WO2021088923A1 - 在生物体内创制新基因的方法及应用 - Google Patents

在生物体内创制新基因的方法及应用 Download PDF

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WO2021088923A1
WO2021088923A1 PCT/CN2020/126747 CN2020126747W WO2021088923A1 WO 2021088923 A1 WO2021088923 A1 WO 2021088923A1 CN 2020126747 W CN2020126747 W CN 2020126747W WO 2021088923 A1 WO2021088923 A1 WO 2021088923A1
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gene
different
promoter
plant
endogenous
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PCT/CN2020/126747
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English (en)
French (fr)
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姜临建
王继尧
莫苏东
陈波
李华荣
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青岛清原化合物有限公司
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Priority to KR1020227014731A priority Critical patent/KR20220066979A/ko
Priority to PE2022000731A priority patent/PE20221178A1/es
Priority to US17/264,367 priority patent/US20220348950A1/en
Priority to MX2022005431A priority patent/MX2022005431A/es
Priority to CA3159360A priority patent/CA3159360A1/en
Priority to AU2020378500A priority patent/AU2020378500A1/en
Priority to BR112022001455A priority patent/BR112022001455A2/pt
Priority to JP2022506562A priority patent/JP2022553598A/ja
Application filed by 青岛清原化合物有限公司 filed Critical 青岛清原化合物有限公司
Priority to EP20884099.1A priority patent/EP4074832A4/en
Priority to IL292654A priority patent/IL292654A/en
Publication of WO2021088923A1 publication Critical patent/WO2021088923A1/zh
Priority to US17/663,075 priority patent/US20230062179A1/en
Priority to CONC2022/0007902A priority patent/CO2022007902A2/es
Priority to ZA2022/06200A priority patent/ZA202206200B/en

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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—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)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the present invention relates to the technical fields of genetic engineering and bioinformatics, in particular to a method and application for creating new genes in organisms without artificial DNA templates.
  • a complete gene expression cassette in an organism includes a promoter, 5'non-coding region (5' UTR), coding region (CDS) or non-coding RNA region (Non-coding RNA), 3'non-coding region ( 3'UTR), terminator and many other elements.
  • Non-coding RNA can perform its biological functions at the RNA level, including rRNA, tRNA, snRNA, snoRNA and microRNA.
  • the CDS region contains exons and introns.
  • RNA After the transcribed RNA is translated into a protein, the amino acids of different segments usually form different domains, and the specific domains determine the intracellular localization and function of the protein (such as nuclear localization signal, chloroplast guide peptide, mitochondrial guide peptide, DNA binding domain, transcription activation domain, enzyme catalytic center, etc.).
  • different segments also have different functions. When one or several elements of a gene change, a new gene will be formed, which may produce new functions. For example, a 1.7Mb chromosome fragment inversion event occurred upstream of the PpOFP1 gene in flat peach, forming a new promoter.
  • Gene editing tools represented by CRISPR/Cas9, etc. can efficiently and accurately generate double-strand breaks (DSB) at specific positions in the organism’s genome, and then repair the double-strand breaks (DSB) through the cell’s own non-homologous end repair or homologous recombination mechanisms. Strand breaks, resulting in site-directed mutations.
  • the current application of gene editing is mainly focused on the editing of the internal elements of a single gene.
  • the editing of the CDS exon region is the most common.
  • the result of editing on the exon usually causes the frameshift mutation of the gene, leading to the loss of the function of the gene.
  • gene editing tools such as CRISPR/Cas9 are also known as gene knockout (ie, gene destruction) tools.
  • the promoter, 5'UTR and other regions can also be knocked out to affect the expression of the gene.
  • These methods are all mutations in existing genes without generating new genes, so it is difficult to meet some production needs.
  • the existing gene editing technology is difficult to achieve up-regulation of gene expression, and it is also very difficult to achieve. It is difficult to change the subcellular localization of the protein or change the functional domain of the protein.
  • inserting a promoter or enhancer sequence at the upstream of an existing gene can change the expression pattern of the gene and generate new traits (Lu et al. 2020. Targeted, efficient sequence insertion and replacement in rice. Nat Biotechnol. DOI: 10.1038/s41587-020-0581-5), but this method requires the provision of exogenous DNA templates, which may be subject to strict regulatory procedures similar to genetically modified crops, which limits its application.
  • the present invention provides a form of generating two or more DNA double-strand breaks at the same time on the combination of specific positions in the organism's genome, and without providing an artificial DNA template. , Methods and applications of creating new genes in organisms.
  • One aspect of the present invention provides a method for creating new genes in organisms, including the following steps:
  • At least two different specific locations in the genome of an organism simultaneously produce DNA breaks, where the specific locations are genomic sites capable of dividing different genetic elements or different protein domains, and the DNA breaks are connected by non-homologous ends ( NHEJ) or homologous repair is connected to each other to generate a new combination of different gene elements or different protein domains that is different from the original genome sequence to form a new gene.
  • NHEJ non-homologous ends
  • homologous repair is connected to each other to generate a new combination of different gene elements or different protein domains that is different from the original genome sequence to form a new gene.
  • the "at least two different specific positions" can be located on the same chromosome or on different chromosomes.
  • the chromosome fragments generated after DNA breaks at two specific positions at the same time may be deleted, inverted or duplicated and doubled after the fragment is repaired; when located on different chromosomes, two specific positions After repair, the DNA breaks generated at these sites may be connected to each other to produce chromosome arm exchange events. These events can be identified and screened by PCR sequencing by designing specific primers.
  • the "at least two different specific positions” may be at least two specific positions on different genes, or at least two different specific positions on the same gene.
  • the transcription directions of the "at least two different genes" can be the same or different (opposite or relative).
  • the "DNA fragmentation" is achieved by delivering a nuclease with targeting properties into the cells of the organism to contact with a specific location of genomic DNA.
  • a nuclease with targeting properties into the cells of the organism to contact with a specific location of genomic DNA.
  • the "nuclease with targeting properties” includes Meganuclease, Zinc finger nuclease (ZFN), TALEN, CRISPR/Cas system.
  • the CRISPR/Cas system can generate more than two DNA double-strand breaks at different positions in the genome through two or more guide RNAs targeting different sequences; Zinc finger nuclease and TALEN systems can target two or more specific position sequences. Design ZFN protein or TALEN protein separately to produce two or more DNA double-strand breaks at the same time.
  • repair results such as deletion, inversion, and doubling may occur; when two breaks are located on two different chromosomes, there may be exchange of chromosomal arms.
  • the deletion, inversion, doubling, exchange, etc. of chromosome fragments at two DNA breaks can recombine different gene elements or protein domains to produce new functional genes.
  • the "nuclease with targeting properties" exists in the form of DNA.
  • the "nuclease with targeting properties" exists in the form of mRNA or protein, rather than in the form of DNA.
  • the method for delivering nucleases with targeting properties into cells is selected from 1) the method of PEG-mediated cell transfection; 2) the method of liposome-mediated cell transfection; 3) ) Electric shock transformation method; 4) Microinjection; 5) Gene gun bombardment; or 6) Agrobacterium-mediated transformation method.
  • the "gene element” includes the promoter of the gene, the 5'non-coding region (5'UTR), the coding region (CDS) or the non-coding RNA region (Non-coding RNA), and the 3'non-coding region (3'UTR). ) And terminator.
  • the combination of the different gene elements is a combination of the promoter of one of the two genes with different expression patterns and the CDS or non-coding RNA region of the other gene.
  • one of the combination of different genetic elements is a strong endogenous promoter in the organism, and the other is a coding region of HPPD, EPSPS, PPO or GH1 gene.
  • the combination of the different gene elements is a combination of the promoter to the 5'UTR region of one of the two genes with different expression patterns and the CDS or non-coding RNA region of the other gene.
  • the "different expression patterns" are differences in gene expression levels.
  • the "different expression pattern” is a tissue-specific difference in gene expression.
  • the "different expression patterns" are specific differences in the developmental stage of gene expression.
  • the combination of different genetic elements is a combination of adjacent genetic elements within the same gene.
  • protein domain refers to a DNA fragment corresponding to a specific functional domain of a protein; it includes, but is not limited to, nuclear localization signal, chloroplast guide peptide, mitochondrial guide peptide, phosphorylation site, methylation site, transmembrane Structural domain, DNA binding domain, transcription activation domain, receptor activation domain, enzyme catalytic center, etc.
  • the combination of the different protein domains is a combination of the localization signal region of one of the two protein coding genes with different subcellular localizations and the mature protein coding region of the other gene.
  • the "different subcellular locations” include, but are not limited to, nuclear location, cytoplasmic location, cell membrane location, chloroplast location, mitochondrial location, or endoplasmic reticulum membrane location.
  • the combination of different protein domains is a combination of two protein domains with different biological functions.
  • the "different biological functions" include, but are not limited to, recognition of specific DNA or RNA conserved sequences, activation of gene expression, binding of protein ligands, binding of small molecule signals, ion binding, or specific enzymatic reactions.
  • the combination of different protein domains is a combination of adjacent protein domains in the same gene.
  • the combination of the gene element and the protein domain is a combination of a protein domain in the same gene and an adjacent promoter, 5'UTR, 3'UTR or terminator.
  • the exchange of promoters of different genes can be achieved through the inversion of chromosome fragments: when the directions of two genes on the same chromosome are different, DNA breaks are generated at specific positions between the promoters and CDS of the two genes. When the region between the fractures is inverted, the promoters of these two genes can be exchanged, and two new genes are produced at both ends of the inverted chromosome segment.
  • the directions of the two genes are different, which can be divided into two situations. One is that the 5'end is inside, that is, the gene direction is opposite, and the other is that the 5'end is outside, that is, the gene direction is opposite.
  • the promoter of the gene is inverted, as shown in Scheme 1 of Fig. 2; when the direction of the gene is opposite, the CDS region of the gene is inverted, as shown in Scheme 1 of Fig. 4.
  • the inverted region can be very short, less than 10kb in length, with no other genes in between; the inverted region can also be very long, reaching 300kb-3Mb, containing hundreds of genes.
  • Another aspect of the present invention provides a method for creating new genes in organisms, including the following steps:
  • DNA at specific locations on at least two different genes is broken to achieve DNA transfer, doubling, inversion or deletion, where a specific element of an endogenous gene and another specific gene
  • NHEJ non-homologous ends
  • the invention also provides a new gene obtained by the method.
  • the new gene may have different promoters and therefore have the expression characteristics of different tissues or different intensities or different developmental stages, or have a new amino acid sequence.
  • the "new amino acid sequence" can be an integral fusion of two or more gene coding regions, a partial fusion of a coding region or a doubling of a partial protein coding region of the same gene.
  • the new gene is an endogenous HPPD, EPSPS, PPO or GH1 gene in a highly expressed organism.
  • the present invention also provides a DNA containing the gene.
  • the present invention also provides a protein or a biologically active fragment thereof encoded by the gene.
  • the present invention also provides a recombinant expression vector, which comprises the gene and a promoter operably linked to it.
  • the present invention also provides an expression cassette containing the gene.
  • the present invention further provides a host cell, which contains the expression cassette.
  • the host cell is a plant cell, animal cell or fungal cell.
  • the present invention additionally provides organisms regenerated using the host cell.
  • the present invention additionally provides an application of the gene in conferring or improving resistance/tolerance traits or growth advantage traits of organisms.
  • the present invention further provides a composition, which comprises:
  • the "different expression patterns" are differences in gene expression levels.
  • the "different expression patterns" are differences in tissue specificity of gene expression.
  • the "different expression patterns" are specific differences in the developmental stage of gene expression.
  • the "different subcellular locations” include, but are not limited to, nuclear location, cytoplasmic location, cell membrane location, chloroplast location, mitochondrial location, or endoplasmic reticulum membrane location.
  • the "different biological functions" include, but are not limited to, recognition of specific DNA or RNA conserved sequences, activation of gene expression, binding of protein ligands, binding of small molecule signals, ion binding, or specific enzymatic reactions.
  • the composition is fused in vivo.
  • the present invention also provides an editing method that does not rely on exogenous DNA donor fragments to increase the expression level of a target endogenous gene in an organism, which includes the following steps: separate the target endogenous gene and the optional Select a specific position between the promoter of the endogenous highly expressed gene and CDS to generate DNA breaks at the same time.
  • the DNA breaks are connected to each other by means of non-homologous end joining (NHEJ) or homologous repair to produce the coding region of the target endogenous gene In vivo fusion with an optional strong endogenous promoter to form a new highly expressed endogenous gene.
  • NHEJ non-homologous end joining
  • This method is named as the editing method of knocking high endogenous genes.
  • the target endogenous gene and the optional endogenous highly expressed gene are located on the same chromosome.
  • the target endogenous gene and the optional endogenous highly expressed gene are located on different chromosomes.
  • Another aspect of the present invention provides an editing method for knocking up endogenous HPPD gene expression in plants, which is to fuse the HPPD gene coding region with a plant endogenous strong promoter in vivo to form a new highly expressed plant endogenous HPPD gene . That is, a specific location is selected between the HPPD gene and the optional endogenous high-expressed gene promoter and CDS to generate DNA breaks at the same time. The DNA breaks are connected to each other through intracellular repair pathways to produce the HPPD gene coding region and the optional The in vivo fusion of the endogenous strong promoter forms a new highly expressed HPPD gene.
  • the strong promoter is preferably the promoter of the ubiquitin2 gene.
  • the invention also provides a highly expressed plant endogenous HPPD gene obtained by adopting the editing method.
  • the present invention also provides a highly expressed rice endogenous HPPD gene with a sequence selected from the following:
  • Another aspect of the present invention provides an editing method for knocking up endogenous EPSPS gene expression in plants, which is to fuse the coding region of the EPSPS gene with the plant endogenous strong promoter in vivo to form a new highly expressed plant endogenous EPSPS gene . That is, a specific position is selected between the EPSPS gene and the optional endogenous high-expressed gene promoter and CDS to generate DNA breaks at the same time. The DNA breaks are connected to each other through intracellular repair pathways to produce the EPSPS gene coding region and the optional The in vivo fusion of the endogenous strong promoter forms a new highly expressed EPSPS gene.
  • the strong promoter is preferably the promoter of the TKT gene.
  • the invention also provides a highly expressed plant endogenous EPSPS gene obtained by adopting the editing method.
  • the present invention also provides a highly expressed rice endogenous EPSPS gene with a sequence selected from the following:
  • Another aspect of the present invention provides an editing method for knocking up the expression of endogenous PPO (PPOX) genes in plants, which is to fuse the coding region of the PPO gene with the strong endogenous promoter of the plant to form a new high-expressing plant.
  • Source PPO gene That is, a specific position is selected between the promoter and CDS of the PPO gene and the optional endogenous high expression gene to generate DNA breaks at the same time.
  • the DNA breaks are connected to each other through the intracellular repair pathway to produce the PPO gene coding region and the optional
  • the in vivo fusion of the endogenous strong promoter forms a new highly expressed PPO gene.
  • the strong promoter is preferably the promoter of the CP12 gene.
  • the strong promoter is preferably the promoter of the ubiquitin10 gene.
  • the invention also provides a highly expressed plant endogenous PPO gene obtained by adopting the editing method.
  • the present invention also provides a highly expressed rice endogenous PPO gene with a sequence selected from the following:
  • SEQ ID NO: 15 SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24.
  • the present invention also provides a DNA containing the HPPD, EPSPS or PPO gene.
  • the present invention also provides a protein or a biologically active fragment thereof encoded by the HPPD, EPSPS or PPO gene.
  • the present invention additionally provides a recombinant expression vector, which comprises the HPPD, EPSPS or PPO gene, and a promoter operably linked to the HPPD, EPSPS or PPO gene.
  • the present invention further provides an expression cassette containing the HPPD, EPSPS or PPO gene.
  • the present invention also provides a host plant cell, which contains the expression cassette.
  • the present invention further provides plants regenerated using the host plant cells.
  • the present invention also provides a method for producing a plant with increased herbicide resistance or tolerance, which comprises regenerating the host plant cell into a plant and/or its derived progeny.
  • the plant with increased herbicide resistance or tolerance is a non-transgenic strain in which a plant regenerated from host plant cells is crossed with a wild-type plant through genetic isolation and exogenous transgenic components are removed.
  • the present invention also provides a herbicide-resistant rice, which comprises one of the above-mentioned high expression rice endogenous HPPD gene, high expression rice endogenous EPSPS gene, high expression rice endogenous PPO gene or any combination thereof.
  • the herbicide-resistant rice is non-transgenic.
  • the present invention additionally provides the application of the highly expressed plant endogenous HPPD, EPSPS or PPO gene to improve the resistance or tolerance of corresponding inhibitory herbicides in plant cells, plant tissues, plant parts or plants.
  • Another aspect of the present invention provides a method for controlling weeds in a plant cultivation site, wherein the plant includes the above-mentioned plant or a plant prepared by the above-mentioned method, and the method comprises applying to the cultivation site an effective weed control The amount of one or more HPPD, EPSPS or PPO inhibitory herbicides.
  • the inventors unexpectedly discovered that by designing gene editing target combinations near specific elements of the target gene, DNA double-strand breaks and spontaneous repair connections are caused, and differences can be achieved at the genome level without the need to provide foreign DNA templates.
  • the targeted combination of genetic elements may produce new functional genes from it, thereby greatly accelerating the production of new genes, and has great application potential in animal and plant breeding and gene function research.
  • genomic refers to all complements of genetic material (genes and non-coding sequences) present in each cell or virus or organelle of an organism, and/or from a parent as a unit (haploid) Inherited complete genome.
  • gene editing refers to strategies and techniques for targeted specific modification of any genetic information or genome of living organisms. Therefore, the term includes editing of gene coding regions, but also includes editing of regions other than gene coding regions of the genome. It also includes editing or modifying the nucleus (if present) and other genetic information of the cell.
  • CRISPR/Cas nuclease can be a CRISPR-based nuclease or a nucleic acid sequence encoding it, including but not limited to: 1) Cas9, including SpCas9, ScCas9, SaCas9, xCas9, VRER-Cas9, EQR-Cas9, SpG- Cas9, SpRY-Cas9, SpCas9-NG, NG-Cas9, NGA-Cas9 (VQR), etc., 2) Cas12, including LbCpf1, FnCpf1, AsCpf1, MAD7, etc., or any variant or derivative of the aforementioned CRISPR-based nuclease
  • the at least one CRISPR-based nuclease contains a mutation compared with the corresponding wild-type sequence, so that the obtained CRISPR-based nuclease recognizes a different PAM sequence.
  • CRISPR-based nuclease is any nuclease that has been identified in a naturally occurring CRISPR system, which is subsequently isolated from its natural background, and which has preferably been modified or combined into a recombinant construct of interest , Suitable as a tool for targeted genome engineering.
  • any CRISPR-based nuclease can be used and optionally reprogrammed or otherwise mutated to suit various embodiments of the invention.
  • CRISPR refers to a sequence-specific genetic manipulation technique that relies on a cluster of regularly spaced short palindrome repeats approach, which is different from RNA interference that regulates gene expression at the transcriptional level.
  • Cas9 nuclease and “Cas9” are used interchangeably herein, and refer to RNA comprising Cas9 protein or fragments thereof (for example, a protein containing the active DNA cleavage domain of Cas9 and/or the gRNA binding domain of Cas9) Guided nuclease.
  • Cas9 is a component of the CRISPR/Cas (clustered regularly spaced short palindrome repeats and related systems) genome editing system, which can target and cleave the DNA target sequence under the guidance of the guide RNA to form a DNA double-strand break (DSB) ).
  • CRISPR/Cas clustered regularly spaced short palindrome repeats and related systems
  • Cas protein or “Cas polypeptide” refers to a polypeptide encoded by a Cas (CRISPR-related) gene.
  • Cas protein includes Cas endonuclease.
  • the Cas protein can be a bacterial or archaeal protein.
  • the type I-III CRISPR Cas protein in this article usually originates from prokaryotes; the type I and type III Cas proteins can be derived from bacteria or archaea species, and the type II Cas protein (ie Cas9) can be derived from bacteria species. .
  • the Cas protein includes Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, C2c3 protein, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, Cas12a, Cas12b, or a combination or complex of these.
  • Cas9 variant or “Cas9 endonuclease variant” refers to a variant of the parent Cas9 endonuclease, wherein when associated with crRNA and tracRNA or with sgRNA, the Cas9 endonuclease variant retains the following Ability: Recognize, bind to all or part of the DNA target sequence and optionally unwind all or part of the DNA target sequence, nick all or part of the DNA target sequence, or cut all or part of the DNA target sequence.
  • Cas9 endonuclease variants include the Cas9 endonuclease variants described herein, wherein the Cas9 endonuclease variant is different from the parent Cas9 endonuclease in the following manner: the Cas9 endonuclease variant The body (when complexed with gRNA to form a polynucleotide-directed endonuclease complex capable of modifying the target site) and the parent Cas9 endonuclease (complexed with the same gRNA to form a complex capable of modifying the same target site) , Polynucleotide-guided endonuclease complexes) have at least one improved characteristic, such as, but not limited to, increased transformation efficiency, increased DNA editing efficiency, decreased off-target cleavage, or any combination thereof .
  • the Cas9 endonuclease variants described herein include variants that can bind to and nick double-stranded DNA target sites when associated with crRNA and tracrRNA or with sgRNA, while those in the parent Cas Dicer, when associated with crRNA and tracrRNA or with sgRNA, can bind at the target site and break (cleavage) the double strand.
  • RNA and gRNA are used interchangeably herein, and refer to a guide RNA sequence used to target a specific gene for correction using CRISPR technology, usually consisting of crRNA and tracrRNA molecules that are partially complementary to form a complex, wherein crRNA Contains a sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and direct the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence.
  • sgRNA single guide RNA
  • single guide RNA and “sgRNA” are used interchangeably herein and refer to the synthetic fusion of two RNA molecules, which contains a crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA) fusion.
  • CRISPR RNA crRNA
  • tracrRNA trans-activating CRISPR RNA
  • the sgRNA may include crRNA or crRNA fragments and tracrRNA or tracrRNA fragments of the type II CRISPR/Cas system that can form a complex with the type II Cas endonuclease, wherein the guide RNA/Cas endonuclease complex can combine the Cas nucleic acid
  • the endonuclease is directed to the DNA target site so that the Cas endonuclease can recognize, optionally bind to the DNA target site, and optionally make the DNA target site nick or cut (introduction of single-stranded or double-stranded breaks) DNA target site.
  • the guide RNA(s) and Cas9 can be delivered to the cell as a ribonucleoprotein (RNP) complex.
  • RNP is composed of purified Cas9 protein complexed with gRNA, and it is well known in the art that RNP can be effectively delivered to various types of cells, including but not limited to stem cells and immune cells (Addgene, Cambridge, MA, Mirus Bio LLC) , Madison, WI).
  • the protospacer adjacent motif (PAM) herein refers to the short nucleotide sequence adjacent to the (targeted) target sequence (prespacer) recognized by the gRNA/Cas endonuclease system. If the target DNA sequence is not adjacent to the appropriate PAM sequence, the Cas endonuclease may not be able to successfully recognize the target DNA sequence.
  • the sequence and length of the PAM herein can be different depending on the Cas protein or Cas protein complex used.
  • the PAM sequence can be any length, but typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 Or 20 nucleotides in length.
  • biology includes animals, plants, fungi, bacteria, and the like.
  • host cell includes plant cells, animal cells, fungal cells, bacterial cells, and the like.
  • plants should be understood as any differentiated multicellular organisms capable of photosynthesis, especially monocotyledonous or dicotyledonous plants, such as: (1) Food crops: Oryza spp., such as rice Oryza sativa, Oryza latifolia, Oryza sativa, Oryza glaberrima; Triticum spp., such as Triticum aestivum, T.
  • Oryza spp. such as rice Oryza sativa, Oryza latifolia, Oryza sativa, Oryza glaberrima
  • Triticum spp. such as Triticum aestivum, T.
  • Turgidumssp .durum Hordeum spp., such as barley (Hordeum vulgare), Arizona barley (Hordeum arizonicum); rye (Secale cereale); Oats (Avena spp.), such as oats (Avena sativa), wild oats (Avena fatua), Bizan oats (Avena byzantina), Avena fatua var.sativa, hybrid oats (Avena hybrida); Echinochloa (Echinochloa spp.), for example, pearl millet (Pennisetum glaucum), sorghum (two-color sorghum (Sorghum) bicolor), sorghum (Sorghum vulgare), triticale, maize or corn, millet, rice (rice), millet, millet, two-color sorghum (Sorghum bicolor), millet, buckwheat (
  • Rubus spp. pear, peach, apricot, plum, bayberry, lemon, kumquat, durian, orange, strawberry (straw berry), blueberry, cantaloupe, melon, date palm, walnut tree, cherry tree; (6) Root crops: cassava (Manihot spp.), sweet potato (Ipomoea batatas), taro (Colocasia esculenta), mustard, onion, water chestnut, sedge, yam; (7) Vegetable crops: Spinach (Spinacia) spp.), Phaseolus spp., Lettuce (Lactuca sativa), Momordica (Momordica spp), Parsley (Petroselinum crispum), Capsicum (Capsicum spp.), Solanum spp.
  • Lycopersicon spp. e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon, pyriforme
  • Macrotyloma spp. kale, Luffa acutangula, lentil, okra, onion, potato, artichoke , Asparagus (asparagus), broccoli (broccoli), Brussels sprouts (Brussels sprouts), cabbage (cabbage), carrot (carrot), cauliflower (cauliflower), celery (celery), collard greens (collar) d greens), summer squash (squash), winter melon (Benincasa hispida), Asparagus officinalis, Apium graveolens, Amaranthus (Amaranthus spp.), Allium (Allium spp.),
  • Poa spp. Poa pratensis (blue grass)
  • Agrostis spp. Agrostis palustris
  • Lolium spp. ryegrass
  • sheep Festuca spp. fescue
  • Zoysia spp. Zoysiajaponica
  • Cynodon spp. Bermuda grass, bermudagrass
  • side Stenotaphrum secunda tum St. Augustine grass
  • Tree crops Pine (Pinus spp.), Salix (Salix sp.), Maple (Acer spp.), Hibiscus (Hibiscus spp.), Eucalyptus (Eucalyptus sp.) ), Ginkgo biloba, Bambusa sp., Populus spp., Prosopis spp., Quercus spp., Phoenix spp.
  • Fagus spp. Ceiba pentandra, Cinnamomum spp., Corchorus sp., Phragmites australis, Physalis spp., Mountain Desmodium (Desmodium spp.), poplar, ivy, poplar, coral tree, ginkgo, oak, Ailanthus altissima, schima superba, holly, platanus, privet, yellow ash, larch, black wattle, masson pine , Simao pine, Yunnan pine, South Asian pine, Chinese pine, Korean pine, black walnut, lemon, sycamore, rose apple, Davidia involucrata, kapok, Javanese kapok, bauhinia, Bauhinia, rain tree, albizia, dragon tooth flower, thorn Tung, Magnolia, Cycad, Lagerstroemia, Conifers, Trees, Shrubs; (16) Nut crops: Brazil Chestnut (Berttan
  • the plant is selected from rice, corn, wheat, soybean, sunflower, sorghum, rape, alfalfa, cotton, barley, millet, sugarcane, tomato, tobacco, cassava, potato, sweet potato, cabbage, cabbage, Cucumber, rose, green dill, watermelon, melon, strawberry, blueberry, grape, apple, citrus, peach, pear, banana, etc.
  • plant includes the whole plant and any progeny, cells, tissues, or parts of the plant.
  • plant part includes any part of a plant, including, for example, but not limited to: seeds (including mature seeds, immature embryos without seed coats, and immature seeds); plant cuttings; plant cells; Plant cell culture; plant organs (e.g., pollen, embryos, flowers, fruits, buds, leaves, roots, stems, and related explants).
  • Plant tissues or plant organs can be seeds, callus, or any other plant cell population organized into structural or functional units.
  • regenerable cells in plant cells or tissue cultures can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silks, flowers, nuts, ears, cobs, shells , Or stem.
  • Plant parts include harvestable parts and parts that can be used to propagate offspring plants.
  • Plant parts that can be used for propagation include, for example, but not limited to: seeds; fruits; cuttings; seedlings; tubers; and rootstocks.
  • the harvestable part of the plant can be any useful part of the plant, including, for example, but not limited to: flowers; pollen; seedlings; tubers; leaves; stems; fruits; seeds; and roots.
  • Plant cells are the structural and physiological units of plants.
  • plant cells include protoplasts and protoplasts with partial cell walls.
  • Plant cells can be in the form of isolated single cells or cell aggregates (e.g., loose callus and cultured cells), and can be part of higher order tissue units (e.g., plant tissues, plant organs, and whole plants) . Therefore, a plant cell can be a protoplast, a cell that produces gametes, or a cell or collection of cells capable of regenerating a whole plant. Therefore, in the embodiments herein, a seed containing multiple plant cells and capable of regenerating into a whole plant is considered a "plant part".
  • protoplast refers to a plant cell whose cell wall is completely or partially removed and whose lipid bilayer membrane is exposed. Typically, protoplasts are isolated plant cells without cell walls, which have the potential to regenerate cell cultures or whole plants.
  • the "offspring" of a plant includes any subsequent generations of the plant.
  • inhibitory herbicide tolerance and “inhibitory herbicide resistance” can be used interchangeably, and both refer to tolerance to and resistance to inhibitory herbicides.
  • Improved tolerance to inhibitory herbicides and “Improved resistance to inhibitory herbicides” mean that the tolerance or resistance to the inhibitory herbicide is increased compared to plants containing wild-type genes.
  • wild type refers to a nucleic acid molecule or protein that can be found in nature.
  • the term "location" includes the site where the plant of the present invention is cultivated, such as soil, and also includes, for example, plant seeds, plant seedlings, and grown plants.
  • weed control effective amount refers to the amount of the herbicide sufficient to affect the growth or development of the target weed, for example, to prevent or inhibit the growth or development of the target weed, or to kill the weed.
  • the effective amount for controlling weeds does not significantly affect the growth and/or development of the plant seeds, plant seedlings or plants of the present invention. A person skilled in the art can determine such an effective amount for controlling weeds through routine experiments.
  • gene includes nucleic acid fragments expressing functional molecules (such as, but not limited to, specific proteins), including regulatory sequences before (5' non-coding sequences) and after (3' non-coding sequences) a coding sequence.
  • a DNA sequence that "encodes" a specific RNA is a DNA nucleic acid sequence that is transcribed into RNA.
  • DNA polynucleotides can encode RNA that is translated into protein (mRNA), or DNA polynucleotides can encode RNA that is not translated into protein (such as tRNA, rRNA, or DNA-targeting RNA; also known as “non-coding” RNA or " ncRNA”).
  • Polypeptide “peptide”, and “protein” are used interchangeably in the present invention and refer to a polymer of amino acid residues.
  • the term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally-occurring amino acids, as well as to naturally-occurring amino acid polymers.
  • the terms "polypeptide”, “peptide”, “amino acid sequence” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxyl And ADP-ribosylation.
  • a “biologically active fragment” refers to a fragment in which one or more amino acid residues are deleted from the N and/or C terminus of a protein while still retaining its functional activity.
  • polynucleotide and “nucleic acid” are used interchangeably and include DNA, RNA or hybrids thereof, which can be double-stranded or single-stranded.
  • nucleotide sequence and “nucleic acid sequence” both refer to the sequence of bases in DNA or RNA.
  • identity refers to sequence similarity to natural nucleic acid sequences. Identity can be evaluated with the naked eye or computer software. Using computer sequence alignment software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. "Partial sequence” means at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of a given sequence.
  • the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO 4 and 1 mM EDTA, and rinsing at 50°C, 2 ⁇ SSC, 0.1% SDS ; It can also be: 50 °C, hybridization in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1 mM EDTA, rinsing in 50 °C, 1 ⁇ SSC, 0.1% SDS; also: 50 °C, in 7% Hybridize in a mixed solution of SDS, 0.5M NaPO 4 and 1mM EDTA, rinse at 50°C, 0.5 ⁇ SSC, 0.1% SDS; also: 50°C, mix in 7% SDS, 0.5M NaPO 4 and 1mM EDTA Hybridization in solution, rinse at 50°C, 0.1 ⁇ SSC, 0.1% SDS; also: 50°C, hybridize in a mixed solution of 7%
  • expression cassette refers to a vector suitable for expression of a nucleotide sequence of interest in a plant, such as a recombinant vector. "Expression” refers to the production of a functional product.
  • the expression of a nucleotide sequence may refer to the transcription of the nucleotide sequence (such as transcription to generate mRNA or functional RNA) and/or the translation of RNA into a precursor or mature protein.
  • the "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be an RNA (such as mRNA) that can be translated.
  • the "expression construct" of the present invention may comprise 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 those normally occurring in nature.
  • the "highly expressed gene” in the present invention refers to a gene whose expression level is higher than that of a common gene in a specific tissue.
  • recombinant expression vector or "DNA construct” are used interchangeably herein and refer to a DNA molecule comprising a vector and at least one insert.
  • Recombinant expression vectors are usually produced for the purpose of expression and/or propagation of inserts or for the construction of other recombinant nucleotide sequences.
  • the insert may be operably or may be inoperably linked to a promoter sequence and may be operably or may be inoperably linked to a DNA regulatory sequence.
  • 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 the coding sequence, and affect the transcription, RNA processing, or processing of the related coding sequence. Stability or translated nucleotide sequence.
  • Plant expression control elements refer to nucleotide sequences that can control the transcription, RNA processing or stability, or translation of a nucleotide sequence of interest in plants.
  • Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
  • Promoter refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment.
  • the promoter is a promoter capable of controlling gene transcription in plant cells, regardless of whether it is derived from plant cells.
  • the promoter can be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.
  • strong promoter is a well-known and widely used term in the art. Many strong promoters are known in the art or can be identified through routine experiments.
  • the activity of the promoter is higher than that of a promoter operatively linked to the nucleic acid molecule to be overexpressed in a wild-type organism, for example, a promoter with higher activity than the promoter of an endogenous gene.
  • the activity of the strong promoter is about 2%, 5%, 8%, 10%, 20%, 30%, 40% higher than the activity of the promoter effectively linked to the nucleic acid molecule to be overexpressed in the wild-type organism.
  • tissue-specific promoter and “tissue-preferred promoter” are used interchangeably, and refer to mainly but not necessarily exclusively expressed in a tissue or organ, and can also be expressed in a specific cell or cell type Promoter.
  • tissue-preferred promoter refers to a promoter whose activity is determined by developmental events.
  • inducible promoters selectively express operably linked DNA sequences in response to endogenous or exogenous stimuli (environment, hormones, chemical signals, etc.).
  • operably linked refers to the connection of regulatory elements (for example, but not limited to, promoter sequences, transcription termination sequences, etc.) to nucleic acid sequences (for example, coding sequences or open reading frames) such that the nucleotides The transcription of the sequence is controlled and regulated by the transcription control element.
  • regulatory elements for example, but not limited to, promoter sequences, transcription termination sequences, etc.
  • nucleic acid sequences for example, coding sequences or open reading frames
  • "Introducing" nucleic acid molecules such as plasmids, linear nucleic acid fragments, RNA, etc.
  • proteins into plants refers to transforming plant cells with the nucleic acid or protein so that the nucleic acid or protein can function in the plant cell.
  • the "transformation” used in the present invention includes stable transformation and transient transformation.
  • “Stable transformation” refers to the introduction of an exogenous nucleotide sequence into the plant genome, resulting in the stable inheritance of the exogenous gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the plant and any successive generations thereof.
  • Transient transformation refers to the introduction of nucleic acid molecules or proteins into plant cells to perform functions without stable inheritance of foreign genes. In transient transformation, the foreign nucleic acid sequence is not integrated into the plant genome.
  • Changing the expression of endogenous genes in organisms includes two aspects: intensity and temporal and spatial characteristics. Intensity includes turn-up (knock-up), turn-down (knock-down), and/or zero (knock-out); temporal and spatial specificity includes time (growth period) specificity and space (tissue) specificity. It also includes inducibility.
  • changing the targeting of the protein for example, is changing the protein localized in the cytoplasm to the chloroplast localization or the nuclear localization.
  • the present invention comprehensively uses the following two different professional fields of information to develop a method for directly creating new genes in organisms, completely changing the conventional use of the original gene editing tools (ie knocking out genes), and realizing the creation of new genes.
  • One is the information in the field of gene editing, that is, when two or more different targets and Cas9 target the biological genome at the same time, there can be different situations such as deletion, inversion, doubling, or inverted doubling combination.
  • the second is information in the field of genomics, that is, the position and distance of different genes in the genome, and the specific positions of different elements in the gene (promoter, 5'UTR, coding region (CDS), different domain regions, terminator, etc.) , Direction, function, expression specific information of different genes, etc. Combining the information of these two different fields, cutting at specific positions of two or more different genes or two or more specific positions within a single gene (specific positions are provided by the field of genomics), combined by deleting, inverting, doubling, and inverting doubling Or the exchange of chromosome arms, etc. (the specific situation is provided by the field of gene editing), forming a new combination of different genetic elements or functional domains, specifically creating new genes in the organism.
  • These new genes created by the present invention are formed by the fusion or recombination of different elements of two or more genes under the action of the spontaneous DNA repair mechanism in the organism to change the expression intensity, temporal and spatial specificity, special functional domains, etc. of the original gene. , Does not contain exogenous transgenes or artificially synthesized genetic elements. Because the new gene fuses two or more different gene elements, this greatly expands the freedom of gene mutation, will produce more abundant and diverse functions, and has a wide range of application prospects. At the same time, these new genes are not linked to gene editing vectors. After removing the vector elements through genetic separation, non-transgenic biological materials containing new genes can be obtained for animal and plant breeding.
  • non-integrated transient editing can be performed by delivery of mRNA or ribonucleic acid protein complex (RNP) to create non-transgenic biological materials containing new genes.
  • RNP ribonucleic acid protein complex
  • This process is non-GMO, and the final edited material does not contain GMO.
  • these new genes can also be obtained through traditional breeding techniques (such as radiation or chemical mutagenesis, etc.).
  • screening with traditional techniques requires the creation of a huge number of random mutant libraries and consumes a lot of time and time.
  • the cost is to screen new functional genes, and the present invention can create new functional genes through bioinformatics analysis combined with gene editing technology, which greatly shortens the breeding cycle. According to the current approval process for gene editing organisms, the method provided by the present invention is not regulated in many countries.
  • the new gene creation technology of the present invention can be used to change the growth, development, resistance, yield and other traits of organisms, and has great application value.
  • the created new genes may have new regulatory elements (such as promoters) to change the original gene's expression intensity, spatiotemporal characteristics, etc., or have new amino acid sequences and thus have new functions.
  • changing the expression of specific genes can increase the resistance of crops to pests such as diseases, pests and weeds, and abiotic stresses such as drought, waterlogging, and salinity, and can also increase yield and improve quality.
  • changing the expression characteristics of growth hormone can significantly change the speed of its growth and development.
  • Figure 1 is a schematic diagram of the creation of a new HPPD gene in rice.
  • Figure 2 is a schematic diagram of the creation of a new EPSPS gene in rice.
  • Figure 3 is a schematic diagram of the creation of a new PPOX gene in Arabidopsis.
  • Figure 4 is a schematic diagram of the creation of a new PPOX gene in rice.
  • Figure 5 shows the sequencing results of the HPPD-duplication protocol for rice protoplast testing.
  • Figure 6 is a vector map of Agrobacterium rice transformation vector pQY2091.
  • Figure 7 shows the PCR electrophoresis results of new gene fragments detected in hygromycin resistant callus transformed with pQY2091.
  • the arrow points to the PCR band of the new gene fused with the UBI2 gene promoter and the HPPD coding region.
  • the numbers are the numbers of different callus samples.
  • M stands for DNA Marker, and the band size is 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 2500bp, 5000bp, 7500bp in order.
  • Figure 8 shows the results of PCR electrophoresis for detecting new gene fragments in the T0 generation seedlings of pQY2091 transformed rice.
  • the arrow points to the PCR band of the new gene fused with the UBI2 gene promoter and the HPPD coding region.
  • the numbers are the numbers of different T0 seedlings.
  • M stands for DNA Marker, and the band size is 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 2500bp, 5000bp, 7500bp in order.
  • Figure 9 shows the test results of the resistance of the QY2091T0 HPPD gene doubled strain to fentrazone.
  • the wild-type Jinjing 818 is on the left, and the HPPD doubling line is on the right.
  • FIG 10 shows the relative expression levels of HPPD and UBI2 genes in the QY2091T0 generation HPPD gene doubling line.
  • 818CK1 and 818CK3 are two wild-type Jinjing 818 control plants; 13M and 20M represent the main tiller leaf samples of QY2091-13 and QY2091-20T0 plants; 13L and 20L represent QY2091-13 and QY2091-20T0 plants for herbicide resistance Samples of secondary tiller leaves tested.
  • Figure 11 is a schematic diagram of possible genotypes of the QY2091T1 generation and binding sites of molecular detection primers.
  • Figure 12 shows the comparison of HPPD doubling sequencing results and predicted doubling sequences detected by QY2091-13 and QY2091-20.
  • Figure 13 shows the results of the herbicide resistance test at the seedling stage of the T1 generation of the QY2091HPPD doubling line.
  • Figure 14 is a schematic diagram of the possible editing event types of rice PPO1 gene chromosome fragment inversion and the binding sites of molecular detection primers.
  • Figure 15 shows the results of EPSPS-inversion detection and sequencing.
  • Figure 16 is a vector map of Agrobacterium rice transformation vector pQY2234.
  • Figure 17 shows the PCR electrophoresis results of new gene fragments for detection of hygromycin resistant callus transformed with pQY2234.
  • the arrow points to the new gene PCR band where the CP12 gene promoter and the PPO1 coding region are fused.
  • the numbers are the numbers of different callus samples.
  • M stands for DNA Marker, and the band size is 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 2500bp, 5000bp, 7500bp in order.
  • FIG 18 shows the resistance test result of the PPO1 gene inversion line 2081 of QY2234T0 generation.
  • the left flowerpot is the wild-type Huaidao No. 5 control
  • the right is the PPO1 inverted strain.
  • Figure 19 shows the relative expression levels of PPO1 and CP12 genes in the QY2234T0 generation PPO1 inverted strain.
  • H5CK1, H5CK2 are two wild-type Huaidao No. 5 control plants; 252M, 304M, 329M represent the primary tiller leaf samples of QY2234-252, QY2234-304, and QY2234-329T0 plants; 252L, 304L, 329L represent the secondary tiller leaf samples .
  • Figure 20 shows the comparison of the PPO1 inversion sequencing result with the predicted inversion sequence of Huaidao 5 background.
  • Figure 21 shows the comparison of the PPO1 inversion sequencing result with the predicted inversion sequence on the background of Jinjing 818.
  • Figure 22 shows the results of the herbicide resistance test at the seedling stage of the T1 generation of the QY2234PPO1 inverted strain.
  • Example 1 Editing method for knocking up endogenous HPPD gene expression in plants by inducing chromosome fragments to double-rice protoplast test
  • HPPD is a key enzyme in the pathway of plant synthesis of chlorophyll, and inhibiting the activity of HPPD enzyme will eventually lead to plant albino chlorosis and death.
  • Many herbicides such as mesotrione and fentrazone are inhibitors of HPPD as the target protein.
  • Increasing the expression level of endogenous HPPD genes in plants should help improve the tolerance of plants to these herbicides.
  • the rice HPPD gene (as shown in SEQ ID NO: 6, in which 1-1067 bp is a promoter, and the rest are expression regions) is located on rice chromosome 2.
  • UBI2 rice Ubiquitin2 gene
  • SEQ ID NO: 5 where 1-2107bp is the promoter and the rest are expression regions
  • UBI2 gene and HPPD The genes are in the same direction on the chromosomes.
  • the UBI2 gene expression intensity in rice leaves is the HPPD gene
  • the UBI2 gene promoter is a strong promoter with constitutive expression.
  • Scheme 1 generates double-strand breaks at the position between the respective promoters of the HPPD and UBI2 genes and the CDS region at the same time. After screening and identification, the event of doubling the region between the two breaks can be obtained.
  • UBI2 starts The coding regions of the gene and HPPD can be fused together to form a new gene.
  • a new gene in which the promoter of UBI2 and the coding region of HPPD are fused can also be formed by two consecutive inversions.
  • guide RNA1 and guide RNA2 are located between the HPPD gene promoter and the CDS region, close to the HPPD protein start codon, and guide RNA3 and guide RNA4 are located between the UBI2 gene promoter and the CDS region, and close to the UBI2 protein start codon.
  • pHUE411 vector https://www.addgene.org/62203/
  • the following primers are designed for the above targets and vector construction is performed.
  • the following two-target combination gene editing vector was constructed.
  • the pCBC-MT1T2 plasmid https://www.addgene.org/50593/
  • BsaI digests the backbone of the pHUE411 vector, cuts the gel to recover, and the target fragment is directly used for the ligation reaction after digestion.
  • T4DNA ligase to ligate the vector backbone and the target fragment, transform the ligation product into Trans5 ⁇ competent cells, pick out different monoclonals for sequencing, after the sequencing is correct, use the Cisco Express High Purity Plasmid Mini Extraction Kit to extract the plasmid to obtain the recombinant plasmid.
  • They are named pQY002065, pQY002066, pQY002067, pQY002068, as follows:
  • pHUE411-HPPD-sgRNA1+3 contains OsHPPD-guide RNA1, guide RNA3 combination
  • pHUE411-HPPD-sgRNA1+4 contains OsHPPD-guide RNA1, guide RNA4 combination
  • pHUE411-HPPD-sgRNA2+3 contains OsHPPD-guide RNA2, guide RNA3 combination
  • pHUE411-HPPD-sgRNA2+4 contains OsHPPD-guide RNA2, guide RNA4 combination
  • rice seedlings for protoplasts.
  • the variety used is Nipponbare.
  • the seeds are provided by the Weeds Department of the College of Plant Protection, China Agricultural University, and the laboratory is expanded.
  • Rice seeds are hulled first, and the hulled seeds are rinsed with 75% ethanol for 1 minute, treated with 5% (v/v) sodium hypochlorite for 20 minutes, and then washed with sterile water for more than 5 times, and then blow-dried in an ultra-clean table.
  • tissue culture bottle containing 1/2MS medium each bottle can contain 20 seeds.
  • Protoplasts can be prepared by incubating at 26°C and 12h light for about 10 days.
  • the protoplast concentration is about 2 ⁇ 10 6 cell/mL.
  • the protoplast DNA needs to be extracted.
  • the specific method is as follows: After centrifugation of the protoplasts, discard the supernatant, and add 500 ⁇ L of DNA extraction solution (recipe: CTAB 20g/L, NaCl 81.82g/L, 100mM Tris-HCl (pH 8.0), 20mM EDTA, 0.2% ⁇ -mercaptoethanol), shake and mix, and incubate in a water bath at 65°C for 1 hour; after cooling the sample after the water bath, add 500 ⁇ L of chloroform, invert and mix and centrifuge at 10,000rpm for 10 minutes; take 400 ⁇ L of supernatant In a new 1.5 ml centrifuge tube, add 1 ml of 70% (v/v) ethanol and precipitate at -20°C for 20 minutes; centrifuge at 12,000 rpm for 15 minutes to precipitate DNA. After the precipitate is dried, add 50 ⁇ L of ultrapure water to dissolve it. , Stored at
  • the length of the PCR product is between 300-1000 bp, among which primer8- The F+primer6-R combination is used to detect the fusion fragment at the middle joint after the chromosome fragment has been doubled, and the expected product length is 630bp.
  • the PCR reaction system is as follows:
  • Example 2 Agrobacterium-mediated transformation was used to create herbicide-resistant rice with chromosome fragments that doubled the expression of endogenous HPPD genes
  • Example 1 Construction of the knock-high editing vector: According to the results of the protoplast test in Example 1, select the high editing efficiency OsHPPD-guide RNA1: 5'GTGCTGGTTGCCTTGGCTGC3' and OsHPPD-guide RNA4: 5'GAAATAATCACCAAACAGAT3' dual target combination, refer to Example 1 Construction of the Agrobacterium transformation vector pQY2091, the vector backbone adopts pHUE411 and rice codon optimization is performed. The vector map is shown in FIG. 6.
  • Agrobacterium transformation Take 1 ⁇ g of rice knock-up editing vector pQY2091 plasmid and add it to 10 ⁇ l Agrobacterium EHA105 heat-shock competent cells (Angyu Biotech, Catalog No. G6040), place on ice for 5 minutes, immerse in liquid nitrogen for quick freezing for 5 minutes, and take out Heat shock at 37°C for 5 minutes, and finally place on ice for 5 minutes.
  • YEB liquid medium (recipe: yeast extract 1g/L, peptone 5g/L, beef extract 5g/L, sucrose 5g/L, magnesium sulfate 0.5g/L), culture in a shaker at 28°C, 200rpm 2 to 3 hours; centrifuge at 3500 rpm for 30 seconds to collect the bacteria, spread the collected bacteria on YEB (kanamycin 50mg/L + rifampicin 25mg/L) plates, and incubate at 28°C for 2 days in an incubator; pick Monoclonal to liquid culture medium, -80 °C preservation of bacteria.
  • Rice callus induction The transformation recipient rice varieties are Huaidao 5 and Jinjing 818, purchased from the seed market in Huaian City, Jiangsu province, and expanded in our laboratory. Take 800-2000 clean rice seeds and hull them, and then wash them with sterile water until the washed water becomes clear, and then disinfect with 70% alcohol for 30 seconds, then add 30 ml of 5% sodium hypochlorite and place it on a horizontal shaker at 50 rpm. After 20 minutes, wash with sterile water 5 times, place on sterile absorbent paper, air-dry the surface water of the seeds, inoculate them on the induction medium and cultivate the callus at 28°C.
  • Induction medium formula: 4.1g/L N6 powder+0.3g/L hydrolyzed casein+2.878g/L proline+2mg/L 2,4-D+3% sucrose+0.1g/L inositol+0.5g Glutamine + 0.45% vegetable gel, pH 5.8.
  • Agrobacterium infection of rice callus select Huaidao No. 5 or Jinjing 818 callus subcultured for 10 days with a diameter of 3 mm, and collect the callus into a 50 ml centrifuge tube; Pour the resuspension of Agrobacterium AAM with an OD600 of 0.2-0.3 into a centrifuge tube containing callus, and place it in a shaker at 28°C at 200 rpm for 20 minutes; when the infection is complete, discard the bacterial solution. Place the callus on sterile filter paper and air-dry for about 20 minutes, and place it on a common medium plate for co-cultivation.
  • the plate is covered with a sterile filter paper soaked in AAM liquid medium containing 100 ⁇ M acetosyringone; co-cultivation 3 After days, wash and remove Agrobacterium (first wash 5 times with sterile water, then wash with 500mg/L cephalosporin antibiotic for 20 minutes), and place it on 50mg/L hygromycin selection medium for selection and culture.
  • the difference between the selection process of conventional rice transformation is that the present invention uses specific primers for the fusion fragments generated after doubling the chromosome fragments to perform molecular identification of hygromycin-resistant callus during the callus selection and culture stage. Double the positive events, select callus containing new genes fused with different genetic elements for differentiation culture, and induce seedling emergence.
  • the specific steps are as follows:
  • Screening medium formula: 4.1g/L N6 powder+0.3g/L hydrolyzed casein+2.878g/L proline+2mg/L 2,4-D+3% sucrose+0.5g glutamine+30mg/L Hygromycin (HYG)+500mg/L cephalosporin (cef)+0.1g/L inositol+0.45% vegetable gel, pH5.8.
  • the callus with positive PCR identification is transferred to a new screening medium for the third round of screening and expansion; after the callus diameter is greater than 5 mm, the expanded callus is subjected to the second round of molecular identification using the 8F+6R primer pair Pick up two rounds of positive yellow-white calli with good growth status and transfer them to differentiation medium for differentiation. Seedlings of about 1 cm can be obtained after 3 to 4 weeks; transfer the differentiated seedlings to rooting medium for rooting Cultivation; after the rooting of the seedlings is subjected to the tempering treatment, they are moved to the pots filled with soil and placed in the greenhouse for cultivation.
  • Differentiation medium formula 4.42g/L MS powder+0.5g/L hydrolyzed casein+0.2mg/L NAA+2mg/L KT+3% sucrose+3% sorbitol+30mg/L hygromycin+0.1g/ L-inositol + 0.45% vegetable gel, pH 5.8.
  • Rooting medium formula 2.3g/L MS powder + 3% sucrose + 0.45% plant gel.
  • HPPD doubled seedlings T0 generation
  • HPPD inhibitor herbicide resistance test T0 generation
  • T0 generation identification doubling event positive transformed seedlings were transplanted into large plastic buckets in the greenhouse and cultivated to expand the T1 generation seeds. After the seedlings start tillering, the tillers are taken from the vigorously growing lines, and they are planted in the same pots with the same period tillers of the wild-type control varieties. After the plant height reaches about 20 cm, the herbicide resistance test is carried out.
  • the herbicide used is fentrazone produced by the company (CAS number: 1622908-18-2), and its field dose is usually 4 grams of active ingredients per mu (4g a.i./mu). In this experiment, the dosage of fentrazone was set to four gradients of 2g, 4g, 8g, and 32g, and a walk-in spray tower was used for spraying.
  • the resistance test results are shown in Figure 9. 5-7 days after application, the wild-type control rice seedlings began to show symptoms of albinism, while the HPPD doubling event each strain remained normal green. After 4 weeks of application, the wild-type rice seedlings were close to death, and all lines in the doubling event continued to remain green and grew normally. The test results show that the HPPD gene-doubled strain has a significantly improved tolerance to bifentrazone.
  • RNA concentration with a microprotein nucleic acid analyzer.
  • the better RNA OD260/OD280 is between 1.8-2.1, lower than 1.8 means serious protein contamination, and higher than 2.1 means serious RNA degradation.
  • the extracted total RNA is reverse transcribed to obtain cDNA.
  • the main process is: first determine the extracted total RNA concentration, and take the amount of 1-4 ⁇ g RNA for reverse transcriptase synthesis of cDNA.
  • cDNA is stored at -20°C.
  • RNA template solution Prepared on ice according to the following table, and place it on the PCR machine for denaturation and annealing reaction. This process is conducive to the denaturation of template RNA and the specific annealing of primers and templates, which can improve the efficiency of reverse transcription.
  • the UBQ5 gene of rice was selected as the internal reference gene, and the synthetic cDNA was used as the template to perform fluorescent quantitative PCR. Use the primers listed in Table 3 to prepare the reaction solution according to Table 4.
  • the Ct value of the target gene minus the Ct value of UBQ5 is ⁇ Ct , and then 2- ⁇ Ct is calculated, which represents the relative expression level of the target gene.
  • 818CK1 and 818CK3 are two wild-type Jinjing 818 Control plants; 13M and 20M represent the primary tiller leaf samples of QY2091-13 and QY2091-20T0 plants; 13L and 20L represent the secondary tiller leaf samples of QY2091-13 and QY2091-20T0 plants for herbicide resistance testing.
  • Example 1 and Example 2 using the chromosome fragment doubling technical solution of Example 1 and Example 2 can also introduce the promoters required for other endogenous genes that need to change the gene expression pattern to create new genes, which can be used in plants. Bacillus-mediated transformation creates new varieties with desired gene expression patterns.
  • the physical distance between the HPPD gene and the UBI2 gene in the wild-type rice genome is 338 kb, as shown in Scheme 1 of Figure 1, the chromosome length between the two chromosome fragments is doubled and the chromosome length increases by 338 kb.
  • UBI2 promoter is generated at the joint of the replicated fragment to drive HPPD
  • the new HPPD gene is highly expressed in the CDS region.
  • HPPD is a key enzyme in the chlorophyll synthesis pathway of plants, there may be chromosomal fragments doubled, deleted, inverted, and edited small fragments at the target site in the T0 generation plants with dual target editing. Mutations and other genotype mosaics. If the HPPD gene is destroyed, for example, the HPPD CDS region is deleted, the plant will have an albino phenotype. Different primer pairs are designed to detect possible genotypes by PCR.
  • Primer 8F TCTGTGTGAAGATTATTGCCACTAGTTC
  • Test 141-F CCCCTTCCCTCTAAAAATCAGAACAG
  • Primer 4R GGGATGCCCTCCTTATCTTGGATC
  • Primer 7R GTGTGGGGGAGTGGATGACAG
  • pg-35S-F TGACGTAAGGGATGACGCAC
  • Primer 8F+Primer 6R is used to detect the fusion fragment of UBI2 promoter and HPPD CDS after chromosome fragment doubling, and the product length is 630bp; Test 141-F+Primer 4R is used to detect chromosome fragments Delete the event, the product length is 222bp.
  • pg-Hyg-R1+pg-35S-F is used to detect the edited vector T-DNA fragment, the product length is 660bp.
  • Example 3 Refer to Example 1 for PCR reaction system, reaction procedure and gel electrophoresis detection.
  • the detection results of doubling and deletion events are shown in Table 7. It can be seen that chromosome fragment doubling events and deletion events are distributed in T1 generation plants, and the proportions of different lines are different. The proportion of doubling events in QY2091-13 is higher than 29/32 The 21/40 of QY2091-20 should be caused by the different chimeric ratios in the T0 generation plants. The test results indicate that the fusion gene produced by doubling is heritable.
  • Pg-Hyg-R1+pg-35S-F primers were used to detect the T-DNA fragment of the editing vector for the above-mentioned T1 seedlings.
  • the results of PCR electrophoresis on QY2091-20-17 and QY2091-13-7 were negative for the T-DNA fragment, and homozygous doubled.
  • the doubling homozygous non-transgenic line can be isolated.
  • the T1 generation of the QY2091 HPPD doubling strain was tested for herbicide resistance at the seedling stage. After surface disinfection of the T1 generation seeds, they germinated on 1/2MS medium with 1.2 ⁇ M fentrazone at 28°C for 16 hours Cultured in light and dark for 8 hours, with wild-type Jinjing 818 as a control.
  • the resistance test results are shown in Figure 13. After 10 days of light culture, the wild-type control rice seedlings showed symptoms of albinism, almost all albino, while the HPPD doubling event lines QY2091-7, 13, 20, 22 had yellowing seedlings and green seedlings Phenotypic separation. According to the aforementioned molecular test results, there is genotype segregation in the T1 generation, and albino seedlings appear without herbicide treatment, while the green seedlings continue to remain green and grow normally after adding 1.2 ⁇ M pyrenpyrin. The test results indicate that the HPPD gene-doubling strain has high resistance to bifentrazone and can be stably inherited to the T1 generation.
  • Example 4 Editing method for knocking up endogenous PPO gene expression by inducing chromosome fragment inversion-rice protoplast test
  • the rice PPO1 also known as PPOX1 gene (as shown in SEQ ID NO: 7, in which 1-1065 bp is the promoter, and the rest are the expression regions) is located on chromosome 1, and the calvin cycle protein CP12 gene (as shown in SEQ ID NO: 8, where 1-2088bp is a promoter, and the rest are expression regions), and its direction is opposite to the PPO1 gene.
  • the expression intensity of CP12 gene in rice leaves is the PPO1 gene
  • the CP12 gene promoter is a strong promoter highly expressed in leaves.
  • guide RNA1 and guide RNA2 are located between the PPO1 gene promoter and the CDS region, close to the PPO1 protein start codon, and guide RNA3 and guide RNA4 are located between the CP12 gene promoter and the CDS region, and close to the CP12 protein start codon.
  • the pCBC-MT1T2 plasmid (https://www.addgene.org/50593/) was used as a template to amplify sgRNA1+3, sgRNA1+4, sgRNA2+3, sgRNA2+4 double target fragments, and construct sgRNA Expression cassette.
  • BsaI digests the backbone of the pHUE411 vector, cuts the gel to recover, and the target fragment is directly used for the ligation reaction after digestion.
  • T4DNA ligase to ligate the vector backbone and the target fragment, transform the ligation product into Trans5 ⁇ competent cells, pick out different monoclonals for sequencing, after the sequencing is correct, use the Cisco Express High Purity Plasmid Mini Extraction Kit to extract the plasmid to obtain the recombinant plasmid.
  • They are named pQY002095, pQY002096, pQY002097, pQY002098, as follows:
  • pHUE411-PPO-sgRNA1+3 contains OsPPO-guide RNA1, guide RNA3 combination
  • pHUE411-PPO-sgRNA2+3 contains OsPPO-guide RNA2, guide RNA3 combination
  • pHUE411-PPO-sgRNA1+4 contains OsPPO-guide RNA1, guide RNA4 combination
  • pHUE411-PPO-sgRNA2+4 contains OsPPO-guide RNA2, guide RNA4 combination
  • the combination of PPO-R2 and CP-R2 is used to amplify the CP12 promoter produced on the right side of the chromosome fragment inverted to drive the new gene fragment of PPO1CDS, and the combination of PPO-F2 and CP-F2 is used to amplify the left side of the inverted gene fragment.
  • the PPO1 promoter drives the new gene segment of CP12CDS.
  • the possible genotypes of dual-target editing and the binding sites of molecular detection primers are shown in Figure 14.
  • PCR and sequencing results showed that the expected new gene driven by the CP12 promoter to express PPO1 was obtained from the transformation of rice protoplasts, and the fusion of the rice CP12 gene promoter and the PPO1 gene expression region can be detected in the genomic DNA of the transformed rice protoplasts.
  • the event shows that the scheme of forming a new PPO gene through the inversion of chromosome fragments is feasible, and it can create a new PPO gene driven by a strong promoter, which is defined as a PPO1 inversion event.
  • Example 5 Agrobacterium-mediated transformation was used to create herbicide-resistant rice with chromosome fragment inversion knocking out endogenous PPO gene expression
  • knock-up editing vector According to the results of protoplast testing, select the OsPPO-guide RNA1: 5'CCATGTCCGTCGCTGACGAG3' and OsPPO-guide RNA4: 5'CGGATTTCTGCGT-GTGATGT3' double target combination with high editing efficiency to construct Agrobacterium
  • the vector backbone adopts pHUE411 and the rice codon optimization is performed.
  • the vector map is shown in Figure 16.
  • the pQY2234 plasmid was used to transform rice callus according to the method described in step 2 of Example 2.
  • the recipient varieties were Huaidao No. 5 and Jinjing 818.
  • the callus selection stage two rounds of molecular identification were performed on hygromycin-resistant callus, and the callus with positive inversion event was selected for differentiation.
  • the stage of callus molecular detection we used the combination of PPO-R2 and CP-R2 to amplify the chromosome fragments generated on the right side after the inversion of the CP12 promoter to drive the new gene fragment of PPO1CDS as the positive standard for the inversion event.
  • the new CP12 gene will be considered after the callus differentiation and emergence.
  • FIG. 17 shows the PCR detection results of the callus numbered 192-259 of Jinjing 818.
  • the QY2234T0 generation identified inversion event positive transformed seedlings were transplanted into large plastic buckets in the greenhouse and cultivated to expand the T1 generation seeds. There are a large number of positive seedlings, so some T0 seedlings and wild-type control varieties with similar growth period and status are selected. After the plant height reaches about 20 cm, the herbicide resistance test is directly carried out.
  • the herbicide used is a highly effective PPO inhibitor herbicide produced by the company (Code 2081, see patent CN202010281666.4). In this experiment, the dosage was set to three gradients of 0.18, 0.4, and 0.6g ai/mu, and the walk-in spray tower was used for spraying.
  • the resistance test results are shown in Figure 18. 3-5 days after application, wild-type control rice seedlings began to wither from the tip of the leaf, necrotic spots appeared on the leaves, and the plants gradually withered, while most of the PPO1 inversion events maintained normal growth. , The leaves have no obvious phytotoxicity. In addition, some strains showed phytotoxicity, which may be caused by the polygenotypic mosaicism of the T0 generation strain editing event and the low expression of PPO1. After 2 weeks of application, wild-type rice seedlings died, and most of the plants in the inversion event continued to remain green and grew normally. The test results show that the PPO1 inverted strain can significantly improve the tolerance of the plant to 2081.
  • the increased resistance of the PPO1 gene inverted line to 2081 is caused by the fusion of the strong promoter of CP12 and the CDS of the PPO1 gene to increase the expression of PPO1. Therefore, the T0 generation lines QY2234-252 and QY2234 with a background of Huaidao 5 were selected. -304, QY2234-329 samples were taken from primary and secondary tillers to detect the expression levels of PPO1 and CP12 genes, and wild-type Huaidao No. 5 was used as a control. Refer to step 6 of Example 2 for the specific operation steps.
  • the rice UBQ5 gene is selected as the internal reference gene, and the fluorescent quantitative primers are as follows: 5'-3'
  • H5CK1 and H5CK2 are two wild-type Huaidao No. 5 control plants, 252M, 304M , 329M represents the primary tiller leaf samples of QY2234-252, QY2234-304, QY2234-329T0 plants, and 252L, 304L, and 329L represent secondary tiller leaf samples.
  • Table 8 The results are shown in Table 8 below:
  • the relative expression levels of PPO1 and CP12 of different strains are shown in Figure 19, and the results show that, unlike the doubling event in Example 2, the gene expression levels of the various strains of the inversion event are significantly different.
  • the expression level of CP12 between the two Huaidao No. 5 CK groups was very different, which may be caused by the different growth stage or seedling stage. Compared with the H5CK2 control group, the expression level of CP12 in the experimental group has a downward trend.
  • the expression levels of 252L and 329M PPO1 increased significantly, the expression levels of 304L, 329L PPO1 increased, and the expression levels of 252M, 304M PPO1 decreased.
  • Example 5 using the chromosome fragment inversion technical solution of Example 4 and Example 5, other endogenous genes that need to change the gene expression pattern can also be fused with the required promoters to create new genes, which can be used in plants.
  • Agrobacterium-mediated transformation creates new varieties with desired gene expression patterns.
  • the physical distance between the PPO1 gene and the CP12 gene of the wild-type rice genome is 911 kb.
  • the chromosome fragment between the two is inverted and the CP12 promoter is generated on the right side to drive the high expression PPO1 new gene in the PPO1CDS region, which may also occur Deletion of chromosome fragments.
  • molecular testing and herbicide resistance test on the T1 generation of the PPO1 inverted strain.
  • PPO-R2 AAGGCTGGAAGCTGTTGGG
  • PPO-F2 CGGACTTATTTCCCACCAGAA
  • pg-35S-F TGACGTAAGGGATGACGCAC
  • the PCR products were electrophoresed on a 1% agarose gel with a voltage of 180V for 10 minutes.
  • test results are shown in Table 9. A total of 48 strains were tested, of which 12 strains were homozygous inversions (2/7/11/16/26/36/37/40/41/44/46/47) and heterozygous inversions 21 plants (1/3/4/5/6/8/9/15/17/20/22/23/24/27/30/31/33/34/39/42/43), homozygous and non-inverted 15 plants (10/12/13/14/18/19/21/25/28/29/32/35/38/45/48). Homozygous inversion: heterozygous inversion: homozygous non-inversion ratio 1:1.75:1.25, approximately 1:2:1, the test results comply with Mendelian law of inheritance, indicating that the new PPO1 gene produced by inversion is heritable .
  • Pg-Hyg-R1+pg-35S-F primers were used to detect the T-DNA fragment of the editing vector for the above-mentioned T1 seedlings.
  • the results of electrophoresis on 16 and 41 were negative for the T-DNA fragment and homozygous inversion. Inversion events can be seen in the T1 generation. It is possible to isolate non-transgenic lines that are homozygous for the inversion.
  • the detection of the genotype of the inversion event focuses on the editing event of the new PPO gene on the right side, and the mutation type with the complete protein coding frame of the PPO1 gene is retained.
  • the editing event at the left CP12 locus does not affect the normal growth of plants through greenhouse and field phenotypic observation. It can be retained.
  • the editing event genotypes detected in the inversion event-positive line are listed below. Seamless means that the sequence of the fusion fragment after inversion is exactly the same as predicted.
  • the QY2234 with Huaidao 5 as the background is successfully inverted.
  • the genotypes are as follows:
  • SEQ ID NO: 20 See SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO for the sequencing results of the above different new PPO1 gene CP12 promoter and PPO1 coding region fusion fragments. :25, SEQ ID NO: 26.
  • the T1 generation of the QY2234/H5-851 PPO1 inverted line was tested for herbicide resistance at the seedling stage.
  • the wild-type Huaidao No. 5 was used as a control, and the seeds of the inverted line T1 were planted at the same time, and the seedlings reached 15 After centimeters, the 2081 treatment was sprayed, and the drug concentration was set to four gradients of 0.3, 0.6, 0.9, 1.2 g ai/mu, and the culture condition was 28°C, 16 hours of light, and 8 hours of darkness.
  • the control has completely died, and some of the T1 seedlings can keep green and continue to grow.
  • the test results indicate that the resistance of the PPO1 gene inverted strain to 2081 can be stably inherited to the T1 generation.
  • Example 7 Editing method for knocking up endogenous EPSPS gene expression in plants
  • EPSPS is a key enzyme in the pathway of aromatic amino acid synthesis in plants and the target of the biocidal herbicide glyphosate.
  • High expression of EPSPS gene can endow plants with resistance to glyphosate.
  • the EPSPS gene (as shown in SEQ ID NO: 4, where 1-1897 bp is the promoter, and the rest are the expression regions) is located on rice chromosome 6, and its upstream gene is transketolase (TKT, as shown in SEQ ID NO: 3 As shown, 1-2091bp is the promoter, and the rest is the expression region), the direction is opposite to the EPSPS gene.
  • the expression intensity of TKT gene in leaves is 20-50 times that of EPSPS gene.
  • the region between the two fractures can be inverted (Scheme 1), or inverted doubling (Scheme 2) Situation.
  • the promoter of the EPSPS gene will be replaced with the promoter of the TKT gene to increase the expression of the EPSPS gene and obtain resistance to glyphosate.
  • the schemes 3, 4, and 5 shown in Figure 2 can also create new EPSPS genes driven by the TKT gene promoter.
  • the gene structure of EPSPS and TKT adjacent and opposite in direction is conserved in monocotyledonous plants (Table 10), and in dicotyledonous plants, the two are also adjacent in the same direction; therefore, this method has versatility in plants.
  • pHUE411 is used as the backbone and the following targets are targeted:
  • the test results showed that the expected inversion event was obtained.
  • the pQY002062 vector transformed protoplast detection inverted sequencing results are shown in SEQ ID NO: 11; the pQY002062 vector transformed protoplast detection deleted sequencing results are shown in SEQ ID No: 12; pQY002093 vector transformed protoplast detection inverted sequencing results are shown in SEQ ID NO: 13; pQY002093 vector transformed protoplast detection deleted sequencing results are shown in SEQ ID NO: 14.
  • Protoporphyrinogen oxidase is one of the main targets of herbicides. High expression of plant endogenous PPO can significantly increase the resistance to PPO inhibitor herbicides.
  • the Arabidopsis PPO gene (as shown in SEQ ID NO:1, where 1-2058bp is the promoter, and the rest are the expression regions) is located on chromosome 4, and the ubiquitin10 gene (as shown in SEQ ID NO: 2 is 1.9M downstream). As shown, 1-2078bp is a promoter, and the rest is an expression region), and the direction is the same as the PPO gene.
  • double-strand breaks are generated at the position between the promoters of PPO and ubiquitin10 genes and the CDS region at the same time. After screening, the double-stranded event between the two breaks can be obtained, namely the promoter of ubiquitin10 and PPO The coding region of the new gene fused together.
  • Scheme 2 shown in Figure 1 it is also possible to create a new gene in which the promoter of ubiquitin10 and the coding region of PPO are fused together.
  • pHEE401E as the backbone (https://www.addgene.org/71287/) to target the following targets:
  • the constructed recombinant plasmid is transformed into Agrobacterium GV3101 competent cells to obtain recombinant Agrobacterium.
  • the inflorescence is luxuriant, and there is no stress response.
  • the first transformation can be carried out with a height of 20cm. If the soil is dry, water properly. The day before transformation, use scissors to subtract the siliques that have grown.
  • the second conversion can be carried out in the same way.
  • the seeds After the seeds are mature, they can be harvested. After harvesting, they can be dried in an oven at 37°C for about a week.
  • the seeds were treated with disinfectant for 5 minutes, washed 5 times with ddH 2 O, and then evenly spread on MS screening medium (containing 30 ⁇ g/ml Hyg, 100 ⁇ g/ml Cef), and placed the medium in a light incubator (temperature 22°C). , 16 hours of light, 8 hours of darkness, light intensity 100-150 ⁇ mol/m 2 /s, humidity 75%), one week later, the positive seedlings are selected and transplanted to the soil.
  • MS screening medium containing 30 ⁇ g/ml Hyg, 100 ⁇ g/ml Cef
  • the target fragments were amplified with detection primers, and 5 ⁇ L of the amplified product was extracted and detected by 1% agarose gel electrophoresis, and then imaged by a gel imager. The remaining products are directly sequenced by the sequencing company.
  • GH Growth hormone
  • the GH1 gene is the growth hormone gene in zebrafish.
  • suitable promoters continuous expression, suitable strength, suitable tissue specificity
  • zebrafish are fused together in vivo through deletion, inversion, doubling, inversion doubling, chromosome transfer, etc., to create a fast-growing fish Variety.

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Abstract

一种在无人工DNA模板的前提下，在生物体内创制新基因的方法及应用。所述方法是通过在生物体基因组中至少两个不同的特定位置上同时产生DNA断裂，其中所述特定位置是能够分割不同基因元件或不同蛋白结构域的基因组位点，所述DNA断裂通过非同源末端连接(NHEJ)或同源修复的方式互相连接，产生所述不同基因元件或不同蛋白结构域之间不同于原始基因组序列的新组合，形成新基因。新基因能够改变生物体的生长、发育、抗性、产量等性状，具有重大应用价值。

Description

在生物体内创制新基因的方法及应用 技术领域
本发明涉及基因工程和生物信息学技术领域，具体而言，涉及一种在无人工DNA模板的前提下，在生物体内创制新基因的方法及应用。
背景技术
一般来讲，生物体内一个完整的基因表达盒包括启动子、5’非编码区(5’UTR)、编码区(CDS)或非编码RNA区(Non-coding RNA)、3’非编码区(3’UTR)、终止子等多个元件。非编码RNA在RNA水平上就能行使其生物学功能，包括rRNA、tRNA、snRNA、snoRNA和microRNA。CDS区域包含外显子和内含子，转录出来的RNA翻译成蛋白之后，不同区段的氨基酸通常会形成不同的结构域(domain)，特异的结构域决定了蛋白的细胞内定位和功能(如核定位信号、叶绿体导肽、线粒体导肽、DNA结合结构域、转录激活结构域、酶催化中心等)。对于非编码RNA来讲，不同的区段也具有不同的功能。当基因的一个或几个元件发生变化，就会形成一个新的基因，可能产生新的功能。例如蟠桃中的PpOFP1基因上游发生一个1.7Mb的染色体片段倒位事件，形成新的启动子，在果实发育S2期，扁桃果实中PpOFP1表达量显著高于圆桃，可以抑制桃子果实的纵向发育，从而形成蟠桃扁平的表型(Zhou et al.2018.A 1.7-Mb chromosomal inversion downstream of a PpOFP1 gene is responsible for flat fruit shape in peach.Plant Biotechnol.J.DOI:10.1111/pbi.13455)。
生物基因组中自然产生新基因需要漫长的进化过程，根据研究，新基因产生的分子机制包括外显子重排、基因复制、逆转座、可移动元件(转座子、反转座子)的整合、基因水平转移、基因融合分裂、从头起源等多种机制，通过新基因起源和功能进化在自然选择作用下在物种中得以保留。已在果蝇、拟南芥、灵长类动物中鉴定出的比较年轻的新基因经计算距今也已有数十万到数百万年历史(Long et al.2012.The origin and evolution of new genes.Methods Mol Biol.DOI:10.1007/978-1-61779-585-5_7)。因此在基因工程和生物育种领域，以植物为例，如果希望在植物中引入新的基因(即便是该新基因的所有基因元件均是来自于该物种自身的不同基因)，目前只能通过转基因技术来实现。即在体外将来自不同基因的元件组装在一起，形成新的基因，再将其通过转基因技术转入植物。其特征是新基因的组装需要在体外进行，产生的是转基因作物。
以CRISPR/Cas9等为代表的基因编辑工具，能够高效且精准地在生物体基因组的特定位置产生双链断裂(DSB)，随后通过细胞自身的非同源末端修复或同源重组的机制修复双链断裂，产生定点突变。基因编辑目前的应用主要集中在针对单个基因内部元件的编辑，其中对CDS外显子区域的编辑最为普遍，在外显子上编辑的结果通常会造成基因的移码突变，导致该基因功能的丧失，为此，CRISPR/Cas9等基因编辑工具，也被称为基因敲除(即破坏基因)工具。除了针对CDS区域，对启动子、5’UTR等区域也可以通过敲除的方式，影响该基因的表达量。这些方法都是对现有基因进行突变，没有产生新的基因，因此难以满足一些生产上的需求，例如对多数基因而言，现有的基因编辑技术很难实现基因表达量的上调，也很难定向改变蛋白的亚细胞定位或改变蛋白的功能结构域。也有文献报道在现有基因的上游定点插入一段启动子或增强子的序列能够改变基因的表达模式从而产生新的性状(Lu et al.2020.Targeted,efficient sequence insertion and replacement in rice.Nat Biotechnol.DOI: 10.1038/s41587-020-0581-5)，但这种方法需要提供外源DNA模板，可能受到类似转基因作物的严格监管程序，限制了其应用。
发明简述
为解决现有技术中存在的上述问题，本发明提供了一种仅通过在生物体基因组特定位置的组合上同时产生两个以上DNA双链断裂的形式，并且在不提供人工DNA模板的情况下，在生物体内创制新基因的方法和应用。
本发明一方面提供了一种在生物体内创制新基因的方法，包括以下步骤：
在生物体基因组中至少两个不同的特定位置上同时产生DNA断裂，其中所述特定位置是能够分割不同基因元件或不同蛋白结构域的基因组位点，所述DNA断裂通过非同源末端连接(NHEJ)或同源修复的方式互相连接，产生所述不同基因元件或不同蛋白结构域之间不同于原始基因组序列的新组合，形成新基因。
在一具体实施方式中，所述的“至少两个不同的特定位置”，可以位于同一条染色体上，也可以位于不同染色体上。当位于同一条染色体上时，两个特定位置处同时发生DNA断裂后产生的染色体片段经修复后可能发生该片段的删除、倒置或复制加倍等事件；当位于不同染色体上时，两个特定位置处产生的DNA断裂经修复后可能相互连接产生染色体臂的交换事件。这些事件可以通过设计特异的引物进行PCR测序鉴定筛选。
在一具体实施方式中，所述的“至少两个不同的特定位置”，可以在至少两个不同基因上的特定位置，也可以在同一基因上的至少两个不同的特定位置。
在一具体实施方式中，所述的“至少两个不同基因”的转录方向可以相同，也可以不同(相反或相对)。
在一具体实施方式中，所述的“DNA断裂”，是通过将具有靶向特性的核酸酶递送到生物体细胞内与基因组DNA特定位置接触实现的。此种DNA断裂与通过传统技术手段(如辐射或化学诱变)产生的DNA断裂无本质差异。
在一具体实施方式中，所述的“具有靶向特性的核酸酶”包括Meganuclease、Zinc finger nuclease(ZFN)、TALEN、CRISPR/Cas系统。
其中，CRISPR/Cas系统可通过两个以上靶向不同序列的引导RNA在基因组不同的位置上，同时产生两处以上DNA双链断裂；Zinc finger nuclease、TALEN系统可以通过针对两个以上特定位置序列分别设计ZFN蛋白或TALEN蛋白，同时产生两处以上DNA双链断裂。当两个断裂位于同一条染色体上时，可能会发生删除、倒置、加倍等修复结果；当两个断裂位于两条不同的染色体上时，则有可能出现染色体臂的交换。两个DNA断裂处染色体片段的删除、倒置、加倍、交换等能够对不同的基因元件或蛋白结构域重新组合产生新的功能基因。
在一具体实施方式中，所述“具有靶向特性的核酸酶”以DNA形式存在。
在另一具体实施方式中，所述“具有靶向特性的核酸酶”以mRNA或蛋白形式存在，而非DNA形式。
在一具体实施方式中，将具有靶向特性的核酸酶递送到细胞内的方法选自1)PEG介导的细胞转染的方法；2)脂质体介导的细胞转染的方法；3)电击转化的方法；4)显微注射；5)基因枪轰击；或6)农杆菌介导的转化方法。
所述“基因元件”，包括基因的启动子、5’非编码区(5’UTR)、编码区(CDS)或非编码RNA区(Non-coding RNA)、3’非编码区(3’UTR)和终止子。
在一具体实施方式中，所述不同基因元件的组合为两个具有不同表达模式的基因其中之一的启动子和另一基因的CDS或非编码RNA区的组合。
在一具体实施方式中，所述不同基因元件的组合中一个为生物内源强启动子，另外一个为HPPD、EPSPS、PPO或GH1基因编码区。
在另一具体实施方式中，所述不同基因元件的组合为两个具有不同表达模式的基因其中之一的启动子至5’UTR区域和另一基因的CDS或非编码RNA区的组合。
在一具体实施方式中，所述“不同表达模式”为基因表达水平强弱的差异。
在另一具体实施方式中，所述“不同表达模式”为基因表达组织特异性的差异。
在另一具体实施方式中，所述“不同表达模式”为基因表达发育时期特异性的差异。
在另一具体实施方式中，所述不同基因元件的组合为同一基因内相邻基因元件的组合。
所述“蛋白结构域”是指对应于蛋白质特定功能结构域的DNA片段；其包括但不限于核定位信号、叶绿体导肽、线粒体导肽、磷酸化位点、甲基化位点、跨膜结构域、DNA结合结构域、转录激活结构域、受体激活结构域、酶催化中心等。
在一具体实施方式中，所述不同蛋白结构域的组合为两个具有不同亚细胞定位的蛋白编码基因其中之一的定位信号区域和另一基因的成熟蛋白编码区域的组合。
在一具体实施方式中，所述“不同亚细胞定位”包括但不限于核定位、胞质定位、细胞膜定位、叶绿体定位、线粒体定位或内质网膜定位。
在另一具体实施方式中，所述不同蛋白结构域的组合为两种不同生物学功能的蛋白结构域的组合。
在一具体实施方式中，所述“不同生物学功能”包括但不限于识别特异DNA或RNA保守序列、激活基因表达、结合蛋白配体、结合小分子信号、离子结合或特异的酶促反应。
在另一具体实施方式中，所述不同蛋白结构域的组合为同一基因内相邻蛋白结构域的组合。
在另一具体实施方式中，所述基因元件和蛋白结构域的组合为同一基因内蛋白结构域与相邻的启动子、5’UTR、3’UTR或终止子的组合。
具体而言，可通过染色体片段倒置实现不同基因启动子的互换：当位于同一条染色体的两个基因的方向不同时，分别在两个基因的启动子和CDS之间选择特定位置产生DNA断裂，断口之间的区域发生倒置，即可实现这两个基因启动子的互换，在倒置染色体片段两端产生了两个新的基因。两个基因的方向不同，分为两种情形，一是5’端在内侧，即基因方向相反，二是5’端在外侧，即基因方向相对。基因方向相反的情况下，倒置的是基因的启动子，如图2方案1所示；基因方向相对的情况下，倒置的是基因的CDS区，如图4方案1所示。倒置的区域可以很短，长度小于10kb，之间没有其它基因；倒置的区域也可以很长，达到300kb-3Mb，包含上百个基因。
也可以通过染色体片段加倍创制新的基因：当位于同一条染色体的两个基因的方向相同时，分别在两个基因的启动子和CDS之间选择特定位置产生DNA断裂，断口之间的区域发生复制加倍，会在加倍片段的连接处形成由下游基因启动子和上游基因CDS区融合而成的新 基因，如图1方案1和图3所示。加倍区域的长度在500bp-5Mb范围内，可以很短，之间没有其它基因，也可以很长，包含上百个基因。这种方法虽然会给原来的两个基因的启动子和CDS区域间造成点突变，但是这种小范围的点突变一般对基因表达特性影响不大，而启动子替换创造出来的新基因则具有新的表达特性。或者在位于同一基因的蛋白结构域两侧选择特定位置产生DNA断裂，断口之间的区域发生复制加倍，会形成特异功能结构域加倍的新基因。
本发明另一方面提供了一种在生物体内创制新基因的方法，包括以下步骤：
通过在生物体基因组或染色体水平上，至少两个不同的基因上特定位置的DNA产生断裂，实现DNA的转移、加倍、倒置或删除，其中，特定的一个内源基因的元件和另外一个特定基因元件，通过非同源末端(NHEJ)或同源修复的方式连接在一起，形成新基因。
本发明还提供一种采用所述方法获得的新基因。
所述的新基因与原基因相比，或具有不同启动子并因此具有了不同组织或不同强度或不同发育时期的表达特性，或具有了新的氨基酸序列。
所述的“新的氨基酸序列”，即可以是两个以上基因编码区的整体融合，也可以是编码区的部分融合或同一基因部分蛋白编码区的加倍。
在一具体实施方式中，所述新基因为高表达生物内源HPPD、EPSPS、PPO或GH1基因。
本发明另外提供一种包含所述基因的DNA。
本发明另外提供一种利用包含所述基因编码的蛋白或其生物活性片段。
本发明另外提供一种重组表达载体，其包含所述的基因，以及与之可操作地连接的启动子。
本发明另外提供一种包含所述基因的表达盒。
本发明另外提供一种宿主细胞，其中包含有所述的表达盒。优选地，所述宿主细胞是植物细胞、动物细胞或真菌细胞。
本发明另外提供采用所述宿主细胞再生成的生物。
本发明另外提供一种所述的基因在赋予或提高生物体抗性/耐受性性状或生长优势性状上的应用。
本发明另外提供一种组合物，其包含：
(a)两个具有不同表达模式的基因其中之一的启动子和另一基因的编码区或非编码RNA区；
(b)两个具有不同表达模式的基因其中之一的启动子至5’非编码区和另一基因的编码区或非编码RNA区；
(c)同一基因内相邻基因元件；
(d)两个具有不同亚细胞定位的蛋白编码基因其中之一的定位信号区域和另一基因的成熟蛋白编码区域；
(e)两种不同生物学功能的蛋白结构域；
(f)同一基因内相邻蛋白结构域；或者，
(g)同一基因内蛋白结构域与相邻的启动子、5’非编码区、3’非编码区或终止子。
在一具体实施方式中，所述“不同表达模式”为基因表达水平强弱的差异。
在一另具体实施方式中，所述“不同表达模式”为基因表达组织特异性的差异。
在另一具体实施方式中，所述“不同表达模式”为基因表达发育时期特异性的差异。
在一具体实施方式中，所述“不同亚细胞定位”包括但不限于核定位、胞质定位、细胞膜定位、叶绿体定位、线粒体定位或内质网膜定位。
在一具体实施方式中，所述“不同生物学功能”包括但不限于识别特异DNA或RNA保守序列、激活基因表达、结合蛋白配体、结合小分子信号、离子结合或特异的酶促反应。
在一具体实施方式中，所述的组合物在体内融合。
特别的，本发明还提供了一种不依赖于外源DNA供体片段，在生物体中提高目标内源基因表达水平的编辑方法，包括以下步骤：分别在所述目标内源基因与可选的内源高表达基因的启动子和CDS之间选择特定位置同时产生DNA断裂，所述DNA断裂通过非同源末端连接(NHEJ)或同源修复的方式互相连接，产生目标内源基因编码区与可选的内源强启动子的体内融合，形成新的高表达内源基因。此种方法命名为敲高内源基因的编辑方法。
在一具体实施方式中，所述目标内源基因与可选的内源高表达基因位于同一染色体上。
在另一具体实施方式中，所述目标内源基因与可选的内源高表达基因位于不同染色体上。
本发明另一方面提供了一种在植物中敲高内源HPPD基因表达的编辑方法，其是将HPPD基因编码区与植物内源强启动子体内融合，形成新的高表达植物内源HPPD基因。即分别在HPPD基因与可选的内源高表达基因的启动子和CDS之间选择特定位置同时产生DNA断裂，所述DNA断裂通过细胞内修复途径互相连接，产生HPPD基因编码区与可选的内源强启动子的体内融合，形成新的高表达HPPD基因。在水稻中，强启动子优选ubiquitin2基因的启动子。
本发明还提供了一种采用所述编辑方法获得的高表达植物内源HPPD基因。
本发明还提供了一种高表达水稻内源HPPD基因具有选自下述的序列：
(1)如SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:18或SEQ ID NO:19所示的核酸序列或其部分序列或其互补序列；
(2)与(1)限定的任一序列具有至少75％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％以上同一性；或
(3)在严谨条件下与(1)或(2)所示序列杂交的核酸序列。
本发明另一方面提供了一种在植物中敲高内源EPSPS基因表达的编辑方法，其是将EPSPS基因编码区与植物内源强启动子体内融合，形成新的高表达植物内源EPSPS基因。即分别在EPSPS基因与可选的内源高表达基因的启动子和CDS之间选择特定位置同时产生DNA断裂，所述DNA断裂通过细胞内修复途径互相连接，产生EPSPS基因编码区与可选的内源强启动子的体内融合，形成新的高表达EPSPS基因。在水稻中，强启动子优选TKT基因的启动子。
本发明还提供了一种采用所述编辑方法获得的高表达植物内源EPSPS基因。
本发明还提供了一种高表达水稻内源EPSPS基因具有选自下述的序列：
(1)如SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14所示的核酸序列或其部分序列或其互补序列；
(2)与(1)限定的任一序列具有至少75％、至少80％、至少85％、至少90％、至少95％、 至少98％或至少99％以上同一性；或
(3)在严谨条件下与(1)或(2)所示序列杂交的核酸序列。
本发明另一方面提供了一种在植物中敲高内源PPO(PPOX)基因表达的编辑方法，其是将PPO基因编码区与植物内源强启动子体内融合，形成新的高表达植物内源PPO基因。即分别在PPO基因与可选的内源高表达基因的启动子和CDS之间选择特定位置同时产生DNA断裂，所述DNA断裂通过细胞内修复途径互相连接，产生PPO基因编码区与可选的内源强启动子的体内融合，形成新的高表达PPO基因。在水稻中，强启动子优选CP12基因的启动子。在拟南芥中，强启动子优选ubiquitin10基因的启动子。
本发明还提供了一种采用所述编辑方法获得的高表达植物内源PPO基因。
本发明还提供了一种高表达水稻内源PPO基因具有选自下述的序列：
(1)如SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25或SEQ ID NO:26所示的核酸序列或其部分序列或其互补序列；
(2)与(1)限定的任一序列具有至少75％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％以上同一性；或
(3)在严谨条件下与(1)或(2)所示序列杂交的核酸序列。
本发明另外提供一种包含所述HPPD、EPSPS或PPO基因的DNA。
本发明另外提供一种利用包含所述HPPD、EPSPS或PPO基因编码的蛋白或其生物活性片段。
本发明另外提供一种重组表达载体，其包含所述HPPD、EPSPS或PPO基因，以及与之可操作地连接的启动子。
本发明另外提供一种包含所述HPPD、EPSPS或PPO基因的表达盒。
本发明另外提供一种宿主植物细胞，其中包含有所述的表达盒。
本发明另外提供采用所述宿主植物细胞再生成的植物。
本发明另外提供一种生产对于除草剂的抗性或耐受性提高的植物的方法，其中包括将所述的宿主植物细胞再生成植物和/或其衍生后代。
在一具体实施方式中，所述对于除草剂的抗性或耐受性提高的植物是将宿主植物细胞再生成的植物与野生型杂交通过遗传分离去除外源转基因成分的非转基因株系。
本发明还提供了一种抗除草剂水稻，其中包含上述高表达水稻内源HPPD基因、高表达水稻内源EPSPS基因、高表达水稻内源PPO基因其中之一或其任意组合。
在一具体实施方式中，所述抗除草剂水稻是非转基因的。
本发明另外提供所述高表达植物内源HPPD、EPSPS或PPO基因在提高植物细胞、植物组织、植物部分或植物的相应抑制性除草剂抗性或耐受性上的应用。
本发明另一方面又提供了一种控制植物栽培场所的杂草的方法，其中所述植物包括上述的植物或者通过上述方法制备的植物，所述方法包括对所述栽培场所施用控制杂草有效量的一种或多种HPPD、EPSPS或PPO抑制性除草剂。
发明人在研究工作中发现，同时进行双靶点或多靶点基因编辑的细胞中，不同靶点处的DNA双链断裂末端有一定的比例产生自发的相互连接，从而产生同一染色体上靶点间片段的 删除、倒位或复制加倍事件，和/或不同染色体靶点之间的染色体片段交换。有文献报道，这种现象在动植物中普遍存在(Puchta et al.2020.Changing local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering.Nat Commun.DOI:10.1038/s41467-020-18277-z；Li et al.2015.Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9.J Mol Cell Biol.DOI:10.1093/jmcb/mjv016)。
本发明人出乎意料地发现，通过在目标基因的特定元件附近设计基因编辑靶点组合，引起DNA双链断裂与自发的修复连接，不需要提供外源DNA模板即可在基因组水平上实现不同基因元件的定向组合，从中可能产生有功能的新基因，从而极大加速新基因的产生，在动植物育种和基因功能研究中都有极大的应用潜力。
发明详述
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在如下文献中有更全面的描述：Sambrook，J.，Fritsch，E.F.和Maniatis，T.，Molecular Cloning：A Laboratory Manual；Cold Spring Harbor Laboratory Press：Cold Spring Harbor，1989。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
本文所用术语“基因组”是指存在于生物体的每个细胞或病毒或细胞器中的遗传物质(基因和非编码序列)的全部互补物，和/或从一个亲本作为一个单位(单倍体)遗传的完整染色体组。
术语“基因编辑”是指针对活体有机体的任何遗传信息或基因组的靶向特异性修饰的策略和技术。因此，术语包括基因编码区的编辑，但也包括除基因组的基因编码区之外的区域的编辑。它还包括编辑或改造核(如果存在)以及细胞的其他遗传信息。
术语“CRISPR/Cas核酸酶”可以是基于CRISPR的核酸酶或编码其的核酸序列，包括但不限于：1)Cas9，包括SpCas9，ScCas9，SaCas9，xCas9，VRER-Cas9，EQR-Cas9，SpG-Cas9，SpRY-Cas9，SpCas9-NG，NG-Cas9，NGA-Cas9(VQR)等，2)Cas12，包括LbCpf1，FnCpf1，AsCpf1，MAD7等，或前述基于CRISPR的核酸酶的任何变体或衍生物，优选其中所述至少一个基于CRISPR的核酸酶与相应的野生型序列相比包含突变，使得所获得的基于CRISPR的核酸酶识别不同的PAM序列。如本文所用，“基于CRISPR的核酸酶”是已经在天然存在的CRISPR系统中鉴定的任何核酸酶，其随后从其天然背景中分离，并且其优选已被修饰或组合成感兴趣的重组构建体，适合作为靶向基因组工程的工具。只要最初的基于野生型CRISPR的核酸酶提供DNA识别，即结合特性，任何基于CRISPR的核酸酶都可以使用并任选重新编程或另外突变以适合本发明的各种实施方案。
术语“CRISPR”指代依赖于成簇规律间隔短回文重复序列途径的序列特异性遗传操纵技术，其不同于RNA干扰在转录水平下调节基因表达。
“Cas9核酸酶”和“Cas9”在本文中可互换使用，指的是包括Cas9蛋白或其片段(例如包含Cas9的活性DNA切割结构域和/或Cas9的gRNA结合结构域的蛋白)的RNA指导的核酸酶。 Cas9是CRISPR/Cas(成簇的规律间隔的短回文重复序列及其相关系统)基因组编辑系统的组分，能在向导RNA的指导下靶向并切割DNA靶序列形成DNA双链断裂(DSB)。
“Cas蛋白”或“Cas多肽”是指由Cas(CRISPR-相关的)基因编码的多肽。Cas蛋白包括Cas内切核酸酶。Cas蛋白可以是细菌或古细菌蛋白。例如，本文中的I-III型CRISPR Cas蛋白通常起源于原核生物；I型和III型Cas蛋白可以源自于细菌或古细菌物种，而II型Cas蛋白(即Cas9)可以源自于细菌种类。Cas蛋白包括Cas9蛋白、Cpf1蛋白、C2c1蛋白、C2c2蛋白、C2c3蛋白、Cas3、Cas3-HD、Cas5、Cas7、Cas8、Cas10、Cas12a、Cas12b或这些的组合或复合物。
“Cas9变体”或“Cas9内切核酸酶变体”是指亲本Cas9内切核酸酶的变体，其中当与crRNA和tracRNA或与sgRNA相缔合时，Cas9内切核酸酶变体保留以下能力：识别、结合DNA靶序列的全部或部分并任选地解旋DNA靶序列的全部或部分、使DNA靶序列的全部或部分产生切口、或切割DNA靶序列的全部或部分。Cas9内切核酸酶变体包括本文所述Cas9内切核酸酶变体，其中所述Cas9内切核酸酶变体不同于亲本Cas9内切核酸酶，其方式为：所述Cas9内切核酸酶变体(当与gRNA复合以形成能够修饰靶位点的、多核苷酸指导的内切核酸酶复合物时)与亲本Cas9内切核酸酶(与相同的gRNA复合以形成能够修饰相同靶位点的、多核苷酸指导的内切核酸酶复合物)相比时具有至少一种改善的特性，例如，但不限于，增加的转化效率、增加的DNA编辑效率、减少的脱靶切割、或其任意组合。
本文所述的Cas9内切核酸酶变体包括当与crRNA和tracrRNA或与sgRNA相缔合时可结合双链DNA靶位点并使双链DNA靶位点产生切口的变体，而亲本Cas内切核酸酶当与crRNA和tracrRNA或与sgRNA相缔合时可在靶位点处结合并使双链断裂(切割)。
“引导RNA”和“gRNA”在本文中可互换使用，指代用于靶向特定基因从而采用CRISPR技术进行校正的引导RNA序列，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互补性以便与该靶序列杂交并且指导CRISPR复合物(Cas9+crRNA+tracrRNA)与该靶序列序列特异性结合的序列。然而，本领域已知可以设计单一引导RNA(sgRNA)，其同时包含crRNA和tracrRNA的特征。
术语“单一引导RNA”和“sgRNA”在本文中可互换使用，并涉及两个RNA分子的合成融合，其中包含可变靶向结构域(与tracrRNA杂交的tracr配对序列连接)的crRNA(CRISPR RNA)与tracrRNA(反式激活CRISPR RNA)融合。sgRNA可以包含可与II型Cas核酸内切酶形成复合物的II型CRISPR/Cas系统的crRNA或crRNA片段和tracrRNA或tracrRNA片段，其中所述引导RNA/Cas核酸内切酶复合物可以将Cas核酸内切酶引导至DNA靶位点，使得Cas核酸内切酶能够识别、任选地结合DNA靶位点、并任选地使DNA靶位点产生切口或切割(引入单链或双链断裂)DNA靶位点。
在某些实施方式中，引导RNA(一个或多个)和Cas9可以作为核糖核蛋白(RNP)复合物递送至细胞。RNP由与gRNA复合的纯化的Cas9蛋白组成，并且在本领域中众所周知RNP可以被有效地递送到多种类型的细胞中，包括但不限于干细胞和免疫细胞(Addgene，Cambridge，MA、Mirus Bio LLC，Madison，WI)。
本文中的原间隔序列毗邻基序(protospacer adjacent motif，PAM)是指与由gRNA/Cas内切核酸酶系统识别的(靶向的)靶序列(前间隔子)邻近的短核苷酸序列。如果靶DNA序列不邻 近合适的PAM序列，则Cas内切核酸酶可能无法成功识别所述靶DNA序列。本文中的PAM的序列和长度可以取决于所使用的Cas蛋白或Cas蛋白复合物而不同。所述PAM序列可以是任何长度，但典型地是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个核苷酸长度。
如本文所使用的，术语“生物”，包括动物、植物、真菌、细菌等。
如本文所使用的，术语“宿主细胞”，包括植物细胞、动物细胞、真菌细胞、细菌细胞等。
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，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(Triticum spp.)，例如普通小麦(Triticum aestivum)、硬粒小麦(T.Turgidumssp.durum)；大麦属(Hordeum spp.)，例如大麦(Hordeum vulgare)、亚利桑那大麦(Hordeum arizonicum)；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)、Avena fatua var.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(Zizania palustris)、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(Elaeis oleifera))、大豆(soybean)、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(Psidium spp.)、石榴(Punica granatum)、苹果属(Malus spp.)、西瓜(Citrullus lanatus)、柑桔属(Citrus spp.)、无花果(Ficus carica)、金桔属(Fortunella spp.)、草莓属(Fragaria spp.)、山楂属(Crataegus spp.)、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(Averrhoa carambola)、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(straw berry)、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(Capsicum spp.)、茄属(Solanum spp.)(例如马铃薯(Solanum tuberosum)、红茄(Solanum integrifolium)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(collard greens)、西葫芦(squash)、冬瓜(Benincasa hispida)、石刁柏(Asparagus officinalis)、旱芹(Apium graveolens)、苋属(Amaranthus spp.)、葱属(Allium spp.)、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(Tropaeolum majus)、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、绿萝；(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、贝母、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、板蓝根、白术；(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(Saccharum spp.))、甜菜(Beta vulgaris)；(13)饮料作物：大叶茶(Camellia sinensis)、茶(Camellia Sinensis)、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(Festuca spp.)(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(Bouteloua var.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(Quercus spp.)、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；(16)坚果作物：巴西栗(Bertholletia excelsea)、栗属(Castanea spp.)、榛属(Corylus spp.)、山核桃属(Carya spp.)、核桃属(Juglans spp.)、阿月浑子(Pistacia vera)、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(Carissa macrocarpa)、菜蓟属(Cynara spp.)、野胡萝卜(Daucus carota)、薯蓣属(Dioscorea spp.)、蔗茅属(Erianthus sp.)、苇状羊茅(Festuca arundinacea)、萱草(Hemerocallis fulva)、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(Olea spp.)、鸟足豆属(Ornithopus spp.)、欧防风(Pastinaca sativa)、接骨木属(Sambucus spp.)、白芥属(Sinapis sp.)、蒲桃属(Syzygium spp.)、鸭茅状摩擦禾(Tripsacum dactyloides)、Triticosecale rimpaui、香堇(Viola odorata)等。
在一具体实施方式中，所述植物选自水稻、玉米、小麦、大豆、向日葵、高粱、油菜、苜蓿、棉花、大麦、谷子、甘蔗、番茄、烟草、木薯、马铃薯、甘薯、白菜、甘蓝、黄瓜、月季、绿萝、西瓜、甜瓜、草莓、蓝莓、葡萄、苹果、柑橘、桃、梨、香蕉等。
如本文所使用的，术语“植物”包括整个植物和任何后代、植物的细胞、组织、或部分。术语“植物部分”包括植物的任何部分，包括，例如但不限于：种子(包括成熟种子、没有种皮的未成熟胚、和不成熟的种子)；植物插条(plant cutting)；植物细胞；植物细胞培养物；植物器官(例如，花粉、胚、花、果实、芽、叶、根、茎，和相关外植体)。植物组织或植物器官 可以是种子、愈伤组织、或者任何其他被组织成结构或功能单元的植物细胞群体。一些植物细胞或组织培养物能够再生出具有该细胞或组织所来源的植物的生理学和形态学特征的植物，并能够再生出与该植物具有基本上相同基因型的植物。与此相反，一些植物细胞不能够再产生植物。植物细胞或组织培养物中的可再生细胞可以是胚、原生质体、分生细胞、愈伤组织、花粉、叶、花药、根、根尖、丝、花、果仁、穗、穗轴、壳、或茎。
植物部分包括可收获的部分和可用于繁殖后代植物的部分。可用于繁殖的植物部分包括，例如但不限于：种子；果实；插条；苗；块茎；和砧木。植物的可收获部分可以是植物的任何有用部分，包括，例如但不限于：花；花粉；苗；块茎；叶；茎；果实；种子；和根。
植物细胞是植物的结构和生理单元。如本文所使用的，植物细胞包括原生质体和具有部分细胞壁的原生质体。植物细胞可以是处于分离的单个细胞或细胞聚集体的形式(例如，松散愈伤组织和培养的细胞)，并且可以是更高级组织单元(例如，植物组织、植物器官、和完整植物)的一部分。因此，植物细胞可以是原生质体、产生配子的细胞，或者能够再生成完整植物的细胞或细胞的集合。因此，在本文的实施方案中，包含多个植物细胞并能够再生成为整株植物的种子被认为是一种“植物部分”。
如本文所使用的，术语“原生质体”是指细胞壁被完全或部分地除去、其脂双层膜裸露的植物细胞。典型地，原生质体是没有细胞壁的分离植物细胞，其具有再生成细胞培养物或整株植物的潜力。
植物“后代”包括植物的任何后续世代。
“抑制性除草剂耐受性”和“抑制性除草剂抗性”两个术语可以互换使用，均指的是对抑制性除草剂的耐受性和对抑制性除草剂的抗性。“抑制性除草剂耐受性提高”和“抑制性除草剂抗性提高”是指对所述抑制性除草剂的耐受性或抗性与含有野生型基因的植物相比提高。
术语“野生型”指的是可以在自然界中被发现存在的核酸分子或蛋白质。
在本发明中，术语“场所”包括栽培本发明植物的场地例如土壤，也包括例如植物种子、植物苗以及长成的植物。术语“控制杂草有效量”指的是除草剂的量足以影响目标杂草的生长或发育，例如阻止或抑制目标杂草的生长或发育，或者杀灭所述杂草。有利地，所述控制杂草有效量不会显著影响本发明植物种子、植物苗或植物的生长和/或发育。本领域技术人员可以通过常规实验确定这样的控制杂草有效量。
术语“基因”包括表达功能性分子(诸如但不限于，特定蛋白质)的核酸片段，包括在编码序列之前(5’非编码序列)和之后(3’非编码序列)的调节序列。
“编码”具体RNA的DNA序列为转录成RNA的DNA核酸序列。DNA多核苷酸可编码翻译成蛋白质的RNA(mRNA)，或DNA多核苷酸可编码不翻译成蛋白质的RNA(例如tRNA、rRNA或靶向DNA的RNA；又称为“非编码”RNA或“ncRNA”)。
“多肽”、“肽”、和“蛋白”在本发明中可互换使用，指氨基酸残基的聚合物。该术语适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的人工化学类似物的氨基酸聚合物，以及适用于天然存在的氨基酸聚合物。术语“多肽”、“肽”、“氨基酸序列”和“蛋白”还可包括修饰形式，包括但不限于糖基化、脂质连接、硫酸盐化、谷氨酸残基的γ羧化、羟化和ADP-核糖基化。
“生物活性片段”是指从蛋白质的N和/或C末端缺失一或多个氨基酸残基而仍保留其功能 活性的片段。
术语“多核苷酸”和“核酸”可以互换使用，包括DNA、RNA或者其杂交体，可以是双链或单链的。
术语“核苷酸序列”和“核酸序列”均是指DNA或RNA中碱基的排列顺序。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的SEQ ID No.9～SEQ ID No.17所示的DNA片段进行突变。那些经过人工修饰的，具有与本发明的前述任一序列所示的DNA片段至少75％同一性的核苷酸，且具有相同功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
术语“同一性”指与天然核酸序列的序列相似性。同一性可以用肉眼或计算机软件进行评价。使用计算机序列比对软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。“部分序列”意味着给定序列的至少5％、10％、20％、30％、40％、50％、60％、70％、80％、90％或95％。
所述严谨条件可为如下：50℃，在7％十二烷基硫酸钠(SDS)、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，2×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.5×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在50℃，0.1×SSC，0.1％SDS中漂洗；还可为：50℃，在7％SDS、0.5M NaPO 4和1mM EDTA的混合溶液中杂交，在65℃，0.1×SSC，0.1％SDS中漂洗；也可为：在6×SSC，0.5％SDS的溶液中，在65℃下杂交,然后用2×SSC,0.1％SDS和1×SSC,0.1％SDS各洗膜一次；也可为：2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；也可为：0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
如本发明所用，“表达盒”“表达载体”和“表达构建体”是指适于感兴趣的核苷酸序列在植物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。
本发明的“高表达基因”是指在特定的组织中表达量高于普通基因的基因。
术语“重组表达载体”或“DNA构建体”在本文中可互换使用，是指包含载体和至少一个插入物的DNA分子。通常出于表达和/或繁殖插入物的目的或出于构建其它重组核苷酸序列而产生重组表达载体。插入物可以可操作的或可以不可操作地连接至启动子序列并且可以可操作的或可以不可操作地连接至DNA调节序列。
“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。
“启动子”指能够控制另一核酸片段转录的核酸片段。在本发明的一些实施方案中，启动子是能够控制植物细胞中基因转录的启动子，无论其是否来源于植物细胞。启动子可以是组成型启动子或组织特异性启动子或发育调控启动子或诱导型启动子。
术语“强启动子”是本领域众所周知的并广泛使用的术语，许多强启动子是本领域已知的，或者可以通过常规实验来鉴定。所述启动子的活性高于与野生型生物中待过量表达的核酸分子有效连接的启动子的活性，例如活性比内源基因的启动子更高的启动子。优选地，强启动子的活性比与野生型生物中待过量表达的核酸分子有效连接的启动子的活性高约2％、5％、8％、10％、20％、30％、40％、50％、60％、70％、80％、90％、100％、150％、200％、250％、300％、350％、400％、450％、500％、600％、700％、800％、900％、1000％或超过1000％。本领域技术人员知晓如何测定启动子活性，并比较不同的启动子的活性。
“组成型启动子”指一般将引起基因在多数细胞类型中在多数情况下表达的启动子。“组织特异性启动子”和“组织优选启动子”可互换使用，并且指主要但非必须专一地在一种组织或器官中表达，而且也可在一种特定细胞或细胞型中表达的启动子。“发育调控启动子”指其活性由发育事件决定的启动子。“诱导型启动子”响应内源性或外源性刺激(环境、激素、化学信号等)而选择性表达可操纵连接的DNA序列。
如本文中所用，术语“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知的。
将核酸分子(例如质粒、线性核酸片段、RNA等)或蛋白质“导入”植物是指用所述核酸或蛋白质转化植物细胞，使得所述核酸或蛋白质在植物细胞中能够发挥功能。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植物基因组中，导致外源基因稳定遗传。一旦稳定转化，外源核酸序列稳定地整合进所述植物和其任何连续世代的基因组中。
“瞬时转化”指将核酸分子或蛋白质导入植物细胞中，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植物基因组中。
改变生物体内源基因的表达包括了强度和时空特性两个方面。强度包括调高(即敲高)、调低(即敲低)和/或调零(即敲除)；时空特异性包括了时间(生育时期)特异性和空间(组织)特异性两种，也包括了诱导性。此外，改变蛋白的靶向性，例如是将定位在细胞质中的蛋白改成叶绿体定位或细胞核定位。
除非另外定义，否则本文使用的所有技术术语和科学术语具有与通过本发明所属领域中的普通技术人员通常理解的相同的含义。虽然还可在本发明的实践或测试中使用类似于或等同于本文所述的那些方法和材料的任何方法和材料，但现在描述优选的方法和材料。
本说明书中引用的所有出版物和专利以引用的方式并入本文如同每个单独的出版物或专利均确切地和单独地指出以引用的方式并入一样，并且以引用的方式并入本文以公开和描述与出版物所引用的相关的方法和/或材料。任何出版物的引用为其公开在申请日之前并且不应该解释为承认本发明没有资格先于现有发明的这种出版物。此外，所提供的出版日期可与实 际出版日期不同，这可能需要独立证实。
除非具体说明或暗示，如本文中所用，术语“一”、“一个/一种”和“所述”表示“至少一个”。本文提到或引用的所有专利、专利申请和出版物整体引入本文作为参考，其引用程度如同单独地个别引用一样。
本发明具有如下优异技术效果：
本发明综合运用了以下两个不同专业领域的信息，开发了在生物体内直接创制新基因的方法，完全改变了原先基因编辑工具的常规用途(即敲除基因)，实现了创制新基因的新用途，特别的，实现了通过基因编辑技术提高目标基因表达量的敲高内源基因的编辑方法。一是基因编辑领域的信息，即当两个以上不同的靶点与Cas9同时靶向生物基因组时，可以出现删除、倒置、加倍或倒置加倍组合等不同情况。二是基因组学领域的信息，即基因组上不同基因的位置，距离，以及基因内不同元件(启动子、5’UTR、编码区(CDS)、不同的结构域区域、终止子等)的具体位置、方向、功能，不同基因的表达特异性信息等。结合这两个不同领域的信息，在两个以上不同基因的特定位置或单个基因内的两个以上特定位置(具体位置由基因组学领域提供)进行切割，通过删除、倒置、加倍、倒置加倍组合或染色体臂的交换等(具体情形由基因编辑领域提供)，形成不同基因元件或功能结构域的新组合，特异地在生物体内创制新基因。
本发明创制的这些新基因是在生物体内自发的DNA修复机制作用下，由两个以上基因的不同元件进行融合或重组从而改变原基因的表达强度、时空特异性、特殊功能结构域等形成的，并不含有外源转基因或者人工合成的基因元件。由于新的基因融合了两种以上不同基因的元件，这极大拓展了基因变异的自由度，会产生更为丰富多样的功能，具有广泛的应用前景。同时，这些新基因与基因编辑载体是不连锁的，通过遗传分离去除载体元件后，即可得到含有新基因的非转基因生物材料用于动植物育种。或者也可以通过mRNA或核糖核酸蛋白复合体(RNP)的递送方式进行非整合的瞬时编辑，创制含有新基因的非转基因生物材料。这个过程是非转基因的，最终得到的编辑材料也不含转基因。在理论和事实上，这些新基因也可以通过传统育种技术(如辐射或化学诱变等)获得，所不同的是，用传统技术进行筛选需要创制数量巨大的随机突变体库并耗费大量时间和成本从中筛选新的功能基因，而本发明能够通过生物信息学分析结合基因编辑技术定向创制新功能基因，大大缩短了育种周期。按照现行的基因编辑生物审批流程，本发明提供的方法在很多国家不受监管。
另外，本发明的新基因创制技术能够用于改变生物体的生长、发育、抗性、产量等多种性状，具有重大应用价值。创制出的新基因或具有新的调控元件(如启动子)从而改变了原基因的表达强度、时空特性等，或具有新的氨基酸序列从而具有了新功能。以农作物为例，改变特定基因的表达可以提高作物对病虫草等有害生物及干旱、水涝、盐碱等非生物胁迫的抗性，也可提高产量，改善品质。以鱼类为例，改变生长激素的表达特性，能够显著改变其生长发育的速度。
附图说明
图1为在水稻中创制新的HPPD基因原理图。
图2为在水稻中创制新的EPSPS基因原理图。
图3为在拟南芥中创制新的PPOX基因原理图。
图4为在水稻中创制新的PPOX基因原理图。
图5为水稻原生质体测试HPPD-duplication方案测序结果。
图6为水稻农杆菌转化载体pQY2091载体图谱。
图7为pQY2091转化水稻潮霉素抗性愈伤检测新基因片段PCR电泳结果。箭头所指为UBI2基因启动子和HPPD编码区融合的新基因PCR条带。数字为不同愈伤样品的编号。M代表DNA Marker，条带大小依次为100bp，250bp，500bp，750bp，1000bp，2000bp，2500bp，5000bp，7500bp。
图8为pQY2091转化水稻T0代幼苗检测新基因片段PCR电泳结果。箭头所指为UBI2基因启动子和HPPD编码区融合的新基因PCR条带。数字为不同T0苗的编号。M代表DNA Marker，条带大小依次为100bp，250bp，500bp，750bp，1000bp，2000bp，2500bp，5000bp，7500bp。
图9为QY2091T0代HPPD基因加倍株系双唑草酮抗性测试结果。同一花盆中左侧为野生型金粳818，右侧为HPPD加倍株系。
图10为QY2091T0代HPPD基因加倍株系HPPD和UBI2基因相对表达量。818CK1，818CK3为两个野生型金粳818对照植株；13M，20M代表QY2091-13，QY2091-20T0植株的主分蘖叶片样品；13L，20L代表QY2091-13，QY2091-20T0植株用于除草剂抗性测试的次级分蘖叶片样品。
图11为QY2091T1代可能的基因型及分子检测引物的结合位点示意图。
图12为QY2091-13、QY2091-20检测HPPD加倍测序结果与预测加倍序列比对。
图13为对QY2091HPPD加倍株系的T1代进行苗期的除草剂抗性检测结果。
图14为水稻PPO1基因染色体片段倒位可能的编辑事件类型及分子检测引物的结合位点示意图。
图15为EPSPS-inversion检测测序结果。
图16为水稻农杆菌转化载体pQY2234载体图谱。
图17为pQY2234转化水稻潮霉素抗性愈伤检测新基因片段PCR电泳结果。箭头所指为CP12基因启动子和PPO1编码区融合的新基因PCR条带。数字为不同愈伤样品的编号。M代表DNA Marker，条带大小依次为100bp，250bp，500bp，750bp，1000bp，2000bp，2500bp，5000bp，7500bp。
图18为QY2234T0代PPO1基因倒位株系2081抗性测试结果。同一处理剂量下左侧花盆为野生型淮稻5号对照，右侧为PPO1倒位株系。
图19为QY2234T0代PPO1倒位株系PPO1和CP12基因相对表达量。H5CK1，H5CK2为两个野生型淮稻5号对照植株；252M、304M、329M代表QY2234-252，QY2234-304，QY2234-329T0植株的主分蘖叶片样品；252L、304L、329L代表次级分蘖叶片样品。
图20为淮稻5号背景的PPO1倒位测序结果与预测倒位序列比对。
图21为金粳818背景的PPO1倒位测序结果与预测倒位序列比对。
图22为对QY2234PPO1倒位株系的T1代进行苗期的除草剂抗性检测结果。
具体实施方式
以下结合实例，对本发明作进一步说明。下面的说明是采用举例的方式，但是本发明的保护范围不应局限于此。
实施例1在植物中通过诱导染色体片段加倍敲高内源HPPD基因表达的编辑方法-水稻原生质体测试
HPPD是植物合成叶绿素通路中的关键酶，抑制HPPD酶的活性最终导致植物白化失绿而死亡。许多除草剂如硝磺草酮、苯唑草酮等是以HPPD为靶标蛋白的抑制剂，提高植物内源HPPD基因的表达水平应该有助于提升植株对该类除草剂的耐受性。水稻HPPD基因(如SEQ ID NO:6所示，其中1-1067bp为启动子，其余为表达区)位于水稻2号染色体上。通过生物信息学分析发现HPPD基因下游约338kb处为水稻Ubiquitin2(以下简称为UBI2)基因(如SEQ ID NO:5所示，其中1-2107bp为启动子，其余为表达区)，UBI2基因与HPPD基因在染色体上的方向相同。根据国际水稻基因组测序计划(International Rice Genome Sequencing Project)提供的水稻基因表达谱数据(http://rice.plantbiology.msu.edu/index.shtml)分析，在水稻叶片中UBI2基因表达强度是HPPD基因的3-10倍，UBI2基因启动子是一个组成型表达的强启动子。
如图1所示，方案1通过同时在HPPD和UBI2基因各自的启动子和CDS区之间的位置产生双链断裂，经过筛选鉴定可以获得两个断口之间的区域加倍的事件，UBI2的启动子和HPPD的编码区可融合在一起形成新基因。此外，按照如图1所示的方案2，通过连续两次倒置也可以形成UBI2的启动子和HPPD编码区融合的新基因。首先对图1所示方案在水稻原生质体系统中进行如下测试：
1.首先将水稻HPPD和UBI2基因组DNA序列输入CRISPOR在线工具(http://crispor.tefor.net/)寻找可用的编辑靶点，经在线评分在HPPD和UBI2基因各自的启动子和CDS区之间选择如下靶点进行测试：
Figure PCTCN2020126747-appb-000001
其中guide RNA1和guide RNA2位于HPPD基因启动子与CDS区之间，靠近HPPD蛋白起始密码子，guide RNA3和guide RNA4位于UBI2基因启动子与CDS区之间，靠近UBI2蛋白起始密码子。
以pHUE411载体(https://www.addgene.org/62203/)为骨架，按照“Xing HL,Dong L,Wang ZP,Zhang HY,Han CY,Liu B,Wang XC,Chen QJ.A CRISPR/Cas9 toolkit for multiplex genome editing in plants.BMC Plant Biol.2014Nov 29；14(1):327”所述方法，针对上述靶点设计以下引物，进行载体构建。
Figure PCTCN2020126747-appb-000002
按上述文献提供的方法，构建如下双靶点组合的基因编辑载体。具体而言，以pCBC-MT1T2质粒(https://www.addgene.org/50593/)为模板分别扩增sgRNA1+3、sgRNA1+4、 sgRNA2+3、sgRNA2+4双靶点片段，构建sgRNA表达盒。BsaI酶切pHUE411载体骨架，切胶回收，目的片段经酶切后直接用于连接反应。采用T4DNA连接酶连接载体骨架与目的片段，转化连接产物至Trans5α感受态细胞，挑取不同的单克隆测序，测序正确后利用思科捷高纯度质粒小量提取试剂盒抽提质粒，得到重组质粒，分别命名为pQY002065、pQY002066、pQY002067、pQY002068，如下所示：
pQY002065   pHUE411-HPPD-sgRNA1+3含OsHPPD-guide RNA1，guide RNA3组合
pQY002066   pHUE411-HPPD-sgRNA1+4含OsHPPD-guide RNA1，guide RNA4组合
pQY002067   pHUE411-HPPD-sgRNA2+3含OsHPPD-guide RNA2，guide RNA3组合
pQY002068   pHUE411-HPPD-sgRNA2+4含OsHPPD-guide RNA2，guide RNA4组合
2.按照如下方法，对上述pQY002065-002068载体制备高纯度、高浓度质粒：
使用Promega中量质粒提取试剂盒(Midipreps DNA Purification System,Promega,A7640)，按操作说明提取质粒。具体步骤为：
(1)向含有卡那霉素的300毫升液体LB培养基中加5毫升大肠杆菌，200rpm，37℃摇菌12–16小时。
(2)将上述菌液置于500毫升离心管中，5,000g离心菌液10分钟，弃上清。
(3)加3毫升细胞重悬液(Cell Resuspension Solution,CRS)重悬细胞沉淀，涡旋混匀。
(4)加3毫升细胞溶解液(Cell Lysis Solution,CLS)，缓慢颠倒混匀时间不宜超过5分钟。
(5)加3毫升中和液(Neutralization Solution)，颠倒混匀，至颜色变清澈透明。
(6)14,000g离心15分钟，如未形成紧实沉淀可再次离心15分钟。
(7)转移上清液至新的50毫升离心管中，避免吸入白色沉淀至离心管中。
(8)加10毫升DNA净化树脂(Purification Resin，用前剧烈摇匀)，混匀。
(9)将Resin/DNA混合液倒入过滤柱中，真空泵负压法(0.05MPa)。
(10)加15毫升洗柱液(Column Wash Solution,CWS)到过滤柱中，抽真空。
(11)加15毫升CWS，重复抽真空一次；溶液全部通过过滤柱后延长抽30s。
(12)切断过滤柱，将其转移至1.5毫升离心管中，12,000g离心2分钟，移除残留液体，将过滤柱转移至新的1.5毫升离心管中。
(13)加200μL预热至70℃的灭菌水，静置2分钟。
(14)12,000g离心2分钟，洗脱质粒DNA；浓度一般在1μg/μL左右。
3.制备水稻原生质体并进行PEG介导转化：
首先准备原生质体用的水稻幼苗，使用的品种为日本晴(Nipponbare)，种子由中国农业大学植物保护学院杂草教研室提供，本实验室扩繁。水稻种子首先去壳，去壳的种子用75％乙醇漂洗1分钟，用5％(v/v)的次氯酸钠处理20分钟，然后用无菌水洗涤5次以上，在超净台中吹干后放在盛有1/2MS培养基的组培瓶中，每瓶可放20粒种子。26℃，12h光照培养10d左右，就可以进行原生质体的制备。
水稻原生质体制备以及PEG介导的转化方法参照“Lin et al.,2018 Application of protoplast technology to CRISPR/Cas9 mutagenesis:from single‐cell mutation detection to mutant plant regeneration.Plant Biotechnology Journal https://doi.org/10.1111/pbi.12870”。具体的步骤如下：
(1)选取幼苗叶鞘部分，用锋利的吉利剃须刀片切成约1mm的碎片，放在0.6M甘露醇和MES培养液(配方：0.6M甘露醇，0.4M MES，pH 5.7)中备用。将全部材料切好后转入20毫升酶解液(配方：1.5％Cellulase R10/RS(YaKult Honsha)，0.5％Mecerozyme R10(YaKult Honsha)，0.5M甘露醇，20mM KCl，20mM MES,pH 5.7，10mM CaCl 2，0.1％BSA，5mMβ-巯基乙醇)中，用锡箔纸包好置于28℃摇床中，50rpm避光酶解约4个小时，最后2分钟将转速提高至100rpm；
(2)酶解结束后，加入等体积的W5溶液(配方：154mM NaCl，125mM CaCl 2，5mM KCl，15mM MES)，水平摇动10秒，释放原生质体。酶解后的细胞经300目的筛子过滤后，150g离心5分钟收集原生质体；
(3)再用W5溶液漂洗细胞两次，150g离心5分钟收集原生质体；
(4)用适量的MMG溶液(配方：3.05g/L MgCl 2，1g/L MES，91.2g/L甘露醇)重悬原生质体，原生质体浓度约为2×10 6cell/mL。
按照如下方法进行原生质体的转化：
(1)取上述200μL MMG重悬的原生质体加入制备好的无内毒素的高质量质粒DNA(10-20μg)，轻弹混匀。
(2)加入等体积的40％(w/v)的PEG溶液(配方：40％(w/v)PEG，0.5M甘露醇，100mM CaCl 2)，轻弹混匀，28℃避光静置15分钟；
(3)诱导转化结束后缓慢加1.5毫升的W5溶液，轻弹混匀细胞，150g离心3分钟收集细胞，重复此步骤一次；
(4)加入1.5毫升W5溶液重悬细胞，置于28℃培养箱中避光培养12-16小时，若用于提取原生质体基因组DNA，需培养48-60小时。
4.进行基因组打靶和新基因产生情况检测：
(1)首先需要提取原生质体DNA。使用CTAB法并进行部分修改，具体的方法如下：原生质体离心后弃上清，加入500μL DNA提取液(配方：CTAB 20g/L，NaCl 81.82g/L，100mM Tris-HCl(pH 8.0)，20mM EDTA，0.2％β巯基乙醇)，振荡混匀，置于65℃水浴锅中孵育1小时；水浴后的样品冷却后加入500μL氯仿，颠倒混匀后10,000rpm离心10分钟；取400μL上清转移到一个新的1.5毫升离心管中，加入1毫升70％(v/v)的乙醇放入-20℃沉淀20分钟；用12,000rpm离心15分钟沉淀DNA，待沉淀晾干后加入50μL超纯水溶解，保存于-20℃备用。
(2)使用下表中的检测引物，扩增含有两侧靶位点的片段或预测的UBI2的启动子和HPPD编码区融合产生的片段，PCR产物长度为300-1000bp之间，其中primer8-F+primer6-R组合用于检测染色体片段加倍后中间接头处的融合片段，预期产物长度为630bp。
Figure PCTCN2020126747-appb-000003
Figure PCTCN2020126747-appb-000004
PCR反应体系如下：
Figure PCTCN2020126747-appb-000005
(3)进行PCR反应，一般反应条件是：
Figure PCTCN2020126747-appb-000006
(4)对PCR反应产物进行1％琼脂糖凝胶电泳检测，结果显示pQY002066和pQY002068转化样品中能够检测到预测的UBI2的启动子和HPPD编码区融合片段630bp阳性条带。
5.将UBI2的启动子和HPPD编码区融合片段阳性样品测序验证，使用OsHPPDduplicated-primer8-F和OsHPPDduplicated-primer6-R引物两端测序，结果如图5所示，显示UBI2基因启动子与HPPD基因表达区能够直接连接，结果表明在pQY002066和pQY002068质粒转化的水稻原生质体基因组DNA中能够检测到水稻UBI2基因启动子与HPPD基因表达区融合的编辑事件，说明通过染色体片段加倍形成新HPPD基因的方案可行，能够创制出强启动子驱动表达的HPPD新基因，定义为HPPD加倍事件。pQY002066载体转化原生质体检测HPPD加倍事件测序结果如SEQ ID NO:9所示；pQY002068载体转化原生质体检测HPPD加倍事件测序结果如SEQ ID NO:10所示。
实施例2通过农杆菌介导转化创制染色体片段加倍敲高内源HPPD基因表达的抗除草剂水稻
1、敲高编辑载体的构建：根据实施例1中原生质体测试的结果，选择编辑效率高的OsHPPD-guide RNA1：5’GTGCTGGTTGCCTTGGCTGC3’和OsHPPD-guide RNA4:5’GAAATAATCACCAAACAGAT3’双靶点组合，参照实施例1构建农杆菌转化载体pQY2091，载体骨架采用pHUE411并进行水稻密码子优化，载体图谱如图6所示。
2、农杆菌转化水稻愈伤：
1)农杆菌转化：取水稻敲高编辑载体pQY2091质粒1μg加入到10μl农杆菌EHA105热击感受态细胞(昂羽生物，货号G6040)，冰上放置5分钟，浸入液氮中速冻5分钟，取出置于37℃热击5分钟，最后放置在冰上5分钟。加入500μl YEB液体培养基(配方：酵母浸膏1g/L，蛋白胨5g/L，牛肉膏5g/L，蔗糖5g/L，硫酸镁0.5g/L)，置于28℃、200rpm摇床中培养2～3小时；3500rpm离心30秒收集菌体，将收集的菌体涂布于YEB(卡那霉素50mg/L+ 利福平25mg/L)平板上，28℃培养箱培养2天；挑取单克隆至液体培养基培养，-80℃保菌。
2)农杆菌的培养：在YEB平板上挑取转化的农杆菌单克隆，加入20毫升YEB液体培养基(卡那霉素50mg/L+利福平25mg/L)，28℃振荡培养至OD600为0.5，5000rpm离心10分钟收集菌落，加入20-40毫升AAM(Solarbio，货号LA8580)液体培养基重悬至OD600为0.2-0.3，再加入乙酰丁香酮(Solarbio，货号A8110)至终浓度200μM，浸染愈伤组织。
3)水稻愈伤组织诱导：转化受体水稻品种为淮稻5号和金粳818，来自江苏淮安市种子市场采购，本实验室扩繁。取800-2000粒干净的水稻种子脱壳，然后用无菌水清洗，直至洗后的水变清澈，再用70％酒精消毒30秒，之后加入30毫升5％次氯酸钠置于水平摇床50rpm振荡20分钟，再用无菌水清洗5次，置于无菌吸水纸，风干种子表面水分，接种于诱导培养基上在28℃下培养愈伤。
诱导培养基配方：4.1g/L N6粉末+0.3g/L水解酪蛋白+2.878g/L脯氨酸+2mg/L 2,4-D+3％蔗糖+0.1g/L肌醇+0.5g谷氨酰胺+0.45％植物凝胶，pH 5.8。
4)农杆菌侵染水稻愈伤：选取继代培养10天，直径为3毫米的淮稻5号或金粳818愈伤组织，将愈伤组织收集至50毫升的离心管中；将已调至OD600为0.2-0.3的农杆菌AAM重悬液倒入含有愈伤组织的离心管中，置于28℃、200转/分摇床中侵染20分钟；侵染完毕，倒掉菌液，将愈伤组织放置于无菌滤纸上风干20分钟左右，置于共同培养基平板上共培养，平板上铺有一张含100μM乙酰丁香酮的AAM液体培养基浸湿的无菌滤纸；共培养3天后，清洗去除农杆菌(先用无菌水洗5遍，再用500mg/L的头孢抗生素清洗20分钟)，置于50mg/L潮霉素筛选培养基上筛选培养。
共同培养基配方：4.1g/L N6粉末+0.3g/L水解酪蛋白+0.5g/L脯氨酸+2mg/L 2,4-D+200μM AS+10g/L葡萄糖+3％蔗糖+0.45％植物凝胶，pH 5.5。
3、潮霉素抗性愈伤的分子鉴定和分化出苗：
与常规水稻转化的筛选过程有所不同的是，本发明利用染色体片段加倍后产生的融合片段的特异性引物，在愈伤筛选培养阶段就可以对潮霉素抗性愈伤进行分子鉴定，确定加倍阳性事件，选择含有融合不同基因元件的新基因的愈伤进行分化培养，诱导出苗。具体操作步骤如下：
1)将共培养后的愈伤组织移至筛选培养基进行第一轮筛选(2周)。筛选培养基配方：4.1g/L N6粉末+0.3g/L水解酪蛋白+2.878g/L脯氨酸+2mg/L 2,4-D+3％蔗糖+0.5g谷氨酰胺+30mg/L潮霉素(HYG)+500mg/L头孢霉素(cef)+0.1g/L肌醇+0.45％植物凝胶，pH5.8。
2)第一轮筛选完毕后将新长出的愈伤移至新的筛选培养基进行第二轮筛选(2周)，在此阶段对新长出的直径大于3毫米的愈伤用镊子夹取少量样品，用实施例1所述CTAB法提取DNA进行第一轮分子鉴定，本实施例对pQY2091载体转化的愈伤，选择OsHPPDduplicated-primer8-F(8F)和OsHPPDduplicated-primer6-R(6R)引物对进行PCR鉴定，反应体系与反应条件参照实施例1，共鉴定350个愈伤，在淮稻5号愈伤中未检测到阳性样品，在金粳818愈伤中检出28个阳性样品，其中部分愈伤PCR检测结果如图7所示。
3)PCR鉴定阳性的愈伤移至新的筛选培养基进行第三轮筛选扩大培养；待愈伤直径大于5毫米之后，对扩大培养的愈伤使用8F+6R引物对进行第二轮分子鉴定，挑取二轮鉴定阳性 的生长状态良好的黄白色愈伤组织转入分化培养基进行分化，3～4周后可以获得1厘米左右的幼苗；将分化出的幼苗移至生根培养基进行生根培养；将生根完成的幼苗进行炼苗处理后，移至装有土壤的花盆中置温室进行培养。分化培养基配方：4.42g/L MS粉末+0.5g/L水解酪蛋白+0.2mg/L NAA+2mg/L KT+3％蔗糖+3％山梨醇+30mg/L潮霉素+0.1g/L肌醇+0.45％植物凝胶，pH 5.8。生根培养基配方：2.3g/L MS粉末+3％蔗糖+0.45％植物凝胶。
4、HPPD加倍苗(T0代)的分子检测：
29个加倍事件阳性愈伤经二轮分子鉴定后共分化得到403株T0代幼苗，对这403株幼苗继续使用8F+6R引物对进行第三轮分子鉴定，其中56株有阳性条带，阳性苗移入温室培养，其中部分T0苗PCR检测结果如图8所示。
5、HPPD加倍苗(T0代)HPPD抑制剂类除草剂抗性测试：
T0代鉴定加倍事件阳性转化苗移栽到温室的大塑料桶里培育以扩繁T1代种子。在幼苗开始分蘖之后，从生长旺盛的株系中掰取分蘖，分别与野生型对照品种的同期分蘖栽种到同一花盆中，待植株高度达到20厘米左右后，进行除草剂抗性测试。所用除草剂为本公司生产的双唑草酮(CAS号：1622908-18-2)，其田间剂量通常是每亩4克有效成分(4g a.i./mu)。本次实验双唑草酮的剂量设置为2g，4g，8g，32g四个梯度，采用步入式喷雾塔施药。
抗性检测结果如图9所示，施药后5天-7天，野生型对照水稻苗开始显现白化症状，而HPPD加倍事件各株系都保持正常绿色。施药4周之后，野生型水稻苗接近死亡，加倍事件各株系都继续保持绿色，生长正常。测试结果说明HPPD基因加倍株系对双唑草酮的耐受性明显提高。
6、HPPD加倍苗(T0代)HPPD基因相对表达量定量检测：
推测HPPD基因加倍株系对双唑草酮的抗性提升是由于UBI2的强启动子与HPPD基因CDS融合提高了HPPD表达量引起的，因此选择T0代株系QY2091-13，QY2091-20，分别对主分蘖和用于除草剂抗性测试的次级分蘖取样检测HPPD和UBI2基因的表达水平，以野生型金粳818为对照。具体步骤如下：
1)总RNA的提取(Trizol法)：
取新鲜叶片0.1-0.3g，液氮研磨至粉末状，每50-100mg组织加入1毫升Trizol试剂对组织进行裂解；将上述组织的Trizol裂解液转入1.5毫升离心管中，在室温(15-30℃)下放置5分钟；按照每1毫升Trizol后加0.2毫升氯仿的量加入氯仿，盖上离心管盖，在手中用力震荡15秒，在室温下(15-30℃)放置2-3分钟后，12000g(4℃)离心15分钟；去上层水相置于新离心管中，按照每1毫升Trizol加0.5毫升异丙醇的量加入异丙醇，在室温下(15-30℃)放置10分钟后，12000g(2-8℃)离心10分钟；弃上清，按照每1毫升Trizol加1毫升75％乙醇进行洗涤，涡旋混合，7500g(2-8℃)离心5分钟，弃上清；让沉淀的RNA在室温下自然干燥30分钟；加入50μl RNase-free水溶解RNA沉淀，通过电泳分析及浓度测定后放于-80℃冰箱保存。
2)RNA电泳分析：
制备浓度为1％的琼脂糖凝胶，然后吸取1μl RNA与1μl 2X Loading Buffer(上样缓冲液)混合后上样，电压设置为180V，电泳时间12分钟，电泳完成后，取出琼脂糖凝胶，在紫外凝胶成像系统中观察片段位置及亮度。
3)RNA纯度检验：
用微量蛋白核酸分析仪测定RNA浓度，较好的RNA OD260/OD280在1.8-2.1之间，低于1.8说明蛋白质污染严重，高于2.1说明RNA降解严重。
4)实时荧光定量PCR
通过专用反转录试剂盒，将提取的总RNA反转录得到cDNA。主要流程为：先测定提取的总RNA浓度，取1-4μg RNA的量用于反转录酶合成cDNA。cDNA于-20℃保存。
①按照下表，在冰上配制RNA模板溶液，置于PCR仪进行变性、退火反应。此过程利于模板RNA的变性以及引物和模板特异性退火，可以提高反转录效率。
表1 反转录变性退火反应体系
Figure PCTCN2020126747-appb-000007
变性、退火反应条件：
Figure PCTCN2020126747-appb-000008
②按表2配制反转录反应体系，合成cDNA：
表2 反转录反应体系
Figure PCTCN2020126747-appb-000009
合成cDNA反应条件：
Figure PCTCN2020126747-appb-000010
③选用水稻的UBQ5基因作为内参基因，以合成的cDNA为模板进行荧光定量PCR。使用表3所列引物，按表4配制反应液。
表3 荧光定量引物序列5’-3’
Figure PCTCN2020126747-appb-000011
Figure PCTCN2020126747-appb-000012
表4 实时定量PCR(Real Time PCR)反应液
Figure PCTCN2020126747-appb-000013
④按表5实时定量PCR反应步骤进行程序反应，反应程序为40个循环
表5 实时定量PCR反应步骤
Figure PCTCN2020126747-appb-000014
5)数据处理及实验结果
如表6所示，以UBQ5作为内参，目的基因的Ct值减去UBQ5的Ct值为ΔCt，之后计算2 -ΔCt，代表目的基因的相对表达水平，818CK1，818CK3为两个野生型金粳818对照植株；13M，20M代表QY2091-13，QY2091-20T0植株的主分蘖叶片样品；13L，20L代表QY2091-13，QY2091-20T0植株用于除草剂抗性测试的次级分蘖叶片样品。
表6 不同基因Ct值及相对表达倍数
  UBQ5 平均值 UBI2 ΔCt 2 -ΔCt 平均值 HPPD ΔCt 2 -ΔCt 平均值
  23.27   17.56 -5.88 58.95   20.81 -2.63 6.20  
  23.55   17.71 -5.73 53.09   21.01 -2.43 5.40  
818CK1 23.51 23.44 17.66 -5.78 55.06 55.70 20.98 -2.47 5.52 5.71
  23.45   17.88 -5.50 45.20   20.93 -2.44 5.43  
  23.19   17.94 -5.44 43.41   21.13 -2.24 4.74  
818CK3 23.49 23.37 17.72 -5.65 50.26 46.29 21.14 -2.24 4.72 4.96
  24.61   19.56 -4.92 30.32   20.23 -4.25 19.07  
  24.27   19.52 -4.96 31.05   20.29 -4.19 18.28  
13M 24.56 24.48 19.16 -5.32 39.97 33.78 20.48 -4.00 15.99 17.78
  23.98   18.76 -5.20 36.70   19.02 -4.94 30.64  
  23.89   18.52 -5.43 43.19   19.07 -4.89 29.56  
13L 24.00 23.96 18.81 -5.14 35.34 38.41 19.07 -4.88 29.45 29.88
  24.34   19.01 -5.40 42.30   19.37 -5.04 32.98  
  24.41   19.07 -5.34 40.64   19.33 -5.09 34.05  
20M 24.49 24.41 19.29 -5.13 35.00 39.32 19.26 -5.16 35.65 34.22
  24.63   19.46 -5.11 34.52   19.88 -4.69 25.83  
  24.67   19.38 -5.19 36.48   19.91 -4.66 25.31  
20L 24.41 24.57 19.42 -5.15 35.61 35.54 19.86 -4.71 26.16 25.77
结果如图10所示，以水稻UBQ5为内参基因，对OsHPPD和UBI2基因的相对表达量进行测定，结果显示，HPPD加倍株系与野生型相比HPPD表达量明显上升，说明融合的UBI2强启动子确实提高了HPPD的表达水平，形成了一个高表达的HPPD新基因，起到了敲高HPPD基因的作用。UBI2的表达量略有下降，可能是由于在启动子区域的编辑产生的小范围突变对基因表达造成一定影响，我们也确实检测到UBI2靶点处的碱基插入、缺失或小片段删除。与野生型相比UBI2和HPPD表达量明显趋于一致，符合理论预期；其中20M样品的HPPD表达量较野生型CK3组最高提高6倍左右。
以上结果证明，根据原生质体中测试有效的染色体片段加倍方案，通过在农杆菌转化和组培过程中引入多轮分子鉴定，我们能够筛选出发生加倍事件的愈伤和转化苗，并且转化苗中产生的新HPPD基因融合的UBI2强启动子确实提高了HPPD基因的表达水平，能够使植株产生对HPPD抑制剂类除草剂双唑草酮的抗性，最高能达到大田剂量的8倍，创制出了敲高内源HPPD基因的抗除草剂水稻。以此为例，采用实施例1，实施例2的染色体片段加倍技术方案也可对其它需要改变基因表达模式的内源基因引入所需的启动子加以融合创造新基因，在植物中可以通过农杆菌介导转化创制具有所需基因表达模式的新品种。
实施例3染色体片段加倍敲高内源HPPD基因表达抗除草剂水稻株系T1代的分子检测和除草剂抗性测试
野生型水稻基因组中HPPD基因与UBI2基因物理距离为338kb，如图1方案1所示，两者间的染色体片段复制加倍后染色体长度增加了338kb在复制片段的接头处产生了UBI2启动子驱动HPPD CDS区的高表达HPPD新基因。为了确认新基因能否稳定遗传以及染色体片段加倍后对遗传稳定性的影响，我们对HPPD加倍株系的T1代进行了分子检测和除草剂抗性测试。
首先我们观察到加倍事件对T0代植株的育性没有明显影响，所有阳性T0株系都能正常结实。继续选择QY2091-13，QY2091-20株系进行T1代苗种植检测。
1、样品准备：
QY2091-13，共种植36株T1代苗，其中27株生长正常，9株白化。取32株提DNA进行检测，编号1-24为正常苗，编号25-32为白化苗。
QY2091-20，共种植44株T1代苗。其中33株生长正常，11株白化。取40株提DNA进行检测，编号1-32正常苗，编号33-40白化苗。
观察到T1代植株中出现白化苗，推测由于HPPD是植物合成叶绿素通路中的关键酶，双靶点编辑的T0代植株中可能存在染色体片段加倍，删除，倒位，编辑靶点处的小片段突变等多种基因型的嵌合，如果HPPD基因被破坏例如HPPD CDS区被删除，则植株会产生白化表型，设计不同的引物对进行PCR检测可能的基因型。
2、PCR分子鉴定：
1)检测引物序列：序列5’-3’
Primer 8F：TCTGTGTGAAGATTATTGCCACTAGTTC
Primer 6R：GAGTTCCCCGTGGAGAGGT
Test 141-F：CCCCTTCCCTCTAAAAATCAGAACAG
Primer 4R：GGGATGCCCTCCTTATCTTGGATC
Primer 3F:CCTCCATTACTACTCTCCCCGATTC
Primer 7R：GTGTGGGGGAGTGGATGACAG
pg-Hyg-R1：TCGTCCATCACAGTTTGCCA
pg-35S-F：TGACGTAAGGGATGACGCAC
2)上述引物结合位点见图11，其中Primer 8F+Primer 6R用于检测染色体片段加倍后UBI2启动子与HPPD CDS的融合片段，产物长度630bp；Test 141-F+Primer 4R用于检测染色体片段删除事件，产物长度222bp。pg-Hyg-R1+pg-35S-F用于检测编辑载体T-DNA片段，产物长度660bp。
3)PCR反应体系，反应程序和凝胶电泳检测参照实施例1。
3、分子检测结果：
加倍和删除事件的检测结果如表7所示，可见T1代植株中染色体片段加倍事件和删除事件均有分布，不同株系之间比例有差异，QY2091-13中加倍事件的比例29/32高于QY2091-20的21/40，应该是由T0代植株中嵌合比例不同引起的，检测结果说明加倍产生的融合基因是可遗传的。
表7 加倍和删除事件的检测结果
QY2091-20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
加倍 + - - - + + + - - - + - - - - - + + + -
删除 - - - - - + - - + - - + - - - - - - - -
  21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
加倍 - + - - + - + + + - + + + - + + + + + -
删除 - - + + - + - - - + + - + - + - - - - +
Figure PCTCN2020126747-appb-000015
对上述T1苗使用pg-Hyg-R1+pg-35S-F引物检测编辑载体T-DNA片段，QY2091-20-17和QY2091-13-7PCR电泳结果为T-DNA片段阴性，纯合加倍，可见加倍事件T1代中能够分离出加倍纯合的非转基因株系。
4、编辑事件测序检测：
对QY2091-20，选择纯合加倍阳性T1代样品1、5、7、11、18、19，对QY2091-13，选择纯合加倍阳性T1代样品1、3、7、9、10、12，测序加倍融合片段，同时扩增左侧HPPD靶点，右侧UBI2样品，送测序检测靶点处的编辑事件。其中Primer 3F+Primer 7R用于检 测左侧HPPD靶点编辑事件，野生型对照产物长度481bp；Primer 8F+Primer 4R用于检测右侧UBI2靶点编辑事件，野生型对照产物长度329bp。
1)加倍事件的基因型：
QY2091-13检测HPPD加倍测序结果见SEQ ID NO:18，QY2091-20检测HPPD加倍测序结果见SEQ ID NO:19，如图12所示，与预测的加倍接头序列比对可见，QY2091-13在接头处插入了一个T碱基，QY2091-20在接头处删除了19个碱基，插入和删除都发生在UBI2的启动子区域，对HPPD蛋白编码区无影响。根据实施例2中的HPPD基因表达量检测结果，这些UBI2启动子融合HPPD CDS区的新HPPD基因表达量明显提高。
2)两侧原始HPPD和UBI2靶点处的编辑事件：
两侧靶点处的编辑事件类型更多样，两个株系的HPPD启动子区存在单个碱基的插入，17个碱基的删除，16个碱基的删除三种编辑类型，UBI2启动子区存在7个碱基的插入，3个碱基的删除两种编辑类型。用于检测取样的T1植株均为绿苗，生长正常，说明这些启动子区域的小范围突变对基因功能无明显影响，可以从后代中选育出抗除草剂的水稻品种。
5、T1代幼苗的除草剂抗性测试：
对QY2091 HPPD加倍株系的T1代进行苗期的除草剂抗性测试，将T1代种子进行表面消毒后，在加入1.2μM双唑草酮的1/2MS培养基上萌发，28℃，16小时光照，8小时黑暗培养，以野生型金粳818为对照。
抗性检测结果如图13所示，光照培养10天后，野生型对照水稻苗显现白化症状，基本全部白化，而HPPD加倍事件株系QY2091-7，13，20，22有黄化苗与绿苗的表型分离。根据前述分子检测结果，T1代中存在基因型分离，不加除草剂处理就有白化苗出现，而绿苗在添加1.2μM双唑草酮之后，继续保持绿色，生长正常。该测试结果说明HPPD基因加倍株系对双唑草酮的高抗性能够稳定遗传到T1代。
实施例4通过诱导染色体片段倒位敲高内源PPO基因表达的编辑方法-水稻原生质体测试
水稻PPO1(也称PPOX1)基因(如SEQ ID NO:7所示，其中1-1065bp为启动子，其余为表达区)位于1号染色体上，其下游911kb处为calvin cycle protein CP12基因(如SEQ ID NO:8所示，其中1-2088bp为启动子，其余为表达区)，其方向与PPO1基因相对。根据国际水稻基因组测序计划(International Rice Genome Sequencing Project)提供的水稻基因表达谱数据(http://rice.plantbiology.msu.edu/index.shtml)分析，在水稻叶片中CP12基因表达强度是PPO1基因的50倍，CP12基因启动子是一个在叶片中高表达的强启动子。
如图4所示方案1，通过在两个基因各自的启动子和CDS区域之间同时造成双链断裂，经过筛选，可以获得两个断口之间区域倒置，将PPO1基因的启动子换成了CP12基因的启动子，提升PPO1基因的表达量，进而获得对PPO抑制剂类除草剂的抗性，选育抗除草剂品种。此外按照图4所示方案2，先倒置再加倍，也可以创制由CP12基因的启动子驱动PPO1的新基因。
1、首先将水稻PPO1和CP12基因组DNA序列输入CRISPOR在线工具(http://crispor.tefor.net/)寻找可用的编辑靶点，经在线评分在PPO1和CP12基因各自的启动子和CDS区之间选择如下靶点进行测试：
Figure PCTCN2020126747-appb-000016
其中guide RNA1和guide RNA2位于PPO1基因启动子与CDS区之间，靠近PPO1蛋白起始密码子，guide RNA3和guide RNA4位于CP12基因启动子与CDS区之间，靠近CP12蛋白起始密码子。
参照实施例1，以pHUE411为骨架，针对上述靶点设计引物构建双靶点载体：
Figure PCTCN2020126747-appb-000017
具体而言，以pCBC-MT1T2质粒(https://www.addgene.org/50593/)为模板分别扩增sgRNA1+3、sgRNA1+4、sgRNA2+3、sgRNA2+4双靶点片段，构建sgRNA表达盒。BsaI酶切pHUE411载体骨架，切胶回收，目的片段经酶切后直接用于连接反应。采用T4DNA连接酶连接载体骨架与目的片段，转化连接产物至Trans5α感受态细胞，挑取不同的单克隆测序，测序正确后利用思科捷高纯度质粒小量提取试剂盒抽提质粒，得到重组质粒，分别命名为pQY002095、pQY002096、pQY002097、pQY002098，如下所示：
pQY002095   pHUE411-PPO-sgRNA1+3含OsPPO-guide RNA1，guide RNA3组合
pQY002096   pHUE411-PPO-sgRNA2+3含OsPPO-guide RNA2，guide RNA3组合
pQY002097   pHUE411-PPO-sgRNA1+4含OsPPO-guide RNA1，guide RNA4组合
pQY002098   pHUE411-PPO-sgRNA2+4含OsPPO-guide RNA2，guide RNA4组合
2、对上述pQY002095-002098载体制备高纯度、高浓度质粒，具体操作参照实施例1步骤2。
3、制备水稻原生质体并对上述载体进行PEG介导转化，具体操作参照实施例1步骤3。
4、进行基因组打靶和新基因产生情况检测，使用下表中的检测引物进行PCR检测，具体操作参照实施例1步骤4。
Figure PCTCN2020126747-appb-000018
其中PPO-R2和CP-R2组合用于扩增染色体片段倒位后右侧产生的CP12启动子驱动 PPO1CDS新基因片段，PPO-F2和CP-F2组合用于扩增倒位后左侧产生的PPO1启动子驱动CP12CDS新基因片段。双靶点编辑可能产生的基因型及分子检测引物的结合位点如图14所示。
5、PCR和测序结果显示，水稻原生质体转化获得了预期的CP12启动子驱动PPO1表达的新基因，转化的水稻原生质体基因组DNA中能够检测到水稻CP12基因启动子与PPO1基因表达区融合的编辑事件，说明通过染色体片段倒位形成新PPO基因的方案可行，能够创制出强启动子驱动表达的PPO新基因，定义为PPO1倒位事件。其中pQY002095载体转化原生质体检测染色体片段倒置测序结果如SEQ ID NO:15所示；pQY002095载体转化原生质体检测染色体片段删除测序结果如SEQ ID NO:16所示；pQY002098载体转化原生质体检测染色体片段倒置测序结果如SEQ ID NO:17所示。
实施例5通过农杆菌介导转化创制染色体片段倒位敲高内源PPO基因表达的抗除草剂水稻
1、敲高编辑载体的构建：根据原生质体测试的结果，选择编辑效率高的OsPPO-guide RNA1：5’CCATGTCCGTCGCTGACGAG3’和OsPPO-guide RNA4：5’CGGATTTCTGCGT-GTGATGT3’双靶点组合，构建农杆菌转化载体pQY2234，载体骨架采用pHUE411并进行水稻密码子优化，载体图谱如图16所示。
2、农杆菌转化水稻愈伤与二轮分子鉴定：
取pQY2234质粒按实施例2步骤2所述方法转化水稻愈伤，受体品种为淮稻5号和金粳818。在愈伤筛选阶段对潮霉素抗性愈伤进行两轮分子鉴定，选择倒位事件阳性愈伤进行分化。在愈伤分子检测阶段，我们用PPO-R2和CP-R2组合扩增染色体片段倒位后右侧产生的CP12启动子驱动PPO1CDS新基因片段作为倒位事件阳性标准，对倒位后左侧的CP12新基因在愈伤分化出苗后再予以考虑。共鉴定淮稻5号愈伤734个，其中倒位事件阳性24个，金粳818愈伤259个，其中倒位事件阳性29个。图17显示了金粳818编号192-259愈伤的PCR检测结果。
3、共53个倒位事件阳性愈伤经二轮分子鉴定后共分化得到9个加倍事件阳性愈伤经二轮分子鉴定后共分化得到1875株T0代幼苗，其中淮稻5号背景768株，金粳818背景1107株，对这1875株幼苗继续使用PPO-R2和CP-R2引物对进行第三轮分子鉴定，其中淮稻5号背景有184株有倒位阳性条带，金粳818背景有350株有倒位阳性条带，阳性苗移入温室培养。
4、PPO1倒位苗(T0代)PPO抑制剂类除草剂抗性测试：
QY2234T0代鉴定倒位事件阳性转化苗移栽到温室的大塑料桶里培育以扩繁T1代种子。阳性苗数量较多，所以选择部分T0苗与生长时期、状态相近的野生型对照品种，待植株高度达到20厘米左右后，直接进行除草剂抗性测试。所用除草剂为本公司生产的高效PPO抑制剂类除草剂
Figure PCTCN2020126747-appb-000019
(代号2081，参见专利CN202010281666.4)，本次实验其剂量 设置为0.18，0.4，0.6g a.i./mu三个梯度，采用步入式喷雾塔施药。
抗性检测结果如图18所示，施药后3-5天，野生型对照水稻苗从叶尖开始干枯，叶片上出现坏死斑，植株逐渐枯死，而PPO1倒位事件多数株系保持正常生长，叶片未见明显药害。另有部分株系出现药害，可能是由于T0代株系编辑事件的多基因型嵌合，PPO1表达量不高引起的。施药2周之后，野生型水稻苗死亡，倒位事件多数株系继续保持绿色，生长正常。测试结果说明PPO1倒位株系能够明显提升植株对2081的耐受性。
5、PPO1倒位苗(T0代)PPO1基因相对表达量定量检测：
推测PPO1基因倒位株系对2081的抗性提升是由于CP12的强启动子与PPO1基因CDS融合提高了PPO1表达量引起的，因此选择淮稻5号背景的T0代株系QY2234-252，QY2234-304，QY2234-329分别取主分蘖和次级分蘖取样检测PPO1和CP12基因的表达水平，以野生型淮稻5号为对照。具体操作步骤参照实施例2步骤6，选用水稻UBQ5基因作为内参基因，荧光定量引物如下：5’-3’
Figure PCTCN2020126747-appb-000020
以UBQ5作为内参，目的基因的Ct值减去UBQ5的Ct值为ΔCt，之后计算2 -ΔCt，代表目的基因相对表达水平，H5CK1、H5CK2为两个野生型淮稻5号对照植株，252M、304M、329M代表QY2234-252，QY2234-304、QY2234-329T0植株的主分蘖叶片样品，252L、304L、329L代表次级分蘖叶片样品。结果见下表8：
表8 不同基因Ct值及相对表达倍数
Figure PCTCN2020126747-appb-000021
Figure PCTCN2020126747-appb-000022
不同株系PPO1和CP12的相对表达量如图19所示，结果显示，与实施例2中的加倍事件不同的是，倒位事件各个株系间的基因表达水平有明显差异。两个淮稻5号CK组之间CP12的表达量差异很大，可能是苗期生长阶段或不同导致的表达量差异。实验组与H5CK2对照组相比CP12的表达量都有下降趋势，其中252L、329M的PPO1的表达水平明显上升，304L、329L PPO1的表达水平有所提高，而252M、304M PPO1的表达量下降。与染色体片段加倍主要提高基因表达水平不同，染色体片段倒位在左右两侧都产生了新基因，在两侧靶点处可能有发生多种编辑事件，同时转录方向的改变可能对基因表达量也有影响，所以T0代植株是复杂的嵌合体，同一植株的主分蘖和次级分蘖之间的基因表达量可能也有明显差异，从定量PCR的结果来看，PPO1倒位事件有较高比率上调PPO1基因的表达，对倒位事件进行抗除草剂筛选能够从中筛选出高表达PPO1抗除草剂的株系。
以上结果证明，根据原生质体中测试有效的染色体片段倒位方案，通过在农杆菌转化和组培过程中引入多轮分子鉴定，我们能够筛选出发生倒位事件的愈伤和转化苗，并且转化苗中产生的新PPO1基因融合的CP12强启动子确实可以提高PPO1基因的表达水平，能够使植株产生对PPO抑制剂类除草剂2081的抗性，创制出了敲高内源PPO基因的抗除草剂水稻。以此为例，采用实施例4，实施例5的染色体片段倒位技术方案也可对其它需要改变基因表达模式的内源基因引入所需的启动子加以融合创造新基因，在植物中可以通过农杆菌介导转化创制具有所需基因表达模式的新品种。
实施例6染色体片段倒位敲高内源PPO1基因表达抗除草剂水稻株系T1代的分子检测和除草剂抗性测试
野生型水稻基因组PPO1基因与CP12基因物理距离为911kb，如图14所示，两者间的染色体片段倒位后在右侧产生了CP12启动子驱动PPO1CDS区的高表达PPO1新基因，也有可能发生染色体片段的删除。为了确认新基因能否稳定遗传以及染色体片段倒位后对遗传稳定性的影响，我们对PPO1倒位株系的T1代进行了分子检测和除草剂抗性测试。
首先我们观察到倒位事件对T0代植株的育性没有明显影响，所有阳性T0株系都能正常结实。选择淮稻5号背景的QY2234/H5-851株系T1代进行检测。
1、样品准备：
QY2234/H5-851，共种植48株T1代苗，均生长正常。
2、PCR分子鉴定：
1)检测引物序列：5’-3’
PPO-R2：AAGGCTGGAAGCTGTTGGG
CP-R2：CTGAGGAGGCGATAAGAAACGA
PPO-F2：CGGACTTATTTCCCACCAGAA
CP-F2：AGGTCTCCTTGAGCTTGTCG
pg-Hyg-R1：TCGTCCATCACAGTTTGCCA
pg-35S-F：TGACGTAAGGGATGACGCAC
2)上述引物结合位点见图14，其中PPO-R2+CP-R2用于检测染色体片段倒位后右侧CP12启动子与PPO1编码区融合片段，产物长度507bp；PPO-F2+CP-F2用于检测染色体片段倒位后左侧PPO1启动子与CP12编码区融合片段，产物长度560bp；PPO-F2+PPO-R2用于检测倒位前左侧PPO靶点，野生型对照产物长度586bp；CP-F2+CP-R2用于检测倒位前右侧CP12靶点，野生型对照产物长度481bp。pg-Hyg-R1+pg-35S-F用于检测编辑载体T-DNA片段，产物长度660bp。
3)PCR反应体系及反应条件：
反应体系(10μL体系)：
Figure PCTCN2020126747-appb-000023
反应条件：
Figure PCTCN2020126747-appb-000024
PCR产物1％琼脂糖凝胶电泳，电压180V，10分钟。
3、分子检测结果：
检测结果如表9所示，共检测48株，其中纯合倒位12株(2/7/11/16/26/36/37/40/41/44/46/47)，杂合倒位21株(1/3/4/5/6/8/9/15/17/20/22/23/24/27/30/31/33/34/39/42/43)，纯合非倒位植株15株(10/12/13/14/18/19/21/25/28/29/32/35/38/45/48)。纯合倒位：杂合倒位：纯合非倒位比例1:1.75:1.25，约合1:2:1，检测结果符合孟德尔遗传定律，说明倒位产生的PPO1新基因是可遗传的。
表9 分子检测结果
Figure PCTCN2020126747-appb-000025
Figure PCTCN2020126747-appb-000026
对上述T1苗使用pg-Hyg-R1+pg-35S-F引物检测编辑载体T-DNA片段，其中16、41电泳结果为T-DNA片段阴性，纯合倒位，可见倒位事件T1代中能够分离出倒位纯合的非转基因株系。
4、编辑事件测序检测：
对倒位事件基因型的检测重点关注右侧新PPO基因的编辑事件，保留PPO1基因蛋白编码框完整的突变类型，对左侧CP12位点编辑事件通过温室和田间表型观察不影响植株正常生长即可保留，以下列出了在倒位事件阳性株系中检测到的编辑事件基因型，无缝代表与预测的倒位后融合片段序列完全一致，淮稻5号为背景的QY2234倒位成功基因型如下：
Figure PCTCN2020126747-appb-000027
其中部分测序峰图与序列比对结果见图20。
金粳818为背景的QY2234倒位成功基因型如下：
Figure PCTCN2020126747-appb-000028
其中部分测序峰图与序列比对结果见图21。
以上不同新PPO1基因CP12启动子与PPO1编码区融合片段测序结果见SEQ ID NO:20，SEQ ID NO:21，SEQ ID NO:22，SEQ ID NO:23，SEQ ID NO:24，SEQ ID NO:25，SEQ ID NO:26。
5、T1代幼苗的除草剂抗性测试：
对QY2234/H5-851 PPO1倒位株系的T1代进行苗期的除草剂抗性测试，以野生型淮稻5号为对照，与倒位株系T1代种子同时种植，幼苗株高达到15厘米后，喷施2081处理，药物浓度设置0.3，0.6，0.9，1.2g a.i./mu四个梯度，培养条件28℃，16小时光照，8小时黑暗。
抗性检测结果如图22所示，施药5天后，野生型对照水稻苗在0.3g a.i./mu剂量下即表现明显药害，从叶尖开始干枯，叶片上出现坏死斑，在0.6g a.i./mu剂量下植株很快枯死。而QY2234/H5-851 T1苗在0.3g a.i./mu剂量下能保持正常生长，叶片未见明显药害，在0.6和0.9g a.i./mu剂量下，部分T1苗出现叶尖干枯的药害表现，但多数T1苗能保持绿色继续生长，而对照基本枯死。在1.2g a.i./mu剂量下，对照已完全死亡，T1苗还有部分能保持绿色继续生长。该测试结果说明PPO1基因倒位株系对2081的抗性能够稳定遗传到T1代。
实施例7在植物中敲高内源EPSPS基因表达的编辑方法
EPSPS是植物中芳香族氨基酸合成通路中的关键酶，是灭生性除草剂草甘膦的靶点。高表达EPSPS基因可以赋予植物对草甘膦的抗性。EPSPS基因(如SEQ ID NO:4所示，其中1-1897bp为启动子，其余为表达区)位于水稻6号染色体上，其上游基因为转酮醇酶transketolase(TKT，如SEQ ID NO:3所示，其中1-2091bp为启动子，其余为表达区)，方向与EPSPS基因相反。TKT基因在叶片中的表达强度是EPSPS基因的20-50倍。如图2所示，通过在两个基因各自的启动子和CDS区域之间同时造成双链断裂，经过筛选，可以获得两个断口之间区域倒置(方案1)，或倒置加倍(方案2)的情形。这两种情形均会将EPSPS基因的启动子换成TKT基因的启动子，提升EPSPS基因的表达量，进而获得对草甘膦的抗性。此外，图2中所示的方案3,4,5也可以分别创制出TKT基因启动子驱动EPSPS的新基因。EPSPS与TKT相邻且方向相对的基因结构在单子叶植物中保守(表10)，在双子叶植物二者也是相邻，方向相同；因此该方法在植物中具有通用性。
表10 不同植物中EPSPS基因与相邻的TKT基因的距离
Figure PCTCN2020126747-appb-000029
为此，以pHUE411为骨架，以如下靶点为目标：
Figure PCTCN2020126747-appb-000030
构建了多个不同的双靶点载体：
Figure PCTCN2020126747-appb-000031
(2)使用下表中的相关检测引物，扩增含有两侧靶位点的片段或预测的UBI2的启动子和HPPD编码区融合产生的片段，产物长度为300-1000bp之间。
Figure PCTCN2020126747-appb-000032
经过原生质转化，测试结果表明获得了预期倒置的事件，如图15所示，pQY002062载体转化原生质体检测倒置测序结果如SEQ ID NO:11所示；pQY002062载体转化原生质体检测删除测序结果如SEQ ID NO:12所示；pQY002093载体转化原生质体检测倒置测序结果如SEQ ID NO:13所示；pQY002093载体转化原生质体检测删除测序结果如SEQ ID NO:14所示。
将这些载体转至农杆菌中，转化水稻愈伤，获得了含有新EPSPS基因的植株，除草剂生测表明该植株对草甘膦除草剂具有明显的抗性。
实施例8在拟南芥中敲高内源PPO基因表达的编辑方法
原卟啉原氧化酶(PPO)是除草剂的主要靶点之一。高表达植物内源PPO，即可显著提升对PPO抑制剂类除草剂的抗性。拟南芥PPO基因(如SEQ ID NO:1所示，其中1-2058bp为启动子，其余为表达区)位于4号染色体上，其下游1.9M处是ubiquitin10基因(如SEQ ID NO:2所示，其中1-2078bp为启动子，其余为表达区)，并且与PPO基因的方向相同。
如图3所示方案，通过同时在PPO和ubiquitin10基因的启动子和CDS区之间的位置产生双链断裂，经过筛选可以获得两个断口之间区域加倍的事件，即ubiquitin10的启动子和PPO的编码区融合在一起的新基因。此外，按照图1中所示的方案2，也可以创制出ubiquitin10的启动子和PPO的编码区融合在一起的新基因。
为此，以pHEE401E为骨架(https://www.addgene.org/71287/)，针对如下靶点：
Figure PCTCN2020126747-appb-000033
按照“Wang ZP,Xing HL,Dong L,Zhang HY,Han CY,Wang XC,Chen QJ.Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation.Genome Biol.2015 Jul 21；16:144.”所述方法，构建双靶点载体：
Figure PCTCN2020126747-appb-000034
按照如下方法转化拟南芥：
(1)农杆菌转化
将构建好的重组质粒转化农杆菌GV3101感受态细胞，得到重组农杆菌。
(2)农杆菌侵染液的制备
1)挑取活化的农杆菌接种于30毫升YEP液体培养基(含25mg/L Rif和50mg/L Kan)中，28℃ 200转/分钟振荡培养过夜至OD600值为1.0-1.5左右。
2)6000转/分钟离心10分钟收集菌体，弃上清。
3)用侵染液(无须调节pH)重新悬浮菌体至OD600＝0.8左右备用。
(3)拟南芥转化
1)植株转化之前注意植株是否生长良好，花序茂盛，无胁迫反应，第一次转化株高20cm即可进行。若土壤干燥可适当浇一些水。转化前一天用剪刀将已长出的角果减去。
2)将待转化植株花序浸泡于上述溶液中30秒-1分钟，期间轻轻搅动，浸润后的植株上应该有一层液体膜。
3)转化完成后植株放于黑暗环境中暗培养24小时，然后取出放于正常光照环境下生长。
4)一周以后即可按同样的方法进行第二次转化。
(4)种子收获
待种子成熟后即可进行收获，收获后置于37℃烘箱一周左右烘干种子。
(5)转基因植株的筛选
种子经过消毒液处理5分钟，用ddH 2O洗5遍后均匀铺于MS筛选培养基(含30μg/毫升Hyg、100μg/毫升Cef)上，将培养基放于光照培养箱培养(温度22℃，16小时光照，8小时黑暗，光照强度100—150μmol/m 2/s，湿度75％)，一周后选取阳性幼苗移栽至土壤。
(6)T1突变植株的检测
(6.1)基因组DNA提取
1)剪取拟南芥叶片约200mg于2毫升离心管中，加入钢珠，利用高通量组织破碎仪进行叶片研磨。
2)待研磨充分后，加入400μL SDS提取缓冲液，上下颠倒混匀，置于65℃水浴锅中孵育15分钟，期间每隔5分钟颠倒混匀一次。
3)13000rpm离心5分钟。
4)吸取300μL上清液转移至一新的1.5毫升离心管中，向离心管中加入等体积经过-20℃预冷的异丙醇，将离心管置于-20℃1小时或者过夜。
5)13000rpm离心10分钟，去掉上清液。
6)向离心管中加入500μL 70％乙醇洗涤沉淀，离心后倒掉洗涤液(注意不要倒掉沉淀)，置于室温晾干后，加入30μL ddH 2O溶解DNA，置于-20℃保存DNA。
(6.2)PCR扩增
以提取的T1植株基因组为模板，用检测引物进行靶点片段扩增，吸取5μL扩增产物经1％琼脂糖凝胶电泳检测，并经凝胶成像仪成像。剩余产物交由测序公司直接进行测序。
测序结果表明，在拟南芥中成功实现了AtPPO1基因加倍，除草剂抗性测试表明加倍的植株具有对PPO除草剂的抗性。
实施例9在斑马鱼中创制具有新表达特性的GH1基因
鱼类中生长激素(growth hormone；GH)基因，控制其生长发育速度。目前通过转基因技术在大西洋三文鱼中高表达GH基因能够将其生长速度显著提升，具有极大的应用价值，但是经过了数十年才通过审批上市。GH1基因是斑马鱼中的生长激素基因。本发明将斑马鱼中合适的启动子(持续表达、强度适宜、组织特异性适宜)在体内通过删除、倒置、加倍、倒置加倍、染色体转移等方式融合在一起，创造出一个生长迅速的鱼类品种。
说明书中提及的所有出版物和专利申请均通过引用并入本文，如同每篇出版物或专利申请被单独、特别地通过引用并入本文一样。
尽管为清楚理解起见，前述发明已通过举例说明和实施例的方式较为详细地进行了描述，但显而易见的是，可以在所附权利要求书的范围内实施某些改变和修改，这样的改变和修改均在本发明的范围之内。

Claims (72)

  1. 一种在生物体内创制新基因的方法，其特征在于，包括以下步骤：
    在生物体基因组中至少两个不同的特定位置上同时产生DNA断裂，其中所述特定位置是能够分割不同基因元件或不同蛋白结构域的基因组位点，所述DNA断裂通过非同源末端连接(NHEJ)或同源修复的方式互相连接，产生所述不同基因元件或不同蛋白结构域之间不同于原始基因组序列的新组合，形成新基因。
  2. 根据权利要求1所述的方法，其特征在于，所述的“至少两个不同的特定位置”可以位于同一条染色体上，也可以位于不同染色体上。
  3. 根据权利要求1或2所述的方法，其特征在于，所述的“至少两个不同的特定位置”可以在至少两个不同基因上的特定位置，也可以在同一基因上的至少两个不同的特定位置。
  4. 根据权利要求3所述的方法，其特征在于，所述的“至少两个不同基因”的转录方向可以相同，也可以不同。
  5. 根据权利要求1-4任意一项所述的方法，其特征在于，所述的“DNA断裂”是通过将具有靶向特性的核酸酶递送到生物体细胞内与基因组DNA特定位置接触实现的。
  6. 根据权利要求5所述的方法，其特征在于，所述的“具有靶向特性的核酸酶”为Meganuclease、Zinc finger nuclease、TALEN或CRISPR/Cas系统。
  7. 根据权利要求5或6所述的方法，其特征在于，所述“具有靶向特性的核酸酶”以DNA形式存在。
  8. 根据权利要求5或6所述的方法，其特征在于，所述“具有靶向特性的核酸酶”以mRNA或蛋白形式存在，而非DNA形式。
  9. 根据权利要求5-8任意一项所述的方法，其特征在于，将具有靶向特性的核酸酶递送到细胞内的方法选自1)PEG介导的细胞转染的方法；2)脂质体介导的细胞转染的方法；3)电击转化的方法；4)显微注射；5)基因枪轰击；或6)农杆菌介导的转化方法。
  10. 根据权利要求1-9任意一项所述的方法，其特征在于，所述“基因元件”包括基因的启动子、5’非编码区、编码区或非编码RNA区、3’非编码区和终止子。
  11. 根据权利要求1-10任意一项所述的方法，其特征在于，所述不同基因元件的组合为两个具有不同表达模式的基因其中之一的启动子和另一基因的编码区或非编码RNA区的组合。
  12. 根据权利要求11所述的方法，其特征在于，所述不同基因元件的组合中一个为生物内源强启动子，另外一个为HPPD、EPSPS、PPO或GH1基因编码区。
  13. 根据权利要求1-10任意一项所述的方法，其特征在于，所述不同基因元件的组合为两个具有不同表达模式的基因其中之一的启动子至5’非编码区和另一基因的编码区或非编码RNA区的组合。
  14. 根据权利要求11或13所述的方法，其特征在于，所述“不同表达模式”为基因表达水平强弱的差异。
  15. 根据权利要求11或13所述的方法，其特征在于，所述“不同表达模式”为基因表达组织特异性的差异。
  16. 根据权利要求11或13所述的方法，其特征在于，所述“不同表达模式”为基因表达 发育时期特异性的差异。
  17. 根据权利要求1-10任意一项所述的方法，其特征在于，所述不同基因元件的组合为同一基因内相邻基因元件的组合。
  18. 根据权利要求1-17任意一项所述的方法，其特征在于，所述“蛋白结构域”是指对应于蛋白质特定功能结构域的DNA片段；优选地，包括核定位信号、叶绿体导肽、线粒体导肽、磷酸化位点、甲基化位点、跨膜结构域、DNA结合结构域、转录激活结构域、受体激活结构域或酶催化中心。
  19. 根据权利要求1-18任意一项所述的方法，其特征在于，所述不同蛋白结构域的组合为两个具有不同亚细胞定位的蛋白编码基因其中之一的定位信号区域和另一基因的成熟蛋白编码区域的组合。
  20. 根据权利要求19所述的方法，其特征在于，所述“不同亚细胞定位”包括核定位、胞质定位、细胞膜定位、叶绿体定位、线粒体定位或内质网膜定位。
  21. 根据权利要求1-18任意一项所述的方法，其特征在于，所述不同蛋白结构域的组合为两种不同生物学功能的蛋白结构域的组合。
  22. 根据权利要求21所述的方法，其特征在于，所述“不同生物学功能”包括识别特异DNA或RNA保守序列、激活基因表达、结合蛋白配体、结合小分子信号、离子结合或特异的酶促反应。
  23. 根据权利要求1-18任意一项所述的方法，其特征在于，所述不同蛋白结构域的组合为同一基因内相邻蛋白结构域的组合。
  24. 根据权利要求1-10和18任意一项所述的方法，其特征在于，所述基因元件和蛋白结构域的组合为同一基因内蛋白结构域与相邻的启动子、5’非编码区、3’非编码区或终止子的组合。
  25. 根据权利要求1-24任意一项所述的方法，其特征在于，所述的生物体是动物、植物或真菌。
  26. 一种采用权利要求1-25任意一项所述方法获得的新基因。
  27. 根据权利要求26所述的新基因，其特征在于，所述的新基因与原基因相比，或具有不同启动子并因此具有了不同时空或不同强度或不同发育时期的表达特性，或具有了新的氨基酸序列；优选地，所述的“新的氨基酸序列”，即可以是两个以上基因编码区的整体融合，也可以是编码区的部分融合或同一基因部分蛋白编码区的加倍。
  28. 根据权利要求26或27所述的新基因，其特征在于，所述新基因为高表达生物内源HPPD、EPSPS、PPO或GH1基因。
  29. 一种包含权利要求26-28任意一项所述基因的DNA。
  30. 一种利用包含权利要求26-28任意一项所述基因编码的蛋白或其生物活性片段。
  31. 一种重组表达载体，其包含权利要求26-28任意一项所述的基因，以及与之可操作地连接的启动子。
  32. 一种包含权利要求26-28任意一项所述基因的表达盒。
  33. 一种宿主细胞，其中包含有权利要求32所述的表达盒；优选地，所述宿主细胞是植 物细胞、动物细胞或真菌细胞。
  34. 采用权利要求33所述宿主细胞再生成的生物。
  35. 一种如权利要求26-28任意一项所述的基因在赋予或提高生物体抗性/耐受性性状或生长优势性状上的应用。
  36. 一种组合物，其包含：
    (a)两个具有不同表达模式的基因其中之一的启动子和另一基因的编码区或非编码RNA区；
    (b)两个具有不同表达模式的基因其中之一的启动子至5’非编码区和另一基因的编码区或非编码RNA区；
    (c)同一基因内相邻基因元件；
    (d)两个具有不同亚细胞定位的蛋白编码基因其中之一的定位信号区域和另一基因的成熟蛋白编码区域；
    (e)两种不同生物学功能的蛋白结构域；
    (f)同一基因内相邻蛋白结构域；或者，
    (g)同一基因内蛋白结构域与相邻的启动子、5’非编码区、3’非编码区或终止子。
  37. 根据权利要求36所述的方法，其特征在于，所述“不同表达模式”为基因表达水平强弱的差异。
  38. 根据权利要求36所述的方法，其特征在于，所述“不同表达模式”为基因表达组织特异性的差异。
  39. 根据权利要求36所述的方法，其特征在于，所述“不同表达模式”为基因表达发育时期特异性的差异。
  40. 根据权利要求36-39任意一项所述的方法，其特征在于，所述“不同亚细胞定位”包括核定位、胞质定位、细胞膜定位、叶绿体定位、线粒体定位或内质网膜定位。
  41. 根据权利要求36-40任意一项所述的方法，其特征在于，所述“不同生物学功能”包括识别特异DNA或RNA保守序列、激活基因表达、结合蛋白配体、结合小分子信号、离子结合或特异的酶促反应。
  42. 根据权利要求36-41任意一项所述的组合物，其在体内融合。
  43. 一种不依赖于外源DNA供体片段，在生物体中提高目标内源基因表达水平的编辑方法，包括以下步骤：
    分别在所述目标内源基因与可选的内源高表达基因的启动子和编码区之间选择特定位置同时产生DNA断裂，所述DNA断裂通过非同源末端连接(NHEJ)或同源修复的方式互相连接，产生目标内源基因编码区与可选的内源强启动子的体内融合，形成新的高表达内源基因。
  44. 根据权利要求43所述的编辑方法，其特征在于，所述目标内源基因与可选的内源高表达基因位于同一染色体上。
  45. 根据权利要求43所述的编辑方法，其特征在于，所述目标内源基因与可选的内源高表达基因位于不同染色体上。
  46. 一种在植物中敲高内源HPPD基因表达的编辑方法，其特征在于，将HPPD基因编码区与植物内源强启动子体内融合，形成新的高表达植物内源HPPD基因。
  47. 根据权利要求46所述的编辑方法，其特征在于，具体包括以下步骤：分别在HPPD基因与可选的内源高表达基因的启动子和编码区之间选择特定位置同时产生DNA断裂，所述DNA断裂通过细胞内修复途径互相连接，产生HPPD基因编码区与可选的内源强启动子的体内融合，形成新的高表达HPPD基因。
  48. 根据权利要求46或47所述的编辑方法，其特征在于，所述植物为水稻，优选地，强启动子为ubiquitin2基因的启动子。
  49. 一种采用权利要求46-48任意一项所述编辑方法获得的高表达植物内源HPPD基因。
  50. 一种高表达水稻内源HPPD基因，其具有选自下述的序列：
    (1)如SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:18或SEQ ID NO:19所示的核酸序列或其部分序列或其互补序列；
    (2)与(1)限定的任一序列具有至少75％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％以上同一性；或
    (3)在严谨条件下与(1)或(2)所示序列杂交的核酸序列。
  51. 一种在植物中敲高内源EPSPS基因表达的编辑方法，其特征在于，将EPSPS基因编码区与植物内源强启动子体内融合，形成新的高表达植物内源EPSPS基因。
  52. 根据权利要求51所述的编辑方法，其特征在于，具体包括以下步骤：分别在EPSPS基因与可选的内源高表达基因的启动子和编码区之间选择特定位置同时产生DNA断裂，所述DNA断裂通过细胞内修复途径互相连接，产生EPSPS基因编码区与可选的内源强启动子的体内融合，形成新的高表达EPSPS基因。
  53. 根据权利要求51或52所述的编辑方法，其特征在于，所述植物为水稻，优选地，强启动子为TKT基因的启动子。
  54. 一种采用权利要51-53任意一项所述编辑方法获得的高表达植物内源EPSPS基因。
  55. 一种高表达水稻内源EPSPS基因，其具有选自下述的序列：
    (1)如SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14所示的核酸序列或其部分序列或其互补序列；
    (2)与(1)限定的任一序列具有至少75％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％以上同一性；或
    (3)在严谨条件下与(1)或(2)所示序列杂交的核酸序列。
  56. 一种在植物中敲高内源PPO基因表达的编辑方法，其特征在于，将PPO基因编码区与植物内源强启动子体内融合，形成新的高表达植物内源PPO基因。
  57. 根据权利要求56所述的编辑方法，其特征在于，具体包括以下步骤：分别在PPO基因与可选的内源高表达基因的启动子和编码区之间选择特定位置同时产生DNA断裂，所述DNA断裂通过细胞内修复途径互相连接，产生PPO基因编码区与可选的内源强启动子的体内融合，形成新的高表达PPO基因。
  58. 根据权利要求56或57所述的编辑方法，其特征在于，所述植物为水稻或拟南芥， 优选地，在水稻中，强启动子为CP12基因的启动子；在拟南芥中，强启动子为ubiquitin10基因的启动子。
  59. 一种采用权利要求56-58任意一项所述编辑方法获得的高表达植物内源PPO基因。
  60. 一种高表达水稻内源PPO基因，其具有选自下述的序列：
    (1)如SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25或SEQ ID NO:26所示的核酸序列或其部分序列或其互补序列；
    (2)与(1)限定的任一序列具有至少75％、至少80％、至少85％、至少90％、至少95％、至少98％或至少99％以上同一性；或
    (3)在严谨条件下与(1)或(2)所示序列杂交的核酸序列。
  61. 一种包含权利要求49、50、54、55、59或60所述基因的DNA。
  62. 一种利用包含权利要求49、50、54、55、59或60所述基因编码的蛋白或其生物活性片段。
  63. 一种重组表达载体，其包含权利要求49、50、54、55、59或60所述的基因，以及与之可操作地连接的启动子。
  64. 一种包含权利要求49、50、54、55、59或60所述基因的表达盒。
  65. 一种宿主植物细胞，其中包含有权利要求64所述的表达盒。
  66. 采用权利要求65所述宿主植物细胞再生成的植物。
  67. 一种生产对于除草剂的抗性或耐受性提高的植物的方法，其中包括将权利要求65所述的宿主植物细胞再生成植物及其衍生后代。
  68. 根据权利要求67所述的方法，其特征在于，所述对于除草剂的抗性或耐受性提高的植物是将宿主植物细胞再生成的植物与野生型杂交通过遗传分离去除外源转基因成分的非转基因株系。
  69. 一种抗除草剂水稻，其包含权利要求50所述高表达水稻内源HPPD基因、权利要求55所述高表达水稻内源EPSPS基因和权利要求60所述高表达水稻内源PPO基因中的一种或两种以上组合。
  70. 根据权利要求69所述的水稻，其是非转基因的。
  71. 如权利要求49、50、54、55、59或60所述基因在提高植物细胞、植物组织、植物部分或植物的相应抑制性除草剂抗性或耐受性上的应用。
  72. 一种控制植物栽培场所的杂草的方法，其中所述植物包括权利要求66所述的植物、通过权利要求67或68的方法制备的植物或者权利要求69或70所述的水稻，所述方法包括对所述栽培场所施用控制杂草有效量的一种或多种HPPD、EPSPS或PPO抑制性除草剂。
PCT/CN2020/126747 2019-11-06 2020-11-05 在生物体内创制新基因的方法及应用 WO2021088923A1 (zh)

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CN114686498A (zh) * 2022-04-22 2022-07-01 中佳源物种(深圳)生物科技有限公司 苹果突变基因及编码蛋白、苹果突变体的制备方法

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