Bombyx mori lysed polysaccharide monooxygenase BmLPMO15-4 gene and application thereof

Abstract

The invention belongs to the technical field of agricultural biology, and relates to a bombyx mori lysed polysaccharide monooxygenase (BmLPMO15-4) gene and application thereof. The bombyx mori BmLPMO15-4 is obtained through cloning and sequencing, detection finds that the bombyx mori BmLPMO15-4 participates in the process that bombyx mori resists BmNPV virus infection, and overexpression of the BmLPMO15-4 can inhibit virus proliferation. The BmLPMO15-4 participates in the BmNPV virus infection resisting process of bombyx mori cells and can enhance the antiviral capacity, and a new thought is provided for developing effective antiviral drugs and screening disease-resistant varieties.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology and relates to the Bombyx mori lytic polysaccharide monooxygenase BmLPMO15-4 gene and its application. Background Art

[0002] The silkworm, Bombyx mori, is an important economic insect and an ideal model organism for studying Lepidoptera insects. The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most common and severe viruses in sericulture production, causing huge economic losses to the sericulture industry every year and seriously affecting the development of the sericulture industry. With the development of biotechnology and the in-depth study of the antiviral mechanism of Bombyx mori, more and more antiviral genes have been discovered and studied, thus promoting the breeding of antiviral Bombyx mori strains.

[0003] The Bombyx mori lytic polysaccharide monooxygenase BmLPMO15-4 is a class of copper-dependent enzymes. Its relatively conserved enzyme active center generally consists of a 3N ligand "histidine scaffold" formed by 2 histidines and 1 tyrosine and 1 divalent metal ion, and the first amino acid at the N-terminus of the mature protein must be histidine. LPMO can catalyze the oxidative cleavage of polysaccharides such as chitin, cellulose, pectin, and starch to provide energy for the body, and can also regulate multiple growth and development processes such as insect molting, peritrophic membrane renewal, and tissue growth. When plant cells are infected by pathogens, LPMO can act as a damage-associated molecular pattern (DAMP) of plants to induce an immune response in plants, causing callose accumulation, an increase in plant hormone levels, and in vivo transcriptional re-editing, etc., ultimately improving the resistance of plants to pathogens. However, the function of BmLPMO15-4 in Bombyx mori remains to be further studied and explored. Summary of the Invention

[0004] Using molecular biology techniques to breed Bombyx mori strains resistant to the Bombyx mori nucleopolyhedrovirus BmNPV and providing more screening platforms for anti-BmNPV drugs are the general pursuit directions of those skilled in the art. In the research on the response of Bombyx mori BmLPMO15-4 to BmNPV virus infection in the host, the present invention discovers that it has an important regulatory effect on virus proliferation. The Bombyx mori BmLPMO15-4 gene has important application value in the breeding of disease-resistant Bombyx mori varieties and the screening of antiviral drugs, and thus the present invention is completed.

[0005] In the first aspect of the present invention, the present invention provides a method for breeding Bombyx mori strains resistant to BmNPV virus, overexpressing the BmLPMO15-4 gene in Bombyx mori to improve the antiviral ability of Bombyx mori. The nucleotide sequence of the Bombyx mori BmLPMO15-4 gene is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.

[0006] In the test of the response of Bombyx mori BmLPMO15-4 to BmNPV virus infection in the host, it was found that this gene plays an important regulatory role in virus proliferation. Overexpression of the BmLPMO15-4 gene in BmN cells inhibits the expression of the BmNPV virus nucleocapsid protein VP39 gene.

[0007] In one aspect of the present invention, a method for screening anti-BmNPV virus drugs is provided. If the drug can significantly increase the level of the BmLPMO15-4 gene in Bombyx mori, then the drug is an anti-BmNPV virus drug.

[0008] In one aspect of the invention, the present invention provides the application of the Bombyx mori BmLPMO15-4 gene in the development of anti-BmNPV virus drugs. The BmLPMO15-4 gene is used to screen anti-BmNPV virus drugs. If the drug can significantly increase the level of the BmLPMO15-4 gene in Bombyx mori, then the drug is an anti-BmNPV virus drug.

[0009] The present invention cloned the Bombyx mori BmLPMO15-4 gene, and through molecular biology techniques, it was found that BmLPMO15-4 is involved in the process of Bombyx mori resisting BmNPV virus infection. By constructing a cell transient overexpression vector pIZT-BmLPMO15-4 and transfecting it into BmN cells, an increased expression of this gene was obtained. The expression of the BmNPV virus nucleocapsid protein gene VP39 was detected by RT-qPCR and Western blotting, and the proliferation of BmNPV virus particles BV labeled with green fluorescent protein EGFP was observed using a fluorescence inverted microscope. The results showed that overexpression of BmLPMO15-4 can inhibit virus proliferation. BmLPMO15-4 is involved in the process of Bombyx mori cells resisting BmNPV virus infection and can enhance the antiviral ability, providing new ideas for the development of effective antiviral drugs and the screening of disease-resistant varieties.

[0010] Beneficial effects

[0011] Through gene knockout and gene overexpression of BmLPMO15-4, the present invention discovered the core role of BmLPMO15-4 in Bombyx mori's resistance to BmNPV infection, laying a foundation and providing a good technical path for breeding Bombyx mori varieties resistant to BmNPV infection and screening anti-BmNPV drugs. Brief description of the drawings

[0012] Figure 1 Spatial and temporal expression analysis of the Bombyx mori BmLPMO15-4 gene in Bombyx mori.

[0013] Figure 2 The Bombyx mori BmLPMO15-4 gene can respond to BmNPV virus infection.

[0014] Figure 3 Construction of the overexpression vector pIZT - BmLPMO15 - 4.

[0015] Figure 4 Inhibitory effect of overexpressed Bombyx mori BmLPMO15 - 4 on the proliferation of BmNPV virus. Specific implementation manners

[0016] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] Example 1: Temporal and spatial expression analysis of Bombyx mori BmLPMO15 - 4 gene

[0018] Using the Bombyx mori genome as a template, the Bombyx mori BmLPMO15 - 4 gene sequence was obtained through bioinformatics analysis, cloning and sequencing. Specific fluorescence quantitative primers and internal reference gene primers were designed. The relevant sequence primers are shown in Table 1 below.

[0019] Table 1: Relevant primer sequences for cloning and detection

[0020]

[0021]

[0022] The nucleotide sequence of the Bombyx mori BmLPMO15 - 4 gene is shown below (SEQ ID NO.1), and the sequence length is 684bp:

[0023] ATGAGAAATATGCAGTTGTCATATTTCCTTGCATTCGTTGCCTTGTTGGCGACTGTTCGTGCACACGGCAGGGTGGTGGAGCCCGCATCTCGAGCTTCGGCCTGGCGTGCCGGCTTCGGCACGAGAATCAACTACGACGACGACGGCATCAACTGCGGCGGCTTCCACCGTCAATGGGAAACTAATAATGGAAAATGCGGCATTTGTGGCGATCCATACGACGATTCGCCTCCTCGGCCGCACGAGTTGGGCGGTGCCTACGGTAATGGCGTCATCGTTGCGAAGTACTCCTCGGGACAGGTCATCGACACCACCGTCGAGATAACCGCCTACCACCGCGGCTATTGGGAATTCAAACTATGCACCGACCCCAGCAATAACGAACAGGAATGCTTCGAGAAATACCTCCTGGAATTAGAAGATGGCGGCACGAAGTATTACCCGAAGAGCAGTGGTCGTTACGACGTGAGGTATCGGCTTCCTGCGGGGGTCTCGTGTGAGCATTGCGTCCTACAATGGACGTACACAGCCGGTAACAACTGGGGTGTTTGCCCCAACGGAACTGGAGCTCTGGGATGCGGGAATCAGGAGACATTCTGGGCATGCACGGATGTTTCGATTAAGCCCGTTGAAGCTTCAGTATCTGGAATGAGCTTACCGATCCGCGTAGCTGATGATAAATGA

[0024] The amino acid sequence encoded by the Bombyx mori BmLPMO15-4 gene is shown below (SEQ ID NO.2), and its sequence consists of 227 amino acid residues:

[0025] MRNMQLSYFLAFVALLATVRAHGRVVEPASRASAWRAGFGTRINYDDDGINCGGFHRQWETNNGKCGICGDPYDDSPPRPHELGGAYGNGVIVAKYSSGQVIDTTVEITAYHRGYWEFKLCTDPSNNEQECFEKYLLELEDGGTKYYPKSSGRYDVRYRLPAGVSCEHCVLQWTYTAGNNWGVCPNGTGALGCGNQETFWACTDVSIKPVEASVSGMSLPIRVADDK

[0026] Samples of eggs, 1st - 5th instar newly molted larvae, pupae, female adults, male adults of the P50 strain of silkworms, and tissues of the epidermis, fat body, hemolymph, head, midgut, Malpighian tubules, ovaries, silk glands, testes, and tracheal plexus of 5th instar day 3 larvae were taken respectively; liquid nitrogen was added to a mortar cooled after dry heat sterilization at 180 °C for 3 h, and the samples were quickly ground into powder. Cellular RNA was extracted by the Trizol - chloroform method, reverse transcribed into cDNA using the NovoScript Plus Reverse Transcription Kit (Novoprotein Scientific Inc.), and real - time fluorescence quantitative PCR (RT - qPCR) analysis was performed according to the instructions of the SYBR qPCR SuperMix Plus reagent. Among them, rnaGAPDH - F / R was used as the primer for the internal reference gene GAPDH, and the specific primer qBmLPMO15 - 4 - F / R of BmLPMO15 - 4 was used to detect the expression level of the BmLPMO15 - 4 gene. Three biological replicates were performed, the melting curve program was the default of the instrument. After the instrument detection was completed, analysis was carried out by the 2 -ΔΔCt method, and significant analysis was performed using IBM SPSS Statistics software. The data were expressed as mean ± standard error. The expression differences at different developmental stages or in different tissues were analyzed by One - way ANOVA, and significant differences (p < 0.05) were indicated by lowercase letters (a, b, c, etc.).

[0027] The RT - qPCR detection results showed that the expression level of BmLPMO15 - 4 was relatively high during the 1st - 5th instar larval stage of silkworms and relatively low during the egg and adult stages ( Figure 1 in A); the detection results in each tissue found that ( Figure 1 in B), the expression level of BmLPMO15 - 4 was the highest in the midgut, followed by the Malpighian tubules and hemolymph. These three tissues are closely related to the immunity and detoxification metabolism of silkworms. Therefore, it is speculated that BmLPMO15 - 4 may respond to the virus infection process.

[0028] Example 2: Expression pattern of Bombyx mori BmLPMO15-4 gene in response to BmNPV virus infection at the cellular level

[0029] BmN cells in good growth state were seeded into six-well plates. When the density reached 70 - 80%, 20 μL of BV-EGFP (1×10 8 pfu / mL) was added to each well. Cell samples were taken at 24, 48, and 72 h after virus infection. Cell RNA was extracted by the Trizol-chloroform method, reverse transcribed into cDNA, and then detected by RT-qPCR. The results showed that when the virus infected the cells, the expression level of BmLPMO15-4 increased significantly (as Figure 2 ), indicating that the Bombyx mori BmLPMO15-4 gene responded to virus infection.

[0030] Example 3: Construction of cell overexpression vector pIZT-BmLPMO15-4

[0031] According to the Bombyx mori BmLPMO15-4 sequence, after selecting appropriate restriction sites, homologous recombination primers: BmLPMO15-4-PIZT-F / R were designed. After the primer design was completed, it was synthesized by Sangon Biotech (Shanghai) Co., Ltd. Using the cDNA of the midgut of P50 Bombyx mori larvae as a template, the target gene was cloned. After detection by agarose gel electrophoresis (as Figure 3 in A), the target band was cut out, and the PCR product was recovered using a gel recovery kit (Sangon Biotech (Shanghai) Co., Ltd.). The pIZT vector was double digested with restriction enzymes BamH1 and EcoR1 and the product was recovered. The target gene was ligated to the digested vector using homologous recombinase, transformed into Escherichia coli competent cell DH5α, plated, and positive clones were screened (as Figure 3 in B) and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the theoretical sequence correctly, indicating that the cell overexpression recombinant vector was successfully constructed (as Figure 3 in C).

[0032] The Escherichia coli containing the recombinant vector was cultured on a large scale, and endotoxin-free plasmid was extracted using an endotoxin-free plasmid midiprep kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). The extracted overexpression plasmid pIZT-BmLPMO15-4 and the control plasmid pIZT empty vector were transfected into BmN cells in good state and appropriate density. After 48 h of transfection, cell samples were collected, and the overexpression efficiency was verified at the transcriptional and translational levels respectively (as Figure 3 in D, E). The results showed that the overexpression efficiency met the expected results, and the recombinant vector could be used for subsequent experiments.

[0033] Example 4: Effect of overexpressing Bombyx mori BmLPMO15-4 on the proliferation of BmNPV virus

[0034] The overexpression plasmid and the control plasmid were transfected into BmN cells respectively. After 48 h, the fresh medium was replaced and virus BV-EGFP was added. The cell samples were collected at 24, 36, and 48 h after virus infection, and DNA and protein samples were extracted. The relative expression level of BmNPV VP39 was detected to represent the virus proliferation. Specific primers q-VP39-F / R were designed using the virus gene VP39 sequence, and specific primers dnaGAPDH-F / R were designed using GAPDH as the internal reference gene. The expression level of the virus VP39 gene was detected by RT-qPCR and Western blot (as shown in A and B below), and the results showed that overexpression of BmLPMO15-4 inhibited the copy number of VP39 compared with the control group; the fluorescence intensity of the virus was observed by an inverted fluorescence microscope to represent the virus proliferation (as shown in C below), and the green fluorescence intensity representing BV in the overexpression group was significantly lower than that in the control group. In summary, overexpression of BmLPMO15-4 at the cell level can inhibit the proliferation of BmNPV. Figure 4 The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as falling within the protection scope determined by the claims submitted for the present invention. Figure 4 In the above, the content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as falling within the protection scope determined by the claims submitted for the present invention.

[0035] The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as falling within the protection scope determined by the claims submitted for the present invention.

Claims

1. A method for breeding silkworms resistant to Bombyx mori nuclear polyhedrosis virus (BmNPV), characterized in that: The BmLPMO15-4 gene is overexpressed in silkworms to improve the antiviral ability of silkworms. The nucleotide sequence of the BmLPMO15-4 gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO:

2.

2. A method for screening anti-BmNPV virus drugs, characterized in that: If the drug can significantly increase the level of the BmLPMO15-4 gene in the silkworm, the drug is an anti-BmNPV virus drug.

3. Application of the silkworm BmLPMO15-4 gene in developing anti-BmNPV virus drugs, characterized in that: The BmLPMO15-4 gene is used to screen anti-BmNPV virus drugs. If the drug can significantly increase the level of the BmLPMO15-4 gene in the silkworm, the drug is an anti-BmNPV virus drug.

4. The use according to claim 3, characterized in that: Overexpression of the BmLPMO15-4 gene in silkworm cells inhibits the expression of the BmNPV virus nucleocapsid protein VP39 gene.

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