Growth-promoting peptides, plant growth regulators, their preparation methods and applications

Abstract

This invention provides a growth-promoting polypeptide, a plant growth regulator, its preparation method, and its application, belonging to the field of plant biology. The amino acid sequence of the growth-promoting polypeptide is: L-V-V-V-N-N-S-G-P-S-P-G-I-G-H. The plant growth regulator using this growth-promoting polypeptide can accelerate the plant growth and development process, with a moderate effect on leaves (no significant difference compared to leaves in a normal growth state), without causing adverse reactions, and the effect is beneficial.

Description

Technical Field

[0001] This invention belongs to the field of plant biology and relates to a growth-promoting polypeptide, a plant growth regulator, its preparation method and application. Background Technology

[0002] Against the backdrop of continued global population growth and increasingly severe climate change, agricultural production faces significant challenges. To address these challenges, improving crop yield and quality has become one of the core objectives of agricultural development. Plant growth regulators are synthetically produced or extracted from microorganisms that regulate plant growth and development. These regulators are widely used to promote crop growth and effectively control plant physiological activities, such as regulating growth rate, promoting flowering, accelerating fruit ripening, and optimizing plant morphology. Currently, research and development on plant growth regulators mainly focuses on some traditional plant hormones and their analogues (such as auxins, gibberellins, cytokinins, naphthaleneacetic acid, ethephon, etc.), plant biostimulants (such as humic acid, chitin and chitosan derivatives, microbial inoculants, etc.).

[0003] With the deepening of scientific research, traditional plant growth regulators are gradually being supplemented and improved by new bioactive substances. In particular, peptides, due to their high bioactivity and low environmental impact, are increasingly attracting the attention of scientists and agricultural technicians. Peptides typically consist of 20 to 100 amino acid residues, possessing diverse structural and functional properties. They are not only essential nutrients in plants but also act as signaling molecules to regulate plant growth and development. Therefore, peptides have great development and utilization value and application prospects in agricultural production. However, research on the application of peptides in crops is currently relatively scarce, especially in promoting plant growth and development. Summary of the Invention

[0004] This invention provides a growth-promoting polypeptide, a plant growth regulator, its preparation method, and its application. The plant growth regulator using this growth-promoting polypeptide can accelerate the plant growth and development process without any other adverse effects.

[0005] To achieve the above objectives, the present invention provides a growth-promoting polypeptide with the amino acid sequence: LVVVNNSGPSPGIGH.

[0006] The present invention also provides a plant growth regulator, wherein the growth-promoting polypeptide described in the above technical solution is the main functional component, and the amino acid sequence of the growth-promoting polypeptide is: LVVVNNSGPSPGIGH.

[0007] Preferably, the concentration of the growth-promoting polypeptide is 0.5-2.0 μM.

[0008] Preferably, the plant growth regulator further includes an organosilicon solution for leaf wetting, wherein the concentration of the organosilicon solution is 1‰.

[0009] The present invention also provides a method for preparing a plant growth regulator according to any of the above technical solutions, comprising the following steps:

[0010] Dissolve the growth-promoting polypeptide powder in water to prepare a 2mM growth-promoting polypeptide stock solution for later use.

[0011] Add the organosilicon solution to deionized water to prepare a 1‰ organosilicon aqueous solution, mix thoroughly and set aside.

[0012] The plant growth regulator containing the growth-promoting polypeptide stock solution was added to the organosilicon aqueous solution and stirred evenly to obtain a plant growth regulator containing the growth-promoting polypeptide.

[0013] Preferably, the concentration of the growth-promoting polypeptide is 0.5-2.0 μM, and the concentration of the organosilicon solution is 1‰.

[0014] Preferably, the prepared growth-promoting polypeptide stock solution should be prepared and used immediately. If it is not used for more than 40 days, the stock solution should be stored at -70°C; if it is used within 40 days, the stock solution should be stored at -20°C; if it is used within one week, it should be stored at 4°C.

[0015] The present invention also provides the application of the plant growth regulator according to any of the above technical solutions in promoting plant growth and development.

[0016] Preferably, when applying the plant growth regulator, it is sprayed directly onto the leaf surface of the plant at a concentration of 0.5-2.0 μM.

[0017] Preferably, the plant is selected from at least one of wheat, Arabidopsis thaliana, lettuce, and tobacco.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0019] 1. The growth-promoting polypeptides used in the plant growth regulators provided by this invention are artificially synthesized polypeptides, which are easy to obtain and can be synthesized in large quantities;

[0020] 2. The plant growth regulator provided by this invention has a simple preparation and application method, is highly operable in the field, and does not require special training for the application personnel. It is convenient and practical to operate.

[0021] 3. The plant growth regulator provided by this invention has a moderate effect on leaves (no significant difference compared with leaves in normal growth state), does not cause adverse reactions, and has beneficial effects. Attached Figure Description

[0022] Figure 1 Spraying with a 1 μM plant growth regulator to promote the aboveground growth of wheat was shown to be effective. A: Wheat growth after 7 days of treatment with the plant growth regulator; B: Wheat growth indicators after 7 days of treatment with the plant growth regulator; * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01).

[0023] Figure 2 Foliar application of a 1 μM growth-promoting peptide to promote the leaf growth of Arabidopsis thaliana is shown in the following figures: A: Foliar application of the 1 μM growth-promoting peptide promotes the aboveground growth of Arabidopsis thaliana; B: Rosette leaf growth of Arabidopsis thaliana 7 days after treatment with the growth-promoting peptide; C: Leaf area of ​​different leaf positions of Arabidopsis thaliana 7 days after treatment with the growth-promoting peptide. * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01).

[0024] Figure 3 Spraying with a 1 μM growth-promoting peptide as a plant growth regulator promotes lettuce leaf growth. A: Growth of hydroponically grown lettuce treated with the growth-promoting peptide for seven days; B: Relevant growth indicators of lettuce after 7 days of treatment with the growth-promoting peptide; * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0025] Figure 4 Tobacco growth was promoted by spraying plant growth regulators with different concentrations of growth-promoting peptides. A: Tobacco growth after 7 days of treatment with different concentrations of growth-promoting peptides; B: Tobacco growth indicators after 7 days of treatment with different concentrations of growth-promoting peptides; different letters indicate significant differences (P<0.05).

[0026] Figure 5 To promote tobacco growth, a plant growth regulator containing 1 μM growth-promoting peptide was applied to the roots. A: Growth of hydroponically grown tobacco 7 days after root application of 1 μM growth-promoting peptide; B: Tobacco growth indicators 7 days after root application of 1 μM growth-promoting peptide; * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01).

[0027] Figure 6 To promote tobacco growth by foliar spraying with a plant growth regulator containing 1 μM growth-promoting peptide five times, where A: phenotypic graphs of tobacco treated with the growth-promoting peptide five times; B: growth indicators of tobacco treated with the growth-promoting peptide five times; * indicates significant difference (P<0.05).

[0028] Figure 7 To promote tobacco growth by foliar spraying with a plant growth regulator containing 1 μM growth-promoting peptide three times, where A: phenotypic graphs of tobacco treated with the growth-promoting peptide three times; B: growth indicators of tobacco treated with the growth-promoting peptide three times; * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01).

[0029] Figure 8 Tobacco growth was promoted by foliar spraying with a 1 μM growth-promoting peptide at different growth stages. A: Phenotypic diagram of tobacco treatments at different growth stages; B: Maximum leaf area of ​​tobacco treatments at different growth stages; ** indicates extremely significant differences (P<0.01). Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] Plant growth regulator, comprising 1 μM growth-promoting polypeptide and organosilicon solution; wherein the organosilicon solution is dissolved in deionized water and the concentration of the organosilicon solution is 1‰.

[0033] The preparation method is as follows:

[0034] Weigh the growth-promoting polypeptide powder and dissolve it in water to prepare a 2mM growth-promoting polypeptide stock solution for later use.

[0035] Take a sample of the organosilicon solution and add it to deionized water to prepare an organosilicon solution with a concentration of 1‰. Mix thoroughly.

[0036] 100 μL of the above-obtained growth-promoting polypeptide stock solution was added to 200 mL of the prepared organosilicon solution and stirred evenly with a glass rod to obtain 200 mL of plant growth regulator.

[0037] Example 2

[0038] Plant growth regulator, comprising 0.5 μM growth-promoting polypeptide and organosilicon solution; wherein the organosilicon solution is dissolved in deionized water and the concentration of the organosilicon solution is 1‰.

[0039] The preparation method is as follows:

[0040] Weigh the growth-promoting polypeptide powder and dissolve it in water to prepare a 2mM growth-promoting polypeptide stock solution for later use.

[0041] Take a sample of the organosilicon solution and add it to deionized water to prepare an organosilicon solution with a concentration of 1‰. Mix thoroughly.

[0042] Add 50 μL of the above-obtained growth-promoting polypeptide stock solution to 200 mL of the prepared organosilicon solution, stir evenly with a glass rod, and obtain 200 mL of plant growth regulator.

[0043] Example 3

[0044] Plant growth regulator, comprising 2 μM growth-promoting polypeptide and organosilicon solution; wherein the organosilicon solution is dissolved in deionized water and the concentration of the organosilicon solution is 1‰.

[0045] The preparation method is as follows:

[0046] Weigh the growth-promoting polypeptide powder and dissolve it in water to prepare a 2mM growth-promoting polypeptide stock solution for later use.

[0047] Take a sample of the organosilicon solution and add it to deionized water to prepare an organosilicon solution with a concentration of 1‰. Mix thoroughly.

[0048] Take 200 μL of the above-obtained growth-promoting polypeptide stock solution and add it to 200 mL of the prepared organosilicon solution. Stir well with a glass rod to obtain 200 mL of plant growth regulator.

[0049] Performance testing

[0050] Indoor test

[0051] Taking Example 1 as an example, the plant growth regulator prepared in Example 1 was applied to the leaves of different plants according to the application method. The leaf area, fresh weight, dry weight, and other growth indicators of the control group with the applied growth-promoting peptide and the control group without the peptide were compared.

[0052] To simulate mature plants grown under field conditions, we used foliar spraying to evaluate plant responses. To test the effects of growth-promoting peptide plant growth regulators on plant growth and development, we treated wheat, Arabidopsis thaliana, and lettuce with the prepared plant growth regulators. In the tobacco test, different concentrations of the growth-promoting peptide plant growth regulator were prepared and applied to tobacco; foliar spraying and root application of the growth-promoting peptide plant growth regulator were used to treat tobacco; and the effect of the growth-promoting peptide plant growth regulator on the growth and development of tobacco was observed by applying the regulators at different numbers of times.

[0053] Treatment 1: Plump Kenong 199 wheat seeds were selected, surface-sterilized, and germinated. The germinated seeds were then transplanted into a 1 / 2 Hoagland nutrient solution (composed of 1000×Calcium Salt Solution and 1 / 2 Hoagland modified nutrient salts, purchased from Beijing Coolaber Company; 0.5 mL of 1000×Calcium Salt Solution and 275.5 mg of 1 / 2 Hoagland modified nutrient salts were dissolved in 1 L of water; this medium provides the essential nutrients for plant growth). When the seedlings reached the one-leaf-one-heart stage, seedlings with uniform growth were selected for further treatment. A control group (water spray) and an experimental group (foliar spray with 1 μM growth-promoting peptide) were established. Spraying was performed once a day for 7 consecutive days, with each treatment replicated 3 times. Aboveground length and root length were measured with a ruler, and fresh weight was determined using a balance. After drying at 60℃ to constant weight, dry weight was measured.

[0054] Treatment 2: Plump wild-type Columbia (Col-0) Arabidopsis thaliana seeds were selected. 25 days after sowing, Arabidopsis thaliana seedlings with uniform growth were selected for treatment. A control group (foliar spraying with water) and an experimental group (foliar spraying with 1 μM growth-promoting peptide) were set up, sprayed once a day for 7 consecutive days. Leaf area was measured using ImageJ image processing software.

[0055] Treatment 3: Select plump, fast-growing lettuce seeds. When the seedlings reach the 5-leaf stage, select those with uniform growth for treatment. Set up a CK group (foliar spraying with water) and an experimental group (foliar spraying with 1μM growth-promoting peptide). Spray once a day for 7 consecutive days. The leaf area was measured using ImageJ image processing software.

[0056] Treatment 4: Plump K326 tobacco seeds were selected and sown in nutrient soil for growth. Once the seedlings reached the large cross stage, seedlings with similar morphology and growth were selected for further treatment. A control group (0 μM growth-promoting peptide sprayed foliarly) and experimental groups (0.5 μM, 1 μM, and 2 μM growth-promoting peptides) were established. Each treatment was repeated three times, with spraying once a day for seven consecutive days. Maximum leaf length and width were measured with a ruler, and fresh weight was determined using a balance. After drying at 60℃ to constant weight, dry weight was measured. Maximum leaf area was calculated using the formula: Maximum leaf area = Maximum leaf length × Maximum leaf width × 0.6345.

[0057] Treatment 5: Plump K326 tobacco seeds were selected and sown in nutrient soil. After germination, they were transplanted into 200 mL of 1 / 2 Hoagland nutrient solution. When the seedlings reached the large cross stage, tobacco seedlings with uniform growth were selected for treatment, setting up a CK group and an experimental group (1 / 2 Hoagland nutrient solution with 100 μL of 2 mM growth-promoting polypeptide stock solution added and thoroughly mixed) and cultured for 7 days. The maximum leaf length and maximum leaf width were measured with a ruler, and the fresh weight was weighed using a balance. After drying at 60℃ to constant weight, the dry weight was measured. The maximum leaf area was calculated using the formula: Maximum leaf area = Maximum leaf length × Maximum leaf width × 0.6345.

[0058] Treatment 6: Plump K326 tobacco seeds were selected and planted in nutrient soil until the seedlings reached the large cross stage. Tobacco seedlings with similar morphology and growth were selected for treatment, with a CK group (foliar spraying with water) and an experimental group (foliar spraying with 1μM growth-promoting peptide). Spraying was performed once a day for 5 consecutive days. Maximum leaf length and width were measured with a ruler, and fresh weight was determined using a balance. After drying at 60℃ to constant weight, dry weight was measured. Maximum leaf area was calculated using the formula: Maximum leaf area = Maximum leaf length × Maximum leaf width × 0.6345.

[0059] Treatment 7: Select plump K326 tobacco seeds for sowing. When the seedlings reach the large cross stage, select tobacco seedlings with similar morphology and growth for treatment. Set up a CK group (foliar spray with water) and an experimental group (foliar spray with 1μM growth-promoting peptide). Spray once every other day for 3 times. After 5 days, the maximum leaf length and maximum leaf width are measured with a ruler, and the fresh weight is weighed with a balance. After drying at 60℃ to constant weight, the dry weight is measured. The maximum leaf area is calculated using the formula: Maximum leaf area = Maximum leaf length × Maximum leaf width × 0.6345.

[0060] Treatment 8: Select plump K326 tobacco seeds for sowing. When the seeds reach the large cross stage, select tobacco seedlings with similar morphology and growth for treatment. Two groups were established: a control group (foliar spray with water) and an experimental group (foliar spray with 1 μM growth-promoting peptide). One application was given; subsequent applications were given once each during the seedling stage and the rosette stage. The promoting effect of three applications of the growth-promoting peptide on the growth and development of tobacco was observed. The maximum leaf area was calculated using the formula: Maximum leaf area = Maximum leaf length × Maximum leaf width × 0.6345.

[0061] The results show that plant growth regulators can promote plant growth and development. For example... Figure 1 As shown in Figure A, after spraying the experimental group of wheat leaves with a 1 μM plant growth regulator containing growth-promoting polypeptide for 7 consecutive days, and spraying the control plants with the same amount of water, it was found that the root length, aboveground part length, root fresh weight, aboveground part fresh weight, root dry weight, and aboveground part dry weight of wheat all increased. Among them, the aboveground part length (P<0.05) and aboveground part weight (P<0.01) of wheat increased significantly, by 7.75% and 26.52%, respectively. Figure 1B). The above results indicate that spraying wheat seedlings with 1 μM of growth-promoting peptides can significantly promote the growth of the aboveground parts of wheat seedlings.

[0062] Arabidopsis thaliana was treated with 1 μM growth-promoting peptides, while control plants were sprayed with an equal amount of water once daily for seven consecutive days. The results showed that the growth-promoting peptide treatment significantly promoted the growth of Arabidopsis thaliana seedlings. Figure 2 A). For example Figure 2 As shown in Figure BC, 1 μM of the growth-promoting peptide increased the leaf area of ​​Arabidopsis thaliana, with the 5th and 6th leaves showing a significant increase compared to the control, increasing by 17.33% (P<0.05) and 80.80% (P<0.01), respectively. These results indicate that spraying Arabidopsis thaliana with 1 μM of the growth-promoting peptide can significantly promote the growth of its aboveground parts.

[0063] Lettuce seedlings were treated with 1 μM growth-promoting peptides, while control plants were sprayed with the same amount of water once a day for seven consecutive days. The results showed that the growth-promoting peptide treatment significantly promoted the growth of lettuce seedlings. Figure 3 A). For example Figure 3 As shown in Figure B, 1 μM of the growth-promoting peptide increased the leaf area, total plant weight, and dry weight of lettuce by 20.96%, 16.04%, and 37.74%, respectively (P<0.01). These results indicate that spraying lettuce with 1 μM of the growth-promoting peptide significantly promotes leaf growth.

[0064] Depend on Figure 4 As shown in Figure A, foliar spraying of different concentrations of growth-promoting peptides can promote the growth of tobacco seedlings, with foliar spraying of 1 μM and 2 μM growth-promoting peptides showing more significant effects. Compared with the control, the maximum leaf area of ​​tobacco seedlings sprayed with 0.5 μM, 1 μM, and 2 μM growth-promoting peptides was significantly increased (P<0.05), by 6.51%, 11.10%, and 10.57%, respectively, compared with the control. Figure 4 B) Considering that the foliar spraying effects of 1μM and 2μM growth-promoting peptides are similar, 1μM growth-promoting peptides were selected as the working concentration of tobacco growth promoter in the later stage.

[0065] Depend on Figure 5 A indicates that both foliar and root application promote the growth of tobacco seedlings. After foliar absorption, the maximum leaf area and aboveground weight of tobacco seedlings treated with foliar spraying were significantly increased (P<0.01), by 33.51% and 45.27% respectively compared to the control. Figure 5 B). After hydroponically grown tobacco seedlings absorb growth-promoting polypeptides in their roots, such as... Figure 5As shown in Figure C, the maximum leaf area, root length, total plant weight, and above-ground part weight were all significantly increased (P<0.05), increasing by 20.69%, 25.43%, 30.01%, and 34.81% respectively compared with the control group. These results indicate that both foliar spraying and continuous root application of the 1 μM growth-promoting peptide to tobacco seedlings can promote seedling growth.

[0066] Depend on Figure 6 A and Figure 7 A indicates that, after 5 days of treatment, spraying with the growth-promoting polypeptide at different frequency all had a growth-promoting effect on tobacco seedlings. Compared with the control, spraying with the growth-promoting polypeptide five times significantly increased the maximum leaf area, total plant weight, and above-ground part weight of tobacco seedlings (P<0.05), increasing by 40.40%, 25.59%, and 32.08%, respectively, compared with the control. Figure 6 B); After three applications of growth-promoting peptides, compared with the control, the maximum leaf area of ​​tobacco seedlings (P<0.01), total plant weight, and above-ground part weight (P<0.05) were significantly increased, increasing by 51.65%, 42.94%, and 45.21% respectively compared with the control. Figure 7 B). The above results indicate that, under the same treatment time, foliar spraying with the growth-promoting peptide for 5 and 3 times both significantly promoted the growth of tobacco seedlings.

[0067] The growth-promoting effect of 1 μM growth-promoting polypeptide was observed by spraying it three times during the tobacco seedling stage, the seedling stage, and the rosette stage. The results are as follows: Figure 8 As shown, continuous foliar spraying of growth-promoting peptides during the large cross stage and seedling stage effectively promoted the growth of the above-ground parts of tobacco. Compared with the control group, the area of ​​the largest tobacco leaf increased by 38.98% (P<0.01). This can reduce the negative impact of the new environment on tobacco after transplanting, help it recover growth quickly, enhance the survival ability of tobacco seedlings after transplanting, rapidly establish a stable growth state, and further improve the nutrient accumulation and quality of leaves.

Claims

1. The application of a plant growth regulator in promoting plant growth and development, characterized in that, The plant growth regulator has growth-promoting polypeptides as its main functional component, and the amino acid sequence of the growth-promoting polypeptides is: LVVVNNSGPSPGIGH; The plant is selected from at least one of wheat, Arabidopsis thaliana, lettuce, and tobacco.

2. The application of the plant growth regulator according to claim 1 in promoting plant growth and development, characterized in that, The concentration of the growth-promoting polypeptide is 0.5-2.0 μM.

3. The application of the plant growth regulator according to claim 2 in promoting plant growth and development, characterized in that, The plant growth regulator also includes an organosilicon solution for leaf wetting, wherein the concentration of the organosilicon solution is 1‰.

4. The application of the plant growth regulator according to claim 2 or 3 in promoting plant growth and development, characterized in that, The preparation method of the plant growth regulator includes the following steps: Dissolve the growth-promoting polypeptide powder in water to prepare a 2mM growth-promoting polypeptide stock solution for later use. Add the organosilicon solution to deionized water to prepare a 1‰ organosilicon aqueous solution, mix thoroughly and set aside. The plant growth regulator containing the growth-promoting polypeptide stock solution was added to the organosilicon aqueous solution and stirred evenly to obtain a plant growth regulator containing the growth-promoting polypeptide.

5. The application of the plant growth regulator according to claim 4 in promoting plant growth and development, characterized in that, The prepared growth-promoting polypeptide stock solution should be prepared immediately before use. If it is not used for more than 40 days, store the stock solution at -70℃; if it is used within 40 days, store the stock solution at -20℃.

6. The application of the plant growth regulator according to claim 4 in promoting plant growth and development, characterized in that, If the prepared growth-promoting polypeptide stock solution is to be used within one week, it should be stored at 4°C.

7. The application of the plant growth regulator according to claim 1 in promoting plant growth and development, characterized in that, When applying the plant growth regulator, it is sprayed directly onto the leaf surface of the plant, and the concentration of the growth-promoting polypeptide is 0.5-2.0 μM.

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