Tobacco endogenous polypeptide nt1318 and application thereof
Patent Information
- Application Number
- CN202611213325.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-11
- Publication Date
- 2026-10-27
Smart Images
Figure CN122978249A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a tobacco endogenous polypeptide Nt1318 and its applications. Background Technology
[0002] Root rot caused by Fusarium oxysporum is a prevalent and significant soil-borne disease in large-scale tobacco cultivation, severely impacting tobacco yield and quality. It is a major cause of economic losses for tobacco farmers and a key factor hindering the stable development of the tobacco industry. Current control measures for tobacco root rot primarily rely on chemical pesticides. However, long-term application easily leads to pesticide resistance in pathogens, and also poses problems such as excessive pesticide residues and environmental pollution. These methods fail to meet the demands of green agriculture and sustainable planting. Therefore, there is an urgent need to develop a new, efficient, safe, and environmentally friendly control technology.
[0003] Plant resistance inducers mainly include chemically synthesized compounds and biological agents. They enhance plant disease resistance by activating the plant's own immune system and are a core technology for green disease control in modern agriculture. Among them, plant endogenous secreted peptides are natural signaling molecules in plants that can participate in regulating plant growth, development, and stress response processes. They possess the advantages of high efficiency, broad spectrum, and environmental friendliness, making them a high-quality research and development direction for green control of crop diseases.
[0004] Existing research indicates that plant-secreted small peptides are core signaling molecules regulating plant immune responses. During pathogen infection, plants release endogenous small peptides into their apoplasts. These peptides are specifically recognized by receptors on the plant cell surface, thereby activating a series of downstream immune defense responses and regulating plant disease resistance. Plant cell surface receptors can not only recognize exogenous molecules secreted by pathogens to trigger immune responses, but also initiate endogenous immune regulation by recognizing danger signaling molecules released by the plant itself. Plant-secreted small peptides play a crucial regulatory role in this endogenous disease resistance pathway.
[0005] Currently, there are no reports on the use of endogenously secreted small peptides from tobacco to control tobacco root rot in existing technologies. Summary of the Invention
[0006] The purpose of this invention is to provide a tobacco endogenous polypeptide Nt1318 and its applications. This addresses the problem in existing technologies of lacking the utilization of endogenously secreted small peptides in tobacco for the control of tobacco root rot.
[0007] In a first aspect, the present invention provides a tobacco endogenous polypeptide Nt1318, which is selected from any of the following: A1) an amino acid sequence as shown in SEQ ID NO.3; A2) an amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence defined in A1); A3) an amino acid sequence having more than 80% sequence identity compared to the amino acid sequence defined in A1) or A2); A4) an amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1) or A2) or A3).
[0008] The tobacco endogenous polypeptide Nt1318 provided by the present invention can be a natural, recombinant or synthetic active polypeptide. The active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants) using recombinant technology.
[0009] In this invention (A4), the linkage can be achieved through direct peptide bond connection or through a linker, using methods conventional in the art. The tags include, but are not limited to: GST (glutathione thioredoxin) tag protein, Trx (thioredoxin) tag protein, His tag protein (His-tag), Flag tag protein, LacZ tag protein, GFP (green fluorescent protein), sfGFP (hyperfolded green fluorescent protein), and HA tag (hemagglutinin tag). Those skilled in the art can select appropriate tag proteins according to actual needs. The use of tags does not alter the function of the target protein (tobacco endogenous polypeptide Nt1318); its purpose is for separation, purification, detection, or tracing. The tags can be separated from the target protein (tobacco endogenous polypeptide Nt1318) using chemical cleavage methods or enzymatic methods known in the art (e.g., introducing protease cleavage sites and using TEV protease to remove the tag).
[0010] In this invention, the inventors discovered that the tobacco endogenous polypeptide Nt1318 can induce a ROS outbreak in tobacco and activate the plant's basic immunity. Simultaneously, it can induce tobacco to develop resistance to Fusarium oxysporum infection and significantly reduce the disease index of Fusarium oxysporum root rot, demonstrating a good control effect against Fusarium oxysporum root rot. Therefore, the tobacco endogenous polypeptide Nt1318 has promising application prospects in the control of Fusarium oxysporum root rot in tobacco.
[0011] Furthermore, the tobacco endogenous peptide Nt1318 contains a highly conserved YISY core functional site, which is an essential recognition element for recognizing tobacco FER receptors, inducing rapid apoplast alkalization, and initiating ROS immune burst. At the same time, the tobacco endogenous peptide Nt1318 contains four conserved cysteine residues, which form intramolecular disulfide bonds to maintain the peptide's spatial conformation and ensure its induced resistance activity.
[0012] In some embodiments, the tobacco endogenous polypeptide Nt1318 is obtained by introducing a recombinant vector containing its encoding gene into an expression host (e.g., E. coli BL21(DE3)), then culturing the recombinant genetically engineered strain and inducing expression to obtain the tobacco endogenous polypeptide Nt1318.
[0013] In a second aspect, the present invention provides a nucleic acid molecule encoding the aforementioned tobacco endogenous polypeptide Nt1318.
[0014] The nucleic acid molecules provided by this invention can be DNA, such as cDNA, genomic DNA, or recombinant DNA; or RNA, such as mRNA or hnRNA; and these nucleic acid molecules can usually be obtained by PCR amplification or artificial synthesis.
[0015] In some embodiments, the nucleic acid molecule is selected from any of the following: B1) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO.4; B2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in B1) and encodes the tobacco endogenous polypeptide Nt1318; B3) a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B1) or B2) and encodes the tobacco endogenous polypeptide Nt1318.
[0016] As used herein, the term "hybridization under stringent conditions" refers to the hybridization of two nucleic acid fragments under standard hybridization conditions as described in the section "Expression of Cloned Genes in E. coli" of *Molecular Cloning: A Laboratory Manual* (1989) (Cold Spring Lane Laboratory, New York, USA). Such conditions include hybridization in 6.0 × SSC at 45 °C, followed by washing in 2 × SSC at 50 °C. To select stringency, the salt concentration in the washing step can be chosen, for example, between 2.0 × SSC at 50 °C for low stringency and 2.0 × SSC at 50 °C for high stringency. Additionally, the temperature in the washing step can be varied between room temperature (approximately 22 °C) for low stringency and 65 °C for high stringency.
[0017] As used herein, the term “sequence identity” can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). Molecules are identical at that position when positions in the compared sequences are occupied by the same bases or amino acids. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. “Sequence identity” of a polynucleotide or amino acid sequence with another sequence having a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.
[0018] In a third aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules.
[0019] The recombinant vector provided by the present invention includes a cloning vector and an expression vector. The cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene. The vector used to construct the expression vector can be at least one of pET-26b(+), pET28a, pET23a, and pET-32a vectors. In the present invention, the expression vector is preferably the pET-32a vector.
[0020] In a fourth aspect, the present invention provides a recombinant cell comprising the above-described nucleic acid molecule or the above-described recombinant vector.
[0021] In some implementations, the method for preparing recombinant cells includes the step of converting the recombinant vector into expression host cells.
[0022] In this invention, the expression host cell is a conventional host cell in the art, as long as the recombinant vector can stably replicate itself and the gene it carries can be effectively expressed. It can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli or yeast. For example, Escherichia coli can be E. coli BL21(DE3), Rosetta(DE3), or BL21(DE3)plysS. In this invention, E. coli BL21(DE3) is preferred as the expression host.
[0023] In a fifth aspect, the present invention provides a method for preparing tobacco endogenous polypeptide Nt1318, comprising the following steps: culturing the above-mentioned recombinant cells, inducing expression to obtain a culture; and isolating the above-mentioned tobacco endogenous polypeptide Nt1318 from the culture.
[0024] In this invention, there are no special requirements for the culture method and culture conditions; it is sufficient to ensure the normal growth of the recombinant cells. Furthermore, the methods for isolating the aforementioned tobacco endogenous polypeptide Nt1318 from the culture are all conventional methods in the art.
[0025] In some embodiments, the culture medium used in the preparation of the tobacco endogenous polypeptide Nt1318 is a protein-expressing culture medium in the art, preferably LB medium.
[0026] In a sixth aspect, the present invention provides an immune induction agent comprising the above-mentioned tobacco endogenous polypeptide Nt1318.
[0027] In some implementations, the immune inducer also includes excipients.
[0028] In this invention, excipients are primarily used to provide a safe, stable, and functional composition. They may also provide methods to dissolve the active ingredient at a desired rate or to promote effective absorption of the active ingredient by plants after acceptance of the composition. Excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. Excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.
[0029] The immune induction agents provided by this invention can be prepared using any method known to those skilled in the art, based on the disclosed information. Examples include, but are not limited to, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.
[0030] In some implementations, the dosage form of the immune inducer includes at least one of solid dosage form, semi-solid dosage form, and liquid dosage form.
[0031] In some implementations, solid dosage forms include at least one of tablets, capsules, powders, pills, granules, and suppositories; semi-solid dosage forms include at least one of ointments, creams, and patches; and liquid dosage forms include at least one of injections, oral liquids, syrups, and inhalants.
[0032] In a seventh aspect, the present invention provides the application of the tobacco endogenous polypeptide Nt1318 as described above, any of the above-described nucleic acid molecules, the above-described recombinant vector, the above-described recombinant cells, the tobacco endogenous polypeptide Nt1318 prepared by the above-described preparation method, or the above-described immune induction agent in the prevention and control of plant root rot.
[0033] In some implementations, plant root rot is caused by Fusarium oxysporum.
[0034] It is understood that the fungal pathogens causing plant root rot can be any conventional fungal pathogens found in the prior art, as long as they can cause plant root rot. For example, in this invention, plant root rot is preferably caused by Fusarium oxysporum.
[0035] In some implementations, the plant includes tobacco.
[0036] It is understood that the plant can be any plant conventional in the prior art, and in this invention, the plant preferably includes tobacco.
[0037] The beneficial effects of this invention are as follows: Unlike existing technologies, the tobacco endogenous polypeptide Nt1318 provided by this invention can induce a ROS outbreak in tobacco and activate the plant's basic immunity; at the same time, it can induce tobacco to develop disease resistance against Fusarium oxysporum infection and significantly reduce the disease index of Fusarium oxysporum root rot in tobacco, thus exhibiting a good control effect on Fusarium oxysporum root rot in tobacco; therefore, this tobacco endogenous polypeptide Nt1318 has good application prospects in the control of Fusarium oxysporum root rot in tobacco. Attached Figure Description
[0038] Figure 1 This is a sequence information diagram of the Nt1318 precursor protein in Example 1 of the present invention. The mature polypeptide sequence is located at the C-terminus of the precursor protein and is underlined. The arrows indicate the signal peptide restriction sites. Figure 2 This is a graph showing the SDS-PAGE detection results of the target protein in Example 2 of the present invention; Figure 3 This is a WB detection result diagram of the target protein in Example 2 of the present invention; Figure 4 This is a graph showing the detection results of ROS burst induced by tobacco endogenous polypeptide Nt1318 in Example 3 of the present invention; Figure 5 This is a graph showing the disease index detection results of tobacco root rot in different treatment groups in Example 4 of the present invention; Figure 6 The results of indoor disease resistance test of tobacco endogenous polypeptide Nt1318 against tobacco Fusarium oxysporum root rot in Example 4 of the present invention are shown. Among them, (A) is a comparison of the aboveground tobacco parts in different treatment groups, (B) is a comparison of the root disease in different treatment groups, and (C) is a statistical result of the length of diseased tobacco roots in different treatment groups. Detailed Implementation
[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.
[0041] In the description of the embodiments of this invention, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this invention, "multiple" means two or more, unless otherwise explicitly defined.
[0042] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.
[0043] In the description of the embodiments of this invention, the term "and / or" is merely a description of the relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following associated objects have an "or" relationship.
[0044] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.
[0045] In this invention, healthy plants of the tobacco variety Yunyan 87 are grown to the 4-5 leaf stage.
[0046] The culture medium used in this invention is as follows: LB medium (liquid): 5 g peptone, 2.5 g yeast extract, 5 g sodium chloride, pH adjusted to 7.0, add ddH2O to bring the volume to 500 mL, autoclave at 121℃ for 30 min.
[0047] In this invention, some of the reagents are prepared as follows: Ampicillin: Prepare a stock solution of 100 mg / mL ampicillin (Amp) using distilled water as a solvent. After filtration through a bacterial filter, store at -20°C. The working concentration is 50-100 μg / mL, i.e., add 100-200 μL of the stock solution to every 200 mL of culture medium.
[0048] 10×TBS: 242 g Tris-base, 80 g NaCl, add ddH2O to bring the volume to 1 L.
[0049] TBST: To prepare TBST, add 0.5 mL of Tween-20 to 1×TBS.
[0050] Protein electrophoresis buffer (1×Running buffer): 3.03 g Tris, 18.8 g glycine, 1 g SDS, add ddH2O to bring the volume to 1 L.
[0051] Protein transfer buffer (I×Transter buffer): 14.4 g glycine, 200 mL methanol, 3.03 g Tris, add ddH2O to bring the volume to 1 L.
[0052] Protein loading buffer and protein marker (10-250 kDa), pre-made protein gel kit, ECL chemiluminescent substrate and PVDF membrane were purchased from BIO-RAD.
[0053] Example 1: Sequence mining of tobacco endogenous polypeptide Nt1318 The precursor protein sequence information of the RALF family endogenous polypeptide Nt1318 was obtained from the genome of tobacco Yunyan 87 (e.g. Figure 1As shown in the figure, further analysis yielded the sequence information of the tobacco endogenous peptide Nt1318. This tobacco endogenous peptide Nt1318 sequence contains a highly conserved YISY core functional site, which is an essential recognition element for recognizing the tobacco FER receptor, inducing rapid apoplast alkalization, and initiating ROS immune burst. The mature peptide of the tobacco endogenous peptide Nt1318 contains four conserved cysteine residues, forming intramolecular disulfide bonds to maintain the peptide's spatial conformation and ensure its induced resistance activity.
[0054] The amino acid sequence of the Nt1318 precursor protein is as follows: MGVPSGLILCVLIGAFFISMAAAGDSGAYDWVMPARSGGGCKGSIGECIAEEEEFELDSESNRRILATKKYISYGALQKNSVPCSRRGASYYNCKPGAQANPYSRGCSAITRCRS(SEQ ID NO.1); The nucleotide sequence of the gene encoding the Nt1318 precursor protein is as follows: atgggagttccttcaggtttgattctttgtgttctgatcggagcttttttcatttcaatggcggcggccggagatagtggggcctacgattgggtgatgccggcgagatctggtgggggatgcaaagggagtatcggagagtgcattgctgaagaagaggagtttgagctggacag tgagtcaaacaggcgcattttagccaccaaaaagtacatcagctatggtgcactgcagaagaacagtgtaccttgttctcgccgtggagcttcgtattacaactgcaaacctggtgctcaggctaatccttactctcgtggatgcagtgctatcactcgttgcaggagttaa(SEQ ID NO.2); The amino acid sequence of the tobacco endogenous polypeptide Nt1318 is as follows: ATKKYISYGALQKNSVPCSRRGASYYNCKPGAQANPYSRGCSAITRCRS(SEQ ID NO.3); The nucleotide sequence of the gene encoding the tobacco endogenous polypeptide Nt1318 is as follows: gccaccaaaaagtacatcagctatggtgcactgcagaagaacagtgtaccttgttctcgccgtggagcttcgtattacaactgcaaacctggtgctcaggctaatccttactctcgtggatgcagtgctatcactcgttgcaggagttaa (SEQ ID NO. 4).
[0055] The theoretical molecular weight of the Nt1318 precursor protein is 12.76 kDa, and the theoretical molecular weight of the tobacco endogenous polypeptide Nt1318 is 5.5 kDa.
[0056] Example 2 Preparation of tobacco endogenous polypeptide Nt1318 2.1 Carrier Construction The prokaryotic expression vector pET-32a was digested with restriction enzymes BamHI and XhoI under the following conditions: 37℃ for 25 min; 80℃ for 20 min. After successful digestion and gel electrophoresis, the artificially synthesized gene fragment encoding the tobacco endogenous polypeptide Nt1318 was subjected to the same restriction enzyme digestion to obtain the target fragment. The vector and the target fragment were then ligated using 2×MultiF Seamless AssemblyMix ligase under the following conditions: 50℃ for 15 min to obtain the ligation product.
[0057] 2.2 Heat shock transformation of Escherichia coli competent cells DH5α Add 1 μL of the ligation product obtained in step 2.1 to DH5α competent cells. Gently tap the bottom of the tube to thoroughly mix the ligation product with the competent cells, then incubate on ice for 25 min. Next, heat shock the cells at 42°C for 45 s, then quickly return them to ice and incubate for 2 min. Afterward, add 500 mL of LB liquid medium to the heat-shocked competent cells and incubate at 37°C and 120 rpm for 45 min. After incubation, spread 50 μL of the medium onto a sterile glass bead plate containing ampicillin. Invert the plate and incubate overnight in the dark at 37°C until single colonies grow. Select 8 single colonies and perform colony PCR. Amplify the colonies using sequencing primers. Check for successful insertion of the target band. Expand the correct clones at 37°C and 200 rpm, extract plasmids, and send them for sequencing. Retain the correctly sequenced plasmids for later use.
[0058] 2.3 Transformation of E. coli competent cells BL21 Thaw competent cells in an ice bath for about 5-10 minutes. Too long a thawing time will affect the transformation efficiency. Add 1 μL of the recombinant plasmid obtained in step 2.2 to the competent cells. Gently tap the bottom of the tube about 2-3 times. Immediately incubate on ice for 30 minutes. Spread the bacterial culture on a plate containing ampicillin and incubate overnight at 37°C and 120 rpm. On the second day, pick single clones to verify the bacterial culture for PCR. After successful transformation, transfer to a 10 mL centrifuge tube and incubate overnight. At the same time, incubate E. coli strains containing the pET-32a empty vector.
[0059] 2.4 Prokaryotic induction of target protein expression Add the well-cultured bacterial suspension to LB liquid medium at a ratio of 1:1000, and incubate at 37°C and 200 r / min for approximately 3 hours. Adjust the OD value accordingly. 600 After adding 0.2 mmol / L IPTG to the culture medium, the culture was induced at 28℃ and 200 r / min for 8 h. The bacterial culture was then transferred to 50 mL centrifuge tubes, and the induced bacterial cells were collected by centrifugation. The bacterial cells were resuspended in chilled PBS, and the operation was performed in an ice bath. The bacterial cells were lysed by sonication (100W) for 5 s, 10 s, and 10 min, with a 2 min interval between each cycle, until the bacterial culture was clear. The culture was then centrifuged at 12000 rpm for 20 min using a centrifuge chilled at 4℃, and the total protein in the supernatant was collected and stored at -80℃ for later use.
[0060] The total protein in the supernatant was detected by SDS-PAGE, and the results are as follows: Figure 2 As shown.
[0061] from Figure 2 As can be seen, a band of the target protein (tobacco endogenous polypeptide Nt1318, containing a carrier tag protein) appears at around 30 kDa, indicating that the tobacco endogenous polypeptide Nt1318 is successfully expressed.
[0062] 2.5 Western blot detection The target protein induced in step 2.4 was collected and subjected to SDS-PAGE gel electrophoresis. After electrophoresis, a Bio-rad semi-dry electroblotting system (Trans-blot) was used. ®Transfer the protein to a PVDF membrane (SD) at 80V for 90 min, drain excess water, and place in 5% skim milk (prepared with 1×TBST) for blocking at room temperature for 2 h. Add primary antibody at a 1:5000 dilution with 1×TBST and incubate at room temperature for 2 h or overnight at 4°C. Wash 6 times with TBST for 5 min each time. Add HRP-labeled secondary antibody at a 1:10000 dilution with 1×TBST and incubate at room temperature for 1 h. Wash 6 times with TBST for 5 min each time. Drain excess water and use Pierce... ® The ECL protein blot substrate was subjected to a colorimetric reaction for 5 min, and imaging and image processing were performed using a Bio-Rad ChemiDoc™ XRS+ instrument. The results are as follows: Figure 3 As shown.
[0063] from Figure 3 As can be seen, a band of the target protein (tobacco endogenous polypeptide Nt1318, containing a carrier tag protein) appears at around 30 kDa, indicating that the tobacco endogenous polypeptide Nt1318 is successfully expressed.
[0064] Example 3 Detection of ROS burst induced by tobacco endogenous peptide Nt1318 A highly sensitive chemiluminescence method was used to monitor the reactive oxygen species (ROS) burst induced by the tobacco endogenous peptide Nt1318. The specific procedures are as follows: Healthy tobacco leaves with uniform growth were selected, and leaf discs with a diameter of 8 mm were prepared using a punch. The leaf discs were floated in a detection buffer containing 100 μmol / L luminol and incubated overnight to eliminate background ROS signals caused by mechanical damage.
[0065] The groups were set as follows: tobacco endogenous polypeptide Nt1318 treatment group (supernatant prepared in step 2.4 of Example 2, with Nt1318 concentration controlled at 60 and 100 μmol / L), empty vector protein negative control group (using the method in Example 2 to induce expression of Escherichia coli strain containing pET-32a empty vector to obtain vector tag protein), Flg22 positive immune induction control group, and sterile water blank control group.
[0066] The different treatment groups were immediately placed in the chemiluminescence analyzer chamber. Within 0-60 min after the addition of the small peptide, the dynamic changes in chemiluminescence intensity were continuously monitored and recorded, with a sampling interval of 10 s. After the experiment, the onset time, peak intensity, peak occurrence time, and total luminescence integral area of the ROS burst were calculated by analyzing the luminescence kinetic curves to comprehensively evaluate the ability and intensity of the tobacco endogenous peptide Nt1318 to induce ROS bursts. At least eight biological replicates were set up for each treatment group to ensure the statistical reliability of the data. ROS generation was expressed in relative light units (RLU) and plotted using Graphpad. The results are shown below. Figure 4 As shown.
[0067] from Figure 4 As can be seen, both Flg22 and Nt1318 induced a rapid and intense ROS burst (peak at 10 min); the negative control group showed no significant signal. These results indicate that Nt1318 possesses the potential to be a plant immune elicitor, capable of inducing ROS bursts, and its activity exhibits a clear dose-dependent effect; the higher the concentration, the stronger the eliciting activity, with a highly significant immune activation advantage at high concentrations.
[0068] Example 4: Indoor disease resistance test of tobacco endogenous polypeptide Nt1318 against Fusarium oxysporum root rot in tobacco. In this embodiment, the indoor resistance effect of the tobacco endogenous polypeptide Nt1318 against *Fusarium oxysporum* root rot was tested. Specifically, the following steps were included: 1) Preparation of pathogen inoculation substrate Sterilized wheat grains were inoculated with Fusarium oxysporum mycelium cakes. After the mycelium had fully grown in the dark at 28°C, the wheat grains and sterilized substrate were mixed at a ratio of 1:50 to prepare diseased soil.
[0069] 2) Tobacco seedling stage treatment methods Healthy five-leaf, one-heart Yunyan 87 tobacco seedlings were selected. The supernatant prepared in step 2.4 of Example 2 was diluted, and the Nt1318 concentration was controlled at 60 μmol / L before root irrigation treatment, with 20 mL of diluted solution applied to each seedling. The control group was treated with an equal amount of empty vector protein (the vector tag protein was obtained by inducing expression in an E. coli strain containing the pET-32a empty vector using the method in Example 2) and PBS buffer. After treatment, the plants were kept moist for 24 h and then transplanted into mold-treated pots. At the same time, a control group of untreated healthy plants was set up.
[0070] 3) Greenhouse cultivation conditions Cultured at 26℃ with a 16-hour light / 8-hour dark cycle.
[0071] 4) Disease classification and prevention efficacy calculation standards The disease grading, disease index, and control efficacy calculation methods for root rot were based on the published research paper by Li XJ et al. (Isolation, identification, and evaluation of the biocontrol potential of aBacillus velezensis strain against tobacco root rot caused by...). Fusarium oxysporum The disease grading system, published in the journal *Journal of Applied Microbiology*, is as follows: Grade 0: No symptoms; Grade 1: Less than 1 / 3 of the plant has wilted leaves; Grade 3: 1 / 3 to 1 / 2 of the total plant area has wilted leaves; Grade 5: 1 / 2 to 2 / 3 of the total plant area has wilted leaves; Grade 7: More than 2 / 3 of the plant area has wilted leaves; Grade 9: Plant dies. The Disease Index (DI) and Control Efficacy (Ce) are calculated using the following formula: This quantifies the control ability of the tobacco endogenous polypeptide Nt1318 against tobacco root rot.
[0072] Disease Index (DI) = ∑(representative value of disease severity level × number of plants affected at that level) / (total number of plants surveyed × representative value of the highest disease severity level) × 100; Prevention and control efficacy (Ce) = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100%; The disease index and control efficacy of tobacco root rot in different treatment groups are shown in Table 1 below. Figure 5 As shown.
[0073] Table 1. Statistical results of disease index and prevention efficacy in different treatment groups.
[0074] From Table 1 and Figure 5 As can be seen, compared with PBS Buffer and pET-32a treatment, the Nt1318 treatment provided by this invention can significantly reduce the disease index of tobacco root rot and improve the control effect of tobacco root rot.
[0075] Indoor disease resistance test results of different treatment groups are as follows: Figure 6 As shown.
[0076] from Figure 6 As can be seen, compared with the Mock control group, tobacco leaf growth and root length were inhibited in other treatment groups due to Fusarium oxysporum infection; however, compared with the pET-32a and PBS buffer groups, tobacco leaf growth and root length were significantly improved in the Nt1318 treatment group. Figure 6(A-6C); The results showed that the tobacco endogenous polypeptide Nt1318 had a good control effect on tobacco Fusarium oxysporum root rot.
[0077] In summary, the tobacco endogenous polypeptide Nt1318 provided by this invention can induce ROS burst in tobacco and activate the plant's basic immunity. At the same time, it can induce tobacco to produce disease resistance to Fusarium oxysporum infection and significantly reduce the disease index of Fusarium oxysporum root rot in tobacco, thus having a good control effect on Fusarium oxysporum root rot in tobacco.
[0078] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.
[0079] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A tobacco endogenous polypeptide Nt1318, characterized in that, The tobacco endogenous polypeptide Nt1318 is selected from any one of the following: A1) The amino acid sequence is shown in SEQ ID NO.3; A2) An amino acid sequence having one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence defined in A1); A3) An amino acid sequence that has more than 80% sequence identity with the amino acid sequence defined by A1) or A2); A4) An amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1), A2), or A3).
2. A nucleic acid molecule encoding the tobacco endogenous polypeptide Nt1318 as described in claim 1.
3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecule is selected from any of the following: B1) Nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO.4; B2) A nucleic acid molecule that hybridizes with the nucleic acid molecule defined in B1) and encodes the tobacco endogenous polypeptide Nt1318 as described in claim 1; B3) is a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B1) or B2) and encodes the tobacco endogenous polypeptide Nt1318 as described in claim 1.
4. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 2 or 3.
5. A recombinant cell, characterized in that, It comprises the nucleic acid molecule of claim 2 or 3 or the recombinant vector of claim 4.
6. A method for preparing tobacco endogenous polypeptide Nt1318, characterized in that, Includes the following steps: The recombinant cells described in claim 5 were cultured and induced to express the desired expression to obtain a culture. The tobacco endogenous polypeptide Nt1318 of claim 1 was isolated from the culture.
7. An immune inducing agent, characterized in that, Includes the tobacco endogenous polypeptide Nt1318 as described in claim 1.
8. The application of the tobacco endogenous polypeptide Nt1318 as described in claim 1, the nucleic acid molecule as described in any one of claims 2-3, the recombinant vector as described in claim 4, the recombinant cell as described in claim 5, the tobacco endogenous polypeptide Nt1318 prepared by the preparation method as described in claim 6, or the immune induction agent as described in claim 7 in the prevention and control of plant root rot.
9. The application according to claim 8, characterized in that, The plant root rot is caused by Fusarium oxysporum.
10. The application according to claim 8 or 9, characterized in that, The plant mentioned includes tobacco.