本文選題：黑腹果蠅 + 壽命��； 參考：《大連醫(yī)科大學(xué)》2016年碩士論文

【摘要】：Foxo作為轉(zhuǎn)錄調(diào)節(jié)因子家族中的一員,可參與多條重要的信號(hào)通路,介導(dǎo)下游靶基因的轉(zhuǎn)錄表達(dá),從而在細(xì)胞分化、代謝和細(xì)胞凋亡等多個(gè)重要的生物進(jìn)程中發(fā)揮作用。同時(shí),Foxo是IIS(Insulin/IGF)信號(hào)通路的關(guān)鍵分子,受到上游Insulin信號(hào)級(jí)聯(lián)通路的磷酸化調(diào)節(jié),介導(dǎo)下游靶基因的表達(dá),參與生物衰老的過程。關(guān)于Foxo調(diào)節(jié)壽命的上游信號(hào)通路研究已有許多,但其轉(zhuǎn)錄調(diào)控下游靶基因的機(jī)制還不清楚。本文利用模式生物果蠅,篩選Foxo轉(zhuǎn)錄調(diào)控的基因,研究Foxo調(diào)控壽命的具體機(jī)制。在GAL4/UAS雙元表達(dá)系統(tǒng)基礎(chǔ)上,利用RU486誘導(dǎo)d Foxo基因在果蠅成蟲脂肪中特異性過表達(dá),將其與野生型果蠅3號(hào)染色體上30株不同基因片段缺失品系果蠅雜交進(jìn)行遺傳篩選,通過Kaplan-Meier生存曲線與野生背景Foxo過表達(dá)的果蠅壽命進(jìn)行比較,根據(jù)Log-Rank分析鑒定Foxo的下游靶基因所在的基因片段缺失品系。選擇上述實(shí)驗(yàn)中影響d Foxo壽命延長的基因片段缺失的果蠅進(jìn)行重復(fù)性試驗(yàn),并進(jìn)一步深入篩選基因缺失片段中可能存在的具體基因。在野生型果蠅成蟲脂肪中過表達(dá)Foxo可以延長果蠅壽命,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果蠅與過表達(dá)Foxo果蠅雜交后,發(fā)現(xiàn)其中基因片段缺失品系7633、6962、7676、7647、8964、9482、27362和24990的后代雄果蠅均可抑制d Foxo過表達(dá)引起的壽命延長的表型,而雌果蠅除7647與9482以外也有同樣的表型,其中以24990與7633最為明顯;而24993、25011和27580則相反,可在其基礎(chǔ)上延長果蠅壽命;3684、7634、7675、7731、7739、8029、8923、9002、9090、9204、9497、9983、24373、24970、24980、26848、25019、25126和26846則對(duì)d Foxo過表達(dá)引起的壽命延長沒有任何影響。繼續(xù)篩選基因片段缺失品系24990,將其中包含的10株小基因缺失片段品系6164、7645、7646、7647、7932、7970、7972、7974、9087、25690進(jìn)行雜交篩選,其中9087、7970、7974和25690可以明顯抑制d Foxo過表達(dá)延長壽命的效果。果蠅Df(3R)Exel6166、Df(3R)Exel8158、Df(3R)ED5610、Df(3R)BSC615基因片段中存在Foxo下游調(diào)控靶基因。本研究結(jié)果為揭示Foxo調(diào)控壽命的基因提供數(shù)據(jù)支持,為深入了解衰老的本質(zhì)以及相關(guān)疾病的研究提供理論線索。
[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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R339.38

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