钝顶节旋藻别藻蓝蛋白基因克隆、分析及重组表达蛋白荧光活性的研究

Cloning,Analyzing and Heterologous Expression of allophycocyanin Gene from Arthrospira platensis FACHB314

为探究蓝藻藻胆体核心色素蛋白-别藻蓝蛋白单个亚基的光学活性和功能,本研究克隆了钝顶节旋藻(Arthrospira platensis FACHB314)中的脱辅基别藻蓝蛋白基因apcAB及其亚基基因apcA和apcB。节旋藻基因组中apcAB全长共1056 bp,由apcA和apcB串联组成,中间间隔84 bp。其中,apcA和apcB基因的全长均为486 bp,且均编码161个氨基酸,预测分子量分别约为17.39和17.33 kDa,它们分别编码的α和β亚基均属于Globin_like超级家族,系统进化树分析显示其氨基酸序列在蓝藻和节旋藻中较为保守。将克隆出的亚基基因apcA、apcB分别同前期A.platensis FACHB314中获得的藻蓝胆素合成相关基因ho和pcyA以及色基裂合酶基因(cpcE、cpcF、cpcU、cpcS、cpcT)共同转化至大肠杆菌中,获得重组别藻蓝蛋白亚基菌株E.coli HPEFUSTA、E.coli HPEFUSTB。SDS-PAGE和Western Blot均显示重组菌株中已成功表达别藻蓝蛋白单亚基。荧光检测结果显示,在600 nm波长激发下,两株重组菌株在640 nm处均出现了别藻蓝蛋白特征荧光发射峰。这些结果表明在重组大肠杆菌中表达的脱辅基别藻蓝蛋白的单亚基可以和藻蓝胆素结合,形成有光学活性的别藻蓝蛋白,这为研究蓝藻中藻胆体的组装和功能提供有效的元件,为别藻蓝蛋白在医药、荧光检测方面的应用奠定了基础。

In order to explore the fluorescence activity and function of allophycocyanin subunits which is the core pigment protein of phycobilisome,apo-allophycocyanin gene(apcAB)and its subunit genes apcA and apcB were cloned from Arthrospira platensis FACHB314 in the study.The length of apcAB was 1056 bp,the apcA and apcB genes are linked together with an interval of 84 bp.The apcA and apcB gene have a total length of 486 bp,encode 161 amino acids,and the predicted molecular weight are 17.39 and 17.33 kDa respectively.Theαandβsubunits belong to Globin_like superfamily.Phylogenetic tree analysis showed that their amino acid sequences were conserved in cyanobacteria and Arthrospira platensis.The genes apcA and apcB were transformed into E.coli together with the previously obtained phycocyanobilin synthesis related genes ho,pcyA and chromophore lyase genes cpcE,cpcF,cpcU,cpcS and cpcT from A.platensis FACHB314.The recombinant allophycocyanin subunits strains E.coli HPEFUSTA,E.coli HPEFUSTB were obtained.SDS-PAGE and Western Blot showed that allophycocyanin subunits in the recombinant strain were successfully expressed.Fluorescence detection results showed that under the excitation of 600 nm wavelength,the characteristic fluorescence emission peaks of allophycocyanin appeared at 640 nm in the recombinant strains.The results showed that the recombinant allophycocyanin subunits from A.platensis FACHB314 combined with phycocyanobilin to express allophycocyanin with fluorescence activity in E.coli.The results provide effective elements for study of the assembly and function of phycobilosome of cyanobacteria,and also lay a foundation for the application of allophycocyanin in medicine and fluorescence detection and so on.