摘要叶绿体是植物体内进行光合作用的场所，是决定作物光合产量形成的根本因素。酪氨酸水解蛋白酶（Clp）是定位于植物叶绿体内的一种重要的水解蛋白酶复合体，在调控叶绿体的生物合成及植物的生长发育上具有十分重要作用。但目前对植物质体Clp基因功能及相关调控机制的研究才刚起步，特别是在单子叶植物中研究更少。基于此，本研究以水稻为材料，通过BLAST序列比对分析搜索拟南芥Clps各个亚基在水稻中的同源基因，进行体外表达和纯化，利用pull-down方法检测水稻叶绿体Clps各蛋白亚基之间的互作关系，研究水稻叶绿体Clps在叶绿体发育中的调控功能及其互作机制。研究结果如下：47738

1、特异性扩增得到的正确目的基因有OsClp11、OsClp6、OsClp8、OsClp5、OsClp2、OsClp4、OsClp10。其中完成表达载体构建的有OsClp11、OsClp6、OsClp8、OsClp5。

2、通过转化BL21（DE3）诱导表达菌株，获得纯化后的OsClp11-GST、OsClp11-MBP、OsClp6-GST和OsClp5-MBP融合蛋白，用于蛋白互作实验。

3、Pull-down实验中我们将OsClp11、OsClp6和OsClp5蛋白两两互作，结果显示这三种蛋白在体外均有相互作用。本实验初步验证了水稻叶绿体中Clps蛋白亚基组分及其互作关系。

Abstract

Chloroplasts is the place of photosynthesis in plants, and it is also the fundamental factors to determine the formation of the photosynthetic crop yields. Clp is an important proteolytic enzyme complex which locates in chloroplast. Besides, it plays a very important role in regulating the biosynthesis and development of chloroplasts. But the current study of plant plastid Clps functions and regulation mechanisms has just started, especially in monocots. In this study, we searched Rice Genome database (http://rice.plantbiology.msu.edu/) for sequences similar to those of the Arabidopsis Clps subunits by BLAST program. Then, we make expression and purification in vitro, and utilize pull-down to detect the interaction between each protein subunits of rice chloroplast Clps. Furthermore, it can contribute to understanding the functions of Clps in chloroplast development and their interaction mechanism. The results are as follows: 

1. We isolated the putative orthologs of rice OsClp1, OsClp2, OsClp4, OsClp5, OsClp6, OsClp8, OsClp9 and OsClp11 based on sequence homology analysis. The prokaryotic expression vectors for the OsClp5, OsClp6, OsClp8 and OsClp11 protein were successfully constructed. 

    2. We purified the fusion protein of OsClp11-GST, OsClp11-MBP, OsClp6-GST and OsClp5-MBP domain with GST（MBP）tag. 

    3. In Pull-down experiments, we make the OsClp11, OsClp6 and OsClp5 protein pairwise interaction, the results show that the three proteins have interaction in vitro. The experiment preliminarily verified the protein subunit component of OsClps and its interaction relationship.

毕业论文关键词：酪氨酸水解蛋白酶；载体构建；诱导表达；蛋白互作

Keyword:  Clp; vector construction; inducible expression; Protein interaction

 目    录

引言 4

1 实验材料 4

1.1水稻材料 4

1.2载体和菌株 5

2 实验方法 5

2.1水稻总RNA的提取 5

2.2 cDNA反转录实验 5

2.3大肠杆菌感受态细胞的制备 6

2.4 Clps蛋白酶亚基基因表达载体构建 6

2.4.1目的基因的PCR扩增 7

2.4.2琼脂糖胶回收