CN117683807A - Efficient fragment assembly and rapid plant cell protoplast transient expression method, system and application - Google Patents
Efficient fragment assembly and rapid plant cell protoplast transient expression method, system and application Download PDFInfo
- Publication number
- CN117683807A CN117683807A CN202311731956.4A CN202311731956A CN117683807A CN 117683807 A CN117683807 A CN 117683807A CN 202311731956 A CN202311731956 A CN 202311731956A CN 117683807 A CN117683807 A CN 117683807A
- Authority
- CN
- China
- Prior art keywords
- gene
- expression cassette
- dna
- transient expression
- sequence
- 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
- 239000012634 fragment Substances 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 56
- 210000001938 protoplast Anatomy 0.000 title claims abstract description 54
- 230000010474 transient expression Effects 0.000 title claims abstract description 39
- 230000014509 gene expression Effects 0.000 claims abstract description 65
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 39
- 230000009466 transformation Effects 0.000 claims abstract description 17
- 239000013598 vector Substances 0.000 claims abstract description 8
- 108020004414 DNA Proteins 0.000 claims description 63
- 238000006243 chemical reaction Methods 0.000 claims description 34
- 239000000047 product Substances 0.000 claims description 25
- 108091005946 superfolder green fluorescent proteins Proteins 0.000 claims description 14
- 238000012408 PCR amplification Methods 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 13
- 108091026890 Coding region Proteins 0.000 claims description 12
- 108700007698 Genetic Terminator Regions Proteins 0.000 claims description 11
- 238000003776 cleavage reaction Methods 0.000 claims description 11
- 230000007017 scission Effects 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 102000012410 DNA Ligases Human genes 0.000 claims description 5
- 108010061982 DNA Ligases Proteins 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 5
- 238000001976 enzyme digestion Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000001681 protective effect Effects 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 4
- 230000004960 subcellular localization Effects 0.000 claims description 4
- 238000003745 diagnosis Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 230000003993 interaction Effects 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- 239000013589 supplement Substances 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 abstract description 29
- 238000000605 extraction Methods 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 2
- 230000004044 response Effects 0.000 abstract description 2
- 238000010276 construction Methods 0.000 abstract 2
- 241000196324 Embryophyta Species 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 10
- 238000011160 research Methods 0.000 description 9
- 230000008569 process Effects 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 5
- 238000010367 cloning Methods 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000002158 endotoxin Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 241000219194 Arabidopsis Species 0.000 description 2
- 108091023045 Untranslated Region Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000001821 nucleic acid purification Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 108020005120 Plant DNA Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8214—Plastid transformation
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- 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
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
-
- 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
- C12N15/8212—Colour markers, e.g. beta-glucoronidase [GUS], green fluorescent protein [GFP], carotenoid
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a high-efficiency fragment assembly method and is applied to a rapid plant cell protoplast transient expression system. Constructing a DNA fragment gene expression cassette in vitro by utilizing a synthetic biology method, and directly carrying out protoplast transformation on the purified DNA fragment gene expression cassette to realize the transient expression of the target gene. Compared with the traditional vector construction and protoplast transient expression system, the invention avoids the complicated procedures of vector construction and plasmid extraction, has the characteristics of short time consumption, quick response, convenient operation and good practicability, can be used for high-throughput high-efficiency gene transient expression, and is a gene transient expression method with great application potential.
Description
Technical Field
The invention belongs to the fields of genetic engineering and molecular biology, and particularly relates to a high-efficiency fragment assembly and rapid plant cell protoplast transient expression method, a system and application.
Background
Because the gene can be expressed in a short time and high efficiency in organisms, a transient expression system (Protoplast-based transient expression system, PTES) based on protoplasts shows great advantages in molecular biology research, and is widely applied to the research on subcellular localization, protein interaction, transcriptional regulation, signal transduction and the like. At present, a protoplast transient expression system is well established and developed in the research of crops such as arabidopsis, tobacco, rice, corn, peanut and the like. In recent years, the application range of the research combining the transient expression of protoplasts with genomics, proteomics, genetic improvement and the like is further widened.
Protoplast transient expression systems include isolation and transformation of protoplasts, wherein protoplast transformation refers to the introduction of exogenous nucleic acid into the protoplasts by way of polyethylene glycol or electroporation. Transient expression of genes in many species relies on plasmid as a vector, delivering the gene expression cassette fused with the plasmid into the protoplast, and DNA does not need to be integrated into the plant genome, but can be expressed rapidly in cells. Although this method can give appropriate transgene expression level to cells, it is necessary to construct gene expression vectors in the early stage and extract high quality plasmids, thereby increasing experimental time and reducing throughput. In the studies, higher conversion efficiencies were required in order to obtain reliable and reproducible results. In addition to the viability and quantity of protoplasts, the concentration of PEG and the transformation incubation time, the quantity and quality of DNA are also critical to the transformation efficiency. However, studies have shown that transformation efficiency drops dramatically as plasmid DNA increases in size from 5kb to 10 kb. Therefore, a large amount of plasmid DNA is required to achieve a certain transformation efficiency. In addition, endotoxin is one of the common pollutants in plasmid DNA extraction, and the presence of endotoxin can cause damage to cells, resulting in a great compromise in protoplast transformation efficiency.
The limitations of the existing protoplast transient expression system have prompted us to develop a simple, rapid, high-throughput gene transient expression method. The DNA fragment gene expression cassette is constructed in vitro by the Golden Gate method, and the purified DNA fragment is directly transformed into protoplast, so that the complex procedures of constructing a vector and extracting plasmids are avoided, the experimental time is greatly shortened, the possibility of bacterial endotoxin pollution is eliminated, and the transformation efficiency is improved. Compared with an expression vector fused with a plasmid, the simple gene expression cassette has the advantage of small fragments, can be prepared in a large amount in a short time through PCR amplification, provides possibility for high-flux high-efficiency gene transient expression, and is a gene transient expression method with very high application potential.
Disclosure of Invention
Aiming at the problems of long time consumption, complex and tedious operation and high cost of transient expression of plant protoplasts in the prior art, the invention provides a high-efficiency fragment assembly method which is applied to a rapid plant cell protoplast transient expression system, and a gene expression cassette prepared based on a synthetic biology method is transformed into the protoplasts, so that the transient expression of genes with short time consumption, quick response and simple operation is realized.
Specifically, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides a method for efficient fragment assembly and rapid transient expression of plant cell protoplasts, comprising the steps of:
s1, using DNA as a template, and obtaining a promoter, a gene coding region and a terminator sequence through PCR amplification;
s2, fusing the promoter, the gene coding region and the terminator DNA fragment into a complete gene expression cassette by utilizing a Golden Gate method;
s3, enriching a complete gene expression cassette by utilizing PCR amplification;
s4, directly carrying out protoplast transformation on the enriched DNA fragment gene expression cassette to realize transient expression of the gene.
In the present invention, a promoter, a gene coding region and a terminator sequence are amplified from a template DNA.
In the present invention, the template DNA may be a buffer containing a DNA substrate, or may be a microorganism, plant tissue, or animal tissue containing DNA.
In the present invention, the buffer containing the template DNA includes, but is not limited to, a linear DNA fragment, a double-stranded DNA fragment, a multi-stranded (three-stranded or more) DNA fragment, a fragment of DNA hybridized with RNA, a circular DNA sequence, a normal natural DNA structure, a modified DNA structure, and the like.
In the present invention, the source of the DNA fragment may be natural animal or plant DNA or microorganism DNA, or may be chemical or biological DNA, including normal DNA structure and modified DNA structure.
In the present invention, the DNA as a substrate includes DNA directly added at the time of reaction, and also includes DNA which has not been directly added before the reaction but occurs during the reaction, such as RT-PCR (Reverse Transcription Polymerase Chain Reaction), in which RNA is reverse transcribed into DNA, and the DNA is used for subsequent amplification.
In the efficient fragment assembly and rapid plant cell protoplast transient expression method of the invention,
preferably, the promoter 35S and the terminator in the step S1 are derived from a pCAMBIA1301 vector, and the gene coding sequence sfGFP is synthesized in the room, and the sequence is shown as SEQ ID NO. 1.
Preferably, in step S1, the pCAMBIA1301 plasmid is used as a template, 35S-F and 35S-R (the sequences are shown as SEQ ID NO.2 and SEQ ID NO. 3) are used as primers, and the 35S promoter (3' -terminal primer has Bsa I cleavage site and protecting base) is amplified by using high-fidelity enzyme (CloneAmp HiFi PCR Premix, takara). The sfGFP gene (with BsaI cleavage site and protecting base at both ends) was amplified using sfGFP synthetic plasmid as template and sfGFP-F and sfGFP-R (sequences shown as SEQ ID NO.4 and SEQ ID NO. 5) as primers. The pCAMBIA1301 plasmid is used as a template, NOS-F and NOS-R (the sequences are shown as SEQ ID NO.6 and SEQ ID NO. 7) are used as primers, and a high-fidelity enzyme (CloneAmp HiFi PCR Premix, takara) is used for amplifying an NOS terminator (a 5' -end primer has Bsa I cleavage site and a protective base).
In the present invention, bsaI is the restriction enzyme selected. Experience has shown that even if BsaI recognition sites are present in the target fragment (gene), subsequent fragment assembly and amplification are not affected.
Preferably, after obtaining the promoter, gene coding region and terminator sequence by PCR amplification, the PCR product is purified by the magnetic bead method (product name: AMPure XP nucleic acid purification kit, cat# A63882).
In the present invention, the promoter, gene coding region and terminator DNA fragment are fused into a complete gene expression cassette by the Golden Gate method.
Preferably, in step S2, the promoter, gene coding region and terminator DNA fragment are mixed with restriction enzymes BsaI and T4DNA ligase to carry out enzyme digestion ligation reaction, and the enzyme digestion ligation reaction system is as follows:
| 10×T4DNAligaseBuffer(NEB) | 1μL |
| 10×CutsmartBuffer(NEB) | 1μL |
| T4DNAligase(NEB) | 1μL |
| BsaⅠ(NEB) | 0.5μL |
| each segment | 100ng |
| Supplement ddH 2 O | To 20 mu L |
Preferably, the cleavage ligation is performed at room temperature for 15min.
In the present invention, PCR amplification is used to enrich for complete gene expression cassettes.
Preferably, in step S3, the assembled product after the reaction in step S2 is diluted 20 times to be used as a PCR reaction template, 35S-F and NOS-R are used as primers, a complete expression cassette is amplified by using high-fidelity enzyme, and then the PCR product is purified by using a magnetic bead method.
Preferably, the whole DNA fragment gene expression cassette amplification system is as follows:
| 2×CloneAmpHiFiPCRPremix(Takara) | 25μL |
| 35S-F | 1μL |
| NOS-R | 1μL |
| assembled product | 1μL |
| ddH 2 O | 22μL |
The reaction procedure: 95℃for 5min, (95℃for 10s,72℃for 1min,35 cycles), 72℃for 5min.
In order to improve the amplification specificity of the expression cassette, the Tm values of the 35S-F primer and the NOS-R primer are set to be more than 68 ℃ in the invention, and the target DNA fragment is amplified by adopting a two-step PCR method, so that the amplification specificity of the DNA fragment expression cassette is greatly improved.
In the invention, the optimized fragment assembly method can realize simultaneous assembly of at most 5 fragments, and the final length can reach more than 6 kb.
Preferably, the DNA fragment gene expression cassette enriched in the step S3 is directly subjected to protoplast transformation to realize transient expression of the gene.
In a second aspect, the invention provides a high efficiency fragment assembly and rapid plant cell protoplast transient expression system comprising an assembly reaction system for a DNA fragment gene expression cassette, a protoplast preparation system, and a protoplast transformation system.
Further, the assembly reaction system of the DNA fragment gene expression cassette comprises a PCR amplification reaction system of a promoter, a gene coding region and a terminator sequence, a Golden Gate reaction system and an enrichment reaction system of the gene expression cassette.
In a third aspect, the present invention provides an application of a high-efficiency fragment assembly and rapid plant cell protoplast transient expression method in exogenous gene expression for non-disease diagnosis and treatment purposes.
In a fourth aspect, the present invention provides an application of a high-efficiency fragment assembly and rapid plant cell protoplast transient expression system in exogenous gene expression for non-disease diagnosis and treatment purposes.
In the present invention, the methods and systems find application in a range of applications including, but not limited to, plant cells, animal cells.
In the invention, the gene transient expression method is applied to related products of gene transient expression in animals and plants, including but not limited to gene subcellular localization related products, gene function research related products, gene interaction related products, non-coding gene function research related products, function research related products of promoter sequences, work research related products of terminator sequences, and function research related products of untranslated region sequences.
In the present invention, the mode of application preferably includes at least one of gene subcellular localization, gene function prediction, gene interaction; more preferably, the gene function prediction includes at least one of non-coding gene function prediction, promoter sequence function prediction, terminator sequence function prediction, and untranslated region sequence function prediction.
Compared with the prior art, the invention has the beneficial effects that:
1. and (3) segment assembly: the Golden Gate reaction system was optimized to reduce the reaction time from the traditional 1.5h (Ma and Liu, CRISPR/Cas9-Based Multiplex Genome Editing in Monocot andDicotPlants, 2016) to 15min.
2. The amplification specificity of the DNA fragment expression cassette is improved: because of the multi-fragment assembly, in order to improve the amplification specificity of the expression cassette, the Tm values of the two primers of 35S-F and NOS-R are set above 68 ℃, and a two-step PCR is adopted to amplify the target DNA fragment.
3. The optimized fragment assembly method can realize simultaneous assembly of at most 5 fragments, and the final length can reach more than 6 kb.
4. In the present invention, bsaI is the restriction enzyme selected. Experience has shown that even if BsaI recognition sites are present in the target fragment (gene), subsequent fragment assembly and amplification are not affected.
5. Under the same molar quantity, the DNA fragment expression cassette and the plasmid expression cassette show the same transformation efficiency and expression quantity, and the experimental purpose can be achieved by only 500ng of the expression cassette at least.
6. The time required for the whole experimental process is shortened from the traditional 7d to 2d.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be described below.
FIG. 1 is a gel diagram of a promoter, sfGFP gene coding region and terminator sequence obtained by PCR amplification, wherein four lanes respectively represent from left to right: DNAmarker; amplifying the 35S promoter fragment by PCR; PCR amplification of sfGFP fragment; PCR amplified NOS terminator fragment.
FIG. 2 is a gel diagram of an sfGFP gene expression fragment prepared by the Golden Gate method, wherein four lanes represent, from left to right: a DNA Marker;35S, sfGFP and NOS mixtures; assembling Golden Gate fragments; amplifying the assembled gene expression cassette.
FIG. 3 shows the result of observing sfGFP fluorescence by a laser confocal microscope. The A panel uses DNA fragments as gene expression cassettes, and the B panel uses plasmids as gene expression cassettes.
FIG. 4 is a comparison of experimental durations of protoplast transient expression performed by two expression cassettes.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention. Unless otherwise indicated, all the experimental procedures used in the examples were conventional; the reagents and materials used are all commercially available.
Example one, assembly of DNA fragment Gene expression cassettes
The promoter 35S and the terminator are derived from pCAMBIA1301 vector, sfGFP is synthesized in the room, and the sequence is shown as SEQ ID NO. 1. The preparation method comprises the following steps:
(1) PCR amplification and DNA purification: the pCAMBIA1301 plasmid is used as a template, 35S-F and 35S-R (the sequences are shown as SEQ ID NO.2 and SEQ ID NO. 3) are used as primers, and a high-fidelity enzyme (CloneAmp HiFi PCR Premix) is used for amplifying a 35S promoter (a 3' -end primer is provided with Bsa I cleavage sites and protective bases). The sfGFP gene (with BsaI cleavage site and protecting base at both ends) was amplified using sfGFP synthetic plasmid as template and sfGFP-F and sfGFP-R (sequences shown as SEQ ID NO.4 and SEQ ID NO. 5) as primers. The pCAMBIA1301 plasmid is used as a template, NOS-F and NOS-R (the sequences are shown as SEQ ID NO.6 and SEQ ID NO. 7) are used as primers, a high-fidelity enzyme (CloneAmp HiFi PCR Premix) is used for amplifying an NOS terminator (the 5' -end primer is provided with BsaI cleavage sites and protective bases), and the glue running result is shown in figure 1. The PCR product was purified by the magnetic bead method (product name: AMPure XP nucleic acid purification kit, cat# A63882). This process takes about 1.5 hours or so.
(2) Golden Gate method DNA fragment fusion and DNA purification:
a) Preparing a 20 mu L enzyme digestion ligation reaction system:
| 10×T4DNAligaseBuffer(NEB) | 1μL |
| 10×CutsmartBuffer(NEB) | 1μL |
| T4DNAligase(NEB) | 1μL |
| BsaⅠ(NEB) | 0.5μL |
| each segment | 100ng |
| Supplement ddH 2 O | To 20 mu L |
b) The reaction solution was allowed to react at room temperature for 15min.
c) The assembled product is diluted 20 times as a PCR reaction template, and 35S-F and NOS-R are used as primers, and a complete expression cassette is amplified by using high-fidelity enzyme.
| 2×CloneAmpHiFiPCRPremix(Takara) | 25μL |
| 35S-F | 1μL |
| NOS-R | 1μL |
| Assembled product | 1μL |
| ddH 2 O | 22μL |
The reaction procedure: 95℃for 5min, (95℃for 10s,72℃for 1min,35 cycles), 72℃for 5min.
d) The PCR product was purified by the magnetic bead method. This process takes about 20 minutes or so.
Example two preparation of plasmid expression cassettes
(1) TOPO cloning: the DNA fragment gene expression cassette prepared above was constructed on pCE3 vector using TOPO cloning kit (product name: ultra-Universal TOPO Cloning Kit, cat# C603) from Norpraise Corp.
(2) Cloning product transformation: e.coli competent cells (DH 5. Alpha.) stored at-80℃were thawed on ice, the clone was added to 50. Mu.l competent cells, gently mixed, left on ice for 30min, then placed on ice for 2min after heat shock in a 42℃water bath for 45 sec. Adding a proper amount of LB liquid medium (without antibiotics) into a centrifuge tube, uniformly mixing, placing into a shaking table at 37 ℃ and 200rpm for resuscitation for 1h, coating the bacterial liquid on an LB flat plate medium containing ampicillin, and culturing at 37 ℃ in an inverted way overnight. This process takes about 1d or so.
(3) Positive plasmid detection and propagation: 8 single colonies are randomly picked from LB plate medium to LB culture solution containing ampicillin for culturing for about 12 hours, plasmids are extracted by an alkaline lysis method, ecoRI is used for enzyme digestion verification, and positive plasmids are selected for sequencing. Single colonies which were correctly sequenced were added to LB medium containing ampicillin for expansion. This process takes about 3d or so.
(4) Transient expression plasmid extraction: plasmids for transient expression were extracted using the QIAGEN large plasmid kit (product name: QIAGEN Plasmid Kits, cat# 12123). This process takes about 3 hours or so.
EXAMPLE three transformation of Arabidopsis protoplasts
(1) Expression cassette concentration setting: the DNA fragment expression cassette was about 2kb in size and the plasmid expression cassette was about 4kb in size, both according to 1:2, and converting the mixture according to the proportion. The DNA fragment expression cassette set up gradients: 0.25 μg, 0.5 μg, 1.25 μg, 2.5 μg and 5 μg; the plasmid expression cassette set up gradients: 0.5 μg, 1.25 μg, 2.5 μg, 5 μg and 10 μg.
(2) Protoplast preparation: taking 4-5 tender leaves of arabidopsis thaliana as a material, carrying out protoplast extraction, placing the leaves on clean filter paper, cutting the leaves into filaments by a blade, placing the filaments into 10mL of enzymolysis liquid, placing the filaments in a dark place, vacuumizing for 30min, and carrying out light-proof enzymolysis for 3-4h at 40 rpm. The enzymatic hydrolysis was terminated by adding 10mL of W5 solution, releasing the protoplasts at 80rpm for 10min, and the solution was filtered through a 75 μm sieve into a round bottom centrifuge tube to collect the protoplasts. Centrifuging at 4deg.C for 2min, removing supernatant, adding pre-cooled W5 solution to resuspend protoplast, centrifuging again for 2min at 4deg.C, removing supernatant, adding pre-cooled W5 solution to re-select protoplast, standing in ice bath for 30min, removing supernatant, adding appropriate amount of MMG solution to re-suspend protoplast, and regulating cell concentration to about 2X105/mL.
(3) Expression cassette transfection protoplasts: mixing 10 μl of the expression cassette with 100 μl, adding 110 μl of LPEG-CaCl2 solution, mixing, and standing at room temperature for 10-20min. The conversion was then terminated by adding 660. Mu.L of W5 solution. 100g, centrifuging at room temperature for 2-3min, discarding supernatant, re-suspending protoplast with 500 μl of W5 solution, and culturing at 20-23deg.C in dark place for 14h. The protoplasts were enriched by discarding 400. Mu.L of supernatant and resuspended in the bottom solution.
(4) Laser confocal microscope observation
Part of the protoplast suspension was pelleted and fluorescence of sfGFP protein was observed with Nikon laser confocal microscopy. All samples observed in the experiment are uniformly observed under a 20-time objective lens, and the settings of the type, the intensity, the gain and the like of the used laser are completely consistent. The results are shown in FIG. 3, wherein FIG. 3 shows the results of transfection of protoplasts with a DNA fragment gene expression cassette and FIG. 3 shows the results of transfection of protoplasts with a plasmid expression cassette. The number of cells having fluorescence was observed to increase with increasing amount of gene expression cassette, and at the same molar amount, the transformed DNA fragment gene expression cassette was almost identical to the fluorescent cells produced by the transformed plasmid expression cassette. More importantly, only 0.5. Mu.g of conversion was needed to observe fluorescent cells.
Compared with the traditional method, the method has the advantages that the segment assembly is very quick, simple and efficient, the reaction time is shortened from traditional 1.5h to 15min, the amplification specificity of the DNA segment expression cassette can be obviously improved, the optimized segment assembly method can realize simultaneous assembly of at most 5 segments, and the final length can reach more than 6 kb. The DNA fragment gene expression cassette assembled by the method can be directly used for transient expression of protoplasts, and the DNA fragment expression cassette and the plasmid expression cassette show the same conversion efficiency and expression quantity under the same molar quantity, and the experimental purpose can be achieved by only 500ng of the expression cassette at least. The optimized system shortens the time required for the whole experimental process from the traditional 7d to 2d.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
Claims (10)
1. An efficient fragment assembly and rapid plant cell protoplast transient expression method is characterized by comprising the following steps:
s1, using DNA as a template, and obtaining a promoter, a gene coding region and a terminator sequence through PCR amplification;
s2, fusing the promoter, the gene coding region and the terminator DNA fragment into a complete gene expression cassette by utilizing a Golden Gate method;
s3, enriching a complete gene expression cassette by utilizing PCR amplification;
s4, directly carrying out protoplast transformation on the enriched DNA fragment gene expression cassette to realize transient expression of the gene.
2. The method according to claim 1, wherein step S1 comprises amplifying the promoter, gene coding region and terminator sequence from a template DNA, which may be a buffer comprising a DNA substrate or a microorganism, plant tissue or animal tissue comprising DNA.
3. The method according to claim 1, wherein the promoter 35S and the terminator of step S1 are derived from pCAMBIA1301 vector, and the gene coding sequence sfGFP is shown in SEQ ID NO. 1.
4. The method according to claim 3, wherein the sequence of the amplification primer 35S-F of the promoter sequence in the step S1 is shown as SEQ ID NO.2, the sequence of the amplification primer 35S-R is shown as SEQ ID NO.3, and the 3' -terminal primer has BsaI cleavage site and protecting base; the amplification primer sfGFP-F of the gene coding sequence sfGFP has a sequence shown in SEQ ID NO.4, the sfGFP-R sequence is shown in SEQ ID NO.5, and both ends of the primer are provided with BsaI cleavage sites and protective bases; the amplification primer NOS-F of the terminator sequence is shown as SEQ ID NO.6, the NOS-R sequence is shown as SEQ ID NO.7, and the 5' -end primer has BsaI cleavage site and protective base.
5. The method according to any one of claims 1 to 4, wherein the cleavage ligation reaction system of step S2 is as follows:
The enzyme digestion ligation reaction was carried out at room temperature for 15min.
6. The method according to claim 1, wherein in step S3, the assembled product after the reaction of step S2 is diluted 20-fold as a PCR reaction template.
7. The method according to claim 5, wherein in step S3, the assembled product after the reaction of step S2 is diluted 20 times as a PCR template, the complete expression cassette is amplified with high fidelity enzyme using 35S-F and NOS-R as primers, and then the PCR product is purified by a magnetic bead method.
8. The method of claim 7, wherein the enrichment of the complete gene expression cassette in step S3 is performed by PCR amplification using the following reaction system:
The reaction procedure: 95℃for 5min, (95℃for 10s,72℃for 1min,35 cycles), 72℃for 5min.
9. The efficient fragment assembly and rapid plant cell protoplast transient expression system is characterized by comprising an assembly reaction system of a DNA fragment gene expression cassette, a protoplast preparation system and a protoplast transformation system, wherein the assembly reaction system of the DNA fragment gene expression cassette comprises a PCR amplification reaction system of a promoter, a gene coding region and a terminator sequence, a Golden Gate reaction system and an enrichment reaction system of the gene expression cassette.
10. Use of a high efficiency fragment assembly and rapid plant cell protoplast transient expression method according to any one of claims 1-8 or a high efficiency fragment assembly and rapid plant cell protoplast transient expression system according to claim 9 for exogenous gene expression for non-disease diagnosis and treatment purposes, wherein said use comprises at least one of gene subcellular localization, gene function prediction, gene interaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311731956.4A CN117683807A (en) | 2023-12-16 | 2023-12-16 | Efficient fragment assembly and rapid plant cell protoplast transient expression method, system and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311731956.4A CN117683807A (en) | 2023-12-16 | 2023-12-16 | Efficient fragment assembly and rapid plant cell protoplast transient expression method, system and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117683807A true CN117683807A (en) | 2024-03-12 |
Family
ID=90128158
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311731956.4A Pending CN117683807A (en) | 2023-12-16 | 2023-12-16 | Efficient fragment assembly and rapid plant cell protoplast transient expression method, system and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117683807A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002036786A2 (en) * | 2000-10-31 | 2002-05-10 | Medicago Inc. | Method of selecting plant promoters to control transgene expression |
| WO2019104770A1 (en) * | 2017-11-29 | 2019-06-06 | 赛业（广州）生物科技有限公司 | Construction method for seamless multi-fragment cloning |
| CN117025663A (en) * | 2023-08-21 | 2023-11-10 | 广东省农业科学院环境园艺研究所 | Gene transient expression method and application |
-
2023
- 2023-12-16 CN CN202311731956.4A patent/CN117683807A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002036786A2 (en) * | 2000-10-31 | 2002-05-10 | Medicago Inc. | Method of selecting plant promoters to control transgene expression |
| WO2019104770A1 (en) * | 2017-11-29 | 2019-06-06 | 赛业（广州）生物科技有限公司 | Construction method for seamless multi-fragment cloning |
| CN117025663A (en) * | 2023-08-21 | 2023-11-10 | 广东省农业科学院环境园艺研究所 | Gene transient expression method and application |
Non-Patent Citations (1)
| Title |
|---|
| YUMING LU 等: "Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay", PLOS ONE, vol. 8, no. 2, 26 February 2013 (2013-02-26), pages 1 - 4 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106701830B (en) | Pig embryo p66 knock-outshcMethod for gene | |
| CN112020554A (en) | Novel CAS9 orthologs | |
| CN108546716A (en) | A kind of genome edit methods | |
| CN108130342B (en) | Cpf 1-based plant genome fixed-point editing method | |
| KR20230021657A (en) | Enzymes containing RUVC domains | |
| JP2012095653A (en) | Promoter for regulating expression of foreign gene | |
| CN110684769B (en) | Ostrea gigas EF-1 alpha promoter, recombinant vector and application thereof | |
| EP2383337B1 (en) | Novel promoter for use in transformation of algae | |
| CN117025663A (en) | Gene transient expression method and application | |
| US8691505B2 (en) | Promoter for use in transformation of algae | |
| CN117683807A (en) | Efficient fragment assembly and rapid plant cell protoplast transient expression method, system and application | |
| AU2022335499A1 (en) | Enzymes with ruvc domains | |
| US6218145B1 (en) | Bacterial expression systems based on plastic or mitochondrial promoter combinations | |
| EP4148129A1 (en) | Directed evolution method based on primary and secondary replicon of gemini virus | |
| CN114891791B (en) | sgRNA of specific targeting canine Rosa26 gene and application thereof | |
| CN109706170A (en) | The expression vector establishment method of arabidopsis FIPV promoter fusion gus gene | |
| CN108486115A (en) | A kind of transfection method and its application for the expression of crayfish Cells Exogenous genetic | |
| CN116987694A (en) | Bidirectional promoter of medaka and application thereof | |
| CN116875601A (en) | GsSRKpro promoter responding to osmotic or ABA stress and application thereof | |
| Griffin | Improving the genetic editing efficiency of CRISPR/Cas9 in maize through intron mediated enhancement (IME) of Cas9 expression. | |
| CN115717154A (en) | Method for knocking in exogenous gene at fixed point in yeast gene | |
| CN117844840A (en) | Plasmid system for efficiently mediating exogenous gene integration and application thereof | |
| CN116478996A (en) | Sweet potato U6 promoter, expression cassette, recombinant expression vector and application thereof | |
| CN116463342A (en) | Application of carnation DcU6 promoter in CRISPR/Cas9 gene editing system and gene editing method | |
| CN114891786A (en) | Dog Rosa26 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 |