Application of Xpac and Det genes as biomarker of drosophila melanogaster to nanoparticle combined cadmium pollution

Abstract

The invention relates to application of Xpac and Det genes as biomarkers of drosophila melanogaster to nanoparticle combined cadmium pollution, which verifies the regulation effect of XPAC and Det on intestinal tract structure, DNA stability and cell apoptosis under the condition of combined pollution by knocking down the XPAC and Det genes. Results show that DNA damage is aggravated after XPAC knock-down, and Det knock-down causes serious intestinal structure damage and skeleton disintegration, which indicates that the XPAC and Det knock-down have functional differentiation in coping with combined pollution. The gene can be used as a key regulatory factor and a potential biomarker of an environmental pollution toxic reaction, and a theoretical basis is provided for pollution toxicological mechanism research and biological prevention and control strategies.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of Xpac and Det genes as biomarkers of Drosophila to nanoparticle combined cadmium pollution. Background Art

[0002] With the widespread presence of nanomaterials and heavy metal pollution in the environment, combined exposure to nanoparticles and cadmium is becoming an increasingly significant environmental health risk. Due to their small particle size, large surface area, and high surface activity, nanoplastics easily combine with heavy metal ions in the environment to form complex pollutants, enhancing their toxicity, bioaccumulation, and ability to penetrate biological barriers. These complex pollutants can enter organisms through ingestion, causing multi-level damage to the intestinal tract, liver, nervous system, and other systems.

[0003] Cadmium is a highly toxic, non-essential heavy metal that can induce reactive oxygen species (ROS) production, mitochondrial dysfunction, DNA damage, and programmed cell death. Existing research has primarily focused on the toxic effects of single pollutants, while systematic studies of the molecular response mechanisms and specific targets of combined NPs and Cd exposure are lacking. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Xpac and Det genes as biomarkers for Drosophila to nanoparticle combined cadmium pollution. By targeted silencing of Xpac and Det genes, their roles in alleviating or exacerbating combined pollution-induced intestinal tissue damage are revealed, providing a new strategy for toxicity assessment and biological control of environmental pollutants.

[0005] To achieve the above objectives, the first aspect of the present invention provides the use of Xpac and Det genes as biomarkers of Drosophila to nanoparticle combined with cadmium pollution.

[0006] Preferably, knocking down the Xpac gene and / or Det gene of Drosophila is used as a biomarker for nanoparticle-combined cadmium pollution.

[0007] A second aspect of the present invention provides a method for constructing a fruit fly model, comprising the following steps: hybridizing the MyO1A-Gal4;nsyb-Gal80 strain fruit flies with the UAS-Xpac-RNAi strain fruit flies and the UAS-Det-RNAi strain fruit flies, respectively, to obtain a MyO1A>Xpac RNAi fruit fly model and a MyO1A>Det RNAi fruit fly model, thereby achieving selective knockdown of the Xpac and Det genes in the fruit fly intestine.

[0008] The second aspect of the present invention provides the use of the above-mentioned Drosophila model in detecting nanoparticle-combined cadmium pollution.

[0009] By knocking down the Drosophila XPAC and Det genes, the present study verified the regulatory effects of XPAC and Det on intestinal structure, DNA stability, and apoptosis under conditions of combined pollution. The results showed that knocking down XPAC exacerbated DNA damage, while knocking down Det led to severe intestinal structural damage and skeletal collapse, indicating that the two genes have differentiated functions in responding to combined pollution. These genes can serve as key regulatory factors and potential biomarkers of environmental pollution toxicity, providing a theoretical basis for research on pollution toxicological mechanisms and biocontrol strategies.

[0010] In this study, knockdown of Xpac exacerbated DNA damage, while knockdown of Det led to cytoskeletal collapse and increased apoptosis, suggesting that these responses to combined toxicity occur through repair and anti-apoptotic pathways, respectively. This study provides key targets for the study of the molecular mechanisms of environmental pollution. Xpac and Det can serve as biomarkers for the combined toxicity of NPs and Cd, and lay the foundation for the development of environmental remediation technologies based on gene regulation. Compared with existing methods for verifying gene function, this study combines the triple indicators of phenotype, cell structure, and DNA damage, improving the sensitivity and specificity of gene function analysis in environmental toxicity assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 qPCR validation of key genes in the Cd + PS-NPs group. (A) FPKM values of key genes. (B) Relative mRNA expression of key genes. Mean ± SD, n = 5. The significance of the differences between the two treatment groups was analyzed using an independent-samples T-test. ns, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0012] Figure 2 Figure 2. Effects of knockdown of Xpac and Det on intestinal morphology and length. (Mean ± SD, n = 15. Differences between the two treatment groups were analyzed using an independent-samples T-test; ns, no significant difference; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.)

[0013] Figure 3 Effects of knocking down Xpac and Det on the cytoskeleton (Phalloidin labels the cytoskeleton (green), DAPI labels the nucleus (blue), and the last column is a fused image. Scale bar: 20 μm, n=10).

[0014] Figure 4 Effects of knocking down Xpac and Det on DNA damage (gH2Av labels DNA-damaged cells (green), the second column is a fused image. DAPI labels cell nuclei (blue), scale bar: 20 μm, n=15).

[0015] Figure 5 Statistics of the DNA damage rate after knocking down Xpac and Det. DETAILED DESCRIPTION

[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0017] The present invention provides a method for constructing a fruit fly model, comprising the following steps: hybridizing a MyO1A-Gal4;nsyb-Gal80 strain fruit fly with a UAS-Xpac-RNAi strain fruit fly and a UAS-Det-RNAi strain fruit fly, respectively, to obtain a MyO1A>Xpac RNAi fruit fly model and a MyO1A>Det RNAi fruit fly model, thereby achieving selective knockdown of the Xpac and Det genes in the fruit fly intestine.

[0018] The gene encoding the Drosophila Xpac gene is CG6358, and the gene encoding the Det gene is CG12265. UAS-Xpac RNAi Drosophila (accession number TH02047.N) and UAS-Det RNAi Drosophila (accession number TH02216.N) were purchased from the Drosophila Resource and Technology Platform of Tsinghua University. The MyO1A-Gal4;nsyb-Gal80 background strain has been maintained in the laboratory for a long time.

[0019] The following experiments were conducted on the constructed fruit fly model:

[0020] (1) Combined pollution treatment and phenotypic observation

[0021] The constructed Drosophila model was treated with combined pollution of PS-NPs and Cd, and the changes in intestinal structure were evaluated.

[0022] 1.1 Treatment Plan: MyO1A>Xpac RNAi Drosophila, MyO1A>Det RNAi Drosophila, and wild-type controls (MyO1A>w^1118) were exposed to the following treatment conditions for 7 days:

[0023] Control group (no contamination); PS-NPs group (20 nm polystyrene nanoparticles, 50 ppm); Cd group (CdCl2, 240 μM); Cd+PS-NPs combined group.

[0024] 1.2 Intestinal morphology observation: After treatment, the Drosophila midgut was dissected and observed using a stereo microscope.

[0025] The midgut of MyO1A>Xpac RNAi fruit flies in the Cd+PS-NPs group was significantly twisted and thinned, showing a "jejunum" phenomenon; the midgut of MyO1A>Det RNAi fruit flies in the Cd and combined groups was shortened by nearly half, the structure became softer, and the transparency increased, with the most significant phenotype.

[0026] 1.3 Cytoskeleton staining analysis: Phalloidin was used to label F-actin, and DAPI was used to label the cell nucleus.

[0027] The intestinal cytoskeleton of Xpac RNAi fruit flies in the Cd and combined groups was broken and disordered; the skeleton of Det RNAi fruit flies was severely missing and the cell nucleus was severely distorted in all treatment groups, especially in the Cd+PS-NPs group.

[0028] 1.3 Cytoskeleton staining analysis: Phalloidin was used to label F-actin, and DAPI was used to label the cell nucleus.

[0029] The intestinal cytoskeleton of Xpac RNAi fruit flies in the Cd and combined groups was broken and disordered; the skeleton of Det RNAi fruit flies was severely missing and the cell nucleus was severely distorted in all treatment groups, especially in the Cd+PS-NPs group.

[0030] (2) DNA damage detection and functional difference analysis

[0031] The effect of gene knockdown on DNA damage levels was analyzed by immunofluorescence staining.

[0032] 3.1 Experimental methods:

[0033] The gH2Av antibody was used to label DNA double-strand breaks in Drosophila midgut tissue. After fixation, blocking, incubation with primary antibodies, and labeling with secondary antibodies, the tissues were imaged using a confocal laser scanning microscope.

[0034] 3.2 Result Analysis:

[0035] In the MyO1A>Xpac RNAi fruit flies in the Cd+PS-NPs group, the DNA damage signal (gH2Av positive) was 27.42% higher than that of the wild type, which was significantly aggravated.

[0036] In MyO1A>Det RNAi flies, DNA damage signals were significantly increased under all four treatments, and the number of positive cells was 2.68 times that of the wild type, indicating that Det is involved in maintaining DNA stability.

[0037] Comparative analysis showed no significant difference in the damage trend between the Xpac knockdown group and the Cd-only exposure group, while the Det knockdown group showed severe damage under all treatment conditions, indicating functional differentiation between the two. These results confirm the key role of Det in maintaining intestinal stability and inhibiting DNA damage, while Xpac plays a supporting regulatory role in DNA repair, and the two exhibit different toxicological responses to nanoparticle and cadmium pollution exposure.

Claims

1. Application of Xpac gene and / or Det gene as biomarkers for Drosophila to nanoparticle combined with cadmium pollution.

2. The use according to claim 1, characterized in that Knockdown of the Drosophila Xpac gene and / or Det gene serves as a biomarker for nanoparticle-associated cadmium pollution.

3. A method for constructing a fruit fly model, characterized in that: The following steps are involved: The MyO1A-Gal4;nsyb-Gal80 strain of fruit flies was hybridized with the UAS-Xpac-RNAi strain of fruit flies and the UAS-Det-RNAi strain of fruit flies, respectively, to obtain the MyO1A>XpacRNAi fruit fly model and the MyO1A>Det RNAi fruit fly model, thereby achieving selective knockdown of the Xpac and Det genes in the fruit fly intestine.

4. Use of the Drosophila model according to claim 3 in detecting nanoparticle-combined cadmium pollution.

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