Cabbage clubroot resistance related gene BraBBE19 and application thereof

Abstract

The invention discloses a Chinese cabbage clubroot resistance related gene BraBBE19 and application thereof. The nucleotide sequence of the Chinese cabbage clubroot resistance related gene BraBBE19 is as shown in SEQ ID No. 1. An overexpression vector of the gene is transformed into Columbia wild type arabidopsis thaliana through an agrobacterium transformation method to obtain a BraBBE19 heterologous overexpression arabidopsis thaliana strain, and disease resistance identification finds that the heterologous overexpression of Chinese cabbage BraBBE19 can enhance the resistance of arabidopsis thaliana to clubroot; and when T-DNA of the BraBBE19 homologous gene AT4G20840 is inserted into a mutant arabidopsis thaliana strain, the resistance to clubroot is reduced. Therefore, the Chinese cabbage BraBBE19 is related to clubroot resistance, and the gene can be applied to molecular breeding of cruciferae crops and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to genes related to clubroot resistance in Chinese cabbage BraBBE19 and their application technologies in regulating clubroot resistance in plants, and particularly relates to a gene related to clubroot resistance in Chinese cabbage BraBBE19 and its application. Background Art

[0002] Clubroot, caused by Plasmodiophora brassicae, is a serious soil-borne disease and is considered one of the most important economic diseases in cruciferous crops. After the host plant is infected by Plasmodiophora brassicae, root nodules will form on the roots, which will disrupt the absorption and transportation of water and nutrients by the plant, resulting in wilting, retarded growth and premature maturity, and further leading to losses in yield and quality. In recent years, frequent outbreaks of clubroot have been reported in cruciferous crop planting areas. Currently, clubroot is widely distributed and affects a large number of cruciferous crops every year, accounting for more than one-third of the cruciferous crop planting area. In agricultural production, clubroot is usually controlled by planting resistant varieties, crop rotation, biological control, soil liming, application of soil fumigants and fungicides, and controlling the sowing date, etc. However, due to the wide variety of pathogens and the long survival time of their dormant spores in the soil, there is still no practice that can provide 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-strain and variety mixtures to diversify the sources of resistance and introduce new sources of resistance, the resistance breakdown caused by strong selection pressure can be delayed, and durability can be imparted to clubroot-resistant plants.

[0003] The berberine bridge enzyme (BBE)-like protein family is a large enzyme family found in bacteria, fungi and plants, and is named after its most characteristic member, berberine bridge enzyme from Eschscholzia californica. With the continuous increase in the number of sequenced plant genomes, more and more genes encoding BBE-like enzymes have been identified in different plant families. Previous studies have observed an abnormally high upregulation of BBE-like enzymes in response to pathogens, and BBE-like enzymes contribute to the expression of the secretory proteome, indicating that they play a role in plant-pathogen interactions. Research has shown that the BBE-like protein family can oxidize and damage the bioactivity of cell wall-derived oligosaccharides, which helps to control the homeostatic level of damage-associated molecular patterns (DAMPs) and prevent their excessive accumulation. For example, at least four BBE-like enzymes in Arabidopsis thaliana are oligogalacturonide (OG) oxidases (OGOX1-4). After OG is oxidized, its ability to activate the immune response is reduced, and it is not easily hydrolyzed by fungal polygalacturonase. Overexpression OGOX1Plants are more resistant to Botrytis cinerea. In addition, another Arabidopsis thaliana BBE-like enzyme, cellodextrin oxidase (CELLOX), is co-expressed with OGOX1 during the immune process and specifically oxidizes cellodextrin (CD). Similarly, the induced activity after CD oxidation is negligible and it is not easily used as a carbon source by Botrytis cinerea. Overexpression of CELLOX plants enhances resistance to Botrytis cinerea. The results show that the BBE-like protein family plays an important role in plant immunity. SUMMARY OF THE INVENTION

[0004] The object of the present invention is to provide a gene related to resistance to clubroot disease of Chinese cabbage BraBBE19 and its application in view of the deficiencies of the prior art.

[0005] The object of the present invention is achieved by the following technical solutions: The present invention provides a gene related to resistance to clubroot disease of Chinese cabbage BraBBE19 , and the BraBBE19 has: (1) The nucleotide sequence shown in SEQ ID No.1; or (2) The nucleotide sequence shown in SEQ ID No.1 with one or more nucleotides substituted, deleted, and / or added; or (3) A nucleotide sequence that hybridizes with the DNA sequence defined in (1) under stringent conditions.

[0006] The present invention provides a method for constructing an overexpression vector of the above-mentioned gene related to resistance to clubroot disease of Chinese cabbage BraBBE19 , which specifically includes: Design specific primers to amplify the nucleotide sequence of the Chinese cabbage BraBBE19 gene; double-digest the pFGC-1008 vector to obtain a linearized vector; ligate the amplified sequence and the linearized vector by homologous recombination to obtain a recombinant product; transform the recombinant product to obtain an overexpression vector of the Chinese cabbage BraBBE19 gene; wherein, the sequences of the primers are the sequences shown in SEQ ID No.2 and SEQ ID No.3.

[0007] The present invention provides a method for identifying a mutant material of the above-mentioned gene related to resistance to clubroot disease of Chinese cabbage BraBBE19 , which specifically includes: Using the DNA of Arabidopsis thaliana to be screened as a template, and performing PCR amplification detection using the three-primer method to identify the homozygous mutant material of the Chinese cabbage BraBBE19 gene; wherein, the sequences of the three primers are respectively as shown in SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6.

[0008] The present invention provides a composition containing the above-mentioned gene related to resistance to clubroot disease of Chinese cabbage BraBBE19The biological material is an expression vector, an expression cassette, a host cell or an engineered bacterium.

[0009] The present invention provides the above-mentioned gene related to resistance to clubroot disease of Chinese cabbage BraBBE19 or its corresponding biological material for use in regulating the disease resistance function of plants.

[0010] The present invention provides the above-mentioned gene related to resistance to clubroot disease of Chinese cabbage BraBBE19 or its corresponding biological material for use in preparing transgenic plants.

[0011] The present invention provides the above-mentioned gene related to resistance to clubroot disease of Chinese cabbage BraBBE19 or its corresponding biological material for use in plant breeding.

[0012] The beneficial effects of the present invention are as follows: The present invention provides a method for constructing an overexpression vector and identifying mutant materials of the gene related to resistance to clubroot disease of Chinese cabbage. The construction method has high specificity and is applicable to BraBBE19 the application of the gene; the present invention transforms the overexpression vector of the BraBBE19 gene into wild-type Arabidopsis thaliana of the Columbia type by the Agrobacterium tumefaciens transformation method to obtain BraBBE19 heterologous overexpression Arabidopsis thaliana lines. Through disease resistance identification, it is found that the heterologous overexpression of Chinese cabbage BraBBE19 will enhance the resistance of Arabidopsis thaliana to clubroot disease, while the resistance of the T-DNA insertion mutant Arabidopsis thaliana lines of the homologous gene BraBBE19 of Chinese cabbage to clubroot disease decreases; therefore, Chinese cabbage BraBBE19 is related to resistance to clubroot disease, and this gene can be applied to molecular breeding of cruciferous crops, having good application prospects. AT4G20840 BraBBE19 Brief Description of the Drawings Brief Description of the Drawings

[0013] Figure 1 BraBBE19 is a schematic diagram of the overexpression vector; is an electrophoretogram for detecting the T-DNA insertion mutant material WiscDsLox432E05 of the Figure 2 BraBBE19 gene; is a comparison diagram of the overall growth of overexpression plants and control plants 21 days after inoculation with Plasmodiophora brassicae (the scale bar is 5 cm); Figure 3 BraBBE19 is a comparison diagram of the root phenotypes of overexpression plants and control plants 21 days after inoculation with Plasmodiophora brassicae (the scale bar is 2 cm); Figure 4 BraBBE19 is a comparison diagram of the root phenotypes of overexpression plants and control plants 21 days after inoculation with Plasmodiophora brassicae (the scale bar is 2 cm); BraBBE19 is a comparison diagram of the root phenotypes of overexpression plants and control plants 21 days after inoculation with Plasmodiophora brassicae (the scale bar is 2 cm); Figure 5Comparison diagram of the overall growth of WiscDsLox432E05 mutant plants and control plants 21 days after inoculation with Plasmodiophora brassicae (scale bar is 5 cm); Figure 6 Comparison diagram of the root phenotypes of WiscDsLox432E05 mutant plants and control plants 21 days after inoculation with Plasmodiophora brassicae (scale bar is 2 cm); Figure 7 21 days after inoculation with Plasmodiophora brassicae BraBBE19 Disease index of overexpression plants, mutant plants and control plants of the gene. Specific implementation mode

[0014] The technical solution of the present invention is illustrated by specific examples. There may be other method steps before, after or between one or more method steps provided by the present invention, and the arrangement order of each method step can also be changed or adjusted. These examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the case of no substantial change in technical content, it should also be regarded as the implementable scope of the present invention.

[0015] For the present invention to 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 only to the embodiments set forth herein.

[0016] The present invention provides a gene related to resistance to clubroot disease of Chinese cabbage BraBBE19 , which is cloned from Chinese cabbage ( Brasica rapa var. pekinensis ) ECD05, and the gene has the sequence shown in SEQ ID No. 1.

[0017] The embodiment of the present invention also provides the application of the above-mentioned gene related to resistance to clubroot disease of Chinese cabbage BraBBE19 in regulating resistance to clubroot disease, which will be specifically described below.

[0018] Example 1: BraBBE19 Construction of overexpression vector

[0019] 1. Synthesis of cDNA: Use the Trizol method to extract the RNA of Chinese cabbage ECD05, reverse transcribe the RNA into cDNA, and use the PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time ) reverse transcription kit of TaKaRa Company, and the specific operation is carried out according to its instruction manual.

[0020] 2. PCR amplification of fragments: Design specific primers, and their sequence information is shown in Table 1; use the KOD One PCR MasterMix from TOYOBO Company TM to amplify the CDS sequence. The PCR reaction system and procedure are carried out according to the instructions. The amplified PCR products are separated by 1% agarose gel electrophoresis. If the length is in line, cut the gel and recover to obtain the target sequence. BraBBE19

[0021] Table 1: Primers used for constructing heterologous expression vectors

[0022] 3. Double digestion of pFGC1008 vector: At the Sal Ⅰ and Kpn Ⅰ sites, perform double digestion on the pFGC1008 vector. The double digestion system is as follows: 4 μL of Buffer, 2 μL each of Sal Ⅰ and Kpn Ⅰ, and make up to 40 μL with double-distilled water (ddH 2 2O). Incubate in a water bath at 37°C for 1 h. The digested products are separated by 1% agarose gel electrophoresis, cut the gel and recover to obtain the linearized vector.

[0023] 4. Homologous recombination ligation: Perform homologous recombination ligation on the above PCR amplified fragment and the linearized vector. For homologous recombination, use the single-fragment homologous recombination kit C112 from Novoprotein. The system is: 4 μL of 5×CEⅡ Buffer, 2 μL of ExaseⅡ, 200 ng of linearized vector, 20 ng of PCR amplified fragment, and make up to 20 μL with ddH 2 2O. The reaction procedure is to react at 37°C for 30 min.

[0024] 5. Transformation of Escherichia coli: Use the freeze-thaw method to transform the product of homologous recombination ligation into Escherichia coli competent cells. After transformation, perform colony PCR verification, extract the target plasmid for sequencing. The plasmid with successful sequence alignment is transformed into Agrobacterium competent cells, and colony PCR is also performed to verify whether there is a target band, and the plasmid is extracted for sequencing. The final obtained BraBBE19 overexpression vector map is as shown in Figure 1 . For the successfully verified bacterial solution, preserve the bacterial strain and store it in a 4°C refrigerator for later use.

[0025] Example 2: Arabidopsis thaliana floral dip transformation and screening of positive transformants

[0026] 1. Transformation of Arabidopsis thaliana by floral dip method: ​Using the Agrobacterium liquid culture verified by sequencing as the mother liquor, Arabidopsis thaliana was transformed by the floral dip method. The method steps are as follows: Add 500 μL of the Agrobacterium liquid containing the target vector to 200 mL of liquid LB medium containing kanamycin and rifampicin (50 mg / L), and shake the bacteria at 28 °C and 200 rpm for about 30 h until the OD 600 reaches 1.2; centrifuge the bacterial liquid at 8000 rpm for 10 min to obtain the Agrobacterium precipitate, and resuspend the bacterial liquid with 200 ml of freshly prepared 5% sucrose; add Silwet L-77 to a final concentration of 200 μL / L, shake at 28 °C and 200 rpm for 2 min to obtain the bacterial liquid; remove the open flowers and siliques from wild-type (WT) Arabidopsis thaliana, immerse the flower buds in the bacterial liquid for 1 min, blot dry the excess bacterial liquid, culture in a humid environment at 25 °C in the dark for 24 h, remove the dark culture and then culture normally, and repeat the floral dip once after one week to improve the transformation efficiency.

[0027] 2. Prepare the sowing medium: Prepare 1 / 2 MS medium. After autoclaving and cooling to 50 - 60 °C, add hygromycin to a final concentration of 90 mg / L in a laminar flow hood, pour solid plates, and store at 4 °C for later use.

[0028] 3. Screen positive transformants: Screen the T 1 generation Arabidopsis thaliana seeds harvested by floral dip on the sowing medium. The method steps are as follows: In a laminar flow hood, disinfect Arabidopsis thaliana seeds with 75% ethanol for 5 min, then wash with sterile water 5 - 6 times, 1 min each time. After that, evenly sow the seeds on the sowing medium, seal the medium with a sealing film, place it in a 22 °C culture room, and transfer the vigorously growing positive plants after two weeks of culture to a phytotron for normal culture.

[0029] Example 3: Screening of T-DNA insertion mutant materials

[0030] 1. Extract DNA by the rapid extraction method: Cut an appropriate amount of Arabidopsis thaliana leaves to be detected, put them into a centrifuge tube, add 200 μL of DNA extraction buffer, add magnetic beads, place them in a tissue disruptor to disrupt the tissue (65 Hz, 120 s) to obtain tissue fluid; transfer all the tissue fluid to a new 1.5 mL centrifuge tube, centrifuge at 13000 rpm for 8 min to obtain the supernatant; prepare a new 1.5 mL centrifuge tube, add 100 μL of isopropanol to each tube, take 100 μL of the supernatant and transfer it to the centrifuge tube, gently shake about 50 times, and let it stand at room temperature (about 25°C) for 5 min; centrifuge at 13000 rpm for 6 min to remove the supernatant; wash the precipitate twice with 1 mL of 70% ethanol, specifically shake up and down 20 times, centrifuge at 13000 rpm for 3 min, discard the supernatant, and repeat once; centrifuge without adding sample for 1 min. After washing, suck out the excess liquid, let the precipitate dry for 5 min, add 25 - 50 μL of ddH 2 O, and store it at -20°C for later use.

[0031] 2. PCR detection by the three-primer method: The three primers are LP, RP, and BP, and their corresponding primer sequence information is shown in Table 2.

[0032] Table 2: Primers used for screening T-DNA insertion mutant materials

[0033] The PCR reaction uses 1.1× T3 Super Mix PCR from Tsingke Biological, and the reaction system is: 44 μL of 1.1×T3super Mix, 2 μL of Template, 2 μL of Primer F, 2 μL of Primer R. The reaction program is: pre-denaturation at 98°C for 2 min 30 s, enter 35 cycles of amplification (denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 10 s), and finally extension at 72°C for 2 min; perform PCR amplification with LP + RP primers and BP + RP primers respectively to obtain PCR products, separate them by 1% agarose gel electrophoresis, and detect the bands. The detection results are as Figure 2 shown, where the M lane is the DL2000 marker, LP+RP and BP+RP are the corresponding primers, and the homozygous plants are shown in the red box. From Figure 2 it can be seen that the plants corresponding to the lanes where the LP + RP primer amplification product has no band and the BP + RP primer amplification product has a band are homozygous mutant materials, and the seeds are harvested for subsequent experiments.

[0034] Example 4: Plasmodiophora brassicae infection and disease experiment and phenotype observation

[0035] 1. Preparation of Plasmodiophora brassicae spore suspension: After the root rot was fully decomposed, it was disinfected with 70% alcohol for 1 min, treated with 10% NaClO for 20 min, and rinsed 3 times with sterile water. The root rot was ground into a homogenate with a juicer and filtered twice with eight layers of sterile gauze. The filtrate was centrifuged at 500 rpm for 5 min, and the black precipitate was discarded, leaving the supernatant and the gray precipitate. A new 50 mL centrifuge tube was used, filled with sterile water to 45 mL, and centrifuged at 4000 rpm for 15 min. The supernatant was discarded. Then, sterile water was added to 40 mL, and centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and this was repeated once. The precipitate was dissolved in 50% sucrose solution and centrifuged at 3100 rpm for 10 min. The supernatant and the outermost off-white precipitate were transferred to a new 50 mL centrifuge tube. Sterile water was added to 45 mL, and centrifuged at 4000 rpm for 10 min. The supernatant was discarded. Sterile water was added to 30 mL, and centrifuged at 3100 rpm for 10 min. The supernatant was discarded, and this was repeated 2 - 3 times. The precipitate was dissolved in 10 - 15 mL of sterile water to obtain a spore suspension, which was stored in a -4℃ refrigerator for later use.

[0036] Use a hemocytometer (1 / 400 mm 2 ), and calculate the concentration of the spore suspension according to the following formula, where the spores on the upper and left lines are counted into the grid: Concentration of spore suspension (cells / mL) = [(Number of dormant spores in 80 small grids of upper left + lower left + middle + upper right + lower right)] / 80 × 400 × 10 4 × dilution factor.

[0037] 2. Infection, disease development and phenotype observation: The overexpressing plants in Example 2, the mutant plants in Example 3 and the corresponding control plants were transplanted into trays filled with substrate. The trays were punched in a 12×6 pattern, with 12 plants as one biological replicate. The overexpressing plants, mutant plants and control plants were each set up with 3 biological replicates. After 5 days of acclimation, a 1 mL spore suspension (concentration 1×10 7 cells / mL) was injected into the roots of each seedling with a sterile syringe. Water was controlled within 48 h after injection to ensure successful infection by the clubroot fungus. After 48 h, normal management was carried out, and the disease incidence was counted 21 days after inoculation.

[0038] The disease was classified into 5 grades: Grade 0, asymptomatic; Grade 1, clubroot mainly in lateral roots, main root asymptomatic; Grade 2, small clubroots appeared on the main root and a few lateral roots; Grade 3, medium and large clubroots appeared on the main root and lateral roots, and plant growth was damaged; Grade 4, severe clubroots appeared on the main root and lateral roots, fine roots were completely destroyed, and plant growth was affected. The disease index calculation formula is: Disease index DI = (1 n 1 + 2 n 2 +3 n3 + 4 n 4 )× 100 / 4 N t , where n 1 to n 4 is the number of plants in the specified grading, N t is the total number of plants counted.

[0039] The results showed that: in the disease resistance identification, BraBBE19 there was no significant difference in the growth of heterologous overexpressing plants and empty vector plants, as Figure 3 shown, but the root disease condition was lighter than that of the empty vector plants, as Figure 4 shown. After statistics, the disease index was significantly lower than that of the empty vector plants, as Figure 7 shown, where * indicates a significant difference at the p = 0.05 level; There was no significant difference in size between the T-DNA inserted Arabidopsis mutant WiscDsLox432E05 and the wild type, but the leaves and stems turned purple, and the stress symptoms were more obvious, as Figure 5 shown, and the disease condition was more serious than that of the wild type, as Figure 6 shown, and the disease index was also significantly higher than that of the wild type, as Figure 7 shown. In summary, the overexpression of Chinese cabbage BraBBE19 can enhance the resistance of Arabidopsis to clubroot disease, while the resistance of the T-DNA inserted WiscDsLox432E05 mutant plants of the homologous gene BraBBE19 of Chinese cabbage AT4G20840 to clubroot disease decreased; Therefore, Chinese cabbage BraBBE19 is closely related to the occurrence of clubroot disease and is a gene related to clubroot disease resistance.

[0040] It should be noted that the materials used in the above examples can be easily obtained from commercial companies if not otherwise specified; Among them, Arabidopsis: Arabidopsis thaliana of Col-0 type; Chinese cabbage ECD05, donated by Professor G. R. Dixon of the University of Reading, UK, and self-propagated and reserved seeds by the laboratory; Diseased roots: collected from diseased plants, washed and stored in a -20°C refrigerator.

[0041] The above are the preferred specific embodiments of the present invention. These embodiments are for better explaining the principles and practical applications of the present invention, so that those skilled in the relevant technical fields can understand and utilize the present invention well. However, these embodiments do not describe all details in detail, and some improvements or modifications can be made on this basis, which are obvious to any person skilled in the art. Therefore, these modifications and improvements made on the basis of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A gene related to cabbage clubroot resistance BraBBE19 , characterized in that, Said BraBBE19 It has the nucleotide sequence shown in SEQ ID No.

1.

2. A cabbage clubroot resistance-related gene according to claim 1 BraBBE19 The method for constructing an overexpression vector is characterized in that: Specifically include: Design of specific primers to amplify Brassica rapa BraBBE19 The nucleotide sequence of the gene; double-enzyme digestion of pFGC-1008 vector to obtain a linearized vector; homologous recombination connection of the amplified sequence and the linearized vector to obtain a recombinant product; transformation of the recombinant product to obtain Chinese cabbage BraBBE19 Gene overexpression vector; wherein the sequences of the primers are the sequences shown in SEQ ID No.2 and SEQ ID No.

3.

3. A cabbage clubroot resistance-related gene according to claim 1 BraBBE19 A method for identifying mutant materials, characterized in that: Specifically include: Using the Arabidopsis DNA to be screened as a template, a three-primer PCR amplification test was performed to identify the cabbage BraBBE19 Homozygous mutant material of a 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.

4. A method comprising the cabbage clubroot resistance-related gene according to claim 1 BraBBE19 The biomaterial is characterized in that The biological material is an expression vector, an expression cassette, a host cell or an engineered bacterium.

5. A cabbage clubroot resistance-related gene according to claim 1 BraBBE19 Or the use of the biomaterial according to claim 4 in regulating the disease resistance function of plants.

6. A cabbage clubroot resistance-related gene according to claim 1 BraBBE19 Or use of the biological material according to claim 4 in preparing transgenic plants.

7. A cabbage clubroot resistance-related gene according to claim 1 BraBBE19 Or the use of the biological material according to claim 4 in plant breeding.

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