Method for controlling pests by expressing dsRNA through intestinal symbiotic bacteria and application

Abstract

The invention provides a method for controlling pests by utilizing intestinal symbiotic bacteria to express dsRNA (double-stranded ribonucleic acid) and application of the method. According to the invention, intestinal symbiotic bacteria are used as a cell factory, and interfering molecules (such as dsRNA) are continuously generated in intestinal tracts of pests to interfere the expression of target genes of the pests, so that the pests are effectively and continuously controlled.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, to a method and application of controlling pests by expressing dsRNA with intestinal symbiotic bacteria. Background Art

[0002] A large number of agricultural and forestry pests (harmful insects) and insect-borne pests pose a great threat to agricultural production and human life and health. For example, they cause crop yield reduction, threaten forestry development, and spread diseases (Pedigo and Rice, 2014, Entomology and pest management (Waveland Press)). In particular, lepidopteran pests cause huge economic losses to crop production and forestry every year.

[0003] Diptera pests, represented by mosquitoes, can spread a variety of diseases, such as malaria, dengue fever and Zika fever, posing a huge threat to human life and health. Due to the lack of effective vaccines and the increasingly serious problem of mosquito resistance, the main method of controlling insect-borne diseases is to cut off their transmission pathways, that is, to reduce the number of mosquitoes. At present, pest control mainly relies on chemical pesticides, but these chemical pesticides will pollute the human living environment, and the problem of pest resistance is also becoming more and more serious, which limits the application of chemical pesticides.

[0004] RNA interference (RNAi) refers to the phenomenon that double-stranded RNA (dsRNA) forms small interfering RNA (siRNA) through shearing to induce gene expression silencing (Grishok, A., 2005, FEBS Lett 579, 5932-5939). At present, RNAi has become a tool for studying gene functions in plants and animals. However, there are few successful cases of using RNAi for pest control, which is due to factors such as the instability of biological molecules themselves and the difficulty of large-scale large-scale deployment, resulting in insufficient results.

[0005] Therefore, finding insecticidal effector factors that are safe, highly effective and not prone to resistance is of great significance for the prevention and control of harmful insects. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application of controlling pests by expressing dsRNA with intestinal symbiotic bacteria.

[0007] In a first aspect of the present invention, a method for controlling (injuring / killing) harmful insects is provided, comprising:

[0008] (1) Providing intestinal symbiotic bacteria, wherein the intestinal engineered bacteria can take the harmful insect as a host and coexist in the intestine of the harmful insect;

[0009] (2) introducing an expression construct of an interfering molecule into the intestinal symbiotic bacteria; the interfering molecule specifically targets the functional gene of the harmful insect;

[0010] (3) The intestinal symbiotic bacteria are applied to the harmful insects, symbiotically coexisting in the intestines of the harmful insects and expressing the interfering molecules, thereby controlling the harmful insects by inhibiting the functional genes of the harmful insects.

[0011] In one or more embodiments, the “applying the intestinal symbiotic bacteria to the harmful insects” includes applying to an object (such as a plant or a place) infested by the harmful insects.

[0012] In one or more embodiments, the functional genes of the harmful insects include: genes that determine the viability of the harmful insects (whose downregulation or silencing reduces the viability of the harmful insects); preferably include genes that regulate physiological functions such as immunity or metabolism (interfering with immune or metabolic physiological functions by regulating the immune signaling pathways or metabolism-related genes of the harmful insects).

[0013] In one or more preferred embodiments, the functional genes include: Toll immune pathway, IMD immune pathway, Jak-STAT immune pathway, juvenile hormone signaling pathway, ecdysone signaling pathway and other related genes; more preferably, the functional genes include: Met, EcR genes.

[0014] In one or more embodiments, the interfering molecules include: interfering molecules that can interfere with the functional genes of the harmful insects and thus affect the survival of the harmful insects.

[0015] In one or more preferred embodiments, the interfering molecule is dsRNA.

[0016] In one or more embodiments, the functional gene includes the Met gene or the EcR gene.

[0017] In one or more embodiments, the interfering molecule is dsRNA, the nucleotide sequence of which is shown in SEQ ID NO: 1, and targets the Met gene (targeting nucleotides 2168-2650 in the coding region of the Met gene).

[0018] In one or more embodiments, the interfering molecule is dsRNA, the nucleotide sequence of which is shown in SEQ ID NO: 2, and it targets the EcR gene (targeting nucleotides 676-1173 of the Met gene coding region).

[0019] In one or more embodiments, the expression of the interfering molecule is driven by a promoter.

[0020] In one or more embodiments, the promoter includes: a constitutive promoter and an inducible promoter.

[0021] In one or more preferred embodiments, the promoter includes (but is not limited to): T5 promoter, T7 promoter, pj23119 promoter.

[0022] In one or more embodiments, the expression construct includes a constitutive promoter, and the interfering molecule is continuously expressed.

[0023] In one or more embodiments, the expression construct comprises an inducible promoter, and the interfering molecule is induced to be expressed.

[0024] In one or more embodiments, the intestinal commensal bacteria include: Serratia, Enterobacter, Acinetobacter, Chromobacterium, and Asaia.

[0025] In one or more preferred embodiments, no endogenous molecules that affect the expression of the interfering molecule exist in the intestinal commensal bacteria.

[0026] In one or more preferred embodiments, nucleases (including RNase III) are absent, inactivated or knocked out in the intestinal commensal bacteria.

[0027] In one or more embodiments, the harmful insects are selected from the following groups of insects: Diptera, Homoptera, Lepidoptera, Coleoptera, Hemiptera, Orthoptera, Hymenoptera, Neuroptera; preferably Diptera, Culicidae.

[0028] In another aspect of the present invention, there is provided an application of modified intestinal symbiotic bacteria for controlling (injuring / killing) harmful insects, or preparing a composition for controlling harmful insects; wherein the intestinal symbiotic bacteria can use the harmful insects as hosts and coexist in the intestines of the harmful insects; the modified intestinal symbiotic bacteria contains an expression construct of an interfering molecule (capable of expressing the interfering molecule); and the interfering molecule specifically targets the functional genes of the harmful insects.

[0029] In another aspect of the present invention, a modified intestinal symbiotic bacterium is provided, which can use the harmful insect as a host and coexist in the intestine of the harmful insect; the modified intestinal symbiotic bacteria contains an expression construct of an interfering molecule (capable of expressing the interfering molecule); the interfering molecule specifically targets the functional gene of the harmful insect.

[0030] In another aspect of the present invention, a composition for controlling (injuring / killing) harmful insects is provided, which comprises the modified intestinal symbiotic bacteria; and a pesticidally acceptable carrier or excipient.

[0031] In one or more embodiments, the composition contains the modified intestinal commensal bacteria at a concentration of 1×10 3 ~1×10 12 pfu; preferably 1×10 4 ~1×10 11 pfu; preferably 1×10 5 ~1×10 10 pfu (e.g. 10 6 , 10 7 , 10 8 , 10 9 pfu).

[0032] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram for designing dsRNA for the Met gene and EcR gene of Anopheles stephensi;

[0034] (A) dsRNA fragment of the Met gene of Anopheles stephensi;

[0035] (B) dsRNA fragment of the Anopheles stephensi EcR gene.

[0036] Figure 2 Bioassay was performed by adding dsRNA of Met and EcR genes into culture water of Anopheles stephensi larvae;

[0037] (A) Schematic diagram of bioassay using Met and EcR gene dsRNA on Anopheles stephensi larvae;

[0038] (B) Survival of Anopheles stephensi larvae treated with Met and EcR gene dsRNA.

[0039] Figure 3 This is the 16S ribosomal DNA sequence of Serratia fonticola, a bacterium symbiotic in the intestine of Anopheles stephensi.

[0040] Figure 4 To construct RNase III knockout Serratia fonticola;

[0041] (A) Schematic diagram of gRNA targeting RNase III gene;

[0042] (B) Genome sequencing results of Serratia fonticola RNase III gene knockout mutant.

[0043] Figure 5 Schematic diagram of dsRNA plasmid expressing Met and EcR genes in Serratia fonticola;

[0044] (A) Using the inducible dsRNA plasmid L4440 as a vector, a map of the plasmid L4T5 that can constitutively express dsRNA in intestinal bacteria was constructed;

[0045] (B) Plasmid map for constitutively expressing Met and EcR gene dsRNA.

[0046] Figure 6 Bioassays were performed to culture Anopheles stephensi larvae using an agar diet containing Serratia fonticola bacteria expressing dsRNA of the Met or EcR genes;

[0047] (A) Schematic diagram of bioassay using Serratia fonticola bacteria expressing Met or EcR gene dsRNA;

[0048] (B) Survival of Anopheles stephensi larvae treated with Serratia fonticola bacteria expressing Met or EcR gene dsRNA. DETAILED DESCRIPTION

[0049] The inventors are committed to the development of safe and effective biological agents for pest control. After in-depth research, they have revealed a new control method for the first time, which uses intestinal symbiotic bacteria as cell factories to continuously produce interfering molecules (such as dsRNA) in the intestines of pests to interfere with the expression of target genes of pests, thereby effectively and continuously controlling pests. At the same time, the present invention also provides a method for using bacteria to constitutively produce interfering molecules continuously.

[0050] the term

[0051] As used in the present invention, the "intestinal symbiotic bacteria" refers to a type of bacteria that can generally coexist in the intestines of insects (including harmful insects) and can be ingested and survive in the insect intestines when the insects feed.

[0052] As used in the present invention, the "modified intestinal symbiotic bacteria" are bacteria with the following characteristics: (a) they can parasitize in the intestines of insects, and (b) they can express interfering molecules; the interfering molecules specifically target the functional genes of the harmful insects described in the present invention, causing damage to the harmful insects.

[0053] As used in the present invention, the "host" and "host" are used interchangeably and refer to insects, especially harmful insects, that can be infected by the modified intestinal symbiotic bacteria.

[0054] As used in the present invention, the "plant" is not particularly limited, as long as the "plant" is capable of or susceptible to insect-borne pathogens or damage by harmful insects (such as the host of the modified intestinal symbiotic bacteria of the present invention), such as various crops, flower plants, herbaceous plants, woody plants, forestry plants, etc. The plant can be, for example (but not limited to): dicotyledonous plants, monocotyledonous plants, or gymnosperms. More specifically, the plant includes (but not limited to): grass plants, cruciferous plants, etc.

[0055] As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein (polypeptide) sequences from different sources, or the relationship between nucleic acids or proteins from different sources and host cells. For example, if the combination of nucleic acid / protein and host cell does not normally occur naturally, the nucleic acid is exogenous to the host cell. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0056] As used in the present invention, the "(expression) construct" or "(expression) construct" refers to a recombinant DNA molecule, which comprises a desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. The "construct" is usually contained in an expression vector.

[0057] As used in the present invention, the "expression cassette" refers to a gene expression system that contains all the necessary elements required to express the target interfering molecule, which generally includes the following elements: a promoter, a gene sequence that produces the interfering molecule in vivo; these elements are operably linked.

[0058] Modified intestinal commensal bacteria

[0059] The modified intestinal symbiotic bacteria described in the present invention are bacteria that can parasitize insect hosts and express interfering molecules, thereby inhibiting the insect hosts or causing disease in the insect hosts.

[0060] A variety of modified intestinal commensal bacteria can be used in the present invention, provided that they express the interfering molecules and are able to parasitize the host.

[0061] The insects involved in the present invention are generally the hosts of the modified intestinal symbiotic bacteria described in the present invention. In a preferred embodiment, the insects include: Diptera, Lepidoptera, Homoptera, Coleoptera, Hemiptera, Orthoptera, Hymenoptera, etc.

[0062] In some preferred embodiments, the modified intestinal commensal bacteria are Serratia.

[0063] Interfering molecules

[0064] In order to improve the efficiency of interfering molecules in inhibiting insects, the present inventors used intestinal symbiotic bacteria as carriers to transform exogenous constructs capable of producing interfering molecules into intestinal symbiotic bacterial cells, so that the bacteria can sustainably express the interfering molecules.

[0065] The technical solution of the present invention targets the interfering molecule to the functional gene in the insect host of the modified intestinal symbiotic bacteria, or interferes with the expression of the functional gene. Although the in vivo system of insects is complex, the inventors have determined, after in-depth research, that the inhibitory target is located at a gene of the juvenile hormone signaling pathway and the ecdysone signaling pathway.

[0066] In a more preferred embodiment of the present invention, the inventor locates the regulatory target at: Met gene or EcR gene. In a more preferred embodiment, the interfering molecule is dsRNA, the nucleotide sequence of which is shown in SEQ ID NO: 1, and the interfering molecule is targeted at Met gene (targeting nucleotides 2168-2650 in the coding region of Met gene); in a more preferred embodiment, the interfering molecule is dsRNA, the nucleotide sequence of which is shown in SEQ ID NO: 2, and the interfering molecule is targeted at EcR gene (targeting nucleotides 676-1173 in the coding region of Met gene).

[0067] The interfering molecules may include shRNA, dsRNA, siRNA, etc., provided that they can target the functional genes in the insect host of the modified intestinal symbiotic bacteria or interfere with the expression of the functional genes. In a preferred embodiment of the present invention, the interfering molecule is dsRNA.

[0068] Modified intestinal commensal bacteria

[0069] The present invention also provides a modified intestinal symbiotic bacterium, which expresses an interfering molecule. The interfering molecule targets a functional gene in an insect host of the modified intestinal symbiotic bacteria and interferes with the expression of the functional gene.

[0070] Typically, a construct is used to genetically engineer the engineered intestinal commensal bacteria, into which an exogenous polynucleotide is introduced to recombinantly express the interfering molecule, preferably dsRNA. The present invention utilizes a construct to express the interfering molecule.

[0071] In the present invention, the full-length sequence of a polynucleotide, a fragment thereof or a construct sequence containing the same can usually be obtained by PCR amplification, recombination or artificial synthesis.

[0072] The present invention also relates to an expression vector comprising the construct of the present invention. The construct encoding the polynucleotide sequence or containing the polynucleotide sequence can be inserted into a recombinant expression vector. "Recombinant expression vectors" suitable for use in the present invention include, but are not limited to, vectors expressed in bacteria. As long as they can replicate and be stable in bacteria, any plasmid or vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene, and a translation control element.

[0073] In the construct of the present invention, in addition to the polynucleotide sequence encoding the interfering molecule, a promoter, etc. is included. The promoter drives the expression of the interfering molecule; more preferably, the promoter includes: a constitutive promoter, an inducible promoter; more preferably, the promoter includes (but is not limited to): T5 promoter, T7 promoter, pj23119 promoter, etc.

[0074] In a specific embodiment of the present invention, dsRNA of the Met and EcR genes of Anopheles stephensi was synthesized in vitro by the T7 transcription system. Bioassay experiments have shown that adding dsRNA of the Met and EcR genes to water can reduce the expression of the Met and EcR genes in Anopheles stephensi larvae through RNAi and reduce the survival time of Anopheles stephensi larvae. RNase III was knocked out on the S.fonticola genome by the CRISPR / Cas9 gene editing method. Then, an expression plasmid was constructed by molecular cloning, and the dsRNA fragments of the Met and EcR genes were expressed in the plasmid using the T5 promoter. The dsRNA fragments containing the Met or EcR gene were cloned into the expression plasmid. The plasmid was transformed into the S.fonticola bacteria with RNase III knocked out, and fed to the Anopheles stephensi larvae. The results showed that the dsRNA expressed by the bacteria can play an RNAi role to reduce the expression of the Met and EcR genes in the Anopheles stephensi larvae and reduce the survival time of the Anopheles stephensi larvae.

[0075] Therefore, the method of using intestinal symbiotic bacteria to act on dsRNA expression vectors proposed in the present invention can be used for dsRNA production and RNAi-based pest control. This method can enable the dsRNA produced by bacteria to exert a lasting effect in the body of pests, and has important application value. Through this strategy, the number of pests can be effectively controlled and the damage to crops and human health by pests can be reduced, providing a feasible solution for agricultural production, ecological protection and public health.

[0076] Composition for controlling harmful insects and control method

[0077] The present invention also provides a composition for controlling harmful insects (such as a pesticide composition, a feed composition), wherein the composition contains: an effective amount of the modified intestinal symbiotic bacteria. More preferably, the composition further contains a pesticide-acceptable carrier.

[0078] The insect control composition of the present invention can be prepared into any suitable dosage form. For example, the form of the composition includes (but is not limited to): powder, granule, oil, emulsion, wettable powder, microcapsule, non-woven fabric agent.

[0079] In preparing the composition, suitable solid diluents include, but are not limited to, diatomaceous earth, corn husks, tricalcium phosphate, cork powder, clays such as kaolin, bentonite or attapulgite, or water-soluble polymers.

[0080] The solid composition may also contain one or more compatible wetting agents, dispersants, emulsifiers or pigments which in the solid state may also act as diluents.

[0081] The liquid composition may be in the form of a solution, suspension or emulsion, or may be encapsulated in a natural or synthetic polymer and may contain a wetting agent, dispersant or emulsifier. Such emulsions, suspensions or solutions may be prepared with aqueous, organic or aqueous-organic diluents for the preparation of water-soluble polymers (and mixtures of the above diluents). In addition, the diluent may contain, for example, an ionic or nonionic wetting agent, dispersant or emulsifier, or a mixture thereof, as described above.

[0082] The composition may be in concentrated form or diluted form. As a preferred embodiment of the present invention, the composition contains 1×10 3 ~1×10 12 The content of the bacteria in the composition can be appropriately increased or decreased according to the actual situation of the dosage form.

[0083] The objects or places suitable for the composition of the present invention to exert their effects (inhibitory effects, killing / killing effects) are not particularly limited, and may be (but not limited to): cereal crops (e.g., corn, wheat, rice, sorghum); ornamental crops, plantation crops and forest trees in fields, forests, plantations, greenhouses, orchards and vineyards, such as: cotton, tobacco, vegetables (e.g., beans, rapeseed, gourds, lettuce, onions, tomatoes, peppers), field crops (e.g., potatoes, beets, peanuts, soybeans, rapeseed), sugarcane, grasslands and forest trees (e.g., corn, sorghum, alfalfa), plantation crops (tea, coffee, cocoa, bananas, oil palm, coconut, rubber, spices), orchards and groves (e.g., stone fruits, citrus, kiwifruit, avocados, mangoes, olives, walnuts), vineyards, ornamental crops in greenhouses, gardens or parks, flowers and shrubs, and trees in forests, plantations and flower gardens (including deciduous trees and evergreen trees).

[0084] The present invention also provides a method for controlling insects, the method comprising: administering an effective amount of the modified intestinal symbiotic bacteria to a subject in need of controlling insects. In a preferred embodiment, the composition is directly administered to a subject in need of controlling insects.

[0085] Suitable methods of applying the compositions to growing plants include solid broadcasting, foliar spraying, liquid irrigation, and the like.

[0086] It will be appreciated by those skilled in the art that the application of the composition must vary with external conditions such as temperature, humidity, area to be treated, and plants to be treated. Therefore, the application ratio should vary within a wide range. The frequency of use of the composition of the present invention can be selected by farmers, disease control professionals, or other skilled in the art based on the desired effect.

[0087] The composition of the present invention is easy to apply, has high killing efficiency on harmful insects, and has no harm to the environment and other animals and plants except insects, and is an environmentally friendly composition.

[0088] In the previous technology in this field, a method of spraying dsRNA to control pests and diseases has been developed based on the mechanism of action of RNAi. However, the direct spraying method requires the use of a large amount of dsRNA, which greatly increases the production and application costs, and the dsRNA released into the environment is unstable and easily degraded, resulting in a short duration of control effect. The method of using Escherichia coli to ferment dsRNA requires the use of reagents such as lactose or isopropyl-β-d-thiogalactoside (IPTG) for induction, and the Escherichia coli that produces dsRNA is not a symbiotic bacteria in the intestine of the pest, and can only be applied once. In addition, since this type of bacteria cannot establish stable colonization in the intestine of the pest, the RNAi effect mediated by live bacteria in the intestine of the pest is limited, and inactivated bacterial agents are usually used for large-scale application. Therefore, the application cost is high, and it is impossible to achieve continuous control of pests. The present invention uses suitable intestinal symbiotic bacteria to change this situation.

[0089] Intestinal symbiotic bacteria are bacteria that stably parasitize in the intestines of animals. Some intestinal symbiotic bacteria can also spread horizontally and vertically within insect populations. Therefore, using intestinal symbiotic bacteria as dsRNA expression vectors can enable symbiotic bacteria that produce dsRNA applied once to colonize in the insect intestines for a long time and continue to produce dsRNA, or spread widely within the pest population. This method can achieve continuous interference with target genes and continuous control of pests, has the advantages of low economic cost and good persistence, and has important scientific significance and application value.

[0090] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0091] Example 1: Design and synthesis of dsRNAs for the Met and EcR genes of Anopheles stephensi

[0092] 1. Synthesis of dsRNA of the Anopheles stephensi Met gene

[0093] According to the CDS sequence of the Met gene of Anopheles stephensi (ASTE005389 in VectorBase), a fragment of about 483 bp was selected, and the sequence is as follows (SEQ ID NO: 1):

[0094] 5'-CGAGCTGCTTTAGCGATCTTATCAATCCCGGCACTGACCTCACTGCGTTGCTAC

[0095] CAAACTCATTCGACGCACTCGATCAATCGCTGCTGACGATGGAAACGACAACGAC

[0096] CATAATACGCGACCGGGTGAGCAACTACTCAAATCTACACGAATCGCAGGACCGC

[0097] TTGGAAAGGATAGTGGAAGAACATCAAGAGCAGCGAGAAATGCTACAGTCAATT

[0098] CAGGAGGAGTACCAACTGCAGATGCAGTCCAACGGGACAGGTGATGCAATAGGA

[0099] CCGGCCGCTGTACATAACAGTGAGGCCCATAGTAGAGGTGTCGGTGGTGGTGGTG

[0100] GTGGTGGCTTAACGACCAACACAACTACCGCTTCACATCTGTACGGTAGCAGTGC

[0101] CGATGCGAATGGAATGTTGTGCAATTCACTAAGCGAGGGACCGATGGGACGCCG

[0102] GAGAGTAGTCAGTACGCTCACCAGCACAACCACAACAACGACGTCAA-3'.

[0103] RNA from Anopheles stephensi was extracted, cDNA was obtained by reverse transcription, and Met dsRNA template was amplified by PCR;

[0104] The PCR upstream primer sequence is:

[0105] 5'-taatacgactcactatagggCGAGCTGCTTTAGCGATCTT-3' (SEQ ID NO: 4);

[0106] The downstream primer sequence is:

[0107] 5'-taatacgactcactatagggTTGACGTCGTTGTTGTGGTT-3' (SEQ ID NO: 5);

[0108] Among them, taatacgactcactataggg is the T7 promoter sequence.

[0109] The PCR product was purified and recovered, and the Met dsRNA template was used as a template to synthesize Met dsRNA through the T7 transcription system ( Figure 1 A).

[0110] 2. Synthesis of dsRNA of Anopheles stephensi EcR gene

[0111] According to the CDS sequence of the EcR gene of Anopheles stephensi (ASTE005081 in VectorBase), a fragment of about 498 bp was selected, and the sequence is as follows (SEQ ID NO: 2):

[0112] 5'-ATCCACTTCCGGCACATAACGGAAATCACCATTCTCACAGTACAACTTATCGTC

[0113] GAGTTCGCGAAGGGACTGCCAGCATTTACCAAGATCCCGCAGGAGGATCAAATTA

[0114] CGTTACTAAAAGCGGTGCTCCAGTGAGGTGATGATGTTGCGAATGGCCCGCCGGTA

[0115] CGACGCCGAAAACCGACTCCATCCTCTTTGCCAACAACCGATCGTACACGCGCGAC

[0116] TCGTACAAGATGGCGGGCATGGCGGACACGATCGAGGACCTGCTACACTTCTGCC

[0117] GGCAGATGTACACGCTCACGGTGGACAACGTCGAGTACGCGCTGCTCACCGCGAT

[0118] CGTCATCTTCTCCGACCGGCCCGGGCTCGAGAAGGCCGAACTGGTCGAGACGATC

[0119] CAGAGCTACTACATCGACACGCTGCGCGTCTACATCCTGAACCGGCACGGGGGCG

[0120] ATCCGAAGTGTAGCGTCACGTTCGCCAAGCTCCTTTCCATACTGACCGAGCTTCG

[0121] GACT-3'.

[0122] RNA from Anopheles stephensi was extracted, cDNA was obtained by reverse transcription, and EcR dsRNA template was amplified by PCR;

[0123] The PCR upstream primer sequence is:

[0124] 5'-taatacgactcactatagggATCCACTTCCGGCACATAAC-3' (SEQ ID NO: 6);

[0125] The downstream primer sequence is:

[0126] 5'-taatacgactcactataggggAGTCCGAAGCTCGGTCAGTA-3' (SEQ ID NO: 7);

[0127] Among them, taatacgactcactataggg is the T7 promoter sequence.

[0128] The PCR product was purified and recovered, and the EcR dsRNA was used as a template to synthesize EcR dsRNA through the T7 transcription system ( Figure 1 B).

[0129] Example 2: dsRNA of Met and EcR genes can kill Anopheles stephensi larvae

[0130] On the first day after hatching, Anopheles stephensi larvae were placed in a culture dish containing deionized water, with 50 larvae in each group. dsRNA of Met and EcR genes were added to the culture dishes of different groups at a final concentration of 10 μg / ml as treatment groups. The control group was added with dsRNA of green fluorescent protein gene (GFP). The survival of the larvae was recorded every day. The rearing conditions of Anopheles stephensi larvae were 28°C, and the larvae were fed with feed.

[0131] The statistical results showed that compared with the larvae of Anopheles stephensi added with GFP dsRNA, the larvae of Anopheles stephensi added with Met and EcR gene dsRNA had a shorter survival time, indicating that the dsRNA of Met and EcR gene can cause the death of Anopheles stephensi larvae ( Figure 2 ).

[0132] Example 3: Isolation of Anopheles stephensi intestinal symbiotic bacteria

[0133] Dissect the midgut tissue of Anopheles stephensi in phosphate buffer solution (PBS). Place the midgut in a 1.5 mL centrifuge tube with PBS and grind it with a grinding rod. Apply the grinding solution on an LB medium plate, and then place the plate in a biochemical incubator at 28°C for culture. After bacterial colonies grow on the LB plate, select a single colony and add it to LB liquid medium for culture.

[0134] Subsequently, bacterial genomic DNA was extracted, and the bacterial 16S ribosomal DNA sequence was amplified and sequenced by PCR. The intestinal symbiotic bacteria were identified as Serratia fonticola ( Figure 3 ; SEQ ID NO:3).

[0135] TAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAG

[0136] TCGAGCGGTAGCACAGGGAGCTTGCTCCTGGGTGACGAGCGGCGGACGGGTGAGTA

[0137] ATGTCTGGGAAACTGCCCGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATAC

[0138] CGCATAACGTCTTCGGACCAAAGTGGGGGACCTTCGGGCCTCACACCATCGGATGTG

[0139] CCCAGATGGGATTAGCTAGTAGGTGAGGTAATGGCTCACCTAGGCGACGATCCCTAG

[0140] CTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTAC

[0141] GGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCC

[0142] GCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGGTT

[0143] CAGTGTTAATAGCACTGTTCATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCC

[0144] GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCG

[0145] TAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGAGCTTAACTTGG

[0146] GAACTGCATTTGAAACTGGCAAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGG

[0147] TGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCC

[0148] TGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATAC

[0149] CCTGGTAGTCCACGCTGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGG

[0150] CTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAA

[0151] ACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAT

[0152] GCAACGCGAAGAACCTTACCTACTCTTGACATCCACAGAACTTTCCAGAGATGGATT

[0153] GGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTG

[0154] AAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCACGT

[0155] CATGGTGGGAACTCAAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGA

[0156] CGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATA

[0157] CAAAGAGAAGCGAACTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCG

[0158] GATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTAGATCAG

[0159] AATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGA

[0160] GTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTG

[0161] ATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCA

[0162] Example 4: Construction of an RNase III knockout intestinal symbiotic bacterium

[0163] The CRISPR / Cas9 system was used to construct RNase III knockout Serratia fonticola, and the gRNA targeting RNase III gene was constructed ( Figure 4 A) plasmid, a plasmid expressing Cas9 protein and a plasmid expressing RNase III gene gRNA were co-transformed into Serratia fonticola.

[0164] gRNA: GCCUGUUUAUUACGAUG (SEQ ID NO:8);

[0165] genome DNA1: ATGAACCCCATCGTAATAAACAGGCTGCAGCGGAAGCTGG (SEQ ID NO: 9);

[0166] The bacterial genome was extracted, the RNase III gene sequence was amplified using PCR, and the amplified product was sequenced.

[0167] By comparing and analyzing the sequencing results, it was confirmed that the RNase III gene in Serratia fonticola had been successfully knocked out ( Figure 4 B).

[0168] Example 5: Construction of intestinal symbiotic bacteria expressing Met and EcR gene dsRNA

[0169] 1. Construction of vector expressing Met gene dsRNA

[0170] The T7 promoter activated by lactose or IPGT in the L4440 plasmid (a common plasmid for bacterial induction of dsRNA) was replaced by the bacteriophage T5 promoter (sequence: 5'-TCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATA-3' (SEQ ID NO: 10)) to construct the constitutive expression of dsRNA expression plasmid L4T5 ( Figure 5 A), L4T5 plasmid produces dsRNA without the need for induction.

[0171] The sequence of the dsRNA fragment of the Met gene of Anopheles stephensi is as described in Example 1. Anopheles stephensi RNA was extracted, reverse transcribed to obtain cDNA, and PCR was used to amplify the Met dsRNA template.

[0172] The PCR upstream primer sequence is:

[0173] 5'-ACAGGAATTGCCGGC AGATCTCGAGCTGCTTTAGCGATCTTATC-3'

[0174] (SEQ ID NO: 11);

[0175] The downstream primer sequence is:

[0176] 5'-TTTCACACAGGAATT GGTACC TTGACGTCGTTGTTGTGGTTG-3' (SEQ ID NO: 12);

[0177] The upstream primer has a BglII restriction site, and the downstream primer has a KpnI restriction site. The PCR fragment and the vector L4T5 are digested with restriction endonucleases BglII and KpnI, respectively. After the digestion reaction, the target fragment and plasmid are purified and recovered. The recovered target fragment and the vector are ligated with T4 DNA ligase to construct the vector L4T5-dsMet( Figure 5 B).

[0178] 2. Construction of vector expressing EcR gene dsRNA

[0179] The sequence of the dsRNA fragment of the Anopheles stephensi EcR gene is as described in Example 1. Anopheles stephensi RNA was extracted, reverse transcribed to obtain cDNA, and PCR was used to amplify the EcR dsRNA template.

[0180] The PCR upstream primer sequence is:

[0181] 5'-ACAGGAATTGCCGGC AGATCT ATCCACTTCCGGCACATAACG-3'(SEQ ID NO:13),

[0182] The downstream primer sequence is:

[0183] 5'-TTTCACACAGGAATT GGTACC AGTCCGAAGCTCGGTCAGTATG-3'

[0184] (SEQ ID NO: 14);

[0185] Among them, the upstream primer has a BglII restriction site, and the downstream primer has a KpnI restriction site. The PCR fragment and the vector L4T5 are digested with restriction endonucleases BglII and KpnI, respectively. After the digestion reaction, the target fragment and plasmid are purified and recovered. The recovered target fragment and the vector are ligated with T4 DNA ligase to construct the vector L4T5-dsEcR ( Figure 5 ).

[0186] 3. Strain construction

[0187] The dsRNA expressing vectors L4T5-dsMet and L4T5-dsEcR were transformed into the RNaseIII knockout Serratia fonticola established in Example 4, respectively, to obtain Serratia fonticola strains S. fonticola-dsMet and S. fonticola-dsEcR expressing Met or EcR gene dsRNA, respectively.

[0188] Example 6: Intestinal symbiotic bacteria expressing Met and EcR gene dsRNA can kill Anopheles stephensi larvae

[0189] The first day after hatching, Anopheles stephensi larvae were placed in a culture dish containing deionized water, with 50 larvae in each group. 5×10 5 The control group was fed with agar mixed feed containing 5×10 5 pfuSerratia fonticola-dsGFP bacteria on agar. Larval survival was recorded daily. Larvae of Anopheles stephensi were reared at 28°C.

[0190] Statistical results showed that compared with Anopheles stephensi larvae fed with agar mixed diet containing Serratia fonticola-dsGFP bacteria, Anopheles stephensi larvae fed with agar mixed diet containing Serratia fonticola-dsMet or Serratia fonticola-dsEcR bacteria had a shorter survival time.

[0191] This indicates that Serratia fonticola expressing Met or EcR gene dsRNA can kill Anopheles stephensi larvae ( Figure 6 ).

[0192] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for controlling harmful insects, comprising: (1) Providing intestinal symbiotic bacteria, wherein the intestinal engineered bacteria can take the harmful insect as a host and coexist in the intestine of the harmful insect; (2) introducing an expression construct of an interfering molecule into the intestinal symbiotic bacteria; the interfering molecule specifically targets the functional gene of the harmful insect; (3) The intestinal symbiotic bacteria are applied to the harmful insects, symbiotically coexisting in the intestines of the harmful insects and expressing the interfering molecules, thereby controlling the harmful insects by inhibiting the functional genes of the harmful insects.

2. The method according to claim 1, characterized in that The functional genes of harmful insects include: genes that determine the survival ability of harmful insects; preferably include genes that regulate physiological functions such as immunity or metabolism; more preferably, the functional genes include: Toll immune pathway, IMD immune pathway, Jak-STAT immune pathway, juvenile hormone signaling pathway, ecdysone signaling pathway and other related genes; more preferably, the functional genes include: Met, EcR genes.

3. The method according to claim 1, characterized in that The interfering molecules include: interfering molecules that can interfere with the functional genes of the harmful insects and thus affect the survival of the harmful insects; preferably, the interfering molecules are dsRNA.

4. The method according to claim 2 or 3, characterized in that The functional gene includes Met gene or EcR gene; Preferably, the interfering molecule is dsRNA, the nucleotide sequence of which is as shown in SEQ ID NO: 1, and targets and interferes with the Met gene; Preferably, the interfering molecule is dsRNA, the nucleotide sequence of which is as shown in SEQ ID NO: 2, and targets and interferes with the EcR gene; More preferably, the expression of the interfering molecule is driven by a promoter; More preferably, the promoter includes: a constitutive promoter, an inducible promoter; more preferably, the promoter includes: a T5 promoter, a T7 promoter, and a pj23119 promoter.

5. The method according to claim 1, characterized in that The intestinal symbiotic bacteria include: Serratia, Enterobacter, Acinetobacter, Chromobacterium, Asaia; preferably, no endogenous molecules that affect the expression of the interfering molecules exist in the intestinal symbiotic bacteria; more preferably, nucleases do not exist, are inactivated or knocked out in the intestinal symbiotic bacteria.

6. The method according to claim 1, characterized in that The harmful insects are selected from the following groups: Diptera, Homoptera, Lepidoptera, Coleoptera, Hemiptera, Orthoptera, Hymenoptera, Neuroptera; preferably Diptera, Culicidae.

7. Use of modified intestinal symbiotic bacteria for controlling harmful insects or preparing a composition for controlling harmful insects; wherein: The intestinal symbiotic bacteria can take the harmful insect as a host and coexist in the intestine of the harmful insect; the modified intestinal symbiotic bacteria contain an expression construct of an interfering molecule; and the interfering molecule specifically targets the functional gene of the harmful insect.

8. A modified intestinal symbiotic bacterium, which can use the harmful insect as a host and coexist in the intestine of the harmful insect; the modified intestinal symbiotic bacteria contains an expression construct of an interfering molecule; the interfering molecule specifically targets the functional gene of the harmful insect.

9. The use according to claim 7 or the intestinal commensal bacteria according to claim 8, characterized in that The functional genes of harmful insects include: genes that determine the survival ability of harmful insects; preferably include genes that regulate physiological functions such as immunity or metabolism; more preferably, the functional genes include: Toll immune pathway, IMD immune pathway, Jak-STAT immune pathway, juvenile hormone signaling pathway, ecdysone signaling pathway and other related genes; more preferably, the functional genes include: Met, EcR genes; or The interfering molecules include: interfering molecules that can interfere with the functional genes of the harmful insects and thus affect the survival of the harmful insects; preferably, the interfering molecules are dsRNA; and / or The intestinal symbiotic bacteria include: Serratia, Enterobacter, Acinetobacter, Chromobacterium, Asaia; preferably, no endogenous molecules that affect the expression of the interfering molecule exist in the intestinal symbiotic bacteria; more preferably, nucleases do not exist, are inactivated or knocked out in the intestinal symbiotic bacteria; and / or The harmful insects are selected from the following groups: Diptera, Homoptera, Lepidoptera, Coleoptera, Hemiptera, Orthoptera, Hymenoptera, Neuroptera; preferably Diptera, Culicidae.

10. A composition for controlling harmful insects, comprising: The modified intestinal commensal bacteria according to claim 8 or 9; and a pesticide-acceptable carrier or excipient.

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