Cytokinin compound with broad biological effects and its preparation method and application

Abstract

The present invention discloses a cytokinin compound with broad biological effects, its preparation method, and application. The cytokinin compound is derived from Fusarium thaliana and is synthesized by the fexA and fexC genes of the cytokinin gene cluster. The compound can be produced by heterologously expressing the fexA and fexC genes in Aspergillus nidulans. Biological activity assays revealed that the compound exhibited superior activity compared to the previously reported cytokinin iP in inducing tobacco callus growth, promoting betalain production in amaranth, inhibiting Arabidopsis taproot growth, and delaying wheat leaf senescence. This provides more efficient and safe cytokinin drug candidates for future agricultural production.

Description

Technical Field

[0001] The present invention relates to the field of agricultural production, in particular to a cytokinin compound with a wide range of biological effects, and also to a preparation method and application of the cytokinin compound. Background Art

[0002] To maintain their growth and development, plants produce trace substances known as phytohormones to regulate their physiological processes. The five classic hormones are auxin, cytokinin (CK), gibberellins (GA), abscisic acid (ABA), and ethylene (ET). Four more plant hormones have been discovered: brassinosteroids (BR), salicylates (SA), jasmonates (JA), and strigolactones (SL). Cytokinins, as an important class of plant growth-regulating hormones, are present in nearly all plant tissues and play crucial roles throughout plant growth and development. These include promoting cell division and expansion, inducing bud differentiation, relieving apical dominance, breaking seed dormancy, promoting seed and bud germination, and regulating nutrient transport. Cytokinins also enhance plant resistance to high salt and high temperature, regulate plant growth under drought conditions, and delay aging in both intact and detached plants. Due to their remarkable biological activity, cytokinins are widely used in agriculture, biotechnology, cosmetics, and medicine. Exploring cytokinins with novel structures and better activities will help enhance crop resistance, help crops better cope with environmental stresses such as drought, pests and diseases, thereby expanding the arable area of ​​crops; promote crop growth and development, thereby increasing crop yield and quality; enhance the natural disease resistance of crops, weaken or avoid the invasion of pathogenic fungi on crops, reduce dependence on chemical pesticides, reduce production costs, and at the same time reduce pollution to the environment. Summary of the Invention

[0003] In view of this, one of the objects of the present invention is to provide a cytokinin compound with broad biological effects; a second object of the present invention is to provide a method for preparing the cytokinin compound with broad biological effects; a third object of the present invention is to provide the use of the cytokinin compound in inducing callus growth; a fourth object of the present invention is to provide the use of the cytokinin compound in promoting the synthesis of betalain in amaranth; a fifth object of the present invention is to provide the use of the cytokinin compound in delaying leaf senescence; and a sixth object of the present invention is to provide the use of the cytokinin compound in eliminating apical dominance in plants.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] A cytokinin compound with a wide range of biological effects, the structure of which is shown in Formula I:

[0006]

[0007] 2. The preparation method of the cytokinin compound with broad biological effects comprises heterologously expressing the gene fexAC from Fusarium expansum 0711 in Aspergillus nidulans, fermenting the heterologous expression strain, and purifying to obtain the cytokinin compound; the nucleotide sequence of the fexA is shown in SEQ ID NO.1; the nucleotide sequence of the fexC is shown in SEQ ID NO.2.

[0008] Preferably, the purification is performed by extracting with acetone:ethyl acetate, concentrating to dryness under reduced pressure, using MCI column chromatography, gradient eluting with methanol-water solution, and collecting the eluate to obtain a component containing compound I.

[0009] Preferably, the volume ratio of acetone to ethyl acetate is 1:3.

[0010] Preferably, the methanol-water solution gradient elution procedure is to use methanol with volume fractions of 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence.

[0011] 3. Application of the cytokinin compound in inducing callus growth.

[0012] 4. Application of the cytokinin compound in promoting betalain synthesis.

[0013] 5. Application of the cytokinin compound in delaying leaf senescence.

[0014] 6. Use of the cytokinin compound in eliminating apical dominance in plants.

[0015] The present invention provides a beneficial effect by disclosing a cytokinin compound with broad biological effects. The compound is derived from Fusarium spp. and is synthesized by the fexA and fexC genes of the cytokinin gene cluster. Compound I can be produced by heterologously expressing the fexA and fexC genes in Aspergillus nidulans. Biological activity assays have shown that Compound I exhibits superior activity to the previously reported cytokinin iP in inducing tobacco callus growth, promoting betalain production in amaranth, inhibiting Arabidopsis taproot growth, and delaying wheat leaf senescence. This provides a more efficient and safe cytokinin candidate for future agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0017] Figure 1 Comparison of the ability of compound I and iP to induce tobacco callus growth (A: picture of callus; B: wet weight of callus);

[0018] Figure 2 Effects of compound I and iP on betalain synthesis (A: results of compound treatment of amaranth cotyledons and hypocotyls; B: betalain content);

[0019] Figure 3 Comparison of the ability of compound I and iP to delay chlorophyll degradation (A: compound treatment results at the leaf tip; B: chlorophyll content);

[0020] Figure 4 Comparison of the ability of compound I and iP to inhibit the growth of Arabidopsis taproot (A: taproot growth; B: measurement of taproot length). DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0022] Example 1. Preparation of Compound I

[0023] Using gDNA of Fusarium expansum 0711 as a template, the primer pairs pANR-fexA-F / R (SEQ ID NO.3 / SEQ ID NO.4) and pANU-fexC-F / R (SEQ ID NO.5 / SEQ ID NO.6) were used to amplify the fexA (SEQ ID NO.1) and fexC (SEQ ID NO.2) gene fragments. Homologous recombination was used to construct fexA and fexC into the BamH I site of the pANR vector and the Not I site of the pANU vector (for pANR and pANU vectors, see Haichun Zeng. Unprecedented [5.5.5.6] Dioxafenestrane Ring Construction in Fungal Insecticidal Sesquiterpene Biosynthesis. Angew. Chem. Int. Ed. 2019, 58, 6569–6573) to obtain recombinant plasmids.

[0024] pANR-fexA-F:5'-ccattaccccgccacatagacacatctaaacaatgacacgcactcacaagcctgc-3' (SEQ ID NO.3);

[0025] pANR-fexA-R:5'-aaagggtatcatcgaaagggagtcatccaatttaaatagatcctgttaaacagctatac-3' (SEQ ID NO.4);

[0026] pANU-fexC-F:5'-gcttcatccccagcatcattacacctcagcaatggcccctcttacattcttgtttc-3' (SEQ ID NO.5);

[0027] pANU-fexC-R:5'-caacacagtggaggacatacccgtaattttctgctcgaaggtaacaaaatcggtcag-3' (SEQ ID NO. 6).

[0028] The two recombinant plasmids were co-transfected into Aspergillus nidulans using protoplast transformation, resulting in efficient expression of the fexA and fexC genes in the fungus. The heterologous expression strain was cultured in CD-ST medium at 25°C. After four days of fermentation, the product was extracted three times with acetone:ethyl acetate (1:3) and concentrated to dryness under reduced pressure. The crude product was fractionated using MCI column chromatography using a methanol-water solvent, eluting at 40%, 50%, 60%, 70%, 80%, 90%, and 100% methanol, with two column volumes of each concentration. Fractions containing Compound I were pooled. Fractions were then re-separated using Sephadex LH-20 (200 mm × 20 mm) using methanol as the solvent. The crude product was further fractionated using preparative liquid chromatography (YMC ODS-A 5μm 120A (10 × 250 mm) column, isocratically eluted with 35% methanol-water to yield 4.5 mg of pure Compound I. The product was dissolved in deuterated DMSO and analyzed by nuclear magnetic resonance (NMR) resonance.

[0029] Identification of compound I: colorless oil; HR TOFMS m / z 218.1036 [M+Na] in positive ion mode + ,C 10 H 11 The calculated value of N5O is 217.0964, indicating that the molecule of this compound is C 10 H 11 N5O, combined with the NMR data, the structure of the compound was determined to be as shown in Formula I.

[0030]

[0031] (400MHz for 1 H NMR, 100 MHz for 13 C NMR)

[0032]

[0033] Example 2: Determination of the activity of compound I in inducing callus growth

[0034] Compound I in Example 1 and control compound iP were tested for their callus growth induction activity. The structure of compound iP is as follows:

[0035]

[0036] The activity assay was performed as follows: 0.1 g of tobacco callus was inoculated into 3 mL of MS medium containing 10 μM compound I or iP, and an equal volume of DMSO was added as a control. The cells were cultured at 24°C in the dark for 4 weeks. The callus size was observed, and the changes in the wet weight of the callus before and after culture were measured. The results were as follows: Figure 1 The results showed that the weight of the compound I-treated group was significantly higher than that of the iP-treated group, indicating that the ability of compound I to induce callus growth was significantly better than that of iP.

[0037] Example 3: Determination of the activity of compound I in promoting betalain production

[0038] The effects of compound I and iP on betalain production in amaranth were determined as follows: Amaranth seeds were sterilized, placed on moistened paper, and cultured in the dark at 25°C for 72 hours. The roots of the seeds were removed under green light in a dark room, and the remaining parts (2 cotyledons and hypocotyls) were placed in a 9 mL culture dish containing 1 mL of culture medium (10 μmol Na2HPO4-KH2PO4, pH 6.8, 5 μmol tyrosine, and 10 μM compound I or iP). The culture was carried out in the dark at 25°C for 48 hours. The results are shown in FIG. Figure 2 As shown in B. Betacyanin was extracted by freezing in 2 mL of 3.33 μM acetic acid, and the absorbance values ​​at 537 nm and 620 nm were measured. The difference between the two values ​​was calculated as the concentration of betacyanin. The results are shown in Figure 2 As shown in Figure B. The results showed that compared with the DMSO control group, compound I could promote the production of betalain, and its activity was comparable to that of iP.

[0039] Example 4: Determination of the activity of compound I in delaying leaf senescence

[0040] The ability of Compound I and iP to slow chlorophyll degradation was determined as follows: After sterilizing wheat seeds, they were placed in a 12 cm Petri dish with a moistened paper disc and cultured in a light incubator (16 h light 15000xL, 70% humidity, 22°C; 8 h dark, 70% humidity, 20°C) for 9 days (the first leaf was fully developed and the second leaf had just grown). 3.5 cm from the tip of the first leaf was cut and placed in 150 μL of water containing 100 μM Compound I or iP (4 leaves per group) and treated in the dark at 25°C for 96 h. The results are shown in the figure. Figure 3 As shown in A. Use 5mL 80% ethanol to treat at 80℃ for 5min to extract the chlorophyll in the leaves, and measure the absorbance value at 665nm, which is the chlorophyll content. The results are shown in Figure 3 As shown in Figure B. The results showed that the chlorophyll content in the compound I treatment group was significantly higher than that in the iP treatment group, indicating that the ability of compound I to delay leaf senescence was significantly better than that of iP.

[0041] Example 5. Determination of the activity of compound I in inhibiting taproot growth

[0042] The effects of Compound I and iP on the growth of the main root of Arabidopsis thaliana were determined as follows: After sterilization, Arabidopsis thaliana seeds were inoculated into 1 / 2MS medium containing 1 μM Compound I or iP, and cultured in a light incubator (16 h light 15000xL, 70% humidity, 22°C; 8 h dark, 70% humidity, 20°C) for 8 days. The growth of the main root was recorded by photographing. The results are shown in the figure below. Figure 4 As shown in A; and the main root length was measured, the result is as follows Figure 4 As shown in Figure B. The results showed that the main root length of the compound I treatment group was significantly shorter than that of the iP treatment group, indicating that compound I was better than iP in inhibiting the growth of the main root of Arabidopsis.

[0043] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A method for preparing a cytokinin compound having a wide range of biological effects, characterized in that: The genes fexA and fexC from Fusarium expansum 0711 were heterologously expressed in Aspergillus nidulans, the heterologous expression strain was fermented, and the cytokinin compound was purified; the nucleotide sequence of the fexA gene is shown in SEQ ID NO.1; the nucleotide sequence of the fexC gene is shown in SEQ ID NO.2; The structure of the cytokinin compound is shown in Formula I: 。 2. The method for preparing the cytokinin compound having broad biological effects according to claim 1, characterized in that: The purification comprises extraction with acetone:ethyl acetate, concentration under reduced pressure, MCI column chromatography, gradient elution with methanol-water solution, and collection of the eluate to obtain a component containing compound I.

3. The method for preparing the cytokinin compound having broad biological effects according to claim 2, characterized in that: The volume ratio of acetone:ethyl acetate is 1:

3.

4. The method for preparing the cytokinin compound having broad biological effects according to claim 2, characterized in that: The procedure of the methanol-water solution gradient elution is to use methanol with volume fractions of 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence.

5. Use of a cytokinin compound in inducing callus growth, characterized in that: The structure of the cytokinin compound is shown in Formula I: 。 6. The use of a cytokinin compound in promoting betalain synthesis, characterized in that: The structure of the cytokinin compound is shown in Formula I: 。 7. The use of a cytokinin compound in delaying leaf senescence, characterized in that: The structure of the cytokinin compound is shown in Formula I: 。 8. Use of a cytokinin compound in eliminating apical dominance in plants, characterized in that: The structure of the cytokinin compound is shown in Formula I: 。

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