Dreb stress resistance fusion protein type crop biostimulant and preparation method thereof

Abstract

The application discloses a DREB stress-resistant fusion protein type crop biological stimulant and a preparation method thereof, and belongs to the technical field of agricultural biological stimulants. The DREB stress-resistant fusion protein type crop biological stimulant is prepared by using a DREB stress-resistant fusion protein with a purity greater than or equal to 95% as a core component, compounding oligosaccharides, compound amino acids, potassium humate, chelated trace elements, an osmotic aid and a stabilizer in specific proportions, and adopting a low-temperature light-avoiding and low-speed stirring special process. The application changes a traditional transgenic application mode of the DREB protein, and the DREB protein expressed in vitro is made into an agricultural preparation, so that crop stress-resistant, photosynthetic and nutrient transport related genes can be targeted activated, and the synergistic effect of gene regulation and nutrient supply is realized. The product has strong stress resistance, remarkable yield increase and quality improvement effects, the components are green and safe, the stability is good, the preparation process is simple, the product is suitable for large-scale production, and the product can be applied to the whole growth period of grain crops, economic crops and fruits and vegetables.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biostimulant technology, specifically referring to a DREB stress-resistant fusion protein-type crop biostimulant and its preparation method. Background Technology

[0002] In agricultural production, biotic and abiotic stresses such as drought, salinity, extreme temperatures, pests and diseases, and soil nutrient imbalances can easily hinder crop growth, ultimately leading to reduced yields and lower quality. Currently, widely used yield-increasing and stress-resistance products mainly include chemical regulators, conventional amino acid fertilizers, humic acid preparations, and microbial inoculants. Chemical regulators are prone to pesticide residue problems and can also damage the soil's microecological environment; conventional biostimulants can only supplement crops with exogenous nutrients and cannot activate crop stress resistance and photosynthetic pathways at the gene level, resulting in poor sustainability of stress resistance and limited yield increases.

[0003] DREB transcription factor is a known key protein for stress resistance in the plant field. Existing research largely focuses on transgenic crop modification, but this application method has limitations and faces significant challenges in widespread adoption. Currently, few technologies exist for processing high-purity DREB recombinant protein prepared in vitro into agricultural spray formulations and precisely combining it with biostimulant components. Conventional biostimulants are mostly simple combinations of oligosaccharides, amino acids, and humic acids, failing to construct a synergistic system that works with the DREB functional protein, and lacking a dedicated preparation process designed for protein activity. Therefore, developing a novel biostimulant with DREB stress-resistant fusion protein as its core, synergistic component effects, and strong process adaptability has significant research value and application implications. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of existing technologies, such as single mechanism of action, poor stress resistance and yield increase effect, limited application of DREB protein, and poor synergy of component compounding, and provides a crop growth stimulant containing DREB stress resistance fusion protein and its preparation method.

[0005] To achieve the above functions, the technical solution adopted by the present invention is as follows: A DREB stress-resistant fusion protein-type crop biostimulant, by weight, is composed of the following components: 1.0-2.0 parts of functional DREB stress-resistant fusion protein, 10-12 parts of oligosaccharide bioactivator, 15-18 parts of complex amino acids, 6-8 parts of potassium humate, 3.0-4.0 parts of trace element chelate, 1.5-2.5 parts of penetration enhancer, 0.4-0.6 parts of stabilizer, and the balance being deionized water.

[0006] Preferably, the functional DREB stress-resistant fusion protein is prepared by a prokaryotic expression system with a purity of ≥95%, and can target and activate key genes of crop DREB, RD, PsbA and NRT, and simultaneously regulate the three major pathways of crop stress-resistant metabolism, photosynthesis and nutrient transport.

[0007] Preferably, the oligosaccharide bioactivator is a compound of chitosan oligosaccharide, seaweed oligosaccharide, and fructooligosaccharide in a mass ratio of 2:1:1, with a degree of polymerization of 2 to 10.

[0008] Preferably, the compound amino acid is composed of glycine, proline, glutamic acid, arginine, and phenylalanine in a mass ratio of 3:2:2:1:1, and the total amino acid content is ≥98%.

[0009] Preferably, the trace element chelate is an EDTA-chelated iron, zinc, boron, and manganese complex, with a chelated trace element content ≥90%; the penetration aid is polysorbate-80; and the stabilizer is a compound of glycerol and sodium benzoate in a mass ratio of 4:1.

[0010] A method for preparing a DREB stress-resistance fusion protein-type crop biostimulant, specifically including the following steps: Preparation of base solution: Add 65% of the total mass of deionized water to the reaction vessel, add potassium humate and compound amino acids at room temperature, and stir for 20-30 minutes until the materials are completely dissolved. Micronutrient compounding: Add trace element chelate, heat to 35-40℃, and stir at a constant temperature for 15-20 minutes; Preparation of bioactivation system: Cool to 25-30℃, add oligosaccharide bioactivator, penetration enhancer and stabilizer in sequence, and stir for 25-35 minutes; Core protein loading: In a dark environment at 20-25℃, add DREB anti-reverse fusion protein at a low stirring speed of 80-100 r / min and stir for 10-15 min; Volumetric homogenization and filtration: Add the remaining deionized water to bring the volume to 100 parts by weight, homogenize at room temperature for 5-10 minutes, and use a 0.22μm sterile microporous filter membrane to complete the sterilization treatment. After passing the test, fill the product to obtain the finished product.

[0011] Compared with the prior art, the present invention achieves the following beneficial effects by adopting the above solution: 1. This invention processes high-purity DREB fusion protein prepared by in vitro expression into a spray formulation, which breaks through the application limitations of transgenic technology and opens up a new application path for DREB protein in the agricultural field. 2. DREB protein achieves gene-level regulation, oligosaccharides activate crop immune function, compound amino acids supplement the substrates required for crop metabolism, potassium humate improves the rhizosphere environment, and multiple components form a synergistic system. The overall effect is far superior to single raw materials or conventional compound systems. It is not a simple mixture of materials. 3. The present invention adopts a preparation method of low temperature, light protection, low speed stirring and sterile filtration, which is a process specially designed to protect the biological activity of proteins. It is different from conventional fertilizer processing operations and can effectively avoid protein denaturation and inactivation. 4. This product can increase crop survival rate by no less than 92% under drought stress, increase plant height by no less than 38% under saline-alkali conditions, and control crop disease incidence rate to within 4.2%; wheat yield can be increased by no less than 15%, tomato soluble solids by no less than 13%, and cotton boll weight by no less than 9.5%. 5. All products use biologically derived raw materials, contain no chemical hormones or harmful residues, meet the requirements of green agricultural production, and have mild overall preparation conditions, simple process, low requirements for production equipment, and are suitable for large-scale continuous production. Detailed Implementation

[0012] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, 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. Example 1:

[0013] A DREB stress-resistant fusion protein-type crop biostimulant, by weight, comprises the following components: 1.5 parts DREB stress-resistant fusion protein, 11 parts oligosaccharide bioactivator, 16 parts compound amino acids, 7 parts potassium humate, 3.5 parts trace element chelate, 2 parts polysorbate-80, 0.5 parts stabilizer, and 58.5 parts deionized water.

[0014] The oligosaccharide bioactivator is a mixture of chitosan oligosaccharide, seaweed oligosaccharide, and fructooligosaccharide in a mass ratio of 2:1:1; the complex amino acid is a mixture of glycine, proline, glutamic acid, arginine, and phenylalanine in a mass ratio of 3:2:2:1:1; the stabilizer is a mixture of glycerol and sodium benzoate in a mass ratio of 4:1; and the purity of the functional DREB gene recombinant protein is ≥95%.

[0015] A method for preparing a DREB stress-resistance fusion protein-type crop biostimulant, comprising the following steps: Preparation of base solution: Add 65% of the total mass of deionized water to the reaction vessel, add potassium humate and compound amino acids at room temperature, and stir for 20-30 minutes until the materials are completely dissolved. Micronutrient compounding: Add trace element chelate, heat to 35-40℃, and stir at a constant temperature for 15-20 minutes; Preparation of bioactivation system: Cool to 25-30℃, add oligosaccharide bioactivator, penetration enhancer and stabilizer in sequence, and stir for 25-35 minutes; Core protein loading: In a light-protected environment at 22°C, add DREB anti-reverse fusion protein at a low stirring speed of 90 r / min and stir for 10-15 min; Volumetric homogenization and filtration: Add the remaining deionized water to bring the volume to 100 parts by weight, homogenize at room temperature for 5-10 minutes, and use a 0.22μm sterile microporous filter membrane to complete the sterilization treatment. After passing the test, fill the product to obtain the finished product. Example 2:

[0016] A DREB stress-resistant fusion protein-type crop biostimulant, by weight, comprises the following components: 1.0 part DREB stress-resistant fusion protein, 10 parts oligosaccharide bioactivator, 15 parts complex amino acids, 6 parts potassium humate, 3 parts trace element chelate, 1.5 parts polysorbate-80, 0.4 parts stabilizer, and 63.1 parts deionized water. The preparation method is the same as in Example 1. Example 3:

[0017] A DREB stress-resistance fusion protein-type crop biostimulant, by weight, comprises the following components: 2.0 parts DREB stress-resistance fusion protein, 12 parts oligosaccharide bioactivator, 18 parts complex amino acids, 8 parts potassium humate, 4 parts trace element chelate, 2.5 parts polysorbate-80, 0.6 parts stabilizer, and 52.9 parts deionized water. The preparation method is the same as in Example 1.

[0018] Comparative Example 1 Referring to the formulation and process of Example 1, the DREB anti-retroviral fusion protein was replaced with an equal weight of deionized water, while the remaining components and operating steps remained unchanged.

[0019] Comparative Example 2 Conventional amino acid biostimulants used in this field were used as control samples.

[0020] Field trial results Five sets of experiments were set up: Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 2. Each set of experiments was repeated three times. The average value of the experimental results was taken, and the experimental data were compiled into a comparison table as follows: Example 1 (Optimal Example) 92.3 38.6 4.2 15.3 13.6 9.7 Example 2 88.7 34.2 5.5 12.8 11.2 8.1 Example 3 90.5 36.1 4.8 14.2 12.5 8.9 Comparative Example 1 (without genomic proteins) 71.2 22.5 9.6 6.3 5.8 4.2 Comparative Example 2 (Commercially Available Regular Products) 63.7 16.3 15.7 4.1 3.2 2.9 The experimental data show that the performance indicators of each embodiment of the present invention are significantly better than those of the two comparative groups. The DREB stress-resistant fusion protein is the core component that enables the product to achieve high stress resistance and stable yield increase. The components exhibit a significant synergistic effect, and the product is not simply a physical mixture. Field observation results show that after applying the product of the present invention, the crop has a well-developed root system, strong photosynthetic capacity, and no excessive vegetative growth or premature aging. The product has good safety and a wide range of applications.

[0021] Meanwhile, field observation results showed that after spraying the product of the present invention, the root vigor, leaf photosynthetic rate and chlorophyll content of the crop were significantly higher than those of the control group. The plants grew vigorously and the crop's resistance and adaptability were greatly improved, making it suitable for large-scale planting and application of various crops.

[0022] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention, and are not actually limited thereto. In short, if those skilled in the art are inspired by this description and design similar embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A DREB stress-resistance fusion protein-type crop biostimulant and its preparation method, characterized in that, By weight, it consists of the following components: 1.0-2.0 parts of functional DREB anti-stress fusion protein, 10-12 parts of oligosaccharide bioactivator, 15-18 parts of complex amino acids, 6-8 parts of potassium humate, 3.0-4.0 parts of trace element chelate, 1.5-2.5 parts of permeation aid, 0.4-0.6 parts of stabilizer, and the balance being deionized water.

2. The biostimulant according to claim 1, characterized in that: The functional DREB stress-resistant fusion protein was prepared using a prokaryotic expression system with a protein purity ≥95%. The DREB stress-resistant fusion protein can target and activate the expression of crop DREB, RD, PsbA, and NRT genes, and regulate crop stress-resistant metabolism, photosynthesis, and nutrient transport processes.

3. The biostimulant according to claim 1, characterized in that: The oligosaccharide bioactivator is composed of chitosan oligosaccharide, seaweed oligosaccharide, and fructooligosaccharide in a mass ratio of 2:1:1, with a degree of polymerization of 2 to 10.

4. The biostimulant according to claim 1, characterized in that: The compound amino acid is composed of glycine, proline, glutamic acid, arginine, and phenylalanine in a mass ratio of 3:2:2:1:1, with a total amino acid content of ≥98%.

5. The biostimulant according to claim 1, characterized in that: The trace element chelate is an EDTA-chelated iron, zinc, boron, and manganese complex, with a chelated trace element content of ≥90%; the penetration aid is polysorbate-80; and the stabilizer is a compound of glycerol and sodium benzoate in a mass ratio of 4:

1.

6. The biostimulant according to any one of claims 1 to 5, characterized in that: This biostimulant can be sprayed or applied to the roots of food crops, cash crops, fruits and vegetables throughout their entire growth period, and has the effects of drought resistance, salt and alkali tolerance, disease resistance, and yield and quality improvement.

7. A method for preparing the biostimulant according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Preparation of base liquid: Add 65% of the total mass of deionized water to the reaction vessel, add potassium humate and compound amino acids under stirring at room temperature, stir at a constant speed for 20-30 minutes until the materials are completely dissolved to obtain a uniform base liquid. (2) Micronutrient compounding: Add micronutrient chelates to the base solution, heat to 35-40℃, stir at constant temperature for 15-20 minutes to fully disperse and chelate the micronutrients, and ensure that no precipitate is formed in the system; (3) Preparation of bioactivation system: Cool down to 25-30℃, add oligosaccharide bioactivator, penetration aid and stabilizer, stir at a constant speed for 25-35 min to obtain a stable bioactivation mixture. (4) Core protein loading: In a dark and sealed environment at 20-25℃, the functional DREB anti-reverse fusion protein is slowly added, and the stirring speed is controlled at 80-100 r / min. Stir at low speed for 10-15 min. (5) Volume adjustment, homogenization and filtration: Add the remaining deionized water to make up to 100 parts by weight, homogenize at room temperature for 5 to 10 minutes, filter and sterilize using a 0.22μm sterile microporous filter membrane, and fill after passing the test to obtain the finished product.

8. The preparation method according to claim 7, characterized in that: In step (4), the stirring speed is kept constant at 90 r / min and the ambient temperature is kept constant at 22℃.