Growth and Physiological Features of Cyanobacterium Anabaena sp. Strain PCC 7120 in a Glucose-Mixotrophic Culture

Mixotrophic growth is one potential mode for mass culture of microalgae and cyanobacteria particularly suitable for the production of high value bioactive compounds and fine chemicals.The typical heterocystous cyanobacterium Anabaena sp.PCC 7120 was grown in the presence of exogenous glucose in light.Glucose improved the cell growth evidently,the maximal specific growth rate under mixotrophic condition（0.38 d 1）being 1.6-fold of that of photoautotrophic growth.Mixotrophy caused a variation in cellular pigment composition,increasing the content of chlorophyll a and decreasing the contents of carotenoid and phycobiliprotein relative to chlorophyll a.Fluorescence emission from photosystem II（PSII）relative to photosystem I was enhanced in mixotrophic cells,implying an increased energy distribution in PSII.Glucokinase（EC 2.7.1.2）activity was further induced in the presence of glucose.The mixotrophic culture was scaled up in a 15 L airlift photobioreactor equipped with an inner and an outer light source.A modified Monod model incorporating the specific growth rate and the average light intensity in the reactor was developed to describe cell growth appropriately.The understanding of mixotrophic growth and relevant physiological features of Anabaena sp.PCC 7120 would be meaningful for cultivation and exploitation of this important cyanobacterial strain.

Influences of Nitrogen-phosphorus Ratio on the Growth and Competition of Chlorella vulga and Anabaena sp. strain PCC

This paper studied the effects of different ratios of nitrogen and phospho- rus on the growth and competition of Anabaena sp. strain PCC and chloralla vul- gads （low nitrogen-phosphorus ratio group： N/P=16：1; Medium low nitrogen-phospho- rus ratio group： N/P=32：1; Medium high nitrogen-phosphorus ratio group： N/P=64：1; High nitrogen-phosphorus ratio group： N/P=320：1）. Results suggested that the largest amount of anabaena sp.strain PCC survived in medium high nitrogen-phosphorus ratio group. The nitrogen-phosphorus ratio showed no significant influences on the growth of Chlorella vulgaris, but it exerted dramatic influences on the growth of Chlore/la vulgaris of the mixed cultivation system. The largest amount of Ch/orel/a vulgaris can be found in the medium-high nitrogen-phosphorus ratio group. The inhi- bition parameter of nitrogen-phosphorus on the algae was distinctive. Anabaena sp. strain PCC had advantages in the competition with the low nitrogen-phosphorus ra- tio and medium-low nitrogen-phosphorus ratio. Potential instability existed between anabaena sp.strain PCC and Chlorella vulgaris when the nitrogen to phosphorus ratio was medium-high and high.

Expression of Mouse MT-1 as a Fusion Protein in Anabaena sp. PCC 7120

To produce mouse metallothionein-1 (mMT-I ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia co/i-cyanobacterium shuttle fusion expression vector, pKG-MT, was constructed. Via this vector, mMT-I cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione-S-trans-ferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS-polyacrylamid gel electrophoresis (SDS-PAGE) showed that the fusion protein GST-MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio-β-D-galactoside (IPTG). Glutatione-S-transferase metallothionein (GST-MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT- I was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G-50. SDS-PAGE demonstrated that the purified mMT- I was the desired protein. The result of ELISA for the purified mMT-I showed that the recovery of mMT- I from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal-binding activity of the purified mMT-I was almost the same as that of wild type MT.

Effect of Iron Deficiency on Heterocyst Differentiation and Physiology of the Filamentous Cyanobacterium Anabaena sp. PCC 7120

The effect of iron deficiency on heterocyst differentiation and some physiological properties of the filamentous cyanobacterium Anabaena sp. PCC 7120 was investigated. Under moderate iron limitation conditions, achieved by addition of iron chelator 2,2′\|Dipyridyl (<80 μmol/L) led to delayed heterocyst differentiation, no heterocyst differentiation was observed under severe iron limitation conditions, when the concentration of 2,2′\|Dipyridyl in the medium was more than 100 μmol/L . It seemed that there are certain iron\|regulated genes or operons whose function is to control heterocyst development. In addition, iron deficiency impaired the growth. Low\|iron cells had a decrease in the quantities of pigment content (chlorophyll and phycocyanin content),the whole cell in vivo absorbance spectra confirmed the decrease, the protein electrophoretic profiles revealed that iron\|deficient cells had less protein bands, with the increase of 2,2′\|Dipyridyl ,the protein bands was more and more less. And differently, iron deficiency also caused an increase of ROS (Reactive Oxygen Species)and SOD activity, it suggests that iron deficiency led to oxidative stress, which generally occured under high\|iron conditions.

Cloning and Characterization of the fecC Gene Necessary for Optimal Growth under Iron-Deficiency Conditions in the Cyanobacterium Anabaena sp. PCC7120

ThefecC gene encoding a putative iron (III) dicitrate transporter was cloned from nitrogen-fixing cyanobacteriumAnabaena sp. PCC 7120, and inactivated. The mutant grows normally in medium with NO 3 ? , NH 4 + or without combined nitrogen. But in iron-deficient medium, the mutant grows slowly. Photosynthetic properties were compared between the mutant and the wildtype strain, the content of photosynthetic pigments in the mutant is lower than that of the wild-type. The results of RT-PCR experiments show that thefecC gene is expressed under iron-deficient conditions, but is not expressed under iron-replete conditions. These results revealed thatfecC gene product is required for optimal growth under iron-deficient conditions inAnabaena sp. PCC 7120. Key words Anabaena sp. PCC 7120 - fecC - iron deficiency - photosynthetic properties - expression CLC number Q 933 Foundation item: Supported by the National Natural Sciences Foundation of China (30070154), the Frontier Science Projects Program of the Institute of Hydrobiology, the Chinese Academy of Sciences (220316), State Key Project on Cyanobacterial Bloom Control in Lake Dianchi (K99-05-35-01)Biography: XU Wen-liang (1974-), male, Ph. D, research direction: molecular genetics of cyanobacteria.

鱼腥藻PCC 7120中all3556蛋白的表达与纯化

琥珀酸半醛脱氢酶在生物体中发挥重要的生理功能。通过BLAST序列分析,发现鱼腥藻PCC 7120中all3556基因编码琥珀酸半醛脱氢酶。实验通过常规PCR从鱼腥藻PCC 7120基因组中克隆了all3556基因,将该基因构建到p ET-28a载体上并在大肠杆菌BL21(DE3)菌体中表达。经过SDS-PAGE电泳,结果表明:all3556蛋白可以在该表达体系中高效可溶性表达。利用镍亲和层析纯化方法得到了纯度大于95%的all3556蛋白,蛋白收率约为27 mg/g菌体。研究为进一步阐明all3556蛋白的催化功能及机制奠定了重要基础。

鱼腥藻PCC7120基因alr0267敲除及其功能的初步研究

【目的】敲除鱼腥藻PCC7120的alr0267基因，并对其功能进行初步研究。[方法】克隆获得鱼腥藻PCC7120alr0267基因部分片段，通过构建敲除载体，使其与目的基因发生单交换同源重组，从而将鱼腥藻PCC7120中的alr0267基因敲除，并对敲除体进行纯化和PCR鉴定，然后对alr0267敲除体和野生型固氮异形胞进行形态观察和统计，同时采用实时荧光定量PCR，在培养不同时间（O，3，8，24h和10d）检测野生型和alr0267基因敲除体中与异形胞功能相关基因all0813、all2736、alr2887的相对表达量。【结果】鱼腥藻PCC7120alr0267基因被成功敲除；在缺氮条件下培养6-12d，敲除体异形胞营养细胞数的均值明显少于野生型；实时定量PCR分析表明，野生型中all0813、all2736、alr2887基因表达量均在培养24h达到最大，敲除体中这3个基因最大相对表达量的出现时间均有所延迟。【结论】alr0267基因敲除导致异形胞分化频率增加，同时影响异形胞的正常发育。

鱼腥蓝细菌PCC 7120中可控降解系统的构建

鱼腥蓝细菌PCC 7120中已有较为成熟的诱导表达系统,但缺乏可控的蛋白降解系统。本研究基于Mesoplasma florum中的Lon蛋白酶(mf-Lon),在蓝细菌中建立可诱导的蛋白降解系统,并通过该系统对关键基因编码产物进行可控降解以研究其生理功能。结果发现降解系统并无预期作用,需进一步改进。

鱼腥藻PCC7120基因asr0757/alr0758的初步研究

为研究鱼腥藻PCC7120(Anabaena sp.)基因asr0757/alr0758在毒素-抗毒素系统中的相关生物学功能,设计了特异性引物,扩增目的片段asr0757和alr0758,将目的基因与p MD18-T载体连接构建克隆载体,并对其进行XhoⅠ和NdeⅠ双酶切,再与表达载体p ET-28a连接构建表达重组菌,重组菌的体外表达在IPTG的诱导下进行。经琼脂糖电泳检测,结果扩增出了大小为210 bp的asr0757和342 bp的alr0758目的基因。经SDS-PAGE电泳检测,表达出相对分子质量分别为8.068 k D和12.534 k D的蛋白质。根据结果可初步认定alr0758为毒素基因,asr0757为抗毒素基因,共同构成鱼腥藻PCC7120毒素-抗毒素系统。

启动子Pcpcβ提高鱼腥藻7120中hGM-CSF基因表达效率的研究

利用聚球藻7002中编码藻蓝蛋白基因(cpcβ)的启动子(Pcpcβ),替代穿梭表达载体pDC-GM中的启动子PpsbA,启动外源人粒巨噬细胞集落刺激因子(hGM-CSF)基因在鱼腥藻7120中的表达。蛋白免疫印迹证实,在含Pcpcβ启动子的cGM藻株中,hGM-CSF基因的表达水平比原先构建的含PpsbA启动子的GM藻株中提高了90%,且该基因的高效表达对宿主细胞的生长未产生明显影响。结果表明,对于在蓝藻中表达hGM-CSF基因而言,Pcpcβ启动子可能是较适宜的强启动子之一。

应用细胞合成计量关系模型分析鱼腥藻7120的混合营养生长

根据文献中鱼腥藻细胞大分子的含量和组成情况 ,计算了细胞合成所需要的小分子单体和前体代谢物的量 ,得到代谢意义上的细胞生物合成计量关系 .利用此计量关系模型 ,对鱼腥藻 712 0细胞在气升式光生物反应器中光自养和混合营养生长的对数生长阶段进行了代谢通量分析 .结果表明 ,葡萄糖的利用影响了细胞初级碳代谢的流动状况 ,混合营养生长过程比光自养生长过程磷酸戊糖途径氧化性分支的反应程度明显加强 .随着培养的进行 ,葡萄糖被细胞用作碳源的比例可能减小 ,而用作能源的比例可能增大 .图 2表 14参

几种因素诱导鱼腥藻7120短藻丝体的形成

丝状蓝藻鱼腥藻 71 2 0 (Anabaena sp.PCC71 2 0 )中可成功表达外源基因 ,但其转化和表达效率不高 ,改变细胞的生理状态可能会影响外源基因的转化和表达效率。将鱼腥藻71 2 0营养藻丝体通过几种因素诱导形成短藻丝体 (具有 2 5个左右细胞 ) ,并对其光合活性进行了测定。结果表明 :红光和高温对鱼腥藻 71 2 0短藻丝体较为有效 ,且红光诱导在 48h时 ,短藻丝体细胞数占总细胞数的比例达到 85 %;DCMU单独诱导效果不明显 ,适当浓度的DCMU+红光诱导时诱导效率略有增加 ;高温以 45℃诱导 1 2 h比例最高 ,约达 87%;高温45℃诱导时 ,对数生长后期的鱼腥藻 71 2 0较易形成短藻丝体。光合活性测定结果显示 ,诱导形成的短藻丝体光合放氧速率比正常营养藻丝体的低 ,这种具有光合放氧能力的短藻丝体显示出作为表达外源基因受体的可能性。

Regulation of pepc gene expression in Anabaena sp. PCC 7120 and its effects on cyclic electron flow around photosystem I and tolerances to environmental stresses

Since pepc gene encoding phosphoenolpyruvate carboxylase （PEPCase） has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregu-lated （forward） or downregulated （reverse） pepc gene in Anabaena sp. PCC 7120. Results from real‐time quantitative polymerase chain reaction （RT‐qPCR）, Western blot and enzymatic analysis showed that PEPCase activity was signifi-cantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I （PSI） activity was inhibited, as reflected by Y（I）, and Y（NA） was elevated, and＆amp;nbsp;dark reduction of P700t was stimulated, indicating enhanced cyclic electron flow （CEF） around PSI in the reverse mutant. Additional y, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recom-binant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with α-32P labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cyto-toxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

Construction of a Shuttle Vector for Heterologous Gene Expression in Escherichia coli and Microalgae Anabaena

Theconstruction of an integrative shuttle expression vector and potential utility was reported inEscherichiacoliandAnabaena(Nostoc) sp. strain PCC 7120. The vector comprised of the following elements: (a) an intergenic non-coding region fromAnabaenato facilitate its genomic integration (b) a strong functional PpsbAIpromoter fromAnabaenafor desired gene expression and (c) neomycin phosphotransferase gene with its own promoter for the selection of transfor-mants. The constructed vectorpAnFP was evaluated by cloning, transfer and expression of thegfpgene encoding green fluorescent protein. When theE.coliandAnabaenasp. strain PCC 7120 were transformed, intensive green fluorescence produced by the products of GFP protein was observed. This result indicated that the integrative shuttle vector pAnFP can be promisingly used in genome transformation for expression of heterologous genes inE.coliand microalgae such asAnabaenaandNostocstrains.

Analysis of Type Ⅱ Toxin-Antitoxin Genes all 3211-asl3212 in Anabaena PCC 7120

The type Ⅱ toxin-antitoxin genes are responsible for the phenotypic switch to a quasi-dormant state that enables cell survival under stresses,a similar function to heterocyst of cyanobacteria. In this paper,we particularly study the role of gene pair all3211-asl3212 under Spectinomycin stress to reveal how the type Ⅱ toxin-antitoxin involved in environmental stress responses. Bioinformatics prediction shows that toxin protein gene All3211 is homologous to Maz F,a member of maz EF family that encoding nucleases. We clone gene all3211-asl3212 into expression vectors to identify its molecular characteristics. Deletion mutant strains of all3211-asl3212 are selected in a tri-parental mating screen. Phenotype comparisons of mutant and wild type reveals no difference of single-deletion-mutants in pigment integrity,the sensitivity to antibiotics,and heterocyst formation. The results show that deletion mutation of single TAS gene pair all3211-asl3212 results in limited effects on the cellular growth of PCC 7120. Thus,we suggest that dosage compensating might be provided from redundant genes or bypass pathways to offset obvious phenotypic differences.

环境因子对转hGM-CSF基因鱼腥藻生长与光合的调节

以转hGM-CSF基因鱼腥藻7120为对象,分别探讨了温度、pH、光强等环境因子和外加碳、氮等基本营养源对转基因藻生长与光合活性的影响.结果表明:当基本环境因子为30℃、pH9.5和90μmol·m-2·s-1光强时,转基因藻生长最快.添加10mmol·L-1的NaHCO3或5g·L-1的葡萄糖对转基因藻的光合活性促进最大;但外加氮源却抑制了转基因藻的光合活性.

人表皮生长因子(hEGF)基因在蓝藻中的表达

人表皮生长因子 (hEGF)是由 5 3个氨基酸组成的蛋白 ,在临床上内服与外敷可促进内外表皮细胞的生长。将人工合成的hEGF基因连接到质粒pRL_489上 ,位于启动子psbA下游。验证连接成功后 ,用三亲接合转移方法将载体pRL_hEGF导入聚球藻Synechococcussp .PCC 70 0 2和鱼腥藻Anabaenasp .PCC 712 0。由于pRL_hEGF没有能在单细胞蓝藻中自主复制的复制子 ,通过筛选 ,hEGF在聚球藻 70 0 2中是整合到蓝藻染色体上进行表达的。用PCR扩增的方法在两种转基因藻中均检测到hEGF基因的存在。放射免疫分析证明 ,hEGF基因在两种转基因藻中均得到了表达。而且 ,在聚球藻 70 0

重组人粒细胞集落刺激因子(rhG-CSF)基因在鱼腥藻中的克隆

为了将rhG-CSF基因在鱼腥藻PCC7120中克隆,用于制备口服制剂,利用DNA重组技术,在不改变阅读框的前提下,将hG-CSF基因进行突变,并插入到pUC-19载体上,构建中间载体pUC-G-CSF;将pUC-G-CSF插入到pRL-489的启动子PpsbA的下游,构建穿梭表达载体pRL-G-CSF;通过三亲接合转移方法,将pRL-G-CSF转入丝状体蓝藻鱼腥藻PCC7120内。本试验得到了有抗生素抗性的鱼腥藻,并用PCR技术检测到rhG-CSF基因在转基因鱼腥藻中存在。

光催化纳米TiO_2治理蓝藻工艺的研究

在波长253.7 nm紫外线C波段(UV-C)照射下,对受试蓝藻进行光催化纳米TiO2氧化反应,探讨反应参数对实验工艺处理效果的影响。对蓝藻生理指标叶绿素a(Chl-a)含量测定显示:在辐照光强0.15 mW/cm2、纳米TiO2浓度100 mg/L下反应,其Chl-a含量下降速率最快;随着辐照剂量、通入空气量(Qair)和反应温度的增大,其Chl-a含量下降速度不断加快;加入1.0 mmol/L H2O2加快了Chl-a含量下降,而加入20 mmol/L抗坏血酸(Vc)则减缓Chl-a含量下降;在偏碱性或低于纳米TiO2零电点(6.25)时,有利于加快Chl-a含量下降。结果表明,光催化纳米TiO2氧化反应能够有效降低受试蓝藻Chl-a含量,在以上参数适宜反应条件下,可以更好提高光催化纳米TiO2治理蓝藻工艺的处理效果。