Estragole and application thereof in inhibiting AtHDA9 activity, improving H3K9ac level and promoting plant growth

Abstract

The invention discloses estragole and application thereof to inhibition of AtHDA9 activity, improvement of H3K9ac level and promotion of plant growth, and belongs to the technical field of plant growth regulators, the monomer component estragole of fennel volatile oil can inhibit the activity of arabidopsis thaliana histone deacetylase (AtHDA9) and improve the content of auxin in plants. Molecular docking and molecular dynamics simulation show that estragole can interact with amino acid residues ASP: 95, PHEE: 202, LEU: 268 and HIS: 174 of AtHDA9, the activity of AtHD9 is inhibited, and the H3K9ac level is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant growth regulators, and specifically relates to estragole and application thereof in inhibiting AtHDA9 activity, increasing H3K9ac levels and promoting plant growth. 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 acetylation is a type of histone modification that mainly regulates the gene expression process in plants. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) work together to regulate histone acetylation modification; HDACs are mainly involved in plant morphological development and response to stresses such as cold, drought, and disease resistance. The latest research shows that HDACs also participate in the repression of transcription in development together with various chromatin remodeling factors and transcription factors. [1] HDAC inhibitors (HDACi) are usually used to study the molecular mechanism of histone deacetylation or promote the treatment of human diseases by inhibiting the catalytic activity of histone deacetylase. Domestic patents have shown that the histone deacetylase inhibitor TMP195 can promote the healing of bone defects in mice, proving that the HDAC inhibitor TMP195 has the potential to treat bone defects and provides new ideas for the preparation of clinical bone defect treatment drugs. [2] ; An oxazole histone deacetylase inhibitor has a good therapeutic effect on solid tumors such as lung cancer [3] ; Histone deacetylase HDAC11 inhibitors can activate the host's own antibacterial immune function and can be used to prepare drugs based on antifungal immunomodulatory therapy [4] A variety of HDACi with different selectivity for HDAC proteins have been developed abroad, and more and more research has been conducted on the application of HDACi in plants. For example, key-2 and SAHA treatments enhance the salt tolerance of Arabidopsis and cassava, respectively. [5,6] The seven HDACis screened by Ueda et al. can also induce salt stress tolerance in Arabidopsis [7] The role of HDAC in root development and callus formation was investigated using HDACi in rice. [8,9]However, how volatile small molecules regulate histone deacetylation as HDACi and their specific functions and mechanisms in plant growth and development are still unclear. Studies have shown that NaCl and hydrogen sulfide can affect the growth of Arabidopsis or wheat by affecting histone deacetylase. [10,11] . This study found that estragole also has the effect of regulating the growth and development of Arabidopsis plants, and has the effect of plant growth regulators. Molecular docking and kinetic simulation results showed that estragole can interact with the amino acid residues ASP:95, PHE:202, LEU:268 and HIS:174 in Arabidopsis histone deacetylase AtHDA9. In vitro enzyme activity and WB experiments showed that it inhibited the activity of AtHDA9 enzyme and increased the level of H3K9ac, indicating that estragole can be used as a new plant-derived HDACi for regulating plant growth.

[0004] References

[0005] [1] Li Tao, Su Huihui, Li Zhenxing, Li Zhiliang, Xu Xiaowan. Research progress on the biological functions of plant histone deacetylases. Molecular Plant Breeding. 2015;13:2912-22.

[0006] [2] Chen Chongwei, Chen Xinping, Li Yue, Wang Shaowei. Application of histone deacetylase inhibitor TMP195 in the preparation of drugs promoting osteogenesis. p.13.

[0007] [3] Zhao Denggao, Zhang Ruiqiang, Ma Yanyan, Zhang Kun, Mo Hualong, Chen Yajun, et al. An oxazole histone deacetylase inhibitor and its preparation method and application. p.11.

[0008] [4] Chen Weilin, Wu Han, Yin Xiaofan. Application of a histone deacetylase HDAC11 inhibitor in the preparation of drugs for preventing and treating fungal infections. p.23.

[0009] [5]Patanun O, Ueda M, Itouga M, Kato Y, Utsumi Y, Matsui A, et al. TheHistone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid AlleviatesSalinity Stress in Cassava. Frontiers in plant science. 2016;7:2039.

[0010] [6] Sako K, Kim JM, Matsui A, Nakamura K, Tanaka M, Kobayashi M, et al. Ky-2, a Histone Deacetylase Inhibitor, Enhances High-Salinity Stress Tolerance in Arabidopsis thaliana. Plant&cell physiology. 2016; 57:776-83.

[0011] [7]Ueda M,Matsui A,Tanaka M,Nakamura T,Abe T,Sako K,et al.TheDistinct Roles of Class I and II RPD3-Like Histone Deacetylases in SalinityStress Response.Plant physiology.2017;175:1760-73.[8]Chung PJ,Kim YS,JeongJS,Park SH,Nahm BH,Kim JK. The histone deacetylase OsHDAC1 epigenetically regulates the OsNAC6 gene that controls seedling root growth in rice. The Plant journal: for cell and molecular biology. 2009; 59:764-76.

[0012] [9] Zhang H, Guo F, Qi P, Huang Y, Xie Y, Xu L, et al. OsHDA710-MediatedHistone Deacetylation Regulates Callus Formation of Rice Mature Embryo. Plant&cell physiology. 2020; 61: 1646-60.

[0013]

[10] Chen Gongchun, Wu Lingge, Lin Juan. NaCl can promote the regulation of HD2 family genes on root growth and development in Arabidopsis. Journal of Fudan University (Natural Science Edition). 1-11.

[0014]

[11] Pei Xinxin, Chen Hongyu, Li Xiao, Liu Huajie, Li Hua. Effects of Hydrogen Sulfide on Plant Root Growth and Histone Deacetylase. Journal of Henan Agricultural Sciences. 2022; 51: 57-64. Summary of the Invention

[0015] Aiming at the deficiencies existing in the prior art, the present invention provides estragole and its application in inhibiting the activity of AtHDA9, increasing the level of H3K9ac, and promoting plant growth.

[0016] The technical solution adopted by the present invention is as follows:

[0017] A volatile substance, which is estragole.

[0018] The present invention also provides an application of the volatile substance according to claim 1, that is, the application of estragole in promoting plant growth.

[0019] Further, the plant is Arabidopsis thaliana.

[0020] Further, the estragole is used as an AtHDA9 inhibitor.

[0021] Further, the estragole can inhibit the activity of AtHDA9, increase the level of H3K9ac, and promote plant growth.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The estragole in the volatile oil of Foeniculum vulgare Mill. is a non-hormonal small molecule, which can promote the increase of plant auxin content. At the same time, after being prepared into nanoemulsion, it can promote plant growth by foliar spraying and root irrigation.

[0024] (2) The present invention finds that estragole can inhibit the activity of AtHDA9, thereby increasing the histone acetylation level, promoting the transcriptional expression of auxin-related genes, and further promoting the growth of Arabidopsis thaliana. Description of the Drawings

[0025] Figure 1 It is a diagram showing the effect of estragole on the growth of Arabidopsis thaliana. The lower half of the figure shows the effects on the primary root length, number of lateral roots, fresh weight, and auxin content in sequence. (*, 0.01 < P < 0.05; **, P < 0.01; ***, P < 0.001 indicate significant differences between different treatment groups at the P < 0.05, 0.01, and 0.001 levels respectively);

[0026] Figure 2 It is a diagram showing the effect of applying estragole at a concentration of 3.37 mM on the growth and development of wild-type Arabidopsis thaliana and deacetylase mutant (HDA9) (primary root length; number of lateral roots; fresh weight of the whole plant; auxin content);

[0027] Figure 3 Effect diagram of estragole on H3K9ac in Arabidopsis thaliana: Immunoblot and immunoblot gray value histogram of H3 and H3K9ac detected from histones isolated after treating Arabidopsis thaliana (WT, hda9) with estragole. H3 was selected as the blank control. (*, 0.01 < P < 0.05; **, P < 0.01 indicate significant differences between different treatment groups at the P < 0.05 and 0.01 levels, respectively. ns, 0.05 < P indicates no significant difference between different treatment groups at the 0.05 < P level);

[0028] Figure 4 Diagrams of molecular docking, molecular dynamics simulation of estragole with AtHDA9 and its effect on the enzyme activity of AtHDA9. (A) 3D binding model of AtHDA9 protein with estragole, including the overall structure, active pocket, and the positional relationship between the monomer compound and amino acids. 2D binding model of AtHDA9 protein with estragole. (B) Time trajectory diagram of the binding model of estragole with AtHDA9. (C) RMSD value of the amino acid backbone atoms of AtHDA9 in the binding model of estragole with AtHDA9 changing over time. (D) Energy decomposition diagram of the binding site of estragole with AtHDA9. (E) Effect of estragole on the activity of AtHDA9 protein. M: Maker; E1 - E5: Waste liquid after BeyoGoldTM His-tag resin adsorbs protein; W1, W12: Waste liquid of His-tag resin after the first and twelfth washes with Wash buffer; CL: Supernatant after ultrasonic disruption of recombinant bacteria induced by IPTG, FT: Flow-through liquid flowing down after obvious solid-liquid stratification. Trichostatin A is an inhibitor of HDAC protein. Detailed implementation manners

[0029] The following describes the detailed implementation manners of the present invention with reference to the accompanying drawings.

[0030] Example 1: Treating Arabidopsis thaliana with estragole

[0031] Prepare an estragole emulsion with a concentration of 3.37 mM using a 2-DMSO-T aqueous solution (1000 / 1 Tween-80 and 20 / 1000 DMSO), and then filter it through a 0.22 μm microporous membrane. Use the 2-DMSO-T aqueous solution as the control solution.

[0032] Place Arabidopsis thaliana seeds in a 4°C refrigerator for "vernalization" for 3 days, and then place the seeds in a 1.5 mL EP tube for surface disinfection. Place the disinfected seeds on a 1 / 8 MS basal medium (Murashige and Skoog basal salt mixture, catalog number: M5524) to germinate and grow. Place the plate vertically at 90°C to allow the roots to grow downward along the agar surface at 22°C with a light intensity of 300 μmol m2 s -1 Under the conditions of 16h light and 8h dark, the plants were grown for 4 days. After germination, filter paper pieces with a diameter of 5mm were placed on the lid of the dish at equal distances. 7μL of artemisinin emulsion and control solution were added to each filter paper piece. Each treatment was repeated 5-7 times. The biomass and hormone content of Arabidopsis thaliana were measured after 20 days of cultivation at 22℃ with 16h light and 8h dark.

[0033] Example 2: Effect of estragole on the auxin content of Arabidopsis

[0034] In order to determine the auxin content of Arabidopsis thaliana after artemisinin 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.

[0035] Example 3: Effects of estragole on H3K9ac in Arabidopsis

[0036] 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.

[0037] Example 4: Molecular docking and molecular dynamics simulation of estragole and AtHDA9

[0038] 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 estragole 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, and molecular docking was performed and the results were calculated using Autodock Vina, and the results were visualized using PyMOL and Ligplot software.

[0039] Example 5: Effect of estragole on AtHDA9 enzyme activity

[0040] In order to determine the activity of AtHDA9, 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). The concentration of the expressed protein is determined using the BCA method (BCA protein concentration determination kit SK1070). The activity of AtHDA9 was measured using the Fluorimetric HDAC Activity AssayKit*Green Fluorescence*(13601) kit.

[0041] This molecular dynamics simulation uses Gromacs2022.3 version 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 305K and normal pressure (1Bar), the force field used Amber 99sb-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 a duration of 100ps. Subsequently, the present invention uses the gmx grompp and gmx mdrun commands to perform molecular dynamics simulations of proteins and small molecules for up to 100ns, and records the conformational information during the simulation. After the simulation is completed, the trajectory is analyzed and the root mean square deviation (RMSD) of the motion trajectory of each amino acid is calculated.

[0042] Figure 1 The figure below shows the effect of estragole on the growth of Arabidopsis thaliana. The lower part shows the effect of main root length, lateral root number, fresh weight, and auxin content. Figure 1 It can be seen that compared with the control group (wild-type Arabidopsis), the monomer (estragole) increased the main root length, lateral root number, fresh weight and auxin content of Arabidopsis plants by 1.45 times, 1.59 times, 1.32 times and 1.24 times, respectively.

[0043] In order to identify the key enzyme that causes a significant increase in the histone acetylation level in Arabidopsis, the present invention simultaneously treats wild-type Arabidopsis and HDAC mutant (hda9) with estragole at a concentration of 3.37 mM. The number of lateral roots, fresh weight, and auxin content of the hda9 mutant treated with estragole did not change significantly compared with those without treatment, and the growth-promoting effect disappeared. It was also found that treatment of wild-type Arabidopsis with estragole resulted in a significant increase in the H3K9ac modification level. Compared with wild-type Arabidopsis, the H3K9ac modification level of the hda9 mutant was significantly increased, but the H3K9ac modification level of the hda9 mutant did not change significantly after treatment with estragole ( Figure 3), indicating that histone deacetylase (AtHDA9) is closely related to the growth-promoting effect of estragole and is the key enzyme that causes changes in the acetylation level of histone H3K9 in Arabidopsis thaliana.

[0044] Using modern molecular technology, we conducted molecular docking between Arabidopsis HDA9 enzyme and estragole. From the molecular docking results, we can see that HDA9 protein and estragole have different binding sites. Using PyMOL to visualize some docking results, the estragole molecule is wrapped by the protein active pocket ( Figure 4 A). Generally, when the binding energy is less than -5 kcal / mol, it indicates that the protein receptor and the compound have certain binding activity and the docking effect is stable. The molecular docking results show that the binding energy between the two is -5.8 kcal / mol, indicating that the two can be stably bound ( Figure 4 A). In the binding model of HDA9 protein and estragole, His174 is a polar amino acid with strong hydrogen bond donor and acceptor capabilities. It can form hydrogen bond interactions with ligands or other protein residues in small molecules, thereby forming a relatively stable hydrogen bond structure with estragole. Molecular dynamics simulation can not only show the dynamic action process of ligand small molecules and receptor proteins, but also evaluate the interaction between ligands and important catalytic sites. In order to verify the stability of the docking complex of estragole and AtHDA9 molecules, the present invention carried out an all-atom molecular dynamics (MD) simulation study. It is not difficult to see from the figure that after the dynamic simulation starts, the protein structure becomes obviously compact, and remains stable throughout the entire process, tightly binding to the ligand, and the small molecule estragole does not escape from the protein pocket ( Figure 4 B). In the whole simulation process, the present invention uses the GROMACS command line gmx_rmsd to estimate the RMSD value of the stability curve of the estragole and AtHDA9 molecular docking complex. The obtained complex RMSD trajectory rises continuously in the whole initial frame (0-30ns) until the RMSD stabilizes around 18ns, and the subsequent trajectory fluctuates around 0.55nm until the MD simulation ends 100ns, indicating that the binding of estragole and AtHDA9 complex has good stability ( Figure 4 C). Subsequently, the present invention analyzed the contribution of each amino acid residue to the intermolecular affinity based on the decomposition of the residue energy. In the binding model of 4-Allylanisol and AtHDA9, the amino acid residues ASP:95, PHE:202, LEU:268 and HIS:174 contributed the most binding energy, which were -63.382 kcal / mol, 21.563 kcal / mol, -19.590 kcal / mol and 10.912 kcal / mol, respectively ( Figure 4D), so it is speculated that these sites may be the key sites for 4-Allylanisol to inhibit AtHDA9, indicating that HDA9 protein has a strong ability to bind to artemisinin and can bind stably, which to a certain extent explains that artemisinin may have a certain effect on the activity of HDA9 protein.

[0045] The present invention uses an HDAC protein activity test kit to determine the effect of estragole on the activity of AtHDA9. First, the target protein AtHDA9 is expressed by prokaryotic protein expression. From the results of WB, it can be seen that the protein has a single band of about 47.72 kb and a high purity ( Figure 4 E). The activity of the expressed target protein AtHDA9 was 1139339RFU / min / g, which was good. After adding the commercial inhibitor Trichostain A, its activity was significantly reduced to 682851RFU / min / g. After being treated with estragole, the protein activity was significantly inhibited, and the protein activity was reduced to 729480RFU / min / g, which shows that estragole can inhibit the activity of AtHDA9, thereby increasing the level of histone acetylation, promoting the transcriptional expression of auxin-related genes, and then promoting the growth of Arabidopsis.

[0046] 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 volatile substance, It is characterized in that The volatile substance is estragole.

2. A use of the volatile substance according to claim 1, It is characterized in that The application of estragole in promoting plant growth.

3. Use of the volatile substance according to claim 2, It is characterized in that The plant is Arabidopsis thaliana.

4. The use of the volatile substance according to claim 2, It is characterized in that The estragole is used as an AtHDA9 inhibitor.

5. Use of the volatile substance according to claim 4, It is characterized in that The estragole can inhibit the activity of AtHDA9, increase the level of H3K9ac and promote plant growth.

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