Plants Tolerant to HPPD Inhibitor Herbicides
The present invention relates to nucleic acid sequences encoding a hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, abbreviated herein as HPPD) obtained from protists belonging to the family Blepharismidae, as well as the proteins encoded thereby, and to a chimeric gene which comprises such nucleic acid sequence, and to the use of such nucleic acid sequences, proteins or chimeric genes for obtaining plants which are tolerant to HPPD inhibitor herbicides.
1 - 16 . (canceled)
17 . A plant, a plant part, a plant cell, or a seed comprising a chimeric gene comprising a coding sequence operably-linked to a plant-expressible promoter, wherein the coding sequence comprises
a) a nucleic acid sequence encoding a Blepharisma japonicum hydroxyphenylpyruvate dioxygenase (HPPD) having the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382; or
b) a nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382, wherein one or more amino acids of SEQ ID No. 4 are substituted at amino acid positions 185, 209, 210, 211, 212, 224, 227, 228, 229, 230, 239, 289, 323, 361, 362, 366, and 367 with an amino acid set forth in the table below:
Position in
Amino acid in
SEQ ID
SEQ ID No. 4
No. 4
Substitutions
Val
185
Thr, Cys, Ala, Gly
Tyr
209
Phe, Ile, Leu, Val, Ala, Gln, Glu, Asp,
Gly, Thr, Ser, Met, Arg, Lys
Trp
210
Ala, Ile, Leu, Ser, Arg, Lys, His, Asp, Glu,
Pro, Gly, Asn
Ser
211
Glu, Thr, Tyr, Phe, His, Gln, Asn, Gly,
Leu, Met, Val, Arg, Ile
Ala
212
Phe, Val, Ile, Leu, Trp, Met, Gln, His
Leu
224
Met, Val
Val
227
Ala, Leu, Met, Ile, Lys, Arg, Gln, Tyr
Val
228
Ala, Thr
Val
229
Leu, Met, Ile, Ala
Ala
230
Ser, Thr, Val, Arg, Lys, Glu, Leu, Ile, Met,
His
Pro
239
Ala, Val, Thr, Asn, Ile,
Leu
289
Met, Ile, Asn
Leu
323
Met
Ile
361
Any except Pro
Gly
362
Ala, Pro, Val, Thr, Met
Ala
366
Glu, Gln, Ser, Val, Phe, Thr
Leu
367
Arg
18 . The plant, the plant part, the plant cell, or the seed of claim 17 , wherein the coding sequence comprises the nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382.
19 . The plant, the plant part, the plant cell, or the seed of claim 17 , wherein the coding sequence comprises the nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382, and wherein one or more amino acids of SEQ ID No. 4 are substituted at amino acid positions 185, 209, 210, 211, 212, 224, 227, 228, 229, 230, 239, 289, 323, 361, 362, 366, and 367 with an amino acid as set forth in the table in claim 17 .
20 . The plant, the plant part, the plant cell, or the seed of claim 18 , wherein the coding sequence comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide position 4 to nucleotide position 1149, or the nucleic acid sequence of SEQ ID No. 3 from nucleotide position 400 to nucleotide position 1542.
21 . The plant, the plant part, the plant cell, or the seed of claim 19 , wherein the coding sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382, and wherein one or more amino acids of SEQ ID No. 4 are substituted at amino acid positions 185, 209, 210, 212, 224, 227, 229, 230, 366, and 367 with the amino acid set forth in the table.
22 . The plant, the plant part, the plant cell, or the seed of claim 19 , wherein the coding sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382, and wherein one or more amino acids of SEQ ID No. 4 are substituted at amino acid positions 221, 228, 239, 289, 323, 361, and 362 with the amino acid set forth in the table.
23 . The plant, the plant part, the plant cell, or the seed of claim 19 , wherein the coding sequence comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382, and wherein one or more amino acids of SEQ ID No. 4 are substituted at amino acid positions 211, 228, 239, 289, 323, 361, and 362 with the amino acid set forth in the following table:
Amino acid in
Position in SEQ ID
SEQ ID No. 4
No. 4
Substitutions
Ser
211
Glu, Thr, Arg, Tyr
Val
228
Ala
Pro
239
Ala, Val, Thr
Leu
289
Met
Leu
323
Met
Ile
361
Ala, Val, Leu, Lys
Gly
362
Ala
24 . The plant, the plant part, the plant cell, or the seed of claim 18 , wherein the chimeric gene further comprises a nucleic acid sequence encoding a transit peptide active in a plant upstream of the HPPD coding sequence, so that a transit peptide/HPPD fusion protein is encoded by the chimeric gene.
25 . The plant, the plant part, the plant cell, or the seed of claim 18 further comprising a chimeric gene encoding a prephenate dehydrogenase (PDH) enzyme.
26 . The plant, the plant part, the plant cell, or the seed of claim 18 further comprising one or more chimeric genes conferring tolerance to
(a) a growth regulator herbicide;
(b) a herbicide inhibiting enzyme, wherein the enzyme is selected from the group consisting of (i) acetolactate synthase, (ii) EPSP synthase, and (iii) glutamine synthase; or
(c) a combination thereof.
27 . The plant, the plant part, the plant cell, or the seed of claim 26 , wherein the growth regulator herbicide is 2,4-D or dicamba.
28 . The plant, the plant part, the plant cell, or the seed of claim 19 , wherein the chimeric gene further comprises a nucleic acid sequence encoding a transit peptide active in a plant upstream of the coding sequence, so that a transit peptide fusion protein is encoded by the chimeric gene.
29 . The plant, the plant part, the plant cell, or the seed of claim 19 further comprising a chimeric gene encoding a prephenate dehydrogenase (PDH) enzyme.
30 . The plant, the plant part, the plant cell, or the seed of claim 19 further comprising one or more chimeric genes conferring tolerance to
(a) a growth regulator herbicide;
(b) a herbicide inhibiting enzyme, wherein the enzyme is selected from the group consisting of (i) acetolactate synthase, (ii) EPSP synthase, and (iii) glutamine synthase; or
(c) a combination thereof.
31 . The plant, the plant part, the plant cell, or the seed of claim 30 , wherein the growth regulator herbicide is 2,4-D or dicamba.
32 . A plant, a plant part, a plant cell, or a seed comprising a chimeric gene comprising a coding sequence operably linked to a plant-expressible promoter, wherein the coding sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, and SEQ ID No. 23.
33 . A chimeric gene comprising a coding sequence operably-linked to a plant-expressible promoter, wherein the coding sequence comprises
a) a nucleic acid sequence encoding a Blepharisma japonicum hydroxyphenylpyruvate dioxygenase (HPPD) having the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382; or
b) a nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382, wherein one or more amino acids of SEQ ID No. 4 are substituted at amino acid positions 185, 209, 210, 211, 212, 224, 227, 228, 229, 230, 239, 289, 323, 361, 362, 366, and 367 with an amino acid set forth in the table below:
Position in
Amino acid in
SEQ ID
SEQ ID No. 4
No. 4
Substitutions
Val
185
Thr, Cys, Ala, Gly
Tyr
209
Phe, Ile, Leu, Val, Ala, Gln, Glu, Asp,
Gly, Thr, Ser, Met, Arg, Lys
Trp
210
Ala, Ile, Leu, Ser, Arg, Lys, His, Asp, Glu,
Pro, Gly, Asn
Ser
211
Glu, Thr, Tyr, Phe, His, Gln, Asn, Gly,
Leu, Met, Val, Arg, Ile
Ala
212
Phe, Val, Ile, Leu, Trp, Met, Gln, His
Leu
224
Met, Val
Val
227
Ala, Leu, Met, Ile, Lys, Arg, Gln, Tyr
Val
228
Ala, Thr
Val
229
Leu, Met, Ile, Ala
Ala
230
Ser, Thr, Val, Arg, Lys, Glu, Leu, Ile, Met,
His
Pro
239
Ala, Val, Thr, Asn, Ile,
Leu
289
Met, Ile, Asn
Leu
323
Met
Ile
361
Any except Pro
Gly
362
Ala, Pro, Val, Thr, Met
Ala
366
Glu, Gln, Ser, Val, Phe, Thr
Leu
367
Arg
34 . The chimeric gene of claim 33 , wherein the coding sequence comprises the nucleic acid sequence encoding a Blepharisma japonicum HPPD having the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382.
35 . The chimeric gene of claim 33 , wherein the coding sequence comprises the nucleic acid sequence encoding the amino acid sequence of SEQ ID No. 4 from amino acid position 2 to amino acid position 382, and wherein one or more amino acids of SEQ ID No. 4 are substituted at amino acid positions 185, 209, 210, 211, 212, 224, 227, 228, 229, 230, 239, 289, 323, 361, 362, 366, and 367 with an amino acid set forth in the table.
36 . A vector comprising the chimeric gene of claim 33 .