摘要：系统获得性抗性(systemic acquired resistance, SAR)是植物抵御病原物侵害的一种机制，信号分子水杨酸(salicylic acid, SA)在其中扮演着重要角色。前人研究发现，病程相关基因非表达子1 (nonexpressor of pathogenesis related genes 1, NPR1)是水杨酸信号通路中非常重要的正调控因子，需要以单体形式进入细胞核激活下游病程相关蛋白(pathogenesis related protein, PR)的基因表达。而入核过程需要核输入载体蛋白(importin, IMP)帮助才能得以完成。PLANTKAP是一类IMP蛋白，但对其在拟南芥中所发挥的功能尚不清楚。实验室前期通过筛选拟南芥imp家族突变体发现，plantkap突变体与水杨酸信号通路有关，且改变了NPR1的亚细胞定位。本文通过克隆拟南芥中NPR1、PLANTKAP、IMPα-3基因，构建基因表达载体，利用酵母双杂交技术进行蛋白互作分析。实验结果表明NPR1、PLANTKAP和IMPα-3之间并不能两两互作，PLANTKAP可能需结合其他转录因子调控水杨酸信号通路。81756

毕业论文关键词：PLANTKAP; NPR1; 酵母双杂交

The importin PLANTKAP contributes to the regulation of disease resistance in Arabidopsis

Abstract：Plants usually induce systemic acquired resistance (SAR) when they are infected by pathogenic microorganisms。 This acquired immunity will be maintained for a long period of time and produce instantaneous broad spectrum defense for the second infection。 Salicylic acid (SA), as a signal molecule, could activate the ankyrin-containing protein NRP1 (nonexpressor of pathogenesis related genes 1) to regulate SAR。 And previous studies have found that NPR1 is in the cytoplasm via the formation of two oligomers; while only the monomer can cross the nuclear envelope to activate downstream pathogenesis related protein (PR) expression。 Importin (IMP) family is essential for this sophisticated nucleocytoplasmic trafficking。 And experiments indicated that PLANTKAP mutant of Arabidopsis thaliana, a member of mutants of the IMP family, was related to the salicylic acid signaling pathway and altered the subcellular localization of NPR1。 So firstly, the genes of NPR1, PLANTKAP and IMPα-3 were cloned to construct vector。 And then yeast two-hybrid system was exerted on the analysis of interaction among these three proteins。 However, the results showed that these three kinds of proteins: PR1, PLANTKAP and IMPα-3, could not interact with each other。 PLANTKAP may be combined with other transcription factors to regulate the signal transduction pathway of salicylic acid。 

Key words: PLANTKAP; NPR1; Yeast two-hybrid system

目  录

摘要1

关键词1

Abstract1

Key words1

引言1

1材料与方法2

1。1实验材料2

1。1。1植物材料及菌株2

1。1。2常用酶2

1。1。3培养基、常用试剂及其配制2

1。2实验方法 3

1。2。1引物设计3

1。2。2拟南芥总RNA的提取3

1。2。3 RT-PCR获得目的基因3

1。2。4回收PCR扩增产物4

1。2。5目的基因及载体的体外连接4

1。2。6转化4

1。2。7重组体的筛选与鉴定4

1。2。8酵母双杂交实验4

2结果与分析5

2。1拟南芥提总RNA琼脂糖凝胶电泳结果5

2。2拟南芥酵母双杂交载体构建 5

2。3 PLANTKAP、NPR1及IMPα-3的互作分析7

3讨论 8

致谢9

参考文献9

拟南芥核输入载体PLANTKAP调控抗病性的机制

真核生物中，由于双层核膜的存在，细胞被分隔为了细胞质和细胞核。其中，细胞质主要是蛋白质合成的场所，而遗传物质的转录以及选择性表达则发生于细胞核内。除了一些小分子物质可以以被动扩散形式进入细胞核，大多数蛋白质出入细胞核实现核内外的物质交换及信号交流需要依靠核质转运相关原件。核输入载体蛋白(importin, IMP)就是一类相对保守的，与核孔选择性运输功能相关的核转运受体蛋白[1-3]，在调控植物生长发育，激素信号转导，环境刺激应答等方面发挥重要作用[4]。它能结合胞质内的核内功能蛋白如转录因子、剪接因子和其他蛋白，通过核孔复合体(nuclear pore complex, NPC)运输到核内[5-7]。