Application of metabotropic glutamate receptor PbMGR in prevention and control of rape clubroot

Abstract

The invention belongs to the field of biopesticides, and relates to application of a metabotropic glutamate receptor PbMGR in prevention and control of rape clubroot. The application of the metabotropic glutamate receptor PbMGR gene in improving the clubroot resistance of plants and / or cultivating clubroot-resistant plants is characterized in that the CDS sequence of the gene is as shown in SEQ ID NO.1. The invention also discloses an application of the metabotropic glutamate receptor PbMGR gene in improving the clubroot resistance of plants and / or cultivating clubroot-resistant plants. According to the invention, G protein coupled receptors (metabotropic glutamate receptors, PbMGR) which are expressed at a high level in each stage are screened from a plasmodiophora brassicae ZJ-1 transcriptome, an RNAi carrier of the gene is constructed, corresponding transgenic plants are obtained, and disease resistance tests of plasmodiophora brassicae show that two PbMGR-RNAi transgenic strains both show good disease resistance phenotypes, and the two PbMGR-RNAi transgenic strains show good disease resistance phenotypes. Therefore, the G protein coupled receptor PbMGR of plasmodiophora brassicae can be used as a gene resource for resisting clubroot.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological pesticides, and relates to application of a metabotropic glutamate receptor PbMGR in prevention and control of rapeseed clubroot. BACKGROUND

[0002] Clubroot is a soil-borne disease caused by Plasmodiophora brassicae, which is a serious threat to the yield and quality of Brassica crops. It has become one of the most harmful diseases to Brassica plants. There is no stable genetic system for Plasmodiophora brassicae, and the pathogenesis mechanism is lagging behind. Moreover, the resting spores of Plasmodiophora brassicae can survive in the soil for a long time, making it difficult to control clubroot disease. Therefore, it is of great significance to find gene resources and germplasm resources resistant to clubroot disease to protect the safe production of Brassica crops. In this study, we used host-induced gene silencing (HIGS) technology to screen for genes resistant to clubroot disease. We designed a vector with a hairpin structure, which carried a specific gene fragment of the pathogen and was transformed into the host plant. The transgenic plants produced corresponding dsRNA and siRNA, which entered the pathogen during the interaction between the host and the pathogen, degraded the pathogen mRNA, silenced the target gene, and protected the host plant from the pathogen (Nunes and Dean 2012, Qi et al 2019, Koch et al 2021). We used HIGS technology to introduce dsRNA targeting thiamine transporters in cotton and successfully generated two stable transgenic lines. Compared with wild-type plants, the colonization and spread of Lecanicillium laxum in the roots of RNAi cotton were significantly reduced, and the disease index was significantly decreased. In field conditions, RNAi transgenic cotton also showed significantly enhanced disease resistance and yield (Wang et al 2024). G protein-coupled receptors (GPCRs) are the largest family of transmembrane protein receptors in organisms, widely found in plants, animals, fungi, and humans. Unlike plants, fungi do not have receptor kinases or receptor-like kinases, and GPCRs play a crucial role in sensing signals and inter-species interactions essential for their survival, thereby activating downstream cAMP and mitogen-activated protein kinase pathways through heterotrimeric G proteins (Jiang et al 2024). G protein-coupled receptors (GPCRs) are a large class of membrane protein receptors. Based on sequence analysis of the human genome, they can be divided into six types: Class A (rhodopsin-like receptors), Class B (secretin receptor family), Class C (metabotropic glutamate receptors), Class D (fungal mating pheromone receptors), Class E (cyclic AMP receptors), and Class F (Frizzled / Smoothened family). GPCRs have diverse biological functions and are involved in regulating various physiological processes.As Jiang Cong et al. explored the systematic characterization of 105 GPCR genes in F. graminearum, the results showed that only 5 (GIV1-GIV5) were up-regulated during plant infection, and played a key role in the virulence of the pathogen (Jiang C, Zhang X, Liu H, Xu J. Mitogen-activated protein kinase signaling in plant pathogenic fungi. PLoS Pathog, 2018, 14: e1006875). There is no report on the relationship between the metabotropic glutamate receptor of the plasmodiophoromycetes and its pathogenicity. SUMMARY

[0003] The purpose of the present application is to overcome the shortcomings of the prior art and provide the application of metabotropic glutamate receptor PbMGR in preventing and controlling the clubroot of oilseed rape.

[0004] The purpose of the present application can be achieved by the following technical solutions:

[0005] The application of metabotropic glutamate receptor PbMGR gene in improving the resistance of plants to clubroot and / or cultivating plants resistant to clubroot, characterized in that the CDS sequence of the gene is shown in SEQ ID NO. 1.

[0006] Preferably, the expression of the metabotropic glutamate receptor PbMGR gene is negatively regulated to improve the resistance of plants to clubroot.

[0007] Preferably, the method for negatively regulating the expression of the metabotropic glutamate receptor PbMGR gene is selected from host-induced gene silencing technology and gene editing technology.

[0008] The application of the substance for negatively regulating the metabotropic glutamate receptor PbMGR gene in improving the resistance of plants to clubroot and / or cultivating plants resistant to clubroot.

[0009] A host-induced gene silencing RNAi vector for the metabotropic glutamate receptor PbMGR gene, wherein the host-induced gene silencing RNAi vector takes the CDS sequence fragment shown in SEQ ID NO. 3 as the interference sequence.

[0010] An engineered bacterium containing the host-induced gene silencing RNAi vector.

[0011] The application of the host-induced gene silencing RNAi vector and the engineered bacterium is as follows:

[0012] 1) increasing the disease resistance of plants to clubroot;

[0013] 2) preparing a product for improving the disease resistance of plants to clubroot;

[0014] 3) breeding plants with improved resistance to plasmodiophora resistance;

[0015] 4) products for breeding plants with improved resistance to plasmodiophora resistance;

[0016] 5) improving a plant with high resistance to plasmodiophora or products for breeding a plant with high resistance to plasmodiophora;

[0017] 7) inhibiting the growth and development of plasmodiophora;

[0018] 8) products for inhibiting the growth and development of plasmodiophora.

[0019] Preferably, the plant is selected from Brassica napus.

[0020] A method for breeding a plant resistant to plasmodiophora, comprising introducing into a plant of interest a host-induced gene silencing RNAi vector targeting the metabotropic glutamate receptor PbMGR gene, to obtain a plant resistant to plasmodiophora, the plant resistant to plasmodiophora being more resistant to plasmodiophora than the plant of interest.

[0021] Preferably, the plant is Brassica napus.

[0022] Beneficial effects:

[0023] The present application screens a G protein-coupled receptor (metabotropic glutamate receptor, PbMGR) expressed at a high level at each stage from the transcriptome of Plasmodiophora brassicae ZJ-1, constructs an RNAi vector of the gene, and obtains a corresponding transgenic plant. Through a plasmodiophora resistance test, it is shown that two PbMGR-RNAi transgenic lines both show a good disease resistance phenotype, indicating that the G protein-coupled receptor PbMGR of plasmodiophora can be used as a gene resource for resistance to plasmodiophora. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 PbGAB2-RNAi fragment amplification, expression amount at different stages, and vector identification

[0025] PbMGR-RNAi sense strand PCR amplification, M: DL 2000 Marker (A). PbMGR-RNAi antisense strand PCR amplification, M: DL 2000 Marker (B). Expression amount of PbMGR-RNAi in the life cycle of plasmodiophora, IN represents the cortical infection period, PZ represents the zoospore period, and RS represents the resting spore period (C). PbMGR-RNAi vector identification: 1: sense strand PCR detection; 2: antisense strand PCR detection; 3: KpnI and SpeI double enzyme digestion plasmid detection; M: DL 2000 Marker (D).

[0026] Figure 2 HIGS screening of transgenic materials

[0027] DNA was extracted from 20 PbMGR-RNAi (A) T0 generation HIGS transgenic materials and identified using specific PCR primers. (B) Phenotype of positive HIGS transgenic materials after 4 weeks of growth.

[0028] Figure 3 Phenotypic and disease index of PbMGR transgenic plants inoculated with *Plasmodiophora*, and expression levels of the target gene and content of *Plasmodiophora* detected by qPCR.

[0029] Three weeks after ZS11 and two different PbMGR transgenic lines were inoculated with *Plasmodium* (each line was inoculated with 1 × 10⁻⁶ dormant spores). 7 Plant phenotype (A) and disease index (B, C) were analyzed using n=20-40 biological replicates. Total RNA (D) and DNA (E) were extracted from the roots of PbMGR-RNAi transgenic lines and wild-type Arabidopsis thaliana 3 weeks after inoculation with *P. t-test*. The content of *P. t-test* in diseased rapeseed roots was quantified by qPCR. The ordinate represents the fold increase in *P. t-test* content relative to ZS11 for different lines. The relative biomass of *P. t-test* was used to measure the expression levels of target genes in diseased rapeseed roots. *P. t-test* was used to analyze differences. Detailed Implementation

[0030] Example 1

[0031] 115 GPCR signaling pathway related genes were identified from the genome of Plasmodiophora brassicae ZJ-1, including 30 encoded GPCRs, 38 encoded G proteins and 47 encoded RGS (G protein signal regulator). Through HMTMM (http: / / www.cbs.dtu.dk / services / TMHMM / ) transmembrane query screening, only 78 of them met the criteria of having 7 transmembrane structures and the N-terminal being extracellular and the C-terminal being intracellular, which is consistent with the typical GPCRs family. Based on the genome, 78 possible GPCR genes have been predicted, then 12 possible GPCR genes were selected for experiment according to the expression amount and comparison in NCBI. PbMGR belongs to one of the GPCRs, and PbMGR is highly expressed in the cortex stage, indicating that this gene may be particularly important for the infection of Plasmodiophora brassicae. It can be studied whether the expression of this gene can inhibit the infection of Plasmodiophora brassicae by inhibiting the expression of this gene. Figure 1 A}.

[0032] Example 2

[0033] 1. Materials and methods

[0034] 1.1 Plant material, strains and plasmids

[0035] Rape: Zhongshuang 11 (ZS11), commercially available seeds. The plant culture conditions are temperature 22℃, humidity 75%. The formula of plant nutrient soil: Finland Kegira peat soil: Jiangsu Peilei substrate: vermiculite = 8:4:1. The pathogenic fungus Plasmodiophora brassicae is from the root system of the root knot disease in Hubei Zhijiang rape test field (Williams No. 1 physiological race) ZJ-1.

[0036] 1.2 Extraction of total DNA of Plasmodiophora brassicae (CTAB method)

[0037] Put the extracted root-knot fungus (collected from the diseased field of Zhejiang Zhujin, identified as the pathogenic root system (Williams 1 physiological race) ZJ-1) into a pre-cooled mortar, and grind it with liquid nitrogen. Transfer the powder into a 2 mL centrifuge tube, add 700 μL of 2% CTAB extraction buffer (preheated at 65°C, 0.2 g powder added to 1 mL buffer), mix well, and incubate at 65°C for 15-30 min. Add 350 μL of chloroform and tris-saturated phenol solution to the mixture, shake well, and centrifuge at 12000 r / min for 15 min. Transfer 500 μL of supernatant to another 2 mL centrifuge tube, add an equal volume of chloroform, and extract again at 12000 r / min for 15 min. Take 450 μL of supernatant, add an equal volume of isopropanol, mix well, and precipitate at -20°C for 10-15 min. Centrifuge at 12000 r / min for 15 min, discard the supernatant, and wash the precipitate twice with 1 mL of 75% ethanol. Dry in a 37°C oven, dissolve the DNA in 30 μL of deionized water (containing 25 μg / mL of RNase A), detect the DNA concentration, and store at -20°C.

[0038] 1.3 Extraction of total RNA from root-knot fungus

[0039] Put the washed and dried root-knot disease root material into a pre-cooled mortar, and grind the tissue into powder under liquid nitrogen. Quickly transfer the frozen tissue powder (50-100 mg) into a 1.5 mL centrifuge tube containing 1 mL of RNA extraction solution Trizol, shake well, and incubate on ice for 5-10 min. Centrifuge at 4°C, 12000 r / min for 10 min, transfer the supernatant to another 1.5 mL centrifuge tube, add 200 μL of chloroform, shake well immediately, and incubate on ice for 5-10 min. After the mixture is layered, centrifuge at 4°C, 12000 r / min for 15 min. Transfer the supernatant to another centrifuge tube, then add an equal volume of isopropanol, shake well, and incubate on ice for 5-10 min. Centrifuge at 4°C, 12000 r / min for 10 min, discard the supernatant, add 75% ethanol, mix well, and resuspend the precipitate. Centrifuge at 4°C, 12000 r / min for 5 min, repeat this step 2 times. Place the centrifuge tube in the centrifuge for 1 min, then carefully aspirate the residual liquid with a pipette, and dry the RNA precipitate at room temperature for 5-15 min. Add 30 μL of DEPC water, dissolve the precipitate, and store at -80°C for later use.

[0040] 1.4 cDNA Synthesis and Target Gene Cloning

[0041] Using Takara Biotech's Prime Scropt 1 st The Strand cDNA synthesis kit was used to synthesize cDNA. The 20 µL mixture was prepared according to the instructions as follows: 2 µg total RNA, 1 µL Oligo(dT)18 Primer (0.5 µg / µL), 10 µL 2X Reaction Mix, 1 µL EasyScript RT / RI Enzyme Mix, 1 µL g DNA Remover, and RNase-free Water, bringing the total volume to 20 µL. The reaction conditions were: incubation at 42°C for 30 min, followed by incubation at 85°C for 5 sec. The synthesized cDNA was stored at -20°C for short-term use in subsequent experiments.

[0042] Primer design: For each gene, a 300-500 bp CDS sequence was selected as an interference sequence. Fragments were compared on NCBI (https: / / www.ncbi.nlm.nih.gov / ) to ensure interference specificity. Furthermore, TAIR (https: / / www.arabidopsis.org / ) was used to confirm the absence of corresponding interference sites in Arabidopsis thaliana, avoiding off-target effects (sequences are in the appendix). Homologous arm primers for restriction enzyme sites were automatically generated using the Novizan introductory cloning website (https: / / bio.vazyme.com / gongju.html). The ds1301 vector sequence and the selected approximately 300 bp gene sequence were input into the website. KpnI and BamHI restriction sites were selected to generate sense primers, and SacI and SpeI restriction sites were selected to generate antisense primers.

[0043] PbMGR positive-strand primers:

[0044] p-PbMGR -F:5'- cccgtgcagctgcggggtaccACAACATCAACTGGAACATTGGC -3'

[0045] p-PbMGR-R: 5'-cgcgtacgtaaggttggatccCCAGCTGCTGCCACATGC-3'

[0046] PbMGR antisense primers:

[0047] o-PbMGR-F: 5'-caattcaattcagtggagctcCCAGCTGCTGCCACATGC-3'

[0048] o-PbMGR-R: 5'- caggactctagacccactagtACAACATCAACTGGAACATTGGC -3'

[0049] DS1301 vector primers (for later identification of colonies grown on plates after homologous recombination)

[0050] V-1-F: 5' -cgttgagtggccctgtttctc-3'

[0051] V-2-F: 5' -gcttcaaattctaatccccaa-3'

[0052] Gene cloning: using cDNA / DNA as template, gene-specific primers with the required homologous arm were used to PCR amplify the target gene. Reaction system: Pfu DNA Polymerase 1 μL, Pfu Buffer 10 μL, High Pured NTPs 2.5 μL, cDNA 2 μL, primers F / R 1 μL each, add ddH2O to 50 μL. Reaction conditions: pre-denaturation 95℃ 2 min, denaturation 94℃ 20 s, annealing 57℃ 20 s, extension 72℃ 15 s / kbp, final extension 72℃ 5 min, 4℃ 2 min. PCR products were electrophoresed using 1.2% agarose gel, the size of the amplified target gene was detected and the bands with the same size were recovered (step details see TaKaRa DNA agarose gel electrophoresis recovery kit).

[0053] 1.5 RNAi expression vector construction of HIGS

[0054] Enzymatic digestion and homologous recombination of sense strand: the empty ds1301 plasmid (disclosed in Host-induced gene silencing of fungal-specific genes of Ustilaginoidea virens confers effective resistance to rice false smut, doi:10.1111 / pbi.13756) was digested with restriction enzymes BamHI and KpnI, and the reaction system was configured as follows: 10 μL of ds1301 vector, 5 μL of 10 x rCutSmart Buffer, 1 μL of BamHI enzyme, 1 μL of KpnI enzyme, and 33 μL of ddH2O. After centrifugation, the system was placed in a 37°C constant temperature water bath for 4 h. Then the target gene was connected to the vector by homologous recombination, and the reaction system was configured as follows: 2 μL of double-digested ds1301 fragment, 1 μL of target gene fragment, 2 μL of Exnase II homologous recombination enzyme, 4 μL of CE II Buffer, and 1 μL of ddH2O. After centrifugation, the system was placed in a 37°C constant temperature water bath for 30 min.

[0055] Intermediate vector and antisense strand recombination: the intermediate vector was double digested with restriction enzymes Sac I and Spel, and the following reaction system was configured: 10 μΐ of ds1301 vector, 5 μΐ of 10 x rCutSmart Buffer, 1 μΐ of Sac I enzyme, 1 μΐ of Spel enzyme, 33 μΐ of ddH2O. After centrifugation, the system was placed in a 37°C constant temperature water bath for 4 h. Then the antisense strand was connected to the intermediate vector by homologous recombination, and the following reaction system was configured: 2 μΐ of double-digested intermediate vector, 1 μΐ of antisense strand fragment, 2 μΐ of Exnase II homologous recombination enzyme, 4 μΐ of CE II Buffer, 1 μΐ of dd H2O. After centrifugation, the reaction system was placed in a PCR for recombination reaction, and after 37°C reaction for 30 min, it was transformed or stored in a -20°C refrigerator for temporary preservation. Heat shock transformation of E. coli DH5a: 10 μΐ of ligation product was added to 50 μΐ of E. coli competent cells, ice bathed for 30 min, 42°C heat shock for 45 s, ice bathed for 2 min, 400 μΐ of SOC medium was added, mixed, and then incubated at 37°C at 200 r / min for 1 h. It was spread on LB plates containing corresponding kanamycin, and inverted in a 37°C incubator for 12 h until single colonies grew. Colony PCR test: a white single colony was picked up with a gun head in 10 μΐ of sterile water, mixed by blowing, and 1 μΐ was taken into 20 μΐ of Super PCR Mix for mixing and PCR. Cycle conditions: (pre-denaturation: 95°C, 2 min; denaturation: 95°C, 15 s; annealing: 56°C, 15 s; extension: 72°C, 15 s / kbp; final extension: 72°C, 5 min; storage: 16°C, 4 min). After the single colony was verified by PCR, it was sent to GenScript for sequencing, and the correct sequencing was used to extract plasmid and transform Agrobacterium.

[0056] 1.6 Agrobacterium-mediated electroporation transformation

[0057] The Agrobacterium competent cells were taken out from -80°C and placed on ice for about 5-10 min, 1 μΐ of plasmid was added to 100 μΐ of competent cells and mixed by sucking, and then added to a dry and clean electroporation cup, and placed for 1 min. The electroporation instrument was opened and adjusted to "Agr". The water on the surface of the electroporation cup was wiped dry, and then electroporation was performed by pressing "Pulse". The preheated SOC was quickly added, and the culture was incubated at 28°C in a 220 r / min shaker for 30 min. Then it was taken out and spread on LB plates containing antibiotics and incubated in a 28°C incubator overnight. After colonies grew, they were identified.

[0058] 1.7 Agrobacterium-mediated chemical transformation

[0059] Take 5 μL RNAi final vector plasmid (about 1-2 μg) into 100 μL Agrobacterium competent cells and mix well; ice bath for 10 min, liquid nitrogen freezing for 5 min, 37℃ water bath for 5 min, ice bath for 2 min; add 800 μL liquid LB medium, 28℃, 200 rpm shaker for 4-5 h; take 200 μL bacterial liquid and spread on solid LB culture dish containing 25 mg / L rifampicin and 50 mg / L kanamycin, 28℃ constant temperature incubator for 48 h; after the culture has bacterial plaque, pick single colony, and the positive detection confirms the successful transformation, then the bacterial liquid can be used for subsequent genetic transformation.

[0060] 1.8 Screening of transgenic plants and inoculation experiment

[0061] Screening of transgenic plants: take sample, add 600 uL fast extraction buffer after grinding, 95℃ metal bath for 15 min, 10000 r / min, 10 min; after cooling to room temperature, take 10 uL supernatant, dilute with 60 uL H2O, and then use for PCR amplification.

[0062] Fast extraction DNA method / CTAB method extracts transgenic plant DNA and uses specific primers for amplification, and the plants with DNA that can be amplified to specific bands are collected for T0 generation seeds. The seeds are planted in culture medium containing hygromycin for screening, and the positive plants can grow normally and are used for detection of disease resistance experiment.

[0063] Inoculation of plasmodiophora: Arabidopsis or rape is cultured for about 2 weeks (14 d), 1 mL of plasmodiophora resting spores with a concentration of 1×10 6 or 1×10 7 / mL is taken with a pipette and irrigated on the roots for inoculation, and the disease index is counted 21 d after inoculation.

[0064] Disease index statistics and grading standards: the disease index of potted rape is counted 4 weeks after inoculation with plasmodiophora: the rape is taken out of the soil with tweezers to minimize damage to the roots. After washing the soil off the roots, the disease index of the plasmodiophora is counted. Disease index (DI) = (1n1+3n2+5n3+7n4) x 100 / 7N t , wherein n1, n3 and ns are the number of Arabidopsis plants with disease severity of 0, 1, 3, 5 and 7, respectively, and N tTotal plant number of Arabidopsis thaliana, control effect (%) = (1- transgenic plant disease index / Col-0 disease index) x 100. According to the five-grade classification standard, 0 grade, no disease; 1 grade, there are very few tumors on lateral roots, and the damage to the main root can be ignored; 3 grade, there are medium to large spherical tumors on the main root; 5 grade, there are serious tumors on the lateral roots and the main root, the main root is completely swollen, and the lateral root is completely damaged; 7 grade, the root is completely swollen and rotten.

[0065] 1.9 Real-time fluorescent quantitative PCR (qPCR)

[0066] Real-time fluorescent quantitative PCR adopts SYBR Green method. According to the method provided by Bio-Rad Company product iTaq™ Universal SYBR® Green Supermix. The quantitative primers are designed by using Beacon designer 8.0 Real-time PCR primer design software. RNA is extracted, and 2 μg of RNA is reversely transcribed into cDNA. The reaction system of real-time fluorescent quantitative PCR (15 μL) is as follows: component iTaq™ Universal SYBR® Green supermix (2X) 7.5 μL, forward primers (10 μM) 0.5 μL, reverse primers (10 μM) 0.5 μL, cDNA 0.5 μL, ddH2O 6 μL. Reaction conditions: 95℃, 10 min; 95℃, 15 sec, 57℃, 15 sec, repeat 45 cycles; 65℃~95℃, each cycle rises by 0.5℃, lasts for 5 sec, 16℃ 1 min. The reaction is carried out on Bio-Rad CFX 96 instrument. After amplification, 2 -∆∆Ct The relative expression amount of the gene is calculated by the method.

[0067] The primers of Plasmodium basionica PbMGR gene are as follows:

[0068] qPCR-PbMGR-F: 5'-ACACGGCCTTCTTCAGCC-3'

[0069] qPCR-PbMGR-R: 5'-TCGGCCACGAACGCGTAA-3'

[0070] The primers of Plasmodium basionica ACTIN gene are as follows:

[0071] Pbactin_qF: 5'-CACCGACTACCTGATGAA-3'

[0072] Pbactin_qR: 5'-CAGCTTCTCCTTGATGTC-3'

[0073] The primers of qBnACTIN7 gene of Brassica napus are as follows:

[0074] qBnACTIN7_qF: 5'-CCCTGGAATTGCTGACCGTA-3',

[0075] qBnACTIN7_qR: 5'-TGGAAAGTGCTGAGGGATGC-3'

[0076] 2. Research results and analysis

[0077] 2.1 Construction of RNAi vector of PbMGR

[0078] According to the materials and methods 1.2 and 1.3, the RNA of Plasmodiophora brassicae was extracted and reversely transcribed into cDNA, and the partial fragment of G protein-coupled receptor PbMGR was amplified by using the cDNA as a template. According to the materials and methods 1.4 and 1.5, the target gene fragment with a size of 300-500 bp was inserted into the vector containing the hairpin structure to construct the RNAi vector. In order to detect whether the RNAi vector is successfully constructed, the specific primers of the sense strand and the antisense strand are used for PCR positive identification, and the recombinant plasmid is detected by double enzyme digestion of Kpnl and Spel (Fig. 1B). Figure 1 One HIGS RNAi vector was successfully constructed.

[0079] 2.2 Creation of PbMGR transgenic Brassica napus

[0080] The constructed RNAi vector was sent to the company for transformation, and the T0 generation transgenic Brassica napus seedlings were obtained. The normally growing plants were transferred to flowerpots, and then transplanted to the transgenic field. The leaf blades of the plants with a size of about 4 weeks were taken, and the DNA was extracted. The specific primers were used for PCR identification of positive plants (Fig. 2A). Figure 2 The results showed that 18 PbMGR-RNAi transgenic plants were obtained. The seeds of the identified positive transgenic plants were collected, which could be used in the subsequent transgenic Brassica napus disease resistance experiments, such as shown in Fig. 2B. The growth phenotype of these transgenic plants was normal, which did not affect the growth of Brassica napus. Figure 2

[0081] 2.3 PbMGR transgenic plants can improve the resistance of Brassica napus to clubroot

[0082] After obtaining the transgenic plants, we wanted to verify the disease resistance of the transgenic materials to Plasmodiophora brassicae. The plants were treated with Plasmodiophora brassicae after growing for 2 weeks, and the root phenotype was observed after growing for 5 weeks. As shown in Fig. 3A, the transgenic plants showed a normal growth phenotype, and the roots were not swollen, while the roots of the wild type plants were swollen. Figure 3 ​As shown in Figure A, wild-type rapeseed variety ZS11 (Double 11) exhibits significant root disease, with swollen and even brown taproots, and absent or minimal lateral roots. The disease index is 64.76. Figure 3 A), and the symptoms of root swelling in all HIGS transgenic rapeseed were significantly reduced, with numerous lateral roots. The disease index of PbMGR-RNAi-5 and PbMGR-RNAi-11 transgenic lines was 45.24 and 44.29, respectively, with control effects of 30.15% and 31.62%. The results indicate that the disease index of HIGS transgenic materials was lower than that of wild type, which can improve plant resistance to clubroot. To verify that the improved resistance of transgenic plants to clubroot was due to siRNA silencing of the target gene in clubroot bacteria, we extracted RNA from rapeseed roots 3 weeks after inoculation. qPCR results showed that the expression level of the target gene in transgenic plants was significantly lower than that in ZS11 ( Figure 3 D), while the root follicle bacteria content of transgenic plants was significantly lower than that of ZS11 ( Figure 3 E), indicating that the transgenic plants have increased resistance to clubroot disease, which is due to the siRNA expressed in the plant inhibiting the expression of the target gene in clubroot bacteria, thereby inhibiting the damage of clubroot bacteria to the plant.

[0083] 3. Summary and Discussion

[0084] Clubroot disease poses a serious threat to the yield and quality of Brassica spp., a type of soil-borne disease. Control efforts are intensive, but chemical control is prone to environmental pollution and can easily lead to resistance in clubroot fungi. While biological control is effective, it is costly and difficult to implement in production. Therefore, creating and breeding resistant varieties can provide an effective measure for clubroot disease control. G protein-coupled receptors (GPCRs) are the largest family of transmembrane protein receptors in organisms, widely found in plants, animals, fungi, and humans. Currently, researchers such as Bi Kai have found that GPCR inhibitor treatment can alleviate the severity of clubroot disease in clubroot fungi, confirming the important influence of the GPCR signaling pathway on clubroot fungi. However, the specific gene functions involved in this pathway have not yet been studied. HIGS technology, based on RNAi, has advantages such as good targeting, long-lasting effect, and short cycle, providing a new approach for creating new resistant germplasm. This study constructed the G protein-coupled receptor PbMGR-RNAi vector and obtained two transgenic plants. The disease resistance test of clubroot showed that they had a good disease resistance phenotype, indicating that the G protein-coupled receptor PbMGR can be used as a gene resource for the green control of clubroot disease in rapeseed.

Claims

1. Metabotropic glutamate receptors PbMGR The use of a gene for increasing the resistance of a plant against clubroot and / or for breeding a plant resistant against clubroot, characterized in that, The CDS sequence of the gene is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, Negative regulation of metabotropic glutamate receptors PbMGR Increased expression of the gene increases plant clubroot.

3. Use according to claim 2, characterized in that, Negative regulation of metabotropic glutamate receptors PbMGR The method of expression of the gene is selected from the group consisting of host-induced gene silencing techniques, gene editing techniques.

4. A metabotropic glutamate receptor according to claim 1 negatively modulated PbMGR Use of a substance of a gene in increasing the resistance of a plant to clubroot and / or breeding a plant resistant to clubroot.

5. A host-induced gene silencing RNAi vector against a metabotropic glutamate receptor PbMGR gene, characterized in that, The host-induced gene silencing RNAi vector uses the CDS sequence fragment shown as SEQ ID NO. 3 as the interference sequence.

6. An engineering bacterium containing the host-induced gene silencing RNAi vector of claim 5.

7. The host-induced gene silencing RNAi vector of claim 5 or the engineering bacterium of claim 6 for use in any of the following: 1) increasing the resistance of a plant to clubroot disease; 2) preparing a product for increasing the resistance of a plant to clubroot disease; 3) cultivating a plant with increased resistance to clubroot disease; 4) preparing a product for cultivating a plant with increased resistance to clubroot disease; 5) improving a plant with high resistance to clubroot disease or preparing a product for a plant with high resistance to clubroot disease; 7) inhibiting the growth and development of Plasmodiophora brassicae; 8) preparing a product for inhibiting the growth and development of Plasmodiophora brassicae.

8. Use according to claim 7, characterized in that, The plant is selected from Brassica napus.

9. A method of breeding a plant resistant to Plasmodium species, characterized in that, comprising introducing into a plant of interest a host-induced gene silencing RNAi vector of claim 5, obtaining a plant resistant to P. palitvcola, said plant resistant to P. palitvcola being more resistant to P. palitvcola than said plant of interest, said RNAi vector targeting a metabotropic glutamate receptor of claim 1 PbMGR gene.

10. The method of claim 9, wherein, The plant is Brassica napus.

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