CN112772662B

CN112772662B - Application of small molecular compound in promoting plant growth and development

Info

Publication number: CN112772662B
Application number: CN202110040713.0A
Authority: CN
Inventors: 郭红卫; 谢银鹏; 姜凯; 朱颖
Original assignee: Southwest University of Science and Technology
Current assignee: Southwest University of Science and Technology
Priority date: 2021-01-13
Filing date: 2021-01-13
Publication date: 2022-02-15
Anticipated expiration: 2041-01-13
Also published as: CN112772662A

Abstract

The invention relates to application of a small molecular compound in promoting plant growth and development. The structural formula of the micromolecule compound is shown as

The small molecular compound can inhibit the amino acidification of plant endogenous auxin by inhibiting the activity of plant GH3 protein, so that the auxin accumulation in the plant body presents original tissue or organ specificity and is beneficial to the growth and development of plants.

Description

Application of small molecular compound in promoting plant growth and development

Technical Field

The invention relates to the technical field of biology, in particular to application of an inhibitor of GH3 protein in regulation and control of plant growth and development.

Background

Auxins are a class of endogenous hormones containing an unsaturated aromatic ring and an acetic acid side chain, and their chemical nature is indoleacetic acid. Indole-3-acetic acid (IAA) is the predominant form of auxin in plants and regulates many aspects of plant growth and development, such as plant cell division and elongation, plant tropism and apical dominance, lateral root formation and adventitious root formation, leaf abscission and fruit development. The regulation of auxin is mainly dependent on the concentration of auxin, and plants are adversely affected by too high or too low auxin concentrations. Therefore, the research on the regulation and control of the auxin concentration is of great significance to the agricultural production application.

After 20 years of research, scientists have made significant research results on the synthesis, transport, signal transduction and the like of indole-3-acetic acid, and developed a plurality of efficient, specific and practical growth regulators through chemical genetics. For example, Kyn and PPBo inhibit auxin synthase TAA1 and YUC, respectively, which in combination can significantly reduce the content of plant endogenous auxin; NPA and NOA inhibit the transport of auxin and break the polar transport of auxin; auxinole as a ghrelin receptor antagonist can inhibit ghrelin signaling and the like. Existing products based on regulating auxin are not tissue or organ specific and auxin is affected in all tissues or organs in plants using these products. However, the growth of each tissue or organ requires an inconsistent concentration of auxin, and the current products do not balance the auxin requirements of each tissue or organ. For example, the growth of main roots, lateral roots and adventitious roots is too high in concentration, the inhibition on the main roots is too strong, the concentration is too low, and the main roots and the adventitious roots cannot be obviously promoted.

Disclosure of Invention

Based on the above, the invention discovers a small molecular compound which has tissue or organ specificity on the growth and development of plants, can balance the requirements of each tissue and organ on auxin by utilizing the distribution mechanism of the endogenous auxin in the growth and development of plants, and solves the problem that the current auxin-based product cannot realize the tissue or organ specificity.

Based on the small molecular compound, the invention provides an application of the small molecular compound in promoting plant growth and development, wherein the structural formula of the small molecular compound is as follows:

the small molecule compound reduces the amino acidification of auxin (endogenous auxin) produced by the plant by inhibiting the activity of the plant GH3 protein, so that the accumulation of the auxin in various tissues or organs of the plant presents self specificity. That is, after the treatment with the small molecule compound, the distribution of auxin in the plant body is still uneven, and the tissue difference still exists. Therefore, the small molecular compound can realize accurate regulation of auxin in plants and is beneficial to the growth and development of the plants.

Furthermore, it was confirmed that the small molecule compound can promote the generation of adventitious roots, promote lateral root generation, promote root hair elongation, and promote hypocotyl elongation in plants, and can produce a phenotype (for example, promote growth of hypocotyl, promote adventitious root generation) superior to that of IAA addition at a low concentration (for example, 0.3 μ M).

In one embodiment, the plant is a dicot or monocot.

In one embodiment, the plant is arabidopsis, tobacco or tomato; and/or, the monocot is rice.

In one embodiment, the method comprises the following steps:

adding the small molecule compound in the process of culturing the plant.

In one embodiment, the small molecule compound is used at a concentration of 0.1 μ M to 5 μ M.

In one embodiment, the small molecule compound is used at a concentration of 0.8 μ M to 5 μ M.

In one embodiment, the plant is grown using a substrate that is

The culture medium or MS culture medium is obtained by adding the small molecular compound.

A method for culturing a plant, the method comprising adding a small molecule compound of the formula:

the application of a small molecule compound in preparing GH3 protein inhibitors, wherein the structure of the small molecule compound is as follows:

an GH3 protein inhibitor, wherein the active ingredient of the GH3 protein inhibitor comprises a small molecule compound shown as a structural formula:

in one embodiment, the GH3 protein inhibitor further comprises an adjuvant selected from at least one of a cosolvent and a preservative.

In one embodiment, the co-solvent is DMSO.

A plant culture medium comprising a basal medium and a small molecule compound of the formula:

in one embodiment, the basal medium is

Medium or MS medium.

In one embodiment, the concentration of the small molecule compound in the plant culture medium is 0.1 μ M to 5 μ M.

In one embodiment, the concentration of the small molecule compound in the plant culture medium is 0.8 μ M to 3 μ M.

Drawings

FIGS. 1 to 4 are the plant phenotype, the statistical result of the length of the main root, the statistical result of the length of the hypocotyl and the statistical result of the number of adventitious roots of the Arabidopsis seedlings of the C9 group, the IAA group and the blank control group in example 1 after 6 days of culture;

FIGS. 5-6 are statistics of plant phenotype and lateral root number after 3 days of growth of Arabidopsis seedlings from group C9, IAA and blank control in example 2 after transfer to treatment plates;

FIGS. 7 and 8 are statistics of root images and root hair lengths of Arabidopsis seedlings of group C9, IAA and blank control in example 3 after being transferred to treatment plates and grown for 48 hours;

FIG. 9 is a fluorescent image of DR5-GFP of Arabidopsis seedlings from group C9 and the blank control in example 4 after culture for 1 day after transfer to a treatment plate;

FIG. 10 shows DR5-GUS expression of Arabidopsis seedlings in C9 group and blank control group in example 4 after culturing for 2 days after transferring to a treatment plate;

FIGS. 11 to 12 are the statistics of plant phenotype and main root length of Arabidopsis seedlings of Kyn + PPBo group, Kyn + PPBo + C9 group, Kyn + PPBo + IAA group and blank control group in example 5 after 6 days of culture;

FIGS. 13 to 14 are statistics of plant phenotype and main root length of Arabidopsis seedlings in IAA group, C9 group, C9+ IAA group and blank control group in example 6 after 6 days of culture;

FIG. 15 shows the effect of 50. mu. M C9 on the enzymatic activity of GH3.5 protein in example 7;

FIG. 16 shows the effect of 50. mu. M C9 on the enzymatic activity of GH3.17 protein in example 7;

FIGS. 17 to 20 are the statistics of plant phenotype, main root length, hypocotyl length and lateral root number of tobacco green seedlings of the present formula in group C9 and blank control group in example 8 after 13 days of culture.

FIGS. 21 to 23 show the phenotype, the length of adventitious roots and the number of adventitious roots of tomato plants in the C9 group, IAA group and blank control group in example 9 after 13 days of culture.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Indole-3-acetic acid (IAA) is an important auxin and plays an important role in the growth and development of plants. Indole-3-acetic acid modulates activity in 3 different ways: homeostasis, polar transport and auxin response. Binding and degradation of molecules such as sucrose and amino acids to indole-3-acetic acid helps to maintain the indole-3-acetic acid homeostasis in plants. When the concentration of the indole-3-acetic acid is increased, a part of the active indole-3-acetic acid is connected with the amino acid to form an indole-3-acetic acid amino acidification product which is stored or degraded through a degradation pathway; when the concentration of the indole-3-acetic acid is reduced, the indole-3-acetic acid amino acidification product can be hydrolyzed into active components by proteolytic enzyme.

The plant GH3 gene family is one of auxin early response genes, and auxin can induce the rapid and transient expression of most genes of the GH3 gene family. The plant GH3 protein has auxin amino-acidification activity. Among them, most of 19 GH3 proteins of arabidopsis thaliana have aminoacid synthase activity of one or more auxins. For example, JARl (GH3.11) binds an amino acid to jasmonic acid, while other GH3 proteins are mainly aminated with indole-3-acetic acid as a substrate.

The research finds that the small molecular compound shown as the following formula has good activity of inhibiting GH3 protein, and through inhibiting the activity of GH3 protein, the amino acid of auxin synthesized by the plant body can be reduced, the auxin in the plant body can be accurately amplified, and the growth and development of the plant can be promoted:

the name of the small molecule compound is: n- [4- [ (6-pyrazol-1-ylpyridazin-3-yl) amino ] phenyl ] -3- (trifloromethyl) benzamide, i.e., N- [4- [ (6-pyrazol-1-ylpyridazin-3-yl) amino ] phenyl ] -3- (trifluoromethyl) benzamide.

Therefore, one embodiment of the present invention provides an application of the above small molecule compound in promoting plant growth and development. The small molecular compound reduces the amino acidification of auxin in plants to promote the growth and development of the plants.

One embodiment of the present invention provides a method for cultivating a plant. Specifically, the plant culture method comprises the step of adding the small molecule compound in the plant culture process. More specifically, the culture method comprises the steps of: and (2) cultivating plants by adopting a matrix, wherein the small molecular compound is added into the matrix.

Specifically, the substrate provides nutrients for the growth and development of plants. Optionally, the substrate comprises a basal medium and the small molecule compound described above. In one embodiment, the basal medium comprises

Solid culture media or

And (4) a culture medium. In an alternative specific example, the basal medium is a solid medium. In another alternative embodiment, the basal medium is a liquid medium. Of course, in other embodiments, the basic medium is not limited to the above list, but may be other media that can be used to cultivate plants, such as soil. The matrix is obtained by adding small molecular compounds based on a basic culture medium.

In this embodiment, the plant is a dicot. Alternatively, the dicot is arabidopsis, tobacco or tomato. Of course, in other embodiments, the dicot is not limited to the above, but may be other dicots. In addition, the GH3 gene family in monocotyledons (such as rice) is highly conserved with the GH3 sequence of arabidopsis thaliana, and a phenotype of rice with increased endogenous auxin, such as suppression of the main root, is also observed after applying the small molecule compound to rice. Therefore, the small molecule compound is also suitable for monocotyledons.

In one embodiment, the small molecule compound is used at a concentration of 0.1. mu.M to 5. mu.M. Herein "use concentration" refers to the concentration of the small molecule compound acting on the plant. In an alternative embodiment, the concentration of the small molecule compound in the matrix is between 0.1 μ M and 5 μ M. Alternatively, the concentration of the small molecule compound in the matrix is 0.1. mu.M, 0.5. mu.M, 1. mu.M, 1.5. mu.M, 2. mu.M, 2.5. mu.M, 3. mu.M, 4. mu.M, or 5. mu.M. Further, the concentration of the small molecule compound in the matrix is 0.5 to 3. mu.M. Further, the concentration of the small molecule compound in the matrix is 0.8 to 3. mu.M.

The small molecule compound is verified to be 0.8-3 mu M in the substrate, so that the elongation of the main root of a seven-day-old arabidopsis green seedling plant can be inhibited, the generation of adventitious roots is promoted, the generation of lateral roots is promoted, the elongation of root hairs is promoted, and the elongation of a hypocotyl is promoted, namely, the small molecule can generate a plurality of phenotypes caused by the increase of endogenous auxin under very low concentration. In addition, the small molecule compound can also enhance the expression of DR5-GFP or DR5-GUS through verification, and the fact that the small molecule compound can activate an auxin signal transduction pathway is also shown.

In one embodiment, the plant is Arabidopsis, tobacco or tomato, and the concentration of the small molecule compound in the substrate is 0.1. mu.M-5. mu.M. Further, the concentration of the small molecule compound in the matrix is 0.5 to 3. mu.M. Further, the concentration of the small molecule compound in the matrix is 0.8 to 3. mu.M.

It will, of course, be appreciated that the light required for growth and development of the plant will need to be imparted during the cultivation of the plant.

The invention also provides a plant culture medium, which comprises the small molecule compound.

Specifically, the plant culture medium comprises a basal culture medium and the small molecule compound. The basic medium is the same as that in the above plant cultivation method, and is not described herein again.

The plant culture medium contains the small molecule compound. Therefore, the plant culture medium can promote the growth and development of plants.

The invention further provides an application of the small molecule compound in preparation of GH3 protein inhibitors. The small molecular compound can obviously inhibit the activity of GH3 protein, so the small molecular compound can be applied to the process of preparing GH3 protein inhibitors.

The invention also provides a GH3 protein inhibitor, and an active ingredient of the GH3 protein inhibitor comprises the small molecule compound.

In one embodiment, the GH3 protein inhibitor further comprises an excipient. Specifically, the auxiliary material is at least one selected from a cosolvent and a preservative.

Because the small molecular compound has poor water solubility, the small molecular compound in the inhibitor can be fully absorbed by plants by adding the cosolvent when preparing the GH3 protein inhibitor. Optionally, the co-solvent is selected from DMSO. Of course, in other embodiments, the co-solvent is not limited to the above, but may be other plant acceptable co-solvents.

Preservatives are used to extend the useful life of GH3 protein inhibitors.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

In the following examples, the structure is as follows

The small molecule compound (2) is abbreviated as "C9", and C9 is synthesized by Shanghai Nafu organism, and is dissolved in DMSO before being added to a culture medium. "μ M" herein means "μmol/L"; PPBo is 4-phenoxyphenylboronic acid; L-Kyn is L-Kynurenine, an auxin synthetic pathway IPA pathway inhibitor; IAA refers to indole-3-acetic acid;

composition of the medium: 2.215g/L MS salt, 15g/L sucrose, 0.5g/L MES, 5g/L plant gel and water, and the pH is 5.8; in the drawings "" means "P<0.5 "," + "means" P<0.01”,“***”“P<0.001”。

Example 1

Effect of different concentrations of C9 on the Main root length, hypocotyl Length and number of adventitious roots in Arabidopsis

(1) Arabidopsis seeds (Columbia type 0, Col-0) were sown on the media of the C9 group, IAA group and blank control group, and cultured for 6 days with at least 30 seeds sown each group. Wherein the culture medium of group C9 contains 0.1. mu.M, 0.3. mu.M, 0.8. mu.M, 1.5. mu.M, 3. mu.M and 5. mu.M of C9

A culture medium; the IAA group medium contained 0.05. mu.M, 0.1. mu.M, 0.3. mu.M and 1. mu.M IAA, respectively

A culture medium; the medium of the blank control group was untreated

And (4) a culture medium. Of course, the other culture conditions were the same for the group C9, IAA and blank control.

(2) After the culture was completed, the growth conditions of the arabidopsis seedlings of each group were observed, the length of the main root, the length of the hypocotyl and the number of adventitious roots of the arabidopsis seedlings treated at different C9 concentrations, the length of the main root, the length of the hypocotyl and the number of adventitious roots of the arabidopsis seedlings treated at different IAA concentrations, and the length of the main root, the length of the hypocotyl and the number of adventitious roots of the untreated arabidopsis seedlings were recorded, and statistical analysis was performed, and the results were shown in fig. 1 to 4.

FIG. 1 shows the plant phenotype of Arabidopsis seedlings from group C9, IAA and blank control after 6 days of culture, and in FIG. 1, the length of the scale bar in the lower right corner is 10 mm. As is clear from FIG. 1, as the concentration of C9 increased, elongation of the main root was suppressed, the hypocotyl length increased, and the number of adventitious roots increased.

FIG. 2 is a statistical analysis result of the main root length of Arabidopsis seedlings in C9 group, IAA group and blank control group after 6 days of culture. As can be seen from fig. 2, the length of the main root of arabidopsis thaliana in the C9 group is smaller than that of the blank control group, and the length of the main root of arabidopsis thaliana seedlings treated by other concentrations is very significantly different from that of the blank control group except for the arabidopsis thaliana seedlings treated by 0.1 μ M in the C9 group; the length of the main root of the arabidopsis thaliana in the IAA group is smaller than that of the blank control group, and the length of the main root of the arabidopsis thaliana seedling treated by all concentrations in the IAA group is very obviously different from that of the blank control group.

FIG. 3 is a statistical analysis result of hypocotyl length of Arabidopsis seedlings of group C9, group IAA and blank control after 6 days of culture. As can be seen from fig. 3, the hypocotyl lengths of arabidopsis thaliana in group C9 were all greater than those of the blank control group, the hypocotyl lengths of arabidopsis thaliana seedlings treated at 0.3 μ M in group C9 were significantly different from those of the blank control group (P <0.05), and the hypocotyl lengths of arabidopsis thaliana seedlings treated at 0.8 μ M, 1.5 μ M, 3 μ M, and 5 μ M in group C9 were significantly different from those of the blank control group; the hypocotyl length of arabidopsis thaliana in the IAA group is smaller than that of the blank control group, but the hypocotyl length of arabidopsis thaliana seedlings treated by all concentrations in the IAA group is not significantly different from that of the blank control group.

FIG. 4 is a statistical analysis result of the number of adventitious roots of Arabidopsis seedlings in C9 group, IAA group and blank control group after 6 days of culture. As can be seen from fig. 4, the numbers of indefinite roots of arabidopsis thaliana treated at different concentrations in the C9 group were all greater than those of the blank control group, and the numbers of indefinite roots of arabidopsis thaliana seedlings treated at different concentrations in the C9 group were very different from those of the blank control group; in the IAA group, the number of adventitious roots of 0.05 mu M treated arabidopsis thaliana is smaller than that of a blank control group, the number of the adventitious roots of 0.1 mu M treated arabidopsis thaliana and the number of the adventitious roots of 0.3 mu M treated arabidopsis thaliana are both larger than that of the blank control group, but no significant difference exists, and the number of the adventitious roots of 1 mu M treated arabidopsis thaliana seedlings and that of the blank control group are both significantly different.

Thus, as can be seen from FIGS. 1 to 4, C9 has tissue specificity for the action of Arabidopsis, not only has inhibitory effect on the primary root, but also has promoting effect on the growth of hypocotyl, the occurrence and growth of adventitious root, and IAA has no promoting effect on the growth of hypocotyl at a high concentration (e.g., 1. mu.M), while C9 has a significant promoting effect at a low concentration (e.g., 0.3. mu.M); c9 also has a distinct advantage over IAA in terms of adventitious root formation.

Example 2

Effect of C9 on the number of lateral roots in Arabidopsis

(1) Will be at

Arabidopsis seedlings (Columbia type 0, Col-0) grown for 5 days on medium and in consistent vigor were randomly grouped into three groups: group C9, IAA and blank control.

(2) Arabidopsis seedlings of group C9 were transferred to a shoot containing 0.8. mu.M and 1.5. mu. M C9

The treated plates of medium were cultured for 3 days. Similarly, Arabidopsis seedlings of the IAA group were transferred to seedlings containing 0.3. mu.M IAA

The treated plates of medium were cultured for 3 days. Arabidopsis seedlings from the blank control group were transferred to treatment plates only

Culture medium (no additional addition of C9, IAA or other substances to the treatment plates) for 3 days. Of course, the other culture conditions were the same for the group C9, IAA and blank control. After the end of the culture, the plant phenotype and the number of lateral roots of each group of Arabidopsis seedlings were recorded, and the results are shown in FIGS. 5 and 6. FIG. 5 shows the plant phenotype of Arabidopsis seedlings from group C9, IAA and blank control (Mock in the figure) after 3 days of growth after transfer to treatment plates, and the length of the scale bar in the lower right corner of FIG. 5 is 10 mm. FIG. 6 is a statistical result of the number of lateral roots of Arabidopsis seedlings in C9 group, IAA group and blank control group (Mock group in the figure) after 3 days of growth after transfer to treatment plates.

As can be seen from fig. 5 and 6, both C9 and IAA had the effect of inhibiting the growth of the main root and promoting the growth of the lateral roots, as compared with the blank control group.

Example 3

Effect of C9 on root Hair Length in Arabidopsis

(1) Will be at

Arabidopsis seedlings (Columbia type 0, Col-0) grown for 5 days and grown consistently on medium (composition of medium: 2.215g/L MS salts, 15g/L sucrose, 0.5g/L MES, 5g/L plant gel and water, pH 5.8) were randomly grouped into three groups: group C9, IAA and blank control.

The treated plates of medium were incubated for 48 hours. Similarly, Arabidopsis seedlings of the IAA group were transferred to seedlings containing 0.3. mu.M IAA

The treated plates of medium were incubated for 48 hours. Arabidopsis seedlings from the blank control group were transferred to treatment plates only (no additional C9, IAA or other substances were added to the treatment plates) and cultured for 48 hours. Of course, the other culture conditions were the same for the group C9, IAA and blank control. After the completion of the culture, the root condition and root hair length of each group of Arabidopsis seedlings were recorded, and the results are shown in FIGS. 7 and 8.

FIG. 7 is an image of the roots of Arabidopsis seedlings from group C9, IAA and blank control (Mock in the figure) after 48 hours of growth after transfer to treatment plates, with the length of the scale bar in the lower right corner of FIG. 7 being 400 μm. FIG. 8 is a statistical result of root hair length of Arabidopsis seedlings in C9 group, IAA group, and blank group (Mock group in the figure) after they were transferred to a treatment plate and grown for 48 hours.

As can be seen from fig. 7 and 8, both C9 and IAA had the effect of promoting the growth of arabidopsis root hair compared to the blank control group.

Example 4

Effect of C9 on the Signal transduction pathway of Arabidopsis auxin

(1) Will be at

Culture medium (C)

Composition of the medium: 2.215g/L MS salt, 15g/L sucrose, 0.5g/L MES, 5g/L plant gel and water, pH 5.8) were randomly divided into two groups, with 5 days of consistent growth of Arabidopsis seedlings (DR5:: GFP/Col-0, DR5:: GUS/Col-0): c9 group and blank control group.

(2) A part of Arabidopsis seedlings of group C9 was transferred to a shoot containing 1.5. mu. M C9

The treated plates of medium were cultured for 1 day. Similarly, a portion of the Arabidopsis seedlings of the blank control group were transferred to only the Arabidopsis seedlings

The plates were incubated for 1 day in medium (no additional C9, IAA or other material was added to the plates). Of course, the other culture conditions were the same for the C9 group and the blank control group. Then, the groups of Arabidopsis seedlings were observed for DR5:, the fluorescence intensity of GFP, and the results are shown in FIG. 9.

(3) Another part of Arabidopsis seedlings from group C9 was transferred to a shoot containing 1.5. mu. M C9

The treated plates of medium were cultured for 2 days. Likewise, a portion of the arabidopsis seedlings from the blank control group were transferred to treatment plates only (no additional C9, IAA or other substances were added to the treatment plates) and cultured for 2 days. Of course, the other culture conditions were the same for the C9 group and the blank control group. Then, GUS (. beta. -glucuronidase) was stained with X-gluc tissue, and expression of DR5-GUS in hypocotyls, main roots and root tips of Arabidopsis seedlings of each group was observed, and the results are shown in FIG. 10.

In fig. 9 and 10, Mock represents the blank control group, and as can be seen from fig. 9 and 10, at the root tip, the DR5-GFP fluorescence intensity of the C9 group is significantly higher than that of the blank control group, C9 enhances the expression of DR5-GUS at the junction of the root tip, lateral root primordium, hypocotyl and root, so C9 can activate the auxin signal transduction pathway, can regulate the growth and development of arabidopsis thaliana by increasing the concentration of endogenous auxin, and the part acted by C9 is mainly at the junction of the lateral root primordium, the hypocotyl and the root.

Example 5

Effect of L-Kyn and PPBo on C9 in Arabidopsis

Arabidopsis seeds (Columbia 0 type, Col-0) were sown on Kyn + PPBo group, Kyn + PPBo + C9 group, Kyn + PPBo + IAA group, and blank control group, each group was sown on at least 30 media, and cultured for 6 days, and each group was sown on at least 30 media. Wherein: the culture medium of the Kyn + PPBo group was 5. mu. M L-Kyn and 5. mu.M PPBo

A culture medium; the culture medium of Kyn + PPBo + C9 group contains 5 mu M L-Kyn, 5 mu M PPBo and 1.5 mu M C9

A culture medium; the culture medium of the Kyn + PPBo + IAA group is a medium containing 5 μ M L-Kyn, 5 μ M PPBo and 0.3 μ M IAA

A culture medium; the blank control group had a medium of

Culture medium, i.e. in the absence of L-Kyn, PPBo, C9 and IAA

Culturing on a culture medium. Of course, the other culture conditions of each group were the same. After the completion of the culture, the growth status of each group of arabidopsis seedlings was observed, and the growth status and the main root length of each group of arabidopsis seedlings were recorded and subjected to statistical analysis, and the results are shown in fig. 11 to 12.

FIG. 11 shows the plant phenotype of Arabidopsis seedlings after 6 days of culture in Kyn + PPBo group, Kyn + PPBo + C9 group, Kyn + PPBo + IAA group and blank control group, in FIG. 11, Mock refers to blank control group, and the length of the scale bar in the lower right corner is 10 mm. Fig. 12 is a statistical plot of the main root length of arabidopsis seedlings for Kyn + PPBo group, Kyn + PPBo + C9 group, Kyn + PPBo + IAA group, and blank control group.

As can be seen from FIGS. 11 and 12, when L-Kyn and PPBo are applied exogenously to inhibit the synthesis of endogenous auxin, the inhibition effect of C9 on the length of the main root is weakened, and after IAA is added exogenously, the inhibition effect of C9 on the length of the root is intensified, which indicates that the action effect of C9 depends on the content of auxin in plants.

Example 6

Effect of IAA in combination with C9 on root systems of Arabidopsis

Arabidopsis seeds (Col-0, Columbia type 0) were sown on the media of IAA group, C9 group, C9+ IAA group and blank control group, and cultured for 6 days, with at least 30 seeds sown per group. Wherein: the IAA group medium was 0.3. mu.M IAA

A culture medium; the culture medium of group C9 contains 1.5. mu. M C9

A culture medium; the medium for the C9+ IAA group was 1.5. mu. M C9 and 0.3. mu.M IAA

A culture medium; the blank control group had a medium of

And (4) a culture medium. Of course, the other culture conditions of each group were the same. After the completion of the culture, the growth status of each group of arabidopsis seedlings was observed, and the growth status and the main root length of each group of arabidopsis seedlings were recorded and subjected to statistical analysis, and the results are shown in fig. 13 to 14.

FIG. 13 shows the plant phenotype of Arabidopsis seedlings in IAA, C9, C9+ IAA and blank control groups after 6 days of culture, and in FIG. 13, Mock refers to the blank control group and the length of the scale bar is 10 mm. Fig. 14 is a statistical plot of the main root length of arabidopsis seedlings from IAA, C9, C9+ IAA and blank controls.

As can be seen from fig. 13 and 14, when 0.3 μ M IAA alone was applied, the primary root length was suppressed by 34%, whereas C9 suppressed the primary root length by 67% in the presence of IAA; the main root lengths of the arabidopsis thaliana in the IAA group, the C9 group and the C9+ IAA group are all smaller than those of the blank control group, and the main root lengths of the arabidopsis thaliana in the IAA group, the C9 group and the C9+ IAA group are greatly different from those of the blank control group.

Example 7

Effect of C9 on the Activity of GH3.5 and GH3.17 proteins

And (3) adopting a liquid chromatography-mass spectrometer to measure the in-vitro enzyme activity kinetic data of the GH3.5 protein and the GH3.17 protein. Reaction system 200. mu.L: 50 μ M C9, 50mM Tris-HCl (pH 7.5), 20mM MgCl₂1mM ATP, 1mM DTT, 2mM IAA, 10. mu.g GH3.5 protein, 0.25 mM-2.5 mM concentration gradient Asp or 1 mM-4 mM concentration gradient Glu. Reacting at 30 ℃ for 30min, adding 20 mu L of concentrated hydrochloric acid to stop the reaction after the reaction is finished, extracting twice by using 200 mu L of ethyl acetate, adding 100 mu L of methanol after vacuum drying, and measuring by using a liquid chromatography-mass spectrometer. The results are shown in FIGS. 15 and 16.

FIG. 15 shows the effect of 50. mu. M C9 on the enzymatic activity of GH3.5 protein, and FIG. 16 shows the effect of 50. mu. M C9 on the enzymatic activity of GH3.17 protein. As can be seen from fig. 15 and fig. 16, C9 can significantly inhibit the activity of GH3.5 protein and GH3.17 protein, further confirming that the target protein of C9 is GH3 protein family (GH3 s). The GH3.5 protein is a protein corresponding to a plant GH3.5 gene, and the GH3.17 protein is a protein corresponding to a plant GH3.17 gene.

Example 8

Effect of C9 on the root System of tobacco

(1) Will be at

The tobacco green seedlings growing for 7 days on the culture medium and having consistent growth vigor are randomly divided into two groups: c9 group and blank control group.

(2) Placing the tobacco green seedling of group C9 in a container containing 0.8 μ M C9

Culturing in culture medium for 6 days; the tobacco seedlings of the present formula of the blank control group are cultured in the absence of C9

Cultured on the medium for 6 days. Of course, of group C9 and blank controlThe other culture conditions were the same.

(3) After the completion of the culture, the growth conditions of the respective groups of the tobacco green plantlets were observed, the main root length, hypocotyl length and number of adventitious roots of the respective groups of the tobacco green plantlets were recorded, and statistical analysis was performed, and the results were shown in fig. 17 to 20.

FIG. 17 shows the phenotype of the present tobacco green plantlets in group C9 and the blank control group after 6 days of culture, in FIG. 17, Mock refers to the blank control group and the length of the scale bar is 10 mm; FIG. 18 is a statistical analysis of the main root length of tobacco seedlings of this formula after 6 days of culture in group C9 and the blank control group; FIG. 19 is a graph showing the statistical analysis of hypocotyl length of tobacco seedlings of this formula after 6 days of culture in groups C9 and a blank control; FIG. 20 is a statistical analysis of the number of lateral roots of tobacco seedlings of this formula in groups C9 and the blank control after 6 days of culture.

As can be seen from fig. 17 to 20, the growth vigor of the tobacco treated by C9 is significantly better than that of the blank control group, and C9 can inhibit the growth of the main root of the present tobacco, promote the increase in the number of lateral roots and the growth of the hypocotyl, and promote the growth and development of the root system of the present tobacco. Thus, C9 was shown to promote hypocotyl elongation, lateral root and adventitious root development with relatively little inhibition of the primary root at very low concentrations; meanwhile, C9 has similar regulation and control effects on other plants except Arabidopsis thaliana, and has a wide application range.

Example 9

C9 production of adventitious roots in tomato

(1) Tomato seedlings which grow for 7 days and have consistent growth vigor are cut off at the roots, and are divided into two groups at random: group C9, IAA and blank control.

(2) The tomatoes of group C9 were mixed in a mixture containing 1.5. mu.M and 3. mu.MC 9

Culturing in culture medium for 6 days; the tomatoes of the IAA group contained 0.3. mu.M and 1. mu.MIAA

Culturing in culture medium for 6 days; the tomatoes in the blank control group are in the tomato without C9

Cultured on the medium for 6 days. Of course, the other culture conditions were the same for the group C9, IAA and blank control.

(3) After the culture was completed, the growth status of each group of tomato seedlings was observed, the number and length of adventitious roots of each group of tomato seedlings were recorded, and statistical analysis was performed, and the results are shown in fig. 21 to 23.

FIG. 21 shows the phenotype of the plants of tomatoes in group C9, IAA and blank control after 6 days in culture, with a scale of 10 mm; FIG. 22 is a statistical analysis of the number of adventitious roots of tomato green seedlings in group C9, group IAA and blank control after 6 days of culture; fig. 23 is a statistical analysis result of the adventitious root length of tomato seedlings after 6 days of culture in C9 group, IAA group and blank control group.

From fig. 21 to 23, it can be seen that the root system of the tomato seedlings treated by C9 is obviously superior to that of the blank control group and IAA group, and C9 can promote the generation and growth of adventitious roots of the tomato with the root system cut off. Thus, C9 was shown to promote the development and growth of adventitious roots at lower concentrations. Meanwhile, C9 has similar regulation and control effects on other plants except Arabidopsis thaliana, and has a wide application range.

In summary, the above examples demonstrate that C9 has tissue specificity for the growth and development of plant root system, and can promote the generation of adventitious root, lateral root, root hair elongation and hypocotyl elongation of arabidopsis green seedling plant, and C9 can generate a phenotype (promote the growth of hypocotyl, promote the generation of adventitious root) better than IAA at very low concentration (e.g. 0.3 μ M); and C9 can activate auxin signal transduction pathway, can obviously inhibit the activity of GH3 protein family, and can act together with exogenous IAA to inhibit root growth. In addition, C9 can promote the growth of plant root system, not only in Arabidopsis, but also in tobacco and tomato.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The application of a small molecular compound in promoting the growth and development of plants, wherein the application comprises promoting the growth of hypocotyl, the generation and growth of adventitious roots, the growth of lateral roots and the growth of root hairs, and the small molecular compound has the structural formula:

the plant is Arabidopsis thaliana, tobacco or tomato.

2. Use according to claim 1, characterized in that it comprises the following steps:

adding the small molecule compound in the process of culturing the plant.

3. The use according to claim 2, wherein the small molecule compound is used at a concentration of 0.1 μ M to 5 μ M.

4. The use according to claim 2, wherein the small molecule compound is used at a concentration of 0.8 μ M to 3 μ M.

5. Use according to claim 2, wherein the plant is cultivated using a substrate which is a culture medium

MS culture medium or MS culture medium is obtained by adding the small molecular compound.

6. A method for culturing a plant, wherein the plant is Arabidopsis, tobacco or tomato, the method comprising adding a small molecule compound of the formula:

7. the application of a small molecule compound in preparing GH3 protein inhibitors, wherein the GH3 protein is GH3.5 protein or GH3.17 protein, and the structure of the small molecule compound is as follows:

8. the use of claim 7, wherein the GH3 protein inhibitor further comprises an adjuvant selected from at least one of a cosolvent and a preservative.

9. Use according to claim 8, wherein the co-solvent is DMSO.

10. The application of the small molecule compound in preparing a plant culture medium is characterized in that the plant culture medium comprises a basal culture medium and the small molecule compound, and the structural formula of the small molecule compound is as follows:

the plant is Arabidopsis thaliana, tobacco or tomato.

11. The use of claim 10, wherein the basal medium is

MS medium or MS medium.

12. The use according to any one of claims 10 to 11, wherein the concentration of the small molecule compound in the plant culture medium is between 0.1 μ M and 5 μ M.

13. The use according to claim 12, wherein the concentration of the small molecule compound in the plant medium is between 0.8 μ Μ and 3 μ Μ.