Application of Arabidopsis S gene AtEIJ1
By inhibiting the expression of the S gene AtEIJ1 of Arabidopsis, the T-DNA insertion method or gene editing technology is used to enhance resistance in Arabidopsis or rapeseed, the problem of prevention and treatment of sclerosis and gray mold is solved, and the genetic resources of disease-resistant varieties are provided.
Patent Information
- Application Number
- CN202510334564.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-20
- Publication Date
- 2025-08-01
AI Technical Summary
The prior art is difficult to effectively prevent and treat sclerosis and grey mildew, especially the lack of functional research on the S gene AtEIJ1 of Arabidopsis, which leads to difficulties in cultivating disease-resistant varieties.
By inhibiting the expression of the Arabidopsis S gene AtEIJ1 with the Arabidopsis genome database number AT2G24860, T-DNA insertion, gene editing or RNAi technology is used to achieve enhanced resistance in Arabidopsis or rapeseed to prevent and treat infection of Scleroticus, Botrytis ale and Pseudomonas syringae tomato pathogenic variants.
The AtEIJ1 mutant of Arabidopsis S gene shows high resistance to Scleroticus and Botrytis aurora, and has strong resistance to Pseudomonas syringa tomato pathogenic variants, significantly reducing disease expansion, and providing a genetic resource against Sclerotica and Sclerotica.
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Figure CN120400173A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of bio - agriculture and relates to the application of the Arabidopsis thaliana S gene AtEIJ1. Background Art
[0002] Sclerotinia sclerotiorum and Botrytis cinerea (caused by Botrytis cinerea) cause serious damage to crops such as rapeseed, and are difficult to control. These two diseases have caused huge economic losses not only in China but also globally. Therefore, how to effectively control Sclerotinia sclerotiorum of rapeseed has become an urgent problem to be solved. In recent years, with the rapid development of molecular biology, molecular breeding techniques have become increasingly mature. At the same time, the research on the interaction mechanism between plants and pathogenic microorganisms has been continuously deepened, and genetic engineering techniques have also been continuously innovated and applied. Using transgenic technology and gene editing technology to cultivate disease - resistant germplasm resources can not only significantly shorten the breeding cycle of resistant varieties, but also may cultivate new varieties with durable broad - spectrum disease resistance. Plant disease - resistance (R) genes can detect pathogen attacks and promote the plant's defense response against pathogens. Different from R genes, there are some sensitive genes (S) in plants, which contribute to the successful infection of plants by pathogens. The confirmed S genes have the following characteristics: promoting the basic compatibility and continuous compatibility between pathogens and plants, and negatively regulating plant immune signal transduction. These genes promote the infection, proliferation and spread of pathogens by inhibiting the plant's disease - resistant response. Therefore, it is expected to obtain materials with broad - spectrum and durable disease resistance by interfering with or disrupting the function of plant S genes. Currently, there is no relevant report on the biological function of the Arabidopsis thaliana S gene AtEIJ1.
[0003] As a model plant, Arabidopsis thaliana is often used to infer the functions of homologous genes in other Brassicaceae plants. Arabidopsis thaliana and rapeseed belong to the Brassicaceae family, and there is a high degree of homology between their genomes. Studying the functions of Arabidopsis thaliana genes can provide important references for the function research of rapeseed homologous genes. For example, the Arabidopsis thaliana AtZntB1 gene has been confirmed to be involved in magnesium ion transport, and the cloning and function verification of its rapeseed homologous gene also show a similar mechanism. After the precursor gene of Arabidopsis thaliana miR395d was introduced into rapeseed by Agrobacterium tumefaciens transformation, the transgenic plants showed sulfur metabolism regulation characteristics similar to those of Arabidopsis thaliana. The genes related to glucosinolate metabolism in Arabidopsis thaliana (such as AOP3, BZO1) and their rapeseed homologous genes (BnMAM1, BnCYP83A1) showed similarity in function and were all involved in the glucosinolate synthesis pathway. Summary of the Invention
[0004] The purpose of the present invention is to provide the application of the Arabidopsis thaliana S gene AtEIJ1 in view of the above - mentioned deficiencies of the prior art.
[0005] The purpose of the present invention can be achieved by the following technical solutions:
[0006] Use of suppressing expression of Arabidopsis thaliana S gene AtEIJ1 with Arabidopsis thaliana genome database number AT2G24860 in enhancing plant resistance to Sclerotinia sclerotiorum, Botrytis cinerea and / or Pseudomonas syringae pv. tomato DC3000. The genomic sequence of AtEIJ1 is shown as SEQ ID NO.1, the cDNA sequence is shown as SEQ ID NO.2, the cds sequence is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO.4.
[0007] Preferably, the plant is selected from Arabidopsis thaliana or Brassica napus.
[0008] Preferably, the suppression of the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion or gene editing.
[0009] Use of suppressing expression of Arabidopsis thaliana S gene AtEIJ1 with Arabidopsis thaliana genome database number AT2G24860 in controlling plant diseases caused by infection of Sclerotinia sclerotiorum, Botrytis cinerea and / or Pseudomonas syringae pv. tomato DC3000.
[0010] Preferably, the plant is selected from Arabidopsis thaliana or Brassica napus.
[0011] Preferably, the suppression of the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion, gene editing or RNAi technology.
[0012] A method for controlling plant diseases caused by infection of Sclerotinia sclerotiorum, Botrytis cinerea and / or Pseudomonas syringae pv. tomato DC3000, which comprises suppressing the expression of Arabidopsis thaliana S gene AtEIJ1 with Arabidopsis thaliana genome database number AT2G24860 in the target plant.
[0013] Preferably, the suppression of the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion, gene editing or RNAi technology.
[0014] Application of Arabidopsis S gene AtEIJ1 with Arabidopsis Genome Initiative number AT2G24860 in constructing transgenic plants resistant to Sclerotinia sclerotiorum, Botrytis cinerea and / or Pseudomonas syringae pv. tomato DC3000.
[0015] Beneficial effects:
[0016] The present invention discovers that mutants of Arabidopsis S gene AtEIJ1 show high resistance to Sclerotinia sclerotiorum and Botrytis cinerea, and have strong disease resistance to Pst. DC3000. It indicates that AtEIJ1 can be used as a potential gene resource for cultivating varieties resistant to Sclerotinia sclerotiorum and Botrytis cinerea in rapeseed. Description of the drawings
[0017] Figure 1 Schematic diagram of the principle for designing T-DNA primers
[0018] Figure 2 Identification of Arabidopsis homozygous mutants (A) Homozygous verification of Arabidopsis mutants. M: DNA Marker; WT: Wild-type Arabidopsis col-0; HZ: Heterozygous lines; HM: Homozygous Lines; LP and RP: Primers on both sides of the T-DNA insertion site on the plant genome; BP: Primer on the T-DNA segment. (B) Expression level of the target gene in its mutants (Three leaves from different plants of the same mutant were collected together, n = 3).
[0019] Figure 3 Growth phenotypes of Arabidopsis homozygous mutants (A) Phenotypes of Arabidopsis at each growth stage. Upper left: Root length phenotype after 7 days of seed germination on 1 / 2 MS medium; Lower left: Phenotype of Arabidopsis at about 4 weeks; Right: Phenotype of Arabidopsis at the bolting and flowering stage; Scale bar is 3 cm. (B) Root length statistics after 7 days of seed germination on 1 / 2 MS medium (n = 5). (C) Thousand-seed weight statistics of Arabidopsis seeds (n = 5).
[0020] Figure 4 Analysis of the resistance of mutants to Sclerotinia sclerotiorum 1980 (A) Leaf phenotypes of Arabidopsis after in vivo inoculation with Sclerotinia sclerotiorum 1980 (36 h). (B) Statistic of lesion area of Arabidopsis after inoculation with 1980 (36 h) (n = 8). (C) Statistic of relative biomass after inoculation with 1980 (36 h). Figure 5Analysis of the resistance of mutants to Botrytis cinerea B05.10 (A) Phenotype of Arabidopsis leaves after inoculation with Botrytis cinerea B05.10 (48 h). (B) Statistical analysis of the lesion area of Arabidopsis after inoculation with B05.10 (48 h) (n = 8). (C) Statistical analysis of the relative biomass expression after inoculation with B05.10 (48 h). Figure 6 Analysis of the resistance of mutants to Pst. DC3000 (A) Phenotype of Arabidopsis after 5 days of inoculation with the bacterium Pst. DC3000. (B) Plate coating of bacteria in Arabidopsis leaves at 0 hpi and 72 h after inoculation with Pst. DC3000 (Take 5 mm leaf margin pieces from the injected leaf and aliquot them into 3 2.0 mL EP tubes). (C) Statistical analysis of the number of bacteria in Arabidopsis leaves at 0 h and 72 h after inoculation. Detailed implementation methods
[0021] Materials and methods
[0022] 1.1 Test materials and culture conditions
[0023] Tested pathogenic microorganisms: Sclerotinia sclerotiorum 1980, Botrytis cinerea B05.10, and Pseudomonas syringae pv. tomato DC3000 (Pst. DC3000) are all stored in the laboratory.
[0024] Tested plants: Arabidopsis thaliana: col-0 (stored in the laboratory), mutant eij1 (AT2G24860) was purchased from
[0025] https: / / www.arashare.cn / index / Product / index.html and constructed using the T-DNA insertion method.
[0026] Culture conditions for pathogenic microorganisms: Sclerotinia sclerotiorum 1980 and Botrytis cinerea B05.10 were activated and cultured on PDA medium at 20 °C. Pst. DC3000 was activated and cultured on King's B (Kana, Rif) medium at 28 °C.
[0027] Plant culture conditions: Arabidopsis was cultured in a plant culture room with the conditions of 22 °C, 12 h of light, 12 h of darkness, and a relative humidity of 75%.
[0028] Example 1 Homozygous verification and phenotype of Arabidopsis mutants
[0029] The Arabidopsis mutant eij1 (AT2G24860) was obtained from
[0030] Purchase at https: / / www.arashare.cn / index / Product / index.html. It is constructed by the T-DNA insertion method. Since the gene sequence is known, it can also be constructed by oneself according to the conventional T-DNA insertion method in the art.
[0031] To determine that the obtained Arabidopsis thaliana seeds are homozygous mutants, analyze the effect of gene deletion on the agronomic traits of the plant itself. Vernalize the Arabidopsis thaliana mutant seeds for 3 days, spot-seed them on 1 / 2 MS medium and grow for 5 - 7 days, then transplant them into nutrient soil. After 3 weeks, extract the DNA of individual mutants respectively, and use the three-primer method (LP, BP, RP) to detect the homozygous genotype of the Arabidopsis thaliana mutant.
[0032] Specific method for homozygous verification of Arabidopsis thaliana mutant: Using the genomic DNA of Arabidopsis thaliana mutant eij1 (AT2G24860) as a template, the extraction method adopts the conventional CTAB method. The specific steps are as follows: Take 100 mg of fresh leaves, place them in a 2.0 mL clean centrifuge tube, add sterilized steel beads, quickly freeze and grind them into dry powder in liquid nitrogen, add 700 μL of 2% CTAB extraction buffer and invert to mix evenly. Place the sample in a 65 °C water bath for 30 min (invert to mix evenly every 10 min), centrifuge at 12000 r / min for 10 min, take 500 μL of the supernatant into a new 2.0 mL tube, add 500 μL of DNA extraction phenol and chloroform respectively, mix well, and centrifuge at 12000 r / min for 10 min. Take 400 μL of the supernatant into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol and mix well, let it stand at -20 °C for 20 - 30 min, centrifuge at 12000 r / min for 10 min and discard the supernatant. Add 700 μL of 75% ethanol to wash the precipitate, centrifuge at 12000 r / min for 1 min, discard the supernatant, repeat once, let the precipitate dry naturally at room temperature, add 30 μL of dd H2O to dissolve it, and take 1 μL as a template for PCR positive detection.
[0033] Use the three-primer method (LP, BP, RP, see Table 1 for details) to detect the homozygous genotype of Arabidopsis thaliana mutant (AT2G24860). The principle of the three-primer method ( Figure 1) Design an upstream primer LP and a downstream primer RP at positions 200 bp - 400 bp on both sides of the insertion site respectively, and design a specific primer BP on the T-DNA insertion fragment. Set up two pairs of reactions, namely the reaction with primers LP and RP, and the reaction with primers BP and RP. In the former reaction, wild-type (WT) and heterozygous mutant plants (Heterozygous lines, HZ) can both amplify bands; in the latter reaction, homozygous mutant plants (Homozygous Lines, HM) and heterozygous mutant plants (HZ) can amplify bands. Since T-DNA insertion has not occurred on both chromosomes of the WT target gene, only the PCR product from LP to RP can be amplified; since T-DNA insertion has occurred on both chromosomes of the HM target gene, the product amplified with BP and RP as primers can be obtained, but because the inserted T-DNA fragment is too long to inhibit the formation of the specific amplification product of the target gene, the PCR product from LP to RP cannot be amplified; since T-DNA has been inserted into only one chromosome of the HZ target gene, products of both reactions can be obtained.
[0034] The results showed that the identified Arabidopsis mutant (AT2G24860) was a homozygous mutant plant ( Figure 2 A). The plants were continuously cultured in the greenhouse. At the same time, leaves of the identified homozygous mutant plants were selected to extract RNA, and the expression level of the mutant target gene was detected by qRT-PCR (the primers for the Arabidopsis EIJ1 gene used were qAtEIJ1, with qAt-UBQ5 as the internal reference, see Table 1 for details). The results showed that the expression level of the homozygous mutant target gene was significantly lower than that of the wild-type col-0 ( Figure 2 B).
[0035] Observation of Arabidopsis mutant phenotypes: Collect seeds from the verified homozygous Arabidopsis. Take some seeds and disinfect them with 75% alcohol for 15 min, wash them more than 3 times with sterile dd H2O in a laminar flow hood, reserve some dd H2O and vernalize it in a 4°C refrigerator for three days. Sow the seeds on 1 / 2MS plates in the laminar flow hood and seal them. Place the seeds in a plant culture room and culture for seven days to observe the differences in root lengths of different mutants. Transplant the seedlings into small cups with nutrient soil (imported soil: domestic soil: vermiculite = 8:4:1), mark them and continue to culture. Observe the leaf phenotypes and plant differences of the mutants in the third and fourth weeks, observe the differences in the bolting periods of the mutants in the fifth and sixth weeks. After bolting, manage the plants in a 25°C culture room and collect seeds.
[0036] The results found that: the root length of the mutant eij1 (AT2G24860) at the seedling stage was significantly shorter than that of the wild-type col-0; there was no obvious difference in the growth status of the mutant plants and the wild-type col-0 around 4 weeks; there was also no significant difference in the mutant phenotypes compared with the wild-type col-0 during the bolting and flowering periods ( Figure 3A and B); At the seed setting stage, the seeds of the mutant eij1 (AT2G24860) and the wild type col-0 were separately harvested, and their 1000-grain weight was measured. It was found that there was no difference between the mutant and the wild type col-0 ( Figure 3 C). The results showed that the mutation of the gene AtEIJ1 had no significant effect on the agronomic traits of Arabidopsis thaliana.
[0037] Example 2 Detection of the resistance of Arabidopsis thaliana mutants to various pathogenic bacteria
[0038] Analysis of the resistance to Sclerotinia sclerotiorum and Botrytis cinerea: Homozygous Arabidopsis thaliana mutants were selected and cultured in small cups containing nutrient soil, and placed in a plant culture room for about four weeks. A hyphal block of wild type Sclerotinia sclerotiorum 1980 or wild type Botrytis cinerea B05.10 was taken with an inoculation needle, and the hyphal surface was inoculated downward onto a PDA medium with a diameter of 90 mm, and placed in a fungal culture room at 20 °C for continuous activation for three generations; A fresh hyphal block was taken from the edge with a puncher and placed on a 2×SY medium with a diameter of 90 mm and a volume of 17.5 mL, and placed in a fungal culture room at 20 °C for 36 h. A hyphal block on the 2×SY medium was taken with a 3 mm puncher and inoculated near the vein on the right side of the leaf of the homozygous Arabidopsis thaliana mutant, and moisture was kept. After 36 h of inoculation with 1980 and 48 h of inoculation with B05.10, the diameter of the lesion was measured by the cross method, and the lesion area was calculated. The experiment was repeated 3 times.
[0039] The results showed that the lesion area of the wild type col-0 was about 91.97 mm2, and the lesion area of the mutant eij1 (AT2G24860) was about 54.21 mm2 ( Figure 4 A and B); The relative biomass per unit area was detected by qPCR (the primers were Arabidopsis thaliana qAt-UBQ5 and Sclerotinia sclerotiorum Ssβ-tubulin, see Table 1 for details). The relative content of the hyphae in the lesion of the wild type col-0 was normalized to 1, and the relative content of the hyphae of the mutant eij1 (AT2G24860) was 0.27 ( Figure 4 C). The above results showed that the lesion area of the mutant eij1 (AT2G24860) was significantly smaller than that of the wild type col-0, and the relative biomass measurement showed that the content of the 1980 hyphae of the mutant eij1 (AT2G24860) was significantly lower than that of the wild type col-0, proving that the mutant eij1 (AT2G24860) had strong resistance to Sclerotinia sclerotiorum 1980.
[0040] After 48 h of inoculation with the hyphal block of Botrytis cinerea B05.10, the statistics were carried out, and it was found that the lesion area of the wild type col-0 was about 92.65 mm 2 and the lesion area of the mutant eij1 (AT2G24860) was about 45.86 mm2 ( Figure 5A and B); the relative biomass per unit area was detected by qPCR (the primers were Arabidopsis thaliana qAt-UBQ5 and Botrytis cinerea Bc-actin, see Table 1 for details), and the results showed that the relative content of hyphae in the mutant eij1 was 0.26( Figure 5 C). The above results showed that the resistance results of inoculating Botrytis cinerea B05.10 were similar to those of inoculating 1980. The lesion area of the mutant eij1 (AT2G24860) was significantly smaller than that of the wild type col-0, and the hyphal content of B05.10 was also significantly lower than that of the wild type col-0, indicating that the mutant eij1 (AT2G24860) also had strong resistance to Botrytis cinerea B05.10.
[0041] To detect whether the homozygous mutants of Arabidopsis thaliana with the target S gene also had resistance to other types of pathogens, the resistance of Arabidopsis thaliana to Pseudomonas syringae pv. tomato Pst.DC3000 was detected under indoor conditions. The specific method was as follows: The Pst.DC3000 bacterial solution was streaked on King's B (Kana, Rif) solid medium and cultured at 28 °C for two days until single colonies grew. The single colonies were taken and cultured in 500 μL King's B (Kana, Rif) liquid medium until turbid, then diluted 1:1000 and cultured overnight until OD600 = 1.0. The bacteria were collected by centrifugation at 4000 r / min for 10 min, washed twice with 10 mM MgCL2 and then resuspended with 10 mM MgCL2 to an OD600 of about 0.1, and then diluted to a concentration of 1×10 5 , and the lower epidermis of Arabidopsis thaliana was injected with a 1 mL syringe, the injection area of the leaves was marked, and samples were taken at 0 h and 72 h after injection to detect the number of bacteria in the leaves. The experiment was repeated three times.
[0042] The leaves of the living wild type col-0 and the mutant eij1 about 4 weeks old were inoculated with Pst.DC3000. After 5 days, observing the phenotypes, it could be seen that there were more yellow spots on the leaves of col-0, the leaves were chlorotic and yellowed, while the leaves of the mutant eij1 (AT2G24860) were less yellowed, and there was no obvious yellowing at the main vein, and the disease condition was lighter than that of the wild type col-0( Figure 6 A). At the same time, the number of bacteria in the leaves at 0 h and 72 h after inoculating Pst.DC3000 was respectively counted, and it was found that the number of bacteria in the leaves of the mutant eij1 (AT2G24860) at 72 h was significantly reduced compared with that of the wild type col-0( Figure 6 B, C). The above results indicated that the mutant eij1 (AT2G24860) had strong disease resistance to Pst.DC3000.
[0043] Table 1 Primer sequences
[0044]
[0045]
Claims
1. Use of suppressing the expression of Arabidopsis thaliana S gene AtEIJ1 with the Arabidopsis thaliana genome database number AT2G24860 in enhancing the resistance of plants to Sclerotinia sclerotiorum, Botrytis cinerea and / or Pseudomonas syringae pv. tomato DC3000.
2. The application according to claim 1, characterized in that, The plant is selected from Arabidopsis thaliana or Brassica napus.
3. The application according to claim 1 or 2, characterized in that, The suppression of the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion method, gene editing or RNAi technology.
4. Use of suppressing the expression of Arabidopsis thaliana S gene AtEIJ1 with the Arabidopsis thaliana genome database number AT2G24860 in preventing and treating plant diseases caused by infection of Sclerotinia sclerotiorum, Botrytis cinerea and / or Pseudomonas syringae pv. tomato DC3000.
5. The application according to claim 4, wherein The plant is selected from Arabidopsis thaliana or Brassica napus.
6. The application according to claim 4 or 5, characterized in that, The suppression of the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion method, gene editing or RNAi technology.
7. A method for preventing and controlling diseases caused by plant infection with Sclerotinia sclerotiorum, Botrytis cinerea and / or Pseudomonas syringae pv. tomato DC3000, characterized in that, Suppress the expression of Arabidopsis thaliana S gene AtEIJ1 with the Arabidopsis thaliana genome database number AT2G24860 in the target plant.
8. The method according to claim 7, wherein The suppression of the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion method, gene editing or RNAi technology.
9. Use of Arabidopsis thaliana S gene AtEIJ1 with the Arabidopsis thaliana genome database number AT2G24860 in constructing transgenic plants resistant to Sclerotinia sclerotiorum, Botrytis cinerea and / or Pseudomonas syringae pv. tomato DC3000.