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AssigneeSyngenta Participations AG
InventorsEdouard Guillaume Lebel, Peter Bernard Heifetz, Stephen Arthur Goff
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The invention provides novel transgenic plants which express trehalose biosynthetic genes, e.g., under control of an inducible promoter, which are developmentally normal, together with methods for improving stress tolerance in said plants, methods of improving food quality, and other methods of making and using the plants of the invention. The invention also provides nucleotide sequences encoded novel trehalose biosynthetic enzymes.
A plant comprising in its plastid genome at least one expression cassette comprising a nucleotide sequence encoding a trehalose 6-phosphate synthase under control of a promoter capable of directing the expression of said nucleotide sequence in a plastid of said plant.
A plastid expression cassette comprising a nucleotide sequence encoding a trehalose 6-phosphate synthase under control of a promoter capable of directing the expression of said nucleotide sequence in a plastid of a plant.
A recombinant vector comprising a nucleotide sequence encoding a trehalose 6-phosphate synthase under control of a promoter capable of directing the expression of said nucleotide sequence in a plastid of a plant.
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Glycosyltransferases: hexosyltransferases
Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for…:…
For plant cells , e.g. plant artificial chromosomes: methods for controlling, regulating or enhancing expression of transgenes in plant cells
Methods for controlling, regulating or enhancing expression of transgenes in plant cells: gene switch
Term ended 4 Mar 2019 (estimated)
The invention relates to expression of trehalose biosynthetic genes and drought resistance in plants.
Trehalose (a-D-glucopyranosyl-[1,1]-a-D-glucopyranoside) is a disaccharide commonly found in lower organisms such as bacteria, fungi and insects where it often accumulates in resting or stationary phase cells and organs. Two enzymatic activities are required for trehalose biosynthesis: a trehalose-6-phosphate synthase catalyses the condensation of UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate and a trehalose-6-phosphate phosphatase phosphorylates trehalose-6-phosphate to trehalose.
Although trehalose can serve as a storage form for reduced carbon, it may play a more significant role as a protectant against the deleterious effects of various abiotic stresses, notably heat and desiccation. Both in vivo and in vitro, trehalose accumulation is correlated with protection of biological macromolecules (particularly membranes and proteins) from dessication, temperature extremes, and osmotic shock. Trehalose produced by fermentation is used commercially in the preservation of enzymes and is registered as a food additive for the stabilization of dehydrated and processed foods.
A. Expression of the trehalose-6-phosphate Synthase and trehalose-6-phosphate Phosphatase Genes in the Plant Cytosol
Preparation of a Chimeric Gene Containing the E. coli trehalose-6-phosphate Synthase Gene Fused to the Tobacco PR-1a Promoter
Plasmid pCGN4467 containing the coding sequence of the E. coli trehalose-6-phosphate synthase gene (OtsA, Kaasen et al. (1994) Gene 145 (1), 9-15, EMBL/Genbank accession number X69160) under the control of a double 35S promoter and fused to the tmI3′ polyadenylation signals (pCGN4467 is a derivative of pCGN1761, EP 0392225) is used as template for PCR with a left-to-right “topstrand” primer including the ATG preceded by a GCC codon and followed by a newly added GCA codon, thus creating a NcoI restriction site at the ATG, and the first 24 bases of the OtsA gene (primer TREA+: GTC AGC CAT GGC AAG TCG TTT AGT CGT AGT ATC TAA C, SEQ ID No:1) and a right-to-left “bottomstrand” primer homologous to positions 392 to 416 downstream of the new ATG (primer TREA−: GCA AAT GGC AAC AGG TGA TAA TCG, SEQ ID No:2). This PCR reaction is undertaken with AmpliTaq DNA polymerase according to the manufacturer's recommendations (Perkin Elmer/Roche, Branchburg, N.J.) for five cycles at 94° C. (30 s), 40° C. (60 s), and 72° C. (30 s) followed by 25 cycles at 94° C. (30 s), 55° C. (60 s) and 72° C. (30 s) and this generated a product of 423 bp containing a NcoI site at its left end and a BamHI site at its right end. The fragment is gel purified using standard procedures, cleaved with NcoI and BamHI (all restriction enzymes are purchased from Promega, Madison, Wis.) and ligated into the NcoI and BamHI sites of pUC21 which is a pUC derivative containing a polylinker with the following restriction sites: SpeI/StuI/XhoI/BgIII/ClaI/NsiI/SphI/NcoI/KpnI/XmaI/SmaI/SacI/EcoRI/BstlBI/HindIII/PstI/MluI/SaII/AatlI/NdeI/BamHI/EcoRV/NotI/EagI/XbaI/SpeI to obtain pUCOTSA.