Disease-resistant application of arabidopsis thaliana AtRTP7 gene

Abstract

The invention discloses a disease-resistant application of an arabidopsis thaliana AtRTP7 gene. The invention relates to application of inhibiting the expression of an arabidopsis thaliana AtRTP7 gene in enhancing the resistance of plants to pathogenic bacteria. The number of the arabidopsis thaliana AtRTP7 gene in an arabidopsis thaliana genome database is AT4G02820. The invention focuses on the S gene AtRTP7 of arabidopsis thaliana, the growth phenotype of the mutant is observed, and inoculation experiment is carried out on the mutant by using sclerotinia sclerotiorum and botrytis cinerea. The result shows that the growth of the AtRTP7 mutant is not obviously influenced, the AtRTP7 mutant shows obvious resistance to sclerotinia sclerotiorum and botrytis cinerea, and the scab area and the pathogenic bacterium biomass of the AtRTP7 mutant are obviously lower than those of a wild plant. Therefore, the S gene AtRTP7 of arabidopsis thaliana can be used as a potential gene resource for green prevention and control of sclerotinia rot of colza.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio-agriculture and relates to the application of the Arabidopsis S gene AtEIJ1. Background Art

[0002] Sclerotinia sclerotiorum of rapeseed and Botrytis cinerea-caused gray mold cause great harm to crops such as rapeseed, and it is relatively difficult to control. How to effectively control Sclerotinia sclerotiorum of rapeseed has become a major problem. In recent years, with the development of molecular biology, molecular breeding techniques have become increasingly mature. At the same time, with the continuous in-depth research on the interaction mechanism between plants and pathogenic microorganisms and the continuous innovation and application of genetic engineering techniques, the cultivation of disease-resistant germplasm resources using transgenic techniques and gene editing techniques can not only further shorten the breeding cycle of resistant varieties, but also is very likely to cultivate durable and broad-spectrum disease-resistant varieties. Plant disease resistance (R) genes have the ability to detect pathogen attacks and promote counterattacks against pathogens. In contrast to R genes, there are some sensitive genes (S) in plants that are conducive to the successful infection of pathogens. Currently, the confirmed S genes have the characteristics of basic compatibility, continuous compatibility, and negative regulation of the immune signal of plants, negatively regulating the disease resistance response of plants and promoting the infection, proliferation, and spread of pathogens. Broad-spectrum and durable disease-resistant materials can be obtained by interfering with or disrupting the function of plant S genes. Currently, there is no relevant report on the biological function of the Arabidopsis S gene AtRTP7.

[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 functional 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 functional 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-mediated 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 S gene AtEIJ1 in view of the above deficiencies of the prior art.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] Application of inhibiting the expression of Arabidopsis thaliana AtRTP7 gene in enhancing plant resistance to pathogenic bacteria, wherein the number of the Arabidopsis thaliana AtRTP7 gene in the Arabidopsis thaliana genome database is AT4G02820. The cDNA sequence of the AtRTP7 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0007] As a preference of the present invention, the plant is selected from Arabidopsis thaliana, rape or tomato.

[0008] As a preference of the present invention, the pathogenic bacteria are selected from pathogenic fungi or bacteria, and the pathogenic fungi are preferably Sclerotinia sclerotiorum and Botrytis cinerea, and the pathogenic bacteria are Pseudomonas syringae pv. tomato.

[0009] As a preference of the present invention, inhibiting the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion method, gene editing or RNAi technology.

[0010] Application of inhibiting the expression of Arabidopsis thaliana AtEIJ1 gene in preventing and controlling plant diseases caused by pathogenic fungi or bacteria, wherein the number of the Arabidopsis thaliana AtRTP7 gene in the Arabidopsis thaliana genome database is AT4G02820.

[0011] As a preference of the present invention, the plant is selected from Arabidopsis thaliana, rape or tomato.

[0012] As a preference of the present invention, the pathogenic fungi are selected from Sclerotinia sclerotiorum and Botrytis cinerea, and the pathogenic bacteria are Pseudomonas syringae pv. tomato.

[0013] As a preference of the present invention, inhibiting the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion method, gene editing or RNAi technology.

[0014] Application of a substance for inhibiting the expression of Arabidopsis thaliana AtRTP7 gene in constructing transgenic plants resistant to pathogenic fungi or bacteria, wherein the number of the Arabidopsis thaliana AtRTP7 gene in the Arabidopsis thaliana genome database is AT4G02820.

[0015] As a preference of the present invention, the pathogenic fungi are selected from Sclerotinia sclerotiorum and Botrytis cinerea, the pathogenic bacteria are Pseudomonas syringae pv. tomato, and the plant is selected from Arabidopsis thaliana, rape or tomato.

[0016] Beneficial effects:

[0017] The present invention focuses on the S gene AtRTP7 of Arabidopsis thaliana. By observing the growth phenotypes of homozygous mutants of this gene constructed by the T-DNA insertion method, inoculation experiments were carried out on them with Sclerotinia sclerotiorum 1980 and Botrytis cinerea B05.10. The results showed that the growth of the AtRTP7 mutants was not significantly affected, and they showed significant resistance to Sclerotinia sclerotiorum and Botrytis cinerea. The lesion area and pathogen biomass were significantly lower than those of wild-type plants. This indicates that the S gene AtRTP7 of Arabidopsis thaliana can be used as a potential gene resource for the green prevention and control of Sclerotinia sclerotiorum in rapeseed. Brief Description of the Drawings

[0018] Figure 1 Schematic diagram of the principle of T-DNA primer design

[0019] Figure 2 Identification of homozygous mutants of Arabidopsis thaliana (A) Homozygous verification of Arabidopsis thaliana mutants. M: DNA Marker; WT: Wild-type Arabidopsis thaliana col-0; HZ: Heterozygous lines (heterozygous mutants); HM: Homozygous Lines (homozygous mutants); 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 of different plants of the same mutant were collected together, n = 3).

[0020] Figure 3 Growth phenotypes of homozygous mutants of Arabidopsis thaliana (A) Phenotypes at each growth stage of Arabidopsis thaliana. Upper left: Root length phenotype after 7 days of seed germination on 1 / 2 MS medium; Lower left: Phenotype of Arabidopsis thaliana at about 4 weeks; Right: Phenotype at the bolting and flowering stage of Arabidopsis thaliana; The 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 thaliana seeds (n = 5).

[0021] Figure 4 Analysis of the resistance of mutants to Sclerotinia sclerotiorum 1980 (A) Leaf phenotypes of Arabidopsis thaliana after in vivo inoculation with Sclerotinia sclerotiorum 1980 (36 h). (B) Lesion area statistics of Arabidopsis thaliana after inoculation with 1980 (36 h) (n = 8). (C) Relative biomass statistics after inoculation with 1980 (36 h).

[0022] Figure 5 Analysis of the resistance of mutants to Botrytis cinerea B05.10 (A) Leaf phenotypes of Arabidopsis thaliana after inoculation with Botrytis cinerea B05.10 (48 h). (B) Lesion area statistics of Arabidopsis thaliana after inoculation with B05.10 (48 h) (n = 8). (C) Relative biomass expression statistics after inoculation with B05.10 (48 h).Figure 6 Analysis of the resistance of mutants to Pst.DC3000 (A) Phenotype of Arabidopsis thaliana after inoculation with the bacterium Pst.DC3000 for 5 days. (B) Plate coating of bacteria in plant leaves at 0 hpi and 72 h after inoculation with Pst.DC3000 (Take 5 mm leaf margin pieces of the injection leaf and dispense them into 3 2.0 mL EP tubes). (C) Bacterial count in Arabidopsis thaliana leaves at 0 h and 72 h after inoculation. Detailed implementation mode

[0023] The test plants and culture conditions involved in the following examples are as follows:

[0024] Test pathogenic microorganisms: Sclerotinia sclerotiorum 1980, Botrytis cinerea B05.10, Pseudomonas syringae pv. tomato DC3000 (Pst.DC3000) are all stored in the laboratory.

[0025] Test plants: Arabidopsis thaliana: col-0 (stored in the laboratory), mutant rtp7 (AT4G02820) can be purchased from

[0026] https: / / www.arashare.cn / index / Product / index.html or Arabidopsis Biological Resource Center (https: / / abrc.osu.edu / ), constructed using the T-DNA insertion method.

[0027] Culture conditions for pathogenic microorganisms: Sclerotinia sclerotiorum 1980 and Botrytis cinerea B05.10 are activated and cultured on PDA medium at 20 °C. Pst.DC3000 is activated and cultured on King's B (Kana, Rif) medium at 28 °C.

[0028] Plant culture conditions: Arabidopsis thaliana is cultured in a plant culture room, and the culture conditions are 22 °C with 12 h of light and 12 h of darkness, and a relative humidity of 75%.

[0029] The qPCR primers used in the following examples are as follows:

[0030] Table 1

[0031]

[0032] Example 1 Homozygous verification and phenotype of Arabidopsis thaliana mutants

[0033] Homozygous verification of Arabidopsis mutants: The seeds of the purchased Arabidopsis mutant rtp7 (AT4G02820) were disinfected and vernalized for 3 days, sown on 1 / 2 MS medium and grown for 7 days, then transplanted into nutrient soil. After 3 weeks, the DNA of single mutant plants was extracted using the conventional CTAB method, and the homozygous genotype of the Arabidopsis mutant was detected by the three-primer method (LP, BP, RP). The principle of the three-primer method is to design an upstream primer LP and a downstream primer RP at positions 200 bp - 400 bp on both sides of the insertion site, and design a specific primer BP on the T-DNA insertion fragment, and design two pairs of reactions, namely the reactions with primers LP, RP and primers BP, RP. In the former reaction, bands can be amplified from wild-type (WT) and heterozygous mutant plants (Heterozygous lines, HZ); in the latter reaction, bands can be amplified from homozygous mutant plants (Homozygous Lines, HM) and heterozygous mutant plants (HZ). Because no T-DNA insertion occurred on both chromosomes of the WT target gene, only the PCR product from LP to RP can be amplified; on both chromosomes of the HM target gene, T-DNA insertion occurred, so 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; for HZ, T-DNA is inserted into only one chromosome of the target gene, so the products of both reactions can be obtained. The results showed that the identified mutant rtp7 (AT4G02820) plants were homozygous mutant plants( Figure 2 A). The plants were continuously cultured in the greenhouse. At the same time, the 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 used for the Arabidopsis RTP7 gene were qAtRTP7, 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).

[0034] Phenotypic Observation of Arabidopsis Mutants: Seeds of verified homozygous Arabidopsis were collected. Part of the seeds were disinfected with 75% alcohol for 15 min, washed with dd H2O more than 3 times, and part of the dd H2O was reserved for vernalization in a 4°C refrigerator for three days. The seeds were sown on 1 / 2MS plates in a laminar flow hood and sealed. The seeds were placed in a plant culture room for seven days to observe the differences in root length of different mutants. The seedlings were transplanted into small cups with nutrient soil (imported soil: domestic soil: vermiculite = 8:4:1), marked, and continued to be cultured. The leaf phenotypes and plant differences of the mutants were observed in the third and fourth weeks, and the differences in the bolting stage of the mutants were observed in the fifth and sixth weeks. After bolting, the plants were cultured in a 25°C culture room and seeds were collected. The results showed that the root length of the mutant rtp7 at the seedling stage was significantly shorter than that of the wild type col-0; there was no obvious difference in the growth state 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 at the bolting and flowering stage ( Figure 3 A and B); At the fruiting stage, the seeds of the mutant and the wild type col-0 were collected separately, and their 1000-grain weights were measured. It was found that there was no difference between the mutant and the wild type col-0 ( Figure 3 C). The results indicated that the mutation of gene AtRTP7 had no significant effect on the agronomic traits of Arabidopsis.

[0035] Example 2 Detection of Resistance of Arabidopsis Mutants to Pathogenic Fungi

[0036] To clarify the ability of homozygous mutants of the target S gene in Arabidopsis to resist plant pathogenic fungi, the resistance of the mutants to two pathogenic fungi, Sclerotinia sclerotiorum 1980 and Botrytis cinerea B05.10, was detected. The homozygous Arabidopsis mutant rtp7 (AT4G02820) was selected and cultured in small cups with nutrient soil, and placed in a plant culture room for about four weeks. A mycelial block of the wild type Sclerotinia sclerotiorum 1980 or the wild type Botrytis cinerea B05.10 was taken with an inoculation needle, and the mycelial surface was inoculated downward onto a 90-mm diameter PDA medium and placed in a 20°C fungal culture room for continuous activation for three generations; A fresh mycelial block was taken from the edge with a punch and placed on a 90-mm diameter 17.5-mL quantitative 2×SY medium and placed in a 20°C fungal culture room for 36 h. A mycelial block on the 2×SY medium was taken with a 2-mm punch and inoculated near the leaf vein on the right side of the homozygous Arabidopsis mutant leaf, and moisture was kept. After 36 h of inoculation with 1980 and 48 h of inoculation with B05.10, the lesion diameter was measured by the cross-cross method and the lesion area was calculated. The experiment was repeated 3 times.

[0037] After 36 h of inoculation with the mycelial block of Sclerotinia sclerotiorum 1980, the statistical results showed that the lesion area of the wild type col-0 was about 119.62 mm 2 or so, and the lesion area of the mutant rtp7 was about 65.77 mm 2 or so ( Figure 4A 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 hyphae in the lesion of wild-type col-0 was normalized to 1, and the relative content of hyphae in mutant rtp7 was 0.45( Figure 4 C). The above results showed that the lesion area of mutant rtp7 was significantly smaller than that of wild-type col-0. The relative biomass determination showed that the hyphal content of mutant rtp7 was significantly lower than that of wild-type col-0, proving that mutant rtp7 had strong resistance to Sclerotinia sclerotiorum 1980.

[0038] After 48 h of inoculation with Botrytis cinerea B05.10 mycelial blocks, the results showed that the lesion area of wild-type col-0 was about 92.65 mm2, and the lesion area of mutant rtp7 was about 46.30 mm2( Figure 5 A 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). The results showed that the relative content of hyphae in mutant rtp7 was 0.24( Figure 5 C). The above results showed that the results of inoculation with Botrytis cinerea B05.10 were similar to those of inoculation with 1980. The lesion area of mutant rtp7 was significantly smaller than that of wild-type col-0, and the B05.10 hyphal content was also significantly lower than that of wild-type col-0, indicating that mutant rtp7 also had strong resistance to Botrytis cinerea B05.10.

[0039] Example 3 Detection of the resistance of Arabidopsis thaliana mutants to pathogenic bacteria

[0040] To detect whether the homozygous mutants of Arabidopsis thaliana with the target S gene were also resistant to other types of pathogenic bacteria, the resistance of Arabidopsis thaliana to Pseudomonas syringae pv. tomato Pst.DC3000 was detected under indoor conditions. 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. A single colony was taken and cultured in 500 μL King’s B (Kana, Rif) liquid medium until it became turbid, and 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 in 10 mM MgCL2 to an OD600 of about 0.1, and then diluted to a concentration of 1×10 5 , and injected into the lower epidermis of Arabidopsis thaliana 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.

[0041] Inoculate the leaves of living wild-type col-0 and mutant rtp7 about 4 weeks old with Pst.DC3000. After 5 days, observe the phenotypes. It can be seen that there are more yellow spots on the leaves of col-0, the leaves turn chlorotic and yellow, while there is less yellowing on the leaves of the mutant, and there is no obvious yellowing at the main veins. The disease incidence is lighter compared with that of the wild-type col-0. Figure 6 A). At the same time, count the number of bacteria in the leaves at 0 h and 72 h after inoculation with Pst.DC3000 respectively, and it is found that the number of bacteria in the leaves of the mutant at 72 h is significantly reduced compared with that of the wild-type col-0. Figure 6 B, C). The above results indicate that the mutant rtp7 has a strong disease resistance to Pst.DC3000.

Claims

1. Use of inhibiting the expression of Arabidopsis thaliana AtRTP7 gene in enhancing plant resistance to pathogenic bacteria, wherein the Arabidopsis thaliana AtRTP7 gene has the number AT4G02820 in the Arabidopsis thaliana genome database.

2. The application according to claim 1, wherein The plant is selected from Arabidopsis thaliana, rapeseed or tomato.

3. The application according to claim 1, wherein The pathogenic bacteria is selected from pathogenic fungi or bacteria. The pathogenic fungi are preferably Sclerotinia sclerotiorum and Botrytis cinerea, and the pathogenic bacteria is Pseudomonas syringae pv. tomato.

4. The application according to claim 1, characterized in that, Inhibiting the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion method, gene editing or RNAi technology.

5. Use of inhibiting the expression of Arabidopsis thaliana AtEIJ1 gene in preventing and controlling plant diseases caused by pathogenic fungi or bacteria, wherein the Arabidopsis thaliana AtRTP7 gene has the number AT4G02820 in the Arabidopsis thaliana genome database.

6. The application according to claim 5, wherein The plant is selected from Arabidopsis thaliana, rapeseed or tomato.

7. The application according to claim 5, wherein The pathogenic fungi are selected from Sclerotinia sclerotiorum and Botrytis cinerea, and the pathogenic bacteria is Pseudomonas syringae pv. tomato.

8. The application according to claim 5, wherein Inhibiting the expression of the Arabidopsis thaliana S gene AtEIJ1 is achieved by T-DNA insertion method, gene editing or RNAi technology.

9. Use of a substance inhibiting the expression of Arabidopsis thaliana AtRTP7 gene in constructing transgenic plants resistant to pathogenic fungi or bacteria, wherein the Arabidopsis thaliana AtRTP7 gene has the number AT4G02820 in the Arabidopsis thaliana genome database.

10. The application according to claim 9, wherein, The pathogenic fungi are selected from Sclerotinia sclerotiorum and Botrytis cinerea, the pathogenic bacteria is Pseudomonas syringae pv. tomato, and the plant is selected from Arabidopsis thaliana, rapeseed or tomato.

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