CN111979236B - Promoter of gene MtGSTs1 of glutathione S-transferase of Populus canula and application - Google Patents

Abstract

The invention discloses a promoter of a glutathione S-transferase MtGSTs1 gene of a populus canescens and application thereof, belonging to the technical field of genetic engineering, wherein the nucleotide sequence of the promoter of the glutathione S-transferase MtGSTs1 gene of the populus canescens is shown as SEQ ID NO. 1. The promoter of the MtGSTs1 gene of the populus canescens is successfully cloned, and experiments prove that the promoter can be used for starting the expression of downstream genes in host cells, particularly can start the expression of the downstream genes under the induction of plant secondary substances, can provide a theoretical basis for a specific action mechanism of the plant secondary substances for the expression regulation and control of the MtGSTs1 gene of the populus canescens, provides a theoretical basis and a gene sequence for a new prevention and control technology of populus trevorax pests, and can also provide an alternative gene for culturing insect cells or transgenic silkworms with higher resistance to toxic substances.

Description

Promoter of gene MtGSTs1 of glutathione S-transferase of Populus canula and application

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a promoter of a glutathione S-transferase gene.

Background

In the long-term evolution, the close interrelationship between plants and insects is largely reflected in the diversity of plant secondary materials, to which insects evolve precise adaptability and resistance. Insect resistance to plant secondary substances mainly includes metabolic resistance, target resistance and behavioral resistance, and the emergence of a series of detoxification enzyme systems is one of the main modes of insect adaptation. Glutathione S-transferases (GSTs), carboxylesterase (Carboxyle-sterase, carE) and cytochrome P450 (Cytochrome P450, CYP 450) are the three most important detoxification enzyme systems in insects. The GSTs belong to important transferase of II-phase metabolic reaction, and the effect of endogenous or exogenous toxic substances is removed through the combination of endogenous glutathione and harmful electrophilic groups and discharge out of the body or the combination of non-covalently combined protein and hydrophobic ligands, thereby playing a role in detoxification.

The promoter is an important cis-element for gene expression regulation, the transcription link is the most main regulation site in the gene expression regulation, and the regulation of the promoter occupies a very important position in the transcription link; the plant secondary biomass can induce the activity of detoxication enzymes such as GSTs in the insect body, and is beneficial to insect detoxification. Thus, it is reasonable to believe that plant secondary organisms are likely to have important regulatory functions for GSTs gene promoters.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention aims to provide a promoter of the gene of the populus canula GSTs, which can be started in host cells under the induction of plant secondary substances. Another objective of the invention is to provide a vector of promoter sequences of the Populus parvifolius GSTs genes, which can promote the expression of downstream genes in host cells. The invention also aims to provide an application method of the vector of the promoter sequence of the cottonwood boat moth GSTs gene, which can be used for improving the resistance of insect cells to toxic substances.

The technical scheme is as follows: in order to solve the problems, the technical scheme adopted by the invention is as follows:

a promoter of a cottonwood boat moth glutathione S-transferase MtGSTs1 gene has a nucleotide sequence shown in SEQ ID NO. 1.

An expression vector contains the promoter of the gene MtGSTs1 of the populus canula glutathione S-transferase.

A host cell comprising said expression vector.

Preferably, the host cell is an insect cell.

The promoter of the gene MtGSTs1 of the glutathione S-transferase of the Populus canna is applied to the promotion of the downstream gene expression in host cells.

The application of the promoter of the gene MtGSTs1 of the glutathione S-transferase of the Populus canna in improving the resistance of insect cells to toxic substances.

The promoter of the cottonwood huckle moth glutathione S-transferase MtGSTs1 gene is applied to the promotion and expression of the MtGSTs1 gene.

Has the advantages that: compared with the prior art, the promoter of the MtGSTs1 gene of the populus canescens is successfully cloned, and experiments prove that the promoter can be used for starting the expression of the downstream gene in host cells, particularly can start the expression of the downstream gene under the induction of plant secondary substances, can provide a theoretical basis for the specific action mechanism of the plant secondary substances on the expression regulation and control of the MtGSTs1 gene of the populus canescens, provides a theoretical basis and a gene sequence for a new control technology of the populus trevorax leaf pests, and can also provide an alternative gene for culturing insect cells or transgenic silkworms with higher resistance to toxic substances.

Drawings

FIG. 1 is a DNA electrophoresis picture of larvae of Populus parva;

FIG. 2 is a diagram showing the result of electrophoresis detection of PCR product of promoter of Myeloptera xylostella MtGSTs 1; in the figure, M: marker,4: the MtGSTs1 promoter;

FIG. 3 is a diagram of: enzyme digestion verification of the PGL4.10-MtGSTs1 promoter recombinant plasmid; in the figure, M: marker,1: PGL4.10,2: PGL4.10 double enzyme digestion, 7: carrying out double enzyme digestion on PGL4.10-MtGSTs1 promoter;

FIG. 4 is a PGL4.10 plasmid map;

FIG. 5 is a diagram showing the result of detecting the promoter activity of the MtGSTs1 gene of Populus canescens;

FIG. 6 is a diagram showing the result of detecting the promoter activity of the MtGSTs1 gene of Populus plutella under the stress of tannin;

FIG. 7 is a diagram showing the result of detecting the promoter activity of the MtGSTs1 gene of Populus plutella under the stress of quercetin;

FIG. 8 is a diagram of the result of detecting the promoter activity of the Marshmania poplara MtGSTs1 gene under the stress of tridecanone.

Detailed Description

The invention is further described with reference to specific examples. The molecular biological experiments, which are not specifically described in the following examples, can be performed by methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or methods conventional in the art, or according to kits and product instructions.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The test insects: poplar small boat moth (Micromelalopha troglotyta) is collected from Ujiang town of Pukou of Nanjing city of Jiangsu province, and is taken back indoors and then raised under the conditions of constant temperature of 26 +/-1 ℃, h light and h dark = 16: 8 and relative humidity of 75%. Dissecting the larva on ice, rapidly freezing in liquid nitrogen, and storing in a refrigerator at-80 deg.C.

Preparation of reagents:

(1) LB liquid culture Medium

Weighing 10g of tryptone and 10g of Yeast extract Yeast by using an electronic balanceExtract 5g, naCl 10g, adjusting pH to 7.0 with NaOH, adding ddH ₂ O constant volume to 1L, and autoclaving at 120 deg.C for 20min.

(2) LB solid medium

Adding 15g agar powder into 1LLB liquid culture medium, and autoclaving at 120 deg.C for 20min.

(3) 50 XTAE electrophoresis buffer 100mL

Tris 24.2g, glacial acetic acid 5.71mL,0.5M EDTA (pH = 8.0) 10mL, plus ddH ₂ O to 100mL.

(4) Ampicillin stock solution (100 mg/mL)

100mg of ampicillin were weighed out and dissolved in 1mL of ddH ₂ Storing at-20 deg.C in O atmosphere.

(5) Preparation of X-Gal solution

20mg of X-Gal was weighed out and dissolved in 1mL of dimethylformamide and stored at-20 ℃.

Example 1: cloning of promoter of glutathione S-transferase gene of Populus mauritiana

1. Cloning of Gene promoter of glutathione S-transferase of Populus canula

1. Design of primers

Designing a joint Primer and a universal Primer according to a chromosome walking method, and designing two specific primers by using Primer Premier 5 software according to a successfully cloned GSTs gene sequence as follows:

Adaptor Primer1：

5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3′，

Nested Adaptor Primer2：5′-ACCAGCCC-3′，

Common1：5′-GTAATACGACTCACTATAGGGC-3′，

Common2：5′-ACTATAGGGCACGCGTGGT-3′，

MtGSTs1 1：5′-AACCTTACATCCTCAAATTCCTGTTTAGTG-3′，

MtGSTs1 2：5′-GACGAGCTTCAGGTTGAGTTGGATGT-3′。

the primers were synthesized by Shanghai Czeri Biotechnology, inc.

2. Extracting genomic DNA of Populus parva by using DNAso Reagent kit (TaKaRa)

(1) Placing the dissected populus canula material into a mortar precooled by liquid nitrogen, adding a proper amount of DNAso Reagent solution, and quickly grinding and homogenizing by using a grinding pestle. (2) The lysate was transferred to a centrifuge tube, allowed to stand at room temperature for 5min, and centrifuged at 12000 Xg for 10min at 4 ℃. (3) And (3) sucking the supernatant into a new centrifugal tube, adding 0.5mL of absolute ethyl alcohol, uniformly mixing for 1-3 min, taking the precipitate into the new centrifugal tube by using a gun head or centrifuging for 2min at 4000 Xg, and pouring out the supernatant. (4) 1mL of 75% ethanol was added along the tube wall, the tube wall was washed gently upside down, and centrifuged at 12000 Xg for 5min at 4 ℃ to discard the ethanol. (5) Drying at room temperature for 10min, adding 50 μ L of sterilized water, dissolving in water for 10min, and storing at-80 deg.C.

20mL of 1 XTAE buffer was added to a DEPC-treated 250mL Erlenmeyer flask, 0.2g of agarose was weighed on an electronic balance, added to the Erlenmeyer flask, mixed, and heated in a microwave oven until completely dissolved. After the solution had cooled slightly to about 50 ℃,2 μ L of dye (GelStain) was added, shaken, poured into a gel-making plate, and a comb was inserted. After the glue solution is solidified, the comb is pulled out, the glue is put into an electrophoresis tank, and 1 mu L of RNA is taken for electrophoresis detection. Set constant voltage 140V, take pictures on an automated gel imaging system after running for 30min. 1 mu L of DNA is used for detecting the concentration and the purity of the DNA by an ultraviolet spectrophotometer.

The electrophoresis result of the extracted Populus plutella DNA is shown in figure 1, and the extracted Populus plutella DNA has an obvious band and is detected by Nano Drop 1000: the concentration of RNA was 6722.3 ng/. Mu.L, OD ₂₆₀ /OD ₂₈₀ The value was 2.04,OD ₂₆₀ /OD ₂₃₀ The value was 2.17. The extracted DNA of the populus canula is better in quality and can be used for subsequent experiments.

3. Enzyme digestion

The appropriate enzyme was selected for digestion using Primer Premier 5 software. The reaction solution (25. Mu.L) was prepared according to the following components: 2 uL 10 XNEBuffer,. Times.uL DNA,0.5 uL enzyme, 22.5-XddH ₂ O; * X: the volume was calculated from the DNA concentration by taking 0.5. Mu.g of Total DNA. Reaction procedures are as follows: forever at 37 ℃.

4. Purification of

Adding equal volume of Tris-saturated phenol into the enzyme digestion product, and oscillating at low speed for 5-10 s. The mixture was centrifuged at room temperature and the supernatant was aspirated into another new centrifuge tube. Adding chloroform with the same volume, and oscillating at low speed for 5-10 s. The mixture was centrifuged at room temperature and the supernatant was aspirated into another new centrifuge tube. Two volumes of 95% ethanol, 1/10 volume of 3M NaOAc (pH = 4.5) was added and shaken at low speed for 5-10 s. Centrifuging at 14000 Xg for 15min at 4 ℃, discarding the supernatant, and washing the precipitate with 80% ethanol. Air drying the precipitate for 10min, adding appropriate amount of sterilized water, dissolving in water for 10min, and storing at-80 deg.C for use.

5. Ligation of adaptor primers

(1) Preparation of adapter primers

10 mu L of each of the long Primer and the short Primer of the Adaptor Primer1 and the Nested Adaptor Primer2 are mixed and put into the just boiled water, and the mixture is cooled to room temperature and then waits for two hours to obtain the mother solution of the Adaptor Primer (Adaptor Primer).

(2) Ligation of adaptor primers

The reaction solution (8. Mu.L) was prepared with the following components: 1.6. Mu.L of 10 Xligation Buffer, 4. Mu.L of DNA, 1.9. Mu.L of Adaptor Primer, 0.5. Mu.L of T4 DNA Ligase.

Reaction procedure: 16 ℃ overnight; 5min at 70 ℃.

(3) Add 72. Mu.L of ddH ₂ O, and storing at-80 ℃ for later use.

6. PCR amplification

First round primer amplification: PCR amplification is performed using the DNA to which the adapter primer is ligated as a template.

A PCR reaction system (25. Mu.L) was prepared with the following components: 1 μ L Template,1 μ L Primer1, 1 μ L Common1, 12.5 μ L Mix Ex-Tag,9.5 μ L ddH ₂ And (O). PCR reaction procedure: 5min at 94 ℃; {94 ℃ 25s,72 ℃ 3min }7 cycles; {94 ℃ 25s,67 ℃ 3min }32 cycles; 7min at 67 ℃;4 ℃ forever.

Second round primer amplification: diluting the first round product by 50 times to be used as a second round product template; a PCR reaction (25. Mu.L) was prepared with the following components: 1 μ L Template 2,1 μ L Primer2, 1 μ L Common2, 12.5 μ L Mix Ex-Tag,9.5 μ L ddH ₂ And (O). PCR reaction procedure: 5min at 94 ℃; {94 ℃ 25s,72 ℃ 3min }5 cycles; {94 ℃ 25s,67 ℃ 3min }30 cycles; 7min at 67 ℃;4 ℃ forever.

The reaction solution was subjected to agarose gel electrophoresis for detection.

7. Gel cutting recovery, connection and transformation of PCR product

The product was recovered using the MINIBest Agarose Gel DNA extraction Kit Ver3.0 recovery Kit (centrifugal column type) from TaKaRa, the procedure was as follows:

(1) The gel containing the desired fragment was excised from the agarose gel (excess was removed as much as possible), weighed accurately, and placed in a sterilized 1.5mL centrifuge tube. (2) Add 300. Mu.L buffer GM to each 100mg agarose gel, and place in 37 ℃ water bath for 3-5min, while gently inverting the tube until the gel mass is completely melted. (3) The solution was observed for color (if not yellow, 10. Mu.L of 3M sodium acetate (pH 5.2) was added and mixed until the solution turned yellow) (4) Spin Column was placed on the Collection Tube. (5) The solution was transferred to Spin Column and centrifuged at 12000rpm for 1min, and the filtrate was discarded. (6) mu.L of Buffer WB was added to Spin Column, centrifuged at 12000rpm at room temperature for 30sec, and the filtrate was discarded. And (7) repeating the steps and washing again. (8) Place Spin Column on the Collection Tube, place Spin Column on a new 1.5mL centrifuge Tube at room temperature, add 30. Mu.L ddH to the center of the Spin Column membrane ₂ And O, standing at room temperature for 1min. (9) Centrifuging at 12000rpm for 1min at room temperature, and collecting 1 μ L of recovered solution for detecting DNA concentration and purity by ultraviolet spectrophotometer.

Connecting: the amplification product was ligated with the pDM19T vector. Ligation composition (5 μ L): 2.5 μ L Solution I buffer, X μ L DNA (recovered), 0.5 μ L pDM19T vector, (2-X) μ L ddH ₂ O; * X: the number of moles of DNA is three times that of the carrier, the volume is calculated according to the DNA concentration, and less than 2. Mu.L of the recovered DNA is made up with water. Reaction procedure: 2h at 16 ℃.

And (3) transformation: (1) mu.L of E.coli competent cells was added to 5. Mu.L of the ligation product, gently mixed and ice-cooled for 30min. (2) Heat shocking in 42 deg.c water bath for 60sec and setting on ice for 3-5 min. (3) Adding 800 μ L SOC culture medium, shaking at 37 deg.C and 150rpm for 1h. (4) Centrifuging at 4000 Xg for 3min, discarding the supernatant, leaving about 150-200 μ L of bacterial solution, and gently blowing and beating the precipitate to mix the precipitate evenly. (5) The bacterial liquid is coated on LB solid plate culture medium with penicillin and X-Gal and inverted at 37 deg.c for over night culture. (6) When colonies appear on the culture dish, picking white single colonies, picking the single colonies into 800 mu L LB liquid medium containing Amp by using a sterile toothpick, and carrying out shake culture at 37 ℃ and 250 ℃.

The transformed bacterial liquid is subjected to PCR detection, and the reaction system is as follows (25 mu L): 1 μ L of bacterial liquid, 1 μ L of Primer2, 1 μ L of Common2, 12.5 μ L of Mix Ex-Tag,9.5 μ L of ddH ₂ O; PCR reaction procedure: 5min at 94 ℃; {94 ℃ 25s,72 ℃ 3min }5 cycles; {94 ℃ 25s,67 ℃ 3min }30 cycles; 7min at 67 ℃;4 ℃ forever. And (4) detecting the reaction solution by electrophoresis, and sending the bacterial solution with the size meeting the target fragment to a biological company for sequencing.

The PCR reaction product was electrophoresed in agarose gel, and the result is shown in FIG. 2,1 promoter of the Dactylopsis poplars GSTs gene was successfully cloned, named as promoter of the Dactylopsis poplars MtGSTs1 gene, with a sequence length of 926bp (containing ATG). The possible transcription initiation sites were predicted by using bioinformatics online prediction software (http:// www. Gene-regulation. Com/pub/programs/alibaba2/index. Html. And http:// www.fruitfly. Org/SEQ _ tools/promoter. Html). According to the prediction results, the length of the promoter sequence of the MtGSTs1 gene was 576bp, as shown in SEQ ID No.1 of the sequence Listing.

Example 2: activity verification of promoter of glutathione S-transferase gene of Artocarpus populi

1. Construction of expression vectors

1) Designing a primer according to a promoter sequence successfully cloned, adding Nhe I and Xho I enzyme cutting sites at the 5' end of the forward and reverse primers through screening, and adding a protective base AA. The primers used for constructing eukaryotic expression donor plasmids of the GST gene promoter of the Populus potamoeba are as follows:

MtGSTs1 gene primer F:5 'AACTCGAGCGACGAAGGCTT-3';

MtGSTs1 gene primer R:5 'AAGCTAGCATTTGCTGCACTATATCA-3'.

The primers were synthesized by Shanghai Czeri Biotechnology, inc.

2) Extracting a promoter plasmid of a cottonwood fruit moth GSTs gene: (1) A single colony was selected from the promoter plate medium of a successful clone, inoculated into 1 to 4mL of LB liquid medium containing antibiotics, and cultured overnight at 37 ℃ (12 to 16 hours). (2) 1-4 mL of overnight culture solution was centrifuged at 12,000rpm for 2min, and the supernatant was discarded. (3) The bacterial pellet was suspended well with 250. Mu.L of Solution I (containing RNase A). (4) Adding 250 mu L Solution II, slightly turning and mixing up and down for 5-6 times to fully crack the thalli and form a transparent Solution. (5) Adding 350 mu L of Solution III precooled at 4 ℃, slightly turning and mixing up and down for 5-6 times until compact aggregate is formed, and then standing for 2min at room temperature. (6) centrifugation is carried out for 10min at room temperature of 12,000rpm, and the supernatant is taken. (7) Spin Column in the kit was mounted on the Collection Tube. (8) The supernatant from operation 6 was transferred to a Spin Column, centrifuged at 12,000rpm for 1min and the filtrate discarded. (9) mu.L of Buffer WA WAs added to the Spin Column, centrifuged at 12,000rpm for 30sec, and the filtrate WAs discarded. (10) mu.L of BufferWB was added to Spin Column, centrifuged at 12,000rpm for 30sec, and the filtrate was discarded. (11) repeating the operation step 10. (12) Spin Column was replaced on the Collection Tube and centrifuged at 12,000rpm for 1min to remove the residual wash. (13) Spin Column was placed on a new 1.5mL centrifuge tube, 50 μ L of Elution Buffer was added to the center of the Spin Column membrane, and left to stand at room temperature for 1min. (14) The DNA was eluted by centrifugation at 12,000rpm for 1min, and 1. Mu.L of the plasmid solution was used in an ultraviolet spectrophotometer to determine the concentration and purity of the DNA.

3) And (3) PCR amplification: a PCR reaction system (25. Mu.L) was prepared using the plasmid as a template and the following components: * X μ L plasmid, 1 μ L forward primer, 1 μ L reverse primer, 12.5 μ L Mix Ex-taq enzyme, (10.5-X) μ L ddH ₂ O; * X: mu.g of plasmid DNA was taken and the volume was calculated from the DNA concentration. PCR reaction procedure: 5min at 94 ℃; {94 ℃ 30s,62 ℃ 30s,72 ℃ 1min30s }25 cycles; 10min at 72 ℃;4 ℃ forever. The reaction solution was subjected to agarose gel electrophoresis.

4) PCR product gel cutting recovery, connection and transformation

The method for recovering the target DNA fragment was the same as in example 1.

Plasmid extraction method is the same as above.

Double enzyme digestion: the extracted plasmid and PGL4.10 vector were subjected to double digestion with Nhe I and Xho I endonucleases, and the digestion reaction system (20. Mu.L) was prepared as follows: * X μ L plasmid, 1 μ L Xho I,1 μ L Nhe I, (18-X) μ L ddH ₂ O; * X: take 1. Mu.g plasmid DNA, calculate the volume based on the DNA concentration. Digestion reaction procedure：37℃ 2h；

Connecting: the target vector PGL4.10 was subjected to double enzymatic digestion with the same enzyme for ligation, with a ligation component composition (10 μ L): mu.L 10 Xligation Buffer,. Mu.L DNA (cleavage product), 0.5. Mu.L PGL4.10 vector, 1. Mu.L T4 DNA Ligase, (7.5-X). Mu.L ddH ₂ O; * X: the number of DNA moles was three times that of the vector and the volume was calculated from the DNA concentration. Reaction procedure: 2h at 16 ℃.

The transformation procedure was as in example 1.

Verification of recombinant plasmids: after sequencing verification, plasmids are extracted for enzyme digestion reaction.

Through sequencing and enzyme digestion verification (figure 3 and figure 4), PGL4.10 and the MtGSTs1 gene promoter successfully construct a recombinant plasmid which can be used for subsequent cell transfection experiments.

2. Transfection of insect Sf9 cells with recombinant plasmids

1) Adherent culture of insect Sf9 cells: insect Sf9 cells were cultured in a cell culture flask using an Sf-900II SFM cell culture medium containing 10% FBS (fetal bovine serum) and antibiotics, incubated at 27 ℃ in a constant temperature biochemical incubator in the dark, and subcultured for 2 to 3 days depending on the cell state.

2) Cryopreservation of insect Sf9 cells: the non-deformed cells with good growth state and less passage number need to be frozen for seed preservation, cells with full wall growth are gently blown down, centrifugation is carried out for 10min at 5000g, and cell precipitation is suspended by precooled cell frozen stock solution (10% DMSO-containing whole cell culture medium). The total amount of cells can be counted using a counting plate before centrifugation or estimated based on the bottom area of the flask to give a final cell concentration of about 1X 10 ⁷ cell/ml. Subpackaging the cell frozen stock solution into ice-bath cell frozen stock tubes at 4 ℃ for 30min; 30min at-20 ℃; 30min at-80 ℃; and finally, placing the freezing tube in liquid nitrogen for storage and standby.

3) Revival of insect Sf9 cells: adding the culture medium into a culture bottle, taking out the cell cryopreservation tube from liquid nitrogen, rapidly bathing in water at 27 ℃, taking out when a small amount of ice remains, disinfecting the outer wall with alcohol, and sucking the cryopreservation liquid into the culture bottle. And (3) carrying out light-resistant culture in a constant-temperature biochemical incubator at 27 ℃, changing the liquid (generally less than 30 min) after the cells adhere to the wall, changing the liquid after 24h of culture, and measuring the cell activity, wherein the successfully recovered cell activity is more than 75%.

4) Using PureYield ^TM Plasmid Midiprep System (Promega) extracts endotoxin-free plasmids:

(1) Single colonies were selected from the recombinant plasmid plate medium and inoculated into 50-100 mL of LB liquid medium containing antibiotics and cultured overnight at 37 ℃ (12-16 h). (2) 5000g of overnight cultured bacterial liquid is taken for centrifugation for 10min, and the supernatant is discarded. (3) The bacterial pellet was suspended thoroughly by adding 3mL of Cell Resuspension Solution. (4) Adding 3mLCell lysine Solution, slightly turning and mixing up and down for 3-5 times, standing for 3min at room temperature to fully crack the thalli to form a transparent Solution. (5) 5mL of precooling Neutralizing Solution with 4 ℃ is added and is gently turned over and mixed for 5 to 10 times. (6) centrifuge at 15000g for 15min at room temperature. (7) The blue cleaning Column was placed on the white Binding Column and placed in a vacuum extractor. (8) The supernatant was taken in a clean Column, passed through the Column by a vacuum apparatus, and the clean Column was discarded. (9) 5mL of Endotoxin Removal Wash was added to the Binding Column and passed through the Column using a vacuum apparatus. (10) 20mL of Column Wash Solution was added to the Binding Column and passed through using a vacuum apparatus. (11) drying the film for 30 to 60sec by a vacuum apparatus. (12) Wiping off alcohol on Binding Column, adding 400-600 μ L of nucleic-Free Water, standing for 1min, and collecting with new EP tube.

5) Adopting liposome transfection method invitrogen cellfection ^R IIreagent transfected cells:

(1) Paving a plate: washing cells to be passaged with 1mL of culture medium containing diabody and serum, adding appropriate amount of culture medium containing diabody and serum to blow up cells, subpackaging to 96-well plate, standing for more than 15min (about 9 × 10 per well) ⁴ One cell). (2) preparing a transfection mixed solution (single-hole dosage): solution A: 1 μ L of liposome, 10 μ L of serum-free antibiotic-free medium; standing at room temperature for 30min; and B, liquid B: 200ng of target plasmid, 20ng of pRL-TK, and 10 mu L of serum-free and antibiotic-free culture medium; adding solution A into solution B, mixing, and standing at room temperature for 30min. (3) transfection: sucking out culture medium from plated cells, adding 80 μ L serum-free and antibiotic-free culture medium, adding 20 μ L transfection mixture, culturing at 27 deg.C for 5 hr, adding 150 μ L serum-containing and double-antibody-containing culture medium, culturing at 27 deg.C for 48 hr, and performing double-antibody reactionAnd (4) detecting fluorescence.

Transferring the promoter recombinant plasmid into an insect Sf9 cell by a liposome transfection method for expression, detecting the activity of luciferase by using double fluorescence after transfection for 48h, wherein the ratio of a firefly fluorescence detection value (Luc) to a Renilla fluorescence detection value (rLuc) is a final result, and analyzing the significant difference of the activity of each promoter relative to a vector control by using variance (P is less than 0.001, and P is less than 0.05), as shown in FIG. 5. The results show that the MtGSTs1 promoter presents a certain significant difference compared with the vector.

Example 3: induced expression of plant secondary biomass to populus canula promoter recombinant plasmid

The main method is the same as example 2, 10 μ L of inducing drug is added when the medium containing serum and double antibody is changed after culturing for 5h at 27 ℃ in the process of cell transfection, three plant secondary biologics of tannin, quercetin and 2-tridecanone are selected in the example to express and induce the promoter of MtGSTs1 gene of populus canescens, ddH is used for inducing ₂ Dissolving tannin in ddH, dissolving quercetin and 2-tridecanone in acetone, and making into medicinal preparations with concentration of 0.01, 0.1 and 1mg/mL respectively ₂ O as a control, quercetin and 2-tridecanone as acetone controls.

1) Tannin with different concentrations is added for expression induction in the process of transfecting Sf9 cells by PGL4.10 and the promoter recombinant plasmid of the MtGSTs1 gene of the Populomala esculenta, and variance analysis is carried out on the luciferase detection result (figure 6). The result shows that under the stress of tannin, the MtGSTs1 gene promoter of the populus canescens is induced to a certain extent, and the expression quantity of the MtGSTs1 gene promoter is obviously increased (P is less than 0.05) under the stress of tannin with the concentration of 0.01mg/mL respectively.

2) Quercetin with different concentrations is added in the process of transfecting Sf9 cells by PGL4.10 and the promoter recombinant plasmid of the MtGSTs1 gene of the Populomala esculenta for expression induction, and variance analysis is carried out on the luciferase detection result (figure 7). The result shows that the activity of the promoter of the MtGSTs1 gene shows a certain reduction (P is less than 0.05) under the stress of quercetin at each concentration, which indicates that the quercetin inhibits the expression of the promoter in Sf9 cells.

3) 2-tridecanone with different concentrations is added in the process of transfecting Sf9 cells by PGL4.10 and the promoter recombinant plasmid of the MtGSTs1 gene of the Populus canna, and variance analysis is carried out on the luciferase detection result (figure 8). The results show that the expression of the MtGSTs1 gene promoter is not remarkably changed under the stress of a certain concentration of 2-tridecanone.

The foregoing description is intended to be illustrative rather than limiting, and it will be understood by those skilled in the art that many modifications, variations, or equivalents may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Nanjing university of forestry

<120> promoter of glutathione S-transferase MtGSTs1 gene of populus canula and application

<130> 100

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 576

<212> DNA

<213> Micromelalopha troglodyta

<400> 1

gagttccacc gtgaccgacg aaggcttcga cgacacgagg gatcctgact gactcactgg 60

gcactcggtt ccgtggtgtc gtccctcacc aacagctcgc cacaaagctg cctgcgggag 120

tagttattgt tatatttaaa actagctgtg cccgcgactt cgtctgcgta gttatcgcgt 180

attctatttg tagctgagta gcgtgatatt atatagccta tactatgacc caacctccag 240

gctatcaaca tgccaaattt caactcaatc cttccagtag tttggctgtg aagctcggac 300

aaacatatac actaaataga ttactgctta ccccctgtga tgagtagcgt gatattatat 360

agcctatact atgaccaagc ctcgtatcta tcaacatgcc aaattacaaa tcaatccgac 420

cagtggtttg gctgtgaagc ttggacaaac ataaatacga acaatcagac aaacagacag 480

acaaaaattc taaaaaccat agatttggca tcagcatcgt ttctagagta cgtaatgatc 540

actttataaa aaaatgtaca gatttttcat cgttac 576

<210> 2

<211> 48

<212> DNA

<213> Adaptor Primer1 Primer sequence (Artificial)

<400> 2

gtaatacgac tcactatagg gcacgcgtgg tcgacggccc gggctggt 48

<210> 3

<211> 8

<212> DNA

<213> Primer2 Primer sequence of Nested adapter (Artificial)

<400> 3

accagccc 8

<210> 4

<211> 22

<212> DNA

<213> Common1 primer sequence (Artificial)

<400> 4

gtaatacgac tcactatagg gc 22

<210> 5

<211> 19

<212> DNA

<213> Common2 primer sequence (Artificial)

<400> 5

actatagggc acgcgtggt 19

<210> 6

<211> 30

<212> DNA

<213> MtGSTs1 primer sequence (Artificial)

<400> 6

aaccttacat cctcaaattc ctgtttagtg 30

<210> 7

<211> 26

<212> DNA

<213> MtGSTs 12 primer sequence (Artificial)

<400> 7

gacgagcttc aggttgagtt ggatgt 26

<210> 8

<211> 20

<212> DNA

<213> MtGSTs 1F primer sequence (Artificial)

<400> 8

aactcgagcg acgaaggctt 20

<210> 9

<211> 24

<212> DNA

<213> MtGSTs 1R primer sequence (Artificial)

<400> 9

aagctagcat ttgctgcact atca 24

Claims (6)

1. A promoter of a gene MtGSTs1 of the glutathione S-transferase of the populus canescens has a nucleotide sequence shown as SEQ ID NO. 1.

2. An expression vector comprising the promoter of the gene MtGSTs1 of glutathione S-transferase of Populus canula as described in claim 1.

3. A host cell comprising the expression vector of claim 2.

4. The host cell of claim 3, wherein the host cell is an insect cell.

5. The use of the promoter of glutathione S-transferase MtGSTs1 gene of Populus canula as claimed in claim 1 for promoting the expression of downstream gene in host cell.

6. The use of promoter of glutathione S-transferase MtGSTs1 gene of Populus canula as claimed in claim 1 for promoting expression of MtGSTs1 gene.

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