Small molecule protein for improving peanut nitrogen fixation and yield and application thereof

Abstract

In view of the problem of the prior art that with the increase of the fertilizer amount, the nitrogen content of the root system gradually increases, the peanut root system gradually reduces the nitrogen fixation capacity, and the fertilizer utilization rate is reduced and the environment is polluted, according to the biological characteristics that peanuts can produce root nodules to fix nitrogen, the application provides a kind of small molecule protein for improving peanut nitrogen fixation and yield and application thereof, the small molecule protein is any one or combination of two or more of 10 kinds of small molecule proteins (ENFP-1 to ENFP-10), and when used, an ENFP aqueous solution containing a Silwet series organosilicon surfactant is prepared. The experiment proves that: leaf spraying ENFP solution can significantly increase the number of peanut root nodules, improve the nitrogen fixation capacity of peanuts, increase the nitrogen content of peanut leaves, and can increase the yield of peanuts, and has a significant yield-increasing effect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a small molecule protein that improves nitrogen fixation and yield in peanuts and its applications. Background Technology

[0002] Peanuts are an important oilseed crop in my country, and increasing their yield is of great significance to ensuring the country's oilseed supply. Most legumes can establish a symbiotic relationship with nitrogen-fixing rhizobia, forming highly efficient "nitrogen-fixing factories"—root nodules. Peanuts, being legumes, also form root nodules in their roots to fix nitrogen and promote growth. Root nodule formation in peanuts begins at the 5-6 leaf stage, mainly concentrated on the taproot and lateral roots formed from the epicotyl. Nodule formation is influenced by internal factors such as different varietal characteristics and external environmental factors such as soil nutrients, soil pH, and soil aeration. Therefore, fully utilizing atmospheric nitrogen to enhance the nitrogen-fixing capacity of peanut roots and increase the nitrogen content in the aboveground parts of the peanut plant are important measures to reduce nitrogen fertilizer application, lower environmental pollution, and increase peanut yield.

[0003] Currently, the nitrogen-fixing capacity of peanut roots is mainly improved by selecting different peanut varieties or adjusting the microenvironment such as nutrients and water in the peanut root system. However, with the increase of fertilizer application, the nitrogen content in the roots gradually increases, and the nitrogen-fixing capacity of peanut roots gradually decreases, leading to a decline in fertilizer utilization and environmental pollution.

[0004] Small molecule peptides play a crucial regulatory role in plant growth, development, stress tolerance, and nutrient absorption. Recent studies have revealed that many small molecule peptides possess hormone-like functions. Current research in plants has also shown that small molecule peptides play an important role in regulating root nodule formation. Therefore, based on a thorough understanding of the functions of small molecule peptides, developing a highly efficient small molecule protein (ENFP, Enhance nitrogen fixation of peanut) to regulate peanut root nodule formation and nitrogen fixation, establishing suitable in vitro application methods, improving peanut root nitrogen fixation capacity, increasing leaf nitrogen content, reducing nitrogen fertilizer application, and increasing peanut yield can provide technical support for ensuring my country's oilseed security. Summary of the Invention

[0005] This invention utilizes bioinformatics techniques to deeply analyze peanut genome information, conducts thorough comparisons and experimental screenings of small molecule peptides in peanuts and other plants, and ultimately identifies 10 small molecule peptides, ENFP-1 to ENFP-10, that can significantly improve peanut nitrogen fixation capacity and yield. Spraying ENFP solution during the peanut's peak flowering period can increase the ENFP content within the peanut plant, thereby enhancing root nitrogen fixation capacity and aboveground nitrogen content, ultimately increasing peanut yield.

[0006] The technical solution of this invention is: a small molecule protein that improves nitrogen fixation and yield in peanuts, characterized in that it is any one or a mixture of two or more of ENFP-1 to ENFP-10, wherein the amino acid sequences of ENFP-1 to ENFP-10 are as follows:

[0007] ENFP-1: AFRPTAPSHSPGSMR

[0008] ENFP-2: DFRPTAPSHSPGSMR

[0009] ENFP-3: NFRPTAPHSPGSMR

[0010] ENFP-4: AFRPTAPGHSPGSMR

[0011] ENFP-5: DFRPTAPGHSPGSMR

[0012] ENFP-6: NFRPTAPGHSPGSMR

[0013] ENFP-7: AFRPTAPGNSPGSMR

[0014] ENFP-8: DFRPTAPGNSPGSMR

[0015] ENFP-9: NFRPTAPGNSPGSMR

[0016] ENFP-10: DFRPTAPGGSPGSMR.

[0017] The aforementioned small molecule proteins ENFP-1 to ENFP-10 can improve the nitrogen fixation capacity of peanut roots, increase the nitrogen content in the aboveground parts, and thus increase peanut yield.

[0018] The method for enhancing the nitrogen fixation capacity of peanut roots using ENFP-1 to ENFP-10 is as follows: Foliar spraying of ENFP solution during the peanut's full flowering period; the ENFP solution is an aqueous solution containing Silwet series organosilicon surfactants and ENFP, with a dosage of 20-30 L per acre for field application. The addition of Silwet series organosilicon surfactants to the ENFP solution promotes the entry of small molecule proteins into peanut leaves, facilitates ENFP-induced production of active substances in peanuts, and enhances the nitrogen fixation capacity of peanut roots.

[0019] The working concentration of the above ENFP solution is: the final concentration of any one or more of ENFP-1 to ENFP-10 is 0.02±0.005 g / L (preferably 0.02 g / L), and the concentration of the Silwet series organosilicon surfactant is 0.01-0.02%.

[0020] The technical effects of this invention are as follows:

[0021] 1. This invention overcomes the problems existing in the prior art. During the peak flowering period of peanuts, foliar spraying of a small molecule protein ENFP solution enhances the nitrogen-fixing capacity of the roots and increases peanut yield. Therefore, using this product can increase peanut yield while reducing the amount of chemical fertilizer used. It not only prevents environmental pollution caused by excessive fertilization but also increases peanut yield, showing broad application prospects.

[0022] 2. Field experiments have shown that foliar spraying of peanut with ENFP solution can increase the number of root nodules, improve nitrogen fixation capacity, and increase leaf nitrogen content, thereby increasing peanut yield. Therefore, the method of this invention not only enhances nitrogen fixation in peanut roots but also increases yield, providing a practical and effective product and method for the efficient and sustainable development of peanut production. Attached Figure Description

[0023] Figure 1 The figure shows the effect of treating greenhouse-grown peanuts with ENFP-1 to ENFP-10 solutions on the nitrogen content of peanut leaves in Experiment 1. The figure shows that all ENFP-1 to ENFP-10 solutions can significantly increase the nitrogen content of peanut leaves.

[0024] Figure 2 The figure shows the effect of treating peanuts grown in the field with ENFP-1 and ENFP-10 solutions on peanut root nodules in Experiment 2. The figure shows that both ENFP-1 and ENFP-10 solutions can significantly increase the number of rhizobium in peanut roots.

[0025] Figure 3 The figure shows the effect of treating peanuts grown in the field with ENFP-1 and ENFP-10 solutions in Experiment 3 on peanut yield. The figure shows that both ENFP-1 and ENFP-10 solutions can significantly increase peanut yield.

[0026] Among them, ENFP solution: an aqueous solution containing silwet L-77 surfactant and ENFP; CK: a water control containing the same concentration of silwet L-77 surfactant. Detailed Implementation

[0027] The technical solution of the present invention and its resulting technical effects will be further described below with reference to specific embodiments and accompanying drawings. The following description is only for explaining the present invention and does not limit the present invention in any way. Any modifications or substitutions made based on the present invention are within the protection scope of the present invention. Unless otherwise specified, the methods described in the present invention are conventional methods in the art. Unless otherwise specified, the reagents described in the present invention are commercially available reagents.

[0028] Example 1: Determination of the amino acid sequence of a small protein

[0029] (1) Determine the amino acid sequence of small protein molecules

[0030] Based on relevant protein sequences found in legumes such as peanuts and alfalfa, and combined with the expression abundance of the protein gene in peanuts, the inventors of this application established a small protein library containing 150 small protein sequences through bioinformatics analysis and substitution of certain key amino acids. Using these 150 small protein sequences as foliar sprays on peanut leaves, 10 small protein sequences were discovered that significantly increased the number of root nodules in peanuts. The C-terminal 6 amino acids of these 10 small protein sequences are conserved and are designated as SPGSMR. These 10 small protein sequences are named ENFP-1 to ENFP-10.

[0031] (2) Synthesis and preservation of small protein molecules

[0032] Small molecule proteins were synthesized using the Fmoc solid-phase peptide synthesis method. The synthesis steps are as follows:

[0033] ① Take 0.3g of Fmoc-C-terminal first amino acid-Wang resin, put it into a centrifuge tube, swell it with DCM for 20 minutes, and filter off the DCM; add 3 times the resin volume of 20% piperidine / DMF solution to dissolve it, and use a nitrogen blower to blow nitrogen for 30 minutes to dry it; add 2 times the resin volume of DMF, wash 5 times, centrifuge and discard the liquid.

[0034] ② Take 0.27 mmol of Fmoc-C-terminal second amino acid -OH, 0.54 mmol of DIPEA, and 0.26 mmol of HBTU, add them to the above centrifuge tube, add an appropriate amount of DMF solvent, and react for 30 minutes to obtain Fmoc-C-terminal second amino acid-C-terminal first amino acid-Wang resin. Amino acid:DIPEA:HBTU:resin = 3:6:2.85:1 (molar ratio); then wash the resin from the previous step three times with 2 times the resin volume of DMF; then add 3 times the resin volume of 20% Pip / DMF solution, purge with nitrogen for 30 minutes, dry under vacuum, remove the Fmoc group, and obtain H2N-second amino acid-first amino acid-Wang resin; finally, wash the resin from the previous step five times with 2 times the resin volume of DMF.

[0035] ③ Repeat step 2. Each time step 2 is repeated, one amino acid can be added. Repeat according to the requirements of small molecule protein sequence to obtain the final target small molecule polypeptide.

[0036] ④ Wash the resin three times with methanol, then add 6 times the volume of cutting fluid (volume ratio: trifluoroacetic acid: anisole: 1,2-ethanedithiol: phenol: water = 87.5%: 5%: 2.5%: 2.5%: 2.5%), shake on a shaker for 2 hours, filter off the resin, precipitate the filtrate with ice-cold anhydrous diethyl ether, and wash the precipitate three times with ice-cold anhydrous diethyl ether. Finally, place the precipitate in a vacuum drying oven and dry at room temperature for 24 hours to obtain 100 mg of crude small molecule protein.

[0037] ⑤ Small molecule proteins with a purity of over 95% were obtained through purification using HPLC.

[0038] First, the crude small molecule protein was analyzed using a C18 analytical column with a fast gradient. The mobile phase was: aqueous phase A: 0.1% TFA / water; organic phase B: 0.1% TFA / acetonitrile; gradient: 5%-70%, 20 minutes. The main peak of the crude protein was observed around 7 minutes.

[0039] Secondly, a C18 preparative column was used to separate the small molecule protein, and the target peak was collected. Mass spectrometry analysis showed that the molecular weight of the collected target peak matched that of the target small molecule protein, and the purity was above 95%, which was considered preliminary acceptable. Then, the collected target peak solution was first rapidly frozen with liquid nitrogen, and then vacuum dried into powder using a lyophilizer. 1 mg of the lyophilized powdered small molecule protein was taken, dissolved in water, and re-analyzed by HPLC and MS. The purity was above 95%, and the molecular weight remained unchanged, which was considered final acceptable.

[0040] These small molecule proteins can be stored at -80℃ for about 2 years and at -20℃ for about 6 months.

[0041] Example 2: Preparation and application of EPW foliar fertilizer

[0042] Take 2g of equal mass of ENFP-1 to ENFP-10 and the above 10 small molecule proteins respectively, dissolve them in 1L of deionized water to prepare 100x stock solution. The stock solution can be stored below -20℃ for 1-2 weeks. If it is stored at room temperature, it needs to be prepared and used immediately.

[0043] When applying it in the field, take 100 ml of ENFP stock solution, dilute it with tap water, and finally dilute it to a final volume of 10 L. Then add 2 ml of silwet L-77 surfactant, mix it evenly, and make the final concentration reach 0.02 g / L. The ENFP working solution contains 2 / 10000 of silwet L-77 surfactant. The solution is used in the following test example.

[0044] Foliar spraying should be carried out during the peak flowering period of peanuts, with a spraying rate of 20-30 L / acre; spraying should be done before 10 am or after 4 pm, and on a day without rain. When spraying, try to ensure that both the upper and lower surfaces of the peanut leaves are evenly covered with ENFP solution.

[0045] Experimental Example 1: Effect of foliar spraying of ENFP solution on nitrogen content in peanut leaves

[0046] Plump Luhua 23 peanut seeds were selected and sown in small boxes (10×10×10cm) containing seedling substrate in a laboratory setting. The seeds were then placed in a greenhouse for germination and growth at 28℃, with 12 hours of light followed by 12 hours of darkness. After germination, the peanuts continued to grow in the greenhouse for 20 days, after which they were treated with ENFP solution by foliar spraying. A control was prepared by spraying water containing the same concentration of Silwet L-77 surfactant. The experiment was repeated three times. Ten days after spraying, the nitrogen content of the peanut leaves was measured. In this experiment, the ENFP solution consisted of 10 small molecule protein solutions containing ENFP-1 to ENFP-10 (preparation method as shown in Example 2).

[0047] Experimental results: The nitrogen content of peanut leaves was measured, and it was found that, compared with the control, spraying ENFP significantly increased the nitrogen content of peanut leaves. Figure 1 It was confirmed that foliar spraying of ENFP solution can significantly increase the nitrogen content of peanut leaves, and the effects of ENFP-1 to ENFP-10 solutions on increasing the nitrogen content of peanut leaves are not significantly different.

[0048] Experimental Example 2: Effects of Foliar Spraying of EPW Solution on Peanut Root Nodules

[0049] Plump Luhua 23 peanut seeds were selected and sown in the field in Jinxiang, Shandong Province on May 25, 2021. Single-seed precision sowing was used at a planting density of 9000 plants / mu (approximately 667 square meters), with equal row spacing of 40 cm. Treatments involving spraying ENFP-1 and ENFP-10 solutions were included, with a control consisting of water sprayed with the same concentration of Silwet L-77 surfactant. The mixture was randomly assigned to a block design and replicated three times. Each plot was 32 m². 2 An 8-row zone was used. During the peak flowering period of peanuts, on a rainless day after 4 pm, 30 L of 0.02 g / L ENFP solution per acre was sprayed on the leaves. Peanuts were harvested on September 17th, and the number of root nodules was counted at harvest. In this experiment, the ENFP solution was a small molecule protein solution containing ENFP-1 and ENFP-10 respectively (preparation method as shown in Example 2). When spraying, efforts were made to ensure that the ENFP solution was evenly sprayed on both sides of the peanut leaves.

[0050] Experimental results: At peanut harvest, peanut roots were dug up, and the number of root nodules per plant was counted. It was found that, compared with the control, spraying with ENFP-1 and ENFP-10 solutions significantly increased the number of root nodules per peanut plant. Figure 2 ).

[0051] Experimental Example 3: Effect of ENFP solution on peanut yield (foliar spraying)

[0052] Plump Luhua 23 peanut seeds were selected and sown in the field in Jinxiang, Shandong Province on May 25, 2021. Single-seed precision sowing was used at a planting density of 9000 plants / mu (approximately 667 square meters), with equal row spacing of 40 cm. Treatments involving spraying ENFP-1 and ENFP-10 solutions were included, with a control consisting of water sprayed with the same concentration of Silwet L-77 surfactant. The mixture was randomly assigned to a block design and replicated three times. Each plot was 32 m². 2 An 8-row area was used. During the peak flowering period of peanuts, on a rainless day after 4 pm, 30 L of 0.02 g / L ENFP solution per acre was sprayed on the leaves. Peanuts were harvested on September 17th, and the yield was recorded. In this experiment, the ENFP solution was a small molecule protein solution containing ENFP-1 and ENFP-10 respectively (preparation method as shown in Example 2). When spraying, efforts were made to ensure that the ENFP solution was evenly sprayed on both sides of the peanut leaves.

[0053] Experimental results: After peanut harvest, peanut yield was measured, and it was found that spraying ENFP-1 and ENFP-10 solutions significantly increased peanut yield compared with the control. Figure 3 ).

Claims

1. A small molecule protein that enhances nitrogen fixation and yield in peanuts, characterized in that it is... A mixture of one or more of ENFP-1 to ENFP-10, wherein the amino acid sequences of ENFP-1 to ENFP-10 are as follows: ENFP-1: AFRPTAPSHSPGSMR ENFP-2: DFRPTAPSHSPGSMR ENFP-3: NFRPTAPHSPGSMR ENFP-4: AFRPTAPGHSPGSMR ENFP-5: DFRPTAPGHSPGSMR ENFP-6: NFRPTAPGHSPGSMR ENFP-7: AFRPTAPGNSPGSMR ENFP-8: DFRPTAPGNSPGSMR ENFP-9: NFRPTAPGNSPGSMR ENFP-10: DFRPTAPGGSPGSMR.

2. An ENFP solution containing the small molecule protein of claim 1, characterized in that, The ENFP solution is an aqueous solution containing one or more of the Silwet series organosilicon surfactants and ENFP-1 to ENFP-10.

3. The ENFP solution as described in claim 2, characterized in that, The final concentration of any one or more of the ENFP-1 to ENFP-10 mixtures is 0.02±0.005 g / L.

4. The ENFP solution as described in claim 3, characterized in that, The concentration of the Silwet series of silicone surfactants is 0.01-0.02%.

5. The application of the small molecule protein of claim 1 or the ENFP solution of any one of claims 2-4 in simultaneously increasing the number of root nodules and the nitrogen content of leaves in peanuts.

6. The application of the small molecule protein of claim 1 or the ENFP solution of any one of claims 2-4 in improving peanut yield.

7. A method for improving nitrogen fixation and yield in peanuts, characterized in that, The ENFP solution described in claim 4 is sprayed on the leaves during the peak flowering period of peanuts.

8. The method for improving nitrogen fixation and yield in peanuts as described in claim 7, characterized in that, When using in the field, spray 20-30L per acre.

Images