Use and method of gene haCHERP in preventing and treating cotton bollworm

Abstract

This invention belongs to the field of biopesticides and relates to the application and method of the gene HaCHERP in the control of cotton bollworm. This invention discovered an RNAi target gene, HaCHERP, that has a lethal effect on cotton bollworm. The encoding CDS sequence of the HaCHERP gene is shown in SEQ ID No. 1. Further research was conducted on RNA biopesticides formulated with the double-stranded RNA of this gene and their control effect on cotton bollworm. The study found that the HaCHERP gene is mainly expressed during the larval stage of insects, and its content is high in the fat body and epidermis. After spraying cotton bollworms with an aqueous solution of dsHaCHERP, the growth and development of cotton bollworms were significantly affected, with a significant reduction in larval and pupal weight. This invention provides a new candidate target for the development of RNA biopesticides for cotton bollworm and also provides valuable exploration for the expansion of green control technologies for lepidopteran pests.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticides and relates to the control of cotton bollworm. Background Technology

[0002] The cotton bollworm (Helicoverpa armigera), belonging to the family Noctuidae in the order Lepidoptera, is a global, polyphagous agricultural pest that infests over 200 host plants, including cotton, corn, tobacco, and tomatoes, posing a serious threat to global agricultural production. Since the 1990s, the cotton bollworm has caused several major outbreaks in my country. Although the subsequent promotion and planting of Bt cotton effectively curbed its damage, the risk of resistance to Bt toxins and traditional chemical pesticides still exists in some areas.

[0003] Currently, the control of cotton bollworm still mainly relies on chemical insecticides, but long-term and extensive use has caused multiple problems: on the one hand, pesticide residues cause environmental pollution and ecosystem damage, while also killing off a large number of the pest's natural enemies, leading to a resurgence of secondary pests; on the other hand, cotton bollworms have developed varying degrees of resistance to various insecticides such as pyrethroids and organophosphates, with some areas even exhibiting high levels of resistance, causing the effectiveness of traditional chemical control to decline continuously. Therefore, there is an urgent need to develop new pest control strategies that are highly targeted, environmentally friendly, and less prone to cross-resistance.

[0004] RNA interference (RNAi) technology offers a new solution to this dilemma. RNAi is a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA). It can precisely target and degrade the mRNA of key genes involved in insect growth, development, metabolism, and reproduction, thereby inhibiting the expression of target genes. Compared with broad-spectrum chemical pesticides, RNAi technology has significant advantages such as high target specificity, high safety for non-target organisms, and good environmental compatibility. In 2023, the world's first sprayable RNA biopesticide received marketing authorization from the US EPA, marking the official transition of RNAi technology from the laboratory to field applications. In 2024, Science magazine named "RNA pesticides in the field" one of the top ten scientific breakthroughs of the year, further confirming the significant importance and promising future of this field.

[0005] However, the practical application of RNAi technology in lepidopteran pests (including the cotton bollworm) still faces challenges. Unlike coleopteran insects, which are highly sensitive to RNAi, most lepidopteran pests have low uptake efficiency of dsRNA and weak intracellular RNAi pathway responses, resulting in insignificant RNAi effects. Furthermore, nucleases secreted by the intestinal symbiotic bacteria of the cotton bollworm can rapidly degrade exogenous dsRNA, further weakening the silencing effect of RNAi. Therefore, screening for RNAi target genes with highly effective lethality against the cotton bollworm and developing stable and effective dsRNA delivery strategies are core scientific issues for the industrial application of RNA biopesticides.

[0006] Against this backdrop, our research group is dedicated to discovering target genes in the cotton bollworm that have key physiological functions and are sensitive to RNAi. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes a gene... HaCHERP Application and methods in the control of cotton bollworm.

[0008] The technical solution of this invention is implemented as follows:

[0009] CHERP (Calcium Homeostasis Endoplasmic Reticulum Protein) is a class of key functional proteins located in the endoplasmic reticulum and involved in the regulation of intracellular calcium signaling. This invention experimentally demonstrates that the HaCHERP gene is highly expressed in the fat body and epidermis of cotton bollworm larvae. Exogenous application of dsHaCHERP significantly inhibits gene expression, leading to phenotypic defects such as larval death, weight loss, and reduced pupal weight, showcasing the gene's promising potential as a candidate target for RNA biopesticides.

[0010] On the one hand, a request is made to protect the application of the silent gene HaCHERP in the control of cotton bollworm, the encoding CDS sequence of the gene HaCHERP being shown in SEQ ID No. 1.

[0011] On the other hand, a request is made to protect the use of the dsRNA of the HaCHERP gene in the control of cotton bollworm, the coding CDS sequence of which is shown in SEQ ID No. 1. The dsRNA comprises the forward sequence and its reverse complementary sequence as shown in SEQ ID No. 2.

[0012] Thirdly, a biological agent for controlling cotton bollworm is provided, the active ingredient of which is the dsRNA of the gene HaCHERP.

[0013] Furthermore, the encoding CDS sequence of the HaCHERP gene is shown in SEQ ID No. 1.

[0014] Preferably, the dsRNA comprises the forward sequence as shown in SEQ ID No. 2 and its reverse complementary sequence. The concentration of the dsRNA is 100-2000 ng / µL.

[0015] Fourthly, a method for controlling cotton bollworms is provided, which involves spraying a solution containing dsRNA of the HaCHERP gene onto the cotton bollworms.

[0016] Furthermore, the dsRNA comprises the forward sequence and its reverse complementary sequence as shown in SEQ ID No. 2. The concentration of dsRNA in the above solution is 100-2000 ng / µL.

[0017] The present invention has the following beneficial effects:

[0018] This invention reveals that the HaCHERP gene is primarily expressed during the larval stage of insects, with high concentrations in the fat body and epidermis. Spraying cotton bollworms with an aqueous solution of dsHaCHERP significantly inhibited HaCHERP gene expression, resulting in a 5-day survival rate of only 52%. This provides a new candidate target for the development of RNA-based biopesticides for cotton bollworms and offers valuable insights for expanding green control technologies for lepidopteran pests. Furthermore, the growth and development of cotton bollworms were significantly affected, with a marked decrease in larval and pupal weight. This invention discloses the full-length sequence, dsRNA sequence, spatiotemporal expression patterns, and effects of this gene. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The expression levels of the HaCHERP gene in the epidermis of cotton bollworm larvae at different instars are shown. 2L1d–2d represents 2nd instar 1–2 days; 3L1d–2d represents 3rd instar 1–2 days; 4Lmo represents 4th instar molting; 4L1d–2d represents 4th instar 1–2 days; 5Lmo represents 5th instar molting; 5L1d–3d represents 5th instar 1–3 days; hy represents the prepupal stage. Data in the figure are mean ± standard deviation; different letters on the bars indicate significant differences (P < 0.05, one-way ANOVA, Tukey multiple comparisons).

[0021] Figure 2 The expression levels of HaCHERP in different tissues of 4th instar larvae of *Bollworm* are shown; where BP: body wall (epidermis); ZF: fat body; ZC: midgut; XLB: hemolymph; JC: testis; MSG: Malpighian tubules; SJ: ganglion; BR: brain. Data in the figure are mean ± standard deviation; different letters on the bars indicate significant differences (P < 0.05, one-way ANOVA, Tukey multiple comparisons).

[0022] Figure 3 The expression level of the target gene in cotton bollworms after spraying dsHaCHERP.

[0023] Figure 4 The survival rate of bollworms after spraying dsHaCHERP.

[0024] Figure 5 The effect of spraying dsHaCHERP on the weight of bollworm larvae.

[0025] Figure 6 To investigate the effect of spraying dsHaCHERP on the pupal body weight of cotton bollworms. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0028] This invention experimentally demonstrates that the HaCHERP gene is highly expressed in the fat body and epidermis of cotton bollworm larvae. Exogenous application of dsHaCHERP significantly inhibits gene expression, leading to phenotypic defects such as larval death, weight loss, and reduced pupal weight, showcasing the gene's potential as a candidate target for RNA biopesticides. This invention provides a lethal target for cotton bollworms based on RNA interference technology. The primer sequences for amplifying the gene and synthesizing its dsRNA are shown in Table 1. The full-length cDNA sequence of the gene is shown in SEQ ID No. 1, and the forward sequence of the synthesized dsRNA is shown in SEQ ID No. 2.

[0029] All data in this experiment were analyzed using DPS 9.5 software, and relevant data graphs were created using Graphpad Prism 9. The data analysis method was Tukey's test. In all data graphs, the height of the bars represents the mean of each group of data. Error bars were drawn based on the standard error of the mean, and lowercase letters indicate the significance (P < 0.05) between different treatment groups at each time point.

[0030] The experimental steps of this invention are described in detail below:

[0031] Example 1: Target gene sequence acquisition and dsHaCHERP synthesis

[0032] Using cotton bollworm as material, total RNA samples were extracted using the conventional Trizol method, purified using conventional methods, and treated with DNase to obtain samples with a concentration ≥300 ng / μl, a total amount ≥6 μg, and an OD260 / 280 of 1.8–2.2. The RNA was then reverse transcribed into cDNA, amplified using primers specific to the HaCHERP gene (Table 1), and the obtained gene fragment was purified. The cDNA was synthesized using the dsRNA synthesis kit (T7 RNA Transcription Kit, T7 RNA Transcription Kit (SJ001)) from Shanghai Zhisheng Yougu Biotechnology Co., Ltd. (See the kit for details.)

[0033] Table 1 Primer sequences involved in this invention

[0034]

[0035] Example 2: Tissue expression profiling analysis of the HaCHERP gene

[0036] Samples of different instars and tissues of cotton bollworms were collected, with three larvae dissected and sampled as one sample, and each tissue sample was collected three times. RNA was extracted from each tissue using the Trizol method, and cDNA was obtained by reverse transcription using the FastKing cDNA First-Strand Synthesis Kit (Tiangen). The RT-PCR reaction system consisted of: 1 µL cDNA, 10 µL SYBR® Premix ExTaq™ II, 0.5 µL each of forward and reverse primers (10 µmol / µL), and 8 µL ddH2O. The reaction program was: 95 °C denaturation for 30 s; 95 °C annealing for 5 s, 60 °C extension for 30 s, for 40 cycles. Each sample was PCR reacted three times. Primer information is shown in Table 1.

[0037] The results are as follows Figure 1 and Figure 2As shown in Figure 1, the HaCHERP gene is expressed in all larval stages of the cotton bollworm. The tissue expression shows that its expression level is higher in the midgut and epidermis (Figure 2).

[0038] Example 3: RNAi and bioassays performed by spraying method

[0039] To evaluate the RNAi effect targeting the HaCHERP gene, this study used dsEGFP as a negative control and treated first-instar cotton bollworm larvae with dsHaCHERP by spraying. The silencing efficiency of the target gene, larval survival rate, larval weight and pupal weight were systematically detected.

[0040] Fifty first-instar larvae of the cotton bollworm were introduced into a rearing box containing feed at the bottom as one treatment group. After the test insects colonized, they were sprayed with 500 µL of dsEGFP (500 ng / µL) or dsHaCHERP (500 ng / µL) using a vacuum micro-sprayer. Samples of the cotton bollworms were taken at 6 h, 12 h, and 24 h after treatment, with three cotton bollworms per sample, and each treatment was repeated three times. The expression level of HaCHERP in cotton bollworms was detected using RT-qPCR. Furthermore, the number of surviving cotton bollworms was counted from 1 to 7 days after treatment, and the larval survival rate was calculated. Larvals that did not respond to external contact were considered dead. At 3, 5, and 7 days after treatment, and during the pupal stage, the weight of the cotton bollworm larvae was measured using an analytical balance, ensuring the larvae were still during weighing. Data were recorded after the analytical balance reading stabilized.

[0041] Compared with the dsEGFP control group, the expression level of HaCHERP in the dsHaCHERP treatment group decreased to 52.3%, 64.8% and 79.5% of that in the control group at 6 h, 12 h and 24 h, respectively (Figure 3), indicating that dsHaCHERP can effectively induce transcriptional silencing of the HaCHERP gene, and the silencing effect was already apparent 6 h after treatment.

[0042] The effect of dsHaCHERP treatment on the survival rate of cotton bollworm larvae was continuously observed. The results showed (Figure 4) that compared with the dsEGFP control group, the survival rate of larvae in the dsHaCHERP treatment group decreased significantly after 5 days of treatment, with a survival rate of only 52%. Moreover, the surviving cotton bollworms grew slowly and could not continue to reproduce, suggesting that the silencing of the HaCHERP gene has a significant lethal effect on larvae.

[0043] Larval weight was measured at 3, 5, and 7 days after treatment. The results (Figure 5) showed that the larval weight of the dsHaCHERP treatment group was significantly lower than that of the dsEGFP control group at all time points, and the difference continued with the extension of treatment time, indicating that HaCHERP gene silencing significantly inhibited larval growth and development.

[0044] After the larvae pupated, the pupal weight of each group was measured. The results showed (Figure 6) that the pupal weight of the dsHaCHERP treatment group was significantly lower than that of the control group, indicating that the silencing of the HaCHERP gene has a sustained effect on the growth and development of the cotton bollworm, not only inhibiting larval growth but also affecting the weight accumulation during the pupal stage.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Silent genes HaCHERP Its application in controlling cotton bollworm is characterized by: The gene HaCHERP The encoded CDS sequence is shown in SEQ ID No.

1.

2. Genes HaCHERP The application of dsRNA in the control of cotton bollworm is characterized by: The gene HaCHERP The encoded CDS sequence is shown in SEQ ID No.

1.

3. The application according to claim 2, characterized in that: The dsRNA includes the forward sequence as shown in SEQ ID No. 2 and its reverse complementary sequence.

4. A biological agent for controlling cotton bollworm, characterized in that: The active ingredient is genes. HaCHERP dsRNA.

5. The biological agent according to claim 4, characterized in that: The gene HaCHERP The encoded CDS sequence is shown in SEQ ID No.

1.

6. The biological agent according to claim 5, characterized in that: The dsRNA includes the forward sequence as shown in SEQ ID No. 2 and its reverse complementary sequence.

7. The biological agent according to claim 5, characterized in that: The concentration of the dsRNA is 100-2000 ng / µL.

8. A method for controlling cotton bollworm, characterized in that: Use containing genes HaCHERP The solution of dsRNA can be sprayed onto cotton bollworms.

9. The method for controlling cotton bollworm according to claim 8, characterized in that: The dsRNA includes the forward sequence as shown in SEQ ID No. 2 and its reverse complementary sequence.

10. The method for controlling cotton bollworm according to claim 9, characterized in that: The concentration of dsRNA in the solution is 100-2000 ng / µL.

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