A clubroot resistance gene BrRLP31 in Chinese cabbage and its application

Abstract

This invention discloses a gene for resistance to clubroot disease in Chinese cabbage. BrRLP31 Its application, the clubroot resistance gene in Chinese cabbage BrRLP31 The nucleotide sequence is shown in SEQ ID No. 1. Functional verification results show that VIGS technology can inhibit endogenous [product / effect] in Chinese cabbage. BrRLP31 After expression, the plant's resistance to clubroot disease was significantly reduced, with both the disease index and clubroot severity being significantly higher than the control material. On the other hand, the Agrobacterium-mediated transformation... BrRLP31 The transgenic Chinese cabbage plants overexpressing the gene were confirmed to have disease resistance. BrRLP31 Heterologous overexpression of [a specific ingredient] significantly improved the plant's resistance to clubroot bacteria. These results indicate that... BrRLP31 The gene plays a key positive regulatory role in the resistance of Chinese cabbage to clubroot disease. Utilizing this gene and its homologous sequences to improve the disease resistance of cruciferous crops and other horticultural plants has important application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and specifically relates to a gene for resistance to clubroot disease in Chinese cabbage. BrRLP31 And its applications. Background Technology

[0002] Cruciferous crops are an important source of vegetables, oilseeds, and feed globally, but their industrial security has long been severely challenged by soil-borne diseases. Among these, *Cladosporium brassicum* (Cladosporium tumefaciens) poses a significant threat. Plasmodiophora brassicae Clubroot, caused by *Cladosporium brassicae*, is considered a persistent problem in the production of this type of crop due to its diverse modes of transmission and difficulty in eradication. *Cladosporium brassicae* is an obligate parasitic, biotrophic pathogen. The pathogen remains dormant in the soil as dormant spores for extended periods, exhibiting strong resistance and a survival time of up to decades. This disease is characterized by its high concealment and explosive spread. Infection occurs in the underground parts of the plant and is difficult to detect with the naked eye in its early stages. During its reproduction within the host, the pathogen disrupts the metabolic balance of endogenous plant hormones (such as auxin and cytokinin), inducing abnormal cell division and excessive proliferation in the roots, leading to irreversible tumor-like deformities (clumps). This altered anatomical structure severely hinders the absorption and transport of water and nutrients by the roots, resulting in stunted growth, wilting, premature aging, and even necrosis of the above-ground parts of the plant. Furthermore, with the increasing mechanization of agriculture, the risk of cross-regional transmission of the pathogen through agricultural machinery has significantly increased, accelerating the spread of the disease.

[0003] Clubroot disease is mainly controlled through traditional methods such as adjusting soil pH, water and fertilizer management, and crop rotation. However, due to the long survival and uneven distribution of dormant spores, the difficulty in pesticide penetration, and high costs, traditional control methods are limited in effectiveness and difficult to sustain, often leading to total crop failure in infected fields. Therefore, discovering disease-resistant genes and breeding durable resistant varieties is currently the most economical, environmentally friendly, and effective way to ensure the safety of cruciferous crops.

[0004] The plant immune system mainly consists of pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). In the ETI pathway, plants recognize effectors secreted by pathogens through R proteins, thereby activating a specific defense response. Most R genes encode proteins containing nucleotide-binding sites (NBS) and leucine-rich repeat (LRR) domains.

[0005] With advancements in genome sequencing technology, researchers have located numerous clubroot resistance (CR) gene loci in the A genome of cruciferous crops. Studies show that these loci are primarily concentrated on chromosomes A03 and A08. For example, loci were found on chromosome A03. CRa , CRb , CRd , Crr3Eleven disease-resistant genes have been identified; these genes have been located on chromosome A08. CRs , Rcr3 , Crr1 (Include Crr1a and Crr1b ), Rcr9 , PbBa8.1 as well as BraPb8.3 Multiple sites, etc. Although as many as 27 disease resistance sites have been reported, only a handful of genes have been successfully cloned and their functions elucidated, with only a handful... Crr1 , CRa , CRd , CRb A few others. However, most of the cloned resistance genes mentioned above belong to the intracellular NBS-LRR family, and functional analysis of receptor-like proteins (RLPs) located on the cell surface in clubroot resistance remains very limited. Researching the application of RLP proteins in clubroot resistance has irreplaceable scientific significance and application value for overcoming the limitations of existing understanding of resistance mechanisms, enriching the clubroot resistance gene resource library, and creating new broad-spectrum and durable resistant germplasm. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a gene for resistance to clubroot disease in Chinese cabbage. BrRLP31 And its applications.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a gene for resistance to clubroot disease in Chinese cabbage. BrRLP31 This gene BrRLP31 For European turnips ( Brassica rapa subsp rapifera The gene cloned from ECD04 has the following characteristics: (1) The nucleotide sequence shown in SEQ ID No. 1; or (2) The nucleotide sequence shown in SEQ ID No. 1 has been substituted, deleted, and / or have one or more nucleotides added; or (3) A nucleotide sequence that hybridizes with the DNA sequence defined in (1) under strict conditions.

[0008] This invention provides the above-mentioned clubroot resistance gene in Chinese cabbage. BrRLP31 The method for constructing a gene silencing vector (VIGS vector) specifically includes: Design specific primers to amplify Chinese cabbage BrRLP31 The gene's specific target fragment, with the sequences of the specific primers shown in SEQ ID No. 3 and SEQ ID No. 4, was obtained. The pTRV2 vector was double-digested to obtain a linearized vector. Homologous recombination ligated the amplified fragment and the linearized vector to obtain the recombinant product. The recombinant product was transformed to obtain the clubroot resistance gene in Chinese cabbage. BrRLP31Gene silencing vector.

[0009] This invention provides the above-mentioned clubroot resistance gene in Chinese cabbage. BrRLP31 Methods for obtaining gene-silenced plants include: The above BrRLP31 The gene silencing vector was introduced into Agrobacterium to prepare a recombinant Agrobacterium bacterial suspension; this suspension was then mixed with an Agrobacterium bacterial suspension containing the pTRV1 vector in a certain proportion, and the mixture was used to infect the leaves of the plants to be tested, and the resulting strains were screened. BrRLP31 Gene-silenced plants with significantly reduced gene expression levels.

[0010] This invention provides the above-mentioned clubroot resistance gene in Chinese cabbage. BrRLP31 The methods for constructing overexpression vectors specifically include: Design specific primers to amplify the gene for resistance to clubroot disease in Chinese cabbage. BrRLP31 The nucleotide sequence of the specific primers is shown in SEQ ID No. 9 and SEQ ID No. 10. The pFGC-1008 vector was double-digested to obtain a linearized vector. Homologous recombination was used to ligate the amplified sequence and the linearized vector to obtain the recombinant product. The recombinant product was transformed to obtain the clubroot resistance gene in Chinese cabbage. BrRLP31 Overexpression vectors.

[0011] This invention provides a gene for resistance to clubroot disease in Chinese cabbage containing the above-mentioned gene. BrRLP31 The biological material is an expression vector, expression cassette, host cell, or engineered bacteria.

[0012] This invention provides the above-mentioned clubroot resistance gene in Chinese cabbage. BrRLP31 The application of its corresponding biological materials in screening plants resistant to clubroot and / or regulating the clubroot resistance function of plants.

[0013] This invention provides the above-mentioned clubroot resistance gene in Chinese cabbage. BrRLP31 Or the application of its corresponding biological materials in the preparation of transgenic plants.

[0014] Furthermore, the application specifically includes: By upregulating the expression of the cabbage gene BrRLP31 To enhance the disease resistance of Chinese cabbage, downregulate or knock out the expression of Chinese cabbage genes. BrRLP31 This weakens the disease resistance of cabbage.

[0015] This invention provides the above-mentioned clubroot resistance gene in Chinese cabbage. BrRLP31 or the application of its corresponding biological materials in plant breeding and / or plant germplasm resource improvement.

[0016] Furthermore, the clubroot disease mentioned is clubroot disease caused by Plasmullerithia brassicae.

[0017] The beneficial effects of this invention are as follows: This invention silences endogenous genes through virus-induced gene silencing (VIGS) technology. BrRLP31 After gene administration, the plants showed increased susceptibility to clubroot disease, with a significantly higher disease index than the control group, and the root swelling symptoms were exacerbated. This invention utilizes Agrobacterium-mediated gene administration... BrRLP31 The overexpression vector was transformed into 'Youqing Sijiu' Chinese cabbage plants to obtain the gene. BrRLP31 Heterologous overexpression lines. Disease resistance identification results showed that the gene... BrRLP31 Overexpression of this gene significantly enhanced the resistance of Chinese cabbage to clubroot. This indicates that the clubroot resistance gene in Chinese cabbage... BrRLP31 Closely related to clubroot disease in cruciferous vegetables, applying this gene to the breeding of Chinese cabbage or other cruciferous vegetables can improve plant germplasm resources and enhance plant disease resistance, showing promising application prospects. Attached Figure Description

[0018] Figure 1 For genes BrRLP31 A schematic diagram of the VIGS carrier; Figure 2 For genes BrRLP31 A schematic diagram of the overexpression vector; Figure 3 For 30-35 days after transplantation BrRLP31 Comparison of relative gene expression levels between gene-silenced plants and control plants; Figure 4 42 days after inoculation with a suspension of *Plasmodiophora stenophylla* spores BrRLP31 Comparison of disease index between gene-silenced plants and control plants; Figure 5 42 days after inoculation with a suspension of *Plasmodiophora stenophylla* spores BrRLP31 Comparison of root symptoms between gene-silenced plants and control plants; Figure 6 For genes BrRLP31 Electrophoresis results of PCR screening of positively expressed overexpressing plants; Figure 7 For genes BrRLP31 Comparison of the relative expression levels of the target gene in positive overexpression plants and control plants; Figure 8 OE- 42 days after inoculation with a suspension of clubroot spores BrRLP31 Comparison of growth status between overexpressing plants and control plants; Figure 9 OE- 42 days after inoculation with a suspension of clubroot spores BrRLP31 Comparison of disease index between overexpressing plants and control plants. Detailed Implementation

[0019] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of apparatuses and methods consistent with some aspects of the invention as detailed in the appended claims.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a,” “the,” and “the” used in this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0021] It should be understood that although the terms first, second, third, etc., may be used in this invention to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, first information may also be referred to as second information without departing from the scope of this invention, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to a determination."

[0022] The present invention will now be described in detail with reference to the accompanying drawings. Unless otherwise specified, the features of the following embodiments and implementations can be combined with each other.

[0023] Unless otherwise specified, all materials used in the examples are readily available from commercial companies. The Arabidopsis thaliana used was of the Col-0 type. European turnip ECD04, a type of Chinese cabbage, was propagated in the laboratory. Preparation of diseased roots: Sufficient quantities of Chinese cabbage were planted, and roots were collected after 24 hours of infection with *Clostridium brassicae* to obtain diseased roots.

[0024] This invention provides a gene for resistance to clubroot disease in Chinese cabbage. BrRLP31 The gene is a cloned gene from the disease-resistant European turnip ECD04, and its gene sequence is shown in SEQ ID No. 1.

[0025] This invention also provides the above-mentioned clubroot resistance gene for Chinese cabbage. BrRLP31 Its application in the resistance of clubroot disease in Chinese cabbage (European turnip ECD04) and Chinese cabbage heart is described in detail below.

[0026] Example 1: Clubroot resistance gene in Chinese cabbage BrRLP31 Construction of gene silencing vector (VIGS vector)

[0027] 1. Prediction of Silent Sequences: Using the SGN-VIGS website to predict silent sequences. BrRLP31 The silenced sequence of the gene was predicted online, and a silenced sequence of 300 bp in length was obtained, which is denoted as SEQ ID No.2.

[0028] 2. Enzyme digestion of pTRV2 vector: The pTRV2 vector... Bam HI and Sma Double digestion at both sites yielded the linearized pTRV2 vector. The digestion system consisted of 4 μL of buffer and approximately 2 μL of recovered product. Sal I and Kpn Add 2 μL of each enzyme, then add double-distilled water to make up to 40 μL, and incubate at 37°C for 1 h.

[0029] 3. Design specific primers: Design specific primers as shown in Table 1, and amplify the predicted 300bp silent sequence using high-fidelity KOD enzyme to obtain PCR amplification products. Subsequently, the PCR amplification products are separated by 1% agarose gel electrophoresis, and gene fragments of the correct length are selected for gel extraction and recovery to obtain the 300bp silent sequence amplified by PCR.

[0030] Table 1: Primers used for VIGS vector construction

[0031] 4. Homologous recombination ligation: The linearized pTRV2 vector and the PCR-amplified 300bp silent sequence were homologously ligated using Novizan's Single Fragment Homologous Recombination Kit C112 to obtain the recombination product.

[0032] The homologous recombination ligation system consisted of: 4 μL 5×CE II Buffer, 2 μL Exase II, 200 ng of double-digested linearized vector (i.e., linearized pTRV2 vector), and 20 ng of... BrRLP31 The 300 bp silent sequence was amplified by PCR (i.e., the 300 bp silent sequence was amplified by PCR), and ddH2O was added to make up to 20 μL; the homologous recombination product was obtained by reacting in a metal bath at 37℃ for 30 min.

[0033] 5. Transformation of Recombinant Products: The recombinant products were transformed into *E. coli* DH5α competent cells using a freeze-thaw method. Specifically, DH5α cells were thawed on ice, the homologous recombinant products were added, and the cells were incubated on ice for 30 min; a heat shock was performed in a 42°C metal bath for 90 s, followed by cooling on ice for 5 min; 1 mL of LB liquid medium was added, and the cells were incubated at 37°C for 1.5 h with a shaker; the cells were centrifuged at 5000 rpm for 1 min, the filtrate was discarded, and approximately 100 μL of the remaining bacterial culture was mixed and plated onto LB solid medium containing kanamycin; the medium was incubated upside down at 37°C overnight. The next day, single colonies obtained from the culture were picked for bacterial PCR detection and verification, and the target plasmid was extracted for sequencing verification. The plasmid that successfully aligned with the sequencing verification was transformed into *Agrobacterium* GV3101 competent cells by electroporation. The successfully transformed bacterial culture showed a band in PCR, and the culture volume was expanded; the plasmid was extracted for sequencing verification, and the successfully verified bacterial culture was preserved, with the mother liquor stored at 4°C for later use. Among these, the *Brassica napus* clubroot resistance gene was identified. BrRLP31 The spectrum of the VIGS carrier is as follows Figure 1 As shown.

[0034] Example 2: Using VIGS carrier to study Chinese cabbage BrRLP31 Gene silencing

[0035] 1. Soaking method for infecting Chinese cabbage: The above-mentioned bacterial culture, verified by sequencing, was used as the mother culture to infect European turnips (Chinese turnips) using the soaking method. B. rapa subsp. rapifera ECD04, the steps are as follows: Add 100 μL of Agrobacterium tumefaciens bacterial suspension containing the target vector to 15 mL of liquid LB containing kanamycin (0.5 μL / mL) and rifampin (1 μL / mL), and incubate at 28℃ and 200 rpm for about 12-16 hours until OD is reached. 600 The bacterial precipitate was obtained by centrifugation at 5000 rpm for 15 min. The precipitate was resuspended in a resuspension solution (RS) prepared by ddH2O, which included 10 mmol / L MES (morpholine ethanesulfonic acid), 10 mmol / L MgCl2 (magnesium chloride), and 150 μmol / L acetylsuccinone. Then, the OD... 600 Adjust the concentration to 1.0 and let it stand at room temperature for 2-3 hours before use. Then, to infect seedlings, mix the bacterial suspension containing pTRV1 with the bacterial suspension containing pTRV1 at a 1:1 volume ratio. pTRV2-BrRLP31 and pTRV2-empty The bacterial suspension (as a control group) was mixed, in which pTRV2-empty The pTRV2 empty vector was used. Chinese cabbage seeds were germinated until the roots were about 1 cm long, then the seed coat was removed and the seeds were placed in the prepared mixed bacterial solution. The solution was then vacuumed (0 Pa, 15 min), and the seeds were transferred to the substrate for normal culture.

[0036] 2. BrRLP31Quantification of gene expression: Cabbage leaves 30-35 days after transplanting were used to extract total RNA using Trizol reagent, followed by reverse transcription to obtain cDNA. The cDNA was obtained using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) provided by TaKaRa, following the manufacturer's instructions. qRT-PCR analysis was then performed. Primers used for qRT-PCR were designed using Primer Premier 6, as shown in Table 2. The reaction mixture consisted of 15 μL: 7.5 μL of SYBR Green MasterMix, 0.3 μL each of forward and reverse primers, 1 μL of template, and 5.9 μL of double-distilled water. The qRT-PCR reaction procedure was: 95℃: 30 s, 40 cycles (95℃: 5 s, 55℃: 45 s). The specificity of the reaction was determined by melting curve analysis. The internal reference gene was [missing information]. BrUBC10 The relative expression level of genes is determined by 2 -ΔΔCt Method calculation (three biological replicates were set at the time of sampling), results are as follows Figure 3 As shown.

[0037] Depend on Figure 3 The qRT-PCR analysis results shown indicate that, compared to the control group, the target gene... BrRLP31 Transcriptional levels were significantly downregulated in VIGS-silenced plants (*) p <0.05).

[0038] Table 2: Primers used for qRT-PCR analysis

[0039] Example 3: Experimental study and phenotypic observation of infection and disease development by *Plasmodiophora brassicae*.

[0040] 1. Preparation of Plasmodium spore suspension:

[0041] 1) Take out the diseased root stored in the -20°C freezer and thaw it.

[0042] 2) Disinfect by soaking in 70% ethanol solution for 1 min.

[0043] 3) Treat with 10% NaClO for 20 min, cover with tin foil to block light, and rinse 3 times with sterile water.

[0044] 4) Use a juicer to extract a homogenate from the root tubers, and filter twice through sterilized gauze. Centrifuge the filtrate at 500 rpm for 5 min, discarding the black precipitate and retaining the supernatant and gray precipitate. Replace with a clean 50 mL centrifuge tube, add sterile water to bring the volume to 45 mL, centrifuge at 4000 rpm for 15 min, and discard the supernatant. Dissolve the precipitate in sterile water, centrifuge at 4000 rpm for 10 min, and repeat once. Dissolve the precipitate in 50% sucrose, centrifuge at 3100 rpm for 10 min, and discard the supernatant. Add sterile water to bring the volume to 45 mL, centrifuge at 4000 rpm for 10 min, discard the supernatant, and repeat 2-3 times. Dissolve the precipitate in 1 mL of sterile water to obtain a high-concentration spore solution, and store at 4°C.

[0045] 2. Microscopic examination of dormant spores of *Cladophora stearothermiae*: A suspension of *Cladophora stearothermiae* spores was aspirated using a pipette and transferred to a hemocytometer (model 1 / 400 mm). 2 Add the suspension to the groove at the edge of the coverslip until the coverslip is filled with the *Plasmodiophora* spore suspension. Count the spores under a microscope, placing them in the grid corresponding to the top and left edges. The spore concentration (spores / mL) is calculated as: (number of dormant spores in the top left + bottom left + center + top right + bottom right total of 5 large grids) / 80 × 400 × 10⁻⁶. 4 × Dilution factor.

[0046] 3. Infection, disease development, and phenotypic observation: When the plants have grown to two true leaves, they are inoculated with a suspension of dormant spores of *Plasmodiophora stylosa* via root injection at a concentration of 1×10⁻⁶. 7 The inoculation rate was 1 mL / mL, with each plant receiving 1 mL. After inoculation, the plants were placed in a greenhouse under standard management, keeping the soil moist to promote disease development. After 42 days of growth under normal conditions, the plants were carefully removed, and the roots were thoroughly cleaned with water. The disease status of the roots was then statistically analyzed and photographed for record-keeping.

[0047] Root disease severity was classified into four grades, from 0 to 3. Grade 0: No root swelling, normal development; Grade 1: No taproot swelling, normal development, small lumps on lateral roots; Grade 2: Swelling of more than 1 / 3 of the total area of ​​both taproots and lateral roots; Grade 3: Significant swelling of taproots and lateral roots, even cracking and rotting. The Disease Index (DI) was calculated using the following formula: Disease index DI = (Number of plants at level 0 * 0 + Number of plants at level 1 * 30 + Number of plants at level 2 * 60 + Number of plants at level 3 * 100) / Total number of plants * 100.

[0048] If DI ≥ 10, it is judged as susceptible; if DI < 10, it is judged as resistant.

[0049] The results of the Disease Index (DI) are as follows: Figure 4 As shown, by Figure 4 It can be seen that, compared with no load pTRV2-empty Compared to the control plants, BrRLP31 The disease index of gene-silenced plants increased significantly (*) p <0.05). Root disease symptoms are as follows: Figure 5 As shown, the scale in the diagram is 5cm, combined with... Figure 5 The phenotypic observations shown indicate that the control plants maintained good growth vigor, while... BrRLP31 Gene-silenced plants exhibited significant growth inhibition and severe clubroot symptoms. These results confirm that endogenous... BrRLP31 Downregulation of this gene significantly increased the plant's susceptibility to clubroot disease. BrRLP31 The silence weakens the plant's disease resistance.

[0050] Example 4: Clubroot resistance gene in Chinese cabbage BrRLP31 Construction of overexpression vectors

[0051] 1. Double digestion of pFGC1008-EGFP vector: Following the method described in Example 1, the digestion was performed... Sal I and Kpn The pFGC1008-EGFP vector was double-digested at site I, and the large fragment product was separated by electrophoresis to recover the linear vector.

[0052] 2. Design of specific primers: Specific primers as shown in Table 3 were designed, and the clubroot resistance gene in Chinese cabbage was amplified using high-fidelity KOD enzyme, following the method described in Example 1. BrRLP31 The coding region sequence (CDS) was obtained, and the PCR amplification products were then separated by 1% agarose gel electrophoresis. The CDS with the correct length was selected for gel extraction and recovery to obtain the amplified CDS sequence.

[0053] Table 3: Primers used for the construction of heterologous overexpression vectors

[0054] 3. Homologous recombination ligation and transformation: Following the homologous recombination ligation method in Example 1, the linear vector of pFGC1008-EGFP and the amplified sequence of CDS were homologously ligated to obtain the recombinant product. The recombinant product was then transformed using the transformation method in Example 1. The final clubroot resistance gene for Chinese cabbage was obtained. BrRLP31 The overexpression vector map is as follows Figure 2 As shown.

[0055] Example 5: Flower infusion transformation of 'Youqing Sijiu' Chinese cabbage and screening of positive transformants

[0056] 1. Transformation of 'Youqing Sijiu' Chinese cabbage using the flower immersion method: Using the above-mentioned bacterial suspension verified by sequencing, along with Agrobacterium GV3101 bacterial suspension containing the pFGC1008 empty vector plasmid (control group), as the mother liquor, Arabidopsis thaliana was transformed using the flower immersion method. The steps were as follows: 500 μL of Agrobacterium suspension containing the target vector was added to 200 mL of liquid LB containing kanamycin and rifampin (50 mg / L), and the mixture was shaken at 28℃ and 200 rpm for approximately 30 h until OD was reached. 600 The concentration was 1.2; Agrobacterium tumefaciens was obtained by centrifugation at 8000 rpm for 10 min, and the bacterial solution was resuspended in 200 mL of 5% sucrose; Silwet-77 was added to a final concentration of 200 μL / L, and the mixture was shaken at 28℃ and 200 rpm for 2 min; a few flowering 'Youqing Sijiu' bok choy plants were reserved to provide pollen for pollination; the pods and open flowers of the remaining plants were removed, and the buds were peeled to expose the stigmas; the plants were placed in a vacuum desiccator so that the stigmas were immersed in the Agrobacterium tumefaciens solution, and after sealing, a vacuum environment was created using a vacuum pump and maintained for 5 min, and the air inlet valve was slowly opened to restore the pressure inside the vacuum desiccator to 0 MPa; the above operation was repeated, and the vacuum was created again for 5 min and restored; after the flowers were immersed, the residual bacterial solution on the plants was wiped off, and the plants were cultured in a moist, dark environment for 24 h; after the dark treatment, the flowers were artificially pollinated using pollen from the reserved flowering plants; after pollination, the plants were transferred to an artificial climate chamber for normal culture, awaiting seed harvest.

[0057] 2. Screening of positive transformants: Harvested T1 generation seeds were germinated and sown. When two true leaves emerged, a small amount of leaves were harvested for DNA extraction. Three pairs of specific detection primers were designed for the target fragment sequence; the primer sequence information is shown in Table 4. Using the extracted plant genomic DNA as a template, PCR amplification was performed using 2×Rapid Taq Master Mix. The reaction system and amplification procedure were performed according to the kit instructions. PCR products were detected by agarose gel electrophoresis. If all three primer pairs amplified specific bands of the expected size, and the sequencing alignment results were correct, the plant was considered a positive plant. RNA was extracted from positive plants, reverse transcribed into cDNA, and the gene expression level was detected by qRT-PCR. Plants with elevated expression levels were used for subsequent experiments.

[0058] Table 4: Primers used for screening positive transformants

[0059] Among them, genes BrRLP31 PCR screening and electrophoresis detection of overexpression-positive transformed plants were performed using three pairs of specific primers designed for the target fragment. The corresponding electrophoresis results are as follows: Figure 6 As shown in the figure, the DNA molecular weight standard (marker) is DL2000, and the samples corresponding to the lanes highlighted in red are the confirmed positive transgenic plants. Subsequently, based on... Figure 6 The PCR screening results shown were used to select confirmed positive plants for qRT-PCR transcriptional level analysis. (Gene) BrRLP31 The quantitative analysis results of the relative expression levels of the target gene in overexpression-positive plants are as follows: Figure 7 As shown, compared with the control plants, the gene... BrRLP31 The relative expression level was significantly upregulated in overexpressing transgenic plants (***) p <0.001).

[0060] Example 6: Identification of disease resistance and phenotypic observation of transgenic Chinese cabbage plants through clubroot inoculation experiment.

[0061] Selected heterologous overexpression Chinese cabbage plants (OE-) with consistent growth from Example 5 BrRLP31 Wild-type control plants (WT) and three biological replicates were set up, each containing 25 plants. When the plants had grown to two true leaves, they were inoculated with a suspension of dormant spores of *Plasmodiophora stearothermum* via root injection at a concentration of 1 × 10⁻⁶. 7 The inoculation rate was 1 mL / mL, with each plant receiving 1 mL. After inoculation, the plants were placed in a greenhouse under standard management, keeping the soil moist to promote disease development. 42 days after inoculation, the plants were carefully dug up, the roots were washed clean of soil, and the root morphology and disease status were observed and recorded. The disease index for each line was calculated based on the grading standard for root swelling.

[0062] In the disease development experiment, the results of the disease development were as follows: Figure 8 As shown, 42 days after inoculation with clubroot fungus, the wild-type control plants exhibited extremely high susceptibility, with roots becoming deformed due to severe swelling and accompanied by significant growth retardation; in contrast, the OE- BrRLP31 Overexpression lines showed no obvious lesions or only mild symptoms; the scale bar in the figure is 3 cm. The disease index for each line is shown below. Figure 9 As shown, it can be seen from this that OE- BrRLP1 The disease index of overexpressing plants was significantly lower than that of wild-type control plants (**) p <0.01). The above results strongly demonstrate that overexpression BrRLP31 The gene significantly improved the plant's resistance to clubroot disease. BrRLP31 It is closely related to the incidence of clubroot disease in Chinese cabbage and is a clubroot resistance gene in Chinese cabbage.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gene for resistance to clubroot disease in Chinese cabbage BrRLP31 The application of biological materials containing this gene in positively regulating the clubroot resistance function of European turnip ECD04 or Chinese cabbage in *Brassica oleracea var. chinensis* is characterized by the following: The gene BrRLP31 The nucleotide sequence is shown in SEQ ID No. 1; the biological material is an expression vector, expression cassette, host cell, or engineered bacteria.

2. The clubroot resistance gene for Chinese cabbage as described in claim 1 BrRLP31 Or the application of biomaterials in the preparation of transgenic European turnip ECD04 or Chinese cabbage with improved resistance to clubroot disease.

3. The clubroot resistance gene for Chinese cabbage as described in claim 1 BrRLP31 Or the application of biological materials in the improvement of germplasm resources for clubroot resistance in European turnip ECD04 or Chinese cabbage.

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