Apis cerana DnaJC28 gene as well as encoding protein and application thereof

Abstract

The invention discloses an apis cerana DnaJC28 gene as well as an encoding protein and application thereof, and belongs to the technical field of cytobiology and molecular biology. The invention finds that apis cerana DnaJC28 is positioned in cytoplasm through research for the first time. The short-term heat stress and the long-term heat stress can induce the expression of the apis cerana DnaJC28, the silent DnaJC28 can reduce the survival rate of the apis cerana under the heat stress, and the heterologous overexpression apis cerana DnaJC28 can increase the survival rate of the drosophila S2 cells under the heat stress. A plurality of antioxidant genes can partially make up the function of DnaJC28 under heat stress, and oxidative damage caused by heat stress to Chinese bees is reduced. The gene can be used as a novel heat-resistant gene target, the gene is over-expressed in other economic animals in the future, the yield and the heat resistance of the gene under the heat stress condition are expected to be improved, and a sustainable solution is provided for livestock production under the climate change.

Description

Technical Field

[0001] This invention belongs to the fields of cell biology and molecular biology, and specifically relates to a species of Chinese honeybee. DnaJC28 Genes and their encoded proteins and their applications. Background Technology

[0002] Heat stress refers to the molecular, physiological, and biochemical stress responses of organisms when exposed to high temperatures exceeding their optimal growth temperature range. Heat stress disrupts the organism's homeostasis, leading to the accumulation of reactive oxygen species (ROS), causing oxidative damage, metabolic disorders, hindered growth and development, and even death. Global climate change has further exacerbated the intensity and frequency of heat stress, drawing significant attention from researchers. Research on heat stress is crucial for ecological balance, food security, and the survival and evolution of organisms, and is a key link in addressing the challenge of global warming. Therefore, screening and identifying heat stress-responsive genes, providing new candidate heat-resistant target genes, and deeply analyzing their mechanisms of response to heat stress are of great significance.

[0003] Heat shock proteins (Hsps) are a key family of proteins in response to heat stress, repairing denatured proteins, preventing protein aggregation, and aiding in proper protein folding. Based on molecular weight (kDa), Hsps are generally classified into six subfamilies: small Hsp (sHsp), Hsp40, Hsp60, Hsp70, Hsp90, and Hsp100. Hsp40 is an important cofactor in the Hsp70 molecular chaperone system. It is worth noting that although Hsps were first discovered in Drosophila due to their heat stress-induced expression, subsequent research has revealed that not all Hsps respond to heat stress. Cellular location and growth environment can influence the ability of Hsps to respond to heat stress. Furthermore, the expression patterns and regulatory mechanisms of the same Hsps protein may differ across species, indicating species-specific Hsps expression.

[0004] Among the Hsps of multiple species, the Hsp40 subfamily has the highest number of genes. For example, 45, 27, 22, 17, 7, and 6 potential genes were found in humans, honeybees, Saccharomyces cerevisiae, Schizothiazonia, Escherichia coli, and fruit flies, respectively. Hsp40 The Hsp40 protein, due to the presence of a J domain (approximately 70 amino acid residues), is often named DnaJ. Based on its structure, DnaJ can be classified into three types: DnaJA, DnaJB, and DnaJC. In many species, DnaJC is far more abundant than DnaJA and DnaJB. Studies have shown that some... DnaJGenes play a crucial role in responses such as disease and stress, and under different stress conditions, different genes... DnaJ Gene expression patterns differ. However, relevant research mainly focuses on... DnaJA and DnaJB China. As the largest quantity DnaJ Members of the Asia family, DnaJC What are its main functions? Which ones? DnaJC Can genes act as key factors in response to heat stress? These questions require further investigation.

[0005] The Chinese honeybee (Apis cerana) is a subspecies of the Oriental honeybee endemic to China. It is highly cold-resistant and adaptable to climates with large temperature differences, particularly suited to mountainous environments. It excels at collecting nectar from scattered sources and is an important species for maintaining my country's ecological balance. The heat tolerance of Chinese honeybees varies across different latitudes in my country. For example, those in low-latitude regions are more heat-resistant and easily adapt to high-temperature environments. Those in mid-latitude regions have moderate heat tolerance and can cope with hot weather. Those in high-latitude regions are less heat-resistant and are more suited to low-temperature environments. Heat stress adversely affects the foraging ability, growth, and development of Chinese honeybees. Therefore, studying the mechanisms of heat stress response in Chinese honeybees, identifying heat-resistant genes, and exploring differences in heat tolerance can not only provide key candidate target genes for breeding heat-resistant bee species but also help Chinese honeybees better adapt to future climate change. Furthermore, candidate target genes in Chinese honeybees can be transferred into other economically important animals through gene editing or transgenic technology, potentially improving their survival ability and yield in the context of global warming, thus possessing significant economic value. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a Chinese honeybee DnaJC28 The research findings of this invention, including the genes, their encoded proteins, and their applications, have important reference value for innovative research on heat-resistant breeding.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the Chinese honeybee. DnaJC28 The application of genes in any of the following (1)-(3): (1) Regulating the survival rate of insects under heat stress conditions; (2) Prepare products that regulate the heat resistance of insects under heat stress; (3) Breed insect varieties with enhanced heat resistance under heat stress conditions; The Chinese honeybee DnaJC28 The gene nucleotide sequence is shown in SEQ ID NO.1, as follows:

[0008] In a second aspect, the present invention provides the Chinese honeybee. DnaJC28 The application of the gene-encoded protein in the following (1) or (2): (1) Regulating the survival rate of insects under heat stress conditions; (2) Prepare products that regulate the heat resistance of insects under heat stress; The Chinese honeybee DnaJC28 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2, as follows: MTIRFNSHRKHLFFKNMLRFNQYNRKILKNIICVPYWEFMLMKRFKHQHNMKKWYQTLEVAEDCEDETLRLAFVYQAKRFHPDSGTSEANATKFSEIETAYRQIRKARIEKKENCTNLPEVEEFDIKHTAPQHRHYLVYNVGIGTHSKRQKLYTMERAQKAVDSVLEHRLKKLQTEERNTLVGMDKQ HAKNIKTRFGMDRLVEDLIQEAMNRGEFKDLPGMGKPLKENTNTRNPYVDFVTYKLNEILIENGFTPEWIQLSKEIREEIQDLRKKLINARSKVGHIPLNYKDEKTWKNIVENFKSKTKEINIKVDKYNLLVPILQKQMLHVKLEDLAKEALSTPAQETLKEKVNSDISVERKNNLLTEMISSIFNK.

[0009] In a third aspect, the present invention provides a product containing the aforementioned Chinese honeybee. DnaJC28 The application of genetic biomaterials in any of the following (1)-(3): (1) Regulating the survival rate of insects under heat stress conditions; (2) Prepare products that regulate the heat resistance of insects under heat stress; (3) Breed insect varieties with enhanced heat resistance under heat stress conditions.

[0010] The biological material is containing the Chinese honeybee. DnaJC28 Gene recombinant vectors, recombinant bacteria, or transgenic cell lines.

[0011] In this application, the heat resistance is specifically manifested in reducing the degree of oxidative damage to insects caused by heat stress and improving the survival rate of insects under heat stress conditions.

[0012] Preferred, by increasing the concentration of Chinese honeybees DnaJC28 Gene expression or increase in Chinese honeybees DnaJC28 The activity of gene-encoded proteins can improve the survival rate of insects under heat stress.

[0013] Due to the Chinese honeybee DnaJC28 Genes can be used to regulate the survival rate of insects under heat stress conditions. For harmful insects, the Chinese honeybee can be used as a potential candidate. DnaJC28 Homologous genes of genes in harmful insects, by downregulating the homologous genes of harmful insects DnaJC28 The expression of homologous genes or the reduction of harmful insects DnaJC28 The activity of homologous gene-encoded proteins can reduce the survival rate of harmful insects under heat stress conditions. Specifically, RNA interference technology can be used to artificially synthesize proteins in harmful insects... DnaJC28 Double-stranded RNA (dsRNA) of homologous genes is fed to harmful insects. After the insects ingest it, it triggers a reaction within the insects. DnaJC28 The silencing of homologous genes reduces the survival rate of harmful insects under heat stress conditions.

[0014] Preferably, the expression of *Apis cerana* is overexpressed by introducing it into the recipient insect species. DnaJC28 Gene recombinant expression vectors were used to breed insect varieties with enhanced heat resistance under heat stress conditions.

[0015] The beneficial effects of this invention are: This invention is the first to discover that both short-term and long-term heat stress can induce erroneous honeybees in the Chinese honeybee. DnaJC28 Expression, silence DnaJC28 It can reduce the survival rate of Chinese honeybees under heat stress, and heterologous overexpression of Chinese honeybees DnaJC28 It can increase the survival rate of Drosophila S2 cells under heat stress. Multiple antioxidant genes can partially compensate for this. DnaJC28 Its function under heat stress reduces oxidative damage caused by heat stress to the Chinese honeybee. Given the Chinese honeybee... DnaJC28 The heat resistance of this gene could be used as a new target for heat resistance genes. Overexpression of this gene in other economically important animals could potentially improve their yield and heat resistance under heat stress. If successfully applied, this could provide a sustainable solution for livestock production in response to climate change. Attached Figure Description

[0016] Figure 1 This diagram shows the subcellular localization of DnaJC28 in the Chinese honeybee; the red color after Cy3 staining indicates the expression of DnaC28, and the blue color after DAPI staining indicates the location of the cell nucleus.

[0017] Figure 2 Chinese honeybee under heat stress DnaJC28 Expression pattern detection map; among which, Figure 2A in the figure represents the result of a 5-hour non-stress treatment at 33°C. DnaJC28 The expression situation, Figure 2 B in the figure represents the result of a 5-hour non-stress treatment at 40°C. DnaJC28 The expression situation, Figure 2 C in the figure represents 43°C after 5 hours of non-stress treatment. DnaJC28 The expression situation, Figure 2 D in the figure represents the result of a 5-hour non-stress treatment at 46°C. DnaJC28 The expression situation, Figure 2 E in the figure represents the result of a 48-hour non-stress treatment at 33°C. DnaJC28 The expression situation, Figure 2 F in the figure represents the result of a 40°C non-stress treatment for 48 h. DnaJC28 The expression of .

[0018] Figure 3 For silence DnaJC28 The effect of heat stress on the response of the Chinese honeybee is shown in the following graph; among them, Figure 3 A in the text represents identification. DnaJC28 The efficiency of silence, Figure 3 B in the figure represents statistical silence. DnaJC28 Survival rate of Chinese honeybees under heat stress conditions.

[0019] Figure 4 Heterologous overexpression of *Apis cerana* under heat stress conditions DnaJC28 The effect of the effect on the state and survival rate of Drosophila S2 cells is shown in the graph; among them, Figure 4 In the figure, A represents heterologous overexpression of *Apis cerana* in Drosophila S2 cells. DnaJC28 The cells were then subjected to heat stress at 37°C. The state of the fruit fly S2 cells after treatment for 0 h, 18 h and 48 h is shown in the figure. Myc: pUAST-6×Myc, Myc-DnaJC28: pUAST-6×Myc-DnaJC28. Figure 4 In the figure, B represents heterologous overexpression of *Honeysuckle chinensis* under heat stress treatment. DnaJC28 The result of trypan blue staining of Drosophila S2 cells at 48 h. The dark blue cells indicated by the red arrow are some of the dead cells after trypan blue staining. Figure 4 In the figure, C represents the difference between heat stress treatment and heterologous overexpression of *Apis cerana* compared to the control group. DnaJC28 The cell mortality rate of Drosophila S2 cells after 48 h is shown in the figure.

[0020] Figure 5 For silence DnaJC28 A graph showing the effect of [the virus] on the expression levels of antioxidant genes in *Apis cerana*. Figure 5 A in the text is CYP4G11 The amount of expression, Figure 5 B in CDK5 The amount of expression, Figure 5 C in the text is CDK5r The amount of expression, Figure 5 D in Trx1 The amount of expression, Figure 5 E in Trx2 The amount of expression, Figure 5 F in Tpx4 The amount of expression, Figure 5 G in Tpx5 The amount of expression, Figure 5 H in Hsp22.6 The amount of expression, Figure 5 The I in MsrB The amount of expression, Figure 5 G in GSTO2 The amount of expression, Figure 5 K in GSTS4 The amount of expression, Figure 5 L in GSTD The amount of expression, Figure 5 M in GSTT1 The amount of expression, Figure 5 N in SOD1 The amount of expression, Figure 5 O in MKK4 The amount of expression, Figure 5 P in CYP336A1 The amount of expression, Figure 5 Q in GSTZ1 The amount of expression, Figure 5 R in TrxR1 The amount of expression, Figure 5 S in p38b The amount of expression, Figure 5 T in Tpx3 The amount of expression. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] As mentioned earlier, many questions remain regarding the adaptability of the Chinese honeybee to heat stress and the specific molecular mechanisms of its heat stress response, requiring further investigation. Due to differences in subcellular localization, developmental stage, and growth environment, different responses to heat stress under various stress conditions will lead to different outcomes. DnaJ Genes also differ in their ability to respond to heat stress.

[0023] Based on this, the present invention provides the Chinese honeybee. DnaJC28 Genes, their encoded proteins, and applications further supplement different DnaJThe study of gene expression patterns, heat tolerance, and participation in heat stress response in Chinese honeybees provides new candidate heat stress response genes for breeding heat-resistant transgenic honeybee varieties.

[0024] Specifically, the Chinese honeybee is a subspecies of the Eastern honeybee. This invention is based on the Eastern honeybee genome published on NCBI. DnaJC28 Nucleotide sequence design and amplification of *Apis cerana* (registration number: LOC108001060) DnaJC28 Specific primers were used to amplify the cDNA of the Chinese honeybee using PCR. DnaJC28 The coding area. The obtained Chinese honeybee DnaJC28 The coding region was constructed into the eukaryotic expression vector pUAST-6×Myc and transfected into Drosophila S2 cells. Immunofluorescence assays and two-photon laser confocal microscopy revealed that the DnaJC28 of the Chinese honeybee was located in the cytoplasm.

[0025] This invention utilizes reverse transcription quantitative PCR (RT-qPCR) to discover that different degrees of heat stress (40°C, 43°C, and 46°C) can induce induced ... DnaJC28 The expression of [the condition]. Furthermore, 4-5 hours after treatment, with increasing heat stress, [the following occurred]. DnaJC28 The degree of induction increases. Furthermore, under both short-term and long-term thermal stress conditions, DnaJC28 The expression levels of all of them increased. These results indicate that... DnaJC28 It plays an important role in the heat stress response of the Chinese honeybee.

[0026] This invention also provides a method to silence Chinese honeybees. DnaJC28 The nucleotide sequence is described. Using this nucleotide sequence to synthesize dsRNA and feeding it to Chinese honeybees can silence them. DnaJC28 Furthermore, this invention provides a silent... DnaJC28 The reduction in the tolerance of Chinese honeybees to heat stress indicates DnaJC2 8. Plays an important role in heat stress. This invention further provides that, compared with the control group, heterologous overexpression of *Apis cerana* plays a crucial role. DnaJC28 It can improve the survival rate of Drosophila S2 cells under heat stress conditions, further illustrating... DnaJC28 It is a key gene in the response to heat stress.

[0027] This invention also provides a silent Chinese honeybee. DnaJC28 Upregulates multiple antioxidant genes ( CYP4G11 , CDK5 , CDK5r , Trx1 , Trx2 , Tpx4 , Tpx5 , Hsp22.6 , MsrB, GSTO2 , GSTS4 , GSTD , GSTT1 , SOD1, MKK4 The transcriptional level of these antioxidant genes is upregulated. Heat stress can induce oxidative damage in honeybees, and these upregulated antioxidant genes may partially compensate for the silencing of other genes. DnaJC28 This function helps reduce oxidative damage to Chinese honeybees caused by heat stress.

[0028] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0029] Example 1: Subcellular localization analysis of DnaJC28 in Honeybee (Apis cerana) 1. Total RNA extraction and first-strand cDNA synthesis from *Apis cerana* Total RNA was extracted from Chinese honeybees using the Trizol method. Then, using this RNA as a template, the first strand of Chinese honeybee cDNA was synthesized using the TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser.

[0030] 2. Construction of pUAST-6×Myc-DnaJC28 eukaryotic expression vector (1) Based on the published genome of the Eastern honeybee DnaJC28 Nucleotide sequence design to amplify Chinese honeybee DnaJC28 The specific primers SEQ ID NO.3-SEQ ID NO.4 were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0031] Upstream primer (SEQ ID NO.3): 5'- AAGGAAAAAA GCGGCCGCGATGCGCATTATATATTTATTGTATA-3' Note: Underlined sequences are protected bases, and bolded sequences are... Not I. Enzyme cleavage site.

[0032] Downstream primer (SEQ ID NO.4): 5'- GC TCTAGATTATTTATTAAAAATCGAAGATATC-3' Note: Underlined sequences are protected bases, and bolded sequences are... Xba I. Enzyme cleavage site.

[0033] (2) Amplification of Chinese honeybee using PCR DnaJC28 The coding region. The PCR system used is as follows: Table 1: PCR System The PCR reaction program used was as follows: 94°C for 5 min, 94°C for 40 sec, 57°C for 30 sec (35 cycles), 72°C for 60 sec, and 72°C for 10 min.

[0034] (3) The PCR products amplified in (2) were subjected to 1% agarose gel electrophoresis and then recovered from the gel. Containing DnaJC28 The gel recovery product and pUAST-6×Myc eukaryotic expression empty vector plasmid were used simultaneously Not I and Xba I. Enzyme digestion. After 1% agarose gel electrophoresis and gel recovery, the digestion products were ligated using T4 DNA ligase. DnaJC28 The pUAST-6×Myc-DnaJC28 eukaryotic expression vector was constructed by ligating it with the empty vector pUAST-6×Myc. Then, the pUAST-6×Myc-DnaJC28 plasmid was extracted using the Gold Standard Ultra-Large-Scale Plasmid Extraction Kit (Kangwei Reagent Company).

[0035] 3. Resuscitation, passage, and transfection of Drosophila S2 cells (1) Preheat the cryopreserved Drosophila S2 cells and Drosophila S2 cell-specific culture medium (iCell) in a 25°C water bath.

[0036] (2) Add the Drosophila S2 cells and culture medium from step (1) to a sterile culture dish and incubate in a sterile incubator (25°C) for the first resuscitation of Drosophila S2 cells. When the cells have filled the dish, divide the cells equally into two sterile culture dishes, add culture medium, and incubate in a sterile incubator (25°C) for the second resuscitation of Drosophila S2 cells. When the cells have filled the dish, transfer 1 mL of Drosophila S2 cells to a new sterile culture dish, add 3 mL of culture medium, and incubate in a sterile incubator (25°C) for passage of Drosophila S2 cells. When the cells have filled the dish, perform the Drosophila S2 cell transfection experiment.

[0037] (3) On a sterile laminar flow hood, add the transfection reagents PEI and pUAST-6×Myc-DnaJC28 to 400 uL of PBS at a mass ratio of 2:1, mix well, and incubate at room temperature for 15 min. Then transfect the PBS into the Drosophila S2 cells that have been passaged in step (2), mix gently, and incubate in a sterile incubator (25°C) for 48 h before performing an immunofluorescence experiment.

[0038] 4. Immunofluorescence assay Collect the transfected cells into centrifuge tubes, centrifuge at 12000 rpm for 3 min, discard the supernatant, and resuspend the pellet in PBS (1 mL). Add 4% formaldehyde (1 mL) to the centrifuge tubes, shake on a shaker for 20 min, centrifuge at 12000 rpm for 3 min, discard the supernatant, and wash the pellet with PBS (1 mL) for 10 min, repeating the wash 3 times. Centrifuge at 12000 rpm for 3 min, discard the supernatant, add PBT (1 mL) to the pellet, and shake for 10 min. Then, centrifuge at 12000 rpm for 1 min, discard the supernatant, and resuspend the pellet in PBS (200 μL). Add mouse primary antibody anti-Myc at a ratio of 1:200, and shake at 4°C for 12 h. Then, centrifuge at 12000 rpm for 3 min, discard the supernatant, and wash the pellet with PBS (1 mL) for 10 min, repeating the wash 3 times. Centrifuge at 12000 rpm for 3 min, discard the supernatant, add PBS (200 μL), and then add Cy3-labeled affinity-purified goat anti-mouse IgG (H+L) secondary antibody at a ratio of 1:200. Incubate at room temperature in the dark for 2 h. Add PBS (300 μL), then add DAPI (1:1000), and incubate at room temperature in the dark for 15 min. Centrifuge at 12000 rpm for 3 min, wash the precipitate with PBS (1 mL) for 10 min, repeating 3 times. Prepare slides and observe the subcellular localization of DnaJC28 using a two-photon laser confocal microscope. Figure 1 It is evident that DnaJC28 in the Chinese honeybee is located in the cytoplasm.

[0039] Example 2: Detection of the effects of heat stress on Chinese honeybees DnaJC28 expression patterns 1. Take 300 foraging bees and divide them into six groups (50 bees in each group). Groups 1-3 were placed in incubators at 46°C, 43°C and 40°C respectively, and samples were taken at 0 h, 1 h, 2 h, 3 h, 4 h and 5 h after treatment. Control group 1 was the fourth group of Chinese honeybees, placed in an incubator at 33°C, and the sampling time was the same as that of groups 1-3.

[0040] The fifth group of bees was placed in an incubator at 40°C, and samples were taken at 0 h, 12 h, 24 h, 36 h and 48 h after treatment; the control group 2 (the sixth group of bees) was placed in an incubator at 33°C, and the sampling time was the same as that of the first group.

[0041] 2. Total RNA was extracted from Chinese honeybees using the Trizol method. Then, using this RNA as a template, the first strand of cDNA was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa).

[0042] 3. Using the first strand of cDNA synthesized in step 2 as a template, perform RT-qPCR using a CFX96™ Real-Time System instrument (Bio-Rad) and a TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) kit (TaKaRa). The internal reference gene is... β-actin (GenBank registration number is HM640276.1).

[0043] 4. The primer sequences used for RT-qPCR are as follows: DnaJC28 Primers: Upstream primer (SEQ ID NO.5): ACAGCACCACAACATCGT; Downstream primer (SEQ ID NO.6): GATCCATGCCAAAACGTG.

[0044] β-actin Primers: Upstream primer (SEQ ID NO.7): TTATATGCCAACACTGTCCTTT; Downstream primer (SEQ ID NO.8): AGAATTGATCCACCAATCCA.

[0045] Depend on Figure 2 It can be seen that under different degrees of thermal stress... DnaJC28 The expression patterns differ; both short-term and long-term heat stress can induce [the expression]. DnJC28 The expression level. Furthermore, 4-5 h post-treatment, with increasing heat stress, DnaJC28 The degree of induction increases.

[0046] Example 3: Exploring Silence DnaJC28 Effects of heat stress on the response of Chinese honeybees 1. Amplification using PCR technology DnaJC28 A portion of the coding region (74-574 nt, SEQ ID NO.9). Simultaneously, 500 bp was amplified. GFP (GenBank registration number U87974) sequence.

[0047] The sequence of SEQ ID NO.9 is as follows: CTCTAGAAGTTGCAGAAGATTGTGAAGATGAGACATTAAGATTAGCATTTGTCTATCAAGCAAAAAGATTCCATCCAGATAGTGGTACATCAGAAGCTAATGCAACTAAATTTTCTGAGATTGAAACTGCTTATAGACAAATTCGTAAAGCAAGAATAGAAAAGAAAGAAAATTGTACAAATCTACCTGAAGTTGAAGAATTTGACATTAAACATACAGCACCACAACATCGTCATTATTTAGTTTATAAT GTAGGTATTGGAACACATAGCAAAAGACAAAAATTATATACAATGGAAAGAGCTCAAAAAGCAGTTGATAGTGTATTGGAACATAGATTAAAAAAATTACAAACTGAAGAACGTAATACATTAGTTGGAATGGATAAACAACATGCAAAAAATATTAAAACACGTTTTGGCATGGATCGTTTAGTAGAAGATTTAATTCAAGAAGCAATGAATAGAGGTGAATTTAAAGATCTACCAGGAATGGGTAAAC.

[0048] Amplification DnaJC28 The primers for the SEQ ID NO.9 sequence are: Upstream primer (SEQ ID NO.10): GGATCC TAATACGACTCACTATAGG CTCTAGAAGTTGCAGAAGATTG; Downstream primer (SEQ ID NO.11): GGATCC TAATACGACTCACTATAGG GTTTACCCATTCCTGGTAGATC.

[0049] The primers used to amplify GFP are: Upstream primer (SEQ ID NO.12): GGATCC TAATACGACTCACTATAGG AGTGGAGAGGGTGAAGGTGA; Downstream primer (SEQ ID NO.13): GGATCC TAATACGACTCACTATAGG GGTAAAAGGACAGGGCCATC.

[0050] Note: The T7 RNA polymerase promoter sequence is the underlined portion.

[0051] 2. Separately, the adhesive recovery process in step 1... DnaJC28 and GFP The PCR products were then used as templates to synthesize RNAi using the T7RiboMAX™ Express RNAi System (Promega). DnaJC28 and GFP dsRNA (dsRNA-DnaJC28 and dsRNA-GFP).

[0052] 3. Thirty foraging bees were collected and divided into two groups (15 bees per group). Each bee in the first group was fed 5 μg of dsRNA-GFP (control group), and each bee in the second group was fed 5 μg of dsRNA-DnaJC28 (experimental group). After two days of feeding, total RNA was extracted from both groups and reverse transcribed into cDNA. Using this cDNA as a template, silencing was detected by RT-qPCR. DnaJC28 The efficiency. (By) Figure 3 As can be seen from A in the data, compared with the control group, dsRNA - DnaJC28 can reduce DnaJC28 The expression level in the Chinese honeybee reached a silencing level. DnaJC28 The effect.

[0053] 4. Identify the Chinese honeybee DnaJC28 After successful silencing with dsRNA-DnaJC28, 60 foraging bees were collected and divided into two groups (30 bees per group). Following the method in step 3, the two groups were fed dsRNA-GFP and dsRNA-DnaJC28 respectively. After two days of incubation, both groups were treated with heat stress (43°C), and mortality was recorded every 0.5 hours. Figure 3 From B, we can know that silence DnaJC28 Reduce the survival rate of Chinese honeybees under heat stress conditions.

[0054] Example 4: Detection of Heterologous Overexpression of Honeybee DnaJC28 Effects on the heat resistance of Drosophila S2 cells 1. Following the method in step 3 of Example 1, the pUAST-6×Myc-DnaJC28 eukaryotic expression vector was transfected into S2 cells of Drosophila, with cells transfected with the empty vector pUAST-6×Myc as a control.

[0055] 2. After culturing the cells from step 1 in a 25°C sterile incubator for 48 h, they were transferred to a 37°C sterile incubator for heat stress treatment. Cell status was observed at 0 h, 18 h, and 48 h after treatment using an inverted white light / fluorescence microscope. Additionally, at 48 h after treatment, both groups of cells were stained with trypan blue using a cell viability assay kit (Beyotime). After trypan blue staining, cell status was observed using a normal white light microscope. Furthermore, the number of dead cells and the total number of cells were counted using a hemocytometer to calculate the cell death rate. Observations of cell status and trypan blue staining results showed that, compared with the control group, heterologous overexpression of *Apis cerana* significantly increased cell viability. DnaJC28 It can significantly reduce the mortality rate of Drosophila S2 cells under heat stress. Figure 4 These results further prove that the Chinese honeybee... DnaJC28 It plays an important role in the thermal stress response.

[0056] Example 5: Analysis of Silent Chinese Honeybees DnaJC28 Effects on the expression levels of other antioxidant genes 1. Take 60 foraging bees and divide them into two groups (30 bees in each group). Feed the two groups of bees dsRNA-GFP and dsRNA-DnaJC28 respectively according to the method in step 3 of Example 3. After two days of culture under normal conditions, store the samples.

[0057] 2. RNA was extracted from two groups of bees, and cDNA was synthesized. Antioxidant genes were detected using RT-qPCR. CYP4G11 , CDK5 , CDK5r , Trx1 , Trx2 , Tpx4 , Tpx5 , Hsp22.6 , MsrB、GSTO2 , GSTS4 , GSTD , GSTT1 , SOD1、MKK4 , CYP336A1 , GSTZ1 , TrxR1 , p38b , Tpx3 The expression level of ). The internal reference gene is β-actin (GenBank registration number is HM640276.1).

[0058] 3. The primer sequences used for RT-qPCR are as follows: Table 2: Primer sequences for RT-qPCR The study results showed that, compared with the control group, silence... DnaJC28 Increase the body of Chinese honeybees CYP4G11 , CDK5 , CDK5r , Trx1 , Trx2 , Tpx4 , Tpx5 , Hsp22.6 , MsrB , GSTO2 , GSTS4 , GSTD , GSTT1 , SOD1 , MKK4 The amount of expression, reduced CYP336A1 , GSTZ1 and TrxR1 The amount of expression, for p38b , Tpx3 The expression level was not affected. Figure 5 ).

[0059] In summary, this invention reveals that DnaJC28 in *Apis chinensis* plays a role in the cytoplasm. Compared to the control group, both short-term and long-term heat stress can induce cytoplasmic hyperthermia in *Apis chinensis*. DnaJC28 The expression; and as the degree of heat stress increases, DnaJC28 The degree of induction increases. Furthermore, this invention provides a method for silencing hysteria in Chinese honeybees. DnaJC28 Nucleotide sequence fragments. And confirmed silencing. DnaJC28 Reduces the survival rate of Chinese honeybees under heat stress. (Silence) DnaJC28 Subsequently, the induced expression of some antioxidant genes may, to some extent, compensate for [the damage caused by these genes]. DnaJC28 This invention aims to reduce oxidative damage to *Apis cerana* caused by heat stress. Subsequently, this invention provides a heterologous overexpression of *Apis cerana*. DnaJC28 It can improve the survival rate of Drosophila S2 cells under heat stress, further confirming... DnaJC28 It plays a crucial role in heat stress and has the potential to drive innovation in heat-resistant breeding. Against the backdrop of global warming, the use of the Chinese honeybee... DnaJC28 The heat resistance of this gene, through gene editing or transgenic technology, could be expressed in other economically important animals, potentially improving their survival rate and yield under heat stress conditions, thus having significant economic value.

[0060] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. Chinese honeybee DnaJC28 The application of genes in any of the following (1)-(3): (1) Regulating the survival rate of insects under heat stress conditions; (2) Prepare products that regulate the heat resistance of insects under heat stress; (3) Breed insect varieties with enhanced heat resistance under heat stress conditions; The Chinese honeybee DnaJC28 A gene is a DNA molecule as shown in i) or ii) below: i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.

2.

2. Chinese honeybee DnaJC28 The application of the gene-encoded protein in the following (1) or (2): (1) Regulating the survival rate of insects under heat stress conditions; (2) Prepare products that regulate the heat resistance of insects under heat stress; The Chinese honeybee DnaJC28 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. Contains the Chinese honeybee as described in claim 1 DnaJC28 The application of genetic biomaterials in any of the following (1)-(3): (1) Regulating the survival rate of insects under heat stress conditions; (2) Prepare products that regulate the heat resistance of insects under heat stress; (3) Breed insect varieties with enhanced heat resistance under heat stress conditions.

4. The application according to claim 3, characterized in that, The biological material is containing the Chinese honeybee. DnaJC28 Gene recombinant vectors, recombinant bacteria, or transgenic cell lines.

5. The application according to claim 1, characterized in that, The heat resistance is specifically manifested in reducing the degree of oxidative damage to insects caused by heat stress and improving the survival rate of insects under heat stress conditions.

6. The application according to claim 1, characterized in that, By increasing the amount of Chinese honeybees DnaJC28 Gene expression or increase in Chinese honeybees DnaJC28 The activity of gene-encoded proteins can improve the survival rate of insects under heat stress.

7. The application according to claim 1, characterized in that, By introducing a gene capable of overexpressing the Chinese honeybee into the recipient insect species. DnaJC28 Gene recombinant expression vectors were used to breed insect varieties with enhanced heat resistance under heat stress conditions.

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