【作者】 关翔宇；

【导师】 唐学玺；

【作者基本信息】 中国海洋大学 ， 生态学， 2008， 博士

【摘要】 蓝藻又称蓝细菌(Cyanobacteria),是藻类植物中最原始的一个门类,是无细胞核,具有植物型放氧光合作用的原核生物。藻胆体作为蓝藻捕光的一种超分子蛋白复合体,由不同种类的藻胆蛋白和连接多肽构成,在光合作用能量吸收和传递方面发挥重要的作用。钝顶螺旋藻(Spirulina platensis,Sp)是一种具有很高营养价值的经济型藻类,其中含有的藻蓝蛋白以其特有的营养和保健价值受到广泛的重视。由于藻蓝蛋白所特有的荧光活性且无毒,可用于荧光标记物以及光动力治疗等领域。本论文借助基因工程技术,实现了具有光学活性藻蓝蛋白的组合生物合成。1.首次实现了在大肠杆菌中利用一个载体完成多个基因的组合生物合成,提供了一种方便高效表达和纯化光学活性藻胆蛋白的新策略。从集胞藻PCC6803 (Synechocystis sp. PCC6803,S6)中克隆了五个基因cpcA(S6)、cpcE、cpcF、ho1及pcyA,构建了载体pCDF-cpcA(S6)-cpcE-cpcF, ho1-pcyA (V1),最终获得了具有光学活性的集胞藻PCC6803藻蓝蛋白α亚基holo-α-PC(S6)。2.基于以上策略的成功,进一步组合生物合成具有光学活性和生物学活性的钝顶螺旋藻C-藻蓝蛋白α亚基(rHHPC)。从Sp基因组DNA中克隆基因cpcA(Sp),构建了载体pCDF-cpcA(Sp)-cpcE-cpcF, ho1-pcyA (V2),以重组菌BL21 (DE3)作为研究对象,对rHHPC进行了5L规模的发酵,菌体密度OD600值达到27。初步摸索了发酵条件,优化了发酵工艺,探索了如何减少发酵过程中包涵体的生成,并对重组产物rHHPC的抗氧化活性进行了研究。结果表明rHHPC具有清除羟自由基和过氧化氢自由基的作用,使其成为一种具有开发潜力的抗氧化剂。3.实现具有光学活性钝顶螺旋藻C-藻蓝蛋白β亚基在大肠杆菌中的组合生物合成。将已知的CpcT及CpeS在S6基因组中进行BLASTP,得到具有高度同源性的基因Slr1649及Slr2049。从Sp基因组DNA中克隆C-藻蓝蛋白β亚基并对其结合色基的82位和153位半胱氨酸进行突变,进而构建了两组质粒分别为: pCDF-cpcB(C153A)-slr2049, ho1-pcyA (V3)和pCDF-cpcB(C82I)-slr2049, ho1-pcyA (V5); pCDF-cpcB(C82I)-slr1649, ho1-pcyA (V4)和pCDF-cpcB(C153I)- slr1649, ho1-pcyA (V6)。结果表明转化了V3和V4质粒的大肠杆菌,经诱导表达后分别产生了具有光学活性cpcB (C153A)-PCB和cpcB(C82I)-PCB,证实了Slr2049和Slr1649分别是催化藻蓝蛋白β亚基82位和153位与色基连接的特异性色基裂合酶。此外,分析和比较了几种本实验室构建的重组藻胆蛋白及天然藻胆蛋白的光谱学性质及差异。以上获得的重组蛋白均采用金属螯合亲和层析的方法,建立起快速高效的纯化工艺,对产物分别进行了SDS-PAGE、色素蛋白锌电泳、吸收和荧光光谱扫描等检测。4.首次对蓝藻连接多肽进行比较基因组学和系统进化分析。在25种蓝藻基因组(20种测序已完成,5种正在进行)中,得到了共192条连接多肽基因序列,包括167条与藻胆蛋白相关的连接多肽(Linker polypeptides)基因序列及25条铁氧化还原-NADP+脱氢酶(Ferredoxin-NADP+ oxidoreductase)基因序列。分析了这192条序列的基因结构、基因组上的定位、保守域和多态性,讨论了这些连接多肽的特征与功能。根据连接多肽的系统进化特征将蓝藻连接多肽分成六类,发现多数的连接多肽与藻胆蛋白聚集成簇,并多与藻胆蛋白的亚基、色基及相关的催化酶共享一个启动子。蓝藻连接多肽的产生、分化和消失归因于蓝藻对不同环境选择压力特别是光适应过程中产生的基因复制、水平基因转移或基因丢失。本论文为光学活性藻蓝蛋白的合成、广泛应用以及人工藻胆体的合成奠定了基础,探讨了藻胆蛋白的荧光活性机理,并通过对藻胆体连接多肽系统进化的研究,加深了对蓝藻光系统功能和进化的认识。更多还原

【Abstract】 Cyanobacteria are among the oldest life on earth with the capacity of oxygenic photosynthesis. As a light-harvesting complex, phycobilisomes （PBSs） play a crucial role in the energy absorbing and transferring of photosynthesis in cyanobacteria. PBSs are highly organized complexes, and are composed of various biliproteins and linker polypeptides. A series of researches on PBSs have been done since they have comprehensive application in the fields of food, medicine and biotechnology, which becomes a hotspot in the research of algae. More stable and single-effect PBSs subunits are urgent for wide applications in the above fields.Five genes cpcA（S6）、cpcE、cpcF、ho1 and pcyA were cloned from Synechocystis sp. PCC6803 （S6） genomic DNA. A vector pCDF-cpcA （S6）-cpcE-cpcF, ho1-pcyA （V1） was successfully constructed resulting in combinational biosynthesis of a fluorescent holo-α-phycocyanin （S6） in Escherichia coli BL21. In addition, a strategy in co-expression of multigenes by using one expression vector was presented, which is a promising method to produce lager-scale fluorescent phycobiliproteins （PBPs） in biotechnological applications.Based on former research about optically active holo-α-PC （S6）, the gene cpcA （Sp） cloned from Spirulina platensis （Sp） genomic DNA, and a new vector pCDF-cpcA （Sp）-cpcE-cpcF, ho1-pcyA （V2） was constructed. The vector could play a role to synthesize PCB, apo-PC （Sp）, and lyase CpcE/F （S6） catalyzing the connection of them in E. coli BL21, which finally produced fluorescentα-subunit C-PC of Sp （rHHPC）. The recombinant E. coli BL21 was cultured to OD₆₀₀ = 27 in a 5L fermentor. The fermentation technique was improved to reduce the production of inclusion bodies. Inhibition effect on hydroxyl and peroxyl radicals makes rHHPC as a potent antioxidant.In order to biosynthesize fluorescent holo-β-PC （Sp） in E. coli BL21, special chromophore lyases genes CpeS and CpcT were carried out BLASTP in S6 genome, and two high homologous genes Slr2049 and Slr1649 were obtained. Sites 82 and 153 inβ-PC （Sp） were site-directed mutated. Two groups of co-expression vectors were constructed and transformed into E. coli BL21, which were pCDF-cpcB（C153A）- slr2049, ho1-pcyA and pCDF-cpcB（C82I）-slr2049, ho1-pcyA, pCDF-cpcB（C82I）- slr1649, ho1-pcyA and pCDF-cpcB（C153I）-slr1649, ho1-pcyA. The result indicates that Slr2049 and Slr1649 catalyzed the 82β- and 153β-PC （Sp） linking to the PCB, respectively. Fluorescent cpcB（C153A）-PCB and cpcB（C82I）-PCB were achieved. In addition, the spectroscopy characteristic between several recombinant and natural PBPs were analyzed.All recombinant proteins were purified by metal chelating affinity chromatography, and were analyzed by SDS-PAGE, chromoprotein Zn2+ electrophoresis, spectrum detect and so on.Recent availability of the whole genome database of cyanobacteria provides us a global and further view on the complex structural PBSs. A total of 192 putative linker genes including 167 putative PBSs-associated linker genes and 25 Ferredoxin-NADP+ oxidoreductase （FNR） genes were detected through whole genome analysis of all 25 cyanobacterial genomes （20 finished and 5 in draft）. A phylogenetic analysis based on protein data demonstrates a possibility of six classes of the linker family in cyanobacteria. Emergence, divergence, and disappearance of cyanobacterial linkers among cyanobacterial species were due to speciation, gene duplication, gene transfer, or gene loss, and acclimation to various environmental selective pressures especially light.This study provides a method for the production of the fluorescent phycocyanin in the research of PBSs construction and biotechnological applications, which would helps to find the mechanism of fluorescent and biological activities in PBPs and approach to construct artificial PBSs. Research on cyanobacterial linker family plays an important role in divergence and evolution of cyanobacterial light-harvesting systems.更多还原