Application of small peptide GhDIRSp1 in enhancing drought resistance of plants in seedling stage

Abstract

The invention belongs to the technical field of biological agriculture, and particularly discloses application of a small peptide GhDIRSp1 in enhancing drought resistance of plants in a seedling stage. According to the application of the small peptide GhDIRSp1 in enhancing the drought resistance of plants in the seedling stage, the amino acid sequence of the small peptide GhDIRSp1 is as shown in SEQ ID NO: 1. The invention discloses an application of a small peptide GhDIRSp1 in enhancing drought resistance of a plant in a seedling stage, the small peptide GhDIRSp1 is applied to a plant root system, elongation of a main root and development of a lateral root of the plant can be effectively promoted, and the drought resistance of the plant is effectively enhanced.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, specifically relating to the application of the small peptide GhDIRSp1 in enhancing the drought resistance of plant seedlings. Background Technology

[0002] Cotton, as a vital global economic crop, is directly and significantly affected by drought stress in terms of yield and quality. In agricultural production, drought has become a key environmental factor restricting cotton seedling growth, boll formation, and fiber quality, especially in water-scarce regions where yield reduction due to drought is more pronounced. Therefore, developing efficient, green, and sustainable drought-resistant technologies for cotton is of great significance for ensuring the stable development of the cotton industry.

[0003] Currently, the main technical approaches to improving cotton drought resistance focus on two directions: first, the application of exogenous chemical substances, such as plant hormones like abscisic acid and jasmonic acid, to regulate cotton's osmotic protection mechanisms and stress responses, thereby enhancing its drought resistance; and second, the application of transgenic technology, which involves introducing drought-resistant genes to improve cotton's genetic characteristics and enhance its drought resistance. However, the large-scale application of exogenous hormones may lead to soil environmental imbalance and easily cause plant growth regulation disorders. Transgenic technology, on the other hand, faces challenges such as long breeding cycles, low gene transformation efficiency, and controversies regarding biosafety, limiting its large-scale application.

[0004] Small peptides, as naturally occurring signaling molecules in plants, possess characteristics such as high bioactivity, low toxicity, easy degradation, and environmental friendliness, making them ideal candidates for developing green drought-resistant agents. In recent years, studies have found that some plant small peptides can enhance plant tolerance to drought stress by regulating root development, stomatal movement, and antioxidant system activity. However, current research on small peptide drought resistance in cotton remains relatively scarce, severely limiting the development and application of cotton small peptide drought-resistant agents. Summary of the Invention

[0005] The present invention aims to provide the application of small peptide GhDIRSp1 in enhancing the drought resistance of plant seedlings. When applied to plant roots, GhDIRSp1 can effectively promote the elongation of the taproot and the development of lateral roots, thereby effectively enhancing the drought resistance of plants.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] Application of small peptide GhDIRSp1 in enhancing drought resistance in plant seedlings, wherein the amino acid sequence of the small peptide GhDIRSp1 is shown in SEQ ID NO:1.

[0008] Preferably, the plant is cotton or Arabidopsis thaliana.

[0009] Preferably, the small peptide GhDIRSp1 is applied externally to plant roots at a concentration of 0.1-1 μM.

[0010] Preferably, the small peptide GhDIRSp1 is applied to the plant seedling stage.

[0011] This invention also provides the application of the small peptide GhDIRSp1 as described above in the preparation of a plant seedling drought resistance enhancer.

[0012] The present invention also provides a plant seedling drought resistance enhancer, the enhancer comprising the small peptide GhDIRSp1 and an agriculturally acceptable carrier.

[0013] Preferably, the concentration of the small peptide GhDIRSp1 in the formulation is 0.1-1 μM.

[0014] The present invention also provides a method for enhancing the drought resistance of plant seedlings using the small peptide GhDIRSp1, comprising the following steps:

[0015] The synthetic peptide GhDIRSp1 was applied topically to the plant roots during the seedling stage, once every 2 days for a total of 3 times.

[0016] Preferably, the plant is cotton or Arabidopsis thaliana.

[0017] Preferably, the application concentration of the small peptide GhDIRSp1 is 0.1-1 μM.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] This invention aims to provide the application of the small peptide GhDIRSp1 in enhancing the drought resistance of plant seedlings. The root-specific expressed small peptide GhDIRSp1 was mined and identified from cotton root LncRNA sequencing data, filling the research gap of this type of functional small peptide in cotton and providing a new candidate molecule for the development of plant drought-resistant small peptides.

[0020] External application of the peptide GhDIRSp1 during the cotton seedling stage effectively increased the length of the taproot, lateral roots, and root surface area under drought conditions, and to some extent reduced the levels of physiological and biochemical indicators such as hydrogen peroxide and malondialdehyde, significantly improving the cotton seedlings' tolerance to drought stress. Application of this peptide to Arabidopsis thaliana effectively promoted taproot elongation and lateral root development, improved its growth status, and enhanced its drought resistance, indicating that the drought-resistant function of this peptide has a certain degree of universality, providing a basis for its application in other plants.

[0021] The externally applied peptide GhDIRSp1 has high bioactivity and low toxicity. It is also easy to apply, has a short cycle, and is easy to operate and promote, thus avoiding soil pollution.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 The root phenotype results of the exogenously applied small peptide GhDIRSp1 in Example 2;

[0024] Figure 2 The results show the root phenotypic data of the exogenously applied small peptide GhDIRSp1 in Example 2, wherein... Figure 2 In the graph, A represents the statistical chart of root length changes. Figure 2 B in the graph represents the lateral root count. Figure 2 C in the graph represents the root mean diameter. Figure 2 D in the graph represents the total surface area of ​​the root.

[0025] Figure 3 This is a diagram showing the aboveground phenotype results of the small peptide GhDIRSp1 applied externally in soil culture under drought stress in Example 3;

[0026] Figure 4 This is a diagram showing the root phenotype results of soil culture with exogenous application of the small peptide GhDIRSp1 under drought stress in Example 3;

[0027] Figure 5 The results show the root phenotypic data of the soil-cultured small peptide GhDIRSp1 under drought stress in Example 3, where... Figure 5 In the graph, A represents the total root length. Figure 5 B in the graph represents the statistical chart of the length of the taproot. Figure 5 C in the graph represents the root system quantity statistics. Figure 5 D in the graph represents the total root area. Figure 5 In the graph, E represents the root average diameter.

[0028] Figure 6 This is a diagram showing the aboveground phenotype results of the exogenous application of the small peptide GhDIRSp1 in soil culture under repeated drought stress experiments in Example 3;

[0029] Figure 7 This is a diagram showing the root phenotype results of the repeated drought stress experiment in Example 3, in which the small peptide GhDIRSp1 was applied externally in soil culture.

[0030] Figure 8 The data pertains to the root phenotype of the exogenous small peptide GhDIRSp1 in soil culture in Example 3. Figure 8 In the graph, A represents the total root length. Figure 8 B in the graph represents the statistical chart of the length of the taproot. Figure 8 C in the graph represents the root system quantity statistics. Figure 8 D in the graph represents the total root area. Figure 8 E in the figure represents the statistical graph of the root average diameter.

[0031] Figure 9 The graph shows the hydrogen peroxide content detection results in Example 3.

[0032] Figure 10 The graph shows the malondialdehyde content detection results in Example 3.

[0033] Figure 11 The phenotypic results of the exogenous peptide GhDIRSp1 in Arabidopsis thaliana in Example 4 are shown.

[0034] Figure 12 This is a graph showing the root index data of the exogenous peptide GhDIRSp1 in Arabidopsis thaliana in Example 4. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0037] Source of experimental materials:

[0038] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0039] Example 1

[0040] The specific experimental protocol for synthesizing the small peptide GhDIRSp1 is as follows:

[0041] The small peptide GhDIRSp1 was synthesized by Nanjing Peptide Research Biotechnology Co., Ltd. The synthesis method employed the Fmoc solid-phase synthesis method, using resin as a carrier. Amino acids were added stepwise from the C-terminus to the N-terminus, and the target polypeptide chain was constructed through cyclic reactions including deprotection, activation, and condensation. Finally, the polypeptide powder was obtained through cleavage and purification.

[0042] The specific steps are as follows: (1) Resin pretreatment: Select loading resin, use DCM to swell and remove Fmoc protecting groups, add Fmoc-AA-OH and DIEA to DCM, react at room temperature, and use ninhydrin detection to detect negative to judge the reaction progress. After the coupling reaction is completed, the reaction solution is dried and washed with DMF. (2) Amino acid condensation cycle: According to the polypeptide sequence, coupling is carried out sequentially from the C-terminus to the N-terminus. The reaction progress is judged by the negative detection of ninhydrin. After the coupling reaction is completed, the reaction solution is dried, washed with DMF and methanol and dried. (3) Cleavage and purification: The polypeptide with all side chain protecting groups removed is obtained by reacting the lysis buffer with the linear peptide resin. The crude linear peptide aqueous solution is filtered with a 0.45μm filter membrane, and the improved crude peptide is purified by high performance liquid chromatography. After high performance liquid purification, the finished peptide liquid with a purity greater than 95% is obtained. The liquid is pre-lyophilized and lyophilized to finally obtain polypeptide powder.

[0043] The amino acid sequence of the small peptide GhDIRSp1 is shown in SEQ ID NO:1.

[0044] SEQ ID NO:1:

[0045] LAYSGTFRKLPVLLRYFWVYWAWGNGLFGLFYKRTWTLI.

[0046] Example 2

[0047] The small peptide GhDIRSp1 powder synthesized in Example 1 was applied to cotton using plant seed bags. Different concentration gradients of small peptide GhDIRSp1 were prepared, and preliminary experiments were conducted to determine the optimal concentration of small peptide GhDIRSp1 for external application.

[0048] Treatment method: 0, 0.1, 0.5, and 1 μM solutions of the small peptide GhDIRSp1 were prepared and applied to plant seed bags for irrigation, once every two days for a total of three applications. Each application consisted of 10 mL per seed bag. Root length and lateral root number were observed and measured starting on day 2 after the end of the irrigated period (referred to as day 0). Root phenotypic data were collected on day 10, and taproot length, lateral root length, root diameter, and root surface area were measured. Results are as follows: Figures 1-2 As shown.

[0049] Depend on Figure 1 It can be seen that when cotton is cultivated in seed bags and 0, 0.1, 0.5 and 1 μM small peptide GhDIRSp1 is applied externally, the root growth is better when 0.5 μM small peptide GhDIRSp1 is applied externally.

[0050] Depend on Figure 2It can be seen that the external application of 0.5 μM small peptide GhDIRSp1 has a better effect on root growth and development. Compared with the control group and the external application concentrations of 0.1 μM and 1 μM, it effectively increased the main root length, lateral root length, root diameter and root surface area of ​​cotton.

[0051] Example 3

[0052] Example 1: The small peptide GhDIRSp1 powder synthesized by exogenous application to soil-grown cotton.

[0053] The specific experimental protocol is as follows: When cotton was cultivated in soil at the cotyledon expansion stage, the synthesized small peptide GhDIRSp1 powder from Example 1 was prepared into solutions of 0, 0.1, 0.5, and 1 μM, respectively. 15 mL of each solution was applied topically to the soil around the roots, once every two days for a total of three times. Other conditions and the environment remained consistent. At the two-leaf-one-heart stage, drought stress was applied. Phenotypic data, root index data, and physiological index measurements were collected when the leaves showed phenotypes. A repeat experiment was conducted with the same protocol. The results are as follows: Figures 3-8 Physiological indicator measurement results are as follows Figures 9-10 As shown.

[0054] Depend on Figure 3 and Figure 6 It can be seen that soil-grown cotton with external application of 0.5 μM GhDIRSp1 showed better aboveground growth under drought conditions.

[0055] Depend on Figure 4 and Figure 7 It can be seen that soil-grown cotton with external application of 0.5 μM GhDIRSp1 showed better root growth under drought conditions.

[0056] Depend on Figure 5 It can be seen that when soil-grown cotton is externally treated with 0.5 μM GhDIRSp1, the length of the taproot and the total surface area of ​​the root system are significantly increased under drought conditions.

[0057] Depend on Figure 8 It can be seen that when soil-grown cotton is externally treated with 0.5 μM GhDIRSp1, the total root length, taproot length, number of roots, and total root surface area are all significantly increased under drought conditions.

[0058] Depend on Figure 9 It can be seen that when soil-grown cotton is treated with 0.5 μM GhDIRSp1 externally, under drought conditions, the hydrogen peroxide content in the roots decreases slightly, while the hydrogen peroxide content in the leaves increases slightly but not significantly. This indicates that the damage to cells caused by drought stress is reduced to some extent.

[0059] Depend on Figure 10The results showed that, under drought conditions, external application of 0.5 μM GhDIRSp1 to soil-grown cotton resulted in a decrease in malondialdehyde content in the roots and a slight but insignificant increase in hydrogen peroxide content in the leaves. This indicates that external application of the small peptide GhDIRSp1 can effectively improve the drought resistance of cotton.

[0060] Example 4

[0061] Based on the optimal application concentration in cotton as described in Example 3, the small peptide GhDIRSp1 was added to Arabidopsis thaliana medium to a final concentration of 0.5 μM. Normal treatment and drought treatment (300 mM mannitol) groups were established. After 14 days of culture, phenotypes were collected and root phenotype indices were statistically analyzed. The results are as follows: Figures 11-12 As shown.

[0062] Depend on Figure 11 It can be seen that the exogenous addition of 0.5 μM GhDIRSp1 peptide to Arabidopsis thaliana culture under 300 mM mannitol treatment can effectively improve the growth and development of Arabidopsis thaliana.

[0063] Depend on Figure 12 It was found that the exogenous addition of 0.5 μM GhDIRSp1 peptide to Arabidopsis culture could increase the number of lateral roots under normal treatment conditions. Under 300 mM mannitol treatment, the main root length, lateral root length and number of Arabidopsis were significantly increased, and the root angle was significantly increased, which effectively improved the drought resistance of Arabidopsis.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of the small peptide GhDIRSp1 for enhancing drought resistance in the seedling stage of plants, characterized in that, The amino acid sequence of the small peptide GhDIRSp1 is shown as SEQ ID NO:

1.

2. Use according to claim 1, characterized in that, The plant is cotton or Arabidopsis.

3. Use according to claim 1, characterized in that, The small peptide GhDIRSp1 is externally applied to the plant root system at a concentration of 0.1-1 μM.

4. Use according to claim 3, characterized in that, The small peptide GhDIRSp1 is applied to the plant seedling stage.

5. The use of the small peptide GhDIRSp1 according to claim 1 in the preparation of a drought resistance enhancer for the plant seedling stage.

6. A plant seedling drought enhancement agent, characterized by, The enhancer comprises the small peptide GhDIRSp1 according to claim 1 and an agriculturally acceptable carrier.

7. The enhancer of claim 6 wherein, The concentration of the small peptide GhDIRSp1 in the preparation is 0.1-1 μM.

8. A method for enhancing drought resistance of seedling stage of plants by using the small peptide GhDIRSp1 according to claim 1, characterized in that, The method comprises the following steps: The small peptide GhDIRSp1 is synthesized, and the small peptide GhDIRSp1 is externally applied to the plant root system at the plant seedling stage, once every 2 days, for a total of 3 times.

9. The method of claim 8, wherein, The plant is cotton or Arabidopsis.

10. The method of claim 8, wherein, The application concentration of the small peptide GhDIRSp1 is 0.1-1 μM.

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