A polypeptide that regulates plant growth and development and its applications

Abstract

This invention discloses a polypeptide for regulating plant growth and development and its applications, relating to the field of biotechnology. Specifically, this invention discloses any one of the following polypeptides: A1) a polypeptide with the amino acid sequence shown in SEQ ID NO:1; A2) a polypeptide with 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 attaching a tag to the N-terminus and / or C-terminus of A1) or A2). This polypeptide can effectively regulate plant growth and development, promoting plant growth and development at low concentrations and inhibiting plant growth and development at high concentrations.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a polypeptide that regulates plant growth and development and its applications. Background Technology

[0002] Roots are vital organs for plants to absorb water and mineral nutrients and to sense and respond to environmental signals. Regulating root growth is essential for root function and is crucial for plant growth, development, and crop yield. Plant peptide hormones are a class of polypeptides composed of several to dozens of amino acids. They function as signaling molecules within plants, regulating various aspects of plant stress and disease resistance, growth, and organ development. They have significant potential applications in agriculture, food processing, and breeding. Therefore, providing a peptide that can effectively regulate plant growth and development is of paramount importance. Summary of the Invention

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

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

[0005] The present invention also provides biomaterials related to the above-mentioned polypeptides.

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

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

[0008] According to a first aspect of the present invention, a polypeptide is any one of A1) to A3):

[0009] A1) A polypeptide with the amino acid sequence shown in SEQ ID NO: 1;

[0010] A2) A polypeptide that regulates plant growth and development by substituting and / or deleting and / or adding one or more amino acid residues into the amino acid sequence shown in SEQ ID NO: 1.

[0011] A3) A fusion peptide obtained by linking a tag to the N-terminus and / or C-terminus of A1) or A2).

[0012] The polypeptides according to embodiments of the present invention have at least the following beneficial effects:

[0013] The peptides in this embodiment can regulate plant growth and development. At low concentrations, they can effectively promote plant growth and development, and promote root growth; at high concentrations, they can effectively inhibit plant growth and development, and inhibit root growth.

[0014] According to some embodiments of the present invention, the tag includes at least one of the tags that facilitate the dissolution, purification, and detection of the polypeptide. It is understood that the polypeptide of the present invention may contain one or more tags; multiple tags may comprise a combination of multiple identical tags, or a combination of multiple different tags. For example: tags facilitating the dissolution of the polypeptide include, but are not limited to, nus tags or maltose-binding protein tags; tags facilitating the purification of the polypeptide include, but are not limited to, strep tags, His tags, GST tags, pelB signal tags, or ompA signal tags; tags facilitating the detection of the polypeptide include, but are not limited to, horseradish peroxidase (HRP) tags, β-galactosidase tags, luciferase tags, green fluorescent protein (GFP) tags, HcRed tags, DsRed tags, or cyan fluorescent protein (CFP) tags.

[0015] 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.

[0016] A reagent according to a second aspect of the present invention comprises the polypeptide described in the first aspect embodiment. Since the reagent employs all the technical solutions of the polypeptide described in the above embodiments, it possesses at least all the beneficial effects brought about by the technical solutions of the above embodiments.

[0017] A biomaterial according to a third aspect of the present invention comprises any one of B1) to B6):

[0018] B1) A nucleic acid molecule encoding the polypeptide described in the first aspect embodiment of the present invention;

[0019] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0020] B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0021] B4) Biological cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3);

[0022] 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);

[0023] 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).

[0024] 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 to initiate transcription of the polypeptide-encoding gene, but also a terminator to terminate transcription of the polypeptide-encoding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0025] According to some embodiments of the present invention, the vector may be a plasmid, a granule, a bacteriophage, or a viral vector.

[0026] 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.

[0027] 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.

[0028] According to some embodiments of the present invention, the biological cell may be a biological cell obtained by introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into a starting biological cell.

[0029] According to some embodiments of the present invention, the biological cells, the transgenic plant tissues, or the transgenic plant organs do not contain reproductive material.

[0030] The use of the polypeptide described in the first aspect embodiment, the reagent described in the second aspect embodiment, or the biomaterial described in the third aspect embodiment according to the fourth aspect embodiment of the present invention in any one of C1) to C3):

[0031] C1) Regulates plant growth and development;

[0032] C2) Prepare products that regulate plant growth and development;

[0033] C3) Plant breeding.

[0034] According to some embodiments of the present invention, the regulation of plant growth and development is to promote plant growth and development or inhibit plant growth and development.

[0035] According to some embodiments of the present invention, promoting plant growth and development includes promoting the growth of plant roots.

[0036] According to some embodiments of the present invention, the inhibition of plant growth and development includes inhibiting the growth of plant roots.

[0037] According to some embodiments of the present invention, the product includes at least one of reagents and kits.

[0038] According to some embodiments of the present invention, the plant is any of the following;

[0039] D1) Monocotyledonous or dicotyledonous plants;

[0040] D2) Cruciferous plants;

[0041] D3) Arabidopsis thaliana plants;

[0042] D4) Arabidopsis thaliana.

[0043] A method for regulating plant growth and development according to a fifth aspect embodiment of the present invention includes the following steps:

[0044] To increase the expression level of the polypeptide described in the first aspect embodiment in plants;

[0045] Alternatively, the polypeptide or the reagent described in the second aspect embodiment may be applied exogenously to the plant.

[0046] According to some embodiments of the present invention, the regulation of plant growth and development is to inhibit plant growth and development; the exogenous application concentration of the polypeptide is not less than 10 μM.

[0047] According to some embodiments of the present invention, the regulation of plant growth and development is to inhibit plant growth and development; the exogenous application concentration of the polypeptide is 10 μM-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.

[0048] According to some embodiments of the present invention, the regulation of plant growth and development is to promote plant growth and development; the exogenous application concentration of the polypeptide is not higher than 1 μM.

[0049] According to some embodiments of the present invention, the regulation of plant growth and development is to promote plant growth and development; the exogenous application 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.

[0050] According to some embodiments of the present invention, the plant is any of the following;

[0051] D1) Monocotyledonous or dicotyledonous plants;

[0052] D2) Cruciferous plants;

[0053] D3) Arabidopsis thaliana plants;

[0054] D4) Arabidopsis thaliana.

[0055] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0056] Figure 1 Effect of 0.1 µM BRP2 on Arabidopsis root growth; A: cultured for 5 days, B: cultured for 7 days;

[0057] Figure 2 The effect of 1 µM BRP2 on Arabidopsis root growth; A: cultured for 5 days, B: cultured for 7 days;

[0058] Figure 3 The effect 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;

[0059] Figure 4 The results of Arabidopsis root length detection after treatment with different concentrations of BRP2 for different numbers of days; A: 0.1 μM BRP2, B: 1 μM BRP2, C: 10 μM BRP2; ns indicates no significant difference, * indicates a significant difference ( p <0.05), *** indicates a highly significant difference ( p <0.001). Detailed Implementation

[0060] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0061] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0062] 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 the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0063] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0064] "Not higher than" means less than or equal to, and should be understood as including the number itself.

[0065] "Not less than" means greater than or equal to, and should be understood as including the number itself.

[0066] The peptide BRP2 was synthesized by Jier Biochemical (Shanghai) Co., Ltd., with a purity of 98.78%.

[0067] The preparation method of 1 mM BRP2 mother liquor is as follows:

[0068] Calibrate the balance before weighing to avoid electrostatic interference. Weigh 1.1740 g of peptide BRP2 using the balance, transfer it to 1 mL of ultrapure water (HPLC grade), vortex for 30 seconds, and then sonicate for 5 minutes to promote the dissolution of peptide BRP2. Filter to remove bacteria, dispense into sterile EP tubes, and store at -80℃ for long-term storage.

[0069] The methods for preparing MS solid culture media with different concentrations of BRP2 are as follows:

[0070] Weigh out 2.16 g MS powder, 14.7 g sucrose, and 3.67 g agar powder. Dissolve the MS powder and sucrose in 490 mL of water, stirring until completely dissolved. Divide the solution into seven 100 mL Erlenmeyer flasks (70 mL / flask), and add 0.52 g agar powder to each flask. Seal the flasks with parchment paper and autoclave them at 121°C for 30 min. Remove the sterilized MS solid culture medium and store it in a 60°C incubator to prevent solidification.

[0071] Using a 100 µL pipette, transfer 70 µL of 1 mM BRP2 stock solution to 70 mL of MS medium and mix well to obtain MS medium with a BRP2 concentration of 1 µM. Using a 10 µL pipette, transfer 7 µL of 1 mM BRP2 stock solution to 70 mL of MS medium and mix well to obtain MS medium with a BRP2 concentration of 0.1 µM. Using a 10 µL pipette, transfer 0.7 µL of 1 mM BRP2 stock solution to 70 mL of MS medium and mix well to obtain MS medium with a BRP2 concentration of 10 nM. While still warm, dispense the medium into petri dishes and store at 4°C after solidification.

[0072] Example 1

[0073] A 10-amino acid polypeptide fragment (named BRP2) was screened from maize using bioinformatics. Its amino acid sequence is shown in SEQ ID NO: 1. The molecular formula of polypeptide BRP2 was calculated using a polypeptide molecular weight calculator to be C1. 51 H 81 N 13 O 15 The theoretical average molecular weight (M) is 1116.25 g / mol.

[0074] PSFPNEVAKK (SEQ ID NO: 1).

[0075] Detection example

[0076] Place an appropriate amount of dried Arabidopsis seeds in a sterile 1.5 mL centrifuge tube, and add 5-10 times the seed volume of 75% alcohol, ensuring the seeds are completely submerged. Place the centrifuge tube on a vortex mixer and vortex at low speed for 1-2 minutes to ensure the seeds are fully in contact with the alcohol. Then let it stand for 10-15 minutes for sterilization, being careful not to sterilize for too long or too short a time, as this will affect seed viability and sterilization effectiveness. In a laminar flow hood, carefully remove the alcohol from the centrifuge tube using a 1000 μL pipette. Add 1 mL of sterile water to the centrifuge tube, and place it on a vortex mixer again. Vortex at low speed for 1-2 minutes to fully dissolve any residual alcohol on the seed surface. Let it stand for 1-2 minutes until the seeds settle, then remove the supernatant of sterile water using a pipette. Repeat the sterile water rinsing steps 3-5 times, ensuring each time that the supernatant is removed as much as possible to minimize alcohol residue, while avoiding removing the seeds. After the final rinse, remove as much sterile water as possible from the centrifuge tube, retaining only the seeds. In a laminar flow hood, add an equal volume of 0.1% (w / v) sterile agar solution to centrifuge tubes containing seeds. Open MS solid medium (Mock) and MS medium plates of different concentrations of BRP2 in the laminar flow hood, with the lids opened at approximately 30-45 degrees to avoid contamination from fully opening them. Using a sterile blue pipette tip, a suitable amount of seed suspension is drawn and evenly spotted onto the surface of the medium. The distance between each spot should be approximately 1 cm, with 50 seeds per plate. Two plates are repeated for each experimental group, for a total sample size of 100 seeds. When spotting, the pipette tip should be as close to the surface of the medium as possible, but not in contact with it, to prevent tip contamination. After spotting, quickly cover the medium and seal it with sealing film to prevent moisture loss and external contamination. Incubate in a plant culture chamber at 22℃ with a light cycle of 16 hours of light followed by 8 hours of darkness. Observe the root growth of Arabidopsis thaliana after 5 and 7 days of culture, and measure and count the root length.

[0077] The results are as follows Figure 1 , Figure 2 As shown.

[0078] After 5 and 7 days of cultivation, the root length of Arabidopsis seedlings treated with 0.1 µM BRP2 was significantly greater than that of the control group. After 5 days of cultivation, the root length of Arabidopsis seedlings treated with 1 µM BRP2 was significantly greater than that of the control group, while after 7 days of cultivation, the root length of Arabidopsis seedlings was almost the same as that of the control group. The root length of Arabidopsis seedlings treated with 10 µM BRP2 was significantly shorter than that of the control group. This indicates that 0.1 µM and 1 µM BRP2 have a promoting effect on the growth and development of Arabidopsis seedlings; and that a higher concentration of 10 µM BRP2 has a significant inhibitory effect on the growth and development of Arabidopsis seedlings.

[0079] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide is any one of A1) to A2): A1) A polypeptide with the amino acid sequence shown in SEQ ID NO: 1; A2) A fusion peptide obtained by attaching a tag to the N-terminus and / or C-terminus of the peptide described in A1).

2. A reagent, characterized in that, Includes the polypeptide described in claim 1.

3. A biomaterial, characterized in that, It is any one of B1) to B4): B1) A nucleic acid molecule encoding the polypeptide of 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 is a prokaryotic cell or a eukaryotic cell; the eukaryotic cell is a fungal, mammalian, or insect cell.

4. The use of the polypeptide of claim 1, the reagent of claim 2, or the biomaterial of claim 3 in any one of C1) to C4): C1) Promotes the growth of Arabidopsis thaliana roots; the exogenous application concentration of the polypeptide is 0.1 μM-1 μM; C2) Inhibits the growth of Arabidopsis thaliana roots; the exogenous application concentration of the polypeptide is not less than 10 μM; C3) Prepare a product that promotes the growth of Arabidopsis thaliana roots; the exogenous application concentration of the polypeptide is 0.1 μM-1 μM. C4) Prepare a product that inhibits the growth of Arabidopsis thaliana roots; the exogenous application concentration of the polypeptide is not less than 10 μM.

5. The application according to claim 4, characterized in that, The product includes at least one of reagents and reagent kits.

6. A method for promoting plant root growth, characterized in that, The plant in question is Arabidopsis thaliana; the steps include the following: The polypeptide of claim 1 or the reagent of claim 2 is applied exogenously to the plant; the concentration of the exogenous application of the polypeptide is 0.1 μM-1 μM.

7. A method for inhibiting plant root growth, characterized in that, The plant in question is Arabidopsis thaliana; the steps include the following: The polypeptide of claim 1 or the reagent of claim 2 is applied exogenously to the plant; the exogenous application concentration of the polypeptide is not less than 10 μM.

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