CN116103301A - Application of transcription factor SfGATAe in green prevention and control of spodoptera frugiperda population - Google Patents

Abstract

The invention discloses application of a transcription factor SfGATAe in green prevention and control of spodoptera frugiperda populations. The invention discovers that the Sf9 cell line insensitive to Cry1Ac toxin overexpresses Spodoptera frugiperda SfGATAe, and can obviously improve the sensitivity of Sf9 cells to Cry1Ac toxin; the expression of SfGATAe is interfered in spodoptera frugiperda larvae, so that the resistance of the spodoptera frugiperda larvae to Cry1Ac toxin can be obviously increased, and therefore, the spodoptera frugiperda transcription factor SfGATAe can be applied to green prevention and control of spodoptera frugiperda populations.

Description

Application of transcription factor SfGATAe in green prevention and control of spodoptera frugiperda population

Technical Field

The invention belongs to the field of insect growth and development regulation and genetic engineering, and relates to application of a transcription factor SfGATAe in green prevention and control of spodoptera frugiperda population.

Background

Bacillus thuringiensis (Bt) is widely used worldwide as a safe, environment-friendly, high-efficiency microbial pesticide for humans and animals. The Bt toxin gene is used as a target gene of transgenic plants to be transferred into crops, so that the use of chemical synthetic pesticides in crops such as corn, cotton, soybean and the like can be effectively reduced, and the healthy development of modern agriculture is promoted. Spodoptera frugiperda (Spodoptera frugiperda) is a major migratory omnivorous pest native to tropical and subtropical areas in america, and has the advantages of wide hosts, strong reproductive capacity, high migration speed, high prevention and control difficulty and serious threat to grain safety of countries around the world. Spodoptera frugiperda invades the western part of Yunnan in China at the end of month 1 in 2019, and then continuously spreads. Bt prevention and control is one of important green prevention and control strategies for preventing spodoptera frugiperda. Transcriptional regulation is one of key links of genetic information expression of living organisms, and relates to important physiological and biochemical processes such as insect metabolism regulation, differentiation and development, immune response, signal transduction and the like. GATA factors are a classical family of transcription factors that mediate the processes of development, differentiation and gene expression of intestinal tissues and cells in insects.

Disclosure of Invention

The invention aims to provide application of an important transcription factor SfGATAe of spodoptera frugiperda in green prevention and control of spodoptera frugiperda population.

The invention discovers that the Sf9 cell line insensitive to Cry1Ac toxin overexpresses Spodoptera frugiperda SfGATAe, and can obviously improve the sensitivity of Sf9 cells to Cry1Ac toxin; the expression of SfGATAe is interfered in spodoptera frugiperda larvae, so that the resistance of the spodoptera frugiperda larvae to Cry1Ac toxin can be obviously increased, and therefore, the spodoptera frugiperda transcription factor SfGATAe can be applied to green prevention and control of spodoptera frugiperda populations.

The aim of the invention is achieved by the following technical scheme:

a transcription factor SfGATAe related to spodoptera frugiperda Cry1Ac toxin resistance has an open reading frame nucleotide sequence shown in SEQ ID NO. 1.

Use of spodoptera frugiperda transcription factor SfGATAe in modulating sensitivity of spodoptera frugiperda to Cry1Ac toxins.

The spodoptera frugiperda transcription factor SfGATAe is applied to green prevention and control of spodoptera frugiperda population.

Use of spodoptera frugiperda transcription factor SfGATAe in the cultivation of spodoptera frugiperda varieties sensitive to Cry1Ac toxins.

Application of spodoptera frugiperda transcription factor SfGATAe in preparing feed or green pesticide for improving sensitivity of spodoptera frugiperda to Cry1Ac toxin.

A feed or pesticide for increasing sensitivity of spodoptera frugiperda to Cry1Ac toxins comprising spodoptera frugiperda transcription factor SfGATAe.

A pesticide for preventing and controlling spodoptera frugiperda, which comprises spodoptera frugiperda transcription factors SfGATAe and Cry1Ac toxin and also comprises a pesticide acceptable excipient.

The invention has the advantages and beneficial effects that: the Spodoptera frugiperda SfGATAe transcription factor regulates and controls the expression of the Bt toxin receptor gene of the insect body, thereby causing the sensitivity of the insect body to the Bt toxin to be changed, and providing important theory and application reference for the green prevention and control of the Spodoptera frugiperda.

Drawings

FIG. 1 is a map of a constructed transcription factor SfGATAe insect cell expression vector.

FIG. 2 is a microscopic view of 10. Mu.g/mL Cry1Ac activated toxin treatment transfected with different plasmid-mediated Sf9 cytopathic effects. Wherein A: normal cell groups without plasmid transfection are treated by 10 mug/mL Cry1Ac, and no pathological cells appear; b: the transient transfection of empty pIE2-EGFP-N1 plasmid group with 10 mug/mL Cry1Ac treatment also has no occurrence of pathological cells; c: a10. Mu.g/mL Cry1Ac treated transiently transfected pIE2-SfGATAe-EGFP recombinant plasmid group had 73.7% diseased cells present.

FIG. 3 shows the detection of the expression levels of transcription factors SfGATAe after injection of dsGFP and dsSfGATAe 24h, 48h, 72h, 96h, respectively, by fluorescence quantitative PCR. The expression quantity of the SfGATAe genes in the interference SfGATAe group is down-regulated, and the detection effect of the interference SfGATAe group is best when the interference SfGATAe gene is interfered for 72 hours.

FIG. 4 is the larval survival rate after seven days of feeding with 40 μg/g and 80 μg/g Cry1Ac toxic feed, respectively, after injection of dsGFP and dsSfGATAe to selected spodoptera frugiperda second instar larvae.

Detailed Description

The advantages and various effects of the present invention will be more clearly presented by the following description of the present invention in conjunction with the examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.

Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.

Unless otherwise specified, the molecular cloning methods, protein expression purification methods, cell culture methods, various detection methods, and the like mentioned in the following schemes are all conventional experimental methods, and can be obtained by querying documents; the relevant reagents used may be purchased from the corresponding reagent suppliers.

Example 1

1. Insect expression recombinant vector construction of Spodoptera frugiperda SfGATAe transcription factor

The spodoptera frugiperda transcription factor SfGATAe gene is constructed into an insect cell line expression vector pIE2-EGFP-N1 (the construction of the pIE2-EGFP-N1 is shown in the prior patent application of the applicant, namely, a recombinant plasmid of an insect cell expression system vector, a preparation method and application thereof, publication No. CN 114540388A) to obtain a fusion plasmid pIE2-SfGATAe-EGFP with the characteristics of an Opie2 promoter, a Corzak sequence, a SfGATAe target gene, an EGFP fluorescent tag, a KanR tag and the like, wherein the plasmid map is shown in figure 1, and the specific construction process is as follows.

(1) Extraction of insect tissue total RNA

Freezing the insect with good living state at low temperature for 5min, dissecting about 3 insect larvae, taking out midgut tissue, washing midgut with PBS buffer solution, placing into clean EP tube without RNase, adding 1mL Trizol solution, sufficiently grinding tissue with homogenizer at low temperature, and grinding for a long time. Centrifuging at 12000 Xg for 5min at 4deg.C, collecting supernatant with a pipette, and discarding precipitate. 200. Mu.L of chloroform (chloroform) was added to the EP tube containing the supernatant, and the mixture was gently mixed with a pipette, and the mixture was ice-bathed for 10 to 15 minutes. The mixture is centrifuged again at a low temperature of 4 ℃ in a refrigerated high-speed centrifuge, the rotation speed is 12000 Xg for 15min, layering phenomenon can occur in the liquid in the EP pipe, the upper water phase is carefully sucked up and placed in another clean EP pipe, and the middle layer or the lower layer can not be sucked up (discarded). To the aqueous phase EP was added 500. Mu.L of isopropanol, and the mixture was gently mixed upside down and allowed to stand at room temperature for 10 minutes. Centrifuging at 4deg.C for 10min at 12000 Xg, removing supernatant, and precipitating white RNA at bottom of EP tube. 1000. Mu.L of 75% cold ethanol was added and the RNA pellet was gently mixed by shaking, and it was possible to find that the white pellet was insoluble in ethanol, but it was not possible to blow it off with a pipette, which had the effect of removing residual isopropanol. Centrifuging at low temperature for 5min at 4deg.C and 8000 Xg with a refrigerated high-speed centrifuge, discarding supernatant, and re-appearing white precipitate of RNA at the bottom of the tube. The EP tube cover is opened and dried at room temperature for 5 to 10 minutes to volatilize residual ethanol, but the residual ethanol cannot be excessively dried. Adding 20-50 mu L of RNase-free water to dissolve RNA precipitate at the bottom of the tube, taking a small amount of sample to measure RNA concentration, and keeping the RNA solution for standby or at-80 ℃.

(2) Synthesis of spodoptera frugiperda larva cDNA

gDNA elimination reaction, reference TaKaRa PrimeScript RT reagent kit with gDNA Eraser instruction manual, genomic DNA removal system: gDNA Eraser 1.0. Mu.L, 5X gDNAEraser buffer 2.0.2.0. Mu.L, total RNA content of about 1. Mu.g/mL, make up RNase Free water to 10.0. Mu.L of the total system, place the whole system in a 42℃water bath, react for 30min, and either spare the reaction or preserve at 4 ℃.

RNA reverse transcription to obtain cDNA: 10.0. Mu.L of the above-mentioned total reaction system was taken, and 5X PrimeScript Buffer, 2, 4.0. Mu.L, primeScript RT Enzyme Mix I, 1.0. Mu.L, RT Primer Mix, 4.0. Mu.L and RNase Free water, 1.0. Mu.L were added so that the total reaction system became 20. Mu.L. Placing the reaction system in a water bath at 37 ℃ for reaction for 15min; then, carrying out water bath at 85 ℃ for 5 seconds to inactivate enzymes; the cDNA obtained after the reaction is ready for use or split charging into clean PCR tubes and preserving at-20 ℃.

(3) Obtaining pIE2-SfGATAe-EGFP recombinant plasmid by homologous recombination and seamless cloning

The description according to pEASY-Uni Seamless Cloning and Assembly Kit is summarized below:

amplification of the target Gene: the target gene (length 182 bp) is amplified by using SEQ ID NO.2 and SEQ ID NO.3 as primers and spodoptera frugiperda cDNA as a template, the length of the target gene fragment with the carrier repeated sequence is 1887bp (annealing temperature is 52 ℃), and the amplified target gene is purified by using a gel recovery kit Gel Extraction Kit (Omega Bio-tek, inc., GA, USA) for later use.

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

SEQ ID NO.3：5’-GGTGGCGACCGGTGGATCACCTCCGCCACCGCCTCCGCGCTGGT ACCCCGCCAGC-3’。

Linearizing a carrier: the vector was amplified using primers SEQ ID No.4 and SEQ ID No.5 to obtain a linearized vector, the pIE2-EGFP-N1 plasmid vector (full length 4709 bp) was amplified to obtain a 4678bp fragment (annealing temperature 60 ℃) and the linearized vector was purified using gel recovery kit Gel Extraction Kit (Omega Bio-tek, inc., GA, USA) for use.

SEQ ID NO.4：5’-GATCCACCGGTCGCCACC-3’，

SEQ ID NO.5：5’-CGAAGCTTGAGCTCGAGATCT-3’。

Homologous recombination: purifying the obtained linearization vector and target gene with linearization vector repeated sequence, connecting according to homologous recombination seamless cloning kit, transforming to E.coli DH5 alpha competence, selecting positive clone to obtain recombinant plasmid pIE2-SfGATAe-EGFP, and specifically, the steps can be referred to as follows

Uni Seamless Cloning and Assembly Kit (Beijing full Jin Shengwu family)Technical Co., ltd.) details of the kit procedure. />

2. Transfection of recombinant plasmid pIE2-SfGATAe-EGFP into insect cells

(1) SF9 cells with good growth state are inoculated into a clean 48-hole cell plate, grace's medium (Grace's Insect cell culture medium) containing 8.0% fetal bovine serum is used for overnight culture in a constant temperature incubator at 28.0 ℃, and the cell transfection can be performed after the density is 60% -80%.

(2) The pIE2-SfGATAe-EGFP wild-type plasmid and pIE2-EGF-N1 empty plasmid used as transfection were aseptically extracted with a plasmid extraction kit at a concentration of 200 ng/. Mu.L or more and at a purity (OD ₂₆₀ /OD ₂₈₀ ) Preferably between 1.75 and 1.85.

(3) Serum-free Grace's medium, 1000. Mu.L, 200. Mu.L, 10. Mu.L pipettes and corresponding sized replacement heads were prepared, 1.5mL clean EP tubing, alcohol lamps and alcohol cotton were placed in a biosafety cabinet and sterilized by UV light for about 30min.

(4) mu.L of serum-free Grace's medium was taken into a sterile 1.5mL EP tube, 1.0. Mu.L of FuGENE HD was added, while 0.25. Mu.g of plasmid was mixed (the volume of transfection reagent to plasmid mass ratio range was recommended to be between 3.0:1.0 and 6.0:1.0).

(5) The mixture added to the 1.5mL EP tube was gently mixed, allowed to stand in the biosafety cabinet for 20min, and 95. Mu.L of serum-free Grace's was added to the above mixture system so that the total volume was 120. Mu.L.

(6) The culture medium in the 48-well plate is discarded, the culture medium is washed three times by serum-free Grace's culture medium, and the reaction solution in the step (5) is added into the washed 48-well plate and is uniformly mixed.

(7) The transfected 48-well plate is placed in a cell constant temperature incubator at 28.0 ℃ for incubation for 3-5 hours, the reaction solution in the 48-well plate is discarded, and 120 mu L of Grace's medium containing 8.0% fetal bovine serum is added.

(8) After the culture medium is changed, the 48-hole cell culture plate is placed in a cell constant temperature incubator at 28.0 ℃ for culturing for 24-48 hours.

3. Transfection of pIE2-SfGATAe-EGFP transcription factor recombinant plasmid mediated SF9 cell virulence determination

(1) After 48h of plasmid transfection, the 48-well plate was removed from the incubator for subsequent toxin treatment experiments.

(2) The Cry1Ac toxin is diluted by a PBS solution by adopting a double dilution method, and the toxins with the concentration of 10 mug/mL are prepared by accurately and uniformly mixing the components in the dilution process.

(3) Media in 48-well plates was aspirated, washed 3 times with locks solution, and 150. Mu.L of 10. Mu.g/mL toxin solution was added to each well.

(4) After 1h of toxin addition, photographs were taken using an eyepiece free inverted microscope.

(5) Toxin treatment transfected SF9 is treated for 1h, photographing and recording are carried out by a fluorescence inversion microscope, the disease number (cell swelling, swelling and rounding are lesions) is recorded under white light, the number of green fluorescent tag protein cells is recorded under fluorescence to be the total number of cells, and the disease rate is calculated, namely, the cytopathic rate = the number of lesions/the total number of cells multiplied by 100%.

(6) Setting three times of repetition, and counting the disease change rate.

(7) As can be seen from FIG. 2, under the treatment of Cry1Ac toxin of 10 μg/mL, the untransfected group and the transfected pIE2-EGF-N1 empty plasmid group have no pathological cells after 1h, and the pathological change rate of SF9 cytopathic rate mediated by the transfected pIE2-SfGATAe-EGFP is remarkably improved (p<0.01). Treatment of pIE2-SfGATAe-EGFP recombinant protein-mediated Sf9 cells with different concentrations of Cry1Ac toxin, cry1Ac toxin concentration of 2. Mu.g/mL cytopathic rate of 14.07%, cry1Ac toxin concentration of 4. Mu.g/mL cytopathic rate of 2.58%, cry1Ac toxin concentration of 8. Mu.g/mL cytopathic rate of 61.14%, cry1Ac toxin concentration of 16. Mu.g/mL cytopathic rate of 83.43%, cry1Ac toxin concentration of 32. Mu.g/mL cytopathic rate of 95.51%, calculation of different concentrations of Cry1Ac toxin versus pIE 2-SfGATAe-EGFP-mediated SF9 cells EC ₅₀ The value is 6.213 mug/mL, and the result shows that after SfGATAe transcription factor is over-expressed in Sf9 cells, the sensitivity of the cells to Cry1Ac toxin is enhanced.

4. Fluorescent quantitative PCR (polymerase chain reaction) detection of change of expression level of transcription factor SfGATAe

(1) dsRNA was synthesized from the transcription factor SfGATAe sequence for interfering with the conserved region length of 518bp. The primer containing the T7 promoter sequence is designed according to the conserved region sequence of the target gene, the DNA fragment of the conserved region is amplified by PCR by taking the three-instar larva cDNA of spodoptera frugiperda as a template, the linear DNA is taken as the template to synthesize dsRNA of the target gene in vitro (the specific method is operated by referring to the specification of the T7 RiboMAXTM Express RNAi System product), and the concentration is determined to be about 1500 ng/. Mu.L.

The GFP gene dSRNA primer of the interference control group was synthesized as follows:

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

SEQ ID NO.7：5’-TAATACGACTCACTATAGGG CTCGATGTTGTGGCGGATC-3’。

the primer for synthesizing the conserved region dSRNA of the interfering target gene SfGATAe is as follows:

SEQ ID NO.8：5’-TAATACGACTCACTATAGGGACAAAGTGGGAGGGGAGCAC-3’，SEQ ID NO.9：5’-TAATACGACTCACTATAGGGCTTCCTCGTCTGGATGCCGTC-3’。

(2) And (5) selecting the spodoptera frugiperda larvae with approximately the same growth vigor, and carrying out ice anesthesia on the larvae at the second age for 30min.

(3) Microinjection was performed between the second and third pair of larvae, with dsRNA injection at 2 μg/head.

dsRNA of interfering EGFP gene is injected as a control group, dsRNA of interfering SfGATAe is injected as each treatment of an experimental group to inject 10 heads of larvae, 3 heads of larva dissection extraction midgut total RNA is respectively extracted 24h, 48h, 72h and 96h after injection, cDNA is obtained through reverse transcription, and the interference efficiency of target genes is detected by fluorescent quantitative PCR.

The following primers were used as the spodoptera frugiperda GAPDH internal reference detection primers:

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

SEQ ID NO.11：5’-CAGACGCCTTCTCTGTGGTT-3’。

the following primers were used as Spodoptera frugiperda SfGATAe detection primers:

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

SEQ ID NO.13：5’-CGTCGTCGATGGGTAGAAGG-3’。

the results are shown in FIG. 3. After 24h, 48h, 72h and 96h of interference of SfGATAe genes, the expression levels of the genes are respectively reduced to 80.65% +/-3.43 (p < 0.01), 70.49% +/-2.79 (p < 0.01), 54.71% +/-3.83 (p < 0.01), 85.47% +/-2.62 (p > 0.05) compared with the control group of interference of GFP, the SfGATAe is obviously reduced after 72h of interference, and the interference efficiency is highest, so that the following virulence experiment is selected for the larva experiment after 72h of interference.

5. Detection of sensitivity of Spodoptera frugiperda populations to toxins following detection of interfering transcription factor SfGATAe

(1) Placing spodoptera frugiperda in an HP3000GS-C intelligent artificial climate box, recovering the spodoptera frugiperda to the optimal state, enabling insects to adapt to the indoor environment of the artificial climate box, controlling parameters of the artificial climate box, and keeping the temperature at 28.0+/-0.5 ℃ and the illumination period ratio at 16L:8D, the humidity is 60+/-5%.

(2) Collecting spodoptera frugiperda second-instar larvae with the same instar and physiological state, placing the larvae in a clean 24-hole plate, and starving for 4-6 hours.

(3) And (3) sealing the prepared insect feed, placing the insect feed in a DHG-9123A type electrothermal constant-temperature air-blast drying oven for 20-30 min, adding dissolved Cry1Ac protoxin into the feed, and uniformly stirring to prepare 40 mug/g and 80 mug/g toxic feed.

(4) dsRNA interfering with EGFP gene was injected as control group, dsRNA interfering with SfGATAe was injected as experimental group, and 60 larvae were injected per treatment.

(5) After 72h of disturbance of the second instar larvae, 40 μg/g and 80 μg/g of toxic feed were fed to 30 larvae of the starved experimental and control groups, each treatment being repeated three times.

(6) The survival rate of each group of insects was counted daily for 7 days, and the death of insects was checked at the same time, and the survival rates of insects in the experimental group and the control group were recorded.

(7) The corrected survival rate of the treatment group was calculated, and the corrected survival rate= (number of survival of treatment group-average number of death of control group)/(total number of treatment group-average number of death of control group) ×100%, and the corrected survival rate was recorded as survival rate.

The results are shown in FIG. 4. The survival rates of the control group after 7 days of feeding toxin 40 mug/g and 80 mug/g are 37.78% + -6.85 and 11.11% + -4.16 respectively. The survival rates of the interference SfGATAe group after 40 mug/g and 80 mug/g for 7 days are 88.89% +/-5.67 and 72.22% +/-8.31, and compared with the control group, the survival rate is obviously improved (p < 0.01).

Claims (10)

1. A spodoptera frugiperda Cry1Ac toxin resistance-related transcription factor SfGATAe, characterized by: the nucleotide sequence of the open reading frame of the transcription factor SfGATAe is shown as SEQ ID NO. 1.

2. Use of the transcription factor SfGATAe of claim 1 for modulating sensitivity of spodoptera frugiperda to Cry1Ac toxins.

3. Use of the transcription factor SfGATAe according to claim 1 for green control of spodoptera frugiperda populations.

4. Use of the transcription factor SfGATAe as claimed in claim 1 for breeding spodoptera frugiperda varieties sensitive to Cry1Ac toxins.

5. Use of the transcription factor SfGATAe of claim 1 in the preparation of a feed for increasing sensitivity of spodoptera frugiperda to Cry1Ac toxins.

6. Use of the transcription factor SfGATAe of claim 1 in the preparation of a green pesticide for increasing sensitivity of spodoptera frugiperda to Cry1Ac toxins.

7. A feed for increasing sensitivity of spodoptera frugiperda to Cry1Ac toxins, characterized in that: comprising the transcription factor SfGATAe of claim 1.

8. A pesticide for increasing sensitivity of spodoptera frugiperda to Cry1Ac toxins, characterized in that: comprising the transcription factor SfGATAe of claim 1.

9. A pesticide for controlling spodoptera frugiperda, which is characterized in that: comprising the transcription factors SfGATAe and Cry1Ac toxins of claim 1.

10. A pesticide according to claim 8 or 9, characterised in that: also comprises an excipient acceptable in pesticides.

Images