【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。更多还原

【Abstract】 To study the biosynthesis mechanism of phycocyanin from Arthrospira,gene cpcA for the apoprotein(phycocyanin α 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 hox1 for heme oxygenase 1 that converts heme to biliverdin was cloned from Synechocystis sp.PCC6803.The cpcA,cpcE and cpcF genes were linked to plasmid pACYCDuet-1,while pcyA and hox1 genes were linked to plasmid pET-24a(+).Then the constructed two plasmids were co-transformed into E.coli BL21(DE3).A fluorescent holo-α-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.更多还原