Abstract
The chloroplasts ofChlamydomonas reinhardtii were transformed using a vector (paadAGUS4.1) that contained a spectinomycin-resistance gene (aadA) as a selectable gene, and bacterialuidA (GUS) as a reporter gene, and pea 4.1 kb D-loop containing sequence. The vector was introduced into the alga through particle gun bombardment. The transformed colonies were screened for the presence of foreign genes by Southern hybridization using GUS,aadA and 4.1 pea Ori probes. Expression ofaadA and GUS genes was detected in all colonies that were grown on spectinomycin. A detailed restriction analysis followed by southern hybridization of total genomic DNA using pea 4.1 kb D-loop as probe indicated that the D-loop sequence can serve in site-specific integration of foreign DNA due to high homology. Restriction analysis of different colonies showed that the foreign DNA was probably present in a mixture population of autonomous segment and integrated in the native chloroplast genome.
Similar content being viewed by others
References
Abel W.O., Knebel W., Koop H.U., Marienfeld J.R., Quader H., Reski R., Schnepf E., Sporlein B.: A cytokinin-sensitive mutant of the mossPhycomitrella patens defective in the chloroplast division.Protoplasma 152, 1–3 (1989).
Blowers A.D., Bogorad L., Shark K.B., Sanford J.C.: Studies onChlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome.Plant Cell 1, 23–32 (1989).
Blowers A.D., Ellmore G.S., Klein U., Bogorad L.: Transcriptional analysis of endogenous and foreign genes in chloroplast trans-formants ofChlamydomonas.Plant Cell 2, 1059–1070 (1990).
Boynton J.E., Gillham N.W., Harris E.H., Hosler J.P., Johnson A.M., Jones A.R., Randolph-Anderson B., Robertson D., Klein T.M., Shark K.B., Sanford J.C.: Chloroplast transformation inChlamydomonas with high velocity microprojectiles.Science 240, 1534–1538 (1988).
Boynton J.E., Gillham N.W.: Chloroplast transformation inChlamydomonas.Meth. Enzymol. 217, 510–536 (1993).
Bradford M.M.: A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of proteindye binding.Anal. Biochem. 72, 248–254 (1976).
Chu N.M., Oishi K.K., Tewari K.K.: Physical mapping of the pea chloroplast DNA and localization of the ribosomal RNA genes.Plasmid 6, 279–292 (1981).
El-Sheekh M.M: Stable transformation of the intact cells of the unicellular green algaChlorella kessleri with high velocity microprojectiles.Biol. Plant. 42, 209–216 (1999).
Gilham N.W., Boynton J.E., Hauser C.R.: Translational regulation of gene expression in chloroplasts and mitochondria.Ann. Rev. Genet. 28, 71–93 (1994).
Goldschmidt-Clermont M.: Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site directed transformation ofChlamydomonas.Nucl. Acids Res. 19, 4083–4089 (1991).
Goldschmidt-Clermont M.: Chloroplast transformation and reverse genetics, pp. 139–149 in J.-D. Rochaix, M. Goldschmidt-Clermont, M. Merchant (Eds):The Molecular Biology of Chloroplast and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht (Netherlands) 1998.
Harris E.H.:The ChlamydomonasSource Book: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego 1989.
Hass M., Dowding J.: Aminoglycoside modifying enzymes.Meth. Enzymol. 43, 611–628 (1975).
Hippler M., Biehler K., Krieger-Liszkay A., Van Dillewjin J., Rochaix J.D.: Limitation in electron transfer in photosystem I donor side mutants ofChlamydomonas reinhardtii. Lethal photo-oxidative damage in high light is overcome in a suppressor strain deficient in the assembly of the light harvesting complex.J. Biol. Chem. 275, 5852–5859 (2000).
Ishikura K., Takaoka Y., Kato K., Sekine M., Yoshida K., Shinmyo A.: Expression of a foreign gene inChlamydomonas reinhardtii chloroplast.J. Biosci. Bioengin. 87, 307–314 (1999).
Jefferson J.G., Kavanagh T.A., Beran M.W.: GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J. 6, 3901–3907 (1987).
Kareiva M.: Transgenic plants on trial.Nature 363, 580–581 (1993).
Kindle K.L., Richards K.L., Stern D.B.: Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation inChlamydomonas reinhardtii.Proc. Nat. Acad. Sci. USA 88, 1721–1725 (1991).
Klein T.M., Wolf E.D., Wu R., Sanford J.C.: High-velocity microprojectiles for delivering nucleic acids into living cells.Nature 327, 70–73 (1987).
Leu S., Schlesinger J., Michaels A., Shavit N.: Complete DNA sequence of theChlamydomonas reinhardtii chloroplastatpA gene.Plant Mol. Biol. 18, 613–616 (1992).
Morais F., Barber J., Nixon P.J.: The chloroplast-encoded α subunit of cytochromeb 559 is required for assembly of the photosystem II complex in both light and dark inChlamydomonas reinhardtii.J. Biol. Chem. 273, 29315–29320 (1998).
Przibilla E., Heiss S., Johanningmeier U., Trebst A.: Site-specific mutagenesis of the D1 subunit of photosystem II in wild-typeChlamydomonas.Plant Cell 3, 169–174 (1991).
Sakamoto W., Kindle K., Stern D.B.:In vivo analysis ofChlamydomonas chloroplastpetD gene expression using stable transformation of β-glucuronidase translational fusions.Proc. Nat. Acad. Sci. USA 90, 497–501 (1993).
Sanford J.C.: The biolistic process.Trends Biotech. 6, 299–302 (1988).
Shimogawara K., Fujiwara S., Grossman A., Usuda H.: High-efficiency transformation ofChlamydomonas reinhardtii by electroporation.Genetics 148, 1821–1828 (1998).
Southern A.: Detection of specific sequence among DNA fragments separated by gel electrophoresis.J. Mol. Biol. 98 503–517 (1975).
Stern D.B., Radwanski E.R., Kindle K.L.: A 3′ stem/loop structure of theChlamydomonas chloroplastatpB gene regulates mRNA accumulationin vivo.Plant Cell 3, 285–297 (1991).
USDA:Agriculture Research Service, United States Developmental Agriculture. Dover Publications Inc., New York 1998.
Zaslavskaia L.A., Lippmeier J.C., Kroth P.G., Grossman A.R., Apt K.E.: Transformation of the diatomPhœodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes.J. Phycol. 36, 379–386 (2000).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
El-Sheekh, M.M. Stable chloroplast transformation inChlamydomonas reinhardtii using microprojectile bombardment. Folia Microbiol 45, 496–504 (2000). https://doi.org/10.1007/BF02818717
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF02818717