Targeted silencing of cucumber genes MLO8 Double-stranded RNA molecules and their applications

Abstract

The invention discloses a method for inhibiting cucumber gene MLO8 The double-stranded RNA molecule expressed and its application. The double-stranded RNA molecule of the present invention has a sequence of one chain as shown in SEQ ID No.1, and a sequence of the other chain as shown in SEQ ID No.2. The present invention also discloses the application of the double-stranded RNA molecule in preventing and controlling plant diseases caused by powdery mildew of the single cyst. The double-stranded RNA molecule of the present invention can be directly sprayed on cucumbers to enhance the resistance of plants to powdery mildew of the single cyst. At the same time, the double-stranded RNA involved in the present invention does not interfere with the growth and development of cucumbers, and using it to prepare biological pesticides can provide an effective way to prevent and control cucumber powdery mildew.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to a method for targeting and silencing endogenous powdery mildew susceptibility genes in cucumbers. MLO8 A double-stranded RNA molecule, and application of the molecule in preventing and controlling plant diseases caused by powdery mildew. Background Art

[0002] Powdery mildew (PM) is caused by Erysiphe graminis Sphaerotheca fuliginea ) is a worldwide disease caused by powdery mildew. Fungicides are one of the effective methods to prevent and control cucumber powdery mildew, but powdery mildew fungi reproduce quickly and have rich genetic variation. They are high-risk pathogens that are easily resistant to commonly used fungicides, resulting in ineffective control. Therefore, exploring new control methods for cucumber powdery mildew is the key to ensuring cucumber yield and quality.

[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 20-30bp. These small RNAs specifically recognize the target gene DNA or mRNA sequence that is complementary to it by mediating the gene silencing complex, causing mRNA cutting, translation inhibition or DNA methylation, and ultimately inhibiting the normal expression of genes (Fire, A. et al. 1998. Nature. 391 (6669): 806). RNAi technology has achieved a lot of results in recent years in plant disease prevention and 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 plant endogenous immune negative regulatory genes (such as disease-susceptibility genes) 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 the endogenous powdery mildew susceptibility gene of cucumber MLO8 With the target as the target, dsRNA that can stably improve the resistance of cucumber to powdery mildew 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 powdery mildew. Summary of the invention

[0006] The present invention aims to provide a powdery mildew susceptibility gene that can effectively inhibit endogenous cucumber MLO8 Expressed double-stranded RNA molecules.

[0007] The second object of the present invention is to provide the double-stranded RNA molecule for inhibiting the endogenous powdery mildew susceptibility gene of cucumber. MLO8 Application in expression.

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

[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 cucumber diseases caused by Erysiphe graminis.

[0011] The sixth object of the present invention is to provide a method for preventing and controlling cucumber diseases caused by Erysiphe graminis using the above double-stranded RNA molecules.

[0012] The technical solution for implementing the present invention is as follows:

[0013] A method for targeting and silencing endogenous powdery mildew susceptibility genes in cucumber MLO8 The double-stranded RNA molecule is characterized in that the double-stranded RNA molecule 1 consists of the nucleic acid sequences shown in SEQ ID No. 1 and SEQ ID No. 2.

[0014] The double-stranded RNA molecules inhibit the endogenous powdery mildew susceptibility gene in cucumber MLO8 Application in expression.

[0015] The double-stranded RNA molecule is used for preventing and controlling cucumber diseases caused by powdery mildew.

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

[0017] The application of pesticides containing the double-stranded RNA molecules in the prevention and treatment of cucumber diseases caused by Erysiphe graminis.

[0018] The method for preventing and controlling cucumber diseases caused by powdery mildew using the double-stranded RNA molecule comprises spraying a solution or preparation containing the double-stranded RNA molecule carrier directly on the surface of cucumber plants.

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

[0020] The method for using the double-stranded RNA molecule of the present invention is as follows: spraying a double-stranded RNA molecule aqueous solution of a suitable concentration (such as about 100 nM) on the surface of cucumber tissue can effectively inhibit the infection and spread of powdery mildew on cucumbers.

[0021] The advantages or beneficial effects of the present invention are:

[0022] (1) The double-stranded RNA molecules of the present invention are effective against endogenous powdery mildew susceptibility genes in cucumber MLO8 The expression of the gene showed an obvious inhibitory effect, which can effectively inhibit the infection of Erysiphe graminis on plants, providing a new and effective way to prevent and control plant diseases caused by Erysiphe graminis.

[0023] (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 Erysiphe graminis, 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 environmental pollution problems, and are beneficial to environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a gel electrophoresis diagram of the double-stranded RNA molecule involved in the present invention. Figure 1 Middle: The bands in the gel image from left to right are DNA Marker, control dsRNA dsGFP and the double-stranded RNA molecule of the present invention.

[0025] Figure 2 It is a gene distribution map of the double-stranded RNA molecules involved in the present invention and other dsRNAs targeting different sites of the same gene. Figure 2 Middle: Cucumbers are grouped based on similar lengths MLO8 ( CxMLO8 ) gene was divided into 4 fragments, and dsRNA was designed for each fragment. A total of 4 dsRNA molecules were obtained, which were numbered dsMLO8-1, dsMLO8-2, dsMLO8-3 and dsMLO8-4.

[0026] Figure 3 The invention discloses the protective effect of the double-stranded RNA molecule and other control treatments on cucumber against powdery mildew. Figure 3Middle: Spraying dsRNA crude extract on cucumber cotyledons 1 day later inoculated with spore suspension of Erysiphe sphaerocephala (10 7 / mL), and the disease index was determined and photographed according to the field efficacy test guidelines after 14 days, including water treatment, control dsRNA (dsGFP), the double-stranded RNA molecule of the present invention, and other dsRNAs targeting different sites of the same gene (dsMLO8-1, dsMLO8-3 and dsMLO8-4).

[0027] Figure 4 The double-stranded RNA molecule of the present invention is applied to cucumber plants. MLO8 Histogram of gene silencing efficiency. Figure 4 Middle: Relative expression of target genes corresponding to dsRNA in leaf tissues 1 day after spraying dsRNA crude extract on cucumber cotyledons. From left to right, they are water control, control dsRNA (dsGFP), dsMLO8-1, double-stranded RNA molecules dsMLO8-2, dsMLO8-3 and dsMLO8-4 of the present invention; after water treatment MLO8 The expression level of the target gene was normalized to 1 and the silencing of the target gene by the double-stranded RNA molecule was calculated. Note: * indicates a significant difference compared with the clear water control. P < 0.05. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1: The present invention relates to a gene for inhibiting endogenous powdery mildew susceptibility in cucumber MLO8 Source of expressed double-stranded RNA molecules

[0030] (1) The sequence of the double-stranded RNA molecule involved in the present invention is derived from cucumber ( Cucumis sativus ) MLO8 of cDNA. MLO8 The cDNA sequence is derived from the gene numbered LOC101212923 in the National Center for Biotechnology Information (NCBI) database.

[0031] (2) Select MLO8 The 520 base sequences numbered LOC101212923 from 401 to 920 in the cDNA sequence database (NCBI) are used as the sequences of the double-stranded RNA molecules involved in the present invention. The sense strand sequence of the double-stranded RNA is shown in Sequence 1 in the sequence list, and the antisense strand sequence is shown in Sequence 2 in the sequence list.

[0032] (3) The above sequences were compared with the genome sequences (NCBI) of common hosts of single-shell powdery mildew such as cucumber to detect the possibility of non-specific silencing of other plant genes. The comparison results showed that the maximum continuous matching between the sequences involved in the present invention and other gene sequences of non-target genes in plants was 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.

[0033] Example 2. Preparation of double-stranded RNA molecules of the present invention

[0034] (1) Double-stranded RNA expression plasmid L4440 contains two bidirectional T7 promoters, which can be transcribed into double-stranded RNA in RNaseⅢ-deficient Escherichia coli. The target fragment was amplified by PCR, and the primers are shown in Table 1. Using the cDNA of cucumber (variety: Xintai Mici) as a template, the amplified fragment was inserted between the two T7 promoters of L4440 plasmid by restriction digestion and ligation ( Sac II and XB I enzyme recognition site), and after verification by PCR and sequencing, a plasmid capable of expressing double-stranded RNA molecules was obtained.

[0035] Table 1 Primer sequences for constructing double-stranded RNA molecules

[0036]

[0037] (2) The plasmid capable of expressing double-stranded RNA molecules obtained in step (1) is transformed into RNase III-deficient Escherichia coli strain HT115 to induce the expression of dsRNA.

[0038] (3) A single colony of the double-stranded RNA molecule expression strain HT115 that was successfully verified by PCR and sequenced was selected, cultured overnight in LB liquid culture medium containing ampicillin, and then stored in a -80°C refrigerator.

[0039] (4) Streak the double-stranded RNA molecule expression strain on an LB plate with a resistance marker, dip a single colony into 5 mL of liquid LB medium, and shake incubate at 37°C at 200 rpm overnight.

[0040] (5) Take 500 µL of the culture medium and add it to 10 mL of LB liquid medium. Incubate the culture medium at 37°C at 230 rpm for about 2.5 h until the OD600 of the culture medium is between 0.5 and 0.8.

[0041] (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.

[0042] (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.

[0043] (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.

[0044] Sequencing showed that the obtained double-stranded RNA molecule had a sense strand sequence as shown in Sequence 1 in the sequence list, and an antisense strand sequence as shown in Sequence 2 in the sequence list.

[0045] Using non-cucumber genes GFP As a control, the control dsRNA can be used to minimize the impact of the control dsRNA on the expression of endogenous genes in cucumber. Consistent with the molecular method of the above-mentioned indicator dsMLO8-2, the GFPF (5'-TCCCCGCGGATGGGCAAGGGCGAGGAACT-3' (SEQ ID No. 11)) and GFPR (5'-GCGGGCCCTCAAGGCGCGCCTGCGG-3' (SEQ ID No. 12)) primer pair was used to generate the GFP from the plasmid pTOR::eGFP (gift from Zhang Xiuguo's research group at Shandong Agricultural University; Fu Li, Phytophthora capsici ( Phytophthora capsici ) 10 pectin lyase gene cloning and pcpel1 Functional study of the amplified e GFP The full-length gene (735 bp) was then inserted between two T7 promoters in the L4440 plasmid to express the control double-stranded RNA dsGFP.

[0046] In order to screen dsRNA with good control activity against cucumber powdery mildew, cucumber MLO8 The gene (full length 1683 bp) was divided into four segments of about 500 bp, and targeted cucumber fragments of similar length were prepared using the same method as dsMLO8-2. MLO8 dsRNAs at other loci of the gene (numbered dsMLO8-1, dsMLO8-3 and dsMLO8-4, respectively; Table 1 shows the primer information used to prepare these dsRNAs. Figure 2 The distribution of gene loci of different dsRNAs is shown in Figure 2). All dsRNAs were screened for high-efficiency disease prevention dsRNAs. MLO8 Compared with other dsRNAs targeting the same gene, the protective effect and gene silencing efficiency of dsMLO8-2 were significantly improved.

[0047] Example 3 Evaluation of the resistance of plants to Erysiphe graminis by applying the double-stranded RNA molecules of the present invention

[0048] The double-stranded RNA molecules were directly sprayed onto the host plant surface (cucumber cotyledons) and then inoculated with a spore suspension of Erysiphe sphaerocephala (10 7 The disease index was determined after 14 days of observation. This method was used to determine the effect of double-stranded RNA in enhancing the resistance of plants to Erysiphe graminis.

[0049] 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.

[0050] The powdery mildew isolates were collected from the Science Park of China Agricultural University (Haidian District, Beijing) and identified according to conventional methods. The preparation method of the spore suspension of the powdery mildew isolate was as follows: cucumber plants with severe disease under natural conditions were selected, powdery mildew fungi were collected from the leaves, fresh powdery mildew fungi spores were brushed into a 50 mL centrifuge tube filled with sterile water with a brush, and Tween 20 was added to the sterile water at 2% (V / V); the powdery mildew fungi conidia were counted under a hemocytometer, and the concentration of the spore suspension was calculated, and the concentration of the conidia suspension was adjusted to 10 7 Pieces / mL.

[0051] Cucumber seedlings were planted in a greenhouse. When the cucumber seedlings grew for one week, 3 mL of the induced double-stranded RNA molecule crude extract was directly sprayed on the cucumber cotyledons. At least 10 cucumber cotyledons were treated, and 3 biological replicates were performed for each treatment. After 1 day, a conidia suspension of powdery mildew (10 7The inoculation method was to evenly inoculate the spore suspension onto the cucumber cotyledons with a brush, and the cucumber plants were placed in an artificial climate box at 25°C, 16 h light, and 85% relative humidity. The disease was investigated on the 14th day after inoculation, and the disease was investigated according to the Guidelines for Field Efficacy Tests of Pesticides (I) Control of Cucumber Powdery Mildew with Fungicides, GB / T17980.30-2000. The incidence rate, disease index, and control effect were calculated on a leaf basis.

[0052] Disease classification standards:

[0053] Level 0, no lesions on leaves;

[0054] Level 1: White powdery spots are initially seen, and the leaf spots account for less than 10% of the entire leaf area;

[0055] Level 3: obvious lesions, thin spore piles, and lesion area accounts for 11%-25% of the entire leaf area;

[0056] Level 5: Yellowing of leaves, with lesions accounting for 26%-50% of the total leaf area;

[0057] Level 7: dense spore piles, severe yellowing of leaves, and lesion area accounting for 51%-75% of the entire leaf area;

[0058] Level 9: The leaves are severely yellowed or dry, and the area of ​​lesions accounts for more than 76% of the total leaf area.

[0059]

[0060]

[0061] like Figure 3 As shown in Table 2, the control effect of dsMLO8-2 on cucumber powdery mildew can reach 52.7%, which is significantly higher than that of the control dsGFP and the targeted cucumber MLO8 dsRNAs at other sites of the gene, therefore the dsRNA involved in the present invention can be used to prevent and control cucumber powdery mildew.

[0062] Table 2. The protective effect of dsMLO8-2 and other dsRNAs on cucumber powdery mildew

[0063]

[0064] Note: Different letters represent significant differences. P < 0.05.

[0065] At the same time, the effect of the dsRNA (dsMLO8-2) of the present invention on the growth and development of cucumber was determined. dsMLO8-2 was sprayed on cucumbers at the three-leaf stage, and clean water was used as a control. After 21 days, the plant height and stem diameter were measured, and the pollen germination rate and pollen tube length of cucumbers were determined.

[0066] Pollen germination determination method: Pick the male cucumber flowers that are in full bloom on the same day, and evenly scatter the pollen in a 15% sucrose liquid medium containing 100 mg / L boric acid. After incubation in an artificial climate box at 25°C and 80% relative humidity for 1.5 hours, observe under a microscope. The pollen germination is recorded based on the standard that the length of the pollen tube germination is greater than the diameter of the pollen grain. Three fields of view are randomly selected to calculate the pollen germination rate. The length of the pollen tube is measured using Image J software.

[0067] Plant height = length from cotyledon node to growing point.

[0068] Stem diameter = diameter 1 cm below the cotyledon node.

[0069] As shown in Table 3, after the dsRNA (dsMLO8-2) of the present invention was sprayed on cucumbers, various growth indicators of the cucumbers were consistent with those of the control group sprayed with water, proving that the dsMLO8-2 of the present invention did not interfere with the growth and development of cucumbers.

[0070] Table 3. Effects of dsMLO8-2 on cucumber growth and development indicators

[0071]

[0072] Note: The same letters represent no significant difference. P < 0.05.

[0073] Example 4: Determination of the expression level of target genes in plants after administration of the double-stranded RNA molecules of the present invention

[0074] The cucumber cotyledons treated with dsRNA crude extract for 1 day were collected, and their RNA was extracted using a kit and reverse transcribed into cDNA. The target gene was determined based on real-time fluorescence quantitative PCR. MLO8 The relative expression level of cucumber Efla The gene was used as the internal reference gene, and the samples were measured with Efla The gene expression levels were normalized and 2 -△△Ct The method was used for statistical analysis. Since dsRNA contains part of the target gene sequence, the target gene sequence in the SIGS mediator should be avoided when determining the target gene expression. The primer sequences are shown in Table 4.

[0075] Table 4. Primers used in determining the effect of the double-stranded RNA molecules of the present invention on the expression of target genes

[0076]

[0077] like Figure 4 As shown, after dsMLO8-2 was sprayed, cucumber MLO8 The gene expression was significantly downregulated compared with the water control, and the silencing efficiency of dsMLO8-2 on the target gene was higher than that of the targeted cucumber MLO8 The above results show that the dsRNA of the present invention can be used to effectively silence cucumber genes. MLO8 Gene.

Claims

1. A targeted silencing method for endogenous powdery mildew susceptibility genes in cucumber MLO8 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. Use of the double-stranded RNA molecule according to claim 1 in interfering with the expression of the endogenous powdery mildew susceptibility gene MLO8 in cucumber.

3. Use of the double-stranded RNA molecule according to claim 1 in preventing and treating cucumber diseases caused by Erysiphe graminis.

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 cucumber diseases caused by Erysiphe graminis.

6. A method for preventing and treating cucumber diseases caused by Erysiphe graminis using the double-stranded RNA molecule of 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 cucumber plants.

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