集胞藻PCC6803细菌光敏色素体外重组和光化学活性研究

Study on the Reconstitution in vitro and Photochemical Activities of Phytochrome from the Synechocystis sp. PCC6803

采用聚合酶链式反应 (PCR)从集胞藻PCC680 3总DNA中扩增出细菌光敏色素全片段cph1及cph1(C 43 5) ,克隆于pBluescriptSK(+ ) ,然后插入表达载体pET3 0a进行高效表达。获得的脱辅基蛋白Cph1、Cph1(C 43 5)在合适的缓冲体系下分别与藻蓝胆素 (PCB)进行体外重组。光化学研究表明 ,两种脱辅基蛋白都能与PCB进行正确的重组 ,重组产物表现出 650～ 70 0nm可逆光致变色效应。锌电泳证实得到的细菌光敏色素辅基色素为PCB 。

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 lik e photoreceptors, collectively called bacteriophytochromes, contain an N-termin al domain homologous to the chromophore-binding domain (CBD) of higher plants a nd a C-terminal domain of histidine kinase domain(HKD). Due to their simple str ucture, bacteriophytochromes broaden the view of phytochrome evolution and provi de us with a simple model to investigate phytochrome-mediated light signal in h igher plants.In this report, the bacteriophytochromes from Synechocystis sp. PCC6803 were investigated. The gene cph1 and its fragment cph1(C-435) were isolated from th e Synechocystis sp. PCC6803 genomic DNA by polymerase chain reaction(PCR) us ing specific primers. Then, the genes were cloned with the vector pBluescript, y ielding plasmids pBlu-cph1 and pBlu-cph1(C-435), before they are subcloned wi th the vector pET30, using the EcoRⅤ and XhoⅠ restriction sites . pBl u-cph1, pBlu-cph1(N_435) were cleaved with SmaⅠ 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℃, and harvested 6 h later after induction with isopropyl thio-β-D-galactoside (IP TG). Then, reconstitution systems were em ployed to study the characteristics of the genes. In the reconstitution sy stem, autoassembly of aprotein of phytochrome with PCB was investigated. The chromophore addi^tion 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 (ΔA max /ΔA min ) of the two fragments differed from each other. It was also shown that PCB was covalently bound to apo_protein via Zn 2+ fluoresc ence SDS_PAGE. After irradiation by light of 700nm, the maximum absorption spec trum o f holo_Cph1 was 650nm. The absorption of it after denaturation in the dar k with ur ea in the presence of hydrochloric acid (pH=2) was 660nm, which was si milar with th at of cis_PCB. In addition, after irradiation by light of 650n m, the maximum absorp tion spectrum of holo_Cphl 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 simil ar with that of trans_PCB. The result showed that the photochromism o f phytochrome resulted from the isomerizaation of chromophore (PCB in this re port). The reconstitution of Cph1(C_435) under the same condition supported the conclusion. Fluorescence emission spectrum of the pro^ducts suggested that bac teriophytochrom e structure with cis_PCB was more stable than that with tr ans_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.