Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase

Claim 2

A nucleic acid fragment comprising a nucleotide sequence encoding plant acetolactate synthase with increased resistance to one or more sulfonylurea herbicides, said nucleotide sequence comprises at least one sub-sequence which encodes one of the substantially conserved amino acid sub-sequences designated A, B, C, D, E, F, and G in Figure 6, the nucleic acid fragment is further characterized in that at least one of the following conditions is met; a) the nucleic acid fragment has a sequence which encodes an amino acid sub-sequence A wherein ε₁ is an amino acid other than alanine, and/or ε₂ is an amino acid other than glycine, b) the nucleic acid fragment has a sequence which encodes an amino acid sub-sequence B wherein α₁ is an amino acid other than proline, c) the nucleic acid gragment has a sequence which encodes an amino acid sub-sequence C wherein δ₂ is an amino acid other than alanine, d) the nucleic acid fragment has a sequence which encodes an amino acid sub-sequence D wherein λ₁ is an amino acid other than lysine, e) the nucleic acid fragment has a sequence which encodes an amino acid sub-sequence E wherein γ₁ is an amino acid other than aspartic acid, f) the nucleic acid fragment has a sequence which encodes an amino acid sub-sequence F wherein β₃ is an amino acid other than tryptophan, and/or β₈ is an amino acid other than valine and/or β₇ is an amino acid other than phenylalanine, and g) the nucleic acid fragment has a sequence which encodes an amino acid sub-sequence G wherein σ₁ is an amino acid other than methionine.

Claim 3

A nucleic acid fragment according to Claim 1, wherein the fragment has a sequence which encodes an amino acid sub-sequence A having the formula PGε₂A wherein: P is proline, G is glycine, ε₂ is an amino acid other than glycine, and A is alanine.

Claim 4

A nucleic acid fragment according to claim 1 or Claim 2, wherein ε₂ is an amino acid selected from the group consisting of serine, threonine or cysteine.

Claim 5

A nucleic acid fragment according to Claim 3, wherein ε₂ is serine.

Claim 6

A nucleic acid fragment according to Claim 1, wherein the fragment has a sequence which encodes an amino acid sub-sequence A having the formula PGGε₁ wherein: P is proline, G is glycine, and ε₁ is an amino acid other than alanine or glycine

Claim 7

A nucleic acid fragment according to Claim 1, wherein the fragment has a sequence which encodes an amino acid sub-sequence B having the formula GQVα₁ wherein: G is glycine, Q is glutamine, V is valine α₁ is an amino acid other than proline.

Claim 8

A nucleic acid fragment according to claim 1 or Claim 6 wherein α₁ is an amino acid selected from the group consisting of alanine, glycine, arginine, lysine, histidine, serine, cysteine, threonine, glutamine and asparagine.

Claim 9

A nucleic acid fragment according to Claim 7, wherein α₁ is alanine, arginine, serine or glutamine.

Claim 10

A nucleic acid fragment according to Claim 1 , wherein the fragment has a sequence which encodes an amino acid sub-sequence C having the formula IGδ₁Dδ₂FQE wherein: I is isoleucine, G is glycine, δ₁ is selected from the group consisting of amino acids, D is aspartic acid, and δ₂ is an amino acid other than alanine, F is phenylalanine, Q is glutamine, and E is glutamic acid.

Claim 11

A nucleic acid fragment according to claim 1 or Claim 9, wherein δ₂ is an amino acid selected from the group consisting of threonine, serine, cysteine, tyrosine, aspartic acid, glutamic acid, tryptophan, histidine, phenylalanine, arginine, and lysine.

Claim 12

A nucleic acid fragment according to Claim 10, wherein δ₂ is threonine, cysteine, aspartic acid, glutamic acid, tryptophan or arginine.

Claim 13

A nucleic acid fragment according to Claim 1 , wherein the fragment has a sequence which encodes an amino acid sub-sequence D having the formula Pλ₁D wherein: P is proline; λ₁ is an amino acid other than lysine; and D is aspartic acid.

Claim 14

A nucleic acid fragment according to claim 1 or Claim 12, wherein λ₁ is an amino acid selected from the group consisting of glycine, alanine, leucine, isoleucine, valine, threonine, serine, cysteine, tyrosine, glutamic acid, aspartic acid, proline, asparagine, glutamine, tryptophan and phenylalanine.

Claim 15

A nucleic acid fragment according to Claim 13, wherein λ₁ is glycine, leucine, threonine, glutamic acid, aspartic acid, asparagine, tryptophan or proline.

Claim 16

A nucleic acid fragment according to Claim 1 , wherein the fragment has a sequence which encodes an amino acid sub-sequence G having the formula MLGσ₁HG wherein M is methionine, L is leucine, G is glycine σ₁ is an amino acid other than methionine, H is histidine, and G is glycine.

Claim 17

A nucleic acid fragment according to claim 1 or Claim 15, wherein σ₁ is an amino acid selected from the group consisting of glutamic acid, aspartic acid, proline, valine, leucine, or isoleucine.

Claim 18

A nucleic acid fragment according to Claim 16, wherein σ₁ is proline, glutamic acid, or valine.

Claim 19

A nucleic acid fragment according to encodes an amino acid subsequence E having the formula RFDγ₁R wherein: R is arginine, F is phenylalanine, D is aspartic acid, and γ₁ is an amino acid other than aspartic acid.

Claim 20

A nucleic acid fragment according to encodes an amino acid subsequence E having the formula RFDγ₁R wherein: R is arginine, F is phenylalanine, D is aspartic acid, and γ₁ is an amino acid other than aspartic acid.

Claim 21

A nucleic acid fragment according to claim 1 or Claim 18, wherein γ₁ is an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, tyrosine, glutamic acid, proline, asparagine, glutamine, lysine, arginine, tryptophan, histidine, and phenylalanine.

Claim 22

A nucleic acid fragment according to Claim 19, wherein γ₁ is glycine, valine, serine, glutamic acid, proline, asparagine, lysine, cysteine or tryptophan.

Claim 23

A nucleic acid fragment according to Claim 1, wherein the fragment has a sequence which encodes an amino acid sub-sequence F having the formula Gβ₁β₈β₂Qβ₃β₄β₅β₆β₇ wherein: G is glycine, β₁, β₂, β₃, β₄, β₅, β₆ and β₇ are selected from the group consisting of of amino acids, Q is glutamine, and β₈ is an amino acid other than valine.

Claim 24

A nucleic acid fragment according to claim 1 or Claim 21, wherein β₁ is methionine, β₃ is tryptophan, β₇ is phenylalanine and β₈ is an amino acid other than valine.

Claim 25

A nucleic acid fragment according to any one of Claims 1, 21 and 22 wherein β₈ is an amino acid selected from the group consisting of alanine and glycine.

Claim 26

A nucleic acid fragment according to Claim 23 wherein β₈ is alanine.

Claim 27

A nucleic acid fragment according to Claim 1, wherein the fragment has a sequence which includes an amino acid sub-sequence F having the formula Gβ₁Vβ₂Qβ₃β₄β₅β₆β₇ wherein: G is glycine, β₁, β₂, β₄, β₅, β₆ and β₇ are selected from the group consisting of amino acids, V is valine, Q is glutamine, and β₃ is an amino acid other than tryptophan or proline.

Claim 28

A nucleic acid fragment according to Claim 1, wherein the fragment has a sequence which includes an amino acid sub-sequence F having the formula Gβ₁Vβ₂Qβ₃β₄β₅β₆β₇ wherein: G is glycine, β₁, β₂, β₃, β₄, β₅ and β₆ are selected from the group consisting of amino acids, V is valine, Q is glutamine, and β₇ is an amino acid other than phenylalanine.

Claim 29

A nucleic acid fragment according to claim 1 or Claim 26 wherein β₇ is an amino acid selected from the group consisting of leucine, valine and isoleucine.

Claim 30

A nucleic acid fragment according to Claim 27, wherein β₇ is leucine.

Claim 31

A nucleic acid fragment encoding plant acetolactate synthase which is capable of being incorporated into a nucleic acid construct suitable for transforming a plant containing wild-type acetolactate synthase which is sensitive to a sulfonylurea herbicide compound, said nucleic acid fragment having at least one point mutation relative to the wild-type nucleic acid fragment encoding plant acetolactate synthase such that upon transformation with said nucleic acid construct said plant contains said nucleic acid fragment and exhibits increased resistance to the application of said sulfonylurea herbicide compound.

Claim 32

A nucleic acid fragment encoding plant acetolactate synthase which is capable of being incorporated into a nucleic acid construct suitable for transforming a plant containing wild-type acetolactate synthase which is sensitive to a sulfonylurea herbicide compound, said nucleic acid fragment having at least one point mutation relative to the wild-type nucleic acid fragment encoding plant acetolactate synthase such that upon transformation with said nucleic acid construct said plant contains said nucleic acid fragment and exhibits increased resistance to the application of said sulfonylurea herbicide compound.

Claim 33

An acetolactate synthase protein encoded by a mucleic acid fragment as claimed in any one of claims 1 to 29.

Claim 34

A nucleic acid construct comprising a nucleic acid fragment as claimed in any way of claims 1 to 29.

Claim 35

A nucleic acid construct as claimed in claim 31 selected from the cloning vectors deposited at the ATCC, Rockville, Maryland, U.S.A. under accession numbers 40237, 67124, 67126 and 67424.

Claim 36

A nucleic acid construct as claimed in claim 31, wherein a nucleic acid fragment encoding plant acetolactate synthase with increased sulfonylurea herbicide resistance as claimed in any one of claims 1 to 29 is linked to a second nucleic acid fragment conferring a second trait such that when said nucleic acid construct is utilized to transform a plant, the expression of herbicide resistance by said plant upon application of sulfonylurea compounds can be utilized to detect the presence of said second nucleic acid fragment in said plant.

Claim 37

A microorganism containing a nucleic acid fragment or nucleic acid construct as claimed in any one of claims 1 to 29 and 31 to 33.

Claim 38

A plant containing a nucleic acid fragment or nucleic acid construct as claimed in any one of claims 1 to 29 and 31 to 33.

Claim 39

A plant as claimed in claim 35 which is a soybean, maize, sugarbeet, sunflower, tobacco, potato, tomato, alfalfa, melon, cotton, lettuce, wheat or rice plant or a Brassica species.

Claim 40

A plant tissue culture transformed with a nucleic acid fragment or nucleic acid construct as claimed in any one of claims 1 to 29 and 31 to 33.

Claim 41

Seed obtained by growing a plant as claimed in claim 35 or claim 36.

Claim 45

A method according to Claim 41 wherein the herbicide is a compound selected from the group consisting of wherein R is H or CH₃; R₁ is Cl, Br, NO₂, C₁-C₄ alkyl, C₂-C₄ alkenyl, CF₃, C₁-C₄ alkoxy, C₁-C₄ haloalkoxy, C₃-C₄ alkenyloxy, C₂-C₄ haloalkenyloxy, C₃-C₄ alkynyloxy, CO₂R₉, CONR₁₀R₁₁, S(O) m R₁₂, OSO₂R₁₂, phenyl SO₂N(OCH₃)CH₃, SO₂NR₁₀R₁₁, R₂ is H, Cl, Br, F, CH₃, NO₂, SCH₃, OCF₂H, OCH₂CF₃ or OCH₃; R₃ is Cl, NO₂, CO₂CH₃, CO₂C₂H₅, SO₂N(CH₃)₂, SO₂CH₃ or SO₂C₂H₅; R₄ is C₁-C₃ alkyl, Cl, Br, NO₂, CO₂R₉, CON(CH₃)₂, SO₂N(CH₃)₂, SO₂N(OCH₃)CH₃ or S(O) m R₁₂; R₅ is C₁-C₃ alkyl, C₄-C₅ cycloalkylcarbon, F, Cl, Br, NO₂, CO₂R₁₄, SO₂N(CH₃)₂, SO₂R₁₂ or phenyl; R₆ is H, C₁-C₃ alkyl, or CH₂CH=CH₂; R₇ is H, CH₃, OCH₃, Cl or Br; R₈ is H, F, Cl, Br, CH₃, OCH₃, CF₃, SCH₃ or OCF₂H; R₉ is C₁-C₄ alkyl, C₃-C₄ alkenyl or CH₂CH₂Cl; R₁₀ is H or C₁-C₃ alkyl; R₁₁ is H or C₁-C₂ alkyl; R₁₂ is C₁-C₃ alkyl; R₁₃ is H or CH₃; R₁₄ is C₁-C₃ alkyl or CH₂CH=CH₂; m is 0, 1 or 2; n is 1 or 2; Q is CH₂, CHCH₃ or NR₁₅; R₁₅ is H or C₁-C₄ alkyl; P is O or CH₂; R₁₆ is H or CH₃; R₁₇ is C(O)NR₁₈R₁₉; R₁₈ is H or CH₃; R₁₉ is CH₃; R₂₀ is H, Cl, F, Br, CH₃, CF₃, OCH₃ or OCF₂H; R₂₁ is H or CH₃; X is CH₃, OCH₃, OC₂H₅ or NHCH₃; Y is CH₃, C₂H₅, OCH₃, OC₂H₅, OCF₂H, OCH₂CF₃, Cl, CH₂OCH₃ or cyclopropyl; Z is CH or N; and their agriculturally suitable salts; provided that a) when Y is Cl, then Z is CH and X is OCH₃; b) when Y is OCF₂H, then Z is CH; c) when J is J-1 and R₁ is OSO₂R₁₂ or phenyl, the Y is other than OCF₂H; d) when J is J-2, then Y is other than OCF₂H or OCH₂CF₃; and e) when J is J-3 and R₄ is S(O) m R₁₂, then Y is other than OCH₂CF₃.

Claim 46

43 . A method according to Claim 41 wherein the herbicide is a compound selected from the group consisting of wherein Ar is R a is C₁-C₄ alkyl, F, Cl, Br, I, NO₂, S(O) p R d , COOR e or CF₃; R b is H, F, Cl, Br, I, C₁-C₄ alkyl or COOR e ; R c is H, C₁-C₄ alkyl, F, Cl, Br, I, CH₂OR d , phenyl, NO₂ or COOR e ; R d is C₁-C₄ alkyl; R e is C₁-C₄ alkyl, C₁-C₄ alkenyl, C₁-C₄ alkynyl, or 2-ethoxyethyl; V is H, C₁-C₃ alkyl, allyl, propargyl, benzyl or C₁-C₃ alkylcarbonyl; X₁, Y₁, and Z₁, are independently H, F, Cl, Br, I, C₁-C₄ alkyl, C₁-C₂ alkylthio or C₁-C₄ alkoxy; and p is 0, 1 or 2.

Claim 47

A method according to Claim 41 wherein the herbicide is a compound selected from the group consisting of R f is C₁-C₄ alkyl; R g is C₁-C₄ alkyl or C₃-C₆ cycloalkyl; A₁ is COOR i , CH₂OH or CHO; R i is H; C₁-C₁₂ alkyl optionally substituted by C₁-C₃ alkyl, C₃-C₆ cycloalkyl or phenyl; C₃-C₅ alkenyl optionally substituted by phenyl or 1-2 C₁-C₃ alkyl, F, Cl, Br or I; or C₃-C₅ alkynyl optionally substituted by phenyl or 1-2 C₁-C₃ alkyl, F, Cl, Br or I; B is H; C(O)C₁-C₆ alkyl or C(O)phenyl optionally substituted by Cl, NO₂ or OCH₃; X₂ is H, F, Cl, Br, I, OH or CH₃; Y₂ and Z₂ are independently H, C₁-C₆ alkyl, C₁-C₆ alkoxy, F, Cl, Br, I, phenyl, NO₂, CN, CF₃ or SO₂CH₃; X₃ is H, C₁-C₃ alkyl, F, Cl, Br, I or NO₂; and L, M, Q and R h are independently H, F, Cl, Br, I, CH₃, OCH₃, NO₂, CF₃, CN, N(CH₃)₂, NH₂, SCH₃ or SO₂CH₃ provided that only one of M or Q may be a substituent other than H, F, Cl, Br, I, CH₃ or OCH₃.