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。

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

The agp gene encoding the ADP-glucose pyrophosphorylase involved in cyanobacterial glycogen synthesis was amplified by PCR. The resulting agp fragment was cloned in plasmid pUC118 to generate plasmid pUCA. Part of the fragment within the agp DNA was deleted and replaced by an erythromycin resistance cassette to generate plasmid pUCAE, which was used to transform the Synechocystis sp. PCC 6803 wild-type strain and a mutant with resistance to erythromycin was obtained. PCR analysis of the genomic DNA from the resulting mutant indicated that the appropriate deletion and insertion indeed had occurred. The cell growth and Chl a, glycogen content in the mutant showed difference from those in the wild-type strain. The obtained biomass as well as the Chl a content in the mutant strain was higher than that of the wild-type strain, which suggested that the photosynthesis efficiency in the agp(-) strain was higher than that in the wild-type strain. No glycogen was found in the mutant, providing evidence for the correction of the mutant in physiological level.

集胞藻Synechocystis sp. PCC 6803 slr1501的克隆及原核表达分析

组蛋白乙酰基转移酶上调表达与抗逆性有一定相关性。集胞藻Synechocystis sp.PCC 6803 Slr1501是一个未知蛋白,预测其属于组蛋白乙酰基转移酶GNAT家族N-乙酰基转移酶。为了进一步明确slr1501基因功能,本研究从集胞藻6803中克隆slr1501基因,成功构建了pET-28a(+)-slr1501原核表达载体,并转化至大肠杆菌BL21(DE3)进行分析。经1 mmol/L IPTG诱导2 h后Slr1501蛋白成功表达,表达量随诱导时间的延长而增加,且主要以包涵体形式表达。Western blot检测结果显示Slr1501重组蛋白特异性表达。本试验结果为进一步研究该基因的功能奠定了基础。

Slr0351的表达及其在Synechocystis sp.PCC 6803中功能的初步研究

Slr0351是Synechocystis sp.PCC 6803中的未知功能蛋白,其同源蛋白广泛存在于各种蓝藻和铁硫杆菌中。为了确定Slr0351的性质,构建了表达质粒p ET-slr0351,并在E.coli中表达Slr0351。在无氧条件下采用亲和层析纯化方法获得了Slr0351,无氧条件下Slr0351呈棕红色,在460 nm处有[2Fe-2S]铁硫簇的特征吸收峰,棕红色Slr0351对氧气敏感,能被连二亚硫酸钠还原,由此表明Slr0351为铁硫蛋白。为了获知slr0351的功能,基于基因同源重组交换的原理,利用Kana抗性片段替换slr0351,将Synechocystis sp.PCC 6803中的slr0351基因缺失,构建了Δslr0351突变体。利用紫外-可见吸收光谱仪扫描了Δslr0351与野生型Synechocystis sp.PCC 6803(WT)的吸收光谱,发现正常光照条件下Δslr0351的叶绿素a仅为WT的68.8%,slr0351的缺失使蓝藻中叶绿素a含量降低。比较了藻细胞在缺乏不同营养元素和不同光照条件下的生长速率差异,与WT相比,Δslr0351具有如下特征:(1)对缺Fe和缺S胁迫条件更敏感;(2)在弱光照条件下Δslr0351的光能利用效率和生长速率更低,该现象与Δslr0351中叶绿素a含量的降低有关。

集胞藻Synechocystis sp. PCC 6803脂质组的分析

以集胞藻Synechocystis sp.PCC 6803为研究对象,研究建立了基于超高效液相色谱耦合串联质谱技术脂质组学分析方法。鸟枪法脂质组学通过电喷雾离子化有效分离油脂粗提物中所含单个脂质分子,在三重四极杆扫描碎片离子,能够利用特征片段离子鉴定光合甘油酯的种类和酰基组成,具有高效、灵敏度高和质量准确度高等优点。对不同光强下生长的Synechocystis sp.PCC 6803细胞的各脂质组分进行了全定量分析,发现单半乳糖甘油二酯(Monogalactosyldiacylglycerol,MGDG)和磷脂酰甘油(Phosphatidyl glycerol,PG)在高光处理的第2小时即显著积累,增长量分别为34.64%和68.49%,其中以含有从头合成、高度饱和的脂肪酸的种类增长最为快速和显著,而后高不饱和度的脂肪酸组成的种类逐渐积累。双半乳糖甘油二脂(Digalactosyldiacylglycerol,DGDG)在各时间点都持续增长,12h后增长量达26.95%,硫代异鼠李糖甘油二酯(Sulfoquinovosyl diacylglycerol,SQDG)的含量则呈现出不断下降的趋势。研究所建立的脂质组学分析方法对进一步研究Synechocystis sp.PCC 6803的脂质代谢及生理功能提供了有力的分析工具。

Synechocystis sp.strain PCC 6803定量PCR模板制备方法的改进

以Synechocystis sp.strain PCC 6803为实验菌株,设计改良制备定量PCR模板的方法;同时,对Synechocystis sp.strain PCC 6803总RNA的Trizol抽提法进行优化。结果显示,利用改良方法抽提得到的RNA结构更完整;同时,利用改良方法制备的模板可以用于后续定量PCR实验;其中,只有高表达量基因适用于半定量PCR,低表达量基因则需要实时荧光定量PCR检测。

Effects of Sodium Thiosulfate on the Occurrence of a Novel Glycolipid in Cyanobacterium Synechocystis sp. PCC 6803 Cells Grown in the Presence of Glucose

A novel lipid occurred when cyanobacterium Synechocystis sp. PCC 6803 cells were grown in BG-11 medium with glucose applied. This lipid was determined to be a glycolipid, designated glycolipid-x (Glyco-x), by staining with alpha-naphthol and concentrated sulfuric acid. The occurrence of Glyco-x accompanies the disappearance of other lipids, especially DGDG. Glyco-x can also be observed in cells grown in BG-11 medium with the application of other carbon sources: fructose, maltose and lactose. Sodium thiosulfate, an effective scavenger of reactive oxygen intermediates, showed strong capability to inhibit glucose-induced occurrence of Glyco-x. In the presence of 0.3% sodium thiosulfate, Glyco-x could only be detected in cells grown in BG-11 medium with 100 mmol/L glucose applied in late-exponential phase. These results suggest that reactive oxygen species might be involved in the occurrence of Glyco-x in cyanobacterium Synechocystis sp. PCC 6803 cells grown in the presence of glucose.

Effects of Na2S2O3 and Glucose on the Compositions of Glycerolipids and Their Fatty Acids in Synechocystis sp. PCC 6803 Cells

Compositions of glycerolipids and fatty acid compositions of glycerolipids were compared among Synechocystis sp. PCC 6803 cells grown in the BG-11 medium containing different concentrations of glucose and Na2S2O3 in this study. It was found that Na2S2O3 can effectively increase the percentage of sulphoquinovosyl diacylglycerol (SQDG) and phosphatidylglycerol (PG) to total membrane lipids and the simultaneous application of glucose with Na2S2O3 can counteract the effect of Na2S2O3. In addition, Na2S2O3 can significantly increase the percentage of palmitic acid (C, 16:0) in fatty acid composition of monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) and decrease the fatty acid unsaturation degree accordingly, and these effects can also be eliminated by glucose. These results indicate that Na2S2O3 can take as a reductant to make membrane lipids in a low unsaturated state, and the simultaneous application of glucose can decrease the reducing power of Na2S2O3. In addition, Na2S2O3 can take as a sulfur donor for the synthesis of SQDG.

Characterization of calcium deposition induced by Synechocystis sp. PCC6803 in BG11 culture medium

Calcium carbonate （CaCO3） crystals in their preferred orientation were obtained in BG11 culture media inoculated with Synechocystis sp. PCC6803 （inoculated BG11）. In this study, the features of calcium carbonate deposition were investigated. Inoculated BGll in different calcium ion concentrations was used for the experimental group, while the BGll culture medium was used for the control group. The surface morphologies of the calcium carbonate deposits in the experimental and control groups were determined by scanning and transmission electron microscopy. The deposits were analyzed by electronic probe micro-analysis, Fourier transform infrared spectrum, X-ray diffraction, thermal gravimetric analysis and differential scanning calorimetry. The results show that the surfaces of the crystals in the experimental group were hexahedral in a scaly pattern. The particle sizes were micrometer-sized and larger than those in the control group. The deposits of the control group contained calcium （Ca）, carbon （C）, oxygen （O）, phosphorus （P）, iron （Fe）, copper （Cu）, zinc （Zn）, and other elements. The deposits in the experimental group contained Ca, C, and O only. The deposits of both groups contained calcite. The thermal decomposition temperature of the deposits in the control group was lower than those in the experimental group. It showed that the CaCO3 deposits of the experimental group had higher thermal stability than those of the control group. This may be due to the secondary metabolites produced by the algae cells, which affect the carbonate crystal structure and result in a close-packed structure. The algae cells that remained after thermal weight loss were heavier in higher calcium concentrations in BGll culture media. There may be more calcium- containing crystals inside and outside of these cells. These results shall be beneficial for understanding the formation mechanism of carbonate minerals.

Effects of heavy metals (Pb^(2+) and Cd^(2+)) on the ultrastructure, growth and pigment contents of the unicellular cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained various initial concentrations of Pb^2＋ and Cd〉 （0, 0.5, 1, 2, 4, 6 and 8 mg/L）. The experiment was conducted for six days and the metal induced alterations in the ultrastructure, growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased （〉4 mg/L Pb^2＋） metal concentration. The photosynthetic apparatus （thylakoid membranes） were found to be the worst affected. Deteriorated or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest concentration （8 mg/L pb^2＋）, the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations （0.2, 3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L pb^2＋）, both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents （chlorophyll a, 13 carotene and phycocyanin） were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb^2＋, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll a, 13 carotene and phycocyanin respectively over the control. Corresponding reductions for the same CdZ＋concentrations were 57.83, 48.94 and 56.90%. Lethal concentration （96 h LC50） values （3.47 mg/L Cd^2＋ and 12.11 mg/L Pb^2＋） indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd^2＋ than Pb^2＋.

Specific genetic variation in two non-motile substrains of the model cyanobacterium Synechocystis sp.PCC 6803

Synechocystis sp. PCC 6803 is a model organism widely used in cyanobacterium biology and biotechnology. To know the genetic background of substrains of Synechocystis sp. PCC 6803 is important for further research and application. In this study, we reported the genome sequences of two non-motile wild-type substrains of Synechocystis sp. PCC 6803 using whole genome resequencing. 55/56 putative single nucleotide polymorphisms(SNPs) and 8/9 Indels(insertion and deletion) were identified. Among these, 47 SNPs were found in both the GT-AR and GT-CH strains, and 8 were unique to GT-AR and 9 were unique to GT-CH. All of these variations were annotated in metabolism pathway referred to KEGG database. Meanwhile, the deletion in s lr0332 gene was detected in these two strains, which attributed to the non-motile phenotype of them and suggested that the insertion in spkA gene was not essential for non-motile phenotype. These resequencing data provide the genetic background information of these two strains and highlighted the microevolution over decades of laboratory cultivation.

Studies on Hemolysis of Hemolysin Produced by Synechocystis sp. PCC 6803

Hemolysin produced by various bacteria,may destroy erythrocyte membranes via a pore-forming mechanism,a deter-gent action,or a lipase activity.Previous to this experiment,the mode of action used by cyanobacterial hemolysin had not been re-ported.To characterize the action mode of hemolysin produced by the wild-type strain of Synechocystis sp.PCC6803,hemolysis of erythrocytes originating from human,mouse,sheep,rabbit and goldfish was studied.The erythrocytes of mouse,sheep and rabbit were sensitive,while those of human and fish were resistant,to this hemolysin.Using rabbit erythrocytes,it was shown that hemoly-sis occurred in two steps:a binding step within the first 10 min of treatment and a lytic step after 30 min.Both binding and lysis were highly temperature-dependent.Effects of erythrocyte density on hemolysis suggest that the hemolysin might target erythrocytes via a multiple-hit mechanism.In the osmotic protection experiment,all tested osmotic protectants,with molecular diameters ranging from 0.9 ?5.66 nm,failed to effectively inhibit hemolysis.Scanning electron micrographs showed that the hemolysin caused protuberances or echinocytes in rabbit erythrocytes,and then disrupted and ruptured the erythrocytes.Characteristics of hemolysis showed distinct differences from other pore-forming mechanisms,suggesting that this hemolysin might act through a detergent-like or lipase mecha-nism,rather than a pore-forming mechanism.

Transcriptomic analysis of Synechocystis sp. PCC6803 under low-temperature stress

In this study, cDNA microarrays were developed from 3569 mRNA reads to analyze the expression profiles of the transcriptomes of Synechocystis sp. PCC6803 under low temperature （LT） stress. Among the genes on the cDNA microarrays, 899 LT-affected genes exhibited a 1.5-fold （or greater） difference in expression compared with the genes from normal unstressed Synechocystis sp. PCC6803. Of the differentially expressed genes, 353 were up-regulated and 246 were down-regulated. The results showed that genes involved in photosynthesis were activated at LT （10℃）, including genes for photosystem I, photosystem II, photosynthetic electron transport, and cytochrome b6/f complex. Moreover, desg, one of four genes that encode the fatty acid desaturases, was also induced by LT. However, the LT conditions to some degree enhanced the transcription of some genes. In addition, LT （10℃） may reduce cellular motility by regulating the transcription of spkA （sll1575）, a serine/threonine protein kinase. The results reported in this study may contribute to a better understanding of the responses of the Synechocystis cell to LT, including pathways involved in photosynthesis and repair.

Biotransformation of 6-deoxypseudoanisatin by Synechocystis sp. PCC6803

Objective:To explore the ability of Synechocystis sp.PCC6803 in transforming 6-deoxypseudoanisatin.Methods:The experiment was performed by incubating 6-deoxypseudoanisatin with the freshwater cyanobacterium Synechocystis sp.PCC6803 under continuous white light at 30C for 5 days.The crude converted product was detected using thin-layer chromatography(TLC)and further analyzed using high-performance liquid chromatography(HPLC)as well as HPLC with electron spray ionization mass spectrometry(HPLC-ESI-MS).Results:TLC results showed that 6-deoxypseudoanisatin was converted into a less polar product.HPLC and MS data indicated that the retention time of the converted product increased in comparison with the standard of 6-deoxypseudoanisatin.Conclusion:Thus,the study appears to demonstrate that Synechocystis sp.PCC6803 can transform 6-deoxypseudoanisatin.The polarity of the converted product is less than that of 6-deoxypseudoanisatin.

集胞藻PCC-6803催化的光合微生物燃料电池性能研究

以集胞藻PCC-6803(Synechocystis PCC-6803)为阳极催化剂搭建直接利用太阳能的双室H-型光合微生物燃料电池(PMFC),通过极化曲线法、交流阻抗法、循环伏安法等电化学方法,开展电极面积比、质子交换膜、内阻等因素对光合微生物燃料电池产电的影响研究。试验结果显示:在PMFC运转过程中,其输出功率稳定,且达到的最大功率密度为72.3 mW/m2;阴阳极面积大小对PMFC产电性能没有显著影响,说明双室光合微生物燃料电池中,质子交换膜传递质子的速率较慢,限制了PMFC发电效能的提高。PMFC启动后,随着生物膜的增长,其欧姆内阻、极化内阻、总内阻都呈现下降的趋势,且欧姆内阻下降的速率小于极化内阻,从而使欧姆内阻占总内阻的比率变大,进一步说明质子交换膜传递质子的速率是限制PMFC发电的关键因素。

集胞蓝细菌PCC6803中非生物胁迫响应的核心基因分析

以集胞蓝细菌PCC6803的多组转录组数据为对象,鉴定蓝细菌在非生物胁迫下共同表达的核心差异基因(CDGs).结果表明,9个对氮、铁、碳、磷缺乏响应的CDGs被鉴定.京都基因和基因组百科全书(KEGG)分析表明,氮代谢、ABC转运蛋白和氨基酸代谢与营养缺乏有关.研究还鉴定了热激处理下共同表达的CDGs,并且代谢通路ABC转运蛋白和碳固定也与热胁迫相关.同时构建了CDGs的蛋白质-蛋白质相互作用网络,以预测基因在应对胁迫可能的响应机制.这些候选基因库将有助于加速理解蓝细菌在应激中的调控模式.

集胞藻6803的混合培养——光照强度和葡萄糖的影响

利用摇瓶研究了混合营养条件下单细胞蓝藻集胞藻6803(Synechocystissp.PCC6803)的生长特性,以及葡萄糖和光照强度对集胞藻6803生长的影响。实验结果表明,在葡萄糖消耗完之前,集胞藻6803的混合营养型生长处于对数生长期,且葡萄糖浓度及光照强度都对集胞藻6803的混合营养型生长有显著影响:在初始葡萄糖浓度097～480g/L范围内,同一光照强度培养下藻细胞的比生长速率随葡萄糖浓度的增大而降低;而在光照强度15～55μE·m-2·s-1范围内,初始葡萄糖浓度相同条件下藻细胞的比生长速率及对葡萄糖的藻体得率都随光照强度的增强而增大,但当光照强度在55～96μE·m-2·s-1时,集胞藻6803混合培养的比生长速率基本不变,出现了光饱和现象。

气升式反应器培养集胞藻6803过程中光能利用特性研究

在气升式光生物反应器中,对集胞藻6803分批培养条件下的光衰减、平均光强、光的分布以及藻细胞生长对光能的利用特性进行分析。结果表明:Lambert-Beer定律可较好描述细胞浓度及光程对光衰减的影响;随着藻细胞密度的增加,光生物反应器内平均光强不断减小,藻细胞的比光能吸收速率和比生长速率也不断降低,反应器中"暗区"体积在培养6d后超过总体积的80%;利用Pirt细胞生长生物能量模型描述比生长速率和比光能吸收速率的关系,在入射光强为58.73KJ/(m2·h),求得基于光能的细胞得率YG和维持系数m分别为9.98×10-3g/kJ和0.445kJ/(g·h)。

用同源重组法将人肝金属硫蛋白突变体ββ基因整合在集胞藻6803中表达

将集胞藻 (Synechocystissp .)PCC 6 80 3上psbB的一段序列 (从 # 6 93bp到 # 2 5 6 3bp)插入pBluescriptKS的多克隆位点 ,构建带有整合平台的载体pKSB。再将人工合成的 ββ基因插入中间载体pRL_439上启动子PpsbA的下游 ,并将pRL_ββ上PpsbA和 ββ基因插入pKSB构建成整合表达载体pKSB_ββ。利用自然转化将整合表达载体导入集胞藻 6 80 3,并通过单交换同源重组使 ββ基因整合到集胞藻 6 80 3基因组DNA上。逐步提高氨苄青霉素浓度 ,筛选得到遗传性状稳定的转基因集胞藻 6 80 3。PCR检测转基因集胞藻 6 80 3,结果证实 ββ基因已整合到集胞藻 6 80 3的染色体上 ;Westernblotting结果表明 ,ββ基因在转基因集胞藻 6 80 3细胞中得到表达 ,ELISA测定表明在 5 0 μmol/L的Zn2 +诱导下 ββ在新鲜集胞藻 6 80 3中的蛋白表达量为 0 .739mg/g ;重金属耐受性实验表明 ,得到能耐受Cu2 +的转基因集胞藻 6 80 3。经原子吸收光谱法测定 ,转基因集胞藻 6 80 3在含低浓度Cu2 +(10 μmol/L)的培养液中对Cu2 +的去除率可达到 82 %

集胞藻6803藻胆体藻蓝蛋白多克隆抗体制备及其初步应用

通过PCR扩增出集胞藻6803 C-PC编码基因cpcA,构建表达质粒pET32a(+)-cpcA,转化大肠菌株BL21(DE3)pLysS,用IPTG诱导表达、经His-tag纯化后免疫日本大耳白兔获得多克隆抗体。间接ELISA法揭示该抗体效价可高达1∶1 025 000;蛋白免疫印迹确定该抗体具有高度特异性。应用该抗体进行蛋白免疫印迹检测,结果发现C-PC蛋白在生长光与高光培养条件下的数量,以及与2个光系统间的连结数量均存在显著差异,为进一步研究藻胆体的杆在响应与适应机制中所承担的重要角色奠定了生化基础。