Target gene and dsRNA for preventing and treating tetranychus urticae and application thereof

Abstract

The invention relates to the technical field of RNA interference biopesticides, in particular to a target gene and dsRNA for preventing and treating tetranychus urticae and application of the target gene and dsRNA. The target gene is selected from at least one of nucleotide sequences of SEQ ID NO.1 to SEQ ID NO.7; the dsRNA molecule is composed of a positive-sense strand and a complementary antisense strand, wherein the positive-sense strand is selected from at least one of nucleotide sequences of SEQ ID NO.8-SEQ ID NO.14. The dsRNA molecule has a nucleotide sequence of SEQ ID NO.12-SEQ ID NO.14. Some gene loci having important influence on the growth and development of the tetranychus urticae in the genome of the tetranychus urticae serve as RNAi target genes, dsRNA molecules are designed for each target gene, and the dsRNA molecules can achieve efficient prevention and control of the tetranychus urticae, are good in sensitivity and high in insecticidal efficiency, have the advantages of being environmentally friendly and the like, and have good application prospects in prevention and control of the tetranychus urticae and control of the tetranychus urticae and control of the tetranychus urticae. Good application prospects are realized.

Description

Technical Field

[0001] This invention relates to the field of RNA interference biopesticide technology, and in particular to target genes, dsRNA, and their applications for the control of two-spotted spider mites. Background Technology

[0002] The two-spotted spider mite (Tetranychus urticae) is a member of the genus Tetranychus in the family Tetranychidae of the order Acari in the class Arachnidae. Also known as the two-spotted spider mite or white spider, it is a highly destructive agricultural pest with a wide host range, causing serious damage to many economic crops such as vegetables, fruit trees, and flowers. It uses its piercing-sucking mouthparts to pierce plant leaf tissue and suck cellular sap. Initially, numerous small yellowish-white spots appear on affected leaves. As the infestation worsens, these spots gradually enlarge and merge, causing the leaves to lose their chlorophyll and turn yellow. In severe cases, the leaves dry out and fall off. Currently, chemical control remains the primary method, but long-term use of chemical pesticides has led to increased resistance in the two-spotted spider mite, reducing control effectiveness and causing environmental pollution and excessive pesticide residues in agricultural products. Biological control has become a research hotspot due to its green, environmentally friendly, and sustainable advantages. However, existing biological control products have shortcomings such as unstable control effects and high costs. Therefore, developing a highly efficient, low-cost, and environmentally friendly biological agent for controlling the two-spotted spider mite is of great significance.

[0003] Currently, the control of two-spotted spider mites mainly relies on the spraying of chemical pesticides. However, the extensive use of chemical pesticides is not only harmful to humans and livestock and causes serious environmental pollution, but also poses a significant challenge to the control of two-spotted spider mites, as the mites have developed resistance to some commonly used chemical insecticides. Therefore, we propose that the control of two-spotted spider mites should follow the plant protection policy of "prevention first, integrated management," combining multiple control methods, utilizing synergists to reduce the dosage of chemical pesticides, strengthening the monitoring of pesticide resistance in two-spotted spider mites, and promptly reducing or prohibiting the use of insecticides in which highly resistant two-spotted spider mites have developed, in order to mitigate or delay the development of pesticide resistance in the mites.

[0004] RNA interference (RNAi) is a widespread gene expression regulation mechanism in organisms, triggered by double-stranded RNA (dsRNA). It blocks the expression of target genes through mRNA degradation, preventing the synthesis of corresponding proteins and ultimately leading to altered biological functions. RNAi technology is highly efficient, specific, and environmentally friendly, playing a crucial role in gene function research and signaling pathway analysis. Furthermore, it has shown great potential in agricultural pest control through direct application, transgenic plant expression, and delivery via microbial vectors.

[0005] Therefore, we propose the target gene, dsRNA, and its application for the control of two-spotted spider mites according to the present invention. Summary of the Invention

[0006] The purpose of this invention is to provide target genes, dsRNAs, and their applications for the control of two-spotted spider mites. Several gene loci in the two-spotted spider mite genome that significantly influence its growth and development are selected as RNAi target genes. Double-stranded RNAi molecules (dsRNAs) are designed for each target gene, and the lethality of the RNAi molecules against two-spotted spider mites is tested. The dsRNA molecules achieve highly efficient control of two-spotted spider mites, exhibiting good sensitivity, high insecticidal efficiency, and numerous advantages such as environmental friendliness, demonstrating promising application prospects.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A target gene for controlling two-spotted spider mites, characterized in that the target gene is selected from at least one of the nucleotide sequences SEQ ID NO.1 to SEQ ID NO.7.

[0009] A dsRNA molecule for controlling two-spotted spider mites, characterized in that the dsRNA molecule consists of a sense strand and a complementary antisense strand, wherein the sense strand is selected from at least one of the nucleotide sequences SEQ ID NO. 8 to SEQ ID NO. 14.

[0010] The method for preparing the dsRNA molecule for controlling two-spotted spider mites is characterized by comprising the following steps:

[0011] S1. Extract total RNA from two-spotted spider mites;

[0012] S2. Using the RNA from step S1 as a template, reverse transcription is performed to synthesize cDNA.

[0013] S3. Using the cDNA from step S2 as a template, design primer sequence pairs containing the T7 promoter sequence, and obtain the target gene fragment by PCR amplification.

[0014] S4. Purify the target gene fragment from step S3 using an adsorption column.

[0015] S5. Using the purified target gene fragment from step S4 as a template, dsRNA is transcribed.

[0016] S6. Purify the dsRNA synthesis product using the phenol-chloroform method.

[0017] Furthermore, in S3, the primer sequence pair includes at least one of the following:

[0018] SEQ ID NO.15 and SEQ ID NO.16;

[0019] SEQ ID NO.17 and SEQ ID NO.18;

[0020] SEQ ID NO.19 and SEQ ID NO.20;

[0021] SEQ ID NO.21 and SEQ ID NO.22;

[0022] SEQ ID NO.23 and SEQ ID NO.24;

[0023] SEQ ID NO.25 and SEQ ID NO.26;

[0024] SEQ ID NO.27 and SEQ ID NO.28.

[0025] Application of target genes with nucleotide sequences of any one of SEQ ID NO.1 to SEQ ID NO.7 in the control of two-spotted spider mites.

[0026] The following applications of dsRNA molecules consisting of a sense strand and a complementary antisense strand, wherein the nucleotide sequence of the sense strand is SEQ ID NO. 8 to SEQ ID NO. 14:

[0027] (1) Preventing and / or controlling two-spotted spider mite infestation;

[0028] (2) Prepare products for the prevention and / or treatment of two-spotted spider mite infestation;

[0029] (3) Promote the death of two-spotted spider mites, and / or, prepare products that promote the death of two-spotted spider mites.

[0030] This invention has at least the following beneficial effects:

[0031] This invention primarily addresses the problems of environmental pollution, increased pesticide resistance in pests, and the impact on non-target organisms encountered by traditional chemical pesticides in the control of two-spotted spider mites. By utilizing gene silencing technology, specifically designing and synthesizing dsRNA targeting genes related to growth, development, and metabolism within the pest, this invention provides a more environmentally friendly, efficient, and specific pest control method. The study focuses on the efficacy of dsRNA (double-stranded RNA) in the control of two-spotted spider mites, examining changes in the pest's phenotypic response and survival and reproductive capabilities after targeted silencing of this gene with dsRNA.

[0032] This invention synthesizes dsRNA for the control of two-spotted spider mites by targeting a specific gene. This dsRNA can silence the target gene. The dsRNA solution is sprayed to allow it to enter the body of the two-spotted spider mite. The dsRNA silences / inhibits the expression of the target gene, promoting the death of the two-spotted spider mite, thereby achieving the goal of controlling the two-spotted spider mite.

[0033] This invention screened and obtained a lethal gene for controlling the two-spotted spider mite, and developed its highly efficient silencing dsRNA, enabling highly effective control of the two-spotted spider mite. The control objective is achieved by utilizing the lethal effect of the dsRNA on the two-spotted spider mite. This method is convenient to operate, has good effectiveness and sensitivity, high insecticidal efficiency, and is environmentally friendly, showing great promise for application. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] Example 1: This example discloses some gene loci in the genome of the two-spotted spider mite that have an important impact on the growth and development of the two-spotted spider mite, as target genes for the prevention and control of the two-spotted spider mite.

[0036] The target genes are selected from SEQ ID NO.1 to SEQ ID NO.7;

[0037] The RNAi molecule for controlling two-spotted spider mites is a dsRNA molecule composed of a sense strand and a complementary antisense strand, wherein the nucleotide sequence of the sense strand is selected from SEQ ID NO.8 to SEQ ID NO.14.

[0038] Specifically, the table below lists the gene number, protein name, protein function, and the corresponding gene, dsRNA, and primer nucleotide sequences. It should be noted that the genes, dsRNA, and primer pairs in this invention all consist of a sense strand and an antisense strand.

[0039] Table 1

[0040]

[0041]

[0042] Example 2: This example uses the above-described method for preparing dsRNA.

[0043] 1. Total RNA extraction from Tetranychus two-spotted mites

[0044] Total RNA was extracted from the two-spotted spider mite using a Total RNA Extractor (Trizol) (Sangon Biotech (Shanghai) Co., Ltd.). The detailed procedure is as follows:

[0045] (1) Grind the frozen two-spotted spider mite sample into powder, add 1 ml of Trizol and shake thoroughly to mix well, then incubate at room temperature for 5 min to allow it to fully lyse.

[0046] (2) Add 200 μl of chloroform, shake to mix, incubate on ice for 10 min, centrifuge at 12000 rpm and 4℃ for 15 min;

[0047] (3) Transfer the supernatant to a new centrifuge tube, add 600 μl of isopropanol and mix thoroughly. Settle at -20℃ for 30 min, centrifuge at 12000 rpm and 4℃ for 10 min, and discard the supernatant.

[0048] (4) Add 1 ml of 75% ethanol to wash the precipitate and tube wall, centrifuge at 12000 rpm and 4℃ for 5 min, discard the supernatant, and repeat the washing twice. Centrifuge at 12000 rpm and 4℃ for 2 min, and aspirate the remaining supernatant.

[0049] (5) Allow the sediment to dry for 5-10 minutes;

[0050] (6) Add 30 μl ddH2O to dissolve the precipitate, determine the concentration using Nanodrop, and store at -80℃ for later use.

[0051] 2. Obtaining the cDNA library of the two-spotted spider mite

[0052] cDNA was synthesized using the MghtyScript first-strand cDNA synthesis Master Mix (genomic DNA removed) kit (Sangon Biotech (Shanghai) Co., Ltd.). Detailed operating steps are as follows:

[0053] (1) Removal of residual genomic DNA

[0054] Prepare the following mixture in an RNase-free centrifuge tube, mix thoroughly by pipetting, and incubate at 42°C for 5-15 minutes.

[0055] Components Usage template RNA 5μg 5×gDNAdigesterMix 3μl <![CDATA[RNaseFreeddH2O]]> to 15μl

[0056] (2) Preparation of reverse transcription reaction system

[0057] Components Usage The reaction solution in the first step 15μl 4×IIIM-MLVRTMix 5μl

[0058] (3) Perform reverse transcription reaction on a PCR instrument under the following conditions.

[0059] temperature time 25℃ 5min 55℃ 15min 85℃ 5min

[0060] After the reaction is complete, place the sample in a -20℃ refrigerator for later use.

[0061] 3. Primer design

[0062] Primers for dsRNA PCR amplification were designed using Primer Premier 5. Primer synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd., and the positive strand sequences of the primers are shown in Table 1. The primers were diluted to a working solution of 10 μM for later use.

[0063] It should be noted that the primer sequence pair consists of a sense strand and an antisense strand, and the nucleotide sequences of the sense strand and antisense strand of the primer sequence pair are selected from SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28.

[0064] 4. Preparation of gene CDS (target gene) sequences

[0065] Gene cloning was performed using 2×Taq Master Mix DNA polymerase (Sangon Biotech (Shanghai) Co., Ltd.), with a reaction volume of 50 μL. Specific ratios are shown in the table below:

[0066] Components Usage 2×TaqMasterMix 25μl <![CDATA[RNaseFreeddH2O]]> 22μl PrimerForward (10mM) 1μl PrimerReverse (10mM) 1μl template cDNA 1μl Total 50μl

[0067] 2. The PCR reaction procedure is as follows:

[0068]

[0069] 5. PCR product recovery

[0070] This experiment used the EZ-10 column PCR product purification kit from Sangon Biotech (Shanghai) Co., Ltd. The specific procedures are as follows:

[0071] (1) Take the adsorption column and the collection tube, add 200 μl of Buffer S, let stand for 1 min, centrifuge at 8000 rpm for 1 min, and discard the liquid in the collection tube;

[0072] (2) Transfer the PCR reaction solution to a 1.5 ml centrifuge tube and add 5 times the volume of Binding Buffer III;

[0073] (3) Transfer the above mixture to the adsorption column treated in step 1, let it stand at room temperature for 2 min, centrifuge at 8000 rpm for 1 min, and discard the liquid in the collection tube.

[0074] (4) Add 500 μl Wash Solution to the adsorption column, centrifuge at 10000 rpm for 1 min, discard the liquid in the collection tube, and put the adsorption column back into the original collection tube;

[0075] (5) Repeat step 4 once;

[0076] (6) Discard the liquid in the collection tube and centrifuge at 10,000 rpm for 1 min to remove residual Wash Solution;

[0077] (7) Place the adsorption column into a clean 1.5ml centrifuge tube, add 30μl ddH2O to the center of the adsorption membrane, incubate at 50℃ for 2min, and centrifuge at 10000rpm for 1min.

[0078] (8) The purified PCR product was stored at -20℃.

[0079] 6. dsRNA synthesis

[0080] This experiment used the Transcript Aid T7 High-Yield Transcription Kit (K0441) (Thermo Fisher Scientific) to synthesize dsRNA. The system was prepared as follows:

[0081]

[0082]

[0083] Mix thoroughly and incubate overnight at 37°C.

[0084] 7. dsRNA purification

[0085] The purification of dsRNA synthesis products using the phenol-chloroform method is as follows:

[0086] (1) Add 2 μl of DNase I to the dsRNA synthesis product, mix well and incubate at 37℃ for 15 min, then add 2 μl of 0.5 MEDTA and react for 10 min;

[0087] (2) Transfer the reaction solution to a 1.5 ml RNase-free centrifuge tube, add 115 μl RNase-free water, add 15 μl 3M sodium acetate (pH = 5.2), and mix thoroughly;

[0088] (3) Add 150 μl of phenol / chloroform mixture (volume ratio 1:1), then add 300 μl of chloroform, mix the reaction solution by inverting it, and centrifuge at 12000 rpm and 4℃ for 10 min.

[0089] (4) Take the supernatant into a new 1.5ml RNase-free centrifuge tube, add 350μl of ethanol, mix well, and let it settle at -20℃ for 1.5h;

[0090] (5) Centrifuge at 12000 rpm and 4℃ for 10 min;

[0091] (6) Discard the supernatant, add 500 μl of 70% ethanol to wash the dsRNA, centrifuge at 12000 rpm and 4℃ for 5 min;

[0092] (7) Discard the supernatant, let stand at room temperature for 20 min until the ethanol has completely evaporated, and add 50 μl RNase-free water to dissolve the precipitate;

[0093] (8) The concentration of dsRNA solution was determined by Nanodrop, and the concentration was adjusted to 100 ng / μl with RNase-free water. It was then stored at -80℃ for later use.

[0094] 8. Bioactivity test of two-spotted spider mite

[0095] Select fresh, uniformly sized, and flat bean sprout leaves, retaining one stem and one leaf, and place them in a hydroponic bottle filled with clean water. Each treatment was repeated three times. Peanut leaves with an appropriate number of adult mites were cut and placed on the back of the prepared bean sprout leaves. After standing for 24 hours, each leaf was inoculated with an average of 50-70 adult mites to ensure uniform mite density. Tween-20 was added to a 100ppm dsRNA aqueous solution to a final concentration of 0.01%. Then, using a precision handheld spray bottle, the solution was evenly sprayed onto the surface of the mite-inoculated bean sprout leaves, with a spray volume of 0.6±0.2mL per leaf, covering both sides evenly. The plants were cultured and observed under conditions of 25±3℃, 30-60%RH, and a photoperiod of 14L:10D. The mortality rate after 6 days was observed and recorded, and the corrected mortality rate was calculated. The results are shown in Table 2.

[0096] Table 2

[0097] dsRNA sequence number SEQ ID NO. mortality rate% 8 82.6 9 84.8 10 83.1 11 71.1 12 89.0 13 88.0 14 64.3

[0098] In summary, this invention primarily addresses the problems of environmental pollution, increased pesticide resistance in pests, and the impact on non-target organisms encountered by traditional chemical pesticides in the control of two-spotted spider mites. By utilizing gene silencing technology, particularly the design and synthesis of dsRNA targeting genes related to growth, development, and metabolism within the pest, this invention provides a more environmentally friendly, efficient, and specific pest control method. The study focuses on the efficacy of dsRNA (double-stranded RNA) in controlling two-spotted spider mites, examining changes in the phenotypic response and survival / reproductive capacity of the pest after targeted silencing of this gene with dsRNA. This invention synthesizes dsRNA for controlling two-spotted spider mites by targeting the gene, and this dsRNA can target and silence the gene. By spraying a dsRNA solution, the dsRNA enters the two-spotted spider mite's body, silencing / inhibiting the expression of the target gene and promoting the death of the two-spotted spider mite, thereby achieving the goal of controlling the two-spotted spider mite. This invention screens and obtains a lethal gene for controlling two-spotted spider mites and develops a highly efficient dsRNA for its silencing, enabling highly effective control of the two-spotted spider mite. The lethal effect of dsRNA on the two-spotted spider mite achieves the control objective. This method is easy to operate, effective and sensitive, highly efficient in killing insects, and environmentally friendly, and has many other advantages, making it a promising application prospect.

[0099] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A target gene for controlling two-spotted spider mites, characterized in that, The target gene is selected from at least one of the nucleotide sequences SEQ ID NO.1 to SEQ ID NO.

7.

2. A dsRNA molecule for controlling two-spotted spider mites, characterized in that, The dsRNA molecule consists of a sense strand and a complementary antisense strand, wherein the sense strand is selected from at least one of the nucleotide sequences SEQ ID NO. 8 to SEQ ID NO.

14.

3. The method for preparing dsRNA molecules for controlling two-spotted spider mites as described in claim 2, characterized in that, Includes the following steps: S1. Extract total RNA from two-spotted spider mites; S2. Using the RNA from step S1 as a template, reverse transcription is performed to synthesize cDNA. S3. Using the cDNA from step S2 as a template, design primer sequence pairs containing the T7 promoter sequence, and obtain the target gene fragment by PCR amplification. S4. Purify the target gene fragment from step S3 using an adsorption column. S5. Using the purified target gene fragment from step S4 as a template, dsRNA is transcribed. S6. Purify the dsRNA synthesis product using the phenol-chloroform method.

4. The preparation method according to claim 3, characterized in that, In S3, the primer sequence pair includes at least one of the following: SEQ ID NO.15 and SEQ ID NO.16; SEQ ID NO.17 and SEQ ID NO.18; SEQ ID NO.19 and SEQ ID NO.20; SEQ ID NO.21 and SEQ ID NO.22; SEQ ID NO.23 and SEQ ID NO.24; SEQ ID NO.25 and SEQ ID NO.26; SEQ ID NO.27 and SEQ ID NO.

28.

5. Application of target genes with nucleotide sequences of any one of SEQ ID NO.1 to SEQ ID NO.7 in the control of two-spotted spider mites.

6. Any of the following applications of dsRNA molecules consisting of a sense strand and a complementary antisense strand, wherein the nucleotide sequence of the sense strand is SEQ ID NO. 8 to SEQ ID NO. 14: (1) Preventing and / or controlling two-spotted spider mite infestation; (2) Prepare products for the prevention and / or treatment of two-spotted spider mite infestation; (3) Promote the death of two-spotted spider mites, and / or, prepare products that promote the death of two-spotted spider mites.

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