Double-stranded RNA molecules for targeted silencing of endogenous genes in tomatoes and their applications

Abstract

The present invention discloses a double-stranded RNA molecule for targeted silencing of endogenous genes in tomatoes and its applications. The double-stranded RNA molecule of the present invention is any one of the following 1)-3): 1) the sequence of one strand is as shown in SEQ ID No. 1, and the sequence of the other strand is as shown in SEQ ID No. 2; 2) the sequence of one strand is as shown in SEQ ID No. 3, and the sequence of the other strand is as shown in SEQ ID No. 4; 3) the sequence of one strand is as shown in SEQ ID No. 5, and the sequence of the other strand is as shown in SEQ ID No. 6. The double-stranded RNA molecule of the present invention can enhance the resistance of plants to Botrytis cinerea by directly spraying it on tomatoes. At the same time, the double-stranded RNA involved in the present invention does not interfere with the growth and development of tomatoes, and its use in the preparation of biopesticides can provide an effective way to control tomato gray mold disease.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly relates to double-stranded RNA molecules targeting and silencing endogenous genes of tomatoes SlACET1 , SlDND1 or SlMLO1 , and the application of these molecules in preventing and controlling plant diseases caused by Botrytis cinerea. Background Art

[0002] Botrytis cinerea ( Botrytis cinerea ) is a necrotrophic pathogen that causes gray mold, and can infect a variety of crops including tomatoes and seriously threaten agricultural production safety. Chemical control is the main means of preventing and controlling gray mold, but currently Botrytis cinerea has developed resistance to a variety of pesticides, so it is urgent to explore new methods for preventing and controlling gray mold.

[0003] RNA interference (RNAi) is a phenomenon of gene silencing induced by double-stranded RNA, which can inhibit gene expression by blocking the transcription or translation of specific genes. RNAi exists ubiquitously in organisms. RNA sequences with double-stranded or hairpin structures formed by DNA transcription can be processed into small RNAs between 20-30 bp. These small RNAs mediate the specific recognition of the gene silencing complex with the target gene DNA or mRNA sequences complementary to them, causing mRNA cleavage, translation inhibition or DNA methylation, and ultimately inhibiting the normal expression of genes (Fire, A. et al. 1998. Nature. 391(6669): 806). In recent years, a large number of achievements have been made in the field of plant disease control by RNAi technology. By directly spraying double-stranded RNA molecules, the key genes of pathogens can be interfered, resulting in a decrease in the fitness of pathogens and a reduction in pathogenicity, which is called SIGS (Spray-Induced Gene Silencing).

[0004] Currently, SIGS mainly uses double-stranded RNA (dsRNA) as a mediator to cross-silence the key genes of pests and diseases by spraying plants, thereby inhibiting the occurrence and development of pests and diseases. There are problems such as low cross-border transfer efficiency of small RNAs formed by plant cells processing dsRNA, which may lead to low cross-border gene silencing efficiency and insignificant pest and disease control effects. Therefore, it may be a more effective strategy to directly target and silence the immune negative regulatory genes in plants by SIGS to improve the resistance of plants to pests and diseases. Currently, there is a lack of research on directly interfering with the expression of plant disease-related genes to enhance their resistance to pests and diseases.

[0005] The present invention uses the endogenous immune negative regulatory genes of tomatoes SlACET1 , SlDND1 and SlMLO1Using [target] as the target, dsRNAs that can stably improve the resistance of tomatoes to Botrytis cinerea were constructed and screened. After retrieval, no relevant literature was found on interfering with the expression of plant disease-susceptible genes based on RNA silencing technology to control plant diseases caused by Botrytis cinerea. Summary of the Invention

[0006] The object of the present invention is to provide three double-stranded RNA molecules that can respectively and efficiently inhibit endogenous genes in tomatoes SlACET1 , SlDND1 and SlMLO1 from expressing.

[0007] The second object of the present invention is to provide the use of the above-mentioned double-stranded RNA molecules in inhibiting the expression of endogenous genes in tomatoes SlACET1 , SlDND1 and SlMLO1 from expressing.

[0008] The third object of the present invention is to provide the use of the above-mentioned double-stranded RNA molecules in controlling plant diseases caused by Botrytis cinerea.

[0009] The fourth object of the present invention is to provide a pesticide containing the above-mentioned double-stranded RNA molecules.

[0010] The fifth object of the present invention is to provide the application of the pesticide containing the above-mentioned double-stranded RNA molecules in controlling plant diseases caused by Botrytis cinerea.

[0011] The sixth object of the present invention is to provide a method for controlling plant diseases caused by Botrytis cinerea using the above-mentioned double-stranded RNA molecules.

[0012] The technical solution to achieve the present invention is as follows:

[0013] Three double-stranded RNA molecules that can respectively target and silence endogenous immune negative regulatory genes in tomatoes SlACET1 , SlDND1 and SlMLO1 from expressing, numbered double-stranded RNA molecules 1 / 2 / 3 respectively. It is characterized in that double-stranded RNA molecule 1 is composed of the nucleic acid sequences shown in SEQ ID No.1 and SEQ ID No.2; double-stranded RNA molecule 2 is composed of the nucleic acid sequences shown in SEQ ID No.3 and SEQ ID No.4; double-stranded RNA molecule 3 is composed of the nucleic acid sequences shown in SEQ ID No.5 and SEQ ID No.6.

[0014] The use of the above-mentioned double-stranded RNA molecules in inhibiting the expression of endogenous genes in tomatoes SlACET1 , SlDND1 and SlMLO1 from expressing is applied.

[0015] Use of the above double-stranded RNA molecule in preventing and controlling plant diseases caused by Botrytis cinerea.

[0016] Pesticide containing the above double-stranded RNA molecule.

[0017] Use of the pesticide containing the above double-stranded RNA molecule in preventing and controlling plant diseases caused by Botrytis cinerea.

[0018] Method for preventing and controlling plant diseases caused by Botrytis cinerea using the above double-stranded RNA molecule, including directly spraying a solution or preparation containing a carrier of the above double-stranded RNA molecule onto the surface of plant tissues.

[0019] The double-stranded RNA molecule involved in the present invention can be produced by prokaryotic expression.

[0020] Method for using the double-stranded RNA molecule involved in the present invention: Spraying an aqueous solution of the double-stranded RNA molecule at a suitable concentration (such as about 100 nM) onto the surface of plant tissues can effectively inhibit the infection and spread of Botrytis cinerea on plants.

[0021] The above plant is preferably tomato.

[0022] Advantages or beneficial effects of the present invention: (1) The double-stranded RNA molecule involved in the present invention shows an obvious inhibitory effect on the expression of three plant immune negative regulatory genes, can effectively inhibit the infection of Botrytis cinerea on plants, and provides a new effective way for preventing and controlling plant diseases caused by Botrytis cinerea. (2) The double-stranded RNA molecule involved in the present invention has strong specificity for the targeted plant immune negative regulatory genes, can theoretically specifically inhibit various plant pathogens including Botrytis cinerea, and there is no problem of drug resistance. (3) The double-stranded RNA molecule involved in the present invention is safe for plants, humans and animals. Its use can effectively reduce the use amount of chemical pesticides, there is no environmental pollution problem, and it is beneficial to environmental protection. Brief Description of the Drawings

[0023] Figure 1 It is a gel electrophoresis diagram of three double-stranded RNA molecules involved in the present invention.

[0024] Legend of the figure: The gel bands are Marker, double-stranded RNA molecules dsACET1-2, dsDND1-1 and dsMLO1-3 from left to right.

[0025] Figure 2 It is a photo of the infection of Botrytis cinerea after the double-stranded RNA molecule involved in the present invention is applied to tomatoes.

[0026] Legend of the figure: After spraying the crude dsRNA extract on tomato leaves for 1 day, inoculating Botrytis cinerea mycelial blocks (5 mm), and the diseased leaves after 4 days (from left to right are control dsRNA, three double-stranded RNA molecules involved in the present invention).

[0027] Figure 3 This is a bar graph showing the size of infected lesions formed on tomatoes after the double-stranded RNA molecules involved in the present invention were applied to tomatoes and inoculated with Botrytis cinerea.

[0028] Figure caption: Tomato leaves were sprayed with dsRNA crude extract 1 day later and then inoculated with Botrytis cinerea hyphae (5 mm). Four days later, the diameter of the lesions on the diseased leaves (from left to right are control dsRNA and the three double-stranded RNA molecules involved in the present invention).

[0029] Figure 4 The present invention relates to the protective effect of double-stranded RNA molecules on Botrytis cinerea after being applied to tomato plants.

[0030] Figure caption: Tomato plants were sprayed with dsRNA crude extract 1 day later and inoculated with Botrytis cinerea conidia suspension (2×10 6 / mL), and 5 days later, the disease incidence of tomatoes was investigated according to the field efficacy test guidelines (Pesticide Inspection and Bioassay Laboratory, Ministry of Agriculture and Rural Affairs of the People's Republic of China, 2000) (from top to bottom, from left to right are water control, control dsRNA, and the three double-stranded RNA molecules involved in the present invention).

[0031] Figure 5 The present invention relates to a bar graph showing the target gene silencing efficiency of the double-stranded RNA molecule after application to tomato plants.

[0032] Figure caption: Relative expression levels of target genes corresponding to dsRNA in tomato leaves one day after spraying dsRNA crude extract (from left to right: control dsRNA, three double-stranded RNA molecules of the present invention; after treatment with dsGFP) SlACET1 、 SlDND1 or SlMLO1 The expression level of the three target genes was normalized to 1 and the relative changes in expression levels of the three target genes were calculated. DETAILED DESCRIPTION

[0033] In the following examples, unless otherwise specified, the reagents used in the examples are all commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0034] Example 1: Sources of the double-stranded RNA molecules for inhibiting the expression of tomato immune negative regulatory genes according to the present invention

[0035] (1) The sequence of the double-stranded RNA molecule 1 (named dsACET1-2) involved in the present invention is derived from SlACET1 The cDNA, double-stranded RNA molecule 2 (designated dsDND1-1) was derived from SlDND1The cDNA of which the sequence of double-stranded RNA molecule 3 (named dsMLO1-3) is derived from SlMLO1 the cDNA. SlACET1 and SlDND1 and SlMLO1 The cDNA sequences are derived from the genes numbered Solyc12g100240, Solyc02g088560 and Solyc04g049090 respectively in the SolGenomics Network (SGN) database.

[0036] (2) Select SlACET1 a 520-base sequence from position 311 to 830 of the cDNA sequence numbered Solyc12g100240 in the cDNA sequence database (SGN) as the sequence of double-stranded RNA molecule 1 involved in the present invention; select SlDND1 a 550-base sequence from position 205 to 754 of the cDNA sequence numbered Solyc02g088560 in the cDNA sequence database (SGN) as the sequence of double-stranded RNA molecule 2 involved in the present invention; select SlMLO1 a 520-base sequence from position 328 to 847 of the cDNA sequence numbered Solyc04g049090 in the cDNA sequence database (SGN) as the sequence of double-stranded RNA molecule 3 involved in the present invention. The sense strand sequence of double-stranded RNA molecule 1 is shown as sequence 1 in the sequence list, and its antisense strand is shown as sequence 2 in the sequence list; the sense strand sequence of double-stranded RNA molecule 2 is shown as sequence 3 in the sequence list, and its antisense strand is shown as sequence 4 in the sequence list; the sense strand sequence of double-stranded RNA molecule 3 is shown as sequence 5 in the sequence list, and its antisense strand is shown as sequence 6 in the sequence list.

[0037] (3) Align the above sequences with the genomic sequences (NCBI) of common hosts of Botrytis cinerea such as tomato and strawberry to detect the possibility of non-specific silencing of other plant genes. The alignment results show that the maximum continuous match of the sequences involved in the present invention with other gene sequences of plant non-target genes is less than 10 bases, and the prediction of siRNAs potentially generated by the sequences shows that the siRNAs generated by the above sequences cannot reach the effective length of gene silencing, excluding the possibility of silencing non-target genes of host plants.

[0038] Example 2. Preparation of the double-stranded RNA molecule of the present invention

[0039] (1) The double-stranded RNA expression plasmid L4440 contains two bidirectional T7 promoters and can transcribe double-stranded RNA in RNaseⅢ-deficient Escherichia coli. The target fragment was amplified by PCR, and the amplification primers are shown in Table 1. Using the cDNA of tomato (variety: Cherry Tomato from Taiwan Province of China, produced by Qianrui Seed Industry) as a template, the amplified fragment was inserted between the two T7 promoters of the L4440 plasmid (between the recognition sites of Sac II and Xba I enzymes) by enzyme digestion and ligation. After verification by PCR and sequencing, the plasmid capable of expressing double-stranded RNA molecules was obtained.

[0040] Table 1 Primer sequences for constructing double-stranded RNA molecules

[0041]

[0042] (2) The plasmid capable of expressing double-stranded RNA molecule 1, double-stranded RNA molecule 2 or double-stranded RNA molecule 3 obtained in step (1) was transformed into the RNaseⅢ-deficient Escherichia coli strain HT115 to induce the expression of dsRNA.

[0043] (3) Single colonies of the HT115 strain expressing double-stranded RNA molecule 1, double-stranded RNA molecule 2 or double-stranded RNA molecule 3 that were verified by PCR and successfully sequenced were selected, cultured overnight in LB liquid medium containing ampicillin, and then stored in a -80℃ refrigerator.

[0044] (4) The expression strains of double-stranded RNA molecule 1, double-stranded RNA molecule 2 or double-stranded RNA molecule 3 were streaked on an LB plate with a resistance marker. A single colony was picked and transferred to 5 mL of liquid LB medium, and cultured overnight at 37℃ with a shaking speed of 200 rpm.

[0045] (5) 500 μL of the cultured bacterial liquid was taken into 10 mL of LB liquid medium and cultured at 37℃ with a shaking speed of 230 rpm for about 2.5 h until the OD600 of the bacterial liquid was between 0.5 and 0.8.

[0046] (6) IPTG with a final concentration of 2 mM was added to the bacterial liquid, and the mixture was cultured at 37℃ with a shaking speed of 230 rpm for 8 h.

[0047] (7) 2 mL of the cultured bacterial liquid was taken into a DNase-free centrifuge tube, and total RNA was extracted using the Trizol method.

[0048] (8) The extracted RNA was subjected to agarose gel electrophoresis, and whether the size of the double-stranded RNA band was consistent with the expectation was determined according to the electrophoresis results. As Figure 1 shown, the electrophoresis band of the double-stranded RNA molecule prepared in this invention was clear, and the size was the expected target size.

[0049] Sequencing showed that for the obtained double-stranded RNA molecules, the sense strand sequence of the double-stranded RNA molecule dsACET1-2 is as shown in Sequence 1 of the sequence list, and its antisense strand is as shown in Sequence 2 of the sequence list; the sense strand sequence of the double-stranded RNA molecule dsDND1-1 is as shown in Sequence 3 of the sequence list, and its antisense strand is as shown in Sequence 4 of the sequence list; the sense strand sequence of the double-stranded RNA molecule dsMLO1-3 is as shown in Sequence 5 of the sequence list, and its antisense strand is as shown in Sequence 6 of the sequence list.

[0050] Example 3. Evaluation of the disease resistance of plants treated with the double-stranded RNA molecules involved in the present invention against Botrytis cinerea

[0051] In this patent, the double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 were directly sprayed onto the surface of host plants (tomato leaves, variety: Cherry Tomato from Taiwan Province, China, produced by Qianrui Seed Industry). One day later, mycelial blocks of Botrytis cinerea (5 mm) were inoculated. Four days later, the diameter of the leaf lesions was observed and measured. According to this method, the effect of double-stranded RNA in enhancing the resistance level of plants against Botrytis cinerea was clarified.

[0052] The Botrytis cinerea strain B05.10 (Staats, Martijn, and Jan AL van Kan. "Genome update of Botrytis cinerea strains B05.10 and T4." (2012): 1413-1414. Preserved by the Laboratory of Fungicide Pharmacology and Pathogen Drug Resistance of China Agricultural University, and identified as Botrytis cinerea through morphological and molecular biological identification. The public can obtain it from China Agricultural University), and the preparation of mycelial blocks was as described in Example 3.

[0053] Crude extraction of double-stranded RNA: According to the induction conditions described in Example 2, the target dsRNA was induced for prokaryotic expression in Escherichia coli in a 100 mL system. After induction, the bacterial solution was centrifuged at 4000 rpm for 30 min at 4 °C to collect the bacterial pellet. The pellet was resuspended with 50 mL of TE buffer, and the OD600 of the resuspended bacterial solution was adjusted to about 1.5. The bacterial solution was disrupted with a high-pressure cell disruptor at a pressure of 0.5 kPa for 2.5 min. The disrupted bacterial solution should be clear, transparent, and have strong fluidity. The concentration of the crude dsRNA extract was measured with an ultra-micro spectrophotometer, and its concentration was adjusted to about 200 ng / μL with TE buffer. 10 μL of the disrupted bacterial solution was subjected to 1% agarose gel electrophoresis, and it was observed that the size of the target dsRNA band was consistent with the expectation, indicating successful preparation of the crude dsRNA extract.

[0054] The double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 were directly sprayed onto the surface of the leaves of the host plant (the third true leaf of tomato, both the front and back sides of the leaf were sprayed), and after 1 day, a mycelial block of Botrytis cinerea was inoculated onto the back side of the leaf, and after 4 days, the diameter of the leaf lesions was observed and measured;

[0055] Collect 4-week-old tomato leaves with similar growth status, and use a 20 mL sprayer sterilized by high temperature and high pressure to spray 5 mL of the double-stranded RNA crude extract onto the front side of the leaves. After the leaves are naturally dried, place them in a humidified petri dish. Each double-stranded RNA crude extract is used to treat 10 leaves. 1 day after the double-stranded RNA is sprayed, inoculate a Botrytis cinerea mycelial block with a diameter of 5 mm onto the front side of the leaf and then place it in a humidified petri dish. After 4 days, the diameter of the lesions is measured by the cross-cross method. The activity of the double-stranded RNA in enhancing the resistance level of plants to Botrytis cinerea is evaluated according to the average diameter of the lesions on the detached leaves.

[0056] As Figure 2 shown, compared with the control, the lesions formed by the infection of Botrytis cinerea on the leaves sprayed with the double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 targeting the susceptible genes of tomato were significantly reduced. The statistical results are shown in Figure 3 , the resistance level of plants to Botrytis cinerea after treatment with the double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 involved in the present invention increased significantly, indicating that the double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 of the present invention can be used for the prevention and control of plant diseases caused by Botrytis cinerea.

[0057] Example 4. Determination of the control effect of plants treated with the double-stranded RNA molecules involved in the present invention against Botrytis cinerea

[0058] Preparation method of Botrytis cinerea conidia suspension: The Botrytis cinerea strain B05.10 was cultured in the dark at 18 °C on a carrot medium plate for about 3 - 5 days, placed under a black light for irradiation for 5 - 7 days, the conidia were washed off with sterilized distilled water, the mycelium was filtered through two layers of gauze, and the spore concentration was adjusted to 2×10 6 per mL with a hemocytometer. 1.5% glucose and 0.5% Tween-20 were added to the spore suspension to improve the success rate of inoculation.

[0059] Plant tomatoes in the greenhouse. When the tomato seedlings grow for 1 month, directly spray 3 mL of the double-stranded RNA molecule crude extract obtained by induction onto the tomato seedlings. Each treatment has 10 tomato seedlings, and each treatment is repeated biologically 3 times. Spray each treatment evenly on both the front and back sides of the leaves until water drips. 1 day later, use the Botrytis cinerea conidia suspension (2×10 6Inoculate at a concentration of [[ID=]], until the leaves are dripping. After the leaf surface dries, keep it moist with a plastic bag, control the temperature at 20 °C, and the relative humidity at 90%. Investigate the disease condition on the 5th day after inoculation. According to the guidelines for field efficacy trials (Biological Assay Laboratory, Institute for the Control of Agrochemicals, Ministry of Agriculture and Rural Affairs of the People's Republic of China, 2000), investigate the disease incidence. Calculate the incidence rate, disease index, and control effect with the leaves as the unit.

[0060] Disease grading standard:

[0061] Grade 0: No disease spots;

[0062] Grade 1: There are 3 disease spots on a single leaf;

[0063] Grade 3: There are 4 - 6 disease spots on a single leaf;

[0064] Grade 5: There are 7 - 10 disease spots on a single leaf;

[0065] Grade 7: There are 11 - 20 disease spots on a single leaf, and some are densely clustered;

[0066] Grade 9: The disease spots on a single leaf are densely clustered and occupy more than 1 / 4 of the leaf area.

[0067] Incidence rate (%) = Number of diseased leaves / Total number of investigated leaves × 100

[0068] Disease index = ∑(Number of diseased leaves at each grade × Representative value of each grade) / (Total number of investigated leaves × Representative value of the most severely diseased grade) × 100

[0069] Control effect (%) = (Disease index of the control group - Disease index of the treatment group) / Disease index of the control group × 100

[0070] As shown in Table 2 and Figure 4 After spraying the double-stranded RNA molecules dsACET1-2, dsDND1-1, or dsMLO1-3 of the present invention, the control effect on Botrytis cinerea is more than 55.3%. The above results indicate that the double-stranded RNA molecules dsACET1-2, dsDND1-1, or dsMLO1-3 of the present invention can be used as a new type of green and safe pesticide for the prevention and control of plant diseases caused by Botrytis cinerea.

[0071] Table 2. Statistical results of greenhouse control efficacy tests of double-stranded RNA molecules related to the present invention against Botrytis cinerea

[0072]

[0073] Example 5. Determination of the expression level of target genes in plants after applying the double-stranded RNA molecules related to the present invention

[0074] Collect tomato leaves 1 day after treatment with the crude dsRNA extract, extract the total RNA of the sample using a kit, and reverse transcribe it into cDNA using a reverse transcription kit. Determine the changes in the expression levels of the corresponding target genes after dsRNA treatment by qRT-PCR. SlACET1 , SlMLO1 and SlDND1 . Take the tomato Efla gene as the internal reference gene, normalize the gene expression levels of each measured sample Efla , and use the 2 -△△Ct method for statistical analysis. Since dsRNA contains partial target gene sequences, the target gene sequences in the SIGS mediator should be avoided during the determination of target gene expression levels. In this experiment, primers for determining the expression levels of target genes were designed for different SIGS mediators, and the primer sequences are shown in Table 3.

[0075] Table 3. Primers used for determining the effect of the double-stranded RNA molecule involved in this invention on the expression level of the target gene

[0076]

[0077] The experimental results are as Figure 5 shown. Compared with the control dsRNA dsGFP, the dsRNAs targeting three tomato genes can significantly silence the corresponding target genes, and the gene silencing efficiency can reach over 50%.

Claims

1. Targeted silencing of tomato endogenous genes SlMLO1 A double-stranded RNA molecule, characterized in that The sequence of one chain is shown as SEQ ID No. 5, and the sequence of the other chain is shown as SEQ ID No.

6.

2. The double-stranded RNA molecule according to claim 1 interferes with the negative regulatory gene of tomato endogenous immunity SlMLO1 Application in expression.

3. Use of the double-stranded RNA molecule according to claim 1 in preventing and treating tomato diseases caused by Botrytis cinerea.

4. A pesticide containing the double-stranded RNA molecule according to claim 1.

5. Use of the pesticide according to claim 4 in preventing and controlling tomato diseases caused by Botrytis cinerea.

6. A method for preventing and controlling tomato diseases caused by Botrytis cinerea using the double-stranded RNA molecule according to claim 1, characterized in that The solution or preparation containing the double-stranded RNA molecule according to claim 1 is directly sprayed on the surface of tomato tissue.

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