Double-stranded RNA molecule of targeted silence tomato endogenous gene SlACET1 or SlDND1 and application of double-stranded RNA molecule

Abstract

The invention discloses a double-stranded RNA molecule of a targeted silence tomato endogenous gene and application of the double-stranded RNA molecule. The double-stranded RNA molecule disclosed by the invention is any one of the following items (1)-(3): (1) the sequence of one strand of the double-stranded RNA molecule is as shown in SEQ ID No.1, and the sequence of the other strand of the double-stranded RNA molecule is as shown in SEQ ID No.2; 2) the sequence of one chain is shown as SEQ ID No.3, and the sequence of the other chain is shown as SEQ ID No.4; and 3) 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. The double-stranded RNA molecule disclosed by the invention can be directly sprayed to tomatoes so as to enhance the resistance of plants to botrytis cinerea. Meanwhile, the double-stranded RNA provided by the invention does not interfere with the growth and development of tomatoes, and an effective way can be provided for preventing and treating tomato gray mold when the double-stranded RNA is used for preparing biopesticides.

Description

Technical Field

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

[0002] Botrytis cinerea ( Botrytis cinerea Botrytis cinerea is a necrotrophic pathogen that causes gray mold, infecting a wide range of crops, including tomatoes, and posing a serious threat to agricultural production. Chemical control is the primary method for gray mold prevention and treatment, but Botrytis cinerea has developed resistance to multiple pesticides, necessitating the exploration of new approaches to gray mold prevention and treatment.

[0003] RNA interference (RNAi) is a gene silencing phenomenon induced by double-stranded RNA, which can inhibit gene expression by blocking the transcription or translation of specific genes. RNAi is ubiquitous in organisms. Double-stranded or hairpin RNA sequences produced by DNA transcription can be processed into small RNAs (20-30 bp). These small RNAs mediate the specific recognition of complementary target gene DNA or mRNA sequences by the gene silencing complex, resulting in mRNA cleavage, translational inhibition, or DNA methylation, ultimately inhibiting normal gene expression (Fire, A. et al. 1998. Nature. 391(6669): 806). RNAi technology has achieved significant success in plant disease control in recent years. Directly spraying double-stranded RNA molecules can interfere with key genes in pathogens, resulting in a decrease in pathogen fitness and reduced virulence, a phenomenon known as SIGS (Spray-Induced Gene Silencing).

[0004] Currently, SIGS primarily uses double-stranded RNA (dsRNA) as a mediator, silencing key genes in pests across kingdoms by spraying them onto plants, thereby suppressing the development of pests and diseases. However, the small RNAs produced by plant cells processing dsRNA have problems with cross-kingdom transmission efficiency, potentially leading to low cross-kingdom gene silencing efficiency and ineffective pest and disease control. Therefore, using SIGS to directly silence endogenous negative immune regulatory genes in plants to enhance plant resistance to pests and diseases may be a more effective strategy. Currently, research on directly interfering with the expression of disease-related genes in plants to enhance pest resistance is lacking.

[0005] The present invention uses tomato endogenous immune negative regulatory gene SlACET1 、 SlDND1 and SlMLO1With the target as the target, dsRNA that can stably improve the resistance of tomatoes to gray mold was constructed and screened. After searching, no relevant literature was found on the use of RNA silencing technology to interfere with the expression of plant disease-susceptible genes to prevent and control plant diseases caused by Botrytis cinerea. Summary of the Invention

[0006] The present invention aims to provide three methods for effectively inhibiting endogenous genes in tomatoes. SlACET1 、 SlDND1 and SlMLO1 Expressed double-stranded RNA molecules.

[0007] The second object of the present invention is to provide the double-stranded RNA molecule for inhibiting tomato endogenous genes SlACET1 、 SlDND1 and SlMLO1 Expressive use.

[0008] The third object of the present invention is to provide the use of the double-stranded RNA molecule in preventing and treating plant diseases caused by Botrytis cinerea.

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

[0010] A fifth object of the present invention is to provide a pesticide containing the double-stranded RNA molecule for use in preventing and controlling plant diseases caused by Botrytis cinerea.

[0011] A sixth object of the present invention is to provide a method for preventing and controlling plant diseases caused by Botrytis cinerea using the double-stranded RNA molecule.

[0012] The technical solutions for implementing the present invention are as follows: Three methods can be used to silence endogenous immune negative regulatory genes in tomatoes SlACET1 、 SlDND1 and SlMLO1 The double-stranded RNA molecules are numbered as double-stranded RNA molecules 1 / 2 / 3, respectively, and are 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; and double-stranded RNA molecule 3 is composed of the nucleic acid sequences shown in SEQ ID No. 5 and SEQ ID No. 6.

[0013] The double-stranded RNA molecules inhibit the endogenous genes of tomato SlACET1 、 SlDND1 and SlMLO1 Application of expressive use.

[0014] Application of the double-stranded RNA molecule in preventing and controlling plant diseases caused by Botrytis cinerea.

[0015] A pesticide containing the above-mentioned double-stranded RNA molecule.

[0016] Application of pesticides containing the double-stranded RNA molecules in the prevention and treatment of plant diseases caused by Botrytis cinerea.

[0017] The method for preventing and controlling plant diseases caused by Botrytis cinerea by using the double-stranded RNA molecule comprises spraying a solution or preparation containing the double-stranded RNA molecule carrier directly on the surface of plant tissue.

[0018] The double-stranded RNA molecules involved in the present invention can be produced by prokaryotic expression.

[0019] The method for using the double-stranded RNA molecule of the present invention is as follows: spraying an aqueous solution of the double-stranded RNA molecule at a suitable concentration (eg, about 100 nM) on the surface of plant tissue can effectively inhibit the infection and spread of Botrytis cinerea on the plant.

[0020] The above-mentioned plant is preferably tomato.

[0021] The advantages or beneficial effects of the present invention are as follows: (1) The double-stranded RNA molecules of the present invention exhibit a significant inhibitory effect on the expression of three plant immune negative regulatory genes, can effectively inhibit the infection of Botrytis cinerea on plants, and provide a new and effective way to prevent and control plant diseases caused by Botrytis cinerea. (2) The double-stranded RNA molecules of the present invention have strong specificity for the targeted plant immune negative regulatory genes, and theoretically can specifically inhibit a variety of plant pathogens including Botrytis cinerea, without the problem of drug resistance. (3) The double-stranded RNA molecules of the present invention are safe for plants, humans and animals, and their use can effectively reduce the use of chemical pesticides, without the problem of environmental pollution, which is beneficial to environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention relates to a gel electrophoresis diagram of three double-stranded RNA molecules.

[0023] Figure caption: From left to right, the gel bands are Marker, double-stranded RNA molecules dsACET1-2, dsDND1-1 and dsMLO1-3.

[0024] Figure 2 The figure shows a photo of Botrytis cinerea infection after the double-stranded RNA molecule of the present invention was applied to tomatoes.

[0025] 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 leaves became diseased (from left to right: control dsRNA and three double-stranded RNA molecules of the present invention).

[0026] Figure 3This 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.

[0027] 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).

[0028] Figure 4 The present invention relates to the protective effect of double-stranded RNA molecules on Botrytis cinerea after being applied to tomato plants.

[0029] 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).

[0030] 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.

[0031] 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

[0032] 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.

[0033] Example 1: Sources of the double-stranded RNA molecules for inhibiting the expression of tomato immune negative regulatory genes according to the present invention (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 SlDND1 The cDNA, double-stranded RNA molecule 3 (designated dsMLO1-3) was derived from SlMLO1 cDNA. SlACET1 、 SlDND1 and SlMLO1 The cDNA sequences were derived from the genes numbered Solyc12g100240, Solyc02g088560, and Solyc04g049090 in the SolGenomics Network (SGN) database.

[0034] (2) Select SlACET1 The 520 base sequences numbered Solyc12g100240 from the cDNA sequence database (SGN) were used as the sequence of the double-stranded RNA molecule 1 of the present invention; SlDND1 The 550 base sequences numbered Solyc02g088560 from the cDNA sequence database (SGN) were used as the sequence of the double-stranded RNA molecule 2 of the present invention; SlMLO1 The 520 base sequences numbered Solyc04g049090, 328-847, in the cDNA sequence database (SGN) were used as the sequence of the 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 listing, and its antisense strand is shown as Sequence 2 in the sequence listing; the sense strand sequence of double-stranded RNA molecule 2 is shown as Sequence 3 in the sequence listing, and its antisense strand is shown as Sequence 4 in the sequence listing; the sense strand sequence of double-stranded RNA molecule 3 is shown as Sequence 5 in the sequence listing, and its antisense strand is shown as Sequence 6 in the sequence listing.

[0035] (3) The above sequences were compared with the genome sequences (NCBI) of common hosts of Botrytis cinerea, such as tomato and strawberry, to test their potential for nonspecific silencing of other plant genes. The comparison results showed that the maximum continuous matches between the sequences involved in the present invention and other gene sequences of non-target plant genes were less than 10 bases, and prediction of potential siRNAs produced by the sequences showed that the siRNAs produced by the above sequences were not long enough to achieve effective gene silencing, thus eliminating the possibility of silencing non-target genes in the host plant.

[0036] Example 2: Preparation of double-stranded RNA molecules of the present invention (1) The double-stranded RNA expression plasmid L4440 contains two bidirectional T7 promoters that can be transcribed into double-stranded RNA in RNase III-deficient Escherichia coli. The target fragment was amplified by PCR, and the amplification primers are shown in Table 1. Using tomato cDNA (variety: cherry tomatoes from Taiwan Province, 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 Sac II and Xba I enzyme recognition sites) by enzyme digestion and ligation. After verification by PCR and sequencing, a plasmid capable of expressing double-stranded RNA molecules was obtained.

[0037] Table 1 Primer sequences for constructing double-stranded RNA molecules

[0038] (2) The plasmid expressing double-stranded RNA molecule 1, double-stranded RNA molecule 2 or double-stranded RNA molecule 3 obtained in step (1) is transformed into RNase III-deficient Escherichia coli strain HT115 to induce expression of dsRNA.

[0039] (3) Select a single colony of the strain HT115 expressing double-stranded RNA molecule 1, double-stranded RNA molecule 2, or double-stranded RNA molecule 3 that has been successfully verified by PCR and sequenced, shake-culture it in LB liquid medium containing ampicillin overnight, and then store it in a -80°C refrigerator.

[0040] (4) Streak the expression strain of double-stranded RNA molecule 1, double-stranded RNA molecule 2, or double-stranded RNA molecule 3 on an LB plate with a resistance marker, dip a single colony into 5 mL of liquid LB medium, and shake at 37°C and 200 rpm overnight.

[0041] (5) Take 500 μL of the culture solution and shake it into 10 mL of LB liquid medium. Incubate the culture at 37°C and 230 rpm for about 2.5 h until the OD600 of the culture solution is between 0.5 and 0.8.

[0042] (6) Add IPTG to a final concentration of 2 mM to the bacterial solution and incubate at 37°C, 230 rpm, and shake for 8 h.

[0043] (7) Take 2 mL of the shake-cultured bacterial solution into an enzyme-free centrifuge tube and extract total RNA using the Trizol method.

[0044] (8) The extracted RNA was subjected to agarose gel electrophoresis, and the size of the double-stranded RNA band was determined to be consistent with the expected value based on the electrophoresis results. Figure 1 As shown, the double-stranded RNA molecules prepared by the present invention have clear electrophoresis bands and are of the expected size.

[0045] Sequencing showed that the obtained double-stranded RNA molecules, the sense chain sequence of the double-stranded RNA molecule dsACET1-2 is shown in sequence 1 in the sequence listing, and its antisense chain is shown in sequence 2 in the sequence listing; the sense chain sequence of the double-stranded RNA molecule dsDND1-1 is shown in sequence 3 in the sequence listing, and its antisense chain is shown in sequence 4 in the sequence listing; the sense chain sequence of the double-stranded RNA molecule dsMLO1-3 is shown in sequence 5 in the sequence listing, and its antisense chain is shown in sequence 6 in the sequence listing.

[0046] Example 3 Evaluation of the resistance of plants to Botrytis cinerea administered with the double-stranded RNA molecules of the present invention This patent investigates the effect of double-stranded RNA (dsACET1-2, dsDND1-1, or dsMLO1-3) on the surface of host plants (tomato leaves, cherry tomatoes from Taiwan, China, produced by Qianrui Seed Industry). One day later, the plants are inoculated with 5mm thick pieces of Botrytis cinerea mycelium. Four days later, the diameter of the leaf lesions is observed and measured. This method is used to determine the effectiveness of double-stranded RNA in enhancing plant resistance to Botrytis cinerea.

[0047] 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. Maintained by the Laboratory of Fungicide Pharmacology and Pathogen Resistance, China Agricultural University, identified morphologically and molecularly as Botrytis cinerea, and publicly available from China Agricultural University), mycelial blocks were prepared as described in Example 3.

[0048] Crude extraction of double-stranded RNA: According to the induction conditions described in Example 2, Escherichia coli was induced to express the target dsRNA in a 100 mL system. The induced bacterial solution was centrifuged at 4000 rpm for 30 min at 4°C to collect the bacterial pellet. Resuspended with 50 mL TE buffer, and the OD600 of the resuspended bacterial solution was adjusted to about 1.5. Use a high-pressure cell disruptor to disrupt the bacterial solution at a pressure of 0.5 kPa for 2.5 minutes. The disrupted bacterial solution should be clear and transparent with strong fluidity. Measure the concentration of the crude dsRNA extract with an ultra-micro spectrophotometer and adjust its concentration to about 200 ng / μL with TE buffer. Take 10 μL of the disrupted bacterial solution for 1% agarose gel electrophoresis, and observe that the size of the target dsRNA band is as expected, that is, the crude dsRNA extract is successfully prepared.

[0049] Double-stranded RNA molecules dsACET1-2, dsDND1-1, or dsMLO1-3 were sprayed directly onto the surface of host plant leaves (the third true leaf of tomato, both the front and back of the leaf were sprayed). One day later, Botrytis cinerea hyphae were inoculated onto the back of the leaf. Four days later, the leaf lesion diameter was observed and measured. Four-week-old tomato leaves with similar growth conditions were collected and 5 mL of crude double-stranded RNA extract was sprayed onto the leaf surface using a 20 mL spray bottle sterilized with high temperature and high pressure. The leaves were allowed to air dry and then placed in a moisture-retaining Petri dish. Ten leaves were treated with each dsRNA extract. One day after dsRNA spraying, 5 mm diameter Botrytis cinerea mycelia were inoculated onto the leaf surface and placed in a moisture-retaining Petri dish. Four days later, the diameter of the lesions was measured using the cross-hatch method. The average diameter of the lesions on the detached leaves was used to evaluate the activity of the double-stranded RNA in enhancing plant resistance to Botrytis cinerea.

[0050] like Figure 2 As shown in Figure 2, compared with the control, the lesions formed by Botrytis cinerea on leaves sprayed with double-stranded RNA molecules targeting tomato disease susceptibility genes dsACET1-2, dsDND1-1 or dsMLO1-3 were significantly reduced. Figure 3 After the leaves were treated with the double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 of the present invention, the resistance level of the plants to Botrytis cinerea was significantly increased, indicating that the double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 of the present invention can be used to prevent and control plant diseases caused by Botrytis cinerea.

[0051] Example 4: Determination of the efficacy of plants treated with the double-stranded RNA molecules of the present invention against Botrytis cinerea Preparation of Botrytis cinerea conidia suspension: Botrytis cinerea strain B05.10 was cultured on carrot culture plates at 18°C ​​in the dark for 3-5 days, then placed under a black light for 5-7 days. The conidia were washed with sterilized distilled water, the mycelia were filtered through two layers of gauze, and the conidia concentration was adjusted to 2 × 10 6 1.5% glucose and 0.5% Tween-20 were added to the spore suspension to increase the success rate of inoculation.

[0052] Tomatoes were grown in a greenhouse. When the seedlings were one month old, 3 mL of the crude double-stranded RNA extract was sprayed directly onto the seedlings. Ten seedlings were treated in each experiment, with three biological replicates per treatment. Each treatment was evenly sprayed on both sides of the leaves, until water dripped. One day later, a conidia suspension of Botrytis cinerea (2 × 10 6 Inoculate the leaves with water dripping from the leaves (1000 ng / mL). After the leaves are dry, place them in plastic bags to retain moisture. The temperature is maintained at 20°C and the relative humidity is 90%. Five days after inoculation, investigate the disease status according to the Field Efficacy Test Guidelines (Laboratory of the Pesticide Control Institute, Ministry of Agriculture and Rural Affairs of the People's Republic of China, 2000). Calculate the incidence rate, disease index, and control efficacy on a leaf-by-leaf basis.

[0053] Disease classification standards: Level 0: no lesions; Level 1: There are 3 lesions on a single leaf; Level 3: 4 to 6 lesions on a single leaf; Level 5: There are 7 to 10 lesions on a single leaf; Level 7: There are 11 to 20 lesions on a single leaf, some of which are densely packed; Level 9: The lesions on a single leaf are densely packed and occupy more than 1 / 4 of the leaf area.

[0054] Incidence rate (%) = number of diseased leaves / total number of leaves surveyed × 100 Disease index = ∑ (number of diseased leaves at each level × representative value of each level) / (total number of leaves surveyed × representative value of the most severe disease level) × 100 Control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100 As shown in Table 2 and Figure 4 As shown, spraying the double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 of the present invention showed a protective effect of more than 55.3% against Botrytis cinerea. 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 green and safe new pesticides for the prevention and treatment of plant diseases caused by Botrytis cinerea.

[0055] Table 2. Statistics of greenhouse control efficacy of the double-stranded RNA molecules of the present invention against Botrytis cinerea

[0056] Example 5: Determination of target gene expression in plants after administration of the double-stranded RNA molecules of the present invention Tomato leaves were collected after 1 day of treatment with dsRNA crude extract. Total RNA of the samples was extracted using a kit and reverse transcribed using a reverse transcription kit to obtain cDNA. The corresponding target genes after dsRNA treatment were determined by qRT-PCR. SlACET1 、 SlMLO1 and SlDND1 The changes in expression levels. Efla The gene is the internal reference gene, and the Efla Gene expression levels were normalized and 2 -△△Ct Statistical analysis was performed using the dsRNA method. Because dsRNA contains a portion of the target gene sequence, amplification of the target gene sequence in the SIGS mediator should be avoided when measuring target gene expression. Primers for measuring target gene expression were designed for different SIGS mediators in this experiment. The primer sequences are shown in Table 3.

[0057] Table 3. Primers used to measure the effect of the double-stranded RNA molecules on the expression of target genes.

[0058] The test results are as follows Figure 5 As shown, compared with the control dsRNA dsGFP, the dsRNAs targeting the three tomato genes can significantly silence the corresponding target genes, and the gene silencing efficiency can reach more than 50%.

Claims

1. Targeted silencing of tomato endogenous genes SlACET1 A double-stranded RNA molecule, characterized in that The sequence of one chain is shown as SEQ ID No. 1, and the sequence of the other chain is shown as SEQ ID No.

2.

2. Targeted silencing of tomato endogenous genes SlDND1 A double-stranded RNA molecule, characterized in that The sequence of one chain is shown as SEQ ID No. 3, and the sequence of the other chain is shown as SEQ ID No.

4.

3. The double-stranded RNA molecule according to claim 1 interferes with the negative regulatory gene of tomato endogenous immunity SlACET1 Application in expression.

4. The double-stranded RNA molecule according to claim 2 interferes with the negative regulatory gene of tomato endogenous immunity SlDND1 Application in expression.

5. Use of the double-stranded RNA molecule according to claim 1 or 2 in preventing and treating tomato diseases caused by Botrytis cinerea.

6. A pesticide containing the double-stranded RNA molecule according to claim 1 or 2.

7. Use of the pesticide according to claim 6 for preventing and controlling tomato diseases caused by Botrytis cinerea.

8. A method for preventing and controlling tomato diseases caused by Botrytis cinerea using the double-stranded RNA molecule according to claim 1 or 2, characterized in that The solution or preparation containing the double-stranded RNA molecule according to claim 1 or 2 is directly sprayed on the surface of tomato tissue.

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