Non-transgenic herbicide resistant plants
11160224 · 2021-11-02
Assignee
Inventors
- Peter R. Beetham (Carlsbad, CA)
- Patricia L. Avissar (East Brunswick, NJ, US)
- Keith A. Walker (San Diego, CA, US)
- Richard A. Metz (Lawrenceville, NJ, US)
Cpc classification
C12N9/1092
CHEMISTRY; METALLURGY
A01H1/06
HUMAN NECESSITIES
C12N15/8213
CHEMISTRY; METALLURGY
International classification
C12N15/82
CHEMISTRY; METALLURGY
A01H1/06
HUMAN NECESSITIES
A01H1/00
HUMAN NECESSITIES
Abstract
The present invention relates to the production of a non-transgenic plant resistant or tolerant to a herbicide of the phosphonomethylglycine family, e.g., glyphosate. The present invention also relates to the use of a recombinagenic oligonucleobase to make a desired mutation in the chromosomal or episomal sequences of a plant in the gene encoding for 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS). The mutated protein, which substantially maintains the catalytic activity of the wild-type protein, allows for increased resistance or tolerance of the plant to a herbicide of the phosphonomethylglycine family, and allows for the substantially normal growth or development of the plant, its organs, tissues or cells as compared to the wild-type plant irrespective of the presence or absence of the herbicide. The present invention also relates to a non-transgenic plant cell in which the EPSPS gene has been mutated, a non-transgenic plant regenerated therefrom, as well as a plant resulting from a cross using a regenerated non-transgenic plant having a mutated EPSPS gene.
Claims
1. A non-transgenic herbicide resistant plant that expresses a mutant EPSPS gene product wherein the mutant EPSPS gene product comprises a Thr to Ile substitution at position Thr.sub.178 in Arabidopsis (SEQ ID NO:2) or at an analogous amino acid residue in an EPSPS paralog, and a Pro to Leu at position Pro.sub.182 in Arabidopsis (SEQ ID NO:2) or at an analogous amino acid residue in an EPSPS paralog, wherein the plant is selected from the group consisting of rice, soybean, flax, canola, sugar beet, wheat, and potato and the herbicide is a phosphonomethylglycine herbicide.
2. The plant according to claim 1 in which the member of the phosphonomethylglycine family is glyphosate.
3. The non-transgenic herbicide resistant plant of claim 1, wherein said plant is potato.
4. The non-transgenic herbicide resistant plant of claim 1, wherein said plant is wheat.
5. The non-transgenic herbicide resistant plant of claim 1, wherein said plant is sugarbeet.
6. A non-transgenic herbicide resistant plant cell that expresses a mutant EPSPS gene product wherein the mutant EPSPS gene product comprises a Thr to Ile substitution at position Thr.sub.178 in Arabidopsis (SEQ ID NO:2) or at an analogous amino acid residue in an EPSPS paralog, and a Pro to Leu at position Pro 182 in Arabidopsis (SEQ ID NO:2) or at an analogous amino acid residue in an EPSPS paralog, wherein the plant cell is selected from the group consisting of a rice cell, a soybean cell, a flax cell, a canola cell, a sugarbeet cell, a wheat cell, and a potato cell and the herbicide is a phosphonomethylglycine herbicide.
7. The plant according to claim 6 in which the member of the phosphonomethylglycine family is glyphosate.
8. The non-transgenic herbicide resistant plant cell of claim 6, wherein said plant cell is a potato cell.
9. The non-transgenic herbicide resistant plant of claim 6, wherein said plant cell is a wheat cell.
10. The non-transgenic herbicide resistant plant of claim 6, wherein said plant cell is a sugarbeet cell.
Description
4. BRIEF DESCRIPTION OF THE FIGURES
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5. DETAILED DESCRIPTION OF THE INVENTION
(9) The present invention is directed to a non-transgenic plant or plant cell having a mutation in the EPSPS gene, which plant has increased resistance or tolerance to a member of the phosphonomethylglycine family and which plant exhibits substantially normal growth or development of the plant, its organs, tissues or cells, as compared to the corresponding wild-type plant or cell. The present invention is also directed to a non-transgenic plant having a mutation in the EPSPS gene, which plant is resistant to or has an increased tolerance to a member of the phosphonomethylglycine family, e.g., glyphosate, wherein the mutated EPSPS protein has substantially the same catalytic activity as compared to the wild-type EPSPS protein.
(10) The present invention is also directed to a method for producing a non-transgenic plant having a mutated EPSPS gene that substantially maintains the catalytic activity of the wild-type protein irrespective of the presence or absence of a herbicide of the phosphonomethylglycine family. The method comprises introducing into a plant cell a recombinagenic oligonucleobase with a targeted mutation in the EPSPS gene and identifying a cell, seed, or plant having a mutated EPSPS gene.
(11) Illustrative examples of a recombinagenic oligonucleobase is found in following patent publications, which are incorporated in their entirety be reference herein: U.S. Pat. Nos. 5,565,350; 5,756,325; 5,871,984; 5,760,012; 5,731,181; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Patent No. PCT/US00/23457; and in International Patent Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO 99/40789.
(12) The plant can be of any species of dicotyledonous, monocotyledonous or gymnospermous plant, including any woody plant species that grows as a tree or shrub, any herbaceous species, or any species that produces edible fruits, seeds or vegetables, or any species that produces colorful or aromatic flowers. For example, the plant may be selected from a species of plant from the group consisting of canola, sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, onion, soya spp, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses, flax, oilseed rape, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nut producing plants insofar as they are not already specifically mentioned.
(13) The recombinagenic oligonucleobase can be introduced into a plant cell using any method commonly used in the art, including but not limited to, microcarriers (biolistic delivery), microfibers, electroporation, microinjection.
(14) The invention is also directed to the culture of cells mutated according to the methods of the present invention in order to obtain a plant that produces seeds, henceforth a “fertile plant”, and the production of seeds and additional plants from such a fertile plant.
(15) The invention is further directed to a method of selectively controlling weeds in a field, the field comprising plants with the disclosed EPSPS gene alterations and weeds, the method comprising application to the field of a herbicide to which the said plants have been rendered resistant.
(16) The invention is also directed to novel mutations in the EPSPS gene that confer resistance or tolerance to a member of the phosphonomethylglycine family, e.g., glyphosate, to a plant or wherein the mutated EPSPS has substantially the same enzymatic activity as compared to wild-type EPSPS.
5.1 Recombinagenic Oligonucleobases
(17) The invention can be practiced with recombinagenic oligonucleobases having the conformations and chemistries described in U.S. Pat. No. 5,565,350 to Kmiec (Kmiec I) and U.S. Pat. No. 5,731,181 (Kmiec II) gene, which are hereby incorporated by reference. Kmiec I teaches a method for introducing specific genetic alterations into a target gene. The recombinagenic oligonucleobases in Kmiec I and/or Kmiec II contain two complementary strands, one of which contains at least one segment of RNA-type nucleotides (an “RNA segment”) that are base paired to DNA-type nucleotides of the other strand.
(18) Kmiec II discloses that purine and pyrimidine base-containing non-nucleotides can be substituted for nucleotides. U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Patent No. PCT/US00/23457; and in International Patent Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO 99/40789, which are each hereby incorporated in their entirety, disclose additional recombinagenic molecules that can be used for the present invention. The term “recombinagenic oligonucleobase” is used herein to denote the molecules that can be used in the methods of the present invention and include mixed duplex oligonucleotides, non-nucleotide containing molecules taught in Kmiec II, single stranded oligodeoxynucleotides and other recombinagenic molecules taught in the above noted patents and patent publications.
(19) In one embodiment, the recombinagenic oligonucleobase is a mixed duplex oligonucleotide in which the RNA-type nucleotides of the mixed duplex oligonucleotide are made RNase resistant by replacing the 2′-hydroxyl with a fluoro, chloro or bromo functionality or by placing a substituent on the 2′-O. Suitable substituents include the substituents taught by the Kmiec II. Alternative substituents include the substituents taught by U.S. Pat. No. 5,334,711 (Sproat) and the substituents taught by patent publications EP 629 387 and EP 679 657 (collectively, the Martin Applications), which are hereby incorporated by reference. As used herein, a 2′-fluoro, chloro or bromo derivative of a ribonucleotide or a ribonucleotide having a 2′-OH substituted with a substituent described in the Martin Applications or Sproat is termed a “2′-Substituted Ribonucleotide.” As used herein the term “RNA-type nucleotide” means a 2′-hydroxyl or 2′-Substituted Nucleotide that is linked to other nucleotides of a mixed duplex oligonucleotide by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II. As used herein the term “deoxyribo-type nucleotide” means a nucleotide having a 2′-H, which can be linked to other nucleotides of a MDON by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II.
(20) In a particular embodiment of the present invention, the recombinagenic oligonucleobase is a mixed duplex oligonucleotide that is linked solely by unsubstituted phosphodiester bonds. In alternative embodiments, the linkage is by substituted phosphodiesters, phosphodiester derivatives and non-phosphorus-based linkages as taught by Kmiec II. In yet another embodiment, each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2′-Substituted Nucleotide. Particular preferred embodiments of 2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy, 2′-propyloxy, 2′-allyloxy, 2′-hydroxylethyloxy, 2′-methoxyethyloxy, 2′-fluoropropyloxy and 2′-trifluoropropyloxy substituted ribonucleotides. More preferred embodiments of 2′-Substituted Ribonucleotides are 2′-fluoro, 2′-methoxy, 2′-methoxyethyloxy, and 2′-allyloxy substituted nucleotides. In another embodiment the mixed duplex oligonucleotide is linked by unsubstituted phosphodiester bonds.
(21) Although mixed duplex oligonucleotide having only a single type of 2′-substituted RNA-type nucleotide are more conveniently synthesized, the methods of the invention can be practiced with mixed duplex oligonucleotides having two or more types of RNA-type nucleotides. The function of an RNA segment may not be affected by an interruption caused by the introduction of a deoxynucleotide between two RNA-type trinucleotides, accordingly, the term RNA segment encompasses such an “interrupted RNA segment.” An uninterrupted RNA segment is termed a contiguous RNA segment. In an alternative embodiment an RNA segment can contain alternating RNase-resistant and unsubstituted 2′-OH nucleotides. The mixed duplex oligonucleotides preferably have fewer than 100 nucleotides and more preferably fewer than 85 nucleotides, but more than 50 nucleotides. The first and second strands are Watson-Crick base paired. In one embodiment the strands of the mixed duplex oligonucleotide are covalently bonded by a linker, such as a single stranded hexa, penta or tetranucleotide so that the first and second strands are segments of a single oligonucleotide chain having a single 3′ and a single 5′ end. The 3′ and 5′ ends can be protected by the addition of a “hairpin cap” whereby the 3′ and 5′ terminal nucleotides are Watson-Crick paired to adjacent nucleotides. A second hairpin cap can, additionally, be placed at the junction between the first and second strands distant from the 3′ and 5′ ends, so that the Watson-Crick pairing between the first and second strands is stabilized.
(22) The first and second strands contain two regions that are homologous with two fragments of the target EPSPS gene, i.e., have the same sequence as the target gene. A homologous region contains the nucleotides of an RNA segment and may contain one or more DNA-type nucleotides of connecting DNA segment and may also contain DNA-type nucleotides that are not within the intervening DNA segment. The two regions of homology are separated by, and each is adjacent to, a region having a sequence that differs from the sequence of the target gene, termed a “heterologous region.” The heterologous region can contain one, two or three mismatched nucleotides. The mismatched nucleotides can be contiguous or alternatively can be separated by one or two nucleotides that are homologous with the target gene. Alternatively, the heterologous region can also contain an insertion or one, two, three or of five or fewer nucleotides. Alternatively, the sequence of the mixed duplex oligonucleotide may differ from the sequence of the target gene only by the deletion of one, two, three, or five or fewer nucleotides from the mixed duplex oligonucleotide. The length and position or the heterologous region is, in this case, deemed to be the length of the deletion, even though no nucleotides of the mixed duplex oligonucleotide are within the heterologous region. The distance between the fragments of the target gene that are complementary to the two homologous regions is identically the length of the heterologous region when a substitution or substitutions is intended. When the heterologous region contains an insertion, the homologous regions are thereby separated in the mixed duplex oligonucleotide farther than their complementary homologous fragments are in the gene, and the converse is applicable when the heterologous region encodes a deletion.
(23) The RNA segments of the mixed duplex oligonucleotides are each a part of a homologous region, i.e., a region that is identical in sequence to a fragment of the target gene, which segments together preferably contain at least 13 RNA-type nucleotides and preferably from 16 to 25 RNA-type nucleotides or yet more preferably 18-22 RNA-type nucleotides or most preferably 20 nucleotides. In one embodiment, RNA segments of the homology regions are separated by and adjacent to, i.e., “connected by” an intervening DNA segment. In one embodiment, each nucleotide of the heterologous region is a nucleotide of the intervening DNA segment. An intervening DNA segment that contains the heterologous region of a mixed duplex oligonucleotide is termed a “mutator segment.”
(24) The change to be introduced into the target EPSPS gene is encoded by the heterologous region. The change to be introduced into the EPSPS gene may be a change in one or more bases of the EPSPS gene sequence or the addition or deletion of one or more bases.
(25) In another embodiment of the present invention, the recombinagenic oligonucleobase is a single stranded oligodeoxynucleotide mutational vector or SSOMV, which is disclosed in International Patent Application PCT/US00/23457, which is incorporated by reference in its entirety. The sequence of the SSOMV is based on the same principles as the mutational vectors described in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO 99/40789. The sequence of the SSOMV contains two regions that are homologous with the target sequence separated by a region that contains the desired genetic alteration termed the mutator region. The mutator region can have a sequence that is the same length as the sequence that separates the homologous regions in the target sequence, but having a different sequence. Such a mutator region can cause a substitution. Alternatively, the homolgous regions in the SSOMV can be contiguous to each other, while the regions in the target gene having the same sequence are separated by one, two or more nucleotides. Such a SSOMV causes a deletion from the target gene of the nucleotides that are absent from the SSOMV. Lastly, the sequence of the target gene that is identical to the homologous regions may be adjacent in the target gene but separated by one two or more nucleotides in the sequence of the SSOMV. Such an SSOMV causes an insertion in the sequence of target gene.
(26) The nucleotides of the SSOMV are deoxyribonucleotides that are linked by unmodified phosphodiester bonds except that the 3′ terminal and/or 5′ terminal internucleotide linkage or alternatively the two 3′ terminal and/or 5′ terminal internucleotide linkages can be a phosphorothioate or phosphoamidate. As used herein an internucleotide linkage is the linkage between nucleotides of the SSOMV and does not include the linkage between the 3′ end nucleotide or 5′ end nucleotide and a blocking substituent, see supra. In a specific embodiment the length of the SSOMV is between 21 and 55 deoxynucleotides and the lengths of the homology regions are, accordingly, a total length of at least 20 deoxynucleotides and at least two homology regions should each have lengths of at least 8 deoxynucleotides.
(27) The SSOMV can be designed to be complementary to either the coding or the non-coding strand of the target gene. When the desired mutation is a substitution of a single base, it is preferred that both the mutator nucleotide be a pyrimidine. To the extent that is consistent with achieving the desired functional result it is preferred that both the mutator nucleotide and the targeted nucleotide in the complementary strand be pyrimidines. Particularly preferred are SSOMV that encode transversion mutations, i.e., a C or T mutator nucleotide is mismatched, respectively, with a C or T nucleotide in the complementary strand.
(28) In addition to the oligodeoxynucleotide the SSOMV can contain a 5′ blocking substituent that is attached to the 5′ terminal carbons through a linker. The chemistry of the linker is not critical other than its length, which should preferably be at least 6 atoms long and that the linker should be flexible. A variety of non-toxic substituents such as biotin, cholesterol or other steroids or a non-intercalating cationic fluorescent dye can be used. Particularly preferred as reagents to make SSOMV are the reagents sold as Cy3™ and Cy5™ by Glen Research, Sterling Va., which are blocked phosphoroamidites that upon incorporation into an oligonucleotide yield 3,3,3′,3′-tetramethyl N,N′-isopropyl substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. Cy3 is the most preferred. When the indocarbocyanine is N-oxyalkyl substituted it can be conveniently linked to the 5′ terminal of the oligodeoxynucleotide through as a phosphodiester with a 5′ terminal phosphate. The chemistry of the dye linker between the dye and the oligodeoxynucleotide is not critical and is chosen for synthetic convenience. When the commercially available Cy3 phosphoramidite is used as directed the resulting 5′ modification consists of a blocking substituent and linker together which are a N-hydroxypropyl, N′-phosphatidylpropyl 3,3,3′,3′-tetramethyl indomonocarbocyanine.
(29) In the preferred embodiment the indocarbocyanine dye is tetra substituted at the 3 and 3′ positions of the indole rings. Without limitation as to theory these substitutions prevent the dye from being an intercalating dye. The identity of the substituents at these positions are not critical. The SSOMV can in addition have a 3′ blocking substituent. Again the chemistry of the 3′ blocking substituent is not critical.
5.2 the Location and Type of Mutation Introduced into the EPSPS Gene
(30) In one embodiment of the present invention, the Arabidopsis thaliana EPSPS gene (see
(31) (i) Leu.sub.173—Phe
(32) (ii) Gly.sub.177—Ala or Ile
(33) (iii) Thr.sub.178—Ile or Val or Leu
(34) (iv) Ala.sub.179—Gly
(35) (v) Met.sub.180—Cys
(36) (vi) Arg.sub.181—Leu or Ser
(37) (vii) Pro.sub.182—Leu or Ser
(38) (viii) Ser.sub.98—Asp
(39) (ix) Ser.sub.255—Ala
(40) (x) Leu.sub.198—Lys.
(41) In another embodiment of the present invention, within the EPSPS gene product, the amino acid residue to be changed is Leu within the contiguous sequence Leu-Tyr-Leu-Gly-Asn (SEQ ID NO:29) and is changed to Phe; or the amino acid residue to be changed is Gly within the contiguous sequence Asn-Ala-Gly-Thr-Ala (SEQ ID NO:30) and is changed to Ala or Ile; or the amino acid to be changed is Thr within the contiguous sequence Ala-Gly-Thr-Ala-Met (SEQ ID NO:31) and is changed to Ile, Val or Leu; or the amino acid to be changed is Ala within the contiguous sequence Gly-Thr-Ala-Met-Arg (SEQ ID NO:32) and is changed to Gly; or the amino acid to be changed is Met within the contiguous sequence Thr-Ala-Met-Arg-Pro (SEQ ID NO:33) and is changed to Cys; or the amino acid to be changed is Arg within the contiguous sequence Ala-Met-Arg-Pro-Leu (SEQ ID NO:34) and is changed to Leu or Ser; or the amino acid to be changed is Pro within the contiguous sequence Met-Arg-Pro-Leu-Thr (SEQ ID NO:35) and is changed to Leu or Ser; or the amino acid to be changed is Ser within a contiguous Pro-Gly-Ser-Lys-Ser (SEQ ID NO:36) and is changed to Asp; or the amino acid to be changed is Ser within the contiguous sequence Ile-Ser-Ser-Gln-Tyr (SEQ ID NO:37) and is changed to Ala; or the amino acid to be changed is Leu within the contiguous sequence Tyr-Val-Leu-Asp-Gly (SEQ ID NO:38) and is changed to Lys. In other embodiments, one or more of the foregoing changes can be made in the EPSPS amino acid sequence.
(42) Alternatively, and/or additionally, the mutation may result in the replacement of any amino acid at positions corresponding to 256, 284-288 and 353-356 with respect to the EPSPS protein depicted in
(43) In specific embodiments of the present invention, the EPSPS gene is mutated at amino acid position 177 in which Gly is replaced by Ala. Another specific embodiment is the substitution of Thr at amino acid position 178 by Ile. A further specific embodiment comprises a mutation at amino acid position 177 in which Gly is replaced by Ala, plus the additional substitution of Thr at amino acid position 178 by Ile. Other specific embodiments of the present invention are directed to mutations at amino acid position 178, in which Thr is replaced by Ile, plus the additional mutation at position 182, in which Pro is replaced by Ser. Other embodiments include the substitution of Gly at amino acid position 177 by Ala, plus the additional mutation at amino acid position 182, in which Pro is substituted by Ser. Other mutated EPSPS sequences comprise the substitution of Gly at position 177 by Ala, plus the substitution at position 178, in which Thr is replaced by Ile, plus the additional substitution of Pro at amino acid position 182 by Ser. Another embodiment is the substitution of Thr at amino acid position 178 by Val and the additional mutation at amino acid position 182, in which Pro is replaced by Ser. A further specific embodiment includes the substitution of Thr at position 178 by Leu, plus the mutation at amino acid position 182, in which Pro is replaced by Ser. A further embodiment includes, the substitution at amino acid position 177 in which Gly is replaced by Ala, plus the substitution of Thr at position 178 by Val. The invention also embodies the substitution at amino acid position 177 in which Gly is replaced by Ala, plus the replacement of Thr at amino acid position 178 by Leu (see
(44) The foregoing mutations in the EPSPS gene were described using the Arabidopsis thaliana EPSPS gene (SEQ ID NO: 1) and protein (SEQ ID NO:2). The present invention also encompasses mutant EPSPS genes of other species (paralogs). However, due to variations in the EPSPS genes of different species, the number of the amino acid residue to be changed in one species may be different in another species. Nevertheless, the analogous position is readily identified by one of skill in the art by sequence homology. For example,
(45) (i) Leu.sub.97—Phe
(46) (ii) Gly.sub.101—Ala or Ile
(47) (iii) Thr.sub.102—Ile or Val or Leu
(48) (iv) Ala.sub.103—Gly
(49) (v) Met.sub.104—Cys
(50) (vi) Arg.sub.105—Leu or Ser
(51) (vii) Pro.sub.106—Leu or Ser
(52) (viii) Ser.sub.23—Asp
(53) (ix) Ser.sub.179—Ala
(54) (x) Leu.sub.122—Lys.
(55) In another embodiment of the present invention, within the Zea mays EPSPS gene product the amino acid residue to be changed is Leu within the contiguous sequence Leu-Phe-Leu-Gly-Asn (SEQ ID NO:39) and is changed to Phe; or the amino acid residue to be changed is Gly within the contiguous sequence Asn-Ala-Gly-Thr-Ala (SEQ ID NO:30) and is changed to Ala or Ile; or the amino acid to be changed is Thr within the contiguous sequence Ala-Gly-Thr-Ala-Met (SEQ ID NO:31) and is changed to Ile, Val or Leu; or the amino acid to be changed is Ala within the contiguous sequence Gly-Thr-Ala-Met-Arg (SEQ ID NO:32) and is changed to Gly; or the amino acid to be changed is Met within the contiguous sequence Thr-Ala-Met-Arg-Pro (SEQ ID NO:33) and is changed to Cys; or the amino acid to be changed is Arg within the contiguous sequence Ala-Met-Arg-Pro-Leu (SEQ ID NO:34) and is changed to Leu or Ser; or the amino acid to be changed is Pro within the contiguous sequence Met-Arg-Pro-Leu-Thr (SEQ ID NO:35) and is changed to Leu or Ser; or the amino acid to be changed is Ser within a contiguous Pro-Gly-Ser-Lys-Ser (SEQ ID NO:36) and is changed to Asp; or the amino acid to be changed is Ser within the contiguous sequence Ile-Ser-Ser-Gln-Tyr (SEQ ID NO:37) and is changed to Ala; or the amino acid to be changed is Leu within the contiguous sequence Tyr-Val-Leu-Asp-Gly (SEQ ID NO:38) and is changed to Lys. In other embodiments, one or more of the foregoing changes can be made in the EPSPS amino acid sequence.
(56) In Brassica napus, the analogous amino acid positions are Leu.sub.169, Gly.sub.173, Thr.sub.174, Ala.sub.175, Met.sub.176, Arg.sub.177, Pro.sub.178, Ser.sub.94, Ser.sub.251 and Leu.sub.194. Thus, the Brassica napus EPSPS amino acid sequence is mutated at one or more of the following amino acid positions and results in one or more of the following substitutions:
(57) (i) Leu.sub.169—Phe
(58) (ii) Gly.sub.173—Ala or Ile
(59) (iii) Thr.sub.174—Ile or Val or Leu
(60) (iv) Ala.sub.175—Gly
(61) (v) Met.sub.176—Cys
(62) (vi) Arg.sub.177—Leu or Ser
(63) (vii) Pro.sub.178—Leu or Ser
(64) (viii) Ser.sub.94—Asp
(65) (ix) Ser.sub.251—Ala
(66) (x) Leu.sub.194—Lys
(67) In another embodiment of the present invention, within the Brassica napus EPSPS gene product the amino acid residue to be changed is Leu within the contiguous sequence Leu-Tyr-Leu-Gly-Asn (SEQ ID NO:29) and is changed to Phe; or the amino acid residue to be changed is Gly within the contiguous sequence Asn-Ala-Gly-Thr-Ala (SEQ ID NO:30) and is changed to Ala or Ile; or the amino acid to be changed is Thr within the contiguous sequence Ala-Gly-Thr-Ala-Met (SEQ ID NO:31) and is changed to Ile, Val or Leu; or the amino acid to be changed is Ala within the contiguous sequence Gly-Thr-Ala-Met-Arg (SEQ ID NO:32) and is changed to Gly; or the amino acid to be changed is Met within the contiguous sequence Thr-Ala-Met-Arg-Pro (SEQ ID NO:33) and is changed to Cys; or the amino acid to be changed is Arg within the contiguous sequence Ala-Met-Arg-Pro-Leu (SEQ ID NO:34) and is changed to Leu or Ser; or the amino acid to be changed is Pro within the contiguous sequence Met-Arg-Pro-Leu-Thr (SEQ ID NO:35) and is changed to Leu or Ser; or the amino acid to be changed is Ser within a contiguous Pro-Gly-Ser-Lys-Ser (SEQ ID NO:36) and is changed to Asp; or the amino acid to be changed is Ser within the contiguous sequence Ile-Ser-Ser-Gln-Tyr (SEQ ID NO:37) and is changed to Ala; or the amino acid to be changed is Leu within the contiguous sequence Tyr-Val-Leu-Asp-Gly (SEQ ID NO:38) and is changed to Lys. In other embodiments, one or more of the foregoing changes can be made in the EPSPS amino acid sequence.
(68) In Petunia hybrida the analogous positions are Leu.sub.169, Gly.sub.173, Thr.sub.1747, Ala.sub.175, Met.sub.176, Arg.sub.177, Pro.sub.178, Ser.sub.94, Ser.sub.251 and Leu.sub.194. Thus, the Petunia hybrida EPSPS amino acid sequence is mutated at one or more of the following amino acid positions and results in one or more of the following substitutions:
(69) (i) Leu.sub.169—Phe
(70) (ii) Gly.sub.173—Ala or Ile
(71) (iii) Thr.sub.174—Ile or Val or Leu
(72) (iv) Ala.sub.175—Gly
(73) (v) Met.sub.176—Cys
(74) (vi) Arg.sub.177—Leu or Ser
(75) (vii) Pro.sub.178—Leu or Ser
(76) (viii) Ser.sub.94—Asp
(77) (ix) Ser.sub.251—Ala
(78) (x) Leu.sub.194—Lys
(79) In another embodiment of the present invention, within the Petunia hybrida EPSPS gene product the amino acid residue to be changed is Leu within the contiguous sequence Leu-Phe-Leu-Gly-Asn (SEQ ID NO:39) and is changed to Phe; or the amino acid residue to be changed is Gly within the contiguous sequence Asn-Ala-Gly-Thr-Ala (SEQ ID NO:30) and is changed to Ala or Ile; or the amino acid to be changed is Thr within the contiguous sequence Ala-Gly-Thr-Ala-Met (SEQ ID NO:31) and is changed to Ile, Val or Leu; or the amino acid to be changed is Ala within the contiguous sequence Gly-Thr-Ala-Met-Arg (SEQ ID NO:32) and is changed to Gly; or the amino acid to be changed is Met within the contiguous sequence Thr-Ala-Met-Arg-Pro (SEQ ID NO:33) and is changed to Cys; or the amino acid to be changed is Arg within the contiguous sequence Ala-Met-Arg-Pro-Leu (SEQ ID NO:34) and is changed to Leu or Ser; or the amino acid to be changed is Pro within the contiguous sequence Met-Arg-Pro-Leu-Thr (SEQ ID NO:35) and is changed to Leu or Ser; or the amino acid to be changed is Ser within a contiguous Pro-Gly-Ser-Lys-Ser (SEQ ID NO:36) and is changed to Asp; or the amino acid to be changed is Ser within the contiguous sequence Ile-Ser-Ser-Gln-Tyr (SEQ ID NO:37) and is changed to Ala; or the amino acid to be changed is Leu within the contiguous sequence Tyr-Val-Leu-Asp-Gly (SEQ II) NO:38) and is changed to Lys. In other embodiments, one or more of the foregoing changes can be made in the EPSPS amino acid sequence.
5.3 the Delivery of Recombinagenic Oligonucleobases into Plant Cells
(80) Any commonly known method can be used in the methods of the present invention to transform a plant cell with a recombinagenic oligonucleobases. Illustrative methods are listed below.
5.3.1 Microcarriers and Microfibers
(81) The use of metallic microcarriers (microspheres) for introducing large fragments of DNA into plant cells having cellulose cell walls by projectile penetration is well known to those skilled in the relevant art (henceforth biolistic delivery). U.S. Pat. Nos. 4,945,050; 5,100,792 and 5,204,253 describe general techniques for selecting microcarriers and devices for projecting them.
(82) Specific conditions for using microcarriers in the methods of the present invention are described in International Publication WO 99/07865. In an illustrative technique, ice cold microcarriers (60 mg/ml), mixed duplex oligonucleotide (60 mg/ml) 2.5 M CaCl.sub.2 and 0.1 M spermidine are added in that order; the mixture gently agitated, e.g., by vortexing, for 10 minutes and let stand at room temperature for 10 minutes, whereupon the microcarriers are diluted in 5 volumes of ethanol, centrifuged and resuspended in 100% ethanol. Good results can be obtained with a concentration in the adhering solution of 8-10 μg/μl microcarriers, 14-17 μg/ml mixed duplex oligonucleotide, 1.1-1.4 M CaCl.sub.2 and 18-22 mM spermidine. Optimal results were observed under the conditions of 8 μg/μl microcarriers, 16.5 μg/ml mixed duplex oligonucleotide, 1.3 M CaCl.sub.2 and 21 mM spermidine.
(83) Recombinagenic oligonucleobases can also be introduced into plant cells for the practice of the present invention using microfibers to penetrate the cell wall and cell membrane. U.S. Pat. No. 5,302,523 to Coffee et al. describes the use of 30×0.5 μm and 10×0.3 μm silicon carbide fibers to facilitate transformation of suspension maize cultures of Black Mexican Sweet. Any mechanical technique that can be used to introduce DNA for transformation of a plant cell using microfibers can be used to deliver recombinagenic oligonucleobases for transmutation.
(84) An illustrative technique for microfiber delivery of a recombinagenic oligonucleobase is as follows: Sterile microfibers (2 μg) are suspended in 150 μl of plant culture medium containing about 10 μg of a mixed duplex oligonucleotide. A suspension culture is allowed to settle and equal volumes of packed cells and the sterile fiber/nucleotide suspension are vortexed for 10 minutes and plated. Selective media are applied immediately or with a delay of up to about 120 hours as is appropriate for the particular trait.
5.3.2 Protoplast Electroporation
(85) In an alternative embodiment, the recombinagenic oligonucleobases can be delivered to the plant cell by electroporation of a protoplast derived from a plant part. The protoplasts are formed by enzymatic treatment of a plant part, particularly a leaf, according to techniques well known to those skilled in the art. See, e.g., Gallois et al., 1996, in Methods in Molecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp et al., 1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa, N.J. The protoplasts need not be cultured in growth media prior to electroporation. Illustrative conditions for electroporation are 3×10.sup.5 protoplasts in a total volume of 0.3 ml with a concentration of recombinagenic oligonucleobase of between 0.6-4 μg/mL.
5.3.3 Whiskers and Microinjection
(86) In yet another alternative embodiment, the recombinagenic oligonucleobase can be delivered to the plant cell by whiskers or microinjection of the plant cell. The so called whiskers technique is performed essentially as described in Frame et al., 1994, Plant J. 6:941-948. The recombinagenic oligonucleobase is added to the whiskers and used to transform the plant cells. The recombinagenic oligonucleobase may be co-incubated with plasmids comprising sequences encoding proteins capable of forming recombinase complexes in plant cells such that recombination is catalyzed between the oligonucleotide and the target sequence in the EPSPS gene.
5.4 Selection of Glyphosate Resistant Plants
(87) Plants or plant cells can be tested for resistance or tolerance to a herbicide using commonly known methods in the art, e.g., by growing the plant or plant cell in the presence of a herbicide and measuring the rate of growth as compared to the growth rate in the absence of the herbicide.
6. EXAMPLE
(88) The following experiments demonstrate the production of mutant Arabidopsis thaliana EPSPS genes which are resistant to the herbicide glyphosate and which allows the plant cells to maintain a growth rate
6.1 Material and Methods
6.1.1 Isolation of Arabidopsis thaliana EPSPS cDNA
(89) A 1.3 kb DNA fragment was amplified by PCR from an Arabidopsis cDNA library using the primers AtEXPEXPM1 and AtEXPEXP2CM-2. The two primers were designed to amplify the cDNA from the mature peptide to the termination codon. The 5′ primer AtEXPEXPM1 contains an XbaI site (underlined) and the 3′ primer AtEXPEXP2CM-2 contains a BglII site (underlined), sites which will be of use for cloning of the fragment into the expression vector.
(90) TABLE-US-00001 AtEXPEXPM1 (SEQ ID NO: 40) 5′-GCTCTAGAGAAAGCGTCGGAGATTGTACTT-3′ AtEXPEXP2CM-2 (SEQ ID NO: 41) 5′-GCAGATCTGAGCTCTTAGTGCTTTGTGATTCTTTCAAGTAC-3′
(91) The PCR band was excised from the agarose gel and purified (GeneClean, Biol). Its sequence was then confirmed as the mature peptide sequence of Arabidopsis thaliana EPSPS gene.
6.1.2 Preparation of the Expression Vector
(92) The EPSPS coding region of the AroE Bacillus subtilis gene was obtained by PCR using the following primers:
(93) TABLE-US-00002 BsAroE5′Xba (SEQ ID NO: 42) 5′-GCGTCTAGAAAAACGAGATAAGGTGCAG-3′ and BsAroE3′BamHI (SEQ ID NO: 43) 5′-GCGGATCCTCAGGATTTTTTCGAAAGCTTATTTAAATG-3′.
(94) The PCR fragment, lacking an initiation codon (ATG), was cloned in-frame to the pACLacIMH6RecA vector by replacing the ORF of RecA by digesting with XbaI and BamHI. PACLacMH6RecA contained the LacI region of Pet21 at positions 1440 to 3176, the MH6 RecA at positions 3809 to 5188, chloramphenicol resistance gene at positions 5445-218 (5446 to 5885 and 1 to 218), and the p15A origin of replication at positions 581 to 1424. The coding region of RecA gene was cloned from E. coli in-frame with the start codon and 6 histidine linker (MH6) behind the LacZ promoter of pUC19.
6.1.3 Cloning of the Arabidopsis EPSPS Gene into Bacterial Expression Vector
(95) The Arabidopsis 1.3 kb PCR fragment was digested with XbaI and BamHI (compatible with BglII) and cloned into the plasmid pACYCLacIMH6EPSPS, in place of the Bacillus gene.
(96) The clones obtained (selected on chloramphenicol) were then sequenced and confirmed positive. One of the confirmed clones (pAtEPS-12) was selected and the junctions between the cDNA and the cloning plasmid were also confirmed to be identical to the expected sequences.
6.1.4 Novel Point Mutations in the EPSPS Gene
(97) Ten different mutants of the Arabidopsis thaliana EPSPS gene were designed, (see
(98) The 353 bp PCR fragments obtained were purified (Qiagen PCR Purification kit) and their sequence confirmed. The fragments were then digested with PstI (underlined in the primer sequences) and BamHI and ligated to the pAtEPS-12 vector, which had itself been previously digested with PstI and BamHI.JM109 (Promega) competent cells were used for the transformation and plated onto chloramphenicol-containing LB plates. Clones from each mutagenesis experiment were then isolated and their sequence confirmed.
6.1.5 Glyphosate Resistance Assays
(99) Electrocompetent cells of SA4247, a LacZ-Salmonella typhi strain, were prepared according to well known procedures (see Current Protocols in Molecular Biology, (Wiley and Sons, Inc.)). 30 μl of SA4247 competent cells were electroporated with 20 ng of each plasmid DNA encoding Arabidopsis wild-type and mutant EPSPS proteins, Bacillus wild-type EPSPS, along with a mock transfection as a control. The settings for electroporation were 25 μF, 2.5 KV and 200 ohms. After electroporation, the cells were transferred into 15 mls culture tube and supplemented with 970 μl of SOC medium. The cultures were incubated for 1½ hours at 37° C. at 225 rpm. 50 μl of each culture were plated onto LB plates containing 17 μg/ml chloramphenicol (in duplicates) and incubated overnight at 37° C. On the following day, 5 colonies of each plate were picked and transferred onto M9 plates and incubated overnight at 37° C.
(100) Colonies from the overnight incubation on solid M9 were inoculated into 4 ml of liquid M9 medium and grown overnight at 37° C. On the following day, 25 ml of liquid M9 medium containing chloramphenicol, IPTG and 17 mM or 0 mM Glyphosate (Aldrich, 33775-7) were inoculated with 1-2 mls of each overnight culture (in duplicates), the starting OD (at 600 nm) was measured and all the cultures were normalized to start at the same OD. An OD measurement was taken every hour for seven hours. As a control of the bacterial growth, a culture of untransformed Salmonella was also inoculated into plain LB medium. In two independent experiments, the clones A.sub.177I.sub.178, A.sub.177V.sub.178, A.sub.177L.sub.178 and I.sub.177 did not grow in M9 medium, therefore the glyphosate-resistance assays could not be performed on them.
6.1.7 Isolation and Purification of the Expressed Protein from Bacterial Clones
(101) One milliliter of overnight culture of each of the bacterial clones is inoculated into 100 ml of liquid LB medium containing chloramphenicol. The cells were allowed to grow at 37° C. until they reached an OD of 0.5-0.7 (approximately 3½ hours). IPTG was then added to the cultures to a concentration of 1.0 mM. The cells were grown five additional hours. They were then pelleted at 4000 rpm for 20 minutes at 4° C.
(102) The isolation and the purification of the His-tagged proteins were performed following the Qiagen Ni-NTA Protein Purification System: Cell lysates and eluates were run in duplicates on 12.5% acrylamide gels. One of the gels was silver-stained for immediate visualization, the second gel was transferred onto Millipore Immobilon-P membrane, and blocked overnight in 5% milk in TBS-T. The membrane was then exposed to Anti-His primary antibody solution (Amersham Pharmacia biotech, cat #37-4710), followed by exposure to Anti-Mouse-IgG secondary antibody solution. (NIF825, from Amersham Pharmacia biotech ECL Western blotting analysis system, cat # RPN2108). Washes and detection reactions were performed according to the manufacturer instructions. Autoradiograms were developed after 5 minutes exposure.
6.2 Results
(103) Cells containing a mutation in the EPSPS gene produced cells that were both resistant to the herbicide glyphosate and that had a substantially similar growth rate in the absence or presence of glyphosate, as compared to the wild-type cells, irrespective of the presence of glyphosate (see
(104) It was also demonstrated that the Arabidopsis clones containing a mutant EPSPS gene expressed the mutant protein at substantially the same level as the wild-type protein (see
(105) The invention claimed and described herein is not to be limited in scope by the specific embodiments, including but not limited to the deposited microorganism embodiments, herein disclosed since these embodiments are intended as illustrations of several aspects of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
(106) A number of references are cited herein, the entire disclosures of which are incorporated herein, in their entirety, by reference.