利用果蝇筛选影响线粒体功能的新基因
发布时间：2019-03-07 20:24
【摘要】：线粒体是真核细胞中普遍存在的细胞器,它参与了许多重要的生理过程。线粒体不仅通过氧化磷酸化为细胞提供大量能量,还调节细胞凋亡过程;此外线粒体也会影响钙离子的稳态和机体衰老过程。结构决定功能,线粒体形态结构的完整性也与它的功能密切相关。线粒体是一个动态的细胞器,它的融合和解聚有利于及时清除损伤的线粒体,避免对细胞产生毒性效应。线粒体功能异常可以导致许多疾病,比如线粒体DNA发生突变可以导致线粒体呼吸链疾病;线粒体蛋白运输机制出现障碍也会导致相关疾病。此外在神经退行性疾病如帕金森病的病人中发现线粒体功能和结构都受到破坏。线粒体的微小结构对它的体内研究造成障碍,但是线粒体通常会在需要能量供给的组织中大量积累比如肌肉组织,因此我们把肌肉组织作为研究线粒体结构功能的载体。果蝇作为一种模式生物,它的生活周期比较短,突变表型也很丰富,比较容易用遗传学、分子生物学和细胞生物学的手段进行操作。果蝇的肌肉组织和哺乳动物的骨骼肌在形态和生理方面有高度的相似性,尤其果蝇胸部的间接飞行肌不仅与哺乳类的骨骼肌在形态类似,而且间接飞行肌中间还有大量管状的线粒体结构,这为研究线粒体形态变化和功能异常提供理想的体内研究模型。本研究中我们把果蝇的运动能力作为筛选线粒体功能异常的首要标准。我们利用肌肉组织特异性表达的Mef2-Gal4果蝇对6972个RNAi果蝇的运动能力进行筛查,初步得到了 141个RNAi品系,与之对应的是139个基因。对初步筛选结果我们在Flybase中进行基因功能分析后挑选出62个RNAi品系进行二次筛选,这次是以果蝇间接飞行肌中肌原纤维间的Mito-GFP变化为主要筛选标准;此外在第二次筛选中,我们把IFMs中肌原纤维形态的变化作为次要筛选标准。综合这两次的筛选结果,我们最终选择表型很明显的CG2508,CG3356去研究它们对线粒体的影响。CG2508,CG3356是E3连接酶,参与蛋白的泛素化过程。蛋白的泛素化是一个酶催化的过程,将泛素链加到相应的靶蛋白上,最终带上泛素标签的靶蛋白可以通过蛋白酶体或溶酶体进而降解。我们从不同的方面分析CG2508,CG3356对线粒体的影响,在线粒体形态方面,在肌肉组织特异性地敲降CG2508,CG3356会引起线粒体形态的改变。在组织学方面,Mef2-Gal4CG2508RNAi和Mef2-Gal4CG3356RNAi果蝇IFMs的形态发生了轻微的损伤,而且Mef2-Gal4CG2508RNAi果蝇的IFMs的损伤是随着年龄的增加而加剧。在ATP水平的检测中,CG2508RNAi、CG3356RNAi的ATP水平都有下降,CG2508RNAi的ATP水平的降低更明显,而且它的ATP水平的降低是是随着年龄的增加而加剧。综上所述,RNAi介导的果蝇筛选的确可以筛选到一些影响线粒体功能的新基因,而这些新基因会影响线粒体形态和功能。但是它们影响线粒体的具体的分子机制可能需要从这些新基因的结构域以及可能与其他一些信号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.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78
，
本文编号：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.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78
，
本文编号：2436430
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