同步化培养后莱茵衣藻生物量和总RNA含量的变化

为了探索莱茵衣藻经光/暗同步化培养后的细胞生长和总RNA的变化规律。本研究检测了16h光/8h暗同步化培养后莱茵衣藻的生物量和总RNA含量的变化规律。结果,在同步化培养结束后的前28h,莱茵衣藻的生物量呈现有节律的阶梯增长;在同步化培养结束后的28～48h,这种阶梯式增长方式逐步消失。在同步化培养结束后的前24h,总RNA含量呈现有节律的峰-谷-峰变化;在同步化培养结束后的24～48h,这种变化幅度逐步减小,节律周期也逐步缩短。对比同步化培养后莱茵衣藻生物量和总RNA含量的变化可以得出,同步化培养后莱茵衣藻的同步化节律仍然可以维持一定时间;但随着连续光培养时间的延长,这种节律逐步消失,通过测定生物量和总RNA含量的变化可以跟踪同步化培养后莱茵衣藻的同步化变化。     

莱茵衣藻中脂质代谢过程研究进展

微藻中脂质代谢产生的化合物,可用于生物燃料、营养品和生物药品的生产,因此具有重要的经济价值。脂质代谢贯穿微藻的全部生命过程,对微藻的生长发育和应对外界胁迫都具有重要意义。微藻与研究较清楚的真菌和陆地植物在脂质代谢过程方面具有相似性。当然,随着微藻脂质代谢相关功能基因逐渐被鉴定,人们发现微藻的脂质代谢也具有区别真菌和陆地植物的独特性,因此针对微藻脂质代谢过程的分析具有重要意义。莱茵衣藻是研究脂质代谢过程的模式生物,已经通过基因组、转录组、蛋白质组和代谢组等方法,对其质体、内质网和过氧化物酶体中进行的脂质合成和分解过程进行了研究。本文总结了近年来莱茵衣藻质体、内质网和过氧化物酶体中脂质代谢过程的研究成果,并进行综合阐述。     

利用Imaging-PAM研究莱茵衣藻对环境变化的响应

本文介绍了Imaging—PAM—M—Series调制叶绿素荧光成像技术在莱茵衣藻活体叶绿素荧光检测中的应用。该方法利用CCD对藻体直接成像,可同时检测多个样品。后期分析可获得图像中任意区域的初始荧光产量（R）、充分暗适应后PSII的最大光化学效率（眠）、PSⅡ光化学能量转化的有效量子产量【Y（Ⅱ）】、光化学淬灭系数（qp）、非光化学淬灭系数（NPQ）等指标。文章以敌草隆[3-（3,4-dichlorophenyl）-1,1-dimethylurea,DCMU]和没食子酸丙酯（propyl gallate,PG）对莱茵衣藻野生型及其抗DCMU突变体的影响为例,说明该技术在莱茵衣藻研究中是可靠的,具有简单、快速、灵敏等特点。     

野生型莱茵衣藻及其不同突变株的抗NaCl能力检测

检测莱茵衣藻的2种野生型CC-124、CC-125和15个不同突变株对NaCl抗性的结果表明,野生型品系CC-124和CC-125对NaCl的抗性达到260 mmo1·L-1,其中叶绿素b缺失的cbn1-48mt 和cbn1-48mt-基因突变株品系对NaCl最为敏感(即对100mmo1·L-1以上浓度的NaC1表现敏感).用紫外线照射诱变法,对野生型品系CC-124进行诱导,初步筛选出对150mmol·L-1NaCl敏感的突变株6个,对350mmol·L-1NaCl有抗性的突变株2个.     

莱茵衣藻基因组文库的构建

以莱菌衣藻CC-849为材料,提取基因组DNA,利用BamHⅠ和BglⅡ对基因组DNA进行酶解,获得了可用于构建基因组文库的6-12 kb的基因组片段,并浓缩至200 ng/μL。该片段与λDNA载体连接,经噬菌体蛋白包装、侵染大肠杆菌XL1-blue后,获得了莱菌衣藻基因组文库。该文库的滴度为2.12×10~5 pfu/mL,共有转化子4.26×10~4个,插入片段的平均长度约为9kb,扩增后基因组文库滴度为9.5×10~6 pfu/mL。     

蓝藻挥发物α-紫罗酮诱导莱茵衣藻细胞程序性死亡

[目的]蓝藻挥发性有机化合物(VOCs)对其他藻类的化感作用可促进蓝藻成为富营养化水体优势种群,本研究旨在以VOCs主要成分α-紫罗酮为例揭示其化感致死机制.[方法]采用α-紫罗酮处理莱茵衣藻,测定藻细胞生长以及致死浓度下藻细胞光合性能、caspase-likes活性和DNA ladders.[结果]采用0.05和0....     

小球藻和莱茵衣藻原生质体的电转化研究

以小球藻及莱茵衣藻原生质体为受体细胞,利用电击法将质粒p CAMBIA1301转入小球藻和莱茵衣藻,摸索电击转化条件并进行分子检测。结果表明:两类藻都对潮霉素敏感,小球藻及莱茵衣藻分别在含25 mg/L和100 mg/L潮霉素的固体培养基上的生长被完全抑制;小球藻和莱茵衣藻原生质体电击转化的最佳电击场强分别为0.8 k V/cm和0.6 k V/cm,最佳脉冲时间均为10 ms;制备原生质体和通过2-脱氧-D-葡萄糖处理可明显提高转化效率;分子检测说明GUS报告基因成功转入两种藻并可稳定遗传。     

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。     

莱茵衣藻中Limp77基因干涉后油脂含量升高

随着能源危机问题日益严重,可再生能源的研究渐渐成为目前研究的热点.微藻生物能源又以众多的优点成为目前可再生能源的研究重点.我们发现,在减氮培养下的莱茵衣藻,其油脂含量增加,Limp77基因的表达量明显下降.Limp77基因编码的是一类CCCH型锌指蛋白,具有通过与DNA、RNA结合来实现转录的调控或通过调控其它基因转录的锌指蛋白来实现转录调控的功能,极可能参与到莱茵衣藻油脂代谢调控中.通过利用RNAi干涉技术构建Limp77基因的干涉载体,并通过玻璃珠法转入莱茵衣藻（Chlamydomonas reinhardtii）2A38中,研究其与油脂相关的生理生化指标的变化.实验结果表明,Limp 77基因明显抑制莱茵衣藻油脂的积累.     

莱茵衣藻Nfr-4突变株中类胡萝卜素含量的变化及其对藻生长的影响

文章对相同条件下培养的莱茵衣藻野生型CC-137和八氢番茄红素脱氢酶(phytoene desaturase,PDS)基因突变株Nfr-4的生长进行分析;并用反相高效液相色谱分析总有色类胡萝卜素以及叶绿素含量变化,结果表明两者生长的差异明显;Nfr-4突变株的单细胞叶绿素和总有色类胡萝卜素含量高于野生型CC-137的。     

Effect of O2:CO2 ratio on the primary metabolism of Chlamydomonas reinhardtii

High oxygen:carbon dioxide ratios may have a negative effect on growth and productivity of microalgae. To investigate the effect of O₂ and CO₂ concentrations and the ratio between these on the metabolism of Chlamydomonas reinhardtii we performed turbidostat experiments at different O₂:CO₂ ratios. These experiments showed that elevated O₂ concentrations and the corresponding increase in the ratio of O₂:CO₂ common in photobioreactors led to a reduction of growth and biomass yield on light with 20–30%. This is most probably related to the oxygenase activity of Rubisco and the resulting process of photorespiration. Using metabolic flux modeling with measured rates for each experiment we were able to quantify the ratio of the oxygenase reaction to the carboxylase reaction of Rubisco and could demonstrate that photorespiration indeed can cause the reduction in biomass yield on light. The calculated ratio of the oxygenase reaction to the carboxylase reaction was 16.6% and 20.5% for air with 2% CO₂ and 1% CO₂, respectively. Thus photorespiration has a significant impact on the biomass yield on light already at conditions common in photobioreactors (air with 2% CO₂). Biotechnol. Bioeng. 2011;108: 2390–2402. © 2011 Wiley Periodicals, Inc.     

Photorespiratory ammonium assimilation in chloroplasts of Chlamydomonas reinhardtii

The effect of CO₂ concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw-15 mutant (lacking cell wall) and in the high CO₂-requiring double mutant cia-3/cw-15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw-15 cells, both the extracellular (CA_ext) and chloroplastic (CA_chl) CA activities increased after transferring cells from media bubbled with 5% CO₂ in air (v/v, high-C_i cells) to 0.03% CO₂ (low-C_i cells), whereas in cia-3/cw-15 cells only the CA_ext was induced after adaptation to low-C_i conditions and the CA_chl activity was negligible. During adaptation to low-C_i conditions in the presence of 1 mM of l-methionine-D,L-sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw-15 in the presence of MSX was lower in cells grown and kept at 5% CO₂ than in high-C_i cells adapted to 0.03% CO₂. The double mutant cia-3/cw-15 excreted photorespiratory ammonium at a higher rate than did cw-15. Total GS activity (GS-1 plus GS-2) increased during adaptation to 0.03% CO₂ in both strains of C. reinhardtii. However, only the activity GS-2, which is located in the chloroplast, increased during the adaptation to low CO₂, whereas the cytosolic GS-1 levels remained similar in high and low-C_i cells. We conclude that: (1) cia-3/cw-15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS-2/GOGAT cycle; and (3) chloroplastic GS-2 concentration changes in response to the variation of environmental CO₂ concentration.     

Blue light delays commitment to cell division in Chlamydomonas reinhardtii

In this study, we describe the effect of red and blue light on the timing of commitment to cell division in Chlamydomonas reinhardtii. The time point and cell size after which cells can complete their cell cycle with one division round were determined for cultures that were exposed to various red and blue light periods. We show that the commitment point of cells grown in blue light is shifted to a later time point and a larger cell size, when compared with cells grown in red light. This shift was reduced when cultures were exposed to shorter blue light periods. Furthermore, this shift occurred only when exposure to blue light started before the cells attained a particular size. We conclude that the critical cell size for cell division, which is the cell size at which commitment to cell division is attained, is dependent on spectral composition.     

Photosynthetic efficiency of Chlamydomonas reinhardtii in attenuated, flashing light

As a result of mixing and light attenuation, algae in a photobioreactor (PBR) alternate between light and dark zones and, therefore, experience variations in photon flux density (PFD). These variations in PFD are called light/dark (L/D) cycles. The objective of this study was to determine how these L/D cycles affect biomass yield on light energy in microalgae cultivation. For our work, we used controlled, short light path, laboratory, turbidostat‐operated PBRs equipped with a LED light source for square‐wave L/D cycles with frequencies from 1 to 100 Hz. Biomass density was adjusted that the PFD leaving the PBR was equal to the compensation point of photosynthesis. Algae were acclimated to a sub‐saturating incident PFD of 220 µmol m^?2 s^?1 for continuous light. Using a duty cycle of 0.5, we observed that L/D cycles of 1 and 10 Hz resulted on average in a 10% lower biomass yield, but L/D cycles of 100 Hz resulted on average in a 35% higher biomass yield than the yield obtained in continuous light. Our results show that interaction of L/D cycle frequency, culture density and incident PFD play a role in overall PBR productivity. Hence, appropriate L/D cycle setting by mixing strategy appears as a possible way to reduce the effect that dark zone exposure impinges on biomass yield in microalgae cultivation. The results may find application in optimization of outdoor PBR design to maximize biomass yields. Biotechnol. Bioeng. 2012; 109: 2567–2574. © 2012 Wiley Periodicals, Inc.     

衣藻叶绿体分裂基因CrFtsZ1在E.coli中的表达

FtsZ蛋白在细菌的分裂中起着重要作用,能够在分裂位点形成一个环状结构而控制细菌的分裂过程。细胞内FtsZ蛋白浓度的明显降低或异常升高均可阻断正常的细胞分裂过程进而导致丝状菌体的产生。为了研究衣藻叶绿体分裂基因ftsZ的功能,构建了衣藻CrFtsZ1的原核表达重组质粒。试验结果表明,衣藻ftsZ的表达严重影响了大肠杆菌的分裂,初步证明衣藻FtsZ蛋白不仅与E．coli FtsZ蛋白在序列上相似,而且也有着相似的功能,同时这一结果也为真核细胞中质体的内共生起源提供了直接的证据。     

Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light

Efficient light to biomass conversion in photobioreactors is crucial for economically feasible microalgae production processes. It has been suggested that photosynthesis is enhanced in short light path photobioreactors by mixing‐induced flashing light regimes. In this study, photosynthetic efficiency and growth of the green microalga Chlamydomonas reinhardtii were measured using LED light to simulate light/dark cycles ranging from 5 to 100 Hz at a light‐dark ratio of 0.1 and a flash intensity of 1000 µmol m⁻² s⁻¹. Light flashing at 100 Hz yielded the same photosynthetic efficiency and specific growth rate as cultivation under continuous illumination with the same time‐averaged light intensity (i.e., 100 µmol m⁻² s⁻¹). The efficiency and growth rate decreased with decreasing flash frequency. Even at 5 Hz flashing, the rate of linear electron transport during the flash was still 2.5 times higher than during maximal growth under continuous light, suggesting storage of reducing equivalents during the flash which are available during the dark period. In this way the dark reaction of photosynthesis can continue during the dark time of a light/dark cycle. Understanding photosynthetic growth in dynamic light regimes is crucial for model development to predict microalgal photobioreactor productivities. Biotechnol. Bioeng. 2011;108: 2905–2913. © 2011 Wiley Periodicals, Inc.     

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.     

Involvement of ethylene and nitric oxide in cell death in mastoparan‐treated unicellular alga Chlamydomonas reinhardtii

This work demonstrates a contribution of ethylene and NO (nitric oxide) in MP (mastoparan)‐induced cell death in the green algae Chlamydomonas reinhardtii. Following MP treatment, C. reinhardtii showed massive cell death, expressing morphological features of PCD (programmed cell death). A pharmacological approach involving combined treatments with MP and ethylene‐ and NO‐interacting compounds indicated the requirement of trace amounts of both ethylene and NO in MP‐induced cell death. By employing a carbon dioxide laser‐based photoacoustic detector to measure ethylene and a QCL (quantum cascade laser)‐based spectrometer for NO detection, simultaneous increases in the production of both ethylene and NO were observed following MP application. Our results show a tight regulation of the levels of both signalling molecules in which ethylene stimulates NO production and NO stimulates ethylene production. This suggests that, in conjunction with the elicitor, NO and ethylene cooperate and act synchronously in the mediation of MP‐induced PCD in C. reinhardtii. To the best of our knowledge, this is the first report on the functional significance of ethylene and NO in MP‐induced cell death.     

Characterization of an l-aspartate aminotransferase activity in Chlamydomonas reinhardtii

The isolation and characterization of an l -aspartate aminotransferase (AAT) activity (EC 2.6.1.1) in the unicellular green alga Chlamydomonas reinhardtii 6145c are reported for the first time. The enzyme transaminates aspartate with the 2-oxoglutarate-glutamate system, and exhibits maximum aminotransferase activity at pH 7.8 and 37°C. It has an Mr of 138 kDa, contains pyridoxal 5'-phosphate, and has a K_m apparent for oxalacetate of 0.55 m M and exhibits positive co-operativity with l -aspartate with an S_0.5 of 2.53 m M and a Hill coefficient of 1.57. In vivo, activity levels were affected by the carbon and nitrogen sources and by the change in the dark-light conditions. All these responses are interpreted in terms of a possible physiological regulation of AAT activity to keep the intracellular pools of glutamate and aspartate within margins compatible with environmental fluctuations.