WO2009142313A1 - Method for searching for gene encoding protein targeted by compound - Google Patents

Abstract

Disclosed is a method for searching for a gene encoding a protein targeted by a compound rapidly and in a simple manner.  Also disclosed is a method for selecting a compound and searching for a gene encoding a protein targeted by the compound.

Description

 Description Method of searching for a gene encoding a target protein of a compound Technical Field

 The present invention relates to a method for searching for a gene encoding a target protein of a compound used in medicine, agricultural chemicals and the like. Background art

 Conventionally, in order to search for proteins and genes targeted by compounds such as pharmaceuticals and agricultural chemicals, mutants that are resistant to the compound are selected from pull-down methods and plant mutants that have point mutations. Methods for identifying genes are used.

 The pull-down method is a method for obtaining a target protein by applying a sample such as a cell extract to a carrier on which a compound is immobilized. For example, Non-Patent Document 1 describes that a target protein (receptor) that binds to tacrolimus is obtained by binding takuguchi rimus on a carrier. However, the pull-down method requires that the compound be synthesized in order to bind the compound to the carrier, that even if the compound can be immobilized on the carrier, the target protein may not bind to the immobilized compound. When the binding force between the target protein and the target protein is weak, it is difficult to purify the target protein, and it is necessary to optimize the conditions for stable purification of the target protein. In particular, it usually takes several months to make a suitable carrier with immobilized compounds.

It takes a year.

A method of selecting a mutant resistant to a compound from plant mutants having the above point mutation and identifying a target protein and its gene is carried out by treating the plant with a mutagen such as ethyl methanesulfonate. This is a method of isolating the causative gene by searching for plants that have acquired resistance to the compound by reducing the binding power between the protein encoded by the mutated gene and the compound. For example, Non-Patent Document 2 includes a medium containing a novel phenyltriazole acetic acid called DAS 7 34, Screening 48,000 Arabidopsis treated with mutagen, selecting resistant mutants that can grow even in the presence of DAS 7 3 4 and performing map-based cloning to identify genes responsible for resistance Is described. This method uses plants whose DNA has been unmethylated by mutagenesis. Therefore, in order to obtain mutants (target mutants) that have target proteins whose binding ability to compounds has been reduced due to accidental mutations, a large number of plants must be screened stochastically. After acquiring a resistant mutant, it is necessary to identify the target gene by map-based cloning, etc., and to acquire multiple alleles and prove that the resistance is due to the mutation of that gene. There are problems such as. In this method, it takes about 1 to 2 years to obtain a target gene candidate.

 In Non-patent Document 3, the mevalonate pathway (MVA pathway) and the non-mevalonate pathway (MEP pathway) are blocked with mevinolin and phosmidomycin, respectively, and over-expressed gene of Arabidopsis thaliana that is resistant to these drugs (activation) The tag line) is selected. The experiments in this document make use of the known actions of mevinolin and HM G—Co A reductase and the known actions of phosmidomycin and D XP reductase isomerase. Further, the activation tag line used in this experiment is a mutant in which DNA containing an enhancer is randomly introduced into the plant genome to overexpress the plant gene. In the activation tag line, the genomic region that can activate transcription covers a wide range before and after the insertion site of the enhancer, and there can be multiple genes in that range, so which gene is activated by the enhancer. There are problems such as difficulty in determining whether or not the gene has been inserted, and if an inserter is inserted into the coding region of a gene, a mutant lacking the function of the gene is generated.

Non-Patent Document 4 includes overexpressing a yeast genomic library in yeast (Saccharorayces cerevi siae) and screening yeast that is resistant to phenylaminopyridine from the gene overexpressing yeast. Is described. In addition, non-patent document Rk 5 t, describes that Mycobacterium avium chemomefifury ¹ ¾r Mycobacterium smegmat is overexpressed and screens bacteria resistant to meflokinin from the gene overexpressing bacteria. Yes. However, In addition, the phenotypes that microorganisms exhibit when compounds are applied to these microorganisms are limited to the suppression of the growth of microorganisms and the change in the content of sugars, proteins, lipids, and the like.

 Non-Patent Document 1 Harding MW. Et al, A receptor for the immuno-suppressant FK506 is a cis-trans peptidyl-prolyl isomerase ", Nature (1989), 341, p.758-760.

 Non-Patent Document 2 Terence A. Walsh, et al, "Chemical Genetic Identification of Glutamine Phosphor ibosylpyrophosphate Amidotransf erase as the Target for a Novel Bleaching Herbicide in Arabidopsis, Plant physiol. (2007), 144, p.1292-1304.

 Non-Patent Document 3 Manuel Rodriguez- concepcion, et al, "Distinct Light-Mediated Pathways Regulate the Biosynthesis and Exchange of Isoprenoid Precursors during Arabidopsis Seedling Development", The Plant Cell (2004), Vol. 16, p.144-156.

 Non-specific S No. 4 Hendrik Luesch, et al, "A Genome-Wide Overexpression Screen in Yeast for Small-Molecule Target Identification, Chemistry & Biology (2004), vol.12, p.55-63.

 Non-Patent Document 5 Lia Danelishvili, et al, "Genomic Approach to Identifying the Putative Target of and Mechanisms of Resistance to Meiloquine in Mycobacteria", Antimicrobial Agents and Chemotherapy (2005), vol.49, No.9, p.3707-3714 Disclosure of the invention

 An object of the present invention is to provide a method for easily and rapidly searching for a gene encoding a protein that is a target of a compound.

 As a result of intensive studies to solve the above problems, the present inventors have completed the present invention by applying a gene function analysis method called Fox hunting system.

The present invention is summarized as follows. [1] The following steps (a) to (d) _:

 (a) introducing a full-length cDNA library into a plant to produce a full-length cDNA overexpression line;

 (b) applying a compound that changes the phenotype of a wild-type plant to an overexpression line,

(c) selecting an overexpression line that exhibits a phenotype different from the altered phenotype of the wild-type plant; and

 (d) A method for searching for a gene encoding a protein targeted by a compound, comprising the step of identifying a full-length cDNA introduced into a selected overexpression line.

[2] The following steps (e) to (i) _:

 (e) applying a group of compounds to a wild-type plant to select a compound that changes the phenotype of the wild-type plant;

 (f) introducing a full-length cDNA library into a plant to produce a full-length cDNA overexpression line;

 (g) applying a compound that changes the phenotype of a wild-type plant to an overexpression line;

(h) selecting an overexpression line that exhibits a phenotype different from the altered phenotype of the wild-type plant; and

 (i) A method for selecting a compound and searching for a gene encoding a target protein of the compound, comprising the step of identifying a full-length cDNA introduced into the selected overexpression line.

 This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2008-133148, which is the basis of the priority of the present application. Brief Description of Drawings

 Figure 1 shows the flow of the verification experiment of action point search using the model compound FSM. Panel (1) shows the whitening of buds of wild-type Arabidopsis when applying FSM at concentrations of 0 // Μ, 100 / ζΜ, 200 μ300 / M, and 400. Panel (2) shows the screening of FSM resistant FOX lines (not whitened).

Figure 2 shows the selection of new whitening agents from the chemical library and the search for their action points. The flow of the cord is shown. Panel (1) shows the whitening of buds of wild-type Arabidopsis when each compound is applied. Panel (2) shows the screening of the PSC 1 resistant FOX line (not whitened). BEST MODE FOR CARRYING OUT THE INVENTION

 1. A method for searching for genes that encode proteins targeted by compounds

 A method for searching for a gene encoding a protein that is a target of the compound of the present invention includes:

(a) introducing a full-length cDNA library into a plant to produce a full-length cDNA overexpression line;

 (b) applying a compound that changes the phenotype of a wild-type plant to an overexpression line,

(c) selecting an overexpression line that exhibits a phenotype different from the altered phenotype of the wild-type plant; and

 (d) identifying a full-length cDNA introduced into the selected overexpression line.

 As used herein, “a protein targeted by a compound” refers to a protein that interacts with a certain compound. The interaction between the compound and the target protein is not particularly limited, and examples include reversible and irreversible binding of the compound to the protein, and the action of the protein metabolizing the compound to change the compound into another structure. It is. In the present specification, a protein targeted by a compound or a gene encoding the same may be referred to as “point of action”.

 The “full-length cDNA overexpression line” used in the present invention is called a Fox hunting system, and it is designed to control the function of a gene from changes in traits caused by introducing and overexpressing full-length cDNA into a plant. It is a line produced by the method to be clarified (see WO 2003 No. 0 1808). In the present specification, a full-length cDNA overexpression line is sometimes referred to as a FOX line.

 The FOX line is a mutation of so-called gain of function, which is created by infecting a plant with a library prepared by connecting full-length cDNA to a strong expression vector and introducing full-length cDNA into the plant. It is a body system.

In the production of the FOX line, the plants into which full-length cDNA is introduced are particularly limited. However, Arabidopsis is preferable. The full-length cDNA to be introduced is not particularly limited, and may be derived from any organism such as a plant, animal, or microorganism. For example, when searching for the site of action of a herbicide, a FOX line in which weed full-length cDNA is introduced into Arabidopsis thaliana can be prepared. In addition, it is not always necessary to prepare the full-length cDNA of the target organism. For example, malaria parasites have a non-mevalonate pathway, but because higher plant plastids also have a non-mevalonate pathway, malaria parasites and plants share enzymes that work in the non-mevalonate pathway. Therefore, FOX lines prepared using a full-length cDNA library of higher plants (including full-length cDNA encoding an enzyme that works in the non-mevalonate pathway) were screened with antimalarial drugs and selected. The full-length cDNA overexpressed in the FOX line can be identified and the site of action of antimalarial drugs can be searched.

 The following describes the FOX line preparation method (step (a)), using Arabidopsis thaliana (Arabidopsis thaliana FOX line) overexpressing the full-length cDNA of Arabidopsis thaliana as an example.

 About 15,000 full-length cDNAs of independent Arabidopsis thaliana were prepared as a pool of cDNA equivalence ratios (referred to as standardization), and this cDNA was used as a promoter, enhancer (transcription enhancer E 2 1, omega sequence, etc. ) Incorporated into a T1 DNA vector having a control region such as a terminator and a selectable marker such as a drug resistance gene. The T-DNA vector was introduced into agrobacterium to produce a complete Arabidopsis full-length cDNA expression library, and after confirming whether the entire cDNA distribution was reflected in the library, Transform Arabidopsis thaliana using pacterium by the floral debating method to create an Arabidopsis transformation line (Arabidopsis FOX line). According to this method, even if a plant is infected with a library of hundreds of millions of clones, only one or two clones are introduced into one plant. Therefore, it is possible to produce a transformation line in which different clones are introduced into a plant. it can. Collect T 1 seeds from the transformed Arabidopsis thaliana,

T 1 seeds are sown and T 2 generation seeds are collected. The Arabidopsis full-length Arabidopsis FOX line that overexpressed Arabidopsis thaliana FOX line and the rice full-length cDNA that overexpressed DNA FOX line are both incorporated by the National Institute of Physical and Chemical Research. Flight number 3 5 1— 0 1 9 8 Available from Hirosawa No. 2 (1), Wako City, Saitama Prefecture, Japan.

 As used herein, “wild type plant” refers to a plant into which full-length cDNA has not been introduced. A “compound that changes the phenotype of a wild-type plant” refers to a compound that changes some phenotype of a wild-type plant when the compound is applied to the wild-type plant. The phenotypic change is not particularly limited, and the plant is whitened in the light, germination inhibition, plant height change (fertility or hypocotyl elongation), true leaf suppression, root elongation change, flowering change , Change in flower color, change in flowering period, sterilization, change in content of sugar, protein, lipid, etc., change in resistance to disease, suppression of changes in plants in plants (extension of hypocotyl hypocotyl) A phenotype that can be observed as a change in the appearance (form) of the plant is preferable. The method of applying the compound to a plant is not particularly limited, a method of sowing seeds in a medium containing the compound, a method of germinating seeds and planting seedlings in a transplanting medium containing the compound, a plant body (leaves, stems, flowers, etc.) ), A method of applying or spraying a compound, a method of growing seedlings in a liquid medium containing the compound, and the like, but a method of sowing seeds in a medium containing the compound is preferable.

 It is preferable to determine the concentration of the compound before applying the compound to the FO X line in step (b). For example, several concentrations of compound can be prepared and applied to wild type plants to determine the concentration. In wild-type plants, the concentration of the compound is a concentration that clearly changes its phenotype, and in the FOX line into which the full-length cDNA encoding the target protein of the compound has been introduced, It is preferable to determine the concentration so that the mold is not changed or the degree of change is low.

A compound that changes the phenotype of a wild type plant is applied to the FOX line (step (b)), and a FOX line that exhibits a phenotype different from the changed phenotype of the wild type plant is selected (step (c)). . An “overexpression line (FOX line) that showed a phenotype different from the altered phenotype of the wild-type plant” means, for example, that if the altered phenotype when the compound is applied to a wild-type plant is whitened, The line is not whitened or has a low degree of whitening. The protein encoded by the full-length cDNA introduced into the line is overexpressed, for example, the excess protein metabolizes the compound and changes the structure of the compound, or the excess protein binds to the compound The It is thought that in becomes resistant to the compound (does not whiten). In addition, if the altered phenotype when the compound is applied to a wild-type plant is dwarfing plant height, it is a line that has not dwarfed or has a low degree of dwarfing.

 Genomic DNA is extracted from the selected FOX line, and PCR is performed using the genomic DNA as a saddle. Primers are designed based on the promoter sequence and the terminator sequence included in the T1 DNA used in FOX line preparation. Using the primer, the base sequence of the amplified product is read with a DNA sequencer, and the full-length cDNA introduced into the selected FOX line is identified (step (d)). The identification can be performed, for example, by searching a nucleotide sequence in a database of NCBI (National Center for Biotechnology Information) or an Arabidopsis gene database of RIKEN.

 In the present invention, further steps may be included in the steps (a) to (d). For example, the cDNA amplified in step (d) is again inserted into TDNA having the above promoter sequence, terminator sequence, etc., and this is reintroduced into a wild type plant of the same type as the plant in the FOX line. Then, it may be confirmed whether the resistance to the compound is reproduced. Alternatively, you can analyze the interaction between the compound and the target protein using known methods. Examples of interaction analysis methods include intermolecular specificity, force enable, and affinity analysis methods using the Biacore system (GE Healthcare, etc.), QCM (Quartz Crystal Microbalance) (Tama Devices Co., Ltd., etc.). Analytical method for examining molecular adhesion, analysis method for examining thermal changes associated with intermolecular interactions using Micro C a 1 (Nippon Siebel Hegner, etc.), — Material analysis by NM R Not.

 2. Method of selecting a compound and searching for a gene encoding a protein targeted by the compound

 The method of the present invention is particularly useful when a compound group (chemical library) whose function or the like is unknown is used as a test compound. According to this method, selecting a candidate compound from the compound group and searching for a gene encoding a protein that is a target of the candidate compound can be performed simultaneously in a series of steps.

Selection of compounds of the present invention and genes encoding proteins that are targets of the compounds How to explore

 (e) applying a group of compounds to a wild-type plant to select a compound that changes the phenotype of the wild-type plant;

 (f) introducing a full-length cDNA library into a plant to produce a full-length cDNA overexpression line;

 (g) applying a compound that changes the phenotype of a wild-type plant to an overexpression line;

(h) selecting an overexpression line exhibiting a phenotype different from the altered phenotype of the wild-type plant; and

 (i) identifying the full-length cDNA introduced into the selected overexpression line.

 In the step (e), a compound group to be applied is not particularly limited, but a compound group such as a chemical library synthesized by a combinatorial chemistry technique and including a derivative and a precursor is preferable. The method of applying the compound to plants and the altered phenotype of wild-type plants are as described above. In the step (e), when the phenotype is plant whitening, the seeds of the wild type plant were sown in a medium containing the compound and a medium not containing the compound, and germinated in the medium containing no compound. When the buds of wild-type plants germinated in the medium containing the compound are whitened, the compounds contained in the medium are selected. In addition, preferably, for the selected compound, the compound concentration when applied to the overexpression line in step (g) is determined in the same manner as described above. Furthermore, optionally, the relationship between the selected compound and the known protein and its gene (known action point) may be examined. The selected compound is applied to a plant overexpressed by introducing the gene, and it is examined whether or not it exhibits a phenotype different from the altered phenotype of the wild-type plant. If the phenotype is different from the altered phenotype of the wild-type plant, it can be assumed that the compound works at a known site of action. If the phenotype is similar to that of a wild-type plant, it can be assumed that the compound works at an unknown site of action.

The steps (f), (g), (h), (i) may be performed in the same manner as the above steps (a), (b), (c), (d), respectively. In addition to these steps, the above-mentioned additional steps (confirmation by reintroducing the identified gene into the plant, analysis of intermolecular interactions using the Biacore system, QCM, or Micro C a 1, NMR Quality analysis).

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

 [Example 1] Production of Arabidopsis FOX line

 In this example, the vector constitutive expression vector p BIG 2 11 13 N (Taji, T. et al. Plant J., 2002.24 (4): pp417-426 and Becker, D. 'et al. Nucleic Acid Res., 1990.18 (1): p BIG 2 1 1 3 SF with S fi I cloning site introduced into p.203) was used.

 (1) Preparation of standardized Arabidopsis full-length cDNA mix

 Full-length cDNA was prepared from Arabidopsis thaliana by the CAP tr a p p e r method. This cDNA was cloned into a site sandwiched by S fi I restriction enzyme cleavage sites of Lamb da ZAP or Lamb dap LC—l—B (Seki M. et al. Plant J., 15, 707-720). (1998)). Using the vector sequence, read the sequences at the 5 'end and 3, and the end of cDNA and perform cDNA grouping.

00 clones were identified (Seki M. et al. Plant Physiol. Biochem. 39, 211-220 (2001)). Next, 0.5 μl was separated from each clone prepared to 50 ng Z / z 1 and mixed in one tube. A 1-minute aliquot of this mixture was taken and transformed into 20 μ 1 of electric competent cell DH10B (Gibco BRL, USA). About 200,000 independent colonies grown on an agar medium containing Amp were mixed, and the plasmid was recovered therefrom. This was a standardized Arabidopsis full-length cDNA mix.

 (2) Construction of Arabidopsis FOX agrobacteria library

 2 μg of standardized Arabidopsis full-length cDNA mix and 700 / zg p B

1 G 2 1 1 3 S F was mixed and then completely cut with S f i I at the same time. After cleavage, the mixture was concentrated by isopropanol precipitation, dissolved in 8 IX 1 of water, 1; u 1 of 10 X buffer and 11 T 4 ligase were mixed, and reacted at 16 ° C. overnight. 2 // 1 reaction solution

40 1 Electric competent cell DH10B was mixed and transformed.

 About 1 independent colony grown on agar containing kanamycin (Km)

50,000 were mixed, and the plasmids were collected from there. Collected 2 μI bra The smudge solution was mixed with 40 μl of Electric competent Agrobacterium cell GV3101 for transformation. Approximately 150,000 independent colonies grown on an agar medium containing Km were suspended in LB liquid medium, glycerol was added to 15%, and stored at −80 ° C. This glycerol solution was used as an Arabidopsis FOX Agrobacterium library.

(3) Arabidopsis FOX line production

 The above Arabidopsis F OX agropacteria library is grown in colonies, suspended in a dipping solution, and then wild-type Arabidopsis (Colombia (C o 1-0)) floral diving. Went. Seed (T 1 seed) is harvested, germinated on an oligotrophic medium B AM containing hygromycin, and only about 15, ◦ 0 0 line plants showing hygromycin resistance are transplanted to the soil, and T 2 seed Got. The following experiment was conducted using the Arabidopsis FOX line produced and established in this way.

 [Example 2] Verification experiment of action point search using model compound phosmidomycin In this example, phosmidomycin (FSM) was used as a model compound, and the verification experiment of the present invention was performed. FSM is known as an antimalarial lead compound, and in plants, it inhibits the non-mevalonate pathway present in the plastids and has the effect of whitening the plant.

 (1) Determination of FSM concentration to whiten Arabidopsis

 Wild-type Arabidopsis thaliana was used to examine the lowest FSM concentrations that clearly whiten Arabidopsis thaliana. Concentration 0; uM, 1 00 M, 2 00 / M, 3 00 μΜ, and 4 0 0 / χΜ on agar medium containing FSM, in the dark to match the timing of germination, 4 The seeds of wild-type Arabidopsis thaliana treated at low temperature at ° C were sown and grown for 2 weeks under 22 ° C light conditions. Wild-type Arabidopsis thaliana germinated on an agar medium with a FSM concentration of 0 μΜ was green, whereas wild-type Arabidopsis thaliana germinated on an agar medium with a concentration of 100 μΜ was yellow-green, and at a concentration of 200 μΜ It was more whitened (Panel (1) in Figure 1). The concentration of FSM used in the screen (2) below was set to 1 ° θ ίΜ.

(2) F SM resistance F ΟΧ line screening Arabidopsis FOX lines were divided into 250 lines and used for FSM resistance line screening. Ten FOX line seeds (T2 seeds) were collected from 10,000 independent lines each, and dispensed into 15 ml conical tubes every 250 lines. To this, 5m 1 of 2% concentration PPM ^™ (Plant Preservative Mixture) solution (Nacalai Tesque) was added and sterilized at 4 ° C in the dark. After low-temperature treatment, the seeds were washed three times with sterilized water, and 0.1% agar solution (8 ml) was added thereto and vortexed. Prepare 40 pieces of LB agar medium (50 mL) containing 1% sucrose containing 100 ppm / iM FSM in a sterile No. 2 petri dish, pour the seed-containing agar solution on each plate, and seed on the plate. Was evenly distributed. After drying the surface until the seeds were fixed, they were grown under 22 ° C light conditions and screened for FSM-resistant FOX lines (lines that were not whitened)

(Panel (2) in Figure 1). As a result, an independent FSM-resistant FOX line was obtained. After 10 days, the FSM-resistant FOX lines were transferred to fresh LB agar plates containing 1% sucrose and grown.

(3) Identification of FSM resistance gene

 Genomic DNA was extracted from the FSM-resistant Arabidopsis FOX line according to a conventional method, and PCR was performed using this as a template. PCR includes 5'-GGAAGTTCATTTATTCGGAGAG-3 '(SEQ ID NO: 1) and 5,-GGCAACAGGATTCAATCTTA-3,

The primer of (SEQ ID NO: 2) was used. The amplification product was sequenced, and a primer of 5, -CCCCCCCCCCCCD-3 '(SEQ ID NO: 3, D represents A, G or T) was used for the sequence. As a result, 1 line out of 11 lines that showed FSM resistance was the target enzyme gene DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase gene, Arabidopsis gene number At5g62790, nucleic acid sequence: SEQ ID NO: 4 The amino acid sequence: SEQ ID NO: 5) was confirmed to be overexpressed. In the other two lines, it was considered that the gene encoding an unknown UGT (glycosylation enzyme) that is the target of FSM may be overexpressed. The other 8 lines were considered to be false positive lines because they did not show FSM resistance in the reproducibility test.

[Example 3] Selection of new whitening agent from chemical library and its action point Search for

 In this example, a new whitening agent was selected from a chemical library, and an experiment was conducted to find a target candidate protein of the selected whitening agent and its gene (action point).

(1) Selection of new whitening agents from chemical libraries

 A chemical library was selected from 10,000 compounds (ChemBridge Corporation) to whiten wild-type white plants. C0STAR (registered trademark) 96-well plate (Sigma Aldrich Co., Ltd.) 10 L of water, 1 nL of compound (final concentration approx. 1 00 // M), 100 μL of MS agar This was dispensed with about 10 grains each of 0.1% agar solution in which seeds of wild-type Arabidopsis thaliana were suspended. The plants were grown for 1 week under 22 ° C light conditions, and we observed whether the buds of wild-type Arabidopsis thaliana were whitened (panel (1) in Fig. 2). The 108 compound was selected as a whitening agent. Next, 33 compounds with unknown functions and new structures were selected from 108 compounds using the chemical substance database (SciFinder).

 (2) Arabidopsis Determination of whitening agent concentration for screening the FOX line 3 Select PSC 1 (molecular weight 350.4, Fig. 2) with the strongest whitening action from the three compounds, and wild type Arabidopsis thaliana Concentrations exhibiting a unique phenotype (whitening) were examined.

 Wild-type Arabidopsis seeds that had been cold-treated on agar media containing various concentrations (1-100 / χ 1) of P S C 1 were sown and grown under 22 ° C light conditions for 2 weeks. As a result, the minimum concentration of PSC1 required to whiten wild-type Arabidopsis thaliana was 20 μΜ, and it was decided to screen the Arabidopsis FOX line at this concentration.

 (3) Whitening agent resistant Arabidopsis FOX line screening

 The FOX line was divided into 250 lines and used for screening. FOX line seeds (T 2 seeds) were sorted from 10,000 independent lines, 10 grains each, and dispensed into 15 ml conical tubes every 250 lines. To this, 5 ml of PPM ™ solution was added, and the mixture was processed at a low temperature at 4 ° C under a certain temperature. After low-temperature treatment, the seeds were washed three times with sterilized water, and 0.1% agar solution (8m 1) was added thereto and vortexed. Sterilization

No. 2 petri dish containing 1% sucrose containing PSC 1 (20 μM) 40 pieces of (4 OmL) were prepared, and the agar solution containing FOX seeds was poured on each of them to disperse the seeds uniformly on the plate. After drying the surface until the seeds were fixed, they were grown under 22 ° C light conditions and screened for PSC 1-resistant Arabidopsis FO X line (not whitened) (Panel in Fig. 2 (2 )). As a result, three PSC 1 resistant FOX lines were obtained from each of the three plates. After 10 days, the PSC 1 resistant FOX line was transferred to a new 1% sucrose LB agar plate and grown.

 (4) Identification of gene encoding P S C 1 resistance protein

 Genomic DNA was extracted from the PSCl-resistant FOX line according to a conventional method, and PCR was performed using this as a template. The primers of SEQ ID NO: 1 and SEQ ID NO: 2 were used for PCR. The amplification product was sequenced, and the primer of SEQ ID NO: 3 was used for the sequence. As a result, all four independent lines showing PSC 1 resistance overexpressed the common function unknown gene At5g61820 (nucleic acid sequence: SEQ ID NO: 6, amino acid sequence: SEQ ID NO: 7). It was isolated as a gene encoding a target candidate protein. Industrial applicability

 According to the present invention, a target protein of a compound and a gene encoding the protein can be searched easily and quickly. The present invention elucidates the action mechanism of a compound and predicts side effects on organisms. Etc. are useful. In addition to the target protein and its gene, the acquisition of various genes involved in resistance to the compound is expected, and it becomes possible to create compound-resistant plants by overexpression of these genes.

 In the present invention, various compounds including compound derivatives or precursors, and compounds with unknown functions can be tested. Therefore, from compounds synthesized by combinatorial chemistry technology, pharmaceuticals, agricultural chemicals, chemicals It is an appropriate method for screening compounds (lead compounds) suitable for genetic studies. In addition, a compound that does not show any action when applied to a certain organism (for example, a microorganism) is converted to the full length c D of the microorganism.

When applied to plants overexpressing NA, the effect may be seen. By derivatizing the compound using the above, it becomes possible to increase the activity of the compound that has been overlooked so far and use it as a microbicide, for example. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

1. The following steps (a) to (d)

 (a) introducing a full-length cDNA library into a plant to produce a full-length cDNA overexpression line;

 (b) applying a compound that changes the phenotype of a wild-type plant to an overexpression line,

(c) selecting an overexpression line that exhibits a phenotype different from the altered phenotype of the wild-type plant; and

 (d) A method for searching for a gene encoding a protein targeted by a compound, comprising the step of identifying a full-length cDNA introduced into a selected overexpression line.

 2. The following steps (e) to (i):

 (e) applying a group of compounds to a wild-type plant to select a compound that changes the phenotype of the wild-type plant;

 (f) introducing a full-length cDNA library into a plant to produce a full-length cDNA overexpression line;

 (g) applying a compound that changes the phenotype of a wild-type plant to an overexpression line;

(h) selecting an overexpression line that exhibits a phenotype different from the altered phenotype of the wild-type plant; and

 (i) A method for selecting a compound and searching for a gene encoding a target protein of the compound, comprising the step of identifying a full-length cDNA introduced into the selected overexpression line.