Application of heat tolerance related gene hsc70-4 of apiscerana and the encoded protein thereof

Abstract

This invention discloses the application of the heat tolerance-related gene Hsc70-4 in the Chinese honeybee and its encoded protein, belonging to the fields of cell biology and molecular biology. This invention verifies the silencing... Hsc70‑4 It can reduce the survival rate of Chinese honeybees under heat stress, and overexpression Hsc70‑4 It can increase the survival rate of Drosophila S2 cells under heat stress. Hsc70‑4 The induced expression of this substance helps reduce oxidative damage caused by heat stress in Chinese honeybees and improves their heat tolerance. Given... Hsc70‑4 Its heat resistance may be improved through genetic engineering in the future. Hsc70‑4 Overexpression in bees or other economically important animals can enhance their heat tolerance and thus increase their yield under heat stress, providing a new gene target for addressing ecological and agricultural challenges in the context of global warming.

Description

Technical Field

[0001] This invention belongs to the fields of cell biology and molecular biology, specifically relating to the application of the heat resistance-related gene Hsc70-4 of the Chinese honeybee and its encoded protein. Background Technology

[0002] As vital insect pollinators, bees are essential for maintaining ecosystem stability, preserving biodiversity, and increasing agricultural yields. Beyond pollination, bees also make significant contributions to the production of honey, beeswax, and royal jelly, further highlighting their economic importance. However, in nature, bee populations are frequently threatened by various environmental stressors, including pests, pesticides, heavy metals, and extreme temperatures. Prolonged exposure to environmental stress can lead to bee mortality, reduced colony numbers, and consequently, adverse effects on beekeeping, the national economy, and ecological balance.

[0003] Among various environmental stresses, heat stress adversely affects the reproduction, growth, development, and foraging behavior of honeybees. Heat stress reduces sperm motility, decreases foraging behavior, causes deformities in the legs, wings, and stingers, accelerates the age at which honeybees begin foraging, and impairs their learning and memory abilities. Prolonged heat stress increases the workload of worker bees, making colonies highly susceptible to decline. Especially with global warming, increased extreme weather events, and widespread greenhouse gas emissions, the risk of heat stress for honeybees is further increased. Therefore, identifying and screening heat stress response genes in honeybees and exploring their mechanisms of coping with heat stress are of significant practical importance for addressing climate change, providing new candidate target genes, and protecting and breeding heat-resistant honeybee germplasm resources.

[0004] Heat shock proteins (Hsps) are key proteins in the response to heat stress. It's worth noting that although Hsps were discovered because of their heat stress-induced expression, not all Hsps respond to heat stress. Based on molecular weight (kDa), Hsps are generally divided into six classes: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, and small Hsps (sHSPs). Hsp70 is a relatively conserved molecular chaperone within the Hsps family. Prokaryotes typically have only one Hsp70. Eukaryotes generally contain multiple Hsp70s, and the number of Hsp70s varies among different eukaryotes. For example, fungi... Blastocladiella emersoniiThe number of Hsp70 molecules in humans, yeast, and Chinese honeybees are 10, 8, 8, and 6, respectively. The Hsp70 structure is conserved, mainly containing three domains: an N-terminal nucleotide-binding domain (NBD), a C-terminal substrate-binding domain (SBD), and a C-terminal variable region. The NBD possesses ATP hydrolysis and binding activity. The SBD contains an α-cap and a β-domain. The α-cap regulates substrate binding, and the β-domain binds the substrate. The C-terminal variable region is not conserved, which may be one reason why different members of the Hsp70 family exhibit non-redundant functions.

[0005] Based on their expression patterns, Hsp70 in cells is classified into two types: constitutive and inducible. Constitutive Hsp70 (Hsc70) is continuously expressed under normal physiological conditions and is not induced by stress. Inducible Hsp70 (Hsp70i) is rapidly induced to express when an organism is subjected to stress, and its expression level returns to low levels after the stress condition subsides. Hsp70 can be localized in the cytoplasm, nucleus, endoplasmic reticulum, and mitochondria, and the localization of different Hsp70 types varies within the cell. Studies have shown that when subjected to stress, inducible Hsp70 localized in the cytoplasm can translocate to the nucleus to exert its function; when the stress condition is removed, Hsp70 located in the nucleus can return to the cytoplasm. These results indicate that inducible Hsp70 plays an important role in the stress response. Furthermore, research shows that Hsp70 is a multifunctional protein, playing not only a role in the stress response but also an important role in protein quality control and disease. However, current research on Hsps mainly focuses on a few species.

[0006] The Chinese honeybee is a bee species endemic to my country, and its genome contains six Hsp70 genes. However, it remains unclear whether these Hsp70 genes are constitutive or inducible under different stress conditions; further investigation is needed to determine whether they all respond to heat stress, as well as their expression patterns, subcellular localization, and specific mechanisms of action. Furthermore, studies have shown that while both yeast and mammals rely on Hsp70 under stress, their mRNA sequences and translational regulatory mechanisms differ significantly, reflecting specific adaptations at the system level in different organisms. Under heat stress, further analysis is needed to clarify the similarities and differences in Hsp70 expression regulation patterns between honeybees and other animals, and whether they exhibit species-specific adaptive characteristics. Summary of the Invention

[0007] In view of the above-mentioned prior art, the purpose of this invention is to provide an application of the heat resistance-related gene Hsc70-4 of the Chinese honeybee and its encoded protein.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, this invention provides heat-resistance-related genes in the Chinese honeybee. Hsc70-4 Application in any of the following (1)-(3):

[0010] (1) Regulating the heat resistance of insects under heat stress conditions;

[0011] (2) Prepare products that regulate the heat resistance of insects under heat stress;

[0012] (3) Breed insect varieties with enhanced heat resistance under heat stress conditions;

[0013] The heat resistance-related genes of the Chinese honeybee Hsc70-4 It is a DNA molecule as shown in i) or ii) below:

[0014] i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0015] ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.2.

[0016] The DNA nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0017]

[0018] The amino acid sequence shown in SEQ ID NO.2 is as follows:

[0019] MAKAPAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTETERLIGDAAKNQVAMNPNNTIFDAKRLIGRRFEDPTVQADMKHWPFTVVNDGGKPKIQVYYKGEAKTFFPEEVSSMVLVKMKETAEAYLGKTVSNAVITVPAYFNDSQRQATKDAG TISGLNVLRIINEPTAAAIAYGLDKKTTSERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNHFVQEFKRKYKKDLTANKRALRRLRTACERAKRTLSSSTQASIEIDSLYEGIDFYTSITRARFEELCADLFRGTLEPVEKSLRDAK MDKAQIHDIVLVGGSTRIPKIQKLLQDFFNGKELNKSINPDEAVAYGAAVQAAILHGDKSEEVQDLLLLDVTPLSLGIETAGGVMTALIKRNTTIPTKQTQTFTTYADNQPGVLIQVYEGERAMTKDNNLLGKFELSGIPPAPRGVPQIEVTFDIDANGILN VSAVDKSTGKENKITITNDKGRLSKEDIERMVNEAEKYRSEDEKQKETIAAKNGLESYCFNMKSTVEDEKLKDKISASDKQVVLDKCNNDIIKWLDANQLADKEEYEHKQKELEAICNPIVTKLYQGTGGMPGGMPGGMPGGFPGAGGGAPGGGASGPTIEEVD

[0020] Preferably, the insect includes honeybees, fruit flies, or mosquitoes.

[0021] In a second aspect, the present invention provides heat-resistance-related genes in the Chinese honeybee. Hsc70-4 The encoded protein is used in either (1) or (2) as follows:

[0022] (1) Regulating the heat resistance of insects under heat stress conditions;

[0023] (2) Prepare products that regulate the heat resistance of insects under heat stress;

[0024] The heat resistance-related genes of the Chinese honeybee Hsc70-4The encoded protein is shown in either (A1) or (A2) below:

[0025] (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2 of the sequence listing;

[0026] (A2) The protein obtained by attaching a protein tag to the N-terminus of the protein defined in (A1).

[0027] In a third aspect, the present invention provides a gene containing the aforementioned heat resistance-related genes of the Chinese honeybee. Hsc70-4 The application of biomaterials in any of the following (1)-(3):

[0028] (1) Regulating the heat resistance of insects under heat stress conditions;

[0029] (2) Prepare products that regulate the heat resistance of insects under heat stress;

[0030] (3) Breed insect varieties with enhanced heat resistance under heat stress conditions.

[0031] The biomaterial contains the heat resistance-related genes of the Chinese honeybee. Hsc70-4 Recombinant vectors, recombinant bacteria, or transgenic cell lines.

[0032] In this application, the heat resistance is specifically manifested in: reducing the content of hydrogen peroxide and protein carbonyl groups in the insect body, increasing the vitamin C content and total antioxidant capacity in the insect body, reducing the degree of oxidative damage to the insect under heat stress, and improving the survival rate of the insect under heat stress conditions.

[0033] In a fourth aspect, this invention provides a method for improving the survival rate of insects under heat stress conditions by upregulating heat-resistance-related genes in the Chinese honeybee. Hsc70-4 The expression or increase of heat-resistant genes in Chinese honeybees Hsc70-4 Enhancing the activity of encoded proteins to improve insect survival rates under heat stress conditions.

[0034] The present invention verified in its embodiments that overexpression of heat-resistance-related genes in honeybees could improve heat resistance. Hsc70-4 It can increase the survival rate of Drosophila S2 cells under heat stress.

[0035] In a fifth aspect, the present invention provides a method for reducing the survival rate of insects under heat stress conditions by downregulating heat-resistance-related genes in the Chinese honeybee. Hsc70-4 The expression or reduction of heat-resistant genes in Chinese honeybees Hsc70-4 The activity of the encoded protein reduces the survival rate of insects under heat stress conditions, further demonstrating... Hsc70-4 It plays an important role in the thermal stress response.

[0036] In a sixth aspect, the present invention provides a method for breeding insect varieties with enhanced heat resistance, comprising: introducing into a recipient insect variety a gene capable of overexpressing heat resistance-related genes in honeybees (Apis cerana). Hsc70-4 The recombinant expression vector was realized.

[0037] The beneficial effects of this invention are:

[0038] This invention discloses heat-resistance-related genes in the Chinese honeybee. Hsc70-4 Applications, through silence Hsc70-4 It can reduce the survival rate of Chinese honeybees under heat stress by overexpressing Hsc70-4 It can increase the survival rate of Drosophila S2 cells under heat stress. Hsc70-4 The induced expression of this substance helps reduce oxidative damage caused by heat stress in Chinese honeybees and improves their heat tolerance. Given... Hsc70-4 Heat resistance, which may be improved through genetic engineering in the future. Hsc70-4 Overexpression in bees or other economically important animals can enhance their heat tolerance and thus increase their yield under heat stress, providing a new gene target for addressing ecological and agricultural challenges in the context of global warming. Attached Figure Description

[0039] Figure 1 Subcellular localization detection diagram of Hsc70-4 in Honeybee (Apis cerana). Cells were hybridized using anti-Flag antibody. Red (stained with Cy3) indicates Hsc70-4 expression. Blue (stained with DAPI) indicates the location of the cell nucleus.

[0040] Figure 2 Gradient thermal stress Hsc70-4 Transcription level detection graph, internal reference gene is β-actin (GenBank Registration No.: HM640276.1); among which Figure 2 In case of no stress (control group) treatment, the treatment was as follows: Hsc70-4 Expression patterns; Figure 2 Medium BD: 40°C (B), 43°C (C), and 46°C (D) heat stress treatment conditions Hsc70-4 Expression patterns;

[0041] Figure 3 :silence Hsc70-4 A graph showing the effect of heat stress on the survival rate of Chinese honeybees. Figure 3 A: Reverse Transcription Quantitative PCR (RT-qPCR) was used to identify dsRNA-Hsc70-4 ( hsc70-4 -)hour Hsc70-4 The efficiency of silence. β-actinAs an internal reference gene, dsRNA-GFP was fed ( gfp The Chinese honeybee was used as a control. Figure 3 B: Statistical silence Hsc70-4 The survival rate of Chinese honeybees under heat stress was measured, with the control group consisting of Chinese honeybees fed dsRNA-GFP.

[0042] Figure 4 Heterogeneous overexpression of Chinese honeybee Hsc70-4 A graph showing the effect of the effect on the heat resistance of Drosophila S2 cells. Figure 4 A: Overexpression of *Apis chinensis* at 0 h, 18 h, and 48 h of heat stress (37°C) treatment Hsc70-4 Effects on the state of Drosophila S2 cells: The control group consisted of Drosophila S2 cells expressing the empty vector pUAST-3×Flag. Flag: pUAST-3×Flag. Flag-Hsc70-4: pUAST-3×Flag-Hsc70-4. Figure 4 B and C: Microscopic observations after trypan blue staining following treatment at 37°C for 48 h (B) and cell death rate results (C). Drosophila S2 cells expressing the empty vector pUAST-3×Flag served as the control group. Dead cells appeared dark blue in trypan blue staining. Red arrows indicate some of the dead cells marked in dark blue.

[0043] Figure 5 :silence Hsc70-4 ( hsc70-4 -) The hydrogen peroxide content in the body of the Chinese honeybee ( Figure 5 (A) Protein carbonyl content ( Figure 5 Vitamin B content ( ) Vitamin C content ( ) [[ID=Z47]]Figure 5 (C) and total antioxidant capacity ( Figure 5 The effect of dsRNA-GFP on the feeding of dsRNA-GFP (D) gfp The Chinese honeybee was used as a control. Detailed Implementation

[0044] 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.

[0045] 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.

[0046] Example 1: Subcellular localization analysis of Hsc70-4 in Honeybee (Apis cerana)

[0047] 1. Extraction of RNA and synthesis of the first strand of cDNA

[0048] Total RNA was extracted from Chinese honeybees using the Trizol method. Then, using this RNA as a template, first-strand cDNA was synthesized using the TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser.

[0049] 2. Put [[ID=Z50]]Hsc70-4 The coding region was constructed onto a eukaryotic expression vector.

[0050] (1) According to the NCBI database, Oriental Honeybee Hsc70-4 The coding region design of (NCBI ID: LOC107994859) amplifies the Chinese honeybee. Hsc70-4 Specific primers were developed. These primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0051] Upstream primer (SEQ ID NO.3):

[0052] 5'- AAGGAAAAAA GCGGCCGATGGCTAAAGCACCTGCAGT-3'

[0053] Note: Underlined sequences are protected bases, and bolded sequences are... Not I. Enzyme cleavage site.

[0054] Downstream primer (SEQ ID NO.4):

[0055] 5'- GC TCTAGATTAATCGACTTCTTCGATGG-3'

[0056] Note: Underlined sequences are protected bases, and bolded sequences are... Xba I. Enzyme cleavage site.

[0057] (2) Using bee cDNA as a template, PCR amplification was performed using the upstream and downstream primers mentioned in (1).

[0058] The PCR system used is as follows:

[0059] Table 1: PCR system:

[0060]

[0061] The PCR reaction procedure used is as follows:

[0062] Table 2: PCR reaction procedure:

[0063]

[0064] (3) The PCR products amplified in (2) were detected by electrophoresis on a 1% agarose gel. Products containing... The fragmented adhesive was cleaved and recycled. The resulting recycled adhesive product and pUAST-3×Flag empty carrier were simultaneously used... Hsc70-4 I and Not I. Enzyme digestion. The digestion products were subjected to 1% agarose gel electrophoresis and gel recovery. Then, T4 DNA ligase was used to ligate... Xba The enzyme digestion gel recovery product of the empty vector pUAST-3×Flag (stored in the laboratory of Qufu Normal University) was ligated to construct pUAST-3×Flag- Hsc70-4 Eukaryotic expression vector. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar (containing ampicillin, 50 mg / L), and incubated at 37°C for 12 h. Positive colonies were selected and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The correctly sequenced pUAST-3×Flag-Hsc70-4 eukaryotic expression vector plasmid was extracted using a high-throughput endotoxin-free plasmid extraction kit (manufactured by Kangwei Reagent Co., Ltd.).

[0065] 3. Resuscitation, passage, and transfection of Drosophila S2 cells

[0066] (1) Use a 25°C water bath to preheat the Drosophila S2 cells (laboratory-preserved) and Drosophila S2 cell culture medium (purchased from iCell).

[0067] (2) Transfer the preheated Drosophila S2 cells to a sterile culture dish with culture medium added in advance, and then incubate them in a sterile incubator at 25°C for the first revival. When the cells have filled the dish, divide the cells into two new sterile culture dishes with culture medium added in advance, mix the cells, and incubate them in a sterile incubator at 25°C for the second revival.

[0068] (3) When the cells from the second resuscitation have filled the dish, mix 1 mL of the cells from the second resuscitation with 3 mL of cell culture medium in a sterile culture dish, and then place the dish in a sterile incubator at 25°C to passage the Drosophila S2 cells. The next day, when the cells have filled the dish, perform the transfection experiment.

[0069] (4) In a sterile cell culture room, pUAST-3×Flag-Hsc70-4 plasmid and transfection reagent PEI were added to 400 uL of PBS at a mass ratio of 1:2. After mixing, the mixture was left to stand at room temperature for 15 min. Then, the mixture was added to the cells that had been passaged in step (3), gently mixed, and transfected into Drosophila S2 cells. After culturing the transfected cells in a sterile incubator at 25°C for 48 h, immunofluorescence experiments were performed.

[0070] 4. Immunofluorescence assay

[0071] Collect the transfected cells into centrifuge tubes, centrifuge at 12000 rpm for 3 min, discard the supernatant, resuspend the pellet in 1 mL PBS and transfer to a new centrifuge tube. Add 1 mL of 4% formaldehyde to the centrifuge tube and shake on a shaker for 20 min. Centrifuge at 12000 rpm for 3 min, discard the supernatant, wash the pellet with 1 mL PBS for 10 min, repeating the wash 3 times. Centrifuge at 12000 rpm for 3 min, discard the supernatant, add 1 mL of PBT to the pellet and shake for 10 min. Centrifuge at 12000 rpm for 1 min, discard the supernatant, resuspend the pellet in 200 μL PBS, add mouse primary antibody anti-Flg 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, wash the pellet with 1 mL PBS for 10 min, repeating the wash 3 times. Centrifuge at 12000 rpm for 3 min, discard the supernatant, add 200 μL of PBS, then add Cy3-labeled affinity-purified goat anti-mouse IgG (H+L) secondary antibody at a ratio of 1:200, and shake in the dark at room temperature for 2 h. Add 300 μL of PBS, then add DAPI (1:1000), and shake in the dark at room temperature for 15 min. Centrifuge at 12000 rpm for 3 min, wash the precipitate with 1 mL of PBS for 10 min, repeat 3 times. Prepare slides and observe the subcellular localization of Hsc70-4 using a two-photon laser confocal microscope. Hsc70-4 It can be seen that Hsc70-4 is located in the cytoplasm.

[0072] Example 2: Detection of different thermal stress gradients Figure 1 expression patterns

[0073] 1. Two hundred foraging bees were selected and divided into four groups of 50 bees each. The first three groups of bees were placed in incubators at 40°C, 43°C, and 46°C, respectively, while the fourth group (control group) was placed in an incubator at 33°C. Samples were taken at 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h after treatment.

[0074] 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 manufactured by TaKaRa.

[0075] 3. Using the synthesized first-strand cDNA 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) (TaKaRa) kit. The internal reference gene is... Hsc70-4 (GenBank Registration No.: HM640276.1).

[0076] 4. The primer sequences used for RT-qPCR are as follows:

[0077] β-actin Primers:

[0078] Upstream primer (SEQ ID NO.5): TCGCTGCTAAGAATGGTCT;

[0079] Downstream primer (SEQ ID NO.6): AACTTGTTTGTCACTGGCG.

[0080] Hsc70-4 Primers:

[0081] Upstream primer (SEQ ID NO.7): TTATATGCCAACACTGTCCTTT;

[0082] Downstream primer (SEQ ID NO.8): AGAATTGATCCACCAATCCA.

[0083] Depend on β-actin It can be seen that, compared with the control group, heat stress treatments at 40°C, 43°C, and 46°C can significantly upregulate [the system / condition]. Figure 2 The expression level. However, under different degrees of thermal stress conditions. Hsc70-4 The expression patterns differ. With prolonged heat treatment time, under heat stress conditions of 40°C and 46°C... Hsc70-4 The expression level was continuously upregulated, and under 46°C heat stress... Hsc70-4 The degree of adjustment should be higher than that of heat treatment at 40°C.

[0084] Example 3: Analysis of Silence Hsc70-4 Impact on the survival rate of Chinese honeybees

[0085] 1. Amplification using PCR technology for silencing Hsc70-4 The fragment (1450-1950 nt, shown in SEQ ID NO.9). Simultaneously amplifying 500 bp... Hsc70-4 (GenBank registration number: U87974) sequence.

[0086] SEQ ID NO.9:

[0087] GGTATTCTTAATGTCTCTGCAGTTGATAAATCTACTGGTAAAGAGAACAAAATCACTATTACTAACGATAAAGGCCGTCTCAGCAAAGAAGATATCGAAAGAATGGTCAACGAAGCTGAGAAATATCGTAGCGAAGACGAAAAACAAAAAGAAACTATCGCTGCTAAGAATGGTCTTGAGTCTTACTGCTTCAACATGAAGAGTACAGTGGAAGACGAGAAGTTGAAAGATAAAATCAGCGCCAGTGACAAACAAGTTGTACTGGATAAATGCAACGATATCATTAAATGGCTCGATGCTAACCAATTAGCTGATAAAGAAGAATATGAACATAAACAGAAAGAATTAGAAGCTATATGCAATCCTATTGTCACAAAATTGTATCAGGGTACTGGTGGGATGCCTGGTGGAATGCCTGGTGGAATGCCTGGTGGCTTCCCAGGAGCTGGTGGCGGTGCTCCTGGTGGTGGTGCATCTGGACCTACCATCGAAGAAGTCGAT。

[0088] Amplification GFP The primers for

[0089] Upstream primer (SEQ ID NO.10):

[0090] GGATCC Hsc70-4 GGTATTCTTAATGTCTCTGCA;

[0091] Downstream primer (SEQ ID NO.11):

[0092] GGATCC TAATACGACTCACTATAGG ATCGACTTCTTCGATGGTAGG.

[0093] The primers for amplifying GFP are:

[0094] Upstream primer (SEQ ID NO.12):

[0095] GGATCC TAATACGACTCACTATAGG AGTGGAGAGGGTGAAGGTGA;

[0096] Downstream primer (SEQ ID NO.13):

[0097] GGATCC TAATACGACTCACTATAGG GGTAAAAGGACAGGGCCATC.

[0098] Note: The T7 RNA polymerase promoter sequence is the underlined portion.

[0099] 2. The amplified PCR products were recovered by gel extraction, and dsRNA-Hsc70-4 and dsRNA-GFP were synthesized using the T7 RiboMAX™ ExpressRNAi System (Promega) as templates.

[0100] 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-Hsc70-4, while each bee in the second group was fed 5 μg of dsRNA-GFP (control group). After two days of feeding, RNA was extracted from both groups and reverse transcribed into cDNA. RT-qPCR was used to verify the silencing effect in *Apis chinensis*. TAATACGACTCACTATAGG The efficiency. (By) Hsc70-4 As shown in section A, compared with the control group, dsRNA - Hsc70-4 can lower Figure 3 The expression level in the Chinese honeybee reached a silencing level. Hsc70-4 The effect.

[0101] 4. It was confirmed that dsRNA-Hsc70-4 can be successfully silenced in Chinese honeybees. Next, 60 foraging bees were collected and divided into two groups (30 bees in each group). Following step 3, the two groups were fed dsRNA-Hsc70-4 and dsRNA-GFP, respectively. After two days of incubation, they were subjected to heat stress at 43°C. Survival rates were recorded every hour until all bees in both groups died. Each treatment was performed in at least three biological replicates. Hsc70-4 According to B, compared with the control group, silence... Hsc70-4 Reduce the survival rate of Chinese honeybees under heat stress.

[0102] Example 4: Analysis of Heterologous Overexpression of Honeybee Figure 3 Effects on the heat stress response of Drosophila S2 cells

[0103] This invention provides a method for heterologous overexpression of *Apis cerana* in Drosophila S2 cells. Hsc70-4 The method is used to further confirm Hsc70-4 Its role in thermal stress response.

[0104] 1. Following the method in step 3 of Example 1, the pUAST-3×Flag-Hsc70-4 eukaryotic expression vector was transfected into Drosophila S2 cells (experimental group), and cells transfected with the empty vector pUAST-3×Flag served as a control. The transfected cells were cultured in a sterile incubator at 25°C for 48 h and then subjected to heat stress treatment.

[0105] 2. Transfer the cells from the experimental and control groups in step 1 to a sterile incubator at 37°C for heat stress treatment. At 0 h, 18 h, and 48 h after treatment, appropriate amounts of cells were collected to prepare slides and photographed under an inverted white light / fluorescence microscope. Furthermore, at 48 h of heat stress treatment, cells from both the experimental and control groups were stained with trypan blue and their cell viability was assessed using a trypan blue staining cell viability assay kit (Beyotime). After trypan blue staining, appropriate amounts of cells were prepared to prepare slides and photographed under an upright white light microscope. Additionally, a hemocytometer was used to count the total number of cells and the number of dead cells after trypan blue staining, and the cell mortality rate was calculated.

[0106] Experimental results showed that, compared with the control group, heterologous overexpression of *Honeysuckle chinensis* significantly improved the efficacy of honeysuckle overexpression. Hsc70-4 It can significantly reduce the mortality rate of Drosophila S2 cells under heat stress. These results further demonstrate the efficacy of Apis cerana. Hsc70-4 It plays an important role in the thermal stress response. Hsc70-4 ).

[0107] Example 5: Detecting Silence Hsc70-4 Effects of hydrogen peroxide, protein carbonyl, vitamin C content and total antioxidant capacity in Chinese honeybees

[0108] 1. Take 60 foraging bees and divide them into two groups (30 bees in each group). Feed the two groups of bees dsRNA-Hsc70-4 and dsRNA-GFP respectively, following the method in step 3 of Example 3. Culture under normal conditions for two days and then store the samples.

[0109] 2. Prepare 10% tissue homogenates from the two groups of samples stored in the previous step using physiological saline. Dilute a small amount of the sample to a 1% tissue homogenate and measure its concentration using a protein quantification kit (Coomassie Brilliant Blue method).

[0110] 3. Take an appropriate amount (10%) of tissue homogenate as needed, and use the following kits to determine the content of hydrogen peroxide, protein carbonyl, vitamin C, and total antioxidant capacity in *Apis cerana*: hydrogen peroxide assay kit (colorimetric method), protein carbonyl content assay kit (UV colorimetric method), vitamin C content assay kit (colorimetric method), and total antioxidant capacity assay kit (colorimetric method). At least three biological replicates should be performed for each assay group. All kits used were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0111] Experimental results show that silence Figure 4 It increased the levels of hydrogen peroxide and protein carbonyl groups in the Chinese honeybee, while decreasing the vitamin C content and total antioxidant capacity in the Chinese honeybee. Hsc70-4 Therefore, this invention verifies silence. Hsc70-4 It will increase the degree of protein damage and exacerbate the oxidative damage of heat stress to Chinese honeybees.

[0112] In summary, this invention provides a method for Hsc70-4 to be localized in the cytoplasm and silenced. Figure 5 To reduce the survival rate of Chinese honeybees under heat stress, overexpression Hsc70-4 Increase the survival rate of Drosophila S2 cells under heat stress. Hsc70-4 Hsc70-4 Hsc70-4 Notes: There seems to be a duplicate tag number ( and [[ID=Z47]], and [[ID=Z50]]) in the original text. I have translated them as they are. If this is an error, it might need to be corrected in the original source. The induced expression of this substance helps reduce oxidative damage caused by heat stress to Chinese honeybees and improve their heat tolerance.

[0113] 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.

[0114] Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. Heat-resistant genes in Chinese honeybees Hsc70-4 Applications in (1) or (2) below: (1) Prepare products that regulate the heat resistance of insects under heat stress; (2) Breed insect varieties with enhanced heat resistance under heat stress conditions; The insect in question is either a honeybee or a fruit fly; The heat resistance-related genes of the Chinese honeybee Hsc70-4 It 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. The application according to claim 1, characterized in that, By introducing genes related to heat tolerance in the Chinese honeybee into recipient insect species... Hsc70-4 We used recombinant expression vectors to cultivate insect varieties with enhanced heat resistance under heat stress conditions.

3. Heat-resistant genes in Chinese honeybees Hsc70-4 Application of the encoded protein in the preparation of products that regulate the heat resistance of insects under heat stress; The insect in question is either a honeybee or a fruit fly; The heat resistance-related genes of the Chinese honeybee Hsc70-4 The encoded protein amino acid sequence is shown in SEQ ID NO.2.

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