Claims
- 1. An isolated polynucleotide comprising the sequence depicted in SEQ ID No. 43.
- 2. A polynucleotide encoding an EPSPS, excluding the cDNA encoding the rice and corn EPSPS, which polynucleotide is complementary to one which when incubated at a temperature of between 65 and 70° C. in 0.1 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.1 strength citrate buffered saline containing 0.1% SDS still hybridises with the sequence depicted in SEQ ID No. 43.
- 3. A polynucleotide encoding an EPSPS obtainable by screening plant genomic DNA libraries with a polynucleotide constituting an intron within the SEQ ID No. 43 sequence.
- 4. An isolated polynucleotide comprising a region encoding a chloroplast transit peptide and a glyphosate resistant 5-enolpyruvylshikimate phosphate synthase (EPSPS) 3′ of the peptide, the said region being under expression control of a plant operable promoter, with the provisos that the said promoter is not heterologous with respect to the said region, and the chloroplast transit peptide is not heterologous with respect to the said synthase.
- 5. A polynucleotide according to claim 2, comprising the following components in the 5′ to 3′ direction of transcription:
(i) at least one transcriptional enhancer being that enhancing region which is upstream from the transcriptional start of the sequence from which the enhancer is obtained and which enhancer per se does not function as a promoter either in the sequence in which it is endogenously comprised or when present heterologously as part of a construct; (ii) the promoter from the rice EPSPS gene; (iii) the rice genomic sequence which encodes the rice EPSPS chloroplast transit peptide; (iv) the genomic sequence which encodes the rice EPSPS; (v) a transcriptional terminator;
wherein the rice EPSPS coding sequence is modified in that a first position is mutated so that the residue at this position is Ile rather than Thr and a second position is mutated so that the residue at this position is Ser rather than Pro, the mutations being introduced into EPSPS sequences which comprise the following conserved region GNAGTAMRPLTAAV in the wild type enzyme such that modified sequence reads GNAGIAMRSLTAAV.
- 6. A polynucleotide according to claim 5, wherein the said enhancing region comprises a sequence, the 3′ end of which is at least 40 nucleotides upstream of the closest transcriptional start of the sequence from which the enhancer is obtained.
- 7. A polynucleotide according to claim 6, wherein the enhancing region comprises a region the 3′ end of which is at least 60 nucleotides upstream of the said closest start.
- 8. A polynucleotide according to claim 5, wherein the said enhancing region comprises a sequence the 3′ end of which is at least 10 nucleotides upstream from the first nucleotide of the TATA consensus of the sequence from which the enhancer is obtained.
- 9. A polynucleotide according to claim 5, comprising first and second transcriptional enhancers.
- 10. A polynucleotide according to claim 9, wherein the first and second enhancers are tandemly present in the polynucleotide.
- 11. A polynucleotide according to claim 5, wherein the 3′ end of the enhancer, or first enhancer, is between about 100 to about 1000 nucleotides upstream of the codon corresponding to the translational start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5′ untranslated region
- 12. A polynucleotide according to claim 5, wherein the 3′ end of the enhancer, or first enhancer, is between about 150 to about 1000 nucleotides upstream of the codon corresponding to the translational start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5′ untranslated region.
- 13. A polynucleotide according to claim 5, wherein the 3′ end of the enhancer, or first enhancer, is between about 300 to about 950 nucleotides upstream of the codon corresponding to the translational start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5′ untranslated region.
- 14. A polynucleotide according to claim 5, wherein the 3′ end of the enhancer, or first enhancer, is between about 770 and about 790 nucleotides upstream of the codon corresponding to the translational start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5′ untranslated region.
- 15. A polynucleotide according to claim 5, wherein the 3′ end of the enhancer, or first enhancer, is between about 300 and about 380 nucleotides upstream of the codon corresponding to the translational start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5′ untranslated region.
- 16. A polynucleotide according to claim 5, wherein the 3′ end of the enhancer, or first enhancer, is between about 320 and about 350 nucleotides upstream of the codon corresponding to the translational start of the EPSPS transit peptide, or the first nucleotide of an intron in the 5′ untranslated region.
- 17. A polynucleotide according to claim 5, wherein the region upstream of the promoter from the rice EPSPS gene comprises at least one enhancer derived from a sequence which is upstream from the transcriptional start of either the CaMV35S or FMV35S promoters.
- 18. A polynucleotide according to claim 5, comprising in the 5′ to 3′ direction a first enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of the GOS 2 promoter and a second enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of either the CaMV35S or FMV35S promoters.
- 19. A polynucleotide according to claim 5, comprising in the 5′ to 3′ direction a first enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of the rice actin promoter and a second enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of either of the FMV35S or CaMV35S promoters.
- 20. A polynucleotide according to claim 5, comprising in the 5′ to 3′ direction a first enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of the barley plastocyanin promoter and a second enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of either the CaMV35S or FMV35S promoters.
- 21. A polynucleotide according to claim 5, comprising in the 5′ to 3′ direction a first enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of the maize polyubuitin promoter and a second enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of either the CaMV35S or FMV35S promoters.
- 22. A polynucleotide according to claim 5, comprising in the 5′ to 3′ direction a first enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of the FMV35S promoter and a second enhancer comprising a transcriptional enhancing region derived from a sequence which is upstream from the transcriptional start of the CaMV35S promoter.
- 23. A polynucleotide according to claim 5, wherein the nucleotides 5′ of the codon which constitutes the translational start of the rice EPSPS chloroplast transit peptide are Kozak preferred.
- 24. A polynucleotide according to claim 5, wherein 5′ of the rice genomic sequence which encodes the rice EPSPS chloroplast transit peptide there is located a non-translated region which comprises a sequence which functions as an intron.
- 25. A polynucleotide according to claim 24 wherein the non-translated region comprises an intron, wherein the intron is the maize ADHI intron.
- 26. A polynucleotide according to claim 25, wherein the intron comprises the sequence depicted in SEQ ID NO. 55.
- 27. A polynucleotide according to claim 5, which comprises a virally derived translational enhancer or other non-viral translational enhancer located within the non translated region 5′ of the rice genomic sequence which encodes the rice EPSPS chloroplast transit peptide.
- 28. A polynucleotide according to claim 5, further comprising regions encoding proteins capable of conferring upon plant material containing it at least one of the following agronomically desirable traits: resistance to insects, fungi, viruses, bacteria, nematodes, stress, dessication, and herbicides.
- 29. A polynucleotide according to claim 28, wherein the herbicide is other than glyphosate.
- 30. A polynucleotide according to claim 28, wherein the insect resistance conferring regions encode crystal toxins derived from Bt, including secreted Bt toxins; protease inhibitors, lectins, Xenhorabdus/Photorhabdus toxins; the fungus resistance conferring regions are selected from the group consisting of those encoding known AFPs, defensins, chitinases, glucanases, Avr-Cf9; the bacterial resistance conferring regions are selected from the group consisting of those encoding cecropins and techyplesin and analogues thereof; the virus resistance regions are selected from the group consisting of genes encoding virus coat proteins, movement proteins, viral replicases, and antisense and ribozyme sequences which are known to provide for virus resistance; the stress, salt, and drought resistance conferring regions are selected from those that encode Glutathione-S-transferase and peroxidase, the sequence which constitutes the known CBF1 regulatory sequence and genes which are known to provide for accumulation of trehalose.
- 31. A polynucleotide according to claim 30, wherein the insect resistance conferring regions are selected from the group consisting of cryIAc, cryIAb, cry3A, Vip 1A, Vip 1B, cystein protease inhibitor, and snowdrop lectin genes.
- 32. A polynucleotide according to claim 2, which is modified in that mRNA instability motifs and/or unwanted splice regions are removed, or crop preferred codons are used so that expression of the thus modified polynucleotide in a plant yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the protein encoding regions of the unmodified polynucleotide in the organism in which they are endogenous.
- 33. A polynucleotide according to claim 32, wherein the degree of identity between the modified polynucleotide and a polynucleotide endogenously contained within the said plant and encoding substantially the same protein is such as to prevent co-suppression between the modified and endogenous sequences.
- 34. A polynucleotide according to claim 33, wherein the said degree is less than about 70%.
- 35. A vector comprising the polynucleotide of claim 2.
- 36. Plant material which has been transformed with the vector of claim 35.
- 37. Plant material which has been transformed with the vector of claim 35, and which has been, or is, further transformed with a polynucleotide comprising regions encoding proteins capable of conferring upon plant material containing it at least one of the following agronomically desirable traits: resistance to insects, fungi, viruses, bacteria, nematodes, stress, desiccation, and herbicides.
- 38. Morphologically normal, fertile whole plants which have been regenerated from the material according to claim 36, progeny seeds or parts.
- 39. Morphologically normal fertile whole plants which comprise the polynucleotide of claim 2 and which result from the crossing of plants which have been regenerated from material transformed with the polynucleotide of claim 2, and plants which have been transformed with a polynucleotide comprising regions encoding proteins capable of conferring upon plant material containing it at least one of the following agronomically desirable traits: resistance to insects, fungi, viruses, bacteria, nematodes, stress, desiccation, and herbicides, the progeny of the resultant plants, their seeds and parts.
- 40. Plants according to claim 38, selected from the group consisting of field crops, fruits and vegetables such as canola, sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, soya spp, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses, flax and oilseed rape, and nut producing plants insofar as they are not already specifically mentioned, their progeny, seeds and parts.
- 41. Maize, wheat or rice plants according to claim 38.
- 42. A method of selectively controlling weeds in a field, the field comprising weeds and plants or progeny according to claim 38, the method comprising application to the field of a glyphosate type herbicide in an amount sufficient to control the weeds without substantially affecting the plants.
- 43. A method according to claim 42, further comprising application to the field either before or after application of the glyphosate herbicide of one or more of the following: a herbicide, insecticide, fungicide, nematicide, bacteriocide and an anti-viral.
- 44. A method of producing plants which are substantially tolerant or substantially resistant to glyphosate herbicide, comprising the steps of:
(i) transforming plant material with the vector of claim 35;(ii) selecting the thus transformed material; and (iii) regenerating the thus selected material into morphologically normal fertile whole plants.
- 45. A method according to claim 44, wherein the transformation involves the introduction of the polynucleotide into the material by: (i) biolistic bombardment of the material with particles coated with the polynucleotide; or (ii) impalement of the material on silicon carbide fibres which are coated with a solution comprising the polynucleotide; or (iii) introduction of the polynucleotide or vector into Agrobacterium and co-cultivation of the thus transformed Agrobacterium with plant material which is thereby transformed and is subsequently regenerated.
- 46. A method according to claim 45, wherein the transformed material is selected by its resistance to glyphosate.
- 47. A method of selecting biological material transformed so as to express a gene of interest, wherein the transformed material comprises the polynucleotide of claim 2 and wherein the selection comprises exposing the transformed material to glyphosate or a salt thereof, and selecting surviving material.
- 48. A method according to claim 47, wherein the biological material is of plant origin.
- 49. A method according to claim 48, wherein the plant is a monocot.
- 50. A method according to claim 49, wherein the monocot is selected from group consisting of barley, wheat, corn, rice, oats, rye, sorghum, pineapple, sugar cane, banana, onion, asparagus, leek,
- 51. A method for regenerating a fertile transformed plant to contain foreign DNA comprising the steps of:
(a) producing regenerable tissue from said plant to be transformed; (b) transforming said regenerable tissue with said foreign DNA, wherein said foreign DNA comprises a selectable DNA sequence, wherein said sequence functions in a regenerable tissue as a selection device; (c) between about one day to about 60 days after step (b), placing said regenerable tissue from step (b) in a medium capable of producing shoots from said tissue, wherein said medium further contains a compound used to select regenerable tissue containing said selectable DNA sequence to allow identification or selection of the transformed regenerated tissue; (d) after at least one shoot has formed from the selected tissue of step (c) transferring said shoot to a second medium capable of producing roots from said shoot to produce a plantlet, wherein the second medium optionally contains the said compound; and (e) growing said plantlet into a fertile transgenic plant wherein the foreign DNA is transmitted to progeny plants in Mendelian fashion, wherein between step (b) and step (c) there is an optional step of placing the transformed material onto callus inducing medium, characterised in that the foreign DNA is, or the selectable DNA sequence comprised by the foreign DNA comprises, the polynucleotide according to claim 2, and the said compound is glyphosate or a salt thereof.
- 52. A method according to claim 51, wherein the plant is a monocot selected from the group consisting of banana, wheat, rice, corn and barley.
- 53. A method according to claim 51, wherein the said regenerable tissue is selected from the group consisting of embryogenic calli, somatic embryos, immature embryos etc.
Priority Claims (11)
Number |
Date |
Country |
Kind |
9930213.5 |
Apr 1999 |
GB |
|
9917834.5 |
Apr 1999 |
GB |
|
9909968.1 |
Apr 1999 |
GB |
|
9917839.4 |
Jul 1999 |
GB |
|
9917840.2 |
Jul 1999 |
GB |
|
9917846.9 |
Jul 1999 |
GB |
|
9917847.7 |
Jul 1999 |
GB |
|
9930204.4 |
Dec 1999 |
GB |
|
9930209.3 |
Dec 1999 |
GB |
|
9930207.7 |
Dec 1999 |
GB |
|
9930200.2 |
Dec 1999 |
GB |
|
Parent Case Info
[0001] This is a continuation of International Application No. PCT/GB00/01573, filed Apr. 20, 2000 (published as WO 00/66748), which claims priority to the following GB national applications: GB 9930213.5, filed Apr. 29, 1999; GB 9917834.5 filed Apr. 29, 1999; GB 9909968.1, filed Apr. 29, 1999; GB 9917839.4, filed Jul. 29, 1999; GB 9917840.2, filed Jul. 29, 1999; GB 9917846.9, filed Jul. 29, 1999; GB 9917847.7, filed Jul. 29, 1999; GB 9930204.4, filed Dec. 21, 1999; GB 9930209.3, filed Dec. 21, 1999; GB 9930207.7, filed Dec. 21, 1999, and GB 9930200.2, filed Dec. 21, 1999. The disclosure of each of these above-listed applications is incorporated by reference in its entirety into the instant disclosure.
Continuations (1)
|
Number |
Date |
Country |
Parent |
PCT/GB00/01573 |
Apr 2000 |
US |
Child |
10012070 |
Oct 2001 |
US |