【作者】 邓勇；

【导师】 米华玲； 沈允钢；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 植物学， 2003， 博士

【摘要】 一般认为，蓝藻和高等植物类囊体膜上都存在 4 种膜蛋白复合体，即 PSII复合体，细胞色素 b6/f 复合体，PSI 复合体和 ATP 合酶复合体。近年来，在蓝藻、高等植物等的类囊体膜上又发现了另一类膜蛋白复合体，称为 NAD(P)H 脱氢酶复合体（NDH），它是一类与线粒体复合体 I 高度同源的多亚基复合体。此复合体在蓝藻中参与呼吸和光合循环电子传递，在高等植物中参与光合循环电子传递和叶绿体呼吸等过程。越来越多的证据表明，NDH 对蓝藻的生理活动甚至生存起着重要的作用，因而对蓝藻 NDH 的结构与生理功能的研究正日益受到重视。然而，目前蓝藻 NDH 复合体的结构与功能研究仍处于起步阶段。本文的研究工作主要集中于 NDH 在蓝藻对低 CO2浓度的响应和适应中的作用、NDH 复合体的分离纯化以及其与藻胆蛋白的相互作用等方面。1．低 CO2 浓度对集胞蓝藻 NDH 复合体的影响 比较了高 CO2（H-cells）和低 CO2（L-cells）下培养的集胞蓝藻 PCC6803中 NAD(P)H 脱氢酶复合体（NDH）的活性以及亚基表达。Western 印迹分析表明，L-cells 所含的 NDH 亚基，NdhH、NdhI 和 NdhK 的量明显比 H-cells 高。非变性凝胶电泳以及活性染色分析显示，L-cells 中，NADPH 专一的 NDH 亚复合体具有更高的 NADPH-氮蓝四唑（NBT）氧化还原酶活性；此外，L-cells 中，NADPH-menadione 氧化还原酶和光系统 I（PSI）驱动的 NADPH 氧化活性也明显比 H-cells 高，这都表明 NDH 的活性在低 CO2下增强了。在 DCMU 和背景远红光存在下，测得的作用光关闭后 P700+的暗中还原初始速率在 L-cells 中明显提高，表明 L-cells 中围绕 PSI 的循环电子传递增强。NDH 的专一抑制剂，鱼藤酮（rotenone）对 L-cells 的作用光关闭后叶绿素荧光瞬时上升的抑制程度比 H-cells 要大得多，这说明在低 CO2浓度下 NDH 介导的循环电子传递明显加强。以上结果表明，NDH 及其介导的循环电子传递均为低 CO2促进，提示 NDH IV<WP=6>参与蓝藻对低 CO2的适应过程。2．集胞蓝藻中 NDH 复合体对 CO2 浓度变化的响应 通过非变性凝胶电泳分离和活性染色，从集胞蓝藻 PCC6803 的细胞粗提物中检测到一个 NADPH 专一的 NDH 亚复合体。当高 CO2浓度培养的蓝藻细胞转为低 CO2浓度培养时，此 NDH 亚复合体的活性迅速提高，同时伴随 NdhK亚基蛋白质表达水平和 PSI 驱动的 NADPH 氧化活性明显增加。而当低 CO2浓度培养的蓝藻转为高 CO2浓度培养时，一开始，此 NDH 亚复合体和 PSI 驱动的 NADPH 氧化活性以及 NdhK 的蛋白质表达水平均有轻微的提高，但随着培养时间的增加，都有不同程度的明显下降。这些结果表明体外的 CO2浓度作为信号可调控 NDH 复合体的活性，同时 NDH 复合体反过来也可参与 CO2吸收的调节。 驱动的 NADPH 氧化活性可以反映 NDH 介导的循环电子传递的活性， PSI因此我们的结果也表明 NDH 介导的 PSI 循环电子传递活性受低 CO2诱导而为高 CO2抑制。综合以上结果，说明 NDH 及其介导的循环电子传递可能参与蓝藻对 CO2浓度变化的响应。3．集胞蓝藻 PCC6803 含疏水亚基的 NAD(P)H 脱氢酶亚复合体的分离 蓝藻 NDH 复合体的分离纯化比较困难。迄今，人们只分离到几个 NDH 亲水亚复合体，含疏水亚基的蓝藻 NDH 亚复合体的分离还未见报道。我们利用离子交换与凝胶过滤层析，从 n-dodecyl β-D-maltoside (DM)处理的集胞蓝藻Synechocystis PCC6803 细胞粗提液中，首次分离到两个包含 NDH 疏水亚基NdhA 的亚复合体。酶活分析表明，分离到的 NDH 亚复合体具有 NADPH-氮蓝四唑(NBT)氧化还原酶活性，以 NADPH 为电子供体可以还原铁氰化钾、二溴百里香醌（DBMIB）、二氯酚靛酚（DCPIP）、duroquinone 以及 UQ-0 等质醌类电子受体。4．集胞蓝藻 PCC6803 中 NAD(P)H 脱氢酶与藻胆蛋白结合的初步证据 通过非变性凝胶电泳（native-PAGE）和 NADPH-氮蓝四唑（NBT）氧化还原酶活性染色，从集胞蓝藻 PCC6803 的细胞粗提物中检测到一个 NADPH 专一的 NAD(P)H 脱氢酶（NDH）亚复合体，Western 印迹分析表明此亚复合体含 V<WP=7>NdhA、NdhB 和 NdhH 但不含 NdhK 和 Fd-NADP+氧化还原酶（FNR）。吸收光谱和 77K 荧光光谱显示此亚复合体中含别藻蓝蛋白和藻蓝蛋白。进一步利用离子交换与凝胶过滤层析从细胞粗提物中分离 NDH 时，发现 NADPH-铁氰化钾氧化还原酶和NADPH-NBT氧化还原酶活性总是与藻胆蛋白共分离，无法分开。分离得到的NDH经 native-PAGE 分离和活性染色后，在凝胶上检测到两条 NDH活性条带，Western 印迹分析显示此两条带均含 NDH 的亚基，表明它们都是NDH 亚复合体。此两 NDH 条带在活性染色前均为蓝色，在非变性条件下将其蛋白质溶出后，吸收光谱以及 77K 和室温荧光光谱均显示此两条带含有藻胆蛋白。本文的结果表明，至少在我们的实验条件下，集胞蓝藻 PCC6803 中 NDH复合体与藻胆蛋白相互结合。更多还原

【Abstract】 It had been known that there are mainly four kinds of complexes, photosystem II (PSII),cytochrome b6/f, photosystem I (PSI) and ATP synthase, in cyanobacterial and higher plantcloroplastic thylakoid membranes. Since mid-1980’s, another protein complex, NAD(P)Hdehydrogenase (NDH) complex, with a high degree homology to mitochondrial complex I hasbeen identified in thylakoid membranes of cyanobacteria and higher plants. NDH complex hasbeen suggested to function as a mediator of both PSI-cyclic and respiratory electron flow tothe intersystem chain in cyanobacteria or of PSI-cyclic and chlororespiratory electron flow inhigher plants. Evidences show that NDH complex has very important physiological functionsin cyanobacteria and is even indispensable to the viability of cyanobacteria. Therefore,presently, more and more attentions are being paid to this area. Up to date, studies on thestructure and functions of NDH complex are still in primary stage. This work mainly focuseson the roles of NDH complex in the responses and acclimation of cyanobacteria to low CO2concentration, the separation and purification of NDH complex and its interaction withphycobiliproteins.1. Effects of low CO2 on cyanobacterial NDH complex The expression and activity of type-1 NAD(P)H dehydrogenase (NDH) was comparedbetween cells of Synechocystis PCC6803 grown in high (H-cells) and low (L-cells) CO2 conditions.Western analysis indicated that L-cells contain higher amounts of the NDH subunits, NdhH, NdhIand NdhK. An NADPH-specific subcomplex of NDH showed higher NADPH-nitroblue tetrazoliumoxidoreductase activity in L-cells. The activities of both NADPH-menadione oxidoreductase and VII<WP=9>light-dependent NADPH oxidation driven by photosystem I were much higher in L-cells than inH-cells. The initial rate of re-reduction of P700+ following actinic light illumination in the presence ofDCMU under background far-red light was enhanced in L-cells. In addition, rotenone, a specificinhibitor of NDH, suppressed the relative rate of post-illumination increase in Chl fluorescence ofL-cells more than that of H-cells, suggesting that the involvement of NDH in cyclic electron flowaround photosystem I was enhanced by low CO2. Taken together, these results suggest that NDHcomplex and NDH mediated-cyclic electron transport are stimulated by low CO2 and function in theacclimation of cyanobacteria to low CO2.2. Response of NDH complex to the alteration of CO2 concentration An NADPH-specific NDH subcomplex was separated by native-polyacrylamide gelelectrophoresis and detected by activity staining from the whole cell extracts of SynechocystisPCC6803. Low CO2 caused increase in the activity of this subcomplex quickly, accompanied by anevident increase in the expression of NdhK and PSI-driven NADPH oxidation activity that canreflect the activity of NDH-mediated cyclic electron transport. During incubation with high CO2,the activities of NDH subcomplex and PSI-driven NADPH oxidation as well as the proteinlevel of NdhK slightly increased at the beginning, but decreased evidently in various degreesalong with incubation time. These results suggest that CO2 concentration in vitro as a signal cancontrol the activity of NDH complex and NDH complex may in turn function in the regulation of CO2uptake3. Separation of hydrophobic NDH subcomplexes from Synechocystis PCC6803 Some efforts have been performed to separate an integrated NA(D)PH dehydrogenase(NDH) complex from cyanobacteria, which proved the purification of NDH complex verydifficult due to its friability in vitro. Until now, no report has ever been showed to illustrate thepurification of a hydrophobic NDH subcomplex from cyanobacteria yet. In this work, two NDHsubcomplexes were separated from n-dodecyl β-D-maltoside (DM)-treated whole cell extractsof Synechocystis PCC6803 by anion exchange chromatography and gel filtration. Bothsubcomplexes contained the hydrophobic subu更多还原