转基因鱼腥藻7120光异养生长的研究

研究了不同有机碳源对转基因鱼腥藻７１２０生长的影响,在此基础上进行了光异养条件的优化,并研究了外源添加剂Ｌ－精氨酸、生长激素２,４－Ｄ与ＫＴ对藻细胞生长的影响。结果表明：蔗糖和葡萄糖能促进藻体的生长;最优碳氮源组合为葡萄糖６ｇ／Ｌ、ＮａＮＯ３１．５ｇ／Ｌ,经高压灭菌;Ｌ－精氨酸不支持藻体的光异养生长;一定含量的２,４－Ｄ与ＫＴ能促进藻体在光异养条件下的后期生长。     

人肿瘤坏死因子α在鱼腥藻7120中的表达

通过三亲结合转移方式,将含人肿瘤坏死因子α（ＴＮＦ－α）ｃＤＮＡ和ｐｓｂＡ启动子的鱼腥藻－大肠杆菌穿梭质粒ＰＤＣ－ＴＮＦ导入单细胞丝状鱼腹藻７１２０中。     

Kranz anatomy is not essential for terrestrial C4 plant photosynthesis.

An important adaptation to CO2-limited photosynthesis in cyanobacteria, algae and some plants was development of CO2-concentrating mechanisms (CCM). Evolution of a CCM occurred many times in flowering plants, beginning at least 15-20 million years ago, in response to atmospheric CO2 reduction, climate change, geological trends, and evolutionary diversification of species. In plants, this is achieved through a biochemical inorganic carbon pump called C4 photosynthesis, discovered 35 years ago. C4 photosynthesis is advantageous when limitations on carbon acquisition are imposed by high temperature, drought and saline conditions. It has been thought that a specialized leaf anatomy, composed of two, distinctive photosynthetic cell types (Kranz anatomy), is required for C4 photosynthesis. We provide evidence that C4 photosynthesis can function within a single photosynthetic cell in terrestrial plants. Borszczowia aralocaspica (Chenopodiaceae) has the photosynthetic features of C4 plants, yet lacks Kranz anatomy. This species accomplishes C4 photosynthesis through spatial compartmentation of photosynthetic enzymes, and by separation of two types of chloroplasts and other organelles in distinct positions within the chlorenchyma cell cytoplasm.     

Photosynthetic bicarbonate utilization inPorphyra haitanensis (Bangiales, Rhodophyta)

The activities of carbonic anhydrase (CA) and photosynthesis of Porphyra haitanensis were investigated in order to see its photosynthetic utilization of inorganic carbon source. Both intra- and extra-cellular CA activities existed in the thallus. CA inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), remarkably depressed the photosynthetic oxygen evolution in seawater of pH 8.2 and 10.0, and EZ showed stronger inhibition than AZ. The observed net photosynthetic rate in seawater of pH 8.2 was much higher than that of CO2 supply theoretically derived from spontaneous dehydration of HCO3-. P. haitanensis also showed a rather high pH compensation point (9.9). The results demonstrated that P. haitanensis could utilize bicarbonate as the external inorganic carbon source for photosynthesis. The bicarbonate utilization was closely associated with dehydration catalyzed by extracellular CA activity. The inorganic carbon composition in seawater could well saturate the photosynthesis of P. haitanensis. The low Km value and compensation points for inorganic carbon reflected the existence of CO2- concentrating mechanism in this alga.     

异形胞发育的蓝细菌DnaA蛋白的表达研究

为了研究DNA复制与Anabaena sp. PCC7120异形胞发育之间的关系.构建了Anabaena PCC7120的复制起始蛋白DnaA的重组表达质粒,在Escherichia coli中诱导其超量表达,纯化后免疫家兔获得抗体,采用Western blotting检测DnaA在营养细胞和异形胞中的表达情况.结果显示,两种不同类型的细胞中都能够检测到DnaA的表达,而且在不同缺氮诱导时间,异形胞中DnaA的表达存在差异.这说明DNA复制起始与异形胞的发育可能存在着一定的关系.     

鱼腥藻PCC 7120染色体上relBE同源基因的克隆及表达

为研究鱼腥藻PCC7120染色体上与relBE同源的基因asl4561和asl4562表达产物的毒素-抗毒素作用,从鱼腥藻细胞中提取总基因组DNA,设计特异引物PCR扩增目的基因asl4561和asl4562,与T载体连接后经XhoI和EcoR I双酶切并与表达载体pET28a（＋）连接,由IPTG诱导表达,并在IPTG浓度和诱导时间上对asl4561基因的表达条件进行了优化.琼脂糖凝胶电泳显示扩增出了264bp的asl4561基因和213bp的asl4562基因,SDS-PAGE检测表明成功表达出15.65kD和13.55kD的两蛋白,当诱导温度28℃、IPTG浓度0.4 mmol/L、诱导时间6h时,asl4561基因表达蛋白在细菌裂解液上清中表达量最大.     

HCO3 - 高亲和转运蛋白操纵子基因在聚球藻7942 CO2浓缩机制中功能的研究

蓝藻 Synechococcus sp.PCC7942 HCO3 - 高亲和转运蛋白操纵子基因 cmpABCD 是其CO2浓缩机制中的调控基因之一.本研究用携带潮霉素B磷酸转移酶基因(hygromycin B pho transferase, hpt) 筛选标记的同源双臂整合载体pUC-HATH转化蓝藻Synechococcus sp.PCC7942,以潮霉素B作为筛选试剂筛选出具潮霉素B抗性的转化藻,运用引物PCR方法证实潮霉素B磷酸转移酶基因表达盒通过质粒pUC-HATH的介导已定点插入蓝藻 Synechococcus sp.PCC7942 基因组中,成功地构建了具有潮霉素B抗性的cmpBCD 基因插入失活突变藻株.并最终通过比较野生藻Synechococcus sp.PCC7942 和突变藻Synechococcus sp.PCC7942 在不同 Na2CO3浓度的改良BG-11培养基中生长特性,探讨了HCO3 -高亲和转运蛋白操纵子 cmpABCD 基因失活对藻体生长的影响.     

海洋浮游藻(Emiliania huxleyi)无机碳利用机理

探讨了海洋浮游藻Emilianiahuxleyi无机碳利用机制以及与藻龄间的关系．结果表明Emilianiahuxleyi细胞在其指数（生长）期和静止（生长）期对无机碳利用的途径不同．指数期细胞是通过HCO-3直接吸收途径,同时还与钙化作用有关,而静止期细胞则是通过CO2吸收途径,存在细胞外碳酸酐酶．     

鱼腥藻铁吸收调节蛋白基因(alr0957)的克隆和表达

依据CyanoBase提供的鱼腥藻PCC7120 furC基因(alr0957)的序列信息设计了一对特异性引物,用Touch-down PCR的方法从基因组DNA中扩增得到大小约450bp的目的片段.通过TA克隆的方法将该片段连接到pMD18-T载体上筛选出重组质粒pMD18-T-fur,然后进行双酶切,纯化furC基因,再连接到原核表达载体pET-28a(+)上,转化表达菌株BL21(DE3).经PCR、双酶切和测序鉴定,对阳性菌株进行IPTG诱导表达,SDS-PAGE检测重组蛋白.结果表明:在25℃条件下经1mmol/L IPTG诱导20h,融合蛋白被成功表达,其分子量约为19 000,为进一步纯化蛋白和对基因的调控功能方面研究奠定了基础.     

鱼腥藻PCC7120α质粒上的parD/E同源基因all7155/asl7156的克隆及表达

为研究鱼腥藻PCC7120质粒上毒素抗毒素系统的蛋白质性质和相互作用,根据NCBI中PCC7120的α质粒上的all7155和asl7156基因数据,通过降落PCR克隆了目的基因(all7155/336 bp,asl7156/258 bp).将目的基因片段连接至p MD18-T构建了克隆载体,蓝白斑筛选了阳性克隆,经双酶切纯化后将目的基因连接至表达载体p ET30a(+),并转入表达菌BL21中,测序后证实构建成功,并利用IPTG诱导进行了表达.通过NCBI的BLAST蛋白比对,发现了all7155和asl7156与已知的Par D/E毒素抗毒素系统同源,可通过对此基因的克隆,表达和蛋白性质来进一步认识Par D/E系统.     

Exogenous carbon acquisition of photosynthesis in Porphyra haitanensis (Bangiales, Rhodophyta) under emersed state

The photosynthetic performances of Porphyra haitanensis thalli were investigated in order to understand its mechanisms for exogenous carbon acquisition during emersion at low tide. The emersed photosynthesis was studied by altering the pH value in the water film on the thalli surface, treating them with carbonic anhydarase inhibitors (acetazolamide and 6-ethoxyzolamide), adjusting the CO2 concentrations in the air, and comparing the theoretical maximum CO2 supply rates within the adherent water film with the observed photosynthetic CO2 uptake rates. It was found that the principal exogenous inorganic carbon source for the photosynthesis of P. haitanensis during emersion was atmospheric CO2. The driving force of CO2 flux across the water film was the CO2 concentration gradient within it. Carbonic anhydrase accelerated both extracellular and intracellular CO2 transport. The emersed photosynthesis of P. haitanensis was limited by the present atmospheric CO2 level, and would be enhanced by atmospheric CO2 rise that would trigger global warming.     

不同光质对嗜热蓝细菌Synechococcus sp. PCC6715生长的影响

使用光合有效光量子数密度(PAR)均为100 μmol/(m²·s)的白光(对照)、红光和蓝光, 对对数生长期的嗜热蓝细菌PCC6715进行培养, 以期探明不同光质对不表达藻红素的蓝细菌光适应生长的影响。结果表明, 在不同光质培养对数生长期PCC6715的过程中, 与白光相比, 干重在蓝光下明显增加, 红光下明显减少; 红、蓝光质对光合色素合成产生影响的开始时间和持续时间有所不同; 最大光能转化效率(F_v/F_m)在蓝光下明显提高, 红光下明显降低, 培养6天后, 红、蓝光条件下的F_v/F_m均趋于稳定。蓝光有利于PCC6715的生长, 红光不利于PCC6715的生长; 同时, PCC6715在红、蓝光质中均产生适应性生长, 藻蓝素的减少使PCC6715在红光中发生适应性生长, 蓝光中PCC6715发生适应性生长是由于藻蓝素的增加。     

Cloning and Characterization of the fecC Gene Necessary for Optimal Growth under Iron-Deficiency Conditions in the Cyanobacterium Anabaena sp. PCC 7120

The fecC gene encoding a putative iron (Ⅲ) dicitrate transporte rwas cloned from nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, and inactivated. The mutant grows normally in medium with NO3^- , NH1^- 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 the fecC gene is expressed under iron-deficient conditions, but is not expressed under iron-replete conditions. These results revealed that fecC gene product is required for optimal growth under iron-deficient conditions in Anabaena sp. PCC 7120.     

A carbon isotope record of CO2 levels during the late Quaternary

Analyses of gases trapped in continental ice sheets have shown that the concentration of CO2 in the Earth's early atmosphere increased from 180 to 280 p.p.m. during the most recent glacial-interglacial transition. This change must have been driven by an increase in the concentration of CO2 dissolved in the mixed layer of the ocean. Biochemical and physiological factors associated with photosynthetic carbon fixation in this layer should lead to a relationship between concentrations of dissolved CO2 and the carbon isotopic composition of phytoplanktonic organic material, such that increased atmospheric CO2 should enhance the difference in 13C content between dissolved inorganic carbon and organic products of photosynthesis. Here we show that a signal related to atmospheric CO2 levels can be seen in the isotope record of a hemipelagic sediment core, which we can correlate with the CO2 record of the Vostok ice core. Calibration of the relationship between isotope fractionation and CO2 levels should permit the extrapolation of CO2 records to times earlier than those for which ice-core records are available.     

Photosynthesis of Populus euphratica and its response to elevated CO2 concentration in an arid environment

The photosynthetic characterization of Populus euphratica and its response to the elevated carbon dioxide concentration ([CO2]) were analyzed based on its net photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), and water use efficiency (WUE) at different groundwater depths measured by a portable gas exchange system (LI-6400) in the lower reaches of the Tarim River. The results showed that the elevation of [CO2] decreased the gs, and increased the Pn, Ci and WUE of P. euphratica. However, the effects of the elevated [CO2] on gs, Pn, Ci and WUE varied considerably with groundwater depth. The response of photosynthesis to rising [CO2] was stronger at the greater groundwater depth (more than 6 m) than that at the shallower groundwater depth (less than 6 m). The critical groundwater depth required to maintain the normal survival of P. euphratica was less than 6 m. When the groundwater depth increased to more than 6 m, P. euphratica encountered moderate water stress, and the plant suffered severe water stress when the groundwater depth increased to more than 7 m.     

Effect of Cl- on photosynthetic bicarbonate uptake in two cyanobacteria Microcystis aeruginosa and Synechocystis PCC6803

Photosynthetic inorganic carbon utilization was investigated in two cyanobacteria Microcystis aeruginosa and Synechocystis PCC6803 grown in standing culture. Photosynthetic rates for the two algae reached about 10 times the theoretical CO₂ supply rate at low dissolved inorganic carbon (DIC) of 100 μmol/L, and the rates were unaffected by the addition of 20 mmol/L Na⁺, indicating that the two algae possessed Na+-independent HCO^-₃ utilization for photosynthesis under low DIC. Their photo- synthetic rates at low DIC were inhibited by higher Cl^- and the degrees of inhibition were increased with the rise of CI^- concentration, and in the presence of Diphenylamine-2-carboxylate (DPC), a reported Cl^- channel inhibitor, the rates decreased by 74%－82%, implying that putative DPC-sensitive Cl^- channels participate in Na+-independent HCO₃^- uptake for photosynthesis. The experiment of intracellular 14C-DIC accumulation for photosynthesis showed that internal DIC pools decreased by about 80% with 200 μmol/L DPC and by 64%－70% with 100 mmol/L Cl^-. The experiment of chlorophyll a fluorescence quenching showed that initial rates and extents of fluorescence quenching obviously decreased with 200 μmol/L DPC or 100 mmol/L Cl^-. The two experiments gave further evidence that putative DPC-sen- sitive Cl^- channels participate in Na+-independent HCO^-₃ uptake for photosynthesis in the two algae grown in standing culture.     

藻蓝蛋白裂合酶催化藻红胆素(PEB)与CpcA和PecA共价偶联

 将由鱼腥藻Anabaenasp.PCC7120中cpcE/F构建的pCOLADuet-cpcE/F质粒和pACYCDuet-pebA质粒、pCDFDuet-hol-pebB质粒、pETDuet-pecA质粒、pETDuet-cpcA质粒转化入大肠杆菌,在IPTG的诱导下,实现了色素蛋白PEB-CpcA和PEB-PecA的成功表达.吸收光谱和荧光光谱研究表明,CpcE/F能够催化PEB共价连接到CpcA和PecA的Cys-84上形成色素蛋白PEB-PecA和PEB-CpcA,色素蛋白的最大吸收峰为555nm,最强荧光峰为570nm.Zn²⁺电泳分析结果表明,体内重组获得了目标色素蛋白,证明由cpcE/F所编码的蛋白裂合酶在体内重组中能够催化PEB与CpcA和PecA的共价偶联反应,生成非天然色素蛋白PEB-CpcA和PEB-PecA.     

光照对坛紫菜生长和无机碳利用的影响

］以坛紫菜Z-61品系为研究材料,分析坛紫菜叶状体和丝状体在三种光强（10,50,500 μmol·m-2·s-1）下的应答。发现:1）坛紫菜叶状体和丝状体的光系统 Ⅱ 最大量子产量及叶绿素a和藻红蛋白的含量都随光强增加而降低,但净光合速率、最大光合速率、胞外碳酸酐酶活性和总碳酸酐酶活性则随光强增加而增加。2）当光强从10 μmol·m-2·s-1增加到500 μmol·m-2·s-1时,坛紫菜叶状体的生长速率约增加77%;丝状体的生长速率先增加后降低,在光强500 μmol·m-2·s-1下达到最低,比在10 μmol·m-2·s-1下显著下降了约90%。3）在三种光强下,坛紫菜叶状体的最大光合速率、光系统 Ⅱ 最大量子产量和生长速率都大幅高于丝状体。这些结果表明:坛紫菜的光系统 Ⅱ、光合作用与生长对光强的应答并不完全一致;叶状体比丝状体更适应高光,这可能是长期进化的结果;光强可以改变坛紫菜对无机碳的吸收。