Application of HpaXpm gene and its protein in plant disease control

Abstract

The present application provides the application of HpaXpm gene and its protein in plant disease prevention and control. The present invention adopts genetic engineering method to construct HpaXpm genetic engineering bacteria, realizes efficient expression of HpaXpm protein in Escherichia coli, and the expressed HpaXpm protein has good biological activity, especially, HpaXpm protein can effectively resist the infection of tomato brown wrinkled fruit virus to plants, prevent or reduce the influence of tomato brown wrinkled fruit virus on plants, is a green, environmentally friendly and multifunctional bio-source pesticide for increasing crop yield, and can be widely used in disease and insect pest control and plant protection as a new type of protein pesticide.

Description

Technical Field

[0001] The present application relates to the technical field of plant disease and insect pest control, and specifically to the application of HpaXpm gene and its protein in plant disease control. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Since the 20th century, mankind has made great progress in agriculture. In particular, the widespread use of chemical pesticides and fertilizers has provided important guarantees for crops to resist pests and diseases and increase agricultural production. However, the extensive use of chemical pesticides and fertilizers has led to a series of problems such as increased resistance to pests and diseases, excessive pesticide residues, deterioration of soil ecology, increased production inputs, and decreased crop quality and yield. The increased difficulty and investment in the development of new pesticides have led to some pests and diseases being unable to be prevented by drugs. It is difficult to ensure the output and quality of agricultural products by relying solely on the investment of agricultural chemicals. Therefore, it is urgent to develop and apply new and green plant protection products to ensure increased agricultural production and income and green development. So far, among the biological pesticides and biological fertilizers discovered in the world, Harpin signaling protein is the safest and most effective new technology product.

[0004] Harpin proteins are a class of natural products that are naturally produced by various plant pathogens and can be secreted into the extracellular space. Harpin proteins are proteins secreted by the type III secretion system of Gram-negative bacteria during the interaction between pathogens and plants. They can act as inducers to stimulate the defense and growth of various non-host plants. In 1992, Wei Zhongmin et al. first isolated a protein that stimulates plant hypersensitive responses from E. amylovora and named it HarpinEa, which is encoded by the hrpN gene. HarpinEa is a hydrophilic protein composed of 403 amino groups with a molecular weight of 44kD. It is rich in glycine and lacks cysteine, and has no quaternary structure. It is heat-stable and will not be inactivated by treatment at 100℃ for 10min. It is sensitive to proteinase K and ultraviolet rays and can cause hypersensitive responses (HR) in tobacco leaves. Based on in-depth research on Harpin proteins, new, efficient and safe biopesticides have been developed abroad. However, in order to improve the resistance, quality and yield levels brought to plants by Harpin protein treatment, further research is needed to identify new Harpin proteins and screen the Harpin proteins that may be most valuable for agricultural applications.

[0005] Tomato brown rugose fruit virus (ToBRFV) belongs to the family Baculoviridae and the genus Tobamovirus. The virus was first discovered in greenhouse-grown tomatoes in Israel in 2014. Like other members of the genus Tobamovirus, ToBRFV has a positive single-stranded RNA genome encapsulated in rod-shaped particles about 300 nm in length and 18 nm in diameter. The virus can be transmitted by contact, propagation materials, seeds, and bumblebees, and is highly contagious. Tomato plants and fruits infected with ToBRFV will show obvious symptoms, including: brown or yellow edges or spots on the leaves of the plants, leaf deformations such as curling or twisting, stunted plant growth, and overall poor growth; brown sunken spots or stripes on the fruits, irregular fruit shapes, possible wrinkling, and abnormal fruit color development, such as mottling or uneven ripening. Infection with ToBRFV can lead to severe reductions in yield, marketability, and fruit quality, posing a persistent and destructive threat to tomato production. Three dominant disease resistance genes have been identified in tomato, Tm-1, Tm-2 and Tm-2 2 , can protect tomato plants from a variety of tomato viruses such as tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV), but they cannot induce resistance to ToBRFV. Currently, the main strategy for managing ToBRFV is to practice good hygiene measures, including the use of disinfected tools and equipment, and to remove infected plants promptly to reduce the risk of virus transmission. Summary of the invention

[0006] The present invention provides an application of HpaXpm gene and its protein in plant disease prevention and control, and in particular provides an application of HpaXpm gene and its protein in resisting infection of tomato brown wrinkled fruit virus on plants, preventing or reducing the influence of tomato brown wrinkled fruit virus on plants. In addition, the present invention also constructs HpaXpm genetic engineering bacteria by genetic engineering method, realizes efficient expression of HpaXpm protein in Escherichia coli, and the yield of HpaXpm protein can reach 192 mg / L, which is conducive to the industrial production of HpaXpm protein.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect of the present invention, there is provided the use of the HpaXpm gene in improving plant resistance to Tomato brown wrinkled fruit virus (ToBRFV).

[0009] In one embodiment of the present invention, the nucleotide sequence of the HpaXpm gene is as shown in SEQ ID NO:1.

[0010] In the second aspect of the present invention, there is provided the use of HpaXpm protein in improving plant resistance to tomato brown wrinkled fruit virus.

[0011] In one embodiment of the present invention, the HpaXpm protein is produced by expression of the HpaXpm gene; in one embodiment, the nucleotide sequence of the HpaXpm gene is as shown in SEQ ID NO:1.

[0012] In one embodiment of the present invention, the amino acid sequence of the HpaXpm protein is as shown in SEQ ID NO:2.

[0013] In the third aspect of the present invention, there is provided the use of a recombinant expression vector expressing the HpaXpm gene in improving plant resistance to tomato brown fruit virus.

[0014] In one embodiment of the present invention, the recombinant expression vector expressing the HpaXpm gene takes the HpaXpm gene or a gene capable of expressing and producing the HpaXpm protein as a target gene.

[0015] In one embodiment of the present invention, the nucleotide sequence of the target gene is as shown in SEQ ID NO: 1, or the target gene is a gene capable of expressing and producing the HpaXpm protein having an amino acid sequence as shown in SEQ ID NO: 2.

[0016] In one embodiment of the present invention, the recombinant expression vector expressing the HpaXpm gene is constructed by inserting the target gene into an expression vector. In one embodiment, the expression vector is a plasmid, preferably pET30a(+).

[0017] In a fourth aspect of the present invention, provided is the use of a recombinant engineered bacterium expressing the HpaXpm gene in improving plant resistance to tomato brown fruit virus.

[0018] In one embodiment of the present invention, the recombinant engineered bacteria expressing the HpaXpm gene carries a target gene, and the target gene is the HpaXpm gene or a gene capable of expressing and producing the HpaXpm protein.

[0019] In one embodiment of the present invention, the nucleotide sequence of the target gene is as shown in SEQ ID NO: 1, or the target gene is a gene capable of expressing and producing the HpaXpm protein having an amino acid sequence as shown in SEQ ID NO: 2.

[0020] In one embodiment of the present invention, the recombinant engineered bacteria expressing the HpaXpm gene contains a recombinant expression vector expressing the HpaXpm gene, and the recombinant expression vector expressing the HpaXpm gene is as described in the third aspect above.

[0021] In one embodiment of the present invention, the recombinant engineered bacteria expressing the HpaXpm gene are obtained by transferring a recombinant expression vector containing the target gene into a host cell. In some embodiments, the host cell is a prokaryotic cell, preferably Escherichia coli.

[0022] In a fifth aspect of the present invention, there is provided a method for improving plant resistance to tomato brown wrinkled fruit virus, comprising applying a HpaXpm protein solution to the plant;

[0023] Preferably, the application is spraying the HpaXpm protein solution onto the leaves, fruits and / or necks of the plants;

[0024] Preferably, the preferred administration concentration of the HpaXpm protein solution is 2-8 μM.

[0025] In the applications of the above various aspects of the present invention, the plant is especially a plant of the Solanaceae family, and is particularly preferably selected from tomato crops and pepper crops.

[0026] In the application of the above-mentioned various aspects of the present invention, the resistance to tomato brown wrinkled fruit virus includes resisting the infection of tomato brown wrinkled fruit virus to plants, and preventing or reducing the impact of tomato brown wrinkled fruit virus on plants, which includes but is not limited to preventing or reducing the appearance of brown or yellow edges or spots on plant leaves, leaf deformation, such as curling or twisting; relieving or alleviating plant growth stagnation and overall poor growth; preventing or reducing the appearance of brown sunken spots or stripes on fruits, irregular fruit shape, wrinkling and abnormal color development, such as spots or uneven ripening, etc.

[0027] Compared with the prior art, the advantages of the present invention include:

[0028] The invention adopts a genetic engineering method to construct an HpaXpm genetic engineering bacterium, realizes efficient expression of HpaXpm protein in Escherichia coli, the yield of HpaXpm protein can reach 192 mg / L, and the expressed HpaXpm protein has good biological activity, especially, the HpaXpm protein can effectively resist the infection of tomato brown wrinkled fruit virus to plants, prevent or reduce the influence of tomato brown wrinkled fruit virus on plants, is a green, environmentally friendly and multifunctional bio-source pesticide for increasing crop yield, and can be widely used in disease and insect pest control and plant protection as a new type of protein pesticide. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings constituting part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. The implementation scheme of the present application is described in detail below in conjunction with the drawings, wherein:

[0030] Figure 1 : Map of pET30a(+)-HpaXpm recombinant expression vector.

[0031] Figure 2 : PCR electrophoresis results of bacterial solution, among which 1-5 are monoclonal colonies.

[0032] Figure 3 : HpaXpm protein purification results, where M: protein molecular weight standard, FT: loading flow-through, W: washing solution, E: eluent.

[0033] Figure 4 : Protein standard curve.

[0034] Figure 5 :Tobacco growth conditions.

[0035] Figure 6 : Growth status of pepper seedlings.

[0036] Figure 7 : Average plant height and leaf area of ​​pepper seedlings; Asterisks indicate significant differences between the HpaXpm group and the PBS group and the hypersensitive protein group, respectively; * indicates p<0.05, ** indicates p<0.01.

[0037] Figure 8 : Plant height of tomatoes treated with different concentrations of HpaXpm protein and hypersensitive protein on the 35th day after inoculation with ToBRFV; the same lowercase letters indicate no significant difference, and different lowercase letters indicate significant difference.

[0038] Fig. 9 : Growth of upper leaves of tomatoes treated with different concentrations of HpaXpm protein and hypersensitive protein on the 35th day after inoculation with ToBRFV. Three tomatoes were selected from each treatment, and the heart leaf and the first leaf adjacent to the heart leaf of each tomato were photographed.

[0039] Fig.10 : The virus accumulation levels in each treatment group on the 35th day after tomatoes treated with different concentrations of HpaXpm protein and hypersensitive protein were inoculated with ToBRFV, where M: protein molecular weight standard, 1: PBS group sample, 2: 8μM hypersensitive protein group sample, 3: 2μM HpaXpm protein group sample, 4: 8μM HpaXpm protein group sample; β-actin is the plant internal reference protein. DETAILED DESCRIPTION

[0040] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0041] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the content described herein can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0042] Example 1 Construction of HpaXpm gene recombinant Escherichia coli expression vector

[0043] 1. Construction of HpaXpm gene recombinant expression vector

[0044] The HpaXpm gene was used as the target gene, the nucleotide sequence of the target gene was shown in SEQ ID NO: 1, and the pET30a(+) plasmid was used as the expression vector to synthesize the recombinant expression vector pET30a(+)-HpaXpm, the structure of which is shown in Figure 1 shown.

[0045] 2. Obtaining engineered Escherichia coli containing the HpaXpm gene

[0046] The pET30a(+)-HpaXpm recombinant expression vector was transformed into Escherichia coli BL21, and positive clones were selected for bacterial liquid PCR. The electrophoresis results were as follows: Figure 2 As shown, there are brighter bands at the corresponding sizes. The corresponding bacterial solution was submitted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequence was compared. The homology with the target gene was 100%, proving that the Escherichia coli engineering bacteria containing the HpaXpm gene were successfully constructed.

[0047] Example 2 Expression and purification of HpaXpm protein

[0048] 1. Expression of HpaXpm protein

[0049] (1) Activating the strain: 10 μL of the E. coli glycerol bacteria containing the HpaXpm gene constructed in Example 1 was inoculated into 10 mL of LB medium containing 5 μL of kanamycin and cultured in a shaking incubator at 37° C. overnight;

[0050] (2) Expansion culture: Take the above activated strain and inoculate it into 200 mL LB medium at a 1.5% inoculum, add 100 μL of kanamycin, and culture at 37°C, 180 rpm, shaking until OD 600nm About 0.6;

[0051] (3) Inducing expression: Add 200 μL IPTG (final concentration 0.1 mM) to 200 mL LB medium, incubate at 37°C, 180 rpm, shake for 4 h, collect the cells by centrifugation (5000 rpm, 10 min, 4°C), and store at -20°C.

[0052] 2. Purification of HpaXpm protein

[0053] Add 2-5 mL of non-denaturing lysis buffer per gram of wet weight of bacterial pellet to fully resuspend the bacteria, add lysozyme to a final concentration of 1 mg / mL and mix well, and place in an ice water bath or on ice for 30 minutes.

[0054] The bacteria were lysed by ultrasonic treatment on ice at a power of 200-300 W, with each ultrasonic treatment lasting 10 s and an interval of 10 s for each treatment, for a total of 80 ultrasonic treatments.

[0055] The HpaXpm protein was purified using a His-tag protein purification kit (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.).

[0056] 3. SDS-PAGE gel electrophoresis verification

[0057] (1) Preparation of separation gel and concentration gel

[0058] The separation gel was prepared using an SDS-PAGE gel rapid preparation kit (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.). After mixing the components in turn, deionized water was added to cover the liquid surface and solidified at room temperature for about 20 minutes. The liquid on the gel was drained and the concentrated gel was poured in the same way. A comb was inserted to avoid bubbles.

[0059] (2) Sample loading and electrophoresis

[0060] Add 5× SDS gel loading buffer to the sample, heat at 98°C for 5-8 minutes, load 10 μL of sample, run electrophoresis at 60V for 30 minutes until bromophenol blue enters the separation gel, increase the voltage to 100V and run until bromophenol blue reaches the bottom.

[0061] (3) Dyeing and bleaching

[0062] Coomassie Brilliant Blue Rapid Staining Solution (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) was used to stain the gel for about 150 minutes, and then the gel was observed and photographed. Figure 3 ).

[0063] 4. Protein content detection

[0064] 4.1 Dialysis

[0065] The purified target protein was dialyzed using a regenerated cellulose dialysis bag (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.), and the dialysate was PBS solution.

[0066] The dialysis bag was placed in a container containing dialysis bag treatment solution (2% NaHCO3, 1 mM EDTA, pH 8.0), treated in a boiling water bath for 10 min, and then thoroughly washed with distilled water for later use.

[0067] The protein sample was placed in a dialysis bag, and the upper and lower ends were clamped. The container containing the dialysis bag was placed at 4°C, and the dialysis solution was changed every 3-4 hours to obtain the product.

[0068] 4.2 Protein concentration determination

[0069] The protein concentration was determined using the Bradford protein assay kit (Keygen Biotechnology).

[0070] Set the concentration gradient of 0mg / mL, 0.125mg / mL, 0.5mg / mL, 0.75mg / mL, and 1mg / mL to prepare the protein concentration standard curve ( Figure 4 ).

[0071] The content of the target protein was determined using Coomassie Brilliant Blue dye. The results showed that the concentration of the HpaXpm protein solution after purification and dialysis was 1307.61 μg / mL. Finally, 192 mg of HpaXpm protein could be obtained by fermenting 1 L of engineered bacteria, that is, the protein yield was 192 mg / L.

[0072] Example 3 Application of HpaXpm protein in plant disease and insect pest control

[0073] The HpaXpm protein was purified according to the method of Example 2 of the present invention, the hypersensitive protein (also called Harpin protein, trade name MessengerR, Kangzhuangsu) was purchased from Hunan Agricultural University Science and Technology Development Co., Ltd., and the tomato brown wrinkled fruit virus (ToBRFV) was donated by Professor Liu Hongmei of the College of Life Sciences of Shandong Agricultural University.

[0074] 1. Explore the concentration of HpaXpm protein and hypersensitive protein that cause tobacco allergic reactions

[0075] 1.1 Test methods

[0076] (1) Protein preparation: Use sterile PBS to dilute HpaXpm protein and hypersensitive protein to 7 different molar concentrations: 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, and 8 μM, respectively.

[0077] (2) Select Nicotiana benthamiana that is in the vigorous growth period (about one month, not yet flowering), and select leaves of uniform size; use a syringe to inhale different concentrations of HpaXpm protein and hypersensitive protein, remove the needle, press the front of the leaf with your finger, and infiltrate the different concentrations of protein from the back of the leaf. After inoculation, use a marker to mark the inoculation site on the leaf; record the inoculation order and position. Each leaf is inoculated with a different concentration of a protein, and three replicates are performed.

[0078] 1.2 Test results

[0079] Seven different concentrations of HpaXpm protein and hypersensitive protein were inoculated on living tobacco leaves. Six days after inoculation, the results showed that when the concentration of HpaXpm protein was 2μM (38.004μg / mL), the leaves produced obvious hypersensitivity reactions (HR), and as the concentration increased, the HR reaction of the leaves became stronger. However, tobacco leaves did not produce HR reactions to hypersensitive proteins ( Figure 5 ).

[0080] 2. HpaXpm protein pepper growth promotion test

[0081] 2.1 Test methods

[0082] Pepper seedlings were sprayed with 2 μM HpaXpm protein and hypersensitive protein solution and PBS buffer until they were suspended but not dripping (9 plants in each group). The plant height and maximum leaf area (correction coefficient 0.785×leaf width×leaf length) of each pepper seedling were measured before and 20 days after treatment; the average growth rate of each treatment group was calculated.

[0083] 2.2 Test results

[0084] 20 days after protein spraying, the plant height of peppers in each test group and PBS group was significantly different ( Figure 6 , Figure 7 ), the average plant height of the HpaXpm protein group was 1.4636 times that of the PBS group and 1.1604 times that of the hypersensitive protein group; the average leaf area of ​​the HpaXpm protein group was 1.5409 times that of the PBS group and 1.4786 times that of the hypersensitive protein group. The maximum leaf area of ​​pepper in the HpaXpm protein group was significantly greater than that of the PBS group and the hypersensitive protein group (p < 0.05), and was 47.86% higher than that of the hypersensitive protein group. In summary, HpaXpm protein has a growth-promoting effect on the growth of pepper seedlings and can increase the plant height and leaf area of ​​pepper.

[0085] Table 2 Plant height and leaf area of ​​pepper seedlings (mean ± standard deviation)

[0086]

[0087] Note: The same lowercase letters in the same row indicate no significant difference (p≥0.05), and different lowercase letters in the same row indicate significant difference (p<0.05); the same uppercase letters in the same row indicate no significant difference (p≥0.01), and different uppercase letters in the same row indicate extremely significant difference (p<0.01).

[0088] 3. Test on HpaXpm protein against tomato brown wrinkled fruit virus

[0089] 3.1 Test methods

[0090] According to Table 2, 4 groups were set up, and protein was sprayed on tomato plants with the same growth potential (10 days after the first true leaf grew). Each treatment group was repeated for 10 plants, and the spraying was continued until the droplets were suspended without dripping. The following treatments were performed:

[0091] Table 2 Treatment methods of different treatment groups

[0092]

[0093] 48 hours after protein spraying, the ToBRFV source was diluted with 1×PBS at 1:30, and the diluted ToBRFV solution was used to rub and inoculate the tomato leaves that had been treated with protein spraying for 48 hours. Each tomato was quantitatively inoculated with 100 μL of virus solution.

[0094] 3.1.1 Measurement of tomato plant height on day 35 after inoculation with ToBRFV

[0095] Use a ruler to measure the distance from the root of tomatoes in each treatment group to the top of the main stem, and calculate the average value and error.

[0096] 3.1.2 Determination of disease index of tomatoes on day 35 after inoculation with ToBRFV

[0097] The tomato heart leaf and the first leaf adjacent to the heart leaf were photographed on the 35th day after inoculation of ToBRFV in each treatment group, with 3 tomatoes in each group.

[0098] According to the classification standard of Tomato Brown Rough Fruit Virus (ToBRFV) disease severity in the literature (Table 3), the disease severity of each plant in each treatment group was counted, and the disease index and relative control effect were calculated.

[0099] Table 3 Tomato disease grade classification

[0100]

[0101] Disease index = [Σ(number of diseased plants at each level × value of the disease level) / (total number of plants surveyed × highest disease level)] × 100

[0102] Relative control effect = [(disease index of control group - disease index of treatment group) / (disease index of control group)] × 100%

[0103] 3.1.3 Western Blot detection of ToBRFV accumulation in tomatoes in different treatment groups

[0104] The leaves of tomatoes at the same leaf position in different treatment groups were collected, ground with liquid nitrogen, and 1×PBS was added at a mass volume ratio of 1:2 to dissolve the samples, and the total protein of the plants was extracted. 15 μL of protein sample was loaded from each treatment group, and the accumulation of ToBRFV coat protein (CP) in tomatoes in different treatment groups was detected by Western Blot, and the grayscale of the target band was quantitatively analyzed using Image J software.

[0105] 3.2 Test results

[0106] 3.2.1 Measurement of tomato plant height on day 35 after inoculation with ToBRFV

[0107] The results showed that after different treatments in each group, on the 35th day after inoculation with ToBRFV (Tomato Brown Fruit Virus), the average plant height of tomatoes in the 8μM HpaXpm protein group was significantly higher than that in the PBS group and the 8μM hypersensitive protein group by 12.23% and 16.83% (p < 0.05) ( Figure 8 ), while there was no significant difference in plant height between the PBS group, 2μM HpaXpm protein group and 8μM hypersensitive protein group (p>0.05). This indicates that at the same concentration, HpaXpm protein can significantly increase tomato plant height compared with hypersensitive protein.

[0108] Table 4 Average plant height of tomatoes in different treatment groups on the 35th day after inoculation with ToBRFV

[0109]

[0110] Note: The same lowercase letters in the same column indicate no significant difference (p≥0.05), and different lowercase letters in the same column indicate significant difference (p<0.05)

[0111] 3.2.2 Determination of disease index of tomatoes after inoculation with ToBRFV

[0112] The leaves of the plants in the PBS group showed severe discoloration, curling and narrowing, while the leaves of the 8μM hypersensitive protein group and the 2μM HpaXpm protein group also showed different degrees of discoloration, curling and narrowing, but the symptoms were milder than those of the PBS group. The tomato plants in the 8μM HpaXpm protein group grew normally compared with the plants in the other treatment groups, and most of the leaves did not show obvious discoloration and curling ( Fig. 9 ).

[0113] The disease index of the plants in the 8μM HpaXpm protein group was the lowest at 42.86, and the disease index of the plants in the PBS group was the highest. The 8μM HpaXpm protein group had the highest control effect, which was about 21.18% higher than that of the 8μM hypersensitive protein group.

[0114] Table 5 Disease index and control effect of tomato friction inoculated with ToBRFV on the 35th day

[0115]

[0116]

[0117] 3.2.2 Western Blot detection of virus accumulation in tomatoes in different treatment groups

[0118] Western Blot results ( Fig.10 ) showed that the virus accumulation in tomatoes in the PBS group was the highest on the 35th day after inoculation with ToBRFV. The results of Image J grayscale analysis showed that the accumulation level of virus CP in the plants of the 8μM hypersensitive protein group was about 80% of that in the PBS treatment group, and the virus accumulation level decreased by 20% compared with the PBS group; the accumulation level of virus CP in the 2μM HpaXpm protein group and the 8μM HpaXpm protein group was about 81% and 11% of that in the PBS group, and the virus accumulation level decreased by 19% and 81% respectively compared with the PBS group. Among them, the accumulation level of virus CP in the 8μM HpaXpm protein group decreased by 69% compared with the 8μM hypersensitive protein group.

[0119] Table 6 Gray values ​​of histones in different treatments

[0120]

[0121] In summary, HpaXpm protein can resist the infection of ToBRFV (Tomato brown wrinkled fruit virus) and prevent and control tomato brown wrinkled fruit disease.

[0122] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. Although the present application is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. HpaXpm The use of a gene in improving plant resistance to tomato brown wrinkled fruit virus is characterized in that: Said HpaXpm The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. Application of HpaXpm protein in improving plant resistance to tomato brown fruit virus, characterized in that: The HpaXpm protein is composed of HpaXpm Gene expression is produced; wherein, HpaXpm The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. The use according to claim 2, characterized in that: The amino acid sequence of the HpaXpm protein is shown in SEQ ID NO:

2.

4. Expression HpaXpm The application of a recombinant expression vector or a recombinant engineering bacterium of a gene in improving plant resistance to tomato brown wrinkled fruit virus is characterized in that: Said HpaXpm The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

5. The use according to claim 4, characterized in that The expression HpaXpm The recombinant expression vector or recombinant engineering bacteria of the gene contains the nucleotide sequence shown in SEQ ID NO: 1 HpaXpm Gene, or contains a gene capable of expressing and producing the HpaXpm protein having an amino acid sequence as shown in SEQ ID NO:

2.

6. The use according to claim 4, characterized in that: The expression HpaXpm The recombinant expression vector of the gene uses pET30a(+) as the expression vector.

7. The use according to claim 4, characterized in that: The expression HpaXpm The gene recombinant engineering bacteria use Escherichia coli as the host cell.

8. The use according to any one of claims 1 to 7, characterized in that The plant is a plant of the Solanaceae family.

9. The use according to claim 8, characterized in that: The plants are tomatoes and peppers.

10. A method for improving plant resistance to tomato brown fruit virus, comprising applying a HpaXpm protein solution to the plant, wherein: The HpaXpm protein is composed of HpaXpm Gene expression produces HpaXpm The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

11. The method according to claim 10, characterized in that The amino acid sequence of the HpaXpm protein is shown in SEQ ID NO:

2.

12. The method according to claim 10, characterized in that The application is to spray the HpaXpm protein solution onto the leaves, fruits and / or necks of the plants.

13. The method according to claim 10, characterized in that The HpaXpm protein solution is administered at a concentration of 2-8 μM.

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