利用果蠅篩選影響線粒體功能的新基因
發(fā)布時(shí)間：2019-03-07 20:24
【摘要】：線粒體是真核細(xì)胞中普遍存在的細(xì)胞器,它參與了許多重要的生理過(guò)程。線粒體不僅通過(guò)氧化磷酸化為細(xì)胞提供大量能量,還調(diào)節(jié)細(xì)胞凋亡過(guò)程;此外線粒體也會(huì)影響鈣離子的穩(wěn)態(tài)和機(jī)體衰老過(guò)程。結(jié)構(gòu)決定功能,線粒體形態(tài)結(jié)構(gòu)的完整性也與它的功能密切相關(guān)。線粒體是一個(gè)動(dòng)態(tài)的細(xì)胞器,它的融合和解聚有利于及時(shí)清除損傷的線粒體,避免對(duì)細(xì)胞產(chǎn)生毒性效應(yīng)。線粒體功能異常可以導(dǎo)致許多疾病,比如線粒體DNA發(fā)生突變可以導(dǎo)致線粒體呼吸鏈疾病;線粒體蛋白運(yùn)輸機(jī)制出現(xiàn)障礙也會(huì)導(dǎo)致相關(guān)疾病。此外在神經(jīng)退行性疾病如帕金森病的病人中發(fā)現(xiàn)線粒體功能和結(jié)構(gòu)都受到破壞。線粒體的微小結(jié)構(gòu)對(duì)它的體內(nèi)研究造成障礙,但是線粒體通常會(huì)在需要能量供給的組織中大量積累比如肌肉組織,因此我們把肌肉組織作為研究線粒體結(jié)構(gòu)功能的載體。果蠅作為一種模式生物,它的生活周期比較短,突變表型也很豐富,比較容易用遺傳學(xué)、分子生物學(xué)和細(xì)胞生物學(xué)的手段進(jìn)行操作。果蠅的肌肉組織和哺乳動(dòng)物的骨骼肌在形態(tài)和生理方面有高度的相似性,尤其果蠅胸部的間接飛行肌不僅與哺乳類(lèi)的骨骼肌在形態(tài)類(lèi)似,而且間接飛行肌中間還有大量管狀的線粒體結(jié)構(gòu),這為研究線粒體形態(tài)變化和功能異常提供理想的體內(nèi)研究模型。本研究中我們把果蠅的運(yùn)動(dòng)能力作為篩選線粒體功能異常的首要標(biāo)準(zhǔn)。我們利用肌肉組織特異性表達(dá)的Mef2-Gal4果蠅對(duì)6972個(gè)RNAi果蠅的運(yùn)動(dòng)能力進(jìn)行篩查,初步得到了 141個(gè)RNAi品系,與之對(duì)應(yīng)的是139個(gè)基因。對(duì)初步篩選結(jié)果我們?cè)贔lybase中進(jìn)行基因功能分析后挑選出62個(gè)RNAi品系進(jìn)行二次篩選,這次是以果蠅間接飛行肌中肌原纖維間的Mito-GFP變化為主要篩選標(biāo)準(zhǔn);此外在第二次篩選中,我們把IFMs中肌原纖維形態(tài)的變化作為次要篩選標(biāo)準(zhǔn)。綜合這兩次的篩選結(jié)果,我們最終選擇表型很明顯的CG2508,CG3356去研究它們對(duì)線粒體的影響。CG2508,CG3356是E3連接酶,參與蛋白的泛素化過(guò)程。蛋白的泛素化是一個(gè)酶催化的過(guò)程,將泛素鏈加到相應(yīng)的靶蛋白上,最終帶上泛素標(biāo)簽的靶蛋白可以通過(guò)蛋白酶體或溶酶體進(jìn)而降解。我們從不同的方面分析CG2508,CG3356對(duì)線粒體的影響,在線粒體形態(tài)方面,在肌肉組織特異性地敲降CG2508,CG3356會(huì)引起線粒體形態(tài)的改變。在組織學(xué)方面,Mef2-Gal4CG2508RNAi和Mef2-Gal4CG3356RNAi果蠅IFMs的形態(tài)發(fā)生了輕微的損傷,而且Mef2-Gal4CG2508RNAi果蠅的IFMs的損傷是隨著年齡的增加而加劇。在ATP水平的檢測(cè)中,CG2508RNAi、CG3356RNAi的ATP水平都有下降,CG2508RNAi的ATP水平的降低更明顯,而且它的ATP水平的降低是是隨著年齡的增加而加劇。綜上所述,RNAi介導(dǎo)的果蠅篩選的確可以篩選到一些影響線粒體功能的新基因,而這些新基因會(huì)影響線粒體形態(tài)和功能。但是它們影響線粒體的具體的分子機(jī)制可能需要從這些新基因的結(jié)構(gòu)域以及可能與其他一些信號(hào)分子的相互作用去研究。
[Abstract]:Mitochondria is a ubiquitous organelle in eukaryotic cells, which is involved in many important physiological processes. The mitochondria not only provide a large amount of energy to the cells through oxidative phosphorylation, but also regulate the cell apoptosis process; in addition, the mitochondria also affect the steady state of the calcium ions and the body aging process. The structural decision function and the integrity of the mitochondrial morphological structure are also closely related to its function. Mitochondria is a dynamic organelle, and its fusion and depolymerization are beneficial to the timely removal of the damaged mitochondria and to avoid the toxic effect on the cells. Abnormal mitochondrial function can lead to a number of diseases, such as mutations in the mitochondrial DNA, which can lead to mitochondrial respiratory chain diseases, and an obstacle to the mitochondrial protein transport mechanism can also lead to related diseases. In addition, mitochondrial function and structure are found to be disrupted in neurodegenerative diseases, such as Parkinson's disease. The microstructure of mitochondria is an obstacle to its in vivo studies, but mitochondria usually accumulate in a large amount of tissue that requires energy supply, such as muscle tissue, so we use muscle tissue as a carrier to study the function of the mitochondrial structure. Drosophila is a kind of model organism, its life cycle is short, the mutation phenotype is also rich, the comparison is easy to operate with the means of genetics, molecular biology and cell biology. the muscle tissue of the fruit flies and the skeletal muscle of the mammal have a high degree of similarity in shape and physiology, especially the indirect flying muscle of the chest of the fly is not only similar to the shape of the skeletal muscle of the mammal, but also a large number of tubular mitochondrial structures in the middle of the indirect flying muscle, This provides an ideal in-vivo study model for the study of mitochondrial morphological changes and functional abnormalities. In this study, we identified Drosophila's ability to exercise as the primary criterion for screening for mitochondrial dysfunction. We screened the motion ability of 6972 RNAi Drosophila by using the Mf2-Gal4 Drosophila, which is specifically expressed by the muscle tissue, and obtained 141 RNAi lines, and 139 genes corresponding to them. After the preliminary screening, we selected 62 RNAi strains for secondary screening after the gene function analysis in the Flybase, which is the main screening standard for the Miteo-GFP change between the myofibrillar fibers in the indirect flying muscle of the Drosophila; and in the second screening, We used the change of myofibrillar morphology in IFMs as a secondary screening criterion. In combination with these two screening results, we finally selected CG2508, CG3356, which had a significant phenotype, to study their effects on mitochondria. CG2508, CG3356 is the E3 ligase and is involved in the ubiquitination of the protein. The ubiquitination of the protein is an enzyme-catalyzed process, and the ubiquitin chain is added to the corresponding target protein, and the target protein with the ubiquitin label can be further degraded by the proteasome or the lysosome. We analyzed the effects of CG2508 and CG3356 on the mitochondria in different aspects. In the form of mitochondria, the specific knockdown of CG2508 and CG3356 in the muscle tissue could cause the change of the mitochondrial morphology. In the histological aspect, the morphology of the Mef2-Gal4CG2508RNAi and the Mef2-Gal4CG3356RNAi Drosophila IFMs has been slightly damaged, and the damage of the IFMs of the Mef2-Gal4CG2508RNAi Drosophila is increased as the age increases. In the detection of ATP level, the ATP level of CG2508RNAi and CG3356RNAi has decreased, and the decrease of ATP level of CG2508RNAi is more obvious, and the decrease of ATP level is increased with the increase of age. In conclusion, RNAi-mediated Drosophila screening can indeed screen a number of new genes that affect the mitochondrial function, and these new genes may affect the mitochondrial shape and function. But their specific molecular mechanisms that affect the mitochondria may need to be studied from the domains of these new genes and possibly with other signal molecules.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q78
，
本文編號(hào)：2436430
[Abstract]:Mitochondria is a ubiquitous organelle in eukaryotic cells, which is involved in many important physiological processes. The mitochondria not only provide a large amount of energy to the cells through oxidative phosphorylation, but also regulate the cell apoptosis process; in addition, the mitochondria also affect the steady state of the calcium ions and the body aging process. The structural decision function and the integrity of the mitochondrial morphological structure are also closely related to its function. Mitochondria is a dynamic organelle, and its fusion and depolymerization are beneficial to the timely removal of the damaged mitochondria and to avoid the toxic effect on the cells. Abnormal mitochondrial function can lead to a number of diseases, such as mutations in the mitochondrial DNA, which can lead to mitochondrial respiratory chain diseases, and an obstacle to the mitochondrial protein transport mechanism can also lead to related diseases. In addition, mitochondrial function and structure are found to be disrupted in neurodegenerative diseases, such as Parkinson's disease. The microstructure of mitochondria is an obstacle to its in vivo studies, but mitochondria usually accumulate in a large amount of tissue that requires energy supply, such as muscle tissue, so we use muscle tissue as a carrier to study the function of the mitochondrial structure. Drosophila is a kind of model organism, its life cycle is short, the mutation phenotype is also rich, the comparison is easy to operate with the means of genetics, molecular biology and cell biology. the muscle tissue of the fruit flies and the skeletal muscle of the mammal have a high degree of similarity in shape and physiology, especially the indirect flying muscle of the chest of the fly is not only similar to the shape of the skeletal muscle of the mammal, but also a large number of tubular mitochondrial structures in the middle of the indirect flying muscle, This provides an ideal in-vivo study model for the study of mitochondrial morphological changes and functional abnormalities. In this study, we identified Drosophila's ability to exercise as the primary criterion for screening for mitochondrial dysfunction. We screened the motion ability of 6972 RNAi Drosophila by using the Mf2-Gal4 Drosophila, which is specifically expressed by the muscle tissue, and obtained 141 RNAi lines, and 139 genes corresponding to them. After the preliminary screening, we selected 62 RNAi strains for secondary screening after the gene function analysis in the Flybase, which is the main screening standard for the Miteo-GFP change between the myofibrillar fibers in the indirect flying muscle of the Drosophila; and in the second screening, We used the change of myofibrillar morphology in IFMs as a secondary screening criterion. In combination with these two screening results, we finally selected CG2508, CG3356, which had a significant phenotype, to study their effects on mitochondria. CG2508, CG3356 is the E3 ligase and is involved in the ubiquitination of the protein. The ubiquitination of the protein is an enzyme-catalyzed process, and the ubiquitin chain is added to the corresponding target protein, and the target protein with the ubiquitin label can be further degraded by the proteasome or the lysosome. We analyzed the effects of CG2508 and CG3356 on the mitochondria in different aspects. In the form of mitochondria, the specific knockdown of CG2508 and CG3356 in the muscle tissue could cause the change of the mitochondrial morphology. In the histological aspect, the morphology of the Mef2-Gal4CG2508RNAi and the Mef2-Gal4CG3356RNAi Drosophila IFMs has been slightly damaged, and the damage of the IFMs of the Mef2-Gal4CG2508RNAi Drosophila is increased as the age increases. In the detection of ATP level, the ATP level of CG2508RNAi and CG3356RNAi has decreased, and the decrease of ATP level of CG2508RNAi is more obvious, and the decrease of ATP level is increased with the increase of age. In conclusion, RNAi-mediated Drosophila screening can indeed screen a number of new genes that affect the mitochondrial function, and these new genes may affect the mitochondrial shape and function. But their specific molecular mechanisms that affect the mitochondria may need to be studied from the domains of these new genes and possibly with other signal molecules.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q78
，
本文編號(hào)：2436430
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