DsRNA of migratory locust gene and application of dsRNA in pest control

Abstract

The invention discloses dsRNA of a migratory locust gene and application of the dsRNA in pest control, and belongs to the technical field of biotechnology and agricultural pest control. The method comprises the following steps of: cloning and sequencing an extraneous gene of migratory locust, so as to obtain the extraneous gene of which the sequence is SEQ ID NO: 1; and then the gene segment with the sequence of SEQ ID NO: 2 is selected and used for synthesis of double-stranded RNA (dsRNA). After the dsRNA of the gene is injected into the body cavity of the migratory locust, a specific extraneous gene can be silenced, so that 83.6% of migratory locust is difficult to exuviate and die, and 16.4% of migratory locust is abnormal in wing development and cannot migrate after exuviating. A novel specific molecular target is provided for pest control based on RNA interference, and a novel technical approach is provided for pest control.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and agricultural pest control technology, specifically relating to a dsRNA of the extradenticle 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, high efficiency and safety, and being green and pollution-free. However, the development of nucleic acid biopesticides urgently needs to overcome the bottleneck of screening for highly efficient and specific insecticidal dsRNA targets. The cuticle is the first line of defense for pests against adverse external environments and pathogens. During the individual development of insects, the old cuticle is constantly shed and a new cuticle is formed. Molting is a biological phenomenon unique to arthropods, while humans and higher mammals lack this biological characteristic. Therefore, the cuticle is of great concern as a safe target for pest control. Developing original molecular targets targeting the insect molting process is the key to solving pesticide resistance and promoting the development of green pesticides.

[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 extradenticle gene to control migratory locusts. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this paper proposes an application of the extradenticle 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 the present invention is a locust extradenticle gene, the nucleotide sequence of which is shown in SEQ ID NO: 1. The open reading frame of this sequence is 1323 bp and encodes 440 amino acids.

[0007] The second aspect of the present invention is a locust extradenticle 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 extradenticle gene, which is synthesized using the locust extradenticle gene fragment described in the second aspect.

[0009] The fourth aspect of the present invention is a method for synthesizing dsRNA of the extradenticle gene of locust 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 extradenticle gene of the locust 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] This invention, by injecting the dsRNA of the extradenticle gene into the body cavity of locusts, can specifically silence the mRNA expression of the extradenticle gene, resulting in 83.6% of locusts experiencing difficulty molting and death, and 16.4% of locusts exhibiting abnormal wing development after molting and being unable to migrate. This provides a new specific molecular target for RNA interference-based pest control and offers a new technical approach for pest control. 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 extradenticle gene mRNA expression (where *P<0.05).

[0017] Figure 2 Effect of 5-year-old 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. dsLmexd). 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 fragment of the extradenticle gene from the locust.

[0020] Based on the transcriptome database of locusts, bioinformatics methods were used to search for the cDNA sequence of the locust extradenticle gene. The full-length cDNA sequence of the locust extradenticle gene was obtained. 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, and 5-instar locust nymphs (half male and half female) were selected and frozen in liquid nitrogen. RNA was extracted using the TaKaRa RNAisoPlus 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 extradenticle 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 extradenticle gene in locusts

[0022] 1) Design of primers for the extradenticle gene dsRNA of the locust

[0023] Based on the sequence of the locust extradenticle 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 extradenticle gene of the locust

[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., dsLmexd) according to the instructions of the T7 RiboMAX™ Express RNAi System (Promega). 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 dsRNA of the extradenticle gene

[0027] 1) Injection of the extradenticle gene dsRNA from locusts

[0028] Fifth instar nymphs of uniform size and health were selected on the first day of instar and 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, and avoiding the spiracles. 10 μg of extradenticle gene dsRNA (dsLmexd) was injected, and a control group of dsGFP (10 μg) was also included, for a total of 43 nymphs. After injection, the nymphs were placed in a 1 L beaker in an artificial climate chamber (light:dark time = 14 h: 10 h, 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 locust extradenticle

[0030] Total RNA was extracted from nymphs injected with dsGFP and dsLmexd 24 h later. Four biological replicates were obtained from both the control and dsLmexd groups, with three nymphs per replicate. The RNA was reverse transcribed into first-strand cDNA, and the relative expression levels of the target gene (Lmextradenticle) 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, Lmextradenticle gene expression was significantly reduced in the dsLmexd group. Figure 1 ).

[0031] 3) Observation of the phenotype of 5th instar locusts after dsRNA injection

[0032] Fifth instar nymphs injected with dsRNA, compared to the dsGFP-injected control group, began molting 7 days later and all successfully molted into adults, exhibiting normal morphology and vitality. In the dsLmexd-injected treatment group, 36 out of 43 nymphs failed to molt and died; 7 nymphs exhibited abnormal wing development after molting and were unable to migrate, with a 100% phenotypic rate. Figure 2 As shown: The old epidermis of the head and thorax can be cracked, but the old and new epidermis cannot be separated, which eventually leads to death, or abnormal wing development after molting.

[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 extradenticle gene in locusts, characterized in that: The dsRNA sequence was synthesized using the locust EGFR gene fragment shown in SEQ ID NO:

2.

2. A method for synthesizing the dsRNA of the extradenticle gene in locusts 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. The application of the dsRNA of the extradenticle gene of locust as described in claim 1 in the control of locusts.

4. The application of the dsRNA of the extradenticle gene in locust control according to claim 3, characterized in that: dsRNA can be prepared into spray insecticides or baits, or transferred into the plant bodies of pests that feed on it.

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