A lethal gene SlCht10 from the beet armyworm and its application

Abstract

This invention discloses a lethal gene SlCht10 of the beet armyworm and its application, belonging to the field of biological control technology. This invention identifies the SlCht10 gene as an RNAi target, constructs a recombinant pET-28a(+) vector with two inversely complementary SlCht10 silencing fragments inserted, transforms it into RNase III-deficient BL21(DE3) to obtain recombinant engineered bacteria with a dsSlCht10 yield of no less than 14.82 μg / mL, prepares a formulation containing this dsRNA, SPc nanocarrier, and surfactant, and sprays the formulation on crop leaves to achieve green, efficient, and large-scale control of the beet armyworm. This effectively solves the problems of existing beet armyworm RNAi control methods, such as the lack of stable, efficient, and specific targets, and the high cost or low yield of dsRNA preparation, which hinders large-scale promotion.

Description

Technical Field

[0001] This invention relates to the application of the SlCht10 gene, recombinant engineered bacteria, RNAi preparations, and RNAi control methods for the beet armyworm, belonging to the field of biological control technology. Background Technology

[0002] The beet armyworm (Spodoptera litura), belonging to the family Noctuidae in the order Lepidoptera, is a major global agricultural pest. It is characterized by its high reproductive capacity, omnivorous diet, voracious feeding habits, and strong migratory ability. It is also highly adaptable to varying temperature and humidity levels, easily causing widespread crop damage. Currently, the mainstream control method for the beet armyworm is chemical control. However, long-term use of chemical pesticides can lead to pesticide resistance in pests, and also poses problems such as pesticide residues and environmental pollution. Therefore, environmentally friendly biological control technologies are an important direction for the development of beet armyworm control.

[0003] RNAi biocontrol technology is a novel pest control technology developed based on the phenomenon of RNA interference. It delivers double-stranded RNA (dsRNA) to pests that target their key functional genes, thereby specifically silencing the expression of the target genes and causing the pests to die. It has advantages such as high targeting specificity, safety of non-target organisms, and low environmental residue. It has been commercially applied in the fields of insect-resistant transgenic crops and pest control sprays, providing a feasible path for green pest control in agriculture.

[0004] Currently, research on RNAi control of the beet armyworm is still in the target screening stage, and no specific RNAi target gene with high lethality against the beet armyworm has been discovered. Furthermore, in terms of dsRNA preparation technology, existing technologies mainly include two routes: in vitro chemical synthesis and prokaryotic expression. In vitro chemical synthesis of dsRNA produces high-purity products, but the synthesis cost is high, making it difficult to meet the large-scale production needs of agricultural applications. In the prokaryotic expression route, the *E. coli* RNase III-deficient expression system is the preferred platform for prokaryotic dsRNA expression. The publicly available pET28-BL21(DE3) RNase III- system uses RNase III-deficient BL21(DE3) *E. coli* as the expression host and pET-28a(+) as the prokaryotic expression vector. Exogenous gene expression is induced by IPTG, and the highest dsRNA yield against target genes of other species is 4.23 μg / mL. Summary of the Invention

[0005] (a) Purpose of the invention

[0006] The purpose of this invention is to provide a highly lethal and specific RNAi target, the SlCht10 gene, for the beet armyworm, along with a matching high-yield, low-cost recombinant engineered bacteria for dsRNA preparation, a dsRNA preparation method, an RNAi agent, and a green control method for the beet armyworm, thereby achieving green, efficient, and large-scale control of the beet armyworm.

[0007] (II) Technical Solution

[0008] In a first aspect, the present invention provides the application of the SlCht10 gene in the control of beet armyworm, wherein the nucleotide sequence of the SlCht10 gene is shown in SEQ ID NO.1.

[0009] Secondly, the present invention provides an application of the SlCht10 gene as an RNAi target in the control of beet armyworm, characterized in that the nucleotide sequence of the SlCht10 gene is shown in SEQ ID NO.1.

[0010] Thirdly, the present invention provides a recombinant engineered bacterium for producing dsRNA targeting the SlCht10 gene of the beet armyworm, wherein the recombinant engineered bacterium is obtained by transforming a recombinant pET-28a(+) expression vector into RNase III-deficient BL21(DE3) Escherichia coli; the recombinant pET-28a(+) expression vector contains two inversely complementary SlCht10 gene silencing fragments; the nucleotide sequence of the SlCht10 gene is shown in SEQ ID NO.1.

[0011] Preferably, the two reverse complementary SlCht10 gene silencing fragments are 585bp and 555bp in length, respectively, and are inserted into the multiple cloning site of the recombinant pET-28a(+) expression vector via EcoRI / XbaI double restriction sites and XhoI / XbaI double restriction sites, respectively.

[0012] Fourthly, the present invention provides an RNAi formulation targeting the beet armyworm, comprising dsRNA targeting the SlCht10 gene of the beet armyworm, an SPc nanocarrier, and a surfactant, wherein the nucleotide sequence of the SlCht10 gene is shown in SEQ ID NO.1.

[0013] Preferably, the mass ratio of dsRNA to SPc nanocarrier is 1:1, and the mass fraction of surfactant is 0.5%.

[0014] Fifthly, the present invention provides a method for preparing dsRNA targeting the SlCht10 gene of the beet armyworm, comprising the following steps:

[0015] S1. The above-mentioned recombinant engineered bacteria were inoculated into LB medium containing kanamycin and cultured.

[0016] S2. Add IPTG inducer to bacterial culture to induce dsSlCht10 expression;

[0017] S3. After induction, the bacterial culture is inactivated, lysozyme is added to break the cell wall, and then nuclease is used to degrade the mixed nucleic acids and purify to obtain dsSlCht10. The nucleotide sequence of dsSlCht10 is shown in SEQ ID NO.2.

[0018] Preferably, in step S1, the bacterial culture is shaken at 37°C until the OD of the bacterial culture is reached. 600 The concentration of the added IPTG inducer was 0.4~0.6; in step S2, the final concentration of the added IPTG inducer was 1 mM, and the culture was induced at 37℃ for 4 h; in step S3, the bacterial culture was inactivated by incubating at 75℃ for 5 min, the final concentration of the added lysozyme was 1.3 mg / mL, and the culture was incubated at 37℃ for 30 min to complete the cell wall disruption.

[0019] Sixthly, the present invention provides a green control method for beet armyworm, wherein the RNAi preparation is uniformly sprayed onto the surface of crop leaves infested by beet armyworm.

[0020] (III) Beneficial Effects

[0021] 1. This invention identifies the SlCht10 gene of the beet armyworm as a specific and effective target for RNAi control. After the dsRNA targeting this gene enters the beet armyworm, it can mediate the RNAi mechanism to specifically silence the expression of the SlCht10 gene, blocking the synthesis of key proteins required for the normal growth and development of the beet armyworm, leading to the death of the beet armyworm. This overcomes the problems of pesticide resistance and pesticide residue pollution caused by the long-term use of current chemical control methods. Compared with traditional chemical control schemes, this invention achieves green and specific control of the beet armyworm. Experimental verification shows that the survival rate of beet armyworms decreased to 56% after 8 days of treatment with in vitro synthesized dsSlCht10.

[0022] 2. This invention constructs a recombinant pET-28a(+) expression vector containing two inversely complementary SlCht10 specific target fragments. Driven by the T7 promoter, the two inversely complementary fragments can be transcribed simultaneously and efficiently and automatically pair to form double-stranded dsSlCht10, avoiding degradation of single-stranded RNA by host nucleases. This solves the problems of low transcription efficiency and easy degradation of dsRNA in existing universal E. coli expression vectors. Compared with existing similar expression vectors, the transcription efficiency of dsSlCht10 is significantly improved, laying a structural foundation for high-yield expression of dsRNA.

[0023] 3. This invention obtains the recombinant engineered bacterium pET-28a(+)-Cht10-BL21(DE3)RNase III- by transforming the above-mentioned recombinant expression vector into RNase III-deficient BL21(DE3) Escherichia coli. The host-deficient RNase III cannot cleave double-stranded RNA, allowing the transcribed dsSlCht10 to accumulate in large quantities in the host cell. This solves the problems of extremely low dsRNA yield in existing E. coli expression systems and the high cost of in vitro chemical synthesis of dsRNA, which prevents large-scale production. Compared with the highest yield of 4.23 μg / mL of existing publicly disclosed similar expression systems, the dsSlCht10 yield of the recombinant engineered bacterium of this invention can reach 14.82 μg / mL, an increase of about 3.5 times. The production cost is significantly reduced compared with in vitro chemical synthesis, which can meet the needs of large-scale production.

[0024] 4. This invention uses crude extract of recombinant engineered bacteria dsSlCht10 to directly formulate SPc nanocarrier composite spraying formulation, omitting the fine purification step of dsRNA. The SPc nanocarrier can protect dsRNA from degradation by nucleases in the environment and assist dsRNA in penetrating the body wall and intestinal wall of Spodoptera litura to exert its effect. This overcomes the problems of high purification cost and low field delivery efficiency of existing RNAi pesticides, which make them difficult to promote and apply. Compared with formulations made using finely purified dsRNA, the application cost is further reduced. Greenhouse experiments have verified that after spraying this formulation for 8 days, the survival rate of Spodoptera litura decreased to 54%, the control efficiency reached 46%, and the effect was stable. Attached Figure Description

[0025] Figure 1 The SlCht10 gene clone sequence diagram, with the start codon and stop codon marked at the points.

[0026] Figure 2 The silencing effect of SlCht10 gene dsRNA on Spodoptera litura.

[0027] Figure 3 Survival rate of Spodoptera litura under dsSlCht10 treatment.

[0028] Figure 4 Morphological changes in the death of Spodoptera litura larvae after treatment with dsRNA-mediated interference target genes.

[0029] Figure 5 (A) PCR verification of recombinant vector pET-28(+)-Cht10; (B) Double enzyme digestion verification of recombinant vector pET-28(+)-Cht10; (C) Verification of expression of dsCht10 in pET-28(+)-Cht10-BL21(DE3)RNase ⅠⅠⅠ- expression system.

[0030] Figure 6 The engineered bacteria expressing dsSlCht10 had a silencing effect on the beet armyworm.

[0031] Figure 7 Based on the indoor control effect of engineered bacteria expressing dsSlCht10 on beet armyworm; (A) before treatment, (B) water treatment, (C) control effect of tobacco plants treated with engineered bacteria expressing dsSlCht10; (D) morphology of beet armyworm treated with water (left) and engineered bacteria expressing dsSlCht10 (right); (E) survival rate of beet armyworm after treatment with recombinant strain. Detailed Implementation

[0032] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] Example 1: Cloning the full-length cDNA of the SlCht10 gene

[0034] Total RNA was extracted from *Spodoptera litura* using the Eastp® Super Total RNA Extraction Kit (Shanghai Promega), and cDNA was synthesized. Primers were designed based on the *Spodoptera litura* transcriptome database, with forward and reverse primers SlCht10-F / SlCht10-R. PCR amplification was performed using cDNA as a template. The PCR reaction mixture consisted of: 12.5 μL Premix Taq enzyme, 1 μL cDNA template, 1 μL each of forward and reverse primers, and 9.5 μL ddH2O. The reaction program was: 95℃ for 30 s; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 35 cycles, followed by 72℃ for 10 min.

[0035] The PCR products obtained by PCR amplification were separated by agarose gel electrophoresis, and the target fragment, namely the full-length cDNA of the SlCht10 gene from *Spodoptera litura*, was purified and recovered. The recovered target fragment was cloned into a pMDTM 19-T vector and transformed into competent DH5α cells. After verification by colony PCR, sequencing was performed. The nucleotide sequence of the SlCht10 gene is shown in SEQ ID NO.1. Figure 1 The start and stop codons are marked in the sequence.

[0036] The upstream and downstream primer sequences for cloning the SlCht10 gene are as follows:

[0037] SlCht10-F:AGTTAACACTGTTCAATATG

[0038] SlCht10-R: GGTATAAGATTAGTTTCTACTA

[0039] The nucleotide sequences of the above primers are shown in SEQ ID NO.4 and 5, respectively.

[0040] Example 2: Insecticidal function test of the SlCht10 target gene of the beet armyworm

[0041] 1. Test insects

[0042] The beet armyworm was obtained from the Guizhou Provincial Tobacco Science Research Institute and was cultured in an artificial climate chamber using artificial feed. The rearing conditions were: temperature 25 ± 1 ℃, relative humidity 70 % ± 5 %, and photoperiod 14L: 10D.

[0043] 2. Design of primers for dsRNA synthesis

[0044] Based on the coding region sequence, the Dicer enzyme recognition site was predicted using the DRSC / TRiP Functional Genomics Resources online platform (https: / / www.flyrnai.org / cgi-bin / RNAi_find_primers.pl) to complete the dsRNA primer design; the T7 transcription promoter sequence (TAATACGACTCACTATAGGG) was introduced into the 5' end of the primer (Table 1).

[0045] Table 1. Primer sequences for GFP and insecticidal target gene SlCht10 in Spodoptera litura.

[0046]

[0047] The nucleotide sequences of the primers are shown in SEQ ID NO.6, 7, 8 and 9, respectively.

[0048] 3. In vitro synthesis of dsRNA of insecticidal target gene SlCht10

[0049] The reaction mixture consisted of 25 µL Prime STAR Max Premix (2X), 1 µL dsRNA forward primer (10 µM), 1 µL dsRNA reverse primer (10 µM), 1 µL cDNA template, and ddH2O to a final volume of 50 µL. The PCR program was as follows: 94 °C pre-denaturation for 5 min, 94 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 45 s, for a total of 35 cycles, followed by a final extension at 72 °C for 7 min. The amplified products were detected by 1% agarose gel electrophoresis. The target band was excised from the gel and recovered, then dissolved in an appropriate amount of ddH2O.

[0050] 3.1 dsRNA Synthesis

[0051] Using the recovered high-concentration DNA with T7 promoters at both ends as a template, dsRNA was synthesized according to the instructions of the 5× MEGAscript™ T7 Kit. The dsRNA synthesis steps are as follows:

[0052] (1) Prepare the following reaction system at room temperature using a 0.2 mL PCR tube without RNase:

[0053]

[0054] (2) Gently blow on the mixture or lightly tap the tube wall to mix the system thoroughly, then micro-centrifuge to collect the mixture at the bottom of the tube, and then let it stand at 37°C overnight (12~16 h).

[0055] (3) Use 1.0 μL of DNaseI to remove residual DNA and ssRNA from the reaction solution, mix gently, and incubate at 37°C for 15 min.

[0056] 3.2 dsRNA purification

[0057] Purify the obtained dsRNA according to the instructions of the MEGAclear™ Kit. Before use, add 20 mL of anhydrous ethanol to the Wash Solution Concentrate reagent, mix well, and label.

[0058] 3.3 dsRNA quality detection

[0059] The purity of the purified product was assessed by 1.0% agarose gel electrophoresis, and the concentration and purity were determined using a NanoDrop™ 2000. After passing the tests, the product was stored at -80°C for later use. The nucleotide sequence of dsSlCht10 is shown in SEQ ID NO.2.

[0060] 4. RNA interference treatment of Spodoptera litura

[0061] The nanocarrier SPc was mixed with dsRNA at a 1:1 mass ratio to achieve a final dsRNA concentration of 2000 ng / μL. Then, 0.5% by volume of surfactant (detergent) was added to form a dsRNA / SPc / detergent complex. Third-instar beet armyworm larvae were completely immersed in 20 μL of the dsRNA / SPc / detergent solution for 10 seconds. Excess liquid was then blotted off with sterile filter paper, and the larvae were transferred to fresh artificial feed for normal rearing. This mixture was then used for subsequent gene expression detection and phenotypic observation.

[0062] 5. Determination of expression level of SlCht10, the insecticidal target gene of Spodoptera litura, after RNA interference.

[0063] Third-instar larvae of the beet armyworm were interfered with using synthesized dsRNA, with dsGFP (the nucleotide sequence of dsGFP is shown in SEQ ID NO. 3) as a control. After 48 h, 10 larvae from each replicate were collected, and the expression level of the target gene was detected using a Bio-Rad C1000 Real-Time PCR system (BIO-RAD, USA). All samples were placed in 1.5 mL RNase-free centrifuge tubes, rapidly frozen in liquid nitrogen, and total RNA was extracted to synthesize cDNA. The RT-qPCR reaction system (20 μL) was prepared according to the TB Green® Premix DimerEraser™ kit (TaKaRa): 10 μL TBGreen Premix Ex Taq, 1 μL each of forward and reverse primers (0.01 mol / L), 1 μL cDNA template, 0.4 μL ROX Reference Dye II, and 6.6 μL DEPC water. Thermal cycling parameters: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 55℃ annealing for 30 s, for a total of 40 cycles. Reactions were performed in 96-well Microseal PCR plates from BIO-RAD Inc. (USA). RT-qPCR results showed that, compared with dsGFP treatment, the expression level of the SlCht10 gene was reduced by 85.75% (…). Figure 2 This indicates that the expression of the insecticidal target gene is suppressed.

[0064] 6. Insecticidal effect of RNA interference with the SlCht10 gene on the beet armyworm.

[0065] The experiment included two control groups: NTC (net water) and dsGFP. The treatment groups were treated with dsRNA of the SlCht10 gene to silence its expression in *Spodoptera litura*. The survival rate of third-instar larvae in both control and treatment groups was recorded within 10 days. The mortality rate in the NTC and dsGFP control groups remained relatively stable within 8 days. Compared to the control, the survival rate of *Spodoptera litura* treated with dsSlCht10 began to decline significantly on day 2, dropping to 56% on day 8, significantly lower than that of the NTC and dsGFP control groups. Figure 3 ), Figure 4 The death morphology of Spodoptera litura larvae after treatment with dsRNA interference target genes.

[0066] Example 3: Construction experiment of dsSlCht10 engineered bacteria

[0067] 1. Test insects and strains

[0068] Spodoptera litura larvae; Beauveria bassiana strain JH19; BL21(DE3) RNase III deletion strain.

[0069] 2. Primer design for engineered bacteria to synthesize dsRNA

[0070] The primers used for the efficient synthesis of dsRNA from the SlCht10 gene of the beet armyworm by engineered bacteria are shown in Table 2. The upstream primer is supplemented with the EcoRI (CGGAATTCCG) or XhoI (CCGCTCGAGC) restriction site sequence, and the downstream primer is supplemented with the XbaI (GCTCTAGAGC) restriction site sequence. EcoSlCht10-F1 and XbaSlCht10-R1 are designed based on two inversely complementary templates.

[0071] Table 2 Primers for dsRNA synthesis by engineered bacteria and primers for identification of recombinant plasmids

[0072]

[0073] The nucleotide sequences of the above primers are shown in SEQ ID NO.10-19, respectively.

[0074] 3. Construction of the Escherichia coli pET28-BL21(DE3) RNase III expression system

[0075] 3.1 PCR amplification and double enzyme digestion of the interference target fragment

[0076] Using the plasmids described above as templates, PCR amplification was performed using primers EcoSlCht10-F1 and XbaSlCht10-R1, and XhoSlCht10-F2 and XbaSlCht10-R2, respectively, yielding gene sequences of 585 bp and 555 bp. After PCR product recovery and purification, the PCR products amplified by EcoSlCht10-F1 and XbaSlCht10-R1 were treated with restriction enzymes EcoRI and XbaI to obtain fragment Cht10-L; the PCR products amplified by EcoSlCht10-F2 and XbaSlCht10-R2 were treated with restriction enzymes XbaI and XhoI to obtain fragment Cht10-R. The enzyme digestion reaction system is as follows:

[0077]

[0078] Eight reaction systems were prepared for each PCR product, with a total volume of 160 μL, and incubated at 37°C for 4 h.

[0079] After enzyme digestion, the PCR products were detected by gel electrophoresis and then recovered using the MiniBEST Agarose Gel DNA Extraction Kit.

[0080] The pET-28a(+) vector was also double-digested with restriction enzymes EcoR I and Xho I. The digestion system is as follows:

[0081]

[0082] A total of 8 reaction systems were prepared, with a total volume of 160 μL, and incubated at 37°C for 4 h.

[0083] The digested plasmids were detected by gel electrophoresis and then recovered from the gel. The recovery method was based on the instructions of the MiniBEST AgaroseGel DNA Extraction Kit.

[0084] 3.2 Ligation of PCR products after enzyme digestion and vector

[0085] The Cht10-L and Cht10-R fragments, recovered and purified after double enzyme digestion, were ligated with the pET-28a(+) vector, recovered and purified after double enzyme digestion, using NEB's T4 ligase. The reaction system is as follows:

[0086]

[0087] 3.3 Transformation of recombinant vectors and PCR detection of positive colonies

[0088] (1) Take 10 μL of ligation product and mix it with 50 μL of BL21(DE3) RNase III- competent cells, incubate on ice for 30 min; heat shock at 42℃ for 1 min and then quickly return to ice for 2 min;

[0089] (2) Add 400 μL of antibiotic-free LB liquid medium and incubate at 37℃ and 200 rpm for 1 h;

[0090] (3) Spread 150 μL of the cultured and revived bacterial solution onto an LB agar plate containing 50 mg / L kanamycin (Kna) using a spreader, and incubate overnight at 37°C with the plate upside down.

[0091] (4) Pick a single colony from the plate into 30 μL of ddH2O, take 1 μL as a template, and perform colony PCR detection using Pet28test-F / R primers;

[0092] (5) Use 1% agarose gel electrophoresis to detect the size of the target band of the PCR product. If the band size is consistent, it means that the vector has been successfully constructed.

[0093] (6) Positive clones after identification were transferred into 5 mL of LB liquid medium containing kanamycin (50 mg / L), and cultured at 37℃ and 200 rpm for 8 h with shaking. Seed culture was then harvested.

[0094] (7) Take 400 μL of bacterial culture and send it to Shanghai Sangon Biotech for sequencing. Compare and analyze the sequencing results.

[0095] The results showed that the PCR product band had a distinct band at approximately 500 bp, which was consistent with the target band size. Figure 5 A) indicates that the carrier was successfully constructed.

[0096] 3.4 Double enzyme digestion verification of the recombinant vector

[0097] (1) To further verify whether the recombinant vector was successfully constructed, plasmids were extracted from the bacterial culture that was correctly sequenced;

[0098] (2) The extracted plasmid was double-digested with endonucleases EcoR I and Xho I. The reaction system and conditions were the same as those for dsRNA synthesis in Example 2.

[0099] (3) The enzyme digestion products were detected by 1% agarose gel electrophoresis. If the observed bright band was 900 bp (Cht10), it indicated that the recombinant vectors pET-28a(+)-CCS-9 and pET-28a(+)-ZFP425 were successfully constructed, and the E. coli pET28-Cht10-BL21(DE3) RNase III- expression system was obtained. The results showed that the PCR product band had a clear band at approximately 900 bp, which was consistent with the target band size ( Figure 5 B), verifying the successful construction of the recombinant vector pET-28(+)-Cht10.

[0100] 4. In vivo induction and extraction of dsRNA

[0101] (1) The seed culture of the verified Escherichia coli pET28-Cht10-BL21(DE3) RNase III- expression system was transferred at a ratio of 1:100 to 25 mL of LB medium containing kanamycin (50 mg / L) and cultured at 37°C and 200 rpm until OD600≈0.4;

[0102] (2) Add IPTG inducer to the bacterial culture to a final concentration of 1 mM, and continue induction at 37℃ and 200 rpm for 4 h;

[0103] (2) The bacterial culture after IPTG induction treatment was inactivated at 75°C for 5 min. Lysozyme with a final concentration of 1.3 mg / mL was added to the solution and incubated at 37°C for 30 min to rupture the cell wall.

[0104] (3) Take 200 μL of the solution after lysozyme treatment, add 3 μL of RNase-Free NAase and 2 ng / μL RNase A Solution, and incubate at 37℃ for 10 min to degrade double-stranded DNA and single-stranded RNA;

[0105] (4) The treated solution was purified into dsRNA using the MEGAclear™ Kit (invitrogen);

[0106] (5) The quality of the purified dsRNA was analyzed by agarose gel electrophoresis, the concentration of dsRNA was detected by Nandrop 2000, and the yield was calculated.

[0107] Figure 5C represents the quality of the dsRNA bands as determined by 1% agarose gel electrophoresis. The purified dsRNA concentration was determined to be 592.8 ng / μL using Nandrop 2000. The dsRNA yield of the pET-28(+)-Cht10-BL21(DE3)RNase ⅠⅠⅠ- expression system was 14.82 μg / mL, meaning that approximately 14.82 μg of dsSlCht10 can be produced per milliliter of E. coli pET-28(+)-Cht10-BL21(DE3)RNase ⅠⅠⅠ- fermentation broth. The nucleotide sequence of dsSlCht10 is shown in SEQ ID NO.2.

[0108] Example 4: Efficacy test of RNAi recombinant strain against tobacco beet armyworm

[0109] 1. Detection of the silencing efficiency of dsRNA synthesized by engineered bacteria on target genes in Spodoptera litura.

[0110] The concentration of total nucleic acid in the solution was roughly estimated by purifying dsRNA from the bacterial culture treated with lysozyme. The nanomaterial (SPc) was mixed with the nucleic acid in the solution at a 1:1 mass ratio, incubated at room temperature for 15 min, and then detergent was added to a final concentration of 0.5% to obtain the complex solution.

[0111] The dsRNA treatment method was the same as in Example 2 for determining the expression level of the insecticidal target gene SlCht10 in *Spodoptera litura* after RNA interference. After 48 h of treatment, total RNA was extracted from 10 live *Spodoptera litura* moths, and cDNA was synthesized via reverse transcription. The RNAi silencing efficiency of the SlCht10 target gene in *Spodoptera litura* was then detected using RT-qPCR. The RT-qPCR reaction system (20 μL) was prepared according to the TB Green® Premix DimerEraser™ kit (TaKaRa): 10 μL TB Green Premix Ex Taq, 1 μL each of forward and reverse primers (0.01 mol / L), 1 μL cDNA template, 0.4 μL ROX Reference Dye II, and 6.6 μL DEPC water. The thermal cycling parameters were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 55℃ annealing for 30 s, for a total of 40 cycles. The reaction was performed in 96-well Microseal PCR plates from BIO-RAD Inc. (USA).

[0112] RNAi treatment of Spodoptera litura larvae revealed that the engineered bacteria expressing dsSlCht10 effectively suppressed the expression of the Cht10 gene, significantly reducing its expression level to 61%. Figure 6 ).

[0113] 2. Analysis of the control effect of engineered bacteria's efficient dsRNA synthesis on beet armyworm in a greenhouse.

[0114] To further investigate the control effect of engineered bacteria expressing highly efficient dsRNA on Spodoptera litura, an indoor chemical control experiment was conducted. Tobacco seedlings were potted in an artificial climate chamber, and 20 one-day-old to third-instar Spodoptera litura larvae were inoculated onto each plant. Three days after inoculation, the engineered bacteria strain that efficiently synthesized dsRNA was centrifuged (6000 g for 5 min), and the supernatant was removed. The bacterial suspension was then resuspended in an equal volume of water, and the cell walls were broken down with lysozyme. A dsRNA / SPc / detergent complex solution was then prepared and sprayed evenly onto the surface of the tobacco leaves inoculated with Spodoptera litura larvae at a rate of 10 mL per plant. Six replicates were performed for each target gene. Water (NTC) was sprayed as a control group. The number of surviving Spodoptera litura larvae on the tobacco plants was observed daily for eight consecutive days. The results showed that the survival rate of dsSlCht10 treatment began to decline significantly after day 2, with the steepest decline occurring between days 5 and 8. The survival rate dropped to 54% on day 8, significantly lower than the NTC (71%) control group. Figure 7 ).

[0115] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of the SlCht10 gene in the control of the beet armyworm, characterized by, The nucleotide sequence of the SlCht10 gene is shown in SEQ ID NO.

1.

2. The application of the SlCht10 gene as an RNAi target in the control of the beet armyworm, characterized by, The nucleotide sequence of the SlCht10 gene is shown in SEQ ID NO.

1.

3. A recombinant engineered bacterium for producing dsRNA targeting the SlCht10 gene of the beet armyworm, characterized in that, The recombinant engineered bacteria were obtained by transforming the recombinant pET-28a(+) expression vector into RNase III-deficient BL21(DE3) Escherichia coli; the recombinant pET-28a(+) expression vector contains two inversely complementary SlCht10 gene silencing fragments; the nucleotide sequence of the SlCht10 gene is shown in SEQ ID NO.

1.

4. The recombinant engineered bacteria according to claim 3, characterized in that, The two reverse complementary silencing fragments of the SlCht10 gene are 585bp and 555bp in length, respectively, and are inserted into the multiple cloning site of the recombinant pET-28a(+) expression vector via EcoRI / XbaI double restriction sites and XhoI / XbaI double restriction sites, respectively.

5. An RNAi formulation targeting the beet armyworm, characterized in that, The invention comprises dsRNA targeting the SlCht10 gene of the beet armyworm, SPc nanocarrier, and surfactant, the nucleotide sequence of which is shown in SEQ ID NO.

1.

6. The RNAi biopesticide formulation targeting the beet armyworm according to claim 5, characterized in that, The mass ratio of dsRNA to SPc nanocarrier is 1:1, and the mass fraction of surfactant is 0.5%.

7. A method for preparing dsRNA targeting the SlCht10 gene of the beet armyworm, characterized in that, Includes the following steps: S1. The recombinant engineered bacteria prepared according to claim 3 or 4 are inoculated into LB medium containing kanamycin and cultured. S2. Add IPTG inducer to bacterial culture to induce dsSlCht10 expression; S3. After induction, the bacterial culture is inactivated, lysozyme is added to break the cell wall, and then nuclease is used to degrade the mixed nucleic acids and purify to obtain dsSlCht10. The nucleotide sequence of dsSlCht10 is shown in SEQ ID NO.

2.

8. The method for preparing dsRNA targeting the SlCht10 gene of *Spodoptera litura* according to claim 7, characterized in that, In step S1, the culture is shaken at 37°C until the bacterial culture OD reaches its maximum. 600 The concentration of the added IPTG inducer was 0.4~0.6; in step S2, the final concentration of the added IPTG inducer was 1mM, and the culture was induced at 37℃ for 4 h; in step S3, the bacterial culture was inactivated by incubating at 75℃ for 5 min, the final concentration of the added lysozyme was 1.3 mg / mL, and the culture was incubated at 37℃ for 30 min to complete the cell wall disruption.

9. A green control method for the beet armyworm, characterized in that, The RNAi formulation described in claim 5 or 6 is uniformly sprayed onto the surface of crop leaves infested by the beet armyworm.

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