AgDoa gene and application thereof in reducing drug resistance of cotton aphid

Abstract

The invention relates to an AgDoa gene and application thereof in reduction of insecticide resistance of cotton aphid, and belongs to the technical field of biological genetic engineering, the AgDoa gene has a DNA sequence as shown in SEQ ID No: 1, and consists 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 consists of 593 amino acids; the AgDoa gene can be used as a target to develop a novel insecticide; the AgDoa gene is knocked down, so that the tolerance of the aphis gossypii glover to the insecticide is reduced, and the aphis gossypii glover can be applied to drug resistance treatment and novel pesticide development.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to AgDoa gene and application thereof in reducing the pesticide resistance of cotton aphid. Background Art

[0002] Cotton aphid (Aphis gossypii Glover) is a worldwide agricultural pest that causes huge losses to cash crops such as cotton. At present, the prevention and control of cotton aphids is mainly based on chemical control. The long-term use of chemical pesticides has caused cotton aphids to develop a high level of resistance to traditional pesticides. At present, the prevention and control of resistant cotton aphids mainly relies on the application of new pesticides, and their potential resistance development also poses a challenge to resistance management.

[0003] After insecticides enter the body of insects, primary metabolism and secondary metabolism will occur, and through a series of biological transformations, their toxicity will eventually be reduced. Among them, cytochrome P450 participates in the primary metabolic process and can catalyze the oxidative metabolism of insecticides. Our laboratory found that cotton aphid cytochrome P450 played an important role in the development of insecticide resistance. How to inhibit the overexpression of P450 and reduce the resistance level of cotton aphids to insecticides is a way to control resistant cotton aphids. In the early stage, it was identified that the expression level of the key resistance P450 gene AgCYP6CY9a was positively regulated by the dual-specific protein kinase gene AgDoa. Therefore, the AgDoa gene has the prospect of being a drug target for reducing the resistance level of cotton aphids and creating new insecticides. Summary of the invention

[0004] The invention aims to provide an AgDoa gene and application thereof in reducing the pesticide resistance of cotton aphid.

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

[0006] The present 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 cotton aphid can regulate downstream key detoxification P450 genes.

[0008] The AgDoa gene can be inhibited to reduce the tolerance of cotton aphids to insecticides, and can be used in the management of cotton aphids' resistance to insecticides.

[0009] The present invention proves that the AgDoa gene expression level is reduced, resulting in a significant downregulation of the expression level of the downstream key P450 gene, and the tolerance of cotton aphids to insecticides is significantly reduced, indicating that the AgDoa gene is an essential gene for cotton aphids to develop resistance to insecticides. Therefore, screening compounds that can prevent the expression of the gene and the expression, modification and positioning of its protein can effectively control the harm of cotton aphids, thereby contributing to the development of new insecticides, that is, an important use of the AgDoa gene provided by the present invention is that the expression of the AgDoa gene and the key structural domain of the protein encoded by it can be used as an important candidate target site for controlling the harm of cotton aphids. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of domain analysis of protein encoded by AgDoa gene;

[0011] Among them: PKc_CLK is the catalytic domain of dual-specificity protein kinase, including ATP binding site, active site and polypeptide substrate binding site.

[0012] Figure 2 This is the electrophoresis diagram of AgDoa linked to PMD19T enzyme digestion identification;

[0013] Figure 3 Electropherogram for dsRNA-AgDoa amplification identification;

[0014] Figure 4 This is a diagram showing the knockdown efficiency of the AgDoa gene in cotton aphids after feeding with dsRNA-AgDoa;

[0015] Among them: the cotton aphid strain fed with dsRNA was from Heze, Shandong. The treatment group was fed with dsRNA-AgDoa, and the control group was fed with the same amount of dsRNA-ECFP as the treatment group. After 48 hours of feeding, the expression level of AgDoa was determined by RT-qPCR. ** indicates significant difference at the P<0.1 level;

[0016] Figure 5 This is the relative expression change of the downstream key P450 gene AgCYP6CY9a after AgDoa was inhibited;

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

[0018] Figure 6 This is a comparison chart of the mortality rate of neonicotinoid insecticides against cotton aphids after AgDoa was suppressed;

[0019] Among them: neonicotinoid insecticides (final concentration: 10mg / L), dsRNA-AgDoa (final concentration: 150ng / μL) and 20% sucrose were used as artificial nutrient solution, and the feeding method was the same as above (the control group was fed with dsRNA-ECFP). Another 80 field strains of cotton aphids from Heze were used for lethal experiments, and 3 replicates were set for each treatment. The mortality rate was recorded after 48 hours. ** indicates significant difference at the P<0.1 level. DETAILED DESCRIPTION

[0020] The present invention is described below with reference to the accompanying drawings.

[0021] The AgDoa gene of the present invention originates from: Heze City, Shandong Province, contact person: Shang Qingli, email: shangqingli@163.com.

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

[0023] The open reading frame of AgDoa gene is 1782bp in length and can encode 593 amino acids. Based on the amino acid sequence of AgDoa of cotton aphid, its domain structure is predicted to have a catalytic domain of dual specific protein kinase PKc_CLK (such as Figure 1 The protein kinase superfamily is mainly composed of the catalytic domains of serine / threonine-specific protein kinases and tyrosine-specific protein kinases, and also includes atypical serine protein kinases RIO kinases, aminoglycoside phosphotransferases and choline kinases, which 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 the selective splicing of genes.

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

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

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

[0027] After the Parafilm membrane was stretched to be thin enough in the clean bench, it was covered to one end of the double pass, and 600 μL of 20% sucrose solution containing dsRNA-AgDoa with a final concentration of 150 ng / μL was added, and the control group was fed with the same concentration of dsRNA-ECFP. After feeding for 48 hours, the expression level of AgDoa was determined by RT-qPCR using the designed specific quantitative primers (5'-CACCTTGCAATGATGGAGCG-3' and 5'-CCAGCAGAGCTGGATTGGTC-3'). Compared with the control group, its expression level decreased by 46% (as shown in Figure 2). Figure 4 The feeding method was the same as above. After 48 h of feeding with dsRNA-AgDoa, the expression of AgCYP6CY9a was measured by RT-qPCR. Compared with the control group (fed with dsRNA-ECFP), its expression level decreased by 42% (Figure Figure 5 shown).

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

[0029] Neonicotinoid insecticides (final concentration: 10 mg / L), dsRNA-AgDoa (final concentration: 150 ng / μL) and 20% sucrose were used as artificial nutrient solution, and the feeding method was the same as above (the control group was fed with dsRNA-ECFP). Another 80 field strains of cotton aphids from Heze were used for lethal experiments, and 3 replicates were set for each treatment. The mortality rate was recorded after 48 hours. The results showed that compared with the control group, the mortality rate after feeding neonicotinoid insecticides was significantly increased, among which thiamethoxam increased by 24.34%, dimethomorph increased by 17.92%, imidacloprid increased by 30.83%, and nitenpyram increased by 37.09% (** indicates significant difference at the P<0.1 level).

[0030] The research results of the present invention show that the AgDoa gene indirectly mediates the process of cotton aphids developing resistance to insecticides. If the expression level of the AgDoa gene is knocked down or the protein encoded by it loses its activity, the tolerance of cotton aphids to insecticides will be significantly reduced, and the toxicity of insecticides to cotton aphids can even be significantly increased.

Claims

1. An AgDoa gene, characterized in that Its DNA sequence is shown in SEQ ID No:

1.

2. Use of the AgDoa gene according to claim 1 in reducing the insecticide resistance of cotton aphid (Aphis gossypii).

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