具荧光活性的节旋藻藻蓝蛋白α亚基在大肠杆菌中的重组表达  

Recombinant Expression of a Fluorescent Phycocyanin Holo-α-subunit from Arthrospira platensis in Escherichia coli

作　　者：衣俊杰[1] 臧晓南[1] 张学成[1] 袁定阳[2] 赵炳然[2] 唐俐[2] 

机构地区：[1]中国海洋大学海洋生命学院,山东青岛266003 [2]国家杂交水稻工程技术研究中心,湖南长沙410125

出　　处：《中国海洋大学学报（自然科学版）》2011年第5期59-62,70,共5页Periodical of Ocean University of China

基　　金：国家转基因生物新品种培育重大专项项目(2008ZX08001-004)资助

摘　　要：为探索节旋藻藻蓝蛋白的生物合成机理,以钝顶节旋藻(Arthrospira platensisFACHB314)基因组DNA为模板克隆了藻蓝蛋白α亚基基因cpcA、催化脱辅基蛋白与色基结合的裂合酶基因cpcE和cpcF,以及催化色基合成的铁氧蛋白氧化还原酶基因pcyA;以Synechocystissp.PCC6803基因组DNA为模板克隆了亚铁血红素氧化酶基因hox1。然后分别将cpcA、cpcE和cpcF基因构建到质粒pACYCDuet-1中,将hox1和pcyA基因构建到质粒pET-24a(+)中,用电击法将二者共同转化E.coliBL21(DE3),经诱导表达得到具有荧光活性的节旋藻藻蓝蛋白α亚基。在590 nm激发波长下,荧光发射峰为637.8 nm,证实了节旋藻自身的裂合酶CpcE和CpcF能够催化色基PCB与藻蓝蛋白脱辅基结合产生有荧光活性的藻蓝蛋白α亚基,为表达有荧光活性的藻蓝蛋白提供理论和实验基础。To study the biosynthesis mechanism of phycocyanin from Arthrospira, gene cpcA for the apoprotein （phycocyanin a subunit）, genes cpcE and cpcF for the heterodimeric lyase that catalyze chromophore attachment, and gene pcyA for 3Z-phycocyanobilin： ferredoxin oxidoreductase that converts biliverdin to PCB were cloned from Arthrospira platensis FACHB314. Gene hozl for heine oxygenase 1 that converts heine to biliverdin was cloned from Synechocystis sp. PCC6803. The cpcA, cpcE and cpcF genes were linked to plasmid pACYCDuet-1, whine pcyA and hoxl genes were linked to plasmid pET-24a（＋）. Then the constructed two plasmids were co-transformed into E. coli BL21 （DE3）. A fluorescent holo-a- phycocyanin was obtained after induction. The fluorescence emission peak of holo-α-phycocyanin was at 637.8 nm （λex=590 nm）, which indicated that CpcE and CpcF of Arthrospira platrensis could catalyze PCB with apoprotein in vivo to produce a fluorescent holo-α-phycocyanin. This study provides theoretical and experimental basis for the expression of a fluorescent phycocyanin.

关 键 词：节旋藻 藻蓝蛋白α亚基基因 裂合酶基因 重组 大肠杆菌 

分 类 号：Q78[生物学—分子生物学]