Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-12-12
1999-11-09
Patterson, Jr., Charles L.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352523, 43525411, 4353201, 536 231, 536 236, 536 241, 536 243, 530350, 530370, 530379, C12N 1500
Patent
active
059812192
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to DNA molecules, particularly from Spinacia oleracea, containing the coding region of a 2-oxoglutarate/malate transporter and the introduction of which into a plant genome modifies the production and transport of carbon skeletons for nitrogen fixation and the transport of the assimilated nitrogen in transgenic plants. The invention furthermore relates to plasmids, yeasts and bacteria containing said DNA molecules, as well as to transgenic plants in which modifications of the activity of the 2-oxoglutarate/malate transporter and thus modifications of the nitrogen and carbon metabolism are brought about by introduction of said DNA molecules. The invention furthermore relates to transgenic plants the photo-respiratory capability of which is influenced by the modification of the activity of the 2-oxoglutarate/malate transporter. The invention also relates to the use of the DNA molecules described which code for a 2-oxoglutarate/malate translocator for the identification of related translocators from Spinacia oleracea and other plants by low-stringency hybridization or by PCR techniques, as well as the use of the 2-oxoglutarate/malate translocator as target for herbicides.
Only plants, bacteria and yeasts are capable of converting on a large scale inorganic nitrogen (nitrate nitrogen) into organically fixed nitrogen (usually in the form of amino acids) by reductively aminating organic carbon compounds. The remainder of the animated world, particularly useful animals and humans, is dependent on plants as primary suppliers of organic nitrogen compounds. Assimilation of inorganic nitrogen in plants by fixation to organic carbon depends on the availability of nitrogen, carbon skeletons and energy. The nitrogen supply of the plant can be influenced by fertilizers. The energy for nitrogen assimilation is derived from the light reaction of photosynthesis or in roots or other non-green tissues from dissimilation and under normal field conditions is no limiting factor.
2-oxoglutarate (.alpha.-ketoglutarate) according to today's knowledge is the primary acceptor of reduced nitrogen in the glutamine synthase/glutamine 2-oxoglutarate aminotransferase (GOGAT) reaction. In this reaction nitrogen (ammonium nitrogen) reduced by glutamine synthase is first transferred onto glutamate under energy consumption. Glutamine is formed. In a sequential reaction the glutamate oxoglutarate aminotransferase (GOGAT; glutamate synthase) catalyzes the transfer of an amino group of glutamine onto 2-oxoglutarate (transamination) while consuming reduction equivalents. Two glutamate molecules are formed.
The entire reaction sequence of the glutamine synthase/GOGAT reaction is localized in the stroma of the plant plastids. These organelles are surrounded by two lipid bilayer membranes, with the exterior having molecular sieve character and being permeable to compounds up to a size of about 10 kD (Flugge and Benz, 1984, FEBS Lett. 169:85-89). The inner membrane is permeable to some smaller compounds such as water, carbon dioxide, oxygen and nitrite, however, not to larger charged molecules such as 2-oxoglutarate. The key compound of the glutamine synthase/GOGAT reaction, the 2-oxoglutarate, must be moved from the cytosol of the plant cell by a specific translocator across the inner membrane of the chloroplast envelope into the stroma of the plastid. Transport of 2-oxoglutarate into the plastids takes place in exchange with malate from the plastids via the 2-oxoglutarate/malate translocator. The malate exported in this process into the cytosol is transported back by a second translocator, the dicarboxylate translocator, which is related with the 2-oxoglutarate/malate translocator in its substrate specificity, in exchange with glutamate. As a result, 2-oxoglutarate is imported into the chloroplast and the end product of the glutamine synthase/GOGAT reaction, glutamate, is exported without a net transport of malate which circulates via both translocator systems ("double translocator", Woo et al., 1987, Plant Physiol. 84
REFERENCES:
EMBL Sequence Database, Rel. 42, Dec. 13, 1994, Acc. No. U13238, Spinacia Oleracea Polka Chloroplast Envelope Membrane 2-Oxoglutarate/Malate Translocator (SODiT1) MRNA, Complete CDs.
Fischer, K., et al., "Cloning and in vivo Expression of Functional Troise Phosphate/Phosphate Translocators from C.sub.3 -and C.sub.4 -Plants: Evidence for the Putative Participation of Specific Amino Acid Residues in the Recognition of Phosphoenolpyruvate," The Plant Journal, 5(2), pp. 215-228 (1994).
Genchi, G., et al., Partial Purification and Reconstruction of the .alpha.-Ketogulatarate Carrier from Corn (Zea mays L. ) Mitochondria, Plant Physiol., 96, pp. 1003-1007 (1991).
Menzlaff, E. and U. Flugge, "Purification and Functional Reconstitution of the 2-oxoglutarate/malate Translocator from Spinach Chloroplasts," Biochim. Biophys. Acta, 1147, pp. 13-18 (1993).
Riesmeier, J.W., et al., "Antisense Repression of the Chloroplast Troise Phosphate Translocator Affects Carbon Partitioning In Transgenic Potato Plants," Proc. Natl. Acad. Sci. 90, pp. 6160-6164 (1993).
Runswick, M.J., et al., "Sequence Of The Bovine 2-Oxoglutarate/Malate Carrier Protein: Structural Relationship To Other Mitochondrial Transport Proteins," Biochemistry, 29, pp. 11033-11040 (1990); and.
Weber, A., et al., "The 2-Oxoglutarate/Malate Translocator of Chloroplast Envelope Membranes: Molecular Cloning of a Transporter Containing a 12-Helix Motif and Expression of the Functional Protein in Yeast Cells," Biochemistry, 34, pp. 2621-2627 (1995).
Fleischman et al. (1995) pir51 Database, Accession No. B64043.
Fischer Karsten
Flugge Ulf-Ingo
Weber Andreas
Haley, Jr. James F.
Hoechst Schering AgrEvo GmbH
Longton Enrique D.
Patterson Jr. Charles L.
Shin Elinor K.
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