A short peptide PSK3, and a preparation method and application thereof

Abstract

The application discloses a short peptide PSK3 and a preparation method and application thereof, and belongs to the technical field of genetic engineering. The application discloses a short peptide PSK3 belonging to the phytosulfokine (PSK) family, and the short peptide PSK3 is prepared by a chemical synthesis method. The short peptide PSK3 is applied to tobacco seedlings, and the PVY virus content in the tobacco can be significantly reduced compared with a control group, indicating that the short peptide PSK3 can be used for improving the resistance of the tobacco to the potato Y virus disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and more particularly to a short peptide PSK3, a preparation method and application thereof. BACKGROUND

[0002] Potato virus Y (PVY) belongs to the family of Potyviridae and the genus of Potyvirus, and has a wide host range, which can infect various solanaceous plants. When infecting tobacco, it can cause symptoms such as vein necrosis, mosaic and dwarfing in tobacco plants, leading to tobacco PVY disease. Tobacco is one of the important economic crops and pillar industries of the national economy in China. In recent years, the tobacco planting area in Hunan Province, as the main production area, has been continuously expanding, but the occurrence of tobacco PVY disease has also shown a worsening trend, which has seriously reduced the yield of tobacco and the quality of cured tobacco leaves.

[0003] For the prevention and control of tobacco PVY disease, the existing technologies include chemical control and microbial control. Chemical control: common agents include 3% hypersensitive protein microgranules, 2% piperacillin, and 5.6% piperacillin · morpholine guanidine, but the control effect depends on the timing of application, and only has inhibitory effect on early infection, has no effect on systemic infection, and has poor spectrum, most of the agents are developed for tobacco mosaic virus (TMV), and have poor effect on PVY. Microbial control: the stability of microbial agents is poor, the environmental adaptability is low, the field control effect fluctuates greatly, the resources of attenuated strains are scarce, and there is a risk of recovery to strong virulence, long-term use may induce virus variation and accelerate the emergence of drug-resistant strains.

[0004] Short peptides are small molecules composed of 2-20 amino acids, which play an important role in plant life activities. Compared with other small molecules, short peptides have the advantages of rich variety, significant activity and easy synthesis. However, there is little research on the use of short peptides in the prevention and control of tobacco PVY disease.

[0005] Therefore, how to develop short peptides for the prevention and control of tobacco PVY disease is a technical problem to be solved by those skilled in the art. SUMMARY

[0006] Therefore, the present application provides a short peptide PSK3, a preparation method and application thereof. The short peptide PSK3 can effectively prevent and control tobacco PVY disease, has obvious inhibitory effect, and is environmentally friendly.

[0007] The first object of the present application is to provide:

[0008] A short peptide PSK3, the amino acid sequence of the short peptide PSK3 is any one of the following:

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

[0010] 2) or an amino acid sequence shown by SEQ ID NO. 1 after one or more substitutions, deletions and / or additions of bases, still encoding the same functional protein;

[0011] The short peptide PSK3 belongs to the plant sulfokine (Phytosulfokine, PSK) family.

[0012] As a preferred technical solution, the molecular weight of the short peptide PSK3 is 1763.947, the average coefficient of hydrophilicity is -0.925, and the theoretical isoelectric point is 9.70.

[0013] Still another purpose of the present application is to provide a nucleic acid molecule, which is a nucleic acid sequence encoding the short peptide PSK3 or a sequence completely complementary to the nucleic acid sequence.

[0014] Still another purpose of the present application is to provide a vector, which comprises the nucleic acid molecule.

[0015] Still another purpose of the present application is to provide a host cell, which is any one of the following:

[0016] 1) comprising the nucleic acid molecule;

[0017] 2) comprising the vector.

[0018] Still another purpose of the present application is to provide a recombinant bacterium, which is any one of the following:

[0019] 1) comprising the nucleic acid molecule;

[0020] 2) comprising the vector;

[0021] 3) comprising the host cell.

[0022] Still another purpose of the present application is to provide the application of the short peptide PSK3, which is any one of the following:

[0023] 1) the application in improving the resistance of tobacco to potato Y virus;

[0024] 2) the application in preparing a medicine for preventing and / or treating tobacco potato Y virus disease.

[0025] Still another purpose of the present application is to provide the application of the nucleic acid molecule, the vector, the host cell or the recombinant bacterium, which is any one of the following:

[0026] 1) the application in the preparation of the short peptide PSK3;

[0027] 2) the application in improving the resistance of tobacco to potato Y virus;

[0028] 3) The use in the preparation of a medicine for preventing and / or treating tobacco PVY disease.

[0029] Still another object of the present application is to provide a medicine for preventing and / or treating tobacco PVY disease, comprising the short peptide PSK3.

[0030] Still another object of the present application is to provide a preparation method of the short peptide PSK3, which is any one of the following:

[0031] 1) prepared by sequence chemical synthesis;

[0032] 2) prepared by in vivo expression in prokaryotes;

[0033] 3) prepared by in vivo expression in eukaryotes.

[0034] According to the above technical solutions, compared with the prior art, the present application has the following beneficial effects:

[0035] The present application finds a short peptide PSK3 belonging to the plant sulfokine (Phytosulfokine, PSK) family through research, and the short peptide PSK3 is prepared by chemical synthesis. After injecting the short peptide PSK3 into seedlings of tobacco, the PVY virus amount of the treatment group is significantly reduced compared with the control group, indicating that the short peptide PSK3 can be used to improve the resistance of tobacco to PVY disease. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0037] Figure 1 are: chromatogram and peak table for preparing the short peptide PSK3.

[0038] Figure 2 are: mass spectrum detection diagram for preparing the short peptide PSK3.

[0039] Figure 3Figure 1 is a PVY resistance identification chart of the short peptide PSK3 injected into seedling tobacco; wherein A is a systemic leaf symptom chart of the seedling tobacco co-injected with PVY and PSK3 small peptide, with H2O as a control, the left is a symptom chart, and the right is a virus fluorescence detection; B is an inoculation leaf symptom chart of the seedling tobacco co-injected with PVY and PSK3 small peptide, with H2O as a control; C is a chart of virus content in the inoculation leaf of the seedling tobacco co-injected with PVY and PSK3 small peptide detected by Western Blotting. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0041] Embodiment 1

[0042] Preparation of the short peptide PSK3

[0043] The short peptide PSK3 is prepared by using Fmoc solid-phase synthesis method. Fmoc-wang resin is used as a solid matrix. 0.5 g of dichlorotrityl chloride resin 60-90 min is swelled by using 10 mL of DMF. 0.36 g of arginine and 100 μL of DIEA are added for reaction for 2 h. 5 mL of methanol and 100 μL of DIEA are added for blocking the unreacted linker. 5 mL of 20% piperidine solution is used for removing the Fmoc protecting group.

[0044] 0.45 g of tryptophan, 0.58 g of PyBoP, 0.15 g of HoBT and 100 μL of DIEA are sequentially added for reaction for 2 h. 5 mL of 20% piperidine solution is used for removing the Fmoc protecting group. The step is repeated. Tryptophan is replaced by 0.42 g of proline, 0.475 g of isoleucine and 0.38 g of leucine (in each case, the amino acid is first reacted with DIEA, and then piperidine is used for removing the Fmoc protecting group) in sequence. Finally, a cleavage agent (TFA: water: triisopropylsilane = 95:2.5:2.5) is used for cleavage. The polypeptide is precipitated in 10 mL of ether. Finally, it is freeze-dried in a freeze dryer for standby use.

[0045] The amino acid sequence of the short peptide PSK3 is NQKIKSNGISYPISSQ, SEQ ID NO. 1.

[0046] It is determined that the molecular weight of the short peptide PSK3 is 1763.947, the average hydrophilic coefficient is -0.925, and the theoretical isoelectric point is 9.70. Figure 1 , Figure 2 )

[0047] The detection process and conditions of chromatography and mass spectrometry are as follows:

[0048] Chromatographic detection process and conditions: in the experiment, pump A is 100% water solution containing 0.1% trifluoroacetic acid, pump B is 100% acetonitrile solution containing 0.1% trifluoroacetic acid, the total flow rate is set to 1.0 mL / min, the detection wavelength is 214 nm, and the analysis column type is SHIMADZU Inertsil ODS-SP (4.6*250 MM, 5 UM). The sample dissolution method is to take 0.5 mg of sample, dissolve it in 10% acetonitrile and 90% water to 0.5 mL, and the injection volume is 30 μL. In addition, at 0.01 minutes, the pump module performs B.Cone operation with a value of 17; at 20.00 minutes, the pump module performs B.Cone operation with a value of 37, so as to carry out chromatographic detection analysis.

[0049] Mass spectrometric detection process and conditions: first prepare the sample, take 0.1 mg of sample, dissolve it in 50% acetonitrile and 50% water, and dilute to 0.5 mL. The mass spectrometric detection conditions are as follows: the injection volume is 1 μL, the interface is ESI, the pre-bias voltage is +4.5 kv, the atomization gas flow rate is 1.50 L / min, the detector is -0.2 kv, the CDL temperature is 250°C, the transmission flow rate is 0.2 mL / min, the CDL voltage is 0 V, the module temperature is 200°C, and the mobile phase concentration is 50% water / 50% methanol.

[0050] Example 2

[0051] Detection of PVY resistance of short peptide PSK3 injected seedling tobacco

[0052] (1) Construction of PVY-GFP (pCamPVY-GZ-GFP) plasmid:

[0053] A: Acquisition of cDNA

[0054] The PVY isolate was taken from the diseased tobacco plants in the field of Guizhou Province, and total RNA was extracted from tobacco leaf tissue using TRIzol reagent (Invitrogen) and treated with RNase-free DNase I. The cDNA was synthesized by reverse transcription using Superscript IV reverse transcriptase and specific primers; the specific primer sequences are as follows:

[0055] 3'-UTR-R: 5'-GTCTCCTGATTGAAGTTTACAGTCAC-3', SEQ ID NO. 2;

[0056] Oligo(dT)18 oligonucleotide primer: 5'-CAGGATCCAAGCTTTTTTTTTTTTTTTTTT-5', SEQ ID NO. 3.

[0057] B: Acquisition of PVY gene fragment

[0058] The cDNA obtained in step A was used as a template to amplify the complete genomic fragment of PVY using primers 5'-UTR-F and 3'-UTR-R with Phusion High-Fidelity DNA Polymerase; the specific primer sequences are as follows:

[0059] 5'-UTR-F: 5'-AAATTAAAACAACTCAATACAACATAAG-3', SEQ ID NO. 4;

[0060] 3'-UTR-R: 5'-GTCTCCTGATTGAAGTTTACAGTCAC-3', SEQ ID NO. 5.

[0061] C: Construction of pPVY-GZ recombinant vector

[0062] 1) Amplification of pCambia0390 vector fragment

[0063] The pCambia0390 backbone containing the 35S promoter was amplified from pCaTVBMV using primers pCam-NOS-F and pCam-35S-R with pCaTVBMV as a template; the specific primer sequences are as follows:

[0064] pCam-NOS-F: 5'-CTTCAATCAGGAGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCGGGAATTCTAAGAGG-3', SEQ ID NO. 6;

[0065] pCam-35S-R: 5'-GAGTTGTTTTAATTTCTCTCCAAATGAAATGAACTTCC-3', SEQ ID NO. 7;

[0066] 2) Construction of pPVY-GZ by seamless cloning

[0067] After PCR amplification, 1 μL DpnI restriction enzyme was added to digest the template plasmid residues at 37°C for 2 hours, and then the PVY genomic fragment obtained in step B was directionally cloned into the pCambia0390 vector using the seamless cloning system, and finally the recombinant plasmid pPVY-GZ was successfully constructed.

[0068] D: Stabilization by insertion of intron

[0069] Inserting plant introns at key sites in the PVY genome to avoid silencing of viral genes in plant cells due to recognition of foreign DNA and to enhance expression stability, as follows:

[0070] A potato intron (GenBank: HM231144, intron 1) was inserted at position 3387 / 3388 of the P3 gene of the potato virus Y genome, and two potato introns (GenBank: A23739, intron 2; X55747, intron 3) were inserted at positions 3967 / 3968 and 5440 / 5441 of the CI gene, respectively.

[0071] 1) Fragment containing intron was amplified in segments

[0072] The specific construction process is as follows: first, the intron fragment was amplified using the following primer pairs, respectively:

[0073] P3-intron1-F: 5'-TATGCAAGTTTACAGGTGAGTTTTCTAATCTGTATCCA-3', SEQ ID NO. 8;

[0074] intron1-P3-R: 5'-ATGTGAAATCGCGTTCAAGTTTGCGCTGAGAATACATCAAGATAAAGAAA-3', SEQ ID NO. 9;

[0075] P3-984F: 5'-ACTTGAACGCGATTTCAC-3', SEQ ID NO. 10;

[0076] CI307-intron2-R: 5'-ATTATCAAATTCACACTTACCTGCTACACTAAGATGAACAGG-3', SEQ ID NO. 11;

[0077] intron2-F: 5'-GTAAGTGTGAATTTGATAATTTGC-3', SEQ ID NO. 12;

[0078] intron2-R: 5'-CTGCAAGAACACCAGTTG-3', SEQ ID NO. 13;

[0079] intron2-CI308-F: 5'-TCCAACTGGTGTTCTTGCAGGATCTGTGCTTTTGATTGAACC-3', SEQ ID NO. 14;

[0080] CI1780-intron3-R: 5'-ATGAATGAAGATTTGCTTACCTGCGGTGTAATCTTTTG-3', SEQ ID NO. 15;

[0081] intron3-F: 5'-GTAAGCAAATCTTCATTCATGA-3', SEQ ID NO. 16;

[0082] intron3-R: 5'-CTGTATAAACCTTAATGAATTAGCG-3', SEQ ID NO. 17;

[0083] Using the above primers, five fragments, intron 1, P3(984)-CI(307) segment, intron 2, CI(308)-CI(1780) segment and intron 3, were obtained by PCR amplification in turn;

[0084] 2) Splicing by overlap extension PCR (SOE-PCR)

[0085] Subsequently, the five fragments were fused into a single continuous fragment by overlap extension PCR technology;

[0086] 3) Linearization of pPVY-GZ vector

[0087] Using primers pCam-intron3-CI-F: 5'-TTAAGGTTTATACAGAGAACATACAAAAACTTGAGAAAGTGAG-3', SEQ ID NO. 18;

[0088] and pCam-intron1-P3-R: 5'-CTGTAAACTTGCATATAGCTCCATG-3', SEQ ID NO. 19;

[0089] The linear vector backbone fragment was obtained by PCR amplification using the pPVY-GZ plasmid constructed in step C as a template;

[0090] 4) Construction of pCamPVY-GZ by seamless cloning

[0091] Finally, the fusion fragment (product of step 2) containing three introns 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.

[0092] E: Insertion of GFP reporter gene

[0093] 1) Acquisition of Sfil-GFP-Sfil recombinant fragment

[0094] GFP(SfiNTN)-R: 5'-GATTGTGTCATTTCCTTGATGGTGCACTTCAT AGGCCGAGGCGGCCTTTTTGTAGAGCTCATCCATGCC-3', SEQ ID NO.21; Sfil-GFP-Sfil recombinant fragment was obtained by PCR amplification, introducing Sfil restriction endonuclease cleavage sites at both ends of the green fluorescent protein (GFP) coding sequence;

[0095] GFP(SfiNTN)-R: 5'-GATTGTGTCATTTCCTTGATGGTGCACTTCAT AGGCCGAGGCGGCCTTTTTGTAGAGCTCATCCATGCC-3', SEQ ID NO.21; Sfil-GFP-Sfil recombinant fragment was obtained by PCR amplification, introducing Sfil restriction endonuclease cleavage sites at both ends of the green fluorescent protein (GFP) coding sequence;

[0096] 2) Vector linearization

[0097] Meanwhile, primers pCamCP-F: 5'-GGAAATGACACAATCGATGC-3', SEQ ID NO.22;

[0098] pCamNIb-R: 5'-ATGGTGCACTTCATAAGTATCGC-3', SEQ ID NO.23;

[0099] The linearized vector backbone fragment was amplified from the recombinant plasmid pCamPVY-GZ obtained in step D;

[0100] 3) Seamless cloning to construct PVY-GFP

[0101] The Sfil-GFP-Sfil fragment was inserted into the linearized vector backbone fragment obtained in step 2) using the seamless cloning system, and the recombinant plasmid PVY-GFP (pCamPVY-GZ-GFP) was successfully constructed.

[0102] (Note: This plasmid was constructed by Professor Li Xiangdong's research group at Shandong Agricultural University and has been disclosed 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.")

[0103] (2) Preparation of PVY-GFP-containing tobacco infiltrate

[0104] The GV3101 competent cells were thawed on ice, 1-5 μL of the PVY-GFP plasmid DNA (concentration ≥ 10 ng / μL) was added, and the mixture was gently mixed and then incubated on ice for 5 minutes. The mixture was then subjected to liquid nitrogen freezing for 5 minutes, 37°C water bath heating for 5 minutes, and ice bath cooling for 5 minutes. Then, 400 μL of antibiotic-free YEP liquid medium was added, and the mixture was incubated at 28°C for 1-2 hours. Then, 200 μL of the bacterial solution was taken and spread on YEP solid medium containing rifampicin (50 μg / ml) and kanamycin (50 μg / ml), and the mixture was incubated at 28°C for 2-3 days (colony diameter 1-2 mm). A single colony was picked and subjected to colony PCR using PVY or GFP specific primers. After the band size was confirmed to be correct, the mixture was incubated in YEP liquid medium containing rifampicin (50 μg / ml) and kanamycin (50 μg / ml) overnight. The mixture was centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded. Tobacco infiltrate was added, and the OD 600 was adjusted to 0.1 to obtain PVY-GFP-containing tobacco infiltrate;

[0105] (3) Preparation of short peptide PSK3 working solution

[0106] The short peptide PSK3 powder was diluted at a ratio of 1:15 to obtain a short peptide PSK3 working solution with a concentration of 188.9702 μM;

[0107] (4) Selecting Nicotiana benthamiana with consistent growth conditions, marking the same order of tobacco leaves with consistent circles for injection range, and dividing the marked Nicotiana benthamiana into a treatment group and a control group. The treatment group was injected with 50 μL of short peptide PSK3, and the control group was injected with 50 μL of sterile water.

[0108] (5) After 6 hours of injection, 100 μL of PVY-GFP-containing tobacco infiltrate was injected, and the tobacco was placed in a greenhouse with a temperature of 26°C, with a day-night rhythm of 16h light / 8h darkness.

[0109] As shown in Figure 3 : After 5 days of inoculation, both the systemic leaves and the inoculated leaves of the virus control group emitted fluorescence under ultraviolet light irradiation, and the amount of virus fluorescence on the treatment group was less than that of the control group. Meanwhile, total leaf proteins were extracted for immunoblotting experiments. The results showed that the viral molecular weight of the treatment group injected with PSK3 was significantly less than that of the control group, indicating that the resistance of tobacco to potato Y virus disease was significantly improved after injection of short peptide PSK3.

[0110] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0111] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A short peptide PSK3, characterized in that, The amino acid sequence of the short peptide PSK3 is shown as SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule has a sequence of a nucleic acid sequence encoding the short peptide PSK3 of claim 1 or a sequence fully complementary to the nucleic acid sequence.

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

4. A host cell, characterized in that, The host cell is any of the following: 1) comprising the nucleic acid molecule of claim 2; 2) comprising the vector of claim 3.

5. A recombinant bacterium, characterized in that, The recombinant bacteria is any of the following: 1) comprising the nucleic acid molecule of claim 2; 2) comprising the vector of claim 3.

6. Use of the short peptide PSK3 according to claim 1, characterized in that, The application is any of the following: 1) application in improving the resistance of tobacco to potato Y virus; 2) application in preparing a medicine for preventing and / or treating tobacco potato Y virus disease.

7. Use of the nucleic acid molecule of claim 2, the vector of claim 3, the host cell of claim 4 or the recombinant bacterium of claim 5, characterized in that, The application is any of the following: 1) application in preparing the short peptide PSK3; 2) application in improving the resistance of tobacco to potato Y virus; 3) application in preparing a medicine for preventing and / or treating tobacco potato Y virus disease.

8. A medicament for preventing and / or treating tobacco virus disease of potato Y, characterized by, The short peptide PSK3 of claim 1.

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