SpringerProtocols: Abstract: Green Alga (Chlamydomonas reinhardtii)

Green Alga (Chlamydomonas reinhardtii)

By: Manchikatla V. Rajam³, S. Vinod Kumar³

Abstract

Full Text

Download PDF (916K)

This protocol describes the Agrobacterium tumefaciens-mediated nuclear transformation of a microalgae Chlamydomonas reinhardtii, using a gene construct carrying the genes coding for β-glucuronidase (gus), green fluorescent protein (gfp), and hygromycin phosphotransferase (hpt). The transformation frequency with this protocol as revealed by hygromycin resistance was many fold higher (about 50-fold) than that of the commonly used glass bead method of transformation. The simplicity of Agrobacterium-mediated gene transfer and the high transformation frequency as well as the precision of T-DNA integration will enable further molecular dissection of this important model organism as well as other algal systems to understand basic plant metabolic processes as well as to exploit the systems for biotechnological applications.

Affiliation(s): (3) Plant Polyamine and Transgenic Research Laboratory, Department of Genetics, University of Delhi - South Campus, Benito Juarez Road, New Delhi, 110 021, India

Book Title: Agrobacterium Protocols Volume 2

Series: Methods in Molecular Biology | Volume: 344 | Pub. Date: Sep-01-2006 | Page Range: 421-433 | DOI: 10.1385/1-59745-131-2:421

Subject: Plant Sciences

Key Words: Algae - Chlamydomonas reinhardtii - genetic transformation - reporter genes - Agrobacterium tumefaciens

References

Download References

1.	Leán-Bañares, R., González-Ballester, D., Galvan, A. and Fernández, E. (2004) Transgenic microalgae as green cell-factories. Trends Biotech. 22, 45–52.

2.	Harris, E.H. (1989) The Chlamydomonas Source Book: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, New York.

3.	Harris, E.H. (2001) Chlamydomonas as a model organism. Ann. Rev. Plant Physiol. Plant Mol. Biol. 52, 363–406.

4.	Den, R.M., Han, M., and Niyogi, K.K. (2001) Functional genomics of plant photosynthesis in the fast lane using Chlamydomonas reinhardtii. Trends Plant Sci. 6, 364–371. <Occurrence Type="DOI"><Handle>10.1016/S1360-1385(01)02018-0</Handle></Occurrence>

5.	Kumar, S.V., Misquitta, R.W., Reddy, V.S., Rao, B.J., and Rajam, M.V. (2004) Genetic transformation of the green alga—Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci. 166, 731–738.

6.	Apt, K.E. Kroth-Pancic, P.G., and Grossman, A.R. (1996) Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol. Gen. Genet. 252, 572–579.

7.	Dunahay, T.G., Jarvis, E.E., and Roessler, P.G. (1995) Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila. J. Phycol. 31, 1004–1012.

8.	Falciatore, A., Casotti, R., Leblanc, C., Abrescia, C., and Bowler, C. (1999) Transformation of nonselectable reporter genes in marine diatoms. Mar. Biotechnol. 1, 239–251.

9.	Shimogawara, K., Fujiwara, S., Grossman, A., and Usuda, H. (1998) High efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148, 1821–1828.

10.	Kindle, K.L., Schnell, R.A., Fernandez, E., and Lefebvre, P.A. (1989) Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J. Cell Biol. 109, 2589–2601.

11.	Kindle, K.L. (1990) High frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 93, 13689–13693.

12.	Dunahay, T.G. (1993) Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. BioTechniques 15, 452–460.

13.	Lohuis, M.R. and Miller, D.J. (1998) Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. Plant J. 13, 427–435.

14.	Hei, Y., Komari, T. and Kumashiro, T. (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundariues of T-DNA. Plant J. 6, 271–282.

15.	Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann. Biochem. 72, 248–254. <Occurrence Type="DOI"><Handle>10.1016/0003-2697(76)90527-3</Handle></Occurrence>

16.	Sambrook, J., Fritsch, E.F., and Maniatis, T. (eds.) (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, New York.

17.	Jefferson, R.A. (1987) Assaying chimeric genes in plants: the uidA gene fusion system. Plant. Mol. Biol. Rep. 5, 387–405.

18.	Doyle, J.J. and Doyle, J.L. (1990) Isolation of plant DNA from fresh tissue. Focus 12, 13–15.

Comments (Loading...)

Post comment/View all comments