Application of Transcription Factor SfGATAe in the Green Prevention and Control of Spodoptera frugiperda Population

Abstract

The present invention discloses the application of the transcription factor SfGATAe in the green prevention and control of the Spodoptera frugiperda population. The present invention discovers that overexpressing SfGATAe of Spodoptera frugiperda in the Sf9 cell line insensitive to the Cry1Ac toxin can significantly improve the sensitivity of Sf9 cells to the Cry1Ac toxin; interfering with the expression of SfGATAe in the larvae of Spodoptera frugiperda can significantly increase the resistance of the larvae of Spodoptera frugiperda to the Cry1Ac toxin. Therefore, the transcription factor SfGATAe of Spodoptera frugiperda can be applied to the green prevention and control of the Spodoptera frugiperda population.

Description

Technical Field

[0001] The present invention belongs to the fields of insect growth and development regulation and genetic engineering, and relates to the application of the transcription factor SfGATAe in the green prevention and control of the Spodoptera frugiperda population. Background Art

[0002] Bacillus thuringiensis (Bt), as a highly efficient microbial insecticide that is safe for humans and animals and environmentally friendly, has been widely used globally. The Bt toxin gene is transferred into crops as a target gene for transgenic plants, which can effectively reduce the use of chemically synthesized insecticides in crops such as corn, cotton, and soybeans, and promote the healthy development of modern agriculture. Spodoptera frugiperda is a major migratory polyphagous pest native to the tropics and subtropics of the Americas. It has a wide range of hosts, strong reproductive ability, and fast migration speed, making it difficult to control. It seriously threatens food security in countries around the world. At the end of January 2019, Spodoptera frugiperda invaded western Yunnan, China, and then continued to spread. Bt control is one of the important green prevention and control strategies for Spodoptera frugiperda. Transcriptional regulation is one of the key links in the expression of genetic information in living organisms, involving important physiological and biochemical processes such as insect metabolism regulation, differentiation and development, immune response, and signal transduction. GATA factors are a classic family of transcription factors that mediate the development, differentiation, and gene expression processes of insect midgut tissues and cells. Summary of the Invention

[0003] The object of the present invention is to provide the application of the important transcription factor SfGATAe of Spodoptera frugiperda in the green prevention and control of the Spodoptera frugiperda population.

[0004] The present invention discovers that overexpressing SfGATAe of Spodoptera frugiperda in the Sf9 cell line that is insensitive to Cry1Ac toxin can significantly improve the sensitivity of Sf9 cells to Cry1Ac toxin; interfering with the expression of SfGATAe in the larvae of Spodoptera frugiperda can significantly increase the resistance of Spodoptera frugiperda larvae to Cry1Ac toxin. Therefore, the transcription factor SfGATAe of Spodoptera frugiperda can be applied to the green prevention and control of the Spodoptera frugiperda population.

[0005] The object of the present invention is achieved by the following technical solutions:

[0006] A transcription factor SfGATAe related to Cry1Ac toxin resistance in Spodoptera frugiperda, whose open reading frame nucleotide sequence is as shown in SEQ ID NO.1.

[0007] The application of the transcription factor SfGATAe of Spodoptera frugiperda in regulating the sensitivity of Spodoptera frugiperda to Cry1Ac toxin.

[0008] The application of the transcription factor SfGATAe of Spodoptera frugiperda in the green prevention and control of the Spodoptera frugiperda population.

[0009] Application of Spodoptera frugiperda transcription factor SfGATAe in cultivating Spodoptera frugiperda varieties sensitive to Cry1Ac toxin.

[0010] Application of Spodoptera frugiperda transcription factor SfGATAe in preparing feed or green pesticide for improving the sensitivity of Spodoptera frugiperda to Cry1Ac toxin.

[0011] A feed or pesticide for improving the sensitivity of Spodoptera frugiperda to Cry1Ac toxin, comprising Spodoptera frugiperda transcription factor SfGATAe.

[0012] A pesticide for controlling Spodoptera frugiperda, comprising Spodoptera frugiperda transcription factor SfGATAe, Cry1Ac toxin, and a pharmaceutically acceptable excipient.

[0013] Advantages and beneficial effects of the present invention: The Spodoptera frugiperda SfGATAe transcription factor regulates the expression of the Bt toxin receptor gene in the insect body, thereby causing a change in the sensitivity of the insect body to the Bt toxin, providing an important theoretical and application reference for the green prevention and control of Spodoptera frugiperda. Description of the Drawings

[0014] Figure 1 It is a map of the insect cell expression vector of the constructed transcription factor SfGATAe.

[0015] Figure 2 It is a microscopic observation diagram of the cytopathic effect of Sf9 cells transfected with different plasmids treated with 10 μg / mL Cry1Ac activated toxin. Among them, A: The normal cell group not transfected with plasmid treated with 10 μg / mL Cry1Ac, and no cytopathic cells appeared; B: The transient transfection of the empty pIE2-EGFP-N1 plasmid group treated with 10 μg / mL Cry1Ac, and no cytopathic cells appeared; C: The transient transfection of the pIE2-SfGATAe-EGFP recombinant plasmid group treated with 10 μg / mL Cry1Ac, and 73.7% cytopathic cells appeared.

[0016] Figure 3 It is the detection of the expression level of the transcription factor SfGATAe in Spodoptera frugiperda injected with dsGFP and dsSfGATAe at 24 h, 48 h, 72 h, and 96 h by fluorescence quantitative PCR. The expression level of the SfGATAe gene in the interference SfGATAe group was down-regulated, and the detection effect was the best at 72 h.

[0017] Figure 4 It is the larval survival rate after selecting the late second instar larvae of Spodoptera frugiperda injected with dsGFP and dsSfGATAe and feeding them with 40 μg / g and 80 μg / g Cry1Ac toxic feed for seven days. Detailed Implementation Modes

[0018] The present invention will be specifically described below in conjunction with embodiments, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0019] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood to have the meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0020] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0021] Unless otherwise specifically mentioned, the molecular cloning methods, protein expression and purification methods, cell culture methods, and various detection methods mentioned in the following schemes are all traditional experimental methods and can be obtained by querying the literature; the relevant reagents used can be purchased from the corresponding reagent suppliers.

[0022] Example 1

[0023] 1. Construction of an insect expression recombinant vector for the SfGATAe transcription factor of Spodoptera frugiperda

[0024] The gene of the SfGATAe transcription factor of Spodoptera frugiperda was constructed into the expression vector pIE2-EGFP-N1 for insect cell lines (the construction of pIE2-EGFP-N1 can be seen in the applicant's prior patent application "An Insect Cell Expression System Vector Recombinant Plasmid and Its Preparation Method and Application", publication number CN114540388A), and the fusion plasmid pIE2-SfGATAe-EGFP with characteristics such as the Opie2 promoter, Corzak sequence, SfGATAe target gene, EGFP fluorescent tag, and KanR tag was obtained. Its plasmid map is as Figure 1 shown, and the specific construction process is as follows.

[0025] (1) Extraction of total RNA from insect tissues

[0026] Freeze the insects in good living condition at low temperature for 5 minutes. Dissect about 3 insect larvae, take out the midgut tissue, wash the midgut with PBS buffer and place it in a clean RNase-free EP tube. Add 1 mL of Trizol solution and grind the tissue thoroughly with a homogenizer at low temperature for a short time. After grinding, set it aside for use. Centrifuge at 4°C in a refrigerated high-speed centrifuge at a speed of 12,000×g for 5 minutes. Use a pipette to transfer the supernatant to another clean EP tube and discard the precipitate. Add 200 μL of chloroform to the EP tube with the supernatant and gently mix with a pipette. Incubate the mixture in an ice bath for 10 - 15 minutes. Centrifuge again at 4°C in a refrigerated high-speed centrifuge at a speed of 12,000×g for 15 minutes. The liquid in the EP tube will show a layered phenomenon. Carefully pipette the upper aqueous phase into another clean EP tube, and do not pipette the middle or lower layer (discard). Mix 500 μL of isopropanol into the taken aqueous phase EP tube, gently mix by inverting up and down, and let it stand at room temperature for 10 minutes. Centrifuge at 4°C in a refrigerated high-speed centrifuge at a speed of 12,000×g for 10 minutes, discard the supernatant, and there is a white RNA precipitate at the bottom of the EP tube. Add 1000 μL of 75% cold ethanol, gently shake to mix the RNA precipitate. It may be found that the white precipitate is insoluble in ethanol, but do not blow it away with a pipette. The purpose of this process is to remove the residual isopropanol. Centrifuge at 4°C and 8000×g for 5 minutes in a refrigerated high-speed centrifuge, discard the supernatant, and the white RNA precipitate reappears at the bottom of the tube. Open the lid of the EP tube and dry it at room temperature for 5 - 10 minutes to volatilize the residual ethanol, but do not over-dry. Add 20 - 50 μL of RNase-free water to the EP tube to dissolve the RNA precipitate at the bottom of the tube. Take a small amount of the sample to measure the RNA concentration. The RNA solution can be used immediately or stored at -80°C.

[0027] (2) Synthesis of cDNA from Spodoptera frugiperda larvae

[0028] For the gDNA elimination reaction, refer to the TaKaRa PrimeScript RT reagent kit with gDNA Eraser instruction manual. The genomic DNA removal system: 1.0 μL of gDNA Eraser, 2.0 μL of 5×gDNA Eraser buffer, about 1 μg / mL of total RNA, and make up to a total volume of 10.0 μL with RNase Free water. Incubate the whole system in a 42°C water bath for 30 minutes. The reaction mixture can be used immediately or stored at 4°C.

[0029] Obtaining cDNA by RNA reverse transcription: Take 10.0 μL of the above total reaction system, add 24.0 μL of 5×PrimeScript Buffer, 1.0 μL of PrimeScript RT Enzyme MixⅠ, 4.0 μL of RT Primer Mix, and 1.0 μL of RNase Free water to make the total reaction system 20 μL. Place the reaction system in a water bath at 37 °C for 15 min; then place it in a water bath at 85 °C for 5 s to inactivate the enzyme; the obtained cDNA after the reaction is reserved for use or aliquoted into clean PCR tubes and stored at -20 °C.

[0030] (3) Obtaining the pIE2-SfGATAe-EGFP recombinant plasmid by seamless cloning using homologous recombination method

[0031] Summarized according to the pEASY-Uni Seamless Cloning and Assembly Kit instruction manual as follows:

[0032] Amplification of the target gene: Using SEQ ID NO.2 and SEQ ID NO.3 as primers, and Spodoptera frugiperda cDNA as the template to amplify the target gene (length 182 bp), obtaining a target gene fragment with vector repeat sequences with a length of 1887 bp (annealing temperature is 52 °C). The amplified target gene is purified using a Gel Extraction Kit (Omega Bio-tek, Inc., GA, USA) and reserved for use.

[0033] SEQ ID NO.2: 5’-AGATCTCGAGCTCAAGCTTCGGCCACCATGGAGAACGTGGCTCA GATGGAGC-3’,

[0034] SEQ ID NO.3: 5’-GGTGGCGACCGGTGGATCACCTCCGCCACCGCCTCCGCGCTGGTACCCCGCCAGC-3’.

[0035] Linearization of the vector: Use primers SEQ ID NO.4 and SEQ ID NO.5 to amplify the vector to obtain a linearized vector. Amplify the pIE2-EGFP-N1 plasmid vector (full length 4709 bp) to obtain a 4678 bp fragment (annealing temperature is 60 °C). The linearized vector is purified using a Gel Extraction Kit (Omega Bio-tek, Inc., GA, USA) and reserved for use.

[0036] SEQ ID NO.4: 5'-GATCCACCGGTCGCCACC-3',

[0037] SEQ ID NO.5: 5'-CGAAGCTTGAGCTCGAGATCT-3'.

[0038] Homologous recombination: The obtained linearized vector and the target gene with the repeated sequence of the linearized vector were purified and ligated according to the homologous recombination seamless cloning kit, and then transformed into competent Escherichia coli DH5α. Positive clones were picked to obtain the recombinant plasmid pIE2-SfGATAe-EGFP. The specific steps can be referred to -Uni Seamless Cloning and Assembly Kit (TransGen Biotech Co., Ltd., Beijing) for the detailed steps of the kit.

[0039] 2. Transfection of the recombinant plasmid pIE2-SfGATAe-EGFP into insect cells

[0040] (1) SF9 cells in good growth state were inoculated into a clean 48-well cell culture plate and cultured overnight in Grace's medium (Grace's Insect cell culture medium) containing 8.0% fetal bovine serum in a constant temperature incubator at 28.0 °C. When the density reached 60% - 80%, the cells were ready for transfection.

[0041] (2) The wild-type plasmid pIE2-SfGATAe-EGFP and the empty plasmid pIE2-EGF-N1 used for transfection were extracted aseptically using a plasmid extraction kit. After extraction, their concentration should be above 200 ng / μL, and the purity (OD 260 / OD 280 ) should be between 1.75 and 1.85.

[0042] (3) Prepare serum-free Grace's medium, 1000 μL, 200 μL, 10 μL pipettes and corresponding pipette tips of appropriate sizes, 1.5 mL clean EP tubes, an alcohol lamp and alcohol cotton, and place them in a biosafety cabinet for ultraviolet sterilization for about 30 min.

[0043] (4) Take 25 μL of serum-free Grace's medium into a sterile 1.5 mL EP tube, add 1.0 μL of FuGENE HD, and mix in 0.25 μg of plasmid (the volume ratio of the transfection reagent to the plasmid mass is preferably in the range of 3.0:1.0 - 6.0:1.0).

[0044] (5) Gently mix the mixture added to a 1.5 mL EP tube, let it stand in the biosafety cabinet for 20 min, and add 95 μL of serum-free Grace's to the above mixture system to make the total volume 120 μL.

[0045] (6) Discard the medium in the 48-well plate, wash it three times with serum-free Grace's medium, and add the reaction solution in step (5) to the washed 48-well plate and mix well.

[0046] (7) Place the transfected 48-well plate in a cell incubator at 28.0 °C and incubate for 3 - 5 h. Discard the reaction solution in the 48-well plate and add 120 μL of Grace's medium containing 8.0% fetal bovine serum.

[0047] (8) After changing the medium, place the 48-well cell culture plate in a cell incubator at 28.0 °C and culture for 24 - 48 h.

[0048] 3. Determination of the virulence of SF9 cells mediated by transfection with the pIE2-SfGATAe-EGFP transcription factor recombinant plasmid

[0049] (1) After 48 h of transfection with the plasmid, take out the 48-well plate from the incubator for subsequent toxin treatment experiments.

[0050] (2) Dilute the Cry1Ac toxin by the two-fold dilution method with PBS solution. Be sure to add the correct amount and mix evenly during the dilution process to prepare a toxin solution with a concentration of 10 μg / mL.

[0051] (3) Aspirate the medium in the 48-well plate, wash it 3 times with Pucks solution, and then add 150 μL of the 10 μg / mL toxin solution to each well.

[0052] (4) Take a photo using an eyepiece-free inverted microscope 1 h after adding the toxin.

[0053] (5) Treat the transfected SF9 with the toxin for 1 h, take photos and record under a fluorescence inverted microscope, record the number of lesions (cell swelling, enlargement, and rounding are considered lesions) under white light, and record the number of cells with green fluorescent tag protein as the total number of cells under fluorescence. Calculate the lesion rate, that is, the cell lesion rate = number of lesions / total number of cells × 100%.

[0054] (6) Set three replicates and count the lesion rate.

[0055] (7) By Figure 2It can be seen that under the treatment of 10 μg / mL Cry1Ac toxin, there were no diseased cells in the untransfected group and the group transfected with the empty plasmid pIE2-EGF-N1 after 1 h. The lesion rate of Sf9 cells mediated by transfection with pIE2-SfGATAe-EGFP increased extremely significantly (p < 0.01). Sf9 cells mediated by the pIE2-SfGATAe-EGFP recombinant protein were treated with different concentrations of Cry1Ac toxin. The cell lesion rate was 14.07% when the Cry1Ac toxin concentration was 2 μg / mL, 2.58% when the Cry1Ac toxin concentration was 4 μg / mL, 61.14% when the Cry1Ac toxin concentration was 8 μg / mL, 83.43% when the Cry1Ac toxin concentration was 16 μg / mL, and 95.51% when the Cry1Ac toxin concentration was 32 μg / mL. The EC 50 value of different concentrations of Cry1Ac toxin on pIE2-SfGATAe-EGFP-mediated Sf9 cells was calculated to be 6.213 μg / mL. The results showed that after the overexpression of the SfGATAe transcription factor in Sf9 cells, the sensitivity of these cells to Cry1Ac toxin was enhanced.

[0056] 4. Detection of changes in the expression level of the transcription factor SfGATAe by fluorescence quantitative PCR

[0057] (1) dsRNA was synthesized according to the sequence of the transcription factor SfGATAe for interfering with the conserved region with a length of 518 bp. Primers containing the T7 promoter sequence were designed according to the conserved region sequence of the target gene. Using the cDNA of the third-instar larvae of Spodoptera frugiperda as a template, the conserved region DNA fragment of about 518 bp was amplified by PCR. Using this linear DNA as a template, dsRNA of the target gene was synthesized in vitro (the specific method was operated according to the product manual of T7 RiboMAXTM Express RNAi System), and the concentration was measured to be about 1500 ng / μL.

[0058] The primers for synthesizing the interfering control group GFP gene dSRNA are as follows:

[0059] SEQ ID NO.6: 5’-TAATACGACTCACTATAGGG ACCTACGGCAAGCTGACCC-3’,

[0060] SEQ ID NO.7: 5’-TAATACGACTCACTATAGGG CTCGATGTTGTGGCGGATC-3’.

[0061] The primers for synthesizing the interfering target gene SfGATAe conserved region dSRNA are as follows:

[0062] SEQ ID NO.8: 5’-TAATACGACTCACTATAGGGACAAAGTGGGAGGGGAGCAC-3’, SEQ ID NO.9: 5’-TAATACGACTCACTATAGGGCTTCCTCGTCTGGATGCCGTC-3’.

[0063] (2) Select second instar late-stage Spodoptera frugiperda larvae with roughly the same growth rate and anesthetize them on ice for 30 min.

[0064] (3) Conduct microinjection between the second-to-last and third-to-last abdominal legs of the larvae, with a dsRNA injection volume of 2 μg / larva.

[0065] Inject dsRNA interfering with the EGFP gene as the control group, and inject dsRNA interfering with SfGATAe as the experimental group. Inject 10 larvae for each treatment. At 24 h, 48 h, 72 h, and 96 h after injection, respectively, extract the total RNA from the midgut of 3 larvae by dissection, obtain cDNA through reverse transcription, and detect the interference efficiency of the target gene by fluorescence quantitative PCR.

[0066] Use the following primers as the internal reference detection primers for Spodoptera frugiperda GAPDH:

[0067] SEQ ID NO.10: 5’-AGATCGCTGTCTTCTGCGAG-3’,

[0068] SEQ ID NO.11: 5’-CAGACGCCTTCTCTGTGGTT-3’.

[0069] Use the following primers as the detection primers for Spodoptera frugiperda SfGATAe:

[0070] SEQ ID NO.12: 5’-GTACAAAGTGGGAGGGGAGC-3’,

[0071] SEQ ID NO.13: 5’-CGTCGTCGATGGGTAGAAGG-3’.

[0072] The results are as Figure 3 shown. After interfering with the SfGATAe gene for 24 h, 48 h, 72 h, and 96 h, its expression level was downregulated to 80.65% ± 3.43 (p < 0.01), 70.49% ± 2.79 (p < 0.01), 54.71% ± 3.83 (p < 0.01), and 85.47% ± 2.62 (p > 0.05) compared with the GFP control group, respectively. The SfGATAe was significantly downregulated after 72 hours of interference, and the interference efficiency was the highest. Therefore, the larvae after 72 h of interference were selected for the following toxicity experiment.

[0073] 5. Detection of the sensitivity of Spodoptera frugiperda population to toxins after interfering with the transcription factor SfGATAe

[0074] (1) Place Spodoptera frugiperda in an HP3000GS-C type intelligent artificial climate chamber to restore it to the best state and make the insects adapt to the indoor environment of the artificial climate chamber. Control the parameters of the artificial climate chamber. The feeding conditions are a temperature of 28.0 ± 0.5 °C, a light cycle ratio of 16L:8D, and a humidity of 60 ± 5%.

[0075] (2) Collect second-instar larvae of Spodoptera frugiperda with consistent instars and physiological states, place them in a clean 24-well plate, and conduct starvation treatment for 4 - 6 h.

[0076] (3) Take the prepared insect feed and place it in a DHG-9123A type electrothermal constant temperature forced air drying oven for 20 - 30 min. Add the dissolved Cry1Ac protoxin to the feed and stir evenly to prepare toxic feeds of 40 μg / g and 80 μg / g.

[0077] (4) Inject dsRNA interfering with the EGFP gene as the control group, and inject dsRNA interfering with SfGATAe as the experimental group. Inject 60 larvae for each treatment.

[0078] (5) After interfering with the late second-instar larvae for 72 h, take the toxic feeds of 40 μg / g and 80 μg / g and feed 30 larvae of the experimental group and the control group that have undergone starvation treatment respectively. Each treatment is repeated three times.

[0079] (6) Count the number of surviving insects in each group every day for 7 days. Check the death situation of the insects at the same time, and record the survival rates of the insects in the experimental group and the control group.

[0080] (7) Calculate the corrected survival rate of the treatment group. Corrected survival rate = (number of surviving insects in the treatment group - average number of dead insects in the control group) / (total number of insects in the treatment group - average number of dead insects in the control group) × 100%. The corrected survival rate is recorded as the survival rate.

[0081] The results are shown in Figure 4 . The survival rates of the control group after feeding toxins of 40 μg / g and 80 μg / g for 7 days are 37.78% ± 6.85 and 11.11% ± 4.16 respectively. While the survival rates of the group interfering with SfGATAe after 7 days with 40 μg / g and 80 μg / g are 88.89% ± 5.67 and 72.22% ± 8.31 respectively, which are significantly higher than those of the control group (p < 0.01).

Claims

1. Use of a transcription factor SfGATAe in regulating the sensitivity of Spodoptera frugiperda to Cry1Ac toxin, characterized in that: The nucleotide sequence of the open reading frame of the transcription factor SfGATAe is shown as SEQ ID NO.

1.

2. Application of a transcription factor SfGATAe in the green prevention and control of the Spodoptera frugiperda population, characterized in that: The nucleotide sequence of the open reading frame of the transcription factor SfGATAe is shown as SEQ ID NO.

1.

3. Use of a transcription factor SfGATAe in cultivating a variety of Spodoptera frugiperda sensitive to Cry1Ac toxin, characterized in that: The nucleotide sequence of the open reading frame of the transcription factor SfGATAe is shown as SEQ ID NO.1.

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