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CN-121975844-A - Rice stripe mosaic virus expression vector, gene silencing vector, preparation method and application

CN121975844ACN 121975844 ACN121975844 ACN 121975844ACN-121975844-A

Abstract

The invention relates to the technical field of genetic engineering, and discloses a Rice Stripe Mosaic Virus (RSMV) expression vector, a gene silencing vector, a preparation method and application. The rice streak mosaic virus vector composition provided by the invention comprises a construct for transcribing and producing virus antigenomic RNA, a construct for expressing trans-acting proteins necessary for virus replication and transcription, and a construct for expressing a virus silencing inhibitor. By utilizing the combination of the components, recombinant RSMV with infection activity can be obtained in the Nicotiana benthamiana cells, and the recombinant virus can be further introduced into a mediator insect electro-optic leafhopper body through microinjection, and inoculated to rice plants through the electro-optic leafhopper to establish systematic infection. The invention provides application of the recombinant viral vector in electro-optic leafhoppers, rice in-vivo expression of exogenous proteins or silencing of endogenous genes and the like. The vector has the advantages of strong carrying capacity of exogenous fragments, high genetic stability and the like.

Inventors

  • LI ZHENGHE
  • WU KAILI

Assignees

  • 浙江大学

Dates

Publication Date
20260505
Application Date
20260210

Claims (10)

  1. 1. A rice streak mosaic virus RSMV expression vector, wherein said expression vector is a composition comprising: a) An expression construct comprising an RSMV genomic polynucleotide sequence and one or more exogenous polynucleotide sequences encoding a polypeptide molecule, said exogenous polynucleotide sequences being operably linked to a repeated RSMV transcription regulatory cis element inserted into said RSMV genomic polynucleotide sequence and forming an independent transcription unit; b) At least one expression construct comprising polynucleotide sequences encoding RSMV nucleocapsid protein N, phosphoprotein P, and RNA polymerase large subunit L, respectively, operably linked to a functional plant promoter; c) At least one expression construct comprising a polynucleotide sequence encoding one or more RNA silencing inhibitors is operably linked to a functional plant promoter.
  2. 2. A rice streak mosaic virus RSMV gene silencing vector, wherein the expression vector is a composition comprising: a) An expression construct comprising an RSMV genomic polynucleotide sequence and one or more exogenous polynucleotide sequences encoding hairpin RNAs, said exogenous polynucleotide sequences being operably linked to a repeated RSMV transcription regulatory cis element inserted into said RSMV genomic polynucleotide sequence and forming an independent transcription unit; b) At least one expression construct comprising polynucleotide sequences encoding RSMV nucleocapsid protein N, phosphoprotein P, and RNA polymerase large subunit L, respectively, operably linked to a functional plant promoter; c) At least one expression construct comprising a polynucleotide sequence encoding one or more RNA silencing inhibitors is operably linked to a functional plant promoter.
  3. 3. The rice streak mosaic virus RSMV expression vector of claim 1 or the rice streak mosaic virus RSMV gene silencing vector of claim 2, wherein the insertion site of the independent transcription unit in a) of the composition is at least one of the N/P, P/P3, P3/M, M/G, G/P6, P6/L gene spacers of the RSMV genome.
  4. 4. The rice streak mosaic virus RSMV expression vector of claim 1 or the rice streak mosaic virus RSMV gene silencing vector of claim 2, wherein the RSMV genomic polynucleotide sequence of a) of the composition is operably linked between a functional plant promoter and a ribozyme, which transcribes to produce a viral antigenomic RNA.
  5. 5. The rice streak mosaic virus RSMV expression vector or the rice streak mosaic virus RSMV gene silencing vector according to claim 4, In the composition a), the functional plant promoter is a cauliflower mosaic virus 35S promoter, and the ribozyme is a hepatitis delta virus antigenome ribozyme; in the b) of the composition, the functional plant promoter is a cauliflower mosaic virus 35S promoter; in the composition c), the RNA silencing inhibitor is one or more selected from tomato bush dwarf virus p19, tobacco etch virus Hc-Pro and barley streak mosaic virus gammar.
  6. 6. A method for preparing a recombinant viral vector having infectivity activity, comprising: The rice stripe mosaic virus RSMV expression vector of claim 1 or the rice stripe mosaic virus RSMV gene silencing vector composition of claim 2, wherein a), b) and c) are co-introduced into a leaf of a nicotiana benthamiana to produce a recombinant viral vector with infection activity.
  7. 7. The application of the recombinant viral vector with infection activity prepared by the preparation method according to claim 6 in expression of exogenous proteins and/or silencing of insect endogenous genes in mediator insects, which is characterized by comprising the following specific steps: The recombinant viral vectors with infectivity are used to inoculate mediator insects and express exogenous proteins and/or silence insect genes.
  8. 8. The use according to claim 7, wherein the mediator insect is an electro-optic leafhopper or a two-point black tail leafhopper; The method for inoculating the mediator insects comprises the step of microinjection of the mediator insects into the crude extract of the benthamiana leaf pieces containing recombinant virus vector particles with infection activity.
  9. 9. The use of the recombinant viral vector with infection activity prepared by the preparation method according to claim 6 for expressing exogenous proteins and/or silencing insect endogenous genes in plant hosts, characterized in that it specifically comprises: inoculating plants with recombinant viral vectors having infectivity and expressing exogenous proteins and/or silencing plant genes.
  10. 10. The use according to claim 9, wherein the plant host is rice or crabgrass; The method for inoculating the plants is to inoculate the plants with mediator insects carrying recombinant viral vectors with infection activity.

Description

Rice stripe mosaic virus expression vector, gene silencing vector, preparation method and application Technical Field The invention belongs to the technical field of genetic engineering, and in particular relates to a rice stripe mosaic virus expression vector, a gene silencing vector, a preparation method and application. Background The rice virus disease threatens the yield and quality of rice for a long time. In recent years, the major etiologies of regional or epidemic rice diseases in east and southeast Asia include reoviridae viruses whose genomes are double stranded RNA (e.g., rice black-streaked dwarf virus, southern rice black-streaked dwarf virus), leukoviridae viruses whose genomes are segmented negative sense RNA (e.g., rice stripe virus), and rhabdoviridae viruses which do not segment negative sense RNA (e.g., rice stripe mosaic virus) (Sasaya et al, 2014, front. Microbiol 4, 409; li et al, 2022, nat Commun 13 (1), 6920). The viruses are mostly transmitted by leafhoppers or plant hoppers in a lasting proliferation mode, and diseases have epidemic characteristics such as outbreak, intermittence, migration and the like, so that large-area yield reduction is often caused, and serious economic loss is caused. Although related rice viruses have important scientific research value, the research on molecular biology is limited by the deletion of a key genetic manipulation tool, namely invasive cloning. To date, no stably operable invasive cloning and reverse genetics system has been successfully constructed against the major epidemic of rice negative-sense RNA viruses and double-stranded RNA viruses. The technical bottleneck severely restricts the deep analysis of viral replication, pathogenicity and host interaction mechanisms, and also restricts the development process of related viruses as biotechnology tools. Plant viral vectors are important platforms for functional genomics research and agricultural biotechnology applications, and can be used for transient over-expression of genes (virus-mediated overexpression, VOX), virus-induced gene silencing (VIGS), and genome editing element delivery (virus-induced genome editing, VIGE) (Peyret al, 2015, plant Biotechnol J13 (8), 1121-1135; zulfiqar, 2023, J Mol Sci 24 (6), 5608, steinberger, 2025, NAT PLANTS, 1241-1251). However, the viral vector systems currently available for rice are still very limited, and the vectors that have been successfully developed are limited to very few DNA viruses and sense strand RNA viruses, such as the rice Douglas baculovirus (rice tungro bacilliform virus, RTBV) and the brome mosaic virus (brome mosaic virus, BMV) (Purkayastha et al, 2010, planta 232 (6), 1531-1540; ding et al, 2006, mol Plant-Microbe interface 19 (11), 1229-1239). The vector is limited by limited bearing capacity or complex genome structure in protein expression application, and is difficult to meet application requirements of exogenous expression of large-capacity genes, multi-gene collaborative expression regulation and control, stable continuous expression and the like. In addition, there are also significant technical limitations to genetic manipulation of insects against plant viral mediators. The traditional gene silencing method which relies on microinjection or oral delivery to synthesize dsRNA in vitro has the defects of short silencing effect, limited efficiency and the like (Jain et al 2020, insects 11 (9), 557; zhu et al 2020, annu Rev Entomol 65 (1), 293-311), and meanwhile, a mature transgenic overexpression technical system capable of stably inheriting is not established for important agricultural insects such as leafhoppers and the like. In recent years, attempts have been made to silence insect endogenous genes using plant viral vectors or host-induced gene silencing strategies that are unable to replicate in insects (Rosa et al, 2018, annu Rev Phytopathol (1), 581-610; bonning et al, 2021, annu Rev Entomol (1), 61-79). However, due to the lack of a proper virus infectious cloning system, the problems of small bearing capacity, low silencing efficiency and the like of the existing vector are generally existed, the application of the related technology is still obviously limited, and a plant-insect cross-border virus vector delivery platform with high efficiency and stability is still lacking. Rice Stripe Mosaic Virus (RSMV) belongs to Rhabdoviridae (Rhabdoviridae), cytoplasmic Rhabdoviridae (cytorhabdovirus), has a genome of single-stranded negative sense RNA, has characteristics of modularization of genome structure, sequential transcription and the like, and can be propagated in a persistent proliferation manner by electro-optic leafhoppers (Recilia dorsalis) and two-point black leafhoppers (Nephotettix virescens) (Yang et al, 2017a, front Microbiol 7, 2140; yang et al, 2017b, front Microbiol 8, 2457; zhao et al, 2019, front Microbiol 9, 3258). The above biological properties provide the potential advantage of RSMV being engineered i