Application of AccFoxD3 gene and its encoded protein in pesticide and heavy metal resistance of Apis cerana cerana

Abstract

This invention discloses the application of the AccFoxD3 gene and its encoded protein in enhancing pesticide and heavy metal resistance in the Chinese honeybee, belonging to the fields of biochemistry and molecular biology. The present invention discovered that the AccFoxD3 gene responds to oxidative stress induced by pesticides and heavy metals in Chinese honeybees; that heterologous expression of AccFoxD3 in Escherichia coli and yeast negatively regulates cellular resistance to external oxidative stress; and that silencing the AccFoxD3 gene improves the antioxidant capacity and pesticide resistance of Chinese honeybees. Therefore, exploring the function of AccFoxD3 in enhancing the oxidative stress resistance of Chinese honeybees induced by pesticides and heavy metals is of great significance.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry and molecular biology, and in particular to the application of the AccFoxD3 gene and its encoded protein in the pesticide and heavy metal resistance of Chinese honeybees. Background Art

[0002] With the development of intensive agriculture, the use of pesticides has become a crucial component of pest and weed control. This process also poses a serious threat to the survival of some non-target organisms. Honeybees, as social insects, are particularly vulnerable. They bring pesticide-contaminated nectar into their hives, exposing the entire colony to long-term pesticide exposure and severely impacting their survival. Furthermore, heavy metal residues in pesticides and environmental heavy metal pollution also significantly impact bee survival. The decline in bee populations poses a serious challenge to the production of insect-pollinated crops worldwide. Studies have shown that pesticide and heavy metal stress can induce oxidative stress in bees, making them one of the most significant environmental factors impacting bee survival.

[0003] Insects have evolved a range of defense mechanisms to cope with oxidative stress caused by these environmental stresses. Studies have shown that transcription factors can alter insect resistance to insecticides by regulating the expression of downstream genes. Among these transcription factors, the Foxhead box protein (Fox) family of transcription factors, which contain a winged helix DNA binding domain (composed of two α-helices and two large loops), has been shown to play an important role in growth and development, immune response, and stress tolerance by regulating the expression of multiple downstream target genes.

[0004] The Fox family of transcription factors is highly conserved across evolution from yeast to humans. Phylogenetic analysis has identified 17 subclasses of Fox family transcription factors, from FoxA to FoxQ. Among them, FoxD3 is a key member of the Fox protein family. Current research on its transcriptional activity focuses primarily on other fundamental research areas, such as mammalian tumorigenesis, chordate embryonic development, and cell damage repair. No studies have been reported in honeybees.

[0005] The Chinese honey bee (Apis cerana cerana), a native bee species in my country, holds significant economic value and plays a crucial role in agricultural development. In recent years, the number of Chinese honey bee colonies has declined significantly due to various environmental stresses, such as pesticides and heavy metals, significantly impacting crop yields and ecosystem stability in my country. Therefore, improving the resistance of Chinese honey bees to pesticides and heavy metals is an urgent issue. Summary of the Invention

[0006] In response to the above-mentioned prior art, the present invention aims to provide the use of the AccFoxD3 gene and its encoded protein for pesticide and heavy metal resistance in the Chinese honey bee. The present invention has discovered that the AccFoxD3 gene responds to oxidative stress induced by pesticides and heavy metals in the Chinese honey bee; that heterologous expression of AccFoxD3 in Escherichia coli and yeast negatively regulates cellular resistance to external oxidative stress; and that silencing the AccFoxD3 gene can enhance the antioxidant capacity and pesticide resistance of the Chinese honey bee. Therefore, exploring the function of AccFoxD3 in protecting the Chinese honey bee from oxidative stress induced by pesticides and heavy metals is of great significance.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides the use of the AccFoxD3 gene as a target gene in the following (1) or (2):

[0009] (1) Regulate the stress resistance of honey bees to pesticides and heavy metals;

[0010] (2) Cultivate bee varieties with enhanced resistance to pesticide and heavy metal stress.

[0011] In the above application, the nucleotide sequence of the AccFoxD3 gene is shown as SEQ ID No.1.

[0012] The second aspect of the present invention provides the use of a protein encoded by the AccFoxD3 gene in regulating the stress resistance of Apis cerana cerana to pesticides and heavy metals.

[0013] In the above application, the amino acid sequence of the protein encoded by the AccFoxD3 gene is shown in SEQ ID No. 2.

[0014] A third aspect of the present invention provides use of a reagent for specifically detecting the AccFoxD3 gene in the preparation of a product for detecting the resistance of honey bees to pesticide and heavy metal stress.

[0015] In the above application, the product uses real-time fluorescence quantitative PCR method to detect the expression level of AccFoxD3 gene.

[0016] In the above application, the product includes: a real-time fluorescence quantitative PCR detection kit.

[0017] In the above application, the real-time fluorescence quantitative PCR detection kit contains: a primer pair for specifically detecting the AccFoxD3 gene, whose sequences are shown in SEQ ID No. 5 and SEQ ID No. 6.

[0018] A fourth aspect of the present invention provides the use of an agent for silencing AccFoxD3 gene expression in the following (1) or (2):

[0019] (1) Improve the stress resistance of honey bees to pesticides and heavy metals;

[0020] (2) Cultivate bee varieties with enhanced resistance to pesticide and heavy metal stress.

[0021] In the above application, the reagent includes: a primer pair for amplifying the AccFoxD3 gene silencing fragment, the sequences of which are shown as SEQ ID No. 13 and SEQ ID No. 14.

[0022] A fifth aspect of the present invention provides the use of the AccFoxD3 gene or a recombinant expression vector containing the AccFoxD3 gene in constructing a cell model sensitive to pesticide stress.

[0023] In the above application, the cell model is an Escherichia coli model or a yeast model.

[0024] Beneficial effects of the present invention:

[0025] This study, published in the journal Nature Communications, demonstrates for the first time that the AccFoxD3 gene in the Chinese honeybee (Apis cerana) is capable of responding to oxidative stress caused by pesticides and heavy metals. Silencing the AccFoxD3 gene in honeybees can improve their antioxidant capacity and survival under pesticide stress. This molecular biology research on honeybee stress resistance provides a theoretical foundation for the conservation of the Chinese honeybee and the breeding of new stress-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure 1 shows the bioinformatics analysis results of the AccFoxD3 protein. Figure A: Amino acid sequence alignment of AccFoxD3 with FoxD3 from other species, with conserved domains underlined. Figure B: Evolutionary relationship analysis of AccFoxD3 and other FoxD3 family proteins from several insects. Figure C: The three-dimensional structure of the conserved domain of FoxD3.

[0027] The results showed that AccFoxD3 is highly conserved across species and contains a conserved 85-amino acid wing-shaped DNA-binding domain unique to the Fox protein family. AccFoxD3 is closely related to AmFoxD3 in Apis mellifera.

[0028] Figure 2Figure 1 shows the expression patterns of AccFoxD3 under stress conditions. Figure A: AccFoxD3 gene expression pattern under glyphosate treatment; B: AccFoxD3 gene expression pattern under paraquat treatment; C: AccFoxD3 gene expression pattern under avermectin treatment; D: AccFoxD3 gene expression pattern under methichloride treatment; E: AccFoxD3 gene expression pattern under mercuric chloride treatment; F: AccFoxD3 gene expression pattern under chromium chloride treatment. β-actin (GenBank accession number HM640276.1) was used as an internal reference gene.

[0029] The results showed that the AccFoxD3 gene responded to oxidative stress caused by pesticides and heavy metals in Chinese honey bees.

[0030] Figure 3 This is the prokaryotic expression and eukaryotic expression experiment of AccFoxD3. Figure A: The inhibition of Escherichia coli growth by cumene hydroperoxide treatment; Figure B: The inhibition of Escherichia coli growth by mercuric chloride treatment. In the figure, No. 1 is ethanol (A) and sterilized ddH2O (B) as controls. In No. 2-5, the concentrations of cumene hydroperoxide and mercuric chloride increase in sequence. Figure C: The expression of recombinant AccFoxD3 protein in yeast; Figure D: The inhibition of yeast growth expressing recombinant AccFoxD3 protein by oxidative stress. The bacterial solution was respectively 10 -2 , 10 -3 , 10 -4 Multiple dilution.

[0031] The results showed that the expression of recombinant AccFoxD3 protein made Escherichia coli and yeast more sensitive to external oxidative stress.

[0032] Figure 4 This image shows the effects of silencing the AccFoxD3 gene on the antioxidant capacity of honey bees under pesticide stress. Figure A: AccFoxD3 gene silencing efficiency in honey bees, with β-actin used as a reference gene; Figure B: Expression of antioxidant-related genes in Apis cerana cerana after AccFoxD3 gene silencing; Figure C: Changes in antioxidant enzyme activity in Apis cerana cerana after AccFoxD3 gene silencing.

[0033] The results showed that the AccFoxD3 gene was successfully silenced, and compared with the control group, the antioxidant capacity of Chinese honeybees was significantly improved after the AccFoxD3 gene was silenced.

[0034] Figure 5This study examined the effects of silencing the AccFoxD3 gene on the survival of honey bees under pesticide stress. Figure A: Survival of Chinese honey bees under pesticide stress after silencing the AccFoxD3 gene; Figure B: Survival of Chinese honey bees under pesticide stress after silencing the AccFoxD3 gene.

[0035] The results showed that the antioxidant capacity of honey bees was improved after AccFoxD3 gene silencing, which enhanced the resistance of honey bees to pesticide stress. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0037] As mentioned earlier, due to the influence of various stresses such as pesticides and heavy metals in the environment, the number of Chinese honey bee colonies has declined significantly, greatly affecting my country's crop yields and the stability of the ecosystem.

[0038] Based on this, the present invention has conducted in-depth research on the stress resistance genes of the Chinese honeybee. The inventors of the present invention have been deeply engaged in the field of honeybee stress resistance for many years. During the long-term research, they found that: the AccFoxD3 gene responds to the oxidative stress caused by pesticides and heavy metals in the Chinese honeybee; after heterologous expression of AccFoxD3 in Escherichia coli and yeast, it negatively regulates the cell's resistance to external oxidative stress; silencing the AccFoxD3 gene can improve the antioxidant capacity and pesticide resistance of the Chinese honeybee, thus proposing the present invention.

[0039] The AccFoxD3 gene is derived from the Chinese honey bee; the nucleotide sequence of the AccFoxD3 gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 2. Specifically,

[0040] SEQ ID No. 1:

[0041]

[0042] SEQ ID No. 2:

[0043] MEPRGMMMSNLSCDGCADSDRDSDSSSMIVDPVSLDKNDLSPSQSSSSPIIPSSPVPPTTTSSRNRGGSRSKKSYSMMSGGNGQQKGNASGQAPSSYGNDKMSSSLIKPPYSYIALITMAILQSPQKKLTLSGICEFIMSRFPYYHDKFPAWQNSIRHNLSLNDCFIKIPREPGNPGKGN YWTLDPLAEDMFDNGSFLRRRKRYKRPPPHYVLRDRAIMATFAICGDRGPCPGGGGGHPGALAYPSAAYLSPPPGLPLLDFSPSTLEALKLGGFLEPPPPLYKPVPITAPPIRQIDPAPTRTTAIPSGHPPAEKKRNFSIDALIGKQAANEQNCGGLLDLSPSEHREIRSQASAFSPLV.

[0044] The protein encoded by the AccFoxD3 gene contains a wing-shaped DNA binding domain unique to the Fox transcription factor family, which is used to bind to downstream target genes and regulate gene transcription.

[0045] The present invention uses real-time fluorescence quantitative PCR to analyze the expression pattern of the AccFoxD3 gene under specific stress conditions (pesticides and heavy metals), revealing that the AccFoxD3 gene responds to oxidative stress induced by pesticides and heavy metals in Apis cerana cerana. Using genetic engineering techniques, prokaryotic and eukaryotic expression vectors for the AccFoxD3 gene were constructed. Analysis of the protein's expression characteristics under specific oxidative stress conditions revealed that expression of the recombinant AccFoxD3 protein increased the sensitivity of Escherichia coli and yeast to external oxidative stress. By constructing a honeybee model in which the AccFoxD3 gene is silenced, it was found that reducing AccFoxD3 gene expression increases the activity of antioxidant-related genes and antioxidant enzymes in the honeybee, thereby enhancing the Apis cerana cerana cerana's resistance to external environmental stress.

[0046] It is known that the AccFoxD3 gene plays an important role in regulating the pesticide and heavy metal resistance of Chinese honey bees, providing a theoretical basis for the protection of Chinese honey bees.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0048] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Experimental methods without detailed conditions were carried out according to conventional test methods or the operating instructions recommended by the supplier.

[0049] Example 1: Cloning and bioinformatics analysis of the Apis cerana cerana AccFoxD3 gene

[0050] Using the cDNA of Apis cerana cerana as a template, primers were designed based on the sequence information at NCBI, as shown in SEQ ID No. 3 and SEQ ID No. 4, and the AccFoxD3 gene fragment was amplified by polymerase chain reaction (PCR). The reaction system was as follows:

[0051]

[0052]

[0053] The PCR reaction procedure is as follows:

[0054]

[0055] The amplified products were separated by 1% agarose gel electrophoresis, and the target fragments were recovered, ligated into the cloning vector pMD19-T, and sequenced for verification.

[0056] The amino acid sequence of the AccFoxD3 protein of the Chinese honey bee was compared with the FoxD3 protein sequences of other species using DNAMAN software, and the evolutionary analysis of the AccFoxD3 protein was performed using MEGA5.0 software.

[0057] The results are as follows Figure 1 As shown, AccFoxD3 has the highest sequence similarity with AmFoxD3 from Apis mellifera and is the closest evolutionary relative. The AccFoxD3 protein sequence contains a wing-like DNA binding domain unique to plug proteins.

[0058] Example 2: Analysis of the expression characteristics of the AccFoxD3 gene under adverse stress conditions

[0059] Honey bees of similar growth conditions were collected and randomly divided into six groups of 50 each. To simulate the field environment in which honey bees live, the bees were exposed to pesticides (glyphosate, paraquat, avermectin, and methicillin) and heavy metals (mercuric chloride and chromium chloride) through feeding. The bees were then placed in an incubator at 33°C and 70% relative humidity. Samples were collected at regular intervals and stored in a -80°C freezer. Total RNA was extracted from the bees using the Trizol method, and cDNA was obtained by reverse transcription.

[0060] Using cDNA as a template, quantitative primers were designed, as shown in SEQ ID No. 5 and SEQ ID No. 6, and real-time fluorescence quantitative PCR analysis was performed to detect the expression pattern of the AccFoxD3 gene under different stress conditions. The reaction system is as follows:

[0061]

[0062] The reaction procedure is as follows:

[0063]

[0064] The experimental results are as follows Figure 2 As shown in the figure, under the conditions of pesticide and heavy metal stress, the expression level of AccFoxD3 gene changed significantly, indicating that AccFoxD3 gene responded to the oxidative stress caused by pesticides and heavy metals in the Chinese honey bee.

[0065] Example 3: Analysis of expression characteristics of recombinant AccFoxD3 protein

[0066] 1. Analysis of prokaryotic expression characteristics of recombinant AccFoxD3 protein.

[0067] Specific primers with BamH1 and Xho1 restriction sites were designed, as shown in SEQ ID No. 7 and SEQ ID No. 8, and PCR amplification was performed using the correctly sequenced plasmid as a template. The recovered product was ligated into the cloning vector pMD19-T, the plasmid was extracted, and after double enzyme digestion, the recovered product was ligated into the prokaryotic expression vector pET-30a(+). After double enzyme digestion verification, the ligation product AccFoxD3-pET-30a(+) and the empty vector pET-30a(+) were respectively transformed into the Escherichia coli expression strain Rosetta, induced by the addition of IPTG, and the protein was extracted and separated by SDS-polyacrylamide gel electrophoresis to detect the expression of the target protein.

[0068] Escherichia coli expressing AccFoxD3 recombinant protein and Escherichia coli expressing pET-30a(+) empty vector (cell number about 5×10 8 ) were spread on LB solid culture medium containing kanamycin (100 mg / L) and cultured inverted at 37°C for one hour. Five circular filter papers with the same diameter (0.6 cm) were placed on the surface of the culture medium in sequence. Isopropyl benzene hydroperoxide and mercuric chloride solutions of different concentrations (0, 20, 40, 60 and 100 mM) were dripped on the filter papers. After inverted culture at 37°C for 8-10 hours, the growth of Escherichia coli was observed, the diameter of the colony growth inhibition zone was measured, and photographed and recorded.

[0069] 2. Analysis of eukaryotic expression characteristics of recombinant AccFoxD3 protein.

[0070] An upstream primer with a BamH1 restriction site and a downstream primer with an Xho1 restriction site and an HA tag (TACCCATACGACGTCCCAGACTACGCT) were designed, as shown in SEQ ID No. 9 and SEQ ID No. 10. Using the correctly sequenced plasmid as a template, PCR amplification was performed to obtain an HA-tagged AccFoxD-HA gene sequence. The PCR amplification product was ligated into a cloning vector, the plasmid was extracted, and after double enzyme digestion, the recovered product was ligated into the eukaryotic expression vector pYES2. After double enzyme digestion verification, the ligation product, AccFoxD-HA-pYES2, and the empty vector pYES2 were respectively transformed into yeast competent cells INVSc1. The cells were induced with 2% galactose, and after protein extraction, the recombinant protein expression was detected by western blotting using an HA antibody (1:1000).

[0071] Yeast expressing AccFoxD3 recombinant protein and yeast expressing pYES2 empty vector were cultured at 10 -2 , 10 -3 , 10 -4 Perform multiple dilutions, aspirate 10 μL of bacterial solution each time, and spot them onto SC / -Ura solid medium containing 2% galactose and SC / -Ura solid medium containing 2% galactose and hydrogen peroxide (2 mM), respectively. Culture at 28°C for 2-3 days, observe the growth of yeast colonies, and take photos to record.

[0072] The experimental results are as follows Figure 3 As shown, Escherichia coli and yeast expressing AccFoxD3 recombinant protein showed more sensitive characteristics to oxidative stress caused by the external environment, which indicates that the expression of AccFoxD3 negatively regulates the oxidative stress resistance of cells.

[0073] Example 4: Effect of silencing the AccFoxD3 gene on oxidative stress resistance in Apis cerana cerana

[0074] A 400-500 bp base sequence (SEQ ID No. 11 and SEQ ID No. 12) was selected from the open reading frames of the AccFoxD3 gene and the GFP gene (GenBank accession number U87974), respectively, and primers (as shown in SEQ ID Nos. 13, 14 and SEQ ID Nos. 15, 16) were designed for PCR amplification to synthesize gene silencing fragments. The amplified products were recovered, ligated into the cloning vector pMD19-T, and transformed into the Escherichia coli cloning strain DH5α for sequencing. The correctly sequenced gene silencing fragments were used as templates for large-scale PCR amplification. The amplified products were recovered and used as transcription templates to synthesize dsRNA-AccFoxD3 and dsRNA-GFP using the T7 RiboMAX™ ExpressRNAi system (Promega, Madison, WI, USA).

[0075] Bees of similar growth status were collected and randomly divided into four groups (50 bees / group):

[0076] Group 1 (ck): fed with 30% sucrose aqueous solution (1 μL);

[0077] Group 2 (water): fed with water (1 μL);

[0078] Group 3 (dsGFP): fed with 4 μg dsRNA-GFP;

[0079] The fourth group (dsAccFoxD3) was fed with 4 μg of dsRNA-AccFoxD3.

[0080] Samples were collected at 12h, 24h and 36h after feeding, RNA was extracted, and the gene silencing efficiency was detected by real-time fluorescence quantitative PCR.

[0081] AccFoxD3 gene-silenced samples and control samples were ground into powder, and total protein was extracted with 0.9% saline. Antioxidant enzyme activities were measured using CAT, POD, and SOD enzyme activity assay kits (developed in Nanjing). Quantitative primers for antioxidant-related genes were designed based on NCBI sequence information, as shown in SEQ.ID.NO.17 and SEQ.ID.NO.18 (AccCAT), SEQ.ID.NO.19 and SEQ.ID.NO.20 (AccGSTO1), SEQ.ID.NO.21 and SEQ.ID.NO.22 (AccTpx1), and SEQ.ID.NO.23 and SEQ.ID.NO.24 (AccSOD1). Real-time fluorescence quantitative PCR was used to detect the expression levels of antioxidant-related genes in honey bees.

[0082] Bees were grouped according to the above criteria and fed sucrose solution, water, dsRNA-GFP, or dsRNA-AccFoxD3 in an incubator for 24 hours. After gene silencing, a 0.02 mg / mL glyphosate solution diluted in sucrose solution was added to the bees' food troughs. The bees were observed for their effects on the pesticides, with mortality rates counted every other day. The bees were photographed on the fourth day after the pesticide exposure.

[0083] The experimental results are as follows Figure 4 and Figure 5 As shown in the results, silencing the AccFoxD3 gene significantly increased the expression levels of antioxidant-related genes and the activity of antioxidant enzymes in honey bees. Furthermore, their resistance to pesticides also increased. This suggests that silencing the AccFoxD3 gene improves the antioxidant capacity of honey bees, thereby enhancing their pesticide resistance.

[0084] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of silencing the AccFoxD3 gene to improve the stress resistance of honey bees to glyphosate pesticide; The nucleotide sequence of the AccFoxD3 gene is shown in SEQ ID No.

1.

2. Application of inactivating the protein encoded by the AccFoxD3 gene in improving the stress resistance of Apis cerana cerana to glyphosate pesticide; the amino acid sequence of the protein encoded by the AccFoxD3 gene is shown in SEQ ID No.

2.

3. Use of a reagent for specifically detecting the AccFoxD3 gene in the preparation of a product for detecting the resistance of honey bees to glyphosate pesticide stress; the nucleotide sequence of the AccFoxD3 gene is shown in SEQ ID No.

1.

4. The use according to claim 3, characterized in that The product uses a real-time fluorescence quantitative PCR method to detect the expression level of the AccFoxD3 gene.

5. The use according to claim 4, characterized in that The product contains: a primer pair for specifically detecting the AccFoxD3 gene, the sequences of which are shown as SEQ ID No. 5 and SEQ ID No.

6.

6. Application of reagents for silencing the AccFoxD3 gene to improve the stress resistance of honey bees to glyphosate pesticide; The nucleotide sequence of the AccFoxD3 gene is shown in SEQ ID No.

1.

7. The use according to claim 6, characterized in that The reagents include: a primer pair for amplifying the AccFoxD3 gene silencing fragment, the sequences of which are shown as SEQ ID No.13 and SEQ ID No.14.

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