蓝细菌光敏色素Alr1966GAF2及其突变体的表达与光化学性质研究  

Expression and photochemical properties of cyanobacteriochrome Alr1966GAF2 and its mutants

作　　者：马琼 向建伟 牟若兰 邓腊青 王运 周明[3] Ma Qiong;Xiang Jian-Wei;Mou Ruo-Lan;Deng La-Qing;Wang Yun;Zhou Ming(Key Laboratory of Biologic Resources Protection and Utilization of Hubei Province, Hubei University for Nationalities,Enshi, Hubei 445000, China;College of Biological Science and Technology, Hubei University for Nationalities, Enshi, Hubei 445000, China;Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China)

机构地区：[1]湖北民族大学生物资源保护与利用湖北省重点实验室,湖北恩施445000 [2]湖北民族大学生物科学与技术学院,湖北恩施445000 [3]华中农业大学农业微生物学国家重点实验室,武汉430070

出　　处：《植物科学学报》2020年第4期551-557,共7页Plant Science Journal

基　　金：恩施州科技计划项目(D20190025);国家自然科学基金(21472055);湖北民族大学高水平科研成果校内培育项目(PY20020);湖北民族大学大学生科研训练项目(X201910517274,X201910517231)。

摘　　要：采用PCR技术从鱼腥藻(Anabaena sp.PCC7120)中扩增蓝细菌光敏色素基因片段alr1966gaf2,将alr1966gaf2插入到pET-30a(+)载体中,构建表达质粒pET-alr1966gaf2。最后将Alr1966GAF2与HO1、PcyA在E.coli BL21(DE3)中共表达获得色素蛋白Alr1966GAF2,并对该蛋白的光化学性质进行分析。结果显示,色素蛋白Alr1966GAF2结合色素为藻蓝胆素(phycoerythrobilin,PCB)或藻紫胆素(phycoviolobilin,PVB),在3种不同吸收态15Z-P428 nm、中间态和15E-P514 nm之间具有顺序可逆光效应。通过定点突变技术将DXCF基序中的保守性Cys突变为Ala,获得了突变体Alr1966GAF2(C72A)。将Alr1966GAF2(C72A)与HO1、PcyA共表达,获得色素蛋白Alr1966GAF2(C72A)。研究结果表明Alr1966GAF2(C72A)结合色素为PCB,Alr1966GAF2(C72A)-PCB具有较强的荧光活性,其荧光量子的产率高达0.11。Alr1966GAF2(C72A)不仅能够共价结合PCB,还可以结合胆绿素(Biliverdin,BV),均具有较强的红色荧光活性。To construct pET-alr1966gaf2,a fragment of alr1966gaf2 was amplified by polymerase chain reaction(PCR)from Anabaena sp.PCC7120,and then inserted into pET-30a(+).For over-expression,pET-alr1966gaf2 was transformed into Escherichia coli BL21(DE3)containing pACYC-ho1-pcyA and biliproteins were co-expressed successfully.Results showed that bili-Alr1966GAF2 had a sequential reversible photoconversion in three different states.We also detected red fluorescence reversible photoconversion of Alr1966GAF2 in 15E-P514 nm/15Z-P428 nm forms.Via site-directed mutagenesis,we mutated conserved Cys into Ala in the conserved DXCF motif of Alr1966GAF2,resulting in Alr1966GAF2(C72A).Alr1966GAF2(C72A)-PCB showed strong red fluorescence and high fluorescence quantum yield of 0.11.Furthermore,Alr1966GAF2(C72A)could bind to phycoerythrobilin(PCB)and biliverdin(BV)covalently,with strong red fluorescence.Therefore,as a red fluorescent protein,Alr1966GAF2(C72A)could be further developed as a fluorescent probe and applied in life sciences.

关 键 词：蓝细菌光敏色素 顺序可逆光效应 红色荧光蛋白 胆绿素 

分 类 号：Q946[生物学—植物学]