WO2022237541A1 - 原卟啉原氧化酶的用途 - Google Patents

原卟啉原氧化酶的用途 Download PDF

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WO2022237541A1
WO2022237541A1 PCT/CN2022/089519 CN2022089519W WO2022237541A1 WO 2022237541 A1 WO2022237541 A1 WO 2022237541A1 CN 2022089519 W CN2022089519 W CN 2022089519W WO 2022237541 A1 WO2022237541 A1 WO 2022237541A1
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Prior art keywords
ppo inhibitor
seq
inhibitor herbicides
protoporphyrinogen oxidase
plants
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PCT/CN2022/089519
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English (en)
French (fr)
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肖翔
宋庆芳
陶青
于彩虹
鲍晓明
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北京大北农生物技术有限公司
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Application filed by 北京大北农生物技术有限公司 filed Critical 北京大北农生物技术有限公司
Priority to CN202280014835.5A priority Critical patent/CN116917486A/zh
Priority to KR1020237037180A priority patent/KR20230162085A/ko
Priority to AU2022275035A priority patent/AU2022275035A1/en
Priority to CA3216814A priority patent/CA3216814A1/en
Priority to JP2023568593A priority patent/JP2024516323A/ja
Priority to IL306115A priority patent/IL306115A/en
Priority to BR112023018976A priority patent/BR112023018976A2/pt
Priority to EP22806510.8A priority patent/EP4339290A1/en
Publication of WO2022237541A1 publication Critical patent/WO2022237541A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
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    • C12Y103/03004—Protoporphyrinogen oxidase (1.3.3.4)

Definitions

  • the present invention relates to the use of a protoporphyrinogen oxidase, in particular to a method and use of a protoporphyrinogen oxidase derived from prokaryotic organisms for endowing plants with tolerance to PPO inhibitor herbicides.
  • the porphyrin biosynthetic pathway is used to synthesize chlorophyll and heme, which play important roles in plant metabolism, and this pathway occurs in chloroplasts.
  • protoporphyrinogen oxidase PPO for short catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX.
  • protoporphyrin IX binds to magnesium via magnesium chelatase to synthesize chlorophyll, or via ferrochelatase to iron to synthesize heme.
  • Herbicides that work by inhibiting PPO include diphenyl ether PPO inhibitor herbicides, oxadiazolone PPO inhibitor herbicides, N-phenylphthalimide PPO inhibitor herbicides, oxazolone PPO inhibitor herbicides, phenylpyrazole PPO inhibitor herbicides, uracil PPO inhibitor herbicides, thiadiazole PPO inhibitor herbicides, triazolone PPO inhibitor herbicides, triazines Ketone PPO inhibitor herbicides and other types of PPO inhibitor herbicides.
  • PPO inhibitors inhibit the enzymatic activity of PPO, resulting in the inhibition of the synthesis of chlorophyll and heme, and the accumulation of the substrate protoporphyrinogen IX, which is rapidly exported from the chloroplast to the cytoplasm , protoporphyrinogen IX in the cytoplasm is converted to protoporphyrin IX under non-enzymatic reactions and further generates highly reactive singlet oxygen ( 1 O 2 ) in the presence of light and oxygen molecules, which can damage cell membranes and Rapidly leads to the death of plant cells.
  • highly reactive singlet oxygen 1 O 2
  • the method for providing PPO inhibitor herbicide-tolerant plants mainly includes: 1) Detoxifying the herbicide by using an enzyme that can convert the herbicide or its active metabolite into a non-toxic product. 2) Overexpression of sensitive PPOs such that, in view of the kinetic constants of this enzyme, a sufficient amount of the target enzyme relative to the herbicide is produced in the plant, so that despite the presence of a PPO inhibitor herbicide, these sensitive PPOs interact with the PPO inhibitor The herbicide is sufficiently active to have sufficient functional enzymes available. 3) To provide a functional PPO which is less sensitive to herbicides or their active metabolites, but which retains the property of catalyzing the oxidation of protoporphyrinogen IX to protoporphyrin IX.
  • a given functional PPO may provide a useful level of tolerance to some PPO inhibitor herbicides
  • the same functional PPO may not be sufficient to provide a commercial level of resistance to a tolerance to a different, more desirable PPO inhibitor herbicide; for example, PPO inhibitor herbicides may differ in the range of weeds they control, their cost of manufacture, and their environmental friendliness. Therefore, new methods for conferring tolerance to PPO inhibitor herbicides to different crops and crop varieties are needed.
  • the object of the present invention is to provide a kind of purposes of protoporphyrinogen oxidase, and described protoporphyrinogen oxidase originates from prokaryote, and transfers the polynucleotide sequence of encoding protoporphyrinogen oxidase described in the present invention Plants have good tolerance to PPO inhibitor herbicides.
  • the present invention provides a method of controlling weeds, comprising applying a herbicide containing an effective dose of a PPO inhibitor to a field where at least one transgenic plant contains in its genome a coded A polynucleotide sequence of protox, said transgenic plant having attenuated plant damage and/or having increased plant yield compared to other plants not having a polynucleotide sequence encoding protox, wherein the protoporphyrinogen oxidase has at least 88% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • the protoporphyrinogen oxidase is selected from the amino acid sequence of the group consisting of SEQ ID NO: 1-14;
  • the transgenic plants include monocotyledonous plants and dicotyledonous plants; more preferably, the transgenic plants are oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa , soybean, chickpea, peanut, sugar beet, cucumber, cotton, rapeseed, potato, tomato or Arabidopsis; further preferably, the transgenic plant is a glyphosate-tolerant plant, and the weed is glyphosate resistant weeds;
  • the PPO inhibitor herbicides include diphenyl ether PPO inhibitor herbicides, oxadiazolone PPO inhibitor herbicides, N-phenylphthalimide imine PPO inhibitor herbicides, oxazoline Ketone PPO inhibitor herbicides, phenylpyrazole PPO inhibitor herbicides, uracil PPO inhibitor herbicides, thiadiazole PPO inhibitor herbicides, triazolone PPO inhibitor herbicides and/or or triazinone PPO inhibitor herbicides;
  • the PPO inhibitor herbicides include oxyfluorfen, saflufenacil, sulfentrazone and/or oxyflufen.
  • polynucleotide sequence of the protoporphyrinogen oxidase has:
  • the transgenic plant further comprises at least one second polynucleotide encoding a second herbicide tolerance protein different from the polynucleotide sequence encoding the protoporphyrinogen oxidase.
  • the second polynucleotide encodes a selectable marker protein, a synthetically active protein, a catabolic active protein, an anti-biotic stress protein, an anti-abiotic stress protein, a male sterility protein, a protein affecting plant yield and/or a protein affecting plant quality .
  • the second polynucleotide encodes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, Glufosinate-ammonium acetyltransferase, alpha-ketoglutarate-dependent dioxygenase, dicamba monooxygenase, 4-hydroxyphenylpyruvate dioxygenase, acetolactate synthase, and/or cytochrome proteins.
  • the herbicides containing an effective dose of PPO inhibitors also include glyphosate herbicides, glufosinate-ammonium herbicides, auxin herbicides, gramineous herbicides, pre-emergence selective herbicides and/or Post-emergence selective herbicide.
  • the present invention also provides a planting combination for controlling the growth of weeds, comprising a PPO inhibitor herbicide and at least one transgenic plant, applying the PPO inhibitor herbicide containing an effective dose to the presence of the A field of at least one transgenic plant comprising in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, the transgenic plant being separated from other plants that do not have a polynucleotide encoding protoporphyrinogen oxidase Plants having reduced plant damage and/or having increased plant yield compared to plants of the sequence wherein said protoporphyrinogen oxidase has at least 88% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 sex;
  • said protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • the protoporphyrinogen oxidase is selected from the amino acid sequence of the group consisting of SEQ ID NO: 1-14;
  • the transgenic plants include monocotyledonous plants and dicotyledonous plants; more preferably, the transgenic plants are oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa , soybean, chickpea, peanut, sugar beet, cucumber, cotton, rapeseed, potato, tomato or Arabidopsis; further preferably, the transgenic plant is a glyphosate-tolerant plant, and the weed is glyphosate resistant weeds;
  • the PPO inhibitor herbicides include diphenyl ether PPO inhibitor herbicides, oxadiazolone PPO inhibitor herbicides, N-phenylphthalimide imine PPO inhibitor herbicides, oxazoline Ketone PPO inhibitor herbicides, phenylpyrazole PPO inhibitor herbicides, uracil PPO inhibitor herbicides, thiadiazole PPO inhibitor herbicides, triazolone PPO inhibitor herbicides and/or or triazinone PPO inhibitor herbicides;
  • the PPO inhibitor herbicides include oxyfluorfen, saflufenacil, sulfentrazone and/or oxyflufen.
  • polynucleotide sequence of the protoporphyrinogen oxidase has:
  • the transgenic plant further comprises at least one second polynucleotide encoding a second herbicide tolerance protein different from the polynucleotide sequence encoding the protoporphyrinogen oxidase.
  • the second polynucleotide encodes a selectable marker protein, a synthetically active protein, a catabolic active protein, an anti-biotic stress protein, an anti-abiotic stress protein, a male sterility protein, a protein affecting plant yield and/or a protein affecting plant quality .
  • the second polynucleotide encodes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, Glufosinate-ammonium acetyltransferase, alpha-ketoglutarate-dependent dioxygenase, dicamba monooxygenase, 4-hydroxyphenylpyruvate dioxygenase, acetolactate synthase, and/or cytochrome proteins.
  • the herbicides containing an effective dose of PPO inhibitors also include glyphosate herbicides, glufosinate-ammonium herbicides, auxin herbicides, gramineous herbicides, pre-emergence selective herbicides and/or Post-emergence selective herbicide.
  • the present invention also provides a method for producing plants tolerant to PPO inhibitor herbicides, comprising introducing a polynucleotide sequence encoding protoporphyrinogen oxidase into the genome of the plant, when containing an effective dose of PPO Inhibitor herbicides are applied to fields where at least said plants are present which have reduced plant damage and/or have increased Plant yield, wherein the protoporphyrinogen oxidase has at least 88% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • the protoporphyrinogen oxidase is selected from the amino acid sequence of the group consisting of SEQ ID NO: 1-14;
  • the introduced method includes genetic transformation method, genome editing method or gene mutation method
  • the plants include monocots and dicots; more preferably, the plants are oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis;
  • the PPO inhibitor herbicides include diphenyl ether PPO inhibitor herbicides, oxadiazolone PPO inhibitor herbicides, N-phenylphthalimide imine PPO inhibitor herbicides, oxazoline Ketone PPO inhibitor herbicides, phenylpyrazole PPO inhibitor herbicides, uracil PPO inhibitor herbicides, thiadiazole PPO inhibitor herbicides, triazolone PPO inhibitor herbicides and/or or triazinone PPO inhibitor herbicides;
  • the PPO inhibitor herbicides include oxyfluorfen, saflufenacil, sulfentrazone and/or oxyflufen.
  • the present invention also provides a method for cultivating plants tolerant to PPO inhibitor herbicides, comprising:
  • Planting at least one plant propagule the genome of the plant propagule includes a polynucleotide sequence encoding protoporphyrinogen oxidase, and the protoporphyrinogen oxidase is selected from the group consisting of SEQ ID NO: 1-14
  • the amino acid sequence of has at least 88% sequence identity;
  • a herbicide comprising an effective amount of a PPO inhibitor to a field comprising at least said plants, harvested with reduced plant damage and/or compared to other plants not having a polynucleotide sequence encoding protoporphyrinogen oxidase Plants with increased plant yield;
  • said protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • the protoporphyrinogen oxidase is selected from the amino acid sequence of the group consisting of SEQ ID NO: 1-14;
  • the plants include monocots and dicots; more preferably, the plants are oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis;
  • the PPO inhibitor herbicides include diphenyl ether PPO inhibitor herbicides, oxadiazolone PPO inhibitor herbicides, N-phenylphthalimide imine PPO inhibitor herbicides, oxazoline Ketone PPO inhibitor herbicides, phenylpyrazole PPO inhibitor herbicides, uracil PPO inhibitor herbicides, thiadiazole PPO inhibitor herbicides, triazolone PPO inhibitor herbicides and/or or triazinone PPO inhibitor herbicides;
  • the PPO inhibitor herbicides include oxyfluorfen, saflufenacil, sulfentrazone and/or oxyflufen.
  • the present invention also provides a method for protecting plants from damage caused by PPO inhibitor herbicides or endowing plants with tolerance to PPO inhibitor herbicides, comprising: The herbicide is applied to a field in the presence of at least one transgenic plant comprising in its genome a polynucleotide sequence encoding protoporphyrinogen oxidase, which is identical to other plants that do not have an encoding protoporphyrinogen oxidase Plants having attenuated plant damage and/or having increased plant yield compared to plants having a polynucleotide sequence of an enzyme wherein the protoporphyrinogen oxidase has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14 At least 88% sequence identity;
  • said protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • the protoporphyrinogen oxidase is selected from the amino acid sequence of the group consisting of SEQ ID NO: 1-14;
  • the transgenic plants include monocotyledonous plants and dicotyledonous plants; more preferably, the transgenic plants are oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa , soybeans, chickpeas, peanuts, sugar beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis;
  • the PPO inhibitor herbicides include diphenyl ether PPO inhibitor herbicides, oxadiazolone PPO inhibitor herbicides, N-phenylphthalimide imine PPO inhibitor herbicides, oxazoline Ketone PPO inhibitor herbicides, phenylpyrazole PPO inhibitor herbicides, uracil PPO inhibitor herbicides, thiadiazole PPO inhibitor herbicides, triazolone PPO inhibitor herbicides and/or or triazinone PPO inhibitor herbicides;
  • the PPO inhibitor herbicides include oxyfluorfen, saflufenacil, sulfentrazone and/or oxyflufen.
  • the present invention also provides a use of protoporphyrinogen oxidase in conferring plant PPO inhibitor herbicide tolerance, said protoporphyrinogen oxidase is selected from SEQ ID NO: 1-
  • the amino acid sequences of the group consisting of 14 have at least 88% sequence identity;
  • said protoporphyrinogen oxidase has at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • said protoporphyrinogen oxidase has at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14;
  • the protoporphyrinogen oxidase is selected from the amino acid sequence of the group consisting of SEQ ID NO: 1-14;
  • the use of said protoporphyrinogen oxidase in conferring tolerance to a PPO inhibitor herbicide in a plant comprises applying an effective dose of a PPO inhibitor herbicide to a field where at least one transgenic plant is present, said A transgenic plant comprising in its genome a polynucleotide sequence encoding said protoporphyrinogen oxidase has attenuated plant damage and/or have increased plant yield;
  • the plants include monocots and dicots; more preferably, the plants are oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis;
  • the PPO inhibitor herbicides include diphenyl ether PPO inhibitor herbicides, oxadiazolone PPO inhibitor herbicides, N-phenylphthalimide imine PPO inhibitor herbicides, oxazoline Ketone PPO inhibitor herbicides, phenylpyrazole PPO inhibitor herbicides, uracil PPO inhibitor herbicides, thiadiazole PPO inhibitor herbicides, triazolone PPO inhibitor herbicides and/or or triazinone PPO inhibitor herbicides;
  • the PPO inhibitor herbicides include oxyfluorfen, saflufenacil, sulfentrazone and/or oxyflufen.
  • polynucleotide sequence of the protoporphyrinogen oxidase has:
  • the PPO inhibitor herbicide can be one or more selected from the group consisting of, but not limited to: , Chlomethoxyfen, Bifenox, Oxyfluorfen, Acifluorfen and its salts and esters, Fomesafen, Lactofenox (lactofen), fluoroglycofen-ethyl, chlorofluorophenyl ether, aclonifen, bifenox, ethoxyfen, chlorintrofen, fluorine Sulfonamide (halosafen)); Oxadiazolones (oxadiazon, oxadiargyl); N-phenylphthalamidoimides (flumioxazin, fluorine flumiclorac-pentyl, cinidon-ethyl); oxazolinones (pentoxazone); phenylpyrazoles (fluazolate , pyrazafen); ura
  • protoporphyrinogen oxidase in the context of the present invention refers to the ability of protoporphyrinogen oxidase to maintain at least a portion of its enzymatic activity in the presence of one or more PPO inhibitor herbicides.
  • the enzymatic activity of protoporphyrinogen oxidase can be measured by any means known in the art, such as by fluorescence, high performance liquid chromatography (HPLC), or mass spectrometry in the presence of one or more PPO inhibitor herbicides (MS) Enzyme activity is determined by measuring the amount of protox product produced or the amount of protox substrate consumed.
  • Herbicide-insensitive can be complete or partial insensitivity to a particular herbicide, and can be expressed as a percentage of tolerance or insensitivity to a particular PPO inhibitor herbicide.
  • herbicide tolerance of plants, seeds, plant tissues or cells or “herbicide-tolerant plants, seeds, plant tissues or cells” means that when herbicides are applied, plants, seeds, plant tissues or cells Ability to resist the action of herbicides.
  • a herbicide-tolerant plant can survive or continue to grow in the presence of the herbicide.
  • Herbicide tolerance of a plant, seed, plant tissue or cell can be measured by comparing the plant, seed, plant tissue or cell to a suitable control.
  • a herbicide can be obtained by applying a herbicide to plants containing a DNA molecule encoding a protein that confers herbicide tolerance (test plants) and plants that do not contain a DNA molecule encoding a protein that confers herbicide tolerance (control plants). ), and then compare the plant injury of the two plants to measure or evaluate herbicide tolerance, wherein the herbicide tolerance of the test plant is indicated by a reduction in the injury rate of the test plant compared to the injury rate of the control plant.
  • Herbicide tolerant plants, seeds, plant tissues or cells exhibit a reduced response to the toxic effects of the herbicide compared to control plants, seeds, plant tissues or cells.
  • herbicide tolerance trait refers to a transgenic trait that confers improved herbicide tolerance on plants compared to wild type plants.
  • Plants that can be produced having the herbicide tolerance trait of the invention include, for example, any plant, including crop plants such as oats, wheat, barley, millet, corn, sorghum, Brachypodium distachyon, rice, tobacco, sunflower, Alfalfa, soybeans, chickpeas, peanuts, sugar beets, cucumbers, cotton, canola, potatoes, tomatoes, and Arabidopsis.
  • DNA molecules of the present invention can be synthesized and modified in whole or in part by methods known in the art, especially where it is necessary to provide sequences for DNA manipulation (such as restriction enzyme recognition sites or recombination-based cloning sites), plant-preferred sequences (such as plant codon usage or Kozak consensus sequences) or in the case of sequences used in DNA construct design (such as spacer or linker sequences).
  • sequences for DNA manipulation such as restriction enzyme recognition sites or recombination-based cloning sites
  • plant-preferred sequences such as plant codon usage or Kozak consensus sequences
  • sequences used in DNA construct design such as spacer or linker sequences.
  • the present invention includes encoding amino acid sequences selected from the group consisting of SEQ ID NO: 1-14 having at least 88% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity DNA of proteins with % sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity and at least 99% sequence identity Molecules, preferably proteins.
  • the term "percent sequence identity” or “% sequence identity” refers to the protein content of a reference sequence or query sequence (or its complementary strand) compared to a test sequence (or its complementary strand) when two sequences are aligned. The percentage of identical amino acids in the sequence.
  • Computer implementations of these mathematical algorithms are available for sequence comparison to determine sequence homology, such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); ALLGN program (version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in GCG Wisconsin Genetics Software Package version 10 (obtained from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Percent sequence identity is expressed as the identity score multiplied by 100.
  • oxyfluorfen refers to 2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-trifluoromethylbenzene, which is a colorless crystal solid. It belongs to diphenyl ethers ultra-low dosage selectivity, pre-emergence and post-emergence contact type PPO inhibitor herbicide, and can be used as emulsifiable concentrate. Weeds are killed mainly by absorbing pesticides through coleoptiles and mesocotyls. Oxyflufen can effectively control weeds in rice, soybeans, corn, cotton, vegetables, grapes, fruit trees and other crop fields.
  • the weeds that can be prevented include but are not limited to barnyard grass, field greens, dry brome, foxtail, Datura, creeping wheatgrass, ragweed, saffron, velvetleaf, crepe cotyledons and broadleaf weeds.
  • the effective dose of oxyfluorfen in the present invention refers to the use of 180-720g ai/ha, including 190-700g ai/ha, 250-650g ai/ha, 300-600g ai/ha or 400-500g ai/ha .
  • the saflufenacil described in the present invention refers to N'-[2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-(trifluoromethyl )-3,6-dihydro-1(2H)-pyrimidine)benzoyl]-N-isopropyl-N-methylsulfamide, in the form of light brown extruded granular solid. It belongs to the uracil class of herbicides that kill PPO inhibitors and can be made into 70% water-dispersible granules. Saflufenacil can effectively control a variety of broad-leaved weeds, including weeds resistant to glyphosate, ALS and triazines. It has a fast killing effect and soil residues will degrade rapidly.
  • the effective dose of saflufenacil in the present invention refers to use at 25-100g ai/ha, including 30-95g ai/ha, 40-90g ai/ha, 50-85g ai/ha or 60-80g ai/ha ha.
  • Flumioxazin described in the present invention refers to 2-[7-fluoro-3,4-dihydro-3-oxo-4-(2-propynyl)-2H-1, 4-Benzoxazin-6-yl]-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione. It belongs to the N-phenylphthalamidoimide category of shoot and leaf absorption type PPO inhibitor herbicides. The commonly used formulations are 50% wettable powder and 48% suspension concentrate. Flurafen propargyl can effectively control annual broad-leaved weeds and some gramineous weeds. It is easy to degrade in the environment and is safe for subsequent crops.
  • the effective dosage of flufenflume is 60-240g ai/ha, including 70-220g ai/ha, 85-200g ai/ha, 90-185g ai/ha or 100-150g ai/ha ha.
  • Sulfentrazone described in the present invention refers to N-(2,4-dichloro-5-(4-difluoromethyl-4,5-dihydro-3-methyl-5-oxo Substitute-1H-1,2,4-triazol-1-yl)phenyl)methanesulfonamide, a brownish-yellow solid. It belongs to the triazolinone PPO inhibitor herbicide, and the commonly used formulations are 38.9% and 44.5% suspension concentrates. Sulfentrazone can be used to control annual broad-leaved weeds such as corn, sorghum, soybeans, peanuts, etc. , grass weeds and sedges, etc.
  • the effective dose of sulfentrazone described in the present invention refers to use at 450-900g ai/ha, including 500-850g ai/ha, 550-700g ai/ha, 500-685g ai/ha or 550-650g ai/ha .
  • the term “resistance” is heritable and allows a plant to grow and reproduce in the presence of herbicides that are normally herbicide effective for a given plant. As will be recognized by those skilled in the art, even if a given plant suffers some degree of damage from herbicide treatment, such as little necrosis, lysis, chlorosis, or other damage, but at least not significantly affects yield, the plant can still be considered “Resistance", that is, the increased ability of a given plant to resist various degrees of herbicide-induced injury, while the same herbicide dose generally results in injury to wild-type plants of the same genotype.
  • the term “tolerance” or “tolerance” in the present invention is broader than the term “resistance” and includes “resistance”.
  • the anti-biotic stress protein in the present invention refers to a protein that resists stress imposed by other organisms, such as insect resistance protein, (virus, bacteria, fungus, nematode) disease resistance protein, etc.
  • the anti-abiotic stress protein in the present invention refers to a protein that resists stress imposed by the external environment, such as a protein that is tolerant to herbicides, drought, heat, cold, freezing, salt stress, oxidative stress, and the like.
  • the protein that affects plant quality refers to the protein that affects the output traits of plants, such as the protein that improves the quality and content of starch, oil, vitamins, etc., and the protein that improves fiber quality.
  • an expression cassette comprising a polynucleotide sequence encoding protoporphyrinogen oxidase may also be expressed in a plant together with at least one protein encoding a herbicide tolerance gene including, but not Limited to, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate oxidoreductase (GOX), glyphosate-N-acetyltransferase (GAT), glyphosate decarboxylase, grass Ammoniumphosphine acetyltransferase (PAT), ⁇ -ketoglutarate-dependent dioxygenase (AAD), dicamba monooxygenase (DMO), 4-hydroxyphenylpyruvate dioxygenase (HPPD), acetyl Lactate synthase (ALS) and/or cytochrome proteins (P450).
  • EPSPS 5-enolpyruvylshikimate-3-phosphate
  • glyphosate refers to N-phosphonomethylglycine and its salts
  • the treatment with “glyphosate herbicide” refers to the treatment with any herbicide preparation containing glyphosate.
  • Commercial formulations of glyphosate include, but are not limited to, (glyphosate as isopropylamine salt), (glyphosate as potassium salt), and (glyphosate as amine salt), (glyphosate as sodium salt) and (Glyphosate as trimethylsulfide salt).
  • the effective dose of glyphosate in the present invention refers to the use of 200-1600g ae/ha, including 250-1600g ae/ha, 300-1600g ae/ha, 500-1600g ae/ha, 800-1500g ae/ha, 1000-1500g ae/ha or 1200-1500g ae/ha.
  • glufosinate-ammonium described in the present invention has another name called glufosinate, which refers to 2-amino-4-[hydroxyl (methyl) phosphono] ammonium butyrate, and the treatment with “glufosinate-ammonium herbicide” refers to the use of any A herbicide formulation containing glufosinate-ammonium was used for treatment.
  • the effective dose of glufosinate-ammonium in the present invention refers to use with 200-800g ae/ha, including 200-750g ae/ha, 250-700g ae/ha, 300-700g ae/ha, 350-650g ae/ha or 400-600g ae/ha.
  • auxin-based herbicides in the present invention mimic or act like natural plant growth regulators called auxins, which affect cell wall plasticity and nucleic acid metabolism, resulting in uncontrolled cell division and growth.
  • Injury symptoms caused by auxin-based herbicides include upward bending or twisting of stems and stalks, cupping or curling of leaves, and abnormal leaf shape and veins.
  • Auxin-based herbicides include, but are not limited to, phenoxy carboxylic acid compounds, benzoic acid compounds, pyridine carboxylic acid compounds, quinoline carboxylic acid compounds, or fenoxetyl compounds.
  • auxin herbicides are dicamba, 2,4-dichlorophenoxyacetic acid (2,4-D), (4-chloro-2-methylphenoxy)acetic acid (MCPA) and/or Or 4-(2,4-dichlorophenoxy)butanoic acid (2,4-DB).
  • Dicamba in the present invention refers to 3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid and their acids and salts. Salts thereof include isopropylamine, diglycolammonium, dimethylamine, potassium and sodium salts. Commercial formulations of dicamba include, but are not limited to, (as DMA salt), (BASF, as DGA salt), VEL-58-CS-11 TM and (BASF, as DGA salt).
  • the 2,4-D described in the present invention is a broad-spectrum, relatively inexpensive and potent broadleaf herbicide that has been used for broad-spectrum broadleaf weed control under both agricultural and non-crop conditions for more than 65 years.
  • 2,4-D has different levels of selectivity in different plants (eg, dicots are more sensitive than grasses).
  • plants metabolize 2,4-D slowly, so different activities of target sites are more likely to explain the different responses of plants to 2,4-D.
  • Plant metabolism of 2,4-D is generally achieved through a two-step metabolism, typically hydroxylation followed by conjugation to amino acids or glucose.
  • the pre-emergence selective herbicides in the present invention include, but are not limited to, acetanilide, acetochlor, acetolactate synthase inhibitors and dinitroaniline.
  • the post-emergence selective herbicides in the present invention include, but are not limited to, nicosulfuron-methyl, rimsulfuron-methyl, and quizalofop-ethyl.
  • Herbicide application rates in the present invention vary with soil structure, pH, organic matter content, tillage system, and weed size, and are determined by checking the herbicide label for the appropriate herbicide application rate.
  • the term "imparting" in the present invention refers to providing a plant with a characteristic or trait, such as herbicide tolerance and/or other desirable traits.
  • heterologous in the context of the present invention means from another source.
  • heterologous refers to any foreign "non-self" DNA, including DNA from another plant of the same species.
  • a soybean PPO gene can be expressed in a soybean plant using a transgenic approach, which is still considered “heterologous” DNA.
  • nucleic acid in the present invention includes reference to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, and, unless otherwise limited, known analogues having the essential properties of natural nucleotides (such as peptide nucleic acids) because they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.
  • nucleic acid when the term "encoding" or “encoded” is used in the context of a specific nucleic acid, it means that the nucleic acid contains the information necessary to direct the translation of the polynucleotide sequence into a specific protein.
  • the information used to encode a protein is specified by the use of codons.
  • a nucleic acid encoding a protein may contain non-translated sequences (eg, introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (eg, in cDNA).
  • the DNA sequence encoding protoporphyrinogen oxidase of the present invention is used to provide plants, plant cells and seeds of the present invention, compared to the same plant that does not contain the DNA sequence encoding protoporphyrinogen oxidase of the present invention ( control plants), which provided better tolerance to various PPO inhibitor herbicides.
  • the gene encoding the protoporphyrinogen oxidase of the present invention is useful for producing plants tolerant to PPO inhibitor herbicides.
  • the gene encoding the protoporphyrinogen oxidase of the present invention is particularly suitable for expression in plants in order to impart herbicide tolerance to plants.
  • polypeptide polypeptide
  • peptide protein
  • polypeptides of the invention can be produced from a nucleic acid disclosed herein or by using standard molecular biology techniques.
  • a truncated protein of the present invention can be produced by expressing a recombinant nucleic acid of the present invention in an appropriate host cell, or alternatively by combining ex vivo methods such as protease digestion and purification.
  • the present invention also provides nucleic acid molecules encoding said protoporphyrinogen oxidase.
  • the invention includes any polynucleotide sequence encoding a protox enzyme having one or more conservative amino acid substitutions relative to said protox enzyme. Conservative substitutions of amino acids that provide functional similarity are well known to those skilled in the art.
  • a sequence having protox inhibitor herbicide tolerance activity and hybridizing under stringent conditions to the gene encoding said protox of the present invention is included in the present invention.
  • these sequences are at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to the sequences of the present invention SEQ ID NO:29-42 and SEQ ID NO:62-64 , 97%, 98%, 99% or greater sequence homology, the gene encoding the protoporphyrinogen oxidase of the present invention does not include SEQ ID NO: 15-28.
  • nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances.
  • a nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if two nucleic acid molecules exhibit perfect complementarity.
  • two nucleic acid molecules are said to exhibit "complete complementarity" when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule.
  • Two nucleic acid molecules are said to be “minimally complementary” if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency” conditions.
  • two nucleic acid molecules are said to be “complementary” if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "high stringency” conditions.
  • Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize to a complementary strand of another matched nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions to promote DNA hybridization for example, treatment with 6.0 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by washing with 2.0 ⁇ SSC at 50° C., are known to those skilled in the art. is well known.
  • the salt concentration in the washing step can be selected from about 2.0 ⁇ SSC, 50°C for low stringency conditions to about 0.2 ⁇ SSC, 50°C for high stringency conditions.
  • the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions.
  • Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed.
  • the stringent conditions of the present invention can be in 6 ⁇ SSC, 0.5% SDS solution, at 65° C. to specifically hybridize with the gene encoding protoporphyrinogen oxidase of the present invention, and then use 2 ⁇ SSC , 0.1% SDS and 1 ⁇ SSC, 0.1% SDS to wash the membrane once.
  • hybridization or “specific hybridization” means that a molecule can only bind, double-stranded or hybridize to a specific polynucleotide sequence under stringent conditions, which is when the sequence exists in a complex Mixtures (e.g., total cells) were performed when DNA or RNA was present.
  • complex Mixtures e.g., total cells
  • substantially identical sequences refer to sequences that have amino acid substitutions, deletions, additions or insertions but do not substantially affect the herbicide tolerance activity, and also include fragments that retain the herbicide tolerance activity.
  • the term "functional activity" or "activity” in the present invention means that the protein/enzyme used in the present invention (alone or in combination with other proteins) has the ability to degrade or reduce the herbicide activity.
  • Plants producing the proteins of the invention preferably produce an "effective amount" of the protein such that when the plant is treated with the herbicide, the protein is expressed at a level sufficient to confer full or partial tolerance to the herbicide (typically used unless otherwise specified) in the plant sex.
  • Herbicides can be used at rates that would normally kill the target plants, normal field rates and concentrations.
  • the plant cells and plants of the invention are protected from growth inhibition or damage caused by herbicide treatment.
  • the transformed plants and plant cells of the present invention are preferably tolerant to PPO inhibitor herbicides, ie, the transformed plants and plant cells are capable of growing in the presence of an effective amount of a PPO inhibitor herbicide.
  • genes and proteins described in the present invention not only include specific exemplary sequences, but also include parts and/or fragments (including compared with full-length proteins and/or terminal deletions) that preserve the active characteristics of the specific exemplary proteins , variants, mutants, variant proteins, substitutes (proteins with substituted amino acids), chimeras and fusion proteins.
  • variant in the present invention means substantially similar sequences.
  • a variant includes deletions and/or additions of one or more nucleotides at one or more internal sites within the reference polynucleotide and/or herbicide tolerance A substitution of one or more nucleotides at one or more sites in a gene.
  • reference polynucleotide or polypeptide in the present invention includes herbicide tolerance polynucleotide sequence or amino acid sequence correspondingly.
  • nucleic acid molecules conservative variants include the polynucleotide sequence (due to the degeneracy of the genetic code) encoding one of the protoporphyrinogen oxidases described herein.
  • Naturally occurring allelic variants such as these can be identified using well known molecular biology techniques, for example using the polymerase chain reaction (PCR) and hybridization techniques outlined below.
  • Variant nucleic acid molecules also include synthetically derived nucleic acid molecules, eg, nucleic acid sequences generated by the use of site-directed mutagenesis but which still encode a protoporphyrinogen oxidase of the invention.
  • variants of a particular nucleic acid molecule of the invention will have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or more sequence identity, as determined by sequence alignment programs and parameters.
  • Variant protein in the present invention means from a reference protein by deletion or addition of one or more amino acids at one or more internal sites in the protoporphyrinogen oxidase and/or in A protein derived from substitution of one or more amino acids at one or more sites in the protoporphyrinogen oxidase.
  • Variant proteins encompassed by the present invention are biologically active, i.e. they continue to have the desired activity of the protox enzyme of the present invention, i.e. still possess the protox activity and/or Herbicide tolerance. Such variants may arise, for example, from genetic polymorphisms or from human manipulation.
  • a biologically active variant of said protoporphyrinogen oxidase of the invention will have at least about 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology as determined by sequence alignment programs and parameters.
  • a biologically active variant of a protein of the invention may differ by as few as 1-15 amino acid residues, as few as 1-10 (such as 6-10), as few as 5 (such as 4, 3 , 2, or even 1) amino acid residues.
  • the nucleic acid sequence encoding the protoporphyrinogen oxidase of the present invention or their variants retaining protox activity can be combined with any nucleic acid of interest Combinations of sequences are superimposed to produce plants with a desired trait.
  • the term "trait" refers to a phenotype resulting from a particular sequence or group of sequences.
  • the nucleic acid sequence encoding protoporphyrinogen oxidase of the present invention or a variant protoporphyrinogen oxidase that retains protoporphyrinogen oxidase activity can be combined with any other polypeptide encoding a desired trait.
  • traits including, but not limited to, resistance to disease, insects, and herbicides, tolerance to heat and drought, shortened crop maturity time, improved industrial processing (e.g., for starch or raw conversion of substances to fermentable sugars) and improved agronomic qualities (eg, high oil content and high protein content).
  • glyphosate resistance e.g. resistant plants or bacteria EPSPS, GOX, GAT
  • glufosinate resistance e.g. PAT, Bar
  • acetolactate synthase (ALS) inhibitory herbicide resistance e.g.
  • phenoxy auxin herbicide resistance such as aryloxyalkanoate dioxygenase-AAD
  • dicamba herbicide resistance e.g. dicamba monooxygenase-DMO
  • bromoxynil resistance e.g. Bxn
  • resistance to phytoene desaturase (PDS) inhibitors resistance to photosystem II inhibitory herb
  • psbA resistance to photosystem I-inhibiting herbicides
  • resistance to 4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicides e.g. HPPD
  • resistance to phenylurea herbicides e.g. CYP76B1
  • dichlorobenzoate degrading enzyme e.g. CYP76B1
  • Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grass weed species.
  • repeated use of glyphosate in glyphosate-tolerant crop and non-crop applications has been (and continues to be) selective for weed succession to naturally more tolerant species or glyphosate-resistant biotypes.
  • Most herbicide resistance management strategies recommend the use of effective rates of tank-mixed herbicide companions that provide control of the same species but have different modes of action as a means of delaying the emergence of resistant weeds.
  • Overlaying the gene encoding protoporphyrinogen oxidase of the present invention with glyphosate tolerance traits can be achieved by allowing the selective use of glyphosate and PPO inhibitor herbicides on the same crop ( glyphosate-resistant weed species in glyphosate-tolerant crops (controlled by one or more PPO inhibitor herbicides) Broadleaf weed species) control.
  • Application of these herbicides can be simultaneous use in tank mixes containing two or more herbicides with different modes of action, single use of a single herbicide composition in sequential applications (e.g.
  • pre-plant, pre-emergence or post-emergence (Use intervals ranging from 2 hours to 3 months), or alternatively, at any time (from within 7 months of planting the crop to when the crop is harvested (or pre-harvest interval for a single herbicide, whichever is shortest) )) uses combinations representing any number of herbicides that can be applied for each compound class.
  • the application range of glyphosate applied to the gene superimposed with glyphosate resistance gene/encoding protoporphyrinogen oxidase of the present invention in crops can be from 250 to 2500g ae/ha; PPO inhibitor herbicide (one or more ) can be in accordance with from 10-1000g ai/ha.
  • the optimal combination of timing for these applications depends on specific conditions, species and environment.
  • Herbicide formulations such as ester, acid or salt formulations or soluble concentrates, emulsifying concentrates or soluble liquids
  • tank mix additives such as adjuvants or compatibilizers
  • Any chemical combination of any of the foregoing herbicides is within the scope of this invention.
  • protoporphyrinogen oxidase of the present invention can be combined with one or more other inputs (such as insect resistance, fungal resistance or stress tolerance) alone or in combination with other herbicide-tolerant crop characteristics. Acceptance, etc.) or output (such as increased yield, improved oil yield, improved fiber quality, etc.) traits are superimposed.
  • the present invention can be used to provide a complete agronomic solution with the ability to flexibly and economically control any number of agronomic pests and improve crop quality.
  • stacked combinations can be produced by any method including, but not limited to, cross-breeding plants or genetic transformation by conventional or topcross methods. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, transgenic plants that include one or more desired traits can be used as targets for the introduction of additional traits by subsequent transformation. These traits can be introduced simultaneously with the polynucleotides of interest provided by any combination of expression cassettes in a co-transformation protocol. For example, if two sequences are to be introduced, the two sequences may be contained in separate expression cassettes (trans) or contained in the same expression cassette (cis).
  • Expression of these sequences can be driven by the same promoter or by different promoters. In some cases, it may be desirable to introduce an expression cassette that suppresses the expression of a polynucleotide of interest. This can be combined with any combination of other suppressor or overexpression cassettes to produce the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be superimposed at a desired genomic location using site-specific recombination systems.
  • the gene encoding the protoporphyrinogen oxidase of the present invention has higher tolerance to PPO inhibitor herbicides, and is the basis for important herbicide-tolerant crops and the possibility of selecting marker characteristics.
  • expression cassette in the present invention refers to a nucleic acid molecule capable of directing the expression of a specific polynucleotide sequence in an appropriate host cell, including a nucleic acid molecule operably linked to a polynucleotide sequence of interest (i.e., a single Or in combination with one or more additional nucleic acid molecules encoding polypeptides endowing desired traits to encode a protoporphyrinogen oxidase of the present invention or a variant protein that retains protoporphyrinogen oxidase activity
  • the promoter of the polynucleotide), the polynucleotide sequence of interest is operably linked to a termination signal.
  • the coding region typically encodes a protein of interest, but may also encode a functional RNA of interest, such as antisense RNA or an untranslated RNA in sense or antisense orientation.
  • An expression cassette comprising the polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be a naturally occurring expression cassette, but must be obtained in a recombinant form useful for heterologous expression.
  • the expression cassette is heterologous to the host, ie, the particular DNA sequence of the expression cassette does not naturally occur in the host cell and must have been introduced into the new host cell by a transformation event.
  • the expression of the polynucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to some specific external stimulus.
  • the promoter is also specific for a particular tissue or organ or developmental stage.
  • the present invention contemplates the expression of a polynucleotide capable of expressing a polynucleotide of interest (i.e., one alone or in combination with one or more additional nucleic acid molecules encoding a polypeptide conferring a desired trait) that encodes a polynucleotide of the present invention.
  • Plants are transformed with an expression cassette of the protoporphyrinogen oxidase or its variant protein that retains protoporphyrinogen oxidase activity.
  • the expression cassette includes a transcription and translation initiation region (i.e. a promoter) and a polynucleotide open reading frame in the 5'-3' transcription direction.
  • the expression cassette may optionally include transcriptional and translational termination regions (ie termination regions) functional in plants.
  • the expression cassette includes a selectable marker gene to allow selection of stable transformants.
  • the expression constructs of the invention may also include a leader sequence and/or a sequence allowing inducible expression of the polynucleotide of interest.
  • the regulatory sequences of the expression cassette are operably linked to the polynucleotide of interest.
  • the regulatory sequences in the present invention include but are not limited to promoters, transit peptides, terminators, enhancers, leader sequences, introns and other regulatory sequences operably linked to the gene encoding the protoporphyrinogen oxidase.
  • the promoter is a promoter that can be expressed in plants, and the "promoter that can be expressed in plants” refers to a promoter that ensures the expression of the coding sequence linked to it in plant cells.
  • a promoter expressible in plants may be a constitutive promoter. Examples of promoters directing constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the corn Ubi promoter, the promoter of the rice GOS2 gene, and the like.
  • the promoter expressible in plants may be a tissue-specific promoter, i.e., the promoter directs expression of the coding sequence at higher levels in some tissues of the plant, such as in green tissues, than in other tissues of the plant (which can be determined by routine RNA assays), such as the PEP carboxylase promoter.
  • the promoter expressible in plants may be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter directing a wound-induced expression pattern means that when a plant is subjected to mechanical or insect-induced wounds, the expression of the coding sequence under the regulation of the promoter is significantly increased compared with normal growth conditions.
  • wound-inducible promoters include, but are not limited to, the promoters of the potato and tomato protease inhibitors (pinI and pinII) and the maize proteinase inhibitor (MPI).
  • the transit peptide (also known as secretion signal sequence or targeting sequence) is to direct the transgene product to a specific organelle or cell compartment.
  • the transit peptide can be heterologous, for example, using the encoding chloroplast transport
  • the peptide sequences target the chloroplast, or the endoplasmic reticulum using the 'KDEL' retention sequence, or the vacuole using the CTPP of the barley lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a poty virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin heavy chain binding protein (BiP); untranslated leader of coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a poty virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancers, Scrophulariaceae mosaic virus (FMV) enhancers, carnation weathering ring virus (CERV) enhancers, cassava vein mosaic virus (CsVMV) enhancers , Mirabilis Mosaic Virus (MMV) Enhancer, Night Scent Yellow Leaf Curl Virus (CmYLCV) Enhancer, Multan Cotton Leaf Curl Virus (CLCuMV), Commelina Yellow Mottle Virus (CoYMV) and Peanut Chlorotic Streak Flower Leaf virus (PCLSV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV Scrophulariaceae mosaic virus
  • CERV carnation weathering ring virus
  • CsVMVMV cassava vein mosaic virus
  • MMV Mirabilis Mosaic Virus
  • CLCuMV Multan Cotton Leaf Curl Virus
  • COYMV Commelina Yellow Mottle Virus
  • PCLSV Peanut Chlorotic Streak Flower Leaf virus
  • the introns include, but are not limited to, the maize hsp70 intron, the maize ubiquitin intron, the Adh intron 1, the sucrose synthase intron, or the rice Act1 intron.
  • such introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.
  • the terminator may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, the polyadenylation signal sequence derived from the nopaline synthase (NOS) gene of Agrobacterium tumefaciens , the polyadenylation signal sequence from the protease inhibitor II (pinII) gene, the polyadenylation signal sequence from the pea ssRUBISCO E9 gene and the polyadenylation signal sequence from the ⁇ -tubulin gene Polyadenylation signal sequence.
  • NOS nopaline synthase
  • pinII protease inhibitor II
  • the “operably linked” in the present invention refers to the linkage of nucleic acid sequences, which allows one sequence to provide the required function for the linked sequence.
  • the "operably linked” in the present invention can be linking a promoter with a sequence of interest, so that the transcription of the sequence of interest is controlled and regulated by the promoter.
  • "Operably linked" when a sequence of interest encodes a protein and expression of that protein is desired means that a promoter is linked to said sequence in such a way that the resulting transcript is efficiently translated. If the junction of the promoter and coding sequence is a transcript fusion and expression of the encoded protein is desired, the junction is made such that the first translation initiation codon in the resulting transcript is that of the coding sequence.
  • the junction of the promoter and coding sequence is a translational fusion and expression of the encoded protein is to be achieved, the junction is made such that the first translation initiation codon contained in the 5' untranslated sequence is fused with the promoter linked in such a way that the resulting translation product is in-frame with the translational open reading frame encoding the desired protein.
  • Nucleic acid sequences that may be "operably linked” include, but are not limited to: sequences that provide gene expression function (i.e., gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, polynucleotides adenylation sites and/or transcription terminators), sequences that provide DNA transfer and/or integration functions (i.e.
  • T-DNA border sequences site-specific recombinase recognition sites, integrase recognition sites
  • provide selection Sequences for sexual function i.e., antibiotic resistance markers, biosynthetic genes
  • sequences that provide scoreable marker function sequences that facilitate sequence manipulation in vitro or in vivo
  • sequences that facilitate sequence manipulation in vitro or in vivo i.e., polylinker sequences, site-specific recombination sequences
  • sequences that provide Sequences of replication function ie bacterial origin of replication, autonomously replicating sequences, centromere sequences.
  • the genome of a plant, plant tissue or plant cell in the present invention refers to any genetic material in a plant, plant tissue or plant cell, and includes nucleus, plastid and mitochondrial genome.
  • plant part or “plant tissue” includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clusters and parts of plants in plants or In complete plant cells, the parts of these plants are such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, cores, ears, cobs, shells, stems, roots, root tips, anthers, etc.
  • the PPO protein of the present invention can be applied to a variety of plants, and the dicotyledonous plants include but are not limited to alfalfa, kidney bean, cauliflower, cabbage, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, zucchini , radish, rape, spinach, soybean, pumpkin, tomato, Arabidopsis, peanut or watermelon; preferably, the dicotyledon refers to cucumber, soybean, Arabidopsis, tobacco, cotton, peanut or rape.
  • the monocotyledons include but are not limited to corn, rice, sorghum, wheat, barley, rye, millet, sugarcane, oats or lawn grasses; preferably, the monocotyledonous plants refer to corn, rice, sorghum, wheat, barley , millet, sugar cane or oats.
  • plant transformation refers to the transformation of a herbicide-resistant or tolerant herbicide-encoding nucleic acid molecule encoding protoporphyrinogen oxidase according to the present invention alone or in combination with a nucleic acid molecule encoding a polypeptide conferring desired traits. or multiple additional nucleic acid molecules combined and cloned into an expression system, it is transformed into a plant cell.
  • the receptor and target expression cassettes of the present invention can be introduced into plant cells by a variety of well-known methods.
  • introducing e.g., a nucleotide construct of interest
  • the term "introducing" is intended to mean that the polynucleotide is provided to the plant in such a way that the polynucleotide acquires a reference to a The approach or realization of the interior of a plant cell.
  • these polynucleotides may be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and may be on the same or different transformation vectors.
  • polynucleotides may thus be introduced into the host cell of interest in a single transformation event, in separate transformation events, or as part of a breeding scheme, for example in plants.
  • the methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, but merely on obtaining access or realization of the polynucleotide(s) to the interior of at least one cell of the plant.
  • Methods known in the art for introducing one or more polynucleotides into a plant include, but are not limited to, transient transformation methods, stable transformation methods, virus-mediated methods, or genome editing techniques.
  • stable transformation refers to the introduction of an exogenous gene into the genome of a plant and its stable integration into the genome of the plant and any successive generations thereof, resulting in stable inheritance of the exogenous gene.
  • transient transformation refers to the introduction of nucleic acid molecules or proteins into plant cells, which perform functions but are not integrated into the plant genome, resulting in the inability to stably inherit foreign genes.
  • genome editing technology refers to a genome modification technology that can perform precise operations on genome sequences and realize gene site-directed mutations, insertions, deletions, and other operations.
  • genome editing technologies mainly include HE (homing endonuclease, homing endonuclease), ZFN technology (Zinc-finger nuclease, zinc finger nuclease), TALEN technology (transcription activator-like effector nuclease, transcription activator-like effector nuclease) ), CRISPR technology (Clustered regulatory interspaced short palindromic repeat, clustered regular interspaced short palindromic repeat).
  • transformation vectors available for plant transformation are known to those skilled in the art, and the genes related to the present invention can be used in combination with any of the above vectors.
  • the choice of vector will depend on the preferred transformation technique and the species of interest for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selectable markers routinely used in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics or related herbicides (this gene was published by Bevan et al. in 1983 in "Natural Science" vol.
  • glufosinate also known as glufosinate; see White et al published in 1990 in “Nucl.AcidsRes” volume 18 page 1062, Spencer et al in 1990 published in "Theor. Appl.Genet “Volume 79, pages 625-631 and U.S. Patents 5,561,236 and 5,276,268) the pat and bar genes for resistance, the hpn gene (Blochinger & Diggelmann, Mol. Cell Biol. 4:2929- 2931) and the dnfr gene conferring resistance to methotrexate (Bourouis et al.
  • Ti plasmid vectors have been utilized for delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles.
  • the weeds refer to plants that compete with cultivated transgenic plants in a field.
  • control and/or "control” in the present invention mean at least direct application (eg by spraying) of an effective dose of a PPO inhibitor herbicide into a field to minimize weed development and/or stop growth.
  • cultivated transgenic plants should be morphologically normal and can be cultured for product consumption and/or production under conventional methods; preferably, have reduced plant damage and /or have increased plant yield. Said having reduced plant damage, the specific manifestations include but not limited to improved stalk resistance, and/or increased grain weight and the like.
  • the "control” and/or “control” effect of the protoporphyrinogen oxidase on weeds can exist independently, and will not be weakened and / or disappear.
  • any tissue of the transgenic plant exists and/or produces simultaneously and/or asynchronously, and the protoporphyrinogen oxidase and/or can be Another substance for controlling weeds, the presence of the other substance neither affects the "control” and/or “control” effect of the protoporphyrinogen oxidase on the weeds, nor can it cause the " The "controlling” and/or “controlling” effect is fully and/or partly achieved by said another substance independently of said protoporphyrinogen oxidase.
  • Plant propagule described in the present invention includes but not limited to plant sexual propagule and plant vegetative propagule.
  • the sexual propagules of plants include but not limited to plant seeds; the vegetative propagules of plants refer to the vegetative organs or certain special tissues of plants, which can produce new plants under in vitro conditions; the vegetative organs or certain special tissues
  • a special tissue includes but not limited to roots, stems and leaves, for example: plants with roots as asexual propagules include strawberry and sweet potato, etc.; plants with stems as asexual propagules include sugarcane and potatoes (tubers) etc.; Propagate plants include aloe vera and begonias.
  • the invention can endow plants with new herbicide resistance traits, and no adverse effects on phenotypes including yield are observed.
  • Plants of the invention are tolerant to, for example, 2x, 3x or 4x typical application levels of at least one of the herbicides tested. Increases in these tolerance levels are within the scope of the present invention.
  • various techniques known in the art can be foreseen for optimization and further development to increase the expression of a given gene.
  • the invention provides a kind of application of protoporphyrinogen oxidase, which has the following advantages:
  • the invention discloses for the first time that protoporphyrinogen oxidases PPO1-PPO14 can show higher tolerance to PPO inhibitor herbicides, and therefore have broad application prospects in plants.
  • the protoporphyrinogen oxidase PPO1-PPO14 of the present invention has strong tolerance to PPO inhibitor herbicides, and it is oxyfluorfen, saflufenacil, and propargylflumecil to 4 times the field concentration and Almost all sulfentrazone at 2 times the field concentration showed high resistance tolerance.
  • Fig. 1 is the structural representation of the Arabidopsis thaliana recombinant expression vector DBN12337 containing the PPO1A nucleotide sequence of the present invention
  • Fig. 2 is a schematic diagram of the structure of the control recombinant expression vector DBN12337N of the present invention
  • Fig. 3 is the structure diagram of the corn recombinant expression vector DBN12354 containing the PPO1B nucleotide sequence of the present invention
  • Fig. 4 is a schematic diagram of the structure of the control recombinant expression vector DBN12354N of the present invention.
  • the first embodiment the acquisition and verification of transgenic Arabidopsis plants
  • the amino acid sequence of Escherichia coli (Escherichia coli) protoporphyrinogen oxidase PPO-EC as shown in SEQ ID NO:43 in the sequence listing; Coding is corresponding to the PPO- of described Escherichia coli protoporphyrinogen oxidase PPO-EC EC nucleotide sequence, as shown in SEQ ID NO:44 in the sequence listing; obtain the PPO-ECA corresponding to the Escherichia coli protoporphyrinogen oxidase PPO-EC according to the common preference codon of Arabidopsis thaliana and soybean Nucleotide sequence, as shown in SEQ ID NO:45 in the sequence listing.
  • the amino acid sequence of Arabidopsis protoporphyrinogen oxidase PPO-AT is corresponding to the PPO-AT nucleus of described Arabidopsis protoporphyrinogen oxidase PPO-AT Nucleotide sequence, as shown in SEQ ID NO:47 in the sequence listing;
  • the PPO-ATA core encoding corresponding to the Arabidopsis protoporphyrinogen oxidase PPO-AT is obtained Nucleotide sequence, as shown in SEQ ID NO:48 in the sequence listing.
  • the amino acid sequence of whitefly symbiotic bacteria (Arsenophonus) protoporphyrinogen oxidase PPO-AP is shown in SEQ ID NO:65 in the sequence listing; coding is corresponding to described whitefly whitefly symbiotic bacteria protoporphyrinogen oxidase PPO -
  • the PPO-AP nucleotide sequence of AP as shown in SEQ ID NO: 66 in the sequence table; according to the common preference codons of Arabidopsis and soybean, the code is corresponding to the protoporphyrinogen oxidation of the whitefly symbiotic bacteria
  • the PPO-APA nucleotide sequence of the enzyme PPO-AP is shown in SEQ ID NO: 67 in the sequence table; according to the maize preference codon, the code is corresponding to the protoporphyrinogen oxidase PPO-
  • the PPO-APB nucleotide sequence of AP is shown in SEQ ID NO
  • PPO1A-PPO14A nucleotide sequence PPO-ECA nucleotide sequence, PPO-ATA nucleotide sequence and PPO-APA nucleotide sequence (SEQ ID NO:29-42, SEQ ID NO:45, SEQ ID NO:45, SEQ The 5' and 3' ends of ID NO:48 and SEQ ID NO:67) are respectively connected with universal adapter primer 1:
  • 5' end universal adapter primer 1 5'-taagaaggagatatacatatg-3' as shown in SEQ ID NO:49 in the sequence listing;
  • the 3' end universal adapter primer 1 5'-gtggtggtggtggtggtgctcgag-3' is shown in SEQ ID NO:50 in the sequence listing.
  • the plant expression vector DBNBC-01 was subjected to a double enzyme digestion reaction using restriction endonucleases Spe I and Asc I, thereby linearizing the plant expression vector, and the digestion product was purified to obtain a linearized DBNBC-01 expression vector backbone (vector backbone: pCAMBIA2301 (CAMBIA organization can provide)), will connect the described PPO1A nucleotide sequence (SEQ ID NO:29) of described universal linker primer 1 and described linearized DBNBC-01 expression vector backbone to carry out recombination reaction, operation steps According to the instructions of Takara's In-Fusion seamless connection product kit (Clontech, CA, USA, CAT: 121416), the recombinant expression vector DBN12337 was constructed, and its structural schematic diagram is shown in Figure 1 (Spec: spectinomycin gene; RB : right border; eFMV: 34S enhancer of Scrophulariaceae mosaic virus (SEQ ID NO:51); prBrCBP: promoter (SEQ ID
  • the recombinant expression vector DBN12337 was transformed into Escherichia coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 ⁇ L Escherichia coli T1 competent cells, 10 ⁇ L plasmid DNA (recombinant expression vector DBN12337), 42 °C water bath for 30 seconds; 37 °C shaking culture 1h (shaking table under 100rpm rotating speed); Then on the LB solid plate (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L L, adjust the pH to 7.5 with NaOH) and culture at 37°C for 12 hours, pick white colonies, and in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, spectinomyces 50 mg/L prime, adjust the pH to 7.5 with NaOH) and cultivate overnight at a temperature of 37°C.
  • LB solid plate tryptone 10g/L, yeast extract 5g/
  • Extract the plasmid by alkaline method centrifuge the bacterial solution at 12000rpm for 1min, remove the supernatant, and use 100 ⁇ l ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylenediaminetetraacetic acid), 50mM glucose , pH8.0) suspension; add 200 ⁇ L freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix, put on ice for 3-5min; add 150 ⁇ L ice-cold Mix solution III (3M potassium acetate, 5M acetic acid) immediately and thoroughly, place on ice for 5-10min; centrifuge at 4°C and 12000rpm for 5min, add 2 times the volume of absolute ethanol to the supernatant, and mix After uniformity, place at room temperature for 5 minutes; centrifuge at 4°C and 12,000 rpm for 5 minutes, discard the supernatant, wash the precipitate with
  • the extracted plasmid was sequenced and identified, and the results showed that the nucleotide sequence between the Spe I and Asc I sites of the recombinant expression vector DBN12337 was the nucleotide sequence shown in SEQ ID NO: 29 in the sequence table, that is, the PPO1A core nucleotide sequence.
  • the PPO2A-PPO14A nucleotide sequence, PPO-ECA nucleotide sequence, PPO-ATA nucleotide sequence and PPO-APA core The nucleotide sequence was recombined with the linearized DBNBC-01 expression vector backbone to obtain the recombinant expression vectors DBN12338 to DBN12353 in sequence. Sequencing verified the correct insertion of the above nucleotide sequences in the recombinant expression vectors DBN12338 to DBN12353.
  • a control recombinant expression vector DBN12337N was constructed, the vector structure of which is shown in Figure 2 (Spec: spectinomycin gene; RB: right border; eFMV: 34S enhancer of Scrophulariaceae mosaic virus ( SEQ ID NO:51); prBrCBP: the promoter of rape eukaryotic elongation factor gene 1 ⁇ (Tsf1) (SEQ ID NO:52); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO:53); EPSPS: 5- Enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO:54); tPsE9: terminator of pea RbcS gene (SEQ ID NO:55); pr35S: cauliflower mosaic virus 35S promoter (SEQ ID NO :59); cPAT: phosphinothric
  • the correctly constructed recombinant expression vectors DBN12337 to DBN12350, DBN12352, DBN12353 and the above-mentioned control recombinant expression vector DBN12337N were respectively transformed into Agrobacterium GV3101 by liquid nitrogen method.
  • the transformation conditions were: 100 ⁇ L Agrobacterium GV3101, 3 ⁇ L plasmid DNA (recombinant Expression vectors DBN12337 to DBN12350, DBN12352, DBN12353, DBN12337N); placed in liquid nitrogen for 10 minutes, 37°C warm water bath for 10 minutes; transformed Agrobacterium GV3101 was inoculated in LB test tubes and cultured for 2 hours at a temperature of 28°C and a rotation speed of 200rpm , be coated on the described LB solid plate containing the rifampicin (Rifampicin) of 50mg/L and the spectinomycin of 50mg/L on the described LB solid plate until growing positive monoclonal, pick monoclonal culture and extract its plasmid, the plasmid of extraction Sequencing identification was carried out, and the results showed that the structures of the recombinant expression vectors DBN12337 to DBN12350, DBN12352, DBN12353 and DBN12337N were completely
  • Wild-type Arabidopsis seeds were suspended in 0.1% (w/v) agarose solution. Suspended seeds were stored at 4°C for 2 days to fulfill the need for dormancy to ensure simultaneous seed germination.
  • the horse manure was mixed with vermiculite and subirrigated with water until moist, allowing the soil mixture to drain for 24 hours.
  • the pretreated seeds were planted on the soil mixture and covered with a moisture hood for 7 days. Seeds were germinated and plants were grown in a greenhouse under long-day conditions (16h light/8h dark) at constant temperature (22°C) constant humidity (40-50%) and light intensity of 120-150 ⁇ mol/m 2 s ⁇ 1 . Start by watering the plants with Hoagland's nutrient solution, followed by deionized water, keeping the soil moist but not soggy.
  • Transform Arabidopsis using the flower soak method Inoculate one or more 15-30 mL precultures of LB broth containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) with the selected Agrobacterium colonies. The preculture was incubated overnight at a temperature of 28° C. with constant shaking at 220 rpm. Each preculture was used to inoculate two 500ml cultures of the YEP broth containing spectinomycin (50mg/L) and rifampicin (10mg/L) and the cultures were incubated overnight at a temperature of 28°C with continuous shaking . Centrifuge at room temperature at about 4000 rpm for 20 min to pellet the cells, and discard the obtained supernatant.
  • T 1 seeds were dried at room temperature for 7 days. Seeds were planted in 26.5 cm x 51 cm germination trays, each receiving 200 mg T1 seeds (approximately 10000 seeds), which had previously been suspended in distilled water and stored at a temperature of 4 °C for 2 days to fulfill the need for dormancy To ensure that the seeds germinate synchronously.
  • Horse manure was mixed with vermiculite and bottom irrigated with water until moist, draining by gravity. Sow the pretreated seeds evenly on the soil mixture with a pipette and cover with a moisture hood for 4-5 days. The hood was removed 1 day before initial transformant selection using a post-emergence spray of glufosinate (selecting for co-transformed PAT genes).
  • T was sprayed with a 0.2% solution of Liberty herbicide (glufosinate-ammonium at 200 g ai/L) at 7 days after planting (DAP) and again at 11 DAP using DeVilbiss compressed air nozzles at a spray volume of 10 mL/pan (703 L/ha). 1 plant (cotyledon stage and 2-4 leaf stage, respectively) to provide an effective amount of glufosinate-ammonium per application of 280 g ai/ha.
  • Surviving plants actively growing plants were identified 4-7 days after the last spraying, and transplanted into 7cm ⁇ 7cm square pots (3-5 per tray) prepared with horse manure and vermiculite, respectively.
  • the transplanted plants were covered with a moisture hood for 3-4 days, and placed in a culture room at a temperature of 22° C. or directly moved into a greenhouse as before. The cover is then removed and the plants are grown at least 1 day prior to testing the ability of the PPO1A-PPO14A nucleotide sequence, the PPO-ATA nucleotide sequence, the PPO-APA nucleotide sequence, and the control vector to provide PPO inhibitor herbicide tolerance.
  • Planted in a greenhouse (temperature 22 ⁇ 5°C, 50 ⁇ 30% RH, 14h light: 10h dark, minimum 500 ⁇ E/m 2 s -1 natural + supplementary light).
  • Transformed Arabidopsis T1 plants were first selected using glufosinate-ammonium herbicide.
  • grade 0 means that the growth status is basically the same as that of spraying blank solvent (water)
  • grade 1 means that the average damage percentage of plants is less than 10%
  • grade 2 means that the average damage percentage of plants is greater than 10%
  • grade 3 means that the average damage percentage of plants is 100%. Plants classified as grade 0 and grade 1 by plant growth status were high-resistant plants, plants classified as grade 2 by plant growth status were medium-low resistant plants, and plants classified as grade 3 by plant growth status were non-resistant plants.
  • the experimental results are shown in Table 1-4.
  • Table 2 the results of the tolerance experiment of Arabidopsis thaliana T1 plants transferred to PPO1A -PPO14A nucleotide sequence, PPO-APA nucleotide sequence, PPO-ATA nucleotide sequence and control vector to saflufenacil
  • Table 3 the results of the tolerance experiment of Arabidopsis thaliana T 1 plants transferred to PPO1A-PPO14A nucleotide sequence, PPO-APA nucleotide sequence, PPO-ATA nucleotide sequence and control vector
  • the second embodiment the acquisition and verification of transgenic soybean plants
  • the recombinant expression vector DBN12337 containing the PPO1A nucleotide sequence, PPO6A nucleotide sequence, PPO12A nucleotide sequence, PPO-ECA nucleotide sequence and PPO-APA nucleotide sequence in the first embodiment 3 DBN12342, DBN12348, DBN12351 and DBN12353 and the control recombinant expression vector DBN12337N in the first embodiment 3 were respectively transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were : 100 ⁇ L of Agrobacterium LBA4404, 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 min; inoculate transformed Agrobacterium LBA4404 in LB test tubes at a temperature of 28 ° C and a rotation speed of 200 rpm Cultivate for 2h, apply on
  • the cotyledon node tissue of the aseptically cultured soybean variety Zhonghuang 13 was co-cultured with the Agrobacterium described in Example 1, so that the recombinant expression vector in Example 1 T-DNA in DBN12337, DBN12342, DBN12348, DBN12351, DBN12353 and control recombinant expression vector DBN12337N (including the 34S enhancer sequence of Scrophulariaceae mosaic virus, the promoter sequence of eukaryotic elongation factor gene 1 ⁇ (Tsf1) in rapeseed, Arabidopsis thaliana Chloroplast transit peptide sequence, 5-enolpyruvate shikimate-3-phosphate synthase gene, pea RbcS gene terminator sequence, Arabidopsis thaliana ubiquitin (Ubiquitin) 10 gene promoter sequence, Arabidopsis thaliana albino or shallow Chloroplast transit peptide, PPO
  • soybean germination medium B5 salt 3.1 g/L, B5 vitamin, sucrose 20 g/L, agar 8 g/L, pH 5.6
  • inoculate the seeds on the germination medium and cultivate according to the following conditions: temperature 25 ⁇ 1°C; photoperiod (light/dark) 16/8h.
  • infectious medium MS salt 2.15g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 40mg/L, 2-morpholineethanesulfonic acid (MES ) 4g/L, zeatin (ZT) 2mg/L, pH5.3
  • Cotyledon node tissue and Agrobacterium co-cultivate a period of time (3 days) (step 2: co-cultivation step).
  • cotyledon Nodal tissue was cultured on solid medium (MS salts 4.3g/L, B5 vitamins, sucrose 20g/L, glucose 10g/L, MES 4g/L, ZT 2mg/L, agar 8g/L, pH 5.6 after the infection step ). After this co-cultivation phase, there can be an optional "recovery" step.
  • the recovery medium (B5 salt 3.1g/L, B5 vitamin, MES 1g/L, sucrose 30g /L, ZT 2mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH5.6) there is at least one known inhibitory Agrobacterium growth Antibiotics (cephalosporin 150-250mg/L), do not add the selection agent of plant transformants (step 3: recovery step).
  • the tissue piece of cotyledonary node regeneration is on solid medium with antibiotics but no selection agent Cultivate to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the tissue pieces regenerated from the cotyledonary nodes are cultivated on the medium containing the selection agent (glyphosate) and select the transformed callus growing (step 4: Selection step).
  • the cotyledonous node regenerated tissue pieces are screened in a selective agent solid medium (B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, 6-benzyl adenine ( 6-BAP) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, N-(phosphonomethyl)glycine 0.25mol/L, pH5 .6) on culture, cause the selective growth of transformed cells.
  • the transformed cells regenerate plant (step 5: regeneration step), preferably, the tissue piece that the cotyledon node regeneration of growth on the substratum that contains selective agent is in Plants were regenerated by culturing on
  • the resistant tissue blocks obtained by screening were transferred to the B5 differentiation medium (B5 salt 3.1g/L, B5 vitamin, MES 1g/L, sucrose 30g/L, ZT 1mg/L, agar 8g/L, cephalosporin 150mg /L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, N-(phosphonomethyl)glycine 0.25mol/L, pH5.6) , cultured and differentiated at 25°C.
  • B5 differentiation medium B5 salt 3.1g/L, B5 vitamin, MES 1g/L, sucrose 30g/L, ZT 1mg/L, agar 8g/L, cephalosporin 150mg /L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, N-(phosphonomethyl)glycine 0.25mol
  • B5 rooting medium B5 salt 3.1g/L, B5 vitamins, MES 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole-3- Butyric acid (IBA) 1mg/L
  • IBA indole-3- Butyric acid
  • soybean plants transferred into the PPO1A nucleotide sequence, the soybean plants transferred into the PPO6A nucleotide sequence, the soybean plants transferred into the PPO12A nucleotide sequence, the soybean plants transferred into the PPO-ECA nucleotide sequence, the transferred About 100 mg of leaves of soybean plants with PPO-APA nucleotide sequence and soybean plants transformed into control vector DBN12337N were used as samples, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the EPSPS gene copy was detected by fluorescent quantitative PCR method of Taqman probe number to determine the copy number of the PPO gene.
  • the wild-type soybean plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.
  • the specific method for detecting the copy number of the EPSPS gene is as follows:
  • Step 11 respectively take the soybean plant transferred to the PPO1A nucleotide sequence, the soybean plant transferred to the PPO6A nucleotide sequence, the soybean plant transferred to the PPO12A nucleotide sequence, and the soybean plant transferred to the PPO-ECA nucleotide sequence 100 mg of leaves of the soybean plants transferred to the PPO-APA nucleotide sequence, the soybean plants transferred to the control vector DBN12337N, and the wild-type soybean plants were ground into a homogenate with liquid nitrogen in a mortar respectively, and 3 samples were taken for each sample. repetitions;
  • Step 12 use Qiagen's DNeasy Plant Mini Kit to extract the genomic DNA of the above sample, and refer to its product manual for specific methods;
  • Step 13 measure the genomic DNA concentration of above-mentioned sample with NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above-mentioned samples to the same concentration value, and the range of the concentration value is 80-100ng/ ⁇ L;
  • Step 15 using the Taqman probe fluorescent quantitative PCR method to identify the copy number of the sample, using the sample with known copy number as the standard, and using the sample of the wild-type soybean plant as the control, each sample is repeated 3 times, and the average Value; Fluorescence quantitative PCR primer and probe sequences are respectively:
  • Primer 1 ctggaaggcgaggacgtcatcaata as shown in SEQ ID NO: 69 in the sequence listing;
  • Primer 2 tggcggcattgccgaaatcgag as shown in SEQ ID NO:70 in the sequence listing;
  • Probe 1 atgcaggcgatgggcgcccgcatccgta as shown in SEQ ID NO:71 in the sequence listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mix contained 45 ⁇ L of each primer at a concentration of 1 mM, 50 ⁇ L of a probe at a concentration of 100 ⁇ M, and 860 ⁇ L of 1 ⁇ TE buffer, and was stored in amber tubes at 4°C.
  • the PCR reaction conditions are:
  • the PPO1A nucleotide sequence, PPO6A nucleotide sequence, PPO12A nucleotide sequence, PPO-ECA nucleotide sequence, PPO-APA nucleotide sequence and the control vector DBN12337N were all It has been integrated into the genome of the detected soybean plants, and the soybean plants that have been transformed into the PPO1A nucleotide sequence, the soybean plants that have been transformed into the PPO6A nucleotide sequence, the soybean plants that have been transformed into the PPO12A nucleotide sequence, and the soybean plants that have been transformed into The soybean plants with the PPO-ECA nucleotide sequence, the soybean plants transformed with the PPO-APA nucleotide sequence and the soybean plants transformed with the control vector DBN12337N all obtained single-copy transgenic soybean plants.
  • the third embodiment the acquisition and verification of transgenic corn plants
  • 5' end universal linker primer 2 5'-ccaagcggccaagctta-3', as shown in SEQ ID NO:72 in the sequence listing;
  • 3' end universal adapter primer 2 5'-tgtttgaacgatcggcgcgcc-3', as shown in SEQ ID NO:73 in the sequence listing.
  • the plant expression vector DBNBC-02 was subjected to a double enzyme digestion reaction with restriction endonucleases Spe I and Asc I, thereby linearizing the plant expression vector, and the enzyme digestion product was purified to obtain a linearized DBNBC-02 expression vector backbone (vector backbone: pCAMBIA2301 (CAMBIA institutions can provide)), the PPO1B nucleotide sequence connected to the universal linker primer 2 and the linearized DBNBC-02 expression vector backbone are subjected to a recombination reaction, and the operation steps are seamlessly connected according to Takara's In-Fusion
  • the product kit (Clontech, CA, USA, CAT: 121416) was carried out according to the instructions, and the recombinant expression vector DBN12354 was constructed, and its vector structure diagram is shown in Figure 3 (Spec: spectinomycin gene; RB: right border; prOsAct1: rice muscle Promoter of kinetin 1 (SEQ ID NO:74); cPAT: pho
  • E. coli T1 competent cells were transformed and its plasmid was extracted by the alkali method, and the extracted plasmid was sequenced and verified.
  • the results showed that the recombinant expression vector DBN12354 contained SEQ ID NO: 62 in the sequence table.
  • the nucleotide sequence shown is the PPO1B nucleotide sequence.
  • the PPO6B nucleotide sequence, PPO12B nucleotide sequence and PPO-APB nucleotide sequence connected to the universal linker primer 2 were respectively combined with the linearized DBNBC-02
  • the expression vector backbone was subjected to recombination reaction, and the recombinant expression vectors DBN12355 to DBN12357 were sequentially obtained. Sequencing verified that the PPO6B nucleotide sequence, PPO12B nucleotide sequence and PPO-APB nucleotide sequence were inserted correctly in the recombinant expression vectors DBN12355 to DBN12357, respectively.
  • the recombinant expression vectors DBN12354 to DBN12357 that have been constructed correctly and the above-mentioned control recombinant expression vector DBN12354N were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L of Agrobacterium Bacteria LBA4404, 3 ⁇ L plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 min; transformed Agrobacterium LBA4404 was inoculated in LB test tubes and cultured at a temperature of 28 ° C and a rotation speed of 200 rpm for 2 h.
  • Agrobacterium LBA4404 Invitrgen, Chicago, USA, CAT: 18313-015
  • the immature embryos of the aseptically cultured maize variety Zong 31 were co-cultured with the Agrobacterium described in Example 2, so as to express the recombinant gene constructed in Example 1.
  • T-DNA in the vectors DBN12354 to DBN12357 and the control recombinant expression vector DBN12354N (including the promoter sequence of rice actin 1, phosphinothricin N-acetyltransferase gene, cauliflower mosaic virus 35S terminator sequence, cauliflower flower Leaf virus 35S promoter sequence, maize heat shock 70kDa intein sequence, Arabidopsis thaliana albino or light green body transit peptide, PPO1B nucleotide sequence, PPO6B nucleotide sequence, PPO12B nucleotide sequence, PPO-APB Nucleotide sequence, terminator sequence of nopaline synthase gene, promoter sequence of maize ubiquitin (Ubiquitin) 1 gene, phosphomannose isomerase gene, terminator sequence of nopaline synthase gene) into corn In the chromosome group, the corn plants transferred to the PPO1B nucleotide sequence, the corn plant transferred to the PPO6B nucle
  • immature immature embryos are isolated from maize and contacted with a suspension of Agrobacterium capable of transforming the PPO1B nucleotide sequence, PPO6B nucleotide sequence, The PPO12B nucleotide sequence or the PPO-APB nucleotide sequence is delivered to at least one cell of one of the young embryos (step 1: infection step).
  • the immature embryos were co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • immature embryos are cultured on solid medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 100mg/L after the infection step. , 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH5.8).
  • solid medium MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 100mg/L after the infection step.
  • 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/L
  • agar 8g/L pH5.8
  • At least one antibiotic known to inhibit the growth of Agrobacterium exists in the plant gel (3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step).
  • immature embryos are cultured on solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selection agent (mannose) and the growing transformed callus is selected (step 4: selection step).
  • the immature embryos are cultured on a solid medium for screening with selective agents (MS salt 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, mannose 12.5g/L, 2,4-dichlorobenzene Oxyacetic acid (2,4-D) 1 mg/L, Phytogel 3 g/L, pH 5.8) resulted in selective growth of transformed cells.
  • selective agents MS salt 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, mannose 12.5g/L, 2,4-dichlorobenzene Oxyacetic acid (2,4-D) 1 mg/L, Phytogel 3 g/L, pH 5.8
  • the callus regenerates into plants (step 5: regeneration step)
  • the callus grown on the medium containing the selection agent is cultured on solid medium (MS differentiation medium and MS rooting medium) to regenerated plants.
  • the resistant callus obtained by screening was transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, mannose 5g/L, Phytogel 3g/L, pH5.8), cultured and differentiated at 25°C.
  • MS differentiation medium MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, mannose 5g/L, Phytogel 3g/L, pH5.8
  • Differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, indole-3-acetic acid 1mg/L, plant gel 3g/L , pH 5.8), cultivated at 25°C to a height of about 10 cm, and moved to the greenhouse for cultivation until firm. In the greenhouse, culture was carried out at 28
  • the corn plants that were transferred to the PPO1B nucleotide sequence, the corn plants that were transferred to the PPO6B nucleotide sequence, and the corn plants that were transferred to the PPO12B nucleotide sequence , the corn plant transformed with the PPO-APB nucleotide sequence and the corn plant transformed with the control vector DBN12354N were detected and analyzed.
  • the copy number of PMI gene was detected by Taqman probe fluorescent quantitative PCR method to determine the copy number of PPO gene.
  • wild-type maize plants were used as a control, and detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.
  • Primer 3 gctgtaagagcttactgaaaaattaaca as shown in SEQ ID NO:79 in the sequence listing;
  • Primer 4 cgatctgcaggtcgacgg as shown in SEQ ID NO: 80 in the sequence listing;
  • Probe 2 tctcttgctaagctgggagctcgatcc is shown in SEQ ID NO: 81 in the sequence listing.
  • Table 8-10 The experimental results are shown in Table 8-10.
  • the protoporphyrinogen oxidase PPO1-PPO14 of the present invention can not only endow Arabidopsis with better tolerance to PPO inhibitor herbicides, especially PPO1, PPO6 and PPO12 can Give Arabidopsis, soybean and corn better tolerance to PPO inhibitor herbicides, therefore, the protoporphyrinogen oxidase PPO1-PPO14 can give plants better tolerance; in terms of herbicides , the present invention discloses for the first time that protoporphyrinogen oxidase PPO1-PPO14 can endow plants with higher tolerance to PPO inhibitor herbicides, and can tolerate at least 4 times the field concentration of oxyfluorfen and phenpyrim Sulfennacil, flufentrafen and sulfentrazone with 2 times the field concentration have broad application prospects in plants.

Abstract

本发明涉及一种原卟啉原氧化酶的用途，所述控制杂草的方法包括将含有有效剂量PPO抑制剂的除草剂施加到存在至少一种转基因植物的田地中，所述转基因植物在其基因组中包含编码原卟啉原氧化酶的多核苷酸序列，所述转基因植物与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。本发明原卟啉原氧化酶PPO1-PPO14对PPO抑制剂除草剂具有较高的耐受性，且含有编码原卟啉原氧化酶多核苷酸序列的植物对PPO抑制剂除草剂的耐受性强，其对4倍大田浓度的乙氧氟草醚、苯嘧磺草胺和丙炔氟草胺以及2倍大田浓度的甲磺草胺几乎全部表现出高抗的耐受性。因此在植物上应用前景广阔。

Description

原卟啉原氧化酶的用途 技术领域
本发明涉及一种原卟啉原氧化酶的用途，特别是涉及一种来源于原核生物的原卟啉原氧化酶赋予植物对PPO抑制剂除草剂具有耐受性的方法及用途。
背景技术
卟啉生物合成途径用于合成在植物代谢中起重要作用的叶绿素和血红素，并且该途径发生在叶绿体中。在该途径中，原卟啉原氧化酶(简称PPO)催化原卟啉原IX氧化成原卟啉IX。在原卟啉IX产生之后，原卟啉IX通过镁螯合酶与镁结合来合成叶绿素，或通过铁螯合酶与铁结合来合成血红素。
通过抑制PPO而发挥作用的除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂、三嗪酮类PPO抑制剂除草剂以及其他类型PPO抑制剂除草剂。在植物中，PPO抑制剂抑制了PPO的酶活性，导致叶绿素和血红素的合成受到抑制，并且导致底物原卟啉原IX的积累，所积累的原卟啉原IX从叶绿体快速输出到细胞质，细胞质中的原卟啉原IX在非酶促反应下转化为原卟啉IX并进一步在光和氧分子的存在下生成高反应性的单线态氧( 1O 2)，它们会破坏细胞膜并迅速导致植物细胞的死亡。
用于提供耐受PPO抑制剂除草剂植物的方法主要包括：1)使用可以将除草剂或其活性代谢物转化成无毒产物的酶，将除草剂脱毒。2)过表达敏感性PPO，使得鉴于此酶的动力学常数，在植物中产生相对于除草剂足够的靶酶量，以致尽管存在PPO抑制剂除草剂，但这些敏感性PPO与该PPO抑制剂除草剂是充分作用的，从而具有足够的可供使用的功能性酶。3)提供一种功能性PPO，该功能性PPO对于除草剂或其活性代谢物是较不敏感的，但其保留了催化原卟啉原IX氧化为原卟啉IX的性质。关于功能性PPO类，虽然一种给定的功能性PPO可以提供一个有用水平的对于一些PPO抑制剂除草剂的耐受性，但是相同的功能性PPO可能不足以提供一个商业化水平的对于一种不同的、更希望的PPO抑制剂除草剂的耐受性；例如，PPO抑制剂除草剂可以在它们控制的杂草范围、它们的制造成本、以及它们的环境友好性方面是不同的。因此，需要用于向不同的作物和作物品种赋予PPO抑制剂除草剂耐受性的新方法。
发明内容
本发明的目的是提供一种原卟啉原氧化酶的用途，所述原卟啉原氧化酶来源于原核生物，且转入编码本发明所述原卟啉原氧化酶的多核苷酸序列的植物对PPO抑制剂除草剂具有较好的耐受性。
为实现上述目的，本发明提供了一种控制杂草的方法，包括将含有有效剂量PPO抑制剂的除草剂施加到存在至少一种转基因植物的田地中，所述转基因植物在其基因组中包含编码原卟啉原氧化酶的多核苷酸序列，所述转基因植物与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量，其中所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
优选地，所述转基因植物包括单子叶植物和双子叶植物；更优选地，所述转基因植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；进一步优选地，所述转基因植物为草甘膦耐受性植物，所述杂草为草甘膦抗性杂草；
优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
优选地，所述原卟啉原氧化酶的多核苷酸序列具有：
(a)编码与选自由SEQ ID NO:1-14具有至少88％序列同一性的氨基酸序列的多核苷酸序列，所述多核苷酸序列不包括SEQ ID NO:15-28；或
(b)SEQ ID NO:29-42或SEQ ID NO:62-64任意一种所示的多核苷酸序 列。
进一步地，所述转基因植物还包括至少一种不同于编码所述原卟啉原氧化酶的多核苷酸序列的编码第二种除草剂耐受性蛋白质的第二种多核苷酸。
所述第二种多核苷酸编码选择标记蛋白质、合成活性蛋白质、分解活性蛋白质、抗生物胁迫蛋白质、抗非生物胁迫蛋白质、雄性不育蛋白质、影响植物产量的蛋白质和/或影响植物品质的蛋白质。
具体地，所述第二种多核苷酸编码5-烯醇丙酮酰莽草酸-3-磷酸合酶、草甘膦氧化还原酶、草甘膦-N-乙酰转移酶、草甘膦脱羧酶、草铵膦乙酰转移酶、α酮戊二酸依赖性双加氧酶、麦草畏单加氧酶、4-羟基苯丙酮酸双加氧酶、乙酰乳酸合酶和/或细胞色素类蛋白质。
可选择地，所述含有有效剂量PPO抑制剂的除草剂还包括草甘膦除草剂、草铵膦除草剂、植物生长素类除草剂、禾本科除草剂、发芽前选择性除草剂和/或发芽后选择性除草剂。
为实现上述目的，本发明还提供了一种控制杂草生长的种植组合，包括PPO抑制剂除草剂和至少一种转基因植物，将含有有效剂量的所述PPO抑制剂除草剂施加到存在所述至少一种转基因植物的田地中，所述转基因植物在其基因组中包含编码原卟啉原氧化酶的多核苷酸序列，所述转基因植物与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量，其中所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
优选地，所述转基因植物包括单子叶植物和双子叶植物；更优选地，所述转基因植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；进一步优选地，所述转基因植物为草甘膦耐受性植物，所述杂草为草甘膦抗性杂草；
优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉 酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
优选地，所述原卟啉原氧化酶的多核苷酸序列具有：
(a)编码与选自由SEQ ID NO:1-14具有至少88％序列同一性的氨基酸序列的多核苷酸序列，所述多核苷酸序列不包括SEQ ID NO:15-28；或
(b)SEQ ID NO:29-42或SEQ ID NO:62-64任意一种所示的多核苷酸序列。
进一步地，所述转基因植物还包括至少一种不同于编码所述原卟啉原氧化酶的多核苷酸序列的编码第二种除草剂耐受性蛋白质的第二种多核苷酸。
所述第二种多核苷酸编码选择标记蛋白质、合成活性蛋白质、分解活性蛋白质、抗生物胁迫蛋白质、抗非生物胁迫蛋白质、雄性不育蛋白质、影响植物产量的蛋白质和/或影响植物品质的蛋白质。
具体地，所述第二种多核苷酸编码5-烯醇丙酮酰莽草酸-3-磷酸合酶、草甘膦氧化还原酶、草甘膦-N-乙酰转移酶、草甘膦脱羧酶、草铵膦乙酰转移酶、α酮戊二酸依赖性双加氧酶、麦草畏单加氧酶、4-羟基苯丙酮酸双加氧酶、乙酰乳酸合酶和/或细胞色素类蛋白质。
可选择地，所述含有有效剂量PPO抑制剂的除草剂还包括草甘膦除草剂、草铵膦除草剂、植物生长素类除草剂、禾本科除草剂、发芽前选择性除草剂和/或发芽后选择性除草剂。
为实现上述目的，本发明还提供了一种产生耐受PPO抑制剂除草剂的植物的方法，包括向植物的基因组中引入编码原卟啉原氧化酶的多核苷酸序列，当含有有效剂量PPO抑制剂的除草剂施加到至少存在所述植物的田地中，所述植物与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量，其中所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的 氨基酸序列；
优选地，所述引入的方法包括遗传转化方法、基因组编辑方法或基因突变方法；
优选地，所述植物包括单子叶植物和双子叶植物；更优选地，所述植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；
优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
为实现上述目的，本发明还提供了一种培养耐受PPO抑制剂除草剂植物的方法，包括：
种植至少一个植物繁殖体，所述植物繁殖体的基因组中包括编码原卟啉原氧化酶的多核苷酸序列，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
使所述植物繁殖体长成植株；
将含有有效剂量PPO抑制剂的除草剂施加到至少包含所述植株的田地中，收获与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
优选地，所述植物包括单子叶植物和双子叶植物；更优选地，所述植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；
优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
为实现上述目的，本发明还提供了一种用于保护植物免受由PPO抑制剂除草剂引起的损伤或赋予植物PPO抑制剂除草剂耐受性的方法，包括将含有有效剂量PPO抑制剂的除草剂施加到存在至少一种转基因植物的田地中，所述转基因植物在其基因组中包含编码原卟啉原氧化酶的多核苷酸序列，所述转基因植物与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量，其中所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
优选地，所述转基因植物包括单子叶植物和双子叶植物；更优选地，所述转基因植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；
优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
为实现上述目的，本发明还提供了一种原卟啉原氧化酶在赋予植物PPO抑制剂除草剂耐受性中的用途，所述原卟啉原氧化酶与选自由SEQ ID NO:1 -14组成的组的氨基酸序列具有至少88％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
优选地，所述原卟啉原氧化酶在赋予植物PPO抑制剂除草剂耐受性中的用途包括将含有有效剂量PPO抑制剂的除草剂施加到存在至少一种转基因植物的田地中，所述转基因植物在其基因组中包含编码所述原卟啉原氧化酶的多核苷酸序列，所述转基因植物与其他不具有编码所述原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量；
优选地，所述植物包括单子叶植物和双子叶植物；更优选地，所述植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；
优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
优选地，所述原卟啉原氧化酶的多核苷酸序列具有：
(a)编码与选自由SEQ ID NO:1-14具有至少88％序列同一性的氨基酸序列的多核苷酸序列，所述多核苷酸序列不包括SEQ ID NO:15-28；或
(b)SEQ ID NO:29-42或SEQ ID NO:62-64任意一种所示的多核苷酸序列。
作为具体的实施方式，PPO抑制剂除草剂也即“PPO抑制剂类除草剂”可以是选自由以下组成的组中的一种或多种，但不限于此：二苯醚类(草枯醚、甲氧除草醚(chlomethoxyfen)、甲羧除草醚(Bifenox)、乙氧氟草醚(oxyfluorfen)、三氟羧草醚及其盐和酯、氟磺胺草醚(Fomesafen)、乳氟禾草灵(lactofen)、乙羧氟草醚(fluoroglycofen-ethyl)、氯氟苯醚、苯草醚 (aclonifen)、治草醚(bifenox)、氯乳醚(ethoxyfen)、氯除草醚(chlorintrofen)、氟硝磺酰胺(halosafen))；噁二唑酮类(噁草酮(oxadiazon)、丙炔噁草酮(oxadiargyl))；N-苯基酞酰胺亚胺类(丙炔氟草胺(flumioxazin)、氟烯草酸(flumiclorac-pentyl)、吲哚酮草酯(cinidon-ethyl))；噁唑啉酮类(环戊噁草酮(pentoxazone))；苯基吡唑类(异丙吡草酯(fluazolate)、吡草醚)；脲嘧啶类(双苯嘧草酮、氟丙嘧草酯、苯嘧磺草胺(saflufenacil))；噻二唑类(噻二唑草胺(thidiazimin)、嗪草酸甲酯(fluthiacet))；三唑啉酮类(唑啶草酮(azafenidin)、甲磺草胺(sulfentrazone)、唑酮草酯(carfentrazone))；三嗪酮类(Trifludimoxazin)；其他类(氟哒嗪草酯(flufenpyr-ethyl)、双唑草腈(pyraclonil))。
本发明中使用的冠词“一种”和“一个”是指一个(种)或多于一个(种)(即至少一个)。例如，“一种要素”表示一种或多种要素(元件)。此外，术语“包括”或其变体例如“包括了”或“包含”应被理解为是指包括一种所述要素、整数或步骤，或一组要素、整数或步骤，但是不排除任何其他要素、整数或步骤，或者成组的要素、整数或步骤。
本发明中术语“除草剂不敏感的”是指在一种或多种PPO抑制剂除草剂的存在下，原卟啉原氧化酶维持其酶活性中的至少一部分的能力。原卟啉原氧化酶的酶活性可通过本领域已知的任何手段来测量，例如在一种或多种PPO抑制剂除草剂的存在下通过荧光、高效液相色谱法(HPLC)或质谱法(MS)测量原卟啉原氧化酶产物的生成量或原卟啉原氧化酶底物的消耗量来测定酶活性。“除草剂不敏感的”可以是对于特定除草剂的完全或部分不敏感性，并且可表达为对于特定PPO抑制剂除草剂的耐受性或不敏感性百分比。
本发明中术语“植物、种子、植物组织或细胞的除草剂耐受性”或“耐除草剂的植物、种子、植物组织或细胞”是指施用除草剂时，植物、种子、植物组织或细胞抵抗除草剂作用的能力。例如，耐除草剂的植物可以在除草剂的存在下存活或继续生长。植物、种子、植物组织或细胞的除草剂耐受性可以通过将植物、种子、植物组织或细胞与合适的对照进行比较来测量。例如，可以通过将除草剂施用于包含编码能够赋予除草剂耐受性的蛋白质的DNA分子的植物(试验植物)和不包含编码能够赋予除草剂耐受性的蛋白质的DNA分子的植物(对照植物)，然后比较两种植物的植物损伤来测量或评估除草剂耐受性，其中试验植物的除草剂耐受性由损伤率与对照植物的损伤率相比降低来指示。与对照植物、种子、植物组织或细胞相比，耐除草剂的植物、种子、植物组织或细胞对除草剂毒性作用表现出的反应降低。术语“除草剂耐受性性状”是指与野生型植物相比赋予植物改善的除草剂耐受性的转基因性状。可以产生的具有本发明的除草剂耐受性性状的植物包括，例如任何植 物，包括作物植物，诸如燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄和拟南芥。
本发明的DNA分子可以完全或部分通过本领域已知的方法合成和修饰，特别是在需要提供用于DNA操作的序列(诸如限制酶识别位点或基于重组的克隆位点)、植物优选序列(诸如植物密码子使用或Kozak共有序列)或用于DNA构建体设计的序列(诸如间隔区或接头序列)的情况下。本发明包括编码选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性、至少90％序列同一性，至少91％序列同一性，至少92％序列同一性，至少93％序列同一性，至少94％序列同一性，至少95％序列同一性，至少96％序列同一性，至少97％序列同一性，至少98％序列同一性和至少99％序列同一性的蛋白质的DNA分子，优选蛋白。术语“序列同一性百分比”或“序列同一性％”是指在两个序列进行比对时，与试验序列(或其互补链)相比，参考序列或查询序列(或其互补链)的蛋白质序列中相同氨基酸的百分比。用于序列对比的方法在本领域中是熟知的并且可以使用数学算法来完成，这些数学算法如Myers and Miller(1988)CABIOS 4:11-17的算法；Smith et al.(1981)Adv.Appl.Math.2:482的局部比对算法；Need eman and Wunsch(1970)J.Mol.Biol.48:443-453的全局比对算法；以及Karlin and Altschul(1990)Proc.Natl.Acad.Sci.USA 872264的算法，如在Karlin and Altschul(1993)Proc.Natl.Acad.Sci.USA 90:5873-5877中的修改。对于序列比较可以利用这些数学算法的电脑实现方式以确定序列的同源性，此类实现方式包括但不限于：PC/Gene程序中的CLUSTAL(从Intelligenetics,Mountain View,California可获得)；ALLGN程序(版本2.0)以及GCG Wisconsin Genetics Software Package版本10中的GAP、BESTFIT、BLAST、FASTA以及TFASTA(从Accelrys Inc.,9685 Scranton Road,San Diego,California,USA获得)。序列同一性百分比表示为同一性分数乘以100。
本发明中所述乙氧氟草醚(oxyfluorfen)，是指2-氯-1-(3-乙氧基-4-硝基苯氧基)-4-三氟甲基苯，为无色结晶固体。属于二苯醚类超低用量选择性、芽前芽后触杀型PPO抑制剂除草剂，可制成乳油使用。杂草主要通过胚芽鞘、中胚轴吸收药剂致死。乙氧氟草醚可以有效防治水稻、大豆、玉米、棉花、蔬菜、葡萄、果树等作物田间的杂草，可防止的杂草包括但不限于，稗草、田菁、旱雀麦、狗尾草、曼陀罗、匍匐冰草、豚草、刺黄花捻、苘麻、田芥菜单子叶和阔叶杂草。
本发明中所述有效剂量乙氧氟草醚是指180-720g ai/ha使用，包括190-700g ai/ha、250-650g ai/ha、300-600g ai/ha或400-500g ai/ha。
本发明中所述苯嘧磺草胺(saflufenacil)，是指N'-[2-氯-4-氟-5-(3-甲基-2,6-二氧-4-(三氟甲基)-3,6-二氢-1(2H)-嘧啶)苯甲酰]-N-异丙基-N-甲基硫酰胺，为浅褐色挤条颗粒状固体。属于脲嘧啶类灭生性PPO抑制剂除草剂，可制成70％水分散颗粒剂型。苯嘧磺草胺可有效防治多种阔叶杂草，包括对草甘膦、ALS和三嗪类产生抗性的杂草，其具有很快的灭生作用且土壤残留会降解迅速。
本发明中所述有效剂量苯嘧磺草胺是指以25-100g ai/ha使用，包括30-95g ai/ha、40-90g ai/ha、50-85g ai/ha或60-80g ai/ha。
本发明中所述丙炔氟草胺(Flumioxazin)，是指2-[7-氟代-3,4-二氢-3-氧代-4-(2-丙炔基)-2H-1,4-苯并嗪-6-基]-4,5,6,7-四氢-1H-异吲哚-1,3(2H)-二酮。属于N-苯基酞酰胺亚胺类幼芽和叶片吸收型PPO抑制剂除草剂，常用剂型为50％可湿性粉剂和48％悬浮剂。丙炔氟草胺可有效防治1年生阔叶杂草和部分禾本科杂草。其在环境中易降解，对后茬作物安全。
本发明中所述有效剂量丙炔氟草胺是指以60-240g ai/ha使用，包括70-220g ai/ha、85-200g ai/ha、90-185g ai/ha或100-150g ai/ha。
本发明中所述甲磺草胺(Sulfentrazone)，是指N-(2,4-二氯-5-(4-二氟甲基-4,5-二氢-3-甲基-5-氧代-1H-1,2,4-三唑-1-基)苯基)甲磺酰胺，为棕黄色固体。属于三唑啉酮类PPO抑制剂除草剂，常用剂型为38.9％、44.5％悬浮剂。甲磺草胺可用于防治玉米、高粱、大豆、花生等田中牵牛、反枝苋、藜、曼陀罗、马唐、狗尾草、苍耳、牛筋草、香附子等1年生阔叶杂草、禾本科杂草和莎草等。
本发明中所述有效剂量甲磺草胺是指以450-900g ai/ha使用，包括500-850g ai/ha、550-700g ai/ha、500-685g ai/ha或550-650g ai/ha。
本发明中，术语“抗性”是可遗传的，并允许植物在除草剂对给定植物进行一般除草剂有效处理的情况下生长和繁殖。正如本领域技术人员所认可的，即使给定植物受到除草剂处理的一定程度损伤，如很少的坏死、溶解、萎黄或其它损伤，但至少没有在产量上有显著影响，植物仍可被认为“抗性”，也即给定植物具有的抵抗除草剂诱导的各种程度损伤的提高的能力，而在同样的除草剂剂量下一般导致相同基因型野生型植物损伤。本发明中术语“耐性”或“耐受性”比术语“抗性”更广泛，并包括“抗性”。
本发明中所述抗生物胁迫蛋白质是指抵抗由其他生物所施加的胁迫的蛋白质，如昆虫抗性蛋白质、(病毒、细菌、真菌、线虫)疾病抗性蛋白质等。
本发明中所述抗非生物胁迫蛋白质是指抵抗外界环境所施加的胁迫的蛋白质，如对除草剂、干旱、热、寒冷、冰冻、盐胁迫、氧化胁迫等具有耐性的蛋白质。
本发明中所述影响植物品质的蛋白质是指影响植物输出性状的蛋白质， 如改进淀粉、油、维生素等质量和含量的蛋白质、提高纤维品质的蛋白质等。
此外，包含编码原卟啉原氧化酶的多核苷酸序列的表达盒在植物中还可以与至少一种编码除草剂耐受性基因的蛋白质一起表达，所述除草剂耐受性基因包括但不限于，5-烯醇丙酮酰莽草酸-3-磷酸合酶(EPSPS)、草甘膦氧化还原酶(GOX)、草甘膦-N-乙酰转移酶(GAT)、草甘膦脱羧酶、草铵膦乙酰转移酶(PAT)、α酮戊二酸依赖性双加氧酶(AAD)、麦草畏单加氧酶(DMO)、4-羟苯基丙酮酸双加氧酶(HPPD)、乙酰乳酸合酶(ALS)和/或细胞色素类蛋白质(P450)。
本发明中所述“草甘膦”是指N-膦酰甲基甘氨酸和它的盐，用“草甘膦除草剂”处理是指使用任何一种含有草甘膦的除草剂制剂进行处理。草甘膦的商业制剂包括但不限于，
Figure PCTCN2022089519-appb-000001
(作为异丙胺盐的草甘膦)、
Figure PCTCN2022089519-appb-000002
(作为钾盐的草甘膦)、
Figure PCTCN2022089519-appb-000003
和
Figure PCTCN2022089519-appb-000004
(作为胺盐的草甘膦)、
Figure PCTCN2022089519-appb-000005
(作为钠盐的草甘膦)和
Figure PCTCN2022089519-appb-000006
(作为三甲基硫盐的草甘膦)。
本发明中所述有效剂量草甘膦是指以200-1600g ae/ha使用，包括250-1600g ae/ha、300-1600g ae/ha、500-1600g ae/ha、800-1500g ae/ha、1000-1500g ae/ha或1200-1500g ae/ha。
本发明中所述“草铵膦”又名草丁膦，是指2-氨基-4-[羟基(甲基)膦酰基]丁酸铵，用“草铵膦除草剂”处理是指使用任何一种含有草铵膦的除草剂制剂进行处理。
本发明中所述有效剂量草铵膦是指以200-800g ae/ha使用，包括200-750g ae/ha、250-700g ae/ha、300-700g ae/ha、350-650g ae/ha或400-600g ae/ha。
本发明中植物生长素类除草剂模拟或如同称为生长素的天然植物生长调节剂起作用，其影响细胞壁可塑性和核酸代谢，从而导致不受控制的细胞分裂和生长。由植物生长素类除草剂引起的损伤症状包括茎和柄的偏上性弯曲或扭曲、叶成杯形或卷曲、以及异常的叶形状和叶脉。植物生长素类除草剂包括但不限于，苯氧基羧酸化合物、苯甲酸化合物、吡啶羧酸化合物、喹啉羧酸化合物或草除灵乙酯化合物。典型地，植物生长素类除草剂为麦草畏、2,4-二氯苯氧基乙酸(2,4-D)、(4-氯-2-甲基苯氧基)乙酸(MCPA)和/或4-(2,4-二氯苯氧基)丁酸(2,4-DB)。
本发明中所述“麦草畏”(Dicamba)是指3,6-二氯-邻-茴香酸或3,6-二氯-2-甲氧基苯甲酸及其酸和盐。其盐包括异丙胺盐、二甘醇铵盐、二甲胺盐、钾盐和钠盐。麦草畏的商业制剂包括但不限于，
Figure PCTCN2022089519-appb-000007
(作为DMA盐)、
Figure PCTCN2022089519-appb-000008
(BASF，作为DGA盐)、VEL-58-CS-11 TM和
Figure PCTCN2022089519-appb-000009
(BASF，作为DGA盐)。
本发明中所述2,4-D是广谱、相对便宜且强力的阔叶除草剂，其已经在农业和非作物条件下用于广谱阔叶杂草控制超过65年。2,4-D对不同植物具有不同水平的选择性(如双子叶植物比禾本科植物更敏感)。通常植物缓慢代谢2,4-D，因此靶位点的不同活性更可能解释植物对2,4-D不同的应答。2,4-D的植物代谢一般通过两步代谢实现，一般是羟基化后接着与氨基酸或葡萄糖缀合。
本发明中所述发芽前选择性除草剂包括但不限于，乙酰苯胺、乙草胺、乙酰乳酸合酶抑制剂和二硝基苯胺。
本发明中所述发芽后选择性除草剂包括但不限于，烟嘧磺隆、砜嘧磺隆、和精喹禾灵。
本发明中除草剂的施用量随土壤结构、pH值、有机物含量、耕作系统和杂草的大小而变化，并且通过查看除草剂标签上合适的除草剂施用量来确定。
本发明中术语“赋予”是指向植物提供特征或性状，如除草剂耐受性和/或其它所希望的性状。
本发明中术语“异源的”是指来自另一个来源。在DNA的背景下，“异源的”是指任何外来的“非自身”DNA，包括来自相同种类的另一个植物的DNA。例如，在本发明中，可以利用转基因的方法在大豆植物中表达大豆PPO基因，该大豆PPO基因仍被认为“异源的”DNA。
本发明中术语“核酸”包括涉及的单链或双链形式的脱氧核糖核苷酸或核糖核苷酸聚合物，并且除非另有限制，包括具有天然核苷酸的基本性质的已知类似物(例如肽核酸)，因为它们以一种类似于天然存在的核苷酸的方式与单链核酸杂交。
本发明中，当术语“编码”或“编码的”用于一种特定核酸的上下文中时，其意指核酸包含必需的信息以指导多核苷酸序列翻译成一种特定蛋白质。用来编码蛋白质的信息通过使用密码子来详细说明。编码蛋白质的核酸可以包含在核酸的翻译区内的非翻译序列(例如内含子)，或者可以缺少此类插入的非翻译序列(例如在cDNA中)。
编码本发明所述原卟啉原氧化酶的DNA序列用于提供本发明的植物、植物细胞以及种子，相比于不包含编码本发明所述原卟啉原氧化酶的DNA序列的相同植物(对照植物)，它们提供了对于多种PPO抑制剂除草剂较好的耐受性。
编码本发明所述原卟啉原氧化酶的基因对于产生耐受PPO抑制剂除草剂的植物是有用的。编码本发明所述原卟啉原氧化酶的基因特别适合在植物中表达，以便向植物赋予除草剂耐受性。
术语“多肽”、“肽”以及“蛋白”在本发明中可互换地使用，指的是氨基酸残 基的聚合物。这些术语应用于氨基酸残基的聚合物，所述氨基酸残基的聚合物中的一个或多个氨基酸残基是一个相应的天然存在的氨基酸的一种人工化学类似物，以及天然存在的氨基酸聚合物。本发明的多肽可以从一种本发明披露的核酸或通过使用标准分子生物学技术来产生。例如，本发明的一种截短的蛋白可以通过在一种适当的宿主细胞中表达本发明的一种重组核酸，或者可选择地通过结合离体方法(如蛋白酶消化和纯化)来产生。
本发明还提供了包括编码所述原卟啉原氧化酶的核酸分子。总体上，本发明包括编码相对于所述原卟啉原氧化酶具有一个或多个保守性氨基酸替换的原卟啉原氧化酶的任何多核苷酸序列。提供功能上相似的氨基酸保守性取代是本领域技术人员所熟知的，以下五组各自包含彼此保守性取代的氨基酸：脂肪族：甘氨酸(G)、丙氨酸(A)、缬氨酸(V)、亮氨酸(L)、异亮氨酸(I)；芳香族：苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)；含硫的：甲硫氨酸(M)、半胱氨酸(C)；碱性的：精氨酸(I)、赖氨酸(K)、组氨酸(H)；酸性的：天冬氨酸(D)、谷氨酸(E)、天冬酰胺(N)、谷氨酰胺(Q)。
因此，具有原卟啉原氧化酶抑制剂除草剂耐受性活性并在严格条件下与本发明编码所述原卟啉原氧化酶的基因杂交的序列包括在本发明中。示例性的，这些序列与本发明序列SEQ ID NO:29-42和SEQ ID NO:62-64至少大约85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更大的序列同源性，本发明编码所述原卟啉原氧化酶的基因不包括SEQ ID NO:15-28。
任何常规的核酸杂交或扩增方法都可以用于鉴定本发明PPO基因的存在。核酸分子或其片段在一定情况下能够与其他核酸分子进行特异性杂交。本发明中，如果两个核酸分子能形成反平行的双链核酸结构，就可以说这两个核酸分子彼此间能够进行特异性杂交。如果两个核酸分子显示出完全的互补性，则称其中一个核酸分子是另一个核酸分子的“互补物”。本发明中，当一个核酸分子的每一个核苷酸都与另一个核酸分子的对应核苷酸互补时，则称这两个核酸分子显示出“完全互补性”。如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在至少常规的“低度严格”条件下退火且彼此结合，则称这两个核酸分子为“最低程度互补”。类似地，如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个核酸分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个核酸分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。
本发明中，基本同源的序列是一段核酸分子，该核酸分子在高度严格条件下能够和相匹配的另一段核酸分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65℃。温度条件和盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明所述严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与编码本发明所述原卟啉原氧化酶的基因发生特异性杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。
本发明中，术语“杂交”或“特异性杂交”是指一种分子在严格条件下仅可与特定的多核苷酸序列结合、双链化或杂交，这是在该序列存在于一种复合混合物(例如，总细胞)DNA或RNA中时进行的。
由于遗传密码子的丰余性，多种不同的DNA序列可以编码相同的氨基酸序列。产生这些编码相同或基本相同的蛋白的可替代DNA序列正在本领域技术人员的技术水平内。这些不同的DNA序列包括在本发明的范围内。所述“基本上相同的”序列是指有氨基酸取代、缺失、添加或插入但实质上不影响除草剂耐受性活性的序列，亦包括保留除草剂耐受性活性的片段。
术语“功能活性”或“活性”在本发明中指本发明用途的蛋白质/酶(单独或与其它蛋白质组合)具有降解或减弱除草剂活性的能力。产生本发明蛋白质的植物优选产生“有效量”的蛋白质，从而在用除草剂处理植物时，蛋白质表达的水平足以给予植物对除草剂(若无特别说明则为一般用量)完全或部分的耐受性。可以以通常杀死靶植物的用量、正常的大田用量和浓度使用除草剂。优选地，本发明的植物细胞和植物被保护免受除草剂处理引起的生长抑制或损伤。本发明的转化植物和植物细胞优选具有PPO抑制剂除草剂的耐受性，即转化的植物和植物细胞能在有效量的PPO抑制剂除草剂存在下生长。
本发明中所述的基因和蛋白质不但包括特定的示例序列，还包括保存了所述特定示例的蛋白质的活性特征的部分和/片段(包括与全长蛋白质相比在内和/或末端缺失)、变体、突变体、变体蛋白质、取代物(有替代氨基酸的蛋白质)、嵌合体和融合蛋白。
本发明术语“变体”意指实质上类似的序列。对于多核苷酸，一种变体包括在参比多核苷酸之内的一个或多个内部位点处的一个或多个核苷酸的缺失和/或添加和/或在除草剂耐受性基因中的一个或多个位点处的一个或多个核苷酸 的替换。本发明中术语“参比多核苷酸或多肽”对应地包括除草剂耐受性多核苷酸序列或氨基酸序列。本发明中术语“天然多核苷酸或多肽”对应地包括天然发生的多核苷酸序列或氨基酸序列。对于核酸分子，保守型变体包括编码本发明所述原卟啉原氧化酶之一的多核苷酸序列(由于遗传密码的简并性)。如这些天然发生的等位基因变体可以使用熟知的分子生物学技术，例如使用以下概述的聚合酶链式反应(PCR)以及杂交技术来鉴定。变体核酸分子还包括合成衍生的核酸分子，例如通过使用定点诱变产生的但是仍然编码本发明的一种原卟啉原氧化酶的核酸序列。通常，本发明的一种特定的核酸分子的变体将具有与该特定的核酸分子至少大约85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同源性，通过序列对比程序和参数来确定同源性。
本发明中“变体蛋白质”意指从一种参比蛋白通过在所述原卟啉原氧化酶中的一个或多个内部位点处的一个或多个氨基酸的缺失或添加和/或在所述原卟啉原氧化酶中的一个或多个位点处的一个或多个氨基酸的替换而衍生的一种蛋白质。由本发明涵盖的变体蛋白是生物学活性的，即它们继续具有本发明所述原卟啉原氧化酶的所希望的活性，即，仍具有所述的原卟啉原氧化酶活性和/或除草剂耐性。此类变体可以产生于例如遗传多态性或产生于人工操作。本发明的一种所述原卟啉原氧化酶的生物活性变体将具有与该所述原卟啉原氧化酶的氨基酸序列的全部的至少大约88％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多的序列同源性，此同源性通过序列比对程序和参数所确定。本发明的一种蛋白质的一种生物活性变体可能不同于以少至1-15个氨基酸残基、少至1-10个(如6-10个)、少至5个(如4、3、2个、或甚至1个)氨基酸残基的蛋白质。
在某些实施例中，编码本发明所述原卟啉原氧化酶或它们的保留了原卟啉原氧化酶活性的变体的原卟啉原氧化酶的核酸序列可以与任何感兴趣的核酸序列的组合进行叠加从而产生具有一种所希望的性状的植物。术语“性状”是指从特定的序列或序列组得到的表型。例如，编码本发明所述原卟啉原氧化酶或保留原卟啉原氧化酶活性的变体的原卟啉原氧化酶的核酸序可以与任何其他的编码赋予一种所希望的性状的多肽的核酸进行叠加，所述性状包括但不限于：对于疾病、昆虫以及除草剂的抗性，对于热和干旱的耐受性，缩短作物成熟时间、改进工业加工(例如，用于将淀粉或生物质转化为可发酵的糖类)以及改进的农艺学品质(例如，高油含量和高蛋白含量)。
本领域技术人员所熟知的，两种或更多作用模式的组合在提高受控杂草谱和/或天然更具耐受性物种或抗性杂草物种上的益处还可扩展到通过人工(转基因或非转基因)在作物中产生除PPO耐受性作物外的除草剂耐受性的 化学品。事实上，可以单独或以多重组合叠加编码以下抗性的性状以提供有效控制或防止杂草演替对除草剂产生抗性能力：草甘膦抗性(如抗性植物或细菌EPSPS、GOX、GAT)、草铵膦抗性(如PAT、Bar)、乙酰乳酸合酶(ALS)抑制性除草剂抗性(如咪唑啉酮、磺酰脲、三唑嘧啶、磺苯胺、嘧啶硫代苯甲酸和其它化学品抗性基因如AHAS、Csrl、SurA等)、苯氧基生长素类除草剂抗性(如芳氧基链烷酸酯双加氧酶-AAD)、麦草畏除草剂抗性(如麦草畏单加氧酶-DMO)、溴草腈抗性(如Bxn)、对八氢番茄红素去饱和酶(PDS)抑制剂的抗性、对光系统Ⅱ抑制性除草剂的抗性(如psbA)、对光系统Ⅰ抑制性除草剂的抗性、对4-羟苯基丙酮酸双加氧酶抑制性除草剂抗性(如HPPD)、对苯脲除草剂的抗性(如CYP76B1)、二氯甲氧苯酸降解酶等等。
草甘膦被广泛地使用，因为它控制非常广谱的阔叶和禾本科杂草物种。然而，在草甘膦耐性作物和非作物应用中重复使用草甘膦已经(而且仍将继续)选择使杂草演替为天然更具有耐性的物种或草甘膦抗性生物型。多数除草剂抗性管理策略建议使用有效用量的罐混除草剂伴侣作为延缓出现抗性杂草的方法，所述除草剂伴侣提供对同一物种的控制，但具有不同的作用模式。将编码本发明所述原卟啉原氧化酶的基因与草甘膦耐性性状(和/或其他除草剂耐性性状)叠加可通过允许对同一作物选择性使用草甘膦和PPO抑制剂除草剂(如乙氧氟草醚、苯嘧磺草胺和丙炔氟草胺)而实现对草甘膦耐性作物中草甘膦抗性杂草物种(被一种或多种PPO抑制剂除草剂控制的阔叶杂草物种)的控制。这些除草剂的应用可以是在含有不同作用模式的两种或更多除草剂的罐混合物中同时使用、在连续使用(如种植前、出苗前或出苗后)中单个除草剂组合物的单独使用(使用的间隔时间范围从2小时到3个月)，或者备选地，可以在任何时间(从种植作物7个月内到收获作物时(或对于单个除草剂为收获前间隔，取最短者))使用代表可应用每种化合类别的任意数目除草剂的组合。
在控制阔叶杂草中具有灵活性是很重要的，即使用时间、单个除草剂用量和控制顽固或抗性杂草的能力。作物中与草甘膦抗性基因/编码本发明所述原卟啉原氧化酶的基因叠加的草甘膦应用范围可以从250至2500g ae/ha；PPO抑制剂除草剂(一种或多种)可按照从10-1000g ai/ha。这些应用的时间的最佳组合取决于具体的条件、物种和环境。
除草剂制剂(如酯、酸或盐配方或可溶浓缩剂、乳化浓缩剂或可溶液体)和罐混添加剂(如佐剂或相容剂)可显著影响给定的除草剂或一种或多种除草剂的组合的杂草控制。任意前述除草剂的任意化学组合均在本发明的范围内。
此外，可以将编码本发明所述原卟啉原氧化酶的基因单独或与其它除草 剂耐受作物特征叠加后再与一种或多种其它输入(如昆虫抗性、真菌抗性或胁迫耐受性等)或输出(如提高的产量、改进的油量、提高的纤维品质等)性状叠加。因此，本发明可用于提供以灵活且经济地控制任何数目的农学害虫的能力和提高作物品质的完整农学解决方案。
这些叠加的组合可以通过任何方法来产生，这些方法包括但不限于：通过常规的或顶交方法的杂交育种植物或遗传转化。如果这些序列是通过遗传转化这些植物来进行叠加的，所感兴趣的多核苷酸序列可以在任何时间并且以任何次序进行组合。例如，包括一个或多个所希望的性状的转基因植物可以用做通过后续转化而引入另外性状的靶标。这些性状可以在一个共转化方案中与由表达盒的任何组合提供的感兴趣的多核苷酸同时引入。例如，如果将引入两个序列，这两个序列可以包含在分开的表达盒(反式)中或包含在相同的表达盒(顺式)中。这些序列的表达可以通过相同的启动子或通过不同的启动子来驱动。在某些情况下，可能希望的是引入一个抑制所感兴趣的多核苷酸的表达的表达盒。这可以与其他的抑制表达盒或过量表达盒的任何组合进行组合以在该植物中产生所希望的性状组合。进一步认识到的是，多核苷酸序列可以在一个所希望的基因组位置处使用位点特异性重组系统进行叠加。
本发明编码所述原卟啉原氧化酶的基因对PPO抑制剂除草剂具有较高的耐受性，是重要的除草剂耐受作物和选择标记物特征可能性的基础。
本发明中术语“表达盒”是指能够在适当的宿主细胞中指引一种特定多核苷酸序列表达的一种核酸分子，包括有效连接到感兴趣的多核苷酸序列(即，一种单独地或与一种或多种编码赋予所希望的性状的多肽的额外的核酸分子相组合而编码一种本发明所述原卟啉原氧化酶或保留了原卟啉原氧化酶活性的变体蛋白的多核苷酸)的启动子，该感兴趣的多核苷酸序列有效连接到终止信号。该编码区通常对一种感兴趣的蛋白质进行编码，但是还可以对一种感兴趣的功能性RNA进行编码，例如在正义或反义方向上的反义RNA或一种非翻译RNA。包含该感兴趣的多核苷酸序列的表达盒可以是嵌合的，意味着至少一个它的组分相对于至少一个它的其他组分是异源的。该表达盒还可以是一种天然发生的表达盒，但一定是以在对异源表达有用的重组体形式而获得的。然而，典型地，该表达盒对于宿主是异源的，即，该表达盒的特定DNA序列并不天然发生于该宿主细胞中，并且必须已通过转化事件而被引入新宿主细胞中。在该表达盒中多核苷酸序列的表达可以是在组成型启动子或诱导型启动子的控制之下，该启动子只有当该宿主细胞暴露于一些特殊的外界刺激时才引发转录。另外，该启动子对于一种特定的组织或器官或发育阶段也是特异性的。
本发明涵盖了用能够表达一种感兴趣的多核苷酸(即一种单独地或与一种或多种编码赋予所希望的性状的多肽的额外的核酸分子相组合而编码一种本发明所述原卟啉原氧化酶或它的保留了原卟啉原氧化酶活性的变体蛋白的多核苷酸)的表达盒来转化植物。该表达盒在5’-3’的转录方向上包括转录和翻译的起始区(即启动子)和多核苷酸开放阅读框。该表达盒可以任选地包括在植物中起作用的转录和翻译终止区(即终止区)。在一些实施方案中，该表达盒包括一种选择标记基因从而允许选择稳定的转化体。本发明的表达构建体还可以包括前导序列和/或允许感兴趣的多核苷酸的诱导型表达的序列。
该表达盒的调控序列有效连接到感兴趣的多核苷酸。本发明中所述调控序列包括但不限于启动子、转运肽、终止子、增强子、前导序列、内含子以及其它可操作地连接到编码所述原卟啉原氧化酶基因的调节序列。
所述启动子为植物中可表达的启动子，所述的“植物中可表达的启动子”是指确保与其连接的编码序列在植物细胞内进行表达的启动子。植物中可表达的启动子可为组成型启动子。指导植物内组成型表达的启动子的示例包括但不限于，来源于花椰菜花叶病毒的35S启动子、玉米Ubi启动子、水稻GOS2基因的启动子等。备选地，植物中可表达的启动子可为组织特异的启动子，即该启动子在植物的一些组织内如在绿色组织中指导编码序列的表达水平高于植物的其他组织(可通过常规RNA试验进行测定)，如PEP羧化酶启动子。备选地，植物中可表达的启动子可为创伤诱导启动子。创伤诱导启动子或指导创伤诱导的表达模式的启动子是指当植物经受机械或由昆虫啃食引起的创伤时，启动子调控下的编码序列的表达较正常生长条件下有显著提高。创伤诱导启动子的示例包括但不限于，马铃薯和西红柿的蛋白酶抑制基因(pinⅠ和pinⅡ)和玉米蛋白酶抑制基因(MPI)的启动子。
所述转运肽(又称分泌信号序列或导向序列)是指导转基因产物到特定的细胞器或细胞区室，对受体蛋白质来说，所述转运肽可以是异源的，例如，利用编码叶绿体转运肽序列靶向叶绿体，或者利用‘KDEL’保留序列靶向内质网，或者利用大麦植物凝集素基因的CTPP靶向液泡。
所述前导序列包含但不限于，小RNA病毒前导序列，如EMCV前导序列(脑心肌炎病毒5’非编码区)；马铃薯Y病毒组前导序列，如MDMV(玉米矮缩花叶病毒)前导序列；人类免疫球蛋白质重链结合蛋白质(BiP)；苜蓿花叶病毒的外壳蛋白质mRNA的不翻译前导序列(AMV RNA4)；烟草花叶病毒(TMV)前导序列。
所述增强子包含但不限于，花椰菜花叶病毒(CaMV)增强子、玄参花叶病毒(FMV)增强子、康乃馨风化环病毒(CERV)增强子、木薯脉花叶病毒 (CsVMV)增强子、紫茉莉花叶病毒(MMV)增强子、夜香树黄化曲叶病毒(CmYLCV)增强子、木尔坦棉花曲叶病毒(CLCuMV)、鸭跖草黄斑驳病毒(CoYMV)和花生褪绿线条花叶病毒(PCLSV)增强子。
对于单子叶植物应用而言，所述内含子包含但不限于，玉米hsp70内含子、玉米泛素内含子、Adh内含子1、蔗糖合酶内含子或水稻Act1内含子。对于双子叶植物应用而言，所述内含子包含但不限于，CAT-1内含子、pKANNIBAL内含子、PIV2内含子和“超级泛素”内含子。
所述终止子可以为在植物中起作用的适合多聚腺苷酸化信号序列，包括但不限于，来源于农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于蛋白酶抑制剂Ⅱ(pinⅡ)基因的多聚腺苷酸化信号序列、来源于豌豆ssRUBISCO E9基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。
本发明中所述“有效连接”表示核酸序列的联结，所述联结使得一条序列可提供对相连序列来说需要的功能。在本发明中所述“有效连接”可以为将启动子与感兴趣的序列相连，使得该感兴趣的序列的转录受到该启动子控制和调控。当感兴趣的序列编码蛋白并且想要获得该蛋白的表达时“有效连接”表示：启动子与所述序列相连，相连的方式使得得到的转录物高效翻译。如果启动子与编码序列的连接是转录物融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得得到的转录物中第一翻译起始密码子是编码序列的起始密码子。备选地，如果启动子与编码序列的连接是翻译融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得5’非翻译序列中含有的第一翻译起始密码子与启动子相连结，并且连接方式使得得到的翻译产物与编码想要的蛋白的翻译开放读码框的关系是符合读码框的。可以“有效连接”的核酸序列包括但不限于：提供基因表达功能的序列(即基因表达元件，例如启动子、5’非翻译区域、内含子、蛋白编码区域、3’非翻译区域、聚腺苷化位点和/或转录终止子)、提供DNA转移和/或整合功能的序列(即T-DNA边界序列、位点特异性重组酶识别位点、整合酶识别位点)、提供选择性功能的序列(即抗生素抗性标记物、生物合成基因)、提供可计分标记物功能的序列、体外或体内协助序列操作的序列(即多接头序列、位点特异性重组序列)和提供复制功能的序列(即细菌的复制起点、自主复制序列、着丝粒序列)。
本发明中所述的植物、植物组织或植物细胞的基因组，是指植物、植物组织或植物细胞内的任何遗传物质，且包括细胞核和质体和线粒体基因组。
在本发明中，术语“植物部分”或“植物组织”包括植物细胞、植物原生质体、植物可以由之再生的植物细胞组织培养物、植物愈伤组织、植物簇以及 在植物或以下植物的部分中完整的植物细胞，这些植物的部分是如胚、花粉、胚珠、种子、叶、花、枝、果实、核、穗、穗轴、外壳、茎、根、根尖、花药等等。
本发明所述PPO蛋白可应用于多种植物中，所述双子叶植物包括但不限于苜蓿、菜豆、花椰菜、甘蓝、胡萝卜、芹菜、棉花、黄瓜、茄子、莴苣、甜瓜、豌豆、胡椒、西葫芦、萝卜、油菜、菠菜、大豆、南瓜、番茄、拟南芥、花生或西瓜；优选地，所述双子叶植物是指黄瓜、大豆、拟南芥、烟草、棉花、花生或油菜。所述单子叶植物包括但不限于玉米、水稻、高粱、小麦、大麦、黑麦、粟、甘蔗、燕麦或草坪草；优选地，所述单子叶植物是指玉米、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦。
在本发明中术语“植物转化”是指将一种抗或耐受除草剂的编码本发明所述原卟啉原氧化酶的核酸分子单独地或与编码赋予所希望的性状的多肽的一种或多种额外的核酸分子结合而克隆到一个表达系统中，它就是被转化到了一种植物细胞中。本发明的受体和目标表达盒可以以多种公知的方法被引入到植物细胞中。在多核苷酸的背景下，术语“引入”(例如，感兴趣的核苷酸构建体)旨在表示以这样一种方式将多核苷酸提供给该植物，使得该多核苷酸获得对一种植物细胞的内部的接近或实现。其中有待引入一个以上的多核苷酸，这些多核苷酸可以作为单个核苷酸构建体的部分而进行组装，或者作为分开的核苷酸构建体，并且可以位于相同的或不同的转化载体上。因此，或作为育种方案的一部分，例如在植物中的在一个单一的转化事件中、在分开的转化事件中可以将这些多核苷酸引入到感兴趣的宿主细胞中。本发明的这些方法并不取决于一种用于引入一个或多个多核苷酸到植物中的具体方法，仅仅是获得这个或这些多核苷酸对于植物的至少一个细胞的内部的接近或实现。在本领域中已知的用于将一个或多个多核苷酸引入到植物中的方法包括但不限于瞬时转化方法、稳定转化方法、病毒介导的方法或基因组编辑技术。
术语“稳定转化”是指将外源基因导入植物基因组，且稳定地整合进该植物及其任何连续世代的基因组中，导致外源基因稳定遗传。
术语“瞬时转化”是指核酸分子或蛋白质导入植物细胞中，执行功能但不整合进植物基因组中，导致外源基因不能稳定遗传。
术语“基因组编辑技术”是指能够对基因组序列进行精确操作，实现基因定点突变、插入、删除等操作的基因组修饰技术。目前基因组编辑技术主要有HE(homing endonuclease，归巢核酸内切酶)、ZFN技术(Zinc-finger nuclease，锌指核酸酶)、TALEN技术(transcription activator-like effector nuclease，转录激活样效应因子核酸酶)、CRISPR技术(Clustered regulatory interspaced short palindromic repeat，成簇规律间隔短回文重复)。
对本领域技术人员而言，用于植物转化的可获得的众多转化载体是已知的，并且与本发明有关的基因可以与任何上述载体结合使用。载体的选择将取决于优选的转化技术以及用于转化的目标种类。对于某些目标种类，可以优选不同的抗生素或除草剂选择标记。在转化中常规使用的选择标记包括赋予对卡那霉素以及相关抗生素或相关除草剂的抗性的nptll基因(此基因被Bevan等人于1983年发表在《自然科学》第304卷184-187页)、赋予对除草剂草丁膦(还被称作草铵膦；参见White等人于1990年发表于《Nucl.AcidsRes》第18卷1062页、Spencer等人于1990年发表于《Theor.Appl.Genet》第79卷625-631页以及美国专利5561236和5276268)抗性的pat和bar基因，赋予对抗生素潮霉素的抗性的hpn基因(Blochinger&Diggelmann,Mol.Cell Biol.4:2929-2931)以及赋予对甲氨蝶呤的抗性的dnfr基因(Bourouis等人1983年于《EMBO J.》第2卷1099-1104页)、赋予对草甘膦的抗性的EPSPS基因(美国专利4940935和5188642)、也赋予对草甘膦的抗性的草甘膦N-乙酰基转移酶(GAT)基因(Castle等人于2004年在《Science》第304卷1151-1154页；美国申请公开专利20070004912，20050246798和20050060767中有描述)以及提供代谢甘露糖的6-磷酸甘露糖异构酶基因(美国专利5767378和5994629有描述)。
用于再生植物的方法在本领域也是熟知的。例如，已经利用Ti质粒载体用于传送外源DNA，以及直接DNA摄入、脂质体、电穿孔、显微注射以及微弹。
本发明中，所述杂草是指在田地中与耕种的转基因植物竞争的植物。
本发明术语“控制”和/或“防治”是指至少将有效剂量的PPO抑制剂除草剂直接施用(例如通过喷雾)到田地中，使杂草发育最小化和/或停止生长。同时，耕种的转基因植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成；优选地，与非转基因的野生型植株相比具有减弱的植物损伤和/或具有增加的植物产量。所述具有减弱的植物损伤，具体表现包括但不限于改善的茎秆抗性、和/或提高的籽粒重量等。所述原卟啉原氧化酶对杂草的“控制”和/或“防治”作用是可以独立存在的，不因其它可“控制”和/或“防治”杂草的物质的存在而减弱和/或消失。具体地，转基因植物(含有编码本发明所述原卟啉原氧化酶的基因)的任何组织同时和/或不同步地，存在和/或产生，所述原卟啉原氧化酶和/或可控制杂草的另一种物质，则所述另一种物质的存在既不影响所述原卟啉原氧化酶对杂草的“控制”和/或“防治”作用，也不能导致所述“控制”和/或“防治”作用完全和/或部分由所述另一种物质实现，而与所述原卟啉原氧化酶无关。
本发明中所述的“植物繁殖体”包括但不限于植物有性繁殖体和植物无性 繁殖体。所述植物有性繁殖体包括但不限于植物种子；所述植物无性繁殖体是指植物体的营养器官或某种特殊组织，其可以在离体条件下产生新植株；所述营养器官或某种特殊组织包括但不限于根、茎和叶，例如：以根为无性繁殖体的植物包括草莓和甘薯等；以茎为无性繁殖体的植物包括甘蔗和马铃薯(块茎)等；以叶为无性繁殖体的植物包括芦荟和秋海棠等。
本发明可赋予植物新除草剂抗性性状，并且未观察到对表型包括产量的不良影响。本发明中植物能耐受住如至少一种受试除草剂2×、3×或4×一般应用水平。这些耐性水平的提高在本发明的范围之内。例如可对本领域已知的多种技术进行可预见到的优化和进一步发展，以增加给定基因的表达。
本发明提供了一种原卟啉原氧化酶的用途，具有以下优点：
1、对除草剂耐受性广。本发明首次公开了原卟啉原氧化酶PPO1-PPO14可以对PPO抑制剂除草剂表现出较高的耐受性，因此在植物上应用前景广阔。
2、对除草剂耐受性强。本发明所述原卟啉原氧化酶PPO1-PPO14对PPO抑制剂除草剂的耐受性强，其对4倍大田浓度的乙氧氟草醚、苯嘧磺草胺和丙炔氟草胺以及2倍大田浓度的甲磺草胺几乎全部表现出高抗的耐受性。
3、对产量影响小。植物对除草剂的耐受性与植物的产量有直接对应关系，高抗植物基本不会受除草剂的影响从而不会影响植物的产量，而中低抗植物的产量相对高抗植物会缩减很多。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明
图1为本发明含有所述PPO1A核苷酸序列的拟南芥重组表达载体DBN12337结构示意图；
图2为本发明对照重组表达载体DBN12337N结构示意图；
图3为本发明含有所述PPO1B核苷酸序列的玉米重组表达载体DBN12354结构示意图；
图4为本发明对照重组表达载体DBN12354N结构示意图。
具体实施方式
下面通过具体实施例进一步说明本发明原卟啉原氧化酶的用途的技术方案。
第一实施例、转基因拟南芥植株的获得和验证
1、获得编码原卟啉原氧化酶的基因
微生物原卟啉原氧化酶PPO1、PPO2、PPO3、PPO4、PPO5、PPO6、PPO7、PPO8、PPO9、PPO10、PPO11、PPO12、PPO13和PPO14的氨基酸序列，如 序列表中SEQ ID NO:1-14所示，编码相应于所述原卟啉原氧化酶PPO1-PPO14的PPO1-PPO14核苷酸序列，如序列表中SEQ ID NO:15-28所示；依据拟南芥和大豆共同偏好性密码子获得编码相应于所述原卟啉原氧化酶PPO1-PPO14的PPO1A-PPO14A核苷酸序列，如序列表中SEQ ID NO:29-42所示；依据玉米偏好性密码子获得编码相应于所述原卟啉原氧化酶PPO1、PPO6和PPO12的PPO1B、PPO6B和PPO12B核苷酸序列，如序列表中SEQ ID NO:62-64所示。
大肠杆菌(Escherichia coli)原卟啉原氧化酶PPO-EC的氨基酸序列，如序列表中SEQ ID NO:43所示；编码相应于所述大肠杆菌原卟啉原氧化酶PPO-EC的PPO-EC核苷酸序列，如序列表中SEQ ID NO:44所示；依据拟南芥和大豆共同偏好性密码子获得编码相应于所述大肠杆菌原卟啉原氧化酶PPO-EC的PPO-ECA核苷酸序列，如序列表中SEQ ID NO:45所示。
拟南芥原卟啉原氧化酶PPO-AT的氨基酸序列，如序列表中SEQ ID NO:46所示；编码相应于所述拟南芥原卟啉原氧化酶PPO-AT的PPO-AT核苷酸序列，如序列表中SEQ ID NO:47所示；依据拟南芥和大豆共同偏好性密码子获得编码相应于所述拟南芥原卟啉原氧化酶PPO-AT的PPO-ATA核苷酸序列，如序列表中SEQ ID NO:48所示。
烟粉虱共生菌(Arsenophonus)原卟啉原氧化酶PPO-AP的氨基酸序列，如序列表中SEQ ID NO:65所示；编码相应于所述烟粉虱共生菌原卟啉原氧化酶PPO-AP的PPO-AP核苷酸序列，如序列表中SEQ ID NO:66所示；依据拟南芥和大豆共同偏好性密码子获得编码相应于所述烟粉虱共生菌原卟啉原氧化酶PPO-AP的PPO-APA核苷酸序列，如序列表中SEQ ID NO:67所示；依据玉米偏好性密码子获得编码相应于所述烟粉虱共生菌原卟啉原氧化酶PPO-AP的PPO-APB核苷酸序列，如序列表中SEQ ID NO:68所示。
2、合成上述核苷酸序列
将所述PPO1A-PPO14A核苷酸序列、PPO-ECA核苷酸序列、PPO-ATA核苷酸序列和PPO-APA核苷酸序列(SEQ ID NO:29-42、SEQ ID NO:45、SEQ ID NO:48和SEQ ID NO:67)的5’和3’端分别连接通用接头引物1：
5’端通用接头引物1：5’-taagaaggagatatacatatg-3’如序列表中SEQ ID NO:49所示；
3’端通用接头引物1：5’-gtggtggtggtggtgctcgag-3’如序列表中SEQ ID NO:50所示。
3、分别构建含有PPO1A-PPO14A核苷酸序列、PPO-ECA核苷酸序列、PPO-ATA核苷酸序列和PPO-APA核苷酸序列的拟南芥重组表达载体
利用限制性内切酶Spe I和Asc I对植物表达载体DBNBC-01进行双酶切 反应，从而对植物表达载体线性化，酶切产物纯化得到线性化的DBNBC-01表达载体骨架(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将连接所述通用接头引物1的所述PPO1A核苷酸序列(SEQ ID NO:29)与所述线性化的DBNBC-01表达载体骨架进行重组反应，操作步骤按照Takara公司In-Fusion无缝连接产品试剂盒(Clontech，CA，USA，CAT：121416)说明书进行，构建成重组表达载体DBN12337，其结构示意图如图1所示(Spec：壮观霉素基因；RB：右边界；eFMV：玄参花叶病毒的34S增强子(SEQ ID NO:51)；prBrCBP：油菜真核延长因子基因1α(Tsf1)的启动子(SEQ ID NO:52)；spAtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:53)；EPSPS：5-烯醇丙酮酸莽草酸-3-磷酸合酶基因(SEQ ID NO:54)；tPsE9：豌豆RbcS基因的终止子(SEQ ID NO:55)；prAtUbi10：拟南芥泛素(Ubiquitin)10基因的启动子(SEQ ID NO:56)；spAtCLP1：拟南芥白化体或浅绿色体转运肽(SEQ ID NO:57)；PPO1A：PPO1A核苷酸序列(SEQ ID NO:29)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:58)；pr35S：花椰菜花叶病毒35S启动子(SEQ ID NO:59)；cPAT：膦丝菌素N-乙酰基转移酶基因(SEQ ID NO:60)；t35S：花椰菜花叶病毒35S终止子(SEQ ID NO:61)；LB：左边界)。
将重组表达载体DBN12337用热激方法转化大肠杆菌T1感受态细胞，其热激条件为：50μL大肠杆菌T1感受态细胞、10μL质粒DNA(重组表达载体DBN12337)，42℃水浴30s；37℃振荡培养1h(100rpm转速下摇床摇动)；然后在含50mg/L壮观霉素(Spectinomycin)的所述LB固体平板(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、琼脂15g/L，用NaOH调pH至7.5)上于温度37℃条件下培养12h，挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、壮观霉素50mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒：将菌液在12000rpm转速下离心1min，去上清液，沉淀菌体用100μl冰预冷的溶液I(25mM Tris-HCl、10mM EDTA(乙二胺四乙酸)、50mM葡萄糖，pH8.0)悬浮；加入200μL新配制的溶液II(0.2M NaOH、1％SDS(十二烷基硫酸钠))，将管子颠倒4次，混合，置冰上3-5min；加入150μL冰冷的溶液III(3M醋酸钾、5M醋酸)，立即充分混匀，冰上放置5-10min；于温度4℃、转速12000rpm条件下离心5min，在上清液中加入2倍体积无水乙醇，混匀后室温放置5min；于温度4℃、转速12000rpm条件下离心5min，弃上清液，沉淀用浓度(V/V)为70％的乙醇洗涤后晾干；加入30μL含RNase(20μg/mL)的TE(10mM Tris-HCl、1mM EDTA，pH8.0)溶解沉淀；于温度37℃下水浴30min，消化RNA；于温度-20℃保存备用。将提取的质粒进行测序鉴定，结果表明重组表达载体DBN12337在Spe I和Asc I位点间的核苷酸序列为序 列表中SEQ ID NO:29所示的核苷酸序列，即所述PPO1A核苷酸序列。
按照上述构建重组表达载体DBN12337的方法，将分别连接所述通用接头引物1的所述PPO2A-PPO14A核苷酸序列、PPO-ECA核苷酸序列、PPO-ATA核苷酸序列和PPO-APA核苷酸序列分别与所述线性化的DBNBC-01表达载体骨架进行重组反应，依次得到重组表达载体DBN12338至DBN12353。测序验证重组表达载体DBN12338至DBN12353中上述核苷酸序列正确插入。
按照上述构建重组表达载体DBN12337的方法，构建对照重组表达载体DBN12337N，其载体结构如图2所示(Spec：壮观霉素基因；RB：右边界；eFMV：玄参花叶病毒的34S增强子(SEQ ID NO:51)；prBrCBP：油菜真核延长因子基因1α(Tsf1)的启动子(SEQ ID NO:52)；spAtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:53)；EPSPS：5-烯醇丙酮酸莽草酸-3-磷酸合酶基因(SEQ ID NO:54)；tPsE9：豌豆RbcS基因的终止子(SEQ ID NO:55)；pr35S：花椰菜花叶病毒35S启动子(SEQ ID NO:59)；cPAT：膦丝菌素N-乙酰基转移酶基因(SEQ ID NO:60)；t35S：花椰菜花叶病毒35S终止子(SEQ ID NO:61)；LB：左边界)。
4、拟南芥重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN12337至DBN12350、DBN12352、DBN12353和上述对照重组表达载体DBN12337N分别用液氮法转化到农杆菌GV3101中，其转化条件为：100μL农杆菌GV3101、3μL质粒DNA(重组表达载体DBN12337至DBN12350、DBN12352、DBN12353、DBN12337N)；置于液氮中10min，37℃温水浴10min；将转化后的农杆菌GV3101接种于LB试管中于温度28℃、转速为200rpm条件下培养2h，涂于含50mg/L的利福平(Rifampicin)和50mg/L的壮观霉素的所述LB固体平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，将提取的质粒进行测序鉴定，结果表明重组表达载体DBN12337至DBN12350、DBN12352、DBN12353、DBN12337N结构完全正确。
5、转基因拟南芥植株的获得
将野生型拟南芥种子悬浮于0.1％(w/v)琼脂糖溶液中。将悬浮的种子在4℃下保存2天以完成对休眠的需要以保证种子同步萌发。用蛭石混合马粪土并用水地下灌溉至湿润，使土壤混合物排水24h。将预处理后的种子种在土壤混合物上并用保湿罩覆盖7天。使种子萌发并在恒温(22℃)恒湿(40-50％)光强度为120-150μmol/m 2s -1的长日照条件(16h光照/8h黑暗)下在温室中培养植物。开始用霍格兰营养液灌溉植物，接着用去离子水灌溉，保持土壤潮湿但不湿透。
使用花浸泡法转化拟南芥。用选取的农杆菌菌落接种一份或多份15-30mL含壮观霉素(50mg/L)和利福平(10mg/L)的LB培养液的预培养物。以220rpm转速将预培养物在温度28℃恒速摇动孵育过夜。每个预培养物用于接种两份500ml含壮观霉素(50mg/L)和利福平(10mg/L)的所述YEP培养液的培养物并将培养物在温度28℃持续摇动孵育过夜。室温以转速约4000rpm离心20min沉淀细胞，弃去得到的上清液。将细胞沉淀轻柔重悬于500mL渗透培养基中，所述渗透培养基含有1/2×MS盐/B5维生素、10％(w/v)蔗糖、0.044μM苄氨基嘌呤(10μL/L(1mg/mL DMSO中的原液))和300μL/L Silvet L-77。将约1月龄的拟南芥植物在含重悬细胞的渗透培养基中浸泡5min，确保浸没最新的花序。接着将拟南芥植物侧面放倒并覆盖，黑暗环境下保湿24h后，在温度22℃以16h光照/8h黑暗的光周期正常培养拟南芥植物。约4周后收获种子。
将新收获的(PPO1A-PPO14A核苷酸序列、PPO-ATA核苷酸序列、PPO-APA核苷酸序列和对照载体DBN12337N)T 1种子在室温干燥7天。将种子种在26.5cm×51cm萌发盘中，每盘接受200mg T 1种子(约10000个种子)，所述种子事先已悬浮于蒸馏水中并在温度4℃下保存2天以完成对休眠的需要以保证种子同步萌发。
用蛭石混合马粪土并用水底部灌溉至湿润，利用重力排水。用移液管将预处理后的种子均匀地种在土壤混合物上，并用保湿罩覆盖4-5天。在使用出苗后喷洒草铵膦(选择共转化的PAT基因)进行最初转化体选择的前1天移去罩。
在7个种植天数后(DAP)并于11DAP再次使用DeVilbiss压缩空气喷嘴以10mL/盘(703L/ha)的喷洒体积用Liberty除草剂(200g ai/L的草铵膦)的0.2％溶液喷洒T 1植物(分别为子叶期和2-4叶期)，以提供每次应用280g ai/ha有效量的草铵膦。在最后喷洒后4-7天鉴定存活株(生长活跃的植物)，并分别移植到用马粪土和蛭石制备的7cm×7cm的方盆中(每盘3-5棵)。用保湿罩覆盖移植的植物3-4天，并如前置于温度22℃培养室中或直接移入温室。接着移去罩并在测试PPO1A-PPO14A核苷酸序列、PPO-ATA核苷酸序列、PPO-APA核苷酸序列和对照载体提供PPO抑制剂除草剂耐受性的能力之前至少1天将植物栽种到温室(温度22±5℃，50±30％RH，14h光照:10h黑暗，最小500μE/m 2s -1天然+补充光)。
6、转基因拟南芥植株的除草剂耐受性效果检测
首先使用草铵膦除草剂选择已转化的拟南芥T 1植株。分别将转入PPO1A核苷酸序列的拟南芥T 1植株(PPO1A)、转入PPO2A核苷酸序列的拟南芥T 1植株(PPO2A)、转入PPO3A核苷酸序列的拟南芥T 1植株(PPO3A)、 转入PPO4A核苷酸序列的拟南芥T 1植株(PPO4A)、转入PPO5A核苷酸序列的拟南芥T 1植株(PPO5A)、转入PPO6A核苷酸序列的拟南芥T 1植株(PPO6A)、转入PPO7A核苷酸序列的拟南芥T 1植株(PPO7A)、转入PPO8A核苷酸序列的拟南芥T 1植株(PPO8A)、转入PPO9A核苷酸序列的拟南芥T 1植株(PPO9A)、转入PPO10A核苷酸序列的拟南芥T 1植株(PPO10A)、转入PPO11A核苷酸序列的拟南芥T 1植株(PPO11A)、转入PPO12A核苷酸序列的拟南芥T 1植株(PPO12A)、转入PPO13A核苷酸序列的拟南芥T 1植株(PPO13A)、转入PPO14A核苷酸序列的拟南芥T 1植株(PPO14A)、转入PPO-ATA核苷酸序列的拟南芥T 1植株(PPO-ATA)、转入PPO-APA核苷酸序列的拟南芥T 1植株(PPO-APA)、转入对照载体的拟南芥T 1植株(对照载体)和野生型拟南芥植株(CK)各24株(播种后18天)分别用3种浓度的乙氧氟草醚(180g ai/ha(1倍大田浓度，1×)、720g ai/ha(4倍大田浓度，4×)和0g ai/ha(水，0×))、3种浓度的苯嘧磺草胺(25g ai/ha(1倍大田浓度，1×)、100g ai/ha(4倍大田浓度，4×)和0g ai/ha(水，0×))、3种浓度的丙炔氟草胺(60g ai/ha(1倍大田浓度，1×)、240g ai/ha(4倍大田浓度，4×)和0g ai/ha(水，0×))和3种浓度的甲磺草胺(450g ai/ha(1倍大田浓度，1×)、900g ai/ha(2倍大田浓度，2×)和0g ai/ha(水，0×))进行喷洒以检测拟南芥的除草剂耐受性。在喷施7天(7DAT)后，根据植株平均损伤百分比等级(植株平均损伤百分比＝叶片损伤面积/叶片总面积×100％)来评价除草剂对每株植株的损伤程度，即药害等级：0级为生长状况和喷施空白溶剂(水)基本一致，1级为植株平均损伤百分比小于10％，2级为植株平均损伤百分比大于10％，3级为植株平均损伤百分比为100％。以植株生长状况划入0级和1级的为高抗植株，以植株生长状况划入2级的为中低抗植株，以植株生长状况划入3级的为不抗植株。实验结果如表1-4所示。
表1、转入PPO1A-PPO14A核苷酸序列、PPO-APA核苷酸序列和PPO-ATA核苷酸序列的拟南芥T 1植株对乙氧氟草醚的耐受性实验结果
Figure PCTCN2022089519-appb-000010
Figure PCTCN2022089519-appb-000011
Figure PCTCN2022089519-appb-000012
对于拟南芥，180g ai/ha乙氧氟草醚除草剂是将敏感植物与具有平均抗性水平的植物区分开来的有效剂量。表1的结果表明，对于不同浓度的乙氧氟草醚，相比于对照载体和CK，PPO1A-PPO14A全部表现出高抗的耐受性，而PPO-APA和PPO-ATA基本不具有耐受性。
表2、转入PPO1A-PPO14A核苷酸序列、PPO-APA核苷酸序列、PPO-ATA核苷酸序列和对照载体的拟南芥T 1植株对苯嘧磺草胺的耐受性实验结果
Figure PCTCN2022089519-appb-000013
Figure PCTCN2022089519-appb-000014
对于拟南芥，25g ai/ha苯嘧磺草胺除草剂是将敏感植物与具有平均抗性水平的植物区分开来的有效剂量。表2的结果表明，相比于对照载体和CK，(1)对于1倍大田浓度的苯嘧磺草胺，PPO1A-PPO14A全部表现出高抗的耐受性，而PPO-APA和PPO-ATA均不具有耐受性；(2)对于4倍大田浓度的苯嘧磺草胺，PPO1A-PPO7A和PPO9A-PPO14A全部表现出高抗的耐受性(PPO8A中仅有2株表现出中低抗的耐受性，其他22株植株全部表现出高抗的耐受性)，而PPO-APA和PPO-ATA均不具有耐受性。
表3、转入PPO1A-PPO14A核苷酸序列、PPO-APA核苷酸序列、PPO-ATA核苷酸序列和对照载体的拟南芥T 1植株对丙炔氟草胺的耐受性实验结果
Figure PCTCN2022089519-appb-000015
Figure PCTCN2022089519-appb-000016
Figure PCTCN2022089519-appb-000017
对于拟南芥，60g ai/ha丙炔氟草胺除草剂是将敏感植物与具有平均抗性水平的植物区分开来的有效剂量。表3的结果表明，对于不同浓度的丙炔氟草胺，相比于对照载体和CK，PPO1A-PPO14A全部表现出高抗的耐受性，而PPO-APA和PPO-ATA基本不具有耐受性。
表4、转入PPO1A-PPO14A核苷酸序列、PPO-APA核苷酸序列、PPO-ATA核苷酸序列和对照载体的拟南芥T 1植株对甲磺草胺的耐受性实验结果
Figure PCTCN2022089519-appb-000018
Figure PCTCN2022089519-appb-000019
对于拟南芥，450g ai/ha甲磺草胺除草剂是将敏感植物与具有平均抗性水平的植物区分开来的有效剂量。表4的结果表明，对于不同浓度的甲磺草胺，相比于对照载体和CK，PPO1A-PPO14A全部表现出高抗的耐受性，而PPO-APA和PPO-ATA均不具有耐受性。
第二实施例、转基因大豆植株的获得和验证
1、重组表达载体转化农杆菌
将第一实施例3中含有所述PPO1A核苷酸序列、PPO6A核苷酸序列、PPO12A核苷酸序列、PPO-ECA核苷酸序列和PPO-APA核苷酸序列的所述重组表达载体DBN12337、DBN12342、DBN12348、DBN12351和DBN12353以及第一实施例3中的对照重组表达载体DBN12337N分别用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)；置于液氮中10min，37℃温水浴10min；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2h，涂于含50mg/L的利福平(Rifampicin)和50mg/L的壮观霉素的所述LB固体平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，将提取的质粒进行测序鉴定，结果表明重组表达载体DBN12337、DBN12342、DBN12348、DBN12351、DBN12353和对照重组表达载体DBN12337N结构完全正确。
2、获得转基因大豆植株
按照常规采用的农杆菌侵染法，将无菌培养的大豆品种中黄13的子叶节组织与本实施例1中所述的农杆菌分别进行共培养，以将本实施例1中重组表达载体DBN12337、DBN12342、DBN12348、DBN12351、DBN12353和对照重组表达载体DBN12337N中的T-DNA(包括玄参花叶病毒的34S增强子序列、油菜真核延长因子基因1α(Tsf1)启动子序列、拟南芥叶绿体转运肽序列、5-烯醇丙酮酸莽草酸-3-磷酸合酶基因、豌豆RbcS基因的终止子序列、拟南芥泛素(Ubiquitin)10基因启动子序列、拟南芥白化体或浅绿色体转运肽、PPO1A核苷酸序列、PPO6A核苷酸序列、PPO12A核苷酸序列、PPO-ECA核苷酸序列、PPO-APA核苷酸序列、胭脂碱合成酶基因的终止子序列、花椰菜花叶病毒35S启动子序列、膦丝菌素N-乙酰基转移酶基因、花椰菜花叶病毒35S终止子序列)转入到大豆染色体组中，分别获得了转入PPO1A核苷酸序列的大豆植株、转入PPO6A核苷酸序列的大豆植株、转入PPO12A核苷酸序列的大豆植株、转入PPO-ECA核苷酸序列的大豆植株、转入PPO-APA核苷酸序列的大豆植株和转入对照载体DBN12337N的大豆植株。
对于农杆菌介导的大豆转化，简要地，将成熟的大豆种子在大豆萌发培养基(B5盐3.1g/L、B5维他命、蔗糖20g/L、琼脂8g/L，pH5.6)中进行萌发，将种子接种于萌发培养基上，按以下条件培养：温度25±1℃；光周期(光/暗)为16/8h。萌发4-6天后取鲜绿的子叶节处膨大的大豆无菌苗，在子叶节下3-4mm处切去下胚轴，纵向切开子叶，去顶芽、侧芽和种子根。用解剖刀的刀背在子叶节处进行创伤，用农杆菌悬浮液接触创伤过的子叶节组织，其中农杆菌能够将所述PPO1A核苷酸序列、PPO6A核苷酸序列、PPO12A核苷酸序列、PPO-ECA核苷酸序列或PPO-APA核苷酸序列分别传递至创伤过的 子叶节组织(步骤1：侵染步骤)，在此步骤中，子叶节组织优选地浸入农杆菌悬浮液(OD 660＝0.5-0.8，侵染培养基(MS盐2.15g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)40mg/L、2-吗啉乙磺酸(MES)4g/L、玉米素(ZT)2mg/L，pH5.3)中以启动接种。子叶节组织与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，子叶节组织在侵染步骤后在固体培养基(MS盐4.3g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、MES 4g/L、ZT 2mg/L、琼脂8g/L，pH5.6)上培养。在此共培养阶段后，可以有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(B5盐3.1g/L、B5维他命、MES 1g/L、蔗糖30g/L、ZT 2mg/L、琼脂8g/L、头孢霉素150mg/L、谷氨酸100mg/L、天冬氨酸100mg/L，pH5.6)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素150-250mg/L)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，子叶节再生的组织块在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，子叶节再生的组织块在含选择剂(草甘膦)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，子叶节再生的组织块在有选择剂的筛选固体培养基(B5盐3.1g/L、B5维他命、MES 1g/L、蔗糖30g/L、6-苄基腺嘌呤(6-BAP)1mg/L、琼脂8g/L、头孢霉素150mg/L、谷氨酸100mg/L、天冬氨酸100mg/L、N-(膦羧甲基)甘氨酸0.25mol/L，pH5.6)上培养，导致转化的细胞选择性生长。然后，转化的细胞再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的子叶节再生的组织块在固体培养基(B5分化培养基和B5生根培养基)上培养以再生植物。
筛选得到的抗性组织块转移到所述B5分化培养基(B5盐3.1g/L、B5维他命、MES 1g/L、蔗糖30g/L、ZT 1mg/L、琼脂8g/L、头孢霉素150mg/L、谷氨酸50mg/L、天冬氨酸50mg/L、赤霉素1mg/L、生长素1mg/L、N-(膦羧甲基)甘氨酸0.25mol/L，pH5.6)上，25℃下培养分化。分化出来的小苗转移到所述B5生根培养基(B5盐3.1g/L、B5维他命、MES 1g/L、蔗糖30g/L、琼脂8g/L、头孢霉素150mg/L、吲哚-3-丁酸(IBA)1mg/L)，在生根培养上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于26℃下培养16h，再于20℃下培养8h。
3、用TaqMan验证转基因大豆植株
取分别转入PPO1A核苷酸序列的大豆植株、转入PPO6A核苷酸序列的大豆植株、转入PPO12A核苷酸序列的大豆植株、转入PPO-ECA核苷酸序列的大豆植株、转入PPO-APA核苷酸序列的大豆植株和转入对照载体DBN12337N的大豆植株的叶片约100mg作为样品，用Qiagen的DNeasy Plant Maxi Kit提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测EPSPS 基因拷贝数以确定PPO基因的拷贝数。同时以野生型大豆植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。
检测EPSPS基因拷贝数的具体方法如下：
步骤11、分别取转入PPO1A核苷酸序列的大豆植株、转入PPO6A核苷酸序列的大豆植株、转入PPO12A核苷酸序列的大豆植株、转入PPO-ECA核苷酸序列的大豆植株、转入PPO-APA核苷酸序列的大豆植株、转入对照载体DBN12337N的大豆植株和野生型大豆植株的叶片各100mg，分别在研钵中用液氮研成匀浆，每个样品取3个重复；
步骤12、使用Qiagen的DNeasy Plant Mini Kit提取上述样品的基因组DNA，具体方法参考其产品说明书；
步骤13、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度；
步骤14、调整上述样品的基因组DNA浓度至同一浓度值，所述浓度值的范围为80-100ng/μL；
步骤15、采用Taqman探针荧光定量PCR方法鉴定样品的拷贝数，以经过鉴定已知拷贝数的样品作为标准品，以野生型大豆植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：
以下引物和探针用来检测EPSPS基因序列：
引物1：ctggaaggcgaggacgtcatcaata如序列表中SEQ ID NO:69所示；
引物2：tggcggcattgccgaaatcgag如序列表中SEQ ID NO:70所示；
探针1：atgcaggcgatgggcgcccgcatccgta如序列表中SEQ ID NO:71所示；
PCR反应体系为：
Figure PCTCN2022089519-appb-000020
所述50×引物/探针混合物包含1mM浓度的每种引物各45μL，100μM浓度的探针50μL和860μL 1×TE缓冲液，并且在4℃，贮藏在琥珀试管中。
PCR反应条件为：
Figure PCTCN2022089519-appb-000021
利用SDS2.3软件(Applied Biosystems)分析数据。
通过分析EPSPS基因拷贝数的实验结果，进而证实PPO1A核苷酸序列、PPO6A核苷酸序列、PPO12A核苷酸序列、PPO-ECA核苷酸序列、PPO-APA核苷酸序列和对照载体DBN12337N均己整合到所检测的大豆植株的染色体组中，而且分别转入PPO1A核苷酸序列的大豆植株、转入PPO6A核苷酸序列的大豆植株、转入PPO12A核苷酸序列的大豆植株、转入PPO-ECA核苷酸序列的大豆植株、转入PPO-APA核苷酸序列的大豆植株和转入对照载体DBN12337N的大豆植株均获得了单拷贝的转基因大豆植株。
4、转基因大豆植株的除草剂耐受性效果检测
取转入PPO1A核苷酸序列的大豆植株(PPO1A)、转入PPO6A核苷酸序列的大豆植株(PPO6A)、转入PPO12A核苷酸序列的大豆植株(PPO12A)、转入PPO-ECA核苷酸序列的大豆植株(PPO-ECA)、转入PPO-APA核苷酸序列的大豆植株(PPO-APA)、转入对照载体的大豆植株(对照载体)和野生型大豆植株(CK)各16株(播种后18天)，分别用3种浓度的苯嘧磺草胺(50g ai/ha(2倍大田浓度，2×)、100g ai/ha(4倍大田浓度，4×)和0g ai/ha(水，0×))、3种浓度的乙氧氟草醚(360g ai/ha(2倍大田浓度，2×)、720g ai/ha(4倍大田浓度，4×)和0g ai/ha(水，0×))和3种浓度的丙炔氟草胺(120g ai/ha(2倍大田浓度，2×)、240g ai/ha(4倍大田浓度，4×)和0g ai/ha(水，0×))进行喷洒以检测大豆植株的除草剂耐受性。按照上述第一实施例6中的方法，在喷施7天(7DAT)后，根据植株平均损伤百分比等级来评价除草剂对每株植株的损伤程度。实验结果如表5-7所示。
表5、转基因大豆植株对苯嘧磺草胺的耐受性实验结果
Figure PCTCN2022089519-appb-000022
Figure PCTCN2022089519-appb-000023
表5的结果表明，(1)相比于对照载体和CK，PPO1A、PPO6A、PPO12A和PPO-ECA均能够对苯嘧磺草胺产生不同程度的耐受性，而PPO-APA对苯嘧磺草胺基本不具有耐受性；(2)对于2倍大田浓度的苯嘧磺草胺，PPO1A、PPO6A和PPO12A的药害等级均为0级，而PPO-ECA中约有44％植株的药害等级为1级；(3)对于4倍大田浓度的苯嘧磺草胺，PPO1A、PPO6A和PPO12A全部表现出高抗的耐受性，而PPO-ECA中约有31％植株表现出中低抗的耐受性。
表6、转基因大豆植株对乙氧氟草醚的耐受性实验结果
Figure PCTCN2022089519-appb-000024
表6的结果表明，相比于对照载体和CK，(1)对于2倍大田浓度的乙氧氟草醚，PPO1A、PPO6A、PPO12A和PPO-ECA均表现出高抗的耐受性，而 PPO-APA中有50％植株不具有耐受性；(2)对于4倍大田浓度的乙氧氟草醚，PPO1A、PPO6A、PPO12A和PPO-ECA均表现出高抗的耐受性，而PPO-APA不具有耐受性。
表7、转基因大豆植株对丙炔氟草胺的耐受性实验结果
Figure PCTCN2022089519-appb-000025
表7的结果表明，对于不同浓度的丙炔氟草胺，相比于对照载体和CK，PPO1A、PPO6A、PPO12A和PPO-ECA全部表现出高抗的耐受性，而PPO-APA对丙炔氟草胺不具有耐受性。
第三实施例、转基因玉米植株的获得和验证
1、构建含有PPO基因的玉米重组表达载体
将上述第一实施例1中所述PPO1B核苷酸序列、PPO6B核苷酸序列、PPO12B核苷酸序列和PPO-APB核苷酸序列的5’和3’端分别连接如下通用接头引物2：
5’端通用接头引物2：5’-ccaagcggccaagctta-3’，如序列表中SEQ ID NO:72所示；
3’端通用接头引物2：5’-tgtttgaacgatcggcgcgcc-3’，如序列表中SEQ ID NO:73所示。
用限制性内切酶Spe I和Asc I对植物表达载体DBNBC-02进行双酶切反应，从而对植物表达载体线性化，酶切产物纯化得到线性化的DBNBC-02表达载体骨架(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将连接所述通用接头引物2的PPO1B核苷酸序列与所述线性化的DBNBC-02表达载体骨架进行重组反应，操作步骤按照Takara公司In-Fusion无缝连接产品试剂盒(Clontech，CA，USA，CAT：121416)说明书进行，构建成重组表达载体DBN12354，其载体结构图如图3所示(Spec：壮观霉素基因；RB：右边界；prOsAct1：水稻肌动蛋白1的启动子(SEQ ID NO:74)；cPAT：膦丝菌素N-乙酰基转移酶基因(SEQ ID NO:60)；t35S：花椰菜花叶病毒35S终止子(SEQ ID NO:61)；pr35S-06：花椰菜花叶病毒35S启动子(SEQ ID NO:75)；iZmHSP70：玉米热休克70kDa蛋白内含子(SEQ ID NO:76)；spAtCLP1：拟南芥白化体或浅绿色体转运肽(SEQ ID NO:57)；PPO1B：PPO1B核苷酸序列(SEQ ID NO:62)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:58)；prZmUbi：玉米泛素(Ubiquitin)1基因的启动子(SEQ ID NO:77)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO:78)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:58)；LB：左边界)。
按照第一实施例3中所述热激方法转化大肠杆菌T1感受态细胞并用碱法提取其质粒，将提取的质粒进行测序验证，结果表明重组表达载体DBN12354中含有序列表中SEQ ID NO:62所示核苷酸序列，即PPO1B核苷酸序列。
按照上述构建重组表达载体DBN12354的方法，将连接所述通用接头引物2的所述PPO6B核苷酸序列、PPO12B核苷酸序列和PPO-APB核苷酸序列分别与所述线性化的DBNBC-02表达载体骨架进行重组反应，依次得到重组表达载体DBN12355至DBN12357。测序验证重组表达载体DBN12355至DBN12357中PPO6B核苷酸序列、PPO12B核苷酸序列和PPO-APB核苷酸序列分别正确插入。
按照上述构建重组表达载体DBN12354的方法，构建对照重组表达载体DBN12354N，其载体结构如图4所示(Spec：壮观霉素基因；RB：右边界；prOsAct1：水稻肌动蛋白1的启动子(SEQ ID NO:74)；cPAT：膦丝菌素N-乙酰基转移酶基因(SEQ ID NO:60)；t35S：花椰菜花叶病毒35S终止子(SEQ ID NO:61)；prZmUbi：玉米泛素(Ubiquitin)1基因的启动子(SEQ ID NO:77)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO:78)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:58)；LB：左边界)。
2、重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN12354至DBN12357以及上述对照重组表达载体DBN12354N用液氮法转化到农杆菌LBA4404(Invitrgen， Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)；置于液氮中10min，37℃温水浴10min；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2h，涂于含50mg/L的利福平(Rifampicin)和50mg/L的壮观霉素的所述LB固体平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，将提取的质粒进行测序鉴定，结果表明重组表达载体DBN12354至DBN12357和DBN12354N结构完全正确。
3、转基因玉米植株的获得
按照常规采用的农杆菌侵染法，将无菌培养的玉米品种综31(Z31)的幼胚与本实施例2中所述的农杆菌共培养，以将本实施例1中构建的重组表达载体DBN12354至DBN12357以及对照重组表达载体DBN12354N中的T-DNA(包括水稻肌动蛋白1的启动子序列、膦丝菌素N-乙酰基转移酶基因、花椰菜花叶病毒35S终止子序列、花椰菜花叶病毒35S启动子序列、玉米热休克70kDa蛋白内含子序列、拟南芥白化体或浅绿色体转运肽、PPO1B核苷酸序列、PPO6B核苷酸序列、PPO12B核苷酸序列、PPO-APB核苷酸序列、胭脂碱合成酶基因的终止子序列、玉米泛素(Ubiquitin)1基因的启动子序列、磷酸甘露糖异构酶基因、胭脂碱合成酶基因的终止子序列)转入到玉米染色体组中，分别获得了转入PPO1B核苷酸序列的玉米植株、转入PPO6B核苷酸序列的玉米植株、转入PPO12B核苷酸序列的玉米植株、转入PPO-APB核苷酸序列的玉米植株和转入对照载体DBN12354N的玉米植株，同时以野生型玉米植株作为对照。
对于农杆菌介导的玉米转化，简要地，从玉米中分离未成熟的幼胚，用农杆菌悬浮液接触幼胚，其中农杆菌能够将所述PPO1B核苷酸序列、PPO6B核苷酸序列、PPO12B核苷酸序列或PPO-APB核苷酸序列传递至幼胚之一的至少一个细胞(步骤1：侵染步骤)。在此步骤中，幼胚优选地浸入农杆菌悬浮液(OD 660＝0.4-0.6，侵染培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖68.5g/L、葡萄糖36g/L、乙酰丁香酮(AS)40mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L，pH5.3))中以启动接种。幼胚与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，幼胚在侵染步骤后在固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)100mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、琼脂8g/L，pH5.8)上培养。在此共培养阶段后，可以有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素)，不添加植物转化体的选择剂(步 骤3：恢复步骤)。优选地，幼胚在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，接种的幼胚在含选择剂(甘露糖)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，幼胚在有选择剂的筛选固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、甘露糖12.5g/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)上培养，导致转化的细胞选择性生长。然后，愈伤组织再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的愈伤组织在固体培养基(MS分化培养基和MS生根培养基)上培养以再生植物。
筛选得到的抗性愈伤组织转移到所述MS分化培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、6-苄基腺嘌呤2mg/L、甘露糖5g/L、植物凝胶3g/L，pH5.8)上，25℃下培养分化。分化出来的小苗转移到所述MS生根培养基(MS盐2.15g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、吲哚-3-乙酸1mg/L、植物凝胶3g/L，pH5.8)上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于28℃下培养16小时，再于20℃下培养8小时。
4、用TaqMan验证转基因玉米植株
按照第二实施例中3用TaqMan验证转基因大豆植株的方法，对分别转入PPO1B核苷酸序列的玉米植株、转入PPO6B核苷酸序列的玉米植株、转入PPO12B核苷酸序列的玉米植株、转入PPO-APB核苷酸序列的玉米植株和转入对照载体DBN12354N的玉米植株进行检测分析。通过Taqman探针荧光定量PCR方法检测PMI基因拷贝数以确定PPO基因的拷贝数。同时以野生型玉米植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。
以下引物和探针用来检测PMI基因序列：
引物3：gctgtaagagcttactgaaaaaattaaca如序列表中SEQ ID NO:79所示；
引物4：cgatctgcaggtcgacgg如序列表中SEQ ID NO:80所示；
探针2：tctcttgctaagctgggagctcgatcc如序列表中SEQ ID NO:81所示。
通过分析PMI基因拷贝数的实验结果，进而证实PPO1B核苷酸序列、PPO6B核苷酸序列、PPO12B核苷酸序列、PPO-APB核苷酸序列和对照载体DBN12354N均己整合到所检测的玉米植株的染色体组中，而且分别转入PPO1B核苷酸序列的玉米植株、转入PPO6B核苷酸序列的玉米植株、转入PPO12B核苷酸序列的玉米植株、转入PPO-APB核苷酸序列的玉米植株和转入对照载体DBN12354N的玉米植株均获得了单拷贝的转基因玉米植株。
5、转基因玉米植株的除草剂耐受性效果检测
取转入PPO1B核苷酸序列的玉米植株(PPO1B)、转入PPO6B核苷酸序列的玉米植株(PPO6B)、转入PPO12B核苷酸序列的玉米植株(PPO12B)、转入PPO-APB核苷酸序列的玉米植株(PPO-APB)、转入对照载体的玉米植株(对照载体)和野生型玉米植株(CK)各16株(播种后18天)，分别用3种浓度的苯嘧磺草胺(50g ai/ha(2倍大田浓度，2×)、100g ai/ha(4倍大田浓度，4×)和0g ai/ha(水，0×))、3种浓度的乙氧氟草醚(360g ai/ha(2倍大田浓度，2×)、720g ai/ha(4倍大田浓度，4×)和0g ai/ha(水，0×))和3种浓度的丙炔氟草胺(120g ai/ha(2倍大田浓度，2×)、240g ai/ha(4倍大田浓度，4×)和0g ai/ha(水，0×))进行喷洒以检测玉米植株的除草剂耐受性。按照上述第一实施例6中的方法，在喷施7天(7DAT)后，根据植株平均损伤百分比等级(植株平均损伤百分比＝叶片损伤面积/叶片总面积×100％)来评价除草剂对每株植株的损伤程度。实验结果如表8-10所示。
表8、转基因玉米植株对苯嘧磺草胺的耐受性实验结果
Figure PCTCN2022089519-appb-000026
表8的结果表明，相比于对照载体和CK，(1)对于2倍大田浓度的苯嘧磺草胺，PPO1B、PPO6B和PPO12B全部表现出高抗的耐受性，而PPO-APB中约有56％植株不具有耐受性；(2)对于4倍大田浓度的苯嘧磺草胺，PPO1B、PPO6B和PPO12B全部表现出高抗的耐受性，而PPO-APB 基本不具有耐受性。
表9、转基因玉米植株对乙氧氟草醚的耐受性实验结果
Figure PCTCN2022089519-appb-000027
表9的结果表明，相比于对照载体和CK，(1)对于2倍大田浓度的乙氧氟草醚，PPO1B、PPO6B和PPO12B全部表现出高抗的耐受性，而PPO-APB中有50％植株不具有耐受性；(2)对于4倍大田浓度的乙氧氟草醚，PPO1B、PPO6B和PPO12B全部表现出高抗的耐受性，而PPO-APB不具有耐受性。
表10、转基因玉米植株对丙炔氟草胺的耐受性实验结果
Figure PCTCN2022089519-appb-000028
Figure PCTCN2022089519-appb-000029
表10的结果表明，对于不同浓度的丙炔氟草胺，相比于对照载体和CK，PPO1B、PPO6B和PPO12B全部表现出高抗的耐受性，而PPO-APB对丙炔氟草胺基本不具有耐受性。
综上所述，就植物而言，本发明所述原卟啉原氧化酶PPO1-PPO14不仅可以赋予拟南芥对PPO抑制剂除草剂较好的耐受性，特别是PPO1、PPO6和PPO12可以赋予拟南芥、大豆和玉米对PPO抑制剂类除草剂较好的耐受性，因此，所述原卟啉原氧化酶PPO1-PPO14可以赋予植物较好的耐受性；就除草剂而言，本发明首次公开了原卟啉原氧化酶PPO1-PPO14可以赋予植物对PPO抑制剂除草剂表现出较高的耐受性，至少可以耐受4倍大田浓度的乙氧氟草醚、苯嘧磺草胺和丙炔氟草胺以及2倍大田浓度的甲磺草胺，因此在植物上应用前景广阔。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (17)

  1. 一种控制杂草的方法，其特征在于，包括将含有有效剂量PPO抑制剂的除草剂施加到存在至少一种转基因植物的田地中，所述转基因植物在其基因组中包含编码原卟啉原氧化酶的多核苷酸序列，所述转基因植物与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量，其中所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
    更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
    进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
    优选地，所述转基因植物包括单子叶植物和双子叶植物；更优选地，所述转基因植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；进一步优选地，所述转基因植物为草甘膦耐受性植物，所述杂草为草甘膦抗性杂草；
    优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
    进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
  2. 根据权利要求1所述控制杂草的方法，其特征在于，所述原卟啉原氧化酶的多核苷酸序列具有：
    (a)编码与选自由SEQ ID NO:1-14具有至少88％序列同一性的氨基酸序列的多核苷酸序列，所述多核苷酸序列不包括SEQ ID NO:15-28；或
    (b)SEQ ID NO:29-42或SEQ ID NO:62-64任意一种所示的多核苷酸序列。
  3. 根据权利要求1或2所述控制杂草的方法，其特征在于，所述转基因植物还包括至少一种不同于编码所述原卟啉原氧化酶的多核苷酸序列的编码 第二种除草剂耐受性蛋白质的第二种多核苷酸。
  4. 根据权利要求3所述控制杂草的方法，其特征在于，所述第二种多核苷酸编码选择标记蛋白质、合成活性蛋白质、分解活性蛋白质、抗生物胁迫蛋白质、抗非生物胁迫蛋白质、雄性不育蛋白质、影响植物产量的蛋白质和/或影响植物品质的蛋白质。
  5. 根据权利要求4所述控制杂草的方法，其特征在于，所述第二种多核苷酸编码5-烯醇丙酮酰莽草酸-3-磷酸合酶、草甘膦氧化还原酶、草甘膦-N-乙酰转移酶、草甘膦脱羧酶、草铵膦乙酰转移酶、α酮戊二酸依赖性双加氧酶、麦草畏单加氧酶、4-羟基苯丙酮酸双加氧酶、乙酰乳酸合酶和/或细胞色素类蛋白质。
  6. 根据权利要求1-5任一项所述控制杂草的方法，其特征在于，所述含有有效剂量PPO抑制剂的除草剂还包括草甘膦除草剂、草铵膦除草剂、植物生长素类除草剂、禾本科除草剂、发芽前选择性除草剂和/或发芽后选择性除草剂。
  7. 一种控制杂草生长的种植组合，其特征在于，包括PPO抑制剂除草剂和至少一种转基因植物，将含有有效剂量的所述PPO抑制剂除草剂施加到存在所述至少一种转基因植物的田地中，所述转基因植物在其基因组中包含编码原卟啉原氧化酶的多核苷酸序列，所述转基因植物与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量，其中所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
    更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
    进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
    优选地，所述转基因植物包括单子叶植物和双子叶植物；更优选地，所述转基因植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；进一步优选地，所述转基因植物为草甘膦耐受性植物，所述杂草为草甘膦抗性杂草；
    优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二 唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
    进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
  8. 根据权利要求7所述控制杂草生长的种植组合，其特征在于，所述原卟啉原氧化酶的多核苷酸序列具有：
    (a)编码与选自由SEQ ID NO:1-14具有至少88％序列同一性的氨基酸序列的多核苷酸序列，所述多核苷酸序列不包括SEQ ID NO:15-28；或
    (b)SEQ ID NO:29-42或SEQ ID NO:62-64任意一种所示的多核苷酸序列。
  9. 根据权利要求7或8所述控制杂草生长的种植组合，其特征在于，所述转基因植物还包括至少一种不同于编码所述原卟啉原氧化酶的多核苷酸序列的编码第二种除草剂耐受性蛋白质的第二种多核苷酸。
  10. 根据权利要求9所述控制杂草生长的种植组合，其特征在于，所述第二种多核苷酸编码选择标记蛋白质、合成活性蛋白质、分解活性蛋白质、抗生物胁迫蛋白质、抗非生物胁迫蛋白质、雄性不育蛋白质、影响植物产量的蛋白质和/或影响植物品质的蛋白质。
  11. 根据权利要求10所述控制杂草生长的种植组合，其特征在于，所述第二种多核苷酸编码5-烯醇丙酮酰莽草酸-3-磷酸合酶、草甘膦氧化还原酶、草甘膦-N-乙酰转移酶、草甘膦脱羧酶、草铵膦乙酰转移酶、α酮戊二酸依赖性双加氧酶、麦草畏单加氧酶、4-羟基苯丙酮酸双加氧酶、乙酰乳酸合酶和/或细胞色素类蛋白质。
  12. 根据权利要求7-11任一项所述控制杂草生长的种植组合，其特征在于，所述含有有效剂量PPO抑制剂的除草剂还包括草甘膦除草剂、草铵膦除草剂、植物生长素类除草剂、禾本科除草剂、发芽前选择性除草剂和/或发芽后选择性除草剂。
  13. 一种产生耐受PPO抑制剂除草剂的植物的方法，其特征在于，包括向植物的基因组中引入编码原卟啉原氧化酶的多核苷酸序列，当含有有效剂量PPO抑制剂的除草剂施加到至少存在所述植物的田地中，所述植物与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量，其中所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基 酸序列具有至少90％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
    更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
    进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
    优选地，所述引入的方法包括遗传转化方法、基因组编辑方法或基因突变方法；
    优选地，所述植物包括单子叶植物和双子叶植物；更优选地，所述植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；
    优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
    进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
  14. 一种培养耐受PPO抑制剂除草剂植物的方法，其特征在于，包括：
    种植至少一个植物繁殖体，所述植物繁殖体的基因组中包括编码原卟啉原氧化酶的多核苷酸序列，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
    使所述植物繁殖体长成植株；
    将含有有效剂量PPO抑制剂的除草剂施加到至少包含所述植株的田地中，收获与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
    更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
    进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的 氨基酸序列；
    优选地，所述植物包括单子叶植物和双子叶植物；更优选地，所述植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；
    优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
    进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
  15. 一种用于保护植物免受由PPO抑制剂除草剂引起的损伤或赋予植物PPO抑制剂除草剂耐受性的方法，其特征在于，包括将含有有效剂量PPO抑制剂的除草剂施加到存在至少一种转基因植物的田地中，所述转基因植物在其基因组中包含编码原卟啉原氧化酶的多核苷酸序列，所述转基因植物与其他不具有编码原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量，其中所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
    更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
    进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
    优选地，所述转基因植物包括单子叶植物和双子叶植物；更优选地，所述转基因植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；
    优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和 /或三嗪酮类PPO抑制剂除草剂；
    进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
  16. 一种原卟啉原氧化酶在赋予植物PPO抑制剂除草剂耐受性中的用途，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少88％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少90％序列同一性；
    优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少95％序列同一性；
    更优选地，所述原卟啉原氧化酶与选自由SEQ ID NO:1-14组成的组的氨基酸序列具有至少99％序列同一性；
    进一步优选地，所述原卟啉原氧化酶选自由SEQ ID NO:1-14组成的组的氨基酸序列；
    优选地，所述原卟啉原氧化酶在赋予植物PPO抑制剂除草剂耐受性中的用途包括将含有有效剂量PPO抑制剂的除草剂施加到存在至少一种转基因植物的田地中，所述转基因植物在其基因组中包含编码所述原卟啉原氧化酶的多核苷酸序列，所述转基因植物与其他不具有编码所述原卟啉原氧化酶的多核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量；
    优选地，所述植物包括单子叶植物和双子叶植物；更优选地，所述植物为燕麦、小麦、大麦、谷子、玉米、高粱、二穗短柄草、水稻、烟草、向日葵、苜蓿、大豆、鹰嘴豆、花生、甜菜、黄瓜、棉花、油菜、土豆、番茄或拟南芥；
    优选地，所述PPO抑制剂除草剂包括二苯醚类PPO抑制剂除草剂、噁二唑酮类PPO抑制剂除草剂、N-苯基酞酰胺亚胺类PPO抑制剂除草剂、噁唑啉酮类PPO抑制剂除草剂、苯基吡唑类PPO抑制剂除草剂、脲嘧啶类PPO抑制剂除草剂、噻二唑类PPO抑制剂除草剂、三唑啉酮类PPO抑制剂除草剂和/或三嗪酮类PPO抑制剂除草剂；
    进一步优选地，所述PPO抑制剂除草剂包括乙氧氟草醚、苯嘧磺草胺、甲磺草胺和/或丙炔氟草胺。
  17. 根据权利要求16所述原卟啉原氧化酶在赋予植物PPO抑制剂除草剂耐受性中的用途，其特征在于，所述原卟啉原氧化酶的多核苷酸序列具有：
    (a)编码与选自由SEQ ID NO:1-14具有至少88％序列同一性的氨基酸序列的多核苷酸序列，所述多核苷酸序列不包括SEQ ID NO:15-28；或
    (b)SEQ ID NO:29-42或SEQ ID NO:62-64任意一种所示的多核苷酸序列。
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