筛选酿酒酵母中与Dsl1复合体亚基互作的细胞自噬相关蛋白
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