盐胁迫状态下莱茵衣藻849光合特性的初步研究

以莱茵衣藻（Chlamydomonas reinhardtii）为研究对象,对其在不同NaCl浓度胁迫条件下的光合特性进行了初步研究。结果表明0.04 mol·L^-1的NaCl对莱茵衣藻的生长无显著影响,而0.075 mol·L^-1的NaCl使莱茵衣藻生长速率下降了50%。在低光照强度下,NaCl胁迫下的衣藻光合速率和呼吸速率在最初7 h内比对照组高;而在较高光照强度下,NaCl胁迫下的衣藻的光合速率和呼吸速率均比对照组低;而且光合速率和呼吸速率升高和下降的幅度与NaCl浓度成正比。     

Effect of red and blue light on acclimation of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> to CO<Subscript>2</Subscript>-limiting conditions

Effects of red (RL) and blue (BL) light on acclimation of the unicellular green alga Chlamydomonas reinhardtii to the low level of ambient CO₂ were studied. C. reinhardtii cells grown at 5% CO₂ and under white light (170 μmol/(m²s)) had a relatively low activity of extracellular carbonic anhydrase (CA), a low affinity for dissolved inorganic carbon, and a low rate of photosynthesis under CO₂-limiting conditions. These cells readily started acclimation to the low CO₂ concentration when they were exposed to atmospheric air (～ 0.03% CO₂) under RL or BL (150 μmol/(m² s) each). The acclimation was manifested in a significant increase in the CO₂-limited rate of photosynthesis, the affinity for dissolved inorganic carbon, and the extracellular CA activity with no difference between RL-and BL-cells. Independently of light quality, the acclimation was completed for 5–7 h after cell exposure to air. As is evident from RL-and BL-dependent changes in the sum of chlorophylls and chlorophyll a/b ratio, transfer of C. reinhardtii cells to air and RL or BL triggered also the process of algal photosynthetic adaptation to light quality. However, this process did not interfere with acclimation to low CO₂ because started 4 h later. On the basis of similarity in the low CO₂-induced changes under RL and BL, it is concluded that acclimation of C. reinhardtii to CO₂-limiting conditions does not depend on light quality.     

衣藻细胞玻璃化超低温保存技术的研究

本研究以衣藻为材料,探讨其玻璃化超低温保存的条件和方法,结果表明,衣藻经含0.25mol/L蔗糖溶液的TAP培养基预培养一天后,在玻璃化冷冻保护剂中脱水5分钟,直接投稿液氮,48小时后快速化冻,去保护剂并用含0.5mol/L蔗糖溶液的TAP培养基境培养一天,再转到ATP培养基暗培养一天,最后置光照条件下恢复培养,其存活率可达31.45％,恢复培养后衣藻细胞的生长规律与未冻存的衣藻相一致。     

Protection against the photo-induced inactivation of the photosystem II complex by abscisic acid

To examine the effect of abscisic acid (ABA) on the photo‐induced inactivation of the photosystem II (PSII) complex, a suspension culture of Chlamydomonas reinhardtii was treated with ABA for 24 h in darkness and then, after removal of ABA, the cells were exposed to strong light at a photon flux density of 2000 μ mol m ^{? 2} s ^{? 1} at various temperatures. The activity of PSII, as estimated in terms of chlorophyll fluorescence and the evolution of oxygen, decreased significantly during the exposure of cells to the strong light, and the extent of the photo‐induced decrease in PSII activity was much greater at lower temperatures. Irrespective of temperature, the decrease in PSII activity in ABA‐treated cells was significantly smaller than that in control cells. Moreover, the recovery of PSII activity from the photo‐inactivated state in ABA‐treated cells was significantly faster than that in control cells. The recovery of PSII activity in both ABA‐treated and control cells was almost entirely prevented by the presence of chloramphenicol. These results indicate that ABA protects the PSII complex in C. reinhardtii against photo‐induced inactivation by accelerating the recovery of this complex.     

Bicarbonate protects the water-oxidizing complex of photosystem II against thermoinactivation in intact <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells

Currently available data about bicarbonate (BC) action on the Mn-containing water-oxidizing complex (WOC) of the photosystem II (PSII) were obtained almost solely in vitro, e.g. on subchloroplast membrane fragments enriched with PSII. To investigate the in vivo BC effect on the PSII donor side, we used the method of dark thermoinactivation of intact Chlamydomonas reinhardtii cells. Photosynthetic activity of PSII was measured as photoinduced changes in the PSII chlorophyll fluorescence yield and as the rate of photosynthetic oxygen evolution. To exclude a “direct” effect of the absence of BC on the PSII activity, before measurements of the photosynthetic activity, the concentration of BC in all samples was equalized by addition of NaHCO₃ to each of them (except for those that contained 5 mM of NaHCO₃ during thermoinactivation) to reach the final concentration of 5 mM. This allowed registering only so-called “irreversible” (i.e., not reversible by subsequent addition of BC) effect of the absence of BC during thermoinactivation. It was shown that, if 5 mM NaHCO₃ was added to the medium before thermoinactivation, the rate of inactivation of the PSII donor side was lower than in BC-depleted medium 1.5-to 2-fold. The obtained results are interpreted as an indication that BC protects the donor side of PSII against thermoinactivation in vivo, in intact C. reinhardtii cells. This proves the correctness of the earlier proposition that BC is an integral constituent of the Mn-containing water-oxidizing complex of PSII. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 3, pp. 342–349. The article was translated by the authors.     

Isolation of a cDNA fragment coding for Chlamydomonas reinhardtii ferredoxin and expression of the recombinant protein in Escherichia coli.

A cDNA clone coding for mature C. reinhardtii ferredoxin has been isolated from a cDNA library using PCR and two oligonucleotide primers based on the N- and C-termini of the protein's amino acid sequence. The nucleotidic sequence of the PCR fragment (299 bp) agreed well with the amino acid sequence since a single conservative substitution (Thr-7 to Ser) could be deduced. The PCR fragment was inserted into the expression vector pTrc 99A, using the incorporated NcoI and BamHI restriction sites and the construction used to transform E. coli (DH5 F′). After subsequent large scale expression and purification of the recombinant protein, biochemical and biophysical analysis have indicated that the product isolated from E. coli is homologous to native ferredoxin isolated from green algae.     

Regulation of the expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in a cyanobacterium, Synechococcus PCC7942

When cyanobacterium cells are grown under extremely low CO₂ concentration, the number of carboxysomes, structures containing ribulose-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), is known to increase. This suggests that Rubisco helps to regulate photosynthesis in cyanobacteria. However, no studies have been done on the changes of Rubisco content and activity in response to the extracellular CO₂ concentration, and no information is available on its effect on photosynthesis. To elucidate the relationship between the expression responses of Rubisco and extracellular CO₂, wild-type cells (Synechococcus PCC7942) and carboxysome-lacking cells were grown under various CO₂ concentrations, and Rubisco activity was determined. In both strains, Rubisco activity increased when the cells were grown under a CO₂ concentration around, or less than, K _1/2(CO₂) of photosynthesis. In carboxysome-lacking cells, Rubisco activity increased five to six times at most, and a simultaneous increase in the rate of photosynthesis was observed. These results suggest that stimulation of expression of Rubisco occurs to compensate for the decrease in the rate of photosynthesis under CO₂-limited conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

The Nostoc-Gunnera symbiosis: carbon fixation and translocation

The in vitro specific activity of ribulose-1,5-bisphosphate carboxylase (Rubisco; EC 4. 1. 1. 39) and the dark and light in vivo CO₂ fixation activities were determined in the cyanobiont of Gunnera . Compared to the free-living isolate Nostoc PCC 9231, the in vitro Rubisco activity was high, while the in vivo CO₂ fixation was very low. Light did not significantly influence CO₂ fixation if the cyanobiont was left in the sliced Gunnera tissues, while a small light stimulation was found for CO₂ fixation of the freshly-isolated cyanobiont. The adjacent non-infected Gunnera tissue showed a very low CO₂ fixation. A rapid translocation of fixed ¹⁴CO₂ from leaves towards apical parts of the plant was apparent, in particular to the symbiotic tissue. The ¹⁴C label appeared mainly in soluble form in this tissue and was rapidly catabolised as shown by ¹⁴C chase experiments. Also, short-term experiments revealed that maximum ¹⁴C accumulation occurred in the symbiotic tissue showing the highest rates of nitrogen fixation (Söderbäck et al. 1990), about 10–15 mm from the plant apex. The data were taken to indicate that there is a modification in the photosynthetic light reaction of the cyanobiont and that the cyanobiont lives heterotrophically in the dark on photo-synthate rapidly delivered from nearby leaves of the host plant.     

外源基因在莱茵衣藻叶绿体中的表达

把莱茵衣藻(Chlamydomonas reinhardtii)叶绿体作为生物反应器来表达外源基因具有广阔的应用前景。人们利用莱茵衣藻叶绿体表达体系已成功表达多种重组蛋白,其中包括人类药用蛋白。综述了莱茵衣藻叶绿体转化的方法、影响外源基因表达的主要因素以及外源基因在莱茵衣藻叶绿体表达研究进展。     

衣藻生物制氢的研究进展

综述了利用衣藻生产氢气作为再生能源的研究进展。分别介绍了衣藻产氢的代谢机理、培养条件、衣藻氢化酶的特性以及利用分子生物学手段、生物信息学手段和生物工程技术提高衣藻生物制氢效率的方法,包括氢化酶的氧耐受性的改造、外源氢化酶基因的表达、影响衣藻产氢的关键基因的筛选、利用缺硫培养基和固定化培养方法提高氢气产量等。最后,还对利用衣藻生物制氢的可行性和经济性进行了分析,对其发展方向提出自己的看法。     

Insertional mutagenesis to isolate acetate-requiring mutants in Chlamydomonas reinhardtii

Abstract An arg 7 mutant of the green alga Chlamydomonas reinhardtii was transformed with pARG7.8, a plasmid bearing the wild-type ARG 7 gene. Out of 4100 arg⁺ transformants selected on an arginine-free medium supplemented with acetate, nine failed to grow on acetate-free medium (ac⁻ mutants). The results of the genetic and molecular analysis of several ac⁻ mutants are in agreement with the hypothesis that they originated from insertion of the incoming plasmid into the nuclear genome. These mutants should constitute valuable tools for isolating the corresponding wild-type genes after plasmid rescue into Escherichia coli .