Monochamus alternatus chitinase-2 and application thereof

Abstract

The application relates to the technical field of gene technology, in particular to a monochamus alternatus chitins degradation key regulation gene and application thereof. MaCht-2 The application provides a monochamus alternatus chitins degradation key regulation gene and application thereof. MaCht-2 MaCht-2 The gene can be used as a novel prevention and treatment target in efficient prevention and treatment of monochamus alternatus. The dsRNA synthesized according to the application has the advantages of strong target specificity, good environmental compatibility and the like, can realize accurate correspondence and targeted regulation in the biological prevention and treatment process of monochamus alternatus, and can develop a novel green and environmentally-friendly RNA pesticide, so that the application prospect is wide.​

Description

Technical Field

[0001] This invention relates to the field of gene technology, and in particular to a key regulatory gene for chitin degradation in the longhorn beetle *Pinus sylvestris*. MaCht-2 And its applications. Background Technology

[0002] Pine pine longhorn beetle Monochamus alternatus It is the main vector insect for pine wilt disease in Asia. Pine wilt disease can occur on a large scale in Asia, along with the pine sawyer beetle and the pine wood nematode. Bursaphelenchus xylophilus The two species have closely related life histories: when the pine sawyer beetle pupates inside the tree, the pine wood nematode's dispersing third instar (L) stage... Ⅲ ) gather in the pupal chamber of the pine beetle, and as the pupae develop, the dispersing third instar larvae (L Ⅲ ) transforms into a dispersing 4th instar larva (L Ⅳ The insects enter the host tree when the longhorn beetle emerges, and then leave the beetle while the adult beetle is feeding or laying eggs on the host tree. Even carrying tens of thousands of pine wood nematodes, the pine sawyer beetle can maintain stable life activities, and the vector-response mechanism involved is key to the "cooperative" relationship between the two.

[0003] Currently, methods for controlling vector insects include biological control, chemical control, attractant (wood) killing, and silvicultural control. While all these methods can achieve some degree of control, they suffer from high costs, difficulty in controlling the duration of control, and environmental pollution. Therefore, finding a safe, reliable, and efficient method for controlling vector insects is urgently needed.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a key regulatory gene for chitin degradation in the longhorn beetle *Pinus sylvestris*. MaCht-2 And its applications. Utilizing MaCht-2 The dsRNA of the gene achieves the control of pine sawyer beetle and pine wood nematode through RNAi-mediated means.

[0006] Specifically, the solution provided by this invention is as follows: This invention provides a chitin degradation gene from the pine longhorn beetle. MaCht-2 , MaCht-2 The nucleotide sequence is shown in SEQ ID NO: 01.

[0007] This invention provides the chitin degradation gene of the pine beetle. MaCht-2 The encoded protein has the amino acid sequence shown in SEQ ID NO: 02.

[0008] This invention provides a method for reducing the normal mobility of the pine sawyer beetle, comprising: inhibiting the gene in the pine sawyer beetle. MaCht-2 Or the expression level and / or activity of the protein.

[0009] This invention provides a method for targeting the gene described in claim 1. MaCht-2 dsRNA.

[0010] The present invention provides a primer set for synthesizing the dsRNA, including a first primer pair and a second primer pair; the upstream primer nucleotide sequence of the first primer pair is shown in SEQ ID NO: 03, and the downstream primer nucleotide sequence is shown in SEQ ID NO: 04; the upstream primer nucleotide sequence of the second primer pair is shown in SEQ ID NO: 05, and the downstream primer nucleotide sequence is shown in SEQ ID NO: 06.

[0011] The present invention provides a method for preparing the dsRNA, which is synthesized using a dsRNA synthesis kit with the nucleotide sequence shown in SEQ ID No: 1 as a template; or, using cDNA of the longhorn beetle as a template, dsRNA is synthesized in vitro after PCR amplification with the primer set.

[0012] This invention provides the application of the dsRNA or its encoded nucleic acid or biological materials containing them in the control of pine wood nematode and / or pine sawyer beetle.

[0013] Preferably, the application method is to introduce the dsRNA into the body of the longhorn beetle.

[0014] Preferably, the method of introduction includes feeding and / or microinjection.

[0015] This invention provides at least one of the following applications of the dsRNA or its encoded nucleic acid or biological materials containing them: (1) Regulating the growth and development of the pine sawyer beetle; (2) Preparation of products that regulate the growth and development of the longhorn beetle; (3) Reduces the normal mobility of the pine saw beetle; (4) Prepare products that reduce the normal mobility of the pine sawyer beetle; (5) Reduce the population size of the pine sawyer beetle; (6) Prepare products that reduce the population size of the pine sawyer beetle; (7) Prepare products for the prevention and control of pine wood nematode; (8) Prepare products for controlling the pine sawyer beetle.

[0016] This invention provides a class of insect-repellent products comprising the dsRNA or its encoded nucleic acid or biological materials containing them; Preferably, the biomaterial includes an expression cassette, a recombinant vector, or a recombinant microorganism.

[0017] Beneficial effects: This invention provides a key regulatory gene for chitin degradation in the longhorn beetle *Pinus sylvestris*. MaCht-2 and its applications. The aforementioned MaCht-2 Genes can serve as a novel control target for the efficient control of the pine sawyer beetle. The dsRNA synthesized in this invention possesses numerous advantages, including strong targeting and good environmental compatibility, enabling precise targeting and regulation in the biological control of the pine sawyer beetle. This allows for the development of novel, environmentally friendly RNA pesticides with broad application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0019] Figure 1 Injecting 5ug, 10ug, or 15ug of the present invention dsMaCht-2 and dsGFP Relative expression levels at 12h, 24h, and 48h.

[0020] Figure 2 Injection for the present invention dsMaCht-2 Phenotypic comparison of *Pseudomonas pulmonarius*, all phenotypes in the figure are those of individuals injected with 5 μg dsRNA. dsGFP, dsMaCht-2 The developmental phenotype after ( ). Detailed Implementation

[0021] Currently, the most widely implemented methods for controlling the pine sawyer beetle in forests are burning infected trees, releasing natural enemy insects, and setting traps. Among these, the released natural enemy insects are mainly the flower beetle. Dastarcus helophoroides The main distribution area of ​​the flower beetle in China is in the north. After being released in the southern regions where pine wilt disease is more severe, it is difficult to establish a population there, requiring a large amount of manpower and resources every year. On the other hand, the longhorn beetles captured by traps may have already completed the process of spreading pine wilt disease, so they cannot achieve the purpose of controlling the spread of pine wilt disease.

[0022] RNA interference (RNAi) refers to the phenomenon where endogenous or exogenous double-stranded RNA (dsRNA) induces highly efficient and specific degradation of homologous mRNA, leading to the silencing or downregulation of target genes. Using RNA interference (RNAi) technology for pest control has become a research hotspot, as this method offers advantages such as short development cycles, high specificity, and environmental friendliness. RNAi technology's high efficiency and relatively good specificity have advanced research into biological gene function and provided new strategies and approaches for pest control. Compared to traditional pest control techniques, RNAi technology offers the following advantages: ① High specificity and precise targeting, without negatively impacting other non-target insects, especially natural enemies, facilitating the implementation of integrated pest management systems. ② The introduced gene sequence has no high sequence similarity in crops and animals, thus not affecting crop growth and ensuring safety for humans and animals. ③ RNA molecules are unstable and easily degraded, leaving no residual pollution problems and ensuring high safety for the ecological environment and humans and animals. ④ Pests do not develop resistance. In recent years, RNAi has been successfully applied to pests of various orders, including Hemiptera, Lepidoptera, Coleoptera, Diptera, and Orthoptera, under laboratory conditions. There are three main methods for introducing RNAi into insects: feeding, injection, and tissue culture. The optimal method must be selected based on the specific experimental requirements. The key to using this method to control pests is identifying target genes that can affect the normal life activities of pests.

[0023] Chitinases are the most common chitin-degrading enzymes, participating in the degradation of chitin in insect tissues and organs such as the epidermis, trachea, and peritrophic membrane. During insect development, the degradation of the old epidermis and the synthesis of the new epidermis are indispensable parts of the insect's life cycle. Abnormal expression of genes related to chitin degradation and synthesis can have fatal consequences for insects. Furthermore, chitinases also participate in the defense against pathogen invasion, primarily involving immune signal regulation, and are an important component of the insect immune system.

[0024] This invention provides a chitinase gene that plays a role in chitin degradation during the growth and development of the pine sawyer beetle. MaCht-2 This invention provides a method for synthesizing corresponding dsRNA and its application in RNAi-mediated pest control. Based on RNA interference technology, the gene is prepared and injected via microinjection into the 3rd and 5th instars and pupae of the pine sawyer beetle to silence the target gene, thereby affecting its normal developmental processes such as molting, pupation, and emergence, resulting in a high mortality rate. This invention confirms... MaCht-2 Genes can serve as a novel control target in the efficient control of the pine sawyer beetle.

[0025] In a more specific embodiment, the technical solution adopted by the present invention includes the following: Firstly, this invention provides a gene related to chitin degradation in the longhorn beetle *Pinus sylvestris*. MaCht-2

[0026] Secondly, this invention provides genes related to the chitin degradation of the aforementioned longhorn beetle. MaCht-2 The encoded protein has the following amino acid sequence: ISQRWLRQKTVCHRCRRFPTQIWL*GIAF*LELPCLLAKQLQKGTFVR*NQLP*TSPGTEEGV*QRKPTTGAGSFNFWIQRSDRRCL*PNDLRANFRFHISDDL*LPWSLGTSNWSCESLVPASQRQISSV*FELHYGIFGISGSP*RKTFDGCSVLRAKFHVNKRYQLWRKCAKFWPGRTRR IYQATWNVGILRSL*QNQKSKMDFKF**CFWRAICL**EPVGWLRRCQFH*RKGKLHQN*RLWRRCRGLDHRPR*LQ*SMLRGNLPASTFFELRTRPYHRQ THQRRLH*TPSTINTRTS*NHNWSRFWSSKFHHRTHTR*MDKQHFKETNYICLVDNYEKAYK*QAHY*SLVDE*TIFVVVI*AINYLEPMVVNVVF (SEQ ID NO:2).

[0027] Based on the above-disclosed sequence, those skilled in the art can obtain proteins with the same function by substituting and / or deleting and / or adding one or more amino acid residues.

[0028] Thirdly, the present invention provides a method for reducing the normal mobility of the pine sawyer beetle.

[0029] The method for reducing the normal mobility of the pine sawyer beetle provided by the present invention includes the step of reducing the expression level and / or activity of the above-mentioned proteins in the pine sawyer beetle, thereby reducing its normal mobility.

[0030] Furthermore, the method for reducing the expression level and / or activity of the aforementioned proteins in the pine sawyer beetle is to introduce a substance that inhibits the expression of the gene encoding the aforementioned proteins in the pine sawyer beetle into its body via microinjection.

[0031] Furthermore, the substance that inhibits the expression of the gene encoding the aforementioned protein in the pine longhorn beetle is a dsRNA that inhibits the expression of the gene encoding the aforementioned protein in the pine longhorn beetle.

[0032] Fourthly, this invention provides a method for targeting the chitinase gene of the longhorn beetle. MaCht-2 The dsRNA, specifically, is composed of molecules that can inhibit... MaCht-2A double-stranded RNA composed of nucleotides that affect gene expression and their inverse complementary nucleotide sequences.

[0033] Fifthly, the present invention provides a primer set for preparing the dsRNA ( MaCht-2 Primer set).

[0034] Furthermore, the primer set includes a first primer pair and a second primer pair; the nucleotide sequence of the upstream primer of the first primer pair is as follows: 5′-GGATCCTAATACGACTCACTATAGGGTCCCAGCGACAAATATCCTC-3′ (SEQ ID NO:3); the nucleotide sequence of the downstream primer of the first primer pair is as follows: 5′-AACGTAAAAGCGGGAAGGTT-3′ (SEQ ID NO:4). The nucleotide sequence of the upstream primer of the second primer pair is as follows: 5′-TCCCAGCGACAAATATCCTC-3′ (SEQ ID NO:5), and the nucleotide sequence of the downstream primer of the second primer pair is as follows: 5′-GGATCCTAATACGACTCACTATAGGGAACGTAAAAGCGGGAAGGTT-3′ (SEQ ID NO:6). The underlined region GGATCCTAATACGACTCACTATAGGG (SEQ ID NO:7) is the T7 RNA polymerase promoter sequence.

[0035] Sixthly, the present invention provides a longhorn beetle. MaCht-2 Methods for preparing gene-specific dsRNA.

[0036] Furthermore, the method for preparing the specific dsRNA includes: (1) Using the cDNA of the longhorn beetle as a template, PCR amplification was performed using the above primers.

[0037] (2) After amplification, gel purification was performed to synthesize dsRNA in vitro.

[0038] Seventhly, the present invention provides a method for inhibiting the growth of pine sawyer beetles. MaCht-2 Application of gene-expressing agents in the control of pine sawyer beetle.

[0039] Furthermore, the product for controlling the pine sawyer beetle according to the present invention contains the above-mentioned inhibitors of the pine sawyer beetle. MaCht-2 The dsRNA expressed by the gene or the nucleotide sequence encoding the dsRNA or biological material containing them.

[0040] This invention introduces the dsRNA, or the nucleotide sequence encoding the dsRNA, or biological material containing them into the body of the pine longhorn beetle to inhibit the pine longhorn beetle.MaCht-2 Gene expression reduces the activity of chitinase in the pine sawyer beetle, causing molting failure and decreased normal mobility, thereby achieving the goal of controlling the pine sawyer beetle.

[0041] Furthermore, in a specific implementation method, dsRNA is introduced into the 3rd instar larvae, 5th instar larvae, and pupae of the pine sawyer beetle via microinjection. After specifically silencing the target gene, the molting process of the pine sawyer beetle is affected, ultimately leading to death.

[0042] Furthermore, the dsRNA injection site is the folds of the second and third abdominal segments of the 3rd and 5th instar larvae, and the junction of the thorax and abdomen of the pupa. The injection dose can be selected as 5 μg, 10 μg, or 15 μg per larva.

[0043] Eighthly, the present invention further provides at least one application of the above-mentioned dsRNA or biological material as follows: (1) Regulate the growth and development of the pine sawyer beetle.

[0044] (2) Prepare products that regulate the growth and development of the pine sawyer beetle.

[0045] (3) Control the pine sawyer beetle.

[0046] (4) Prepare products for controlling the pine sawyer beetle.

[0047] (5) Reduce the normal mobility of the pine saw beetle.

[0048] (6) Prepare products that reduce the normal mobility of the pine saw beetle.

[0049] (7) Reduce the population size of the pine longhorn beetle.

[0050] (8) Prepare products that reduce the population size of the pine sawyer beetle.

[0051] In a ninth aspect, the present invention also provides at least one of the following biomaterials: (1) The nucleic acid molecule that encodes the above dsRNA.

[0052] (2) An expression cassette containing the nucleic acid molecule, a recombinant vector or a recombinant microorganism.

[0053] By utilizing the above-described technical solution provided by this invention, the present invention has the following beneficial effects: First, the dsRNA synthesized in this invention has many advantages such as strong targeting and good environmental compatibility, which can achieve precise response and targeted regulation in the biological control of pine sawyer beetle.

[0054] Furthermore, the dsRNA of the present invention can be prepared by biosynthesis and can be sustainably processed during the production process, thereby enabling the development of novel green and environmentally friendly RNA pesticides.

[0055] Furthermore, the dsRNA synthesis method provided by this invention can serve as a research reference for similar studies. Those skilled in the art can easily use known methods to mutate the nucleotide sequence encoding the aforementioned protein. Artificially modified nucleotides that have a similar nucleotide sequence to the protein isolated by this invention, as long as they encode the aforementioned protein and have the same function, are all derived from and equivalent to the nucleotide sequence of this invention.

[0056] Furthermore, the application of the above-mentioned dsRNA products and methods in the control of the pine sawyer beetle also falls within the scope of protection of this invention.

[0057] Furthermore, this invention clones from the pine sawyer beetle. MaCht-2 The gene was synthesized and the corresponding specific double-stranded RNA interference sequence fragment dsRNA was prepared. The dsRNA was then introduced into the *Pseudomonas pineinae* longhorn beetle using an injection method. MaCht-2 RNAi was performed on the gene. Experiments demonstrated that the specific double-stranded RNA interference fragment dsRNA successfully interfered with the mRNA expression level of the corresponding gene in the pine sawyer beetle, leading to molting failure and affecting mobility by 100%, demonstrating significant interference efficiency. This indicates that the gene plays a crucial role in the growth and development of the pine sawyer beetle, and this invention also provides a new approach for developing novel insecticides for this beetle.

[0058] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0059] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0060] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0061] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0062] In the following examples, the insects used in the tests were all obtained from dissection of *Pinus massoniana* in Quannan County, Ganzhou City, Jiangxi Province, and were reared to the required developmental stage in the quarantine laboratory of Beijing Forestry University. The rearing process was as follows: After the larvae of the pine sawyer beetle collected from the wild were disinfected, they were placed in an artificial climate chamber. The rearing conditions were a temperature of 25±1℃, a humidity of 60%-70%, and a photoperiod of L:D=0:24 h. They were reared using artificial feed until the required developmental stage was reached.

[0063] Example 1 This embodiment provides a longhorn beetle. MaCht-2 Gene cloning methods (1) Extraction and amplification of the full-length RNA sequence from the longhorn beetle: Total RNA was extracted from the pine longhorn beetle using the MolPure® Cell / Tissue Total RNA Kit according to the manufacturer's instructions. The RNA was then reverse transcribed into cDNA and amplified by PCR using primers designed using Primer-BLAST (Primer designing tool (nih.gov)).

[0064] The upstream primer sequence for amplification is: 5′-ATTAGTCAGCGATGGCTCCG-3′ (SEQ ID NO:8).

[0065] The downstream primer sequence for amplification is: 5′-AGAAGACGACGTTGACCACC-3′ (SEQ ID NO:9).

[0066] After amplification, a full-length sequence of 1138 bp was obtained (SEQ ID NO:1).

[0067] The PCR amplification system consisted of: 25 μL of PCR 2 × Phanta Max Buffer (Novizan), 2 μL each of forward and reverse primers (10 μM concentration), 1 μL of cDNA template, and 20 μL of ddH2O.

[0068] The reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; 72℃ extension for 5 min.

[0069] (2) Recovery and cloning of PCR products: PCR products were electrophoresed on a 1% agarose gel prepared by TAE. The gel fragment containing the target band was then cut off with a blade and placed into a sterile centrifuge tube. The target band was then purified using a gel extraction kit (Axygen) according to the manufacturer's instructions. The ligation product was then obtained by ligating the PEASY-Blunt3 vector. This ligation product was added to competent cells for cloning and plate plating.

[0070] (3) Sequencing: The positive clones were sent to Beijing Ruibo Company for sequencing.

[0071] Sequencing results showed that a 1138 bp DNA fragment was amplified, and its nucleotide sequence is shown in SEQ ID NO:1.

[0072] Example 2 This embodiment provides a longhorn beetle. MaCht-2 Methods for preparing dsRNA of the gene and control gene.

[0073] (1) Primers were designed based on the cloned gene fragment, and the T7 promoter was introduced at the 5′ end. PCR amplification was performed using the two primer pairs shown in SEQ ID NO:3-6 above. The template used for amplification was the cDNA from Example 1. The two amplification products obtained were confirmed by electrophoresis and could be used as forward and reverse templates for dsRNA synthesis (refer to the Novizan T7RNAi Transcription Kit instructions). The concentration of dsRNA was detected by a micro spectrophotometer, and 1 μL of dsRNA was confirmed by 1% agarose gel electrophoresis and stored at -80℃ for later use.

[0074] (2) Control group GFP The dsRNA of the gene. The synthesis method is as described above. MaCht-2 The dsRNA of a gene. The template is containing... GFP Plasmids containing gene fragments.

[0075] The first primer set has the following sequence: forward primer sequence 5′-GGATCCTAATACGACTCACTATAGGGACGTAAACGGCCACAAGTTC-3′ (SEQ ID NO:10), and reverse primer sequence 5′-TGTTCTGCTGGTAGTGGTCG-3′ (SEQ ID NO:11). The second primer set has the following sequence: forward primer sequence 5′-ACGTAAACGGCCACAAGTTC-3′ (SEQ ID NO:12), and reverse primer sequence 5′-GGATCCTAATACGACTCACTATAGGGTGTTCTGCTGGTAGTGGTCG-3′ (SEQ ID NO:13).

[0076] Example 3 This embodiment provides a longhorn beetle. MaCht-2 The application of genes and the detection of their silencing effects.

[0077] (1) Take the synthesized in Example 2. MaCht-2 dsRNA of genes ( dsMaCht-2 )and GFP dsRNA of a gene (denoted as dsGFP 5 μg, 10 μg, and 15 μg were injected via microinjection into the second and third abdominal folds of 3rd and 5th instar larvae of the pine sawyer beetle, and into the thorax-abdomen junction of the pupae. At least 5 larvae were injected in each independent experiment for phenotypic observation. dsGFP This served as the control group. At 24h, 48h, and 72h after injection, the whole larvae were flash-frozen in liquid nitrogen and then stored at -80℃ for later use.

[0078] (2) Total RNA was extracted from the larvae of the pine sawyer beetle using Yeasen total RNA extraction reagent (refer to the instructions for the cell / tissue total RNA extraction kit). The RNA was then reverse transcribed into cDNA using Yeasen's Hifair® Ⅱ 1st Strand cDNA Synthesis Kit. This cDNA was then used as a template for quantitative real-time RT-PCR detection after injection. MaCht-2 Gene expression levels.

[0079] qRT PCR involves MaCht-2 The primer sequences are F: 5′- CCCCTGCTTCCAGGTTTAGG′ (SEQ ID NO:14), R: 5′- GATCCGCTACCGGTTCTCTG-3′ (SEQ ID NO:15).

[0080] The amplification program was as follows: 95℃ pre-denaturation for 1 min; 94℃ denaturation for 100 s; 60℃ annealing for 20 s; 35 cycles.

[0081] The results were adopted using 2 -ΔΔCT The method is used for analysis. Actin The gene is used as an internal reference gene, and the gene sequence of the internal reference gene is as follows: F: 5′-TGGGTATGGAATCTTGCGGT-3′ (SEQ ID NO:16), R: 5′-GGCCCTGATTTCCTTTTGCA-3′ (SEQ ID NO:17).

[0082] injection dsMaCht-2 The interference from the pine sawyer beetle after injection is shown in Table 1. dsMaCht-2 and dsGTP Post-gene expression levels, such as Figure 1 As shown, the phenotypic comparison results are as follows: Figure 2 As shown in Table 1, the formula for calculating the percentage of insects affecting mobility is: (severe deformities + larval / pupaary mortality) / number of injected insects × 100%.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for controlling pine wood nematode and / or pine sawyer beetle using dsRNA, characterized in that, The dsRNA targets the chitin degradation gene of the longhorn beetle. MaCht-2 The MaCht-2 The nucleotide sequence is shown in SEQ ID NO: 01; The dsRNA is obtained by amplifying the nucleotide sequence of the template shown in SEQ ID NO: 01 using a primer set, wherein the primer set includes a first primer pair and a second primer pair; the upstream primer nucleotide sequence of the first primer pair is shown in SEQ ID NO: 03, and the downstream primer nucleotide sequence is shown in SEQ ID NO: 04; the upstream primer nucleotide sequence of the second primer pair is shown in SEQ ID NO: 05, and the downstream primer nucleotide sequence is shown in SEQ ID NO:

06.

2. The use of the dsRNA of claim 1 or biological materials containing them in the control of pine wood nematode and / or pine sawyer beetle.

3. The application according to claim 2, characterized in that, The dsRNA is introduced into the body of the longhorn beetle, and the introduction method includes feeding or microinjection.

4. At least one of the following applications of the dsRNA of claim 1 or biological materials containing it: (1) Reduces the normal mobility of the pine saw beetle; (2) To prepare products that reduce the normal mobility of the pine sawyer beetle; (3) Reduce the population size of the pine sawyer beetle; (4) Prepare products that reduce the population size of the pine sawyer beetle; (5) Prepare products for controlling the pine sawyer beetle.

5. An insect-repellent product, characterized in that, The dsRNA of claim 1 or biological material containing them.

6. The insect-repellent product according to claim 5, characterized in that, The biomaterials include expression cassettes, recombinant vectors, or recombinant microorganisms.

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