摘要在新近发现的光合绿丝菌Roseiflexus castenholzii中不具有该类光合细菌模式种Chloroflexus aurantiacus中存在的外周捕光天线chlorosome（绿小体），所以更适合研究其中的光合电子传递的机理。在R。 castenholzii中存在一种被推测用来代替细胞周质中可溶性电子传递体的蓝铜蛋白Auracyanin（金色蓝素），但目前对于其结构和功能还缺乏相关的实验研究证据。
本文第一部分采用分子生物学的相关方法和手段对光合绿丝菌R。 castenholzii中的全长Auracyanin基因进行了如下研究：（1）对Auracyanin基因进行生物信息学的分析，主要是在NCBI资源网站的各种数据库和分析软件中对其全长DNA进行各种分析。（2）利用试剂盒分离研究物种的总基因组DNA。（3）基于生物信息分析设计特异引物，以分离的总基因组为模板，利用PCR来扩增目的基因后与线性化载体pEASY-E2连接形成重组质粒，并转化至感受态大肠杆菌中。本文第二部分采用蛋白质化学的相关技术对蓝铜蛋白Auracyanin进行了如下研究：（4）利用大肠杆菌表达系统，探索异源表达的最适宜条件，扩大培养条件，最终获得高产量的可溶形式的目的蛋白。（5）通过重力柱亲和层析、离子交换层析和凝胶过滤等蛋白质分离纯化方法，从离心收获的大肠杆菌细胞的超声波破碎液中分离纯化出目标蛋白质。（6）进行Tricine SDS-PAGE和Western Blotting等分析纯化后蛋白的纯度以及鉴定目的蛋白。本文中进行诱导表达的条件和蛋白纯化的方法及条件等可为进一步蛋白的三维结晶奠定坚实的基础。76557
Abstract Roseiflexus castenholzii does not have peripheral light-harvesting complex chlorosome which exists in the model species Chloroflexus aurantiacus as a newly discovered species in filamentous anoxygenic phototrophs, so it is very suitable for researching the mechanism of photosynthetic electron transfer process of filamentous anoxygenic phototrophs。 There is a kind of special blue copper protein Auracyanin which is presumed to instead of soluble electron transport carriers in filamentous anoxygenic phototrophs, but the related experimental research evidences about its structure and function are scarce so far。
In the first part of this project, we use some methods and means of molecular biology to research the full Auracyanin gene in R。 castenholzii。 The investigation include the following experiments: (1) the bioinformatics analysis of Auracyanin gene were conducted, mainly including all kinds of analysis of its full length DNA by some various databases and analysis software in NCBI resource website。 (2) Based on the analysis of bioinformatics, specific primers were designed to amplify target gene by PCR with isolation of the total genome as template。 Then it connected with linear carrier pEASY-E2 to form a recombinant plasmid and transformed into the competent Escherichia coli。 The second part of this study, we utilized protein chemistry technology to research blue copper protein Auracyanin。 The investigation include the following experiments: (4) We explored the most suitable conditions for heterologous expression and expanded culture condition to obtain high yields of soluble forms of protein in E。 coli expression system。 (5) We isolated and purified the target protein from the cell lysate by methods of protein purification such as centrifugation, column affinity chromatography, ion exchange chromatography, and gel filtration。 (6) We used Tricine SDS-PAGE and Western Blotting to analyze the purity of purified target protein。 The expression conditions and methods for protein purification reported in this report set up a robust framework for following protein crystallization and structure analysis。
毕业论文关键词：金色蓝素； 克隆； 表达； 光合绿丝菌
Keywords: Auracyanin; gene clone; heterologous expression; phototrophic bacterium 光合绿丝菌金色蓝素基因的克隆和异源表达:http://www.youerw.com/shengwu/lunwen_87836.html