CN117264953A

CN117264953A - dsRNA for inhibiting rice stripe virus, and preparation method and application thereof

Info

Publication number: CN117264953A
Application number: CN202311164495.7A
Authority: CN
Inventors: 兰莹; 赵弘巍; 周彤; 陈盼; 杜琳琳; 徐乐; 李晨羊; 石学超
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2023-09-08
Filing date: 2023-09-08
Publication date: 2023-12-22

Abstract

The invention discloses dsRNA for inhibiting pathogenicity of rice stripe virus and application thereof. The dsRNA disclosed by the invention targets the gene of the RSV3 pc3 protein, so that the replication and expression level of the rice stripe virus in a plant body are inhibited, and the occurrence of rice stripe disease is effectively reduced. The dsRNA disclosed by the invention is inhibited before virus infection, so that the pathogenicity of rice stripe viruses is greatly reduced; the biological antiviral agent prepared by using the dsRNA disclosed by the invention has the advantages of obvious plant disease resistance improving effect, safety, environmental friendliness, simple preparation process and stronger applicability.

Description

dsRNA for inhibiting rice stripe virus, and preparation method and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to dsRNA for inhibiting rice stripe virus (Rice stripe virus, RSV) and a preparation method and application thereof.

Background

The rice stripe disease is a rice virus disease caused by the persistent transmission of RSV from the medium Laodelphax striatellus through eggs. Typical symptoms of rice plants infected by rice stripe viruses are formation of chlorosis stripe spots or plaques, and the typical symptoms are roughly classified into leaf rolling types and leaf spreading types, wherein the leaf rolling types show core leaf chlorosis, twist and arc ring sagging, severe core leaf death, leaf spreading type disease leaves do not twist, and yellow-green alternate stripes appear on leaf surfaces. Most of the plants with diseases cannot normally heading, so that the yield is seriously reduced. In 2000, rice stripe disease is widely popular in japonica rice planting areas at the downstream of Yangtze river in China, generally the yield is reduced by 20% -30%, the incidence rate of serious field blocks reaches 80% -90%, the yield loss is 7-8, and some grains are not harvested, so that serious threat is formed to agricultural production. In addition to damaging rice, RSV infects more than 80 grasses such as wheat, barley, oats, corn, and the like.

RNA interference (RNAi) is a process by which dsRNA (double-stranded RNA) recognizes mRNA having a homologous complementary sequence and inhibits its expression and even degrades its molecule, also known as gene silencing, which plays a very important role in eukaryotic protection against biotic stress. There are a large number of small cross-border RNAs in nature that are transmitted bi-directionally between plant-pathogens. dsRNA derived from pathogenic bacteria pathogenic genes is sprayed on the surface of a plant, so that an RNA silencing system in the plant can be induced, the expression of virus genes is inhibited, the pathogenicity of the virus genes is reduced, and the plant is effectively protected from being damaged by pathogenic bacteria. Therefore, the dsRNA based on the RSV genome is explored for preventing and treating viral diseases, the problems of lack of prevention and treatment medicaments for viral diseases in production, pollution caused by insect prevention and pesticide application, scarcity of resistant variety resources and the like are solved, and the method becomes a new means for researching green and environment protection.

Disclosure of Invention

The invention aims to: the first object of the present invention is to provide a dsRNA for inhibiting rice stripe virus by targeting a gene encoding rice stripe virus RSV3 pC3 protein.

The invention also solves the technical problem of providing the application of the dsRNA in inhibiting rice stripe virus.

The technical scheme is as follows: the RNA sequence of the dsRNA for inhibiting the pathogenicity of the RSV is shown as SEQ ID NO. 3.

The preparation method of the dsRNA comprises the following steps: PCR amplification is carried out by taking cDNA of RSV as a template and using a primer pair with a nucleotide sequence shown as SEQ ID NO. 1-2 to obtain a dsRNA fragment with a nucleotide sequence shown as SEQ ID NO.4, and the dsRNA fragment is connected to an expression vector to construct a recombinant vector; introducing a recombinant expression vector containing the dsRNA fragment into escherichia coli HT115 for culture; dsRNA production was induced by IPTG, and the bacterial liquid was collected by culture.

Wherein the expression vector is a commercially available pet28 vector.

Further, upon induction with IPTG, the initial concentration of E.coli HT115 was OD ₆₀₀ =0.4-0.8; IPTG concentration is 0.5-1.0mmol/mL; the culture time is 4-5h.

The dsRNA is applied to preparation of medicines for preventing and treating rice stripe disease.

Further, the rice stripe disease is caused by RSV.

Further, the concentration of the dsRNA is 1.20-2.0 mug/. Mu.L.

The agent of the invention can be sprayed on the leaves or stems of rice.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:

1. the dsRNA targets the gene for encoding the RSV3 pc3 protein of the rice stripe virus, and can play a role in preventing and treating rice stripe disease in early virus infection;

2. the bacterial induction dsRNA has low cost, high yield, simple process, strong specificity, obvious antiviral effect, safety and environmental protection, and ensures that the RNAi technology is better applied to the prevention and treatment of rice stripe disease.

Drawings

FIG. 1 selects the conserved domains (both ends) of segments;

FIG. 2 clustered distribution of small RNAs of non-conserved domains of selected segments;

FIG. 3 is a bacterial induction dsRNA electrophoresis pattern;

FIG. 4 is a graph of symptoms and bars of leaf blight disease of rice stripe in inoculated RSV strain treated with dsRNA of the present invention and non-inoculated control Wuyunj No.3 (WYJ 3), wherein the significant differences (p-value < 0.05) are shown;

FIG. 5 is a bar graph of the incidence of rice stripe disease analysis for each treatment group after treatment with dsRNA at different concentrations and virus inoculation according to the present invention;

FIG. 6 is a bar graph of the analysis of different disease grade phenotypes and different disease grade numbers for each treatment and control after treatment with dsRNA at different concentrations and virus inoculation according to the present invention;

FIG. 7 shows a western blot expression pattern of rice stripe virus proteins treated with dsRNA according to the invention, with Actin as a reference;

FIG. 8 is a bar graph showing the analysis of rice stripe virus content relative to expression in treated and control rice leaves after dsRNA treatment and virus inoculation according to the present invention.

Detailed Description

The technical solution of the present invention will be further described by way of example with reference to the accompanying drawings.

The materials and instruments used in the following examples are all commercially available, the rice material used is Wuyujing No.3, and the virus-carrying Laodelphax striatellus population carrying RSV is stored and screened by the plant protection institute of research institute of Agents for viral disease resistance, the institute of agricultural sciences, jiangsu province, where the applicant of the present invention is located.

The cDNA coding sequence of the RSV3 of the invention is derived from the National Center for Biotechnology Information databasehttps:// www.ncbi.nlm.nih.gov/gene) under the number NC003776.1Is a gene of (a). Firstly, the sequence is respectively compared with rice genome sequences (http:// rice. Plant biology. Msu/; http:// www.gramene, org /) in two rice genome databases through conservative comparison, so that the enrichment sections of the conservative regions at the two ends are eliminated, and the silent rice genes are eliminatedIs possible (fig. 1). And then, according to analysis, virus small RNAs of inoculated samples are distributed on a genome, and small RNA distribution areas enriched in high abundance are selected, so that the silencing efficiency is improved (figure 2). Finally, 327 bp base sequence in RSV3 was successfully amplified as a transcription template for the dsRNA of interest in the present invention.

The RNA sequence of the dsRNA molecule derived from RSV3 is shown as SEQ ID No.3, and the cDNA coding sequence is shown as SEQ ID No. 4.

SEQ ID No.3：

SEQ ID No.4

The cDNA coding sequence of RSV3 is derived from the gene numbered NC_003776.1 in the database of National Center for Biotechnology Information (https:// www.ncbi.nlm.nih.gov/nucleic /). The base sequences 1207-1608 of the cDNA coding sequence of ORF2 were selected as template sequences for the dsRNA of the invention. Again, this sequence was aligned with the rice genome sequences (http:// rice. Plant biology. Msu. Edu/; http:// www.gramene.org /) in the two rice genome databases, respectively, and the likelihood of nonspecific silencing of the rice genes was examined. The comparison result shows that the dsRNA molecule of the invention can not reach the effective length of RNA silencing rice genome, and further eliminates the possibility that the fragment interferes with silencing rice genes.

The dsRNA molecules of the present invention (the cDNA sequence of which is shown in SEQ ID No. 4) were amplified from the cDNA of RSV3 ORF3 saved by the institute of plant protection, proc. Natl.Acad. Jiang Susheng.

The invention classifies the symptoms of leaf blight of rice into the following categories by referring to the industry standard NYT2055/2011 of Ministry of agriculture, namely the identification technical Specification of leaf blight of rice variety:

grade 0, asymptomatic;

grade I, with slight yellow-green mottle and mottle symptoms, the disease leaves are not curled, and the plant grows normally;

stage II, the chlorosis and the development on the disease leaves are connected into irregular yellow-white or yellow-green strip spots, the disease leaves are not curled or slightly curled, and the growth is basically normal;

and III, seriously fading the diseased leaves, twisting the curled diseased leaves, and causing the diseased leaves to have yellowing wither symptoms, and causing the plants to be withered in a pseudo-withered heart shape or dead whole plants.

Example 1: preparation of bacterial induced expression dsRNA preparation

(1) Construction of vectors and recombinants:

1.1 extraction of rice stripe virus RNA: the leaf of the diseased rice plant was collected and the intermediate RNA was extracted by TRIzol (TaKaRa) method. The specific operation is as follows:

1.1.1, uniformly grinding a proper amount of rice plant leaves by using liquid nitrogen, and rapidly transferring the ground powdery or liquid substances into a 1.5mL RNase-free centrifuge tube.

1.1.2, 1mL of Trizol is rapidly sucked into a centrifuge tube, 200 mu L of chloroform is added into the centrifuge tube, and the liquid in the tube is stirred and mixed uniformly and then kept stand for 5min at 4 ℃.

1.1.3, 4℃and 12000rpm for 15min.

1.1.4, the supernatant after centrifugation was sucked into a new 1.5mL RNase-free centrifuge tube, 600. Mu.L isopropyl alcohol was added into the centrifuge tube, and the centrifuge tube was allowed to stand at-80℃for 2 hours.

1.1.5, 4 ℃,12000rpm for 15min, discarding the supernatant.

1.1.6, 1mL of 75% ethanol solution was pipetted into a centrifuge tube, the pellet resuspended by pipetting, and centrifuged at 12000rpm for 3-5min at 4℃and the supernatant discarded.

1.1.7, repeating the step 1.6 once, then carrying out air separation once, and sucking residual liquid by using a liquid transfer device.

1.1.8, drying the precipitate until the precipitate is transparent, adding 30 mu L of RNase-free Water into a centrifuge tube, and sucking and beating the precipitate by a pipette to fully dissolve the precipitate, thus obtaining the total RNA of the leaf blades of the rice plants.

1.2 transcription of Rice stripe Virus template, RNA concentration was adjusted to 500 ng/. Mu.L, primeScript was selected ^TM RT reagent Kit gDNA Eraser (TaKaRa) kit reverse transcribes the total RNA extracted into cDNA. Specific primers containing two cleavage sites of Sac I and Xho I are designed according to sequence information, and PCR amplification is carried out by taking cDNA of RSV3 as a template. The primer sequences were as follows:

F：5’-CGAGCTCGGCTCTACTAGAGAGTGAACAAT-3’(SEQ ID NO.1)；

R：5’-CCGCTCGAGATGGGAAAGTGAAGACCACAATC-3’(SEQ ID NO.2)。

the underlined part is the site of the introduced cleavage.

1.3, detecting the amplified product by 1.0% agarose gel electrophoresis, showing that a single band exists at about 400bp, and the size of the single band is consistent with that of the expected sequence, and performing gel recovery and purification.

1.4 double restriction fragments were purified together with a laboratory-stored pet28 vector (purchased from the commercial company Tianzenzetiand z, mianyang, china) and the restriction fragments and vector were ligated using T4 DNA Ligase (TaKaRa) to obtain recombinant plasmids.

1.5 preparation of GFP dsRNA fragments for control treatment reference is made to Annete Niehl et al, new Phytologist. ( Double-stranded RNAs induce a pattern-triggered immune signaling pathway in plants.New Phytologist Volume 211, issue3, https: (v)/doi. Org/10.1111/nph.13944 )

1.6, transforming the recombinant plasmid into Escherichia coli competent HT115 preserved in a laboratory to obtain a desired recombinant, and preserving glycerol bacteria.

(2) Bacteria induce expression of dsRNA. The specific operation is as follows:

2.1 Induction of expression of dsRNA

The stored glycerinum containing the target gene is prepared according to the following steps: the volume ratio of the culture solution is 1:100 (12.5. Mu.g/mL Tet) ⁺ ，50μg/mL Amp ⁺ ) In LB sterile culture medium. Placing in a constant temperature shaking table at 37 ℃ and 220rpm overnight for preliminary culture.

In an ultra-clean workbench, the primary culture liquid is sucked up according to the volume ratio of 4:100 in a proportion of 400mL of liquid LB sterile culture medium (500 mL conical flask) containing ampicillin and tetracycline resistances (concentration as above), and placing in a constant temperature shaker at 37℃and expanding culture at 220rpm for 2h to OD ₆₀₀ ＝0.4-0.8。

Sterile IPTG was added to the super clean bench at a final concentration of 1.0mmol/L, and the culture was continued for 5 hours under the above conditions.

2.2 extraction of total RNA from E.coli HT115 using TRIzol (TaKaRa) method, for specific procedures reference is made to example 1, step 1.1.

2.3 purification and characterization of induced dsRNA

2.3.1 Total RNA from E.coli was treated with DNase I (final concentration 200U/ml) at 37℃for 30min and then RNase A (final concentration 100. Mu.g/. Mu.l) at 37℃for 30min.

2.3.2, measuring the concentration of dsRNA by using a spectrophotometer, wherein the yield of total RNA extracted by the method can reach 5mg per 200ml bacterial liquid, and carrying out agarose gel electrophoresis on the dsRNA to verify the size of a band so as to confirm whether the dsRNA is successfully synthesized. The results are shown in FIG. 3, confirming successful synthesis of correct dsRNA. According to the estimated result of the electrophoresis diagram, the dsRNA content in the expression system of the invention accounts for 40% -60% of the total RNA, namely, the yield of bacterial liquid per 200ml can reach 2-3mg, the original concentration can reach 2-4 mug/mu l, and the yield is improved by two times compared with the conventional level. The final concentration of dsRNA was adjusted to 1.5. Mu.g/. Mu.l with DEPC water and stored as test treatment fluid.

Example 2: in vivo inoculation of RSV in rice and detection of the pathogenicity and expression level of dsRNA in inhibiting RSV

(1) The extraction of rice stripe virus RNA is described in step 1.1 of example 1.

(2) Rice inoculation RSV

Healthy Wuyujing No.3 rice seedlings are cultivated in a growing cup (35 strains per cup), when the seedlings grow to a leaf stage of 1.5, the finally expressed dsRNA product is mixed with RNase-free water, the concentration is regulated to 1.5 mug/mu l, 800 mu l of the mixture is sprayed per cup, and the total amount of 1200 mu g is uniformly sprayed on leaf surfaces and stems. Substitution of dsRNA solution with RNaseF reee ddH ₂ O and GFP dsRNA at a concentration of 1.5. Mu.g/. Mu.l as non-viralTreatment control of dsRNA. After 24 hours of treatment, the rice is inoculated with 3-4-year old Laodelphax striatellus population with high toxicity, the insect quantity of each cup of rice seedling to be inoculated is calculated according to 1.5 head insects of each effective plant, the Laodelphax striatellus with corresponding insect quantity is sucked by a simple insect sucking device, and 58 head insects are inoculated in each cup (taking 90 percent of the toxic rate as an example). The high virulent population of Laodelphax striatellus used in this example was a high affinity population of Laodelphax striatellus with virulent (RSV) obtained by multiple generation screening in the laboratory, and derived from Liu Haijian et al (2007) and Zhou Tong et al (Liu Qing Jian, cheng Zhaobang, wang Yue, wei Bangqing, ren Chunmei, zhou Yijun, fan Yongjian, 2007. Laodelphax striatellus transmitted Rice stripe virus Industy. Jiangsu agricultural journal, 23 (5): 492-494; zhou Tong, fan Yongjian, cheng Zhaobang, et al. Study of Rice stripe disease resistance identification methods [ J]Plant protection 2008.34 (6): 77-80). And (5) after 48 hours of inoculation, the Laodelphax striatellus is swept out, then the seedlings are transplanted into a test field, and the disease condition is observed. After 5d the phenotype was observed by sampling. 3 strains of materials are mixed for each treatment, and the incidence of living organisms is detected after 14 days.

(3) In vivo RNA extraction and quantitative expression

Total RNA of the above rice samples was extracted using TRIzol reagent (Invitrogen), and the expression level of RSV was analyzed by real-time quantitative PCR after reverse transcription in accordance with steps 1.1 and 1.2 of reference example 1.

As can be seen from FIG. 4, plants that were re-infected with RSV after treatment with the dsRNA of the present invention, were treated with H ₂ The incidence of treated samples was significantly reduced compared to GFP dsRNA control. As can be seen from FIG. 6, the relative expression level of RSV in the dsRNA treated rice plants was significantly reduced compared to the control.

From this, the above examples and data demonstrate that the dsRNA of the present invention has significant effects in inhibiting the pathogenicity of rice stripe disease and regulating the expression of rice stripe virus in plants.

EXAMPLE 3 Effect of aqueous dsRNA concentration on control Effect

dsRNA prepared in example 1 was dissolved in an aqueous solution to prepare aqueous solutions with concentrations of 0.5, 1.0, 1.5, 2.0 and 2.5. Mu.g/. Mu.l, and 800. Mu.l of each cup was uniformly sprayed on rice leaves, and after 24 hours of treatment, RSV (1.5 heads/seedling of effective virus carrying insects) was inoculated. And (5) after 48 hours of inoculation, the Laodelphax striatellus is swept out, the seedlings are transplanted into a test field, and the disease condition is observed after 14-20 days.

Table 1: optimal control concentration test for dsRNA-RSV3 aqueous solution

dsRNA concentration (μg/μl)	Prevention and control effect
		0.5	64.45％
1.0	100.0
		1.5	94.6
2.0	84.2
		2.5	47.4

As can be seen from Table 1 and FIG. 5, the optimal control concentration was 1.0. Mu.g/. Mu.l. When the dsRNA concentration is 0.5 mug/mu l compared with 1.0 mug/mu l, the control level is obviously different, and the resistance improving effect is not obvious. There was no significant difference in control levels when the concentration of aqueous dsRNA solutions was between 1.0-2.0 μg/μl. When the concentration of dsRNA is up to 2.5 mug/mul, the prevention and treatment effect is not obviously improved, and the cost is increased.

As can be seen from FIG. 6, the number of disease stages II-III in the rice plants is reduced with the increase of the dsRNA concentration, which indicates that the disease symptoms of the plants are reduced and the resistance level is increased.

Example 4 detection of dsRNA having inhibitory Effect on Rice stripe Virus

Cultivating healthy Wuyujing No.3 rice seedling in a growing cup (35 plants per cup), and selecting dsRNA and DEPC-H with final concentration of 1.0 μg/μl when the seedling grows to 1.5 leaf stage ₂ The mixture of O is added into a spray can treated by RNase scavenger (product No. 3090-250 of the Biotechnology Co., ltd., beijing Tianze) according to the volume of 800 μl per cup, and is uniformly sprayed on the leaf surface of the Wuyujing No.3 rice plant in the 1.5 leaf period. After the treatment, the pre-inoculated rice seedlings with the Wuyujing No.3 are transferred into a constant temperature culture room at 26 ℃ for 24 hours, and then RSV artificial inoculation is carried out, and the inoculation step is shown in example 2.

Experiments prove that after dsRNA is sprayed on plants, inoculated rice is moved to an identification garden, the virus expression level in the plants is detected, and Westen blot and Q-PCR results of FIG. 7 and FIG. 8 show that after the treatment of RSV 3dsRNA, the plants are treated with H ₂ In vivo virus content was reduced in the plants of WYJ3 (WYJ 3) with martial transport for 7 days, 14 days, and 21 days (7 dpi,14dpi, and 21 dpi) compared to GFP dsRNA treatment.

By combining the results, the dsRNA has good effect of preventing and treating rice stripe viruses and has wide market application prospect.

Claims

1. A dsRNA for inhibiting pathogenicity of rice stripe virus is characterized in that the sequence of the dsRNA is shown as SEQ ID NO. 3.

2. The method of producing dsRNA of claim 1, comprising the steps of: PCR amplification is carried out by taking cDNA of RSV as a template and using a primer pair with a nucleotide sequence shown as SEQ ID NO. 1-2 to obtain a dsRNA fragment with a nucleotide sequence shown as SEQ ID NO.4, and the dsRNA fragment is connected to an expression vector to construct a recombinant vector; introducing a recombinant expression vector containing the dsRNA fragment into escherichia coli HT115 for culture; dsRNA production was induced by IPTG, and the bacterial liquid was collected by culture.

3. The method of claim 2, wherein the expression vector is pet28 vector.

4. The method of claim 2, wherein the initial concentration of e.coli HT115 is OD ₆₀₀ = 0.4-0.8。

5. The method of claim 2, wherein the IPTG concentration is 0.5-1.0mmol/mL.

6. The method of claim 2, wherein the incubation time is 4-5h.

7. The use of the dsRNA as defined in claim 1 for preventing and treating rice stripe disease.

8. The use according to claim 7, wherein the dsRNA is at a concentration of 1.0-2.0 μg/μl.

9. The use according to claim 7, characterized in that it comprises spraying an aqueous dsRNA solution onto rice leaves or stems.