集胞藻PCC 6803类铁氧还蛋白Slr1205的功能研究

类铁氧还蛋白 (ferredoxin-like, Fd-like) 在高等植物中具有调控叶绿体发育等多种重要的生理功能,但在蓝藻中的生物功能尚未被发现。通过比较集胞藻PCC 6083编码Fd-like蛋白基因的敲除突变株Δslr1205与野生型 (WT) 在不同碳源和光周期条件下的生理生化表型,分析Slr1205在集胞藻中的功能。结果显示,在高CO2浓度自养、混合营养和光异养时,Δslr1205的生长速率低于WT,而在空气中自养条件下并无差异。与此相对应,混合营养和光异养时Δslr1205比WT的呼吸速率低,与呼吸作用密切相关的NDH-1L复合体的含量少。Δslr1205在所有测试的条件下有较高的类胡萝卜素以及偏黄的表型。这些数据表明,Fd-like蛋白Slr1205的缺失造成在碳源充足条件下的生长速率下降,这可能是由于呼吸作用下调导致供能不足。研究结果为今后深入研究蓝藻Fd-like蛋白奠定了基础,为开展光合作用和呼吸作用的调节机制研究探索了新方向。     

Identification of a cyanobacterial CRR6 protein,Slr1097, required for efficient assembly of NDH‐1 complexes in Synechocystis sp. PCC 6803

Despite significant progress in clarifying the subunit compositions and functions of the multiple NADPH dehydrogenase (NDH‐1) complexes in cyanobacteria, the subunit maturation and assembly of their NDH‐1 complexes are poorly understood. By transformation of wild‐type cells with a transposon‐tagged library, we isolated three mutants of Synechocystis sp. PCC 6803 defective in NDH‐1‐mediated cyclic electron transfer and unable to grow under high light conditions. All the mutants were tagged in the same slr1097 gene, encoding an unknown protein that shares significant homology with the Arabidopsis protein chlororespiratory reduction 6 (CRR6). The slr1097 product was localized in the cytoplasm and was required for efficient assembly of NDH‐1 complexes. Analysis of the interaction of Slr1097 with 18 subunits of NDH‐1 complexes using a yeast two‐hybrid system indicated a strong interaction with NdhI but not with other Ndh subunits. Absence of Slr1097 resulted in a significant decrease of NdhI in the cytoplasm, but not of other Ndh subunits including NdhH, NdhK and NdhM; the decrease was more evident in the cytoplasm than in the thylakoid membranes. In the ?slr1097 mutant, NdhH, NdhI, NdhK and NdhM were hardly detectable in the NDH‐1M complex, whereas almost half the wild‐type levels of these subunits were present in NDH‐1L complex; similar results were observed in the NdhI‐less mutant. These results suggest that Slr1097 is involved in the maturation of NdhI, and that assembly of the NDH‐1M complex is strongly dependent on this factor. Maturation of NdhI appears not to be crucial to assembly of the NDH‐1L complex.     

增强型绿色荧光蛋白在集胞藻6803中的表达

利用聚球藻7942热休克基因groESL的启动子和报告基因egfp,构建了表达载体pUC-Tegfp并转化集胞藻6803,并通过所制备抗体对转基因藻进行蛋白免疫印迹检测.结果发现,在转基因藻株T-egfp的细胞粗提液中含有能与eGFP抗体特异结合的蛋白质,表明外源增强型绿色荧光蛋白基因(egfp)在集胞藻6803中成功表达.     

Proteomic analysis of heterotrophy in Synechocystis sp. PCC 6803

To provide an insight into the heterotrophic metabolism of cyanobacteria, a proteomic approach has been employed with the model organism Synechocystis sp. PCC 6803. The soluble proteins from Synechocystis grown under photoautotrophic and light-activated heterotrophic conditions were separated by 2-DE and identified by MALDI-MS or LC-MS/MS analysis. 2-DE gels made using narrow- and micro-range IPG strips allowed quantitative comparison of more than 900 spots. Out of 67 abundant protein spots identified, 13 spots were increased and 9 decreased under heterotrophy, representing all the major fold changes. Proteomic alterations and activity levels of selected enzymes indicate a shift in the central carbon metabolism in response to trophic change. The significant reduction in light-saturated rate of photosynthesis as well as in the expression levels of rubisco and CO(2)-concentrating mechanism proteins under heterotrophy indicates the down-regulation of the photosynthetic machinery. Alterations in the expression level of proteins involved in carbon utilization pathways refer to enhanced glycolysis, oxidative pentose phosphate pathway as well as tricarboxylic acid cycle under heterotrophy. Proteomic evidences also suggest an enhanced biosynthesis of amino acids such as histidine and serine during heterotrophic growth.     

A large-scale protein protein interaction analysis in Synechocystis sp. PCC6803.

Protein-protein interactions (PPIs) play crucial roles in protein function for a variety of biological processes. Data from large-scale PPI screening has contributed to understanding the function of a large number of predicted genes from fully sequenced genomes. Here, we report the systematic identification of protein interactions for the unicellular cyanobacterium Synechocystis sp. strain PCC6803. Using a modified high-throughput yeast two-hybrid assay, we screened 1825 genes selected primarily from (i) genes of two-component signal transducers of Synechocystis, (ii) Synechocystis genes whose homologues are conserved in the genome of Arabidopsis thaliana, and (iii) genes of unknown function on the Synechocystis chromosome. A total of 3236 independent two-hybrid interactions involving 1920 proteins (52% of the total protein coding genes) were identified and each interaction was evaluated using an interaction generality (IG) measure, as well as the general features of interacting partners. The interaction data obtained in this study should provide new insights and novel strategies for functional analyses of genes in Synechocystis, and, additionally, genes in other cyanobacteria and plant genes of cyanobacterial origin.     

groESL启动子提高集胞藻6803外源egfp基因表达效率的研究

利用聚球藻7942中热激蛋白基因groESL的启动子(PgroESL)驱动外源egfp基因在集胞藻6803(Syn-echocystis sp.PCC6803)中的表达,通过蛋白免疫印迹技术研究不同温度条件下该外源基因的表达情况。结果表明,42℃诱导30min后,PgroESL启动子能显著提高转基因藻Pg中外源egfp基因的表达,使外源基因的表达量比正常温度条件下提高3.4倍。研究表明,聚球藻7942中groESL操纵子的启动子区域是一类可以受温度诱导的强启动子,能够显著提高宿主细胞中外源基因的表达效率。     

硫代硫酸钠对蓝细菌Synechocystis sp. PCC 6803生长在含葡萄糖培养基中所产生的一种新糖脂的影响

当蓝细菌Synechocystis sp. PCC 6803在添加葡萄糖的BG-11培养基中培养时,细胞出现了一种新脂.这种脂经浓硫酸/α-萘酚染色反应证实为糖脂,记为糖脂-x.这一糖脂的出现伴随着其他脂、尤其是双半乳糖甘油二酯含量的下降.此外,在添加果糖、麦芽糖、乳糖等其他碳源的培养基中生长的细胞中也检测到这一糖脂.活性氧猝灭剂硫代硫酸钠加入到培养基中能有效地抑制糖脂x的出现.当在BG-11培养基中加入0.3%硫代硫酸钠后,糖脂x仅能在培养基中添加高浓度(100 mmol/L)的葡萄糖且细胞生长处于后指数期时检测到.这些结果表明蓝细菌Synechocystis sp. PCC 6803细胞在含葡萄糖的培养基中生长出现的一种新糖脂可能与活性氧有关.     

集胞藻Synechocystissp.PCC6803利用葡萄糖生长与光合能量转化的关系

用PAM叶绿素荧光仪测定了饥饿细胞、光自养细胞和混合营养细胞的叶绿素荧光 ,并对 3种类型细胞的荧光参数 :PSⅡ实际光化学效率 φⅡ和还原型质醌Q-A 进行了比较。用双重转盘磷光机测定了光自养细胞和混合营养细胞的毫秒延迟发光。根据叶绿素荧光动力学分析和毫秒延迟发光的结果及光合电子传递抑制剂 3,4_二氯苯基二甲脲 (DCMU)、二溴百里香醌 (DBMIB)对集胞藻 6 80 3(Synechocystissp .PCC 6 80 3)混合营养生长影响进行了分析 ,集胞藻 6 80 3混合营养培养的生长速率显著高于光自养培养的原因可能在于一是外源葡萄糖没有抑制反而是促进了混合营养细胞的光自养生长 ,二是呼吸基质向质醌库提供电子 ,使光合机构的能量转化加强 ,从而促进了集胞藻6 80 3细胞的利用葡萄糖的合成代谢。     

蓝细菌6803agp基因的分子克隆和缺失突变株的构建

PCR扩增了蓝细菌集胞藻6803(Synechocystis sp.PCC6803)的agp基因（编码ADP－葡萄糖焦磷酸羧化酶）,进一步以pUC118为载体将其克隆到大肠杆菌中,构建了pUCA质粒。通过DNA体外重组,以红霉素抗性基因部分取代agp基因片段,构建了既含agp基因上游及下游序列、又携带选择性标记－红霉素抗性的pUCAE质粒。该质粒转化野生型集胞藻6803细胞,获得了能在含红霉素的培养基上正常生长的agp基因缺失突变株。对该突变株基因组DNA进行PCR扩增,验邝了其基因结构的正确性。突变株细胞生长速度较野生型细胞快,胞内的叶绿素含量比野生型细胞高,表明该突变株具有较高的光合效率。在突变株中未检测到糖原的存在,进一步从生理水平上验证了突变株构建的正确性。     

集胞藻6803NdhO蛋白多克隆抗体制备及其初步应用

蓝藻NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸.迄今为止,人们在蓝藻细胞中已鉴定出17种NDH-1复合体亚基(NdhA-NdhQ).最近,人们还获得了NdhO亚基的缺失突变株.然而,人们对NdhO亚基的研究还不充份,至今仍不清楚它的功能角色.通过PC...     

Effect of light fluctuations on photosynthesis and metabolic flux in Synechocystis sp. PCC 6803

In nature, photosynthetic organisms are exposed to fluctuating light, and their physiological systems must adapt to this fluctuation. To maintain homeostasis, these organisms have a light fluctuation photoprotective mechanism, which functions in both photosystems and metabolism. Although the photoprotective mechanisms functioning in the photosystem have been studied, it is unclear how metabolism responds to light fluctuations within a few seconds. In the present study, we investigated the metabolic response of Synechocystis sp. PCC 6803 to light fluctuations using ¹³C-metabolic flux analysis. The light intensity and duty ratio were adjusted such that the total number of photons or the light intensity during the low-light phase was equal. Light fluctuations affected cell growth and photosynthetic activity under the experimental conditions. However, metabolic flux distributions and cofactor production rates were not affected by the light fluctuations. Furthermore, the estimated ATP and NADPH production rates in the photosystems suggest that NADPH-consuming electron dissipation occurs under fluctuating light conditions. Although we focused on the water–water cycle as the electron dissipation path, no growth effect was observed in an flv3-disrupted strain under fluctuating light, suggesting that another path contributes to electron dissipation under these conditions.     

Molecular Cloning and Construction of agp Gene Deletion-mutant in Cyanobacterium Synechocystis sp. PCC 6803

Nine compounds were isolated from Elsholtzia blanda (Benth.) Benth. Their　structures were identified with spectral and chemical methods as follows: 5,6-dihydro-6-styry-2-pyrone (1), friedelin (2), 4-hydroxy-3-methoxystyrene (3), 5,2′-dimethoxy-6,7-methylene dioxyflavanone (4), 5-hydroxy-7-methoxy-6-O-［α- L -rhamnopyranosyl(1→2)-β- D -fucopyranosyl］ flavone glycoside (5), 5,5′-dihydroxy-7-acetoxyl-6,8,3″,3″-tetramethylpyran　(3′,4′)　flavone (6), 5,5′-dihydroxy-7-(α-methyl) butyroxyl-6,8,3″,3″-tetramethylpyran (3′,4′) flavone (7), 5,5′-dihydroxy-6,7-methylenedioxy-8,3″,3″-trimethylpyran　(3′,4′)　flavone (8), glucosyringic acid (9). Among them, 6, 7 and 8 are new compounds, named as sifanghaoine Ⅰ,Ⅱ and Ⅲ, respectively.     

Relationship Between the Growth of Synechocystis sp. PCC 6803 on Medium with Glucose and the Photosynthetic Energy Transformation

Measurements of chlorophyll fluorescence kinetics from dark-starved cells, light-grown cells and mixotrophic cells of Synechocystis sp. PCC 6803 were obtained using a pulse amplitude modulation (PAM) fluorometer. Photosystem Ⅱ photochemical efficiency Ⅱand the extent of reduction of Q－A in the three kinds of cells described above were compared. The millisecond delayed light emission (MDLE) of light-grown cells and mixotrophic cells were also detected. On the basis of the analysis of fluorescence kinetic parameters, comparison of the slow phase of MDLE and the influence of inhibitors of photosynthetic electron transport3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) on the mixotrophic growth of Synechocystis sp. PCC 6803, it was concluded that the reasons for higher growth rate under mixotrophic than that under photoautotrophic might be that glucose promoted the photoautotrophic growth of mixotrephic cells and the donation of eletrons to the plastoquinone pool from the respiratory substance and the transform of energy was promoted by photosynthetic system, which provided the energy needed by anabolism of cells caused by the glucose added to the medium.      

Structural analysis of four large plasmids harboring in a unicellular cyanobacterium, Synechocystis sp. PCC 6803.

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 consists of a single chromosome and several plasmids of different sizes, and the nucleotide sequences of the chromosome and three small plasmids (5.2 kb, 2.4 kb, and 2.3 kb) have already been sequenced. We newly determined the nucleotide sequences of four large plasmids, which have been identified in our laboratory (pSYSM:120 kb, pSYSX:106 kb, pSYSA:103 kb, and pSYSG:44 kb). Computer-aided analysis was performed to explore the genetic information carried by these plasmids. A total of 397 potential protein-encoding genes were predicted, but little information was obtained about the functional relationship of plasmids to host cell, as a large portion of the predicted genes (77%) were of unknown function. The occurrence of the potential genes on plasmids was divergent, and parA was the only gene common to all four large plasmids. The distribution data of a Cyanobacterium-specific sequence (HIP1: 5'-GCGATCGC-3') suggested that respective plasmids could have originated from different cyanobacterial strains.     

Cloning and expression of a prokaryotic sucrose-phosphate synthase gene from the cyanobacterium Synechocystis sp. PCC 6803

Sucrose is one of several low-molecular-weight compounds that cyanobacteria accumulate in response to osmotic stress and which are believed to act as osmoprotectants. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains a 2163 bp open reading frame (ORF) that shows similarity to genes from higher plants encoding sucrose-phosphate synthase (SPS), the enzyme responsible for sucrose synthesis. The deduced amino acid sequence shows 35–39% identity with known higher-plant SPS sequences. The putative Synechocystis sps gene was cloned from genomic DNA by PCR amplification and expressed as a His₆-tagged amino-terminal fusion protein in Escherichia coli. The expressed protein was purified and shown to be a functional SPS enzyme, confirming the identity of the ORF, which is the first sps gene to be cloned from a prokaryotic organism. The Synechocystis SPS has a molecular mass of 81.5 kDa, which is smaller than the typical higher-plant SPS subunit (117–119 kDa), and lacks the phosphorylation site motifs associated with light- and osmotic stress-induced regulation of SPS in higher plants. The enzyme has K_m values for UDPG1c and Fru6P of 2.9 mM and 0.22 mM, respectively, with a V_max of 17 mol per minute per mg protein and a pH optimum of 8.5. Unlike the higher-plant enzyme, ADPG1c, CDPG1c and GDPG1c can substitute for UDPG1c as the glucosyl donor with K_m values of 2.5, 7.2 and 1.8 mM, respectively. The enzyme is activated by Mg²⁺ but not by G1c6P, and is only weakly inhibited by inorganic phosphate. The purified protein was used to raise a high-titre antiserum, which recognises a low-abundance 81 kDa protein in Synechocystis sp. PCC 6803 extracts. There was no apparent increase in expression of the 81 kDa protein when the cells were exposed to moderate salt stress, and SPS activity was very low in extracts from both unstressed and salt- stressed cells. These results and the lack of evidence for sucrose accumulation in Synechocystis sp. PCC6803 lead to the conclusion that expression of the sps gene plays no obvious role in adaptation to osmotic stress in this species.     

ggpS基因过表达对集胞藻PCC 6803甘油葡萄糖苷和甘油合成的影响

为了研究甘油葡萄糖苷磷酸合成酶(GgpS)在集胞藻PCC 803甘油葡萄糖苷和甘油合成中的作用,本研究在前期获得高产甘油葡萄糖苷藻株的基础上分别过量表达来自于集胞藻PCC 6803自身和聚球藻PCC7002的甘油葡萄糖苷磷酸合成酶基因ggpS,并测定了在不同浓度NaCl胁迫时突变藻株的甘油葡萄糖苷和甘油积累量。结果发现获得的突变株甘油葡萄糖苷合成没有提高,但是甘油合成显著增强。此外,当培养基NaCl浓度从600 mmol/L提高到900 mmol/L时,集胞藻PCC 6803自身ggpS过表达藻株的甘油合成进一步提高75%。这些结果显示了GgpS在将碳代谢流导入集胞藻甘油合成途径中的作用。研究成果也为进一步通过基因工程改造提高集胞藻甘油葡萄糖苷和甘油合成效率奠定了基础。     

硫代硫酸钠和葡萄糖对Synechocystis sp.PCC 6803细胞甘油酯及其脂肪酸组成的影响

对生长在添加有不同浓度的葡萄糖、硫代硫酸钠培养基中的蓝细菌Synechocystis sp.PCC 6803中的甘油酯及其脂肪酸组成进行比较.结果表明:硫代硫酸钠能有效地增加膜脂中硫代异鼠李糖二酰基甘油(SQDG)和磷脂酰甘油(PG)的百分含量,培养基中同时添加葡萄糖时能抵消硫代硫酸钠的这一效应.此外,硫代硫酸钠能显著增加单半乳糖甘油二酯(MGDG)、双半乳糖甘油二酯(DGDG)中十六碳酸(C16:0)的百分含量,这一效应也能为葡萄糖消除.硫代硫酸钠不能显著地改变SQDG中C16:0的百分含量,加入葡萄糖时能降低C16:0的百分含量.这些结果说明硫代硫酸钠可能充当一种还原剂使膜脂处于一种低的不饱和状态,同时加入葡萄糖时能降低硫代硫酸钠的还原力.此外,硫代硫酸钠还可作为SQDG合成中的硫供体.     

Growth kinetics and mathematical modeling of Synechocystis sp. PCC 6803 under flashing light

In photobioreactors and natural systems, microalgae are subjected to rapidly changing light intensities (LI) due to light attenuation and mixing. A controlled way to study the effect of rapidly changing LI is to subject cultures to flashing light. In this study, series of flashing-light experiments were conducted using Synechocystis sp. PCC6803 with constant overall average LI of approximately 84 μmol·m⁻²·s⁻¹ and relative times in the light and dark varied. The results were also compared with simulated results using a mathematical model including an absorbed pool of light energy, photoacclimation, and photoinhibition. With equal time in light and dark, the specific growth rate (μ) systematically decreased with increasing light duration, and µ decreased further when the ratio of light to dark was decreased. The model captured both trends with the mechanistic explanation that when the light duration was very short the changes in the pool of absorbed LI were smoothed out across the light and dark periods, whereas longer durations caused the biomass to experience discrete light and dark conditions that lead to reduced light absorption, more energy loss to nonphotochemical quenching, and more photodamage. These growth effects were accentuated as the ratio of light to dark decreased.     

小鼠金属硫蛋白在聚胞藻中的金属诱导表达与纯化

应用蓝藻类金属硫蛋白基因启动子(smt O-P)的金属诱导性,在单细胞的聚胞藻PCC 6803中表达小鼠金属硫蛋白结构基因(mMT-1 cDNA)。在大肠杆菌HB 101中构建含有smt O-P和mMT1 cDNA的穿梭表达载体pKT-MRE,经质粒转移,链霉素筛选,Southern和Western杂交分析鉴定得稳定的转基因工程藻落。同时,做小批量锌诱导表达,并纯化了外源蛋白,5L培养液含鲜藻重5.0g,得到3.5mg mMT-1;转基因藻在高金属浓度下的耐受性测定表明,外源基因的表达提高了蓝藻对金属离子的抗性,约为野生藻的2倍。