A plant small molecule peptide, a plant small peptide precursor gene and its application

Abstract

The present invention relates to the field of biotechnology, and more specifically to a plant small molecule peptide, a plant small peptide precursor gene, and their applications. The present invention provides a plant small molecule peptide, the amino acid sequence of which is shown in SEQ ID NO: 2; and a plant small peptide precursor gene, the gene sequence of which is shown in SEQ ID NO: 1. The plant small peptide precursor gene and its complete or partial amino acid sequence can significantly improve plant resistance to living pathogens such as grape downy mildew and pepper phytophthora.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a plant small molecule peptide, a plant small peptide precursor gene and applications thereof. Background Art

[0002] Small peptides (small peptides for short) are a class of small peptides produced by plants that act as signaling molecules and are usually composed of 20 amino acid residues. Existing reports have shown that small peptides play an important role in regulating plant growth (flowering, fertilization, stomatal opening and closing, etc.), responding to plant biotic adversities (pathogen or virus invasion, insect and herbivorous animal bites, etc.) and abiotic adversities (drought, high temperature, high salt, cold, and nutrient deficiency, etc.).

[0003] The mature peptide segments of plant small peptides are small, typically consisting of 20 amino acid residues, with a very small number of precursors exceeding 120 amino acid residues in length. These peptides are typically present at very low physiological concentrations in plants, yet the mature peptide segments are highly conserved among homologous genes. As signaling molecules, plant small peptides play a key role in short-range cell-to-cell communication. They interact as ligands with corresponding receptor kinase molecules on the cell membrane, thereby activating downstream genes in the pathway or initiating related signal transduction processes.

[0004] Plant small peptides are classified as secretory or non-secretory based on whether they can be secreted. Secretory peptides include extracellular post-translationally modified secretory peptides and extracellular cysteine-rich secretory peptides. They are primarily secreted into the extracellular space by free diffusion and play a decisive role in the fate of neighboring cells. Extracellular post-translationally modified secretory peptides are characterized by post-translational modifications regulated by specific transferases, resulting in small peptide segments, typically less than 20 amino acids. These peptides are initially translated into precursor peptides of approximately 100 amino acids with an N-terminal secretion signal. They then undergo at least one post-translational modification, such as tyrosine sulfation, proline hydroxylation, and hydroxyproline arabinosylation, before finally forming biologically functional mature peptides through the action of processing enzymes located in the endoplasmic reticulum and Golgi apparatus. Extracellular cysteine-rich secretory peptides, another type of secretory signal peptide, are characterized by the presence of an even number of cysteine ​​residues (4-16) in their functional domains, forming intramolecular disulfide bonds. These residues are essential for the proper conformational folding of the mature peptide. Extracellular cysteine-rich peptides are typically much larger and positively charged than post-translationally modified peptides (less than 160 amino acids). Similar to extracellular post-translationally modified peptides, cysteine-rich peptides also contain a conserved N-terminal signal peptide that guides the peptide to the correct cellular location through the secretory pathway, and their C-terminal mature peptides are also highly conserved.

[0005] Small peptides are produced by plants themselves and can promote plant growth and disease resistance. They are considered green, pollution-free, and biopesticides with significant application prospects. The precursor genes that synthesize small peptides in plants have significant scientific and economic value. These precursor genes can be used to genetically improve crop yield, quality, and stress resistance through molecular genetics (transgenics). Alternatively, engineered plants or bacteria can be used to directly synthesize small peptide precursors in vitro, process them, and then spray them directly on plants.

[0006] Some small peptides and their precursor genes have been widely used. For example, the bacterial flagellin flg22, which can enhance plant immunity, is often exogenously administered to enhance or induce plant immunity. Some plant small peptide precursor genes have also been shown to promote plant growth or enhance plant immunity through transgenic means. Furthermore, using these small peptide precursor gene sequences, precursor proteins synthesized in plants or in vitro (by engineered bacteria) can be proteolytically digested to produce mature small peptides.

[0007] At present, agricultural production mainly uses chemical agents (pesticides) to control the effects of crop pathogens on crops. The chemical composition and molecular structure of these synthetic chemical agents are generally divided into inorganic, organochlorine, organophosphorus, carbamate, thiocarbamate, substituted urea, triazine, phenoxycarboxylic acid, phenol, organosulfur, organoarsenic, pyrethroid and other categories. In addition to being toxic to plant pathogens, these pesticides are also toxic to humans and environmental organisms (mainly some beneficial insects and animals). At the same time, many pesticides have stable structures and are not easily degraded. They accumulate in large quantities in soil and surface water, resulting in a lasting impact on the environment. These chemicals also exist as a screening pressure for natural selection, promoting the accumulation of a large number of harmful mutations related to drug resistance in pathogens, thereby leading to the generation of superbugs. The small molecule peptide synthesis gene for protection in the present invention is an endogenous small peptide synthesis gene of grapes. All components are inherent to the plant itself, have no toxic effects on humans, and as a polypeptide can be relatively easily degraded by soil microorganisms. It acts directly on the plant itself, promoting plant immunity on multiple levels and avoiding the accumulation of pathogen mutations caused by a single inhibitory effect. Summary of the Invention

[0008] The present invention aims to provide a plant small molecule peptide and its application, the specific scheme is as follows:

[0009] A plant small molecule peptide, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0010] The plant small molecule peptide is obtained by connecting amino acids encoded by codons of the plant small peptide precursor gene VvprePIP through a peptide chain. The sequence of the gene VvprePIP is shown in SEQ ID NO: 1.

[0011] The plant small molecule peptide is used to improve plant disease resistance (for example, resistance to grape downy mildew in grapes, resistance to pepper phytophthora in tobacco, and other living pathogens).

[0012] The plant peptide precursor gene (VvprePIP) of the present invention and its complete or partial amino acid sequence (i.e., mature peptide) can significantly improve plant resistance to living pathogens such as Plasmopara viticola and Phytophthora capsici. Heterologous expression of this gene (VvprePIP) in plants can promote the production of mature small peptides (PIPs), activating basal plant immunity, including the salicylic acid signaling pathway that enhances plant immunity, promoting ROS production, and the production of callose, which restricts pathogen growth. Expressing this peptide precursor gene (VvprePIP) in grape leaves can significantly improve grape resistance to Plasmopara viticola, while expressing it in tobacco can significantly improve tobacco resistance to Phytophthora capsici. Because this peptide precursor gene can promote basal immune resistance, it has the potential to provide broad-spectrum disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The resistance of LUC (control) and VvprePIP to grape downy mildew.

[0014] Figure 2 The resistance of LUC (control) and VvprePIP to Phytophthora capsici in tobacco. DETAILED DESCRIPTION

[0015] 1. Obtaining the sequence of the grape peptide precursor gene VvprePIP

[0016] (1) Cut 0.2 g of young grape leaves into a sample tube, freeze them directly in liquid nitrogen, grind them into powder, add 900 μL of CTAB extract (2% CTAB, 2 mol / L NaCl, 2.5% PVP, 100 mmol / L Tris-HCl (pH 8.0), 25 mmol / L EDTA (pH 8.0), 0.2% mercaptoethanol) preheated at 65°C, mix thoroughly, and incubate at 65°C.

[0017] (2) Incubate for 30 minutes, add 700 μL of chloroform and extract for 2 minutes, centrifuge at 13,000 g for 10 minutes, and aspirate the supernatant.

[0018] (3) Add 700 μL of chloroform and extract for 2 minutes. Centrifuge at 13,000 g for 10 minutes and aspirate the supernatant.

[0019] (4) Accurately aspirate 600 μL of the supernatant, add 272 μL of 8 mol / L LiCl (final LiCl concentration is 2.5 mol / L), and precipitate at -20°C for 2 hours.

[0020] (5) Centrifuge at 13,000 g for 15 minutes at 4°C, discard the supernatant, add 1 mL of 70% ethanol, and let it stand at room temperature for 30 minutes.

[0021] (6) Centrifuge at 13,000 g for 5 minutes at 4°C, discard the supernatant, add 1 mL of 70% ethanol, and let it stand at room temperature for 30 minutes.

[0022] (7) Centrifuge at 13,000 g for 5 minutes at 4°C and discard the supernatant.

[0023] (8) Add 500 μL SSTE and incubate at 65°C for 10 minutes.

[0024] (9) After cooling to room temperature, add 500 μL of chloroform and extract for 2 minutes. Centrifuge at 13,000 g for 10 minutes and aspirate 450 μL of supernatant.

[0025] (10) Add 45 μL of ice-cold 3 mol / L sodium acetate (pH 5.4), mix well, add 900 μL of ice-cold anhydrous ethanol, and precipitate at -20°C for 30 min.

[0026] (11) Centrifuge at 13,000 g for 15 minutes at 4°C, discard the supernatant, add 1 mL of 70% ethanol, and let stand at room temperature for 30 minutes.

[0027] (12) Centrifuge at 13,000 g for 5 minutes at 4°C, discard the supernatant, add 1 mL of 70% ethanol, and let it stand at room temperature for 30 minutes.

[0028] (13) Centrifuge at 13,000 g for 5 minutes at 4°C, discard the supernatant, and dry the pellet at room temperature for 5 minutes.

[0029] (14) Add 15 μL of RNase-free ultrapure water and dissolve on ice for 20 minutes.

[0030] (15) The RNA concentration was detected using an ultra-micro nucleic acid analyzer (Hangzhou Aosheng Instrument Co., Ltd., Nano-400).

[0031] (16) 3 μg of RNA sample was aspirated, and the volume was adjusted to 12 μL by adding RNase-free ultrapure water. 3 μL of RNase-free DNase was added and incubated at 42°C for 5 min.

[0032] (17) After the incubation, the mixture was placed on ice and immediately added with 5°C reverse transcriptase (Shanghai Yisheng Biotechnology Co., Ltd., 11141ES60). The mixture was incubated at 25°C for 2 minutes, at 42°C for 30 minutes, and denatured at 85°C for 15 seconds.

[0033] (18) The reverse transcription product is directly used for gene cloning and the gene cloning primers are designed:

[0034] Forward primer: ATGCGCCCCTTTAATGTG Reverse primer: TCAGTGGGGCATGCCGTCGAC

[0035] (19) Fastpfu (Shanghai Shenger Biotechnology) was used for gene cloning.

[0036] The reaction system is:

[0037]

[0038] The reaction procedure is:

[0039]

[0040] (20) PCR products were directly recovered after electrophoresis using the gel recovery kit from Shanghai Bioengineering Company.

[0041] (21) DNA sequencing of PCR products to obtain the complete gene sequence:

[0042] >VvprePIP

[0043] ATGGCTAGAGCACTCAAATTTGTGAGCTTCCTCTTCCTTGTTCTGGTGTTGAACTCCAT

[0044] TGCCATCCATGGCCGCCCCTTTAATGTGTTGAAGAAGCCGCGAGGCCCTGATGTGAGG

[0045] AGATGAGGGGATTCTTTGATGGGTTGTCTCTTGGAGCCATCAAGCAATCAGGACCAAGC

[0046] CCTGGTAATGGACACAAATTCACCAACGCTGGAACACTTGGAGGAATCAAGGACTCGGG

[0047] TCCCAGCCCTGGTAATGGACACAAATTCACCAACGCTGGAACACTTGGAGGAATCAAGG

[0048] The protein sequence encoded by ACTCGGGTCCCAGCCCTGGTGAGGGACACAAGTATGTCGACGGCATGCCCCACTGA is:

[0049] >VvprePIP

[0050] MARALKFVSFLFLLVLVLNSIAIHGRPFNVLKKPRGPDGEEMRGFFDGLSLGAIKQSGPS PGNGHKFTNAGTLGGIKDSGPSPGNGHKFTNAGTLGGIKDSGPSPGEGHKYVDGMPH*

[0051] The small peptide precursor encoded by this gene belongs to the PIP family and has three conserved SGPS (threonine-glycine-proline-serine)-GH (glycine-histidine) motifs.

[0052] 2. Grape peptide precursor gene VvprePIP Plant expression vector construction

[0053] (1) VvprePIP Construct into a plant expression vector, and at the same time construct the luciferase gene LUC gene without disease resistance function into a plant expression vector as a control. Design nested primers with restriction enzyme cutting sites:

[0054]

[0055]

[0056] (2) High-fidelity DNA polymerase Fastpfu (Shanghai Shenger Biotechnology) was used to amplify the gene using the previous PCR product as a template.

[0057] The reaction system is:

[0058]

[0059] The reaction procedure is:

[0060]

[0061] (3) The PCR products were directly recovered using the gel recovery kit of Shanghai Bioengineering Company after direct electrophoresis.

[0062] (4) The plant expression vector pHB-FLAG (double FLAG protein tag driven by the double cauliflower virus 35S promoter) was digested with restriction endonucleases (NEB). The digestion reaction and system are as follows:

[0063] The reaction system is:

[0064]

[0065] Enzyme digestion was performed at 37°C for 2 hours, followed by denaturation at 65°C for 5 minutes and ice bath for 5 minutes.

[0066] (5) The PCR product was constructed into a plant expression vector using DNA homologous recombination (Nanjing Novozyme Medical Technology Co., Ltd.; C112-02). The homologous recombination reaction and system are as follows:

[0067]

[0068] The reaction was allowed to proceed at 37°C for 30 minutes and terminated by placing in an ice bath.

[0069] (6) The homologous recombination reaction products were directly transformed into Escherichia coli DH5a using the CaCl2 heat shock transformation method. After incubation at 37°C for 30 minutes, the cells were coated with LB culture plates containing kanamycin and incubated at 37°C for 12 hours.

[0070] (7) Single colonies that grew were taken for DNA sequencing and identification. The correct single colonies were sequenced and identified, and the plasmids were extracted using the SPARKeasy high-purity plasmid small-scale rapid extraction kit (Shandong SPARK Biotechnology Co., Ltd.; AD0102-C).

[0071] (8) The constructed plant expression vector was transformed into Agrobacterium tumefaciens GV3101 by electroporation. After the transformed Agrobacterium was cultured in a 30°C incubator for 2 hours, it was spread on LB culture plates containing kanamycin (50 mg / L) and rifampicin (10 mg / L) and cultured in a 30°C incubator for 48 hours. Normally growing monoclonal Agrobacterium tumefaciens colonies were used for disease resistance phenotype identification.

[0072] 3. Grape peptide precursor gene VvprePIP Improving grape resistance to Plasmoparaviticola

[0073] (1) Monoclonal Agrobacterium tumefaciens was cultured in 5 mL of LB medium containing kanamycin (50 mg / L) and rifampicin (10 mg / L) for 12 hours.

[0074] (2) Centrifuge the Agrobacterium culture at 5000 g for 5 minutes and discard the supernatant.

[0075] (3) Resuspend the cells in sterile water, centrifuge at 5000 g for 5 minutes, and discard the supernatant.

[0076] (4) Resuspend Agrobacterium tumefaciens in grape transformation buffer (10 mM magnesium chloride, 10 mM morpholineethanesulfonic acid, 100 μM acetosyringone, 0.3% surfactant Silwet L-77, pH adjusted to 5.7 using NaOH) and adjust the concentration of Agrobacterium tumefaciens to OD 600 =0.60-0.80.

[0077] (5) Immerse the young grape branches in the bacterial solution and use a vacuum pump to treat the solution at -0.80 MPa for 5 minutes. Quickly return the pressure to normal. Repeat this vacuum infiltration treatment three times, each time for 5 minutes.

[0078] (6) After transient transformation, the grape branches were washed twice with clean water and the grape materials were returned to the culture room and continued to grow for 5 days.

[0079] (7) The leaves that were transformed were removed and made into 1 cm leaf discs. Each leaf disc was inoculated with 20 μL of 10 5 After culturing the conidia of grape downy mildew in a 25°C incubator for 24 hours, the excess bacterial liquid was removed. After continuing to culture in a 25°C incubator for 6 days, the bacterial count was counted to determine the effect of the small peptide precursor gene on grape downy mildew resistance.

[0080] Compared with the control material, the small peptide precursor gene VvprePIP It can significantly inhibit the growth of grape downy mildew ( Figure 1 A), reduced the sporangium production of grape downy mildew by 75% (the amount of leaf disc fungi expressing the LUC gene in the control group was 2.40×10 4 The number of leaf disc fungi expressing prePIP gene was 0.59×10 4 indivual, Figure 1 B) Improve the resistance of grapes to downy mildew (Plasmodium vitis). 4. Grape peptide precursor gene VvprePIP Improving tobacco's resistance to Phytophthora capsici

[0081] (1) Monoclonal Agrobacterium tumefaciens was cultured in 5 mL of LB medium containing kanamycin (50 mg / L) and rifampicin (10 mg / L) for 12 hours.

[0082] (2) Centrifuge the Agrobacterium culture at 5000 g for 5 minutes and discard the supernatant.

[0083] (3) Resuspend the cells in sterile water, centrifuge at 5000 g for 5 minutes, and discard the supernatant.

[0084] (4) Resuspend Agrobacterium tumefaciens in tobacco transformation buffer (10 mM magnesium chloride, 10 mM morpholineethanesulfonic acid, 100 μM acetosyringone, pH adjusted to 5.7 with NaOH) and adjust the concentration of Agrobacterium tumefaciens to OD 600 =0.60-0.80.

[0085] (5) Carrying small peptide precursor gene VvprePIP The Agrobacterium bacterial solution and the transformation solution of Agrobacterium tumefaciens carrying the LUC gene were injected into tobacco using a needle-less syringe using the pressure infiltration method, and then cultured at 25°C in the dark for 2 days.

[0086] (6) Remove the instantaneously transformed leaves and place them directly on wet filter paper, avoiding facing upward. Place a 0.3 cm diameter Phytophthora capsici disk in the center of the corresponding injection area. Keep the leaves moisturized and place them in the dark at 25°C for 2 days. Then take pictures and use Image J software to count the plaque area.

[0087] (7) The grape peptide precursor gene VvprePIP can significantly inhibit the growth of pepper fungus on tobacco ( Figure 2 A) Reduce the lesion area of ​​Phytophthora capsici on tobacco by 50% and improve the resistance of tobacco to Phytophthora capsici.

[0088] The above description is only a preferred embodiment of the present invention. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the principles of the present invention. These modifications and improvements should also be regarded as within the scope of protection of the present invention.

Claims

1. An application of a plant small peptide precursor gene, the gene sequence of which is shown in SEQ ID NO: 1, characterized in that: Expressing the plant peptide precursor gene in grape leaves improves the resistance of grapes to grape downy mildew.

2. Application of a plant small peptide precursor gene, the gene sequence of which is shown in SEQ ID NO: 1, characterized in that: Expressing plant peptide precursor genes in tobacco improves tobacco resistance to tobacco pepper phytophthora.

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