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摘要：细胞自噬是真核细胞内一种物质降解途径，以文持细胞稳态的重要生理过程。该过程通过降解细胞内的组分来文持细胞内的生理平衡帮助细胞度过逆境。细胞自噬在生物体的生长发育、免疫防御、细胞程序性死亡、肿瘤抑制等方面有着非常重要的作用。Dsl1复合体是真核细胞中定位于内质网的大分子多亚基复合体，在高尔基体至内质网的囊泡运输中发挥重要作用。本实验室前期研究结果显示，Dsl1复合体各亚基同时也参与细胞自噬过程，但它们参与细胞自噬的调控机制并不清楚。本研究以酿酒酵母为研究对象，通过酵母双杂交技术筛选与Dsl1复合体亚基可能存在互作的细胞自噬相关蛋白，进而探讨Dsl1复合体参与细胞自噬的分子机制。本研究结果将为阐明Dsl1复合体参与细胞自噬的调控机制提供一定的基础。25628
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毕业论文关键词：细胞自噬；Dsl1复合体；酵母双杂交
Screening of Dsl1 complex subunits interacting with autophagy related proteins in Saccharomyces cerevisiae
Abstract：Autophagy is a substance degradation process in eukaryotic cells that plays an important physiological process to maintain cellular homeostasis. By eliminating obsolete or damaged cytoplasmic materials, autophagy is critical in maintaining intracellular homeostasis under stress conditions. Autophagy plays an important role in biological growth and development, immune defense, cell programmed cell death, tumor suppression and so on. The Dsl1 complex is a large molecule subunit complex localized in the endoplasmic reticulum in eukaryotic cells and plays an important role in the vesicle transport of the Golgi apparatus to the endoplasmic reticulum. Previous studies in our laboratory showed that the subunits of the Dsl1 complex also participated in the process of autophagy, but their mechanisms involved in autophagy were not clear. In this study, Saccharomyces cerevisiae as the research object, through the yeast two hybrid screening and Dsl1 complex subunit may exist autophagy related protein interaction, and to explore the molecular mechanism of Dsl1 complex involved in autophagy. The results of this study will provide a basis for elucidating the mechanism of Dsl1 complex involved in autophagy.
Key words: Autophagy；Dsl1 complex；Yeast two hybrid
目 录
摘要 3
关键词 3
Abstract. 3
Key words 3
引言 3
1 材料与方法 5
1．1 菌株与质粒 5
1．1．1 酵母菌株 5
1．1．2 质粒 6
1．2 酵母总DNA的提取 6
1．3 质粒的提取 6
1．4 DSL1,DSL3和TIP20的克隆 6
1．4．1 高保真酶扩增目的基因片段 6
1．4．2 目的基因及载体的双酶切 7
1．4．3 酶连 8
1．4．4 大肠杆菌感受态的热击转化法转化 8
1．4．5 菌落PCR 8
1．4．6 阳性单克隆菌落质粒的酶切验证 9
1．4．7 重组质粒的测序 9
1．5 酵母转化方法 9
1．6 pACT2-DSL1和pACT2-TIP20的功能检测 9
1．7 酵母菌双杂交 10
2 结果与分析 10
2．1 克隆Dsl1复合体各亚基基因DSL1、DSL3、TIP20至酵母双杂交载体pACT2 10
2．2 pACT2-DSL1、pACT2-TIP20能够分别抑制突变体dsl1-22和tip20-5对高温的敏感性 12
2．3 酵母双杂交筛选Dsl1复合体亚基与细胞自噬相关蛋白之间的互作关系 12