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基于转录组学技术探究中华绒螯蟹胚胎发育期间的基因和通路表达特征

发布时间:2018-07-23 09:03
【摘要】:甲壳动物作为无脊椎动物最大的类群之一,具有丰富的生物多样性以及复杂的生活史。而胚胎阶段作为甲壳动物重要的发育时期,其经历了一系列的基因发育调控和表达模式的变化,然而,到目前为止,相关甲壳动物胚胎发育阶段的分子生物学机理研究并不充分,尤其是在中华绒螯蟹等十足目动物中。本文以长江水系中华绒螯蟹胚胎在受精卵(El),卵裂期(E2),囊胚期(E3),原肠期(E4),卵内无节幼体期(E5),眼点期(E6),心跳期(E7)和初孵幼体期(Z1)为研究对象,进行转录组测序,从中获得了一批生长发育相关的重要功能基因,初步解析了这一发育过程中重要生理现象的分子基础。论文的主要研究结果如下:1.基于Illumina Hiseq2000测序平台,获得了总共108,338,422条原始双端测序读段,经过数据预处理和Trinity软件拼接组装,共获得60,985个非冗余基因,平均长度是614bp,其中有30,488条在Nr,Swiss-Prot,GO,COG,KEGG等数据库中得到了注释。通过对8个胚胎发育时期的转录组数据进行统计分析,我们获得了在整个河蟹胚胎发育阶段基因表达谱,同时,我们利用层级聚类、主成分分析和相关性分析等方法将8个胚胎样品分为4个时期,与河蟹胚胎发育过程中的前期(El-E2),中期(E3-E5),后(E6-E7)以及初孵幼体(Z1)时期相对应。对河蟹相邻发育时期的转录组数据集进行差异基因分析和富集分析,我们发现,发育前期与中期共获得1,123个差异基因,它们大量富集在细胞分化和发育相关过程,有机物的代谢与运输以及内源性刺激反应等方面;从发育中期到后期共获得3,422个差异表达基因,它们在外源性生物降解和代谢,脂类代谢,氨基酸代谢,调控过程,肌肉的发生与发育,肌蛋白和肌原纤维组装等过程中显著富集;从胚胎发育后期到初孵幼体共获得1,729差异基因,它们主要富集到翻译、代谢过程、肌原纤维组装和肌肉发育、有机物的分解过程、转运和蛋白质稳定等过程。此外,我们鉴别出3,303个在整个胚胎发育阶段持续表达的基因,它们在整个发育阶段的表达量变化较为剧烈,表现出一定的时空表达特性。2.将获得的河蟹胚胎发育转录组数据集与河蟹草图基因组,河蟹EST数据以及三种节肢动物黑腹果蝇、家蚕和淡水枝角蚤基因组等数据进行比较,初步分析了河蟹转录组数据的序列信息和特征,为研究河蟹胚胎阶段相关功能基因的序列结构,选择性剪切事件等打下基础。3.根据组学数据,我们鉴别了发育相关基因(包含794条转录本),代谢相关基因(包含1 382条转录本)以及免疫相关的基因(包含662条转录本)等3大类群的基因数据集。其中鉴别出10个母体效应基因:aristaless,bicaudal,Goosecoid,Homeobox protein OTX2-A,nanos,polehole,pumilio,staufen,tudor和vasa;间隔基因(gap genes)共有6个,分别为bicaudal,crocodile,empty spiracles,giant,knirps-like以及krüppel基因;对控基因(pair-rule genes)共有8个,分别为bowel,even skipped,hairy,odd,odd skipped,runt,sloppy paired 2以及sob基因;体节极性基因(segment-polarity genes)共有14个,分别为armadillo,cap'n'collar,cubitus interruptus,frizzled,fused,gooseberry,invected,lines,pangolin,patched,roadkill,smoothened,wnt以及wntless基因;背腹分化基因(dorsoventral patterning genes)共有5个,分别为capicua,pipe,slalom,snail和twist基因;Hox基因Dfd,Dll,labial,Msx,pb,Scr和Ubx等。我们利用胚胎发育转录组数据集鉴别出了刺猬,JAK-STAT通路,MAPK,TGF-β,Toll样受体,NOD样受体、Wnt等调节胚胎形成和发育调控中起着关键作用信号通路,分析了通路中的基因在不同时期的表达情况。此外,我们根据中华绒螯蟹胚胎发育转录组数据中脂质代谢相关基因和的表达信息,绘制了胚胎发育阶段的代谢网路,这些可视化注释信息为深入了解胚胎发育的代谢路径提供了有利的资源。
[Abstract]:As one of the largest groups of invertebrates, crustaceans have rich biological diversity and complex life history. As an important developmental period of crustaceans, the embryonic stage has undergone a series of changes in the regulation of gene development and expression patterns. The study of subbiological mechanism is not sufficient, especially in the animal of Eriocheir sinensis. In this paper, the embryo of Eriocheir sinensis in the Changjiang River system was studied in El, E2, E3, E4, E5, E6, E7 and Z1. A number of important functional genes related to growth and development are obtained, and the molecular basis of important physiological phenomena in this development process is preliminarily analyzed. The main research results of this paper are as follows: 1. based on the Illumina Hiseq2000 sequencing platform, a total of 108338422 original double end sequence reading segments were obtained by data preprocessing and Trinity software spelling. 60985 non redundant genes were obtained, with an average length of 614bp, and 30488 of them were annotated in the Nr, Swiss-Prot, GO, COG, KEGG and other databases. By statistical analysis of the transcriptional data of the 8 embryonic development period, we obtained the gene expression profiles at the stage of embryo development throughout the river crab. At the same time, we used the hierarchy. 8 embryos samples were divided into 4 periods by clustering, principal component analysis and correlation analysis, which corresponded to the early stage (El-E2), middle stage (E3-E5), post (E6-E7) and early hatchle (Z1) period of the embryo development of the river crab. There are 1123 different genes in the early and middle stages, which are enriched in the cell differentiation and development related processes, the metabolism and transport of organic matter and the endogenous stimulus response. From metaphase to late development, 3422 differentially expressed genes are obtained. They are degraded and metabolizing exogenous organisms, lipid metabolism, amino acid metabolism, and modulation. The process, the occurrence and development of muscle, the process of muscle protein and myofibrillar assembly are enriched, and 1729 different genes are obtained from the late embryonic development to the early hatchlings. They are mainly enriched in the process of translation, metabolism, myofibrillar assembly and muscle development, the process of organic matter decomposition, transport and protein stability. We identified 3303 genes that were expressed continuously throughout the development stage of the embryo, and their expression changes in the whole development stage were more intense, showing a certain spatio-temporal expression characteristic.2. will obtain the data set of the crab embryo development transcriptional group and the river crab sketching genome, the EST data of the river crab, and the three species of the orodrosophila melanogaster and the silkworm. Compared with the genomic data of the freshwater horns flea, the sequence information and characteristics of the data of the river crab transcriptional group were preliminarily analyzed. In order to study the sequence structure of the related functional genes in the embryonic stage of the river crab and the selective shear events,.3. based on the data of the group, we identified the related genes (including 794 transcripts), and the metabolic related related genes. Gene data set (including 1382 transcripts) and immune related genes (including 662 transcripts), which identified 10 parent effect genes: aristaless, Bicaudal, Goosecoid, Homeobox protein OTX2-A, nanos, polehole, pumilio, Staufen, Tudor and vasa; the interval gene was 6, respectively. Al, crocodile, empty spiracles, giant, knirps-like and Kr u ppel genes. The control genes (pair-rule genes) are 8, respectively. Frizzled, fused, gooseberry, Invected, lines, pangolin, patched, roadkill, smoothened, Wnt, and Wntless genes. Hedgehog, JAK-STAT pathway, MAPK, TGF- beta, Toll like receptor, NOD like receptor, Wnt and so on regulate the key signaling pathways in the regulation of embryonic formation and development, and analyze the expression of genes in the pathway in different periods. In addition, we are based on the table of lipid metabolism related genes and tables in the embryo development of Eriocheir sinensis. Information has been drawn to map the metabolic network of embryonic development. These visual annotation information provide a favorable resource for understanding the metabolic pathways of embryonic development.
【学位授予单位】:上海海洋大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:S917.4

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