JP4161597B2 - Method for analyzing agonist activity against cytokinin receptors - Google Patents

Description

【００５８】
（実施例１０）サイトカイニン受容体に対するアゴニスト活性を有する物質の探索方法（その１）
実施例７で得られた形質転換細胞TM182-AHK2、TM182-AHK2Δ及びTM182-AHK3をDOLU+Gal培地200mlに植菌し、30℃で36時間前培養を行い、この培養液をDOLU＋Glu培地でOD600＝0.100に希釈し、さらにそれをDOLU＋Glu培地で200分の１に希釈したものを、希釈前培養液とした。
96穴プレートの各ウエルに、濃度10mMに調製した被験物質（Abscisic acidまたは6-Benzyl aminopurine）のDMSO溶液をDOLU+Glu培地にて100倍に希釈した溶液（100μＭ）を20μl添加したアッセイプレートを準備した。同時に、DMSO 20μlをDOLU+Glu培地にて100倍に希釈した溶液を20μl入れたアッセイプレートを対照区として準備した。
上記の希釈前培養液を200μlずつ両アッセイプレートの各ウエルに添加し、30℃で24時間培養した後、プレートリーダーを用いて各ウエルの濁度を測定した。該濁度を対照区における濁度と比較することにより、被験物質のサイトカイニン受容体に対するアゴニスト活性を検定した。表２に、各被験物質を添加した各形質転換細胞培養液のOD620の吸光度を示した。6-Benzyl aminopurineを添加した試験区における濁度が対照区における濁度に比べて高い値を示したことから、6-Benzyl aminopurineを、サイトカイニン受容体に対するアゴニスト活性物質として選抜した。
【００５９】
【表２】

【００６０】
（実施例１１）サイトカイニン受容体に対するアゴニスト活性物質の探索方法（その２）
実施例７で得られた形質転換細胞TM182-AHK2、TM182-AHK2Δ及びTM182-AHK3 をDOLU+Gal培地200mlに植菌し、30℃で30時間前培養を行う。実施例１０で選抜されたサイトカイニン受容体に対するアゴニスト活性物質の供試濃度が10ｎＭから100μＭとなるように種々変化させて添加したDOLU+Glu寒天培地に、前培養した形質転換細胞（TM182-AHK2、TM182-AHK2Δ及びTM182-AHK3）10μlをスポットして30℃で30時間培養する。培養終了後、形質転換細胞（TM182-AHK2、TM182-AHK2Δ及びTM182-AHK3）の増殖状態が観察される最低供試濃度から当該供試物質のサイトカイニン受容体に対するアゴニスト活性の強度を検定・確認する。
【００６１】
以下に、本発明において使用される培地の組成を記す。
（ａ）DOLU+GLU培地
Bacto-yeast nitrogen base without amino acids 6.7g
Glucose 20g
Drop-out mix（ｘ） 2.0g
Distilled water 1000ml
（ｂ）DOLU+GAL培地
Bacto-yeast nitrogen base without amino acids 6.7g
Glucose 20g
Drop-out mix（ｘ） 2.0g
Distilled water 1000ml
（ｘ）Drop-out mix:
Adenine 0.5g Lysine 2.0g
Alanine 2.0g Methionine 2.0g
Arginine 2.0g para-Aminobenzoic acid 0.2g
Asparagine 2.0g Phenylalanine 2.0g
Aspartic acid 2.0g Proline 2.0g
Cysteine 2.0g Serine 2.0g
Glutamine 2.0g Threonine 2.0g
Glutamic acid 2.0g Tryptophan 2.0g
Glycine 2.0g Tyrosine 2.0g
Histidine 2.0g Valine 2.0g
Inositol 2.0g Isoleucine 2.0g
Distilled water 1000ml
（ｃ）DOLU+GLU寒天培地
培地（ａ）に２％（Ｗ／Ｖ）の寒天が添加された固体培地。
（ｄ）DOLU+GAL寒天培地
培地（ｃ）に２％（Ｗ／Ｖ）の寒天が添加された固体培地。
【００６２】
【発明の効果】
本発明によって、サイトカイニン受容体に対するアゴニスト活性の分析方法が提供可能となり、さらに当該分析方法を使用することでサイトカイニン受容体に対するアゴニスト活性を有する物質を少量のサンプルでかつ迅速に見出す方法等が提供可能となる。
【００６３】
「配列表フリーテキスト」
配列番号９
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１０
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１１
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１２
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１３
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１４
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１５
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１６
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１７
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１８
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号１９
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号２０
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号２１
PCRのために設計されたオリゴヌクレオチドプライマー
配列番号２２
PCRのために設計されたオリゴヌクレオチドプライマー
【００６４】
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for analyzing agonist activity of a test substance with respect to a cytokinin receptor.
[0002]
[Prior art]
Cytokinin is a plant hormone related to cell division and differentiation of higher plants, induces division of higher plant cells, differentiates from callus and medulla to stalks and leaves, prevents leaf yellowing, fallen leaves, fallen fruits, etc. It is an important physiologically active substance that is known to exhibit such actions [Cytokinins: Chemistry, Activity, and Function, CRC Press (1994)].
[0003]
[Problems to be solved by the invention]
Substances with cytokinin-like activity include plant growth regulators, such as fruit tree apples, fruit drop prevention agents such as mandarin oranges, rice, wheat and other lodging prevention agents by regulating plant height, and sweetness of fruits after harvest It can be used as an enhancer.
As a method of finding a substance having such cytokinin-like activity, a method of directly observing and evaluating a physiological change by spraying a test substance directly on a plant individual can be used. This method requires preparing a test substance in an amount sufficient to be sprayed directly on a plant individual, and also takes a great deal of time to grow the plant individual and to observe and evaluate the physiological change of the plant individual after spraying the test substance. There was a problem that required. Therefore, development of various methods for rapidly finding a substance having cytokinin-like activity with a small amount of a test substance has been demanded.
[0004]
[Means for Solving the Problems]
As a result of intensive studies under such circumstances, the present inventors have found a protein that functions as a cytokinin receptor, and by using the cytokinin receptor, it is possible to analyze the agonist activity of a test substance against the cytokinin receptor. The present inventors have found that it is possible to quickly search for a substance having cytokinin-like activity with a small amount of sample by using this analysis method.
[0005]
That is, the present invention
1. A method for analyzing agonist activity against a cytokinin receptor,
(1) a first step of bringing a test substance into contact with a transformed cell into which a DNA encoding one of the following cytokinin receptors (hereinafter sometimes referred to as the present cytokinin receptor) has been introduced; and
(2) Second step of measuring the presence or amount of intracellular signal transmission from the cytokinin receptor expressed in the transformed cell after the first step.
(Hereinafter, also referred to as the present analysis method);
(A) a cytokinin receptor having the amino acid sequence represented by SEQ ID NO: 2
(B) a cytokinin receptor having the amino acid sequence represented by SEQ ID NO: 4
(C) A cytokinin receptor that lacks a part of the transmembrane region of the plurality of transmembrane regions of the natural cytokinin receptor and has at least one transmembrane region
(D) Of the amino acid sequence represented by SEQ ID NO: 2, a cytokinin receptor comprising the amino acid sequence represented by amino acid numbers 196 to 1176
(E) a cytokinin receptor comprising the amino acid sequence represented by amino acid numbers 50 to 1176 among the amino acid sequence represented by SEQ ID NO: 2
(F) a cytokinin receptor comprising the amino acid sequence represented by amino acid numbers 32 to 1036 among the amino acid sequence represented by SEQ ID NO: 4
(G) an extracellular region of the cytokinin receptor that is homologous to each other, a transmembrane region of the cytokinin receptor, and a histidine kinase region of the cytokinin receptor, and a histidine kinase receiver region that is heterogeneous to the region; Chimeric cytokinin receptor composed of
(H) Cytokinin receptor having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of (a), (b), (d), (e) or (f)
2. The transformed cell is a cell having a function in which cell growth is directly controlled by intracellular signal transmission from a cytokinin receptor, and the presence or absence of the intracellular signal transmission or the amount thereof is determined by the transformed cell. The analysis method according to item 1 above, wherein the growth amount is measured as an index;
3. The transformed cell is a transformed cell in which a DNA encoding the cytokinin receptor is introduced into a host cell improved so as to have a histidine kinase activity lower than the cell-specific histidine kinase activity. The analysis method according to item 1 above;
4). Introducing DNA encoding the cytokinin receptor into a host cell improved so that the transformed cell has histidine kinase activity lower than the cell-specific histidine kinase activity by deleting one or more histidine kinases The analysis method according to item 1 above, which is a transformed cell obtained;
5. 2. The analysis method according to item 1 above, wherein the transformed cell is a transformed cell obtained by introducing a DNA encoding the cytokinin receptor into a host cell that does not have a cytokinin receptor;
6). The analysis method according to item 1, wherein the transformed cell is yeast;
7). The analysis method according to item 1 above, wherein the transformed cell is budding yeast;
8). The presence or absence or amount of intracellular signal transduction from cytokinin receptors measured for the transformed cells in contact with each test substance by analyzing the agonist activity of two or more different test substances by the analysis method according to claim 1 A method for assaying agonist activity against a cytokinin receptor, comprising evaluating the agonist activity against the cytokinin receptor of the substance based on the difference obtained by comparing
9. 9. The assay method according to item 8 above, wherein at least one of the two or more different substances is a substance that does not have an agonist activity for the cytokinin receptor;
10. A method for searching an agonist active substance for a cytokinin receptor, comprising selecting a substance having an agonist activity for a cytokinin receptor based on the agonist activity for the cytokinin receptor evaluated by the assay method according to the preceding item 8;
11. A plant growth regulator comprising, as an active ingredient, a substance selected by the search method according to item 10;
Is to provide.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
The cytokinin receptor specifically binds to cytokinins such as purine cytokinins such as kinetin and zeatin, urea cytokinins such as N-phenyl-N ′-(4-pyridyl) urea, and two-component regulatory system ( Alternatively, it is a protein having a function of controlling the growth and differentiation of higher plant cells by an intracellular signal transmission mechanism called His to Asp phosphorelay system). The cytokinin receptor used in the present invention belongs to the histidine kinase family, and includes an extracellular region, a transmembrane region, a histidine kinase region (having a His residue that has histidine kinase activity in the cell and serves as an active site). Region) and a receiver region (region having an acceptor for transphosphorylation and holding an Asp residue as an active site).
[0007]
Specific examples of the cytokinin receptor include, for example, a cytokinin receptor having the amino acid sequence represented by SEQ ID NO: 2 or 4, and one or more amino acids deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2 or 4 Alternatively, a cytokinin receptor having an added amino acid sequence, a cytokinin comprising an amino acid sequence encoded by a DNA hybridizing under stringent conditions with a DNA having a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or 4 Examples thereof include a receptor, a transmembrane region partially deleted cytokinin receptor described later, a chimeric cytokinin receptor described later, and the like. Here, the “plural amino acids” means more specifically about 2 to about 20 amino acids, for example, 2 to about 10 amino acids, 2 to 5 amino acids. Can be given as The “amino acid sequence in which one or more amino acids are deleted, substituted or added” is, for example, 80% or more, preferably 90% or more, compared to the amino acid sequence before deletion, substitution or addition, More preferably, an amino acid sequence having a sequence identity of 95% or more can be mentioned as an example.
These “amino acid deletion, substitution or addition” and “80% or more sequence identity” are derived from the processing that the protein having the amino acid sequence shown in SEQ ID NO: 2 or 4 undergoes in the cell, and the protein Mutations that occur naturally due to species differences, individual differences, differences between tissues, etc. are included.
In the present invention, “sequence identity” refers to sequence identity and homology between two DNAs or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the DNA or protein to be compared may have an addition or a deletion (for example, a gap) in an optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. The sequence identity is measured using sequence analysis software, specifically, an analysis tool provided by Vector NTI, GENETYX-MAC, or a public database, for example, the homepage address http It is generally available at: //www.ddbj.nig.ac.jp.
[0008]
Regarding the above-mentioned “hybridizes under stringent conditions”, the hybridization used here is, for example, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, cold. It can be carried out according to the usual method described in the publication of the Spring Harbor Laboratory press (Cold Spring Harbor Laboratory press). “Stringent conditions” means, for example, after a hybrid is formed at 65 ° C. in a 6 × SSC (1.5M NaCl, 0.15M trisodium citrate solution is 10 × SSC) solution. And conditions such as washing with 1 × SSC at room temperature. The salt concentration in the washing step can be selected, for example, from 1 × SSC at room temperature (low stringency conditions) to 0.1 × SSC at room temperature (high stringency conditions). The temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 68 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.
[0009]
(Preparation of transformed cells into which DNA encoding this cytokinin receptor has been introduced)
A transformed cell into which the DNA encoding the cytokinin receptor has been introduced contains a DNA encoding the cytokinin receptor, that is, a DNA having a base sequence encoding the amino acid sequence of the cytokinin receptor as follows: Thus, it can be obtained by introducing and expressing in a host cell. Hereinafter, an example of a method for producing the transformed cell will be described.
[0010]
(1) Preparation of cDNA
First, for example, according to the method described in J., Sambrook, E., F., Frisch, T., Maniatis, Molecular Cloning 2nd edition, total RNA from plants such as higher plants To prepare. Specifically, for example, after collecting a part of the tissue from a higher plant such as a monocotyledonous plant such as rice, corn, or wheat, or a dicotyledonous plant such as tobacco, soybean, or Arabidopsis, the tissue is placed in liquid nitrogen. After freezing in mortar, physically grind in a mortar, etc. (a) To the obtained ground product, a solution containing guanidine hydrochloride and phenol or a solution containing SDS and phenol is obtained to obtain total RNA. A method, (b) a method in which a solution containing guanidine thiocyanate is added to the above-mentioned ground product, and CsCl is further added and centrifuged to obtain total RNA may be used. For this operation, for example, a commercially available kit such as ISOGEN (manufactured by Nippon Gene) or RNeasy Total RNA Purification Kit (manufactured by QIAGEN) can also be used.
[0011]
Next, mRNA is prepared from the total RNA. For example, a method using hybridization of oligo dT chain bonded to cellulose or latex and poly A chain of mRNA can be used. For this operation, for example, a commercially available kit such as mRNA Purification Kit (Amersham Pharmacia), Oligotex ™ -dT30 <Super> (Takara Shuzo) can be used.
Further, cDNA is prepared using the mRNA thus prepared (mRNA having a poly A chain). For example, cDNA may be prepared by annealing an oligo dT chain or a random primer to mRNA and then allowing reverse transcriptase to act on it. Furthermore, a double-stranded cDNA can be prepared by allowing RNaseH or DNA Polymerase I to act on the cDNA. For this operation, commercially available kits such as SMART ™ PCR cDNA Synthesis Kit (Clontech), cDNA Synthesis Kit (Takara Shuzo), cDNA Synthesis Kit (Amersham Pharmacia), ZAP-cDNA Synthesis Kit (Stratagene), etc. Can be used.
[0012]
(2) Cloning
From the thus prepared cDNA, for example, a polymerase chain reaction (hereinafter referred to as PCR) using a DNA having a partial base sequence of the base sequence shown in SEQ ID NO: 1 or 3 as a primer, or SEQ ID NO: 1 or 3 The DNA encoding the cytokinin receptor can be obtained by a hybridization method using a DNA having a partial base sequence of the base sequence represented by the above as a probe.
[0013]
When PCR is used, it is based on a base sequence of about 20 bp to about 40 bp, for example, a base sequence selected from the 5 ′ untranslated region and the 3 ′ untranslated region of the base sequence represented by SEQ ID NO: 1 or 3, respectively. The designed and synthesized DNA can be used as a primer set. As the primer set, for example, a primer set may be a set of DNA consisting of the base sequence shown by SEQ ID NO: 9 and DNA consisting of the base sequence shown by SEQ ID NO: 10. The PCR reaction solution to be used may be prepared by adding the reaction solution specified by the kit to 250 ng of cDNA. The PCR reaction conditions can be appropriately changed depending on the primer set to be used. For example, the temperature is kept at 94 ° C. for 2 minutes, then kept at about 8 ° C. for 3 minutes, then at 94 ° C. for 30 seconds and at 55 ° C. Repeat this for 5 to 10 cycles under the conditions of repeating 40 cycles of 30 seconds at 72 ° C for 4 minutes, or 94 ° C for 5 seconds and then 72 ° C for 4 minutes, and then at 94 ° C for 5 seconds. A cycle in which the temperature is kept and then kept at 70 ° C. for 4 minutes is defined as one cycle, and this can be repeated 20 to 40 cycles. For the operation, for example, Takara Heraculase ^TM (Manufactured by Takara Shuzo), DNA polymerase included in Advantage cDNA PCR Kit (manufactured by Clontech), TAKARA Ex Taq (manufactured by Takara Shuzo), PLATINUMTM PCR SUPER Mix (manufactured by Lifetech Oriental), etc. it can.
[0014]
When the hybridization method is used, cloning can be performed according to the method described in, for example, cloning and sequencing: plant biotechnology experiment manual (Watanabe, Sugiura, 1989).
As a probe to be used, DNA having a partial base sequence of the base sequence represented by SEQ ID NO: 1 or 3 (chain length of about 200 bases to about 500 bases) is synthesized, and the DNA is, for example, Random Primed DNA Labeling Kit (Boehringer), Random Primer DNA Labeling Kit Ver.2 (Takara Shuzo), ECL Direct Nucleic Acid Labeling and Ditection System (Amersham Pharmacia), Megaprime DNA-labelling system (Amersham Pharmacia), etc. were used. It can be obtained by radioisotope labeling or fluorescent labeling according to a known method. Examples of hybridization conditions include stringent conditions. Specifically, for example, 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate), 5 × Denhardt's solution (0.1% (w / v) Ficoll 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BSA), 0.5% (w / v) SDS and 100 μg / ml denatured salmon sperm DNA or 100 μg / ml denatured salmon sperm DNA Incubate in DIG EASY Hyb solution (Boehringer Mannheim) at 65 ° C, then incubate for 15 minutes at room temperature in the presence of 1x SSC (0.15M NaCl, 0.015M trisodium citrate) and 0.5% SDS The conditions can be raised twice and further maintained at 68 ° C. for 30 minutes in the presence of 0.1 × SSC (0.015 M NaCl, 0.0015 M trisodium citrate) and 0.5% SDS.
To obtain DNA encoding the cytokinin receptor of Arabidopsis thaliana, first use the Arabidopsis thaliana cDNA library phage solution (approximately 1000000 pfu) as a template, and TAKARA LA taq ^TM (Takara Shuzo Co., Ltd.) may be used to amplify the DNA used as a probe by PCR using the DNA shown in SEQ ID NO: 11 and the DNA shown in SEQ ID NO: 12 as a primer set, and obtain this. The PCR reaction solution to be used may be prepared by adding the reaction solution specified by the kit to 250 ng of the DNA library.
As PCR reaction conditions, for example, the sample is kept at 94 ° C. for 2 minutes, then kept at 8 ° C. for 3 minutes, and then cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 5 minutes are repeated 40 cycles. Thus, conditions for amplification can be raised.
Next, by using the amplified and obtained DNA as a template and using the Megaprime DNA-labelling system (manufactured by Amersham Pharmacia), using the reaction solution specified by the kit ³² Probes labeled with P can be made. Colony hybridization was performed according to the usual method using the probe thus prepared, and 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate), 5 × Denhardt's solution (0.1% (w / v) Ficoll) 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BSA), 0.5% (w / v) SDS and 100 μg / ml denatured salmon sperm DNA or DIG EASY Hyb containing 100 μg / ml denatured salmon sperm DNA Incubate in solution (Boehringer Mannheim) at 65 ° C, then incubate twice for 15 minutes at room temperature in the presence of 1x SSC (0.15M NaCl, 0.015M trisodium citrate) and 0.5% SDS, Furthermore, clones that hybridize to the probe can be obtained by incubating at 68 ° C. for 30 minutes in the presence of 0.1 × SSC (0.015 M NaCl, 0.0015 M sodium citrate) and 0.5% SDS.
[0015]
The DNA encoding this cytokinin receptor can be obtained, for example, based on the nucleotide sequence represented by SEQ ID NO: 1 or 3, for example, the phosphite triester method (Hunkapiller, M. et al., Nature, 310, 105 , 1984) and the like, and can also be prepared by chemical synthesis of nucleic acids.
[0016]
The DNA encoding the cytokinin receptor obtained as described above is described in `` Molecular Cloning: A Laboratory Manual 2nd edition '' (1989), Cold Spring Harbor Laboratory Press, `` Current Protocols In Molecular Biology '' (1987), John What is necessary is just to clone to a vector according to the normal method described in Wiley & Sons, Inc. ISBN0-471-50338-X etc. Examples of the vector used include pBlueScriptII vector (Stratagene), pUC18 / 19 vector (Takara Shuzo), TA cloning vector (Invitrogen) and the like.
The base sequence of the cloned DNA is determined by Maxam Gilbert method (for example, described in Maxam, AM & W. Gilbert, Proc. Natl. Acad. Sci. USA, 74, 560, 1977, etc.) or Sanger method ( For example, Sanger, F. & ARCoulson, J. Mol. Biol., 94, 441, 1975, Sanger, F, & Nicklen and ARCoulson., Proc. Natl. Acad. Sci. USA, 74, 5463, 1977 etc. Etc.). Commercially available kits such as Termo Seqenase II dye terminator cycle sequencing kit (Amersham Pharmacia) and Dye Terminator Cycle Sequencing FS Ready Reaction Kit (PE Biosystems Japan) can be used for this operation.
[0017]
(3) Construction of expression vector
The expression vector for the DNA encoding this cytokinin receptor is constructed by a conventional method (for example, Molecular Cloning 2nd edition, written by J., Sambrook, E., F., Frisch, T., Maniatis, Molecular Cloning 2nd edition). ), The method described in Cold Spring Harbor Laboratory press (Cold Spring Harbor Laboratory press) etc.).
For example, a vector that can be used in the host cell to be transformed, for example, a vector that contains genetic information that can be replicated in the host cell and can be propagated autonomously, and can be isolated and purified from the host cell. Yes, and a vector that may have a detectable marker (specifically, when a bacterium such as Escherichia coli is used as a host cell, for example, plasmid pUC119 (manufactured by Takara Shuzo Co., Ltd.) or phagemid pBluescriptII (Stratagene) In the case of using yeast as a host cell, for example, plasmid pACT2 (manufactured by Clontech) etc. may be used, and in the case of using a plant cell as a host cell, for example, plasmid pBI221. (Clontech) or the like may be used) and the DNA encoding the cytokinin receptor may be incorporated.
[0018]
The DNA encoding the cytokinin receptor is expressed in the host cell by incorporating the promoter capable of functioning in the host cell into the vector in a form that can function in the upstream of the DNA encoding the cytokinin receptor. It is possible to construct an expression vector that makes it possible. Here, “to bind in a functional manner” means that the promoter and the DNA encoding the cytokinin receptor are expressed in a host cell so that the DNA encoding the cytokinin receptor is expressed under the control of the promoter. It means to combine. Examples of promoters that can function in the host cell include, for example, when the host cell is Escherichia coli, the E. coli lactose operon promoter (lacP), the tryptophan operon promoter (trpP), the arginine operon promoter (argP), and the galactose operon. Promoters (galP), tac promoter, T7 promoter, T3 promoter, λ phage promoters (λ-pL, λ-pR), and the like. When the host cell is yeast, the ADH1 promoter (where the ADH1 promoter is, for example, the yeast expression vector pAAH5 carrying the ADH1 promoter and the terminator, available from Washington Research Fundation, Ammerer et al., Method in Enzymology, 101 part (p.192-201)] by the usual genetic engineering method The ADH1 promoter is included in US Patent Application No. 299,733 to Washington Research Fundation and is used for industrial and commercial purposes in the United States. In the case of using with the above, a license from the right holder is required.) When the host cell is a plant cell, for example, nopaline synthase gene (NOS) promoter, octopine synthase gene (OCT) promoter, cauliflower mosaic virus (CaMV) -derived 19S promoter, CaMV-derived 35S promoter, etc. it can.
[0019]
When the DNA encoding the cytokinin receptor is incorporated into a vector having a promoter that can function in the host cell in advance, the promoter possessed by the vector and the DNA encoding the cytokinin receptor can function. What is necessary is just to insert DNA which codes this cytokinin receptor downstream from the said promoter so that it may couple | bond together in a form. For example, the aforementioned plasmid pACT2 for yeast has an ADH1 promoter, and if a DNA encoding the cytokinin receptor is inserted downstream of the ADH1 promoter of the plasmid, the DNA encoding the cytokinin receptor is, for example, An expression vector that can be expressed in yeast such as CG1945 (manufactured by Clontech) can be constructed.
[0020]
(4) Preparation of transformed cells
By introducing the constructed expression vector into a host cell according to a normal method, a transformed cell used in the present invention can be produced. Examples of host cells used for producing transformed cells include bacteria, yeast, plant cells and the like. Examples of bacteria include bacteria belonging to Escherichia coli, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, and the like. Examples of the yeast include budding yeast and fission yeast, and more specifically, yeasts belonging to the genus Saccharomyces, Schizosaccharomyces, and the like. Examples of plant cells include BY-2 strain, which is a cultured cell of tobacco, and BMS strain, which is a cultured cell of corn (Black Mexican Sweet).
As a method for introducing the expression vector into the above host cell, a normal introducing method according to the host cell to be transformed can be applied. For example, when a bacterium is used as a host cell, the usual introduction such as the calcium chloride method and the electroporation method described in “Molecular Cloning” (J. Sambrook et al., Cold Spring Harbor, 1989), etc. By using the method, the expression vector can be introduced into a host cell. When yeast is used as the host cell, the expression vector can be introduced into the host cell by using, for example, Yeast transformation kit (manufactured by Clontech) based on the lithium method. When plant cells are used as host cells, for example, an Agrobacterium infection method (JP-B-2-58917 and JP-A-60-70080), an electroporation method to protoplasts (JP-A-60-251887 and JP-A-60-251887) The expression vector can be introduced into a host cell by using a conventional introduction method such as JP-A-5-68575) or particle gun method (Japanese Patent Publication No. 5-508316 and JP-A-63-258525).
[0021]
(Transformed cells expressing a transmembrane region partially deleted cytokinin receptor-transmembrane frequency variant cytokinin receptor)
The cytokinin receptor used in the present invention is one in which some of the transmembrane regions (generally about 2 to 4) possessed by the natural cytokinin receptor are missing. In addition, a cytokinin receptor having at least one transmembrane region (in the present application, it may be referred to as a transmembrane region partially deleted cytokinin receptor) can also be mentioned. Here, the “natural cytokinin receptor” means a cytokinin receptor having an amino acid sequence that is present at the highest frequency among organisms of the same species, and is generally called a wild type cytokinin receptor. It is what.
More specifically, such a transmembrane region partially deleted cytokinin receptor has a part of the transmembrane region, for example, about 1 to 2 transmembrane regions deleted from the natural cytokinin receptor. And a cytokinin receptor that retains a smaller number of transmembrane regions than the number of transmembrane regions possessed by the natural cytokinin receptor. The structure of the transmembrane region of the cytokinin receptor is available from, for example, http://www.ch.embnet.org/software/TMPRED_form.html for the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. It can be estimated using prediction software or the like.
[0022]
Specifically, among the amino acid sequence represented by SEQ ID NO: 2, a cytokinin receptor (two transmembrane regions) consisting of the amino acid sequence represented by amino acid numbers 196 to 1176, among the amino acid sequence represented by SEQ ID NO: 2, A cytokinin receptor consisting of the amino acid sequence represented by amino acid numbers 50 to 1176 (three transmembrane regions), and a cytokinin receptor consisting of the amino acid sequence represented by amino acid numbers 32 to 1036 among the amino acid sequences represented by SEQ ID NO: 4 ( 2 transmembrane regions), a cytokinin receptor comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of these cytokinin receptors, for example, one at the amino terminus of these amino acid sequences Rhino consisting of an amino acid sequence with methionine added Kainate receptor, and the like can be given.
[0023]
The above DNA encoding the cytokinin receptor is, for example, a DNA encoding a natural cytokinin receptor, wherein the base sequence encoding a part of the transmembrane region of the receptor is deleted, and the natural cytokinin receptor It can be constructed by cleaving and joining using ordinary genetic engineering techniques so that a base sequence encoding a number of transmembrane regions smaller than the number of transmembrane regions of the body is retained.
The production of a transformed cell into which the DNA encoding the cytokinin receptor is introduced may be in accordance with the above-mentioned “(production of a transformed cell into which the DNA encoding the cytokinin receptor is introduced)”.
[0024]
(Transformed cells that express chimeric cytokinin receptors)
As the cytokinin receptor used in the present invention, the extracellular region of the cytokinin receptor, the transmembrane region of the cytokinin receptor and the histidine kinase region of the cytokinin receptor, which are homologous to each other, are heterogeneous to the above regions. And a chimeric cytokinin receptor composed of a histidine kinase receiver region. Specifically, for example, extracellular region derived from cytokinin receptor of CRE1, AHK2, AHK3, transmembrane region and histidine kinase region, and receiver region derived from histidine kinase encoded by Sln1 gene of budding yeast And a chimeric cytokinin receptor composed of:
[0025]
Proteins of the histidine kinase family including cytokinin receptors have the following sequences in common with plants and microorganisms such as higher plants. That is, the extracellular region, the transmembrane region (usually about 2 to 4), the histidine kinase region having histidine kinase activity in the cell and holding the His residue serving as the active site, and the receptor for transphosphorylation And a receiver region that retains an Asp residue serving as an active site. In the above chimeric cytokinin receptor, the extracellular region, the transmembrane region and the histidine kinase region are all derived from the same cytokinin receptor, whereas the receiver region is different from the former cytokinin receptor. It is important that it is derived from a family protein.
The receiver region of the chimeric cytokinin receptor only needs to have a function of receiving signal transmission from the histidine kinase region and transmitting it to the next step. The extracellular region of the cytokinin receptor, the transmembrane region In addition, any function can be used as long as it can complement or improve the function originally possessed by the receiver region that is homologous to the region consisting of the histidine kinase region.
Examples of such a receiver region include a receiver region possessed by a microorganism-derived histidine kinase (for example, a receiver region possessed by a histidine kinase derived from a microorganism such as yeast or Escherichia coli, and more specifically, a Sln1 gene derived from a budding yeast. Encoded in the receiver region of the encoded histidine kinase (see, for example, the amino acid sequence shown in SEQ ID NO: 7), in the receiver region of the histidine kinase encoded in the Salmonella-derived Chey gene, and in the RcsC gene, which is an E. coli hybrid sensor. Histidine kinase has a receiver region (Maeda T et al. Nature: 369 242-245 (1994), for example, refer to the amino acid sequence shown in SEQ ID NO: 8), which is encoded by the Phks gene involved in cell cycle control of fission yeast Receiver region of histidine kinase ( Shieh, JC et al, Gene Dev. 11, 1008-1022 (1997)) and the like can be used.
[0026]
Here, the histidine kinase region of the cytokinin receptor is, for example, a region existing downstream of the transmembrane region present at the most C-terminal side among the plurality of transmembrane regions, and which is the Annual Review of Genetics 23: 311-336 (1989), Microbiological Reviews 53 (4): 450-490 (1989), Science 262: 539-544 (1993) and Science 274: 982-985 (1996) This region is characterized by having five conserved motifs common to histidine kinase. For example, in the cytokinin receptor (AHK2) having the amino acid sequence represented by SEQ ID NO: 2, the 587th amino acid to 844th amino acid of SEQ ID NO: 2 It is a region including amino acids, and in the cytokinin receptor (AHK3) having the amino acid sequence represented by SEQ ID NO: 4, it is a region including amino acids from the 450th amino acid to 700th amino acid of SEQ ID NO: 4, and is represented by SEQ ID NO: 6 Be From 449 th amino acids of the cytokinin receptor (CRE1) in SEQ ID NO: 6 having the amino acid sequence up to 714 amino acid is a region including a.
The receiver region of the cytokinin receptor is, for example, a region that exists between the histidine kinase region and the C-terminus of the cytokinin receptor, and is described in Annual Review of Genetics 23: 311-336 (1989), Science 262: 539- 544 (1993) and Science 274: 982-985 (1996), a region characterized by having three conserved motifs common to common histidine kinases. For example, in AHK2, 891 of SEQ ID NO: 2 The region from the 1st amino acid to the 1163rd amino acid, AHK3 is the region from the 746th amino acid to the 1018th amino acid of SEQ ID NO: 4, and CRE1 is the region from the 763rd amino acid to SEQ ID NO: 6 This region includes up to amino acids.
At least one of the extracellular regions is a region existing between the transmembrane region present on the most C-terminal side and the transmembrane region located on the N-terminal side of the transmembrane region (region involved in cytokinin recognition) In addition, as described in Plant and Cell Physiology 42 (2): 231-235 (2001), it is a region conserved by 50% or more among the three members AHK2, AHK3, and CRE1, and in AHK2, SEQ ID NO: 2 From 259th amino acid to 536th amino acid, AHK3 is a region from amino acid 120 to 399th amino acid of SEQ ID NO: 4, and CRE1 is from the 132nd amino acid of SEQ ID NO: 6. This region includes up to the 398th amino acid.
[0027]
DNA encoding the chimeric cytokinin receptor is prepared by encoding the extracellular region of the cytokinin receptor, the transmembrane region of the cytokinin receptor, the histidine kinase region of the cytokinin receptor and the receiver region of the histidine kinase, respectively. These should be constructed by inserting appropriate linkers as necessary so that stop codons do not appear in the middle and without shifting the frame, and linking them using conventional genetic engineering techniques. Can do. DNA encoding the extracellular region of the cytokinin receptor and the transmembrane region of the cytokinin receptor, and the DNA encoding the extracellular region of the cytokinin receptor, the transmembrane region of the cytokinin receptor and the histidine kinase region of the cytokinin receptor Alternatively, it may be prepared as a single molecule of DNA and used to construct a DNA encoding a chimeric cytokinin receptor.
[0028]
The DNA encoding each region can be prepared using a known method. For example, in the case of producing by PCR, first, an oligonucleotide having a base sequence of the 5 ′ end region (5 ′ end primer) of DNA encoding each region to be amplified and a base sequence of the 3 ′ end region are complementary. Design and synthesize oligonucleotides (3'-side primers) each having a specific base sequence. The primer is usually an oligonucleotide of about 14 bases to 35 bases, and the 5 'end of this primer is connected to DNA amplified by PCR or to the DNA and vector. A restriction enzyme recognition sequence that can be used in some cases may be provided. Next, an amplification reaction may be performed using the primers and a cDNA library as a template under normal reaction conditions used for PCR. As a template used when preparing a DNA encoding the extracellular region of cytokinin receptor, the transmembrane region of cytokinin receptor or the histidine kinase region of cytokinin receptor, for example, a cDNA derived from a plant such as a higher plant can be used. You can raise a library. Examples of the template used in the preparation of DNA encoding the histidine kinase receiver region include a microorganism-derived cDNA library or total DNA prepared by a conventional method.
[0029]
The production of a transformed cell into which the DNA encoding the chimeric cytokinin receptor has been introduced is in accordance with the above-mentioned “(production of a transformed cell into which the DNA encoding the cytokinin receptor has been introduced)”. Good.
[0030]
(Intracellular signal transmission system related to cytokinin)
In the present invention, in order to determine the presence or amount of intracellular signal transduction from the cytokinin receptor expressed in the transformed cells prepared as described above, it is used to prepare transformed cells. An intracellular signal transmission system inherent in the host cell may be used. Alternatively, a DNA encoding a regulator and / or mediator responsible for an intracellular signal transmission function called a two-component regulatory system may be introduced into and expressed in the host cell and used as an intracellular signal transmission system. Examples of available Two-Component regulatory systems include five receptors ETR1, ETR2, ERS1, ERS2, and EIN4 possessed by Arabidopsis (Chang et al. Science 262: 539-544 (1993), Hua et al. Science). 269: 1712-1714 (1995), Sakai et al. Plant Cell Physiol 39: 1232-1239 (1998)) and AtHK1 (Urao Plant Cell 11: 1743-1754 (1999)) with osmotic pressure sensor function Two-Component regulatory system etc. can be mentioned.
[0031]
Moreover, the host cell improved so that it may have histidine kinase activity lower than the histidine kinase activity intrinsic | native to a host cell can also be used as a host cell used in order to produce such a transformed cell. For example, host cells that have been modified to have a histidine kinase activity that is lower than the cell-specific histidine kinase activity by deleting one or more histidine kinases from a natural host cell. Low histidine kinase activity means that the amount of phosphate group transferred from the His residue, which is the active site of the histidine kinase region, to the Asp residue, which is the active site of the receiver region, results in intracellular signal transduction. The amount of decreases. In host cells modified to have a histidine kinase activity lower than that inherent to the host cell, for example, changes in growth, changes in shape, changes in shape, changes in the amount of biosynthesis of a specific substance in the cell Changes in the metabolic rate of certain substances may occur. Specifically, strains (Maeda T et al. Nature: 369 242-245 (1994)) lacking the Sln1 gene encoding a protein having an osmotic pressure sensor function derived from budding yeast such as Saccharomyces cerevisiae Can do. Since this strain lacks the histidine kinase present in Saccharomyces cerevisiae, the amount of growth decreases, and when this cytokinin receptor is introduced, the presence or absence of intracellular signal transmission from the receptor or the amount thereof is determined by the host cell. Can be detected more clearly as an index. Also, examples of preferable embodiments include an RcsC gene-deficient strain that is a hybrid sensor derived from E. coli, a Phks gene-deficient strain involved in cell cycle control of fission yeast, and the like.
[0032]
(Method of analyzing agonist activity for cytokinin receptor)
As a specific example of the first step of contacting a test substance with a transformed cell into which DNA encoding the cytokinin receptor has been introduced in the method for analyzing agonist activity with respect to the cytokinin receptor, for example, the transformed cell Can be cultured in a medium containing a test substance. The transformed cells may be cultured in any form, such as liquid culture that is cultured in a liquid medium, or solid culture that is cultured on a solid medium obtained by adding agar or the like to the liquid medium. The concentration of the test substance in the medium can be, for example, about 1 nM to about 1 mM, and preferably about 10 nM to about 100 μM. The culture time can be, for example, from 1 hour to about 3 days, and preferably from about 25 hours to about 2 days. In addition, when analyzing the agonist activity with respect to a cytokinin receptor, the culture medium containing a test substance should just use a cytokinin non-added culture medium.
After the first step, the presence or amount of intracellular signal transduction from the cytokinin receptor expressed in the transformed cell is measured, and the agonist activity against the cytokinin receptor is analyzed using the obtained measurement value as an index. To do.
[0033]
Specifically, for example, the TM182 (sln1Δ) strain (Maeda T et al) which is a Sln1 gene-deficient strain into which the PTP2 Tyrosine phosphatase gene (Ota et al, Proc. NAsci. USA, 89, 2355-2359 (1992)) has been introduced. Nature: 369 242-245 (1994)) as a host cell (ie, a transformed cell having a function in which cell growth is directly controlled by intracellular signal transduction from a cytokinin receptor) Is used, the agonist activity against the cytokinin receptor should be measured using the growth amount of the transformed cell in a medium (agar medium or liquid medium) using glucose as a carbon source, for example, DOLU + Glu medium. That's fine. When using DOLU + Glu medium (medium that does not contain a substance having agonist activity for cytokinin receptor) added with the test substance, the test substance that can grow the transformed cells is evaluated to have agonist activity for cytokinin receptor. can do. As a control, it may be examined that the growth of the transformed cells in a medium using galactose instead of glucose as a carbon source, for example, DOLU + Gal medium, is observed regardless of the presence or absence of the test substance.
[0034]
In addition, a transformed cell produced using fission yeast, which is a Phks gene-deficient strain, as a host cell (ie, a transformed cell having a function of directly controlling cell growth by intracellular signal transduction from a cytokinin receptor). When used, the division mode of the fission yeast may be observed under a microscope. When a medium containing a test substance and not containing a substance having agonist activity for cytokinin receptors is used, it can be evaluated that the test substance capable of normally dividing and proliferating the transformed cells has agonist activity for cytokinin receptors. it can.
When using transformed cells prepared using RcsC gene-deficient E. coli into which cps-LacZ has been introduced as host cells, the color development of X-Gal may be observed on an agar medium or liquid medium (Suzuki et al. Plant Cell Physiol 42: 107-113 (2001)). When a medium containing a test substance and not containing a substance having agonist activity for cytokinin receptors is used, it can be evaluated that the test substance capable of coloring the transformed cells in blue has agonist activity for cytokinin receptors. .
[0035]
Further, according to the method for analyzing agonist activity with respect to the cytokinin receptor, two or more different substances (for example, at least one of the two or more different substances is a substance having no agonist activity with respect to the cytokinin receptor). And the difference obtained by comparing the presence or absence of intracellular signal transduction from the cytokinin receptor and the amount thereof measured for transformed cells in contact with each test substance. The agonist activity of the substance against the cytokinin receptor can also be evaluated and assayed.
[0036]
And the agonist active substance with respect to a cytokinin receptor can also be searched by selecting the substance which has the agonist activity with respect to the cytokinin receptor based on the agonist activity with respect to the cytokinin receptor evaluated by the above-mentioned assay method.
[0037]
The agonist active substance selected by the search method described above may be used as an active ingredient of a plant growth regulator.
Examples of plants to be treated with the above plant growth regulator include ornamental plants such as flower buds and foliage plants, crops such as cereals, vegetables and fruit trees, textile plants, trees and turf.
The plant growth regulator is usually mixed with a solid carrier, a liquid carrier, etc., and if necessary, a surfactant, other formulation adjuvants, etc. are added to the emulsion, wettable powder, suspending agent, aqueous solvent, etc. It is used in the formulation. These preparations generally contain 0.5 to 90% by weight, preferably 1 to 80% by weight, of an agonist active substance for the cytokinin receptor.
[0038]
Examples of solid carriers used in the formulation include clays (kaolinite, diatomaceous earth, synthetic silicon hydroxide, fubasami clay, bentonite, acidic clay), talc, and other inorganic minerals (sericite, quartz powder, sulfur powder, Activated carbon, calcium carbonate, etc.) Fine fertilizers and particulates such as chemical fertilizers (ammonium sulfate, phosphorous acid, ammonium nitrate, ammonium chloride, urea, etc.) can be mentioned. Ketones (acetone, methyl ethyl ketone, cyclohexanone, etc.), aromatic hydrocarbons (toluene, xylene, ethylbenzene, methylnaphthalene, etc.), non-aromatic hydrocarbons (hexane, cyclohexane, kerosene, etc.), esters (ethyl acetate, acetic acid, etc.) Butyl), nitriles (acetonitrile, isobutyronitrile, etc.), ET S (dioxane, diisopropyl ether), acid amides (dimethylformamide, dimethylacetamide), halogenated hydrocarbons (dichloroethane, trichlorethylene, etc.) and the like.
Examples of the surfactant include alkyl sulfates, alkyl sulfonates, alkyl aryl sulfonates, alkyl aryl ethers and polyoxyethylenates thereof, polyethylene glycol ethers, polyhydric alcohol esters, sugar alcohol derivatives, etc. Is mentioned.
Other formulation adjuvants include, for example, casein, gelatin, polysaccharides (starch, gum arabic, cellulose derivatives, alginic acid, etc.), lignin derivatives, bentonite, synthetic water-soluble polymers (polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, etc.) ) And the like, PAP (isopropyl acid phosphate), BHT (2,6-tert-butyl-4-methylphenol), BHA (2- / 3-tert-butyl-4-methoxyphenol), Stabilizers such as vegetable oils, mineral oils, fatty acids and fatty acid esters can be mentioned.
[0039]
The agonist active substance for the cytokinin receptor formulated in this way, as it is or diluted with water etc., spraying treatment to the stem foliage, branch leaves, flower seeds of the plant, soaking treatment to the fruit, Used for fruit application. Moreover, the said plant growth regulator is processed once or several times with respect to the object plant.
When the above plant growth regulator is used for the purpose of inhibiting fruit fall, for example, the plant growth regulator is diluted in water and sprayed on the fruit and branches of fruit trees before harvesting.
When the above plant growth regulator is used for the purpose of suppressing the fall of cotton balls, for example, the plant growth regulator is diluted in water and sprayed onto cotton balls and foliage before harvesting.
The plant growth regulator may be treated on a growing plant or on a harvested plant.
[0040]
The application amount of the agonist active substance for the cytokinin receptor in the above plant growth regulator may vary depending on the preparation form, application time, application method, application place and target plant, but is usually 1 to 8000 g per hectare. In addition, the concentration used when the plant growth regulator is diluted in water may vary depending on the preparation form, application time, application method, application location, target plant, but is generally 0.0001 to 1000 mM, preferably Is 0.001 to 10 mM.
[0041]
Next, an example in which an agonist active substance for a cytokinin receptor is formulated into the above plant growth regulator is shown as a formulation example below. In the following examples, parts represent parts by weight.
Formulation Example 1
A wettable powder is obtained by thoroughly pulverizing and mixing 50 parts of an agonist active substance for cytokinin receptor, 3 parts of calcium lignin sulfonate, 2 parts of sodium lauryl sulfate, and 45 parts of synthetic hydrous silicon oxide.
Formulation Example 2
70 parts of an agonist active substance for cytokinin receptor, 3 parts of calcium lignin sulfonate, 2 parts of sodium lauryl sulfate and 25 parts of synthetic silicon hydroxide are thoroughly pulverized and mixed to obtain a wettable powder.
Formulation Example 3
Suspending agent by mixing 40 parts of agonist active substance for cytokinin receptor, 3 parts of polyoxyethylene sorbitan monooleate, 3 parts of CMC (carboxymethylcellulose) and 52 parts of water, and wet-grinding until particle size is 5 microns or less Get.
[0042]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.
[0043]
(Reference Example 1) Preparation of an Arabidopsis cDNA phage library for CER1 cloning
The seeds of the Wassilewskija line of Arabidopsis were sterilized with 70% ethyl alcohol for 1 minute and further sterilized with 1.5% sodium hypochlorite for 10 minutes. After thoroughly washing with sterilized water, GM medium (4.3g Murashige and Skoog's basal salt mixture, 1% ssucrose, 10ml of 5% MES-KOH (pH5.7), 0.3% Phytagel ^TM (SIGMA)) for 2 weeks, 5 g of plant was obtained. This was frozen in liquid nitrogen and then physically ground with a mortar. 10 ml of extraction buffer (200 mM Tris-HCl (pH 8.5), 100 mM NaCl, 10 mM EDTA, 0.5% SDS, 14 mM β-mercaptoethanol) and 10 g of phenol were added to the obtained ground product. After stirring with a Voltex mixer, 10 ml of chloroform was further added and stirred vigorously, followed by centrifugation at 10,000 rpm for 20 minutes. LiCl was added to the recovered aqueous layer to a final concentration of 2M, left at −80 ° C. for 3 hours, thawed, and centrifuged at 10,000 rpm for 20 minutes to recover the precipitate. Dissolve the collected precipitate in 2 ml of TE (10 mM Tris-HCl (pH 8.0) 1 mM EDTA), add 0.2 ml of 3 M sodium acetate (pH 5.2) and 5 ml of ethanol, and centrifuge to precipitate the RNA. It was collected. The recovered precipitate (RNA) is ^TM RNA with polyA bound was extracted using dT30super (Roche Japan).
A phage cDNA library from the extracted polyA-bound RNA was prepared using a ZAP-cDNAR Synthesis Kit (Stratagene) according to the instructions. The titer of the prepared phage cDNA library was 500000 PFU.
[0044]
(Reference Example 2) Preparation of CRE1 DNA probe
Using the phage solution (about 1000000PFU) of the phage cDNA library prepared in Reference Example 1 as a template, TAKARA LA Taq ^TM Using a kit (Takara Shuzo Co., Ltd.), PCR was carried out using the DNA shown in SEQ ID NO: 11 and the DNA shown in SEQ ID NO: 12 as primers to amplify the DNA. Details are described below.
A PCR reaction solution is prepared by adding a reaction composition such as dNTP to phage 1000000pfu and primer DNA 0.2 μM each according to the kit instructions. The PCR conditions are kept at 94 ° C. for 2 minutes, and then kept at 94 ° C. for 30 seconds. The target DNA fragment was amplified by PCR under amplification conditions in which a cycle of 30 seconds at 60 ° C. and 40 minutes at 68 ° C. was repeated 40 times. Next, using the amplified DNA fragment as a template, Megaprime DNA-labelling system kit (manufactured by Amersham Pharmacia) ³² A probe labeled with P was prepared. The reaction solution (25 μl) was prepared by adding 32 PdCTP 2.0 MBq to 25 ng of the amplified DNA fragment and adding the reaction composition specified in the kit. The labeling reaction was performed at 37 ° C. for 10 minutes.
[0045]
(Reference Example 3) Acquisition of a phage cDNA clone carrying the CRE1 gene
The target CRE1 gene was cloned by plaque hybridization using the probe prepared in Reference Example 2. Details are described below.
Using the phage cDNA library prepared in Reference Example 1, plaques were formed according to the instructions of the ZAP-cDNAR Synthesis Kit. After adsorbing DNA from the formed plaques on a nitrocellulose filter, the DNA was immobilized on the filter by ultraviolet treatment. Using the filter thus prepared, 6 × SSC (0.9 M NaCl, 0.09 M sodium citrate), 5 × Denhardt's solution (0.1% (w / v) Ficoll 400, 0.1% (w / v) polyvinyl Pyrrolidone, 0.1% BSA), 0.5% (w / v) SDS and 100 μg / ml denatured salmon sperm DNA in DIG EASY Hyb solution (Boehringer Mannheim) containing 100 μg / ml denatured salmon sperm DNA Incubate 2 times for 15 minutes at room temperature in the presence of 1 × SSC (0.15M NaCl, 0.015M sodium citrate) and 0.5% SDS, and further 0.1 × SSC (0.015M NaCl, 0.0015M citric acid). Sodium) and 0.5% SDS were obtained by incubation at 68 ° C. for 30 minutes to obtain a hybridized phage cDNA clone.
[0046]
(Reference Example 4) Cloning of CRE1 cDNA
Using the cDNA of the phage cDNA clone obtained in Reference Example 3 as a template, PCR is performed using the DNA shown in SEQ ID NO: 13 and the DNA shown in SEQ ID NO: 14 as primers, and a DNA having the base sequence shown in SEQ ID NO: 5 Was amplified. Details are described below. PCR reaction was carried out using Herculase Enhanced DNA Polymerase (TOYOBO) at 94 ° C for 1 minute, followed by 25 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 4 minutes. Was done below. The PCR reaction solution (50 μl) was prepared by adding a reaction composition such as dNTP to 500 ng of cDNA of a cDNA clone of the phage and 100 ng of primer DNA according to the kit instructions.
In this way, the target DNA fragment was amplified.
[0047]
(Reference Example 5) Construction of CRE1 expression vector
The yeast expression vector p415CYC1 (available from Munberg et al. Gene: 156 119-122 (1995), ATCC library (No. 87382)) was cleaved with the restriction enzyme SmaI and then used as a reference using T4 DNA Ligase. The DNA having the base sequence represented by SEQ ID NO: 5 obtained in Example 4 was ligated to the CYC1 promoter sequence of the expression vector p415CYC1 so that the target protein was expressed in yeast. It was confirmed using an automatic base sequencer that the inserted DNA sequence was in the correct orientation and the DNA sequence was the base sequence shown in SEQ ID NO: 5, and expression plasmid p415CYC-CRE1 was obtained.
[0048]
(Example 1) Cloning of AHK3 (AAF99730) cDNA
The seeds of the Wassilewskija line of Arabidopsis were sterilized with 70% ethyl alcohol for 1 minute and further sterilized with 1.5% sodium hypochlorite for 10 minutes. After thoroughly washing with sterilized water, GM medium (4.3g Murashige and Skoog's basal salt mixture, 1% ssucrose, 10ml of 5% MES-KOH (pH5.7), 0.3% Phytagel ^TM (SIGMA)) was cultured for 2 weeks to obtain 5 g of plants. This was frozen in liquid nitrogen and then physically ground with a mortar. 10 ml of extraction buffer (200 mM Tris-HCl (pH 8.5), 100 mM NaCl, 10 mM EDTA, 0.5% SDS, 14 mM β-mercaptoethanol) and 10 g of phenol were added to the obtained ground product. After stirring with a Voltex mixer, 10 ml of chloroform was further added and stirred vigorously, followed by centrifugation at 10,000 rpm for 20 minutes. LiCl was added to the recovered aqueous layer to a final concentration of 2M, left at −80 ° C. for 3 hours, thawed, and centrifuged at 10,000 rpm for 20 minutes to recover the precipitate. Dissolve the collected precipitate in 2 ml of TE (10 mM Tris-HCl (pH 8.0) 1 mM EDTA), add 0.2 ml of 3 M sodium acetate (pH 5.2) and 5 ml of ethanol, and centrifuge to precipitate the RNA. It was collected. Furthermore, from 40 μg of recovered precipitate (RNA), 30 units of FPLCpure ^TM Dnase I (Amersham-Pharmacia) and 60 units of Superace (Ambion) were added to remove mixed genomic DNA, which was then purified by phenol / chloroform treatment and ethanol treatment. Next, RT-PCR reaction was performed using the purified RNA as a template and oligo (dT) 12-18 (amersham-Pharmacia) as a primer. The RT-PCR reaction was performed at 42 ° C. for 40 minutes using Superscript II (GIBCO BRL). The PT-PCR reaction solution was prepared according to the method specified in the Superscript II instructions.
In this way, the target cDNA was amplified.
A PCR reaction was performed using the amplified cDNA as a template and the DNA shown in SEQ ID NO: 16 and the DNA shown in SEQ ID NO: 17 as primers to amplify the DNA having the base sequence shown in SEQ ID NO: 3. The PCR reaction was carried out using Herculase Enhanced DNA Polymerase (TOYOBO) at 94 ° C for 1 minute, under amplification conditions of 41 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 4 minutes. It was conducted. The PCR reaction solution (50 μl) was prepared by adding a reaction composition such as dNTP to 500 ng of template DNA and 100 ng of primer DNA according to the kit instructions.
In this way, the target DNA fragment was amplified.
[0049]
(Example 2) Construction of pHM-1
p415CYC1 (Munberg et al. Gene: 156 119-122 (1995), available from ATCC library (No. 87382)) was digested with restriction enzymes SpeI and BamHI, and then T4 DNA Ligase was used. A synthetic DNA fragment (linker) shown in FIG. 15 was inserted, and restriction enzyme sites SacII, ApaI and NheI were newly added to plasmid p415CYC1 to construct pHM-1.
[0050]
(Example 3) Construction of AHK3 expression vector
After cleaving pHM-1 with restriction enzymes SalI and SacII, using T4 DNA Ligase, the DNA having the base sequence shown in SEQ ID NO: 3 is linked to the CYC1 promoter sequence of expression vector pHM-1, It was incorporated so that the target protein was expressed in yeast. It was confirmed by using an automatic base sequencer that the sequence and orientation of the inserted DNA were correct, and an expression plasmid p415CYC-AHK3 was obtained.
[0051]
(Example 4) Cloning of AHK2 (BAB09274) cDNA
Using the DNA fragment prepared in Example 1 (cDNA adjusted by reverse transcription from total RNA) as a template, a PCR reaction was performed using the DNA shown in SEQ ID NO: 19 and the DNA shown in SEQ ID NO: 20 as primers. A DNA having the base sequence indicated by No. 1 was amplified. PCR reaction was carried out using TAKARA Pfu Turbo denature (Takara Shuzo) at 94 ° C for 1 minute, followed by 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 4 minutes. Was done below. The PCR reaction solution (50 μl) was prepared by adding a reaction composition such as dNTP to 500 ng of template DNA and 50 ng of primer DNA according to the instructions.
In this way, the target DNA fragment was amplified.
[0052]
Example 5 Cloning of AHK2 (BAB09274) Δ cDNA
Using the DNA fragment prepared in Example 1 (cDNA prepared by reverse transcription from total RNA) as a template, a PCR reaction was carried out using the DNA shown in SEQ ID NO: 21 and the DNA shown in SEQ ID NO: 22 as primers. Of the base sequence represented by No. 1, DNA having a base sequence in which ATG was added to the 5 ′ end of the base sequence represented by base numbers 586 to 3531 was amplified. PCR reaction was carried out using TAKARA Pfu Turbo denature (Takara Shuzo) at 94 ° C for 1 minute, followed by 30 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 4 minutes. Was done below. The PCR reaction solution (50 μl) was prepared by adding a reaction composition such as dNTP to 500 ng of template DNA and 50 ng of primer DNA according to the instructions.
In this way, the target DNA fragment was amplified.
[0053]
(Example 6) Construction of AHK2 and AHK2Δ expression vectors
After cleaving pHM-1 with restriction enzymes SacII and NheI, the DNA fragments obtained in Example 4 and Example 5 were ligated to the CYC1 promoter sequence of expression vector pHM-1 using T4 DNA Ligase. The protein was incorporated so that the target protein was expressed in yeast. It was confirmed by using an automatic base sequencer that the base sequence and orientation of the inserted DNA were correct, and expression plasmids p415CYC-AHK2 and p415CYC-AHK2Δ were obtained.
[0054]
(Example 7) Production of transformed cells TM182-CRE1, TM182-AHK2, TM182-AHK2Δ and TM182-AHK3
Using the obtained expression plasmids p415CYC-CRE1 (Reference Example 5), p415CYC-AHK2 (Example 6), p415CYC-AHK2Δ (Example 6) and p415CYC-AHK3 (Example 3), One TM182 (sln1Δ) (Maeda T et al. Nature: 369 242-245 (1994)) was transformed. The transformation was performed according to VII. Library Transformation & Screening Protocols described on page 22 of CLONTECH: MATCHMAKER Two-Hybrid System 3 User Manual using a polyethylene glycol / lithium acetate (PEG / LiAc) -mediated transformation method. In the resulting transformed cells, the transformed yeast TM182-CRE1, TM182-AHK2, TM182-AHK2Δ and TM182 are selected by selecting transformed yeasts that grow on DOLU + Gal medium, taking advantage of the loss of leucine auxotrophy. -Made AHK3.
[0055]
(Example 8) Cytokinin response of transformed cells TM182-CRE1, TM182-AHK2, TM182-AHK2Δ and TM182-AHK3 (Part 1)
10 μl of the culture solution of transformed cells TM182-CRE1, TM182-AHK2, TM182-AHK2Δ and TM182-AHK3 prepared in Example 7 (about 800 individuals of yeast), DOLU + Glu agar supplemented with 10 μM of trans-zeatin It was spotted on the medium and cultured at 30 ° C. for 30 hours. After completion of the culture, the growth state of the transformed cells was observed, and photographs were taken with a digital camera.
As a result, TM182-CRE1, TM182-AHK2, TM182-AHK2Δ and TM182-AHK3 are all transformed cells in the case of DOLU + Glu agar medium supplemented with trans-zeatin. The degree of growth was higher than that in the case of Glu agar medium. This indicates that transformed cells respond to cytokinin and can grow on DOLU + Glu agar. It was also confirmed that the transformed cells can grow on DOLU + Gal medium regardless of the presence or absence of trans-zeatin.
[0056]
(Example 9) Cytokinin response of transformed cells TM182-CRE1, TM182-AHK2Δ and TM182-AHK2 (part 2)
The cultures of transformed cells TM182-CRE1, TM182-AHK2Δ and TM182-AHK2 transformed cells obtained in Example 7 were used in the DOLU + containing cytokinin at various concentrations (1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM). It was spotted on a Glu agar medium and cultured at 30 ° C. for 30 hours. After completion of the culture, the growth state was observed, and photographs were taken with a digital camera. Table 1 shows the minimum concentrations of trans-zeatin and cis-zeatin at which each transformed cell was able to grow.
[0057]
[Table 1]

[0058]
Example 10 Method for Searching for Substance having Agonist Activity for Cytokinin Receptor (Part 1)
The transformed cells TM182-AHK2, TM182-AHK2Δ and TM182-AHK3 obtained in Example 7 were inoculated into 200 ml of DOLU + Gal medium, pre-cultured at 30 ° C. for 36 hours, and this culture solution was OD600 in DOLU + Glu medium. = 0.100 and further diluted 1: 200 with DOLU + Glu medium was used as the pre-dilution culture solution.
An assay plate in which 20 μl of a solution (100 μM) obtained by diluting a DMSO solution of a test substance (Abscisic acid or 6-Benzyl aminopurine) prepared to a concentration of 10 mM 100-fold in DOLU + Glu medium is added to each well of a 96-well plate Got ready. At the same time, an assay plate containing 20 μl of a solution obtained by diluting 20 μl of DMSO 100-fold with DOLU + Glu medium was prepared as a control group.
200 μl of the above pre-dilution culture solution was added to each well of both assay plates, cultured at 30 ° C. for 24 hours, and then the turbidity of each well was measured using a plate reader. By comparing the turbidity with the turbidity in the control group, the test substance was tested for agonist activity against the cytokinin receptor. Table 2 shows the absorbance of OD620 of each transformed cell culture solution to which each test substance was added. Since the turbidity in the test group to which 6-Benzyl aminopurine was added was higher than that in the control group, 6-Benzyl aminopurine was selected as an agonist active substance for the cytokinin receptor.
[0059]
[Table 2]

[0060]
(Example 11) Method for searching for agonist active substance for cytokinin receptor (part 2)
The transformed cells TM182-AHK2, TM182-AHK2Δ and TM182-AHK3 obtained in Example 7 are inoculated into 200 ml of DOLU + Gal medium and pre-cultured at 30 ° C. for 30 hours. Transformed cells (TM182-AHK2, pre-cultured in DOLU + Glu agar medium added with various changes so that the test concentration of the agonist active substance for the cytokinin receptor selected in Example 10 was changed from 10 nM to 100 μM. 10 μl of TM182-AHK2Δ and TM182-AHK3) are spotted and incubated at 30 ° C. for 30 hours. After completion of the culture, test and confirm the strength of agonist activity against the cytokinin receptor of the test substance from the lowest test concentration at which the growth state of the transformed cells (TM182-AHK2, TM182-AHK2Δ and TM182-AHK3) is observed .
[0061]
The composition of the medium used in the present invention is described below.
(A) DOLU + GLU medium
Bacto-yeast nitrogen base without amino acids 6.7g
Glucose 20g
Drop-out mix (x) 2.0g
Distilled water 1000ml
(B) DOLU + GAL medium
Bacto-yeast nitrogen base without amino acids 6.7g
Glucose 20g
Drop-out mix (x) 2.0g
Distilled water 1000ml
(X) Drop-out mix:
Adenine 0.5g Lysine 2.0g
Alanine 2.0g Methionine 2.0g
Arginine 2.0g para-Aminobenzoic acid 0.2g
Asparagine 2.0g Phenylalanine 2.0g
Aspartic acid 2.0g Proline 2.0g
Cysteine 2.0g Serine 2.0g
Glutamine 2.0g Threonine 2.0g
Glutamic acid 2.0g Tryptophan 2.0g
Glycine 2.0g Tyrosine 2.0g
Histidine 2.0g Valine 2.0g
Inositol 2.0g Isoleucine 2.0g
Distilled water 1000ml
(C) DOLU + GLU agar medium
Solid medium in which 2% (W / V) agar is added to medium (a).
(D) DOLU + GAL agar medium
Solid medium in which 2% (W / V) agar is added to medium (c).
[0062]
【The invention's effect】
According to the present invention, it is possible to provide a method for analyzing an agonist activity for a cytokinin receptor, and further, by using the analysis method, a method for quickly finding a substance having an agonist activity for a cytokinin receptor in a small amount of sample can be provided. It becomes.
[0063]
"Sequence Listing Free Text"
SEQ ID NO: 9
Oligonucleotide primers designed for PCR
SEQ ID NO: 10
Oligonucleotide primers designed for PCR
SEQ ID NO: 11
Oligonucleotide primers designed for PCR
SEQ ID NO: 12
Oligonucleotide primers designed for PCR
SEQ ID NO: 13
Oligonucleotide primers designed for PCR
SEQ ID NO: 14
Oligonucleotide primers designed for PCR
SEQ ID NO: 15
Oligonucleotide primers designed for PCR
SEQ ID NO: 16
Oligonucleotide primers designed for PCR
SEQ ID NO: 17
Oligonucleotide primers designed for PCR
SEQ ID NO: 18
Oligonucleotide primers designed for PCR
SEQ ID NO: 19
Oligonucleotide primers designed for PCR
SEQ ID NO: 20
Oligonucleotide primers designed for PCR
SEQ ID NO: 21
Oligonucleotide primers designed for PCR
SEQ ID NO: 22
Oligonucleotide primers designed for PCR
[0064]
[Sequence Listing]

Claims (10)

A method for analyzing agonist activity against a cytokinin receptor,
(1) a first step in which a test substance is brought into contact with a transformed cell into which a DNA encoding any of the following cytokinin receptors has been introduced; and (2) expression in the transformed cell after the first step. And a second step of measuring the presence or amount of intracellular signal transduction from the cytokinin receptor.
(A) a cytokinin receptor having the amino acid sequence shown in SEQ ID NO: 2 (b) a cytokinin receptor having the amino acid sequence shown in SEQ ID NO: 4
( D) a cytokinin receptor comprising the amino acid sequence represented by amino acid numbers 196 to 1176 in the amino acid sequence represented by SEQ ID NO: 2 (e) represented by amino acid numbers 50 to 1176 among the amino acid sequences represented by SEQ ID NO: 2 the cytokinin receptor having the amino acid sequence (f) the amino acid sequence as shown in SEQ ID NO: 4, cytokinin receptors having the amino acid sequence represented by amino acid numbers 32 1036
( H) a cytokinin receptor having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of (a), (b), (d), (e) or (f) above   The transformed cell is a cell having a function in which cell growth is directly controlled by intracellular signal transmission from a cytokinin receptor, and the presence or absence of the intracellular signal transmission or the amount thereof is determined by the transformed cell. The analysis method according to claim 1, wherein the growth amount is measured as an index. A transformed cell obtained by introducing a DNA encoding one of the following cytokinin receptors into a host cell improved so that the transformed cell has a histidine kinase activity lower than the cell-specific histidine kinase activity. The analysis method according to claim 1.
(A) a cytokinin receptor having the amino acid sequence shown in SEQ ID NO: 2 (b) a cytokinin receptor having the amino acid sequence shown in SEQ ID NO: 4
( D) a cytokinin receptor comprising the amino acid sequence represented by amino acid numbers 196 to 1176 in the amino acid sequence represented by SEQ ID NO: 2 (e) represented by amino acid numbers 50 to 1176 among the amino acid sequences represented by SEQ ID NO: 2 the cytokinin receptor having the amino acid sequence (f) the amino acid sequence as shown in SEQ ID NO: 4, cytokinin receptors having the amino acid sequence represented by amino acid numbers 32 1036
( H) a cytokinin receptor having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of (a), (b), (d), (e) or (f) above The transformed cell encodes one of the following cytokinin receptors in a host cell improved so that the transformed cell has a histidine kinase activity lower than the cell-specific histidine kinase activity by deleting one or more histidine kinases. The analysis method according to claim 1, which is a transformed cell into which the DNA to be transformed is introduced.
(A) a cytokinin receptor having the amino acid sequence shown in SEQ ID NO: 2 (b) a cytokinin receptor having the amino acid sequence shown in SEQ ID NO: 4
( D) a cytokinin receptor comprising the amino acid sequence represented by amino acid numbers 196 to 1176 in the amino acid sequence represented by SEQ ID NO: 2 (e) represented by amino acid numbers 50 to 1176 among the amino acid sequences represented by SEQ ID NO: 2 the cytokinin receptor having the amino acid sequence (f) the amino acid sequence as shown in SEQ ID NO: 4, cytokinin receptors having the amino acid sequence represented by amino acid numbers 32 1036
( H) a cytokinin receptor having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of (a), (b), (d), (e) or (f) above 2. The analysis method according to claim 1, wherein the transformed cell is a transformed cell obtained by introducing a DNA encoding any of the following cytokinin receptors into a host cell not having a cytokinin receptor. .
(A) a cytokinin receptor having the amino acid sequence shown in SEQ ID NO: 2 (b) a cytokinin receptor having the amino acid sequence shown in SEQ ID NO: 4
( D) a cytokinin receptor comprising the amino acid sequence represented by amino acid numbers 196 to 1176 in the amino acid sequence represented by SEQ ID NO: 2 (e) represented by amino acid numbers 50 to 1176 among the amino acid sequences represented by SEQ ID NO: 2 the cytokinin receptor having the amino acid sequence (f) the amino acid sequence as shown in SEQ ID NO: 4, cytokinin receptors having the amino acid sequence represented by amino acid numbers 32 1036
( H) a cytokinin receptor having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of (a), (b), (d), (e) or (f) above   The analysis method according to claim 1, wherein the transformed cell is yeast.   The analysis method according to claim 1, wherein the transformed cell is a budding yeast.   The presence or absence or amount of intracellular signal transduction from cytokinin receptors measured for the transformed cells in contact with each test substance by analyzing the agonist activity of two or more different test substances by the analysis method according to claim 1 A method for assaying agonist activity against a cytokinin receptor, comprising evaluating the agonist activity against the cytokinin receptor of the substance based on a difference obtained by comparing the above.   9. The assay method according to claim 8, wherein at least one of the two or more different test substances is a substance that does not have an agonist activity for the cytokinin receptor.   A method for searching for an agonist active substance for a cytokinin receptor, comprising selecting a substance having agonist activity for a cytokinin receptor based on the agonist activity for the cytokinin receptor evaluated by the assay method according to claim 8.