Bombyx mori BmLIS1 gene and application thereof

Abstract

The invention provides a bombyx mori BmLis1 gene and application thereof, and belongs to the technical field of gene engineering. A BmLis1 gene is knocked out and overexpressed in a BmN cell, it is found that the overexpressed bombyx mori BmLis1 gene can inhibit proliferation and replication of BmNPV, the full-length sequence of the bombyx mori BmLis1 gene is shown as SEQ ID No.3, and the amino acid sequence of protein coded by the bombyx mori BmLis1 gene is shown as SEQ ID No.4; the bombyx mori BmLis1 gene can be used for preparing bombyx mori virus prevention and treatment drugs, and a new target is provided for prevention, control and treatment of BmNPV.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a BmLIS1 gene and an application thereof. Background Art

[0002] The silkworm (Bombyx mori) is an important economic insect of the family Bombycidae, order Lepidoptera. As a core biological resource for the silk industry, its silk production supports the global silk manufacturing industry and holds irreplaceable traditional economic value. However, the sericulture industry has long been plagued by viral diseases, most notably Bombyx mori Nucleopolyhedrovirus (BmNPV). The Bombyx mori Nucleopolyhedrovirus (BmNPV), which causes blood-borne pus disease, is one of the most serious diseases in silkworm production, causing significant economic losses to the global sericulture industry annually. BmNPV is a double-stranded circular DNA virus and a representative species of the family Baculoviridae. Upon invading the host, BmNPV modulates multiple host cellular processes, including apoptosis, the cell cycle, the DNA damage response, RNAi pathways, and energy metabolism, creating an internal environment conducive to viral replication and proliferation. After invading host cells, Bombyx mori nuclear polyhedrosis virus (BmNPV) can disrupt the host cell cycle by regulating the expression of host genes through the expression of viral proteins, thereby arresting the host cell cycle at the G2 / M phase. Currently, no effective method for preventing and treating BmNPV has been discovered. The elucidation of silkworm antiviral mechanisms and the identification of antiviral genes are relatively slow, hindering the breeding of resistant silkworm varieties. Therefore, it is crucial to identify a key gene that influences silkworm resistance to the virus. This gene could serve as a proliferation inhibitor of BmNPV and be used in the preparation of silkworm antiviral drugs, providing a new target for BmNPV prevention, control, and treatment. Summary of the Invention

[0003] In view of this, one of the objects of the present invention is to provide a full-length sequence of the silkworm BmLis1 gene that can regulate BmNPV infection of BmN cells. Overexpression of BmLis1 at the cellular level can significantly inhibit the proliferation of BmNPV, indicating that this gene is a key gene affecting the silkworm's resistance to viruses. It can be used as a proliferation inhibitor of silkworm nuclear polyhedrosis virus and used to prepare silkworm antiviral drugs; the second object of the present invention is to provide the use of the silkworm BmLis1 gene in the preparation of a reagent for regulating BmNPV infection of BmN cells.

[0004] In order to achieve the above object, the present invention provides the following technical solutions: 1. A silkworm BmLis1 gene capable of regulating BmNPV infection of BmN cells, wherein the amino acid sequence of the protein encoded by the BmLis1 gene is shown in SEQ ID No. 4.

[0005] Preferably, the nucleotide sequence encoded by the silkworm BmLis1 gene is shown as SEQ ID No.3.

[0006] 2. The use of the silkworm BmLis1 gene in the preparation of a reagent for regulating BmNPV infection of BmN cells. Overexpression of the silkworm BmLis1 gene can inhibit the proliferation and replication of BmNPV in BmN cells, and knockout of the silkworm BmLis1 gene can promote the proliferation and replication of BmNPV in BmN cells.

[0007] Preferably, in the present invention, the method for overexpressing the silkworm BmLis1 gene is to construct a recombinant vector for overexpression of the BmLis1 gene.

[0008] Preferably, the overexpression recombinant vector is obtained by inserting the silkworm BmLis1 gene between the KpnI and XbaI restriction sites of the pIZ-V5 / His vector.

[0009] Preferably, the method for knocking out the silkworm BmLis1 gene is to construct a knockout vector of the BmLis1 gene based on the CRISPR / Cas9 system.

[0010] Preferably, the knockout vector comprises Cas9 expressed by an IE1 promoter and sgRNA expressed by a U6 promoter, and the target sequence of the sgRNA is shown in SEQ ID No. 13-15.

[0011] The present invention has the following beneficial effects: The invention discloses the silkworm BmLis1 gene, which has an inhibitory effect on the Bombyx mori nuclear polyhedrosis virus (BmNPV). By knocking out and overexpressing the BmLis1 gene in BmN cells, it was demonstrated that overexpression of the BmLis1 gene can significantly inhibit the proliferation and replication of BmNPV. Therefore, the BmLis1 gene is of great value in the research of antiviral molecular breeding in silkworms. Overexpression of this gene can be used to enhance the antiviral capacity of silkworms, providing a new target for the prevention, control, and treatment of BmNPV. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration: Figure 1 Analysis of the expression characteristics of the BmLis1 gene (A: tissue expression of the BmLis1 gene; B: expression of the BmLis1 gene at different time points after viral infection); Figure 2 Effects of overexpression of BmLis1 on the proliferation and replication of BmNPV (A: Schematic diagram of the construction of the BmLis1 gene overexpression vector; B: RT-PCR detection of BmLis1 gene overexpression; C: Effects of overexpression of BmLis1 on the transcription levels of genes related to BmNPV proliferation and replication; D: Effects of overexpression of BmLis1 on the expression level of BmNPV VP39 protein); Figure 3 Effects of BmLis1 gene knockout on BmNPV proliferation (A: Schematic diagram of BmLis1 gene knockout vector construction; B: RT-PCR detection of BmLis1 gene knockout; C: Effects of BmLis1 knockout on the transcription levels of genes related to BmNPV proliferation and replication; D: Effects of BmLis1 knockout on the expression level of BmNPV VP39 protein). Figure 4 The effect of BmLis1 gene on cell cycle (A: Flow cytometry was used to detect the effect of BmLis1 on cell cycle; B: RT-PCR was used to detect the effect of overexpression / knockout of BmLis1 on the transcription level of BmCyclin B, a key gene in the G2 / M phase of cells). DETAILED DESCRIPTION

[0013] Below with reference to accompanying drawing, preferred embodiment of the present invention is described in detail.The test method that does not specify specific conditions in embodiment is usually carried out according to normal condition or according to the condition that reagent manufacturer recommends.It is particularly important to point out that the embodiment of the present invention specification is only for helping to understand the present invention, and they do not have any limiting effect, and promptly the present invention can also have other embodiment except the example of specification.Therefore, any technical scheme that adopts equivalent replacement or equivalent conversion form to form, all in protection scope of the present invention.

[0014] Example 1. Analysis of expression characteristics of the BmLis1 gene (1) Tissue expression of the BmLis1 gene RNA was extracted from the midgut, fat body, hemolymph, head, gonads and other tissues of 5-instar 1-day-old silkworms. The reverse-transcribed cDNA was used as a template to perform RT-PCR analysis using primers shown in SEQ ID No. 1 and 2 to analyze the expression of the BmLis1 gene in various tissues. The results are shown in Figure 2. Figure 1 , as shown in A. As can be seen from the figure, the BmLis1 gene is highly expressed in the head and gonads of the silkworm, indicating that the expression of the BmLis1 gene is tissue-specific in the silkworm.

[0015] RT-BmLis1-F: TCCGTCATCAGACTTCAGAAAA (SEQ ID No. 1) RT-BmLis1-R:GTGTCCAGTGAGACAAAACTTC (SEQ ID No. 2) (2) Expression of the BmLis1 gene at different time points during viral infection After BmN cells were infected with BmNPV, cell samples were collected at 0h, 12h, 24h, 48h and 72h to extract RNA. The reverse transcribed cDNA was used as a template to perform RT-PCR analysis using primers shown in SEQ ID No. 1 and 2 to analyze the expression level of the BmLis1 gene at different time points of BmNPV infection. The results are shown in Figure 2. Figure 1 As shown in Figure , B, the expression level of the BmLis1 gene decreased with the increase of infection time, indicating that the BmLis1 gene may be involved in the regulatory process of the host response to BmNPV infection.

[0016] Example 2: Overexpression of the BmLis1 gene inhibits the proliferation and replication of BmNPV To determine the role of the BmLis1 gene (lissencephaly-1 homolog, NCBI ID: 101736689) in the proliferation and replication of BmNPV and to identify the function of BmLis1 in regulating viral proliferation, we constructed an overexpression vector (plZ-V5 / His-Lisl) for the silkworm BmLis1 gene. The BmLis1 overexpression vector construction process was as follows: a silkworm cDNA library was used as a template and PCR amplification was performed using the primers shown in SEQ ID Nos. 5 and 6. The amplified PCR product was analyzed by agarose gel electrophoresis and the target fragment (SEQ ID No. 3) was recovered and purified using a gel extraction kit (OMEGA). The commercial vector pIZ-V5 / His (ThermoFisher Scientific Inc.) was digested with KpnI and XbaI and ligated with the target fragment (containing the HA tag, KpnI and XbaI restriction sites) of Lis1 to obtain plZ-V5 / His-Lisl. Figure 2 , as shown in A. The transfected cells were collected and RNA was extracted. Then, RT-PCR was performed using primers shown in SEQ ID No. 1 and 2 to analyze the expression of the BmLis1 gene after transfection of the overexpression vector. The results showed that the expression level of the BmLis1 gene was significantly increased, indicating that the vector can be used for subsequent experiments, such as Figure 2 , as shown in B.

[0017] BmLis1Protein:MKMVLSQRQREELNKAIADYLGSNGYNDALEAFKKEADMTGEMDRKFGGLLEKKWTSVIRLQKKVMELESKLSEAQKEFIEGAPTRAKRTPTE WIPRPPEKFCLTGHRATITKVIFHPVFSLMVSSSEDATIKVWDFESGEYERTLKGHTDSVQDIAFDHHGKLLVSCSADMSIKLWDFNQTFECIKTMHGHDHNVSSVA FSPSGDIVYSASRDKTIKAWEVATGYCIKTYKGHREWVRMVRVSPDGTLLASCSNDQTVRIWNADTNECKMELREHEHVVECVSWAPEAAAAAINEAAGGDNRRANH TGPFLASGSRDKSVKIWEVSTGQCLATLVGHDNWVRGAAWHPGGRYLLTASDDKTLRVWDVAHTRCLKTLYAHHHFATSLDFHRSLPYVISGSVDQTVKVWECR (SEQ ID No.4) BmLis1-HA-KpnI-F:ggggtaccATGTACCCATACGATGTTCCAGATTACGCTAAAATGGTGTTGTCACAA (SEQ ID No. 5) BmLis1-HA-XbaI-R:gctctagaCTATACCCATACGATGTTCCAGATTACGCTGCGACACTCCCAAACT (SEQ ID No. 6) To identify the effect of the BmLis1 gene on BmNPV proliferation, this study analyzed the proliferation characteristics of BmNPV after overexpression of the BmLis1 gene by RT-PCR and Western Blot. The results showed that after overexpression of the BmLis1 gene, the transcription levels of the immediate early gene ie1 and the early gene gp64 related to BmNPV proliferation and replication were significantly downregulated (SEQ ID No. 7-12). Figure 2 , as shown in C. Western blot analysis of viral nucleocapsid protein VP39 also down-regulated expression, as shown in Figure 2 , D. The above results show that overexpression of BmLis1 has a significant inhibitory effect on the proliferation of BmNPV.

[0018] RT-ie1-F:AAGAAGGAGGACGGCAGCAT (SEQ ID No.7) RT-ie1-R:ATCTCGCCAGAAATCCAATAAAAC (SEQ ID No.8) RT-gp64-F:ATGCTAAGAACGCCAACAGAAG (SEQ ID No.9) RT-gp64-R:TTGTAACAAATCCATGCCCAC (SEQ ID No.10) RT-SW22934-F:TTCGTACTGGCTCTTCTCGT (reference gene, SEQ ID No. 11) RT-SW22934-R:CAAAGTTGATAGCAATTCCCT (SEQ ID No.12) Example 3: Knocking out the BmLis1 gene promotes BmNPV proliferation and replication To further determine the function of the BmLis1 gene in regulating viral proliferation and replication, this study constructed a BmLis1 gene knockout vector pSL1180-IE1-Cas9-U6-sgLis1 based on the CRISPR / Cas9 system. Construction process of pSL1180-IE1-Cas9-U6-sgLis1 vector: pSL1180-lE1-Cas9-Ser-U6-sgRNA (Zhanqi Dong, QiQin, Zhigang Hu, Peng Chen, Liang Huang, Xinling Zhang, Ting Tian, Cheng Lu, Minhui Pan. Construction of a One-Vector Multiplex CRISPR / Cas9 Editing Systemto Inhibit Nucleopolyhedrovirus Replication in Silkworms .VIROLOGICA SINICA, 2019, 34(4): 444-453.) vector was digested with BglII, and sgLis1 (SEQ ID No.13-14) with BglII restriction site was annealed and ligated to the vector to obtain pSL1180-IE1-Cas9-U6-sgLis1, as shown in Figure 5. Figure 3 , as shown in A. The transfected cells were collected and RNA was extracted. Then, the expression of BmLis1 gene after transfection of knockout vector was analyzed by RT-PCR using primers shown in SEQ ID No. 1 and 2. The results showed that the transcription of BmLis1 gene was significantly inhibited, indicating that the vector can be used for subsequent experiments, such as Figure 3 , as shown in B.

[0019] sgLis1-F:AAGTGGTTGTCACAACGGCAACGAG (SEQ ID No. 13) sgLis1-R:AAACCTCGTTGCCGTTGTGACAACC (SEQ ID No. 14) To further examine the effect of knocking out the BmLis1 gene on the proliferation and replication of BmNPV, this study analyzed the proliferation characteristics of BmNPV after knocking out the BmLis1 gene by RT-PCR and Western Blot. The results showed that the transcription levels of the immediate early gene ie1 and the early gene gp64 related to BmNPV proliferation and replication were significantly upregulated after knocking out the BmLis1 gene (SEQ ID No. 7-12). Figure 3 , as shown in C. Western Blot analysis showed that the viral nucleocapsid protein VP39 was also up-regulated, as shown in Figure 3 As shown in Figure , D, the above results indicate that knockout of BmLis1 significantly promotes the proliferation of BmNPV.

[0020] Example 4: Effect of the BmLis1 gene on the cell cycle To further explore the function of the BmLis1 gene, flow cytometry was used to detect the effect of the BmLis1 gene on cell cycle distribution. The results showed that after overexpression of the BmLis1 gene, the number of cells in the G2 / M phase was significantly reduced, and after knocking out the BmLis1 gene, the proportion of cells in the G2 / M phase was significantly increased. Figure 4 , as shown in A. The above results indicate that the BmLis1 gene is involved in the regulation of the cell cycle. To explore how the BmLis1 gene affects the cell cycle and regulates the G2 / M phase of cells, the BmLis1 gene was overexpressed and knocked out in BmN cells, and the changes in the transcription level of the key gene BmCyclin B in the G2 / M phase of cells were analyzed by RT-PCR (SEQ ID No.15-16). The results showed that the expression level of BmCyclin B was upregulated after overexpression of BmLis1, and the expression level of BmCyclin B was downregulated after knocking out the BmLis1 gene, as shown in Figure 2. Figure 4 , as shown in Figure B. The above results further indicate that the BmLis1 gene may affect the G2 / M phase of cells by regulating the expression level of the transgene BmCyclin B, which is a key cycle in the G2 / M phase of cells, thereby inhibiting the proliferation and replication of BmNPV.

[0021] RT-BmCyclin BF:TGTCAAAAATGTTATTCAGCC (SEQ ID No. 15) RT-BmCyclin BR:TTTCCGTAAAAGAGTCAGTTCC (SEQ ID No. 16) The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A silkworm BmLis1 gene capable of regulating BmNPV infection of BmN cells, characterized in that: The protein amino acid sequence of the BmLis1 gene is shown in SEQ ID No.

4.

2. The silkworm BmLis1 gene capable of regulating BmNPV infection of BmN cells according to claim 1, characterized in that The nucleotide sequence encoded by the silkworm BmLis1 gene is shown in SEQ ID No.

3.

3. Use of the silkworm BmLis1 gene according to claim 1 in preparing a reagent for regulating BmNPV infection of BmN cells, characterized in that: Overexpression of the silkworm BmLis1 gene can inhibit the proliferation and replication of BmNPV in BmN cells, while knockout of the silkworm BmLis1 gene can promote the proliferation and replication of BmNPV in BmN cells.

4. The use according to claim 3, characterized in that The method for over-expressing the silkworm BmLis1 gene is to construct a recombinant vector for over-expressing the BmLis1 gene.

5. The use according to claim 4, characterized in that The overexpression recombinant vector is obtained by inserting the silkworm BmLis1 gene between the KpnI and XbaI restriction enzyme cutting sites of the pIZ-V5 / His vector.

6. The use according to claim 3, characterized in that The method for knocking out the silkworm BmLis1 gene is to construct a knockout vector of the BmLis1 gene based on the CRISPR / Cas9 system.

7. The use according to claim 6, characterized in that The knockout vector comprises Cas9 expressed by an IE1 promoter and an sgRNA expressed by a U6 promoter, and the target sequence of the sgRNA is shown in SEQ ID No. 13-15.

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