Application of gene BmTsp.D in suppressing silkworm nucleopolyhedrovirus

Abstract

This application discloses the application of the gene BmTsp.D in the inhibition of silkworm nucleopolyhedrovirus. It belongs to the field of genetic engineering technology. Based on preliminary screening of silkworm genome data, this application identified and characterized a four-transmembrane protein called BmTsp.D. Through overexpression and knockdown experiments of the BmTsp.D gene, the inhibitory effect of the gene BmTsp.D on nucleopolyhedrovirus was clarified, providing technical support for the development of nucleopolyhedrovirus inhibitory genes.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to the application of the gene BmTsp.D in inhibiting silkworm nucleopolyhedrovirus. Background Technology

[0002] The silkworm, also known as the domesticated silkworm, is a silk-spinning insect with high economic value. It feeds on mulberry leaves, and its cocoons are used to reel silk, a precious textile raw material primarily used for weaving silk fabrics. Silk is an excellent textile material and also has wide applications in military and electrical industries. The silkworm's pupae, moths, and excrement can also be utilized comprehensively, serving as raw materials for various chemical and pharmaceutical industries, and can also be used as plant fertilizer.

[0003] Because BmNPV poses a serious threat to sericulture, researchers have been trying to find the key genes that inhibit BmNPV and improve the resistance of silkworms to BmNPV by upregulating or downregulating key resistance genes through transgenic methods. However, there is currently little research on BmNPV inhibitory genes.

[0004] Tetraspan proteins, as highly conserved membrane receptors, are important members of the transmembrane 4 superfamily (TM4SF). They can bind to specific molecular chaperone proteins or another tetraspan protein to form tetraspan protein-rich microdomains (TEMs or TERMs). Tetraspan proteins regulate physiological processes such as cell signaling, cell adhesion, and membrane fusion, and play a crucial role in viral recognition and entry, syncytial formation, and viral particle release. Among 33 human tetraspan proteins, CD151, CD82, CD81, CD63, CD9, Tspan9, and Tspan7 have been found to be associated with viral infection.

[0005] Therefore, how to provide a gene that inhibits silkworm nucleopolyhedrovirus is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the application of the gene BmTsp.D in inhibiting silkworm nucleopolyhedrovirus, clarifying that the silkworm gene BmTsp.D encodes a novel four-span membrane protein, and through overexpression and knockdown experiments of this gene, the inhibitory effect of this gene on silkworm nucleopolyhedrovirus is clarified, providing technical support for the prevention and control of silkworm nucleopolyhedrovirus.

[0007] The first objective of this invention is to provide the application of the gene BmTsp.D in inhibiting silkworm nucleopolyhedrovirus, wherein the nucleotide sequence of the gene BmTsp.D is any one of the following:

[0008] 1) The nucleotide sequence of the gene BmTsp.D contains the sequence shown in SEQ ID NO.2; 2) The nucleotide sequence of the gene BmTsp.D encodes the amino acid sequence shown in SEQ ID NO.1; 3) The nucleotide sequence of the gene BmTsp.D shown contains a nucleotide sequence that has more than 90% homology with the sequence shown in SEQ ID NO.2, and the nucleotide sequence encodes the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence that still has the same enzyme function after the amino acid sequence shown in SEQ ID NO.1 has been replaced, deleted and / or added to one or more amino acids.

[0009] As a preferred technical solution, the gene BmTsp.D encodes a tetraspan membrane protein; the amino acid sequence of the tetraspan membrane protein is any one of the following: 1) The amino acid sequence of the tetratransmembrane protein encoded by the gene BmTsp.D is shown in SEQ ID NO.1; 2) The amino acid sequence of the tetratransmembrane protein encoded by the gene BmTsp.D is composed of an amino acid sequence that still has the same enzymatic function after substitution, deletion and / or addition of one or more amino acids of the amino acid sequence shown in SEQ ID NO.1.

[0010] Another object of the present invention is to provide: a biomaterial, said biomaterial being any of the following: 1) Expression cassettes containing the gene BmTsp.D; 2) A recombinant vector containing the gene BmTsp.D, or a recombinant vector containing the expression cassette described in 1); 3) Transgenic cell lines containing the gene BmTsp.D, or transgenic cell lines containing the recombinant vector described in 2); 4) Recombinant bacteria containing the gene BmTsp.D, or recombinant bacteria containing the expression cassette described in 1), or recombinant bacteria containing the recombinant vector described in 2).

[0011] As a preferred technical solution, 2) the recombinant vector is an overexpression vector.

[0012] Another object of the present invention is to provide: the application of the above-mentioned biomaterial, wherein the application is any one of the following: 1) Application in the preparation of reagents for inhibiting silkworm nucleopolyhedrovirus; 2) Application in the preparation of drugs for the treatment and / or prevention of silkworm nucleopolyhedrovirus; 3) Application in silkworm breeding resistant to nucleopolyhedrovirus; 4) Application in improving the ability of silkworms to resist nucleopolyhedrovirus.

[0013] Another object of the present invention is to provide: an siRNA for use in gene BmTsp.D knockdown, the nucleotide sequence of said siRNA being shown in SEQ ID NO.9.

[0014] Another object of the present invention is to provide a method for improving the resistance of silkworms to nucleotropic multiviruses, wherein an overexpression vector based on the gene BmTsp.D is constructed and introduced into silkworms to overexpress the gene BmTsp.D in the silkworms.

[0015] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: Based on preliminary screening of silkworm genome data, this application identifies and characterizes a tetraspan membrane protein called BmTsp.D. Through overexpression and knockdown experiments of the BmTsp.D gene, the inhibitory effect of the BmTsp.D gene on nucleopolyhedrovirus was clarified, providing technical support for the development of nucleopolyhedrovirus inhibitory genes. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 For: Expression profile analysis of BmTsp.D; A: The relative mRNA expression level of BmTsp.D gene in five tissues was studied; B: The induced expression profile of BmTsp.D gene in the midgut of silkworm after BmNPV infection was studied; This indicates that p < 0.05. This indicates that p < 0.01. p < 0.0001, ns indicates no significant difference.

[0018] Figure 2 The study investigated the effect of BmTsp.D overexpression on BmNPV infection. A: Infected cells after overexpression were observed under a high-resolution fluorescence microscope, including images under white light (transmitted light) and images showing the green fluorescent protein (GFP) signal (scale bar: 100 µm). B: Transcriptional levels were analyzed 48 hours after transfection with the BmTsp.D overexpression vector. C: The effect of BmTsp.D overexpression on VP39 gene expression levels was analyzed at different time points after viral infection. This indicates that p < 0.05. This indicates that p < 0.01. p < 0.001, ns indicates no significant difference.

[0019] Figure 3 The study aimed to investigate the effect of BmTsp.D knockdown on viral infection. A: Infected cells after BmTsp.D knockdown were observed under a fluorescence microscope using a white light microscope (transmitted light) and a green fluorescent protein (GFP) channel. The scale bar was 100 µm. B: Transcriptional levels in BmN cells transfected with the BmTsp.D gene were analyzed 48 hours after viral infection. C: Transcriptional levels of the VP39 gene were detected at different time points after gene knockdown. p < 0.01, ns indicates no significant difference. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Silkworm larvae: The p50 variety of silkworm was selected and provided by the Sericulture Research Institute of Jiangsu University of Science and Technology. They were cultured at 26℃±1℃, relative humidity of 75%±5%, and photoperiod of 12 hours light / 12 hours dark for later use. Silkworm ovarian BmN cell line: purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd., cultured on TC-100 (Beijing Livining Biotechnology Co., Ltd.) medium supplemented with 10% (v / v) fetal bovine serum (FBS) (Gibco), and 0.1% penicillin and streptomycin, at a constant temperature of 27°C for later use.

[0022] BmNPV virus: The BmNPV tagged with EGFP (BmNPV-EGFP) was used. (The recombinant baculovirus BmNPV-EGFP expressing enhanced green fluorescent protein (EGFP) was constructed and preserved in our laboratory. The specific construction process is as follows: specific primers were designed to amplify the EGFP gene (with BamHI and XhoI sites at both ends, respectively), and it was constructed into the pFastBac1 plasmid, expressed using a polyhedral promoter without fusion with any protein). It was used to assess viral infection and transmission; the BmNPV virus was suspended in sterile water (1.0 × 10⁻⁶). 7OB / mL), for later use.

[0023] Example 1 Sequence characteristics and bioinformatics analysis of BmTsp.D We conducted a preliminary screening of tetraspan membrane proteins in the silkworm genome database, identified and characterized a typical tetraspan membrane protein, named BmTsp.D, for further research. Its specific amino acid and nucleotide sequences are as follows: BmTsp.D: MTLPPPARPAHPAAHRAMRYYRIWIYACNGALLLGALAFCAAAGRALSDYRRALVPGLGIAQPGFLYGYAALPVQAGLLQLLGCLAALRLSERMLNAYWLALLALLVGDAAIGVYWAFRFERVCRELRPQLRLRLARDYDTDVDFAEAWDRLQREQRCCGVTGPSDFATFNKTTLPASCCRIPAHT PAVTPTLLAVTSATSPGPFTPVHCAPHTAACAERLLVWLRRTADALFVLGYCVIAFLKLCFLGILRYEIKEMIQKIRILRSELGAGELLDGSPMHGGPLSQTIIVNAVNVNGGVRAHGGSPERAGERDALLGRASEACSRRSTHDEPLGPKTINGNNNCEMRELARGEYRPLAADSAATRI, SEQID NO.1; The nucleotide sequence encoding the aforementioned tetratransmembrane protein BmTsp.D is as follows: The inferred amino acid sequence, molecular weight, and isoelectric point were predicted using the EXPASY (Expert Protein Analysis System) website (http: / / www.expasy.org); potential signal peptides were predicted using the SignalP-5.0 server (http: / / www.cbs.dtu.dk / services / SignalP / ).

[0024] The results showed that the ORF of BmTsp.D encodes 367 amino acids, has no signal peptide, and the molecular weight of the mature protein is 39.94 kDa with an isoelectric point of 9.12.

[0025] Example 2 Construction of overexpression vectors and synthesis of small interfering RNA (siRNA) (1) RNA extraction and cDNA synthesis The experiment was set up with two groups: a treatment group and a control group, as follows: Treatment group: Silkworm p50 strain larvae were soaked in BmNPV virus suspension (1.0 × 10⁻⁶). 7 Feed with fresh mulberry leaves (OB / mL); Control group: Silkworm p50 strain larvae were fed with mulberry leaves soaked in sterile water; Forty-eight hours after infection, on the first day of the fifth instar, p50 strain larvae from both the treatment and control groups were collected, anesthetized on ice, and dissected. Tissue samples were collected, including those from the head, midgut, fat body, Malpighian tubules, and hemolymph (three samples were collected for each tissue, and each sample came from three larvae in the same batch). Total RNA was extracted from the above samples using TRIzol reagent (Beijing Tiangen Biotech Co., Ltd.).

[0026] To determine the specific biological function of BmTsp.D, the total RNA was collected and reverse transcribed into cDNA. Then, its tissue expression pattern and the inducible expression pattern after viral infection were detected by qRT-PCR. The silkworm BmGAPDH gene was selected as an internal control. The results are as follows: Figure 1 As shown.

[0027] The primer sequences for BmTsp.D amplification are as follows: 5'-AGAGCAGCGTTGTTGTGGTGTT-3', SEQ ID NO.3; 5'-CCTGGCGAAGTTGCGGAAGTAA-3', SEQ ID NO.4; The primer sequences for GAPDH amplification are as follows: 5'-TTCATGCCACAACTGCTACA-3', SEQ ID NO.5; 5'-AGTCAGCTTGCCATTAAGAG-3', SEQ ID NO.6; The amplification program was as follows: pre-denaturation at 95℃ for 10 min, followed by 40 cycles of denaturation at 95℃ for 15 s, annealing at 60℃ and extension for 1 min. Amplification reaction system: 1 μL cDNA template, 0.5 μL primer F, 0.5 μL primer R, 5 μL 2×SYBR premixExTaq™, 3 μL ddH2O.

[0028] Results analysis: BmTsp.D was transcribed in the head, midgut, fat body, Malpighian tubules, and hemolymph. Furthermore, the transcriptional level of BmTsp.D in the midgut significantly increased at 24 and 72 hours after viral infection.

[0029] First-strand cDNA for subsequent experiments was synthesized using the FastKing RT kit (Beijing Tiangen Biotech Co., Ltd.).

[0030] (2) Construction of overexpression vector and synthesis of small interfering RNA (siRNA) A: Construction of overexpression vectors Using the cDNA obtained in step (1) as a template, specific amplification primer sequences were designed to clone the ORF region of BmTsp.D. The specific primer sequences are as follows: F: 5'-TAGGTACCATGACGCTGCCCGCCGCCCGC-3', SEQ ID NO.7; R: 5'-GCTCTAGACCGATTCGAGTCGCCGCACT-3', SEQ ID NO.8; Amplification reaction program: 94℃ pre-denaturation for 3 min, followed by 38 cycles of 94℃ denaturation for 30 s, 56℃ annealing for 30 s, extension for 1 min, and then 72℃ extension for 10 min. Amplification reaction system: cDNA template 1 μL, primer F 1 μL, primer R 1 μL, 2×SYBRpremix ExTaq™ 10 μL, ddH2O 7 μL.

[0031] The amplified PCR product was ligated with the pMD-19T vector (commercially available from Takara), and the ligation was verified by sequencing to obtain the recombinant pMD-19T vector containing the target gene. The recombinant pMD-19T vector and the empty pIZT / V5-His plasmid (purchased from Invitrogen) were double-digested with Kpn I and Xba I, respectively, at 37°C for at least 4 h. After digestion, the target fragment and the vector were recovered and ligated using T4 ligase. The ligation product was transformed into DH5α competent cells, and positive clones were obtained and verified by double digestion. The constructed vector was then sequenced and verified to obtain the overexpression vector. The empty vector was used as a control. B: Synthesis of small interfering RNA (siRNA) The siRNA targeting BmTsp.D was synthesized by Suzhou Jima Pharmaceutical Technology Co., Ltd., and its specific sequence is as follows: siRNA-Tsp.D: 5'-GCGAGAGAUUACGAUACAGTT-3', SEQ ID NO.9; siRNA-NC: 5'-UUCUCCGAACGUGUCACGUTT-3', SEQ ID NO.10 (negative control).

[0032] (3) Cell transfection and BmNPV infection BmN cells were seeded at an appropriate density in 12-well plates and cultured overnight. Next, 1.6 µg of the overexpression vector or 80 pm of siRNA oligonucleotide was added to serum-free TC-100 medium, adjusting the volume to 100 µL. Simultaneously, 4 µL of GP transfection reagent (Germ Pharmaceuticals, Suzhou, China) was added to 100 µL of serum-free medium, and the two were then mixed to prepare the transfection mixture. The transfection mixture was incubated at room temperature for 15 min, and then added to 12-well plates for cell transfection. BmNPV-EGFP (MOI 3) was then added to transfected BmN cells for infection. Immunofluorescence of BmTsp.D was analyzed and fluorescence intensity was captured using an inverted fluorescence microscope (Ti-E, Nikon, Tokyo, Japan) at 24, 48, and 72 hours post-infection. Simultaneously, cells were harvested at these three time points to extract total RNA, and the effects of overexpression / knockdown on BmNPV infection and replication were analyzed using qRT-PCR. The specific experimental procedure is as follows: Immunofluorescence analysis of BmTsp.D BmTsp.D (polyhistidine tag) overexpressing / knockdown BmN cells were placed on cell culture slides and cultured overnight. The cells were then washed three times with PBS, fixed with 4% paraformaldehyde for 20 min, and washed with sterile PBS. The cells were permeabilized three times at room temperature with 1% Triton X-100 (prepared in PBS) for 20 min each time, followed by three washes with PBS. The permeabilized cells were then blocked for 1 h with 5% bovine serum albumin (BSA) blocking solution (prepared in PBST). Subsequently, the cells were incubated overnight at 4°C with mouse monoclonal anti-polyhistidine antibody (1:800 in 0.5% BSA). After three washes with PBS, the cells were incubated for 2 h at room temperature with goat anti-mouse antibody and goat anti-mouse 488 (1:500 in 0.5% BSA). The cells were then washed with PBS, the nuclei were stained with DAPI (0.5 µg / mL) for 15 min, and then washed three times again with PBS. Finally, the stained cells were fixed onto cell sheets and photographed under a fluorescence microscope (Ti-E, Nikon, Tokyo, Japan). Cells transfected with the empty vector pIZT / V5-His were used as controls.

[0033] qRT-PCR analysis of the effects of overexpression / knockdown on BmNPV infection and replication qRT-PCR digestion was performed using UltraSYBR reagent (Kangwen Biotechnology Co., Ltd. (Beijing)); qRT-PCR detection was performed using a LightCycler 96 PCR instrument (Roche (Basel, Switzerland)). Each reaction was performed in 3 biological replicates and 2 technical replicates. The relative expression level of each mRNA was determined using the 2-ΔΔCt method. Total RNA levels were calibrated using silkworm glyceraldehyde-3-phosphate dehydrogenase (BmGAPDH) as an internal control.

[0034] Forty-eight hours after transfection with the overexpression vector pIZT / V5-His-BmTsp.D, the transcriptional level of BmTsp.D was detected by qRT-PCR. The transcription of the control group (pIZT / V5-His empty plasmid) represented the expression of endogenous BmTsp.D.

[0035] The amplification program was as follows: pre-denaturation at 95℃ for 10 min, followed by 40 cycles of denaturation at 95℃ for 15 s, annealing at 60℃ and extension for 1 min. Amplification reaction system: 1 μL cDNA template, 0.5 μL primer F, 0.5 μL primer R, 5 μL 2×SYBR premix ExTaq™, 3 μL ddH2O; Amplification primer sequences: BmTsp.D amplification primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4; GAPDH amplification primer sequences are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0036] The results showed that overexpression significantly increased the transcriptional level of BmTsp.D ( Figure 2 B). Furthermore, observation of the green fluorescence intensity of BmNPV tagged with EGFP showed that the green fluorescence density of the BmTsp.D overexpression group was weaker than that of the control group at 48 hours post-infection, and the difference was more pronounced at 72 hours. Figure 2 A).

[0037] In addition, the BmNPV viral nucleocapsid protein gene VP39 was selected, and viral replication ability was detected by qRT-PCR experiment.

[0038] The amplification program was as follows: pre-denaturation at 95℃ for 10 min, followed by 40 cycles of denaturation at 95℃ for 15 s, annealing at 60℃ and extension for 1 min. Amplification reaction system: 1 μL cDNA template, 0.5 μL primer F, 0.5 μL primer R, 5 μL 2×SYBR premixExTaq™, 3 μL ddH2O; Amplification primer sequences: The GAPDH amplification primer sequences are shown in SEQ ID NO5 and SEQ ID NO.6; The primer sequences for VP39 amplification are as follows: 5'-CAACTTTTTGCGAAACGACTT-3', SEQ ID NO.11; 5'-GGCTACACCTCCACTTGCTT-3', SEQ ID NO.12; The results showed that, compared with the control group, the expression level of VP39 was significantly decreased at 48 hours and 72 hours after viral infection. Figure 2 C).

[0039] In summary, the results indicate that overexpression of BmTsp.D can significantly inhibit the proliferation of BmNPV.

[0040] The transcriptional level of BmTsp.D was reduced using siRNA-mediated knockdown, and the replication level of BmNPV was detected. Figure 3 As shown in Figure B, qRT-PCR results indicated that, compared to the control group (pIZT / V5-His empty plasmid), BmN cells transfected with siRNA targeting the BmTsp.D gene significantly downregulated the transcriptional level of this gene. Furthermore, at 48 hours post-viral infection, the treated group cells showed a greater distribution of green fluorescence than the control group (…). Figure 3A). Meanwhile, compared to the siNC-transfected control group, the treatment group showed significantly increased VP39 expression at 24 and 48 hours post-BmNPV infection. However, at 72 hours post-infection, there was no significant difference in fluorescence intensity or VP39 expression levels between the two groups ( Figure 3 C). This may be due to the reduced knockdown effect of siRNA at this time.

[0041] In summary, the results indicate that knockdown of BmTsp.D can promote the proliferation of BmNPV.

[0042] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0043] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of gene BmTsp.D in the preparation of drugs to inhibit silkworm nucleopolyhedrovirus, characterized in that, The gene BmTsp.D encodes a tetraspan membrane protein; the amino acid sequence of the tetraspan membrane protein is shown in SEQ ID NO.

1.

2. The application of the gene BmTsp.D according to claim 1 in the preparation of a drug to inhibit silkworm nucleopolyhedrovirus, characterized in that, The nucleotide sequence of the gene BmTsp.D is any one of the following: 1) The nucleotide sequence of the gene BmTsp.D contains the sequence shown in SEQ ID NO.2; 2) The nucleotide sequence of the gene BmTsp.D shown contains a nucleotide sequence that has more than 90% homology with the sequence shown in SEQ ID NO.2, and the nucleotide sequence encodes the amino acid sequence shown in SEQ ID NO.

1.

3. An application of a biomaterial, wherein the biomaterial is as follows: (1) An expression cassette containing the gene BmTsp.D as described in claim 1; (2) A recombinant vector containing the gene BmTsp.D as described in claim 1, or a recombinant vector containing the expression cassette as described in (1); (3) A transgenic cell line containing the gene BmTsp.D as described in claim 1, or a transgenic cell line containing the recombinant vector as described in (2); (4) A recombinant bacterium containing the gene BmTsp.D as described in claim 1, or a recombinant bacterium containing the expression cassette as described in (1), or a recombinant bacterium containing the recombinant vector as described in (2); characterized in that, The application is any one of the following: 1) Application in the preparation of reagents for inhibiting silkworm nucleopolyhedrovirus; 2) Application in the preparation of drugs for the treatment and / or prevention of silkworm nucleopolyhedrovirus; 3) Application in the breeding of silkworms resistant to nucleopolyhedrovirus.

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