Application of cucumber CsMYB60 gene in enhancing cucumber bacterial angular leaf spot resistance

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of a cucumber CsMYB60 gene in enhancing cucumber bacterial angular leaf spot resistance. According to the invention, the CsMYB60 gene is cloned in the cucumber, and the over-expression of the CsMYB60 gene in the cucumber can obviously improve the resistance of the cucumber to the bacterial angular leaf spot, which is of great significance to the safe production of the cucumber. Therefore, the CsMYB60 gene provided by the invention can be introduced into a cucumber plant as a target gene, and the resistance of the cucumber to the bacterial angular leaf spot is improved by changing the expression quantity of the cucumber CsMYB60 gene in the cucumber plant and increasing the expression of a resistance gene; in cucumber breeding, cucumber plants with relatively high expression of the cucumber CsMYB60 gene can be screened by virtue of a PCR (Polymerase Chain Reaction) technical detection means, so that a cucumber variety with relatively high bacterial angular leaf spot resistance is bred. The application method is safe and reliable, and has high agricultural application value.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to application of cucumber CsMYB60 gene in enhancing resistance to bacterial angular leaf spot of cucumber. Background Art

[0002] Cucumber (Cucumis sativus L.) is a dicotyledonous herb belonging to the genus Cucurbitaceae, also known as thorny melon and cucumber. Currently, it is mainly cultivated in greenhouses and facilities. Due to the continuous cropping mode and the special climatic conditions in the facility cultivation, cucumber plants are more susceptible to various pathogens. One of the most serious cucumber diseases is bacterial angular leaf spot of cucumber, which is caused by infection with Pseudomonas syringae pv. Lachrymans. Bacterial angular leaf spot can occur in both the seedling stage and the adult stage of cucumber, causing great harm to cucumber roots, stems, flowers, leaves, fruits and other tissues, resulting in reduced production or even total crop failure. Therefore, constructing and breeding disease-resistant high-quality varieties is a more economical and environmentally friendly way to efficiently produce cucumbers.

[0003] Since bacterial angular leaf spot has a huge impact on cucumber yield, in the production mode of continuous cucumber greenhouse planting, once infected, the yield will be reduced by 10-30%, and in severe cases it can reach 50% or even total crop failure. Therefore, prevention is the main approach to bacterial angular leaf spot in cucumber, supplemented by comprehensive management. Under the current planting model, the best prevention and control system should be the use of new disease-resistant germplasm, supplemented by comprehensive measures such as strengthening management and reasonable crop rotation.

[0004] Therefore, it is of great significance to analyze the molecular regulatory network of resistance to bacterial angular leaf spot of cucumber, discover potential key resistance genes, and further explore the role of related key resistance genes in the response of cucumber to bacterial angular leaf spot pathogens through modern molecular biology and genetics techniques. Summary of the invention

[0005] In order to solve the problems that cucumbers are extremely susceptible to bacterial angular leaf spot in current facility cultivation due to the continuous cropping mode and the special climatic conditions in facility cultivation, resulting in a significant decrease in yield and serious use of pesticides, the present invention provides the use of the cucumber CsMYB60 gene in enhancing the resistance of cucumbers to bacterial angular leaf spot.

[0006] The technical solution of the present invention is as follows:

[0007] The invention discloses an application of cucumber CsMYB60 gene in enhancing resistance of cucumber to bacterial angular leaf spot. The nucleotide sequence of the cucumber CsMYB60 gene is shown in SEQ ID NO.1.

[0008] The invention discloses an application of cucumber CsMYB60 gene in breeding cucumber varieties resistant to bacterial angular leaf spot disease. The nucleotide sequence of the cucumber CsMYB60 gene is shown in SEQ ID NO.1.

[0009] The invention discloses an application of CsMYB60 protein encoded by cucumber CsMYB60 gene in enhancing resistance of cucumber to bacterial angular leaf spot. The amino acid sequence of the CsMYB60 protein is shown in SEQ ID NO.2.

[0010] In the above application, by overexpressing the cucumber CsMYB60 gene, the transcription level of immune-related genes is increased, thereby enhancing the resistance of cucumber to bacterial angular leaf spot.

[0011] Preferably according to the present invention, the immune-related genes include CsFRK1, CsWRKY30 and CsPER5 genes.

[0012] A method for enhancing the resistance of cucumber to bacterial angular spot disease or breeding a cucumber variety resistant to bacterial angular spot disease, using transgenic technology to overexpress the cucumber CsMYB60 gene in cucumber plants, and cultivating transgenic bacterial angular spot disease-resistant cucumber plants containing the overexpressed cucumber CsMYB60 gene.

[0013] Preferably, according to the present invention, the specific steps of the method are as follows:

[0014] (a) constructing an overexpression vector containing the CsMYB60 gene;

[0015] (b) transferring the overexpression vector containing the CsMYB60 gene into Agrobacterium competent cells to obtain Agrobacterium engineered bacteria containing the CsMYB60 gene;

[0016] (c) Transforming the engineered Agrobacterium described in step (b) into cucumber plants to obtain transgenic cucumber plants containing the overexpressed CsMYB60 gene.

[0017] Further preferably, the CsMYB60 gene in step (a) is inserted into pCAMBIA1300 to construct an overexpression vector.

[0018] Beneficial effects:

[0019] The present invention enhances the resistance of cucumber to bacterial angular spot disease by constructing a cucumber CsMYB60 gene overexpression vector and overexpressing it in cucumber plants. The statistical analysis of the bacterial angular spot disease pathogen infection experimental data proves that after 3 days of infection, the leaf disease of the overexpressed plant is significantly weaker than that of the wild-type plant, and the colony count in the overexpressed plant extract is significantly lower than that of the wild-type. It shows that overexpression of the CsMYB60 gene improves the resistance of cucumber to bacterial angular spot disease pathogens and plays a positive regulatory role in the biological process of cucumber resisting bacterial angular spot disease. The present invention clones the CsMYB60 gene in cucumber, and finds that overexpression of the CsMYB60 gene in cucumber can significantly improve the resistance of cucumber to bacterial angular spot disease, which is of great significance to the safe production of cucumber. Therefore, the CsMYB60 gene provided by the present invention can be introduced into cucumber plants as a target gene, and the expression of resistance genes can be increased by changing the expression amount of the cucumber CsMYB60 gene in the cucumber plants, thereby improving the resistance of cucumbers to bacterial angular leaf spot; and in cucumber breeding, PCR technology detection means can be used to screen cucumber plants with relatively high expression of the cucumber CsMYB60 gene, thereby breeding cucumber varieties with strong resistance to bacterial angular leaf spot. The application method is safe and reliable, and has strong agricultural application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the gel electrophoresis diagram of the amplified target fragment of cucumber CsMYB60 gene.

[0021] Figure 2 This is a bar graph showing the expression level of CsMYB60 gene detected by qRT-PCR.

[0022] Figure 3 This is the electrophoresis diagram of Western Blot detection of CsMYB60 protein expression.

[0023] Figure 4 This is a comparison of the leaf phenotypic states of the plants after Psl induction.

[0024] Figure 5 This is a statistical bar chart of the number of bacteria in plants after Psl induction.

[0025] Figure 6 This is a bar graph showing the transcription levels of relevant immune genes in plants in response to Psl. DETAILED DESCRIPTION

[0026] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, the culture medium and experimental conditions in the examples are conventional culture medium and experimental conditions, or are performed according to the conditions recommended in the manufacturer's instructions. The experimental materials, reagents, etc. used in the examples, unless otherwise specified, can all be obtained from commercial sources.

[0027] In the following examples, the bacterial angular spot pathogen used, namely, Pseudomonas syringae pv. Lachrymans (Psl), was isolated from diseased leaves with typical symptoms of cucumber bacterial angular spot in the greenhouse of Shandong Agricultural University. The above biological materials are only used to repeat the relevant experiments of the present invention and cannot be used for other purposes. Other technicians in this field can purchase the above strains from Shandong Agricultural University or from commercial channels to repeat the relevant experiments of the present invention.

[0028] Example 1

[0029] Cloning of Cucumber CsMYB60 Gene and Construction of Overexpression Vector

[0030] (1) The cucumber variety selected is Xintai Michai, which has strong growth potential and produces rooted cucumbers at the 4th to 5th nodes. The cucumbers are club-shaped, symmetrical, green, densely thorned, and have longitudinal ridges, but they are not obvious. The cucumber seeds were sown in a solar greenhouse and cultivated and managed in the usual way. When the plants began to produce cucumbers, the young leaves were quickly frozen with liquid nitrogen. The RNA of the young cucumber leaves was extracted using the Trizol (purchased from Nanjing Novozyme Co., Ltd.) extraction method, and the cDNA was obtained by reverse transcription using the reverse transcription kit of Nanjing Novozyme Co., Ltd. and stored at -20°C for later use.

[0031] (2) Design amplification primers with restriction enzyme cleavage sites for the CsMYB60 gene. The primer sequences and the restriction enzyme cleavage sites they carry are shown in Table 1 (the underlined sequences are restriction enzyme cleavage site sequences):

[0032] Table 1. Primer sequences and restriction sites of cucumber CsMYB60 gene

[0033]

[0034] (3) Using the cDNA of cucumber leaves as a template, the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase was used to amplify the coding region sequence of the CsMYB60 gene with restriction sites. The PCR reaction system is shown in Table 2:

[0035] Table 2. PCR reaction system

[0036]

[0037] The PCR reaction program is shown in Table 3:

[0038] Table 3. PCR reaction program

[0039]

[0040]

[0041] The three steps of denaturation, annealing and extension were set for 35 cycles to obtain the PCR amplification fragment of the CsMYB60 gene.

[0042] (4) Recovering the target fragment: Take 20 μL of the PCR amplification product and perform electrophoresis on an agarose gel with a mass volume ratio of 1.3%. The test results are as follows: Figure 1 As shown, the target fragment was cut and operated according to the instructions of the gel recovery kit (Beijing Tiangen Biochemical) to obtain the target gene fragment, which was connected to the pEASY-Blunt Simple cloning vector. The connection system is shown in Table 4:

[0043] Table 4. Connection system

[0044]

[0045] After the reaction system is mixed, it is connected in a PCR instrument at 25°C for 30 minutes to obtain a connection product.

[0046] (5) Transformation and screening: Take out the E. coli DH5α competent cells from the -80°C refrigerator, place them on ice to thaw, transfer 50 μL of the E. coli competent cells and the ligation product described in step (4) into a 1.5 mL centrifuge tube on a sterile operating table, mix gently, and place on ice for 30 min; turn on the water bath and heat shock in a 42°C water bath for 90 s, then immediately place on ice for 3 min, and keep the centrifuge tube still during this operation; add 900 μL of liquid LB culture medium to the sterile operating table, place at 37°C, and shake at 200 rpm for 1 h to recover; centrifuge at 5000 rpm for 3 min, discard 900 μL of supernatant, resuspend the cells with the remaining culture medium, and evenly spread them on an LB solid culture medium plate containing 100 mg / L kanamycin, and culture them upside down at 37°C overnight until monoclonal colonies grow; pick the positive monoclonal colonies for sequencing, and the nucleotide sequence of the cucumber CsMYB60 gene obtained by sequencing is shown in SEQ ID NO.1, with a molecular weight of 942 bp.

[0047] (6) Extracting plasmid: Use a small plasmid extraction kit to extract the positive plasmid. Use restriction endonucleases Smal and Spel to double-digest the positive plasmid and the plant expression vector pCAMBIA1300-GFP; after constant temperature digestion at 37°C for 30 minutes, recover the target gene fragment and the linear expression vector. Mix the target gene fragment and the linear expression vector and add T4 DNA ligase to connect. The connection system is shown in Table 5:

[0048] Table 5. Connection system

[0049]

[0050] After the ligation system is mixed, it is connected in a PCR instrument at 16° C. for 12 h to obtain a ligation product.

[0051] (7) Transformation and screening of positive clones: The ligation product of step (6) was transformed and screened by shaking the bacteria, extracting, and verifying the plasmid enzyme digestion. The result was a 942 bp clone with Figure 1 The bands were basically the same in size, indicating that the pCAMBIA1300-CsMYB60-GFP overexpression vector was successfully constructed.

[0052] (8) Agrobacterium competent transformation: After taking out Agrobacterium EHA105 competent cells from -80℃ and partially thawing them on ice, add 8μL of pCAMBIA1300-CsMYB60-GFP overexpression vector to every 50μL and mix well. Then, treat them on ice, in liquid nitrogen, in a 37℃ water bath, and in an ice bath for 5 min each. Then, add 700μL of antibiotic-free YEP liquid medium, shake and culture at 28℃ for 2h, centrifuge, and retain 100μL of supernatant to resuspend the cells and spread them on a YEP solid plate containing 100mg / L kanamycin and 50mg / L rifampicin. Invert and culture at 28℃ for 2-3d, screen for positive monoclonal colonies, and obtain Agrobacterium positive transformants, which are Agrobacterium engineered bacteria EHA105 containing the pCAMBIA1300-CsMYB60-GFP overexpression vector. The bacterial liquid is stored at -80℃ for future use.

[0053] Example 2 Genetic transformation and transgenic verification of cucumber CsMYB60 gene

[0054] Through the Agrobacterium-mediated cucumber genetic transformation method, the Agrobacterium engineering bacteria EHA105 containing the pCAMBIA1300-CsMYB60-GFP overexpression vector was used to infect cucumber cotyledons and screened with differentiation medium and rooting medium. At the same time, in order to clarify the expression of the CsMYB60 gene in transfected cucumbers, the expression level of the CsMYB60 gene in XTMC (wild-type plants) and OE-1, OE-2 (overexpression plants) was detected by qRT-PCR, Western Blot and other technologies. The specific steps are as follows:

[0055] (1) Select full cucumber seeds, soak them in water for 2 hours, peel off the seed coat, sterilize them with 75% ethanol for 30 seconds on a sterile table, sterilize them with 4.0% NaClO for 13 minutes, and finally rinse them with sterilized ultrapure water for 4 times. Sow the seeds on MS culture medium plates with high-temperature sterilized tweezers and culture them in the dark for 2 days. After germination and root growth, infect the cotyledons.

[0056] (2) Cut the cucumber cotyledons, separate the two cotyledons (without the growth point) with tweezers, and place them in MS liquid culture medium; shake culture (28°C, 220rpm) the Agrobacterium engineering bacteria EHA105 containing the pCAMBIA1300-CsMYB60-GFP overexpression vector for about 12 hours, centrifuge to separate the Agrobacterium, suspend it in the MS liquid culture medium containing the cucumber cotyledons, and vacuum infect it, shaking it all the time, the infection time is 13 minutes, and after infection, place it on a co-culture medium plate and culture it in the dark for 2 days;

[0057] The components of the above co-culture medium are: 30 g / L sucrose, 4.4 g / L MS culture medium, 0.5 mg / L 6-benzyladenine (6-BA), 2.22 g / L Maryong Phytagel plantcell II, 1 mg / L abscisic acid (ABA); pH value is 5.8.

[0058] (3) After 2 days of dark culture, the cucumber cotyledons were transferred to differentiation medium plates to screen for resistant calli, and the medium was replaced every 7 days;

[0059] The above differentiation medium comprises 100 mg / L kanamycin (Kan) and 400 mg / L cephalosporin (Cef) added to the co-culture medium.

[0060] (4) After about 15 days, a green callus will grow on one end of the cotyledon. When a seedling grows on the callus, it will be cultured for rooting until the root system is fully developed in about 20 to 30 days.

[0061] The culture medium components used in the above-mentioned rooting culture are: 30 g / L sucrose, 4.4 g / L MS culture medium, 2.22 g / L Marryong Phytagel plantcell II, 400 mg / L Cef; pH value is 5.7.

[0062] (5) After the root system is well developed and the growth is good, open the culture bottle cap and add 2 mL of sterile water to keep it moist. Then put it in a transparent plastic bag and harden it for 3 days to obtain transgenic seedlings.

[0063] (6) The transgenic seedlings with the CsMYB60 gene were moved to the substrate (nutrient soil and vermiculite 1:1) for hardening. Non-transgenic plants were selected as controls. When hardening the seedlings in the substrate, note that to prevent water loss, a transparent plastic bag can be placed on the seedlings. After two days, a small hole was torn on the top of the plastic bag. The tear should be gradually enlarged until the hardening is completed after 10 days. Then the seedlings are moved to the field and managed as usual.

[0064] (7) Cucumber transgenic verification was performed by qRT-PCR and immunoblotting experiments. The qRT-PCR reaction system is shown in Table 6, and the amplification program is shown in Table 7:

[0065] Table 6. qRT-PCR reaction system

[0066]

[0067] Table 7. qRT-PCR amplification program

[0068]

[0069] The denaturation and annealing / extension steps were set for 40 cycles.

[0070] The qRT-PCR test results are as follows Figure 2 As shown in Figure 2, the expression levels of CsMYB60 gene in overexpression plants OE-1 and OE-2 were significantly higher than that in wild-type plant XTMC. The results of Western Blot electrophoresis are shown in Figure 2. Figure 3 As shown, α-actin was used as the internal reference protein, and GFP antibody detection showed that the overexpression plants OE-1 and OE-2 were successfully obtained (the presence of fusion protein GFP proved that the CsMYB60 gene was successfully transferred).

[0071] Example 3 Analysis of resistance to bacterial angular leaf spot of cucumber

[0072] (1) Overexpression of CsMYB60 gene enhances resistance of cucumber to bacterial angular leaf spot

[0073] In order to clarify whether overexpression of CsMYB60 gene can enhance the resistance of cucumber to bacterial angular leaf spot pathogen (Psl), diseased leaves with typical symptoms of cucumber bacterial angular leaf spot were collected in the greenhouse of Shandong Agricultural University and placed in 50 mL centrifuge tubes. The surface was first rinsed with sterile distilled water indoors, 10% sodium hypochlorite was added to the sterile tissue culture table for disinfection for 4 min, and then rinsed three times with sterile distilled water; then the diseased leaves were crushed with a sterile glass rod, the more broken the better, 2 mL of sterile water was added to mix, a sterile inoculation needle was dipped in the pathogen juice, and streaked on KB solid culture medium, and cultured in the dark at 28 ° C for 1 day until colonies grew; a single colony was picked and inoculated in KB liquid culture medium at 28 ° C for 12 h to obtain Psl bacterial solution, and the concentration of Psl bacterial solution was adjusted to 1×10 8 CFU / mL, the wild-type plants XTMC and overexpressing plants seedlings in the true leaf stage were treated by artificial spraying, all the sprayed leaves were placed under the same conditions without being separated from the main body culture, and the growth status of the leaves of each strain was observed regularly. Figure 4 The results showed that after infection with Psl3d, the leaf disease severity of the overexpressing plants was significantly weaker than that of XTMC. Figure 5As shown in the figure, the Psl colony forming unit (CFU) counting results showed that the Psl bacterial proliferation of the overexpressing plants was significantly lower than that of XTMC. The above experimental results indicate that overexpression of the CsMYB60 gene improves the resistance of cucumber to bacterial angular leaf spot pathogens and plays a positive regulatory role in the biological process of cucumber resistance to bacterial angular leaf spot.

[0074] (2) Effect of overexpression of CsMYB60 gene on expression of related defense genes

[0075] Defense genes FRK1, WRKY30 and PER5 all play an important role in plant immune responses, especially in response to MAMPs and local immune activation after cell damage. To further clarify the reasons for the enhanced resistance of overexpressing plants to Psl, the expression levels of CsFRK1, CsWRKY30 and CsPER5 genes were detected. Figure 6 As shown in the results, the expression levels of CsFRK1, CsWRKY30 and CsPER5 genes in XTMC and overexpression plants were upregulated to varying degrees after 2h of response to Psl. However, the upregulation of related immune genes in overexpression plants was more significant than that in XTMC, indicating that overexpression of CsMYB60 gene can increase the transcription level of immune-related genes and enhance the resistance of cucumber to bacterial angular leaf spot. Based on the existing research results, it is speculated that CsWRKY30 gene may be involved in the SA-mediated signal pathway transduction, and then participate in the biological process of cucumber resistance to bacterial angular leaf spot; CsFRK1 gene is involved in the biological defense response of cucumber; CsPER5 gene responds to biological stress and participates in immune regulation.

Claims

1. Application of cucumber CsMYB60 gene in enhancing resistance to bacterial angular leaf spot of cucumber, the nucleotide sequence of the cucumber CsMYB60 gene is shown in SEQ ID NO.

1.

2. Application of cucumber CsMYB60 gene in breeding cucumber varieties resistant to bacterial angular leaf spot, the nucleotide sequence of the cucumber CsMYB60 gene is shown in SEQ ID NO.

1.

3. Application of the CsMYB60 protein encoded by the cucumber CsMYB60 gene in enhancing resistance to bacterial angular leaf spot in cucumber, the amino acid sequence of the CsMYB60 protein being shown in SEQ ID NO.

2.

4. The use according to any one of claims 1 to 3, characterized in that: By overexpressing the cucumber CsMYB60 gene, the transcription level of immune-related genes was increased, thereby enhancing the resistance of cucumber to bacterial angular leaf spot.

5. The use according to claim 4, characterized in that The immune-related genes include CsFRK1, CsWRKY30 and CsPER5 genes.

6. A method for enhancing resistance to bacterial angular leaf spot of cucumber or breeding a cucumber variety resistant to bacterial angular leaf spot, characterized in that: The cucumber CsMYB60 gene according to claim 1 or claim 2 is overexpressed in cucumber plants by using transgenic technology, and transgenic cucumber plants resistant to bacterial angular leaf spot containing the overexpressed cucumber CsMYB60 gene are cultivated.

7. The method according to claim 6, characterized in that The specific steps are as follows: (a) constructing an overexpression vector containing the CsMYB60 gene; (b) transferring the overexpression vector containing the CsMYB60 gene into Agrobacterium competent cells to obtain Agrobacterium engineered bacteria containing the CsMYB60 gene; (c) Transforming the engineered Agrobacterium described in step (b) into cucumber plants to obtain transgenic cucumber plants containing the overexpressed CsMYB60 gene.

8. The method according to claim 7, characterized in that The CsMYB60 gene in step (a) is inserted into pCAMBIA1300 to construct an overexpression vector.

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