Construction of immune receptor-single-chain antibody fusion protein and application of immune receptor-single-chain antibody fusion protein in prevention and treatment of PVY and ToBRFV

Abstract

The invention discloses an immune receptor-single-chain antibody fusion protein and application of the immune receptor-single-chain antibody fusion protein in prevention and treatment of potato virus Y (pota virus Y, PVY) and tomato brown fruit ruffle virus (ToBRFV), and belongs to the technical field of biological prevention and treatment of viral diseases. According to the invention, a modified immune receptor Sr35 (Sr3527) and a single-chain antibody scFvM1 or scFv16 are fused to create an immune receptor-single-chain antibody fusion protein, and the immune receptor-single-chain antibody fusion protein can specifically recognize PVY coat protein (CP) or CP of ToBRFV, activate host resistance and generate hypersensitive response (HR); after being expressed in plants, the bacillus subtilis can inhibit infection of PVY or ToBRFV, and has a remarkable prevention and treatment effect on PVY or ToBRFV.

Description

Technical Field

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

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

[0003] Potato virus Y (PVY) is a plant virus that widely harms crops such as potatoes and tobacco. It belongs to the genus Potyvirus. PVY can cause symptoms such as mosaic and curling of potato leaves and necrosis of tubers, seriously affecting 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, seriously affecting the appearance and quality of tomato fruits. The coat protein (CP) is the 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 the replication and movement of the virus. Currently, there are relatively few resistance genes for the prevention and control of 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 currently include site-directed mutagenesis, domain replacement, and reconstruction of immune receptors. However, there are currently no reports on the creation of synthetic immune receptors by fusing immune receptors with single-chain antibodies to combat diseases caused by PVY and ToBRFV. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide an immune receptor-single-chain antibody fusion protein and its application in preventing and treating PVY and ToBRFV diseases. △27 ) was fused with a single-chain antibody that specifically recognizes PVY or ToBRFV CP to create an immune receptor-single-chain antibody fusion protein that can specifically recognize CP PVY or CP ToBRFV, triggering a hypersensitive response (HR); after being expressed in plants, it can inhibit the infection of PVY and ToBRFV, and has a significant preventive and control effect on diseases caused by PVY and ToBRFV.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides an immune receptor-single chain antibody fusion protein, which is composed of a modified immune receptor gene Sr35 △27 It is fused with the gene encoding the 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 by targeting the conserved regions of plant viruses, and the monoclonal antibody or the CDR region of the monoclonal antibody is selected as the single-chain antibody.

[0010] The specifically identified plant viruses include, but are not limited to, tobacco mosaic virus and potato Y virus.

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

[0012] Preferably, the immunoreceptor-single-chain antibody fusion protein is any one of the following proteins (A1)-(A3):

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

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

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

[0016] The present invention truncates the C-terminal 27 amino acid sequence of the protein encoded by the immune receptor gene Sr35 from wheat (Triticum monococcum) to obtain the modified immune receptor gene Sr35. △27 ; Then Sr35 △27 The gene encoding the immunoreceptor-single-chain antibody fusion protein was fused with the gene encoding the single-chain antibody scFvM1 or scFv16 to construct a gene encoding the immunoreceptor-single-chain antibody fusion protein, which was named Sr35. △27 -scFvM1 or Sr35 △27 -scFv16.

[0017] Among the above genes, a protein tag gene refers to a polypeptide that is fused and expressed with a target protein using in vitro DNA recombination techniques to facilitate the expression, detection, tracing, and / or purification of the target protein. To facilitate purification of the protein in (A1), a tag may be attached to the amino or carboxyl terminus of the protein in (A1). The tag may be Poly-His (typically six HHHHHH), HA (YPYDVPDYA), FLAG (DYKDDDDK), or c-myc (EQKLISEEDL), among others.

[0018] The second aspect of the present invention provides the use of the above-mentioned immunoreceptor-single-chain antibody fusion protein in the following (1) or (2):

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

[0020] (2) Preparation of 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 the immune receptor-single-chain antibody fusion protein in these plants, the plant's resistance to PVY and ToBRFV can be improved. Furthermore, the immune receptor-single-chain antibody fusion protein can inhibit the systemic infection of PVY and ToBRFV in the plant, thereby improving the prevention and control of diseases caused by PVY and ToBRFV.

[0022] The third aspect of the present invention provides a gene encoding the above-mentioned immunoreceptor-single-chain antibody fusion protein, wherein the gene is a DNA molecule shown in any one of the following i)-iv):

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

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

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

[0026] iv) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 12 except iii).

[0027] The fourth aspect of the present invention provides a recombinant expression vector or genetically engineered bacteria containing the above-mentioned gene.

[0028] The fifth aspect of the present invention provides the use of the above-mentioned gene encoding the immune receptor-single-chain antibody fusion protein, the recombinant expression vector containing the above-mentioned 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 is linked into an expression vector to construct a recombinant expression vector, which is then transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation; and the Agrobacterium strain is used to infect plants.

[0033] Preferably, the expression vector is plCH86966; the gene encoding the immune receptor-single-chain antibody fusion protein is linked downstream of the 35S promoter to drive 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] Beneficial effects of the present invention:

[0037] (1) The present invention modified the immune receptor gene Sr35, deleted the bases encoding the C-terminal 27 amino acids of Sr35, and constructed the modified immune receptor gene Sr35 △27 Sr35 △27 It still has the potential to stimulate the host immune response and induce local hypersensitivity reaction (HR).

[0038] (2) The present invention uses Sr35 △27The gene encoding the immune receptor-single-chain antibody fusion protein was fused with the gene encoding a single-chain antibody that specifically recognizes plant viruses to obtain a gene encoding an immune receptor-single-chain antibody fusion protein. This fusion protein can effectively inhibit the infection of PVY and ToBRFV in plants, thereby improving the control effect of PVY and ToBRFV. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 :Sr35 △27 -scFvM1 can recognize CP PVY Triggering cellular hypersensitivity reaction; Figure A. Sr35 in different injection areas of common tobacco leaves WT With AvrSr35, Sr35 WT With CP PVY 、Sr35 WT with empty vector (EV), Sr35 △27 -scFvM1 and AvrSr35, Sr35 △27 -scFvM1 and CP PVY 、Sr3 5△27 -hypersensitive phenotype produced by co-expression of scFvM1 and EV for 72 hours; Figure B. Western blot detection of protein accumulation of different combinations in tobacco leaves after 72 hours of co-expression, Sr35 WT 、Sr35 △27 -scFvM1, AvrSr35, and CP PVY All were expressed in fusion with HA tag.

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

[0041] Figure 3 :Sr35 △27-scFv16 can recognize CP ToBRFV Triggering cellular hypersensitivity reaction; Figure A. Sr35 in different injection areas of common tobacco leaves WT With AvrSr35, Sr35 WT With CP ToBRFV 、Sr35 WT with empty vector (EV), Sr35 △27 -scFv16 and AvrSr35, Sr35 △27 -scFv16 and CP ToBRFV 、Sr35 △27 -hypersensitive phenotype after 72 hours of co-expression of scFv16 and EV; Figure B. Western blot detection of protein accumulation of different combinations in tobacco leaves after 72 hours of co-expression, Sr35 WT 、Sr35 △27 -scFv16, AvrSr35, and CP ToBRFV All were expressed in fusion with HA tag.

[0042] Figure 4 :Sr35 △27 -scFv16 can recognize CP and induce cellular immune response against ToBRFV; in Figure A, Sr35 in different injection areas of Nicotiana benthamiana leaves △27 -scFv16 with ToBRFV and Sr35 WT HR phenotype after 4 days of co-expression with ToBRFV, empty vector (EV) and ToBRFV; Figure B. Western blot analysis of CP in different injection areas ToBRFV Accumulation level. DETAILED DESCRIPTION

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

[0044] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0045] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Experimental methods without detailed conditions were carried out in accordance with conventional test methods or the operating instructions recommended by the supplier.

[0046] The PVY used in the examples of the present invention has an NCBI accession number of X97895.1, ToBRFV has an NCBI accession number of MT018320, Sr35 has an NCBI accession number of KC573058.1, and AvrSr35 has an NCBI accession number of MF474174. PVY is described in a current journal (doi:10.1093 / plphys / kiad612).

[0047] The plCH86966 vector and the pICH47811 vector are described in a 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 immunoreceptor-single-chain antibody fusion protein

[0049] 1. Construction of the gene Sr35 encoding the 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 from wheat (Triticum monococcum) is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0052] The 27 amino acids encoding the C-terminus of the immune receptor gene Sr35 were deleted to construct the mutant 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) Construction of the gene scFvM1 encoding the single-chain antibody:

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

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

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

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

[0058] The amino acid sequence of the single-chain antibody scFv16 that 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] The 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, the nucleotide sequence of which is shown in SEQ ID NO.11; encoding 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 recognizes CP PVY Induce hypersensitivity reaction

[0061] 1. Test method:

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

[0063] Using Agrobacterium-mediated transient expression system, vector pICHSr35, pICHSr35 △27 -scFvM1, pICHavrSr35, pICHCP PVY and EV were transformed into Agrobacterium GV3101. Subsequently, the Agrobacterium resuspension containing the vector pICHSr35 was mixed with the Agrobacterium resuspension containing pICHavrSr35, pICHCP PVY , EV Agrobacterium resuspension in a volume ratio of 1:1; △27 -scFvM1 Agrobacterium resuspension was mixed with pICHavrSr35, pICHCP PVY The Agrobacterium resuspensions of EV and EV were mixed in a volume ratio of 1:1; a total of 6 Agrobacterium resuspension combinations were obtained and injected into tobacco (Nicotiana tabacum) leaves respectively, so that the two were co-expressed in tobacco cells.

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

[0065] 2. Test results:

[0066] The results are as follows Figure 1 As shown in A, in tobacco leaves, Sr35 WT Co-expression of AvrSr35 and Sr35 induced a strong HR; △27 -scFvM1 and AvrSr35 and Sr35 △27 -scFvM1 and CP PVY When co-expressed, it can also induce a strong HR response; Sr35 WT With CP PVY 、Sr35 WT With EV, Sr35 △27 -scFvM1 co-expression with EV did not produce 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 show that Sr35 △27 -scFvM1 fusion protein can sense CP PVY Activate the host immune response and produce HR.

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

[0068] 1. Test method:

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

[0070] We used Agrobacterium-mediated transient expression system to express Sr35-scFvM1 vector pICHSr35 △27 -scFvM1 and the vector pCBPVY encoding PVY were transformed into Agrobacterium GV3101. Then, the Agrobacterium resuspension containing Sr35-scFvM1 and PVY was mixed in a volume ratio of 1:1, and the mixed Agrobacterium was infiltrated into Nicotiana benthamiana leaves to co-express Sr35-scFvM1 and PVY in tobacco cells. As a control group, we used the same operation to transform Sr35 WT and PVY, as well as EV and PVY, 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 HR. WT Co-expression with PVY and EV+PVY did not produce significant HR ( Figure 2 A) Western blot results showed that compared with Sr35 WT and empty vector pICH86966, Sr35-scFvM1 could significantly reduce the accumulation of PVY ( Figure 2 B) We inserted the GFP gene into the PVY genome, so that we can analyze the movement of the virus in the plant by observing GFP under ultraviolet light. WTIn the plants co-expressing PVY and EV+PVY, obvious green fluorescence was observed in both the inoculated leaves and the systemic leaves. △27 No obvious green fluorescence was observed in the inoculated leaves and systemic leaves of the plants co-expressing -scFvM1 and PVY, indicating that Sr35 △27 -scFvM1 inhibits the systemic movement of PVY ( Figure 2 C) Western blot detection system leaf results showed that PVY and Sr35 WT CP accumulation was detected in leaves of plants co-expressing PVY and EV, but PVY and Sr35 △27 No CP accumulation was detected in leaves of the -scFvM1 co-expressing plant system ( Figure 2 D).

[0073] Example 4: Sr35 △27 -scFv16 recognizes CP ToBRFV Induce hypersensitivity reaction

[0074] 1. Test method:

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

[0076] Using Agrobacterium-mediated transient expression system, vector pICHSr35, pICHSr35 △27 -scFv16, pICHavrSr35, pICHCP PVY and EV were transformed into Agrobacterium GV3101. Subsequently, the Agrobacterium resuspension containing the vector pICHSr35 was mixed with the Agrobacterium resuspension containing pICHavrSr35, pICHCP ToBRFV , EV Agrobacterium resuspension in a volume ratio of 1:1; △27 -scFv16 Agrobacterium resuspension was mixed with pICHavrSr35, pICHCP ToBRFVThe Agrobacterium resuspensions of EV and EV were mixed in a volume ratio of 1:1; a total of 6 Agrobacterium resuspension combinations were obtained and injected into tobacco (Nicotiana tabacum) leaves respectively, so that the two were co-expressed in tobacco cells.

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

[0078] 2. Test results:

[0079] The results are as follows Figure 3 As shown, in tobacco leaves, Sr35 WT Co-expression of AvrSr35 and Sr35 induced a strong HR; △27 -scFv16 and AvrSr35 and Sr35 △27 -scFv16 and CP ToBRFV When co-expressed, it can also induce a strong HR response; Sr35 WT With CP ToBRFV 、Sr35 WT With EV, Sr35 △27 -scFv16 and EV co-expression did not produce HR response. The above results show that Sr35 △27 -scFv16 fusion protein can sense CP ToBRFV Activate the host immune response and produce HR.

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

[0081] 1. Test method:

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

[0083] We used Agrobacterium-mediated transient expression system to express Sr35-scFv16 in pICHSr35 △27-scFv16 and the vector pCBToBRFV encoding ToBRFV were transformed into Agrobacterium GV3101 respectively. Then, the Agrobacterium resuspension containing Sr35-scFv16 and ToBRFV was mixed in a volume ratio of 1:1. The mixed Agrobacterium was infiltrated into Nicotiana benthamiana leaves to co-express Sr35-scFv16 and ToBRFV in tobacco cells. As a control group, we used the same operation to transform Sr35 WT and ToBRFV, as well as EV and ToBRFV, were co-transformed into Agrobacterium for co-expression.

[0084] 2. Test results:

[0085] Four days after vaccination, co-expression of Sr35-scFv16 and ToBRFV induced a strong HR. WT Co-expression with ToBRFV and EV+ToBRFV did not produce significant HR ( Figure 4 A) Western blot results showed that compared with Sr35 WT and 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 the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. An immunoreceptor-single-chain antibody fusion protein, characterized in that: Modified immune receptor Sr35 △27 It is fused with a single-chain antibody that specifically recognizes plant viruses; The modified immune receptor Sr35 △27 The amino acid sequence is shown in SEQ ID NO.

3.

2. The immunoreceptor-single-chain antibody fusion protein according to claim 1, characterized in that The single-chain antibody that specifically recognizes plant viruses is a single-chain antibody scFvM1 or a single-chain antibody scFv16; the amino acid sequence of the single-chain antibody scFvM1 is shown in SEQ ID NO.6, which is used to specifically recognize PVY; the amino acid sequence of the single-chain antibody scFv16 is shown in SEQ ID NO.9, which is used to specifically recognize ToBRFV.

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

4. Use of the immunoreceptor-single-chain antibody fusion protein according to any one of claims 1 to 3 in the following (1) or (2): (1) Improve plant resistance to PVY and / or ToBRFV; (2) Preparation of drugs for the prevention and treatment of PVY and / or ToBRFV.

5. The use according to claim 4, characterized in that The plants include: potato, tobacco, tomato, pepper, wheat, corn, soybean and / or cotton.

6. A gene encoding an immunoreceptor-single-chain antibody fusion protein, characterized in that: The coding gene is a DNA molecule shown in any one of the following i)-iv): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.7; ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 8 other than i); iii) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.11; iv) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 12 except iii).

7. A recombinant expression vector or genetically engineered bacteria containing the coding gene according to claim 6.

8. Use of the gene encoding the immune receptor-single-chain antibody fusion protein according to claim 6 or the recombinant expression vector or genetically engineered bacteria according to claim 7 in the following (1) or (2): (1) Improve plant resistance to PVY and / or ToBRFV; (2) Cultivate plant varieties resistant to PVY and / or ToBRFV.

9. A method for improving plant resistance to plant viruses, characterized in that: The following steps are involved: The gene encoding the immune receptor-single-chain antibody fusion protein according to claim 6 is linked to an expression vector to construct a recombinant expression vector, which is then transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation; and the Agrobacterium strain is used to infect plants.

10. The method according to claim 9, characterized in that The plant virus is PVY or ToBRFV.

Images