CN119040320A

CN119040320A - Application of spodoptera litura SlTl-2 gene in pest control

Info

Publication number: CN119040320A
Application number: CN202410960569.6A
Authority: CN
Inventors: 余小强; 李锦�; 邓心悦; 李琪琳
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2024-07-17
Filing date: 2024-07-17
Publication date: 2024-11-29
Anticipated expiration: 2044-07-17
Also published as: CN119040320B

Abstract

The invention belongs to the technical field of pest control, discloses application of a spodoptera litura SlTl-2 gene in pest control, and particularly discloses application of a SlTl-2 inhibitor in pest control or preparation of a product for pest control, wherein the nucleotide sequence of SlTl-2 is shown as SEQ ID NO. 1. The invention discloses a method for preventing and controlling prodenia litura by targeting the nocturnal emission SlTl-2 gene, wherein the method is characterized in that the inhibitor SlTl-2 is used for targeting and downregulating SlTl-2 (inhibiting SlTl-2 expression), degrading SlTl-2 and inhibiting SlTl-2 activity, so that the sensitivity of the prodenia litura to pesticides (such as Bt toxins) is improved, the purpose of preventing and controlling the prodenia litura is achieved, and in the prevention and control process, the drug resistance is not generated, the product is harmless to people and livestock, and the environment is not polluted.

Description

Application of spodoptera litura SlTl-2 gene in pest control

Technical Field

The invention belongs to the technical field of pest control, and particularly relates to application of a spodoptera litura SlTl-2 gene in pest control.

Background

Prodenia litura (Spodoptera litura), belonging to the family Lepidoptera, is a omnivorous agricultural pest widely distributed in tropical, subtropical and temperate regions. The larvae feed on more than 300 agricultural crops such as corn, rice, wheat, cotton, potato and the like, so that serious economic loss and agricultural damage are caused. With the recent twenty years of climate warming and pest invasion, the hazard of prodenia litura in south China is more serious, and especially in fowls and Yunnan and other places, the prodenia litura has become a main agricultural pest. The prodenia litura has the characteristics of short generation period, large spawning quantity, long flying distance and overeating, so that the prodenia litura floods and disasters worldwide.

Double-stranded RNA (dsRNA) is an RNA molecule having two complementary base sequences. RNA interference (RNAi) is a conserved biological response to double-stranded RNA (dsRNA) that prevents protein translation by targeted degradation of mRNA, inducing sequence-specific gene silencing. RNAi is applied in the agricultural field, and is used for improving crop quality, increasing yield, resisting diseases and insect pests and the like. Because of the wide use of chemical agriculture, problems of environmental pollution, food safety, drug resistance and the like are brought, and RNAi as an important biological control means becomes a widely studied pest control and pest comprehensive treatment technology. Bt toxin (Bacillus thuringiensis toxin) is a protein toxin produced by Bacillus thuringiensis (Bacillus thuringiensis) and has insecticidal activity against insects. With Bts pesticides, the pests are also made to have drug resistance, so that the insecticidal effect is reduced. Therefore, research on the combination of RNAi and Bt toxins has important practical application value.

Toll gene is an important immune-related gene, originally found in Drosophila (Drosophila melanogaster). Toll protein encoded by Toll gene is a transmembrane protein and plays a key role in regulating immune response in drosophila and other insects. Toll protein was originally thought to be primarily involved in embryo axis formation in embryonic development, but later studies have shown that it also plays an important role in immune responses. The observation of Toll in Drosophila immunization and development, respectively, gave a Nobel prize twice. When Drosophila is infected or invaded, toll proteins are activated, which in turn initiate a series of signaling pathways that promote the production of immune-related molecules such as antimicrobial peptides, thereby enhancing resistance of Drosophila. In addition to studies in Drosophila, toll genes and their homologs are also widely studied in other organisms, including mammals and plants. In mammals, toll genes are also involved in immune responses, regulating inflammatory responses and immune cell activation. In plants, the Toll gene is associated with a disease resistance response in plants. In summary, toll genes and their encoded receptors play an important regulatory role in the immune system of organisms, helping them to fight against exogenous pathogens and maintain the homeostasis and health of the organism. However, whether the Toll gene plays a similar function in prodenia litura has not been reported yet.

Disclosure of Invention

It is an object of a first aspect of the present invention to provide the use of SlTl-2 inhibitors for controlling pests or for preparing products for controlling pests.

It is an object of a second aspect of the present invention to provide the use of SlTl-2 inhibitors to promote the sensitivity of pests to pesticides or to prepare products which promote the sensitivity of pests to pesticides.

The object of the third aspect of the present invention is to provide a dsRNA.

The object of the fourth aspect of the present invention is to provide a biomaterial associated with the dsRNA of the third aspect of the present invention.

The object of the fifth aspect of the invention is to provide a product.

The object of the sixth aspect of the present invention is to provide the use of the dsRNA of the third aspect of the present invention, the biomaterial of the fourth aspect of the present invention and/or the product of the fifth aspect of the present invention for or in the manufacture of a product for promoting the sensitivity of a pest to a pesticide.

The object of the seventh aspect of the present invention is to provide a method of controlling pests or a method of promoting the sensitivity of pests to pesticides.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided the use of SlTl-2 inhibitors for controlling pests or for the preparation of a product for controlling pests, the nucleotide sequence of SlTl-2 being as shown in SEQ ID NO. 1.

In some embodiments of the invention, the SlTl-2 inhibitor is at least one of an agent that inhibits SlTl-2 activity, an agent that degrades SlTl-2, or an agent that reduces SlTl-2 expression levels, and further an agent that reduces SlTl-2 expression levels.

In some embodiments of the invention, the agent that reduces SlTl-2 expression levels is at least one of (1) - (3):

(1) siRNA, dsRNA, miRNA, ribozyme or shRNA targeting SlTl-2;

(2) A nucleic acid molecule encoding siRNA, dsRNA, miRNA, ribozyme or shRNA of claim 1 that targets SlTl-2;

(3) An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule of (2).

In some embodiments of the present invention, the SlTl-2 inhibitor is at least one of (4) to (6):

(4) A dsRNA targeting SlTl-2;

(5) A nucleic acid molecule encoding the dsRNA targeting SlTl-2 of (4);

(6) An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule of (5).

In some embodiments of the invention, the dsRNA comprises a double stranded RNA consisting of the nucleotide sequence set forth in SEQ ID NO. 2 and the nucleotide sequence set forth in the reverse complement thereof.

In some embodiments of the invention, the pest includes a lepidopteran insect.

In some embodiments of the invention, the pest is one or more of prodenia litura, tobacco astromoth, cotton bollworm, plutella xylostella, asparagus caterpillar, and spodoptera frugiperda.

In a second aspect of the invention there is provided the use of a SlTl-2 inhibitor for promoting the sensitivity of a pest to a pesticide or for the preparation of a product for promoting the sensitivity of a pest to a pesticide, the nucleotide sequence of SlTl-2 being as shown in SEQ ID NO 1.

(1) siRNA, dsRNA, miRNA, ribozyme or shRNA targeting SlTl-2;

(4) A dsRNA targeting SlTl-2;

(5) A nucleic acid molecule encoding the dsRNA targeting SlTl-2 of (4);

In some embodiments of the invention, the pest includes a lepidopteran insect.

In some embodiments of the invention, the pesticide includes, but is not limited to, toxins that are toxic to pests (e.g., bt toxins, cry toxins, nereistoxin), compounds (e.g., fluo Lei Lana, tetrazolium tebufenozide, thiamethoxam, acetylfipronil, cyclosporin a), drugs (e.g., polygonum cuspidatum root, purslane extract), or proteins (e.g., WBY-7.06 protein).

In a third aspect of the present invention, there is provided a dsRNA comprising a double stranded RNA consisting of the nucleotide sequence shown in SEQ ID NO. 2 and the nucleotide sequence shown in the reverse complement thereof.

In a fourth aspect of the invention there is provided a biomaterial associated with the dsRNA of the third aspect of the invention, the biomaterial comprising any one of 1) to 12):

1) A nucleic acid molecule encoding a dsRNA of the third aspect of the invention;

2) An expression cassette comprising 1) the nucleic acid molecule;

3) A vector comprising 1) the nucleic acid molecule;

4) A vector comprising 2) the expression cassette;

5) A transgenic cell line comprising 1) said nucleic acid molecule;

6) A transgenic cell line comprising 2) said expression cassette;

7) A transgenic cell line comprising 3) the vector;

8) A transgenic cell line comprising 4) the vector;

9) A recombinant microorganism comprising 1) said nucleic acid molecule;

10 A recombinant microorganism comprising 2) said expression cassette;

11 A recombinant microorganism containing 3) the vector;

12 Recombinant microorganism containing 4) the vector

In some embodiments of the invention, the transgenic cell line does not comprise propagation material.

In some embodiments of the invention, the vector is a plasmid vector, phagemid, viral vector, cellular vector, phage, cosmid, F cosmid, artificial chromosome. The plasmid vector may be an optional plasmid, the viral vector may be an optional virus, and the cellular vector does not include propagation material.

In a fifth aspect of the invention, there is provided a product comprising the dsRNA of the third aspect of the invention and/or the biomaterial of the fourth aspect of the invention.

In some embodiments of the invention, the product is used to promote pest sensitivity to pesticides or to control pests.

In some embodiments of the invention, the product comprises at least one of an agent, a drug, and an insecticide.

In some embodiments of the invention, the product further comprises a pharmaceutically acceptable carrier including, but not limited to, diluents, buffers, suspensions, emulsions, granules, encapsulates, excipients, fillers, binders, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, or adsorption carriers.

In some embodiments of the invention, the pest includes a lepidopteran insect.

In a sixth aspect of the invention there is provided the use of a dsRNA of the third aspect of the invention, a biomaterial of the fourth aspect of the invention and/or a product of the fifth aspect of the invention in or for the manufacture of a product for promoting the sensitivity of a pest to a pesticide.

In some embodiments of the invention, the pest includes a lepidopteran insect.

In a seventh aspect of the present invention, there is provided a method of controlling pests or a method of promoting the sensitivity of pests to pesticides, comprising the step of reducing the expression level and/or activity of SlTl-2 in the pests.

In some embodiments of the invention, the nucleotide sequence of SlTl-2 is shown as SEQ ID NO. 1.

In some embodiments of the invention, the step of reducing the amount of expression and/or activity of SlTl-2 in the pest is introducing the dsRNA of the third aspect of the invention, the biomaterial of the fourth aspect of the invention, the product of the fifth aspect of the invention into the pest.

In some embodiments of the invention, the means of introduction comprises oral feeding or injection.

In some embodiments of the invention, the oral feeding comprises pre-application, spraying, or water of food, pest habitat (soil, area, material or environment in which the pest is growing or may grow, or material, cultivated plants, plant propagation material (such as seeds), soil, surfaces or spaces) to be protected from attack or infestation by the pest, containing the dsRNA of the third aspect of the invention, the biological material of the fourth aspect of the invention and/or the product of the fifth aspect of the invention.

In some embodiments of the invention, the pest includes a lepidopteran insect.

In some preferred embodiments of the invention, the pest is one or more of spodoptera litura, tobacco astromoth, cotton bollworm, plutella xylostella, spodoptera exigua and spodoptera frugiperda.

In some more preferred embodiments of the invention, the pest is prodenia litura.

The beneficial effects of the invention are as follows:

The invention discloses a method for preventing and controlling prodenia litura by targeting the nocturnal emission SlTl-2 gene, wherein the method is characterized in that the inhibitor SlTl-2 is used for targeting and downregulating SlTl-2 (inhibiting SlTl-2 expression), degrading SlTl-2 and inhibiting SlTl-2 activity, so that the sensitivity of the prodenia litura to pesticides (such as Bt toxins) is improved, the purpose of preventing and controlling the prodenia litura is achieved, and in the prevention and control process, the drug resistance is not generated, the product is harmless to people and livestock, and the environment is not polluted.

Further, a specific dsRNA which can effectively improve the sensitivity of the prodenia litura to pesticides (such as Bt toxins) is designed according to the prodenia litura SlTl-2 gene, and after the dsRNA of the prodenia litura SlTl-2 is fed to the prodenia litura, the expression of the SlTl-2 gene is inhibited. When this gene is inhibited, feeding Bt toxins significantly accelerates the death of prodenia litura larvae. The research result provides targets and techniques for improving the insecticidal efficiency of pesticides such as Bt toxins and the like.

Drawings

FIG. 1 shows the L4440 vector map and multiple cloning sites.

Figure 2 is a plot of survival of larvae after feeding prodenia litura larvae dsRNA and Bt toxins.

FIG. 3 shows the interference effect of qRT-PCR detection on dsRNA fed.

FIG. 4 shows the effect of qRT-PCR on the anti-peptide genes Acttacin (A), cecropin (B), glovirin (C), lebocin (D) and VIRESCEIN (E) fed dsRNA.

Detailed Description

The following describes the present invention in further detail by way of specific examples.

It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

EXAMPLE 1 double-stranded RNA (dsRNA) Synthesis and expression

In the example, dsRNA capable of reducing the gene SlTl-2 of prodenia litura is synthesized and expressed by escherichia coli.

(1) Design of dsRNA of spodoptera litura SlTl-2 gene

Toll gene is an important gene family, which contains abundant homologous genes in species, and the inventor identifies a Toll homologous gene in prodenia litura and names SlTl-2. The complete sequence of the prodenia litura SlTl-2 gene is obtained by comparing and searching the genes of the prodenia litura in NaTlonal Center for Biotechnology InformaTlon database (NCBI), and the RNA sequence is shown as SEQ ID NO. 1.

TGCACTGCAGTTTAGCGGCAGACGTGCGCTGAGACGGACGCGTGATCTCGTCCCTCCACTAATTGATTAATTTGTGTGTTGTTTTCGTGCGAACTAATTAGAGTTTTTATTTATTAAACTCATTGAAAGTGGTTTTGCTTTGTGAAGTTGTTGGCTTGATTTGATTATTAACGTTGATGTGTGGCACATAGCAAGATGCGGTTCCTACTACTGCTAGCTCTAGCCCCGGCCGCGCTCGCACTATATTGTCCATCAAACTGCTACTGCGAGCTTTATGATGAACTGGAATACAGATGCAAGTCTGGCAGCAACAAAGTAACATATAACGCGAGGAACAACGAGTATGTTAACATAGAATGTGAGGGTGCAAACCTCACCTGCGCAGATTTCCCTCAGGTCAATTTTGCCAATAACCAAACCCTCCCGTCCCTCTGGCTGAAGAAATGCCCTCCTTCCATCATCCCCTGCCTGAATGACGCGCTGATGACGTCATCAATAAGTTTTGTCACTTTCACGAAGTTGCGAGCACCTATGCGAGCTGATGATGTAGAGCATCTTGGAGAGGTCCAGGAGATCATGATATCTGAGGGCCCAAAAGACCCACTACCACCATCAGCTCTCGAGAAGCTCCCAACTCTTCGACGGCTGCGAATCACCAGCACTCCAGTGACCCTCGACCTATCCACTTTCGCCCAGTCTCCAGGGTTGGAGTACTTGGAGCTATCAGACCCTTTGATCACGGAGATCCCCTCGGGGATGTTTGCTAGTCTCCACAAGTTGAAGATGCTTAACTTTTGGGGGAACAGTTTGAGTGACGTGGAGGTGGATTCTTTGTTAGGTCTGGACTCGTTAGAAAAGCTATCGTTCACCATGAGTCGTCTGGCAAATATATCTCCTGGAGCATTCAAACACCTGCCGAAGTTAAAGTCTCTGTACATAATGAAGAATAGAATCGAAGAGATCGCTCCAGGACTGTTGACTAACTTGACTCAACTGGAAAATATAACCATACAATTAAACGACATCAAGTTACATTTGCATTCAAATTCGATTACCAATCTGCCTGGATTAAAGAATCTTATAATCAAGGACTGCTTCGCTGAATTACCGAAAGACTTGATCACCGGCTGTGAGGCCTTACGCACTTTGGACCTCTCCCAAAACCGGATAACAGCTATACCGGATAATTTTTTCAAAGATTTGAAGAATTTAGAACATCTCGATTTGAGCTACAATAGAATTGCCAAATTGGATAGTGGAGTTTTATCGCCTTTGAAACAATTGAAATATTTGAACCTGGACCGAAATCACTTGGAGGTGTTGCCTGAATTCTTCTTCGCTGGTCTAAGGAAATTGGAAGAACTATCGATTAACGAAAATTTACTCACTACGATAAATGCCTTAGCGTTCCAAGGCGCAACGTCTTTGCAAACAATATCAATGATGGGAAACAAATTGCAGCTAAAATCTCAAGAACAAATCGAATCTGCATACAATATGGGCTATGAAGTGTATTCACCGTTCAACACTTTGATCGAGTTAAAAAAGTTGACATTGAGTCGAAATAATATCACAACCATTTTTGACGATTGGAGAATCGTGTTCACCAATTTGGAAGTATTGGATTTGTCTCACAACCGTATTGAGAATTTAAATCCGGTCAACTATCAGTTCCTAAGCAAAAAGGTAACAGTGGACTTGCAGTATAATAATATAAGTACTGTAGGCATGTACCAACCAGCTGTGTCGATTGACCATTCGGAATCAGAAATACAATCAAACACCATATTTCTGTTGGACCACAACCCTTTCAACTGCGATTGTCATATATATAACTTCGTTATGCGTCTCCGGGGGAAAAAACCTGCCTCCGAACCGAATTTTAACGTAGGCGACGCTGAATGTAAATCCCCAGACCACTTAAACGGGGATTTGTTGAGAAATGTATCCCCATCCCACTTATTATGCGAAGAAATGCTGCCTGAAGTTCGATTTAATTGTAATTCTGTTCTGGTGAGACCAGAGTTCAATGATTTGTCCTATGATTGTGATTATCTGCCACCTTCTTTCCCTGATCTTGCAGAATATGGCGTTCAAGAACTACGTCTAAAATTAAAGCACCCACCAAAAGATCTAAGCAACTTATCTCTAAGTTTACTAAACTTAACTAATATAGGTTTACAAAAAATCCCATTCGAACCGTCAAAATTGGTGAAAGTAATCGATTTTTCGAATAATAGTTTAACAAAAATTCCTGTTCGATTCGTAAAATCGAATACGACGTTGTATTTATCGCATAATCCGTTCGACTGTGGTTGTTTTTCTAAAGATGACATTGTAGCTTTAAAAGGTAGACGTAGTATCGTGAAAGATTATGAGTCCGTTGGCTGTTCCGACGGTGTCTTAGTGAAAGAGCTAGATATTCCCGCCCTATGTTACACTCAAACTTTAGTTGCAGAAATCGGCGGCATATTCGTATTTTTACTAATTTTATTCATACTAGCATTCATCTTTTCTCGACAAATTGTTGATTACTGTGGACACAGACTACTTCCTTGTTGGTACATCGACGACCCAGACAATATAGTCAAAGCATACGATATTTTCGTTTCCTACGCCCACCAAGATCAAAAATACGTCGACAAGTTACTGCCCAAACTAGAAAACGACTTCAAATTGAAAGTCTGTGTCCACTACCGAGACTGGGAAGTCGGTGATTTCATCCCCGATCAAATTCATCGGTCGGTATCGAATTCTCGGAAAACTATTATTTTATTATCGAATCATTTTCTCGATTCGACGTTTGCGAATATGGAGTTTAGGACGGCTCATAACTTGGCTTTGAAGGAAGGTCGGGAAAGAGTGATTTTGATCCTTCTGGAAGACGTAAGTAAGCATGAGAAGTTGTCTGAAGAGTTGAAATATCATATGAAGATGAATACGTACCTGACTTGGGATGACATTCGCTTCGATGATAAATTAAAACGTCGTACGATCCCGCAAAAATATAATAAGAAGAAATTCGTTGCGCCGGCCATTTTGAAACCAATTTTCAGGCAAGCTACTGAAAATAATTTGAAGAAAGCATTAGATGTTCACTTGAATAGTTCTGGGCAACTTGTAAATATGGCTCAGAATAAGAAAAATATCGAAAGTGTATAATTTTTTAGCAATTATTCCAAAAAAGGACTTCTGAAAGTCTATTGTTTTCGTGAACCTCTATTTTACCAAGAACCCCCAACCCTTAAAAGGCTGACCACGCACTTGTAACGCCTCTGGTGATCCAGGTGTCCATGGGCGACGACGATTGCTTACCATCAGGTGATCCGTCTGCTCGTTTACCGATATCACCTAACGGTAAAGTTTATGTCATTGAGTTCACCTCACGCTGAGTAACTCTAAATTAAGGGATGTTTTAGTGGCTCTCTTAAATTTTAAGTTACCCCTAATCTAAGTGGCACTTAATGATGTTTTGGTTTTACCAAGCTACAAATGACACCTAACGACAAAGGCAGTTAGTTAAGATTTTCCCTGACGTTAAGTAACTCTTAGTTAAGTGATGGTTTAGGGGGTGGTTAGTGGAAACGAAACGTTAGTCTTTTAAGTCATTATAGACTAAAAAATAATTTTTGGAAGTCTAAAATTAAAGCATTTATTTGAATTAAACTGTCAAACACCGCGAGGTCACCGGTGACCGCAACCTATTGTTCTTTAATTGTGTCTATAATGACGGTACTCGACGAGTTAAACCGTCAGGAACTTTCGTCCGTCAGTGAAGGTAGGTGCTCGTTCCAAAGTGTAAATTCCTTTAAAAAATAAGCAATTATAGTGTAAAAAATAGAATTTTATATGAATACTATCTCTTGGCCGCGATAATAAATATAATTACGGGGTATGCCAATTAAAAACCCAGTTAATCCGTCAGTTAGGTAACGAAAAAACAT(SEQ ID NO:1).

And (3) treating the exon region of the spodoptera litura SlTl-2 gene by using NCBI, and designing a dsRNA targeting sequence to finally obtain the optimal dsRNA targeting sequence.

The dsRNA targeting sequence is as follows:

AGCATACGATATTTTCGTTTCCTACGCCCACCAAGATCAAAAATACGTCGACAAGTTACTGCCCAAACTAGAAAACGACTTCAAATTGAAAGTCTGTGTCCACTACCGAGACTGGGAAGTCGGTGATTTCATCCCCGATCAAATTCATCGGTCGGTATCGAATTCTCGGAAAACTATTATTTTATTATCGAATCATTTTCTCGATTCGACGTTTGCGAATATGGAGTTTAGGACGGCTCATAACTTGGCTTTGAAGGAAGGTCGGGAAAGAGTGATTTTGATCCTTCTGGAAGACGTAAGTAAGCATGAGAAGTTGTCTGAAGAGTTGAAATATCATATGAAGATGAATACGTACCTGACTTGGGATGACATTCGCTTCGATGATAAATTAAAACGTCGTACGATCCCGCAAAAA(SEQ ID NO:2).

The primers for designing the dsRNA targeting sequence of SlTl-2 are as follows:

The upstream primer L4440-dsTl-2-F:5'-GCTCTAGAGCATACGATATTTTCGTTT-3' (SEQ ID NO: 3);

The downstream primer L4440-dsTl-2-R:5'-CGCTGCAGTTTTTGCGGGATCGTACGA-3' (SEQ ID NO: 4).

GFP gene was silenced by synthesis of dsRNA primers targeting GFP (Green fluorescent protein ) gene, as a control.

The primers for designing the dsRNA targeting sequence of GFP are as follows:

the upstream primer L4440-dsGFP-F5'-CGAGCTCAAGTTCAGCGTGTCCG-3' (SEQ ID NO: 5);

the downstream primer L4440-dsGFP-F5'-GCTGCAGCACCTTGATGCCGTTC-3' (SEQ ID NO: 6).

(2) Recombinant vector construction of escherichia coli expression dsRNA

After specific primers related to RNAi experiments are obtained, a specific region is obtained through PCR, and is subjected to digestion and connection to an L4440 plasmid, wherein the L4440 plasmid is a plasmid capable of efficiently expressing dsRNA (a plasmid map is shown in figure 1), a connection product is transformed into the competence of escherichia coli HT115 (DE 3), the exact positive bacteria are identified by double digestion, sequencing is carried out by the engine company, and comparison analysis of DNA sequences is carried out by adopting SnapGene software. The method comprises the following specific steps:

step 1, preparation of prodenia litura sample cDNA

1) Putting the prodenia litura larvae into a centrifuge tube, adding two small steel balls and 500 mu L Tritol (Takara) reagent into each tube, and putting into a grinder for grinding

2) Adding 200 mu L of mixed reagent of chloroform and isoamyl alcohol with the volume ratio of 24:1 into each centrifuge tube, and standing on ice for 5min after violent shaking;

3) Symmetrically placing the sample into a centrifugal machine for centrifugation, and sucking about 500 mu L of supernatant to a newly precooled centrifugal tube;

4) Adding 400-500 mu L of isopropanol, uniformly mixing, and standing for 4 hours in a refrigerator at-20 ℃;

5) Centrifuging the centrifuge tube in a centrifuge, discarding the supernatant, and finally adding 1mL of 75% ethanol;

6) And 7, after centrifugation, repeating the step 7. Removing the supernatant, drying in air for 5-10 min, and finally adding a proper amount of DEPC-ddH ₂ O to dissolve RNA.

7) RNA samples were then formulated according to the reverse transcription kit (purchased from Nanjiazan corporation) protocol.

8) Mix and enzyme in the kit and RNA are prepared in a reaction tube, and mixed evenly by shaking, and after centrifugation, placed in a PCR instrument to set the program to 50 ℃, reacted for 15min, then 85 ℃ and reacted for 5sec. After the reaction, the cDNA was stored in a-20℃refrigerator.

Step 2, construction of recombinant vector of spodoptera litura dsRNA

1) The cDNA synthesized in the step1 is used as a template, mRNA sequences of the prodenia litura SlTl-2 are cloned through PCR, a reaction system is shown in a table 1, and the setting of a PCR reaction program is shown in a table 2. The PCR products were detected by 1% agarose gel electrophoresis. DL2000 DNA MARKER,110V,30min electrophoresis was selected and observed with a DNA gel imager.

TABLE 1PCR reaction System

TABLE 2PCR reaction procedure

2) DNA gel recovery

The gel strip with correct gel position is cut off, and DNA is recovered. Gel purification was performed using Gel-DNA recovery kit (Omega Bio-tek), and reference was made to Omega Bio-tek Gel ExtracTlon Kit recovery kit instructions (catalog number: D2500).

3) Double enzyme digestion reaction

The L4440 plasmid was subjected to a double cleavage reaction. The double enzyme digestion reaction system is referred to as Thermo FISHER SCIENTLFIC company, the reaction system is shown in Table 3, and the reaction solution is placed in a 37 ℃ water bath for reaction for 3 hours.

TABLE 3 double cleavage reaction System

4) Ligation of the fragment of interest to the vector

The digested vectors were separately subjected to agarose gel electrophoresis, and the plasmid without digestion was used as a control. The digested vector was Gel-purified using Gel-DNA recovery kit (Omega Bio-tek), and the kit instructions (catalog number: D2500) were referred to by Omega Bio-tek Gel ExtracTlon Kit.

The recovered vector and fragment were subjected to ligation according to the specification of Takara company T4 DNA LIGASE (catalog number: 2011A) in a 1:3 molar ratio system. The prepared reaction solution was allowed to join at 16 ℃ overnight.

5) Conversion of ligation products

The ligation product was added to competent cells of E.coli HT115 (DE 3) and ice-bathed for 30min, the mixture was heat-shocked at 42℃for 90s and rapidly placed on ice for 5min, 700. Mu.L LB liquid medium was added, shaking culture was performed for 1h at 37℃and 200rpm, the supernatant approximately 600. Mu.L was discarded from the upper layer by centrifugation (5000 rpm,5 min), the pellet was resuspended, the bacterial suspension was spread on LB plates containing ampicillin (Amp) in an ultra clean bench, incubated overnight at 37℃with inversion, and finally sent to sequencing company for sequencing.

(3) Preparation of dsRNA

HT115 (DE 3) single colonies containing L4440-dsRNA, which were sequenced correctly, were inoculated into LB liquid medium (containing Amp 50. Mu.g/mL and Tet 12.5. Mu.g/mL), incubated on a 37℃shaker for 15h, then inoculated into about 500mL fresh LB liquid medium (containing Amp 50. Mu.g/mL and Tet 12.5. Mu.g/mL, inoculum size 1:100), and 0.1mM/L isopropyl beta-D-1-thiopyran galactoside (IPTG) was added to induce dsRNA expression when OD600 reached 0.4.

Example 2 Spodoptera litura larva bioassay

(1) Prodenia litura larva survival rate determination

HT115 (DE 3) bacteria containing L4440-dsRNA (prepared from example 1) were grown up, centrifuged at 5000rpm for 10min, and then resuspended in 0.05M PBS (pH 7.4) at a ratio of 20:1 (20 Xconcentration), the concentration of expressed dsRNA being about 0.5. Mu.g/. Mu.L. About 1cm ³ of artificial diet, covered with HT115 (DE 3) bacterial suspension expressing L4440-dsRNA (i.e., dsSlTl-2) and dsGFP (control group) etc. were taken, and two-instar larvae were placed on the artificial diet of each treatment group, and for each treatment 60 larvae were used. Each treatment was repeated three times. Fresh feed is replaced and bacterial liquid is added every 24 hours, and after continuous ingestion for 48 hours, a plurality of whole insects are collected for each group to be one part, and the process is repeated three times. Larvae were placed in each treatment group, and after 48 hours of feeding, the crude Bt extract was added to an artificial feed (30. Mu.g of the crude Bt extract was added to 0.5cm ³ of artificial feed; the artificial feed formulation was 100g of soybean meal, 80g of wheat germ, 26g of yeast powder, 8g of casein, 8g of vitamin C, 1g of choline chloride, 2g of sorbic acid, 0.2g of cholesterol, 0.2g of inositol and 26g of agar powder, dissolved in 1000mL of ultrapure water), and the survival rate was counted every 12 hours.

DsGFP sequence:

CAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTG(SEQ ID NO:7).

The results are shown in figure 2, which shows that after dsSlTl-2 is fed with the Bt toxin of the larvae, the death rate of the larvae is obviously improved.

(2) RNAi silencing efficiency detection of spodoptera litura SlTl-2 gene

Treating prodenia litura larvae according to the experimental process in (1), collecting 6 prodenia litura whole worms fed with dsSlTl-2 and dsGFP hours for extracting total RNA in each group, reversely transcribing the total RNA into cDNA, and respectively detecting the relative expression quantity of a target gene (SlTl-2) and an internal reference gene (RP 49) by adopting a qRT-PCR method. Each group was set with 3 biological replicates, each biological replicate with 3 nymphs.

The specific process is as follows:

step 1 extraction and reverse transcription of tissue Total RNA the procedure of example 1 was followed.

Step 2 real-time fluorescent quantitative PCR (qRT-PCR)

The quantitative PCR primer of SlTl-2 gene is designed on NCBI website, and the primer is synthesized by Beijing engine company by taking RP49 gene of spodoptera litura as reference. The primers used in the experiments are shown in Table 4. Real-time fluorescent quantitative PCR (qRT-PCR) detection was performed using cDNA obtained by reverse transcription (diluted 5-fold) as a template and RP49 as an internal reference gene. Instrument procedure and system settings were made with reference to qRT-PCR instructions for nanjing novzan, three replicates per sample, quantification system as shown in table 5, amplification curve selection two-step procedure, procedure set as shown in table 6.

TABLE 4 primer sequences

TABLE 5qRT-PCR reaction System

TABLE 6qRT-PCR reaction procedure

Step 3 data processing of qRT-PCR

The relative expression of the target gene is calculated by using RP49 gene as reference and using 2-delta Ct method. The difference in expression of the target gene between the different samples was analyzed using T-test in GRAPHPAD PRISM software. p.ltoreq.0.05 is denoted by x, p.ltoreq.0.01 is denoted by x, and p.ltoreq.0.001 is denoted by x.

As a result, as shown in FIG. 3, after 48h in E.coli fed larvae expressing dsSlTl-2, the expression level of SlTl-2 gene was significantly down-regulated, indicating that feeding dsSlTl-2 can successfully knock down the expression of SlTl-2 gene (FIG. 3).

Example 3 Effect of RNAi silencing of Spodoptera SlTl-2 Gene on antibacterial peptide Gene

Antibacterial peptides (antimicrobial peptide, AMPs) refer to a class of polypeptides with antibacterial activity, also known as host defenses peptides, produced by induction in organisms, which are widely found in most organisms and have the effect of protecting the host from infectious agents. They have broad-spectrum antibacterial and immunomodulatory activity against infectious bacteria (gram positive and gram negative), viruses and fungi.

In this example, expression of SlTl-2 gene was silenced by RNAi, and knock down of SlTl-2 was found to accelerate death of the Cry toxins by Spodoptera litura. Further, after successful knockdown of the expression level of SlTl-2 gene, the insect bodies were treated with Cry1Ca toxin to detect changes in expression of the antimicrobial peptide gene downstream of the immune pathway. And collecting 6 whole worms fed with dsSlTl-2 and dsGFP hours of prodenia litura for extracting total RNA in each group, reversely transcribing the total RNA into cDNA, and respectively detecting the relative expression quantity of the antibacterial peptide genes (Acttacin, cecropin, gloverin, lebocin and VIRESCEIN) and the internal reference gene (RP 49) by adopting a qRT-PCR method, wherein the primer sequences are shown in the table 4. The detection procedure was as in example 2.

As a result, as shown in fig. 4, after the expression of SlTl-2 gene was knocked down, cry1Ca toxin-induced expression of Acttacin, cecropin, lebocin and VIRESCEIN four antibacterial peptide genes was significantly suppressed, whereas the expression of Gloverin gene, although immunity was significantly suppressed, had a tendency to decrease. This indicates that SlTl-2 can participate in response of the prodenia litura to Cry1Ca, and that the knock-down of SlTl-2 can reduce expression of the prodenia litura antibacterial peptide under the stress of Cry1Ca, so that sensitivity of pests to toxins is promoted, and the effect of pest control is achieved.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

The application of the SlTl-2 inhibitor in pest control or pest control product preparation, wherein the nucleotide sequence of SlTl-2 is shown as SEQ ID NO. 1.
Use of a sltl-2 inhibitor for promoting the sensitivity of a pest to a pesticide or for the preparation of a product for promoting the sensitivity of a pest to a pesticide, wherein the nucleotide sequence of SlTl-2 is shown in SEQ ID No. 1.
3. The use according to claim 1 or 2, wherein the SlTl-2 inhibitor is at least one of an agent that inhibits SlTl-2 activity, an agent that degrades SlTl-2, or an agent that reduces SlTl-2 expression levels.
4. The use according to claim 3, wherein the substance for reducing SlTl-2 expression level is at least one of (1) to (3):

(1) siRNA, dsRNA, miRNA, ribozyme or shRNA targeting SlTl-2;

(2) A nucleic acid molecule encoding siRNA, dsRNA, miRNA, ribozyme or shRNA of claim 1 that targets SlTl-2;

(3) An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule of (2).
5. The use of claim 4, wherein the SlTl-2 inhibitor is at least one of (4) - (6):

(4) A dsRNA targeting SlTl-2;

(5) A nucleic acid molecule encoding the dsRNA targeting SlTl-2 of (4);

(6) An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule of (5);

Preferably, the dsRNA comprises double-stranded RNA consisting of the nucleotide sequence shown in SEQ ID NO. 2 and the nucleotide sequence shown in the reverse complement thereof;

preferably, the pest includes lepidopteran insects.
6. A dsRNA comprising a double stranded RNA consisting of a nucleotide sequence shown in SEQ ID No.2 and a nucleotide sequence shown in its reverse complement.
7. A biomaterial associated with the dsRNA of claim 6, said biomaterial comprising any one of 1) to 12):

1) A nucleic acid molecule encoding the dsRNA of claim 6;

2) An expression cassette comprising 1) the nucleic acid molecule;

3) A vector comprising 1) the nucleic acid molecule;

4) A vector comprising 2) the expression cassette;

5) A transgenic cell line comprising 1) said nucleic acid molecule;

6) A transgenic cell line comprising 2) said expression cassette;

7) A transgenic cell line comprising 3) the vector;

8) A transgenic cell line comprising 4) the vector;

9) A recombinant microorganism comprising 1) said nucleic acid molecule;

10 A recombinant microorganism comprising 2) said expression cassette;

11 A recombinant microorganism containing 3) the vector;

12 A recombinant microorganism containing the vector of 4).
8. A product comprising the dsRNA of claim 6 and/or the biomaterial of claim 7;

Preferably, the product is used to promote the sensitivity of pests to pesticides or to control pests.
9. Use of the dsRNA of claim 6, the biomaterial of claim 7 and/or the product of claim 8 for promoting the sensitivity of a pest to a pesticide or for the preparation of a product promoting the sensitivity of a pest to a pesticide.
10. A method for controlling pests or promoting the sensitivity of pests to pesticides, comprising the step of reducing the expression level and/or activity of SlTl-2 in the pests;

preferably, the nucleotide sequence of SlTl-2 is shown as SEQ ID NO. 1;

Preferably, the step of reducing the expression level and/or activity of SlTl-2 in a pest is to introduce the dsRNA of claim 6, the biological material of claim 7, the product of claim 8 into the pest;

preferably, the pest includes lepidopteran insects.