WO2020038384A1 - 具有提高的糖含量的植物 - Google Patents

具有提高的糖含量的植物 Download PDF

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WO2020038384A1
WO2020038384A1 PCT/CN2019/101697 CN2019101697W WO2020038384A1 WO 2020038384 A1 WO2020038384 A1 WO 2020038384A1 CN 2019101697 W CN2019101697 W CN 2019101697W WO 2020038384 A1 WO2020038384 A1 WO 2020038384A1
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plant
gene
uorf
uorf2
increase
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PCT/CN2019/101697
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French (fr)
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高彩霞
张华伟
司小敏
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中国科学院遗传与发育生物学研究所
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Priority to CN201980055293.4A priority Critical patent/CN112969791B/zh
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the invention relates to the field of plant genetic engineering. Specifically, the present invention relates to a method for producing a plant having an increased sugar content, preferably a non-transgenic plant, and a plant having an increased sugar content produced by this method, preferably a non-transgenic plant, and progeny thereof. More specifically, the present invention relates to disrupting the uORF in plants that mediates sucrose-induced translation inhibition through gene editing, thereby increasing sugar content in plants.
  • Tomato Solanum lycopersicum
  • Tomato is a widely cultivated crop in the world, which can be used as a vegetable and a fruit. At present, consumers are increasingly demanding the flavor of tomatoes.
  • the sweetness of tomato fruit is an important part of improving tomato flavor.
  • the sweetness of tomato fruits depends on the sucrose content in the sugar accumulation.
  • Sucrose is the working sugar of tomatoes transported from leaf sources to the fruit bank, but most tomato varieties are mainly glucose and fructose, but the sucrose content is very low. The reason is that sucrose can be quickly converted into glucose and fructose by metabolic enzymes such as invertase. Therefore, the decomposition of sucrose is the main link of sugar accumulation in tomato fruits.
  • sucrose content in Arabidopsis and the expression of the Arabidopsis transcription factor AtbZIP11 gene is a negative feedback regulatory relationship (Fatemeh Rahmani et al., Plant Physiology, July 2009, Vol. 150, pp. 1356-1367 ).
  • Sucrose can be used as a signal molecule to inhibit the translation of Arabidopsis AtbZIP11 gene transcripts. This phenomenon is called sucrose-induced suppression of translation (SIRT).
  • SIRT sucrose-induced suppression of translation
  • the 5 'untranslated region of AtbZIP11 has open reading frames (ORFs) that can be translated. These open reading frames are called uORF (upstream open reading frames).
  • the 5 'untranslated region of the AtbZIP11 gene has four uORFs, and studies have shown that the conserved uORF2 (the second uORF of AtbZIP11) is necessary for SIRT.
  • the sucrose concentration in the cell is high, it will be sensed by the uORF2 of the gene, thereby inhibiting the downstream AtbZIP11 gene expression, and then affect the sucrose synthesis pathway, so that the sucrose content in the body is stabilized at a certain level.
  • the invention provides a method of producing a plant having an increased sugar content, the method comprising introducing into the plant a gene editing system that targets a uORF that mediates sucrose-induced translation inhibition (SIRT), wherein The introduction of the gene editing system results in a mutation in the uORF, and the mutation results in a decrease or deletion of the expression of the polypeptide encoded by the uORF, or the mutation results in a decrease or deletion of the activity of the polypeptide encoded by the uORF.
  • SIRT sucrose-induced translation inhibition
  • the SIRT-mediated uORF-encoded polypeptide comprises at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about About 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 99%, and even at least about 99% sequence identity .
  • the mutation comprises a substitution, deletion, or addition of one or more nucleotides.
  • the gene editing system is selected from the group consisting of a zinc finger nuclease system, a TALEN system, and a CRISPR system.
  • the CRISPR system is a CRISPR-Cas9 system.
  • the gene editing system is introduced by transient transformation, thereby generating non-transgenic plants with increased sugar content.
  • the gene editing system is introduced by stable transformation, whereby an exogenous nucleotide sequence encoding a component of the gene editing system is integrated into the plant genome.
  • the method further comprises obtaining a non-transgenic plant without an integrated exogenous nucleotide sequence by genetic isolation.
  • the plant is selected from tomato, strawberry, Arabidopsis, tobacco, rice, corn, barley, sorghum, wheat, potato, carrot, sweet pepper, watermelon, cantaloupe, apple, pear, grape, citrus, Orange, grapefruit, cherry, lychee, red dragon fruit, peach, money orange, plum, apricot, mango, fig, cantaloupe, hawthorn, banana, bayberry, blueberry, beet, kiwi, kale, pineapple.
  • the plant is tomato.
  • the uORF that mediates SIRT is the uORF2 of the SlbZIP1 gene, for example, the uORF2 of the SlbZIP1 gene encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.
  • the CRISPR system comprises at least one guide RNA that targets the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 in uORF2 of the SlbZIP1 gene; preferably, all The CRISPR system includes two types of guide RNAs, which target the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 in uORF2 of the SlbZIP1 gene, respectively.
  • the uORF that mediates SIRT is the uORF2 of the SlbZIP2 gene, for example, the uORF2 of the SlbZIP2 gene encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6.
  • the invention provides a plant or progeny thereof produced by a method of the invention, preferably the plant or progeny thereof is non-transgenic.
  • Figure 1 shows the function of uORF in the 5'leader sequence of tomato SlbZIP1 gene in a transient experiment.
  • uORF SlbZIP1 is a wild-type 5'leader sequence;
  • uorf SlbZIP1 -A1, uorf SlbZIP1 -A2, and uorf SlbZIP1 -3 each mutate three start codons AUG in the 5'leader sequence to AAA.
  • the left picture shows the activity of LUC / REN, which means the translation level of LUC;
  • the right picture shows the transcript level of LUC / REN, and REN is the reference gene.
  • Figure 2 shows the sequencing results of the SlbZIP1 gene structure (containing the 5 'and 3' untranslated regions) and the T0 mutant uORF2.
  • the underlined part is the sgRNA target sequence, and the bold part is PAM.
  • Fig. 3 shows the results of determination of the sugar content of the uORF2 mutant TO plant.
  • Figure 4 shows the growth phenotype of a uORF2 mutant TO plant (scale bar 6cm).
  • Figure 5 shows the fruit phenotype of the uORF2 mutant TO plant.
  • Figure 6 shows the sequencing results of the SlbZIP1 gene structure (containing the 5 'and 3' untranslated regions) and the T1 mutant uORF2.
  • the underlined part is the sgRNA target sequence, and the bold part is PAM.
  • Figure 7 shows the growth phenotype (8cm scale), flowering fruit phenotype (scale 4cm) and fruit phenotype (scale 1.5cm) of uORF2 mutant T1 plants.
  • Figure 8 shows changes in uorf SlbZIP1 transcript levels in different tissues of mutant T1 plants.
  • Figure 9 shows the sugar content determination results of the uORF2 mutant T1 plant.
  • the Arabidopsis transcription factor AtbZIP11 has been shown to control sugar content in plants and is subject to sucrose-induced translational suppression (SIRT).
  • SIRT sucrose-induced translational suppression
  • the use of fruit-specific promoters requires transgenic tomato plants, which raises public concerns about their safety.
  • the present inventors have discovered that in situ destruction of tomato SlbZIP1 gene-mediated uORF by gene editing technology can significantly increase the sugar content in tomato fruits. And, it is unexpected that although the SIRT of the SlbZIP1 gene is also constitutively deleted, the normal growth of tomato plants is not affected, which is of great significance in agriculture. More importantly, the use of gene editing technology can obtain non-transgenic tomato plants with increased fruit sugar content, which can eliminate the safety issues related to genetic modification. This is the first time in the art that the sugar content in plants can be increased by disrupting the uORF that mediates SIRT in situ by using gene editing techniques.
  • the invention provides a method of generating a plant having an increased sugar content, the method comprising introducing into the plant a gene editing system that targets a uORF that mediates sucrose-induced translational suppression (SIRT), wherein the introduction of the gene editing system results in a mutation in the uORF, and the mutation results in a decrease or deletion of the expression of the polypeptide encoded by the uORF, or the mutation results in a decrease or deletion of the activity of the polypeptide encoded by the uORF .
  • the sucrose-induced translational suppression (SIRT) is reduced or eliminated.
  • sucrose means a carbohydrate that can provide a sweet taste, including but not limited to sucrose, glucose, fructose.
  • the "sugar content” refers to the total sugar content.
  • the sucrose content refers to the sucrose content.
  • the sucgar content refers to a glucose content.
  • the “sugar content” refers to a fructose content.
  • the uORF which mediates sucrose-induced translation inhibition (SIRT), is a conserved uORF identified from the 5 'untranslated region of the S-type basic leucine zipper (bZIP) transcription factor in plants whose expression is regulated by SIRT. Also called sucrose control uORF (Sucrose Control uORF, SC-uORF), which plays a negative feedback role in plant sugar accumulation (Anika Wiese et al., The Plant Cell, July 2004, Vol. 16, pp. 1717-1729; Sagor et al., Plant Biotechnology Journal (2016) 14, pp. 1116-1126).
  • sucrose control uORF Sucrose Control uORF, SC-uORF
  • Such regulatory uORFs are present in the bZIP-encoding genes of the plant genome, but are absent in other organisms.
  • the uORF may be the uORF from AtbZIP11 of Arabidopsis thaliana, TBZ17 in tobacco, SlbZIP1 and SlbZIP2 of tomato, such as the second uORF (uORF2) of the 5 'untranslated region.
  • Plants from which the uORF that mediates SIRT can come from include, but are not limited to, tomato, strawberry, Arabidopsis, tobacco, rice, corn, barley, sorghum, wheat, potato, carrot, sweet pepper, watermelon, cantaloupe, apple, pear, Grape, citrus, orange, grapefruit, cherry, lychee, red dragon fruit, peach, mandarin orange, plum, apricot, mango, fig, cantaloupe, hawthorn, banana, bayberry, blueberry, beet, kiwi, kale, pineapple.
  • plants to which the method of the present invention can be applied include, but are not limited to, tomato, strawberry, Arabidopsis, tobacco, rice, corn, barley, sorghum, wheat, potato, carrot, sweet pepper, watermelon, melon, apple, pear, grape , Citrus, orange, grapefruit, cherry, lychee, red dragon fruit, peach, golden tangerine, plum, apricot, mango, fig, cantaloupe, hawthorn, banana, bayberry, blueberry, beet, kiwi, kale, pineapple.
  • the plant is tomato.
  • the SIRT-mediated uORF-encoded polypeptide comprises at least about 50%, at least about 55%, at least about 60%, at least about 65% with SEQ ID NO: 2 (uORF2 corresponding to tomato SlbZIP1) %, At least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 99%, or even at least about Amino acid sequence with 99% sequence identity.
  • Sequence "identity” has art-recognized meaning, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions thereof can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a specific region of the molecule.
  • identity is well known to the skilled person (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988) ).
  • the plant is tomato and the uORF that mediates SIRT is the uORF2 of the SlbZIP1 gene.
  • the nucleotide sequence of the SlbZIP1 gene is available from Gene ID 543618 in Genbank.
  • uORF2 of the SlbZIP1 gene encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2.
  • the plant is tomato
  • the uORF that mediates SIRT is the uORF2 of the SlbZIP2 gene.
  • the nucleotide sequence of the SlbZIP2 gene is available from Gene ID: 543618 in Genbank.
  • uORF2 of the SlbZIP2 gene encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6.
  • the plant is Citrus clementina
  • the SIRT-mediated uORF is the uORF of the money orange LOC18047493 gene, which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7.
  • the plant is Prunus persica
  • the uORF that mediates SIRT is the uORF of the peach LOC18767264 gene, which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8.
  • Suitable uORFs that mediate SIRT can also be, for example, those described in Anika Wiese et al. (The Plant Cell, July 2004, Vol. 16, pp. 1717-1729).
  • the mutations in the uORF include substitutions, deletions, or additions of one or more nucleotides.
  • the mutation causes the translation initiation codon in the uORF to be deleted or becomes a translation stop codon, or the strong translation initiation codon of the uORF is mutated to a weak translation initiation codon, so that the uORF encodes a
  • the polypeptide cannot be translated or the level of translation is reduced.
  • the mutation may be a frameshift mutation such that the polypeptide encoded by the uORF cannot be translated correctly (for example, truncated, or the active site is mutated or deleted), and the activity is reduced or deleted.
  • "activity of a uORF-encoded polypeptide” means the ability of the polypeptide to mediate sucrose-induced translational suppression (SIRT).
  • Gene editing also known as genome editing, uses sequence-specific nucleases or derivatives thereof to make nucleotide insertions, deletions, or substitutions in the genome of an organism. Gene editing usually results in a site-specific double-strand break (DSB) at a desired location in the genome, and then introduces the desired DNA insertions, deletions, or substitutions in the process of repairing the DSB.
  • DSB site-specific double-strand break
  • gene editing also covers base editing techniques that do not involve DSB.
  • gene editing systems are known in the art.
  • the invention does not specifically limit the gene editing system used as long as it is capable of realizing the mutation.
  • gene editing systems suitable for use in the present invention include, but are not limited to, a zinc finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, and a CRISPR system.
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR CRISPR
  • a "zinc finger nuclease” is an artificial restriction enzyme prepared by fusing a zinc finger DNA binding domain and a DNA cleavage domain.
  • a single zinc finger DNA binding domain of a ZFN typically contains 3-6 individual zinc finger repeats, and each zinc finger repeat can recognize a unique sequence such as 3 bp. By combining different zinc finger repeats, different genomic sequences can be targeted.
  • a "transcriptional activator-like effector nuclease” is a restriction enzyme that can be engineered to cleave a specific DNA sequence. It is usually obtained by fusing the DNA binding domain of a transcriptional activator-like effector (TALE) with a DNA cleavage domain. preparation. TALE is engineered to bind almost any desired DNA sequence.
  • TALE transcriptional activator-like effector
  • CRISPR (regularly, interspaced, short, paindromic, repeats, clustered, regularly spaced, short palindromic repeats) system” usually contains two components that can form a sequence-specific complex: a CRISPR nuclease and a corresponding guide RNA.
  • CRISPR nuclease generally refers to a nuclease present in a naturally occurring CRISPR system, as well as modified forms thereof, variants thereof (including nickase mutants), or catalytically active fragments thereof.
  • CRISPR nucleases can recognize, bind, and / or cleave target nucleic acid structures by interacting with guide RNA.
  • the term covers any nuclease or functional variant based on the CRISPR system that enables gene editing in cells.
  • CRISPR nucleases A variety of functional variants of CRISPR nucleases are known in the art, such as highly specific variants or nickase variants, or fusion proteins thereof with cytidine deaminase or adenosine deaminase. Those skilled in the art know how to select a suitable CRISPR nuclease functional variant to achieve the purpose of the present invention.
  • the CRISPR nuclease used in the CRISPR gene editing system of the present invention can be selected from, for example, Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3 or C2c2 proteins, or functional variants of these nucleases.
  • the CRISPR nuclease includes a Cas9 nuclease or a variant thereof.
  • the Cas9 nuclease or variant-based CRISPR gene editing system is also referred to herein as the CRISPR-Cas9 system.
  • the Cas9 nuclease may be a Cas9 nuclease from a different species, such as spCas9 from S. pyogenes.
  • An exemplary amino acid sequence of spCas9 is shown in SEQ ID NO: 9.
  • the Cas9 nuclease variant may include, for example, a highly specific variant of Cas9 nuclease, such as Cas9 nuclease variant eSpCas9 (1.0) (K810A / K1003A / R1060A), eSpCas9 (1.1) (K848A / K1003A / R1060A), and a Cas9 nuclease variant SpCas9-HF1 (N497A / R661A / Q695A / Q926A) developed by J. Keith Joung et al.
  • the Cas9 nuclease variant may further include Cas9 nickase (nCas9), wherein one of the two subdomains (HNH nuclease subdomain and RuvC subdomain) of the DNA cleavage domain of Cas9 nuclease. Is inactivated to form a nicking enzyme.
  • a Cas9 nicking enzyme can be used in combination with two gRNAs targeted upstream and downstream of the sequence to be edited to achieve the deletion of the sequence to be edited or to replace the sequence to be edited in the presence of the donor sequence.
  • the CRISPR nuclease may further include a Cpf1 nuclease or a variant thereof such as a highly specific variant.
  • the Cpf1 nuclease may be a Cpf1 nuclease from a different species, such as a Cpf1 nuclease from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceae bacteria ND2006.
  • the Cpf1 nuclease-based CRISPR gene editing system is also referred to herein as the CRISPR-Cpf1 system.
  • the CRISPR nuclease may further include a base editor.
  • a base editor is typically a fusion protein containing a deaminase and a CRISPR nuclease that lacks DNA-cleaving activity.
  • CRISPR nucleases that lack DNA cleavage activity include, but are not limited to, Cas9 nicking nuclease (nCas9), nuclease-dead Cas9 nuclease (dCas9), or nuclease-dead Cpf1 nuclease (dCpf1). Nuclease-dead Cas9 nuclease (dCas9) or nuclease-dead Cpf1 nuclease (dCpf1) completely lacks DNA-cleaving activity.
  • a variety of CRISPR nucleases are known in the art that lack DNA cutting activity.
  • deaminase refers to an enzyme that catalyzes a deamination reaction.
  • the deaminase refers to cytosine deaminase, which can accept single-stranded DNA as a substrate and can catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuria, respectively. Pyrimidine.
  • the deaminase refers to adenine deaminase, which is capable of accepting single-stranded DNA as a substrate and is capable of catalyzing adenosine or deoxyadenosine (A) to form inosine (I).
  • adenine deaminase or adenine deaminase that accept single-stranded DNA as a substrate are known in the art, such as APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, or
  • APOBEC1 deaminase activation-induced cytidine deaminase
  • CDA1 APOBEC3G
  • the DNA-dependent adenine deaminase disclosed by Nicloe M. Gaudelli et al. (Doi: 10.1038 / nature24644, 2017).
  • base editing in the target nucleotide sequence can be achieved, such as C to T conversion or A to G Conversion.
  • a variety of base editors are known in the art, and those skilled in the art know how to select a suitable base editor to achieve the purpose of the present invention.
  • sequence-specific nucleases used for gene editing in the present invention may also include subcellular localization signals (such as nuclear localization signals), peptide linkers , Can detect labels and other components.
  • subcellular localization signals such as nuclear localization signals
  • peptide linkers such as peptide linkers
  • Can detect labels and other components such as nuclear localization signals
  • CRISPR nucleases in CRISPR base editing systems often contain one or more nuclear localization signals (NLS) to facilitate their entry into the nucleus and enable editing of chromosomal DNA.
  • NLS nuclear localization signals
  • gRNA and “guide RNA” are used interchangeably and refer to RNA capable of forming a complex with a CRISPR nuclease and being able to target the complex to a target sequence due to a certain complementarity with the target sequence molecule.
  • gRNA is usually composed of crRNA and tracrRNA molecules that are partially complementary to form a complex, where the crRNA contains sufficient identity with the target sequence to guide the CRISPR complex (Cas9 + crRNA + tracrRNA) with The target sequence is a sequence that specifically binds.
  • sgRNA unidirectional RNA
  • CRISPR genome editing system based on Cpf1
  • gRNA is usually only composed of mature crRNA molecules, where the crRNA contains a sequence that is sufficiently identical to the target sequence in order to guide the complex (Cpf1 + crRNA) to specifically bind to the target sequence.
  • Cpf1 + crRNA complex
  • the plant is tomato
  • the CRISPR system includes at least one guide RNA that targets the nucleus shown in SEQ ID NO: 3 or SEQ ID NO: 4 in uORF2 of the SlbZIP1 gene.
  • the CRISPR system includes two guide RNAs, which respectively target the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 in uORF2 of the SlbZIP1 gene.
  • the gene editing system may be introduced into a plant in various forms.
  • CRISPR nucleases and guide RNA can be produced in vitro and assembled into ribonucleoproteins (RNPs), which are then introduced into plants.
  • RNPs ribonucleoproteins
  • expression constructs encoding all components of the CRIPSR system can be introduced into a plant and the components can then be expressed in plant cells.
  • expression constructs encoding some components of the CRIPSR system, as well as other components generated in vitro can be introduced into plants simultaneously.
  • the CRISPR gene editing system can be introduced into a plant in at least one of the following i) to v):
  • At least one expression construct comprising a nucleotide sequence encoding a CRISPR nuclease, and at least one guide RNA;
  • At least one CRISPR nuclease at least one CRISPR nuclease, and at least one expression construct comprising a nucleotide sequence encoding at least one guide RNA;
  • At least one expression construct comprising a nucleotide sequence encoding a CRISPR nuclease, and at least one expression construct comprising a nucleotide sequence encoding at least one guide RNA;
  • At least one expression construct comprising a nucleotide sequence encoding a CRISPR nuclease and at least one nucleotide sequence encoding at least one guide RNA.
  • expression construct refers to a vector, such as a recombinant vector, suitable for expression of a nucleotide sequence of interest in an organism. "Expression” refers to the production of a functional product.
  • expression of a nucleotide sequence may refer to transcription of the nucleotide sequence (eg, transcription to produce mRNA or functional RNA) and / or translation of the RNA into a precursor or mature protein.
  • the "expression construct” of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, may be an RNA (such as mRNA) capable of translation.
  • the "expression construct" of the present invention may contain regulatory sequences and nucleotide sequences of interest from different sources, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from that which is generally naturally occurring.
  • "Regulatory sequence” and “regulatory element” are used interchangeably and refer to the upstream (5 'non-coding sequence), middle or downstream (3' non-coding sequence) of a coding sequence, and affect the transcription, RNA processing or Stability or translated nucleotide sequence.
  • a plant expression regulatory element refers to a nucleotide sequence capable of controlling transcription, RNA processing or stability, or translation of a nucleotide sequence of interest in a plant. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • "Introducing" a nucleic acid molecule (eg, an expression construct, etc.) and / or protein into a plant refers to transforming a plant with the nucleic acid and / or protein so that the nucleic acid and / or protein can function in the plant.
  • the nucleic acid and / or protein can be used to transform an isolated plant cell or tissue, and then a plant can be regenerated from the transformed cell or tissue.
  • the base editing system of the present invention can be transformed into specific parts on whole plants, such as leaves, stem tips, pollen tubes, young ears or hypocotyls. This is particularly suitable for the transformation of plants which are difficult to carry out tissue culture regeneration.
  • Methods that can be used to introduce nucleic acid molecules and / or proteins into plants or plant cells include, but are not limited to: gene gun method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube channels And ovary injection.
  • transformation includes stable transformations and transient transformations.
  • Stable transformation refers to the introduction of a foreign nucleotide sequence into the genome, resulting in stable inheritance of the foreign nucleotide sequence. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the plant's genome and can be passed on to any successive generation thereof.
  • Transient transformation refers to the introduction of a nucleic acid molecule or protein into a cell to perform a function without the stable inheritance of a foreign gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.
  • the gene editing system is introduced by transient transformation, thereby generating non-transgenic plants with increased sugar content.
  • the introduction of the gene editing system is performed in the absence of selection pressure, thereby avoiding the integration of exogenous nucleotide sequences in the plant genome.
  • the gene editing system of the present invention is transformed into isolated plant cells or tissues, and then the transformed plant cells or tissues are regenerated into whole plants, preferably, in the absence of selective pressure. Said regeneration, that is, in the tissue culture process does not use any selection agent (such as antibiotics, herbicides, etc.) against the selection gene carried on the expression vector. Without the use of a selection agent, the regeneration efficiency of the plant can be improved, and a gene-edited plant containing no exogenous nucleotide sequence can be obtained.
  • RNA molecules can achieve gene editing in plant cells, and then be degraded by the cells, avoiding the integration of foreign nucleotide sequences in the plant genome.
  • the gene editing system is introduced by stable transformation, whereby an exogenous nucleotide sequence encoding a component of the gene editing system is integrated into the plant genome.
  • Stable transformation may increase the efficiency of screening transformants.
  • the progeny of the obtained transformed plant can be genetically isolated to obtain a non-transgenic plant without an integrated exogenous nucleotide sequence.
  • the method of gene editing only the components of the gene editing (such as sequence-specific nucleases and / or guide RNAs) can be introduced or produced in plant cells to modify the target sequence, and Such modifications can be stably inherited without the need for the gene editing system to persist in plants. This can avoid the integration of exogenous nucleotide sequences in the plant genome, thereby having higher biological safety.
  • components of the gene editing such as sequence-specific nucleases and / or guide RNAs
  • the present invention provides a plant and its progeny produced by the method described above of the present invention, which have an increased sugar content.
  • the plant and its progeny are non-transgenic.
  • the present invention provides a plant having an increased sugar content, which comprises a mutation in a uORF that mediates SIRT relative to a wild-type plant, said mutation causing a decrease in the expression of a polypeptide encoded by the uORF or A deletion, or the mutation results in a reduction or deletion in the activity of the uORF-encoded polypeptide.
  • the plant is non-transgenic.
  • the mutation is generated by introducing into the plant a gene editing system that targets the uORF.
  • the introduction of the gene editing system and / or the gene editing system is as defined above.
  • the mutation is introduced by cross breeding.
  • the sugar content of a plant or tissue or organ of the invention is increased compared to a corresponding wild-type plant or tissue or organ thereof (eg, uORF that mediates SIRT is not mutated).
  • tissue or organ with increased sugar content includes, but is not limited to, fruits, leaves, roots, stems, tubers, and the like.
  • Corresponding wild-type plant means a plant produced by the method of the invention or a wild-type plant from which the plant of the invention is derived, for example, the uORF in the wild-type plant that mediates SIRT is not mutated.
  • the total sugar content in the plant or its tissue or organ of the present invention is increased by at least about 10%, by at least about 20 %, Increase at least about 30%, increase at least about 40%, increase at least about 50%, increase at least about 60%, increase at least about 70%, increase at least about 80%, increase at least about 90%, increase at least about 100%, An increase of at least about 125%, an increase of at least about 150%, an increase of at least about 175%, an increase of at least about 200% or more.
  • the sucrose content in the plant or tissue or organ thereof of the present invention is increased by at least about 10%, compared to the corresponding wild-type plant or tissue or organ thereof (eg, the uORF that mediates SIRT is not mutated), About 20%, increase at least about 30%, increase at least about 40%, increase at least about 50%, increase at least about 60%, increase at least about 70%, increase at least about 80%, increase at least about 90%, increase at least about 100 %, Increase at least about 125%, increase at least about 150%, increase at least about 175%, increase at least about 200% or more.
  • the fructose content in the plants or tissues or organs of the present invention is increased by at least about 10%, by at least about 10%, About 20%, increase at least about 30%, increase at least about 40%, increase at least about 50%, increase at least about 60%, increase at least about 70%, increase at least about 80%, increase at least about 90%, increase at least about 100 %, Increase at least about 125%, increase at least about 150%, increase at least about 175%, increase at least about 200% or more.
  • the glucose content in the plant or tissue or organ of the invention is increased by at least about 10%, compared to the corresponding wild-type plant or tissue or organ thereof (eg, uORF that mediates SIRT is not mutated), About 20%, increase at least about 30%, increase at least about 40%, increase at least about 50%, increase at least about 60%, increase at least about 70%, increase at least about 80%, increase at least about 90%, increase at least about 100 %, Increase at least about 125%, increase at least about 150%, increase at least about 175%, increase at least about 200% or more.
  • the plants of the invention have comparable (similar or substantially similar) conditions under substantially the same or the same growth conditions compared to the corresponding wild-type plant (e.g., the uORF that mediates SIRT is not mutated).
  • the same) growth parameters and / or morphological parameters include, but are not limited to, growth rate, leaf size, leaf thickness, fruit size, fruit weight, plant height, seed germination rate, and the like.
  • the invention provides a method for plant breeding, comprising
  • the present invention provides a method for producing a tomato (Solanum lycopersicum) plant with increased fruit sugar content, the method comprising introducing into a tomato plant a gene editing system of uORF2 targeting the tomato SlbZIP1 and / or SlbZIP2 gene
  • a gene editing system of uORF2 targeting the tomato SlbZIP1 and / or SlbZIP2 gene
  • the introduction of the gene editing system results in a mutation in uORF2 of the SlbZIP1 and / or SlbZIP2 gene, and the mutation results in a decrease or deletion in the expression of the uORF2-encoded polypeptide, or the mutation results in the uORF2-encoded Decrease or absence of polypeptide activity.
  • the introduction of the gene editing system and / or the gene editing system is as defined above.
  • uORF2 of the SlbZIP1 gene encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, uORF2 of the SlbZIP2 gene encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6.
  • the CRISPR system comprises at least one guide RNA that targets the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 in uORF2 of the SlbZIP1 gene.
  • the CRISPR system includes two guide RNAs, and the two guide RNAs respectively target the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 in uORF2 of the SlbZIP1 gene.
  • the invention provides a tomato plant or progeny thereof produced by the method of the invention, which has an increased fruit sugar content.
  • the tomato plant with increased fruit sugar content is non-transgenic.
  • the present invention provides a tomato plant having increased fruit sugar content, which comprises a mutation in uORF2 of the SlbZIP1 gene and / or the SlbZIP2 gene relative to a wild-type tomato plant, the mutation causing the uORF2
  • the expression of the encoded polypeptide is reduced or deleted, or the mutation results in a decrease or deletion of the activity of the uORF2 encoded polypeptide.
  • the tomato plant is non-transgenic.
  • the mutation is generated by introducing into a tomato plant a uORF2 gene editing system that targets the SlbZIP1 gene and / or the SlbZIP2 gene.
  • the introduction of the gene editing system and / or the gene editing system is as defined above.
  • the mutation is introduced by cross breeding.
  • the tomato plant of the present invention having an increased sugar content can be derived from different tomato varieties, including but not limited to the cultivar M82 (Solanum Lycopersicum cv. M82).
  • the total sugar content in the fruit of the tomato plant of the present invention is increased by at least about 10%, by at least about 20%, Increase at least about 30%, increase at least about 40%, increase at least about 50%, increase at least about 60%, increase at least about 70%, increase at least about 80%, increase at least about 90%, increase at least about 100%, increase at least About 125%, an increase of at least about 150%, an increase of at least about 175%, an increase of at least about 200% or more.
  • the sucrose content in the fruit of the tomato plant of the invention is increased by at least about 10%, by at least about 20%, compared to a wild-type tomato plant from which it is derived (eg, uORF2 of the SlbZIP1 gene is not mutated), At least about 30%, increase at least about 40%, increase at least about 50%, increase at least about 60%, increase at least about 70%, increase at least about 80%, increase at least about 90%, increase at least about 100%, increase at least about 125%, an increase of at least about 150%, an increase of at least about 175%, an increase of at least about 200% or more.
  • a wild-type tomato plant from which it is derived eg, uORF2 of the SlbZIP1 gene is not mutated
  • the fructose content in the plants or tissues or organs of the present invention is increased by at least about 10%, compared to corresponding wild-type plants or tissues or organs thereof (eg, uORF that mediates SIRT is not mutated), About 20%, at least about 30% increase, at least about 40% increase, at least about 50% increase, at least about 60% increase, at least about 70% increase, at least about 80% increase, at least about 90% increase, at least about 100 increase %, Increase at least about 125%, increase at least about 150%, increase at least about 175%, increase at least about 200% or more.
  • the glucose content in the plant or tissue or organ of the invention is increased by at least about 10%, compared to the corresponding wild-type plant or tissue or organ thereof (eg, uORF that mediates SIRT is not mutated), About 20%, increase at least about 30%, increase at least about 40%, increase at least about 50%, increase at least about 60%, increase at least about 70%, increase at least about 80%, increase at least about 90%, increase at least about 100 %, Increase at least about 125%, increase at least about 150%, increase at least about 175%, increase at least about 200% or more.
  • tomato plants with increased fruit sugar content of the present invention are at substantially the same or the same growth conditions as compared to corresponding wild-type tomato plants (e.g., the uORF that mediates SIRT is not mutated).
  • Example 1 Transient experiments to verify the function of uORF in the 5’leader sequence of tomato SlbZIP1 gene
  • the wild-type form of the 5'leader sequence of the SlbZIP1 gene and the uORF mutant form (that is, three start codons AUG were mutated to AAA and named uorfSlbZIP1-A1, uorfSlbZIP1-A2, uorfSlbZIP1-A3) were constructed in LUC Upstream area. These plasmids were transferred into tomato protoplasts for 48 hours of expression, and LUC / REN transcripts and translation levels were determined.
  • Vector name and source pGreenII 0800-LUC (Hellens, R.P.etal. Transient expression vectors for functional geometrics, quantification of promoter activity and RNA silencing inplants.Plant methods1, 13 (2005))
  • Protoplast preparation and transformation 2 to 3 weeks old tomato seedlings grown on solid medium were cut into filaments and the tomato protoplast cells were isolated by enzymatic hydrolysis. The constructed plasmid was then transformed by PEG-mediated transformation Methods Protoplasts were transferred into protoplast cells, and the protoplasts were collected after dark culture at 23 ° C for 48 hours.
  • REN Renilla luciferase
  • LOC firefly luciferase
  • RNA levels Protoplasts were collected after 48 hours of dark culture, and the RNA levels in cells were extracted with TRIzol reagent and reversed to cDNA, followed by quantification by qRT-PCR.
  • the tomato variety used in this experiment was M82 (Solanum Lycopersicum cv. M82).
  • sgRNA-1 and sgRNA-2 targeted to the uORF were designed so that the sequence to be mutated contains the second and third start codons .
  • the two sgRNAs were simultaneously constructed into the pKSE401 binary vector backbone (Addgene: 62202; it also mediates the expression of SpCas9) to obtain pKSE401-SlbZIP1uORF-2sgRNA.
  • This vector plasmid was transferred into Agrobacterium tumefaciens strain LBA4404 for transformation of M82 tomato plants.
  • sgRNA-1 target sequence AATGCGAACTCGACGCATG (SEQ ID NO: 3).
  • sgRNA-2 target sequence AAACACATAGAACCAGTAC (SEQ ID NO: 4).
  • Agrobacterium carrying the plasmid pKSE401-SlbZIP1uORF-2sgRNA obtained in 1.1 was used to transform tomato variety M82. Cotyledons of tomato seedlings were used as recipients for Agrobacterium-mediated transformation. Regenerated tomato plants were obtained by tissue culture after transformation. Planting conditions: carried out in a tomato cultivation room, the temperature is 25-28 ° C, the light cycle is 16 hours light / 8 hours dark, and the light intensity is about 600 ⁇ mol m -2 s -1 , which is routinely managed.
  • the obtained transgenic plants were positively identified.
  • the genomic DNA of the transgenic plant was extracted, and the genomic DNA transformed with pKSE401-SlbZIP1uORF-2sgRNA was amplified by PCR using specific primers capable of amplifying SlbZIP1-uORF2. Ligation of PCR products to Clone the vector and pick positive clones for Sanger sequencing.
  • the tomato mutant with S0ZIP1-uORF2 obtained in Example 2 was used as the T0 mutant.
  • the mutant material and the corresponding wild-type (WT) plants were cultured to obtain mature tomato fruits, respectively.
  • Three mature fruits were randomly selected from wild type and T0 mutant plants, 1 g of fresh weight tomato fruits were taken, and total sugar was extracted 3 times at 80 ° C with 15 ml of 80% ethanol.
  • T0-14, T0-15 and T0-29 mutant plants were grown in the same growth environment as the WT plants, and the differences in their growth indexes were observed.
  • Planting conditions carried out in a tomato cultivation room, the temperature is 25-28 ° C, the light cycle is 16 hours light / 8 hours dark, and the light intensity is about 600 ⁇ mol m -2 s -1 , which is routinely managed.
  • the germination rate of the seeds of the TO plant was measured.
  • the germination experiment was performed as follows: the dried tomato seeds were stored in a petri dish containing wet filter paper for about 3 days to germinate, and transferred to a 32-well tomato seedling tray when the seeds were exposed to bacon.
  • the nursery soil is vermiculite: peat (1: 1) and mixed evenly.
  • the temperature is 25-28 ° C, 16 hours of light, 20-22 ° C at night, and 8 hours of darkness. As shown in Table 1 below, there was no significant difference in seed germination rates between the mutants and the WT T1 seeds.
  • Seeds are harvested from the TO plants of the foregoing examples, that is, T1 seeds are obtained, the seeds are germinated and the genotype is identified, and a stable inherited homozygous uorf SlbZIP1 mutant is obtained (as shown in FIG. 6). Five individuals from each line were selected for passaging and subsequent experiments. The lines uorf SlbZIP1 -1, uorf SlbZIP1 -2, and uorf SlbZIP1 -4 were identified as non-transgenic mutant vaccines.
  • the non-transgenic plant identification method includes: designing three specific primers for amplifying fragments of different lengths based on the sequence inserted between the RB and LB of the genome of the Agrobacterium transformation vector pKSE401-SlbZIP1uORF-2sgRNA.
  • the mutant DNA is amplified. If the target fragment is not amplified, it is a plant that does not contain the transgene, and if the target band can be amplified, it is a plant that contains the transgene.
  • the T1 mutant plant and the WT plant were grown in the same growth environment, and the differences in their growth indexes were observed.
  • Planting conditions carried out in a tomato cultivation room, the temperature is 25-28 ° C, the light cycle is 16 hours light / 8 hours dark, and the light intensity is about 600 ⁇ mol m -2 s -1 , which is routinely managed.
  • the sweetness in the fruit is mainly caused by the accumulation of sugar. Therefore, normal-growing tomato fruits were randomly selected from mature fruits in the control and the uorf SlbZIP1 mutant, and 1 g of fresh fruit was weighed. The sugar content was extracted and measured ( Method described above). The results ( Figure 9) showed that the total sugar (the sum of sucrose, glucose, and fructose) in the mutant increased by 23% to 39%, glucose increased by 13% to 34%, and fructose increased by 43% to 46% compared to the control , While the sucrose content was not significantly different from the control. The main reason for the difference between sucrose and the control may be that the sucrose accumulated in the fruit is quickly converted to glucose and fructose by the invertase.

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Abstract

一种产生具有提高的糖含量的植物，优选非转基因植物的方法，以及通过此方法产生的具有提高的糖含量的植物，优选非转基因植物，及其后代。更具体而言，通过基因编辑破坏植物中介导蔗糖诱导的翻译抑制的uORF，从而提高植物中的糖含量。

Description

具有提高的糖含量的植物 技术领域
本发明涉及植物基因工程领域。具体而言，本发明涉及一种产生具有提高的糖含量的植物，优选非转基因植物的方法，以及通过此方法产生的具有提高的糖含量的植物，优选非转基因植物，及其后代。更具体而言，本发明涉及通过基因编辑破坏植物中介导蔗糖诱导的翻译抑制的uORF，从而提高植物中的糖含量。
发明背景
番茄(Solanum lycopersicum)是一种世界上广泛栽培的作物，其可作蔬菜又可作水果。目前，消费者对番茄的风味要求越来越高。而番茄果实的甜度是改善番茄风味的重要环节。番茄果实的甜度取决于糖分积累中的蔗糖含量。蔗糖是番茄从叶片源运输到果实库的运转糖，但多数番茄品种果实中主要是葡萄糖和果糖，蔗糖含量却很低。其原因是蔗糖会通过代谢酶如转化酶等将其迅速的转换为葡萄糖和果糖。因此蔗糖的分解是番茄果实中糖积累的主要环节。有研究通过参与糖代谢或信号途径过程中的关键基因如敲除蔗糖水解酶基因来提高番茄果实的糖含量，但效果并不是很明显。也有研究通过环境胁迫(如干旱或高盐胁迫)来影响碳水化合物的新陈代谢和碳水化合物从源到库器官的运输增加番茄果实中的甜度，但是其机制目前并不是很明确。因此到目前为止还没有出现一种有效且非转基因的提高番茄果实中的甜度(糖含量)的方法。
已有研究证明拟南芥体内的蔗糖含量与拟南芥转录因子AtbZIP11基因的表达之间是负反馈调节关系(Fatemeh Rahmani et al.,Plant Physiology,July 2009,Vol.150,pp.1356-1367)。蔗糖可以作为一种信号分子抑制拟南芥AtbZIP11基因转录本的翻译，这种现象被称为蔗糖诱导的翻译抑制(sucrose-induced repression of translation，SIRT)。AtbZIP11的5’非翻译区中有能够被翻译的开放阅读框(ORF)，这些开放阅读框称为uORF(upstream open reading frame)。AtbZIP11基因的5’非翻译区有4个uORF，有研究证明其中保守的uORF2(AtbZIP11的第二个uORF)对于SIRT是必须的。当细胞内的蔗糖浓度较高时，会被该基因的uORF2感知，从而抑制下游AtbZIP11基因的表达，进而影响蔗糖合成途径，使机体内的蔗糖含量稳定在某一个特定水平。
之前的研究显示，烟草中AtbZIP11的直系同源物tbz17也表现出uORF介导的SIRT(Thalor et al.,PLoS One,(2012),Volume 7,Issue 3,e33111)。组成型过表达tbz17ORF的转基因烟草植物叶片中蔗糖水平比野生型植物大约高出3-4倍。然而，转基因烟草植物的生长严重受损。
此后，在番茄中也鉴定了AtbZIP11的同源基因SlbZIP1和SlbZIP2，在其上游分别包含有4个和3个uORF，其中第二个保守的uORF即uORF2参与SIRT。Sagor等人已 经证明(Plant Biotechnology Journal(2016)14,pp.1116-1126)，用果实特异性启动子在番茄果实中仅过表达SlbZIP1基因的ORF区域(不包括uORF2)，由于缺少uORF2介导的负反馈，可使细胞内的糖持续上升，最终使番茄果实甜度提高1.5倍。然而，该策略需要转基因。
本领域仍然需要具有正常生长表型和提高的糖含量的植物(如番茄植物)，特别是非转基因植物(如非转基因番茄植物)。
发明简述
在一方面，本发明提供一种产生具有提高的糖含量的植物的方法，所述方法包括将靶向介导蔗糖诱导的翻译抑制(SIRT)的uORF的基因编辑系统导入所述植物，其中所述基因编辑系统的导入导致所述uORF中的突变，且所述突变导致所述uORF编码的多肽的表达减少或缺失，或所述突变导致所述uORF编码的多肽的活性的降低或缺失。
在一些实施方案中，所述介导SIRT的uORF编码的多肽包含与SEQ ID NO:2具有至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约99％、甚至至少约99％序列相同性的氨基酸序列。
在一些实施方案中，所述突变包括一或更多个核苷酸的取代、缺失或添加。
在一些实施方案中，所述基因编辑系统选自锌指核酸酶系统、TALEN系统和CRISPR系统。在一些实施方案中，所述CRISPR系统是CRISPR-Cas9系统。
在一些实施方案中，通过瞬时转化导入所述基因编辑系统，由此产生具有提高的糖含量的非转基因植物。
在一些实施方案中，通过稳定转化导入所述基因编辑系统，由此编码所述基因编辑系统组分的外源核苷酸序列被整合进所述植物基因组。在一些实施方案中，所述方法进一步包括通过遗传分离获得不含整合的外源核苷酸序列的非转基因植物。
在一些实施方案中，所述植物选自番茄、草莓、拟南芥、烟草、水稻、玉米、大麦、高粱、小麦、马铃薯、胡萝卜、甜椒、西瓜、香瓜、苹果、梨、葡萄、柑橘、橙子、柚子、樱桃、荔枝、红龙果、桃、金钱橘、李子、杏、芒果、无花果、哈密瓜、山楂、香蕉、杨梅、蓝莓、甜菜、猕猴桃、甘蓝、菠萝。
在一些具体实施方案中，所述植物是番茄。在一些实施方案中，所述介导SIRT的uORF是SlbZIP1基因的uORF2，例如，所述SlbZIP1基因的uORF2编码包含SEQ ID NO:2所示氨基酸序列的多肽。在一些实施方案中，所述CRISPR系统包含至少一种向导RNA，所述向导RNA靶向SlbZIP1基因的uORF2中SEQ ID NO:3或SEQ ID NO:4所示核苷酸序列；优选地，所述CRISPR系统包含两种向导RNA，所述两种向导RNA分别靶向SlbZIP1基因的uORF2中SEQ ID NO:3和SEQ ID NO:4所示核苷酸序列。在一些实施方案中，所述介导SIRT的uORF是SlbZIP2基因的uORF2，例如，所述SlbZIP2基因的uORF2编码包含SEQ ID NO:6所示氨基酸序列的多肽。
在另一方面，本发明提供一种通过本发明的方法产生的植物或其后代，优选地，所述植物或其后代是非转基因的。
附图简述
图1示出瞬时实验验证番茄SlbZIP1基因的5’leader序列中uORF的功能。uORF SlbZIP1是野生型5’leader序列；uorf SlbZIP1-A1，uorf SlbZIP1-A2，uorf SlbZIP1-3是分别将5’leader序列中的三个起始密码子AUG突变为AAA。左图为LUC/REN的活性，即代表LUC的翻译水平；右图代表LUC/REN的转录本水平，REN为内参基因。
图2示出SlbZIP1基因结构(含5’和3’非翻译区)以及T0突变体uORF2的测序结果。划线部分为sgRNA靶序列，加粗的为PAM。
图3示出uORF2突变体T0植物的糖含量测定结果。A：总糖含量；B：蔗糖含量；C：葡萄糖含量；D：果糖含量。
图4示出uORF2突变体T0植物的生长表型(比例尺为6cm)。
图5示出uORF2突变体T0植物的果实表型。A：果实大小与成熟度(比例尺为6mm)；B：果实发育和种子发育(比例尺为7mm)；C：果实重量。
图6示出SlbZIP1基因结构(含5’和3’非翻译区)以及T1突变体uORF2的测序结果。划线部分为sgRNA靶序列，加粗的为PAM。
图7示出uORF2突变体T1植物的生长表型(比例尺为8cm)、开花结果表型(比例尺为4cm)以及果实表型(比例尺为1.5cm)。
图8示出突变体T1植物不同组织中uorf SlbZIP1转录本水平变化。
图9示出uORF2突变体T1植物的糖含量测定结果。
发明详述
拟南芥转录因子AtbZIP11已经被证明在植物内控制糖含量并且受到蔗糖诱导的翻译抑制(SIRT)。在烟草中，在缺少SIRT下组成型过表达AtbZIP11的同源物TBZ17，尽管糖含量提高，但植物生长受到严重影响。这促使研究人员在番茄中使用果实特异性启动子过表达缺少SIRT的番茄SlbZIP1基因，结果实现番茄果实糖含量提高，并保持了植物正常生长。然而，使用果实特异性启动子需要进行对番茄植物进行转基因，会增加公众对其安全性的担忧。
本发明人发现，通过基因编辑技术原位破坏番茄SlbZIP1基因介导SIRT的uORF，能够显著提高番茄果实中的糖含量。并且，令人意想不到的是，尽管SlbZIP1基因的SIRT也是组成型被缺失，番茄植物的正常生长却不受影响，这在农业上具有重大意义。更为重要的是，使用基因编辑技术可以获得具有提高的果实糖含量的非转基因番茄植物，能够消除关于转基因的安全性问题。这是本领域第一次证明可以通过使用基因编辑技术原位破坏介导SIRT的uORF，提高植物中的糖含量。
因此，在一方面，本发明提供一种产生具有提高的糖含量的植物的方法，所述方法 包括将靶向介导蔗糖诱导的翻译抑制(SIRT)的uORF的基因编辑系统导入所述植物，其中所述基因编辑系统的导入导致所述uORF中的突变，且所述突变导致所述uORF编码的多肽的表达减少或缺失，或所述突变导致所述uORF编码的多肽的活性的降低或缺失。优选地，所述蔗糖诱导的翻译抑制(SIRT)被降低或被消除。
如本发明所用，术语“糖”意指可以提供甜味的碳水化合物，包括但不限于蔗糖、葡萄糖、果糖。在一些实施方案中，所述“糖含量”指的是总糖含量。在一些实施方案中，所述“糖含量”指的是蔗糖含量。在一些实施方案中，所述“糖含量”指的是葡萄糖含量。在一些实施方案中，所述“糖含量”指的是果糖含量。
介导蔗糖诱导的翻译抑制(SIRT)的uORF是从植物中其表达受SIRT调控的S类碱性亮氨酸拉链(basic leucine zipper，bZIP)转录因子5’非翻译区鉴定出来的保守uORF，也称作蔗糖控制uORF(Sucrose Control uORF，SC-uORF)，其在植物糖积累中起负反馈作用(Anika Wiese et al.,The Plant Cell,July 2004,Vol.16,pp.1717-1729；Sagor et al.,Plant Biotechnology Journal(2016)14,pp.1116-1126)。此类调节性uORF存在于植物基因组的bZIP编码基因，但在其它生物体中不存在。例如，所述uORF可以是来自拟南芥的AtbZIP11，烟草中的TBZ17，番茄的SlbZIP1和SlbZIP2的uORF，例如5’非翻译区的第二个uORF(uORF2)。
介导SIRT的uORF在植物中非常保守(Anika Wiese et al.,The Plant Cell,July 2004,Vol.16,pp.1717-1729；Sagor et al.,Plant Biotechnology Journal(2016)14,pp.1116-1126)。所述介导SIRT的uORF可以来自的植物包括但不限于番茄、草莓、拟南芥、烟草、水稻、玉米、大麦、高粱、小麦、马铃薯、胡萝卜、甜椒、西瓜、香瓜、苹果、梨、葡萄、柑橘、橙子、柚子、樱桃、荔枝、红龙果、桃、金钱橘、李子、杏、芒果、无花果、哈密瓜、山楂、香蕉、杨梅、蓝莓、甜菜、猕猴桃、甘蓝、菠萝。
因此，本发明的方法可以应用的植物包括但不限于番茄、草莓、拟南芥、烟草、水稻、玉米、大麦、高粱、小麦、马铃薯、胡萝卜、甜椒、西瓜、香瓜、苹果、梨、葡萄、柑橘、橙子、柚子、樱桃、荔枝、红龙果、桃、金钱橘、李子、杏、芒果、无花果、哈密瓜、山楂、香蕉、杨梅、蓝莓、甜菜、猕猴桃、甘蓝、菠萝。在一些具体实施方式中，所述植物是番茄。
在一些实施方案中，所述介导SIRT的uORF编码的多肽包含与SEQ ID NO:2(对应于番茄SlbZIP1的uORF2)具有至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约99％、甚至至少约99％序列相同性的氨基酸序列。
序列“相同性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或其区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着所述分子的特定区域测量序列相同性。(参见，例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis of  Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987；and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991)。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。
在一些具体实施方案中，所述植物是番茄，且所述介导SIRT的uORF是SlbZIP1基因的uORF2。在一些实施方案中，SlbZIP1基因的核苷酸序列可从Genbank中的Gene ID 543618获得。例如，所述SlbZIP1基因的uORF2编码包含SEQ ID NO:2所示氨基酸序列的多肽。
在一些具体实施方案中，所述植物是番茄，且所述介导SIRT的uORF是SlbZIP2基因的uORF2。在一些实施方案中，SlbZIP2基因的核苷酸序列可从Genbank中的Gene ID:543618获得。例如所述SlbZIP2基因的uORF2编码包含SEQ ID NO:6所示氨基酸序列的多肽。
在一些具体实施方案中，所述植物是金钱橘(Citrus clementina)，且所述介导SIRT的uORF是金钱橘LOC18047493基因的uORF，其编码包含SEQ ID NO:7所示氨基酸序列的多肽。
在一些具体实施方案中，所述植物是桃(Prunus persica)，且所述介导SIRT的uORF是桃LOC18767264基因的uORF，其编码包含SEQ ID NO:8所示氨基酸序列的多肽。
合适的介导SIRT的uORF还可以例如是Anika Wiese et al.(The Plant Cell,July 2004,Vol.16,pp.1717-1729)中描述的那些。
在一些实施方案中，所述uORF中的突变包括一或更多个核苷酸的取代、缺失或添加。例如，所述突变导致uORF中的翻译起始密码子缺失或变为翻译终止密码子，或者导致所述uORF的强翻译起始密码子突变为弱翻译起始密码子，使得所述uORF编码的多肽不能被翻译或翻译水平下降。或者，所述突变可以是移码突变，使得所述uORF编码的多肽不能被正确翻译(例如被截短，或活性位点被突变或缺失)，从而活性被降低或缺失。如本文所用，“uORF编码的多肽的活性”意指所述多肽介导蔗糖诱导的翻译抑制(SIRT)的能力。
“基因编辑”，也称为基因组编辑，其使用序列特异性核酸酶或其衍生物在生物体基因组中进行核苷酸插入、缺失或取代。基因编辑通常通过在基因组中期望的位置导致位点特异性双链断裂(DSB)，然后在修复DSB的过程中引入期望的DNA插入、缺失或取代。然而，基因编辑也涵盖不涉及DSB的碱基编辑技术。
本领域已知多种基因编辑系统。本发明并不特别限制所使用的基因编辑系统，只要其能够实现所述突变。例如，适于本发明使用的基因编辑系统包括但不限于锌指核酸酶(ZFN)系统、转录激活因子样效应物核酸酶(TALEN)系统和CRISPR系统。本领域技术人员能够根据本申请的教导选择或设计合适的基因编辑系统。
“锌指核酸酶”是通过将锌指DNA结合结构域与DNA切割结构域融合而制备的 人工限制性酶。ZFN的单个锌指DNA结合结构域通常含有3-6个单独的锌指重复序列，每个锌指重复序列可以识别例如3bp的独特序列。通过组合不同的锌指重复序列，可以靶向不同的基因组序列。
“转录激活因子样效应物核酸酶”是可以经工程化而可以切割特定DNA序列的限制性酶，通常通过将转录激活因子样效应物(TALE)的DNA结合结构域与DNA切割结构域融合而制备。TALE经工程化后可以结合几乎任何想要的DNA序列。
“CRISPR(Clustered regularly interspaced short palindromic repeats，成簇的规律间隔的短回文重复序列)系统”通常包含可以形成具有序列特异性的复合物的两种组分：CRISPR核酸酶和相应的向导RNA。
如本文所用，术语“CRISPR核酸酶”通常指在天然存在的CRISPR系统中存在的核酸酶，以及其修饰形式、其变体(包括切口酶突变体)、或其催化活性片段。CRISPR核酸酶可以通过与向导RNA一起相互作用来识别、结合和/或切割靶核酸结构。该术语涵盖基于CRISPR系统的能够在细胞内实现基因编辑的任何核酸酶或其功能性变体。本领域已知多种CRISPR核酸酶的功能性变体，例如高特异性变体或切口酶变体，或其与胞苷脱氨酶或腺苷脱氨酶的融合蛋白等。本领域技术人员知晓如何选择合适的CRISPR核酸酶功能性变体以实现本发明的目的。
本发明的CRISPR基因编辑系统使用的CRISPR核酸酶例如可以选自Cas3、Cas8a、Cas5、Cas8b、Cas8c、Cas10d、Cse1、Cse2、Csy1、Csy2、Csy3、GSU0054、Cas10、Csm2、Cmr5、Cas10、Csx11、Csx10、Csf1、Cas9、Csn2、Cas4、Cpf1、C2c1、C2c3或C2c2蛋白，或这些核酸酶的功能性变体。
在一些实施方案中，所述CRISPR核酸酶包括Cas9核酸酶或其变体。基于Cas9核酸酶或其变体的CRISPR基因编辑系统在本文也称作CRISPR-Cas9系统。所述Cas9核酸酶可以是来自不同物种的Cas9核酸酶，例如来自化脓链球菌(S.pyogenes)的spCas9。spCas9的示例性氨基酸序列如SEQ ID NO:9所示。
所述Cas9核酸酶变体例如可以包括Cas9核酸酶的高特异性变体，例如Feng Zhang等人的Cas9核酸酶变体eSpCas9(1.0)(K810A/K1003A/R1060A)、eSpCas9(1.1)(K848A/K1003A/R1060A)，以及J.Keith Joung等人开发的Cas9核酸酶变体SpCas9-HF1(N497A/R661A/Q695A/Q926A)。
或者，所述Cas9核酸酶变体还可以包括Cas9切口酶(nCas9)，其中Cas9核酸酶的DNA切割结构域中的两个亚结构域(HNH核酸酶亚结构域和RuvC亚结构域)之一被失活而形成切口酶。在一些实施方案中，可以利用Cas9切口酶与靶向待编辑序列上下游的两种gRNA组合，实现待编辑序列的缺失，或在供体序列存在下实现待编辑序列的替换。
在一些实施方案中，所述CRISPR核酸酶还可以包括Cpf1核酸酶或其变体例如高 特异性变体。所述Cpf1核酸酶可以是来自不同物种的Cpf1核酸酶，例如来自Francisella novicida U112、Acidaminococcus sp.BV3L6和Lachnospiraceae bacterium ND2006的Cpf1核酸酶。基于Cpf1核酸酶或其变体的CRISPR基因编辑系统在本文也称作CRISPR-Cpf1系统。
在一些实施方案中，所述CRISPR核酸酶还可以包括碱基编辑器(base editor)。碱基编辑器通常是包含脱氨酶和缺失DNA切割活性的CRISPR核酸酶的融合蛋白。
如本发明所用，“缺失DNA切割活性的CRISPR核酸酶”包括但不限于Cas9切口核酸酶(nCas9)、核酸酶死亡的Cas9核酸酶(dCas9)或核酸酶死亡的Cpf1核酸酶(dCpf1)。核酸酶死亡的Cas9核酸酶(dCas9)或核酸酶死亡的Cpf1核酸酶(dCpf1)完全缺失DNA切割活性。本领域已知多种缺失DNA切割活性的CRISPR核酸酶。
如本发明所用，“脱氨酶”是指催化脱氨基反应的酶。在本发明一些实施方式中，所述脱氨酶指的是胞嘧啶脱氨酶，其能够接受单链DNA作为底物并能够催化胞苷或脱氧胞苷分别脱氨化为尿嘧啶或脱氧尿嘧啶。在本发明一些实施方式中，所述脱氨酶指的是腺嘌呤脱氨酶，其能够接受单链DNA作为底物并能够催化腺苷或脱氧腺苷(A)形成肌苷(I)。本领域已知多种合适的接受单链DNA作为底物的胞嘧啶脱氨酶或腺嘌呤脱氨酶，例如APOBEC1脱氨酶、激活诱导的胞苷脱氨酶(AID)、APOBEC3G、CDA1，或者例如Nicloe M.Gaudelli等人(doi：10.1038/nature24644，2017)所公开的DNA依赖型腺嘌呤脱氨酶。
通过使用缺失DNA切割活性的CRISPR核酸酶与脱氨酶融合(形成所谓的“碱基编辑器”)，可以实现靶核苷酸序列中的碱基编辑，例如C至T的转换或A至G的转换。本领域已知多种碱基编辑器，且本领域技术人员知晓如何选择合适的碱基编辑器以实现本发明的目的。
本发明中用于基因编辑的序列特异性核酸酶，例如锌指核酸酶、转录激活因子样效应物核酸酶或CRISPR核酸酶等，还可以包含亚细胞定位信号(如核定位信号)、肽接头、可检测标签等元件。例如，CRISPR碱基编辑系统中的CRISPR核酸酶通常包含一个或多个核定位信号(NLS)，以促进其进入细胞核，实现对染色体DNA的编辑。
如本文所用，“gRNA”和“向导RNA”可互换使用，指的是能够与CRISPR核酸酶形成复合物并由于与靶序列具有一定互补性而能够将所述复合物靶向靶序列的RNA分子。例如，在基于Cas9的CRISPR基因编辑系统中，gRNA通常由部分互补形成复合物的crRNA和tracrRNA分子构成，其中crRNA包含与靶序列具有足够相同性以便指导CRISPR复合物(Cas9+crRNA+tracrRNA)与该靶序列序列特异性地结合的序列。然而，本领域已知可以设计单向导RNA(sgRNA)，其同时包含crRNA和tracrRNA的特征。而在基于Cpf1的CRISPR基因组编辑系统中，gRNA通常仅由成熟crRNA分子构成，其中crRNA包含有与靶序列具有足够相同性的序列以便指导复合物(Cpf1+crRNA)与该靶序列序列特异性结合。基于所使用的CRISPR核酸酶和待编辑的靶序列(如介导SIRT的uORF)设计合适的gRNA属于本领域技术人员的能力范围内。
在一些具体实施方式中，所述植物是番茄，且所述CRISPR系统包含至少一种向导RNA，所述向导RNA靶向SlbZIP1基因的uORF2中SEQ ID NO:3或SEQ ID NO:4所示核苷酸序列；优选地，所述CRISPR系统包含两种向导RNA，所述两种向导RNA分别靶向SlbZIP1基因的uORF2中SEQ ID NO:3和SEQ ID NO:4所示核苷酸序列。
在本发明的实施方案中，所述基因编辑系统可以以各种形式导入植物。例如，对于CRISPR系统，CRISPR核酸酶和向导RNA可以在体外产生并组装成核糖核蛋白(RNP)，然后导入植物。或者，可以将编码所述CRIPSR系统的所有组分的表达构建体导入植物，然后在植物细胞中表达出所述组分。或者，可以将编码所述CRIPSR系统的部分组分的表达构建体以及体外生成的其它组分同时导入植物。
因此，本发明的实施方案中，所述CRISPR基因编辑系统可以以下述i)至v)中至少一种形式导入植物中：
i)至少一种CRISPR核酸酶，和至少一种向导RNA；
ii)至少一种包含编码CRISPR核酸酶的核苷酸序列的表达构建体，和至少一种向导RNA；
iii)至少一种CRISPR核酸酶，和至少一种包含编码至少一种向导RNA的核苷酸序列的表达构建体；
iv)至少一种包含编码CRISPR核酸酶的核苷酸序列的表达构建体，和至少一种包含编码至少一种向导RNA的核苷酸序列的表达构建体；
v)至少一种包含编码CRISPR核酸酶的核苷酸序列和至少一种编码至少一种向导RNA的核苷酸序列的表达构建体。
如本发明所用，“表达构建体”是指适于感兴趣的核苷酸序列在生物体中表达的载体如重组载体。“表达”指功能产物的产生。例如，核苷酸序列的表达可指核苷酸序列的转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。本发明的“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者，在一些实施方式中，可以是能够翻译的RNA(如mRNA)。本发明的“表达构建体”可包含不同来源的调控序列和感兴趣的核苷酸序列，或相同来源但以不同于通常天然存在的方式排列的调控序列和感兴趣的核苷酸序列。“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5’非编码序列)、中间或下游(3’非编码序列)，并且影响相关编码序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。植物表达调控元件指的是能够在植物中控制感兴趣的核苷酸序列转录、RNA加工或稳定性或者翻译的核苷酸序列。调控序列可包括但不限于启动子、翻译前导序列、内含子和多腺苷酸化识别序列。
将核酸分子(例如本发明的表达构建体等)和/或蛋白质“导入”植物是指用所述核酸和/或蛋白质转化植物，使得所述核酸和/或蛋白质在植物中能够发挥功能。例如，可以用所述核酸和/或蛋白质转化分离的植物细胞或组织，然后从所述经转化的细胞或组织再生植物。或者，可以将本发明的碱基编辑系统转化至完整植物上的特定部位，例如叶片、茎尖、花粉管、幼穗或下胚轴。这特别适合于难以进行组织培养再生的植物的转化。
可用于将核酸分子和/或蛋白质导入植物或植物细胞的方法包括但不限于：基因枪法、PEG介导的原生质体转化、土壤农杆菌介导的转化、植物病毒介导的转化、花粉管通道法和子房注射法。
本发明所用的“转化”包括稳定转化和瞬时转化。“稳定转化”指将外源核苷酸序列导入基因组中，导致外源核苷酸序列稳定遗传。一旦稳定转化，外源核酸序列稳定地整合进所述植物的基因组中并且可以传递至其任何连续世代。“瞬时转化”指将核酸分子或蛋白质导入细胞中，执行功能而没有外源基因稳定遗传。瞬时转化中，外源核酸序列不整合进基因组中。
在一些实施方案中，通过瞬时转化导入所述基因编辑系统，由此产生具有提高的糖含量的非转基因植物。
例如，所述基因编辑系统的导入在不存在选择压力下进行，从而避免外源核苷酸序列在植物基因组中的整合。在一些具体实施方案中，将本发明的基因编辑系统转化至分离的植物细胞或组织，然后使所述经转化的植物细胞或组织再生为完整植物，优选地，在不存在选择压力下进行所述再生，也即是，在组织培养过程中不使用任何针对表达载体上携带的选择基因的选择剂(如抗生素、除草剂等)。不使用选择剂可以提高植物的再生效率，获得不含外源核苷酸序列的经基因编辑的植物。
直接将体外表达的蛋白质和/或体外转录的RNA分子转化至植物或植物细胞也是可能的。所述蛋白质和/或RNA分子能够在植物细胞中实现基因编辑，随后被细胞降解，避免了外源核苷酸序列在植物基因组中的整合。
在一些实施方案中，通过稳定转化导入所述基因编辑系统，由此编码所述基因编辑系统组分的外源核苷酸序列被整合进所述植物基因组。稳定转化可能提高筛选转化体的效率。之后，可以使所获得的经转化的植物的后代进行遗传分离，获得不含整合的外源核苷酸序列的非转基因植物。
总言之，通过基因编辑的方法，只需在植物细胞中导入或产生所述基因编辑的组分(如序列特异性核酸酶和/或向导RNA)即可实现对靶序列的修饰，并且所述修饰可以稳定遗传，无需所述基因编辑系统在植物中持续存在。这样可以避免外源核苷酸序列在植物基因组中的整合，从而具有更高生物安全性。
在另一方面，本发明提供了通过本发明上述的方法产生的植物及其后代，其具有提高的糖含量。优选地，所述植物及其后代是非转基因的。
在另一方面，本发明提供了一种具有提高的糖含量的植物，其相对于野生型植物，在介导SIRT的uORF中包含突变，所述突变导致所述uORF编码的多肽的表达减少或缺失，或所述突变导致所述uORF编码的多肽活性的降低或缺失。优选地，所述植物是非转基因的。
在一些实施方案中，所述突变通过向所述植物导入靶向所述uORF的基因编辑系统产生。所述基因编辑系统和/或所述基因编辑系统的导入如上文所定义。在另一些实施方案中，所述突变通过杂交育种导入。
在一些实施方案中，相比于相应的野生型植物或其组织或器官(例如，介导SIRT的uORF未被突变)，本发明的植物或其组织或器官的糖含量增加。所述糖含量增加的组织或器官包括但不限于果实、叶、根、茎、块茎等。“相应的野生型植物”意指通过本发明的方法产生的植物或本发明的植物所衍生自的野生型植物，例如所述野生型植物中介导SIRT的uORF未被突变。
相比于相应的野生型植物或其组织或器官(例如，介导SIRT的uORF未被突变)，本发明的植物或其组织或器官中的总糖含量增加至少大约10％、增加至少大约20％、增加至少大约30％、增加至少大约40％、增加至少大约50％、增加至少大约60％、增加至少大约70％、增加至少大约80％、增加至少大约90％、增加至少大约100％、增加至少大约125％、增加至少大约150％、增加至少大约175％、增加至少大约200％或更高。优选地，相比于相应的野生型植物或其组织或器官(例如，介导SIRT的uORF未被突变)，本发明的植物或其组织或器官中的蔗糖含量增加至少大约10％、增加至少大约20％、增加至少大约30％、增加至少大约40％、增加至少大约50％、增加至少大约60％、增加至少大约70％、增加至少大约80％、增加至少大约90％、增加至少大约100％、增加至少大约125％、增加至少大约150％、增加至少大约175％、增加至少大约200％或更高。优选地，相比于相应的野生型植物或其组织或器官(例如，介导SIRT的uORF未被突变)，本发明的植物或其组织或器官中的果糖含量增加至少大约10％、增加至少大约20％、增加至少大约30％、增加至少大约40％、增加至少大约50％、增加至少大约60％、增加至少大约70％、增加至少大约80％、增加至少大约90％、增加至少大约100％、增加至少大约125％、增加至少大约150％、增加至少大约175％、增加至少大约200％或更高。优选地，相比于相应的野生型植物或其组织或器官(例如，介导SIRT的uORF未被突变)，本发明的植物或其组织或器官中的葡萄糖含量增加至少大约10％、增加至少大约20％、增加至少大约30％、增加至少大约40％、增加至少大约50％、增加至少大约60％、增加至少大约70％、增加至少大约80％、增加至少大约90％、增加至少大约100％、增加至少大约125％、增加至少大约150％、增加至少大约175％、增加至少大约200％或更高。
在一些实施方案中，相比于相应的野生型植物(例如，介导SIRT的uORF未被突变)，本发明的植物在基本上相同或相同的生长条件下具有可比较的(类似的或基本上相同)的生长参数和/或形态参数，包括但不限于生长速率、叶片大小、叶片厚度、果实大小、果实重量、株高、种子萌发率等。
在另一方面，本发明提供了一种植物育种方法，包括
a)提供第一植物，其通过本发明上述方法产生；
b)提供其中介导SIRT的uORF未被突变的第二植物；和
c)使所述第一植物和所述第二植物杂交。
在一具体方面，本发明提供一种产生具有提高的果实糖含量的番茄(Solanum lycopersicum)植物的方法，所述方法包括将靶向番茄SlbZIP1和/或SlbZIP2基因的uORF2 的基因编辑系统导入番茄植物，所述基因编辑系统的导入导致所述SlbZIP1和/或SlbZIP2基因的uORF2中的突变，且所述突变导致所述uORF2编码的多肽的表达减少或缺失，或所述突变导致所述uORF2编码的多肽的活性的降低或缺失。所述基因编辑系统和/或所述基因编辑系统的导入如上文所定义。
在一些实施方式中，所述SlbZIP1基因的uORF2编码包含SEQ ID NO:2所示氨基酸序列的多肽。在一些实施方式中，所述SlbZIP2基因的uORF2编码包含SEQ ID NO:6所示氨基酸序列的多肽。
在一些实施方案中，所述CRISPR系统包含至少一种向导RNA，所述向导RNA靶向SlbZIP1基因的uORF2中SEQ ID NO:3或SEQ ID NO:4所示核苷酸序列。优选地，所述CRISPR系统包含两种向导RNA，所述两种向导RNA分别靶向SlbZIP1基因的uORF2中SEQ ID NO:3和SEQ ID NO:4所示核苷酸序列。
在另一方面，本发明提供一种通过本发明的方法产生的番茄植物或其后代，其具有提高的果实糖含量。在一些优选实施方案中，所述具有提高的果实糖含量的番茄植物是非转基因的。
在另一方面，本发明提供了一种具有提高的果实糖含量的番茄植物，其相对于野生型番茄植物，在SlbZIP1基因和/或SlbZIP2基因的uORF2中包含突变，所述突变导致所述uORF2编码的多肽的表达减少或缺失，或所述突变导致所述uORF2编码的多肽活性的降低或缺失。优选地，所述番茄植物是非转基因的。
在一些实施方案中，所述突变通过向番茄植物导入靶向SlbZIP1基因和/或SlbZIP2基因的uORF2的基因编辑系统产生。所述基因编辑系统和/或所述基因编辑系统的导入如上文所定义。在另一些实施方案中，所述突变通过杂交育种导入。
本发明的具有提高的糖含量(如果实糖含量)的番茄植物可以衍生自不同的番茄品种，包括但不限于栽培种M82(Solanum lycopersicum cv.M82)。
相比于相应的野生型番茄植物(例如，SlbZIP1基因和/或SlbZIP2基因的uORF2未被突变)，本发明的番茄植物的果实中的总糖含量增加至少大约10％、增加至少大约20％、增加至少大约30％、增加至少大约40％、增加至少大约50％、增加至少大约60％、增加至少大约70％、增加至少大约80％、增加至少大约90％、增加至少大约100％、增加至少大约125％、增加至少大约150％、增加至少大约175％、增加至少大约200％或更高。优选地，相比于其衍生自的野生型番茄植物(例如，SlbZIP1基因的uORF2未被突变)，本发明的番茄植物的果实中的蔗糖含量增加至少大约10％、增加至少大约20％、增加至少大约30％、增加至少大约40％、增加至少大约50％、增加至少大约60％、增加至少大约70％、增加至少大约80％、增加至少大约90％、增加至少大约100％、增加至少大约125％、增加至少大约150％、增加至少大约175％、增加至少大约200％或更高。优选地，相比于相应的野生型植物或其组织或器官(例如，介导SIRT的uORF未被突变)，本发明的植物或其组织或器官中的果糖含量增加至少大约10％、增加至少大约20％、增加至少大约30％、增加至少大约40％、增加至少大约50％、增加至少大约60％、 增加至少大约70％、增加至少大约80％、增加至少大约90％、增加至少大约100％、增加至少大约125％、增加至少大约150％、增加至少大约175％、增加至少大约200％或更高。优选地，相比于相应的野生型植物或其组织或器官(例如，介导SIRT的uORF未被突变)，本发明的植物或其组织或器官中的葡萄糖含量增加至少大约10％、增加至少大约20％、增加至少大约30％、增加至少大约40％、增加至少大约50％、增加至少大约60％、增加至少大约70％、增加至少大约80％、增加至少大约90％、增加至少大约100％、增加至少大约125％、增加至少大约150％、增加至少大约175％、增加至少大约200％或更高。
在一些实施方案中，相比于相应的野生型番茄植物(例如，介导SIRT的uORF未被突变)，本发明的具有提高的果实糖含量的番茄植物在基本上相同或相同的生长条件下具有可比较的(类似的或基本上相同)的生长参数和/或形态参数，包括但不限于生长速率、叶片大小、叶片厚度、果实大小、果实重量、株高、种子萌发率等。
实施例
实施例1、瞬时实验验证番茄SlbZIP1基因的5’leader序列中uORF的功能
为了检测SlbZIP1的第二个uORF(uORF2)是否和拟南芥中AtbZIP11中uORF2具有类似的功能，分别将SlbZIP1uORF2的三个起始密码子AUG突变为AAA以使该起始密码子失去起始翻译功能，从而探索uORF2对SlbZIP1基因的作用。利用双荧光素酶报告系统(如图1所示)，海肾荧光素酶(REN)和萤火虫荧光素酶(LUC)分别利用花椰菜花叶病毒的35S启动子驱动表达，其中REN是内参对照。将SlbZIP1基因的5’leader序列的野生型形式和uORF突变形式(即分别将三个起始密码子AUG突变为AAA，并分别命名为uorfSlbZIP1-A1、uorfSlbZIP1-A2、uorfSlbZIP1-A3)构建在LUC的上游区域。将这些质粒分别转入番茄原生质体中进行48小时的表达，测定LUC/REN的转录本和翻译水平。
1.1实验材料和方法
载体名称和来源：pGreenII 0800-LUC(Hellens,R.P.et al.Transient expression vectors for functional genomics,quantification of promoter activity and RNA silencing in plants.Plant Methods 1,13(2005))
原生质体制备及转化：选取2～3周大的生长在固体培养基上的番茄幼苗，将其切成细丝并用酶解法分离番茄原生质体细胞，然后将构建好的质粒通过PEG介导的转化方法转入原生质体细胞中，在23℃条件下黑暗培养48小时后收集原生质体。
荧光测定方法：通过双荧光素酶报告基因检测系统在荧光检测仪上对细胞中的相应载体的海肾荧光素酶(REN)和萤火虫荧光素酶(LUC)的表达活性进行测定，其中所得的LUC/REN的比值即为目标报告基因LUC的相对表达量。
mRNA水平的测定：收集黑暗培养48小时后的原生质体，用TRIzol试剂提取细胞 中的RNA水平，并将其反转为cDNA，之后用qRT-PCR进行定量。
1.2实验结果
结果发现当把第二个AUG突变为AAA时，可以有效增强下游基因的活性且不影响其转录本水平(图1)。说明该基因的uORF能够抑制下游基因的翻译水平，其中第二个起始密码子AUG在所述抑制中具有重要的作用。
实施例2、通过基因编辑敲除SlbZIP1的uORF2
本实验使用的番茄品种为M82(Solanum lycopersicum cv.M82)。
2.1.sgRNA的设计
为了能够完全破坏SlbZIP1的uORF2翻译形成的多肽，同时设计了2种靶向该uORF的sgRNA(sgRNA-1和sgRNA-2)，使得待突变的序列包含第二个和第三个起始密码子。将该2种sgRNA同时构建进pKSE401双元载体骨架(Addgene:62202；其同时介导SpCas9的表达)获得pKSE401-SlbZIP1uORF-2sgRNA。将该载体质粒转入根癌农杆菌菌株LBA4404内，用于M82番茄植物的转化。
SlbZIP1-uORF2序列：
Figure PCTCN2019101697-appb-000001
(备注：方框部分为sgRNA；划线部分为PAM；斜体所示为起始密码子)
SlbZIP1-uORF2多肽的氨基酸序列：MIHMRRVRIMHSFSVVFLYWFYVFS(SEQ ID NO:2)。
sgRNA-1靶序列：AATGCGAACTCGACGCATG(SEQ ID NO:3)。
sgRNA-2靶序列：AAACACATAGAACCAGTAC(SEQ ID NO:4)。
2.2.转化获得突变体
用1.1中所获得的带有质粒pKSE401-SlbZIP1uORF-2sgRNA的农杆菌转化番茄品种M82。以番茄幼苗的子叶作为受体进行农杆菌介导的转化。转化后经过组织培养方法获得再生番茄植株。植物种植条件：在番茄培养间进行，气温为25-28℃，光照周期为16小时光照/8小时黑暗，光照强度约为600μmol m -2s -1，常规管理。
2.3.检测突变体
对获得的转基因植株进行阳性鉴定。提取转基因植株基因组DNA，用能够扩增SlbZIP1-uORF2的特异引物对转化有pKSE401-SlbZIP1uORF-2sgRNA的基因组DNA进行PCR扩增。将PCR产物连接至
Figure PCTCN2019101697-appb-000002
克隆载体上并挑取阳性克隆进行Sanger 测序。
结果如图2所示。获得了SlbZIP1-uORF2突变的T0突变体植物。示例性的T0突变体(T0-14、T0-15和T0-29)的测序结果如图2所示，它们的uORF2的起始密码子被破坏，且产生移码突变，从而可以阻止形成保守的uORF2多肽。
实施例3、SlbZIP1-uORF2敲除的T0突变体植物的获得和表型分析
3.1.糖分测定
以实施例2中所获得SlbZIP1-uORF2破坏的T0突变体番茄为材料。分别将突变体材料和相应的野生型(WT)植物进行培养至得到成熟番茄果实。在野生型和T0突变体植物中分别随机选取3个成熟果实，取1g鲜重番茄果实，并用15ml的80％乙醇分3次在80℃提取其中的总糖。吸取50ul糖溶液冻干，并使用MSTFA(N-甲基-N-(三甲基硅烷)三氟乙酰胺)进行衍生，之后用GC-MS(气相色谱-质谱联用仪)对突变体和野生型果实中的各种糖分(蔗糖、葡萄糖、果糖)进行测定并进行差异性分析。
结果如图3所示。突变体果实中的总糖含量分别提高至野生型果实的245.77％，188.85％和175.62％。其中T0突变体果实中蔗糖、葡萄糖和果糖的含量也相对于野生型果实显著提高。
3.2.生长表型
使T0-14、T0-15和T0-29突变体植物与WT植物在同一生长环境里面生长，观察其各项生长指标的差异。植物种植条件：在番茄培养间进行，气温为25-28℃，光照周期为16小时光照/8小时黑暗，光照强度约为600μmol m -2s -1，常规管理。
如图4所示，突变体植物与WT植物的生长无明显差异。如图5A所示，突变体和WT的果实大小与成熟度无明显差异。如图5B所示，突变体和WT的果实发育、种子发育无明显差异。WT与突变体植物分别随机选取3株称量所结番茄果实，并进行差异性分析如图5C所示，果实重量无明显差异。
此外，测量T0植物的种子的萌发率。萌发实验如下进行：将保存干燥的番茄种子，放入存有润湿滤纸的培养皿里催芽3天左右，待种子露出培根时转移至32孔番茄育苗盘中。育苗土为蛭石:草炭(1:1)均匀混合。温度为25-28℃，16小时光照，夜间20-22℃，8小时黑暗。如下表1所示，突变体和WT的T1代种子萌发率无显著差异。
表1. T0突变体和WT的种子的萌发率
Figure PCTCN2019101697-appb-000003
实施例4、SlbZIP1-uORF2敲除的T1突变体植物的获得和表型分析
从前述实施例的T0代植物收获种子，即获得T1代种子，将该种子进行萌发并鉴定 基因型，获得稳定遗传的纯合uorf SlbZIP1突变体(如图6所示)。每个株系分别选取5株个体进行传代以及后续的实验，其中株系uorf SlbZIP1-1、uorf SlbZIP1-2、uorf SlbZIP1-4经鉴定均为非转基因突变体苗。非转基因植物鉴定方法包括：针对农杆菌转化载体pKSE401-SlbZIP1uORF-2sgRNA插入到基因组的RB与LB之间的序列设计3对扩增不同长度片段的特异引物，利用这三对引物分别对所获得的突变体DNA进行扩增，如果没有扩增出来目的片段，则为不包含转基因的植株，如果能够扩增出目的条带则为包含有转基因的植株。
4.1.生长表型
使T1突变体植物与WT植物在同一生长环境里面生长，观察其各项生长指标的差异。植物种植条件：在番茄培养间进行，气温为25-28℃，光照周期为16小时光照/8小时黑暗，光照强度约为600μmol m -2s -1，常规管理。
如图7所示，突变体植物与WT植物的生长无明显差异。如图7A所示，突变体生长表型与对照没有明显差异。如图7B所示，突变体开花和结果均正常，与对照无明显差异。如图7C所示，突变体果实大小和成熟情况与对照无明显差异。此外，如下表2所示，突变体和WT的种子的萌发率无显著差异。
表2、T1突变体和WT的种子的萌发率
Figure PCTCN2019101697-appb-000004
4.2.uorf SlbZIP1转录本水平变化
通过用qRT-PCR对突变体和对照中果实(图8A)或其他部位(如花)(图8B)的uorf SlbZIP1转录本水平进行测定，并以Slactin作为内参基因，其结果显示，突变体与对照中的uorf SlbZIP1的转录本水平一致，无明显差异。
4.3.uorf SlbZIP1突变体糖含量变化
果实中的甜度主要由糖分的积累所致，因而从对照和uorf SlbZIP1突变体中成熟的果实中分别随机选取生长正常的番茄果实，称取1g新鲜的果实，并提取并测定其中的糖分(方法如上所述)。结果(图9)发现突变体中的总糖(蔗糖、葡萄糖、果糖之和)相比于对照增加了23％～39％，葡萄糖增加了13％～34％，果糖增加了43％～46％，而蔗糖含量则与对照无明显差异。其中蔗糖与对照无差异的主要原因可能是果实中积累的蔗糖迅速被蔗糖酶转化为葡萄糖和果糖。
上述实验结果说明破坏SlbZIP1的uORF2，可以解除uORF2对SlbZIP1基因表达的抑制，从而使番茄果实中的糖分含量显著提高。同时令人意想不到的是，uORF2突变体植物的生长指标和野生型植物基本保持一致。这尤其适合在提高甜度的同时保证番茄 产量，在农业上具有重要意义。此外，对所获得的后代进行筛选，去除基因编辑元件，还可获得不含转基因的甜度提高的番茄品种。
Figure PCTCN2019101697-appb-000005

Claims (14)

  1. 一种产生具有提高的糖含量的植物的方法，所述方法包括将靶向介导蔗糖诱导的翻译抑制(SIRT)的uORF的基因编辑系统导入所述植物，其中所述基因编辑系统的导入导致所述uORF中的突变，且所述突变导致所述uORF编码的多肽的表达减少或缺失，或所述突变导致所述uORF编码的多肽的活性的降低或缺失。
  2. 权利要求1的方法，所述介导SIRT的uORF编码的多肽包含与SEQ ID NO:2具有至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约99％、甚至至少约99％序列相同性的氨基酸序列。
  3. 权利要求1或2的方法，其中所述突变包括一或多个核苷酸的取代、缺失或添加。
  4. 权利要求1-3中任一项的方法，其中所述基因编辑系统选自锌指核酸酶系统、TALEN系统和CRISPR系统。
  5. 权利要求4的方法，其中所述CRISPR系统是CRISPR-Cas9系统。
  6. 权利要求1-5中任一项的方法，其中通过瞬时转化导入所述基因编辑系统，编码所述基因编辑系统组分的外源核苷酸序列不整合进所述植物基因组，由此产生具有提高的糖含量的非转基因植物。
  7. 权利要求1-6中任一项的方法，其中通过稳定转化导入所述基因编辑系统，由此编码所述基因编辑系统组分的外源核苷酸序列被整合进所述植物基因组。
  8. 权利要求7的方法，其进一步包括通过遗传分离获得不含整合的外源核苷酸序列的非转基因植物。
  9. 权利要求1-8中任一项的方法，其中所述植物选自番茄、草莓、拟南芥、烟草、水稻、玉米、大麦、高粱、小麦、马铃薯、胡萝卜、甜椒、西瓜、香瓜、苹果、梨、葡萄、柑橘、橙子、柚子、樱桃、荔枝、红龙果、桃、金钱橘、李子、杏、芒果、无花果、哈密瓜、山楂、香蕉、杨梅、蓝莓、甜菜、猕猴桃、甘蓝、菠萝。
  10. 权利要求9的方法，其中所述植物是番茄。
  11. 权利要求10的方法，其中所述介导SIRT的uORF是SlbZIP1基因的uORF2，例如，所述SlbZIP1基因的uORF2编码包含SEQ ID NO:2所示氨基酸序列的多肽。
  12. 权利要求11的方法，其中所述CRISPR系统包含至少一种向导RNA，所述向导RNA靶向SlbZIP1基因的uORF2中SEQ ID NO:3或SEQ ID NO:4所示核苷酸序列；优选地，所述CRISPR系统包含两种向导RNA，所述两种向导RNA分别靶向SlbZIP1基因的uORF2中SEQ ID NO:3和SEQ ID NO:4所示核苷酸序列。
  13. 权利要求10的方法，其中所述介导SIRT的uORF是SlbZIP2基因的uORF2，例如，所述SlbZIP2基因的uORF2编码包含SEQ ID NO:6所示氨基酸序列的多肽。
  14. 一种通过权利要求1-13中任一项的方法产生的植物或其后代，优选地，所述植物或其后代是非转基因的。
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