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

Abstract

The present invention relates to the technical field of plant gene engineering, and specifically relates to a target gene for inhibiting RBSDV virus proliferation, and an interfering ribonucleic acid thereof. The target gene is a combination of rice black streaked dwarf virus (RBSDV) S1 and S6 proteins, and the expression level of the virus is reduced by means of silencing the target gene via RNAi, thereby achieving the aim of controlling rough dwarf diseases.

Description

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

[0001] The present application relates to the technical field of plant genetic engineering, in particular to a target gene for inhibiting proliferation of RBSDV virus and interfering ribonucleic acid thereof. BACKGROUND

[0002] Maize rough dwarf disease is a maize virus disease caused by insects such as planthoppers and leafhoppers, and is also a worldwide maize disease. Currently, four viruses of the Reoviridae family, Fijivirus genus, i.e. 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), can cause maize rough dwarf disease worldwide. They are all double-stranded RNA viruses, and the viral genome contains ten strands, which can infect various gramineous crops such as rice, maize, wheat, barley, oat, and sorghum.

[0003] In China, maize rough dwarf disease is mainly caused by rice black streaked dwarf virus (RBSDV). The virus generally overwinters on wheat fields or gramineous weeds such as loose silkybush and dogtail grass, and can also overwinter in the body of the virus-transmitting insect planthopper. After the temperature rises in the second year, the planthopper carrying the virus multiplies and replicates in the wheat field, and after the wheat matures and is harvested in May and June, the virus-carrying planthopper population migrates to the newly grown maize and spreads the virus, causing maize rough dwarf disease. Maize rough dwarf disease was first discovered in Israel in 1945, in China in 1954, and began to occur in large areas in the country's maize planting areas around 2000, with the most serious damage in North China and the coastal areas of Jiangsu. Maize can be infected with rough dwarf disease throughout the growing season, with the most severe damage occurring at the seedling stage, generally at 5-6 leaves, and more obvious dwarfing at 9-10 leaves. The height of the diseased plants is less than half of the normal plants, and the male ears of the plants with mild disease appear to be poorly developed, with less pollen, while the female ears are short, with less silk and fewer seeds. The plants with severe disease have male ears that cannot be pulled out or have few branches and no pollen, and the female ears are deformed and have few or no seeds, which seriously affects the yield of maize. Maize rough dwarf disease is a very difficult-to-control maize disease, and once infected with the virus, it is difficult to cure. Maize plants that have been infected with the virus also cannot recover to normal growth, so maize rough dwarf disease is also known as "maize cancer".

[0004] RNA interference (RNAi) is also known as RNA silencing, nucleic acid silencing, a phenomenon of gene silencing induced by double-stranded RNA (dsRNA) in molecular biology, which mechanism is to inhibit gene expression by hindering the transcription of specific genes. The phenomenon of RNA interference is found in many eukaryotes such as animals, plants and fungi. When the double-stranded RNA homologous to the coding region of endogenous mRNA is introduced into the organism, the double-stranded RNA will be recognized and cut by Dicer nuclease in eukaryotes to produce many 21-24 nt siRNAs (small interfering RNA). Then each siRNA is unwound to form two single-stranded siRNAs, and the primer strand will form an RNA-induced silencing complex (RISC) with AGO (Argonaute) and other proteins. The RISC binds to the mRNA homologous to it, resulting in the degradation of the mRNA and the inhibition of the expression of the gene. The RNA interference technology is often used to study the function of endogenous genes in plants and is also a common means for preventing and treating plant viral diseases.

[0005] SUMMARY

[0006] In view of the serious harm of maize rough dwarf disease to corn production and the potential of RNA interference technology in preventing and treating plant viral diseases, the present application provides a target gene for inhibiting the proliferation of RBSDV virus and an interfering ribonucleic acid thereof.

[0007] The specific scheme adopted by the present application is as follows:

[0008] A RBSDV virus target gene, the target gene is a combination of rice black-streaked dwarf virus RBSDV S1 and S6 proteins.

[0009] 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.

[0010] In another specific embodiment, the nucleotide sequences encoding RBSDV S1 and S6 proteins are shown in SEQ ID NO: 5 and 7, respectively. The present application also provides an interfering ribonucleic acid designed according to the target gene, which can inhibit the proliferation of the RBSDV virus.

[0011] In one specific embodiment, the interfering ribonucleic acid comprises at least any one of the following:

[0012] a nucleotide sequence having at least 90%, 95%, 98%, 98.5%, 99%, 99.5%, 99.8%, or 100% identity to 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 contiguous nucleotides of the coding sequence of RBSDV S1, S2, S3, S6, S7, and / or S10, or the reverse complement thereof.

[0013] In another embodiment, the interfering ribonucleic acid is a combination of SEQ ID NO: 9 and SEQ ID NO: 11, or the reverse complement thereof. In one embodiment, each of the sense sequences of the interfering ribonucleic acid is on a different RNA strand from its corresponding antisense sequence in the interfering ribonucleic acid.

[0014] In one embodiment, each of the sense sequences and the antisense sequence of the interfering ribonucleic acid are on a single RNA strand that loops back on itself to form a hairpin structure.

[0015] The present application also provides an expression cassette comprising a polynucleotide encoding the interfering ribonucleic acid under the control of operably linked regulatory sequences.

[0016] The present application also provides a recombinant vector capable of expressing at least one of the interfering ribonucleic acids.

[0017] The present application also provides a method of creating an RBSDV virus resistant plant and the transgenic plants obtained by constructing an RNAi vector expressing the interfering ribonucleic acid targeting the target gene, transforming a transgenic plant to produce resistance to the virus infection, the interfering ribonucleic acid capable of inhibiting the proliferation of RBSDV.

[0018] The present application also provides a method of increasing the resistance of a plant to RBSDV virus, comprising introducing the expression cassette or the recombinant vector or the construct comprising the expression of the interfering ribonucleic acid into the plant.

[0019] The present application also provides a composition for controlling RBSDV virus and its use for preventing and / or controlling the proliferation of RBSDV virus, comprising at least one of the interfering ribonucleic acids and an auxiliary ingredient that maintains the biological activity of the interfering ribonucleic acid.

[0020] The present application also provides a method for controlling RBSDV virus, wherein a plant infected by or at risk of being infected by the plant virus and / or the vicinity of the plant is contacted with an effective amount of the composition.

[0021] In one embodiment, the plant is corn, wheat, rice, sorghum, millet or barley.

[0022] The transgenic corn expressing RBSDV S1 chain and S6 chain double-stranded RNA provided by the present application achieves true high resistance, no virus and no virus transmission, which not only guarantees the growth and development of the transgenic corn itself and hinders the spread of rough dwarf disease, but also builds a solid "firewall" for preventing the spread and diffusion of rice black-streaked dwarf virus RBSDV to rice, wheat, sorghum, barley, millet and other other crops.

[0023] Detailed description of the invention

[0024] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Meanwhile, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0025] The "interfering ribonucleic acid" of the present application encompasses any type of RNA molecule capable of down-regulating 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.

[0026] The interfering ribonucleic acid of the present application achieves specific down-regulation of target gene expression by binding to a target sequence within the target gene. The binding occurs due to base pairing between the complementary regions of the interfering RNA and the target sequence.

[0027] The term "dsRNA" or "double-stranded RNA" used in the present application relates to two strands of antiparallel polyribonucleic acid held together by base pairing, and can be used interchangeably. The two strands can be the same length or different lengths, as long as there is sufficient sequence homology between the two strands of the double-stranded structure formed with more than 80%, 90%, 95% or 100% complementarity.

[0028] Any dsRNA molecule can be used according to the teachings of the present application, as long as it is amplified by RNA-dependent RNA polymerase (RDRP). The dsRNA molecule can be naturally occurring or synthetic. The dsRNA can be synthesized by any method known in the art, including enzymatic synthesis or solid-phase synthesis.

[0029] The present invention relates to dsRNAs of different lengths, with the shorter form, x, being less than or equal to 50 bp (e.g. 17-50), referred to as siRNA or miRNA. Double stranded RNA molecules of 51-600 or even longer are referred to herein as dsRNA, which can be further processed to produce siRNA molecules.

[0030] In the present invention, the dsRNA molecule can act as a precursor to active siRNA molecules that bind to a range of Ago proteins (Argonaute) to form RNA-induced silencing complexes (RISC), which bind to the RNA transcript of the target gene under the guidance of the siRNA, resulting in the degradation or translational inhibition of the target gene's RNA transcript, and the target protein cannot be efficiently produced. dsRNA molecules present in the environment of an organism or its cells can be taken up by the organism and processed by the enzyme DICER to yield siRNA molecules. Alternatively, the dsRNA molecule can be produced in vivo, i.e. transcribed from one or more polynucleotides encoding the dsRNA present within a cell (e.g. a bacterial cell or a plant cell), and processed by DICER within the host cell after uptake of the longer precursor dsRNA. The dsRNA can be formed from two separate (sense and antisense) RNA strands that anneal by means of complementary base pairing. Alternatively, the dsRNA can be a single strand that is capable of refolding on itself to form a hairpin RNA or stem loop structure. In the case of a single RNA, the double-stranded region or "stem" is formed from two regions or segments of the RNA that are substantially the reverse complement of each other and have sufficient complementarity to allow a double-stranded region to form. There can be one or more functional double-stranded silencing elements in this "stem region" of the molecule. The reverse complement regions are typically separated by a region or segment of the RNA referred to as the "loop" region. This region can comprise any nucleotide sequence that imparts sufficient flexibility to allow self-pairing to occur between the flanking complementary regions of the RNA, which overall is substantially single-stranded and acts as a spacer sequence between the reverse complement sequences.

[0031] The term "siRNA" refers to a small inhibitory RNA duplex (typically between 17-30 base pairs, but also longer base pairs such as 31-50bp) that induces the RNA interference (RNAi) pathway.

[0032] The term "sense sequence", also known as "sense strand" or "plus strand", is the strand of a gene that is identical to the base sequence of the transcribed product mRNA (except that T is used instead of U).

[0033] The sequence in a gene that is complementary to the sense sequence is referred to as the "antisense sequence". In a DNA molecule comprising a plurality of genes, the sense strand of each gene is not necessarily on the same DNA deoxynucleotide strand, i.e. on one strand there are both the sense strands of some genes and the antisense strands of other genes. The "antisense sequence" comprised by the dsRNA molecule according to the present application is an RNA sequence that is complementary to the corresponding sense sequence.

[0034] The term "partial sequence" refers to at least 20 contiguous nucleotides of the sequence. For example, a "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 contiguous nucleotides of the sequence.

[0035] As used herein, the term "hybridizing sequence" is a polynucleotide strand that binds to a substantially complementary complementary strand through base pairing. Two nucleotide sequences are at least 80% complementary in their nucleotide sequence. Preferably, 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 over their entire length. The term "antisense RNA" refers to a RNA transcript that is complementary to all or part of a mRNA that is normally produced in the cells of an organism of interest. The complementarity of the antisense RNA can be to any portion of a particular gene transcript (i.e., a portion of the 5' non-coding sequence, 3' untranslated sequence, intron, or coding sequence).

[0036] RNA interference, also known as "RNAi", has emerged as a genetic tool to accelerate plant biotechnology research. RNAi is a conserved component of gene regulation processes that exists in all eukaryotes. For RNAi in plants, the alternative term "post-transcriptional gene silencing" (PTGS) is used. PTGS begins with the processing or cleavage of a precursor double-stranded RNA by a RNase III-like enzyme called Dicer (Baulcombe, Nature 431 :356-363. 2004) into short, about 20-25 ribonucleotides long, single- or double-stranded interfering RNAs (siRNAs) or micro interfering RNAs (miRNAs). The siRNAs or miRNAs are incorporated into an RNA-induced silencing complex (RISC) that recognizes and degrades complementary messenger RNA (mRNA) molecules, thus leading to a substantial reduction in RNA levels and effectively reducing the expression of the corresponding gene, i.e. the production of the target protein, whose functional activities are affected.

[0037] The term "genome" as used herein refers to the totality of genetic material (genes and non-coding sequences) present in each cell of an organism or virus or organelle, and / or the complete set of chromosomes inherited as a unit (haploid) from one parent.

[0038] The term "transgene" is used herein to describe genetic material that has been or will be artificially introduced into the genome of a host organism and is transmitted to the progeny of that host. A transgene will typically comprise a polynucleotide comprising non-coding and / or coding sequences that typically, but not necessarily, affect or cause an activity (e.g., modulation of transcription or translation, production of a nucleotide sequence comprising coding and / or non-coding sequences, etc.).

[0039] The term "gene" includes a nucleic acid segment that expresses a functional molecule such as, but not limited to, a particular protein, including regulatory sequences before (5' non-coding sequences) and after (3' non-coding sequences) the coding sequence.

[0040] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide can encode RNA that is translated into a protein (mRNA), or a DNA polynucleotide can encode RNA that is not translated into a protein (e.g., tRNA, rRNA, or RNA that targets DNA; also known as "non-coding" RNA or "ncRNA").

[0041] The terms "polynucleotide," "nucleotide," and "nucleic acid" are used interchangeably and include DNA, RNA, or hybrids thereof, which can be single-stranded or double-stranded. "Nucleic acid" can also contain non-naturally occurring or altered bases, which allow for proper read through by a polymerase without reducing the expression of the polypeptide encoded by the nucleic acid.

[0042] The terms "nucleotide sequence" and "nucleic acid sequence" both refer to the order of bases in DNA or RNA, referring to both the sense and anti-sense strands of nucleic acids, either existing as separate single strands or in duplexes.

[0043] One of ordinary skill in the art can readily employ known methods, such as methods of directed evolution and point mutation, to mutate the DNA segments of the present application. Those artificially modified nucleotides having at least 90%, 95%, 98%, 98.5%, 99%, 99.5%, or 99.8% identity to the DNA segments as set forth in the foregoing sequences of the present application, and having the same function, are derived from the nucleotide sequences of the present application and are equivalent to the sequences of the present application.

[0044] The term "identity" refers to sequence similarity with a native nucleic acid sequence. Identity can be assessed by eye or by computer software. Using computer sequence alignment software, identity between two or more sequences can be expressed as a percentage (%) that can be used to assess identity between related sequences. "Reverse complement" refers to a sequence that is complementary to the original polynucleotide sequence but in the opposite orientation.

[0045] "Complementary" polynucleotides are those which are capable of base pairing according to the rules of Watson-Crick complementarity. In particular, base pairs will form between purines and pyrimidines, including guanine and cytosine pairing (G:C) and adenine and thymine (A:T) in the case of DNA or uracil (A:U) in the case of RNA. It is understood that two polynucleotides can hybridize to each other even if they are not perfectly complementary to each other, as long as each has at least one region that is substantially complementary to the other.

[0046] The term "expression" as used herein in reference to a gene sequence refers to the translation of the coding sequence into a polypeptide. Inhibition of gene expression can be detectable at the mRNA level, at the polypeptide level, or both. Methods for assessing changes in mRNA levels or protein levels of a gene are well known in the art. For example, changes in mRNA levels of a gene can be assessed by quantitative real-time PCR (RT-qPCR) or Northern blotting.

[0047] The term "plant" is used herein to mean a whole plant at any stage of development and parts or derivatives thereof, thus including, for example, a plant cell, a population of plant cells, a plant tissue (e.g., meristematic tissue, callus), a plant organ (e.g., stem, leaf, root, ovule, stamen), a reproductive form or reproductive part of a plant (e.g., seed, tuber, cutting, gametophyte, sporophyte, pollen, microspore, embryo). A plant cell or population of plant cells can be isolated from (e.g., in suspension culture) or comprised in a plant tissue, a plant organ, or a whole plant at any stage of development.

[0048] In one particular embodiment, the plant is maize, wheat, rice, sorghum, millet, or barley.

[0049] In the present application, "target" and "target sequence" can be used interchangeably, i.e., can be selected from any suitable region or nucleotide sequence of a 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.

[0050] "Target gene" or "targeted gene" as used interchangeably herein refers to any sequence intended to be down-regulated in a virus. The virus is controlled by down-regulating the targeted gene, for example, by disrupting a biological process of virus multiplication. Thus, preferred targeted genes include, but are not limited to, genes that play a key role in the transcription of mRNA, protein, and DNA or RNA synthesis. When the expression of a viral targeted gene is down-regulated or inhibited (e.g., by interfering with viral mRNA with dsRNA or antisense RNA to inhibit the expression of the targeted gene), the multiplication of the virus is prevented, the viral load in the host cell is reduced; or the ability of the virus to infect a plant or crop species is reduced, thereby protecting the plant or crop species from the virus. "Multiplication" as used herein refers to the biological process by which a virus replicates its nucleic acid in a host cell, relying on the host cell's enzyme system, raw materials, and energy. The multiplication of the virus in the host cell affects the normal growth of the host (e.g., plant). For example, the multiplication of MRDV / RBSDV in a crop (e.g., rice, corn) cell causes stunted growth, dwarfing, short and thick internodes, and results in reduced crop yield.

[0051] The terms "infest," "infect," "invade," and / or "invade" are used interchangeably throughout the specification.

[0052] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the application. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are now described.

[0053] All publications and patents cited in this specification are herein incorporated 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 in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date of this application. Nothing in this regard should be construed as a

[0054] Unless otherwise defined, the terms "a," "an," and "the" as used herein refer to "at least one." All patents, patent applications, and publications cited or referred to in this disclosure are incorporated herein by reference in their entirety as to the disclosure and description of methods and / or materials. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 shows the position of various dsRNAs on a target gene.

[0056] Figure 2 qPCR-RBSDV-S5 vector schematic diagram.

[0057] Figure 3 Standard curve of Ct value versus copy number.

[0058] Figure 4 Maize rough dwarf virus resistance identification-virus load statistics.

[0059] Figure 5 Maize rough dwarf virus resistance identification-straw height statistics.

[0060] Sequence listing DETAILED DESCRIPTION

[0061] Example I, selection of RBSDV RNA interference target genes and their double-stranded RNA sequences

[0062] The S1 chain (encoding RNA-dependent RNA polymerase), S2 chain (encoding the main core structural protein), S6 chain (encoding RNA silencing suppressor), and S10 chain (encoding outer coat protein) encoding key functional proteins in the genome of rice black-streaked dwarf virus RBSDV were selected as target genes for RNA interference, with their amino acid sequences shown as SEQ ID NOs: 1-4 and the corresponding nucleotide sequences shown as SEQ ID NOs: 5-8.

[0063] The conserved region sequences of the above target genes were used as double-stranded RNA sequences for RNA interference, and were named as 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 positions on the target genes are shown in Figure 1.

[0064] In addition to designing double-stranded RNAs that individually silence the conserved regions of the above 4 chains, double-stranded RNAs that simultaneously silence the conserved regions of two of the chains were also designed, and these combined double-stranded RNA sequences were 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), respectively.

[0065] Example 2, Design and construction of RNA interference vector

[0066] The expression vector pCambia1300 was linearized by double digestion with restriction enzymes Avrll and Mlul (purchased from NEB), then the double-stranded RNA sequence, ST-LS1 sequence, and reverse complementary sequence of the double-stranded RNA sequence were amplified respectively, and the three fragments were ligated into the linearized expression vector by seamless cloning to construct the final RNA interference vector. The RNA interference vectors corresponding to the double-stranded RNA 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 were 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 corn receptor was transformed with the above expression vectors, and the positive transformants were selected for subsequent testing after identification. The maize rough dwarf disease was transmitted to maize by artificially feeding the RBSDV-carrying white-backed planthoppers, and the specific method was as follows:

[0070] (1) Corn seeding

[0071] In addition to the need to seed each RNA interference transgenic material, the transformed receptor variety and the diseased control variety Zhengdan 958 also needed to be seeded, and 100 plants of each material were seeded.

[0072] (2) Virus inoculation

[0073] The corn seedlings were grown to the two-leaf stage (two fully expanded leaves) and then inoculated with the virus, using the method of inoculating virus-carrying white-backed planthoppers on individual seedlings. From the above-seeded transgenic materials, transformed receptors, and Zhengdan 958, 50 plants were selected for virus transmission by virus-carrying white-backed planthoppers, and at least 3 white-backed planthoppers were ensured for each corn single plant (effective inoculation amount = inoculation amount x virus-carrying rate), and the virus-carrying white-backed planthoppers were transmitted for 3 days.

[0074] (3) Insect removal

[0075] The infected small brown planthoppers are removed after 3 days of transmission, and insecticides are sprayed on all transmission materials. After 7-10 days of observation, no residues or newly hatched small brown planthoppers are confirmed for field planting.

[0076] (4) Planting and management

[0077] The non-infected materials and infected materials are planted in the field at the same time, and corn is managed with regular water and fertilizer. During the entire growth period, attention is paid to preventing vector insects such as small brown planthoppers and leafhoppers to ensure that the maize rough dwarf disease in the test plot does not spread.

[0078] 2. Investigation of maize rough dwarf disease

[0079] The investigation is carried out when the disease level of the susceptible control (Zhengdan 958) reaches level 7 or above during the corn filling period. The virus load and plant height data are measured. The resistance evaluation standard of maize rough dwarf disease is provided in the Agricultural Industry Standard of the People's Republic of China, NY / T 1248.13-2016 Technical Specification for Identification of Corn Disease Resistance Part 13: Rough Dwarf Disease.

[0080] (1) RBSDV virus load detection of infected corn

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

[0082] Average molecular weight MW (g / mol) = number of plasmid base pairs (bp) x 660 (dalton / bp)

[0083] The standard plasmid with a concentration of 10 10 copies / μl is gradient diluted to 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , and 10 2 copies / μl, respectively. The standard plasmids at the above concentrations are subjected to RT-PCR together with the test samples to obtain the standard curve of the relationship between Ct value and copy number, as shown in Figure 3.

[0084] The leaves of 10 materials with the most serious disease were selected from the transgenic materials of all the RNA interference vectors, the transformed corn receptor, and the inoculated materials of Zhengdan 958. The leaves of 10 non-inoculated materials were taken as blank controls. The leaf sample size was about 0.2 g. The RNA was extracted from the leaves and reverse transcribed. 500 ng of template RNA was added into 10 μl of reverse transcription system. 1 μl of the obtained cDNA was used for RT-PCR. Finally, the standard plasmid copy number corresponding to each plant was calculated according to the standard curve, as the viral load. The average viral load of each material was calculated and counted.

[0085] The average viral load of each material was taken as the logarithmic value. The final results showed that the viral loads of the diseased control Zhengdan 958 and the wild-type transformed corn receptor were both more than 10 6 copies / μl. The viral load of the transgenic material of the RNA interference vector RBSDV_S1-dsRNA1+RBSDV_S6-dsRNA1 (No. 5) tended to be 0, and the resistance of the corn rough dwarf disease reached the level close to immunity. The viral loads of the transgenic materials of other RNA interference vectors could still be detected, and the values of the viral loads were different depending on the resistance levels (see Fig. 4).

[0086] (2) Investigation of corn plant height and disease

[0087] In the corn filling period, the plant height of the diseased control Zhengdan 958, the transformed corn receptor, and the transgenic materials of the above RNA interference vectors was investigated (Fig. 5). The results showed that the transgenic material of vector 5 showed high resistance (HR), and the plant height of the non-inoculated and inoculated materials had no significant difference. The transgenic materials of other vectors also showed resistance to different degrees, but the plant height was reduced to different degrees.

[0088] Table 1 Investigation results of corn plant height and disease

[0089] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application and not to limit. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application.

Claims

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

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

3. An interfering ribonucleic acid, comprising, It is designed according to the target gene of claim 1 or 2, and can inhibit the proliferation of the RBSDV virus.

4. The interfering ribonucleic acid of claim 3, wherein, It comprises at least any one of the following: a nucleotide sequence or its reverse complement sequence having at least 90%, 95%, 98%, 98.5%, 99%, 99.5%, 99.8% or 100% identity to 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 continuous nucleotides of 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 complement sequence.

5. The interfering ribonucleic acid of claim 3 or 4, wherein each of the sense sequences is on a different RNA strand from its corresponding antisense sequence in the interfering ribonucleic acid.

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

7. An expression cassette comprising, A polynucleotide comprising a polynucleotide encoding the interfering ribonucleic acid of any one of claims 3-6 under the control of operably linked regulatory sequences.

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

9. A method of creating an RBSDV virus-resistant plant and the transgenic plant obtained thereby, by constructing an RNAi vector expressing an interfering ribonucleic acid targeting the target gene of claim 1 or 2, which is capable of inhibiting the proliferation of RBSDV, transforming a transgenic plant to produce resistance to infection by the virus; preferably, the interfering ribonucleic acid is as claimed in any one of claims 3-6.

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

11. A composition for controlling RBSDV virus and its use for preventing and / or controlling the proliferation of RBSDV virus, comprising at least one of the interfering ribonucleic acids of any one of claims 3-6 and an auxiliary ingredient that maintains the biological activity of the interfering ribonucleic acid.

12. A method for controlling RBSDV virus, wherein a plant infected or at risk of being infected by said plant virus and / or the vicinity of said plant is contacted with an effective amount of a composition according to claim 11.

13. The method of claim 9, 10 or 12 or the transgenic plant of claim 9, wherein, Said plant is corn, wheat, rice, sorghum, millet, oat or barley; preferably, said plant is corn.

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