發(fā)布時(shí)間：2018-06-16 14:48

  本文選題：盤基網(wǎng)柄菌 + gp150��； 參考：《華東師范大學(xué)》2005年碩士論文

【摘要】：盤基網(wǎng)柄菌(Dietyostelium discoideum)是一種低等的真核原生生物,在營養(yǎng)豐富的條件下以阿米巴單細(xì)胞形式吞噬細(xì)菌為食,行二分裂方式繁殖生長。一旦食物匱乏,細(xì)胞由自由生活狀態(tài)進(jìn)入多細(xì)胞發(fā)育階段。約100,000個細(xì)胞在趨化信號cAMP的誘導(dǎo)下聚集成一個多細(xì)胞結(jié)構(gòu),形態(tài)發(fā)生和細(xì)胞分化的結(jié)果是形成孢子來幫助有機(jī)體度過惡劣環(huán)境。盤基網(wǎng)柄菌與后生動物有許多共同的細(xì)胞生理生化反應(yīng),如胞漿移動、吞噬作用、趨化性、信號轉(zhuǎn)導(dǎo)以及一些發(fā)育事件如細(xì)胞分類、模式形成和細(xì)胞類型決定等。在其它模式有機(jī)體中,這些細(xì)胞行為和生化反應(yīng)并不都能很好表現(xiàn)出來,因此盤基網(wǎng)柄菌已作為一種獨(dú)特的模式生物來研究細(xì)胞運(yùn)動、細(xì)胞類型分化以及眾多發(fā)育事件的分子機(jī)理。 實(shí)驗(yàn)以從加拿大多倫多大學(xué)引進(jìn)的盤基網(wǎng)柄菌野生型KAX-3細(xì)胞株和突變型AK127(gp150缺失)細(xì)胞株為材料。剝奪食物觸發(fā)多細(xì)胞的發(fā)育后,野生KAX-3細(xì)胞經(jīng)歷趨化性聚集、細(xì)胞丘形成和蛞蝓體階段最后形成子實(shí)體,發(fā)育順利完成。而突變AK127細(xì)胞由于不能表達(dá)gp150蛋白,盡管發(fā)育早期細(xì)胞趨化性聚集,但到疏松聚集階段發(fā)育停滯下來,細(xì)胞的分化被鎖住,最后細(xì)胞解聚。gp150為細(xì)胞丘內(nèi)的形態(tài)發(fā)生建立一個粘附微環(huán)境,同時(shí)它也參與調(diào)節(jié)細(xì)胞類型專一化和分化的信號途徑。gp150是一種細(xì)胞表面膜蛋白,它作為粘附分子,推測介導(dǎo)細(xì)胞之間的信號傳遞,調(diào)節(jié)細(xì)胞內(nèi)部的酶促反應(yīng),開啟發(fā)育所需基因的表達(dá),進(jìn)而推進(jìn)發(fā)育進(jìn)程。突變AK127多細(xì)胞發(fā)育停滯,在基因表達(dá)上必然與野生型KAX-3細(xì)胞存在差異。gp150介導(dǎo)的信號途徑影響了哪些發(fā)育所需基因的表達(dá),以及這些基因在盤基網(wǎng)柄菌發(fā)育中的作用正是本論文的主要研究內(nèi)容。 采用mRNA差異顯示法來分析盤基網(wǎng)柄菌野生型KAX-3細(xì)胞與突變型AK127細(xì)胞在多細(xì)胞發(fā)育階段基因的表達(dá)差異。提取多細(xì)胞發(fā)育重要階段(發(fā)育后12h、14h和16h)的兩種細(xì)胞總RNA,反轉(zhuǎn)錄合成cDNA第一鏈,進(jìn)行不同錨定引物與任意引物組合的差異顯示。瓊脂糖凝膠電泳圖譜上觀察到兩種細(xì)胞間存在明顯的差異表達(dá)條帶。選取5條特征的差異條帶,進(jìn)行回收和克隆,酶切鑒定。Northern雜交檢測,最終確定3個差異片段:d12b、d14a和d16a。片段測序并運(yùn)用NCBI數(shù)據(jù)庫系統(tǒng)對這3個片段分別進(jìn)行核苷酸和氨基酸序列同源性信息查詢。分析結(jié)果表明:d12b與已知基因同源性低,氨基酸序列比對沒有找到同源性高的蛋白;d14a與已知基因同源性低,能在數(shù)據(jù)庫中查找到已翻譯完成的d14a氨基酸序列,通過SMART軟件進(jìn)行蛋白質(zhì)檢索,最終得知d14a序列可能對應(yīng)的蛋白質(zhì)為IgC2;d16a與盤基網(wǎng)柄菌線粒體DNA有較高同源性,氨基酸序列比對發(fā)現(xiàn)它與盤基網(wǎng)柄菌線粒體核糖體蛋白S4(Dd-mtRPS4)有同源性。Dd-mtRPS4在盤基網(wǎng)柄菌單
[Abstract]:Dietyostelium discoideum is a kind of low eukaryotic protozoa which feeds on amoeba single cell phagocytic bacteria and propagates and grows in a dimitotic manner. Once food is scarce, cells move from free living to multicellular development. About 100000 cells were induced by chemotactic signal camp to form a multicellular structure. The result of morphogenesis and cell differentiation was the formation of spores to help the organism through the harsh environment. There are many common cellular physiological and biochemical responses such as cytoplasmic mobility, phagocytosis, chemotaxis, signal transduction and some developmental events such as cell classification, pattern formation and cell type determination. In other model organisms, these cell behaviors and biochemical responses are not well demonstrated, so the Phaeopsis discus has been used as a unique model organism to study cell movement. Cell type differentiation and the molecular mechanism of many developmental events. The wild-type KAX-3 cell lines and mutant AK127Gp150 deletion cell lines from the University of Toronto, Canada, were used as materials. After deprivation of food triggered the development of many cells, the wild KAX-3 cells experienced chemotactic aggregation, cell mound formation and final fruiting body formation in slug stage, and the development of wild KAX-3 cells was completed successfully. But the mutant AK127 cells could not express gp150 protein, although the chemotactic aggregation of the cells at the early stage of development, but at the stage of loose aggregation, the differentiation of the cells was blocked. Finally, cell deaggregation.gp150 establishes an adhesion microenvironment for morphogenesis in the cell colliculus. At the same time, it is involved in regulating the signal pathway of cell type specificity and differentiation. Gp150 is a cell surface membrane protein, which acts as an adhesion molecule. We speculated to mediate the signal transduction between cells, regulate the enzymatic reaction in the cells, turn on the expression of genes needed for development, and then promote the development process. The gene expression of the mutant AK127 cells is stagnant, and the signaling pathway mediated by gp150 may influence the expression of which genes are needed for development, and the gene expression is different from that of the wild-type KAX-3 cells. The role of these genes in the development of Staphylococcus discus is the main research content in this paper. MRNA differential display was used to analyze the difference of gene expression between wild-type KAX-3 cells and mutant AK127 cells. Two kinds of cell total RNAs were extracted at the important stage of multi-cell development (12h ~ 14h and 16h after development). The first strand of cDNA was synthesized by reverse transcription and the difference between different anchored primers and arbitrary primer combinations was displayed. There were obvious differentially expressed bands between the two kinds of cells on agarose gel electrophoresis. Five characteristic differential bands were selected for recovery and cloning. Northern blot was used to identify the three differential fragments: d12bnd14a and d16a. The nucleotide and amino acid sequence homology information of the three fragments were queried by NCBI database system. The results showed that the homology of the two genes was low, and the amino acid sequence alignment did not show that the protein with high homology was of low homology with the known gene, and the translated d14a amino acid sequence could be found in the database. The result of protein retrieval by smart software showed that the possible protein corresponding to d14a sequence was IgC2D16a, which had high homology with mitochondrial DNA of P. dissecta. Amino acid sequence alignment revealed that it was homologous to S4Dd-mtRPS4. Dd-mtRPS4 was found to be homologous to S4Dd-mtRPS4.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q75

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