WO2022042446A1 - 一种提高植物中维生素c含量的方法 - Google Patents

一种提高植物中维生素c含量的方法 Download PDF

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WO2022042446A1
WO2022042446A1 PCT/CN2021/113825 CN2021113825W WO2022042446A1 WO 2022042446 A1 WO2022042446 A1 WO 2022042446A1 CN 2021113825 W CN2021113825 W CN 2021113825W WO 2022042446 A1 WO2022042446 A1 WO 2022042446A1
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mutant
utr
utr nucleotide
plants
plant
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French (fr)
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李彦莎
梁亚峰
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山东舜丰生物科技有限公司
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    • 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
    • C12N15/1137—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 against enzymes
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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)
    • C12N15/8241—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
    • C12N15/8243—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 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07069—GDP-L-galactose phosphorylase (2.7.7.69)

Definitions

  • the invention relates to the field of botany, and more particularly to a method for increasing the content of vitamin C in plants.
  • Vitamin C (Vitamin C/ascorbic acid, also known as L-ascorbic acid) is an essential substance in most organisms. It not only plays an important role in regulating the reduction potential of cells, but also assists in the normal functioning of key enzymes in many important biological reactions. factor. However, humans lost the ability to synthesize vitamin C by themselves during the evolution process, so this essential vitamin can only be obtained through food intake. If the intake of vitamin C is insufficient, scurvy will occur.
  • the object of the present invention is to provide a method for increasing the content of vitamin C in plants.
  • an isolated first 5'-UTR nucleotide mutant, the first 5'-UTR nucleotide mutant and the first parental 5'-UTR nucleotide Compared with the absence of a fragment of 50-80bp, preferably 60-70bp, the optimal length of 68bp, the sequence of the first parent 5'-UTR nucleotide is shown in SEQ ID NO.:1.
  • the deleted nucleic acid fragment includes the nucleic acid sequence shown in SEQ ID NO.:2.
  • the deleted nucleic acid fragment is the nucleotide of the nucleotide sequence shown in SEQ ID NO.: 2, a fragment thereof, or a conservative variant nucleotide thereof.
  • the mutant is a nucleotide having the nucleotide sequence shown in SEQ ID NO.: 3, a fragment thereof, or a conservative variant nucleotide thereof.
  • nucleotide sequence of the mutant is shown in SEQ ID NO.:3.
  • the remaining nucleotide sequence of the mutant is the same as that of SEQ ID NO.:1
  • the sequences shown are the same or substantially the same.
  • the substantially same number is at most 50 (preferably 1-40, more preferably 1-30, more preferably 1-15, more preferably 1-5 nucleotides) are not identical, wherein the difference includes substitution, deletion or addition of nucleotides.
  • the first parent 5'-UTR is a protein in the cell genome that is related to GDP-L-galactose phosphorylase 2-like (also referred to as GGP2 or LsGGP2 herein) (GDP-L-galactose phosphorylase 2-
  • GGP2 GGP2 or LsGGP2 herein
  • LsGGP2 GDP-L-galactose phosphorylase 2-
  • the NCBI accession number of the like protein is LOC111907963) the nucleotide sequence related to the expression regulation.
  • the first parent 5'-UTR is located upstream of the initiation codon encoding the GGP2 nucleotide sequence in the genome of the cell.
  • the cells include eukaryotic cells and prokaryotic cells.
  • the cells are derived from plants, animals or microorganisms.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plant is selected from the group consisting of Compositae, Poaceae, Leguminosae, Cruciferae, or a combination thereof.
  • the plant is selected from the group consisting of rice, corn, tobacco, sorghum, wheat, barley, corn, soybean, Arabidopsis, potato, tomato, lettuce, rape, cabbage, quinoa, or its combination.
  • the first parent 5'-UTR is derived from a monocotyledonous plant or a dicotyledonous plant.
  • the first parent 5'-UTR is derived from one or more plants selected from the group consisting of Compositae, Poaceae, Leguminosae, and Cruciferae.
  • the first parent 5'-UTR is derived from one or more plants selected from the group consisting of rice, corn, tobacco, sorghum, wheat, barley, corn, soybean, Arabidopsis, Potatoes, tomatoes, lettuce, canola, cabbage, quinoa.
  • the mutant is derived from a monocotyledonous plant or a dicotyledonous plant.
  • the mutant is derived from one or more plants selected from the group consisting of Compositae, Poaceae, Leguminosae, and Cruciferae.
  • the mutant is derived from one or more plants selected from the group consisting of rice, corn, tobacco, sorghum, wheat, barley, corn, soybean, Arabidopsis, potato, tomato, lettuce , rapeseed, cabbage, quinoa.
  • the first parent 5'-UTR is derived from lettuce (Lactuca sativa).
  • the mutant is derived from lettuce (Lactuca sativa).
  • the mutant is selected from the following group:
  • the mutant is formed by mutation of the 5'-UTR nucleotide shown in SEQ ID NO.1.
  • the mutant is DNA.
  • the deletion is achieved by means of gene editing, gene knockout, or gene deletion.
  • the gene editing method is selected from the group consisting of Crispr, Cre-lox, ZFNs, TALENs, homologous recombination, or a combination thereof.
  • a second aspect of the present invention provides a mutant combination, the mutant combination comprising:
  • the nucleic acid fragment deleted in the second 5'-UTR nucleotide mutant comprises SEQ ID NO.: The nucleic acid sequence shown in 5.
  • the deleted nucleic acid fragment is the nucleotide of the nucleotide sequence shown in SEQ ID NO.: 5, a fragment thereof, or a conservative variant nucleotide thereof.
  • the second 5'-UTR nucleotide mutant is a nucleotide having the nucleotide sequence shown in SEQ ID NO.: 6, a fragment thereof, or a conservative variant nucleotide thereof .
  • nucleotide sequence of the second 5'-UTR nucleotide mutant is shown in SEQ ID NO.:6.
  • the remaining nucleotide sequence of the mutant is the same as that of SEQ ID NO.:
  • the sequences shown in 4 are identical or substantially identical.
  • the substantially same number is at most 50 (preferably 1-40, more preferably 1-30, more preferably 1-15, more preferably 1-5 nucleotides) are not identical, wherein the difference includes substitution, deletion or addition of nucleotides.
  • the second parent 5'-UTR is the NCBI registration of the GDP-L-galactose phosphorylase 2 (also referred to as GGP1 or LsGGP1 herein) (GDP-L-galactose phosphorylase 2 protein) in the cell genome No. LOC111899617) expression regulation related nucleotide sequence.
  • the second parent 5'-UTR is located upstream of the initiation codon encoding the GGP1 nucleotide sequence in the genome of the cell.
  • the cells include eukaryotic cells and prokaryotic cells.
  • the cells are derived from plants, animals or microorganisms.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plant is selected from the group consisting of Compositae, Poaceae, Leguminosae, Cruciferae, or a combination thereof.
  • the plant is selected from the group consisting of rice, corn, tobacco, sorghum, wheat, barley, corn, soybean, Arabidopsis, potato, tomato, lettuce, rape, cabbage, quinoa, or its combination.
  • the first and second parent 5'-UTRs are each independently derived from a monocotyledonous plant or a dicotyledonous plant.
  • first and second parent 5'-UTRs are each independently derived from one or more plants selected from the group consisting of Compositae, Poaceae, Leguminosae, and Cruciferae.
  • the first and second parent 5'-UTRs are each independently derived from one or more plants selected from the group consisting of rice, corn, tobacco, sorghum, wheat, barley, corn, Soybeans, Arabidopsis, potatoes, tomatoes, lettuce, canola, cabbage, quinoa.
  • the first and second 5'-UTR nucleotide mutants are each independently derived from a monocotyledonous plant or a dicotyledonous plant.
  • the first and second 5'-UTR nucleotide mutants are each independently derived from one or more plants selected from the group consisting of: Compositae, Poaceae, Leguminosae, Cruciferae Floral plants.
  • the first and second 5'-UTR nucleotide mutants are each independently derived from one or more plants selected from the group consisting of rice, corn, tobacco, sorghum, wheat, Barley, Corn, Soybean, Arabidopsis, Potato, Tomato, Lettuce, Canola, Cabbage, Quinoa.
  • first and second parent 5'-UTRs are each independently derived from lettuce (Lactuca sativa).
  • the first and second 5'-UTR nucleotide mutants are each independently derived from lettuce (Lactuca sativa).
  • the second 5'-UTR nucleotide mutant is selected from the following group:
  • the second 5'-UTR nucleotide mutant is formed by mutation of the 5'-UTR nucleotide shown in SEQ ID NO. 4.
  • the mutant is DNA.
  • the deletion is achieved by means of gene editing, gene knockout, or gene deletion.
  • the gene editing method is selected from the group consisting of Crispr, Cre-lox, ZFNs, TALENs, homologous recombination, or a combination thereof.
  • the third aspect of the present invention provides a nucleic acid construct comprising the 5'-UTR nucleotide mutant described in the first aspect of the present invention or a combination of mutants described in the second aspect of the present invention.
  • the nucleic acid construct further comprises one or more selected from the group consisting of a promoter, a GGP2 or GGP1 protein encoding gene, an enhancer, a terminator or a tag gene.
  • the promoter is selected from the group consisting of a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a strong promoter.
  • the promoter is selected from the following group: UBI, UBQ, 35S, Actin, SPL, CmYLCV, YAO, CDC45, rbcS, rbcL, PsGNS2, UEP1, TobRB7, Cab, 6, U3, U6a, U6b, U6c, U6-1, U3b, U3d, U6-26, U6-29, H1, or a combination thereof.
  • the GGP2 or GGP1 protein-encoding gene is derived from plants or microorganisms.
  • the GGP2 or GGP1 protein-encoding gene is derived from a monocotyledonous plant or a dicotyledonous plant.
  • the GGP2 or GGP1 protein-encoding gene is derived from rice, corn, tobacco, sorghum, wheat, barley, corn, soybean, Arabidopsis, potato, tomato, lettuce, rape, cabbage, quinoa .
  • the GGP2 or GGP1 protein-encoding genes include wild-type and mutant genes.
  • a fourth aspect of the present invention provides a nucleic acid construct for gene editing, the nucleic acid construct capable of mutating a first parental 5'-UTR nucleotide (SEQ ID NO.: 1) to a first 5'- A UTR nucleotide mutant; and/or a second parental 5'-UTR nucleotide (SEQ ID NO.: 4) is mutated to a second 5'-UTR nucleotide mutant, wherein the first 5'- UTR nucleotide mutants have fragments of 50-80 bp (preferably, 60-70 bp, most preferably, 68 bp) in length compared to the first parental 5'-UTR nucleotide (SEQ ID NO.: 1) Deletion; the second 5'-UTR nucleotide mutant has 80-100 bp compared with the second parent 5'-UTR nucleotide (SEQ ID NO.: 4), preferably 90-95 bp, most Deletion of the optimal 91
  • the nucleic acid construct includes a gene editing protein.
  • the gene editing protein is selected from the group consisting of CRISPR, TALEN, ZFN, or a combination thereof.
  • the CRISPR protein is selected from the group consisting of Cas9, Cas12, Cas13, or a combination thereof.
  • the nucleic acid construct further includes gRNA.
  • At least part of the sequence of the gRNA can be complementary to the target DNA, and the gRNA can form a functional complex with the CRISPR protein.
  • the gRNA is selected from SEQ ID NO.:7 and/or SEQ ID NO.:8.
  • the gene editing protein includes the gene editing protein required by CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.
  • the fifth aspect of the present invention provides a vector, the vector contains the 5'-UTR nucleotide mutant described in the first aspect of the present invention or the mutant combination described in the second aspect of the present invention or the third aspect of the present invention.
  • the nucleic acid construct of the aspect or the nucleic acid construct of the fourth aspect of the invention is provided.
  • the vector includes an expression vector, a shuttle vector, and an integration vector.
  • the vector is a plant expression vector.
  • the vector is an expression vector that can transfect or transform plant cells.
  • the carrier is an Agrobacterium Ti carrier.
  • the construct is integrated into the T-DNA region of the vector.
  • the carrier is circular or linear.
  • the vector further comprises a selectable marker expression cassette.
  • the sixth aspect of the present invention provides a genetically engineered cell, the genetically engineered cell contains the vector of the fifth aspect of the present invention or the mutant of the first aspect of the present invention or the second aspect of the present invention is integrated into the genome Said mutant combination or the nucleic acid construct of the third aspect of the present invention or the fourth aspect of the present invention.
  • the cells are eukaryotic cells, such as yeast cells, animal cells or plant cells, or prokaryotic cells, such as Escherichia coli.
  • the cells are plant cells.
  • the cells contain a higher content of vitamin C.
  • the genetically engineered cell is a combination of the mutants described in the first aspect of the present invention or the mutants described in the second aspect of the present invention or the third aspect of the present invention by a method selected from the following group.
  • the nucleic acid construct described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention are introduced into cells: Agrobacterium transformation method, gene gun method, microinjection method, electric shock method, ultrasonic method and polyethylene glycol ( PEG) mediated method.
  • the plants include angiosperms and gymnosperms.
  • the gymnosperms are selected from the group consisting of: Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cedaraceae, Cycadaceae, Tricuspidaceae firaceae, yewaceae, ephedraaceae, marigenaceae, monotype, centenaceae, or a combination thereof.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the herb is selected from the group consisting of: Compositae, Solanaceae, Poaceae, Leguminosae, or a combination thereof.
  • the woody plant is selected from the group consisting of Actiniaceae, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of: Cruciferae, Poaceae, Leguminosae, Solanaceae, Actiniaceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, Compositae, or a combination thereof.
  • the plant is selected from the group consisting of Arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, barley, wheat, potato, tomato, rapeseed, lettuce, cabbage, quinoa, sorghum or a combination thereof.
  • the seventh aspect of the present invention provides a method for increasing the content of Vc in a cell, the method comprising introducing the 5'-UTR nucleotide mutant of the first aspect of the present invention or the 5'-UTR nucleotide mutant of the second aspect of the present invention into the cell Said mutant combination or the step of the nucleic acid construct of the third aspect of the invention or the fourth aspect of the invention.
  • the method includes the step of introducing the mutant described in the first aspect of the present invention or a combination of mutants described in the second aspect of the present invention into a cell using gene editing technology.
  • the gene editing technology is selected from the group consisting of CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.
  • the gene editing technology includes a base editor.
  • introducing a 5'-UTR nucleotide mutant or the mutant combination comprises expressing the 5'-UTR nucleotide mutant or mutant combination in a cell,
  • the mutant or mutant combination is expressed through an expression vector, or the mutant or mutant combination is integrated into the plant genome for expression or expression after mutating the cell's endogenous 5'-UTR.
  • the VC content in the cells containing the mutant is increased by ⁇ 4 times, preferably, ⁇ 5 times, more preferably, ⁇ 8 times, compared to the VC content in the wild-type cells of the same species. times.
  • the VC content in the cells containing the mutant is increased by 4-15 times, preferably, 4-10 times, more preferably, compared with the VC content in the wild-type homologous cells, 7-10 times.
  • An eighth aspect of the present invention provides a method for improving plants, the method comprising the steps of:
  • step (b) regenerating the plant cells, plant tissues and plant parts in step (a) into plants.
  • the method comprises the step of introducing the mutant of the first aspect of the present invention or a combination of mutants of the second aspect of the present invention into a cell using gene editing technology.
  • the introduction refers to that the cell contains the mutant of the first aspect of the present invention or a combination of the mutants of the second aspect of the present invention.
  • the gene editing technology is selected from the group consisting of CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.
  • the gene editing technology includes a base editor.
  • the introduction of a 5'-UTR nucleotide mutant or mutant combination comprises expressing the 5'-UTR nucleotide mutant or mutant combination in a cell, for example, by The expression vector expresses the mutant or mutant combination, or integrates the mutant or mutant combination into the plant genome for expression or expresses after mutating the endogenous 5'-UTR of the cell.
  • step (a) the method comprises the following steps:
  • Agrobacterium carrying an expression vector is provided, and the expression vector contains the 5'-UTR nucleotide mutant or combination of mutants;
  • step (1) (2) contacting a plant cell, plant tissue, plant part with the Agrobacterium in step (1), so that the 5'-UTR nucleotide mutant or combination of mutants or an active fragment thereof is transferred into a plant cell, and integrated into the chromosome of a plant cell;
  • step (a) the plant cell, plant tissue, and plant part are modified by gene editing technology, so that the first 5'-UTR in the plant cell, plant tissue, and plant part is There is a deletion of a fragment of 50-80 bp (preferably, 60-70 bp, more preferably, 68 bp) in nucleotides compared to the first parental 5'-UTR nucleotide (SEQ ID NO.: 1).
  • step (a) the plant cell, plant tissue, and plant part are modified by gene editing technology, so that the first 5'-UTR in the plant cell, plant tissue, and plant part is There is a deletion of a fragment of 50-80 bp (preferably, 60-70 bp, more preferably, 68 bp) in nucleotides compared to the first parental 5'-UTR nucleotide (SEQ ID NO.: 1); and
  • the second 5'-UTR nucleotide mutant has 80-100 bp (preferably, 90-95 bp, more preferably 90-95 bp) compared to the second parental 5'-UTR nucleotide (SEQ ID NO.: 4). , 91bp) length of fragment deletion.
  • the gene editing technology is selected from the group consisting of CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.
  • the gene editing technology includes a base editor.
  • the improving the plant comprises increasing the content of vitamin C in the plant.
  • the method is used to improve the performance of vitamin C content in plants.
  • the method is used to regulate the expression of endogenous GGP2 and/or GGP1 genes.
  • the plants include angiosperms and gymnosperms.
  • the gymnosperms are selected from the group consisting of: Cycadaceae, Podocarpaceae, Araucariaceae, Pinaceae, Cedaraceae, Cycadaceae, Tricuspidaceae firaceae, yewaceae, ephedraaceae, marigenaceae, monotype, centenaceae, or a combination thereof.
  • the plants include monocotyledonous plants and dicotyledonous plants.
  • the plants include herbaceous plants and woody plants.
  • the herb is selected from the group consisting of: Compositae, Solanaceae, Poaceae, Leguminosae, or a combination thereof.
  • the woody plant is selected from the group consisting of Actiniaceae, Rosaceae, Moraceae, or a combination thereof.
  • the plant is selected from the group consisting of: Compositae, Cruciferae, Poaceae, Leguminosae, Solanaceae, Actiniaceae, Malvaceae, Paeoniaceae, Rosaceae, Liliaceae, or a combination thereof.
  • the plant is selected from the group consisting of Arabidopsis, rice, cabbage, soybean, tomato, corn, tobacco, barley, wheat, potato, tomato, rapeseed, lettuce, cabbage, quinoa, sorghum or a combination thereof.
  • the method further comprises the step of: testing the performance of the increased amount of vitamin C content on the plant cells, plant tissues, plant parts or plants.
  • the increase (C1) of the vitamin C content of the plant improved by the method is compared with the increase (C2) of the vitamin C content of the wild-type same plant, C1/C2 ⁇ 4, which is higher than Preferably C1/C2 ⁇ 5, preferably C1/C2 ⁇ 7.
  • the ninth aspect of the present invention provides a reagent comprising the mutant described in the first aspect of the present invention, the mutant combination described in the second aspect of the present invention, and the nucleic acid described in the third aspect of the present invention or the fourth aspect of the present invention.
  • the agent is used to increase the content of vitamin C in plant cells, plant tissues or plants.
  • a tenth aspect of the present invention provides a kit comprising the reagents described in the eighth aspect of the present invention.
  • the kit further includes labels or instructions.
  • the eleventh aspect of the present invention provides the mutant described in the first aspect of the present invention, the mutant combination described in the second aspect of the present invention, the nucleic acid construct described in the third aspect of the present invention or the fourth aspect of the present invention, the present invention
  • content of vitamin C or for the preparation of (a) an agent or kit for increasing the content of vitamin C in a plant, and/or (b) a plant having an increased content of vitamin C.
  • a twelfth aspect of the present invention provides a method for identifying high-content vitamin C plants, comprising:
  • step (i) it is determined by sequencing whether the plant sample has the mutant described in the first aspect of the present invention or the mutant combination described in the second aspect of the present invention or the first aspect of the present invention.
  • Figure 1 shows the Cas9 editor, where AtU6 and 35 promoters are promoters, gRNAs are guide RNAs, and NOS terminators are terminators.
  • nucleotides in the 5'-end untranslated region (5'-UTR) of GGP2 and/or GGP1 genomes in plant cells were mutated (ie, large fragment deletions) , which can regulate the expression of plant endogenous GGP2 and/or GGP1 genes, thereby increasing the content of vitamin C in plants.
  • the present inventors have completed the present invention.
  • parental nucleotide refers to a nucleic acid molecule that can be found in nature, including a wild-type nucleic acid molecule that has not been artificially modified, or a nucleic acid molecule that has been artificially modified but does not contain the mutation site of the present invention. , and its nucleotides can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be obtained by deducing the nucleotide sequence.
  • the "parent plant” is a plant containing the parent nucleotide or polypeptide.
  • the “parent nucleotide” can be extracted from the parent plant according to techniques well known to those skilled in the art, or obtained by chemical synthesis. The sequence of the parent 5'-UTR nucleotide is shown, for example, as SEQ ID No. 1 or 4.
  • isolated refers to the separation of a substance from its original environment (in the case of a natural substance, the original environment is the natural environment).
  • the original environment is the natural environment.
  • polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in the natural state. .
  • the terms “homology” or “identity” are used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by an adenine, or both A position in each of the polypeptides is occupied by a lysine)
  • the molecules are identical at that position.
  • the identity of two nucleotide sequences can be confirmed by the BLAST algorithm (Altannet al., 1990, Mol. Biol. 215:403-10) obtained from the National Center for Biotechnology Information (NCBI), using The default parameters are ok.
  • plant should be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, such as: (1) Foodstuffs Crops: Oryza spp., such as Oryza sativa, Oryza latifolia, Oryza sativa, Oryza glaberrima; Triticum spp., such as common Wheat (Triticumaestivum), Durum (T.Turgidumssp.durum); Hordeum (Hordeum spp.), e.g.
  • Hordeum Hordeum (Hordeum vulgare), Arizona Barley (Hordeumarizonicum); Rye (Secale cereale); Avena (Avena spp.) ), e.g. Avena sativa, Avena fatua, Avena byzantina, Avenafatuavar. sativa, Avena hybrida; Echinochloa spp., e.g.
  • Glycine spp. such as Glycine max, Soja hisspida, Soja max, Vicia spp., Vigna spp., Pisum spp.), kidney beans (field bean), Lupinus (Lupinus spp.), Vicia, capers (Tamarindus indica), lentils (Lens culinaris), Mucuna (Lathyrus spp.), lentils Genus (Lablab), broad
  • Lycopersicon spp. e.g. Tomato (Lycopersicon esculentum), Tomato (Lycopersicon lycopersicum), Pear Tomato (Lycopersicon pyriforme), Macrotyloma spp., Kale, Luffa acutangula, lentil, okra, onion, potato (potato), artichoke (artichoke), asparagus (asparagus), broccoli (broccoli), Brussels sprouts (Brussels sprouts), cabbage (cabbage), carrot (carrot), cauliflower (cauliflower), celery (c elery), kale (collardgreens), zucchini (squash), winter squash (Benincasa hispida), Asparagus officinalis, celery (Apium g ra veolens), amarant
  • Agrostis spp. Agrostis palustris
  • Lolium spp. ryegrass
  • Festucaspp. fescue
  • Zoysia spp. Zoysiajaponica
  • Cynodon spp. Bermudagrass
  • bermudagrass Stenotaphrum secunda tum
  • Paspalum spp. Baja grass
  • Eremochloa ophiuroides Baja grass
  • Carpetgrass Species Axonopus spp.
  • Carpet Grass Bouteloua dactyloides (Bison grass), Bouteloua var.
  • Fagus spp. Ceiba pentandra, Cinnamomum spp., Corchorus sp., Phragmites australis, Physalis spp., Mountain Desmodium spp., Poplar, Ivy, Aspen, Coral Tree, Ginkgo, Quercus, Ailanthus, Ailanthus, Holly, Sycamore, Privet, Larix, Larch, Black Wax, Masson Pine , Simao pine, Yunnan pine, South Asian pine, Chinese pine, Korean pine, black walnut, lemon, sycamore, Pu Tao, Dove tree, kapok, Java kapok, Bauhinia bauhinia, sheep's hoof, rain tree, acacia, dragon tooth flower, thorn Tung, Magnolia, Cycas, Lagerstroemia, Conifers, Trees, Shrubs; (1 6) Nut Crops: Brazil Chestnut (Bertholletia excelsea), Chestnut (Ber
  • plant tissue or “plant part” includes plant cells, protoplasts, plant tissue cultures, plant callus, plant pieces as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, Branches, seedlings, fruits, pits, ears, roots, root tips, anthers, etc.
  • the term "gene editing” technology mainly includes CRISPR technology, TALEN technology and ZFN technology.
  • the gene editing tools referred to in CRISPR technology include guideRNA, Cas proteins (such as Cas9, Cpf1, Cas12b, etc.), and Cas proteins can recognize and cut target DNA under the guidance of guideRNA.
  • Gene editing tools referred to in TALEN technology are restriction enzymes that can cut specific DNA sequences and include a TAL effector DNA binding domain and a DNA cleavage domain.
  • Gene editing tools referred to in ZFN technology are also restriction enzymes that can cut specific DNA sequences, including a zinc finger DNA binding domain and a DNA cleavage domain.
  • the Crispr protein of the present invention refers to a class of proteins with nuclease activity in the CRISPR system, which is a CRISPR associated (CRISPR associated) protein, and a preferred CRISPR includes Cas9, Cas12, Cas13 and mutants that maintain activity, Preferably, the CRISPR protein is Cas9 and its active mutants.
  • the present invention can use Cas9, Cas12, Cas13 and other types of Cas proteins that have been found so far.
  • Cas proteins in the present invention include naturally occurring proteins or recombinant proteins.
  • Cas protein includes not only any protein identified before the filing date, but also proteins that can be determined to have the activity of the protein based on currently known knowledge and whose activity can be identified after the filing date.
  • gRNA is also referred to as guide RNA (guide RNA).
  • guide RNA guide RNA
  • At least part of the sequence of the gRNA can be complementary to the target DNA, and the gRNA can form a functional complex with the Cas protein.
  • the sequence of the gRNA is not particularly limited and varies according to the sequence of the target DNA to be edited, and the gRNA may be one type or a combination of various types.
  • UTR the full name of untranslated region
  • the UTR referred to in the present invention refers to the sequence in the DNA molecule as a standard to describe its structure and function.
  • the untranslated region includes the 5'-end untranslated region and the 3'-end untranslated region. It is believed that the untranslated region is related to the regulation of gene expression.
  • the 5' untranslated region starts at the transcription start site and ends at the nucleotide before the start codon and can contain elements that control gene expression through regulatory elements. The diversity of the 5' untranslated region leads to its genetic changes in expression.
  • the 5'-UTR of the present invention can be the untranslated region in the genome including introns, or it can be the coding sequence of the untranslated region of mature mRNA (such as removing introns).
  • the 5'-UTR described in the present invention is preferably a nucleotide sequence located in the range of 1-250bp upstream of the start codon of the encoding gene of GGP2 and/or GGP1 protein, preferably 1-200bp, preferably 1-100bp.
  • the present invention provides an isolated 5'-UTR nucleotide mutant that exists compared to the parental 5'-UTR nucleotide (SEQ ID NO.: 1) 50-80bp (preferably, 60-70bp, more preferably, 68bp) length of fragment deletion;
  • mutation of parental 5'-UTR nucleotides can regulate the expression of endogenous GGP2 and/or GGP1 genes in plants, thereby increasing the content of vitamin C in plants.
  • the present invention also provides a mutant combination comprising:
  • combinatorial mutation of parental 5'-UTR nucleotides can regulate the expression of endogenous GGP2 and/or GGP1 genes in plants, thereby increasing the content of vitamin C in plants.
  • the mutant or mutant combination of the present invention is capable of increasing, increasing or promoting the gene encoding GGP2 and/or GGP1 protein or the expression level of GGP2 and/or GGP1 protein in the cell, such as promoting the transcription of the gene encoding GGP2 and/or GGP1 protein, Increase the amount of mRNA, thereby increasing the content of GGP2 and/or GGP1 protein in plants.
  • the degree of improvement of the gene encoding GGP2 and/or GGP1 protein or the expression level of GGP2 and/or GGP1 protein according to the present invention can be described by selecting different reference materials according to different improvement techniques.
  • the reference can be the same wild-type non-transgenic cell that does not contain the mutant of the present invention; if the cell containing the 5'-UTR of the present invention is obtained by mutating the endogenous UTR, the reference can be The same unmutated wild-type cell (ie, the parental cell from which the UTR was derived).
  • the person skilled in the art is familiar with the selection of a reasonable reference in different situations.
  • the mutants of the present invention are capable of conferring or increasing the content of vitamin C in plant cells, plant tissues, plant parts or plants. By introducing the nucleotide mutant or mutant combination of the present invention into a plant, the content of vitamin C in the plant is increased.
  • the mutant is shown in SEQ ID NO.: 3 or 6.
  • polynucleotide mutants of the present invention are preferably provided in isolated form, more preferably, purified to homogeneity.
  • the full-length sequences of the mutants of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis, and can also be obtained by natural or induced recombination or mutation in natural or artificial sequences.
  • primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercial cDNA libraries or cDNAs prepared by conventional methods known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splicing the amplified fragments together in the correct order.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the mutant of the present invention can be obtained completely by chemical synthesis. This mutant sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the polynucleotides of the present invention.
  • the RACE method RACE-cDNA Rapid Amplification of cDNA Ends
  • the primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods.
  • Amplified DNA/RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the 5'-UTR nucleotide mutation of the present invention also includes active fragments, variants, derivatives and analogs thereof, which can be modified by any substitution, deletion, mutation, addition, modification (such as methylation) of nucleotides , demethylation).
  • the invention also includes mutants of one or more other nucleotide substitution derivatives, so long as the mutant form does not significantly affect the desired function and biological activity of the 5'-UTR nucleotide mutation of the invention.
  • the variant generally has higher homology (identity) compared with the sequence shown in SEQ ID NO.: 3 or 6, preferably, the mutant has a higher homology (identity) with SEQ ID NO.:
  • the homology of the sequences shown in 3 or 6 is at least 80%, preferably at least 85%-90%, more preferably at least 95%, and most preferably at least 98% or 99%.
  • the 5'-UTR nucleotide mutants of the present invention are not limited to combination with other elements to form nucleic acid constructs, such as promoter sequences, genes encoding GGP2 and/or GGP1 proteins, enhancer sequences, terminator sequences, tag genes, Sequence combinations such as leader sequence, polyadenylation sequence, etc.
  • the GGP2 and/or GGP1 protein may be derived from any species, including animals, plants, microorganisms, preferably plants, especially monocotyledonous or dicotyledonous plants.
  • the parental GGP2 and/or GGP1 proteins of the present invention are derived from Asteraceae, especially lettuce.
  • the promoter of the present invention may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutated, truncated and hybrid promoters, and may be homologous or heterologous to the host cell of genes encoding extracellular or intracellular polypeptides.
  • a promoter native to p-hydroxyphenylpyruvate dioxidase, or a heterologous promoter active in plants is preferably used.
  • the promoter may be constitutively expressed, or may be inducible. Examples of promoters include, for example, histone promoters, rice actin promoters, plant viral promoters such as cauliflower mosaic virus promoters, and the like.
  • the present invention also provides an expression vector, which contains the nucleic acid construct encoding the 5'-UTR nucleotide mutant or mutant combination of the present invention or the third or fourth aspect of the present invention.
  • the expression vector also contains at least one origin of replication to achieve self-replication.
  • the choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be an autonomously replicating vector, ie a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome or artificial chromosome.
  • the vector may contain any elements that ensure self-replication.
  • the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it is integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or transposons can be used.
  • the vector may also be a vector for gene editing of 5'-UTR nucleotides endogenous to the host cell.
  • Vectors can be of the type such as plasmids, viruses, cosmids, phages, etc., which are well known to those skilled in the art and are numerously described in the art.
  • the expression vector in the present invention is a plasmid.
  • the expression vector may also contain one or more selectable marker genes for use in selection of host cells containing the vector.
  • selectable markers include genes encoding dihydrofolate reductase, or genes conferring neomycin resistance, genes conferring resistance to tetracycline or ampicillin, and the like.
  • Vectors suitable for use in the present invention include commercially available plasmids such as but not limited to: pCambia, pBR322 (ATCC37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, WI, USA) ) pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), among
  • expression vectors comprising the 5'-UTR nucleotide mutant sequences of the invention or in combination with other elements to form nucleic acid constructs. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like.
  • the 5'-UTR nucleotide mutant sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • Expression vectors also include a ribosome binding site for translation initiation and a transcription terminator.
  • Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on a promoter to enhance transcription of a gene.
  • the present invention also provides host cells comprising the 5'-UTR nucleotide mutants, mutant combinations, nucleic acid constructs or expression vectors of the present invention.
  • a vector comprising a 5'-UTR nucleotide mutant or combination of mutants is introduced into a host cell such that the vector exists as part of a chromosomal integrant or as a self-replicating extrachromosomal vector as described earlier, or the vector can be directed against the host cell. Endogenous 5'-UTR for gene editing.
  • the host cell can be any host cell familiar to those skilled in the art, including prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells (such as agricultural crops and forestry plants).
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as plant cells (such as agricultural crops and forestry plants).
  • Plant cells such as agricultural crops and forestry plants.
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, animal cells, and the like.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging and the like.
  • Plants can also be transformed using methods such as Agrobacterium transformation or biolistic transformation, such as the leaf disk method.
  • the transformed plant cells, tissues or organs can be regenerated into plants by conventional methods, thereby obtaining plants with increased vitamin C content.
  • the present invention also provides a method for improving plants, which specifically comprises introducing the 5'-UTR nucleotide mutants or mutant combinations of the present invention or their nucleic acid constructs or expression vectors into plant cells, plant tissues, and plant parts, and then The plant cells, plant tissues, plant parts are regenerated into plants.
  • the introduction method is as described above, including transforming an expression vector containing a 5'-UTR nucleotide mutant or a combination of mutants, or gene editing technology can be used to directly modify the 5' of GGP2 and/or GGP1 in the genome of the target plant.
  • the 'terminal untranslated region (5'-UTR) is edited to regulate the expression of endogenous GGP2 and/or GGP1 genes and improve plant traits.
  • the regeneration method includes tissue culture, greenhouse culture or soil cultivation and the like.
  • the improved plant traits include increasing or imparting or enhancing the content of vitamin C in the plant.
  • the present invention finds for the first time that the expression of endogenous GGP2 and/or GGP1 gene can be changed by mutating the 5'-end untranslated region (5'-UTR) of GGP2 and/or GGP1 genome, thereby increasing the level of vitamin C in plants content.
  • 5'-UTR 5'-end untranslated region
  • the present invention provides for the first time a 5'-UTR mutant and a combination thereof that can increase the vitamin C content in plants.
  • the present invention obtains lettuce with an extremely significant increase in vitamin C content (up to nearly 10 times the original content) through a series of screening and identification in the early stage, which greatly increases the nutritional value of the vegetable.
  • sequence of the first 5' UTR of the lettuce is shown in SEQ ID NO.: 1, which is located at the 5' end of the endogenous LsGGP2 gene of lettuce.
  • a gRNA (DNA sequence shown in SEQ ID NO.: 7) was designed in the uORF upstream of endogenous LsGGP2 in lettuce, cloned into an editor (shown in Figure 1), and an editor targeting the 5'UTR upstream of endogenous LsGGP2 in lettuce was formed .
  • the edited plant LsGGP2 gene upstream 5'UTR has a fragment deletion, and the deleted nucleic acid fragment is shown in SEQ ID NO.: 2. After the fragment deletion The sequence of the 5'UTR is shown in SEQ ID NO.:3.
  • Vc content measurement is as follows:
  • Sample preparation Take two 0.1 g samples, add extraction buffer 1 and 2 ml and 1 mL of extraction buffer respectively, and measure immediately after homogenizing and mixing.
  • Standard curve inject the standard series of working solutions into the liquid chromatograph respectively, measure the corresponding peak area, draw the standard curve with the concentration of the standard working solution as the abscissa and the peak area as the ordinate.
  • the sample solution was injected into the liquid chromatograph, and the retention time was used to characterize it, and the peak area was recorded at the same time, and the concentration of vitamin C in the solution to be tested was obtained according to the standard curve.
  • the content of vitamin C in the sample is calculated according to formula (1)
  • X the content of vitamin C in the sample, in milligrams per kilogram (mg/kg);
  • V Total volume of the tested sample, in milliliters (mL);
  • the calculation results are expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions, and the results are rounded to two digits after the decimal point.
  • the mutation types of edited plants are shown in Table 1.
  • the Vc content of the above-mentioned LS-1 mutant plants is greatly increased compared with the wild type (wt), and the increase times are about 7-10 times, and the highest can reach 10 times.
  • the mutant type shown in SEQ ID NO.: 3 of the first 5' UTR of lettuce can endow plants with high Vc content, and the present invention has important application value in cultivating high Vc plants.
  • sequence of the second 5' UTR of the lettuce is shown in SEQ ID NO.: 4, which is located in the endogenous LsGGP1 (GDP-L-galactose phosphorylase 2) (NCBI accession number is LOC111899617) gene of lettuce 5' end.
  • the gRNA (DNA sequence shown in SEQ ID NO.: 8) was designed in the endogenous second 5'UTR of lettuce, cloned into the editor, and the editor targeting the second 5'UTR of lettuce was formed.
  • the mutant type shown in SEQ ID NO.: 6 of the second 5' UTR of lettuce can endow plants with high Vc content, and the present invention has important application value in cultivating high Vc plants.
  • the mutant LS-1 plant shown in SEQ ID NO.: 3 of the first 5' UTR is crossed with the mutant shown in SEQ ID NO.: 6 of the second 5' UTR.
  • the double mutant plant LS-4 was screened in the progeny. Wherein LS-4 contains the first 5'UTR mutant as shown in SEQ ID NO.:3 and the second 5'UTR mutant as shown in SEQ ID NO.:6.
  • the second 5'UTR of the mutant type as shown in SEQ ID NO.:6 can further improve the high Vc content brought by the first 5'UTR of the mutant type, and compared with the wild type, there is a more significant improve.

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Abstract

提供了一种植物细胞中GGP2和/或GGP1基因组5'端非翻译区(5'-UTR)核苷酸突变体提高植物中维生素C含量的方法，所述突变体与亲本5'-UTR核苷酸相比存在50-80bp长度的片段的缺失，亲本5'-UTR核苷酸的序列如SEQ ID NO:1所示，所述缺失的片段包括SEQ ID NO:2所示的序列。

Description

一种提高植物中维生素C含量的方法 技术领域
本发明涉及植物学领域，更具体地涉及一种提高植物中维生素C含量的方法。
背景技术
维生素C(Vitamin C/ascorbic acid，又称L-抗坏血酸)是大多数生物体内的必需物质，它不仅在调控细胞还原势方面发挥重要作用，而且还是很多重要生物学反应关键酶正常发挥作用的辅助因子。然而，人类在进化过程中失去了自身合成维生素C的能力，所以这一必需维生素的获取只能通过食物的摄入，如果维生素C摄取量不足便会引起坏血病的发生。
因此，本领域技术人员迫切需要开发能够提高植物中维生素C含量的方法。
发明内容
本发明的目的在于提供一种能够提高植物中维生素C含量的方法。
在本发明的第一方面，提供了一种分离的第一5’-UTR核苷酸突变体，所述第一5’-UTR核苷酸突变体与第一亲本5’-UTR核苷酸相比存在50-80bp，较佳地，60-70bp，最佳的68bp长度的片段的缺失，所述第一亲本5’-UTR核苷酸的序列如SEQ ID NO.:1所示。
在另一优选例中，所述缺失的核酸片段包括SEQ ID NO.:2所示的核酸序列。
在另一优选例中，所述缺失的核酸片段为SEQ ID NO.:2所示核苷酸序列的核苷酸、其片段、或其保守性变异核苷酸。
在另一优选例中，所述突变体为具有SEQ ID NO.:3所示核苷酸序列的核苷酸、其片段、或其保守性变异核苷酸。
在另一优选例中，所述突变体的核苷酸序列如SEQ ID NO.:3所示。
在另一优选例中，所述的突变体除50-80bp，较佳地，60-70bp，最佳的68bp长度的片段的缺失外，其余的核苷酸序列与SEQ ID NO.:1所示的序列相同或基本相同。
在另一优选例中，所述的基本相同是至多有50个(较佳地为1-40个，更佳地为1-30个、更佳地1-15个、更佳地1-5个)的核苷酸不相同，其中，所述的不相 同包括核苷酸的取代、缺失或添加。
在另一优选例中，所述第一亲本5’-UTR为细胞基因组中与GDP-L-galactose phosphorylase 2-like蛋白(本文中又称为GGP2或者LsGGP2)(GDP-L-galactose phosphorylase 2-like蛋白的NCBI登录号为LOC111907963)表达调控相关的核苷酸序列。
在另一优选例中，所述第一亲本5’-UTR位于细胞基因组中编码GGP2核甘酸序列的起始密码子的上游。
在另一有优选例中，所述细胞包括真核细胞和原核细胞。
在另一有优选例中，所述细胞来源于植物、动物或微生物。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物选自下组：菊科、禾本科、豆科、十字花科植物、或其组合。
在另一优选例中，所述植物选自下组：水稻、玉米、烟草、高粱、小麦、大麦、玉米、大豆、拟南芥、马铃薯、番茄、生菜、油菜、白菜、藜麦、或其组合。
在另一优选例中，所述第一亲本5’-UTR来源于单子叶植物或双子叶植物。
在另一优选例中，所述第一亲本5’-UTR来源于选自下组的一种或多种植物：菊科、禾本科、豆科、十字花科植物。
在另一优选例中，所述第一亲本5’-UTR来源于选自下组的一种或多种植物：水稻、玉米、烟草、高粱、小麦、大麦、玉米、大豆、拟南芥、马铃薯、番茄、生菜、油菜、白菜、藜麦。
在另一优选例中，所述突变体来源于单子叶植物或双子叶植物。
在另一优选例中，所述突变体来源于选自下组的一种或多种植物：菊科、禾本科、豆科、十字花科植物。
在另一优选例中，所述突变体来源于选自下组的一种或多种植物：水稻、玉米、烟草、高粱、小麦、大麦、玉米、大豆、拟南芥、马铃薯、番茄、生菜、油菜、白菜、藜麦。
在另一优选例中，所述的第一亲本5’-UTR来源于生菜(Lactuca sativa)。
在另一优选例中，所述的突变体来源于生菜(Lactuca sativa)。
在另一优选例中，所述突变体选自下组：
(a)序列如SEQ ID NO.:3所示的多核苷酸；
(b)核苷酸序列与SEQ ID NO.:3所示序列的同源性≥80％(较佳地≥90％，更佳 地≥95％，最佳地≥98％)的多核苷酸；
(c)与(a)-(b)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述突变体为SEQ ID NO.1所示的5’-UTR核苷酸经突变形成的。
在另一优选例中，所述突变体为DNA。
在另一优选例中，所述缺失通过基因编辑、基因敲除、基因缺失的方式实现。
在另一优选例中，所述基因编辑方式选自下组：Crispr、Cre-lox、ZFNs、TALENs、同源重组、或其组合。
本发明第二方面提供了一种突变体组合，所述突变体组合包括：
(a)第一5’-UTR核苷酸突变体，所述5’-UTR核苷酸突变体与第一亲本5’-UTR核苷酸(SEQ ID NO.:1)相比存在50-80bp(较佳地，60-70bp，最佳地，68bp)长度的片段的缺失；
(b)第二5’-UTR核苷酸突变体，所述5’-UTR核苷酸突变体与第二亲本5’-UTR核苷酸(SEQ ID NO.:4)相比存在80-100bp，较佳地，90-95bp，最佳的91bp长度的片段的缺失。
在另一优选例中，相对于第二亲本5’-UTR核苷酸(SEQ ID NO.:4)，第二5’-UTR核苷酸突变体中缺失的核酸片段包括SEQ ID NO.:5所示的核酸序列。
在另一优选例中，所述缺失的核酸片段为SEQ ID NO.:5所示核苷酸序列的核苷酸、其片段、或其保守性变异核苷酸。
在另一优选例中，所述第二5’-UTR核苷酸突变体为具有SEQ ID NO.:6所示核苷酸序列的核苷酸、其片段、或其保守性变异核苷酸。
在另一优选例中，所述第二5’-UTR核苷酸突变体的核苷酸序列如SEQ ID NO.:6所示。
在另一优选例中，所述的突变体除80-100bp(较佳地，90-95bp，最佳地，91bp)长度的片段的缺失外，其余的核苷酸序列与SEQ ID NO.:4所示的序列相同或基本相同。
在另一优选例中，所述的基本相同是至多有50个(较佳地为1-40个，更佳地为1-30个、更佳地1-15个、更佳地1-5个)的核苷酸不相同，其中，所述的不相同包括核苷酸的取代、缺失或添加。
在另一优选例中，所述第二亲本5’-UTR为细胞基因组中与GDP-L-galactose phosphorylase 2(本文中又称为GGP1或者LsGGP1)(GDP-L-galactose  phosphorylase 2蛋白的NCBI登录号为LOC111899617)表达调控相关的核苷酸序列。
在另一优选例中，所述第二亲本5’-UTR位于细胞基因组中编码GGP1核甘酸序列的起始密码子的上游。
在另一有优选例中，所述细胞包括真核细胞和原核细胞。
在另一有优选例中，所述细胞来源于植物、动物或微生物。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物选自下组：菊科、禾本科、豆科、十字花科植物、或其组合。
在另一优选例中，所述植物选自下组：水稻、玉米、烟草、高粱、小麦、大麦、玉米、大豆、拟南芥、马铃薯、番茄、生菜、油菜、白菜、藜麦、或其组合。
在另一优选例中，所述第一、第二亲本5’-UTR各自独立的来源于单子叶植物或双子叶植物。
在另一优选例中，所述第一、第二亲本5’-UTR各自独立的来源于选自下组的一种或多种植物：菊科、禾本科、豆科、十字花科植物。
在另一优选例中，所述第一、第二亲本5’-UTR各自独立的来源于选自下组的一种或多种植物：水稻、玉米、烟草、高粱、小麦、大麦、玉米、大豆、拟南芥、马铃薯、番茄、生菜、油菜、白菜、藜麦。
在另一优选例中，所述第一、第二5’-UTR核苷酸突变体各自独立的来源于单子叶植物或双子叶植物。
在另一优选例中，所述第一、第二5’-UTR核苷酸突变体各自独立的来源于选自下组的一种或多种植物：菊科、禾本科、豆科、十字花科植物。
在另一优选例中，所述第一、第二5’-UTR核苷酸突变体各自独立的来源于选自下组的一种或多种植物：水稻、玉米、烟草、高粱、小麦、大麦、玉米、大豆、拟南芥、马铃薯、番茄、生菜、油菜、白菜、藜麦。
在另一优选例中，所述的第一、第二亲本5’-UTR各自独立的来源于生菜(Lactuca sativa)。
在另一优选例中，所述的第一、第二5’-UTR核苷酸突变体各自独立的来源于生菜(Lactuca sativa)。
在另一优选例中，所述第二5’-UTR核苷酸突变体选自下组：
(a)序列如SEQ ID NO.:6所示的多核苷酸；
(b)核苷酸序列与SEQ ID NO.:6所示序列的同源性≥80％(较佳地≥90％，更佳地≥95％，最佳地≥98％)的多核苷酸；
(c)与(a)-(b)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述第二5’-UTR核苷酸突变体为SEQ ID NO.4所示的5’-UTR核苷酸经突变形成的。
在另一优选例中，所述突变体为DNA。
在另一优选例中，所述缺失通过基因编辑、基因敲除、基因缺失的方式实现。
在另一优选例中，所述基因编辑方式选自下组：Crispr、Cre-lox、ZFNs、TALENs、同源重组、或其组合。
本发明第三方面提供了一种核酸构建物，所述核酸构建物包括本发明第一方面所述的5’-UTR核苷酸突变体或本发明第二方面所述的突变体组合。
在另一优选例中，所述核酸构建物还包括选自下组中的一种或多种：启动子、GGP2或GGP1蛋白编码基因、增强子、终止子或标签基因。
在另一优选例中，所述的启动子选自下组：组成型启动子、组织特异性启动子、诱导型启动子、或者强启动子。
在另一优选例中，所述启动子选自下组：UBI、UBQ、35S、Actin、SPL，CmYLCV、YAO、CDC45、rbcS、rbcL、PsGNS2、UEP1、TobRB7、Cab、6、U3、U6a、U6b、U6c、U6-1、U3b、U3d、U6-26、U6-29、H1、或其组合。在另一优选例中，所述GGP2或GGP1蛋白编码基因来源于植物或微生物。
在另一优选例中，所述GGP2或GGP1蛋白编码基因来源单子叶植物或双子叶植物。
在另一优选例中，所述的GGP2或GGP1蛋白编码基因来源于水稻、玉米、烟草、高粱、小麦、大麦、玉米、大豆、拟南芥、马铃薯、番茄、生菜、油菜、白菜、藜麦。
在另一优选例中，所述的GGP2或GGP1蛋白编码基因包括野生型和突变型基因。
本发明第四方面提供了一种用于基因编辑的核酸构建物，所述核酸构建物能够使第一亲本5’-UTR核苷酸(SEQ ID NO.:1)突变为第一5’-UTR核苷酸突变体；和/或第二亲本5’-UTR核苷酸(SEQ ID NO.:4)突变为第二5’-UTR核苷酸突变体，其中所述第一5’-UTR核苷酸突变体与第一亲本5’-UTR核苷酸(SEQ ID NO.:1)相比存在50-80bp(较佳地，60-70bp，最佳地，68bp)长度的片段的缺 失；所述第二5’-UTR核苷酸突变体与第二亲本5’-UTR核苷酸(SEQ ID NO.:4)相比存在80-100bp，较佳地，90-95bp，最佳的91bp长度的片段的缺失。
在另一优选例中，所述核酸构建物包括基因编辑蛋白。
在另一优选例中，所述基因编辑蛋白选自下组：CRISPR、TALEN、ZFN、或其组合。
在另一优选例中，所述CRISPR蛋白选自下组：Cas9、Cas12、Cas13、或其组合。
在另一优选例中，所述核酸构建物还包括gRNA。
在另一优选例中，所述gRNA的至少部分序列能够与靶DNA互补，且gRNA能够与CRISPR蛋白形成功能性复合体。
在另一优选例中，所述gRNA选自SEQ ID NO.:7和/或SEQ ID NO.:8。
在另一优选例中，所述的基因编辑蛋白包括CRISPR基因编辑体系、易错PCR、基因重组、TALEN和ZFN所需要的基因编辑蛋白。
本发明第五方面提供了一种载体，所述的载体含有本发明第一方面所述的5’-UTR核苷酸突变体或本发明第二方面所述的突变体组合或本发明第三方面所述的核酸构建物或本发明第四方面所述的核酸构建物。
在另一优选例中，所述载体包括表达载体、穿梭载体、整合载体。
在另一优选例中，所述的载体为植物表达载体。
在另一优选例中，所述的载体为可转染或转化植物细胞的表达载体。
在另一优选例中，所述的载体为农杆菌Ti载体。
在另一优选例中，所述的构建物整合到所述载体的T-DNA区。
在另一优选例中，所述载体是环状的或线性的。
在另一优选例中，所述的载体还包含一筛选标记表达盒。
本发明第六方面提供了一种基因工程细胞，所述的基因工程细胞含有本发明第五方面所述的载体或基因组中整合有本发明第一方面所述的突变体或本发明第二方面所述的突变体组合或本发明第三方面或本发明第四方面所述的核酸构建物。
在另一优选例中，所述细胞为真核细胞，如酵母细胞或动物细胞或植物细胞，或原核细胞，如大肠杆菌。
在另一优选例中，所述的细胞为植物细胞。
在另一优选例中，所述的细胞含有更高含量的维生素C。
在另一优选例中，所述的基因工程细胞是用选自下组的方法将本发明第一方面 所述的突变体或本发明第二方面所述的突变体组合或本发明第三方面或本发明第四方面所述的核酸构建物或本发明第五方面所述的载体导入细胞的：农杆菌转化法、基因枪法、显微注射法、电击法、超声波法和聚乙二醇(PEG)介导法。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物包括草本植物和木本植物。
在另一优选例中，所述草本植物选自下组：菊科、茄科、禾本科植物、豆科植物、或其组合。
在另一优选例中，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、或其组合。
在另一优选例中，所述植物选自下组：十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、菊科、或其组合。在另一优选例中，所述的植物选自下组：拟南芥、水稻、白菜、大豆、番茄、玉米、烟草、大麦、小麦、马铃薯、番茄、油菜、生菜、白菜、藜麦、高粱或其组合。
本发明第七方面提供了一种提高细胞中Vc含量的方法，所述方法包括向所述细胞中引入本发明第一方面所述5’-UTR核苷酸突变体或本发明第二方面所述的突变体组合或本发明第三方面或本发明第四方面所述的核酸构建物的步骤。
在另一优选例中，所述方法包括利用基因编辑技术在细胞中引入本发明第一方面所述的突变体或本发明第二方面所述突变体组合的步骤。
在另一优选例中，所述的基因编辑技术选自下组：CRISPR基因编辑体系、易错PCR、基因重组、TALEN和ZFN。
在另一优选例中，所述基因编辑技术包括碱基编辑器。
在另一优选例中，所述的方法中，引入5’-UTR核苷酸突变体或所述突变体组合包括将5’-UTR核苷酸突变体或突变体组合在细胞中进行表达，例如，通过表达载体对所述突变体或突变体组合进行表达的，或者将所述突变体或突变体组合整合到植物基因组上进行表达或将细胞内源性5’-UTR突变后进行表达。
在另一优选例中，相对于野生型同种细胞中的VC含量，含有所述突变体的细胞中的VC含量提高了≥4倍，较佳地，≥5倍，更佳地，≥8倍。
在另一优选例中，相对于野生型同种细胞中的VC含量，含有所述突变体的细胞中的VC含量提高了4-15倍，较佳地，4-10倍，更佳地，7-10倍。
本发明第八方面提供了一种改良植物的方法，所述的方法包括步骤：
(a)提供一植物细胞、植物组织或植物部分，向所述植物细胞、植物组织、植物部分中引入本发明第一方面所述的5’-UTR核苷酸突变体或本发明第二方面所述的突变体组合或本发明第三方面或本发明第四方面所述的核酸构建物；
(b)将步骤(a)中的植物细胞、植物组织、植物部分再生成植株。
在另一优选例中，所述方法包括利用基因编辑技术在细胞中引入本发明第一方面所述突变体或本发明第二方面所述突变体组合的步骤。
在另一优选例中，所述引入指细胞中含有本发明第一方面所述突变体或本发明第二方面所述突变体组合。
在另一优选例中，所述的基因编辑技术选自下组：CRISPR基因编辑体系、易错PCR、基因重组、TALEN和ZFN。
在另一优选例中，所述基因编辑技术包括碱基编辑器。
在另一优选例中，所述的引入5’-UTR核苷酸突变体或突变体组合包括将所述5’-UTR核苷酸突变体或突变体组合在细胞中进行表达，例如，通过表达载体对所述突变体或突变体组合进行表达的，或者将所述突变体或突变体组合整合到植物基因组上进行表达或将细胞内源性5’-UTR突变后进行表达。
在另一优选例中，在步骤(a)中，所述方法包括以下步骤：
(1)提供携带表达载体的农杆菌，所述的表达载体含有所述的5’-UTR核苷酸突变体或突变体组合；
(2)将植物细胞、植物组织、植物部分与步骤(1)中的农杆菌接触，从而使所述5’-UTR核苷酸突变体或突变体组合或其活性片段转入植物细胞，并且整合到植物细胞的染色体上；和
(3)选择已转入所述5’-UTR核苷酸突变体或突变体组合或其活性片段的植物细胞、植物组织、植物部分。
在另一优选例中，在步骤(a)中，利用基因编辑技术改造所述植物细胞、植物组织、植物部分，从而使所述植物细胞、植物组织、植物部分中的第一5’-UTR核苷酸与第一亲本5’-UTR核苷酸(SEQ ID NO.:1)相比存在50-80bp(较佳地，60-70bp，更佳地，68bp)长度的片段的缺失。
在另一优选例中，在步骤(a)中，利用基因编辑技术改造所述植物细胞、植物 组织、植物部分，从而使所述植物细胞、植物组织、植物部分中的第一5’-UTR核苷酸与第一亲本5’-UTR核苷酸(SEQ ID NO.:1)相比存在50-80bp(较佳地，60-70bp，更佳地，68bp)长度的片段的缺失；和
所述第二5’-UTR核苷酸突变体与第二亲本5’-UTR核苷酸(SEQ ID NO.:4)相比存在80-100bp(较佳地，90-95bp，更佳地，91bp)长度的片段的缺失。
在另一优选例中，所述的基因编辑技术选自下组：CRISPR基因编辑体系、易错PCR、基因重组、TALEN和ZFN。
在另一优选例中，所述基因编辑技术包括碱基编辑器。
在另一优选例中，所述改良植物包括提高植物中维生素C的含量。
在另一优选例中，所述方法用于提高植物中维生素C含量的性能。
在另一优选例中，所述方法用于调控内源性GGP2和/或GGP1基因的表达。
在另一优选例中，所述植物包括被子植物和裸子植物。
在另一优选例中，所述裸子植物选自下组：苏铁科(Cycadaceae)、罗汉松科(Podocarpaceae)、南洋杉科(Araucariaceae)、松科(Pinaceae)、杉科、柏科、三尖杉科、红豆杉科、麻黄科、买麻藤科、单型科、百岁兰科、或其组合。
在另一优选例中，所述植物包括单子叶植物和双子叶植物。
在另一优选例中，所述植物包括草本植物和木本植物。
在另一优选例中，所述草本植物选自下组：菊科、茄科、禾本科植物、豆科植物、或其组合。
在另一优选例中，所述木本植物选自下组：猕猴桃科、蔷薇科、桑科、或其组合。
在另一优选例中，所述植物选自下组：菊科、十字花科植物、禾本科植物、豆科植物、茄科、猕猴桃科、锦葵科、芍药科、蔷薇科、百合科、或其组合。
在另一优选例中，所述的植物选自下组：拟南芥、水稻、白菜、大豆、番茄、玉米、烟草、大麦、小麦、马铃薯、番茄、油菜、生菜、白菜、藜麦、高粱或其组合。
在另一优选例中，所述方法还包括步骤：对所述植物细胞、植物组织、植物部分或植物，测试其维生素C含量增加量的性能。
在另一优选例中，所述方法改良的植物的维生素C含量的增加量(C1)与野生型的同种植物的维生素C含量的增加量(C2)相比，C1/C2≥4，较佳地C1/C2≥5，较佳地C1/C2≥7。
本发明第九方面提供了一种试剂，含有本发明第一方面所述的突变体、本发明第二方面所述的突变体组合、本发明第三方面或本发明第四方面所述的核酸构建物、本发明第五方面所述的载体、或本发明第六方面所述的基因工程细胞。
在另一优选例中，所述试剂用于提高植物细胞、植物组织或植物中的维生素C的含量。
本发明第十方面提供了一种试剂盒，包括本发明第八方面所述的试剂。
在另一优选例中，所述试剂盒还包括标签或说明书。
本发明第十一方面提供了本发明第一方面所述的突变体、本发明第二方面所述的突变体组合、本发明第三方面或本发明第四方面所述的核酸构建物、本发明第五方面所述的载体、本发明第六方面所述的基因工程细胞、本发明第九方面所述的试剂、或本发明第十方面所述的试剂盒的用途，用于提高植物中的维生素C的含量，或用于制备(a)提高植物中维生素C含量的试剂或试剂盒，和/或(b)维生素C含量提高的植物。
本发明第十二方面提供了一种鉴定高含量维生素C植物的方法，包括：
(i)鉴定植物样本是否具有本发明第一方面所述的突变体或本发明第二方面所述的突变体组合或本发明第三方面所述的核酸构建物或本发明第五方面所述的载体。
在另一优选例中，在步骤(i)中，通过测序确定所述植物样本中是否具有本发明第一方面所述的突变体或本发明第二方面所述的突变体组合或本发明第三方面所述的核酸构建物或本发明第五方面所述的载体。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了Cas9编辑器，其中AtU6和35 promoter为启动子，gRNA为向导RNA，NOS terminator为终止子。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现，将植物细胞中GGP2和/ 或GGP1基因组的5’端非翻译区(5’-UTR)的核苷酸进行突变(即大片段缺失)，可调节植物内源性GGP2和/或GGP1基因的表达，从而提高植物中维生素C的含量。在此基础上，本发明人完成了本发明。
术语
术语“亲本核苷酸”指的是可以在自然界中被发现存在的核酸分子，其包括未经人工改造的野生型核酸分子，也可以包括经过人工改造但不含有本发明突变位点的核酸分子，其核苷酸可以通过基因工程技术来获得，如基因组测序、聚合酶链式反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。所述“亲本植物”即含有亲本核苷酸或多肽的植物。所述“亲本核苷酸”可以根据本领域技术人员所熟知的技术从亲本植物中进行提取，亦可通过化学合成的方法获得。所述亲本5’-UTR核苷酸的序列，例如SEQ ID No.1或4所示。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
术语“同源性”或“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。例如两个核苷酸序列的同一性可以通过以下方式确认：从美国国立生物技术信息中心(NCBI)获得的BLAST运算法则(Altschulet al.,1990,Mol.Biol.215:403-10)，使用默认参数确定。
在本发明中，“植物”应理解为能够进行光合作用的任何分化的多细胞生物，在包括处于任何成熟或发育阶段的作物植物，特别是单子叶或双子叶植物，例如：(1)粮食作物：稻属(Oryza spp.)，例如稻(Oryza sativa)、阔叶稻(Oryza latifolia)、水稻(Oryza sativa)、光稃稻(Oryza glaberrima)；小麦属(Trit icum spp.)，例如普通小麦(Triticumaestivum)、硬粒小麦(T.Turgidumssp.durum)；大麦属(Hordeum spp.)，例如大麦(Hordeum vulgare)、亚利桑那大麦(Hordeumarizonicum)；黑麦(Secale cereale)；燕麦属(Avena spp.)，例如燕麦(Avena sativa)、野燕麦(Avena fatua)、比赞燕麦(Avena byzantina)、 Avenafatuavar.sativa、杂种燕麦(Avena hybrida)；稗属(Echinochloa spp.)，例如，珍珠粟(Pennisetum glaucum)、高粱(两色高粱(Sorghum bicolor)、高粱(Sorghum vulgare))、黑小麦、玉蜀黍或玉米、粟、稻(rice)、谷子、糜子、两色蜀黍(Sorghum bicolor)、黍子、荞麦属(Fagopyrum spp.)、黍(Panicum miliaceum)、小米(Setaria italica)、沼生菰(Zizaniapalustris)、埃塞俄比亚画眉草(Eragrostis tef)、稷(Panicum miliaceum)、龙爪稷(Eleusine coracana)；(2)豆类作物：大豆属(Glycine spp.)，例如大豆(Glycine max)、黄豆(Soja hispida)、Soja max)、野豌豆属(Vicia spp.)、豇豆属(Vigna spp.)、豌豆属(Pisum spp.)、芸豆(field bean)、羽扇豆属(Lupinus spp.)、蚕豆属(Vicia)、酸豆(Tamarindus indica)、兵豆(Lens culinaris)、山黧豆属(Lathyrus spp.)、扁豆属(Lablab)、蚕豆、绿豆、红豆、鹰嘴豆；(3)油料作物：花生(Arachis hypogaea)、落花生属(Arachis spp)、胡麻属(Sesamum spp.)、向日葵属(Helianthus spp.)(例如向日葵(Helianthus annuus))、油棕属(Elaeis)(例如油棕(Eiaeis guineensis)、美洲油棕(Elaeisoleifera))、大豆(soy bean)、油菜(Brassicanapus)、芸苔、芝麻、芥菜(Brassicajuncea)、油菜籽油菜(oilseedrape)、油茶、油棕、油橄榄、蓖麻、欧洲油菜(Brassica napus L.)、卡诺拉油菜(canola)；(4)纤维作物：剑麻(Agave sisalana)、棉属(棉花、海岛棉(Gossypium barbadense)、陆地棉(Gossypium hirsutum))、红麻、剑麻、蕉麻、亚麻(Linum usitatissimum)、黄麻、苎麻、大麻(Cannabis sativa)、火麻；(5)水果类作物：枣属(Ziziphus spp.)、香瓜属(Cucumis spp.)、鸡蛋果(Passiflora edulis)、葡萄属(Vitis spp.)、越桔属(Vaccinium spp.)、西洋梨(Pyrus communis)、李属(Prunus spp.)、番石榴属(Psidiumspp.)、石榴(Punicagranatum)、苹果属(Malusspp.)、西瓜(Citrulluslanatus)、柑桔属(Citrusspp.)、无花果(Ficuscarica)、金桔属(Fortunella s pp.)、草莓属(Fraga ria s pp.)、山楂属(Cra taeg us s pp.)、柿树属(Diospyros spp.)、红仔果(Eugenia unifora)、枇杷(Eriobotrya japonica)、龙眼(Dimocarpus longan)、番木瓜(Carica papaya)、椰子属(Cocos spp.)、阳桃(Averrhoacarambola)、狲猴桃属(Actinidia spp.)、扁桃(Prunus amygdalus)、芭蕉属(Musa spp.)(香蕉)、鳄梨属(Persea spp.)(鳄梨(Persea americana))、番石榴(Psidium guajava)、曼密苹果(Mammea americana)、芒果(Mangifera indica)、橄榄(油橄榄(Oleaeuropaea))、番木瓜(Caricapapaya)、椰子(Cocos nucifera)、凹缘金虎尾(Malpighia emarginata)、 人心果(Manilkara zapota)、菠萝(Ananas comosus)、番荔枝属(Annona spp.)、柑桔树(柑桔属物种(Citrus spp.))、波罗蜜属(Artocarpus spp.)、荔枝(Litchi chinensis)、茶藨子属(Ribes spp.)、悬钩子属(Rubus spp.)、梨、桃、杏、梅、杨梅、柠檬、金橘、榴莲、橙、草莓(strawbe rry)、蓝莓、哈密瓜、甜瓜、椰枣、胡桃树、樱桃树；(6)根茎类作物：木薯属(Manihot spp.)、甘薯(Ipomoea batatas)、芋(Colocasia esculenta)、榨菜、洋葱、荸荠、油莎草、山药；(7)蔬菜类作物：菠菜属(Spinacia spp.)、菜豆属(Phaseolus spp.)、莴苣(Lactuca sativa)、苦瓜属(Momordica spp)、欧芹(Petroselinum crispum)、辣椒属(Ca psicum s pp.)、茄属(Solanum s pp.)(例如马铃薯(Solanum tuberosum)、红茄(Solanumintegrifolium)或蕃茄(Solanum lycopersicum))、蕃茄属(Lycopersicon spp.)(例如西红柿(Lycopersicon esculentum)、蕃茄(Lycopersicon lycopersicum)、梨形蕃茄(Lycopersicon pyriforme))、硬皮豆属(Macrotyloma spp.)、无头甘蓝(kale)、棱角丝瓜(Luffa acutangula)、小扁豆(lentil)、秋葵(okra)、洋葱(onion)、马铃薯(potato)、洋蓟(artichoke)、芦笋(asparagus)、西兰花(broccoli)、球芽甘蓝(Brussels sprouts)、卷心菜(cabbage)、胡萝卜(carrot)、花椰菜(cauliflower)、芹菜(celery)、羽衣甘蓝(collardgreens)、西葫芦(squash)、冬瓜(Benincasa hispida)、石刁柏(Asparagus officinalis)、旱芹(Apium g ra veolens)、苋属(Ama ranthus s pp.)、葱属(Allium s pp.)、秋葵属(Abelmoschus spp.)、苦苣(Cichorium endivia)、南瓜属(Cucurbita spp.)、芫荽(Coriandrum sativum)、埃塞俄比亚芥(B.carinata)、萝卜(Rapbanus sativus)、芸苔属(Brassica)物种(例如例如欧洲油菜(Brassica napus)、芜菁亚种(Brassica rapa ssp.)、卡诺拉油菜(canola)、油籽油菜(oilseed rape)、芜菁油菜(turnip rape)、芥菜、甘蓝、黑芥、油菜籽油菜)、孢子甘蓝、茄科植物(茄子)、甜椒、黄瓜、丝瓜、白菜、油菜、甘蓝、葫芦、韭菜、莲、藕、生菜；(8)花卉作物：小金莲花(Tropaeolum minus)、金莲花(Tropaeolummajus)、美人蕉(Canna indica)、仙人掌属(Opuntia spp.)、万寿菊属(Tagetes spp.)、兰花、文殊兰、君子兰、朱顶红、玫瑰、月季、茉莉花、郁金香、樱花、牵牛花、金盏花、荷花、雏菊、康乃馨、矮牵牛花、郁金香、百合、梅花、水仙、迎春、报春、瑞香、山茶、白玉兰、紫玉兰、琼花、君子兰、海棠、牡丹、芍药、丁香、杜鹃、西洋杜鹃、含笑、紫荆、棣棠、锦带花、连翘、云南黄馨、金雀花、仙客来、蝴蝶兰、石斛、风信子、鸢尾、马蹄莲、金盏菊、百枝莲、四季海棠、吊钟海棠、竹节海棠、 天竺葵；(9)药用作物：红花(Carthamus tinctorius)、薄荷属(Mentha spp.)、波叶大黄(Rheum rhabarbarum)、番红花(Crocus sativus)、枸杞、玉竹、黄精、知母、麦冬、川贝、郁金、砂仁、何首乌、大黄、甘草、黄芪、人参、三七、五加、当归、川芎、北柴胡、曼佗罗、洋金花、薄荷、益母草、藿香、黄芩、夏枯草、除虫菊、银杏、金鸡纳树、天然橡胶树、苜蓿、胡椒；(10)原料作物：橡胶、蓖麻(Ricinus communis)、油桐、桑、忽布、桦、桤木、漆树；(11)牧草作物：冰草属(Agropyron spp.)、车轴草属(Trifolium spp.)、芒(Miscanthus sinensis)、狼尾草属(Pennisetum sp.)、虉草(Phalaris arundinacea)、柳枝稷(Panicum virgatum)、草原草(prairiegrasses)、印度草(Indiangrass)、大须芒草(Big bluestem grass)、梯牧草(Phleum pratense)、草皮草(turf)、莎草科(高山嵩草、脚苔草(Carex pediformis)、低苔草)、苜蓿、梯牧草、紫花苜蓿、草木犀、紫云英、柽麻、田菁、红萍、水葫芦、紫穗槐、羽扇豆、三叶草、沙打旺、水浮莲、水花生、黑麦草；(12)糖料作物：甘蔗(甘蔗属物种(Saccharumspp.))、甜菜(Beta vulgaris)；(13)饮料作物：大叶茶(Camellia sinensis)、茶(CamelliaSinensis)、茶树(tea)、咖啡(咖啡属物种(Coffea spp.))、可可树(Theobroma cacao)、蛇麻花(啤酒花)；(14)草坪植物：固沙草(Ammophila arenaria)、早熟禾属(Poa spp.)(草地早熟禾(Poa pratensis)(蓝草))、剪股颖属物种(Agrostis spp.)(剪股颖、匍匐剪股颖(Agrostis palustris))、黑麦草属物种(Lolium spp.)(黑麦草)、羊茅属物种(Festucaspp.)(羊茅)、结缕草属物种(Zoysia spp.)(结缕草(Zoysiajaponica))、狗牙根属物种(Cynodon spp.)(百慕大草、狗牙根)、侧钝叶草(Stenotaphrum secunda tum)(圣奥古斯丁草)、雀稗属物种(Paspalum spp.)(巴哈草)、假俭草(Eremochloa ophiuroides)(百足草)、地毯草属物种(Axonopus spp.)(地毯草)、指形垂穗草(Bouteloua dactyloides)(野牛草)、垂穗草属变种物种(Boutelouavar.spp.)(格兰马草)、马唐(Digitariasanguinalis)、香附子(Cyperusrotundus)、短叶水蜈蚣(Kyllingabrevifolia)、阿穆尔莎草(Cyperusamuricus)、加拿大飞蓬(Erigeroncanadensis)、天胡荽(Hydrocotylesibthorpioides)、鸡眼草(Kummerowiastriata)、地锦(Euphorbiahumifusa)、耕地堇菜(Violaarvensis)、白颖苔草、异穗苔草、草皮草(turf)；(15)树木作物：松属(Pinus spp.)、柳属(Salix sp.)、槭树属(Acer spp.)、木槿属(Hibiscus spp.)、桉属(Eucalyptus sp.)、银杏(Ginkgo biloba)、箣竹属(Bambusa sp.)、杨属(Populus spp.)、牧豆树属 (Prosopis spp.)、栎属(Quercusspp.)、刺葵属(Phoenix spp.)、山毛榉属(Fagus spp.)、吉贝(Ceiba pentandra)、樟属(Cinnamomum spp.)、黄麻属(Corchorus sp.)、南方芦苇(Phragmites australis)、酸浆属(Physalis spp.)、山蚂蝗属(Desmodium spp.)、杨、常春藤、白杨、珊瑚树、银杏、栎类、臭椿、木荷、冬青、悬铃木、女贞、大叶黄扬、落叶松、黑荆树、马尾松、思茅松，云南松、南亚松、油松、红松、黑胡桃、柠檬、悬铃木、蒲桃、珙桐、木棉、爪哇木棉、洋紫荆、羊蹄甲、雨树、合欢、龙牙花、刺桐、广玉兰、苏铁、紫薇、针叶树、乔木、灌木；(1 6)坚果作物：巴西栗(Bertholletia excelsea)、栗属(Castanea spp.)、榛属(Corylus spp.)、山核桃属(Caryaspp.)、核桃属(Juglansspp.)、阿月浑子(Pistaciavera)、腰果(Anacardium)、occidentale)、澳洲坚果(全缘叶澳洲坚果(Macadamia integrifolia))、碧根果、夏威夷果、开心果、巴旦木以及产生坚果的植物；(17)其他：拟南芥、臂形草、蒺藜草、大狗尾草、牛筋草、Cadaba farinosa、藻类(algae)、Carex elata、观赏植物、大果假虎刺(Carissamacrocarpa)、菜蓟属(Cynara spp.)、野胡萝卜(Daucus carota)、薯蓣属(Dioscoreaspp.)、蔗茅属(Erianthus sp.)、苇状羊茅(Festuca arundinacea)、萱草(Hemerocallisfulva)、百脉根属(Lotus spp.)、Luzula sylvatica、紫苜蓿(Medicago sativa)、草木樨属(Melilotus spp.)、黑桑(Morus nigra)、烟草属(Nicotiana spp.)、木犀榄属(Oleaspp.)、鸟足豆属(Ornithopus spp.)、欧防风(Pastinaca sativa)、接骨木属(Sambucusspp.)、白芥属(Sina pis sp.)、蒲桃属(Syzygium spp.)、鸭茅状摩擦禾(Tripsacumdactyloides)、Triticosecale rimpaui、香堇(Viola odorata)等。
在本发明中，术语“植物组织”或“植物部分”包括植物细胞、原生质体、植物组织培养物、植物愈伤组织、植物块以及植物胚、花粉、胚珠、种子、叶、茎、花、枝、幼苗、果实、核、穗、根、根尖、花药等。
在本发明中，术语“基因编辑”技术主要包括CRISPR技术、TALEN技术、ZFN技术。CRISPR技术中所指基因编辑工具包括guideRNA、Cas蛋白(如Cas9、Cpf1、Cas12b等)，Cas蛋白在guideRNA的引导下可识别并切割靶标DNA。TALEN技术中所指的基因编辑工具是可以切割特定DNA序列的限制酶，其包括一个TAL效应子DNA结合结构域和一个DNA切割结构域。ZFN技术中所指的基因编辑工具也是可以切割特定DNA序列的限制酶，其包括一个锌指DNA结合结构域与一个DNA切割结构域。本领域技术人员熟知，将编码基因编辑工具的核苷酸及其他调控元件构建于适 宜的载体中，再转化细胞，可以实现对细胞内基因组的编辑，所述编辑的类型包括基因敲除、插入、碱基编辑。
在本发明中，本发明的Crispr蛋白是指CRISPR系统中具有核酸酶活性的一类蛋白，其为CRISPR关联(CRISPR associated)蛋白，优选的CRISPR包括Cas9、Cas12、Cas13及保持活性的突变体，优选的，所述CRISPR蛋白为Cas9及其保持活性的突变体。本发明可使用目前已经发现的Cas9、Cas12、Cas13等多种类型的Cas蛋白。本发明中的Cas蛋白包括天然存在的蛋白或重组蛋白。另外，Cas蛋白不仅包含本申请日之前已鉴定的任何蛋白，还包含基于目前已知的知识能够确定可能具有该蛋白活性，并在本申请日之后鉴定其活性的蛋白。
在本发明中，gRNA也称为导向RNA(guide RNA)。gRNA的至少部分序列能够与靶DNA互补，且gRNA能够与Cas蛋白形成功能性复合体。gRNA的序列不特别限定，根据待编辑的靶DNA的序列而变化，并且gRNA可以是一种，也可以是多种不同类型的组合。
UTR
UTR，全称untranslated region，通常是指成熟mRNA位于编码区(CDS)上游不被翻译为蛋白质的区域，本发明所指的UTR为以DNA分子中的序列为标准描述其结构及功能。非翻译区包括5’端非翻译区和3’端非翻译区，研究认为非翻译区与基因表达调控有关。5′非翻译区从转录起始位点开始，在起始密码子的前一个核苷酸处结束，可以包含通过调控元件控制基因表达的元件，5′非翻译区的多样性会导致其基因表达的变化。
本发明所述的5’-UTR可以是包含内含子在内的基因组中的非翻译区，也可以是成熟mRNA非翻译区的编码序列(如去掉内含子)。本发明所述的5’-UTR优选是位于GGP2和/或GGP1蛋白的编码基因起始密码子上游1-250bp，优选1-200bp，优选1-100bp范围内的核苷酸序列。
本发明提供了一种分离的5’-UTR核苷酸突变体，所述5’-UTR核苷酸突变体与亲本的5’-UTR核苷酸(SEQ ID NO.:1)相比存在50-80bp(较佳地，60-70bp，更佳地，68bp)长度的片段的缺失；
且对亲本5’-UTR核苷酸进行突变可调节植物内源性GGP2和/或GGP1基因的表达，从而提高植物中维生素C的含量。
本发明还提供了一种突变体组合，所述突变体组合包括：
(a)第一5’-UTR核苷酸突变体，所述5’-UTR核苷酸突变体与第一亲本的5’-UTR核苷酸(SEQ ID NO.:1)相比存在50-80bp(较佳地，60-70bp，更佳地，68bp)长度的片段的缺失；
(b)第二5’-UTR核苷酸突变体，所述5’-UTR核苷酸突变体与第二亲本5’-UTR核苷酸(SEQ ID NO.:4)相比存在80-100bp(较佳地，90-95bp，更佳地，91bp)长度的片段的缺失；
且对亲本5’-UTR核苷酸进行组合突变可调节植物内源性GGP2和/或GGP1基因的表达，从而提高植物中维生素C的含量。
本领域技术人员可以理解，对于核苷酸位置采用不同方式描述不会引起对本发明内容的误解。
本发明突变体或突变体组合能够提高、增加或促进细胞中GGP2和/或GGP1蛋白的编码基因或GGP2和/或GGP1蛋白的表达水平，如促进GGP2和/或GGP1蛋白的编码基因的转录，增加mRNA的量，从而增加植物体内GGP2和/或GGP1蛋白的含量。本发明对GGP2和/或GGP1蛋白的编码基因或GGP2和/或GGP1蛋白的表达水平的改进程度可依据改进技术的不同而选择不同的参照物进行描述，如通过转基因技术获得含有本发明5’-UTR突变体的细胞，其参照物可以为不含有本发明突变体的同种野生型非转基因细胞；如通过突变内源性UTR获得含有本发明5’-UTR的细胞，其参照物可以为同种未突变的野生型细胞(即UTR来源的亲本细胞)。本领域技术人员熟知在不同的情况下选择合理的参照物。
本发明的突变体能够赋予或提高植物细胞、植物组织、植物部分或植物中的维生素C的含量。通过向植物中引入本发明所述的核苷酸突变体或突变体组合，从而提高植物中的维生素C的含量。
优选地，所述的突变体如SEQ ID NO.:3或6所示。
本发明多核苷酸突变体优选以分离的形式提供，更佳地，被纯化至均质。
本发明的突变体的全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得，也可通过天然或人工序列中天然或诱导的重组或突变方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常 是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到本发明的突变体。然后可将该突变体序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明的所述5’-UTR核苷酸突变还包括其活性片段、变体、衍生物和类似物，其可以通过核苷酸的任何取代、缺失、突变、添加、修饰(如甲基化、去甲基化)而产生。
本发明还包括一个或多个其他核苷酸取代衍生物的突变体，只要该突变形式不显著影响本发明5’-UTR核苷酸突变所需功能和生物活性即可。应理解，所述变体与SEQ ID NO.:3或6所示的序列相比，通常具有较高的同源性(相同性)，优选地，所述的突变体与SEQ ID NO.:3或6所示序列的同源性至少为80％，较佳地至少为85％-90％，更佳地至少为95％，最佳地至少为98％或99％。
核酸构建物、表达载体、植物改良
本发明的5’-UTR核苷酸突变体不限于与其他元件组合形成核酸构建物，如与启动子序列、GGP2和/或GGP1蛋白的编码基因、增强子序列、终止子序列、标签基因、前导序列、多聚腺苷酸化序列等序列组合。所述GGP2和/或GGP1蛋白可以来源于任何物种，包括动物、植物、微生物，优选植物，特别是单子叶或双子叶植物。优选地，本发明的亲本GGP2和/或GGP1蛋白来源于菊科，尤其是生菜。
本发明所述的启动子可以是在选定宿主细胞内显示转录活性的任何核苷酸序列，包括突变的、截断的和杂合的启动子，并且可能获子与宿主细胞同源或异源的编码细胞外或细胞内多肽的基因。作为在植物细胞或植物中表达的启动子，优选使用对羟苯基丙酮酸双氧化酶天然的启动子，或者在植物中具有活性的异源启动子。所述启动子可以是组成型表达的，或者可以是诱导型表达的。启动子的实例包括例如组蛋白启动子，水稻肌动蛋白启动子，植物病毒启动子例如花椰菜花叶病毒启动 子等。
本发明还提供了一种表达载体，其中包含有编码本发明的5’-UTR核苷酸突变体或突变体组合或本发明第三方面或第四方面所述的核酸构建物。表达载体中还至少含有一个复制起点，以实现自我复制。载体的选择通常取决于载体与该载体待引入之宿主细胞的相容性。载体可能是自主复制载体，即作为染色体外实体存在的载体，它的复制不依赖于染色体的复制，例如质粒、染色体外元件、微型染色体或人工染色体。该载体可能包含保证自我复制的任何元件。或者，所述载体可能是当引入宿主细胞时被整合入基因组中并与其所整合入的染色体一起复制的载体。此外，可使用单个载体或质粒或者一起包含待引入宿主细胞基因组之总DNA的两个或更多个载体或质粒，或者转座子。或者，所述载体也可以是对宿主细胞内源性的5’-UTR核苷酸进行基因编辑的载体。
载体可以是例如质粒、病毒、粘粒、噬菌体等类型，它们是本领域技术人员所熟知的，在本领域中众多描述。优选地，本发明中的表达载体是质粒。表达载体中也可以含有一个或多个可选择标记基因以便用于选择包含载体的宿主细胞。这种可选择的标记包括编码二氢叶酸还原酶的基因，或赋予新霉素耐受性的基因，赋予对四环素或氨苄青霉素耐受性的基因等。
本发明中适用的载体包括可从商业渠道获得的质粒，例如但不限于：pCambia，pBR322(ATCC37017)，pKK223-3(Pharmacia Fine Chemicals，Uppsala，Sweden)，GEM1(Promega Biotec，Madison，WI，USA)pQE70，pQE60，pQE-9(Qiagen)，pD10，psiX174pBluescript II KS，pNH8A，pNH16a，pNH18A，pNH46A(Stratagene)，ptrc99a，pKK223-3，pKK233-3，pDR540，pRIT5(Pharmacia)，pKK232-8，pCM7，pSV2CAT，pOG44，pXT1，pSG(Stratagene)，pSVK3，pBPV，pMSG，和pSVL(Pharmacia)等。
本领域的技术人员熟知的方法能用于构建含本发明的5’-UTR核苷酸突变体序列或其与其他元件组合形成核酸构建物的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的5’-UTR核苷酸突变体序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
本发明还提供了包含本发明5’-UTR核苷酸突变体、突变体组合、核酸构建物或表达载体的宿主细胞。将包含5’-UTR核苷酸突变体或突变体组合的载体引入宿主细胞中使得载体作为染色体整合体的一部分存在或如早先所述作为自我复制的染色体外载体存在，或者载体可以对宿主细胞内源性的5’-UTR进行基因编辑。宿主细胞可以是本领域技术人员熟悉的任何宿主细胞，包括原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞(如农作物和林业植物的细胞)。代表性例子有：大肠杆菌，链霉菌属、农杆菌；真菌细胞如酵母；植物细胞、动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得维生素C含量提高的植物。
本发明还提供了一种改良植物的方法，具体包括向植物细胞、植物组织、植物部分中引入本发明5’-UTR核苷酸突变体或突变体组合或其核酸构建物或表达载体，再将所述植物细胞、植物组织、植物部分再生为植株。所述的引入方法如前文所述，包括转化含有5’-UTR核苷酸突变体或突变体组合的表达载体，也可以利用基因编辑技术直接对目标植物基因组中的GGP2和/或GGP1的5’端非翻译区(5’-UTR)进行编辑，从而调控内源性GGP2和/或GGP1基因的表达，改良植物的性状。所述的再生的方法包括组织培养、温室培养或土壤栽培等方式。所述改良植物的性状包括提高或赋予或增强植物中的维生素C的含量。
本发明的主要优点包括：
(a)本发明首次发现，通过突变GGP2和/或GGP1基因组5’端非翻译区(5’-UTR)，可改变内源性GGP2和/或GGP1基因的表达，从而提高植物中维生素C的含量。
(b)本发明首次提供了一种可以提高植物中维生素C含量的5’-UTR突变体及其组合。
(c)本发明通过前期一系列筛选以及鉴定，获得了维生素C含量极显著提高(提高到原有含量的将近10倍)的生菜，极大的增加了该蔬菜的营养价值。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非有特别说明，否则本发明实施例中的试剂和材料均为市售产品。
实施例1.突变第一5’UTR，可以显著提高生菜中的Vc含量
1、构建靶向生菜内源LsGGP2(GDP-L-galactose phosphorylase 2-like)(NCBI登录号为LOC111907963)上游uORF的编辑器
本实施例中，所述生菜的第一5’UTR的序列如SEQ ID NO.:1所示，其位于生菜内源LsGGP2基因的5’端。
在生菜内源LsGGP2上游uORF中设计gRNA(DNA序列如SEQ ID NO.:7所示)，克隆至编辑器(如图1所示)，形成靶向生菜内源LsGGP2上游5’UTR的编辑器。
2、生菜遗传转化及植株鉴定
以绿蝶生菜品种作为实验材料，把以上构建好的编辑器通过农杆菌转化，基因编辑植株。通过PCR及测序对进行鉴定，筛选到了生菜的编辑植株LS-1，所述编辑植株LsGGP2基因上游5’UTR发生了片段缺失，缺失的核酸片段如SEQ ID NO.:2所示，片段缺失后的5’UTR的序列如SEQ ID NO.:3所示。
3、亲本型植株及编辑植株LS-1中Vc含量的测量
Vc含量测量的方法如下：
a.试样制备：取样品0.1g两份，分别加入提取缓冲液1和提取缓冲液2ml和1mL，经均质混匀后，立即测定。
b.标准曲线：将标准系列工作液分别注入液相色谱仪中，测定相应的峰面积，以标准工作液的浓度为横坐标，以峰面积为纵坐标，绘制标准曲线。
c.试样溶液的测定
将试样溶液注入液相色谱仪中，以保留时间定性，同时记录峰面积，根据标准曲线得到待测液中维生素C的浓度。
d.分析结果的表述
试样中维生素C含量按公式(1)计算
Figure PCTCN2021113825-appb-000001
式中：
X——试样中维生素C的含量，单位为毫克每千克(mg/kg)；
C——试样溶液中维生素C的质量浓度，单位为微克每毫升(μg/mL)；
V——被测试样总体积，单位为毫升(mL)；
m——称取试样的质量，单位为克(g)；
1000——换算系数。
计算结果以重复性条件下获得的两次独立测定结果的算术平均值表示，结果保留小数点后两位数字。
编辑植株的突变类型如表1所示。
表1编辑植株的突变类型
Figure PCTCN2021113825-appb-000002
选择了LS-1的几个植株，测定了其Vc含量，如下表2所示。
表2不同生菜植株的Vc含量
生菜植株 Vc(ppm)
WT 367.78
LS-1 2654.23
LS-1 3366.97
LS-1 3675.04
LS-1 2803.97
如表2所示，上述LS-1突变植株的Vc含量相对于野生型(wt)，有大幅度的提高，提高倍数在7-10倍左右，最高可以达到10倍。
4、实验结论
生菜第一5’UTR的如SEQ ID NO.:3所示的突变型，可以赋予植物高Vc含量，本发明在培育高Vc植物中具有重要应用价值。
实施例2.突变第二5’UTR，可以提高生菜中的Vc含量
本实施例中，所述生菜的第二5’UTR的序列如SEQ ID NO.:4所示，其位于生菜内源LsGGP1(GDP-L-galactose phosphorylase 2)(NCBI登录号为LOC111899617)基因的5’端。
1、构建靶向生菜内源LsGGP1上游uORF的编辑器
在生菜内源第二5’UTR中设计gRNA(DNA序列如SEQ ID NO.:8所示)，克隆至编辑器，形成靶向生菜第二5’UTR的编辑器。
2、生菜遗传转化及植株鉴定
以绿蝶生菜品种作为实验材料，把以上构建好的编辑器通过农杆菌转化，基因编辑植株。通过PCR及测序对进行鉴定，筛选到生菜的编辑植株LS-2，其中LS-2在第二5’UTR缺失连续91bp的片段，缺失片段序列如SEQ ID NO.:5所示，片段缺失后的第二5’UTR核酸序列如SEQ ID NO.:6。
3、亲本型植株及编辑植株Vc含量的测量
编辑植株的突变类型以及Vc含量如表3所示。
表3.编辑植株的突变类型以及Vc含量
Figure PCTCN2021113825-appb-000003
结果如表3所示，如突变型第二5’UTR植株相对于亲本型来说，Vc含量有2-3倍的提高。
4、实验结论
生菜第二5’UTR的如SEQ ID NO.:6所示的突变型，可以赋予植物高Vc含量，本发明在培育高Vc植物中具有重要应用价值。
实施例3.突变型第一5’UTR和突变型第二5’UTR双突变，可以显著提高生菜中的Vc含量
1、将第一5’UTR的如SEQ ID NO.:3所示的突变型LS-1植株与第二5’ UTR的如SEQ ID NO.:6所示的突变型进行杂交。在后代中筛选双突植株LS-4。其中LS-4同时包含如SEQ ID NO.:3所示的第一5’UTR突变型和如SEQ ID NO.:6所示第二5’UTR突变型。
2、亲本型植株及编辑植株Vc含量的测量
编辑植株的突变类型以及Vc含量如表4所示。
表4.编辑植株的突变类型以及Vc含量
Figure PCTCN2021113825-appb-000004
4、实验结论
如表4所示,如SEQ ID NO.:6所示的突变型第二5’UTR可以进一步提高突变型第一5’UTR带来的高Vc含量，与野生型相比，有更显著的提高。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (12)

  1. 一种分离的第一5’-UTR核苷酸突变体，其特征在于，所述第一5’-UTR核苷酸突变体与第一亲本5’-UTR核苷酸相比存在50-80bp，较佳地，60-70bp，最佳的，68bp长度的片段的缺失，所述第一亲本5’-UTR核苷酸的序列如SEQ ID NO.:1所示，优选的，所述缺失的片段包括SEQ ID NO.:2所示的序列。
  2. 一种突变体组合，其特征在于，所述突变体组合包括：
    (a)第一5’-UTR核苷酸突变体，所述第一5’-UTR核苷酸突变体与第一亲本5’-UTR核苷酸相比存在50-80bp，较佳地，60-70bp，最佳地，68bp长度的片段的缺失；所述第一亲本5’-UTR核苷酸的序列如SEQ ID NO.:1所示；优选的，所述缺失的片段包括SEQ ID NO.:2所示的序列；
    (b)第二5’-UTR核苷酸突变体，所述第二5’-UTR核苷酸突变体与第二亲本5’-UTR核苷酸相比存在80-100bp，较佳地，90-95bp，最佳的91bp长度的片段的缺失；所述第二亲本5’-UTR核苷酸的序列如SEQ ID NO.:4所示；优选的，所述缺失的片段包括SEQ ID NO.:5所示的序列。
  3. 一种核酸构建物，其特征在于，所述核酸构建物包括权利要求1所述的5’-UTR核苷酸突变体或权利要求2所述的突变体组合。
  4. 一种用于基因编辑的核酸构建物，其特征在于，所述核酸构建物能够使第一亲本5’-UTR核苷酸突变为第一5’-UTR核苷酸突变体；和/或第二亲本5’-UTR核苷酸突变为第二5’-UTR核苷酸突变体，其中所述第一5’-UTR核苷酸突变体与第一亲本5’-UTR核苷酸相比存在50-80bp长度的片段的缺失；所述第二5’-UTR核苷酸突变体与第二亲本5’-UTR核苷酸相比存在80-100bp长度的片段的缺失；所述第一亲本5’-UTR核苷酸的序列如SEQ ID NO.:1所示；所述第二亲本5’-UTR核苷酸的序列如SEQ ID NO.:4所示。
  5. 一种载体，其特征在于，所述的载体含有权利要求1所述的5’-UTR核苷酸突变体或权利要求2所述的突变体组合或权利要求3所述的核酸构建物或权利要求4所述用于基因编辑的核酸构建物。
  6. 一种基因工程细胞，其特征在于，所述的基因工程细胞含有权利要求5所述的载体或基因组中整合有权利要求1所述的突变体或权利要求2所述的突变体组合或权利要求3或4所述的核酸构建物。
  7. 一种提高细胞中Vc含量的方法，其特征在于，所述方法包括向所述细胞中 引入权利要求1所述5’-UTR核苷酸突变体或权利要求2所述的突变体组合或权利要求3或4所述的核酸构建物的步骤；
    优选的，所述方法包括利用基因编辑技术在细胞中引入权利要求1所述5’-UTR核苷酸突变体或权利要求2所述的突变体组合的步骤。
  8. 一种改良植物的方法，其特征在于，所述的方法包括步骤：
    (a)提供一植物细胞、植物组织或植物部分，向所述植物细胞、植物组织、植物部分中引入权利要求1所述的5’-UTR核苷酸突变体或权利要求2所述的突变体组合或权利要求3或4所述的核酸构建物；
    (b)将步骤(a)中的植物细胞、植物组织、植物部分再生成植株。
  9. 一种试剂，其特征在于，含有权利要求1所述的突变体、权利要求2所述的突变体组合、权利要求3或4所述的核酸构建物、权利要求5所述的载体、或权利要求6所述的基因工程细胞。
  10. 一种试剂盒，其特征在于，包括权利要求8所述的试剂。
  11. 权利要求1所述的突变体、权利要求2所述的突变体组合、权利要求3或4所述的核酸构建物、权利要求5所述的载体、权利要求6所述的基因工程细胞、权利要求9所述的试剂、或权利要求10所述的试剂盒的用途，其特征在于，用于提高植物中的维生素C的含量，或用于制备提高植物中维生素C含量的试剂或试剂盒，或用于制备维生素C含量提高的植物。
  12. 一种鉴定高含量维生素C植物的方法，其特征在于，包括：
    (i)鉴定植物样本是否具有权利要求1所述的突变体或权利要求2所述的突变体组合或权利要求3所述的核酸构建物或权利要求5所述的载体。
PCT/CN2021/113825 2020-08-24 2021-08-20 一种提高植物中维生素c含量的方法 WO2022042446A1 (zh)

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