JP2008208113A - Agent which has activity of inhibiting intracellular signaling from plant-derived cytokinin receptor of its cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for controlling growth or differentiation of plants and to provide a technique for searching a chemical substance having useful biological activity clarifying a target, namely a method, etc., for screening the chemical substance with activity to a specific target in order to chemically control the target part. <P>SOLUTION: The method for searching for a chemical substance promoting the growth of a root of a plant comprises measuring the level of intracellular signaling from a cytokinin receptor in a system where a cell having the cytokinin receptor derived from a plant is brought into contact with a chemical substance having an agonistic activity on the receptor and a substance to be tested, comparing the level of intracellular signaling measured in the preceding step with a level of intracellular signaling measured in the absence of the chemical substance and determining the chemical substance as being a chemical substance promoting the growth of a root of a plant based on the difference obtained by the comparison. 6-Chloro-2-ethoxycarbonylmethylamino-4-phenylquinazoline, etc., was found as the active substance by the method. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a drug that controls the growth or differentiation of a plant, and relates to a drug that has an activity of inhibiting intracellular signal transmission from a plant-derived cytokinin receptor possessed by a cell.

  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 foliage, prevents yellowing of leaves, fallen leaves and fallen fruits, breaks down top priority It is an important physiologically active substance that is known to exhibit such actions (see Non-Patent Document 1, for example). As a method for controlling a physiological phenomenon caused by cytokinin, a method of giving cytokinin from the outside, a method of controlling cytokinin biosynthesis in the plant, a method of controlling cytokinin metabolism in the plant, and the like have been considered.

  By the way, the chemical substance which becomes an active ingredient of a plant growth regulator has hitherto been found by random screening in which a test chemical substance is directly applied to a plant and its biological activity is assayed. In this case, in order to predict the safety of the chemical substance and the burden on the environment, after the chemical substance having a useful biological activity is identified, the action mechanism of the chemical substance is effective. There was a need to study in detail at the molecular level what the chemicals act on.

Cytokinins: Chemistry, Activity, and Function, CRC Press (1994)

  For example, in the situation as described above, which is related to the search and development of chemical substances that are active ingredients of plant growth regulators, a method for searching for a chemical substance having a useful biological activity with a clear target, that is, a target site is defined. For the purpose of chemical regulation, we tried to develop a method for screening chemical substances with activity against specific targets.

（式中、R及びXは同一又は異なって置換されていてもよい炭化水素基、NR¹R²で表される基、OR³で表される基、S(O)_mR⁴で表される基、ニトロ基又はハロゲン原子を示し、
ここでR¹は水素原子又は置換されていてもよい炭化水素基を示し、
R²は水素原子、置換されていてもよい炭化水素基、NR⁵R⁶で表される基（ここでR⁵及びR⁶は同一もしくは異なり水素原子又は置換されていてもよいC1-6アルキル基を示す）又はOR⁷で表される基（ここでR⁷は水素原子又は置換されていてもよいC1-6アルキル基を示す）を示し、又はR¹及びR²が隣接する窒素原子と一緒になって置換されていてもよい環状アミノ基を示し、
R³及びR⁴はそれぞれ置換されていてもよい炭化水素基を示し、
lは0〜1の整数を示し、
mは0〜２の整数を示し、
nは0〜４の整数を示し、
nが2以上の場合Xは同一又は異なっていてもよく、
Arは置換されていてもよいアリール基又は置換されていてもよいヘテロアリール基を示す）で表される化合物又はその農学的に許容される塩を含有することを特徴とする植物成長調節剤；
（２６）lが1であり、Rが置換されていてもよい炭化水素基である前項２５記載の植物成長調節剤；
（２７）ｌが１であり、Rがハロゲン原子又はオキソ基で置換されていてもよいC1-3アルキル基である前項２５記載の植物成長調節剤；
（２８）置換されていてもよい炭化水素基がハロゲン原子又はオキソ基で置換されていてもよいC1-3アルキル基である前項２６記載の植物成長調節剤；
（２９）lが1であり、RがNR¹R²で表される基である前項２５記載の植物成長調節剤；
（３０）R¹が水素原子又はC1-3アルキル基を示し、R²が水素原子、アミノ基、C1-3アルキルアミノ基、ジC1-3アルキルアミノ基、アミジノ基、C1-3アルコキシ基、フェニル基、C1-3アシル基、C1-6アルキル基、C3-6アルケニル基又はC3-6アルキニル基を示し、ここで前記のフェニル基は1〜3個の同じもしくは異なるC1-3アルキル基で置換されていてもよく、前記のフェニル基、アシル基、アルキル基、アルケニル基及びアルキニル基はハロゲン原子、ヒドロキシル基、C1-3アルコキシ基、ヒドロキシC1-3アルコキシ基、カルボキシル基、C1-3アルコキシカルボニル基、カルバモイル基、アミノ基、C1-3アルキルアミノ基、ジC1-3アルキルアミノ基、メルカプト基、C1-3アシルチオ基、シアノ基、フリル基及びテトラヒドロフリル基の中から選ばれる1〜3個の同じもしくは異なる置換基で置換されていてもよく、又はR¹及びR²が隣接する窒素原子と一緒になってピロリジノ基、ピペリジノ基又はモルホリノ基を示す前項２９記載の植物成長調節剤；
（３１）R¹が水素原子を示し、R²が水素原子、ホルミル基、C1-6アルキル基、C3-6アルケニル基又はC3-6アルキニル基を示し、ここで前記のアルキル基、アルケニル基及びアルキニル基はヒドロキシル基、メトキシ基、メトキシカルボニル基、エトキシカルボニル基、シアノ基及びフリル基の中から選ばれる置換基で置換されていてもよい前項２９記載の植物成長調節剤；
（３２）lが1であり、RがOR³で表される基である前項２５記載の植物成長調節剤；
（３３）R³がアミノ基で置換されていてもよいC1-3アルキル基である前項３２記載の植物成長調節剤；
（３４）lが1であり、RがS(O)_mR⁴で表される基である前項２５記載の植物成長調節剤；
（３５）R⁴がアミノ基又はヒドロキシル基で置換されていてもよいC1-3アルキル基であり、mが0である前項３４記載の植物成長調節剤；
（３６）lが1であり、Rがハロゲン原子である前項２５記載の植物成長調節剤；
（３７）ハロゲン原子が塩素原子である前項３６記載の植物成長調節剤；
（３８）nが1〜2であり、XがC1-3アルキル基、C1-3アルコキシ基、C1-3ハロアルキル基、シアノ基、ハロゲン原子又はニトロ基である前項２５〜３７いずれか一項記載の植物成長調節剤；
（３９）Xが塩素原子、臭素原子又はニトロ基であり、Xの置換位置が6位及び／又は8位である前項３８記載の植物成長調節剤；
（４０）Arがハロゲン原子又はC1-3アルキル基で置換されていてもよいフェニル基である前項２３〜３７いずれか一項記載の植物成長調節剤；
（４１）前項２５〜４０いずれか一項記載の植物成長調節剤の有効量を、植物又はその生息場所に施用することを特徴とする植物成長調節方法；
等を提供するものである。 As a result of intensive studies under such circumstances, the inventor is an agent that controls plant growth or differentiation and has an activity of inhibiting intracellular signal transduction from plant-derived cytokinin receptors possessed by cells. The present inventors have found a drug characterized by
That is, the present invention
(1) A drug that regulates plant growth or differentiation and has an activity of inhibiting intracellular signal transduction from a plant-derived cytokinin receptor possessed by cells;
(2) The agent according to item 1 above, wherein the agent that controls plant growth or differentiation is a plant growth regulator;
(3) The drug according to item 1 above, wherein the drug that controls plant growth or differentiation is a drug that controls plant growth;
(4) The agent according to item 1 above, wherein the agent that controls plant growth or differentiation is an agent that controls plant cell differentiation;
(5) The drug according to item 3 above, wherein the drug for controlling the growth of the plant body is a drug for controlling the growth of plant buds;
(6) The agent according to item 5 above, wherein the control of plant bud growth is suppression of buds and the like;
(7) The agent according to item 5 above, wherein the control of plant bud growth is flower bud suppression or the like;
(8) The drug according to item 3 above, wherein the drug that controls the growth of the plant is a drug that promotes plant seedling establishment;
(9) The drug according to claim 3, wherein the drug that controls the growth of the plant is a drug that promotes the tillering of the plant;
(10) The agent according to item 3 above, wherein the agent that controls plant growth is an agent that promotes plant root growth;
(11) The plant-derived cytokinin receptor of the cell is a cytokinin receptor selected from the following group A;
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and A cell having a cytokinin receptor selected from the following group A, wherein the protein (12) having an activity that functions as an inine receptor has an activity that inhibits intracellular signal transduction from a plant-derived cytokinin receptor; 11. The drug according to any one of 1 to 10 above, which has an activity of inhibiting intracellular signal transmission from the cytokinin receptor in a contact system with a substance having agonist activity with respect to the cytokinin receptor. ;
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and As a protein (13) active ingredient having an activity that functions as an inine receptor, it contains a chemical substance that inhibits intracellular signal transmission from a plant-derived cytokinin receptor that the cell has, or an agriculturally acceptable salt thereof. A plant growth regulator characterized by:
(14) In the contact system between the chemical substance, a cell having a cytokinin receptor selected from the following group A, a substance having agonist activity with respect to the cytokinin receptor, and the chemical substance, the cytokinin 14. The plant growth regulator according to item 13 above, which is a chemical substance having an activity of inhibiting intracellular signal transduction from a receptor;
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and Substances having agonistic activity towards the protein (15) a cytokinin receptor having the activity of functioning as a delegate receptor, plant growth regulators of item 14, wherein the trans zeatin;
(16) The chemical substance is present in a contact system of a cell having a cytokinin receptor selected from the following group A, 0.6 ppm transzeatin, and 2 ppm of the chemical substance. 14. A plant growth regulator according to item 13 above, which is a chemical substance having an activity of inhibiting intracellular signal transduction from the cytokinin receptor than in the case of
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and A protein having an activity that functions as an inine receptor (17) wherein the chemical substance comprises a cell having a cytokinin receptor selected from the following group A, 0.6 ppm of transzeatin, and 2 ppm of the chemical substance. 14. The plant growth regulator according to item 13 above, which is a chemical substance having an activity of inhibiting 90% or more of intracellular signal transmission from the cytokinin receptor in the contact system as compared with the case where the chemical substance is not present. ;
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and Protein having an activity of functioning as a delegate receptor (18) A method for searching a chemical substance having the ability to promote the growth of plant roots,
<1> Intracellular from the cytokinin receptor in a contact system of a cell having a cytokinin receptor selected from the following group A, a substance having agonist activity with respect to the cytokinin receptor, and a test substance A first step of measuring the amount of signal transmission, and <2> the amount of intracellular signal transmission measured by the first step and the amount of intracellular signal transmission in the absence of the chemical substance. A second step of selecting a chemical having the ability to promote plant root growth based on the difference
A method characterized by comprising:
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and A protein having an activity that functions as an inin receptor (19) A cell having a cytokinin receptor is a transformed cell into which a polynucleotide comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 has been introduced. 19. The search method according to item 18 above, characterized by
(20) The searching method according to item 18 above, wherein the cell having a cytokinin receptor is a transformed yeast cell into which a polynucleotide comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 has been introduced. ;
(21) The search method according to the above item 18, 19 or 20, wherein the substance having agonist activity with respect to a cytokinin receptor is transzeatin;
(22) A plant growth regulator comprising, as an active ingredient, a chemical substance selected by the search method according to the preceding item 18, 19, 20, or 21 or an agriculturally acceptable salt thereof;
(23) A method for regulating plant growth, which comprises applying an effective amount of the plant growth regulator described in the preceding item 13, 14, 15, 16, 17, or 22 to a plant or its habitat;
(24) a chemical substance having the ability to promote the growth of plant roots by specifying the chemical substance having the ability to promote the growth of plant roots by the search method according to the preceding item 18, 19, 20 or 21; A plant growth control method comprising contacting with a plant;
(25) General formula (I) as an active ingredient

(In the formula, R and X are the same or different hydrocarbon groups which may be substituted, a group represented by NR ¹ R ² , a group represented by OR ³ and represented by S (O) _m R ^4. A group, a nitro group or a halogen atom,
Here, R ¹ represents a hydrogen atom or an optionally substituted hydrocarbon group,
R ² is a hydrogen atom, an optionally substituted hydrocarbon group, a group represented by NR ⁵ R ⁶ (wherein R ⁵ and R ⁶ are the same or different, a hydrogen atom or an optionally substituted C 1-6 alkyl Or a group represented by OR ⁷ (wherein R ⁷ represents a hydrogen atom or an optionally substituted C 1-6 alkyl group), or R ¹ and R ² are adjacent nitrogen atoms Together with a cyclic amino group which may be substituted,
R ³ and R ⁴ each represents an optionally substituted hydrocarbon group,
l represents an integer of 0 to 1,
m represents an integer of 0 to 2,
n represents an integer of 0 to 4,
when n is 2 or more, Xs may be the same or different;
Ar represents an aryl group which may be substituted or a heteroaryl group which may be substituted, or a plant growth regulator characterized by containing an agriculturally acceptable salt thereof;
(26) The plant growth regulator according to the above item 25, wherein l is 1 and R is a hydrocarbon group which may be substituted;
(27) The plant growth regulator according to the above item 25, wherein l is 1, and R is a C1-3 alkyl group optionally substituted with a halogen atom or an oxo group;
(28) The plant growth regulator according to the above item 26, wherein the hydrocarbon group which may be substituted is a C1-3 alkyl group which may be substituted with a halogen atom or an oxo group;
(29) The plant growth regulator according to item 25, wherein l is 1 and R is a group represented by NR ¹ R ² ;
(30) R ¹ represents a hydrogen atom or a C1-3 alkyl group, R ² represents a hydrogen atom, an amino group, a C1-3 alkylamino group, a diC1-3 alkylamino group, an amidino group, a C1-3 alkoxy group, Represents a phenyl group, a C1-3 acyl group, a C1-6 alkyl group, a C3-6 alkenyl group or a C3-6 alkynyl group, wherein said phenyl group is 1 to 3 identical or different C1-3 alkyl groups; The above phenyl group, acyl group, alkyl group, alkenyl group and alkynyl group may be substituted with halogen atom, hydroxyl group, C1-3 alkoxy group, hydroxy C1-3 alkoxy group, carboxyl group, C1-3 alkoxy 1-3 selected from carbonyl group, carbamoyl group, amino group, C1-3 alkylamino group, di-C1-3 alkylamino group, mercapto group, C1-3 acylthio group, cyano group, furyl group and tetrahydrofuryl group Same if 30. The plant growth regulator according to 29 above, which may be substituted with different substituents, or R ¹ and R ² together with the adjacent nitrogen atom represent a pyrrolidino group, piperidino group or morpholino group;
(31) R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, a formyl group, a C 1-6 alkyl group, a C 3-6 alkenyl group or a C 3-6 alkynyl group, wherein the alkyl group, alkenyl group and 30. The plant growth regulator according to item 29, wherein the alkynyl group may be substituted with a substituent selected from a hydroxyl group, a methoxy group, a methoxycarbonyl group, an ethoxycarbonyl group, a cyano group, and a furyl group;
(32) The plant growth regulator according to the above item 25, wherein l is 1 and R is a group represented by OR ³ ;
(33) The plant growth regulator according to the above item 32, wherein R ³ is a C1-3 alkyl group optionally substituted with an amino group;
(34) The plant growth regulator according to the above item 25, wherein l is 1 and R is a group represented by S (O) _m R ⁴ ;
(35) The plant growth regulator according to the above item 34, wherein R ⁴ is a C1-3 alkyl group optionally substituted with an amino group or a hydroxyl group, and m is 0;
(36) The plant growth regulator according to the above item 25, wherein l is 1 and R is a halogen atom;
(37) The plant growth regulator according to the above item 36, wherein the halogen atom is a chlorine atom;
(38) Any one of the preceding items 25 to 37, wherein n is 1 to 2, and X is a C1-3 alkyl group, a C1-3 alkoxy group, a C1-3 haloalkyl group, a cyano group, a halogen atom or a nitro group. Plant growth regulators of
(39) The plant growth regulator according to 38 above, wherein X is a chlorine atom, a bromine atom or a nitro group, and the substitution position of X is at the 6-position and / or 8-position;
(40) The plant growth regulator according to any one of the items 23 to 37, wherein Ar is a phenyl group optionally substituted with a halogen atom or a C1-3 alkyl group;
(41) A plant growth regulation method comprising applying an effective amount of the plant growth regulator according to any one of the above items 25 to 40 to a plant or a habitat thereof;
Etc. are provided.

  According to the present invention, an agent for controlling plant growth or differentiation, and a method for searching for a chemical substance having a useful biological activity in which a target is clarified, that is, for the purpose of chemically adjusting a target site, It is possible to provide a method for screening a chemical substance with activity against a target.

  Hereinafter, the present invention will be described in detail.

In the present invention, the term “plant” is used in a broad sense to indicate an organism such as grass or tree that has a root and has a fixed life. It is a concept that includes such as. Specifically, it shows organisms such as higher plants that can play important roles such as plant fixation in the soil, absorption of moisture and nutrients from the outside through the organs of the roots, and ornamental plants such as flowers and foliage plants And crops such as cereals, vegetables and fruit trees, fiber plants, trees, and turf plants. Specifically, for example, cereals such as rice and corn, turf such as bentgrass and cucumber, cucumbers such as tomatoes, peppers, peppers, watermelons, cucumbers, pumpkins, melons, cucumbers such as watermelons, cabbage, broccoli, Vegetables such as Chinese cabbage, raw vegetables such as celery, parsley and lettuce, spicy vegetables, green onions such as leeks, onions and garlic, beans such as soybeans, green beans, peas and azuki bean, fruit vegetables such as strawberries, radish, turnip, Carrots, straight roots such as burdock, potatoes such as taro, potato, sweet potato, Chinese yam, soft vegetables such as asparagus, spinach, honeybee, petals such as eustoma, stock, carnation, chrysanthemum, rapeseed, groundnut, etc. Oil crops, sugar crops such as sugar cane and sugar beet, fiber crops such as cotton and rush, black Forage crops such as bars and sorghum, deciduous fruit trees such as apples, pears, grapes, peaches and chestnuts, citrus fruits such as mandarin oranges, lemons and grapefruits, woody species such as satsuki, azalea and cedar .
In the present invention, “growth” of a plant means an already existing vegetative organ in the process from the initial development of a plant starting with seed germination to the growth and development of roots, stems and leaves, from flower formation to seed maturation. This is a general process in which (roots, stems, leaves) are newly created and stacked. For example, germination, root elongation, bud elongation, stem elongation, and formation and elongation of buds and buds Branch and leaf development, flower bud formation and flowering, fruiting, seed maturation and the like.
In the present invention, “differentiation” of a plant means that plant tissues such as roots, stems and leaves are formed from callus which is a plant cell population that has acquired totipotency (redifferentiation), or roots, stems, Callus formation (dedifferentiation) from cells of plant tissues such as leaves.
In the present invention, “control of bud growth” of a plant refers to promoting or suppressing the growth of apical buds or buds, for example, starting the growth of buds whose growth is suppressed by the top bud dominance. In other words, it is possible to suppress the growth of buds that start growing by excising the top buds, or to suppress the growth of normal top buds.
In the present invention, the “agent that controls plant growth or differentiation” is an agent that can control plant growth or differentiation by treating the plant with various methods. By controlling the growth or development of plants, the growth and development of useful plants such as crops can be controlled to increase the initial growth, improve the quality, increase the yield, stabilize the yield even under poor conditions, and produce The above labor can be saved, and the “agent for controlling plant growth or differentiation” can be used as a “plant growth regulator”.

  In the present invention, the “root” of a plant means, in the case of dicotyledonous plants and gymnosperms, a main root developed from a young root in a seed embryo, a side root that branches off from the main root, and the like. In the case of monocotyledonous plants, it refers to larvae (seed roots) in seed embryos, adventitious roots (so-called beard roots) formed in the upper part after seed root growth stops, side roots branching out from adventitious roots, etc. . In addition, root epidermis cells may be referred to as root hairs extending long outward. Plant roots are organs that play an important role for plants, such as plant fixation in soil, absorption of moisture and nutrients from the outside. Plant roots are also very important as a plant hormone production site. In agriculture, many crops grow with seeds. Therefore, uniform establishment of seedlings at the early stage of plant growth is a very important factor that leads to high quality and high yield. By promoting the growth of plant roots, it is possible to improve the survival rate to soil, improve productivity or quality by improving seedling establishment, early weed control by improving seedling establishment, and increase the efficiency of seed supply sources. Various advantages are expected. In addition, improvement in rooting is expected to improve drought stress tolerance or pest tolerance, and to reduce fertilizer amount by improving nutrient absorption capacity.

  In the present invention, “root growth” of a plant means that the root length, the number of roots, or the amount, thickness, activity, etc. of the root increase due to cell division, elongation, weight increase, etc. Say.

  In the present invention, “promoting root growth” of the plant means that the root growth of the plant is more active than before, the length of the root, the number of roots is increased, or the amount of root compared to the case of no treatment. , Increase in thickness, activity, etc.

  In the present invention, the “agent that promotes plant root growth” is an agent that can promote plant root growth by treating the plant by various methods. By promoting the growth of plant roots, the growth and development of useful plants such as agricultural crops can be controlled to increase initial growth, improve quality, increase yield, stabilize yield even under poor conditions, and produce The above labor can be saved, and the “agent for promoting the growth of plant roots” can be used as a “plant growth regulator”.

Here, “seedling establishment” means that after seeded seeds have emerged, the seedlings of the transplanted plant settle in the soil in a state that allows normal growth as a plant body, or grow normally. That means. By promoting seedling establishment, the initial growth of the plant is improved, and a healthy plant can be grown. In addition, an increase in the final yield is expected by increasing the number of plants that grow healthy. For example, in direct sowing cultivation of rice, budding and seedling establishment are unstable, and it is generally difficult to always secure a certain number of seedling establishments. The chemical | medical agent which accelerates | stimulates the seedling establishment of this invention can promote seedling establishment, and can improve the efficiency of rice direct sowing cultivation. The “seed establishment rate” refers to the total number of seeds sown or the ratio of seedlings to the total number of seedlings of transplanted plants. For example, in the case of rice, it can be defined by the following formula in the present specification: .
Formula: [Seedling establishment rate (%)] = [Number of seedlings with leaf tips appearing on the water surface] / [Number of seeding] × 100

  “Tillering” of a plant refers to branching with the growth of the plant or its branch, for example, a side branch in the case of gramineous plants. The promotion of tillering includes that the time of tillering is advanced and that the number of tillers is increased. For example, under the stress conditions such as low temperature, high temperature, and dryness, if the tillering time is advanced by using the plant growth regulator of the present invention and healthy seedlings can be secured early, damage of the seedlings from stress should be avoided. Can do. For example, when the tillering time is advanced, the cultivation period of the plant is shortened. Moreover, since the tiller formation of plants directly affects the number of spikes, an increase in yield can be expected depending on the cultivation conditions.

Auxin active substances may exhibit undesirable properties such as uneven growth of leaves, twisting of stems, cracking of stems, induction of roots, etc., depending on the type of plant, auxin treatment concentration, and the like.
Cytokinin active substances are considered to have both properties of plant root growth suppression and growth promotion (for example, PNAS 101 (23): 8821-8826 (2004)). To use it practically, we still have to wait for a lot of knowledge. However, considering the role of cytokinin in plants in detail, it is usually difficult to regulate the amount of cytokinin in the plant body (particularly to reduce the amount of endogenous cytokinin in the plant). Researchers who are familiar with the field are not easy.
It is considered that endogenous cytokinin in plants can be negatively controlled by inhibiting cytokinin signaling and weakening sensitivity to cytokinin. For example, cytokinin signaling can be inhibited by mutating the cytokinin receptor itself, and a mutant of the cytokinin receptor has been isolated [The Plant Cell 16: 1365-1377 (2004), PNAS 101 (23 ): 8821-8826 (2004)]. However, in these mutants, it is difficult to control the degree of inhibition stepwise, and the single mutant of cytokinin receptor shows a phenotype similar to that of the wild type, whereas the triple mutant It shows elongation inhibition and poor growth of plants.

  In the present invention, the “plant-derived cytokinin receptor that a cell has” refers to a cytokinin receptor present in a plant. The cytokinin receptor specifically binds to cytokinins such as purine cytokinins such as kinetin and zeatin and urea cytokinins such as N-phenyl-N ′-(4-pyridyl) urea, and two-component regulatory system ( Alternatively, it is a protein having a function of controlling the growth and differentiation of higher plant cells by an intracellular signal transmission mechanism called His to Asp phosphorelay system). The cytokinin receptor used in the present invention belongs to the histidine kinase family, and includes an extracellular region, a transmembrane region, a histidine kinase region (having a His residue that has histidine kinase activity in the cell and serves as an active site). Region) and a receiver region (region having an acceptor for transphosphorylation and holding an Asp residue as an active site).

The two-component regulatory system is an information-reception / intracellular signal transmission mechanism widely used in eubacteria, archaea, fungi, and plants. Histidine kinase that acts as a receptor in this mechanism has an input region that accepts a signal on the N-terminal side, and a region called a transmitter region that is involved in phosphoryl group transfer on the C-terminal side. When the input region senses a signal, the His residue in the transmitter region (the histidine kinase region of the cytokinin receptor described above) is autophosphorylated. This phosphate group transfers while alternately phosphorylating a specific conserved His and Asp residue, and finally phosphorylates the Asp residue in the receiver region of the protein called a response regulator. The phosphate group may be delivered directly from the histidine kinase to the response regulator, or may be delivered to the response regulator through several stages of phosphate group transfer. There are many simple two-component regulatory systems like the former in prokaryotes. On the other hand, multi-step phosphoryl group transfer is frequently observed in eukaryotes, and such eukaryotic histidine kinases are often accompanied by a receiver region. Further, a phosphate group transfer mediator is also involved in the transfer of the phosphate group. Response regulator phosphorylation controls the activity of the associated output region. The output region is often a transcriptional control factor.
In plant cytokinin receptors, a receiver region is associated with the same molecule. That is, in the cytokinin receptor bound to cytokinin, it is known that the transfer of the phosphate group from this His residue to the intramolecular Asp residue occurs following the autophosphorylation of the His residue in the molecule. . Furthermore, it has been clarified that this phosphate group is transferred to the Asp residue of the response regulator via the His residue of the phosphate group transfer mediator. For example, in Arabidopsis thaliana, it has been clarified that the phosphate group is transferred from the cytokinin receptors CRE1, AHK2, and AHK3 to the response regulator via the phosphate group transfer mediator AHP.

As genes encoding cytokinin receptors, Arabidopsis thaliana (CRE1: accession No.AB049934, AHK2: accession No.AB046869, AHK3: accession No.AB046870), periwinkle (Catharanthus roseus, accession No.AY0920) ), Rice (Oryza sativa, accession No.AY572461), maize (Zea mays, ZmHK1: accession No. AB042270, ZmHK2: accession No. AB102956, ZmHK3a: accession No. AB102957, ZmHK3b: accession No. AB121445), etc. The nucleotide sequence of is known. Such a gene with a known base sequence is a base sequence corresponding to the vicinity of the amino terminus and a base corresponding to the vicinity of the carboxy terminus of the protein encoded by the genome DNA or cDNA of the organism having the target gene as a template. It can be amplified by PCR using a primer prepared based on the sequence and isolated. A gene encoding a cytokinin receptor can also be obtained from plants other than those described above. First, mRNA is prepared from the target plant, cDNA is synthesized using reverse transcriptase using the mRNA as a template, and this is incorporated into a phage vector such as ZAPII or a plasmid vector such as pUC to produce a cDNA library. . Using this cDNA library as a template, PCR is performed using primers designed and synthesized based on base sequences well conserved among genes with known base sequences as described above, thereby encoding cytokinin receptors. A DNA fragment containing at least a part of the gene can be amplified. A cDNA library is screened using this DNA fragment as a probe, and positive clones are selected. By determining the base sequence of the DNA of the selected clone, it can be confirmed that the gene encodes the target cytokinin receptor.
All three Arabidopsis cytokinin receptors (CRE1, AHK2, AHK3) are histidine kinases with a receiver region in the same molecule. The homology of the amino acid sequence between these three members is high, and in particular, the homology of the amino acid sequence in the extracellular region considered to bind to cytokinin is high. In the recombinant yeast described later, these three types of cytokinin receptors all caused intracellular signal transmission in response to cytokinin, and the activity of the cytokinin receptor could be confirmed. Cytokinin receptors of other plants are also histidine kinases, and their amino acid sequences are highly homologous to the amino acid sequences of Arabidopsis cytokinin receptors.
Table 1 shows the identity of the amino acid sequence of the cytokinin receptor CRE1 of Arabidopsis thaliana with that of other cytokinin receptors.
Preferred “plant-derived cytokinin receptors possessed by cells” include, for example, an amino acid sequence having 45% or more, preferably 49% or more, more preferably 53% or more amino acid sequence with the amino acid sequence of Arabidopsis cytokinin receptor CRE1 And a protein having an activity of functioning as a cytokinin receptor.

“Intracellular signal transmission from a plant-derived cytokinin receptor of a cell” is information transmission performed by transfer of a phosphate group starting from autophosphorylation of a cytokinin receptor to which cytokinin is bound as described above. That is, in plant cells, cytokinin response signals are transmitted by the transfer of the phosphate group from the cytokinin receptor, which is autophosphorylated by binding to cytokinin, to the phosphate transfer mediator, and from there to the response regulator. The Similarly, in a recombinant cell having a plant-derived cytokinin receptor, intracellular signal transduction from the cytokinin receptor is performed by transfer of a phosphate group, but a phosphate group transfer mediator and a response regulator are derived from a host cell. It may be a thing. Some are all host-derived, and some are host-derived. In addition, there may be no phosphate group transfer mediator. For example, in a recombinant budding yeast having a cytokinin receptor, intracellular signal transduction from a plant-derived cytokinin receptor is carried out from a cytokinin receptor to which cytokinin is bound and self-phosphorylated from a host budding yeast cell-derived phosphorous. This is performed by transferring a phosphate group to Ssk1, which is a response regulator derived from a host budding yeast cell, via Ypd1, which is an acid group transfer mediator. In addition, in recombinant fission yeast having cytokinin receptors, intracellular signal transduction from plant-derived cytokinin receptors is transferred to host-fission yeast-derived response regulator Mcs4 via the host-transferred yeast-derived phosphate group transfer mediator Spy1. Transmitted by transfer of phosphate groups. In addition, in recombinant Escherichia coli having a cytokinin receptor, intracellular signal transmission from a plant-derived cytokinin receptor is transferred to the host E. coli-derived response regulator RcsB via a phosphate group transfer mediator YojN. Transmitted by the transition.
One way to measure the presence or amount of intracellular signal transduction from plant-derived cytokinin receptors is to control transcription by a response regulator located downstream of intracellular signal transduction from plant-derived cytokinin receptors. There is a method for measuring the presence and amount of target gene expression. In this method, in addition to directly measuring the presence / absence and amount of target gene expression, a host cell is used with a reporter plasmid prepared by linking a reporter gene such as a fluorescent protein gene or beta-galactosidase gene to the promoter region of the target gene. To measure the presence / absence and amount of reporter gene expression using fluorescence and color development as an index, and to measure and observe changes in the number of cells and changes in cell traits related to target gene expression There is a way to do it. For example, the above-mentioned recombinant budding yeast uses cytokinin-dependent recombinant budding yeast growth as an indicator, recombinant fission yeast uses cytokinin-dependent recombinant fission yeast size as an indicator, and recombinant E. coli targets The presence / absence and amount of intracellular signal transmission from a plant-derived cytokinin receptor can be measured using color development resulting from expression of a beta-galactosidase gene bound to the promoter region of gene cps as an index. In addition, in Arabidopsis thaliana transformed with a reporter plasmid linked to the promoter region of type A response regulators ARR5 and ARR6, cytokinin-dependent fluorescence and color development are used as indicators of intracellular signal transduction from cytokinin receptors. Presence / absence and quantity can also be measured. The measurement method as described above is, for example, Higuchi et al, Nature 409, 1060-1063 (2001); Suzuki et al, Plant Cell Physiol. 42, 107-113 (2001); Hwang and Sheen, Nature 413, 383- 389 (2001).
Among various methods for measuring intracellular signal transduction from plant-derived cytokinin receptors possessed by cells as described above, a method for efficiently measuring mechanically and quantitatively is dependent on the cytokinin dependence of recombinant budding yeast. It is desirable to measure the turbidity of the liquid medium with a spectrophotometer. Specifically, a method described in Japanese Patent Publication (JP 2003-079393) or the like can be given.

  “Activity to inhibit intracellular signal transduction from plant-derived cytokinin receptors possessed by cells” refers to the ability of cells to reduce intracellular signal transduction from plant-derived cytokinin receptors. That is, it means the ability to reduce the amount of phosphate groups transferred from the cytokinin receptor to the response regulator, beginning with autophosphorylation of the cytokinin receptor. Specifically, the ability to inhibit the histidine kinase activity of the cytokinin receptor, the ability to inhibit the phosphate transfer from the cytokinin receptor to the phosphate group transfer mediator, the phosphate group from the phosphate transfer mediator to the response regulator Examples include the ability to inhibit metastasis and the ability to inhibit the transcriptional control of response regulators. More specifically, the ability of the cytokinin receptor to inhibit the histidine kinase activity depends on whether or not the binding between the substance having cytokinin agonist activity and the cytokinin receptor is inhibited. The ability to inhibit intracellular signal transduction from the cytokinin receptor by a mechanism that determines the presence or absence of inhibition, resulting in the ability to inhibit the histidine kinase activity of the cytokinin receptor. In this case, one of the methods for investigating that the test substance inhibits the binding between the substance having cytokinin agonist activity and the cytokinin receptor is a method using a radiolabeled substance having cytokinin agonist activity and the cytokinin receptor. There is. For example, a substance having cytokinin agonist activity labeled with tritium, a radioisotope of hydrogen, and having high radioactivity and a cytokinin receptor protein prepared from a recombinant yeast introduced with a cytokinin receptor gene are appropriately used. Cytokinin agonist labeled with the tritium bound to the cytokinin receptor and given high radioactivity by allowing the cytokinin receptor protein to be collected on a glass filter and measuring the radioactivity after coexisting in the buffer A substance that has cytokinin agonist activity and cytokinin receptor can be detected using the decrease in the measured value of radioactivity when an active substance can be detected and the test substance coexists in the buffer. That the test substance inhibits. Then, a test substance is added to the reaction system for measuring the presence or amount of intracellular signal transmission from the plant-derived cytokinin receptor, and the test substance gives intracellular signal transmission from the plant-derived cytokinin receptor. You can investigate the impact.

  “A drug having an activity of inhibiting intracellular signal transduction from a plant-derived cytokinin receptor possessed by a cell” refers to inhibiting intracellular signal transduction from a plant-derived cytokinin receptor possessed by a cell. It is a drug containing an active substance as an active ingredient.

  In the present invention, “the agent for controlling plant growth or differentiation, characterized in that it has an activity of inhibiting intracellular signal transduction from a plant-derived cytokinin receptor possessed by cells” means the measurement method described above It is a drug whose ability to inhibit intracellular signal transduction from a plant-derived cytokinin receptor possessed by a cell is specified, and means a drug capable of controlling the growth or differentiation of a plant. Desirably, the drug includes a drug in which the drug that controls plant growth or differentiation is a plant growth regulator.

In the present invention, the “plant growth regulator” refers to an agent having the ability to control the growth or differentiation of the plant.
As one of methods for measuring the ability to control the growth or differentiation of a plant, in addition to the method disclosed in the present invention, for example, there is a method for measuring a root growth promoting activity on the above-mentioned plant. Specifically, for example, it can be measured according to the following method.
A standard garden culture medium having the following composition (see Table 2) is prepared. Dispense 4 μl of chemical DMSO solution into the cluster tube to a final concentration of 0.001 ppm to 10 ppm, and add 600 μl of sterilized horticultural standard medium to the cluster tube and mix. After seeding 10-20 Arabidopsis seeds per tube and culturing at 22 ° C. in a light place for 10 days, the average main root length is measured. The average value of 8 iterations is obtained, and the root growth rate is obtained by the following formula.

Root growth rate (%) = (Average main root length of chemical treatment section) / (Average main root length of control section) x 100
A reagent that exhibits a significantly high root growth rate can be said to have root growth promoting activity. More preferably, it can be determined that a reagent with a root growth rate of 120% or more has a root growth promoting activity.

The plant growth regulator in this invention contains the chemical substance which inhibits the intracellular signal transmission from the cytokinin receptor derived from the plant which a cell has as an active ingredient, or its agriculturally acceptable salt.
In the present invention, “agronomically acceptable salt” refers to a salt in a form in which the production and application are not impossible with respect to the production as a plant growth regulator and the application of the product. It may also be a salt. Specific examples of such salts include mineral salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate and phosphate, formate, acetate, propionate, sulphate, and the like. Acid salt, malonate, succinate, fumarate, maleate, lactate, malate, tartrate, citrate, methanesulfonate, ethanesulfonate, and other organic acid salts, aspartic acid Salts, acid addition salts such as acidic amino acid salts such as glutamate, alkali metal salts (sodium salt, potassium salt etc.) and alkaline earth metal salts (magnesium salt etc.), metal salts such as aluminum salt, and methylamine, Examples include addition salts and ammonium salts with organic bases such as ethylamine and ethanolamine, and basic amino acids such as lysine and ornithine.

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, the cytokinin signal transduction inhibitor 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 hydrous silicon oxide, fubasami clay, bentonite, acidic clay), talc, and other inorganic minerals (sericite, quartz powder, sulfur powder, Active powder, calcium carbonate, etc.), fine powders and granular materials such as chemical fertilizers (ammonium sulfate, phosphorous acid, ammonium nitrate, ammonium chloride, urea, etc.) can be mentioned. 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, isobutyronitrile, etc.), ether S (dioxane, diisopropyl ether), acid amides (dimethylformamide, dimethylacetamide), halogenated hydrocarbons (dichloroethane, trichlorethylene, etc.) and the like.
Examples of the surfactant include alkyl sulfates, alkyl sulfonates, alkyl aryl sulfonates, alkyl aryl ethers and polyoxyethylenates thereof, polyethylene glycol ethers, polyhydric alcohol esters, sugar alcohol derivatives and the like. Can be mentioned.
Other formulation adjuvants include, for example, casein, gelatin, polysaccharides (starch, gum arabic, cellulose derivatives, alginic acid, etc.), lignin derivatives, bentonite, synthetic water-soluble polymers (polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylic acid, etc.) ) And the like, PAP (isopropyl acid phosphate), BHT (2,6-tert-butyl-4-methylphenol), BHA (2- / 3-tert-butyl-4-methoxyphenol), Stabilizers such as vegetable oil, mineral oil, fatty acid, fatty acid ester and the like can be mentioned.

  In the present invention, “a plant growth regulator comprising a chemical substance that inhibits intracellular signal transduction from a plant-derived cytokinin receptor possessed by a cell or an agriculturally acceptable salt thereof as an active ingredient” And by containing, as an active ingredient, a chemical substance or its agriculturally acceptable salt whose ability to inhibit intracellular signal transduction from a plant-derived cytokinin receptor possessed by a cell is measured by the above-described measurement method. A drug that can control the growth or differentiation of plants. Desirably, the chemical substance has an ability to inhibit intracellular signal transmission from the cytokinin receptor in the recombinant budding yeast contact system of a cytokinin receptor and a substance having agonist activity with respect to the cytokinin receptor. The chemical substance which has can be mentioned. More preferably, in the recombinant budding yeast contact system of a cytokinin receptor and a substance having agonistic activity with respect to the cytokinin receptor, the concentration of transzeatin is 0.6 ppm and the concentration of the chemical is present. In the case of 2 ppm or more, a chemical substance having an ability to inhibit the activity of the cytokinin receptor to be lower than in the case where the chemical substance is not present can be mentioned. More preferably, in the recombinant budding yeast contact system of a cytokinin receptor and a substance having agonist activity with respect to the cytokinin receptor, the concentration of transzeatin is 0.6 ppm and the concentration of chemical is 2 ppm. In the above case, a chemical substance having an ability to inhibit the cytokinin receptor activity to be 90% or more lower than the case where the chemical substance is not present can be mentioned.

In the present invention, “a test substance has an ability to promote the growth of plant roots, comprising (1) a cell having a plant-derived cytokinin receptor selected from the following group A, and the cytokinin receptor: In a contact system between a substance having agonist activity on the body and a test substance, the activity (or presence or absence of intracellular signal transmission or the amount thereof) that inhibits intracellular signal transmission from the cytokinin receptor is measured. One step, and (2) a second step of evaluating the ability of the substance to promote the growth of plant roots based on the difference obtained by comparing the activity measured in the first step with the activity in the control, "The method characterized by having" the above-mentioned first step and second step among various methods for assaying the ability of the test substance to promote the growth of plant roots. The method characterized by having.
Here, “Group A” means
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and It indicates a protein having an activity of functioning as a delegate receptor.

In the first step, the cytokinin receptor is contacted with a cell having cytokinin receptors derived from various plants, a substance having agonist activity with respect to the cytokinin receptor, and a test substance. This is a step of measuring the activity of inhibiting intracellular signal transmission (or the presence or absence of intracellular signal transmission or the amount thereof). The second step is a step of evaluating the ability of the substance to promote plant root growth based on the difference obtained by comparing the activity measured in the first step with the activity in the control. Here, for example, when the test substance is added to the reaction system in a state in which the test substance is dissolved in a solvent, the control means a test group to which only the solvent in which the test substance is dissolved is added.
The plant-derived cytokinin receptor used by the test substance having the first step and the second step, which is used in the test method for the ability to promote plant root growth, is shown in the group A. It is a protein. Among the above-mentioned group A proteins, the amino acid sequences of the proteins shown in (b), (c), (d), and (e) may be found between the amino acid sequences shown in (a). Differences include deletion, substitution, addition, etc. of some amino acids. These include, for example, deletion due to processing that the protein having the amino acid sequence shown in (a) above undergoes in the cell. In addition, it occurs due to gene mutations that occur naturally due to species differences or individual differences of the organism from which the protein is derived, or gene mutations that are artificially introduced by site-specific mutagenesis, random mutagenesis, mutagenesis, etc. Amino acid deletions, substitutions, additions and the like are included.
The number of amino acids subjected to such deletion, substitution, addition, etc. may be any number within the range in which the histidine kinase activity of the cytokinin receptor can be found. Examples of amino acid substitution include substitution with an amino acid that is similar in terms of hydrophobicity, charge, pK, steric features, and the like. Specific examples of such substitution include (1) glycine, alanine; (2) valine, isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine, glutamine, (4) serine, threonine; ) Lysine, arginine; (6) substitution within groups such as phenylalanine, tyrosine and the like.
As a technique for artificially performing such amino acid deletion, addition, or substitution (hereinafter, sometimes collectively referred to as amino acid modification), for example, a site for DNA encoding the amino acid sequence shown in (a) There is a technique in which specific mutagenesis is performed and then this DNA is expressed by a conventional method. Here, as a site-specific mutagenesis method, for example, a method utilizing amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), a PCR method using a mutagenesis primer Etc. In addition, as a technique for artificially modifying amino acids, for example, a technique in which mutation is randomly introduced into DNA encoding the amino acid sequence shown in (a), and then this DNA is expressed by a conventional method is also exemplified. It is done. Here, as a method for introducing mutations at random, for example, a DNA encoding one of the above amino acid sequences is used as a template, and a primer pair capable of amplifying the full length of each DNA is used, and dATP and dTTP used as a substrate are used. , DGTP, dCTP, the method of performing PCR under the reaction conditions in which the respective addition concentrations are changed from normal, or the reaction conditions in which the concentration of Mg ²⁺ that promotes the polymerase reaction is increased more than usual. . Examples of such PCR techniques include the method described in Method in Molecular Biology, (31), 1994, 97-112. Moreover, the method described in WO0009682 can also be mentioned.

  Here, “sequence identity” refers to identity between two base sequences or two amino acid sequences. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the entire region of the sequence to be compared. Here, in the optimal alignment of the base sequence or amino acid sequence to be compared, addition or deletion (for example, a gap or the like) may be allowed. Such sequence identity can be determined, for example, by FASTA [Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 4, 2444-2448 (1988)], BLAST [Altschul et al., Journal of Molecular Biology, 215, 403- 410 (1990)], CLUSTAL W [Thompson, Higgins & Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)] and the like. The above program is, for example, the DNA Data Bank of Japan [International DNA Data Bank operated within the Center for Information Biology and DNA Data Bank of Japan (CIB / DDBJ)]. It is generally available on the homepage (http://www.ddbj.nig.ac.jp). Sequence identity can also be determined using commercially available sequence analysis software. Specifically, for example, GENETYX-WIN Ver.5 (manufactured by Software Development Co., Ltd.) is used, and the homology is obtained by the Lipman-Pearson method [Lipman, DJ and Pearson, WR, Science, 227, 1435-1441, (1985)] It can be calculated by creating an alignment by performing sex analysis.

“Stringent conditions” described in (e) include: Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory) For example, a solution containing 6 × SSC (a solution containing 1.5M NaCl and 0.15M trisodium citrate is 10 × SSC) The conditions were such that a hybrid was formed at 45 ° C. and then washed with 2 × SSC at 50 ° C. (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6). Can be mentioned. The salt concentration in the washing step can be selected from, for example, conditions from 2 × SSC (low stringency conditions) to 0.2 × SSC (high stringency conditions). The temperature in the washing step can be selected from conditions from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions), for example. It is also possible to change both the salt concentration and the temperature.
These proteins are, of course, proteins having an activity that functions as a cytokinin receptor, but more desirably, when aligned with the amino acid sequence shown in SEQ ID NO: 1 so as to obtain the maximum sequence identity. The amino acid residues corresponding to positions (I) 459 and (II) 973 of the amino acid sequence shown in Fig. 1 are the following amino acid residues; (I) 459 is histidine, and (II) 973 is aspartic acid. A protein is used. Here, “alignment with the amino acid sequence represented by SEQ ID NO: 1 so as to obtain the maximum sequence identity” means that the amino acid represented by SEQ ID NO: 1 using the above-mentioned programs such as FASTA, BLAST, CLUSTAL W, etc. It means that the sequence identity analysis of a plurality of target amino acid sequences including the sequence is performed and aligned. By aligning a plurality of sequences by such a method, it is possible to determine the positions of homologous amino acid residues in each amino acid sequence regardless of insertion or deletion in the amino acid sequence. The homologous position is considered to exist at the same position in the three-dimensional structure, and it can be presumed that the homologous position has a similar effect on the specific function of the target protein. For example, known cytokinin receptors, including the cytokinin receptors whose sequences are disclosed in the present invention, are sequenced when aligned with the amino acid sequence shown in SEQ ID NO: 1 so as to obtain the maximum sequence identity of the amino acid sequence. The amino acid residues corresponding to positions (I) 459 and (II) 973 of the amino acid sequence shown by No. 1 are the following amino acid residues: (I) histidine at position 459 and (II) position 973 at aspartic acid It is.

An active ingredient of a drug that controls the growth or differentiation of a plant can be searched for, for example, by measuring the ability to promote root growth relative to the root of the plant.
It is also possible to search for substances having the ability to control plant growth or differentiation by a method for assaying the ability to promote plant root growth using plant-derived cytokinin receptors possessed by the cells. . Specifically, using the above-described method for assaying the ability to promote plant root growth using the cytokinin receptor, the test substance has the ability to promote plant root growth over a certain value, or a certain value When the following is specified, a substance having the ability to promote plant root growth can be searched by selecting the substance.
In addition, since the substance selected by the search method has the ability to control the growth or differentiation of plants, it can be a plant growth regulator containing the substance or its agriculturally acceptable salt as an active ingredient.

Specific examples of substances that inhibit cytokinin information transmission include cytokinin antagonists and cytokinin agonists.
The cytokinin signal transduction inhibitor selected by the above-described searching method can promote the growth of the root part of the plant. The root growth refers to the extension of the main root, the extension of the side root, the extension of the root hair, and the like.
The root growth promoting activity of the cytokinin signal transduction inhibitor selected by the above search method can be assayed using, for example, the following method. Although it may vary depending on the type of plant and assay method, for example, aqueous solutions, hydroponic culture media, and tissue culture media prepared with various concentrations of cytokinin signal transduction inhibitor in the range of 0.0001 to 100 ppm are prepared. In the case of a petri dish test, the above solution is squeezed into a filter paper laid in the petri dish, and plant seeds are placed on the floor. In the case of a sachet test, the above solution is soaked in cardboard in a bag in which growth of roots such as a seed growth bag can be observed, and plant seeds are sown. In addition, a solid medium in which agarose, agar, etc. are added to the above solution is prepared in a plastic petri dish or plastic centrifuge tube, seeded with plant seeds, and cultured at a temperature of 10 to 30 ° C. for a certain period of time in a light place The length of the main root or the side root, the number of side roots, the root wet weight, the root dry weight, etc. are measured.
The cytokinin signal transduction inhibitor selected by the above search method may be used as a plant growth regulator.

R, X, R ¹ , R ² , R ³ , R ⁴ , R ⁵ in the compound represented by the general formula (I) used in the present invention (hereinafter sometimes referred to as compound (I)), In the groups represented by R ⁶ and R ⁷
Examples of the “hydrocarbon group” include an aliphatic hydrocarbon group, a monocyclic saturated hydrocarbon group, an aromatic hydrocarbon group, and the like, and those having 1 to 16 carbon atoms are preferable. Specifically, for example, an alkyl group, alkenyl group, alkynyl group, cycloalkyl group, aralkyl group, aryl group and the like are used.
The “alkyl group” is preferably, for example, a lower alkyl group, and specifically, for example, C1-6 such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl, pentyl, hexyl and the like. An alkyl group or the like is used.
The “alkenyl group” is preferably, for example, a lower alkenyl group, and specifically, for example, C 2-6 alkenyl groups such as vinyl, 1-propenyl, allyl, isopropenyl, butenyl and isobutenyl are used.
The “alkynyl group” is preferably, for example, a lower alkynyl group, and specifically, for example, C 2-6 alkynyl groups such as ethynyl, propargyl and 1-propynyl are used.
The “cycloalkyl group” is preferably, for example, a lower cycloalkyl group, and specifically, for example, a C 3-6 cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like are used. The “aralkyl group” is preferably a C7-11 aralkyl group such as benzyl or phenethyl, and specifically, for example, a benzyl group is used.
The “aryl group” is preferably, for example, a C 6-14 aryl group such as phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, and 2-anthryl, and specifically, for example, a phenyl group is used.

As the substituent that the “hydrocarbon group” and the “C1-6 alkyl group” of the “optionally substituted hydrocarbon group” and the “optionally substituted C1-6 alkyl group” may have, For example, a halogen atom (specifically, for example, fluorine, chlorine, bromine, iodine, etc.), a nitro group, a cyano group, a hydroxyl group, a lower alkyl group (specifically, for example, methyl, ethyl, propyl, isopropyl, butyl) , Isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl and the like C1-6 alkyl group), lower alkoxy groups (specifically, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, C1-6 alkoxy groups such as isobutoxy, pentyloxy, hexyloxy, etc.), amino groups, mono-lower alkylamino groups (specifically, methylamino, for example) Mono-C1-6 alkylamino groups such as ethylamino, etc.), di-lower alkylamino groups (specifically, di-C1-6 alkylamino groups such as dimethylamino, diethylamino etc.), imino groups, A carboxyl group, a lower alkylcarbonyl group (specifically, for example, a C1-6 alkylcarbonyl group such as acetyl or propionyl), a lower alkoxycarbonyl group (specifically, for example, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl) C1-6 alkoxycarbonyl groups, etc.), carbamoyl groups, thiocarbamoyl groups, mono-lower alkylcarbamoyl groups (specifically, mono-C1-6 alkylcarbamoyl groups such as methylcarbamoyl, ethylcarbamoyl etc.), di A lower alkylcarbamoyl group (specifically, for example, dimethyl Di-C1-6 alkylcarbamoyl groups such as carbamoyl and diethylcarbamoyl), arylcarbamoyl groups (specifically, for example, C6-10 arylcarbamoyl groups such as phenylcarbamoyl and naphthylcarbamoyl), aryl groups (specifically, For example, C6-10 aryl groups such as phenyl and naphthyl), aryloxy groups (specifically, for example, C6-10 aryloxy groups such as phenyloxy and naphthyloxy), heterocyclic groups (specifically, for example, 2-, 3-thienyl, 2- or 3-tetrahydrothienyl, 2- or 3-furyl, 2- or 3-tetrahydrofuryl, 1-, 2- or 3-pyrrolyl, 1-, 2- or 3-pyrrolidinyl 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isoxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 3-, 4- or 5- Pyrazolyl, 2-, 3- or 4-pyrazolidinyl, 2-, 4- or 5-imidazolyl, 4- or 5-1H-1,2,3-triazolyl, 3- or 5-1,2,4-triazolyl, 5-1H- or 5-2H-tetrazolyl, 2-, 3- or 4-pyridyl, 2-, 4- or 5-pyrimidinyl, 1-, 2- or 3-thiomorpholinyl, 1-, 2- or 3-morpholinyl 1-, 2-, 3- or 4-piperidino, 2-, 3- or 4-piperidyl, 2-, 3- or 4-thiopyranyl, 2-, 3- or 4-4H-1,4-oxazinyl, 2-, 3- or 4-4H-1,4-thiazinyl, 1,3-thiazinyl, 1- or 2-piperazinyl, 3-, 5- or 6-1, 2,4-triazinyl, 2-1 , 3,5-triazinyl, 3- or 4-pyridazinyl, 2-pyrazinyl, etc.), lower alkylcarbonylamino groups (specifically, for example, C1-6 alkylcarbonyl such as acetylamino) Amino groups, etc.), mercapto groups, C1-6 alkylthio groups (specifically, for example, C1-6 alkylthio groups such as methylthio), alkylsulfinyl groups (specifically, for example, C1-6 alkylsulfinyl groups such as methylsulfinyl) ), An alkylsulfonyl group (specifically, for example, a C1-6 alkylsulfonyl group such as methylsulfonyl), an arylthio group (specifically, for example, a C6-10 arylthio group such as phenylthio), an arylsulfinyl group (specifically, For example, C6-10 arylsulfinyl group such as phenylsulfinyl), arylsulfonyl group (specifically, C6-10 arylsulfonyl group such as phenylsulfonyl), oxo group, thioxo group and the like are used.
Here, an acyl group (specifically, for example, formyl, acetyl, propionyl, pivaloyl, acryloyl, benzoyl, etc.) is a kind of hydrocarbon group substituted with an oxo group, and the above-mentioned “optionally substituted hydrocarbon” Group ”and“ optionally substituted C 1-6 alkyl group ”.

“Hydrocarbon group” and “C1-6 alkyl group” of “optionally substituted hydrocarbon group” and “optionally substituted C1-6 alkyl group” are the positions where the above substituents can be substituted. 1 or more, preferably 1 to 3 may be substituted, and when the substituent is a halogen atom, it may be substituted up to the maximum possible number, and when the number of substituents is 2 or more Each substituent may be the same or different.
The above substituent is a lower alkyl group, lower alkoxy group, mono-lower alkylamino group, di-lower alkylamino group, lower alkylcarbonyl group, lower alkoxycarbonyl group, mono-lower alkylcarbamoyl group, di-lower alkylcarbamoyl group. Group, arylcarbamoyl group, aryl group, aryloxy group, heterocyclic group, lower alkylcarbonylamino group, alkylthio group, alkylsulfinyl group, alkylsulfonyl group, arylthio group, arylsulfinyl group, arylsulfonyl group, etc. These substituents are halogen atoms (specifically, for example, fluorine, chlorine, bromine, iodine, etc.), nitro groups, cyano groups, hydroxyl groups, C1-4 alkoxy groups (specifically, for example, methoxy, ethoxy, propoxy, etc.) , Isopropyloxy, butoxy , Isobutyloxy), aryl group, oxo group or the like, and the above substituents are arylcarbamoyl group, aryl group, aryloxy group, heterocyclic group, C6-10 arylthio, C6 In the case of -10 arylsulfinyl, C6-10 arylsulfonyl and the like, these substituents are C1-4 alkyl groups (specifically, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl) Etc.) may be substituted by 1 to 3 units.
As the “C 1-6 alkyl group”, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl, pentyl, hexyl and the like are used.
As the “C1-3 alkyl group” (including the C1-3 alkylamino group and the C1-3 alkyl group of the group shown as the diC1-3 alkylamino group), methyl, ethyl, propyl and isopropyl are used.
As the “C 3-6 alkenyl group”, for example, 1-propenyl, allyl, isopropenyl, butenyl, isobutenyl and the like are used.
As the “C 3-6 alkynyl group”, for example, propargyl, 1-propynyl and the like are used.
As the “C 1-3 alkoxy group” (including the C 1-3 alkyl group of the group shown as hydroxy C 1-3 alkoxy group and C 1-3 alkoxycarbonyl group), methoxy, ethoxy, propoxy, isopropyloxy and the like are used.
Examples of the “C1-3 acyl group” (including the C1-3 acyl group of the group shown as the C1-3 acylthio group) include formyl, acetyl, propionyl and the like.
As the “C 1-3 haloalkyl group”, for example, chloromethyl, trifluoromethyl, 2-bromoethyl, 2,2,2-trifluoroethyl, 2,2,3,3,3-pentafluoropropyl and the like are used. .
Examples of the “cyclic amino group” represented by “cyclic amino group in which R ¹ and R ² may be substituted together with the adjacent nitrogen atom” include, for example, 1-aziridinyl, pyrrolidino, piperidino, morpholino, thio Morpholino and the like are used, and examples of the substituent include 1 to 3 C1-3 alkyl groups, C1-3 alkoxy groups, hydroxyl groups and the like as described above.
In the group represented by Ar,
As the “aryl group”, for example, as a part of the “hydrocarbon group” of the groups represented by the above, R, X, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ The aryl group shown is used,
As the “heteroaryl group”, for example, 2- or 3-thienyl, 2- or 3-furyl, 1-, 2- or 3-pyrrolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isoxazolyl, 2-, 4- or 5-thiazolyl, 3-, 4- or 5-isothiazolyl, 3-, 4- or 5-pyrazolyl, 2-, 4- or 5-imidazolyl, 4- or 5-1H -1,2,3-triazolyl, 3- or 5-1,2,4-triazolyl, 5-1H- or 5-2H-tetrazolyl, 2-, 3- or 4-pyridyl, 2-, 4- or 5 -Pyrimidinyl, 1- or 2-piperazinyl, 3-, 5- or 6-1,2,4-triazinyl, 2-1,3,5-triazinyl, 3- or 4-pyridazinyl, 2-pyrazinyl, etc. Used.
Examples of the substituents of these “aryl group” and “heteroaryl group” include the above-described R, X, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ^7. The groups exemplified as the substituents of “optionally substituted hydrocarbon group” and “optionally substituted C1-6 alkyl group” are used.

（式中、Xは置換されていてもよい炭化水素基、NR¹R²で表される基、OR³で表される基、S(O)_mR⁴で表される基、ニトロ基またはハロゲン原子を示し、
ここでR¹は水素原子または置換されていてもよい炭化水素基を示し、
R²は水素原子、置換されていてもよい炭化水素基、NR⁵R⁶で表される基（ここでR⁵およびR⁶は同一もしくは異なり水素原子または置換されていてもよいC1-6アルキル基を示す）またはOR⁷で表される基（ここでR⁷は水素原子または置換されていてもよいC1-6アルキル基を示す）を示し、又はR¹およびR²が隣接する窒素原子と一緒になって置換されていてもよい環状アミノ基を示し、
R³およびR⁴はそれぞれ置換されていてもよい炭化水素基を示し、
mは0〜２の整数を示し、
nは0〜４の整数を示し、
nが2以上の場合Xは同一または異なっていてもよく、
Arは置換されていてもよいアリール基または置換されていてもよいヘテロアリール基を示す）で表される化合物である。 In the general formula (I), the compound in which l is 0 is a compound in which the 2-position of the quinazoline skeleton is unsubstituted, that is, the general formula (I-1)

(In the formula, X represents an optionally substituted hydrocarbon group, a group represented by NR ¹ R ² , a group represented by OR ³ , a group represented by S (O) _m R ⁴ , a nitro group or Represents a halogen atom,
Here, R ¹ represents a hydrogen atom or an optionally substituted hydrocarbon group,
R ² is a hydrogen atom, an optionally substituted hydrocarbon group, a group represented by NR ⁵ R ⁶ (wherein R ⁵ and R ⁶ are the same or different, a hydrogen atom or an optionally substituted C 1-6 alkyl Or a group represented by OR ⁷ (wherein R ⁷ represents a hydrogen atom or an optionally substituted C 1-6 alkyl group), or R ¹ and R ² are adjacent to a nitrogen atom Together with a cyclic amino group which may be substituted,
R ³ and R ⁴ each represents an optionally substituted hydrocarbon group,
m represents an integer of 0 to 2,
n represents an integer of 0 to 4,
when n is 2 or more, Xs may be the same or different;
Ar represents an aryl group which may be substituted or a heteroaryl group which may be substituted.

（式中、RおよびXは同一または異なって置換されていてもよい炭化水素基、NR¹R²で表される基、OR³で表される基、S(O)_mR⁴で表される基、ニトロ基またはハロゲン原子を示し、
ここでR¹は水素原子または置換されていてもよい炭化水素基を示し、
R²は水素原子、置換されていてもよい炭化水素基、NR⁵R⁶で表される基（ここでR⁵およびR⁶は同一もしくは異なり水素原子または置換されていてもよいC1-6アルキル基を示す）またはOR⁷で表される基（ここでR⁷は水素原子または置換されていてもよいC1-6アルキル基を示す）を示し、又はR¹およびR²が隣接する窒素原子と一緒になって置換されていてもよい環状アミノ基を示し、
R³およびR⁴はそれぞれ置換されていてもよい炭化水素基を示し、
mは0〜２の整数を示し、
nは0〜４の整数を示し、
nが2以上の場合Xは同一または異なっていてもよく、
Arは置換されていてもよいアリール基または置換されていてもよいヘテロアリール基を示す）で表される化合物である。 In the general formula (I), the compound in which l is 1 is a compound in which the 2-position of the quinazoline skeleton is substituted with R, that is, the general formula (I-2)

(In the formula, R and X are the same or different hydrocarbon groups which may be substituted, a group represented by NR ¹ R ² , a group represented by OR ³ and represented by S (O) _m R ⁴ A group, a nitro group or a halogen atom,
Here, R ¹ represents a hydrogen atom or an optionally substituted hydrocarbon group,
R ² is a hydrogen atom, an optionally substituted hydrocarbon group, a group represented by NR ⁵ R ⁶ (wherein R ⁵ and R ⁶ are the same or different, a hydrogen atom or an optionally substituted C 1-6 alkyl Or a group represented by OR ⁷ (wherein R ⁷ represents a hydrogen atom or an optionally substituted C 1-6 alkyl group), or R ¹ and R ² are adjacent to a nitrogen atom Together with a cyclic amino group which may be substituted,
R ³ and R ⁴ each represents an optionally substituted hydrocarbon group,
m represents an integer of 0 to 2,
n represents an integer of 0 to 4,
when n is 2 or more, Xs may be the same or different;
Ar represents an aryl group which may be substituted or a heteroaryl group which may be substituted.

Compound (I) may be in the form of the “agronomically acceptable salt” described above.
When the compound (I) has one or more asymmetric centers, the compound has two or more stereoisomers (specifically, for example, enantiomers, diastereomers, etc.). The compound of the present invention includes all of these stereoisomers and a mixture of any two or more thereof.
When the compound (I) has geometric isomerism based on a double bond or the like, the compound contains two or more geometric isomers (specifically, for example, E / Z or trans / cis isomers, S-trans / S-cis isomers, etc.). Compound (I) includes all of these geometric isomers and mixtures of any two or more thereof.

As a preferable aspect of compound (I), the aspect shown below is mentioned, for example.
In general formula (I):
(1) A compound wherein l is 1 and R is an optionally substituted hydrocarbon group.
(2) The compound according to (1), wherein l is 1, and R is a C1-3 alkyl group which may be substituted with a halogen atom or an oxo group.
(3) The compound according to (1), wherein the hydrocarbon group which may be substituted is a C1-3 alkyl group which may be substituted with a halogen atom or an oxo group.
(4) A compound wherein l is 1 and R is a group represented by NR ¹ R ² .
(5) R ¹ represents a hydrogen atom or a C1-3 alkyl group, R ² represents a hydrogen atom, an amino group, a C1-3 alkylamino group, a diC1-3 alkylamino group, an amidino group, a C1-3 alkoxy group, Represents a phenyl group, a C1-3 acyl group, a C1-6 alkyl group, a C3-6 alkenyl group or a C3-6 alkynyl group, wherein said phenyl group is 1 to 3 identical or different C1-3 alkyl groups; The phenyl group, acyl group, alkyl group, alkenyl group and alkynyl group which may be substituted are halogen atom, hydroxyl group, C1-3 alkoxy group, hydroxy C1-3 alkoxy group, carboxyl group, C1-3 alkoxy 1-3 selected from carbonyl group, carbamoyl group, amino group, C1-3 alkylamino group, di-C1-3 alkylamino group, mercapto group, C1-3 acylthio group, cyano group, furyl group and tetrahydrofuryl group Same or May be substituted with different substituents, or the compound according to (4), wherein R ¹ and R ² together with the adjacent nitrogen atom represent a pyrrolidino group, piperidino group or morpholino group.
(6) R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, a formyl group, a C 1-6 alkyl group, a C 3-6 alkenyl group or a C 3-6 alkynyl group, wherein the alkyl group, alkenyl group and The compound according to (4), wherein the alkynyl group may be substituted with a substituent selected from a hydroxyl group, a methoxy group, a methoxycarbonyl group, an ethoxycarbonyl group, a cyano group, and a furyl group.
(7) A compound in which l is 1 and R is a group represented by OR ³ .
(8) The compound according to (7), wherein R ³ is a C1-3 alkyl group which may be substituted with an amino group.
(9) A compound wherein l is 1 and R is a group represented by S (O) _m R ⁴ .
(10) The compound according to (9), wherein R ⁴ is a C1-3 alkyl group which may be substituted with an amino group or a hydroxyl group, and m is 0.
(11) A compound wherein l is 1 and R is a halogen atom.
(12) The compound according to (11), wherein the halogen atom is a chlorine atom.
(13) A compound wherein n is 1 to 2, and X is a C1-3 alkyl group, a C1-3 alkoxy group, a C1-3 haloalkyl group, a cyano group, a halogen atom or a nitro group.
(14) The compound according to (13), wherein X is a chlorine atom, a bromine atom or a nitro group, and the substitution position of X is at the 6-position and / or 8-position.
(15) The compound wherein Ar is a phenyl group optionally substituted with a halogen atom or a C1-3 alkyl group.

Compound (I) contains a known compound and can be produced by a known method or a method analogous thereto.
For example, compound (Ia) in which l is 1 and R is a chlorine atom can be produced, for example, by heating compound (II) with phosphorus oxychloride by the following production method 1.

（式中の記号は前記と同意義を示す。） Reference example: JP 62-145073
Production method 1

(The symbols in the formula are as defined above.)

The reaction may be performed in a solvent that does not hinder the reaction, but usually an excess amount of phosphorus oxychloride (5 to 30 equivalents relative to compound (II)) is used without a solvent. The reaction temperature is usually 80 to 200 ° C, preferably 90 ° C to reflux (105 ° C). The reaction time is usually 0.1 to 96 hours, preferably 0.5 to 5 hours, more preferably 0.5 to 2 hours. When the progress of the reaction is slow even under reflux, a pressure-resistant sealed container can be used, heated to about 200 ° C., and reacted under pressure (eg, 1.1 to 100 atm).
The production, l is 1, and R is a group R ^a other than halogen atoms (Ib) is, for example, by the following production method 2, by reacting the compound (III) with the compound (IV) can do.

（式中、Yは脱離基（例えば、フッ素、塩素、臭素、ヨウ素等のハロゲン原子、例えば、メタンスルホニルオキシ基、ベンゼンスルホニルオキシ基、p-トルエンスルホニルオキシ基等のアルカン又はアレーンスルホニルオキシ基，例えばメタンスルホニル、ベンゼンスルホニル、フェニルメタンメタンスルホニル等のアルカン、アレーン又はアレーンアルカンスルホニル基等）を示し、R^aはRと同意義であるがハロゲン原子ではなく、他の記号は前記と同意義を示す。） Reference example: Journal of Chemical Society of Japan, 1973, p. 1944; JP-A-58-88369
Production method 2

(In the formula, Y represents a leaving group (for example, a halogen atom such as fluorine, chlorine, bromine or iodine, for example, an alkane or arenesulfonyloxy group such as a methanesulfonyloxy group, a benzenesulfonyloxy group, or a p-toluenesulfonyloxy group) , For example, alkane such as methanesulfonyl, benzenesulfonyl, phenylmethanemethanesulfonyl, etc., arene or arenealkanesulfonyl groups, etc.), R ^a is the same as R, but is not a halogen atom, and other symbols are as defined above Is shown.)

The reaction may be performed without a solvent or a solvent may be used. Examples of the solvent include aliphatic hydrocarbons such as pentane, hexane, heptane, petroleum ether, and cyclohexane, esters such as methyl acetate, ethyl acetate, ethyl formate, and ethyl propionate, acetone, and methyl ethyl ketone. Ketones, diethyl ether, methyl tert-butyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran, dioxane, and other ethers, methanol, ethanol, isopropanol, and other nitriles, acetonitrile, propionitrile, and other nitriles, dimethylformamide, dimethylacetamide Acid amides, cyclic amides such as 1-methyl-2-pyrrolidone, phosphoric acid amides such as hexamethylphosphoramide, cyclic ureas such as 1,3-dimethyl-2-imidazolidinone, dimethyl sulfoxide Sulfoxides such as sulfolane, sulfones such as sulfolane, halogenated hydrocarbons such as dichloromethane, chloroform, 1,2-dichloroethane, carbon tetrachloride, aromatic amines such as pyridine, picoline, lutidine, quinoline, and mixtures thereof Examples thereof include a solvent, water, and a mixed solvent of these with water.
When a mixed solvent with water is used and the reaction is not homogeneous, a phase transfer catalyst (for example, quaternary ammonium salts such as benzyltriethylammonium chloride and tetrabutylammonium bromide, crown ethers such as 18-crown-6) Etc.) may be used.
Examples of the base include alkali metal alcoholates such as sodium ethylate, sodium methylate, potassium tert-butoxide, such as pyridine, picoline, lutidine, quinoline, triethylamine, diisopropylethylamine, 4-dimethylaminoviridine, N, N- Organic bases such as dimethylaniline, for example, inorganic bases such as potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, such as lithium hydride, sodium hydride, potassium hydride, etc. Examples of the metal hydride include organic lithium reagents such as butyl lithium and lithium diisopropylamide.
The amount of the base used is not particularly limited as long as it does not adversely affect the reaction, and a large excess amount can also be used as a solvent.
When compound (IV) is an amine, an excess amount of compound (IV) can also be used as a base and a solvent.
The reaction temperature is usually −50 to 200 ° C., preferably room temperature to 150 ° C. The reaction time is generally 0.1 to 96 hours, preferably 0.1 to 72 hours, more preferably 0.1 to 24 hours.
When the compound (IV) is a low-boiling compound such as ammonia, methylamine, ethylamine, or when the reaction proceeds slowly, use a pressure-resistant sealed container and heat to about 40 to 150 ° C. under pressure (for example, The reaction can also be carried out at 1.1 to 100 atm).

Compound (II) contains a known compound and can be produced by a known method or a method analogous thereto.
For example, it can be produced by reacting compound (V) with compound (VI) and methyl chlorocarbonate according to the following Reference Production Method 1.

（式中、Zは塩素、臭素、ヨウ素等のハロゲン原子を示し、他の記号は前記と同意義を示す。） Reference example: Tetrahedron 42, 3697 (1986)
Reference manufacturing method 1

(In the formula, Z represents a halogen atom such as chlorine, bromine or iodine, and other symbols have the same meaning as described above.)

In this reaction, compound (V) and compound (VI) are first reacted. Usually using a solvent, for example, aliphatic hydrocarbons such as pentane, hexane, heptane, petroleum ether, aromatic hydrocarbons such as benzene, toluene, xylene, diethyl ether, methyl tertbutyl ether, diisopropyl ether, dibutyl ether , Ethers such as tetrahydrofuran and dioxane, or a mixture of two or more of these.
Compound (VI) is generally used in an amount of 1 to 5 equivalents, preferably 2 to 2.5 equivalents, relative to compound (V).
The reaction temperature at this stage is usually 40 to 100 ° C, preferably 50 to 70 ° C.
The reaction time is usually 0.2 to 96 hours, preferably 0.5 to 24 hours, more preferably 1 to 3 hours.
The methyl chlorocarbonate is then reacted. Methyl chlorocarbonate is usually used in an amount of 1 to 5 equivalents, preferably 1 to 2 equivalents, relative to compound (V). In this stage, it is preferable to first cool to 0 to 20 ° C., add methyl chlorocarbonate, and then heat. The reaction temperature at the time of heating is usually 40 to 200 ° C, preferably 50 to 70 ° C. The reaction time is generally 0.2 to 96 hours, preferably 0.5 to 10 hours, more preferably about 1 to 3 hours.
Compound (II) is prepared by reacting compound (VII) with trichloroacetyl chloride by the following Reference Production Method 2 to produce compound (VIII), and then reacting compound (VIII) with ammonia or a weakly acidic substance of ammonia. It can also be produced by reacting with a salt.

（式中の記号は前記と同意義を示す。） Reference example: Chem. Pharm. Bull., 26, 1633 (1978)
Reference manufacturing method 2

(The symbols in the formula are as defined above.)

In the first half of the reaction, compound (VII) is reacted with trichloroacetyl chloride in the presence of a base. Although the reaction may be carried out without a solvent, it is usually carried out in the presence of a solvent. As such a solvent, as described in Production Example 2, aliphatic hydrocarbons, aromatic hydrocarbons, esters, ketones, ethers, nitriles, acid amides, cyclic amides, phosphoric acid amides, Cyclic ureas, sulfoxides, sulfones, halogenated hydrocarbons, etc., or mixtures thereof are used. As the base, bases as described in Production Example 2 are used, and organic bases and inorganic bases are particularly preferable, and triethylamine is generally used.
The reaction temperature is usually −50 to 100 ° C., preferably 0 to 20 ° C. The reaction time is generally about 0.1 to 96 hours, preferably 0.2 to 5 hours, more preferably 0.5 to 3 hours.
In the latter half of the reaction, the compound (VIII) produced in the first half of the reaction is reacted with ammonia or a compound that generates ammonia in the system. As such a compound, a salt with a weakly acidic substance of ammonia such as ammonium carbonate, ammonium formate, or ammonium acetate is used. In the reaction, a solvent as described in Production Example 2 is usually used. Preferred solvents are acid amides, cyclic amides, phosphoric acid amides, cyclic ureas, sulfoxides, sulfones and the like.
The reaction temperature is usually 20 to 200 ° C, preferably 50 to 120 ° C.
The reaction time is generally 0.2 to 96 hours, preferably 0.5 to 10 hours, more preferably 1 to 5 hours.

Compound (III) contains a known compound and can be produced by a known method or a method analogous thereto.
For example, when Y is a chlorine atom, compound (III) is compound (Ia) contained in compound (I), and can be produced by the method described above.

  Compound (IV), compound (V), compound (VI) and compound (VII) are generally known compounds and are either commercially available or can be produced by known methods. In particular, compound (VI) is a compound called a Grignard reagent, and a commercially available product may be used, but it can be produced by a known method before use and used as it is without isolation and purification.

  Each of the compounds produced by the above production methods 1 and 2 and reference production methods 1 and 2 can be obtained by known means such as concentration, vacuum concentration, extraction, phase transfer, crystallization, recrystallization, chromatography and the like. Can be isolated and purified.

The plant growth regulation in the present invention is usually performed by applying an effective amount of a plant growth regulator to the plant or its habitat.
When the plant growth regulator of the present invention is used for agriculture and forestry, the application amount is usually 0.01 to 1000 g in an amount of 1000 m ² . When the plant growth regulator is formulated into emulsions, wettable powders, flowables, microcapsules, etc., it is usually diluted with water so that the active ingredient concentration is 0.001 to 10,000 ppm. When the plant growth regulator is formulated into granules, powders, etc., it is usually applied as it is.
The plant growth regulator of the present invention thus formulated is treated with the soil for the purpose of promoting the growth of plant roots in the soil of the cultivated land, for example, as it is or diluted with water or the like. May be used. Moreover, it can also be used by the method of wrapping the resin formulation processed into the sheet form, the string form, etc. around the crop, stretching it around the crop, and / or laying it on the soil surface of the plant stock. In addition, the plant growth regulator of the present invention can be used by treating the plant with foliage treatment or shoots, and treating the planted hole or stock at the time of planting or planting the plant seedling before planting the crop seedling. Can also be used. The plant growth regulator is treated once or a plurality of times for the target plant.
When the plant growth regulator of the present invention is used by treating foliage to the plant body or when used by treating the soil, the treatment amount may vary depending on the preparation form, application time, application method, application location, target plant. The amount is usually 0.1 to 10,000 g per hectare. In addition, the concentration used when the plant growth regulator is diluted in water may vary depending on the preparation form, application time, application method, application location, target plant, but is generally 0.001 to 10,000 ppm, preferably Is 0.01 to 1000 ppm.
In addition, the plant growth regulator of the present invention can be used by treating the plant before transplantation. In the case of directly absorbing the plant before transplantation, the root or whole of the plant can be used by immersing it in a solution diluted or suspended to 0.001 ppm to 10000 ppm as a use concentration.
The formulated plant growth regulator of the present invention can be used by directly treating the seed of the target plant. For example, a method of immersing seed in a plant growth regulator of the present invention prepared by adjusting the concentration of an active ingredient in the plant growth regulator of the present invention to 1 to 10000 ppm, a plant seed in the plant growth regulator of the present invention. Examples include a method of spraying or smearing the plant growth regulator of the present invention having an active ingredient concentration of 1 to 10,000 ppm and a method of dressing the plant growth regulator of the present invention on plant seeds.
Moreover, the plant growth regulator of this invention may be mixed and used for the hydroponic liquid in hydroponics, and may be used as one of the culture medium components in tissue culture. When used for hydroponics, it can be used by dissolving or suspending in a culture medium for hydroponics such as garden trials that is usually used in the range of 0.001 ppm to 10000 ppm as the concentration in the medium. Moreover, when using at the time of a tissue culture or cell culture, it can melt | dissolve or use in the culture medium for plant tissue cultures, such as MS culture medium used normally, as a density | concentration in a culture medium in the range of 0.001 ppm-10000 ppm. In this case, a saccharide as a carbon source, various plant hormones, and the like can be appropriately added according to a conventional method.

The plant growth regulator of the present invention can also be used with other fungicides, insecticides, acaricides, nematicides, herbicides, plant growth regulators and / or fertilizers.
These application rates and application concentrations vary depending on the type of formulation, application time, application location, application method, plant type, expected effect level, etc., and may be increased or decreased without regard to the above range. Can be selected as appropriate.
The plant growth regulator can be used in the plant growth regulation method described above.
On the other hand, the plant evaluated by the method for testing the ability of the test substance to promote plant root growth, comprising the first step and the second step using a cytokinin receptor selected from the group A. It is also possible to identify a substance that has the ability to promote root growth of plants and to promote plant root growth by contacting the plant with a substance that has the ability to promote root growth of the identified plants. is there. As a method of bringing a plant into contact with a substance having the ability to promote root growth of the plant specified here, the above-mentioned preparation methods, application methods, and the like can be used.

The plant growth regulator of the present invention can be used for the purpose of improving seedling establishment and survival rate by promoting root growth during direct sowing cultivation of rice. The plant growth regulator of the present invention can also be used for the purpose of promoting root growth during rice seedling box cultivation. In addition, the plant growth regulator of the present invention can be used for the purpose of improving the greening of golf courses and improving heat resistance and drought resistance. In crops such as soybean, corn, and wheat, it can be used for the purpose of improving rooting, improving productivity by establishing early seedling establishment, or reducing herbicides. In the cultivation of tomatoes and peppers, it can be used for the purpose of improving the survival rate at the time of transplantation. In the production of seedlings such as vegetables, the use of the plant growth regulator of the present invention can be expected to improve the efficiency of machine transplantation by establishing uniform seedling establishment.
Using the plant growth regulator of the present invention, it is possible to control the apical dominance of plants. For example, the plant growth regulator of the present invention can be used to suppress buds such as tobacco and roses. In addition, it can be used as a flowering agent by controlling the formation of flower buds such as fruit crops and flowering plants. In addition, increasing the number of flower buds can increase the yield of fruit crops or improve the quality of flowering plants. In addition, the number of branches can be reduced or increased by suppressing the growth of branches of fruit tree crops and the like, and the number of branches can be increased and used for the control of tree growth.
The plant growth regulator of the present invention can be used for tissue culture techniques such as dedifferentiation into callus or redifferentiation from callus. For example, callus formation from plant tissues can be promoted using the plant growth regulator of the present invention. Moreover, the redifferentiation efficiency from somatic embryos, such as soybean, can be improved.
The plant growth regulator of the present invention can be used to control plant senescence. For example, the plant growth regulator of the present invention can be used to suppress cut flower senescence and improve flower life of flower plants such as carnations. It can also be used to suppress the maturation of vegetables, fruits and the like. In addition, it can prevent leaf senescence in paddy rice seedlings and the like and grow healthy seedlings. Moreover, leaf senescence can be promoted by processing before harvesting plants such as cotton.

The plant-derived cytokinin receptor consisting of the amino acid sequence shown in the group B can be used as a research tool. For example, it can be used as a research tool for conducting research such as testing of the ability to promote the growth of roots of the plant and the search for chemical substances having the ability to control the growth or differentiation of plants. In addition, for example, in a study analyzing the action mechanism of a drug acting on a cytokinin receptor, the cytokinin receptor can be used as a research tool.
Further, a polynucleotide encoding the amino acid sequence shown in the group B, a polynucleotide having a complementary nucleotide sequence thereto, and a partial base of the polynucleotide encoding the amino acid sequence shown in the group B A polynucleotide having a base sequence complementary to the sequence or a partial base sequence thereof, and a polypeptide comprising the base sequence represented by SEQ ID NO: 3 or 4 can be used as a research tool. For example, a part of these functions as a polynucleotide used in the method for producing a cytokinin receptor as described above. In addition, a part of the method is used for obtaining the polynucleotide shown in the polynucleotide group B using PCR or obtaining the polynucleotide shown in the polynucleotide group B using hybridization as described above. It can be used as an important research tool.
In particular, when performing screening for plant growth regulators, it can be used as an experimental tool for experiments performed for screening. Specifically, it is used as an experimental tool for experiments conducted in conducting tests of the ability to promote the growth of roots of the aforementioned plants, searching for chemical substances having the ability to control the growth or differentiation of plants, etc. can do.

  Furthermore, the present invention relates to a test substance, data information relating to the activity of the test substance, which inhibits intracellular signal transmission from a plant-derived cytokinin receptor of a cell (or the presence or absence of intracellular signal transmission or the amount thereof). Means for inputting / accumulating / managing (hereinafter also referred to as means a), means for querying / searching the data information based on desired conditions (hereinafter also referred to as means b), and It also includes a system (hereinafter also referred to as the present invention system) characterized by comprising means (hereinafter also referred to as means c) for displaying and outputting the inquiry / searched results. .

  First, the means a will be described. As described above, the means a stores the input information after inputting the data information relating to the activity of the cell to inhibit intracellular signal transduction from the plant-derived cytokinin receptor of the cell, as described above. It is a means to manage. Such information is input by the input unit 1 and stored in the normal storage unit 2. As an input means, what can input the said information, such as a keyboard and a mouse | mouth, is mentioned, for example. When the input, storage, and management of the information are completed, the process proceeds to the next means b. For the storage and management of the information, information having a data structure is input using hardware such as a computer and software such as an OS and database management, and an appropriate storage device such as a flexible disk, magneto-optical, or the like. By storing in a computer-readable recording medium such as a disk, CD-ROM, DVD-ROM, or hard disk, a large amount of data may be stored and managed efficiently.

  The means b will be described. The means b is a means for inquiring and searching the data information stored and managed by the means a based on conditions for obtaining a desired result as described above. For such information, if the condition for inquiry / search is input by the input means 1 and the information that matches the condition is selected from the information stored in the normal storage means 2, the process proceeds to the next means c. The selected result is normally stored in the storage unit 2 and can be displayed by the display / output unit 3.

  The means c will be described. The means c is a means for displaying and outputting the result of inquiry / search as described above. Examples of the display / output means 3 include a display, a printer, and the like, and the result may be displayed on a display device of a computer or output on paper by printing or the like.

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

Synthesis Examples and Reference Synthesis Examples are shown below to describe the compound (I) used in the present invention more specifically, but the compound (I) is not limited to these examples.
In Synthesis Examples and Reference Synthesis Examples, “room temperature” usually indicates 10-30 ° C. “ ¹ H NMR” means proton nuclear magnetic resonance spectrum, measured with JEOL JNM-AL400 spectrometer (400 MHz) using tetramethylsilane as internal standard, and chemical shift (δ) in ppm. Indicated. “Mp” indicates a melting point, and was measured with a Mettler FP61 melting point measuring apparatus.
The symbols used in the following synthesis examples, reference synthesis examples, and Tables 3 to 7 have the following meanings. “CDC1 ₃ ”: deuterated chloroform, “DMSO-d ₆ ”: deuterated dimethyl sulfoxide, “s”: singlet, “d”: doublet, “t”: triplet, “q”: quartet, “dd”: double doublet, “M”: multiplet, “br”: broad (wide), “J”: coupling constant, “Me”: methyl, “Et”: ethyl, “Pr”: propyl, “i-Pr”: isopropyl, “T-Bu”: tertiary butyl, “Ph”: phenyl, “Ac”: acetyl, “THF”: tetrahydrofuran, “DMF”: N, N-dimethylformamide, “DMSO”: dimethyl sulfoxide, “MTBE”: Methyl tertiary butyl ether

Synthesis Example 1 (Production of 2,8-dichloro-4-phenylquinazoline (Compound No. Ia1-11))
6.65 g of phosphorus oxychloride was added to 1.24 g of 8-chloro-4-phenyl-2 (1H) -quinazolinone (Compound No. II-11), and the mixture was stirred at 95 ° C. for 1 hour. The obtained reaction solution was poured into 200 ml of ice water, and sodium bicarbonate was added to adjust the pH to 9. Next, the precipitated crystals were collected by filtration. The recovered product was recrystallized from ethanol to obtain 1.07 g of the title compound. Mp. 154.4 ° C. ¹ H NMR (CDCl ₃ ): 7.52-7.65 (4H, m), 7.76-7.80 (2H, m), 8.02-8.08 (2H, m).

Synthesis Example 2 (Production of 2-amino-6,8-dichloro-4-phenylquinazoline (Compound No. Ic12-4))
A mixture of 300 mg of 2,6,8-trichloro-4-phenylquinazoline (Compound No. Ia1-12), 30 g of 28% aqueous ammonia solution and 6 ml of acetonitrile was reacted at 105 ° C. for 1.5 hours in a pressure resistant reactor. The obtained reaction solution was cooled and then poured into 100 ml of water. The mixture was extracted with 60 ml of ethyl acetate, and the resulting extract was concentrated. The obtained residue was purified by silica gel column chromatography (chloroform) to obtain 230 mg of the title compound. Mp.212.7 ° C. ¹ H NMR (CDCl ₃ ): 5.56 (2H, br.s), 7.54-7.60 (3H, m), 7.63-7.68 (2H, m), 7.74 (1H, d, J = 2.3 Hz), 7.79 (1H , d, J = 2.3 Hz).

Synthesis Example 3 (Production of 6-chloro-2-furfurylamino-4-phenylquinazoline (Compound No. Ic3-16))
A mixture of 275 mg of 2,6-dichloro-4-phenylquinazoline (Compound No. Ia1-3) and 486 mg of furfurylamine was stirred at 85 ° C. for 40 minutes, and then poured into 50 ml of water. The mixture was extracted with ethyl acetate, and the resulting extract was washed with water and concentrated. The obtained residue was recrystallized from ethanol to obtain 280 mg of the title compound. Mp.142.0 ℃. ¹ H NMR (CDCl ₃ ): 4.77 (2H, d, J = 5.6 Hz), 5.70 (1H, br. T, J = 5.6 Hz), 6.29-6.32 (2H, m), 7.36 ( 1H, dd, J = 1.8, 0.9 Hz), 7.53-7.70 (7H, m), 7.78 (1H, d, J = 2.2 Hz).

Synthesis Example 4 (Production of 6-chloro-2-ethoxycarbonylmethylamino-4-phenylquinazoline (Compound No. Ic3-33))
To 550 mg of 2,6-dichloro-4-phenylquinazoline (Compound No. Ia1-3) and 419 mg of glycine ethyl ester hydrochloride were added 3 ml of DMF and 607 mg of triethylamine, and the mixture was stirred at 85 ° C. for 5.5 hours. The obtained reaction solution was poured into 100 ml of water, extracted with ethyl acetate, and the extract was concentrated. The obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate = 6: 1-3: 1) to give 503 mg of the title compound. Mp. 146.1 ° C. ¹ H NMR (CDCl ₃ ): 1.30 (3H, t, J = 7.2 Hz), 4.25 (2H, q, J = 7.2 Hz), 4.31 (2H, d, J = 5.2 Hz), 5.97 (1H, br. s), 7.54-7.63 (5H, m), 7.67-7.70 (2H, m), 7.79 (1H, s).

Synthesis Example 5 (Production of 6-chloro-2-methoxyamino-4-phenylquinazoline (Compound No. Ic3-32))
A mixture of 275 mg of 2,6-dichloro-4-phenylquinazoline (Compound No. Ia1-3), 10 ml of acetonitrile and 334 mg of methoxyamine hydrochloride is added dropwise at room temperature to a mixture obtained by adding 506 mg of triethylamine at a room temperature. The vessel was charged and reacted at 105 ° C. for 3.5 hours. After cooling, the reaction solution was poured into 100 ml of water. The mixture was extracted with ethyl acetate, and the resulting extract was washed with water and concentrated. The obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate = 3: 1) to obtain 156 mg of crude crystals. Further, this was recrystallized from ethyl acetate to obtain 55 mg of the title compound. Mp. 173.9 ° C. ¹ H NMR (CDCl ₃ ): 3.98 (3H, s), 7.47-7.72 (6H, m), 7.87 (1H, d, J = 9.0 Hz), 7.89 (1H, d, J = 2.2 Hz), 8.03 ( 1H, br.s).

Synthesis Example 6 (Production of 6-chloro-2- (2-hydroxyethylthio) -4-phenylquinazoline (Compound No. Ie3-1))
To a solution of 86 mg of 2-mercaptoethanol in 7.5 ml of DMF, 44 mg of sodium hydride (60%) was added, and this was stirred at room temperature for 30 minutes. To the reaction mixture was added 275 mg of 2,6-dichloro-4-phenylquinazoline (Compound No. Ia1-3), and the mixture was further stirred at room temperature for 2 hours. The reaction solution was poured into 100 ml of water, extracted with ethyl acetate, and the resulting extract was concentrated. The obtained residue was purified by silica gel column chromatography (ethyl acetate: hexane = 1: 5) to obtain 266 mg of the title compound. Mp. 124.4 ° C. ¹ H NMR (CDCl ₃ ): 3.50 (2H, t, J = 5.5 Hz), 3.64 (1H, br. T), 4.07 (2H, q-like, J = 5.5 Hz), 7.57-7.61 (3H, m ), 7.71-7.74 (2H, m), 7.77 (1H, dd, J = 8.9, 2.3 Hz), 7.85 (1H, d, J = 8.9 Hz), 7.97 (1H, d, J = 2.3 Hz).

Synthesis Example 7 (Production of 6-chloro-2-formamide-4-phenylquinazoline (Compound No. Ic3-35))
48 mg of sodium hydride (60%) was added to a solution of 54 mg of dry formamide in 5 ml of DMF, and this was stirred at room temperature for 30 minutes. After adding 275 mg of 2,6-dichloro-4-phenylquinazoline (Compound No. Ia1-3) to the reaction mixture, the mixture was heated to 85 ° C. and stirred for 3 hours. The obtained reaction solution was poured into 100 ml of water, extracted with ethyl acetate, and the extract was concentrated. The obtained residue was purified by silica gel column chromatography (ethyl acetate: hexane = 1: 3) to obtain 64 mg of the title compound. Mp.252.1 ° C. ¹ H NMR: 7.59-7.66 (3H, m), 7.72-7.81 (3H, m), 7.88 (1H, d, J = 8.8 Hz), 8.02 (1H, d, J = 2.0 Hz), 8.37 (1H, br.d, J = 10.4 Hz), 9.71 (1H, d, J = 10.4 Hz).

Synthesis Example 8 (Production of 6-chloro-2- (1-methylhydrazino) -4-phenylquinazoline (Compound No. Ic3-27))
A mixture of 275 mg of 2,6-dichloro-4-phenylquinazoline (Compound No. Ia1-3) and 461 mg of monomethylhydrazine was stirred at 85 ° C. for 30 minutes. After cooling the obtained reaction product, 200 ml of water was added thereto. The precipitated crystals were collected by filtration, and the recovered product was purified by column chromatography (ethyl acetate) to obtain 225 mg of the title compound. Mp. 155.9 ° C. ¹ H NMR (CDCl ₃ ): 3.52 (3H, s), 4.65 (2H, s), 7.54-7.63 (5H, m), 7.70-7.74 (2H, m), 7.80 (1H, d, J = 2.0 Hz ).

Synthesis Example 9 (Production of 2- (2-aminoethoxy) -6-chloro-4-phenylquinazoline (Compound No. Id3-1))
2,6-Dichloro-4-phenylquinazoline (Compound No. Ia1-3) 500 mg and N- (2-hydroxyethyl) phthalimide 382 mg were reacted according to Synthesis Example 6 to give 6-chloro-4-phenyl-2. 380 mg of-(2-phthalimidoethoxy) quinazoline was obtained. Mp. 154.7 ° C. ¹ H NMR (CDCl ₃ ): 4.25 (2H, t, J = 5.8 Hz), 4.84 (2H, t, J = 5.8 Hz), 7.53-7.60 (3H, m), 7.67-7.71 (3H, m), 7.72-7.76 (3H, m), 7.78-7.82 (2H, m), 7.97 (1H, d, J = 2.2 Hz).
A mixture of 380 mg of 6-chloro-4-phenyl-2- (2-phthalimidoethoxy) quinazoline, 70 mg of hydrazine hydrate and 6 ml of ethanol was heated to reflux for 2 hours. After adding 1.2 ml of water to the resulting reaction solution, ethanol was distilled off. After adding 1.5 ml of concentrated hydrochloric acid to the obtained residue, this was heated under reflux for 1 hour. The obtained reaction product was poured into a saturated aqueous sodium bicarbonate solution under cooling, extracted with ethyl acetate, and the extract was concentrated to obtain 240 mg of the title compound. Mp. 134.9 ° C. ¹ H NMR (CDCl ₃ ): 3.59-3.63 (2H, m), 3.84 (2H, t, J = 4.6 Hz), 6.15 (2H, br.s), 7.53-7.59 (5H, m), 7.63-7.67 (2H, m), 7.75 (1H, d, J = 1.2 Hz).

Synthesis Example 10 (Production of 2- (2-acetylthioethylamino) -6-chloro-4-phenylquinazoline (Compound No. Ic3-14))
To a solution of 292 mg of triphenylphosphine in 3 ml of dehydrated THF, 563 mg of a 40% toluene solution of diisopropylcarbodiimide was added dropwise under ice cooling, followed by stirring for 20 minutes. A solution of 167 mg of 6-chloro-2- (2-hydroxyethylamino) -4-phenylquinazoline (Compound No. Ic3-1) in 2 ml of dehydrated THF was added dropwise to the resulting suspension dropwise under ice cooling, and 127 mg of thioacetic acid was added directly. It was dripped. The resulting yellow transparent solution was stirred for 1 hour under ice-cooling and at room temperature for 1 hour, then poured into saturated aqueous sodium bicarbonate, extracted with chloroform, and the extract was concentrated. The obtained residue was purified by silica gel column chromatography (chloroform: ethyl acetate = 10: 1) to obtain 170 mg of the title compound. Mp.128.9 ° C. ¹ H NMR (CDCl ₃ ): 2.34 (3H, s), 3.22 (2H, t, J = 6.5 Hz), 3.73 (2H, q, J = 6.5 Hz), 5.74 (1H, br. T, J = 6.5 Hz), 7.53-7.62 (5H, m), 7.65-7.70 (2H, m), 7.77 (1H, s).

２８ｍｇを得た。Mp.159.2℃。¹H NMR (CDCl₃): 3.02 (4H, t, J=6.4 Hz), 3.89 (4H, q, J=6.4 Hz), 5.81 (2H, br. t, J=6.4 Hz), 7.51-7.61 (10H, m), 7.63-7.68 (4H, m), 7.75 (2H, d, J = 2.2Hz)。 Synthesis Example 11 (6-Chloro-2- (2-mercaptoethylamino) -4-phenylquinazoline (Compound No. Ic3-13) and bis [2- (6-chloro-4-phenyl-2-quinazolinyl) aminoethyl ] Disulfide (Compound No. Ic3-15))
A mixture of 108 mg of 2- (2-acetylthioethylamino) -6-chloro-4-phenylquinazoline (Compound No. Ic3-14), 2 ml of ethanol and 362 mg of 10% aqueous sodium hydroxide solution was heated under reflux at room temperature for 2 hours. After stirring for 30 minutes, insoluble matters were collected from the reaction solution by filtration. The recovered product (solid) was purified by silica gel column chromatography (chloroform: ethyl acetate = 10: 1) to obtain 50 mg of 6-chloro-2- (2-mercaptoethylamino) -4-phenylquinazoline. Mp.165.9 ° C. ¹ H NMR (CDCl ₃ ): 1.46 (1H, t, J = 8.5 Hz), 2.84 (2H, q-like, J = 7.2 Hz), 3.66 (2H, q-like, J = 6.4 Hz), 5.79 ( 1H, br. T, J = 5.4 Hz), 7.55-7.57 (3H, m), 7.60 (2H, s), 7.66-7.69 (2H, m), 7.77-7.78 (1H, m). Next, bis [2- (6-chloro-4-phenyl-2-quinazolinyl) aminoethyl] disulfide (compound represented by the following formula)

28 mg was obtained. Mp.159.2 ° C. ¹ H NMR (CDCl ₃ ): 3.02 (4H, t, J = 6.4 Hz), 3.89 (4H, q, J = 6.4 Hz), 5.81 (2H, br. T, J = 6.4 Hz), 7.51-7.61 ( 10H, m), 7.63-7.68 (4H, m), 7.75 (2H, d, J = 2.2 Hz).

Examples of compounds that can be produced in the same manner as in the above synthesis examples and commercially available compounds are shown in Table 3, Table 4, Table 5, and Table 6 (including the compounds produced in the above synthesis examples).
The annotations “a)”, “b)”, “c)” and “d)” in Tables 4 and 5 are as follows.

a) The structure was described in Synthesis Example 11.
b) ¹ H NMR (CDCl ₃ ): 1.66-1.75 (1H, m), 1.86-2.08 (3H, m), 3.57-3.64 (1H, m), 3.75-3.83 (2H, m), 3.89-3.59 ( 1H, m), 4.13-4.20 (1H, m), 5.70 (1H, br.s), 7.52-7.60 (5H, m), 7.64-7.69 (2H, m), 7.75-7.76 (1H, m).
c) ¹ H NMR (CDCl ₃ ): 1.22 (3H, t, J = 7.0 Hz), 3.55 (2H, q, J = 7.0 Hz), 3.68 (2H, t, J = 5.2 Hz), 3.78 (2H, q-like, J = 5.2 Hz), 5.75 (1H, br.t), 7.53-7.60 (5H, m), 7.65-7.69 (2H, m), 7.75-7.77 (1H, m).
d) ¹ H NMR (CDCl ₃ ): 1.22 (3H, t, J = 7.0 Hz), 1.96 (2H, quintet, J = 6.3 Hz), 3.50 (2H, q, J = 7.0 Hz), 3.58 (2H, t, J = 6.3 Hz), 3.67 (2H, q-like, J = 6.3 Hz), 5.65 (1H, br.t), 7.53-7.60 (5H, m), 7.65-7.69 (2H, m), 7.74 -7.76 (1H, m).

Reference Synthesis Example 1 (Production of 8-chloro-4-phenyl-2 (1H) -quinazolinone (Compound No. II-11))
To 7.10 g of phenylmagnesium bromide (32% THF solution), a solution of 953 mg of 2-amino-3-chlorobenzonitrile in 7 ml of THF was added dropwise at room temperature, and the mixture was heated to reflux for 30 minutes. To the obtained reaction product, 885 mg of methyl chlorocarbonate was added dropwise under ice cooling, and then the mixture was heated to reflux for 40 minutes. The obtained reaction liquid was poured into 40 ml of 2N hydrochloric acid while cooling, and then 8 g of sodium bicarbonate and 20 ml of MTBE were added thereto and stirred. The precipitated crystals were then collected by filtration to obtain 1.30 g of the title compound. ¹ H NMR (DMSO-d ₆ ): 7.24 (1H, t, J = 8.0 Hz), 7.57-7.70 (6H, m), 7.90-7.93 (1H, m), 11.45 (br.s).

Reference Synthesis Example 2 (4-Phenyl-6-trifluoromethyl-2 (1H) -quinazolinone (Compound No. II-6))
762 mg of 2-amino-5-trifluoromethylbenzophenone was dissolved in 10 ml of chloroform, 349 mg of triethylamine was added dropwise thereto, and then 627 mg of trichloroacetyl chloride was added dropwise under ice cooling. After stirring at the same temperature for 30 minutes, 50 ml of water was added thereto for liquid separation. The organic layer was collected and the collected organic layer was concentrated. The obtained residue is purified by silica gel column chromatography (hexane: ethyl acetate = 5: 1) to obtain 1.08 g of 2′-benzoyl-2,2,2-trichloro-4′-trifluoromethylacetanilide. It was. ¹ H NMR (CDCl ₃ ): 7.53-7.58 (2H, m), 7.66-7.75 (3H, m), 7.90-7.95 (2H, m), 8.81 (1H, d, J = 8.6 Hz), 12.38 (1H , br.s).
A mixture of 1.08 g of 2′-benzoyl-2,2,2-trichloro-4′-trifluoromethylacetanilide, 10 ml of DMSO and 1.18 g of ammonium acetate was stirred at 75 ° C. for 1 hour. After cooling, 100 ml of water was added thereto, and the precipitated crystals were collected by filtration. The obtained recovered product was dissolved in a mixed solution of hexane: ethyl acetate = 1: 1, and then dehydrated with anhydrous magnesium sulfate and concentrated to obtain 765 mg of the title compound. ¹ H NMR (CDCl ₃ ): 7.58-7.68 (3H, m), 7.75 (1H, d, J = 8.8 Hz), 7.79-7.83 (2H, m), 7.93 (1H, dd, J = 8.8, 1.7 Hz ), 8.17 (1H, br.s), 13.37 (1H, br.s).

Examples of compounds that can be produced in the same manner as in the above Reference Synthesis Examples are shown in Table 7 (including the compounds produced in the above Reference Synthesis Examples).
The annotations “a)” to “i)” in Table 7 are as follows.

a) ¹ H NMR (DMSO-d ₆ ): 7.27 (1H, dd, J = 7.7, 1.0 Hz), 7.38 (1H, dd, J = 8.3, 1.1 Hz), 7.43-7.55 (5H, m), 7.69 (1H, t, J = 8.1 Hz), 12.18 (1H, br.s).
b) ¹ H NMR (DMSO-d ₆ ): 7.35 (1H, dd, J = 9.2, 2.7 Hz), 7.43 (1H, dd, J = 9.2, 4.8 Hz), 7.58-7.74 (6H, m), 12.05 (1H, br.s).
c) ¹ H NMR (DMSO-d ₆ ): 7.35 (1H, d, J = 9.2 Hz), 7.59-7.71 (6H, m), 7.91 (1H, dd, J = 9.2, 2.2 Hz), 12.08 (1H , br.s).
d) It was described in Reference Synthesis Example 2 for ¹ H NMR.
e) ¹ H NMR (CDCl ₃ ): 3.78 (3H, s), 7.25-7.27 (1H, m), 7.36-7.40 (1H, m), 7.54-7.62 (4H, m), 7.81-7.85 (2H, m), 13.37 (1H, br.s).
f) ¹ H NMR (DMSO-d ₆ ): 7.48 (1H, d, J = 8.4 Hz), 7.60-7.68 (3H, m), 7.71-7.74 (2H, m), 8.05 (1H, s), 8.08 -8.12 (1H, m), 12.36 (1H, br.s).
g) (Not isolated and purified.)
h) ¹ H NMR Reference synthesis example 1
i) ¹ H NMR (DMSO-d ₆ ): 7.52-7.72 (6H, m), 8.10-8.12 (1H, m), 11.69 (1H, br.s).

Example 1 (Preparation of Arabidopsis cDNA Phage Library for CER1 Cloning)
The seeds of the Wassilewskija line of Arabidopsis were sterilized with 70% ethyl alcohol for 1 minute and further sterilized with 1.5% sodium hypochlorite for 10 minutes. Rinse well with sterilized water, then in GM medium (4.3g Murashige and Skoog's basal salt mixture, 1% sucrose, 10ml of 5% MES-KOH (pH5.7), 0.3% Phytagel ^TM (SIGMA)) for 2 weeks By culturing, 5 g of a plant was obtained. This was frozen in liquid nitrogen and then physically ground with a mortar. To the obtained ground product, 10 ml of extraction buffer (200 mM Tris-HCl (pH 8.5), 100 mM NaCl, 10 mM EDTA, 0.5% SDS, 14 mM β-mercaptoethanol) and 10 g of phenol were added. After stirring the mixture with a Voltex mixer, 10 ml of chloroform was added thereto and stirred vigorously. Subsequently, the obtained mixture was centrifuged at 10,000 rpm for 20 minutes, and the aqueous layer was recovered. LiCl was added to the recovered aqueous layer to a final concentration of 2M, and this was left at -80 ° C for 3 hours. After the obtained frozen product was thawed, it was centrifuged at 10,000 rpm for 20 minutes to collect the precipitate. Dissolve the collected precipitate in 2 ml of TE (10 mM Tris-HCl (pH 8.0) 1 mM EDTA), add 0.2 ml of 3 M sodium acetate (pH 5.2) and 5 ml of ethanol, and centrifuge to remove the RNA. Collected as a precipitate. Next, the recovered precipitate (RNA) was subjected to Oligotex ^™ dT30super (manufactured by Nippon Roche) to extract RNA bound with polyA from the precipitate.
A phage cDNA library from the extracted polyA-bound RNA was prepared according to the kit instructions using ZAP-cDNARSynthesis Kit (Stratagene). The titer of the prepared phage cDNA library was 500000 PFU.

Example 2 (Preparation of DNA probe of CRE1)
Using the TAKARA LA Taq ^TM kit (manufactured by Takara Shuzo Co., Ltd.) using the phage solution (about 1000000 PFU) of the phage cDNA library prepared in Example 1 as a template, the DNA shown in SEQ ID NO: 3 and SEQ ID NO: 4 PCR was performed using the obtained DNA as a primer to amplify the DNA. Details are described below.
A PCR reaction solution is prepared by adding a reaction composition such as dNTP to phage 1000000pfu and primer DNA 0.2 μM each according to the kit instructions. The PCR conditions are kept at 94 ° C. for 2 minutes, and then kept at 94 ° C. for 30 seconds. The target DNA fragment was amplified by PCR under amplification conditions in which a cycle of 30 seconds at 60 ° C. and 40 minutes at 68 ° C. was repeated 40 times. Next, using the amplified DNA fragment as a template, a probe labeled with ³² P was prepared using a Megaprime DNA-labelling system kit (Amersham Pharmacia). 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 (Acquisition of a phage cDNA clone carrying the CRE1 gene)
The target CRE1 gene was cloned by plaque hybridization using the DNA probe prepared in Example 2. Details are described below.
Using the cDNA phage library prepared in Example 1, plaques were formed according to the instructions of the ZAP-cDNAR Synthesis Kit. After adsorbing DNA from the formed plaques on a nitrocellulose filter, the DNA was immobilized on the filter by ultraviolet treatment. Using the filter thus prepared, 6 × SSC (0.9 M NaCl, 0.09 M sodium citrate), 5 × Denhardt's solution (0.1% (w / v) Ficoll 400, 0.1% (w / v) polyvinyl Pyrrolidone, 0.1% BSA), 0.5% (w / v) SDS and 100 μg / ml denatured salmon sperm DNA or 65 ° C. in DIG EASY Hyb solution (Boehringer Mannheim) containing 100 μg / ml denatured salmon sperm DNA Incubate twice for 15 minutes at room temperature in the presence of 1x SSC (0.15M NaCl, 0.015M sodium citrate) and 0.5% SDS, and further 0.1x SSC (0.015M NaCl, 0.0015M citric acid) Sodium) and 0.5% SDS were obtained by incubation at 68 ° C. for 30 minutes to obtain a hybridized phage cDNA clone.

Example 4 (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 shown in SEQ ID NO: 5 and the DNA shown in SEQ ID NO: 6 as primers, and a DNA having the base sequence shown in SEQ ID NO: 5 Was amplified. Details are described below.
PCR reaction was carried out using Herculase Enhanced DNA Polymerase (TOYOBO) at 94 ° C for 1 minute, followed by 25 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 4 minutes. Was done below. The PCR reaction solution (50 μl) was prepared by adding a reaction composition such as dNTP to 500 ng of cDNA of a cDNA clone of the phage and 100 ng of primer DNA according to the kit instructions.
In this way, the target DNA fragment was amplified.

Example 5 (Construction of CRE1 expression plasmid)
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 fragment obtained in Example 4 (DNA having the base sequence shown in SEQ ID NO: 2) 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 fragment had the correct base sequence and the base sequence was the base sequence represented by SEQ ID NO: 2, and the expression plasmid p415CYC-CRE1 was obtained. .

Example 6 (Preparation of transformed cell TM182-CRE1 and transformed cell TM182-p415CYC1)
Using the expression plasmid p415CYC-CRE1 obtained in Example 5 and the yeast expression vector p415CYC1, respectively, TM182 (sln1Δ), which is a Sln1 gene-deficient strain (Maeda T et al. Nature: 369 242-245 (1994)) Was transformed. The transformation was performed according to VII. Library Transformation & Screening Protocols described on page 22 of CLONTECH: MATCHMAKER Two-Hybrid System 3 User Manual using a polyethylene glycol / lithium acetate (PEG / LiAc) -mediated transformation method. In the obtained transformed cells, the transformed cells TM182-CRE1 and TM182-p415CYC1 were transformed by selecting transformed yeasts that grow on DOLU + Gal medium, taking advantage of the loss of leucine auxotrophy. Obtained.

Example 7 (Method for Searching for Chemical Substances Inhibiting Intracellular Signal Transmission from Plant-derived Cytokinin Receptors in Cells)
The transformed cell TM182-CRE1 and transformed cell TM182-p415CYC1 obtained in Example 6 were inoculated into 10 ml of DOLU + Gal medium, pre-cultured at 30 ° C. for 18 hours, and this was used as a preculture solution. This preculture was diluted with DOLU + Glu medium for transformed cells TM182-CRE1 and with DOLU + Gal medium for transformed cells TM182-p415CYC1 so that OD600 = 0.1. Each pre-dilution culture was used.
An assay plate was prepared in which 1 μl of a test substance prepared to 200 ppm with dimethyl sulfoxide (DMSO) was added to each well of a 96-well plate. At this time, a control group in which 1 μl of DMSO was added was set in some wells. As assay plates, an assay plate for transformed cell TM182-CRE1 and an assay plate for transformed cell TM182-p415CYC1 were prepared.
A solution of trans-zeatin (cytokinin) dissolved in DMSO at 10000 ppm was diluted 50-fold with DOLU + Gul medium to 200 ppm. This 200 ppm trans-zeatin solution was added to each of the above predilution culture solutions at a volume of 3/1000 to finally prepare each predilution culture solution containing 0.6 ppm trans-zeatin. 100 μl of each pre-dilution medium containing 0.6 ppm of trans-zeatin was added to each well of each transformed cell assay plate, cultured at 30 ° C. for 24 hours, and then each plate was read using a plate reader. Well turbidity (OD600) was measured. By comparing the turbidity with the turbidity in the control group, the activity of the test substance to inhibit intracellular signaling from the plant-derived cytokinin receptor of the cells was assayed. The results are shown in Tables 8 and 9.
Regarding transformed cell TM182-CRE1, a chemical that inhibits intracellular signal transduction from plant-derived cytokinin receptors in cells with a test substance whose turbidity in the test group was lower than that in the control group. Selected as a substance. However, for the transformed cell TM182-p415CYC1, test substances that showed a turbidity in the test group that was lower than the turbidity in the control group at least as much as in the transformed cell TM182-CRE1, were toxic to yeast. It was not selected as a chemical substance that inhibits intracellular signal transduction from plant-derived cytokinin receptors possessed by cells.

(Relative growth of test plot relative to control plot) [%] = [(turbidity of test plot) − (blank turbidity)] / [(turbidity of control plot) − (blank turbidity)] × 100

(Activity to inhibit intracellular signal transduction from cytokinin receptor derived from plant possessed by cells) [%] = 100− (relative growth degree of test plot relative to control plot)

Example 8 (Method for assaying concentration responsiveness of chemical substance that inhibits intracellular signal transduction from plant-derived cytokinin receptor possessed by cells)
With respect to the chemical substance selected in Example 7 (inhibiting intracellular signal transduction from plant-derived cytokinin receptors possessed by cells), the same test as in Example 7 was carried out by changing the test concentration. Other than changing the test concentration of the chemical substance selected in Example 7 (inhibiting intracellular signal transduction from the plant-derived cytokinin receptor in the cells) to 0.06 ppm to 6 ppm, Under the same conditions as in Example 7, the transformed cells TM182-CRE1 and TM182-p415CYC1 obtained in Example 6 were cultured. DMSO was used to adjust the concentration of the chemical substances tested. From the lowest test concentration at which the growth state of the transformed cell TM182-CRE1 was not observed after completion of the culture, or from the concentration-responsive growth inhibition curve prepared by the method described below, from the plant-derived cytokinin receptor of the cell The concentration responsiveness of the activity that inhibits intracellular signal transduction was assayed.
In preparing a concentration-responsive growth inhibition curve, first, the relative growth rate was calculated as follows.

(Relative growth rate) [%] = (B) / (A) × 100
(A) = [(0.06 ppm turbidity of test group) − (blank turbidity)] / [(control turbidity) − (blank turbidity)]
(B) = [(turbidity of each test group) − (blank turbidity)] / [(turbidity of control group) − (blank turbidity)]

  Then, a graph (refer to FIG. 1, left figure: transformed cell TM182-CRE1, right figure: transformed cell TM182-p415CYC1) with the X axis as the concentration of the tested chemical substance and the Y axis as the relative growth degree is shown. A concentration-responsive growth inhibition curve was prepared.

Example 9 (Root growth promoting activity test)
A garden standard culture medium having the following composition (see Table 10) was prepared. Dispense 4 μl of chemical DMSO solution into the cluster tube to a final concentration of 0.001 ppm to 10 ppm, and further dispense 600 μl of sterilized horticultural standard medium, and then mix the resulting solution thoroughly. . 10-20 Arabidopsis seeds per cluster tube were sown in the cluster tube and cultured for 10 days at 22 ° C in the light. Then, the length of the main roots (average main roots) generated from the seeds of white seeds was measured. . By calculating the average value of 8 iterations and calculating the root growth rate using the following formula, those that showed a significant root growth rate (for example, a root growth rate of 120% or more) can be determined as having root growth promoting activity. It was.
Moreover, as a detailed result, the value which showed the highest root growth rate in the said final concentration was shown in Table 11 and Table 12.

Root growth rate (%) = (Average main root length of chemical treatment section) / (Average main root length of control section) x 100

Example 10 (Evaluation of root growth promoting activity of cytokinin signaling inhibitor using lettuce)
For the chemical substance IC3-1 and chemical substance Ic7-1 selected in Example 7 (inhibiting intracellular signal transduction from plant-derived cytokinin receptors in cells), lettuce (Lactuca sativa Red wave) was used. The elongation promotion activity of the main root was evaluated. After preparing chemical substances in various concentration aqueous solutions (0.6ppm, 1.2ppm, 2.5ppm, 5ppm, 10ppm, 20ppm, each containing 0.1% DMSO), add 1ml to a filter paper with a diameter of 50mm in a 60φ plastic petri dish, 30 lettuce seeds were sown on the plastic petri dish. After culturing at 22 ° C. for 4 days in a bright place, the length of the main root was measured. The average value of 3 repetitions was calculated | required and the root growth rate was calculated | required by the following formula.

Root growth rate (%) = (average main root length of chemical treatment group) / (average main root length of control group) x 100-100

  The results are shown in FIGS. Chemical substance Ic3-1 has a concentration of 0.6 to 2.5 ppm and 15 to 17% (see Fig. 2), and chemical substance Ic7-1 has a concentration of 10 to 20 ppm and 10 to 17% (see Fig. 3). Main root elongation was observed. As a result of Dunnett's test, it was judged that there was a significant difference at the significance level of 5% in all treatment sections, and it was shown that there was a remarkable root growth promoting effect.

Example 11 (Evaluation of Root Growth Promoting Activity of Cytokinin Signaling Inhibitor Using Rice)
Rice (Oriza sativa L. japonica) was used for chemical substance Ic3-1 and chemical substance Ic7-1 selected in Example 7 (inhibiting intracellular signal transduction from plant-derived cytokinin receptors possessed by cells). The main root elongation promoting activity was evaluated. After preparing chemical substances in various concentrations (10 ppm, 25 ppm, each containing 0.1% DMSO), 17 ml of the chemical solution in a seed growth bag (177 mm x 163 mm, manufactured by Dairika Kogyo) for root growth observation Soaked in cardboard, three rice seeds were sown on the cardboard. The sachet was placed in a plastic container and sealed, and after culturing at 25 ° C. in a light place for 7 days, the length of the main root was measured. The average value of 3 repetitions was calculated | required and the root growth rate was calculated | required by the following formula.

Root growth rate (%) = (average main root length of chemical treatment group) / (average main root length of control group) x 100-100

  The results are shown in FIGS. The chemical substance Ic3-1 showed a main root elongation of 10% at 17 ppm and 20% at 25 ppm (see FIG. 4). In addition, chemical substance Ic7-1 was found to have a main root elongation of 10% at 17 ppm and 19% at 25 ppm (see FIG. 5). As a result of Dunnett's test, it was judged that there was a significant difference at the significance level of 5% in all treatment sections, and it was shown that there was a remarkable root growth promoting effect.

Example 12 (Evaluation of root growth promoting activity of cytokinin signal transduction inhibitory substance by rice seed treatment)
Rice (Oriza sativa L. japonica) was used for chemical substance Ic3-1 and chemical substance Ic7-1 selected in Example 7 (inhibiting intracellular signal transduction from plant-derived cytokinin receptors possessed by cells). The main root elongation promoting activity by seed treatment was evaluated. Chemical substances were dissolved in acetone to prepare a 10,000 ppm solution. After 300 μl of the prepared acetone chemical substance solution was put into an Eppendorf tube, 15 seeds were put into the Eppendorf tube, and then the resulting mixture was stirred and mixed for about 30 seconds. The seed thus treated with the medicine was spread on a filter paper and dried.
Approximately 820 ml of culture soil was placed in a plastic container (diameter 120 mm, height 97 mm) with a hole in the bottom, and seeds treated with the drug as described above were sown 15 by each. The seeds were cultivated in the dark at 30 ° C. for 4 days, and then cultivated in a greenhouse for 35 days. The root part of the plant thus cultivated was cut out and the attached soil was washed away, and then lyophilized. Next, the weight of the root after lyophilization was measured. Three tests were performed for each treatment group, and the average value was obtained. The results are shown in FIG.
The root dry weight per strain increased to 119% in the control group in the case of Compound Ic3-1 and 126% in the case of Compound Ic7-1, and a remarkable root growth promoting effect was confirmed in any compound. It was. In the case of the auxin compound IAA, the root weight was suppressed to 87% of the control group, and the root growth inhibitory effect was recognized.

Example 13 (Evaluation of plant differentiation promoting activity of cytokinin signal transduction inhibitor using Arabidopsis hypocotyl)
For the chemical substance Ic3-1 and chemical substance Ic7-1 selected in Example 7 (inhibiting intracellular signal transduction from the plant-derived cytokinin receptor of the cell), the hypocotyl of Arabidopsis thaliana Columbia was used. The plant differentiation promoting activity was evaluated by examining adventitious root formation activity.
First, an agar medium for seeding Arabidopsis thaliana was prepared. The components of the agar medium are 1 liter of mixed salt for Murashige-Skoog plant medium (Wako Pure Chemical Industries) per liter of aqueous solution, 10% sucrose, 5% MES adjusted to pH 5.7 with potassium hydroxide (2- (N-Morpholino) ethanesulfonic acid) aqueous solution 10 ml, inositol 100 mg, vitamin stock solution (thiamine hydrochloride 10 g, pyridoxine hydrochloride 1 g, nicotinic acid 1 g) per liter of aqueous solution 1 ml, agar 8 g. Next, the agar medium was autoclaved at 120 ° C. for 20 minutes, and then dispensed into a circular petri dish to solidify.
Next, put Arabidopsis seeds with a seed volume of about 25 μl into a 1.5 ml tube, add 1 ml of a solution obtained by diluting sodium hypochlorite solution (Nacalai Tesque) 10 times with sterile distilled water, and add a tube mixer (TOMY , MT-360, MIXING SPEED10) was sterilized with stirring for about 1 minute. Centrifuge in a tabletop centrifuge to settle the seeds, remove the diluted sodium hypochlorite solution from the tube, add 1 ml of sterile distilled water, and then stir for about 1 minute with the tube mixer The seeds were washed. After centrifuging with a tabletop small centrifuge to settle the seeds, the washed water was removed from the tube. The washing operation was repeated 3 times. The washed seeds were sown on a circular petri dish agar medium and germinated and grown in a dark place at 22 ° C.
Meanwhile, a chemical substance was dissolved in DMSO to prepare a 10,000 ppm solution. Furthermore, it diluted with DMSO and the solution of 6000 ppm, 2000 ppm, 600 ppm, and 200 ppm was prepared. Then, 2 μl of the prepared chemical substance DMSO solution of one kind and one concentration per well was dispensed into each hole of the 12-well multiplate (Sumitomo Bakelite). Instead of the chemical DMSO solution, 2 μl of DMSO was placed in the hole set as the control group. Next, transzeatin was dissolved in DMSO to prepare a 10,000 ppm solution. This was diluted with sterile distilled water to prepare a 10 ppm solution. An agar medium having the above composition was prepared, and after autoclaving, the solution was cooled to about 50 ° C., and this 10 ppm transzeatin solution was added and mixed to a final concentration of 0.01 ppm. 2 ml of this transzeatin-containing agar medium was dispensed and solidified in each well of a 12-well multiplate where the chemical substance DMSO solution or DMSO had been dispensed.
The seedlings of Arabidopsis thaliana germinated and grown in the dark at 22 ° C. were cut at the hypocotyl part, and the hypocotyl side with the cotyledon was used for the following measurement of adventitious root formation activity. The hypocotyl on the side with the root was discarded. Specifically, hypocotyls with cotyledons were inserted into the agar medium in each hole of a 12-well multiplate from the cut part to about 5 mm. As a result of allowing the multiplate to stand at 22 ° C. for 16 hours in a light place (8 hours in a dark place), after 1 week, an agar medium containing the chemical substance Ic3-1 (final concentrations of 6 ppm, 2 ppm, 0.6 ppm, 0.2 ppm) The formation of adventitious roots was confirmed from the hypocotyl portion inserted in all test sections) and the hypocotyl portion inserted in the agar medium containing the chemical substance Ic7-1 (final concentrations of 6 ppm and 2 ppm test sections). In the control group to which DMSO was added instead of the chemical DMSO solution, adventitious root formation could not be confirmed from the hypocotyl part. Hypocotyl inserted into agar medium containing chemical substance Ic3-1 with final concentration of 0.6 ppm, hypocotyl inserted into agar medium containing chemical substance Ic7-1 with final concentration of 2 ppm, and embryo inserted into agar medium in control group The test results in the case of the shaft are shown in FIG.

Example 14 (Measurement of root growth promoting activity of cytokinin signal transduction inhibitor using rice)
For the chemical substance Ic3-3, root elongation promoting activity was measured using rice (Oriza sativa japonica Nipponbare).
First, an acetone chemical solution containing a predetermined concentration of a chemical substance was prepared, and then diluted 100 times with distilled water to prepare a chemical solution having a concentration of 10 ppm (containing 1% acetone). Three seeds that had been germinated by soaking in water for 2 days were sown in 288-well plug trays, and 500 μl of the chemical solution was perfused into the soil. After covering the soil, the plug tray was placed in a plastic bag and left for 3 days in an artificial weather room (30 ° C., dark place). After removing the plastic bag from the plug tray, it was left to stand for 4 days under light conditions of light / dark place = 16h / 8h while constantly watering the bottom. After growing rice seeds in this way, grown rice was obtained. The obtained rice roots were washed, and the total root length was analyzed using a root length measurement image analyzer WinRHIZO (manufactured by Regent Instruments). In the group treated with chemical substance Ic3-3, the root elongation was significantly promoted compared to the control group (UTC) using only acetone in the soil irrigation treatment. A photograph is shown in FIG. In addition, as a result of analysis using a root length measurement image analyzer, an increase in total root length was observed in the chemical Ic3-3 treatment group. The results are shown in FIG.

Example 15 (CRE1 cDNA cloning Part 2)
Using the expression plasmid p415CYC-CRE1 obtained in Example 5 as a template, PCR is performed using the DNA shown in SEQ ID NO: 7 and the DNA shown in SEQ ID NO: 8 as primers, and a DNA having the base sequence shown in SEQ ID NO: 2 Was amplified. Details are described below.
The PCR reaction was carried out using KOD Plus DNA Polymerase (TOYOBO) at 94 ° C for 2 minutes, followed by 30 cycles of 94 ° C for 15 seconds, 58 ° C for 30 seconds, and 68 ° C for 3 minutes and 30 seconds. Performed under amplification conditions. The PCR reaction solution (50 μl) was prepared by adding a reaction composition such as dNTP to 500 ng of plasmid p415CYC-CRE1 and 100 ng of each primer DNA according to the kit instructions.
The target DNA fragment thus amplified was cloned into the pCR-Blunt II-TOPO vector (Invitrogen) according to the instructions attached to the kit. At this time, the target DNA fragment was inserted into the pCR-Blunt II-TOPO vector so that the base sequence represented by SEQ ID NO: 7 was close to the T7 promoter and the base sequence represented by SEQ ID NO: 8 was close to the Sp6 promoter. It was confirmed using an automatic base sequencer that the base sequence of the inserted DNA fragment was in the correct orientation and that the base sequence was the base sequence shown in SEQ ID NO: 2.

Example 16 (Construction of CRE1 expression plasmid)
P425GPD (available from Munberg et al. Gene: 156 119-122 (1995), ATCC library (No. 87359)), a yeast expression vector, was cleaved with the restriction enzyme BamHI and then used with T4 DNA Ligase. The DNA fragment obtained in Example 15 (DNA having the base sequence shown in SEQ ID NO: 2) was ligated to the GPD promoter sequence of the expression vector p425GPD, so that the target protein was expressed in yeast. It was confirmed by using an automatic base sequencer that the inserted DNA fragment had the correct base sequence and the base sequence was the base sequence represented by SEQ ID NO: 2, and the expression plasmid p425GPD-CRE1 was obtained. .

Example 17 (Preparation of transformed cell TM182-p425GPD-CRE1)
The expression plasmid obtained in Example 16 was used to transform TM182 (sln1Δ) (Maeda T et al. Nature: 369 242-245 (1994)), which is a Sln1 gene-deficient strain. Transformation was performed using S. cerevisiae Direct Transformation Kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.) according to the attached manual. In the obtained transformed cells, transformed cells TM182-p425GPD-CRE1 were obtained by selecting transformed yeasts that grow on DOLU + Gal medium by utilizing the disappearance of leucine auxotrophy.
Further, transformed cells TM182-p425GPD were obtained in the same manner using the yeast expression vector p425GPD.

Example 18 (Preparation of membrane protein fraction containing CRE1)
After transforming the transformed cells TM182-p425GPD-CRE1 obtained in Example 17 with glass beads, a membrane protein fraction containing CRE1 was prepared by an ultracentrifuge. Details are described below.
The transformed cell TM182-p425GPD-CRE1 obtained in Example 17 was inoculated into 100 ml of DOLU + Gal medium and cultured at 30 ° C. for 16 hours to obtain a culture solution having an OD600 = 1.4. The culture solution was dispensed into a 50 ml centrifuge tube and centrifuged at 4 ° C. and 7,400 × g for 5 minutes to recover the cells of the transformed cell TM182-p425GPD-CRE1. The cells were resuspended in phosphate buffer (adjusted to pH 7.0 by mixing 50 mM aqueous sodium dihydrogen phosphate solution and 50 mM aqueous disodium hydrogen phosphate solution at a ratio of 4 to 6) cooled to 4 ° C. The cells were dispensed into 2 ml tubes and centrifuged at 1,000 × g for 5 minutes at 4 ° C. to recover the cells of transformed cells TM182-p425GPD-CRE1. The cells were resuspended in a phosphate buffer cooled to 4 ° C., and centrifuged at 1,000 × g for 5 minutes at 4 ° C. to recover transformed cells TM182-p425GPD-CRE1. This bacterial cell is 3 times the amount of phosphate buffer containing DTT (dithiothreitol) and PMSF (phenylmethylsulfonyl fluoride) (5 mM DTT and 0.5 mM PMSF added to the aforementioned phosphate buffer) Then, 250 μl of glass beads (diameter: 0.25 to 0.5 mm) were added in advance, and 200 μl each was dispensed into a 1.5 ml tube that had been cooled to 4 ° C. These 1.5 ml tubes were stirred at the maximum output of a microtube mixer (TOMY, MT-360) for 30 seconds and then cooled in ice for 1 minute twice. Further, these 1.5 ml tubes were stirred for 30 seconds with an output (SPEED METER) 2000 of a multi-bead shocker (Yasui Kikai Co., Ltd., MB-200), and then cooled in ice for 1 minute three times. Next, these 1.5 ml tubes were centrifuged at 1,500 × g for 10 minutes at 4 ° C. to recover the supernatant. The supernatant was transferred to a new 1.5 ml tube and centrifuged at 10,000 × g for 3 minutes at 4 ° C. to recover the supernatant. This supernatant was centrifuged at 100,000 × g for 1 hour at 4 ° C. using an ultracentrifuge to collect the precipitate. This precipitate was dissolved in a phosphate buffer containing 1% sucrose monocaprate (1% sucrose monocaprate added to the aforementioned phosphate buffer), and the membrane protein fraction of transformed cells TM182-p425GPD-CRE1 did.
Further, a membrane protein fraction of the transformed cell TM182-p425GPD was prepared in the same manner.

Example 19 (Method for investigating that a chemical substance inhibits the binding of cytokinin to a cytokinin receptor)
Using the membrane protein fraction of the transformed cell TM182-p425GPD-CRE1 obtained in Example 18 and cytokinin labeled with tritium, a radioisotope of hydrogen, and having high radioactivity, the test substance is cytokinin. Has been shown to inhibit binding to the cytokinin receptor. Details are described below.
Cytokinin labeled with tritium, a radioactive isotope of hydrogen, has high radioactivity, [3H] N-6- (isopent-2-enyl) Adenine (hereinafter referred to as Radiolabeled 2IP) manufactured by Amersham Biosciences. Used). Specific radioactivity is 74.0GBq / mmol, and radioactivity concentration is 37.0MBq / ml.
First, the membrane protein fraction of the transformed cell TM182-p415CYC1 was diluted with phosphate buffer so that the membrane protein fraction would be 100 μg and set concentration, and 1 μl of the test substance diluted with DMSO so that the concentration would be set in phosphate buffer. To prepare 100 μl of a reaction solution. Next, this reaction solution was allowed to stand in ice for 1 hour and then filtered through a glass filter GF / B (manufactured by Whatman) to collect a membrane protein fraction of the transformed cell TM182-p425GPD-CRE1. The glass filter was immersed in a liquid scintillation cocktail Ultima Gold (manufactured by Perkin Elmer), and the radioactivity was measured with a liquid scintillation counter. Moreover, the same test was performed using the membrane protein fraction of the transformed cell TM182-p425GPD as a control. In order to eliminate the influence of radioactivity due to non-specific binding of radiolabeled 2IP, the radioactivity value obtained in the test using the membrane protein fraction of the transformed cell TM182-p425GPD-CRE1 was used. The radioactivity value obtained in the test using the membrane protein fraction of p425GPD was subtracted. The test results are shown in FIG.
The radioactivity decreased when the test substance was transzeatin or Ic3-4, compared to the absence of the test substance (adding only DMSO used as the solvent for the test substance) and the test substance being abscisic acid.

Example 20 (Evaluation of Rice Seedling Promoting Activity of Cytokinin Signaling Inhibitory Substance Treated with Seeds in Flooded Direct Sowing Test)
About the chemical substance Ic3-3 selected in Example 7 (inhibiting intracellular signal transduction from plant-derived cytokinin receptors possessed by cells), rice (Oryza sativa japonica Nipponbare) was treated with seeds and subjected to direct sowing conditions. The rice seedling-promoting activity of the chemical substance was evaluated by growing the seedlings and examining the seedling establishment rate.
First, 5% (v / v) Color Coat Red (BECKER UNDERWOOD), 5% (v / v) CF-Clear (BECKER UNDERWOOD), 0.42% (v / v) Maxim-XL (Syngenta) A blank slurry solution was prepared. Dissolve 6.25mg, 12.5mg, 25mg and 50mg of chemical substance Ic3-3 per 1.3ml Blank slurry solution, respectively, and use the solution with Hege11 seed treatment equipment (Hans-Ulrich Hege) per 50g seed Treated at a rate of 1.3 ml of chemical-slurry solution (corresponding to 0.125 mg, 0.25 mg, 0.5 mg and 1 mg / g seed, respectively).
Next, a concrete pot (50 cm × 50 cm) installed outdoors was set to a flooding depth of 5 cm, and 50 seeds were sprayed per pot. As a result of investigating the number of seedlings whose leaf tips appeared on the water surface on the 23rd day after sowing, the treatment concentration of 0.125 to 1 mg / g seed increased the seedling establishment rate compared to the Blank slurry treatment group. Admitted. Table 13 shows the test results (average values) for each treatment group (4 repetitions).
[Seedling establishment rate (%)] = [number of seedlings with leaf tips appearing on the water surface] / [number of seeding] × 100

Example 21 (Evaluation of rice tillering promoting activity of seed-treated cytokinin signal transduction inhibitor in dry rice direct sowing test)
About the chemical substance Ic3-3 selected in Example 7 (inhibiting intracellular signal transduction from plant-derived cytokinin receptors possessed by cells), rice (Oryza sativa japonica Nipponbare) was treated with seeds under direct seeding conditions in dry rice fields. By growing and examining the number of tillers, the activity of promoting the tillering of chemicals to rice was evaluated.
First, 5% (v / v) Color Coat Red (BECKER UNDERWOOD), 5% (v / v) CF-Clear (BECKER UNDERWOOD), 0.42% (v / v) Maxim-XL (Syngenta) A blank slurry solution was prepared. 11.5 mg of chemical substance Ic3-3 was dissolved per 1.3 ml of Blank slurry solution, and the solution was treated at a rate of 1.3 ml per 50 g seed using a Hege11 seed treatment device (manufactured by Hans-Ulrich Hege) ( Equivalent to 0.25 mg / g seed).
Next, seeds treated with seeds were sown in 1 / 5000a Wagner pots, 20 grains per pot at a seeding depth of 1 cm, and cultivated in a greenhouse. On the 10th day after sowing, the inundation depth was set at 5 cm and the cultivation was continued. As a result of investigating the number of tillers on the 30th day after sowing, an increase in the number of tillers per strain was observed as compared with the Blank slurry treatment group. FIG. 11 shows the test results (average value) of each treatment section (3 repetitions).

The composition of the medium used in the present invention is described below.
(A) DOLU + Glu medium
Bacto-yeast nitrogen base without amino acids 6.7g
Glucose 20g
SC-HIS-LEU-URA (Q-BIOgene) 1.66g
Histidine 0.076g
Distilled water 1000ml
(B) DOLU + Gal medium
Bacto-yeast nitrogen base without amino acids 6.7g
Glucose 20g
SC-HIS-LEU-URA (Q-BIOgene) 1.66g
Histidine 0.076g
Distilled water 1000ml

  According to the present invention, an agent for controlling plant growth or differentiation, and a method for searching for a chemical substance having a useful biological activity in which a target is clarified, that is, for the purpose of chemically adjusting a target site, It is possible to provide a method for screening a chemical substance with activity against a target.

FIG. 1 is a graph showing the results of assaying the concentration responsiveness of chemical substances that inhibit intracellular signal transduction from plant-derived cytokinin receptors possessed by cells in Example 8. The data line in the figure represents a concentration-responsive growth inhibition curve in which the X axis is the concentration of the tested chemical substance and the Y axis is the relative growth degree. The left figure in the figure (three sub-diagrams in the left vertical column) shows the results of the test system using the transformed cell TM182-CRE1, and the right figure (three sub-diagrams in the right vertical column). (Figure) shows the results of a test system using transformed cells TM182-p415CYC1). Further, the upper, middle and lower diagrams in the figure show the results in the test system using the chemical substance Ic7-1, the chemical substance Ic3-1 and the chemical substance Ic3-3 from the top. FIG. 2 is a graph showing the results of evaluating the root growth promoting activity of a cytokinin signal transduction inhibitor using lettuce in Example 10. The data line in the figure represents a concentration-responsive growth promotion curve with the X axis as the concentration of the tested chemical substance (chemical substance Ic3-1) and the Y axis as the root growth rate (%). . FIG. 3 is a diagram showing the results of evaluating the root growth promoting activity of a cytokinin signal transduction inhibitor using lettuce in Example 10. The data line in the figure represents a concentration-responsive growth promotion curve with the X axis as the concentration of the tested chemical substance (chemical substance Ic7-1) and the Y axis as the root growth rate (%). . FIG. 4 shows the results of evaluating the root growth promoting activity of a cytokinin signal transduction inhibitor using rice in Example 11. The data line in the figure represents a concentration-responsive growth promotion curve with the X axis as the concentration of the tested chemical substance (chemical substance Ic3-1) and the Y axis as the root growth rate (%). . FIG. 5 shows the results of evaluating the root growth promoting activity of a cytokinin signal transduction inhibitor using rice in Example 11. The data line in the figure represents a concentration-responsive growth promotion curve with the X axis as the concentration of the tested chemical substance (chemical substance Ic7-1) and the Y axis as the root growth rate (%). . FIG. 6 is a graph showing the results of evaluating the root growth promoting activity of a cytokinin signal transduction inhibitory substance by rice seed treatment in Example 12. In the figure, “UTC” indicates the result in the control group using only acetone in the seed treatment, and “IAA” indicates the result in the test system using the auxin compound IAA. . FIG. 7 is a graph showing the results of measuring adventitious root formation activity of cytokinin signal transduction inhibitor using the hypocotyl of Arabidopsis thaliana in order to evaluate plant differentiation promoting activity in Example 13. The “control group” in the figure shows the result of the control group described in Example 13 on the agar medium. FIG. 8 is a graph showing the results of measuring the root growth promoting activity of a cytokinin signal transduction inhibitor using rice in Example 14. “UTC” in the figure indicates the result in the control group using only acetone in the soil irrigation treatment. FIG. 9 is a diagram showing the results of analyzing the root growth promoting activity of a cytokinin signal transduction inhibitor using rice in Example 14 using a root length measurement image analyzer. In the data line in the figure, the X-axis represents the concentration of the chemical substance tested (chemical substance Ic3-3), and the Y-axis represents the total root length for each root diameter (total root length). “UTC” in the figure indicates the result in the control group using only acetone in the soil irrigation treatment. The numerical value (L) in the legend indicates the root diameter (mm). FIG. 10 shows the test results obtained by verifying that the test substance inhibits the binding of cytokinin to the cytokinin receptor in Example 19. “DMSO only” in the figure is a control group not containing a test substance, and is obtained by adding only DMSO used as a solvent for the test substance instead of the DMSO solution of the test substance. "T-Zeatin" is a test substance added with transzeatin, "Ic3-4" is a test substance added with Ic3-4, and "ABA" is a test substance added with abscisic acid It is. FIG. 11 is a diagram showing the results of evaluation of rice tillering promoting activity of seed-treated cytokinin signal transduction inhibitor in the dry seeding direct seeding test in Example 21. In the figure, “Ic3-3” means that the test substance Ic3-3 is used as a blank slurry solution for seed treatment, and “Blank slurry treatment” means that a blank slurry is used for seed treatment instead of the Ic3-3 solution. It is the figure which showed the number of tillers per share when there was.

SEQ ID NO: 3
Oligonucleotide primer designed for PCR SEQ ID NO: 4
Oligonucleotide primer designed for PCR SEQ ID NO: 5
Oligonucleotide primer designed for PCR SEQ ID NO: 6
Oligonucleotide primer designed for PCR SEQ ID NO: 7
Oligonucleotide primer designed for PCR SEQ ID NO: 8
Oligonucleotide primers designed for PCR

Claims (41)

An agent that controls plant growth or differentiation and has an activity of inhibiting intracellular signal transduction from a plant-derived cytokinin receptor possessed by a cell. The agent according to claim 1, wherein the agent that controls plant growth or differentiation is a plant growth regulator. The agent according to claim 1, wherein the agent that controls plant growth or differentiation is an agent that controls plant growth. The agent according to claim 1, wherein the agent that controls plant growth or differentiation is an agent that controls plant cell differentiation. The agent according to claim 3, wherein the agent for controlling the growth of a plant body is an agent for controlling the growth of a bud of a plant. 6. The agent according to claim 5, wherein the control of plant bud growth is suppression of bud buds and the like. 6. The agent according to claim 5, wherein the control of plant bud growth is flower bud suppression or the like. 4. The agent according to claim 3, wherein the agent that controls the growth of the plant body is an agent that promotes plant seedling establishment. The agent according to claim 3, wherein the agent that controls the growth of the plant body is an agent that promotes tillering of the plant. The agent according to claim 3, wherein the agent that controls the growth of a plant body is an agent that promotes the growth of plant roots. The plant-derived cytokinin receptor which a cell has is a cytokinin receptor selected from the following group A, The chemical | medical agent as described in any one of Claims 1-10 characterized by the above-mentioned.
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and Protein having an activity of functioning as a delegate receptor A cell having a cytokinin receptor selected from the following group A having an activity of inhibiting intracellular signal transduction from a plant-derived cytokinin receptor possessed by a cell; and a substance having an agonist activity with respect to the cytokinin receptor The agent according to any one of claims 1 to 10, which has an activity of inhibiting intracellular signal transmission from the cytokinin receptor in the contact system.
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and Protein having an activity of functioning as a delegate receptor A plant growth regulator comprising, as an active ingredient, a chemical substance that inhibits intracellular signal transduction from a plant-derived cytokinin receptor of a cell or an agriculturally acceptable salt thereof. In the contact system between the chemical substance, a cell having a cytokinin receptor selected from the following group A, a substance having agonist activity for the cytokinin receptor, and the chemical substance, The plant growth regulator according to claim 13, which is a chemical substance having an activity of inhibiting intracellular signal transduction.
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and Protein having an activity of functioning as a delegate receptor 15. The plant growth regulator according to claim 14, wherein the substance having agonist activity for a cytokinin receptor is transzeatin. Compared to the case where the chemical substance is not present in a contact system of a cell having a cytokinin receptor selected from the following group A, 0.6 ppm of transzeatin and 2 ppm of the chemical substance. The plant growth regulator according to claim 13, which is a chemical substance having an activity of inhibiting intracellular signal transduction from the cytokinin receptor.
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and Protein having an activity of functioning as a delegate receptor Compared to the case where the chemical substance is not present in a contact system of a cell having a cytokinin receptor selected from the following group A, 0.6 ppm of transzeatin and 2 ppm of the chemical substance. The plant growth regulator according to claim 13, which is a chemical substance having an activity of inhibiting 90% or more of intracellular signal transduction from the cytokinin receptor.
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and Protein having an activity of functioning as a delegate receptor A method for searching for a chemical substance capable of promoting the growth of plant roots,
(1) Intracellular from the cytokinin receptor in a contact system of a cell having a cytokinin receptor selected from the following group A, a substance having agonist activity with respect to the cytokinin receptor, and a test substance A first step of measuring the amount of signal transmission, and (2) obtained by comparing the amount of intracellular signal transmission measured in the first step with the amount of intracellular signal transmission in the absence of the chemical substance. A second step of selecting a chemical having the ability to promote plant root growth based on the difference
A method characterized by comprising:
<Group A>
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) an amino acid sequence represented by SEQ ID NO: 1, comprising an amino acid sequence in which one or more amino acids have been deleted, added or substituted, and a cytokinin receptor A protein having an activity that functions as (c) a protein comprising an amino acid sequence having 45% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and having an activity that functions as a cytokinin receptor (d) SEQ ID NO: 2 A protein comprising the amino acid sequence encoded by the nucleotide sequence represented by (e) a polynucleotide that hybridizes under stringent conditions with a polynucleotide having complementarity to the polynucleotide having the nucleotide sequence represented by SEQ ID NO: 2 Consisting of the amino acid sequence encoded by and Protein having an activity of functioning as a delegate receptor The search method according to claim 18, wherein the cell having a cytokinin receptor is a transformed cell into which a polynucleotide comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 has been introduced. The search method according to claim 18, wherein the cell having a cytokinin receptor is a transformed yeast cell into which a polynucleotide comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 has been introduced. 21. The search method according to claim 18, 19 or 20, wherein the substance having agonist activity for a cytokinin receptor is transzeatin. A plant growth regulator comprising a chemical substance selected by the search method according to claim 18, 19, 20, or 21 or an agriculturally acceptable salt thereof as an active ingredient. A method for regulating plant growth, comprising applying an effective amount of the plant growth regulator according to claim 13, 14, 15, 16, 17, or 22 to a plant or a habitat thereof. A chemical substance having the ability to promote the growth of roots of a plant identified by the search method according to claim 18, 19, 20, or 21, and the chemical substance having the ability to promote the growth of roots of the identified plant and the plant A method for regulating plant growth, comprising contacting As an active ingredient, the general formula (I)

(In the formula, R and X are the same or different hydrocarbon groups which may be substituted, a group represented by NR ¹ R ² , a group represented by OR ³ and represented by S (O) _m R ⁴ A group, a nitro group or a halogen atom,
Here, R ¹ represents a hydrogen atom or an optionally substituted hydrocarbon group,
R ² is a hydrogen atom, an optionally substituted hydrocarbon group, a group represented by NR ⁵ R ⁶ (wherein R ⁵ and R ⁶ are the same or different, a hydrogen atom or an optionally substituted C 1-6 alkyl Or a group represented by OR ⁷ (wherein R ⁷ represents a hydrogen atom or an optionally substituted C 1-6 alkyl group), or R ¹ and R ² are adjacent to a nitrogen atom Together with a cyclic amino group which may be substituted,
R ³ and R ⁴ each represents an optionally substituted hydrocarbon group,
l represents an integer of 0 to 1,
m represents an integer of 0 to 2,
n represents an integer of 0 to 4,
when n is 2 or more, Xs may be the same or different;
Ar represents an optionally substituted aryl group or an optionally substituted heteroaryl group), or an agriculturally acceptable salt thereof. 26. The plant growth regulator according to claim 25, wherein l is 1 and R is an optionally substituted hydrocarbon group. 26. The plant growth regulator according to claim 25, wherein l is 1, and R is a C1-3 alkyl group optionally substituted with a halogen atom or an oxo group. 27. The plant growth regulator according to claim 26, wherein the hydrocarbon group which may be substituted is a C1-3 alkyl group which may be substituted with a halogen atom or an oxo group. The plant growth regulator according to claim 25, wherein l is 1 and R is a group represented by NR ¹ R ² . R ¹ represents a hydrogen atom or a C1-3 alkyl group, R ² represents a hydrogen atom, an amino group, a C1-3 alkylamino group, a diC1-3 alkylamino group, an amidino group, a C1-3 alkoxy group, a phenyl group, C1-3 acyl group, C1-6 alkyl group, C3-6 alkenyl group or C3-6 alkynyl group, wherein the phenyl group is substituted with 1 to 3 identical or different C1-3 alkyl groups. The phenyl group, acyl group, alkyl group, alkenyl group and alkynyl group may be a halogen atom, hydroxyl group, C1-3 alkoxy group, hydroxy C1-3 alkoxy group, carboxyl group, C1-3 alkoxycarbonyl group, 1 to 3 identical groups selected from carbamoyl group, amino group, C1-3 alkylamino group, di-C1-3 alkylamino group, mercapto group, C1-3 acylthio group, cyano group, furyl group and tetrahydrofuryl group if 30. The plant growth regulator according to claim 29, which may be substituted with different substituents, or R ¹ and R ² together with the adjacent nitrogen atom represent a pyrrolidino group, piperidino group or morpholino group. R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, a formyl group, a C 1-6 alkyl group, a C 3-6 alkenyl group or a C 3-6 alkynyl group, wherein the alkyl group, alkenyl group and alkynyl group are 30. The plant growth regulator according to claim 29, which may be substituted with a substituent selected from a hydroxyl group, a methoxy group, a methoxycarbonyl group, an ethoxycarbonyl group, a cyano group and a furyl group. The plant growth regulator according to claim 25, wherein l is 1 and R is a group represented by OR ³ . R ³ is a plant growth regulator according to claim 32 wherein the optionally substituted C1-3 alkyl group with an amino group. The plant growth regulator according to claim 25, wherein l is 1 and R is a group represented by S (O) _m R ⁴ . The plant growth regulator according to claim 34, wherein R ⁴ is a C1-3 alkyl group which may be substituted with an amino group or a hydroxyl group, and m is 0. 26. The plant growth regulator according to claim 25, wherein l is 1 and R is a halogen atom. The plant growth regulator according to claim 36, wherein the halogen atom is a chlorine atom. 38. The plant according to any one of claims 25 to 37, wherein n is 1 to 2, and X is a C1-3 alkyl group, a C1-3 alkoxy group, a C1-3 haloalkyl group, a cyano group, a halogen atom or a nitro group. Growth regulator. 39. The plant growth regulator according to claim 38, wherein X is a chlorine atom, a bromine atom or a nitro group, and the substitution position of X is at the 6-position and / or 8-position. The plant growth regulator according to any one of claims 25 to 39, wherein Ar is a phenyl group which may be substituted with a halogen atom or a C1-3 alkyl group. A method for regulating plant growth, comprising applying an effective amount of the plant growth regulator according to any one of claims 25 to 40 to a plant or its habitat.

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