Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
AssigneeMonsanto Technology LLC
InventorsGerard F. Barry, Ganesh M. Kishore, Stephen R. Padgette, William C. Stallings
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Abstract
Genes encoding Class II EPSPS enzymes are disclosed. The genes are useful in producing transformed bacteria and plants which are tolerant to glyphosate herbicide. Class II EPSPS genes share little homology with known, Class I EPSPS genes, and do not hybridize to probes from Class I EPSPS's. The Class II EPSPS enzymes are characterized by being more kinetically efficient than Class I EPSPS's in the presence of glyphosate. Plants transformed with Class II EPSPS genes are also disclosed as well as a method for selectively controlling weeds in a planted transgenic crop field.
Claims149
An isolated DNA molecule which encodes an EPSPS enzyme having the sequence of SEQ ID NO:3.
A recombinant, double-stranded DNA molecule comprising in sequence:
- a) a promoter which functions in plant cells to cause the production of an RNA sequence;
- b) a structural DNA sequence that causes the production of an RNA sequence which encodes a EPSPS enzyme having the sequence domains:
- -R-X1-H-X2-E-(SEQ ID NO:37), in which
- X1 is G, S, T, C, Y, N, Q, D or E;
- X2 is S or T;
- and
- -G-D-K-X3-(SEQ ID NO:38), in which
- X3 is S or T;
- and
- -S-A-Q-X4-K-(SEQ ID NO:39), in which
- X4 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V;
- and
- -N-X5-T-R-(SEQ ID NO:40), in which
- X5 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V,
- provided that when X1 is D, X2 is T, X3 is S, and X4 is V, then X5 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, S, T, W, Y, or V;
- and
- c) a 3′ non-translated region which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence;
- where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the encoded EPSPS enzyme to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule.
A method of producing genetically transformed plants which are tolerant toward glyphosate herbicide, comprising the steps of:
- a) inserting into the genome of a plant cell a recombinant, double-stranded DNA molecule comprising:
- i) a promoter which functions in plant cells to cause the production of an RNA sequence,
- ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence domains:
- -R-X1-H-X2-E-(SEQ ID NO:37), in which
- X1 is G, S, T, C, Y, N, Q, D or E;
- X2 is S or T;
- and
- -G-D-K-X3-(SEQ ID NO:38), in which
- X3 is S or T;
- and
- -S-A-Q-X4-K-(SEQ ID NO:39), in which
- X4 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V;
- and
- -N-X5-T-R-(SEQ ID NO:40), in which
- X5 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, provided that when X1 is D, X2 is T, X3 is S, and X4 is V, then X5 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, S, T, W, Y or V;
- and
- iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence;
- where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the polypeptide to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule;
- b) obtaining a transformed plant cell;
- and
- c) regenerating from the transformed plant cell a genetically transformed plant which has increased tolerance to glyphosate herbicide.
A method for selectively controlling weeds in a field containing a crop having plant crop seeds or plants comprising the steps of:
- a) planting the crop seeds or plants which are glyphosate-tolerant as a result of a recombinant double-stranded DNA molecule being inserted into the crop seed or plant, the DNA molecule having:
- i) a promoter which functions in plant cells to cause the production of an RNA sequence,
- ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence domains:
- -R-X1-H-X2-E-(SEQ ID NO:37), in which
- X1 is G, S, T, C, Y, N, Q, D or E;
- X2 is S or T;
- and
- -G-D-K-X3-(SEQ ID NO:38), in which
- X3 is S or T;
- and
- -S-A-Q-X4-K-(SEQ ID NO:39), in which
- X4 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V;
- and
- -N-X5-T-R-(SEQ ID NO:40), in which
- X5 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V, provided that when X1 is D, X2 is T, X3 is S, and X4 is V, then X5 is A, R, N, D, C, Q, E, G, H, I, L, K, M, F, S, T, W, Y or V;
- and
- iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence
- where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the EPSPS enzyme to enhance the glyphosate tolerance of the crop plant transformed with the DNA molecule;
- and
- b) applying to the crop and weeds in the field a sufficient amount of glyphosate herbicide to control the weeds without significantly affecting the crop.
A glyphosate-tolerant plant comprising a the DNA molecule of claims 5, 8 or 10 claim 131 in which:
- (a) the promoter is selected from the group consisting of CaMV 35S and FMV 35S promoters;
- (b) the structural DNA sequence encodes:
- ;
- (i) a chloroplast transit peptide selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17;
- and
- (ii) an EPSPS enzyme selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, SEQ ID NO:42 and SEQ ID NO:44 ;
- and
- (c) the 3′ non-translated region is selected from the group consisting of the NOS 3′ and the E9 3′ non-translated regions.
A transgenic soybean plant which contains a heterologous gene which encodes an EPSPS enzyme having a Km for phosphoenolpyruvate (PEP) between 1 and 150 μM and a Ki(glyphosate)/Km(PEP) ratio between about 2 and 500, said plant exhibiting tolerance to N-phosphonomethylglycine herbicide at a rate of 1 lb/acre without significant yield reduction due to herbicide application.
An isolated DNA molecule that encodes a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme having the sequence of SEQ ID NO:70.
A recombinant, double-stranded DNA molecule comprising in sequence:
- a) a promoter which functions in plant cells to cause the production of an RNA sequence;
- b) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:70;
- and
- c) a 3′ non-translated region that functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence;
- where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the encoded EPSPS enzyme to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule.
A method of producing a genetically transformed plant which is tolerant toward glyphosate herbicide, comprising the steps of:
- a) inserting into the genome of a plant cell a recombinant double-stranded DNA molecule comprising:
- i) a promoter that functions in plant cells to cause the production of an RNS sequence;
- ii) a structural DNA sequence that causes the production of an RNS sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:
- 70;
- and
- iii) a 3′ non-translated DNA sequence that functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNS sequence;
- wherein the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the polypeptide to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule;
- b) obtaining a transformed plant cell;
- and
- c) regenerating from the transformed plant cell a genetically transformed plant which has increased tolerance to glyphosate herbicide.
A glyphosate-tolerant plant cell comprising a DNA sequence encoding an EPSPS enzyme having the sequence of SEQ ID NO:
- 70.
A glyphosate-tolerant plant comprising a DNA sequence encoding an EPSPS enzyme having the sequence of SEQ ID NO:
- 70.
A method for selectively controlling weeds in a field containing a crop having planted crop seeds or plants comprising the steps of:
- a) planting the crop seeds or plants which are glyphosate-tolerant as a result of a recombinant double-stranded DNA molecule being inserted into the crop seed or plant, the DNA molecule having:
- i) a promoter which functions in plant cells to cause the production of an RNA sequence,
- ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:70;
- and
- iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence,
- where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the EPSPS enzyme to enhance the glyphosate tolerance of the crop plant transformed with the DNA molecule;
- and
- b) applying to the crop and weeds in the field a sufficient amount of glyphosate herbicide to control the weeds without significantly affecting the crop.
A recombinant, double-stranded DNA molecule comprising in sequence:
- a) a promoter which functions in plant cells to cause the production of an RNA sequence;
- b) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:
- 7;
- c) a 3′ non-translated region which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence;
- where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the encoded EPSPS enzyme to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule.
A method of producing genetically transformed plants which are tolerant toward glyphosate herbicide, comprising the steps of:
- a) inserting into the genome of a plant cell a recombinant, double-stranded DNA molecule comprising:
- i) a promoter which functions in plant cells to cause the production of an RNA sequence,
- ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7;
- and
- iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence;
- where the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the polypeptide to enhance the glyphosate tolerance of a plant cell transformed with the DNA molecule;
- b) obtaining a transformed plant cell;
- and
- c) regenerating from the transformed plant cell a genetically transformed plant which has increased tolerance to glyphosate herbicide.
A glyphosate-tolerant plant cell comprising an EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.
A glyphosate-tolerant plant comprising an EPSPS enzyme having the sequence of SEQ ID NO:
- 3, SEQ ID NO:
- 5 or SEQ ID NO:
- 7.
A method for selectively controlling weeds in a field containing a crop having planted crop seeds or plants comprising the steps of:
- a) planting the crop seeds or plants which are glyphosate-tolerant as a result of a recombinant double-stranded DNA molecule being inserted into the crop seed or plant, the DNA molecule having:
- i) a promoter which functions in plant cells to cause the production of an RNA sequence,
- ii) a structural DNA sequence that causes the production of an RNA sequence which encodes an EPSPS enzyme having the sequence of SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7;
- and
- iii) a 3′ non-translated DNA sequence which functions in plant cells to cause the addition of a stretch of polyadenyl nucleotides to the 3′ end of the RNA sequence,
- wherein the promoter is heterologous with respect to the structural DNA sequence and adapted to cause sufficient expression of the EPSPS enzyme to enhance the glyphosate tolerance of the crop plant transformed with the DNA molecule;
- and
- b) applying to the crop and weeds in the field a sufficient amount of glyphosate herbicide to control the weeds without significantly affecting the crop.
Dependencies are inferred from the claim text.