Double-stranded RNA molecule of targeted silence tomato endogenous gene 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 , SldNd1 or SlMLO1 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 ) is a necrotrophic pathogen that causes gray mold. It can infect a variety of crops including tomatoes and seriously threaten agricultural production safety. Chemical control is the main means of gray mold control, but currently Botrytis cinerea has developed resistance to a variety of agents, so it is urgent to explore new methods for gray mold control.

[0003] RNA interference (RNAi) is a gene silencing phenomenon induced by double-stranded RNA, which can inhibit gene expression by hindering the transcription or translation of specific genes. RNAi is ubiquitous in organisms. The double-stranded or hairpin RNA sequence formed by DNA transcription can be processed into small RNAs between 20-30 bp. These small RNAs specifically recognize the complementary target gene DNA or mRNA sequence by mediating the gene silencing complex, causing mRNA cleavage, translation inhibition or DNA methylation, and ultimately inhibiting the normal expression of the gene (Fire, A. et al. 1998. Nature. 391(6669): 806). In recent years, RNAi technology has achieved a lot of results in plant disease control. By directly spraying double-stranded RNA molecules, key genes of pathogens can be interfered with, resulting in a decrease in the fitness of pathogens and a reduction in pathogenicity, which is called SIGS (Spray-Induced Gene Silencing).

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

[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 interfering with the expression of plant disease-susceptible genes based on RNA silencing technology 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 Use of expression.

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

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

[0010] The 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] The 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 solution for implementing the present invention is as follows: Three methods for targeting and silencing 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, 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.

[0013] The double-stranded RNA molecules inhibit the endogenous gene 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 double-stranded RNA molecule.

[0016] The 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 molecule 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 of a suitable concentration (such as 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 involved in the present invention show obvious inhibitory effects on the expression of three plant immune negative regulatory genes, and can effectively inhibit the infection of Botrytis cinerea on plants, providing a new and effective way to prevent and control plant diseases caused by Botrytis cinerea. (2) The double-stranded RNA molecules involved in the present invention have strong specificity for targeted plant immune negative regulatory genes, and theoretically can specifically inhibit a variety of plant pathogens including Botrytis cinerea, and there is no problem of drug resistance. (3) The double-stranded RNA molecules involved in the present invention are safe for plants, humans and animals, and their use can effectively reduce the use of chemical pesticides, without environmental pollution problems, 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: The gel bands from left to right 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 for 1 day and then inoculated with Botrytis cinerea hyphae (5 mm). Four days later, the leaves became diseased (from left to right: control dsRNA, three double-stranded RNA molecules involved in the present invention).

[0026] Figure 3This is a bar graph showing the size of infection spots formed when tomatoes were inoculated with Botrytis cinerea after the double-stranded RNA molecules involved in the present invention were applied to the tomatoes.

[0027] Figure caption: Tomato leaves were sprayed with dsRNA crude extract for 1 day and then inoculated with Botrytis cinerea hyphae (5 mm). The diameter of lesions on diseased leaves 4 days later (from left to right are control dsRNA and three double-stranded RNA molecules involved in the present invention).

[0028] Figure 4 The invention relates to the protective effect of the double-stranded RNA molecule 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 situation of tomatoes was investigated according to the field efficacy test guidelines (Pesticide Inspection and Bioassay Laboratory of the Ministry of Agriculture and Rural Affairs, 2000) (from top to bottom, from left to right are water control, control dsRNA, and 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 it is applied to tomato plants.

[0031] Figure caption: Relative expression levels of target genes corresponding to dsRNA in leaf tissues 1 day after spraying dsRNA crude extract on tomato leaves (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 then 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 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) of the present invention is derived from SlACET1 The cDNA sequence of the double-stranded RNA molecule 2 (named dsDND1-1) was derived from SldNd1 The cDNA of the double-stranded RNA molecule 3 (named dsMLO1-3) was derived from SlMLO1 of 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 A total of 520 base sequences numbered Solyc12g100240 from the cDNA sequence database (SGN) are 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) are used as the sequence of the double-stranded RNA molecule 2 of the present invention; SlMLO1 The 520 base sequences numbered Solyc04g049090 No. 328-847 in the cDNA sequence database (SGN) of the invention are used as the sequence of the double-stranded RNA molecule 3 involved in the present invention. The sense strand sequence of the double-stranded RNA molecule 1 is shown in Sequence 1 in the sequence list, and its antisense strand is shown in Sequence 2 in the sequence list; the sense strand sequence of the double-stranded RNA molecule 2 is shown in Sequence 3 in the sequence list, and its antisense strand is shown in Sequence 4 in the sequence list; the sense strand sequence of the double-stranded RNA molecule 3 is shown in Sequence 5 in the sequence list, and its antisense strand is shown in Sequence 6 in the sequence list.

[0035] (3) The above sequences were compared with the genome 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 comparison results showed that the maximum continuous matches between the sequences involved in the present invention and other gene sequences of non-target genes in plants were less than 10 bases, and the prediction of siRNAs potentially generated by the sequences showed that the siRNAs generated by the above sequences could not reach the effective length of gene silencing, thus excluding the possibility of silencing non-target genes of host plants.

[0036] Example 2: Preparation of double-stranded RNA molecules of the present invention (1) Double-stranded RNA expression plasmid L4440 contains two bidirectional T7 promoters, which can be transcribed in RNaseⅢ-deficient Escherichia coli to generate double-stranded RNA. The target fragment was amplified by PCR, and the amplification primers are shown in Table 1. Using the cDNA of tomato (variety: Taiwan Shengnu, produced by Qianrui Seed) 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 restriction digestion and ligation. After verification by PCR and sequencing, a plasmid that can express double-stranded RNA molecules was obtained.

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

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

[0039] (3) 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 was successfully verified by PCR and sequenced was selected, cultured overnight in LB liquid medium containing ampicillin, and then stored 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-culture at 37°C and 200 rpm overnight.

[0041] (5) Take 500 μL of the culture solution and add it to 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 bacterial culture solution after shaking and place it in an enzyme-free centrifuge tube, and extract total RNA using the Trizol method.

[0044] (8) The extracted RNA is subjected to agarose gel electrophoresis, and the size of the double-stranded RNA band is determined based on the electrophoresis results to determine whether it meets expectations. 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 list, and its antisense chain is shown in sequence 2 in the sequence list; the sense chain sequence of the double-stranded RNA molecule dsDND1-1 is shown in sequence 3 in the sequence list, and its antisense chain is shown in sequence 4 in the sequence list; the sense chain sequence of the double-stranded RNA molecule dsMLO1-3 is shown in sequence 5 in the sequence list, and its antisense chain is shown in sequence 6 in the sequence list.

[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 sprays double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 directly onto the surface of the host plant (tomato leaves, variety: Taiwan Shengnu, produced by Qianrui Seed Industry) and then inoculates Botrytis cinerea hyphae (5 mm) 1 day later. The diameter of the leaf lesions is observed and measured 4 days later. This method is used to clarify the effect of double-stranded RNA in enhancing the resistance level of plants 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. It is preserved by the Laboratory of Fungicide Pharmacology and Pathogen Resistance of China Agricultural University, has been identified by morphology and molecular biology, and is confirmed to be Botrytis cinerea. The public can obtain it from China Agricultural University). The mycelial block is 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. Resuspend with 50 mL TE buffer and adjust the OD600 of the resuspended bacterial solution 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. The concentration of the dsRNA crude extract was measured with an ultra-micro spectrophotometer, and its concentration was adjusted to about 200 ng / μL with TE buffer. Take 10 μL of the disrupted bacterial solution for 1% agarose gel electrophoresis, and it was observed that the size of the target dsRNA band was as expected, that is, the dsRNA crude extract was successfully prepared.

[0049] The double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 were directly sprayed on the surface of the host plant leaves (the third true leaf of tomato, both the front and back of the leaf were sprayed), and then the hyphae of Botrytis cinerea were inoculated on the back of the leaf 1 day later. After 4 days, the diameter of the leaf lesions was observed and measured; Tomato leaves of similar growth status were collected for 4 weeks, and 5 mL of double-stranded RNA crude extract was sprayed on the front of the leaves using a 20 mL spray bottle sterilized with high temperature and high pressure. After the leaves were naturally dried, they were placed in a moisturizing culture dish. Ten leaves were treated with each dsRNA crude extract. One day after dsRNA spraying, a 5 mm diameter Botrytis cinerea mycelium block was inoculated on the front of the leaves and placed in a moisturizing culture dish. After 4 days, the diameter of the lesions was measured by the cross method. The activity of double-stranded RNA in enhancing the resistance level of plants to Botrytis cinerea was evaluated based on the average diameter of the lesions on the detached leaves.

[0050] like Figure 2 As shown in Figure 2, compared with the control, the lesions formed by Botrytis cinerea infection on leaves sprayed with double-stranded RNA molecules dsACET1-2, dsDND1-1 or dsMLO1-3 targeting tomato disease susceptibility genes 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 plant 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 for the prevention and treatment of plant diseases caused by Botrytis cinerea.

[0051] Example 4: Determination of the protective effect of plants treated with the double-stranded RNA molecules of the present invention against Botrytis cinerea Preparation method of Botrytis cinerea conidia suspension: Botrytis cinerea strain B05.10 was cultured in the dark at 18°C ​​on a carrot medium plate for about 3-5 days, then placed under a black light for 5-7 days, the conidia were washed with sterilized distilled water, the mycelium was filtered with two layers of gauze, and the conidia concentration was adjusted to 2×10 using a hemocytometer. 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 planted in a greenhouse. When the tomato seedlings were grown for one month, 3 mL of the induced double-stranded RNA molecule crude extract was directly sprayed on the tomato seedlings. Ten tomato seedlings were treated in each treatment, and three biological replicates were performed for each treatment. Each treatment was evenly sprayed on both sides of the leaves until dripping. After 1 day, a conidia suspension of Botrytis cinerea (2×10 6 / mL) and the dripping water on the leaves was used as the degree. After the leaf surface was dried, plastic bags were used to keep moisture, and the temperature was controlled at 20℃ and the relative humidity was 90%. The disease was investigated on the 5th day after inoculation. The disease situation was investigated according to the field efficacy test guidelines (Laboratory of the Pesticide Testing Institute of the Ministry of Agriculture and Rural Affairs, 2000), and the incidence rate, disease index and control effect were calculated based on the leaf.

[0053] Disease classification standards: Level 0: no lesions; Level 1: A single leaf has 3 lesions; Level 3: There are 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 investigated × 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 serious 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 protective efficacy of the double-stranded RNA molecules of the present invention against Botrytis cinerea

[0056] Example 5: Determination of the expression level of target genes in plants after administration of the double-stranded RNA molecules of the present invention Tomato leaves treated with dsRNA crude extract for 1 day were collected, total RNA of the samples was extracted using a kit, and cDNA was obtained by reverse transcription using a reverse transcription kit. The corresponding target genes after dsRNA treatment were determined by qRT-PCR. SlACET1 , SlMLO1 and SldNd1 The change of expression level. Efla The gene is the internal reference gene, and the Efla Gene expression levels were normalized and 2 -△△Ct The method was statistically analyzed. Since dsRNA contains part of the target gene sequence, the target gene sequence in the SIGS mediator should be avoided when measuring the target gene expression. In this experiment, primers for measuring the target gene expression were designed for different SIGS mediators. The primer sequences are shown in Table 3.

[0057] Table 3. Primers used in determining the effect of the double-stranded RNA molecules of the present invention on the expression of target genes

[0058] The test results are as follows Figure 5 As shown, compared with the control dsRNA dsGFP, the dsRNA 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. 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.

4. The double-stranded RNA molecule of claim 1 interferes with tomato endogenous immune negative regulatory genes SlACET1 Application in expression.

5. The double-stranded RNA molecule of claim 2 interferes with tomato endogenous immune negative regulatory genes SldNd1 Application in expression.

6. The double-stranded RNA molecule of claim 3 interferes with the negative regulation gene of tomato endogenous immunity SlMLO1 Application in expression.

7. Use of the double-stranded RNA molecule according to any one of claims 1 to 3 in preventing and controlling tomato diseases caused by Botrytis cinerea.

8. A pesticide comprising the double-stranded RNA molecule according to any one of claims 1 to 3.

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

10. A method for controlling tomato diseases caused by Botrytis cinerea using the double-stranded RNA molecule according to any one of claims 1 to 3, characterized in that The solution or preparation containing the double-stranded RNA molecule according to any one of claims 1 to 3 is directly sprayed on the surface of tomato tissue.

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