酿酒酵母中SNARE蛋白Use1影响细胞自噬蛋白Atg8定位的研究
毕业论文关键词：SNARE蛋白；Use1；细胞自噬；Atg8
Study on the Location of Autophagy Protein Atg8 Affected by SNARE Protein Use1 in Yeast
Abstract： Autophagy is a physiological response for cells' own survival when they are facing harsh environment. The purpose of this reaction is providing nutrition and energy for themselves under harsh environment through wrapping, transporting and degrading some degradation products. SNARE protein is a key protein of regulating vesicle trafficking and it plays an important role in the fusion process of transport vesicles and target membrane. Recent studies have found that some SNARE proteins regulate the autophagy process simultaneously. This study conducted a research on SNARE protein Use1 located in endoplasmic reticulum and found that autophagy protein Atg8 located abnormally in use1-10AA mutant. But after putting Use1 into use1-10AA mutant, the location of Atg8 restored normally. Besides, the protein Use1 was found that it did not have co-localization with ATG8 directly by checking the location of Use1. This result provides an experimental basis for the study of the function of SNARE protein USE1 in autophagy.
Key words: SNARE protein；use1；autophagy；atg8
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1材料与方法2
1.1实验器材 2
1.1.1菌株2
1.1.2质粒2
1.2主要试剂和方法 2
1.2.1主要试剂2
1.2.2主要仪器2
1.3实验方法3
1.3.1突变体的获得3
1.3.2 检测细胞自噬蛋白Atg8在use1-10AA中的定位3
1.3.2.1整合GFP-ATG8质粒至酵母基因组3
1.3.2.2荧光观察3
1.3.3 USE1基因的克隆3
1.3.3.1克隆USE1至pRS4233
1.3.3.2 pRS423-USE1的功能检测6
1.3.4检测Use1和Atg8的共定位7
1.3.4.1克隆USE1至pRS416-GFP7
1.3.4.2 pRS416-USE1的功能检测10
2结果与分析 10
2.1 GFP-Atg8在use1-10AA中定位异常10
2.2 Use1抑制use1-10AA突变体中的自噬缺陷表型11
2.2.1 克隆USE1至pRS423质粒12
2.2.2 pRS423-USE1的功能检测12
2.2.3 pRS423-USE1能够抑制use1-10AA的细胞自噬缺陷12
2.3 Use1与Atg8间无共定位13
2.3.1 克隆USE1至pRS416-GFP质粒13
2.3.2 Use1与Atg8之间的共定位检测14
3讨论 15
致谢16
参考文献16
酿酒酵母中SNARE蛋白Use1影响细胞自噬蛋白Atg8定位的研究
细胞自噬是真核生物必需的一种高度保守的亚细胞降解途径，在真核生物细胞内细胞自噬被分为三种类型：巨自噬、微自噬和分子伴侣介导的自噬(酵母菌中不存在这种类型)[1]。其中巨自噬是科学家常常所指的自噬类型，它的机制被研究的较为透彻，按照惯例，本文所指的自噬皆为巨自噬。巨自噬指的是真核生物细胞内，一些错误折叠或衰老的蛋白、衰老的细胞器等细胞内的待降解物被一个脂质化的双层膜包裹起来，最终形成一个球状的完整的双层膜结构的自噬体，它在细胞内不同蛋白的作用下将被转运至溶酶体（酵母中为液泡）进行降解。这一过程中，双层膜自噬体的形成是细胞自噬的关键步骤，而双层膜也不是在自噬体形成位置上新合成，最新的研究表明这些双层膜结构可能来自于线粒体膜，内质网膜或高尔基体膜等具有膜结构的细胞器[2]。