A short peptide PRP1, its preparation method and application

Abstract

This application discloses a short peptide PRP1, its preparation method, and its application. It belongs to the field of genetic engineering technology. This application identifies a short peptide derived from an unknown protein that can regulate tobacco resistance to Potato Virus Y (PVY), named PVY resistance peptide (PRP1). Its amino acid sequence is ATKHIVGSIVAS. The short peptide PRP1 was prepared through chemical synthesis. Injecting this short peptide into tobacco seedlings can effectively reduce PVY virus levels and improve tobacco resistance to PVY.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to a short peptide PRP1, its preparation method, and its applications. Background Technology

[0002] Potato virus Y (PVY) belongs to the family Theoviridae and the genus Theoviridae. It has a wide host range, infecting various Solanaceae plants. When infecting tobacco, it can cause symptoms such as leaf vein necrosis, mosaic patterns, and stunted growth, leading to tobacco potato virus Y disease. Hunan Province, as a major tobacco-producing area, has seen a continuous expansion of tobacco planting area in recent years, but the occurrence of tobacco potato virus Y disease has also become more severe, seriously reducing tobacco yield and the quality of cured tobacco leaves.

[0003] Existing technologies for the control of tobacco and potato virus Y (PVY) include chemical control and microbial control. Chemical control: Commonly used agents include 3% hypersensitive protein microparticles, 2% pyrimethamine, and 5.6% pyrimethamine·morpholine guanidine. However, their efficacy depends on the timing of application, they only inhibit early infection, are ineffective against systemic infection, and have poor broad-spectrum activity. Most agents are developed for tobacco mosaic virus (TMV) and are not effective against PVY. Microbial control: Microbial preparations have poor stability, low environmental adaptability, large fluctuations in field efficacy, scarce resources of attenuated strains, and the risk of reverting to strong virulence. Long-term use may induce viral mutations and accelerate the emergence of drug-resistant strains.

[0004] Short peptides are a class of small molecules composed of 2-20 amino acids that play an important role in plant life activities. Compared with other small molecules, short peptides have significant advantages such as rich variety, significant activity, and ease of synthesis. However, there is currently little research on the role of short peptides in the control of tobacco and potato virus Y disease.

[0005] Therefore, how to develop short peptides for the prevention and control of tobacco and potato Y virus disease is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a short peptide PRP1, its preparation method and application. The short peptide PRP1 can effectively prevent and control tobacco and potato virus Y disease, with obvious inhibitory effect and environmental friendliness.

[0007] The primary purpose of this application is to provide:

[0008] A short peptide PRP1, wherein the amino acid sequence of said short peptide PRP1 is any one of the following:

[0009] 1) As shown in SEQ ID NO.1;

[0010] 2) Or an amino acid sequence that encodes the same functional protein after substitution, deletion and / or addition of one or more bases as shown in SEQ ID NO.1.

[0011] As a preferred technical solution, the short peptide PRP1 has a molecular weight of 1182.65, an average hydrophilicity coefficient of 0.933, and a theoretical isoelectric point of 8.80.

[0012] Another object of this application is to provide a nucleic acid molecule, said nucleic acid molecule being a nucleic acid sequence encoding the aforementioned short peptide PRP1 or a sequence that is completely complementary to said nucleic acid sequence.

[0013] Another object of this application is to provide a vector comprising the above-described nucleic acid molecules.

[0014] Another object of this application is to provide a host cell, said host cell being any of the following:

[0015] 1) Includes the aforementioned nucleic acid molecules;

[0016] 2) Including the aforementioned carriers.

[0017] Another object of this application is to provide: a recombinant bacterium, said recombinant bacterium being any one of the following:

[0018] 1) Includes the aforementioned nucleic acid molecules;

[0019] 2) Includes the aforementioned carriers;

[0020] 3) Including the host cells mentioned above.

[0021] Another object of this application is to provide: the application of the above-mentioned short peptide PRP1, wherein the application is any one of the following:

[0022] 1) Application in improving tobacco resistance to Potato Virus Y;

[0023] 2) Application in the preparation of drugs for the prevention and / or treatment of tobacco potato virus Y disease.

[0024] Another object of this application is to provide the application of the above-mentioned nucleic acid molecule, the above-mentioned vector, the above-mentioned host cell, or the above-mentioned recombinant bacteria, wherein the application is any one of the following:

[0025] 1) Application in the preparation of short peptide PRP1;

[0026] 2) Application in improving tobacco resistance to Potato Virus Y;

[0027] 3) Application in the preparation of drugs for the prevention and / or treatment of tobacco potato virus Y disease.

[0028] Another object of this application is to provide a medicine for the prevention and / or treatment of tobacco potato virus Y disease, comprising the aforementioned short peptide PRP1.

[0029] Another object of this application is to provide a method for preparing the short peptide PRP1, wherein the method is any one of the following:

[0030] 1) Prepared by sequential chemical synthesis;

[0031] 2) Prepared through in vivo expression in prokaryotes;

[0032] 3) Prepared through in vivo expression in eukaryotic organisms.

[0033] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention identifies a short peptide derived from an unknown protein that can regulate tobacco resistance to Potato Virus Y disease, named PVY resistance peptide (PRP1). Its amino acid sequence is ATKHIVGSIVAS. The short peptide PRP1 was prepared by chemical synthesis. Injecting this short peptide into tobacco seedlings can effectively reduce the amount of PVY virus and improve tobacco resistance to Potato Virus Y disease. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0036] Figure 1 The chromatogram and peak table for the preparation of the short peptide PRP1 are shown below.

[0037] Figure 2 Here is a mass spectrometry image of the prepared short peptide PRP1.

[0038] Figure 3 Figure 1: Identification of PVY resistance in tobacco seedlings after injection of short peptide PRP1; A: Symptoms of inoculated leaves after co-injection of PVY and PRP1 short peptide in tobacco seedlings; H2O was used as a control, with the left side injected with short peptide and the right side injected with H2O; B: Virus content in inoculated leaves after co-injection of PVY and PRP1 short peptide in tobacco seedlings using Western blotting. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0040] Example 1

[0041] Preparation of short peptide PRP1

[0042] The short peptide PRP1 was prepared by Fmoc solid-phase synthesis, using Fmoc-wang resin as the solid matrix. The synthesis sequence was from the carboxyl terminus to the N-terminus (amino terminus).

[0043] The synthesis method is as follows:

[0044] Swell 0.5g of dichlorotriphenylmethyl chloride resin with 10mL of DMF for 60-90min, add 0.36g of arginine and 100μL of DIEA and react for 2h. Then add 5mL of methanol and 100μL of DIEA to block the unreacted linker. Remove the Fmoc protecting group with 5mL of 20% piperidine solution.

[0045] Subsequently, 0.45 g tryptophan, 0.58 g PyBoP, 0.15 g HoBT, and 100 μL DIEA were added sequentially and reacted for 2 h. Then, the Fmoc protecting group was removed using 5 mL of 20% piperidine solution. This step was repeated, with tryptophan replaced sequentially with 0.42 g proline, 0.475 g isoleucine, and 0.38 g leucine (each time, the amino acid was reacted with DIEA first, followed by piperidine removal of the Fmoc protecting group). Finally, the peptide was cleaved using a cleavage agent (TFA: water: triisopropylsilane = 95:2.5:2.5), precipitated in 10 mL of diethyl ether, and then lyophilized for later use.

[0046] The amino acid sequence of the short peptide PRP1 is ATKHIVGSIVAS, SEQ ID NO.1.

[0047] The molecular weight of the short peptide PRP1 was determined to be 1182.65, with an average hydrophilicity coefficient of 0.933 and a theoretical isoelectric point of 8.80 (Figure 1). Figure 2 );

[0048] Detection procedures and conditions for chromatography and mass spectrometry:

[0049] Chromatographic detection procedure and conditions: In the experiment, pump A was a 100% aqueous solution containing 0.1% trifluoroacetic acid, and pump B was a 100% acetonitrile solution containing 0.1% trifluoroacetic acid. The total flow rate was set to 1.0 mL / min, the detection wavelength was 214 nm, and the analytical column type was SHIMADZUInertsil ODS-SP (4.6 × 250 mm, 5 μm). The sample dissolution method was to take 0.5 mg of sample, dissolve it in 10% acetonitrile and 90% water to a final volume of 0.5 mL, and inject the solution in 30 μL. Additionally, at 0.01 minutes, the pump module performed a B. Cone operation, with a value of 17; at 20.00 minutes, the pump module performed a B. Cone operation, with a value of 37, for chromatographic detection and analysis.

[0050] Mass spectrometry detection procedure and conditions: First, prepare the sample. Take 0.1 mg of sample, dissolve it in 50% acetonitrile and 50% water, and bring the volume to 0.5 mL. Mass spectrometry detection conditions are as follows: injection volume 1 μL, interface ESI, pre-bias voltage +4.5 kV, nebulizer gas flow rate 1.50 L / min, detector -0.2 kV, CDL temperature 250 °C, transfer flow rate 0.2 mL / min, CDL voltage 0 V, module temperature 200 °C, and mobile phase concentration 50% water / 50% methanol.

[0051] Example 2

[0052] PVY resistance detection in tobacco seedlings injected with short peptide PRP1

[0053] (1) Construction of PVY-GFP (pCamPVY-GZ-GFP) plasmid:

[0054] A: cDNA Acquisition

[0055] PVY isolates were obtained from diseased tobacco plants in the field of Guizhou Province. Total RNA was extracted from tobacco leaf tissue using TRIzol reagent (Invitrogen), and the tissue was treated with RNase-free DNase I. cDNA was synthesized by reverse transcription using SuperScript IV reverse transcriptase and specific primers. The specific primer sequences are as follows:

[0056] 3'-UTR-R: 5'-GTCTCCTGATTGAAGTTTACAGTCAC-3', SEQ ID NO.2;

[0057] Oligo(dT)18 oligonucleotide primer: 5'-CAGGATCCAAGCTTTTTTTTTTTTTTTTTT-5', SEQ ID NO.3.

[0058] B: Obtaining the PVY gene fragment

[0059] Using the cDNA obtained in step A as a template, the complete PVY genome fragment was amplified using primers 5'-UTR-F and 3'-UTR-R with Phusion high-fidelity DNA polymerase; the specific primer sequences are as follows:

[0060] 5'-UTR-F: 5'-AAATTAAAACAACTCAATACAACATAAG-3', SEQ ID NO.4;

[0061] 3'-UTR-R: 5'-GTCTCCTGATTGAAGTTTACAGTCAC-3', SEQ ID NO. 5.

[0062] C: Construction of pPVY-GZ recombinant vector

[0063] 1) Amplification of the pCambia0390 vector fragment

[0064] Using pCaTVBMV as a template, the pCambia0390 backbone containing the 35S promoter was amplified from pCaTVBMV using primers pCam-NOS-F and pCam-35S-R; the specific primer sequences are as follows:

[0065] pCam-NOS-F: 5'-CTTCAATCAGGAGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCGGGAATTCTAAGAGG-3', SEQ ID NO.6;

[0066] pCam-35S-R: 5'-GAGTTGTTTAATTTCTCTCCAAATGAAATGAACTTCC-3', SEQ ID NO.7;

[0067] 2) Seamless cloning and building pPVY-GZ

[0068] After PCR amplification, 1 μL of DpnI restriction endonuclease was added, and the mixture was digested at 37°C for 2 hours to remove template plasmid residues. The PVY genome fragment obtained in step B was then directionally cloned into the pCambia0390 vector using a seamless cloning system, and the recombinant plasmid pPVY-GZ was successfully constructed.

[0069] D: Stabilization is achieved by inserting introns.

[0070] Plant introns were inserted at key sites in the PVY genome to prevent viral genes from being recognized as foreign DNA and silenced in plant cells, thereby enhancing expression stability, as follows:

[0071] A potato intron was inserted at position 3387 / 3388 of the P3 gene in the potato virus Y genome (GenBank: HM231144, intron 1), and two potato introns were inserted at positions 3967 / 3968 and 5440 / 5441 of the CI gene (GenBank: A23739, intron 2; X55747, intron 3).

[0072] 1) Segmented amplification of fragments containing introns

[0073] The specific construction process is as follows: First, the intron fragments are amplified using the following primer pairs:

[0074] P3-intron1-F: 5'-TATGCAAGTTTACAGGTGAGTTTTCTAATCTGTATCCA-3', SEQ IDNO.8;

[0075] intron1-P3-R: 5'-ATGTGAAATCGCGTTCAAGTTTGCGCTGAGAATACATCAAGATAAAGAAA-3', SEQ ID NO.9;

[0076] P3-984F: 5'-ACTTGAACGCGATTTCAC-3', SEQ ID NO.10;

[0077] CI307-intron2-R: 5'-ATTATCAAATTCACACTTACCTGCTACACTAAGATGAACAGG-3', SEQID NO.11;

[0078] intron2-F: 5'-GTAAGTGTGAATTTGATAATTTGC-3', SEQ ID NO.12;

[0079] intron2-R: 5'-CTGCAAGAACACCAGTTG-3', SEQ ID NO.13;

[0080] intron2-CI308-F: 5'-TCCAACTGGTGTTTCTTGCAGGATCTGTGCTTTTGATTGAACC-3', SEQID NO.14;

[0081] CI1780-intron3-R: 5'-ATGAATGAAGATTTGCTTACCTGCGGTGTAATCTTTTG-3', SEQ IDNO.15;

[0082] intron3-F: 5'-GTAAGCAAATCTTCATTCATGA-3', SEQ ID NO.16;

[0083] intron3-R: 5'-CTGTATAAACCTTAATGAATTAGCG-3', SEQ ID NO.17;

[0084] Using the primers described above, five fragments were obtained sequentially by PCR amplification: intron 1, P3(984)-CI(307) region, intron 2, CI(308)-CI(1780) region, and intron 3.

[0085] 2) Overlap extension PCR (SOE-PCR)

[0086] Subsequently, overlap extension PCR technology was used to fuse these five fragments into a single continuous fragment;

[0087] 3) Linearization of pPVY-GZ vector

[0088] Primer pCam-intron3-CI-F: 5'-TTAAGGTTTATACAGAGAACATACAAAAACTTGAGAAAGTGAG-3', SEQ ID NO.18;

[0089] and pCam-intron1-P3-R: 5'-CTGTAAACTTGCATATAGCTCCATG-3', SEQ ID NO.19;

[0090] Using the pPVY-GZ plasmid constructed in step C as a template, the linear vector backbone fragment was amplified by PCR.

[0091] 4) Seamlessly clone and build pCamPVY-GZ

[0092] Finally, the fusion fragment containing three introns (product of step 2) was directionally inserted into the linearized vector (product of step 3) using a seamless cloning system, and the recombinant plasmid pCamPVY-GZ was successfully constructed.

[0093] E: Insertion of GFP reporter gene

[0094] 1) Obtaining the SfiI-GFP-SfiI recombinant fragment

[0095] Primers GFP(SfiNTN)-F: 5'-TATGAAGTGCACCATCAAGGAAATagggccattacggccATGAGTAAAGGAGAAGAACTTTTCAC-3', SEQ ID NO.20;

[0096] And GFP(SfiNTN)-R: 5'-GATTGTGTCATTTCCTTGATGGTGCACTTCATAGGCCGAGGCGGCCTTTTTGTAGAGCTCATCCATGCC-3', SEQ ID NO.21; By PCR amplification, SfiI restriction endonuclease sites were introduced at both ends of the green fluorescent protein (GFP) coding sequence to obtain the SfiI-GFP-SfiI recombinant fragment;

[0097] 2) Carrier linearization

[0098] Simultaneously, primer pCamCP-F: 5'-GGAAATGACACAATCGATGC-3', SEQ ID NO.22, was used;

[0099] and pCamNIb-R: 5'-ATGGTGCACTTCATAAGTATCGC-3', SEQ ID NO. 23;

[0100] A linearized vector backbone fragment was amplified from the recombinant plasmid pCamPVY-GZ obtained in step D;

[0101] 3) Seamless cloning and construction of PVY-GFP

[0102] Using a seamless cloning system, the SfiI-GFP-SfiI fragment was directionally inserted into the linearized vector backbone fragment obtained in step 2), and the recombinant plasmid PVY-GFP (pCamPVY-GZ-GFP) was successfully constructed.

[0103] (Note: This plasmid was constructed by Professor Li Xiangdong's research group at Shandong Agricultural University and has been published in the paper "Development and application of a full-length infectious clone of potato virus Y isolate belonging to SYR-I strain, De-Jie Cheng".)

[0104] (2) Preparation of tobacco infusion containing PVY-GFP

[0105] Thaw GV3101 competent cells on ice, add 1-5 μL of PVY-GFP plasmid DNA (concentration ≥10 ng / μL), mix gently, and incubate on ice for 5 minutes. Then, perform liquid nitrogen freezing for 5 minutes, heat shock at 37°C for 5 minutes, and ice cooling for 5 minutes. Add 400 μL of antibiotic-free YEP liquid medium and incubate at 28°C with shaking for 1-2 hours. Spread 200 μL of the bacterial culture onto YEP solid medium containing rifampicin (50 μg / ml) and kanamycin (50 μg / ml) and incubate upside down at 28°C for 2-3 days (colon diameter 1-2 mm). Single colonies were picked and colony PCR was performed using PVY or GFP specific primers. After confirming the band size, the colonies were incubated overnight in YEP liquid medium containing rifampicin (50 μg / ml) and kanamycin (50 μg / ml). The cultures were then centrifuged at 5000 rpm for 10 min, the supernatant was discarded, tobacco infusion was added, and the OD... 600 The concentration was adjusted to 0.1, and a tobacco impregnation solution containing PVY-GFP was prepared.

[0106] (3) Preparation of working solution for short peptide PRP1

[0107] Short peptide PRP1 powder was dissolved in water and the working concentration was adjusted to 234.9734 μM to prepare short peptide PRP1 working solution.

[0108] (4) Select tobacco plants with consistent growth conditions, mark the injection range of the same size circle on both sides of the tobacco leaves of the same position of each tobacco plant, inject 50 μL of short peptide PRP1 working solution into the left treatment group of the marked tobacco plants, and inject 50 μL of sterile water into the right control group.

[0109] (5) Six hours after injection, inject 100 μL of tobacco infusion containing PVY-GFP and place the tobacco in a greenhouse at 26°C with a circadian rhythm of 16 hours of daylight / 8 hours of darkness for cultivation.

[0110] like Figure 3 As shown: 5 days after inoculation, the virus fluoresced under ultraviolet light, and the amount of virus in the left treatment group was significantly less than that in the right control group; at the same time, leaf protein was extracted for immunoblotting test. The results showed that the viral molecular weight in the PRP1-injected treatment group was significantly less than that in the control group, indicating that the injection of short peptide PRP1 significantly improved the resistance of tobacco to potato virus Y.

[0111] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0112] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A short peptide PRP1, characterized in that, The amino acid sequence of the short peptide PRP1 is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The sequence of the nucleic acid molecule is the nucleic acid sequence encoding the short peptide PRP1 of claim 1.

3. A vector, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, The host cell can be any of the following: 1) Includes the nucleic acid molecule as described in claim 2; 2) Includes the carrier as described in claim 3.

5. A recombinant bacterium, characterized in that, The recombinant bacteria is any one of the following: 1) Includes the nucleic acid molecule as described in claim 2; 2) Includes the carrier as described in claim 3.

6. The application of the short peptide PRP1 according to claim 1, characterized in that, The application is any one of the following: 1) Application in improving tobacco resistance to Potato Virus Y; 2) Application in the preparation of drugs for the prevention and / or treatment of tobacco potato virus Y disease.

7. The application of the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4, or the recombinant bacteria of claim 5, characterized in that, The application is any one of the following: 1) Application in the preparation of short peptide PRP1; 2) Application in improving tobacco resistance to Potato Virus Y; 3) Application in the preparation of drugs for the prevention and / or treatment of tobacco potato virus Y disease.

8. A drug for the prevention and / or treatment of tobacco potato virus Y disease, characterized in that, Includes the short peptide PRP1 as described in claim 1.

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