Application of VdGH7-6 protein in improving plant resistance and / or inducing plant immune response

Abstract

The invention provides application of VdGH7-6 protein in improving plant resistance and / or inducing plant immune response, a method for improving plant resistance and / or inducing plant immune response and application of the VdGH7-6 protein in preparing pesticide. The amino acid sequence of the VdGH7-6 protein is as shown in SEQ ID NO. 1. The VdGH7-6 protein can efficiently induce plant ROS outbreak, callose deposition, expression of resistance-related genes and other immunoreactions, and has important significance in agricultural production.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular, to the use of VdGH7-6 protein in improving plant resistance and / or inducing plant immune response. Background Art

[0002] In order to break through the plant cell wall defense barrier, pathogens secrete a series of enzymes (CWDEs) for degrading cell wall components during the infection of host plants, helping them to invade the host and obtain nutrients. However, CWDEs or plant molecules themselves can also act as activators to induce plant immune responses, such as stomatal closure, cell wall callose precipitation, reactive oxygen species (ROS) burst, and intracytoplasmic Ca 2+. 2+ Among the many plant defense responses induced by pathogens, ROS plays an important role and is an important means for plants to resist pathogen infection. Therefore, starting from pathogen CWDEs can provide a new perspective for plant resistance gene mining and disease-resistant plant breeding.

[0003] Current research on the pathogenicity of Verticillium dahliae has identified a variety of CWDEs-like proteins. Due to the diversity of the ways in which CWDEs proteins interact with hosts, there are differences in the efficiency of inducing plant immune responses. Therefore, it is of great significance to discover host immune activation proteins that better meet production needs. Summary of the invention

[0004] In order to efficiently induce plant immune responses such as ROS burst, callose deposition, and resistance-related gene expression, the present disclosure provides the use of VdGH7-6 protein in improving plant resistance and / or inducing plant immune response, a method for improving plant resistance and / or inducing plant immune response, and the use of VdGH7-6 protein in the preparation of pesticides.

[0005] In order to achieve the above objectives, the first aspect of the present disclosure provides the use of VdGH7-6 protein in improving plant resistance and / or inducing plant immune response, and the amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1.

[0006] Optionally, the improving the resistance of the plant comprises: improving the resistance of the plant to at least one pathogen selected from Staphylococcus cinerea and Verticillium dahliae;

[0007] The inducing the immune response of the plant includes: inducing the reduction of the harm of at least one pathogen selected from Staphylococcus cinerea and Verticillium dahliae to the plant.

[0008] Optionally, the VdGH7-6 protein is used at a concentration of 20-40 μM, with an optimal concentration of 25 μM.

[0009] Optionally, the plant comprises tobacco.

[0010] In another aspect, the present disclosure provides a method for improving plant resistance and / or inducing plant immune response, the method comprising the following steps:

[0011] Applying the VdGH7-6 protein to target plants, and / or introducing a gene encoding the VdGH7-6 protein into target plants;

[0012] The amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1;

[0013] The nucleotide sequence of the gene encoding the VdGH7-6 protein is shown in SEQ ID NO.2.

[0014] Optionally, the gene encoding the VdGH7-6 protein is introduced into the target plant via a plant expression vector.

[0015] Optionally, the plant expression vector comprises pGR107.

[0016] Optionally, the improving the resistance of the plant comprises: improving the resistance of the plant to at least one pathogen selected from Staphylococcus cinerea and Verticillium dahliae;

[0017] Optionally, inducing an immune response in the plant includes: inducing a reduction in the harm of at least one pathogen selected from Staphylococcus cinerea and Verticillium dahliae to the plant.

[0018] On the other hand, the present disclosure provides the use of VdGH7-6 protein in the preparation of pesticides. Optionally, the pesticide is used to improve plant resistance and / or induce plant immune response. The amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1.

[0019] Optionally, the plant comprises tobacco.

[0020] Through the above technical scheme, the present disclosure provides VdGH7-6 protein that acts on stimulating plant immune responses, which can efficiently induce plant ROS burst, callose deposition, resistance-related gene expression and other immune responses at a relatively low usage concentration, while significantly enhancing plant resistance and immune capacity, providing a new idea for improving plant resistance.

[0021] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the experimental results of the VdGH7-6 protein inducing ROS response in plant leaves in Example 1 of the present disclosure.

[0023] Figure 2 This is a diagram of the lesion area on the leaves of plants treated with VdGH7-6 protein and GFP in Example 2 of the present disclosure.

[0024] Figure 3 This is a diagram of the experimental results of VdGH7-6 protein inducing plant resistance gene expression in Example 2 of the present disclosure.

[0025] Figure 4 This is a diagram of the experimental results of VdGH7-6 protein inducing the expression of plant disease resistance-related genes in Example 3 of the present disclosure. DETAILED DESCRIPTION

[0026] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0027] In a first aspect, the present disclosure provides the use of VdGH7-6 protein in improving plant resistance and / or inducing plant immune response, and the amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1.

[0028] The inventors of the present disclosure have found through extensive research that after injecting VdGH7-6 protein into plant leaves, such as tobacco leaves, the production of ROS and the accumulation of callose can be detected in the leaves, and the expression levels of some immune response-related genes in the injected plants are also significantly upregulated. Therefore, VdGH7-6 protein can be used to improve plant resistance and / or induce plant immune response.

[0029] According to the present disclosure, the improving plant resistance comprises: improving the plant resistance to at least one pathogenic bacteria selected from Staphylococcus cinerea and Verticillium dahliae;

[0030] The inducing the immune response of the plant includes: inducing the reduction of the harm of at least one pathogen selected from Staphylococcus cinerea and Verticillium dahliae to the plant.

[0031] According to the present disclosure, the use concentration of the VdGH7-6 protein is 20-40 μM, preferably 25 μM. The VdGH7-6 protein provided by the present disclosure can significantly improve plant resistance and induce plant immune response at a lower use concentration.

[0032] According to the present disclosure, the plant comprises tobacco.

[0033] In another aspect, the present disclosure provides a method for improving plant resistance and / or inducing plant immune response, the method comprising the following steps:

[0034] Applying the VdGH7-6 protein to a target plant, and / or introducing a gene encoding the VdGH7-6 protein into a target plant;

[0035] The amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1;

[0036] The nucleotide sequence of the gene encoding the VdGH7-6 protein is shown in SEQ ID NO.2.

[0037] According to the present disclosure, the gene encoding the VdGH7-6 protein is introduced into the target plant via a plant expression vector.

[0038] According to the present disclosure, the plant expression vector includes PGR107.

[0039] According to the present disclosure, the improving plant resistance comprises: improving the plant resistance to at least one pathogenic bacteria selected from Staphylococcus cinerea and Verticillium dahliae;

[0040] According to the present disclosure, the inducing immune response of the plant includes: inducing reduction of the harm of at least one pathogenic bacteria selected from Verticillium dahliae and Staphylococcus cinerea to the plant.

[0041] On the other hand, the present disclosure provides the use of VdGH7-6 protein in the preparation of pesticides, wherein the pesticides are used to improve plant resistance and / or induce plant immune response, and the amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1.

[0042] According to the present disclosure, the plant comprises tobacco.

[0043] The present disclosure is further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0044] The VdGH7-6 protein involved in the embodiment of the present disclosure has an amino acid sequence as shown in SEQ ID NO. 1. The preparation method of the VdGH7-6 protein includes: constructing a recombinant expression vector, using Escherichia coli for prokaryotic expression, the expression vector has a maltose binding protein MBP tag, and using an MBP TrapHP pre-packed column for purification to obtain the target protein.

[0045] SEQ ID NO.1:

[0046] MKTNFGVLLAFLAAARAQQACTSQAETHPPLTWQKCTGTGGSSCTNVSGSVVLDSNWRWTHTVSGSTN

[0047] CYDGNKWTASCGTDGTTCAQQCCVDGADYANTYGVTTSGNALTLKFVTKHQYGTNIGSRVYLMENDT

[0048] KYQAFNLLGNEFTFDVDVSKTGCGLNGALYFVSMDLDGGVSKYSTNKAGAKYGTGYCDSQCPRDVKFI

[0049] GGVANVAGWVPAADDPNSGVGNLGACCAEMDIWEANKISTAYTPHPCQNNAYHTCQGDKCGGTYSA

[0050] DRYGGDCDPDGCDFNSYRQGDKTFYGEGTGFKLNTAQKLTVVTQFLRSGGSTGDLSEIKRFYVQNGVV

[0051] FANSQSTVAGNPGNSLTPAFCKAQKTAFGDQNIFDQRGGFAQTSDAVSKPMVLVMSLWNDHYANMLW

[0052] LDSTFPTTSTGPGAERGSCPTTSGTPADTETNQANATVQFSNIKFGPIGSTFNQGAVSPPSGGTTVVAPPPA

[0053] TTLATSTRAAVTTTTRAAPPTTTTASSGGAAAPKWGQCGGQGWTGPTTCVAGSTCQAQNQWYSQCL

[0054] SEQ ID NO.2:

[0055]

[0056] Example 1

[0057] VdGH7-6 protein stimulates plant immune response:

[0058] (1) VdGH7-6 protein induces ROS burst in plants

[0059] Four-week-old tobacco was selected and about 20 μL of 25 μM glycoside hydrolase VdGH7-6 protein was injected from the back of the leaf using a 1 mL syringe (without the needle). 1×PBS (pH 7.5) solution was used as a negative control. 6 hours after injection, the leaves were rinsed with deionized water, placed in 1×DAB dye solution, and incubated at 25°C in the dark for 8 hours; the leaves were rinsed with deionized water to remove the residual dye solution, and after decolorization with 95% ethanol, they were observed under a stereo microscope.

[0060] Results: Compared with the 1×PBS control, the DAB dye staining was obvious in the area of ​​tobacco leaves injected with VdGH7-6 protein, indicating that a large amount of reactive oxygen species accumulated, such as Figure 1 shown.

[0061] The above experiments showed that glycoside hydrolase VdGH7-6 can induce ROS burst in tobacco leaves.

[0062] Example 2

[0063] VdGH7-6 induces tobacco resistance to Botrytis cinerea:

[0064] About 20 μL of 25 nM glycoside hydrolase VdGH7-6 protein was injected into 4-week-old tobacco leaves from the back of the leaves using a 1 mL syringe without a needle. 1× PBS (pH 7.5) was used as a negative control. 12-24 hours after injection, a needle was used to puncture the injection area and 5 μL of 5×10 6 spores / mL were dropped just above the eye of the needle and cultured at 25°C and 80% humidity. The diameter of the lesions of each treatment was observed and measured 3-5 days after inoculation, and the biomass was detected. Figure 2-3 As shown, the lesion area of ​​the experimental leaves is ≈210mm 2 ; The area of ​​lesions on the control leaves is ≈813mm 2 .

[0065] The above results indicate that VdGH7-6 protein can induce tobacco resistance to Botrytis cinerea, and the lesion area is reduced by about 74.17%, which significantly improves the plant's resistance to the pathogen.

[0066] Example 3

[0067] Transient transformation of VdGH7-6 protein induces expression of resistance-related genes in tobacco leaves:

[0068] Select 4-week-old tobacco and adjust the concentration of VdGH7-6 transiently transformed with Agrobacterium pGR107-VdGH7-6 to OD 600 0.5-0.8, 200 μL of Agrobacterium solution was injected from the back of the leaf using a 1 mL syringe (without the needle). Agrobacterium with the same concentration and transient expression of pGR107-GFP was used as a negative control. 48 hours after injection, leaves in the injection area were collected, total RNA was extracted, and the transcription level of resistance-related genes was detected by quantitative PCR.

[0069] Results: Compared with the control, the transcription levels of disease resistance-related genes in the tobacco leaves injected with pGR107-VdGH7-6 were significantly increased. Figure 4 shown.

[0070] The above experiments showed that glycoside hydrolase VdGH7-6 induced the expression of tobacco resistance genes.

[0071] It can be seen that applying VdGH7-6 protein to plants can stimulate plant immune response in a short time, and a strong plant immune response can be produced in the protein injection area within 6 hours: active oxygen burst, callose deposition, etc.; a strong plant immune response can be quickly stimulated at a lower working concentration.

[0072] The preferred embodiments of the present disclosure are described in detail above; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0073] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0074] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. Use of VdGH7-6 protein in improving plant resistance and / or inducing plant immune response, characterized in that: The amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, wherein: Improving the resistance of the plant comprises: improving the resistance of the plant to at least one pathogen selected from Staphylococcus cinerea and Verticillium dahliae; The inducing the immune response of the plant includes: inducing the reduction of the harm of at least one pathogen selected from Staphylococcus cinerea and Verticillium dahliae to the plant.

3. The use according to claim 1, wherein: The VdGH7-6 protein is used at a concentration of 20-40 μM, preferably 25 μM.

4. The use according to claim 1, wherein: The plant comprises tobacco.

5. A method for improving plant resistance and / or inducing plant immune response, characterized in that: The method comprises the following steps: Applying the VdGH7-6 protein to a target plant, and / or introducing a gene encoding the VdGH7-6 protein into a target plant; The amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the VdGH7-6 protein is shown in SEQ ID NO.

2.

6. The method according to claim 5, wherein: The gene encoding the VdGH7-6 protein is introduced into the target plant via a plant expression vector.

7. The method according to claim 5, wherein: The plant expression vector includes pGR107.

8. The method according to claim 5, wherein: Improving the resistance of the plant comprises: improving the resistance of the plant to at least one pathogen selected from Staphylococcus cinerea and Verticillium dahliae; The inducing the immune response of the plant includes: inducing the reduction of the harm of at least one pathogen selected from Staphylococcus cinerea and Verticillium dahliae to the plant.

9. The use of VdGH7-6 protein in the preparation of pesticides, characterized in that: The pesticide is used to improve plant resistance and / or induce plant immune response, and the amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.

1.

10. The use according to claim 9, wherein: The plant comprises tobacco.

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