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Stage:29
From Master 2 Recherche Biosciences Végétales
Laboratoire d'accueil : Surfaces Cellulaires et Signalisation chez les Végétaux (UMR UPS-CNRS 5546)
Equipe d'accueil : Régulation transcriptionnelle et formation du xylème (J Grima-Pettenati)
Encadrant(e)(s) : Grima-Pettenati et Wang / Mail : grima@scsv.ups-tlse.fr /Tél : 05 62 19 35 13 /Mail : wang@scsv.ups-tlse.fr / Tel 05 62 19 35 51
The main objective of this M2R project, which will be continued by PhD project, is to characterize the mechanisms underlying the regulatory network ruled by the transcription factors EgMYB1 and EgMYB2 during wood formation in Eucalyptus. The target plant Eucalyptus is the first tree species for industrial plantations in the world; it is also adapted to genomic approaches thanks to its small genome size (the genome sequence will publicly released in 2010), the availability of BAC libraries, genetic maps and QTLs.
Wood, which is mainly composed of lignified secondary walls (SW), constitutes the most abundant biomass on earth. Eucalyptus plantations which grow very fast and produce high yield of biomass, are a major component of economic activity in Europe. They supply high quality pulpwood for a large pulp and paper industry and are increasingly becoming important sources of bio-energy and so through the use of dedicated bio-energy tree crops or complementary bio-ethanol production avenues. Although the SW biosynthetic pathway has been well characterized biochemically and genetically, our current knowledge about the signals and transcriptional regulators that are responsible for turning on the SW biosynthetic program, is still limited (Demura and Fukuda, 2007). We have recently characterized in Eucalyptus, two R2R3 MYB transcription factors (EgMYB1 and EgMYB2) preferentially expressed in xylem and playing antagonist roles in the regulation of the SW biogenesis. This original situation offers a great opportunity to study the hierarchical and combinatorial control of the wood formation process.
The project M2R is aimed to detect potential protein partners that act together with EgMYB1 and EgMYB2 to perform their regulatory role. We will construct a two-hybrid yeast library using mRNA extracted from Eucalyptus xylem tissue to identify potential partners of EgMYB1 and EgMYB2. A newly improved approach of yeast two-hybrid, called the “split protein sensor system”, will be used, which permits the identification of interacting partners that are involved in transcriptional regulation (Laser et al. 2000). Moreover, newly improvements to avoid false negative and false positive results, which are the most limiting factor of two-hybrid approach, will be used (Tafelmeyer et al. 2004, Dirnberger et al. 2008). The co-immuno-precipitation will be also used as a complementation approach. The putative interactions will be validated in vivo using the “bimolecular fluorescence complementation technique” (BiFC, Bracha-Drori et al. 2004), in collaboration with the cell imaging platform.
Main Techniques :
Molecular biology : RNA extraction, 2Y hybrid library construction; cloning in Gateway, PCR, Q-RT-PCR...
Biochemistry : immuno-precipitation
Cellular biology and imagery: BiFC
Tissue culture: genetic transformation of plant cells
Relevant references of the host team:
Rengel D, San Clémente H, Servant F, Ladouce N, Paux E, Wincker P, Couloux A, Sivadon P, Grima-Pettenati J (2009) A new genomic resource dedicated to wood formation in Eucalyptus. BMC Plant Biology 9:36 (IF=4.03)
Legay S, Lacombe E, Goicoechea M, Briere C, Seguin A, Mackay J, Grima-Pettenati J (2007) Molecular characterization of EgMYB1, a putative transcriptional repressor of the lignin biosynthetic pathway. Plant Sci 173:542–549 (IF =1.795)
Bedon F, Grima-Pettenati J, MacKay J (2007) Conifer R2R3-MYB transcription factors: sequence analyses and gene expression in wood-forming tissues of white spruce (Picea glauca). BMC Plant Biology 7:17 (IF =3.23)
Foucart C, Paux E, Ladouce N, San-Clemente H, Grima-Pettenati J, Sivadon P (2006) Transcript profiling of a xylem vs phloem cDNA subtractive library identifies new genes expressed during xylogenesis in Eucalyptus. New Phytol 170:739-752 (IF = 5.25)
Goicoechea M, Lacombe E, Legay S, Milhaevic S, Rech P, Jauneau A, Lapierre C, Pollet B., Verhaegen D, Chaubet-Gigot N, Grima-Pettenati J (2005) EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis. Plant J 43:553-567 (IF = 6.75)
Paux, E., Carocha, V., Marques, C., Mendes de Sousa, A., Borralho, N., Sivadon, P., and Grima-Pettenati, J. (2005). Transcript profiling of Eucalyptus xylem genes during tension wood formation. New Phytol 167, 89-100.
Paux, E., Tamasloukht, M., Ladouce, N., Sivadon, P., and Grima-Pettenati, J. (2004). Identification of genes preferentially expressed during wood formation in Eucalyptus. Plant Mol Biol 55, 263-280.
Boudet, A.M., Kajita, S., Grima-Pettenati, J., and Goffner, D. (2003). Lignins and lignocellulosics: a better control of synthesis for new and improved uses. Trends Plant Sci 8, 576-581.
Bibliography
Demura, T., and Fukuda, H. (2007). Transcriptional regulation in wood formation. Trends Plant Sci 12, 64-70.
Dirnberger D, Messerschmid M, Baumeister R (2008) An optimized split-ubiquitin cDNA-library screening system to identify novel interactors of the human Frizzled 1 receptor. Nucleic Acids Res. 36: e37.
Laser H, Bongards C, Schüller J, Heck S, Johnsson N, Lehming N. (2000) A new screen for protein interactions reveals that the Saccharomyces cerevisiae high mobility group proteins Nhp6A/B are involved in the regulation of the GAL1 promoter. Proc Natl Acad Sci USA 5: 13732-13737.
Tafelmeyer P, Johnsson N, Johnsson K (2004) Transforming a (?/?)8-Barrel Enzyme into a Split-Protein Sensor through Directed Evolution. Chem Biol 11: 681-689.