果蝇中影响dFOXO调控寿命功能的相关基因的鉴定

发布时间：2018-05-19 10:23

本文选题：黑腹果蝇 + 寿命　；参考：《大连医科大学》2016年硕士论文

【摘要】：Foxo作为转录调节因子家族中的一员,可参与多条重要的信号通路,介导下游靶基因的转录表达,从而在细胞分化、代谢和细胞凋亡等多个重要的生物进程中发挥作用。同时,Foxo是IIS(Insulin/IGF)信号通路的关键分子,受到上游Insulin信号级联通路的磷酸化调节,介导下游靶基因的表达,参与生物衰老的过程。关于Foxo调节寿命的上游信号通路研究已有许多,但其转录调控下游靶基因的机制还不清楚。本文利用模式生物果蝇,筛选Foxo转录调控的基因,研究Foxo调控寿命的具体机制。在GAL4/UAS双元表达系统基础上,利用RU486诱导d Foxo基因在果蝇成虫脂肪中特异性过表达,将其与野生型果蝇3号染色体上30株不同基因片段缺失品系果蝇杂交进行遗传筛选,通过Kaplan-Meier生存曲线与野生背景Foxo过表达的果蝇寿命进行比较,根据Log-Rank分析鉴定Foxo的下游靶基因所在的基因片段缺失品系。选择上述实验中影响d Foxo寿命延长的基因片段缺失的果蝇进行重复性试验,并进一步深入筛选基因缺失片段中可能存在的具体基因。在野生型果蝇成虫脂肪中过表达Foxo可以延长果蝇寿命,不同基因片段缺失品系3486、6962,7633、7634、7647、7675、7676、7731、7739、8029、8923、8964、9002、9090、9204、9482、9497、9983、24373、24970、24980、24990、24993、25011、25019、25126、26846、26848、27342、27580果蝇与过表达Foxo果蝇杂交后,发现其中基因片段缺失品系7633、6962、7676、7647、8964、9482、27362和24990的后代雄果蝇均可抑制d Foxo过表达引起的寿命延长的表型,而雌果蝇除7647与9482以外也有同样的表型,其中以24990与7633最为明显;而24993、25011和27580则相反,可在其基础上延长果蝇寿命;3684、7634、7675、7731、7739、8029、8923、9002、9090、9204、9497、9983、24373、24970、24980、26848、25019、25126和26846则对d Foxo过表达引起的寿命延长没有任何影响。继续筛选基因片段缺失品系24990,将其中包含的10株小基因缺失片段品系6164、7645、7646、7647、7932、7970、7972、7974、9087、25690进行杂交筛选,其中9087、7970、7974和25690可以明显抑制d Foxo过表达延长寿命的效果。果蝇Df(3R)Exel6166、Df(3R)Exel8158、Df(3R)ED5610、Df(3R)BSC615基因片段中存在Foxo下游调控靶基因。本研究结果为揭示Foxo调控寿命的基因提供数据支持,为深入了解衰老的本质以及相关疾病的研究提供理论线索。
[Abstract]:As a member of transcription regulator family, Foxo is involved in many important signaling pathways, mediates the transcription and expression of downstream target genes, and thus plays an important role in many important biological processes such as cell differentiation, metabolism and apoptosis. At the same time, Foxo is the key molecule of the insulin / IGF signal pathway, which is regulated by phosphorylation of upstream Insulin signaling cascade pathway, mediates the expression of downstream target gene, and participates in the process of biological senescence. There have been many studies on upstream signaling pathway of Foxo regulation life, but the mechanism of its transcription and regulation of downstream target genes is unclear. In this paper, a model organism, Drosophila melanogaster, was used to screen genes for Foxo transcriptional regulation and to study the specific mechanism of Foxo regulation longevity. On the basis of GAL4/UAS duplex expression system, d Foxo gene was specifically overexpressed in adult fat of Drosophila melanogaster by RU486. It was hybridized with 30 strains of Drosophila melanogaster with different gene fragment deletion on chromosome 3 of Drosophila melanogaster for genetic screening. The survival curve of Kaplan-Meier was compared with that of Drosophila melanogaster overexpressed in wild background Foxo. The gene fragment deletion lines of downstream target gene of Foxo were identified by Log-Rank analysis. Drosophila melanogaster, which affected the prolongation of the life span of d Foxo, was selected for repetitive experiments, and the specific genes that might exist in the deletion fragments were screened further. Overexpression of Foxo in adult fat of wild type Drosophila melanogaster can prolong the life span of Drosophila melanogaster. After crossing with the over-expressed Foxo, the line with different gene fragment deletion, 34866 6962, 763 774, 776 775, 776 775, 776 775, 777 751, is crossed with the over-expressed Foxo. It was found that the male Drosophila melanogaster with gene fragment deletion line 7633, 6962, 767, 776, 747, 8964, 9482, 27362 and 24990 could inhibit the prolongation of the longevity induced by the overexpression of d Foxo, while the female Drosophila also had the same phenotype except for 7647 and 9482, especially 24990 and 7633, among which 24990 and 7633 had the same phenotype. However, on the contrary, 24993C 25011 and 27580 could prolong the lifespan of Drosophila melanogaster on the basis of it, 36847634 / 77731/ 77399 / 80298923900290990 / 94987 / 94987 / 243737 / 2497024980268484 / 2501925126 and 26846 had no effect on the prolongation of life span induced by the overexpression of d Foxo. The gene fragment deletion strain 24990 was continuously screened, and the 10 small gene deletion lines 6164N76455, 76465, 76476, 7932, 7970, 79742, 79742, 90874, 25690 were selected for hybridization. Among them, 9087, 797, 797, 774 and 25690 could significantly inhibit the prolongation of d Foxo overexpression, and the survival time of d Foxo overexpression was significantly inhibited by 9 087, 797, 774 and 25690, respectively. The down-regulation target gene of Foxo was found in the gene fragment of Drosophila melanogaster DfC3RN Exel6166 (DfC666), Exel8158DfC3RN3RD56010 and DfC615 (DfC615). The results of this study provide data support for revealing genes that regulate longevity of Foxo and provide theoretical clues for further understanding of the nature of aging and related diseases.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R339.38

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