鱼腥藻7120响应NaCl胁迫的光合特性

NaCl胁迫处理丝状蓝藻鱼腥藻7120后光合特性的变化表明;鱼腥藻7120的净光合放氧速率和呼吸速率随NaCl浓度的程式高而降低,且浓度低于0.4mol/LNaCl时的降幅比高于0.4mol/LNaCl时的降幅小,加入0.4%(W?V)的蔗糖后可提高盐胁迫后的鱼腥藻7120的光合放氧速率,吸收光谱测定结果表明盐胁迫没有改变鱼腥藻7120的光合色素组成,但导致藻胆蛋白的总含量降低,类胡萝卜素含量增加。低温荧光发射光谱测定表明盐胁迫后改变了光能在两个光系统之间的分配。由藻胆蛋白吸收的光能向光Ⅱ传递受阻。荧光动力学分析表明光系统Ⅱ的光化学效率随盐浓度的增加而降低。表现出与光合放氧速率的一致性。     

鱼腥藻PCC7120细胞液泡的初步研究

从保存３个月以上的老化培养物中直接检查到游离液泡。液泡为标准圆球状,完全透明,大小相差极为悬殊,多数大型液泡吞噬了数个衰老藻细胞。采用低渗酶解,渗透冲击,低渗酶解和渗透冲击相结合从培养３个月以上,２个月,１个月,１８ｄ,１０ｄ及２ｄ的藻丝细胞都分离到液泡。液泡略大于细胞,泡内无吞噬物。培养３ｄ的藻丝有１５％的细胞分离到液泡。其他多种蓝藻也分离到同样的液泡。     

几种因素诱导鱼腥藻7120短藻丝体的形成

丝状蓝藻鱼腥藻7120（Anabaena sp.PCC7120）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7120藻丝体通过几种因素诱导形成短藻丝体（具有25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7120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12h比例最高,约达87％;高温45℃诱导时,对数生长后期的鱼腥藻7120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。     

在鱼腥藻7120中建立反义glnA系统

应用反义技术对鱼腥藻７１２０的内源ｇｌｎＡ基因的表达进行调控,首次获得了人工反义系统的蓝藻品系。先从编码谷酰胺合成酶的基因ｇｌｎＡ中取得部分结构基因片段,与表达质粒载体ｐＲＬ－４３９及穿梭质粒载体ｐＤＣ－８相连接。通过酶切鉴定筛选出反向克隆的穿梭表达质粒ｐＤＣ－ＡＴＧＳ,然后应用三亲接合转移法把它鱼腥藻７１２０。     

鱼腥藻Anabaena PCC7120原生质球和液泡的同步诱导

植物和真菌的液泡,都是通过原生质体途径被分离后才得以深入研究.要分离蓝藻液泡,首先要求制备蓝藻原生质体(球),而这一技术在蓝藻方面长期不过关.最近十年来,作者在这方面进行了不断的努力,先后在蓝藻原生质球制备1、培养再生和融合2等方面获得进展,这方面的研究导致了蓝藻液泡的重新发现3.所发现的液泡由无机盐诱导产生,经电镜检查为单位膜所包围,故确定为液泡.借助较为成功的原生质球制备技术,首先分离了无机盐诱导形成的液泡,在此基础上进一步发现和分离了非诱导液泡4,在分离非诱导液泡的试验过程中发现了原生质球和液泡的同步诱导作用.       

转基因鱼腥藻7120适宜生长条件的初步研究

以前的研究报导了将人肿瘤坏死因子基因(TNF-α)成功转入鱼腥藻7120中,这项研究在实验室小规模范围内探讨了其转TNF-α基因鱼腥藻7120的适宜生长条件。结果表明,转基因鱼腥藻7120最适生长温度在25～30℃,最适pH7.0～7.5。不同光照强度下的净光合放氧速率测定结果表明,转基因鱼腥藻7120与野生型具有一致的光饱和点和光补偿点,且适合在10～700μE.m-2.s-1下生长。外加有机碳源(4g/L葡萄糖)一定程度上可以增加转基因鱼腥藻7120生长速度。对不同藻龄转基因鱼腥藻7120中TNF-α的累积量的检测发现:生长8d左右时转基因鱼腥藻7120中TNF-α累积量达到最大值。这将为产业化生产、适时收获转基因蓝藻提供理论指导,同时讨论了葡萄糖对转基因鱼腥藻7120代谢的调节。     

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.     

鱼腥藻PCC7120基因a115292和alr0647的初步研究

通过BLAST软件分别对藻胆蛋白裂合酶（biliprotein lyase）编码基因cpcS和cpcT进行同源搜索分析,在鱼腥藻（Anabaena）PCC7120中获取了同源基因all5292和alt0647。同源分析发现,这两个基因所编码氨基酸序列与其相对应的裂合酶氨基酸序列相似程度分别达到53．4％和61．4％。随后,对这两个基因进行了初步研究。结果显示：All5292和Alr0647无论单独还是共同表达均没有裂合酶催化藻蓝胆素PCB结合到藻蓝蛋白（phycocyanin）或藻红蓝蛋白（phycoerythrocyanin）β亚基上的功能。通过在不同生理条件下对鱼腥藻PCC7120的培养,还对这两个基因的调控表达进行了初步的探索。结果表明：all5292和alr0647的表达与氮源的缺乏与否有联系,在氮胁迫条件下两个基因均进行了转录而在氮源充足的情况下则没有表达。     

鱼腥藻PCC7120基因all5292和alr0647的初步研究

通过BLAST软件分别对藻胆蛋白裂合酶(biliprotein lyase)编码基因cpcS和cpcT进行同源搜索分析,在鱼腥藻(Anabaena)PCC7120中获取了同源基因all5292和alt0647.同源分析发现,这两个基因所编码氨基酸序列与其相对应的裂合酶氨基酸序列相似程度分别达到53.4%和61.4%.随后,对这两个基因进行了初步研究.结果显示:All5292和Air0647无论单独还是共同表达均没有裂合酶催化藻蓝胆素PCB结合到藻蓝蛋白(phycocyanin)或藻红蓝蛋白(phycoerythrocyanin)B亚基上的功能.通过在不同生理条件下对鱼腥藻PCC7120的培养,还对这两个基因的调控表达进行了初步的探索.结果表明:all5292和alt0647的表达与氮源的缺乏与否有联系,在氮胁迫条件下两个基因均进行了转录而在氮源充足的情况下则没有表达.     

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。     

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。     

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。     

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.     

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.     

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。     

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。     

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.     

小鼠金属硫蛋白-I在鱼腥藻7120中的融合表达及其纯化(英文)

为了提高小鼠金属硫蛋白-I(mMT-I)在鱼腥藻7120(Anabaena sp.PCC 7120)中的表达量、便于表达产物的分离纯化,构建了新的穿梭融合表达载体pKG-MT。通过pKG-MT,mMT-I cDNA在tac启动子的调控下,以与谷胱甘肽转硫酶(GST)C-末端相融合(GST-MT)的形式在鱼藻中表达。SDS-PAGE结果显示在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下GST-MT在鱼腥藻中表达。经谷胱甘肽亲合层析,从转基因藻中分离、纯化得到GST-MT,利用GSTC-末端的凝血酶酶切位点,用凝血酶对GST-MT进行柱上酶切,经Sephadex G50除去凝血酶得到mMT-I。SDS-PAGE表明纯化得到所要的目标产物;ELISA测定结果显示从每克转基因藻(鲜重)中可纯化得到0.9 mg mMT-I;原子吸收测定表明纯化得到的mMT-I的镉离子结合能力接近于天然MT。     

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.