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。     

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.     

不同培养条件下莱茵衣藻细胞中SGAT酶活性的变化

利用模式单细胞植物莱茵衣藻,研究不同培养条件下细胞中丝氨酸：乙醛酸氨基转移酶活性的变化情况。结果表明：莱茵衣藻SGAT酶活性的最适pH介于5￣7之间,当pH高于7以后,酶活性逐渐下降;随着细胞密度增加,SGAT酶活性降低;光强可显著影响SGAT酶活性,在一定光强范围内,随着光照强度的增加,酶活性增强;乙酸作为莱茵衣藻的唯一异养碳源也会影响SGAT酶活性,两者间呈正相关;提高氧浓度,显著地提高了细胞内SGAT的酶活性;当二氧化碳浓度增加时,细胞内SGAT的酶活性也略有升高;40℃高温和15℃低温处理后,SGAT酶活性均降低。此外,提高氧浓度时细胞内Gly含量增加,Ser含量减少,Gly/Ser的比值从0.79提高到1.49。     

Ubiquitin metabolism in Chlamydomonas reinhardtii following cold shock

The present work characterizes parameters of ubiquitin turnover in Chlamydomonas reinhardtii Dangeard growing under constant temperature conditions and after an exposure to cold shock. The ratio of free and conjugated ubiquitin to total protein and the rate constant of ubiquitin synthesis and conjugation increased about 2-fold during the first 4 h after cold treatment, whereas the rate constant of ubiquitin degradation reached its maximum 8 h after treatment. The half-life of ubiquitin calculated from the constant of degradation decreased from 6 h to 3.5 h during the first 4 h after completion of the cold treatment. The rate constant of ubiquitin deconjugation did not change after cold treatment. The ratio of free to conjugated ubiquitin decreased temporarily to approximately 8 immediately after cold treatment and increased back to its original value of about 11 at 2 h after cold treatment. These observations raise questions regarding the regulatory mechanisms of ubiquitin synthesis and degradation.     

CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii

Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C. reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2-inducible gene 5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2-exposed algal mutant deficient in the light-activated protein kinase Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon.     

Effect of nitrogen starvation on ammonium assimilation by Chlamydomonas reinhardtii

In nitrogen-starved Chlamydomonas reinhardtii , wild type, strain 21 gr cells, consumption of nitrate, nitrite and ammonium may occur in the dark in the absence of an added carbon source. Consumption of ammonium in the dark was about 25% higher than in the light, while consumption of nitrate or nitrite in the dark was lower than in the light.
N starvation produced a linear increase with time in the intracellular level of glutamine synthetase (GS, EC 6.3.2.1) and glutamate synthase (NADH-GOGAT, EC 1.4.1.14 and ferredoxin-GOGAT, EC 1.4.7.1) activities in C. reinhardtii . The effect on GS₁ (3-fold) and NADH-GOGAT (4.5-fold) was higher than that on GS₂ (1.5-fold) and ferredoxin-GOGAT (1.5-fold).
Experiments with methylammonium, L-methionine-D, L-sulfoximine (MSX) and azaserine suggest that: 1) Ammonium itself decreases the intracellular levels of glutamine synthetase and ferredoxin-glutamate synthase activities; and 2) a metabolite resulting from ammonium assimilation by the alga may be a negative modulator of NADH-glutamate synthase activity.     

Effect of Immobilization on the Kinetic and Stability Properties of O-Acetyl-L-Serine Sulfhydrylase from: Chlamydomonas reinhardtii

The O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) from Chlamydomonas reinhardtii has been immobilized either by ionic binding to DEAE-cellulose or by covalent bonding to alkylamine silica and to vinyl acetate-divinylethylene urea copolymers. The immobilized enzyme had improved stability and showed sigmoidal kinetic behaviour with respect to O-acetyl-L-serine, but without major alterations in the corresponding apparent Km value or in the inhibitory effects of this substrate observed with the enzyme in solution. In addition, significant changes in optimum pH and reaction temperature for O-acetyl-L-serine sulfhydrylase activity were observed.     

Effect of culture conditions on the isocitrate dehydrogenase and isocitrate lyase activities in Chlamydomonas reinhardtii

In the green alga Chlamydomonas reinhardtii , nitrogen staravation induced a reversible increase (2-fold) in NAD-isocitrate dehydrogenase (NAD-IDH; EC 1.1.1.41) and NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO₂, the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD-IDH activity was detected, a 2-fold increase of NADP-IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD- and NADP-IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD-IDH increased in the last 4 h of the dark period, NADP-IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle.     

CrPP2C蛋白影响衣藻细胞鞭毛过渡区结构

目的:在莱茵衣藻细胞中构建并筛选鞭毛组装缺陷突变体,克隆缺陷基因,探索其对鞭毛组装的影响。方法:使用带有巴龙霉素(Paromomycin)抗性的基因片段随机插入衣藻细胞基因组中,通过性状筛选和基因序列分析获得与CrPP2C(Chlamydomonas reinhardtii type 2C protein phosphatase)基因相关的鞭毛异常突变体,根据突变体基本生物学性状和生化分析对CrPP2C基因的功能进行分析。结果:采用电转法成功获得衣藻细胞鞭毛缺陷相关突变体,部分细胞具有短鞭毛,部分细胞则不具有鞭毛;通过RESDA-PCR(restriction enzyme site-directed amplification PCR)对突变体基因序列分析,鞭毛缺陷性状由CrPP2C基因遭到破坏导致;把含有完整CrPP2C基因的重组质粒通过电转法导入突变体后,其鞭毛几乎恢复为野生型长度,并可检测到PP2C-HA融合蛋白的表达;观察鞭毛再生,突变体鞭毛只能再生为原有长度;使用药物处理使鞭毛缩短,突变体鞭毛能正常解聚;电镜检测突变体的鞭毛显微结构,发现过渡区的Y形结构缺陷。结论:CrPP2C基因的破坏导致鞭毛过渡区结构缺失,影响鞭毛组装过程,不组装鞭毛或组装短鞭毛。     

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene （apcA and apcB） into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.     

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。     

Recombination and Heterologous Expression of Aliophycocyanin Gene in the Chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires theintroduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins ormetabolic pathways.In order to accomplish the expression of multiple genes in a single transformationevent,we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonasreinhardtii chloroplast expression vector,resulting in papc-S.The constructed vector was then introducedinto the chloroplast of C.reinhardtii by micro-particle bombardment.Polymerase chain reaction and Southernblot analysis revealed that the two genes had integrated into the chloroplast genome.Western blot andenzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria couldbe correctly expressed in the chloroplasts of C.reinhardtii.The expressed foreign protein in transformantsaccounted for about 2%-3% of total soluble proteins.These findings pave the way to the reconstitution ofmulti-subunit proteins or metabolic pathways in transgenic C.reinhardtii chloroplasts in a single transformationevent.     

Kinetic modeling of the photosynthetic growth of Chlamydomonas reinhardtii in a photobioreactor

The aim of this study was to establish and validate a model for the photosynthetic growth of Chlamydomonas reinhardtii in photobioreactors (PBRs). The proposed model is based on an energetic analysis of the excitation energy transfer in the photosynthesis apparatus (the Z-scheme for photosynthesis). This approach has already been validated in cyanobacteria (Arthorspira platensis) and is extended here to predict the volumetric biomass productivity for the microalga C. reinhardtii in autotrophic conditions, taking into consideration the two metabolic processes taking place in this eukaryotic microorganism, namely photosynthesis and respiration. The kinetic growth model obtained was then coupled to a radiative transfer model (the two-flux model) to determine the local kinetics, and thereby the volumetric biomass productivity, in a torus PBR. The model was found to predict PBR performances accurately for a broad set of operating conditions, including both light-limited and kinetic growth regimes, with a variance of less than 10% between experimental results and simulations.     

Protein Disulfide Isomerase 2 of Chlamydomonas reinhardtii Is Involved in Circadian Rhythm Regulation

Protein disulfide isomerases （PDIs） are known to play important roles in the folding of nascent proteins and in the formation of disulfide bonds. Recently, we identified a PDI from Chlamydomonas reinhardtii （CrPDI2） by a mass spectrometry approach that is specifically enriched by heparin affinity chromatography in samples taken during the night phase. Here, we show that the recombinant CrPDI2 is a redox-active protein. It is reduced by thioredoxin reductase and catalyzes itself the reduction of insulin chains and the oxidative refolding of scrambled RNase A. By immunoblots, we confirm a high-amplitude change in abundance of the heparin-bound CrPDI2 during subjective night. Interestingly, we find that CrPDI2 is present in protein complexes of different sizes at both day and night. Among three identified interac- tion partners, one （a 2-cys peroxiredoxin） is present only during the night phase. To study a potential function of CrPDI2 within the circadian system, we have overexpressed its gene. Two transgenic lines were used to measure the rhythm of phototaxis~ In the transgenic strains, a change in the acrophase was observed. This indicates that CrPDI2 is involved in the circadian signaling pathway and, together with the night phase-specific interaction of CrPDI2 and a peroxiredoxin, these findings suggest a close coupling of redox processes and the circadian clock in C. reinhardtii.     

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。     

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。     

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。