| 摘要 | 第1-5页 |
| ABSTRACT | 第5-10页 |
| Chapter 1 Introductions | 第10-21页 |
| ·Background | 第10-11页 |
| ·Sulfur uptake and assimilation | 第11-15页 |
| ·Sulfur uptake and assimilation pathway in plants | 第11-12页 |
| ·Sulfate assimilation pathway and cysteine biosynthesis in Escherichia coli | 第12-13页 |
| ·Metabolic model for the uptake and assimilation of sulfur in A.ferrooxidans | 第13-15页 |
| ·ATP sulfurylase | 第15-17页 |
| ·APS (adenosine 5'-phosphosulfate) reductase | 第17-18页 |
| ·Cysteine synthase | 第18-20页 |
| ·The objective and main content of the present study | 第20-21页 |
| ·The objective of the present study | 第20页 |
| ·The main content of the present study | 第20-21页 |
| Chapter 2 Materials and methods | 第21-31页 |
| ·Materials | 第21页 |
| ·Methods | 第21-25页 |
| ·Cloning of the Gene from A.ferrooxidans ATCC23270 | 第21-22页 |
| ·Double Digestion of Vector with Two Restriction Enzymes | 第22页 |
| ·Purification of PCR Product | 第22-23页 |
| ·Double Digestion of PCR Product with Two Restriction Enzymes | 第23页 |
| ·Purification of Digested Vector | 第23页 |
| ·Purification of Digested PCR Product | 第23-24页 |
| ·Ligation Reaction | 第24页 |
| ·Transformation with Electroporation | 第24-25页 |
| ·Screen out the positive colony and sequenced | 第25页 |
| ·Site-Directed Mutagenesis | 第25-27页 |
| ·The mutant primer of the cysH | 第25页 |
| ·The mutant primer of the cysH | 第25-26页 |
| ·Mutant PCR reaction | 第26页 |
| ·Digestion of mutant PCR product with restriction enzymes | 第26页 |
| ·Transformation and Identification of Positive Colony | 第26-27页 |
| ·Expression of Recombinant wild-type and mutant Proteins in E.coli | 第27-28页 |
| ·Purification of wild type and mutant proteins from A.ferrooxidans | 第28页 |
| ·UV-Scanning of the proteins | 第28页 |
| ·EPR Spectra | 第28-29页 |
| ·Iron-sulfur cluster assembly in vitro | 第29页 |
| ·Molecular structure modeling of the monomer adenylylsulfate reductase | 第29页 |
| ·ATP sulfurylase activity assays | 第29-30页 |
| ·APS reductase activity assays | 第30页 |
| ·cysteine synthase activity assays | 第30-31页 |
| Chapter 3 Expression, purification and characterization of ATP sulfu rylases | 第31-36页 |
| ·The study of cysD-1 gene expression difference from A.ferrooxidans ATCC23270 | 第31-32页 |
| ·Cloning of cysD-1 Gene from A.ferrooxidans | 第32-33页 |
| ·Expression and Purification of cysD-1 from A.ferroodoxins | 第33-34页 |
| ·UV-Scanning and Fluorescence of cys D-1 | 第34-35页 |
| ·Conclusions | 第35-36页 |
| Chapter 4 Expression, purification and characterization of APS reductase | 第36-44页 |
| ·The study of cysH gene expression difference from A.ferrooxidans ATCC23270 | 第36-37页 |
| ·Cloning of APS reductase Gene from A.ferrooxidans | 第37-38页 |
| ·Expression and Purification of APS reductase from A.ferrooxidans | 第38-39页 |
| ·UV-Scanning and EPR spectra of APS reductase | 第39-40页 |
| ·Role of conserved cysteines of APS reductase in iron-sulfur cluster binding | 第40-42页 |
| ·Molecular modeling of APS reductase from A.ferrooxidans | 第42页 |
| ·Iron-sulfur cluster assembly from APS reductase | 第42-43页 |
| ·conclusions | 第43-44页 |
| Chapter 5 Expression, purification and characterization of Cysteine synthase | 第44-52页 |
| ·Cloning of OASS Gene from A.ferrooxidans | 第44-45页 |
| ·Expression and Purification of OASS from A.ferroodoxins | 第45-47页 |
| ·UV-Scanning and Fluorescence of OASS | 第47-48页 |
| ·The PLP cofator was ligated active sites of the enzymes | 第48-50页 |
| ·Interraction of serine acetyltransferase(SAT) with O-acetylserine sulfhdrylase | 第50页 |
| ·In summry | 第50-52页 |
| Chapter 6 Conclusions | 第52-53页 |
| Reference | 第53-60页 |
| Thanks | 第60-61页 |
| 硕士期间发表论文 | 第61页 |
| 参加的科研项目及学术活动 | 第61页 |
