阅读说明：本技术 一种提高棉花抗旱性的多肽类抗旱剂及其应用 (Polypeptide drought-resistant agent for improving cotton drought resistance and application thereof ) 是由 孔祥强 胡秋月 唐薇 罗振 卢合全 尹静 秦澜 于 2021-09-17 设计创作，主要内容包括：本发明公开了一种提高棉花抗旱性的多肽类抗旱剂及其应用。本发明的多肽类抗旱剂的核心成分是小分子多肽DRP-C,其氨基酸序列为RRVPNGPDPIHN。本发明首次以人工合成小分子多肽DRP-C作为棉花抗旱剂的核心成分,利用叶片喷施的方式,通过诱导棉花体内ABA合成基因表达的方式,提高棉花体内ABA含量,提高棉花抗旱能力。试验表明：施加DRP-C水溶液后,通过提高棉花本身ABA提高了棉花的抗旱性、干旱胁迫下的存活率,促进了干旱胁迫下棉花的生长。(The invention discloses a polypeptide drought-resistant agent for improving cotton drought resistance and application thereof. The core component of the polypeptide drought resistant agent is small molecular polypeptide DRP-C, and the amino acid sequence of the polypeptide drought resistant agent is RRVPNGPDPIHN. The artificially synthesized small molecular polypeptide DRP-C is used as a core component of the cotton drought-resistant agent for the first time, the ABA content in cotton is increased by using a leaf spraying mode and inducing ABA synthetic gene expression in cotton, and the drought-resistant capability of cotton is improved. The test shows that: after the DRP-C aqueous solution is applied, the drought resistance of the cotton and the survival rate under drought stress are improved by improving the ABA of the cotton, and the growth of the cotton under drought stress is promoted.)

1. The application of the small molecule polypeptide DRP-C in improving the drought resistance of crops is disclosed, wherein the amino acid sequence of the small molecule polypeptide DRP-C is RRVPNGPDPIHN, and the crops are cotton, wheat, corn, peanut, millet or sorghum.

2. A polypeptide drought-resistant agent for improving the drought resistance of cotton, which is characterized in that the main active ingredient of the agent is the small-molecule polypeptide DRP-C as claimed in claim 1.

3. The polypeptide drought-resistant agent for improving cotton drought resistance of claim 2, wherein the drought-resistant agent is an aqueous solution of DRP-C, and the final concentration of DRP-C is 5-20 μ M.

4. The polypeptide drought resistant agent for improving cotton drought resistance of claim 3, wherein the final concentration of DRP-C is 10 μ M.

5. The method for applying the polypeptide drought-resistant agent as claimed in claim 3 or 4, wherein the application is directly carried out on the leaf surface when the cotton is about to suffer from drought stress.

6. The method of claim 5, wherein the drought-resistant agent is applied to cotton planted indoors, and the drought-resistant agent is prepared into 10 μ M aqueous solution of DRP-C by directly using tap water, and sprayed on leaves.

7. The method for applying the polypeptide drought-resistant agent as claimed in claim 5, wherein the drought-resistant agent is prepared into 200 times of storage liquid for cotton planted in a field, and the storage liquid is directly diluted in the field and sprayed on leaf surfaces; or directly preparing 10 mu M of DRP-C working solution in the field, and spraying the leaf surfaces by using a large field sprayer or an unmanned aerial vehicle.

8. The method for applying the polypeptide drought-resistant agent as claimed in claim 5, wherein the small molecular polypeptide DRP-C is applied to the field simultaneously with the foliar fertilizer and/or other drought-resistant agents.

9. The method of using the polypeptide drought resistant agent of claim 8, wherein the final concentration of DRP-C +2mM CaCl is 10 μ M in the field₂+2mM KCl for combined application to raise drought resisting effect.

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a polypeptide drought-resistant agent for improving drought resistance of cotton and application thereof.

Background

The fresh water resources in China are scarce, the total amount is about 28000 billions of cubic meters, and the per-capita water resource amount is only 1/4 which is the average level in the world. Nearly half of our country's land is stressed by drought and semi-drought. Drought stress inhibits plant growth, severely affecting crop yield. Under drought stress, on one hand, the plants promote the root systems to absorb water by synthesizing and accumulating osmotic protective substances and other strategies, and on the other hand, transpiration is reduced by regulating and controlling the closure of stomata, so that the drought resistance of the plants is improved, and the drought stress is relieved. The plant hormones ABA (abscisic acid) and JA (jasmonic acid) and the like play important roles in regulating stomata to open and close and improving the drought resistance of plants. At present, exogenous application of hormones such as ABA and JA and chemical substances such as lanthanum chloride and calcium chloride can improve the drought resistance of plants, overexpression of synthetic genes such as ABA and JA can improve the content of endogenous ABA and JA of the plants, and the drought resistance of the plants can also be improved. However, ABA is easy to decompose under the action of light and is high in price, the effect is poor in a mode of irrigating ABA through leaves or roots, the ABA synthesis key gene is over-expressed in target crops, the ABA content in transgenic crops is increased, and the generated crops are transgenic, so that the transgenic safety evaluation problem is involved. Therefore, the applicant develops a way to improve the drought resistance of plants by spraying small-molecule polypeptide on the leaves to solve the above problems.

Small molecular polypeptides are substances with hormone-like functions, and more researches show that: the small molecular polypeptide has the functions of improving the resistance of plants to abiotic stress (salt stress, drought stress and the like) and biotic stress (various diseases), regulating and controlling the shedding of plant fruits, regulating and controlling the formation of root nodule bacteria of leguminous plants, regulating and controlling the absorption of nutrients by the plants and the like. Recent studies have found that: the polypeptide substance can induce the expression of ABA synthetic gene NCED in plants, and improve the ABA content of the overground parts of the plants, thereby improving the drought resistance of the plants. Therefore, the method finds the plant endogenous micromolecule polypeptide capable of improving the drought resistance of crops, carries out artificial synthesis, and sprays root or leaf sources to the crops, and has important significance for enhancing the drought resistance of the crops and improving the yield.

Disclosure of Invention

Based on the method, the invention provides a polypeptide drought-resistant agent for improving the drought resistance of cotton and application thereof. The invention firstly screens plant endogenous micromolecule polypeptide DRP-C sequences with higher drought resistance activity, adds water to prepare drought resistance agent for foliage spraying, can improve the drought resistance of plants, can be suitable for crops such as cotton, wheat, corn, peanut, millet or sorghum, and is particularly suitable for cotton drought resistance.

The invention provides a polypeptide drought-resistant agent for improving cotton drought resistance, which comprises an active ingredient of a small molecular polypeptide DRP-C (dry drought polypeptide of cotton) with 12 amino acids, wherein the amino acid sequence of the polypeptide drought-resistant agent is RRVPNGPDPIHN.

The preparation method of the drought resistant agent comprises the following steps: preparing DRP-C powder into DRP-C aqueous solution with final concentration of 5-20 μ M, preferably 10 μ M; or preparing the DRP-C into 10mM mother liquor, storing at low temperature (4 ℃), diluting into working solution of DRP-C10 mu M when in use, and then spraying crops such as cotton on leaf surfaces.

The invention also provides an application method of the polypeptide drought-resistant agent, which comprises the following steps: the drought-resistant agent is used for cotton planted indoors, tap water can be directly used for preparing the polypeptide drought-resistant agent, and the components and the final concentration of the drought-resistant agent are 10 mu M DRP-C. When the water content of the indoor culture crop soil reaches 60%, the leaves are sprayed with the polypeptide drought-resistant agent; the cotton for field planting can be prepared into 200 times of storage liquid by drought resistance agent, and can be directly diluted for use in the field, or can be prepared into 10 mu M of working solution of DRP-C in the field, and can be sprayed on the leaf surface by using a large field sprayer or an unmanned aerial vehicle, and the spraying amount is about 10L/mu.

The polypeptide drought-resistant agent can be applied to fields together with other leaf fertilizers, drought-resistant agents and other substances, is convenient to apply, and provides an effective method for improving the drought-resistant capability of crops. Preferably, the drought-resistant agent comprises a main functional component DRP-C and can be added with low-concentration CaCl₂And KCl, calcium ions and potassium ions play an important role in salt and drought resistance of plants, and the two substances can be effectively added into the drought resistance agentPromoting the expression of drought-resistant related genes and further improving the drought-resistant capability of crops. Which is added with low concentration of CaCl₂And KC1 comprises the following components: 10 μ M DRP-C +2mM CaCl₂+2mM KCl。

The invention has the following beneficial effects:

1. the invention adopts synthetic artificial polypeptide DRP-C for foliage spraying for the first time, and improves the drought resistance of crops by improving the ABA content in plants. The problems that ABA is easy to decompose under visible light, the price is high and the direct application effect is not good in the prior art are solved; the limitation that the ABA content is improved in a transgenic mode and safety evaluation is required is solved, and a foundation is laid for industrial application of the ABA content.

2. The DRP-C polypeptide sequence which is a key drought-resistant component synthesized by the invention is elaborately designed by the inventor of the application, and the polypeptide is proved to be capable of effectively inducing ABA synthetic gene expression in crops and improving the ABA content in the crops, so that the drought-resistant capability of the crops is improved. The test shows that: after the drought-resistant agent is applied, the drought resistance and the survival rate under drought stress of cotton are improved by improving the ABA of the cotton, and the growth of the cotton under drought stress is promoted.

3. The drought-resistant agent has simple components, can be used after being diluted by water, is convenient to prepare and use, has good safety and prominent drought-resistant effect, can be applied to production in a large area, relieves the drought stress of crops, and reduces the economic loss caused by the drought stress.

Drawings

FIG. 1 shows the effect of the treatment of one leaf of the test example by spraying the polypeptide drought-resistant agent on the ABA content of cotton. Note: CK is a clear water control place, and DRP-C is a drought-resistant agent treatment;

FIG. 2 shows the growth of cotton after 6 days of the test example of one leaf sprayed with the polypeptide drought-resistant agent. Note: CK is clear water contrast, and DRP-C is drought-resistant agent treatment;

FIG. 3 is a graph showing the effect of applying a polypeptide drought-resistant agent to one leaf of a test case on the survival rate of cotton. Note: CK is clear water contrast, and DRP-C is drought-resistant agent treatment;

FIG. 4 shows the effect of spraying polypeptide drought-resistant agent on the dry weight of a single plant of field cotton on both leaf surfaces in the experimental example. Note: CK is clear water contrast, and DRP-C is drought-resistant agent treatment.

Detailed Description

The technical solution and the technical effects thereof are further explained in the following with reference to the specific embodiments and the accompanying drawings, and the following description is only for explaining the present invention, but not limiting the present invention in any way, and any changes or substitutions based on the present invention are within the protection scope of the present invention. The methods of the present invention are conventional in the art unless otherwise specified. The reagents of the present invention are commercially available unless otherwise specified.

EXAMPLE I determination of the amino acid sequence of a DRP-C polypeptide and Synthesis of a DRP-C polypeptide

(1) Determination of DRP-C amino acid sequence

The inventor searches a database of small molecule polypeptides in plants, finds a large number of small molecule polypeptides, combines a drought stress induced expression mode and sequence characteristics of related genes in cotton, performs related analysis and research of bioinformatics, and finally determines that a DRP-C sequence with high activity suitable for cotton is as follows: RRVPNGPDPIHN are provided.

(2) DRP-C polypeptide Synthesis and preservation

The required polypeptide DRP-C is synthesized by a polypeptide synthesis biological company by using a solid phase synthesis method, and purified by using a reverse high performance liquid chromatography and a low temperature freeze drying method to obtain DRP-C polypeptide powder with the purity of more than 95%. The DRP-C polypeptide powder is stored at-80 deg.C for a long period. The prepared working solution mother liquor of the drought-resistant agent can be stored for 1 month at 4 ℃.

Example two, drought resistance agent formulation and application

The DRP-C polypeptide synthesized in example was prepared into a mother liquor with a concentration of 10mM DRP-C using tap water, and stored at 4 ℃ for further use.

For the cotton seedlings planted in the field cultivated by indoor soil, the working solution flow for preparing 1L of drought resistance agent is as follows: firstly, 0.5L of tap water is added into a 1 container, 1ml of 10mM DRP-C solution is added, the volume is fixed to 1L, and the mixture is fully and uniformly mixed to obtain 10 mu M DRP-C aqueous solution. The working solution with large demand can be multiplied according to the method. The prepared working solution of the drought resistant agent is directly and uniformly sprayed on the leaves by a sprayer.

The first test example: influence of the drought resistance agent on ABA content of cotton leaves and survival rate under drought

Taking a plastic pot with the diameter of 10cm and the height of 12cm, and filling the plastic pot with a seedling culture substrate; uniformly sowing 15 full No. K836 delinted seeds in a matrix at a sowing depth of 2 cm; the opening of the plastic pot is covered with a transparent plastic film, and the plastic pot is placed in a greenhouse (24-30 ℃) for sprouting and seedling emergence. Removing the plastic film after seedling alignment, thinning the seedlings until the cotton seedlings grow to have the first true leaves, placing the seedlings in a greenhouse until each pot contains 5 cotton seedlings with consistent growth vigor, illuminating for 14 hours, darkness for 10 hours, temperature of 28 ℃, humidity of 50 percent, and continuing to grow. Irrigating tap water 1 time every 3 days, performing saturation irrigation when the cotton seedling grows to 3-4 true leaves, and then not irrigating.

The test is set to 6 repetitions, each repetition is respectively 5 cotton seedlings, and the drought resistance agent treatment group and the clear water control group are respectively 30 cotton seedlings. Spraying drought-resistant agent DRP-C (10 mu M DRP-C aqueous solution) on leaves on the 3 rd day after saturation irrigation for treatment; leaves were sprayed with equal volume of clear water as control. After 1 day of treatment, taking the leaves to measure the ABA content in the leaves, and finding that the ABA content in the leaves treated by spraying the drought-resistant agent is obviously higher than that of the leaves treated by clear water (figure 1); after 6 days of treatment, the leaves of the control cotton seedlings are found to have wilting phenotype, but the leaves of the cotton seedlings treated by the drought-resistant agent grow normally (figure 2), then tap water is used for saturated irrigation, the survival rate of the cotton seedlings is counted on the 3 rd day after irrigation, the survival rate of the cotton seedlings treated by the drought-resistant agent is found to be remarkably higher than that of the control (as shown in figure 3, the survival rate of a drought-resistant agent treatment group is 73%, and the survival rate of a clear water control group is 37%), and the drought-resistant agent treatment obviously enhances the drought-resistant capability of the cotton.

Test example two: influence of foliage spray drought-resisting agent on drought resistance of field cotton

And (3) sowing the full and full cotton K836 seeds in the Amelanchier-free field in 5-month and 2-day 2020. Spraying drought resistant agent (10 mu M DRP-C aqueous solution) and clear water for contrast treatment, randomly arraying in blocks, and repeating for 3 times. Cell area 20m²The method adopts 4-row area, and plants are planted at equal row spacing, the row spacing is 76cm, and the planting spacing is 15 cm. And performing daily management on cotton according to local cotton planting habits. In the early flowering stage, in the soilWhen the cotton plants with the soil water content of about 60 percent of the maximum water content feel drought stress and are continuously in a rainless period of 1 week, after 4 pm, the cotton plants are sprayed with a drought-resistant agent on leaves, and the cotton plants are sprayed with clear water as a contrast. After spraying, the cotton treated by the control group and the DRP-C is not watered, and after 2 weeks of spraying, the dry weight of the cotton plants is measured, and the growth vigor of the cotton seedlings treated by the drought-resistant agent is found to be obviously better than that of the control group, and the dry weight of the cotton seedlings is obviously higher than that of the control group (as shown in figure 4), which indicates that the drought-resistant agent treatment enhances the drought-resistant capability of the cotton.