Application of calmodulin binding protein CBP60g or coding gene CBP60g thereof in prevention and treatment of clubroot

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to application of a calmodulin binding protein CBP60g or a coding gene CBP60g thereof in prevention and treatment of clubroot, and the amino acid sequence of the calmodulin binding protein CBP60g is shown as SEQ ID NO.1. The invention also relates to a preparation method of the calmodulin binding protein CBP60g. By overexpressing the CBP60g in the arabidopsis thaliana, the resistance of the CBP60g to the plasmodiophora brassicae can be recovered at high temperature, the CBP60g is also verified in the crop oilseed rape, and the normal growth, yield and quality of the oilseed rape are not influenced, so that the CBP60g gene can resist the plasmodiophora brassicae at high temperature; the restoring force of the crops to immune response of high-temperature composite stress is increased, and a strategy is provided for molecular breeding for coping with climate change.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to application of calmodulin binding protein CBP60g or its encoding gene CBP60g in the prevention and treatment of clubroot disease. Background Art

[0002] Greenhouse gas emissions are increasing year by year, and compound heat waves and drought extreme weather will become more frequent. Climate conditions greatly affect the interaction between plants and pathogens, increasing the probability of plant disease epidemics. As a biotrophic protozoan, root knot fungi can cause devastating root knot disease, which is more severe at high temperatures, causing serious yield losses in cruciferous crops. Therefore, it is urgent to provide technical means to effectively prevent and control root knot disease under high temperatures. Summary of the invention

[0003] The purpose of the present invention is to provide the application of calmodulin binding protein CBP60g and / or encoding gene CBP60g in the prevention and treatment of clubroot disease, and the disease resistance of Arabidopsis and rapeseed to clubroot fungi can be enhanced under high temperature conditions by promoting the expression of calmodulin binding protein CBP60g.

[0004] The present invention provides application of calmodulin binding protein CBP60g and / or encoding gene CBP60g in prevention and treatment of clubroot disease; the amino acid sequence of calmodulin binding protein CBP60g is shown in SEQ ID NO.1.

[0005] In a specific implementation, the nucleotide sequence encoding the gene CBP60g is shown as SEQ ID NO.2.

[0006] In a specific implementation, the root knot disease prevention and control includes: enhancing the plant's resistance to root knot fungi under high temperature conditions.

[0007] In a specific implementation, the application also includes: increasing the resilience of the plant's immune response to high temperature combined stress.

[0008] In a specific implementation, the plant includes: a cruciferous plant.

[0009] In a specific implementation, the cruciferous plants include: Arabidopsis thaliana and / or rapeseed.

[0010] In a specific implementation, it is characterized in that the high temperature is 20-35°C.

[0011] The present invention provides a method for preventing and treating clubroot disease, comprising the following steps: promoting the expression of a coding gene CBP60g in a target plant or increasing the content of a calmodulin binding protein CBP60g in the target plant.

[0012] In a specific implementation, the promotion process further includes: constructing an overexpression vector containing the coding gene CBP60g.

[0013] In a specific implementation, the basic backbone vector of the overexpression vector includes pGWB517.

[0014] Beneficial effects: The present invention provides the use of calmodulin binding protein CBP60g and / or encoding gene CBP60g in the prevention and treatment of root knot disease; the amino acid sequence of calmodulin binding protein CBP60g is shown in SEQ ID NO. 1. Calmodulin binding protein CBP60g described in the present invention is a key transcription factor of ICS1, and its expression is inhibited at high temperature, resulting in impaired plant immunity. By overexpressing CBP60g in Arabidopsis, its resistance to root knot fungi can be restored at high temperature, which is also verified in the crop rapeseed and does not affect the normal growth, yield and quality of rapeseed, indicating that the CBP60g gene can resist root knot fungi at high temperature, increase the resilience of crop immune response to high temperature composite stress, and provide a strategy for molecular breeding to cope with climate change. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure shows the effect of overexpression of CBP60g on the growth phenotype of rapeseed; A: the growth phenotype of transgenic CBP60g rapeseed after 8 weeks of growth (from left to right: WT, A1-5, A1-20); B: the expression level of CBP60g in A1-5 and A1-20 families detected by qPCR;

[0016] Figure 2 This is the effect diagram of overexpression of CBP60g on rapeseed yield traits; A: plant height data of rapeseed during flowering period, B: thousand-grain weight of rapeseed, C: oil content of rapeseed;

[0017] Figure 3 The phenotypes of transgenic CBP60g rapeseed after inoculation with root knot fungi at different temperatures (from left to right are WT, A1-5, A1-20, top is 28℃, bottom is 22℃);

[0018] Figure 4 The statistical diagram of the incidence of rapeseed inoculated with root-knot fungi at different temperatures; A: the proportion of each disease level after inoculation with root-knot fungi; B: the content of root-knot fungi in the diseased roots of rapeseed quantified by qPCR;

[0019] Figure 5 The effect diagram of overexpression of CBP60g on the growth phenotype of Arabidopsis thaliana; A: Growth phenotype of overexpression of CBP60g Arabidopsis thaliana, B: CBP60g expression level of WT and transgenic Arabidopsis thaliana;

[0020] Figure 6The phenotypes of Arabidopsis thaliana inoculated with root-knot fungi at different temperatures are shown in Figure 1. The phenotypes of Col-0 (top) after inoculation with root-knot fungi at 22°C (left) and 28°C (right), and the phenotypes of Arabidopsis thaliana overexpressing CBP60g (bottom) after inoculation with root-knot fungi at 22°C (left) and 28°C (right);

[0021] Figure 7 The following are the disease statistics of Arabidopsis thaliana inoculated with root-knot fungi at different temperatures, where A: the proportion of each disease level after inoculation with root-knot fungi; B: the experimental results of the root-knot fungi content determination after inoculation with root-knot fungi. DETAILED DESCRIPTION

[0022] The present invention provides the use of calmodulin binding protein CBP60g and / or encoding gene CBP60g in the prevention and treatment of clubroot disease; the amino acid sequence of calmodulin binding protein CBP60g is shown in SEQ ID NO.1:

[0023] MKIRNSPSFHGGSGYSVFRARNLTFKKVVKKVMRDQSNNQFMIQMENMIRRIVREEIQRSLQPFLSSSCVSMERSRSETPSSRSRLKLCFINSPPSSIFTGSKIEAEDGSPLVIELVDATTNTLVSTGPFSSSRVELVPLNADFTEESWTVEGFNRNILTQREGKRPLLTGDLTVMLKNGVGVITGDIAFSDNSSWTRSRKFRLGAKLTGD GAVEEARSEAFGCRDQRGESYKKHHPPCPSDEVWRLEKIAKDGVSATRLAERKILTVKDFRRLYTVNRNELHNIIGAGVSKKTWNTIVSHAMDCVLDETECYIYNA NTPGVTTLLFNSVYELIRVSFNGNDIQNLDQPILDQLKAEAYQNLNRITAVNDRTFVGHPQRSLQCPQDPGFVVTCSGSQHIDFQGIQYVMVMVLSLLIYLYLTID.

[0024] In the specific implementation of the present invention, the nucleotide sequence encoding the gene CBP60g is shown in SEQ ID NO.2:

[0025]

[0026] In the specific implementation of the present invention, the clubroot disease control includes: enhancing the plant's resistance to clubroot fungi under high temperature conditions. In one embodiment, the expression of calmodulin binding protein CBP60g is inhibited at high temperatures, resulting in impaired plant immunity, and overexpression of CBP60g in Arabidopsis can restore its resistance to clubroot fungi at high temperatures.

[0027] In the specific implementation of the present invention, the plant includes a cruciferous plant; for example, in one embodiment, the cruciferous plant refers to Arabidopsis thaliana, and in another embodiment, the cruciferous plant rapeseed, of course, can also be other cruciferous plants, and both can achieve similar technical effects as the embodiments of the present invention.

[0028] In a specific embodiment of the present invention, the high temperature is 20-35°C, such as 24-28°C. In one embodiment of the present invention, the high temperature is 28°C.

[0029] In the specific implementation of the present invention, the application also includes: increasing the resilience of the plant's immune response to high temperature combined stress. The present invention can restore its resistance to root knot fungi at high temperature by overexpressing CBP60g in Arabidopsis, which has also been verified in the crop rapeseed and has no effect on the normal growth, yield and quality of rapeseed.

[0030] The present invention provides a method for preventing and treating clubroot disease, comprising the following steps: promoting the expression of a coding gene CBP60g in a target plant or increasing the content of a calmodulin binding protein CBP60g in the target plant.

[0031] In the specific implementation of the present invention, the coding gene CBP60g was cloned into pGWB517 by Gateway reaction (Invitrogen) to construct an overexpression vector containing the coding gene CBP60g. The present invention can enhance the disease resistance of Arabidopsis and rapeseed to root knot fungi under high temperature conditions by overexpressing CBP60g, and increase the resilience of the immune response of crops to high temperature combined stress.

[0032] To further illustrate the present invention, the application of the calmodulin binding protein CBP60g and / or the encoding gene CBP60g provided by the present invention in the prevention and treatment of clubroot disease is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1 Acquisition of transgenic rapeseed

[0034] Extraction of total RNA from rapeseed: Take 0.1g of leaves, grind them into powder quickly in liquid nitrogen, transfer the powder to a 1.5mL EP tube, add 1mL Trizol, shake vigorously and mix, and place on ice for 15min; add 200μL chloroform, shake vigorously and mix, and place on ice for 15min; centrifuge at 4℃, 12000rpm for 15min; carefully transfer the supernatant to a 1.5mL centrifuge tube, add isopropanol equal to the volume of the supernatant, mix, and place on ice for 30min. Centrifuge at 4℃, 12000rpm for 15min; carefully discard the supernatant, wash twice with 1mL 75% alcohol; place the centrifuge tube in a centrifuge and empty for 1min, then aspirate the supernatant as thoroughly as possible, dry at room temperature for 3-5min to completely evaporate the alcohol, add 30μL DEPC water, and store at -80℃ for later use after the RNA precipitate is dissolved.

[0035] Vector construction: RNA was used as a template for reverse transcription using a reverse transcription kit (Novozyme). The system conditions were 5×All-in-oneqRTSuperMix 4 μL, EnzymeMix 1 μL, RNA 2 μg, and ddH 2 O was added to 20 μL. The reaction program conditions were 50℃15min, 85℃5s. The cDNA product generated by the reaction was stored at 4℃ for later use. Using cDNA as a template, specific primers (Kim, JH, Castroverde, CDM, Huang, S. et al. Increasing theresilience of plant immunity to a war ming climate. Nature 607, 2022, 339–344) were used to amplify the target gene. The reaction system conditions were Pfu DNA Polymerase 1 μL, Fast Pfu Buffer polymerase 10 μL, High Purified NTPs 2.5 μL, cDNA 2 μL, primers F / R 1 μL each, and ddH 2 O was added to 50 μL. The reaction conditions were pre-denaturation at 95°C for 2 min, denaturation at 94°C for 20 s, annealing at 57°C for 20 s, extension at 72°C for 15 s, 35 cycles, final extension at 72°C for 5 min, and storage at 4°C for 2 min. The PCR product was recovered after agarose gel electrophoresis, and some samples were sent for sequencing. It was cloned into pGWB517 through Gateway reaction (Invitrogen), and a single colony was picked and sent to the company for sequencing after positive PCR verification. The correct sequencing was used to extract the plasmid and transform it into the target Agrobacterium.

[0036] Agrobacterium culture: 3 days after rape sowing, streak a single colony of the target Agrobacterium; 5 days after sowing, take a single colony to 2mL double resistance culture medium and shake overnight for 12 hours; 6 days after sowing, use liquid LB to culture the target bacteria. Use a 20mL glass bottle to add 10mL resistance LB culture medium (resistance LB culture medium: 1L of culture medium contains 10g of peptone, 5g of yeast extract, 5g of sodium chloride, 10g of agar powder, 50mg of rifampicin antibiotics and 50mg of hygromycin) and 1mL of target Agrobacterium, and culture at 28℃ in a shaker at 180-220rpm for about 4-6 hours.

[0037] Preparation and infection of explants: prepare M1 medium, add acetosyringone AS (final concentration 100 μM) to the medium after it cools down, and obtain M1 (AS+) medium for use; add AS (final concentration 100 μM) to DM solution to obtain DM (AS+) solution; measure bacterial OD with a spectrophotometer 600 value, about 0.5 in LB is better; prepare bacterial night, pipette 2mL of cultured bacterial liquid into a sterile centrifuge tube, centrifuge at 3000rpm for 3min, and discard the supernatant; add 2mL DM (AS+) liquid, centrifuge at 3000rpm for 3min, and discard the supernatant; add 2mL DM (AS+) liquid to obtain DM bacterial night, and put it in a 4℃ refrigerator for use; add 18mL DM culture medium to the explant cutting dish in advance, move the explants into it after cutting, pour in 2mL DM bacterial liquid, soak for 15min, shake every 3min, and repeat 4 to 5 times; when the infection is at the 10th minute, use a pipette to suck off the DM bacterial night, spread the explants on sterile filter paper, blow for 3min in the clean bench to suck away and evaporate the excess bacterial liquid on the explants. The explants were transferred to M1 (AS+) medium and placed in dim light at 22-24°C. After 22-26 hours of co-culture, the explants were transferred to M2 medium for 14-18 days, and callus formation was induced and then cultured normally under light. After 24 days of culture, the explants were transferred to M3 medium, and then subcultured every 2-3 weeks until green buds appeared. The green buds with complete growth points were transferred to M4 medium to grow and root, and the rooted T0 plants were transferred to soil for normal growth.

[0038] Verification and screening of transgenic plants: After extracting rapeseed DNA for PCR verification, the T0 generation was self-pollinated to produce the T1 generation, and two pairs of primers for HPT and AtCBP60g genes (see: Kim, JH, Castro verde, CDM, Huang, S. et al. Increasing there immunity of plant immunity to a warming climate. Nature 607, 2022, 339-344) were used for identification. And quantitative primers (gene number AT5G26920) and rapeseed internal reference primers (Kim, JH, Castro verde, CDM, Huang, S. et al. Increasing there immunity of plant immunity to a warming climate. Nature 607, 2022, 339-344) were used for qPCR screening. The expression level of CBP60g in A1-5 and A1-20 families was detected by qPCR. The expression data of genes analyzed by qPCR were normalized with the rapeseed BnaGDI1 gene. The expression data of genes analyzed by qPCR were normalized with the rapeseed BnaGDI1 gene. Statistical analysis was performed using one-way analysis of variance (significance was set at P ≤ 0.05). n = 3 biological replicates; data are presented as mean ± sd. Families with high and stable expression levels in the T1 generation were screened (see Tables 1 and Figure 1 Middle B). Self-pollination produces the T2 generation, which is further used to obtain the T3 generation, and the T3 generation is used for experiments.

[0039] Table 1 Expression of CBP60g in A1-5 and A1-20 families detected by qPCR

[0040]

[0041] Test Example 1: Rapeseed root knot fungus inoculation test

[0042] Extraction of dormant spores of root knot fungus: wash the swollen part of the root of rapeseed, cut the swollen tissue into small pieces with a knife, add an appropriate amount of sterile water and break it in a homogenizer; filter the plant fragments with 8 layers of gauze, centrifuge at 5000rpm for 5min, the spores are in the precipitate, and remove the supernatant; suspend the precipitate with 50% sucrose solution on a vortex instrument, and centrifuge at 5000rpm for 5min; collect the supernatant containing dormant spores, transfer it to another clean sterile centrifuge tube, add distilled water equal to the supernatant, mix well, centrifuge at 5000rpm for 5min, remove the supernatant, suspend the precipitate at the bottom of the tube with sterile distilled water, repeat this step (from collecting the supernatant containing dormant spores to suspending the precipitate at the bottom of the tube with sterile distilled water) 3 times to wash out the sucrose in the dormant spores; add sterile distilled water to the washed spore precipitate, vortex the spore precipitate to suspend the spores and store them at 4°C for standby use.

[0043] Inoculation test of root knot fungi at different temperatures: When rapeseed was cultured at 22℃ for 12 days, it was placed in 22℃ and 28℃ incubators for 2 days and then the root knot fungi were diluted to 1×10 7 / mL, use a pipette to water and inoculate 1mL to the roots of the plants, continue to culture in the incubator for 2 days, then transfer to a culture room at 22℃ for normal culture. Calculate the disease index 25 days after inoculation with root-knot fungi.

[0044] Statistical standard for clubroot disease index: The five-level grading standard for rapeseed is: Level 0, no disease; Level 1, very small tumors mainly on the lateral roots, and the damage to the main root is negligible; Level 3, the main root and a few lateral roots are covered with tiny tumors; Level 5, medium to large spherical tumors on the main root; Level 7, the root is completely enlarged.

[0045] Test Example 2 Test on the relative biomass determination of diseased rapeseed root knot fungus

[0046] Extraction of total DNA of root-knot fungus: When inoculating root-knot fungus, each family will inoculate 20 rapeseeds, and the disease index will be counted and photos will be taken after the disease occurs. Among these diseased seedlings, 3 representative seedlings from each family will be selected for DNA extraction and qPCR detection of the relative biomass of root-knot fungus. Take 3 representative diseased root systems, clean them, grind them into powder in liquid nitrogen, transfer them to 2mL enzyme-free centrifuge tubes, add 800μL of 2% CTAB separation buffer preheated in a 65℃ water bath; bathe in a 65℃ water bath for 30min, mix every 5min; add 400μL chloroform and 400μL DNA extraction phenol (absorb the liquid in the lower layer), mix, and centrifuge at 12000rpm for 15min; aspirate the supernatant to a 1.5mL enzyme-free centrifuge tube, add an equal amount of chloroform to the supernatant, mix, and centrifuge at 12000rpm for 15min; aspirate the supernatant to a 1.5mL enzyme-free centrifuge tube, add an equal amount of isopropanol to the supernatant, mix, and place on ice for 30min; centrifuge at 12000rpm for 15min; carefully discard the supernatant, and use 1mL Wash twice with 75% alcohol; place the centrifuge tube in a centrifuge and empty it for 1 minute, then aspirate the supernatant as thoroughly as possible. After the alcohol has completely evaporated, add 40 μL of RNase water, and store at -20°C until the DNA precipitate is dissolved.

[0047] Determination of relative biomass of root-knot bacteria in the root system: After the concentration of the extracted DNA was determined according to the extraction steps of the total DNA of root-knot bacteria, it was diluted to 200 ng / μL. The diluted DNA was used as a template to detect the content of the root-knot bacteria reference gene (gene encoding AY452179.1) relative to the reference gene of rapeseed (Kim, JH, Castroverde, CDM, Huang, S. et al. Increasing the resilience of plant immunity to a warming climate. Nature 607, 2022, 339–344) by qPCR reaction. The qPCR system is as follows: SYBR GREEN MIX 5μL, Forward primers 0.5μL, Reverse primers 0.5μL, CDNA 2μL, ddH 2O2μL. The reaction conditions were 95℃, 2min, 95℃, 15s, 60℃, 30s, 72℃, 20s, repeated 35-40 cycles; 65-95℃ to draw the melting curve (5s / 0.5℃). After the amplification was completed, the relative expression of the gene was calculated using the 2-ΔΔCt method. The content of root-knot bacteria in the diseased roots of rapeseed was quantified by qPCR, and the ordinate indicated the multiple of the root-knot bacteria content of different strains relative to WT. The relative biomass of root-knot bacteria was represented by the content of the root-knot bacteria ACTIN gene relative to the rapeseed BnaGDI1 gene. One-way analysis of variance was used for statistical analysis (significance set at P≤0.05). Among them, n=3 biological replicates; data are expressed as mean±sd.

[0048] Test Example 3 Determination of Thousand-Grain Weight and Oil Content of Rapeseed

[0049] The seeds of the mutants were harvested when they matured after 210 days of growth in the T3 generation, and then threshed after natural air drying. The thousand seeds of each plant were weighed, that is, the weight of 1000 seeds was weighed with a 1 / 1000 scale. Statistical analysis was performed using one-way analysis of variance (significance set at P ≤ 0.05). Among them, n ≥ 6 biological replicates; data are expressed as mean ± sd.

[0050] The oil content of 2-3 g seeds of 5 randomly selected plants from all wild-type (WT) and mutant rapeseed was determined using near infrared spectroscopy (NIR Systems 3750), and statistical analysis was performed using one-way analysis of variance (significance set at P ≤ 0.05). Among them, n = 5 biological replicates; data are expressed as mean ± sd.

[0051] The method of test examples 1 to 3 was used to measure Example 1, and the results are shown in Figure 1 .

[0052] Transgenic CBP60g rapeseed was obtained through genetic transformation, and the expression of CBP60g was detected by qPCR to select two families with high and stable expression levels: A1-5, A1-20 ( Figure 1 ). And at the flowering stage (about 180 days after the rapeseed plant grew), the plant heights of A1-5 and A1-20 were measured (the distance from the root base to the highest point of the rapeseed plant was measured with a tape measure) and there was no significant difference compared with the wild type WT (Westar) ( Figure 2 Middle A) shows that there is no difference in growth phenotype between rapeseed overexpressing CBP60g and WT. It can be seen that the introduction of CBP60g will not affect the normal growth phenotype and yield of the plant under normal growth conditions.

[0053] According to the method of Experimental Example 1, root knot fungus was inoculated to detect whether overexpression of CBP60g could enhance rapeseed's resistance to root knot fungus at high temperature. The experimental results showed that A1-5 and A1-20 were significantly higher than WT at 22°C and 28°C (see Figure 3 ) were significantly reduced, and the biological content of root knot fungi in the diseased roots detected by qPCR in Experiment 2 also corresponded to the disease index and was significantly lower than that of WT ( Figure 4 ).

[0054] At the same time, according to the above statistics, the thousand-grain weight and oil content of rapeseed of A1-5 and A1-20 after harvest were not significantly different from those of WT ( Figure 2 (B, C)

[0055] These results indicate that overexpression of CBP60g in rapeseed can enhance its resistance to root-knot fungi at both high temperature (28°C) and normal temperature (22°C) without affecting its normal growth and yield quality.

[0056] Example 2 Obtaining transgenic Arabidopsis

[0057] The CBP60g-overexpressing Arabidopsis material was provided by Professor Xin Xiufang (Wan, D., Li, R., Zou, B. et al. Calmodulin-binding protein CBP60g is a positive regulator of both disease resistance and drought tolerance in Arabidopsis. Plant Cell Rep 31, 2012, 1269–1281.) 。

[0058] The experiment was carried out in the same manner as in Experiment 1, except that the CBP60g primer and the Arabidopsis internal reference Actin2 primer (see: Wan, D., Li, R., Zou, B. et al. Calmodulin-binding protein CBP60g is a positive regulator of both disease resistance and drought tolerance in Arabidopsis. Plant Cell Rep 31, 1269–1281) were used to select a family OE-16 ( Figure 2 A and C).

[0059] Test Example 4 Arabidopsis root knot fungus inoculation test

[0060] Extraction of dormant spores of root knot fungus: wash the swollen part of the root of Arabidopsis thaliana, cut the swollen tissue into small pieces with a knife, add an appropriate amount of sterile water and break it in a homogenizer; filter the plant fragments with 8 layers of gauze, centrifuge at 5000rpm for 5min, the spores are in the precipitate, and remove the supernatant; suspend the precipitate with 50% sucrose solution on a vortex instrument, and centrifuge at 5000rpm for 5min; collect the supernatant containing dormant spores, transfer it to another clean sterile centrifuge tube, add an equal amount of distilled water, mix it, centrifuge it at 5000rpm for 5min, remove the supernatant, suspend the precipitate at the bottom of the tube with sterile distilled water, repeat this step (from collecting the supernatant containing dormant spores to suspending the precipitate at the bottom of the tube with sterile distilled water) 3 times to wash out the sucrose in the dormant spores; add sterile distilled water to the washed spore precipitate, vortex the spore precipitate to suspend the spores and store it at 4°C for use.

[0061] Inoculation test of root knot fungi at different temperatures: Arabidopsis thaliana was cultured at 22°C for 12 days, and then placed in 22°C and 28°C incubators for 2 days, and then root knot fungi were diluted to 1×10 7 / mL, use a pipette to water and inoculate 1mL to the roots of the plants, continue to culture in the incubator for 2 days, then transfer to a culture room at 22℃ for normal culture. Calculate the disease index 25 days after inoculation with root-knot fungi.

[0062] Statistical standard for clubroot disease index: The five-level grading standard for Arabidopsis is as follows: Level 0, no disease; Level 1, very small tumors mainly on the lateral roots, and the damage to the main root is negligible; Level 2, the main root and a few lateral roots are covered with tiny tumors; Level 3, medium to large spherical tumors on the main root; Level 4, serious tumors on the lateral roots and main root, the main root is completely swollen, and the fine roots on the lateral roots are completely damaged; Level 5, the roots are completely swollen and rotten.

[0063] Test Example 2 Test on the relative biomass determination of Arabidopsis thaliana root knot fungus

[0064] Extraction of total DNA of root knot fungus: Take 3 representative (T3) diseased roots from each plant family, clean them, grind them into powder in liquid nitrogen, transfer them to a 2mL enzyme-free centrifuge tube, add 800μL of 2% CTAB separation buffer preheated in a 65℃ water bath; bathe in a 65℃ water bath for 30min, mix every 5min; add 400μL chloroform and 400μL DNA extraction phenol (absorb the liquid in the lower layer), mix, and centrifuge at 12000rpm for 15min; aspirate the supernatant to a 1.5mL enzyme-free centrifuge tube, add an equal amount of chloroform to the supernatant, mix, and centrifuge at 12000rpm for 15min; aspirate the supernatant to a 1.5mL enzyme-free centrifuge tube, add an equal amount of isopropanol to the supernatant, mix, and place on ice for 30min; centrifuge at 12000rpm for 15min; carefully discard the supernatant, and use 1mL Wash twice with 75% alcohol; place the centrifuge tube in a centrifuge and empty it for 1 minute, then aspirate the supernatant as thoroughly as possible. After the alcohol has completely evaporated, add 40 μL of RNase water, and store at -20°C until the DNA precipitate is dissolved.

[0065] Determination of relative biomass of root-knot bacteria in the root system: After the concentration of the extracted DNA according to the extraction steps of the total DNA of root-knot bacteria is determined, it is diluted to 200 ng / μL. The diluted DNA is used as a template to detect the content of the root-knot bacteria internal reference gene (gene encoding AY452179.1) relative to the Arabidopsis internal reference gene (gene encoding AT3G18780) by qPCR reaction. The qPCR system is as follows: SYBR GREEN MIX 5μL, Forward primers 0.5μL, Reverse primers 0.5μL, CDNA 2μL, ddH 2 O 2μL. The reaction conditions were 95℃, 2min, 95℃, 15s, 60℃, 30s, 72℃, 20s, repeated 35-40 cycles; 65-95℃ to draw the melting curve (5s / 0.5℃). After the amplification was completed, the relative expression of the gene was calculated using the 2-ΔΔCt method. The content of root-knot bacteria in the diseased roots of Arabidopsis was quantified by qPCR, and the ordinate represents the multiple of the root-knot bacteria content of different strains relative to Col-0. The relative biomass of root-knot bacteria is represented by the content of the root-knot bacteria ACTIN gene relative to the Arabidopsis gene. Statistical analysis was performed using one-way analysis of variance (significance set at P≤0.05). Among them, n=3 biological replicates; data are expressed as mean±sd.

[0066] The test results of Example 2 are shown in Figures 5 to 7 .

[0067] from Figures 5 to 7It can be seen that OE-16 can enhance resistance to root-knot fungi at both high temperature (28°C) and normal temperature (22°C). The relative biomass of root-knot fungi was significantly reduced in the overexpression family. In the experiment of inoculating root-knot fungi, the disease index of transgenic plants was lower than that of wild type at both high and low temperatures, indicating that resistance can be enhanced at both high and low temperatures. These results indicate that overexpression of CBP60g can enhance Arabidopsis' resistance to root-knot fungi at both high and normal temperatures.

[0068] It can be seen that the calmodulin binding protein CBP60g is a key transcription factor of ICS1, and its expression is inhibited at high temperatures, resulting in impaired plant immunity. Overexpression of CBP60g in Arabidopsis can restore its resistance to root-knot fungi at high temperatures. The same has been verified in the crop rapeseed and has no effect on the normal growth, yield and quality of rapeseed, indicating that the CBP60g gene can be used to resist root-knot fungi at high temperatures, providing a strategy for molecular breeding to cope with climate change.

[0069] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of calmodulin binding protein CBP60g and / or encoding gene CBP60g in the prevention and treatment of clubroot disease; the amino acid sequence of calmodulin binding protein CBP60g is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence encoding the gene CBP60g is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The clubroot disease prevention and control comprises: enhancing the disease resistance of plants to clubroot fungi under high temperature conditions.

4. The use according to claim 1, characterized in that: The application also includes: increasing the resilience of the plant's immune response to high temperature combined stress.

5. The use according to claim 3 or 4, characterized in that: The plants include: cruciferous plants.

6. The use according to claim 5, characterized in that: The cruciferous plants include: Arabidopsis thaliana and / or rapeseed.

7. The use according to claim 3 or 4, characterized in that: The high temperature is 20-35°C.

8. A method for preventing and treating clubroot, characterized in that: The following steps are involved: The method promotes the expression of the coding gene CBP60g in the target plant or increases the content of the calmodulin binding protein CBP60g in the target plant.

9. The method according to claim 8, characterized in that The promotion method also includes: constructing an overexpression vector containing the coding gene CBP60g.

10. The method according to claim 9, characterized in that The basic backbone vector of the overexpression vector includes pGWB517.

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