WO2002079469A1

WO2002079469A1 - Mold polynucleotide arrays

Info

Publication number: WO2002079469A1
Application number: PCT/JP2002/003267
Authority: WO
Inventors: Keietu Abe; Katuya Gomi; Tasuku Nakajima; Yohei Yamagata; Humihiko Hasegawa; Yasutaka Iguchi
Original assignee: Tohoku Techno Arch Co., Ltd.
Priority date: 2001-03-30
Filing date: 2002-04-01
Publication date: 2002-10-10
Also published as: JPWO2002079469A1; WO2002081701A1; JP3619844B2

Abstract

A method of detecting gene expression characterized by comprising immobilizing a probe containing the full base sequence or a part thereof of a mold-origin nucleic acid, hybridizing a labeled polynucleotide sample to be detected with the immobilized probe as described above, and then detecting a signal from the hybridization product thus obtained.

Description

Description Filamentous fungal polynucleotide array

The present invention relates to a filamentous fungus polynucleotide array having a probe containing the entire or partial nucleotide sequence of a filamentous fungus-derived nucleic acid immobilized on a base, and a method for detecting gene expression using the polynucleotide array. The polynucleotide array of the present invention is a device for quantitatively monitoring the gene expression of a filamentous fungus. Specifically, the polynucleotide array of the present invention is obtained by spotting one or more types of cDNA which is a gene expressed by a filamentous fungus (for example, koji mold), and labeling the gene extracted from the target mold to form an array. This device analyzes the frequency of gene expression by performing the above hybridization. Background art

Fungi such as Aspergillus, Penicillium, Fusarium, Trichoderma and Mucor exist in the filamentous fungi, and are considered to be important molds because they are involved in the fermentation industry, human and cereal infections, etc. I have. Aspergillus sp. Includes other species used in the fermentation industry, as well as those that are primarily involved in diseases or diseases such as human or animal infections. Therefore, in order to quickly achieve high efficiency and optimization of fermentation production, comprehensive expression analysis of useful genes involved in growth status, substance production, etc., is necessary. Comprehensive expression analysis of genes involved in human infection is necessary to identify human infectious disease molds and to develop antifungal agents. From the viewpoint of epidemic control, comprehensive analysis of the expression of genes involved in cereal infections, and comprehensive analysis of genes involved in toxin production, from the viewpoint of epidemic control, to identify infected molds and develop antifungal agents Expression analysis is needed.

Until now, there has been no method for simultaneously and directly monitoring the gene expression of many kinds of ^^ perg ZZ ^ fungi, which are particularly important in the fermentation industry, medical care, and agriculture. Therefore, the gene expression function itself or the expressed substance (useful yeast produced from koji mold) Useful secondary products, such as proteins and antibiotics, are monitored by professional technicians (artisans, quality control technicians, doctors, etc.) using visual aroma, etc., and organic acids and esters in products are It is monitored by observation based on analysis of components, identification by cultivation of isolated bacteria from infected sites, separation and measurement of metabolites such as toxins, etc.Based on experience, growth conditions and fermentation functions are monitored. We have estimated its association with expression, disease state, and cereal contamination. However, the data obtained by these conventional methods are not objective and cannot be directly observed, making it difficult to quantify them in most cases.If automation and labor saving are difficult, There were many. Furthermore, in the case of identifying metabolites, the chromatographic separation and quantification method ensures objectivity and can be quantified. However, not only is the analysis technique complicated, but it is difficult to identify only a small number of substances. Disclosure of the invention

An object of the present invention is to provide a polynucleotide array having a filamentous fungal gene immobilized on a support, and a method for detecting a gene using the polynucleotide array.

The present inventors have conducted intensive studies to solve the above problems, and as a result, by immobilizing a polynucleotide isolated from a filamentous fungus on a base, produced a polynucleotide array useful for detection of filamentous fungi, The present invention has been completed. That is, the present invention is as follows.

(1) A polynucleotide array in which probes containing all or part of the nucleotide sequence of a nucleic acid derived from a filamentous fungus are immobilized on a support.

The nucleic acid may be at least one selected from the group consisting of a C source-added culture condition, a C source deficient culture condition, a maltose culture condition, an alkaline culture condition, a solid culture condition, a high temperature culture condition, a solid low temperature culture condition, and a spore germination culture condition. One example is a polynucleotide containing a cDNA region prepared from a cDNA library derived from a filamentous fungus cultured under one condition. The nucleic acid may be genomic DNA, cDNA, RNA or oligonucleotide DNA.

The filamentous fungi include Aspergillus, Penicillium, Fusarium, Examples include microorganisms belonging to the genus Trichoderma or the genus Mucor. In the embodiment of the present invention, the microorganisms belonging to the genus Aspergillus include Aspergillus oryzae, A. sp. At least one selected from the group consisting of Nomius, Aspergillus fumigatus and Aspergillus nidulans, preferably Aspergillus oryzae, Aspergillus zoya, Aspergillus parasites, Aspergillus flavus and Aspergillus At least one member selected from the group consisting of, more preferably Aspergillus oryzae.

The support used in the polynucleotide array of the present invention includes, for example, cell-opening polymer, nylon polymer, glass, non-porous material or porous material.

(2) After labeling the target polynucleotide for detection, it is hybridized with the probe immobilized on the polynucleotide array of (1) above, and a signal is detected from the obtained hybridization product. A method for detecting a target polynucleotide.

In the above detection method, the target polynucleotide for detection is derived from a filamentous fungus, and the polynucleotide is labeled with a fluorescent label, a radioactive label, an electronic label, or a multistage modified label. The target polynucleotide for detection is, for example, genomic DNA, cDNA, RNA or oligonucleotide DNA. The filamentous fungus is at least one of microorganisms belonging to the genera Aspergillus, Penicillium, Fusarium, Trichoderma or Mucor. Microorganisms belonging to the genus Aspergillus are the same as those exemplified in (1).

In the detection method of the present invention, 20 to 100,000 types of probes are immobilized on the polynucleotide array.

(3) A method for identifying and / or distinguishing a filamentous fungus-related species or a mutant thereof using the detection result obtained by the detection method shown in (2) as an index.

(4) Useful substance or harmful substance candidate samples to be screened After coexisting with at least one of them, the nucleic acid is isolated and labeled from the filamentous fungus, and the labeled nucleic acid is hybridized with the probe immobilized on the polynucleotide array of the above (1). A method for detecting a signal from a product of a shion and screening for useful and / or harmful substances using the obtained detection result as an index.

The useful or harmful substance includes a drug.

(5) Isolating and labeling nucleic acids from filamentous fungi cultured under different culture conditions, hybridizing the labeled nucleic acids with the probes immobilized on the polynucleotide array of (1) above, and obtaining the resulting hybridization. A method for detecting a signal from a product and optimizing the expression of the target gene of the filamentous fungus using the obtained detection result as an index. Hereinafter, the present invention will be described in detail.

1.

The present invention seeks to solve the above-mentioned problems by utilizing the base sequences of a large number of genes of filamentous fungi, especially koji molds. That is, a large number of hybridization probes having the base sequence according to the present invention are prepared, and more specifically, nucleic acids are aligned and immobilized as probes on a solid phase substrate as a support, and the amount of DNA derived from filamentous fungi is determined. By measuring the expression level of the gene and the gene, the growth state of the filamentous fungus and the expression of the fermentation function of the fungus can be directly and accurately measured. Furthermore, in the analysis of closely related bacterial species, for example, Aspergillus fumiga

Gene expression during human infection, gene expression during cereal infections such as Aspergillus flavus (n yZav;) and Aspergillus parasticus (n pasC), which are cereal-infected molds, or production of metabolites including toxins It is also possible to accurately measure the gene expression related to the disease. It can also be used to monitor gene expression in fermentation-producing strains such as Aspergillus nigaichi (n ').

The polynucleotide immobilized on the polynucleotide array of the present invention covers almost all of the nucleotide sequence of a gene expressed under various typical fermentation production and growth conditions. It is considered to be effective for detecting bacterial genes. As a result, automation that was extremely difficult In addition to labor saving, fermentation production reliability is improved and standardization is achieved. This will allow a significant reduction in production costs.

2. Polynucleotide array

(1) Preparation of polynucleotide for immobilization

In the present invention, the term “polynucleotide array” refers to an array in which nucleic acids, which are polynucleotides, are immobilized as probes on a base, which is a support.

A polynucleotide array can be prepared by dissolving a polynucleotide in a solvent to prepare a polynucleotide solution, and fixing the prepared polynucleotide solution to the surface of a substrate. For example, the polynucleotide solution is placed by abutting the tip surface of the polynucleotide solution dispensing needle on the surface of the substrate, and then fixed. Alternatively, it can be produced by directly synthesizing it on the substrate surface.

The polynucleotide to be used may be one derived from a filamentous fungus. Examples of the filamentous fungi include microorganisms belonging to the genera Aspergillus, Penicillium, Fusarium, Trichoderma, and Mucor. The type of the microorganism is not particularly limited, but includes the following.

(i) Aspergillus

Industrial species: Aspergillus oryzae, Aspergillus sojae), Aspergillus niger, a black koji, Aspergillus awamori, Aspergillus awaschi

These industrial species are used in the production of pharmaceuticals, chemicals, enzymes or foods. Human infectious species: Aspergillus fumigatus spergUlus fumigatus ")

Grain infectious species: Aspergillus flavus, Aspergillus parasiticus) ヽ Aspergillus nomius

Experimental model species: Aspergillus

(ii) Penicillium Industrial species: Penicillium chrysogenum, a penicillin-producing bacterium, Penicillium notatum, Penicillium griseofulvum, a glyceofulvin-producing bacterium for the treatment of athlete's foot, griseofulvin Penicillium roqueforti Henryum J ヾ nove J Lena (Penicillium camembefti) Grain and fruit contaminants: Penicillium citricum Penicillium 'Pisnicillium' expansum)

(iii)

Industrial species: Fusarium vencnatum j for microbial proteins (mycop mouth tin)

Infected plant species: Fusarium fum solani {Fusarium

(iv) Trichoderma

Industrial species: Trichoderma reese, a cellulase-producing bacterium, Trichoderma reese, Hilia (Trichoderma viri.de j

(iv) Mucor genus

Industrial species: Mucor pusillus, a clotting enzyme-producing bacterium-\ Mucor mie iei)

(V) Rhizopus

Industrial species: Rhizopus delemar (Dhicopus delemar), Rhizopus nigricans (Rhizopus nigricans) 菌 Rhizopus oryzae (Rhizopus oryzae), Rhizopus (Thizopus oligosporus)

(vi) Monascus Industrial species: Monascus' anca Monascus α or β), Monascus puriier (Momwc rii & er), Monascus no J resoleso (Monascus purpureus)

These fungi are used to produce red wine and tofu, produce red pigments, or produce drugs to treat cholesterol. Examples of the type of the polynucleotide (nucleic acid) include DNA (deoxyliponucleic acid) and RNA (liponucleic acid), and any of DNA fragments derived from chromosomal DNA, RNA (such as mRNA), cDNA, and oligonucleotides may be used. The oligonucleotide refers to a product obtained by subjecting all or a part of the above-described chromosomal DNA fragment, mRNA, or cDNA to a chemical treatment, or a product obtained by chemically synthesizing based on the above-described nucleic acid sequence. For example, PCR amplification products, restriction enzyme digested DNA and the like can be mentioned.

It may also be a DNA fragment containing a part of a specific gene, an mRNA having an abnormal transcription level, or a cDNA derived from the mRNA. The nucleic acid is dissolved in a solvent to prepare a nucleic acid-containing solution (called a polynucleotide solution).

Furthermore, nucleic acids can be prepared from the filamentous fungi after culturing the filamentous fungi under various culture conditions. Cultivation conditions include C source-added culture conditions, C source-deficient culture conditions, maltose culture conditions, alkaline culture conditions, solid culture conditions, high-temperature culture conditions, solid low-temperature culture conditions, and spore germination culture conditions. One or a combination of them can be used for culturing.

C source-added culturing conditions refer to culturing conditions when culturing in a medium containing a carbon source, such as 0% in YPD medium (1% yeast extract, 2% Bacto-pepton, 0.04% adenine sulfate, 2% glucose). Shaking culture (180 rpm) for 22 hours at ° C.

C source-deficient culture conditions refer to culture conditions when cultured in a medium that does not contain a carbon source. For example, after culturing in YPD medium (1% yeast extract, 2% Bacto-pepton, 0.04% adenine sulfate, 2% glucose) at 30 for 22 hours, collect the cells by filtration and wash them with distilled water. Then, transfer the cells to a CD medium (Czapek-Dox) that does not contain a carbon source such as glucose (Nakajima K et al. Curr Genet. 2000 May; 37 (5): 322-7) and further culture for 8 hours To do something.

Maltose culture conditions refer to the culture conditions when cultured in a medium containing maltose. means. For example, the cells are cultured in an ACM medium (2% Malt extract, 0.1% Bacto-pepton, 2% Glucose) with shaking for 24 hours (180 rpm), and the cells are collected by filtration and collected in a natural medium (1% Bacto-pepton, 0.5%). _{_{% KH 2 P0 4, 0.1%}} NAN0 3, washed 0.05% MgS0 ₄ · 7Η in ₂ theta), those that 37 ° C, 4 hours further cultured with shaking at natural medium containing the cells 2% Maltose Point.

The alkaline culture condition means a culture condition when the culture is performed in a pH range of 7 to 11. For example, put sterilized cellophane on a CD (Czapek-Dox) agar medium adjusted to pHIO, inoculate a conidium of the oryz eRIB40 strain, and culture at 30 ° C for 3 days.

The solid culture conditions are as follows: 5 x 10 ⁶ conidia are suspended in 4 ml of water and added to 5 g of wheat bran (10 ⁶ / g-bran), and the mixture is allowed to stand at 30 ° C for 27 hours. Refers to what is cultured. High-temperature culture conditions refer to culture conditions when culture is performed at a temperature in the range of 30 to 42 ° C. For example, it refers to a medium cultured with shaking at 37 ° C. for 24 hours in the above YPD medium. The solid low-temperature culturing conditions refer to culturing conditions when culturing at a temperature in the range of 30 to 15 ° C. in a medium having a composition containing cereals for wheat, rice or soy sauce koji. For example, 15 g of water is added to 10 g of defatted soybeans to absorb water, and 10 g of roasted cracked wheat is added and stirred. This is placed in a 500 ml Erlenmeyer flask, covered with a biosilico stopper, and autoclaved for 30 minutes. After cooling, 1 × 10 ⁵ Aspergillus oryzae RIB40 is inoculated at a concentration of 1 × 10 ⁵ spores / g. After inoculation, koji is made at 30 ° C (gas phase) for 34 hours and then at 25 ° C for 3 hours.

The spore germination culture conditions refer to the culture conditions when spores are cultivated in a medium containing the germinated cells. For example, conidia are obtained by culturing them on PD (potato dextrose agar) plates at 28 ° C for 8 days. Germination bacterial cells, conidia SP liquid medium (3.5% soluble starch, 2% _{polypeptone, 0 · 5% ΚΗ 2 ΡΟ} 4, 0.5%, MgS0 4 - 7H 2 0) in cultured 30 ° C, 12 hours obtain. The type or number of polynucleotides used may be any number as long as it is 20 or more (species), but is in the range of 20 to 100,000 (species), preferably 100 to 100,000 (species). It is. The length of the polynucleotide itself can be arbitrarily set as long as it can function as a probe. For example, 10 to 50,000 bases, preferably 10 20002000 bases, more preferably 10-1000 bases.

(2) Arrangement of polynucleotide solution on support

A number of polynucleotide solutions are placed on the surface of the polynucleotide array support. As the support, a membrane such as a cellulose membrane (cellulose polymer) or a nylon membrane (nylon polymer), a glass support, a nonporous support or a porous support can be used. Non-porous or porous support materials include plastics (eg, polyethylene, polypropylene, polystyrene, etc.). In the present invention, a glass support is preferable because it is low in cost, easy to achieve high density, and excellent in planar characteristics. Specifically, a slide glass (a slide glass for DNA Microarray Kit) manufactured by Nippon Laser Electronics Co., Ltd. is used. be able to. The surface of the support is positively charged. As the positive charge treatment, the surface of the support is subjected to amination treatment with poly-relysin, aminopropyltriethoxysilane, or the like so as to be positively charged. Next, a polynucleotide solution is placed on the surface of the support. The immobilization of the polynucleotide on the surface of the support is not limited to this. For example, there is a combination of an aminated polynucleotide with a support having surface characteristics capable of covalently binding. A specific example is Surrmodics' 3D-Link. A commercially available arrangement device such as a GTM AS Stamp (manufactured by Nippon Laser Electronics Co., Ltd.) can be used. This device controls the movement of the dispensing head in the XYZ axis direction with respect to the surface of the support so that the tip surface of each dispensing needle in the dispensing head comes into contact for about 0.5 to 1 second, and various types of The polynucleotide solution can be arranged in a matrix. In this arrangement, the dispensing head is controlled to move in the X-Y axis direction so that the distance between the spots is about 100 to 200 m. However, the arrangement method is not limited to this.

The support is not limited to a flat one, but may be a bead-like support. In that case, different polynucleotide species are retained for each bead.

(3) Immobilization of polynucleotide

After drying a large number of polynucleotide solutions arranged on the surface of the support, the polynucleotide is immobilized by irradiation with ultraviolet light of about 50 to 120 mJ. Thereafter, the non-arranged region of the polynucleotide on the surface of the support is subjected to a blocking treatment. blocking The treatment is performed by immersing the entire support on which the polynucleotide is immobilized in a blocking solution containing a predetermined amount of a succinic anhydride, N-methylpyrosinone and sodium borate solution. Specifically, a blocking solution (mixed solution of Microarray Kit A2, A3, A4) manufactured by Nippon Laser Electronics Co., Ltd. can be used. The support removed from the blocking solution is immersed in hot water at 95 to 99 ° C and left to stand. After immersion in ethanol, the surface of the support is air-dried.

3. Gene detection

(1) Filamentous fungi to be analyzed

Total RNA and mRNA to be detected can be prepared from hyphae and spores of filamentous fungi. As described above, the types of filamentous fungi are Aspergillus oryzae, Aspergillus sojae, Aspergillus niger Aspergillus awamor Aspergillus kawacni Aspergillus fumigatus Aspergillus flavus Aspergillus parasiticus Aspergillus nomius Aspergillus nidu. The gene is prepared from at least one of the above filamentous fungi. For preparation of the target gene, any known method (eg, freezing cells in liquid nitrogen, pulverizing, and then extracting total RNA in the presence of an RNase inhibitor) can be employed (Nakajima K et al. Curr Genet). 2000 May; 37 (5): 322-7), and a commercially available total RNA extraction kit such as ISOGEN manufactured by JEIN. Purification of mRNA from total RNA can be performed using oligo (dT) -immobilized resin or beads such as MESSAGE MAKER manufactured by Promega.

(2) Gene labeling

In the method of the present invention, a polynucleotide to be detected is labeled so that a detection result can be easily obtained. Examples of the label include a fluorescent label, a radioactive label, an electronic label and a multi-stage modified label.

Fluorescent labeling is a labeling method that enzymatically or chemically introduces a fluorescent substance into the polynucleotide to be analyzed (one get), and includes ROX, TET, FAM, IRD, cyanine (Cy3, Cy5, Cy3.5), ALEX, and the like. For example, Cy3 is colored green and Cy5 is colored red, which is convenient when you want to distinguish between two types of detection targets. Commercially available It is also possible to use a labeling kit, such as Takara Shuzo's Label IT non-RI Labeling.

Radiolabeling is a labeling method that introduces a radioactive nucleic acid or a radioactive functional group into a nucleic acid to be analyzed.For labeling substances, dNTPs and nucleic acids containing ³² P, ³³ P, ³⁵ S, C, ¾, and ¹²⁵ I are used. A substance containing a reactive functional group that can be introduced is given. It is also possible to use a commercially available labeling kit, for example, Amersham's random primer-labeling kit ゃ terminal labeling kit manufactured by Pharmacia.

Electronic labeling is a method in which a reactive substance is added to a double-stranded polynucleotide that has been hybridized between a probe and a target, and the double-stranded site is detected by an electrochemical detection method. is there. One such material is the charged Yin-ichi-karator.

The term “multi-stage modified label” means a label in which a fluorescent label or a radiolabel is not a one-step reaction but a two- or more-step reaction. In general, in one-step modification, the labeled compound may be difficult to be incorporated into the evening polynucleotide. Therefore, a substance that is reactive with the labeled compound in the first step and that is easily incorporated into the target is introduced, and the second and subsequent steps incorporate the labeled compound in advance. The labeling efficiency is increased by reacting the compound with a reactive substance. It is also possible to use commercially available labeling kits, such as Clontech's Atlas Glass Fluorescent Labeling Kit and NEN's Micromax TSA Labeling Kit.

(3) Hybridization between probe and target gene

A probe (polynucleotide immobilized on a support) is reacted with a target (a solution containing the target gene) to perform hybridization. The hybridization conditions are set so that specific hybrids are formed and non-specific hybrids are not formed. For example, conditions are set under which DNA having high homology (homology is 60% or more, preferably 80% or more) is hybridized. Specifically, the sodium concentration is 100-900 mM (2-20 X SSC, preferably 4-10 X SSC), preferably 150-900 mM, and the temperature is 37-68 ° C, preferably 42-68 ^. is there. (4) Detection

After the hybridization, the signal of the labeling substance is measured (detected) using a predetermined detector. For fluorescent labels, use fluorescence image analyzers, for radiolabels, radiographic or radiological image analyzers, for electronic labels, electrochemical detectors, and for multi-stage modified labels, use them according to the type of label. The signal can be measured using the instrument.

4. Identification and / or identification of closely related filamentous fungi or variants thereof

In the present invention, a species related to a filamentous fungus or a mutant thereof can be identified and / or distinguished based on the above detection results.

Closely related species are those having a DNA homology of 40% or more, preferably 50% or more, and more preferably 70% or more.

Mutants are homologous bacteria (DNA homology is almost 100%) and have a limited number (one to several) of bases with deletions, additions, or substitutions of bases. Alternatively, deletion of a limited number of genes (one to several) may be used. Alternatively, some genes have a new constant number (one to several) of genes added to the genome.

Competitive hybridization of the labeled genomic DNA to the polynucleotide array in the two strains is regarded as a closely related species, and the use of oligo DNA etc. to hybridize single nucleotide changes In a polionucleotide array designed to be detected as a change in the condition, a mutation can be identified by the presence or absence of hybridization.

5. Screening of useful or harmful substances, analysis of useful (harmful) substance production conditions In the present invention, useful substances can be screened based on the above detection results. Useful substances include drugs, enzymes, proteins and the like. Hazardous substances include mutagens, carcinogens, environmental hormones, and heavy metals. To screen a drug, a useful or harmful substance as a screening candidate is given to Aspergillus mold, which provides a probe for a polynucleotide array. MRNA is prepared from the co-cultured cells and the cells cultured without providing useful or harmful substances, and fluorescent labeling is performed, and gene expression response analysis is performed using a polynucleotide array. At this time, by selecting and analyzing the genes that responded to the expression, it is possible to estimate the mechanism of action of the drug or to use it as a criterion for selecting a lead compound for the synthesis of a useful drug. is there.

Further, in the present invention, it is possible to optimize the conditions for producing a useful substance by analyzing an expression response including not only an enzyme gene but also a transcription control factor. On the other hand, polynucleotide array analysis is also effective in suppressing the production of toxic substances such as toxins. In other words, when toxin production-related genes (including genes for transcription control factors) are immobilized on a polynucleotide array, it is possible to search for culture conditions that suppress their expression and to screen for expression-suppressing substances. It is possible. The various uses of the detection results are described below.

(1) Outline of analysis method for genes that responded to expression:

As a result of measurement using a detection device, the gene intensity of the gene that responds under certain conditions is quantified using analysis software (eg, ArrayPro or ScanAlyze) to determine the gene expression level. In this case, standardize based on the expression level of the control gene as a control to obtain a quantitative value. Statistical analysis was performed using specialized analysis software (such as GeneSpring and GeneMaths) to analyze the expression information data of many genes, which was used to elucidate gene expression control networks and map metabolic pathway maps. The expression pattern of the gene can be clarified.

(2) Criteria for estimating the mechanism of action of drugs:

The genes whose gene expression level has changed under the conditions given the drug are mapped to a metabolic pathway diagram using analysis software. As a result, if many genes with altered expression levels are mapped to the metabolic pathways of the same line, it can be estimated that the drug acts specifically on that metabolic pathway.

If a gene specific to Aspergillus genus responds to the expression, the responding gene is an essential gene, and if it is negatively (possibly positive) regulated by the screening candidate substance, screening for an antifungal agent Provide information. (3) Criteria for selecting lead compounds:

By mapping to metabolic pathway diagrams of genes whose expression responses have changed under culture conditions using lead compound candidates, compounds that are presumed to act on previously unknown pathways can be identified. It is possible to screen as a compound.

(4) Determination of efficacy

It can also be used to determine the efficacy of a drug based on the intensity of the expression response and time series analysis. For example, when the expression response is examined using a certain drug, if the expression level of the acting gene fluctuates greatly, it can be determined that the drug effect used is extremely strong.

(5) Criteria for judging that the substance is harmful:

This analysis method can be used for screening not only useful drugs but also toxic substances such as poisons. In other words, for polynucleotide arrays in which homologous genes in AsergiZZ fungi of cell essential genes, which are conserved in mammals, are immobilized, screening for substances that inhibit their expression requires screening of drugs. It is also possible, and they provide a toxicological screening system for mammals and the like. For example, when a gene group whose expression response fluctuates greatly when given a certain substance is a DNA repair-related gene such as uvs or md, it can be determined that the substance is a harmful substance having mutagenicity.

(6) Screening for secondary metabolites

In order to screen unknown useful enzymes and useful (harmful) secondary metabolites produced by Aspergillus mold itself, first, it is recognized that the culture conditions are such that enzyme induction and useful (harmful) secondary metabolite production are observed. Analyze gene expression differences under no culture conditions. The responding gene is narrowed down using the mapped metabolic pathway map as an index. In the process, the gene of the useful enzyme itself, or the production of a useful enzyme or a useful (harmful) secondary metabolite Genes that control production can be identified.

For example, if the gene that is expressed or suppressed only under culture conditions that produce kojic acid used in whitening cosmetics is a gene that encodes a DNA-binding protein, the DNA-binding protein gene is It is determined to be an important gene that controls acid production, and is screened as a gene that regulates the production of industrially useful secondary metabolites.

(7) Optimization of target gene expression

In addition, regarding the fermentative production of useful substances by fungi of the genus ^ ¾ e iZZ, gene expression analysis using a polynucleotide array under various culture conditions and control culture conditions revealed that the target gene of the filamentous fungus was Expression can be optimized.

Filamentous fungi are cultured under any of the aforementioned culture conditions (C source-added culture conditions, C source-deficient culture conditions, maltose culture conditions, alkaline culture conditions, solid culture conditions, high-temperature culture conditions, solid low-temperature culture conditions, or spore germination cultures). Condition), the target gene can be expressed by examining under which culture conditions the expression of a particular gene was promoted and under what culture conditions the expression of that gene was suppressed. It is possible to select cultivation conditions that are optimal for the purpose.

Here, “optimization” means setting culture conditions that achieve the maximum or minimum yield by artificially controlling the expression of one or more genes involved in the production of a certain substance. For example, in soy sauce koji production, when hybridization is performed between several conditions of soy sauce solid culture, and conditions that result in the highest expression signal of the protea-peptidase gene are found. Is optimized for maximizing the expression of the enzyme enzyme, which is important for soy sauce production. In the case of unnecessary genes, it can be said that minimization is to find conditions that minimize the expression signal of the target gene in the polynucleotide array by comparing the culture conditions. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of the polynucleotide array of the present invention.

FIG. 2 is a schematic diagram of the polynucleotide array of the present invention. FIG. 3 is a schematic diagram of the polynucleotide array of the present invention.

FIG. 4 is a photograph of an image showing the result of detecting the expression intensity of the or ore gene (cDNA clone fed with a C source) in the polynucleotide array of the present invention. FIG. 5 is a photograph of the polynucleotide array of the present invention. FIG. 6 is a photograph of an image showing the results of detecting the expression intensity of the oryzae gene (c source-deficient vegetative cDNA clone). FIG. 6 shows the m′ger gene (C source deficient) in the polynucleotide array of the present invention. FIG. 7 is a photograph of an image showing the results of detection of the expression intensity of oligotrophic cultured cDNA clones. FIG. 7 is a diagram showing an outline of a polynucleotide array.

FIG. 8A is a view showing a clone mounted on the polynucleotide array (Nos. 1 to 4 of the block sequence shown in FIG. 7).

FIG. 8B is a diagram showing the clones mounted (Nos. 5 to 8 in the block sequence shown in FIG. 7).

FIG. 8C is a diagram showing polynucleotide clones: clones carried therein (Nos. 9 to 12 in the block sequence shown in FIG. 7).

FIG. 8D is a diagram showing a polynucleotide array: clones on board (Nos. 13 to 16 in the block sequence shown in FIG. 7).

FIG. 8E is a diagram showing clones mounted on the polynucleotide array (Nos. 17 to 20 in the block sequence shown in FIG. 7).

FIG. 8F is a diagram showing a clone mounted on the polynucleotide array (the block sequence Nos. 21 to 24 shown in FIG. 7).

FIG. 8G is a diagram showing clones mounted on the polynucleotide array (blocks 25 to 28 in the block sequence shown in FIG. 7).

FIG. 8H is a diagram showing clones mounted on the polynucleotide array (the block sequence Nos. 29 to 32 shown in FIG. 7).

FIG. 9 is a photograph showing the result of gene expression of Aspergillus oryzae. The green color indicates the gene (Cy3-labeled) obtained by culturing with the C source, and the red color indicates the gene (Cy5-labeled) obtained by culturing with the C source deficient.

Fig. 10 shows a comparison between the results of detection of gene expression by DNA polynucleotide and the results of Northern analysis of the glycolytic enzyme gene of Aspergillus oryzae. It is.

Figure 11 shows

3 is a photograph showing growth and gene expression results of mr. 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] Preparation of Aspergillus mold DNA for preparation of polynucleotide array In this example, aspergillus mold, Aspergillus oryzae was used as an example to prepare DNA for a polynucleotide array. A cDNA library was prepared from the Aspergillus oryzae RIB40 strain donated by the National Tax Agency's Brewing Institute by the method described below, and cDNAs from multiple different libraries were isolated and used as DNA for immobilizing polynucleotide arrays. .

1. Total RNA was extracted from about 50 g of Aspergillus oryzae cultured in a polypeptone liquid medium containing glucose (culture conditions with a C source), and mRNA was extracted using a resin or beads on which oligo dT was immobilized. Extracted. CDNA was synthesized using this mRNA as type III and oligo dT as a primer. Subsequently, double-stranded cDNA was synthesized using partial digestion of the RNA strand of the heteroduplex with RNaseH and DNA strand elongation reaction with DNA polymerase. An EcoRI adapter was ligated to this cDNA and ligated to an E. coli plasmid vector such as pBluescript SKII + to transform E. coli. After appropriately diluting the mixture, apply it evenly to the agar medium to form a large number of independent, hundreds to thousands or more colonies on the agar medium.

2. In the same manner as above, besides the polypeptone liquid medium containing glucose, the liquid medium containing maltose, the liquid medium containing maltose (maltose culture condition), the liquid medium containing no carbon source (culture condition lacking C source), Aspergillus was cultured under alkaline culture medium (alkaline culture conditions) and solid culture medium (solid culture conditions) such as bran, and a library was prepared from the cultures. Also, a polypeptone liquid medium containing glucose In addition to the usual culture at 30 ° C, a library was prepared under culture conditions at 35-45 ° C, preferably at 37 ° C (high-temperature culture conditions). For this purpose, a library was prepared from the conditions (solid-state low-temperature culture conditions) in which the temperature of the koji cells was grown at 30 ° C, and then the temperature was lowered to 20 to 25 ° C, preferably 25 ° C. In addition, a library was prepared for cells (spore germination culture conditions) immediately before spore germination but before mycelium formation.

3. A large number of colonies from each of the above libraries were cultured overnight in an independent liquid medium, and plasmid DNA was extracted and purified from E. coli cells. Using this plasmid as type III, a DNA fragment derived from Aspergillus oryzae inserted into the plasmid was amplified using primers located near the vector cloning site, such as M13 universal and reverse primers. The DNA fragment was amplified by directly treating the cells after heat-treating the cells.

The amplification reaction was performed using the attached PCR buffer, 0.25; M PCR primer, 250 ^ M dNTP, about 10 ng of type I plasmid, and about 1.25 U of Taq DNA polymerase. 30 cycles of shuttle PCR of denaturation at 94 ° C for 1 minute, annealing and extension at 68 ° C for 2 minutes were performed.

4. Using cDNA fragments prepared from each of the above libraries, each was independently immobilized on a solid support. An array was prepared by selecting 86 clones from the cultured source cDNA clones with the C source (Figures 1 and 4). In addition, 86 clones were selected from C-source-deficient cultured cDNA clones, and arrays were prepared (Figures 2 and 5). Furthermore, 82 cDNA clones derived from cultured cells deficient in the C source were selected to prepare an array for use (Figs. 3 and 6).

[Example 2] Preparation of mold polynucleotide array of genus Aspergillus

In this example, the polynucleotide solution prepared in Example 1 was used as an example of mold of the genus Aspergills, and a method of preparing a polynucleotide array is described below using cDNA as a probe.

1. The polynucleotide was dissolved in a solvent to prepare a polynucleotide solution. Poly The nucleotide was dissolved using a spot buffer (DNA Microarray KitAl solution) manufactured by Nippon Laser Electronics. In this example, the Aspegill oryzfle koji mold cDNA prepared in Example 1 was prepared in a spot buffer so as to have a concentration of 0.1 to 1 g / 1, preferably 0.5 ig / 111, and The support was spotted in the manner described. In this example, the isolated clone of the cDNA library prepared in Example 1 was used as a spot, and the nucleotide function of the clone was determined as necessary to estimate the gene function.

2. A number of polynucleotide solutions were spotted by abutting the tip of the dispensing needle against the surface of the support. Specifically, a slide glass (a slide glass dedicated to DNA Microarray Kit) manufactured by Nippon Laser Electronics Co., Ltd. was used. The surface of the support was subjected to a surface treatment with poly-L-lysine or aminopropyltriethoxysilane or the like to make it positively charged.

3. A polynucleotide solution was spotted on the surface of the support. GTMAS Stamp (manufactured by Nippon Laser Electronics Co., Ltd.) was used as the spotting device.

4. The surface of the support having the polynucleotide immobilized thereon was irradiated with ultraviolet light of about 50 to 120 mJ using a UV crosslinker (UVC500 manufactured by Hoefer) to immobilize the polynucleotide. Thereafter, the pH was adjusted to 5 to 6 with a solution containing 1 to 1.5 wt / vol% succinic anhydride, 90 vol / vol% N-methylpyrosinone and 10 vol / vol% 0.2 M sodium borate (pH: 8.0). The entire support on which the polynucleotide was immobilized was immersed in the blocking solution for about 15 minutes. Specifically, a blocking solution (mixed solution of Microarray Kit A2, A3, A4) manufactured by Nippon Laser Electronics Co., Ltd. was used. The support removed from the block solution was immersed in 95-99 hot water and left for about 60 seconds. Thereafter, the support was immersed in ethanol, and the support surface was air-dried to prepare a polynucleotide array. [Example 3] Detection of gene expression using Aspergillus mold polynucleotide array (1)

The detection of the remaining expression of Aspergillus oryzfle and ypergi〃i «'ger by a polynucleotide array is shown as an example.

1. Culture of Aspergillus filamentous fungi

A spore suspension (1 × 10 ⁷ / ml) was prepared from a mold colony stored on a Maltz agar medium (malt extract 2%, peptone 0.1%, glucose 2%, agar 1.5%). YPD medium in an Erlenmeyer flask of 1L (2% yeast extract, Pact peptone 4%, glucose 4%) and 200ml is prepared, the spore suspension lml (l X 10 ⁷ cells) were seeded, 30, between 10pm Shaking culture was performed at 100 rpm on a rotary shaker. Thereafter, 100 ml of the cells were collected, washed, dehydrated, and stored at -80 until RNA extraction as donor cells provided with the C source. The remaining 100 ml of cells are aseptically collected, washed, and dehydrated, and then the minimum volume is adjusted to minimum medium (NaNO ₃ 0.3%, KC1 0.2%, KH ₂ P0 ₄ 0.1% prepared in tap water and supplied with Mg and Fe) The resulting mixture was transferred to a 500 ml Erlenmeyer flask containing 100 ml and further cultured at 30 ° C. for 8 hours on a rotary shaker (100 rpm). C source-deficient cultured cells were collected, washed and dehydrated and stored at -80 ° C until RNA extraction. '

2. Total RNA extraction

Approximately 1.5 g (wet weight) of the freezing bacterium prepared in 1 was sufficiently crushed in a mortar containing liquid nitrogen, and the crushed cells were dispensed into 5 ml of ISOGEN (manufactured by Nippon Gene) in 15 ml. Into a Falcon tube (Falcon) and mixed well. After leaving at room temperature for 5 minutes, lml of cloper form was added and mixed well. After leaving at room temperature for 3 minutes, centrifugation was performed at 7,500 xg at 4 ° C for 15 minutes. The upper layer (aqueous phase) was collected, transferred to a 15 ml Falcon tube containing 2.5 ml of isopropanol, mixed, and allowed to stand at room temperature for 10 minutes. The mixture was centrifuged at 7,500 xg and 4 ° C for 10 minutes, and the RNA was recovered as a precipitate. After removing isopropanol in the supernatant, 5 ml of 70% ethanol was added to the precipitate, lightly suspended, and then centrifuged again at 7,500 × _g at 4 ° C. for 5 minutes to collect RNA as a precipitate and air-dried. RNA was dissolved in TE or RNase-free water (DEPC-treated water) 100-500 / xl to make a total RNA solution. Dilute 1 ^ -tal RNA solution 10- to 500-fold with TE or RNase free water and absorb at 260 nm and 280 nm. The degree was measured. High purity RNA with OD260 / OD280 values in the range of 1.8-2.0 was used in the following experiments. The total RNA solution was stored at -80 ° C until use. In this procedure, all the instruments and reagents used were RNAase-free. For details, refer to the manual attached to ISOGEN.

3. Purification of mRNA

Preparation of mRNA from total RNA was performed using MESSAGE MAKER manufactured by Promega, and the method followed the attached manual. The total RNA prepared in 2 was subjected to the following procedure to purify the mRNA. .

(1) Sample preparation

1) Transfer 2 mg of total RNA to a 15 ml Falcon tube, and add RNase-free water to 3.6 ml (to a final concentration of 0.55 mg / ml)

2) Incubate at 65 ° C for 5 minutes and quench on ice

3) Add 360M of 5M NaCl (to a final concentration of 0.5M NaCl)

4) Mix Oligo (dT) Cellulose Suspensio bottle well and add 2 ml to the solution obtained in 3).

5) Mix this sample solution well and heat it at 37 ° C for 10 minutes

6) Preheat RNase-free water for elution to 65 ° C

(2) Purification treatment

1) Put the sample solution directly into the syringe and push it off with the plunger

2) Put 5 ml of Wash Buffer 1 into the disposable cup and suck with a syringe

3) Suspend the liquid in the syringe well and drain the liquid.

4) Put 5 ml of Wash Buffer 2 into the cup of the disposer and suck with a syringe

5) Re-suspend the liquid in the syringe again and drain the liquid.

(3) mRNA elution

1) Add 2ml of RNase free water for elution heated to 65 ° C to the cup of disposable

2) Take up with a syringe, suspend well, and pour the solution into a 15ml Falcon tube.

3) Save this syringe for the second purification. (4) Purification treatment (second time)

1) Incubate the eluted sample solution at 65 ° C for 5 minutes, and rapidly cool on ice.

2) Add 5M NaCl 200/1 to make sample solution (final concentration 0.5M NaCl)

3) Suction the syringe used for the first purification and wash well with the attached Wash Buffer 1.

4) Put the sample solution in the disposable cup and suspend with a syringe.

5) Leave the syringe at room temperature for 10 minutes, then drain the liquid

6) Process in the same way as (2) to (5) in (3) above

(5) Elution treatment and ethanol precipitation

1) Process in the same way as (1) and 2) in (3) above

2) Centrifuge at 2,600G, 4 ° C for 3 minutes to remove contaminated Oligo (dT) Cellulose

3) Add so that the final concentration is 50 glycogen / ml, 0.75M ammonium acetate

4) Add 2 volumes of ethanol (-20 ° C) and put 1 晚

5) Centrifuge at 2,600G, 4 ° C for 30 minutes and rinse with 75% ethanol

6) After centrifugation at 2,600G, 4 ° C for 10 minutes, dry and dissolve in 10-30 l of ΤΕ or RNase free water.

4. Evening get labeling

A cDNA fluorescently labeled using the mRNA prepared in 3 above was prepared as a target probe for a polynucleotide array.

Add 25mer oligo (dT) primer 91 (4.5 g, adjust the concentration to 0.5 Ug / β)) to 2 to 4 g of poly (A) RNA (mRNA), and add TE to reduce the total volume. 15.4 1. After denaturation at 70 for 10 minutes, the mixture was quenched on ice, and the following reagents were added to perform fluorescent labeling by reverse transcription at 42 ° C for 1 hour. The reverse transcriptase used was Superscript manufactured by Gibco, and the DTT used was Superscript II.

5 X Superscript buffer 6 l

DTT 3 H 1

dNTP (25mMdA, G, C, 10mMdT) 0.6 H 1 Cy3- dUTP (or Cy5- dUTP) 3 β 1

Superscriptll 2 1

Further, Superscriptll 2 iI was added and reacted at 42 ° C. for 1 hour, and then TE270 Z1 was added. Cy3-dUTP and Cy5-dUTP were manufactured by Amersham-Pharmacia, and one of mRNA derived from a C-cell-supplied culture cell and one derived from a C-source-deficient culture cell was Cy3-dUTP, and the other was Cy5-dUTP. The reaction was performed. After the reaction, the Cy3 reaction solution and the Cy5 reaction solution were mixed, transferred to a Microcon-30 (manufactured by Millipore), and concentrated by centrifugation until the volume of the solution reached approximately ΙΟl (Eppendorf centrifuge 12,000 rpm, about 10 minutes ). TE400 1 was added to the upper part of the Microcon-30 forcep, and the mixture was centrifuged again until the liquid volume reached about 10 1 (12,000 rpm, about 15 minutes). The Microcon-30 cup was removed, inserted upside down into a new tube, and centrifuged at 3,000 rpm for 3 minutes. The following reagents were added to make the total amount about 301.

yeast tRNA llil (10 g, ΤΕ to adjust the concentration to 25 mg / 2.5 ml) polydA 4 Ι (4lig, TE to adjust the concentration to 5 U / 200 1)

20XSSC 0.85 / 11

1% SDS 1.5 1

TE about 13.

The approximately 301 overnight get for a polynucleotide array containing cDNA labeled with Cy3 or Cy5 was stored at -20 ° C in the dark until use.

5.Detection

The overnight get solution labeled with the fluorescent dye in 4 was denatured at 100 ° C for 1 minute, and left at room temperature for 30 minutes to obtain single-stranded labeled cDNA. A target solution of 7.5 1 was placed on a 22 mm × 22 mm cover glass, and covered with the polynucleotide array of the Aspergillus oryzae produced in Example 2 so that air bubbles did not enter. Slide glasses were placed in a wet box, and left overnight at 65 ° C to perform drying without drying. Dip slightly in 2XSSC / 0.1% SDS solution, gently shake off the force berglas, and gently shake in the solution 2-3 times. Then, it was shaken for 2 minutes in the 1 × SSC solution. Furthermore, it was left still in a 0.2XSSC solution for 2 minutes. After gently applying dry nitrogen gas to the slide surface and drying the slide, The analysis was performed using GTMAS Scanll manufactured by Laser Electronics. 6.Analysis results

1) Gene expression analysis in oryzae C source-supplemented medium and C source-deficient medium

86 clones were arbitrarily selected from the oryzae cDNA library prepared under the culture conditions with the C source in Examples 1 and 1, and a polynucleotide array was prepared by the method described in Example 2 (Figures 1 and 2). reference). In addition, 86 clones were selected from the C source-deficient culture conditions described in Examples 1 and 2, and a polynucleotide array was prepared in the same manner. Oryzae C source-provided cDNA from cells was fluorescently labeled with Cy5 (red), and cDNA from cells lacking C source was fluorescently labeled with Cy3 (green). Using these labeled cDNAs as targets, analysis was performed using a polynucleotide array. For clones showing characteristics in fluorescence intensity, the nucleotide sequence of the clone was determined, and the gene was estimated from a overnight base search using Blast or the like.

In the cDNA clone polynucleotide array derived from the culture conditions provided with the C source, about 70% of the spots, the fluorescence intensity derived from Cy5 (red) is higher than the fluorescence intensity derived from Cy3 (green), and the expression intensity is low. Some spots were observed at the same level (array arrangement shown in Fig. 1, array image shown in Fig. 4).

Analysis of the nucleotide sequence of the clone with high Cy5 fluorescence intensity revealed that, for example, clones with 1 row and 7 columns (fluorescence intensity of 7894 (Cy5) and 1452 (Cy3)) were homologs of the ^ ccaromyc cerevisiae HEM13 gene (SEQ ID NO: 1). ), 2 rows and 1 column (fluorescence intensity 5506 (Cy5) and 1117 (Cy3)) are Aspergillus oryzae pyruvate decarpoxylase gene (SEQ ID NO: 2), 3 rows and 3 columns (fluorescence intensity 6910 (Cy5) and 1351 (Cy5) Cy3)) was a glyceraldehyde-3-phosphate dehydrogenase monogene (SEQ ID NO: 3). It was a transcriptional regulator (prf: 2305380B) of Han niger (prf: 2305380B), which had strong fluorescence intensity at 7 rows and 3 columns (fluorescence intensity 3653 (Cy5) and 8736 (Cy3)). Six rows and three columns (fluorescence intensity 7451 (Cy5) and 5834 (Cy3)), in which both fluorescence of Cy3 and Cy5 showed the same intensity, were the histone H3 gene (SEQ ID NO: 5).

In addition, cDNA clones derived from C source-deficient culture conditions In 50% of the spots, the fluorescence intensity of Cy3 was higher than that of Cy5, and the other spots showed weaker expression intensity or the fluorescence intensity of the two dyes was observed to be comparable ( Array arrangement shown in Fig. 2, array image shown in Fig. 5). The clones with 5 rows and 1 column (fluorescence intensity 1862 (Cy5) and 4321 (Cy3)) with high Cy3 expression intensity were enolase gene (SEQ ID NO: 6), 6 rows and 7 columns (fluorescence intensity 1117 (Cy5) and 3267 (Cy3)) was the aldase gene (SEQ ID NO: 7). 3 rows and 6 columns (fluorescence intensity 4953 (Cy5) and 1951 (Cy3)), which had strong Cy5 fluorescence, were presumed to be a heat shock protein gene (SEQ ID NO: 8). The fourth row and the sixth column (fluorescence intensity 4381 (Cy5) and 3953 (Cy3)) in which both fluorescence of Cy3 and Cy5 are almost the same was estimated to be a cytoplasmic serine protease protein (SEQ ID NO: 9).

2) Gene expression analysis in ger C source supplemented medium and C source deficient medium

Similarly to 1), a polynucleotide array was prepared in which 82 clones selected from the oyzae C source-deficient culture conditions were prepared (array arrangement shown in FIG. 3, array image shown in FIG. 6). Using Aspergillus niger FGSC A798 strain donated by Fungal Genetic Stock Center, U.S.A., cDNA derived from cells with C source was fluorescently labeled with Cy3 and cDNA from cells lacking C source was labeled with Cy5. As a get, analysis using a polynucleotide array was performed. For clones showing characteristics in fluorescence intensity, the nucleotide sequence of the clone was determined, and the gene was estimated from a database search using Blast or the like. However, in the case of hybridization targeting fluorescently labeled cDNA derived from niger, the temperature of hybridization depends on the type of clones arranged on the array. It is necessary to set to. In this example, the test was performed at 53 ° C.

As a result, clones in the first row and second column (fluorescence intensity 3017 (Cy3) and 1153 (Cy5)) and the first row and column 10 (fluorescence intensity 3203 (Cy3) and 1050 (Cy5)), which had strong fluorescence in Cy3 As a result of its nucleotide sequence analysis, was a β-1,3-glucanase gene homolog (SEQ ID NO: 10) and a sconB gene homolog (SEQ ID NO: 11) of Aspergillus nidulans, respectively. 4 rows and 4 columns (Fluorescence intensity 1543 (Cy3) and 4751 (Cy5)), 4 rows and 5 columns (Fluorescence intensity 1123 (Cy3) and 4213 (Cy5)), 6 rows and 6 columns ( The fluorescence intensities 917 (Cy3) and 2981 (Cy5)) are the homologues of Aspergillus nidulans alcohol dehydrogenase gene (SEQ ID NO: 12), a yeast translation factor SU1 gene homolog (SEQ ID NO: 13), and 4 erg Z ^ oryze phosphoglycerate kinase gene (SEQ ID NO: 14).

From the above detection results, it was shown that it is possible to capture the gene expression response of the heterologous filamentous fungus niger using the cDNA array of cDNA or zae.

[Example 4] Detection of gene expression using Aspergillus mold polynucleotide array (2)

From the cDNA libraries derived from various media of Example 1, 2170 amounts of various independent clones were selected, and the same polynucleotide array in which each clone was spotted in duplicate was prepared (2000 clone array). FIG. 7 (panel A) shows a schematic diagram of the polynucleotide array used in this example, and FIGS. 8A to 8H show the list of clones mounted (indicated by library Accession No.). FIGS. 8A to 8H show clones of genes contained in the 1st to 32nd segments of the block sequence of FIG. 7A, and 1 to 32 shown in FIGS. 8A to 8H show the block sequence of FIG. Corresponding to the number. Figure 7 Panel B is an expanded view of each section in Figure 7A, with 12 or 10 spots in columns and 12 (3, 5, 6) spots in rows. Row of

“(3,5,6)” indicates that the spots in the 11th and 12th columns are 3 spots (10-12 rows, for example, No. 5 in FIG. 8B), 5 spots (8-12 rows, for example, FIG. 8D No. 15) or 6 spots (lines 7 to 12, for example, No. 7 in FIG. 8B). Each clone shown in Fig. 8 is spotted in two rows. For example, in the first section of FIG. 8A, the positions where the clone “JZ1823” is spotted are at row 1 column 1 and row 1 column 2 in FIG. 7B. The gene expression analysis of Aspergillus oryzae ^ Aspergillus niger was performed for kX ~ Y, 2000 chronoarray.

1) Gene of oryzae in C source-supplemented medium and C source-deficient medium

CDNA derived from the C source-supplied cells of oryzae was fluorescently labeled with Cy3, and cDNA derived from the C source-deficient cells was fluorescently labeled with Cy5. Using these labeled cDNAs as targets, analysis was performed using a 2000 clone array. The results are shown in FIG. FIG. 9 shows the flow arrangement of the block arrangement of FIG. is there. Therefore, the first section of the block arrangement is in the upper left of FIG. 9, and the fourth section is in the upper right of FIG. As a characteristic example of the analysis results, the gene specifically expressed in the C source deficiency state was the aldolase gene, and most of the other glycolytic genes were expressed in the C source state. Figure 10 shows a comparison between Northern analysis and polynucleotide array analysis projected onto a metabolic map. In FIG. 10, the substances in the boxes are the names of the enzymes that act in the glycolysis system, and the MIX numbers described on the right side of the boxes represent the independent EST clones grouped by the class ring. The results of the polynucleotide array of the genes encoding each enzyme and the results of the Northern analysis were in good agreement.

2) Growth time and gene expression in medium with rC source

The Aspergillus niger FGSCA798 strain donated by Fungal Genetic Stock Center in the United States was cultured for 16 hours or 26 hours in a C source-supplied medium. CDNA prepared from mRNA derived from 16 cultured cells was labeled with Cy3, and cDNA derived from 26 hours cultured cells was fluorescently labeled with Cy5. Using these labeled cDNAs as targets, analysis using a 2000 clone array was performed. For clones showing characteristics in fluorescence intensity, the nucleotide sequence of the clone was determined, and the gene was estimated from a database search using Blast or the like. However, in the case of hybridization targeting fluorescence-labeled cDNA derived from niger, the hybridization temperature depends on the type of clones arranged on the array. It is necessary to set to. In this embodiment, the temperature was set at 53 ° C. Fig. 11 shows an example of hybridization. In the 16-hour culture, Zn transport protein and xylosidase (Zn protein, alpha-xylosidase) were specifically expressed, and in the 26-hour culture, α-amylase and glutamate-ammonium ligase were specifically expressed.

From the above detection results, it was shown that the present polynucleotide array can be effectively used for gene expression tests of heterologous filamentous fungi such as not only oryzae, but also heterozygous fungi, even when the number of loaded genes is increased. This is the description of PCT Application No. PCT / JP01 / 02823, which is the priority document of the present application. And / or the contents described in the drawings. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial applicability

According to the present invention, there is provided a polynucleotide array having a polynucleotide derived from a filamentous fungus immobilized thereon and a method for detecting a gene. The polynucleotide array of the present invention realizes simultaneous, simultaneous hybridization analysis of many polynucleotide species of filamentous fungi, which has been extremely difficult in the past. As a result, it is possible to achieve automation and labor saving of gene expression analysis, precise identification or identification of related species, mutants and the like. In addition, this system can be used for advanced optimization of fermentable production, and it also provides a screening system for useful (harmful) substances.

Claims

The scope of the claims

1. A polynucleotide array in which probes containing all or part of the nucleotide sequence of a nucleic acid derived from a filamentous fungus are immobilized on a support.

2. The nucleic acid is selected from the group consisting of a C source-added culture condition, a C source-deficient culture condition, a maltose culture condition, an alkaline culture condition, a solid culture condition, a high temperature culture condition, a solid low temperature culture condition, and a spore germination culture condition. 2. The polynucleotide array according to claim 1, which is a polynucleotide containing a cDNA region produced from a cDNA library derived from a filamentous fungus cultured under at least one selected condition.

3. The polynucleotide array according to claim 1, wherein the nucleic acid is genomic DNA, cDNA, RNA, or oligonucleotide DNA.

4. The polynucleotide array according to claim 1, wherein the filamentous fungus is a microorganism belonging to the genus Aspergillus, Penicillium, Fusarium, Trichoderma or Mucor.

5. Microorganisms belonging to the genus Aspergillus are Aspergillus oryzae, Aspergillus zoja, Aspergillus niger, Aspergillus amori, Aspergillus ryuti, Aspergillus parasites, Aspergillus flavus, Aspergillus, Aspergillus, Aspergillus The polynucleotide array according to claim 4, wherein the polynucleotide array is at least one selected from the group consisting of Aspergillus nidulans.

6. The poly according to claim 4, wherein the microorganism belonging to the genus Aspergillus is at least one selected from the group consisting of Aspergillus oryzae, Aspergillus socia, Aspergillus parastics, Aspergillus flavus, and Aspergillus nomius. Nucleotide array.

7. The polynucleotide array according to claim 4, wherein the microorganism belonging to the genus Aspergillus is Aspergillus oryzae.

8. The polynucleotide array according to any one of claims 1 to 7, wherein the support is a cellulose polymer, a nylon polymer, glass, a non-porous material, or a porous material.

9. After labeling the target polynucleotide for detection, it is hybridized with the probe immobilized on the polynucleotide array according to any one of claims 1 to 8, and a signal is detected from the obtained hybridization product. A method for detecting a target polynucleotide.

10. The detection method according to claim 9, wherein the label is a fluorescent label, a radioactive label, an electronic label, or a multi-stage modified label.

11. The detection method according to claim 9 or 10, wherein the target polynucleotide for detection is genomic DNA, cDNA, RNA, or oligonucleotide DNA.

12. The detection method according to any one of claims 9 to 11, wherein the type of the probe is 20 to 100,000.

13. The detection method according to any one of claims 9 to 12, wherein the target polynucleotide for detection is derived from a filamentous fungus.

14. The detection method according to claim 13, wherein the filamentous fungus is at least one of microorganisms belonging to the genera Aspergillus, Penicillium, Fusarium, Trichoderma or Mucor.

15. Microorganisms belonging to the genus Aspergillus are Aspergillus' orize, Aspergillus soja, Aspergillus niger, Aspergillus amori, Aspergillus sp. 15. The detection method according to claim 14, wherein the method is at least one selected from the group consisting of Aspergillus nidulans.

16. The detection method according to claim 14, wherein the microorganism belonging to Aspergillus で is at least one selected from the group consisting of Aspergillus oryzae, Aspergillus zoya, Aspergillus'parasitics, Aspergillus flavus, and Aspergillus nomius.

17. The detection method according to claim 14, wherein the microorganism belonging to the genus Aspergillus is Aspergillus oryzae.

18. A method for identifying and / or discriminating a closely related filamentous fungus or a mutant thereof using the detection result obtained by the detection method according to any one of claims 9 to 17 as an index.

19. After a candidate sample of a useful substance or a harmful substance to be screened is allowed to coexist with at least one of the filamentous fungi, the nucleic acid is isolated and labeled from the filamentous fungus, and the labeled nucleic acid is claimed. And hybridizing with the probe immobilized on the polynucleotide array according to any one of (1) to (8), detecting a signal from the obtained eight hybridization products, and screening for useful and / or harmful substances using the obtained detection result as an index. Method.

20. The method according to claim 19, wherein the useful or harmful substance is a drug.

21.Isolating and labeling each nucleic acid from filamentous fungi cultured under different culture conditions, hybridizing the labeled nucleic acid with the probe immobilized on the polynucleotide array according to any one of claims 1 to 8, A method for detecting a signal from the obtained hybridization product and optimizing the expression of the target gene of the filamentous fungus using the obtained detection result as an index.