Application of CsaMLO8 gene in improving cucumber root knot nematode disease resistance

Abstract

The invention discloses an application of a CsaMLO8 gene in improving resistance of cucumber southern root knot nematode disease, and also discloses an application of the CsaMLO8 gene in genetic improvement and molecular breeding of cucumber southern root knot nematode disease resistance traits. The research result shows that the resistance of cucumber to the root knot nematode disease can be obviously enhanced by reducing the expression quantity of the CsaMLO8 gene or losing the function of the CsaMLO8 gene. Theoretical support is provided for cucumber meloidogyne incognita resistant breeding, and the achievement of the invention has important significance for ensuring the quality and high and stable yield of cucumbers, and has certain application value in the field of cucumber molecular breeding.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of a CsaMLO8 gene in improving resistance to cucumber root-knot nematode disease. Background Art

[0002] Cucumber (Cucumis sativus L.) is a widely planted vegetable crop in my country and plays an important role in rural revitalization. Root-knot nematodes (Meloidogyne spp.) are common soil-borne diseases in cucumber production, which seriously restrict the growth and development of cucumbers and can cause a 10%-50% reduction in cucumber yield (Kumar, V., Khan, MR, and Walia, RK (2020). Crop Loss Estimations due to Plant-Parasitic Nematodes in Major Crops in India. National Academy Science Letters 43, 409-412; Sasser, J. (1979). Economic importance of Meloidogyne in tropical countries). Among the root-knot nematodes that harm cucumbers in my country, southern root-knot nematodes are the dominant species and are the most harmful to cucumbers (Kayani, MZ, Mukhtar, T., Hussain, MA, and Ul-Haque, MI (2013). Infestation assessment of root-knot nematodes (Meloidogyne spp.) associated with cucumber in the Pothowar region of Pakistan. Crop Protection 47, 49-54). However, the control of southern root-knot nematodes in cucumber cultivation and production mainly relies on the frequent application of highly toxic chemical nematicides, which seriously affects the food safety of cucumber products and also pollutes the soil ecological environment. Other control measures such as grafting and biological control methods have certain limitations. In addition, the lack of southern root-knot nematode resistant materials in cultivated cucumber resources has caused a bottleneck in the breeding of cucumbers resistant to southern root-knot nematode (Gu Xingfang, Zhang Shengping, Feng Lanxiang, et al. 2005. Identification and screening of cucumber virus disease resistant materials. Chinese Vegetables, (6): 21-23). ​​Mining and identifying genes related to southern root-knot nematode resistance in cucumbers will provide important gene resources for cucumber disease resistance breeding, which is of great significance for ensuring the quality and high and stable yield of cucumbers, and has certain application value in the field of cucumber molecular breeding. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is how to improve the resistance of cucumber to southern root-knot nematode disease and / or the application of cucumber's resistance to southern root-knot nematode disease in genetic improvement and molecular breeding. The use of virus-induced gene silencing (VIGS) technology to downregulate the expression of the CsaMLO8 gene can improve the resistance of cucumber to southern root-knot nematode disease, and can be applied to the genetic improvement and molecular breeding of cucumber's resistance to southern root-knot nematode disease. The use of gene editing (CRISPR / Cas9) technology to inactivate the function of the CsaMLO8 gene can improve the resistance of cucumber to root-knot nematode disease, and can be applied to the genetic improvement and molecular breeding of cucumber's resistance to root-knot nematode disease.

[0004] In order to solve the above technical problems, the present invention discloses the CsaMLO8 gene in improving the resistance of cucumber to southern root-knot nematode disease, wherein the CsaMLO8 gene is selected from any one of the following four types:

[0005] (1) The full-length DNA sequence of the CsaMLO8 gene is shown in SEQ ID NO: 1;

[0006] (2) The full-length CDS sequence of the CsaMLO8 gene is shown in SEQ ID NO: 2;

[0007] (3) any DNA molecule whose base sequence has 90% or more homology with the sequence shown in SEQ ID NO: 1 and encodes the amino acid sequence shown in SEQ ID NO: 3;

[0008] (4) Any DNA molecule whose base sequence has 90% or more homology with the sequence shown in SEQ ID NO: 2 and encodes the amino acid sequence shown in SEQ ID NO: 3.

[0009] On the other hand, the present application discloses the use of the CsaMLO8 gene in the genetic improvement and molecular breeding of cucumber resistance to southern root-knot nematode disease, wherein the CsaMLO8 gene is selected from any one of the following four types:

[0010] (1) The full-length DNA sequence of the CsaMLO8 gene is shown in SEQ ID NO: 1;

[0011] (2) The full-length CDS sequence of the CsaMLO8 gene is shown in SEQ ID NO: 2;

[0012] (3) any DNA molecule whose base sequence has 90% or more homology with the sequence shown in SEQ ID NO: 1 and encodes the amino acid sequence shown in SEQ ID NO: 3;

[0013] (4) Any DNA molecule whose base sequence has 90% or more homology with the sequence shown in SEQ ID NO: 2 and encodes the amino acid sequence shown in SEQ ID NO: 3.

[0014] In the above application, the expression of CsaMLO8 gene in the target plant is inhibited.

[0015] In some embodiments, the expression of the CsaMLO8 gene is inhibited by gene silencing or gene knockout.

[0016] The steps for gene silencing of the CsaMLO8 gene are as follows:

[0017] 1) constructing an Agrobacterium tumefaciens strain containing a VIGS silencing vector of the CsaMLO8 gene;

[0018] 2) Injecting the Agrobacterium tumefaciens strain into cucumber leaves to obtain CsaMLO8 gene silenced plants.

[0019] In step 2), the obtained CsaMLO8 gene-silenced plants are subjected to normal growth management. Two weeks later, young leaves of the CsaMLO8 gene-silenced plants are collected, RNA is extracted, and the expression level of the CsaMLO8 gene is identified. Plants with significantly downregulated expression levels are used for subsequent inoculation identification.

[0020] More specifically, the preparation method of the Agrobacterium tumefaciens strain containing the CsaMLO8 gene VIGS silencing vector is as follows:

[0021] (a) Total RNA was extracted from young cucumber leaves;

[0022] (b) reverse transcribing the cucumber total RNA obtained in step (a) into cDNA;

[0023] (c) The present invention performs sequence analysis (SEQ ID NO: 2) on the full length CDS of cucumber CsaMLO8 gene (Csa5G623470), designs a silencing fragment of the target gene on the online website https: / / vigs.solgenomics.net / , designs primers with homology arms based on the silencing fragment, and uses the cDNA in step (b) as a template for PCR amplification. The PCR product is connected to the linearized VIGS vector using a homologous recombinase, and the connection product is transformed into Escherichia coli. The CsaMLO8 gene VIGS silencing vector is obtained by monoclonal PCR and sequencing verification. The vector is transformed into Agrobacterium to obtain an Agrobacterium tumefaciens strain carrying the CsaMLO8 gene VIGS silencing vector.

[0024] Preferably, the cucumber leaf is preferably a cucumber cotyledon, but is not limited to a cotyledon. The cucumber explant test material is Xintai Mici.

[0025] The steps for knocking out the CsaMLO8 gene are:

[0026] (a) constructing an Agrobacterium tumefaciens strain containing a CRISPR / Cas9 vector containing the CsaMLO8 gene;

[0027] (b) Transforming the target cucumber explants through the Agrobacterium tumefaciens strain to prepare a mutant plant with CsaMLO8 gene knockout.

[0028] Wherein, the cucumber explant is cucumber cotyledon, and the cucumber explant test material is cucumber CU2.

[0029] The preparation method of Agrobacterium tumefaciens strain containing CRISPR / Cas9 vector of CsaMLO8 gene is as follows:

[0030] a) Extracting total RNA from young cucumber leaves;

[0031] b) reverse transcribing the cucumber total RNA obtained in step a) into cDNA;

[0032] c) Designing the target sequence of the cucumber CsaMLO8 gene, wherein the target sequence sgRNA-CsaMLO8 is as shown in SEQ ID NO: 6, constructing a recombinant expression vector containing sgRNA and Cas9 protein, and transforming the correctly identified recombinant vector into Agrobacterium tumefaciens to form an Agrobacterium tumefaciens strain containing a CRISPR / Cas9 vector containing the CsaMLO8 gene.

[0033] Among them, the obtained CsaMLO8 gene knockout mutant plants were subjected to normal growth management, self-pollinated to obtain homozygous mutant plants, and sufficient seeds were harvested for subsequent inoculation and identification.

[0034] Furthermore, CsaMLO8 gene-silenced plants and CsaMLO8 gene-knockout mutant plants were inoculated with southern root-knot nematodes, and the number of root knots, the number of egg masses and the root weight of the plants were recorded.

[0035] The specific operation of inoculating with southern root-knot nematodes is as follows: when the cucumber plants grow to two leaves and one heart, inoculate with southern root-knot nematodes.

[0036] The inoculated southern root-knot nematodes may be southern root-knot nematodes in the J2 stage with infective activity.

[0037] The duration of inoculation with southern root-knot nematode treatment can be 35 days.

[0038] Beneficial effects: The present application found that the decrease in CsaMLO8 gene expression or the loss of CsaMLO8 gene function can significantly enhance the resistance of cucumber to root-knot nematode disease. The present invention provides theoretical support for the breeding of cucumbers resistant to southern root-knot nematodes. The invention is of great significance for ensuring the quality and high and stable yield of cucumbers, and has certain application value in the field of cucumber molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the result of the identification of CsaMLO8 silenced plants. Fluorescence quantitative identification of the expression of CsaMLO8 gene in silenced plants. pV190 is the plant injected with an empty vector, which serves as the control group; pV190::MLO8 is the plant injected with V190::CsaMLO8 infection solution, which serves as the experimental group. This experiment includes three repetitions.

[0040] Figure 2 The phenotypic identification results of CsaMLO8 silenced plants, root node number / root weight ( Figure 2 A) and egg mass / root weight ( Figure 2 B) Statistical results, pV190 refers to plants injected with an empty vector, serving as the control group; pV190::MLO8 refers to plants injected with V190::CsaMLO8 infection solution, serving as the experimental group. This experiment included three replicates;

[0041] Figure 3 The results of phenotypic identification of CsaMLO8 knockout mutants are shown in Figure 2. Figure 3 A is the statistical result of root knot number / root weight (g) and egg mass number / root weight (g), Figure 3 B is the root phenotype diagram. Bar=1cm, WT is the wild type control; mlo8 CR1 With mlo8 CR2 Knockout mutant. DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent. The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The experimental materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0043] Example 1. Cultivation and collection of southern root-knot nematodes.

[0044] (1) The southern root-knot nematode was provided by the Disease Research Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The nematodes were propagated on water spinach in a glass greenhouse, and the room temperature was maintained at 26°C during the day and 22°C at night. The nematodes developed and reproduced in the root system of water spinach.

[0045] (2) After about 45 days, remove the matrix from the water spinach roots, clean them, remove the egg masses from the roots with tweezers, and place them in a centrifuge tube filled with distilled water.

[0046] (3) Soak the harvested egg mass in a 0.5% sodium hypochlorite aqueous solution for 5 minutes, and then rinse it with distilled water for 5 to 10 times. The purpose of this step is to disinfect the egg mass.

[0047] (4) Place the sterilized egg mass in a culture dish, add an appropriate amount of distilled water, and place it in an incubator for incubation to hatch the second-instar larvae (J2). The incubator temperature is 28°C.

[0048] (5) Collect the liquid every two days and add distilled water for incubation.

[0049] (6) Stir the nematode suspension evenly, pipette 10 μL onto a glass slide, observe and count the number of nematodes under an optical microscope, calculate the average value, repeat three times, count 6 times each time. Estimate the total number of nematodes based on the final average value. The collected nematodes are used in Examples 2 and 3.

[0050] Example 2: Obtaining the cucumber CsaMLO8 gene VIGS silenced strain.

[0051] (1) Total RNA was extracted from young cucumber leaves using the MiniBEST Plant RNA Extraction kit (Takara). The total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara).

[0052] (2) Download the full CDS of cucumber CsaMLO8 gene (Csa5G623470) (SEQ ID NO: 2) from the Cucurbit Genomics Database (CuGenDB). Enter SEQ ID NO: 2 on the https: / / vigs.solgenomics.net / online website, select the cucumber reference genome, click the 'Run VIGSAnalisis' button, and obtain the CsaMLO8 specific fragment 144 bp (SEQ ID NO: 4). Design primers with homology arms for the selected specific fragment. The primers are as follows:

[0053] VIGs-CsaMLO8-144F (SEQ ID NO: 5): 5'-CAGGACTTTACTTAATGGATCCCGCTTTCGTGTCGGCATATG-3'

[0054] VIGs-CsaMLO8-144R (SEQ ID NO: 6): 5'-TAGACCTATAACTGGATCCTTGGTTTCATCCTCCCAGGC-3'

[0055] Using the cDNA in step (1) as a template, perform PCR amplification.

[0056] (3) VIGS vector pV190 was kindly donated by Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences. The VIGS expression vector was digested with restriction endonuclease BamHI and recovered to obtain a linearized vector.

[0057] (4) Using homologous recombinase, the PCR product obtained in step (2) was connected to the linearized VIGS vector obtained in step (3), and the connected product was transformed into Escherichia coli. The pV190::CsaMLO8 silencing vector was obtained by single clone PCR and sequencing verification. The pV190::CsaMLO8 recombinant silencing vector plasmid was extracted and transformed into Agrobacterium GV3101 to obtain the Agrobacterium tumefaciens strain carrying the CsaMLO8 gene VIGS silencing vector.

[0058] (5) The pV190::CsaMLO8 Agrobacterium tumefaciens obtained in step (4) was spread on a LB solid medium containing kanamycin and rifampicin, and cultured at 28 degrees for 2 days. A single clone was selected and placed in a LB liquid culture containing kanamycin and rifampicin, and cultured overnight at 28°C and 200 rpm to make the bacterial solution OD600 value reach 1.0-1.2, thereby obtaining a pV190::CsaMLO8 Agrobacterium tumefaciens bacterial solution.

[0059] (6) Centrifuge the pV190::CsaMLO8 Agrobacterium tumefaciens culture obtained in step (5) at 6000 rpm for 5 minutes, carefully discard the supernatant, and retain the lower bacterial cells. Then suspend the lower bacterial cells with MmA Buffer (10 mmol / LMgcl2, 10 mmol / LMES and 200 μmol / LAS) to obtain a pV190::CsaMLO8 pre-infection solution with OD600 = 0.8-1.2, and place the pre-infection solution in the dark for 2 hours to 3 hours.

[0060] (7) Use a 1 ml sterile syringe needle to pierce the back of the two cotyledons of the "one leaf, one heart" stage cucumber. Then use the syringe without the needle to absorb the pV190::CsaMLO8 infection solution obtained in step (6) and inject it into the cotyledons. During the injection, push the syringe carefully so that the infection solution enters more than 80% of the cotyledons.

[0061] (8) After two weeks of normal culture, roots were taken for RNA extraction and quantitative fluorescence PCR was used to identify the silenced plants. Plants injected with the empty vector pV190 infection solution were used as controls. Plants with significantly downregulated expression levels were CsaMLO8 gene VIGS silenced strains.

[0062] The primers for fluorescent quantitative PCR were designed according to the gene sequence as follows:

[0063] CsaMLO8qp-F (SEQ ID NO: 7): 5'-TCGTTGGGAAGAGTTGCCTC-3'

[0064] CsaMLO8qp-R (SEQ ID NO: 8): 5'-CGAGCAGGACGTGTTATGGA-3'

[0065] Cucumber Ubiqutin gene was used as an internal reference gene and as a control. The primers are as follows:

[0066] UBI-F (SEQ ID NO: 9): 5'-CCTTATTGACCAACCAGTAGT-3'

[0067] UBI-R (SEQ ID NO: 10): 5'-GGACAATGTTGATTTCCTCG-3'

[0068] The results are as follows Figure 1 As shown, compared with the control, the expression of CsaMLO8 gene in the silenced strain injected with pV190::CsaMLO8 was significantly downregulated, indicating that the cucumber CsaMLO8 gene silenced strain was obtained.

[0069] (5) The cucumber CsaMLO8 gene silenced strain and the control plants were inoculated with about 600 J2 nematodes per plant, and watered normally during the period. The growth conditions were: day and night temperature of 26°C / 22°C.

[0070] (6) After 35 days, gently clean the roots, remove the substrate, count the number of root knots and egg masses on the root system of each plant, measure the root weight, and take photos for record. The number of root knots / root weight and the number of egg masses / root weight are used as indicators of disease. The smaller the number of root knots / root weight and the number of egg masses / root weight, the stronger the plant's resistance; the larger the number of root knots / root weight and the number of egg masses / root weight, the weaker the plant's resistance.

[0071] The specific statistical methods are as follows:

[0072] Wash the cucumber roots to be counted, place the roots in a tray filled with tap water, expand the roots, carefully record the number of root knots and egg masses of each root under the light, and take photos. After completion, take the roots out of the water, absorb the water on the root surface with filter paper, weigh them, and record the root weight. Enter the data into Excel to calculate the number of root knots / root weight and the number of egg masses / root weight.

[0073] The root knot phenotype observation and root knot number statistics are shown in Figure 2 . Compared with the control, the number of knots and egg masses per gram of root in the CsaMLO8 gene-silenced strain of cucumber was significantly less than that in the control, indicating that the CsaMLO8 gene-silenced strain had enhanced resistance to southern root-knot nematodes, indicating that the CsaMLO8 gene negatively regulates the resistance to cucumber root-knot nematodes. In this example, the CsaMLO8 knockout mutant plants had enhanced resistance to root-knot nematodes, indicating that CsaMLO8 can be applied to the breeding of cucumbers resistant to root-knot nematodes.

[0074] Example 3: Preparation and identification of cucumber CsaMLO8 knockout mutant plants.

[0075] (1) Design the sgRNA sequence of cucumber CsaMLO8 gene on the CRISPR-GE website (http: / / skl.scau.edu.cn / ): sgRNA-CsaMLO8.

[0076] sgRNA sequence (SEQ ID NO: 11): 5'-GACTATGGTTCGAGGCGTA-3'

[0077] (2) The synthesized sgRNA forward and reverse primers were annealed to form a double-stranded sgRNA containing a sticky end adapter, which was diluted 10 times and then ligated with the sgRNA-Cas9 backbone vector that had been digested with BbsI under the action of T4 ligase at 16°C overnight. 5ul of the ligation product was transformed into E. coli DH5α competent cells. After overnight culture at 37°C in LB solid medium containing 50mg / L kanamycin, a single clone was picked and PCR was performed with detection primers F and R. The positive single clone was sequenced and identified to obtain a positive clone. The sequencing primer was detection primer F. The positive plasmid with correct sequencing was named sgRNA-CsaMLO8-Cas9.

[0078] Detection primer F (SEQ ID NO: 12): 5'-TGTCCCAGGATTAGAATGATTAGGC-3'

[0079] Detection primer R (SEQ ID NO: 13): 5'-CCCCAGAAATTGAACGCCGAAGAAC-3'

[0080] (3) The CRISPR / Cas9 vector sgRNA-CsaMLO8-Cas9 obtained in step (2) is transferred into Agrobacterium tumefaciens EHA105 to obtain an Agrobacterium strain containing a CRISPR / Cas9 vector containing a specific sgRNA.

[0081] (4) The Agrobacterium strain obtained in step (3) was transformed into the cotyledons of cucumber CU2, and candidate transgenic plants were preliminarily screened using GFP. Specific primers were designed upstream and downstream of the sgRNA sequence of the CsaMLO8 gene to detect changes in the DNA sequence of the CsaMLO8 gene. The specific operation was to extract DNA from the leaves of the transgenic plants and perform PCR amplification using the DNA as a template.

[0082] CsaMLO8checkF (SEQ ID NO: 14): 5'-CCTTGTTGATGGTATCGTGAA-3';

[0083] CsaMLO8checkR (SEQ ID NO: 15): 5'-CGAAGATGAAAATATGGAGCT-3';

[0084] The PCR product was sequenced and the sequencing results were compared with the CsaMLO8 gene of wild-type cucumber to detect whether there was a base mutation at the position of sgRNA. The plants with the changed CsaMLO8 gene were selected and recorded as T0 gene-edited plants, named mlo8 CR1 With mlo8 CR2 The T0 generation plants were planted in a glass greenhouse and self-pollinated to obtain T1 seeds. The T1 plants were tested for gene editing using the same method as above, homozygous CsaMLO8 knockout mutants were identified, and T2 seeds were harvested by self-pollination.

[0085] (5) When the CsaMLO8 knockout mutant plants and wild-type (WT) plants grew to two leaves and one heart, they were inoculated with about 500 J2 nematodes per plant, and watered normally during the period. The growth conditions were: day and night temperature of 26°C / 22°C.

[0086] (6) The method for investigating the disease of southern root-knot nematodes is the same as step 6 in Example 2.

[0087] The root knot phenotype observation and root knot number statistics are shown in Figure 3. Compared with WT, the number of knots and egg masses per gram of root in CsaMLO8 knockout mutant plants was significantly less than that in wild type, indicating that CsaMLO8 knockout mutant plants have enhanced resistance to root-knot nematodes, again demonstrating that the CsaMLO8 gene negatively regulates resistance to cucumber root-knot nematodes. In this example, CsaMLO8 knockout mutant plants have enhanced resistance to root-knot nematodes, indicating that CsaMLO8 can be applied to breeding of cucumber root-knot nematode resistance.

[0088] The present invention provides a method and idea for the application of the CsaMLO8 gene in improving resistance to cucumber root-knot nematode disease. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. Application of CsaMLO8 gene in improving resistance of cucumber to southern root-knot nematode disease, including: The CsaMLO8 gene is selected from any one of the following four: (1) The full-length DNA sequence of the CsaMLO8 gene is shown in SEQ ID NO: 1; (2) The full-length CDS sequence of the CsaMLO8 gene is shown in SEQ ID NO: 2; (3) Any DNA molecule whose base sequence has 90% or more homology with the sequence shown in SEQ ID NO: 1 and encodes the amino acid sequence shown in SEQ ID NO: 3; (4) Any DNA molecule whose base sequence has 90% or more homology with the sequence shown in SEQ ID NO: 2 and encodes the amino acid sequence shown in SEQ ID NO:

3.

2. Application of CsaMLO8 gene in genetic improvement and molecular breeding of cucumber resistance to southern root-knot nematode disease, including: The CsaMLO8 gene is selected from any one of the following four: (1) The full-length DNA sequence of the CsaMLO8 gene is shown in SEQ ID NO: 1; (2) The full-length CDS sequence of the CsaMLO8 gene is shown in SEQ ID NO: 2; (3) Any DNA molecule whose base sequence has 90% or more homology with the sequence shown in SEQ ID NO: 1 and encodes the amino acid sequence shown in SEQ ID NO: 3; (4) Any DNA molecule whose base sequence has 90% or more homology with the sequence shown in SEQ ID NO: 2 and encodes the amino acid sequence shown in SEQ ID NO:

3.

3. The use according to claim 1 or 2, characterized in that: When used, the expression of the CsaMLO8 gene in the target plant is inhibited.

4. The use according to claim 3, characterized in that: The expression of CsaMLO8 gene was inhibited by gene silencing or gene knockout.

5. The use according to claim 4, characterized in that: The steps for gene silencing of the CsaMLO8 gene are: 1) constructing an Agrobacterium tumefaciens strain containing the VIGS silencing vector of the CsaMLO8 gene; 2) Injecting the Agrobacterium tumefaciens strain into cucumber leaves to obtain CsaMLO8 gene silenced plants.

6. The use according to claim 5, characterized in that: In step 2), the obtained CsaMLO8 gene-silenced plants are subjected to normal growth management. Two weeks later, young leaves of the CsaMLO8 gene-silenced plants are collected, RNA is extracted, and the expression level of the CsaMLO8 gene is identified. Plants with significantly downregulated expression levels are used for subsequent inoculation identification.

7. The use according to claim 4, characterized in that: The steps for knocking out the CsaMLO8 gene are: (a) constructing an Agrobacterium tumefaciens strain containing a CRISPR / Cas9 vector containing the CsaMLO8 gene; (b) The Agrobacterium tumefaciens strain is used to mediate transformation into target cucumber explants to prepare mutant plants with CsaMLO8 gene knockout.

8. The use according to claim 7, characterized in that: The cucumber explant is cucumber cotyledon, and the cucumber explant test material is cucumber CU2.

9. The use according to claim 7, characterized in that: The preparation method of Agrobacterium tumefaciens strain containing CRISPR / Cas9 vector of CsaMLO8 gene is as follows: a) Extracting total RNA from young cucumber leaves; b) reverse transcribing the cucumber total RNA obtained in step a) into cDNA; c) Designing the target sequence of the cucumber CsaMLO8 gene, wherein the target sequence sgRNA-CsaMLO8 is as shown in SEQ ID NO: 6, constructing a recombinant expression vector containing sgRNA and Cas9 protein, and transforming the correctly identified recombinant vector into Agrobacterium tumefaciens to form an Agrobacterium tumefaciens strain containing a CRISPR / Cas9 vector containing the CsaMLO8 gene.

10. The use according to claim 7, characterized in that: The obtained CsaMLO8 gene knockout mutant plants were grown and managed normally, and homozygous mutant plants were obtained by self-pollination, and sufficient seeds were harvested for subsequent inoculation and identification.

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