Application of pnpub35 protein and its encoding gene in regulating resistance of plants to fusarium wilt

Abstract

The present application relates to the field of biotechnology, and in particular to the application of PnPUB35 protein and its coding gene in regulating the resistance of plants to fusarium wilt. The present application provides a pepper PnPUB35 protein and its gene PnPUB35, which are related to the resistance of pepper to fusarium wilt and contain a U-box domain E3 ubiquitin ligase. The expression of pepper PnPUB35 in the roots of pepper increases after inoculation with Fusarium solani. The resistance of pepper PnPUB35 silenced plants to Fusarium solani is weakened, and the resistance of Arabidopsis PnPUB35 overexpressed plants to Fusarium solani is enhanced. The PnPUB35 gene of the present application plays a positive regulation role in the resistance of pepper to fusarium wilt, and can be used as an important candidate gene for breeding pepper resistant to fusarium wilt.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the PnPUB35 protein and its encoding gene in regulating plant resistance to Fusarium wilt. Background Technology

[0002] Fusarium wilt is a significant disease affecting pepper cultivation, causing substantial economic losses to the pepper industry and severely impacting the stable development of the global pepper industry. However, the pathogen causing fusarium wilt, *Fusarium solani*, can survive for extended periods in the soil and can act as a primary source of infection, reinfecting the pepper roots and causing disease. Infected plants exhibit yellowing and leaf drop from top to bottom, with necrosis of the vascular bundles in the underground roots and near-ground stems, making control extremely difficult.

[0003] Plant U-box proteins (PUBs), a family of proteins containing U-box domains, belong to the E3 ubiquitin ligase family. PUBs assist in the ubiquitination and degradation of their substrate proteins, playing a crucial role in plant immune signaling pathways. Studies have shown that PUBs directly participate in regulating crop resistance to pathogens in crops such as rice, tomato, and cotton. For example, rice OsPUB44 activates the PTI response, positively regulating rice resistance to bacterial blight; cotton GhPUB17 negatively regulates cotton resistance to Verticillium wilt. However, research on the role of PUBs in pepper disease resistance is limited.

[0004] Because the pathogen causing pepper wilt can survive in the soil for extended periods, current control primarily relies on chemical pesticides, which are inefficient and environmentally harmful. Therefore, studying the function of the pepper U-box protein PnPUB35 using molecular disease resistance breeding techniques has become a crucial approach to solving the problem of pepper resistance to wilt.

[0005] To date, it remains unclear whether PnPUB35 is associated with resistance to wilt in pepper. The completion of functional studies on PnPUB35 will help to further understand the molecular mechanisms of wilt resistance in pepper, provide new gene resources for molecular breeding of pepper wilt resistance, and offer new ideas for the control of wilt in pepper. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide the application of PnPUB35 protein and its encoding gene in regulating plant wilt resistance. The present invention has found that PnPUB35 protein or its encoding gene can be used for plant wilt resistance.

[0007] This invention provides the application of at least one of the following ① to ⑤ in regulating plant resistance to Fusarium wilt.

[0008] ① PnPUB35 protein;

[0009] ② A protein that has one or more amino acids substituted, deleted, or added to the amino acid sequence of PnPUB35 protein, and has the same or similar function as PnPUB35 protein.

[0010] ③ The nucleic acid molecule encoding the protein described in ① or ②;

[0011] ④ A nucleic acid molecule that has one or more nucleotides substituted, deleted, or added in the nucleotide sequence of the nucleic acid molecule described in ③, and that encodes a protein with the same or similar functions;

[0012] ⑤ A substance that can regulate the level or activity of at least one of ① to ④.

[0013] In some embodiments, the PnPUB35 protein has the amino acid sequence shown in SEQ ID NO:1, and the PnPUB35 protein encoding gene has the nucleotide sequence shown in SEQ ID NO:2.

[0014] In some embodiments, the plant includes at least one of pepper, Arabidopsis thaliana, rice, tomato, and cotton.

[0015] This invention provides an agent for improving plant resistance to wilt disease, comprising at least one of the following (1) to (5):

[0016] (1) Expression vectors that upregulate the PnPUB35 protein encoding gene;

[0017] (2) A recombinant host containing (1);

[0018] (3) Nucleic acid molecules that upregulate the expression of the PnPUB35 protein-encoding gene;

[0019] (4) Strong promoters for the expression of the PnPUB35 protein-coding gene;

[0020] (5) Preparations that promote the transcription of the PnPUB35 protein-encoding gene;

[0021] (6) PnPUB35 protein-encoding gene activity promoter.

[0022] This invention provides an agent for reducing plant resistance to Fusarium wilt, comprising at least one of the following: 1) to 5)

[0023] 1) Expression vectors that knock out, knock down, or silence the PnPUB35 protein-coding gene;

[0024] 2) Recombinant hosts containing (1);

[0025] 3) Nucleic acid molecules that downregulate the expression of the PnPUB35 protein-encoding gene;

[0026] 4) Terminator or transposon for the expression of the PnPUB35 protein-coding gene;

[0027] 5) Agents that inhibit the transcription of the PnPUB35 protein-encoding gene;

[0028] 6) Inhibitor of PnPUB35 protein-encoding gene activity.

[0029] This invention provides the application of the preparations that enhance plant resistance to Fusarium wilt or the preparations that reduce plant resistance to Fusarium wilt in regulating plant resistance to Fusarium wilt.

[0030] This invention provides a method for improving plant resistance to Fusarium wilt, wherein the preparation for improving plant resistance to Fusarium wilt upregulates the expression of the PnPUB35 protein-encoding gene, the transcription of the PnPUB35 protein-encoding gene, and the level and / or activity of endogenous PnPUB35 protein in plants.

[0031] This invention provides a method for reducing plant resistance to Fusarium wilt, wherein the agent for reducing plant resistance to Fusarium wilt is used to knock out, knock down or silence the expression of the PnPUB35 protein-encoding gene, inhibit the transcription of the PnPUB35 protein-encoding gene, and reduce the level and / or activity of endogenous PnPUB35 protein in plants.

[0032] This invention provides an agent for predicting plant resistance to Fusarium wilt, comprising:

[0033] Preparations for detecting the transcriptional level of the PnPUB35 protein-encoding gene; and / or

[0034] A formulation for detecting the expression level or activity of the PnPUB35 protein-encoding gene.

[0035] This invention provides a method for identifying plant resistance to Fusarium wilt, which involves using the agent for predicting plant resistance to Fusarium wilt to detect the transcriptional level of the PnPUB35 protein-encoding gene in plants or to detect the expression level or activity of the PnPUB35 protein-encoding gene.

[0036] The beneficial effects of this invention are:

[0037] This invention provides a protein and its gene, PnPUB35, containing an E3 ubiquitin ligase with a U-box domain, associated with resistance to Fusarium wilt in pepper. The expression level of PnPUB35 increases in pepper roots after inoculation with Fusarium solani. Plants with silenced PnPUB35 in pepper show reduced resistance to Fusarium solani, while plants overexpressing PnPUB35 in Arabidopsis show enhanced resistance. The PnPUB35 gene of this invention plays a positive regulatory role in pepper resistance to Fusarium wilt and can serve as an important candidate gene for breeding pepper with resistance to Fusarium wilt. Attached Figure Description

[0038] Figure 1 This figure shows the expression of PnPUB35 in pepper, where ** represents p < 0.01.

[0039] Figure 2 The figure shows the expression of PnPUB35 in pepper roots under the stress of Fusarium solani. ** represents p<0.01 and *** represents p<0.001.

[0040] Figure 3 The figure shows the silencing status of PnPUB35 in pepper as detected by quantitative fluorescence detection. ** represents p<0.01.

[0041] Figure 4 This shows the silencing status of PnPUB35 in pepper detected by RT-PCR;

[0042] Figure 5 This shows the disease incidence after inoculation with pathogens following silencing of the pepper PnPUB35 gene;

[0043] Figure 6 The figure shows the disease index after inoculation with pathogens after silencing the pepper PnPUB35 gene. * represents p<0.05.

[0044] Figure 7 This shows the isolation of pathogens from the stems of pepper plants with the PnPUB35 gene silenced after inoculation.

[0045] Figure 8 The figure shows the biomass of pathogens in the stems of pepper plants with the PnPUB35 gene silenced after inoculation. ** represents p<0.01.

[0046] Figure 9 This shows the accumulation of callose in the leaves of pepper plants with the PnPUB35 gene silenced after inoculation.

[0047] Figure 10 The test showed a positive result in transgenic Arabidopsis thaliana overexpressing PnPUB35.

[0048] Figure 11 This shows the disease incidence of PnPUB35-overexpressing transgenic Arabidopsis thaliana after inoculation with pathogens;

[0049] Figure 12 The figure shows the incidence of disease in PnPUB35 overexpressing transgenic Arabidopsis thaliana after inoculation with pathogens. * represents p<0.05.

[0050] Figure 13 The figure shows the pathogen biomass analysis after PnPUB35 overexpression in transgenic Arabidopsis thaliana. * indicates p<0.05. Detailed Implementation

[0051] This invention provides the application of the PnPUB35 protein and its encoding gene in regulating plant resistance to Fusarium wilt. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0052] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0053] Example 1: Expression levels of PnPUB35 in various tissues of pepper plants

[0054] Fresh pepper root, stem, leaf, flower and fruit samples were collected from the pepper plantation (variety "Reyin 1") of the Spice and Beverage Research Institute of the Chinese Academy of Tropical Agricultural Sciences, and RNA was extracted from each tissue.

[0055] The gene information for PnPUB35, a gene associated with resistance to wilt in black pepper, is derived from the article "The chromosome-scale reference genome of black pepper provides insight into piperine biosynthesis. Nat Commun, 10, 4702." The genome assembly and annotation files are available at the following website: File name: Piper_nigrum_genome.tar.gz (Black pepper chromosome-level reference genome file).

[0056] Specifically, the CDS gene sequence of PnPUB35, a gene associated with resistance to wilt in pepper, is shown in SEQ ID NO: 2:

[0057]

[0058] The U-box type E3 ubiquitin ligase (also known as the PnPUB35 protein) encoded by the pepper gene PnPUB35 has the amino acid sequence shown in SEQ ID NO: 1 (containing a conserved U-box domain, as shown in bold):

[0059] MEIGSALEEENRLSSLAFQTITVAVCGNKSSKHALKWALDKFIPEGRLIFKLLYVRPTIAIVPTPMGISIPISKVRDDIAAAYKKEIEWQTDAMLLPFKNLCNKRQVEAEAIAIEADDVAEAISSEVSKCLFTTLVIGASSRNLLTRKFKGREMSSRILDCVPNFCTVYTVSNDRLLSVRHSTSDIILNRGEKTDCGMSVSS DASQTSSSQAASEGTSPNSSTEYSYNCPSSLPLQRYQALLTVNQALMNRKANMVDVHRSKQSLCPSEEDDMTSISDYSEMPYGGSLISSSRSIETDDFSIVSDQPSSSNRLAEASMSGSEMDLNFELERLRLELKHLNGIYTMAQTEKMEASLQLDELSKRRTVEAIALREINLREEQARELAELQKERNEVARREAELVTEC AWREALDKEKAEIQAARDKKGRLNLEKSLMNIDQKYKRFSWEEIESATSSFSDTNKIGIGAYGTVYKCKLAHTIVAVKVLHSFEGQIKRTFQQELDILSSIHHPHLLLLLGACPEHGCVVYEYMDNGSLEDRLFRKDNTPSIPWFHRFRIAWEIAAALAYLHDAKPKQIVHRDLKPSNILLDQKLFSKIGDVGLSTLLPSDNS SVASIYKETAPIGTLCYIDPEYQRSGLVSPKSDLYAFGIVVMQLLTAKPPMALAPIVETALEEGNLAGILDADAGQWPLEETQELALLALSCTELRRRRDRPDLSSQLLPNLKRLKDFAEMANDSSTLVPAAPPSHFICPILKDVMNDPCVAADGYTYDRTAMEIWLNMNDKSPVTNSTLPHKHLIPNYTLLSAITEWRTRNRS

[0060] Primers for quantitative fluorescence analysis of PnPUB35 were designed based on its gene and CDS sequences: qPnPUB35-F: AGCACCCATTGTGGAGACTG (SEQ ID NO: 3), qPnPUB35-R: CTGAGTGCCAGAAGAGCCAA (SEQ ID NO: 4). The pepper gene PnMLF1 was used as an internal reference gene for quantitative fluorescence analysis: qPnMLF1-F: CGAGAGGGCCGATCATTGAA (SEQ ID NO: 5), qPnMLF1-R: GGCTCATCGCTGGATCTGAA (SEQ ID NO: 6). The expression of PnPUB35 in various pepper tissues was detected.

[0061] like Figure 1 As shown, PnPUB35 is expressed in all tissues of pepper, with the lowest expression level in the fruit and the highest expression level in the stem, indicating that PnPUB35 plays an important role in the growth and development of pepper.

[0062] Example 2: Expression level of PnPUB35 in pepper roots after pathogen treatment

[0063] Pepper 'Reyin No. 1' was planted in vermiculite sandy soil and inoculated with a 1×10⁻⁶ spore suspension of Fusarium rotundifolium WN-1 using the root soaking method. 7 Pepper roots were soaked in a spore suspension for 15 minutes (CFU / mL), and root samples were collected at different time points to extract RNA. The expression of PnPUB35 after pathogen treatment was then detected.

[0064] like Figure 2 As shown, the expression level of PnPUB35 increased after pathogen infection, reaching its peak after 24 hours. This indicates that PnPUB35 is induced to express at high levels by Fusarium solani, and may be involved in regulating the resistance of pepper to Fusarium wilt.

[0065] Example 3: Studying the function of PnPUB35 using virus-mediated gene silencing (VIGS) technology.

[0066] 3.1 The Silence of PnPUB35 in Pepper

[0067] Primers for the PnPUB35 silencing vector were designed according to the VIGS primer design principle: 156-PnPUB35-F: GGTTACCGAATTCTCTAGATGCGTCCCAAATTTTTGTAC (SEQ ID NO: 7), 156-PnPUB35-R: GCCCGGGCCTCGAGACGCGTGAGCTCTCAACAATGCTTGATACCG (SEQ ID NO: 8). The PnPUB35 silencing fragment was amplified using the pepper "Hot Primer 1" cDNA as a template to construct the silencing vector pYL-156-PnPUB35, which was then transformed into Agrobacterium GV3101. After mixing the Agrobacterium culture with the pYL-156-PnPUB35 vector and an equal volume of the Agrobacterium culture with the helper vector pYL-192, and allowing it to stand, the OD was adjusted... 600 To 1.0, pepper leaves were injected using a needleless syringe. A mixture of pYL-156 empty vector Agrobacterium and an equal volume of pYL-192 auxiliary vector Agrobacterium was used as a control. After injection, the plants were cultured in the dark for 24 h, then placed under normal light at 28°C. The expression level of PnPUB35 in the silenced plants was detected using quantitative real-time PCR and RT-PCR to assess the silencing effect. Primers for RT-PCR were designed based on the gene sequence and CDS sequence of PnPUB35: PnPUB35-F: ATGGAAATTGGGAGTGCATT (SEQ ID NO: 9), PnPUB35-R: AGATCTGTTTCTGGTCCTCC (SEQ ID NO: 10). The pepper gene PnMLF1 is used as an internal reference gene. The primers for its RT-PCR are: PnMLF1-F: ATGGAGAGGGGAGGCGGGG (SEQ ID NO: 11)G, PnMLF1-R: TTTCTTTGCGCTAGAGAAGT (SEQ ID NO: 12).

[0068] like Figure 3 As shown in the figure, the results of quantitative real-time PCR showed that, compared with the empty vector control plant TRV:00, the expression level of PnPUB35 in the silent plant TRV:PnPUB35 was significantly reduced.

[0069] like Figure 4 As shown in the figure, the RT-PCR results showed that, compared with the empty vector control plant TRV:00, the expression of PnPUB35 in the silenced plant TRV:PnPUB35 was significantly reduced, indicating that PnPUB35 silencing was successful.

[0070] 3.2 Study on disease resistance of silent plants

[0071] Select plants with good silencing effects, and when they have grown 4 new leaves, inoculate them with Fusarium spore solution (concentration 1×10⁻⁶) using the root soaking method. 7 After soaking in CFU / mL solution for 15 minutes, the inoculated plants were transplanted into sterile nutrient soil and placed in a 28℃ greenhouse under normal light conditions. Disease severity was assessed three times at 4, 6, and 8 weeks post-inoculation. Each strain was tested three times individually, with three replicates. The number of diseased plants was counted based on leaf disease incidence. Disease severity was categorized into four levels: 0, 1 (less than 33% of leaves affected), 2 (more than 33% but less than 66% of leaves affected), 3 (more than 66% but less than 99% of leaves affected), and 4 (100%). The assessed data were calculated using the disease index formula, as follows:

[0072] Disease index = [(0·n0 + 1·n1 + 2⋅n2 + 3⋅n3 + 4⋅n4) / 4⋅n] × 100%

[0073] Note: n0-n4 represent the number of plants for each disease level, and n represents the total number of plants surveyed each time.

[0074] like Figure 5 As shown, the disease phenotype after inoculation was observed. Compared with the control plant TRV:00, the silent plant TRV:PnPUB35 showed increased leaf drop and significantly enhanced disease severity after inoculation.

[0075] like Figure 6 As shown, the results of the survey at 4, 6 and 8 weeks after inoculation showed that the disease index of the control plant TRV:00 after inoculation was 26.27, 32.10 and 38.63, respectively; and the disease index of the silent plant TRV:PnPUB35 after inoculation was 35.80, 49.76 and 64.71, respectively.

[0076] 3.3 Isolation of fungi from stems of silent plants after inoculation

[0077] To investigate the fungal recovery of pepper plant stems inoculated with different strains, pepper stems were collected 8 weeks after inoculation. The stems were disinfected by soaking in sodium hypochlorite and alcohol, then rinsed with sterile water. They were then cut into 1 cm segments, placed on PDA plates, and incubated in a 25°C incubator under darkness. Mycelial growth was observed after 3 days. The experiment was repeated three times.

[0078] like Figure 7 As shown, the number of stem segments from which pathogens were isolated in silent plants was significantly higher than that in control plants, indicating that the pathogen content in silent plants was high.

[0079] 3.4 Detection of fungal biomass in the stems of silent plants after inoculation

[0080] To detect changes in root fungal biomass in silent plants after inoculation, this experiment used qRT-PCR to determine the biomass of *Fusarium solani* in the stems of silent plants after inoculation. Total DNA was extracted from pepper stems 8 weeks after inoculation, and relative quantification was performed using the *Fusarium solani*-specific FsTEF gene as the target fragment and pepper PnMLF1 as the internal reference gene for quantitative PCR. TEF-F: ATGGGTAAGGARGACAAGAC (SEQ ID NO: 13), TEF-R: GGARGTACCAGTSATCTG (SEQ ID NO: 14).

[0081] like Figure 8 As shown, the biomass of pathogens in the stems of silent plants was significantly higher than that in control plants.

[0082] 3.5 Detection of callose in leaves of silent plants after inoculation

[0083] To detect callose deposition in the leaves of VIGS plants after inoculation, this experiment used toluidine blue staining to detect callose deposition in the leaves of inoculated VIGS plants. Leaf samples were selected 48 h after inoculation. After immersion in a mixture of alcohol and glacial acetic acid (V:V=3:1) for 3 h, the samples were then soaked in 70% alcohol and 50% alcohol for 2 h each, followed by overnight incubation in water. The leaves were then placed in 10% NaOH solution for 2 h, and finally stained with 0.01% toluidine blue solution for 3 h. Callose deposition was observed under UV excitation light using a fluorescence microscope.

[0084] like Figure 9 As shown, when pepper leaves were stained with aniline blue, the amount of callose accumulation in the silenced plants was lower than that in the control under ultraviolet excitation light, indicating that the immune resistance of pepper was reduced after PnPUB35 silencing.

[0085] Example 4: Overexpression of PnPUB35 in Arabidopsis thaliana enhances resistance to Fusarium wilt.

[0086] 4.1 Preparation of PnPUB35 overexpression transgenic Arabidopsis

[0087] Using the cDNA of pepper "Hot Guide 1" as a template, the CDS sequence of the PnPUB35 gene was amplified, and the plant overexpression vector pCAMBIA2300-PnPUB35 was constructed. The vector was then transformed into Agrobacterium GV3101, and the PnPUB35 protein was overexpressed in Arabidopsis thaliana by the flower dipping method. After three consecutive generations of resistance screening and molecular verification, positive transgenic seedlings were detected.

[0088] like Figure 10 As shown, three PnPUB35-positive transgenic lines were obtained by Western blot analysis.

[0089] 4.2 Identification of resistance in transgenic Arabidopsis thaliana

[0090] The root-immersion method was used to soak T3 generation positive overexpressing transgenic Arabidopsis thaliana in a Fusarium solani WN-1 spore suspension (concentration 1×10⁻⁶). 6 After soaking in a solution of CFU / mL for 2 minutes, the plants were transplanted into sterile nutrient soil and cultured normally at 25°C in a greenhouse. Three weeks later, the disease incidence was assessed, and the disease index was recorded. Diseased plants were collected, total DNA was extracted, and the fungal biomass within the plants was measured.

[0091] like Figure 11 As shown, the disease phenotype of the transgenic lines after inoculation was significantly milder than that of the wild-type lines.

[0092] like Figure 12 As shown, the survey results indicate that the incidence rates of transgenic Arabidopsis thaliana were 29.09%, 36.59%, and 40.45%, respectively, while the incidence rate of wild-type strains was 71.54%. Transgenic Arabidopsis thaliana exhibited significant resistance to Fusarium wilt.

[0093] like Figure 13 As shown in the results, the fungal biomass in transgenic Arabidopsis thaliana was significantly lower than that in wild-type strains.

[0094] The PnPUB35 gene of this invention plays a positive regulatory role in pepper resistance to Fusarium wilt and can be used as an important candidate gene for breeding pepper to resist Fusarium wilt.

[0095] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of PnPUB35 protein in improving resistance of Arabidopsis thaliana to Fusarium wilt: The amino acid sequence of the PnPUB35 protein is shown as SEQ ID NO: 1, and the nucleotide sequence of the PnPUB35 protein coding gene is shown as SEQ ID NO:

2.

2. Formulations for increasing resistance to fusarium wilt in Arabidopsis thaliana, characterized in that, At least one of the following (1)~(4) is included: (1) an expression vector for up-regulating the PnPUB35 protein coding gene; (2) a recombinant host containing (1); (3) a nucleic acid molecule for up-regulating the expression of the PnPUB35 protein coding gene; The amino acid sequence of the PnPUB35 protein is shown as SEQ ID NO: 1, and the nucleotide sequence of the PnPUB35 protein coding gene is shown as SEQ ID NO:

2.

3. A method for increasing resistance to fusarium wilt in Arabidopsis thaliana, characterized in that, The preparation of claim 2 up-regulates the expression of the PnPUB35 protein coding gene, the transcription of the PnPUB35 protein coding gene, the level and / or activity of the endogenous PnPUB35 protein in Arabidopsis thaliana.

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