但操作过的胚胎在体内存活率与自然发育的胚胎相比降低很多,主要表现就是胚胎附植失败。胚胎附植需要胚胎与母体子宫间复杂的相互作用,子宫对于附植乃至妊娠成功都是至关重要的。因此,对山羊胚胎附植阶段子宫中基因表达情况进行研究,能够为提高山羊胚胎附植能力提供理论基础。转录组是指特定组织或细胞在某一功能状态下转录出来的所有RNA总和,包括编码蛋白的mRNA和非编码RNA,转录组可以从整体水平上反应细胞中基因表达情况及其调控规律。反刍动物中,对绵羊和牛胚胎附植阶段转录组研究分析已有一部分进展。目前山羊全基因组序列已经获得,本文对胚胎附植阶段山羊子宫转录组进行了测序及分析,并从中发现一些可能参与山羊胚胎附植的关键基因,为深入了解山羊胚胎附植机制提供基础。选取健康的黑龙江普通山羊,在妊娠第6(D6)、第16(D16)、第19(D19)天取子宫组织,每个时间点取三只羊以排除个体差异。RNA-Seq检测结果初步分析显示,妊娠第16天较第6天有1121个基因有差异,其中638上调,483下调;妊娠第19天较第6天有2555个差异基因,其中1183上调,1372下调;妊娠第19天较第16天有452个差异基因,其中124个上调,328个下调。通过GO分析发现差异基因主要与乳腺芽形态发生、d UMP代谢过程、糖原代谢过程调节、脱氧核糖核苷酸分解代谢过程、病毒包膜、CRLF-CLCF1复合物、神经介素B受体结合、类固醇21单加氧酶活性等有关,Pathway分析发现主要参与烟酸和烟酰胺代谢、RNA聚合酶、嘧啶代谢、糖脂生物合成以及缬氨酸、亮氨酸和异亮氨酸的生物合成等途径。选择部分基因进行实时荧光定量PCR和原位杂交验证,验证结果与RNA-Seq结果一致。结论:得到在山羊胚胎附植阶段差异表达的基因,荧光定量PCR和原位杂交验证测序结果可靠,得到的数据可以用于后续试验分析,为后续试验打下基础。
[Abstract]:Embryo attachment refers to the process that the embryo contacts with the endometrium of the mother and establishes the structural connection so as to realize the material exchange. Capra hircusi is a ruminant. Ruminant embryos are attached to the surface, that is, the embryo does not invade the uterine matrix. Instead, they fuse with endometrial epithelial cells and form cotyledon placentas. Placenta is used to realize the communication and material exchange between fetus and mother. Embryo implantation mainly involves the interaction between embryonic trophodermal cells and endometrial epithelial cells due to the characteristics of surface attachment. It makes goats become a favorable model to study the interaction between mother and fetus during embryo implantation. As one of the oldest species in the world, goat is an important part of herbivorous animal husbandry in China, in the dairy industry, meat industry. Modern embryo engineering technology has been widely used in the breeding of fine goat breeds. However, the survival rate of the operated embryos in vivo is much lower than that of the naturally developed embryos, the main manifestation of which is the failure of embryo implantation. Embryo implantation requires complex interaction between the embryo and the mother uterus. Uterus is very important for the success of implantation and pregnancy. Therefore, the gene expression in the uterus of goat embryo implantation is studied. Transcriptome refers to the sum of all RNA transcribed by specific tissues or cells in a certain functional state. Including mRNA and non-coding RNAs that encode proteins, transcriptome can respond to gene expression and its regulation in ruminants at the overall level. Some progress has been made in the analysis of the transcriptome of sheep and bovine embryos during the implantation stage. At present, the whole genome sequence of goat has been obtained. In this paper, the transcriptome of goat uterus in the stage of embryo implantation has been sequenced and analyzed. Some key genes which may be involved in the implantation of goat embryos were found, which provided a basis for further understanding of the mechanism of goat embryo implantation. Select healthy Heilongjiang ordinary goats in the 6th trimester of pregnancy. The uterine tissue was collected on day 16 and day 19, and three sheep were collected at each time point to rule out individual differences. The results of RNA-Seq analysis showed that: 1. On the 16th day of gestation, there were 1121 genes different from those on the 6th day, of which 638 were up-regulated and 483 down-regulated. There were 2555 differentially expressed genes on the 19th day of gestation compared with the sixth day, of which 1183 genes were up-regulated and 1372 down-regulated. There were 452 differentially expressed genes on the 19th day of pregnancy compared with the 16th day, of which 124 genes were up-regulated and 328 down-regulated. Through go analysis, it was found that the differential genes were mainly related to the metabolism of mammary gland morphogenetic d UMP. Regulation of glycogen metabolism, deoxyribonucleotide catabolism, viral envelope CRLF-CLCF1 complex, IL-B receptor binding, steroid 21 monooxygenase activity, etc. Pathway analysis showed that it was mainly involved in the metabolism of nicotinic acid and nicotinamide, pyrimidine metabolism, glycolipid biosynthesis and valine. The biosynthesis of leucine and isoleucine. Some genes were selected for real-time fluorescence quantitative PCR and in situ hybridization. Conclusion: the differentially expressed genes were obtained in the implantation stage of goat embryos. The results of fluorescence quantitative PCR and in situ hybridization were reliable. The obtained data can be used for the analysis of subsequent experiments and lay the foundation for subsequent tests.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S827

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