Application of chitin deacetylase gene PbCDA in prevention and control of rape clubroot

Abstract

The invention discloses an application of a chitin deacetylase gene PbCDA in prevention and control of rape clubroot. According to the invention, a chitin deacetylase gene CDA (Chitin Deacetylase, CDA) which possibly has important influence on the pathogenic process of plasmodiophora brassicae is screened out from a gene bank of plasmodiophora brassicae ZJ-1, and an RNAi carrier of the chitin deacetylase gene CDA is constructed by using an HIGS technology and is transformed into double 11 in rape. The PbCDA-RNAi transgenic rape is subjected to a plasmodiophora brassicae disease resistance experiment, and the result shows that the PbCDA-RNAi transgenic rape has remarkable resistance to plasmodiophora brassicae, so that the gene can provide a new gene resource for green prevention and resistance of rape clubroot.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of bio-agriculture, and relates to application of a chitin deacetylase gene PbCDA in prevention and control of clubroot of Brassica napus. BACKGROUND

[0002] Clubroot caused by Plasmodiophora brassicae is one of the most devastating diseases of cruciferous crops in the world. Plasmodiophora brassicae is a specific parasite of cruciferous plants, causing host root swelling and forming tumors of different sizes, affecting nutrient and water absorption, and causing yield reduction, which seriously threatens the production safety of cruciferous crops such as Brassica napus. Host-induced gene silencing (HIGS) technology is a new technology that can study the interaction between host plants and fungi based on VIGS and RNAi technology. When the pathogen invades the host plant, the siRNA released by the host plant silences the target gene of the pathogen, reduces the pathogen infection, and enhances the plant resistance (Nowara et al 2010). Currently, HIGS technology has been applied in Fusarium graminearum, Puccinia striiformis, Botrytis cinerea, Verticillium dahliae, and Magnaporthe oryzae (Qiao et al 2021, Chen et al 2022). Wang et al. degraded six genes (CRZ1, PMC1, MAGB, LHS1, CYP51A, and CYP51B) that play an important role in the pathogenicity of Magnaporthe oryzae in rice through HIGS, and the pathogenicity of transgenic rice plants was significantly reduced except for PMC1 and LHS1 (Wang and Dean 2022). Yi et al. used HIGS to silence MSTRG.4380.1, reducing the virulence of Magnaporthe oryzae to wheat (Yi et al 2023).

[0003] Chitin deacetylase is an enzyme that can convert chitin into chitosan, chitin is one of the main components of cell wall, when pathogenic bacteria infect plants, chitin deacetylase can modify chitin in cell wall, change its structure and properties, so as to help pathogenic bacteria escape the immune recognition of plants. Filamentous fungi modify chitin on cell wall by secreting chitin deacetylase to produce deacetylated chitin, which can reduce the activity of plant chitinase (Blair et al 2006). Cladosporium fulvum is a fungal pathogen of tomato, it can secrete two proteins ECP6 and AVR4, which can change the recognition of host plants to chitin (van den Burg et al 2006, Sanchez-Vallet et al 2013). The resting spore wall of Plasmodiophora contains chitin (Moxham and Buczacki 1983), while higher animals and plants do not contain chitin, and chitin metabolism related enzymes can be used as potential targets of green pesticides. SUMMARY

[0004] The purpose of the present application is to provide the application of chitin deacetylase gene CDA in preventing and controlling the clubroot of Brassica napus, aiming at the above-mentioned deficiencies of the prior art.

[0005] The purpose of the present application can be realized by the following technical solutions.

[0006] The application of inhibiting or silencing the chitin deacetylase gene PbCDA of Plasmodiophora in preventing and controlling the clubroot of Brassica napus, wherein the CDS sequence of the chitin deacetylase gene PbCDA of Plasmodiophora is shown in SEQ ID NO. 1.

[0007] Preferably, the inhibition or silencing of the chitin deacetylase gene PbCDA of Plasmodiophora is realized by host-induced gene silencing technology.

[0008] The application of the substance for inhibiting or silencing the chitin deacetylase gene PbCDA of Plasmodiophora is selected from any one of the following:

[0009] (1) preventing and controlling the clubroot of Brassica napus;

[0010] (2) improving the resistance of Brassica napus to the clubroot;

[0011] (3) cultivating new varieties of Brassica napus resistant to the clubroot of Brassica napus.

[0012] Preferably, the substance for inhibiting or silencing the chitin deacetylase gene PbCDA of Plasmodiophora in claim 1 is selected from host-induced gene silencing RNAi vector, gene editing system, siRNA, dsRNA, and common RNAi vector targeting the gene.

[0013] Further preferably, the substance inhibiting or silencing the Plasmodiophora brassicae chitin deacetylase gene PbCDA in claim 1 is selected from a host-induced gene silencing RNAi vector against the gene, the host-induced gene silencing RNAi vector taking the CDS sequence fragment shown in SEQ ID NO. 2 as an interference sequence.

[0014] A host-induced gene silencing RNAi vector silencing the Plasmodiophora brassicae chitin deacetylase gene PbCDA, the host-induced gene silencing RNAi vector taking the CDS sequence fragment shown in SEQ ID NO. 2 as an interference sequence.

[0015] An engineered bacterium containing the RNAi vector.

[0016] The RNAi vector, the engineered bacterium in claim 7, is used in any of the following applications:

[0017] (1) preventing and controlling the clubroot of rape;

[0018] (2) improving the resistance of rape to the clubroot;

[0019] (3) cultivating new varieties of rape resistant to the clubroot of rape.

[0020] A method for cultivating a plant resistant to Plasmodiophora brassicae, comprising introducing the host-induced gene silencing RNAi vector into a target plant to obtain a plant resistant to Plasmodiophora brassicae, the plant resistant to Plasmodiophora brassicae being more resistant to Plasmodiophora brassicae than the target plant, the RNAi vector targeting the Plasmodiophora brassicae chitin deacetylase gene PbCDA.

[0021] Advantages:

[0022] The present application screens a chitin deacetylase gene CDA (Chitin Deacetylase, CDA) which may have an important influence on the pathogenic process of Plasmodiophora brassicae from the gene library of Plasmodiophora brassicae ZJ-1, constructs an RNAi vector thereof by using HIGS technology, and transforms the RNAi vector into double 11 of rape. The transgenic rape is subjected to a Plasmodiophora brassicae disease resistance experiment, and the results show that the PbCDA-RNAi transgenic rape has significant resistance to Plasmodiophora brassicae, which indicates that the gene can provide a new gene resource for the green prevention and resistance of rape clubroot. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 PbCDA expression profile and recombinant plasmid construction and identification

[0024] Expression of PbCDA in the life cycle of Ustilaginoidea virens ZJ-1 (A): IN represents the cortical infection stage, PZ represents the zoospore stage, and RS represents the resting spore stage; PbCDA sense strand clone (B); PbCDA antisense strand clone (C).

[0025] Figure 2 Identification of HIGS transgenic rapeseed

[0026] The leaf DNA of 7 T1 generation transgenic plants of PbCDA-RNAi was extracted, and specific primers of the gene were used for identification.

[0027] Figure 3 Enhanced resistance of transgenic rapeseed to Ustilaginoidea virens

[0028] Phenotype of PbCDA-RNAi transgenic rapeseed inoculated with Ustilaginoidea virens for 30 d (A), Bar = 2 cm; Disease index of PbCDA-RNAi transgenic rapeseed inoculated with Ustilaginoidea virens for 30 d (B and C), n = 28 biological replicates; Relative biomass of Ustilaginoidea virens in diseased root system detected by qPCR (D), the relative biomass of Ustilaginoidea virens was calculated by the content of Ustilaginoidea virens ACTIN gene relative to the content of rapeseed ACTIN2 gene; The expression of target gene was determined by using specific primers with actin gene as internal reference; The silencing level of PbCDA in diseased root system was detected by qPCR (E); One-way ANOVA was used, and different lowercase letters represent significant differences (P ≤ 0.05). DETAILED DESCRIPTION

[0029] Example 1

[0030] 1. Materials and methods

[0031] 1.1 Plant materials, strains and plasmids

[0032] The rapeseed variety Zhong 11 was from the laboratory preservation. The pathogenic fungus Ustilaginoidea virens was from the diseased root system of the root rot disease in Zhijiang, Hubei (Williams 1 physiological race) ZJ-1. The Escherichia coli DH5a strain was from the laboratory preservation. The Agrobacterium tumefaciens GV3101 was from the laboratory preservation. The plasmid vector ds1301 (Host-induced gene silencing of fungal-specific genes of Ustilaginoidea virens confers effective resistance to rice false smut, doi: 10.1111 / pbi.13756) was publicly disclosed.

[0033] 1.2 Extraction of Plasmodiophora brassicae chlamydospores

[0034] Plasmodiophora brassicae extraction: The frozen swollen roots were taken out and thawed, washed with clean water, then treated with 70% ethanol for 2 min, and then treated with 10% hydrogen peroxide for 1 h to disinfect the root surface, and finally washed with sterile water for 5 times. The treated roots were cut into small pieces with a knife and put into a homogenizer with appropriate amount of sterile water for homogenization. The homogenized residue was filtered with 8 layers of gauze, and the filtrate was divided into clean 50 mL centrifuge tubes. The filtrate was centrifuged at 8000 rpm / min for 3 min, the supernatant was discarded, the precipitate was resuspended with sterile water, then centrifuged, and the supernatant was discarded after repeating the operation twice. The precipitate was resuspended with 50% (w / v) sucrose solution, centrifuged at 8000 rpm / min for 3 min, and the supernatant containing chlamydospores was collected. An equal volume of sterile water was added, mixed well, and then centrifuged at 8000 rpm / min for 3 min, and the supernatant was discarded. The precipitate was resuspended with sterile water, centrifuged at 8000 rpm / min for 3 min, and the supernatant was discarded. The precipitate was resuspended with appropriate amount of sterile water, and stored in the refrigerator at 4°C in the dark.

[0035] Chlamydospore disinfection: The Plasmodiophora brassicae chlamydospore solution was centrifuged at 3000 rpm / min for 5 min, the supernatant was discarded, and 12.5 mL of freshly prepared 2% chloramine T solution was added to resuspend the precipitate. After standing at room temperature for 20 min, it was centrifuged at 3000 rpm / min for 5 min, and the supernatant was discarded. The precipitate was resuspended with sterile water, centrifuged at 3000 rpm / min for 5 min, and the supernatant was discarded. This operation was repeated 3 times to wash the chloramine T from the chlamydospores. To the chlamydospore precipitate, 5 mL of vancomycin hydrochloride, antistaphylococcal substance sulfate, and cefotaxime sodium with final concentrations of 1 mg / L, 1 mg / L, and 6 mg / L, respectively, were added to resuspend the precipitate, and centrifuged at 3000 rpm / min for 5 min, and the supernatant was discarded. The precipitate was resuspended with sterile water, centrifuged at 3000 rpm / min for 5 min, and the supernatant was discarded. This operation was repeated 3 times. Finally, the chlamydospores were resuspended with appropriate amount of sterile water, counted with a hemocytometer, and stored in the refrigerator at 4°C in the dark for future use.

[0036] 1.3 Extraction of Plasmodiophora brassicae DNA

[0037] Take 2 mL of the root galls or spore suspension, cut into small pieces and grind into powder in liquid nitrogen. For spore suspension, centrifuge at 3000 rpm / min for 5 min, discard the supernatant, and freeze in liquid nitrogen. Then add 2 steel beads of 3 mm into the tube and grind in a grinder at 45 Hz for 2 min. Add 700 μL of 2% CTAB preheated at 65°C into the ground powder, and incubate in a water bath at 65°C for 30 min, with shaking every 10 min. Add 350 μL of Tris-saturated phenol solution and chloroform into the tube, shake for 1 min, and centrifuge at 12000 rpm / min for 10 min. Take 500 μL of the supernatant and mix with equal volume of chloroform, and let it stand for layering. Take 500 μL of the supernatant and mix with equal volume of isopropanol, and let it stand for 15-30 min. Centrifuge at 12000 rpm / min for 10 min, discard the supernatant. Add 500 μL of 75% ethanol, centrifuge at 12000 rpm / min for 5 min, discard the supernatant, and repeat this operation twice. Dry the residual ethanol, and add 40 μL of RNase-free water to dissolve the precipitate, and store at -20°C.

[0038] 1.4 Extraction of RNA from Plasmodiophora brassicae

[0039] Take the root galls or spore suspension, cut into small pieces and grind into powder in liquid nitrogen. For spore suspension, centrifuge at 3000 rpm / min for 5 min, discard the supernatant, and freeze in liquid nitrogen. Then add 2 steel beads of 3 mm into the tube and grind in a grinder at 45 Hz for 2 min. Add 1 mL of Trizol into the powder, vortex to mix, and add 200 μL of chloroform for extraction, vortex to shake, and let it stand on ice for 15 min until obvious layering. Centrifuge at 12000 rpm / min for 15 min at 4°C, and transfer the upper aqueous phase into a new 1.5 mL enzyme-free tube, add equal volume of isopropanol, shake to mix, and incubate on ice for 10 min. Centrifuge at 12000 rpm / min for 15 min at 4°C, discard the supernatant. Wash the precipitate with 75% ethanol prepared with DEPC water, centrifuge at 12000 rpm / min for 5 min at 4°C, and repeat twice. Dry the residual ethanol, let it stand to dry, add 50 μL of DEPC water to dissolve the precipitate, and store at -80°C.

[0040] 1.5 Reverse transcription of RNA into cDNA

[0041] The synthesis of cDNA was performed using the Prime Scropt 1st Strand cDNA synthesis kit from Takara Bio Company, and a 20 μL system was configured according to the instructions as follows: 4 μL of 5×All-in-one qRT SuperMix, 1 μL of Enzyme Mix, 1 pg-1 μg of total RNA, and finally RNase-free Water was added to 20 μL, incubated at 50°C for 15 min in the PCR instrument, incubated at 85°C for 5 sec, and the synthesized cDNA was stored at -20°C for standby.

[0042] 1.6 Target gene cloning

[0043] Target gene fragment selection: a 300-500 bp CDS sequence (SEQ ID NO. 2) was selected from the gene as the interference sequence, and the selected fragment was aligned on NCBI (https: / / www.ncbi.nlm.nih.gov / ) to ensure the specificity of the interference, and its corresponding interference site in Brassica napus double 11 was determined through the TAIR (https: / / www.arabidopsis.org / ) website to avoid off-target.

[0044] Primer design: the homologous arm primers for enzyme digestion sites were designed using the Vazyme cloning website (https: / / crm.vazyme.com), and the sense strand primer of the target gene fragment was designed at the KpnI-BamHI enzyme digestion site, and the antisense strand primer of the target gene fragment was designed at the SacI-SpeI enzyme digestion site, with ds1301 as the carrier. The designed primers are as follows:

[0045] PbCDA sense strand construction primer:

[0046] CDA-dS1301-F: 5'-cccgtgcagctgcggggtaccCAGGCCGACATCGTAGGGC-3'

[0047] CDA-dS1301-R: 5'-cgcgtacgtaaggttggatccGGTGGTGCTGCCAACGTACA-3'

[0048] PbCDA antisense strand construction primer:

[0049] CDA-RC-dS1301-F: 5'-caattcaattcagtggagctcGGTGGTGCTGCCAACGTACA-3'

[0050] CDA-RC-dS1301-R: 5'-caggactctagacccactagtCAGGCCGACATCGTAGGGC-3'

[0051] Prepare 50 μL of gene amplification system: 10 μL of 6x FastPfu Buffer, 1 μL of FastPfu, 1 μL of Primer F / R, 5 μL of dNTPs, 2 μL of DNA / cDNA, and finally add RNase-free Water to 50 μL. Mix the system, and then pre-denature at 98°C for 2 min in the PCR instrument, denature at 98°C for 30 sec, anneal at 59°C for 30 sec, extend at 72°C for 1 min, 35 cycles, then extend at 72°C for 5 min, and finally cool at 16°C for 2 min. Use 1.2% agarose gel electrophoresis to detect the size of the amplified target gene fragment, and then recover the target fragment of the correct size (the steps are described in the TaKaRa DNA agarose gel electrophoresis recovery kit).

[0052] 1.7 Extraction of E. coli plasmid

[0053] Pour the fresh E. coli liquid containing the ds1301 vector into a 2 mL centrifuge tube, centrifuge at 12000 rpm / min for 2 min, and discard the supernatant. Add 200 μL of Solution I (10 mM EDTA, 25 mM Tris-HCl, 10 mM Glucose) to the precipitate, and mix well. Add Solution II (200 μL of 200 mM NaOH and 200 μL of 1% SDS), mix well by gently inverting up and down for 10-20 times, and the solution will become stringy. Add 200 μL of Solution III (29.442 g of KAc and 11.5 mL of anhydrous acetic acid, and dilute to 100 mL), mix well, and a white precipitate will be observed. Add 400 μL of chloroform, stand for 1 min, centrifuge at 12000 rpm / min for 10 min, mix 600 μL of supernatant with an equal volume of isopropanol, centrifuge at 12000 rpm / min for 10 min, and discard the supernatant. Wash the precipitate with 1 mL of 75% ethanol, centrifuge at 12000 rpm / min for 3 min, discard the supernatant, and repeat this operation twice. Remove the residual ethanol from the tube, dry at 37°C, add 50 μL of RNase-free Water, and store at -20°C.

[0054] 1.8 Construction of PbCDA-RNAi vector

[0055] Positive strand homologous recombination: ds1301 empty plasmid was digested by restriction enzyme Kpnl and BamHI, the enzyme digestion system was as follows: 20 μL ds1301 plasmid, 5 μL 10 x rCutSmart Buffer, 1 μL Kpnl and BamHI, and RNase-free Water was added to 50 μL, and the system was mixed and then incubated at 37°C for 30 min. The digested ds1301 vector was subjected to positive strand homologous recombination with the target gene fragment, and the recombination reaction system was as follows: 2 μL 5 x CEII Buffer, 1 μL exnase II, 2 μL digested vector, 1 μL target fragment, and RNase-free Water was added to 10 μL, and mixed and then incubated at 37°C for 30 min in a PCR instrument.

[0056] E. coli heat shock transformation: the recombinant plasmid was added to 50 μL E. coli competent DH5a, mixed and then incubated at ice bath for 30 min, 42°C heat shock for 45 sec, and then incubated at ice bath for 2 min. 1 mL LB medium was added, and the mixture was incubated at 37°C at 220 r / min for 30 min or more, centrifuged at 5000 rpm / min for 5 min, and most of the supernatant was discarded. About 100 μL of resuspended bacteria were left and spread on LB plates with Kana (50 μg / mL) resistance, and incubated at 37°C overnight.

[0057] Transformation identification: 8 single colonies grown on the plates were randomly selected, and LB medium with Kana (50 μg / mL) resistance was used for overnight culture at 37°C on a shaker. Then, the bacterial liquid was subjected to PCR using a Mix system, and the PCR system was as follows: 5 μL 5 x Rapidtaq Mix, 0.5 μL Primer F / R, 1 μL bacterial liquid, and RNase-free Water was added to 10 μL. The reaction program was 95°C pre-denaturation for 3 min, 95°C denaturation for 30 sec, 59°C annealing for 15 sec, 72°C extension for 5 min, 30 cycles, and then 72°C extension for 5 min, and finally 16°C cooling for 2 min. The PCR product was detected by 1.2% agarose gel electrophoresis, and the band with correct size of the target fragment was sent to the company for sequencing for further verification.

[0058] Antisense strand homologous recombination: the plasmid correctly constructed by the positive strand was extracted, and the plasmid was digested by restriction enzymes SacI and SpeI. Then, the antisense strand was subjected to homologous recombination with the digested plasmid, and E. coli heat shock transformation and identification were performed, which were the same as described above.

[0059] 1.9 Rape transformation and identification

[0060] The complete constructed vector was sent to the company for transformation of Brassica napus double 11, and the transgenic Brassica napus was returned after growing up. The green and healthy leaves were selected to extract DNA, and then specific primers of the target gene were used for identification. Mix system was used for identification: 5 μL 5×Rapid taq Mix, 0.5 μL Primer F / R, 1 μL DNA, and RNase-free Water was added to 10 μL. The reaction program was 95°C pre-denaturation for 3 min, 95°C denaturation for 30 sec, 59°C annealing for 15 sec, 72°C extension for 5 min, 30 cycles, then 72°C extension for 5 min, and 16°C cooling for 2 min, and then the reaction was ended. The PCR product was detected by 1.2% agarose gel electrophoresis, and the correct band was the positive transformant.

[0061] 1.10 Disease resistance test of transgenic Brassica napus inoculated with Plasmodiophora brassicae

[0062] The seeds of the previously identified transgenic Brassica napus were collected, and after disinfection, they were uniformly dispersed on 1 / 2 MS medium with hygromycin resistance. After 7 days, 30 seedlings with consistent growth were selected and transplanted into soil. After a week, 1 mL of 1×10 7 CFU / mL of Plasmodiophora brassicae spore solution was inoculated per plant. The control was Brassica napus double 11. After 28 days, the roots were removed and washed to count the disease. The root rot disease index grading standard: 0 level: the plant grows well, and there is no tumor on the main and lateral roots; 1 level: the lateral roots have small tumors, and the main roots have no tumors; 3 level: the main roots have small tumors; 5 level: the main and lateral roots have moderate tumors; 7 level: the main and lateral roots have large tumors, and there are a small amount of root hairs; 9 level: the main root tumors are large or even rotten, and there are very few or no root hairs. Disease index = Σ (disease level x number of plants of the corresponding disease level) x 100 / (highest level x total number of plants), control effect (%) = (control disease index - transgenic plant disease index) x 100 / control disease index.

[0063] 1.11 Detection of the relative biomass of Plasmodiophora brassicae in the roots of diseased plants

[0064] The roots of diseased Brassica napus at 28 days after inoculation were used as experimental materials. Three diseased roots of each family were taken as a sample, and total DNA was extracted from the diseased roots of Brassica napus using the CTAB method. The DNA concentration was determined using Nanodrop, and the total DNA concentration was diluted to 200 ng / μL. The diluted DNA was used as a template to detect the content of the reference gene of Plasmodiophora brassicae relative to the reference gene of Brassica napus using qPCR reaction. The relative biomass of Plasmodiophora brassicae was represented by the content of the Actin gene of Plasmodiophora brassicae relative to the qBnACTIN7 gene of Brassica napus.

[0065] Plasmodiophora brassicae actin gene:

[0066] Pbactin-qF: 5'-CACCGACTACCTGATGAA-3'

[0067] Pbactin-qR: 5'-CAGCTTCTCCTTGATGTC-3'

[0068] Rape qBnACTIN7 gene:

[0069] qBnACTIN7-qF: 5'-GCACCCTGTTCTTCTTACGGA-3'

[0070] qBnACTIN7-qR: 5'-GTGAGACACACCATCACCAGA-3'

[0071] 1.12 Detection of the level of PbCDA silencing in inoculated rape

[0072] Total RNA was extracted from the roots of diseased rape using Trizol reagent. 1 μg of RNA was used for reverse transcription to obtain 20 μL of cDNA, and 2 μL of the cDNA was used as a template for qPCR experiments. The reaction system for real-time fluorescent quantitative PCR (10 μL) was as follows: iTaq™ Universal SYBR® Green supermix (2X) 5 μL, Forward primers (10 μM) 0.5 μL, Reverse primers (10 μM) 0.5 μL, cDNA 2 μL, ddH2O 3 μL. The reaction conditions were as follows: 95 °C, 10 min; 95 °C, 15 sec, 57 °C, 15 sec, repeated for 45 cycles; 65 °C~95 °C, each cycle increased by 0.5 °C for 5 sec, 16 °C for 1 min. The reaction was performed on a Bio-Rad CFX 96 instrument. After amplification, the melting curve was analyzed using 2 -∆∆Ct The relative expression of the gene was calculated by the method.

[0073] Plasmodiophora actin gene:

[0074] Pbactin-qF: 5'-CACCGACTACCTGATGAA-3'

[0075] Pbactin-qR: 5'-CAGCTTCTCCTTGATGTC-3'

[0076] Plasmodiophora CDA gene:

[0077] PbCDA-qF: 5'-GAATCCAAGTCGCTGACGTCG-3'

[0078] PbCDA-qR: 5'-GTCGTGGACGAACATGTGGTC-3'

[0079] 2. Results

[0080] 2.1 Construction of PbCDA-RNAi vector

[0081] PbCDA was highly expressed in the three stages of P. thornei, especially in the cortical infection stage (Fig. 1A). We extracted RNA from P. thornei and reverse transcribed it into cDNA. Then we cloned a 360 bp fragment of PbCDA using the cDNA as a template and inserted it into the vector ds1301 to construct the RNAi vector. To detect whether the PbCDA-RNAi vector was successfully constructed, we used specific primers for the sense and antisense strands to perform PCR positive identification (Fig. 1B, C), and the results showed that it was successfully constructed. Figure 1 Figure 1 2.2 Identification of transgenic rapeseed

[0082] 2.2 Identification of transgenic rapeseed

[0083] We moved the normally growing rapeseed into the soil and then extracted the leaf DNA to identify the positive plants using specific primers (Fig. 2A). Finally, we obtained 7 positive transgenic plants. Figure 2 2.3 Increased resistance of PbCDA-RNAi transgenic rapeseed to P. thornei

[0084] To verify the disease resistance of PbCDA-RNAi transgenic rapeseed to P. thornei, we inoculated the roots of the transgenic rapeseed with P. thornei when it was two weeks old, and observed the root growth phenotype after four weeks. As shown in Fig. 3A, the disease of Zhongshuang 11 was significantly more severe than that of the transgenic rapeseed. The main root of Zhongshuang 11 was significantly swollen, and the fibrous root was almost absent, while most of the main roots of the PbCDA-RNAi transgenic rapeseed were smaller, the lateral roots were almost not swollen, and there were more fibrous roots. The control effects of PbCDA-1 and PbCDA-20 transgenic rapeseed reached 26.8% and 56.1%, respectively, indicating that the PbCDA-RNAi transgenic rapeseed can significantly improve the resistance to P. thornei (Fig. 3B, C). To verify that the resistance of the PbCDA-RNAi transgenic rapeseed to P. thornei is due to the silencing of the target gene in P. thornei by SiRNA, we collected the diseased rapeseed roots to extract their DNA and RNA to detect the relative biomass of P. thornei in the roots and the silencing level of PbCDA. The qPCR results showed that the relative biomass of P. thornei in the roots of the PbCDA-RNAi transgenic rapeseed was significantly reduced (Fig. 3D), and the expression level of the target gene was significantly lower than that of Zhongshuang 11 (Fig. 3E).

[0085] Figure 3 Figure 3 Figure 3 Figure 3 ​​​​​E), all of which indicated that the silencing of target gene by SiRNA enhanced the resistance of PbCDA-RNAi transgenic Brassica napus to P. brassicae.

[0086] 3 Summary and discussion

[0087] Brassica is one of the most important genera of Brassicaceae, including hundreds of agricultural crops such as oilseed rape, Chinese cabbage, radish, etc. Many varieties of them can be infected by P. brassicae. P. brassicae is difficult to control due to its soil-borne characteristics, and its chlamydospores can survive in soil for 8-12 years or even longer, which accumulates mutations and promotes the emergence of new physiological races, and also makes it more likely to develop new resistance to chemical pesticides. The resistance to P. brassicae was significantly enhanced after the silencing of PbCDA by HIGS, indicating that PbCDA can be used as a green resource for the prevention and control of oilseed rape clubroot.

Claims

1. The application of inhibiting or silencing the chitin deacetylase gene PbCDA from *Plasmodium* in the control of clubroot disease in rapeseed, characterized in that... The CDS sequence of the *Plasmodium* chitin deacetylase gene PbCDA is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The inhibition or silencing of the PbCDA chitin deacetylase gene in *Plasmodium* is achieved through host-induced gene silencing technology.

3. The application of a substance that inhibits or silences the PbCDA gene of *Plasmodium* chitin deacetylase as described in claim 1, characterized in that, Choose from any of the following: (1) Control of clubroot disease in rapeseed; (2) Improve rapeseed's resistance to clubroot; (3) Develop new rapeseed varieties resistant to clubroot disease.

4. The application according to claim 3, characterized in that, The substance used to inhibit or silence the PbCDA chitin deacetylase gene of *Plasmodium* as described in claim 1 is selected from host-induced gene silencing RNAi vectors, gene editing systems, siRNA, dsRNA, and ordinary RNAi vectors.

5. The application according to claim 4, characterized in that, The substance used to inhibit or silence the PbCDA chitin deacetylase gene of *Plasmodium* in claim 1 is selected from a host-induced gene silencing RNAi vector targeting the gene, wherein the host-induced gene silencing RNAi vector uses the CDS sequence fragment shown in SEQ ID NO.2 as an interfering sequence.

6. A host-induced gene silencing RNAi vector for silencing the *PbCDA* chitin deacetylase gene of *P. 1* as described in claim 1, characterized in that, The host-induced gene silencing RNAi vector uses the CDS sequence fragment shown in SEQ ID NO.2 as the interference sequence.

7. Engineered bacteria containing the RNAi vector of claim 6.

8. Any one of the following applications of the RNAi vector of claim 6 and the engineered bacteria of claim 7: (1) Control of clubroot disease in rapeseed; (2) Improve rapeseed's resistance to clubroot; (3) Develop new rapeseed varieties resistant to clubroot disease.

9. A method for cultivating plants resistant to clubroot pathogen, characterized in that, The method includes introducing the host-induced gene silencing RNAi vector of claim 6 into a target plant to obtain a clubroot-resistant plant, wherein the clubroot-resistant plant has higher resistance to clubroot than the target plant, and the RNAi vector targets the clubroot chitin deacetylase gene PbCDA of claim 1.

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