SFGH gene and application of SFGH gene in plant virus resistance

Abstract

The invention relates to the technical field of genetic engineering breeding, in particular to an SFGH gene and application of the SFGH gene in plant antivirus, solves the problems that anti-disease gene resources are limited, and all crop-virus combinations are difficult to cover, guarantees normal growth of plants and improves economic benefits of the plants, and the SFGH gene comprises the SFGH gene overexpressed in the plants, the nucleic acid sequence of the SFGH gene is as shown in SEQ ID NO. 1. The SFGH gene and the SFGH protein provided by the invention can activate downstream SA-mediated broad-spectrum antiviral defense by recognizing virus-coded CRP (C-reactive protein), reducing glutathione modification of the SFGH gene and the SFGH protein, improving enzyme activity of the SFGH gene and the SFGH protein, improving GSH content in cells and activating broad-spectrum antiviral defense through breeding, and have great application value and practical significance for realizing broad-spectrum resistance of CRP-coded plant viruses.

Description

SFGH gene and its application in plant antiviral therapy Technical Field

[0001] This invention relates to the field of genetic engineering breeding technology, and in particular to an SFGH gene and its application in plant antiviral activity. Background Technology

[0002] Plant viral diseases, one of the three major diseases affecting crop yield, are often referred to as "plant cancer." Due to the obligate parasitic nature of viruses, traditional chemical pesticides are ineffective against them, resulting in huge economic losses to agricultural production every year. Currently, the main control measures for viral diseases include chemical control to kill the vector insects that transmit viruses in order to block the spread of viruses, and traditional breeding to cultivate crop varieties with high resistance.

[0003] However, while chemical control is a common method in agricultural production, it has significant limitations: it can only indirectly control diseases by killing vector insects, and its effect on existing viral diseases is minimal. Furthermore, some plant viruses can be transmitted through non-vector pathways such as mechanical friction and seed transmission, which cannot be blocked by chemical pesticides. Long-term use of chemical agents can also easily lead to pesticide resistance in insect vectors, resulting in decreased subsequent control effectiveness. At the same time, the design, synthesis, and large-scale spraying of chemical pesticides require substantial economic costs; excessive use not only creates a significant environmental burden but also indirectly affects human health through the food chain. Moreover, due to the weak specificity of pesticides, beneficial organisms may be killed while controlling harmful insects, causing significant damage to the farmland ecosystem. In addition, traditional breeding methods for highly resistant crop varieties are time-consuming and inefficient, typically requiring multiple generations of hybridization and screening, making it difficult to quickly respond to the sudden outbreaks and spread of viral diseases in the field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to provide an SFGH gene and its application in plant antiviral therapy, solving the problem of limited disease resistance gene resources that make it difficult to cover all crop-virus combinations, ensuring normal plant growth, and improving their economic benefits.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In the first aspect of the present invention, an SFGH gene is provided, wherein the nucleotide sequence of the gene is any one of the following: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) the nucleotide sequence encoding a protein with the same function obtained by substituting, deleting or inserting one or more nucleotides as shown in SEQ ID NO.1.

[0006] In a second aspect, the present invention provides an SFGH protein, wherein the amino acid sequence of the SFGH protein is any one of the following: (1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; (2) a protein derived from SEQ ID NO.2 that has the same function as the amino acid sequence of SEQ ID NO.2 obtained by substitution, deletion and / or insertion of one or more amino acid residues.

[0007] A third aspect of the present invention provides a plasmid containing the above-mentioned SFGH gene, comprising an SFGH plasmid formed by inserting the nucleic acid sequence of the SFGH gene into the pBIN219 vector via a KpnI restriction site and an XbaI restriction site.

[0008] In a fourth aspect, the present invention provides a method for constructing the above-mentioned plasmid, comprising the following steps: designing specific primers according to the nucleotide sequence shown in SEQ ID NO.1, wherein the primer pair sequences are shown in SEQ ID NO.3 and SEQ ID NO.4; amplifying the SFGH sequence fragment by PCR; ligating the amplified fragment into the pMDC32-3×Flag plasmid digested with BamHI and SpeI by recombination ligation; subsequently amplifying the SFGH-3×Flag gene by PCR; ligating it into the pBIN219 plasmid digested with KpnI and XbaI by restriction enzyme digestion ligation; and performing coliform transformation, single colony streaking, and colony PCR on the ligation product; selecting positive clones for shaking culture and plasmid extraction.

[0009] According to a fifth aspect of the present invention, a transgenic antiviral plant is provided comprising the above-described SFGH gene or the above-described plasmid or comprising at least one of a plant virus vector capable of expressing the SFGH gene, an Agrobacterium Ti plasmid-derived vector, or a bacterial artificial chromosome.

[0010] According to a sixth aspect of the present invention, the use of the SFGH gene or SFGH protein in the prevention and control of plant viruses is provided.

[0011] The SFGH gene and SFGH protein provided in this invention can recognize virus-encoded CRP, thereby activating salicylate (SA)-mediated broad-spectrum resistance, and have significant application value and practical significance. SFGH protein belongs to a class of proteins with activity in hydrolyzing S-formylglutathione. SFGH catalyzes the hydrolysis of S-formylglutathione into formic acid and a reducing small molecule, glutathione (GSH). GSH can activate SA-mediated antiviral defense. Glutathioneization of SFGH at position 60 (cysteine) can inhibit its enzymatic activity, while SFGH can sense virus-encoded CRP, thereby reducing its own glutathioneization modification and increasing its own enzymatic activity, thus activating SA-mediated antiviral defense.

[0012] The SFGH gene described in this invention and its application in plant antiviral therapy offer new insights for disease-resistant breeding in agriculture. Introducing the SFGH gene into crops using genetic engineering techniques can enhance crop resistance to various viruses, reduce the occurrence of viral diseases, and improve crop yield and quality. Based on the antiviral mechanism of the SFGH protein, novel antiviral biological agents can be developed, providing new means for virus control. Attached Figure Description

[0013] Figure 1 shows the SFGH-OE type Nicotiana Bunsenii plant and SFGH in Example 3 of the present invention. S152A -OE type Nicotiana Bunsenii plants and SFGH-KO type Nicotiana Bunsenii plants; Figure 2 shows the growth and development phenotypes of SFGH-OE type Nicotiana Bunsenii plants and SFGH-KO type Nicotiana Bunsenii plants in Example 4 of the present invention. S152A The effects of -OE type and SFGH-KO type of Nicotiana Bunsenifolia plants on BSMV infection; Figure 3 shows the experimental results of broad-spectrum antiviral activity of SFGH-KO type Nicotiana Bunsenifolia plants in Example 5 of this invention. Detailed Implementation

[0014] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0016] Example 1: SFGH-OE type (SFGH gene overexpression) and SFGH S152A -OE type (SFGH S152A Construction of transgenic plasmids (gene overexpression): SFGH-OE transgene and SFGH S152A -OE transgenic strains were cultured in a G10 native tobacco background using the pBin219 vector with KpnI and XbaI restriction sites. First, SFGH and SFGH... S152A The gene fragment was inserted into the BamHI and SpeI restriction sites of the pMDC32-3×Flag vector, and then SFGH-3×Flag and SFGH were amplified by PCR. S152A-3×Flag gene fragment, and inserted into the KpnI and XbaI restriction sites of pBIN219 after digestion with KpnI and XbaI.

[0017] Example 2: Construction of SFGH-KO (SFGH gene knockout) transgenic plasmid. DNA fragments corresponding to sgRNAs were synthesized and used as forward and reverse complement primers. PCR amplification was performed using pCDC-T1T2 plasmid as a template. The PCR product was digested with BsaI and ligated into BsaI-digested pKSE401 using T4 DNA ligase to obtain pKSE401-SFGH.

[0018] Example 3: SFGH-OE type, SFGH S152A -Cultivation of OE and SFGH-KO type plants (1) Verification of transgenic vectors Before transgenic experiments, the vectors used are verified. Transient expression is performed to detect whether the protein is expressed normally. After verification, transgenic experiments can be carried out.

[0019] (2) Symbiotic Culture ① Preparations before transgenic culture: Prepare scalpels, forceps, glass petri dishes, filter paper, and sterilize with MiliQ sterile water; ② Shake the bacteria at night, and transfer them to 50 ml LB medium at a ratio of 1:50 at 9:00 am the next morning, add the appropriate antibiotics, and incubate at 28°C and 220 rpm on a shaker; ③ Start measuring the bacterial concentration 6 hours after transfer. When the OD of the bacterial solution... 600 Subsequent experiments were conducted when the bacterial culture OD was between 0.6 and 0.8; ④ When the bacterial culture OD 600 When the concentration approaches 0.6, collect leaves from the greenhouse. The best leaves to collect are the 3rd and 4th true leaves of the 6-leaf stage of Nicotiana benzi. After collection, rinse both sides of the leaves with deionized water to remove dust. Prepare four petri dishes. Place the first dish in a 5-fold diluted sodium hypochlorite solution, and the following three dishes in sterilized water. Soak the prepared leaves in the sodium hypochlorite solution for 3 minutes, turning them several times to ensure thorough disinfection. Rinse the leaves in the three water-filled dishes to remove any remaining sodium hypochlorite. After rinsing, place the leaves in a clean petri dish for later use. Use a scalpel to cut 0.5 × 0.5 cm² explants from each leaf, cutting four per leaf. Make several small incisions on the explants with the scalpel tip to facilitate Agrobacterium infection. Pour the shaken bacterial solution into a clean petri dish, place the incision-filled explants inside, pressing them below the liquid surface, and soak for 10 minutes. min; ⑨ Take a clean filter paper, remove the explant and place it on the filter paper, then absorb the bacterial solution; ⑩ Place a clean filter paper on the symbiotic culture medium, place the explant with the bacterial solution absorbed face up on the filter paper, cover it with the lid, seal it with sealing film, and incubate upright in the dark for 48 h.

[0020] (3) Differentiation culture ① Pour differentiation medium into the tissue culture bottle, filling it to 1 / 4 full; ② After the medium solidifies, use tweezers to dig two shallow pits on the surface of the medium, fill them with the scooped-out fragments of medium, remove the explant from the symbiotic medium, place it in the shallow pit, and gently press it with tweezers to ensure full contact between the explant and the medium; ③ After processing one explant, thoroughly burn the tweezers with an alcohol lamp before processing another explant; ④ After the transfer, seal the opening and culture in the tissue culture room, changing the differentiation medium once a week for three weeks, then every 10 days thereafter, until small buds grow on the explant.

[0021] (4) Rooting culture ① Pour rooting medium into the tissue culture bottle, filling it to 1 / 3 full; ② Take out the explant with the sprout and place it on a clean petri dish. Use the tip of a knife to gently cut the bottom of the sprout and cut it off; ③ Use tweezers to hold the leaves of the sprout and insert the sprout into the rooting medium. Three sprouts can be inserted into each tissue culture bottle; ④ After cutting each sprout, the scalpel and tweezers need to be thoroughly burned over an alcohol lamp.

[0022] (5) Transplanting into soil ① When the top of the seedling in the isorooting medium reaches the sealing film of the tissue culture bottle, it can be transplanted into the soil; ② One and a half days before transplanting, remove the sealing film and harden the seedlings overnight; ③ Pour the seedlings with the culture medium into water, gently rub off the culture medium, rinse the culture medium under running water, remove any bad leaves from the seedlings, and plant them in the soil; ④ After transplanting, cover the seedlings with a large plastic lid. After the seedlings have improved in growth, you can open the lid, take samples to test for transgenic status, and wait for the seedlings to be seeded and propagated.

[0023] Example 4: SFGH-OE type plants, SFGH S152A To clarify the effect of SFGH on BSMV infection in SFGH-OE and SFGH-KO type plants, the infection status of BSMV in SFGH-OE and SFGH-KO type plants was investigated. S152A BSMV was inoculated onto OE and SFGH-KO type plants using the Agrobacterium tumefaciens infiltration method. γb-sfGFP11 BSMV γb-sfGFP11 Agrobacterium OD per chain 600 =0.05. Virus infection was observed and photographed at approximately 14 days, and Western blot analysis was performed on the virus accumulation in inoculated and systemic leaves. The results are shown in Figure 2. Compared to the control group G10 (Nicotiana Bunsenata plants transformed into super-folder GFP at β-sheets 1-10), virus infection was significantly weakened in SFGH-OE plants, significantly enhanced in SFGH-KO plants, and... S152AThe viral infection in the -OE type plants showed no significant change (Figure 2A). Western blot results showed that, compared with the control group, the accumulation of viral protein on the virus-inoculated leaves and systemically infected leaves of the SFGH-OE type plants was significantly reduced, while the accumulation of viral protein in the SFGH-KO type plants was significantly increased, and the accumulation of viral protein in the SFGH-KO type plants was significantly increased. S152A The accumulation of viral proteins in the -OE genotype plants showed no significant change (Figures 2B, 2C, 2D, and 2E), and the molecular-level detection results were consistent with the phenotypic observations. These results further demonstrate that SFGH negatively regulates BSMV infection.

[0024] Example 5: Broad-spectrum antiviral activity of SFGH protein was achieved by inoculating SFGH-KO type plants with LRSV using the Agrobacterium tumefaciens infiltration method. γb-3×Flag LRSV (Beetroot ringspot virus) and BNYVV-GFP (beet necrosis yellow vein virus) were inoculated, and viral infection was observed and photographed at 10 and 15 days after inoculation. Western blot analysis was also performed on virus-infected leaves. The results showed that compared to the control group (G10 Nicotiana spp.), LRSV and BNYV infection symptoms were significantly enhanced in SFGH-KO type plants (Figures 3A and 3B). Western blot analysis showed that, compared to the control group, the viral protein accumulation of LRSV and BNYV in SFGH-KO type plants was significantly increased (Figures 3C and 3D). Molecular-level detection results were consistent with phenotypic observations. These results further indicate that SFGH protein negatively regulates LRSV and BNYV infection.

[0025] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0026] Finally, it should be noted that those skilled in the art can implement this invention in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without requiring unnecessary experiments. Although specific embodiments are given in this invention, it should be understood that further modifications can be made to the invention. In summary, according to the principles of this invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some basic features can be applied within the scope of the following appended claims.

Claims

1. An SFGH gene, characterized in that, The nucleotide sequence of the gene is any one of the following: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) the nucleotide sequence encoding a protein with the same function obtained by substituting, deleting or inserting one or more nucleotides as shown in SEQ ID NO.

1.

2. An SFGH protein, characterized in that, The amino acid sequence of the SFGH protein is any one of the following: (1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; (2) a protein derived from SEQ ID NO.2 that has the same function as the amino acid sequence of SEQ ID NO.2 obtained by substituting, deleting and / or inserting one or more amino acid residues.

3. A plasmid comprising the SFGH gene of claim 1, characterized in that, This includes the SFGH plasmid formed by inserting the SFGH gene nucleic acid sequence into the pBIN219 vector via KpnI and XbaI restriction sites.

4. A method for constructing the plasmid according to claim 3, characterized in that, The procedure includes the following steps: Specific primers are designed based on the nucleotide sequence shown in SEQ ID NO.1, with primer pair sequences shown in SEQ ID NO.3 and SEQ ID NO.

4. The SFGH sequence fragment is amplified by PCR. The amplified fragment is ligated into the pMDC32-3×Flag plasmid digested with BamHI and SpeI via recombination ligation. Subsequently, the SFGH-3×Flag gene is amplified by PCR and ligated into the pBIN219 plasmid digested with KpnI and XbaI via restriction enzyme digestion and ligation. The ligation product is then subjected to coliform transformation, single colony streaking, and colony PCR. Positive clones are selected for shaking culture and plasmid extraction.

5. A transgenic antiviral plant comprising the SFGH gene of claim 1 or the plasmid of claim 3, or comprising at least one of a plant virus vector capable of expressing the SFGH gene, an Agrobacterium Ti plasmid-derived vector, or a bacterial artificial chromosome.

6. The use of an SFGH gene or SFGH protein in the control of plant viruses.

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