鱼腥藻PCC7120藻红蓝蛋白操纵子A、E和F基因的克隆、表达和活性分析  

CLONING EXPRESSION AND ACTIVITY ANALYSIS OF A,E,F GENES OF ANABAENA PCC 7120 PEC-OPERON

作　　者：孙亚楠[1] 武栋[2] 周明[1] 赵开弘[1] 

机构地区：[1]华中科技大学环境科学与工程学院,武汉430074 [2]华中科技大学生命科学与技术学院,武汉430074

出　　处：《水生生物学报》2004年第3期275-278,共4页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金 (3 0 2 70 3 2 6和 90 2 0 10 0 1)资助

摘　　要：通过PCR技术从鱼腥藻PCC712 0总DNA中扩增藻红蓝蛋白A基因 (pecA)、E基因 (pecE)、F基因 (pecF) ,将pecA、pecE、pecF分别克隆于pBluescript,然后将pecA、pecE、pecF基因分别插入表达载体pET30a进行高效表达。pecA表达产物藻红蓝蛋白α亚基脱辅基蛋白PecA与藻蓝胆素PCB在pecE/pecF表达产物裂合异构酶PecE/PecF催化下进行体外重组 ,产物经提纯后 ,用紫外可见吸收光谱和荧光光谱检测 ,结果表明生成的色素藻胆蛋白具有藻红蓝蛋白α 亚基所特有的光谱性质和可逆光致变色性质。Phycobiliproteins are brilliantly coloured,intensely fluorescent chromoproteins,which consist of apoproteins and covalently bound linear tetra pyrroles (phycobilins) as prosthetic groups. Phycoerythrocyanin (PEC) is an integral component of the phycobilisome (Pbsome) in several species of cyanobacteria. However,unlike other biliproteins, isolated PEC shows a pronounced reversible photochemistry,which is related to the α subunit (α PEC). In this study,the genes pecA , pecE ,and pecF from Anabaena PCC 7120 were cloned with vector pBluescript,yielding plasmids pBlu pec A,pBlu pec E and pBlu pec F, respectively. All constructions were verified by sequencing. These genes were subcloned into vector pET30a using the EcoRV and XhoⅠrestriction sites. pBlu PecA, pBlu PecE and pBlu PecF were cleaved with SmalⅠand Xho Ⅰ, and the released genes were ligated to the large pET30a fragment. The E. coli [strain BL21(DE3)]cells containing recombinant pET30a were grown in Luria Bertani medium at 20℃, and harvested 6h after induction with isopropyl thio β D galactoside(IPTG). His tagged PecA,E and F were purified separately by metal ion chelating affinity chromatography on chelating sepharose charged with Ni 2+ . The reconstitution system consists of PCB,His 6 PecA,His 6 PecE,His 6 PecF,and 2 mercaptoethano1. Incubating over night at room temperature,the product of reconstruction investigated by UV vis absorption and fluorescene spectrophotometry appeared an absorption at 565nm,which is the absorption maximum of integral α PEC containing the PVB chromophore. After irradiation at 570 nm,the band at 565 nm decreased,and a new absorption band arose at 505 nm. Some previous experiments showed that the α PEC synthase consists of two proteins,PecE and PecF,in Mastigocladus laminosus. Under catalysis of the over expressed PecE and PecF,PCB can be conVerted to PVB,and bound covalently to apo α PEC to give native α PEC. The classical phototransformati

关 键 词：鱼腥藻 PCC7120 藻红蓝蛋白 操纵子 基因 克隆 表达 体外重组 裂合异构酶 

分 类 号：Q949.2[生物学—植物学] Q78