Application of piperazosin hydrochloride as strigolactone inhibitor

Abstract

The invention discloses an application of prazosin hydrochloride as a strigolactone inhibitor. The piperazosin hydrochloride provided by the scheme of the invention can be used as a plant strigolactone inhibitor for promoting plant branching, regulating the length of a plant hypocotyl and promoting gene expression of key inhibition factors SMXL6, SMXL7 and SMXL8 of strigolactone signal transduction pathways; the plant growth regulator can be applied to various plants, is small in environmental pollution, can be applied to agricultural production in a large scale, and achieves the effects of promoting plant branching, realizing more flowering and more fruiting, improving the stress resistance of the plants, avoiding diseases caused in the pruning process and saving labor consumption.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of prazosin hydrochloride as a strigolactone inhibitor. Background Art

[0002] Strigolactone (SL) is a multifunctional plant hormone that was first identified from cotton root secretions. It is a sesquiterpenoid compound that stimulates the germination of the seeds of the malignant parasitic weed Strigolactone. It was later proved to be ubiquitous in plants. Strigolactone has the ability to induce the germination of seeds of two root parasitic plants, Strigolactone and Orobanche, such as inducing the germination and parasitism of parasitic weeds. Strigolactone inhibits the germination of plant axillary buds and can synergistically regulate plant branching / tillering with plant hormones such as auxin and cytokinin, becoming a new means of regulating crop plant type. In addition, SL also affects root growth, root hair elongation, nodule formation, photosynthetic efficiency and nitrogen and phosphorus response.

[0003] Strigolactone plays an important role in regulating plant / crop growth and development and environmental response. It shows great application potential in crop plant shape, yield formation, resistance improvement, quality improvement and efficiency enhancement. Therefore, exploring and developing SL plant growth regulators (including inhibitors and promoters) and applying them to agricultural production will surely produce huge social and economic benefits. However, the existing strigolactone and its analogs have high production costs and are not suitable for large-scale use in agricultural production. At present, there is no strigolactone plant growth regulator technical on the market, and there are still too few SL inhibitors that can be used in agricultural production. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a use of prazosin hydrochloride as a strigolactone inhibitor.

[0005] The invention also provides a method for promoting plant branching.

[0006] The invention also provides a method for regulating the length of plant hypocotyls.

[0007] According to one aspect of the present invention, the use of prazosin hydrochloride as a strigolactone inhibitor is proposed.

[0008] In some embodiments of the present invention, the strigolactone inhibitor can be used to regulate the expression of inhibitory factors of the strigolactone signaling pathway.

[0009] In some embodiments of the present invention, the inhibitor of the strigolactone signaling pathway includes SMXL6, SMXL7 and / or SMXL8.

[0010] In some embodiments of the present invention, the strigolactone inhibitor can be used to promote plant branching and / or regulate plant hypocotyl length.

[0011] In some embodiments of the invention, the plant comprises a plant of the family Cruciferae.

[0012] In some embodiments of the invention, the plant is Arabidopsis thaliana.

[0013] According to a second aspect of the present invention, a method for promoting plant branching is provided, the method comprising the following steps: applying a solution containing prazosin hydrochloride to the plant.

[0014] In some embodiments of the present invention, the effective concentration of prazosin hydrochloride in the solution is 1-100 μM.

[0015] In some embodiments of the present invention, the effective concentration of prazosin hydrochloride in the solution is 10-80 μM.

[0016] In some embodiments of the present invention, the effective concentration of prazosin hydrochloride in the solution is 50-80 μM.

[0017] In some embodiments of the present invention, the application amount of the solution is 0.1-3 mL / plant.

[0018] In some embodiments of the present invention, the application amount of the solution is 0.1-0.5 mL / plant.

[0019] In some embodiments of the invention, the plant comprises a plant of the family Cruciferae.

[0020] In some embodiments of the invention, the plant is Arabidopsis thaliana.

[0021] According to a third aspect of the present invention, a method for regulating plant hypocotyl length is provided, the method comprising the following steps: applying a solution containing prazosin hydrochloride to the plant.

[0022] In some embodiments of the present invention, the effective concentration of prazosin hydrochloride in the solution is 1-100 μM.

[0023] In some embodiments of the present invention, the effective concentration of prazosin hydrochloride in the solution is 10-80 μM.

[0024] In some embodiments of the present invention, the application amount of the solution is 0.1-3 mL / plant.

[0025] In some embodiments of the present invention, the application amount of the solution is 0.1-0.5 mL / plant.

[0026] In some embodiments of the invention, the plant comprises a plant of the family Cruciferae.

[0027] In some embodiments of the invention, the plant is Arabidopsis thaliana.

[0028] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the prazosin hydrochloride of the present invention can be used as a plant monocotyledon inhibitor to promote plant branching, regulate plant hypocotyl length, and promote the expression of SMXL6, SMXL7, and SMXL8 genes, which are key inhibitors of the monocotyledon signal transduction pathway; and it can be applied to a variety of plants, has little pollution to the environment, and can be used on a large scale in agricultural production, so as to promote plant branching, more flowering and fruiting, and improve plant resistance to adversity, while avoiding diseases caused during pruning and saving labor consumption. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels.

[0030] Reagents and Materials:

[0031] Preparation of Prazosin-HCl: Accurately weigh 0.042 g of analytical grade Prazosin-HCl, dissolve it in DMSO, and then add water to make up to 10 mL to prepare a 10 mM Prazosin-HCl reagent stock solution.

[0032] Example 1

[0033] This example provides the use of prazosin hydrochloride as a strigolactone inhibitor, and the specific verification process is as follows:

[0034] 1. Effects of different concentrations of Prazosin-HCl on Arabidopsis hypocotyls

[0035] Experimental plan: Take wild-type Arabidopsis seeds from Columbia, soak and disinfect them in 75% ethanol for 15 minutes, wash them with sterilized deionized water 3-5 times, and then plant them on MS solid medium containing different concentrations of Pra-Hcl (1, 2 and 5 μM respectively). Put them in a 4°C refrigerator for vernalization for 3 days, and then place them at 22°C, 16h light and 8h low light conditions for 7 days, and use image J software to measure the length of the hypocotyl. The control group MOCK was planted on MS solid medium without Pra-Hcl. Each group of samples was tested 15 times.

[0036] Table 1

[0037]

[0038] The results are shown in Table 1. It can be seen from the table that as the concentration of Prazosin-HCl increases, the length of the hypocotyl increases significantly. The results show that Prazosin-HCl can effectively increase the length of the hypocotyl of Arabidopsis seeds.

[0039] 2. Effect of Prazosin-HCl on the number of Arabidopsis branches

[0040] About 15 days after Arabidopsis was transplanted into a small pot, bolting began. At this time, Arabidopsis with consistent growth was selected. Six small pots (30 plants) were grouped together. The experimental group was sprayed with Prazosin-HCl treatment solutions of different concentrations (10, 30, 50 and 80 μM), and the control group was sprayed with the same amount of deionized water. The standard was that the reagent on the surface of Arabidopsis leaves did not gather into a stream and flow down (about 30 plants were sprayed with 10 mL). Spraying was done when bolting just began, once a day, and sprayed for 3 consecutive days. During the entire growth period, it was cultured at 22°C, 16h light and 8h dark environment, and the nutrient soil was kept moist. The number of branches of 15 Arabidopsis plants was randomly recorded during the fruit pod stage.

[0041] Table 2

[0042]

[0043] The results of the branch number test of 15 Arabidopsis strains are shown in Table 2. It can be seen from the table that the branch number of Arabidopsis sprayed with Prazosin-HCl increased significantly compared with the control group. The results showed that Prazosin-HCl can effectively increase the branch number of Arabidopsis.

[0044] 3. Detection of key inhibitory factors in the strigolactone signaling pathway

[0045] Take about 100 mg of plant seedlings (2. The effect of razosin-HCl on the number of Arabidopsis branches in Arabidopsis seedlings. When they just started to bolt, they were treated with Prazosin-HCl at a final concentration of 10 μM and then sampled for 4 hours (only the first day was taken)) and placed in a mortar with liquid nitrogen, quickly ground into powder, and transferred to a 1.5 mL RNase-free EP tube. Add 1 mL of Trizol extract to each tube, blow and mix with a gun, vortex and oscillate for 30 seconds, and place on ice for 10 minutes. Add 250 μL of chloroform and vortex to mix, let stand on ice for 5 minutes, and centrifuge at 12000 rpm at 4°C for 5 minutes. Transfer the supernatant to a new pre-cooled EP tube, add 0.6 times the volume of pre-cooled isopropanol, invert to mix, and let stand on ice for 10 minutes. Centrifuge at 12000 rpm at 4°C for 5 minutes and discard the supernatant. Add 75% ethanol to wash the precipitate 2-3 times (add 1mL 75% ethanol to resuspend the washed precipitate. Centrifuge at 12000rpm for 5min at 4℃), and discard the excess supernatant. Dry in a clean bench for about 10min, add 30-50μL RNase-free water to dissolve the RNA. Measure the RNA concentration on an enzyme-labeled instrument, and evaluate the RNA quality by 1% agarose gel electrophoresis. Finally, divide the RNA into 1-2μg per tube and store at -80℃.

[0046] cDNA synthesis: According to the instructions of VazymeHiScript II Strand cDNA Synthesis Kit. The reaction system was prepared on ice, and the components were as follows: 1 μg total RNA, 4 μL 4×g DNA wiper mix, 1 μL OligodT, 1 μL Random Hexamers, supplemented with Nuclease-Free H 2 O to 16μL. React at 42℃ for 2min. Add the following components to the reaction system: 10×RT Mix 2μL, HiScript II Enzyme Mix 2μL, and mix well. The reaction conditions are: annealing temperature 25℃, duration 5min, extension temperature 50℃, duration 15min, inactivation at 85℃ for 2min, and then cDNA is synthesized. Dispense cDNA into small tubes and store at -20℃.

[0047] Fluorescence quantitative qPCR reaction: According to the instructions of VazymeChamQ SYBR qPCR Master Mix Kit, the primer sequences are shown in Table 3, the reaction system is shown in Table 4, and the reaction procedure is shown in Table 5. -△△ct Calculate the relative gene expression.

[0048] Table 3

[0049]

[0050]

[0051] Table 4

[0052] Components Dosage Primer 1 (10 mM) (forward primer as described in Table 3) 0.5μL Primer 2 (10 mM) (reverse primer as described in Table 3) 0.5μL 2×ChamQ SYBR qPCR Master Mix 4μL Template (cDNA) 1μL ddH2O Make up to 10 μL

[0053] Table 5

[0054]

[0055] Table 6

[0056]

[0057] The results are shown in Table 6. It can be seen from Table 6 that after Arabidopsis thaliana was treated with Prazosin-HCl, the expression levels of SMXL6, SMXL7, and SMXL8 genes, which are key inhibitors of the strigolactone signaling pathway, were significantly increased. The results showed that Prazosin-HCl effectively inhibited the content of strigolactone in plants and could be used as a strigolactone inhibitor.

[0058] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of prazosin hydrochloride as a monolactone inhibitor.

2. The use according to claim 1, characterized in that: The strigolactone inhibitor can be used to regulate the expression of inhibitory factors of the strigolactone signal transduction pathway; Preferably, the inhibitor of the strigolactone signaling pathway includes SMXL6, SMXL7 and / or SMXL8.

3. The use according to claim 1, characterized in that: The strigolactone inhibitor can be used to promote plant branching and / or regulate plant hypocotyl length.

4. The use according to claim 3, characterized in that: The plants include cruciferous plants; Preferably, the plant comprises Arabidopsis thaliana.

5. A method for promoting plant branching, characterized in that: The method comprises the following steps: applying a solution containing prazosin hydrochloride to plants.

6. The method according to claim 5, characterized in that The effective concentration of prazosin hydrochloride in the solution is 1-100 μM.

7. The method according to claim 5, characterized in that The application amount of the solution is 0.1-3 mL / plant.

8. A method for regulating plant hypocotyl length, characterized in that: The method comprises the following steps: applying a solution containing prazosin hydrochloride to plants.

9. The method according to claim 8, characterized in that The effective concentration of prazosin hydrochloride in the solution is 1-100 μM.

10. The method according to claim 8, characterized in that The application amount of the solution is 0.1-3 mL / plant.

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