BraBBE19 gene related to clubroot resistance in Chinese cabbage and its application

Abstract

The invention discloses a cabbage clubroot resistance-related gene BraBBE19 and its application, cabbage clubroot resistance-related genes BraBBE19 The nucleotide sequence of is shown in SEQ ID No. 1. The overexpression vector of the gene was transformed into Columbia wild-type Arabidopsis thaliana by Agrobacterium transformation method to obtain BraBBE19 Heterologous overexpression of Arabidopsis thaliana strains was found to be resistant to disease. BraBBE19 Heterologous overexpression of enhances Arabidopsis resistance to clubroot; BraBBE19 Homologous genes AT4G20840 The T-DNA insertion mutant Arabidopsis thaliana lines showed reduced resistance to clubroot. BraBBE19 This gene is related to clubroot resistance and can be used in molecular breeding of cruciferous crops with good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering technology, and specifically relates to cabbage clubroot resistance-related genes BraBBE19 The technical field of its application in regulating plant clubroot disease resistance, in particular to a cabbage clubroot disease resistance-related gene BraBBE19 and its applications. Background Art

[0002] Clubroot, caused by the fungus Brassicae, is a serious soil-borne disease and is considered one of the most economically important diseases of cruciferous crops. Infection by the fungus causes root nodules to form on the host plant's roots, impairing the plant's absorption and transport of water and nutrients, leading to wilting, stunted growth, and premature maturity, resulting in yield and quality losses. In recent years, frequent outbreaks of clubroot have been reported in cruciferous crop areas. Clubroot is widespread, affecting a significant portion of crucifer crops annually, accounting for over one-third of the total area cultivated with these crops. Common agricultural strategies for clubroot control include planting resistant varieties, crop rotation, biological control, soil liming, application of soil fumigants and fungicides, and controlled sowing dates. However, due to the diverse nature of the pathogen and the long survival of its dormant spores in the soil, no single approach currently offers a sustainable solution for clubroot control. The use of resistant varieties is a major approach in the current exploration of clubroot management. By developing varieties with multi-gene resistance and gene pyramids, as well as using multi-line and variety mixtures, diversifying the sources of resistance and introducing new sources of resistance, the collapse of resistance caused by strong selection pressure can be delayed, and clubroot-resistant plants can be given durability.

[0003] The berberine bridge enzyme (BBE)-like protein family is a large family of enzymes found in bacteria, fungi and plants. It is named after its most characterized member, the berberine bridge enzyme from California poppy. With the increasing number of sequenced plant genomes, more and more genes encoding BBE-like enzymes have been identified in different plant families. Previous studies have observed abnormally high upregulation of BBE-like enzymes in response to pathogens, and BBE-like enzymes contribute to the expression of secretory proteomes, indicating that they play a role in plant-pathogen interactions. Studies have shown that the BBE-like protein family can oxidize and damage the biological activity of cell wall-derived oligosaccharides, which helps control the steady-state level of damage-associated molecular patterns (DAMPs) and prevent their excessive accumulation. For example, at least four BBE-like enzymes in Arabidopsis are oligogalacturonate (OG) oxidases (OGOX1-4). After OG is oxidized, its ability to activate immune responses is reduced, and it is not easily hydrolyzed by fungal polygalacturonases. Overexpression of OG reduces the ability of OG to activate immune responses. OGOX1Plants with BBE-like enzymes are more resistant to Botrytis cinerea. In addition, another Arabidopsis BBE-like enzyme, cellodextrin oxidase (CELLOX), is involved in the immune process. OGOX1 Coordinated expression specifically oxidizes cellodextrin (CD). Similarly, the induced activity of CD after oxidation is negligible and it is not easily used as a carbon source by Botrytis cinerea. CELLOX The results showed that the BBE-like protein family plays an important role in plant immunity. Summary of the Invention

[0004] The purpose of the present invention is to provide a cabbage clubroot resistance-related gene to address the deficiencies of the prior art. BraBBE19 and its applications.

[0005] The object of the present invention is achieved through the following technical solutions: The present invention provides a cabbage clubroot resistance-related gene BraBBE19 , BraBBE19 have:

[0006] (1) the nucleotide sequence shown in SEQ ID No. 1; or

[0007] (2) The nucleotide sequence shown in SEQ ID No. 1 is substituted, deleted and / or added with one or more nucleotides; or

[0008] (3) A nucleotide sequence that hybridizes to the DNA sequence defined in (1) under stringent conditions.

[0009] The present invention provides the cabbage clubroot resistance-related gene BraBBE19 The method for constructing an overexpression vector specifically comprises:

[0010] Design of specific primers to amplify Brassica rapa BraBBE19 The nucleotide sequence of the gene was obtained; the pFGC-1008 vector was double-enzyme digested to obtain a linearized vector; the amplified sequence and the linearized vector were connected by homologous recombination to obtain a recombinant product; the recombinant product was transformed to obtain Chinese cabbage BraBBE19 A gene overexpression vector; wherein the primer sequences are the sequences shown in SEQ ID No. 2 and SEQ ID No. 3.

[0011] The present invention provides the cabbage clubroot resistance-related gene BraBBE19 The method for identifying mutant materials specifically includes:

[0012] Using the DNA of Arabidopsis thaliana to be screened as a template, a three-primer PCR amplification test was performed to identify the cabbage BraBBE19Homozygous mutant material of the gene; wherein the sequences of the three primers are shown as SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6 respectively.

[0013] The present invention provides a gene containing the cabbage clubroot resistance related gene BraBBE19 The biological material is an expression vector, an expression cassette, a host cell or an engineered bacterium.

[0014] The present invention provides the cabbage clubroot resistance-related gene BraBBE19 Or the application of its corresponding biomaterials in regulating plant disease resistance function.

[0015] The present invention provides the cabbage clubroot resistance-related gene BraBBE19 Or the use of corresponding biological materials in preparing transgenic plants.

[0016] The present invention provides the cabbage clubroot resistance-related gene BraBBE19 Or the application of its corresponding biological materials in plant breeding.

[0017] The beneficial effects of the present invention are as follows: the present invention provides cabbage clubroot resistance-related genes BraBBE19 The overexpression vector construction and mutant material identification method has high specificity and is suitable for BraBBE19 Application of genes; the present invention will BraBBE19 The overexpression vector of the gene was transformed into Columbia wild-type Arabidopsis thaliana by Agrobacterium transformation. BraBBE19 Heterologous overexpression of Arabidopsis thaliana strains was found to be resistant to disease. BraBBE19 Heterologous overexpression of BraBBE19 Homologous genes AT4G20840 The T-DNA insertion mutant Arabidopsis lines showed reduced resistance to clubroot; therefore, BraBBE19 This gene is related to clubroot resistance and can be used in molecular breeding of cruciferous crops with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 for BraBBE19 Schematic diagram of the overexpression vector;

[0019] Figure 2 for BraBBE19 Electrophoresis diagram of the T-DNA insertion mutant material WiscDsLox432E05;

[0020] Figure 3 21 days after inoculation of Brassica rapa BraBBE19 Comparison of the overall growth of overexpression plants and control plants (scale is 5 cm);

[0021] Figure 4 21 days after inoculation of Brassica rapa BraBBE19 Comparison of root phenotypes between overexpressing plants and control plants (scale bar is 2 cm);

[0022] Figure 5 This is a comparison of the overall growth of the WiscDsLox432E05 mutant plants and the control plants 21 days after inoculation with Brassica rapa (scale bar is 5 cm);

[0023] Figure 6 The figure shows the comparison of root phenotypes of WiscDsLox432E05 mutant plants and control plants 21 days after inoculation with Brassica rapa (scale bar is 2 cm);

[0024] Figure 7 21 days after inoculation of Brassica rapa BraBBE19 Disease index of gene overexpression plants, mutant plants and control plants. DETAILED DESCRIPTION

[0025] The technical solutions of the present invention are described through specific examples. Other method steps may be present before, after, or between one or more method steps provided by the present invention, and the order of the method steps may be changed or adjusted. These embodiments are merely for illustrating the present invention and are not intended to limit the scope of the present invention. In the absence of substantial changes in the technical content, they should also be considered as the applicable scope of the present invention.

[0026] In order that the present invention may be more thoroughly understood, detailed exemplary embodiments will be described, but it should be understood that the present invention can be implemented in other forms and is not limited to only the embodiments set forth herein.

[0027] The present invention provides a cabbage clubroot resistance-related gene BraBBE19 , which was cloned from Brassica rapa ( Brasica rapa var. pekinensis ) ECD05, the gene has the sequence shown in SEQ ID No.1.

[0028] The present invention also provides the cabbage clubroot resistance-related gene BraBBE19 The application in regulating clubroot disease resistance is described in detail below.

[0029] Example 1: BraBBE19 Construction of overexpression vector

[0030] 1. Synthesis of cDNA:

[0031] RNA from Chinese cabbage ECD05 was extracted using the Trizol method and reverse transcribed into cDNA using TaKaRa's PrimeScript reverse transcription reagent. TM RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription kit. Specific operations were performed according to the instructions.

[0032] 2. PCR amplified fragments:

[0033] Specific primers were designed, and their sequence information is shown in Table 1; KOD One TM PCR MasterMix amplification BraBBE19 The CDS sequence of the gene was obtained, and the PCR reaction system and procedure were carried out according to the instructions. The amplified PCR products were separated by 1% agarose gel electrophoresis, and the target sequence was obtained by gel tapping and recovery.

[0034] Table 1: Primers used for construction of heterologous expression vectors

[0035]

[0036] 3. Double enzyme digestion of pFGC1008 vector:

[0037] exist Sal Ⅰ and Kpn The pFGC1008 vector was double-digested at site I. The enzyme digestion system was as follows: Buffer 4 μL, Sal Ⅰ and Kpn Ⅰ 2 μL each, double distilled water (ddH2O) to 40 μL, 37 ℃ water bath for 1 h, the enzyme digestion products were separated by 1% agarose gel electrophoresis, the gel was cut and recovered to obtain the linearized vector.

[0038] 4. Homologous recombination ligation:

[0039] The PCR amplified fragment and the linearized vector were ligated by homologous recombination using the Novozymes Single Fragment Homologous Recombination Kit C112. The system consisted of: 4 μL 5× CEⅡ Buffer, 2 μL ExaseⅡ, 200 ng of linearized vector, 20 ng of PCR amplified fragment, and ddH2O to a final volume of 20 μL. The reaction was incubated at 37°C for 30 min.

[0040] 5. Transformation of E. coli:

[0041] The homologous recombination ligation product was transformed into Escherichia coli competent cells using the freeze-thaw method. After transformation, the bacterial solution was PCR verified, the target plasmid was extracted and sequenced, and the plasmid with successful sequence alignment was transformed into Agrobacterium competent cells. The bacterial solution was PCR verified to see if there was a target band, and the plasmid was extracted and sequenced. The final result was BraBBE19 Overexpression vector map Figure 1 As shown, the successfully verified bacterial culture was preserved and placed in a 4°C refrigerator for later use.

[0042] Example 2: Arabidopsis floral dipping transformation and screening of positive transformants

[0043] 1. Transformation of Arabidopsis thaliana by floral dipping method:

[0044] The Agrobacterium culture liquid that was successfully verified by sequencing was used as the mother liquid to transform Arabidopsis thaliana by the floral dip method. The steps are as follows: 500 μL of the Agrobacterium culture liquid containing the target vector was added to 200 mL of liquid LB medium containing kanamycin and rifampicin (50 mg / L), and the culture was shaken at 28°C and 200 rpm for about 30 h until the OD 600 The bacterial solution was centrifuged at 8000 rpm for 10 min to obtain the Agrobacterium precipitate, and the bacterial solution was resuspended in 200 ml of freshly prepared 5% sucrose; Silwet L-77 was added to a final concentration of 200 μL / L, and the culture was shaken at 200 rpm at 28°C for 2 min to obtain the bacterial solution; the open flowers and siliques of wild-type (WT) Arabidopsis thaliana were removed, and the flower buds were immersed in the bacterial solution for 1 min. The excess bacterial solution was aspirated and the plants were cultured in a moist environment at 25°C for 24 h. After the dark culture was removed, the plants were cultured normally. After one week, the flower buds were immersed once more to improve the transformation efficiency.

[0045] 2. Prepare the seeding medium:

[0046] Prepare 1 / 2 MS medium, sterilize by high-pressure steam, and cool to 50-60°C. Add hygromycin to a final concentration of 90 mg / L in a clean bench, pour onto a solid plate, and store at 4°C for later use.

[0047] 3. Screening of positive transformants:

[0048] The T1 generation Arabidopsis seeds harvested by soaking flowers were screened on the sowing culture medium. The method steps are as follows: In a clean bench, the Arabidopsis seeds were disinfected with 75% alcohol for 5 minutes, then washed with sterile water 5-6 times, each washing for 1 minute. After the end, they were evenly sown on the sowing culture medium, and the culture medium was sealed with sealing film and placed in a culture room at 22°C. After two weeks of culture, the healthy positive plants were removed and placed in an artificial climate room for normal culture.

[0049] Example 3: Screening of T-DNA insertion mutant materials

[0050] 1. Rapid extraction method for DNA extraction:

[0051] Cut an appropriate amount of Arabidopsis leaves to be tested, place them in a centrifuge tube, add 200 μL of DNA extraction buffer, add magnetic beads, and disrupt the tissue using a microtome (65 Hz, 120 s) to obtain tissue fluid. Transfer the entire tissue fluid to a new 1.5 mL centrifuge tube and centrifuge at 13,000 rpm for 8 min to obtain the supernatant. Prepare new 1.5 mL centrifuge tubes, add 100 μL of isopropanol to each tube, transfer 100 μL of the supernatant to each tube, gently shake approximately 50 times, and let stand at room temperature (approximately 25°C) for 5 min. Centrifuge at 13,000 rpm for 6 min and remove the supernatant. Wash the pellet twice with 1 mL of 70% ethanol: shake up and down 20 times, centrifuge at 13,000 rpm for 3 min, discard the supernatant, and repeat this cycle. Vortex for 1 min. After washing, aspirate the excess liquid, air-dry the pellet for 5 min, add 25-50 μL of ddH2O, and store at -20°C until ready for use.

[0052] 2. Three-primer PCR detection:

[0053] The three primers are LP, RP, and BP, and their corresponding primer sequence information is shown in Table 2.

[0054] Table 2: Primers used for screening of T-DNA insertion mutants

[0055]

[0056] The PCR reaction used Qingke Bio's 1.1× T3 Super Mix PCR. The reaction system consisted of 44 μL 1.1× T3 Super Mix, 2 μL Template, 2 μL Primer F, and 2 μL Primer R. The reaction procedure was as follows: pre-denaturation at 98°C for 2 min 30 s, followed by 35 cycles of amplification (denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 10 s), and a final extension at 72°C for 2 min. PCR amplification was performed using LP + RP primers and BP + RP primers, respectively. PCR products were obtained and separated by 1% agarose gel electrophoresis, and bands were detected. The test results are shown in Figure 2. Figure 2 As shown, the M lane is the DL2000 marker, LP+RP and BP+RP are the corresponding primers, and the red box shows the homozygous plant. Figure 2 As can be seen from the figure, the lanes where the product amplified by LP + RP primers had no bands and the lanes where the product amplified by BP + RP primers had bands corresponded to plants that were homozygous mutant materials and were collected for subsequent experiments.

[0057] Example 4: Brassica rapa infection and phenotypic observation

[0058] 1. Preparation of Brassica rapa spore suspension:

[0059] After the diseased roots were fully decomposed, they were disinfected with 70% alcohol for 1 min, treated with 10% NaClO for 20 min, and rinsed with sterile water three times; the diseased roots were squeezed into a homogenate with a juicer and filtered twice using eight layers of sterile gauze; the filtrate was centrifuged at 500 rpm for 5 min, the black precipitate was discarded, and the supernatant and gray precipitate were retained; a new 50 mL centrifuge tube was replaced with sterile water to 45 mL, centrifuged at 4000 rpm for 15 min, and the supernatant was discarded; sterile water was added to 40 mL, centrifuged at 4000 rpm for 10 min, and the supernatant was discarded, and the mixture was repeated once; the precipitate was dissolved in 50% sucrose solution, centrifuged at 3100 rpm for 10 min, and the supernatant and the outermost off-white precipitate were placed in a new 50 mL centrifuge tube; sterile water was added to 45 mL, centrifuged at 4000 rpm for 10 min, and the supernatant was discarded; sterile water was added to 30 mL, centrifuged at 3100 rpm for 10 min, discard the supernatant, repeat 2-3 times; dissolve the precipitate in 10-15 mL of sterile water to obtain a spore suspension, and store it in a -4°C refrigerator for later use.

[0060] Use a hemocytometer (1 / 400 mm 2 ) Calculate the concentration of the spore suspension according to the following formula, where the spores on the upper and left lines are included in the grid: Concentration of spore suspension (spores / mL) = [number of dormant spores in 80 grids (upper left + lower left + middle + upper right + lower right)] / 80 × 400 × 10 4 × dilution factor.

[0061] 2. Infection, morbidity and phenotypic observation:

[0062] The overexpression plants of Example 2, the mutant plants of Example 3, and the corresponding control plants were transplanted into trays filled with substrate. The trays were perforated in a 12×6 pattern, with 12 plants per biological replicate. Three biological replicates were set for each of the overexpression plants, mutant plants, and control plants. After 5 days of seedling acclimatization, 1 mL of spore suspension (at a concentration of 1×10 7 The soil was maintained at 48 h post-inoculation, with water control for 48 h to ensure successful infection by the root knot fungus. Normal management was performed 48 h later, and disease incidence was recorded 21 days after inoculation.

[0063] The disease is graded into 5 categories: Grade 0: no symptoms; Grade 1: root swelling mainly in lateral roots, with no symptoms in the main root; Grade 2: small root swelling in the main root and a few lateral roots; Grade 3: medium to large root swelling in the main root and lateral roots, with plant growth impaired; Grade 4: severe root swelling in the main root and lateral roots, with fine roots completely destroyed, and plant growth affected. The disease index calculation formula is: Disease Index DI = (1 n1+ 2 n 2+3 n 3+ 4 n 4) × 100 / 4 N t ,in n 1 to n 4 is the number of plants in a given class, N t is the total number of plants counted.

[0064] The results showed that: in the disease resistance identification, BraBBE19 There was no significant difference in growth between heterologous overexpression plants and empty vector plants. Figure 3 As shown in Figure 2, the root disease is milder than that of the empty-loaded plants. Figure 4 As shown in the figure, the disease index was statistically significantly lower than that of the empty plants. Figure 7 As shown, * indicates significant difference at the p=0.05 level; T-DNA insertion mutant WiscDsLox432E05 has no significant difference in size from the wild type, but the leaves and stems turn purple, and the stress symptoms are more obvious, as shown in Figure 5 As shown, the disease is more serious than that of the wild type, as Figure 6 As shown in Figure 2, the disease index is also significantly higher than that of the wild type. Figure 7 As shown. In summary, cabbage BraBBE19 Overexpression of BraBBE19 Homologous genes AT4G20840 The resistance of the WiscDsLox432E05 mutant plants to clubroot disease was reduced; therefore, BraBBE19 It is closely related to the incidence of clubroot disease and is a gene related to clubroot disease resistance.

[0065] It should be noted that the materials used in the above examples, unless otherwise specified, can be readily obtained from commercial companies; Arabidopsis thaliana: Col-0 type Arabidopsis thaliana; Brassica rapa ECD05, donated by Professor GR Dixon of the University of Reading, UK, and propagated in the laboratory; diseased roots: collected from diseased plants, washed, and stored in a -20°C refrigerator.

[0066] The foregoing describes preferred embodiments of the present invention. These examples are intended to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. However, these embodiments do not describe all details, and improvements and modifications based on these embodiments are readily apparent to those skilled in the art. Therefore, such modifications and improvements based on the present invention are intended to fall within the scope of the present invention.

Claims

1. A gene related to cabbage clubroot resistance BraBBE19 The application of the invention in regulating the function of resisting clubroot disease caused by Brassica rapa is characterized in that: described BraBBE19 The nucleotide sequence is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The cabbage clubroot resistance-related gene BraBBE19 The method for constructing an overexpression vector specifically comprises: Design of specific primers to amplify Brassica rapa BraBBE19 The nucleotide sequence of the gene was obtained; the pFGC-1008 vector was double-enzyme digested to obtain a linearized vector; the amplified sequence and the linearized vector were connected by homologous recombination to obtain a recombinant product; the recombinant product was transformed to obtain Chinese cabbage BraBBE19 A gene overexpression vector; wherein the primer sequences are the sequences shown in SEQ ID No. 2 and SEQ ID No. 3.

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