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WO2025007760A1 - Construction et utilisation d'un outil d'expression de cellules végétales - Google Patents

Construction et utilisation d'un outil d'expression de cellules végétales Download PDF

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WO2025007760A1
WO2025007760A1 PCT/CN2024/100713 CN2024100713W WO2025007760A1 WO 2025007760 A1 WO2025007760 A1 WO 2025007760A1 CN 2024100713 W CN2024100713 W CN 2024100713W WO 2025007760 A1 WO2025007760 A1 WO 2025007760A1
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sequence
protein
expression
promoter
nucleic acid
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Chinese (zh)
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王勇
田晨菲
李建华
孙雨伟
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Center for Excellence in Molecular Plant Sciences of CAS
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Center for Excellence in Molecular Plant Sciences of CAS
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • 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)
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    • 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
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    • 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
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
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    • 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
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    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to the field of synthetic biology, and in particular to the creation and use of expression tools in plant cells.
  • Plants use carbon dioxide as a carbon source and are able to perform photosynthetic autotrophy. Plant cells have a wealth of secondary metabolic synthesis pathways and can produce a variety of precursors for the synthesis of complex compounds.
  • plants as synthetic biology chassis also have the advantages of high safety, easy large-scale cultivation and production. With the development of plant synthetic biology technology, more and more functional therapeutic proteins and active small molecules have been successfully synthesized in plant chassis. The most widely used plant chassis in synthetic applications is tobacco. It has the advantages of a short growth cycle and large biomass.
  • ZMapp a special antibody drug for the treatment of Ebola virus, was produced in tobacco, which was able to cure seriously ill Ebola patients in clinical trials; in 2022, Medicago's new crown vaccine produced using tobacco was as effective as 71.6% against the new crown mutant strain, and has now been approved for marketing by the Canadian Ministry of Health.
  • bxn's transgenic tobacco has been approved for commercial cultivation in the UK; NtQPT1's transgenic tobacco has also passed safety verification in the United States and has been commercially cultivated.
  • An effective solution is to introduce artificial gene expression regulatory tools to achieve the regulation of the expression pattern and expression intensity of the target gene.
  • Synthetic biology is based on rational design, and through the integration and assembly of components, artificial nucleic acid/protein sequence tools with specific functions are constructed.
  • Artificially synthesized expression regulatory tools have shown superior and more controllable functional traits in microbial cells than natural gene sequences.
  • a highly active gene expression tool can help improve the economic benefits of producing functionally active proteins in plant cells and promote the commercial application of plant chassis.
  • the present invention provides an artificial transcription activation expression tool, which can improve the expression of target proteins in plant cells.
  • the first aspect of the present invention provides a chimeric promoter comprising a protein binding region and a core promoter sequence.
  • the protein binding region is a nucleotide region that binds an activator protein.
  • the protein binding region comprises 1-8 tandem repeats of the sequence shown in SEQ ID NO: 1, positions 1-36.
  • the number of repeats is 5 and/or 6.
  • the core promoter sequence comprises an Act2 core promoter sequence, preferably an Act2 core promoter sequence of Arabidopsis thaliana.
  • the core promoter element is a fragment of the Act2 promoter of Arabidopsis thaliana comprising the sequence shown in SEQ ID NO: 1, positions 43-243. The fragment is 200-1000 bp, preferably 200-500 bp.
  • the protein binding region and the core promoter sequence are directly linked or operably linked via a linker.
  • the protein binding region is located upstream of the core promoter sequence.
  • the distance between the protein binding region and the core promoter sequence is 0-300 nt, preferably 0-216 nt.
  • the chimeric promoter sequence has a sequence shown in any one of SEQ ID NOs: 1-8 or a variant thereof having at least 90% sequence identity thereto and retaining the protein binding function of SEQ ID NOs: 1-8.
  • the second aspect of the present invention provides a fusion protein comprising a nucleic acid binding domain and a transcription activation domain, wherein the nucleic acid binding domain is a protein or domain capable of binding to nucleic acid, including a LexA binding protein or a peptide segment thereof containing a LexA recognition domain.
  • the nucleic acid binding domain has the amino acid sequence of SEQ ID NO: 9, positions 1-87, or a homologous sequence having at least 90% sequence identity therewith and retaining the nucleic acid recognition function.
  • the homologous sequence is from the family Enterobacteriaceae or from the genus Escherichia.
  • the transcriptional activation domain is selected from VP16, DELL, ERF2, One or more of Med2, P300, TV, VP64, VPR; preferably VPR or TV; more preferably VPR.
  • the nucleic acid binding domain and the transcription activation domain are directly connected or connected via a linker peptide.
  • the fusion protein further comprises a nuclear localization signal (NLS), preferably a plant cell nuclear localization signal amino acid sequence.
  • NLS nuclear localization signal
  • the sequence of the nuclear localization signal is as shown in SEQ ID NO: 10, positions 1-8.
  • the fusion protein has a sequence shown in any one of SEQ ID NOs: 9-17 or a variant thereof having at least 90% sequence identity thereto and retaining the function of SEQ ID NOs: 9-17.
  • the present invention also provides a nucleic acid construct comprising (1) the chimeric promoter described in any embodiment of the present invention and (2) the coding sequence of the fusion protein described in any embodiment of the present invention.
  • the nucleic acid construct is a vector, preferably a non-viral vector, more preferably a vector suitable for replication, transcription and/or expression in plants.
  • the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.
  • the nucleic acid construct comprises a first expression cassette and a second expression cassette
  • the first expression cassette comprises the coding sequence of the fusion protein
  • the second expression cassette comprises the chimeric promoter
  • the first expression cassette and the second expression cassette are located on the same vector or on different vectors.
  • the first expression cassette comprises the coding sequence of the fusion protein, a promoter located upstream of the fusion protein, and a terminator located downstream of the fusion protein.
  • the promoter of the fusion protein in the first expression frame is a promoter suitable for initiating expression in plants, for example, selected from P_StUbi, P_MAS, P_AtRbcsl, P_CsVMV, P_Ocs, P_rbc.
  • the sequences of the promoters are shown in SEQ ID NOs: 18-23, respectively.
  • the terminator in the first expression frame is a terminator capable of terminating expression in plants.
  • the terminator is selected from T_AtHsp, T_RbcS1, T_MAS, T_AtAct2, T_Ubq and T_35S.
  • the terminator sequence is shown in SEQ ID NO: 29.
  • the second expression cassette further comprises a region for inserting exogenous sequences, such as a cloning site, located downstream of the chimeric promoter.
  • the second expression cassette further comprises a terminator located downstream of the chimeric promoter.
  • the terminator is selected from T_AtHsp, T_RbcS1, T_MAS, T_AtAct2, T_Ubq and T_35S.
  • the terminator sequence is as shown in SEQ ID NO: 24.
  • the second expression cassette further comprises a coding sequence of a target protein, and the coding sequence of the target protein is expressed by the chimeric promoter.
  • the coding sequence of the target protein is located downstream of the chimeric promoter.
  • the coding sequence of the target protein is located in the cloning site.
  • the coding sequence of the target protein comprises the coding sequence of one or more proteins selected from EGFP protein, West Nile virus NS1 protein, Zika virus NS1 protein, and Miraculin.
  • the second expression cassette comprises the chimeric promoter, a cloning site, and a terminator sequence located downstream of the cloning site.
  • the second expression cassette comprises the chimeric promoter, a target protein coding sequence, and a terminator sequence located downstream of the target protein.
  • a fusion protein according to any embodiment of the present invention and an expression cassette having a chimeric promoter according to any embodiment of the present invention, or,
  • the host cell comprises Escherichia coli or Agrobacterium.
  • the host cell is not a plant cell.
  • the present invention also provides a kit comprising the chimeric promoter, fusion protein, nucleic acid construct, and/or host cell described in any embodiment of the present invention.
  • the present invention also provides a method for expressing a target protein in a cell, wherein the cell comprises: (1) a fusion protein according to any embodiment of the present invention and (2) an expression vector having a chimeric promoter according to any embodiment of the present invention.
  • the coding sequence of the target protein is operably linked to the chimeric promoter, and the method includes the step of incubating the cells under conditions where the fusion protein induces the chimeric promoter to start expression.
  • the conditions are conditions suitable for the growth of the cells.
  • the cell comprises a nucleic acid construct described herein.
  • the method comprises: (1) introducing the coding sequence of the target protein into the nucleic acid construct described herein, wherein the coding sequence of the target protein is expressed by the chimeric promoter; (2) transferring the nucleic acid construct into a cell; (3) incubating the cell under conditions suitable for the expression of the nucleic acid construct.
  • the coding sequence of the target protein is introduced downstream of the chimeric promoter in the second expression frame, preferably, introduced into the cloning site downstream of the chimeric promoter.
  • the conditions suitable for the expression of the nucleic acid construct are conditions under which the first expression frame is expressed and the expressed fusion protein can induce the chimeric promoter to initiate the expression of the target nucleic acid. In one or more embodiments, the conditions are conditions suitable for cell growth.
  • the method comprises: (1) introducing the coding sequence of the target protein into a second expression frame comprising a chimeric promoter as described in any embodiment herein, located downstream of the chimeric promoter, (2) introducing the second expression frame into a cell, the cell comprising the fusion protein of any embodiment herein or containing a first expression frame, the first expression frame comprising the coding sequence of the fusion protein as described in any embodiment herein; (3) incubating the cell under conditions suitable for expression of the second expression frame.
  • the second expression frame further comprises a terminator and an optional cloning site.
  • the coding sequence of the target protein is located in the cloning site.
  • the conditions suitable for expression of the second expression frame are conditions under which the fusion protein can induce the chimeric promoter to initiate expression, or conditions under which the first expression frame is expressed and the expressed fusion protein can induce the chimeric promoter to initiate expression of the coding sequence of the target protein. In one or more embodiments, the conditions are conditions suitable for cell growth.
  • the coding sequence of the target protein comprises the coding sequence of one or more proteins selected from EGFP protein, West Nile virus NS1 protein, Zika virus NS1 protein, and Miraculin.
  • the cell is a plant cell.
  • the present invention also provides a method for obtaining a plant expressing a target protein, comprising:
  • nucleic acid construct described herein which contains the coding sequence of the target protein, into a plant organ or tissue to obtain a plant tissue or organ transformed with the nucleic acid construct, wherein the coding sequence of the target protein is expressed by the chimeric promoter;
  • the coding sequence of the target protein is located in the second expression frame and is expressed by the chimeric promoter.
  • step (1) (2) contacting the plant tissue or organ with the Agrobacterium in step (1), so that the coding sequence of the target protein is transferred into and integrated into the chromosome of the plant cell;
  • the present invention introduces artificial promoter nucleotide sequences and artificially activated fusion proteins into plant cells, utilizes activated transcription circuits to increase the expression of endogenous and exogenous proteins in plant cells, and achieves the expression pattern and intensity of target genes that cannot be achieved by natural promoters, which helps to improve the economic benefits of producing functional active proteins in plant cells and promote the commercial application of plant chassis.
  • Figure 1 is a diagram of the structure of the EGFP protein expression plasmid.
  • FIG. 2 is a diagram showing the structure of the West Nile virus NS1 protein expression plasmid.
  • Figure 3 is a diagram of the structure of the Zika virus NS1 protein expression plasmid.
  • FIG. 4 is a diagram showing the structure of the Miraculin expression plasmid.
  • FIG5 shows the expression intensity of EGFP/mCherry produced by different APBD copy numbers detected by an ELISA instrument.
  • the data show the numerical ratio of EGFP and mCherry.
  • FIG. 6 shows the expression intensity of EGFP/mCherry produced by different transcription activating proteins detected by an ELISA instrument.
  • Figure 7 shows the detection of EGFP/mCherry expressed by the VPR transcriptional activation system by an ELISA instrument Expression intensity.
  • FIG8 is the detection of EGFP protein expression based on the VPR transcription activation system.
  • FIG. 9 is a Western blot showing the expression of West Nile virus NS1 protein obtained using a transcription activation system.
  • FIG. 10 is a Western blot showing the expression of Zika virus NS1 protein obtained using a transcription activation system.
  • FIG. 11 is a Western blot showing the expression of Miraculin obtained by using a transcription activation system.
  • the inventors provide a chimeric promoter comprising a protein binding region and a core promoter sequence.
  • the protein binding region comprises 1-8 repeats of the sequence shown in SEQ ID NO: 1, positions 1-36. Preferably, the number of repeats is 5 and/or 6.
  • the core promoter sequence is an Act2 promoter.
  • the Act2 promoter of Arabidopsis thaliana is an Act2 promoter.
  • the transcriptional activation domain binding nucleotide sequence and the core promoter sequence nucleotide sequence can be directly connected or connected through a joint.
  • the joint is an enzyme cutting site that is convenient for genetic manipulation.
  • the "joint" can include GTCGAG or GTCGAGAAGGG.
  • the protein binding region is usually located upstream of the core promoter sequence, with a distance of 0-300 nt, preferably 0-216 nt, from the core promoter sequence.
  • the chimeric promoter has a sequence shown in any one of SEQ ID NOs: 1-8 or a variant thereof having at least 90% sequence identity thereto and retaining the function of SEQ ID NOs: 1-8.
  • the present invention also provides a fusion protein comprising a nucleic acid binding domain that binds to a specific nucleotide sequence and a transcription activation domain that exerts transcriptional activation.
  • domain used is a level located between the super secondary structure and the tertiary structure, and is an independent folding unit within the tertiary structure of the protein, usually a combination of several super secondary structure units.
  • the nucleic acid binding domain is a LexA recognition sequence domain.
  • Transcription activation domain is a protein molecule that can recruit RNA polymerase II to initiate gene transcription.
  • the transcription activation domain is selected from VP16, DELL, ERF2, Med2, P300, TV, VP64, VPR.
  • the sequence of VP16 may be as shown in SEQ ID NO: 9 amino acids 93-215 or a homologous sequence or variant thereof having at least 90%, at least 95%, at least 98%, at least 99% sequence identity with transcriptional activation function.
  • the sequence of DELL may be as shown in SEQ ID NO: 11 amino acids 100-123 or a homologous sequence or variant thereof having at least 90%, at least 95%, at least 98%, at least 99% sequence identity with transcriptional activation function.
  • the sequence of ERF2 may be as shown in SEQ ID NO: 12 amino acids 100-130 or a homologous sequence or variant thereof having at least 90%, at least 95%, at least 98%, at least 99% sequence identity with transcriptional activation function.
  • the sequence of Med2 may be as shown in SEQ ID NO: 13 amino acids 100-529 or a homologous sequence or variant thereof having at least 90%, at least 95%, at least 98%, at least 99% sequence identity with transcriptional activation function.
  • the sequence of P300 may be as shown in SEQ ID NO: 14, amino acids 100-732, or a homologous sequence or variant thereof having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto having a transcriptional activation function.
  • the sequence of TV may be as shown in SEQ ID NO: 15, amino acids 100-549, or a homologous sequence or variant thereof having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto having a transcriptional activation function.
  • the sequence of VP64 may be as shown in SEQ ID NO: 16, amino acids 100-149, or a homologous sequence or variant thereof having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto having a transcriptional activation function.
  • the sequence of VPR may be as shown in SEQ ID NO: 17, amino acids 100-481, or a homologous sequence or variant thereof having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto having a transcriptional activation function. body.
  • homologous sequence refers to a sequence from a species of the same order, family, genus, or species as the species of the source sequence and having the sequence identity.
  • VP16 is derived from human herpesvirus, and its homologous sequence is from the family Herpesviridae
  • DELL and p300 are derived from humans, and their homologous sequences are from the order Primates
  • ERF2 is derived from Arabidopsis, and its homologous sequence is from the order Cruciferae, the family Cruciferae, or the genus Arabidopsis
  • Med2 is derived from Saccharomyces cerevisiae, and its homologous sequence is from the order Saccharomyces, the family Saccharomyces, and the genus Saccharomyces.
  • the nucleic acid binding domain and the transcription activation domain can be directly connected or operably connected through a polypeptide sequence.
  • the polypeptide sequence is a GGGS polypeptide sequence.
  • the fusion protein further comprises a nuclear localization signal amino acid sequence (NLS) for guiding the protein into the nucleus through the nuclear pore complex.
  • NLS nuclear localization signal amino acid sequence
  • the plant cell nuclear localization signal amino acid sequence Preferably, the sequence of the nuclear localization signal is as shown in SEQ ID NO: 10, positions 1-8.
  • the fusion protein has a sequence shown in any one of SEQ ID NOs: 9-17 or a variant thereof having at least 90% sequence identity thereto and retaining the function of SEQ ID NOs: 9-17.
  • the present invention also provides a nucleic acid construct for cloning or expressing a genetic function such as a gene, comprising (1) a chimeric promoter as described in any embodiment of the present invention and (2) a coding sequence of a fusion protein as described in any embodiment of the present invention.
  • the nucleic acid construct is a vector, preferably a non-viral vector, more preferably a vector suitable for replication, transcription and/or expression in plants.
  • the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.
  • the nucleic acid construct comprises a first expression cassette and a second expression cassette
  • the first expression cassette comprises the coding sequence of the fusion protein
  • the second expression cassette comprises the chimeric promoter
  • the first expression cassette comprises the coding sequence of the fusion protein, a promoter sequence located upstream of the fusion protein, and a terminator sequence located downstream of the fusion protein.
  • the promoter of the fusion protein is a promoter suitable for initiating expression in plants, for example, selected from P_StUbi (SEQ ID NO: 18), P_MAS (SEQ ID NO: 19), P_AtRbcs1 (SEQ ID NO: 20), P_CsVMV (SEQ ID NO: 21), P_Ocs (SEQ ID NO: 22), P_rbc (SEQ ID NO: 23).
  • the terminator of the fusion protein is a terminator suitable for terminating expression in plants, including but not limited to terminators such as T_RbcS1 (SEQ ID NO: 29), T_AtHsp, T_MAS, T_AtAct2, T_Ubq and T_35S.
  • terminators such as T_RbcS1 (SEQ ID NO: 29), T_AtHsp, T_MAS, T_AtAct2, T_Ubq and T_35S.
  • the second expression cassette also includes a cloning site.
  • the cloning site is a DNA sequence containing one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20) restriction enzyme sites, and is the location where the exogenous gene is inserted.
  • the cloning site can be a DNA sequence containing a pair of type II s restriction enzyme sites (e.g., Bsa I and BsmB I).
  • type II s restriction enzyme sites e.g., Bsa I and BsmB I.
  • the second expression frame also contains a terminator, which is a terminator suitable for terminating expression in plants, including but not limited to terminators such as T_RbcS1 (SEQ ID NO: 29), T_AtHsp, T_MAS, T_AtAct2, T_Ubq and T_35S.
  • a terminator suitable for terminating expression in plants, including but not limited to terminators such as T_RbcS1 (SEQ ID NO: 29), T_AtHsp, T_MAS, T_AtAct2, T_Ubq and T_35S.
  • the second expression cassette comprises the chimeric promoter and a terminator downstream thereof. In one or more embodiments, the second expression cassette comprises the chimeric promoter, a cloning site and a terminator from 5' to 3'. In one or more embodiments, the second expression cassette comprises the chimeric promoter, a target protein coding sequence and a terminator from 5' to 3'.
  • the second expression frame further comprises a coding sequence (target nucleic acid) of a target protein, and the target nucleic acid is expressed by the chimeric promoter.
  • the target nucleic acid is located downstream of the chimeric promoter. In one or more embodiments, the target nucleic acid is located in the cloning site.
  • the target nucleic acid comprises a coding sequence selected from EGFP protein, West Nile virus NS1 protein, Zika virus NS1 protein, and Miraculin.
  • the amino acid sequence of EGFP protein is shown in SEQ ID NO: 28, and the amino acid sequence of West Nile virus NS1 protein is shown in SEQ ID NO: 25, positions 31-382.
  • the amino acid sequence of Zika virus NS1 protein is shown in SEQ ID NO: 26, positions 31-382.
  • the amino acid sequence of Miraculin is shown in SEQ ID NO: 27, positions 1-220.
  • the polypeptide or protein described herein may also have a signal peptide or endoplasmic reticulum retention sequence to facilitate expression and/or secretion.
  • the sequence of the signal peptide is well known in the art, such as the sequence shown in SEQ ID NO: 25, positions 1-30 or the KDEL sequence.
  • the polypeptide or protein may also have a tag that facilitates identification or purification, such as a Flag tag.
  • the amino acid sequences of West Nile virus NS1 protein, Zika virus NS1 protein and Miraculin containing a signal peptide, a Flag tag and a KDEL sequence are shown in SEQ ID NO: 25-27.
  • a typical expression vector comprises an expression control sequence that can be used to regulate the expression of a desired nucleic acid sequence, and is operably linked to the nucleic acid sequence of the present invention or its complementary sequence.
  • operably connection means that the nucleotide sequence of interest is connected to the regulatory sequence in a manner that allows the expression of the nucleotide sequence.
  • DNA sequence can be effectively connected to an appropriate promoter in the expression vector to guide mRNA synthesis. As long as it can be replicated and stable in the host (especially plant cells), any plasmid and vector can be used.
  • An important feature of an expression vector is that it usually contains regulatory sequences, including but not limited to origins of replication, promoters, marker genes, translation control elements, ribosome binding sites for translation initiation, and transcription terminators, etc.
  • nucleic acid molecules polynucleotides
  • nucleic acids are used interchangeably.
  • promoters include: lac or trp promoters of Escherichia coli; ⁇ phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retroviruses and other known promoters that can control gene expression in prokaryotic or eukaryotic cells (especially plant cells) or their viruses.
  • the promoter of the fusion protein is selected from P_StUbi, P_MAS, P_AtRbcs1, P_CsVMV, P_Ocs, P_rbc.
  • various nucleic acids and regulatory sequences can be linked together to produce a recombinant expression vector that may include one or more convenient restriction sites that allow insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites.
  • the coding sequences of the various transcriptional activation domains described herein are located in the vector so that the coding sequences are operably linked to appropriate regulatory sequences for expression.
  • the expression vector can be any vector (such as a plasmid or virus) that can be easily subjected to recombinant DNA methods and can result in expression of the coding sequence of the transcriptional activation domain described herein.
  • the choice of the vector generally depends on the compatibility of the vector with the host cell into which the vector is introduced.
  • the vector can be a linear or closed circular plasmid.
  • the vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome.
  • the vector can include any means for ensuring self-replication.
  • the vector can be a vector that is integrated into the genome and replicated together with the chromosome into which it has been integrated when introduced into the host cell.
  • a single vector or vectors that together contain the total DNA to be introduced into the genome of the host cell may be used.
  • the expression vector preferably contains one or more selectable markers that allow easy selection of cells for transformation, transfection, transduction, etc. Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophy to auxotrophy, etc.
  • the expression vector contains elements that allow the vector to be integrated into the host cell genome or the vector to replicate autonomously in the cell independently of the genome.
  • More than one copy of the coding sequence of the present invention can be inserted into the host cell to increase the yield of the gene product.
  • the increase in the number of polynucleotide copies can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selection marker gene and the polynucleotide, wherein the cell containing the amplified copy of the selection marker gene and thus the additional copy of the polynucleotide can be screened by culturing the cell in the presence of an appropriate selection agent.
  • the nucleic acid construct described herein comprises a first expression cassette and a second expression cassette, wherein the first expression cassette comprises, from 5' to 3', a promoter as shown in any one of SEQ ID NOs: 18-23, a coding sequence for a polypeptide as shown in any one of SEQ ID NOs: 9-17, and a terminator as shown in SEQ ID NO: 29.
  • the second expression cassette comprises, from 5' to 3', a chimeric promoter as shown in any one of SEQ ID NOs: 1-8, and a terminator as shown in SEQ ID NO: 24, and optionally comprises a cloning site and/or a target protein coding sequence as shown in SEQ ID NOs: 25-28 between the chimeric promoter and the terminator.
  • the nucleic acid construct described herein comprises a first expression cassette and a second expression cassette, wherein the first expression cassette comprises, from 5' to 3', a chimeric promoter as shown in any one of SEQ ID NOs: 1-8 (preferably SEQ ID NO: 6), a coding sequence of a protein as shown in any one of SEQ ID NOs: 25-28, and a terminator as shown in SEQ ID NO: 24, and the second expression cassette comprises, from 5' to 3', a promoter as shown in any one of SEQ ID NOs: 18-23 (preferably SEQ ID NO: 18), a coding sequence of a polypeptide as shown in any one of SEQ ID NOs: 9-17 (preferably SEQ ID NO: 17), and a terminator as shown in SEQ ID NO: 29.
  • the first expression cassette comprises, from 5' to 3', a chimeric promoter as shown in any one of SEQ ID NOs: 1-8 (preferably SEQ ID NO: 6), a coding sequence of a protein as shown in
  • the present invention also provides a host cell, which comprises or has integrated into its genome (1) the fusion protein described in any embodiment herein and (2) an expression cassette having a chimeric promoter described in any embodiment herein, or comprises the nucleic acid construct described in any embodiment herein.
  • Host cells can be used as recipients of vectors.
  • Host cells can be "transfected” or “transformed,” which refers to the process by which exogenous nucleic acids are transfected or transduced into host cells.
  • Transformed cells include the primary subject cell and its progeny.
  • the terms "engineered” and “recombinant” cells or host cells often refer to cells into which exogenous nucleic acid sequences, such as vectors, have been introduced. Thus, recombinant cells can be used with different Naturally occurring cells containing the introduced recombinant nucleic acid are differentiated.
  • the fusion protein coding sequence described in the present invention can be obtained by PCR amplification, recombination or artificial synthesis methods known in the art.
  • the primer sequences and templates disclosed herein can be used for amplification.
  • the vector is transformed into a host cell to produce a protein or peptide including a fusion protein.
  • This transfer process can be carried out using conventional techniques known to those skilled in the art such as transformation or transfection.
  • the host cell of the present invention refers to a cell that can receive and accommodate a recombinant DNA molecule, and is the site of recombinant gene amplification.
  • the ideal recipient cell should meet two conditions of easy access and proliferation.
  • the "host cell” of the present invention may include prokaryotic cells and eukaryotic cells.
  • the host cell of the present invention includes Escherichia coli and Agrobacterium. In one or more embodiments, the host cell is not a plant cell.
  • the host cells can be transformed using methods well known in the art. For example, when the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, and the steps used are well known in the art. Another method is to use MgCl2 . If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the present invention also provides a kit comprising the chimeric promoter, fusion protein, nucleic acid construct, and/or host cell described in any embodiment of the present invention.
  • the present invention also provides a method for expressing a target nucleic acid in a cell (e.g., a plant cell), the cell comprising: (1) a fusion protein according to any embodiment of the present invention and (2) an expression cassette having a chimeric promoter according to any embodiment of the present invention, the target nucleic acid being operably linked to the chimeric promoter, the method comprising the step of incubating the cell under conditions where the fusion protein induces the chimeric promoter to initiate expression. Typically, the conditions are conditions suitable for the growth of the cell.
  • the cell comprises a nucleic acid construct as described herein.
  • the cell expresses the fusion protein through the nucleic acid construct described herein. Therefore, the method for expressing a target nucleic acid comprises: (1) introducing a target nucleic acid into the nucleic acid construct described herein, wherein the target nucleic acid is expressed by the chimeric promoter; (2) introducing the nucleic acid into the nucleic acid construct described herein; (3) incubating the cells under conditions suitable for expression of the nucleic acid construct. In one or more embodiments, the target nucleic acid is introduced downstream of the chimeric promoter in the second expression frame, preferably, into the cloning site.
  • the conditions suitable for expression of the nucleic acid construct are conditions under which the first expression frame is expressed and the expressed fusion protein can induce the chimeric promoter to initiate expression of the target nucleic acid. In one or more embodiments, the conditions are conditions suitable for cell growth.
  • the cell already constitutively expresses the fusion protein, or has been transformed with an expression cassette or vector that expresses the fusion protein.
  • the method of expressing a target nucleic acid comprises: (1) introducing the target nucleic acid into a second expression cassette comprising a chimeric promoter as described in any embodiment herein; (2) introducing the second expression cassette into a cell, the cell comprising the fusion protein as described in any embodiment herein or comprising a first expression cassette comprising a coding sequence for the fusion protein as described in any embodiment herein; (3) incubating the cell under conditions suitable for expression of the second expression cassette.
  • the second expression cassette also includes a cloning site and a terminator, and the target nucleic acid is located in the cloning site.
  • the cloning site and terminator are as described elsewhere in this article.
  • the target nucleic acid includes a coding sequence of one or more proteins selected from EGFP protein, West Nile virus NS1 protein, Zika virus NS1 protein, and Miraculin protein.
  • the conditions suitable for the expression of the second expression cassette are the conditions under which the fusion protein described herein can induce the chimeric promoter to start expression, or the conditions under which the first expression cassette is expressed and the expressed fusion protein can induce the chimeric promoter to start expression of the target nucleic acid.
  • the conditions are conditions suitable for cell growth.
  • the present invention also provides a method for expressing a target nucleic acid in a plant, comprising: (1) transferring the nucleic acid construct described herein containing the target nucleic acid into a plant organ or tissue to obtain a plant tissue or organ transformed with the nucleic acid construct; the target nucleic acid is expressed by the chimeric promoter; and (2) regenerating the plant tissue or organ obtained in step (1) into a plant strain.
  • the target nucleic acid is located in the second expression frame and is expressed by the chimeric promoter.
  • the method comprises the steps of: (a) providing an Agrobacterium carrying an expression vector, wherein the expression vector contains the nucleic acid construct of the present invention; (b) contacting the plant tissue or organ with the Agrobacterium in step (a), thereby transferring and integrating the target nucleic acid into the chromosome of the plant cell; (c) selecting the plant tissue or organ into which the target nucleic acid is transferred; and (d) regenerating the plant tissue or organ in step (c) into a plant.
  • PCR polymerase chain reaction
  • plasmid extraction kit used were all products of Axygen in the United States; PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) polymerase kit and polymerase chain reaction (PCR) high-fidelity enzyme PrimeSTAR Max DNA Polymerase were products of Takara Bio (TAKARA) in Japan; restriction endonucleases were all products of NEB.
  • T4 ligase was a product of Thermo Scientific.
  • Escherichia coli DH10B was used for gene cloning, and Agrobacterium tumefaciens GV3101 strain was used for transient transformation of tobacco leaves.
  • the intermediate gene fragment was constructed using the pEASY-Blunt cloning vector plasmid from Transgen.
  • the pCF001 and pCF161 vectors were kept in the laboratory and used for the assembly of the transcription activation system.
  • PCR was performed using Arktik Thermal Cycler (Thermo Fisher Scientific); constant temperature culture was performed using ZXGP-A2050 constant temperature incubator (Zhicheng) and ZWY-211G constant temperature culture oscillator (Zhicheng); centrifugation was performed using 5418R high-speed refrigerated centrifuge and 5418 small centrifuge (Eppendorf); OD600 was detected using UV-1200 UV-visible spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.); EGFP and mCherry were detected using a multifunctional microplate reader (Thermo Scientific). Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Genes were synthesized by Shanghai Biotechnology Co., Ltd.
  • Example 1 Construction of an artificial promoter reporter plasmid carrying a LexA recognition site
  • primers shown in Table 1 Design the primers shown in Table 1. Using primers Act2-SX-F/200-AtAct2-R and PBS100 plasmid as template, PCR amplify the cpAct promoter sequence with BsaI, SacI and XhoI at the 5' end and BsaI at the 3' end. Load the sequence into the vector pEASY to obtain the pEASY-cpAct2 plasmid. The primer annealing method was used to construct promoters with different copies of LexA protein recognition sites. Using the primer pair LexAPBD-F/LexAPEB-R containing the LexA recognition sequence (APBD), primers were mutually linked.
  • APBD LexA recognition sequence
  • Complementary pairing (TE buffer, 95°C reaction for 5min) can form APBD double-stranded fragments with SacI and XhoI restriction endonucleases at both ends.
  • the APBD double-stranded fragment is connected to the plasmid pEASY-cpAct2 fragment after SacI and XhoI restriction digestion to obtain pEASY-A1cpAct2.
  • the pEASY-A1cpAct2 plasmid is further digested by SacI and XhoI, and the APBD double-stranded fragment can be loaded to obtain pEASY-A2cpAct2.
  • artificial promoters containing 3-8 APBDs can be constructed in sequence.
  • the nucleotide sequence of APBD1-AtAct2 is shown in SEQ ID NO: 1; the nucleotide sequence of APBD2-cpAct2 is shown in SEQ ID NO: 2; the nucleotide sequence of APBD3-cpAct2 is shown in SEQ ID NO: 3; the nucleotide sequence of APBD4-cpAct2 is shown in SEQ ID NO: 4; the nucleotide sequence of APBD5-cpAct2 is shown in SEQ ID NO: 5; the nucleotide sequence of APBD6-cpAct2 is shown in SEQ ID NO: 6; the nucleotide sequence of APBD7-cpAct2 is shown in SEQ ID NO: 7; and the nucleotide sequence of APBD8-cpAct2 is shown in SEQ ID NO: 8;
  • the vector pCF161, the artificial promoter plasmid constructed above, the reporter gene EGFP plasmid PBS70, and the terminator plasmid PBS43 were digested with the endonuclease BsaI, and the target fragments were recovered and then T4 ligated to obtain the reporter plasmid pCF189-pCF197 with 0-8 LexA recognition sites.
  • PCR reaction conditions 98°C pre-denaturation for 2min, then 98°C denaturation for 10s, 55°C annealing for 15s, 72°C extension for 20s, 25 cycles, agarose gel electrophoresis detection, gel cutting to recover target fragments.
  • Enzyme digestion reaction conditions 37°C reaction for 1h, gel cutting to recover target fragments.
  • T4 ligation conditions 22°C reaction for 1h.
  • the assembled sequence is as follows, wherein the single horizontal line represents the recognition sequence of the type 2 restriction endonuclease BsaI, the curve represents the sticky end sequence produced after enzyme cleavage, and the double horizontal line refers to the corresponding functional nucleic acid sequence used in the implementation scheme.
  • Different transcription activation modules were replaced based on the NA-VP16 transcription factor sequence.
  • the primer pair LexA-N-F/LexA-N-R was used to amplify NA fragments with homologous sequences adapted to different transcription activation domains.
  • Different transcription factor primer pairs were used, and the synthesized sequences were used as templates to amplify different transcription activation domain sequences with homologous sequences adapted to NA fragments.
  • the amino acid sequence of the LexA-VP16 fusion protein is shown in SEQ ID NO: 9, wherein the amino acid sequence of the VP16 transcription activation domain is shown in the amino acid sequence at positions 93 to 215 of SEQ ID NO: 9;
  • amino acid sequence of the Nls-LexA-VP16 fusion protein is shown in SEQ ID NO: 10, wherein the amino acid sequence of NLS is shown in the amino acid sequence at positions 1 to 8 of SEQ ID NO: 10;
  • amino acid sequence of the Nls-LexA-ERF2 fusion protein is shown in SEQ ID NO: 12, wherein the amino acid sequence of the ERF2 transcription activation domain is shown in the amino acid sequence at positions 100 to 130 of SEQ ID NO: 12;
  • amino acid sequence of the Nls-LexA-Med2 fusion protein is shown in SEQ ID NO: 13, wherein the amino acid sequence of the Med2 transcription activation domain is shown in the amino acid sequence of positions 100-529 of SEQ ID NO: 13;
  • amino acid sequence of the Nls-LexA-P300 fusion protein is shown in SEQ ID NO: 14, wherein the amino acid sequence of the P300 transcription activation domain is shown in the amino acid sequence of positions 100-732 of SEQ ID NO: 14;
  • amino acid sequence of the Nls-LexA-TV fusion protein is shown in SEQ ID NO: 15, wherein the amino acid sequence of the TV transcriptional activation domain is shown in the amino acid sequence of positions 100-549 of SEQ ID NO: 15;
  • amino acid sequence of the Nls-LexA-VP64 fusion protein is shown in SEQ ID NO: 16, wherein the amino acid sequence of the VP64 transcription activation domain is shown in the amino acid sequence at positions 100 to 149 of SEQ ID NO: 16;
  • amino acid sequence of the Nls-LexA-VPR fusion protein is shown in SEQ ID NO: 17, wherein the amino acid sequence of the VPR transcriptional activation domain is shown in the amino acid sequence of positions 100-481 of SEQ ID NO: 17;
  • the vector pCF001, promoter plasmid PBS90, pEASY plasmid containing various transcription factor genes, and terminator plasmid PBS78 were digested with endonuclease BsaI. After recovering the target fragments, T4 ligation was performed to obtain expression plasmids pCF206, pCF208, pCF215, pCF216, pCF217, pCF218, pCF219, pCF220, and pCF221 carrying different artificial transcription factors.
  • Example 3 Artificial promoters with different APBD copy numbers are used to express EGFP protein
  • the VP16-based transcription activator expression plasmids pCF206, pCF208 and the reporter plasmids pCF189-197 carrying 0-8 APBD copies were transformed into Agrobacterium GV3101 competent cells (see Table 3).
  • LB solid medium kanamycin 50 ⁇ g/mL, gentamicin 2550 ⁇ g/mL, rifampicin 50 ⁇ g/mL
  • kanamycin 50 ⁇ g/mL, gentamicin 2550 ⁇ g/mL, rifampicin 50 ⁇ g/mL was cultured at 30°C for 36 h.
  • a single clone was picked and transferred to 2 mL LB liquid medium (kanamycin 50 ⁇ g/mL, gentamicin 2550 ⁇ g/mL, rifampicin 50 ⁇ g/mL).
  • the transcription activator expression plasmids pCF206, pCF208, pCF215, pCF216, pCF217, pCF218, pCF219, pCF220, pCF221 and the reporter plasmids pCF194 and pCF195 carrying 5/6 copies of APBD were transferred into Agrobacterium GV3101 competent cells (see Table 4), and 4-week-old tobacco leaves were infected according to the method in Example 3. After 3 days, tobacco samples were collected to detect the expression intensity of EGFP and mCherry. The test results are shown in Figure 6. Different transcription factor activators obtained different The activation expression intensity of VPR was the highest, followed by TV.
  • the vector pCF001, promoter plasmid (Table 5), pEASY plasmid containing transcription factor N gene (NAVP16, NAVPR, NATV), and terminator plasmid PBS78 were digested with endonuclease BsaI. After recovering the target fragments, T4 ligation was performed to obtain expression plasmids pCF210-214 and pCF222-231 with artificial transcription factors of different expression intensities.
  • the plasmids pCF206, pCF208, pCF210-214, pCF222-231 expressing transcriptional activators such as VP16 and the reporter plasmids pCF194 and pCF195 carrying 5/6 copies of APBD were transferred into Agrobacterium GV3101 competent cells (see Table 6), and 4-week-old tobacco leaves were infected according to the method in Example 3. Tobacco samples were collected 3 days later to detect the expression intensity of EGFP and mCherry. The test results are shown in Figure 7. The intensity of EGFP expressed based on the transcriptional activation system is generally higher than the intensity of EGFP expressed directly using the natural promoter.
  • the primers in Table 7 were designed using plasmid pCF218 (pCF001-P_90-NAVPR-T_78) as a template.
  • the plasmid pCF232 (pCF218-BsaI) was obtained by homologous recombination of the two gene fragments obtained by PCR.
  • the homologous recombination reaction conditions 37°C for 30min.
  • Example 8 VPR-activated artificial transcriptional activation expression plasmid vector is used to express EGFP protein
  • the vector pCF232, artificial promoter plasmid pEASY-A6cpAct2, EGFP protein plasmid PBS70, and terminator plasmid PBS78 were digested with endonuclease BsaI, and the target fragment was recovered and then T4 ligated to obtain the expression plasmid pCF235 expressing EGFP using the artificial transcription system.
  • the plasmid pCF233 (pCF001-P_90-GFP-T_43) expressing EGFP using promoter PBS90 was used as a control for comparison.
  • the blank control pCF232, the artificial transcription expression plasmid pCF235 and the pCF233 with the strongest expression activity of the natural promoter were transferred into Agrobacterium GV3101 competent cells (see Table 8).
  • Four-week-old tobacco leaves were infected according to the method in Example 3. After 3 days, 0.2 g of tobacco sample was collected, and the samples were quickly frozen in liquid nitrogen and ground into powder by a grinder. Protein was extracted using protein extraction buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% Glycerol, 1 mM EDTA, and 1 mM PMSF was added before use).
  • Example 9 VPR-activated artificial transcriptional activation expression plasmid vector for expressing West Nile virus NS1 protein
  • SWFK The gene plasmid (SWFK) of West Nile virus_NS1 with BsaI restriction sites at both ends was directly synthesized by JEW.
  • SWFK The gene plasmid
  • JEW The gene plasmid (SWFK) of West Nile virus_NS1 with BsaI restriction sites at both ends was directly synthesized by JEW.
  • PR1a signal peptide at the front end of West Nile virus NS1 and a 3 ⁇ Flag tag and KDEL sequence at the end.
  • SWFK sequence is shown in SEQ ID NO: 25, wherein the signal peptide sequence of PR1a is shown in the amino acid sequence of positions 1-30 of SEQ ID NO: 25; the 3 ⁇ Flag tag and KDEL sequence are shown in the amino acid sequence of positions 390-414 of SEQ ID NO: 25.
  • the vector pCF232, artificial promoter plasmid pEASY-A6cpAct2, synthetic SWFK, and terminator plasmid PBS78 were digested with endonuclease BsaI, and the target fragment was recovered and then connected by T4 to obtain the expression plasmid pCFP7 expressing SWFK in the artificial transcription system.
  • Plasmid pCFP2 (pCF001-P_90-SWFK-T_43) expressing SWFK using promoter PBS90 and plasmid pCFP001 (pCF001-P_83-SWFK-T_98) expressing SWFK using vector pEAQ-HT were used as controls for protein expression for comparison.
  • the artificial transcription expression plasmid pCFP7 and pCFP1 and pCFP2 with high expression activity of natural promoter were transferred into Agrobacterium GV3101 competent cells (see Table 9), and 4-week-old tobacco leaves were infected according to the method in Example 3. Proteins were extracted according to the method in Example 8 to prepare protein samples. After running SDS-PAGE gel, protein expression was detected by Western blot (Flag antibody purchased from Sigma). The expression results are shown in Figure 9. The NS1 protein concentration obtained by using the transcription activation system is much higher than that of the NS1 protein expressed using the natural promoter.
  • Example 10 VPR-activated artificial transcriptional activation expression plasmid vector for expressing Zika virus NS1 protein
  • Zika_NS1 gene (SZFK) plasmid carrying BsaI restriction sites at both ends was directly synthesized by Jierui Co.
  • Zika_NS1 In order to facilitate expression in plants, we designed Zika_NS1 with a PR1a signal peptide at the front end and a 3 ⁇ Flag tag and KDEL sequence at the end.
  • SZFK sequence is shown in SEQ ID NO: 26, wherein the signal peptide sequence of PR1a is shown in the amino acid sequence of positions 1-30 of SEQ ID NO: 26; the 3 ⁇ Flag signal peptide and KDEL signal peptide sequences are shown in the amino acid sequence of positions 390-414 of SEQ ID NO: 26.
  • the vector pCF232, artificial promoter plasmid pEASY-A6cpAct2, synthetic SZFK, and terminator plasmid PBS78 were digested with endonuclease BsaI, and the target fragment was recovered and then connected by T4 to obtain the expression plasmid pCFP8 expressing SZFK in the artificial transcription system.
  • the plasmid pCFP5 (pCF001-P_90-SZFK-T_43) expressing SZFK using promoter PBS90 and the plasmid pCFP4 (pCF001-P_83-SZFK-T_98) expressing SZFK using vector pEAQ-HT were used as high expression controls for comparison.
  • the vectors pCF001, pEASY-A6cpAct2, SZFK, and PBS78 were digested with the endonuclease BsaI, and the target fragments were recovered and then ligated with T4 to obtain the expression plasmid pCFP006 of SZFK without artificial transcription factors as a negative control.
  • the blank control pCF001 and pCFP006 (pCF001-A6-cpAct2-SZFK-T_43), the artificial transcription expression plasmid pCFP8, and the pCFP4 and pCFP5 with high expression activity of the natural promoter were transferred into Agrobacterium GV3101 competent cells (see Table 10), and the 4-week-old tobacco leaves were infected according to the method in Example 3, and the protein was extracted according to the method in Example 8 to prepare the protein sample. After running the SDS-PAGE gel, the protein expression was detected by Western blot (the antibody was from the Flag antibody of SIGMA Company, the item number is: A8592). The expression results are shown in Figure 10. The NS1 protein concentration obtained by using the transcription activation system is much higher than the NS1 protein expressed using the natural promoter.
  • Example 11 VPR-activated artificial transcriptional activation expression plasmid vector used to express Miraculin
  • the plasmid containing the gene (MIRF) of Miraculin with BsaI restriction sites at both ends was directly synthesized by JEWELL.
  • MIRF gene of Miraculin with BsaI restriction sites at both ends
  • the MIRF sequence is shown in SEQ ID NO: 27, wherein the 3 ⁇ Flag tag and KDEL sequence are shown in the amino acid sequence of positions 228-249 of SEQ ID NO: 26.
  • the vector pCF232, artificial promoter plasmid pEASY-A6cpAct2, synthetic MIRF, and terminator plasmid PBS78 were digested with endonuclease BsaI, and the target fragment was recovered and then connected by T4 to obtain the expression plasmid pCFP12 expressing SZFK using the artificial transcription system.
  • the plasmid pCFP10 (pCF001-P_90-MIRF-T_43) expressing MIRF using the promoter PBS90 and the plasmid pCFP9 (pCF001-P_83-MIRF-T_98) expressing MIRF using the vector pEAQ-HT were used as high expression controls for comparison.
  • the vectors pCF001, pEASY-A6cpAct2, MIRF, and PBS78 were digested with endonuclease BsaI, and the target fragments were recovered and then subjected to T4 ligation to obtain the expression plasmid pCFP011 of MIRF without artificial transcription factor as a negative control.
  • the blank control pCF001 and pCFP011 (pCF001-A6-cpAct2-MIRF-T_43), the artificial transcription expression plasmid pCFP012, and the pCFP009 and pCFP010 with high expression activity of the natural promoter were transferred into Agrobacterium GV3101 competent cells (see Table 11), and 4-week-old tobacco leaves were infected according to the method in Example 2. Proteins were extracted according to the method in Example 8 to prepare protein samples. After running the SDS-PAGE gel, the protein expression was detected by Western blot (the antibody was from the Flag antibody of SIGMA Company, item number: A8592). The expression results are shown in Figure 11. The miraculin protein concentration obtained using the transcription activation system is much higher than the miraculin protein expressed using the natural promoter.

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Abstract

L'invention concerne la construction et l'utilisation d'un outil d'expression de cellules végétales. Plus particulièrement, l'invention concerne une construction d'acide nucléique, comprenant un promoteur chimère et une séquence codante d'une protéine de fusion, le promoteur chimère comprenant une région de liaison à la protéine et une séquence de promoteur central, et la protéine de fusion comprenant un domaine de liaison à l'acide nucléique et un domaine d'activation transcriptionnelle. L'outil d'expression peut améliorer l'expression de protéines cibles dans des cellules végétales.
PCT/CN2024/100713 2023-07-03 2024-06-21 Construction et utilisation d'un outil d'expression de cellules végétales Pending WO2025007760A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100071086A1 (en) * 2008-09-12 2010-03-18 Mendel Biotechnology, Inc. Polysome-mediated cell type-, tissue type- or condition-enhanced transcript profiling
CN102190731A (zh) * 2010-03-09 2011-09-21 中国科学院上海生命科学研究院 用人工转录因子诱导产生多能干细胞
CN102643852A (zh) * 2011-02-28 2012-08-22 华东理工大学 光可控的基因表达系统
CN104531702A (zh) * 1999-12-16 2015-04-22 孟山都技术有限公司 新型植物表达构建物
US20190203214A1 (en) * 2016-06-20 2019-07-04 Algentech Sas Protein production in plant cells
CN110172477A (zh) * 2019-05-17 2019-08-27 中国科学院遗传与发育生物学研究所 一种优化的获得无选择标记转基因植物的化学诱导删除表达载体及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104531702A (zh) * 1999-12-16 2015-04-22 孟山都技术有限公司 新型植物表达构建物
US20100071086A1 (en) * 2008-09-12 2010-03-18 Mendel Biotechnology, Inc. Polysome-mediated cell type-, tissue type- or condition-enhanced transcript profiling
CN102190731A (zh) * 2010-03-09 2011-09-21 中国科学院上海生命科学研究院 用人工转录因子诱导产生多能干细胞
CN102643852A (zh) * 2011-02-28 2012-08-22 华东理工大学 光可控的基因表达系统
US20190203214A1 (en) * 2016-06-20 2019-07-04 Algentech Sas Protein production in plant cells
CN110172477A (zh) * 2019-05-17 2019-08-27 中国科学院遗传与发育生物学研究所 一种优化的获得无选择标记转基因植物的化学诱导删除表达载体及其应用

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