【摘要】：甲殼動物作為無脊椎動物最大的類群之一,具有豐富的生物多樣性以及復雜的生活史。而胚胎階段作為甲殼動物重要的發(fā)育時期,其經(jīng)歷了一系列的基因發(fā)育調(diào)控和表達模式的變化,然而,到目前為止,相關(guān)甲殼動物胚胎發(fā)育階段的分子生物學機理研究并不充分,尤其是在中華絨螯蟹等十足目動物中。本文以長江水系中華絨螯蟹胚胎在受精卵(El),卵裂期(E2),囊胚期(E3),原腸期(E4),卵內(nèi)無節(jié)幼體期(E5),眼點期(E6),心跳期(E7)和初孵幼體期(Z1)為研究對象,進行轉(zhuǎn)錄組測序,從中獲得了一批生長發(fā)育相關(guān)的重要功能基因,初步解析了這一發(fā)育過程中重要生理現(xiàn)象的分子基礎(chǔ)。論文的主要研究結(jié)果如下:1.基于Illumina Hiseq2000測序平臺,獲得了總共108,338,422條原始雙端測序讀段,經(jīng)過數(shù)據(jù)預處理和Trinity軟件拼接組裝,共獲得60,985個非冗余基因,平均長度是614bp,其中有30,488條在Nr,Swiss-Prot,GO,COG,KEGG等數(shù)據(jù)庫中得到了注釋。通過對8個胚胎發(fā)育時期的轉(zhuǎn)錄組數(shù)據(jù)進行統(tǒng)計分析,我們獲得了在整個河蟹胚胎發(fā)育階段基因表達譜,同時,我們利用層級聚類、主成分分析和相關(guān)性分析等方法將8個胚胎樣品分為4個時期,與河蟹胚胎發(fā)育過程中的前期(El-E2),中期(E3-E5),后(E6-E7)以及初孵幼體(Z1)時期相對應。對河蟹相鄰發(fā)育時期的轉(zhuǎn)錄組數(shù)據(jù)集進行差異基因分析和富集分析,我們發(fā)現(xiàn),發(fā)育前期與中期共獲得1,123個差異基因,它們大量富集在細胞分化和發(fā)育相關(guān)過程,有機物的代謝與運輸以及內(nèi)源性刺激反應等方面;從發(fā)育中期到后期共獲得3,422個差異表達基因,它們在外源性生物降解和代謝,脂類代謝,氨基酸代謝,調(diào)控過程,肌肉的發(fā)生與發(fā)育,肌蛋白和肌原纖維組裝等過程中顯著富集;從胚胎發(fā)育后期到初孵幼體共獲得1,729差異基因,它們主要富集到翻譯、代謝過程、肌原纖維組裝和肌肉發(fā)育、有機物的分解過程、轉(zhuǎn)運和蛋白質(zhì)穩(wěn)定等過程。此外,我們鑒別出3,303個在整個胚胎發(fā)育階段持續(xù)表達的基因,它們在整個發(fā)育階段的表達量變化較為劇烈,表現(xiàn)出一定的時空表達特性。2.將獲得的河蟹胚胎發(fā)育轉(zhuǎn)錄組數(shù)據(jù)集與河蟹草圖基因組,河蟹EST數(shù)據(jù)以及三種節(jié)肢動物黑腹果蠅、家蠶和淡水枝角蚤基因組等數(shù)據(jù)進行比較,初步分析了河蟹轉(zhuǎn)錄組數(shù)據(jù)的序列信息和特征,為研究河蟹胚胎階段相關(guān)功能基因的序列結(jié)構(gòu),選擇性剪切事件等打下基礎(chǔ)。3.根據(jù)組學數(shù)據(jù),我們鑒別了發(fā)育相關(guān)基因(包含794條轉(zhuǎn)錄本),代謝相關(guān)基因(包含1 382條轉(zhuǎn)錄本)以及免疫相關(guān)的基因(包含662條轉(zhuǎn)錄本)等3大類群的基因數(shù)據(jù)集。其中鑒別出10個母體效應基因:aristaless,bicaudal,Goosecoid,Homeobox protein OTX2-A,nanos,polehole,pumilio,staufen,tudor和vasa;間隔基因(gap genes)共有6個,分別為bicaudal,crocodile,empty spiracles,giant,knirps-like以及krüppel基因;對控基因(pair-rule genes)共有8個,分別為bowel,even skipped,hairy,odd,odd skipped,runt,sloppy paired 2以及sob基因;體節(jié)極性基因(segment-polarity genes)共有14個,分別為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個,分別為capicua,pipe,slalom,snail和twist基因;Hox基因Dfd,Dll,labial,Msx,pb,Scr和Ubx等。我們利用胚胎發(fā)育轉(zhuǎn)錄組數(shù)據(jù)集鑒別出了刺猬,JAK-STAT通路,MAPK,TGF-β,Toll樣受體,NOD樣受體、Wnt等調(diào)節(jié)胚胎形成和發(fā)育調(diào)控中起著關(guān)鍵作用信號通路,分析了通路中的基因在不同時期的表達情況。此外,我們根據(jù)中華絨螯蟹胚胎發(fā)育轉(zhuǎn)錄組數(shù)據(jù)中脂質(zhì)代謝相關(guān)基因和的表達信息,繪制了胚胎發(fā)育階段的代謝網(wǎng)路,這些可視化注釋信息為深入了解胚胎發(fā)育的代謝路徑提供了有利的資源。
[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.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4

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