Application of accl(2)efl gene in regulating the resistance of apismellifera ligustica to heavy metals

Abstract

The application discloses application of an Accl(2)efl gene in regulating the heavy metal resistance of Apis cerana, and belongs to the technical field of molecular biology. The application is based on the discovery that the Accl(2)efl gene is significantly induced to express when Apis cerana is subjected to heavy metal (Cd and Hg) stress, and for the first time, a bee stress biomarker is combined with an environmental pollution detection biological index. The research also proves that silencing the Accl(2)efl gene can reduce the tolerance of Apis cerana to heavy metals (Cd and Hg). Through application of bee stress biomarker and environmental pollution detection biological index, a theoretical basis is established for identifying new bee stress biomarkers and environmental pollution detection biological indexes.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically to the application of the Accl(2)efl gene in regulating the heavy metal resistance of the Chinese honeybee. Background Technology

[0002] Honeybees, as vital pollinators, play a crucial role in the reproduction of many wild plants and are essential for ensuring adequate yields of approximately 85% of food crops. However, since the 1990s, the number of honeybee species recorded annually by the Global Biodiversity Information Facility has been declining, posing a significant threat to biodiversity, ecosystem sustainability, and global food security. In addition to interference from infectious pathogens such as viruses and parasites, industrial activities and technological advancements are driving changes in global social and economic structures, leading to the emergence of various environmental pollutants, including pesticides and heavy metals. These factors present increasingly severe challenges to the survival of honeybees.

[0003] Cadmium (Cd) and mercury (Hg) are highly toxic heavy metal pollutants that pose significant risks to humans, plants, and animals. Their primary entry into the environment is through the discharge of industrial and agricultural waste. As typical heavy metals, the concentrations of Cd and Hg in the environment are reliable indicators of environmental pollution.

[0004] The Chinese honeybee (Apis cerana cerana), endemic to China, possesses exceptional cold resistance, a small size, and high flight speed. It excels at collecting nectar from scattered sources and exhibits significant advantages in disease resistance and coping with extreme weather. Furthermore, the Chinese honeybee has a wide foraging range (approximately 7 square kilometers). Environmental pollutants such as pesticides and heavy metals can enter bee colonies through various products collected by bees, including pollen, propolis, and wax (not honey), seriously threatening the survival of the population. Unlike pesticides and other stressors that directly or indirectly cause bee death or subfatal effects, heavy metals accumulate in bees and their products, posing a potentially serious threat to human health.

[0005] Protein lethal proteins (l2efl) belong to the α-crystallin-hsps_p23-like superfamily and are key members of small heat shock proteins (sHsps). They were isolated from embryonic lethal mutants and are therefore called protein lethal proteins. Among them, sHsps are also known as "forgotten molecular chaperone proteins" because they were discovered later than other HSP families. They function independently of ATP and are the first line of defense for cells. sHSPs can form large oligomers under stress-induced conditions to prevent irreversible denaturation of substrate proteins and protect cells from oxidative stress damage. All homologs of l(2)efl (except GenBank accession number LOC410857) can be induced by heat stress. Based on the strong induction of various stressors by some l(2)efl genes, the quantification of these sHSPs is expected to provide the best biomarkers for bee stress. However, the functions of many homologs of lethal proteins (lethal(2)essentialforlifeinApis cerana cerana, Accl(2)efl) in Chinese honeybees are unknown and need further investigation.

[0006] In recent years, bees have been extensively studied as ideal biomarkers for environmental pollution. On the one hand, extensive research on bee stress biology has identified many new biomarkers that reflect the stress levels of individual bees, which is crucial for assessing the relative stress of individual bees or entire colonies. On the other hand, biomarkers play a key role in identifying and evaluating environmental pollution, and selecting appropriate organisms is essential for effective environmental monitoring. Therefore, in-depth research into the effects of Accl(2)efl gene expression levels on the health and survival of individual bees, and its relationship with tolerance to heavy metals cadmium (Cd) and mercury (Hg), will help identify new bee stress biomarkers and biomarkers for detecting environmental pollution.

[0007] In summary, how to provide a gene in the Chinese honeybee that resists Cd and Hg heavy metal stress, and how to combine honeybee stress biomarkers with environmental pollution detection bioindicators, are problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides the application of the Accl(2)efl gene in regulating the heavy metal resistance of the Chinese honeybee.

[0009] The purpose of this invention is to provide the application of the Accl(2)efl gene in the resistance of Chinese honeybees to heavy metals (Cd and Hg). This invention found that the Accl(2)efl gene was significantly induced to express under various abiotic stresses, including 4℃, 44℃, UV radiation, heavy metals (CdCl2 and HgCl2), and pesticides (flufenoxam, phoxim, and paraquat). Heterologous overexpression of the Accl(2)efl gene in *E. coli* enhanced the tolerance of *E. coli* cells to heavy metals (CdCl2 and HgCl2). Silencing the Accl(2)efl gene significantly reduced the activity of antioxidant enzymes in Chinese honeybees, ultimately leading to excessive accumulation of Cd and Hg and a sharp decline in survival rate. Therefore, exploring the function of the Accl(2)efl gene in the tolerance of Chinese honeybees to heavy metals Cd and Hg is of great significance for identifying new biomarkers of bee stress and bioindicators for environmental pollution detection.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] Applications of the Accl(2)efl gene in (1), (2) or (3) below:

[0012] (1) Regulate the tolerance of Chinese honeybees to heavy metals;

[0013] (2) As a biological indicator for detecting environmental pollution;

[0014] (3) Breed honeybee varieties with enhanced resistance to heavy metal stress;

[0015] The nucleotide sequence of the Accl(2)efl gene is shown in SEQ ID No. 1.

[0016] Application of the protein encoded by the Accl(2)efl gene in regulating the resistance of Chinese honeybees to heavy metal stress.

[0017] Furthermore, the amino acid sequence of the protein encoded by the Accl(2)efl gene is shown in SEQ ID No. 2.

[0018] Application of silencing the Accl(2)efl gene in constructing a bee model with reduced tolerance to heavy metal stress.

[0019] Application of overexpression of Accl(2)efl gene in constructing a cell model with enhanced tolerance to heavy metal stress.

[0020] Furthermore, the cell model is an Escherichia coli model.

[0021] Furthermore, the heavy metals are Cd and Hg.

[0022] Primer pairs for specific detection of the Accl(2)efl gene, the nucleotide sequences of which are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0023] A kit for detecting the resistance of bees to Cd and Hg heavy metal stress, including the primer pairs described above.

[0024] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0025] This invention, based on the discovery that the Accl(2)efl gene is significantly induced in expression in Chinese honeybees under heavy metal (Cd and Hg) stress, is the first to combine bee stress biomarkers with environmental pollution detection bioindicators. The study also demonstrated that silencing the Accl(2)efl gene reduces the tolerance of Chinese honeybees to heavy metals (Cd and Hg). By applying molecular biology and biochemical research techniques for bee stress resistance, this invention lays a theoretical foundation for identifying new bee stress biomarkers and environmental pollution detection bioindicators. Attached Figure Description

[0026] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 The following are the bioinformatics analysis results of the protein encoded by the Accl(2)efl gene in Example 1 of this invention. Among them, a is the amino acid sequence alignment result of Accl(2)efl with l(2)efl protein in other insect species, and the conserved domains and predicted secondary structures have been marked; b is the evolutionary relationship and conserved domain analysis of Accl(2)efl protein in other insect species; c is the three-dimensional structure of Accl(2)efl and its conserved domains.

[0028] Figure 2The relative expression levels of the Accl(2)efl gene under environmental stress conditions in Example 2 of this invention are shown below. Specifically, a represents the result under cold stress (4℃); b represents the result under normal temperature (16℃); c represents the result under heat stress (44℃); d represents the result under ultraviolet radiation; e represents the result under cadmium chloride (CdCl2) treatment; f represents the result under mercuric chloride (HgCl2) treatment; g represents the result under permethrin treatment; h represents the result under phorate treatment; i represents the result under paraquat treatment; and β-actin (GenBank accession number HM640276.1) is used as an internal reference gene.

[0029] Figure 3 The results of the in vitro stress resistance study of Accl(2)efl protein in Example 3 of this invention are shown. Among them, a is the expression pattern map of Accl(2)efl protein under environmental stress (CdCl2, HgCl2 and paraquat) treatment conditions; b is the growth inhibition of Escherichia coli expressing recombinant Accl(2)efl protein under environmental stress (CdCl2, HgCl2 and paraquat) treatment conditions, where 1, 2, 3, 4 and 5 represent 5 concentrations from low to high.

[0030] Figure 4 This invention describes the detection of the ability of silencing the Accl(2)efl gene in Chinese honeybees to resist Cd and Hg heavy metals in Example 4 of this invention. Specifically, a) is the silencing efficiency of the Accl(2)efl gene in honeybees injected with dsRNA-Accl(2)efl detected by RT-qPCR, with β-actin as an internal reference gene; b) shows honeybees with the Accl(2)efl gene silenced after treatment with CdCl2 and HgCl2; c) shows the Cd concentration in hemolymph after CdCl2 treatment; d) shows the survival rate after CdCl2 treatment; e) shows the Hg concentration in hemolymph after HgCl2 treatment; and f) shows the survival rate after HgCl2 treatment.

[0031] Figure 5 This invention describes the determination of the effect of silencing the Accl(2)efl gene on the antioxidant enzyme activity in Chinese honeybees in Example 5 of this invention. In this example, a represents the result of peroxidase activity determination; b represents the result of catalase activity determination; and c represents the result of superoxide dismutase activity determination. Honeybees fed with dsRNA-GFP were used as the control group, and a blank control group was also set up. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] As mentioned above, Cd and Hg are two common and seriously harmful heavy metal pollutants, requiring strengthened pollution prevention and monitoring to protect the ecological environment and human health. Cd and Hg heavy metal pollution also threatens the survival of the Chinese honeybee, thereby affecting the entire ecosystem and agricultural production.

[0034] Based on this, the present invention aims to study the genes in the Chinese honeybee that resist Cd and Hg heavy metal stress, and to give full play to the role of honeybees as ecological indicators by combining honeybee stress biomarkers with environmental pollution detection bioindicators.

[0035] In this invention, we first demonstrated that the Accl(2)efl gene was induced to express in Chinese honeybees after exposure to Cd and Hg heavy metal stress; and found that heterologous overexpression of the Accl(2)efl gene in Escherichia coli enhanced tolerance to Cd and Hg heavy metals; finally, silencing the Accl(2)efl gene resulted in an abnormal increase in Cd and Hg concentrations in the hemolymph and a significant decrease in survival rate in Chinese honeybees exposed to Cd and Hg heavy metal stress, thus proposing this invention.

[0036] The Accl(2)efl gene originates from the Chinese honeybee; the nucleotide sequence of the Accl(2)efl gene is shown in SEQ ID No. 1, and the amino acid sequence of its encoded protein is shown in SEQ ID No. 2. Details are as follows:

[0037] , SEQ ID No.1.

[0038] MSVVPLIFRDWWDDFERPVSRLMDQHFGRGLNRDDLLSRFSDISFDRPLRSIFRDRYYRPWRNVTCQPSSGSSTIQLDNKDNFQVILDVQQFSPEEITVKTVGNNVIVEAKHEERQDEHGFVSRQFIRRYVLPPSHDVINITSSLSSDGVLTITAPKKGETSSGDERIIEIVKTGEPAGKSIKVETTTEEK, SEQ ID No. 2.

[0039] The protein encoded by the Accl(2)efl gene contains an α-crystal domain (ACD) consisting of 80 amino acid residues (78–157), which plays a crucial role in maintaining molecular chaperone activity.

[0040] This invention analyzes the transcriptional expression profile of the Accl(2)efl gene under Cd and Hg heavy metal stress using real-time quantitative reverse transcription PCR, revealing that the Accl(2)efl gene is induced in *Apis cerana* under Cd and Hg stress. A prokaryotic expression vector for the Accl(2)efl gene in *E. coli* was constructed using genetic engineering techniques. Under Cd and Hg stress, the expression characteristics of the Accl(2)efl protein were analyzed, showing that the expression of recombinant Accl(2)efl protein enhanced the tolerance of *E. coli* to Cd and Hg. A bee model silencing the Accl(2)efl gene was constructed by injecting dsRNA-Accl(2)efl, reducing the expression of the Accl(2)efl gene in *Apis cerana*. This resulted in abnormally high Cd and Hg concentrations in the hemolymph, significantly decreased antioxidant capacity, and increased mortality in *Apis cerana* under Cd and Hg stress.

[0041] In conclusion, the Accl(2)efl gene plays a crucial role in enhancing the resistance of Chinese honeybees to Cd and Hg heavy metals, which provides a theoretical basis for the protection of Chinese honeybees and its application as an ecological indicator.

[0042] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0043] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Test methods without specified detailed conditions are performed according to conventional test methods or the operating instructions recommended by the supplier.

[0044] The bees processed in the embodiments of this invention are all Chinese honeybees.

[0045] Example 1

[0046] Bioinformatics analysis of the Accl(2)efl gene in the Chinese honeybee

[0047] 1. Cloning of the Accl(2)efl gene in the Chinese honeybee

[0048] Total RNA was extracted from bees using TaKaRa's RNAiso Plus reagent. Reverse transcription was then performed using the Evo M-MLV reverse transcription reagent premix kit. The total volume was 10 μL, and the reaction mixture is as follows:

[0049] Table 1 Reverse Transcription System

[0050]

[0051]

[0052] The reaction system should be prepared on ice. After gentle mixing, react at 37°C for 15 min, then at 85°C for 5 s, and finally terminate the reaction at 4°C. The reaction is then terminated, and the final product is used as a template.

[0053] Since the Chinese honeybee and the Italian honeybee are two closely related species, the sequence variation of the same gene is very small between the two species. Therefore, based on the gene sequence of the Italian honeybee l(2)efl in GenBank (GenBank accession number LOC410857), primers for gene sequencing were designed using Primer 5.0 software, as shown in SEQ ID No. 5 and SEQ ID No. 6. The details are as follows:

[0054] atgtcggtcgtgcctctg, SEQ ID No.5;

[0055] tttttcttctgtagtcgtttctactttga, SEQ ID No. 6.

[0056] The Accl(2)efl gene fragment was amplified by polymerase chain reaction (PCR) in the following reaction system (25 μL):

[0057] 5 μL cDNA, 2.5 μL 10×PCR buffer, 1 μL dNTP, 1 μL template, 1 μL each of forward and reverse primers, 0.25 μL LA-Taq DNA polymerase, and 13.25 μL double-distilled water.

[0058] Amplification program: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 40 s, 50℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min; store at 4℃ until analysis.

[0059] The PCR products were purified by 1% agarose gel electrophoresis and gel cutting, cloned into the T1 vector, and finally the identified positive clones were sent to Shanghai Sangon Biotech for sequencing verification.

[0060] 2. Bioinformatics analysis of the Accl(2)efl gene in Honeybee (Apis cerana)

[0061] The open reading frame of the Accl(2)efl gene was translated into an amino acid sequence using DNAMAN 6.0 software to obtain the protein. The Accl(2)efl gene was then compared with other species using Blast homology on the NCBI website (http: / / www.ncbi.nlm.nih.gov / ). Multiple alignments of the Accl(2)efl gene sequences from different species were performed using MEGA 5 software, and a phylogenetic tree was constructed using the NJ (neighbor-joining) method. The conserved domains of Accl(2)efl were analyzed using the MEME (https: / / meme-suite.org / meme / tool-s / meme) online tool. The secondary structure of the protein was predicted using the SWISS-MODEL (https: / / swissmodel.expasy.org / interactive) online tool.

[0062] 3. Test Results

[0063] The test results are as follows Figure 1 As shown, the protein encoded by the Accl(2)efl gene contains an α-crystal domain (ACD) consisting of 80 amino acid residues (78–157), which plays a crucial role in maintaining molecular chaperone activity. Phylogenetic analysis shows that it exhibits high similarity and conservation among most bee species.

[0064] Example 2

[0065] Analysis of the expression characteristics of the Accl(2)efl gene under environmental stress conditions

[0066] A batch of Chinese honeybees was randomly selected from normal bee colonies and randomly divided into 10 groups of 50 bees each. They were housed in specially designed indoor wooden boxes (CN 204047589 U) and placed in a constant temperature and humidity incubator (temperature 34℃, humidity 60% ± 10%), fed with a 50% sucrose solution. After 24 hours of acclimatization, a non-biological stress experiment was conducted.

[0067] The first group of bees was placed in an incubator at a temperature of 33°C (control group);

[0068] The second, third, and fourth groups of bees were placed in incubators at temperatures of 4℃, 16℃, and 44℃, respectively (experimental groups);

[0069] The fifth group was placed in an incubator at 33℃, with the ultraviolet lamp (254nm, 30mJ / cm²) turned on. 2 );

[0070] Groups 6 and 7 were placed in an incubator at 33°C and subjected to heavy metal stress treatment by freely feeding on a 50% sucrose solution of CdCl2 and HgCl2 (final concentration of CdCl2 and HgCl2 was 1.0 μg / mL).

[0071] Groups 8, 9, and 10 were placed in an incubator at 33°C and subjected to pesticide stress treatment by freely feeding on a 50% sucrose solution of fluchlorfon, phoxim, and paraquat (with a final concentration of 20 mg / L for fluchlorfon, phoxim, and paraquat).

[0072] Samples were taken at specific time intervals, flash-frozen in liquid nitrogen, and stored at -80°C. Total mRNA was extracted using the Trizol method, and then immediately reverse transcribed into cDNA using a reverse transcription kit. After adjusting the cDNA concentration of each sample to the same level, the samples were stored at -20°C for later use. Real-time quantitative reverse transcription PCR was performed, with 1000 ng of cDNA added to a 20 μL quantitative PCR system. The relative expression level of the target gene was detected using a 7500 Real-Time PCR instrument (ABI 7500, USA) following the instructions of the quantitative PCR kit (TaKaRa).

[0073] Reaction program: Pre-denaturation 95℃, 10s; denaturation 95℃, 5s; annealing 60℃, 40s, 40 cycles; melting curve addition, 1 cycle.

[0074] The reference sequences for primer design of the target gene were obtained from the NCBI database, and the primer sequences are shown in SEQ ID No. 3 and SEQ ID No. 4:

[0075] cggaagagattacggtgaaga, SEQ ID No. 3;

[0076] tgagatggaggcaagacgta, SEQ ID No. 4.

[0077] The test results are as follows Figure 2 As shown, the Accl(2)efl gene in the Chinese honeybee responds to oxidative stress caused by environmental stress. Under low temperature conditions (4℃), its expression level continues to increase with the increase of stress time; under high temperature stress (44℃), it shows a trend of first decreasing and then increasing; under heavy metal (CdCl2 and HgCl2) treatment, the transcription level increases significantly and reaches the highest level in 2-3 hours; with the continuous stress of pesticides (flufenoxam, phoxim and paraquat), the expression of Accl(2)efl is in a state of rapid increase.

[0078] Example 3

[0079] Study on the in vitro stress resistance of Accl(2)efl protein

[0080] 1. Analysis of the expression characteristics of Accl(2)efl protein under environmental stress conditions

[0081] Design a pair of upstream and downstream primers with HindIII restriction sites, as shown in SEQ ID No. 7 and SEQ ID No. 8:

[0082] aagctt atgtcggtcgtgcc(HindⅢ), SEQ ID No.7;

[0083] aagctt tttttcttctgtagtcgttt(HindⅢ), SEQ ID No. 8.

[0084] Then, PCR was performed using cDNA as a template; the target fragment was obtained and recovered, and then the target fragment and the prokaryotic expression vector pET30a(+) were digested with HindIII, and the digested target fragment and vector fragment were recovered by agarose gel electrophoresis; the two were ligated with ligase, and after transformation and identification, the positive clone pET-30a(+)-Accl(2)efl was obtained. The recombinant pET-30a(+)-Accl(2)efl plasmid was transformed into BL21(DE3) Escherichia coli competent cells and incubated with shaking at 37℃ for 2-3 hours to allow OD 600 The concentration was reached to 0.2–0.5. Subsequently, *E. coli* cells were induced to express the protein with IPTG at a final concentration of 1 mM / L, and SDS-PAGE analysis was performed. 0.5 ml of purified Accl(2)elf protein was used as the antigen, mixed with an equal volume of Freund's complete adjuvant, and 1 ml was subcutaneously injected into the abdomen of mice. One week after the initial immunization, 0.5 ml of antigen was mixed with an equal volume of Freund's incomplete adjuvant, and 1 ml was subcutaneously injected into the mice again. This process was repeated twice every week. After the fourth immunization, a large amount of blood was collected from the mouse eyeballs, and serum was extracted as anti-Accl(2)efl antibody.

[0085] The samples treated with CdCl2, HgCl2, or paraquat in Example 2 were homogenized with a mixture of protease inhibitors in RIPA buffer for Western blot analysis. After centrifugation at 14000×g for 15 minutes, the supernatant was collected, and the protein concentration was determined using the Micro BCA Protein Assay Kit from Kangwei Century. The protein supernatant was adjusted to a final concentration of 2 μg / μL with RIPA buffer and boiled for 10 minutes. The denatured protein mixture (40 μg each) was run on a 4–12% polyacrylamide gel and transferred to a PVDF membrane. The membrane was blocked with NcmBlot rapid blocking buffer from Xinsemide and incubated sequentially with primary antibody (anti-Accl(2)efl antibody, 1:100 or anti-α-tubulin, 1:2000) and secondary antibody, and detected using NcmECL Ultra from Xinsemide.

[0086] 2. Prokaryotic expression characteristics analysis of recombinant Accl(2)efl protein

[0087] The *E. coli* cells expressing Accl(2)efl as described in Example 3 were seeded onto LB agar plates for an antibacterial assay. The control group consisted of *E. coli* cells containing the pET-30a(+) empty vector. After incubation at 37°C for 1 hour, five circular filter papers with a diameter of 6 mm were placed on the plate surface, and 2 μL of HgCl2 (0, 20, 40, 60, 80 mg / L), CdCl2 (0, 20, 40, 60, 80 mM / L), and paraquat (0, 50, 100, 200, 300 mM / L) solutions were added sequentially. The agar plates were incubated overnight at 37°C, and the diameter of the surrounding inhibition zone was measured and recorded, and photographs were taken to record the data.

[0088] The test results are as follows Figure 3 As shown, the expression level of Accl(2)efl protein increased under stress from CdCl2, HgCl2, and paraquat. The inhibition zone assay results showed that, compared with the control group, the diameter of the inhibition zone in the HgCl2 and CdCl2-treated media significantly decreased with increasing concentration. These results all indicate that Accl(2)efl plays a role in responding to HgCl2 and CdCl2-induced oxidative stress.

[0089] Example 4

[0090] The effect of silencing the Accl(2)efl gene on the tolerance of Chinese honeybees to heavy metals Cd and Hg.

[0091] The base sequences (SEQ ID No. 9 and SEQ ID No. 10) of the Accl(2)efl gene and the GFP gene (GenBank accession number U87974) are as follows:

[0092] atgtcggtcgtgcctctgatatttcgcgattggtgggacgatttcgaacgtccagtgtcccgattgatggaccaacacttcggcagaggattgaatcgagacgacttgttatcacggttttccgatatcagcttcgatagacctctacgctcgatatttcgcgacagatattaccgtccgtggagaaacgtgacatgtcaaccctcgagcggatccagcacgattcaactcgacaataaggacaacttccaggtaatactggacgttcaacaattctcaccggaagagattacggtgaagacggtcggtaataatgttattgtcgaagcaaaacacgaggagagacaagacgagcacggatttgtcagccgacaattcatacggagatacgtcttgcctccatctcacgacgtgatcaacattacctcgagtctatcttcagatggtgttttaactatcacggcacccaaaaagggagaaacatctagtggagatgaacgaatcattgaaatcgtgaaaacaggagaaccagcgggtaaatcgatcaaagtagaaacgactacagaagaaaaa,SEQ ID No.9。

[0093] agtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaagcggcacgacttcttcaagagcgccatgcctgagggatacgtgcaggagaggaccatcttcttcaaggacgacgggaactacaagacacgtgctgaagtcaagtttgagggagacaccctcgtcaacaggatcgagcttaagggaatcgatttcaaggaggacggaaacatcctcggccacaagttggaatacaactacaactcccacaacgtatacatcatggccgacaagcaaaagaacggcatcaaagccaacttcaagacccgccacaacatcgaagacggcggcgtgcaactcgctgatcattatcaacaaaatactccaattggcgatggccctgtccttttacc, SEQ ID No. 10.

[0094] Primers containing a T7 promoter are designed, as set forth in SEQ ID No. 11 and 12, and SEQ ID No. 13 and 14.

[0095] taatacgactcactatagggcgaatgtcggtcgtgcctctg (T7 Promoter), SEQ ID No. 11;

[0096] taatacgactcactatagggcgatttttcttctgagtcgtttctactttga (T7 Promoter), SEQ ID No. 12;

[0097] taatacgactcactatagggcgaagtggagagggtgaag (T7 Promoter), SEQ ID No. 13;

[0098] taatacgactcactatagggcgaggtaaaaggacagggc (T7 Promoter), SEQ ID No. 14.

[0099] PCR amplification was performed separately. Then, using the PCR amplification products as templates, the T7 high-efficiency transcription kit (TransGenBiotech) was used to transcribe them into dsRNA-Accl(2)elf and dsRNA-GFP. The reaction system is as follows:

[0100] Table 2. High-efficiency T7 transcription system

[0101]

[0102] After reacting the above system at 37°C for 16 hours, 2 μL of DNase I was added, and the reaction was continued at 37°C for 0.5 hours. Finally, the reaction was terminated with 1 μL of EDTA (500 mM, pH 8.0), and the system was stored at -80°C for later use.

[0103] Five healthy bee colonies were selected, and dsRNA-Accl(2)efl (8 μg), dsRNA-GFP (8 μg), and an equal volume of water were injected into the junction of the first and second abdominal segments to interfere with RNA. Water and dsRNA-GFP served as controls. Samples were collected every 12 hours and immediately frozen in liquid nitrogen and stored at -80℃. The knockout efficiency of the Accl(2)efl gene was detected by RT-qPCR. Subsequently, the Chinese honeybees with the Accl(2)efl gene silenced were exposed to Cd and Hg heavy metal stress (3 μg / mL) again. The serum Cd and Hg concentrations were measured using inductively coupled plasma mass spectrometry (YS EXT8600, Thermo Fisher Scientific, USA), and the survival rate was calculated.

[0104] The test results are as follows Figure 4 As shown, the expression level of the Accl(2)efl gene was significantly reduced in Chinese honeybees 36 h after injection of dsRNA-Accl(2)efl. Compared with the control group (water and dsRNA-GFP), silencing the Accl(2)efl gene caused an abnormal increase in the concentration of Cd and Hg in the hemolymph of Chinese honeybees and increased the mortality rate of bees under Cd and Hg heavy metal stress, reducing the bees' resistance to Cd and Hg heavy metals.

[0105] Example 5

[0106] The effect of silencing the Accl(2)efl gene on the antioxidant capacity of the Chinese honeybee

[0107] The Accl(2)efl gene silencing sample and the control sample (prepared in the same way as in Example 4) were added to an appropriate amount of PBS buffer and placed in a 2mL centrifuge tube. The tissue homogenate was broken up and homogenized. The sample was centrifuged at 5,000g for 5 minutes at 4℃. The supernatant was collected and divided into two parts. One part was used to determine the protein concentration according to the BCA Protein Assay Kit instructions for the calibration of enzyme activity data. The other part was used to detect the activities of peroxidase, catalase and superoxide dismutase in Chinese honeybees using the enzyme-linked immunosorbent assay (ELISA) double antibody sandwich method to determine its antioxidant capacity.

[0108] The test results are as follows Figure 5 As shown, compared with the control group, silencing the Accl(2)efl gene significantly reduced the activity of peroxidase, catalase and superoxide dismutase in Chinese honeybees, thus reducing the antioxidant capacity of honeybees.

[0109] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0110] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. silencing Accl(2)efl application of a gene in the construction of a model of honeybee with reduced tolerance under heavy metal stress, characterized in that, The Accl(2)efl The nucleotide sequence of the gene is shown in SEQ ID No. 1 ; The heavy metal is Cd and Hg.

2. overexpression Accl(2)efl application of the genes in the construction of cell models with enhanced tolerance to heavy metal stress, characterized in that, The Accl(2)efl The nucleotide sequence of the gene is shown as SEQ ID No. 1 ; The cell model is an E. coli model; The heavy metal is Cd and Hg.

Images