CN111979234B - Promoter of gene MtGSTz1 of glutathione S-transferase of Populus parva and application - Google Patents
Promoter of gene MtGSTz1 of glutathione S-transferase of Populus parva and application Download PDFInfo
- Publication number
- CN111979234B CN111979234B CN201910431713.6A CN201910431713A CN111979234B CN 111979234 B CN111979234 B CN 111979234B CN 201910431713 A CN201910431713 A CN 201910431713A CN 111979234 B CN111979234 B CN 111979234B
- Authority
- CN
- China
- Prior art keywords
- gene
- promoter
- mtgstz1
- populus
- glutathione
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
- C12N9/1088—Glutathione transferase (2.5.1.18)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
- C12Y205/01018—Glutathione transferase (2.5.1.18)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Abstract
The invention discloses a promoter of a glutathione S-transferase MtGSTz1 gene of a Populus parviana and application thereof, belonging to the technical field of genetic engineering, wherein the nucleotide sequence of the promoter of the glutathione S-transferase MtGSTz1 gene of the Populus parviana is shown as SEQ ID NO. 1. The promoter of the MtGSTz1 gene of the Populus canula 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 the specific action mechanism of the plant secondary substances on the expression regulation and control of the MtGSTz1 gene of the Populus canula, 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
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 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, CYP450) 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 with 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 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 parvifolius GSTs, which can be promoted in host cells under the induction of plant secondary biomass. 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 populus canula 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 populus canula glutathione S-transferase MtGSTz1 gene has a nucleotide sequence shown in SEQ ID NO. 1.
An expression vector contains the promoter of the gene MtGSTz1 of the Populus parva glutathione S-transferase.
A host cell contains the expression vector.
Preferably, the host cell is an insect cell.
The promoter of the glutathione S-transferase MtGSTz1 gene of the Populus canula is applied to starting the expression of downstream genes in host cells.
The application of the promoter of the populus canula glutathione S-transferase MtGSTz1 gene in improving the resistance of insect cells to toxic substances.
The application of the promoter of the cottonwood moth glutathione S-transferase MtGSTz1 gene in promoting expression of the MtGSTz1 gene is provided.
Has the beneficial effects that: compared with the prior art, the promoter of the MtGSTz1 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 the specific action mechanism of the plant secondary substances on the expression regulation and control of the MtGSTz1 gene of the populus canescens, provides a theoretical basis and a gene sequence for a new control technology of the populus canescens defoliators, and also can 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 an electrophoresis detection map of PCR products of promoter of Mariothis virescens MtGSTz 1; in the figure, M: marker, 5: the MtGSTz1 promoter;
FIG. 3 is a diagram showing the results of enzyme digestion verification of the PGL4.10-MtGSTz1 promoter recombinant plasmid; in the figure, M: marker, 1: PGL4.10, 2: PGL4.10 double enzyme digestion, 3: PGL4.10-MtGSTz1, 4: PGL4.10-MtGSTz1 single enzyme digestion, 5: carrying out double enzyme digestion on PGL4.10-MtGSTz1 promoter;
FIG. 4 is a PGL4.10 plasmid map;
FIG. 5 is a diagram showing the result of detecting the promoter activity of the MtGSTz1 gene of Populus canula;
FIG. 6 is a diagram showing the result of detecting the promoter activity of the promoter of the Marshall plusia xylostella MtGSTz1 gene under the stress of tannin;
FIG. 7 is a diagram showing the result of detecting the promoter activity of the Mariothis virescens MtGSTz1 gene under the stress of quercetin;
FIG. 8 is a diagram showing the result of detecting the promoter activity of the Marsupenaeus populi MtGSTz1 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 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 boat moth (Micromelalopha troglodyta) is collected from Wujiang town of Pukou district of Nanjing, Jiangsu province, and taken back indoors, and then is kept at a constant temperature of 26 +/-1 ℃, and h light: feeding under the conditions of 16: 8 of darkness and 75% of relative humidity. 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 Trypone, 5g of Yeast Extract Yeast Extract and 10g of NaCl10g by using an electronic balance, adjusting the pH value to 7.0 by using NaOH, and adding ddH 2 O constant volume is 1L, and autoclaving is carried out at 120 deg.C for 20 min.
(2) LB solid culture medium
Adding 15g agar powder into 1LLB liquid culture medium, and autoclaving at 120 deg.C for 20 min.
(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 100 mL.
(4) Ampicillin stock solution (100mg/mL)
100mg of ampicillin are weighed out and dissolved in 1ml ddH 2 Storing at-20 deg.C in O.
(5) Preparation of X-Gal solution
20mgX-Gal was weighed out and dissolved in 1mL of dimethylformamide and stored at-20 ℃.
Example 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 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′，
MtGSTz1 1∶5′-CTTGTCTCTTCCAGGTAGTGCATTATGTTC-3′，
MtGSTz1 2∶5′-ATGGGATCTCCTTCAAGTTGAGTGCG-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 DNAssoReagent liquid, 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 by gentle inversion, 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 well, and heated in a microwave oven until completely dissolved. After the solution was 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. And (3) after the glue solution is solidified, pulling out the comb, putting the glue into an electrophoresis tank, and taking 1 mu LRNA for electrophoresis detection. Set constant voltage 140V, take pictures on an automated gel imaging system after running for 30 min. 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 strip and is detected by Nano Drop 1000: the concentration of RNA was 6722.3 ng/. mu.L, OD 260/ OD 280 A value of 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 Premier 5 software. The reaction solution (25. mu.L) was prepared as follows: 2. mu.L of 10 XNEBuffer,. mu.L of DNA, 0.5. mu.L of enzyme, 22.5-XddH 2 O; x: a volume was calculated from the DNA concentration using 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. Twice the volume of 95% ethanol, 1/10 volumes 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 of the long Primer and the short Primer of the Nested adapter 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 adapter Primer.
(2) Ligation of adaptor primers
The reaction solution (8. mu.L) was prepared by the following components: 1.6 μ L10 Xligation Buffer, 4 μ LDNA, 1.9 μ LAdaptor Primer, 0.5 μ L T4DNA Ligase.
Reaction procedure: 16 ℃ overhead night; 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. The PCR reaction (25. mu.L) was prepared as follows: 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 ℃ for 25s, 72 ℃ for 3min }7 cycles; {94 ℃ for 25s, 67 ℃ for 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; the PCR reaction (25. mu.L) was prepared as follows: 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 ℃ for 25s, 72 ℃ for 3min }5 cycles; {94 ℃ for 25s, 67 ℃ for 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 minibestagagose Gel dnaextraction Kit ver3.0 recovery Kit (centrifugal column type) from TaKaRa corporation, 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 (pH5.2) 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 BufferWB was added to Spin Column, centrifuged at 12000rpm at room temperature for 30sec, and the filtrate was discarded. (7) Repeating the above 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 1 min. (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 pDM19T vector. Ligation composition (5 μ L): 2.5 μ L of solution I buffer, X μ L of DNA (recovered), 0.5 μ L of pDM19T vector, (2-X) μ L of ddH 2 O; x: the DNA was three times the number of moles of the vector, the volume was calculated based on the DNA concentration, and less than 2. mu.L of the recovered DNA was made up with water. Reaction procedure: 16 ℃ for 2 h.
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 30 min. (2) And thermally shocking the mixture in a water bath at 42 ℃ for 60sec, and immediately placing the mixture on ice for 3-5 min. (3) Adding 800 μ LSOC culture medium, shaking at 37 deg.C and 150rpm for 1 h. (4) Centrifuging at 4000 Xg for 3min, discarding the supernatant, leaving about 150-200 mu L of bacterial solution, and gently blowing and beating the precipitate to uniformly mix the precipitate. (5) The bacterial liquid is coated on LB solid plate culture medium with penicillin and X-Gal, and is inverted at 37 ℃ for overnight culture. (6) When the bacteria appear on the culture dish, selecting white single colony, selecting the single colony into 800 mu L LB liquid culture medium containing Amp by using a sterile toothpick, and culturing at 37 ℃ under 250-rotation shaking table.
The transformed bacterial liquid was subjected to PCR detection in the following reaction system (25. mu.L): 1 μ L of bacterial liquid, 1 μ L of Primer2, 1 μ L of LCommon 2, 12.5 μ L of LMix Ex-Tag，9.5μLddH 2 And O. PCR reaction procedure: 5min at 94 ℃; {94 ℃ for 25s, 72 ℃ for 3min }5 cycles; {94 ℃ for 25s, 67 ℃ for 3min }30 cycles; 7min at 67 ℃; 4 ℃ forever. And (4) 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 result is shown in FIG. 2, the promoter of 1 Populus mauritiana GSTs gene was successfully cloned, named as the promoter of the Populus mauritiana MtGSTz1 gene, the sequence length of which was 1142bp (containing ATG). The possible transcription initiation sites are predicted by using bioinformatics online prediction software (http:// www.Gene-regulation. com/pub/programs/alibaba2/index. html. and http:// www.fruitfiy.org/SEQ _ tools/promoter. htm1), and according to the prediction result, the length of the promoter sequence of the MtGSTz1 gene is 543bp, 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:
MtGSTz1 gene primer F: 5'-AACTCGAGGGCACGCGTG-3', respectively;
MtGSTz1 gene primer R: 5'-AAGCTAGCTTGTAAGTCGGTATGTATGTAA-3' are provided.
The primers were synthesized by Shanghai Czeri Biotechnology, Inc.
2) Extracting a promoter plasmid of a cottonwood huckle moth GSTs gene: (1) a single colony is selected from a successfully cloned promoter plate culture medium and inoculated into 1-4 mL of LB liquid culture medium containing antibiotics, and the culture is carried out overnight at 37 ℃ (12-16 h). (2) 1-4 mL of overnight culture broth 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 5-6 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 the Solution III up and down for 5-6 times until compact aggregates are formed, and then standing the aggregates for 2min at room temperature. (6) Centrifuge at 12,000rpm for 10min at room temperature and collect the supernatant. (7) Spin Column in the kit was mounted on the Collection Tube. (8) The supernatant from operation 6 was transferred to Spin Column, centrifuged at 12,000rpm for 1min, and the filtrate was 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) 700 μ L of BufferWB was added to Spin Column, centrifuged at 12,000rpm for 30sec, and the filtrate was discarded. (11) Operation step 10 is repeated. (12) The 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 an ElutionBuffer was added to the center of the Spin Column membrane, and allowed to stand at room temperature for 1 min. (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: using the plasmid as a template, a PCR reaction system (25. mu.L) was prepared by the following components: XuL plasmid, 1 uL forward primer, 1 uL reverse primer, 12.5 uL Mix Ex-taq enzyme, (10.5-X) uL 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 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.
The extraction method of the plasmid added with the enzyme cutting site promoter is the same as the above.
Double enzyme digestion: the extracted plasmid was digested with the PGL4.10 vector with Nhe I and Xho I endonucleases, and the digestion reaction system (20. mu.L) was prepared as follows: XuL plasmid, 1 uL Xho I, 1 uL Nhe I, (18-X) uL ddH 2 O; x: take 1. mu.g plasmid DNA, calculate the volume based on 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): mu.L 10 × Ligation Buffer, X. mu.L DNA (digestion product), 0.5. mu.L PGL4.10 vector, 1. mu.LL T4DNA Ligase，(7.5-X)μL ddH 2 O; x: the DNA was three times as many moles as 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), the PGL4.10 and the MtGSTz1 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: culturing the insect Sf9 cells in a cell culture bottle by using an Sf-900II SFM cell culture medium containing 10% FBS (fetal bovine serum) and antibiotics, culturing in a constant-temperature biochemical incubator at 27 ℃ in a dark place, and subculturing for 2-3 d according to the cell state.
2) Cryopreservation of insect Sf9 cells: the undeformed cells with good growth state and less passage times need to be frozen and reserved, cells which are full of adherent cells are gently blown down, the cells are centrifuged for 10min at 5000g, and cell precipitates are suspended by precooled cell frozen stock solution (whole cell culture medium containing 10% DMSO). 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 30 min; 30min at-20 ℃; 30min at-80 ℃; and finally, placing the freezing tube in liquid nitrogen for storage and standby.
3) Resuscitation of insect Sf9 cells: adding the culture medium into a culture bottle, taking out the cell cryopreservation tube from liquid nitrogen, rapidly carrying out water bath at 27 ℃, taking out the cell cryopreservation tube after shaking until a small amount of ice remains, disinfecting the outer wall with alcohol, and absorbing the cryopreservation liquid into the culture bottle. And (3) performing light-tight culture in a constant-temperature biochemical incubator at 27 ℃, changing the liquid (generally less than 30min) after the cells are attached to the wall, changing the liquid after culturing for 24h, 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) selecting a single colony from a recombinant plasmid plate culture medium and inoculating the single colony to 50-100 mL of L containing antibioticsAnd B, culturing the cells in the liquid culture medium at 37 ℃ overnight (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 the mixture up and down for 3-5 times, and standing the mixture at room temperature for 3min to fully crack the thalli to form a transparent Solution. (5) 5mL of 4 ℃ precooled Neutralizationsolution is added and mixed by gently turning up and down for 5-10 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 30-60 sec by using a vacuum device. (12) Wiping off alcohol on the Binding Column, adding 400-600 mu 1 nucleic-Free Water, standing for 1min, and collecting with a new EP tube.
5) Adopting liposome transfection method invitrogen cellfection R II reagent transfected cells
(1) Paving a plate: washing cells to be passaged with 1mL of culture medium containing double antibody and serum, adding appropriate amount of culture medium containing double antibody and serum to blow up cells, packaging into 96-well plate, standing for more than 15min (9 × 10 per well) 4 One cell). (2) Preparing a transfection mixed solution (single-hole dosage) A solution: 1 μ L of liposomes, 10 μ L of serum-free and antibiotic-free medium; standing at room temperature for 30 min; 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 30 min. (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 to insect Sf9 cells for expression by a liposome transfection method, the luciferase activity is detected by double fluorescence after transfection for 48h, the ratio of the firefly fluorescence detection value (Luc) to the renilla fluorescence detection value (rLuc) is the final result, and the significant difference of the activity of each promoter relative to the vector control is analyzed by variance (P is less than 0.001, P is less than 0.05), as shown in FIG. 5. The results show that MtGSTz1 does not show a significant difference compared to the vector.
Example 3: inducible expression of plant secondary biomass on MtGSTz1 gene promoter recombinant plasmid
The main method is the same as example 2, in the process of cell transfection, 10 microliter of inducing medicine is added when the culture medium containing serum and double antibody is changed after the culture is carried out for 5 hours at 27 ℃, three plant secondary biologics of tannin, quercetin and 2-tridecanone are selected in the experiment to express and induce the promoter of the gene of the populus canescens GSTs, ddH is used for inducing the promoter of the gene of the populus canescens GSTs 2 Dissolving tannin with ddH, dissolving quercetin and 2-tridecanone with O, and respectively preparing into medicinal preparations with concentration of 0.01, 0.1 and 1mg/mL 2 O is a control, and quercetin and 2-tridecanone are 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 Mycoplasma gondii MtGSTz1 gene, and variance analysis is carried out on the luciferase detection result (figure 6). The result shows that under the stress of tannin, the promoter expression of the MtGSTz1 gene of the populus canescens is induced to a certain degree, wherein the promoter expression of the MtGSTz1 gene is obviously increased under the stress of tannin at the concentrations of 0.01 and 0.1mg/mL respectively (P is less than 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 populus canula MtGSTz1 gene, and variance analysis is carried out on the luciferase detection result (figure 7). The result shows that the MtGSTz1 gene promoter shows higher expression level (P < 0.05) relative to the control group under the stress of 0.1mg/mL quercetin.
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 populus canula MtGSTz1 gene, and variance analysis is carried out on the luciferase detection result (figure 8). The result shows that the expression level of the MtGSTz1 gene promoter shows a significant increase under the stress of 2-tridecanone with the concentration of 0.01mg/mL (P < 0.05).
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 MtGSTz1 gene of Populus canula and application thereof
<130> 100
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 543
<212> DNA
<213> Micromelalopha troglodyta
<400> 1
cttgcatgcc tgcaggtcga cgattactat agggcacgcg tggtcgacgg cccgggctgg 60
tctcaaataa taaattacat tcaaataata tgtcttttgt aaattgatat gaacgctata 120
cttacatttt taagtcccaa atttactgcc ttcgcttttg ttttctttaa acttttagca 180
ccagctacct tgaagacgtt ggcattagga ccctttttct tatttttccc cattatcctt 240
ctttattact aaaatgaatt cgtgaagaaa aataaatcca aatttaacct caaaacaatg 300
aaatgtcaat tatgacaatc acatgtctta tttggaaacg ttttattaat attagaaaga 360
aagcatggat ttaaaaacat tttctatttt gggaaattta ttacatgata atgattttaa 420
tttaaaaata tccaaaacat atgggtgaat atttatagag aaacaaatta acgattgaag 480
aaaaatcaca gagagatata ataaaacgct aatgtaaaat aggcgactgc tggctgcaat 540
tag 543
<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
<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> MtGSTz 11 primer sequence (Artificial)
<400> 6
cttgtctctt ccaggtagtg cattatgttc 30
<210> 7
<211> 26
<212> DNA
<213> MtGSTz 12 primer sequence (Artificial)
<400> 7
atgggatctc cttcaagttg agtgcg 26
<210> 8
<211> 18
<212> DNA
<213> MtGSTz 1F primer sequence (Artificial)
<400> 8
<210> 9
<211> 30
<212> DNA
<213> MtGSTz 1R primer sequence (Artificial)
<400> 9
aagctagctt gtaagtcggt atgtatgtaa 30
Claims (6)
1. A promoter of a populus canula glutathione S-transferase MtGSTz1 gene has a nucleotide sequence shown in SEQ ID NO. 1.
2. An expression vector comprising the promoter of the gene MtGSTz1 for 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 MtGSTz1 of Populus canula as claimed in claim 1 in promoting the expression of downstream genes in host cells.
6. The use of promoter of glutathione S-transferase MtGSTz1 of Populus canula as claimed in claim 1 for promoting expression of MtGSTz1 gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910431713.6A CN111979234B (en) | 2019-05-22 | 2019-05-22 | Promoter of gene MtGSTz1 of glutathione S-transferase of Populus parva and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910431713.6A CN111979234B (en) | 2019-05-22 | 2019-05-22 | Promoter of gene MtGSTz1 of glutathione S-transferase of Populus parva and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111979234A CN111979234A (en) | 2020-11-24 |
| CN111979234B true CN111979234B (en) | 2022-09-23 |
Family
ID=73437048
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910431713.6A Active CN111979234B (en) | 2019-05-22 | 2019-05-22 | Promoter of gene MtGSTz1 of glutathione S-transferase of Populus parva and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111979234B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160524A (en) * | 2013-03-27 | 2013-06-19 | 西南大学 | Bombyx mori glutathione-S-transferase BmGSTe4 gene |
-
2019
- 2019-05-22 CN CN201910431713.6A patent/CN111979234B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160524A (en) * | 2013-03-27 | 2013-06-19 | 西南大学 | Bombyx mori glutathione-S-transferase BmGSTe4 gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111979234A (en) | 2020-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107502608A (en) | Construction method and application for sgRNA, ALDH2 gene delection cell line for knocking out people's ALDH2 genes | |
| US20180171302A1 (en) | Genetically Modified IPS Cells That Carry a Marker to Report Expression of Neurogenin3, TPH2, FOXO1 and/or Insulin Genes | |
| CN107760707A (en) | A kind of foundation for the self-activation Gal4/UAS system expression boxes for strengthening gene expression | |
| CN113234723B (en) | Japanese eel cytokine IL-6 gene promoter and application thereof | |
| CN111979234B (en) | Promoter of gene MtGSTz1 of glutathione S-transferase of Populus parva and application | |
| Zieler et al. | Intron‐dependent stimulation of marker gene expression in cultured insect cells | |
| CN110747199B (en) | Bee stress-resistance related gene NF-Y and application thereof | |
| CN103233010B (en) | Large yellow croaker antimicrobial peptide hepcidin gene promoter sequence and application thereof | |
| CN111979231B (en) | Promoter of glutathione S-transferase MtGSTd2 gene of populus canula and application | |
| CN111979235B (en) | Promoter of glutathione S-transferase MtGSTt1 gene of populus canula and application | |
| CN111979236B (en) | Promoter of gene MtGSTs1 of glutathione S-transferase of Populus canula and application | |
| CN111979232B (en) | Promoter of gene MtGSTo1 of glutathione S-transferase of populus canula and application | |
| CN113201541B (en) | Japanese eel transcription factor AP-1 gene promoter and application thereof | |
| CN115779084A (en) | Application of preparation for activating pig TUSC1 gene expression in preparation of pig pseudorabies virus infection resisting medicine | |
| CN106811484A (en) | A kind of ox PDHB gene adenovirus interference carrier builds and its authentication method | |
| CN108384790A (en) | The method of the identification regulation and control active controlling element of FATP1 gene promoter transcriptions | |
| CN104059141B (en) | A kind of structure of mouse RANKL mutant and its expression vector, expression and application | |
| CN113717973B (en) | Japanese eel liver expression antibacterial peptide LEAP2 gene promoter and application thereof | |
| CN113564169B (en) | Japanese eel antibacterial peptide Cathelicidin2 gene promoter and application thereof | |
| CN109321575B (en) | Pig adipose tissue specific promoter and application thereof | |
| CN113388626B (en) | Application of novel coronavirus NSP13 gene | |
| CN111206034B (en) | New application of pig GADD45a gene and construction and application of high-expression cell line | |
| CN113584085B (en) | Lentiviral vector for suspension cells and application thereof | |
| CN102628061A (en) | HBD3 mammary gland specific expression vector and constructed recombinant cell | |
| CN113564165A (en) | Cell strain for intracellular editing key gene of pseudorabies virus and construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |