光合玫瑰菌新型电子传递蛋白ActB亚基的克隆和异源表达

摘要嗜热不产氧光合玫瑰菌Roseiflexus castenholzii的细胞膜中存在一种独特的膜蛋白复合体ACⅢ，它在该细菌的光合电子传递链中取替了其它类型光合生物中的细胞色素bc1或b6f复合体所发挥的作用。ACⅢ复合体由多个亚基组成，含有多个铁硫中心和c型血红素，分布在几种光合细菌和其它细菌中，参与光合电子传递与呼吸电子传递过程。ActB亚基是ACⅢ蛋白复合体的一个关键亚基，其结构和功能仍存许多疑惑。为了深入了解ACⅢ复合体的空间结构及ActB亚基的功能，阐明ACⅢ复合体在电子传递链中的工作机制，我们开展了本项研究。47731

本研究以提取的光合玫瑰菌的基因组DNA为模板，根据JGI数据库中actB基因的序列设计了特异性引物，通过PCR扩增目的片段后，把actB基因直接与新型表达质粒载体pEASY-E2连接，转化到大肠杆菌Trans1-T1进行重组构建体的筛选，用菌落PCR和测序方法确定阳性克隆，然后提取质粒转化到表达宿主细胞大肠杆菌BL21（DE3），用IPTG进行诱导表达，获得了目标蛋白质。本研究将为嗜热光合玫瑰菌ACⅢ蛋白复合体亚基的结构和功能研究提供了基础材料。

毕业论文关键词：光合玫瑰菌； ACⅢ蛋白复合体； ActB亚基；基因克隆；表达与纯化

Abstract The filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii possesses an novel electron transfer protein complex called Alternative Complex Ⅲ(ACⅢ) instead of the cytochrome bc1 or b6f type complex found in other known groups of phototrophs. Alternative Complex Ⅲ function as menaquinone: electron acceptor oxidoreductase activity in the photosynthetic electron transfer chain. It was a multisubunit integral membrane protein complex and contains protein subunits with multiple Fe-S centers and c-type hemes. ACⅢ is found in a perse group of bacteria, including both phototrophic and nonphototrohpic taxa. ACⅢ involved both in aerobic and photosynthetic electron transport chains. ActB is the most important subunit of the ACⅢ complex, there are lots of debates about its structure and role in ACⅢ complex. Evolution of photosynthesis will become clearer when more is understood about the structure and mechanism of the ACⅢ complex.

This study based on the analysis of genome of Roseiflexus castenholzii in JGI database , specific primers were designed to clone the actB gene by PCR . The cloned genes were ligated to new type of expression vector pEASY-E2 . The construct was transformed into the expression host E.coli BL21(DE3) and the target protein was expressed after induction and isolated by affinity chromatography and analyzed by SDS-PAGE and Western Blotting. These results pave the foundation of further research on ACⅢ complex in photosynthetic bacteria.

Key words：Roseiflexus castenholzii；Alternative Complex Ⅲ；ActB subunit；Gene cloning；Expression and purification

第一章引言1

1.1 光合细菌概述.1

1.1.1 光合作用的生物.1

1.1.2 光合细菌的分类.1

1.1.3 光合玫瑰菌Roseiflexus castenholzii2

1.1.4 丝状不产氧光合细菌的光合机制2

1.2 ACⅢ蛋白复合体概述.3

1.2.1 细胞色素bc1或b6f复合体3

1.2.2 ACⅢ蛋白复合体.3

1.2.3 ACⅢ的亚基组成.3

1.2.4 ACⅢ的结构4

1.3 论文研究意义及技术路线..5

1.3.1 实验研究意义..5

1.3.2 实验设计思路..5

1.3.3 技术路线.6 光合玫瑰菌新型电子传递蛋白ActB亚基的克隆和异源表达:http://www.751com.cn/shengwu/lunwen_49925.html