CN111733181A - Construction method of biosafety ABCG11 gene overexpression vector - Google Patents
Construction method of biosafety ABCG11 gene overexpression vector Download PDFInfo
- Publication number
- CN111733181A CN111733181A CN202010332370.0A CN202010332370A CN111733181A CN 111733181 A CN111733181 A CN 111733181A CN 202010332370 A CN202010332370 A CN 202010332370A CN 111733181 A CN111733181 A CN 111733181A
- Authority
- CN
- China
- Prior art keywords
- enzyme digestion
- pmi
- flag
- gene
- vector
- 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.)
- Pending
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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
- C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a construction method of a biosafety ABCG11 gene overexpression vector, which comprises the following steps: (1) after the fusion gene vector plasmid is subjected to double enzyme digestion, carrying out gel running on the enzyme digestion product; (2) carrying out step-by-step enzyme digestion on the safety marker gene and the target gene expression vector plasmid; carrying out double enzyme digestion on the epidermis specific promoter and the vector plasmid, taking pmi as a biosafety marker gene, carrying out step-by-step enzyme digestion on the gene and the vector plasmid, and connecting and transforming to obtain the vector. The contained pmi gene is a coding gene of a key carbohydrate metabolism enzyme 6-phosphomannose isomerase and is used as a novel safe selective marker gene, and the novel safe selective marker gene has the advantages of high selection, high transformation and strong safety which cannot be matched with other selective marker genes, and can not bring potential harm to the health and ecological balance of human beings and livestock; in addition, the promoter is an epidermal specific promoter, and can drive the target gene to specifically express in epidermal cells on the overground part of the plant, so that the drought resistance of the plant is improved.
Description
Technical Field
The invention relates to a construction method of a gene over-expression vector, in particular to a construction method of a biosafety type ABCG11 gene over-expression vector.
Background
Since 1973 recombinant DNA technology became available, plant genetic engineering has rapidly progressed. To date, over 100 transgenic crops have been successfully obtained, 25 of them have been approved for commercial production to bring multiple benefits to farmers and social development, including agricultural, environmental, economic, health and social benefits (James, 2015). In plant genetic engineering, plant expression vectors play a role as vectors to transform target genes and marker genes into target improved crops. Among them, the currently used selection marker genes of antibiotics and herbicides have raised public question about the safety of transgenic plants, and such marker genes may bring potential harm to the health, ecological balance and the like of human beings and livestock, and are one of the most important factors influencing the commercialization of transgenic plants. In addition, since the target gene (ZxABCG11) is a protein related to the lipid secretion of stratum corneum, studies have shown that the constitutive promoter 35s driving the expression of the lipid-related genes CER6 and WXP1 causes the growth and development of plants to be delayed and even wax deposition to fail (Zhang et al, 2005).
Disclosure of Invention
In order to solve the problems, the invention provides a construction method of a biosafety type ABCG11 gene overexpression vector.
The invention is realized by the following technical scheme: a construction method of a biosafety ABCG11 gene overexpression vector comprises the following steps: (1) after the fusion gene ZxABCG11-FLAG and pCAMBIA1302 carrier plasmid are subjected to double enzyme digestion, carrying out gel running on the enzyme digestion product, recovering the obtained target fragment and carrying out connection transformation on the enzyme digestion carrier to obtain a target plant expression carrier pCAMBIA 1302-ZxBCG 11-FLAG; (2) carrying out step-by-step enzyme digestion on safety marker gene pmi and pCAMBIA1302-ZxABCG11-FLAG vector plasmids, respectively recovering, connecting and transforming corresponding target fragments after the enzyme digestion is finished, and obtaining a biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector; (3) carrying out double enzyme digestion on an epidermal specific promoter AtCER6 and pCAMBIA1302-ZxABCG11-FLAG-pmi vector plasmid, respectively recovering and connecting and transforming corresponding target fragments after the enzyme digestion is finished, and finally obtaining a biosafety ABCG11 gene overexpression vector pCAMBIA1302-AtCER 6-ZxBCG 11-FLAG-pmi, wherein the pmi in the step (2) is a biosafety marker gene, and the vector obtained after the gene and the pCAMBIA 1302-ZxBCG 11-FLAG vector plasmid are subjected to stepwise enzyme digestion and are connected and transformed is: the biosafety pCAMBIA 1302-ZxACG 11-FLAG-pmi expression vector comprises the following steps:
s11, step-by-step enzyme digestion of the safety marker gene pmi:
step-by-step enzyme digestion 1: pMD19-T-pmi plasmid is taken as a template, F is CATGCCATGGATGCAAAAACTCATTAACTCAGTGC; GGGTNACCTTACAGCTTGTTGTAAACACGCGCT is used as a primer to carry out amplification to obtain a pmi recovered product, and the pmi recovered product is cut by Nco I enzyme for 4h in a water bath kettle at 37 ℃;
step-by-step enzyme digestion 2: then, the product recovered by the step-by-step enzyme digestion 1 is digested by Bst E II for 4 hours in a water bath kettle at 37 ℃;
step-by-step enzyme digestion of S12, pCAMBIA1302-ZxABC 11-FLAG vector plasmid:
step-by-step enzyme digestion 1: taking the plant expression vector pCAMBIA1302-ZxABCG11-FLAG in the step (1) as a template, and carrying out enzyme digestion for 4h by using Nco I in a water bath kettle at 37 ℃;
step-by-step enzyme digestion 2: then, the product recovered by the step-by-step enzyme digestion 1 is digested by Bst E II for 4 hours in a water bath kettle at 37 ℃;
s13, connecting the safety marker gene pmi with the enzyme digestion product recovered from the pCAMBIA1302-ZxABCG11-FLAG vector plasmid by using T4 ligase, and transforming in Escherichia coli DH5 alpha to obtain a vector: the biosafety pCAMBIA 1302-ZxACG 11-FLAG-pmi expression vector;
the other technical scheme adopted by the invention is as follows: a construction method of a biosafety ABCG11 gene overexpression vector is characterized in that a promoter AtCER6 in the step (3) is an epidermis-specific promoter, the promoter and a biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector plasmid are subjected to double enzyme digestion, connected and transformed to obtain the vector: an ultra-expression vector of the ABCG11 gene with biological safety. The method comprises the following steps:
s21, promoter AtCER6 double enzyme digestion:
using arabidopsis DNA as a template, F: CCCAAGCTTCTTCGATATCGGTTGTTGACG;
TCCCCCGGGCGTCGGAGAGTTTTAATGTAT is used as a primer to carry out amplification to obtain an AtCER6 recovered product, and enzyme digestion is carried out for 1h in a water bath kettle at 30 ℃ by using Hind III and Sma I, and then enzyme digestion is carried out for 1h at 37 ℃;
s22, double digestion of the biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi vector plasmid:
taking the biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector in the step (2) as a template, carrying out enzyme digestion on Hind III and Sma I for 1h in a water bath kettle at 30 ℃, and then carrying out enzyme digestion for 1h at 37 ℃;
s23, connecting the enzyme digestion products recovered by the epidermis-specific promoter AtCER6 and the biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector plasmid by using T4 ligase, and transforming in escherichia coli DH5 alpha to obtain a vector: the biosafety pCAMBIA1302-AtCER6-ZxABCG11-FLAG-pmi overexpression vector.
Compared with the prior art, the invention has the beneficial effects that:
the pmi gene contained in the plant vector constructed by the invention is a coding gene of carbohydrate metabolism key enzyme 6-phosphomannose isomerase, and as a novel safety selection marker gene, the plant vector has the advantages of high selection, high transformation, strong safety and the like which are not comparable to other selection marker genes, and can not bring potential harm to the health, ecological balance and the like of human beings and livestock; in addition, the AtCER6 promoter is an epidermis-specific promoter, and can drive the specific expression of the target gene ZxABCG11 in the epidermal cells of the overground parts of the plants, so that the drought resistance of the plants is improved. In view of the above, the vector constructed by the invention is a biosafety type overexpression vector, and the application of the vector provides a theoretical basis for cultivating transgenic pasture with strong drought resistance and popularizing the transgenic pasture.
Drawings
FIG. 1 is a flow chart of the construction of pCAMBIA1302-AtCER6-ZxABCG11-FLAG-pmi vector of the present invention;
FIG. 2 is a fusion of the ZxABCG11 of the present invention with a FLAG tag;
FIG. 3 is the gel electrophoresis chart of pCAMBIA1302-ABCG11-FLAG bacterial liquid of the present invention;
FIG. 4 is the enzyme cutting electrophoresis detection diagram of pCAMBIA1302-ABCG11-FLAG of the present invention;
FIG. 5 is an amino acid alignment of the sequencing results of the present invention with the ZxABCG11 calibration sequence;
FIG. 6 is a pCAMBIA1302-ABCG11-FLAG-pmi bacterial fluid gel electrophoresis chart of the present invention;
FIG. 7 is a diagram of the enzyme digestion electrophoresis detection of pCAMBIA1302-ABCG11-FLAG-pmi of the present invention;
FIG. 8 is an amino acid alignment of the sequencing results of the present invention with a pmi correction sequence;
FIG. 9 is a gel electrophoresis diagram of AtCER6: ZxABCG11-FLAG: 35s: pmi bacterial liquid of the present invention;
FIG. 10 is a graph showing the restriction enzyme digestion electrophoresis detection of AtCER6: ZxABCG11-FLAG: 35s: pmi in the present invention;
FIG. 11 is an amino acid alignment of the sequence results of the present invention with the corrected sequence of the AtCER6 promoter.
Detailed Description
The technical scheme in the embodiment of the invention will be made clear below by combining the attached drawings in the embodiment of the invention; fully described, it is to be understood that the described embodiments are merely exemplary of some, but not all, embodiments of the invention and that all other embodiments, which can be derived by one of ordinary skill in the art based on the described embodiments without inventive faculty, are within the scope of the invention.
A construction method of a biosafety ABCG11 gene overexpression vector comprises the following materials and methods before construction:
1. preparation of culture medium, antibiotics and reagents
The method comprises the following steps: preparation of 50mg/mL gentamicin sulfate: 2.25g Gent's mycin powder in 45mL ddH2Filtering the O medium filter membrane, and subpackaging in a 1.5mL centrifuge tube;
step two: preparation of 50mg/mL Rifampicin: 2.25g Rif mycin powder in 45mL ddH2Filtering the O medium filter membrane, and subpackaging in a 1.5mL centrifuge tube;
step three: 50mg/mL kanamycin preparation: 2.25g Kan mycin powderDissolved in 45mL ddH2In O, the mixture was filtered and dispensed into 1.5mL centrifuge tubes.
Step four: preparing a DNA extraction buffer solution mother solution: ddH at 800mL2Dissolving 121g Tris base in O, adjusting pH to required pH8.0 with concentrated HCl, controlling pH at 42mL, adding water to desired volume of 1000mL, subpackaging, autoclaving, weighing 292.2g NaCl, and adding ddH2O to 1000mL, autoclaving for use, in 400mL ddH2Adding 90.05g of disodium ethylene diamine tetraacetate dihydrate into the O, stirring and dissolving, adjusting the pH value to 8.0 by using NaOH, fixing the volume to 500mL, and sterilizing under high pressure; 10g of SDS powder was dissolved in water to 100 mL.
Step five: dissolving a primer: centrifuging the dry powder at 12000rpm for 2min at 4 deg.C, and adding ddH2Preparing the O into mother liquor, and diluting by 10 times for use.
Step six: 1/2Hoagland nutrient solution: the appropriate drug concentration is 0.6 mu M CuSO4，0.05μMNa2MoO4·2H2O，0.05mM H3BO3And Ca (NO)3)2·4H2O，1.6μM ZnSO4·7H2O，0.01mM MnCl2·4H2O，2mM KNO3，0.5mM Fe-citrate·5H2O、MgSO4·7H2O and KH2PO4pH 5.5-5.7, using 1M citric acid [ HOC (CH)2COOH)2COOH]And 1M Tris [ NH ] Tris (hydroxymethyl) aminomethane2(CH2OH)3]。
Step seven: LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and 10g/L agar.
2. Cultivation of plant material
The method comprises the following steps: soaking overlord seed in 5% sodium hypochlorite, oscillating at 100rpm/min until seed coat is whitened, cleaning, soaking at room temperature for 1d, transferring into 4 deg.C refrigerator for germination for 1d, germinating on filter paper for 2-3d, planting germinated seed in quartz sand, irrigating 1/2Hoagland nutrient solution, culturing at (28 + -2)/(23 + -2) deg.C in culture room with light intensity of 800mol · m-2s-1The photoperiod is 16/8 h.d-1Relative humidity of about 60%, culturing for 3 weeks, and collecting young leaf for RNAExtracting;
step two: respectively sterilizing wild Col-0 seed of Arabidopsis thaliana in 75% alcohol and 5% sodium hypochlorite for 3min, cleaning with sterile water for 6-8 times, soaking in dark at 4 deg.C for vernalization for 2-3d, inoculating the vernalized seed on 1/2MS plate with pH of 5.7, and performing sterile culture under illumination time of 8h/d and illumination intensity of 200 μmol/m-2s-1The cloning of the AtCER6 promoter was performed after 2 weeks of culture at a day temperature of 22. + -.2 ℃ and a night temperature of 20. + -.2 ℃ with a humidity of about 65%.
3. Total RNA extraction
The method comprises the following steps: quickly and fully grinding a proper amount of Bawang young leaves in liquid nitrogen;
step two: adding 850 microliter of lysis agent 50mg sample within 1min, uniformly mixing, and vortexing or oscillating for 30 s;
step three: standing at 23 + -2 deg.C for 8min, mixing with 200 μ L24: 1 chloroform-isoamyl alcohol by vortex, vortex for 20-30s, standing at room temperature for 3min, and centrifuging at 12000rpm at 4 deg.C for 10 min;
step four: gently transfer 400-450. mu.L of the RNA-containing upper phase to a CS filtration column for 4min at 12000 rpm;
step five: gently transferring the supernatant into a new RNase-free centrifuge tube, adding 200-225 μ L of anhydrous ethanol, mixing, transferring the homogenate to a CR3 adsorption column, centrifuging at 4 ℃ and 14000rpm for 45s, and pouring off the waste liquid;
step six: adding 350 μ L RW1 solution to remove protein, centrifuging at 12000rpm for 45s, and pouring out waste liquid;
step seven: adding 80 μ L DNaseI working solution (10 μ L DNaseI stock solution and 70 μ L RDD solution mixed solution, ready for use) and standing at 23 + -2 deg.C for 15 min;
step eight: adding 350 μ L RW1 solution, centrifuging at 12000rpm for 45s, and removing waste liquid;
step nine: adding 500 μ L RW for rinsing, standing at 23 + -2 deg.C for 2min, centrifuging at 12000rpm for 45s, and pouring out waste liquid;
step ten: repeating the step 9;
step eleven: idling at 2000rpm for 2min, pouring the waste liquid, and standing at 23 +/-2 ℃ for several minutes until the rinsing liquid is dried;
step twelve: placing the adsorption column in a clean centrifugal tube without RNase, suspending and dropwise adding 30 μ L ddH to the middle part of the adsorption membrane2O, standing at 23 +/-2 ℃ for 2min, and centrifuging at 12000rpm for 2 min;
step thirteen: evaluating the purity and concentration of the RNA by using a NanoDrop1000 spectrophotometer, and selecting the RNA with an OD260/OD280 value of 1.8-2.1 and an OD260/OD230 value of 2.0-2.2 for the next experiment;
fourteen steps: running 5 mu L of the selected RNA solution for electrophoresis, and if the brightness of 28SrRNA is 1.5-2.0 times of that of 18SrRNA, indicating that the RNA integrity is high;
step fifteen: the RNA solutions in accordance with steps 13 and 14 were stored at-80 ℃ until use.
4. Reverse transcription into cDNA
The method comprises the following steps: sequentially adding 0.5-2 μ g of Tyrophora ovata total RNA, 1 μ L of Oligo and 10 μ L of RNase-free ddH2O；
Step two: performing instantaneous centrifugation after light mixing;
step three: cooling in water bath at 65 deg.C for 5min on ice for 30s, and centrifuging at 4 deg.C;
step four, adding 4 mu L of 5 × ReactionBuffer and 1 mu L of dNTPmix with the concentration of 10 mmol.L on ice-1And an RNaseINHIBITor concentration of 1. mu.L (20U. mu.L)-1) 1. mu.L of M-MuLVRT (20U. mu.L)-1)；
Step five: water bath at 42 deg.c for 1 hr;
step six: cooling in 70 deg.C water bath for 10min, and storing in-80 deg.C refrigerator;
5. extraction of DNA
The method comprises the following steps: adding 300 mu L of SDS extraction buffer solution into a 1.5mL sterile centrifuge tube;
step two: taking a proper amount of arabidopsis thaliana/medicago truncatula leaf tissues, and putting the arabidopsis thaliana/medicago truncatula leaf tissues into the centrifugal tube;
step three: fully grinding the blade by using a grinding rod;
step four: centrifuging at 7500rpm for 1min, and transferring 250 μ L of supernatant into a clean centrifuge tube;
step five: adding 250 μ L isopropanol with the same volume, mixing gently, precipitating at room temperature for 5min, centrifuging at 12000rpm for 5min, pouring off supernatant, turning over on filter paper, and quickly sucking water in the tube;
step six: adding 75% ethanol with the same volume, slowly washing, and centrifuging at maximum rotation speed for 5 min;
step seven: abandoning the supernatant, opening a pipe cover, idling at the maximum rotating speed for 2min, and sucking the redundant liquid by a gun;
step eight: blowing until the precipitate is completely air-dried for 20-30 min;
step nine: add 50. mu.L ddH to the precipitate2Dissolving O for 10min or more, and slightly scraping on a plate frame without stirring;
step ten: centrifuging at 5000rpm for 1min, collecting supernatant, and storing at-20 deg.C.
6. Primer design and Synthesis
According to the previous ZxABCG11 gene, the safety marker pmi gene and the AtCER6 promoter sequence, a corresponding sequence is introduced into Primer5.0, specificity design is carried out according to the required conditions, and SmaI and XhoI, NcoI and BstEII, HindIII and SmaI enzyme cutting sites and protective bases are added to the 5' end of the corresponding primer. The primer sequences are as follows:
examples
Construction of biologically safe ABCG11 gene overexpression vector (fig. 1):
1. construction of target plant expression vector pCAMBIA1302-ZxABCG11-FLAG
(1) Taking the Bawang cDNA as a template and P1 and P2 as specific primers, and carrying out amplification on the ZxABCG11-FLAG fusion gene;
(2) propagation of plant expression vector plasmid pCAMBIA 1302:
taking the DH5 alpha bacterial liquid of the preserved plant expression vector pCAMBIA1302 from a refrigerator at the temperature of-80 ℃, streaking on an LB + Kan + Gent + Rif solid culture medium, carrying out overnight culture at the temperature of 37 ℃ until a single colony is clear and visible, randomly selecting the single colony by using a sterilizing gun head to be placed in 5mL of LB liquid culture medium containing antibiotics Kan + Gent + Rif at the speed of 180rpm and the temperature of 37 ℃ for 12-16h, and using the bacterial liquid for plasmid extraction;
(3) purifying and recovering the amplified products of the fusion gene ZxABCG11-FLAG and the plant expression vector plasmid pCAMBIA1302 in the steps (1) and (2), and performing double enzyme digestion by SmaI and XhoI respectively, wherein the enzyme digestion system is as follows:
(4) after the fusion gene ZxABCG11-FLAG and the plant expression vector plasmid pCAMBIA1302 are subjected to enzyme digestion, loading the enzyme digestion product on a recovery gel, running the gel, then cutting down a target band under a UV lamp respectively, and then recovering and purifying the cut-down band by using a gel recovery kit;
(5) and (3) connecting enzyme digestion products: by T4The prepared target gene and the vector are connected for 2d at 4 ℃ by ligase, and the connection system is as follows:
| 10×Buffer | 1μL |
| pCAMBIA1302 vector | 1μL |
| T4Ligase | 1μL |
| Fusion gene ZxABCG11-FLAG | 1μL |
| ddH2O | 6μL |
(6) Conversion of ligation products: adding the ligation product of (5) above to 80. mu.L DH 5. alpha. competence, standing on ice for 20min, heat-shocking in 42 ℃ water bath for 1min30s, immediately standing on ice for 1-2min without shaking, adding 900. mu.L SOC for culturing, shaking bacteria at 37 ℃ and 200rpm for 1h, centrifuging at 4000rpm in a centrifuge for 1min, collecting supernatant, leaving about 100. mu.L of bacteria solution, plating LB + Kan, and finally culturing overnight at 37 ℃;
(7) selecting positive clones: selecting a single colony in 300 mu L LB + Kan liquid culture medium, centrifuging at 37 ℃ and 200rpm for 2h, and carrying out PCR detection on bacterial liquid by using P1 and P2 as primers according to a 20 mu L rTaq system; then adding 250 mu L of the bacterial liquid into a 5mLLB + Kan liquid culture medium at 180rpm and 37 ℃ for 12-16h, and after shaking, preserving the bacteria and sequencing by using 15-20% of glycerol. Finally, the target gene expression vector pCAMBIA1302-ZxABCG11-FLAG is obtained.
| 10×Buffer | 2μL |
| dNTP | 1.6μL |
| P1 | 0.4μL |
| P2 | 0.4μL |
| Bacterial liquid | 0.4μL |
| rTaq | 0.1μL |
| ddH2O | 15.1μL |
2. Construction of biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector
(1) Amplifying the safety marker gene pmi by taking the pMD19-T-pmi plasmid as a template and P3 and P4 as primers;
(2) purifying and recovering the amplification products of the safety marker gene pmi and the target gene expression vector pCAMBIA 1302-ZxACG 11-FLAG, and performing step-by-step enzyme digestion by using NcoI and BstEII, wherein the enzyme digestion system is as follows:
(3) after the safety marker gene pmi and the target gene expression vector pCAMBIA1302-ZxABCG11-FLAG are subjected to enzyme digestion, loading the enzyme digestion product on recovered gel, carrying out gel running, then cutting down a target band under a UV lamp respectively, and then recovering and purifying the cut down band by using a gel recovery kit;
(4) and (3) connecting enzyme digestion products: by T4The target gene is connected with the carrier at 4 ℃ for 2d by ligase, and the connection system is as follows:
(5) conversion of ligation products: adding the ligation product in (4) above into 80 μ L DH5 α competence, standing on ice for 20min, heat-shocking in 42 deg.C water bath for 1min30s, immediately standing on ice for 1-2min without shaking, adding 900 μ L SOC for culturing, shaking bacteria at 37 deg.C and 200rpm for 1h, centrifuging at 4000rpm at room temperature in a centrifuge for 1min, collecting supernatant, removing about 100 μ L of bacteria solution, plating LB + Kan, and culturing overnight at 37 deg.C;
(6) selecting positive clones: selecting a single colony in 300 mu L LB + Kan liquid culture medium, centrifuging at 37 ℃ and 200rpm for 2h, and carrying out PCR detection on bacterial liquid by using P3 and P4 as primers according to a 20 mu L rTaq system; then adding 250 mu L of the bacterial liquid into a 5mLLB + Kan liquid culture medium at 180rpm and 37 ℃ for 12-16h, and after shaking, preserving the bacteria and sequencing by using 15-20% of glycerol. Finally, obtaining the biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector.
| 10×Buffer | 2μL |
| dNTP | 1.6μL |
| P3 | 0.4μL |
| P4 | 0.4μL |
| Bacterial liquid | 0.4μL |
| rTaq | 0.1μL |
| ddH2O | 15.1μL |
3. Construction of biosafety pCAMBIA1302-AtCER6-ZxABCG11-FLAG-pmi overexpression vector
(1) Using arabidopsis DNA as a template and P5 and P6 as primers to amplify a promoter AtCER 6;
(2) purifying and recovering the amplification products of the promoter AtCER6 and the expression vector pCAMBIA1302-ZxABCG11-FLAG-pmi, and performing double enzyme digestion by HindIII and SmaI, wherein the enzyme digestion system is as follows:
(3) after the promoter AtCER6 and the pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector are subjected to enzyme digestion, loading the enzyme digestion product on recovery gel, carrying out gel running, cutting down a target band under a UV lamp respectively, and then recovering and purifying the cut-down band by using a gel recovery kit;
(4) and (3) connecting enzyme digestion products: by T4Ligase the promoter was ligated to the vector at 4 ℃ for 2d in the following system:
(5) conversion of ligation products: adding the ligation product in (4) above into 80 μ L DH5 α competence, standing on ice for 20min, heat-shocking in 42 deg.C water bath for 1min30s, immediately standing on ice for 1-2min without shaking, adding 900 μ L SOC for culturing, shaking bacteria at 37 deg.C and 200rpm for 1h, centrifuging at 4000rpm at room temperature in a centrifuge for 1min, collecting supernatant, removing about 100 μ L of bacteria solution, plating LB + Kan, and culturing overnight at 37 deg.C;
(6) selecting positive clones: selecting a single colony in 300 mu L LB + Kan liquid culture medium, centrifuging at 37 ℃ and 200rpm for 2h, and carrying out PCR detection on bacterial liquid by using P5 and P6 as primers according to a 20 mu L rTaq system; then adding 250 mu L of the bacterial liquid into a 5mLLB + Kan liquid culture medium at 180rpm and 37 ℃ for 12-16h, and after shaking, preserving the bacteria and sequencing by using 15-20% of glycerol. Finally, the biosafety pCAMBIA1302-AtCER6-ZxABCG11-FLAG-pmi overexpression vector is obtained.
4. Results and analysis
Referring to FIG. 2, fusion primers F: GCGAAGGAACTCTTGAAAGAACATG are designed; TCATTTGTCGTCATCGTCTTTGTAGTCCCTTCTGCTTGCTCGATTTGATC, fusing the FLAG tag with the ZxABCG11 gene by a PCR method, and sequencing. The results show that the two genes are well fused.
Referring to FIG. 3, the ZxABCG11-FLAG fusion gene was incorporated into a plant expression vector, and the fusion gene was incorporated into a plant expression vector by SmaI and XhoI double digestion. PCR detection of bacterial liquid shows that a 2000 bp strip is consistent with 2127bp of fusion gene.
Referring to FIGS. 4-5, the restriction enzyme assay shows that the two bands in columns 1-7 and 11 are in proper positions, and the translated amino acid sequence of the sequencing result is consistent with the result of the amino acid translation of the calibration sequence in the previous laboratory.
Referring to FIGS. 6-8, the pmi gene was constructed into the vector, and the pmi gene was constructed into the plant expression vector by double digestion with NcoI and BstEII. The PCR detection, enzyme digestion detection and sequencing comparison of the bacterial liquid are all suitable.
Referring to FIGS. 9-11, the AtCER6 promoter was incorporated into the vector, and the AtCER6 promoter was incorporated into the vector by double digestion with HindIII and SmaI.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (2)
1. A construction method of a biosafety ABCG11 gene overexpression vector comprises the following steps: (1) after the fusion gene ZxABCG11-FLAG and pCAMBIA1302 carrier plasmid are subjected to double enzyme digestion, carrying out gel running on the enzyme digestion product, recovering the obtained target fragment and carrying out connection transformation on the enzyme digestion carrier to obtain a target plant expression carrier pCAMBIA 1302-ZxBCG 11-FLAG; (2) carrying out step-by-step enzyme digestion on safety marker gene pmi and pCAMBIA1302-ZxABCG11-FLAG vector plasmids, respectively recovering, connecting and transforming corresponding target fragments after the enzyme digestion is finished, and obtaining a biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector; (3) carrying out double enzyme digestion on an epidermal specific promoter AtCER6 and pCAMBIA1302-ZxABCG11-FLAG-pmi vector plasmid, respectively recovering and connecting and transforming corresponding target fragments after the enzyme digestion is finished, and finally obtaining a biosafety ABCG11 gene overexpression vector pCAMBIA1302-AtCER 6-ZxBCG 11-FLAG-pmi, which is characterized in that pmi in the step (2) is a biosafety marker gene, and the carrier obtained by carrying out step-by-step enzyme digestion and connection transformation on the gene and pCAMBIA 1302-ZxBCG 11-FLAG vector plasmid is: the biosafety pCAMBIA 1302-ZxACG 11-FLAG-pmi expression vector comprises the following steps:
s11, step-by-step enzyme digestion of the safety marker gene pmi:
step-by-step enzyme digestion 1: pMD19-T-pmi plasmid is taken as a template, F is CATGCCATGGATG CAAAAACTCATTAACTCAGTGC; GGGTNACCTTACAGCTTGTTG TAAACACGCGCT is used as a primer to carry out amplification to obtain a pmi recovered product, and the pmi recovered product is cut by Nco I enzyme for 4h in a water bath kettle at 37 ℃;
step-by-step enzyme digestion 2: then, the product recovered by the step-by-step enzyme digestion 1 is digested by Bst E II for 4 hours in a water bath kettle at 37 ℃;
step-by-step enzyme digestion of S12, pCAMBIA1302-ZxABC 11-FLAG vector plasmid:
step-by-step enzyme digestion 1: taking the plant expression vector pCAMBIA1302-ZxABCG11-FLAG in the step (1) as a template, and carrying out enzyme digestion for 4h by using Nco I in a water bath kettle at 37 ℃;
step-by-step enzyme digestion 2: then, the product recovered by the step-by-step enzyme digestion 1 is digested by Bst E II for 4 hours in a water bath kettle at 37 ℃;
s13, connecting the safety marker gene pmi with the enzyme digestion product recovered from the pCAMBIA1302-ZxABCG11-FLAG vector plasmid by using T4 ligase, and transforming in Escherichia coli DH5 alpha to obtain a vector: the biosafety pCAMBIA 1302-ZxACG 11-FLAG-pmi expression vector.
2. The method for constructing the biosafety ABCG11 gene overexpression vector as claimed in claim 1, wherein the promoter AtCER6 in step (3) is an epidermal specific promoter, and the vector obtained by performing double digestion and ligation transformation on the promoter and biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector plasmid is: an ultra-expression vector of the ABCG11 gene with biological safety. The method comprises the following steps:
s21, promoter AtCER6 double enzyme digestion:
using arabidopsis DNA as a template, F: CCCAAGCTTCTTCGATATCGGTTGTT GACG; TCCCCCGGGCGTCGGAGAGTTTTAATGTAT is used as a primer to carry out amplification to obtain an AtCER6 recovered product, and enzyme digestion is carried out for 1h in a water bath kettle at 30 ℃ by using Hind III and Sma I, and then enzyme digestion is carried out for 1h at 37 ℃;
s22, performing double enzyme digestion on the biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi vector plasmid;
taking the biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector in the step (2) as a template, carrying out enzyme digestion on HindIII and Sma I for 1h in a water bath kettle at 30 ℃, and then carrying out enzyme digestion for 1h at 37 ℃;
s23, connecting the enzyme digestion products recovered by the epidermis-specific promoter AtCER6 and the biosafety pCAMBIA1302-ZxABCG11-FLAG-pmi expression vector plasmid by using T4 ligase, and transforming in escherichia coli DH5 alpha to obtain a vector: the biosafety pCAMBIA1302-AtCER6-ZxABCG11-FLAG-pmi overexpression vector.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010332370.0A CN111733181A (en) | 2020-04-24 | 2020-04-24 | Construction method of biosafety ABCG11 gene overexpression vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010332370.0A CN111733181A (en) | 2020-04-24 | 2020-04-24 | Construction method of biosafety ABCG11 gene overexpression vector |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111733181A true CN111733181A (en) | 2020-10-02 |
Family
ID=72646728
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010332370.0A Pending CN111733181A (en) | 2020-04-24 | 2020-04-24 | Construction method of biosafety ABCG11 gene overexpression vector |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111733181A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1863917A (en) * | 2003-10-07 | 2006-11-15 | 植物遗传学与作物培养研究所 | Promoter for the epidermis-specific transgenic expression in plants |
| CN101208433A (en) * | 2005-06-03 | 2008-06-25 | 莱布尼茨植物遗传学和栽培植物研究所 | Promoter for epidermis-specific, pathogen-inducible transgenic expression in plants |
| CN101463393A (en) * | 2009-01-12 | 2009-06-24 | 天津大学 | Method for screening transgenic plate with safety marker gene |
| CN102787132A (en) * | 2011-05-19 | 2012-11-21 | 华中农业大学 | Method for constructing transgenic Arabidopsis plant by phosphomannose-isomerase (PMI) gene of yeast |
| CN107058336A (en) * | 2017-02-22 | 2017-08-18 | 兰州大学 | A kind of ZxABCG11 genes and application thereof |
| CN107207573A (en) * | 2014-08-26 | 2017-09-26 | 加利福尼亚大学董事会 | Super quick ABA acceptors |
-
2020
- 2020-04-24 CN CN202010332370.0A patent/CN111733181A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1863917A (en) * | 2003-10-07 | 2006-11-15 | 植物遗传学与作物培养研究所 | Promoter for the epidermis-specific transgenic expression in plants |
| CN101208433A (en) * | 2005-06-03 | 2008-06-25 | 莱布尼茨植物遗传学和栽培植物研究所 | Promoter for epidermis-specific, pathogen-inducible transgenic expression in plants |
| CN101463393A (en) * | 2009-01-12 | 2009-06-24 | 天津大学 | Method for screening transgenic plate with safety marker gene |
| CN102787132A (en) * | 2011-05-19 | 2012-11-21 | 华中农业大学 | Method for constructing transgenic Arabidopsis plant by phosphomannose-isomerase (PMI) gene of yeast |
| CN107207573A (en) * | 2014-08-26 | 2017-09-26 | 加利福尼亚大学董事会 | Super quick ABA acceptors |
| CN107058336A (en) * | 2017-02-22 | 2017-08-18 | 兰州大学 | A kind of ZxABCG11 genes and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| 宋扬等: "植物组织特异性启动子研究", 《生物技术通报》 * |
| 宋扬等: "植物组织特异性启动子研究", 《生物技术通报》, no. 06, 26 December 2007 (2007-12-26), pages 21 - 24 * |
| 朱璐等: "拟南芥ABC转运类蛋白家族的分子进化、表达模式和蛋白功能网络预测分析", 《植物生理学报》 * |
| 朱璐等: "拟南芥ABC转运类蛋白家族的分子进化、表达模式和蛋白功能网络预测分析", 《植物生理学报》, vol. 48, no. 12, 20 December 2012 (2012-12-20), pages 1151 - 1166 * |
| 田野: "霸王ZxABCG11的功能验证及其对紫花苜蓿（Medicago sativa L.）的遗传转化", 《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》 * |
| 田野: "霸王ZxABCG11的功能验证及其对紫花苜蓿（Medicago sativa L.）的遗传转化", 《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》, no. 02, 15 February 2018 (2018-02-15), pages 3 * |
| 陈伟等: "植物表皮蜡质及相关基因研究进展", 《植物生理学报》 * |
| 陈伟等: "植物表皮蜡质及相关基因研究进展", 《植物生理学报》, vol. 52, no. 08, 20 August 2016 (2016-08-20), pages 1117 - 1127 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754957B (en) | OsSCAMP13 gene, encoding protein, application of stress resistance and acquisition method | |
| CN109081865B (en) | Phyllostachys pubescens PeVQ28 protein and coding gene and application thereof | |
| CN107012147B (en) | Drought and/or high-salt induction promoter SlWRKY8P from tomato and application thereof | |
| CN108486149B (en) | Application of cucumber CsWRKY50 gene in enhancing cucumber downy mildew resistance | |
| CN104404043B (en) | Promoter of gene Me094 related to bacterial-blight resistance of Oryza meyeriana | |
| CN109748959B (en) | Anthocyanin synthesis related protein SlANT1L, and coding gene and application thereof | |
| CN116656698B (en) | Application of corn gene Zm00001d018037 in improving drought resistance of monocotyledonous crops | |
| CN115896128B (en) | Tobacco nitrate transporter NtNPF6.13, coding gene and application thereof | |
| CN110904106A (en) | Application of cymbidium goeringii miR159b in enhancing plant cold sensitivity | |
| CN113337522B (en) | Application of cotton GhNFYC4 gene in promoting plant flowering | |
| CN115058433B (en) | Tobacco leaf yellowing regulatory gene NtMYB2, protein and application thereof | |
| CN116083445A (en) | CrBZR1 gene and application thereof | |
| CN111961124B (en) | Plant precocity protein and coding gene and application thereof | |
| CN111304220B (en) | Cymbidium CgWRKY3 gene and application thereof | |
| CN111304222B (en) | Cymbidium CgWRKY11 gene and application thereof | |
| CN111733181A (en) | Construction method of biosafety ABCG11 gene overexpression vector | |
| CN110627887B (en) | Application of SlTLFP8 protein and related biological material thereof in regulation and control of tomato drought resistance | |
| CN110615833A (en) | Plant phosphorus transport protein ZmPT4 and coding gene and application thereof | |
| CN116004646B (en) | Tobacco NtSWEET gene and application thereof | |
| CN115704036B (en) | Tobacco NtDSR1 gene and application thereof | |
| CN115806999B (en) | Tobacco NtEIJ gene and application thereof | |
| CN115058432B (en) | Tobacco NtWRKY51 gene and application thereof in regulation and control of bacterial wilt resistance of tobacco | |
| CN115011631B (en) | Protein for regulating drought resistance of corn at seedling stage, and coding gene and application thereof | |
| CN116004647B (en) | Tobacco NtSWEET gene and application thereof | |
| CN115704035B (en) | Tobacco NtDSR2 gene 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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201002 |
|
| RJ01 | Rejection of invention patent application after publication |