果蝇gish基因影响果蝇精巢生殖干细胞维持的研究

发布时间：2018-06-17 11:46

本文选题：果蝇 + 雄性不育系　；参考：《安徽师范大学》2016年硕士论文

【摘要】：干细胞是一类具有自我更新与分化潜能细胞类群,该特点决定了干细胞在生物个体的胚胎发育、造血再生、组织修复等多种生理过程中发挥重要作用。生殖干细胞作为干细胞家族的一员,在生殖器官的发育、生殖系统的稳态调节以及配子发生等过程中发挥重要作用。果蝇精巢生殖干细胞(Germline Stem Cells,GSCs)为探究干细胞维持与分化的调控机理提供了非常理想的研究模型。Gilgamesh(gish)基因编码一种丝氨酸/苏氨酸激酶,属于酪蛋白激酶I(Casein Kinase 1,CK1)家族,主要在神经细胞、生殖细胞以及肌肉胞中显著表达,Gish在神经胶质细胞迁移、精子发生过程以及嗅觉学习等过程中都发挥着重要功能。在雄性不育系果蝇的遗传学筛选过程中,我们发现,与同期的野生型果蝇相比,果蝇gish基因缺失突变体的精巢生殖干细胞数量出现严重缺失。以此为线索,本课题围绕gish基因调控精巢生殖干细胞维持这一科学问题进行了一系列研究。首先,采用免疫组织化学技术对果蝇精巢生殖干细胞(Germline Stem Cells,GSCs)进行标记,研究了野生型果蝇Oregon与三组gish基因突变体(gishKG03891/gish04895、gishKG03891/gishMI08417、gish04895/gishMI08417)果蝇的精巢GSCs在羽化后不同天数的数量。结果显示,野生型果蝇羽化后第1d、10d、20d精巢GSCs的平均数量分别为8.1个、7.7个、6.6个;而三组gish突变体羽化后在这三个时间点的GSCs的平均数目分别为6.5个、5.6个、3.7个。此结果表明,gish突变体精巢生殖干细胞的数目与野生型相比出现了严重缺失,故初步认定gish基因突变导致精巢生殖干细胞缺失。其次,依据果蝇数据库(www.Flybase.org)中提供的果蝇全基因组信息,合成引物,采用RT-PCR技术对果蝇精巢中gish不同转录本的编码序列进行克隆并测序;采用实时荧光定量PCR技术检测了gish不同转录本在果蝇精巢中的表达情况;构建了转基因载体并且进行显微注射。结果如下:(1)测序得到果蝇精巢中8条gish编码框的实际序列。(2)依据果蝇数据库,克隆所得的8条gish序列中,其中6种gish(B、E、F、I、K、L)拥有共同的翻译起始点,2种gish(A、C)拥有共同的翻译起始点。荧光定量PCR(q PCR)结果表明,6种gish(B、E、F、I、K、L)的表达水平显著高于2种gish(A、C)的表达水平(26倍)。此结果暗示了gishF等6种转录本在精巢GSCs的维持中承担重要的功能。(3)qPCR的结果也表明,与野生型相比,三组突变体gishKG03891/gish04895、gishKG03891/gishMI08417、gish04895/gishMI08417果蝇中gish基因的表达水平显著降低。其中,gishKG03891/gishMI08417突变体精巢中gish基因的表达量相对最低,该突变体的表型也是最强,gish基因相对表达量高低趋势与其突变体表型强弱吻合。(4)借助显微注射技术,获得gish转基因果蝇品系。此结果为下一步基因挽救试验奠定基础。再者,利用P{UASp-gishF}转基因果蝇品系,通过与P{NosP-gal4}工具株品系果蝇杂交,对突变体进行了表型挽救试验。统计结果显示,三组挽救组gish突变体羽化后第1d、10d、20d精巢GSCs的平均数量分别为7.0个、7.0个、5.8个,恢复到正常水平。遗传挽救试验及前期的表型统计结果充分表明,gish基因影响果蝇精巢生殖干细胞的维持。最后,研究了gish突变背景下bam与dad基因的表达模式。通过GFP抗体免疫荧光染色,bam与dad基因的表达模式与野生型无明显差异。该结果暗示了gish基因不是位于bam及dad基因的上游发挥作用,由此推测gish可能是位于bam与dad基因的下游或平行于BMP信号通路发挥功能。综上结果都表明,gish基因在维持果蝇精巢干细胞过程中发挥功能,但其具体调控机制以及与其它信号通路的关系还有待进一步研究。
[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.
【学位授予单位】：安徽师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q492

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