【作者】 付学军；

【导师】 杨官品； 孙力；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 多管藻(Polysiphonia urceolata Grev)是海洋大型红藻之一,红藻的藻胆体以超分子复合体形式附着在类囊体膜上作为光系统Ⅱ(photosystemⅡ, PSⅡ)的捕光色素蛋白复合体为PSⅡ传递光能。目前,光合作用光反应机制研究以原核蓝藻、绿色植物等为主要研究对象,捕光色素复合体研究主要在蓝藻、绿藻及高等植物中进行。红藻色素—蛋白复合体研究远落后于蓝藻、绿藻及高等植物。比较深入的研究工作主要在单细胞红藻-紫球藻(Porphyridium cruentum)中进行。红藻藻蓝蛋白(phycocyanin, PC)和别藻蓝蛋白(allophycocyanin, AP)研究更少。红藻藻胆体特征对深入认识其结构、功能及光系统进化非常重要。本研究围绕多管藻藻胆体的分离纯化方法及藻胆蛋白的性质等进行了研究。采用两次蔗糖密度超速离心法分离纯化了多管藻藻胆体,确立了高效制备多管藻藻胆体的方法；利用层析法及非变性聚丙烯酰胺凝胶电泳分离纯化了多管藻藻胆体中含量较高的藻红蛋白(phycoeryanin, PE)和含量较低的藻蓝蛋白和别藻蓝蛋白；研究了纯化的藻胆蛋白的光谱特性、等电点等,提出了藻胆蛋白在藻胆体中的聚合模式。主要研究内容和结果如下：1.多管藻藻胆体分离纯化利用超声破碎,增溶,两次蔗糖密度梯度超速离心分离纯化藻胆体。结果表明以终浓度为2%(v/v)的NP-40增溶1.5 h,用1.0mol/L-2.0mol/L蔗糖密度和1.0mol/L蔗糖进行两次蔗糖密度梯度超速离心(138000×g,3.5h),获得多管藻的完整藻胆体(F675/F576=5.25),多管藻完整的藻胆体粒径在210 nm左右。2.藻胆蛋白分离纯化比较了分离纯化多管藻R-藻红蛋白(R-PE)的不同方法。结果表明先经过Sephadex G-150分子筛柱层析,再经离子交换柱层析是纯化多管藻藻胆体中R-PE的最有效手段,可使其A565/A280=4.95。多管藻R-藻蓝蛋白(R-PC)经过Sephadex G-150分子筛柱层析和离子交换柱层析后,再经过非变性聚丙烯酰胺电泳(7%(w/v),pH7.5的分离胶,3%(w/v),pH5.5的浓缩胶)纯化,使R-PC的A618/A280=5.25。R-PE和R-PC离子交换柱层析的洗脱条件分别是：R-PE采用0-400 mmol/L的NaCl溶液(25 mmol/L的PBS缓冲液配制)共500 ml进行梯度洗脱；R-PC用50-400 mmol/L的NaCl溶液(25 mmol/L的PBS缓冲液配制)共500 ml线性梯度洗脱。R-PC离子交换后,分别用400 mmol/LNaCl溶液和1.5 mol/L NaCl溶液洗脱离子交换柱,又洗脱下两个组分,分别进行非变性聚丙烯酰胺电泳(5%(w/v),pH7.5的分离胶,4%(w/v),pH5.5的浓缩胶),可分离出别藻蓝蛋白。3.藻胆蛋白的特征分析对纯化的R-PE, R-PC和AP进行了光谱性质,等电点,分子量等分析。结果如下：R-PE的等电点在pH4.7左右；分子量为247kDa; SDS-PAGE的结果显示R-PE有α,β,γ,γ’四种亚基(SDS-PAGE的条件为13-21%(w/v),pH9.0的分离胶和4%(w/v),pH6.8的浓缩胶)；R-PE有两种不同的六聚体形式(α17.6β19.2)3γ29.5(α17.6β19.2)3和(α17.6β19.2)3γ31.0(α17.6β19.2)3,两种聚合体的等电点非常接近；变性等电聚焦电泳结果显示R-PE亚基的等电点在pH5.0-5.8之间,α/β的等电点为pH5.0和pH5.8。R-PC的等电点在pH5.7；SDS-PAGE的结果表明R-PC有α、β和β’三种亚基,没有γ亚基和无色多肽(SDS-PAGE的条件为12%-21%(w/v),pH8.8的梯度分离胶和4%(w/v),pH6.8的浓缩胶)；R-PC两种不同的六聚体形式(α17.5β21.3)6和(α17.5β22.6)6；R-PC的α/β亚基的等电点为pH5.1和pH5.2。AP的等电点在pH5.5, SDS-PAGE的结果表明AP有α、β两种亚基(SDS-PAGE的条件为13%(w/v),pH 9.5的分离胶和4%(w/v),pH6.8的浓缩胶)；经两次非变性电泳透析得到的AP1和由400 mmol/L NaCl洗下组分经非变性电泳得到的AP2有两种无色多肽,它们都有两种聚合体形式(α17β18.5)3L1和(α17β18.5)3 L2,由1.5 mol/L NaCl洗下组分经非变性电泳得到的AP3没有无色多肽,只有一种聚合体形式(α17β18.5)3。研究结果为深入了解大型红藻藻胆体中藻胆蛋白特征、藻胆体结构及其与蓝藻、单细胞红藻藻胆体的差异；为藻类叶绿体进化,光合作用机理及光系统的微观结构研究提供有价值的研究基础,为海洋红藻的应用提供理论依据。更多还原

【Abstract】 Polysiphonia urceolata is one of the marine red macro algae. Phycobilisomes of red algae are in the form of macromolecular complexes attached to the stromatic side of the thylakoid membrane, which harvest the sunlight energy and transfer it to photosystemⅡ(PSⅡ).At present, prokaryotic cyanobacteria and green plants have been the main objects of study on the light reaction center of photosynthesis mechanism. Divers studies have been carried out using cyanobacteria, green algae and high plants as materials. The research of pigment-protein complexes of red algae is far behind those of cyanobacteria, green algae and high plants. In red algae, studies are mainly in unicellular Porphyridium cruentum. Report on the structure and function of phycobilisomes of multicellular red algae is scarce. Research about phycocyanin (PC) and allophycocyanin (AP) is even less. Phycocyanin and allophycocyanin are the components of the phycobilisomes, and their content is low in phycobilisomes. Study of phycobilisome composition of red algae, is very meaningful to the deep understanding of the structure and function of phycobilisome and evolution of photosynthetic systems.This study focuses on the separation and purification method of phycobilisomes from P. urceolata and the properties and the characteristics of phycobiliprotein. A two step sucrose density ultracentrifugation method has been used to separate and purify the phycobilisomes of P. urceolata. This is an efficient method of P. urceolata phycobilisomes preparation. The phycobiliprotein, phycoeryanin (PE), PC and AP of phycobilisomes from P. urceolata were separated and purified by combanation of chromatographies and native polyacrylamide gel electrophores. Spectral properties, isoelectric point and strctural characteristics of phycobiliproteins were studied. The main contents and results of the study are as follows: 1. Separation and purification of phycobilisomes from P. urceolataThe use of ultrasonication, solubilization and two-step sucrose density gradient ultracentrifugation purified phycobilisomes. The results showed that solubilization with 2% NP-40 (final concentration, v/v) for 1.5 h and then a two-step ultra-centrifugation (138,000×g,3.5 h) with 1.0-2.0 mol/L sucrose density gradient first and 1.0 mol/L sucrose solution later can obtain phycobilisomes with highly pure (F675/F576=5.25) and complete. Diameter of the phycobilisomes is about 21 Onm.2. Purification of phycobiliprotein from phycobilisomesComparisonat different isolation and purification methods of R-PE found that Sephadex G-150 column chromatography in combination with ion exchange chromatography is effective for purifing phycoerythrin of phycobilisomes. A565/A280 of the purified R-PE is arrived at 4.95.R-PC was purified by native polyacrylamide gel electrophoresis with 7%(w/v) of separation gel (pH7.5) and 3%(w/v) of stack gel (pH5.5) after purified by the Sephadex G-150 column chromatography and ion exchange column chromatography. A618/A280 of the purified R-PC is arrived at 5.25.The elution conditions of ion exchange chromatography are that R-PE is eiuted by NaCl solution of 0-400 mmol/L linear gradient (25 mmol/L of PBS buffer preparation) with 500ml volumn and R-PC is eiuted by L NaCl solution of 50-400 mmol/L linear gradient (25mmol/L of PBS buffer preparation) with 500ml volumn. After R-PC was eluted from ion exchange column, AP can be isolated by native polyacrylamide gel electrophoresis with 5%(w/v) of separation gel (pH7.5) and 4%(w/v) of stack gel (pH5.5) from components which eluted by 400mmol/LNaCl andl.5mol/LNaCl from the ion exchange column respectively.3. Analysis of phycobiliprotein of phycobilisomesSpectral properties, isoelectric point, structure characteristic of purified R-PE, R-PC and AP have been researched. The results are as follows:The isoelectric point of R-PE is about pH4.7; molecular weight is 247kDa; R-PE contains four kinds of subunitsα,β,γ,γ’by SDS-PAGE with 13-21%(w/v) separating gel (pH9.0) and 4%(w/v) stacking gel(pH6.8). It has two different hexamer forms (α17.6β19.2)3γ29.5 (α17.6β19.2)3 and (α17.6β19.2)3γ31.0 (α17.6β19.2)3 and two hexamers have very close isoelectric points. The isoelectric points of R-PE subunits are pH5.0-5.8 and isoelectric points ofα/βsubunit are pH5.0 and pH5.8.The isoelectric point of R-PC is pH5.7. R-PC has three kinds of subunitsα,βandβ’, has noγ-subunit and no colorless polypeptide by SDS-PAGE with 12%-21%(w/v) separating gel (pH8.8) and 4%(w/v) stacking gel (pH6.8). R-PC has two different hexamer forms (α17.5β21.3)6 and (α17.5β22.6)6. The isoelectric point ofα/βsubunit of R-PC is pH5.1 and pH5.2.The isoelectric point of AP is pH5.5. Results of SDS-PAGE with 13%(w/v) separating gel (pH 9.5),4%(w/v) stacking gel (pH6.8) show that AP has two kinds of chromospheres-carrying subunitsα,β. and quadratic AP1 (which dialyzed after second native-PAGE) and AP2 (which obtained by native-PAGE from fraction eluted by 400mmol/L NaCl) either have two colorless peptides. They all have two kinds of polymer form (α17β18.5)3L1, and (α17β18.5)3L2, AP3 (which obtained by native-PAGE from fraction eluted 1.5mol/L NaCl) has no colorless peptides. AP3 only has one type of polymer form(α17β18.5)3.Polymer form of phycobiliprotein of phycobilisomes was proposed in this study. It helps the understanding of the characteristic of phycobiliprotein and the structure of phycobilisomes of red algae and differences of phycobilisomes with cyanobacteria and unicellular algal. It can provide valuable research results for the evolution of algal chloroplasts, photosynthesis mechanism and micro-structure of photosystem.It also can provide theoretical basis for the application of marine red algae.更多还原