WO2003080842A1 - Gene d'enzyme detoxifiant de la zearalenone et transformant dans lequel le gene est transfere - Google Patents

Gene d'enzyme detoxifiant de la zearalenone et transformant dans lequel le gene est transfere Download PDF

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WO2003080842A1
WO2003080842A1 PCT/JP2003/003602 JP0303602W WO03080842A1 WO 2003080842 A1 WO2003080842 A1 WO 2003080842A1 JP 0303602 W JP0303602 W JP 0303602W WO 03080842 A1 WO03080842 A1 WO 03080842A1
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zearalenone
protein
gene
seq
dna
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PCT/JP2003/003602
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English (en)
Japanese (ja)
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Isamu Yamaguchi
Makoto Kimura
Naoko Ando
Arisa Nishiyama
Tetsuko Fukuda
Hideaki Kakeya
Hiroyuki Osada
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Riken
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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
    • C12N9/14—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/32—Antioestrogens
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8259—Phytoremediation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • the present invention relates to a protein capable of purifying mycotoxin-infected plants and a gene encoding the same.
  • a known disease of the plant is Fusarium head blight of wheat, which is infected with phytopathogenic fungi and damages the plant, causing a large loss in yield.
  • the disease has been widespread due to recent warm and humid climate change.
  • wheat scabs which were widespread around the world, were incurable diseases that could not be affected even in North America where advanced intensive agriculture is conducted. It became. For this reason, red mold has recently been taken up as a major problem in Europe and the United States, and national measures are being taken.
  • Protecting wheat, an important crop, from the threat of Fusarium head blight is an important issue in order to secure a stable and secure food supply on a global scale.
  • Fusarium head blight is a plant disease caused by a plant called Fusarium, which infects grasses such as wheat, corn, and rice. More than seventeen species of Fusarium, including Fusarium graminea rum, have been isolated and reported as Fusarium mycobacteria (Fusarium Mycotoxins, iaxonomy and Pathogeni city J. Che 1 Rows). ky3 ⁇ 4, Elsevier Science Ltd., (1989) p. 1-39). If a plant is infected with Fusarium head blight, the yield and quality of the grain will be significantly reduced, which will be economically hurt, and the accumulation of mycotoxin toxin in the grain will cause food hygiene problems. Is caused.
  • red blight is a double threat to food supply.
  • pesticides such as tebuconazole are used to control Fusarium head blight, it is not very practical due to the possibility of emergence of resistant bacteria, increased labor and cost, difficulty in timing of application, and pesticide persistence. is not. Therefore, efforts have been made to cultivate varieties resistant to red blight (“Plant disease resistance from the molecular level”, supervision: Tetsuka Yamada, Isao Shimamoto Yuichiro Watanabe, Shujunsha, Japan, ( 1997) p. 90-97).
  • Trichothecene toxins are also protein synthesis inhibitors and enhance infectivity as a virulence factor when bacteria are infected.
  • a gene that blocks the protein synthesis inhibitory activity of the trichothecene toxin Japanese Patent Application Laid-Open No. 2000-32985
  • the trichothecene toxin are extracellularly excreted. Pumping genes (Alexander, J, "Molecular & general genetics” (1999) 261, p. 977-984) have been reported.
  • Zearalenone [6- (10-hydroxy-6-oxo-trans-1-onedecenyl)-/ 3-ratatatone resorcylate] is a mycotoxin with estrogenic activity produced by Fusarium spp. It is.
  • Zearalenone is an endocrine disruptor that has estrogenic activity and can cause toxic symptoms and reproductive harm to humans and livestock who take it (Etienne, M and Jammali, M. Journal of animal science ( 1982) 55, p. 1-10).
  • An object of the present invention is to provide a gene encoding a protein that detoxifies zearalenone accumulated in plants due to infection with Fusarium head blight.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, succeeded in isolating a protein having an activity of degrading zearalenone, and a gene encoding the protein, and completed the present invention. Reached.
  • the present invention is as follows.
  • the protein in [1] may be characterized in that zearalenones are compounds having estrogenic activity.
  • zearalenones include zearalenone, zearalenol, j8-zearalenol, h-zearalanore, ⁇ -zearalanol, 2,4-0-dimethyl- ⁇ -hydroxyzearalenone, and 6-amino-zearalenone. It may be at least one selected from the group consisting of non-, zearalanone and 6-acetyl-] 3-zearalenol.
  • the protein of [1] may be characterized in that the action of suppressing toxicity is a degrading action. The effect of suppressing toxicity may be to generate a compound having no estrogenic activity. Further, the protein of [1] may have an activity of pH 6 to 11, preferably pH 9 to 10.5.
  • a gene comprising the following DNA (a) or (b):
  • DNA encoding a protein having an action of suppressing the toxicity of zearalenones [4] A gene comprising the following DNA (a) or (b):
  • the genes in [2:] to [4] may be characterized in that zearalenones are compounds having estrogenic activity.
  • Zearalenones include zearalenone, hy-zearalenone, ⁇ -zearalenol, ⁇ -zearalanone, J3-zearalanol, 2,4-0-dimethyl / le- ⁇ -hydroxyzearalenone, 6-amino- It may be at least one selected from the group consisting of zearalenone, zearalanone and 6-acetyl-j3-zealarenol.
  • the genes in [2;] to [4] may be characterized in that the action of suppressing toxicity is a degradation effect. Product that suppresses toxicity The use may be to produce a compound having no estrogenic activity.
  • the transformant may be one in which a recombinant vector has been introduced into a cell selected from the group consisting of Escherichia coli, yeast cells, and gramineous plant cells.
  • a method for producing a protein which comprises culturing the transformant of [6] and collecting, from the resulting culture, a protein having an activity of suppressing toxicity of zearalenones.
  • a method for detoxifying zearalenones which comprises applying the antidote of [8] or [9] to zearalenones.
  • ⁇ IV The resistance of wheat (Triticum aestivum L.) to Fusarium head blight is roughly classified into the following four types: ⁇ IV:
  • Type I Resistance to fungal invasion (depending on flowering characteristics, shape of ears, etc.)
  • Type II Resistance to fungal growth after invasion (hyphal elongation)
  • Type III Resistance exerted in the grain (reduction of mold damage to seeds)
  • Type IV resistance based on reduced mycotoxin accumulation.
  • genes involved in type IV resistance that is, resistance based on the reduction of mycotoxin accumulation in infected plants, are not only useful in protecting plant individuals from red blight, but also from plants. It is considered to be very useful in ensuring the safety of the produced grains as food. That is, Fusarium
  • the ability to isolate and identify a gene encoding an enzyme capable of detoxifying mycotoxins, such as zearalenone, that accumulates in grains due to bacterial infection, would be very useful in protecting plants from red blight. It was thought to be.
  • the present invention has been completed based on such an idea.
  • Zearalenone in the present invention basically indicates the chemical formula shown in FIG.
  • the zearalenone detoxifying enzyme in the present invention can use other zearalenones as substrates, other than zearalenone itself of the chemical formula shown in FIG. That is, the zearalenone detoxifying enzyme of the present invention is a protein having an action of suppressing the toxicity of zearalenones by an enzymatic reaction using the zearalenones as a substrate.
  • Examples of zearalenones that can be used as a substrate by zearalenone detoxifying enzyme include zearalenone analogs in addition to zearalenone.
  • zearalenone analog means a compound having a zearalenone skeleton having a lactone ring composed of 14 carbon atoms, such as ⁇ -zearalenone, / 3-zealalenone, ⁇ -zearalanone, / 3-zearalanone, and 2 , 4 - ⁇ -Dimethinole- ⁇ -hydroxyzearalenone, 6-amino-zearalenone, zearalanone, 6-acetyl- -zearalenol and the like.
  • the zearalenones that can be used as a substrate by the zearalenone detoxifying enzyme are preferably compounds having an estrogenic activity.
  • the above zearalenones will be referred to as “zearalenone” for convenience.
  • toxicity of zearalenones or “toxicity of zearalenone” refers to toxicity brought to an individual plant by accumulation of zearalenone, for example, cell toxicity, and furthermore, human ingestion of zearalenone.
  • inhibiting the toxicity of zearalenone means that the degree of the toxicity indicated by the action of zearalenone is reduced, preferably that the toxicity becomes undetectable or lost. means.
  • detoxification has the same meaning as “suppress the toxicity of zearalenone”.
  • the action of suppressing the toxicity of zearalenone (class) is the chemical structure of zearalenone itself By suppressing the toxicity as described above. The action of suppressing the toxicity of zearalenone may be specifically caused by decomposing or cleaving zearalenone, or may be effected by chemically modifying zearalenone. .
  • the action of suppressing the toxicity of zearalenone may be one that produces a compound having no estrogenic activity using zearalenone as a substrate.
  • the action of suppressing the toxicity of zearalenone is preferably measured as an action of reducing the estrogenic activity.
  • Estrogen-like activity in the present invention means binding to an estrogen receptor and promoting the growth of a human cancer cell line MCF-7 in vitro. This estrogenic activity can be easily measured, for example, by a test as described in Example 9 herein.
  • strains available from a distribution agency were screened for their ability to detoxify zearalenone, and strains having zearalenone detoxifying enzymes were isolated.
  • the cells to be screened may be any of animal cells, plant cells, fungal cells and bacterial cells, but are preferably fungal cells.
  • Those skilled in the art can easily obtain a cell line (eg, a fungal strain) from a distributing institution based on the catalog number of each distributing institution.
  • AKU Ferty of Agriculture, Kyoto University, Kyoto, Japan
  • ATCC American Type Culture Collection Rockville, US A
  • HUT Ferty of Engineering, Hiroshima University, Hiroshima, Japan
  • IAM Institute of Applied Microbiology, University of Tokyo, Japan
  • IF0 ⁇ institute for Fermentation Osaka, Japan
  • JCM Japan Collection of Microorganisms, RIKEN
  • information such as culture conditions is provided for each cell line available from the distributing organization, and by referring to this information, those skilled in the art can easily culture the cell line.
  • the following methods can be used.
  • zearalenone for each strain. Cultivation according to a culture method known to a human. The culture is then extracted, for example, in black-mouthed form. For example, when screening for zearalenone resolution is performed as zearalenone detoxification, the extract is subjected to thin layer chromatography as follows:
  • TLC TLC analysis.
  • zearalenone a strain in which spots having the same mobility as the zearalenone standard do not appear and spots having a mobility distinct from zearalenone appear, and cells having the ability to detoxify zearalenone (for example, strains).
  • extraction and purification of zearalenone detoxification enzyme are performed from the cells selected as described above.
  • the extraction and purification of the enzyme from the cells can be performed using any technique known to those skilled in the art. For example, it may be performed by column elution separation and TLC analysis as described below.
  • the cell line is cultured in a medium supplemented with zearalenone (for fungi, for example, YG medium) according to a culture method known to those skilled in the art.
  • zearalenone for fungi, for example, YG medium
  • the cultured cells are collected, the cells are crushed with liquid nitrogen or the like, and cell debris is spun down by centrifugation to obtain supernatant.
  • the supernatant is fractionated with ammonium sulfate, and the obtained protein solution is dialyzed to obtain a crude enzyme solution.
  • This crude enzyme solution can be applied to, for example, a HiTrapQ column (Pharmacia), eluted and separated for further purification.
  • Each of the eluted fractions thus obtained is subjected to an in vitro enzyme reaction test for zearalenone, and thereafter, it can be examined by TLC whether or not it has an enzyme reaction activity for zearalenone.
  • zearalenone degradation test add zearalenone to each eluted fraction and incubate at 37 ° C.
  • zearalenone and Z or a degradation product thereof are extracted from each of the samples, and subjected to TLC.
  • spots with a mobility clearly different from that of zearalenone are detected on TLC.
  • zearalenone detoxifying enzyme is an enzyme that changes the molecular structure of zearalenone in a manner different from degradation
  • zearalenone is used for a sample obtained using an elution fraction containing such an enzyme. Clearly different mobility spots are detected.
  • the Rf value is generally used as an index of this mobility.
  • the Rf value is the sample
  • the distance from the development origin applied to the sample to the center of the color spot of the sample (the average travel distance of the compound) is the distance from the development origin to the development tip of the development solution (the maximum travel distance of the development solution).
  • Rf Is defined as the value divided by This Rf value varies depending on the affinity of the compound with the adsorbent applied to thin-layer chromatography and the solubility of the compound in the developing solution. This is useful for identifying compounds.
  • the eluted fraction identified in this way which can detect spots having a mobility clearly different from that of zearalenone upon addition of zearalenone, is subjected to a further purification step.
  • Further purification steps include FPLC separation using a gel filtration column, and those using an ion exchange column.By repeating these various protein purification steps, a desired protein can be obtained with high purity. .
  • the partial amino acid sequence of zearalenone detoxifying enzyme obtained in 1 above is determined in the present invention.
  • the purified zearalenone detoxification enzyme is fragmented with a protease such as lysyl endopeptidase (eg, TAKA RA, Kyoto, JAPAN).
  • the reaction mixture is separated for each peptide fragment by HPLC.
  • HPLC HPLC
  • Such an HPLC operation may be usually performed according to the manufacturer's instructions.
  • the peptide fragment separated by HPLC and the zearalenone detoxifying enzyme itself purified above are respectively applied to a protein sequencer, and the amino acid sequence is determined by Edman degradation.
  • a primer for PCR amplification of DNA encoding zearalenone detoxification enzyme is designed. Design a degenerate 5 'primer based on the N-terminal amino acid sequence and a degenerate 3' primer based on the amino acid sequence obtained from the peptide fragment based on the partial amino acid sequence determined above .
  • the peptide fragment used in designing the primer may be any of those separated by the above HPLC, but it is preferable to use a primer sequence that minimizes the degeneracy pattern.
  • Type II used for PCR amplification has the ability to detoxify zearalenone.
  • P Use cDNA from cells that leak.
  • This cDNA can be obtained by culturing cells capable of detoxifying zearalenone, extracting total RNA or mRNA from the culture by a conventional method, and further synthesizing it by RT-PCR. By subjecting the cDNA thus obtained to PCR amplification using the above primer set, a partial DNA fragment of the gene encoding zearalenone detoxification enzyme can be obtained.
  • As the PCR reaction conditions for example, 30 cycles of 94 ° C (30 seconds), 55 ° C (30 seconds) and 72 ° C (1 minute) may be performed. The size of the amplified product obtained is confirmed by agarose gel electrophoresis.
  • an appropriate one of the PCR amplification products obtained as described above for example, the one having the largest size is selected, cloned into an appropriate vector, and subjected to DNA sequence determination.
  • DNA sequencing can be performed by a conventional method.
  • ABI PRISM (R) 377 DNA sequencer and ABI kit are used according to the protocol provided by the manufacturer. You may.
  • RACE rapid amplification at ion of cDNA ends
  • the base sequence of the entire coding region of the zearalenone detoxifying enzyme gene of the present invention can be determined.
  • the zearalenone detoxification enzyme gene isolated and sequenced according to the above-described method 1 or 2 has the nucleotide sequence shown in SEQ ID NO: 1.
  • the amino acid sequence deduced from the nucleotide sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2.
  • the amino acid sequence deduced from the nucleotide sequence of SEQ ID NO: 1 was identical to the amino acid sequence determined by the Edman method.
  • the amino acid sequence represented by SEQ ID NO: 2 was searched in public databases (Swiss-Prot and GenBank), no corresponding sequence was found, so that this zearalenone detoxifying enzyme was a novel one. It was judged.
  • this zearalenone detoxifying enzyme gene (SEQ ID NO: 1) is a zearalenone detoxifying enzyme whose gene product (SEQ ID NO: 2) degrades zearalenone to produce a degradation product having no estrogenic activity.
  • the confirmation was performed in a system using human breast cancer cell MCF-7, as shown in 7 below.
  • the zearalenone detoxifying enzyme gene of the present invention is a DNA having the nucleotide sequence shown in SEQ ID NO: 1.
  • This DNA should be obtained, for example, by PCR-amplifying the cDNA obtained above as a ⁇ type using primers designed based on SEQ ID NO: 1, and extracting and purifying the amplified DNA fragment by a conventional method. Can be.
  • the zearalenone detoxifying enzyme gene of the present invention more generally encodes a protein having an action of suppressing the toxicity of zearalenone.
  • the zearalenone detoxifying enzyme gene of the present invention is not limited to the DNA comprising the base sequence shown in SEQ ID NO: 1.
  • the gene of the present invention may encode a protein consisting of the amino acid sequence shown in SEQ ID NO: 2.
  • the gene of the present invention has one or more (preferably 1 to 10, more preferably several) amino acids in the amino acid sequence shown in SEQ ID NO: 2 as long as it has an action of suppressing the toxicity of zearalenone. It may encode a protein consisting of a deleted, substituted or added amino acid sequence.
  • a gene encoding a protein is also included in the gene of the present invention.
  • a DNA capable of hybridizing under stringent conditions with a DNA encoding the amino acid sequence shown in SEQ ID NO: 2 or a DNA consisting of a sequence complementary to a part thereof, The gene encoding the protein having the action of suppressing the toxicity of zearalenone is also included in the gene of the present invention.
  • Stringent conditions refer to conditions under which a so-called specific hybrid is formed.
  • nucleic acids having high homology that is, DNAs having a homology of 90% or more, preferably 95% or more, and DNAs encoding a protein having an action of suppressing the toxicity of zearalenone hybridize
  • the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM
  • the temperature is 25 to 70 ° (: preferably, 50 to 70 ° C, Preferably, it is a condition at 55 to 65 ° (:, a formamide concentration of 0 to 50%, preferably 20 to 50%, more preferably 35 to 45%.
  • the washing condition of the filter after hybridization is The conditions are a sodium salt concentration of 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and a temperature of 50 to 70 ° C, preferably 55 to 70 ° C, more preferably 60 to 65 ° C. Such cases can be included in the "stringent conditions" of the present invention.
  • the nucleotide sequence of the gene of the present invention is then subjected to chemical synthesis, or to PCR using a cloned cDNA, cDNA library or genomic DNA library as type III, or to a DNA having the nucleotide sequence.
  • the gene of the present invention can be obtained by hybridizing the fragment to a cDNA library or a genomic DNA library as a probe.
  • the organism from which the cDNA library or the genomic DNA library is derived is not particularly limited, but is preferably an organism belonging to a fungus.
  • a gene encoding a protein having a function of suppressing the toxicity of zearalenone, which is a mutant form of the gene of the present invention can also be synthesized.
  • a protein which is a mutant of the gene of the present invention, which has a function to suppress the toxicity of zearalenone, and which produces a degradation product having no estrogen-like activity by site-directed mutagenesis or the like can also be synthesized.
  • mutant form of the gene of the present invention by site-directed mutagenesis or the like, which has the effect of suppressing the toxicity of zearalenone, and has a pH of 6 to 11, preferably pH of 9 to 10. It is also possible to synthesize a gene encoding a protein having an activity in step 5.
  • a known method such as the Kunkel method and the Gapped duplex method or a method similar thereto can be employed.
  • a mutagenesis kit using site-directed mutagenesis for example, Mutan-K TAKARA
  • Mutan-G TAKARA
  • TAKARA's LA PCR in vitro Mutagenesis Mutations are introduced using a series kit. 4. Construction of recombinant vector
  • the gene of the present invention described in 3 above is preferably cloned into a vector to prepare a recombinant vector for subsequent operations.
  • the recombinant vector of the present invention can be obtained by ligating (inserting) the zearalenone detoxifying enzyme gene of the present invention to an appropriate vector.
  • the vector for inserting the zearalenone detoxifying enzyme gene is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA.
  • plasmid DNA examples include Escherichia coli-derived plasmids (eg, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), and yeast-derived plasmids (eg, Phage DNA; L phage (Charon4A, Charon21A, EMBL3, EMB L4, gtl0, Lgtll, LZAP, etc.).
  • retroviruses or animals such as Vincenius innoles, innores, and insects such as noculois / les, innores vectors can also be used.
  • the purified DNA is cut with an appropriate restriction enzyme, inserted into an appropriate vector DNA restriction enzyme site or a multi-cloning site, and ligated to the vector. The method is adopted.
  • the zearalenone detoxification enzyme gene needs to be incorporated into a vector so that the function of the gene is exerted.
  • the recombinant vector of the present invention is preferably prepared as a recombinant expression vector in which a zearalenone detoxification enzyme gene is incorporated into a vector so that the gene is expressed as a protein having good activity in a host.
  • various commercially available expression vectors corresponding to many host organisms can be used as the vector of the present invention.
  • Such expression vectors usually include various elements essential for expression in the host organism, such as a transcription promoter, a terminator, and a ribosome binding site, as well as a selection marker that indicates that the vector is retained in the cell.
  • Cis-elements such as polylinker, henno, and ss1 to easily introduce genes into the vector in the correct orientation, splicing signal, poly-A addition signal, ribosome binding sequence (SD sequence), secretion factor sequence, etc. useful Unique sequences are linked as necessary.
  • the expression vector containing a secretory factor sequence compatible with the host organism is used.
  • the recombinant protein can be secreted into the medium. This technique is useful because the recombinant protein can be purified directly from the culture supernatant.
  • the secretory factor sequence may be one that can be specifically cleaved with a specific protease or the like from the protein encoded by the gene incorporated in the vector after secretion into the culture supernatant, and removed.
  • the selectable marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like.
  • the zearalenone detoxification enzyme gene is ligated to the vector as described above in such a position and orientation that it can be appropriately expressed.
  • the gene of the present invention can also be directly introduced into the genome of a host organism by a homologous recombination method.
  • an appropriate targeting vector incorporating the gene of the present invention is prepared.
  • a vector that can be used for this purpose a known gene targeting vector such as Cre-- ⁇ can be used.
  • such a targeting vector incorporating the gene of the present invention is also included in the recombinant vector of the present invention. 5. Introduction of zearalenone detoxification enzyme gene into plants
  • Transgenic plants can be obtained by introducing the recombinant vector prepared as described in 4 above into a plant.
  • Plants to be transformed in the present invention include whole plants, plant organs (eg, leaves, petals, stems, roots, seeds, etc.), and plant tissues (eg, epidermis, phloem, soft tissue, xylem, vascular bundle, It refers to either a palisade tissue, spongy tissue, etc.) or plant culture cells (eg, callus).
  • the plant used for the transformation is preferably a plant in which the accumulation of zearalenone is observed in the plant due to the infection of Fusarium spp., Which is a scab of Fusarium.
  • plants into which the zearalenone detoxifying enzyme gene of the present invention is introduced include corn, wheat, rye, rye, rice, and the like. Are preferred. Examples of plants used for transformation include the following.
  • Eggplant tomato eggplant (Solanum melongena ishi), tomato (Lycopersicon escu ⁇ entum Mi l 1), bell pepper (Capsicum annuum L. var.angulosum ki ll.), Tofu-zong (Capsi cum annuum L-), Tanoko (Nicotiana tabacum L.)
  • Brassica tomato Arabidopsis thaliana, Brassica (Brassica ca mpestris), Chinese cabbage (Brassica pekinensis Rupr-), Kyahe (Brassica ole racea L. var.capitata L.)
  • Guicon Renicon sativus L
  • Nata (Brassi ca campestri s L., B. napus L.)
  • Rice paddy maize (Zea mays), rice (Oryza sativa), wheat (Triticum ae stivum L.), wom (Hordeum vulgare L.)
  • Legumes Soybean (Glycine max), Azuki (Vigna angularis Willd.), Bean (Phaseolus vulgaris.), Broad bean (Vicia faba L.)
  • Periaceae Cucumber (Cucumis sativus L.), Melon (Cucumis melo L.), Watermelon (Citrullus vulgaris Schrad.), Cabochia (C. moschata Duch., C. maxima Duch.)
  • Lily family Leek (Allium fistulosum L.), onion (Allium cepa L.), -la (Allium tuberosum Rottl.), Garlic (Allium sativum L.), Asunoragasu (Asparag us officinalis L.)
  • Perilla fritters Perilla (Perilla frutescens Britt. Var. Crispa)
  • Chrysanthemum Chrysanthemum raorifolium, Chrysanthemum coron arium L. s Lettuce (Lactuca sativa L. var. Capitata L.)
  • Gentian geese Gentian (Gentiana scabra Bunge var. Buergeri Maxim.) Nadesico geese: Carnation (Dianthus caryophyllus L.)
  • the above recombinant vector can be introduced into a plant by a conventional transformation method, for example, an agrobacterium method, a particle gun method, a PEG method, an electoral poration method, or the like.
  • agrobacterium method when used, the constructed plant expression vector is introduced into an appropriate agrobacterium, for example, Agrobacterium tumefaciens, and this strain is transformed into rice power.
  • the transgenic plants can be obtained by inoculation and infection.
  • the plant, plant organ, or plant tissue itself may be used as it is, may be used after preparing a section, or may be used after preparing a protoplast .
  • the sample thus prepared can be processed using a gene transfer apparatus (for example, PDS-1000 (BIO-RAD) or the like).
  • the treatment conditions vary depending on the plant or sample, but usually the pressure is about 450 to 2000 psi and the distance is about 4 to 12 cm.
  • the recombinant vector is introduced into the culture cell by a Partique Noregan method, an electoral poration method, or the like. At this time, it may be possible to cause homologous recombination to the plant genome using a targeting vector.
  • the tumor tissue, shoots, hairy roots, etc. obtained as a result of the transformation can be directly used for cell culture, tissue culture or organ culture, or by using a conventionally known plant tissue culture method.
  • Plant hormones eg, auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.
  • Plant hormones can be regenerated into plants by administration of various concentrations.
  • Whether or not the gene has been integrated into the plant can be confirmed by a PCR method, a Southern hybridization method, a Northern hybridization method, or the like.
  • PCR can be performed under the same conditions as those used to amplify the cDNA fragment inserted into the recombinant vector.
  • agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis By performing staining and the like, staining with Chidium bromide, SYBR Green solution, and the like, and detecting the amplified product as a single band, the transformation can be confirmed.
  • amplification products can also be detected by performing PCR using primers previously labeled with a fluorescent dye or the like. Furthermore, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence or an enzymatic reaction can also be adopted.
  • the transgenic plant into which the zearalenone detoxifying enzyme gene of the present invention prepared as described above has been introduced is capable of producing zearalenone detoxifying enzyme in a plant body, thereby producing the toxicity of zearalenone produced by infection with Fusarium head blight. Can be suppressed.
  • zearalenone detoxifying enzyme Preparation of transformant and production of zearalenone detoxifying enzyme using the transformant
  • a transformant transformed cell into which the zearalenone detoxifying enzyme gene of the present invention is introduced is prepared and cultured.
  • zearalenone detoxifying enzyme can be produced.
  • the present invention also relates to such a transformant and a method for producing zearalenone detoxifying enzyme using the transformant.
  • any of bacteria such as Escherichia coli and Bacillus subtilis, yeast cells, insect cells, animal cells (for example, mammalian cells), plant cells, and the like may be used.
  • Escherichia coli, yeast cells or grass plants cells More specifically, for example, for the control of Fusarium head blight on grasses, it is preferable to use grass cells.
  • E. coli or yeast cells are preferably used.
  • yeast cells are preferably used.
  • Transformants can be selected according to a standard method, but usually, the method for culturing the transformant of the present invention using a selection marker incorporated in the recombinant vector used is suitable for culturing a host organism. Normal one used It is done according to the law.
  • a medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or a yeast cell as a host contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host microorganism.
  • a natural medium or a synthetic medium can be used as long as the medium can be efficiently used.
  • antibiotics such as ampicillin or tetracycline may be added to the medium.
  • an inducer may be added to the medium as necessary.
  • a microorganism transformed with an expression vector using the Lac promoter a microorganism transformed with an expression vector using the trp promoter such as isopropyl-1-thio-D-galactoside (IPTG) can be used.
  • IPTG isopropyl-1-thio-D-galactoside
  • IAA indoleacetic acid
  • the culture conditions are not particularly limited, but are preferably performed under conditions suitable for the host organism used for transformation.
  • the zearalenone detoxifying enzyme is produced in the cells or cells, the cells or cells are disrupted.
  • the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like to obtain a supernatant.
  • the resulting solution contains zearalenone detoxifying enzyme.
  • zearalenone detoxifying enzyme can be produced using a cell-free translation system instead of performing transformation.
  • a cell-free translation system is an in vitro transcription-translation system that adds reagents such as amino acids required for translation to a suspension obtained by mechanically disrupting the structure of the host organism's cells. Alternatively, it constitutes an in vitro translation system.
  • kits that can be advantageously used are commercially available.
  • the produced zearalenone detoxifying enzyme can be used alone or by a common biochemical method used for isolating and purifying proteins, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. When used in an appropriate combination, it can be isolated and purified from the above-mentioned culture (in a cell lysate, a culture, or a supernatant thereof) or a cell-free translation system solution. However, in some cases, for example, the culture supernatant is concentrated with an ultrafiltration filter or the like.
  • the crude enzyme solution obtained by shrinking or dialyzing after ammonium sulfate fractionation may be used as it is for zearalenone detoxification.
  • zearalenone detoxifying enzyme of the present invention has sufficient activity in a highly alkaline environment at pH 9.5. It was shown that. Further, ZHD101 has P H 7. 0 even some activity, whereas pH 4. At low pH, such as below 5 was found to be non-reversibly deactivated. As described above, the zearalenone detoxifying enzyme of the present invention exhibits, as one feature, a bias toward the alkaline side at an optimum pH. That is, the zearalenone detoxifying enzyme of the present invention has activity at pH 6 to 11, preferably at pH 9 to 10.5.
  • the toxicity of zearalenone can be suppressed by using the zearalenone detoxification enzyme of the present invention and cells or transgenic plants containing the zearalenone detoxification enzyme gene.
  • a reaction solution containing the zearalenone detoxification enzyme of the present invention or a culture of cells containing the zearalenone detoxification enzyme gene may be directly applied to a solution containing zearalenone or zearalenone.
  • the present invention may be applied to a material (plant, grain, fruit, soil, natural or artificial base, etc.) to which zearalenone is attached.
  • cultivation in an area where there is a risk of Fusarium head blight infection may reduce zearalenone contamination, or soil contaminated with zearalenone
  • cultivation in environmental water may suppress the toxicity of zearalenone contained in the surrounding environment.
  • a transformant having a zearalenone detoxifying enzyme gene is cultured, and an enzyme solution obtained from the culture is added to a solution containing zearalenone.
  • the enzyme reaction is allowed to proceed by incubating the mixture at, for example, 37 ° C.
  • the reaction product is analyzed by, for example, TLC, it can be confirmed that zearalenone is consumed and products having different mobilities are generated. In this case, it can be considered that the activity of zearalenone detoxifying enzyme was shown.
  • the transformant is cultured in the presence of zearalenone, and an extract is obtained from the culture.
  • the extract is analyzed by, for example, TLC, and the results indicate that zearalenone is consumed and products with different mobilities are generated, it is considered that the activity of zearalenone detoxifying enzyme was indicated. it can.
  • the compound produced from zearalenone by the catalytic reaction of the zearalenone detoxifying enzyme of the present invention can be subjected to NMR analysis and mass spectrometry (FAB-MS, EI-MS, etc.). By performing these determinations, the chemical structure of the compound can be determined.
  • NMR analysis and mass spectrometry FAB-MS, EI-MS, etc.
  • the estrogen-like activity of a compound produced from zearalenone (referred to as zearalenone-derived detoxification product) by the catalytic reaction of the zearalenone detoxifying enzyme of the present invention can be measured.
  • zearalenone-derived detoxification product does not have estrogen-like activity
  • the zearalenone-derived detoxification enzyme has an action of suppressing the toxicity of zearalenone.
  • the estrogen-like activity can be measured using, as an index, the cell growth promoting effect on human breast cancer cells MCF-7.
  • the cell growth-promoting effect on human breast cancer cells MCF-7 can be measured as follows. First, human breast cancer cells, MCF-7, were converted to, for example, phenol red, L-glutamine (2 mM), penicillin (50 units / ml), streptomycin (50 g / ml) and 10% fetal serum. (FCS) in the added RPMI-1640 medium, cultured at 37 ° C for humidified air containing 5% C0 2. The cultured cells are inoculated with, for example, 5 ⁇ 10 3 cells / well in RPMI medium without phenol red.
  • FCS fetal serum.
  • the medium was replaced with the same type of medium, and the test compound , Zaralenone-derived detoxification product, or 17-estradiol at various concentrations, and further culture for 120 hours.
  • the number of cells is evaluated by a color reaction.
  • the sodium salt of 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfofenyl) -2-tetrazolium is used.
  • Some WST-8 TM Na kalai Tesque, Kyoto
  • the addition of 17-estradiol as a test compound increases the cell number on average by about 250% compared to a control without any addition.
  • the cell number is comparable to that of 17] 3-estradiol.
  • the ratio of the number of cells ( ⁇ ) when the test compound is added to the number of cells ( ⁇ ) when the test compound is not added [(B) / (A)] ⁇ 100 (%); In the present invention, this value is referred to as the cell growth promoting ratio.) If the force is 0% to 150%, preferably 80% to 120%, it is assumed that the test compound has no cell growth promoting effect.
  • the absence of the cell growth promoting effect of the test compound can be judged to have no estrogen-like activity. Therefore, when the above-mentioned zearalenone-derived detoxification product is added as a test compound, if the cell growth promoting action is not observed, the zearalenone-derived detoxification product is a compound having no estrogenic activity. In this manner, if it is confirmed that the zearalenone-derived detoxification product does not have estrogen-like activity, the zearalenone-detoxification enzyme that has produced the zearalenone-derived detoxification product can use a zearalenone-based compound that has no estrogen-like activity. It shows that it can be created.
  • the zearalenone detoxification enzyme of the present invention is a protein having an action of suppressing the toxicity of zearalenone. it can.
  • the determination that the zearalenone detoxifying enzyme of the present invention has the activity of suppressing the toxicity of zearalenone is limited only to the confirmation that the zearalenone-derived detoxification product has no estrogenic activity. It is not specified.
  • FIG. 1 is a photograph showing TLC data obtained by detecting the hydrolysis activity of a culture supernatant derived from the IF07063 strain on zearalenone. Lane 1, culture supernatant sample; Lane 2, negative control; Lane 3, zearalenone standard sample.
  • FIG. 2 is a view showing an HPLC profile of ZHD101 fragmented by peptide with lysylendopeptidase.
  • FIG. 3 shows a PCR primer set for amplifying a partial DNA fragment of ZHD101 based on the amino acid sequence.
  • FIG. 4 is a photograph showing the results of SDS-PAGE of recombinant ZHD101 expressed by the T7 transcription / expression system in E. coli DE3.
  • Lane 1 is a homogenate sump of wild-type DE3
  • lane 2 is a homogenate sample of DE3 (transformant) having pET12-zhdlOl
  • lane 3 is a precipitate sample of a homogenate of the transformant
  • lane 4 is a transformant
  • Lane 5 is the ZHD101 directly extracted and purified from IF07063 strain
  • Lane 6 is the molecular weight marker.
  • FIG. 5 is a photograph showing the results of TLC of recombinantly expressed ZHD101.
  • Lane 1 is a homogenate of wild-type DE3 treated with zearalenone
  • lane 2 is a homogenate of DE3 (transformant) having pET12-zhdlOl treated with zearalenone
  • lane 3 is a standard zearalenone.
  • FIG. 6 is a photograph showing TLC data in which the hydrolysis activity of ZHD101 for ⁇ -zararail and -zararail was detected.
  • Lane 1 ZHD101 treated ⁇ -zararail sample; lane 2, negative control for ⁇ -zararail; lane 3, ⁇ -zararail standard, lane 4, ZHD101 treated _ zararail sample; lane 5, negative for ⁇ -zararail Control; Lane 6, _ Zararail standard.
  • FIG. 7 shows, using a chemical formula, that zearalenone is degraded by the zearalenone degrading enzyme ZHD101 to generate a zearalenone degradation product.
  • FIG. 8 shows the cell growth promoting effect of zearalenone and a zearalenone degradation product on human breast cancer cells MCF-7.
  • Added zearalenone ( ⁇ ) or zearalenone decomposition The number of cells after incubation at 37 ° C for 120 hours against the concentration of the product (country) is shown. The results are shown as the average value SD.
  • FIG. 9 is a schematic diagram showing the steps of constructing the zhdlOl gene transfer vector pWheat-egfp :: zhdl01.
  • FIG. 10 is a photograph of a transformant (transgenic callus) into which the egfp :: zhdl01 gene has been introduced, observed under a fluorescence microscope.
  • FIG. 11 is a photograph of a regenerated rice plant into which the egfp :: zhdl01 gene has been introduced.
  • FIG. 12 is a photograph showing the result of the easter blot analysis of a transgenic individual into which the egfp :: zhdl01 gene has been introduced.
  • the leftmost is the molecular weight marker
  • lane 1 is a sample extracted from wild-type rice leaves
  • lane 2 is regenerated No. 14
  • lane 3 is regenerated No. 54
  • lane 4 is regenerated No. 68
  • lane 5 is regenerated No. 71
  • Lane 6 shows the results of regenerated body No. 76
  • lane 7 shows the results of regenerated body No. 79
  • lane 8 shows the results of regenerated body No. 79
  • lane 9 shows the results of recombinant EGFP :: ZH D101 protein.
  • FIG. 13 is a photograph showing the result of TLC analysis of the extract from the medium in which the egfp :: zhdl01 gene-introduced suspension cultured cells were cultured.
  • Lane 1 is the zearalenone standard
  • Lane 2 is the extract from the culture medium containing zearalenone in which the transfected cells have been cultured
  • Lane 3 is the extract from the culture medium containing zearalenone in which wild-type cells have been cultured
  • Lane 4 contains zearalenone.
  • the extract from the LS medium, lane 5 shows the result of the extract from the LS medium alone.
  • FIG. 14 is a graph showing changes in the amount of zearalenone in the medium during the culture period.
  • “ ⁇ ”, “ ⁇ ” and “X” indicate the amount of zearalenone in the culture medium of egfp :: zhdl01-introduced suspension culture cells
  • “Hata”, “mouth” and “ ⁇ ” indicate the wild-type suspension culture cells.
  • the amount of zearalenone in the culture medium, and the open triangle indicates the amount of zearalenone in the cell-free zearalenone-containing medium.
  • ⁇ and ⁇ indicate the amount of zearalenone in the medium
  • ⁇ and ⁇ indicate the amount of zearalenone contained in the cells
  • X and ⁇ ⁇ indicate the amount of residual zearalenone.
  • Example 1 Isolation of microorganisms having zearalenone resolution
  • Antibiotic isolates 209 and 31 microbial strains were collected by IFO (Institute for Fermentation, Osaka, Japan) and JAI (Japan Collection of Microorganisms, RIKEN). Obtained from. Each of these strains was randomly screened for zearalenone resolution.
  • YG medium (0.5% yeast extract, 2% glucose, pH 7.0) containing 100 ppm zearalenone (Sigma, St. Louis, MI) was inoculated with each strain and allowed to stand at room temperature for 1 week. Cultured. The culture was centrifuged at 6, OOOrpm for 5 minutes to separate the culture supernatant, and the culture supernatant was extracted with a black-mouthed form. This extract was concentrated, placed on a TLC plate, and developed in a developing tank using 80:20 chloroform: acetone as a solvent. This TLC plate was detected under a UV lamp (UV 254 nm).
  • FIG. 1 shows the TLC data for the IF07063 strain in this screening.
  • Lane 3 of FIG. 1 shows spots of zearalenone obtained by applying zearalenone (Sigma, St. Louis, MI) as a standard sample as a control.
  • Lane 2 shows, as a negative control, a spot of zearalenone, which was obtained by applying a buffer incubated with zearalenone (Sigma, St. Louis, MI).
  • zearalenone Sigma, St. Louis, MI
  • the lane 1 does not show spots of similar mobility to the spots of zearalenone shown in lanes 2 and 3, but instead shows spots of lower mobility than the spots. I have.
  • the Rf value of zearalenone shown in lanes 2 and 3 was approximately 0.8, whereas the Rf value of the material shown in lane 1 was approximately 0.2.
  • Lane 1 shows only the smaller mobility spots This means that the molecular structure of zearalenone was changed by the hydrolysis activity of the culture supernatant of the IF07063 strain corresponding to lane 1. Therefore, the culture supernatant of the IF07063 strain corresponding to lane 1 may contain zearalenone-degrading enzyme.
  • IF07063 strain was identified as a candidate strain having zearalenone resolution.
  • This IF07063 strain was obtained from IFO (Institute for Ferraentation, Osaka, Japan) and is classified as Clonos tachys rosea.
  • the IFO Biological Resources Database provides more detailed information on this bacterium, and based on that information, this strain can be used, for example, at 24 ° C for potato sucrose agar (PSA medium; potato 200 g, sucrose 20 g, Culture can be performed under the conditions of 1 L of distilled water, 20 g of agar, and pH 5.6).
  • Clonostachys rosea IF07063 strain was placed in 100 ml of YG medium (0.5% yeast extract, 2% g lucose, pH 7.0) containing 100 ppm of zearalenone (Sigma, St. Louis, Ml) at room temperature. For one week. Thereafter, the culture was subcultured in 1 L of the above YG medium containing 25 ppm of zearalenone, and cultured at room temperature for 1 week. Cells were collected by filtration, crushed with liquid nitrogen, and further sonicated. The cell debris was then spun down with 5, OOOXg, and ammonium sulfate was added to the supernatant at a saturation of 40-60%.
  • YG medium 0.5% yeast extract, 2% g lucose, pH 7.0
  • zearalenone Sigma, St. Louis, Ml
  • the precipitate obtained by centrifugation at 10,000 ⁇ g for 1 hour was permeated at 4 ° C. against 10 mM Tris-HCl (pH 7.5).
  • the dialyzed sample was then applied to a HiTrapQ column and eluted with 10 mM Tris-HCl (pH 7.5) showing a linear concentration gradient of NaCl (0-: IM, 5 ml / min, 20 min).
  • each of the eluted fractions obtained here was subjected to an in vitro zearalenone hydrolysis test, and then the hydrolysis activity on zearalenone was examined by TLC.
  • zearalenone hydrolysis test zearalenone 25 was added to each eluted fraction37. Incubation was carried out at ⁇ C (100 mM Tris-HCl (pH 9.5), total volume ⁇ ). Next Then, the reaction mixture was extracted with black form and a part of the extract was collected.
  • zearalenone As a negative control, a sample obtained by adding 25 ⁇ g of zearalenone (Sigma, St. Louis, Ml) to the buffer and incubating at 37 ° C. was applied. As a result, spots of zearalenone were observed. However, when the eluted fraction sample was used, no spot having the same mobility as the spot of zearalenone in the control and the negative control was observed, and instead, a spot having a lower mobility than zearalenone was found. Was observed. Therefore, the protein contained in the eluted fraction may be zearalenone degrading enzyme.
  • the individual fractions of the hornworm hornworm were further separated by FP1 (AKTA Explorer, Amersham Pharmacia Biotech, Buckingharashire, UK).
  • FPLC separation the elution fraction was applied to a gel filtration column (Superdex75 HR 10/30, Amersham Pharmacia Biotech), and eluted with 10 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl.
  • These eluted fractions obtained by this FPLC were also subjected to the above-mentioned TLC to further select a catalytic fraction. The same results as above were obtained in TLC using the eluted fraction obtained from this FPLC.
  • the selected catalytic fraction was applied to an ion exchange column (MonoQ, Amersham Pharmacia 'Biotech), followed by lOmM Tris-HCl ( P ) with a linear concentration gradient of NaCl (0-0.5M). According to H7.5), elution was performed at a flow rate of 0.4 ml / min for 50 minutes.
  • the purification process using this ion exchange column MonoQ was repeated twice.
  • the above T of the catalytic fraction LC selection was also performed after the first MonoQ purification step, and the selected eluted fractions were subjected to the second MonoQ purification step to obtain further purified proteins.
  • the purified protein obtained in the second MonoQ purification step was used as zearalenone-degrading enzyme in subsequent experiments. This protein was named ZHD101.
  • ZHD101 purified according to Example 2 was digested with lysyl endopeptidase (TAKARA, Kyoto, JAPAN) at 37 ° C. for 1 hour with addition of 2 M urea.
  • the reaction mixture was separated for each peptide fragment by HPLC using a reverse-phase column, VP304-1251 (Senshu Kagaku, Tokyo, JAPAN).
  • the HPLC was performed under the following conditions: a flow rate of 1 ml / min, a concentration gradient of B from 0% to 60% (A: 0.1% aqueous solution of trifluoroacetic acid, B: 0.08% trifluoroacetic acid). Acetonitrile solution).
  • the peptide was detected at 225 nm.
  • ZHD1 of the present invention Primers for PCR amplification of 01 were designed.
  • the 5 ′ primer corresponds to four types of 5 ′ primers based on the N-terminal amino acid sequence
  • the 3 ′ primer corresponds to peaks p-32.5 and P ⁇ 34.5 obtained by HPLC in Example 3.
  • Eight kinds of 3 'primers ( Figure 3) based on the amino acid sequence of each peptide were designed and chemically synthesized.
  • PCR amplification was performed under the following conditions using the above-prepared cDNA as type III using 32 different primer pairs combining these four 5 ′ primers and eight 3 ′ primers: 94 ° 30 cycles of C (30 seconds), 55 ° C (30 seconds) and 72 ° C (1 minute). The size of the amplified fragment of the obtained amplification product was confirmed by agarose gel electrophoresis.
  • the amplified fragment obtained by the combination of N 2 (5 ′ primer) and 34.5 ⁇ 2 (3 ′ primer) was the largest, about 800 bp.
  • the amplified fragment obtained by the combination of ⁇ 1-4 (5 ′ primer) and 32.5-:!-32.5-3 (3 ′ primer) was about 550 bp.
  • a DNA base sequence was determined for an approximately 800 bp fragment obtained by combining N 2 (5 ′ primer) and 34.5 ⁇ 2 (3 ′ primer).
  • the approximately 800 bp PCR amplification product was ligated into pGEM-TEasy Better (Promega, Madison, WI). Using this recombinant vector, E.
  • coli DH5a (T0Y0B0, Osaka, JAPAN) was transformed by the heat shock method.
  • a recombinant vector was purified from a culture obtained by appropriately culturing the transformant using a plasmid purification kit (M0 BIO, Solana Beach, CA).
  • Primer 1 5'-GGG CTT CCC ACG CAG AGC CTC CAG ATC CTT AAC—3, (for first PCR of 5'-RACE) (SEQ ID NO: I 5 )
  • Primer 2 5'-CTC CGA GCC TCC AGA CAC GTC GTT CAA CAT TAC-3 '(for nested PCR of 5, 1 RACE) (SEQ ID NO: 16)
  • Primer 3 5'-ACC GCT GTG CTC GAA GAC GAG GAA ATC TCA AAG— 3 '(3'—for first PCR of RACE) (SEQ ID NO: 17)
  • Primer 4 5'-GTA ATG TTG AAC GAC GTG TCT GGA GGC TCG GAG-3 '(for nested PCR of 3, 1 RACE) (SEQ ID NO: 18)
  • SEQ ID NO: 1 shows the entire nucleotide sequence of zhdlOl thus obtained.
  • the putative amino acid sequence encoded by SEQ ID NO: 1 is shown in SEQ ID NO: 2.
  • the amino acid sequence shown in SEQ ID NO: 2 completely matched the partial amino acid sequence of the fragmented ZHD101 peptide determined in Example 3.
  • the amino acid sequence shown in SEQ ID NO: 2 was composed of 264 amino acids, and its molecular weight was calculated to be 28,751 Da.
  • RNA prepared in (1) of Example 4 was used.
  • RT-PCR amplification was performed as type III.
  • RT-PCR includes Superscript First-Strand Synthesis
  • the plasmids were digested with Ndel and BamHI, and a zhdlOl fragment was obtained by separation by agarose gel electrophoresis.
  • the zhdlOl fragment was inserted into a pET12a vector that had been digested and purified with Ndel and BamHI in advance.
  • the ligation product was transformed into DH5 °; and the colonies were selected by ampicillin resistance to obtain transformants.
  • a recombinant vector purified from a culture obtained by appropriately culturing the transformant using a plasmid purification kit (MO BI0, Solana Beach, CA) was subjected to DNA sequencing, and the nucleotide sequence of zhdlOl was determined. confirmed.
  • DE3 T0Y0B0
  • pET12-zhdlOl plasmid incorporating zhdlOl whose correct nucleotide sequence was confirmed, and transformants were selected based on ampicillin resistance.
  • a recombinant expression vector was obtained from the transformant in the same manner as described above.
  • a single roller of DE3 (T0Y0B0) transformant containing the recombinant expression vector prepared in Example 5 was inoculated into a cycle Glo medium (Funakoshi) until the 0D fraction reached 0.6.
  • the cells were cultured at 37 ° C for several hours.
  • ImM IPTG was added and the cell culture was cultured at room temperature. The next day, cells were spun down and sonicated. Then, SDS-PAGE confirmed that the recombinant ZHD101 was expressed in DE3.
  • Fig. 4 shows the results.
  • FIG. 4 shows the results of SDS-PAGE obtained by applying the following sample to a 12.5% SDS-PAGE gel.
  • Lane 1 is a homogenate samples of the wild-type DE3
  • Lane 2 PETL 2 - is DE3 having ZhdlOl (Chi words, crude recombinant ZHD101) homogenate samples (transformant)
  • Lane 4 is a soluble fraction of the homogenate of the transformant
  • Lane 5 is ZHD101 directly extracted and purified from the IF07063 strain obtained in Example 2.
  • Fig. 4 in the transformant, a protein that was not expressed in the wild type was expressed (lanes 1 and 2), and the protein expressed in the transformant was also secreted into the culture supernatant. (Lane 4), and the protein expressed in the transformant. It has the same molecular weight as ZHD101 extracted and purified directly from IF07063 strain
  • the hydrolysis activity of the crude recombinant ZHD101 derived from the transformant was detected by TLC. Wild-type DE3 homogenate and pET12- the homogenate 10 mu 1 of DE3 having ZhdlOl (transformant), was added to Zeararenon respectively Eta 2 0 at and Fi pull up the respective 100 ⁇ 1 (lOOmM Tris- HCl ( pH 9.5)) and incubated at 37 ° C for 4 hours. The reaction mixture was extracted in the same manner as above and applied to TLC plates (silica gel 60 F 254 ). The results are shown in FIG.
  • FIG. 5 shows lane 1 in which the homogenate of wild-type DE3 was treated with zearalenone, lane 2 in which the homogenate of pET12-zhdlOl-containing DE3 (transformant) was treated with zearalenone, and lane 3 with zearalenone standard.
  • 3 shows the results of TLC applying the product.
  • TLC performs extraction with black port Holm from the reaction treatment liquid, a portion of the extract was collected, charged TLC plates (Merck, Shirikagenore 60 F 254), a black hole Holm as Solvent: acetone 80: 20 (Volume ratio) and deployed in the deployment tank. This TLC plate was detected under a UV lamp (UV 254 nm). The results in FIG.
  • ZHD101 had the same zearalenone-degrading activity as ZHD101 isolated directly from the IF07063 strain.
  • the optimum pH of ZHD101 was found to be biased toward the alkaline side. That is, in the above-mentioned analysis of the hydrolysis activity of ZHD101, the enzyme reaction condition after the addition of zearalenone was a highly alkaline pH of 9.5, but ZHD101 sufficiently exhibited the enzyme activity. Thus, the optimum activity of ZHD101 was pH 9 to 10.5, but the activity was as high as pH 7.0. ZHD101 was also found to be irreversibly inactivated at low pH below pH 4.5.
  • Example 7 Degradation of zearalenone derivative by zearalenone degrading enzyme
  • ⁇ which is a zearalenone derivative, ⁇ -se larenone ⁇ , / 3 -zeararenol; -Zearalenol or -Zearalenol), and ZHD101 (ammonium sulfate fraction derived from the IF07063 strain described above at 10 mM to 10 mM Tri s - HC1 (pH 7 ⁇ 5) those dialyzed in) to was added to 37 ° C De ⁇ incubated respectively (lOOraM Tri s- HCl (pH 9. 5), the total capacity ⁇ ⁇ ⁇ ). The reaction solution was extracted with a black hole form and subjected to TLC in the same manner as described above (FIG. 6).
  • Example 8 Isolation and characterization of degradation products generated from zearalenone by zearalenone degrading enzyme
  • the crude extract of zearalenone degradation product obtained by the above hydrolysis test using ZHD101 was subjected to TLC and developed using precoated silica gel 60 F 254 plate (0.25 mm thick, 20 x 20 cm, Merck). The liquid was developed using Clos form: acetone 80:20 (volume ratio). Spots on the TLC plate were detected under UV (254 nra).
  • Acetone-d 6 at 29.8 ppm and acetone-d 5 at 2.04 ppm were used as internal standards for 13 C and 1 H NMR, respectively. Chemical shifts were recorded as ⁇ values.
  • the multiplicity of the 13 C MiR signal was determined by DEPT.
  • 2D NMR spectra (PFG-DQFC0SY, PFG-HMQC and PFG-HMBC) were measured by JEOL ECP-500 using JEOL standard pulse sequence, and collected data were processed by JEOL standard software.
  • the FAB-MS spectrum was measured using a glycerol matrix on a JEOL JMSH X-110 mass spectrometer, and the EI-MS spectrum was measured using a JMS-SX102 mass spectrometer.
  • the estrogenic activity of the zearalenone degradation product obtained according to the above example was measured.
  • Estrogenic activity was measured as a cell growth promoting effect on human breast cancer cells MCF-7.
  • human breast cancer cells MCF-7 were converted to phenol red, L-glutamine (2 mM), ⁇ -cillin (50 units / ml), streptomycin (50 ⁇ g / ml) and 10% ⁇ fetal serum ( FCS) was added RPMI-1640 medium (SIGMA, St. Louis, at MO), and incubated at 37 ° C for a 5% C0 2 in including humidified air.
  • FCS RPMI-1640 medium
  • the FCS used in this experiment was treated with dextran-treated charcoal.
  • the cultured cells were inoculated with 5 ⁇ 10 3 cells per well in a 96-well plate in a phenol red-free RPMI medium containing 10% charcoal-degraded FCS.
  • the medium was replaced with the same kind of medium.
  • various concentrations of zearalenone, zearalenone degradation product, or 17-estradiol as a test compound were added, and the cells were further cultured for 120 hours. Then The number of cells was evaluated by the following color reaction.
  • the color-forming reactions include 2- (2-methoxy-4-diphenyl) -3- (4-diphenyl) -5- (2,4-disulfophenyl) -2H-tetrazo WST-8 TM (Nakalai Tesque, Kyoto), a sodium salt of lithium, was added to the culture, and the cultured cells were further cultured at 37 ° C for 4 hours.
  • the absorbance (A 45 ) of each well was determined by using a Wal lac 1420 Manoretirabeno counter (Amersham Biosciences).
  • a known estrogen, 17-estradiol 0.1 nM
  • the zearalenone (chemical formula: FIG. 7) according to the present invention has the same cell number as 17-estradiol, and thus has the same cell growth promoting activity as 17-estradiol, ie, an estrogenic activity.
  • the zearalenone degradation product of the present invention (chemical formula: FIG.
  • the zearalenone degradation product of the present invention does not show estrogenic activity. Therefore, the zearalenone-degrading enzyme ZHD101 of the present invention degrades zearalenone to a degradation product having no estrogen-like activity, thereby losing the estrogenic activity of zearalenone, that is, suppressing the toxicity of zearalenone. It was shown that you can.
  • the GFP-zhd-5 'primer (5'-ATG CG C ACT CGC AGC ACA ATC TCG-3' (SEQ ID NO: 21)
  • GFP-zhd-3 'primer 5′-T GT ACC GTT CAA AGA TGC TTC TGC-3 ′ (SEQ ID NO: 22)
  • a recombinant plasmid in which zhdl01 was incorporated into the pGEM-TEasy vector (Promega, Madison, Wis.) Prepared in Example 5 was type III.
  • a DNA fragment containing the entire zhdlO l gene was prepared by PCR amplification.
  • the PCR conditions used were as follows: 30 cycles of 30 seconds at 94 ° C, 1 minute at 57 ° C, and 1 minute at 72 ° C.
  • a synthetic linker (5′-GTA CAG GC C CGG GCC GC-3, (SEQ ID NO: 23), 5′-GGC CGC GGC CCG GGC GT) is inserted between BsrG I-Not I of pEGFP (Clontech). -3, (SEQ ID NO: 24)) was inserted to create pEGFP-SrfI.
  • the obtained PCR product was ligated to the SrfI site of the pEGFP-SrfI vector for integration into the downstream of the egfp gene, and a pEGFP-zhdlOl clone was prepared. Furthermore, the Nco I-Not I fragment (referred to as “egfp :: zhdl01” in this specification) excised from the pEGFP-zhdlOl clone was transferred to a pWheat vector (pDM302 (Genes. Genet. Syst., 72) containing the Act I promoter. , 63-69, 1997) was replaced with Tril01 (J. Biol.
  • Fig. 9 shows a schematic diagram of the vector construction process.
  • Example 11 Introduction of egfp :: zhdl01 gene into cereals using a particle gun
  • the recombinant vector pWheat-egfp :: zhdl01 obtained in Example 10 was used together with the selection marker, bialaphos resistance gene bar, as follows: A monocotyledonous plant was introduced into a callus derived from a ripe embryo of rice by the following procedure.
  • Rice mature seed embryo (also called mature embryo) is sterilized with 40% hypochlorous acid.
  • LS medium containing 70 ppm kanamycin, 70 ppm cefotaxime, and 2 ppm 2,4_D (2,4-dichlorophenoxyacetic acid) (Callus induction medium) (LS medium: LINSMAIER SK00G Medium 1, Invitrogen) was transplanted and cultured for 6 to 7 days to induce callus.
  • Canoleth was placed in an LS medium containing 2 ppm 2,4-D and 0.4 M mannitol, and pWheat-egfp :: zhdl01 and bar genes, which were coated on gold particles, respectively, were introduced using a particle gun.
  • the transgenic calli were transplanted to the LS medium (selection medium) containing 2 ppm 2, 4-D and 5 ppm bialaphos the next day, and then subcultured every two weeks to a fresh selection medium. Then, under a fluorescence microscope, calli which exhibited fluorescence by GFP (green fluorescent protein) and grew selectively were judged as transformants into which the egfp :: zhdl01 gene had been introduced (FIG. 10).
  • LS medium containing 1 mg / m 1 AA ( ⁇ -naphthalene acetate), 2 mg / ml BA (benzyladenine) and 30 g / 1 sorbitol (Regeneration medium).
  • lane 3 is regenerated body No 54
  • lane 4 is regenerated body No 68
  • lane 5 is regenerated body No 71
  • lane 6 is regenerated body No. 76
  • lane 7 is regenerated body No 79
  • lane 8 is regenerated body No 79
  • lane 9 is recombinant EGFP :: ZHD101.
  • regenerated cells No. 14, No. 54, No. 68, No. 76 and No. 79 proteins not found in the wild type were detected. This protein had the same molecular weight as the recombinant EGFP :: ZHD101. From these results, it was shown that the EGFP :: ZHD101 protein was expressed in five regenerants (No 14, No 54, No 68, No 76 and No 79).
  • Example 13 Degradation of zearalenone by suspension culture cells into which egfp :: zhdl01 was introduced Zaralenone degradation test was carried out using suspension culture cells of egfp :: zhdl01 into rice according to the following procedure.
  • egfp:: zhdl01 introduced suspension culture cells, e g fp prepared in Example 1 1:: zhdl01 callus is introduced, 2 ppm 2,4 D - (2, 4-dichloro-phenoxyethanol acetate) It was prepared by transplantation into the LS liquid medium containing LS. Similarly, wild-type suspension-cultured rice cells were similarly prepared by transplanting wild-type calli into LS liquid medium containing 2 ppm 2,4-D (2,4-dichlorophenoxyacetic acid). .
  • Lane 13 shows the results of this TLC analysis.
  • Lane 1 is a zearalenone standard
  • lane 2 is an extract from a zearalenone-containing LS medium cultured with egfp :: zhdl01 transfected cells
  • lane 3 is an extract from a zearalenone-containing LS medium cultured wild-type cells
  • lane 4 is zearalenone An extract from the LS medium containing LS medium
  • lane 5 shows an extract from the LS medium without zearalenone.
  • the extract from the culture medium in which the egfp :: zhdl01-transfected cells were cultured spots showing zearalenone disappeared, suggesting that zearalenone in the culture medium was degraded.
  • zearalenone quantification of zearalenone was performed 6 days after the start of the culture.
  • the remaining amount of zearalenone is considered to be the total amount of the amount present in the medium, the amount adhering to the cell surface, and the amount taken up into the cells. Quantification was performed for both zearalenone and the amount.
  • zearalenone was quantified in extracts from the medium three days after the start of the culture. Further, as a control, zearalenone was quantified in an extract from a cell-free LS medium containing zearalenone 6 days after the start of culture.
  • RIDASCREEN FAST Zearalenon R-Biophar
  • Table 1 shows the amount of zearalenone quantified 6 days after the start of the culture.
  • the amount of residual zearalenone in Table 1 was calculated as the total value of the amount of zearalenone in the medium and the amount of zearalenone contained in the cells.
  • the amounts of residual zearalenone in the culture medium of egfp :: zhdl01-transfected cells and wild-type cells were 6.41 / g and 260.85 ⁇ , respectively.
  • the amount of residual zearalenone in the culture medium of egfp :: zhdl01-introduced cells was about 1/117 of the amount of zearalenone at the start of the culture of 750 / ⁇ g, and was about 1/117 of the amount of residual zearalenone in the culture medium of wild-type cells. / 40.
  • the amount of residual zearalenone in the zearalenone-containing LS medium also decreased to 52.5 / zg, which was the result of the precipitation of zearalenone after culturing for 6 days.
  • the culture medium of egfp :: zhdl01-introduced cells and wild-type cells No such precipitation phenomenon was observed.
  • FIG. 14 shows the change in the amount of zearalenone in the medium based on the above results.
  • the amount of zearalenone in the culture medium of egfp :: zhdl01-transfected cells was 750 tg at the start of culture, 29.5 / _ig 3 days after the start of culture, and 0.76 ⁇ g 6 days after the start of culture. And decreased over time. Therefore, it was confirmed that zearalenone in the medium was degraded during the culture period.
  • the protein of the present invention can advantageously suppress the toxicity of zearalenone.
  • the gene of the present invention can be used for expressing the protein.
  • the transformants and transgenic plants into which the gene of the present invention has been introduced can be advantageously used for the purpose of efficiently degrading and detoxifying zearalenone contained in the surrounding environment. Sequence listing free text
  • SEQ ID Nos: 3 to 24 are synthetic DNAs.
  • N in SEQ ID NOs: 3 to 14 is a, t, c or g.

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Abstract

L'invention concerne un gène d'enzyme détoxifiant de la zéaralenone ; une enzyme de détoxification à la zéaralenone ; un vecteur recombinant contenant ledit gène ; une plante transgénique contenant ledit gène ; un transformant contenant le vecteur recombinant ; un procédé de production d'une protéine ainsi que l'utilisation du transformant ; un agent de détoxification à la zéaralenone ; et enfin un procédé de détoxification à la zéaralenone.
PCT/JP2003/003602 2002-03-25 2003-03-25 Gene d'enzyme detoxifiant de la zearalenone et transformant dans lequel le gene est transfere WO2003080842A1 (fr)

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WO2012113827A1 (fr) 2011-02-24 2012-08-30 Eucodis Bioscience Gmbh Enzymes de traitement d'aliments pour animaux
CN107099521A (zh) * 2017-05-09 2017-08-29 中国农业科学院农产品加工研究所 一种耐酸性玉米赤霉烯酮解毒酶及其编码基因与应用
CN109825484A (zh) * 2017-11-23 2019-05-31 吉林中粮生化有限公司 玉米赤霉烯酮水解酶zhd101突变体及利用该突变体水解玉米赤霉烯酮的方法

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KR101280811B1 (ko) 2010-11-15 2013-07-02 (주)진바이오텍 제아라레논 분해 활성을 갖는 미생물, 이를 이용한 제아라레논 분해 방법 및 사료첨가제
AT514775B1 (de) * 2013-08-28 2017-11-15 Erber Ag Polypeptid zur hydrolytischen Spaltung von Zearalenon und/oder Zearalenon Derivaten, isoliertes Polynukleotid davon sowie Zusatzstoff enthaltend das Polypeptid
KR101494624B1 (ko) * 2013-11-07 2015-02-24 동아대학교 산학협력단 제아라레논 분해 활성을 가지는 아그로마이세스 속 m15 균주 및 이의 용도
KR102108744B1 (ko) * 2017-10-20 2020-05-08 대한민국 제아라레논 분해 활성을 가지는 클로노스타치스 로세 균주 및 이의 용도
CN110029095B (zh) * 2019-04-15 2022-06-07 南京工业大学 一种玉米赤霉烯酮降解酶及其应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012113827A1 (fr) 2011-02-24 2012-08-30 Eucodis Bioscience Gmbh Enzymes de traitement d'aliments pour animaux
CN107099521A (zh) * 2017-05-09 2017-08-29 中国农业科学院农产品加工研究所 一种耐酸性玉米赤霉烯酮解毒酶及其编码基因与应用
CN107099521B (zh) * 2017-05-09 2020-08-25 中国农业科学院农产品加工研究所 一种耐酸性玉米赤霉烯酮解毒酶及其编码基因与应用
CN109825484A (zh) * 2017-11-23 2019-05-31 吉林中粮生化有限公司 玉米赤霉烯酮水解酶zhd101突变体及利用该突变体水解玉米赤霉烯酮的方法
CN109825484B (zh) * 2017-11-23 2022-06-28 吉林中粮生化有限公司 玉米赤霉烯酮水解酶zhd101突变体及利用该突变体水解玉米赤霉烯酮的方法

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