Construction of immune receptor-single chain antibody fusion protein and its application in prevention and treatment of pvy and tobrfv

Abstract

The application discloses an immune receptor-single-chain antibody fusion protein and application thereof in prevention and treatment of potato virus Y (PVY) and tomato brown rugose fruit virus (ToBRFV), and belongs to the technical field of biological prevention and treatment of viral diseases. The application fuses the modified immune receptor Sr35 (Sr35 △27 ) with single-chain antibodies scFvM1 or scFv16 to create an immune receptor-single-chain antibody fusion protein, which can specifically recognize the CP of PVY or the CP of ToBRFV, activate host resistance, and produce a hypersensitive response (HR); and after expression in plants, the fusion protein can inhibit the infection of PVY or ToBRFV and has a significant prevention and treatment effect on PVY or ToBRFV.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for viral diseases, specifically to an immune receptor-single-chain antibody fusion protein and its application in controlling plant diseases caused by PVY and ToBRFV. Background Technology

[0002] Through long-term co-evolution with pathogens, plants have developed a highly efficient immune system to resist pathogen infection. Intracellular immune receptors are key components of the plant immune system, and the precise regulation of their expression and activity is crucial for plant disease resistance, growth, and development. Immune receptors activate the plant immune response by recognizing avirulence genes (Avr) secreted by pathogens.

[0003] Potato virus Y (PVY) is a plant virus that widely affects crops such as potatoes and tobacco, belonging to the genus Potatovirus Y. PVY can cause symptoms such as mosaic and curling of potato leaves and tuber necrosis, severely impacting potato yield and quality. Tomato brown rugose fruit virus (ToBRFV) belongs to the genus Tobamovirus. ToBRFV is a newly emerging virus that can cause brown spots, wrinkling, and deformation of tomato fruits, severely affecting the appearance and quality of the fruit. The coat protein (CP) is a key protein that is first recognized by the host when the virus enters the cell. In addition to participating in the assembly of virus particles, it also plays an important role in viral replication and movement. Currently, there are relatively few resistance genes for controlling PVY and ToBRFV.

[0004] Targeted modification of immune receptors, enabling precise regulation of their recognition spectrum, has become an effective strategy for cultivating virus-resistant plants. Common targeted modification methods include site-directed mutagenesis, domain substitution, and remodeling of immune receptors. However, there are currently no research reports on creating synthetic immune receptors by fusing them with single-chain antibodies to control diseases caused by PVY and ToBRFV. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention aims to provide an immune receptor-single-chain antibody fusion protein and its application in the prevention and control of PVY and ToBRFV diseases. This invention utilizes a modified immune receptor Sr35 (Sr35... △27 By fusing with single-chain antibodies that specifically recognize PVY or ToBRFV CP, an immune receptor-single-chain antibody fusion protein was created, which can specifically recognize CP. PVY or CP ToBRFVIt induces a hypersensitive response (HR); when expressed in plants, it can inhibit the infection of PVY and ToBRFV, and has a significant control effect on diseases caused by PVY and ToBRFV.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an immune receptor-single-chain antibody fusion protein, comprising a modified immune receptor gene Sr35. △27 It is formed by fusing with a gene that encodes a single-chain antibody that specifically recognizes plant viruses;

[0008] The modified immune receptor gene Sr35 △27 The encoded amino acid sequence is shown in SEQ ID NO.3.

[0009] Preferably, the single-chain antibody that specifically recognizes plant viruses can be prepared as a monoclonal antibody targeting a conserved region of the plant virus, and the monoclonal antibody or the CDR region of the monoclonal antibody can be selected as the single-chain antibody.

[0010] Plant viruses that are specifically identified include, but are not limited to, Tobacco Mosaic Virus (TMV) viruses and Potato Virus Y (PVY) viruses.

[0011] In a preferred embodiment of the present invention, the single-chain antibody that specifically recognizes plant viruses is either scFvM1, a single-chain antibody that recognizes PVY CP, or scFv16, a single-chain antibody that recognizes ToBRFV CP. The amino acid sequence of the single-chain antibody scFvM1 is shown in SEQ ID NO.6, and it specifically recognizes PVY; the amino acid sequence of the single-chain antibody scFv16 is shown in SEQ ID NO.9, and it specifically recognizes ToBRFV.

[0012] Preferably, the immune receptor-single-chain antibody fusion protein is any one of the proteins shown in (A1)-(A3) below:

[0013] (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.8 of the sequence listing;

[0014] (A2) A protein consisting of the amino acid sequence shown in SEQ ID NO.12 of the sequence listing;

[0015] (A3) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1) or (A2).

[0016] This invention truncates the C-terminal 27 amino acid sequence of the Sr35 immune receptor gene, derived from wheat (Triticum monococcum), to obtain a modified immune receptor gene Sr35. △27 Then Sr35 △27 By fusing with genes encoding single-chain antibodies scFvM1 or scFv16, a gene encoding an immune receptor-single-chain antibody fusion protein was constructed and named Sr35. △27 -scFvM1 or Sr35 △27 -scFv16.

[0017] Among the aforementioned genes, the protein tag gene refers to a polypeptide expressed by fusing it with the target protein using in vitro DNA recombination technology, facilitating the expression, detection, tracing, and / or purification of the target protein. Specifically, to facilitate the purification of the protein in (A1), a tag can be attached to the amino or carboxyl terminus of the protein in (A1). The tag can be Poly-His (typically six HHHHHH), HA (YPYDVPDYA), FLAG (DYKDDDDK), or c-myc (EQKLISEEDL), etc.

[0018] In a second aspect, the present invention provides the use of the above-described immune receptor-single-chain antibody fusion protein in either (1) or (2) below:

[0019] (1) Improve plant resistance to PVY and / or ToBRFV;

[0020] (2) Prepare drugs for the prevention and treatment of PVY and / or ToBRFV.

[0021] In the above applications, the plants include, but are not limited to, the following crops: potatoes, tobacco, tomatoes, peppers, wheat, corn, soybeans, and / or cotton. By expressing an immune receptor-single-chain antibody fusion protein in the above plants, the plant's resistance to PVY and ToBRFV can be improved; moreover, the immune receptor-single-chain antibody fusion protein can also inhibit systemic infection of PVY and ToBRFV in plants, thereby improving the control effect against diseases caused by PVY and ToBRFV.

[0022] A third aspect of the invention provides a gene encoding the aforementioned immune receptor-single-chain antibody fusion protein, said gene being any of the DNA molecules shown in i)-iv) below:

[0023] i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.7;

[0024] ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO. 8;

[0025] iii) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.11;

[0026] iv) DNA molecules other than iii) encoding the amino acid sequence shown in SEQ ID NO.12.

[0027] In a fourth aspect, the present invention provides a recombinant expression vector or genetically engineered bacteria containing the above-mentioned genes.

[0028] In a fifth aspect, the present invention provides the use of the gene encoding the immune receptor-single-chain antibody fusion protein, the recombinant expression vector containing the gene, or the genetically engineered bacteria in the following (1) or (2):

[0029] (1) Improve plant resistance to PVY and / or ToBRFV;

[0030] (2) Cultivate plant varieties resistant to PVY and / or ToBRFV.

[0031] A sixth aspect of the present invention provides a method for improving plant resistance to plant viruses, comprising the following steps:

[0032] The gene encoding the immune receptor-single-chain antibody fusion protein was ligated into an expression vector to construct a recombinant expression vector. The recombinant expression vector was then transformed into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation. The Agrobacterium strain was then used to infect plants.

[0033] Preferably, the expression vector is plCH86966; the gene encoding the immune receptor-single-chain antibody fusion protein is inserted downstream of the 35S promoter to drive the efficient expression of the fusion protein in plants.

[0034] Preferably, the Agrobacterium competent cells are Agrobacterium tumefaciens GV3101.

[0035] Preferably, the plant virus is PVY or ToBRFV.

[0036] The beneficial effects of this invention are:

[0037] (1) In this invention, the immune receptor gene Sr35 was modified by deleting 27 amino acids from the C-terminus of Sr35 to construct the modified immune receptor gene Sr35. △27 Sr35 △27 It still has the potential to elicit a host immune response, thereby inducing a local hypersensitivity reaction (HR).

[0038] (2) This invention uses Sr35 △27A gene encoding a single-chain antibody that specifically recognizes plant viruses was fused with a gene to obtain a gene encoding an immune receptor-single-chain antibody fusion protein. This fusion protein can effectively inhibit PVY and ToBRFV infection in plants, thereby improving the control efficacy against PVY and ToBRFV. Attached Figure Description

[0039] Figure 1 Sr35 △27 -scFvM1 can recognize CP PVY Inducing cellular hypersensitivity; Figure A shows different injection areas of ordinary tobacco leaves, where Sr35... WT Compared with AvrSr35 and Sr35 WT With CP PVY Sr35 WT Compared with empty carrier (EV), Sr35 △27 -scFvM1 and AvrSr35, Sr35 △27 -scFvM1 and CP PVY Sr3 5△27 The hypersensitive phenotype resulting from co-expression of scFvM1 and EV for 72 hours; the figure shows the protein accumulation of different combinations in tobacco leaves after 72 hours of co-expression, as detected by B. Western blot. Sr35 WT Sr35 △27 -scFvM1, AvrSr35 and CP PVY All are expressed in fusion with the HA tag.

[0040] Figure 2 Sr35 △27 -scFvM1 can recognize CP-induced cellular immune responses against PVY; in the figure, A. Sr35 in different injection areas of the tobacco leaf of Nicotiana benthamiana. △27 -scFvM1 and PVY, Sr35 WT The HR phenotypes were generated 4 days after co-expression with PVY, empty vector (EV), and PVY; the figure shows B. Western blot analysis of CP in different injection regions. PVY Accumulation level; C.Sr35 in the figure △27 -scFvM1 and PVY, Sr35 WT The green fluorescence phenotype observed in inoculated leaves and systemic leaves of plants co-expressed with PVY or empty vector (EV) and PVY 8 days after inoculation; D. Western blot analysis of CP in inoculated leaves (indicated by yellow arrows) and systemic leaves (indicated by white arrows) of different co-expressed plants on day 8 after inoculation. PVY The level of accumulation.

[0041] Figure 3 Sr35 △27-scFv16 can recognize CP ToBRFV Inducing cellular hypersensitivity; Figure A shows different injection areas of ordinary tobacco leaves, where Sr35... WT Compared with AvrSr35 and Sr35 WT With CP ToBRFV Sr35 WT Compared with empty carrier (EV), Sr35 △27 -scFv16 and AvrSr35, Sr35 △27 -scFv16 and CP ToBRFV Sr35 △27 The hypersensitive phenotype induced by co-expression of scFv16 and EV for 72 hours is shown in the figure; the protein accumulation of different combinations after 72 hours of co-expression in tobacco leaves was detected by B. Western blot, Sr35 WT Sr35 △27 -scFv16, AvrSr35 and CP ToBRFV All are expressed in fusion with the HA tag.

[0042] Figure 4 Sr35 △27 -scFv16 can recognize CP-induced cellular immune responses against ToBRFV; Figure A shows different injection areas of *Tobacco Benzoinus* leaves, where Sr35... △27 -scFv16 and ToBRFV, Sr35 WT The HR phenotypes were generated 4 days after co-expression with ToBRFV, empty vector (EV), and ToBRFV; the figure shows B. Western blot analysis of CP in different injection regions. ToBRFV Accumulation level. Detailed Implementation

[0043] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0044] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0045] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein:

[0046] The PVY used in this embodiment of the invention has the following NCBI accession number: X97895.1; ToBRFV has the NCBI accession number: MT018320; Sr35 has the NCBI accession number: KC573058.1; and AvrSr35 has the NCBI accession number: MF474174. PVY is described in existing journal literature (doi:10.1093 / plphys / kiad612).

[0047] The plCH86966 and pICH47811 vectors are described in the journal article (A modular cloning system for standardized assembly of multigene constructs. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. PLoS One. 2011 Feb 18; 6(2):e16765. doi:10.1371 / journal.pone.0016765.10.1371 / journal.pone.0016765 PubMed 21364738).

[0048] Example 1: Construction of a gene encoding an immune receptor-single-chain antibody fusion protein

[0049] 1. Construct the gene Sr35 encoding an immune receptor-single-chain antibody fusion protein. △27 -scFvM1:

[0050] (1) Modification of the immune receptor gene Sr35:

[0051] The nucleotide sequence of the immune receptor gene Sr35, derived from wheat (Triticum monococcum), is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0052] The mutant Sr35 was constructed by deleting 27 amino acids from the C-terminus of the immune receptor gene Sr35. △27 Sr35 △27 The encoded amino acid sequence is shown in SEQ ID NO.3; mutant Sr35 △27 The nucleotide sequence is shown in SEQ ID NO.4.

[0053] (2) Construct the gene scFvM1 encoding a single-chain antibody:

[0054] The VH and VL sequences encoding monoclonal antibody M1 were fused to construct the gene encoding single-chain antibody scFvM1, the nucleotide sequence of which is shown in SEQ ID NO.5; the amino acid sequence of single-chain antibody scFvM1 is shown in SEQ ID NO.6.

[0055] (3) Constructing Sr35 △27 -scFvM1:

[0056] The mutant Sr35 △27 The nucleotide sequence of [a gene] was fused with the nucleotide sequence of scFvM1 to create the fusion gene Sr35. △27 -scFvM1, whose nucleotide sequence is shown in SEQ ID NO.7, encodes the immune receptor-single-chain antibody fusion protein Sr35. △27 The amino acid sequence of -scFvM1 is shown in SEQ ID NO.8.

[0057] 2. Construct the gene Sr35 encoding an immune receptor-single-chain antibody fusion protein. △27 -scFv16:

[0058] The amino acid sequence of the single-chain antibody scFv16, which specifically recognizes ToBRFV, is shown in SEQ ID NO.9, and the nucleotide sequence of the scFv16 gene is shown in SEQ ID NO.10.

[0059] Encoding mutant Sr35 △27 The nucleotide sequence of Sr35 (SEQ ID NO.4) was fused with the nucleotide sequence encoding scFv16 (SEQ ID NO.10) to create the fusion gene Sr35. △27 -scFv16, whose nucleotide sequence is shown in SEQ ID NO.11; encodes the immune receptor-single-chain antibody fusion protein Sr35. △27 The amino acid sequence of -scFv16 is shown in SEQ ID NO.12.

[0060] Example 2: Sr35 △27 -scFvM1 identifies CP PVY Inducing hypersensitivity reaction

[0061] 1. Test method:

[0062] The immune receptor gene Sr35 WT (SEQ ID NO.1) was ligated into the pICH86966 vector to construct the vector pICHSr35; the fusion gene Sr35 was then incorporated into the vector. △27 -scFvM1 was incorporated into the pICH86966 vector to construct the pICHSr35 vector. △27-scFvM1; The gene encoding AvrSr35 was ligated into the pICH47811 vector to construct the vector pICHavrSr35; The gene encoding PVYCP (CP) was ligated into the vector pICH47811 to construct the vector pICHavrSr35. PVY The pICH47811 vector was incorporated to construct the pICHCP vector. PVY The pICH86966 vector was used as the empty vector (EV).

[0063] Using an Agrobacterium-mediated transient expression system, the vectors pICHSr35 and pICHSr35 were expressed. △27 -scFvM1, pICHavrSr35, pICHCP PVY EVs were transformed into Agrobacterium GV3101. Subsequently, Agrobacterium resuspension containing the vector pICHSr35 was reacted with Agrobacterium containing pICHavrSr35 and pICHCP, respectively. PVY Agrobacterium tumefaciens resuspension of EV was mixed at a 1:1 volume ratio; the pICHSr35 carrier was added. △27 Agrobacterium resuspension containing -scFvM1 was reacted with pICHavrSr35 and pICHCP, respectively. PVY Agrobacterium tumefaciens resuspension of EV was mixed at a 1:1 volume ratio; a total of 6 Agrobacterium tumefaciens resuspension combinations were obtained, which were injected into tobacco (Nicotiana tabacum) leaves to co-express both in tobacco cells.

[0064] After 72 hours of co-expression, the hypersensitivity phenotype at the injection site was observed; at the same time, Western blot was used to detect the protein accumulation of different combinations after 72 hours of co-expression in tobacco leaves.

[0065] 2. Test Results:

[0066] The results are as follows Figure 1 As shown in Figure A, Sr35 in tobacco leaves WT Co-expression with AvrSr35 can induce strong HR; Sr35 △27 -scFvM1 and AvrSr35 and Sr35 △27 -scFvM1 and CP PVY Co-expression can also induce a strong HR response; Sr35 WT With CP PVY Sr35 WT Compared to EV and Sr35 △27 -scFvM1 co-expression with EV did not elicit a HR response. Western blot results showed that Sr35 △27 -scFvM1 and Sr35 △27 -scFvM1, AvrSr35 and CP PVYAll can be expressed normally. Figure 1 B). The above results indicate that Sr35 △27 -scFvM1 fusion protein can sense CP PVY It activates the host's immune response, producing HR.

[0067] Example 3: Sr35 △27 -scFvM1 inhibits PVY infection

[0068] 1. Test method:

[0069] Sr35 WT (SEQ ID NO.1) was ligated into the pICH86966 vector to construct the vector pICHSr35; the fusion gene Sr35 was then incorporated into the vector. △27 -scFvM1 was incorporated into the pICH86966 vector to construct the pICH Sr35 vector. △27 -scFvM1; pCBPV Y vector was obtained from the literature doi:10.1093 / plphys / kiad612; pICH86966 vector was used as empty vector (EV).

[0070] We used an Agrobacterium-mediated transient expression system to express the vector pICHSr35 encoding Sr35-scFvM1. △27 Sr35-scFvM1 and the vector pCBPVY encoding PVY were transformed into Agrobacterium GV3101. Then, Agrobacterium resuspensions containing Sr35-scFvM1 and PVY were mixed at a 1:1 volume ratio, and the resulting Agrobacterium was infiltrated into tobacco leaves to co-express Sr35-scFvM1 and PVY in tobacco cells. As a control group, the same procedure was performed, transforming Sr35-scFvM1 and PVY separately into pCBPVY. WT PVY and EV were co-transformed into Agrobacterium for co-expression.

[0071] 2. Test Results:

[0072] Four days after inoculation, co-expression of Sr35-scFvM1 and PVY induced a strong heart rate (HR). WT No significant HR was observed with co-expression with PVY or EV+PVY. Figure 2 A). Western blot results show that, compared to Sr35 WT Compared with the empty vector pICH86966, Sr35-scFvM1 can significantly reduce the accumulation of PVY. Figure 2 B). We inserted the GFP gene into the PVY genome, allowing us to analyze viral movement in plants by observing GFP under UV light. Eight days after inoculation, Sr35... WTObvious green fluorescence was observed in both inoculated leaves and systemic leaves of plants co-expressing PVY and EV+PVY, but Sr35... △27 No obvious green fluorescence was observed in the inoculated leaves of plants co-expressing -scFvM1 and PVY, as well as in the systemic leaves, indicating that Sr35 △27 -scFvM1 suppresses system movement in PVY. Figure 2 C). Western blot analysis of the blades showed that PVY and Sr35... WT CP accumulation was detected in the leaves of both PVY and EV co-expression plant systems, but not in PVY and Sr35. △27 No CP accumulation was detected in the leaves of the -scFvM1 co-expressing plant system. Figure 2 D).

[0073] Example 4: Sr35 △27 -scFv16 identifies CP ToBRFV Inducing hypersensitivity reaction

[0074] 1. Test method:

[0075] The immune receptor gene Sr35 WT (SEQ ID NO.1) was ligated into the pICH86966 vector to construct the vector pICHSr35; the fusion gene Sr35 was then incorporated into the vector. △27 -scFv16 was ligated into the pICH86966 vector to construct the pICHSr35 vector. △27 -scFv16; The gene encoding AvrSr35 was ligated into the pICH47811 vector to construct the vector pICHavrSr35; The gene encoding ToBRFV CP (CP) was ligated into the vector pICH47811. ToBRFV The pICH47811 vector was incorporated to construct the pICHCP vector. ToBRFV The pICH86966 vector was used as the empty vector (EV).

[0076] Using an Agrobacterium-mediated transient expression system, the vectors pICHSr35 and pICHSr35 were expressed. △27 -scFv16, pICHavrSr35, pICHCP PVY EVs were transformed into Agrobacterium GV3101. Subsequently, Agrobacterium resuspension containing the vector pICHSr35 was reacted with Agrobacterium containing pICHavrSr35 and pICHCP, respectively. ToBRFV Agrobacterium tumefaciens resuspension of EV was mixed at a 1:1 volume ratio; the pICHSr35 carrier was added. △27 Agrobacterium resuspension containing -scFv16 was reacted with pICHavrSr35 and pICHCP, respectively. ToBRFVAgrobacterium tumefaciens resuspension of EV was mixed at a 1:1 volume ratio; a total of 6 Agrobacterium tumefaciens resuspension combinations were obtained, which were injected into tobacco (Nicotiana tabacum) leaves to co-express both in tobacco cells.

[0077] After 72 hours of co-expression, the hypersensitivity phenotype at the injection site was observed; at the same time, Western blot was used to detect the protein accumulation of different combinations after 72 hours of co-expression in tobacco leaves.

[0078] 2. Test Results:

[0079] The results are as follows Figure 3 As shown, in tobacco leaves, Sr35 WT Co-expression with AvrSr35 can induce strong HR; Sr35 △27 -scFv16 and AvrSr35 and Sr35 △27 -scFv16 and CP ToBRFV Co-expression can also induce a strong HR response; Sr35 WT With CP ToBRFV Sr35 WT Compared to EV and Sr35 △27 Co-expression of -scFv16 and EV did not elicit a HR response. These results indicate that Sr35 △27 -scFv16 fusion protein can sense CP ToBRFV It activates the host's immune response, producing HR.

[0080] Example 5: Sr35 △27 -scFv16 inhibits ToBRFV infection

[0081] 1. Test method:

[0082] immune receptor Sr35 WT (SEQ ID NO.1) was ligated into the pICH86966 vector to construct the vector pICHSr35; the fusion gene Sr35 was then incorporated into the vector. △27 -scFv16 was ligated into the pICH86966 vector to construct the pICHSr35 vector. △27 -scFv16; ToBRFV is ligated into the pCB301 vector to construct the vector pCBToBRFV; the pCBToBRFV vector is from the literature: doi:10.1111 / mpp.13115; the pICH86966 vector is used as the empty vector (EV).

[0083] We used an Agrobacterium-mediated transient expression system to express the vector pICHSr35 encoding Sr35-scFv16. △27Sr35-scFv16 and the vector pCBToBRFV encoding ToBRFV were transformed into Agrobacterium GV3101 cells. Then, Agrobacterium resuspensions containing Sr35-scFv16 and ToBRFV were mixed at a 1:1 volume ratio. The mixed Agrobacterium was then infiltrated into tobacco leaves to co-express Sr35-scFv16 and ToBRFV in tobacco cells. As a control group, the same procedure was performed, transforming Sr35-scFv16 and ToBRFV separately. WT It was co-expressed in Agrobacterium with ToBRFV and EV and ToBRFV.

[0084] 2. Test Results:

[0085] Four days after inoculation, co-expression of Sr35-scFv16 and ToBRFV induced a strong heart rate (HR). WT Co-expression with ToBRFV and EV+ToBRFV did not produce significant HR ( Figure 4 A). Western blot results show that, compared to Sr35 WT Compared with the empty vector pICH86966, Sr35-scFv16 can significantly reduce the accumulation of ToBRFV. Figure 4 B).

[0086] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An immune receptor-single-chain antibody fusion protein, characterized in that, Modified immune receptor Sr35 △27 It is formed by fusing with single-chain antibodies that specifically recognize plant viruses; The modified immune receptor Sr35 △27 The amino acid sequence is shown in SEQ ID NO. 3; The single-chain antibody that specifically recognizes plant viruses is either single-chain antibody scFvM1 or single-chain antibody scFv16; the amino acid sequence of single-chain antibody scFvM1 is shown in SEQ ID NO. 6, and it is used to specifically recognize PVY; the amino acid sequence of single-chain antibody scFv16 is shown in SEQ ID NO. 9, and it is used to specifically recognize ToBRFV.

2. The immune receptor-single-chain antibody fusion protein according to claim 1, characterized in that, The immune receptor-single-chain antibody fusion protein is any one of the following proteins (A1)-(A3): (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.8 of the sequence listing; (A2) A protein consisting of the amino acid sequence shown in SEQ ID NO.12 of the sequence listing; (A3) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1) or (A2).

3. Applications of immune receptor-single-chain antibody fusion proteins in the following (1) or (2): (1) Improve plant resistance to PVY; (2) Preparation of drugs for the prevention and treatment of PVY; The immune receptor-single-chain antibody fusion protein is Sr35. △27 -scFvM1, whose amino acid sequence is shown in SEQ ID NO.8; The plant in question is tobacco.

4. Applications of immune receptor-single-chain antibody fusion proteins in the following (1) or (2): (1) Improve plant resistance to ToBRFV; (2) Preparation of drugs for the prevention and treatment of ToBRFV; The immune receptor-single-chain antibody fusion protein is Sr35. △27 -scFv16, whose amino acid sequence is shown in SEQ ID NO.12; The plant in question is tobacco.

5. The gene encoding an immune receptor-single-chain antibody fusion protein, characterized in that, The encoding gene is any of the DNA molecules shown in i)-iv) below: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.7; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO. 8; iii) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.11; iv) DNA molecules other than iii) encoding the amino acid sequence shown in SEQ ID NO.

12.

6. A recombinant expression vector or genetically engineered bacteria containing the encoding gene of claim 5.

7. The application of the gene encoding the immune receptor-single-chain antibody fusion protein, or a recombinant expression vector or genetically engineered bacteria containing the gene encoding the immune receptor-single-chain antibody fusion protein, in the following (1) or (2): (1) Improve plant resistance to PVY; (2) Cultivate plant varieties resistant to PVY; The gene encoding the immune receptor-single-chain antibody fusion protein is any one of the DNA molecules shown in i)-ii): i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.7; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO. 8; The plant in question is tobacco.

8. The application of the gene encoding the immune receptor-single-chain antibody fusion protein, or a recombinant expression vector or genetically engineered bacteria containing the gene encoding the immune receptor-single-chain antibody fusion protein, in the following (1) or (2): (1) Improve plant resistance to ToBRFV; (2) Cultivate plant varieties resistant to ToBRFV; The gene encoding the immune receptor-single-chain antibody fusion protein is any one of the DNA molecules shown in iii)-iv) below: iii) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.11; iv) DNA molecules other than iii) encoding the amino acid sequence shown in SEQ ID NO. 12; The plant in question is tobacco.

Images