Plant growth regulator and application thereof

Abstract

The invention discloses a plant growth regulator and application thereof, and relates to application of lemongrass volatile oil monomer citronellol as a novel plant source HDACi for regulating and controlling plant growth in promoting plant growth and root development. The lemongrass volatile oil monomer component citronellol can increase the content of auxin and inhibit the activity of arabidopsis thaliana histone deacetylase (AtSRT1). Molecular docking and molecular dynamics simulation show that citronellol can inhibit the activity of AtSRT1 and improve the H3K9ac level through interaction with amino acid residues PHE-64, ARG: 65, MET: 1 and ILE: 214 in Arabidopsis thaliana histone deacetylase AtSRT1.

Description

Technical Field

[0001] The invention belongs to the technical field of regulators, and in particular relates to a plant growth regulator and application thereof. Background Art

[0002] Auxin is the earliest discovered plant hormone, which is widely involved in various processes of plant growth and development, such as embryonic development, root, stem and leaf morphogenesis, fruit development and tropism, etc. It is mainly synthesized in young buds, leaves and developing seeds, and then transferred to other plant organs to perform functions. Among them, indoleacetie acid (IAA) is the main endogenous active auxin of plants.

[0003] Histone deacetylation is one of the most critical epigenetic mechanisms in eukaryotes, regulating various genes involved in plant growth, development, and responses to environmental stress. HDAC inhibitors (HDACi) are often used to study the molecular mechanisms of histone deacetylation or promote the treatment of human diseases by inhibiting the catalytic activity of histone deacetylases. However, how volatile small molecules as HDACi regulate histone deacetylation and their specific functions and mechanisms in plant growth and development are still unclear. Molecular docking and kinetic simulation results show that citronellol can inhibit the activity of AtSRT1 and increase the level of H3K9ac by interacting with amino acid residues PHE:64, ARG:65, MET:1, and ILE:214 in Arabidopsis histone deacetylase AtSRT1. Citronellol can be used as a new plant-derived HDACi for regulating plant growth. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a plant growth regulator and application thereof.

[0005] The technical solution adopted by the present invention is:

[0006] A plant growth regulator, characterized in that the regulator is an AtSRT1 inhibitor.

[0007] Furthermore, the regulator is a volatile substance that can inhibit AtSRT1 enzyme activity, increase H3K9ac levels and promote plant growth.

[0008] Furthermore, the volatile substance is citronellol.

[0009] Furthermore, the citronellol is used in promoting plant growth.

[0010] Furthermore, the citronellol is used as a novel plant-derived HDACi for regulating plant growth.

[0011] Furthermore, the citronellol is used to promote the increase of plant auxin content.

[0012] Furthermore, the citronellol is used to prepare a nanoemulsion, and the nanoemulsion promotes plant growth through leaf spraying and root irrigation.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] 1. Citronellol, a component of lemongrass volatile oil discovered by the present invention, as a non-hormone small molecule, can promote plant growth;

[0015] 2. The citronellol found in the present invention can promote the increase of plant growth hormone content;

[0016] 3. The nanoemulsion prepared by the present invention using citronellol, a component of lemongrass volatile oil, can be sprayed on leaves and irrigated to promote plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a graph showing the effect of citronellol on the growth of Arabidopsis thaliana;

[0018] Figure 2 This is a graph showing the effect of citronellol on the growth of AtSirt1 mutant strains;

[0019] Figure 3 This is a graph showing the effect of citronellol on AtSRT1 enzyme activity;

[0020] Figure 4 This is a diagram showing the effect of citronellol on H3K9ac in Arabidopsis;

[0021] Figure 5 Molecular docking and molecular dynamics simulation diagram of citronellol and AtSRT1. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings.

[0023] Example 1: Citronellol treatment of Arabidopsis thaliana

[0024] A monomer emulsion with a concentration of 3.2 mM was prepared with a 2-DMSO-T aqueous solution (1000 / 1 Tween-80 and 20 / 1000 DMSO), and then filtered with a 0.22 μm microporous filter membrane. The 2-DMSO-T aqueous solution was used as a control solution.

[0025] Arabidopsis seeds were placed in a 4°C refrigerator for 3 days for vernalization, and then placed in a 1.5 mL EP tube for surface sterilization. The sterilized seeds were placed on 1 / 8 MS basal medium (Murashige and Skoog basal salt mixture, catalog number: M5524) for germination and growth. The plate was placed vertically at 90°C to allow the roots to grow downward along the agar surface, 22°C, and the light intensity was 300 μmol m 2 s -1 The Arabidopsis thaliana was grown for 4 days under the conditions of 16 h of light and 8 h of darkness. After germination, filter paper pieces with a diameter of 5 mm were placed on the lid of the dish at equal distances. 7 μL of monomer emulsion and control solution were added to each filter paper piece. Each treatment was repeated 5-7 times. The Arabidopsis thaliana biomass was measured after 20 days of cultivation under the conditions of 16 h of light and 8 h of darkness at 22°C.

[0026] Example 2: Effect of citronellol on AtSirt1 enzyme activity

[0027] In order to determine the activity of AtSRT1, the present invention uses gene recombination technology to transfer the target gene into an expression vector (pET28a, kan+), and expresses the protein through Escherichia coli (BL21), and the concentration of the expressed protein is determined using the BCA method (BCA protein concentration determination kit SK1070). The activity of AtSRT1 is determined using the Epigenase Universal SIRT Activity / inhibition Assay Kit (P-4037).

[0028] Example 3: Effect of citronellol on H3K9ac in Arabidopsis

[0029] Histone extraction kit Histones were prepared according to the instructions. 15% SDS-PAGE gel was used for separation, and H3 lysine acetylation / status was detected by immunoblotting using anti-H3K9ac (Abcam, Cat#: ab16635) and anti-H3 (PTM-1002) antibodies. Anti-H3 immunoblotting was used as a control. Band intensity was quantified using Image J software.

[0030] Example 4: Effect of citronellol on the auxin content of Arabidopsis

[0031] In order to determine the hormone content of Arabidopsis thaliana after lemongrass volatile oil and monomer treatment, the Arabidopsis thaliana samples were ground into homogenate at a ratio of 1 g fresh tissue + 1 mL PBS, and analyzed using an enzyme-linked immunosorbent assay (ELISA) kit.

[0032] Example 5: Molecular docking and molecular dynamics simulation of citronellol and AtSirt1

[0033] Autodock Vina was used for molecular docking simulation. First, the 3D structure of AtSRT1 protein was downloaded from the Unipot official website (https: / / www.uniprot.org / uniprotkb / Q9FE17 / entry#names_and_taxonomy), and then the 3D structure of citronellol was downloaded from the Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ) database. The protein receptor was dehydrated, hydrogenated, and energy minimized using the AutoDock Tool software. Autodock Vina was used to perform molecular docking and calculate the results, and PyMOL and Ligplot software were used to visualize the results. This molecular dynamics simulation used Gromacs 2022.3 to perform molecular dynamics simulation on the receptor protein with the lowest binding energy with the ligand molecule in the molecular docking results. The simulation conditions were carried out at a static temperature of 310K and normal pressure (1Bar), the force field used Amber99sb-ildn, the solvent was water molecules (Tip3p water model), and the total charge of the simulation system was neutralized by adding an appropriate amount of Na+. The molecular dynamics simulation system first uses the steepest descent method to minimize energy, and then performs 100,000 steps of isothermal isochoric ensemble (NVT) equilibrium and isothermal isobaric ensemble (NPT) equilibrium, respectively, for 100 ps. Finally, the free molecular dynamics simulation is run, which has a total of 5,000,000 steps and a total duration of 100 ns. After the simulation is completed, the trajectory is analyzed and the root mean square deviation (RMSD) of the trajectory of each amino acid is calculated.

[0034] Figure 1 The effect of citronellol on the growth of Arabidopsis thaliana is shown in Figure 2. Figure 1 It can be seen that the main root length, lateral root number, and fresh weight of Arabidopsis thaliana treated with citronellol increased significantly by 1.54, 1.5, and 1.59 times, respectively, with a significant growth-promoting effect. In addition, citronellol can also significantly increase the content of auxin in Arabidopsis thaliana, which increased by 1.3 times after treatment with citronellol.

[0035] Figure 2 The effect of citronellol on the growth of AtSirt1 mutant strains is shown in Figure 2. Figure 2 It can be seen that the growth-promoting effect is significant after treatment with citronellol; however, the main root length, number of lateral roots, fresh weight, etc. of the AtSRT1 mutant treated with citronellol have no significant differences compared with those without treatment, and the growth-promoting effect disappears, indicating that AtSRT1 is closely related to the growth-promoting effect of citronellol and is the key enzyme that causes changes in the acetylation level of histones in Arabidopsis.

[0036] Figure 3The figure shows the effect of citronellol on the activity of AtSRT1 enzyme. The SIRT protein activity test kit was used to determine the effect of citronellol on the activity of AtSRT1. First, the target protein AtSRT1 was expressed through prokaryotic protein expression. The activity of the expressed target protein AtSRT1 was 367598RFU / min / g, with good activity. After adding the commercial inhibitor nicotinamide, its activity was significantly reduced to 129789RFU / min / g. After treatment with citronellol, the protein activity was significantly inhibited, and the protein activity was reduced to 258303RFU / min / g, which shows that citronellol can inhibit the activity of AtSRT1, thereby increasing the level of histone acetylation, promoting the transcriptional expression of related genes, and then promoting the growth of Arabidopsis.

[0037] Figure 4 This is the effect of citronellol on H3K9ac in Arabidopsis. There is no significant change in the H3K9ac level of the AtSRT1 mutant before and after citronellol treatment, which further proves that AtSRT1 is closely related to the growth-promoting effect of citronellol and is a key enzyme that causes changes in histone acetylation levels in Arabidopsis.

[0038] Figure 5 The molecular docking and molecular dynamics simulation diagram of citronellol and AtSRT1. Generally, when the binding energy is less than -5kacl / mol, it means that the protein receptor has a certain binding activity with the compound and the docking effect is stable. The molecular docking results show that the binding energy of citronellol and AtSRT1 is -6.2kacl / mol ( Figure 5 A), indicating that citronellol is stably bound to AtSRT1. Subsequently, the present invention analyzes the contribution of each amino acid residue to the intermolecular affinity based on the decomposition of the energy of the residues. In the binding model of citronellol and AtSRT1, the amino acid residues PHE:64, ARG:65, MET:1 and ILE:214 contribute the most binding energy, which are -1.113kacl / mol, -1.379kacl / mol, -0.710kacl / mol and -0.993kacl / mol, respectively ( Figure 5 B), so it is speculated that these sites may be the key sites for citronellol to inhibit AtSRT1.

[0039] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A plant growth regulator, characterized in that The modulator is an AtSRT1 inhibitor.

2. The plant growth regulator according to claim 1, characterized in that The regulator is a volatile substance that can inhibit AtSRT1 enzyme activity, increase H3K9ac levels and promote plant growth.

3. The plant growth regulator according to claim 2, characterized in that The volatile substance is citronellol.

4. The plant growth regulator according to claim 1, characterized in that The application of the citronellol in promoting plant growth.

5. The plant growth regulator according to claim 3, characterized in that The citronellol is used as a novel plant-derived HDACi for regulating plant growth.

6. The plant growth regulator according to claim 4, characterized in that The application of the citronellol in promoting the increase of plant growth hormone content.

7. The plant growth regulator according to claim 3, characterized in that The citronellol is used to prepare a nanoemulsion, and the nanoemulsion promotes plant growth through leaf spraying and root irrigation.

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