發(fā)布時(shí)間：2018-06-17 11:46

  本文選題：果蠅 + 雄性不育系��； 參考：《安徽師范大學(xué)》2016年碩士論文

【摘要】：干細(xì)胞是一類具有自我更新與分化潛能細(xì)胞類群,該特點(diǎn)決定了干細(xì)胞在生物個(gè)體的胚胎發(fā)育、造血再生、組織修復(fù)等多種生理過程中發(fā)揮重要作用。生殖干細(xì)胞作為干細(xì)胞家族的一員,在生殖器官的發(fā)育、生殖系統(tǒng)的穩(wěn)態(tài)調(diào)節(jié)以及配子發(fā)生等過程中發(fā)揮重要作用。果蠅精巢生殖干細(xì)胞(Germline Stem Cells,GSCs)為探究干細(xì)胞維持與分化的調(diào)控機(jī)理提供了非常理想的研究模型。Gilgamesh(gish)基因編碼一種絲氨酸/蘇氨酸激酶,屬于酪蛋白激酶I(Casein Kinase 1,CK1)家族,主要在神經(jīng)細(xì)胞、生殖細(xì)胞以及肌肉胞中顯著表達(dá),Gish在神經(jīng)膠質(zhì)細(xì)胞遷移、精子發(fā)生過程以及嗅覺學(xué)習(xí)等過程中都發(fā)揮著重要功能。在雄性不育系果蠅的遺傳學(xué)篩選過程中,我們發(fā)現(xiàn),與同期的野生型果蠅相比,果蠅gish基因缺失突變體的精巢生殖干細(xì)胞數(shù)量出現(xiàn)嚴(yán)重缺失。以此為線索,本課題圍繞gish基因調(diào)控精巢生殖干細(xì)胞維持這一科學(xué)問題進(jìn)行了一系列研究。首先,采用免疫組織化學(xué)技術(shù)對(duì)果蠅精巢生殖干細(xì)胞(Germline Stem Cells,GSCs)進(jìn)行標(biāo)記,研究了野生型果蠅Oregon與三組gish基因突變體(gishKG03891/gish04895、gishKG03891/gishMI08417、gish04895/gishMI08417)果蠅的精巢GSCs在羽化后不同天數(shù)的數(shù)量。結(jié)果顯示,野生型果蠅羽化后第1d、10d、20d精巢GSCs的平均數(shù)量分別為8.1個(gè)、7.7個(gè)、6.6個(gè);而三組gish突變體羽化后在這三個(gè)時(shí)間點(diǎn)的GSCs的平均數(shù)目分別為6.5個(gè)、5.6個(gè)、3.7個(gè)。此結(jié)果表明,gish突變體精巢生殖干細(xì)胞的數(shù)目與野生型相比出現(xiàn)了嚴(yán)重缺失,故初步認(rèn)定gish基因突變導(dǎo)致精巢生殖干細(xì)胞缺失。其次,依據(jù)果蠅數(shù)據(jù)庫(www.Flybase.org)中提供的果蠅全基因組信息,合成引物,采用RT-PCR技術(shù)對(duì)果蠅精巢中g(shù)ish不同轉(zhuǎn)錄本的編碼序列進(jìn)行克隆并測(cè)序;采用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)了gish不同轉(zhuǎn)錄本在果蠅精巢中的表達(dá)情況;構(gòu)建了轉(zhuǎn)基因載體并且進(jìn)行顯微注射。結(jié)果如下:(1)測(cè)序得到果蠅精巢中8條gish編碼框的實(shí)際序列。(2)依據(jù)果蠅數(shù)據(jù)庫,克隆所得的8條gish序列中,其中6種gish(B、E、F、I、K、L)擁有共同的翻譯起始點(diǎn),2種gish(A、C)擁有共同的翻譯起始點(diǎn)。熒光定量PCR(q PCR)結(jié)果表明,6種gish(B、E、F、I、K、L)的表達(dá)水平顯著高于2種gish(A、C)的表達(dá)水平(26倍)。此結(jié)果暗示了gishF等6種轉(zhuǎn)錄本在精巢GSCs的維持中承擔(dān)重要的功能。(3)qPCR的結(jié)果也表明,與野生型相比,三組突變體gishKG03891/gish04895、gishKG03891/gishMI08417、gish04895/gishMI08417果蠅中g(shù)ish基因的表達(dá)水平顯著降低。其中,gishKG03891/gishMI08417突變體精巢中g(shù)ish基因的表達(dá)量相對(duì)最低,該突變體的表型也是最強(qiáng),gish基因相對(duì)表達(dá)量高低趨勢(shì)與其突變體表型強(qiáng)弱吻合。(4)借助顯微注射技術(shù),獲得gish轉(zhuǎn)基因果蠅品系。此結(jié)果為下一步基因挽救試驗(yàn)奠定基礎(chǔ)。再者,利用P{UASp-gishF}轉(zhuǎn)基因果蠅品系,通過與P{NosP-gal4}工具株品系果蠅雜交,對(duì)突變體進(jìn)行了表型挽救試驗(yàn)。統(tǒng)計(jì)結(jié)果顯示,三組挽救組gish突變體羽化后第1d、10d、20d精巢GSCs的平均數(shù)量分別為7.0個(gè)、7.0個(gè)、5.8個(gè),恢復(fù)到正常水平。遺傳挽救試驗(yàn)及前期的表型統(tǒng)計(jì)結(jié)果充分表明,gish基因影響果蠅精巢生殖干細(xì)胞的維持。最后,研究了gish突變背景下bam與dad基因的表達(dá)模式。通過GFP抗體免疫熒光染色,bam與dad基因的表達(dá)模式與野生型無明顯差異。該結(jié)果暗示了gish基因不是位于bam及dad基因的上游發(fā)揮作用,由此推測(cè)gish可能是位于bam與dad基因的下游或平行于BMP信號(hào)通路發(fā)揮功能。綜上結(jié)果都表明,gish基因在維持果蠅精巢干細(xì)胞過程中發(fā)揮功能,但其具體調(diào)控機(jī)制以及與其它信號(hào)通路的關(guān)系還有待進(jìn)一步研究。
[Abstract]:Stem cells are a class of cells with self renewal and differentiation potential, which determines that stem cells play an important role in a variety of physiological processes, such as embryonic development, hematopoiesis, and tissue repair, as a member of the stem cell family, the development of reproductive organs, the homeostasis of the reproductive system and the coordination of the reproductive stem cells. Germline Stem Cells (GSCs) provides a very ideal research model,.Gilgamesh (GISH) gene encoding a serine / threonine kinase, which belongs to the family of casein kinase I (Casein Kinase 1, CK1), mainly in God. In the process of glial cell migration, spermatogenesis and olfactory learning, Gish plays an important role in the migration of glial cells, the process of spermatogenesis and the learning of olfactory. In the genetic screening process of the male sterile Drosophila, we found that the sperms of the Drosophila melanogaster Gish gene deletion mutant were compared with the wild type fruit flies in the same period. The number of reproductive stem cells is seriously missing. This is the clue. This subject has conducted a series of studies on the Gish gene regulation of spermary stem cells for the maintenance of this scientific problem. First, the Germline Stem Cells (GSCs) was labeled with immunohistochemical technique, and the wild type Drosophila melanogaster Oregon and three were studied. The number of GISH gene mutants (gishKG03891/gish04895, gishKG03891/gishMI08417, gish04895/gishMI08417) in the spermary of Drosophila melanogaster GSCs in different days after emergence. The results showed that the average number of GSCs of 1D, 10d and 20d in the wild type fruit fly was 8.1, 7.7 and 6.6, respectively, while the three Gish mutants were produced at these three times. The average number of GSCs points was 6.5, 5.6 and 3.7. The results showed that the number of GISH mutant spermary stem cells had a serious loss compared with the wild type. Therefore, it was preliminarily identified that the Gish gene mutation leads to the deletion of the spermary stem cells. Secondly, the whole genome letter of Drosophila fruit fly provided in the Drosophila data base (www.Flybase.org). The encoding sequence of different transcripts of GISH in Drosophila spermary was cloned and sequenced by RT-PCR technique. The expression of GISH different transcripts in the spermary of Drosophila melanogaster was detected by real-time fluorescence quantitative PCR, and the transgenic carrier was constructed and microinjected. The results were as follows: (1) 8 of the fruit fly sperms were sequenced. The actual sequence of the Gish coding frame. (2) in the 8 Gish sequences derived from the Drosophila database, 6 kinds of GISH (B, E, F, I, K, L) have a common translation starting point, and 2 Gish (A, C) have a common translation starting point. The expression level of the 6 kinds of fluorescent quantitative expressions shows that the expression level of the 6 kinds is significantly higher than that of the 2 kinds of expressions. Level (26 times). This result suggests that 6 transcripts, such as gishF, have important functions in the maintenance of the spermary GSCs. (3) the results of qPCR also showed that the expression level of the Gish gene in the three mutant gishKG03891/gish04895, gishKG03891/gishMI08417, gish04895/gishMI08417 Drosophila melanogaster was significantly lower than that of the wild type. GishKG03891/gishMI, among them, gishKG03891/gishMI. 08417 the expression of GISH gene in the mutant spermary is the lowest, and the phenotype of the mutant is the strongest. The trend of the relative expression of the Gish gene is consistent with the mutant phenotype. (4) the Gish transgene causality fly strain is obtained by the microinjection technique. The result is the basis for the next step gene rescue test. Furthermore, the use of P{UASp-gish The F} transgene causality fly strain, through the hybridization with the P{NosP-gal4} tool strain of Drosophila melanogaster, carried out a phenotypic salvage test. The results showed that the average number of 1D, 10d, 20d spermary GSCs in the three groups of rescue groups was 7, 7 and 5.8, respectively, to the normal level. Genetic rescue test and the earlier phenotypic series. The results fully show that the Gish gene affects the maintenance of the spermary stem cells in the Drosophila spermary. Finally, the expression patterns of BAM and dad genes in the Gish mutation background are studied. The expression pattern of the bam and dad genes is not significantly different from the wild type by GFP antibody immunofluorescence staining. The results suggest that the Gish gene is not in the upstream of the bam and dad genes. It is suggested that Gish may be located downstream or parallel to the BMP signaling pathway in the bam and dad genes. The results show that the Gish gene plays a role in the maintenance of Drosophila spermary stem cells, but the specific regulatory mechanism and the relationship with other signaling pathways are still to be further studied.
【學(xué)位授予單位】：安徽師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q492

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