Application of cis-aconitase in prevention and control of rape clubroot

Abstract

The invention discloses an application of targeted cis-aconitase in prevention and control of clubroot of rape. According to the invention, cis-aconitase (Aconitase, Aco) with high-level expression at each stage is screened from a plasmodiophora brassicae ZJ-1 transcriptome, then an RNAi carrier of the cis-aconitase is constructed through an HIGS technology, the RNAi carrier is transformed into wild type arabidopsis thaliana, then the resistance of transgenic arabidopsis thaliana to clubroot is tested, and the result shows that PbAco has remarkable resistance to plasmodiophora brassicae, and PbAco can be used for preparing the plasmodiophora brassicae. Therefore, the cis-aconitase in the plasmodiophora brassicae can be used as a gene resource for resisting the clubroot of the cruciferae.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of aconitase in the prevention and control of rapeseed clubroot. Background Art

[0002] Rapeseed clubroot, caused by the fungus Plasmodiophora brassicae, is a soil-borne disease that primarily harms Brassica crops in the Brassicaceae family, such as rapeseed, Chinese cabbage, and cabbage. The disease infects plant roots, forming tumor-like structures that severely impair the root system's absorption and transport functions, leading to stunted plant growth, yellowing and wilting of leaves, and even complete plant death, posing a serious threat to the safe production of vegetables and oilseed crops. Host-induced gene silencing (HIGS) is an RNAi-based technique. A dsRNA or hairpin-shaped dsRNA construct targeting a specific pathogen gene is transformed into the host plant. Transgenic plants produce dsRNA and siRNA, which enter the plant pathogen during host-pathogen interactions. The siRNA degrades the pathogen's mRNA, protecting the host plant from the pathogen. In recent years, this technique has made significant progress in crop disease resistance breeding, showing promising applications, particularly in plant disease control.

[0003] Aconitase is a key enzyme in the tricarboxylic acid cycle, primarily catalyzing the reversible isomerization reaction between citrate and isocitrate. This reaction is a pivotal step in energy metabolism in the TCA cycle, connecting the oxidation of sugars, lipids, and amino acids with ATP synthesis. Furthermore, aconitase participates in the regulation of cellular iron homeostasis through its iron-sulfur clusters and isomers, making it a crucial molecule connecting energy metabolism and metal ion homeostasis.

[0004] In this study, aconitase (Aco), which was highly expressed at all stages, was screened from the transcriptome of the root-knot fungus ZJ-1. Then, its RNAi vector was constructed using the HIGS technology and transformed into wild-type Arabidopsis thaliana. The resistance of transgenic Arabidopsis to root-knot fungus was tested. The results showed that PbAco had significant resistance to Brassicae root-knot fungus, indicating that aconitase in root-knot fungus can be used as a genetic resource for resistance to crucifer root-knot disease. Summary of the Invention

[0005] In view of the shortcomings of the existing problems, the present invention aims to provide an application of aconitase in the prevention and control of rapeseed clubroot.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] In a first aspect, the present invention protects an aconitase PbAco of a root-knot fungus, wherein the aconitase PbAco is any one of the following proteins:

[0008] (C1) a protein with an amino acid sequence as shown in SEQ ID No. 2;

[0009] (C2) a fusion protein obtained by connecting a tag protein to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 2;

[0010] (C3) a protein having the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2;

[0011] (C4) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID No. 2 and having the same function.

[0012] In order to facilitate purification or detection of the protein in (C1), a tag protein may be connected to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 2.

[0013] In a specific embodiment, in (C3), the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0014] In a specific embodiment, in (C3), the protein can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0015] In a specific embodiment, in (C3), the gene encoding the protein can be obtained by deleting one or several codons for amino acid residues from the DNA sequence shown in SEQ ID No. 1, and / or performing missense mutations of one or several base pairs, and / or linking the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0016] In a specific embodiment, "homology" includes amino acid sequences having 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher homology to the amino acid sequence shown in SEQ ID No. 2 of the present invention.

[0017] In a second aspect, the present invention protects a biomaterial related to the aforementioned aconitase PbAco, wherein the biomaterial is any one of the following:

[0018] (B1) the gene encoding the aforementioned aconitase;

[0019] (B2) an expression cassette containing the gene described in (B1);

[0020] (B3) a recombinant vector containing the gene described in (B1), or a recombinant vector containing the expression cassette described in (B2);

[0021] (B4) a recombinant microorganism containing the gene described in (B1), a recombinant microorganism containing the expression cassette described in (B2), or a recombinant microorganism containing the recombinant vector described above;

[0022] (B5) a transgenic plant cell line containing the gene described in (B1), or a transgenic plant cell line containing the expression cassette described in (B2);

[0023] (B6) transgenic plant tissue containing the gene described in (B1), or transgenic plant tissue containing the expression cassette described in (B2);

[0024] (B7) A transgenic plant organ containing the gene described in (B1), or a transgenic plant organ containing the expression cassette described in (B2).

[0025] (C1) A recombinant vector for inhibiting or silencing the expression of the gene encoding aconitase described above.

[0026] In a specific embodiment, the gene (B1) is any one of the following genes:

[0027] 1) The nucleotide sequence is the gene shown in SEQ ID No. 1;

[0028] 2) a gene having 75% or more identity with the nucleotide sequence defined in 1);

[0029] 3) A gene that hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2).

[0030] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that are 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identical to the nucleotide sequences of the present invention. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences. The aforementioned 75% or higher identity can be 80%, 85%, 90% or higher identity.

[0031] The nucleic acid molecule of the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0032] The vectors described herein are well known to those skilled in the art, including but not limited to plasmids, phages (such as lambda phage or M13 filamentous phage), cosmids (i.e., cosmids), Ti plasmids, or viral vectors, and specifically, ds1301 plasmids.

[0033] The microorganism described herein can be yeast, bacteria, algae or fungi, and can specifically be Agrobacterium GV3101.

[0034] In a specific embodiment, the recombinant vector for inhibiting or silencing the expression of the gene encoding aconitase described above is an interference vector PbAco-RNAi. The CDS sequence used to construct the PbAco-RNAi vector is shown in SEQ ID No.3.

[0035] In a third aspect, the present invention also protects any of the following applications of the aforementioned aconitase or the aforementioned biomaterial:

[0036] (D1) Prevention and control of plant clubroot;

[0037] (D2) preparing products for preventing and controlling clubroot;

[0038] (D3) Cultivate plants resistant to clubroot.

[0039] In a specific embodiment, the plant is Arabidopsis thaliana or a Brassica crop of the family Cruciferae.

[0040] In a more specific embodiment, the cruciferous Brassica crop is rapeseed, cabbage, kale, etc.

[0041] In a fourth aspect, the present invention protects a method for preventing and controlling plant clubroot disease, wherein the method inhibits or silences the expression level of the aconitase described above.

[0042] In a fifth aspect, the present invention protects a method for cultivating plants resistant to clubroot, wherein the method obtains plants resistant to clubroot by inhibiting or silencing the expression level of the aconitase described above.

[0043] In a specific embodiment, the inhibition or silencing of the expression of the aforementioned aconitase is achieved by introducing the interference vector PbAco-RNAi for silencing aconitase into the recipient plant.

[0044] The recipient plant is a host plant of the root knot fungus, specifically Arabidopsis thaliana or a Brassica crop of the family Cruciferae.

[0045] In a more specific embodiment, the cruciferous Brassica crop is rapeseed, cabbage, kale, etc.

[0046] Beneficial effects

[0047] The application of aconitase provided by the present invention in the prevention and control of rapeseed clubroot has the following beneficial effects compared with the existing technology: the present invention constructs a vector of aconitase PbAco in root-knot bacteria through HIGS technology to obtain transgenic strains, and root-knot bacteria disease resistance detection finds that they have good disease resistance, indicating that root-knot bacteria aconitase PbAco can be used as a genetic resource for green prevention and control of root-knot bacteria in cruciferous Brassica crops such as rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The expression level and recombinant plasmid identification of PbAco at different stages; Figure 1 A: The expression level of PbAco gene in the life cycle of root knot fungus in transcriptome data. IN indicates the cortical infection stage, PZ indicates the zoospore stage, and RS indicates the dormant spore stage. Figure 1 B: M: DL 2000; 1: positive strand PCR detection; 2: antisense strand PCR detection; 3: KpnI and SpeI double enzyme digestion plasmid detection.

[0049] Figure 2 For screening of PbAco-RNAi transgenic materials.

[0050] Figure 3 The phenotype and disease index of PbAco-RNAi transgenic plants inoculated with root knot fungus, as well as the expression of target genes and the content of root knot fungus detected by qPCR; 3 weeks after Col-0 and two PbAco-RNAi transgenic lines were inoculated with root knot fungus (1×107 dormant spores per plant), the phenotype of the aboveground parts of the plants ( Figure 3 A) underground phenotype ( Figure 3 B) and disease index ( Figure 3 C and 3D), n = 32-36 biological replicates; the content of Rhizobium in diseased roots of Arabidopsis was quantified by qPCR. The ordinate represents the multiple of the Rhizobium content of different lines relative to Col-0. The relative biomass of Rhizobium is determined by the content of the Rhizobium ACTIN gene relative to the Arabidopsis ACTIN2 gene ( Figure 3 E); Quantification of PbAco silencing levels in diseased roots of Arabidopsis thaliana by qPCR ( Figure 3 F). DETAILED DESCRIPTION

[0051] The present invention is further described in detail below with reference to the examples. Reagents or instruments used without manufacturer's indication are considered to be conventional products that can be purchased on the market.

[0052] The present invention screened out aconitase (Aco) that is highly expressed at all stages from the ZJ-1 transcriptome of the root knot fungus, then constructed its RNAi vector using the HIGS technology, and transformed it into wild-type Arabidopsis thaliana. The resistance of the transgenic Arabidopsis to root knot disease was then tested. The results showed that PbAco had significant resistance to Brassica root knot fungus, indicating that aconitase in root knot fungus can be used as a gene resource for resistance to crucifer root knot disease. Among them, the CDS sequence of aconitase PbAco is shown in SEQ ID NO.1; the amino acid sequence is shown in SEQ ID NO.2. The following examples illustrate preferred specific embodiments of the present invention, but the present invention is not limited thereto.

[0053] Test materials

[0054] Plant material: Arabidopsis thaliana (Col-0), obtained from the laboratory. Plant culture conditions were a temperature of 22°C and a relative humidity of 75%. Plant nutrient soil formulation: Finnish Kaijila peat soil: Jiangsu Peilei substrate: vermiculite = 8:4:1. The pathogenic root knot fungus was derived from a root system infected with clubroot (Williams race 1) ZJ-1 in a rapeseed experimental field in Zhijiang, Hubei. Escherichia coli DH5α strain was obtained from this laboratory. Agrobacterium tumefaciens GV3101 was obtained from this laboratory. Plasmid vectors: ds1301 and others were obtained from this laboratory.

[0055] Example 1 Extraction of total DNA of root knotweed fungi

[0056] Add the extracted dormant spores of root knot fungus or the swollen tissue of root knot disease to a pre-cooled mortar and grind it into powder with liquid nitrogen. Transfer the powder to a 2 mL centrifuge tube and add 700 μL of 2% preheated at 65°C. Extract the buffer solution with CTAB, quickly invert it upside down to mix, place it in a 65℃ water bath for 30min, and gently invert it every 10min to mix; add 350μL of chloroform and tris-saturated phenol solution to the mixture, shake it for 1min, and centrifuge it at 12000r / min for 10min; transfer 600μL of the supernatant to a 1.5mL centrifuge tube, add an equal volume of chloroform and mix it, let it stand for stratification, take 450μL of the supernatant, add an equal volume of isopropanol, mix it, and then precipitate it at -20℃ for 10-15min; centrifuge it at 12000r / min for 10min, discard the supernatant; add 500mL of 75% ethanol to wash the precipitate, centrifuge it at 12000r / min for 3-5min, and repeat this step twice; place it in a 37℃ oven to dry, dissolve the DNA with 30-50μL of DEPC water, test the DNA purity and concentration, and store it at -20℃.

[0057] Example 2 Total RNA Extraction of Plasmodium

[0058] Add clubroot swollen tissue to a pre-cooled mortar and pestle, grind it thoroughly into powder with liquid nitrogen, transfer the powder to a 2 mL RNase-free centrifuge tube, add 1 mL Trizol, vortex and mix immediately, and let it stand on ice for 10 minutes; add 200 μL of chloroform extraction, mix by inversion, and let it stand on ice for 15 minutes; centrifuge at 12000 r / min at 4°C for 15 minutes; transfer 600 μL of the upper aqueous phase to a new enzyme-free centrifuge tube, add an equal volume of pre-cooled isopropanol, vortex and mix, ice bath for 5 minutes, and settle at -20°C for 10 minutes; centrifuge at 12000 r / min at 4°C for 10 minutes and discard the supernatant; wash the precipitate with 75% ethanol prepared with DEPC water, centrifuge at 12000 r / min at 4°C for 5 minutes, discard the supernatant, and repeat this step twice; aspirate the residual liquid, let it stand at room temperature for 5-10 minutes to evaporate the residual alcohol; add 30-50 μL RNA was dissolved in DEPC water, and the purity and concentration of RNA were tested and stored at -20°C.

[0059] Example 3 cDNA Synthesis

[0060] After extracting high-quality RNA from Rhizoctonia solani, prepare a 20 μL reverse transcription system using an enzyme-free PCR tube: 1 μg to 1 μg of total RNA, 4 μL of 5× All-in-one qRT SuperMix, 1 μL of Enzyme Mix, and RNase-free water to a total volume of 20 μL. The reaction procedure is: incubate at 42°C for 15 minutes and heat inactivate at 85°C for 5 seconds to obtain the target cDNA.

[0061] Example 4 Target gene cloning

[0062] Selection of target gene fragments: A CDS sequence of approximately 300-500 bp was selected as the interference sequence. The fragment was compared at NCBI (https: / / www.ncbi.nlm.nih.gov / ) to ensure interference specificity. TAIR (https: / / www.arabidopsis.org / ) was used to confirm the absence of corresponding interference sites in Arabidopsis to avoid off-target effects. The CDS sequence used to construct the PbAco-RNAi vector is shown in SEQ ID NO. 3.

[0063] Primer design: The primers for the homology arms of the restriction enzyme sites were automatically generated using the Novagen entry cloning website (http: / / crm.vazyme.com. / cetool / simple.htm), and other conventional primers were independently designed with the help of Snapgene software.

[0064] PCR amplification of target genes: The enzymes, dNTPs, and other reagents used for gene amplification in this experiment were purchased from Takara Biotechnology. A 50 μL PCR system was prepared using DNA / cDNA as a template by adding 2 μL of DNA / cDNA, 10 μL of 2× TransStart FastPfu Buffer, 4 μL of 2.5 mM dNTPs, 1 μL of each Primer F / R, 1 μL of TransStart FastPfu DNA Polymerase, and 31 μL of ddH2O.

[0065] The PCR reaction procedure was as follows: initial denaturation at 95°C for 2 minutes, denaturation at 95°C for 20 seconds, annealing at 59°C for 20 seconds, extension at 72°C for 1 minute, 35 cycles followed by a further extension at 72°C for 5 minutes, and cooling at 16°C for 2 minutes before termination. PCR products were electrophoresed on a 1.2% agarose gel to determine the size of the target gene amplified, and bands of consistent target size were recovered from the gel (see the Omega DNA Agarose Gel Electrophoresis Recovery Kit for detailed procedures).

[0066] Example 5 Escherichia coli plasmid extraction

[0067] Single colonies were picked and placed in LB liquid medium containing Kana resistance, shaken and cultured at 37°C 220 r / min overnight, and 2 mL of bacterial solution was collected into a 2 mL EP tube; centrifuged at 12000 r / min at room temperature for 1 min, and the supernatant was discarded; 200 μL of solution I (10 mM EDTA, 25 mM Tris-HCl, 10 mM Glucose) was added and mixed by vortexing; 200 μL of solution II (200 mM NaOH, 1% SDS), gently invert up and down 10-20 times, let it stand for 2 minutes; then add 200 μL of solution III (29.442 g KAc, 11.5 mL anhydrous acetic acid, dilute to 100 mL), invert and mix; add 400 μL of chloroform, invert and mix, and centrifuge at 12,000 rpm for 10 minutes; transfer 650 μL of the supernatant to a new EP tube, add an equal volume of isopropanol, mix, and place it at -20°C for 10 minutes; centrifuge at 12,000 rpm for 10 minutes and discard the supernatant; add 500 μL of 75% ethanol to wash the precipitate, centrifuge at 12,000 rpm for 5 minutes, discard the supernatant, and repeat this step twice; place the centrifuge tube back into the centrifuge for a short centrifuge of 30 seconds, remove the remaining liquid with a pipette, and place it in an oven or at room temperature to dry until the precipitate becomes transparent, add 30-50 μL of RNase-free water to dissolve the precipitate, and store at -20°C.

[0068] Example 6 RNAi expression vector construction

[0069] Sense strand homologous recombination:

[0070] The empty vector ds1301 plasmid was double-digested with the restriction endonucleases BamHI and KpnI. The reaction mixture was prepared as follows: 20 μL of ds1301 vector plasmid, 5 μL of 10× rCutSmart Buffer, 1 μL of BamHI enzyme, 1 μL of KpnI enzyme, and 23 μL of ddH2O. After preparation, the mixture was briefly centrifuged and placed in a 37°C water bath for 30 minutes. After digestion, the plasmid was confirmed by electrophoresis on a 1.2% agarose gel and the gel was recovered.

[0071] The target gene and vector were then linked via homologous recombination. The reaction system was prepared as follows: 3 μL of double-digested ds1301 fragment, 1 μL of target gene fragment, 1 μL of Exnase II Mix homologous recombination enzyme, 2 μL of 5× CE II Buffer, and 3 μL of ddH2O. After preparation, the system was briefly centrifuged and the PCR reaction was performed at 37°C for 30 minutes.

[0072] Heat shock transformation of E. coli:

[0073] The competent cells of Escherichia coli DH5α were taken out from -80℃ and placed on ice to thaw. 10μL of the product after the recombination reaction was completed was added to 40μL of the competent cells and gently pipetted to mix. After ice bath for 30min, heat shock was performed at 42℃ for 90sec, and ice bath was performed again for 2-3min. Then 800μL of antibiotic-free LB liquid culture medium was added and placed at 37℃, shaken at 220r / min for 30min, centrifuged at 5000r / min at room temperature for 5min, the supernatant was poured out, and the bacteria were resuspended with the remaining culture medium. Finally, it was spread on the LB plate containing Kana resistance. After drying, it was sealed completely and placed upside down at 37℃ incubator for overnight culture.

[0074] Among them, PbAco positive chain primer:

[0075] p-PbAco-F: 5'-cccgtgcagctgcggggtaccCGATTCTGGCTCGAGCAAAGT-3', as shown in SEQ IDNO.4;

[0076] p-PbAco-R: 5'-cgcgtacgtaaggttggatccCCGTCAGGTGGACACCGA-3', as shown in SEQ ID NO.5;

[0077] PCR verification of positive transformants:

[0078] Single colonies from eight plates were randomly selected and cultured overnight in Kana-resistant LB medium at 37°C on a shaker. Fresh bacterial culture was used as template, and the reaction system was prepared as follows: 1 μL of fresh bacterial culture, 0.5 μL of each Primer F / R, and 5 μL of 2× Rapid Taq Mix. The PCR reaction program was as follows: denaturation at 95°C for 5 min, melting at 95°C for 20 sec, annealing at 59°C for 10 sec, and extension at 72°C for 15 sec, repeated 32 times, followed by extension at 72°C for 2 min. PCR products were analyzed by electrophoresis on a 1.2% agarose gel, and cultures showing the desired band were sequenced. Verified cultures were amplified, mixed with 1 mL of fresh culture in 40% glycerol, and stored frozen at -20°C.

[0079] Recombination of intermediate vector and antisense strand:

[0080] Double-digest the intermediate vector with the restriction endonucleases SacI and SpeI. Prepare the following reaction mixture: 20 μL of the intermediate vector, 5 μL of 10× rCutSmart Buffer, 1 μL of SacI, 1 μL of SpeI, and 23 μL of ddH2O. Briefly centrifuge the mixture and place it in a 37°C water bath for 30 minutes. After digestion, perform electrophoresis on a 1.2% agarose gel to verify plasmid cleavage and recover the plasmid.

[0081] The intermediate vector and antisense strand were then linked via homologous recombination. The following reaction system was prepared: 3 μL of double-digested intermediate vector, 1 μL of antisense strand gene fragment, 1 μL of Exnase II homologous recombination enzyme, 1 μL of CE II buffer, and 5 μL of dd H2O. After brief centrifugation, the PCR reaction was performed at 37°C for 30 minutes.

[0082] Among them, PbAco antisense strand primer:

[0083] o-PbAco-F: 5′-caattcaattcagtggagctcCCGTCAGGTGGACACCGA-3′, as shown in SEQ ID NO. 6;

[0084] o-PbAco-R: 5'-caggactctagaccccactagtCGATTCTGGCTCGAGCAAAGT-3', as shown in SEQ ID NO.7.

[0085] Example 7 Agrobacterium transformation

[0086] Take out the Agrobacterium competent cells GV3101 from -80℃ and place them on ice for about 5-10 minutes to thaw them. Then add the plasmid to be transformed into 50 μL competent cells, mix them by gently pipetting, and let them stand on ice for 10 minutes, in liquid nitrogen for 5 minutes, in a 37℃ water bath for 5 minutes, and in an ice bath for 5 minutes. Then add 800 μL of antibiotic-free LB liquid culture medium and place it at 28℃, shake it at 200 r / min for 2-3 hours, centrifuge it at 6000 r / min at room temperature for 1 minute, discard the supernatant, and leave about 100 μL of bacterial liquid at the bottom. Gently blow to resuspend the precipitate, and finally spread it on an LB plate containing Kana and Rif resistance. After drying, seal it completely and place it upside down at 28℃ incubator for 2 days.

[0087] Example 8 Agrobacterium-mediated transformation of Arabidopsis thaliana (floral dip method)

[0088] This experiment used the floral dipping method to transform Arabidopsis thaliana. Agrobacterium containing the target gene vector was cultured on a shaker at 28°C, 200 rpm, for 12-16 hours to obtain 50 mL of fresh Agrobacterium culture. The culture was centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded. The Agrobacterium cells were collected and suspended in 100 mL of a 5% (w / v) sucrose solution. 20 μL of the surfactant Silwet L-77 was added to a final concentration of 0.02%. Well-grown Arabidopsis plants were selected, and all white flowers and fruit pods were removed. The cut Arabidopsis inflorescences were immersed in the Agrobacterium culture solution and gently shaken for approximately 15-30 seconds. This procedure was repeated once after 1-3 hours. The treated Arabidopsis plants were incubated in the dark, moisturized, and then transferred to a normal culture environment. After 7-10 days, the above procedure was repeated once more to complete the transformation. Mature Arabidopsis seeds were collected and stored for later use.

[0089] Example 9 Screening of transgenic plants

[0090] T0 generation Arabidopsis seeds, harvested by dipping flowers, were placed in a 1.5 mL centrifuge tube and disinfected with 84 disinfectant: sterile water at a ratio of 1:10. Mix thoroughly by inverting the tubes. After 10 minutes, the disinfectant was discarded and the seeds were washed 6–7 times with sterile water to ensure complete removal of the disinfectant. 1 mL of sterile water was added to the washed seeds and vernalized at 4°C for 2 days. 1 / 2 MS plates containing hygromycin resistance were prepared and the vernalized seeds were evenly spread on them. After air drying, the plates were sealed and placed in a culture chamber for incubation. After 8–11 days, the growth of the Arabidopsis plants was observed, and seedlings showing significantly better growth than the others were selected and transplanted to nutrient soil. When the plants were approximately 4 weeks old, DNA from the transgenic plants was extracted using the CTAB method and amplified using specific primers to verify whether the plants were transformants.

[0091] Example 10 Statistics and analysis of the incidence of transgenic materials

[0092] Pipette 1mL of a 1×10 7 spores / mL of dormant root knotweed fungi were inoculated onto the roots of 10-day-old transgenic plants. Three weeks later, the disease index was calculated. The transgenic plants were removed from the soil with tweezers, minimizing root damage. After rinsing the soil off the roots, the clubroot disease index was calculated. Disease index = 100 × ∑ (number of diseased roots at each level × representative value for each level) / (total number of roots surveyed × highest representative value). Control efficacy (%) = (1 - disease index of transgenic plants / disease index of controls) × 100. The clubroot disease index is graded from 0 to 5: Grade 0: Good plant growth, no tumors; Grade 1: Small tumors on lateral roots, not affecting main root growth; Grade 2: Small tumors on main root; Grade 3: Moderate tumors on both main and lateral roots; Grade 4: Large tumors on both main and lateral roots, with a small number of lateral roots; Grade 5: Large or blackened and rotten main roots, with few or no lateral roots, severely impacting plant growth or even causing death.

[0093] Example 11 Real-time fluorescence quantitative PCR (Quantitative PCR, qPCR)

[0094] Real-time fluorescence quantitative PCR was performed using the SYBR Green method. TM Universal Green Supermix was used. Quantitative primers were designed using Beacon Designer 8.0 Real-time PCR Primer Design Software. RNA was extracted from the samples, and 2 μg of RNA was reverse transcribed into cDNA.

[0095] The real-time fluorescence quantitative PCR (15 μL) reaction system is as follows:

[0096] Component iTaq TM Universal Green supermix (2X) 7.5μL, Forward primers (10μM) 0.5μL, Reverse primers (10μM) 0.5μL, cDNA 0.5μL, dd H2O 6μL.

[0097] Reaction conditions: 95°C, 10 min; 95°C, 15 sec, 57°C, 15 sec repeated 45 times; 65°C to 95°C, 0.5°C rise per cycle, 5 sec, 16°C for 1 min. The reaction was performed on a Bio-Rad CFX 96 instrument. After the amplification was completed, 2 -ΔΔCtMethods The relative expression of genes was calculated.

[0098] Among them, the primers used for PbAco are:

[0099] qPCR-PbAco-F: 5′-CGCCGGTATCCTGACTGT-3′, as shown in SEQ ID NO. 10;

[0100] qPCR-PbAco-R: 5′-GGCGCTCGTTAAACGGG-3′, as shown in SEQ ID NO.11.

[0101] The primers for the ACTIN gene of Plasmodium falciparum are:

[0102] Pbactin_qF: 5′-CACCGACTACCTGATGAA-3′, as shown in SEQ ID NO.12.

[0103] Pbactin_qR: 5′-CAGCTTCTCCTTGATGTC-3′, as shown in SEQ ID NO.13.

[0104] The primers for the Arabidopsis thaliana ACTIN2 gene are:

[0105] ACTIN2_qF: 5′-GCACCCTGTTCTTCTTACGGA-3′, as shown in SEQ ID NO.14.

[0106] ACTIN2_qR: 5′-GTGAGACACACCATCACCAGA-3′, as shown in SEQ ID NO.15.

[0107] Here are the results:

[0108] 1. Construction of PbAco-RNAi vector

[0109] With reference to the methods of Examples 1 and 2, RNA from root-knot bacteria was extracted and reverse transcribed into cDNA. A partial fragment of aconitase PbAco was amplified using the cDNA template. According to the methods of Examples 4 and 6, a 300-500 bp targeted gene fragment was inserted into a vector containing a hairpin structure to construct an RNAi vector. In order to detect whether the RNAi vector was successfully constructed, positive identification was performed using specific primers for the sense and antisense strands, respectively, and the recombinant plasmid was double-digested with KpnI and SpeI for detection ( Figure 1 ), and successfully constructed the HIGS RNAi vector.

[0110] Among them, the primers for positive chain PCR detection and PbAco-RNAi vector identification are:

[0111] V-1-F: 5′-cgttgagtggccctgtttctc-3′, as shown in SEQ ID NO. 8;

[0112] p-PbAco-R: 5'-cgcgtacgtaaggttggatccCCGTCAGGTGGACACCGA-3', as shown in SEQ ID NO.5.

[0113] Antisense strand PCR detection and primers for PbAco-RNAi vector identification are:

[0114] V-2-F: 5′-gcttcaaattctaatccccaa-3′, as shown in SEQ ID NO. 9;

[0115] o-PbAco-R: 5'-caggactctagaccccactagtCGATTCTGGCTCGAGCAAAGT-3', as shown in SEQ ID NO.7.

[0116] 2. Creation of PbAco-RNAi Transgenic Arabidopsis

[0117] Referring to the method of Example 7, the HIGS RNAi vector was introduced into wild-type Arabidopsis Col-0 by Agrobacterium transformation to obtain T0 transgenic Arabidopsis seeds. After preliminary screening using 1 / 2MS plates containing hygromycin resistance, normally growing plants were transferred to pots. DNA was extracted from leaves of plants about 4 weeks old, and positive plants were identified using PCR with specific primers ( Figure 2 A), the results showed that 16 PbAco-RNAi transgenic plants were obtained. The seeds of the transgenic plants with positive identification results were collected and screened again with hygromycin to obtain stable genetic T2 generation transgenic seeds, such as Figure 2 As shown in B, the growth phenotype of these transgenic plants is the same as that of wild-type Arabidopsis, and does not affect the normal growth and development of Arabidopsis.

[0118] Among them, DNA of 16 PbAco-RNAi T1 generation HIGS transgenic materials was extracted and identified using specific PCR primers. The primers used for PbAco-RNAi were: V-2-F: 5'-gcttcaaattctaatccccaa-3' (SEQ ID NO. 9) and o-PbAco-R: 5'-caggactctagacccactagtCGATCTGGCTCGAGCAAAGT-3' (SEQ ID NO. 7).

[0119] 3. PbAco-RNAi transgenic plants can improve Arabidopsis resistance to clubroot

[0120] After obtaining the transgenic plants, a pot experiment was conducted to verify the resistance of the transgenic materials to clubroot. After the plants grew for 2 weeks, the roots were inoculated with clubroot fungi. After 5 weeks of growth, the growth phenotype and root phenotype of the plants were observed. Figure 3 As shown in A, the roots of wild-type Col-0 were obviously diseased, with the main and lateral roots swollen and the fibrous roots very short. Disease levels 3 and 4 accounted for 15% of the total, and the disease index was 32.8 ( Figure 3 B), while all HIGS-transgenic Arabidopsis plants showed significantly reduced root swelling symptoms, with slightly swollen taproots and lateral roots and long fibrous roots. The disease index of the PbAco-RNAi-1 and PbAco-RNAi-3 transgenic lines was 18.01 and 12.5, respectively, representing control efficacy of 45.0% and 61.9%, respectively. These results indicate that the disease index of HIGS-transgenic materials was significantly lower than that of the wild type, demonstrating improved resistance to crucifer clubroot.

[0121] In summary, the present invention constructs a vector of aconitase PbAco in root-knot fungus through HIGS technology to obtain transgenic strains. Through root-knot fungus disease resistance detection, it is found that they have good disease resistance, indicating that root-knot fungus aconitase PbAco can be used as a genetic resource for green prevention and control of rapeseed clubroot disease.

[0122] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. Aconitase from a root-knot fungus PbAco, Aconitase PbAco Any of the following proteins: (C1) a protein having an amino acid sequence as shown in SEQ ID No. 2; (C2) a fusion protein obtained by connecting a tag protein to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 2; (C3) a protein having the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2; (C4) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID No. 2 and having the same function.

2. Aconitase according to claim 1 PbAco Related biological materials, the biological material is any one of the following: (B1) the gene encoding the aconitase described above; (B2) an expression cassette containing the gene described in (B1); (B3) a recombinant vector containing the gene described in (B1) or a recombinant vector containing the expression cassette described in (B2); (B4) a recombinant microorganism containing the gene described in (B1), or a recombinant microorganism containing the expression cassette described in (B2), or a recombinant microorganism containing the recombinant vector described; (B5) A transgenic plant cell line containing the gene described in (B1), or a transgenic plant cell line containing the expression cassette described in (B2); (B6) transgenic plant tissue containing the gene described in (B1), or transgenic plant tissue containing the expression cassette described in (B2); (B7) A transgenic plant organ containing the gene described in (B1), or a transgenic plant organ containing the expression cassette described in (B2); (C1) A recombinant vector for inhibiting or silencing the expression of the gene encoding the aconitase according to claim 1.

3. The biomaterial according to claim 2, characterized in that (B1) The gene is any of the following genes: 1) The nucleotide sequence is the gene shown in SEQ ID No. 1; 2) A gene having 75% or more identity with the nucleotide sequence defined in 1); 3) A gene that hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2).

4. The biomaterial according to claim 2, wherein The recombinant vector for inhibiting or silencing the expression of the gene encoding aconitase according to claim 1 is an interference vector PbAco-RNAi, which is constructed PbAco The CDS sequence used in the -RNAi vector is shown in SEQ ID No.

3.

5. Use of the aconitase according to claim 1 or the biomaterial according to any one of claims 2 to 4 in any of the following applications: (D1) Prevention and control of plant clubroot; (D2) Preparation of products for the prevention and control of clubroot; (D3) Cultivate plants that are resistant to clubroot.

6. The use according to claim 5, characterized in that The plant is Arabidopsis thaliana or a Brassica crop of the family Cruciferae.

7. A method for preventing and controlling clubroot disease in plants, characterized in that: The method is achieved by inhibiting or silencing the expression level of the aconitase according to claim 1.

8. A method for cultivating plants resistant to clubroot, characterized in that: The method is achieved by inhibiting or silencing the expression level of the aconitase according to claim 1.

9. The method according to claim 7 or 8, characterized in that The inhibition or silencing of the expression of the aconitase according to claim 1 is achieved by introducing the interference vector PbAco-RNAi for silencing aconitase into the recipient plant.

10. The method according to claim 9, wherein The CDS sequence used in the interference vector PbAco-RNAi is shown in SEQ ID No.3.

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