A method for inhibiting viral replication in rice by targeting viral small RNA

Abstract

The application discloses a method for inhibiting rice virus replication by targeting small RNA of virus, and belongs to the technical field of biology. The small RNA of virus is a single-stranded RNA molecule, and the nucleotide sequence is SEQ ID No. 1. The application provides experimental evidence that the expression of the small RNA molecule can be inhibited, and the replication of rice stripe virus in rice can be inhibited.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for inhibiting viral replication in rice by targeting small viral RNA. BACKGROUND

[0002] Rice is an important food crop in China, and it often encounters complex and diverse biological and abiotic stresses during its life cycle, and viral diseases are particularly serious, known for their destructive and explosive nature. To date, there have been as many as 14 viral diseases that have broken out in rice, among which the more serious pathogens mainly include rice stripe virus (RSV), rice black-streaked dwarf virus (RBSDV), southern rice black-streaked dwarf virus (SRBSDV), etc., all of which are efficiently transmitted by insects as vectors. Among them, RSV is transmitted by the planthopper in a persistent propagative manner, causing rice stripe leaf blight to break out in rice planting areas, causing serious losses to rice production (S. Toriyama, Rice stripe virus: prototype of a new group of viruses that replicate in plants and insects. Microbiol Sci 3, 347-351 (1986).; T. Y. Wei et al., Genetic diversity and population structure of rice stripe virus in China. J. Gen. Virol. 90, 1025-1034 (2009).). The prevention and control of rice viral diseases is very difficult, and the existing commercial agents are mostly insecticides targeting insect vectors, or immune inducers targeting crops, and there is still a lack of targeted drugs that can efficiently block viral replication or transmission. In the face of the demand for green ecological construction, the identification and development of key reagents or technologies targeting viruses have become an important demand to ensure the stable yield of food and economic crops.

[0003] RNA interference (RNAi) is an important antiviral defense mechanism in plants and invertebrates (S. W. Ding, O. Voinnet, Antiviral immunity directed by small RNAs. Cell 130, 413-426 (2007).; R. W. Carthew, E. J. Sontheimer, Origins and Mechanisms of miRNAs and siRNAs. Cell 136, 642-655 (2009).). It has been found that endogenous microRNAs (miRNAs) and small interfering RNAs (siRNAs) in insects can regulate the proliferation of viruses in insects (C. Kutter, P. Svoboda, miRNA, siRNA, piRNA: Knowns of the unknown. RNA Biol 5, 181-188 (2008).). Interestingly, viruses not only change the expression pattern of small RNAs in the vector insects or hosts, but also use their synthesis mechanism to produce virus-derived small RNAs, including virus miRNAs (vmiRNAs) and virus siRNAs (vsiRNAs). These virus-derived small RNAs, by targeting the genes of the virus itself or the genes of the vector and host, finely regulate the infection process of the virus, showing a potential new way of viral control.Among them, the vmiRNA encoded by the virus often occurs in the terminal structure of the viral genome, which can promote virus proliferation (M. Hussain et al., West Nile virus encodes a microRNA-like small RNA in the 3' untranslated region which up-regulates GATA4 mRNA and facilitates virus replication in mosquito cells. Nucleic Acids Res 40, 2210-2223 (2012).), and can also play an antiviral role (M. Hussain, R. J. Taft, S. Asgari, An insect virus-encoded microRNA regulates viral replication. J Virol 82, 9164-9170 (2008).), and the vsiRNA plays a broad-spectrum antiviral role (W. Zhao, Q. Li, M. Sun, Y. Xiao, F. Cui, Interaction between endogenous microRNAs and virus-derived small RNAs controls viral replication in insect vectors. PLoS Pathog 18, e1010709 (2022).).

[0004] The nucleotides at the ends of the genome of all segmented negative-strand RNA viruses are highly conserved, and the complementarity of about 10 nucleotides at the 3' and 5' ends can form a handle structure, which is a key to initiate viral RNA synthesis (A. Pflug, D. Guilligay, S. Reich, S. Cusack, Structure of influenza A polymerase bound to the viral RNA promoter. Nature 516, 355-360 (2014).). As a representative member of segmented negative-strand RNA viruses, RSV contains four single-stranded genomic RNA segments, and each RNA segment has about 20 conserved nucleotides at the 3' and 5' ends (M. Takahashi, S. Toriyama, Y. Kikuchi, T. Hayakawa, A. Ishihama, Complementarity between the 5'- and 3'-terminal sequences of rice stripe virus RNAs. J Gen Virol 71, 5 (1990).).

[0005] Therefore, exploring the synergistic regulation of rice stripe virus terminal vsiRNA on rice stripe virus has an important role in preventing and treating rice stripe virus. SUMMARY

[0006] The technical problem to be solved by the present application is to identify the vsiRNA of rice stripe virus. The technical problem to be solved is not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0007] The present inventors' previous studies have shown that RSV can produce vsiRNA in Laodelphax striatellus and rice, and the terminal region of RSV genome is also a hotspot for vsiRNA production (M. Yang et al. , Rice stripe virus-derived siRNAs play different regulatory roles in rice and in the insect vector Laodelphax striatellus . BMC Plant Biol18, 219 (2018)). Considering the conservation of the sequence at the end of the genome of negative-strand RNA viruses, the inventors speculated that the vsiRNAs derived from the end of the viral genome might have a synergistic role in regulating viral infection. Therefore, in this study, the synergistic regulatory effect of the vsiRNAs derived from the end of the viral genome on the proliferation of the virus was studied using rice - planthopper - RSV as the research object, and a gene-edited rice targeting vsiRNAs was constructed based on the research results, and the anti-viral effect thereof was detected, thereby providing a potential rice germplasm resource for the prevention and control of RSV.

[0008] To solve the above technical problems, the present application provides the following technical solutions:

[0009] The present application provides a viral small RNA or a modifier thereof, wherein the viral small RNA is a single-stranded RNA molecule, and the nucleotide sequence of the viral small RNA is SEQ ID No. 1 in the sequence listing.

[0010] The present application provides a viral small RNA or a modifier thereof, wherein the viral small RNA is a single-stranded RNA molecule, and the viral small RNA includes single-stranded RNA molecules with nucleotide sequences of SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively.

[0011] The term "modifier" refers to a product obtained by modifying the single-stranded RNA molecule, and various modification methods can be selected, including one or a combination of several selected from ribose modification, base modification, and phosphate backbone modification, etc.

[0012] The present application also provides a short tandem target mimic, which can specifically bind to the aforementioned viral small RNA, and the short tandem target mimic is a double-stranded DNA molecule.

[0013] The nucleotide sequence of the short tandem target mimic is SEQ ID No. 4.

[0014] The present application also provides a rice stripe virus inhibitor, which contains a substance that can specifically bind to the aforementioned viral small RNA or a biological material that produces the substance.

[0015] In the above-mentioned rice stripe virus inhibitor, the rice stripe virus inhibitor can be a preparation that inhibits the replication of the rice stripe virus in host cells.

[0016] In the above-mentioned rice stripe virus inhibitor, the substance is a short tandem target mimic (STTM).

[0017] In the above-mentioned inhibitor of rice stripe virus, the short tandem target mimic is a single-stranded DNA molecule, the nucleotide sequence of the viral small RNA is SEQ ID No. 1, and the sequence of the binding site is 5'-ACACAAAGUCCAGCUAAGGAAAACAA-3'.

[0018] In the above-mentioned inhibitor of rice stripe virus, the nucleotide sequence of the short tandem target mimic is SEQ ID No. 4.

[0019] In the above-mentioned inhibitor of rice stripe virus, the biological material is a gene encoding the short tandem target mimic, an expression cassette containing the gene, or a vector containing the gene.

[0020] The present application provides a biological material, which is any one of the following:

[0021] A1) a nucleic acid molecule encoding the aforementioned short tandem target mimic,

[0022] A2) an expression cassette, a recombinant vector, a recombinant microorganism, or a transgenic plant cell line, a transgenic plant tissue, or a transgenic plant organ containing the nucleic acid molecule of A1).

[0023] The carrier used by the recombinant vector can be specifically a 35S-pBWA(V)HS plasmid, and the recombinant vector is specifically obtained by replacing the small fragment between the BsaI and Eco31I enzyme digestion recognition sites of the 35S-pBWA(V)HS vector with the nucleotide sequence shown in SEQ ID No. 4 while keeping other nucleotide sequences unchanged.

[0024] The microorganism can be any strain capable of expressing the recombinant vector, and can be specifically Agrobacterium tumefaciens.

[0025] The present application provides a method for cultivating a plant resistant to rice stripe virus, which comprises introducing a substance targeting the aforementioned viral small RNA or the aforementioned biological material into a target plant, thereby improving the resistance of the target plant to rice stripe virus, obtaining a plant resistant to rice stripe virus, and the plant resistant to rice stripe virus has higher resistance to rice stripe virus than the target plant.

[0026] The present application provides a method for inhibiting viral replication, which comprises introducing a substance targeting the aforementioned viral small RNA or the aforementioned biological material into a target plant, thereby inhibiting the replication of rice stripe virus in the target plant.

[0027] The aforementioned single-stranded RNA molecule or its modification is also within the protection scope of the present application for the preparation of an inhibitor of rice stripe virus or for the inhibition of rice stripe virus.

[0028] The application of the aforementioned biomaterials in the preparation of rice stripe virus inhibitors or in the inhibition of rice stripe virus also falls within the scope of protection of this invention.

[0029] The plant is any one of the following:

[0030] G1) Monocotyledons;

[0031] G2) Plants of the order Poales;

[0032] G3) Gramineae plants;

[0033] G4) Oryza genus;

[0034] G5) Rice.

[0035] The advantage of this invention is that this study uses RSV-gray planthopper-rice as the research object, and the results show that the vsiRNA derived from the end of the RSV virus genome has a synergistic regulatory effect on virus proliferation. Inhibiting the expression of the aforementioned RNA molecules can inhibit the replication of rice stripe virus in rice. Attached Figure Description

[0036] Figure 1 Characterization of vsiRNAs derived from the RSV genome ends. Figure caption: (A) Schematic diagram of three RSV-derived siRNAs. These three vsiRNAs originate from the 3' end of RSV complementary genomic RNA (vcRNA). Red arrows indicate the direction and position of the vsiRNA sequence. These three vsiRNAs share 11 nucleotides, marked in red. (B) Gene encoding the viral nucleocapsid protein (NP) in virus-free (N) and virus-carrying (V) third-instar nymphs. NP Compared to internal reference genes EF2 The expression levels of vsiRNA and the RNA levels of vsiRNA relative to U6snRNA were compared using a t-test. P < 0.05; **, P < 0.001. (C) Using biotin-labeled LNA oligonucleotide probes, vsiR-8401, vsiR-7607, and vsiR-5532 were identified in third-instar nymphs without virus (N) and carrying virus (V) by Northern blotting. (DE) Different time points after inoculating non-virus nymphs with crude RSV extract by artificial injection method. NP The relative RNA levels (D) and the relative RNA levels of the three vsiRNAs (E). (F) Virus in rice leaves fed by infected planthoppers for different number of days. NP Compared to rice UBQ10 RNA levels, and the RNA levels of the three vsiRNAs relative to rice U6snRNA. Different letters indicate significant differences in the Tukey multiple comparison test.

[0037] Figure 2 The three vsiRNAs promote RSV accumulation in N. lugens. (A)-(C) Relative RNA levels of the three vsiRNAs and NP in non-toxic N. lugens 6 days after injection of RSV crude extract mixed with vsiR-8401 activator (A), vsiR-7607 activator (B), vsiR-5532 activator (C), or the three vsiRNAs (3vsiR) activator (D). (E) Western blot analysis of NP protein levels in (D) samples using anti-NP monoclonal antibody. (F)-(H) Relative RNA levels of the three vsiRNAs and NP in non-toxic N. lugens 6 days after injection of RSV mixed with vsiR-8401 inhibitor (F), vsiR-7607 inhibitor (G), or vsiR-5532 inhibitor (H). (I) Western blot analysis of NP protein levels in (F)-(H) samples using anti-NP monoclonal antibody (n=3). Tubulin was measured as an internal control using anti-tubulin polyclonal antibody. The gray values represent the relative optical density of NP to tubulin. NC, negative control. Comparison was made using t test. NS, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0038] Figure 3 The cross-influence of each vsiRNA inhibitor on the expression of the other two vsiRNAs. (A) RNA levels of vsiR-8401 and vsiR-5532 relative to U6 snRNA in non-toxic N. lugens 6 days after inoculation of RSV crude extract mixed with vsiR-7607 inhibitor. (B) RNA levels of vsiR-7607 and vsiR-5532 relative to U6 snRNA after inoculation of vsiR-8401 inhibitor (n=8). (C) RNA levels of vsiR-8401 and vsiR-7607 relative to U6 snRNA after inoculation of vsiR-5532 inhibitor (n=7 or 8). Comparison was made using t test. NS, not significant. *, P < 0.05; ***, P < 0.001.

[0039] Figure 4 The three vsiRNAs promote RSV replication in rice. (A) and (B) Relative RNA levels of the three vsiRNAs and NP(C) Western blot analysis of NP protein levels in (B) samples using anti-NP monoclonal antibody (n = 3). Tubulin was detected using anti-Tubulin polyclonal antibody as a loading control. The gray value represents the relative value of NP to Tubulin. (D) Morbidity of WT and STTM8401 rice after 7 days of continuous feeding on RSV-carrying planthoppers. Five rice seedlings per replicate, 6 replicates in total. t-test was used for comparison. NS, no significant difference. *, P < 0.05; **, P < 0.01.

[0040] Figure 5 Economic traits detection of STTM8401 line. (A) Comparison of plant height between mature wild type and STTM8401 rice line, scale bar, 50 cm. (B) Thousand-grain weight of wild type and STTM8401 line. (C-D) Width (C) and length (D) of dehulled grain of wild type and STTM8401 line. Scale bar, 5 mm. Different letters indicate statistically significant difference. DETAILED DESCRIPTION

[0041] The application will be further described in conjunction with the preferred embodiments thereof, given only by way of illustration of the present application, and not to limit the scope of the present application. The following examples provided as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any way of limiting the present application.

[0042] The experimental methods in the following examples are all routine methods, unless otherwise specified, according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0043] The quantitative test in the following examples sets up three repeated experiments, and the results are averaged.

[0044] O. sativa subsp. japonica Nipponbare for generating knockout or overexpression lines was from Wuhan Beryon Biotech Co., Ltd.

[0045] Non-viruliferous small brown planthopper, small brown planthopper (hereinafter referred to as viruliferous small brown planthopper) carrying rice stripe virus (RSV): described in the non-patent literature "W. Zhao, P. Yang, L. Kang, F. Cui, Different pathogenicities of Rice stripe virus from the insect vector and from viruliferous plants. New Phytol 210, 196-207 (2016)." which is available to the public from the Institute of Zoology, Chinese Academy of Sciences, and this biological material is only used for repeating the relevant experiments of the present application and cannot be used for other purposes. The aforementioned non-viruliferous small brown planthopper and viruliferous small brown planthopper populations were respectively reared in glass bottles in an insect chamber, and the rearing conditions were 24°C, 16 hours light / 8 hours dark. Every three months, the small brown planthoppers carrying RSV were screened by dot-ELISA using self-made anti-NP monoclonal antibodies to maintain the viruliferous rate of the viruliferous strain at more than 95%.

[0046] The following examples use GraphPad Prism version 8.0 software to process data, and the experimental results are expressed as mean ± standard deviation. Student's t-test was used for pairwise comparison, and P<0.05 (*) indicates significance; one-way ANOVA with Tukey's multiple comparison test was used for multiple comparisons, and different letters indicate significant differences.

[0047] Example 1, Identification of vsiRNA at the 3' end of the RSV genome

[0048] I. Design of vsiRNA

[0049] This example screened three vsiRNAs from the 3' end of the RSV genome, vsiR-8401 (23 nt), vsiR-7607 (22 nt), and vsiR-5532 (22 nt), with the following specific information:

[0050] vsiR-8401: 5'-UUGUUUUCCUCUGGACUUUGUGU-3' (corresponding to the sequence SEQ ID No. 1 in the sequence listing, U is represented by T in SEQ ID No. 3, and the other nucleotides are the same as the sequence);

[0051] vsiR-7607: 5'-UUAUAUACCCAGGACUUUGUGU-3' (corresponding to the sequence of SEQ ID No. 2 in the sequence listing, U is represented by T in SEQ ID No. 3, and the other nucleotides are the same as the sequence);

[0052] vsiR-5532: 5'-UAUUUUACCCAGGACUUUGUGU-3' (corresponding to the sequence of SEQ ID No. 3 in the sequence listing, U is represented by T in SEQ ID No. 3, and the other nucleotides are the same as the sequence).

[0053] The RSV viral genome contains four RNA strands, and the above three vsiRNAs are from the complementary strands of RNA1-RNA3 (NCBI numbers MF287955, MF287954, and MF287953), respectively. The above vsiR-8401 is from the complementary strand vcRNA1 of the viral genome RNA1, the vsiR-7607 is from the complementary strand vcRNA2 of the viral genome RNA2, and the vsiR-5532 is from the complementary strand vcRNA3 of the viral genome RNA3. Figure 1 Central A).

[0054] II. Material preparation

[0055] 1. Viruliferous and non-viruliferous white-backed planthopper samples

[0056] Both viruliferous and non-viruliferous white-backed planthopper populations were obtained from a long-term screening of virus preservation populations in the laboratory, and were divided into non-viruliferous insects (N line, non-viruliferous insects) and viruliferous insects (V line, viruliferous insects) infected with RSV. Wujujing rice seedlings were planted in a 1 L glass beaker, and when the rice seedlings grew to 2-3 cm, the white-backed planthoppers were fed in them, and the bottle mouth was covered with a fine nylon mesh. New rice seedlings were replaced in time, and water was appropriately and timely added. The white-backed planthopper feeding temperature was 25°C, the relative humidity was maintained at 15%-25%, and the light cycle was 16 h:8 h (light:dark).

[0057] To ensure that the viruliferous planthopper strain can maintain a high rate of drug carrying, the viruliferous planthopper strain is screened every 3 months. The female viruliferous planthopper to be born is raised in a single bottle, and after the nymphs are hatched, 5 offspring planthoppers are randomly taken from each bottle for detection. The presence of RSV in the nymphs of planthoppers is detected by ELISA method using NP monoclonal antibody. The detection results by dot enzyme-linked immunoassay (W. Zhao, P. Yang, L. Kang, F. Cui, Different pathogenicities of Rice stripe virus from the insect vector and from viruliferous plants. New Phytol 210, 196-207 (2016).) show that all 5 nymphs in each bottle are viruliferous, and the female offspring of the planthopper in the bottle are used as a viruliferous population for further culture and can be used for subsequent experiments.

[0058] 2. Extraction of RSV crude extract

[0059] Take 80 fourth instar nymphs of high viruliferous planthoppers, wash several times with 1xPBS buffer (pH 7.4), and place in a 1.5 mL EP tube. Add 100 μL PBS buffer and grind with a grinder, operating on ice. After grinding, centrifuge at 4°C for 15 minutes at 12000 g. After centrifugation, the supernatant is removed to a new EP tube, and the centrifugation step is repeated 3-5 times until a uniform and clear supernatant is obtained, which is the RSV crude extract. The concentration of the shell protein NP in the virus crude extract is 0.2 μg / μL as determined by ELISA.

[0060] 3. Artificial injection of virus into non-viruliferous planthoppers

[0061] (1) Use a pipette to suck up several fourth instar non-viruliferous planthoppers, and temporarily place them in a 1.5 mL EP tube on ice to freeze the planthoppers into a coma.

[0062] (2) Cut the needle of the glass needle according to the size of the hind leg of the planthopper, and then fill the needle with mineral oil, without air bubbles.

[0063] (3) Using a microinjector (Nanoliter 2000), install a glass needle, discharge the mineral oil in the glass needle, suck the previously extracted RSV crude extract, inject 23 nL of RSV crude extract into the non-toxic planthopper nymphs, and inject the same volume of non-toxic planthopper crude extract as a control. If it is necessary to inject vsiRNA agonists or inhibitors at the same time, the vsiRNA agonists or inhibitors are injected at a volume ratio of 1:1 with the RSV crude extract, ensuring that the final working concentration of the agonists or antagonists is 250 μM. After injection, the planthopper nymphs are placed in fresh rice seedlings for further culture for subsequent experiments.

[0064] III. qPCR detection of the relative expression levels of the viruliferous planthopper NP genes, vsiR-8401, vsiR-7607 and vsiR-5532

[0065] The RNA of the planthoppers was extracted by Trizol method (Invitrogen, Carlsbad, CA, USA).

[0066] When detecting the enrichment degree of vsiRNA, the cDNA was synthesized by using miRcute enhanced miRNA cDNA first strand synthesis kit (Tiangen, Beijing, China), and then the content of vsiRNA was quantitatively detected by using miRcute enhanced miRNA fluorescent quantitative detection kit (Tiangen). In Table 1, vsiR8401-q-F and the Reverse Primer provided in the kit were used as a pair of primers to detect the relative expression amount of vsiR8401 in the sample. vsiR7607-q-F and the Reverse Primer provided in the kit were used as a pair of primers to detect the relative expression amount of vsiR7607 in the sample. vsiR5532-q-F and the Reverse Primer provided in the kit were used as a pair of primers to detect the relative expression amount of vsiR5532 in the sample. LsU6 -F and LsU6 -R were used to detect the relative expression amount of snRNA U6 gene in the planthoppers U6The genes used were internal reference genes for calculating the relative expression levels of vsiR8401, vsiR7607, and vsiR5532 in the planthopper. The reaction mixture was as follows: 7 μL sterile ddH2O, 10 μL 2×miRcute Plus miRNA Premix, 0.5 μL of forward primer (10 μM), and 0.5 μL of reverse universal primer (10 μM, provided in the kit). After mixing and centrifuging, the mixture was carefully added to the quantitative wells. The cDNA solution was diluted 10-fold, and 2 μL was added to the corresponding quantitative wells. The membrane was sealed and centrifuged. The reaction program was as follows: 95℃ for 15 min (initial template denaturation); 94℃ for 20 s (template denaturation during PCR cycles), 60℃ for 34 s (annealing, extension), 45 cycles. The melting curve analysis program was: 94℃ for 5 s, 60℃ for 1 min, and 97℃ for 5 consecutive light exposures.

[0067] When detecting gene expression levels, cDNA from planthoppers and plants was synthesized using Promega's M-MLV Reverse Transcriptase, and the virus was detected using Roche's SYBR Green Master. NP Gene content. NP -F and NP -R is used to detect in samples NP The relative expression level of genes. EF2 -qF and EF2 -qR is used to detect elongation factor 2 in the brown planthopper. EF2 The relative expression level of ). EF2 To calculate the number of planthoppers NP Internal reference gene for relative gene expression levels. The reaction system was as follows: 7 μL of sterile ddH2O, 10 μL of SYBR Green I Master, and 0.5 μL each of forward and reverse primers (10 μM). After mixing and centrifugation, the mixture was added to a quantitative plate. Subsequently, 2 μL of the appropriately diluted cDNA solution was added to the corresponding quantitative wells, the plates were carefully sealed, and briefly centrifuged. The reaction program was performed in a LightCycler 480 quantitative instrument (Roche). The PCR amplification program was: 95℃ for 15 min (pre-denaturation); 95℃ for 10 s (denaturation), 58℃ for 20 s (annealing), 72℃ for 20 s (extension), for 40 cycles. The melting curve plotting program was: 95℃ for 5 s (denaturation), 65℃ for 60 s (annealing), and 40℃ for 10 s.

[0068] Quantitative settings 6-8 biological replicates, normal distribution of data using independent sample t test statistical analysis, not in line with the normal distribution of Wilcoxon rank sum test method, multiple comparisons using one-way ANOVA statistical, and using Tukey test.

[0069] Table 1

[0070]

[0071] Results as shown in Figure 1 B, only with toxic gray cicadas (V) Figure 1 B in the V) can be successfully detected NP Gene, vsiR8401, vsiR7607 and vsiR5532, in non-toxic gray cicadas, the expression of the above (N in B) can not be detected. Figure 1

[0072] Four, Northern blotting detection of vsiR-8401, vsiR-7607 and vsiR-5532 expression in toxic and non-toxic gray cicadas

[0073] 1. Probe design

[0074] The biotin-labeled LNA oligonucleotide probes designed for the foregoing three vsiRNAs are as follows (+ represents LNA modification):

[0075] vsiR-8401-LNA probe: 5'biotin-ACA+CAA+AGU+CCA+GAG+GAA+AAC+AA-biotin 3 '.

[0076] vsiR-7607-LNA probe: 5'biotin-ACA+CAA+AGU+CCU+GGG+UAU+AUA+A-biotin 3 '.

[0077] vsiR-5532-LNA probe: 5'biotin-ACA+CAA+AGU+CCA+GGG+CAU+UUG-biotin 3 '.

[0078] U6 snRNA-LNA probe: 5'biotin-CUA+AUC+UUC+UCU+GUA+UCG+UUC+C-biotin 3 '.

[0079] 2. Northern blotting

[0080] Total RNA was extracted from toxic and non-toxic gray cicadas and subjected to Northern detection, the specific method is as follows: ​

[0081] (1) Prepare 15% (w / v) polyacrylamide gel.

[0082] (2) Load the RNA sample into the gel well, and run the electrophoresis at 180 V for 45 min in lx TBE.

[0083] (3) Soak the gel in 0.5x TBE for about 10 min.

[0084] (4) Stain the gel with SYBR Gold (Invitrogen) for 10-20 min under UV light to observe the RNA electrophoresis.

[0085] (5) Wash the gel with 0.5x TBE for 5 min, and soak the membrane and two pieces of filter paper in 0.5x TBE for about 10 min.

[0086] (6) Install the membrane transfer device in the following order: positive electrode - sponge - filter paper - membrane - gel - filter paper - sponge - negative electrode, and draw the positions of the markers on the gel on the positively charged nylon membrane (BrightStar™-Plus Positively Charged Nylon Membrane, Invitrogen; AM10102) with a pencil.

[0087] (7) Transfer the membrane at 300 mA for one hour (in an ice bath).

[0088] (8) Crosslink the membrane under UV light at 1200 energy value for 300 seconds (with the RNA side of the membrane facing up).

[0089] (9) Bake the membrane at 80°C for 30 min (with two pieces of filter paper sandwiched in the middle) to ensure that the RNA is fixed on the membrane.

[0090] (10) Place the membrane in the hybridization solution, and add the LNA oligonucleotide probes of biotin-labeled vsiR-8401, vsiR-7607, vsiR-5532, and U6 snRNA, and hybridize overnight at 37°C.

[0091] (11) Wash the membrane twice with 2x SSC + 0.1% SDS at 42°C for 30 min each time.

[0092] (12) Block the membrane with Blocking Buffer for 15 min, and shake slowly on a shaker.

[0093] (13) Dilute the HPR-labeled streptavidin (product of Biodragon, product number BF06185X) 100-fold in Blocking Buffer, and incubate the membrane in the antibody solution for one hour.

[0094] (14) Wash the membrane with 1x Wash solution for 4 times, 5 min each.

[0095] (15) HRP color development, SuperSignal West Femto chemiluminescent reagent was used.

[0096] Results are shown in Figure 1 Fig. 4C, the vsiR-8401, vsiR-7607 and vsiR-553 were all expressed in the infected N. lugens.

[0097] V. Viruses in the infected N. lugens and infected rice at different time points after inoculation of RSV crude extract NP Relative expression levels of the vsiRNA-8401, vsiRNA-7607 and vsiRNA-5532 in the infected N. lugens and infected rice

[0098] Total RNA was extracted from the aforementioned infected N. lugens (including the infected N. lugens at 4 days, 6 days, 8 days, 12 days, 14 days, 16 days and 18 days after injection of RSV crude extract) and infected rice (including the infected rice that was taken by the infected N. lugens for 4 days, 8 days and 12 days), and cDNA was synthesized by using M-MLV Reverse Transcriptase of Promega Company. When detecting the enrichment degree of the vsiRNA in the infected rice, the snRNA U6 gene of the rice (the primers were -F and -R in Table 1) was used as the internal reference instead of the snRNA U6 gene of the N. lugens, and the expression level of the vsiRNA in the infected rice was detected, and the snRNA U6 gene of the rice (the primers were -F and -R in Table 1) was used as the internal reference instead of the vsiRNA-8401 gene of the N. lugens, and the expression level of the vsiRNA-8401 in the infected rice was detected, and the snRNA U6 gene of the rice (the primers were -F and -R in Table 1) was used as the internal reference instead of the vsiRNA-7607 gene of the N. lugens, and the expression level of the vsiRNA-7607 in the infected rice was detected, and the snRNA U6 gene of the rice (the primers were -F and -R in Table 1) was used as the internal reference instead of the vsiRNA-5532 gene of the N. lugens, and the expression level of the vsiRNA-5532 in the infected rice was detected, and the rest of the operations were the same as those in “III. qPCR detection of the expression levels of the vsiRNA-8401, vsiRNA-7607 and vsiRNA-5532 in the infected N. lugens and non-infected N. lugens”. OsU6 OsU6 NP UBQ10 UBQ10 UBQ10 EF2 NP

[0099] Results are shown in Figure 1 Fig. 4D-F, the three viral siRNAs could be detected in the infected N. lugens and rice, but not in the non-infected samples. In addition, with the extension of the infection time of the N. lugens, the viral content (the RNA level of the vsiRNA) increased significantly first, then reached a plateau, and then decreased slightly (Fig. 4D), and the change trend of the contents of the three vsiRNAs was similar to that of the vsiRNA (Fig. 4E). NP Figure 1 NP Figure 1

[0100] ​​​​​​​​​​​​Example 2, Anti-viral effect of viral terminal small RNA in insects

[0101] Each experiment was repeated three times, and the groups were as follows:

[0102] activator-NC group: The fourth instar non-toxic planthoppers were injected with RSV crude extract and activator-NC (the sequence of the sense strand is 5'-UUCUCCGAACGUGUCACGUTT-3', and the sequence of the antisense strand is 5'-ACGUGACACGUUCGGAGAATT-3'), and the injection method was as described in "II. Material Preparation" in Example 1. The vsiRNA activator or inhibitor was injected in a volume ratio of 1:1 with the RSV crude extract, and the total injection volume was 23 nL per planthopper. The injected planthopper nymphs were placed in fresh rice seedlings for further culture, and were used in subsequent experiments. After 6 days of injection, the following experiments were performed: the relative expression levels of the genes, vsiR-8401, vsiR-7607, and vsiR-5532 in the planthoppers infected with RSV were detected according to "III. Detection of the relative expression levels of the genes, vsiR-8401, vsiR-7607, and vsiR-5532 in the planthoppers" in Example 1. NP NP The relative expression levels of the genes, vsiR-8401, vsiR-7607, and vsiR-5532 in the planthoppers were detected according to "III. Detection of the relative expression levels of the genes, vsiR-8401, vsiR-7607, and vsiR-5532 in the planthoppers" in Example 1. Each group included 8 planthoppers.

[0103] activator-vsiRNA, the activator is an artificially synthesized double-stranded RNA, wherein the sequence of the sense strand is the sequence of the corresponding vsiRNA, and the sequence of the antisense strand is not completely complementary to the sequence of the sense strand.

[0104] activator-8401 group: activator-8401 (the sequence of the sense strand is 5'-UUGUUUUCCUCUGGACUUUGUGU-3', and the sequence of the antisense strand is 5'-ACAAAGUCCAGAGGAAAACAAUU-3') was used to replace activator-NC, and the relative expression levels of the genes, vsiR-8401 in the planthoppers infected with RSV were detected according to the operation of the activator-NC group. NP

[0105] activator-7607 group: activator-7607 (the sequence of the sense strand is 5'-UUAUAUACCCAGGACUUUGUGU-3', and the sequence of the antisense strand is 5'-ACAAAGUCCUGGGUAUAUAAUU-3') was used to replace activator-NC, and the relative expression levels of the genes, vsiR-7607 in the planthoppers infected with RSV were detected according to the operation of the activator-NC group. NP

[0106] ​​​activator-5532 group: activator-NC was replaced by activator-5532 (the sequence of sense strand is 5'-UAUUUUACCCAGGACUUUGUGU-3', and the sequence of antisense strand is 5'-ACAAAGUCCUGGGUAAAAUAUU-3'), and the operation of the activator-NC group was referred to detect the relative expression level of NP genes, vsiR-5532 in viruliferous planthoppers.

[0107] vsiRNA (3vsiR) group: RSV crude extract and activator-3vsiR (activator-8401, activator-7607 and activator-5532, mixed at the same ratio) were injected into the fourth instar non-viruliferous planthoppers, and the injection method referred to "II. Material preparation" in Example 1. After 6 days of injection, the following experiments were performed:

[0108] ① The relative expression level of genes, vsiR-8401, vsiR-7607 and vsiR-5532 in viruliferous planthoppers was detected by referring to "III. qPCR detection of viruliferous planthoppers and non-viruliferous planthoppers" in Example 1. NP genes, vsiR-8401, vsiR-7607 and vsiR-5532 in viruliferous planthoppers. NP genes, vsiR-8401, vsiR-7607 and vsiR-5532 in viruliferous planthoppers.

[0109] ② The total protein of viruliferous planthoppers was collected and extracted using 1xPBS buffer, and then Western blotting analysis of the protein level of NP was performed using anti-NP monoclonal antibody (prepared by the inventors' research group, see non-patent document "W. Zhao, P. Yang, L. Kang, F. Cui, Different pathogenicities of Rice stripe virus from the insect vector and from viruliferous plants. New Phytol 210, 196-207 (2016).") and gray scale analysis was performed using Image J. The internal reference was Tubulin antibody (product of Abeam Company, product number ab15568).

[0110] inhibitor-NC group: inhibitor-NC (the sequence is: 5'-CAGUACUUUUGUGUAGUACAA-3') was used to replace activator-NC, and the operation of activator-NC group and vsiRNA (3vsiR) group was referred to detect the relative expression level of NPThe relative expression levels of genes, vsiR-8401, vsiR-7607 and vsiR-5532, and the protein level of NP.

[0111] The inhibitor of vsiRNA is a single-stranded RNA molecule, and its sequence is complementary to that of vsiRNA.

[0112] Inhibitor-8401 group: Inhibitor-8401 (sequence: 5'-ACACAAAGUCCAGAGGAAAACAA-3') was used to replace inhibitor-NC, and the same procedure as the inhibitor-NC group was followed to detect infected planthoppers. NP The relative expression levels of the gene and vsiR-8401.

[0113] Inhibitor-7607 group: Inhibitor-7607 (sequence: 5'-ACACAAAGUCCUGGGUAUAUAA-3') was used instead of inhibitor-NC, and the same procedure as the inhibitor-NC group was followed for detecting infected planthoppers. NP The relative expression levels of the gene and vsiR-7607.

[0114] Inhibitor-5532 group: Inhibitor-5532 (sequence: 5'-ACACAAAGUCCUGGGUAAAAUA-3') was used instead of inhibitor-NC, and the same procedure as the inhibitor-NC group was followed for detecting infected planthoppers. NP The relative expression levels of the gene and vsiR-5532.

[0115] Experimental results: Compared with the negative control NC group (activator-NC group), the expression levels of vsiRNA agonists were significantly increased after injection of vsiRNA agonists, but the viral expression levels remained unchanged. NP No significant changes were observed at the RNA level. Figure 2 (A, B, and C). After injecting a mixture of the three vsiRNA agonists, regardless of whether vsiRNA or... NP The RNA content of all samples was significantly increased. Figure 2 In the middle D), Western spectroscopy revealed a significant increase in NP protein levels. Figure 2 (E). Subsequently, using the same method, while injecting the crude RSV extract, inhibitors of vsiRNA were also injected. It was found that compared with the negative control NC group (inhibitor-NC group), all three vsiRNAs decreased significantly after the injection of inhibitors. Figure 2Figs. 3A-3H show the results of the experiment of Example 2. Fig. 3A shows the results of the experiment of Example 2, Fig. 3B shows the results of the experiment of Example 2, Fig. 3C shows the results of the experiment of Example 2, Fig. 3D shows the results of the experiment of Example 2, Fig. 3E shows the results of the experiment of Example 2, Fig. 3F shows the results of the experiment of Example 2, Fig. 3G shows the results of the experiment of Example 2, and Fig. 3H shows the results of the experiment of Example 2. Figure 2 Fig. 4 shows the results of the experiment of Example 3.

[0116] Fig. 5 shows the results of the experiment of Example 4.

[0117] The experiment was repeated three times, and each time the groups were divided as follows:

[0118] inhibitor-NC group: RSV crude extract and inhibitor-NC were injected into the non-toxic planthoppers at the fourth instar, and the injection method was the same as that described in “II. Material Preparation” of Example 1. The injection amount of RSV crude extract was 23 nL / inhibitor-8401 group: inhibitor-8401 was used to replace inhibitor-NC, and the relative expression levels of vsiR-7607 and vsiR-5532 in the virus-carrying planthoppers were detected according to the operation of the inhibitor-NC group.

[0119] inhibitor-7607 group: inhibitor-7607 was used to replace inhibitor-NC, and the relative expression levels of vsiR-8401 and vsiR-5532 in the virus-carrying planthoppers were detected according to the operation of the inhibitor-NC group.

[0120] inhibitor-5532 group: inhibitor-5532 was used to replace inhibitor-NC, and the relative expression levels of vsiR-8401 and vsiR-7607 in the virus-carrying planthoppers were detected according to the operation of the inhibitor-NC group.

[0121] The experimental results show that the inhibition of each vsiRNA can down-regulate the accumulation of RSV in the planthoppers, but only the up-regulation of the three vsiRNAs can promote the replication of the virus. Based on the fact that the three vsiRNAs have the same 11 nt nucleotide sequence, the inventors speculate that the interference with one of the vsiRNAs can affect the expression of the other two vsiRNAs. In order to verify this hypothesis, the inventors injected RSV crude extract and the inhibitors of the three vsiRNAs into non-toxic planthoppers, respectively, and quantitatively determined the contents of the other two vsiRNAs after 6 days of injection. The inventors found that the interference with vsiR-7607 can achieve specific interference without affecting the other vsiRNAs. Figure 3 Fig. 4A shows the results of the experiment of Example 3, Fig. 4B shows the results of the experiment of Example 3, Fig. 4C shows the results of the experiment of Example 3, and Fig. 4D shows the results of the experiment of Example 3. Figure 3 Fig. 4A shows the results of the experiment of Example 3, Fig. 4B shows the results of the experiment of Example 3, Fig. 4C shows the results of the experiment of Example 3, and Fig. 4D shows the results of the experiment of Example 3.Figure 3 Based on the experimental results, the subsequent rice knockout lines of vsiR-8401 will be constructed to achieve the purpose of interfering with three kinds of vsiRNAs.

[0122] Example 4, Antiviral effect of viral terminal small RNA in rice

[0123] I. Preparation of vsiRNA-8401 silenced rice (STTM8401)

[0124] STTM is an artificially synthesized short tandem target mimic with a specific sequence of about 48 oligonucleotides in the middle and target miRNA binding sites at both ends. This sequence can bind to the target miRNA to form a non-complete complementary double-strand, thereby effectively preventing the miRNA from binding to the target gene, making the miRNA silenced (J. Yan et al., Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. Plant Cell 24, 415-427 (2012).). This technology has been widely used in rice. In this study, STTM technology was used to knock out vsiRNA in rice. STTM8401 transgenic rice was constructed by Wuhan Boyuan Biotechnology Co., Ltd. The specific experimental method is as follows:

[0125] The recombinant Agrobacterium containing the recombinant vector 35S-pBWA(V)HS-vsiR-8401 was obtained by transforming the recombinant vector 35S-pBWA(V)HS-vsiR-8401 into Agrobacterium tumefaciens. The obtained rice seedlings were T0 generation, which were cultivated to T2 generation, and STTM-8401 transgenic lines T2 generation plants were obtained. Among them, the recombinant vector 35S-pBWA(V)HS-vsiR-8401 is a recombinant vector obtained by replacing the small fragment between the BsaI and Eco31I enzyme recognition sites of the 35S-pBWA(V)HS vector with the DNA (STTM) of nucleotide sequence SEQ ID No. 4 and keeping other nucleotide sequences unchanged. SEQ ID No. 4: 5'-ACACAAAGTCCAGCTAAGGAAAACAAgttgttgttgttatggtctaatttaaatatggtctaaagaagaagaatACACAAAGTCCAGCTAAGGAAAACAA-3'.

[0126] II. Inoculation of RSV-carrying white-backed planthoppers

[0127] Six replicates of five rice seedlings each. Three-week-old seedlings of T2 generation of STTM-8401 transgenic lines and wild type Nipponbare (WT) were inoculated with one microcage per leaf, which contained 10 RSV-carrying third instar nymphs (i.e. the nymphs from hatching to the completion of the third molt of the white-backed planthopper nymphs) for 7 days. After 7 days, the remaining rice and the fed leaves were collected, and the following experiments were performed:

[0128] ① The relative expression levels of genes, vsiR-8401, vsiR-7607 and vsiR-5532 in the viruliferous planthoppers were detected according to the "detection of the relative expression levels of genes, vsiR-8401, vsiR-7607 and vsiR-5532 in viruliferous planthoppers" in Example 1. NP NP The relative expression levels of genes, vsiR-8401, vsiR-7607 and vsiR-5532 in the viruliferous planthoppers were detected according to the "detection of the relative expression levels of genes, vsiR-8401, vsiR-7607 and vsiR-5532 in viruliferous planthoppers" in Example 1.

[0129] ② The total protein of the rice leaves was collected and extracted using 1x PBS buffer, and then Western blotting analysis of the protein level of NP was performed using anti-NP monoclonal antibody and gray scale analysis was performed using Image J. The internal reference was Actin antibody (EASYBIO product, product number BE0028).

[0130] ③ The incidence of WT and STTM8401 rice was calculated as follows: each leaf of wild type (WT) and STTM8401 transgenic rice plants was fixed by a microcage with 10 three instar planthopper nymphs carrying the virus for 7 days. After removing the planthoppers, the rice leaves were placed in a greenhouse at 28°C to observe the leaf symptoms. Five plants were used for each group of replicates, and six replicates were performed to calculate the incidence of disease.

[0131] Experimental results: compared with the negative control NC group, the contents of the three vsiRNAs in the STTM8401 transgenic rice were significantly reduced, and this synergistic down-regulation was probably due to the similarity of the three vsiRNA sequences (Fig. 1A). In addition, the virus was significantly down-regulated at the RNA level and the protein level, indicating that the vsiRNA also played a role in promoting the virus in the rice (Fig. 1B and C). The inventors continued to cultivate the remaining rice after 7 days of feeding to 30 days and observed the symptoms, and found that the onset time of STTM8401 was delayed compared with the wild type, and the incidence of disease was much lower than that of the wild type, which again indicated that after knocking out the vsiRNA, the replication level of the virus in the plant was down-regulated (Fig. 1D). Figure 4 NP Figure 4 Figure 4

[0132] Example 5, Analysis of economic traits of STTM8401 transgenic plants​​​​​

[0133] Experimental method: The T2 generation plants of STTM-8401 transgenic lines and wild type Nipponbare (WT) rice were planted in the greenhouse under the conditions of 28°C, 70% humidity, 12 hours light / 12 hours darkness. From planting to harvesting the seeds, the plant height, 1000-grain weight, length and width of the dehulled rice grains of each line were counted. The experiment was repeated three times, and each time the transgenic lines and wild type were planted in 6-8 pots, with 2-3 plants of rice in each pot.

[0134] Experimental results: Compared with the wild type WT, the plant height of STTM8401 had no obvious difference (Fig. 1A), the 1000-grain weight had a slight upward trend, but the difference was not significant (Fig. 1B), and the length and width of the dehulled rice grains were similar to those of WT (Fig. 1C and D). Figure 5 Figure 5 Figure 5

[0135] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application.​​​

Claims

1. A small RNA or a modification thereof of a virus, characterized in that, The viral small RNA is a single-stranded RNA molecule, and the nucleotide sequence of the viral small RNA is SEQ ID No. 1 in the sequence listing.

2. Short tandem target mimics characterized in that, The short tandem target mimic is a double-stranded DNA molecule capable of specifically binding to the viral small RNA as defined in claim 1.

3. The short-tandem-target mimic of claim 2, wherein, The nucleotide sequence of the short tandem target mimic is SEQ ID No.

4.

4. A biological material, which is any one of the following: A1) a nucleic acid molecule encoding the short tandem target mimic as defined in claim 2 or 3; A2) an expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule as defined in A1).

5. The biomaterial of claim 4, wherein, The nucleotide sequence of the nucleic acid molecule is SEQ ID No.

4.

6. A method for breeding a plant resistant to Rice stripe virus, comprising introducing a substance targeting to inhibit the viral small RNA as defined in claim 1 or the biological material as defined in claim 4 or 5 into a plant of interest, thereby increasing the resistance of the plant of interest to Rice stripe virus, to obtain a plant resistant to Rice stripe virus, which has a higher resistance to Rice stripe virus than the plant of interest.

7. A method for inhibiting the replication of a virus, comprising introducing a substance targeting to inhibit the viral small RNA as defined in claim 1 or the biological material as defined in claim 4 into a plant of interest, thereby inhibiting the replication of Rice stripe virus in the plant of interest.

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