Application of BraA08g039497E gene from Brassica rapa ECD04 in improving resistance to root knotweed

Abstract

The invention discloses the application of BraA08g039497E gene from European turnip ECD04 in improving root knot fungus resistance, and belongs to the field of gene cloning technology. The nucleotide sequence of the BraA08g039497E gene is shown in SEQ ID No.1 or SEQ ID No.2, and the amino acid sequence of its encoded protein is shown in SEQ ID No.3; the target gene is located on the A08 chromosome of European turnip ECD04, and its physical position is 16663120-16666537. The invention discloses the disease resistance function of BraA08g039497E gene from European turnip ECD04 to root knot fungus, and the invention can select and breed varieties resistant to root knot fungus through the positioning interval where the gene is located, thereby shortening the breeding process.

Description

Technical Field

[0001] The invention relates to the technical field of gene cloning, and in particular to application of a BraA08g039497E gene derived from European turnip ECD04 in improving resistance to root knotweed. Background Art

[0002] Clubroot is a worldwide soil-borne disease caused by the fungus Plasmodiophora brassicae, which can infect almost all cultivated cash crops of the genus Brassica, including rapeseed, Chinese cabbage, kale, cauliflower, mustard, radish, etc. (Chai, AL, Xie, XW, Shi, YX and Li, BJ (2014) Special Issue: Research status of clubroot (Plasmodiophora brassicae) on cruciferous crops in China. Canadian Journal of Plant Pathology, 36, 142-153.). Clubroot not only consumes a large amount of nutrients required for normal plant growth, but also causes the roots of plants to swell after infection with clubroot, and the root swelling also limits the plant's absorption of water and mineral nutrients (Howard, RJ, Strelkov, SE and Harding, MW (2010) Clubroot of cruciferous crops–new perspectives on an old Canadian Journal of Plant Pathology, 32, 43-57.). The initial symptoms of clubroot are stunted growth and yellowing of the aboveground parts of the plant, especially wilting in hot weather at noon. Severely infected plants will die directly, seriously affecting crop yield and quality (Rai, HH, Devkota, TR, Shrestha, SM and Gahatraj, S. (2019) A review on clubroot of crucifers: symptoms, life-cycle of pathogen, factors affecting severity, and management strategies. Archives of Agricμlture and Environmental Science, 4, 342-349.).

[0003] The root knot fungus has a great impact on cruciferous crops. Taking rapeseed as an example, according to incomplete statistics, the area of ​​rapeseed root knot disease in my country is about 667,000 hm2. 2 The above are mainly concentrated in different regions of Sichuan, Hubei, Hunan, Yunnan and Anhui provinces, and the infected areas are constantly expanding (Wang Yanyan, Yang Zhiquan, Yang Qingyong, et al. Genetic improvement and application of rapeseed clubroot resistance [J]. Journal of Huazhong Agricultural University, 2021, 40(2): 1-5). Rapeseed in the affected areas has suffered a serious reduction in yield or even a total crop failure. With the continuous improvement of agricultural mechanization, this soil-borne disease is spreading explosively to rapeseed producing areas across the country, and has become the number one killer affecting the safe production of the rapeseed industry.

[0004] Breeding and planting disease-resistant varieties is currently considered to be the most economical and effective approach, but root-knot fungi are mostly mixed in the field, have strong reproductive capacity, and can mutate rapidly (Le Boldus, JM, Manolii, VP, Turkington, TK and Strelkov, SE (2012) Adaptation to Brassica Host Genotypes by a Single-Spore Isolate and Popμlation of Plasmodiophora brassicae (Clubroot). Plant Disease, 96, 833-838). Therefore, long-term planting of a single resistant rapeseed variety is very likely to result in loss of resistance. At present, exploring new clubroot resistance resources and sites is particularly important for breeding new varieties with durable resistance (Hirani, AH, Gao, F., Liu, J., Fu, G., Wu, C., Yuan, Y., et al. (2016) Transferring clubroot resistance from Chinese cabbage (Brassica rapa) to canola (B. napus). Canadian Journal of Plant Pathology, 38, 82-90). The main source of resistance in Brassica comes from turnip. As of 2022, a total of 28 resistance sites have been located, of which CRa and Crr1a have been successfully cloned and functionally verified. In addition, Yang et al. also sequenced and assembled the resistant source material ECD04 containing multiple disease resistance sites from scratch, integrated the 28 reported clubroot resistance sites into 15 sites, and predicted 62 disease resistance candidate genes of the NBS-LRR\RLK\RLP type (Yang, Z., Jiang, Y., Gong, J., Li, Q., Dun, B., Liu, D., et al. (2022) R gene triplication confers European fodder turnip with improved clubroot resistance. Plant Biotechnol J, 20, 1502-1517), which laid a good foundation for the cloning and functional verification of clubroot resistance genes. Summary of the invention

[0005] The purpose of the present invention is to provide an application of the BraA08g039497E gene derived from European turnip ECD04 in improving root knot fungus resistance to solve the problems existing in the above-mentioned prior art. The present invention verifies the disease resistance function of the BraA08g039497E gene against root knot fungus, and can provide the application of the BraA08g039497E gene in basic research on resistance to root knot fungus disease, disease-resistant breeding and molecular marker-assisted selection. Varieties resistant to root knot fungus can be bred through the location interval where the gene is located, thereby shortening the breeding process.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of the BraA08g039497E gene in conferring resistance to root knotweed bacteria. The nucleotide sequence of the BraA08g039497E gene is shown as SEQ ID No.1 or SEQ ID No.2, and the amino acid sequence of the encoded protein is shown as SEQ ID No.3.

[0008] Furthermore, the BraA08g039497E gene is located on the A08 chromosome of European turnip ECD04, and its physical position is 16663120-16666537.

[0009] The present invention also provides an application of the BraA08g039497E gene in clubroot disease resistance breeding. The BraA08g039497E gene is used in clubroot disease resistance breeding of Brassica plants through transgenic or sexual hybridization. The Brassica plants include rapeseed, cabbage, kale or radish. The nucleotide sequence of the BraA08g039497E gene is shown in SEQ ID No.1 or SEQ ID No.2.

[0010] The present invention also provides the use of the BraA08g039497E gene and a marker developed therefrom in molecular marker-assisted selection of Brassica plants, wherein the Brassica plants include rapeseed, Chinese cabbage, kale or radish, and the nucleotide sequence of the BraA08g039497E gene is shown in SEQ ID No.1 or SEQ ID No.2.

[0011] The present invention also provides a primer for amplifying the full length of the BraA08g039497E gene, comprising a forward primer as shown in SEQ ID No.4 and a reverse primer as shown in SEQ ID No.5.

[0012] The present invention also provides a primer for constructing a genetic transformation vector of the BraA08g039497E gene, comprising a forward primer as shown in SEQ ID No.6 and a reverse primer as shown in SEQ ID No.7.

[0013] The present invention also provides a primer for positive detection of BraA08g039497E gene, comprising a forward primer shown in SEQ ID No.8 and a reverse primer shown in SEQ ID No.9.

[0014] The present invention also provides a primer for detecting BraA08g039497E gene Quantitative Real-time PCR (Q-PCR), comprising a forward primer shown in SEQ ID No.10 and a reverse primer shown in SEQ ID No.11.

[0015] The present invention discloses the following technical effects:

[0016] The invention discloses that the BraA08g039497E gene derived from European turnip ECD04 has a resistance function to Chinese root knot fungus No. 4 physiological race, and through the transformation system mediated by Agrobacterium rhizogenes, the root overexpression is verified with Brassica napus Huashuang No. 5 as the background, thereby verifying the disease resistance function of the BraA08g039497E gene to root knot fungus. The invention can provide an application of the BraA08g039497E gene in basic research on resistance to root knot fungus disease, disease-resistant breeding and molecular marker-assisted selection, and breed varieties resistant to root knot fungus through the location interval where the gene is located, thereby shortening the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 : The cloning pattern of BraA08g039497E gene in the pCAMBIA3301 plant expression vector in the embodiment of the present invention;

[0019] Figure 2 : Agarose gel image of the full-length PCR amplification of the BraA08g039497E gene in the embodiment of the present invention, M is a 250 bp DNA ladder, 1-3 is the amplified target band, and the band size is 3332 bp;

[0020] Figure 3 :Agarose gel image of BraA08g039497E positive clone detection, M is DL 2000 DNA Marker, 1-21 are colony PCR detection bands, the positive band size is 916bp;

[0021] Figure 4 : The phenotype of the dominant physiological race 4 of Chinese root knotweed fungus after the BraA08g039497E gene is overexpressed in the Brassica napus receptor material Huashuang 5 in the embodiment of the present invention, with Huashuang 5 as the susceptible control and H5R as the resistant control, from left to right are H5R, grade 0, grade 1, grade 2, grade 3, and Huashuang 5;

[0022] Figure 5 : Agarose gel image of target gene detection in the root system of BraA08g039497E transformed plant in the embodiment of the present invention, M is DL 2000 DNA Marker, N is negative control, P is positive control, 1-10 are target gene PCR detection bands, and the target fragment size is 916bp;

[0023] Figure 6 : Q-PCR statistical diagram of the relative expression of the target gene in the root system of the BraA08g039497E transformed plant in the embodiment of the present invention, R represents a disease-resistant plant, S1 represents a susceptible plant at level 1, S2 represents a susceptible plant at level 2, S3 represents a susceptible plant at level 3, H5R is a disease-resistant control, and H5S is a susceptible control;

[0024] Figure 7 :Prediction map of conserved domains of BraA08g039497E gene, predicted using Conserved Domain Database in NCBI. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] 1. Clone the BraA08g039497E gene and amplify the target gene by PCR. The primer sequences used to amplify the BraA08g039497E genome are:

[0032] Forward primer (SEQ ID No. 4): 5'-gtgtgaacagaacatacttgtgtg-3'

[0033] Reverse primer (SEQ ID No. 5): 5'-ctactcgtcgagccaatctaaac-3'

[0034] The homologous recombination method was used to connect to the modified pCAMBIA3301 (Hyg resistance) plant expression vector as the backbone to construct an expression vector driven by CaMV35S ( Figure 1 ), and finally transferred into K599 Agrobacterium competent cells, as detailed below:

[0035] The BraA08g039497E gene was amplified using the genome of donor material Brassica rapa ECD04 as a template.

[0036] The PCR reaction amplification primers consist of three parts: the homology arm of the pCAMBIA3301 vector, the restriction site, and the homology sequence of the target gene, which is convenient for subsequent gene cloning operations.

[0037] The forward primer sequence (SEQ ID No.6) is

[0038] 5'-acgggggactcttgaccatgggtgtgaacagaacatacttgtgtg-3'

[0039] The reverse primer sequence (SEQ ID No.7) is

[0040] 5'-ggggaaattcgagctggtcaccctactcgtcgagccaatctaaac-3'

[0041] The CTAB method was used to extract the DNA of European turnip ECD04, and Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novozyme Biotechnology Co., Ltd.) was used to amplify the target fragment.

[0042] 50μl reaction system: 2×Phanta Max Buffer 25μl, Forward Primer (10μM) 2μl, Reverse Primer (10μM) 2μl, dNTP Mix (10mM each) 1μl, DNA template 2μl, ddH2O 19μl.

[0043] PCR reaction conditions: 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 3 min 30 s, 34 cycles; extension at 72°C for 5 min; 25°C for 5 min.

[0044] The amplified product was detected by 1% agarose gel electrophoresis, and the expected band size was 3332 bp ( Figure 2 ).

[0045] 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.

[0046] The modified pCAMBIA3301 (Hyg resistance) plant expression vector was digested with enzymes.

[0047] Enzyme digestion system: Buffer 6μl, plasmid DNA 50μl, NcoI FastDigest enzyme(s) 1μl, BstEII FastDigest enzyme(s) 1μl, ddH2O 2μl.

[0048] 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.

[0049] 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.).

[0050] Transfer the connected product into DH5α E. coli competent cells: a. Thaw the clone competent cells on ice. b. Take 10μl of the recombinant product and add it to 100μl of the competent cells, gently tap the tube wall to mix (do not oscillate to mix), and let it stand on ice for 30 minutes. c. Heat shock in a 42℃ water bath for 45 seconds, and immediately place it on ice to cool for 2-3 minutes. d. Add 700μl LB medium (without antibiotics), and shake at 37℃ for 1 hour (speed 200-250r / min). e. Preheat the Kana-resistant LB plate solid culture medium in a 37℃ incubator. f. Centrifuge at 5000r / min for 5 minutes, discard 600μl of supernatant, resuspend the tube bacteria, and gently spread it on the plate containing Kana resistance with a sterile coating stick. g. Incubate inverted in a 37℃ incubator for 12-16 hours.

[0051] For positive identification of monoclonal clones, pick a single clone on the transformation plate and perform positive detection.

[0052] Positive detection forward primer sequence (SEQ ID No.8): 5'-agtgatcatcaacaacggcatg-3'

[0053] Positive detection reverse primer sequence (SEQ ID No.9): 5'-tgacaccgcgcgcgataat-3'

[0054] Bacterial liquid PCR 12μl reaction system: 2×Taq Master Mix (Dye Plus) 6μl, Forward Primer (10μM) 1μl, Reverse Primer (10μM) 1μl, bacterial liquid 2μl, ddH2O 2μl.

[0055] 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 60°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.

[0056] The PCR reaction product was detected by 1% agarose gel electrophoresis, and the band at 916 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.

[0057] 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:

[0058] Take K599 competent cells and melt them on ice. Add 500ng of recombinant plasmid to 100μl competent cells. Place on ice for 30min, transfer to liquid nitrogen for 5min, place in a 37℃ water bath for 5min, place on ice for 5min, add 400μl of non-antibiotic LB, and incubate on a shaker at 28℃ for 1 hour. Take 100μl of transformation product and spread it on a resistant LB plate with Kana and Str. Incubate at 28℃ for 36-48h.

[0059] 2. Agrobacterium rhizogenes-mediated transformation system

[0060] The successfully transformed Agrobacterium rhizogenes was propagated and shaken, and monoclonal plaques were picked, and 400μl of LB containing kana and str resistance was added, and placed in a 28℃ shaker at 200r / min overnight. 100-150μl of the 400μl bacterial solution after small shaking was evenly spread on the plate containing kana and str antibiotics, and placed in a 28℃ constant temperature box for 24h until the plate was full of bacteria. The bacteria were scraped into 100ml MS liquid culture medium, AS was added, and the OD value was adjusted to 0.4-0.6 to prepare the infection solution. The material grown for 7d under light was cut at about 0.5cm 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℃ shaker at 90r / min for infection for 45min, 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), and placed in a dark greenhouse for dark culture for 2-3d.

[0061] 3. Inoculation system of Brassica napus strain (Huashuang 5)

[0062] The soil method of inoculation was used with root-knot fungi from Zhijiang, Hubei Province, China. (a) The diseased root tissues stored in a -20°C refrigerator were thawed and rotted at room temperature (25°C) for 1 day. (b) The diseased tissues were rinsed with clean water, and an appropriate amount of ddH2O was added. The roots were beaten into a homogenate using a juicer to release dormant spores (Chinese root-knot fungi No. 4 physiological subspecies). (c) The soil method was used for inoculation of rapeseed. The mycorrhizae and substrate soil were mixed in a ratio of 1:20. (d) A 50-hole pot was used for inoculation of rapeseed. 2 / 3 of the substrate soil was spread on the bottom of the pot, and the top layer was spread with soil. (e) The infected plants were transplanted onto the soil, covered with a layer of plastic wrap, and the plastic wrap was covered for about 3 days. The plants were cultured at a temperature of 24°C and a humidity of 60%-80%, and watered every two days.

[0063] 4. Investigation of disease status after vaccination of Huashuang 5 overexpressing BraA08t039497E

[0064] The phenotype of the strains after inoculation is divided into three levels, namely level 0, level 1, level 2, and level 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.

[0065] Calculation method of incidence and disease-related index:

[0066] Strain incidence rate = (number of susceptible plants / total number of plants investigated) × 100%;

[0067] Strain disease index = [∑ (number of diseased plants at each level × corresponding level) / total number of plants surveyed × highest level value] × 100.

[0068] According to the investigation, after Huashuang 5 overexpressed BraA08g039497E, the incidence rate dropped to 50% and the disease index dropped to 28 (see Figure 4 and Table 1).

[0069] Table 1 Identification results of Huashuang 5 overexpressing BraA08t039497E followed by Chinese root knotweed 4 physiological race

[0070]

[0071] Note: Huashuang 5 (H5S) was used as the susceptible control, and H5R was used as the resistant control.

[0072] 5. The roots of BraA08g039497E plants inoculated with root-knot bacteria were positively identified.

[0073] 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. 8-9).

[0074] 20μl PCR amplification system, 2×Taq Master Mix (Dye Plus) 10μl, Forward Primer (10μM) 1μl, Reverse Primer (10μM) 1μl, DNA template 2μl, ddH2O 6μl.

[0075] PCR reaction conditions: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 15s, annealing at 60℃ 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 916bp ( Figure 5 ).

[0076] 6. Expression of BraA08g039497E after transformation into Huashuang 5

[0077] 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 BraA08g039497E plants after inoculation, and the RNA concentration was measured.

[0078] According to the concentration of sample RNA, adjust the sample concentration with RNase-free ddH2O to make the total RNA injection volume 12μl, add 4×gDNA Wiper Mix 4μl, mix gently with a pipette, and incubate at 42℃ for 2min.

[0079] 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.

[0080] Quantitative Real-time PCR was used to analyze the expression of BraA08g039497E gene. CEDesign was used to design quantitative primers for BraA08g039497E gene. The sequences of quantitative primers are as follows:

[0081] Forward quantitative primer (SEQ ID No.10): 5'-TCATGTCCCTGAACTCCAC-3'

[0082] Reverse quantitative primer (SEQ ID No. 11): 5'-TTGGGAAGAGGTTGGAGATG-3'

[0083] qPCR reaction system: qPCR SYBR Green Master Mix 11.8μl, F / R Primer 0.6μl, template cDNA 2μl, ddH2O to 15μl.

[0084] 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.

[0085] The expression of BraA08g039497E in transgenic plants showed that the relative expression of this gene in Huashuang 5, which was overexpressed and showed complete disease resistance, was higher than that in Huashuang 5 and susceptible transformed plants ( Figure 6 ).

[0086] 7. The physical location of the root-knot fungus resistance gene BraA08g039497E on the A08 chromosome is 16663120-16666537. The BraA08g039497E gene can exert its disease resistance function after being overexpressed in the roots, giving the susceptible material Huashuang No. 5 the ability to resist root-knot fungus. The nucleotide sequence of the BraA08g039497E gene is shown in SEQ ID No. 1 and SEQ ID No. 2, and the amino acid sequence of its encoded protein is shown in SEQ ID No. 3, which has a typical NBS-LRR domain (this domain can recognize pathogen effect factors and activate the plant's immune response) ( Figure 7 ), so this gene has the function of resisting root knot fungus.

[0087] 8. Application of BraA08g039497E gene in clubroot resistance breeding and marker-assisted selection

[0088] As an NBS-LRR gene, BraA08g039497E gene enables rapeseed transgenic materials to obtain better resistance to clubroot, resist the infection of Chinese root-root fungus No. 4 physiological subspecies, and effectively protect the yield of rapeseed in the field contaminated by root-root fungus. In addition to its anti-root-root effect on Brassica napus, BraA08g039497E has little effect on other agronomic traits, indicating that this gene can be used as a resistance gene to carry out resistance breeding of Brassica plants such as rapeseed, cabbage, cabbage and radish through genetic engineering. Molecular marker-assisted breeding based on the full-length sequence of the gene (including the upstream sequence and the coding region) and markers developed with other sequences linked to this gene can also greatly accelerate the breeding process of clubroot resistance.

[0089] 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. Use of the BraA08g039497E gene in conferring resistance to root knotweed on Brassica plants, characterized in that: The nucleotide sequence of the BraA08g039497E gene is shown in SEQ ID No. 1; the root-knot fungus is the Chinese root-knot fungus physiological race 4; and the Brassica plant is rapeseed, Chinese cabbage or cabbage.

2. Application of BraA08g039497E gene in breeding for clubroot disease resistance, characterized in that: The BraA08g039497E gene is used in the breeding of Brassica plants for resistance to clubroot disease through transgenic or sexual hybridization. The Brassica plants are rapeseed, Chinese cabbage or cabbage. The nucleotide sequence of the BraA08g039497E gene is shown in SEQ ID No.

1. The pathogen of clubroot disease is the physiological race 4 of Chinese clubroot.

3. Application of BraA08g039497E gene in molecular marker-assisted selection of Brassica plants for resistance to root knotweed fungus, characterized in that: The Brassica plant is rapeseed, Chinese cabbage or cabbage, the nucleotide sequence of the BraA08g039497E gene is shown in SEQ ID No. 1; and the root-knot fungus is the Chinese root-knot fungus physiological race 4.

4. The use according to claim 3, characterized in that: The primers used to amplify the full length of the BraA08g039497E gene are the forward primer shown in SEQ ID No.4 and the reverse primer shown in SEQ ID No.

5.

5. The use according to claim 3, characterized in that: The primers used to construct the BraA08g039497E gene genetic transformation vector are the forward primer shown in SEQ ID No.6 and the reverse primer shown in SEQ ID No.

7.

6. The use according to claim 3, characterized in that: The primers used for positive detection of BraA08g039497E gene are the forward primer shown in SEQ ID No.8 and the reverse primer shown in SEQ ID No.

9.

7. The use according to claim 3, characterized in that: The primers used for Q-PCR detection of BraA08g039497E gene are the forward primer shown in SEQ ID No.10 and the reverse primer shown in SEQ ID No.11.

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