Development of a New Thermostable Photosynthetic Bactria by Ricombinant DNA

为了培育新的耐高温光合细菌，我们研究了光合反应热稳定性的来源，并利用DNA重组技术克隆了光合反应中心基因进行点突变。耐高温光合细菌蛋白质结构的比较（Chloroflevus aurantiacus，Chromatium tepidum）和嗜温菌（Chromatium vinosm，Rhodobacter sphaeroides）的分离和鉴定，为了解其起源提供了一把钥匙。从嗜热紫色硫细菌Chromatium tepidum的全细胞中提取基因组DNA，并对该基因组DNA的结构进行了分析。由限制性内切酶Sau 3Al消化的基因组DNA，通过体外包装和转染至大肠杆菌细胞来产生基因库（由800个菌落组成）。选择了一个编码光合作用初级反应的光合反应中心蛋白的克隆，通过DNA碱基序列分析，对反应中心蛋白进行了纯化和结晶试验，以获得所需的三维结构，为定点突变提高热稳定性奠定基础。

In order to develop a new thermostable photosynthetic bacterium, we have investigated the origin of the thermostability in the photosynthetic reaction, and cloning of the photosynthetic reaction center gene for point mutation by recombinat DNA technique.Comparison of protein structures between thermostable photosynthetic bacteria (Chloroflevus aurantiacus, Chromatium tepidum), and mesophilic ones (Chromatium vinosm, Rhodobacter sphaeroides) gave a key to understand the origin. Thermostabel reaction center protein were revealed to have extra structures to keep their thrtiary structure rigid.Genomic DNA was extracted from whole cell of a thermophilic purple sulfur bacterium Chromatium tepidum. From genomic DNA digested by a restriction enzyme Sau 3Al, a gene bank (consist of 800 colonies) was produced by in vitro packaging and transfection to a Ecoli cell. A clone which encodes the photosynthetic reaction center protein for the primary reaction of the photosynthesis was selected and under analysis of the DNA base sequence.Purification of the reaction center protein and trial for crystallization was carried out in order to get three dimensinal structures which are needed to pin point the place to make point mutation for improving thermal stability.