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。     

Membrane lipid biosynthesis in Chlamydomonas reinhardtii: ethanolaminephosphotransferase is capable of synthesizing both phosphatidylcholine and phosphatidylethanolamine

Phosphatidylethanolamine, but not phosphatidylcholine, is found in Chlamydomonas reinhardtii. A cDNA coding for diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase (EPT) was cloned from C. reinhardtii. The C. reinhardtii EPT appears phylogenetically more similar to mammalian aminoalcoholphosphotransferases than to those of yeast and the least close to those of plants. Similar membrane topography was found between the C. reinhardtii EPT and the aminoalcoholphosphotransferases from mammals, yeast, and plants. A yeast mutant deficient in both cholinephosphotransferase and ethanolaminephosphotransferase was complemented by the C. reinhardtii EPT gene. Enzymatic assays of C. reinhardtii EPT from the complemented yeast microsomes demonstrated that the C. reinhardtii EPT synthesized both PC and PE in the transformed yeast. The addition of either unlabeled CDP-ethanolamine or CDP-choline to reactions reduced incorporation of radiolabeled CDP-choline and radiolabeled CDP-ethanolamine into phosphatidylcholine and phosphatidylethanolamine. EPT activity from the transformed yeast or C. reinhardtii cells was inhibited nearly identically by unlabeled CDP-choline, CDP-ethanolamine, and CMP when [14C]CDP-choline was used as the primary substrate, but differentially by unlabeled CDP-choline and CDP-ethanolamine when [14C]CDP-ethanolamine was the primary substrate. The Km value of the enzyme for CDP-choline was smaller than that for CDP-ethanolamine. This provides evidence that C. reinhardtii EPT, similar to plant aminoalcoholphosphotransferase, is capable of catalyzing the final step of phosphatidylcholine biosynthesis, as well as that of phosphatidylethanolamine in the Kennedy pathway.     

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii with a Carbonic Anhydrase-Directed Photoaffinity Label

A carbonic anhydrase (CA)-directed photoaffinity reagent, 125I-labeled p-aminomethylbenzenesulfonamide-4-azidosalicylamide,was synthesized and shown to derivatize periplasmic CA in the unicellular green alga Chlamydomonas reinhardtii. The photoderivatization of purified C. reinhardtii periplasmic CA or intact C. reinhardtii cells with the reagent resulted in the modification of the large (37 kD) subunit of the enzyme. Photoderivatization of proteins in lysed C. reinhardtii cells also resulted in the specific labeling of a polypeptide of 30 kD. Centrifugation of the cell extract prior to photoaffinity labeling revealed that the labeled peptide was present predominantly in a particulate fraction. The photoaffinity-labeled 30-kD polypeptide was not observed in extracts from a mutant of C. reinhardtii that is believed to be deficient in an intracellular form of CA. These results provide evidence that the 30-kD polypeptide, which is photoaffinity labeled in lysed C. reinhardtii cells, is an intracellular form of CA.     

Kinetics of excitation trapping in intact Photosystem I of Chlamydomonas reinhardtii and Arabidopsis thaliana

We measured picosecond time-resolved fluorescence of intact Photosystem I complexes from Chlamydomonas reinhardtii and Arabidopsis thaliana. The antenna system of C. reinhardtii contains about 30-60 chlorophylls more than that of A. thaliana, but lacks the so-called red chlorophylls, chlorophylls that absorb at longer wavelength than the primary electron donor. In C. reinhardtii, the main lifetimes of excitation trapping are about 27 and 68 ps. The overall lifetime of C. reinhardtii is considerably shorter than in A. thaliana. We conclude that the amount and energies of the red chlorophylls have a larger effect on excitation trapping time in Photosystem I than the antenna size.     

134 Cryopreservation of Chlamydomonas Reinhardtii (Chlorophyceae): A Cause of Low Viability at High Cell Density

Cryopreservation provides a convenient method for long term storage of living organisms. Current protocols allow the successful cryopreservation of a wide range of algae, although many strains remain recalcitrant to cryopreservation. Chlamydomonas reinhardtii , a species utilized in many molecular and biochemical studies, survives cryopreservation best at low cell density. We show that reduced viability at higher cell densities is caused by the accumulation of a substance released from C. reinhardtii into the culture medium during cryopreservation. A mutant strain of C. reinhardtii (cw10) with a greatly reduced cell wall did not release a substance inhibitory to wild type or cw10 C. reinhardtii during cryopreservation, and could be cryopreserved with the same viability regardless of cell density. The inhibitory substance is small (mw<1300), polar, heat-stable and organic. Chlamydomonas moewusii Gerloff and Chlamydomonas zebra Korschikov ex Pascher both produce substances that reduce the viability of cryopreserved C. reinhardtii . However, neither is affected by the inhibitory substance produced by themselves or C. rienhardtii. Pandorina morum (Müller) Bory and Volvox carteri f. nagariensis Iyengar are colonial Volvocalean algae related to C. reinhardtii that cannot be successfully cryopreserved. They both generate substances that inhibit C. reinhardtii during cryopreservation. The identification of the substance inhibitory to C. reinhardtii during cryopreservation should explain why this alga cryopreserves best at a low cell density, and may lead to protocols that facilitate the more successful cryopreservation of C. reinhardtii and related algae.     

Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.     

The chloroplast protein translocation complexes of Chlamydomonas reinhardtii: a bioinformatic comparison of Toc and Tic components in plants, green algae and red algae

The recently completed genome of Chlamydomonas reinhardtii was surveyed for components of the chloroplast protein translocation complexes. Putative components were identified using reciprocal BlastP searches with the protein sequences of Arabidopsis thaliana as queries. As a comparison, we also surveyed the new genomes of the bryophyte Physcomitrella patens, two prasinophyte green algae (Ostreococcus lucimarinus and Ostreococcus tauri), the red alga Cyanidioschizon merolae, and several cyanobacteria. Overall, we found that the components of the import pathway are remarkably well conserved, particularly among the Viridiplantae lineages. Specifically, C. reinhardtii contained almost all the components found in A. thaliana, with two exceptions. Missing from C. reinhardtii are the C-terminal ferredoxin-NADPH-reductase (FNR) binding domain of Tic62 and a full-length, TPR-bearing Toc64. Further, the N-terminal domain of C. reinhardtii Toc34 is highly acidic, whereas the analogous region in C. reinhardtii Toc159 is not. This reversal of the vascular plant model may explain the similarity of C. reinhardtii chloroplast transit peptides to mitochondrial-targeting peptides. Other findings from our genome survey include the absence of Tic22 in both Ostreococcus genomes; the presence of only one Toc75 homolog in C. merolae; and, finally, a distinctive propensity for gene duplication in P. patens.     

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。     

Identification of mitochondrial proteins in membrane preparations from Chlamydomonas reinhardtii.

Preparations enriched in Chlamydomonas reinhardtii thylakoids have proven useful in the study of photosynthesis. Many of their polypeptides however remain unidentified. We report here on three of those, h1 (34 kDa), h2 (11 kDa), and P3 (63 kDa). h1, h2, and P3 are present in all tested mutants of C. reinhardtii lacking either one or several of the photosynthetic chain complexes or depleted in thylakoid membranes. h2 is an ascorbate-reducible, soluble c550-type cytochrome encoded in the nucleus. It cross-reacts immunologically with mitochondrial cytochromes c from various sources and contains a hexapeptide encoded in C. reinhardtii cytochrome c cDNA. P3, a nuclear-encoded peripheral protein, cross-reacts with various ATP synthase beta subunits. Its N-terminal sequence is encoded in C. reinhardtii mitochondrial beta subunit cDNA. h1 behaves as an integral hemoprotein; it is absent in a mitochondrial mutant that carries a deletion in apocytochrome b gene. We conclude that C. reinhardtii mitochondrial membranes copurify with thylakoid membranes. h1 is part of the cytochrome bc1 complex, h2 is cytochrome c, and P3 is the beta subunit of mitochondrial ATP synthase.     

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.     

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.     

Green algal extracellular products regulate antialgal toxin production in a cyanobacterium

Many bacterial genes and virulence factors are regulated by interbacterial and/or host–parasite chemical signals. We demonstrate that toxin production by a free-living freshwater cyanobacterium is regulated in part by the presence of extracellular products of a eukaryotic green alga. In growth experiments, extracellular products made by the cyanobacterium Anabaena flos-aquae contained both anatoxin and microcystin, and significantly reduced the yield of Chlamydomonas reinhardtii , a green alga. Based on experiments in which we added purified toxins to C. reinhardtii cultures, we believe that microcystin was responsible for the growth reduction. A. flos-aquae produced anatoxin constitutively when grown alone, but anatoxin concentration increased in the presence of C. reinhardtii elicitors. Microcystin accumulation depended on the growth phase; however, high concentrations of C. reinhardtii extracellular products completely inhibited microcystin accumulation. Our results demonstrate that cyanobacterial toxin production may be regulated by complex growth phase-dependent and environmental chemical cues, and suggest that secreted chemicals can mediate the outcome of competition between the cyanobacterium A. flos-aquae and the green alga C. reinhardtii .     

A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii†

The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.     

Extensive gene rearrangements in the chloroplast DNAs of Chlamydomonas species featuring multiple dispersed repeats

We have constructed a physical and gene map for the chloroplast DNA (cpDNA) of the unicellular green alga Chlamydomonas gelatinosa, a close relative of Chlamydomonas reinhardtii. At 285 kb, the C. gelatinosa cpDNA is 89 kb larger than its C. reinhardtii counterpart. The alterations in the order of 77 genes on the cpDNAs of these green algae are attributable to nine inversions and one event of expansion/contraction of the inverted repeat. These rearrangements are much more extensive than those previously reported between the cpDNAs of the closely related Chlamydomonas moewusii and Chlamydomonas pitschmannii. Because the divergence level of the C. gelatinosa and C. reinhardtii chloroplast-encoded large subunit rRNA gene sequences is equivalent to that of the corresponding C. moewusii and C. pitschmannii sequences, our results may suggest that, in the same period of time, there have been more numerous rearrangements in the lineage comprising C. gelatinosa and C. reinhardtii than in the lineage comprising C. moewusii and C. pitschmannii. Alternatively, given that substitution rates in chloroplast genes are not necessarily uniform across lineages, the extensive rearrangements between the C. gelatinosa and C. reinhardtii cpDNAs may reflect a longer divergence period for this pair of Chlamydomonas species compared to that for the C. moewusii/C. pitschmannii pair. We have also found that, like its C. reinhardtii homologue but unlike its C. moewusii and C. pitschmannii counterparts, the C. gelatinosa cpDNA features a large number of dispersed repeated sequences that are readily detectable by Southern blot hybridization with homologous fragment probes. Assuming that the two pairs of closely related Chlamydomonas species diverged at about the same time, these data suggest that the susceptibility of Chlamydomonas cpDNAs to rearrangements is correlated with the abundance of repeated sequences. Preliminary characterization of a 345-bp C. gelatinosa cpDNA region containing a repeated sequence by both DNA sequencing and Southern blot analysis has revealed no sequence homology between this region and the cpDNAs of C. reinhardtii and other Chlamydomonas species.      

Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.     

Chlamydomonas reinhardtii as a new model system for studying the molecular basis of the circadian clock

The genome of the unicellular green alga Chlamydomonas reinhardtii has both plant-like and animal-like genes. It is of interest to know which types of clock genes this alga has. Recent forward and reverse genetic studies have revealed that its clock has both plant-like and algal clock components. In addition, since C. reinhardtii is a useful model organism also called "green yeast", the identification of clock genes will make C. reinhardtii a powerful model for studying the molecular basis of the eukaryotic circadian clock. In this review, we describe our forward genetic approach in C. reinhardtii and discuss some recent findings about its circadian clock.     

Transformation of Chlamydomonas reinhardtii CW-15 with the hygromycin phosphotransferase gene as a selective marker

To transform Chlamydomonas reinhardtii Dang. Cells, plasmid pCTVHyg was constructed with the use of the Escherichia coli hygromycin phosphotransferase gene (hpt) controlled by the SV40 early promoter. Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10(3) hygromycin-resistant (HygR) clones per 10(6) recipient cells. The exogenous DNA integrated in the Ch. reinhardtii nuclear genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was expressed as an unstable character. Codon usage was compared for the hpt gene and Ch. reinhardtii nuclear genes. The results testified that codon usage bias, which is characteristic of Ch. reinhardtii, is not the major factor affecting foreign gene expression. The advantages of the selective system for studying Ch. reinhardtii transformation with heterologous genes are discussed.     

The chloroplast ATP synthase in Chlamydomonas reinhardtii. I. Characterization of its nine constitutive subunits

We have characterized the subunit composition of the chloroplast ATP synthase from Chlamydomonas reinhardtii by means of a comparison of the polypeptide deficiencies in a mutant defective in photophosphorylation, with the polypeptide content in purified coupling factor (CF)1 and CF1.CF0 complexes. We could distinguish nine subunits in the enzyme, four of which were CF0 subunits. Further characterization of these subunits was undertaken by immunoblotting experiments, [14C]dicyclohexylcarbodiimide binding and analysis of their site of translation. In particular, we were able to show the presence of an as yet unidentified delta subunit in CF1 from C. reinhardtii. We have identified a 70-kDa peripheral membrane protein in the thylakoid membranes of C. reinhardtii, which is immunologically related to the beta subunit of CF1. We discuss its conceivable ATPase function with respect to the Ca2+-dependent ATPase activity previously reported in the thylakoid membranes from C. reinhardtii.     

A cosmid vector containing a dominant selectable marker for cloning Chlamydomonas genes by complementation

Cosmid vectors containing a dominate selectable marker (ble) for complementation cloning of genes in Chlamydomonas reinhardtii were created. The usefulness of these vectors, which differ in the orientation of the ble cassette, was demonstrated by transforming C. reinhardtii to phleomycin resistance, by constructing a large library (approximately 5 x 10(5) recombinants) in one of them using DNA from a C. reinhardtii mutant, and by transforming C. reinhardtii with recombinant cosmid clones and pools.