Application of BmMOF gene of acetyltransferase of silkworm

Abstract

The application discloses application of Bombyx mori acetyltransferase BmMOF gene. A full-length CDS sequence of a gene BmMOF affecting Bombyx mori resistance is cloned. Through a manner of overexpression and siRNA interference expression of a transient expression vector of the BmMOF gene in Bombyx mori BmN cells, influence of the BmMOF on a virus mRNA transcription level, a membrane fusion protein expression level and an amplification level under a virus infection condition is explored, and a detection result proves that overexpression of the BmMOF in the BmN cells significantly inhibits virus proliferation, and knocking down the BmMOF expression promotes BmNPV gene transcription and protein expression. The application provides a new idea for Bombyx mori antiviral breeding, improvement of antiviral infection capacity and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and protein function technology, and designs the application of the silkworm acetyltransferase BmMOF gene, specifically disclosing the application of the silkworm acetyltransferase BmMOF gene in regulating baculovirus BmNPV infection. Background Technology

[0002] The silkworm is a type insect of the order Lepidoptera, characterized by its exclusively mulberry leaf-feeding diet. It is an economically important insect completely domesticated from the primitive silkworm. Bombyx mori nucleopolyhedrovirus (BmNPV) is the pathogen causing hemorrhagic septicemia in silkworms, and the viral diseases it causes result in significant economic losses to the sericulture industry. Currently, BmNPV control is limited to physical disinfection of the rearing environment and cannot effectively solve the silkworm disease problem. With in-depth research into the interaction between the virus and host cells, more and more antiviral proteins are being discovered, and their antiviral mechanisms are currently a research hotspot.

[0003] MOF (Males absent on the first), also known as MYST1 or KAT8, is a member of the MYST family of histone acetyltransferases. As key regulators in the epigenetic system, MOFs participate in various cellular processes as chromatin modification complexes, playing crucial roles in gene transcription, DNA damage repair, chromosome homeostasis, cell cycle, apoptosis, and tumorigenesis. In mammalian cells, MOFs are enriched in gene promoters and coding regions, exhibiting transcriptional activation functions. During cellular stress responses, MOFs, as key maintainers of the transcriptional regulatory network, directly bind to the promoters of cell cycle-regulating genes, activating their expression to protect the corresponding cells. Regarding target protein recognition, MOFs exhibit strong substrate selectivity, specifically catalyzing the acetylation of histone H4 at position 16 (H4K16), weakening the interaction between H4 and the H2A-H2B acid pocket, thereby altering chromatin structure and activating gene transcription. Furthermore, MOFs can also target non-histone substrates, such as the tumor suppressor protein p53. MOFs activate the expression of p53-dependent apoptosis genes by catalyzing acetylation at the p53 K120 site, ultimately inducing apoptosis. Summary of the Invention

[0004] The first objective of this invention is to address the lack of effective means for the defense of silkworm cells against viral invasion. Through research on the role of silkworm acetyltransferase MOF in viral infection, it has been found that MOF plays an important role in regulating baculovirus proliferation. Therefore, the application of the above-mentioned gene is developed to provide new ideas for antiviral breeding of silkworms and improving their resistance to viral infection.

[0005] The application of the silkworm BmMOF gene in breeding silkworm varieties resistant to silkworm nucleopolyhedrovirus (MPV) involves overexpressing the BmMOF gene to enable silkworms to acquire resistance to MPV; the nucleotide sequence of the BmMOF gene is shown in SEQ ID NO:1.

[0006] Preferably, the amino acid sequence of the protein encoded by the BmMOF gene is shown in SEQ ID NO:2.

[0007] As a preferred method, overexpression of BmMOF inhibits the proliferation of BmNPV virus.

[0008] As a preferred embodiment, overexpression of BmMOF can inhibit the transcriptional expression of BmNPV virus gp41 gene mRNA, reduce the expression of BmNPV fusion protein GP64, and increase the acetylation level of BmMOF protein substrate H4K16.

[0009] The second objective of this invention is to provide a method for breeding silkworm varieties resistant to silkworm nucleopolyhedrovirus, wherein the silkworm acquires the ability to resist silkworm nucleopolyhedrovirus by overexpressing the BmMOF gene in the silkworm; the nucleotide sequence of the silkworm BmMOF gene is shown in SEQ ID NO:1.

[0010] A third objective of this invention is to provide the application of the silkworm BmMOF gene in the preparation of a drug that inhibits the proliferation of silkworm nucleopolyhedrovirus, wherein the nucleotide sequence of the silkworm BmMOF gene is shown in SEQ ID NO:1.

[0011] A fourth objective of this invention is to provide a silkworm nucleopolyhedrovirus inhibitor, comprising a drug that inhibits the expression of the silkworm BmMOF gene, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0012] The beneficial effects of this invention are as follows:

[0013] This invention utilizes genetic engineering and cell biology techniques to demonstrate that the silkworm acetyltransferase MOF participates in the baculovirus infection process and regulates viral proliferation. The regulatory role of the silkworm MOF protein in BmNPV infection was verified in silkworm ovarian cells through overexpression and RNA interference.

[0014] This invention cloned a silkworm BmMOF gene that regulates viral infection. The BmMOF protein was highly expressed in BmN cells by constructing a eukaryotic transient expression vector pIEx-1-BmMOF. The expression of the MOF protein in BmN cells was knocked down using siRNA. Recombinant plasmids and siRNA were transfected into cells, with the pIEx-1 empty vector as a control. Using the BmNPV structural gene gp41 as a molecular marker, the viral genome replication level in cells was detected. It was found that overexpression of BmMOF significantly inhibited viral genome replication, while knockdown of BmMOF promoted viral genome replication. Western spectroscopy was used to analyze the expression of the BmMOF protein. Blotting analysis of the viral fusion protein GP64 revealed that overexpression of BmMOF significantly reduced GP64 expression and increased the acetylation level of the substrate H4K16, while knockdown of BmMOF promoted viral protein expression. Infection of BmN cells with BmNPV labeled with green fluorescent protein (eGFP) and observation of viral amplification using fluorescence microscopy showed that overexpression of BmMOF significantly inhibited BmNPV proliferation, while knockdown of BmMOF promoted viral proliferation. These results indicate that BmMOF plays a crucial role in the resistance of silkworm cells to BmNPV infection, significantly enhancing cellular antiviral capabilities. This has significant theoretical and practical value for elucidating the host-virus game mechanism, improving antiviral silkworm strains using genetic engineering, and creating new silkworm germplasm resources. Attached Figure Description

[0015] Figure 1 Construction of the transient eukaryotic expression vector pIEx-1-BmMOF; wherein, A is the PCR amplification product of the BmMOF gene; B is the enzyme digestion verification result; C is the map of the recombinant vector constructed in this invention.

[0016] Figure 2 This study analyzed the tissue-specific and spatiotemporal expression distribution characteristics of BmMOF. Among them, A represents the expression of BmMOF in the hemolymph, midgut, Malpighian tubules, gonads, silk glands, trachea, fat body, head, and epidermal tissues of silkworms during the fifth instar; B represents the expression characteristics of BmMOF in the egg stage, the first to fifth instar larval stages, the pupal stage, and the adult stage.

[0017] Figure 3 Analysis of the effects of BmNPV on BmMOF expression patterns; where A represents the effect of BmNPV infection on BmMOF transcriptional levels; and B represents the effect of BmNPV infection on BmMOF protein expression and the acetylation level of its substrate H4K16.

[0018] Figure 4The study investigated the effect of BmMOF overexpression on BmNPV proliferation. Specifically, A represents the effect of BmMOF overexpression on the transcriptional level of the baculovirus gene gp41; B represents the effect of BmMOF overexpression on the expression of the baculovirus fusion protein GP64; and C and D represent the effects of BmMOF overexpression on viral amplification.

[0019] Figure 5 The study investigated the effect of BmMOF knockdown on BmNPV proliferation. A represents the validation of siRNA interference efficiency; B represents the effect of BmMOF knockdown on the transcriptional level of the baculovirus gene gp41; C represents the effect of BmMOF knockdown on the expression of the baculovirus fusion protein GP64; and D and E represent the effect of BmMOF knockdown on viral amplification. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0021] Sequence ID NO:1 is the nucleotide sequence of the silkworm BmMOF gene, with a length of 1326 bp. The specific sequence of SEQ ID NO:1 is shown below:

[0022]

[0023] The sequence listing SEQ ID NO:2 is the amino acid sequence of the silkworm MOF protein, which contains 422 amino acid residues.

[0024] The sequence SEQ ID NO:2 is shown below:

[0025] MAKGDKELEKPIVNNELPNPECRSTDNEDSESVPEQPLDIGEHYLVRRSDESWHPAEIIQSRYSTAESCYEYYVHYVGYDRRLDEWVSRHRVMSDRFDVCEQSNNNINCD HLLTDKSGRKITRNQKRKHDEINHVQKTYAEMDPTTAALEKEHEAITKVKYIDRIQIGKYEIDTWYFSPYPDEYGKQSKLWLCEYCLKYMRMEKTYRYHLSECTARQPQGN EIYRKGTIAIFEADGKEHKIYCQNLCLLAKLFLDHKTLYFDIEQFLFYILCEVDKQGAHLVGYFSKEKDSPEGNNVACILTLPPYQRQGYGKLLIAFSYELSRLEQVVGS PEKPLSDLGKLSYRSYWSYVLLEVLSASRGTLSIKDLSQMTGISQTDIISTLQSMNMVKYWKGQHVICVTPKIVAEQLASPHFKKPRLSIDPSALRWTPPSKQGNSAKAKK

[0026] The list of primers used in the following examples is shown in Table 1.

[0027] Table 1 Primer Information

[0028]

[0029]

[0030] Example 1: Cloning of the BmMOF gene fragment in silkworm and construction of a eukaryotic expression vector

[0031] Based on the silkworm cell MOF gene sequence downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / 100101212), after selecting appropriate restriction enzyme sites, homologous arm primers (5'→3') were designed according to the pIEx-1 vector sequence.

[0032] Upstream primer BmMOF-HindⅢ-F: see SEQ ID NO:3.

[0033] Downstream primer BmMOF-HindⅢ-R: see SEQ ID NO:4.

[0034] Total RNA was extracted from silkworm BmN cells using the traditional method of Trizol-chloroform extraction. The Plus Reverse Transcription Kit (Nearshore Protein Technology Co., Ltd.) was used to reverse transcribe cDNA, which was then used to amplify the MOF gene fragment using the high-fidelity PCR enzyme KOF FX. The PCR amplification conditions were: 95℃ for 5 min pre-denaturation, 95℃ for 30 s denaturation, 58℃ for 30 s annealing, 68℃ for 1 min extension, 30 cycles, 68℃ for a final extension for 10 min, and then incubation at 12℃. The amplified product ( Figure 1 A) The size of the target fragment and primer specificity were detected by 1% agarose gel electrophoresis. The pIEx-1 vector was then digested with HindIII nuclease and cleaned for recovery. The PCR one-step directional cloning kit ligates two enzyme digestion products at a specific molar ratio, transforms them into TG1 competent cells, plates them, screens positive clones, and verifies them through enzyme digestion. Figure 1 B) The sample was then sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing peak diagram was viewed and the sequencing results were compared using Snap Gene software. The sequencing results showed that the silkworm BmMOF gene sequence (SEQ ID NO:1) was successfully cloned, consisting of 1326 bp of deoxyribonucleic acid. The recombinant transient expression vector pIEx-1-BmMOF was successfully constructed. Figure 1 C).

[0035] Example 2: Tissue-specific and spatiotemporal expression distribution characteristics of BmMOF in silkworms

[0036] Silkworms were raised under optimal temperature and humidity conditions using the diapause-type Qiufeng × Baiyu strain. RNA samples were extracted from various tissues and metamorphic developmental stages, and cDNA was synthesized via reverse transcription. Specific qPCR primers (5'→3') were designed based on the BmMOF gene sequence.

[0037] qBmMOF-F: See SEQ ID NO:5.

[0038] qBmMOF-R: See SEQ ID NO:6.

[0039] Design Bmβ-Actin-specific qPCR primers as a control gene. The primer sequence (5'→3') is as follows:

[0040] qBmβ-Actin-F: See SEQ ID NO:9.

[0041] qBmβ-Actin-R: See SEQ ID NO:10.

[0042] The expression level of BmMOF mRNA was detected using qRT-PCR technology. The qPCR amplification system was 20 μL. SYBR qPCR SuperMix plus 10 μL, forward and reverse primers 0.2-1 μM each (final concentration), template 1-2 μL, and sterile water to a final volume of 20 μL. PCR amplification program: 95℃ for 1 min, 95℃ for 10 s, 60℃ for 30 s, 40 cycles.

[0043] Samples were collected from silkworm eggs, larvae (day 3 of instar 1-5), pupae, and adults. For day 3 of instar 5 larvae, hemolymph, midgut, Malpighian tubules, gonads, silk glands, trachea, fat body, head, and skin tissue were dissected. The samples were rapidly ground into powder in a sterile mortar with an appropriate amount of liquid nitrogen. RNA was extracted from each sample and reverse transcribed to synthesize cDNA. qPCR detection was performed using BmMOF-specific primers qBmMOF-F / R. Bmβ-Actin was used as an internal reference gene, and its specific qPCR primers qBmβ-Actin-F / R were used as controls. The experiment was performed in triplicate (biological replicates) and in triplicate (technical replicates) for each sample. 2 -△△Ct The relative changes in gene expression were analyzed using a statistical method, and significance analysis was performed using IBM SPSS statistical software.

[0044] qPCR analysis showed that BmMOF mRNA was most abundant in the trachea and epidermis, followed by the head, with small amounts also distributed in hemolymph, midgut, and fat body, but its expression was low in Malpighian tubules, gonads, and silk glands. Figure 2 A), (p<0.01). Furthermore, the expression characteristics of BmMOF mRNA at different developmental stages were analyzed ( Figure 2 B) It was demonstrated that the BmMOF transcription level was highest in larvae on day 3 of the second instar (L2D3). Except for the fifth instar, it was generally transcribed in all other larval stages. There was also a small amount of expression in the pupal and adult stages, but almost no expression in the egg stage. The differences between the expression levels were significant (p<0.01).

[0045] Example 3: Analysis of the effect of BmNPV on the expression pattern of BmMOF protein in silkworm

[0046] After passage, cells in good growth condition were seeded into 12-well plates and cultured overnight at 27°C. Cells were infected with BmNPV at a multiplicity of infection (MOI) of 10 for different time periods. Cell pathogenesis was observed under a microscope. qRT-PCR was performed using the silkworm BmMOF gene-specific primer qBmMOF-F / R and the internal reference gene Bmβ-Actin-specific primer qBmβ-Actin-F / R. The results are shown below. Figure 3As shown in Figure A, the results indicate that BmNPV infection of BmN cells did not significantly alter the transcriptional level of BmMOF. Western blotting was performed using MOF-specific antibodies and their substrate H4K16 acetylation antibody, and the results are as follows. Figure 3 As shown in Figure B, the expression level of BmMOF shows a significant downward trend with the extension of viral infection time, and the acetylation level of its substrate H4K16 also gradually decreases; indicating that BmNPV infection can directly affect the expression of BmMOF protein and the acetylation level of its substrate H4K16.

[0047] Example 4: Effect of overexpression of silkworm BmMOF on baculovirus proliferation

[0048] Based on the principle of homologous recombination, a transient eukaryotic expression vector pIEx-1-BmMOF was constructed. pIEx-1-BmMOF was transfected into BmN cells, with an empty pIEx-1 vector as a control. Total RNA and protein samples were extracted from cells 48 and 72 hours after viral infection. The viral genome replication level in cells was detected using the BmNPV structural gene gp41 as a molecular marker. Specific qPCR primers (5'→3') were designed based on the viral gene gp41 sequence.

[0049] qgp41-F: See SEQ ID NO:7.

[0050] qgp41-R: See SEQ ID NO:8.

[0051] qRT-PCR results are as follows Figure 4 As shown in Figure A, the results indicate that overexpression of BmMOF inhibits viral genome replication; Western blotting was used to detect the expression level of the viral fusion protein GP64. Figure 4 B) indicates that BmMOF overexpression can reduce the expression of BmNPV fusion protein while increasing the acetylation level of substrate H4K16; cells were infected with BmNPV labeled with eGFP after BmMOF overexpression, and the viral proliferation level at different infection stages was observed using fluorescence microscopy. The results are as follows: Figure 4 As shown in C and D, compared with the control group, overexpression of BmMOF inhibited the proliferation of BmNPV (p<0.01); the overall results indicate that overexpression of BmMOF significantly inhibited viral proliferation and played an antiviral role in the viral infection process.

[0052] Example 5: Effect of BmMOF knockdown on baculovirus proliferation

[0053] To investigate the effect of BmMOF knockdown on baculovirus replication, siRNA target sequences composed of RNA bases were designed based on the BmMOF sequence. The forward and reverse strands were synthesized separately, mixed in equimolar amounts, and annealed using a PCR instrument to form double strands. Three pairs of siRNAs were designed and synthesized, with the following sequences:

[0054] siRNA-1 sense: see SEQ ID NO:11.

[0055] siRNA-1 antisense: see SEQ ID NO:12.

[0056] siRNA-2sense: see SEQ ID NO:13.

[0057] siRNA-2 antisense: see SEQ ID NO:14.

[0058] siRNA-3sense: see SEQ ID NO:15.

[0059] siRNA-3 antisense: see SEQ ID NO:16.

[0060] BmMOF expression in BmN cells was inhibited by interfering with the expression of the target protein. 50 nM siRNA was transfected into BmN cells, and protein samples were extracted after 72 hours. Western blotting was used to detect BmMOF protein expression in silkworms. The primer pair with the best interference efficiency was selected for subsequent experiments. The experimental results are as follows: Figure 5 As shown in Figure A, it is demonstrated that siRNA-3 knockdown of BmMOF protein expression is the most significant, and the acetylation level of BmMOF protein substrate H4K16 is also significantly reduced, further indicating that siRNA-3 knockdown of BmMOF expression is the most efficient.

[0061] To investigate the relationship between BmMOF and viral replication, siRNA-3 was transfected into BmN cells for 72 hours. After viral infection for 48 and 72 hours, samples were collected, total RNA was extracted, and cDNA was synthesized via reverse transcription. Changes in viral gene transcription levels were detected using qRT-PCR. Figure 5 As shown in Figure B, the results indicate that knockdown of BmMOF increases the BmNPV genome replication level; simultaneously, protein samples were extracted, and Western blotting was used to detect the expression level of viral GP64 protein. Figure 5 C) indicates that knocking down BmMOF significantly increases viral protein expression; after transfection with siRNA, the addition of BmNPV virus fused with eGFP and the interference for 48 and 72 hours, followed by recording viral amplification under a fluorescence inverted microscope, are shown in the following figures. Figure 5As shown in D and E, the interference group showed a stronger fluorescence signal compared to the control group (p<0.01); the above experimental results indicate that knocking down BmMOF can promote viral replication and proliferation.

Claims

1. Silkworm BmMOF The application of genes in breeding silkworm varieties resistant to silkworm nucleopolyhedrovirus is characterized by, Through overexpression BmMOF The gene endows silkworms with resistance to silkworm nucleopolyhedrovirus; BmMOF The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The BmMOF The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, overexpression BmMOF Inhibits the proliferation of BmNPV virus.

4. The application according to claim 3, characterized in that, overexpression BmMOF Able to inhibit BmNPV virus gp41 Transcriptional expression of gene mRNA.

5. The application according to claim 3, characterized in that, overexpression BmMOF It can reduce the expression of BmNPV fusion protein GP64.

6. The application according to claim 3, characterized in that, overexpression BmMOF It can increase the acetylation level of BmMOF substrate H4K16.

7. A method for breeding silkworm varieties resistant to silkworm nucleopolyhedrovirus, characterized in that, By overexpression in silkworms BmMOF Genes enable silkworms to acquire resistance to silkworm nucleopolyhedrovirus; the silkworms BmMOF The nucleotide sequence of the gene is shown in SEQ ID NO:1.

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