A root knot fungus resistance gene and its application

Abstract

The present invention discloses a root-knot resistance gene and its application, and relates to the field of gene engineering technology. The nucleotide sequence of the root-knot resistance gene is shown in SEQ ID No. 1. The present invention has found that the gene has a resistance function to the Chinese root-knot bacteria No. 4 physiological race, and through the transformation system mediated by Agrobacterium rhizogenes, the root overexpression is verified with the cabbage-type rapeseed Huashuang No. 5 as the background, thereby verifying the resistance function of the gene to the root-knot bacteria. The present invention also provides the application of the gene in basic research on resistance to root-knot bacteria disease, disease-resistant breeding and molecular marker-assisted selection. By breeding varieties resistant to root-knot bacteria through the localization interval where the gene is located, the breeding process can be shortened.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, in particular to a root knotweed resistance gene and application thereof. Background Art

[0002] Clubroot is a soil-borne disease caused by Brassicae root-knot fungus and a parasite of the crucifer family. It is highly contagious and poses a serious threat to the production safety of cruciferous crops. Brassicae root-knot fungus was first considered a fungus, but its division and growth and development states are clearly different from those of fungi. With the development of sequencing technology, researchers have classified it as Brassicae root-knot fungus of the genus Brassicae in the protist Foraminifera kingdom.

[0003] Cruciferous crops include many species such as rapeseed, radish, cabbage, Chinese cabbage, mustard and other crops, which occupy a very important position in the field of cash crops in my country. With the continuous spread of root-knot fungi, root-knot disease occurs in most parts of my country. After root-knot fungi infect cruciferous plants, the nutrients originally used by the plants for their own growth and development will be consumed by pathogens. At the same time, it causes swelling of the roots of the plants, affects the life activities of the root system, causes the above-ground parts of the plants to gradually slow down and wither, and eventually leads to a decrease in crop yields or even a complete failure. The dormant spores of root-knot fungi can survive in the field for up to 20 years. At present, there are traces of root-knot disease in more than 60 countries and regions in the world, causing a global yield loss of 10%-15%. In my country, more than 3.2-4 million hectares of farmland are contaminated each year, and the yield loss reaches 20-30%. In addition to affecting yield, studies have shown that root-knot disease has an adverse effect on the quality of crops. For example, the oil content of seeds of infected plants is reduced by about 2%-6%. Therefore, formulating reasonable and effective prevention and control measures is of great significance to the production safety of cruciferous crops.

[0004] Breeding resistant varieties to clubroot is considered one of the most cost-effective and environmentally friendly measures. As early as the 1990s, the screening of disease-resistant resources in the Cruciferae family began internationally. Due to the limited resistance resources in Brassica napus, researchers simultaneously screened the resistance resources of Brassica rapa (AA), Cabbage (CC) and Raphanus sativus (RR), and transferred the resistance to Brassica napus through breeding, interspecific hybridization or synthesis of new species. At present, the mining of CR sites is mainly concentrated in the A, B and C genomes of Brassica and the R genome of Raphanus sativus, and resistance sites have been identified in all of them.

[0005] Agrobacterium rhizogenes, also known as root-generating rhizobia, is a soil bacterium of the Rhizobium family that can induce plant wounds to produce hairy roots. The Ti plasmid of Agrobacterium rhizogenes carries the rol gene with dominant root-inducing properties, which can quickly insert and integrate the target gene into the host plant chromosome by infecting plant wounds. Since the root-generating transformation system is reliable, reproducible, and efficient, the transformation system mediated by Agrobacterium rhizogenes has been widely used in gene function research in recent years, which has laid a good foundation for the cloning and functional verification of anti-clump knot disease genes. Summary of the invention

[0006] The purpose of the present invention is to provide a root-knot bacteria resistance gene and its application to solve the problems existing in the above-mentioned prior art. The gene has a resistance function to root-knot bacteria and can be used to cultivate plant varieties resistant to root-knot bacteria.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an anti-root-knot fungus gene, and the nucleotide sequence of the anti-root-knot fungus gene is shown as SEQ ID No.1.

[0009] The present invention also provides an anti-root-knot bacteria protein, the amino acid sequence of the protein is shown in SEQ ID No.2.

[0010] The present invention also provides a recombinant vector, comprising the above-mentioned root knot fungus resistance gene.

[0011] The present invention also provides a host cell, comprising the above-mentioned recombinant vector.

[0012] Furthermore, the initial strain of the host cell is Agrobacterium rhizogenes K599.

[0013] The present invention also provides the use of the above-mentioned root-knot fungus resistance gene, recombinant vector or host cell in improving the root-knot fungus resistance of plants.

[0014] Furthermore, the plant is a plant of the genus Brassica of the family Cruciferae.

[0015] The present invention also provides a method for improving the resistance of plants to root-knot fungi, comprising the steps of transforming the root-knot fungi resistance gene into the plant to obtain a transgenic plant over-expressing the gene.

[0016] Furthermore, the plant is a plant of the genus Brassica of the family Cruciferae.

[0017] The present invention also provides an application of primers for detecting the above-mentioned root-knot fungus resistance gene in detecting the root-knot fungus resistance of a plant. The higher the expression level of the root-knot fungus resistance gene in the plant, the higher the root-knot fungus resistance of the plant.

[0018] The present invention discloses the following technical effects:

[0019] The invention discloses that the ResyR05g6066 gene derived from radish-kale LJ has a resistance function to Chinese root knot fungus No. 4 physiological race, and through a transformation system mediated by Agrobacterium rhizogenes, the root overexpression is verified with Brassica napus Huashuang No. 5 as the background, thereby verifying the resistance function of the ResyR05g6066 gene to root knot fungus. The invention also provides the application of the ResyR05g6066 gene in basic research on resistance to root knot fungus disease, disease-resistant breeding and molecular marker-assisted selection, and breeding varieties resistant to root knot fungus can be bred through the localization interval where the gene is located, which can shorten the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The figure shows the cloning pattern of ResyR05g6066 gene in pCAMBIA1305.3 plant expression vector;

[0022] Figure 2 The figure is an agarose gel image after full-length PCR amplification of the ResyR05g6066 gene; M is DL 5000 DNA Ladder, 1-2 is the amplified target band, and the band size is 2469 bp;

[0023] Figure 3 This is an agarose gel image of ResyR05g6066 positive clone detection, where M is DL 2000DNAMarker, 1-24 are colony detection bands, and the band size is 840bp;

[0024] Figure 4 This is the phenotype of the dominant physiological race 4 of Chinese root knotweed fungus after the ResyR05g6066 gene was overexpressed in the receptor material Huashuang 5 of Brassica napus. Huashuang 5 was used as the susceptible control and Huashuang 5R as the resistant control. From left to right, they are Huashuang 5R (H5R), Huashuang 5 (H5S), grade 0 and grade 3;

[0025] Figure 5 This is an agarose gel image of the target gene detection in the root system of Huashuang No. 5 plant transformed with ResyR05g6066 gene; M is DL 2000DNAMarker; N is the negative control; P is the positive control; 1-12 are transformed plants, and the target fragment size is 840bp;

[0026] Figure 6 This is the Q-PCR statistical chart of the relative expression of the target gene in the roots of ResyR05g6066-transformed plants. 0-1, 0-2, 0-3 and 0-4 are disease-resistant plants, 2-1, 2-2, 2-3 and 2-4 are susceptible level 2 plants, 3-1, 3-2, 3-3 and 3-4 are susceptible level 3 plants, and H5S-1, H5S-2 and H5S-3 are susceptible controls. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The physical position of the ResyR05g6066 gene referred to in the present invention on the R05 chromosome is 45720529-45722967bp. The ResyR05g6066 gene can exert its resistance function after overexpression in the root, and endow the susceptible material Huashuang No. 5 with the ability to resist root knot fungi. The nucleotide sequence of the ResyR05g6066 gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2, which has a typical RLP domain and has the function of resisting root knot fungi.

[0033] SEQ ID No.1:

[0034]

[0035] SEQ ID No.2:

[0036] MQYLWRDLSFFLFSFFLIPQLSFSCPPDQRQSLLEFKNLLTQAIKNHSTADLGGLKTWRPNSDCCKWQLVRCNTRSLQVIELNLNSLVLSGSVSSSLLRPVMRINSLAMLDVSSNYMQGEIPGDGFANLTRLISLDMSSNIFNGSIPPQLFSLKTLERLDLSMNVIGGTLSGEIKALKNLHELVLEENLIEGEIPPEIGSLTELRKLSLKNNKFSGPIPLSLSKLTNLEVLDLQSNSISFGIPEGIGSLVNLSTLSLSRNNLSGGIPSSFQKLANLKTLELENNHGLSGEIPTWLFSLEKLKILRLGGNKLQWNKNGSVSALSKLTHLSLRSCGLEGNIPDWLKNQTSLLFLDLSMNRLEGSFPKWLADLKIGTIIMSDNRLSGSLPPSLFQTFNLSVLSLSRNNFSGQIPDTIGETAVMILMLSENNFSGPVPKSITMVYRLLLLDLSKNKLSGEFPRFSPLSLLAVLDISSNEFSGDVPASFGVTTIMLSMSQNNFSGEFPQDFKNLPFLMHLDLHDNRISGGIPSGLSPSLEVLSLRNNSIKGSIPEDISNLTSLKVLDLSDNELDGSLPSSLGNLPGMIVSPTSNSSSFSFVLSFSIEIQDLAVNWKNSKQGLSNRNLYLYTLLDLSKNKLQGELPASLGNLRSLKLLNLSYNDISGLIPKSFGGLEKIEVLDLSHNNLSGEIPKTLSNLGELNTLGLSNNKLTGPIPESPQLDRLNDPNIYANNSKLCGMQIQVPCFNNATQTEPAEEEEEDESFFSWKAALIGCPCGFLVAVSVMYLVGYFSRPPPPPRRSLKNREGFKKMKTSSP。

[0037] Example 1

[0038] 1. Cloning of the target gene and construction of the expression vector

[0039] The ResyR05g6066 gene was cloned and the target gene was amplified by PCR. Since there were a lot of A bases near the ResyR05g6066 gene, the front and back sequences were amplified by 30 bp in total. The primer sequences used to amplify the ResyR05g6066 genome were:

[0040] Forward primer (SEQ ID No. 3): 5′-cacaaaacaaaaagcctgaga-3′;

[0041] Reverse primer (SEQ ID No. 4): 5'-gtctcaaggagacgaagtttt-3'.

[0042] The modified pCAMBIA1305 (CaMV35S promoter and terminator sequence were inserted into pCAMBIA1305, and the GUS gene was transformed into red3, and finally named pCAMBIA1305.3) plant expression vector was used as the backbone to construct an expression vector driven by CaMV35S for the target gene ( Figure 1 ), and finally transferred into K599 Agrobacterium competent cells, as detailed below:

[0043] The ResyR05g6066 gene was amplified using the genome of the donor material Raphanus sativus-Brassica alboglabra LJ (published in the following document: Chen HG and Wu JS. Characterization of fertile amphidiploid between Raphanus sativus and Brassica alboglabra and the crossability with Brassica species. Genetic Resources and Crop Evolution, 2007b, 55: 143-150) as a template.

[0044] The PCR reaction amplification primers consist of three parts: the homology arm of the pCAMBIA1305.3 vector, the restriction site, and the homology sequence of the target gene, which is convenient for subsequent gene cloning operations.

[0045] Forward primer sequence (SEQ ID No.5):

[0046] 5'-agaggacacgaattctctagacacaaaacaaaaagcctgaga-3';

[0047] Reverse primer sequence (SEQ ID No.6):

[0048] 5'-cgcctcgagcccgggtctagagtctcaaggagacgaagtttt-3'.

[0049] The CTAB method was used to extract DNA from radish-kale LJ, and Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novozyme Biotechnology Co., Ltd.) was used to amplify the target fragment.

[0050] 50μL reaction system: 2×Phanta Max Buffer 25μL, forward primer (10μM) 2μL, reverse primer (10μM) 2μL, dNTPMix (10mM each) 1μL, DNA template 2μL and ddH 2 O 18 μL.

[0051] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 2.5 min, 34 cycles; extension at 72°C for 5 min; 25°C for 5 min.

[0052] The amplified product was detected by 1% agarose gel electrophoresis, and the expected band size was 2469 bp ( Figure 2 ).

[0053] The amplified target fragment was recovered using a DNA Purification Kit (Tiangen Biochemical Technology Co., Ltd.) to recover the PCR stock solution and measure the concentration of the target fragment after purification and recovery.

[0054] The pCAMBIA1305.3 plant expression vector was digested with enzymes.

[0055] Enzyme digestion system: 1 buffer 6μL, plasmid DNA 50μL, XbaⅠFastDigest enzyme(s) 1μL and ddH 2 2 μL of HO.

[0056] The reaction solution was centrifuged and mixed, and placed in a 37°C water bath for 30 minutes. After the reaction, 1% agarose gel electrophoresis was performed, and the gel was cut and recovered using a DNA Purification Kit (Tiangen Biochemical Technology Co., Ltd.) to measure the concentration.

[0057] The target gene fragment and the enzyme-cut plant expression vector were connected using ClonExpress II One Step Cloning Kit (Nanjing Novogene Biotechnology Co., Ltd.).

[0058] Transform the ligated product into DH5α E. coli competent cells:

[0059] a. Thaw cloning competent cells on ice;

[0060] b. Take 10 μL of the recombinant product and add it to 100 μL of competent cells, flick the tube wall to mix (do not oscillate to mix), and let it stand on ice for 30 minutes;

[0061] c. After heat shock at 42℃ water bath for 45s, immediately place on ice to cool for 5min;

[0062] d. Add 700 μL LB medium (without antibiotics) and shake at 37°C for 1 h (speed 200 r / min);

[0063] e. Preheat the Kana-resistant LB plate solid culture medium in a 37°C incubator;

[0064] f. Centrifuge at 5000 rpm for 5 min, discard 600 μL of supernatant, resuspend the remaining cells, and spread them evenly on a plate containing Kana resistance using a sterile spreader stick;

[0065] g. Incubate inverted in a 37°C incubator for 16 hours.

[0066] For positive identification of monoclones, pick a single clone on the transformation plate and perform positive identification of bacterial solution PCR.

[0067] Positive detection forward primer sequence (SEQ ID No. 7): 5′-caagatctctctgccgacagt-3′;

[0068] Positive detection reverse primer sequence (SEQ ID No. 8): 5′-cctatctctggaggaatctc-3′;

[0069] Bacterial liquid PCR 12μL reaction system: 2×Taq Master Mix (Dye Plus) 6μL, forward primer (10μM) 1μL, reverse primer (10μM) 1μL, bacterial liquid 2μL and ddH 2 2 μL of HO.

[0070] The PCR reaction conditions of the bacterial solution were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 1 min, 34 cycles; extension at 72°C for 5 min; and 25°C for 5 min.

[0071] The PCR reaction product was detected by 1% agarose gel electrophoresis, and the band at 840 bp was a positive single clone ( Figure 3 ), and the transformed DH5α bacterial solution of the positive clones was directly sent to Wuhan Qingke Biotechnology Co., Ltd. for first-generation sequencing.

[0072] The clones whose sequencing results were completely consistent with the genome sequence were selected for expansion and culture, and the plasmids were extracted using TIANprep RapidMini Plasmid Kit (Tiangen Biochemical Technology Co., Ltd.). The plasmids were transformed into Agrobacterium rhizogenes K599, and the specific steps were as follows:

[0073] Take K599 competent cells and melt them on ice. Add 1 μg of recombinant plasmid to 100 μL competent cells, place on ice for 30 minutes, transfer to liquid nitrogen and place for 5 minutes, place in a 37°C water bath for 5 minutes, place on ice for 5 minutes, add 400 μL of non-antibiotic LB, and incubate on a shaker at 28°C for 1 hour; take 100 μL of transformation product and spread it on a resistant LB plate with Kana and Str, and culture at 28°C for 36 hours. Pick a single clone for detection, and the primers and test steps used are consistent with the above positive identification of E. coli.

[0074] 2. Agrobacterium rhizogenes-mediated transformation system

[0075] The successfully transformed Agrobacterium rhizogenes was propagated and shaken, and monoclonal plaques were picked, and 400 μL of LB containing Kana and Str resistance was added, and placed in a 28°C shaker at 200 r / min overnight. 100 μL of the 400 μL bacterial solution after small shaking was evenly spread on the plate containing Kana and Str antibiotics, and placed in a 28°C incubator for 24 hours until the plate was full of bacteria. The bacteria were scraped into 100 mL MS liquid culture medium, AS was added, and the OD value was adjusted to 0.6 to prepare the infection solution. The material grown for 7 days under light was cut at about 0.5 cm below the cotyledon node, and the cotyledons with petioles were retained. The cut material was placed in the infection solution, and placed in a 28°C shaker at 90 r / min for infection for 45 minutes, then taken out, the residual bacterial solution was absorbed with filter paper, and moved to a dish (with two layers of MS-soaked filter paper), placed in a dark greenhouse for dark culture for 2 days, and then moved to the soil.

[0076] 3. Inoculation system of the cabbage rapeseed variety Huashuang 5

[0077] The root-root fungus used for indoor inoculation is the Chinese root-root fungus race 4 (Ji Haiwen, Ren Li, Chen Kunrong, Xu Li, Liu Fan, Sun Chaochao, Li Jun, Liu Shengyi, Fang Xiaoping. Identification of the main physiological races and varieties of rapeseed clubroot pathogens [J]. Chinese Journal of Oil Crops, 2013, 35(03): 301-306.).

[0078] Huashuang No. 5, Huashuang No. 5 overexpressing ResyR05g6066 (H5S+35S-ResyR05g6066) and Huashuang 5R (disease resistance control) were inoculated with indoor bacteria for clubroot disease resistance. The greenhouse soil method was used for inoculation: first, the mycorrhizae frozen at -20°C were thawed at room temperature, ground with a homogenizer, and the mycorrhizae and substrate soil were mixed at a ratio of 1:20. The mixed soil was placed in a dark environment at 25°C and sealed for 48 hours. The greenhouse culture conditions were 16L / 8D, 24°C, and 60%-80% humidity. The disease condition can be investigated after 30 days of culture.

[0079] 4. Investigation of the disease status after inoculation of Huashuang 5 overexpressing ResyR05g6066

[0080] The phenotype of the strains after inoculation is divided into three levels, namely 0, 1, 2, and 3. Level 0 shows that there is no tumor on the root and the root system develops normally; Level 1 shows that there are a few tumors on the lateral roots or the main root is slightly swollen; Level 2 shows that the main root has a tumor, with obvious large nodules or spherical masses; Level 3 shows that the main root has a large tumor in the shape of a spindle, and the plant is short and stops growing.

[0081] Calculation method of incidence and disease-related index:

[0082] Strain incidence rate = (number of susceptible plants / total number of plants investigated) × 100%;

[0083] Strain disease index = [∑ (number of diseased plants at each level × corresponding level) / total number of plants surveyed × highest level value] × 100.

[0084] According to the investigation, after Huashuang 5 overexpressed ResyR05g6066, the incidence rate dropped to 70.7% and the disease index dropped to 53 (see Figure 4 and Table 1).

[0085] Table 1 Identification results of the physiological race of China No. 4 after Huashuang No. 5 overexpressed ResyR05g6066 and was inoculated with bacteria

[0086]

[0087] 5. The roots of ResyR05g6066 plants inoculated with the physiological subspecies 4 of root-knot fungus were positively identified.

[0088] The roots of the plants inoculated with root-knot bacteria were washed, and a certain amount of samples were taken to extract DNA using the CTAB method, and the DNA was detected using positive detection primers (SEQ ID No. 7-8).

[0089] 20 μL PCR amplification system, 2× Taq Master Mix (Dye Plus) 10 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, DNA template 2 μL and ddH 2 2 μL of HO.

[0090] PCR reaction conditions: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 15s, annealing at 57℃ for 15s, extension at 72℃ for 1min, 34 cycles; extension at 72℃ for 5min; 25℃ for 5min, and finally detection by 1% agarose gel electrophoresis. The target band is at the position of 840bp ( Figure 5 ).

[0091] 6. Expression of ResyR05g6066 after transformation into Huashuang 5

[0092] use Super total RNA extraction kit (Promega (Beijing) Biotechnology Co., Ltd.) was used to extract total RNA from root samples of Huashuang No. 5 transformed ResyR05g6066 plants after inoculation and measure RNA concentration.

[0093] Depending on the concentration of sample RNA, use RNase-free ddH 2 Adjust the sample concentration to a consistent total RNA injection volume of 12 μL, add 4 μL of 4×g DNA Wiper Mix, mix gently with a pipette, and incubate at 42°C for 2 min.

[0094] Add 4 μL of 5×HiScript II qRT SuperMix II directly to the above reaction tube, mix gently by pipetting, and then perform reverse transcription reaction: 50°C for 15 min, 85°C for 5 s.

[0095] Quantitative Real-time PCR was used to analyze the expression of the ResyR05g6066 gene. The quantitative primers of the ResyR05g6066 gene were designed using CE Design. The sequences of the quantitative primers are as follows:

[0096] Forward quantitative primer (SEQ ID No. 9): 5′-ctcttgcccaccagaccaaa-3′;

[0097] Reverse quantitative primer (SEQ ID No. 10): 5'-caatctgagtttggcctcca-3'.

[0098] qPCR reaction system: qPCR SYBR Green Master Mix 11.8 μL, forward / reverse primer 0.6 μL, template cDNA 2 μL, ddH 2 Add 0.1% HO to make up to 15 μL.

[0099] qPCR reaction program: 95°C for 5 min; 95°C for 10 s, 56°C for 20 s, 72°C for 20 s, 40 cycles, melting curve stage default setting 1.

[0100] Expression of ResyR05g6066 in transgenic plants. The relative expression of this gene in Huashuang 5 was higher than that in Huashuang 5 and susceptible transformed plants. Figure 6 ).

[0101] 7. Application of ResyR05g6066 gene in clubroot resistance breeding and marker-assisted selection

[0102] As an RLP gene, the ResyR05g6066 gene enables rapeseed transgenic materials to obtain better resistance to clubroot, resist the infection of Chinese root-root fungus race 4, and effectively protect the yield of rapeseed in the field contaminated by root-root fungus, indicating that this gene can be used as a resistance gene to carry out resistance breeding of Brassica plants such as rapeseed, cabbage, kale and radish through genetic engineering. Molecular marker-assisted breeding based on the full-length sequence of the gene (including upstream sequences and coding regions) and markers developed with other sequences linked to this gene can also greatly accelerate the breeding process of clubroot resistance.

[0103] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A root-knot bacteria resistance gene, characterized in that The nucleotide sequence of the anti-root-knot bacteria gene is shown in SEQ ID No.

1.

2. An anti-rootroot fungus protein, characterized in that The amino acid sequence of the anti-root-knot protein is shown in SEQ ID No.

2.

3. A recombinant vector, characterized in that: The invention comprises the anti-root-knot bacteria gene according to claim 1.

4. A host cell, characterized in that Comprising the recombinant vector according to claim 3.

5. The host cell according to claim 4, characterized in that The initial strain of the host cell is Agrobacterium rhizogenes K599.

6. Use of the anti-root-knot bacteria gene according to claim 1, the recombinant vector according to claim 3, or the host cell according to claim 4 or 5 in improving the anti-root-knot bacteria performance of plants; The plant is a plant of the genus Brassica of the family Cruciferae; The root-knot fungus is the Chinese root-knot fungus physiological race 4.

7. A method for improving the resistance of plants to root knotweed fungi, characterized in that: The method comprises the steps of transforming the root knotweed resistance gene of claim 1 into the plant to obtain a transgenic plant overexpressing the gene; The plant is a plant of the genus Brassica of the family Cruciferae; The root-knot fungus is the Chinese root-knot fungus physiological race 4.

8. Use of a primer for detecting the anti-rootroot fungus gene as claimed in claim 1 in detecting the anti-rootroot fungus performance of a plant, characterized in that: The higher the expression level of the anti-root-knot fungus gene in the plant, the higher the anti-root-knot fungus performance of the plant; The plant is a plant of the genus Brassica of the family Cruciferae; The root-knot fungus is the Chinese root-knot fungus physiological race 4.

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