WO2003080842A1 - Zearalenone-detoxifying enzyme gene and transformant having the gene transferred thereinto - Google Patents

Abstract

A zearalenone-detoxifying enzyme gene; a zearalenone-detoxifying enzyme; a recombinant vector containing the gene; a transgenic plant containing the gene; a transformant containing the recombinant vector; a process for producing a protein with the use of the transformant; a zearalenone-detoxifying agent; and a method of detoxifying zearalenones.

Description

 Zearalenone detoxification enzyme gene and transformant into which the gene has been introduced

 The present invention relates to a protein capable of purifying mycotoxin-infected plants and a gene encoding the same.

 Background technology

 The stable supply of food and environmental protection have become important research issues for plant science research in the coming days. On the other hand, in recent years, herbicide resistance and pest

Various types of recombinant crops that have been imparted with useful traits such as resistance have been produced.

 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. In particular, in the 1990s, wheat scabs, which were widespread around the world, were incurable diseases that could not be devastated 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). ky¾, 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. Thus, red blight is a double threat to food supply. Although 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). In order to put the varieties resistant to red blight in commercial varieties, first find suitable wild varieties that exhibit resistance, and repeat the backcrossing to introduce only the favorable traits of resistance into the commercial variety, Breeding is carried out by selecting progeny that are resistant and have high yield and quality. However, this classical method took a lot of time and also had problems such as the emergence of bacteria that break the resistance (“Plant disease resistance from the molecular level”, supervision: Tetsuharu Yamada Shimamoto Isao Yuichiro Watanabe, Shujunsha, Japan, (1997), p. 90-97).

It is also known that mycotoxin trichothecene toxin accumulates in grains due to infection with E. coli. Trichothecene toxins are also protein synthesis inhibitors and enhance infectivity as a virulence factor when bacteria are infected. As a method for preventing the toxicity of the trichothecene toxin, a gene that blocks the protein synthesis inhibitory activity of the trichothecene toxin (Japanese Patent Application Laid-Open No. 2000-32985) and the trichothecene toxin are extracellularly excreted. Pumping genes (Alexander, J, "Molecular & general genetics" (1999) 261, p. 977-984) have been reported. However, a gene that can inactivate the trichothecene toxin itself has not yet been found.Similarly, infection with E. coli can accumulate zearalenone in the grains of cereals such as corn and wheat. (Pittet, A., "Revue Medicine Veterinaire" Ecole Nat ionale Veterinaire de Toulouse, Hufuns, 998 998) 149, p. 479-492). Zearalenone [6- (10-hydroxy-6-oxo-trans-1-onedecenyl)-/ 3-ratatatone resorcylate] is a mycotoxin with estrogenic activity produced by Fusarium spp. It is. When a Fusarium bacterium infects a plant, zearalenone is produced and accumulated in the grains of cereal crops before and after harvest (Pittet, A., "Revue Medicine Veterinaire" Ecole Nationale Veterinaire de Toulouse , France, (1998) 149, p. 479-492 ). 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). For sialalenone and its metabolites, binding to human estrogen receptor (Miks icek, RJ, The Journal oi steroid biochemistry and molecular biology "(1994) 49, p. 13-160) and in vitro (Makela, S., et al., "Environmental Health Perspectives" (1994) 102, p. 572-578) have been reported to promote the growth of the human breast cancer cell line MCF-7.

 It is very important to solve the problem of zearalenone contamination of cereals in protecting plants from red blight. However, no effective method has yet been reported for effective detoxification of zearalenone, either in chemical or enzymatic methods. Disclosure of the invention

 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.

 That is, the present invention is as follows.

 [1] The following protein (a) or (b):

 (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2

 (b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having an action of suppressing toxicity of zearalenones

Here, the protein in [1] may be characterized in that zearalenones are compounds having estrogenic activity. Examples of zearalenones include zearalenone, zearalenol, j8-zearalenol, h-zearalanore, β-zearalanol, 2,4-0-dimethyl-δ-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.

 [2] A gene encoding the following protein (a) or (b):

 (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2

 (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having an action of suppressing toxicity of zearalenones

 [3] A gene comprising the following DNA (a) or (b):

 (a) DNA comprising the nucleotide sequence of SEQ ID NO: 1

 (b) hybridizing under stringent conditions with DNA consisting of a nucleotide sequence complementary to all or part of the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, and

DNA encoding a protein having an action of suppressing the toxicity of zearalenones [4] A gene comprising the following DNA (a) or (b):

 (a) DNA encoding the amino acid sequence shown in SEQ ID NO: 2

 (b) hybridizing under stringent conditions with DNA consisting of a nucleotide sequence complementary to all or part of the DNA encoding the amino acid sequence shown in SEQ ID NO: 2, and inhibiting the toxicity of zearalenones DNA encoding protein

Here, the genes in [2:] to [4] may be characterized in that zearalenones are compounds having estrogenic activity. Zearalenones include zearalenone, hy-zearalenone, β-zearalenol, α-zearalanone, J3-zearalanol, 2,4-0-dimethyl / le-δ-hydroxyzearalenone, 6-amino- It may be at least one selected from the group consisting of zearalenone, zearalanone and 6-acetyl-j3-zealarenol. Further, 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.

[5] A recombinant vector containing the gene of [ ² ] to [4].

 [6] A transformant comprising the recombinant vector of [5].

 Here, 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.

 [7] 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.

[8] An antidote containing the transformant of [ ⁶ ].

[9] An antidote containing the protein of [1].

[10] A method for detoxifying zearalenones, which comprises applying the antidote of [8] or [9] to zearalenones.

 [11] [2 :! A transgenic plant comprising the gene according to [4]. Hereinafter, the present invention will be described in detail.

 The resistance of wheat (Triticum aestivum L.) to Fusarium head blight is roughly classified into the following four types: ~ IV:

 [1] Type I: Resistance to fungal invasion (depending on flowering characteristics, shape of ears, etc.) [2] Type II: Resistance to fungal growth after invasion (hyphal elongation)

 [3] Type III: Resistance exerted in the grain (reduction of mold damage to seeds)

 [4] Type IV: resistance based on reduced mycotoxin accumulation.

 These resistances are thought to be governed by many different genes. Therefore, it was thought that if the function of the gene related to Fusarium head blight resistance could be artificially regulated, plants could be given resistance to Fusarium head blight.

In particular, 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. However, 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. The term zearalenone analog means a compound having a zearalenone skeleton having a lactone ring composed of 14 carbon atoms, such as α-zearalenone, / 3-zealalenone, α-zearalanone, / 3-zearalanone, and 2 , 4 -◦-Dimethinole-δ-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. Hereinafter, the above zearalenones will be referred to as “zearalenone” for convenience.

In the present invention, “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. Toxicity to mammals, including livestock, such as toxicity based on endocrine disrupting activities, especially estrogen-like activity, more specifically, for example, enlargement of the female genital tract, enlargement of the mammary gland, abortion, It refers to inhibition of embryonic development, decreased number of fetuses, reproductive disorders, cavity prolapse, rectal prolapse, teratogenicity, carcinogenicity, etc. In the present invention, "inhibiting the toxicity of zearalenone (s)" means that the degree of the toxicity indicated by the action of zearalenone is reduced, preferably that the toxicity becomes undetectable or lost. means. In the present invention, “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. . In particular, the action of suppressing the toxicity of zearalenone may be one that produces a compound having no estrogenic activity using zearalenone as a substrate. In the present invention, 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.

1. Isolation of zearalenone detoxifying enzyme

In the present invention, a wide variety of 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 (Faculty of Agriculture, Kyoto University, Kyoto, Japan), ATCC (American Type Culture Collection Rockville, US A), HUT (Faculty 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) and the like. In addition, 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. In order to screen for the ability to detoxify zearalenone, for example, but not limited to, the following methods can be used. Transfer the obtained cells to a medium (for example, YG medium for fungi) containing 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) analysis. In the TLC analysis, 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). ) Can be selected as a candidate.

 Subsequently, 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.

 First, 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. Next, 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.

 For example, in an in vitro zearalenone degradation test, add zearalenone to each eluted fraction and incubate at 37 ° C. Next, zearalenone and Z or a degradation product thereof are extracted from each of the samples, and subjected to TLC. In the fraction containing zearalenone-degrading enzyme, spots with a mobility clearly different from that of zearalenone are detected on TLC. When zearalenone detoxifying enzyme is an enzyme that changes the molecular structure of zearalenone in a manner different from degradation, similarly, 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).

) 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. .

2. Isolation of zearalenone detoxification enzyme gene

 In order to isolate the gene encoding zearalenone detoxifying enzyme obtained in 1 above, first, for example, the partial amino acid sequence of zearalenone detoxifying enzyme is determined in the present invention. For amino acid sequencing, 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. Such an HPLC operation may be usually performed according to the manufacturer's instructions. Next, 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.

 Next, 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.

 Furthermore, 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. For example, ABI PRISM (R) 377 DNA sequencer and ABI kit (Applied Biosystems, Foster City, CA, USA) are used according to the protocol provided by the manufacturer. You may.

 Furthermore, in order to determine the base sequence of the 5′-end and 3′-end DNA regions of the partial DNA fragment of the zearalenone detoxifying enzyme gene, RACE (rapid amplification at ion of cDNA ends) was used. Can be implemented. The DNA fragment amplified by RACE is cloned in the same manner as described above, and used for DNA sequencing.

 Thus, the base sequence of the entire coding region of the zearalenone detoxifying enzyme gene of the present invention can be determined.

3. The zearalenone detoxifying enzyme gene of the present invention

In one embodiment of the present invention, 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. In this embodiment, 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. Furthermore, when 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. Furthermore, it was confirmed that 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.

 Therefore, in one embodiment of the present invention, 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 铸 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. Furthermore, 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.

In addition, a DNA that can hybridize under stringent conditions with DNA consisting of a sequence complementary to the base sequence shown in SEQ ID NO: 1 or a part thereof, and has an action of suppressing the toxicity of zearalenone. A gene encoding a protein is also included in the gene of the present invention. Similarly, 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. For example, 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, Lower homology Conditions under which the nucleic acids do not hybridize to each other. More specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM, and 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%. Furthermore, 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.

 Once the nucleotide sequence of the gene of the present invention has been determined, it 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.

 Furthermore, by a site-directed mutagenesis method or the like, 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. In addition, 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. Encoding genes can also be synthesized. Furthermore, it is a 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.

In order to introduce a mutation into a gene, a known method such as the Kunkel method and the Gapped duplex method or a method similar thereto can be employed. For example, using a mutagenesis kit using site-directed mutagenesis (for example, Mutan-K TAKARA) or Mutan-G (TAKARA), or using 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.

 Examples of the plasmid DNA 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.). In addition, retroviruses or animals such as Vincenius innoles, innores, and insects such as noculois / les, innores vectors can also be used.

 To insert the zearalenone detoxifying enzyme gene into a vector, first, 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. In particular, 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. For this purpose, 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. When a transformant is produced by using a recombinant expression vector and the recombinant protein is produced by culturing the transformant, 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. Furthermore, 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. . Examples of 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.

 Further, the gene of the present invention can also be directly introduced into the genome of a host organism by a homologous recombination method. In that case, an appropriate targeting vector incorporating the gene of the present invention is prepared. As a vector that can be used for this purpose, a known gene targeting vector such as Cre--οχΡ can be used. In the present specification, 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. Although not limited thereto, 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 丄 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.) _N Guicon (Raphanus 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.)

 Hinoregao geese: Samaimo (Ipomoea batatas)

 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.)

 / Kura Hakuto: Nokura (Rose hybrida Hort.), Strawberry (Fragaria x ananassa Duch.) Rutaceae: Citrus unshiu, Sansho (Zanthoxylura piperitum DC.) Eucalyptus globulus Labill)

 Willow Doo: Poplar (Populas nigra L. var. Ital ica Koehne)

 Families: Spinach (Spinacia oleracea L.), Sugar beet (Beta vulgaris)

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. For example, when the agrobacterium method is 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.

 When the particle gun method is used, 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.

 When a plant culture cell is used as a host, for the transformation, 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.) 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. For example, prepare DNA from transgenic plants, design DNA-specific primers, and perform PCR. PCR can be performed under the same conditions as those used to amplify the cDNA fragment inserted into the recombinant vector. After that, 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. In addition, 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.

6. Preparation of transformant and production of zearalenone detoxifying enzyme using the transformant In the present invention, a transformant (transformed cell) into which the zearalenone detoxifying enzyme gene of the present invention is introduced is prepared and cultured. Thus, 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.

 For transformation, 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. In the present invention, it is particularly preferable to use 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. In the production of zearalenone detoxifying enzyme, E. coli or yeast cells are preferably used. In food production using yeast cell fermentation, yeast cells are preferably used.

For the transformation, generally used techniques, for example, a calcium phosphate method, an electroporation method, a lipofection method, a particle gun method, a PEG method and the like can be applied. 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. For example, 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. Either a natural medium or a synthetic medium can be used as long as the medium can be efficiently used. If necessary, antibiotics such as ampicillin or tetracycline may be added to the medium.

 When culturing a microorganism transformed with an expression vector using an inducible promoter, an inducer may be added to the medium as necessary. For example, when culturing 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. When culturing is used, indoleacetic acid (IAA) or the like may be added to the medium.

 The culture conditions are not particularly limited, but are preferably performed under conditions suitable for the host organism used for transformation.

 After culturing, if the zearalenone detoxifying enzyme is produced in the cells or cells, the cells or cells are disrupted. On the other hand, when the target protein is produced outside the cells or cells, 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.

 In the present invention, 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. As a cell-free 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.

Further, a protein having an amino acid sequence represented by SEQ ID NO: 2 (ZHD101; see Example), which is one embodiment of the 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.

7. Detoxification of zearalenone by zearalenone detoxifying enzyme

 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. In order to suppress the toxicity of zearalenone, 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. Alternatively, the present invention may be applied to a material (plant, grain, fruit, soil, natural or artificial base, etc.) to which zearalenone is attached. When using transgenic plants containing the zearalenone detoxification enzyme gene, cultivation in an area where there is a risk of Fusarium head blight infection may reduce zearalenone contamination, or soil contaminated with zearalenone Alternatively, cultivation in environmental water (groundwater, sewage, rainwater, river water or cultivation water, etc.) may suppress the toxicity of zearalenone contained in the surrounding environment.

In order to confirm the activity of the zearalenone detoxifying enzyme of the present invention, first, 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. When 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. In order to confirm the zearalenone detoxification activity of the transformant having the zearalenone detoxification enzyme gene of the present invention, first, the transformant is cultured in the presence of zearalenone, and an extract is obtained from the culture. When 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. These analytical methods are known and can be performed according to the manufacturer's instructions of the NMR spectrometer and the mass spectrometer. A general textbook on NMR analysis and mass spectrometry is, for example, “Explanation and Exercise of UV, IR, IR, and MS for Spectrum Analysis for Organic Chemistry” (M. Hesse, Tokyo Chemical Doujinshi) And the like.

 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. When it is confirmed that the zearalenone-derived detoxification product does not have estrogen-like activity, it is determined that the zearalenone-derived detoxification enzyme has an action of suppressing the toxicity of zearalenone. Good. Various techniques available to those skilled in the art can be used to measure the estrogen-like activity. For example, 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. After incubating this at 37 ° C for 20 hours, 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. Next, the number of cells is evaluated by a color reaction. For the 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 ™ (Na kalai Tesque, Kyoto) can be used. Finally, the absorbance of each Ueru (A _450), measured by ί column e iiWal lac 1420 Manorechira base Honoré counter (Araersham Biosci e nces). In the present invention, 17-estradiol (0.1 ηΜ), which is a known ester, can be used as a control without limitation.

It has been found that 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. When zearalenone is added, the cell number is comparable to that of 17] 3-estradiol. In the present invention, the ratio of the number of cells (Β) when the test compound is added to the number of cells (Α) 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. And, in the present invention, 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. That is, by confirming that the zearalenone-derived detoxification product does not have an estrogen-like activity, it is possible to confirm that the zearalenone detoxification enzyme of the present invention is a protein having an action of suppressing the toxicity of zearalenone. it can. However, 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. BRIEF DESCRIPTION OF THE FIGURES

 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, and lane 4 is a transformant Lane 5 is the ZHD101 directly extracted and purified from IF07063 strain, and 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, and lane 3 is a standard zearalenone.

FIG. 6 is a photograph showing TLC data in which the hydrolysis activity of ZHD101 for α-zararail and -zararail was detected. Lane 1, ZHD101 treated α-zararail sample; lane 2, negative control for _α -zararail; lane 3, α-zararail standard, lane 4, ZHD101 treated _ zararail sample; lane 5, negative for β-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 (擊) 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, and 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, and 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. In the graph, “♦”, “◊” and “X” indicate the amount of zearalenone in the culture medium of egfp :: zhdl01-introduced suspension culture cells, and “Hata”, “mouth” and “酾” 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 ぴ indicate the amount of zearalenone in the medium, ◊ and ぴ indicate the amount of zearalenone contained in the cells, and X and ぴ 騸 indicate the amount of residual zearalenone. This description includes part or all of the contents as disclosed in the description of Japanese Patent Application No. 2002-84139 and Japanese Patent Application No. 2003-248760, which are the basis of the priority of the present application. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

 [Example 1] Isolation of microorganisms having zearalenone resolution

 (1) strains to be screened

 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.

 (2) Screening of microbial strains for zearalenone resolution

100 ml of 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). As a result, spots that could be considered as degradation products of zearalenone were detected in IF07063 strain. Figure 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). In lane 1, when a sample extracted from the culture supernatant of the IF07063 strain was applied, spots that were considered to be degradation products of zearalenone were detected. 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.

 Based on such TLC results, 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).

[Example 2] Extraction and purification of zearalenone-degrading enzyme from IF07063 strain

 (1) Extraction and purification of zearalenone degrading enzyme

 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%. 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. In the zearalenone hydrolysis test, zearalenone 25 was added to each eluted fraction37. Incubation was carried out at 晚 C (100 mM Tris-HCl (pH 9.5), total volume ΙΟΟμΙ). Next Then, the reaction mixture was extracted with black form and a part of the extract was collected.

Was charged into a TLC plate (Merck, silica gel 60 F ₂₅₄ ), and developed in a developing tank using 80:20 (volume ratio) of form: acetone as a solvent. This TLC plate was detected under a UV lamp (UV 254 nm). As a result, it was possible to select an eluted fraction in which spots that could be considered as degradation products of zearalenone could be detected. The detection results for spots that appeared to be degradation products of zearalenone were the same as in Figure 1. As a control, zalalenone (Sigma, St. Louis, Ml) was applied as a standard sample, and spots of zearalenone were observed. As a negative control, a sample obtained by adding 25 μg 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. In addition, when a sample using an elution fraction different from the lyophilic fraction was applied, only spots having the same mobility as zearalenone were shown. The eluted fraction (catalytic fraction) that could contain the zearalenone degrading enzyme thus identified was subjected to the next FPLC separation for further purification of the enzyme.

 The individual fractions of the hornworm hornworm were further separated by FP1 (AKTA Explorer, Amersham Pharmacia Biotech, Buckingharashire, UK). In the 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.

In addition, 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.

[Example 3] Partial amino acid sequencing of zearalenone degrading enzyme

 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.

 As a result of HPLC, seven main peaks (p-12, p-18, p-24.5, p-32.5, p-34.5, p-36.5, p-39) appeared (Fig. 2). Therefore, each of these seven main peaks was eluted at 12, 18, 24.5, 32.5, 34.5, 36.5, and 39 minutes and collected manually. For these peptides and whole protein, auto-tank according to the manufacturer's instructions. Amino acid sequencing by Edman degradation was performed using a quality sequencer, Procise HT 492 (Applied Biosystems, Inc., Foster City, CA).

[Example 4] Isolation of zearalenone degrading enzyme gene

 Clonostachys rosea IF07063 strain in 100 ml of YG medium (0.5% yeast extract, 2% g lucose, pH 7.0) containing 100 ppm of zearalenone (Sigma, St. Louis, MI) And cultured for 1 week at room temperature. From this culture, total RNA was extracted and purified using the RNeasy Mini kit (Qiagen GmbH, Hi 1 den Germany). Using this total RNA as type II, cDNA was synthesized by reverse premature PCR (RT-PCR) using a Superscript First-Strand Synthesis System for RT-PCR (Invitrogen, Groningen, The Nethrlands).

Next, based on the partial amino acid sequence of ZHD101 determined in Example 3, 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, and 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.

 As a result, the amplified fragment obtained by the combination of N 2 (5 ′ primer) and 34.5−2 (3 ′ primer) was the largest, about 800 bp. On the other hand, the amplified fragment obtained by the combination of Ν 1-4 (5 ′ primer) and 32.5-:!-32.5-3 (3 ′ primer) was about 550 bp. In this example, 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). First, 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).

For DNA sequencing, use the ABI PRISM (R) 377 DNA sequencer incorporating the analysis software Sequencing analysis v. 3. 4. (Applied Biosystems) and the ABI kit (Applied Biosystems, Foster City, CA, USA). In order to determine the nucleotide sequence of the 5'-end and 3'-end DNA regions of zhdlOl, which was performed by a conventional method using the above recombinant vector as a sample according to the protocol provided by the manufacturer, SMART RACE cDNA amplification was performed. RACE (rapid amplification of cDNA ends) was performed using a kit (Clontech, Palo Alto, CA). All procedures were performed according to the protocol provided by the manufacturer. In this example, the following primers were designed and chemically synthesized for use in the 5′-RACE PCR reaction and the 3′-RACE PCR reaction. 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)

 The DNA base sequence of the amplified DNA fragment was determined in the same manner as described above. 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.

Furthermore, a homology search was performed on a protein database (Swiss-Prot and GenBank) using the BLASTP program on the NCBI Internet site. This search revealed that ZHD101 of the present invention was a novel protein. The present inventors also performed a search for possible catalytic sites against a conserved domain database (RPS-BLAST). As a result, ZHD101 had a high homology with the α / hydrolase fold at an E value of 4 × e- ¹¹ (Ollis, et al. (1992) Protein Eng. 5, 197-221).

[Example 5] Preparation of recombinant vector and preparation of transformant

 In order to obtain a DNA fragment containing the entire coding region of zhdlOl, 5 'primer (5'-GCC CAT ATG CGC ACT CGC AGC ACA ATC-3' (SEQ ID NO: 19); Ndel site is underlined) and Using the 3 'primer (5'-TCG GAT CCG AGC TAT CGT GAG CAG TG-3' (SEQ ID NO: 20); BamHI site is underlined), the total RNA prepared in (1) of Example 4 was used. RT-PCR amplification was performed as type III. RT-PCR includes Superscript First-Strand Synthesis

(Invitrogen, Groningen, The Nethrlands) according to the manufacturer's instructions. Was used to synthesize cDNA. This amplification product was ligated into the pGEM-TEasy vector.

 Subsequently, the plasmids were digested with Ndel and BamHI, and a zhdlOl fragment was obtained by separation by agarose gel electrophoresis. Next, 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) was transformed using a plasmid (pET12-zhdlOl) 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.

[Example 6] Production of zearalenone-degrading enzyme using transformant

 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. To induce the recombinant protein, 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 3 transformants Lane 4 is a soluble fraction of the homogenate of the transformant, and Lane 5 is ZHD101 directly extracted and purified from the IF07063 strain obtained in Example 2. As shown in 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

(Lane 5) showed that the transformant expressed and secreted the same protein as ZHD101. The molecular weight of ZHD101 was about 30 kD.

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 μ 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.

Figure 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. 5 were similar to the results in FIG. Therefore, it was confirmed that the recombinant 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. This irreversible deactivation was confirmed by the following experiment. First, a ZHD101-containing ammonium sulfate fraction to which 1M phosphate buffer (1M) was added in an amount of 1Z10 was adjusted to pH 4.5 and pH 5.5, and they were placed on ice for 30 minutes. These samples were each dialyzed against a chromatographic Champa one inside in rapidly ^{10mM Tris-HCl (pH 7.} 5), subsequently subjected to in vitro hydrolysis test for Zeararenon (pH 9. 5) Analysis by TLC showed that the sample adjusted to pH 5.5 showed zearalenone decomposition activity, but the sample adjusted to pH 4.5 did not show zearalenone decomposition activity. In other words, ZHD101 placed at pH 4.5 does not show its enzymatic activity even if it is subsequently reacted at an optimum pH, so it is irreversibly inactivated under pH 4.5. It turned out to be.

[Example 7] Degradation of zearalenone derivative by zearalenone degrading enzyme

Β, which is a zearalenone derivative, β-se larenone α, / 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晚incubated respectively (lOOraM Tri s- HCl (pH 9. 5), the total capacity ΙΟΟ μ Ι). The reaction solution was extracted with a black hole form and subjected to TLC in the same manner as described above (FIG. 6). As a control, a standard of Q; zearalenol (lanes 3 and 6 in FIG. 6) and a negative control in which α, j3-zearalenol was added to a buffer and the ink was added (lanes 2 and 5 in FIG. 6) were used. . As a result, spots of α,] 3-Zearalenol found in the control and the negative control were completely lost under UV (254 nm) by ZHD101 treatment, and no degradation products could be detected (Fig. 6 lanes 1 and 4). From this result, it can be inferred that the decomposed product was not detected on the TLC plate under UV, or that it was too soluble in water to be extracted with black form. The complete disappearance of the substrate suggests that ZHD101 probably caused the degradation of the zearalenone derivative α, zearalenol.

[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).

The degradation products purified in this way were subjected to 丽 R analysis. Marauder R Skull, JE By 0L ECP- 500 spectrometer, Aseton - in d ₆ the ¹³ C NMR at 125 MHz, NMR was measured at 50 0 MHz.

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 δ 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 following are the measurement results of the FAB-MS spectrum, EI-MS spectrum and NMR spectrum.

FAB-MS (m / z: positive): 293 (M + H) ⁺

^{EI-MS .: 292 (M +} , 8%), 274 (M + - H 2 0, 24%), 162 (100%), 161 (86%), 112 (30%) -. Negation R (500 MHz, acetone _{- d 5, δ ppm);} 8. 12 (2H, br s, 2 - OH and 4- OH), 6. 38 (2H , d, 2. 0 Hz, H - 1及Pi H - 5), 6.25 (1H, d, 16.5 Hz, H—), 6.13 (1H, ddd, 6.8, 7.1, 16.5 Hz, H-2,), 6. 03 (1H, d, 2.0 Hz, H-3), 3.69 (1H, m, H-10,), 3.41 (1H, br d, 3.8 Hz, 10'-OH), 2.48 (2H, t, 7.2 Hz, H-5), 2.43 (2H, t, 7.1 Hz, H-7 '), 2.17 (2H, ra, H-3') ), 1.70 (2H, m, H-4,), 1.58 (2H, m, H-8 '), 1.37 (2H, m, H-9,), 1.10 (3H, d, 6.2 Hz, H-11,)

1 ³ C- NMR (125 MHz, acetone _{- d 6, δ ppm);} 210. 47 (s, C- 6,), 159. 49 (s, C- 2 and C- 4), 140. 73 ( s, C-1 6), 131.53 (d, C-), 130.56 (d, C-2,), 105.49 (d, C-1 and C-5), 102.42 (d , C-3), 67.34 (d, C-10), 43.11 (t, C-7,), 42.15 (t, C-5 '), 39.65 (t, C- 9,), 33.00 (t, C—3 ′), 24.09 (t, C—1, 4), 24.03 (q, C—11 ′), 20.93 (t, C—8 ′) ).

Additional mass spectrometry by (FAB- MS及Pi EI- MS) and NMR spectrum were determined the molecular formula of an peptidase Ararenon degradation products with C ₁₇ H ₂₄ 0 _4. ! Δ 6.38 (d), 3.69 (m) and 3.41 (br d), which are characteristic peaks when compared with zearalenone in the 1 H NMR spectrum, are C-10 ′, C Assigned to -1 and 10'-OH. This means that the ester bond in zearalenone was hydrolyzed and subsequently decarboxylated. To taste. C '- geometry in 1, high binding constants: was determined with E configuration Homeshiyon based on (1 ^{6 5} Hz.). By ¹³ C NMR spectra, one ketone, three sp ² 4-valent carbon atoms, 17 carbon atoms, including one sp ² methine carbon atom and one methyl carbons, above Zeararenon degradation products Was confirmed to be present. PFG- DQ from FC0SY及Pi PFG- HMQC data, each pro Tonsupin'ne' network and all single bonds of ¹ H - evaluation of binding ¹³ C. The long-range bond observed in the PFG-HMBC spectrum is due to the fact that the ketone group (δ 210.47) is located at C-6 'in the above-mentioned zearalenone degradation product, This indicates that a deletion of the carboxyl group at -12 'occurs. This was also supported by the chemical shift and multiplicity at C-1 (d, δ 105.49) and C-10 '(d, δ 67.34). Based on the above analysis, the overall structure of the above-mentioned zearalenone degradation product was determined to be 1- (3,5-dihydroxyl-propyl) -10-hydroxy-1-l-decene-6, -one. The chemical structure is shown in FIG. From this analysis result, it was found that zearalenone was degraded by the zearalenone degrading enzyme ZHD101 of the present invention to produce a zearalenone degradation product, and the reaction was described by a chemical formula (FIG. 7).

[Example 9] Estrogenic activity of zearalenone degradation product

 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.

First, human breast cancer cells MCF-7 were converted to phenol red, L-glutamine (2 mM), ぺ -cillin (50 units / ml), streptomycin (50 μg / ml) and 10% ゥ 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. 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. After culturing this at 37 ° C for 20 hours, the medium was replaced with the same kind of medium. To this culture, 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 ™ (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). In this example, a known estrogen, 17-estradiol (0.1 nM) was used as a control. When 17β-estradiol is added as a test compound, the number of cells is increased by an average of about 250% compared to a control in which nothing is added. 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. On the other hand, the zearalenone degradation product of the present invention (chemical formula: FIG. 7) hardly increased the cell number even at a concentration 1000 times higher than zearalenone, and did not show a cell growth promoting effect (FIG. 8). . That is, 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.

[Example 10] Construction of zhdl01 gene transfer vector

Based on the zhdlOl DNA sequence (SEQ ID NO: 1), the GFP-zhd-5 'primer (5'-ATG CG C ACT CGC AGC ACA ATC TCG-3' (SEQ ID NO: 21)) and GFP-zhd-3 'primer ( 5′-T GT ACC GTT CAA AGA TGC TTC TGC-3 ′ (SEQ ID NO: 22)) was designed and chemically synthesized. Using these primers and Vent polymerase (New England Biolabs), 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. Separately from this, 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. Chem., 273, 1654-1661, 1998), and Hind III_BstXI was deleted to create an Nco I site. Vector) NC. The ligation was performed at the I-NotI site to construct a recombinant vector pWheat-egfp :: zhd101 for gene transfer. 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. After washing, 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 (α-naphthalene acetate), 2 mg / ml BA (benzyladenine) and 30 g / 1 sorbitol (Regeneration medium). The regenerated plants were acclimated 2-3 weeks after transplantation to an LS medium (selective rooting medium) containing 5 ppm bialaphos to obtain a regenerated body (FIG. 11). . [Example 12] Expression of EGFP :: ZHD101 in transformed rice

 Western blot analysis was performed to examine whether the EGFP :: ZHD101 protein was expressed in the regenerated rice obtained by introducing the egfp :: zhdlOl gene.

 0.1 g of the regenerated leaves were ground in liquid nitrogen, and 100 μl of extraction buffer was added and mixed well. This was centrifuged (15,000 rpm, 5 min) to recover the supernatant, which was used as a crude protein. The obtained crude protein was applied to a 10% SDS-polyatarylamide gel at 20 g per lane and electrophoresed. After the electrophoresis, the protein was transferred to a PVDF membrane, and the GFP :: ZHD101 fusion protein was detected with an anti-gfp antibody. The results are shown in FIG. Lane 1 is a sample extracted from wild-type rice leaves, lane 2 is regenerated body No. 14, lane 3 is regenerated body No 54, lane 4 is regenerated body No 68, lane 5 is regenerated body No 71, and lane 6 is regenerated body No. 76, lane 7 is regenerated body No 79, lane 8 is regenerated body No 79, and lane 9 is recombinant EGFP :: ZHD101. In 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). . Next, about 50 mg of the suspension cultured cells and 50 ppra zearalenone-containing LS added with 750 g of zearalenone were added to each of the thus-prepared egfp :: zhdl01-introduced suspension culture cells and wild-type suspension culture cells. The medium was added to 15 ml of the medium, and cultured with shaking at 26 ° C. After 3 days from the start of the culture, extract the extract containing zearalenone from each of the above media using a black-mouthed form, place the medium extract on a TLC plate, and develop with a black-mouthed form: acetone (80:20). Detection was performed below. Extracts similarly extracted from the zearalenone-containing LS medium and the zearalenone-free LS medium were used as controls. Figure 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, and lane 4 is zearalenone An extract from the LS medium containing LS medium, and lane 5 shows an extract from the LS medium without zearalenone. Extracts from the medium in which wild-type cells were cultured and the LS medium containing zearalenone were found to have the same spots as the mobility of the zearalenone standard, indicating that zearalenone remained in the medium. Was. On the other hand, in 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.

 Therefore, next, to determine the amount of remaining 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. In order to confirm that zearalenone in the medium was degraded, 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. For the above quantification of zearalenone, RIDASCREEN FAST Zearalenon (R-Biophar) was used according to the instruction manual.

Table 1 shows the amount of zearalenone quantified 6 days after the start of the culture. table 1

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 μ, respectively. In addition, 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. As shown in Fig. 14, 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 μg 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.

These data indicate that transformed cells into which the egfp :: zhdl01 gene has been introduced can strongly degrade zearalenone in the medium. This result indicates that the transgenic rice plant into which egfp :: zhdl01 has been introduced has zearalenone resolution. All publications, patents and patent applications cited herein are incorporated by reference in their entirety. And are incorporated herein by reference. Industrial applicability

 Use of the protein of the present invention can advantageously suppress the toxicity of zearalenone. Further, the gene of the present invention can be used for expressing the protein. Furthermore, 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.

Claims

Scope of Claim The following (a) or (b) protein.

 (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2

 (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having an action of suppressing toxicity of zearalenones

 2. The protein according to claim 1, wherein the zearalenone is a compound having an estrogenic activity.

 3 The zearalenones are zearalenone, γ-zearalenol, / 3-zearalenol, α-zearalanol, j8-zearalanol, 2,4-0-dimethyl-δ-hydroxy-zearalenone, 6-amino-zearalenone, zearalanone and 6-acetyl The protein according to claim 1, which is at least one selected from the group consisting of-j3-zearalenol.

4. The method according to claim 1, wherein the effect of suppressing toxicity is a decomposition effect.

5. The protein according to any one of claims 1 to 4, wherein the action of suppressing toxicity is to produce a compound having no estrogenic activity.

 The protein according to any one of claims 1 to 5, which has an activity at 6ρΗ6 to 11.

 7. The protein according to claim 6, which has an activity at 7 ρ Η 9 to 10.5.

8 A gene encoding the following protein (a) or (b):

 (a) a protein consisting of the amino acid sequence of SEQ ID NO: 2

 (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having an action of suppressing toxicity of zearalenones

 9 A gene consisting of the following DNA (a) or (b):

 (a) DNA comprising the nucleotide sequence of SEQ ID NO: 1

(b) complementary to all or part of the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 Encoding a protein that hybridizes under stringent conditions with DNA consisting of a specific base sequence and that has a function of suppressing the toxicity of zearalenones

 10. A gene consisting of the following DNA (a) or (b):

 (a) DNA encoding the amino acid sequence shown in SEQ ID NO: 2

 (b) an ability to hybridize under stringent conditions to DNA consisting of a nucleotide sequence complementary to all or part of the DNA encoding the amino acid sequence shown in SEQ ID NO: 2, and to suppress the toxicity of zearalenones DNA encoding a protein having

11. The zearalenone is a compound having estrogenic activity.

The gene according to any one of 8 to 10.

12. The zearalenones are zearalenone, α-zearalenone, β-zearalenol, α-zearalanol,] 3-zearalanol, 2,4-0-dimethyl-δ-hydroxyzearalenone, 6-amino-zearalenone, zearalanone and The gene according to any one of claims 8 to 11, wherein the gene is at least one selected from the group consisting of 6-acetinol- / 3-zelarenol.

 13. The gene according to any one of claims 8 to 12, wherein the action of suppressing toxicity is a decomposition action.

 14. The gene according to any one of claims 8 to 13, wherein the action of suppressing toxicity is to generate a compound having no estrogen-like activity.

 15. A thread and recombinant vector containing the gene according to any one of claims 8 to 14.

16. A transformant comprising the recombinant vector according to claim 15.

 17. The transformant according to claim 16, wherein the recombinant vector has been introduced into a cell selected from the group consisting of Escherichia coli, yeast cells, and gramineous plant cells.

18. A method for producing a protein, comprising culturing the transformant according to claim 16 or 17, and collecting a protein having an action of suppressing toxicity of zearalenones from the obtained culture.

19. An antidote containing the transformant according to claim 16 or 17.

20. An antidote containing the protein according to any one of claims 1 to 7.

A method for detoxifying zearalenones, which comprises applying the antidote according to claim 19 or 20 to zearalenones.

A transgenic plant comprising the gene according to any one of claims 8 to 14.