WO2017215329A1 - 除草剂抗性蛋白质、其编码基因及用途 - Google Patents

除草剂抗性蛋白质、其编码基因及用途 Download PDF

Info

Publication number
WO2017215329A1
WO2017215329A1 PCT/CN2017/079661 CN2017079661W WO2017215329A1 WO 2017215329 A1 WO2017215329 A1 WO 2017215329A1 CN 2017079661 W CN2017079661 W CN 2017079661W WO 2017215329 A1 WO2017215329 A1 WO 2017215329A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
gene
dicamba
herbicide
tolerant
Prior art date
Application number
PCT/CN2017/079661
Other languages
English (en)
French (fr)
Inventor
何健
姚利
贾兴军
谢香庭
吴业春
陶青
丁德荣
Original Assignee
北京大北农生物技术有限公司
南京农业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京大北农生物技术有限公司, 南京农业大学 filed Critical 北京大北农生物技术有限公司
Priority to US16/308,852 priority Critical patent/US11365425B2/en
Publication of WO2017215329A1 publication Critical patent/WO2017215329A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
    • 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
    • A01G13/00—Protecting plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
    • C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
    • C12Y105/0102—Methylenetetrahydrofolate reductase [NAD(P)H] (1.5.1.20)

Definitions

  • the present invention relates to a herbicide resistance protein, a gene encoding the same, and a use thereof, in particular to a protein which is resistant to the herbicide dicamba, a gene encoding the same and use thereof.
  • Dicamba is one of the most effective and environmentally friendly herbicides that have been used by farmers for more than 40 years. Dicamba can be used to control corn, sorghum, millet, pasture, hay, pasture, sugar cane, asparagus, turf and grass seed crops.
  • dicamba can harm many commercial crops and dicots such as soybeans, cotton, peas, potatoes, sunflowers and canola, these crops/plants are low level
  • the dicambas are particularly sensitive. Despite this, dicamba is still effective in controlling weed growth and is important.
  • DMO dicamba monooxygenase
  • the present invention provides a gene comprising:
  • the stringent conditions may be hybridization in a solution of 6 x SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) at 65 ° C, followed by 2 x SSC, 0.1% SDS and 1 x SSC, The membrane was washed once for each 0.1% SDS.
  • 6 x SSC sodium citrate
  • SDS sodium dodecyl sulfate
  • the present invention also provides an expression cassette comprising the gene under the control of an operably linked regulatory sequence.
  • the regulatory sequence is a chloroplast transit peptide, and the chloroplast transit peptide is operably linked to the gene.
  • the nucleotide sequence of the chloroplast transit peptide has the nucleotide sequence set forth in SEQ ID NO: 7.
  • the present invention also provides a recombinant vector comprising the gene or the expression cassette.
  • the present invention also provides a method for increasing the tolerance of a herbicide, comprising: a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or a protein encoded by the expression cassette in a plant and at least A protein different from the amino acid sequence composition shown in SEQ ID NO: 2 or a second protein of the protein encoded by the expression cassette is expressed together.
  • the second protein is glyphosate-tolerant protein, glufosinate-tolerant protein, alpha-ketoglutarate dioxygenase, 4-hydroxyphenylpyruvate dioxygenase, acetolactate Synthase, cytochrome protein or protoporphyrinogen oxidase.
  • expression of a MTHFR66 herbicide resistance protein in a transgenic plant may be accompanied by expression of one or more glyphosate-tolerant proteins and/or glufosinate-tolerant proteins. Co-expression of such more than one herbicide-tolerant protein in the same transgenic plant can be achieved by genetically engineering the plant to contain and express the desired gene.
  • one plant first parent
  • the second plant second parent
  • Phosphine-tolerant protein Progeny plants expressing all of the genes introduced into the first parent and the second parent are obtained by hybridization of the first parent and the second parent.
  • the present invention also provides a method of selecting a transformed plant cell, comprising: transforming a plurality of plant cells with the gene or the expression cassette, and allowing expression of the gene or the expression cassette
  • the cells are grown by transforming the cells, and the cells are cultured at a herbicide concentration that kills the untransformed cells or inhibits the growth of the untransformed cells, which is dicamba.
  • the present invention also provides a method of controlling weeds comprising: applying an effective amount of a dicamba herbicide to a field planting a plant, the plant comprising the gene, the expression cassette or the recombinant Carrier.
  • the present invention also provides a method for protecting a plant from damage caused by a herbicide, comprising: introducing the gene, the expression cassette or the recombinant vector into a plant to produce a plant after introduction A herbicide tolerance protein sufficient to protect it from dicamba damage.
  • the present invention also provides a method for conferring tolerance to a plant dicamba herbicide, comprising: introducing the gene, the expression cassette or the recombinant vector into a plant.
  • the present invention also provides a glyphosate tolerance tolerance in a field of glyphosate-tolerant plants.
  • a method of grass comprising: administering an effective amount of dicamba to a field planted with a glyphosate-tolerant plant comprising the gene, the expression cassette or the recombinant vector.
  • the present invention also provides a method of producing a dicamba tolerant plant comprising introducing the gene or the expression cassette into the genome of the plant to produce a dicamba tolerant plant.
  • the method for producing a dicamba-tolerant plant comprises: producing a dicamba-tolerant plant by selfing or hybridizing the parent plant, the parent plant and/or the second plant comprising The gene or the expression cassette, the dicamba tolerant plant inherited the gene or the expression cassette from the parental plant and/or the second plant.
  • the present invention also provides a method of cultivating a plant which is tolerant to a dicamba herbicide, comprising:
  • Planting at least one plant seed comprising the gene or the expression cassette in the genome of the plant seed;
  • the plants are sprayed with an effective dose of dicamba herbicide, and plants having reduced plant damage compared to other plants not having the gene or the expression cassette are harvested.
  • the plant is soybean, cotton, corn, rice, wheat, sugar beet or sugar cane.
  • the present invention also provides a use of a methyltetrahydrofolate reductase tolerant to a dicamba herbicide having the amino acid sequence of SEQ ID NO: 2.
  • the present invention also provides a use of a plant-tolerant dicamba herbicide producing methyltetrahydrofolate reductase having the amino acid represented by SEQ ID NO: 2. sequence.
  • introducing the gene or the expression cassette or the recombinant vector into a plant, in the present invention, introducing foreign DNA into a plant cell, and conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-emission Bombardment, direct DNA uptake into protoplasts, electroporation or whisker silicon-mediated DNA introduction.
  • the present invention can increase the tolerance of plants to oxidative stress, including but not limited to, providing plants with dicamba or dicamba monooxygenase-mediated metabolism or analogs thereof to improve herbicide tolerance of plants Receptivity, for example, by metabolizing dicamba to DCSA.
  • Dicamba means 3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid and its acids and salts.
  • the salts thereof include isopropylamine salt, diethylene glycol ammonium salt, dimethylamine salt, potassium salt and sodium salt.
  • Commercial preparations of dicamba include, but are not limited to, (as a DMA salt), (BASF, as DGA salt), VEL-58-CS-11TM and (BASF, as a DGA salt).
  • RHOs Rieske non-heme-type oxidase
  • DMO GeneBank: AY786443.1
  • RHO type oxidase (2) cytochrome P450, a superfamily of ferrous heme-thiolate proteins involved in the metabolism of endogenous substances and exogenous substances including drugs and environmental compounds Many methoxy-containing aromatic compounds Methylase is this type; (3) Anaerobic tetrahydrofolate-dependent demethylase, found in anaerobic bacteria such as Moorellathermoacetica, involved in lignin degradation intermediates such as syringic acid And anaerobic degradation of vanillic acid, it has also been found that anaerobic tetrahydrofolate-dependent demethylase can also anaerobic degradation of dicamba; (4) aerobic tetrahydrofolate-dependent demethylase (Sphingomonas paucimobilis SYK -6), involved in the anaerobic degradation of lignin degradation intermediates such as syringic acid and vanillic acid, but there is no such report that aerobic tetrahydrof
  • MTHFR is a 5,10-methylenetetrahydrofolate reductase, methylenetetrahydrofolate reductase protein-encoding gene, which is mainly used to convert 5,10-methylenetetrahydrofolate into a biologically functional 5-A in the folate metabolic pathway. Tetrahydrofolate and its reverse reaction.
  • the gene of the present invention has a property of allowing the use of a dicamba herbicide in plants after plant expression, in which the inherent tolerance is not present or insufficient to allow the use of the dicamba herbicide. Furthermore, the MTHFR66 gene of the present invention can provide protection against dicamba herbicides in plants when natural tolerance is insufficient to allow selectivity.
  • the amount applied to the field is from about 0.0025 lbs/acre (lb/a) to about 20 lb/a dicamba, more typically from 0.25 lb/a to 12 lb/a. Combining different chemical classes and herbicides with different modes of action and ranges in the same field (continuous or tank mix) can provide control of most potential weeds that require herbicide control.
  • Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grass weed species.
  • repeated use of glyphosate in glyphosate-tolerant crops and non-crop applications has (and will continue to be) selected to succeed weeds as naturally more tolerant species or glyphosate tolerant biotypes.
  • Most herbicide tolerance management strategies recommend the use of an effective amount of a tank-mixed herbicide companion as a means of delaying the emergence of tolerant weeds that provide control of the same species but have different modes of action.
  • MTHFR66 gene with glyphosate tolerance traits (and/or other herbicide tolerance traits) can achieve glyphosate tolerance in glyphosate-tolerant crops by allowing selective use of glyphosate and dicamba for the same crop Control of phosphine-tolerant weed species (wideleaf weed species controlled by dicamba).
  • the use of these herbicides can be used simultaneously in a tank mix of two or more herbicides containing different modes of action, for individual use of individual herbicide compositions in continuous use (eg, before planting, before emergence or after emergence).
  • the interval used ranges from 2 hours to 3 months), or alternatively, at any time (from 7 months from planting to when harvesting crops (or for pre-harvest intervals for individual herbicides, the shortest) ))
  • Flexibility in controlling broadleaf weeds is important, namely the time of use, the amount of individual herbicides, and the ability to control stubborn or tolerant weeds.
  • the glyphosate application in the crop with the glyphosate tolerance gene/MTHFR66 gene can range from 250 to 2500 g ae/ha; dicamba can range from 0.25 lb/a to 12 lb/a. The optimal combination of time for these applications depends on the specific conditions, species and environment.
  • Herbicide formulations such as ester, acid or salt formulations or soluble concentrates, emulsified concentrates or solvables
  • tank mix additives such as adjuvants or compatibilizers
  • Any chemical combination of any of the foregoing herbicides is within the scope of the invention.
  • glyphosate tolerance eg resistance
  • Affected plants or bacteria EPSPS GOX, GAT
  • glufosinate tolerance such as PAT, Bar
  • phenoxy auxin tolerance such as 2,4-D, metformin tolerance genes such as AAD-1, AAD-12, etc.
  • acetolactate synthase (ALS) inhibitory herbicide tolerance eg imidazolinone, sulfonylurea, triazole pyrimidine, sulfanilide, pyrimidine thiobenzoic acid and other chemicals resistant
  • Resistant genes such as AHAS, Csrl, SurA, etc., bromooxanitride tolerance (eg Bxn), tolerance to HPPD (4-hydroxyphenylpyruvate dioxygenase) enzyme inhibitor, octahydr
  • some other preferred ALS inhibitors include triazolopyrimidine sulfanilide (chlorosulfasalin, diclofenac, sulffenapyr, sulfazinamide, and pyrimidotriazole sulfonamide), Pyrimidine thiobenzoic acid and flucarbazone.
  • Some preferred HPPD inhibitors include mesotrione, isoxaflutole and sulcotrione.
  • Some preferred PPO inhibitors include propargylamine, flufenacetate, oxazolone, mesalil and diphenyl ethers (eg, acifluorfen, flufenazone, flufenacil and Oxyfluorfen).
  • the MTHFR66 gene can be superimposed alone or in combination with other herbicide-tolerant crop characteristics followed by one or more other inputs (such as insect tolerance, fungal tolerance or stress tolerance) or output (eg increased yield) , improved oil quantity, improved fiber quality, etc.) superimposed traits.
  • the present invention can be used to provide a complete agronomic solution that flexibly and economically controls the ability of any number of agronomic pests and enhances crop quality.
  • the MTHFR66 gene of the present invention is capable of degrading dicamba herbicides and is the basis for important herbicide tolerance to crop and selection marker characteristics.
  • the present invention allows transgenic expression to control the herbicide combination of almost all broadleaf weeds.
  • the MTHFR66 gene can be used as an excellent herbicide tolerant to crop traits such as other herbicide tolerant crop traits (eg glyphosate tolerance, glufosinate tolerance, phenoxy auxin tolerance, ALS inhibitors (such as imidazolinones, sulfonylureas, triazolopyrimidine sulfonamides, tolerance, bromoxynil tolerance, HPPD inhibitor tolerance, PPO inhibitor tolerance, etc.) and insect tolerance Traits (Cry1Ab, Cry1F, Vip3, other B. thuringiensis proteins or non-Bacillus-derived insect-tolerant proteins, etc.) are superimposed.
  • the MTHFR66 gene can be used as a selection marker to aid in the selection of primary transformants of plants genetically engineered with another gene or gene population.
  • the herbicide-tolerant crop traits of the present invention can be used in new combinations with other herbicide-tolerant crop traits including, but not limited to, glyphosate tolerance. These trait combinations result in new methods of controlling weed species due to newly acquired tolerance or inherent tolerance to herbicides such as glyphosate.
  • the scope of the invention includes a novel method of controlling weeds using a herbicide wherein the herbicide is rendered resistant by the enzyme in the transgenic crop Sex.
  • the invention is applicable to a variety of plants such as Arabidopsis thaliana, tobacco, soybean, cotton, rice, corn and alfalfa.
  • the invention is also applicable to a variety of other monocotyledons (such as forage grasses or turfgrass) and dicotyledonous crops (such as alfalfa, clover, arbor species, etc.).
  • dicamba or other MTHFR66 substrate
  • dicamba can be more aggressively used in moderately tolerant grass crops, and the increased tolerance afforded by the trait will provide growers with more effective dosages and wider application. Time to use these herbicides without the risk of crop damage.
  • the genome of a plant, plant tissue or plant cell as referred to in the present invention refers to any genetic material within a plant, plant tissue or plant cell, and includes the nucleus and plastid and mitochondrial genomes.
  • the "tolerance” and the “resistance” as described in the present invention are heritable and allow plants to grow and multiply in the case where the herbicide is subjected to a general herbicide effective treatment of a given plant. As recognized by those skilled in the art, plants can be considered “tolerant” or “resistant” even if the plants are significantly damaged by herbicide treatment.
  • the term “tolerance” in the present invention is broader than the term “tolerance” and includes “tolerance” as well as the ability of a particular plant to have an increased resistance to herbicide-induced damage to various degrees, while in the same herbicide The dose generally results in damage to the same genotype of wild type plants.
  • polynucleotides and/or nucleotides described herein form a complete "gene" encoding a protein or polypeptide in a desired host cell.
  • polynucleotides and/or nucleotides of the invention can be placed under the control of regulatory sequences in a host of interest.
  • Regulatory sequences of the invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the MTHFR66 gene.
  • the promoter is a promoter expressible in a plant
  • the "promoter expressible in a plant” refers to a promoter which ensures expression of a coding sequence linked thereto in a plant cell.
  • a promoter expressible in a plant can be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the maize ubi promoter, the promoter of the rice GOS2 gene, and the like.
  • a promoter expressible in a plant may be a tissue-specific promoter, ie the promoter directs the expression level of the coding sequence in some tissues of the plant, such as in green tissue, to be higher than other tissues of the plant (through conventional The RNA assay is performed), such as the PEP carboxylase promoter.
  • a promoter expressible in a plant can be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-inducible expression pattern means that when the plant is subjected to mechanical or wounding by insect foraging, the expression of the coding sequence under the control of the promoter is significantly improved compared to normal growth conditions.
  • wound-inducible promoters include, but are not limited to, promoters of protease inhibitory genes (pin I and pin II) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, including but not limited to the Arabidopsis thaliana chloroplast transit peptide AtCTP2, or the endoplasmic reticulum is targeted using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a small RNA viral leader sequence, such as an EMCV leader sequence (encephalomyocarditis) Virus 5' non-coding region); potato Y virus leader sequence, such as MDMV (maize dwarf mosaic virus) leader sequence; human immunoglobulin heavy chain binding protein (BiP); alfalfa mosaic virus coat protein mRNA Translation leader sequence (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • the enhancer includes, but is not limited to, a cauliflower mosaic virus (CaMV) enhancer, a figwort mosaic virus (FMV) enhancer, a carnation weathering ring virus (CERV) enhancer, and a cassava vein mosaic virus (CsVMV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathering ring virus
  • CsVMV cassava vein mosaic virus
  • MMV Purple Jasmine Mosaic Virus
  • MMV Yellow Jasmine Mosaic Virus
  • CmYLCV Night fragrant yellow leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV Acanthus yellow mottle virus
  • PCLSV peanut chlorotic line flower Leaf virus
  • the introns include, but are not limited to, maize hsp70 introns, maize ubiquitin introns, Adh introns 1, sucrose synthase introns, or rice Actl introns.
  • the 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, a polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene. a polyadenylation signal sequence derived from the protease inhibitor II (pin II) gene, a polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and a gene derived from the ⁇ -tubulin gene. Polyadenylation signal sequence.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • operably linked refers to the joining of nucleic acid sequences that allow one sequence to provide the function required for the linked sequence.
  • the "operably linked” in the present invention may be such that the promoter is ligated to the sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter.
  • Effective ligation when a sequence of interest encodes a protein and is intended to obtain expression of the protein means that the promoter is ligated to the sequence in a manner that allows efficient translation of the resulting transcript.
  • the linker of the promoter to the coding sequence is a transcript fusion and it is desired to effect expression of the encoded protein, such ligation is made such that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • the linkage of the promoter to the coding sequence is a translational fusion and it is desired to effect expression of the encoded protein, such linkage is made such that the first translation initiation codon and promoter contained in the 5' untranslated sequence Linked and linked such 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 for gene expression functions (ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly Adenylation site and/or transcription terminator), sequences that provide DNA transfer and/or integration functions (ie, T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide options Sexually functional sequences (ie, antibiotic resistance markers, biosynthetic genes), sequences that provide for the function of scoring markers, sequences that facilitate sequence manipulation in vitro or in vivo (ie, polylinker sequences, site-specific recombination sequences) and A sequence that provides replication (ie, a bacterial origin of replication, an autonomously replicating sequence, a centromeric sequence).
  • gene expression functions ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein
  • the present invention confers new herbicide tolerance traits on plants and does not observe adverse effects on phenotype including yield.
  • the plants of the invention are tolerant to an application level of at least one of the tested herbicides. These levels of tolerance are within the scope of the invention. For example, predictable optimizations and further developments can be made to a variety of techniques known in the art to increase expression of a given gene.
  • the herbicide resistance protein is an MTHFR66 amino acid sequence as shown in SEQ ID NO: 2 in the Sequence Listing.
  • the herbicide resistance gene is a MTHFR66 nucleotide sequence as shown in SEQ ID NO: 1 in the Sequence Listing.
  • the herbicide resistance gene is for use in a plant, and may comprise, in addition to the coding region of a protein encoded by the MTHFR66 nucleotide sequence, other elements, such as a coding region encoding a transit peptide, a protein encoding a selectable marker, or conferring The coding region of an insect-tolerant protein.
  • the MTHFR66 herbicide resistance protein of the present invention is resistant to dicamba herbicides.
  • the plant of the present invention contains exogenous DNA in its genome, and the exogenous DNA comprises a MTHFR66 nucleotide sequence which is protected from the herbicide by expressing an effective amount of the protein.
  • An effective amount refers to a dose that is undamaged or slightly damaged.
  • the plants should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product.
  • the present invention provides a herbicide resistance protein, a gene encoding the same and use thereof, and has the following advantages:
  • the MTHFR66 gene of the invention can degrade the dicamba herbicide, optimize the MTHFR66 gene using the preferred codons of maize and soybean, making it particularly suitable for expression in plants; optimizing the MTHFR66 gene can confer tolerance to the transgenic plant dicamba herbicide, and Optimizing the expression of the MTHFR66 gene in chloroplasts enhances the tolerance of transgenic plants to dicamba herbicides.
  • the application prospect is broad.
  • the herbicide resistance protein MTHFR66 of the present invention is methyltetrahydrofolate reductase, which is different from the known dicamba tolerant gene, and thus can expand the application range of the dicamba tolerant type on plants.
  • FIG. 1 is a diagram showing the SDS-PAGE electrophoresis of a protein expressed by the MTHFR66 gene of the herbicide resistance protein, the coding gene thereof and the use thereof in the expression host strain BL21 (DE3);
  • FIG. 3 is an HPLC chromatogram of the degradation of dicamba by the MTHFR66 protein of the herbicide-resistant protein, the gene encoding the same, and the use thereof for the expression of the MTHFR66 protein under different concentrations of tetrahydrofolate;
  • Figure 4 is an HPLC chromatogram of the MTHFR66 protein metabolism 5-methyltetrahydrofolate induced by the herbicide resistance protein, the coding gene thereof and the use thereof;
  • Figure 5 is a first-order mass spectrum of an intermediate product for the metabolism of 5-methyltetrahydrofolate of the MTHFR66 protein induced by the herbicide-resistant protein, the gene encoding the same, and the use thereof;
  • Figure 6 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing the optimized MTHFR66 nucleotide sequence of the herbicide resistance protein, the gene encoding the same, and the use thereof;
  • Figure 7 is a flow chart showing the construction of a recombinant expression vector DBN111101 containing the optimized MTHFR66 nucleotide sequence of the herbicide resistance protein, the coding gene thereof and the use thereof;
  • Figure 8 is a schematic diagram showing the structure of a recombinant expression vector DBN111101N containing a natural MTHFR66 nucleotide sequence of the herbicide resistance protein, its coding gene and use thereof;
  • Figure 9 is a diagram showing the herbicide tolerance effect of the herbicide-resistant protein, the gene encoding the same, and the use of the transgenic Arabidopsis T1 plant of the present invention.
  • Figure 10 is a flow chart showing the construction of a recombinant expression vector DBN-HT130066 containing the optimized MTHFR66 nucleotide sequence of the herbicide resistance protein, its coding gene and use thereof;
  • Figure 11 is a schematic diagram showing the structure of a recombinant expression vector DBN-HT130066N containing a native MTHFR66 nucleotide sequence of the herbicide resistance protein, its coding gene and use thereof.
  • the herbicide-resistant protein of the present invention the gene encoding the same, and the use of the same are described below by way of specific examples.
  • the first embodiment in vitro high-efficiency expression and functional identification of methyltetrahydrofolate reductase MTHFR66
  • Primer 1 5-GGAATTCCATATGGGCTCGCCCGTTATGG-3 (underlined as NdeI restriction site), as shown in SEQ ID NO: 4 in the Sequence Listing;
  • Primer 2 5-CCGCTCGAGGTGCTTTCGAGCGTAGTCAG-3 (underlined as XhoI restriction site, as shown in SEQ ID NO: 5 in the Sequence Listing;
  • the MTHFR66 gene was amplified using the PCR amplification system described below:
  • the template DNA (i.e., the native MTHFR66 nucleotide sequence) is set forth in SEQ ID NO: 3 of the Sequence Listing.
  • the PCR reaction conditions were: denaturation at 98 ° C for 1 min; then entering the following cycles: denaturation at 98 ° C for 15 s, annealing at 55 ° C for 15 s, extension at 72 ° C for 1 min for a total of 29 cycles; finally extending at 72 ° C for 10 min, cooling to room temperature.
  • the PCR amplification product and the bacterial expression vector pET-29a(+) were digested with restriction endonucleases NdeI and XhoI, respectively, and the cut MTHFR66 nucleotide sequence fragment and the digested bacterial expression vector pET-29a ( +) Enzyme-linked, the enzyme-linked product was transformed into the expression host strain BL21 (DE3) to obtain a recombinant microorganism BL21 (MTHFR66).
  • LB medium tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, kanamycin 100 mg/L, pH adjusted to 7.5 with NaOH.
  • IPTG isopropyl thiogalactoside
  • Enzyme reaction system 300 ⁇ L: containing 1 mM substrate (dicamba), 0.2 mg MTHFR66, 1 mM tetrahydrofolate (THF), buffer system was 100 mM Tris-HCl (pH 8.0), temperature at 30 ° C in a water bath The reaction was carried out for 1 hour and then left in boiling water for 1 min to terminate the reaction. The reaction solution was freeze-dried, 300 ⁇ L of methanol was added to dissolve the lyophilizate, and the amount of 3,6-dichlorosalicylic acid (DCSA), the intermediate metabolite of dicamba, was detected by high performance liquid chromatography (HPLC).
  • An enzyme activity unit is defined as the amount of enzyme required to degrade dexamethasone to produce 1 nmol of product DCSA within 1 min at pH 8.0 at a temperature of 30 ° C, expressed as U.
  • Enzyme-active reaction system 300 ⁇ L: Tris-HCl containing 1 mM substrate (dicamba), 0.2 mg MTHFR66 and 0.01 mM, 0.02 mM, 0.05 mM and 1 mM tetrahydrofolate (THF), respectively, buffer system at a concentration of 100 mM pH 8.0), the reaction was carried out in a water bath at a temperature of 30 ° C for 1 hour, and then left in boiling water for 1 min to terminate the reaction. The reaction solution was freeze-dried, 300 ⁇ L of methanol was added to dissolve the lyophilizate, and the amount of DCSA produced by the intermediate product of dicamba was detected by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • Enzyme reaction system 300 ⁇ L: containing 0.2 mg of MTHFR66, 1 mM 5-methyltetrahydrofolate (5-CH3-H4F), buffer system of 100 mM Tris-HCl (pH 8.0), temperature at 30 ° C in a water bath The reaction was carried out for 1 hour and then left in boiling water for 1 min to terminate the reaction. The reaction solution was freeze-dried, and the dried product was added to 300 ⁇ L of 0.1 mol/L. KH2PO4 (pH 6.8, 1% ascorbic acid, 0.1% ⁇ -mercaptoethanol) was dissolved, filtered through a filter (pore size 0.22 ⁇ m), and detected by high performance liquid chromatography.
  • KH2PO4 pH 6.8, 1% ascorbic acid, 0.1% ⁇ -mercaptoethanol
  • the liquid chromatographic conditions were: KH2PO4 (pH 3.0) with a mobile phase of 0.05 mol/L: acetonitrile (90:10, V/V), Zorbax C218ODS Spherex reverse phase column (5 ⁇ m, 4.6 mm ⁇ 250 mm, Agilent, USA).
  • the column temperature was 23 ° C
  • the UV detector the measurement wavelength was 298 nm
  • the injection volume was 20 ⁇ L
  • the flow rate was 1.0 mL/min.
  • the external standard method is quantified by peak area.
  • the metabolites were identified by HPLC-MS (high performance liquid chromatography and mass spectrometry) under the following conditions: mobile phase 0.05 mol/L KH2PO4 (pH 3.0): acetonitrile (90:10, V/V), Zorbax XDB- C18, 5 cm x 0.46 cm, 1.8 mm reverse phase column (5 [mu]m, 4.6 mm x 250 mm, Agilent, USA) with a flow rate of 0.25 mL/min.
  • the MS analysis used the ESI mode and the detector was an Agilent G6410B Triple Quad Mass Spectrometer.
  • the amino acid sequence (289 amino acids, as shown in SEQ ID NO: 2 in the Sequence Listing) of the methyltetrahydrofolate reductase MTHFR66 is maintained unchanged, and the amino acid corresponding to the methyltetrahydrofolate reductase MTHFR66 is encoded.
  • the sequence of the MTHFR66 nucleotide sequence (870 nucleotides) was codon optimized.
  • the strategy of codon optimization transformation mainly includes: based on the preferred codons of monocotyledonous corn and dicotyledon soybeans, the transformation of unstable sequences, and the improvement of G+C content.
  • the natural gene has a low G+C content and a high A+T content.
  • +T is rich in regions, similar to the TATA box in the gene promoter, which results in aberrant transcription of the gene; on the other hand, the polyadenylation signal sequence (AAUAAA) in the transcribed mRNA, associated with mRNA splicing Small RNA complementary sequences cause RNA instability.
  • the modified gene sequence also changes the unstable structure of DNA and transcription into mRNA, thus ensuring the normal translation of the protein; on the other hand, applying the preferred password of corn and soybean.
  • the natural gene sequence is engineered to exclude restriction enzyme sites and modifications of some sequences.
  • the optimized MTHFR66 nucleotide sequence was obtained, and the optimized MTHFR66 nucleotide sequence contained 870 nucleotides, encoding 289 amino acids, and the nucleotide sequence thereof is shown in SEQ ID NO: 1 in the Sequence Listing.
  • the optimized MTHFR66 nucleotide sequence was synthesized by Nanjing Kingsray Biotechnology Co., Ltd.; the optimized MTHFR66 nucleotide sequence (SEQ ID NO: 1) was also ligated to the 5' end of the SacI cleavage site. The 3' end of the MTHFR66 nucleotide sequence (SEQ ID NO: 1) is also ligated with a KasI cleavage site.
  • the fourth embodiment the construction of a recombinant expression vector of Arabidopsis thaliana and a recombinant expression vector for transforming Agrobacterium
  • the synthetic optimized MTHFR66 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the procedure was carried out according to the Promega product pGEM-T vector specification to obtain a recombinant cloning vector DBN01-T.
  • the construction process is shown in Figure 6 (wherein Amp represents the ampicillin resistance gene; f1 represents the origin of replication of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; and T7 is the T7 RNA polymerase promoter).
  • mMTHFR66 is the optimized MTHFR66 nucleotide sequence (SEQ ID NO: 1); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ L E. coli T1 competent cells, 10 ⁇ L of plasmid DNA (recombinant) Cloning vector DBN01-T), water bath at 42 ° C for 30 seconds; shaking culture at 37 ° C for 1 hour (shake at 100 rpm), coated with IPTG (isopropylthio- ⁇ -D-galactoside) and X -gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) ampicillin (100 mg/L) LB plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl) 10 g/L, agar 15 g/L, adjusted to pH 7.5 with NaOH) and grown overnight.
  • heat shock method Transgen, Beijing, China, CAT: CD501
  • White colonies were picked and cultured in LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, ampicillin 100 mg/L, pH adjusted to 7.5 with NaOH) at 37 °C. overnight.
  • the plasmid was extracted by alkaline method: the bacterial solution was centrifuged at 12000 rpm for 1 min, the supernatant was removed, and the precipitated cells were pre-cooled with 100 ⁇ l of ice (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose.
  • the TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was dissolved in the precipitate; the RNA was digested in a water bath at 37 ° C for 30 min; and stored at -20 ° C until use.
  • the positive clone was verified by sequencing.
  • the result showed that the optimized MTHFR66 nucleotide sequence inserted in the recombinant cloning vector DBN01-T was represented by SEQ ID NO: 1 in the sequence listing.
  • Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases SacI and KasI, respectively, and the cut optimized MTHFR66 nucleotide sequence fragment was inserted into Between the SacI and KasI sites of the expression vector DBNBC-01, the construction of the vector by conventional enzymatic cleavage method is well known to those skilled in the art, and the recombinant expression vector DBN111101 is constructed, and the construction process thereof is shown in Fig.
  • the recombinant expression vector DBN111101 was transformed into E. coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 ⁇ L of E. coli T1 competent cells, 10 ⁇ L of plasmid DNA (recombinant expression vector DBN111101), 42 ° C water bath for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake shake at 100 rpm); then LB solid plate containing 50 mg/L kanamycin (trypeptin 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g) /L, adjust the pH to 7.5 with NaOH and incubate at 37 °C for 12 hours, pick white colonies, in LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, Kanamycin 50 mg/L was adjusted to pH 7.5 with NaOH and incubated overnight at 37 °C.
  • the plasmid was extracted by an alkali method.
  • the extracted plasmid was digested with restriction endonucleases SacI and KasI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the SacI and KasI sites of the recombinant expression vector DBN111101 was SEQ ID in the sequence listing. NO: The nucleotide sequence shown in Figure 1, which optimizes the MTHFR66 nucleotide sequence.
  • the recombinant cloning vector DBN01-T containing the optimized MTHFR66 nucleotide sequence was constructed according to the method of 1 in the present Example, and the native MTHFR66 nucleotide sequence (SEQ ID NO: 3) was used to construct a nucleotide sequence containing the native MTHFR66.
  • Recombinant cloning vector DBN01R-T The positive clone was verified by sequencing, and the result showed that the nucleotide sequence of the native MTHFR66 inserted in the recombinant cloning vector DBN01R-T was the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing, that is, the natural MTHFR66 nucleotide sequence was correctly inserted. .
  • the recombinant expression vector DBN111101N containing the native MTHFR66 nucleotide sequence was constructed using the native MTHFR66 nucleotide sequence according to the method of constructing the recombinant expression vector DBN111101 containing the optimized MTHFR66 nucleotide sequence as described in Example 2, and the vector structure thereof was as follows.
  • Figure 8 vector backbone: pCAMBIA2301 (available from CAMBIA); Kan: kanamycin gene; RB: right border; prAtUbi10: Arabidopsis Ubiquitin 10 gene promoter (SEQ ID NO: 6) AtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 7); MTHFR66: native MTHFR66 nucleotide sequence (SEQ ID NO: 3); tNos: terminator of the nopaline synthase gene (SEQ ID NO: 8) ; prCaMV35S: cauliflower mosaic virus 35S promoter (SEQ ID NO: 9); PAT: glufosinate acetyltransferase gene (SEQ ID NO: 10); tCaMV35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 11) ); LB: left border).
  • nucleotide sequence of the native MTHFR66 inserted in the recombinant expression vector DBN111101N was the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, that is, the native MTHFR66 nucleotide sequence was correctly inserted.
  • the transformation conditions were: 100 ⁇ L Agrobacterium GV3101, 3 ⁇ L plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; the transformed Agrobacterium GV3101 was inoculated into LB tubes Incubate for 2 hours at a temperature of 28 ° C and a rotation speed of 200 rpm, and apply it to an LB plate containing 50 mg/L of rifampicin and 50 mg/L of kanamycin until a positive monoclonal is grown, and a single monoclonal is picked.
  • the plasmid was cultured and extracted, and DBN111101 and DBN111101N were digested with restriction endonucleases, and the results showed that the recombinant expression vectors DBN111101 and DBN111101N were completely
  • Wild type Arabidopsis seeds were suspended in a 0.1% agarose solution. The suspended seeds were stored at 4 ° C for 2 days to complete the need for dormancy to ensure simultaneous seed germination. Mix the horse dung with vermiculite and irrigate with water to wetness to drain the soil mixture for 24 hours. The pretreated seeds were planted on a soil mixture and covered with a moisturizing hood for 7 days. Seeds were germinated and plants were grown in a greenhouse under constant temperature (22 ° C) constant humidity (40-50%) long day conditions (16 hours light / 8 hours dark) with a light intensity of 120-150 [mu]mol/m2 sec. Start irrigating the plants with Hoagland nutrient solution, then irrigate with deionized water to keep the soil moist but not soaked.
  • Arabidopsis thaliana was transformed using flower soaking.
  • One or more 15-30 mL precultures of YEP broth containing kanamycin (100 mg/L) and rifampicin (10 mg/L) were inoculated with selected Agrobacterium colonies. The culture was incubated overnight at 28 ° C with constant shaking at 220 rpm.
  • Each preculture was used to inoculate two 500 ml cultures of YEP medium containing kanamycin (100 mg/L) and rifampicin (10 mg/L) and the culture was incubated overnight at 28 °C with constant shaking.
  • the cells were pelleted by centrifugation at about 8700 x g for 10 minutes at room temperature, and the resulting supernatant was discarded.
  • the cell pellet was gently resuspended in 500 mL osmotic medium containing 1/2 x MS salt/B5 vitamin, 10% (w/v) sucrose, 0.044 ⁇ M benzylaminopurine (10 ⁇ L/L (1 mg/) Stock solution in mL DMSO)) and 300 ⁇ L/L Silvet L-77. Plants of about 1 month old were soaked in the medium for 15 seconds to ensure that the latest inflorescences were immersed. The sides of the plants were then placed downside and covered (transparent or opaque) for 24 hours, then washed with water and placed vertically. The plants were cultured at 22 ° C with a photoperiod of 16 hours light / 8 hours dark. Seeds were harvested after about 4 weeks of soaking.
  • T1 seeds Freshly harvested (optimized MTHFR66 nucleotide sequence and native MTHFR66 nucleotide sequence) T1 seeds were dried at room temperature for 7 days. Seeds were seeded in 26.5 x 51 cm germination trays, each receiving 200 mg T1 seeds (about 10,000 seeds), which were previously suspended in 40 mL of 0.1% agarose solution and stored at 4 ° C for 2 days to complete dormancy Need to ensure that seeds are germinated simultaneously.
  • the pretreated seeds (each 40 mL) were evenly planted on the soil mixture with a pipette and covered with a moisturizing hood for 4-5 days. The hood was removed 1 day prior to the initial transformant selection using glufosinate (selected co-transformed PAT gene) after emergence.
  • T1 was sprayed with a 0.2% solution of Liberty herbicide (200 g ai/L glufosinate) at a spray volume of 10 mL/disc (703 L/ha) after 7 days of planting (DAP) and again at 11 DAP using a DeVilbiss compressed air nozzle. Plants (coronal stage and 2-4 leaf stage, respectively) were provided to provide an effective amount of 280 g ai/ha of glufosinate per application. Surviving strains (plants that are actively growing) were identified 4-7 days after the last spraying and transplanted to 7 cm x prepared with horse manure and vermiculite. 7cm square pots (3-5 trees per plate).
  • Liberty herbicide 200 g ai/L glufosinate
  • the transplanted plants were covered with a moisturizing hood for 3-4 days and placed in a 22 ° C culture chamber as before or directly into the greenhouse. The hood was then removed and the plants were planted in the greenhouse at least 1 day prior to testing for the ability of the MTHFR66 gene to provide dicamba herbicide tolerance (22 ⁇ 5 ° C, 50 ⁇ 30% RH, 14 hours light: 10 hours dark, minimum 500 ⁇ E /m2s1 natural + supplement light).
  • the first Arabidopsis transformation was carried out using the MTHFR66 gene.
  • the T1 transformants were first selected from the untransformed seed background using a glufosinate selection protocol.
  • the recombinant expression vector DBN111101 was transformed into a chloroplast-transferred Arabidopsis thaliana plant (At mMTHFR66) optimized for the MTHFR66 nucleotide sequence, and the recombinant expression vector DBN111101N was transformed into a chloroplast-transferred native MTHFR66 nucleotide sequence.
  • Arabidopsis plants Arabidopsis plants (At MTHFR66).
  • T1 seeds of about 20,000 At mMTHFR66 were screened, and 197 T1 positive transformants (PAT gene) were identified, with a transformation efficiency of about 1.0%; T0 seeds of about 20,000 At MTHFR66 were screened, and 182 T1 seeds were identified.
  • Generation positive transformants (PAT gene) about 0.91% conversion efficiency.
  • Arabidopsis thaliana T1 plants (At-mMTHFR66) which are optimized for the MTHFR66 nucleotide sequence, Arabidopsis T1 plants (At-MTHFR66) transfected with the native MTHFR66 nucleotide sequence, and wild-type Arabidopsis plants (after seeding) 18 days) Test for herbicide tolerance in dicamba.
  • Plant tolerance was measured after spraying for 7 days and 14 days: after 7 days, the growth condition and the blank solvent (water) were consistently classified as high-resistance plants, and after 7 days, the rosettes of the rosettes were classified as medium-resistant plants, and after 14 days, they could not be pumped.
  • the moss is classified as a low-resistance plant, and the death after 14 days is not resistant to the plant. Since each Arabidopsis thaliana T1 plant is an independent transformation event, a significant difference in individual T1 response can be predicted at a given dose. The results are shown in Table 1 and Figure 9.
  • Recombinant cloning vector DBN01-T and expression vector DBNBC-02 were digested with restriction endonucleases SacI and KasI, respectively, and the cut optimized MTHFR66 nucleotide sequence fragment was inserted into Between the SacI and KasI sites of the expression vector DBNBC-02, the construction of the vector by conventional enzymatic cleavage method is well known to those skilled in the art, and the recombinant expression vector DBN-HT130066 is constructed, and the construction process thereof is shown in FIG.
  • the recombinant expression vector DBN-HT130066 was transformed into E. coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 ⁇ L of E. coli T1 competent cells, 10 ⁇ L of plasmid DNA (recombinant expression vector DBN-HT130066), and a water bath at 42 ° C for 30 seconds.
  • the extracted plasmids were digested with restriction endonucleases SacI and KasI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence of the recombinant expression vector DBN-HT130066 between the SacI and KasI sites was in the sequence listing.
  • the recombinant expression vector DBN-HT120066 containing the optimized MTHFR66 nucleotide sequence was constructed according to the method of 1 in the present Example, and the recombinant cloning vector containing the native MTHFR66 nucleotide sequence according to the third embodiment of the present invention was used.
  • DBN01R-T a recombinant expression vector DBN-HT130066N containing the native MTHFR66 nucleotide sequence was constructed, and its vector structure is shown in Figure 11 (vector backbone: pCAMBIA2301 (available by CAMBIA); Kan: kanamycin gene; RB: Right border; prUbi: maize Ubiquitin 1 gene promoter (SEQ ID NO: 12); AtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 7); MTHFR66: native MTHFR66 nucleotide sequence (SEQ ID NO: 3); tNos: terminator of the nopaline synthase gene (SEQ ID NO: 8); PMI: phosphomannose isomerase gene (SEQ ID NO: 13); LB: left border).
  • vector backbone pCAMBIA2301 (available by CAMBIA)
  • Kan kanamycin gene
  • RB Right border
  • prUbi maize Ubiquitin
  • nucleotide sequence of the native MTHFR66 inserted in the recombinant expression vector DBN-HT130066N was the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, that is, the native MTHFR66 nucleotide sequence was correctly inserted.
  • the recombinant expression vectors DBN-HT130066 and DBN-HT130066N, which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium LBA4404, 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, and warmed at 37 ° C for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated into LB tubes and incubated at a temperature of 28 ° C and a rotation speed of 200 rpm for 2 hours.
  • Agrobacterium LBA4404 Invitrgen, Chicago, USA, CAT: 18313-015
  • the immature embryo of the aseptically cultured maize variety Heise 31 (Z31) is co-cultured with the Agrobacterium described in the seventh embodiment of the present invention, to the seventh embodiment of the present invention.
  • T-DNA in the recombinant expression vectors DBN-HT130066 and DBN-HT130066N constructed in the 1 and 2 were transferred into the maize genome to obtain a maize plant (Zm-mMTHFR66) that was mapped to the chloroplast and transferred to the optimized MTHFR66 nucleotide sequence and the maize into the native MTHFR66 nucleotide sequence.
  • Plant (Zm-MTHFR66); wild type maize plants were used as controls at the same time.
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with Agrobacterium suspension, wherein Agrobacterium can transfer the optimized MTHFR66 nucleotide sequence and the native MTHFR66 nucleotide sequence. At least one cell to one of the young embryos (step 1: infection step).
  • the immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • the immature embryo is in solid medium after the infection step (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, acetosyringone (AS) 100 mg/L) It was cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step.
  • the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg /
  • At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in L, plant gel 3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step).
  • the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selective agent (mannose) and the selected transformed callus is selected.
  • a selective agent mannose
  • the immature embryo is screened in solid medium with selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene)
  • MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene Incubation of oxyacetic acid (2,4-D) 1 mg/L, plant gel 3 g/L, pH 5.8) resulted in selective growth of transformed cells.
  • the callus regenerates the plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) Recycled plants.
  • the selected resistant callus was transferred to the MS differentiation medium (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, 6-benzyl adenine 2 mg/L, mannose) 5 g/L, plant gel 3 g/L, pH 5.8), cultured and differentiated at 25 °C.
  • the differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, indole-3-acetic acid 1 mg/L, plant gel 3 g/L) , pH 5.8), cultured at 25 ° C to a height of about 10 cm, moved to a greenhouse to grow to firm. In the greenhouse, the cells were cultured at 28 ° C for 16 hours and then at 20 ° C for 8 hours.
  • the specific method for detecting the copy number of the PMI gene is as follows:
  • Step 11 Take 100 mg of each of the maize plants transformed with the MTHFR66 nucleotide sequence, the maize plants transfected with the native MTHFR66 nucleotide sequence, and the wild-type maize plants, respectively, and homogenize them with liquid nitrogen in a mortar. , each sample takes 3 repetitions;
  • Step 12 Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product manual for the specific method;
  • Step 13 Determine the genomic DNA concentration of the above sample using NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / ⁇ L;
  • Step 15 The Taqman probe real-time PCR method is used to identify the copy number of the sample, and the sample with the known copy number is used as a standard, and the sample of the wild type corn plant is used as a control, and each sample has 3 replicates, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:
  • Primer 3 CCGGGTGAATCAGCGTTT is shown in SEQ ID NO: 14 in the Sequence Listing;
  • Probe 1 TGCCGCCAACGAATCACCGG as shown in SEQ ID NO: 16 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 probe at a concentration of 100 ⁇ M and 860 ⁇ L of 1 ⁇ TE buffer, and was stored in an amber tube at 4°C.
  • the PCR reaction conditions are:
  • the experimental results showed that the optimized MTHFR66 nucleotide sequence and the native MTHFR66 nucleotide sequence were integrated into the genome of the tested maize plants, and transferred to the maize plants optimized for the MTHFR66 nucleotide sequence and transferred to the native MTHFR66 nucleoside.
  • Transgenic maize plants containing a single copy of the MTHFR66 gene were obtained from the acid sequence maize plants.
  • the maize plants transformed into the optimized MTHFR66 nucleotide sequence, the maize plants transferred to the native MTHFR66 nucleotide sequence, and the wild-type maize plants (V5-V6 period) were tested for herbicide resistance effects on dicamba.
  • Maize plants transfected with optimized MTHFR66 nucleotide sequence maize plants transfected with native MTHFR66 nucleotide sequence and T1 hybrid plants of wild-type maize plants (V5-V6 period), and dicamba herbicide (4480g) Ae/ha, 8 times field concentration) and white solvent (water) spray.
  • the development of the supporting roots was counted 21 days after spraying.
  • Zm-mMTHFR66 has 3 strains (S1, S2 and S3), Zm-MTHFR66 has 2 strains (S4 and S5), and wild type (CK) has 1 strain; from each strain, 10- 15 strains were tested. The results are shown in Table 2.
  • the results in Table 2 indicate that the optimized MTHFR66 gene confers a high level of tolerance to the transgenic maize plant dicamba herbicide (since the monocot itself has some resistance to the dicamba herbicide and thus exhibits high levels of resistance); Zm-MTHFR66, Zm-mMTHFR66 was able to produce higher dicamba herbicide tolerance, indicating that the MTHFR66 gene can be enhanced by plant codon optimization to enhance the tolerance of maize plants to dicamba herbicides; There is no dicamba herbicide tolerance.
  • the MTHFR66 protein of the present invention can degrade the dicamba herbicide, optimize the MTHFR66 gene using the preferred codons of maize and soybean, making it particularly suitable for expression in plants; optimizing the MTHFR66 gene can give the transgenic plants better wheatgrass weeding Tolerance to the agent; at the same time, the herbicide tolerance protein MTHFR66 of the present invention also has methyltetrahydrofolate reductase activity, which is different from the known dicamba tolerant gene, and thus can expand the dicamba tolerant type on the plant. Application range.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Environmental Sciences (AREA)
  • Toxicology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

提供了一种针对除草剂麦草畏的抗性蛋白质、其编码基因及用途，该基因包括：（a）编码SEQ ID NO:2所示的氨基酸序列的核苷酸序列；或（b）在严格条件下与（a）限定的核苷酸序列互补的核苷酸序列；或（c）SEQ ID NO:1所示的核苷酸序列。

Description

除草剂抗性蛋白质、其编码基因及用途 技术领域
本发明涉及一种除草剂抗性蛋白质、其编码基因及用途，特别是涉及一种对除草剂麦草畏具有耐受性的蛋白质、其编码基因及用途。
背景技术
杂草可以迅速耗尽土壤中作物和其它目的植物所需要的有价值的养分。尽管目前可以得到耐受除草剂草甘膦、草丁膦、2,4-D和其它除草剂处理的转基因植物，但是还存在空白区域，如所控制的杂草范围、开发额外的除草剂耐受性作物等。此外，耐受上述除草剂的杂草的出现(尽管通常是局部的和可变的)造成了对额外或备选的杂草控制措施的需要。
已经证明耐受除草剂性状在商业上是有价值的，因此需要增加耐受其它除草剂的植物和管理难以控制的杂草种类的选项，以避免过度依赖任何单一除草剂，尤其需要针对环境友好的并且在控制杂草方面高度有效的除草剂而开发除草剂耐受性。麦草畏是有效且环境友好的除草剂之一，其已经被农民使用40多年了，麦草畏可用于控制玉米、高粱、小米、牧草、干草、牧场、甘蔗、芦笋、草皮和草籽作物中一年生和多年生阔叶杂草和几种窄叶杂草；与此同时，麦草畏可以伤害许多商业作物和双子叶植物，如大豆、棉花、豌豆、马铃薯、向日葵和油菜，上述作物/植物对于低水平的麦草畏都是特别敏感的。尽管如此，麦草畏在控制杂草生长中仍然是有效的，并且是重要的。
已报道从嗜麦芽假单胞菌(Pseudomonas maltophilia)分离了编码麦草畏单加氧酶(DMO)的基因，其为铁氧还蛋白依赖型并赋予对麦草畏的耐受性。DMO参与将除草剂麦草畏(3,6-二氯-邻-茴香酸)转化为无毒的3,6-二氯水杨酸(DCSA)，表达DMO基因的植物具有对麦草畏的耐受性。
由于目前发现的麦草畏耐受型基因均极其相似，所以需要更多新型的麦草畏耐受型基因以避免过度依赖一种麦草畏耐受型基因，使麦草畏-麦草畏耐受型作物在商业上具有更广阔的应用空间。
发明内容
本发明的目的是提供一种除草剂抗性蛋白质、其编码基因及用途，所述MTHFR66蛋白在植物中对麦草畏除草剂具有较高的耐受性。
为实现上述目的，本发明提供了一种基因，包括：
(a)编码SEQ ID NO:2所示的氨基酸序列的核苷酸序列；或
(b)在严格条件下与(a)限定的核苷酸序列互补的核苷酸序列；或
(c)SEQ ID NO:1所示的核苷酸序列。
所述严格条件可为在6×SSC(柠檬酸钠)、0.5％SDS(十二烷基硫酸钠)溶液中，在65℃下杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。
为实现上述目的，本发明还提供了一种表达盒，包含在有效连接的调控序列调控下的所述基因。
进一步地，所述调控序列为叶绿体转运肽，所述叶绿体转运肽与所述基因有效连接。
优选地，所述叶绿体转运肽的核苷酸序列具有SEQ ID NO:7所示的核苷酸序列。
为实现上述目的，本发明还提供了一种包含所述基因或所述表达盒的重组载体。
为实现上述目的，本发明还提供了一种增加耐受除草剂范围的方法，包括：将SEQ ID NO:2所示的氨基酸序列组成的蛋白质或所述表达盒编码的蛋白质在植物中与至少一种不同于SEQ ID NO:2所示的氨基酸序列组成的蛋白质或所述表达盒编码的蛋白质的第二种蛋白质一起表达。
进一步地，所述第二种蛋白质为草甘膦耐受性蛋白质、草铵膦耐受性蛋白质、α酮戊二酸双加氧酶、4-羟苯基丙酮酸双加氧酶、乙酰乳酸合酶、细胞色素类蛋白质或原卟啉原氧化酶。
在本发明中，MTHFR66除草剂抗性蛋白质在一种转基因植物中的表达可以伴随着一个或多个草甘膦耐受性蛋白质和/或草铵膦耐受性蛋白质的表达。这种超过一种的除草剂耐受性蛋白质在同一株转基因植物中共同表达可以通过遗传工程使植物包含并表达所需的基因来实现。另外，一种植物(第1亲本)可以通过遗传工程操作表达MTHFR66除草剂抗性蛋白质，第二种植物(第2亲本)可以通过遗传工程操作表达草甘膦耐受性蛋白质和/或草铵膦耐受性蛋白质。通过第1亲本和第2亲本杂交获得表达引入第1亲本和第2亲本的所有基因的后代植物。
为实现上述目的，本发明还提供了一种选择转化的植物细胞的方法，包括：用所述基因或所述表达盒转化多个植物细胞，并在允许表达所述基因或所述表达盒的转化细胞生长，而杀死未转化细胞或抑制未转化细胞生长的除草剂浓度下培养所述细胞，所述除草剂为麦草畏。
为实现上述目的，本发明还提供了一种控制杂草的方法，包括：对种植植物的大田施用有效剂量的麦草畏除草剂，所述植物包含所述基因、所述表达盒或所述重组载体。
为实现上述目的，本发明还提供了一种保护植物免受由除草剂引起的损伤的方法，包括：将所述基因、所述表达盒或所述重组载体导入植物，使导入后的植物产生足够保护其免受麦草畏损害量的除草剂耐受性蛋白质。
为实现上述目的，本发明还提供了一种赋予植物麦草畏除草剂耐受性的方法，包括：将所述基因、所述表达盒或所述重组载体导入植物。
为实现上述目的，本发明还提供了一种控制草甘膦耐性植物的大田中草甘膦耐受性杂 草的方法，包括：对种植草甘膦耐受性植物的大田施用有效剂量的麦草畏，所述草甘膦耐受性植物包含所述基因、所述表达盒或所述重组载体。
为实现上述目的，本发明还提供了一种产生麦草畏耐受性植物的方法，包括向所述植物的基因组中引入所述基因或所述表达盒，以产生麦草畏耐受性植物。
具体地，所述产生麦草畏耐受性植物的方法包括：通过将亲本植物自交或与第二种植物杂交而产生麦草畏耐受性植物，所述亲本植物和/或第二种植物包含所述基因或所述表达盒，所述麦草畏耐受性植物遗传了来自所述亲本植物和/或第二种植物的所述基因或所述表达盒。
为实现上述目的，本发明还提供了一种培养对麦草畏除草剂具有耐受性的植物的方法，包括：
种植至少一粒植物种子，所述植物种子的基因组中包括所述基因或所述表达盒；
使所述植物种子长成植株；
用有效剂量麦草畏除草剂喷洒所述植株，收获与其他不具有所述基因或所述表达盒的植株相比具有减弱的植物损伤的植株。
在上述技术方案的基础上，优选地，所述植物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
为实现上述目的，本发明还提供了一种甲基四氢叶酸还原酶耐受麦草畏除草剂的用途，所述甲基四氢叶酸还原酶具有SEQ ID NO:2所示的氨基酸序列。
为实现上述目的，本发明还提供了一种产生甲基四氢叶酸还原酶的植物耐受麦草畏除草剂的用途，所述甲基四氢叶酸还原酶具有SEQ ID NO:2所示的氨基酸序列。
将所述的基因或所述的表达盒或所述的重组载体导入植物，在本发明中为将外源DNA导入植物细胞，常规转化方法包括但不限于，农杆菌介导的转化、微量发射轰击、直接将DNA摄入原生质体、电穿孔或晶须硅介导的DNA导入。
本发明可以增加植物对于氧化应激的耐受性，包括但不限于，向植物群提供麦草畏或麦草畏单加氧酶所介导代谢的产物或其类似物，以改善植物的除草剂耐受性，例如，通过将麦草畏代谢为DCSA。
本发明“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 PCTCN2017079661-appb-000001
(作为DMA盐)、
Figure PCTCN2017079661-appb-000002
(BASF，作为DGA盐)、VEL-58-CS-11TM和
Figure PCTCN2017079661-appb-000003
(BASF，作为DGA盐)。
目前已经报道的含甲氧基芳烃化合物脱甲基酶有4种，分别是(1)RHOs(Rieske非血红素型氧化酶)，降解麦草畏的DMO(GenBank:AY786443.1)属于三组份RHO类型的氧化酶；(2)细胞色素P450，是一类亚铁血红素—硫醇盐蛋白的超家族，它参与内源性物质和包括药物、环境化合物在内的外源性物质的代谢，很多含甲氧基芳烃化合物脱 甲基酶就是这一类型；(3)厌氧型四氢叶酸依赖型脱甲基酶，发现于厌氧细菌如热醋穆尔氏菌(Moorellathermoacetica)中，参与木质素降解中间产物如丁香酸和香草酸的厌氧降解，也有研究发现厌氧型四氢叶酸依赖型脱甲基酶也可以厌氧降解麦草畏；(4)好氧型四氢叶酸依赖型的脱甲基酶(Sphingomonas paucimobilis SYK-6)，参与木质素降解中间产物如丁香酸和香草酸的厌氧降解，但目前还没有这类好氧型四氢叶酸依赖型脱甲基酶能降解麦草畏的报道。
MTHFR为5,10-methylenetetrahydrofolate reductase，亚甲基四氢叶酸还原酶蛋白编码基因，主要作用是在叶酸代谢通路中将5,10-亚甲基四氢叶酸转化为具有生物学功能的5-甲基四氢叶酸及其逆反应。
本发明所述基因用于植物表达后具有允许在植物中使用麦草畏除草剂的特性，所述植物中固有耐性不存在或不足以允许使用麦草畏除草剂。此外，本发明所述MTHFR66基因可以在天然耐性不足以允许选择性时在植物中提供对麦草畏除草剂的防护。向田地使用的施用量是大约0.0025磅/英亩(lb/a)到大约20lb/a麦草畏，更通常从0.25lb/a至12lb/a。在同一大田里(连续或罐混组合地)组合不同化学类别和具有不同作用模式和范围的除草剂可以提供对大多数需要除草剂控制的潜在杂草的控制。
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耐受性生物型。多数除草剂耐受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耐受性杂草的方法，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MTHFR66基因与草甘膦耐性性状(和/或其他除草剂耐受性性状)叠加可通过允许对同一作物选择性使用草甘膦和麦草畏而实现对草甘膦耐受性作物中草甘膦耐受性杂草物种(被麦草畏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耐受性杂草的能力。作物中与草甘膦耐受性基因/MTHFR66基因叠加的草甘膦应用范围可以从250至2500g ae/ha；麦草畏可按照从0.25lb/a至12lb/a。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。
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耐受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)在作物中产生除草甘膦耐性作物外的除草剂耐性的化学品。事实上，可以单独或以多重组合叠加编码以下耐受性的性状以提供有效控制或防止杂草演替对任意前述类别的除草剂的耐受性的能力：草甘膦耐受性(如耐受性植物或细菌EPSPS、GOX、GAT)、草铵膦耐受性(如PAT、Bar)、苯氧基生长素耐受性(如2,4-D、二甲四氯耐受性基因如AAD-1、AAD-12等)乙酰乳酸合酶(ALS)抑制性除草剂耐受性(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耐受性基因如AHAS、Csrl、SurA等)、溴草腈耐受性(如Bxn)、对HPPD(4-羟苯基丙酮酸双加氧酶)酶抑制剂的耐受性、对八氢番茄红素去饱和酶(PDS)抑制剂的耐受性、对光系统Ⅱ抑制性除草剂的耐受性(如psbA)、对光系统Ⅰ抑制性除草剂的耐受性、对原卟啉原氧化酶Ⅸ(PPO)抑制性除草剂耐受性(如PPO-1)、对苯脲除草剂的耐受性(如CYP76B1)、二氯甲氧苯酸降解酶等等。
关于其他除草剂，一些其它优选的ALS抑制剂包括三唑嘧啶磺苯胺(氯酯磺草胺、双氯磺草胺、唑嘧磺草胺、磺草唑胺和嘧啶并三唑类磺胺)、嘧啶硫代苯甲酸和氟唑磺隆。一些优选的HPPD抑制剂包括甲基磺草酮、异恶唑草酮和磺草酮。一些优选的PPO抑制剂包括丙炔氟草胺、氟丙嘧草酯、唑草酮、甲磺草胺和二苯醚(如三氟羧草醚、氟磺胺草醚、乳氟禾草灵和乙氧氟草醚)。
此外，可以将MTHFR66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(如昆虫耐受性、真菌耐受性或胁迫耐性等)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。
本发明MTHFR66基因能降解麦草畏除草剂，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。
本发明可进行转基因表达，可以控制几乎所有阔叶杂草的除草剂组合。MTHFR66基因可作为优秀的除草剂耐受作物性状与例如其它除草剂耐受作物性状(如草甘膦耐受性、草铵膦耐受性、苯氧基生长素耐受性、ALS抑制剂(如咪唑啉酮类、磺酰脲类、三唑并嘧啶磺酰胺类)耐受性、溴草腈耐受性、HPPD抑制剂耐受性、PPO抑制剂耐受性等)和昆虫耐受性性状(Cry1Ab、Cry1F、Vip3、其它苏云金芽孢杆菌蛋白质或非芽孢杆菌属来源的昆虫耐受性蛋白等)叠加。此外，MTHFR66基因可作为选择标记物辅助选择用另一个基因或基因群遗传改造的植物的原代转化体。
本发明的除草剂耐性作物性状可用在与其它除草剂耐性作物性状(包括但不限于草甘膦耐性)的新组合中。由于对除草剂(如草甘膦)的新获得的耐受性或固有的耐性，这些性状组合产生控制杂草物种的新方法。因此，除了除草剂耐性作物性状，本发明的范围包括使用除草剂控制杂草的新方法，其中通过转基因作物中的所述酶产生对所述除草剂的耐 性。
本发明可应用于多种植物中，如拟南芥、烟草、大豆、棉花、稻、玉米和芸薹。本发明还可用于多种其它单子叶(如牧草禾本科或草坪草禾本科)和双子叶作物(如苜蓿、三叶草、乔木物种等)。类似的，麦草畏(或其它MTHFR66底物)可更积极地用于耐性适中的禾本科作物中，由此性状得到的提高的耐性将为种植者提供能以更有效的用量和更广的施用时间来使用这些除草剂而无作物损伤风险的可能性。
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。
本发明中所述“耐受性”和所述“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，即使植物受到除草剂处理的一定损伤程度明显，植物仍可被认为“耐受性”或“抗性”。本发明中术语“耐性”比术语“耐受性”更广泛，并包括“耐受性”，以及特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。
本发明中所述的多核苷酸和/或核苷酸形成完整“基因”，在所需宿主细胞中编码蛋白质或多肽。本领域技术人员很容易认识到，可以将本发明的多核苷酸和/或核苷酸置于目的宿主中的调控序列控制下。
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所述MTHFR66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(pin Ⅰ和pin Ⅱ)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，包括但不限于拟南芥叶绿体转运肽AtCTP2，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(即抗生素耐受性标记物、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)。
本发明可赋予植物新除草剂耐受性性状，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1倍的应用水平。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。
本发明中，所述除草剂抗性蛋白质为MTHFR66氨基酸序列，如序列表中SEQ ID NO:2所示。所述除草剂抗性基因为MTHFR66核苷酸序列，如序列表中SEQ ID NO:1所示。所述除草剂抗性基因为用于植物，除了包含由MTHFR66核苷酸序列编码的蛋白质的编码区外，也可包含其他元件，例如编码转运肽的编码区、编码选择性标记的蛋白质或赋予昆虫耐受性的蛋白质的编码区。
本发明中MTHFR66除草剂抗性蛋白质对麦草畏除草剂具有耐性。本发明中的植物，在其基因组中含有外源DNA，所述外源DNA包含MTHFR66核苷酸序列，通过表达有效量的该蛋白而保护其免受除草剂的威胁。有效量是指未损伤的或轻微损伤的剂量。同时，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。
本发明提供了一种除草剂抗性蛋白质、其编码基因及用途，具有以下优点：
1、对除草剂耐受性强。本发明MTHFR66基因可以降解麦草畏除草剂，优化MTHFR66基因采用玉米和大豆的偏好密码子，使其特别适合在植物中表达；优化MTHFR66基因可以赋予转基因植物麦草畏除草剂耐受性，且所述优化MTHFR66基因定位于叶绿体中表达可以增强转基因植物对麦草畏除草剂的耐受性。
2、应用前景广阔。本发明除草剂抗性蛋白质MTHFR66为甲基四氢叶酸还原酶，其不同于已知的麦草畏耐受型基因，因此可以扩大麦草畏耐受型在植物上应用范围。
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为本发明除草剂抗性蛋白质、其编码基因及用途的MTHFR66基因在表达宿主菌BL21(DE3)中表达的蛋白SDS-PAGE电泳图；
图2为本发明除草剂抗性蛋白质、其编码基因及用途的诱导表达的MTHFR66蛋白降解麦草畏的HPLC图谱；
图3为本发明除草剂抗性蛋白质、其编码基因及用途的诱导表达的MTHFR66蛋白在不同浓度的四氢叶酸条件下降解麦草畏的HPLC图谱；
图4为本发明除草剂抗性蛋白质、其编码基因及用途的诱导表达的MTHFR66蛋白代谢5-甲基四氢叶酸的HPLC图谱；
图5为本发明除草剂抗性蛋白质、其编码基因及用途的诱导表达的MTHFR66蛋白代谢5-甲基四氢叶酸的中间产物鉴定一级质谱图；
图6为本发明除草剂抗性蛋白质、其编码基因及用途的含有优化MTHFR66核苷酸序列的重组克隆载体DBN01-T构建流程图；
图7为本发明除草剂抗性蛋白质、其编码基因及用途的含有优化MTHFR66核苷酸序列的重组表达载体DBN111101构建流程图；
图8为本发明除草剂抗性蛋白质、其编码基因及用途的含有天然MTHFR66核苷酸序列的重组表达载体DBN111101N结构示意图；
图9为本发明除草剂抗性蛋白质、其编码基因及用途的转基因拟南芥T1植株除草剂耐受性效果图；
图10为本发明除草剂抗性蛋白质、其编码基因及用途的含有优化MTHFR66核苷酸序列的重组表达载体DBN-HT130066构建流程图；
图11为本发明除草剂抗性蛋白质、其编码基因及用途的含有天然MTHFR66核苷酸序列的重组表达载体DBN-HT130066N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除草剂抗性蛋白质、其编码基因及用途的技术方案。
第一实施例、甲基四氢叶酸还原酶MTHFR66的体外高效表达及功能鉴定
1、细菌表达载体的构建和重组微生物获得
(1)MTHFR66基因的PCR扩增
设计一对引物：
引物1：5-GGAATTCCATATGGGCTCGCCCGTTATGG-3(下划线为NdeI酶切位点)，如序列表中SEQ ID NO:4所示；
引物2：5-CCGCTCGAGGTGCTTTCGAGCGTAGTCAG-3(下划线为XhoI酶切位点，如序列表中SEQ ID NO:5所示；
用下述PCR扩增体系扩增MTHFR66基因：
Figure PCTCN2017079661-appb-000004
模板DNA(即天然MTHFR66核苷酸序列)如序列表中SEQ ID NO:3所示。PCR反应条件为：98℃变性1min；然后进入下列循环：98℃变性15s，55℃退火15s，72℃延伸1min，共29个循环；最后72℃延伸10min，冷却至室温。
(2)细菌表达载体的构建和重组微生物获得
用限制性内切酶NdeI和XhoI分别酶切上述PCR扩增产物和细菌表达载体pET-29a(+)，将切下的MTHFR66核苷酸序列片段与酶切后的细菌表达载体pET-29a(+)进行酶连，将酶连产物转化到表达宿主菌BL21(DE3)，获得重组微生物BL21(MTHFR66)。
2、MTHFR66蛋白在大肠杆菌中的表达及纯化
所述重组微生物BL21(MTHFR66)在100mL的LB培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，卡那霉素100mg/L，用NaOH调pH至7.5)中培养至浓度为OD600nm＝0.6-0.8，加入浓度为0.4mM的异丙基硫代半乳糖苷(IPTG)，在温度16℃下诱导20小时。离心，收集菌体，用20ml Tris-HCl buffer(100mM，pH 8.0)重悬菌体，超声破碎(X0-900D ultrasonic processor ultrasonic processor,30％intensity)10min，然后离心，收集上清，用镍离子亲和层析柱对MTHFR66蛋白进行纯化，用SDS-PAGE蛋白电泳检测纯化结果，条带大小和理论预测的条带大小(31.79kDa)一致(如图1所示)。
3、测定MTHFR66蛋白的酶活力
酶活反应体系(300μL)：含有1mM底物(麦草畏)、0.2mg MTHFR66、1mM四氢叶酸(THF)，缓冲体系为浓度100mM的Tris-HCl(pH 8.0)，温度30℃下在水浴锅中反应1小时，然后在沸水中放置1min，终止反应。反应液冷冻干燥后加入300μL甲醇溶解冻干物，高效液相色谱(HPLC)检测麦草畏中间代谢产物3,6-二氯水杨酸(DCSA)的生成量。一个酶活力单位定义为：在pH 8.0、温度30℃条件下1min内降解麦草畏生成1nmol产物DCSA所需要酶的量，以U表示。
上述实验结果表明：纯化后的MTHFR66蛋白能在1小时内产生0.15mM的DCSA，MTHFR66蛋白的比酶活为3.75U/mg(如图2所示)。
4、测定MTHFR66蛋白在微量四氢叶酸存在条件下的麦草畏脱甲基功能
酶活反应体系(300μL)：含有1mM底物(麦草畏)、0.2mg MTHFR66和分别含0.01mM、0.02mM、0.05mM和1mM四氢叶酸(THF)，缓冲体系为浓度100mM的Tris-HCl(pH 8.0)，温度30℃下在水浴锅中反应1小时，然后在沸水中放置1min，终止反应。反应液冷冻干燥后加入300μL甲醇溶解冻干物，高效液相色谱(HPLC)检测麦草畏中间代谢产物DCSA的生成量。
上述实验结果表明：纯化后的MTHFR66蛋白在0.01mM的四氢叶酸存在时，就能够将麦草畏脱甲基形成DCSA，在0.5mM的四氢叶酸存在时能够将1mM的麦草畏全部脱甲基产生DCSA(如图3所示)。
第二实施例、甲基四氢叶酸还原酶MTHFR66的水解酶功能鉴定及产物鉴定
1、MTHFR66蛋白的水解酶功能测定
酶活反应体系(300μL)：含有0.2mg MTHFR66、1mM 5-甲基四氢叶酸(5-CH3-H4F)，缓冲体系为浓度100mM的Tris-HCl(pH 8.0)，温度30℃下在水浴锅中反应1小时，然后在沸水中放置1min，终止反应。反应液冷冻干燥，干燥物加入300μL的0.1mol/L的 KH2PO4(pH 6.8，1％抗坏血酸、0.1％β-巯基乙醇)溶解，用滤膜(孔径0.22μm)过滤，采用高效液相色谱进行检测。液相色谱条件为：流动相为0.05mol/L的KH2PO4(pH 3.0):乙腈(90:10，V/V)，Zorbax C218ODS Spherex反相柱(5μm，4.6mm×250mm，Agilent，USA)，柱温为23℃，紫外检测器，测定波长为298nm，进样量为20μL，流速为1.0mL/min。外标法按峰面积定量。
上述HPLC结果表明：纯化后的MTHFR66蛋白能够在没有电子受体NAD+存在的条件下，将5-甲基四氢叶酸转化成其他的物质(如图4所示)。
2、产物鉴定
代谢产物通过HPLC-MS(高效液相色谱和质谱联用)进行鉴定，条件为：流动相为0.05mol/L的KH2PO4(pH 3.0):乙腈(90:10，V/V)，Zorbax XDB-C18，5cm×0.46cm，1.8mm反相柱(5μm，4.6mm×250mm，Agilent，USA)，流速为0.25mL/min。MS分析使用ESI模式，检测器为Agilent G6410B Triple Quad Mass Spectrometer。
结果表明：纯化后的MTHFR66蛋白能够在没有电子受体NAD+的条件下将5-甲基四氢叶酸水解产生四氢叶酸(如图5所示)。
第三实施例、基因序列的优化和合成
1、植物优化序列的获得
保持所述甲基四氢叶酸还原酶MTHFR66的氨基酸序列(289个氨基酸，如序列表中SEQ ID NO:2所示)不改变，对编码相应于所述甲基四氢叶酸还原酶MTHFR66的氨基酸序列的MTHFR66核苷酸序列(870个核苷酸)进行密码子优化改造。
密码子优化改造的策略主要包括：依据单子叶植物玉米和双子叶植物大豆的偏好密码子、不稳定序列的改造、G+C含量的提高等。天然基因的G+C含量较低，A+T含量很高，一方面，如果直接将天然基因序列导入植物基因组中可能会被误认为是植物基因调控序列，同时在这些天然基因中会出现A+T富含区域，类似于基因启动子中的TATA盒，这些区域则会导致基因的异常转录；另一方面，在转录的mRNA中聚腺苷酸化信号序列(AAUAAA)、与mRNA剪接相关的小RNA互补序列会导致RNA不稳定。因此，改造后的基因序列除了有较高的G+C含量外，还改变DNA和转录成mRNA中出现的不稳定结构，从而保证蛋白的正常翻译；再一方面，应用玉米和大豆的偏好密码子改造天然基因序列，排除酶切位点和一些序列的修饰。
基于以上优化策略，得到优化MTHFR66核苷酸序列，优化MTHFR66核苷酸序列共含有870个核苷酸，编码289个氨基酸，其核苷酸序列如序列表中SEQ ID NO:1所示。
2、合成优化MTHFR66核苷酸序列
优化MTHFR66核苷酸序列由南京金斯瑞生物科技有限公司合成；合成的所述优化MTHFR66核苷酸序列(SEQ ID NO:1)的5’端还连接有SacI酶切位点，所述优化MTHFR66核苷酸序列(SEQ ID NO:1)的3’端还连接有KasI酶切位点。
第四实施例、拟南芥重组表达载体的构建及重组表达载体转化农杆菌
1、构建含有优化MTHFR66核苷酸序列的拟南芥重组克隆载体
将合成的优化MTHFR66核苷酸序列连入克隆载体pGEM-T(Promega，Madison，USA，CAT：A3600)上，操作步骤按Promega公司产品pGEM-T载体说明书进行，得到重组克隆载体DBN01-T，其构建流程如图6所示(其中，Amp表示氨苄青霉素抗性基因；f1表示噬菌体f1的复制起点；LacZ为LacZ起始密码子；SP6为SP6RNA聚合酶启动子；T7为T7RNA聚合酶启动子；mMTHFR66为优化MTHFR66核苷酸序列(SEQ ID NO:1)；MCS为多克隆位点)。
然后将重组克隆载体DBN01-T用热激方法转化大肠杆菌T1感受态细胞(Transgen，Beijing，China，CAT：CD501)，其热激条件为：50μL大肠杆菌T1感受态细胞、10μL质粒DNA(重组克隆载体DBN01-T)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)，在表面涂有IPTG(异丙基硫代-β-D-半乳糖苷)和X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)的氨苄青霉素(100mg/L)的LB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)上生长过夜。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，氨苄青霉素100mg/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。
提取的质粒经SacI和KasI酶切鉴定后，对阳性克隆进行测序验证，结果表明重组克隆载体DBN01-T中插入的所述优化MTHFR66核苷酸序列为序列表中SEQ ID NO:1所示的核苷酸序列，即优化MTHFR66核苷酸序列正确插入。
2、构建含有优化MTHFR66核苷酸序列的拟南芥重组表达载体DBN111101
用限制性内切酶SacI和KasI分别酶切重组克隆载体DBN01-T和表达载体DBNBC-01(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的优化MTHFR66核苷酸序列片段插到表达载体DBNBC-01的SacI和KasI位点之间，利用常规的酶切方法构建载体是本领域技术人员所熟知的，构建成重组表达载体DBN111101，其构建流程如图7所示(Kan：卡那霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基 因启动子(SEQ ID NO:6)；AtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:7)；mMTHFR66：优化MTHFR66核苷酸序列(SEQ ID NO:1)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:8)；prCaMV35S：花椰菜花叶病毒35S启动子(SEQ ID NO:9)；PAT：草丁膦乙酰转移酶基因(SEQ ID NO:10)；tCaMV35S：花椰菜花叶病毒35S终止子(SEQ ID NO:11)；LB：左边界)。
将重组表达载体DBN111101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DBN111101)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)；然后在含50mg/L卡那霉素(Kanamycin)的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12小时，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，卡那霉素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。将提取的质粒用限制性内切酶SacI和KasI酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN111101在SacI和KasI位点间的核苷酸序列为序列表中SEQ ID NO:1所示核苷酸序列，即优化MTHFR66核苷酸序列。
3、构建含有天然MTHFR66核苷酸序列的拟南芥重组表达载体DBN111101N
按照本实施例中1所述的构建含有优化MTHFR66核苷酸序列的重组克隆载体DBN01-T的方法，利用天然MTHFR66核苷酸序列(SEQ ID NO:3)构建含有天然MTHFR66核苷酸序列的重组克隆载体DBN01R-T。对阳性克隆进行测序验证，结果表明重组克隆载体DBN01R-T中插入的天然MTHFR66核苷酸序列为序列表中SEQ ID NO:3所示的核苷酸序列，即天然MTHFR66核苷酸序列正确插入。
按照本实施例中2所述的构建含有优化MTHFR66核苷酸序列的重组表达载体DBN111101的方法，利用天然MTHFR66核苷酸序列构建含有天然MTHFR66核苷酸序列的重组表达载体DBN111101N，其载体结构如图8所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Kan：卡那霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQ ID NO:6)；AtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:7)；MTHFR66：天然MTHFR66核苷酸序列(SEQ ID NO:3)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:8)；prCaMV35S：花椰菜花叶病毒35S启动子(SEQ ID NO:9)；PAT：草丁膦乙酰转移酶基因(SEQ ID NO:10)；tCaMV35S：花椰菜花叶病毒35S终止子(SEQ ID NO:11)；LB：左边界)。对阳性克隆进行测序验证，结果表明重组表达载体DBN111101N中插入的天然MTHFR66核苷酸序列为序列表中SEQ ID NO:3所示的核苷酸序列，即天然MTHFR66核苷酸序列正确插入。
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DBN111101和DBN111101N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)；置于液氮中10分钟，37℃温水浴10分钟；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2小时，涂于含50mg/L的利福平(Rifampicin)和50mg/L的卡那霉素的LB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，用限制性内切酶酶切DBN111101和DBN111101N后进行酶切验证，结果表明重组表达载体DBN111101和DBN111101N结构完全正确。
第五实施例、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％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24小时。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/m2秒的长日照条件(16小时光照/8小时黑暗)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含卡那霉素(100mg/L)和利福平(10mg/L)的YEP培养液的预培养物。以220rpm将培养物在28℃恒速摇动孵育过夜。每个预培养物用于接种两份500ml含卡那霉素(100mg/L)和利福平(10mg/L)的YEP培养液的培养物并将培养物在28℃持续摇动孵育过夜。室温以约8700×g离心10分钟沉淀细胞，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月龄的植物在培养基中浸泡15秒，确保浸没最新的花序。接着将植物侧面放倒并覆盖(透明或不透明)24小时，接着用水洗涤并竖直放置。在22℃以16小时光照/8小时黑暗的光周期培养植物。浸泡约4周后收获种子。
将新收获的(优化MTHFR66核苷酸序列和天然MTHFR66核苷酸序列)T1种子在室温干燥7天。将种子种在26.5×51cm萌发盘中，每盘接受200mg T1种子(约10000个种子)，所述种子事先已悬浮于40mL 0.1％琼脂糖溶液并在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，利用重力排水。用移液管将预处理后的种子(每个40mL)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T1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 x 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22℃培养室中或直接移入温室。接着移去罩并在测试MTHFR66基因提供麦草畏除草剂耐受性的能力之前至少1天将植物栽种到温室(22±5℃，50±30％RH，14小时光照：10小时黑暗，最小500μE/m2s1天然+补充光)。
第六实施例、转基因拟南芥植株的除草剂耐受性效果检测
用MTHFR66基因进行首次拟南芥转化。首先使用草铵膦选择方案从未转化种子背景中选择T1转化体。转化重组表达载体DBN111101的为定位于叶绿体的转入优化MTHFR66核苷酸序列的拟南芥植株(At mMTHFR66)，转化重组表达载体DBN111101N的为定位于叶绿体的转入天然MTHFR66核苷酸序列的拟南芥植株(At MTHFR66)。筛选了约20000个At mMTHFR66的T1种子，并鉴定了197株T1代阳性转化子(PAT基因)，约1.0％的转化效率；筛选了约20000个At MTHFR66的T1种子，并鉴定了182株T1代阳性转化子(PAT基因)，约0.91％的转化效率。将转入优化MTHFR66核苷酸序列的拟南芥T1植株(At-mMTHFR66)、转入天然MTHFR66核苷酸序列的拟南芥T1植株(At-MTHFR66)和野生型拟南芥植株(播种后18天)对麦草畏进行除草剂耐受性效果检测。
分别将转入优化MTHFR66核苷酸序列的拟南芥T1植株、转入天然MTHFR66核苷酸序列的拟南芥T1植株和野生型拟南芥植株用麦草畏(560g ae/ha，1倍大田浓度)和空白溶剂(水)喷洒。喷施7天和14天后统计植株耐受性情况：7天后生长状况和空白溶剂(水)一致的划为高抗植株，7天后有莲座叶卷曲的划为中抗植株，14天后仍不能抽苔的划为低抗植株，14天后死亡的划为不抗植株。由于每株拟南芥T1植株是独立的转化事件，可以预计在给定剂量内个体T1应答的显著差异。结果如表1和图9所示。
表1、转基因拟南芥T1植株除草剂耐受性实验结果
Figure PCTCN2017079661-appb-000005
对于拟南芥，560g ae/ha麦草畏是将敏感植物与具有平均耐受性水平的植物区分开来的有效剂量。表1和图9的结果表明：优化MTHFR66基因赋予个体拟南芥植物麦草畏除 草剂耐受性(只有部分植株具有耐受性的原因是由于T1代植物插入位点是随机的，因而耐受性基因的表达水平有差异，表现出耐受性水平的差异)；相比于At-MTHFR66的T1植株，At-mMTHFR66的T1代拟南芥后代部分能够产生更高的麦草畏除草剂耐受性，表明所述MTHFR66基因经植物密码子优化可以增强拟南芥植物对麦草畏除草剂的耐受性；而野生型拟南芥则不具有麦草畏除草剂耐受性。
第七实施例、玉米重组表达载体的构建及重组表达载体转化农杆菌
1、构建含有优化MTHFR66核苷酸序列的玉米重组表达载体DBN-HT130066
用限制性内切酶SacI和KasI分别酶切重组克隆载体DBN01-T和表达载体DBNBC-02(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的优化MTHFR66核苷酸序列片段插到表达载体DBNBC-02的SacI和KasI位点之间，利用常规的酶切方法构建载体是本领域技术人员所熟知的，构建成重组表达载体DBN-HT130066，其构建流程如图10所示(Kan：卡那霉素基因；RB：右边界；prUbi：玉米Ubiquitin(泛素)1基因启动子(SEQ ID NO:12)；AtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:7)；mMTHFR66：优化MTHFR66核苷酸序列(SEQ ID NO:1)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:8)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO:13)；LB：左边界)。
将重组表达载体DBN-HT130066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DBN-HT130066)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)；然后在含50mg/L卡那霉素的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12小时，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，卡那霉素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。将提取的质粒用限制性内切酶SacI和KasI酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN-HT130066在SacI和KasI位点间的核苷酸序列为序列表中SEQ ID NO:1所示核苷酸序列，即优化MTHFR66核苷酸序列。
2、构建含有天然MTHFR66核苷酸序列的玉米重组表达载体DBN-HT130066N
按照本实施例中1所述的构建含有优化MTHFR66核苷酸序列的重组表达载体DBN-HT120066的方法，利用本发明第四实施例中3所述的含有天然MTHFR66核苷酸序列的重组克隆载体DBN01R-T，构建含有天然MTHFR66核苷酸序列的重组表达载体DBN-HT130066N，其载体结构如图11所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Kan：卡那霉素基因；RB：右边界；prUbi：玉米Ubiquitin(泛素)1基因启动子(SEQ ID NO:12)；AtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:7)；MTHFR66：天然MTHFR66核苷酸序列(SEQ ID NO:3)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:8)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO:13)；LB：左边界)。对阳性克隆 进行测序验证，结果表明重组表达载体DBN-HT130066N中插入的天然MTHFR66核苷酸序列为序列表中SEQ ID NO:3所示的核苷酸序列，即天然MTHFR66核苷酸序列正确插入。
3、玉米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DBN-HT130066和DBN-HT130066N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10分钟，37℃温水浴10分钟；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2小时，涂于含50mg/L的利福平(Rifampicin)和50mg/L的卡那霉素的LB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，用限制性内切酶酶切DBN-HT130066和DBN-HT130066N后进行酶切验证，结果表明重组表达载体DBN-HT130066和DBN-HT130066N结构完全正确。
第八实施例、转基因玉米植株的获得及验证
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)的幼胚与本发明第七实施例中3所述的农杆菌共培养，以将本发明第七实施例中1和2构建的重组表达载体DBN-HT130066和DBN-HT130066N中的T-DNA(包括玉米Ubiquitin1基因的启动子序列、优化MTHFR66核苷酸序列、天然MTHFR66核苷酸序列、AtCTP2叶绿体转运肽序列、PMI基因和Nos终止子序列)转入到玉米染色体组中，获得了定位于叶绿体的转入优化MTHFR66核苷酸序列的玉米植株(Zm-mMTHFR66)和转入天然MTHFR66核苷酸序列的玉米植株(Zm-MTHFR66)；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，其中农杆菌能够将优化MTHFR66核苷酸序列和天然MTHFR66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(OD660＝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。
2、用TaqMan验证转基因玉米植株
分别取转入优化MTHFR66核苷酸序列的玉米植株和转入天然MTHFR66核苷酸序列的玉米植株的叶片约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PMI基因的拷贝数来确定转基因玉米植株的拷贝数。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。
检测PMI基因拷贝数的具体方法如下：
步骤11、分别取转入优化MTHFR66核苷酸序列的玉米植株、转入天然MTHFR66核苷酸序列的玉米植株和野生型玉米植株的叶片各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，以野生型玉米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PMI基因：
引物3：CCGGGTGAATCAGCGTTT如序列表中SEQ ID NO:14所示；
引物4：GCCGTGGCCTTTGACAGT如序列表中SEQ ID NO:15所示；
探针1：TGCCGCCAACGAATCACCGG如序列表中SEQ ID NO:16所示；
PCR反应体系为：
Figure PCTCN2017079661-appb-000006
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 PCTCN2017079661-appb-000007
利用SDS2.3软件(Applied Biosystems)分析数据。
实验结果表明，优化MTHFR66核苷酸序列和天然MTHFR66核苷酸序列均己整合到所检测的玉米植株的染色体组中，而且转入优化MTHFR66核苷酸序列的玉米植株和转入天然MTHFR66核苷酸序列的玉米植株均获得了含有单拷贝MTHFR66基因的转基因玉米植株。
第九实施例、转基因玉米植株的除草剂抗性效果检测
将转入优化MTHFR66核苷酸序列的玉米植株、转入天然MTHFR66核苷酸序列的玉米植株和野生型玉米植株(V5-V6时期)分别对麦草畏进行除草剂抗性效果检测。
分别取转入优化MTHFR66核苷酸序列的玉米植株、转入天然MTHFR66核苷酸序列的玉米植株和野生型玉米植株的T1代杂合植株(V5-V6时期)，并用麦草畏除草剂(4480g ae/ha，8倍大田浓度)和空白溶剂(水)喷洒。喷施21天后统计支撑根发育情况。Zm-mMTHFR66共3个株系(S1、S2和S3)，Zm-MTHFR66共2个株系(S4和S5)，野生型的(CK)共1个株系；从每个株系选10-15株进行测试。结果如表2所示。
表2、转基因玉米T1植株除草剂抗性实验结果
Figure PCTCN2017079661-appb-000008
Figure PCTCN2017079661-appb-000009
表2的结果表明：优化MTHFR66基因赋予转基因玉米植物麦草畏除草剂的高水平耐受性(由于单子叶植物本身对麦草畏除草剂具有一定抗性，因而表现出高水平抗性)；相比于Zm-MTHFR66，Zm-mMTHFR66能够产生更高的麦草畏除草剂耐受性，表明所述MTHFR66基因经植物密码子优化可以增强玉米植物对麦草畏除草剂的耐受性；而野生型玉米植株则不具有麦草畏除草剂耐受性。
综上所述，本发明MTHFR66蛋白可以降解麦草畏除草剂，优化MTHFR66基因采用玉米和大豆的偏好密码子，使其特别适合在植物中表达；优化MTHFR66基因可以赋予转基因植物更好的麦草畏除草剂耐受性；同时本发明除草剂耐受性蛋白质MTHFR66还具有甲基四氢叶酸还原酶活性，其不同于已知的麦草畏耐受型基因，因此可以扩大麦草畏耐受型在植物上应用范围。
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 (18)

  1. 一种基因，其特征在于，包括：
    (a)编码SEQ ID NO:2所示的氨基酸序列的核苷酸序列；或
    (b)在严格条件下与(a)限定的核苷酸序列互补的核苷酸序列；或
    (c)SEQ ID NO:1所示的核苷酸序列。
  2. 一种表达盒，其特征在于，包含在有效连接的调控序列调控下的权利要求1所述基因。
  3. 根据权利要求2所述表达盒，其特征在于，所述调控序列为叶绿体转运肽，所述叶绿体转运肽与权利要求1所述基因有效连接。
  4. 根据权利要求3所述表达盒，其特征在于，所述叶绿体转运肽的核苷酸序列具有SEQ ID NO:7所示的核苷酸序列。
  5. 一种包含权利要求1所述基因或权利要求2-4任一项所述表达盒的重组载体。
  6. 一种增加耐受除草剂范围的方法，其特征在于，包括：将SEQ ID NO:2所示的氨基酸序列组成的蛋白质或权利要求2-4任一项所述表达盒编码的蛋白质在植物中与至少一种不同于SEQ ID NO:2所示的氨基酸序列组成的蛋白质或权利要求2-4任一项所述表达盒编码的蛋白质的第二种蛋白质一起表达。
  7. 根据权利要求6所述增加除草剂耐受性的方法，其特征在于，所述第二种蛋白质为草甘膦耐受性蛋白质、草铵膦耐受性蛋白质、α酮戊二酸双加氧酶、4-羟苯基丙酮酸双加氧酶、乙酰乳酸合酶、细胞色素类蛋白质或原卟啉原氧化酶。
  8. 一种选择转化的植物细胞的方法，其特征在于，包括：用权利要求1所述基因或权利要求2-4任一项所述表达盒转化多个植物细胞，并在允许表达所述基因或所述表达盒的转化细胞生长，而杀死未转化细胞或抑制未转化细胞生长的除草剂浓度下培养所述细胞，所述除草剂为麦草畏。
  9. 一种控制杂草的方法，其特征在于，包括：对种植植物的大田施用有效剂量的麦草畏除草剂，所述植物包含权利要求1所述基因、权利要求2-4任一项所述表达盒或权利要求5所述重组载体。
  10. 一种保护植物免受由除草剂引起的损伤的方法，其特征在于，包括：将权利要求1所述基因、权利要求2-4任一项所述表达盒或权利要求5所述重组载体导入植物，使导入后的植物产生足够保护其免受麦草畏损害量的除草剂耐受性蛋白质。
  11. 一种赋予植物麦草畏除草剂耐受性的方法，其特征在于，包括：将权利要求1所述基因、权利要求2-4任一项所述表达盒或权利要求5所述重组载体导入植物。
  12. 一种控制草甘膦耐受性植物的大田中草甘膦耐受性杂草的方法，其特征在于，包括：对种植草甘膦耐受性植物的大田施用有效剂量的麦草畏，所述草甘膦耐受性植物包含权利要求1所述基因、权利要求2-4任一项所述表达盒或权利要求5所述重组载体。
  13. 一种产生麦草畏耐受性植物的方法，其特征在于，包括向所述植物的基因组中引入权利要求1所述基因或权利要求2-4任一项所述表达盒，以产生麦草畏耐受性植物。
  14. 根据权利要求13所述产生麦草畏耐受性植物的方法，其特征在于，包括：通过将亲本植物自交或与第二种植物杂交而产生麦草畏耐受性植物，所述亲本植物和/或第二种植物包含权利要求1所述基因或权利要求2-4任一项所述表达盒，所述麦草畏耐受性植物遗传了来自所述亲本植物和/或第二种植物的所述基因或所述表达盒。
  15. 一种培养对麦草畏除草剂具有耐受性的植物的方法，其特征在于，包括：
    种植至少一粒植物种子，所述植物种子的基因组中包括权利要求1所述基因或权利要求2-4任一项所述表达盒；
    使所述植物种子长成植株；
    用有效剂量麦草畏除草剂喷洒所述植株，收获与其他不具有所述基因或所述表达盒的植株相比具有减弱的植物损伤的植株。
  16. 根据权利要求6-15任一项所述方法，其特征在于，所述植物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
  17. 一种甲基四氢叶酸还原酶耐受麦草畏除草剂的用途，其特征在于，所述甲基四氢叶酸还原酶具有SEQ ID NO:2所示的氨基酸序列。
  18. 一种产生甲基四氢叶酸还原酶的植物耐受麦草畏除草剂的用途，其特征在于，所述甲基四氢叶酸还原酶具有SEQ ID NO:2所示的氨基酸序列。
PCT/CN2017/079661 2016-06-18 2017-04-07 除草剂抗性蛋白质、其编码基因及用途 WO2017215329A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/308,852 US11365425B2 (en) 2016-06-18 2017-04-07 Resistant protein for use in herbicide, encoding gene and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610440763.7A CN105925590B (zh) 2016-06-18 2016-06-18 除草剂抗性蛋白质、其编码基因及用途
CN201610440763.7 2016-06-18

Publications (1)

Publication Number Publication Date
WO2017215329A1 true WO2017215329A1 (zh) 2017-12-21

Family

ID=56830282

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/079661 WO2017215329A1 (zh) 2016-06-18 2017-04-07 除草剂抗性蛋白质、其编码基因及用途

Country Status (4)

Country Link
US (1) US11365425B2 (zh)
CN (1) CN105925590B (zh)
AR (1) AR108656A1 (zh)
WO (1) WO2017215329A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105925590B (zh) * 2016-06-18 2019-05-17 北京大北农生物技术有限公司 除草剂抗性蛋白质、其编码基因及用途
CN108330116B (zh) * 2018-02-07 2020-05-05 北京大北农生物技术有限公司 除草剂耐受性蛋白质、其编码基因及用途
CN111139238B (zh) * 2019-10-14 2021-08-03 中国农业科学院生物技术研究所 除草剂麦草畏的降解基因dicX3及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008095972A1 (en) * 2007-02-09 2008-08-14 Basf Plant Science Gmbh Compositions and methods using rna interference targeting mthfr - like genes for control of nematodes
CN101501196A (zh) * 2006-06-06 2009-08-05 孟山都技术有限公司 修饰的麦草畏单加氧酶及其使用方法
CN102242133A (zh) * 2010-05-14 2011-11-16 昆山科腾生物科技有限公司 麦草畏耐受型的单加氧酶基因及其应用
CN102776217A (zh) * 2012-07-16 2012-11-14 中国药科大学 一种提高l-5-甲基四氢叶酸累积量的生物合成方法
CN105925590A (zh) * 2016-06-18 2016-09-07 北京大北农生物技术有限公司 除草剂抗性蛋白质、其编码基因及用途

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7022896B1 (en) * 1997-04-04 2006-04-04 Board Of Regents Of University Of Nebraska Methods and materials for making and using transgenic dicamba-degrading organisms
US7884262B2 (en) 2006-06-06 2011-02-08 Monsanto Technology Llc Modified DMO enzyme and methods of its use
US20100162428A1 (en) * 2007-05-31 2010-06-24 Basf Plant Science Gmbh Method Of Excising A Nucleic Acid Sequence From A Plant Genome
BRPI0817560B1 (pt) * 2007-09-27 2022-09-20 Dow Agrosciences Llc Proteínas de dedo de zinco (zfp) engenheiradas que têm como alvo genes de 5-enolpiruvil shiquimato-3-fosfato sintase, nuclease de dedo de zinco (zfn), vetor, bem como métodos para introdução de um transgene no genoma de uma célula de planta, para expressar o produto de um transgene em uma célula de planta, para deleção de uma sequência de gene de epsps a partir de um locus de gene epsps no genoma de uma célula de planta, e para modular expressão de um gene de epsps
CN104611304B (zh) * 2014-12-22 2017-05-17 北京大北农生物技术有限公司 除草剂耐受性蛋白质、其编码基因及用途
CN104630162B (zh) * 2014-12-22 2017-07-25 北京大北农生物技术有限公司 除草剂耐受性蛋白质、其编码基因及用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101501196A (zh) * 2006-06-06 2009-08-05 孟山都技术有限公司 修饰的麦草畏单加氧酶及其使用方法
WO2008095972A1 (en) * 2007-02-09 2008-08-14 Basf Plant Science Gmbh Compositions and methods using rna interference targeting mthfr - like genes for control of nematodes
CN102242133A (zh) * 2010-05-14 2011-11-16 昆山科腾生物科技有限公司 麦草畏耐受型的单加氧酶基因及其应用
CN102776217A (zh) * 2012-07-16 2012-11-14 中国药科大学 一种提高l-5-甲基四氢叶酸累积量的生物合成方法
CN105925590A (zh) * 2016-06-18 2016-09-07 北京大北农生物技术有限公司 除草剂抗性蛋白质、其编码基因及用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHRISTAL, A.S. ET AL.: "Purification and Properties of NADH-Dependent 5, 10-Methylenetetrahydrofolate Reductase (MetF) from Escherichia coli", JOURNAL OF BACTERIOLOGY, vol. 181, no. 3, 28 February 1999 (1999-02-28), pages 718 - 725, XP055447884, ISSN: 0021-9193 *
HONG, LI ET AL.: "Methylene Tetrahydrofolate Reductase Regulates the Growth, Development and Pathogenicity of the Rice Blast Fungus Magnaporthe Oryzae", ACTA PHYTOPATHOLOGICA SINICA, vol. 45, no. 3, 30 June 2015 (2015-06-30), pages 270 - 279, ISSN: 0412-0914 *

Also Published As

Publication number Publication date
CN105925590B (zh) 2019-05-17
US11365425B2 (en) 2022-06-21
US20190249187A1 (en) 2019-08-15
CN105925590A (zh) 2016-09-07
AR108656A1 (es) 2018-09-12

Similar Documents

Publication Publication Date Title
EP3425046B1 (en) Herbicide tolerant protein, encoding gene and use thereof
US9464117B2 (en) Herbicide-resistant proteins, encoding genes, and uses thereof
US10633669B2 (en) Herbicide-resistant protein, encoding gene and use thereof
US9462805B2 (en) Herbicide-resistant proteins, encoding genes, and uses thereof
WO2021051265A1 (zh) 突变的羟基苯丙酮酸双加氧酶多肽、其编码基因及用途
WO2017161914A1 (zh) 除草剂耐受性蛋白质的用途
WO2017161913A1 (zh) 除草剂耐受性蛋白质的用途
WO2017161915A1 (zh) 除草剂耐受性蛋白质的用途
CA2975773C (en) Herbicide-resistant protein, encoding gene and use thereof
CA2976060C (en) Herbicide-resistant protein, encoding gene and use thereof
WO2017215329A1 (zh) 除草剂抗性蛋白质、其编码基因及用途
WO2022237541A1 (zh) 原卟啉原氧化酶的用途
WO2023108495A1 (zh) 突变的羟基苯丙酮酸双加氧酶多肽、其编码基因及用途
BR102013033387A2 (pt) Proteína resistente a herbicida, gene codificante e uso dos mesmos

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17812441

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17812441

Country of ref document: EP

Kind code of ref document: A1