目录
摘要II
关键词II
AbstractIII
引言
引言1
1材料与方法3
1.1实验材料3
1.1.1 植物材料 3
1.1.2 菌株与载体 3
1.1.3 酶、试剂与化学药品 3
1.2 实验仪器 3
1.3 实验方法 3
1.3.1 拟南芥的种植管理3
1.3.2 质粒的构建4
1.3.3 大肠杆菌感受态的制备与转化4
1.3.4大肠杆菌质粒的提取5
1.3.5 农杆菌感受态的制备与转化 5
1.3.6 萤火虫荧光素酶（LCI）实验6
1.3.7 GST Pulldown验证6
1.3.8 遗传分析12
1.3.9 tctp1/+ 突变体花粉体外萌发12
2 结果与分析14
2.1萤火虫荧光素酶（LCI）实验验证COG复合体亚基与TCTP1的互作14
2.2 GST Pulldown验证COG3亚基与TCTP1的互作15
2.3 tctp1/+ 突变体的鉴定和遗传分析16
2.4 tctp1/+ 花粉管表型观察17
3 讨论18
3.1蛋白质互作实验的单一性问题18
3.2 TCTP1与COG复合体互作的生物学意义18
3.3 遗传学与花粉萌发实验揭示tctp1相关表型18
致谢18
参考文献19拟南芥TCTP1与COG3亚基互作及其在花粉管极性生长中的功能研究
摘 要
在花粉管极性生长的过程中，从高尔基体合成的多糖与细胞壁合成相关的酶类通过囊泡运输到达花粉管的顶端，从而保证了花粉管的正常生长。在这期间，一系列蛋白质正确的相互作用保证着囊泡运输的正确进行。在哺乳动物与酵母中，保守的八亚基复合体COG（Conserved Oligomeric Golgi）复合体参与了高尔基体驻留蛋白的逆向运输与定位。本实验室前期通过IPMS技术发现，拟南芥COG复合体与翻译控制肿瘤蛋白（T *51今日免费论文网|www.51jrft.com +Q: ^351916072* 
ranslationally Controlled Tumor Protein 1, TCTP1）可能存在互作，但是互作的亚基以及互作的生物学意义尚待探索。在此基础上，本课题利用结合萤火虫荧光素酶互补成像实验（Firefly Luciferase Complementation Imaging Assay, LCI）与体外GST Pulldown实验，证明了是COG复合体的COG3亚基与TCTP1互作，而COG复合体的其他亚基并不与TCTP1存在直接的互作。遗传分析和花粉表型观察表明，拟南芥tctp1突变影响了花粉管的正常生长，进而导致雄性不育，该表型与cog3突变花粉的表型类似。因此，TCTP1与COG复合体的互作与花粉管的极性生长相关。
PRELIMINARY STUDY ON THE INTERACTION OF TCTP1 AND COG3 SUBUNIT IN ARABIDOPSIS AND THE FUNCTION OF POLARIZED GROWTH IN POLLEN TUBE
ABSTRACT
During the polarized growth of pollen tube, in order to maintain the normal growth of pollen tube, polysaccharides and enzymes associated with the synthesis of cell walls from Golgi are transported to the top of pollen tube by vesicles. The correct interaction of proteins ensures the proper vesicle transport. In mammalian and yeast, Conserved Oligomeric Golgi (COG) complex participates in the reverse transport and localization of Golgi resident proteins. The previous experimental results by IPMS in our laboratory have found that there perhaps exit the interaction of COG complex and Translationally Controlled Tumor Protein 1 (TCTP1), but the interacting subunit and the biological significance remain to be investigates. On the basis of the previous experimental findings, combining the Firefly Luciferase Complementation Imaging Assay (LCI) and GST Pulldown in vitro, we confirmed that it is COG3 that mediates the interaction of TCTP1 and COG complex, and the other subunits do not interact with TCTP1 directly. Genetic analysis and the phenotype of the pollen tubes reveal that the tctp1 mutation affect the normal growth of pollen tube, leading to the male sterility. This phenotype is similar to the phenotype of cog3 mutant pollen. Consequently, the interaction between TCTP1 and COG complex is associated with the polarized growth of pollen tube.

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