Target gene for inhibiting RBSDV virus proliferation and interfering ribonucleic acid thereof

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a target gene for inhibiting RBSDV (Radial Basis Syndrome Development Virus) proliferation and interfering ribonucleic acid thereof. The target gene is a combination of S1 and S6 proteins of rice black-streaked dwarf virus (RBSDV), and the expression quantity of the virus is reduced by silencing the target gene through RNAi, so that the purpose of preventing and treating rough dwarf virus is achieved.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to a target gene for inhibiting the proliferation of RBSDV virus and its interfering ribonucleic acid. Background Technology

[0002] Maize rough dwarf virus (MRDV) is a viral disease of maize transmitted by insects such as planthoppers and leafhoppers. It is also a global maize disease. Currently, four viruses in the family Reoviridae and genus Fijivirus can cause MRDV worldwide: Maize Rough Dwarf Virus (MRDV), Rice Black Streaked Dwarf Virus (RBSDV), South Rice Black Streaked Dwarf Virus (SRBSDV), and Mal de río cuarto virus (MRCV). All of them are double-stranded RNA viruses with ten strands in their genome, and they can infect a variety of gramineous crops such as rice, maize, wheat, barley, oats, and sorghum.

[0003] In China, maize rough dwarf disease is mainly caused by rice black-streaked dwarf virus (RBSDV). The virus typically overwinters in wheat fields or on gramineous weeds such as *Alopecurus aequalis* and *Setaria viridis*, and can also overwinter in the vector insect, the planthopper. The following year, as temperatures rise, the planthoppers carry the virus and multiply and replicate in wheat fields. After wheat matures and is harvested in May and June, the infected planthopper population migrates to newly grown maize, spreading the virus and causing maize rough dwarf disease. Maize rough dwarf disease was first discovered in Israel in 1945 and in my country in 1954. Around 2000, it began to occur on a large scale in maize-growing areas across the country, with the most severe damage in North China and the coastal areas of Jiangsu. Corn rough dwarf disease can be infected throughout the entire growth cycle, with the seedling stage being the most severely affected. Symptoms typically appear when the plant has 5-6 leaves, and the stunting effect is more pronounced at the 9-10 leaf stage, with diseased plants reaching less than half the height of healthy plants. In mildly affected plants, the tassels are underdeveloped with little pollen, and the female ears are short with few silks and few kernels. In severely affected plants, the tassels fail to emerge, or if they do, they have very few branches and no pollen. The female ears are deformed, empty, or have very few kernels, severely impacting corn yield. Corn rough dwarf disease is extremely difficult to control. Once infected, it is very difficult to cure, and infected corn plants cannot recover to normal growth. Therefore, corn rough dwarf disease is also known as "corn cancer."

[0004] RNA interference (RNAi), also known as RNA silencing or nucleic acid silencing, is a molecular biological phenomenon induced by double-stranded RNA (dsRNA) to suppress gene expression by inhibiting the transcription of specific genes. RNA interference has been observed in many eukaryotes, including animals, plants, and fungi. When a double-stranded RNA homologous to the coding region of endogenous mRNA is introduced into an organism, it is recognized and cleaved by the Dicer ribozyme in the eukaryote, producing many 21-24 nt siRNAs (small interfering RNAs). Each siRNA then unwinds to form two single-stranded siRNAs. The primer strands of these single-stranded siRNAs form an RNA-induced silencing complex (RISC) with proteins such as AGO (Argonaute). The RISC binds to its homologous mRNA, causing the mRNA to degrade and thus inhibiting gene expression. RNA interference technology is frequently used to study the function of endogenous genes in plants and is also a common method for controlling plant viral diseases. Invention Summary

[0005] In view of the serious damage of maize rough dwarf disease to maize production and the potential of RNA interference technology in the control of plant viral diseases, this invention provides a target gene and its interfering ribonucleic acid for inhibiting the proliferation of RBSDV virus.

[0006] The specific solution adopted in this invention is as follows:

[0007] A target gene of RBSDV virus, wherein the target gene is a combination of the S1 and S6 proteins of rice black-streaked dwarf virus RBSDV.

[0008] In one specific embodiment, the amino acid sequences of the target genes RBSDV S1 and S6 proteins are shown in SEQ ID NO: 1 and 3, respectively.

[0009] In another specific embodiment, the nucleotide sequences encoding the RBSDV S1 and S6 proteins are shown in SEQ ID NO: 5 and 7, respectively. The present invention also provides an interfering ribonucleic acid, designed according to the aforementioned target genes, capable of inhibiting the proliferation of the RBSDV virus.

[0010] In one specific embodiment, the interfering ribonucleic acid comprises at least any one of the following:

[0011] A nucleotide sequence or its reverse complementary sequence having at least 20, at least 25, at least 50, at least 100, at least 150, at least 200, at least 400, at least 500, at least 800, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, or all consecutive nucleotides having at least 90%, 95%, 98%, 98.5%, 99%, 99.5%, 99.8%, or 100% identity with the coding sequences of RBSDV S1, S2, S3, S6, S7, and / or S10.

[0012] In another specific embodiment, the interfering ribonucleic acid is a combination of SEQ ID NO: 9 and SEQ ID NO: 11 or their reverse complementary sequence. In one specific embodiment, each sense sequence of the interfering ribonucleic acid is located on an RNA strand different from its corresponding antisense sequence within the interfering ribonucleic acid.

[0013] In one specific embodiment, the sense sequences and antisense sequences of the interfering ribonucleic acid are located on a single RNA strand that loops back into itself to form a hairpin structure.

[0014] The present invention also provides an expression cassette comprising a polynucleotide encoding the interfering ribonucleic acid under the control of an effectively linked regulatory sequence.

[0015] The present invention also provides a recombinant vector capable of expressing at least one of the interfering ribonucleic acids.

[0016] The present invention also provides a method for creating plants resistant to RBSDV virus and the transgenic plants obtained therefrom, which are obtained by constructing an RNAi vector expressing the interfering ribonucleic acid that targets the target gene, and transforming it to produce transgenic plants to generate resistance to the virus infection, wherein the interfering ribonucleic acid can inhibit the proliferation of RBSDV.

[0017] The present invention also provides a method for improving plant resistance to RBSDV virus infection, comprising introducing the expression cassette, the recombinant vector, or a construct containing the expression of the interfering ribonucleic acid into the plant.

[0018] The present invention also provides compositions for controlling RBSDV virus and their use for preventing and / or controlling RBSDV virus proliferation, comprising at least one of the interfering ribonucleic acids and an adjuvant component for maintaining the biological activity of the interfering ribonucleic acid.

[0019] The present invention also provides a method for controlling RBSDV virus, wherein a plant infected with or at risk of infection by the plant virus and / or the vicinity of the plant is brought into contact with an effective amount of the composition.

[0020] In one specific embodiment, the plant is corn, wheat, rice, sorghum, millet, or barley.

[0021] The transgenic maize expressing double-stranded RNA of RBSDV S1 and S6 strands provided by this invention achieves true high resistance, no virus transmission, and no virus transmission. This not only ensures the growth and development of the transgenic maize itself and hinders the spread of rice black-streaked dwarf virus, but also builds a solid "firewall" to prevent the spread of rice black-streaked dwarf virus RBSDV to other gramineous crops such as rice, wheat, sorghum, barley, and millet. Invention Details

[0023] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0024] The "interfering RNA" of this invention encompasses any type of RNA molecule capable of downregulating or "silencing" the expression of a target gene, including but not limited to sense RNA, antisense RNA, siRNA, miRNA, dsRNA, hairpin RNA, etc. Methods for determining functional interfering RNA molecules are well known in the art and have been disclosed.

[0025] The interfering RNA of this invention achieves specific downregulation of target gene expression by binding to the target sequence within the target gene. This binding occurs due to base pairing between the complementary regions of the interfering RNA and the target sequence.

[0026] As used in this invention, the terms "dsRNA" or "double-stranded RNA" refer to two antiparallel strands of a polynucleotide held together by base pairing and are used interchangeably. The two strands may be of the same length or different lengths, provided that there is sufficient sequence homology between the two strands forming a double-stranded structure with at least 80%, 90%, 95%, or 100% complementarity over the full length.

[0027] Any dsRNA molecule described herein may be used in accordance with the teachings of this invention, provided that it is amplified by an RNA-dependent RNA polymerase (RDRP). The dsRNA molecule may be naturally occurring or synthetic. dsRNA may be synthesized using any method known in the art, including enzymatic synthesis or solid-phase synthesis.

[0028] This invention relates to dsRNAs of different lengths, wherein shorter forms, i.e., x shorter than or equal to 50 bp (e.g., 17-50), are referred to as siRNAs or miRNAs. Double-stranded RNA molecules of 51-600 or even longer are referred to herein as dsRNAs, which can be further processed to produce siRNA molecules.

[0029] In this invention, dsRNA molecules can act as precursors to active siRNA molecules. These active siRNA molecules bind to a series of Ago proteins (Argonautes) to form an RNA-induced silencing complex (RISC). Guided by the siRNA, the RISC binds to the RNA transcript of the target gene, leading to degradation or translational repression of the target gene's RNA transcript, preventing the effective production of the target protein. dsRNA molecules present in an organism or its cellular environment can be taken up by the organism and processed by an enzyme called DICER to obtain siRNA molecules. Alternatively, dsRNA molecules can be produced in vivo, i.e., transcribed from one or more polynucleotides encoding the dsRNA present in cells (e.g., bacterial or plant cells), and processed by DICER within the host cell after ingestion of a longer precursor dsRNA. The dsRNA can be formed from two separate (sense and antisense) RNA strands annealed by complementary base pairing. Alternatively, the dsRNA can be a single strand capable of refolding itself to form hairpin RNA or stem-loop structures. In the case of an RNA molecule, a double-stranded region, or "stem," is formed by two regions or segments that are essentially anticomplementary sequences to each other and have sufficient complementarity to allow the formation of a double-stranded region. One or more functional double-stranded silencing elements may be present in this "stem region" of the molecule. The anticomplementary regions are typically separated by a region or segment in the RNA called a "loop." This region can contain any nucleotide sequence that provides sufficient flexibility to allow self-pairing between the flanking complementary regions of the RNA; overall, the loop region is essentially single-stranded and acts as a spacer sequence between the anticomplementary sequences.

[0030] The term "siRNA" refers to a small, repressive RNA duplex (usually between 17 and 30 base pairs, but longer duplexes such as 31 to 50 bp) that induces RNA interference (RNAi) pathway.

[0031] The term "sense sequence," also known as "sense chain" or "sense sequence," refers to the chain in a gene that has the same base sequence as the mRNA transcript (except that T is used instead of U).

[0032] In a gene, the sequence complementary to the sense sequence is called an "antisense sequence." In a DNA molecule containing multiple genes, the sense strands of each gene are not all on the same DNA deoxyribonucleotide chain; that is, a single chain contains both the sense strands of some genes and the antisense strands of others. The "antisense sequence" contained in the dsRNA molecule described in this invention is an RNA sequence complementary to the corresponding sense sequence.

[0033] The term "partial sequence" refers to at least 20 consecutive nucleotides of the sequence. For example, "partial sequence" refers to at least 25, at least 50, at least 100, at least 200, at least 400, at least 600, at least 800, and more consecutive nucleotides of the sequence.

[0034] As used in this invention, the term "hybrid sequence" refers to a polynucleotide chain linked to a substantially complementary complementary chain via base pairing. The two nucleotide sequences are at least 80% complementary in their nucleotide sequences. Preferably, the two nucleotide sequences are at least 85%, at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99% complementary throughout their entire length. The term "antisense RNA" refers to an RNA transcript that is wholly or partially complementary to the mRNA normally produced in the cells of the target organism. The complementarity of antisense RNA can be complementarity with any portion of a particular gene transcript (i.e., a portion of the 5' non-coding sequence, the 3' untranslated sequence, an intron, or a coding sequence).

[0035] RNA interference, also known as “RNAi,” has emerged as a genetic tool to accelerate plant biotechnology research. RNAi is a conserved component of gene regulatory processes present in all eukaryotes. For RNAi in plants, the alternative term “post-transcriptional gene silencing” (PTGS) is used. PTGS begins by processing or cleaving precursor double-stranded RNA into short, approximately 20–25 ribonucleotide-long, single- or double-stranded interfering RNA (siRNA) or micro-interfering RNA (miRNA) by an RNase III-like enzyme called Dicer (Baulcombe, Nature 431:356–363.2004). Incorporating siRNA or miRNA into the RNA-induced silencing complex (RISC), which recognizes and degrades complementary messenger RNA (mRNA) molecules, results in a significant reduction in RNA levels and effectively reduces the expression of the corresponding gene, i.e., reduces the production of the target protein, whose functional activities are affected.

[0036] As used in this article, “genome” refers to the complete complement of genetic material (genes and non-coding sequences) present in every cell, virus, or organelle of an organism, and / or the complete set of chromosomes inherited as a unit (haploid) from a parent.

[0037] The term "transgenic" is used herein to describe genetic material that has been or will be artificially introduced into the genome of a host organism and passed on to the host's offspring. Transgenic materials will typically contain polynucleotides, which include non-coding and / or coding sequences that generally, but not necessarily, affect or cause activity (e.g., regulation of transcription or translation, the production of nucleotide sequences containing coding and / or non-coding sequences, etc.).

[0038] The term "gene" includes a segment of nucleic acid that expresses a functional molecule (such as, but not limited to, a specific protein), including regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence).

[0039] The DNA sequence that “encodes” a specific RNA is the DNA nucleic acid sequence that is transcribed into RNA. DNA polynucleotides can encode RNA (mRNA) that is translated into proteins, or DNA polynucleotides can encode RNA that is not translated into proteins (such as tRNA, rRNA, or RNA that targets DNA; also known as “non-coding” RNA or “ncRNA”).

[0040] The terms "polynucleotide," "nucleotide," and "nucleic acid" are used interchangeably and include DNA, RNA, or their hybrids, which can be double-stranded or single-stranded. A "nucleic acid" may also contain non-naturally occurring or altered bases that allow for proper reading by polymerases without reducing the expression of the polypeptide encoded by that nucleic acid.

[0041] The terms "nucleotide sequence" and "nucleic acid sequence" both refer to the arrangement of bases in DNA or RNA, and refer to the sense and antisense strands of nucleic acids that exist as a single strand or in a dimer.

[0042] Those skilled in the art can readily mutate the DNA fragments of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have at least 90%, 95%, 98%, 98.5%, 99%, 99.5%, or 99.8% identity with the DNA fragments shown in the foregoing sequences of the present invention, and have the same function, are all derived from and equivalent to the nucleotide sequences of the present invention.

[0043] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated visually or using computer software. Using computer sequence alignment software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. A "reverse complementary sequence" is a sequence that is complementary to the original polynucleotide sequence but in the opposite direction.

[0044] "Complementary" polynucleotides are those that can pair bases according to the Watson-Crick complementarity rule. Specifically, base pairs will form between purines and pyridines, including guanine pairing with cytosine (G:C) and adenine with thymine in the case of DNA (A:T) or uracil in the case of RNA (A:U). It is understood that two polynucleotides can hybridize even if they are not perfectly complementary, as long as each has at least one region substantially complementary to the other.

[0045] As used herein with respect to gene sequences, the term "expression" refers to the translation of a coding sequence into a polypeptide. Inhibition of gene expression can become detectable at the mRNA level, at the polypeptide level, or both. Methods for assessing changes at the mRNA or protein levels of a gene are well known in the art. For example, changes at the mRNA level of a gene can be assessed by quantitative real-time PCR (RT-qPCR) or RNA blotting.

[0046] The term "plant" is used herein to refer to the whole plant and its parts or derivatives at any developmental stage, and therefore includes, for example, plant cells, plant cell groups, plant tissues (e.g., meristems, callus), plant organs (e.g., stems, leaves, roots, ovules, stamens), and reproductive forms or parts of a plant (e.g., seeds, tubers, cuttings, gametophytes, sporophytes, pollen, microspores, embryos). Plant cells or plant cell groups may be isolated from plant tissues, plant organs, or the whole plant at any developmental stage (e.g., in suspension culture) or contained within plant tissues, plant organs, or the whole plant at any developmental stage.

[0047] In one specific embodiment, the plant is corn, wheat, rice, sorghum, millet, or barley.

[0048] In this invention, "target site" and "target sequence" can be used interchangeably, meaning they can be selected from any suitable region or nucleotide sequence of the target gene or its RNA transcript. For example, the target sequence can be located within the 5'UTR or 3'UTR of the target gene or RNA transcript, or within an exon or intron region of the gene.

[0049] The terms "target gene" or "target gene" used in this invention are interchangeable and refer to any sequence intended to be downregulated in a virus. Viral infection is controlled by downregulating target genes, for example, by disrupting the biological processes of viral replication. Therefore, preferred target genes include, but are not limited to, genes that play a key role in mRNA transcription, protein and DNA or RNA synthesis. When the expression of a viral target gene is downregulated or suppressed (e.g., by interfering with viral mRNA through dsRNA or antisense RNA targeting the viral gene), viral replication is prevented, reducing the viral load in host cells; or the ability of the virus to infect plant or crop species is reduced, thereby protecting the plant or crop species from viral harm. "Replication" as used in this invention refers to the biological process by which a virus replicates its nucleic acid using the host cell's enzyme system, raw materials, and energy after invading a host cell. Viral replication within host cells can affect the normal growth of the host (e.g., plants). For example, MRDV / RBSDV replication within crop (e.g., rice, corn) cells can cause stunted growth, dwarfing, and short, thick internodes, leading to reduced crop yield.

[0050] The terms “intrusion,” “infection,” “infringement,” and / or “attack” are generally used interchangeably throughout the text.

[0051] Unless otherwise defined, 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 invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are described hereafter.

[0052] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials relating to the publications referenced. Any reference to a publication is made prior to the filing date and should not be construed as an admission that the invention does not predate such publication. Furthermore, the publication date provided may differ from the actual publication date, which may require independent verification.

[0053] Unless specifically stated or implied, as used herein, the terms “a,” “an,” and “described” mean “at least one.” All patents, patent applications, and publications mentioned or cited herein are incorporated herein by reference in their entirety as if they were individually cited separately. Attached Figure Description

[0054] Figure 1 A schematic diagram showing the position of each dsRNA on the target gene.

[0055] Figure 2 Schematic diagram of the qPCR-RBSDV-S5 vector.

[0056] Figure 3 A standard curve plot showing the relationship between Ct value and copy number.

[0057] Figure 4 Identification of resistance to maize rough dwarf virus - viral load statistics.

[0058] Figure 5 Identification of resistance to maize rough dwarf virus - plant height statistics.

[0059] sequence list

[0060] serial number name SEQ ID NO:1 RBSDVS1 amino acid sequence SEQ ID NO:2 RBSDVS2 amino acid sequence SEQ ID NO:3 RBSDVS6 amino acid sequence SEQ ID NO:4 RBSDVS10 amino acid sequence SEQ ID NO:5 RBSDVS1 nucleotide sequence SEQ ID NO:6 RBSDVS2 nucleotide sequence SEQ ID NO:7 RBSDVS6 nucleotide sequence SEQ ID NO:8 RBSDVS10 nucleotide sequence SEQ ID NO:9 RBSDV_S1-dsRNA1 SEQ ID NO:10 RBSDV_S2-dsRNA1 SEQ ID NO:11 RBSDV_S6-dsRNA1 SEQ ID NO:12 RBSDV_S10-dsRNA1 Detailed Implementation

[0061] Example 1: Selection of RBSDV RNA interference target genes and their double-stranded RNA sequences

[0062] In the genome of rice black-streaked dwarf virus (RBSDV), the S1 chain (encoding RNA-dependent RNA polymerase), S2 chain (encoding major core structural protein), S6 chain (encoding RNA silencing repressor), and S10 chain (encoding capsid protein) were selected as target genes for RNA interference. Their amino acid sequences are shown in SEQ ID NO:1-4, and their corresponding nucleotide sequences are shown in SEQ ID NO:5-8.

[0063] The conserved regions of the target genes were used as double-stranded RNA sequences for RNA interference, and named RBSDV_S1-dsRNA1 (SEQ ID NO:9), RBSDV_S2-dsRNA1 (SEQ ID NO:10), RBSDV_S6-dsRNA1 (SEQ ID NO:11), and RBSDV_S10-dsRNA1 (SEQ ID NO:12), respectively. Their locations on the target genes are as follows: Figure 1 As shown.

[0064] In addition to designing double-stranded RNAs that individually silence the conserved regions of the four strands mentioned above, we also designed combined double-stranded RNAs that simultaneously silence the conserved regions of two strands. These combined double-stranded RNA sequences are RBSDV_S1-dsRNA1(SEQ ID NO:9)+RBSDV_S6-dsRNA1(SEQ ID NO:11), RBSDV_S10-dsRNA1(SEQ ID NO:12)+RBSDV_S6-dsRNA1(SEQ ID NO:11), RBSDV_S1-dsRNA1(SEQ ID NO:9)+RBSDV_S2-dsRNA1(SEQ ID NO:10), and RBSDV_S2-dsRNA1(SEQ ID NO:10)+RBSDV_S6-dsRNA1(SEQ ID NO:11).

[0065] Example 2: Design and Construction of RNA Interference Vector

[0066] The expression vector pCambia1300 was linearized by double digestion with restriction endonucleases AvrII and MluI (purchased from NEB). Then, the double-stranded RNA sequence, ST-LS1 sequence, and reverse complementary sequence of the double-stranded RNA sequence were amplified. These three fragments were then ligated into the linearized expression vector using seamless cloning to construct the final RNA interference vector. The RNA interference vectors corresponding to the double-stranded RNAs RBSDV_S1-dsRNA1, RBSDV_S2-dsRNA1, RBSDV_S6-dsRNA1, RBSDV_S10-dsRNA1, RBSDV_S1-dsRNA1+RBSDV_S6-dsRNA1, RBSDV_S10-dsRNA1+RBSDV_S6-dsRNA1, RBSDV_S1-dsRNA1+RBSDV_S2-dsRNA1, and RBSDV_S2-dsRNA1+RBSDV_S6-dsRNA1 are numbered 1-8, respectively.

[0067] Example 3: Inoculation Management and Resistance Identification of Maize Rough Dwarf Disease

[0068] 1. Inoculation management of maize rough dwarf disease

[0069] The above expression vectors were used to transform maize receptors, and positive transformed seedlings were selected for further testing after identification. Maize rough dwarf disease was transmitted to maize using artificially reared planthoppers carrying RBSDV. The specific method was as follows:

[0070] (1) Corn planting

[0071] In addition to sowing the various RNA interference transgenic materials, it is also necessary to sow the transformation recipient variety and the susceptible control variety Zhengdan 958, with 100 plants sown for each material.

[0072] (2) Virus inoculation

[0073] Inoculation with the virus began when corn seedlings reached the two-leaf stage (both leaves fully expanded), using a single-plant inoculation method with infected planthoppers. Fifty plants were selected from the aforementioned transgenic materials, transformation recipients, and Zhengdan 958 for virus-infected planthopper transmission. Each corn plant was guaranteed to have at least 3 planthoppers (effective inoculation count = inoculation count × virus infection rate). Virus transmission with infected planthoppers lasted for 3 days.

[0074] (3) Pest control

[0075] Three days after the infected planthoppers transmit the virus, remove them and spray all the virus-transmitting materials with insecticide. Observe for 7 to 10 days. Once it is confirmed that there are no residues or newly hatched planthoppers, the plants can be transplanted into the field.

[0076] (4) Planting and Management

[0077] Both uninoculated and inoculated materials were planted in the field simultaneously. Corn was managed with conventional water and fertilizer. Throughout the growing season, attention was paid to preventing vector insects such as planthoppers and leafhoppers to ensure that corn rough dwarf disease in the experimental plots did not spread.

[0078] 2. Survey on maize rough dwarf disease

[0079] The investigation was conducted during the grain-filling stage of maize, when the disease severity of the susceptible control (Zhengdan 958) reached level 7 or above (if the disease severity of Zhengdan 958 did not reach level 7 or above, the experiment was invalid). Virus load and plant height were measured. The resistance evaluation criteria for maize rough dwarf disease are detailed in the People's Republic of China Agricultural Industry Standard "NY / T 1248.13-2016 Technical Specification for Identification of Maize Disease and Pest Resistance Part 13: Maize Rough Dwarf Disease".

[0080] (1) Detection of RBSDV viral load in inoculated corn

[0081] Viral load was detected using RT-PCR absolute quantification. The standard quality plasmid for absolute quantification was pUC57 ligated to the S5 fragment of rice black-streaked dwarf virus RBSDV. The vector map is shown below. Figure 2 The concentration of the standard quality grains was measured using a spectrophotometer. The copy number concentration of the original standard quality grains was calculated using the following formula, and the concentration was adjusted to 10. 10 copies / μl.

[0082]

[0083] Average molecular weight MW (g / mol) = number of plasmid base pairs (bp) × 660 (dalton / bp)

[0084] With a concentration of 10 10 The standard quality particles were serially diluted to 10 copies / μl. 7 10 6 10 5 10 4 10 3 10 2 The standard quality plasmids at various concentrations were used in RT-PCR along with the test samples to obtain a standard curve showing the relationship between Ct value and copy number. Figure 3 As shown.

[0085] Leaves from the 10 most severely infected plants were selected from all transgenic materials with RNA interference vectors, transformed maize recipients, and Zhengdan 958 inoculated materials. Leaves from 10 uninoculated materials were also taken as blank controls. Each leaf sample was approximately 0.2g in size. RNA was extracted from the leaves and reverse transcribed. 500ng of template RNA was added to a 10μl reverse transcription system. 1μl of the obtained cDNA was used for RT-PCR. Finally, the copy number of the standard plasmid corresponding to each plant was calculated according to the standard curve and used as the viral load. The average viral load of each material was calculated and statistically analyzed.

[0086] The logarithmic values ​​of the average viral load for each material were taken, and the final results showed that the viral load of the infected control Zhengdan 958 and the wild-type transformed maize receptor both exceeded 10. 6 The viral load of transgenic material with RNA interference vector RBSDV_S1-dsRNA1+RBSDV_S6-dsRNA1 (number 5) was close to 0 copies / μl, indicating near-immunity against maize rough dwarf disease. In contrast, transgenic materials with other RNA interference vectors still showed detectable viral loads, with the viral load varying depending on the level of resistance (see [link to relevant documentation]). Figure 4 ).

[0087] (2) Investigation on corn plant height and disease status

[0088] During the grain-filling stage of maize, plant height was surveyed for the disease-susceptible control Zhengdan 958, transgenic materials transformed with the maize recipient, and transgenic materials containing the aforementioned RNA interference vector. Figure 5 The results showed that the transgenic material of vector 5 exhibited high resistance (HR), and there was no significant difference in plant height between the uninoculated and inoculated materials. Although the transgenic materials of other vectors also showed varying degrees of resistance, their plant height was reduced to varying degrees.

[0089] Table 1. Results of the survey on maize plant height and disease status.

[0090] Material Number Double-stranded RNA (Inoculated / Uninoculated plant height) x 100% stunted disease grade Resistance level Zhengdan 958 (none) 17.2％ 9 High Sensitivity HS Transforming maize receptors (none) 34.6％ 7 Feel S 1 RBSDV_S1-dsRNA1 90.5％ 1 High anti-HR 2 RBSDV_S2-dsRNA1 86.4％ 1 High anti-HR 3 RBSDV_S6-dsRNA1 45.6％ 7 Feel S 4 RBSDV_S10-dsRNA1 88.2％ 1 High anti-HR 5 RBSDV_S1-dsRNA1+RBSDV_S6-dsRNA1 100.4％ 1 High anti-HR 6 RBSDV_S10-dsRNA1+RBSDV_S6-dsRNA1 93.1％ 1 High anti-HR 7 RBSDV_S1-dsRNA1+RBSDV_S2-dsRNA1 91.6％ 1 High anti-HR 8 RBSDV_S2-dsRNA1+RBSDV_S6-dsRNA1 91.3％ 1 High anti-HR

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. An RBSDV viral target gene, characterized in that, The target gene is a combination of the S1 and S6 proteins of rice black-streaked dwarf virus (RBSDV).

2. The target gene according to claim 1, characterized in that, The amino acid sequences of RBSDV S1 and S6 proteins are shown in SEQ ID NO: 1 and 3, respectively; preferably, the nucleotide sequences encoding RBSDV S1 and S6 proteins are shown in SEQ ID NO: 5 and 7, respectively.

3. An interfering RNA, characterized in that, Designed according to the target gene described in claim 1 or 2, it is capable of inhibiting the proliferation of the RBSDV virus.

4. The interfering ribonucleic acid according to claim 3, characterized in that, It includes at least one of the following: A nucleotide sequence or its reverse complementary sequence having at least 20, at least 25, at least 50, at least 100, at least 150, at least 200, at least 400, at least 500, at least 800, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500 or all of the consecutive nucleotides having at least 90%, 95%, 98%, 98.5%, 99%, 99.5%, 99.8% or 100% identity with the coding sequences of RBSDV S1 and S6, respectively. Preferably, the interfering ribonucleic acid is a combination of SEQ ID NO: 9 and SEQ ID NO: 11 or its reverse complementary sequence.

5. The interfering ribonucleic acid according to claim 3 or 4, wherein each sense sequence is located on an RNA strand different from its corresponding antisense sequence in the interfering ribonucleic acid.

6. The interfering RNA according to any one of claims 3-5, wherein each sense sequence and the antisense sequence are located on a single RNA strand that loops back onto itself to form a hairpin structure.

7. An expression box, characterized in that, The polynucleotide encoding any one of claims 3-6, contained under the regulation of a regulatory sequence with effective linkage.

8. A recombinant vector capable of expressing at least one of the interfering ribonucleic acids according to any one of claims 3-6.

9. A method for creating plants resistant to RBSDV virus and the transgenic plants obtained therefrom, wherein the transgenic plants are obtained by constructing an RNAi vector expressing interfering ribonucleic acid targeting the target gene as described in claim 1 or 2, and transforming the transgenic plants to produce resistance to the virus infection, wherein the interfering ribonucleic acid can inhibit the proliferation of RBSDV; preferably, the interfering ribonucleic acid is as described in any one of claims 3-6.

10. A method for improving plant resistance to RBSDV virus infection, characterized in that, This includes introducing the expression cassette of claim 7, the recombinant vector of claim 8, or a construct containing the interfering ribonucleic acid of any one of claims 3-6 into a plant.

11. A composition for controlling RBSDV virus and its use for preventing and / or controlling RBSDV virus proliferation, comprising at least one of the interfering ribonucleic acids according to any one of claims 3-6 and an adjuvant component for maintaining the biological activity of said interfering ribonucleic acid.

12. A method for controlling RBSDV virus, wherein a plant infected with or at risk of infection by the plant virus and / or the vicinity of the plant is brought into contact with an effective amount of the composition of claim 11.

13. The method according to claim 9, 10, or 12, or the transgenic plant according to claim 9, characterized in that, The plant is corn, wheat, rice, sorghum, millet, oats, or barley; preferably, the plant is corn.

Images