盐泽螺旋藻藻蓝蛋白裂合酶CpcS的分子克隆及功能研究  

Molecular cloning and function study of the phycocyanin lyase CpcS from Spirulina subsalsa

作　　者：盛怡 李慧敏 伍贤军 杨红 朱咏莉 李萍萍[1,2] SHENG Yi;LI Hui-min;WU Xian-jun;YANG Hong;ZHU Yong-li;LI Ping-ping(College of Biology and the Environment,Nanjing Forestry University,Nanjing,Jiangsu 210037,China;Collaborative Innovation Center of Sustainable Forestry in Southern China of Jiangsu Province,Nanjing Forestry University,Nanjing,Jiangsu 210037,China)

机构地区：[1]南京林业大学生物与环境学院,江苏南京210037 [2]南京林业大学江苏省南方现代林业协同创新中心,江苏南京210037

出　　处：《农业现代化研究》2019年第6期1048-1055,共8页Research of Agricultural Modernization

基　　金：国家自然科学基金项目(41977354);江苏省自然科学基金青年基金(BK20150884);江苏省高校优势学科建设工程项目(PAPD)~~

摘　　要：为研究盐泽螺旋藻(Spirulina subsalsa)藻蓝蛋白裂合酶CpcS的催化功能,首先通过PCR技术从S. subsalsa FACHB351基因组DNA中扩增藻蓝蛋白裂合酶的编码基因SscpcS,构建表达质粒pCDFDuet-SscpcS,然后再与脱辅基蛋白和色素合成酶表达质粒pETDuet-SscpcB-Ssho1::SspcyA共同转入大肠杆菌BL21(DE3),并经IPTG(Isopropyl β-D-Thiogalactoside,异丙基硫代半乳糖苷)诱导重组合成藻蓝蛋白。PCR产物测序表明SscpcS扩增成功;双酶切检测和SDS-PAGE电泳分析表明质粒pCDFDuet-SscpcS构建成功,且能表达目的蛋白。重组藻蓝蛋白PCB-CpcB细胞产物为深蓝色;纯化后的色素蛋白展现620 nm的最大吸收峰和646 nm的最大荧光发射峰;色素蛋白通过锌离子染色,在紫外线照射下展现明显荧光。该研究成功克隆源自盐泽螺旋藻的藻蓝蛋白裂合酶SsCpcS的编码基因,其表达产物SsCpcS能有效催化藻蓝蛋白的生物合成。此研究为S. subsalsa藻蓝蛋白的重组合成及抗氧化试剂的研制奠定基础,也为探明盐泽螺旋藻中CpcS的催化机理提供科学依据。To study the catalytic function of the phycocyanin lyase CpcS from the Spirulina subsalsa, the gene SscpcS encoding the phycocyanin lyase was amplified from S. subsalsa FACHB351 genomic DNA by using the polymerase chain reaction(PCR)technology. The expression plasmid pCDFDuet-SscpcS was constructed and co-expressed with the genes encoding apo-protein and bilin synthetase in E. coli BL21(DE3). DNA sequencing analysis of PCR products showed that the gene SscpcS was successfully amplified. The double enzyme digestion analysis and SDS-PAGE analysis indicated that the plasmid pCDFDuet-SscpcS was successfully constructed and the target protein was produced. The co-expressed cells were dark blue. The purified recombinant biliprotein exhibited the maximum absorption peak at 620 nm and the maximum fluorescence emission peak at 646 nm. The obvious fluorescence of the recombinant biliprotein was observed under ultraviolet irradiation after Zn2+ staining. The results showed that the gene SscpcS encoding the phycocyanin lyase from the Spirulina subsalsa was cloned successfully and the lyase SsCpcS efficiently catalyzed the biosynthesis of phycocyanin. The results obtained in this research provided a foundation for developing the recombinant phycocyanin antioxidant reagents and a reference for understanding the catalytic mechanism of the CpcS from S. subsalsa.

关 键 词：盐泽螺旋藻 裂合酶 藻蓝蛋白 重组表达 生物合成 

分 类 号：Q71[生物学—分子生物学]