鱼腥藻细菌光敏色素的分子设计和光化学活性

鱼腥藻细菌光敏色素的分子设计和光化学活性 Molecular Design and Photochemical Activities of Bacterial Phytochrome from Anabaena sp.PCC7120 李坚 LI Jian 1 2 first-author 赵开弘 ZHAO Kai-hong 1 2 董依然 DONG Yi-ran 1 2 孙亚楠 SUN Ya-nan 1 2 华中科技大学环境科学与工程学院 华中科技大学环境科学与工程学院 College of Environmental Science and Engineering,Huazhong University of Science and Technology College of Environmental Science and Engineering,Huazhong University of Science and Technology 华中科技大学环境科学与工程学院,湖北,武汉,430074 华中科技大学环境科学与工程学院,湖北,武汉,430074 采用PCR技术从鱼腥藻PCC7120中扩增出细菌光敏色素C端分别缺失234和275个氨基酸的突变基因aphA（C-234）、aphA（C-275）,克隆于pBluescript SK（+）,将前者插入表达载体pET30a进行表达,在后者的N端连接一段跨膜片段（ts）,融合基因转入表达载体pGEMD中进行表达。获得的两种脱辅基蛋白在合适的条件下分别与藻蓝胆素（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 7 00 nm可逆光致变色效应.锌电泳证实与脱辅基蛋白连接的辅基色素为PCB,酸性尿素变性实验证明可逆光致变色效应来源于PCB在不同波长光照下的顺反异构化. Two C terminus-truncated genes of cyanobacterial phytochrome of Anabaena sp.PCC7120,i.e.aphA（C-234）and aphA（C-275） were amplified via PCR.Both gene fragments were cloned into vector pBluescript SK（+）,then the former was inserted into vector pET30a for efficient expression,the latter was N terminus-ligated with a transmembrane sequence from glycophorin A,the fused gene was cloned with vector pGEMD for expression.Proper reconstitution systems are employed for the two kinds of the expressed apo-proteins with phycocyanin（PCB） and phycocyanin（PC）,respectively.Photochemical activities indicated that after the reconstitution reactions they could both form correct reconstitution products.They were red/infrared（R/FR） photochromic.It was also shown that PCB was covalently bound to apo-protein via Zn 2+ fluorescence SDS- PAGE.After irradiation by light of 650 nm and 700 nm,the sample absorption after denaturation in the dark with urea in the presence of hydrochloric acid（pH 2） changed correspondingly.The result showed that the photochromism of the phytochromes resulted from the isomerization of PCB. 细菌光敏色素 可逆光致变色 体外重组 基因克隆 基因表达 bacterial phytochromes photochromism reconstitution in vitro gene clone gene overexpression Q78 国家自然科学基金资助项目（9020100130270326） 2005-06-01 2021-04-01 6