It is widely distributed, and is often isolated from soil and freshwater samples. Chloroplast transformation of platymonas tetraselmis plos. Stable chloroplast transformation in chlamydomonas reinhardtii. The development of technologies for the stable genetic transformation of plastid chloroplast genomes has been a boon to both basic and applied research. Chloroplast transformation in wheat was achieved by bom bardment of.
Bombardment of three mutants of the chloroplast atpb gene of chlamydomonas reinhardtii with high velocity tungsten microprojectiles that were coated with cloned chloroplast dna carrying the wildtype gene permanently restored the photosynthetic capacity of the algae. Am improved procedure for the isolation of chloroplast dna. I usually resuspend the cells in about 125 the original volume in autolysin. Boynton je, gillham nw, harris eh, hosler jp, johnson am, jones ar, randolphanderson bl, robertson d, klein tm, shark kb etal. Aug 16, 2016 culturing chlamydomonas reinhardtii alg app008. Since transformation of the plastid genome in chlamydomonas and tobacco became possible boynton et al. Pdf recent achievements obtained by chloroplast transformation. Hybrid transcription system for controlled plastid transgene expression. Chlamydomonas reinhardtii provides a system particularly amenable to analyze the structurefunction relationship of photosystem i. Chloroplast transformation of platymonas tetraselmis. Stable plastid transformation of rice, a monocot cereal. Tools and techniques for chloroplast transformation of. This article describes the procedures for chloroplast transformation.
By using a method in which cellwalldeficient chlamydomonas reinhardtii cells were agitated in the presence of dna, glass beads, and polyethylene glycol, nuclear transformation rates of approximately 103 transformants per micrograms of plasmid dna were achieved. Techniques and capabilities for chloroplast transformation in chlamydomonas reinhardtii. Pdf use of the ptxd gene as a portable selectable marker. Boynton je, gillham nw, harris eh, hosler jp, johnson am, jones ar, randolphanderson bl, robertson d, klein tm, shark kb, et al. Use of the ptxd gene as a portable selectable marker for chloroplast transformation in chlamydomonas reinhardtii. American association for the advancement of science. Simple operation and high transformation efficiency makes it a favorable way for plastid or chloroplast transformation. In brief, 10 ml of a preculture were inoculated into fresh 400 ml tap medium and grown to. Metabolic pathway engineering by transgene expression from the plastid chloroplast genome offers significant attractions, including straightforward multigene engineering by pathway expression from operons, high transgene expression levels, and increased transgene containment due to maternal inheritance of plastids in most crops. Chloroplast transformation in chlamydomonas with high. A d blowers, l bogorad, k b shark, and j c sanford biological laboratories, harvard university, cambridge, massachusetts 028.
Chlamydomonas reinhardtii chloroplasts as protein factories. Chlamydomonas reinhardtii, green alga, is favourable organism for chloroplast transformation and used a model system for number of reasons. Utr sequences with the original mutations fail to bind a 20kd protein, one of five proteins that bind to leaders of several chloroplast genes. Chloroplast transformation in chlamydomonas with high velocity. The chloroplast organelle of plant and algal cells contains its own genetic. T1 a simple method for chloroplast transformation in chlamydomonas reinhardtii. Chloroplast transformation in chlamydomonas with high velocity microprojectiles. In recent years, the applications of omics technologies in algae have emerged as a strategic research front. The green eukaryotic alga chlamydomonas reinhardtii has been a long.
A simple method for chloroplast transformation in chlamydomonas. We have mapped this gene by transformation of a deletion mutant with a particle gun. With the advent of nuclear transformation in chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed. Dna delivery process utilizing high velocity microprojectiles 6 circumvents these problems, probably by direct introduction of many copies of a gene into the chloroplast. Chlamydomonas reinhardtii, plastid structure, chloroplast. We have established a high efficiency method for transforming the unicellular, green alga chlamydomonas reinhardtii by electroporation. Plastid transformation and its application in metabolic. Chloroplast ribosomal protein s7 of chlamydomonas binds to. Mutational analysis of eggplant latent viroid rna processing. Factors influencing gene delivery into zea mays cells by high.
Chloroplast transformation for engineering of photosynthesis maureen r. A protocol for expression of foreign genes in chloroplasts. The availability of efficient nuclear and chloroplast transformation in chlamydomonas provides specific advantages for the study of chloroplast biogenesis, photosynthesis, and. Plastid transformation has achieved striking implementation since its first accomplishment in a unicellular green alga, chlamydomonas reinhardtii, followed by stable plastid transformation in tobacco, a wellknown model flowering plant. Directed chloroplast transformation in chlamydomonas reinhardtii. Tools for regulated gene expression in the chloroplast of chlamydomonas article in methods in molecular biology clifton, n. The cell walllacking mutant cw15 of the unicellular green alga chlamydomonas reinhardtii was transformed by electroporation using plasmid pctvhyg, which was constructed with the hygromycin phosphotransferase genehpt as the selective marker and the tn5 transposon of escherichia coli under the control of the virus sv40 early gene promoter. Factors influencing the efficiency of dna delivery into suspension culture cells of zea mays by the particle bombardment process were studied using a chimeric gene coding for the production of. Pdf the chloroplasts of chlamydomonas reinhardtii were transformed using a vector. Recent achievements obtained by chloroplast transformation.
Stable chloroplast transformation of immature scutella and. We describe a simple and efficient method for separating these dna fractions by using a sodium iodide gradient in combination with the dnabinding dye, bisbenzimide. Stable chloroplast transformation was first accomplished in 1989 when boynton and coworkers restored the photosynthetic capacity of a c. Pdf tools for chloroplast transformation in chlamydomonas. At least one additional chloroplast locus tsca is required for transsplicing of exons 1 and 2. Y takahashi, m goldschmidtclermont, s y soen, l g franzen, and j d rochaix. This high frequency of cotransformation will allow any cloned gene to be introduced into chlamydomonas. The objective of this research was to establish a chloroplast transformation technique for platymonas tetraselmis subcordiformis. Mutation of residue threonine2 of the d2 polypeptide and. Tools for chloroplast transformation in chlamydomonas. Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inchlamydomonas reinhardtii. Recent achievements obtained by chloroplast transformation article pdf available in plant methods 1 december 2017 with 822 reads how we measure reads. The d2 polypeptide of the photosystem ii psii complex in the green alga chlamydomonas reinhardtii is thought to be reversibly phosphorylated.
Novel control elements in the alpha1 tubulin gene promoter from chlamydomonas reinhardii. Nuclear and chloroplast transformation in chlamydomonas. The nitrate reductase gene from wildtype chlamydomonas was used to complement a mutation in the corresponding gene of a strain. In most transformants of one of the mutants, a fragment with a 2. Chlamydomonas reinhardtii chloroplast transformation.
Highfrequency nuclear transformation of chlamydomonas. Transgenic chloroplasts offer unique advantages in plant biotechnology, including high level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of. Generation of microalga chlamydomonas reinhardtii expressing. Subsequently, lowfrequency transformation of the nuclear genome was achieved with the same method 810, s.
Reversegenetic studies of chloroplast genes in the green alga chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. It is in chlamydomonas that chloroplast transformation was first achieved boynton et al. Directed chloroplast transformation in chlamydomonas. Viroids of the family avsunviroidae, such as eggplant latent viroid elvd, contain hammerhead ribozymes and replicate in the chloroplasts of the host plant through an rnabased symmetrical rollingcircle mechanism in which oligomeric rnas of both polarity are processed to monomeric linear rnas by cleavage and to monomeric circular rnas by ligation. Svab and maliga, 1993, researchers have exploited the technology to understand how plastid genes are regulated, to determine the function of plastid gene products, to produce large amounts of particular endogenous or foreign proteins or to alter photosynthesis or. Chlamydomonas reinhardtii as a new model system for studying. Employing the gfp gene as a reporter and the bar gene as a selectable marker, transformation vectors of p. Electroporation of strains cc3395 and cc425, cell wallless mutants devoid of argininosuccinate lyase encoded by arg7, in the presence of the plasmid pjd67. Because of the rapidity and simplicity of plastid transformation of chlamydomonas. High levels of steadystate crcpadh1 transcripts were detected in the homoplasmic transformants. This process is also proving effective for introducing dna into the nuclei of higher plants 7 and for nuclear 8 and mitochondrial 9 gene transformation of yeast. Chloroplast transformation in chlamydomonas with high velocity microprojectiles pp.
They have the power and raw materials that can be used as sophisticated biological factories. Hitherto, a total of 18 species of flowering plants have been reported to obtain the homoplastomic plants via stable plastid. Stable genetic transformation of tomato plastids and. Tools for regulated gene expression in the chloroplast of. The isolation of chloroplast dna fromchlamydomonas reinhardtii requires the efficient separation of this atrich genome from the gcrich nuclear genome by densitygradient centrifugation. The scientometric analysis of the research on the algal. The availability of routine techniques for the genetic manipulation of the chloroplast genome of chlamydomonas. Pegmediated and agrobacterium mediated transformation method was also employed in the early days. The transformation of the unicellular alga chlamydomonas. First attempts to express recombinant proteins in c. Studies on chlamydomonas chloroplast transformation.
Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Highefficiency generation of fertile transplastomic. Stable nuclear transformation of chlamydomonas using the. Boynton je, gillham nw, harris eh, hosler jp, johnson am, jones ar, randolphanderson bl, robertson d, klein tm, shark kb 1988 chloroplast transformation in chlamydomonas with high velocity microprojectiles.
Full text the full text of this article is available as a pdf 2. To submit a comment for a journal article, please use the space above and note the following. A small chloroplast rna may be required for transsplicing in. A simple, lowcost method for chloroplast transformation. Chloroplast transformation for engineering of photosynthesis. First successful chloroplast transformation was performed in chlamydomonas reinhardtii by particle bombardment method. N2 photosystem psi is a multisubunit pigmentprotein complex that uses light energy to transfer electrons from plastocyanin to ferredoxin. Boynton je1, gillham nw, harris eh, hosler jp, johnson. Jun 10, 1988 chloroplast transformation in chlamydomonas with high velocity microprojectiles. Boynton je1, gillham nw, harris eh, hosler jp, johnson am, jones ar, randolphanderson bl, robertson d, klein tm, shark kb, et al. We have established a highefficiency method for transforming the unicellular, green alga chlamydomonas reinhardtii by electroporation. Introduction to chlamydomonas and its laboratory use vol.
Chloroplasts play a great role for sustained wellbeing of life on the planet. Chlamydomonas reinhardtii using microprojectile bombardment. C chloroplast transformation in chlamydomonas with high velocity microproject. Electroporation of strains cc3395 and cc425, cell wallless mutants devoid of argininosuccinate lyase encoded by arg7, in the presence of the plasmid pjd67 which contains arg7 was used to optimize conditions for the introduction of exogenous. Genetic engineering of thylakoid protein complexes by. Edison in 1880 and published by aaas, today ranks as the worlds largest circulation general science journal. Utr of the chloroplast rps7 gene in chlamydomonas reduce expression of reporter genes. Moreover, the overall efficiency of transformation should be high enough to permit isolation of genes from genomic libraries by complementation of stable nuclear mutants. Studies on chlamydomonas chloroplast transformation plant cell. Plastid transformation and its application in metabolic engineering. Science 240, 15341538, a nonphotosynthetic, acetaterequiring mutant strain of chlamydomonas reinhardtii with a 2.
Chloroplast transformation for engineering of photosynthesis, journal of experimental botany. Bombardment of three mutants of the chloroplast atpb gene of chlamydomonas reinhardtii with highvelocity tungsten microprojectiles that were coated with cloned chloroplast dna carrying the wildtype gene permanently restored the photosynthetic capacity of the algae. Electroporation of strains cc3395 and cc425, cell wallless mutants devoid of argininosuccinate lyase encoded by arg7, in the presence of the plasmid pjd67 which contains arg7 was used to optimize conditions for the introduction of exogenous dna. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. The green alga chlamydomonas reinhardtii and some other algal. Stable transformation of the intact cells of the unicellular green algachlorella kessleri with high velocity microprojectiles. Chloroplast transformation the first chloroplast transformation was reported in chlamydomonas using high velocity micro projectiles by biolistic delivery of naked dna that integrated into the genome through homologous targeting boynton et al. Chloroplast transformation techniques can be used to study on plastid genetic and molecular biology and transformation of foreign genes into chloroplast genome. By analogy to higher plants, the phosphorylation site is likely to be at residue threonine2 thr2. Chlamydomonas reinhardtii as the photosynthetic yeast. We have investigated the role of d2 phosphorylation by constructing two mutants in which residue thr2 has been replaced by either. The availability of efficient nuclear and chloroplast transformation in chlamydomonas provides specific advantages for the study of chloroplast biogenesis, photosynthesis, and nuclearchloroplast genome interactions.
Pdf stable chloroplast transformation in chlamydomonas. They are rich in energy as they have lots of pigmentprotein complexes capable of collecting sunlight, in sugar produced by photosynthesis and in minerals imported from the plant cell. Highefficiency transformation of chlamydomonas reinhardtii. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. Construction of a novel vector for the nuclear transformation of the. Harris eh et al 1988 chloroplast transformation in chlamydomonas with high velocity microprojectiles. A small chloroplast rna may be required for transsplicing. If desired, treat cells with gamete autolysin prepared as described in the chlamydomonas sourcebook for approximately 45 minutes.
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