Small peptide sp07 for regulating drought tolerance of crops and application thereof

Abstract

The application discloses a small peptide SP07 for regulating drought resistance of crops and application thereof, and the amino acid sequence of the small peptide SP07 is shown as SEQ ID NO. 1. The small peptide SP07 is obtained by a chemical synthesis method, seedling stage corn in drought is treated by spraying, and the survival rate of the corn is significantly improved compared with a control. The small peptide SP07 is sprayed in a field under normal growth and drought environment, the flowering period of the corn is advanced, and the yield-related traits of the corn are significantly improved. In addition, wheat under drought stress can be promoted to flower in advance by spraying the small peptide SP07, and the small peptide SP07 can be used for improving drought resistance of crops.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and botany, and particularly relates to a small peptide SP07 for regulating drought tolerance of crops and application thereof. BACKGROUND

[0002] Maize (Zea may L) is an annual plant of Gramineae with cross-pollination, and is an important food crop and also an important feed and industrial raw material. Maize is the world's largest food crop, and its production is seriously threatened by drought. With the change of natural environment, drought events occur more frequently, and the threat of drought to maize production is also more serious. Previous studies have shown that the application of specific small molecules can improve the drought tolerance of plants to achieve the purpose of water-saving and drought resistance. The drought-resistant agent based on specific small molecules as active ingredients has a wide range of use scenarios and can produce significant economic, social and environmental benefits.

[0003] Small peptides are a class of small molecular substances with a length of 2-100 amino acids. Small peptides play an important role in plant life activities and have high application value. Compared with other small molecules, small peptide compounds have the characteristics of rich types, obvious effect and easy synthesis, and are ideal objects for screening and developing drought-resistant agents. However, at present, there are still very limited small peptide molecules reported to participate in regulating drought tolerance of crops. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art and provides a small peptide SP07 for regulating drought tolerance of crops and application thereof. When the small peptide SP07 is applied, it can improve the drought tolerance of crops at the seedling stage and reduce the yield loss of crops under drought.

[0005] To achieve the above-mentioned purpose, the technical solutions designed by the present application are as follows:

[0006] The present application provides a small peptide SP07 for regulating drought tolerance of crops, and the amino acid sequence of the small peptide SP07 is shown in SEQ ID NO. 1.

[0007] Further, the molecular weight of the small peptide SP07 is 1580.694, the average coefficient of hydrophilicity is -0.52, and the theoretical isoelectric point is 3.88.

[0008] The small peptide obtained by the present application can be obtained by sequence chemical synthesis or expressed in prokaryotes (such as Escherichia coli) or eukaryotes (such as yeast).

[0009] It should be clear that one skilled in the art can make substitutions, deletions and / or additions of one or more amino acids to the amino acid sequence disclosed in the present application, and the resulting mutant sequence has more than 90% sequence alignment homology. Therefore, the present application also includes the derived peptides with high homology and activity obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1. In addition, it should be understood that, in view of the characteristics of codon degeneracy and codon bias of species, one skilled in the art can use codons suitable for expression in a specific species to express the sequence as needed. Therefore, the present application also includes nucleotide sequences encoding the above-mentioned sequences.

[0010] The present application also provides the use of the above-mentioned small peptide SP07 in the preparation of a corn drought-resistant agent.

[0011] The present application also provides a drought-resistant agent for improving the drought resistance of corn, wherein the drought-resistant agent contains the small peptide SP07; and the drought-resistant agent is an aqueous solvent, and the content of the small peptide SP07 is 0.5-2 μmol / L.

[0012] Further, the content of the small peptide SP07 in the drought-resistant agent is 1 μmol / L.

[0013] The present application has the following beneficial effects:

[0014] The small peptide SP07 is obtained by a chemical synthesis method, and is sprayed on corn seedlings in drought, and the survival rate of the corn is significantly improved compared with the control (plants sprayed only with the solvent). Spraying the small peptide SP07 on corn in normal growth and drought environment, the corn flowering period is advanced, and the corn yield-related traits are significantly improved. In addition, spraying the small peptide SP07 on wheat under drought stress can promote early flowering, indicating that the small peptide SP07 can be used to improve the drought resistance of crops. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A chromatogram for detecting the small peptide SP07;

[0016] Figure 2 A mass spectrum for detecting the small peptide SP07;

[0017] Figure 3 A drought resistance identification chart of the small peptide SP07 sprayed on corn in the seedling stage. DETAILED DESCRIPTION

[0018] The present application will be further described in detail below in conjunction with specific examples, so as to be understood by one skilled in the art.

[0019] Example 1 Chemical synthesis of the small peptide SP07

[0020] The polypeptide synthesis adopts Fmoc solid-phase synthesis method, and Fmoc-wang resin is used as a solid matrix. The synthesis sequence is from the C-terminal to the N-terminal. In the synthesis process, 20% piperidine solution is used for Fmoc deprotection, and HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HoBt (1-hydroxybenzotriazole) and DIPEA (N,N-diisopropylethylamine) solution is used for amino acid coupling. In the synthesis process, DMF (dimethylformamide) is used to wash the resin sufficiently to remove excess reactants and avoid side reactions affecting the subsequent synthesis process. The general steps of the Fmoc solid-phase synthesis process include resin activation, Fmoc deprotection, amino acid coupling, resin cleavage, polypeptide separation and purification, and identification, etc., and are as follows:

[0021] 1) Resin activation: the calculated Fmoc-wang resin is weighed and placed in a 10 mL polypeptide solid-phase reaction tube, 5 mL DMF is added to swell the resin, and the time is 30 min. The DMF is removed by vacuum filtration.

[0022] 2) Fmoc deprotection: 5 mL of 20% piperidine / DMF solution (v / v) is added to the reaction tube, and the resin is contacted sufficiently. The reaction is carried out on a mixing instrument, and the reaction is carried out twice, each for 10 min. The reaction solution is removed by vacuum filtration, and the resin is washed with DMF and then the DMF is removed.

[0023] 3) Amino acid coupling: the resin is 1 equivalent, the amino acid used in the coupling process is 4 equivalents, HBTU and HoBt are 4 equivalents, and DIPEA is 8 equivalents. The reaction is carried out in DMF at room temperature for 45 min. After each amino acid coupling, Kjeldahl reagent is used for detection to determine the completeness of the reaction.

[0024] The composition of Kjeldahl reagent: A liquid: 2.5 g of indanone solid is dissolved in 50 mL of anhydrous ethanol; B liquid: 20 g of phenol is dissolved in 5 mL of anhydrous ethanol; C liquid: 1 mL of 0.001 M potassium cyanide is dissolved in 49 mL of redistilled pyridine.

[0025] Detection method: take a few grains of resin particles in a small glass tube with a small spoon, add 2-3 drops of A, B and C, heat for 1 min until the solution boils. If the resin color is light yellow and no blue color appears, the reaction is complete; if the resin color turns blue (if proline and phenylalanine are coupled, the resin color is orange red), the reaction is incomplete, and the coupling step needs to be repeated until the resin detection does not change color.

[0026] After the reaction is completed, the resin in the reactor is washed with DMF and DCM, and the reactor containing the resin is placed in a vacuum drying box to dry all the solvents.

[0027] 4) Resin cleavage: The dried resin was transferred to a clean chicken heart bottle, a small magnet was added, 5 mL cleavage solution (ratio of trifluoroacetic acid (TFA): pure water: triisopropylsilane (TIS): ethanedithiol (EDT) = 91:3:3:3) was added, and the resin was stirred to make full contact with the cleavage solution. The reaction was carried out at room temperature for at least 3 h. After the reaction was completed, the reaction was transferred to a filter tube, the filtrate was collected in a 50 mL centrifuge tube, and most of the TFA was removed by blowing nitrogen. After pre-cooling with ethyl ether, 30 mL was added for precipitation, centrifugation was carried out at 5000 rpm for 5 min, the supernatant was removed, and the precipitate was washed with ethyl ether. The operation was repeated 4 times, and the final precipitate was the crude polypeptide, which was vacuum dried.

[0028] 5) Polypeptide separation, purification and identification: reverse phase high performance liquid chromatography was used for liquid phase analysis of the polypeptide. The specific steps are as follows: first, prepare the mobile phase, A phase is ultrapure water (containing 0.05% TFA), B phase is 80% acetonitrile solution (v / v) (containing 0.05% TFA), and then filter with a 0.22 μm filter and ultrasonic degassing. A certain amount of polypeptide crude powder was weighed and dissolved with A phase to a concentration of 3 mg / mL, and then filtered with a 0.22 μm filter. A C18 semi-preparative column was used, and elution was carried out according to a certain elution gradient by reverse phase liquid chromatography. The detection wavelengths were 215 nm and 254 nm, and the target product was collected. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for polypeptide molecular weight identification, and the identification showed that:

[0029] The amino acid sequence of the small peptide SP07 is shown in SEQ ID NO. 1:

[0030] AAAAAAAPADDNRGLLL;

[0031] The molecular weight of the small peptide SP07 is 1580.694, the average hydrophilic coefficient is -0.52, and the theoretical isoelectric point is 3.88 Figure 1 Figure 2 ).

[0032] 6) The collected target product was freeze-dried after rotary evaporation to obtain a polypeptide pure product, which was SP07 powder.

[0033] Preparation of drought resistance agent

[0034] The preparation method of the drought resistance agent for improving the drought resistance of plants comprises the following steps:

[0035] The SP07 powder was dissolved in single distilled water, and the working concentration was diluted to 1 μmol / L, which was the drought resistance agent.

[0036] ​Example 3: Drought resistance test of corn seedlings after spraying with drought-resistant agent

[0037] Equal amounts of substrate (peat: potting soil: coarse sand = 1:1:1) were filled into 11 cm square pots (length × width × height = 11 cm × 10 cm). Four plants of the maize hybrid Zhengdan 958 were planted in each pot. The plants were grown in a growth chamber at a temperature of 22-26℃ with a diurnal rhythm of 16 h day / 8 h dark. Watering was stopped when the plants reached the 4-leaf stage, and a drought treatment was initiated. Soil moisture content was monitored. When the soil moisture content dropped to approximately 20%, the plants were divided into a treatment group and a control group with essentially the same soil moisture content.

[0038] The treatment group was sprayed with a drought-resistant agent, while the control group was sprayed with an equal amount of solvent (water). The amount sprayed was approximately 3 ml per plant. Rewatering was performed approximately 5 days after the soil moisture content dropped below 10%.

[0039] like Figure 3 As shown in the 16 replicates, after three rehydration cycles, all plants in both the treatment and control groups either survived or died, indicating that the drought stress in this experiment was either too mild or too severe. After 12 rehydration cycles, the plants in the treatment group showed a higher survival rate, while after one rehydration cycle, the survival rates in the treatment and control groups were the same. This indicates that the exogenous application of drought-resistant agents significantly improved the plants' ability to resist drought stress (P = 0.0004).

[0040] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A small peptide SP07 for regulating drought tolerance of crops, characterized in that: The amino acid sequence is shown as SEQ ID NO.

1.

2. Use of the small peptide SP07 according to claim 1 in the preparation of a corn drought-resistant agent.

3. A drought-resistant agent for improving the drought resistance of corn, characterized in that: The drought-resistant agent contains the small peptide SP07 according to claim 1, and the drought-resistant agent is a water solvent, and the content of the small peptide SP07 is 0.5-2 μmol / L.

4. The agent against drought according to claim 3, characterized in that: In the drought-resistant agent, the content of the small peptide SP07 is 1 μmol / L.

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