Target gene of lasioderma serricorne and dsrna thereof in preventing and treating lasioderma serricorne

Abstract

The present application relates to a kind of Lasioderma serricorne target genes, selected from: Lasioderma serricorne LsCarE gene, Lasioderma serricorne LsCASK gene, Lasioderma serricorne LsMRP1 gene, the combination of Lasioderma serricorne LsCASK gene and Lasioderma serricorne LsMRP1 gene, the combination of Lasioderma serricorne LsCarE gene and Lasioderma serricorne LsCASK gene, the combination of Lasioderma serricorne LsCarE gene and Lasioderma serricorne LsMRP1 gene, the combination of Lasioderma serricorne LsCASK gene and Lasioderma serricorne LsMR1P gene and Lasioderma serricorne LsCASK gene, the nucleotide sequence of the described Lasioderma serricorne LsCarE gene is as shown in SEQ ID NO:1, the nucleotide sequence of the described Lasioderma serricorne LsCASK gene is as shown in SEQ ID NO:2, the nucleotide sequence of the described Lasioderma serricorne LsMRP1 gene is as shown in SEQ ID NO:3.The present application also provides corresponding dsRNA and the application of dsRNA in the prevention and treatment of Lasioderma serricorne.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and biopesticide technology, and in particular to the application of tobacco beetle target genes and their dsRNA in the control of tobacco beetle. Background Technology

[0002] The RNAi phenomenon was discovered in 1998 by Fire et al., whose research demonstrated that double-stranded RNA (dsRNA) can silence the expression of target genes, earning them the Nobel Prize in 2006. Since then, it has been widely used as a tool for gene function research, especially in plants and animals where genetic manipulation tools are not yet fully developed. In addition, it can also serve as a valuable tool for targeted pesticide and pharmaceutical development (Perrimon et al., 2010).

[0003] In the field of agricultural pest and disease control, two studies in 2002 found that silencing specific target genes could lead to abnormal insect development, embryonic malformation, and even death (Bettencourt et al., 2002; Bucher et al., 2002). This marked the beginning of the application of RNAi technology in entomological research and application. Two reports in 2007 confirmed that expressing insect dsRNA in transgenic plants could achieve insecticidal effects (Baum et al., 2007; Mao et al., 2007). These two studies provided strong evidence for the application of RNAi technology in pest control. Pesticides developed using this technology are called RNA biopesticides, also known as nucleic acid pesticides, RNA pesticides, or RNA interference agents. They are novel biopesticides developed based on RNA interference technology. Their core component is a polynucleotide that can specifically bind to the mRNA transcribed from the target gene in the target organism (Wang and Jin, 2017b; Wang et al., 2019; Hu et al., 2019). RNA biopesticides can specifically silence the expression of target genes, exhibiting high efficiency and specificity (Baum et al., 2007; Mao et al., 2007; Zhang et al., 2015). The principle involves using specific fragments of endogenous functional genes, synthesizing them in vitro, and then introducing them into the target species to inhibit gene expression, thereby hindering gene function and ultimately affecting the growth, development, and even death of the target species (Zhu and Palli, 2020). Due to its species-specificity, ease of target development, and easy degradation, it possesses most of the functions required for green pesticides, attracting the attention of numerous scientists and pesticide companies, and is hailed as the third revolution in pesticide production history. Currently, many international pesticide companies, such as Bayer-Monsanto, Dow AgroSciences, and Syngenta, are utilizing this technology, investing significant human and material resources in targeted insecticide research and development. Reportedly, corresponding products are already on the market or about to be launched (Head et al., 2017).

[0004] On June 15, 2017, the U.S. Environmental Protection Agency (EPA) approved MON87411, the world's first insect-resistant maize expressing insect dsRNA. Currently, most of the scientific questions surrounding dsRNA as a novel type of biopesticide have been largely resolved; the key to subsequent research and application lies in screening for effective insecticidal target genes.

[0005] However, there are many problems to be solved in the development of RNA biopesticides, such as the low efficiency of single targets; although the technology is highly precise, the difficulty in controlling the diversity of pest outbreaks in complex ecological environments hinders its widespread application.

[0006] Tobacco A [ Lasioderma serricorne [Fabricius] belongs to the family Crotalariae in the order Coleoptera. It is a global storage pest with a complex diet. It can damage grains, beans, and oilseeds in granaries; and especially in tobacco warehouses, it damages stored tobacco leaves, cigarettes, cigars, and other tobacco products. Its larvae can bore into tobacco leaves during storage, causing mold and resulting in serious losses to the tobacco industry. Because of its wide-ranging diet, the tobacco beetle can damage tobacco, tea, cereals, beans, dried dates, oilseeds, animal and plant specimens, cocoa beans, leather, and rattan and bamboo products, with tobacco products being the most severely affected. This insect particularly favors aging tobacco leaves and can enter the interior of cigarettes along with the processed tobacco, bore into the tobacco shreds, and even perforate the cigarette paper. The insect's carcasses and excrement contaminate tobacco leaves and tobacco products, seriously affecting the usability of tobacco leaves and the quality of cigarettes. Summary of the Invention

[0007] The main objective of this invention is to address the above-mentioned problems by providing a tobacco beetle target gene and its dsRNA that enhance the control effect of RNA biopesticides in the control of tobacco beetle.

[0008] To achieve the above objectives, a first aspect of the present invention provides a tobacco A target gene, characterized in that it is selected from: tobacco A LsCarE Genes, tobacco A LsCASK Genes, tobacco A LsMRP1 Genes, tobacco A LsCASK Genes and Tobacco A LsMRP1 Gene combination, tobacco A LsCarE Genes and Tobacco A LsCASK Gene combination, tobacco A LsCarE Genes and Tobacco A LsMRP1 Gene combination, tobacco A LsCASK Genes and Tobacco A LsMRP1 Genes and Tobacco A LsCASK The combination of genes, the tobacco A LsCarE The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 7. The tobacco A... LsCASK The nucleotide sequence of the gene is shown in SEQ ID NO: 2, and the amino acid sequence is shown in SEQ ID NO: 8. The tobacco A... LsMRP1The nucleotide sequence of the gene is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 9. SEQ ID NO: 1 to SEQ ID NO: 3 are the nucleotide sequences of the complete coding region (CDS) of the corresponding gene.

[0009] A second aspect of the invention provides the dsRNA of the tobacco A target gene.

[0010] Preferably, the dsRNA is one or more of a first dsRNA, a second dsRNA, and a third dsRNA, wherein the first dsRNA is tobacco methyl ether. LsCarE The second dsRNA of the gene is tobacco A. LsCASK The dsRNA of the gene, wherein the third dsRNA is tobacco A. LsMRP The dsRNA of the gene; or, the dsRNA is a fourth dsRNA, the fourth dsRNA being tobacco A. LsCarE Genes, tobacco A LsCASK Genes, tobacco A LsMRP Two or three dsRNAs in a gene are spliced ​​together in tandem to form dsRNA.

[0011] Better location, tobacco A LsCarE The nucleotide sequence of the dsRNA of the gene is shown in SEQ ID NO: 4, tobacco A. LsCASK The nucleotide sequence of the dsRNA of the gene is shown in SEQ ID NO: 5, tobacco A. LsMRP1 The nucleotide sequence of the dsRNA of the gene is shown in SEQ ID NO: 6. For cases where multiple genes jointly suppress multiple targets, tobacco A is used as an example. LsCarE Genes and LsCASK Taking genes as an example, SEQ ID NO:4 and SEQ ID NO:5 can be spliced ​​and tandem to obtain the targeted tobacco A. LsCarE Genes and LsCASK The dsRNA of the gene can also be tobacco A. LsCarE dsRNA of the gene and tobacco A LsCASK Both dsRNA pairing and gene pairing can simultaneously inhibit tobacco acetylcholine (TAC). LsCarE Genes and LsCASK Gene.

[0012] Preferably, each individual target gene fragment is amplified separately using PCR technology or spliced ​​target gene fragments are amplified using overlap PCR technology for the synthesis of corresponding dsRNA.

[0013] A third aspect of the invention provides the application of the dsRNA in the prevention and treatment of tobacco beta-associated venom.

[0014] Ideally, this is achieved through feeding.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects:

[0016] This invention screened effective insecticidal target genes against the harmful tobacco beetle and developed a multi-gene, multi-target inhibition technology using overlapping PCR. This technology can not only improve the lethality of a single species and reduce the amount of dsRNA used, but also be extended to simultaneously inhibit multiple genes of multiple target species, thereby realizing a strategy of controlling multiple species with a single application. Attached Figure Description

[0017] Figure 1 A to Figure 1 E represents the RNAi effect on mature tobacco beetle larvae in Example 1 of this invention. LsCASK , LsMRP1 , LsMRP2 , LsMRP3 , LsMRP4 Gene silencing efficiency analysis.

[0018] Figure 2 This refers to the effect of RNAi on the survival of tobacco A in Example 1 of the present invention, wherein... Figure 2 A is LsCarE The effect of RNAi on the survival of tobacco beta; Figure 2 B is LsCASK The effect of RNAi on the survival of tobacco beta; Figure 2 C is LsMRP1 The effect of RNAi on the survival of tobacco beta.

[0019] Figure 3 The figure shows the effect of different dsRNA compound feedings on the mortality rate of tobacco beetle in Example 2 of the present invention. "dsMRP" in the figure refers to dsLsMRP1.

[0020] Figure 4 A and Figure 4 B represents ds in Embodiment 2 of the present invention. LsCarE +ds LsCASK After compounding, the target gene LsCarE , LsCASK The results of RNAi efficiency analysis.

[0021] Figure 5 A and Figure 5 B represents ds in Embodiment 2 of the present invention. LsCarE +ds LsMRP1 After compounding, the target gene LsCarE ds LsMRP1 The RNAi efficiency analysis results are shown in the figure. "dsMRP" refers to dsLsMRP1, and "LsMRP" refers to "LsMRP1".

[0022] Figure 6 A and Figure 6 B represents ds in Embodiment 2 of the present invention. LsCASK +ds LsMRP1 After compounding, the target gene LsCASK ds LsMRP1 The RNAi efficiency analysis results are shown in the figure. "dsMRP" refers to dsLsMRP1, and "LsMRP" refers to "LsMRP1".

[0023] Figure 7 A, Figure 7 B and Figure 7 C represents ds in Embodiment 2 of the present invention. LsCarE +ds LsCASK +ds LsMRP1 After compounding, the target gene LsCarE , LsCASK ds LsMRP1 The RNAi efficiency analysis results are shown in the figure. "dsMRP" refers to dsLsMRP1, and "LsMRP" refers to "LsMRP1".

[0024] Figure 8 A to Figure 8 Figure D shows the effect of feeding dsRNA synthesized from different gene sequences on the mortality rate of tobacco A in Example 3 of this invention. In the figure, "MRP" refers to "LsMRP1".

[0025] Figure 9 A and Figure 9 B represents the embodiment 3 of the present invention. LsCarE-CASK After sequence splicing, dsRNA was synthesized and fed to tobacco A, targeting the gene. LsCarE , LsCASK The results of RNAi efficiency analysis are shown in the figure.

[0026] Figure 10 A and Figure 10 B represents the embodiment 3 of the present invention. LsCarE-MRP After sequence splicing, dsRNA was synthesized and fed to tobacco A, targeting the gene. LsCarE ds LsMRP1 The figure shows the RNAi efficiency analysis results. In the figure, "MRP" and "LsMRP" both refer to "LsMRP1".

[0027] Figure 11 A and Figure 11 B represents the embodiment 3 of the present invention. LsCASK-MRP After sequence splicing, dsRNA was synthesized and fed to tobacco A, targeting the gene. LsCASK ds LsMRP1The figure shows the RNAi efficiency analysis results. In the figure, "MRP" and "LsMRP" both refer to "LsMRP1".

[0028] Figure 12 A, Figure 12 B and Figure 12 C represents the embodiment 3 of the present invention. LsCarE-CASK-MRP1 After sequence splicing, dsRNA was synthesized and fed to tobacco A, targeting the gene. LsCarE , LsCASK ds LsMRP1 The figure shows the RNAi efficiency analysis results. In the figure, "MRP" and "LsMRP" both refer to "LsMRP1".

[0029] Figure 13 This is a schematic diagram of the splicing of the three sequences LsCarE, LsCASK, and LsMRP1 in Embodiment 3 of the present invention. Detailed Implementation

[0030] To provide a clearer understanding of the technical content of this invention, the following embodiments are provided in detail. However, it is important to note that these descriptions are merely for further illustrating the features and advantages of this invention, and not for limiting the scope of the claims.

[0031] This invention primarily relates to the application of RNA biopesticides developed using RNA interference technology in pest control. Unless otherwise specified, the reagents and methods involved in the examples are those commonly used in the art.

[0032] Example 1

[0033] Screening target genes and RNAi efficiency of single target genes

[0034] Using the BLAST tool, the seven pre-screened gene sequences were compared with the Tobacco A genome database for sequence similarity, identifying five that might be related to... LsCarE Candidate genes exhibiting functional interactions are: peripheral plasma membrane protein CASK isoform X5 and four multidrug resistance-associated proteins (multidrug resistance-associated protein 1, multidrug resistance-associated protein 2, probable multidrug resistance-associated protein lethal 3, and multidrug resistance-associated protein 4), which are abbreviated as: LsCASK , LsMRP1 , LsMRP2 , LsMRP3 , LsMRP4 .

[0035] RNAi efficiency analysis

[0036] Feeding with dsRNA to screen for key lethal genes in tobacco beetle

[0037] Third-instar larvae with relatively uniform growth and development were selected and placed in culture boxes of uniform size, with 30 tobacco beetle larvae per box. These larvae were pretreated by starvation for 15 hours to enhance their feeding activity. For the treatment group, 20 µL of crude dsRNA extract (1500 ng / µL) was evenly applied to the surface of 5 pieces of oatmeal (4 µL per piece), and after air-drying, they were placed in the culture box. Simultaneously, tobacco beetles fed with oatmeal coated with dsGFP (1500 ng / µL), which does not target any endogenous genes of the tobacco beetle, served as a control to eliminate non-specific interference. The experiment was conducted in triplicate. During the observation period, larval mortality was systematically recorded every 48 hours, and detailed phenotypic changes, including growth retardation, abnormal molting, and mortality, were recorded to comprehensively evaluate the RNAi interference effect.

[0038] Real-time quantitative PCR

[0039] After feeding tobacco beta bacteria with dsRNA for 7 consecutive days, samples were taken, RNA was extracted and reverse-engineered into cDNA, and then... LsRPL18 and LsEF1ɑ This gene serves as an internal reference. The expression level of the lethal gene in tobacco A was detected by qPCR after RNAi. The primers used are shown in Table 3 below, and the specific operating procedure is as follows:

[0040] (1) Total RNA was extracted according to the method for total RNA extraction, and three biological replicates were set up for each sample. Reverse transcription was performed according to the method in the reverse transcription reaction procedure.

[0041] Total RNA extraction:

[0042] The experiments were conducted using TRIzol® Reagent (Shanghai Polang Biotechnology Co., Ltd.) and strictly followed the instructions in the experimental manual (strictly low temperature and REase-free conditions):

[0043] Sample grinding: The sample, which was flash-frozen in liquid nitrogen, was thoroughly ground using a grinding column (soaked in DEPC and sterilized at high temperature before use), 1 mL of TRIzol was added, mixed well, and allowed to stand on ice for 5 min.

[0044] Chloroform extraction: Add 300 μL of chloroform, shake well to mix, and let stand on an ice box for 5 minutes (chloroform must be stored in a sealed, cool, and dark place, as chloroform easily generates highly toxic gas when exposed to light in the air. This step must be carried out in a fume hood).

[0045] Precipitation: The mixture from (2) was centrifuged at 4°C and 12,000 rpm for 20 min in a low-temperature high-speed centrifuge. After centrifugation, the mixture was divided into three phases (the lower layer was an organic phase such as phenol / chloroform, the middle layer was a white interface, and the upper layer was a colorless aqueous phase). All the RNA was dissolved in the upper aqueous phase. 500 μL of the upper aqueous phase was transferred to a new centrifuge tube, 330 μL of pre-cooled isopropanol was added, and the mixture was shaken and mixed. The sample was placed in a 4°C refrigerator for 10 min to allow the RNA to precipitate.

[0046] After removing the sample, centrifuge at 4℃ and 12000 rpm for 20 min, carefully discard the supernatant, and then centrifuge again for 30 s (4℃ and 12000 rpm).

[0047] Washing the precipitate: Rinse with 1000 μL of RNase-free 75% ethanol, gently shake to suspend the RNA precipitate and ensure that the salt ions in the RNA precipitate are fully dissolved, centrifuge at 12000 rpm for 10 min at 4 °C, discard the supernatant and rinse again with 75% ethanol.

[0048] Dissolving the precipitate: After centrifuging at 12000 rpm for 10 min at 4℃, discard the supernatant (you can centrifuge the centrifuge tube for 30 s and use a pipette to remove the residual liquid). Then dry the centrifuge tube at room temperature for 2-3 min, add an appropriate amount of Nuclease-free Water to dissolve, and you will get the total RNA sample.

[0049] Sample testing: The absorbance was measured using a spectrophotometer (Nanodrop), and the quality of the extracted total RNA was determined by 1% agarose gel electrophoresis. RNA samples that passed the test were stored at -80℃ for later use.

[0050] Reverse transcription reaction:

[0051] Use the ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO) kit and follow the instructions for experimental procedures. For first-time use, mix the 4xDN Master Mix and gDNA Remover in a 50:1 ratio.

[0052] RNA denaturation: 2 µg of RNA sample was denatured in a 65°C water bath for 5 min and immediately placed on ice to cool.

[0053] gDNA removal reaction: Prepare the reaction solution as shown in Table 1 on ice, gently mix the reaction solution, and incubate at 37°C for 5 min.

[0054] ;

[0055] The reverse transcription reaction is shown in Table 2 below:

[0056] ;

[0057] After gently mixing the prepared reaction solution, proceed with the reverse transcription reaction according to the procedure in Table 3 below.

[0058] ;

[0059] After the reaction was completed, the sample was diluted 10 times and stored in a -20°C refrigerator for later use.

[0060] (2) Dilute the prepared tobacco A cDNA template 4 times and mix the samples according to the amplification system in Table 4 below (the entire operation is carried out on ice, and the mixture is gently mixed to avoid generating air bubbles; because the fluorescent dye needs to be protected from light, the PCR 96-well plate or eight-tube strip used is for the purpose of fluorescence quantitative experiment):

[0061] (3) Real-time PCR reaction was performed using a two-step PCR procedure. The reaction extent is shown in Table 5 below.

[0062] ;

[0063] ;

[0064] (4) After the reaction is complete, the primer quality is determined based on the amplification curve and melting curve of Real Time PCR.

[0065] (5) To eliminate individual differences, each experimental group consisted of 5 larvae that survived the treatment; each gene sample was tested 3 times.

[0066] dsRNA synthesis

[0067] dsRNA was synthesized using the T7 RNA Transcription Kit (ZS-SJ001) (Shanghai Zhisheng Yougu Biotechnology Co., Ltd.), and the specific operations were performed according to the instructions.

[0068] Remove the required reagents and place them on ice to thaw naturally, and prepare the reaction system as shown in Table 6 below.

[0069] ;

[0070] Gently mix the reaction solution and incubate at 37°C for 3-5 hours, then incubate at 72°C for 10 minutes, and allow to cool naturally until annealed to form dsRNA.

[0071] Take 1 µl of the reaction product and use agarose gel electrophoresis to detect the synthesis of the transcription product.

[0072] dsRNA purification

[0073] (1) Vortex or invert the Bind MagBeads several times to mix thoroughly. Add an equal amount of Bind MagBeads to the sample to be purified, pipette until thoroughly mixed, and incubate at room temperature for 8 minutes.

[0074] (2) Place the sample on the magnetic rack and magnetically attract it for 5 minutes. After the magnetic beads are completely gathered, carefully remove the supernatant (the centrifuge tube is still placed on the magnetic rack).

[0075] (3) Keep the sample on the magnetic rack, rinse the magnetic beads with 80% ethanol, incubate at room temperature for 30s, and carefully remove the supernatant; repeat the previous step, rinse twice in total, and remove as much residual liquid as possible.

[0076] (4) Keep the sample on the magnetic rack and open the lid to dry the magnetic beads for 3-5 minutes at room temperature to allow the residual ethanol and other liquids to evaporate completely.

[0077] (5) Remove the sample from the magnetic rack, add 40~50µl of Nnclease-free Water, gently mix with a pipette, and incubate at room temperature for 3 min.

[0078] (6) Place the sample on the magnetic rack and wait for the magnetic beads to completely aggregate (about 2 minutes). Carefully aspirate the supernatant into a new centrifuge tube. This solution is the purified dsRNA sample.

[0079] dsRNA quality testing: The concentration and purity of dsRNA were determined using NanoDrop. dsRNA samples that passed the agarose gel electrophoresis test were diluted with Nnclease-free Water to 1500 ng / µl and stored at -80°C for later use.

[0080] Data Analysis

[0081] Relative expression level reference 2 -ΔΔCt Method: ΔCT value of treatment group = CT value of target gene - CT value of internal reference gene; ΔCT value of control group = CT value of target gene - CT value of internal reference gene; relative expression level = 2 - (ΔCT value of treatment group - ΔCT value of control group); significance analysis of differences between data was performed by T. Test, and the corresponding results were obtained by calculating the mean and the standard deviation (SD) between data. The analysis results were plotted using GraphPad Prism 8.

[0082] The results are as follows Figure 1 A to Figure 1As shown in E, compared with the control group fed dsGFP (fed dsGFP that cannot target any endogenous gene of tobacco beta), continuous feeding with three target gene dsRNAs ( LsCASK , LsMRP1 , LsMRP3 7 days later, tobacco A LsCASK , LsMRP1 , LsMRP3 The mRNA expression levels of these cells were significantly downregulated (P<0.05), while LsMRP2, LsMRP4 There was no significant change in expression levels.

[0083] Tobacco beetle larvae were fed with dsRNA of three genes and the control group dsGFP for 22 days (mortality was recorded every 2 days). The results are as follows: Figure 2 A to Figure 2 As shown in Figure C, tobacco beetle larvae fed dsGFP in the control group molted and pupated normally, while those fed dsGFP... LsCarE The mortality rate of tobacco beetle larvae was 84.4 ± 5.09% (P < 0.05), significantly higher than that of the control group; meanwhile, feeding ds LsCASK ds LsMRP1 The larvae of the tobacco beetle also died in large numbers. The mortality rates of the larvae fed for 22 days were 36.7±8.8% and 43.3±6.6%, respectively, significantly higher than the control group (P<0.05). Some of the surviving larvae failed to pupate normally, which was consistent with the mortality rate of the control group. LsCarE The processing results were consistent.

[0084] Example 2

[0085] Feeding the dsRNAs of the three target genes in pairs

[0086] The mortality rate of tobacco beetle was determined using the same procedure as in Example 1, except that the concentration of each dsDNA was 750 ng / µL.

[0087] like Figure 3 As shown in Figure A, compared to feeding with dsGFP, feeding with dsGFP alone at a concentration of 750 ng / µL significantly reduced the effectiveness of dsGFP. LsCarE Or 750 ng / µL ds LsCASK At that time, the mortality rate of tobacco-induced cancer was significantly higher (30.00±0%, 23.33±1.73%) (P<0.05). Meanwhile, the mortality rate of ds... LsCarE With ds LsCASK (750 ng / µL ds) LsCarE +750ng / µL ds LsCASK When the compound was fed to tobacco beetle, the mortality rate reached 53.33±1%, which was significantly different from the control group (P<0.05).

[0088] like Figure 3 As shown in B, compared with feeding dsGFP, feeding alone with dsGFP at a concentration of 750 ng / µL was significantly better. LsCarE Or 750 ng / µL ds LsMRP1 At that time, the mortality rate of tobacco A was significantly increased (30.00±0%, 28.89±2.08%) (P<0.05), and the ds LsCarE With ds LsMRP1 When tobacco beetles were fed a compound of 750 ng / µL + 750 ng / µL, the mortality rate reached 47.78 ± 4.16%, which was significantly different from the control group (P < 0.05).

[0089] like Figure 3 As shown in C, compared with feeding dsGFP, feeding 750 ng / µL dsGFP alone... LsCASK Or 750 ng / µLds LsMRP1 Compared with dsGFP, the mortality rate of tobacco beetle was significantly increased (23.33±1.73%, 28.89±2.08%) (P<0.05). LsCASK With ds LsMRP1 When tobacco beetles were fed a compound of 750 ng / µL + 750 ng / µL, the mortality rate reached 61.11 ± 1.53%, which was significantly different from the control group (P < 0.05).

[0090] Feeding with a combination of dsRNAs from three target genes

[0091] The mortality rate of tobacco beetle after compound feeding was detected using the same steps as in Example 1, except that the feeding concentration of each dsDNA was 500 ng / µL.

[0092] like Figure 3 As shown in Figure D, compared to feeding with dsGFP, feeding with 500 ng / µL dsGFP alone... LsCarE 500ng / µLds LsCASK Or 500 ng / µL ds LsMRP1 Compared with dsGFP, the mortality rate of tobacco beetles was significantly increased (28.28±1.53%, 23.33±1%, 27.78±4.04%) (P<0.05). LsCarE ds LsCASK ds LsMRP1 (500ng / µLds) LsCarE +500ng / µL ds LsCASK +500ng / µLds LsMRP When the compound was fed to tobacco beetle, the mortality rate reached 67.78±1.53%, which was significantly different from the control group (P<0.05).

[0093] The RNAi efficiency of the target gene was analyzed using the same method as in Example 1, and the expression level of the lethal gene in tobacco A was detected after RNAi.

[0094] like Figure 4 A and Figure 4 As shown in B, using ds LsCarE +ds LsCASK (Final concentration 1500 ng / µL) After being fed a mixture of tobacco beetles for 7 days, the tobacco beetles showed... LsCarE and LsCASK Gene expression levels were significantly downregulated, by 44.28% and 75.60% respectively compared with the control group dsGFP (P<0.05).

[0095] like Figure 5 A and Figure 5 As shown in B, using ds LsCarE +ds LsMRP1 (Final concentration 1500 ng / µL) After being fed a mixture of tobacco beetles for 7 days, the tobacco beetles showed... LsCarE , LsMRP Gene expression levels were significantly downregulated, by 43.52% and 56.65% respectively compared with the control group dsGFP (P<0.05).

[0096] like Figure 6 A and Figure 6 As shown in B, using ds LsCASK +ds LsMRP1 (Final concentration 1500 ng / µL) After being fed a mixture of tobacco beetles for 7 days, the tobacco beetles showed... LsCASK Gene expression levels were significantly downregulated, down by 75.83% compared to the control group dsGFP. LsMRP There was no significant difference in expression levels (P<0.05).

[0097] like Figure 7 A and Figure 7 As shown in B, using ds LsCarE +ds LsCASK + dsLsMRP1 (Final concentration 1500 ng / µL) After being fed a mixture of tobacco beetles for 7 days, the tobacco beetles showed... LsCarE , LsMRP1 Gene expression levels were significantly downregulated, by 97.63% and 59.12% respectively compared to the control group dsGFP. (This refers to the expression of tobacco A.) LsCASK There was no significant difference in expression levels (P<0.05).

[0098] Example 3

[0099] The sequences of the three target genes were spliced ​​together to synthesize dsRNA for feeding.

[0100] Sequence splicing between three target genes using overlap PCR technology

[0101] Primer Design: Design three pairs of primers. The first pair should have the R-terminal primer containing a 20-base sequence of another DNA fragment at its 3' end (CarE-CASK-R). The middle primer should have the F-terminal primer containing a 20-base sequence corresponding to the CarE fragment at its 5' end (CarE-CASK-F), and the R-terminal primer containing a 20-base sequence corresponding to the MRP fragment at its 3' end (CASK-MRP1-R). The third pair of primers should have the F-terminal containing a 20-base sequence corresponding to the CASK fragment at its 5' end (CASK-MRP1-F). Figure 13 Illustration. The primer sequences designed using overlap PCR technology in this embodiment are shown in Table 7 below.

[0102] ;

[0103] Amplifying the three DNA fragments separately: Using specially designed primers, the three DNA fragments were amplified separately by PCR, followed by gel recovery. High-fidelity enzymes were used for amplification to minimize errors. The amplification systems are shown in Tables 8 to 10 below.

[0104] ;

[0105] ;

[0106] ;

[0107] Mixing the three DNA fragments: The three DNA fragments obtained from the above amplification are mixed together. They will anneal to each other through overlapping regions to form hybrid chains. The amplification system is shown in Table 11 below.

[0108] ;

[0109] The PCR program is shown in Table 12 below, and 8 cycles are performed.

[0110] ;

[0111] Note: To improve the success rate, the annealing temperature and number of cycles in PCR can be adjusted. Generally, the annealing temperature should be lower than the Tm value of the overlapping region, and the number of cycles should be less than in conventional PCR to reduce nonspecific bands.

[0112] Overlap PCR: PCR amplification of the mixed DNA fragments was performed using CarE-F and MRP-R as external primers. For the first 8 cycles, no external primers were added; only template DNA and high-fidelity enzyme were used for primer extension. Afterward, external primers were added, and PCR amplification continued (30 cycles).

[0113] After amplification, the target fragment of the expected size was checked by gel electrophoresis and then sent to Shanghai Platinum Biotech Co., Ltd. for sequencing analysis.

[0114] After the sequencing verification was successful, the fragment was ligated into a plasmid, and dsRNA was synthesized using the T7 RNA Transcription Kit (ZS-SJ001) (Shanghai Zhisheng Yougu Biotechnology Co., Ltd.).

[0115] The impact of tobacco A mortality rate

[0116] The same steps as in Example 1 were used to detect the mortality rate of tobacco beetle after compound feeding.

[0117] like Figure 8 A to Figure 8 As shown in C, feed ds LsCarE-CASK ds LsCarE-MRP ds LsCASK-MRP After 21 days, the mortality rate of tobacco beetle was significantly higher than that of the control group fed with dsGFP (P<0.05), at 74.44±2.87%, 72.22±2.87% and 75.56±0.47%, respectively.

[0118] like Figure 8 As shown in D, feed ds LsCarE-CASK-MRP At the same time, the mortality rate of tobacco A was also significantly higher than that of the control group (P<0.05), reaching 71.11±1.25%.

[0119] These results indicate that RNAi treatment targeting carboxylesterase and its associated genes significantly increased the mortality rate of tobacco beta.

[0120] Effects of target gene expression levels

[0121] Using the same method as in Example 1, the expression level of lethal genes in Tobacco A after RNAi was detected.

[0122] like Figure 9 A and Figure 9 As shown in B, feed ds LsCarE-CASK 7 days after tobacco A, LsCarE , LsCASK Gene expression levels were significantly downregulated, by 45.77% and 88.68% respectively compared with the control group dsGFP (P<0.05).

[0123] like Figure 10 A and Figure 10 As shown in B, feed ds LsCarE-MRP 7 days after tobacco A, LsCarE , LsMRP Gene expression levels were significantly downregulated, down by 75.05% and 77.89% compared with the control group dsGFP (P<0.05).

[0124] like Figure 11 A and Figure 11 As shown in B, feed ds LsCASK-MRP 7 days after tobacco A, LsCASK, LsMRP1 Gene expression levels were significantly downregulated, by 82.11% and 69.39% compared to the control group dsGFP (P<0.05).

[0125] like Figure 12 A to Figure 12 As shown in C, feed ds LsCarE-CASK-MRP 7 days after tobacco A, LsCarE , LsMRP1, LsCASK Gene expression levels were significantly downregulated, by 37.51%, 85.32%, and 46.60% respectively compared with the control group dsGFP (P<0.05).

[0126] The above embodiments demonstrate a synergistic effect between the research gene and related genes of this invention. Through a multi-gene strategy, methods such as dsRNA co-occurrence or sequence splicing are used to improve RNAi inhibition efficiency and reduce production costs. Results show that feeding tobacco beetle (LsCarE, dsLsCASK, and dsLsMRP1) for 22 days after co-occurrence significantly reduced the mortality rate to 67.78 ± 1.53%, indicating a significant improvement in mortality after RNAi treatment of a single related gene. Furthermore, splicing LsCASK and LsMRP1 into dsRNA using overlap PCR technology resulted in a tobacco beetle mortality rate of 75.56 ± 0.47%. This demonstrates that using a multi-target approach for pest control can significantly improve control effectiveness, provides more potential targets for RNAi technology, and offers new insights into optimizing the application of RNAi in different species.

[0127] Therefore, the multi-target strategy can improve the control effect by selecting related genes in a single species, or select different targets in multiple species for tandem control, and control multiple diseases and pests at the same time with a single application. This invention focuses more on the scope of application of this method itself.

[0128] Therefore, this invention demonstrates that gene tandem technology can effectively improve the lethality of pests and reduce the dosage of dsRNA. This technology can address the problem of low lethality of single genes against pests, and can also achieve a strategy of interfering with multiple gene targets in a single production. This provides a basis for improving the control effect of RNA biopesticides, expanding the application scope of RNA biopesticides, and increasing the range of control efficacy without changing the advantages of this technology in precise control.

[0129] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, this specification should be considered illustrative rather than restrictive.

Claims

1. A dsRNA of a target gene of Lasioderma serricorne, characterized in that, The dsRNA is for the gene of the tobacco beetle LsCarE the gene of the tobacco beetle The dsRNA is for the gene of the tobacco beetle LsCASK the gene of the tobacco beetle The dsRNA is for the gene of the tobacco beetle LsMRP1 the gene of the tobacco beetle The dsRNA is for the corn rootworm LsCarE dsRNA for the corn rootworm LsCASK dsRNA for the corn rootworm The dsRNA is for the tobacco beetle LsCarE The dsRNA is for the tobacco beetle LsMRP1 The dsRNA is for the tobacco beetle The dsRNA is for the tobacco beetle LsCASK The dsRNA is for the tobacco beetle LsMRP1 The dsRNA is for the tobacco beetle The dsRNA is for the corn rootworm LsCASK dsRNA for the corn rootworm LsCarE dsRNA for the corn rootworm LsMRP1 dsRNA for the corn rootworm The dsRNA is for the corn rootworm LsCarE gene and the corn rootworm LsCASK dsRNA formed by the combination of the gene splicing strings The dsRNA is for the corn rootworm LsCarE gene and the corn rootworm LsMRP1 dsRNA formed by the combination of the gene splicing strings The dsRNA is for the Western corn rootworm LsCASK gene and the Western corn rootworm LsMRP1 dsRNA formed by the combination of the gene splicing string The dsRNA is for the corn rootworm LsCASK gene, corn rootworm LsCarE gene and corn rootworm LsMRP1 dsRNA formed by the combination of the gene splicing strings Lasioderma serricorne LsCarE The nucleotide sequence of the dsRNA of the gene is shown as SEQ ID NO: 4, Lasioderma serricorne LsCASK The nucleotide sequence of the dsRNA of the gene is shown as SEQ ID NO: 5, Lasioderma serricorne LsMRP1 The nucleotide sequence of the dsRNA of the gene is shown as SEQ ID NO:

6.

2. The dsRNA of claim 1, wherein, Each single target gene fragment is amplified separately by PCR technique or spliced target gene fragments are amplified by overlapping PCR technique for synthesizing corresponding dsRNA.

3. The dsRNA of claim 1 is used for preventing and treating Lasioderma serricorne.

4. Use according to claim 3, characterized in that, Through feeding.

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