JP2005130854A - Transformed cell co-expressing cytokinin receptor and cytokinin biosynthesis enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop and provide a method for analyzing the activity for controlling biosynthesis of cytokinin in the test article and rapidly searching substance having activity for controlling biosynthesis of cytokinin with a small amount of sample. <P>SOLUTION: A transformed cells or the like, comprising a polynucleotide having a base sequence encoding an amino acid sequence of a cytokinin receptor and a polynucleotide having a base sequence encoding an amino acid sequence of a cytokinin biosynthesis enzyme. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a transformed cell co-expressing a cytokinin receptor and a cytokinin biosynthetic enzyme.

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 known to exhibit such actions (see Non-Patent Document 1). In recent years, the key enzyme of plant cytokinin biosynthesis is isopentenyl transferase, and this enzyme converts the isopentenyl group of dimethylallyl diphosphate to adenosine-5′-triphosphate and / or adenosine-5′-bis. It has become clear that it is transferred to phosphoric acid (see Non-Patent Document 2, Non-Patent Document 3, and Patent Document 1).
Substances that have the activity to control the biosynthesis of cytokinins include plant growth regulators such as fruit tree apples, fruit drop prevention agents such as mandarin oranges, and lodging inhibitors such as rice and wheat by adjusting plant height. It can be used as a sweetness enhancer for later fruits, a bud inhibitor for tobacco and roses, and the like.
As a method for finding a substance having an activity for controlling the biosynthesis of cytokinin, a method for directly observing and evaluating a physiological change by spraying a test substance directly on a plant individual can be used.

Cytokinins: Chemistry, Activity, and Function, CRC Press (1994) Identification of Plant Cytokinin Biosynthtic Enzymes as Dimethylallyl Diphosphate: ATP / ADP Isopentenyltransferases.Plant & Cell Physiol. (2001) 42,677-685 Identification of Genes Encoding Adenylate Isopentenyltransferase, a Cytokinin Biosynthesis Enzyme, in Arabidopsis thaliana. J. Biol. Chem. (2001) 276, 26405-26410 WO2002072818A1 Genes encoding proteins participating in cytokinin synthesis

  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 an activity to control cytokinin biosynthesis with a small amount of a test substance has been demanded.

  As a result of intensive investigations under such circumstances, the present inventors analyzed the activity of controlling the biosynthesis of cytokinin as a test substance, and quickly and rapidly added a substance having the activity of controlling the biosynthesis of cytokinin in a small sample. Thus, the present inventors have found a transformed cell co-expressing a cytokinin receptor and a cytokinin biosynthetic enzyme for the purpose of searching for the present invention.

That is, the present invention
1. A transformed cell into which a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin receptor and a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin biosynthetic enzyme are introduced (hereinafter referred to as the transformed cell of the present invention). May be noted);
2. The transformed cell according to item 1 above, wherein the cytokinin receptor is any of the following cytokinin receptors (a) having the amino acid sequence represented by SEQ ID NO: 2 and represented by cytokinin receptor (b) SEQ ID NO: 4 A cytokinin receptor (c) having the amino acid sequence shown in SEQ ID NO: 6 and a part of the cytokinin receptor (d) transmembrane region deleted, Among the amino acid sequences represented by amino acid sequences 196 to 1176, among the amino acid sequences represented by amino acid sequences 196 to 1176 among the amino acid sequences represented by SEQ ID NO: 4 having one or more cytokinin receptors, Cytokinin receptor comprising the amino acid sequence represented by amino acid numbers 50 to 1176 (g Among the amino acid sequences shown in SEQ ID NO: 6, the cytokinin receptor (h) consisting of the amino acid sequences shown in amino acid numbers 32 to 1036 (h) the extracellular region of the cytokinin receptor, which is derived from homology to each other, the transmembrane region of the cytokinin receptor And a histidine kinase region of a cytokinin receptor and a histidine kinase receiver region heterozygous for the three regions (i) (a), (b), ( c) Cytokinin receptor having an amino acid sequence in which one or a plurality of amino acids are added, deleted and / or substituted with other amino acids in the amino acid sequence of the cytokinin receptor of c), (e), (f) or (g) (J) Cytokinin reception of (a), (b) or (c) above Cytokinin receptor having the amino acid sequence encoded by the nucleotide sequence of a polynucleotide and a polynucleotide hybridizing under stringent conditions with a nucleotide sequence having complementarity to the base sequence encoding the amino acid sequence of a;
3. 3. The transformed cell according to item 1 or 2, wherein the cytokinin biosynthetic enzyme is isopentenyl transferase;
4). 3. The transformed cell according to item 1 or 2, wherein the cytokinin biosynthetic enzyme is any of the following cytokinin biosynthetic enzymes (a) a cytokinin biosynthetic enzyme (b) having the amino acid sequence represented by SEQ ID NO: 8 ) Cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 10 (c) Cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 12 (d) Cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 14 ( e) Cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 16 (f) Cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 18 (g) Cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 20 (H) (a), (b), (c), (d), ( ), (F) or (g), the cytokinin biosynthetic enzyme having an amino acid sequence in which one or more amino acids are added, deleted and / or substituted by another amino acid in the amino acid sequence of the cytokinin biosynthetic enzyme (i) A base sequence complementary to the base sequence encoding the amino acid sequence of the cytokinin biosynthetic enzyme of (a), (b), (c), (d), (e), (f) or (g) A cytokinin biosynthetic enzyme comprising an amino acid sequence encoded by a base sequence of a polynucleotide that hybridizes with a polynucleotide having a stringent condition;
5). 5. The transformed cell according to any one of items 1 to 4 above, wherein the transformed cell is yeast;
6). 5. The transformed cell according to any one of 1 to 4 above, wherein the transformed cell is budding yeast;
Etc. are provided.

  According to the present invention, a co-expressing transformed cell of a cytokinin receptor and a cytokinin biosynthetic enzyme can be provided, and an analysis method of activity for controlling cytokinin biosynthesis can be provided by using the transformed cell. By using the analysis method, it is possible to provide a method for quickly finding a substance having an activity of controlling cytokinin biosynthesis with a small amount of sample.

Hereinafter, the present invention will be described in detail.
Since cytokinin has an isopentenyl group or a hydroxylated isopentenyl group, it has long been thought that cytokinin is synthesized in plants by isopentenylation of some adenine skeleton. In recent years, isopentenylation of adenosine-5′-triphosphate and / or adenosine-5′-diphosphate using dimethylallyl diphosphate as an isopentenyl group donor is a rate-limiting step in plant cytokinin biosynthesis. It became clear that there was. Such an isopentenyl transferase is a typical example of the cytokinin biosynthetic enzyme used in the present invention.

Specific examples of isopentenyl transferase include, for example, a cytokinin biosynthetic enzyme having the amino acid sequence represented by any of SEQ ID NOs: 8, 10, 12, 14, 16, 18 or 20, SEQ ID NOs: 8, 10 , 12, 14, 16, 18 or 20, a cytokinin biosynthetic enzyme having an amino acid sequence in which one or more amino acids are added, deleted and / or substituted by other amino acids in the amino acid sequence shown in any one of SEQ ID NOs: A polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence that is complementary to the base sequence encoding the amino acid sequence represented by any of 8, 10, 12, 14, 16, 18, or 20. Cytokinin biosynthetic enzyme consisting of amino acid sequence encoded by base sequence It can be mentioned. 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 added, deleted, and / or substituted with other amino acids” includes, for example, an amino acid sequence before addition, deletion, and / or substitution with other amino acids. On the other hand, an amino acid sequence having sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more can be mentioned as an example.
These “amino acids are added, deleted and / or replaced by other amino acids” and “80% or more sequence identity” include any of SEQ ID NOs: 8, 10, 12, 14, 16, 18 or 20. This includes processing that is carried out in a cell by a protein having the amino acid sequence shown, naturally occurring mutations due to species differences, individual differences, differences between tissues, etc. from which the protein is derived.

  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, for example, the histidine kinase family, and includes an extracellular region, a transmembrane region, a histidine kinase region (a His residue that has histidine kinase activity in the cell and serves as an active site). And a protein composed of a receiver region (a region having a phosphate transfer receptor and an Asp residue serving as an active site).

Specific examples of such a cytokinin receptor include, for example, a cytokinin receptor having an amino acid sequence represented by any of SEQ ID NOs: 2, 4, or 6, and any one of SEQ ID NOs: 2, 4, or 6. A cytokinin receptor having an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted by another amino acid in the amino acid sequence, a base encoding the amino acid sequence represented by any of SEQ ID NOs: 2, 4 or 6 A cytokinin receptor comprising an amino acid sequence encoded by a base sequence of a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the sequence, and a transmembrane region partially deleted cytokinin described later Receptors, chimeric cytokinin receptors described later, etc. It can gel. 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 added, deleted, and / or substituted with other amino acids” is, for example, 80% or more of the amino acid sequence before deletion, substitution, or addition, An amino acid sequence having a sequence identity of preferably 90% or more, more preferably 95% or more can also be mentioned as an example.
These “amino acids are added, deleted and / or replaced by other amino acids” and “80% or more sequence identity” include a protein having an amino acid sequence represented by any of SEQ ID NOs: 2, 4 or 6. It includes mutations that occur naturally due to intracellular processing, species differences in the organism from which the protein is derived, individual differences, differences between tissues, and the like.
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 the 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.

  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, that a hybrid is formed at 65 ° C. in a 6 × SSC (1.5M NaCl, 0.15M trisodium citrate solution is 10 × SSC) solution. Then, conditions such as washing with 1 × SSC at room temperature can be mentioned. The salt concentration in the washing step can be selected, for example, from 1 × SSC room temperature conditions (low stringency conditions) to 0.1 × SSC room temperature conditions (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.

Next, preparation of a transformed cell into which a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin receptor and a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin biosynthetic enzyme are introduced will be described.
A transformed cell into which a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin receptor and a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin biosynthetic enzyme have been introduced is used to convert the amino acid sequence of the cytokinin receptor. A polynucleotide having a base sequence encoding and a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme can be obtained, for example, by introducing them into a host cell as follows.

(1) Preparation of cDNA First, according to the method described in J., Sambrook, E., F., Frisch, T., Maniatis, Molecular Cloning 2nd edition, higher plants, etc. Total RNA is prepared from plants and the like. 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 with 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. Method (b) A method in which a solution containing guanidine thiocyanate is added to the above-mentioned ground product, CsCl is further added, and total RNA is obtained by centrifugation and the like may be used. For this operation, for example, a commercially available kit such as ISOGEN (Nippon Gene Co., Ltd.) or RNeasy Total RNA Purification Kit (Qiagen Co., Ltd.) can be used.

Then, 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 Bioscience Co., Ltd.), Oligotex ™ -dT30 <Super> (Takara Bio Inc.) can be used.
Furthermore, cDNA is prepared using the mRNA thus prepared (mRNA having a poly A chain). For example, after annealing an oligo dT chain or a random primer to mRNA, cDNA may be prepared by acting reverse transcriptase. Furthermore, double-stranded cDNA can be prepared by allowing RNaseH and DNA PolymeraseI to act on the cDNA. For this operation, SMARTTM PCR cDNA Synthesis Kit (Nippon Becton Dickinson Clontech Company), cDNA Synthesis Kit (Takara Bio Inc.), cDNA Synthesis Kit (Amersham Biosciences Inc.), ZAP-cDNA Synthesis Kit (Stratagene) Commercially available kits such as can be used.

(2) Cloning Polymerase chain reaction (hereinafter referred to as PCR) using, as a primer, a DNA having a partial base sequence of the base sequence represented by any one of SEQ ID NOS: 1, 3 or 5 from the thus prepared cDNA. Or a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin receptor by a hybridization method using a DNA having a partial base sequence of the base sequence represented by any of SEQ ID NOs: 1, 3 or 5 as a probe Can be obtained. Similarly, for example, PCR using a DNA having a partial base sequence of the base sequence represented by any of SEQ ID NOs: 7, 9, 11, 13, 15, 17 or 19 as a primer, or SEQ ID NOs: 7, 9, 11, A polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme is obtained by a hybridization method using as a probe a DNA having a partial base sequence of the base sequence shown in any one of 13, 15, 17 or 19 be able to.

When PCR is used, 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, 3, or 5 respectively. DNA designed and synthesized based on this can be used as a primer set. As the primer set, for example, a set of DNA consisting of the base sequence shown by SEQ ID NO: 23 and DNA consisting of the base sequence shown by SEQ ID NO: 24 can be cited as a primer set. 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 the cycle of 30 seconds at 72 ° C for 4 minutes for about 40 cycles, or heat at 94 ° C for 5 seconds and then at 72 ° C for 4 minutes for 5 to 10 cycles, 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 taken as one cycle, and this can be repeated 20 to 40 cycles. Such operations include, for example, Takara Heraculase ^TM (Takara Bio Inc.), DNA polymerase included in Advantage cDNA PCR Kit (Nippon Becton Dickinson Clontech Company), TAKARA Ex Taq (Takara Bio Inc.), Platinum PCR SuperMix Commercially available kits such as (Invitrogen Corporation) can be used.

When the hybridization method is used, cloning can be performed according to the method described in, for example, cloning and sequence: plant biotechnology experiment manual (Watanabe, Sugiura, edited by Rural Bunkasha, 1989).
As a probe to be used, a DNA having a partial base sequence of the base sequence represented by SEQ ID NO: 1, 3 or 5 (a chain length of about 200 bases to about 500 bases) is synthesized, and the DNA is, for example, Random Primed DNA. Labeling Kit (Roche Diagnostics Inc.), Random Primer DNA Labeling Kit Ver.2 (Takara Bio Inc.), ECL Direct Nucleic Acid Labeling and Ditection System (Amersham Biosciences Inc.), Megaprime DNA-labelling system ( It can be obtained by radioisotope labeling or fluorescent labeling according to a known method using Amersham Bioscience Co., Ltd.).
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. In the presence of 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 in DIG Easy Hyb solution (Roche Diagnostics) containing 100 μg / ml denatured salmon sperm DNA, and then kept at 65 ° C., then 1 × SSC (0.15 M NaCl, 0.015 M tricitrate) (Sodium) and 0.5% SDS in the presence of 2 times of incubation at room temperature for 15 minutes, and further 0.1 × SSC (0.015M NaCl, 0.0015M In the presence of phosphate trisodium) and 0.5% SDS, can be exemplified conditions that kept 30 minutes at 68 ° C..
In order to obtain a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin receptor derived from Arabidopsis thaliana, first, TAKARA LA taq ^TM (Takara Bio Inc.) using an Arabidopsis thaliana cDNA library phage solution (about 1000000 pfu) as a template. The DNA used as a probe may be amplified by PCR using the DNA shown in SEQ ID NO: 25 and the DNA shown in SEQ ID NO: 26 as a primer set, and obtained. Similarly, in order to obtain a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme derived from Arabidopsis thaliana, first, TAKARA LA taq ^TM (Takara) DNA used as a probe may be amplified by performing PCR using Bio Inc. and obtained. 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 temperature is kept at 94 ° C. for 2 minutes, then kept at 8 ° C. for 3 minutes, and then a cycle of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 5 minutes is repeated 40 cycles. Thus, conditions for amplification can be raised.
Next, using the amplified and obtained DNA as a template and using the Megaprime DNA-labelling system (Amersham Biosciences Co., Ltd.), a probe labeled with ³² P is prepared by using the reaction solution specified by the kit. be able to. Colony hybridization was carried out using the probe thus prepared according to the usual method, 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 100 μg / Incubate at 65 ° C. in DIG Easy Hyb solution (Roche Diagnostics) containing ml denatured salmon sperm DNA, then 1 × SSC (0.15 M NaCl, 0.015 M trisodium citrate) and 0 Incubate twice for 15 minutes at room temperature in the presence of 5% SDS, and then add 0.1 × SSC (0.015M NaCl, 0.0015M sodium citrate Arm) and in the presence of 0.5% SDS, can be obtained clones hybridized to the probe by incubating 30 minutes at 68 ° C..

  In addition, the polynucleotide having the base sequence encoding the amino acid sequence of the cytokinin receptor and the polynucleotide having the base sequence encoding the amino acid sequence of the cytokinin biosynthetic enzyme are, for example, any one of SEQ ID NOs: 1, 3 and 5, respectively. On the basis of the nucleotide sequence represented by SEQ ID NO: 7, 9, 11, 13, 15, 17 or 19, for example, the phosphite triester method (Hunkapiller, M. et al. , Nature, 310, 105, 1984) and the like, and can also be prepared by performing chemical synthesis of nucleic acids.

The polynucleotide having the base sequence encoding the amino acid sequence of cytokinin receptor and the polynucleotide having the base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme obtained as described above are "Molecular Cloning: A Laboratory Manual". 2nd edition '' (1989), Cold Spring Harbor Laboratory Press, `` Current Protocols In Molecular Biology '' (1987), John Wiley & Sons, Inc.ISBN0-471-50338-X, etc. To clone. Examples of the vector used include pBlueScriptII vector (Stratagene), pUC18 / 19 vector (Takara Bio Inc.), TA cloning vector (Invitrogen Corp.) 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.). For this operation, for example, a commercially available kit such as Termo Sequenase II dye terminator cycle sequencing kit (Amersham Biosciences), BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems Japan) can be used.

(3) Construction of expression vector Construction of a vector expressing a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and a vector expressing a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme , Conventional methods (eg, J., Sambrook, E., F., Frisch, T., Maniatis, Molecular Cloning 2nd edition), published by Cold Spring Harbor Laboratory press Or the like).
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 E. coli is used as a host cell, for example, plasmid pUC119 (Takara Bio Inc.), phagemid pBluescriptII (Stratagene), etc.) When yeast is used as a host cell, for example, plasmid pACT2 (Nippon Becton Dickinson Co., Ltd., Clontech Company) or the like may be used. pBI221 (Nippon Becton Dickinson Co., Ltd., Clontech Company) etc.) It may be constructed by incorporating a polynucleotide having a base sequence encoding the amino acid sequence of the receptor or a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme.

  The amino acid sequence of the cytokinin receptor is incorporated into the vector in such a manner that a promoter capable of functioning in the host cell is operably linked upstream of the polynucleotide having the base sequence encoding the amino acid sequence of the cytokinin receptor. An expression vector capable of expressing a polynucleotide having a base sequence encoding in a host cell can be constructed. Similarly, cytokinin biosynthesis is achieved by incorporating a promoter capable of functioning in the host cell into a functioning form upstream of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme. An expression vector capable of expressing in a host cell a polynucleotide having a base sequence encoding the amino acid sequence of the enzyme can be constructed. Here, “to bind in a functional manner” means a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin receptor under the control of a promoter in a host cell and a base encoding the amino acid sequence of a cytokinin biosynthetic enzyme. A polynucleotide having a base sequence encoding the amino acid sequence of the promoter and cytokinin receptor, and a polynucleotide having a base sequence encoding the amino acid sequence of the promoter and cytokinin biosynthetic enzyme so that the polynucleotide having the sequence is expressed Means to combine. Examples of promoters that can function in the host cell include, for example, when the host cell is E. 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 promoter (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, examples include nopaline synthase gene (NOS) promoter, octopine synthase gene (OCT) promoter, cauliflower mosaic virus (CaMV) -derived 19S promoter, CaMV-derived 35S promoter, and the like. it can.

  In addition, a polynucleotide having a base sequence encoding the amino acid sequence of the cytokinin receptor and a polynucleotide having a base sequence encoding the amino acid sequence of the cytokinin biosynthetic enzyme are incorporated into a vector having in advance a promoter that can function in the host cell. In this case, the promoter possessed by the vector and the cytokinin biosynthetic enzyme so that the promoter possessed by the vector and the polynucleotide having the base sequence encoding the amino acid sequence of the cytokinin receptor are bound in a functional manner. Each of the polynucleotides having a base sequence encoding the amino acid sequence of the cytokinin receptor downstream of the promoter so that the polynucleotide having a base sequence encoding the amino acid sequence is operably linked. The amino acid sequence of de or cytokinin biosynthesis enzyme may be inserted a polynucleotide having a nucleotide sequence encoding. For example, the aforementioned yeast plasmid pACT2 has an ADH1 promoter and encodes a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin receptor downstream of the ADH1 promoter of the plasmid or an amino acid sequence of a cytokinin biosynthetic enzyme. A polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin receptor or a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin biosynthetic enzyme, for example, CG1945 An expression vector that can be expressed in yeast such as Nippon Becton Dickinson Clontech Co., Ltd. can be constructed.

(4) Preparation of transformed cell The transformed cell used in the present invention can be prepared by introducing the constructed expression vector into a host cell according to a usual method. 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 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 the BY-2 strain, which is a cultured cell of tobacco, and the 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 introduction 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. The expression vector can be introduced into a host cell by using the method. When yeast is used as the host cell, the expression vector can be introduced into the host cell by using, for example, a Yeast transformation kit (Nippon Becton Dickinson Clontech Company) 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).

Next, a transformed cell into which a polynucleotide having a base sequence encoding the amino acid sequence of the transmembrane region partially deleted cytokinin receptor is introduced and its production will be described.
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.

  Specifically, among the amino acid sequence represented by SEQ ID NO: 4, 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: 4, A cytokinin receptor consisting of the amino acid sequence shown by amino acid numbers 50 to 1176 (3 transmembrane regions), and a cytokinin receptor consisting of the amino acid sequence shown by amino acid numbers 32 to 1036 among the amino acid sequence shown by SEQ ID NO: 6 ( Two transmembrane regions), cytokinin receptors consisting of amino acid sequences in which one or more amino acids are added, deleted and / or replaced by other amino acids in the amino acid sequences of these cytokinin receptors, such as these amino acid sequences One methionine is added to the amino terminus of Cytokinin receptor or the like consisting of the amino acid sequence can be exemplified.

The polynucleotide having the base sequence encoding the amino acid sequence of the above cytokinin receptor is, for example, a polynucleotide having the base sequence encoding the amino acid sequence of the natural cytokinin receptor, and a part of the transmembrane region of the receptor. A polynucleotide comprising a base sequence encoding the amino acid sequence of a transmembrane region in which the number of transmembrane regions is smaller than the number of transmembrane regions possessed by a natural cytokinin receptor Can be constructed by cleaving and joining using ordinary genetic engineering techniques.
Preparation of a transformed cell into which a polynucleotide having a base sequence encoding the amino acid sequence of the cytokinin receptor and a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme are introduced is performed as described above. In the method described in the description of the preparation of transformed cells into which a polynucleotide having a base sequence encoding the amino acid sequence of the receptor and a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme are introduced. Just like that.

Next, a transformed cell obtained by introducing a polynucleotide having a base sequence encoding the amino acid sequence of a chimeric cytokinin receptor and its production will be described.
Cytokinin receptors used in the present invention include an extracellular region of cytokinin receptors that are homologous to each other, a transmembrane region of cytokinin receptor, and a histidine kinase region of cytokinin receptor, heterogeneous to the above three regions. And a chimeric cytokinin receptor composed of a histidine kinase receiver region (which may be referred to as a chimeric cytokinin receptor in the present application). Specifically, for example, the extracellular region derived from the cytokinin receptor of CRE1, AHK2, AHK3, the transmembrane region and the histidine kinase region, and the receiver region derived from the histidine kinase encoded by the Sln1 gene of budding yeast And a chimeric cytokinin receptor composed of:

Proteins of the histidine kinase family including cytokinin receptors have the following amino acid 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 comprising 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. Receiver region of the encoded histidine kinase (see, for example, the amino acid sequence shown in SEQ ID NO: 21), receiver region of the histidine kinase encoded by the Salmonella-derived Chey gene, encoded by 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, see amino acid sequence shown in SEQ ID NO: 22), histidine encoded by Phks gene involved in cell cycle control of fission yeast Receiver region of kinase (Shieh, JC et al, Gene Dev. 11, 1008-1022 (1997)) and the like can be used.

Here, the histidine kinase region of the cytokinin receptor refers to, for example, a region present downstream of the transmembrane region present at the most C-terminal side among a plurality of transmembrane regions, 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) A region 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: 4, the 587th amino acid to 844th amino acid of SEQ ID NO: 4 The cytokinin receptor (AHK3) having the amino acid sequence represented by SEQ ID NO: 6 is a region including from the 450th amino acid to 700th amino acid of SEQ ID NO: 6, Is a region containing from 449 th amino acids of the cytokinin receptor (CRE1) in SEQ ID NO: 2 having the amino acid sequence shown in ID NO: 2 to 714 amino acid.
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, SEQ ID NO: 4 A region including the 891st amino acid to the 1163rd amino acid, AHK3 is a region including the 746th amino acid to the 1018th amino acid of SEQ ID NO: 6, and CRE1 is the region including the 763rd amino acid of SEQ ID NO: 2 to 1038 It is a region including the amino acid up to the first amino acid.
At least one of the extracellular regions is a region existing between the transmembrane region present at the most C-terminal side and the transmembrane region present at one N-terminal side from 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 AHK2, AHK3, and CRE1, and in AHK2, SEQ ID NO: 4 The region from amino acid 259 to amino acid 536 of AHK3 is a region including from amino acid 120 to amino acid 399 of SEQ ID NO: 6 in AHK3, and from amino acid 132 of SEQ ID NO: 2 in CRE1 This region includes up to the 398th amino acid.

  A polynucleotide having a base sequence encoding the amino acid sequence of a chimeric cytokinin receptor is an extracellular region of a cytokinin receptor, a transmembrane region of a cytokinin receptor, a histidine kinase region of a cytokinin receptor, and a receiver region of a histidine kinase. Each of the polynucleotides encoding the amino acid sequence of the region is prepared, and an appropriate linker is inserted as necessary so that a stop codon does not appear in the middle and the frame is not shifted. Can be constructed by concatenating using a statistical approach. A polynucleotide having a base sequence encoding the amino acid sequence of the extracellular region of cytokinin receptor and the transmembrane region of cytokinin receptor, the extracellular region of cytokinin receptor, the transmembrane region of cytokinin receptor, and the cytokinin receptor The polynucleotide encoding the amino acid sequence of the histidine kinase region may be prepared as a single molecule polynucleotide and used to construct a polynucleotide having a base sequence encoding the amino acid sequence of the chimeric cytokinin receptor.

  In addition, the polynucleotide which codes the amino acid sequence of each said area | region can each be produced using a well-known method. For example, in the case of producing by PCR, first, an oligonucleotide (5 ′ side primer) having a base sequence of the 5 ′ end region of a polynucleotide having a base sequence encoding the amino acid sequence of each region to be amplified and 3 Design and synthesize oligonucleotides (3'-side primers) each having a nucleotide sequence complementary to the nucleotide sequence of the 'end region'. The primer may be an oligonucleotide of usually about 14 bases to about 35 bases, and further, on the 5 ′ end side of this primer, there is a ligation between DNAs amplified by PCR or a ligation between these DNAs and a 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 for preparing a polynucleotide having a base sequence encoding the amino acid sequence of the extracellular region of the cytokinin receptor, the transmembrane region of the cytokinin receptor or the histidine kinase region of the cytokinin receptor, for example, higher A library of cDNA derived from plants such as plants can be mentioned. In addition, as a template used in the preparation of a polynucleotide having a base sequence encoding the amino acid sequence of the histidine kinase receiver region, for example, a cDNA library or total DNA derived from a microorganism prepared by a usual method is used. can give.

  The preparation of a transformed cell into which a polynucleotide having a base sequence encoding the amino acid sequence of the chimeric cytokinin receptor and a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme are introduced as described above. The method described in the description of the preparation of transformed cells into which a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme are introduced Follow the above.

Next, an intracellular signal transmission system related to cytokinin will be described.
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 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.

  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, is low. The amount of transmission is reduced. In host cells modified to have histidine kinase activity lower than that inherent to the host cell, for example, changes in growth, changes in shape, changes in shape, biosynthesis of specific substances in the cell Changes, changes in the metabolism of specific substances, etc. 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.

Next, a method for analyzing the activity controlling the biosynthesis of cytokinin will be described.
In the method for analyzing activity controlling cytokinin biosynthesis, a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme are introduced. As an example of a specific step of bringing the test substance into contact with the transformed cell, there can be mentioned, for example, a method of culturing the transformed cell in a medium containing the 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, about 1 hour to about 3 days, and preferably about 25 hours to about 2 days.
After the step, the presence or amount of intracellular signal transduction from the cytokinin receptor expressed in the transformed cells is measured, and the activity to control cytokinin biosynthesis is analyzed using the obtained measured value as an index. To do.

  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. al. Nature: 369 242-245 (1994)) as a host cell (ie, a transformed cell having a function of directly controlling cell growth by intracellular signal transduction from a cytokinin receptor) ), The activity to control cytokinin biosynthesis using the growth of the transformed cell in a medium (agar medium or liquid medium) using glucose as a carbon source, for example, DOHLU + Glu medium. Can be measured. DOHLU + Glu medium with test substance added, based on the growth of the transformed cells when using DOHLU + Glu medium without test substance (medium that does not contain a substance having the activity to control cytokinin biosynthesis) It can be evaluated that a test substance capable of changing the growth of the transformed cell when a medium is used has an activity to control cytokinin biosynthesis. Furthermore, the TM182 (sln1Δ) strain, which is a Sln1 gene-deficient strain into which the PTP2 Tyrosine phosphatase gene has been introduced, is used as a host cell, and a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme is not introduced. When the growth of transformed cells into which only a polynucleotide having a base sequence encoding an amino acid sequence is introduced is constant regardless of the presence or absence of the test substance in DOLU + Glu medium and DOLU + Glu medium with cytokinin added In addition, the test substance has an activity to control cytokinin biosynthesis, and in addition to the activity to control cytokinin biosynthesis, cells from cytokinin receptors are different from those controlling cytokinin biosynthesis. It may not have the activity to increase or decrease the amount of internal signal transmission or to control the growth of yeast by other methods. It can be evaluated to have a high sex. In addition, galactose was used in place of glucose as a carbon source as a control for verifying whether the test substance has an activity to control yeast growth by another method unrelated to cytokinin biosynthesis or reception. The growth of the TM182 (sln1Δ) strain, which is a Sln1 gene-deficient strain into which the PTP2 Tyrosine phosphatase gene has been introduced, in a medium (for example, DOLU + Gal medium) may be examined regardless of the presence or absence of the test substance.

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. The test substance that can change the mitotic growth of the transformed cell when the medium to which the test substance is added is used as a reference based on the division and growth of the transformed cell when the medium without the test substance is used. It can be evaluated as having the activity of controlling biosynthesis.
When using transformed cells prepared using RcsC gene-deficient E. coli cells into which cps-LacZ has been introduced as host cells, the color development of X-Gal may be observed in an agar medium or liquid medium (Suzuki et al. Plant Cell Physiol 42: 107-113 (2001)). The test substance that can change the color of the transformed cell when the medium with the test substance is used on the basis of the color of the transformed cell when the medium without the test substance is used as a reference is the biosynthesis of cytokinin It can be evaluated that it has the activity to control

  Further, according to the method for analyzing the activity controlling the biosynthesis of cytokinin, at least one of the two or more different substances (for example, at least one of the two or more different substances has an activity of controlling the biosynthesis of cytokinin. The amount of intracellular signal transduction from the cytokinin receptor measured for transformed cells in contact with each test substance, and the amount thereof. Based on the difference obtained by comparing the above, the activity of the substance to control the biosynthesis of cytokinin can also be evaluated and tested.

  Then, a substance having an activity to control the biosynthesis of cytokinin is selected by selecting a substance having an activity to control the biosynthesis of cytokinin based on the activity to control the biosynthesis of cytokinin evaluated by the above assay method. You can also

A substance having an activity of controlling the biosynthesis of cytokinin selected by the above search method 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 by formulating it. In these preparations, a substance having an activity of controlling the biosynthesis of cytokinin is generally contained in an amount of 0.5 to 90% by weight, preferably 1 to 80% by weight.

Examples of solid carriers used for 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 granular materials such as chemical fertilizers (ammonium sulfate, phosphorous acid, ammonium nitrate, ammonium sulfate, urea, etc.), and examples of liquid carriers include water, alcohols (methanol, ethanol, etc.) ), Ketones (acetone, methyl ethyl ketone, cyclohexanone, etc.)
, Aromatic hydrocarbons (toluene, xylene, ethylbenzene, methylnaphthalene, etc.), non-aromatic hydrocarbons (hexane, cyclohexane, kerosene, etc.), esters (ethyl acetate, butyl acetate, etc.), nitriles (acetonitrile, iso Butyronitrile), ethers (dioxane, diisopropyl ether, etc.), acid amides (dimethylformamide, dimethylacetamide, etc.), halogenated hydrocarbons (dichloroethane, trichloroethylene, etc.) and the like.
Surfactants include, for example, alkyl sulfates, alkyl sulfonates, alkyl aryl sulfonates, alkyl aryl ethers and polyoxyethylenates thereof, polyethylene glycol ethers, polyhydric alcohol esters, sugar alcohol derivatives. Etc.
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). 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.

The substance having the activity to control the biosynthesis of cytokinin formulated in this way is diluted with water or the like as it is, and is applied to the foliage part, branch leaves part, flower part of the plant, to the fruit. It is used for soaking treatment and application to fruit. Moreover, the said plant growth regulator is processed once or several times with respect to the object plant.
When the plant growth regulator is used for the purpose of suppressing the fall of fruits, for example, the plant growth regulator is diluted in water and sprayed onto fruit parts and branches and leaves 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.

  The application amount of the substance having the activity of controlling the biosynthesis of cytokinin in the above plant growth regulator may vary depending on the preparation form, application time, application method, application place, target plant, but is usually 1 to 8000 g per hectare. is there. 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 generally the concentration used for the above substances is 0.0001. ˜1000 mM, desirably 0.001 to 10 mM.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these.

  First, an example in which a substance having an activity to control cytokinin biosynthesis 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 a substance having an activity of controlling the biosynthesis of cytokinin, 3 parts of calcium lignin sulfonate, 2 parts of sodium lauryl sulfate, and 45 parts of synthetic hydrous hydroxide.

Formulation Example 2
70 parts of a substance having an activity of controlling cytokinin biosynthesis, 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
Mix 40 parts of a substance with activity to control the biosynthesis of cytokinin, 3 parts of polyoxyethylene sorbitan monooleate, 3 parts of CMC (carboxymethylcellulose) and 52 parts of water and wet mill until the particle size is 5 microns or less. To obtain a suspension.

Example 1 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 1): Preparation of an Arabidopsis cDNA phage library for CER1 cloning)
The seed of the Arabidopsis Wassilewskija line was 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 (4.3 g Murashige and Skoog's basal salt mixture, 1% sucrose, 10 ml of 5% MES-KOH (pH 5.7), 0.3% Phytagel ^™ (SIGMA)) After 2 weeks of cultivation, 5 g of plant was obtained. This was frozen in liquid nitrogen and then physically ground with a mortar. A mixture of 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 was 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 so as to have a final concentration of 2 M, left at −80 ° C. for 3 hours, thawed, and centrifuged at 10,000 rpm for 20 minutes to recover the precipitate. The recovered precipitate was dissolved in 2 ml of TE (10 mM Tris-HCl (pH 8.0) 1 mM EDTA), 0.2 ml of 3 M sodium acetate (pH 5.2) and 5 ml of ethanol were added and centrifuged. RNA was recovered as a precipitate. Furthermore, RNA with which polyA was bound was extracted from the recovered precipitate (RNA) using Oligotex ^™ dT30super (Roche Diagnostics).
A phage cDNA library was prepared from the extracted polyA-bound RNA using a ZAP-cDNAR Synthesis Kit (Stratagene) according to the instructions. The titer of the prepared phage cDNA library was 500000 PFU.

Example 2 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 1): Preparation of a DNA probe for CRE1)
Using the TAKARA LA Taq ^TM kit (Takara Bio Inc.) using the phage solution (about 1000000 PFU) of the phage cDNA library prepared in Example 1 as a template, the DNA represented by SEQ ID NO: 25 and the DNA represented by SEQ ID NO: 26 PCR reaction was performed using the prepared DNA as a primer to amplify the DNA. Details are described below.
A PCR reaction solution was prepared by adding a reaction composition such as dNTP to phage 1000000 pfu and primer DNA 0.2 μM each according to the kit instructions. As a PCR condition, the DNA fragment of interest is obtained by performing PCR under amplification conditions in which a cycle of 94 ° C. for 2 minutes, 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 5 minutes is repeated 40 cycles. Was amplified. Next, using the amplified DNA fragment as a template, a probe labeled with ³² P was prepared using a Megaprime DNA-labelling system kit (Amersham Biosciences). 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.

Example 3 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 1): Acquisition of a phage cDNA clone carrying the CRE1 gene)
The target CRE1 gene was cloned by plaque hybridization using the probe prepared in Example 2. Details are described below.
Using the phage cDNA library prepared in Example 1, plaques were formed according to the instructions of the ZAP-cDNAR Synthesis Kit. After adsorbing DNA from the formed plaque on a nitrocellulose filter, the DNA was fixed 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% DIG Easy Hyb in the presence of (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 After incubating in a solution (Roche Diagnostics Inc.) at 65 ° C. for 15 minutes at room temperature in the presence of 1 × SSC (0.15 M NaCl, 0.015 M sodium citrate) and 0.5% SDS. Incubate twice and incubate at 68 ° C for 30 minutes in the presence of 0.1 x SSC (0.015M NaCl, 0.0015M sodium citrate) and 0.5% SDS. The phage cDNA clone which hybridizes was obtained.

Example 4 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 1): Cloning of CRE1 cDNA)
Using the cDNA of the phage cDNA clone obtained in Example 3 as a template, PCR was performed using the DNA represented by SEQ ID NO: 23 and the DNA represented by SEQ ID NO: 24 as primers, whereby the base sequence represented by SEQ ID NO: 1 was obtained. Amplified DNA was amplified. Details are described below.
The PCR reaction was performed by using Herculase Enhanced DNA Polymerase (Toyobo Co., Ltd.), incubating at 94 ° C. for 1 minute, repeating 25 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 4 minutes. Performed under conditions. The PCR reaction solution (50 μl) was prepared by adding a reaction composition such as dNTP to 500 ng of the cDNA of the phage cDNA clone and 100 ng of each primer DNA according to the kit instructions.
In this way, the target DNA fragment was amplified.

Example 5 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 1): 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 with T4 DNA Ligase. The DNA having the base sequence represented by SEQ ID NO: 1 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 by 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: 1, and the expression plasmid p415CYC-CRE1 was obtained.

Example 6 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 2): Cloning of AHK3 (AAF99730) cDNA)
The seed of the Arabidopsis Wassilewskija line was 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 (4.3 g Murashige and Skoog's basal salt mixture, 1% sucrose, 10 ml of 5% MES-KOH (pH 5.7), 0.3% Phytagel ^™ (SIGMA)) Was cultured for 2 weeks to obtain 5 g of a plant. This was frozen in liquid nitrogen and then physically ground with a mortar. A mixture of 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 was 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 so as to have a final concentration of 2 M, left at −80 ° C. for 3 hours, thawed, and centrifuged at 10,000 rpm for 20 minutes to recover the precipitate. The collected precipitate was dissolved in 2 ml of TE (10 mM Tris-HCl (pH 8.0) 1 mM EDTA), and then 0.2 ml of 3 M sodium acetate (pH 5.2) and 5 ml of ethanol were added and centrifuged. RNA was collected as a precipitate. Further, from 40 μg of the collected precipitate (RNA), 30 units of FPLCpure ^™ DnaseI (Amersham Biosciences) and 60 units of Superace (Ambion) were added to remove the mixed genomic DNA, which was then added with phenol / chloroform. Purification by treatment and ethanol treatment. Next, RT-PCR reaction was performed using the purified RNA as a template and oligo (dT) 12-18 (Amersham Biosciences) as a primer. The RT-PCR reaction was performed at 42 ° C. for 40 minutes using SuperScript II Rnase H-Reverse Transcriptase (Invitrogen Corporation). The RT-PCR reaction solution was prepared according to the method specified in the SuperScript II Rnase H-Reverse Transcriptase manual.
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: 27 and the DNA shown in SEQ ID NO: 28 as primers to amplify the DNA having the base sequence shown in SEQ ID NO: 5. The PCR reaction was carried out using Herculase Enhanced DNA Polymerase (Toyobo Co., Ltd.) for 1 minute at 94 ° C, followed by 41 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 4 minutes. Performed under conditions. A 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 each of primer DNA according to the kit instructions.
In this way, the target DNA fragment was amplified.

Example 7 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (part 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 to obtain SEQ ID NO: 29 and PHM-1 was constructed by inserting the synthetic DNA fragment (linker) shown by 30 and adding restriction enzyme sites SacII, ApaI and NheI to p415CYC1.

Example 8 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 2): Construction of AHK3 expression vector)
After the pHM-1 constructed in Example 7 was cleaved with restriction enzymes SalI and SacII, a DNA having the base sequence shown in SEQ ID NO: 5 was converted to CYC1 of the expression vector pHM-1 using T4 DNA Ligase. By binding to a promoter sequence, it was incorporated so that the target protein was expressed in yeast. It was confirmed using an automatic base sequencer that the sequence and orientation of the inserted DNA were correct, and an expression plasmid p415CYC-AHK3 was obtained.

Example 9 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 2): Cloning of AHK2 (BAB09274) cDNA)
Using the DNA fragment prepared in Example 6 (cDNA prepared by reverse transcription from total RNA) as a template, a PCR reaction was performed using the DNA represented by SEQ ID NO: 31 and the DNA represented by SEQ ID NO: 32 as primers, A DNA having the base sequence indicated by No. 3 was amplified. The PCR reaction was carried out using TAKARA Pfu Turbo denature (TAKARA BIO INC.) For 1 minute at 94 ° C., followed by 30 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 4 minutes. Performed under conditions. A 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.

Example 10 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 2): Cloning of AHK2 (BAB09274) Δ cDNA)
Using the DNA fragment prepared in Example 6 (cDNA prepared by reverse transcription from total RNA) as a template, a PCR reaction was performed using the DNA represented by SEQ ID NO: 33 and the DNA represented by SEQ ID NO: 34 as primers, Of the base sequence represented by No. 3, a DNA having a base sequence obtained by adding ATG to the 5 ′ end of the base sequence represented by base numbers 586 to 3531 was amplified. The PCR reaction was carried out using TAKARA Pfu Turbo denature (TAKARA BIO INC.) For 1 minute at 94 ° C., followed by 30 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 4 minutes. Performed under conditions. A 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.

Example 11 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin receptor and preparation of a vector expressing the polynucleotide (Part 2): Construction of AHK2 and AHK2Δ expression vectors)
After cleaving pHM-1 constructed in Example 7 with restriction enzymes SacII and NheI, the DNA fragments obtained in Example 9 and Example 10 were subjected to CYC1 promoter sequence of pHM-1 using T4 DNA Ligase. The target protein was incorporated so as to be 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.

Reference Example 1 (Preparation of transformed cells TM182-CRE1, TM182-AHK2, TM182-AHK2Δ and TM182-AHK3)
Using the resulting expression plasmids p415CYC-CRE1 (Example 5), p415CYC-AHK2 (Example 11), p415CYC-AHK2Δ (Example 11) and p415CYC-AHK3 (Example 8), A TM182 (sln1Δ) (Maeda T et al. Nature: 369 242-245 (1994)) was transformed. Transformation is performed using the polyethylene glycol / lithium acetate (PEG / LiAc) -mediated transformation method, which is described on page 22 of Matchmaker Two-Hybrid System 3 User Manual of Clontech Company, Nippon Becton Dickinson Co., Ltd. VII. Library Transformation & Screening Performed according to Protocols. 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.

Reference Example 2 (Cytokinin response of transformed cells TM182-CRE1, TM182-AHK2, TM182-AHK2Δ and TM182-AHK3 (Part 1))
10 μl of a culture solution of transformed cells TM182-CRE1, TM182-AHK2, TM182-AHK2Δ and TM182-AHK3 prepared in Reference Example 1 (approximately 800 individuals of yeast) and 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.

Reference Example 3 (Cytokinin response of transformed cells TM182-CRE1, TM182-AHK2Δ and TM182-AHK2 (2))
The culture solution of the transformed cells TM182-CRE1, TM182-AHK2Δ and TM182-AHK2 transformed cells obtained in Reference Example 1 contains 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.

Reference Example 4 (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 Reference Example 1 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.
In each well of a 96-well plate, a test substance (Abscisic acid or 6-Benzyl aminopurine) dimethyl sulfoxide (hereinafter referred to as DMSO) solution prepared to a concentration of 10 mM is diluted 100-fold with DOLU + Glu medium. An assay plate to which 20 μl of 100 μM) was added was prepared. At the same time, an assay plate containing 20 μl of a solution obtained by diluting 20 μl of DMSO 100 times with DOLU + Glu medium was prepared as a control.
200 μl of the above pre-dilution culture solution was added to each well of both assay plates and 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 agonist activity of the test substance against the cytokinin receptor was assayed. 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 the turbidity in the control group, 6-Benzyl aminopurine was selected as an agonist active substance for the cytokinin receptor.

Reference Example 5 (Method for Searching Agonist Active Substance for Cytokinin Receptor (Part 2))
The transformed cells TM182-AHK2, TM182-AHK2Δ and TM182-AHK3 obtained in Reference Example 1 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 agonist active substance for 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 cultured 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 .

Example 12 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthesis enzyme and preparation of a vector expressing the polynucleotide: cloning of AtIPT4 cDNA)
The seed of the Arabidopsis Wassilewskija line was sterilized with 70% ethyl alcohol for 1 minute and further sterilized with 1.5% sodium hypochlorite for 10 minutes. After washing this well with sterilized water, GM medium (4.3 g Murashige and Skoog's basal salt mixture, 1% sucrose, 10 ml of 5% MES-KOH (pH 5.7), 0.3% Phytagel ^™ (SIGMA)) Was cultured for 20 days to obtain 5 g of a plant. This was frozen in liquid nitrogen and then physically ground in a 1.5 ml tube. 100 μl of 2 × CTAB (2% (w / v) Cetyl trimethyl ammonium bromide (hereinafter referred to as CTAB)), 0.1 M Tris-HCl (pH 8.0), 1.4 M NaCl, % (W / v) PVP). After standing at 60 ° C. for 30 minutes, 100 μl of a chloroform / isoamyl alcohol solution (chloroform: isoamyl alcohol = 24: 1) was added and stirred with a Voltex mixer. Then, it centrifuged at 20 degreeC and 15000 rotation for 20 minutes. 75 μl of the upper layer after centrifugation was transferred to a 0.6 ml tube in which 75 μl of isoamyl alcohol had been put in advance, and left at 25 ° C. for 20 minutes. Thereafter, the precipitate was recovered by centrifugation at 15,000 rpm for 20 minutes at 20 ° C. 300 μl of 70% ethanol was added to the collected precipitate and stirred with a Voltex mixer. Thereafter, the precipitate was recovered by centrifuging at 4 ° C. and 15000 rpm for 15 minutes. The collected precipitate was allowed to stand at 37 ° C. for 5 minutes, and 30 μl of T10E0.2 (10 mM Tris-HCl (pH 8.0), 0.2 mM EDTA (pH 8.0)) was added to dissolve the precipitate to obtain a genomic DNA solution. Got.
Next, a DNA having the base sequence represented by SEQ ID NO: 11 is obtained by performing a PCR reaction using the obtained genomic DNA as a template and the DNA represented by SEQ ID NO: 35 and the DNA represented by SEQ ID NO: 36 as primers. Was amplified. The PCR reaction was carried out using Herculase Enhanced DNA Polymerase (Toyobo Co., Ltd.), kept at 94 ° C. for 1 minute, repeated for 40 cycles of 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, 72 ° C. for 60 seconds, Finally, it was carried out under amplification conditions of incubation at 72 ° C. for 1 minute. The PCR reaction solution (50 μl) was prepared by adding a reaction composition such as dNTP to 100 ng of template DNA and 100 ng of each of the primer DNA according to the kit instructions.
In this way, the target DNA fragment was amplified.

Example 13 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme and preparation of a vector expressing the polynucleotide: Construction of an AtIPT4 expression vector)
The yeast expression vector p423ADH (Munberg et al. Gene: 156 119-122 (1995), available from ATCC library (No. 87371)) was cleaved with the restriction enzyme SmaI and then used with T4 DNA Ligase. The DNA having the base sequence represented by SEQ ID NO: 11 obtained in Example 12 was ligated to the ADH promoter sequence of the expression vector p423ADH so that the target protein was expressed in yeast. It was confirmed by 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: 11, and expression plasmid p423ADH-IPT4 was obtained.
Similarly, a yeast expression vector p423CYC1 (available from Munberg et al. Gene: 156 119-122 (1995), ATCC library (No. 87379)) was cleaved with the restriction enzyme SmaI, and then T4 DNA Ligase was used. Then, the DNA having the base sequence represented by SEQ ID NO: 11 obtained in Example 12 was linked to the CYC1 promoter sequence of the expression vector p423CYC1 so that the target protein was expressed in yeast. The inserted DNA sequence was in the correct orientation and the DNA sequence was confirmed to be the base sequence shown in SEQ ID NO: 11 using an automatic base sequencer to obtain an expression plasmid p423CYC1-IPT4.

Example 14 (Preparation of polynucleotide having base sequence encoding amino acid sequence of cytokinin biosynthetic enzyme and preparation of vector expressing the polynucleotide: cloning of AtIPT5 cDNA)
In the same manner as in Example 12, a genomic DNA obtained from a plant of the Wassilewskija strain of Arabidopsis thaliana was used as a template to perform a PCR reaction using the DNA represented by SEQ ID NO: 37 and the DNA represented by SEQ ID NO: 38 as primers, A DNA having the base sequence represented by SEQ ID NO: 13 was amplified. The PCR reaction was carried out using Herculase Enhanced DNA Polymerase (Toyobo Co., Ltd.), kept at 94 ° C. for 1 minute, repeated at 35 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 90 seconds, Finally, it was carried out under amplification conditions of incubation at 72 ° C. for 1 minute. A PCR reaction solution (50 μl) was prepared by adding a reaction composition such as dNTP to 100 ng of template DNA and 100 ng of primer DNA according to the kit instructions.
In this way, the target DNA fragment was amplified.

Example 15 (Preparation of a polynucleotide having a base sequence encoding the amino acid sequence of cytokinin biosynthetic enzyme and preparation of a vector expressing the polynucleotide: Construction of an AtIPT5 expression vector)
The yeast expression vector p423ADH (Munberg et al. Gene: 156 119-122 (1995), available from ATCC library (No. 87371)) was cleaved with the restriction enzyme SmaI and then used with T4 DNA Ligase. The DNA having the base sequence represented by SEQ ID NO: 13 obtained in Example 14 was ligated to the ADH promoter sequence of the expression vector p423ADH so that the target protein was expressed in yeast. The inserted DNA sequence was in the correct orientation and the DNA sequence was confirmed to be the base sequence shown in SEQ ID NO: 13 using an automatic base sequencer to obtain the expression plasmid p423ADH-IPT5.
Similarly, a yeast expression vector p423CYC1 (available from Munberg et al. Gene: 156 119-122 (1995), ATCC library (No. 87379)) was cleaved with the restriction enzyme SmaI, and then T4 DNA Ligase was used. Then, the DNA having the base sequence represented by SEQ ID NO: 13 obtained in Example 14 was linked to the CYC1 promoter sequence of the expression vector p423CYC1 so that the target protein was expressed in yeast. The inserted DNA sequence was in the correct orientation and the DNA sequence was confirmed to be the base sequence shown in SEQ ID NO: 13 using an automatic base sequencer to obtain the expression plasmid p423CYC1-IPT5.

Example 16 (Preparation of transformed cells of the present invention: transformed cells TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5, TM182-CYC1-ADHIPT5, TM182-CRE1-CYC1IPT4, TM182-CRE1-ADHIPT4 , TM182-CRE1-CYC1IPT5, TM182-CRE1-ADHIPT5, TM182-AHK2-CYC1IPT4, TM182-AHK2-ADHIPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5)
Commercially available p415CYC1, p415CYC-CRE1 obtained in Example 5, p415CYC-AHK2 obtained in Example 11, one of the three expression plasmids, p423ADH-IPT4 obtained in Example 13, p423CYC1- TM182 (sln1Δ) (Maeda), which is a Sln1 gene-deficient strain, was used simultaneously with a total of two expression plasmids, IPT4 and any one of the four expression plasmids p423ADH-IPT5 and p423CYC1-IPT5 obtained in Example 15. T et al. Nature: 369 242-245 (1994)). Transformation is performed using the polyethylene glycol / lithium acetate (PEG / LiAc) -mediated transformation method, which is described on page 22 of Matchmaker Two-Hybrid System 3 User Manual of Clontech Company, Nippon Becton Dickinson Co., Ltd. VII. Library Transformation & Screening Performed according to Protocols. In the transformed cells obtained, the transformed yeast TM182-CYC1-CYC1IPT4, TM182 is selected by selecting transformed yeast that grows in DOHLU + Gal medium by utilizing the loss of leucine auxotrophy and histidine auxotrophy. -CYC1-ADHIPT4, TM182- CYC1-CYC1IPT5, TM182- CYC1-ADHIPT5, TM182-CRE1-CYC1IPT4, TM182-CRE1-ADHIPT4, TM182-CRE1-CYC1IPT5, TM182-CRE1-ADHIPT5, TM182-AHK2-CYC1IPT4 -ADHIPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5 were prepared.

Example 17 (Preparation of transformed cells of the present invention: transformed cells TM182-AHK2Δ-CYC1IPT4, TM182-AHK2Δ-ADHIPT4, TM182-AHK2Δ-CYC1IPT5, TM182-AHK2Δ-ADHIPT5, TM182-AHK3-CYC1IPT4, TM182-AHK3-ADHIPT4 , TM182-AHK3-CYC1IPT5 and TM182-AHK3-ADHIPT5)
Any one of the two expression plasmids p415CYC-AHK2Δ obtained in Example 11 and p415CYC-AHK3 obtained in Example 8, and p423ADH-IPT4 and p423CYC1-IPT4 obtained in Example 13, Example TM182 (sln1Δ) (Maeda T et al.), Which is a Sln1 gene-deficient strain, was used simultaneously with a total of two expression plasmids, one of the four expression plasmids p423ADH-IPT5 and p423CYC1-IPT5 obtained in Step 15. Nature: 369 242-245 (1994)). Transformation is performed using the polyethylene glycol / lithium acetate (PEG / LiAc) -mediated transformation method, which is described on page 22 of Matchmaker Two-Hybrid System 3 User Manual of Clontech Company, Nippon Becton Dickinson Co., Ltd. VII. Library Transformation & Screening Perform according to Protocols. In the transformed cells obtained, the transformed cells TM182-AHK2Δ-CYC1IPT4 are selected by selecting transformed yeasts that grow on DOHLU + Gal medium, taking advantage of the loss of leucine auxotrophy and histidine auxotrophy. TM182-AHK2Δ-ADHIPT4, TM182-AHK2Δ-CYC1IPT5, TM182-AHK2Δ-ADHIPT5, TM182-AHK3-CYC1IPT4, TM182-AHK3-ADHIPT4, TM182-AHK3-CYC1IPT5 and TM182-AHK3-ADHIPT5 are produced.

Example 18 (Use of transformed cells of the present invention: transformed cells TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5, TM182-CYC1-ADHIPT5, TM182-CRE1-CYC1IPT4, TM182-CRE1-ADHIPT4 TM182-AHK2-CYC1IPT4, TM182-AHK2-ADHIPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5)
Transformed cells prepared in Example 16 TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5, TM182-CYC1-ADHIPT5, TM182-CRE1-CYC1IPT4, TM182-CRE1-ADHIPT4, TM182-AHK2- 10 μl of culture solution of CYC1IPT4, TM182-AHK2-ADHIPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5 (about 800 individuals) was spotted on DOHLU + Glu agar medium and cultured at 30 ° C for 30 hours It was. 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-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5 and TM182-CYC1-ADHIPT5 did not grow, but TM182-CRE1-CYC1IPT4, TM182-CRE1-ADHIPT4, TM182-AHK2-CYC1IPT4 TM182-AHK2-ADHIPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5 grew. In the case of DOHLU + Glu agar medium supplemented with 10 μM trans-zeatin, TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5 and TM182-CYC1-ADHIPT5 did not grow. TM182-CRE1-CYC1IPT4, TM182-CRE1-ADHIPT4, TM182-AHK2-CYC1IPT4, TM182-AHK2-ADHIPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5 grew. This is because the transformed cells TM182-CRE1-CYC1IPT4, TM182-CRE1-ADHIPT4, TM182-AHK2-CYC1IPT4, TM182-AHK2-ADHIPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5 are synthesized by themselves. In response, it indicates that growth on DOHLU + Glu agar is possible. In addition, it was also confirmed that all these 10 types of transformed cells can grow on DOHLU + Gal medium regardless of the presence or absence of trans-zeatin.

Example 19 (Use of transformed cells of the present invention: transformed cells TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5, TM182-CYC1-ADHIPT5, TM182-CRE1-CYC1IPT4, TM182-CRE1-ADHIPT5 Culture)
Transformed cells TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5, TM182-CYC1-ADHIPT5, TM182-CRE1-CYC1IPT4 and TM182-CRE1-ADHIPT5 obtained in Example 16 in DOHLU + Gal medium Inoculate to 200 ml, pre-culture at 30 ° C. for 36 hours, dilute this culture solution to OD600 = 0.200 with DOHLU + Glu medium, and further dilute it to 200 times with DOHLU + Glu medium. It was.
An assay plate was prepared by adding 1 μl of a DMSO solution of a test substance (Abscisic acid or 6-Benzyl aminopurine) adjusted to a concentration of 200 ppm to each well of a 96-well plate. At the same time, an assay plate containing 1 μl of DMSO was prepared as a control group.
100 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. Table 3 shows the absorbance of OD600 of each transformed cell culture solution to which each test substance was added. TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5 and TM182-CYC1-ADHIPT5 did not grow in any test substance, but TM182-CRE1-CYC1IPT4 and TM182-CRE1-ADHIPT5 It also grew in the test substance. In addition, TM182-CRE1-CYC1IPT4 and TM182-CRE1-ADHIPT5 grew faster on the medium added with 6-Benzylaminopurine than the medium added with DMSO alone. These facts indicate that transformed cells TM182-CRE1-CYC1IPT4 and TM182-CRE1-ADHIPT5 respond to cytokinin synthesized by themselves and can grow in DOHLU + Glu medium and that transformed cells TM182-CRE1-CYC1IPT4 It shows that the growth rate of TM182-CRE1-ADHIPT5 increases or decreases depending on the amount of cytokinin.

Example 20 (Use of transformed cells of the present invention: transformed cells TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5, TM182-CYC1-ADHIPT5, TM182-CRE1-ADHIPT4, TM182-AHK2-CYC1IPT4 , TM182-AHK2-CYC1IPT5, TM182-AHK2-ADHIPT5 culture)
Transformed cells obtained in Example 16 TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5, TM182-CYC1-ADHIPT5, TM182-CRE1-ADHIPT4, TM182-AHK2-CYC1IPT4, TM182-AHK2- CYC1IPT5 and TM182-AHK2-ADHIPT5 are inoculated into 200 ml of DOHLU + Gal medium and pre-cultured at 30 ° C. for 36 hours. The one diluted to 1 was used as a pre-dilution culture solution.
An assay plate was prepared by adding 1 μl of a DMSO solution of a test substance (Abscisic acid or 6-Benzyl aminopurine) adjusted to a concentration of 200 ppm to each well of a 96-well plate. At the same time, an assay plate containing 1 μl of DMSO was prepared as a control group.
100 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. Table 4 shows the absorbance of OD600 of each transformed cell culture solution to which each test substance was added. Although TM182-CYC1-CYC1IPT4, TM182-CYC1-ADHIPT4, TM182-CYC1-CYC1IPT5 and TM182-CYC1-ADHIPT5 did not grow in any of the test substances, TM182-CRE1-ADHIPT4, TM182-AHK2-CYC1IPT4, TM182- AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5 grew on any test substance. In addition, TM182-CRE1-ADHIPT4, TM182-AHK2-CYC1IPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5 grew faster in the medium added with 6-Benzylaminopurine than the medium added with DMSO alone. These facts indicate that transformed cells TM182-CRE1-ADHIPT4, TM182-AHK2-CYC1IPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5 respond to cytokinin synthesized by themselves and can grow in DOHLU + Glu medium. This shows that the growth rate of the transformed cells TM182-CRE1-ADHIPT4, TM182-AHK2-CYC1IPT4, TM182-AHK2-CYC1IPT5 and TM182-AHK2-ADHIPT5 increases or decreases depending on the amount of cytokinin.

Example 21 (Utilization of transformed cells of the present invention: method for searching for a substance having an activity to control cytokinin biosynthesis)
The transformed cell TM182-CRE1-ADHIPT5 obtained in Example 16 was inoculated into 200 ml of DOHLU + Gal medium, pre-cultured at 30 ° C. for 36 hours, and this culture solution was diluted with DOHLU + Glu medium to OD600 = 0.200, Furthermore, what diluted it 200 times with DOHLU + Glu culture medium is used as a culture solution before dilution. Also, TM182 (sln1Δ), a Sln1 gene-deficient strain into which the PTP2 Tyrosine phosphatase gene was introduced, was inoculated into 200 ml of DOLU + Gal medium, pre-cultured at 30 ° C. for 36 hours, and this culture solution was OD600 = 0.100 in DOLU + Gal medium. In addition, the cell line obtained by diluting it to 1: 200 with DOLU + Gal medium and the transformed cell TM182-CRE1 obtained in Reference Example 1 were inoculated into 200 ml of DOLU + Gal medium and incubated at 30 ° C. for 36 hours. Pre-culture is performed, and this culture solution is diluted to OD600 = 0.100 with DOLU + Glu medium, and further diluted 1: 200 with DOLU + Glu medium is used as a pre-dilution culture solution. A pre-dilution culture solution of the transformed cell TM182-CRE1 is also prepared at the same time as a pre-dilution culture solution to which 6-benzylaminopurine (cytokinin) having a final concentration of 2 ppm is added.
Prepare an assay plate in which 1 μl of a DMSO solution of a test substance prepared to a concentration of 200 ppm is added to each well of a 96-well plate. At the same time, an assay plate containing 1 μl of DMSO is prepared as a control group.
100 μl of the above pre-dilution culture solution is added to each well of both assay plates, cultured at 30 ° C. for 24 hours, and then the turbidity of each well is measured using a plate reader. A test substance added to a well in which the growth rate of the transformed cell TM182-CRE1-ADHIPT5 is increased or decreased as compared with the control group to which only DMSO was added is selected as a substance having an activity to control cytokinin biosynthesis. I can do it. At this time, growth of TM182 (sln1Δ) in the well to which the test substance added was added to the well in which the growth rate of the transformed cell TM182-CRE1-ADHIPT5 was increased or decreased compared to the control group to which DMSO alone was added. When the rate does not increase or decrease as compared with the control group to which only DMSO is added, the test substance has an activity for controlling cytokinin biosynthesis and / or a cytokinin receptor by a method different from that for controlling cytokinin biosynthesis. Has the activity of increasing or decreasing the amount of intracellular signal transduction from the cell, and the activity of controlling cytokinin biosynthesis and / or the amount of intracellular signal transduction from the cytokinin receptor by a method different from controlling cytokinin biosynthesis In addition to the activity to be increased or decreased, it can be selected as a substance that has a high possibility of not having an activity to control the growth of yeast. Further, the growth rate of the transformed cell TM182-CRE1-ADHIPT5 increased or decreased compared to the control group to which DMSO alone was added. The well of the transformed cell TM182-CRE1 in the well to which the test substance was added was added. When the growth rate does not increase or decrease compared with the control group to which only DMSO is added, the test substance has an activity to control cytokinin biosynthesis, and in addition to the activity to control cytokinin biosynthesis, It can be selected as a substance that has a high possibility of not having an activity to control growth.

The composition of the medium used in the present invention is described below.
(A) DOLU + Glu medium

(B) DOLU + Gal medium

(X) Drop-out mix:

(C) A solid medium in which 2% (W / V) agar is added to DOLU + Glu agar medium (a).

(D) A solid medium in which 2% (W / V) agar is added to DOLU + Gal agar medium (b).

(E) DOHLU + Glu medium

(F) DOHLU + Gal medium

(Y) Drop-out mix:

（ｇ）DOHLU+Glu寒天培地
培地（ｅ）に２％（Ｗ／Ｖ）の寒天が添加された固体培地。
（ｈ）DOHLU+Gal寒天培地
培地（ｆ）に２％（Ｗ／Ｖ）の寒天が添加された固体培地。

(G) Solid medium obtained by adding 2% (W / V) agar to DOHLU + Glu agar medium (e).
(H) Solid medium obtained by adding 2% (W / V) agar to DOHLU + Gal agar medium (f).

  According to the present invention, a co-expressed transformed cell of a cytokinin receptor and a cytokinin biosynthetic enzyme can be provided, and by using the transformed cell, it is possible to provide an analysis method of activity that controls the biosynthesis of cytokinin. By using the analysis method, it is possible to provide a method for quickly finding a substance having an activity of controlling cytokinin biosynthesis with a small amount of sample.

SEQ ID NO: 23
Oligonucleotide primer designed for PCR SEQ ID NO: 24
Oligonucleotide primer designed for PCR SEQ ID NO: 25
Oligonucleotide primer designed for PCR SEQ ID NO: 26
Oligonucleotide primer designed for PCR SEQ ID NO: 27
Oligonucleotide primer designed for PCR SEQ ID NO: 28
Oligonucleotide primer designed for PCR SEQ ID NO: 29
Oligonucleotide SEQ ID NO: 30 designed as a linker
Oligonucleotide designed as linker SEQ ID NO: 31
Oligonucleotide primer designed for PCR SEQ ID NO: 32
Oligonucleotide primer designed for PCR SEQ ID NO: 33
Oligonucleotide primer designed for PCR SEQ ID NO: 34
Oligonucleotide primer designed for PCR SEQ ID NO: 35
Oligonucleotide primer designed for PCR SEQ ID NO: 36
Oligonucleotide primer designed for PCR SEQ ID NO: 37
Oligonucleotide primer designed for PCR SEQ ID NO: 38
Oligonucleotide primers designed for PCR

Claims (6)

  A transformed cell into which a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin receptor and a polynucleotide having a base sequence encoding the amino acid sequence of a cytokinin biosynthetic enzyme are introduced. The transformed cell according to claim 1, wherein the cytokinin receptor is any of the following cytokinin receptors.
(A) Cytokinin receptor having the amino acid sequence represented by SEQ ID NO: 2 (b) Cytokinin receptor having the amino acid sequence represented by SEQ ID NO: 4 (c) Cytokinin receptor having the amino acid sequence represented by SEQ ID NO: 6 (d ) Cytokinin receptor in which part of the transmembrane region is deleted and having at least one transmembrane region (e) Among the amino acid sequence represented by SEQ ID NO: 4, amino acid numbers 196 to 1176 The cytokinin receptor (g) consisting of the amino acid sequence represented by amino acid numbers 50 to 1176 among the amino acid sequence represented by amino acid sequence represented by (f) SEQ ID NO: 4 and the amino acid sequence represented by SEQ ID NO: 6 Of these, a cytoplasm comprising the amino acid sequence represented by amino acid numbers 32 to 1036 Kinin receptor (h) extracellular region of cytokinin receptor that is homologous to each other, transmembrane region of cytokinin receptor, histidine kinase region of cytokinin receptor, and histidine that is heterogeneous to the above three regions A chimeric cytokinin receptor comprising a kinase receiver region (i) in the amino acid sequence of the cytokinin receptor of (a), (b), (c), (e), (f) or (g) Cytokinin receptor having an amino acid sequence in which one or a plurality of amino acids are added, deleted and / or substituted with other amino acids (j) The amino acid sequence possessed by the cytokinin receptor of (a), (b) or (c) above A polynucleotide and a stringer having a base sequence complementary to the base sequence encoding Cytokinin receptor having the amino acid sequence encoded by the nucleotide sequence of a polynucleotide hybridizes under preparative conditions,   The transformed cell according to claim 1 or 2, wherein the cytokinin biosynthetic enzyme is isopentenyl transferase. The transformed cell according to claim 1 or 2, wherein the cytokinin biosynthetic enzyme is any of the following cytokinin biosynthetic enzymes.
(A) cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 8 (b) cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 10 (c) cytokinin biosynthesis having the amino acid sequence represented by SEQ ID NO: 12 Enzyme (d) Cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 14 (e) Cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 16 (f) Cytokinin biosynthesis having the amino acid sequence represented by SEQ ID NO: 18 Synthase (g) Cytokinin biosynthetic enzyme having the amino acid sequence represented by SEQ ID NO: 20 (h) (a), (b), (c), (d), (e), (f) or (g) In the amino acid sequence of the cytokinin biosynthetic enzyme, one or more amino acids are added, deleted and / or young Or a cytokinin biosynthetic enzyme having an amino acid sequence substituted by another amino acid (i) the cytokinin of the above (a), (b), (c), (d), (e), (f) or (g) Cytokinin biosynthetic enzyme comprising an amino acid sequence encoded by a base sequence of a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to the base sequence encoding the amino acid sequence of the biosynthetic enzyme   5. The transformed cell according to claim 1, wherein the transformed cell is yeast. 5. The transformed cell according to claim 1, wherein the transformed cell is budding yeast.