Lepidoptera insect Caspase-5 gene and application thereof

Abstract

The invention provides a lepidoptera insect Caspase-5 gene and application of the lepidoptera insect Caspase-5 gene to improvement of insecticidal toxicity of baculovirus to lepidoptera pests, the nucleotide sequence of an open reading frame of the gene is SEQ ID NO: 1, and the amino acid sequence of encoded protein of the gene is SEQ ID NO: 2. The SeCaspase-5 screened by the invention has a function of promoting apoptosis, and the insecticidal toxicity of the virus polyhedrosis OB on beet armyworms can be improved. Therefore, by constructing the recombinant virus bacmid carrying the SeCaspase-5 gene and transfecting insect cells, the obtained progeny virus polyhedrosis OB is further fed to beet armyworm larvae, and the insecticidal toxicity of the recombinant virus to pests can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological agriculture and related industries, and specifically relates to a lepidopteran insect Caspase-5 gene and application thereof. Background Art

[0002] Most species of Lepidoptera are important pests in agriculture and forestry, among which the beet armyworm (Spodoptera exigua) is a representative species that is widely distributed and causes serious damage worldwide. The beet armyworm has a very wide diet. It is reported that it can feed on more than 170 species of plants from up to 35 families. It can not only harm vegetable crops such as peppers, sweet peppers, and tomatoes, but also grain crops such as corn and peanuts. Years of records have found that beet armyworm outbreaks have been recorded in more than half of my country's provinces, cities, or municipalities, which has continuously and seriously affected my country's agricultural development and agricultural economic level. At present, the prevention and control of Lepidoptera pests mainly uses chemical pesticides, but chemical pesticides are prone to environmental pollution, pesticide residues, and pests are prone to develop resistance, which seriously affects the environment and the life and health of humans and animals. Therefore, biological control of pests as a green prevention and control strategy has attracted much attention.

[0003] Baculovirus is a natural microorganism that only infects invertebrates. Therefore, it is safe for humans and animals and environmentally friendly. It has been developed as a safe insecticide and widely used in the biological control of lepidopteran pests. However, baculovirus insecticides also have disadvantages such as narrow host range, slow insecticidal speed and low field toxicity, so they are not easily accepted by farmers, which seriously limits the application of baculovirus. Summary of the invention

[0004] The present invention aims to provide a lepidopteran insect Caspase-5 gene and its application, namely a beet armyworm Caspase-5 (SeCaspase-5) gene, and its application in improving the insecticidal toxicity of baculovirus to lepidopteran pests.

[0005] The invention provides a beet armyworm SeCaspase-5 gene; the nucleotide sequence of the gene open reading frame (ORF) is SEQ ID NO: 1, and the amino acid sequence of the encoded protein is SEQ ID NO: 2.

[0006] In another aspect, the present invention provides a viral shuttle plasmid bacmid, which is constructed by inserting the nucleic acid fragment of the beet armyworm SeCaspase-5 gene into the viral genome;

[0007] As a specific record of the embodiment, the virus is a baculovirus;

[0008] The present invention also provides another use of the beet armyworm SeCaspase-5 gene, which is to improve the insecticidal toxicity of the virus to lepidopteran pests;

[0009] In another aspect, the present invention provides a method for improving the insecticidal toxicity of a virus to lepidopteran pests, wherein the method comprises increasing the expression level of the above-mentioned beet armyworm SeCaspase-5 gene in a host cell;

[0010] As a specific description of the embodiment, the method of increasing the expression level of the Spodoptera exigua SeCaspase-5 gene in the host cell is to transfer the shuttle plasmid bacmid into the host cell.

[0011] The present invention identifies the DNA sequence and protein sequence of the SeCaspase-5 gene, and constructs the genome bacmid of the recombinant Autographa californica multiplenucleopolyhedrovirus (AcMNPV) carrying the SeCaspase-5 gene, and successfully obtains the recombinant virus polyhedron (occlusion body, OB) of the corresponding shear body by transfecting cells. SeCaspase-5 has a pro-apoptosis function, can promote cell apoptosis during the AcMNPV infection of cells, and can improve the insecticidal toxicity of the virus polyhedron OB to beet armyworm. Therefore, by constructing the recombinant virus bacmid carrying the SeCaspase-5 gene and transfecting insect cells, the obtained progeny virus polyhedron OB is further fed to the beet armyworm larvae, which can improve the insecticidal toxicity of the recombinant virus to pests. The present invention enriches the knowledge about Caspase-5 of lepidopteran insects and its involvement in regulating baculovirus-induced cell apoptosis, and also provides an effective way to improve the insecticidal toxicity of baculovirus, which is of great significance for the efficient use of baculovirus for biological control of lepidopteran pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1The amino acid sequence alignment and protein functional domain analysis of SeCaspase-5 and other Lepidoptera Caspase-5 were performed. The results showed that SeCaspase-5 contained a pro-domain located at the N-terminus, which was separated from the C-terminus of the protein by the first cleavage site. The pro-domain contained a CARD (caspase recruitment domain) domain with the function of recruiting other caspases; and the C-terminus of the protein contained a large subunit and a small subunit, which were separated by the second cleavage site. The large subunit contained a substrate binding site with the motif "SSHG" and an enzyme active site with the motif "QMCRG", while the small subunit contained a substrate binding site with the motif "RDGKLG".

[0013] Figure 2 This is a diagram of the construction and identification analysis of SeCaspase-5 recombinant virus bacmid, Figure 2 A is a schematic diagram of the construction of SeCaspase-5 recombinant virus bacmid. GFP (green fluorescent tag) was fused to the C-terminus of SeCaspase-5 and then cloned into the genomic bacmid of AcMNPV. Figure 2 B is the PCR identification analysis of SeCaspase-5 recombinant virus bacmid. The bacmid carrying SeCaspase-5 was amplified by PCR using the universal primer M13 / pUCR and the upstream primer of SeCaspase-5 gene, and a GFP fragment of 2476 bp (control) and a SeCaspase-5 of 3835 bp were obtained, respectively, indicating that the bacmid was constructed correctly.

[0014] Figure 3 The results show that no apoptosis was observed in cells transfected with GFP recombinant bacmid from 24 to 96 hours post transfection (h pt), and all cells produced virus polyhedra OB (indicated by red arrows), while apoptosis was observed in cells transfected with SeCaspase-5 recombinant bacmid (indicated by black arrows), and virus polyhedra OB were only observed in a small number of cells 72 hours after transfection.

[0015] Figure 4To investigate the effect of SeCaspase-5 on the insecticidal toxicity of the recombinant virus AcMNPV, OBs carrying the recombinant virus GFP (control) or SeCaspase-5 were fed to third-instar larvae of Spodoptera exigua. Each larva was fed 1×10 6 After the larvae finished eating the poisoned feed, the number of deaths of Spodoptera exigua larvae was counted every day, and the survival rate was calculated. The results showed that the survival rate of the larvae fed with SeCaspase-5 recombinant virus polyhedron OB was significantly lower than that of the larvae fed with GFP recombinant virus polyhedron OB (the significant difference analysis showed that P<0.0001). DETAILED DESCRIPTION

[0016] Baculovirus can be used to construct recombinant baculovirus carrying foreign genes or lacking specific genes, so as to improve the yield and virulence of recombinant viruses. However, the inherent genetic characteristics and infection mechanism of baculovirus currently affect its insecticidal virulence.

[0017] The beet armyworm SeCaspase-5 gene screened by the present invention has the function of promoting the apoptosis of beet armyworm cells. The SeCaspase-5 gene is cloned into the genome shuttle plasmid bacmid of the polyhedrosis virus AcMNPV and transfected into the beet armyworm Se-3 cells. The apoptosis analysis shows that the overexpression of SeCaspase-5 can cause the apoptosis of the beet armyworm Se-3 cells and delay the production of the virus polyhedron OB to a certain extent. The yield of the virus polyhedron OB in the cells is collected and measured, and the result shows that the overexpression of SeCaspase-5 reduces the yield of the virus polyhedron OB. The virus polyhedron OB is further fed to the 3rd instar larvae of the beet armyworm, and the survival rate of the susceptible larvae is counted. The result shows that SeCaspase-5 improves the insecticidal toxicity of its recombinant virus polyhedron OB.

[0018] The eggs of Spodoptera exigua used in the embodiments of the present invention were purchased from Keyun Company, and the larvae were hatched and bred by the Shandong Sino-US Invertebrate Cell Culture and Cell Engineering Cooperative Research Center and the Shandong Provincial Key Laboratory for Integrated Control of Plant Diseases and Insect Pests. The Se-3 cells of Spodoptera exigua were constructed and preserved from the laboratory of the inventor; the competent cells of Escherichia coli (DH10Bac) containing the AcMNPV genome shuttle plasmid bacmid were purchased from Invitrogen. The pMD18-T vector and restriction enzymes BamHI and EcoRI were purchased from Takara; the transfection reagent Cellfection II was purchased from Invitrogen; the 6-well and 12-well cell culture plates were purchased from Nice Life Sciences Co., Ltd.; primer synthesis and gene sequencing were performed by Shanghai Shenggong Biotechnology Co., Ltd.

[0019] The present invention is described below in conjunction with specific embodiments and drawings.

[0020] Example 1: Cloning and Identification of SeCaspase-5

[0021] 1. Gene cloning of SeCaspase-5

[0022] By analyzing the genome of Spodoptera exigua SeCaspase-5, gene-specific full-length primers were designed, and PCR amplification was performed using the cDNA of Spodoptera exigua Se-3 cells as a template. The PCR primer sequences used are as follows:

[0023] 5′-AAT GGATCC ATGCAGAGAGAACACAGAGAAGC-3′(BamHI),

[0024] 5′-AAT GAATTC TTACTCGTAGAGGCCAGGGTG-3′(EcoRI).

[0025] The PCR product was separated by agarose gel electrophoresis to obtain a gene fragment of about 1350 bp in size. After gel recovery, it was double-digested with restriction endonucleases BamHI and EcoRI and ligated into the vector pMD18-T. After clone screening and DNA sequencing, the complete ORF of the SeCaspase-5 gene was obtained, which was 1362 bp in size.

[0026]

[0027] The amino acid sequence of the encoded protein is as follows (SEQ ID NO:2):MQREHREAAIRSNFTSLVERTDLDSVVTALYEKGVFSEQMIEPFRNNSELERDRKRRLYMDVTRRGPHAFAHLVDALGELGYWDLVRDLDPDSPFSFSSQQRFNPIPQPT TRTRTADNDNFHSLSTSNRPRAEPNRNSTQLQSAPPPPPPAVDNDNSPEKVTAPPFIVKKSTKFMEDDDTKDLKLYRTRGRNRGILLDFTYTEFDNNNIEEFRNGVDVDCNNLKYL FDELGFRRLGYLNLTKTETMETLKSLNNVLVNIESVFIVVSSHGYERPHSSDTDVRCKDGQLISLYDIMTYFNNRNMPALIGVPKVFIFQMCRGSSADYAWQSSSAHAPAQMPMP AGDVVYDGQPMAPASAATSALHLYDRPRQTPLYSDILIAHSTLPGLVAHRDGKLGSWYIQALCEVFAARAHDCHVEKLFTLVDKRMQDKFKVQTSSVDRWGFNKRLYLHPGLYE.

[0028] 2. Phylogenetic analysis of SeCaspase-5

[0029] Using the amino acid sequence of SeCaspase-5 as a template, the amino acid sequences of other lepidopteran insect Caspase-5 and Drosophila Dronc were searched and downloaded from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The phylogenetic analysis of SeCaspase-5 and other lepidopteran insect Caspase-5-based Drosophila Dronc was performed using Mega 5.0 software, and a phylogenetic tree was constructed. The results showed that the similarity between SeCaspase-5 and other lepidopteran insect Caspase-5 was only more than 70%.

[0030] 3. Amino acid sequence alignment and protein functional domain prediction of SeCaspase-5

[0031] The amino acid sequences of SeCaspase-5 and other Lepidoptera Caspase-5 were further compared and analyzed using the ClustalOmega website (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo) and GeneDoc software. The protein functional domain of SeCaspase-5 was predicted using the SWISS-MODEL website (https: / / swissmodel.expasy.org / interactive). Figure 1 As shown in the figure. The full length of SeCaspase-5 contains 453 amino acid residues, including the N-terminal pro-domain, the C-terminal large subunit and the small subunit, which are separated by two cleavage sites. A death domain (caspase activation and recruitment domain, CARD) is predicted in the pro-domain of the protein, the large subunit contains a substrate binding site with the motif "SSHG" and an enzyme active site with the motif "QMCRG", and the small subunit contains a substrate binding site with the motif "RDGKLG".

[0032] Example 2: Effect of SeCaspase-5 on Baculovirus AcMNPV Infection

[0033] 1. Construction of SeCaspase-5 recombinant virus bacmid

[0034] The pMD18-T vector plasmid of SeCaspase-5 was double-digested with restriction endonucleases BamHI and EcoRI, and a green fluorescent protein tag (GFP) was fused to its C-terminus. The obtained gene fragment was ligated into the donor plasmid pFastBac of the Bac-to-Bac system (Invitrogen), and then the donor plasmid was further transformed into DH10Bac competent cells containing AcMNPV genome bacmid, and the recombinant virus bacmid was constructed by homologous recombination (the schematic diagram of bacmid construction is shown in Figure 2 A), and then the recombinant virus bacmid was extracted according to the Bac-to-Bac system manual and used as a template to generate the bacmid using the universal primer M13 / pUCR (5'-CGCCAGGGTTTTCCCAGTCAC-3') and the gene-specific full-length primer of SeCaspase-5 (5'-AAT GGATCCATGCAGAGAGAACACAGAGAAGC–3') was used for PCR amplification to identify whether the constructed recombinant virus bacmid was correct, and the recombinant virus bacmid carrying the GFP gene was used as a control. The results of PCR product electrophoresis analysis are shown in Figure 2 As shown in B, a GFP fragment of 2476 bp and a SeCaspase-5-GFP gene fragment of 3835 bp were obtained, respectively, indicating that the recombinant viral genome bacmid of SeCaspase-5 was constructed correctly and the correct recombinant viral genome SeCaspase-5 bacmid was identified.

[0035] 2. Analysis of cell apoptosis induced by SeCaspase-5 recombinant virus bacmid transfection

[0036] Spodoptera exigua Se-3 cells were inoculated into 6-well cell culture plates, with 1×10 6 After the cells adhered to the wall, the cells were transfected with the correctly constructed recombinant virus bacmids of GFP and SeCaspase-5 using the transfection reagent Cellfectin II. 4 μg of bacmid DNA was transfected into each well. At different time points after transfection (24, 48, 72, 96 hours post transfection, h pt), the fluorescence expression, cell morphology and the production of recombinant virus polyhedral OB in the cells were observed under a fluorescence microscope, and photos were taken. The results are shown in Figure 3 As shown, from 24 to 96 hours after transfection, cells transfected with GFP bacmid all expressed green fluorescence, and there was no apoptosis. Virus polyhedra OB (indicated by red arrows) were observed in all cells, and the number of OBs increased with the extension of infection time; while cells transfected with SeCaspase-5 bacmid all observed apoptosis (indicated by black arrows) from 24 to 72 hours after transfection, and virus polyhedra OB were observed in a small number of cells 72 hours after transfection. This indicates that the expression of SeCaspase-5 induces apoptosis.

[0037] 3. Analysis of insecticidal toxicity of SeCaspase-5 recombinant virus

[0038] 96 hours after the above-mentioned SeCaspase-5 recombinant virus bacmid transfection, the cell pellet was collected and the cells were broken by ultrasound to completely release the virus polyhedron OB therein, and the yield of the recombinant virus OB of different splicing bodies was counted using a hemocytometer. Then, the GFP recombinant virus polyhedron OB was used as a control to feed the third-instar larvae of Spodoptera exigua, and each larva was fed 1×10 6OBs were used to count the number of dead beet armyworm larvae every day and calculate the survival rate. Figure 4 As shown, the survival rate of larvae fed with SeCaspase-5 recombinant virus polyhedron OB was significantly lower than that of larvae fed with GFP recombinant virus polyhedron OB.

[0039] Taken together, these results indicate that overexpression of SeCaspase-5 can induce cell apoptosis during AcMNPV infection, which provides an effective way to improve the insecticidal toxicity of baculovirus during production.

Claims

1. A SeCaspase-5 gene from Spodoptera exigua, It is characterized in that The amino acid sequence of the protein encoded by the SeCaspase-5 gene is SEQ ID NO:

2.

2. The SeCaspase-5 gene according to claim 1, It is characterized in that The SeCaspase-5 gene has an open reading frame nucleotide sequence of SEQ ID NO:

1.

3. Use of the SeCaspase-5 gene according to claim 1 in the preparation of a preparation for improving the insecticidal toxicity of a virus against lepidopteran pests.

4. The use according to claim 3, It is characterized in that The preparation is a viral shuttle plasmid.

5. A viral shuttle plasmid, It is characterized in that The viral shuttle plasmid is constructed by inserting the nucleic acid fragment of the SeCaspase-5 gene described in claim 1 into the viral genome.

6. The viral shuttle plasmid according to claim 5, It is characterized in that The virus is a baculovirus.

7. A method for increasing the insecticidal toxicity of a virus against lepidopteran pests, It is characterized in that The method is to increase the expression level of the SeCaspase-5 gene described in claim 1 in the host cell to increase the insecticidal toxicity of the virus to lepidopteran pests.

8. The method according to claim 7, It is characterized in that The method is to transfer the shuttle plasmid of the virus according to claim 5 into a host cell.

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