Growth promoting polypeptide, plant growth regulator as well as preparation method and application of plant growth regulator

Abstract

The invention provides a growth promoting polypeptide, a plant growth regulator and a preparation method and application thereof, and belongs to the field of plant biology. The amino acid sequence of the growth promoting polypeptide is as follows: L-V-V-V-N-N-S-G-P-S-P-G-I-G-H. The invention further discloses a preparation The plant growth regulator adopting the growth-promoting polypeptide can accelerate the growth and development process of plants, has moderate influence on leaves (has no obvious difference compared with leaves in a normal growth state), does not cause adverse reaction, and has beneficial effects.

Description

Technical Field

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

[0002] In the current context of continuous global population growth and increasingly severe climate change, agricultural production faces major challenges. In order to meet these challenges, improving crop yield and quality has become one of the core goals of agricultural development. Plant growth regulators are chemical substances that are artificially synthesized or extracted from microorganisms and have a regulatory effect on 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. At present, the research and development of plant growth regulators mainly focuses on some traditional plant hormones and their analogues (such as auxin, gibberellin, cytokinin, naphthyl acetic acid, ethephon, etc.), plant biostimulants (such as humic acid, chitin and chitosan derivatives, microbial agents, etc.).

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

[0004] The present invention provides a growth-promoting polypeptide, a plant growth regulator, and a preparation method and application thereof. The plant growth regulator using the growth-promoting polypeptide can accelerate the growth and development process of plants without other additional adverse performances.

[0005] In order to achieve the above-mentioned purpose, the present invention provides a growth-promoting polypeptide, whose amino acid sequence is: LVVVNNSGPSPGIGH.

[0006] The present invention also provides a plant growth regulator, wherein the growth-promoting polypeptide described in the above technical scheme 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 also includes an organosilicon solution for wetting the leaf surface, wherein the concentration of the organosilicon solution is 1‰.

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

[0010] Dissolve the growth-promoting polypeptide powder in water to prepare a growth-promoting polypeptide mother solution with a concentration of 2 mM for later use;

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

[0012] The growth-promoting polypeptide mother solution is added into the organic silicon 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 mother solution should be prepared before use. If it is not used for more than 40 days, the mother solution should be stored at -70°C; if it is used within 40 days, the mother 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 a use of the plant growth regulator according to any of the above technical solutions in promoting plant growth and development.

[0016] Preferably, during application, the plant growth regulator is sprayed directly on the surface of plant leaves at a spraying concentration of 0.5-2.0 μM.

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

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

[0019] 1. The growth-promoting polypeptide used in the plant growth regulator provided by the present invention is an artificially synthesized polypeptide, which is easy to obtain and can be synthesized in large quantities;

[0020] 2. The preparation and use methods of the plant growth regulator provided by the present invention are simple, and the field operability is strong. The application personnel do not need to undergo special training, and the operation is convenient and practical;

[0021] 3. The plant growth regulator provided by the present invention has a moderate effect on leaves (no significant difference compared with leaves in a normal growth state), does not cause adverse reactions, and has a beneficial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Spraying a plant growth regulator containing 1 μM growth-promoting peptide promoted the growth of the aboveground part of wheat, where A: wheat growth after 7 days of treatment with growth-promoting peptide; B: wheat growth index after 7 days of treatment with growth-promoting peptide; * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01);

[0023] Figure 2 The plant growth regulator spraying treatment with 1 μM growth-promoting peptide promoted the growth of Arabidopsis leaves, where A: 1 μM growth-promoting peptide spraying on the leaves promoted the growth of the aboveground part of Arabidopsis; B: The growth of Arabidopsis rosette leaves 7 days after the growth-promoting peptide spraying treatment; C: The leaf area of ​​Arabidopsis at different leaf positions 7 days after the growth-promoting peptide treatment; * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01);

[0024] Figure 3 Spraying a plant growth regulator containing 1 μM growth-promoting peptide promoted the growth of lettuce leaves, where A: growth of hydroponic lettuce after seven days of treatment with growth-promoting peptide; B: growth indicators of lettuce after seven days of treatment with growth-promoting peptide; * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0025] Figure 4 Spraying plant growth regulators with different concentrations of growth-promoting peptides promoted tobacco growth, where 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 Plant growth regulators that applied 1 μM growth-promoting peptides to the roots promoted tobacco growth, where A: growth of hydroponic tobacco after 7 days of root application of 1 μM growth-promoting peptides; B: tobacco growth indicators after 7 days of root application of 1 μM growth-promoting peptides; * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01);

[0027] Figure 6 The plant growth regulator of 1 μM growth-promoting peptide was sprayed on the leaves for 5 times to promote tobacco growth, where A: Tobacco phenotype after 5 treatments of growth-promoting peptide; B: Tobacco growth index after 5 treatments of growth-promoting peptide; * indicates significant difference (P<0.05);

[0028] Figure 7 The plant growth regulator of 1 μM growth-promoting peptide was sprayed on the leaves three times to promote tobacco growth, where A: Tobacco phenotype after three times of growth-promoting peptide treatment; B: Tobacco growth index after three times of growth-promoting peptide treatment; * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01);

[0029] Figure 8 The plant growth regulator of 1 μM growth-promoting peptide was sprayed on the leaves at different periods to promote tobacco growth, where A: phenotype of tobacco treated at different periods; B: maximum leaf area of ​​tobacco treated at different periods; ** indicates extremely significant difference (P<0.01). DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] A plant growth regulator comprises a 1 μM growth-promoting polypeptide and an organosilicon solution; 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 growth-promoting polypeptide mother solution with a concentration of 2 mM for later use;

[0035] Take the organosilicon solution and add it to deionized water to prepare a solution with an organosilicon concentration of 1‰, and mix thoroughly;

[0036] Take 100 μL of the growth-promoting polypeptide mother solution obtained above and add it to 200 mL of the prepared organosilicon solution, stir evenly with a glass rod to obtain 200 mL of plant growth regulator.

[0037] Example 2

[0038] The plant growth regulator comprises 0.5 μM growth-promoting polypeptide and organosilicon solution; 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 growth-promoting polypeptide mother solution with a concentration of 2 mM for later use;

[0041] Take the organosilicon solution and add it to deionized water to prepare a solution with an organosilicon concentration of 1‰, and mix thoroughly;

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

[0043] Example 3

[0044] A plant growth regulator comprises a 2μM growth-promoting polypeptide and an organosilicon solution; 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 growth-promoting polypeptide mother solution with a concentration of 2 mM for later use;

[0047] Take the organosilicon solution and add it to deionized water to prepare a solution with an organosilicon concentration of 1‰, and mix thoroughly;

[0048] Take 200 μL of the growth-promoting polypeptide mother solution obtained above and add it to 200 mL of the prepared organosilicon solution, stir evenly 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 used to treat leaves of different plants according to the application method, and the growth indicators such as leaf area, fresh weight, and dry weight of the control group sprayed with the growth-promoting polypeptide and the control group without the addition were compared.

[0052] In order to simulate mature plants growing under field conditions, we used foliar spraying to evaluate the plant response. In order to test the effect of growth-promoting polypeptide plant growth regulators on plant growth and development, wheat, Arabidopsis, and lettuce were treated with the prepared plant growth regulators. In the test on tobacco, different concentrations of growth-promoting polypeptide plant growth regulators were prepared and treated with tobacco; the growth-promoting polypeptide plant growth regulators were used for foliar spraying and root application; and the growth-promoting polypeptide plant growth regulators were sprayed on tobacco at different times to observe the growth-promoting effect of tobacco.

[0053] Treatment 1: Select full-grained wheat seeds of Kenong 199, germinate after surface disinfection, and transplant into 1 / 2 Hoagland nutrient solution (composed of 1000×Calcium Salt Solution and 1 / 2Hoagland modified nutrient salts, purchased from Beijing Coolaber Company, 0.5mL 1000×Calcium Salt Solution and 275.5mg 1 / 2Hoagland modified nutrient salts are dissolved in 1L water when used. This culture medium can be used as a nutrient solution to provide the necessary elemental nutrients for plant growth). When the seeds grow to one leaf and one heart, select seedlings with consistent growth for treatment. Set up CK group (spraying water) and experimental group (spraying 1μM growth-promoting peptide on the leaves), spray once a day for 7 consecutive days, and repeat each treatment 3 times. The length of the aboveground part and the length of the root were measured by a ruler, the fresh weight was weighed with a balance, and the dry weight was measured after drying at 60℃ to constant weight.

[0054] Treatment 2: Select plump wild-type Columbia (Col-0) Arabidopsis seeds, and select Arabidopsis seedlings with consistent growth 25 days after sowing for treatment. Set up the CK group (foliar spraying of water) and the experimental group (foliar spraying of 1 μM growth-promoting peptide) once a day for 7 consecutive days. Image J image processing software was used to measure the leaf area.

[0055] Treatment 3: Select full and fast-growing lettuce seeds, and select seedlings with the same growth when they grow to the 5-leaf stage for treatment. Set up the CK group (foliar spraying of water) and the experimental group (foliar spraying of 1 μM growth-promoting peptide) once a day for 7 consecutive days. Image J image processing software was used to measure the leaf area.

[0056] Treatment 4: Select full K326 tobacco seeds and sow them in nutrient soil for growth. When the seedlings grow to the large cross stage, select tobacco seedlings with basically the same morphology and growth for treatment; set up the CK group (0μM growth-promoting peptide sprayed on the leaves) and the experimental group (0.5μM growth-promoting peptide, 1μM growth-promoting peptide and 2μM growth-promoting peptide). Each treatment of the experiment was repeated 3 times, sprayed once a day, and sprayed continuously for 7 days. The maximum leaf length and maximum leaf width were measured by a ruler, the fresh weight was weighed with a balance, and the dry weight was measured after drying at 60℃ to constant weight. The maximum leaf area was calculated according to the formula: maximum leaf area = maximum leaf length × maximum leaf width × 0.6345.

[0057] Treatment 5: Select full K326 tobacco seeds and sow them in nutrient soil. After germination, transplant them into 200mL1 / 2Hoagland nutrient solution. When the seedlings grow to the large cross stage, select tobacco seedlings with consistent growth for treatment. Set up CK group and experimental group (1 / 2Hoagland nutrient solution is added with 100μL of 2mM growth-promoting polypeptide mother solution and mixed thoroughly) and culture for 7 days. The maximum leaf length and maximum leaf width were measured by a ruler, and the fresh weight was weighed with a balance. The dry weight was measured after drying at 60℃ to constant weight. The maximum leaf area was calculated according to the formula: maximum leaf area = maximum leaf length × maximum leaf width × 0.6345.

[0058] Treatment 6: Select full K326 tobacco seeds and plant them in nutrient soil until the big cross stage. Select tobacco seedlings with basically the same morphology and growth for treatment. Set up the CK group (foliar spraying of water) and the experimental group (foliar spraying of 1 μM growth-promoting peptide) and spray once a day for 5 consecutive days. The maximum leaf length and maximum leaf width were measured by a ruler, the fresh weight was weighed with a balance, and the dry weight was measured after drying at 60°C to constant weight. The maximum leaf area was calculated according to the formula: Maximum leaf area = maximum leaf length × maximum leaf width × 0.6345.

[0059] Treatment 7: Select full K326 tobacco seeds for sowing, wait until the seedlings grow to the large cross stage, select tobacco seedlings with basically the same morphology and growth for treatment, set up the CK group (foliar spraying of water) and the experimental group (foliar spraying of 1 μM growth-promoting peptide) and spray once every other day, spray 3 times. After 5 days, the maximum leaf length and maximum leaf width were measured by a ruler, the fresh weight was weighed with a balance, and the dry weight was measured after drying at 60°C to constant weight. The maximum leaf area was calculated according to the formula: Maximum leaf area = maximum leaf length × maximum leaf width × 0.6345.

[0060] Treatment 8: Select full K326 tobacco seeds for sowing, grow to the big cross stage, select tobacco seedlings with basically the same morphology and growth for treatment, set up CK group (foliar spraying of water) and experimental group (foliar spraying of 1μM growth-promoting peptide), spray once; subsequently spray once at the seedling stage and cluster stage. Observe the effect of spraying growth-promoting peptides three times on the growth of tobacco. The maximum leaf area is calculated according to the formula: maximum leaf area = maximum leaf length × maximum leaf width × 0.6345.

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

[0062] Arabidopsis thaliana was treated with 1 μM growth-promoting peptide, and the control plants were sprayed with the same amount of water, once a day, for 7 consecutive times. The results showed that the growth-promoting peptide treatment could significantly promote the growth of Arabidopsis thaliana seedlings ( Figure 2 A). Figure 2 As shown in BC, 1 μM growth-promoting peptide can increase the area of ​​Arabidopsis leaves, and the leaves at the 5th and 6th leaf positions are significantly increased compared with the control, increasing by 17.33% (P<0.05) and 80.80% (P<0.01), respectively. The above results show that spraying 1 μM growth-promoting peptide on Arabidopsis can significantly promote the growth of the aboveground part of Arabidopsis.

[0063] The lettuce seedlings were treated with 1 μM growth-promoting peptides, and the control plants were sprayed with the same amount of water, once a day, for 7 consecutive times. The results showed that the growth-promoting peptide treatment can significantly promote the growth of lettuce seedlings ( Figure 3 A). Figure 3 As shown in Figure B, 1 μM growth-promoting peptide can increase the leaf area, whole plant weight and dry weight of lettuce by 20.96%, 16.04% and 37.74% respectively (P<0.01). The above results show that spraying 1 μM growth-promoting peptide on lettuce can significantly promote the growth of lettuce leaves.

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

[0065] Depend on Figure 5 A shows that both foliar application and root application can promote the growth of tobacco seedlings. After leaf absorption, the maximum leaf area and aboveground weight of tobacco seedlings sprayed with foliar spray increased significantly (P<0.01), increasing by 33.51% and 45.27% respectively compared with the control ( Figure 5 B) After the hydroponic tobacco seedlings absorb the growth-promoting peptides in their roots, Figure 5As shown in C, the maximum leaf area, root length, whole plant weight and aboveground weight were significantly increased (P<0.05), increasing by 20.69%, 25.43%, 30.01% and 34.81% respectively compared with the control group. The above results show that spraying the tobacco seedlings with 1 μM growth-promoting peptides on the leaves and continuously applying them to the roots can promote the growth of tobacco seedlings.

[0066] Depend on Figure 6 A and Figure 7 A shows that after 5 days of treatment, spraying different times of growth-promoting peptides has a growth-promoting effect on tobacco seedlings. Compared with the control, the maximum leaf area, whole plant weight and aboveground weight of tobacco seedlings after spraying 5 times of growth-promoting peptides were significantly increased (P<0.05), which increased by 40.40%, 25.59% and 32.08% respectively compared with the control ( Figure 6 B); Compared with the control, the maximum leaf area (P<0.01), whole plant weight and aboveground weight (P<0.05) of tobacco seedlings sprayed with growth-promoting peptides three times were significantly increased, increasing by 51.65%, 42.94% and 45.21% respectively compared with the control ( Figure 7 B). The above results show that under the same treatment time, foliar spraying of growth-promoting peptides 5 times and 3 times can significantly promote the growth of tobacco seedlings.

[0067] The growth-promoting effect of 1 μM growth-promoting peptide was observed by spraying three times at the tobacco cross stage, seedling stage and cluster stage. Figure 8 As shown in the results, continuous foliar spraying of growth-promoting peptides during the large cross stage and seedling stage has a good promoting effect on the growth of the aboveground part of tobacco. Compared with the control group, the area of ​​the largest tobacco leaf increased by 38.98% (P<0.01). It can reduce the negative impact of the new environment on tobacco after transplanting, help to quickly resume growth, enhance the survival ability of tobacco seedlings after transplanting, quickly establish a stable growth state, and further improve the nutrient accumulation and quality of the leaves.

Claims

1. A growth-promoting polypeptide, It is characterized in that Its amino acid sequence is: LVVVNNSGPSPGIGH.

2. Plant growth regulators, It is characterized in that The main functional component of the invention is the growth-promoting polypeptide described in claim 1, and the amino acid sequence of the growth-promoting polypeptide is: LVVVNNSGPSPGIGH.

3. The plant growth regulator according to claim 2, It is characterized in that The concentration of the growth-promoting polypeptide is 0.5-2.0 μM.

4. The plant growth regulator according to claim 2 or 3, It is characterized in that The plant growth regulator also includes an organic silicon solution for wetting the leaf surface, wherein the concentration of the organic silicon solution is 1‰.

5. A method for preparing a plant growth regulator according to any one of claims 2 to 4, It is characterized in that The steps include: Dissolve the growth-promoting polypeptide powder in water to prepare a growth-promoting polypeptide mother solution with a concentration of 2 mM for later use; Add the organosilicon solution into deionized water to prepare a 1‰ organosilicon aqueous solution, mix thoroughly and set aside; The growth-promoting polypeptide mother solution is added into the organic silicon aqueous solution and stirred evenly to obtain a plant growth regulator containing the growth-promoting polypeptide.

6. The preparation method according to claim 5, It is characterized in that The concentration of the growth-promoting polypeptide is 0.5-2.0 μM, and the concentration of the organosilicon solution is 1‰.

7. The preparation method according to claim 6, It is characterized in that The prepared growth-promoting polypeptide mother solution must be prepared before use. If it is not used for more than 40 days, store the mother solution at -70°C; if it is used within 40 days, store it at -20°C; if it is used within one week, store it at 4°C.

8. Use of the plant growth regulator according to any one of claims 2 to 4 in promoting plant growth and development.

9. The use according to claim 8, It is characterized in that When applied, the plant growth regulator is sprayed directly on the leaf surface of the plant at a spraying concentration of 0.5-2.0 μM.

10. The use according to claim 8 or 9, It is characterized in that The plant is selected from at least one of wheat, Arabidopsis, lettuce and tobacco.

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