Dong Yi-Ran, Ran Yong, Zhao Kai-Hong, Zhou Ming
School of Environmental Science & Engineering, Huazhong University of Science and Technology, Wuhan 430074, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Mar;20(2):238-44.
Genomic DNA sequence analysis of phytochrome like photoreceptors in a number of bacteria revealed several open reading frames (ORFs) encoding proteins with amino acid sequences homologous to plant phytochromes. The phytochrome like photoreceptors, collectively called bacteriophytochromes, contain an N-terminal domain homologous to the chromophore-binding domain (CBD) of higher plants and a C-terminal domain of histidine kinase domain( HKD). Due to their simple structure, bacteriophytochromes broaden the view of phytochrome evolution and provide us with a simple model to investigate phytochrome-mediated light signal in higher plants. In this report, the bacteriophytochromes from Synechocystis sp. PCC6803 were investigated. The gene cph1 and its fragment cph1 (C-435) were isolated from the Synechocystis sp. PCC6803 genomic DNA by polymerase chain reaction(PCR) using specific primers. Then, the genes were cloned with the vector pBluescript, yielding plasmids pBlu-cphl and pBlu-cph1 ( C-435), before they are subcloned with the vector pET30, using the EcoRV and Xho I restriction sites. pBlu-cph1, pBlu-cph1 (N-435) were cleaved with Sma I and Xho I, and the released genes were ligated to the pET30a fragment. The E. coli [strain BL21 (DE3)] cells containing recombinant pET30a were grown in medium RB at 20 degrees C, and harvested 6 h later after induction with isopropyl thio-beta-D-galactoside (IPTG). Then, reconstitution systems were employed to study the characteristics of the genes. In the reconstitution system, autoassembly of aprotein of phytochrome with PCB was investigated. The chromophore addition was an autocatalytic process. Reconstitution products were red/infrared (R/FR) photochromic, which was similar to that of the phytoehrome in higher plants. How ever, the spectral change ratios (deltaAmax/deltaAmin) of the two fragments differed from each other. It was also shown that PCB was covalently bound to apo-protein via Zn2+ fluoresc ence SDS-PAGE. After irradiation by light of 700 nm, the maximum absorption spectrum o f holo-Cphl was 650nm. The absorption of it after denaturatior in the dark with ur ea in the presence of hydrochloric acid (pH = 2) was 660nm, which was similar with th at of cis-PCB. In addition, after irradiation by light of 650nm, the maximum absorption spectrum of holo-Cph1 was 700nm. The absorption of it after denaturation in the dark with urea in the presence of hydrochloric acid (pH = 2) was 600nm, which was similar with that of trans-PCB. The result showed that the photochromism of phytochrome resulted from the isomerizaation of chromophore (PCB in this report). The reconstitution of Cph1 (C-435) under the same condition supported the conclusion. Fluorescence emission spectrum of the products suggested that bacteriophytochrom e structure with cis-PCB was more stable than that with trans-PCB. The new reconstitution system in this report sets a base for the application of phytochrome as photochromic biomaterials in biosensors. In addition, phytochrome shows great potential in food, cosmetic and biological engineering, etc.
对多种细菌中类光敏色素光感受器的基因组DNA序列分析揭示了几个开放阅读框（ORF），这些开放阅读框编码的蛋白质其氨基酸序列与植物光敏色素同源。这些类光敏色素光感受器统称为细菌光敏色素，包含一个与高等植物生色团结合结构域（CBD）同源的N端结构域和一个组氨酸激酶结构域（HKD）的C端结构域。由于其结构简单，细菌光敏色素拓宽了我们对光敏色素进化的认识，并为我们提供了一个简单的模型来研究高等植物中光敏色素介导的光信号。在本报告中，对集胞藻属PCC6803中的细菌光敏色素进行了研究。使用特异性引物通过聚合酶链反应（PCR）从集胞藻属PCC6803基因组DNA中分离出基因cph1及其片段cph1（C - 435）。然后，将这些基因与载体pBluescript进行克隆，得到质粒pBlu - cphl和pBlu - cph1（C - 435），之后利用EcoRV和Xho I限制性酶切位点将它们与载体pET30进行亚克隆。用Sma I和Xho I酶切pBlu - cph1、pBlu - cph1（N - 435），并将释放的基因连接到pET30a片段上。含有重组pET30a的大肠杆菌[菌株BL21(DE3)]细胞在20℃的RB培养基中生长，并在用异丙基硫代 - β - D - 半乳糖苷（IPTG）诱导6小时后收获。然后，采用重组系统来研究这些基因的特性。在重组系统中，研究了光敏色素蛋白与PCB的自组装。生色团的添加是一个自催化过程。重组产物具有红/红外（R/FR）光致变色特性，这与高等植物中的光敏色素类似。然而，两个片段的光谱变化率（deltaAmax/deltaAmin）彼此不同。通过锌离子荧光SDS - PAGE也表明PCB通过共价键与脱辅基蛋白结合。在700nm光照射后，全Cphl的最大吸收光谱为650nm。在盐酸（pH = 2）存在下用尿素在黑暗中变性后，其吸收值为660nm，这与顺式PCB的吸收值相似。此外，在650nm光照射后，全Cph1的最大吸收光谱为700nm。在盐酸（pH = 2）存在下用尿素在黑暗中变性后，其吸收值为600nm，这与反式PCB的吸收值相似。结果表明，光敏色素的光致变色是由生色团（本报告中的PCB）的异构化引起的。在相同条件下Cph1（C - 435）的重组支持了这一结论。产物的荧光发射光谱表明，具有顺式PCB的细菌光敏色素结构比具有反式PCB的结构更稳定。本报告中的新重组系统为将光敏色素作为光致变色生物材料应用于生物传感器奠定了基础。此外，光敏色素在食品、化妆品和生物工程等领域显示出巨大潜力。
