WO2006132270A1 - Gène résistant à un herbicide - Google Patents

Gène résistant à un herbicide Download PDF

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Publication number
WO2006132270A1
WO2006132270A1 PCT/JP2006/311415 JP2006311415W WO2006132270A1 WO 2006132270 A1 WO2006132270 A1 WO 2006132270A1 JP 2006311415 W JP2006311415 W JP 2006311415W WO 2006132270 A1 WO2006132270 A1 WO 2006132270A1
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Prior art keywords
hppd
gene
herbicide
resistant
seq
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PCT/JP2006/311415
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English (en)
Japanese (ja)
Inventor
Fumihiko Sato
Hiromichi Minami
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Kyoto University
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Publication of WO2006132270A1 publication Critical patent/WO2006132270A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • the present invention relates to an herbicide-resistant enzyme, particularly an enzyme resistant to a herbicide targeting p-hydroxypyruvate dioxygenase.
  • HPPD p-Hydroxypyruvate dioxygenase
  • HPP p-hydroxyphenyl viruvate
  • molecular oxygen p-hydroxyphenyl viruvate
  • this enzyme is involved in the biosynthesis of blast quinone, ⁇ -tocopherol (vitamin E), etc. (Fig. 1).
  • this enzyme has recently been found to be a molecular target for several herbicides.
  • HPPD gene The full-length or partial sequence of a gene encoding HPPD of a plant (hereinafter also referred to as HPPD gene) is carrot, Arabidopsis thaliana, barley, corn, rice, , Ryegrass, oshinokedasa, akinoenokorogusa, ohishiba, sorghum (Non-Patent Document 1)
  • Herbicides that inhibit HPPD inhibit plastoquinone biosynthesis in plants, resulting in decreased carotenoids, chloroplast development arrest and whitening.
  • a decrease in plastoquinone content in plants is thought to affect the photosynthetic electron transport system and cause plant growth suppression symptoms.
  • Tocopherol another product of the plastoquinone biosynthetic system, acts as a free radical scavenger.
  • a decrease in tocopherol content in plants causes lipid peracids, which lead to necrotic symptoms.
  • a decrease in tocopherol content is thought to cause a decrease in the photosynthetic ability of plants!
  • HPPD inhibitors are thought to kill weeds by such various actions. Therefore, HPPD is extremely effective as a molecular target for powerful herbicides.
  • Herbicides that inhibit HPPD are triketone (cyclohexanedi) according to chemical structure. On), isoxazole, pyrazole, and bicyclooctanedione (BOD) systems.
  • pyrazole pyrazolate is known to have high herbicidal activity. Pyrazolate is hydrolyzed to form active detosyl virazolate (hereinafter referred to as DTP) as its herbicide (Fig. 2).
  • DTP active detosyl virazolate
  • Fig. 2 Bicyclooctanedione
  • BOD bicyclooctanedione
  • Patent Documents 1 and 2 Enzymes resistant to herbicides targeting HPPD have been reported (Patent Documents 1 and 2).
  • the degree of herbicide resistance of the HPPD disclosed in Patent Document 1 is 2 to 4.5 times as low as that of conventionally known HP PD.
  • the HPPD disclosed in Patent Document 2 is also considered to be only several times more resistant than the conventionally known HPPD, and the HPPD is a mutant enzyme in which a mutation is introduced into wild-type HPPD.
  • a method known in the art for creating an HPPD resistant plant is to increase the amount of enzyme and impart resistance by overexpressing HPPD in the plant body.
  • Met A method for producing a herbicide-resistant plant by using a wild-type enzyme derived from a plant whose enzyme itself has high herbicide resistance has been successful so far.
  • HPPD gene is useful as a tocopherol and plastoquinone biosynthetic gene, it is not only useful for the production of herbicide-resistant plants, but is also useful for Tocophere mouths such as plastoquinone. It is also useful as a gene for producing useful compounds.
  • Patent Document 1 Special Table 2004— No. 528821
  • Patent Document 2 Special Table 2001-522608
  • Non-patent document 1 Plant growth regulation, vol.27, No.2, pl46-155, 2002
  • An object of the present invention is to provide HPPD having extremely high herbicide resistance.
  • the present inventors have found that olene cultured cells are resistant to DTP (detosyl virazolate), which is the active body of pyrazolate, and olene force also has the ability to express HPPD gene. Released. The present inventors further expressed the HPPD gene in Escherichia coli and measured the activity thereof. As a result, it was revealed that the HPPD gene exhibits a resistance about 500 times higher than that of conventionally known HPPD. The HPPD also showed high resistance to a hydrolyzate of benzobicyclone.
  • DTP detosyl virazolate
  • the present invention relates to
  • a gene encoding a herbicide-resistant p-hydroxyphenylbiruvate dioxygenase enzyme which comprises a polynucleotide having the first to 1293th nucleotides of the nucleotide sequence represented by SEQ ID NO: 1;
  • a p-hydroxyphenylpyruvate dioxygenase enzyme that is hybridized under stringent conditions with a polynucleotide having the first to 1293th nucleotides of the nucleotide sequence represented by SEQ ID NO: 1 and resistant to herbicides A gene comprising a polynucleotide encoding.
  • the open reading frame (ORF) of the herbicide-resistant HPPD according to the present invention has a base number of 1 to 1290. Accordingly, the present invention provides a gene consisting of nucleotides 1-1290 of SEQ ID NO: 1.
  • the coding region containing 3 bases of the ORF and stop codon consists of nucleotides 1 to 1293. As long as the present invention includes a region corresponding to nucleotide numbers 1 to 1290 of SEQ ID NO: 1, any gene is included in the range.
  • the present invention relates to a polynucleotide that is hybridized under stringent conditions with a polynucleotide having the 1st to 1293th nucleotides of the nucleotide sequence represented by SEQ ID NO: 1 and that encodes herbicide-resistant HPPD.
  • a gene containing is also provided.
  • stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5x SSC or equivalent stringency hybridization conditions.
  • the present invention also relates to a nucleotide sequence ability in which one or several bases or base pairs are deleted, substituted or added in the polynucleotide having the first to 1293th nucleotide strengths of the nucleotide sequence represented by SEQ ID NO: 1. And a gene comprising a polynucleotide encoding the herbicide-resistant HPPD is also provided.
  • “one or several bases or base pairs” means a number of bases that can be substituted, deleted, inserted, and Z or added by a known mutagenesis method such as site-directed mutagenesis. Or base pair.
  • the present invention also shows a homology of 70% or more at the nucleotide level with the polynucleotide having the first to 1293th nucleotides of the nucleotide sequence represented by SEQ ID NO: 1, and encodes herbicide-resistant HP PD. Also provided is a gene comprising the polynucleotide.
  • the nucleotide sequence homology is preferably 75% or more, more preferably 80% or more, 85% or more, 90% or more, and still more preferably 92% or more, 95% or more, 98% or more. Sequence identity can be determined by FASTA search (Pearson WR and DJ Lipman (1988) Proc. Natl. Acad. Sci. USA. 85: 2444-2448) or BLASTN search.
  • the present invention provides the following (c) or (d) p-hydroxyphenyl-birubinate dioxygenase enzyme:
  • P-hydroxyphenylpyruvate dioxygenase that has the ability to align amino acids in which one or several amino acids are deleted, replaced or added in the amino acid sequence represented by SEQ ID NO: 2 and is resistant to herbicides enzyme.
  • mutant protein includes a protein having a mutation artificially introduced by a known mutant protein production method or the like, and a protein having a mutation in comparison with a naturally-occurring similar isolated SEQ ID NO: 2. Including.
  • the present invention comprises a protein having the amino acid sequence ability represented by SEQ ID NO: 2, an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, And a protein that acts as a herbicide-resistant HPPD and a protein that exhibits 72% or more homology at the amino acid level with a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 and that acts as a herbicide-resistant HPPD. provide. Furthermore, the present invention provides a protein encoded by the HPPD gene of the present invention.
  • the term “one or several amino acids” has the same meaning as described above.
  • amino acid sequence homology is preferably 75% or more, more preferably 80% or more, 85% or more, 90% or more, and still more preferably 95% or more, 98% or more. Sequence identity is determined by FASTA search (Pea rson WR and DJ Lipman (1988) Proc. Natl. Acad. Sci. USA. 85: 2444- 2448) and BL
  • the herbicide-resistant HPPD of the present invention includes herbicides for herbicides targeting HPPD, such as pyrazole pyrazolate (DTP which is the active body) and bicyclooctanedione benzobicyclon. Useful for imparting resistance to plants.
  • herbicides for herbicides targeting HPPD such as pyrazole pyrazolate (DTP which is the active body) and bicyclooctanedione benzobicyclon.
  • the herbicide-resistant HPPD provided by the present invention is detosyl virazolate.
  • the herbicide resistant HPPD provided by the present invention is derived from allene.
  • the present invention also provides a recombinant vector containing the HPPD gene of the present invention, a transformed plant cell containing the recombinant vector, and a transformed plant containing the transformed plant cell.
  • Specific examples of the plant to be transformed include soybean, rice, rapeseed, potato, potato, tomato, tobacco, barley, wheat, corn and the like, preferably soybean and rice.
  • the present invention provides a method for producing an HPPD-inhibiting herbicide-resistant plant comprising the steps of transforming a plant cell with an HPPD gene, and regenerating the transformed plant cell to obtain a plant body. provide.
  • transformed plant includes all transformed plant bodies, plant organs, plant tissues and plant culture cells.
  • the present invention is also a method for controlling weeds in a field where a transformed plant is cultivated, and is effective for controlling target weeds without substantially changing the transformed product.
  • a method comprising applying to a field an amount of an HPPD-inhibiting herbicide.
  • the HPPD-inhibiting herbicide is pyrazolate and benzobicyclon.
  • HPPD gene of the present invention it is possible to confer resistance to a herbicide that inhibits HPPD. Since herbicides that inhibit HPPD are known as non-selective inhibitors, the HPPD gene of the present invention confers herbicide resistance to a wide range of crops.
  • the HPPD gene of the present invention may be useful for metabolic engineering of these useful compound biosynthesis systems.
  • the present invention is expected to reduce the weeding work.
  • the recombinant microorganism or recombinant plant containing the HPPD gene of the present invention can be useful in the production of useful vitamins such as tocopherol z plastoquinone.
  • FIG. 1 is a diagram showing HPPD-catalyzed reaction and inhibition of plastoquinone biosynthesis.
  • FIG. 2 is a diagram showing the structure of an HPPD inhibitor.
  • a Pyrazolate is hydrolyzed to form active detosyl virazolate (DTP).
  • DTP active detosyl virazolate
  • b Triketone type HPPD inhibitor.
  • FIG. 3 is a graph showing the effect of DTP on the growth of cultured tobacco cells. DTP showed significant growth inhibition in tobacco.
  • FIG. 4 is a graph showing the effect of DTP on the growth of ollen cultured cells. In the wings, DTP had no inhibitory effect.
  • FIG. 5 is a diagram showing a partial comparison of HPPD sequences isolated from allenes and HPPD sequences isolated from other species. In the figure, triangles indicate iron binding residues. The active site is indicated by a frame.
  • FIG. 6 is a diagram showing a systematic line of HPPD.
  • FIG. 7 is a diagram showing SDS-PAGE of allene HPPD expressed in E. coli.
  • Lane 1 molecular weight marker
  • lane 2 control vector (pET-21d)
  • lane 3 HPPP D 0 arrow indicates predicted production of recombinant HPPD.
  • FIG. 8 is a diagram showing LC-MS analysis of a reaction product by recombinant HPPD.
  • FIG. 9 is a graph showing the inhibitory effect of pyrazolate and DTP on HPPD derived from carrot cultured cells. Not an inhibitor! The activity is 4.9 nmol homogentisic acid Z-min Zmg protein. ⁇ indicates pyrazolate, and ⁇ indicates DTP. This figure shows that the IC of DTP for carrot HPPD is 13 nM (Hiroshi Matsumoto et al., 200
  • FIG. 10 is a diagram showing the effect of DTP on allen HPPD.
  • the enzyme activity in the absence of inhibitor was 3.8 nmol Z min Zmg protein. From the inhibition curve, it can be seen that our HPPD IC is 6.75 ⁇ (about 500 times that of carrot).
  • FIG. 11 is a graph showing the effect of benzobicyclone hydrolyzate on olene HPPD. From the inhibition curve, it can be seen that the IC of Aulen HPPD is 1.2 ⁇ .
  • herbicide-resistant HPPD is a herbicide that targets HPPD, for example, DTP's IC power against HPPD. Conventionally known! /, HPPD (Garcia,
  • HPPD that is 50 times or more, more preferably 100 times or more, and even more preferably 500 times or more.
  • IC is a herbicide that inhibits HPPD enzyme activity by 50%, for example,
  • the concentration of DTP It is the concentration of DTP. Therefore, even when a herbicide in a concentration substantially sufficient to kill plants such as weeds not containing HPPD of the present invention is applied, the plant having the HPP D of the present invention, preferably useful for agriculture. Crops do not die. Therefore, the herbicide-resistant HPPD of the present invention makes it possible to weed only weeds that do not kill useful crops.
  • the present invention provides a herbicide resistant HPPD gene.
  • the present inventors isolated the HP PD gene from cultured cells of Coptis japonica of the family Ranunculaceae and sequenced it (SEQ ID NO: 1).
  • the HPPD gene of the present invention is preferably a polynucleotide which is a polynucleotide comprising the bases 1 to 1293 of the base sequence shown in SEQ ID NO: 1, so long as the part corresponding to this part is included.
  • the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 can be obtained from a cDNA library prepared by a conventionally known method.
  • the present invention also includes a gene encoding a protein functionally equivalent to the protein acting as the herbicide-resistant HPPD of SEQ ID NO: 2.
  • herbicide resistant HPPD The protein functionally equivalent to the protein acting as ⁇ has the same biological function as the protein of SEQ ID NO: 2, i.e., has HPPD enzyme activity and has the herbicide resistance defined above. ⁇ ⁇ that it is a protein to show.
  • the gene of the present invention includes, for example, a mutation encoding a protein having an amino acid sequence ability in which one or several amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2. Bodies, derivatives, variants and homologs.
  • the number of bases mutated in comparison with the sequence of SEQ ID NO: 1 in the DNA constituting the HPPD gene of the present invention is within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids, at the amino acid level. More preferably, it causes mutation within 5 amino acids (for example, within 3 amino acids, within 2 amino acids).
  • the method for obtaining the HPPD gene of the present invention is not particularly limited, and a conventionally known method is employed.
  • the gene may be excised from a genomic DNA or cDNA library with an appropriate restriction enzyme and purified. That is, the herbicide-resistant HPPD gene of the present invention includes genomic DNA, cDNA, and chemically synthesized DNA. Methods for preparing genomic DNA and cDNA are well known to those skilled in the art.
  • the genomic DNA encoding the HPPD of the present invention for example, extracts genomic DNA from plant cells or tissue, and creates a genomic library (vectors include plasmids, phages, cosmids, BACs, PACs, etc.).
  • a probe prepared based on DNA encoding the protein of the present invention SEQ ID NO: 1
  • the library can be prepared by a method for screening (for example, colony hybridization or plaque hybridization).
  • the HPPD gene of the present invention can also be prepared by a method in which a primer specific to the sequence of SEQ ID NO: 1 is prepared and PCR is performed using this primer.
  • the cDNA encoding the HPPD of the present invention can be synthesized by, for example, synthesizing cDNA based on mRNA extracted from plant cells or tissue, and inserting it into a vector such as ⁇ ZAP to prepare a cDNA library. It can be prepared by a screening method (eg, colony hybridization or plaque hybridization), or by PCR.
  • the herbicide-resistant HPPD gene of the present invention can be used for mass production of useful vitamins such as tocopherol Z plastoquinone by enhancing the biosynthesis pathway of tocopherol Z plastoquinone.
  • the HPPD gene of the present invention is derived from ollen cultured cells. Therefore, it is possible to clone the full-length cDNA of the herbicide-resistant HPPD gene from plants other than olen using the HPPD gene derived from ollen.
  • a conventionally known method can be used and is not particularly limited.
  • whether or not the obtained gene codes for a protein having a function as a herbicide-resistant HPPD can be determined in a manner generally performed by those skilled in the art.
  • the protein power HPP and oxygen power encoded by the obtained gene have HPPD enzyme activity that catalyzes the formation of homogentisic acid can be confirmed by enzyme activity measurement methods well known to those skilled in the art. .
  • the HPPD enzyme activity is then measured in the presence of various concentrations of herbicide, such as DTP, and the concentration of herbicide (IC) that inhibits the enzyme activity by 50% is determined.
  • An evaluation of 50 can be performed as described in the examples.
  • the gene of the present invention can be isolated from many plant powers according to a conventional method.
  • the gene of the present invention can be obtained by chemical synthesis using a general method such as the phosphite triester method (H. Hunkapiller et al, Nature, vol. 310, p.10 5-111, 1984). You can also.
  • a base sequence having homology to the base sequence of the polynucleotide of the present invention is selected.
  • a base sequence homology search using an algorithm such as BLASTN can be suitably used.
  • an HPPD gene is obtained from a plant after the genome is made into a database
  • a conventional hybridization method using a conventionally known DNA library can also be used.
  • a genomic library or cDNA library is prepared using an appropriate cloning vector, and hybridization is performed using at least a part of the polynucleotide of SEQ ID NO: 1 as a probe. And a step of detecting a fragment that hybridizes to the probe.
  • the HPPD gene of the present invention is also useful as a probe.
  • the region used for the probe preferably contains a sequence specific to the HPPD gene.
  • the length of the polynucleotide used as the probe is preferably lOObp or more.
  • a hybridization reaction is preferably performed under stringent conditions.
  • stringent hybridization conditions are as described above, 6M urea, 0.4% SDS, 0.5x SSC conditions, or a stringency hybridization equivalent thereto. Refers to a condition.
  • Higher stringency conditions, such as 6M urea, 0.4% SDS, O.lx SSC Genes can be isolated efficiently.
  • the isolated gene is considered to have high homology at the amino acid level with the amino acid sequence of the protein of the present invention (SEQ ID NO: 2).
  • high homology refers to sequence identity of at least 72% or more, more preferably 75% or more, more preferably 80% or more (for example, 85% or more) at the amino acid level.
  • Sequence identity can be determined by FASTA search (Pearson WR and DJ Lipman (1988) Proc. Natl. Acad. Sci. USA. 85: 2444- 2448) or BLASTP search.
  • a method for producing a transformed plant that expresses the gene of the present invention comprises the steps of inserting the HPPD gene of the present invention into an appropriate vector, introducing it into a plant cell, and transforming plant cells obtained thereby. Including playing.
  • the transformed cell of the present invention is a cell into which the HPPD gene or recombinant vector of the present invention has been introduced.
  • the gene or recombinant vector has been introduced means that the gene or recombinant vector is introduced into the host in an expressible manner by the action of the gene or recombinant vector, eg, a promoter, by a known genetic manipulation technique. .
  • the transformed cell of the present invention can be obtained by directly introducing the HPPD gene of the present invention into a host, or by introducing a vector incorporating the gene into the host.
  • the host is not particularly limited, and examples include plants for producing herbicide-resistant plants, and microorganisms such as yeast and Escherichia coli for biosynthesis of useful vitamins. Although transformation may be transient, it is preferably one in which the HPPD gene is stably integrated.
  • the obtained gene When the obtained gene is introduced into a plant, it is used by being linked to a promoter capable of causing expression in the plant.
  • promoters include, for example, the 35S promoter of cauliflower mono-mosaic virus (CaMV35S promoter), the promoter of nopaline synthetase, the promoter of the small subunit of ribulose diphosphate canoleboxylase Z-oxygenase, and the like.
  • Various methods known to those skilled in the art may be used to produce the transformed cells of the present invention. Such methods include, for example, the polyethylene glycol method, electo-portion polarization. Method, method using agro-batterium, particle gun method and the like. Plant regeneration from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant cells (Toki et al., Plant Physiol. 100: 1503-1507 (1995)). reference). For example, as a method for producing a transformed plant, a gene is introduced into a protoplast using polyethylene glycol and the plant is regenerated (Datta, SK (1995) In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.
  • the present invention also includes a plant cell into which the gene of the present invention has been introduced, a plant containing the cell, progeny and clones of the plant, and propagation material derived therefrom.
  • the vector used for plant cell transformation is not particularly limited as long as the transgene can be expressed in the cell.
  • vectors having inducible promoters eg, cauliflower mosaic virus 35S promoter
  • inducible promoters eg, ethylene-responsive PR5d promoter, water stress-inducible rd29
  • drug resistance genes include ampicillin resistance gene, kanamycin resistance gene, and hygromycin resistance gene.
  • replication origins examples include replication origins derived from Ti or Ri plasmids.
  • DNA linked to a promoter can be directly introduced into plant cells using a microinjection method, an electopore position method, a polyethylene glycol method, a fusion method, or the like.
  • the DNA can also be incorporated into a plant vector for gene transfer into a plant and indirectly introduced into a plant cell via a virus or bacterium capable of plant infection.
  • cauliflower mosaic virus, diemi-virus, tobacco mosaic virus, brom mosaic virus, etc. can be used as powerful viruses, and as bacteria, Agrobacterium tumefaciens (hereinafter abbreviated as A.
  • Agrobacterium 'Rhizogenes' or the like can be used.
  • Examples of plasmids used for gene introduction into plants by the agrobacterium method using A. umefaciens include pBI101 and pBI121 (both from Clontech).
  • plant cells into which a vector is introduced include various forms of plant cells, such as suspension culture cells, protoplasts, leaf sections, and callus.
  • the method for producing an HPPD-inhibiting herbicide-resistant plant comprising the steps of transforming a plant cell with an HPPD gene, and regenerating the transformed plant to obtain a plant body according to the present invention. According to this, a plant having high herbicide resistance can be obtained.
  • ollen and other various plants for example, poppy family (no, nabisiso, poppy), gramineous plant (rice, corn), solanaceous plant (tomato, potato), legumes, etc. It is also possible to mass-produce tocopherol Z plastoquinone from (such as soybean).
  • Protein of the present invention gene encoding the same, and use thereof
  • the protein of the present invention includes a protein consisting of the amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence ability in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2. And a protein that acts as a herbicide-resistant HPPD enzyme, or a protein having amino acid sequence ability represented by SEQ ID NO: 2 and amino Includes proteins that show 72% or greater homology at the acid level and act as herbicide-resistant HPPD enzymes.
  • the protein of the present invention may be fused with a known tag such as HA or Flag for facilitating protein purification or detection at the end, or may be fused with a heterologous protein. Yes.
  • the protein of the present invention may be subjected to various modifications such as N-glycosylamine.
  • the protein according to the present invention may be obtained by recombinant production as described below, or may be isolated and purified from various sources including plant cells.
  • a general method for preparing recombinant HPPD is to insert the HPPD gene of the present invention into an appropriate expression vector, introduce the vector into an appropriate cell, culture the transformed cell, and express the expressed protein. Including purification.
  • the recombinant protein may be expressed as a fusion protein with another protein for the purpose of facilitating purification or the like.
  • Examples of fusion proteins include fusion proteins with maltose-binding proteins (vector pMAL series released by New England BioLabs, USA) and fusion proteins with glutathione-S-transferase (GST) (vectors released by Amersham Pharmacia Biotech).
  • GST glutathione-S-transferase
  • pGEX series fusion proteins with histidine tags (Novagen pET series).
  • host cells include Escherichia coli, yeast, animal cells, plant cells, and insect cells that are not particularly limited as long as they are suitable for the expression of recombinant proteins.
  • Various methods known to those skilled in the art can be used to introduce the vector into the host cell. For example, for introduction into E. coli, introduction methods using calcium ions (Mandel, M. & Higa, A. Journal of Molecular Biology, 53, 158-162 (1970), Hanahan, D. Journal of Molecular Biology 166, 557-580 (1983)).
  • the recombinant protein expressed in the host cell can be purified and recovered from the host cell or its culture supernatant by methods known to those skilled in the art. When the recombinant protein is expressed as a fusion protein with a maltose binding protein, it can be easily recovered by affinity purification.
  • an antibody that binds to the protein can be prepared.
  • a polyclonal antibody contains purified protein of the present invention or a part thereof. It can be prepared by immunizing animals such as herons and collecting blood after a certain period of time. Monoclonal antibodies can be obtained by fusing antibody-producing cells and bone tumor cells from animals immunized with the above protein or a part thereof to isolate single clone cells (hypridoma) that produce the desired antibodies. It can be prepared by obtaining an antibody from the cells. The antibody thus obtained can be used for purification and detection of the protein of the present invention.
  • the present invention includes an antibody that binds to the protein of the present invention.
  • Fig. 3 is a graph showing the growth of tobacco cultured cells in the presence of DTP
  • Fig. 4 is a graph showing the growth of cultured olene cells in the presence of DTP.
  • the present inventors isolated full length cDNA encoding the allen cell force HPPD.
  • the present inventors provide the nucleotide sequence of HPPD cDNA derived from allene.
  • the nucleotide sequence (1293 bp) encodes a 430 amino acid residue protein with a molecular weight of 47.3 kDa and has a specific C-terminal region similar to HPPD from other species.
  • HPPD p-hydroxyphenol biruvic acid
  • the herbicide resistance of the HPPD of the present invention which is dramatically higher than the value for PPD, was about 500 times higher than the HPPD derived from a conventionally known plant (carrot).
  • the IC value for the hydrolyzate of the herbicide benzobicyclon was 1.2 ⁇ .
  • Oulen cultured cells (156-SMT) and tobacco sputum cells were used.
  • Ouren cells are 10
  • the cells were cultured in LS (Linsmaier-Skoog) liquid medium containing ⁇ M naphthalene acetic acid and 0.01 ⁇ 6-benzyladen.
  • Tobacco cells were cultured in LS liquid medium containing 10 ⁇ cocoons and 1 ⁇ ⁇ of strength rice.
  • Suspended cultured cells in a 100 ml Erlenmeyer flask, 20 ml of medium in a rotary shaker at 90 rpm, 25 ° C, ollen cells in the dark, tobacco cells in 3000 lux, 16-hour light conditions Maintained.
  • the herbicide DTP was added to the liquid medium as a methanol solution at various concentrations.
  • the growth of the cultured cells was measured as the total weight of the whole yarn and weave in one 100 ml Erlenmeyer flask after 3 weeks treatment with DTP.
  • p-Hydroxypyruvic acid was purchased from Aldrich. Homogentisic acid was purchased from Sigma. DTP was acquired from Sankyo Pharmaceutical. The benzobicyclone hydrolyzate was obtained from S.D.
  • the deduced protein sequence was aligned using Clustal W and Boxshade (www.ch.embnet.org/software/BOX form.html.). Clustal W was also used to calculate the phylogenetic tree.
  • PCR includes the following Oligonucleotides were used:
  • PCR was performed under the following conditions: first denaturation step, 2 minutes, 94 ° C; 15 seconds, 94 ° C denaturation, 30 seconds, 56 ° C annealing, 2 minutes, 68 ° C DNA 30 cycles consisting of elongation and the last 68 ° C for 5 minutes, KOD-plus DNA polymerase (Toyobo) was used for this.
  • the PCR fragment was subcloned into the pET21d vector (Novagen) cut with Ncol and EcoRI restriction enzymes.
  • the Allen HPPD cDNA fragment was placed under the control of the T7 polymerase promoter in pET21d. Both strands of the DNA insert were sequenced to confirm that mutations were introduced during PCR amplification.
  • Ollen HPPD expression plasmid (pET21d) was introduced into E. coli BL21 (DE3). These recombinant E. coli cells were cultured in 100 ml of LB medium containing 100 mM ampicillin at 30 ° C. and 120 rpm. IPTG (isopropyl- ⁇ -D-thiogalatatoside) is used as the OD force of E. coli.
  • the final concentration was 1 mM.
  • the cells were further cultured at 30 ° C for 8 hours.
  • Recombinant proteins were separated by SDS-polyacrylamide gel electrophoresis containing 10% acrylamide. Protein samples were treated with 2XSDS-PAGE sample solution (0.125 M Tris-HC1, pH 6.8, 20% glycerol, 10% 2-mercaptoethanol, 4% SDS). The unfolded gel Stained with Coomassie Brilliant Blue R-250. The protein concentration was measured by the Bradford method using urchin serum albumin as a standard.
  • HPPD activity was measured by HPLC and LC-MS.
  • the 100 L standard enzyme reaction mixture consisted of: 100 mM potassium phosphate buffer, pH 6.5, 50 mM ascorbic acid, 10 L (37.18 g desalted protein) enzyme preparation and 200 M P-Hydroxyphenyl viruvate (pH 7.7).
  • concentration of p-hydroxyphenol biruvic acid was set to various concentrations from 2 ⁇ to 200 ⁇ in the measurement of kinetic parameters.
  • the herbicide DTP was added to the solution as a solution of 0.5% dimethyl sulfoxide (DMSO).
  • reaction mixture was incubated for 5 minutes at 30 ° C.
  • the reaction was stopped by the addition of triclonal acetic acid (final concentration 2%).
  • the reaction products were quantified using reverse phase HPLC (equipped with Shimadzu LC-10A system).
  • TCA supernatant (50 ⁇ 1 or 20 ⁇ 1) was injected onto a C18 column and eluted at a flow rate of 0.8 mL / min.
  • the mobile phase was 10 mM acetic acid-methanol (85: 15 [Vol / Vol]).
  • the HPPD activity was based on measurement of the amount of homogentisic acid formed by HPLC and UV absorbance at 280 nm. Homogentisic acid was quantified by measuring the peak area. The peak area was converted into the amount of homogentisic acid using a calibration curve. The formation of the product homogentisic acid was confirmed by LC-MS (LC-MS-2010, Shimadzu Corp., negative mode, solvent 20 mM ammonium acetate pH 5.5, flow rate 0.5 ml / min).
  • HPPD inhibition experiment using benzobicyclone hydrolyzate was carried out in the same manner as the inhibition experiment using DTP, except for the following points.
  • inhibitor benzobicyclone hydrolyzate and enzyme solution 100 mM potassium phosphate buffer, pH 6.5, 50 mM ascorbic acid, 10 ⁇ L (37.18 ⁇ g of desalted protein) enzyme preparation
  • concentrations of inhibitor benzobicyclone hydrolyzate and enzyme solution 100 mM potassium phosphate buffer, pH 6.5, 50 mM ascorbic acid, 10 ⁇ L (37.18 ⁇ g of desalted protein) enzyme preparation
  • the substrate 200 M p-hydroxyfurrubic acid (pH 7.7) was added to the enzyme solution and the enzyme reaction was carried out for 10 minutes.
  • the formation of the product homogentisic acid was LC-MS ( LC-MS-2010, Shimadzu Corporation, negative mode, solvent was 20 mM ammonium acetate pH 5.5, flow rate was 0.5 ml / min).
  • a cDNA library was also prepared for the capacity of allen cells to produce large amounts of alkaloids, and 1014 ESTs were obtained.
  • a BLAST search (hppt: ⁇ www.ncbi.nlmgov / blast /) showed that these sequenced clones included HPPD clones! /.
  • the cDNA insert in this HPPD clone was approximately 1539 bp in length and had a poly (A) tile at its 3, end.
  • Several amino acid domains were highly conserved between the deduced amino acid sequence of the DNA insert of the Allen HPPD clone and the Arabidopsis sequence, and these two domain sequences showed 80% identity to each other.
  • allen HPPD was a partial cDNA encoding the C-terminal part of HPPD.
  • the isolated allen HPPD was 2183 bp long and contained an ORF encoding a 430-amino acid polypeptide (molecular weight 47.3 kDa)!
  • the Orlen HPPD ORF was sandwiched between a 208 bp 5 'untranslated region and a 3' untranslated region containing 194 bp poly (A).
  • the alignment of Aulen HPPD with other species of HPPD ( Figure 5) showed a high identity with HPPD of other plants, with the highest identity with Arabidopsis thaliana being 71.0%. The identity with humans (30.5%) and S. avermilitis (32.4%) was low.
  • HPPD Various species of HPPD, including olene, were highly conserved in the C-terminal region of the protein. Amino acid residues believed to be active sites of proteins from maize and Arabidopsis thaliana were also present in the Allen HPPD sequence! And, like HPPD from other plants, olene HPPD also has an N-terminal extension of the amino acid! /, But the extension in this olene was shorter than in other plants. Plant HPPD is thought to be significantly different from bacterial and mammalian HPPD at the N-terminus in that it has an extension of at least 30 amino acids. These residues may be targeting signals to the intracellular compartment, i.e. transport signals to the chloroplast.
  • the amino acid sequence suggests that the isolated cDNA encodes allen HPPD.
  • a recombinant protein was produced in E. coli.
  • the present inventors constructed an expression vector that produces recombinant protein in Escherichia coli.
  • the present inventors introduced an Ncol site into cDNA so as to match the start codon in the expression vector pET-21d for E. coli. This construct was then introduced into E. coli cells to induce the production of recombinant protein.
  • the crude E. coli enzyme extract was desalted with a PD-10 column, and the desalted solution was used for detection of HPPD activity. SDS / PAGE analysis clearly showed that transgenic expression was induced.
  • the molecular weight of the subunit was estimated to be approximately 51 kDa ( Figure 7).
  • E. coli carrying the pET-HPPD construct showed a dark brown color in both liquid and solid media, whereas E. coli carrying an empty pET_21d vector did not show such a dark brown color (data Not shown). Similar dark brown coloration is also observed by HPPD of other origins. Has been observed.
  • This dye is the product of the acid polymerization of homogentisic acid synthesized by recombinant HPPD. Clearly, the reaction mixture was subjected to HPLC analysis and the recombinant E. coli enzyme crude extract produced a compound that behaved exactly like the homogentisic acid standard! ( Figure 8). The control E. coli lysate containing the empty pET-21d vector did not produce this peak at the same position.
  • the inventors optimized the pH conditions for the HPPD reaction using p-hydroxyfurrubic acid as a substrate. Enzyme assembly at various pHs revealed that the optimal pH for the conversion of HPP to homogentisic acid was approximately 6.5. Time course studies have shown that homogentisic acid accumulation is linear for at least 10 minutes in the presence of 10 1 desalting enzyme (37.2 ⁇ g protein) and 200 ⁇ ⁇ -hydroxyphenol-birubinic acid. did. The optimum temperature was 30 ° C.
  • the substrate affinity of recombinant HPPD was measured by HPLC analysis using HPP as a substrate.
  • DTP is known to act as an HPPD inhibitor. Matsumoto et al. (2002) showed that DTP inhibited HPPD at an IC value of 13 nmol / L by HPPD assay using highly active extracts from carrot cultured cells ( Figure 9). .
  • benzobicyclone hydrolyzate In order to detect the effect of benzobicyclone hydrolyzate on recombinant olene HPPD, the present inventors added various concentrations of benzobicyclone hydrolyzate solution to the enzyme reaction solution, and then became a substrate p- Hydroxyphenylpyruvic acid was added. Enzyme activity was expressed as relative activity based on the peak area of homogentisic acid and the activity without inhibitor benzobicyclone hydrolyzate as 100%. Benzobicyclone hydrolyzate inhibited allen HPPD with an IC value of 1.2 ⁇ (Fig. 11).
  • HPPD of the present invention exhibits herbicide resistance and is very useful.
  • HP PD and HPPD gene of the present invention can be used for the production of plastoquinone Z tocopherol.

Abstract

L’invention concerne un p-hydroxyphénylpyruvate dioxygénase présentant une résistance élevée contre un herbicide. L’invention concerne également un gène (a) ou (b) : (a) un gène codant une enzyme p-hydroxyphénylpyruvate dioxygénase résistante à un herbicide qui comprend un polynucléotide comprenant du nucléotide 1 au nucléotide 1293 de la séquence nucléotide illustrée par SEQ ID NO:1 ; ou (b) un gène comprenant un polynucléotide capable d’hybridiser avec un polynucléotide comprenant du nucléotide 1 au nucléotide 1293 de la séquence nucléotide illustrée par SEQ ID NO:1 dans des conditions stringentes et qui code une enzyme p-hydroxyphénylpyruvate dioxygénase résistante à un herbicide.
PCT/JP2006/311415 2005-06-10 2006-06-07 Gène résistant à un herbicide WO2006132270A1 (fr)

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