Recombinant insect baculovirus capable of efficiently killing spodoptera frugiperda and application of recombinant insect baculovirus

Abstract

The invention belongs to the technical field of insect biological control, and particularly relates to a recombinant insect baculovirus PH-SVT / AC capable of efficiently killing spodoptera frugiperda and application of the recombinant insect baculovirus PH-SVT / AC. The recombinant insect baculovirus PH-SVT / AC is preserved in the China General Microbiological Culture Collection Center on August 7, 2024, the preservation number is CGMCC No.46096, the preservation address is No.3, courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the recombinant insect baculovirus is classified and named as an autographa californica nuclear polyhedrosis virus. The recombinant insect baculovirus PH-SVT / AC has a remarkable prevention and control effect on the spodoptera frugiperda, and has a good application prospect in biological prevention and control of the spodoptera frugiperda.

Description

Technical Field

[0001] The invention belongs to the technical field of insect biological control, and specifically relates to a recombinant insect baculovirus PH-SVT / AC capable of efficiently killing fall armyworm and an application thereof. Background Art

[0002] Spodopterafrugiperda (JESmith), also known as "Fall Armyworm", belongs to the Noctuidae family of the Lepidoptera order. It has the characteristics of a wide host range, high reproductive capacity, strong migration ability, rapid development of pesticide resistance, wide suitable habitat, and serious sudden damage. Spodopterus frugiperda is native to the subtropical and tropical regions of America. At the end of 2018, Spodopterus frugiperda invaded Yunnan, my country from Myanmar. As of September 2022, Spodopterus frugiperda has accumulated 35.31 million mu in 846 counties in 27 provinces in my country (https: / / www.natesc.org.cn), posing a huge threat to my country's food security.

[0003] The fall armyworm has a wide host range and can feed on more than 353 kinds of plants. It likes to eat corn. One generation lasts about 30 days (28℃), which can be extended to 60-90 days in colder months. The egg stage is 3-4 days, and the larvae have 6 instars. The younger larvae gather together to cause damage, and the older ones disperse. After the 4th instar, they enter a period of gluttony and show self-mutilation habits. The mature larvae pupate in the soil, and the pupal stage is 7-37 days. The average lifespan of adults is about 10 days, and the reproductive organs can only mature after nutrition is supplemented. After the fall armyworm damages corn, the yield loss of corn is 15% to 73%.

[0004] Autographa california multiple nuclear polyhedrosis virus (AcMNPV) is a model virus of the genus Baculovirus and has been widely used in recombinant protein expression and biological pest control. AcMNPV can infect more than 50 species of Lepidoptera, and insecticides based on AcMNPV have been developed as commercial products in many countries. The site of action of AcMNPV infecting Lepidoptera larvae is the midgut. After being orally ingested by Lepidoptera larvae, its polyhedron acts in the midgut and enters the insect body fluids after passing through the two barriers of the peritrophic membrane and the basement membrane. Therefore, AcMNPV has the potential to be used as a microbial insecticide to control fall armyworm, but AcMNPV has problems such as slow insecticidal speed and low infectivity to the host.

[0005] Bacillus thuringiensis (Bt) is a Gram-positive (G+) bacterium belonging to the Bacillaceae family and the genus Bacillus cereus. It is a naturally occurring insect pathogenic bacterium. As a microbial pesticide with high ecological safety, Bt has been widely used in the world. The reason why Bt can be used as a biological insecticide to control agricultural and forestry pests is that it can form insecticidal proteins that specifically kill insects, including insecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (VIP). ICPs play an important role in the biological control of harmful insects, but due to their long-term and large-scale use, many insects have developed strong resistance to them. VIP proteins usually do not exist in crystalline form, but compared with Cry proteins, VIP proteins have a wide insecticidal spectrum. Summary of the invention

[0006] In order to give full play to the advantages of AcMNPV and VIP in killing fall armyworm, the present invention discloses a recombinant insect baculovirus PH-SVT / AC that can efficiently kill fall armyworm and its application. Experiments have shown that after expressing the Vip3A gene, the toxicity of AcMNPV to fall armyworm larvae is significantly improved, and the expression of the Vip3A gene on the surface of the virus further improves the killing effect of the virus on fall armyworm larvae. The recombinant insect baculovirus PH-SVT / AC has a significant control effect on fall armyworm and has a good application prospect in the biological control of fall armyworm.

[0007] The technical solution adopted by the present invention is as follows: A recombinant insect baculovirus PH-SVT / AC that can effectively kill fall armyworm, wherein the recombinant insect baculovirus PH-SVT / AC has been deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on August 7, 2024, with a deposit number of CGMCC No.46096, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Autographa california multiple nuclear polyhedrosisvirus; The genome of the recombinant insect baculovirus PH-SVT / AC contains the ph gene and the svt gene, the nucleotide sequence of the ph gene is shown in SEQ ID NO.2, and the nucleotide sequence of the svt fusion gene is shown in SEQ ID NO.3; The svt gene is formed by inserting the signal peptide sp sequence of the baculovirus envelope glycoprotein gp64 into the N-terminal of the Vip3A gene sequence and fusion with the structural domain tmd sequence inserted into the C-terminal. The nucleotide sequence of the Vip3A gene is shown in SEQ ID NO.1.

[0008] A method for preparing the above-mentioned recombinant insect baculovirus PH-SVT / AC comprises the following steps: 1) cloning the ph gene into the pFastBac Dual vector to obtain the transfer vector pFastBac-Dual-ph; the nucleotide sequence of the ph gene is shown in SEQ ID NO.2; 2) cloning the svt gene into the transfer vector pFastBac-Dual-ph to obtain the transfer vector pFastBac-Dual-ph-svt; the nucleotide sequence of the svt gene is shown in SEQ ID NO.3; 3) The transfer vector pFastBac-Dual-ph-svt was transformed into DH10Bac to obtain the recombinant Bacmid plasmid; 4) The recombinant Bacmid plasmid was transfected into Sf9 insect cells to obtain the recombinant insect baculovirus PH-SVT / AC expressing Vip3A protein on the surface.

[0009] Use of the above-mentioned recombinant insect baculovirus PH-SVT / AC in controlling fall armyworm; The application is: feeding the recombinant insect baculovirus PH-SVT / AC to the fall armyworm.

[0010] The use of the above-mentioned recombinant insect baculovirus PH-SVT / AC in the preparation of a preparation for controlling fall armyworm.

[0011] A preparation for controlling fall armyworm, comprising the above-mentioned recombinant insect baculovirus PH-SVT / AC.

[0012] A method for enhancing the toxicity of Autographa californica nuclear polyhedrosis virus to fall armyworm, the method comprising: recombining the Vip3A gene into the Autographa californica nuclear polyhedrosis virus genome, thereby improving the toxicity of Autographa californica nuclear polyhedrosis virus to fall armyworm; the nucleotide sequence of the Vip3A gene is shown in SEQ ID NO.1; Preferably, the method comprises: expressing the Vip3A protein on the surface of the Autographa californica nuclear polyhedrosis virus; the Vip3A protein is encoded by the Vip3A gene, and the nucleotide sequence of the Vip3A gene is shown in SEQ ID NO.1.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes the advantages of AcMNPV and VIP in killing fall armyworm, expresses VIP on the surface of AcMNPV, and successfully obtains the recombinant insect baculovirus PH-SVT / AC that can effectively kill fall armyworm. The half lethal concentration LC of the recombinant insect baculovirus PH-SVT / AC to fall armyworm is 50 1.065×10 6 PIB / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Schematic diagram of the construction of recombinant Bacmid plasmid. A: ph / AcBac; B: ph-vip / AcBac; C: ph-svt / AcBac.

[0015] Figure 2 :Pathological changes of sf9 cells after transfection of recombinant Bacmid plasmid. A: normal sf9 cells; B: sf9 cells transfected with ph / AcBac; C: sf9 cells transfected with ph-vip / AcBac; D: sf9 cells transfected with ph-svt / AcBac.

[0016] Figure 3 : Morphology of dead fall armyworm larvae after feeding recombinant insect baculovirus. A: fed with sterile water (CK); B: fed with PH / AC; C: fed with PH-VIP / AC; D: fed with PH-SVT / AC. DETAILED DESCRIPTION

[0017] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0018] The wild-type Autographacalifornia multiplenuclear polyhedrosis virus (AcMNPV) and pFastBac-Dual vector bacteria used in the following examples are preserved in the laboratory of the inventor of this patent. The fall armyworm egg masses used in the following examples were collected from corn fields in the suburbs of Fuzhou City, Fujian Province, and artificially raised and bred for more than 10 generations indoors to establish a laboratory population. The breeding conditions are: photoperiod L:D=16:8, relative humidity (70±5)%, temperature (26±1)℃, and light intensity 18000lx; larvae were raised in potted corn in 40cm×40cm×40cm insect cages.

[0019] Example 1: Obtaining recombinant insect baculovirus PH / AC, PH-VIP / AC, PH-SVT / AC 1. Construction of transfer vectors pFastBac-Dual-ph, pFastBac-Dual-ph-vip, and pFastBac-Dual-ph-svt The primer pair Ac-PH FW: 5'-ATGCCGGATTATTCATACCGTC-3' and Ac-PH RW: 5'-TTAATACGCCGGACCAGTGAAC-3' for amplifying the ph gene (SEQ ID NO.2) was designed, and the target fragment was cloned from the wild-type AcMNPV by PCR. The amplified fragment was cloned into the vector pMD19-T. After sequencing identification, the ph gene was further cloned into the pFastBac-Dual vector to obtain the transfer vector pFastBac-Dual-ph.

[0020] The Vip3A gene (SEQ ID NO.1) was artificially chemically synthesized, and XhoI and NheI restriction sites were introduced at the 5' and 3' ends, respectively. The Vip3A gene was then cloned into the transfer vector pFastBac-Dual-ph and double enzyme digestion was performed for identification. The results showed that a fragment consistent with the expected size appeared, indicating that the Vip3A gene was successfully cloned into the transfer vector pFastBac-Dual-ph, and the transfer vector pFastBac-Dual-ph-vip was obtained.

[0021] The Vip3A gene sequence (SEQ ID NO. AAC37036) and the signal peptide (sp) and transmembrane domain (tmd) sequences of AcMNPV GP64 were artificially synthesized, and the gene fragments sp, Vip3A and tmd were sequentially connected by overlapping PCR to form a fusion gene svt (SEQ ID NO. 3). -Uni Seamless Cloning and Assembly Kit (Uni Seamless Cloning and Assembly Kit) was used to clone the fusion gene svt into the transfer vector pFastBac-Dual-ph to obtain the transfer vector pFastBac-Dual-ph-svt.

[0022] 2. Obtaining recombinant insect baculovirus PH / AC, PH-VIP / AC, PH-SVT / AC The transfer vectors pFastBac-Dual-ph, pFastBac-Dual-ph-vip, and pFastBac-Dual-ph-svt were transformed into DH10Bac competent cells, respectively. After 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline and blue-white screening, single colonies were picked for propagation, and Bacmid DNA was extracted. After PCR identification, recombinant Bacmids containing ph gene, ph gene and Vip3A gene, ph gene and svt gene were obtained and named ph / AcBac, ph-vip / AcBac, and ph-svt / AcBac, respectively. Figure 1 ).

[0023] The recombinant Bacmid DNA was transfected into sf9 cells. At 48h after transfection, the cells began to grow and stopped growing. At 72h, the cells began to fall off. >96h, most cells fell off and began to lyse. A large number of polyhedral particles were seen in the sf9 cells transfected with the three Bacmids. After the sf9 cells were infected with the recombinant virus expressing the Vip3A gene, many cells fused. This indicates that the recombinant insect baculovirus was successfully obtained, which was named PH / AC, PH-VIP / AC, PH-SVT / AC ( Figure 2 ).

[0024] The recombinant insect baculovirus PH-SVT / AC was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on August 7, 2024, with the deposit number CGMCC No.46096. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named Autographa california multiple nuclear polyhedrosis virus.

[0025] Example 2: Pathogenicity of recombinant insect baculoviruses PH / AC, PH-VIP / AC, and PH-SVT / AC to fall armyworm larvae 1. Pathogenicity of recombinant insect baculovirus PH / AC, PH-VIP / AC, and PH-SVT / AC The supernatants of Sf9 cells infected with recombinant insect baculoviruses PH / AC, PH-VIP / AC, and PH-SVT / AC were collected respectively. Healthy 5th instar larvae of Spodoptera frugiperda were selected, disinfected with 70% alcohol, and injected with the virus at the intersegmental membrane. Then, the larvae were placed at 25°C for 4-5 days, and the hemolymph was collected to extract the virus polyhedron. The virus polyhedron suspension was prepared at a concentration of 10 8PIBs / ml. Cut corn leaves with the same thickness and relatively flat surface into square pieces with a length and width of 3 cm, immerse the cut leaves in the virus suspension for 10 seconds (the blank control is immersed in sterile water), take them out and feed the 5th instar larvae of fall armyworm after the surface moisture is slightly dry. From the beginning of virus feeding, the midgut tissue of the 5th instar larvae of fall armyworm was taken every 24 hours for 7 consecutive days to observe the pathological changes of the intestine.

[0026] The results showed that on the third day after virus inoculation, some third-instar larvae of S. frugiperda showed typical AcMNPV infection symptoms; on the fifth day, most third-instar larvae of S. frugiperda turned black and died. The symptoms of death of the third-instar larvae of S. frugiperda infected with the three recombinant insect baculoviruses were similar ( Figure 3 ).

[0027] 2. Toxicity of recombinant insect baculovirus PH / AC, PH-VIP / AC, and PH-SVT / AC to fall armyworm larvae. The third instar larvae of fall armyworm were fed with different concentration gradients of recombinant insect baculovirus PH / AC, PH-VIP / AC, and PH-SVT / AC. It was found that when the concentration of the virus was lower than 10 8 PIB / ml, with the increase of virus concentration, the mortality rate of the three recombinant viruses to the third instar larvae of Spodoptera frugiperda increased significantly (p < 0.05). 8 PIB / ml, it began to approach the peak value, and the concentration was higher than 10 8 When PIB / ml, the mortality rates of PH / AC and PH-VIP / AC were not much different, about 80%. Compared with the first two, the mortality rate of recombinant insect baculovirus PH-SVT / AC on the third instar larvae of Spodoptera frugiperda was significantly increased (p < 0.05), about 90%.

[0028] The number of larval deaths was counted and the LC of the virus was calculated using SPSS22.0 50 and LC 90 The results showed that the LC of recombinant insect baculovirus PH / AC, PH-VIP / AC, PH-SVT / AC 50 They are 1.008×10 7 PIB / ml, 3.44×10 6 PIB / ml, 1.065×10 6 PIB / ml, LC 90 They are 8.701×10 9 PIB / ml, 5.212×10 9 PIB / ml, 4.408×10 8 PIB / ml (Table 1). The virulence of the recombinant insect baculovirus expressing the Vip3A gene was significantly enhanced, and the LC of PH-VIP / AC50 1 / 3 of PH / AC, LC 90 Slightly lower than PH / AC, the lethality rate is about 1 day earlier; LC of PH-SVT / AC 50 1 / 10 of PH / AC, LC 90 The rate of death was about 2 days earlier than that of PH / AC, which indicated that the toxicity of AcMNPV to fall armyworm larvae was significantly enhanced after the expression of Vip3A gene, and the expression of Vip3A gene on the surface of the virus further enhanced the killing effect of the virus on fall armyworm larvae.

[0029] Table 1 Virulence determination of recombinant insect baculovirus against the third instar larvae of Spodoptera frugiperda The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A recombinant insect baculovirus PH-SVT / AC that can effectively kill fall armyworm, characterized in that: The recombinant insect baculovirus PH-SVT / AC was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 7, 2024, with a deposit number of CGMCC No.46096 and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. It was classified and named Autographa californica nuclear polyhedrosis virus ( Autographa california multiplenuclearpolyhedrosis virus).

2. The recombinant insect baculovirus PH-SVT / AC according to claim 1, characterized in that: The genome of the recombinant insect baculovirus PH-SVT / AC contains ph Gene, svt Gene, ph The nucleotide sequence of the gene is shown in SEQ ID NO.

2. svt The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. The recombinant insect baculovirus PH-SVT / AC according to claim 2, characterized in that: Said svt Gene in Vip3A The N-terminal of the gene sequence is inserted into the signal peptide sp sequence of the baculovirus envelope glycoprotein gp64 and the C-terminal is inserted into the transmembrane domain tmd sequence to fuse. Vip3A The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. A method for preparing the recombinant insect baculovirus PH-SVT / AC according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) ph The gene was cloned into the pFastBac Dual vector to obtain the transfer vector pFastBac-Dual-ph; ph The nucleotide sequence of the gene is shown in SEQ ID NO.2; 2) svt The gene was cloned into the transfer vector pFastBac-Dual-ph to obtain the transfer vector pFastBac-Dual-ph-svt; Said svt The nucleotide sequence of the gene is shown in SEQ ID NO.3; 3) The transfer vector pFastBac-Dual-ph-svt was transformed into DH10Bac to obtain the recombinant Bacmid plasmid; 4) The recombinant Bacmid plasmid was transfected into Sf9 cells to obtain the recombinant insect baculovirus PH-SVT / AC expressing Vip3A protein on the surface.

5. Use of the recombinant insect baculovirus PH-SVT / AC according to any one of claims 1 to 3 in controlling fall armyworm.

6. The use according to claim 5, characterized in that: The recombinant insect baculovirus PH-SVT / AC was fed to Spodoptera frugiperda.

7. Use of the recombinant insect baculovirus PH-SVT / AC according to any one of claims 1 to 3 in the preparation of a formulation for controlling fall armyworm.

8. A preparation for controlling fall armyworm, characterized in that: The preparation comprises the recombinant insect baculovirus PH-SVT / AC according to any one of claims 1 to 3.

9. A method for enhancing the toxicity of Autographa californica nuclear polyhedrosis virus to Spodoptera frugiperda, characterized in that: Will Vip3A The gene is recombined into the genome of the Autographa californica nuclear polyhedrosis virus, thereby increasing the toxicity of the Autographa californica nuclear polyhedrosis virus to the fall armyworm; Vip3A The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that: The Vip3A protein is expressed on the surface of Autographa californica nuclear polyhedrosis virus; the Vip3A protein is Vip3A The gene encodes Vip3A The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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