Agdoa gene and application thereof in reducing resistance of cotton aphids

Abstract

The AgDoa gene and application thereof in reducing the pesticide resistance of cotton aphids belong to the technical field of biological genetic engineering, the DNA sequence of the AgDoa gene is shown as SEQ ID No:1, and the AgDoa gene is composed of 1782 nucleotides; the amino acid sequence of the protein coded by the AgDoa gene is shown as SEQ ID No:2, and the protein is composed of 593 amino acids; the AgDoa gene can be used as a target to develop a new pesticide; the tolerance of the cotton aphids to the pesticide is reduced by knocking down the AgDoa gene, and the AgDoa gene can be applied in the resistance management and development of new pesticides.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the AgDoa gene and its application in reducing cotton aphid resistance. Background Technology

[0002] The cotton aphid (Aphis gossypii Glover) is a global agricultural pest that causes enormous losses to cotton and other cash crops. Currently, control of cotton aphids mainly relies on chemical control. However, the long-term use of chemical insecticides has led to high levels of resistance in cotton aphids to traditional pesticides. Control of resistant cotton aphids currently depends primarily on the application of novel insecticides, while the potential development of resistance poses a challenge to resistance management.

[0003] After insecticides enter an insect's body, they undergo primary and secondary metabolism, ultimately reducing their toxicity through a series of biological transformations. Cytochrome P450 participates in the primary metabolic process and catalyzes the oxidative metabolism of insecticides. Our laboratory has discovered that cytochrome P450 in cotton aphids plays a crucial role in the development of insecticide resistance. Inhibiting the overexpression of P450 to reduce insecticide resistance levels in cotton aphids is one approach to managing resistant cotton aphids. Previous studies have identified that the expression level of the key resistance P450 gene AgCYP6CY9a is positively regulated by the bispecific protein kinase gene AgDoa. Therefore, the AgDoa gene shows promise as a target for reducing cotton aphid resistance levels and for the development of novel insecticides. Summary of the Invention

[0004] This invention aims to provide an AgDoa gene and its application in reducing cotton aphid resistance.

[0005] The gene for regulating pesticide resistance provided by this invention is derived from the cotton aphid and named AgDoa, whose DNA sequence is shown in SEQ ID No:1. This DNA sequence is the open reading frame of the AgDoa gene and consists of 1782 nucleotides.

[0006] This invention provides a protein encoded by the AgDoa gene, the amino acid sequence of which is shown in SEQ ID No:2, and the sequence consists of 593 amino acids.

[0007] The AgDoa gene from the cotton aphid can regulate the downstream key detoxification P450 gene.

[0008] Suppressing the AgDoa gene reduces the cotton aphid's tolerance to insecticides and can be applied to manage the cotton aphid's resistance to insecticides.

[0009] This invention demonstrates that reduced AgDoa gene expression leads to a significant downregulation of the downstream key P450 gene expression and a significant decrease in cotton aphid tolerance to insecticides, indicating that the AgDoa gene is essential for cotton aphids to develop insecticide resistance. Therefore, screening for compounds that can inhibit the expression, modification, and localization of this gene and its encoded protein can effectively control cotton aphid damage, thereby contributing to the development of novel insecticides. One important application of the AgDoa gene provided in this invention is that the expression of the AgDoa gene and the key structural domains of its encoded protein can serve as important candidate target sites for controlling cotton aphid damage. Attached Figure Description

[0010] Figure 1 A schematic diagram illustrating the domain analysis of the protein encoded by the AgDoa gene;

[0011] Wherein: PKc_CLK is the catalytic domain of a dual-specific protein kinase, which includes an ATP-binding site, an active site, and a polypeptide substrate-binding site.

[0012] Figure 2 Electrophoresis image for AgDoa-linked PMD19T enzyme digestion identification;

[0013] Figure 3 Electrophoresis diagram for dsRNA-AgDoa amplification and identification;

[0014] Figure 4 A graph showing the knockdown efficiency of the AgDoa gene in cotton aphids after feeding with dsRNA-AgDoa.

[0015] The cotton aphid strains fed with dsRNA were from Heze, Shandong. The treatment group was fed dsRNA-AgDoa, while the control group was fed the same amount of dsRNA-ECFP as the treatment group. AgDoa expression was measured by RT-qPCR 48 h after feeding. ** indicates a significant difference at the P<0.1 level.

[0016] Figure 5 This is a graph showing the change in the relative expression levels of the downstream key P450 gene AgCYP6CY9a after AgDoa is suppressed.

[0017] The feeding method was the same as above. After feeding dsRNA for 48 hours, the expression level of AgCYP6CY9a was measured by RT-qPCR; ** indicates that the difference was significant at the P<0.1 level.

[0018] Figure 6 A graph showing the mortality rate of neonicotinoid insecticides against cotton aphids after AgDoa inhibition;

[0019] In this study, neonicotinoid insecticides (final concentration: 10 mg / L), dsRNA-AgDoa (final concentration: 150 ng / μL), and 20% sucrose were used as artificial nutrient solutions. The feeding method was the same as above (the control group was fed dsRNA-ECFP). An additional 80 cotton aphids of field strains from Heze were used in the lethality experiment. Each treatment was repeated in triplicate, and mortality rates were recorded after 48 hours. ** indicates statistical significance at the P < 0.1 level. Detailed Implementation

[0020] The present invention will now be described in conjunction with the accompanying drawings.

[0021] The AgDoa gene of this invention originated from Heze City, Shandong Province. Contact person: Shang Qingli, email: shangqingli@163.com.

[0022] Example 1: Domain analysis of the protein encoded by the AgDoa gene

[0023] The AgDoa gene has an open reading frame length of 1782 bp, encoding 593 amino acids. Based on the amino acid sequence of the cotton aphid AgDoa, its domains are predicted to include the catalytic domain PKc_CLK (e.g., a dual-specific protein kinase). Figure 1 (As shown). The protein kinase superfamily mainly consists of the catalytic domains of serine / threonine-specific protein kinases and tyrosine-specific protein kinases. It also includes atypical serine protein kinases such as RIO kinase, aminoglycoside phosphotransferase, and choline kinase. These proteins catalyze the transfer of the v-phosphate group of ATP to the hydroxyl group of specific substrates, such as serine, threonine, or tyrosine residues in proteins, thereby regulating alternative splicing of genes.

[0024] Example 2: Amplification of AgDoa gene and dsRNA-AgDoa

[0025] Based on the mRNA sequence of the cotton aphid AgDoa, specific primers (5-ATGTTTCCTCCGGATATGAA-3 and 5-TCATCTGGACAGTGAATGTG-3) were designed to amplify the ORF. PCR amplification was performed using Ex-Taq polymerase according to the designed specific primers. The amplified target fragment was analyzed by 1% agarose gel electrophoresis, revealing a band of 1782 bp in length (e.g., ...). Figure 2(As shown), and then recovered. The recovered fragment was ligated into the PMD19T vector to obtain a recombinant plasmid. Using the recombinant plasmid as a template, specific primers were used to amplify (5'-GGTGtaatacgactcactataggTCCAAACCCTTGGTGAAGGT-3' and 5'-GGTGtaatacgactcactataggATATGCCGCACTTGGTCAAC-3') dsRNA-AgDoa. The target fragment was amplified, and the band with a length of 313 bp was analyzed by 1% agarose gel electrophoresis and then recovered (as shown). Figure 3 (As shown). The recovered fragments were used to synthesize dsRNA-AgDoa.

[0026] Example 3: Knockdown efficiency of dsRNA-AgDoa and detection of expression levels of key downstream P450 after knockdown

[0027] In a clean bench, a Parafilm membrane was stretched to a sufficiently thin thickness and placed over one end of the double-pass filter. 600 μL of a 20% sucrose solution containing dsRNA-AgDoa at a final concentration of 150 ng / μL was added. The control group was fed the same concentration of dsRNA-ECFP. After 48 h of feeding, AgDoa expression was measured by RT-qPCR using designed specific quantitative primers (5'-CACCTTGCAATGATGGAGCG-3' and 5'-CCAGCAGAGCTGGATTGGTC-3'). Compared to the control group, its expression level decreased by 46% (e.g., ...). Figure 4 (As shown). The feeding method was the same as above. After feeding dsRNA-AgDoa for 48 h, the expression level of AgCYP6CY9a was measured by RT-qPCR. Compared with the control group (fed dsRNA-ECFP), its expression level decreased by 42% (as shown in the figure). Figure 5 (As shown).

[0028] Example 4: Bioassay of neonicotinoid insecticides for cotton aphids after AgDoa gene knockdown

[0029] Neonicotinic acid insecticides (final concentration: 10 mg / L), dsRNA-AgDoa (final concentration: 150 ng / μL), and 20% sucrose were used as artificial nutrient solutions. The feeding method was the same as above (the control group was fed dsRNA-ECFP). An additional 80 cotton aphids of field strains from Heze were used in a lethality experiment, with three replicates for each treatment. Mortality rates were recorded after 48 hours. The results showed that compared with the control group, the mortality rates were significantly increased after feeding neonicotinic acid insecticides. Specifically, thiamethoxam increased by 24.34%, dinotefuran by 17.92%, imidacloprid by 30.83%, and acetamiprid by 37.09% (** indicates a significant difference at the P<0.1 level).

[0030] The results of this invention indicate that the AgDoa gene indirectly mediates the process by which cotton aphids develop resistance to insecticides. If the expression level of the AgDoa gene is knocked down or its encoded protein loses activity, the cotton aphid's tolerance to insecticides will be significantly reduced, and it may even significantly increase the toxicity of insecticides to cotton aphids.

Claims

1. Knocking down cotton aphids via RNAi ( Aphis gossypii In ) AgDoa The application of this gene in reducing the resistance of cotton aphids to imidacloprid, thiamethoxam, dinotefuran, and acetamiprid is characterized by: AgDoa The DNA sequence of the gene is shown in SEQ ID No: 1.

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