EP1032681A1 - Chimera gene having a light dependent promoter providing tolerance to hppd inhibitors - Google Patents
Chimera gene having a light dependent promoter providing tolerance to hppd inhibitorsInfo
- Publication number
- EP1032681A1 EP1032681A1 EP98954565A EP98954565A EP1032681A1 EP 1032681 A1 EP1032681 A1 EP 1032681A1 EP 98954565 A EP98954565 A EP 98954565A EP 98954565 A EP98954565 A EP 98954565A EP 1032681 A1 EP1032681 A1 EP 1032681A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- chimeric gene
- plants
- promoter
- hppd
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 53
- 239000003112 inhibitor Substances 0.000 title abstract description 11
- 230000001419 dependent effect Effects 0.000 title description 4
- 241000196324 Embryophyta Species 0.000 claims abstract description 99
- 239000004009 herbicide Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 23
- 230000001105 regulatory effect Effects 0.000 claims abstract description 20
- 238000013518 transcription Methods 0.000 claims abstract description 12
- 230000035897 transcription Effects 0.000 claims abstract description 12
- 108090000790 Enzymes Proteins 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 10
- 230000009466 transformation Effects 0.000 claims abstract description 8
- 230000008488 polyadenylation Effects 0.000 claims abstract description 4
- 230000001276 controlling effect Effects 0.000 claims abstract description 3
- 230000002363 herbicidal effect Effects 0.000 claims description 12
- 239000013598 vector Substances 0.000 claims description 10
- 230000008685 targeting Effects 0.000 claims description 9
- 108091026890 Coding region Proteins 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 7
- 239000013612 plasmid Substances 0.000 claims description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 6
- 244000020551 Helianthus annuus Species 0.000 claims description 5
- 235000003222 Helianthus annuus Nutrition 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 5
- 108010003581 Ribulose-bisphosphate carboxylase Proteins 0.000 claims description 4
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 239000012190 activator Substances 0.000 claims description 3
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 3
- 231100000816 toxic dose Toxicity 0.000 claims description 3
- 108010034715 Light-Harvesting Protein Complexes Proteins 0.000 claims description 2
- 108700023158 Phenylalanine ammonia-lyases Proteins 0.000 claims description 2
- 108090000051 Plastocyanin Proteins 0.000 claims description 2
- 229930002875 chlorophyll Natural products 0.000 claims description 2
- 235000019804 chlorophyll Nutrition 0.000 claims description 2
- 229930002868 chlorophyll a Natural products 0.000 claims description 2
- 229930002869 chlorophyll b Natural products 0.000 claims description 2
- NSMUHPMZFPKNMZ-VBYMZDBQSA-M chlorophyll b Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C=O)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 NSMUHPMZFPKNMZ-VBYMZDBQSA-M 0.000 claims description 2
- 238000010367 cloning Methods 0.000 claims description 2
- 239000013599 cloning vector Substances 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims description 2
- 238000003306 harvesting Methods 0.000 claims description 2
- 230000010076 replication Effects 0.000 claims description 2
- 230000003612 virological effect Effects 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims 2
- 102000014914 Carrier Proteins Human genes 0.000 claims 1
- 108091008324 binding proteins Proteins 0.000 claims 1
- 238000009331 sowing Methods 0.000 claims 1
- 231100000331 toxic Toxicity 0.000 claims 1
- 230000002588 toxic effect Effects 0.000 claims 1
- 101100339555 Zymoseptoria tritici HPPD gene Proteins 0.000 abstract 2
- 230000002103 transcriptional effect Effects 0.000 abstract 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 26
- 241000208125 Nicotiana Species 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 17
- 210000001519 tissue Anatomy 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 9
- 239000005571 Isoxaflutole Substances 0.000 description 8
- OYIKARCXOQLFHF-UHFFFAOYSA-N isoxaflutole Chemical compound CS(=O)(=O)C1=CC(C(F)(F)F)=CC=C1C(=O)C1=C(C2CC2)ON=C1 OYIKARCXOQLFHF-UHFFFAOYSA-N 0.000 description 8
- 229940088649 isoxaflutole Drugs 0.000 description 8
- 231100000674 Phytotoxicity Toxicity 0.000 description 7
- 238000010276 construction Methods 0.000 description 6
- 108010033040 Histones Proteins 0.000 description 5
- 238000011069 regeneration method Methods 0.000 description 5
- 108700001094 Plant Genes Proteins 0.000 description 4
- 241000723792 Tobacco etch virus Species 0.000 description 4
- 240000008042 Zea mays Species 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 210000002706 plastid Anatomy 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 241000589516 Pseudomonas Species 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 240000002791 Brassica napus Species 0.000 description 2
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 2
- ZAQJHHRNXZUBTE-NQXXGFSBSA-N D-ribulose Chemical compound OC[C@@H](O)[C@@H](O)C(=O)CO ZAQJHHRNXZUBTE-NQXXGFSBSA-N 0.000 description 2
- ZAQJHHRNXZUBTE-UHFFFAOYSA-N D-threo-2-Pentulose Natural products OCC(O)C(O)C(=O)CO ZAQJHHRNXZUBTE-UHFFFAOYSA-N 0.000 description 2
- 244000000626 Daucus carota Species 0.000 description 2
- 235000002767 Daucus carota Nutrition 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 2
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
- 244000061176 Nicotiana tabacum Species 0.000 description 2
- 241000589540 Pseudomonas fluorescens Species 0.000 description 2
- 241001233957 eudicotyledons Species 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 208000037824 growth disorder Diseases 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XUHGTGGPZFJRMF-UHFFFAOYSA-N 1,3-dihydropyrazole-2-carboxylic acid Chemical class OC(=O)N1CC=CN1 XUHGTGGPZFJRMF-UHFFFAOYSA-N 0.000 description 1
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- HUTNOYOBQPAKIA-UHFFFAOYSA-N 1h-pyrazin-2-one Chemical class OC1=CN=CC=N1 HUTNOYOBQPAKIA-UHFFFAOYSA-N 0.000 description 1
- ZWSNPZPIJHQHGA-UHFFFAOYSA-N 2-(1-methylcyclopropanecarbonyl)-3-[2-methylsulfonyl-4-(trifluoromethyl)phenyl]-3-oxopropanenitrile Chemical compound C=1C=C(C(F)(F)F)C=C(S(C)(=O)=O)C=1C(=O)C(C#N)C(=O)C1(C)CC1 ZWSNPZPIJHQHGA-UHFFFAOYSA-N 0.000 description 1
- ZTTKDUXKVPEXCG-UHFFFAOYSA-N 2-cyano-3-cyclopropyl-1-(2-mesyl-4-trifluoromethylphenyl)propan-1,3-dione Chemical compound CS(=O)(=O)C1=CC(C(F)(F)F)=CC=C1C(=O)C(C#N)C(=O)C1CC1 ZTTKDUXKVPEXCG-UHFFFAOYSA-N 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- 241000208173 Apiaceae Species 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 235000007319 Avena orientalis Nutrition 0.000 description 1
- 244000075850 Avena orientalis Species 0.000 description 1
- 235000021537 Beetroot Nutrition 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000016680 Dioxygenases Human genes 0.000 description 1
- 108010028143 Dioxygenases Proteins 0.000 description 1
- 241000219146 Gossypium Species 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 101150012639 HPPD gene Proteins 0.000 description 1
- 102100034535 Histone H3.1 Human genes 0.000 description 1
- 101001067844 Homo sapiens Histone H3.1 Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 240000001140 Mimosa pudica Species 0.000 description 1
- 235000016462 Mimosa pudica Nutrition 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 239000005618 Sulcotrione Substances 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- 229960004261 cefotaxime Drugs 0.000 description 1
- AZZMGZXNTDTSME-JUZDKLSSSA-M cefotaxime sodium Chemical compound [Na+].N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C([O-])=O)=O)C(=O)\C(=N/OC)C1=CSC(N)=N1 AZZMGZXNTDTSME-JUZDKLSSSA-M 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 208000006278 hypochromic anemia Diseases 0.000 description 1
- 230000001965 increasing effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- CTAPFRYPJLPFDF-UHFFFAOYSA-N isoxazole Chemical compound C=1C=NOC=1 CTAPFRYPJLPFDF-UHFFFAOYSA-N 0.000 description 1
- 150000002545 isoxazoles Chemical class 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 125000004170 methylsulfonyl group Chemical group [H]C([H])([H])S(*)(=O)=O 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000006870 ms-medium Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 231100001222 nononcogenic Toxicity 0.000 description 1
- 108010058731 nopaline synthase Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000012882 rooting medium Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- PQTBTIFWAXVEPB-UHFFFAOYSA-N sulcotrione Chemical compound ClC1=CC(S(=O)(=O)C)=CC=C1C(=O)C1C(=O)CCCC1=O PQTBTIFWAXVEPB-UHFFFAOYSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
Definitions
- the present invention relates to a chimeric gene having a light-dependent promoter operably linked to a gene coding for a HPPD (hydroxy phenyl pyruvate dioxygenase), conferring improved tolerance to herbicides inhibitors of 1 ⁇ PPD to a normally sensitive plant cell and plant .
- HPPD hydroxy phenyl pyruvate dioxygenase
- the invention also relates to plant cells and palntes transformed with the chimeric gene according to the invention, a method for transforming plant cells and plants and a method for cultivating transformed plants in which an herbicide inhibitor of HPPD is applied to eliminate weeds.
- the herbicides targeting HPPD are in particular isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5 424 276) in particular isoxaflutole, a selective herbicide corn, diketonitriles (EP 496 630, EP 496 631), in particular 2-cyano-3-cyclopropyl-1- (2-S ⁇ 2 CH3-4-CF3 phenyl) propane- 1, 3 -dione and 2- cyano-3-cyclopropyl-l- (2-S ⁇ 2 CH3 ⁇ 4-2.3 CI2 phenyl) propane- 1, 3-dione, triketones (EP 625 505, EP 625 508, US 5,506,195), in particular sulcotrione or again the pyrazolinates.
- isoxazoles EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5 424 27
- LD promoter lightly dependent promoter
- the present invention therefore firstly relates to a chimeric gene comprising at least one elementary chimeric gene containing, in the direction of transcription, 5 'regulatory elements necessary for its transcription in plants, at least one heterologous coding part. comprising a coding sequence coding for an enzyme conferring on plants tolerance to herbicides which inhibit HPPD and at least one terminator or polyadenylation regulatory sequence, the 5 'regulatory elements ensuring the transcription of the elementary chimeric gene at the tissue level chlorophylliens, the said 5 'regulatory elements preferably comprising at least one promoter regulatory sequence of the LD promoter type.
- LD promoter any gene promoter coding for peptides whose transcription is induced by light which is functional as a promoter in plant cells.
- LD promoters can be of bacterial, viral or vegetable origin. Such promoters are in particular described by Terzaghi & coll. (Light-regulated transcription, Annu. Rev. Plant Physiol. Plant Mol. Biol, 1995, 46: 445-474) the content of which is incorporated herein by reference.
- LD promoter useful according to the invention, there will be mentioned more particularly the promoter of a gene for the small subunit of plant ribulose-biscarboxylase (rbcs), of the protein "light-harvesting chlorophyll a / b binding" (LHCP) , plastocyanine (pet E) and phenylalanine ammonia lyase (pal).
- rbcs ribulose-biscarboxylase
- LHCP light-harvesting chlorophyll a / b binding
- plastocyanine plastocyanine
- pal phenylalanine ammonia lyase
- a promoter regulatory sequence which promotes the overexpression of the coding sequence in chlorophyllian tissues, such as for example, that comprising at least one functional fragment of the promoter of the small rbcs subunit (SSU) d 'a plant, more particularly isolated from Helianthus annuus as described in US patent 5,559,024.
- SSU small rbcs subunit
- the sequence of the SSU promoter of Helianthus annuus comprises the DNA sequence represented by the sequence identifier No. 1 (SEQ ID NO: 1) or a sequence homologous to said sequence . More preferably, the sequence of the SSU promoter consists of the DNA sequence represented by the sequence identifier No. 1.
- homologous a DNA sequence having one or more sequence modifications with respect to the reference DNA sequence described by the sequence identifier No. 1, and reproducing the function of this sequence. These modifications can be obtained according to the usual mutation techniques, or alternatively by choosing the synthetic oligonucleotides which can be used in the preparation of said sequence by hybridization.
- the degree of homology will be at least 70% relative to the reference sequence, preferably at least 80%, more preferably at least 90%.
- plant cell any cell derived from a monocotyledonous plant and which may constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of monocotyledonous plants, monocotyledonous plants or seeds.
- plant means any differentiated multicellular organism capable of photosynthesis, more particularly monocotyledonous or dicotyledonous plants, preferably from cultures intended or not intended for animal or human food, such as, for example, wheat, barley, oats, rice, corn, sorghum, sugarcane, soybeans, rapeseed, cotton, tobacco, beetroot or vegetable or flower crops.
- promoter regulatory sequence of the LD type other regulatory sequences, which are located between the promoter and the coding sequence, such as enhancer trancription activators, as for example the translation activator of the tobacco etch virus (VTE) described in the article by Carrington and Freed, 1990; J. Virol.
- enhancer trancription activators as for example the translation activator of the tobacco etch virus (VTE) described in the article by Carrington and Freed, 1990; J. Virol.
- sequences coding for transit peptides either single or double, and in this case optionally separated by an intermediate sequence, that is to say comprising, in the direction of transcription, a sequence coding for a transit peptide of a plant gene coding for an enzyme with plastid location, a sequence part of the mature N terminal part of a plant gene coding for an enzyme with localization plastid, then a sequence coding for a second transit peptide of a plant gene coding for a plastid localized enzyme, consisting of a sequence part of the mature N terminal part of a plant gene coding for a plastid localized enzyme , as described in patent application EP 508 909.
- HPPD As coding sequence for an enzyme conferring on plants tolerance to herbicides which inhibit HPPD, it is possible in particular to use all those known to confer plant tolerance on certain inhibitors of HPPD such as the sequences coding for a HPPD described in patent application WO 96/38567 and in patent application FR 9714264 filed on November 7, 1997.
- This HPPD can be of any kind.
- this sequence can be of bacterial origin, such as in particular the genus Pseudomonas or also of vegetable origin, such as in particular of monocotyledonous or dicotyledone plant, in particular Arabidopsis or umbelliferae such as for example carrot (Daucus carota). It can be native or wild or possibly mutated while fundamentally retaining a herbicidal tolerance property against HPPD inhibitors, such as herbicides of the isoxazole family or that of the triketones or pyrazinolates.
- HPPD inhibitors such as herbicides of the isoxazole family or that of the triketones or pyrazinolates.
- any corresponding sequence of bacterial origin such as for example the terminator nos dAgrobacterium tumefaciens, or of plant origin, such as for example a histone terminator as described in the application, can be used.
- European EP 633,317 As terminating or polyadenylation regulatory sequence, any corresponding sequence of bacterial origin, such as for example the terminator nos dAgrobacterium tumefaciens, or of plant origin, such as for example a histone terminator as described in the application, can be used.
- European EP 633,317 As terminating or polyadenylation regulatory sequence, any corresponding sequence of bacterial origin, such as for example the terminator nos dAgrobacterium tumefaciens, or of plant origin, such as for example a histone terminator as described in the application.
- the present invention also relates to a cloning or expression vector for the transformation of a plant cell or a monocotyledonous or dicotyledonous plant.
- the vector according to the invention comprises, in addition to the above chimeric gene, at least one origin of replication.
- This vector can consist of a plasmid, a cosmid, a bacteriophage or a virus, transformed by the introduction of the chimeric gene according to the invention.
- Such vectors for transforming plant cells and monocotyledonous plants are well known to those skilled in the art and widely described in the literature.
- the vector for transforming plant cells or plants according to the invention is a plasmid.
- the subject of the invention is also a process for transforming plant cells by integration of at least one nucleic acid fragment or a chimeric gene as defined above, a transformation which can be obtained by any suitable known means with the vector according to the invention.
- a series of methods involves bombarding cells or protoplasts with particles to which the DNA sequences are attached. Another series of methods consists in using a chimeric gene as a means of transfer into the plant. inserted into a Ti d grobacterium tumefaciens or Ri plasmid from Agrobacterium rhizogenes. Other methods can be used such as micro-injection or electroporation, or even direct precipitation using PEG.
- Another subject of the present invention is plant cells or plants, transformed tolerant to herbicides targeting HPPD and containing at least one chimeric gene according to the invention defined above.
- the present invention also relates to plants containing transformed cells, in particular plants regenerated from transformed cells.
- the regeneration is obtained by any suitable process which depends on the nature of the species.
- the present invention also relates to the transformed plants resulting from the culture and / or the crossing of the regenerated plants above, as well as the seeds of transformed plants.
- the present invention also relates to a method of controlling weeds in a surface of a field comprising seeds or plants transformed with the chimeric gene according to the invention, which method consists in applying in said field surface a toxic dose for the said weeds of a herbicide targeting HPPD, without however substantially affecting the seeds or plants transformed with the said chimeric gene according to the invention.
- the present invention also relates to a process for cultivating plants transformed according to the invention with a chimeric gene according to the invention, which process consists in planting the seeds of said transformed plants in a surface of a field suitable for the cultivation of said plants, to apply to said surface of said field a toxic dose for weeds of a herbicide targeting HPPD defined above in the presence of weeds, without substantially affecting said seeds or said plants then harvest the cultivated plants when they reach the desired maturity and possibly separate the seeds from the harvested plants.
- the application of the herbicide targeting HPPD can be carried out according to the invention, both in pre-planting, pre-emergence and post-emergence of the crop.
- herbicide within the meaning of the present invention is meant a herbicidal active material alone or associated with an additive which modifies its effectiveness such as for example an agent increasing activity (synergist) or limiting activity (in English safener).
- an agent increasing activity for example an agent increasing activity (synergist) or limiting activity (in English safener).
- limiting activity in English safener.
- the above herbicides are associated in a known manner with the adjuvants of formulations usually used in agrochemistry. The various aspects of the invention will be better understood with the aid of the experimental examples below.
- Example 1 Construction of a chimeric gene with an HPPD sequence and an LD promoter. To confer plant tolerance to herbicides that inhibit HPPD, a chimeric gene called pRPA-RD-2005 is constructed:
- HPPD HPPD will then be localized in the chloroplast and the gene is mainly expressed in chlorophyll tissues.
- PRPA-RD-2005 is a binary vector of pRPA-BL-150A ⁇ 2 type (EP 508 909) containing an HPPD expression cassette: promoter of the small subunit of ribulose discarboxylase-OTP-HPPD gene-terminator our
- pRPA-RD-2004 - pRD-207 is a pBluescript SK (-) (stratagene catalog # 21 2206) containing the gene for nopaline synthase (terminator nos).
- PRD-207 is used as the basic vector for the construction of pRPA-RD-2004
- - pRD-208 contains the OTP / HPPD cassette: nos. It is obtained from the plasmid pRPA S, described in WO 96/38567, digested with Xbal / ClaI, treatment with polymerase pfu type. The cassette is introduced into the open pRD-207 by a Sali digestion, klenow treatment.
- the promoter of the small subunit of the ribulose biscarboxylase from Helianthus annuus comes from the plasmid pRD-127 described in WO 96/38567 digested Ncol / Xbal is introduced into pRD-208. This construction constitutes pRPA-RD-2004
- Example 2 Processing of PBD6 industrial tobacco.
- Transformation The vector is introduced into the non-oncogenic strain of Agrobacterium EHA 101 or LBA 4404 (Hood et al, 1987) carrying the cosmid pTVK 291 (Komari et al, 1986). The transformation technique is based on the procedure of Horsh R. et al. (1985) Science, 227, 1229-1231.
- Regeneration The regeneration of PBD6 tobacco (from SEITA France) from leaf explants is carried out on a Murashige and Skoog (MS) base medium comprising 30g / l of sucrose as well as 350 mg / 1 of cefotaxime and 1 mg / 1 diketonitrile derived from isoxaflutole or 10 mg / 1 l- [4- (trifluoromethyl) -2- (methylsulfonyl) phenyl] -2-cyano- 3- (l-methylcyclopropyl) -propan-1, 3- dione, another HPPD inhibitor.
- MS Murashige and Skoog
- the leaf explants are taken from plants in the greenhouse or in vitro and transformed using the leaf disc technique (Science 1985, Vol 227, p.1229-1231) in three successive stages: the first includes the induction of shoots on a medium MS supplemented with 30g / l of sucrose containing 0.05mg / l of naphthylacetic acid (ANA) and 2 mg / 1 of benzylaminopurine (BAP) for 15 days and 1 mg / 1 of isoxaflutole.
- ANA naphthylacetic acid
- BAP benzylaminopurine
- the green shoots formed during this stage are then developed by culture on an MS medium supplemented with 30 g / 1 of sucrose and 1 mg / 1 of isoxaflutole or 10 mg / 1 of 1- [4- (trifluoromethyl) -2 - (methylsulfonyl) phenyl] -2-cyano-3- (1-methylcyclopropyl) -propan 1,3-dione but containing no hormone, for 10 days.
- the transformed tobacco seedlings were acclimated in the greenhouse (60% relative humidity; temperature: 20 ° C at night and 23 ° C during the day) for three weeks then treated with 4- [4- (trifiuoromethyl) -2- (methylsulfonyl) benzoyl] -5-cyclopropylisoxazole (isoxafutole).
- the control tobacco, unprocessed and treated with isoxafutole at the doses of 400 g / ha develops in about 72 hours chloroses, which intensify to progress to very pronounced necrosis in a week (covering about 80% of the leaves final).
- the greenhouse tolerance of 2005 tobacco is compared to that of COI 1 tobacco, transformed with the pRPA-V chimeric gene described in patent application WO 96/38567 and that of unprocessed tobacco. Description of tobacco trials.
- Example 4 The comparative tolerance tests of Example 4 are reproduced in the field for the same tobacco 2005, COI 1 and PBD6.
- Young plants from seedlings were transplanted individually in miniballs to be transplanted a second time in the field.
- the post treatment was carried out one week later at doses of isoxaflutole of 0/100/200/300/400/500/600 g / ha.
- plants transformed with the pRPA-RD-2005 gene comprising the SSU promoter have better tolerance to HPPD inhibitors than those transformed with the pRPA-V gene comprising the double histone promoter combined with TEV "enhancer" of the state of the art.
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Catching Or Destruction (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention concerns a chimera gene comprising at least an elementary chimera gene including, in the transcriptional direction, elements regulating in 5' necessary for its transcription into plants, at least one heterologous coding portion comprising a sequence coding for an enzyme providing plants with tolerance to herbicides inhibitors of HPPD and at least one terminator or polyadenylation regulating sequence, wherein the elements regulating in 5' ensure the transcription of the elementary chimera gene at the level of chlorophyllian tissues. The invention also concerns the transformation of plants and the plants transformed with said chimera gene. It further concerns a method for growing transformed plants which consists in applying a HPPD inhibitor for controlling weeds.
Description
Gène chimère ayant un promoteur lumière dépendant conférant la tolérance aux inhibiteurs de 1ΗPPD Chimeric gene having a light-dependent promoter conferring tolerance to 1ΗPPD inhibitors
La présente invention a pour objet un gène chimère ayant un promoteur lumière dépendant lié de manière fonctionnelle à un gène codant pour une HPPD (hydroxy phényl pyruvate dioxygénase), conférant une tolérance améliorée aux herbicides inhibiteurs de 1ΗPPD à une cellule végétale et une plante normalement sensibles. L'invention concerne également les cellules végétales et palntes transformées avec le gène chimère selon l'invention, un procédé de transformation des cellules végétales et des plantes et un procédé de culture des plantes transformées dans lequel on applique un herbicide inhibiteur de l'HPPD pour éliminer les mauvaises herbes.The present invention relates to a chimeric gene having a light-dependent promoter operably linked to a gene coding for a HPPD (hydroxy phenyl pyruvate dioxygenase), conferring improved tolerance to herbicides inhibitors of 1ΗPPD to a normally sensitive plant cell and plant . The invention also relates to plant cells and palntes transformed with the chimeric gene according to the invention, a method for transforming plant cells and plants and a method for cultivating transformed plants in which an herbicide inhibitor of HPPD is applied to eliminate weeds.
Les herbicides ayant pour cible l'HPPD sont notamment les isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5 424 276) en particulier l'isoxaflutole, herbicide sélectif du maïs, les dicétonitriles (EP 496 630, EP 496 631), en particulier la 2-cyano-3-cyclopropyl-l-(2-Sθ2 CH3-4-CF3 phényl) propane- 1, 3 -dione et la 2-cyano-3-cyclopropyl-l-(2-Sθ2 CH3~4-2,3 CI2 phényl) propane- 1, 3-dione, les tricétones (EP 625 505, EP 625 508, US 5,506,195), en particulier la sulcotrione ou encore les pyrazolinates. Des gènes codant pour une HPPD conférant une tolérance à ces herbicides et les plantes transgéniques contenant ce gène montrent une tolérance significative aux dits herbicides sont décrits dans la demande de brevet WO 96/38567 et dans la demande de brevet FR 97 14264 déposée le 7 novembre 1997, dont le contenu est ici inclus par référence.The herbicides targeting HPPD are in particular isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682 659, US 5 424 276) in particular isoxaflutole, a selective herbicide corn, diketonitriles (EP 496 630, EP 496 631), in particular 2-cyano-3-cyclopropyl-1- (2-Sθ2 CH3-4-CF3 phenyl) propane- 1, 3 -dione and 2- cyano-3-cyclopropyl-l- (2-Sθ2 CH3 ~ 4-2.3 CI2 phenyl) propane- 1, 3-dione, triketones (EP 625 505, EP 625 508, US 5,506,195), in particular sulcotrione or again the pyrazolinates. Genes coding for a HPPD conferring tolerance to these herbicides and the transgenic plants containing this gene show significant tolerance to said herbicides are described in patent application WO 96/38567 and in patent application FR 97 14264 filed on November 7 1997, the content of which is included here by reference.
Jusqu'à maintenant tous les essais effectués pour conférer une bonne tolérance aux inhibiteurs de l'HPPD l'ont été en utilisant des promoteurs « constitutifs » ou permettant une expression dans toutes sortes de tissus végétaux, tissus racinaires, tissus foliaires, zones en croissance plus ou moins rapides. Pour ce faire des plantes dicotylédones comme le tabac, Arabidopsis thaliana, le colza et le soja ont été transformées soit avec: - le gène chimère pRP TUp to now, all the attempts made to confer good tolerance on HPPD inhibitors have been done using “constitutive” promoters or allowing expression in all kinds of plant tissues, root tissues, leaf tissues, growing areas. more or less fast. To do this, dicotyledonous plants such as tobacco, Arabidopsis thaliana, rapeseed and soybeans have been transformed with either: - the pRP T chimera gene
- le gène chimère pRP V- the pRP V chimeric gene
Ces essais nous ont permis de confirmar que ce type d'expression permet dans les dicotylédones d'obtenir une très bonne tolérance.
Des plantes monocotylédones comme le mais ont également été transformées avec le gène chimère pRPA-RD-1004 (demande de brevet PCT/FR97/01256 déposée le 10 juillet 1997): promoteur histone Intron 1 de OTP Région codante de l'HPPD Terminateur nos H3C4 de maïs AdhlThese tests allowed us to confirm that this type of expression allows dicots to obtain very good tolerance. Monocotyledonous plants like maize have also been transformed with the chimeric gene pRPA-RD-1004 (patent application PCT / FR97 / 01256 filed July 10, 1997): histone promoter Intron 1 of OTP Coding region of HPPD Terminator nos H3C4 Adhl corn
On a maintenant trouvé qu'on pouvait conférer à une cellule végétale et à une plante une tolérance herbicide améliorée par sur-expression de l'HPPD par l'intermédiaire d'un promoteur " lumière dépendant " ( appelé ci-après promoteur LD). L'utilisation de ces promoteurs LD conduit à l'expression du gène chimère dans les tissus chlorophylliens ou " tissus verts " sans aucune expression dans les tissus non chlorophylliens ou une expression très faible dans les tissus non chlorophylliens comme les tissus racinaires, une telle expression étant suffisante pour améliorer la tolérance des plantes transformées aux herbicides inhibiteurs de l'HPPD.We have now found that it is possible to confer on a plant cell and on a plant an improved herbicidal tolerance by overexpression of HPPD via a "light dependent" promoter (hereinafter called LD promoter). The use of these LD promoters leads to the expression of the chimeric gene in chlorophyllian tissues or "green tissues" without any expression in non-chlorophyllian tissues or a very weak expression in non-chlorophyllian tissues such as root tissues, such expression being sufficient to improve the tolerance of the transformed plants to herbicides that inhibit HPPD.
La présente invention a donc d'abord pour objet un gène chimère comprenant au moins un gène chimère élémentaire contenant , dans le sens de la transcription, des éléments de régulation en 5' nécessaires à sa transcription dans les plantes, au moins une partie codante hétérologue comprenant une séquence codante codant pour une enzyme conférant aux plantes la tolérance aux herbicides inhibiteurs de l'HPPD et au moins une séquence de régulation terminatrice ou de polyadenylation, les éléments de régulation en 5' assurant la transcription du gène chimère élémentaire au niveau des tissus chlorophylliens, les dits éléments de régulations en 5' comprenant de préférence au moins une séquence de régulation promotrice de type promoteur LD.The present invention therefore firstly relates to a chimeric gene comprising at least one elementary chimeric gene containing, in the direction of transcription, 5 'regulatory elements necessary for its transcription in plants, at least one heterologous coding part. comprising a coding sequence coding for an enzyme conferring on plants tolerance to herbicides which inhibit HPPD and at least one terminator or polyadenylation regulatory sequence, the 5 'regulatory elements ensuring the transcription of the elementary chimeric gene at the tissue level chlorophylliens, the said 5 'regulatory elements preferably comprising at least one promoter regulatory sequence of the LD promoter type.
Par « promoteur LD », on entend selon l'invention tout promoteur de gène codant pour des peptides dont la transcription est induite par la lumière qui est fonctionnel comme promoteur dans les cellules végétales. Ces promoteurs LD peuvent être d'origine bactérienne, virale ou végétale. De tels promoteurs sont notamment décrits par Terzaghi & coll. (Light-regulated transcription, Annu. Rev. Plant Physiol. Plant Mol. Biol, 1995, 46:445-474) dont le contenu est incorporé ici par référence. Comme promoteur LD utile selon l'invention, on citera plus particulièrement le promoteur d'un gène de la petite sous unité de ribulose-biscarboxylase (rbcs) de plante, de la protéine « light-harvesting chlrorophyll a/b binding » (LHCP), de la plastocyanine (pet E) et de la phénylalanine ammonia lyase (pal). De préférence on a recours à une séquence de régulation promotrice qui favorise la surexpression de la séquence codante dans les tissus chlorophylliens, telle que par exemple, celle comprenant au moins un fragment fonctionnel du promoteur de la petite sous unité de la rbcs (SSU) d'une plante, plus particulièrement isolé d'Helianthus annuus tel que décrit dans lebrevet US 5,559,024.
Parmi les promoteurs S SU, on peut également citer le promoteur S SU de pétunia décrit dans le brevet US 4,962,028.By "LD promoter" is meant according to the invention any gene promoter coding for peptides whose transcription is induced by light which is functional as a promoter in plant cells. These LD promoters can be of bacterial, viral or vegetable origin. Such promoters are in particular described by Terzaghi & coll. (Light-regulated transcription, Annu. Rev. Plant Physiol. Plant Mol. Biol, 1995, 46: 445-474) the content of which is incorporated herein by reference. As LD promoter useful according to the invention, there will be mentioned more particularly the promoter of a gene for the small subunit of plant ribulose-biscarboxylase (rbcs), of the protein "light-harvesting chlorophyll a / b binding" (LHCP) , plastocyanine (pet E) and phenylalanine ammonia lyase (pal). Preferably, a promoter regulatory sequence is used which promotes the overexpression of the coding sequence in chlorophyllian tissues, such as for example, that comprising at least one functional fragment of the promoter of the small rbcs subunit (SSU) d 'a plant, more particularly isolated from Helianthus annuus as described in US patent 5,559,024. Among the S SU promoters, mention may also be made of the S SU promoter for petunia described in US Pat. No. 4,962,028.
Par fragment fonctionnel, on entend selon l'invention toute séquence d'ADN issue de la séquence du promoteur SSU reproduisant la fonction de la séquence d'où elle est issue.By functional fragment is meant according to the invention any DNA sequence originating from the sequence of the SSU promoter reproducing the function of the sequence from which it is derived.
Selon un mode préférentiel de réalisation de l'invention, la séquence du promoteur SSU d'Helianthus annuus comprend la séquence d'ADN représentée par l'identificateur de séquence n° 1 (SEQ ID NO:l) ou une séquence homologue de ladite séquence. Plus préférentiellement, la séquence du promoteur SSU consiste en la séquence d'ADN représentée par l'identificateur de séquence n° 1.According to a preferred embodiment of the invention, the sequence of the SSU promoter of Helianthus annuus comprises the DNA sequence represented by the sequence identifier No. 1 (SEQ ID NO: 1) or a sequence homologous to said sequence . More preferably, the sequence of the SSU promoter consists of the DNA sequence represented by the sequence identifier No. 1.
Par " homologue ", on entend selon l'invention une séquence d'ADN présentant une ou plusieurs modifications de séquence par rapport à la séquence d'ADN de référence décrite par l'identificateur de séquence n° 1, et reproduisant la fonction de cette séquence. Ces modifications peuvent être obtenues selon les techniques usuelles de mutation, ou encore en choisissant les oligonucléotides synthétiques pouvant être employés dans la préparation de ladite séquence par hybridation. De manière avantageuse, le degré d'homologie sera d'au moins 70 % par rapport à la séquence de référence, de préférence d'au moins 80 %, plus préférentiellement d'au moins 90 %.By "homologous" is meant according to the invention a DNA sequence having one or more sequence modifications with respect to the reference DNA sequence described by the sequence identifier No. 1, and reproducing the function of this sequence. These modifications can be obtained according to the usual mutation techniques, or alternatively by choosing the synthetic oligonucleotides which can be used in the preparation of said sequence by hybridization. Advantageously, the degree of homology will be at least 70% relative to the reference sequence, preferably at least 80%, more preferably at least 90%.
Par "cellule végétale", on entend selon l'invention toute cellule issue d'une plante monocotyledone et pouvant constituer des tissus indifférenciés tels que des cals, des tissus différenciés tels que des embryons, des parties de plantes monocotyledones, des plantes monocotyledones ou des semences.By "plant cell" is meant according to the invention any cell derived from a monocotyledonous plant and which may constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of monocotyledonous plants, monocotyledonous plants or seeds.
On entend par " plante " selon l'invention, tout organisme multicellulaire différencié capable de photosynthèse, plus particulièrement des plantes monocotyledones ou dicotylédones, de préférence de culture destinées ou non à l'alimentation animale ou humaine, comme par exemple le blé, l'orge, l'avoine, le riz, le maïs, le sorgho, la canne à sucre, le soja, le colza, le coton, le tabac, la betterave ou encore desplantes de cultures maraichères ou florales.The term "plant" according to the invention means any differentiated multicellular organism capable of photosynthesis, more particularly monocotyledonous or dicotyledonous plants, preferably from cultures intended or not intended for animal or human food, such as, for example, wheat, barley, oats, rice, corn, sorghum, sugarcane, soybeans, rapeseed, cotton, tobacco, beetroot or vegetable or flower crops.
Selon l'invention, on peut également utiliser, en association avec la séquence de régulation promotrice de type LD, d'autres séquences de régulation, qui sont situées entre le promoteur et la séquence codante, telles que des activateurs de trancription "enhancer", comme par exemple l'activateur de traduction du virus etch du tabac (TEV) décrit dans l'article de Carrington and Freed, 1990; J. Virol. 64: 1590-1597, ou des séquences codant pour des peptides de transit, soit simples, soit doubles, et dans ce cas éventuellement séparés par une séquence intermédiaire, c'est à dire comprenant, dans le sens de la transcription, une séquence codant pour un peptide de transit d'un gène végétal codant pour une enzyme à localisation plastidiale, une partie de séquence de la partie mature N terminale d'un gène végétal codant pour une enzyme à localisation
plastidiale, puis une séquence codant pour un second peptide de transit d'un gène végétal codant pour une enzyme à localisation plastidiale, constituée d'une partie de séquence de la partie mature N terminale d'un gène végétal codant pour une enzyme à localisation plastidiale, tels que décrit dans la demande demande de brevet EP 508 909. Comme séquence codante pour une enzyme conférant aux plantes la tolérance aux herbicides inhibiteurs de l'HPPD, on peut notamment utiliser toutes celles connues pour conférer la tolérance de plantes à certains inhibiteurs de l'HPPD telles que les séquences codant pour une HPPD décrites dans la demande de brevet WO 96/38567 et dans la demande de brevet FR 9714264 déposée le 7 novembre 1997. Cette HPPD peut être de toute nature.According to the invention, it is also possible to use, in association with the promoter regulatory sequence of the LD type, other regulatory sequences, which are located between the promoter and the coding sequence, such as enhancer trancription activators, as for example the translation activator of the tobacco etch virus (VTE) described in the article by Carrington and Freed, 1990; J. Virol. 64: 1590-1597, or sequences coding for transit peptides, either single or double, and in this case optionally separated by an intermediate sequence, that is to say comprising, in the direction of transcription, a sequence coding for a transit peptide of a plant gene coding for an enzyme with plastid location, a sequence part of the mature N terminal part of a plant gene coding for an enzyme with localization plastid, then a sequence coding for a second transit peptide of a plant gene coding for a plastid localized enzyme, consisting of a sequence part of the mature N terminal part of a plant gene coding for a plastid localized enzyme , as described in patent application EP 508 909. As coding sequence for an enzyme conferring on plants tolerance to herbicides which inhibit HPPD, it is possible in particular to use all those known to confer plant tolerance on certain inhibitors of HPPD such as the sequences coding for a HPPD described in patent application WO 96/38567 and in patent application FR 9714264 filed on November 7, 1997. This HPPD can be of any kind.
Plus particulièrement cette séquence peut être d'origine bactérienne, telle que notamment le genre Pseudomonas ou encore d'origine végétale, telle que notamment de plante monocotylédone ou dicotyledone, notamment dArabidopsis ou d'ombellifères comme par exemple la carotte (Daucus carota). Elle peut être native ou sauvage ou éventuellement mutée tout en gardant fondamentalement une propriété de tolérance herbicide contre les inhibiteurs de l'HPPD, tels que les herbicides de la famille des isoxazoles ou de celle des tricétones ou des pyrazinolates.More particularly, this sequence can be of bacterial origin, such as in particular the genus Pseudomonas or also of vegetable origin, such as in particular of monocotyledonous or dicotyledone plant, in particular Arabidopsis or umbelliferae such as for example carrot (Daucus carota). It can be native or wild or possibly mutated while fundamentally retaining a herbicidal tolerance property against HPPD inhibitors, such as herbicides of the isoxazole family or that of the triketones or pyrazinolates.
Comme séquence de régulation terminatrice ou de polyadenylation, on peut utiliser toute séquence correspondante d'origine bactérienne, comme par exemple le terminateur nos dAgrobacterium tumefaciens, ou encore d'origine végétale, comme par exemple un terminateur d'histone tel que décrit dans la demande européenne EP 633 317.As terminating or polyadenylation regulatory sequence, any corresponding sequence of bacterial origin, such as for example the terminator nos dAgrobacterium tumefaciens, or of plant origin, such as for example a histone terminator as described in the application, can be used. European EP 633,317.
La présente invention concerne également un vecteur de clonage ou d'expression pour la transformation d'une cellule végétale ou d'une plante monocotylédone ou dicotyledone. Le vecteur selon l'invention comprend outre le gène chimère ci-dessus, au moins une origine de réplication. Ce vecteur peut être constitué par un plasmide, un cosmide, un bactériophage ou un virus, transformés par l'introduction du gène chimère selon l'invention. De tels vecteurs de transformation de cellules végétales et de plantes monocotyledones sont bien connus de l'homme du métier et largement décrits dans la littérature. De manière préférentielle, le vecteur de transformation des cellules végétales ou des plantes selon l'invention est un plasmide.The present invention also relates to a cloning or expression vector for the transformation of a plant cell or a monocotyledonous or dicotyledonous plant. The vector according to the invention comprises, in addition to the above chimeric gene, at least one origin of replication. This vector can consist of a plasmid, a cosmid, a bacteriophage or a virus, transformed by the introduction of the chimeric gene according to the invention. Such vectors for transforming plant cells and monocotyledonous plants are well known to those skilled in the art and widely described in the literature. Preferably, the vector for transforming plant cells or plants according to the invention is a plasmid.
L'invention a encore pour objet un procédé de transformation des cellules végétales par intégration d'au moins un fragment d'acide nucléique ou un gène chimère tels que définis ci-dessus, transformation qui peut être obtenue par tout moyen connu approprié avec le vecteur selon l'invention.The subject of the invention is also a process for transforming plant cells by integration of at least one nucleic acid fragment or a chimeric gene as defined above, a transformation which can be obtained by any suitable known means with the vector according to the invention.
Une série de méthodes consiste à bombarder des cellules ou des protoplastes avec des particules auxquelles sont accrochées les séquences d'ADN. Une autre série de méthodes consiste à utiliser comme moyen de transfert dans la plante un gène chimère
inséré dans un plasmide Ti d grobacterium tumefaciens ou Ri d'Agrobacterium rhizogenes. D'autres méthodes peuvent être utilisées telles que la micro-injection ou l'électroporation, ou encore la précipitation directe au moyen de PEG.A series of methods involves bombarding cells or protoplasts with particles to which the DNA sequences are attached. Another series of methods consists in using a chimeric gene as a means of transfer into the plant. inserted into a Ti d grobacterium tumefaciens or Ri plasmid from Agrobacterium rhizogenes. Other methods can be used such as micro-injection or electroporation, or even direct precipitation using PEG.
L'homme du métier fera le choix de la méthode appropriée en fonction de la nature de la cellule végétale ou de la plante.Those skilled in the art will choose the appropriate method depending on the nature of the plant cell or the plant.
La présente invention a encore pour objet les cellules végétales ou plantes, transformées tolérantes aux herbicides ayant pour cible l'HPPD et contenant au moins un gène chimère selon l'invention défini ci-dessus.Another subject of the present invention is plant cells or plants, transformed tolerant to herbicides targeting HPPD and containing at least one chimeric gene according to the invention defined above.
La présente invention a encore pour objet les plantes contenant des cellules transformées, en particulier les plantes régénérées à partir des cellules transformées. La régénération est obtenue par tout procédé approprié qui dépende de la nature de l'espèce.The present invention also relates to plants containing transformed cells, in particular plants regenerated from transformed cells. The regeneration is obtained by any suitable process which depends on the nature of the species.
Pour les procédés de transformation des cellules végétales et de régénération des plantes, on citera notamment les brevets et demandes de brevet suivants: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159, EP 604 662, EP 672 752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442 174, EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 et WO 95/06128.For the processes of transformation of plant cells and regeneration of plants, the following patents and patent applications may be mentioned: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159, EP 604 662, EP 672 752 , US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253,175 EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 and WO 95/06128.
La présente invention concerne également les plantes transformées issues de la culture et/ou du croisement des plantes régénérées ci-dessus, ainsi que les graines de plantes transformées.The present invention also relates to the transformed plants resulting from the culture and / or the crossing of the regenerated plants above, as well as the seeds of transformed plants.
La présente invention concerne également un procédé de contrôle des mauvaises herbes dans une surface d'un champ comprenant des graines ou des plantes transformées avec le gène chimère selon l'invention, lequel procédé consiste à appliquer dans la dite surface du champ une dose toxique pour les dites mauvaises herbes d'un herbicide ayant pour cible l'HPPD, sans toutefois affecter de manière substantielle les graines ou plantes transformée avec le dit gène chimère selon l'invention.The present invention also relates to a method of controlling weeds in a surface of a field comprising seeds or plants transformed with the chimeric gene according to the invention, which method consists in applying in said field surface a toxic dose for the said weeds of a herbicide targeting HPPD, without however substantially affecting the seeds or plants transformed with the said chimeric gene according to the invention.
La présente invention concerne également un procédé de culture des plantes transformées selon l'invention avec un gène chimère selon l'invention lequel procédé consiste à planter les graines des dites plantes transformées dans une surface d'un champ approprié pour la culture des dites plantes, à appliquer sur la dite surface du dit champ une dose toxique pour les mauvaises herbes d'un herbicide ayant pour cible l'HPPD défini ci-dessus en cas de présence de mauvaises herbes, sans affecter de manière substantielle les dites graines ou les dites plantes transformées, puis à récolter les plantes cultivées lorsquelles arrivent à la maturité souhaitée et éventuellement à séparer les graines des plantes récoltées.
Dans les deux procédés ci-dessus, l'application de l'herbicide ayant pour cible l'HPPD peut être faite selon l'invention, tant en présemis, en prélevée qu'en postlevée de la culture.The present invention also relates to a process for cultivating plants transformed according to the invention with a chimeric gene according to the invention, which process consists in planting the seeds of said transformed plants in a surface of a field suitable for the cultivation of said plants, to apply to said surface of said field a toxic dose for weeds of a herbicide targeting HPPD defined above in the presence of weeds, without substantially affecting said seeds or said plants then harvest the cultivated plants when they reach the desired maturity and possibly separate the seeds from the harvested plants. In the above two methods, the application of the herbicide targeting HPPD can be carried out according to the invention, both in pre-planting, pre-emergence and post-emergence of the crop.
Par herbicide au sens de la présente invention on entend une matière active herbicide seule ou associée à un additif qui modifie son efficacité comme par exemple un agent augmentant l'activité (synergiste) ou limitant l'activité (en anglais safener). Bien entendu, pour leur application pratique, les herbicides ci-dessus sont associée de manière en soi connue aux adjuvants de formulations utilisés habituellement en agrochimie Les différents aspects de l'invention seront mieux compris à l'aide des exemples expérimentaux ci-dessous.By herbicide within the meaning of the present invention is meant a herbicidal active material alone or associated with an additive which modifies its effectiveness such as for example an agent increasing activity (synergist) or limiting activity (in English safener). Of course, for their practical application, the above herbicides are associated in a known manner with the adjuvants of formulations usually used in agrochemistry. The various aspects of the invention will be better understood with the aid of the experimental examples below.
Exemple 1: Construction d'un gène chimère avec une séquence HPPD et un promoteur LD. Pour conférer la tolérance de plantes aux herbicides inhibant l'HPPD, on construit un gène chimère appelé pRPA-RD-2005 :Example 1: Construction of a chimeric gene with an HPPD sequence and an LD promoter. To confer plant tolerance to herbicides that inhibit HPPD, a chimeric gene called pRPA-RD-2005 is constructed:
Il consiste à mettre la partie codante du gène de l'HPPD de P. fluorescens A32 sous le contrôle du promoteur de la petite sous unité de la RuBisCO isolé d'Helianthus annuus (US 5,559,024), du peptide de transit optimisé (OTP) (EP 508 909), lui même suivi de la région codante de l'HPPD de Pseudomonas fluorescens (WO 96/38567) à son tour suivie du terminateur nos dAgrobacterium tumefaciens. L'HPPD sera alors localisée dans le chloroplaste et le gène s'exprime essentiellement dans les tissus chlorophylliens.It consists in putting the coding part of the HPPD gene of P. fluorescens A32 under the control of the promoter of the small subunit of RuBisCO isolated from Helianthus annuus (US 5,559,024), of the optimized transit peptide (OTP) ( EP 508 909), itself followed by the coding region of the HPPD of Pseudomonas fluorescens (WO 96/38567) in turn followed by the terminator nos dAgrobacterium tumefaciens. HPPD will then be localized in the chloroplast and the gene is mainly expressed in chlorophyll tissues.
Le pRPA-RD-2005 est un vecteur binaire de type pRPA-BL-150Aα2 (EP 508 909) contenant une cassette d'expression de l'HPPD: promoteur de la petite sous unité de la ribulose discarboxylase-OTP-gène HPPD-terminateur nosPRPA-RD-2005 is a binary vector of pRPA-BL-150Aα2 type (EP 508 909) containing an HPPD expression cassette: promoter of the small subunit of ribulose discarboxylase-OTP-HPPD gene-terminator our
-Pour le construire, on a utilisé le pRPA-RD 2004 qui contient la cassette d'expression de l'HPPD-To build it, we used pRPA-RD 2004 which contains the HPPD expression cassette
Construction du pRPA-RD-2004 - pRD-207 est un pBluescript SK(-) (stratagène catalog#21 2206) contenant le gène de la nopaline synthase (terminateur nos). Le pRD-207 est utilisé comme vecteur de base pour la construction du pRPA-RD-2004Construction of pRPA-RD-2004 - pRD-207 is a pBluescript SK (-) (stratagene catalog # 21 2206) containing the gene for nopaline synthase (terminator nos). PRD-207 is used as the basic vector for the construction of pRPA-RD-2004
- pRD-208 contient la cassette OTP/HPPD:nos. Il est obtenu à partir du plasmide pRPA S, décrit dans WO 96/38567, digéré par Xbal/Clal, traitement à la polymérase type pfu. La cassette est introduite dans le pRD-207 ouvert par une digestion Sali, traitement klenow.- pRD-208 contains the OTP / HPPD cassette: nos. It is obtained from the plasmid pRPA S, described in WO 96/38567, digested with Xbal / ClaI, treatment with polymerase pfu type. The cassette is introduced into the open pRD-207 by a Sali digestion, klenow treatment.
- Le promoteur de la petite sous unité de la ribulose biscarboxylase d'Helianthus annuus (SSU HA; SEQ ID NO:l) provient du plasmide pRD-127 décrit dans WO
96/38567 digéré Ncol/Xbal est introduit dans le pRD-208. Cette construction constitue le pRPA-RD-2004- The promoter of the small subunit of the ribulose biscarboxylase from Helianthus annuus (SSU HA; SEQ ID NO: 1) comes from the plasmid pRD-127 described in WO 96/38567 digested Ncol / Xbal is introduced into pRD-208. This construction constitutes pRPA-RD-2004
Construction du pRD-2005 - Pour construire le pRPA-RD-2005, on ouvre le vecteur pRPA-BL-150Aα2 par les enzymes de restriction Xbal/HindIII auquel on insert la cassette d'expression de l'HPPD du pRPA-RD-2004 (décrit ci-dessus)par les même enzymes Il a donc comme structure:Construction of pRD-2005 - To construct pRPA-RD-2005, the vector pRPA-BL-150Aα2 is opened by the restriction enzymes Xbal / HindIII to which the HPPD expression cassette of pRPA-RD-2004 is inserted. (described above) by the same enzymes It therefore has the following structure:
Exemple 2: Transformation du tabac industriel PBD6.Example 2: Processing of PBD6 industrial tobacco.
Afin de déterminer l'efficacité du ce gène chimère de l'exemple 1, il a été transféré dans du tabac industriel PBD6 selon les procédures de transformation et de régénération déjà décrites dans la demande de brevet EP 508 909.In order to determine the efficiency of this chimeric gene of Example 1, it was transferred into industrial tobacco PBD6 according to the transformation and regeneration procedures already described in patent application EP 508 909.
1) Transformation: Le vecteur est introduit dans la souche non oncogène dAgrobacterium EHA 101 ou LBA 4404 (Hood et al, 1987) porteuse du cosmide pTVK 291(Komari et al, 1986). La technique de transformation est basée sur la procédure de Horsh R. et al. (1985) Science, 227, 1229-1231.1) Transformation: The vector is introduced into the non-oncogenic strain of Agrobacterium EHA 101 or LBA 4404 (Hood et al, 1987) carrying the cosmid pTVK 291 (Komari et al, 1986). The transformation technique is based on the procedure of Horsh R. et al. (1985) Science, 227, 1229-1231.
2) Régénération: La régénération du tabac PBD6 (provenance SEITA France) à partir d'expiants foliaires est réalisée sur un milieu de base Murashige et Skoog (MS) comprenant 30g/l de saccharose ainsi que 350 mg/1 de cefotaxime et 1 mg/1 du dicétonitrile dérivant de l'isoxaflutole ou 10 mg/1 de l-[4-(trifluorométhyl)-2-(méthylsulfonyl)phényl]-2-cyano- 3-(l-méthylcyclopropyl)-propan-l,3-dione, autre inhibiteur de l'HPPD. Les explants foliaires sont prélevés sur des plants en serre ou in vitro et transformés selon la technique des disques foliaires (Science 1985,Vol 227,p.1229- 1231) en trois étapes successives: la première comprend l'induction des pousses sur un milieu MS additionné de 30g/l de saccharose contenant 0.05mg/l d'acide naphtylacétique (ANA) et 2 mg/1 de benzylaminopurine (BAP) pendant 15 jours et 1 mg/1 d'isoxaflutole. Les pousses vertes formées au cours de cette étape sont ensuite développées par culture sur un milieu MS additionné de 30 g/1 de saccharose et 1 mg/1 d'isoxaflutole ou 10 mg/1 de l-[4- (trifluorométhyl)-2-(méthylsulfonyl)phényl]-2-cyano-3-(l-méthylcyclopropyl)-propan- 1,3-dione mais ne contenant pas d'hormone, pendant 10 jours. Puis on prélève des pousses développées et on les cultive sur un milieu d'enracinement MS à teneur moitié en sels, vitamines et sucres et 1 mg/1 d'isoxaflutole ou 10 mg/1 de l-[4-(trifluorométhyl)-
2-(méthylsulfonyl)phényl]-2-cyano-3-(l-méthylcyclopropyl)-propan-l ,3-dione et ne contenant pas d'hormone. Au bout d'environ 15 jours, les pousses enracinées sont passées en terre.2) Regeneration: The regeneration of PBD6 tobacco (from SEITA France) from leaf explants is carried out on a Murashige and Skoog (MS) base medium comprising 30g / l of sucrose as well as 350 mg / 1 of cefotaxime and 1 mg / 1 diketonitrile derived from isoxaflutole or 10 mg / 1 l- [4- (trifluoromethyl) -2- (methylsulfonyl) phenyl] -2-cyano- 3- (l-methylcyclopropyl) -propan-1, 3- dione, another HPPD inhibitor. The leaf explants are taken from plants in the greenhouse or in vitro and transformed using the leaf disc technique (Science 1985, Vol 227, p.1229-1231) in three successive stages: the first includes the induction of shoots on a medium MS supplemented with 30g / l of sucrose containing 0.05mg / l of naphthylacetic acid (ANA) and 2 mg / 1 of benzylaminopurine (BAP) for 15 days and 1 mg / 1 of isoxaflutole. The green shoots formed during this stage are then developed by culture on an MS medium supplemented with 30 g / 1 of sucrose and 1 mg / 1 of isoxaflutole or 10 mg / 1 of 1- [4- (trifluoromethyl) -2 - (methylsulfonyl) phenyl] -2-cyano-3- (1-methylcyclopropyl) -propan 1,3-dione but containing no hormone, for 10 days. Then take developed shoots and cultivate them on an MS rooting medium with half content of salts, vitamins and sugars and 1 mg / 1 of isoxaflutole or 10 mg / 1 of l- [4- (trifluoromethyl) - 2- (methylsulfonyl) phenyl] -2-cyano-3- (1-methylcyclopropyl) -propan-1, 3-dione and containing no hormone. After about 15 days, the rooted shoots are put in the ground.
Exemple 3: Tolérance du tabac transformé avec le gène chimère pRPA-RD-2005EXAMPLE 3 Tolerance of Tobacco Transformed with the Chimeric Gene pRPA-RD-2005
Au sortir de l'étape in-vitro de l'exemple 2, les plantules de tabac transformées ont été acclimatées à la serre (60% d'humidité relative; température: 20°C la nuit et 23 °C la jour) pendant trois semaines puis traitées au 4-[4-(trifiuorométhyl)-2- (méthylsulfonyl)benzoyl]-5-cyclopropylisoxazole (isoxafutole). Le tabac témoin, non transformé et traité à l'isoxafutole à la doses de 400 g/ha, développe en environ 72 heures des chloroses, qui s'intensifient pour évoluer vers des nécroses très prononcées en une semaine (couvrant environ 80% des feuilles terminales).At the end of the in vitro step of Example 2, the transformed tobacco seedlings were acclimated in the greenhouse (60% relative humidity; temperature: 20 ° C at night and 23 ° C during the day) for three weeks then treated with 4- [4- (trifiuoromethyl) -2- (methylsulfonyl) benzoyl] -5-cyclopropylisoxazole (isoxafutole). The control tobacco, unprocessed and treated with isoxafutole at the doses of 400 g / ha, develops in about 72 hours chloroses, which intensify to progress to very pronounced necrosis in a week (covering about 80% of the leaves final).
Le tabac transformé correspondant, ci-après tabac 2005, qui surexprime l'HPPD de R. fluorescens, est très bien protégé contre un traitement à l'isoxafutole à la dose de 400 g/ha.The corresponding processed tobacco, hereafter 2005 tobacco, which overexpresses the HPPD of R. fluorescens, is very well protected against treatment with isoxafutole at a dose of 400 g / ha.
Exemple 4; Tolérance en serre du tabac 2005Example 4; Tobacco greenhouse tolerance 2005
Dans cet exemple on compare la tolérance en serre du tabac 2005 à celle du tabac COI 1, transformé avec le gène chimère pRPA-V décrit dans la demande de brevet WO 96/38567 et à celle du tabac non transformé. Description des essais tabac.In this example, the greenhouse tolerance of 2005 tobacco is compared to that of COI 1 tobacco, transformed with the pRPA-V chimeric gene described in patent application WO 96/38567 and that of unprocessed tobacco. Description of tobacco trials.
Des graines de chacun de ces types de tabac sont semées en terrine et le jour même un traitement à des doses d'isoxaflutole de 0/30/200/400 g/ha appliqué donc en pré-emergence sont effectués. Constructions testées : Coll: Double histone~TEV~OTP~HPPD Pseudomonas -Nos 2005: SSU-OTP-HPPD Pseudomonas -Nos PBD6: non transformé. BilanSeeds of each of these types of tobacco are sown in the ground and the same day a treatment with doses of isoxaflutole of 0/30/200/400 g / ha applied therefore in pre-emergence are carried out. Constructions tested: Coll: Double histone ~ TEV ~ OTP ~ HPPD Pseudomonas -Nos 2005: SSU-OTP-HPPD Pseudomonas -Nos PBD6: not transformed. Balance sheet
Pour le tabac PBD6, la germination se fait normalement à 0 g /ha et aucune germination n'a lieu dès la dose de 30 g/ ha. Si par hasard une plantule arrive à levée elle est blanche et meurt très rapidement.For PBD6 tobacco, germination is normally done at 0 g / ha and no germination takes place at the dose of 30 g / ha. If by chance a seedling emerges it is white and dies very quickly.
Pour des graines issues d'un tabac COI 1, la levée est normale et sans symptômes de phytotoxicité pour les doses allant de 0 à 200 g/ ha. A 400 g/ha, les graines germent, la levée a lieu normalement mais il y a un retard de croissance ou " stunting ", assez net par rapport aux plantes non traitées.
Pour des graines issues d'un tabac 2005, la levée est normale et sans symptômes de phytotoxicité pour les doses allant de 0 à 400 g/ ha. A aucune des trois doses d'isoxaflutole, en serre, ne provoque de symptôme de phytotoxicité ou de " stunting ".For seeds from a COI 1 tobacco, emergence is normal and without phytotoxicity symptoms for doses ranging from 0 to 200 g / ha. At 400 g / ha, the seeds germinate, emergence takes place normally but there is a growth retardation or "stunting", quite clear compared to untreated plants. For 2005 tobacco seeds, emergence is normal and without phytotoxicity symptoms for doses ranging from 0 to 400 g / ha. At none of the three doses of isoxaflutole, in the greenhouse, does it cause symptoms of phytotoxicity or "stunting".
Exemple 5: Tolérance au champ du tabac du tabac 2005Example 5: Tolerance in the field of tobacco tobacco 2005
Les essais comparatifs de tolérance de l'exemple 4 sont reproduits au champ pour les même tabacs 2005, COI 1 et PBD6.The comparative tolerance tests of Example 4 are reproduced in the field for the same tobacco 2005, COI 1 and PBD6.
Description des essais tabac.Description of tobacco trials.
Des jeunes plantes issues de semis ont été repiquées individuellement en minimottes pour être une deuxième fois repiquées au champ. Le traitement de post a été réalisé une semaine après à des doses d'isoxaflutole de 0/100/200/300/400/500/600 g/ha.Young plants from seedlings were transplanted individually in miniballs to be transplanted a second time in the field. The post treatment was carried out one week later at doses of isoxaflutole of 0/100/200/300/400/500/600 g / ha.
BilanBalance sheet
Le diagramme ci-dessous comparant les tabacs de type COU et 2005, montre clairement que le promoteur SSU confère une meilleure tolérance que le double histone associé au TEV " enhancer ".The diagram below comparing tobacco of the COU type and 2005, clearly shows that the promoter SSU confers better tolerance than the double histone associated with the TEV "enhancer".
2005 COU [J Wild type2005 COU [J Wild type
Ainsi, 13 jours après traitement à 400g/ha, on observe 15 % de phytotoxicité sur les tabacs COI 1 alors que les tabacs 2005 ne présentent pas ou peu de phytotoxicité.Thus, 13 days after treatment at 400 g / ha, 15% of phytotoxicity is observed on COI 1 tobacco, while 2005 tobacco has little or no phytotoxicity.
De même, 20 jours après un traitement à 400g/ha, on observe plus de 20 % de phytotoxicité sur les tabacs COI 1 alors que les tabacs 2005 présentent moins de 10% de phytotoxicité.Likewise, 20 days after a 400g / ha treatment, more than 20% phytotoxicity is observed on COI 1 tobacco, while 2005 tobacco has less than 10% phytotoxicity.
Dans les conditions de culture en champs, les plantes transformées avec le gène pRPA-RD-2005 comprenant le promoteur SSU ont une meilleure tolérance aux inhibiteurs de l'HPPD que celles transformées avec le gène pRPA-V comprenant le promoteur double histone combiné au TEV " enhancer " de l'état de la technique.
Under field culture conditions, plants transformed with the pRPA-RD-2005 gene comprising the SSU promoter have better tolerance to HPPD inhibitors than those transformed with the pRPA-V gene comprising the double histone promoter combined with TEV "enhancer" of the state of the art.
Claims
1. Gène chimère comprenant au moins un gène chimère élémentaire conprenant, dans le sens de la transcription, des éléments de régulation en 5' nécessaires à sa transcription dans les plantes, au moins une partie codante hétérologue comprenant une séquence codante codant pour une enzyme conférant aux plantes la tolérance aux herbicides inhibiteurs de l'HPPD et au moins une séquence de régulation terminatrice ou de polyadenylation, caractérisé en ce que les éléments de régulation en 5' assurent la transcription du gène chimère élémentaire au niveau des tissus chlorophylliens. 1. Chimeric gene comprising at least one elementary chimeric gene comprising, in the direction of transcription, 5' regulatory elements necessary for its transcription in plants, at least one heterologous coding part comprising a coding sequence coding for an enzyme conferring to plants tolerance to HPPD-inhibiting herbicides and at least one terminator or polyadenylation regulatory sequence, characterized in that the 5' regulatory elements ensure transcription of the elementary chimeric gene at the level of chlorophyll tissues.
2. Gène chimère selon la revendication 1, caractérisé en ce que les dits éléments de régulations en 5' comprenent au moins une séquence de régulation promotrice de type promoteur LD.2. Chimeric gene according to claim 1, characterized in that said 5' regulatory elements comprise at least one promoter regulatory sequence of the LD promoter type.
3. Gène chimère selon la revendication 2, caractérisé en ce que le promoteur LD est d'origine bactérienne, virale ou végétale. 3. Chimeric gene according to claim 2, characterized in that the LD promoter is of bacterial, viral or plant origin.
4. Gène chimère selon la revendication 3, caractérisé en ce que le promoteur LD est choisi parmi une séquence fonctionnelle du promoteur d'un gène de la petite sous unité de la RuBisCO de plante, de la light-harvesting chlrorophyll a/b binding protéine (LHCP), de la plastocyanine (pet E) et de la phénylalanine ammonia lyase (pal). 4. Chimeric gene according to claim 3, characterized in that the LD promoter is chosen from a functional sequence of the promoter of a gene of the small subunit of plant RuBisCO, of the light-harvesting chlorophyll a/b binding protein (LHCP), plastocyanin (pet E) and phenylalanine ammonia lyase (pal).
5. Gène chimère selon la revendication 4, caractérisé en ce que le promoteur LD comprend un fragment fonctionnel du promoteur de la petite sous unité de la RuBisCO d'une plante.5. Chimeric gene according to claim 4, characterized in that the LD promoter comprises a functional fragment of the promoter of the small subunit of RuBisCO of a plant.
6. Gène chimère selon la revendication 5, caractérisé en ce que le promoteur LD comprend un fragment fonctionnel fonctionnel du promoteur de la petite sous unité de la RuBisCO d'Helianthus annuus .6. Chimeric gene according to claim 5, characterized in that the LD promoter comprises a functional fragment of the promoter of the small subunit of RuBisCO of Helianthus annuus.
1. Gène chimère selon la revendication 6, caractérisé en ce que le promoteur LD comprend la séquence d'ADN représentée par l'identificateur de séquence n° 1 (SEQ ID NO:l) ou une séquence homologue de ladite séquence.1. Chimeric gene according to claim 6, characterized in that the LD promoter comprises the DNA sequence represented by sequence identifier No. 1 (SEQ ID NO:l) or a homologous sequence of said sequence.
8. Gène chimère selon l'une des revendications 1 à 7, caractérisé en ce que le gène chimère élémentaire comprend en association avec les éléments de régulation en8. Chimeric gene according to one of claims 1 to 7, characterized in that the elementary chimeric gene comprises in association with the regulatory elements in
5' au moins une séquence de régulation, située entre le promoteur et la séquence codante, choisie parmi les activateurs de trancription et/ou les séquences codant pour des peptides de transit.5' at least one regulatory sequence, located between the promoter and the coding sequence, chosen from transcription activators and/or sequences coding for transit peptides.
9. Gène chimère selon l'une des revendications 1 à 8, caractérisé en ce que la séquence codant pour une enzyme conférant aux plantes la tolérance aux herbicides inhibiteurs de l'HPPD est choisie parmi les séquences codant pour une HPPD d'origine bactérienne ou d'origine végétale.
9. Chimeric gene according to one of claims 1 to 8, characterized in that the sequence coding for an enzyme conferring on plants tolerance to HPPD-inhibiting herbicides is chosen from the sequences coding for an HPPD of bacterial origin or of plant origin.
10. Vecteur de clonage ou d'expression pour la transformation d'une cellule végétale ou d'une plante monocotylédone ou dicotyledone, caractérisé en ce qu'il comprend outre le gène chimère selon l'une des revendications 1 à 9, au moins une origine de réplication. 10. Cloning or expression vector for the transformation of a plant cell or a monocotyledonous or dicotyledonous plant, characterized in that it comprises, in addition to the chimeric gene according to one of claims 1 to 9, at least one origin of replication.
11. Vecteur selon la revendication 10, carctérisé en ce qu'il est un plasmide.11. Vector according to claim 10, characterized in that it is a plasmid.
12. Procédé de transformation des cellules végétales, caractérisé en ce que l'on intègre dans ladite cellule végétale au moins un gène chimère selon l'une des revendications 1 à 9.12. Process for transforming plant cells, characterized in that at least one chimeric gene according to one of claims 1 to 9 is integrated into said plant cell.
13. Cellule végétale transformée tolérante aux herbicides ayant pour cible l'HPPD, caratérisée en ce qu'elle comprend au moins un gène chimère selon l'une des revendications 1 à 9.13. Transformed plant cell tolerant to herbicides targeting HPPD, characterized in that it comprises at least one chimeric gene according to one of claims 1 to 9.
14. Plante transformée tolérante aux herbicides ayant pour cible l'HPPD, caractérisée en ce qu'elle comprend des cellules transformées selon la revendication 13.14. Transformed plant tolerant to herbicides targeting HPPD, characterized in that it comprises transformed cells according to claim 13.
15. Plante transformée selon la revendication 14, caractérisée en ce qu'elle est régénérée régénérée à partir des cellules transforméesselon la revendication 14.15. Transformed plant according to claim 14, characterized in that it is regenerated regenerated from the cells transformed according to claim 14.
16. Plante transformée tolérante aux herbicides ayant pour cible l'HPPD, caractérisée en ce qu'elle est issue de la culture et/ou du croisement des plantes régénérées selon la revendication 15.16. Transformed plant tolerant to herbicides targeting HPPD, characterized in that it comes from the cultivation and/or crossing of regenerated plants according to claim 15.
17. Graines de plantes transformées selon l'une des revendications 14 à 16. 17. Transformed plant seeds according to one of claims 14 to 16.
18. Procédé de contrôle des mauvaises herbes dans une surface d'un champ comprenant des graines ou des plantes transformées selon l'une des revendications 14 à 17, caractérisé en ce que l'on applique sur la dite surface du champ une dose toxique pour les dites mauvaises herbes d'un herbicide ayant pour cible l'HPPD, sans toutefois affecter de manière substantielle les dites graines ou plantes transformées. 18. Method for controlling weeds in a surface of a field comprising seeds or transformed plants according to one of claims 14 to 17, characterized in that a toxic dose is applied to said surface of the field to said weeds of a herbicide targeting HPPD, without however substantially affecting said seeds or transformed plants.
19. Procédé de culture des plantes transformées selon l'une des revendications 14 à 16, caractérisé en ce qu'il consiste à planter les graines selon la revendication 17 dans une surface d'un champ approprié pour la culture des dites plantes transformées, à appliquer sur la dite surface du dit champ une dose toxique pour les mauvaises herbes d'un herbicide ayant pour cible l'HPPD en cas de présence de mauvaises herbes, sans affecter de manière substantielle les dites graines ou les dites plantes transformées, puis à récolter les plantes transformées cultivées lorsquelles arrivent à la maturité souhaitée et éventuellement à séparer les graines des plantes transformées récoltées.19. Method for cultivating transformed plants according to one of claims 14 to 16, characterized in that it consists of planting the seeds according to claim 17 in an area of a field suitable for the cultivation of said transformed plants, at applying to said surface of said field a dose toxic to weeds of a herbicide having as its target HPPD in the event of the presence of weeds, without substantially affecting said seeds or said transformed plants, then harvesting the transformed plants cultivated when they reach the desired maturity and possibly to separate the seeds from the transformed plants harvested.
20. Procédé selon l'une des revendications 18 ou 19, caractérisé en ce que l'on applique l'herbicide ayant pour cible l'HPPD en présemis et/ou en prélevée et/ou en postlevée de la culture.
20. Method according to one of claims 18 or 19, characterized in that the herbicide whose target is HPPD is applied in pre-sowing and/or pre-emergence and/or post-emergence of the crop.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9714591 | 1997-11-17 | ||
FR9714591A FR2771104B1 (en) | 1997-11-17 | 1997-11-17 | CHIMERIC GENE HAVING A DEPENDENT LIGHT PROMOTER GIVING TOLERANCE TO HPPD INHIBITORS |
PCT/FR1998/002414 WO1999025842A1 (en) | 1997-11-17 | 1998-11-13 | Chimera gene having a light dependent promoter providing tolerance to hppd inhibitors |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1032681A1 true EP1032681A1 (en) | 2000-09-06 |
Family
ID=9513607
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP98954565A Withdrawn EP1032681A1 (en) | 1997-11-17 | 1998-11-13 | Chimera gene having a light dependent promoter providing tolerance to hppd inhibitors |
Country Status (11)
Country | Link |
---|---|
EP (1) | EP1032681A1 (en) |
AR (1) | AR017630A1 (en) |
AU (1) | AU747634B2 (en) |
BR (1) | BR9815628A (en) |
CA (1) | CA2309880A1 (en) |
CO (1) | CO4950591A1 (en) |
FR (1) | FR2771104B1 (en) |
ID (1) | ID21668A (en) |
MA (1) | MA24818A1 (en) |
WO (1) | WO1999025842A1 (en) |
ZA (1) | ZA9810498B (en) |
Families Citing this family (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2815969B1 (en) | 2000-10-30 | 2004-12-10 | Aventis Cropscience Sa | TOLERANT PLANTS WITH HERBICIDES BY METABOLIC BYPASS |
CN102741415A (en) | 2010-02-02 | 2012-10-17 | 拜尔农科股份公司 | Soybean transformation using hppd inhibitors as selection agents |
WO2013026740A2 (en) | 2011-08-22 | 2013-02-28 | Bayer Cropscience Nv | Methods and means to modify a plant genome |
AU2014211570A1 (en) | 2013-01-29 | 2015-07-23 | The University Court Of The University Of Glasgow | Methods and means for increasing stress tolerance and biomass in plants |
CA2957921A1 (en) | 2014-07-22 | 2016-01-28 | Nmc, Inc. | Improved carbon fixation systems in plants and algae |
WO2016050512A1 (en) | 2014-10-03 | 2016-04-07 | Bayer Cropscience Nv | Methods and means for increasing stress tolerance and biomass in plants |
US11666012B2 (en) | 2016-03-16 | 2023-06-06 | Basf Se | Plants comprising wheat G-type cytoplasmic male sterility restorer genes, molecular markers and uses thereof |
CA3017995A1 (en) | 2016-03-16 | 2017-09-21 | Antje ROHDE | Plants comprising wheat g-type cytoplasmic male sterility restorer genes, molecular markers and uses thereof |
EP3429335A1 (en) | 2016-03-16 | 2019-01-23 | Basf Se | Plants comprising wheat g-type cytoplasmic male sterility restorer genes, molecular markers and uses thereof |
CN109715811A (en) | 2016-07-18 | 2019-05-03 | 巴斯夫欧洲公司 | Include wheat G type cytoplasmic male sterilty restoring gene, the plant of molecular marker and application thereof |
AU2017300617A1 (en) | 2016-07-18 | 2019-02-07 | Basf Se | Plants comprising wheat G-type cytoplasmic male sterility restorer genes, molecular markers and uses thereof |
US20190225974A1 (en) | 2016-09-23 | 2019-07-25 | BASF Agricultural Solutions Seed US LLC | Targeted genome optimization in plants |
BR112020014168A2 (en) | 2018-01-12 | 2020-12-08 | Basf Se | PROTEIN, ISOLATED NUCLEIC ACID, RECOMBINANT GENE, VECTOR, HOSTING CELL, PLANT, PART OF PLANT OR WHEAT SEED, METHODS OF PRODUCTION, WHEAT PRODUCT, FLOUR, WHOLE MUSHROOM, STARCH, MUSHROOMS AND MUSHROOMS. AND / OR SELECTING A WHEAT PLANT |
CA3099621A1 (en) | 2018-05-25 | 2019-11-28 | Basf Se | Plants comprising wheat g-type cytoplasmic male sterility restorer genes and uses thereof |
EA202092647A1 (en) | 2018-05-25 | 2021-07-08 | Басф Се | PLANTS CONTAINING RESTORER GENES FOR CYTOPLASMATIC MALE STERILITY G-TYPE WHEAT, AND THEIR APPLICATION |
WO2019224359A1 (en) | 2018-05-25 | 2019-11-28 | Basf Se | Plants comprising wheat g-type cytoplasmic male sterility restorer genes and uses thereof |
WO2019234231A1 (en) | 2018-06-08 | 2019-12-12 | Basf Se | Plants comprising wheat g-type cytoplasmic male sterility restorer genes and uses thereof |
WO2023118541A1 (en) | 2021-12-22 | 2023-06-29 | Basf Se | Regulatory nucleic acid molecules for modifying gene expression in cereal plants |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2629098B1 (en) * | 1988-03-23 | 1990-08-10 | Rhone Poulenc Agrochimie | CHEMICAL GENE OF HERBICIDE RESISTANCE |
FR2734842B1 (en) * | 1995-06-02 | 1998-02-27 | Rhone Poulenc Agrochimie | DNA SEQUENCE OF A HYDROXY-PHENYL PYRUVATE DIOXYGENASE GENE AND OBTAINING PLANTS CONTAINING A HYDROXY-PHENYL PYRUVATE DIOXYGENASE GENE, TOLERANT TO CERTAIN HERBICIDES |
US6087563A (en) * | 1996-01-29 | 2000-07-11 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Cloned arabidopsis p-hydroxyphenyl pyruvic acid dioxygenase DNA |
FR2751347B1 (en) * | 1996-07-16 | 2001-12-07 | Rhone Poulenc Agrochimie | CHIMERIC GENE WITH MULTIPLE HERBICIDE TOLERANCE GENES, PLANT CELL AND PLANT TOLERANT WITH MULTIPLE HERBICIDES |
-
1997
- 1997-11-17 FR FR9714591A patent/FR2771104B1/en not_active Expired - Fee Related
-
1998
- 1998-11-13 AU AU11628/99A patent/AU747634B2/en not_active Ceased
- 1998-11-13 BR BR9815628-4A patent/BR9815628A/en not_active IP Right Cessation
- 1998-11-13 WO PCT/FR1998/002414 patent/WO1999025842A1/en not_active Application Discontinuation
- 1998-11-13 CA CA002309880A patent/CA2309880A1/en not_active Abandoned
- 1998-11-13 EP EP98954565A patent/EP1032681A1/en not_active Withdrawn
- 1998-11-16 ID IDP981498A patent/ID21668A/en unknown
- 1998-11-16 AR ARP980105795 patent/AR017630A1/en unknown
- 1998-11-17 ZA ZA9810498A patent/ZA9810498B/en unknown
- 1998-11-17 MA MA25353A patent/MA24818A1/en unknown
- 1998-11-17 CO CO98067540A patent/CO4950591A1/en unknown
Non-Patent Citations (1)
Title |
---|
See references of WO9925842A1 * |
Also Published As
Publication number | Publication date |
---|---|
AU747634B2 (en) | 2002-05-16 |
MA24818A1 (en) | 1999-12-31 |
FR2771104B1 (en) | 2000-12-08 |
ZA9810498B (en) | 1999-05-24 |
ID21668A (en) | 1999-07-08 |
FR2771104A1 (en) | 1999-05-21 |
WO1999025842A1 (en) | 1999-05-27 |
AU1162899A (en) | 1999-06-07 |
AR017630A1 (en) | 2001-09-12 |
CA2309880A1 (en) | 1999-05-27 |
BR9815628A (en) | 2000-10-24 |
CO4950591A1 (en) | 2000-09-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0633317B1 (en) | Isolated DNA sequence able to function as a termination region in chimeric genes for plant transformation | |
CA2261094C (en) | Chimera gene with several herbicide resistant genes, plant cell and plant resistant to several herbicides | |
AU2016399292B2 (en) | Herbicide tolerant protein, encoding gene and use thereof | |
EP1032681A1 (en) | Chimera gene having a light dependent promoter providing tolerance to hppd inhibitors | |
WO1996038567A2 (en) | Dna sequence of a gene of hydroxy-phenyl pyruvate dioxygenase and production of plants containing a gene of hydroxy-phenyl pyruvate dioxygenase and which are tolerant to certain herbicides | |
CA2315677C (en) | Maize h3c4 promoter associated with first rice actin intron, chimeric gene containing it and transformed plant | |
EP4032976A1 (en) | Mutant hydroxyphenylpyruvate dioxygenase polypeptide, encoding gene thereof and use thereof | |
FR2961375A1 (en) | OVERPRODUCTION OF JASMONIC ACID IN TRANSGENIC PLANTS | |
EP3110832B1 (en) | Plants with increased yield and method for producing said plants | |
WO2023206126A1 (en) | Use of protoporphyrinogen oxidase | |
EP4339290A1 (en) | Use of protoporphyrinogen oxidase | |
KR100480479B1 (en) | Method for improving herbicide resistance of crops by expressing protoporphyrinogen oxidase both in chloroplasts and mitochondria and method for selecting transgenic cell lines using the said gene as herbicide-resistant selection marker | |
CA3236161A1 (en) | Mutated hydroxyphenylpyruvate dioxygenase polypeptide, and coding gene and use thereof | |
FR2796954A1 (en) | Fusion protein of hydroxyphenylpyruvate dioxygenase, useful for imparting herbicide resistance to plants, includes signal for non-cytoplasmic or non-plast localization | |
EP1315801A1 (en) | Hydroxy-phenyl pyruvate dioxygenase fused with a signal peptide, dna sequence and use for obtaining plants containing herbicide-tolerant plants | |
FR2796394A1 (en) | New SGS3 gene from Arabidopsis thaliana, useful for increasing virus resistance in plants and, when inhibited, for increasing transgene expression | |
WO2001055407A1 (en) | Novel sgs2 plant gene and use thereof | |
FR2796395A1 (en) | NEW PLANT SGS3 GENE AND ITS USE |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20000508 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU NL PT SE |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: BAYER CROPSCIENCE S.A. |
|
17Q | First examination report despatched |
Effective date: 20030624 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20050813 |