DsRNA of migratory locust nubbin gene and application of dsRNA in pest control
By designing and synthesizing dsRNA of the locust nubbin gene, its gene expression was silenced, leading to abnormal wing development. This solved the problem of the lack of effective RNA interference technology for the control of locusts in the existing technology, and achieved a specific control effect on locusts.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANXI UNIV
- Filing Date
- 2026-01-20
- Publication Date
- 2026-04-21
AI Technical Summary
There is a lack of effective RNA interference techniques for controlling locusts, an intercontinental agricultural pest, especially methods targeting the nubbin gene, which have not been reported.
The dsRNA of the locust nubbin gene was designed and synthesized. It was then amplified by PCR and synthesized by transcription. It was prepared into a spray insecticide or bait and injected into the locust to silence the expression of the nubbin gene, resulting in abnormal wing development and preventing its migration.
It has achieved specific control of locusts, with 100% of locusts exhibiting abnormal wing development after molting and being unable to migrate, providing a new molecular target for pest control.
Smart Images
Figure CN121896232A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and agricultural pest control technology, specifically relating to a dsRNA of the nubbin gene of the locust and its application in pest control. Background Technology
[0002] Nucleic acid biopesticides utilize the dsRNA of pests or pathogens themselves to silence target gene transcription, thereby inhibiting the growth and development of pests or pathogens and achieving the purpose of pest and disease control. Compared with traditional pesticides, nucleic acid biopesticides have advantages such as strong insecticidal specificity, a wide range of target selection, and being green and safe. Nucleic acid biopesticides have become the core products of the "third revolution in the history of pesticide development." However, the research and development of nucleic acid biopesticides urgently needs to overcome the bottleneck of screening for highly efficient and specific insecticidal dsRNA targets. Insects are the only group of invertebrates with wings. Wings, as important flight organs, develop from the ectoderm and show great advantages in insects' search for food, avoidance of disasters, seeking mates, and expanding their activity range.
[0003] The migratory locust (Locusta migratoria) is an intercontinental agricultural pest, mainly distributed across Asia, Europe, Africa, and Australia. It is characterized by its explosive, gregarious, and migratory nature; once it occurs, it not only affects a wide area but also spreads rapidly and causes severe damage. Currently, there are no reports on RNA interference technology using the nubbin gene to control this pest. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides an application of the nubbin gene and its dsRNA in pest control.
[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:
[0006] The first aspect of this invention is a locust nubbin gene, the nucleotide sequence of which is shown in SEQ ID NO: 1. The open reading frame of this sequence is 1242 bp and encodes 413 amino acids.
[0007] The second aspect of the present invention is a locust nubbin gene fragment, the nucleotide sequence of which is shown in SEQ ID NO: 2.
[0008] A third aspect of the present invention is a dsRNA of the locust nubbin gene, which is synthesized using the locust nubbin gene fragment described in the second aspect.
[0009] The fourth aspect of the present invention is a method for synthesizing dsRNA of the locust nubbin gene as described in the third aspect, comprising the following steps: designing an upstream primer as shown in SEQ ID NO: 3 and a downstream primer as shown in SEQ ID NO: 4 according to the sequence SEQ ID NO: 1, and then obtaining the product as shown in SEQ ID NO: 2 by PCR amplification, which is then purified, transcribed and synthesized into dsRNA.
[0010] The fifth aspect of the present invention is the application of the dsRNA of the locust nubbin gene described in the third aspect in pest control.
[0011] Furthermore, dsRNA can be prepared into sprayable insecticides or baits, or transferred into the plant bodies ingested by pests.
[0012] Furthermore, the pest is the locust.
[0013] Compared with the prior art, the present invention has the following advantages:
[0014] The present invention provides a novel specific molecular target for RNA interference-based pest control, and offers a new technical approach for pest control. After the dsRNA of the nubbin gene in the locust is injected into the body cavity of the locust, it can specifically silence the mRNA expression of the nubbin gene in the locust, and cause 100% of the locusts to have abnormal wing development after molting and be unable to migrate. Attached Figure Description
[0015] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0016] Figure 1 Effect of 5-instar locusts injected with dsRNA synthesized by SEQ ID NO:2 on nubbin gene mRNA expression (where *P<0.05).
[0017] Figure 2 Effect of 5-instar locusts injected with dsRNA synthesized by SEQ ID NO:2 on locust phenotype (left side is control injected with dsGFP, right side is dsRNA synthesized by SEQ ID NO:2, i.e. dsLmnubbin). Detailed Implementation
[0018] To gain a deeper understanding of this invention, we will provide a comprehensive and detailed description. However, this invention has various implementations and is not limited to the specific examples listed herein. These examples are presented to enhance a full understanding of the disclosure of this invention.
[0019] Example 1: Obtaining the full-length cDNA sequence and gene fragments of the locust nubbin gene
[0020] Based on the transcriptome database of locusts, bioinformatics methods were used to search for the cDNA sequence of the locust nubbin gene, obtaining the full-length cDNA sequence. Primers were designed using Primer Premier 5.0 software to verify the full-length cDNA sequence, and the sequence was synthesized at Shanghai Bioengineering Co., Ltd. Four healthy, uniformly sized, half-male and half-female nymphs of the fifth instar locust were selected and frozen in liquid nitrogen. RNA was extracted according to the TaKaRa RNAiso Plus kit, and the extracted RNA was reverse transcribed into first-strand cDNA using the HiScript III qRT SuperMix (Nanjing Novizan Biotechnology Co., Ltd.) instructions. Using this cDNA as a template, and with the designed primers, the full-length cDNA sequence of the nubbin gene was amplified by PCR. The obtained product was purified, cloned, and transformed into *E. coli*, and then sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequence is SEQ ID NO: 1.
[0021] Example 2: Synthesis of dsRNA from the nubbin gene in locusts
[0022] 1) Design of primers for the dsRNA of the locust nubbin gene
[0023] Based on the sequence of the locust nubbin gene obtained in Example 1 (SEQ ID NO: 1), dsRNA primers were designed using Primer Premier 5.0 software. The primer sequences are SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and both the upstream and downstream primers carry the T7 promoter sequence. All primers were synthesized by Shanghai Bioengineering Co., Ltd.
[0024] 2) Synthesis of dsRNA in the locust nubbin gene
[0025] Using the upstream and downstream primers of SEQ ID NO: 3 and SEQ ID NO: 4, PCR amplification was performed on the cDNA template of Example 1. The amplified PCR product, with the sequence SEQ ID NO: 2, was purified using the FastPure Gel DNA Extraction MiniKit (Vazyme) and then transcribed in vitro to synthesize dsRNA (i.e., dsLmnubbin) according to the instructions of the T7 RiboMAX™ Express RNAi System (Promega) kit. Quantification was performed using a NANODROP 2000 (Thermo Scientific) to achieve a final concentration of 2 μg / μL. It was stored at -80°C for later use.
[0026] Example 3: Experiment on lethality of 5th instar locusts by nubbin gene dsRNA
[0027] 1) Injection of the nubbin gene dsRNA from locusts
[0028] Fifth instar nymphs of uniform size and health status, selected on the third day, were injected with the dsRNA synthesized in Example 2. A 25 μl microsyringe was used for injection; excessive force was avoided during injection. The injection point was the junction of the second and third abdominal segments on the lateral side, following the direction of blood flow, avoiding the spiracles. The injection volume of dsRNA was 10 μg, and a control group of dsGFP (10 μg) was also included. Each group consisted of 10 nymphs, with 3 biological replicates, for a total of 30 nymphs. After injection, the nymphs were placed in 1 L beakers in an artificial climate chamber (light:dark time = 14h:10h, temperature 30±2℃, humidity 60%), provided with fresh wheat seedlings and appropriate light, and sprayed with water to maintain humidity.
[0029] 2) Detection of gene silencing in 5th instar locusts (nubbin gene)
[0030] Total RNA was extracted from nymphs injected with dsGFP and dsLmnubbin 24 h later. Four biological replicates were obtained from both the control and dsLmnubbin groups, with three nymphs per replicate. The RNA was reverse transcribed into first-strand cDNA, and the relative expression levels of the target gene (Lmnubbin) and the housekeeping gene (β-actin) were detected using RT-qPCR to calculate their silencing efficiency. The results showed that, compared with the control group, the expression of the Lmnubbin gene in nymphs injected with dsLmnubbin was significantly reduced. Figure 1 ).
[0031] 3) Observation of the phenotype of 5th instar locusts after dsRNA injection
[0032] Fifth instar nymphs injected with dsRNA, and those in the dsGFP-injected control group, began molting 7 days later and all successfully molted into adults. After molting, the adults exhibited normal morphology and vitality. In the dsLmnubbin-injected treatment group, all 30 nymphs molted but exhibited abnormal wing development, were unable to migrate, and displayed the following phenotype: Figure 2 As shown.
[0033] Contents not described in detail in this specification are prior art known to those skilled in the art. Although illustrative specific embodiments of the invention have been described above to facilitate understanding by those skilled in the art, it should be understood that the invention is not limited to the scope of the specific embodiments. Various modifications are readily apparent to those skilled in the art as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of this invention are protected.
Claims
1. A dsRNA of the locust nubbin gene, characterized in that: The dsRNA sequence was synthesized using the nubbin gene fragment shown in SEQ ID NO:
2.
2. A method for synthesizing dsRNA of the locust nubbin gene as described in claim 1, characterized in that: The process includes the following steps: designing an upstream primer as shown in SEQ ID NO: 3 and a downstream primer as shown in SEQ ID NO: 4 based on the sequence SEQ ID NO: 1, and then obtaining the product as shown in SEQ ID NO: 2 by PCR amplification, which is then purified, transcribed, and synthesized into dsRNA.
3. A primer pair for synthesizing the dsRNA of the locust nubbin gene as described in claim 1, characterized in that, It includes an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
4. The application of the dsRNA of the locust nubbin gene as described in claim 1 in pest control.
5. The application of the dsRNA of the locust nubbin gene in pest control according to claim 4, characterized in that: dsRNA can be prepared into spray insecticides or baits, or transferred into the plant bodies of pests that feed on it.