Application of helicase PbDDX15-like in prevention and control of clubroot

Abstract

The invention belongs to the field of biological agriculture, and relates to application of helicase PbDDX15-like in prevention and control of clubroot. According to the invention, a core gene of plasmodiophora brassicae ZJ-1 is compared with a brewer's yeast protein sequence, a gene DEAD-box RNA helicase (DEAD-box RNA helicase 15-like, PbDDX15-like) which may have a lethal effect on plasmodiophora brassicae is screened out, an RNAi vector of the gene is constructed by using an HIGS technology, and the RNAi vector is transformed into wild type arabidopsis thaliana. The PbDDX15-like-RNAi transgenic arabidopsis thaliana is subjected to a plasmodiophora brassicae disease resistance experiment, and the result shows that the PbDDX15-like-RNAi transgenic arabidopsis thaliana has remarkable resistance to plasmodiophora brassicae, so that the protein can provide a new gene resource for green prevention and resistance of the rape clubroot.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bio-agriculture, and relates to the application of helicase 15-like (PbDDX15-like) in the prevention and control of clubroot. BACKGROUND

[0002] Clubroot caused by Plasmodiophora brassicae, which is soil-borne, is the most destructive disease of cruciferous crops in the world. The infection of P. brassicae in the root system of cruciferous crops can cause root swelling and loss of root function, thereby affecting the development of the aboveground part and causing huge yield loss, and in severe cases, there is no harvest at all. 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 infects the host plant, the siRNA released by the host plant silences the target gene of the pathogen, reduces the infection of the pathogen, and enhances the resistance of the plant (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, which significantly reduced the pathogenicity of transgenic rice plants except for PMC1 and LHS1 (Wang and Dean 2022). Yi et al. used HIGS to silence MSTRG.4380.1, which reduced the virulence of the root rot fungus to wheat (Yi et al 2023).

[0003] RNA helicases unwind double-stranded regions of RNA molecules in an ATP-dependent manner. The energy released from ATP hydrolysis is used to unwind RNA or remove proteins bound to RNA. DEAD-box RNA helicases are central to RNA metabolism and gene expression. These RNA-binding proteins can prevent non-productive rearrangements of nascent RNA or non-specific interactions with other accessory proteins. These enzymes are also important for structural rearrangements of RNA and RNA-protein complexes and are involved in regulating many cellular structures and functions, such as RNA folding, RNA strand dissociation, protein displacement from RNA, or stabilization of RNA-protein complexes. These proteins also play a role in RNA synthesis, RNA decay, RNA processing, and ribosome biogenesis (Ashaq 2024). SUMMARY

[0004] The application aims at solving the above problems in the prior art and providing an application of helicase 15-like (PbDDX15-like) in preventing and controlling clubroot.

[0005] The application aims at solving the above problems in the prior art and providing an application of helicase 15-like (PbDDX15-like) in preventing and controlling clubroot.

[0006] The DEAD-box RNA helicase encoding gene PbDDX15-like has a nucleotide sequence as shown in SEQ ID NO. 1.

[0007] The recombinant vector contains the DEAD-box RNA helicase encoding gene PbDDX15-like or a fragment thereof as claimed in claim 1.

[0008] Preferably, the recombinant vector is an RNAi vector of the PbDDX15-like gene constructed based on the HIGS technology.

[0009] Preferably, the recombinant vector contains a sense strand and an antisense strand of the interference fragment as shown in SEQ ID NO. 2.

[0010] The application of the DEAD-box RNA helicase encoding gene PbDDX15-like in improving the resistance of plants to clubroot.

[0011] Preferably, the method for inhibiting or silencing the DEAD-box RNA helicase encoding gene PbDDX15-like is selected from the host-induced gene silencing technology and the gene editing technology.

[0012] Further preferably, the plant is Arabidopsis thaliana or Brassica napus.

[0013] The application of the substance for inhibiting or silencing the DEAD-box RNA helicase encoding gene PbDDX15-like in cultivating plants with resistance to clubroot.

[0014] Preferably, the application of the recombinant vector is in cultivating plants with resistance to clubroot.

[0015] Further preferably, the plant is Arabidopsis thaliana or Brassica napus.

[0016] Advantages:

[0017] The application compares the core gene of Plasmodiophora brassicae ZJ-1 with the protein sequence of Saccharomyces cerevisiae, and screens out a gene DEAD-box RNA helicase 15-like (PbDDX15-like) which may have a lethal effect on Plasmodiophora brassicae. We use the HIGS technology to construct the RNAi carrier of the gene, and transform it into wild-type Arabidopsis. The transgenic Arabidopsis is subjected to the Plasmodiophora brassicae disease resistance experiment, and the results show that the PbDDX15-like-RNAi transgenic Arabidopsis has significant resistance to Plasmodiophora brassicae, which indicates that the protein can provide a new gene resource for the green resistance of the oilseed rape clubroot. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Expression profile of PbDDX15-like and construction and identification of recombinant plasmid

[0019] The expression amount of PbDDX15-like in the life cycle of Plasmodiophora brassicae ZJ-1 (A), IN represents the cortical infection period, PZ represents the zoospore period, and RS represents the resting spore period; PbDDX15-like sense strand cloning (B); PbDDX15-like antisense strand cloning (C); PbDDX15-like-RNAi carrier identification (D), 1: sense strand construction detection; 2: antisense strand construction detection; 3: KpnI and SpeI double enzyme digestion plasmid detection.

[0020] Figure 2 Identification of HIGS transgenic plants

[0021] Leaf DNA of 7 T1 generation transgenic plants of PbDDX15-like-RNAi is extracted, and specific primers of the gene are used for identification (A); growth phenotype of wild-type Arabidopsis Col-0 and PbDDX15-like-RNAi positive transformation plants for 5 weeks (B), Bar = 2 cm.

[0022] Figure 3 Enhanced resistance of transgenic Arabidopsis to Plasmodiophora brassicae

[0023] Phenotypic characteristics of PbDDX15-like transgenic Arabidopsis thaliana inoculated with *Plasmodiophora* 21 days after inoculation (A), Bar = 2 cm; Disease index of PbDDX15-like transgenic Arabidopsis thaliana inoculated with *Plasmodiophora* 21 days after inoculation (B and C), n = 20 biological replicates; Relative biomass of *Plasmodiophora* in diseased roots was detected by qPCR (D), the relative biomass of *Plasmodiophora* was determined by the content of *Plasmodiophora* ACTIN gene relative to *Arabidopsis* ACTIN2 gene; Using the actin gene as an internal control, the expression level of the target gene was determined using specific primers, and the silencing level of PbDDX15-like in diseased roots was detected by qPCR (E); One-way ANOVA was used (P ≤ 0.05), * indicates significant difference. Detailed Implementation

[0024] The plant materials, strains, and plasmids involved in the following examples

[0025] Arabidopsis thaliana Col-0, Escherichia coli DH5α, and Agrobacterium tumefaciens GV3101 are all conventional strains already disclosed in this field.

[0026] The ds1301 vector is disclosed in Host-induced gene silencing offungal-specific genes of Ustilaginoidea virensconfers effective resistance to rice false smut, doi:10.1111 / pbi.13756.

[0027] The pathogen, *Cladophora mycoides*, was found in the root system (Williams 1 physiological race) ZJ-1 of clubroot disease in a rapeseed experimental field in Zhijiang, Hubei Province.

[0028] Example 1

[0029] By comparing the core gene of *Plasmodiophora* ZJ-1 with the protein sequence of *Saccharomyces cerevisiae*, 10 genes that may have a lethal effect on *Plasmodiophora* were screened, one of which was PbDDX15-like. Analysis of the *Plasmodiophora* ZJ-1 transcriptome data showed that PbDDX15-like was expressed at high levels during both the zoospore and dormant spore stages of *Plasmodiophora*. Figure 1 A).

[0030] Example 2

[0031] 1. Method

[0032] 1.1 Extraction of dormant spores of *Plasmodiophora stylosa*

[0033] Clubroot extraction: Thaw the frozen, swollen roots, wash them thoroughly with clean water, treat them with 70% ethanol for 2 minutes, then treat them with 10% hydrogen peroxide for 1 hour to disinfect the root surface, and finally rinse them thoroughly with sterile water 5 times. Cut the treated roots into small pieces and homogenize them thoroughly with an appropriate amount of sterile water. Filter the homogenized residue through 8 layers of gauze, and aliquot the filtrate into clean 50 mL centrifuge tubes. Centrifuge the filtrate at 8000 rpm / min for 3 minutes, discard the supernatant, resuspend the precipitate with sterile water, centrifuge again, and repeat the process twice, discarding the supernatant each time. Resuspend the precipitate in 50% (w / v) sucrose solution, centrifuge at 8000 rpm / min for 3 minutes, collect the supernatant containing dormant spores, add an equal volume of sterile water, mix thoroughly, centrifuge at 8000 rpm / min for 3 minutes, and discard the supernatant. The spores were washed by resuspending the precipitate in sterile water, centrifuged at 8000 rpm for 3 min, and the supernatant was discarded. This operation was repeated 3 times to thoroughly wash away the sucrose in the spores. After resuspending the spore precipitate in an appropriate amount of sterile water, it was stored in a refrigerator at 4°C in the dark.

[0034] Sterilized dormant spores: Centrifuge the dormant spore solution of *Plasmodiophora* at 3000 rpm / min for 5 min, discard the supernatant, add 12.5 mL of freshly prepared 2% chloramine-T solution to the tube to resuspend the precipitate, let stand at room temperature for 20 min, then centrifuge at 3000 rpm / min for 5 min, discard the supernatant. Add sterile water to resuspend the precipitate, centrifuge at 3000 rpm / min for 5 min, discard the supernatant, and repeat this operation 3 times to wash away chloramine-T from the spores. Add 5 mL each of vancomycin hydrochloride, antidimethicone sulfate, and cefotaxime sodium to the spore precipitate at final concentrations of 1 mg / L, 1 mg / L, and 6 mg / L to resuspend the precipitate, centrifuge at 3000 rpm / min for 5 min, discard the supernatant. Add sterile water to resuspend the precipitate, centrifuge at 3000 rpm / min for 5 min, discard the supernatant, and repeat this operation 3 times. Finally, add an appropriate amount of sterile water to resuspend the spores, calculate the concentration using a hemocytometer, and store at 4℃ protected from light for later use.

[0035] 1.2 DNA extraction from *Plasmodiophora*

[0036] Pipe 2 mL of *Plasmodiophora* spores into a 2 mL centrifuge tube, centrifuge at 3000 rpm for 5 min, discard the supernatant, and freeze in liquid nitrogen. Then, add two 3 mm steel balls to the tube and grind at 45 Hz for 2 min. Add 700 μL of preheated 2% CTAB (65℃) to the ground powder, incubate in a 65℃ water bath for 30 min, shaking every 10 min. Add 350 μL of Tris-saturated phenol solution and chloroform to the tube, shake for 1 min, centrifuge at 12000 rpm for 10 min, and mix 500 μL of the supernatant with an equal volume of chloroform. Allow to separate into layers. After separation, mix 500 μL of the supernatant with an equal volume of isopropanol, allow to stand for 15-30 min, centrifuge at 12000 rpm for 10 min, and discard the supernatant. Add 500 μL of 75% ethanol, centrifuge at 12000 rpm / min for 5 min, discard the supernatant, and repeat the operation twice. Aspirate the residual ethanol, air dry, dissolve in 40 μL of RNase-free water, and store at -20℃ for later use.

[0037] 1.3 RNA extraction from *Plasmodium*

[0038] Take swollen roots or spores from the plant. Cut the roots into small pieces and grind them into powder in liquid nitrogen. Centrifuge the spores at 3000 rpm for 5 min, discard the supernatant, and freeze in liquid nitrogen. Then add two 3 mm steel balls to the tube and grind at 45 Hz for 2 min. Add 1 mL of Trizol to the powder, vortex to mix, add 200 μL of chloroform for extraction, vortex, and incubate on ice for 15 min until clear separation occurs. Centrifuge at 12000 rpm for 15 min at 4°C, transfer the upper aqueous phase to a new 1.5 mL enzyme-free tube, add an equal volume of isopropanol, vortex to mix, incubate on ice for 10 min, centrifuge at 12000 rpm for 15 min at 4°C, and discard the supernatant. Wash the precipitate with 75% ethanol prepared with DEPC water, centrifuge at 12000 rpm for 5 min at 4°C, and repeat twice. Blot off any residual ethanol, allow to stand and air dry, add 50 μL of DEPC water to dissolve the precipitate, and freeze at -80℃ for later use.

[0039] 1.4 RNA reverse transcription into cDNA

[0040] cDNA was synthesized using the Prime Scropt 1st Strand cDNA synthesis kit from Takara Biotech. The 20 µL system was prepared 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 bring the total to 20 µL. The system was incubated in a PCR instrument at 50 °C for 15 min and then at 85 °C for 5 sec. The synthesized cDNA was stored at -20 °C for later use.

[0041] 1.5 Cloning of the target gene

[0042] Target gene fragment selection: A 300-500 bp CDS sequence (SEQ ID NO.2) was selected from the PbDDX15-like gene as an interfering sequence. The selected fragment was compared with NCBI (https: / / www.ncbi.nlm.nih.gov / ) to ensure the specificity of the interference. The TAIR website (https: / / www.arabidopsis.org / ) was used to confirm that there was no corresponding interfering site in Arabidopsis thaliana to avoid off-target effects.

[0043] Primer design: Primers for homologous arms of restriction sites were designed using the Novizan Cloning website (https: / / crm.vazyme.com). Using ds1301 as a vector, the sense primers for the target gene fragment were designed at the KpnI-BamHI restriction site, and the antisense primers for the target gene fragment were designed at the SacI-SpeI restriction site.

[0044] PbDDX15-like primers for constructing the positive chain:

[0045] PbDDX15-like-F: 5'-cccgtgcagctgcggggtaccCGTTGCTCGAGTCGCTTGA-3'

[0046] PbDDX15-like-R: 5'-cgcgtacgtaaggttggatccACTTCTCGATACGCTCCTCCTG-3'

[0047] PbDDX15-like antisense strand primers:

[0048] PbDDX15-like-F: 5'-caattcaattcagtggagctcACTTCTCGATACGCTCCTCCTG-3'

[0049] PbDDX15-like-R: 5'-caggactctagaccccactagtCGTTGCTCGAGTCGCTTGA-3'

[0050] Prepare a 50 μL gene amplification system: 10 μL of 6×FastPfu Buffer, 1 μL of FastPfu, 1 μL each of Primer F / R, 5 μL of dNTPs, 2 μL of DNA / cDNA, and finally add RNase-free water to bring the total to 50 μL. After mixing the system, incubate in a PCR instrument with 98℃ pre-denaturation for 2 min, 98℃ denaturation for 30 sec, 59℃ annealing for 30 sec, 72℃ extension for 1 min, for 35 cycles, followed by a final extension at 72℃ for 5 min, and a cooling to 16℃ for 2 min to terminate the process. Detect the amplified size of the target gene fragment using 1.2% agarose gel electrophoresis, and then recover the correctly sized bands from the gel (see TaKaRa's DNA Agarose Gel Electrophoresis Recovery Kit for detailed steps).

[0051] 1.6 Escherichia coli plasmid extraction

[0052] Pour fresh E. coli culture containing the ds1301 vector into a 2 mL centrifuge tube, centrifuge at 12000 rpm 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 vortex to mix. Add Solution II (200 μL 200 mM NaOH, 200 μL 1% SDS), gently invert 10-20 times to mix, and the solution should form a stringy consistency when the cap is opened. Add 200 μL of Solution III (29.442 g KAc, 11.5 mL anhydrous acetic acid, bring the volume to 100 mL), mix, and a white precipitate should be observed. Add 400 μL of chloroform, let stand for 1 min, centrifuge at 12000 rpm for 10 min, take 600 μL of the supernatant and mix with an equal volume of isopropanol, centrifuge at 12000 rpm 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 the operation twice. Remove any residual ethanol from the tube, dry at 37°C, add 50 μL of RNase-free water, and store at -20°C.

[0053] 1.7 Construction of PbDDX15-like RNAi vector

[0054] Homologous recombination of the positive strand: The ds1301 empty vector was digested with restriction endonucleases KpnI and BamHI. The digestion system was as follows: 20 μL ds1301 plasmid, 5 μL 10×rCutSmart Buffer, 1 μL KpnI and BamHI, and RNase-free water was added to a final volume of 50 μL. The mixture was incubated at 37°C for 30 min. The digested ds1301 vector was then subjected to homologous recombination of the positive strand with the target gene fragment. The recombination reaction system was as follows: 2 μL 5×CEⅡ Buffer, 1 μL exnase Ⅱ, 2 μL digested vector, 1 μL target fragment, and RNase-free water was added to a final volume of 10 μL. The mixture was incubated at 37°C for 30 min.

[0055] Heat shock transformation of *E. coli*: The recombinant plasmid was added to 50 μL of competent *E. coli* DH5α cells, mixed well, and incubated on ice for 30 min. The cells were then heat-shocked at 42℃ for 45 sec, followed by an ice incubation for 2 min. 1 mL of LB medium was added, and the cells were incubated at 37℃ on a shaker at 220 r / min for at least 30 min. After centrifugation at 5000 rpm for 5 min, most of the supernatant was discarded, and approximately 100 μL of the resuspended cells was plated onto LB agar plates containing Kana (50 μg / mL) antibiotics. The plates were incubated overnight at 37℃.

[0056] Transformant identification: Eight single colonies grown on plates were randomly selected and cultured overnight in LB culture medium supplemented with kanamycin (50 μg / mL) at 37°C using a shaker. Then, bacterial PCR was performed using a mixed system: 5 μL 5×Rapidtaq Mix, 0.5 μL Primer F / R, 1 μL bacterial culture, and RNase-free water to a final volume of 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, followed by another 72°C extension for 5 min, and a final cooling to 16°C for 2 min. PCR products were detected by 1.2% agarose gel electrophoresis. Bands with the correct target fragment size were sent to the company for further sequencing verification.

[0057] Homologous recombination of the antisense strand: The plasmid with the correct sense strand was extracted, digested with restriction endonucleases SacI and SpeI, and then the antisense strand was homologously recombinated with the digested plasmid. The E. coli heat shock transformation and identification were performed in the same manner as described above.

[0058] 1.8 Agrobacterium-mediated transformation in Arabidopsis thaliana (flower-dipping method)

[0059] Agrobacterium transformation: Add 1 μL of the complete plasmid to 40 μL of competent GV3101 cells, gently mix, and incubate on ice for 10 min, then in liquid nitrogen for 5 min, incubate at 37°C for 5 min, and incubate on ice for 5 min. Add 800 μL of antibiotic-free LB medium and incubate at 28°C with shaking for 2-3 h. Centrifuge at 6000 rpm / min for 1 min, resuspend the cells in the bottom 100 μL of supernatant, and spread the bacterial culture on LB plates containing Kana (50 μg / mL) and Rif (25 μg / mL) antibiotics. Incubate at 28°C for 2 days. Verify the transformants using a mixed PCR system; a correct band indicates successful transformation.

[0060] Arabidopsis flower-dipping transformation: Transformed Agrobacterium was cultured overnight at 28°C with shaking in LB medium supplemented with Kana (50 μg / mL) and Rif (25 μg / mL) resistance. The fresh bacterial culture was centrifuged at 5000 rpm / min for 10 min, and the supernatant was discarded. The precipitate was resuspended in a 5% sucrose solution, and surfactant Silwet L-77 was added to a final concentration of 0.02%. Healthy Arabidopsis plants were selected, and their white flowers and pods were removed. The inflorescences were immersed in the prepared bacterial solution for 15-30 seconds, and this process was repeated once after 1 h. The treated Arabidopsis were then kept in the dark under moist conditions for 16 h, followed by normal culture. The above steps were repeated after 7-10 days until the plants matured, and the seeds were harvested.

[0061] 1.9 Screening and Identification of Transgenic Plants

[0062] After disinfection and vernalization, T0 generation seeds collected from flower dips were evenly spotted onto 1 / 2 MS plates treated with hygromycin (50 μg / mL). Seedlings that still showed normal germination and growth were selected after 7-10 days and transplanted into soil for culture. Once the plants had matured, DNA was extracted from healthy green leaves and identified using primers specific to the target gene. The identification process used a mix system: 5 μL 5×Rapid Taq Mix, 0.5 μL Primer F / R, 1 μL DNA, and RNase-free water to a final volume of 10 μL. The reaction program was: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 sec, 59℃ annealing for 15 sec, 72℃ extension for 5 min, 30 cycles, followed by another 72℃ extension for 5 min, and a final cooling to 16℃ for 2 min. PCR products were detected using 1.2% agarose gel electrophoresis; a correct band indicated a positive transformant.

[0063] 1.10 Disease resistance test of transgenic Arabidopsis thaliana inoculated with Plasmodium falciparum

[0064] Seeds of previously identified T1 generation Arabidopsis thaliana were collected, sterilized, and vernalized. They were then evenly dispersed on 1 / 2 MS plates treated with hygromycin. Seven days later, 40 seedlings with uniform growth were selected and transplanted into soil. One week later, each seedling was inoculated with 1 mL of 1×10⁻⁶ mol / L mol / L agar. 7 CFU / mL of *Plasmodiophora stenopsis* spores, with wild-type *Arabidopsis thaliana* as the control.

[0065] Col-0, after 21 days, the roots were removed, washed, and the disease condition was assessed. The disease severity index grading standard for clubroot is as follows: Grade 0: Plants are growing well, with no tumors on either the main or lateral roots; Grade 1: Small tumors on lateral roots, no tumors on the main root; Grade 2: Small tumors on the main root; Grade 3: Medium-sized tumors on both the main and lateral roots; Grade 4: Large tumors on both the main and lateral roots, with a few fibrous roots; Grade 5: Large tumors on the main root, even rotting, with very few or no fibrous roots, severely affecting plant growth and even causing death. Disease index = Σ(disease grade × number of plants corresponding to that disease grade) × 100 / (highest grade × total number of plants), control effect (%) = (control disease index - transgenic plant disease index) × 100 / control disease index.

[0066] 1.11 Detection of relative biomass of clubroot bacteria on the roots of diseased plants

[0067] Roots of Arabidopsis thaliana exhibiting disease at 21 days post-inoculation were used as experimental materials. Three diseased roots from each family were collected as one sample. Total DNA was extracted from the diseased roots using the CTAB method. DNA concentration was determined using Nanodrop, and the total DNA concentration was diluted to 200 ng / μL. Using the diluted DNA as a template, the content of the *Plasmodiophora stylosa* internal reference gene relative to the *Arabidopsis thaliana* internal reference gene was detected using qPCR. The relative biomass of *Plasmodiophora stylosa* was expressed as the content of the *Plasmodiophora stylosa* Actin gene relative to the *Arabidopsis thaliana* AtACTIN2 gene.

[0068] Plasmodium actin gene:

[0069] Pbactin_qF:5'-CACCGACTACCTGATGAA-3'

[0070] Pbactin_qR: 5'- CAGCTTCTCCTTGATGTC-3'

[0071] Arabidopsis thaliana AtACTIN2 gene:

[0072] ATactin2_qF: 5'-GCACCCTGTTCTTCTTACGGA-3'

[0073] ATactin2_qR:5'-GTGAGACACACCATCACCAGA-3'

[0074] 1.12 Detection of PbDDX15-like silencing levels in inoculated Arabidopsis thaliana

[0075] Total RNA was extracted from the roots of diseased Arabidopsis thaliana using Trizol reagent. 1 μg of RNA was reverse transcribed to obtain 20 μL of cDNA, and 2 μL was used as a template for qPCR. The real-time quantitative PCR (10 µL) reaction system 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. Reaction conditions: 95℃, 10 min; 95℃, 15 sec, 57℃, 15 sec, repeated for 45 cycles; 65℃~95℃, increasing by 0.5℃ per cycle for 5 sec, 16℃ for 1 min. The reaction was performed on a Bio-Rad CFX 96 instrument. After amplification, 2... -∆∆Ct The method calculates the relative expression level of genes.

[0076] Plasmodium actin gene:

[0077] Pbactin_qF:5'-CACCGACTACCTGATGAA-3'

[0078] Pbactin_qR: 5'- CAGCTTCTCCTTGATGTC-3'

[0079] DDX15-like gene in clubroot bacteria:

[0080] PbDDX15-like-qF: 5'-CGTTGCTCGAGTCGCTTGATA-3'

[0081] PbDDX15-like-qR:5'-CGGTGCACAGAGTTCACAAGAT-3'

[0082] 2. Research Results

[0083] 2.1 Construction of PbDDX15-like-RNAi vector

[0084] RNA was extracted from *Plasmodiophora* and reverse transcribed into cDNA. A 300 bp fragment of PbDDX15-like RNA was then cloned using the cDNA as a template and inserted into the ds1301 vector to construct an RNAi vector. To verify the successful construction of the PbDDX15-like RNAi vector, we performed positive identification using specific primers for the sense and antisense strands, and detected the positive result by double digestion of the recombinant plasmid with KpnI and SpeI. Figure 1 (B) The results show that the construction was successful.

[0085] 2.2 Creation of HIGS Transgenic Arabidopsis

[0086] Referring to method 1.8, the PbDDX15-like-RNAi vector was transformed into wild-type Arabidopsis thaliana using the flower-dipping method. The resulting T0 generation Arabidopsis seeds were screened on hygromycin-resistant 1 / 2 MS plates. Normally growing plants were transplanted into soil, and leaf DNA was subsequently extracted. Positive plants were identified by PCR using specific primers. Figure 2 A), ultimately obtaining 7 positive transformant plants. Seeds from the identified positive transformant plants were collected and screened again on 1 / 2 MS plates containing hygromycin resistance. After transplanting into soil, stable T2 generation transgenic seeds were collected. When the T2 generation transgenic seeds were simultaneously planted with wild-type Arabidopsis thaliana Col-0, no significant difference in growth phenotype was found between the transgenic plants and wild-type Arabidopsis thaliana Col-0. Figure 2 B) indicates that PbDDX15-like does not affect the normal growth of Arabidopsis thaliana.

[0087] 2.3 Increased resistance to clubroot bacteria in PbDDX15-like-RNAi transgenic Arabidopsis thaliana

[0088] To verify the resistance of PbDDX15-like-RNAi transgenic plants to clubroot fungus, clubroot fungus was inoculated into the roots of transgenic Arabidopsis thaliana two weeks after growth, and root growth phenotype was observed three weeks later. Figure 3 As shown in Figure A, root disease in wild-type Arabidopsis thaliana Col-0 was significantly more severe than in transgenic Arabidopsis thaliana. Almost all of the main and lateral roots of Col-0 were diseased, with almost no fibrous roots, and grades 4 and 5 accounted for 75% of the total. In contrast, the roots of transgenic Arabidopsis thaliana were mostly not rotten, with smaller swelling of the main root, almost no swelling of the lateral roots, and more fibrous roots; grades 4 and 5 accounted for only 10-20% of the total. The disease index of Col-0 was 79, while the disease index of transgenic Arabidopsis thaliana PbDDX15-like-RNAi-1 was 62, and the disease index of PbDDX15-like-RNAi-2 was 57, with control effects of 21.5% and 27.8%, respectively, indicating that transgenic Arabidopsis thaliana can improve resistance to clubroot fungus. Figure 3B, C). To verify that the resistance of this transgenic Arabidopsis to clubroot bacteria was due to silencing of target genes within the clubroot bacteria by siRNA, we collected DNA and RNA from diseased Arabidopsis roots and detected the relative biomass of clubroot bacteria and the silencing level of PbDDX15-like genes. qPCR results showed that the relative biomass of clubroot bacteria in the roots of the transgenic Arabidopsis was significantly reduced (B, C). Figure 3 D) and the expression level of the target gene was significantly lower than that of Col-0 ( Figure 3 E), in summary, this demonstrates that silencing the target gene of *Plasmodiophora* using siRNA enhances the resistance of transgenic *Arabidopsis thaliana* to *Plasmodiophora*.

[0089] 3. Summary and Discussion

[0090] Brassica genus is one of the most important genera in the Brassicaceae family, encompassing hundreds of agricultural crops such as rapeseed, Chinese cabbage, and radish. Many of these varieties can be infected by clubroot fungus. Clubroot fungus is difficult to control due to its soil-borne transmission characteristics, and its dormant spores can survive in the soil for 8-12 years or even longer, allowing for mutation accumulation and promoting the emergence of new physiological races, making it easier to develop new resistance to chemical pesticides. Silencing the lethal gene PbDDX15-like in clubroot fungus using HIGS technology significantly enhanced resistance to clubroot fungus, indicating that PbDDX15-like can serve as a green resource for the control of clubroot disease in rapeseed.

Claims

1. DEAD-box RNA helicase encoding gene PbDDX15-like Its characteristics are, The nucleotide sequence is shown in SEQ ID NO.

1.

2. Contains the DEAD-box RNA helicase encoding gene as described in claim 1. PbDDX15-like Or a recombinant vector of its fragments.

3. The recombinant vector according to claim 2, characterized in that, The recombinant vector is constructed based on HIGSA technology. PbDDX15-like RNAi vectors for genes.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector contains the sense and antisense strands of the interfering fragment shown in SEQ ID NO.

2.

5. Suppress or silence the DEAD-box RNA helicase encoding gene as described in claim 1 PbDDX15-like Application in improving plant resistance to clubroot.

6. The application according to claim 5, characterized in that, The inhibition or silencing of the DEAD-box RNA helicase-encoding gene as described in claim 1 PbDDX15-like The methods are selected from host-induced gene silencing technology and gene editing technology.

7. The application according to claim 6, characterized in that, The plant in question is either Arabidopsis thaliana or rapeseed.

8. Suppressing or silencing the DEAD-box RNA helicase-encoding gene as described in claim 1 PbDDX15-like The application of substances in cultivating plants resistant to clubroot disease.

9. The application according to claim 8, characterized in that, The application of the recombinant vector according to claim 3 or 4 in the cultivation of plants with clubroot resistance.

10. The application according to claim 8 or 9, characterized in that, The plant in question is either Arabidopsis thaliana or rapeseed.

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