Plant immunomodulatory gene RP, expression vector and application thereof

Abstract

This invention discloses a plant immune regulatory gene RP, its expression vector, and its applications. The inventors discovered a plant immune regulatory factor gene RP in potato, cloned its corresponding sequence, and transformed it into Nicotiana benthamiana using Agrobacterium competent cells. This verified that RP could be successfully expressed in other plants, and that it could be further expressed by Phytophthora infestans (a pathogenic fungus). Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ) and Phytophthora indicum ( Phytophthora parasitica The pathogenicity of this gene was verified, demonstrating that its expression effectively enhances the disease resistance of Nicotiana benthamiana. The experimental results indicate that this gene can serve as a candidate gene for breeding disease-resistant varieties, providing theoretical support and genetic resources for the control of plant diseases.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and in particular to a plant immune regulation gene RP, its expression vector, and its applications. Background Technology

[0002] Phytophthora and other pathogens pose a significant threat to plants, often causing devastating diseases known as "plant plague." These pathogens (such as Phytophthora infestans causing potato late blight) can infect leaves, stems, fruits, and roots, causing water-soaked lesions that rapidly necrotize and rot, especially in young, tender parts. Their destructive nature lies in their short incubation period, frequent reinfections, and rapid spread under suitable conditions, leading to substantial yield reductions or even crop failure, resulting in economic losses of hundreds of billions of yuan annually to agricultural production. Phytophthora overwinters primarily as mycelium or oospores in diseased plant debris or soil, spreading through rainwater and irrigation water. High humidity and warm conditions are particularly conducive to their outbreaks, and the pathogen is prone to mutation and the development of drug resistance. It may also disguise itself to evade the recognition of crop resistance genes, further complicating control efforts.

[0003] Current prevention and control strategies emphasize the integrated application of multiple methods to address the challenges. The primary measure is to select disease-resistant varieties (such as potato varieties carrying the specific resistance gene R9) and combine this with agricultural management practices such as raised bed cultivation, drainage and flood prevention, reasonable planting density, and crop rotation to reduce the risk of disease. Although chemical control relies on fungicides (such as metalaxyl-mancozeb, dimethomorph, copper-based fungicides, etc.), long-term use faces the problem of increased pathogen resistance (for example, metalaxyl has become ineffective against some late blight pathogens). Therefore, it is necessary to pay attention to the rotation of fungicides and precise application, and to remove diseased plants at the early stage of disease to reduce the source of infection. Meanwhile, biological control and green technologies are receiving increasing attention, including the application of biocontrol bacteria (such as Bacillus), plant immune inducers (such as BcIEB1 and RcCDI1), or "plant vaccine" technology to activate the plant's own resistance and to achieve reduced pesticide use and increased efficiency when used in combination with low-dose fungicides. In addition, disease monitoring and early warning systems (such as potato late blight early warning equipment) can improve the timeliness of control, and classified control strategies such as seed treatment and the application of resistance inducers can reduce costs and improve efficiency. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a plant immune regulation gene RP.

[0005] Another object of the present invention is to provide a plant immune regulatory protein RP.

[0006] Another object of the present invention is to provide an expression vector for the above-mentioned plant immune regulation gene RP.

[0007] Another object of the present invention is to provide the application of the above-mentioned plant immune regulation gene RP and its expression vector.

[0008] The objective of this invention is achieved through the following technical solution: A plant immune regulatory protein RP is at least one of the following: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (b) Analogs of the amino acid sequence shown in SEQ ID NO: 1 that still have the effect of enhancing plant immunity and disease resistance by substitution, insertion or deletion of one or more amino acids.

[0009] The aforementioned plant immune regulatory protein RP can enhance the plant's immune resistance to disease.

[0010] The nucleotide sequence of the encoding gene of the aforementioned plant immune regulatory protein RP is obtained according to the codon coding rules; preferably, the nucleotide sequence is shown in SEQ ID NO.2.

[0011] A plant immune regulation gene RP, the nucleotide sequence of which is at least one of the following: (a) The nucleotide sequence shown in SEQ ID NO.2; (b) Analogs of the nucleotide sequences in (a) above, obtained by base insertion, deletion, or substitution, that still have the effect of enhancing plant immunity and disease resistance.

[0012] An expression vector carrying the plant immune regulation gene RP, comprising the nucleotide sequence shown in SEQ ID NO.2.

[0013] The vector backbone of the expression vector carrying the plant immune regulation gene RP is pBin-3HA.

[0014] An Agrobacterium that expresses the plant immune regulation gene RP in plants, comprising the above-mentioned expression vector carrying the plant immune regulation gene RP.

[0015] The above-mentioned plant immune regulatory protein RP, plant immune regulatory gene RP, expression vector carrying plant immune regulatory gene RP, and Agrobacterium expressing plant immune regulatory gene RP in plants are used to enhance plant immunity and disease resistance.

[0016] The above-mentioned plant immune regulatory protein RP, plant immune regulatory gene RP, expression vector carrying plant immune regulatory gene RP, and the application of Agrobacterium expressing plant immune regulatory gene RP in plants in the preparation of plant immune inducers.

[0017] The above-mentioned plant immune-regulating protein RP, plant immune-regulating gene RP, expression vector carrying plant immune-regulating gene RP, and Agrobacterium expressing plant immune-regulating gene RP in plants are used in the screening of disease-resistant varieties.

[0018] A method for enhancing plant immunity and disease resistance includes the following steps: (1) The nucleotide sequence of the plant immune regulation gene RP was integrated into the expression vector and transformed into Agrobacterium to obtain Agrobacterium expressing the plant immune regulation gene RP in plants; (2) Agrobacterium tumefaciens expressing the plant immune regulation gene RP in plants was applied to plants to activate the plant's immune disease resistance.

[0019] The application to plants refers to the application to plant leaves.

[0020] The plant in question is Nicotiana benthamiana.

[0021] A biological agent that enhances the immune resistance of plants includes Agrobacterium, which expresses the plant immune regulation gene RP in plants.

[0022] The Agrobacterium mentioned is Agrobacterium GV3101.

[0023] The present invention has the following advantages and effects compared with the prior art: The inventors discovered a plant immune regulatory factor gene RP in potatoes. After cloning the corresponding sequence, they used Agrobacterium competent cells to transform it into Nicotiana benthamiana, verifying that it could be successfully expressed in other plants, and subsequently after being infected by Phytophthora infestans (…). Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ) and Phytophthora indicum ( Phytophthora parasitica The pathogenicity of this gene was verified, demonstrating that its expression effectively enhances the disease resistance of Nicotiana benthamiana. The experimental results indicate that this gene can serve as a candidate gene for breeding disease-resistant varieties, providing theoretical support and genetic resources for the control of plant diseases. Attached Figure Description

[0024] Figure 1 The above image shows the results of the Western blot protein detection experiment in Example 3; the top image shows the electrophoresis bands, and the bottom image shows the results of Ponceau S staining. Figure 2 The images show the phenotypic results of leaves after inoculation with Phytophthora capsici in Example 3; the left image is a photograph taken under visible light, and the right image is a photograph taken under ultraviolet fluorescence. Figure 3 The images show the phenotypic results of leaves after inoculation with Phytophthora intolis in Example 3; the left image is a photograph under visible light, and the right image is a photograph under ultraviolet fluorescence. Figure 4 The images show the phenotypic results of leaves after inoculation with Phytophthora in Example 3; the left image is a photograph taken under visible light, and the right image is a photograph taken under ultraviolet fluorescence. Figure 5This is a statistical chart of the diameter of lesions inoculated with Phytophthora capsici in Example 3; where GFP treatment is a negative control, and the lesion diameter is in centimeters. Figure 6 This is a statistical chart of the diameter of lesions inoculated with Phytophthora intolis in Example 3; where GFP treatment is a negative control, and the lesion diameter is in centimeters. Figure 7 This is a statistical chart of the diameter of lesions inoculated with pathogenic Phytophthora in Example 3; where GFP treatment is a negative control, and the lesion diameter is in centimeters. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0026] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0027] The vector pBin-3HA is a pBin vector carrying an HA fusion tag, which has been disclosed in the literature Zheng L, Yu Y, Zheng Y, Wang Y, Wu N, Jiang C, Zhao H, Niu D. Long small RNA76113 targets CYCLICNUCLEOTIDE-GATED ION CHANNEL 5 to repress disease resistance in rice. PlantPhysiol. 2024 Feb 29;194(3):1889-1905. doi: 10.1093 / plphys / kiad599. PMID:37949839; PMCID: PMC10904327.

[0028] The tested strain of Phytophthora infectivity ( Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ) and Phytophthora indicum ( Phytophthora parasitica It is preserved by the Potato Research Laboratory of the Crop Research Institute of Guangdong Academy of Agricultural Sciences.

[0029] Example 1: Acquisition of the RP gene and construction of the expression vector 1.1 Obtaining the target gene Potato leaves were collected, flash-frozen in liquid nitrogen, and then cooled and ground in a mortar. Potato RNA was extracted and purified according to the recommended method of the Tiangen Total RNA Extraction and Purification Kit. cDNA was synthesized by reverse transcription using Oligo(dT) primers, following the method provided by the Novizan RNA Reverse Transcription Kit. Using the cDNA as a template, PCR amplification was performed using upstream and downstream primers RP-F / RP-R to obtain the amplified product. After successful sequencing verification, the RP gene fragment was obtained.

[0030] The PCR reaction system is as follows: 2.5 μL 10× PCR reaction buffer; 1.5 μL 1.5 mM MgCl2; 0.5 μL 2.5 mM dNTPs; 0.25 μL Taq DNA polymerase (5.0 U / μL); 0.5 μL primers; 0.5 μL template; sterile water to a final volume of 25 μL.

[0031] The reaction procedure is as follows: Pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 1:30 min, 35 cycles; extension at 72℃ for 10 min; storage at 4℃.

[0032] RP-F: 5'-CGATAGGGTACCCCCATGGCTCCAAAGAAGGGT-3'; RP-R: 5'-GGATCCGTCGACCCGTTCAATCTCTGGGCAGC-3'.

[0033] 1.2 Construction of pBin3-HA:RP expression vector The empty pBin3-HA vector plasmid was digested with SmaI. The cDNA and vector were ligated using a homologous recombinase from Novizuma.

[0034] The reaction system is as follows: 2 μL 10×CEⅡ reaction buffer; 2 μL RP gene fragment recovered by PCR; 2 μL pBin3-HA empty vector digested with enzymes; 1 μL homologous recombinase; sterile water to a final volume of 10 μL.

[0035] The reaction procedure was as follows: react at 37℃ for 30 min. The ligation product was transformed into *E. coli* DH5α, and transformants were screened on Kans resistant medium. Positive clones were selected, and plasmids were extracted for colony PCR identification. The identified positive clones were sequenced, and those that were correct yielded the pBin3-HA:RP expression vector.

[0036] In addition, the pBin3-HA:GFP expression vector was prepared by referring to the above method.

[0037] 1.3 Obtaining Agrobacterium competent cells Agrobacterium GV3101 strain was streaked on LB solid medium plates and incubated upside down at 28°C for 18-20 h. Single colonies were placed in LB liquid medium (containing 100 mg / L rifampin) and incubated with shaking at 28°C for 18-20 h. 100 times the volume of antibiotic-free bacterial solution was added to the bacterial culture and incubated with shaking at 28°C until the OD600 reached 0.3-0.5. After cooling on ice, the culture was centrifuged at 4000 rpm for 5 min at 4°C and the supernatant was discarded. 20 mmol / L CaCl2 solution was added to suspend the precipitate, and the culture was centrifuged at 4000 rpm for 5 min at 4°C and the supernatant was discarded. 20 mmol / L CaCl2 solution was added again to suspend the bacterial precipitate for later use.

[0038] In addition, following the above method, the vector pBin3-HA:GFP containing the GFP gene and competent cells were prepared.

[0039] Example 2: Transformation of competent cells 2.1 Experimental Materials The tested strain of Phytophthora infectivity ( Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ) and Phytophthora indicum ( Phytophthora parasitica The strain was preserved by the Potato Research Laboratory of the Crop Research Institute, Guangdong Academy of Agricultural Sciences. It was stored on 10% V8 solid medium at 10℃.

[0040] 2.2 Preparation of the tested Nicotiana benthamiana seedlings Ben's tobacco ( Nicotiana benthamiana The seeds were sown in plastic flowerpots (d=10cm) filled with vermiculite (2-4mm) and placed in a greenhouse with 14 hours of light (strong light) and 10 hours of darkness. After 7 days of growth, when two true leaves had emerged, the seeds were transplanted to a culture soil with a vermiculite-black soil-to-potassium ratio of 5:1 and grown for 30 days. Leaves from the same 3rd, 4th, and 5th leaf positions were used to express the experimental gene.

[0041] 2.3 pBin3-HA:RP expression vector transformed into Agrobacterium The pBin3-HA:RP vector was transformed into Agrobacterium cells using electroporation. The electroporation cuvettes were washed three times with 70% ethanol, and then the ethanol was completely air-dried. 100 ng of the expression vector was added to competent Agrobacterium cells and incubated on ice for 30 min. The competent Agrobacterium cells were then transferred to electroporation cuvettes and transformed using 2.5 kV. After electroporation, the competent Agrobacterium cells were added to 400 μL of LB broth and incubated at 28°C with shaking for 2 h. 20 μL of the bacterial culture was evenly spread onto a solid LB agar plate containing 50 mg kanamycin and 25 mg rifampicin, and incubated upside down at 28°C for 48 h. Single colonies were picked for colony PCR, and positive clones were obtained. Sequencing confirmed the positive clones, confirming the presence of Agrobacterium carrying the pBin3-HA:RP expression vector.

[0042] In addition, Agrobacterium carrying the pBin3-HA:GFP expression vector was prepared by referring to the above method.

[0043] 2.4 Transient expression of pBin3-HA:RP protein in Nicotiana benthamiana The above positive clones were placed in LB liquid medium and cultured at 28℃ with shaking for 30 h. The bacterial solution was collected, centrifuged at 8000 rpm for 2 min, and washed three times with 10 mM MgCl2. Finally, the bacterial solution was diluted with 10 mM MgCl2 to an OD600 value of 0.3. The bacterial solution was mixed 1:1 with the bacterial solution containing the silencing repressor p19, and injected into the left and right sides of the same Tobacco Bengal leaf with a 1 mL syringe. (The expression vector Agrobacterium containing the p19 gene silencing repressor of the Tomato dwarf virus gene can improve the expression effect of Agrobacterium after co-injection. For the transient expression method, please refer to the literature "Optimization of transient expression conditions of Agrobacterium-mediated pCB302-3 vector in Tobacco Bengal [J]. Journal of Huazhong Agricultural University, 2015, 34(3):5.DOI:10.13300 / j.cnki.hnlkxb.2015.03.018.").

[0044] In addition, following the experimental method described above, Agrobacterium suspension containing the pBin3-HA:GFP expression vector was prepared and injected into other leaves as controls using the same method, with 3 replicates per group.

[0045] Example 3: Expression and Verification of Disease Resistance 3.1 Western blot experiment To demonstrate that the transferred vector expressed the target protein and that plant immunity was induced by this protein, Western blot (WB) experiments were performed on tobacco samples after injection. In Example 2, section 2.4, 48 hours after injection, leaves were collected as samples for total protein extraction. The samples were then analyzed using standard WB methods. SDS-PAGE gels were prepared, electrophoresed, transferred to PVDF membranes, blocked with skim milk for 1.5–3 hours, hybridized with HRP-conjugated HA antibody (Wuhan Sanying, catalog number: 66006) for 1–3 hours, washed three times with TBST buffer, and then subjected to chemiluminescence detection.

[0046] The results are as follows Figure 1 As shown, the corresponding protein expression product was detected in the total protein extracted from the leaves injected with the expression vector, proving that the expression vector was successfully expressed in tobacco leaves and achieved the desired effect.

[0047] 3.2 Detection of resistance to Nicotiana benthamiana by transient expression of GFP and RP proteins Leaves expressing the confirmed protein were cut and placed in a filter paper-insulated tray. Holes were punched along the edge of the *Phytophthora capsici* or *Phytophthora nicotine* plates using a 7 mm punch. The fungal discs were then placed symmetrically on either side of the *N. benthamiana* leaves, with the mycelial surface facing down. The trays were placed in a 25°C incubator in the dark for 36 hours. The leaves were then removed and irradiated with a handheld UV fluorometer. The areas that darkened in color were the infected areas of *Phytophthora capsici* or *Phytophthora nicotine*. The diameter of the infected area was measured with a ruler. The leaf surface expressing the GFP protein served as a control. The effects of the two proteins on resistance to *N. benthamiana* were compared.

[0048] Phytophthora pathogenica was inoculated using a spore suspension method. Spores were collected from the Phytophthora pathogenica culture medium, diluted with sterile water, and the spore concentration was adjusted to 1×10⁻⁶. 6 With a concentration of approximately CFU / mL, 1 mL of suspension was dropped onto each side of the leaf of *Nicotiana benthamiana* to be tested. The leaf was then incubated in the dark at 18°C ​​for 4 days, and the test was performed using the method described above.

[0049] 3.3 Results and Discussion The results of the tobacco disease resistance test are as follows: Figures 2-7 As shown in the experimental results, the lesions on the leaves with the RP gene were smaller when infected by various Phytophthora fungi. Biostatistical analysis showed that there was a significant difference in the lesion area on the leaves treated with the RP gene, and the infected area on the leaves with the RP gene was smaller, proving that the gene can effectively improve the disease resistance of Nicotiana benthamiana.

[0050] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a plant immune regulatory protein RP in enhancing plant immunity and disease resistance, screening disease-resistant varieties, and preparing plant immune inducers, characterized in that: The amino acid sequence of the plant immune regulatory protein RP is shown in SEQ ID NO.1; The plant in question is Nicotiana benthamiana; The aforementioned disease resistance refers to resistance to pathogens caused by Phytophthora infestans; The plant immune inducer is a preparation that enhances the plant's resistance to Phytophthora infestans; The aforementioned *Phytophthora* includes pathogenic *Phytophthora* (… Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ) and Phytophthora indicum ( Phytophthora parasitica At least one of the following.

2. The application of a plant immune regulation gene RP in enhancing plant immunity and disease resistance, screening disease-resistant varieties, and preparing plant immune inducers, characterized in that: The nucleotide sequence of the plant immune regulation gene RP is shown in SEQ ID NO.2; The plant in question is Nicotiana benthamiana; The aforementioned disease resistance refers to resistance to pathogens caused by Phytophthora infestans; The plant immune inducer is a preparation that enhances the plant's resistance to Phytophthora infestans; The aforementioned *Phytophthora* includes pathogenic *Phytophthora* (… Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ) and Phytophthora indicum ( Phytophthora parasitica At least one of the following.

3. The application of an expression vector carrying the plant immune regulation gene RP in enhancing plant immunity and disease resistance, screening disease-resistant varieties, and preparing plant immune inducers, characterized in that: The expression vector carrying the plant immune regulation gene RP includes the nucleotide sequence shown in SEQ ID NO.2; The plant in question is Nicotiana benthamiana; The aforementioned disease resistance refers to resistance to pathogens caused by Phytophthora infestans; The plant immune inducer is a preparation that enhances the plant's resistance to Phytophthora infestans; The aforementioned *Phytophthora* includes pathogenic *Phytophthora* (… Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ) and Phytophthora indicum ( Phytophthora parasitica At least one of the following.

4. The application of Agrobacterium expressing the plant immune regulation gene RP in plants in enhancing plant immunity and disease resistance, screening disease-resistant varieties, and preparing plant immune inducers, characterized in that: The Agrobacterium that expresses the plant immune regulation gene RP in plants includes the expression vector carrying the plant immune regulation gene RP as described in claim 3; The plant in question is Nicotiana benthamiana; The aforementioned disease resistance refers to resistance to pathogens caused by Phytophthora infestans; The plant immune inducer is a preparation that enhances the plant's resistance to Phytophthora infestans; The aforementioned *Phytophthora* includes pathogenic *Phytophthora* (… Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ) and Phytophthora indicum ( Phytophthora parasitica At least one of the following.

5. A method for enhancing plant immunity and disease resistance, characterized in that... Includes the following steps: (1) The nucleotide sequence of the plant immune regulation gene RP was integrated into the expression vector and transformed into Agrobacterium to obtain Agrobacterium expressing the plant immune regulation gene RP in plants; (2) Agrobacterium, which expresses the plant immune regulation gene RP in plants, is applied to plants to activate the plant's immune disease resistance; The nucleotide sequence of the plant immune regulation gene RP is shown in SEQ ID NO.2; The application to plants refers to the application to plant leaves; The plant in question is Nicotiana benthamiana; the disease resistance described refers to resistance to Phytophthora blight. The aforementioned *Phytophthora* includes pathogenic *Phytophthora* (… Phytophthora infestans ), Phytophthora capsici ( Phytophthora capsici ) and Phytophthora indicum ( Phytophthora parasitica At least one of the following.

Images