Application of CaBEH2 gene in enhancing resistance of pepper to phytophthora blight

Abstract

The application provides application of a CaBEH2 gene in enhancing resistance of a pepper to a blight, and belongs to the technical field of plant genetic engineering. The CaBEH2 gene is overexpressed, the cDNA sequence of the CaBEH2 gene is shown as SEQ ID NO:1, the CaBEH2 gene is used as a target gene, overexpression of the CaBEH2 gene is induced by a genetic engineering method, resistance of the pepper to a pepper phytophthora is enhanced, and resistance of the pepper to a blight is enhanced; the blight is a disease caused by the pepper phytophthora. The application provides a pepper blight-related gene CaBEH2 and application thereof, and overexpression of the CaBEH2 gene can enhance the resistance of the pepper plant to the blight, and provides a favorable basis for improving plant disease resistance and cultivating a disease-resistant variety.

Description

Technical Field

[0001] This invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of the CaBEH2 gene in enhancing the resistance of peppers to blight. Background Technology

[0002] CaBEH2 belongs to the BES1 / BZR1 family, a key transcription factor in the BR (Brassinosteroids) signaling pathway. This family mediates BR signaling to regulate plant growth and development, such as cell elongation and division, flowering, ovule number, and seed size. The BES1 / BZR1 family plays a multidimensional role in plant immunity by integrating hormone signals and regulating resistance gene expression. Studies have found that pathogen-associated molecular pattern (PAMP) sensing promotes BES1 phosphorylation. In PAMP-triggered immunity (PTI), BES1, as a direct substrate of pathogen-induced MPK6 (Mitogen-activated Protein Kinase 6), is phosphorylated and participates in regulating the plant's immune response to pathogens. Furthermore, in extremely resistant pepper varieties, BES1 / BZR1 family genes are upregulated after inoculation with Phytophthora.

[0003] Phytophthora blight of peppers is a soil-borne disease caused by Phytophthora capsici. It is characterized by rapid infection and rapid onset, leading to large-scale outbreaks within a short period and causing reduced yields or even total crop failure. Phytophthora capsici has a wide host range, infecting not only peppers but also tomatoes and tobacco (Solanaceae), cucumbers, watermelons and pumpkins (Cucurbitaceae), and common beans, green beans, and yellow-podded beans (Fabaceae), causing severe losses. Furthermore, the disease caused by Phytophthora capsici is a typical multi-cycle disease, spreading and spreading through the production of various spore forms, including oospores, sporangia, zoospores, and chlamydospores. While chemical control is feasible in the short term once an outbreak occurs, it easily causes environmental pollution, and long-term use of chemical agents can lead to plant resistance. One approach to controlling Phytophthora capsici is to select and cultivate disease-resistant varieties, and identifying disease-resistant genes is key to this breeding process. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide the application of the CaBEH2 gene in enhancing the resistance of chili peppers to blight, aiming to solve at least one technical problem in the background art.

[0005] This invention is implemented as follows:

[0006] The first aspect of this invention provides the application of the CaBEH2 gene in enhancing the resistance of pepper to blight, by overexpressing the CaBEH2 gene, the cDNA sequence of which is shown in SEQ ID NO:1.

[0007] Furthermore, using the CaBEH2 gene as a target gene, the overexpression of the CaBEH2 gene is induced through genetic engineering methods to enhance the resistance of pepper to Phytophthora capsici and to the blight disease; the blight disease is a disease caused by Phytophthora capsici.

[0008] Furthermore, the CaBEH2 gene is induced to overexpress in pepper using an overexpression recombinant vector. The construction steps of the overexpression recombinant vector include:

[0009] Primer pairs were designed using the cDNA sequence of the CaBEH2 gene shown in SEQ ID NO:1 as a template, the template was amplified, and the amplified fragment was recovered by gel excision to obtain a DNA fragment for overexpression of the CaBEH2 gene.

[0010] The vector pHR containing the CaMV 35S promoter was digested with the restriction endonuclease EcoRI, and the vector backbone was recovered by gel excision.

[0011] The DNA fragment and vector backbone were ligated via homologous recombination and transformed into Escherichia coli DH5α to obtain a recombinant vector for overexpression of the CaBEH2 gene.

[0012] Furthermore, the sequences of the primer pairs are as follows:

[0013] Forward primer: 5'-GATGACGATGACAAGGAATTCATGACGGCCTGCGGTGG-3', as shown in SEQ ID NO:4;

[0014] Reverse primer: 5'-GTCCTTGTAATCCATGAATTCTTAAGCACGTGCCTTTGCATTACCA-3', as shown in SEQ ID NO:5.

[0015] A second aspect of the present invention provides the application of a recombinant vector in enhancing the resistance of chili peppers to Phytophthora blight, the recombinant vector containing the CaBEH2 gene, the cDNA sequence of which is shown in SEQ ID NO:1; by overexpressing the CaBEH2 gene in chili peppers, the resistance of chili peppers to Phytophthora capsici is enhanced, thereby enhancing the resistance of chili peppers to Phytophthora blight; the Phytophthora blight is a disease caused by Phytophthora capsici.

[0016] A third aspect of the present invention provides a method for enhancing the resistance of chili peppers to blight, the method comprising: overexpressing the CaBEH2 gene, wherein the cDNA sequence of the CaBEH2 gene is shown in SEQ ID NO:1.

[0017] Compared with the prior art, the present invention provides a pepper blight-related gene CaBEH2 and its application. Overexpression of the CaBEH2 gene can enhance the resistance of pepper plants to blight, providing a favorable foundation for improving plant disease resistance and breeding disease-resistant varieties. Attached Figure Description

[0018] Figure 1 This is a diagram showing the CaBEH2 gene expression of TRV:00 and TRV:CaBEH2 in Example 1 of the present invention;

[0019] Figure 2 This is a bright field and trypan blue staining image of a leaf 3 days after inoculation with Phytophthora capsici in Example 1 of the present invention.

[0020] Figure 3 This is a comparison of the lesion area on detached leaves three days after inoculation with Phytophthora capsici in Example 1 of the present invention;

[0021] Figure 4 This is a diagram showing the CaBEH2 gene expression of HR and CaBEH2-HR in Example 2 of the present invention;

[0022] Figure 5 This is a bright field and trypan blue staining image of a leaf 3 days after inoculation with Phytophthora capsici in Example 2 of the present invention.

[0023] Figure 6 This is a comparison of the lesion area on the leaves 3 days after inoculation with Phytophthora capsici in Example 2 of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] Example 1

[0026] This embodiment is a pepper disease resistance experiment. The CaBEH2 gene was silenced in pepper leaves using virus-induced gene silencing (VIGS) technology. The leaves of the CaBEH2 gene-silenced pepper plants were then inoculated with *Phytophthora capsici*. The susceptibility of the CaBEH2 gene-silenced pepper plants to blight was assessed by statistically analyzing the area of ​​leaf lesions, confirming that silencing the CaBEH2 gene weakens the pepper plant's resistance to blight.

[0027] 1.1 Experimental Materials

[0028] Using the chili pepper variety 'Hangjiao 12' as material, RNA was extracted from its leaf tissue and reverse transcribed into cDNA, the sequence of which is shown in SEQ ID NO:1. This cDNA served as a template for the subsequent construction of the CaBEH2 gene silencing recombinant vector and the overexpression recombinant vector. The plant material was grown under conditions set with a light intensity of 200 μmol·m². -2 ·s -1 In a greenhouse with a temperature of 25℃ / 19℃ and a photoperiod of 12h.

[0029] 1.2 Construction of a recombinant vector for silencing the CaBEH2 gene

[0030] Primer pairs were designed using the CaBEH2 gene sequence from the Solanaceae genome database SGN (https: / / solgenomics.net / ) as a template.

[0031] The sequence of the forward primer of this primer pair is as follows:

[0032] 5'-TGAGTAAGGTTACCGAATTCTCTAGAGTCAGCTTCCAGACGGTG-3', as shown in SEQ ID NO:2;

[0033] The sequence of the reverse primer of this primer pair is:

[0034] 5'-TTTAATGTCTTCGGGACATGCCCGGGACAGCAACTTCATGTATTCTCTCG-3', as shown in SEQ ID NO:3.

[0035] Using the primer pairs described above, a 218bp specific fragment was amplified using cDNA from the chili pepper variety 'Hangjiao 12' as a template. The amplified fragment was recovered by gel digestion, and the tobacco brittle virus vector pTRV2 was digested with restriction endonucleases Xba I and Sma I. The vector backbone was recovered by gel digestion, and the two recovered products were ligated by homologous recombination and transformed into Escherichia coli DH5α. Sequencing yielded the recombinant vector pTRV2:CaBEH2 with the CaBEH2 gene silenced.

[0036] 1.3 Silencing the CaBEH2 gene in pepper plants

[0037] The recombinant vector pTRV2:CaBEH2, which silences the CaBEH2 gene, was transferred into Agrobacterium tumefaciens competent cells GV3101, and then activated and amplified to prepare Agrobacterium tumefaciens culture pTRV2:CaBEH2; the vector pTRV1, which expresses TRV, was transferred into Agrobacterium tumefaciens competent cells GV3101, and then activated and amplified to prepare Agrobacterium tumefaciens culture pTRV1; the vector pTRV2 was transferred into Agrobacterium tumefaciens competent cells GV3101, and then activated and amplified to prepare Agrobacterium tumefaciens culture pTRV2:00 empty vector.

[0038] Using pepper plants that have grown to the cotyledon stage as the target, the bacterial solutions of Agrobacterium tumefaciens pTRV1 and Agrobacterium tumefaciens pTRV2:CaBEH2 were mixed at a ratio of 1:1 and used to infect the cotyledons of 'Hangjiao 12' pepper plants to obtain pepper plants with the CaBEH2 gene silenced, which were named TRV:CaBEH2.

[0039] A mixture of pTRV1 Agrobacterium tumefaciens bacterial suspension and pTRV2:00 empty vector Agrobacterium tumefaciens bacterial suspension at a 1:1 ratio was used to infect pepper plants as a negative control, which was named TRV:00.

[0040] The relative expression levels of the CaBEH2 gene in leaves of TRV:00 and TRV:CaBEH2 were detected, and the results are as follows: Figure 1 As shown, by Figure 1 It can be seen that CaBEH2 gene silencing was successfully achieved in TRV:CaBEH2. Figure 1 Different letters in the middle column indicate statistically significant differences (P<0.05).

[0041] 1.4. Culture of Phytophthora capsici

[0042] Phytophthora capsici was inoculated onto PDA medium (200 g / L potato + 20 g / L glucose + 17 g / L agar, diluted to 1000 mL) and incubated in the dark for 4–7 days to activate it. The medium containing Phytophthora capsici mycelia was then broken into mycelial blocks using a sterile punch and placed in Petri dishes containing 10% V8 medium (100 mL V8 vegetable juice + 1 g CaCO3 + 17 g agar, diluted to 1000 mL). The mixture was incubated in the dark at 28°C for 7 days to induce sporangium production. 10 mL of sterile water was added to the Petri dish containing mycelia, and the mixture was refrigerated at 40°C for 30 min. Then, the mixture was brought to room temperature (25°C–28°C) and incubated for 20 min to stimulate sporangium release. The mycelia on the surface of the medium were gently scraped off with a sterile spreader, and the filtered liquid was collected as the spore suspension. The number of spores was counted using a hemocytometer, and the spore suspension was diluted to the required concentration (1×10⁻⁶). 5 zoospores·mL -1 )

[0043] 1.5. Inoculation of isolated leaves with silenced CaBEH2 gene by Phytophthora capsici and analysis of results

[0044] Leaves of the TRV:CaBEH2 and TRV:00 pepper plants obtained in Section 1.2 above were placed on 0.8% agar medium (8g agar powder diluted to 1000mL) to maintain humidity. 20μL of a 1×10⁻⁶ agar solution was dropped next to the widest vein of each leaf. 5 zoospores·mL -1 Suspension of Phytophthora capsici spores.

[0045] Three days later, the area of ​​lesions was measured using the following method: Trypan blue staining solution (95% alcohol: lactol = 2:1 by volume) was preheated for 1 minute, then the leaves were immersed in the solution and boiled for 2 minutes. The leaves were then removed and placed in a petri dish containing trypan blue staining solution for approximately 12 hours. The staining solution was then poured out, and a decolorizing agent (900g chloral hydrate dissolved in 600mL deionized water) was added. The decolorizing agent was changed every 12 hours until the leaves became transparent. Finally, the leaves were rinsed in 95% alcohol to remove the decolorizing agent, and the area of ​​dead cells in the leaves was measured and recorded. Lactol was prepared by mixing 10mL lactic acid, 9.3mL phenol, 10mL glycerol, 10mL trypan blue, and 10mL ddH₂O.

[0046] Among them, the bright field and trypan blue stained images of the detached leaves 3 days later are as follows: Figure 2 As shown, the area of ​​the lesion is as follows Figure 3 As shown. Figure 2 and Figure 3 The results showed that pepper plants with silenced CaBEH2 gene had significantly larger leaf lesions than pepper plants without silenced CaBEH2 gene. Figure 3Different letters in the middle column indicate statistically significant differences (P<0.05).

[0047] Example 2

[0048] Example 2 is a disease resistance experiment of pepper. The CaBEH2 gene was overexpressed in pepper leaves using transient overexpression technology, and then the pepper leaves with overexpressed CaBEH2 gene were inoculated with Phytophthora capsici. The resistance and susceptibility of pepper leaves with overexpressed CaBEH2 gene to Phytophthora capsici were determined by statistically analyzing the area of ​​leaf lesions, further clarifying that overexpression of CaBEH2 enhances the resistance of pepper leaves to Phytophthora capsici.

[0049] 2.1 Experimental Materials

[0050] The same experimental materials as in Example 1, Section 1.1.

[0051] 2.2 Construction of a recombinant vector for transient overexpression of the CaBEH2 gene

[0052] Primer pairs were designed using the CaBEH2 gene sequence from the Solanaceae genome database SGN (https: / / solgenomics.net / ) as a template.

[0053] The sequence of the forward primer of this primer pair is as follows:

[0054] 5'-GATGACGATGACAAGGAATTCATGACGGCCTGCGGTGG-3', as shown in SEQ ID NO: 4;

[0055] The sequence of the reverse primer of this primer pair is:

[0056] 5'-GTCCTTGTAATCCATGAATTCTTAAGCACGTGCCTTTGCATTACCA-3', as shown in SEQ ID NO:5.

[0057] A 957 bp full-length fragment of the CaBEH2 gene was amplified using cDNA from the chili variety 'Hangjiao 12' as a template. The amplified fragment was recovered by gel digestion and digested with the restriction endonuclease EcoRI. The vector pHR containing the CaMV 35S promoter was recovered by gel digestion and the vector backbone was recovered. The two recovered products were ligated by homologous recombination and transformed into Escherichia coli DH5α. Sequencing yielded the recombinant vector pCaBEH2-HR, which overexpresses the CaBEH2 gene.

[0058] 2.3 Overexpression of the CaBEH2 gene in chili pepper plants

[0059] The recombinant vector pCaBEH2-HR, which overexpresses the CaBEH2 gene, was transformed into competent Agrobacterium GV3101 and infected the leaves of 'Hangjiao 12' pepper plants to obtain pepper leaf material with transient overexpression of the CaBEH2 gene, which was named CaBEH2-HR.

[0060] Pepper plant leaves were infected with Agrobacterium tumefaciens culture containing pHR as a negative control, and this culture was named HR.

[0061] The relative expression levels of the CaBEH2 gene in HR and CaBEH2-HR leaves were detected, and the results are as follows: Figure 4 As shown, by Figure 4 It can be seen that CaBEH2 gene overexpression was successfully achieved in CaBEH2-HR. Figure 4 Different letters in the middle column indicate statistically significant differences (P<0.05).

[0062] 2.4. Culture of Phytophthora capsici

[0063] The same as the *Phytophthora capsici* culture in Example 1, 1.4.

[0064] 2.5. Inoculation of detached leaves with overexpression of the CaBEH2 gene with Phytophthora capsici and analysis of the results.

[0065] Leaves of the CaBEH2-HR and HR pepper plants obtained in Section 2.3 above were placed on 0.8% agar medium (8g agar powder diluted to 1000mL) to maintain humidity. 20μL of a 1×10⁻⁶ agar solution was dropped next to the widest vein of each leaf. 5 zoospores·mL -1 Suspension of Phytophthora capsici spores.

[0066] Three days later, the area of ​​the lesions was measured using the same method as in Example 1 above. The brightfield and trypan blue stained images of the detached leaves three days later are shown below. Figure 5 As shown, the area of ​​the lesion is as follows Figure 6 As shown. Figure 5 and Figure 6 The results showed that pepper plants with transient overexpression of the CaBEH2 gene had significantly smaller leaf lesions. Figure 6 Different letters in the middle column indicate statistically significant differences (P<0.05).

[0067] Examples 1 and 2 demonstrate that the CaBEH2 gene, as a positive regulator, can confer resistance to Phytophthora capsici and the diseases it causes in different plants. As a target gene, the CaBEH2 gene can be applied to: (1) detecting the expression level of the CaBEH2 gene or preparing molecular markers for use in breeding peppers to combat diseases; (2) preparing products resistant to Phytophthora capsici, such as reagents and drugs that promote CaBEH2 gene expression or protein activity; and (3) cultivating transgenic plants resistant to Phytophthora capsici, such as introducing nucleic acid molecules that promote the overexpression of the CaBEH2 gene into target plants.

[0068] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of the CaBEH2 gene in enhancing the resistance of pepper to blight, characterized by, The CaBEH2 gene is overexpressed in chili peppers, and the cDNA sequence of the CaBEH2 gene is shown in SEQ ID NO:1; the disease is caused by Phytophthora capsici.

2. The application of the CaBEH2 gene according to claim 1 in enhancing the resistance of chili peppers to blight, characterized in that, By inducing overexpression of the CaBEH2 gene through genetic engineering methods, the resistance of pepper to Phytophthora capsici and the resistance of pepper to Phytophthora capsici disease are enhanced.

3. The application of the CaBEH2 gene according to claim 2 in enhancing the resistance of chili peppers to blight, characterized in that, The CaBEH2 gene was induced to overexpress in pepper using a recombinant vector for overexpression. The construction steps of the recombinant vector for overexpression included: Primer pairs were designed using the cDNA sequence of the CaBEH2 gene shown in SEQ ID NO:1 as a template, the template was amplified, and the amplified fragment was recovered by gel excision to obtain a DNA fragment for overexpression of the CaBEH2 gene. The vector pHR containing the CaMV 35S promoter was digested with the restriction endonuclease EcoRI, and the vector backbone was recovered by gel excision. The DNA fragment and vector backbone were ligated via homologous recombination and transformed into Escherichia coli DH5α to obtain a recombinant vector for overexpression of the CaBEH2 gene.

4. The application of the CaBEH2 gene according to claim 3 in enhancing the resistance of chili peppers to blight, characterized in that, The sequences of the primer pair are as follows: Forward primer: 5'-GATGACGATGACAAGGAATTCATGACGGCCTGCGGTGG-3', as shown in SEQ ID NO:4; Reverse primer: 5'-GTCCTTGTAATCCATGAATTCTTAAGCACGTGCCTTTGCATTACCA-3', as shown in SEQ ID NO:

5.

5. The application of recombinant vectors in enhancing the resistance of chili peppers to blight, characterized in that, The recombinant vector contains the CaBEH2 gene, the cDNA sequence of which is shown in SEQ ID NO:

1. By overexpressing the CaBEH2 gene in peppers, the peppers' resistance to Phytophthora capsici is enhanced, as is their resistance to Phytophthora blight. Phytophthora blight is a disease caused by Phytophthora capsici.

6. A method for enhancing the resistance of chili peppers to blight, characterized in that, The method includes: overexpressing the CaBEH2 gene in chili peppers, wherein the cDNA sequence of the CaBEH2 gene is shown in SEQ ID NO:1; and the disease is a disease caused by Phytophthora capsici.

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