摘    要嗜热光合细菌Chloroflexus aurantiacus是一种不放氧的原核生物，对其光合作用反应历程的研究将有助于阐明光合作用的进化和开发利用太阳能提供新思路。其特异捕光天线——绿小体被膜上存在一些蛋白复合物，发挥着重要的功能，CsmM是主要之一。本研究从克隆有关的基因入手，采用分子生物学和蛋白质化学技术对CsmM蛋白展开如下研究：从已完成测序的JGI全基因组中找出CsmM基因序列，运用生物信息学分析方法，对蛋白的基本性质进行分析，设计特异引物，提取细菌基因组DNA作为模板进行PCR扩增，然后进行TA克隆，将阳性重组质粒转化至大肠杆菌BL21（DE3）中进行诱导表达，优化异源表达条件获得高效表达。89783

毕业论文关键词：光合细菌；绿小体；CsmM蛋白；基因克隆； 异源表达

Abstract Chloroflexus aurantiacus is the typical species of thermophilic filamentous green bacteria which did not evolve oxygen during its photosynthesis。 The study on the mechanism of its photosynthetic reaction is important to illustrate the evolution of life on the earth and develop the new methods of solar energy utilization。 The unique light-harvesting antenna in Chloroflexus aurantiacus is called chlorosome。 There are several pigment-protein complexes located in the envelope of chlorosome such as CsmM, which is the crucial component of the light energy transfer system。 

In this project, we began with cloning related target gene, and utilized molecular biology and protein chemistry technologies to do the following experiments: Analysis of genome of Chloroflexus aurantiacus in JGI databank; By using several bioinformatics method, the basic properties of the CsmM protein were analyzed。 Based on the bioinformatics results, the specific primers were designed and used to clone the target gene by PCR with extracted genome DNA as template。 The purified PCR product was ligated by TA cloning method and the positive recombinants were selected and transformed into the expression host cell strain E。coli BL21(DE3)。 Multiple condition screening experiments were carried out to optimize the heterologous expression of the target protein。 

Keyword: Photosynthetic bacteria; Chlorosome; CsmM protein; Gene cloning; Heterologous expression

目   录

一、引言 4

（一）不放氧光合细菌概述 4

1 光合细菌分类 4

2 不放氧光合细菌分类 4

3 光合细菌与植物的光合系统的异同 4

（二）嗜热橙色绿丝菌Chloroflexus aurantiacus概述 4

（三）绿小体概述 5

（四）研究意义和技术路线 6

1 研究意义 6

2 实验基本内容 6

3 技术路线 6

二、被膜蛋白CsmM的基因克隆 7

（一）实验方法 7

1 目的片段PCR扩增 7

2 PCR产物的凝胶回收 8

3 重组质粒pEASY-E2-CsmM-sGFP的构建 8

4 重组质粒pEASY-E2-CsmM-sGFP转化到感源Q于W优E尔A论S文R网wwW.yOueRw.com 原文+QQ75201,8766 受态细胞Trans1-T1 8

5 菌液检测 9

6 重组质粒pEASY-E2-CsmM-sGFP的提取