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  本文關(guān)鍵詞：山羊胚胎附植階段子宮轉(zhuǎn)錄組測序及附植相關(guān)基因的篩選　出處：《東北農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 山羊 胚胎附植 子宮 RNA-Seq 粘附

【摘要】：胚胎附植是指胚胎與母體子宮內(nèi)膜接觸并建立結(jié)構(gòu)上的聯(lián)系,從而實(shí)現(xiàn)物質(zhì)交流的過程,在哺乳動物生殖過程中起到十分關(guān)鍵的作用。山羊(Capra hircus)屬反芻動物,反芻動物胚胎為表面附植,即胚胎不侵入到子宮基質(zhì),而是與子宮內(nèi)膜上皮細(xì)胞融合并形成子葉胎盤,通過胎盤實(shí)現(xiàn)胎兒與母體間的交流及物質(zhì)交換。胚胎附植主要涉及胚胎滋養(yǎng)外胚層細(xì)胞和子宮內(nèi)膜上皮細(xì)胞之間的相互作用,由于其表面附植的特點(diǎn),使得山羊成為研究胚胎附植階段母胎界面相互作用的有利模型。此外山羊作為世界上最古老的物種之一,是我國草食畜牧業(yè)的重要組成部分,在乳業(yè)、肉業(yè)、纖維等產(chǎn)業(yè)中具有不可忽視的地位�，F(xiàn)代胚胎工程技術(shù)在山羊優(yōu)良品種的繁育中已得到廣泛應(yīng)用,但操作過的胚胎在體內(nèi)存活率與自然發(fā)育的胚胎相比降低很多,主要表現(xiàn)就是胚胎附植失敗。胚胎附植需要胚胎與母體子宮間復(fù)雜的相互作用,子宮對于附植乃至妊娠成功都是至關(guān)重要的。因此,對山羊胚胎附植階段子宮中基因表達(dá)情況進(jìn)行研究,能夠?yàn)樘岣呱窖蚺咛ジ街材芰μ峁├碚摶A(chǔ)。轉(zhuǎn)錄組是指特定組織或細(xì)胞在某一功能狀態(tài)下轉(zhuǎn)錄出來的所有RNA總和,包括編碼蛋白的mRNA和非編碼RNA,轉(zhuǎn)錄組可以從整體水平上反應(yīng)細(xì)胞中基因表達(dá)情況及其調(diào)控規(guī)律。反芻動物中,對綿羊和牛胚胎附植階段轉(zhuǎn)錄組研究分析已有一部分進(jìn)展。目前山羊全基因組序列已經(jīng)獲得,本文對胚胎附植階段山羊子宮轉(zhuǎn)錄組進(jìn)行了測序及分析,并從中發(fā)現(xiàn)一些可能參與山羊胚胎附植的關(guān)鍵基因,為深入了解山羊胚胎附植機(jī)制提供基礎(chǔ)。選取健康的黑龍江普通山羊,在妊娠第6(D6)、第16(D16)、第19(D19)天取子宮組織,每個時間點(diǎn)取三只羊以排除個體差異。RNA-Seq檢測結(jié)果初步分析顯示,妊娠第16天較第6天有1121個基因有差異,其中638上調(diào),483下調(diào);妊娠第19天較第6天有2555個差異基因,其中1183上調(diào),1372下調(diào);妊娠第19天較第16天有452個差異基因,其中124個上調(diào),328個下調(diào)。通過GO分析發(fā)現(xiàn)差異基因主要與乳腺芽形態(tài)發(fā)生、d UMP代謝過程、糖原代謝過程調(diào)節(jié)、脫氧核糖核苷酸分解代謝過程、病毒包膜、CRLF-CLCF1復(fù)合物、神經(jīng)介素B受體結(jié)合、類固醇21單加氧酶活性等有關(guān),Pathway分析發(fā)現(xiàn)主要參與煙酸和煙酰胺代謝、RNA聚合酶、嘧啶代謝、糖脂生物合成以及纈氨酸、亮氨酸和異亮氨酸的生物合成等途徑。選擇部分基因進(jìn)行實(shí)時熒光定量PCR和原位雜交驗(yàn)證,驗(yàn)證結(jié)果與RNA-Seq結(jié)果一致。結(jié)論:得到在山羊胚胎附植階段差異表達(dá)的基因,熒光定量PCR和原位雜交驗(yàn)證測序結(jié)果可靠,得到的數(shù)據(jù)可以用于后續(xù)試驗(yàn)分析,為后續(xù)試驗(yàn)打下基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S827

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