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CN-121975003-A - SPCSV-RNase3 antagonistic protein mutant, encoding gene, expression vector, creation method and application

CN121975003ACN 121975003 ACN121975003 ACN 121975003ACN-121975003-A

Abstract

The application belongs to the fields of biotechnology, protein engineering and plant protection, and in particular relates to an antagonistic protein mutant for cultivating sweet potato compound virus disease (SPVD) resistant plants and application thereof. The key factor in SPVD pathogenesis is the RNase3 protein encoded by sweet potato chlorosis dwarf virus (SPCSV), which acts as an RNA silencing inhibitor (RSS) to disrupt host immunity. The application provides an SPCSV-RNase3 antagonistic protein mutant, which is derived from a natural antagonistic protein, namely a sweet potato trypsin inhibitor (IbSPLTI-a). The application successfully creates and screens high activity inhibition mutants (such as IbSPLTI-a-m 0805) by analyzing a complex structure model of wild-type IbSPLTI-a and RNase3 (constructed by AlphaFold2, for example) and modifying IbSPLTI-a by utilizing a protein directed evolution strategy, in particular by adopting a hot spot amino acid scanning and protein surface design strategy. In vivo and in vitro functional assays indicate that the mutant (e.g., ibSPLTI-a-m 0805) has significantly enhanced binding affinity to SPCSV-RNase3 and exhibits a viral accumulation inhibition effect superior to that of wild-type IbSPLTI-a. The application also provides a nucleic acid sequence encoding the mutant, a plant expression vector containing the sequence, and a method for cultivating SPVD-resistant transgenic plants (especially sweet potato) by using the sequence. The application provides a core gene resource and a technical path for the genetic improvement of SPVD and the development of novel antiviral protein preparations.

Inventors

  • YU YICHENG
  • WANG XIAO
  • SUN JIAN
  • PAN ZHIYUAN

Assignees

  • 江苏师范大学

Dates

Publication Date
20260505
Application Date
20251113

Claims (12)

  1. 1. A mutant SPCSV-RNase3 antagonistic protein, characterized in that the mutant is derived from sweet potato trypsin inhibitor (IbSPLTI-a) and comprises at least one amino acid mutation.
  2. 2. The mutant SPCSV-RNase3 antagonistic protein according to claim 1, wherein the mutant is IbSPLTI-a-m0805 and has the amino acid sequence shown in SEQ ID NO. 1.
  3. 3. The mutant SPCSV-RNase3 antagonistic protein according to claim 1, wherein the amino acid sequence of the sweet potato trypsin inhibitor (IbSPLTI-a) is shown in SEQ ID NO. 2.
  4. 4. A DNA molecule encoding a protein mutant according to any one of claims 1 to 3.
  5. 5. The DNA molecule of claim 4, wherein the nucleotide sequence is shown in SEQ ID NO. 3.
  6. 6. A plant expression vector comprising the DNA molecule of claim 4 or 5 operably linked downstream of a plant promoter.
  7. 7. Plant expression vector according to claim 6, characterized in that the vector is an agrobacterium-mediated plant expression vector, the vector backbone is preferably a pCambia-or pr-series vector, and the promoter is preferably a constitutive promoter, such as a CaMV 35S promoter.
  8. 8. A method of growing a transgenic plant having resistance to sweet potato complex virus disease (SPVD), comprising the steps of: (1) Obtaining the plant expression vector of claim 6 or 7; (2) Introducing the vector obtained in step (1) into a plant cell, tissue or organ to obtain a transgenic plant; (3) Screening and propagating the obtained transgenic plants; (4) SPVD resistance identification is carried out on the transgenic plants obtained by screening.
  9. 9. The method according to claim 8, wherein the method of introducing in the step (2) is an Agrobacterium-mediated method, the Agrobacterium strain is preferably EHA105 or GV3101, and the plant cell, tissue or organ is preferably embryogenic callus cells of sweetpotato.
  10. 10. A transgenic plant having integrated into its genome the plant expression vector of claim 6 or 7 and expressing the protein mutant of any one of claims 1 to 3.
  11. 11. Use of a protein mutant according to any one of claims 1 to 3 for the preparation of a composition or biological agent for inhibiting the activity of SPCSV-RNase 3.
  12. 12. A method for creating a mutant of an SPCSV-RNase3 antagonistic protein, comprising the steps of: (1) The direct interaction of wild type IbSPLTI-a with SPCSV-RNase3 was verified by experimental means (e.g., yeast two-hybrid, bimolecular fluorescent complementation, co-immunoprecipitation, GST Pull-down); (2) Constructing a three-dimensional complex structure model of the wild IbSPLTI-a and the SPCSV-RNase 3; (3) Determining one or more mutation sites on IbSPLTI-a using hotspot amino acid scanning and protein surface design strategies based on the model; (4) Mutagenesis is carried out on the sites to create a mutant library; (5) Screening the mutant library to obtain mutants with enhanced binding affinity to RNase3 and more effective inhibition of the RSS activity of RNase 3.

Description

SPCSV-RNase3 antagonistic protein mutant, encoding gene, expression vector, creation method and application Technical Field The invention belongs to the fields of molecular biology, protein engineering and plant breeding, and in particular relates to a novel antagonistic protein mutant with sweet potato compound virus disease (SPVD) resistance, which is created by utilizing a molecular biology technology and artificial intelligence-assisted protein design, and application of the antagonistic protein mutant in plant genetic improvement and breeding work. Background Sweet potato virus disease is the most serious disease faced by the sweet potato industry. The sweet potato compound virus disease (Sweet potato virus disease, SPVD) is a destructive virus disease caused by the synergistic infection of sweet potato pinnate virus (Sweet potato feathery mottle virus, SPFMV) and sweet potato chlorosis dwarf virus (Sweet potato chlorotic stunt virus, SPCSV), and forms a serious threat to global sweet potato production. The disease is rapid in onset, and can cause serious shrinkage, fading and dwarfing of sweet potato leaves, so that yield loss is up to 50% -100%, and especially the damage of sweet potato main producing areas such as Africa, asia and the like is extremely large. The typical characteristic of SPVD is its strong synergistic pathogenic effect, namely that when two viruses (SPFMV/SPCSV) are independently infected by sweet potato, the disease phenotype of the plant is not obvious or only slight symptoms occur, and when SPFMV and SPCSV are jointly infected by sweet potato, obvious viral synergistic effect occurs, so that the sweet potato plant shows serious symptoms such as leaf distortion, deformity, chlorosis, clear pulse, plant dwarfing and the like. During this process, the SPFMV virus copy number rose rapidly, about 600-fold as compared to when sweetpotato was infected alone, SPFMV being a distinct beneficiary, and SPCSV functioning as a "helper". Studies have shown that the RNase3 protein encoded by SPCSV is a key causative agent of this synergistic effect. SPCSV-RNase3 is a type III RNA endonuclease (RNase 3) encoded in the SPCSV genome, which has dsRNA-specific endonuclease activity. vsiRNAs (virus-DERIVED SMALL INTERFERING RNAS) mediated RNA silencing is one of the most important mechanisms of plant defense against RNA viruses. SPCSV-RNase3 is used as a powerful RNA silencing inhibitor (RSS) and can effectively destroy RNAi antiviral immunity line of plant foundation. It can specifically cleave vsiRNAs of the base vsiRNAs of 21-22 bases to 14 bases in host cells, resulting in a decrease or even loss of silencing ability of the host antiviral RNA, thereby creating favorable conditions for rapid accumulation of helper viruses such as SPFMV, and ultimately leading to the occurrence of serious disorders. At present, no technical means for effectively preventing and treating the sweet potato compound virus disease SPVD exists. The improvement of sweet potato SPVD resistance by conventional biotechnology breeding all relies on methods of exogenous viral fragment-induced activation of the sweet potato plant's own RNA silencing system (i.e., RNAi). However, as the vegetative propagation of sweet potato in tuber propagation and cutting causes the virus to accumulate year by year in plants after it infects SPVD, a series of silencing suppressors (especially RNase 3) encoded by the virus inhibit the RNA silencing system that has been activated, resulting in the final burst of SPVD. Therefore, the anti-SPVD technology based on plant RNAi mechanism is not suitable for inhibiting RNase 3-mediated virus synergistic effect, and cannot fundamentally improve the resistance of sweet potatoes to SPVD. In recent years, artificial Intelligence (AI) driven protein engineering and directed evolution strategies provide new schemes for plant disease-resistant breeding. The use of high-precision structural prediction models (e.g., alphaFold 2) to resolve virus-host protein interactions has become a powerful tool for high-throughput screening and dynamic verification. Therefore, development of a novel technique for directly inhibiting the activity of RNase3 protein, so that it loses the function of RNA Silencing Suppressor (RSS) and is useful for genetic improvement of SPVD resistance is very urgent. Disclosure of Invention The inventor of the present invention found through research that sweet potato natural antagonistic protein, namely sweet potato trypsin inhibitor (IbSPLTI-a), has direct interaction with SPCSV-RNase3 and can inhibit the RSS activity thereof. However, the affinity and inhibition efficiency of natural proteins are often limited and insufficient to provide complete protection under field conditions. Therefore, the invention utilizes an artificial intelligence aided protein directed evolution strategy to modify IbSPLTI-a so as to create a mutant with stronger RNase3 inhibition activity and provide a brand-new