CN111979231B - Promoter of glutathione S-transferase MtGSTd2 gene of populus canula and application - Google Patents

Abstract

The invention discloses a promoter of a glutathione S-transferase MtGSTd2 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 MtGSTd2 gene of the populus canescens is shown as SEQ ID NO. 1. The promoter of the MtGSTd2 gene of the populus canger 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 MtGSTd2 gene of the populus canger, provides a theoretical basis and a gene sequence for a new prevention and control technology of poplar leaf-eating pests, and can also provide an alternative gene for culturing insect cells or transgenic silkworms with higher resistance to toxic substances.

Description

Promoter of glutathione S-transferase MtGSTd2 gene of populus canula and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a promoter of a glutathione S-transferase MtGSTd2 gene of a populus canescens and application thereof.

Background

In long-term evolution, the close interrelationship between plants and insects is largely reflected in the diversity of plant secondary materials, and insects evolve with precise adaptability and resistance to adapt to it. 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 influence of endogenous or exogenous toxic substances is relieved by combining endogenous glutathione with harmful electrophilic groups and discharging the combination in vitro or combining non-covalently combined protein and hydrophobic ligands, thereby playing a role in detoxification.

The promoter is an important cis-element for gene expression regulation, a 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 substance can induce the activity of detoxifying 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. The invention also aims to provide a vector of a promoter sequence of the populus canula GSTs gene, which can start 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 canula glutathione S-transferase MtGSTd2 gene has a nucleotide sequence shown in SEQ ID NO. 1.

An expression vector contains the promoter of the gene MtGSTd2 of the populus canula glutathione S-transferase.

A host cell contains the expression vector.

Preferably, the host cell is an insect cell.

The promoter of the gene MtGSTd2 of the cottonwood huckle glutathione S-transferase is applied to promoting the expression of downstream genes in host cells.

The application of the promoter of the gene MtGSTd2 of the glutathione S-transferase of the populus canula in improving the resistance of insect cells to toxic substances.

Application of promoter of glutathione S-transferase MtGSTd2 gene of populus canula in promoting expression of MtGSTd2 gene

Has the beneficial effects that: compared with the prior art, the promoter of the MtGSTd2 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 MtGSTd2 gene of the populus canescens, provides a theoretical basis and a gene sequence for a new control technology of the populus canescens defoliators, 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 of larvae of Populus plutella;

FIG. 2 is a diagram showing the result of electrophoresis detection of PCR products of the promoter MtGSTd2 of Populus mauritiana; in the figure, M: marker,2: the MtGSTd2 promoter;

FIG. 3 is a diagram showing the results of enzyme digestion verification of the MtGSTd2 promoter recombinant plasmid; in the figure, M: marker,1: PGL4.10,2: PGL4.10 double enzyme digestion, 8: carrying out double enzyme digestion on PGL4.10-MtGSTd2 promoter;

FIG. 4 is a PGL4.10 plasmid map;

FIG. 5 is a diagram showing the result of detecting the promoter activity of the MtGSTd2 gene of Populus mauritiana;

FIG. 6 is a diagram showing the result of detecting the promoter activity of the MtGSTd2 gene of Populus plutella under the stress of tannin;

FIG. 7 is a diagram showing the result of detecting the promoter activity of the promoter of the Mariothis virescens MtGSTd2 gene under the stress of quercetin;

FIG. 8 is a diagram of the result of detecting the promoter activity of the promoter of the Mariothis virescens MtGSTd2 gene under the stress of tridecanone.

Detailed Description

The invention is further described with reference to specific examples. The molecular biology 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. Sambuchok, or by 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.

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

Preparation of reagents:

(1) LB liquid culture Medium

Weighing 10g of tryptone by using an electronic balance,yeast Extract 5g, naCl 10g, pH adjusted to 7.0 with NaOH, and ddH ₂ O constant volume to 1L, and autoclaving at 120 deg.C for 20min.

(2) LB solid culture 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 And O is metered to 100mL.

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

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

(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. Design of primers

Designing a joint primer and a universal primer according to a chromosome walking method, and designing two specific primers by using PrimerPremier 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′，

MtGSTd21：5′-ATCGTCTACTATCGTGGGAATGGTGTG-3′，

MtGSTd2 2：5′-GACGAGCTTCAGGGTTGAGTTGGATGT-3′。

the primers were synthesized by Shanghai Czeri Biotechnology, inc.

2. Extracting genomic DNA of Populus plutella by using DNAiso Reagent kit (TaKaRa)

(1) Placing the dissected Populus mauritiana 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) Add 1mL of 75% ethanol along the tube wall, wash the tube wall gently upside down, centrifuge at 12000 Xg for 5min at 4 ℃ and 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 l.times.TAE 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 well, and heated to complete dissolution in a microwave oven. 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 DNA of the Populus parviana is shown in figure 1, and the DNA has obvious bands 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 plutella xylostella is better in quality and can be used for subsequent experiments.

3. Enzyme digestion

The appropriate enzyme was selected for digestion using Primer Premier5 software. The reaction solution (25. Mu.L) was prepared with 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 procedure: 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) were 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 long Primer and short Primer of the Adaptor Primer1 and the Nested Adaptor Primer2 are mixed, put into water which is just boiled, and cooled to room temperature 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 μ L10 × Ligation Buffer,4 μ L DNA,1.9 μ L adapter Primer,0.5 μ L T 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 }7cycles; {94 ℃ 25s,67 ℃ 3min }32cycles; 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 }5cycles; {94 ℃ 25s,67 ℃ 3min }30cycles; 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 products

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 for 1min at 12000rpm and the filtrate 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: 16 ℃ for 2h.

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) Jiang Junye was plated on LB solid plate medium supplemented with penicillin and X-Gal, and cultured overnight at 37 ℃ by inversion. (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 ℃.

PCR detection of the transformed bacterial liquidThe reaction system was measured as follows (25. Mu.L): mu.L of bacterial solution, 1. Mu.L of Primer2, 1. Mu.L of Common2, 12.5. Mu.L of Mix Ex-Tag, 9.5. Mu.L of ddH ₂ And O. PCR reaction procedure: 5min at 94 ℃; {94 ℃ 25s,72 ℃ 3min }5cycles; {94 ℃ 25s,67 ℃ 3min }30cycles; 7min at 67 ℃;4 ℃ forever. And (3) carrying out electrophoresis detection on the reaction solution, 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 results are shown in FIG. 2,1 promoter of the Dactylopsis poplars GSTs gene was successfully cloned, named as promoter of the Dactylopsis poplars MtGSTd2 gene, and the sequence length was 908 (containing ATG). The possible transcription start sites are 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). The length of the promoter sequence of the MtGSTd2 gene is 642 according to the prediction result, and is shown as SEQ ID NO.1 in the sequence table.

Example 2: activity verification of promoter of glutathione S-transferase gene of Populus mauritiana

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 populus canula GST gene promoter are as follows:

MtGSTd2F：5′-AACTCGAGAGATTACTATAGGGCACG-3′；

MtGSTd2R：5′-AAGCTAGCAAAAATTCCTCAACATAAA-3′。

the primers were synthesized by Shanghai Czeri Biotechnology, inc.

2) Extracting a promoter plasmid of a cottonwood huckle moth GSTs gene: (1) Single colonies were picked from the successfully cloned promoter plate medium and inoculated into 1-4 mL LB liquid medium containing antibiotics and cultured overnight at 37 ℃ (12-16 h). (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 of Solution II, slightly turning and mixing the mixture up and down for 5 to 6 times to ensure that the thalli are fully cracked to 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 Buffer WB 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 }25cycles; 10min at 72 ℃;4 ℃ forever. The reaction solution was subjected to agarose gel electrophoresis for detection.

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 was as above.

Double enzyme digestion: the extracted plasmid and PGL4.10 vector are subjected to double enzyme digestion by using Nhe I and Xho I endonucleases, and an enzyme digestion reaction system (20 mu L) is prepared according to the following components: * X μ L plasmid, 1 μ L Xho I,1 μ L Nhe I, (18-X) μ L ddH ₂ O; * X: mu.g of plasmid DNA was taken and the volume was calculated from the DNA concentration. And (3) enzyme digestion reaction program: 2h at 37 ℃;

connecting: the target vector PGL4.10 was double digested with the same enzymes for ligation, with the ligation components consisting of (10 μ L): 1 μ L10 × Ligation BufferL DNA (enzyme digestion product), 0.5. Mu.L PGL4.10 vector, 1. Mu. L T4DNA Ligase, (7.5-X). Mu.L ddH ₂ O; * X: the number of moles of DNA is three times that of the carrier and the volume is calculated from the DNA concentration. Reaction procedure: 16 ℃ for 2h.

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 MtGSTd2 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 cell amount can be counted by 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 deg.C 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) Selection of sheets from recombinant plasmid plating MediumThe colonies were inoculated into 50 to 100mL of LB liquid medium containing antibiotics and cultured overnight at 37 ℃ (12 to 16 hours). (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) The liposome transfection method is adopted to obtain the invitrogen cellinfection ^R II reagent transfected cells:

(1) Plate paving: 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 Individual cells). (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 the culture medium in the plated cells, adding 80 mu L of serum-free and antibiotic-free culture medium, adding 20 mu L of transfection mixed solution, culturing at 27 ℃ for 5h, then replacing 150 mu L of serum-containing and double-antibody-containing culture medium, culturing at 27 ℃ for 48h, and performing double fluorescence detection.

The promoter recombinant plasmid is transferred into an insect Sf9 cell for expression by a liposome transfection method, the activity of luciferase is detected by using double fluorescence 48h after transfection, the ratio of a firefly fluorescence detection value (Luc) to a Renilla fluorescence detection value (rLuc) is the final result, and the significant difference of the activity of each promoter relative to a vector control is analyzed by using variance (P is less than 0.001, and P is less than 0.05), as shown in figure 5. The result shows that the MtGSTd2 gene promoter has very strong activity and extremely strong significance compared with a vector.

Example 3 inducible expression of Poplar canula moth promoter recombinant plasmids by plant secondary Biomass

The main method is the same as the embodiment 2, 10 mul of inducing medicine is added when the culture medium containing serum and double antibody is changed after the cell is cultured for 5h at 27 ℃ in the process of cell transfection, three plant secondary organisms of tannin, quercetin and 2-tridecanone are selected in the embodiment to express and induce the promoter of the MtGSTd2 gene of the populus canescens, and ddH is used for expression and induction 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 MtGSTd2 gene of the populus canescens, and variance analysis is carried out on the luciferase detection result (figure 6). The result shows that under tannin stress, the MtGSTd2 gene promoter of the populus canescens is induced to a certain extent, and the expression level of the MtGSTd2 gene promoter is obviously increased under tannin stress at the concentration of 0.01 and 0.1mg/mL (P < 0.05).

2) Quercetin with different concentrations is added in the process of transfecting Sf9 cells by PGL4.10 and the promoter recombinant plasmid of the MtGSTd2 gene of the populus canger, expression induction is carried out, and variance analysis is carried out on the luciferase detection result (figure 7). The result shows that the stress of 0.1mg/mL quercetin plays a remarkable induction role in the expression of the MtGSTd2 gene promoter in Sf9 cells (P < 0.05).

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 Mycoplasma gondii MtGSTd2 gene, and variance analysis is carried out on the luciferase detection result (figure 8). The result shows that the expression of the MtGSTd2 gene promoter is remarkably inhibited 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 appreciated 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 MtGSTd2 gene of populus canula and application

<130> 100

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 642

<212> DNA

<213> Micromelalopha troglodyta

<400> 1

gctcggtacc cggggatcct ctagagatta ctatagggca cgcgtggtcg acggcccggg 60

ctggtctgcg agtggcgcac gctgttgact aacggatgag gttctgacga cagaccgaca 120

gatggcgaaa aatttttttt tagccttgat aatttataat aattttattt cccatattgt 180

tcattgtttt gtgaaggtct gtatagcacg ctcgtttggc agaaatttct atcactttgt 240

attacagcct agcttagctg gttgctggca tttgagttgt catgctctct aaaaaatcct 300

gacaatggtg gcgttgtgtg tcgggttgtc cacttcccta gaaaatggtt gaggtgagca 360

gtggctgaca cacacgtcat gcttgtgaac aggcgccatt gctcgggatt ggtcatctca 420

cttgtcattt taactttcca ttgctacgag ccttcttcca tagatctaaa gtgtgctatc 480

tatctatgta ttgaatcatt gtttggcgat aaaagagtaa ctgtagagtt tcttactggt 540

tcttctctac agagtcaact ttacgaaccg gcggtaactg taattgtatt tgacgattca 600

tctatactaa tatataaagc tgaagagttt gtttgtttgg tt 642

<210> 2

<211> 48

<212> DNA

<213> Adaptor Primer1(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> 27

<212> DNA

<213> MtGSTd2 primer sequence (Artificial)

<400> 6

atcgtctact atcgtgggaa tggtgtg 27

<210> 7

<211> 26

<212> DNA

<213> MtGSTd2 primer sequence (Artificial)

<400> 7

gacgagcttc aggttgagtt ggatgt 26

<210> 8

<211> 26

<212> DNA

<213> MtGSTd 2F primer sequence (Artificial)

<400> 8

aactcgagag attactatag ggcacg 26

<210> 9

<211> 27

<212> DNA

<213> MtGSTd 2R primer sequence (Artificial)

<400> 9

aagctagcaa aaattcctca acataaa 27

Claims (6)

1. A promoter of a gene MtGSTd2 of a glutathione S-transferase of a populus canescens, the nucleotide sequence of which is shown as SEQ ID NO. 1.

2. An expression vector comprising the promoter of the gene MtGSTd2 of glutathione S-transferase from 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 the glutathione S-transferase, mtGSTd2, of the Populus canula as claimed in claim 1 to promote the expression of downstream genes in host cells.

6. The use of promoter of glutathione S-transferase MtGSTd2 gene of Populus canula as claimed in claim 1 for promoting the expression of MtGSTd2 gene.

Images