鱼腥藻7120细胞液泡内含物的初步测定

对鱼腥藻7120细胞液泡内含物中4种水溶性的物质进行了测定,液泡中4种物质占整个细胞中4种物质的比例分别为:蛋白质:14.1％;还原糖:34.4％;核酸:28.5％;藻青蛋白12.1％。     

CO2对转基因鱼腥藻生长的影响

为了进一步探索转基因鱼腥藻高密度培养的方法,在小型气升式反应器中研究了CO2对转基因鱼腥藻7120培养的影响。结果发现转基因鱼腥藻培养过程中通入5％ CO2能促进藻细胞生长,12d生物量提高7．44％,由于光照限制,不能大幅提高15d收获生物量,但生长周期能缩短近20％;而高浓度(10％)的CO2抑制转基因鱼腥藻的生长。CO2是通过调节pH值和影响碳源利用来影响藻细胞生长的,合适浓度的CO2有利于转基因鱼腥藻的培养。     

生物反应器培养转基因鱼腥藻的研究

在反应器中研究了转人TNF-α基因鱼腥藻7120(Anabaena sp．PCC7120,pDC-TNF)的培养。结果表明气升式反应器适合于转基因鱼腥藻的培养。气升式反应器中通气量和光照是主要的影响因素,观察到1L罐中最适通气量为60～75L／h,最适光照强度为1200lx,此时在25℃混养,光照时间／黑暗时间为12h／12h,15d生物量干重大于3g／L,TNF表达水平约占总可溶蛋白的22％,达到了摇瓶培养水平。实验发现添加维生素B1 300μg／L、B12 200μg／L和生物素4μg／L时,生产周期为12d,缩短20％,表达水平相同。培养过程通入含有5％CO2的空气,能促进生长,缩短生产周期,但收获生物量不受影响。从添加维生素和通入CO2的培养结果证明反应器中培养时,光照是限制性因素,当反应器系统一定时,最终生物量有一个最大值,如需进一步提高产量,必须设法改变光照系统。     

转TNF-α基因鱼腥藻7120质粒稳定性研究

为了实现转基因鱼腥藻培养生产TMF的目的,讲究了转基因鱼腥藻的稳定性。影印法证实转TNF-α。基因鱼腥藻7120能保持质粒分配稳定性。比较无选择压力下连续传代的转丛因鱼醒藻7120在不同培养基中的生长和外源基因表达,证实没有发生质粒部分缺失,但转基因鱼腥藻在无选择压力下会降低重组质粒拷贝数。在培养过程中,种子培养越含有的新霉素可以保持生产过程质粒稳定,这可以大火减少新霉素用量。     

蓝细菌断裂DNA聚合酶DnaE的体内/外反式剪接分析

蓝细菌Anabaena PCC7120的DNA聚合酶DnaE由2个断裂基因dnaENI和dnaECI编码.通过纯化它们的部分基因在大肠杆菌中的表达产物DnaENI′和DnaECI′来免疫家兔获得抗体,用于对断裂的DnaE在体内和体外的反式剪接活性进行鉴定.在体外实验中,将2个断裂的DNA聚合酶DnaENI′和DnaECI′混合,进行反应,并利用它们的抗体对反应产物进行鉴定;在体内实验中,利用以上抗体对Anabaena PCC7120细胞总蛋白进行免疫杂交分析.实验结果表明,蓝细菌AnabaenaPCC7120中断裂的2个DnaE多肽在体内和体外均能进行反式剪接.体外实验中,纯化的DnaENI′和DnaECI′的混合反应产生新的蛋白产物,既有成熟的反式剪接产物DnaEN′-,又有剪切了内含肽后的副产物DnaEN′和DnaE,且DTT的存在对剪接反应具有促进作用;此外体内实验中,在Anabaena PCC7120细胞中既能检测到完整的DnaE,也能检测到未作用的DnaENI,但未能检测到DnaECI.     

细胞周期蛋白依赖性激酶抑制剂Roscovitine对3种蓝藻生长和活性的影响

为了明确蓝藻中丝氨酸/苏氨酸激酶的功能是否与调控细胞的生长分裂相关,以丝状鱼腥藻7120、单细胞集胞藻6803和聚球藻7002为对象,利用OD750光吸收测定和MTT方法研究了不同浓度丝氨酸苏氨酸激酶抑制剂roscovitine对其生长和脱氢酶活性的影响。结果表明:4 h roscovitine处理后对鱼腥藻7120和集胞藻6803生长量影响不大,对聚球藻7002的生长有促进作用。4 h roscovitine的处理对鱼腥藻7120有浓度依赖的显著抑制活性,对集胞藻6803的活性无影响,但是却促进聚球藻7002的活性。药物作用4 d后,7120的生长和活性均显著降低,并有浓度效应;6803的生长量较对照减少,但活性变化不明显;聚球藻7002的生长和活性均未受影响。显微观察结果显示,roscovitine对3种细胞形态没有影响,但药物作用4 d后的7120藻丝体较短。结果表明丝氨酸/苏氨酸抑制剂roscovitine影响丝状藻7120的生长和活性。     

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PCC 7120中克隆,用于制备口服制剂,利用DNA重组技术,在不改变阅读杠的前提下,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PCC 7120内。本试验得到了有抗生素性的鱼腥藻,并用PCR技术检测到rhG-CSF基因在转基因鱼腥藻中存在。     

Genome-wide expression analysis of the responses to nitrogen deprivation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

A heterocyst is a terminally differentiated cell of cyanobacteria which is specialized in dinitrogen fixation. Heterocyst differentiation in Anabaena sp. strain PCC 7120 is triggered by deprivation of combined nitrogen in the medium. Although various genes that are upregulated during heterocyst differentiation have been reported, most studies to date were limited to individual or a small number of genes. We prepared microarrays in collaboration with other members of the Anabaena Genome Project. Here we report on the genome-wide expression analysis of the responses to nitrogen deprivation in Anabaena. Many unidentified genes, as well as previously known genes, were found to be upregulated by nitrogen deprivation at various time points. Three main profiles of gene expression were found: genes expressed transiently at an early stage (1-3 hr) of nitrogen deprivation, genes expressed transiently at a later stage (8 hr), and genes expressed when heterocysts are formed (24 hr). We also noted that many of the upregulated genes were physically clustered to form 'expressed islands' on the chromosome. Namely, large, continuous genomic regions containing many genes were upregulated in a coordinated manner. This suggests a mechanism of global regulation of gene expression that involves chromosomal structure, which is reminiscent of eukaryotic chromatin remodelling. The possible implications of this global regulation are discussed.     

鱼腥藻7120遗传转化的研究进展

鱼腥藻7120作为模式生物被广泛用于光合、固氮、进化、代谢等基本生命现象的研究。近几年, 对其基因工程的研究使人们看到它在医药、环保、能源等方面的应用潜力, 但表达效率低是其发展的瓶颈。为了提高其表达效率, 研究者从鱼腥藻7120的载体(包括启动子、复制子、选择标记基因等)的改进、目的基因的优化(密码子和SD序列)、宿主的改善、转化方法的改变等方面进行了大量探索, 除了用于功能基因的研究, 已经有几十个外源基因在鱼腥藻7120中表达。除了研究载体, 诱变鱼腥藻7120形成有利于外源基因表达的突变体和摸索转基因蓝藻最佳生长条件和表达条件, 可能是新的发展方向。     

Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120.

The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.     

Regulation of CO on heterocyst differentiation and nitrate uptake in the cyanobacterium Anabaena sp. PCC 7120

AIMS: The aim of the present investigation was to study the effects of different inorganic carbon and nitrogen sources on nitrate uptake and heterocyst differentiation in the culture of cyanobacterium Anabaena sp. PCC 7120. METHODS AND RESULTS: Anabaena was cultivated in media BG11 containing combined nitrogen and supplementary NaHCO3 or CO2. Cell growth, heterocyst differentiation, nitrate reductase (NR, EC 1.7.7.2), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and NO uptake were analysed. The cells cultivated in BG11(0) medium with aeration were taken as reference. Experimental results showed that the differentiation frequency of heterocysts when the cells were cultivated with elevated CO2 was higher than that of the cells grown with air or bicarbonate. Heterocysts appeared unexpectedly when CO2 was introduced into the medium containing nitrate. However, no heterocysts emerged when CO2 was added to medium containing NH or urea, or when NaHCO3 was supplied to the medium with nitrate. Both nitrate uptake rate and nitrate reduction enzyme activity were depressed by the supplement of CO2 to the culture. The activity of G6PDH was enhanced with the increase in heterocyst differentiation frequency. CONCLUSION: CO2 might compete with NO for energy and electrons in the uptake process and CO2 appears favoured. This led to a high intracellular C/N ratio and a relative N limitation. So the process of heterocyst differentiation was activated to supplement nitrogen uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided an attractive possibility to form more heterocysts by rapid growth of Anabaena cells cultivated in the medium containing nitrate in order to increase nitrogen fixation and hydrogen production.     

Transcription of a “silent” cyanobacterial psbA gene is induced by microaerobic conditions

Cyanobacteria, contrary to higher plants, have a small psbA gene family encoding the reaction centre D1 protein subunit of photosystem II, the first macromolecular pigment-protein complex of the photosynthetic electron transport chain. Modulation of expression of multiple psbA genes in the family allows cyanobacteria to adapt to changing environmental conditions. To date, two different strategies for regulation of the psbA genes have emerged. One, characterized in Synechocystis PCC6803 and Gloeobacter violaceus PCC7421 involves the increased expression of one type of D1 protein to cope with the increased rate of damage. The other strategy, in Synechococcus PCC7942 and Anabaena PCC7120, is to replace the existing D1 with a new D1 form for the duration of the stress. However, most of the psbA gene families characterized to date contain also a divergent, apparently silent psbA gene of unknown function. This gene, present in Synechocystis, Anabaena and Thermosynechococcus elongatus BP-1 was not induced by any stress condition applied so far. Our data shows a reversible induction of the divergent psbA gene during the onset of argon-induced microaerobic conditions in Synechocystis, Anabaena and Thermosynechococcus elongatus. The unitary functional response of three unrelated cyanobacterial species, namely the induction of the expression of the divergent psbA gene as a reaction to the same environmental cue, indicates that these genes and the protein they encode are part of a specific cellular response to microaerobic conditions. There are no specific primary structure similarities between the different microaerobic inducible D1 forms, designated as D1′. Only three amino acid residues are consistently conserved in D1′. These modifications are: G80 to A, F158 to L and T286 to L. In silico mutation of the published D1 structure from Thermosynechococcus did not reveal major modifications. The point by point effects of the mutations on the local environment of the PSII structure are also discussed.     

UV-C光催化纳米TiO2对蓝藻生长影响的研究

研究了UV-C光催化纳米TiO2对蓝藻生长的影响。从生理上分析了UV-C光催化纳米TiO2具有促进蓝藻中鱼腥藻7120体内活泼态氧化物O2,.OH和H2O2的产生,抑制藻体中过氧化氢酶(CAT)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)和超氧歧化酶(SOD)在内的抗氧化酶活性,降低可溶性蛋白(Soluble Pr)、藻蓝蛋白(C-PC)、叶绿素a(C-Chl a)和类胡萝卜素(C-Carotenoids)含量,最终表现为藻细胞生长代谢中光合速率和呼吸速率迅速降低,细胞增殖中遗传物质核酸含量下降,并出现几乎达到100%的细胞致死率;同时,通过显微镜观察发现,受试蓝藻细胞形态结构发生了明显变化。可以看出,UV-C光催化纳米TiO2能够有效抑制蓝藻生长。     

环境因子对转基因鱼腥藻培养的影响

摇瓶中对转TNF-α基因鱼腥藻7120（Anabaenasp.PCC7120,pDC-TNF)混合培养条件进行了研究,在含蔗糖9g/L,NaNO32.25g/L的BG-11培养基混合培养时,得到最适培养条件接种量5%,光照强度为1000Lux,光/暗周期（光照时间/黑暗时间）12h/12h,温度25-30℃,自然初始pH值,100mL摇瓶装液量40mL,转基因鱼腥藻15d生物量可达到3g/L以上,可溶性蛋白含量接近30%,TNF表达水平大于22%,与自养相比,生物量增加82.06%,表达水平提高38.79%,证明混合营养型培养是转rhTNF-α基因鱼腥藻7120实现高密度,高表达培养的途径。     

碳氮源对转基因鱼腥藻Anabaena sp.PCC7120培养的影响

对碳源、氮源种类和用量对转rhTNF-α基因鱼腥藻7120（Anabaena sp.PCC7120）培养的影响进行了研究,发现最适碳源为蔗糖,最适氮源为NaNO3,最佳用量分别为9g/L和2．25g/L,此时生物量远高于自养方式,达2．52g/L,比相同条件下在BG－11培养基培养高71．66％,ATNF－α表达量为16％－22％,生物活性为10^5U/mg。     

植物生长调节剂对转基因鱼腥藻7120生长与外源基因表达的影响

适宜浓度的IBA、6-BA、赤霉酸和氯化胆碱均促进转基因鱼腥藻7120生长,提高其生物量,不影响外源基因表达,但从总体上讲高浓度植物生长调节剂则抑制藻细胞生长,降低外源基因表达水平.这些植物生长调节剂混合施用促进转基因鱼腥藻生长效果不如单一因子好,外源基因表达水平也略有下降.     

Paired cloning vectors for complementation of mutations in the cyanobacterium<Emphasis Type="Italic"> Anabaena</Emphasis> sp. strain PCC 7120

The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetF _A ) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.     

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. PCC7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregulated(forward) or downregulated(reverse) pepc gene in Anabaena sp. PCC 7120. Results from real-time quantitative polymerase chain reaction(RT-q PCR), Western blot and enzymatic analysis showed that PEPCase activity was significantly 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 Rdark reduction of P700 t was stimulated, indicating enhanced cyclic electron flow(CEF) around PSI in the reverse mutant.Additionally, 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.     

Structure of the oxidized long-chain flavodoxin from Anabaena 7120 at 2 A resolution.

The structure of the long-chain flavodoxin from the photosynthetic cyanobacterium Anabaena 7120 has been determined at 2 A resolution by the molecular replacement method using the atomic coordinates of the long-chain flavodoxin from Anacystis nidulans. The structure of a third long-chain flavodoxin from Chondrus crispus has recently been reported. Crystals of oxidized A. 7120 flavodoxin belong to the monoclinic space group P2(1) with a = 48.0, b = 32.0, c = 51.6 A, and beta = 92 degrees, and one molecule in the asymmetric unit. The 2 A intensity data were collected with oscillation films at the CHESS synchrotron source and processed to yield 9,795 independent intensities with Rmerg of 0.07. Of these, 8,493 reflections had I > 2 sigma and were used in the analysis. The model obtained by molecular replacement was initially refined by simulated annealing using the XPLOR program. Repeated refitting into omit maps and several rounds of conjugate gradient refinement led to an R-value of 0.185 for a model containing atoms for protein residues 2-169, flavin mononucleotide (FMN), and 104 solvent molecules. The FMN shows many interactions with the protein with the isoalloxazine ring, ribityl sugar, and the 5'-phosphate. The flavin ring has its pyrimidine end buried into the protein, and the functional dimethyl benzene edge is accessible to solvent. The FMN interactions in all three long-chain structures are similar except for the O4' of the ribityl chain, which interacts with the hydroxyl group of Thr 88 side chain in A. 7120, while with a water molecule in the other two. The phosphate group interacts with the atoms of the 9-15 loop as well as with NE1 of Trp 57. The N5 atom of flavin interacts with the amide NH of Ile 59 in A. 7120, whereas in A. nidulans it interacts with the amide NH of Val 59 in a similar manner. In C. crispus flavodoxin, N5 forms a hydrogen bond with the side chain hydroxyl group of the equivalent Thr 58. The hydrogen bond distances to the backbone NH groups in the first two flavodoxins are 3.6 A and 3.5 A, respectively, whereas in the third flavodoxin the distance is 3.1 A, close to the normal value. Even though the hydrogen bond distances are long in the first two cases, still they might have significant energy because their microenvironment in the protein is not accessible to solvent.(ABSTRACT TRUNCATED AT 400 WORDS)