Polypeptide for regulating plant growth and development and application thereof

Abstract

The invention discloses a polypeptide for regulating plant growth and development and application thereof, and relates to the technical field of biology. The invention specifically discloses any polypeptide from A1) to A3) as shown in the following: A1) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; a2) a polypeptide which is obtained by substitution and / or deletion and / or addition of more than one amino acid residue on the amino acid sequence as shown in SEQ ID NO: 1 and has the function of regulating plant growth and development; the polypeptide can be used for effectively regulating and controlling the growth and development of plants, promoting the growth and development of the plants at low concentration and inhibiting the growth and development of the plants at high concentration.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide for regulating plant growth and development and applications thereof. Background Art

[0002] Roots are crucial organs for plants to absorb water and mineral nutrients and sense and respond to environmental signals. Regulating root growth facilitates root function and is crucial for plant growth and development, as well as crop yield. Plant peptide hormones, a class of peptides composed of a few to dozens of amino acids, function as signaling molecules within plants, regulating stress and disease resistance, growth, and organ development. They hold enormous potential for application in agriculture, the food industry, and breeding. Therefore, developing a peptide that can effectively regulate plant growth and development is crucial. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a polypeptide that can effectively regulate plant growth and development.

[0004] The present invention also provides a reagent comprising the above polypeptide.

[0005] The present invention also provides biological materials related to the above polypeptide.

[0006] The present invention also provides applications of the above polypeptides, reagents or biomaterials.

[0007] The invention also provides a method for regulating plant growth and development.

[0008] A polypeptide according to the first aspect of the present invention, wherein the polypeptide is any one of A1) to A3): A1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; A2) a polypeptide having the function of regulating plant growth and development obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1; A3) A fusion polypeptide obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0009] The polypeptide according to the embodiments of the present invention has at least the following beneficial effects: The polypeptides of the examples can regulate plant growth and development. At low concentrations, they can effectively promote plant growth and development, including plant root growth; at high concentrations, they can effectively inhibit plant growth and development, including plant root growth.

[0010] According to some embodiments of the present invention, the tag includes at least one tag that facilitates the solubilization, purification, and detection of the polypeptide. It is understood that the polypeptide of the present invention may include one or more tags; multiple tags may include a combination of multiple identical tags or a combination of multiple different tags. For example, tags that facilitate the solubilization of the polypeptide include, but are not limited to, a nus tag or a maltose binding protein tag; tags that facilitate the purification of the polypeptide include, but are not limited to, a strep tag, a His tag, a GST tag, a pelB signal tag, or an ompA signal tag; tags that facilitate the detection of the polypeptide include, but are not limited to, a horseradish peroxidase (HRP) tag, a β-galactosidase tag, a luciferase tag, a green fluorescent protein (GFP) tag, an HcRed tag, a DsRed tag, or a cyan fluorescent protein (CFP) tag.

[0011] According to some embodiments of the present invention, the polypeptide can be artificially synthesized, or its encoding gene can be synthesized first and then biologically expressed.

[0012] A reagent according to an embodiment of the second aspect of the present invention includes the polypeptide described in the embodiment of the first aspect. Since the reagent adopts all the technical solutions of the polypeptide of the above embodiment, it has at least all the beneficial effects brought by the technical solutions of the above embodiment.

[0013] A biomaterial according to an embodiment of the third aspect of the present invention includes any one of B1) to B6): B1) a nucleic acid molecule encoding the polypeptide described in the embodiments of the first aspect of the present invention; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) a biological cell containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3); B5) transgenic plant tissue containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3); B6) A transgenic plant organ containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3).

[0014] According to some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the polypeptide in a host cell. This DNA may include not only a promoter for initiating transcription of the polypeptide-encoding gene, but also a terminator for terminating transcription of the polypeptide-encoding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0015] According to some embodiments of the present invention, the vector may be a plasmid, a cosmid, a phage or a viral vector.

[0016] According to some embodiments of the present invention, the recombinant vector may be a recombinant vector obtained by inserting a DNA molecule encoding the polypeptide into the multiple cloning site of the vector.

[0017] According to some embodiments of the present invention, the biological cells include prokaryotic cells and eukaryotic cells. The prokaryotic cells include bacteria or algae. The eukaryotic cells include fungi, mammalian cells, or insect cells.

[0018] According to some embodiments of the present invention, the biological cell may be a biological cell obtained by introducing B1) the nucleic acid molecule, B2) the expression cassette, or B3) the recombinant vector into a starting biological cell.

[0019] According to some embodiments of the present invention, the biological cell, the transgenic plant tissue or the transgenic plant organ does not contain reproductive material.

[0020] Use of the polypeptide described in the first embodiment, the reagent described in the second embodiment, or the biomaterial described in the third embodiment according to the fourth aspect of the present invention in any one of C1) to C3): C1) Regulate plant growth and development; C2) Preparation of products for regulating plant growth and development; C3) Plant breeding.

[0021] According to some embodiments of the present invention, the regulating plant growth and development is promoting plant growth and development or inhibiting plant growth and development.

[0022] According to some embodiments of the present invention, the promoting plant growth and development includes promoting the growth of plant roots.

[0023] According to some embodiments of the present invention, inhibiting plant growth and development includes inhibiting the growth of plant roots.

[0024] According to some embodiments of the present invention, the product includes at least one of a reagent and a kit.

[0025] According to some embodiments of the present invention, the plant is any one of the following: D1) Monocots or dicots; D2) Cruciferous vegetables; D3) Arabidopsis thaliana; D4) Arabidopsis thaliana.

[0026] According to a fifth aspect of the present invention, a method for regulating plant growth and development comprises the following steps: increasing the expression level of the polypeptide described in the embodiment of the first aspect in the plant; Alternatively, the polypeptide or the agent described in the embodiment of the second aspect is exogenously applied to the plant.

[0027] According to some embodiments of the present invention, the regulating plant growth and development is inhibiting plant growth and development; and the exogenously applied concentration of the polypeptide is not less than 10 μM.

[0028] According to some embodiments of the present invention, the regulating plant growth and development is inhibiting plant growth and development; and the exogenously applied concentration of the polypeptide is 10 μM to 100 μM. For example, it can be 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, or 100 μM.

[0029] According to some embodiments of the present invention, the regulating plant growth and development is promoting plant growth and development; and the exogenously applied concentration of the polypeptide is no higher than 1 μM.

[0030] According to some embodiments of the present invention, the regulating plant growth and development is promoting plant growth and development; and the exogenously applied concentration of the polypeptide is 0.1 μM-1 μM. For example, it can be 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, or 1 μM.

[0031] According to some embodiments of the present invention, the plant is any one of the following: D1) Monocots or dicots; D2) Cruciferous vegetables; D3) Arabidopsis thaliana; D4) Arabidopsis thaliana.

[0032] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Effects of 0.1 µM BRP2 on Arabidopsis root growth; A: 5 days of culture, B: 7 days of culture; Figure 2 Effects of 1 μM BRP2 on Arabidopsis root growth; A: 5 days of culture, B: 7 days of culture; Figure 3Effects of 10 μM BRP2 on Arabidopsis root growth; A: MS group cultured for 5 days, B: BRP2 group cultured for 5 days, C: MS group cultured for 7 days, D: BRP2 group cultured for 7 days; Figure 4 Results of Arabidopsis root length test after treatment with different concentrations of BRP2 for different days; A: 0.1 μM BRP2, B: 1 μM BRP2, C: 10 μM BRP2; ns indicates no significant difference, * indicates significant difference ( p <0.05), *** indicates a very significant difference ( p <0.001). DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described 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 those skilled in the art without creative work are all within the scope of protection of the present invention.

[0035] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0036] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0037] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0038] “Not higher than” means less than or equal to, and should be understood as including the number itself.

[0039] “Not less than” means greater than or equal to and should be understood as including the number itself.

[0040] The peptide BRP2 was commissioned to be synthesized by Jier Biochemical (Shanghai) Co., Ltd. with a purity of 98.78%.

[0041] A 1 mM BRP2 stock solution was prepared as follows: Before weighing, calibrate the balance to avoid electrostatic interference. Weigh 1.1740 g of peptide BRP2 using a balance and transfer to 1 mL of ultrapure water (HPLC grade). Vortex for 30 seconds and sonicate for 5 minutes to dissolve the peptide BRP2. Filter sterilize and aliquot into sterile EP tubes for long-term storage at -80°C.

[0042] The preparation method of MS solid culture medium with different concentrations of BRP2 is as follows: Weigh 2.16 g MS powder, 14.7 g sucrose, and 3.67 g agar powder, respectively. Dissolve the MS powder and sucrose in 490 mL of water, stirring until completely dissolved. Divide the mixture equally into seven 100 mL Erlenmeyer flasks (70 mL / flask). Add 0.52 g agar powder to each flask. Seal the flasks with parchment paper and sterilize them in an autoclave at 121°C for 30 min. Remove the sterilized MS solid medium and store it in a 60°C incubator to prevent solidification.

[0043] Use a 100 µL pipette to transfer 70 µL of the 1 mM BRP2 stock solution to 70 mL of MS medium and stir to obtain a 1 µM BRP2 concentration in MS medium. Use a 10 µL pipette to transfer 7 µL of the 1 mM BRP2 stock solution to 70 mL of MS medium and stir to obtain a 0.1 µM BRP2 concentration in MS medium. Use a 10 µL pipette to transfer 0.7 µL of the 1 mM BRP2 stock solution to 70 mL of MS medium and stir to obtain a 10 nM BRP2 concentration in MS medium. Aliquot the medium into culture dishes while hot and store at 4°C after solidification.

[0044] Example 1 A polypeptide fragment consisting of 10 amino acids (named: BRP2) was screened from corn through bioinformatics, and its amino acid sequence is shown in SEQ ID NO: 1. The molecular formula of the polypeptide BRP2 was calculated using a peptide molecular weight calculator: 51 H 81 N 13 O 15 , theoretical average molecular weight (M) = 1116.25 g / mol.

[0045] PSFPNEVAKK (SEQ ID NO: 1).

[0046] Test example Place an appropriate amount of dried Arabidopsis seeds in a sterile 1.5 mL centrifuge tube and add 75% alcohol (5-10 times the volume of the seeds) to ensure they are completely submerged. Place the tube on a vortex shaker and vortex at low speed for 1-2 minutes to allow the seeds to fully contact the alcohol. Then, let it rest for 10-15 minutes to disinfect. Be careful not to overdo or underdosing, as this will affect seed viability and disinfection effectiveness. In a clean bench, carefully remove the alcohol from the tube using a 1000 μL pipette. Add 1 mL of sterile water to the tube and place it on a vortex shaker again. Vortex at low speed for 1-2 minutes to fully dissolve any alcohol remaining on the seed surface. Then, let it rest for 1-2 minutes. Once the seeds have settled, remove the top layer of sterile water with a pipette. Repeat this sterile water wash procedure 3-5 times, ensuring that the top layer is completely removed each time to minimize alcohol residue and avoid removing the seeds. After the final wash, remove as much sterile water as possible from the tube, retaining only the seeds. In a clean bench, add an equal volume of 0.1 (w / v)% sterile agar solution to the centrifuge tube containing the seeds. In the clean bench, open the MS solid medium (mock) and MS medium plates containing different concentrations of BRP2. Open the lids of the culture dishes at an angle of approximately 30-45 degrees to avoid contamination caused by complete opening. Use a sterile blue pipette tip to draw up an appropriate amount of seed suspension and evenly spot the seeds on the surface of the culture medium. Keep the spacing between each spot approximately 1 cm. Each dish contains 50 seeds, with two replicates per experimental group, for a total of 100 seeds. When spotting, keep the pipette tip as close to the surface of the culture medium as possible, but avoid contact with it to prevent contamination. After spotting, quickly cover the culture dish and seal it with parafilm to prevent water loss and external contamination. Cultivate the seeds in a plant culture room at 22°C under fluorescent light with a 16-hour light / 8-hour dark cycle. Root growth of the Arabidopsis thaliana plants was observed on days 5 and 7, and root length was measured and counted.

[0047] The results are as follows Figure 1 、 Figure 2 shown.

[0048] After 5 and 7 days of culture, the root length of Arabidopsis seedlings treated with 0.1 µM BRP2 was significantly longer than that of the control group. After 5 days of culture, the root length of Arabidopsis seedlings treated with 1 µM BRP2 was significantly longer than that of the control group, while the root length of Arabidopsis seedlings treated with 10 µM BRP2 was almost the same as that of the control group. This indicates that 0.1 µM and 1 µM BRP2 promote the growth and development of Arabidopsis seedlings, while the higher concentration of 10 µM BRP2 has a significant inhibitory effect on the growth and development of Arabidopsis seedlings.

[0049] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A polypeptide, characterized in that The polypeptide is any one of A1) to A3): A1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; A2) a polypeptide having the function of regulating plant growth and development obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1; A3) A fusion polypeptide obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

2. A reagent, characterized in that Comprising the polypeptide of claim 1.

3. A biomaterial, characterized in that Including any of B1) to B4): B1) a nucleic acid molecule encoding the polypeptide according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) A biological cell containing the nucleic acid molecule described in B1), the expression cassette described in B2, or the recombinant vector described in B3; the biological cell does not contain any propagation material.

4. Use of the polypeptide according to claim 1, the reagent according to claim 2, or the biomaterial according to claim 3 in any one of C1) to C3): C1) Regulate plant growth and development; C2) Preparation of products for regulating plant growth and development; C3) Plant breeding.

5. The use according to claim 4, characterized in that The product includes at least one of a reagent and a kit.

6. The use according to claim 4, characterized in that The plant is any one of the following: D1) Monocots or dicots; D2) Cruciferous vegetables; D3) Arabidopsis thaliana; D4) Arabidopsis thaliana.

7. A method for regulating plant growth and development, characterized in that: The steps include: increasing the expression level of the polypeptide according to claim 1 in a plant; Alternatively, the polypeptide or the agent according to claim 2 is exogenously applied to the plant.

8. The method according to claim 7, characterized in that The regulating plant growth and development is inhibiting plant growth and development; the exogenously applied concentration of the polypeptide is not less than 10 μM.

9. The method according to claim 7, characterized in that The regulating plant growth and development is promoting plant growth and development; the exogenously applied concentration of the polypeptide is no higher than 1 μM.

10. The method according to claim 7, characterized in that The plant is any one of the following: D1) Monocots or dicots; D2) Cruciferous vegetables; D3) Arabidopsis thaliana; D4) Arabidopsis thaliana.

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