WO2018082611A1 - 在植物细胞内表达外源基因的核酸构建物及其应用 - Google Patents

在植物细胞内表达外源基因的核酸构建物及其应用 Download PDF

Info

Publication number
WO2018082611A1
WO2018082611A1 PCT/CN2017/109119 CN2017109119W WO2018082611A1 WO 2018082611 A1 WO2018082611 A1 WO 2018082611A1 CN 2017109119 W CN2017109119 W CN 2017109119W WO 2018082611 A1 WO2018082611 A1 WO 2018082611A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
gene
construct
expression cassette
gfp
Prior art date
Application number
PCT/CN2017/109119
Other languages
English (en)
French (fr)
Inventor
朱健康
王木桂
陆钰明
毛妍斐
Original Assignee
中国科学院上海生命科学研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201710061566.9A external-priority patent/CN108018306B/zh
Application filed by 中国科学院上海生命科学研究院 filed Critical 中国科学院上海生命科学研究院
Publication of WO2018082611A1 publication Critical patent/WO2018082611A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a nucleic acid construct for expressing a foreign gene in a plant cell and uses thereof.
  • CRISPR clustered regular interspaced short palindromic repeats
  • the mechanism of action of the above three gene-directed editing tools has similarities.
  • the nucleic acid cleavage enzyme is directed to the gene locus to be edited by the recognition sequence, and the subsequent cleavage causes double strand breaks of the DNA sequence (double strands break, DSB), activates the DNA repair pathway of the host cell, by means of non-homologous end-joining (NHEJ), or by homologous recombination (HR) when the homologous template is nearby The way to fix double-chain gaps.
  • NHEJ non-homologous end-joining
  • HR homologous recombination
  • TALEN or CRISPR-mediated gene targeting has also been successful in plants such as rice, Arabidopsis, and tobacco (Zhang et al., 2012; Feng et al., 2013; Shan et al., 2013), of course, these Gene targeting events are achieved at the protoplast level. But for plant varieties such as rice and Arabidopsis, Rebuilding plants through protoplasts is almost impossible at the moment, and only other techniques can be developed to overcome this difficulty.
  • Gao Caixia's research group of the Institute of Genetics and Development of the Chinese Academy of Sciences reported that the TALEN system was used to bind to single-stranded small-segment DNA donor molecules and co-transformed into wheat immature embryos by gene gun, realizing two positions in the genome.
  • the research group used a circular plasmid as a donor, and used the rice EPSPS gene as a target to achieve homologous recombination and directional insertion of the target gene by co-transformation between the gene gun and the Cas9 system.
  • the success rate of the experiment is still not high, only About 2% (Li et al., 2016).
  • the gene gun transformation method requires complex instrument consumables, and often causes a large number of foreign fragments to be randomly inserted into the genome.
  • a conventional transformation with Agrobacterium has sufficient target success rate, is suitable for most functional genes, and utilizes broad-spectrum screening markers such as the anti-hygromycin gene. Or targeted anti-kanamycin gene for targeted screening.
  • the present invention provides a tool suitable for carrying a large amount of expression of a foreign gene in a plant cell, and provides a simple and efficient method for achieving homologous recombination or site-directed knocking of an exogenous fragment in a plant genome.
  • the ability of WDV to replicate large amounts of exogenous fragments, while efficiently producing a DSB near the target site using efficient site-cutting nucleases such as ZFN, Talen, CRISPR/Cas9 or Cpf1 can be efficiently performed by targeted screening. Realize homologous recombination or directed insertion of genes in plant cells.
  • nucleic acid construct having a structure of formula I from 5' to 3':
  • A, B, C, D, E, F, G, and H are respectively elements for constituting the construct
  • Each "-" is independently a bond or nucleotide linkage sequence
  • A is the first long gene spacer sequence
  • B is an optional 5' homology arm sequence
  • C is a foreign gene expression cassette
  • D is an optional 3' homology arm sequence
  • E is an optional second expression cassette
  • F is a short gene spacer sequence
  • G is the coding sequence of a replication protein of the corn genus virus
  • H is the second long gene spacer sequence
  • the first long gene spacer sequence, the short gene spacer sequence, and the second long gene spacer sequence are derived from a genus Mastrevirus.
  • the zebravirus includes Wheat Dwarf Virus (WDV), Bean Yellow Dwarf Virus (BeYDV), Corn Line Virus (MSV), and Tobacco Yellow Dwarf Virus (TYDV).
  • WDV Wheat Dwarf Virus
  • BeYDV Bean Yellow Dwarf Virus
  • MSV Corn Line Virus
  • TYDV Tobacco Yellow Dwarf Virus
  • the exogenous gene expression cassette has a structure of formula II from 5' to 3':
  • C1, C2, and C3 are elements for constituting the exogenous gene expression cassette, respectively;
  • Each "-" is independently a bond or nucleotide linkage sequence
  • C1 is an optional first promoter sequence
  • C2 is the coding sequence of a foreign protein
  • C3 is a terminator sequence.
  • the exogenous gene expression cassette has the structure of Formula II, and the construct does not contain Element B, Element D, and Element E.
  • the exogenous gene expression cassette has the structure of Formula II, and the sequence of the element A is shown in positions 1-409 of SEQ ID NO.: 1.
  • the first promoter and terminator are a plant promoter and a plant terminator, respectively.
  • the first promoter comprises a 35S, Ubi, UBQ, SPL promoter or a combination thereof.
  • the terminator comprises a NOS terminator, a Poly A terminator.
  • the exogenous gene expression cassette has a structure of formula III from 5' to 3':
  • Ci, Cii, Ciii, Civ are elements for constituting the exogenous gene expression cassette, respectively;
  • Each "-" is independently a bond or nucleotide linkage sequence
  • Ci is the coding sequence of the first foreign protein
  • Cii is a 2A sequence or an IRES sequence
  • Ciii is the coding sequence of a second foreign protein (such as a selection marker);
  • Civ is a terminator sequence.
  • the first foreign protein coding sequence in element Ci does not comprise a start codon.
  • the first foreign protein coding sequence in element Ci does not comprise a stop codon.
  • the first foreign protein coding sequence in element Ci does not comprise a start codon and a stop codon.
  • the N-terminus of the first foreign protein further comprises a flexible amino acid linker sequence.
  • the element Cii has a self-shearing function.
  • the 2A sequence is as shown at positions 733-798 of SEQ ID NO.: 2.
  • the coding sequence for the second foreign protein (e.g., a selection marker) in element Ciii does not comprise a start codon.
  • the screening marker comprises a hygromycin resistance gene (Hyg), a kanamycin resistance gene (NptII), a GFP gene, an Amp resistance gene, a glufosinate resistance gene (Bar). ).
  • the exogenous gene expression cassette has a structure of formula III, and said element A is a modified first long intergenic spacer sequence, said element A having no transcriptional drive capability.
  • sequence of the element A is shown as positions 1-329 of SEQ ID NO.: 1.
  • the exogenous gene expression cassette has a structure of formula III, and the construct contains element B, element D and element E which are not.
  • the exogenous gene expression cassette has the structure of formula III, and the construct contains element B and element D which are not.
  • the second expression cassette is an expression cassette for a targeted site recognition sequence of a site-directed nuclease.
  • the site-cutting nuclease comprises a zinc finger ribonuclease (ZFN), a transcriptional activator-like effector nuclease (TALEN), a regular cluster Interspersed cis-linked proteins (Cas) and Cpf1 (CRISPR from Prevotella and Francisella 1).
  • ZFN zinc finger ribonuclease
  • TALEN transcriptional activator-like effector nuclease
  • Cas regular cluster Interspersed cis-linked proteins
  • CRISPR from Prevotella and Francisella 1).
  • the recognition sequence expression cassette comprises an sgRNA expression cassette.
  • the sgRNA expression cassette comprises the following elements in order from 5' to 3':
  • CRISPR targeting site recognition sequence small guid RNA, sgRNA
  • the second promoter comprises: OsU3, OsU6a, OsU6b, OsU6c, AtU6-1, AtU3b, AtU3d, AtU6-1, AtU6-29.
  • the CRISPR targeting site recognition sequence is 17-22 nt in length.
  • the CRISPR targeting site sg is as set forth in SEQ ID NO.: 3 or SEQ ID NO.: 4, 1-20nt.
  • annular replicon can be formed between the element A and the element H.
  • the total length of the element B, the element C, the element D and the element E is less than 3000 bp.
  • the total length of the element B, the element C, the element D and the element E is 0-100 bp, preferably 0-3000 bp.
  • the element B and the element D have a length of 0-1000 bp, preferably 400-800 bp.
  • said element B and element D are homologous arm sequences flanking the targeting site of said site-directed nuclease.
  • the element G comprises a replication protein sequence Rep and a replication protein sequence RepA.
  • the foreign protein comprises: ⁇ -glucuronidase (GUS), Green Fluorescent Protein (GFP) , Yellow Fluorescent Protein (YFP), Red Fluorescent Protein (RFP).
  • GUS ⁇ -glucuronidase
  • GFP Green Fluorescent Protein
  • YFP Yellow Fluorescent Protein
  • RFP Red Fluorescent Protein
  • sequence of element F is as shown in positions 422-608 of SEQ ID NO.: 1.
  • sequence of the element G is as shown at positions 60917 to 5050 of SEQ ID NO.: 1.
  • sequence of said element H is shown as positions 1751-2159 of SEQ ID NO.: 1.
  • the nucleotide ligation sequence has a length of from 1 to 100 bp, preferably from 1 to 50 bp; more preferably from 1 to 20 bp.
  • the nucleotide linker sequence comprises a restriction endonuclease recognition sequence.
  • the construct is for expressing a foreign gene in a plant cell.
  • a vector comprising the construct of the first aspect of the invention.
  • the vector is a plant expression vector.
  • the vector is a pCambia vector.
  • the construct is integrated into the T-DNA region of the pCambia vector.
  • the Cas9 expression cassette is also integrated into the T-DNA region (i.e., both the WDV replication region and the Cas9 expression cassette are integrated).
  • the Cas9 expression cassette contains the Ubi promoter, the Cas9 intact CDS and the NOS terminator.
  • the vector further comprises a screening marker expression cassette.
  • the vector further comprises a cleavage nuclease expression cassette.
  • the vector further comprises an expression cassette for a targeted site recognition sequence of a cleavage nuclease.
  • the selection marker expression cassette, the expression cassette of the targeting site recognition sequence, and the site-directed cleavage nuclease expression cassette comprise a promoter sequence and a terminator sequence.
  • a genetically engineered cell comprising the construct of the first aspect of the invention, or the genome thereof integrated with one or more of the constructs of the first aspect of the invention Things.
  • the genome of the cell incorporates one or more elements C of the construct.
  • the cell is a plant cell.
  • the plant is selected from the group consisting of a gramineous plant, a leguminous plant, and a cruciferous plant.
  • the plant comprises: Arabidopsis thaliana, wheat, barley, oats, corn, rice, sorghum, millet, soybean, peanut, tobacco, and tomato.
  • the genetically engineered cell is introduced into the cell by the method according to the first aspect of the invention by a method selected from the group consisting of Agrobacterium transformation, gene gun, microinjection, and electric shock. Method, ultrasonic method and polyethylene glycol (PEG) mediated method.
  • a method of preparing a transgenic plant cell comprising the steps of:
  • step (i) the plant cell is further transfected with a construct comprising a site-directed cleavage nuclease coding sequence to effect cleavage of the chromosome of the plant cell.
  • the method further comprises: transfecting the plant cell with an expression cassette of a cleavage target site recognition sequence of a nuclease, thereby performing chromosomes on the plant cell Fixed point cutting.
  • the coding sequence of the site-directed nuclease is integrated into the genome of the plant cell.
  • the site of the site-directed cleavage is located near the site of the site-directed recombination.
  • the site-cutting nuclease is a Cas protein.
  • the Cas protein comprises a Cas9 protein.
  • the transfection is performed using an Agrobacterium transformation method or a gene gun bombardment method.
  • a method of preparing a transgenic plant cell comprising the steps of:
  • the construct is present in the transgenic plant cell in free cyclized form.
  • the construct has a copy number of ⁇ 100, preferably ⁇ 400.
  • a method of preparing a transgenic plant comprising the steps of:
  • the transgenic plant cell prepared by the method of the fourth aspect of the invention or the method of the fifth aspect of the invention is regenerated into a plant body, thereby obtaining a transgenic plant.
  • a transgenic plant prepared by the method of the sixth aspect of the invention, or the plant cell of the plant comprising the first aspect of the invention
  • the construct or genome thereof is integrated with one or more of the constructs of the first aspect of the invention.
  • FIG 1 shows the genomic structure of wheat dwarf virus (WDV). Specifically including long gene spacers (long Intergenic region (LIR), short intergenic region (SIR), complex two replicase proteins (Rep and RepA), and Coat Protein (CP) and Move Protein (Move Protein, MP).
  • LIR long Intergenic region
  • SIR short intergenic region
  • RepA complex two replicase proteins
  • CP Coat Protein
  • Move Protein Move Protein, MP
  • the engineered WDV can remove the sequence of the coated protein and the migratory protein to load the foreign gene fragment (shaded portion).
  • Figure 2 shows the structure of the pCambia-WDV-GUS transformant.
  • a complete GUS expression gene (containing the 35S promoter, the CDS and NOS terminator of the GUS gene) was cloned between the LIR and SIR regions of the engineered virus, and the integrated viral sequence was cloned into the T- of pCambia-1300. DNA region (between LB and RB).
  • Figure 3 shows the structure of the WDV-GUS gene replicon. After the pCambia-WDV-GUS transformant is introduced into the plant cell, the region between the two LIRs forms a circular replicon and is largely replicated.
  • Figure 4 shows the structure of the pCambia-WDV-GFP transformant.
  • a complete GFP-expressing gene (containing the 35S promoter, CDS and NOS terminator of the GFP gene) was cloned between the LIR and SIR regions of the engineered virus, and the integrated viral sequence was cloned into the T- of pCambia-1300. DNA region (between LB and RB).
  • the arrow is the primer binding site for detecting circularized replicons.
  • Figure 5 shows the structure of the WDV-GFP gene replicon. After the pCambia-WDV-GFP transformant is introduced into the plant cell, the region between the two LIRs forms a circular replicon and is largely replicated. The arrow is the primer binding site for detecting circularized replicons.
  • Figure 8 shows the detection of the presence of the circulated replicon of the WDV-GFP gene by PCR. Primer binding sites for circularized PCR detection are shown in Figures 4 and 5.
  • Figure 9A shows the number of copies of the WDV-GFP gene replicon detected.
  • Figure 9B shows the number of copies of the WDV-GFP gene replicon detected.
  • Figure 12 shows the structure of the pCambia-WDV-GFP (LIR) transformant.
  • the 35S promoter of the GFP gene cassette was removed and driven directly by the LIR of WDV.
  • Figure 14 shows a design for site-specific insertion of the GFP gene into the rice genome using WDV replication.
  • the GFP-P2A-NptII gene cassette is located between the left and right homology arms.
  • Figure 15 shows a design for the insertion of a GFP gene at the 3' end of the rice OsACT1 site by means of WDV replication.
  • FIG 17 is a schematic diagram showing the GFP-P2A-NptII gene cassette site-directed insertion into the 3' end of the rice OsACT1 gene (top) and the wild type OsACT1 gene (bottom).
  • ACT-F1/GFP-R1, NptII-F1/ACT-R1, and ACT-F1/ACT-R1 are PCR primers for 5' fusion border, 3' fusion border and endogenous OsGST gene, respectively.
  • Figure 18 shows the results of PCR detection of the integration of foreign genes in the OsACT1 locus of some rice T0 plants.
  • the binding sites for the 5' and 3' fusion boundaries and the endogenous gene PCR detection primers are shown in Figure 17. It can be seen that P1 and P2 are positive plants, and N1 and N2 are negative plants.
  • Figure 19 shows the sequencing results of the rice OsACT1 integration sequence and the recombination boundary.
  • Blue is the sequence of the left homology arm
  • light green is the sequence of the right homology arm
  • red thick TAA is the OsACT1 translation stop codon
  • light brown is the related sequence of the exogenous GFP-P2A-NptII gene cassette
  • 12 strains The sequencing results of positive plants are indicated in black.
  • the s9-related sg sequence, the PAM sequence, and the site in which the DSB is generated are shown in the figure.
  • Figure 20 shows a design for the insertion of a GFP gene at the 3' end of the rice OsGST gene by means of WDV replication.
  • Figure 21 is a schematic diagram showing the integration of the GFP-P2A-NptII gene cassette (top) and the wild-type OsGST gene (bottom) at the 3' end of the rice OsGST gene.
  • GST-F1/GFP-R1, NptII-F1/GST-R1, and GST-F1/GST-R1 are PCR primers for 5' fusion border, 3' fusion border and endogenous OsGST gene, respectively.
  • Figure 22 shows the results of PCR detection of foreign genes integrated into the OsGST locus of some rice T0 plants.
  • the binding sites for the 5' and 3' fusion border PCR detection primers are shown in Figure 22.
  • the number 6 is positive for the plants, and the others are negative plants.
  • Figure 23 shows the sequencing results of the rice OsGST integration sequence and the recombination boundary.
  • Light red is the sequence of the left homology arm
  • light blue is the sequence of the right homology arm
  • red thick TAA is the OsGST translation stop codon
  • light brown is the related sequence of the exogenous GFP-P2A-NptII gene cassette
  • 3 Sequencing results of strain-positive plants are indicated in black.
  • the s9-related sg sequence, the PAM sequence, and the site in which the DSB is generated are shown in the figure.
  • Figure 24 shows a schematic representation of the nucleic acid construct of the WDV expression cassette and the Cas9 expression cassette in the same T-DNA region, which can be used to directional knock-in of the wild type rice into the GFP-P2A-NptII gene cassette.
  • the present inventors have extensively and intensively studied, and for the first time, unexpectedly discovered a nucleic acid construct which expresses a foreign gene in a plant cell and its use.
  • the present invention transforms a DNA virus of the genus Corn genus, specifically, a wheat dwarf virus (WDV), as a tool for replicating a foreign gene or a donor fragment in a plant cell.
  • WDV wheat dwarf virus
  • the present invention provides a tool suitable for carrying a large amount of expression of a foreign gene in a plant cell, and a method for increasing the efficiency of homologous recombination or site-directed knocking of the exogenous fragment in the plant genome.
  • the invention also provides vectors, plant cells and plants comprising the exogenous construct.
  • homologous arm refers to a flanking sequence that is identical to a genomic sequence flanking a foreign sequence to be inserted on a targeting vector, for identifying and recombining a region.
  • foreign gene refers to an exogenous DNA molecule that acts in a phased manner. Can be used for this
  • the foreign gene to be applied is not particularly limited, and includes various exogenous genes commonly used in the field of transgenic animals. Representative examples include, but are not limited to, a ⁇ -glucuronidase gene, a red fluorescent protein gene, a green fluorescent protein gene, a lysozyme gene, a salmon calcitonin gene, a lactoferrin, or a serum albumin gene.
  • screening marker gene refers to a gene used for screening a transgenic cell or a transgenic animal in a transgenic process
  • the screening marker gene useful in the present application is not particularly limited, and includes various screening marker genes commonly used in the transgenic field, representative examples. These include, but are not limited to, the hygromycin resistance gene (Hyg), the kanamycin resistance gene (NPTII), the neomycin gene, or the puromycin resistance gene.
  • the term "expression cassette” refers to a stretch of polynucleotide sequences comprising a gene to be expressed and a sequence component that expresses the desired element.
  • the term “screening marker expression cassette” refers to a polynucleotide sequence comprising a sequence encoding a selection marker and a sequence component expressing the desired element.
  • the components required for expression include a promoter and a polyadenylation signal sequence.
  • the selection marker expression cassette may or may not contain other sequences including, but not limited to, enhancers, secretion signal peptide sequences, and the like.
  • the promoter suitable for the exogenous gene expression cassette and the selection marker gene expression cassette may be any of the common promoters, and it may be a constitutive promoter or an inducible promoter.
  • the promoter is a constitutive strong promoter, such as the 35S promoter and other plant promoters suitable for eukaryotic expression.
  • plant promoter refers to a nucleic acid sequence capable of initiating transcription of a nucleic acid in a plant cell.
  • the plant promoter may be derived from a plant, a microorganism (such as a bacterium, a virus) or an animal, or a synthetic or engineered promoter.
  • plant terminator refers to a terminator capable of stopping transcription in a plant cell.
  • the plant transcription terminator may be derived from a plant, a microorganism (such as a bacterium, a virus) or an animal, or a synthetic or engineered terminator. Representative examples include (but are not limited to): Nos terminator.
  • Cas protein refers to a nuclease.
  • a preferred Cas protein is the Cas9 protein.
  • Typical Cas9 proteins include, but are not limited to, Cas9 derived from Streptococcus pyogenes SF370.
  • the term "coding sequence of a Cas protein” refers to a nucleotide sequence that encodes a Cas protein having cleavage activity.
  • the skilled artisan will recognize that because of the degeneracy of the codon, a large number of polynucleotide sequences can encode the same polypeptide. .
  • the skilled person will also recognize that different species have a certain preference for codons, and may optimize the codons of the Cas protein according to the needs of expression in different species. These variants are all referred to by the term "Cas protein.
  • the coding sequence is specifically covered.
  • the term specifically encompasses a full-length sequence substantially identical to the Cas gene sequence, as well as a sequence encoding a protein that retains the function of the Das protein.
  • plant includes whole plants, plant organs (such as leaves, stems, roots, etc.), seeds and plant cells, and progeny thereof.
  • the kind of plant which can be used in the method of the present invention is not particularly limited, and generally includes any higher plant type which can be subjected to transformation techniques, including monocots, dicots, and gymnosperms.
  • 2A polypeptide coding sequence and "2A sequence” refer to a protease-independent self-cleaving amino acid sequence found in a virus, similar to an IRES, which enables simultaneous expression of two promoters using 2A. Genes. It is also widely found in various eukaryotic cells. Unlike IRES, the amount of downstream protein expression does not decrease. However, after the cleavage, the 2A polypeptide residue is integrated with the upstream protein, and a Furin protease cleavage site (four basic amino acid residues, such as a basic amino acid residue, can be added between the upstream protein and the 2A polypeptide. Arg-Lys-Arg-Arg) completely excises the 2A polypeptide residue from the upstream protein end.
  • the present invention provides a nucleic acid construct for expressing a foreign gene in a plant cell, which is a transformation of wheat dwarf virus (WDV) using its long gene spacer (LIR), short gene spacer (SIR) and Replicon proteins (Rep and RepA) enable large-scale replication of donor fragments in plant cells.
  • WDV wheat dwarf virus
  • LIR long gene spacer
  • SIR short gene spacer
  • RepA Replicon proteins
  • the construct of the invention is as described in the first aspect of the invention.
  • the construction of the present invention is carried out by inserting the construct of the present invention into an exogenous vector, especially a vector suitable for transgenic plant manipulation.
  • the transgenic plant cells are prepared by transforming the vector of the present invention into plant cells to mediate the integration of the plant cell chromosomes by the vector of the present invention.
  • transgenic plant cells of the present invention are regenerated into plant bodies to obtain transgenic plants.
  • WDV wheat dwarf virus
  • LIR long intergenic region
  • SIR short intergenic region
  • Rep and RepA complex two replication proteins
  • MP Move Protein
  • LIR long gene spacer
  • SIR short gene spacer
  • Rep and RepA replication protein
  • a complete GUS expression gene cassette and a GFP expression gene cassette (both containing the 35S promoter-CDS-NOS terminator) were cloned into BamHI and SpeI restriction sites, respectively.
  • the integrated viral sequence was cloned into the T-DNA region of pCambia-1300 (Fig. 2, 4), and the callus of Nipponbare was transformed by Agrobacterium infection.
  • the GUS gene expression cassette and the GFP gene expression cassette form a circular replicon and replicate in large numbers (Fig. 3, 5).
  • the transformed callus was stained by GUS or observed under a fluorescence microscope, and the results showed that the expression intensity of GUS or GFP replicated by WDV was significantly higher than that of ordinary pCambia-GUS or pCambia-GFP (Fig. 6, 7).
  • pCambia-GFP was extracted The DNA of the callus transformed with pCambia-WDV-GFP was detected by specific primer amplification to detect the Rep fragment in pCambia-WDV-GFP transformation and the fragment which was produced only after circularization (Fig. 8).
  • GFP/Taq-P2A-NptII/Hyg gene cassette that can be fused to the 3' end of the plant function gene.
  • a gene cassette includes a functional sequence such as GFP which can be used as a subcellular localization and dynamic tracing of the target gene, or a tag such as 3XFlag, which can be used as a protein for isolating and purifying the target gene.
  • the latter part of the gene cassette is a broad-spectrum screening marker such as the anti-hygromycin gene (Hyg) or the anti-kanamycin gene (NptII).
  • Hyg anti-hygromycin gene
  • NptII anti-kanamycin gene
  • the GFP therein contains only the CDS, and does not contain a transcriptional promoter (such as 35S) and a transcription terminator (such as NOS), and the translation initiation codon ATG and the translation stop codon TAA are also removed.
  • the front end of the GFP coding sequence is ligated to a stretched flexible amino acid sequence (along with the BamHI restriction site), which is a bridge connecting the target gene and the GFP gene, avoiding interference between the two encoded proteins, depending on the type of target gene Flexible design.
  • the 2A polypeptide coding sequence with self-shearing function, and the 2A polypeptide coding sequence is followed by the restriction enzyme cleavage site KpnI to ligate the NptII coding sequence.
  • the translation initiation codon ATG of the NptII coding sequence is also removed. , but retain its translation stop codon TGA.
  • the GFP-P2A-NptII gene cassette is fused to the 3' end of a functional gene in the plant genome, the GFP and NptII coding sequences can follow the expression and translation of the gene.
  • the GFP-P2A-NptII gene cassette which is not fused to a specific sequence, does not function because it does not have a transcriptional promoter and a translation initiation codon ATG.
  • the directional insertion event can be screened by the action of the NptII gene to avoid interference with a large number of random insertion events, and the GFP gene can select the callus with GFP signal when screening rice positive callus clones.
  • the GFP gene can select the callus with GFP signal when screening rice positive callus clones.
  • the WDV replication region and a DNA fragment expressing a site-directed cleavage nuclease are introduced into a plant receptor, and the above two fragments can be integrated into a T-DNA region of pCambia.
  • the introduction methods include, but are not limited to, gene gun method, microinjection method, electric shock method, ultrasonic method, and polyethylene glycol (PEG)-mediated method.
  • Receptor plants include, but are not limited to, rice, soybean, tomato, corn, tobacco, wheat, sorghum, and the like.
  • the invention does not require large-scale Agrobacterium transformation and subsequent
  • the tissue culture and screening identification work can be obtained by routine tissue culture to obtain sufficient fixed-point knock-in plants.
  • SEQ ID NO.: 1 shows the structural sequence LIR-SIR-Rep-LIR of the engineered WDV.
  • the 1-109th bit is the first LIR sequence
  • the 422-608th bit is the SIR sequence
  • the 60 thirteenth to the 1950th is the Rep sequence
  • the 1751-2159th bit is the second LIR sequence.
  • SEQ ID NO.: 2 shows the sequence of the EGFP-P2A-NptII gene cassette.
  • the 19th-732th position is the CDS sequence of EGFP
  • the 733-798th position is the sequence of P2A
  • the 8055-199th position is the sequence of NptII
  • the 1st-18th position is the sequence of translation of the flexible amino acid.
  • BmHI is indicated by a straight line
  • KpnI is indicated by a downward wavy line
  • PstI is indicated by a dotted line.
  • the above restriction sites are set for convenient cloning.
  • SEQ ID NO.: 5 shows the sequence of rice OsACT1 site-specific insertion into the EGFP-P2A-NptII gene cassette.
  • Capital letters indicate the relevant sequence of OsACT1, the sequence of which is referred to the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml), accession number: LOC_Os03g50885.
  • Lower case letters indicate the exogenously inserted EGFP-P2A-NptII gene cassette sequence.
  • the underlined wavy line is a 5' homology arm sequence and the underlined line is a 3' homology arm sequence.
  • PAM sequence of sg
  • the 2336-2338-bit bold TAA is the OsACT1 translation stop codon.
  • the 1st position is the binding site of the primer ACT-F1; the 1207-1228 is the binding site of the primer GFP-R1; the 1765-1784 is the binding site of the primer NPTII-F1; the 2990-3009 It is the binding site of the primer ACT-R1.
  • SEQ ID NO.: 6 shows the sequence of rice OsGST site-specific insertion into the EGFP-P2A-NptII gene cassette.
  • Capital letters indicate the relevant sequence of OsGST, the sequence of which is referred to the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml), accession number: LOC_Os05g02530.
  • Lower case letters indicate the exogenously inserted EGFP-P2A-NptII gene cassette sequence.
  • the underlined wavy line is a 5' homology arm sequence and the underlined line is a 3' homology arm sequence.
  • PAM sequence of sg
  • the bold TAA at positions 2374-2376 is the OsGST translation stop codon.
  • the 1st position is the binding site of the primer GST-F1; the 1236-1257 is the binding site of the primer GFP-R1; the 1794-1813 is the binding site of the primer NPTII-F1; the 3032-3053 It is the binding site of the primer GST-R1.
  • the invention can be applied to the field of plant genetic engineering for transforming various plants, especially agricultural plants and forestry plants of economic value.
  • the transformed virus does not have a serious impact on the growth and development of the plant in the contemporary transformation, and the modified target gene can be normally transmitted to the next generation, and the viral sequence integrated into the T-DNA region and inserted into the genome Can be separated by selfing.
  • a GFP-P2A-NptI I gene cassette was inserted into the CDS end of the rice endogenous OsACT1 gene.
  • the CRISPR-Cas9 targeting site (sg) was designed and tested at the CDS end of the endogenous OsACT1 gene in rice, and the callus of the rice variety Nipponbare was transformed and the regenerated plant population was obtained.
  • the target site was amplified and sequenced.
  • a high proportion of various knockout mutants (Table 1, Figure 19) demonstrate that this sg can efficiently cleave DSB in the target sequence.
  • the underlined sequence is a PAM sequence.
  • a 500 bp homology arm was amplified on the left and right sides of the target site (Fig. 19), and ligated to GFP-P2A by BamHI and PstI restriction sites, respectively.
  • the CRISPR expression cassette OsU6-ACT1sg-sgRNA was then cloned into the 3' homology arm end by SpeI cleavage site.
  • the entire 5' homology arm-GFP-P2A-NptII gene cassette-3' homology arm-sgRNA expression cassette was integrated into the LIR and SIR replication regions of WDV.
  • the entire WDV expression cassette was cloned into the T-DNA region of pCambia-1300 (Fig. 15).
  • the WDV expression cassette was carried by Agrobacterium tumefaciens into the callus of the rice variety Nipponbare which had integrated the Cas9 gene, and the transformed callus was transferred to the screening culture containing the G418 screening agent after 3 days of co-culture. After 15 days as a screening cycle, the callus of the positive clone was obtained after two rounds of continuous screening, and the callus of GFP luminescence was observed under a fluorescence microscope (Fig. 16). Or the transformed callus is directly transferred to a differentiation medium containing G418 screening agent to regenerate the plant after one cycle of screening, and is planted at T0. Samples were extracted from the strains to detect the integration of foreign genes.
  • the CRISPR-Cas9 targeting site was designed at the CDS end of the rice endogenous OsGST gene, and the callus of the rice variety Nipponbare was transformed and the regenerated plant population was obtained. Sampling and sequencing of the target site revealed a high proportion of various sites. Knockout of the mutant indicated that the sg was able to efficiently guide the Cas9 protein to cleave in the target sequence to produce DSB (Table 1, Figure 23).
  • a 500 bp homology arm was amplified on the left and right sides of the target site, and ligated to the GFP-P2A-NptII gene cassette by BamHI and PstI restriction sites, respectively.
  • the WDV expression cassette was carried by Agrobacterium tumefaciens into the callus of the rice variety Nipponbare which had integrated the Cas9 gene, and the transformed callus was transferred to the screening culture containing the G418 screening agent after 3 days of co-culture. After one cycle of screening, the callus was directly transferred to a differentiation medium containing G418 screening agent to regenerate the plant, and the DNA was sampled and extracted in the T0 generation plant population to detect the integration of the foreign gene.
  • the Cas9 expression cassette (containing the Ubi promoter, the Cas9 intact CDS and the NOS terminator) was further integrated into the T-DNA region in which the WDV is located in Examples 1 and 2. Furthermore, a 35S polyA transcription terminator was placed between the WDV replication region and the Cas9 expression cassette in addition to the WDV replication region, and to prevent possible interference of the LIR to the Cas9 expression cassette (Fig. 24).
  • the nucleic acid construct was transformed into the callus of the wild type rice variety Nipponbare by means of Agrobacterium transformation, and the transformed callus was transferred to a screening culture containing the G418 screening agent after 3 days of co-cultivation. After 15 days of screening, the plants were directly transferred to a differentiation medium containing a G418 screening agent, and the DNA was sampled and extracted in the T0 generation plant population to detect the integration of the foreign gene.

Abstract

提供了一种在植物细胞内复制或表达外源基因的核酸构建物及其应用。还提供了包含该构建物的载体、植物细胞和植物。

Description

在植物细胞内表达外源基因的核酸构建物及其应用 技术领域
本发明涉及生物技术领域，更具体地涉及一种在植物细胞内表达外源基因的核酸构建物及其应用。
背景技术
在植物细胞内进行基因同源重组或定向插入一直是一个难题，为突破这个瓶颈前人已经做了大量尝试，如在2002年，Terada等人提出了基于农杆菌介导的同源重组技术(Terada et al.,2002)，这套技术体系不依赖于所改造基因的功能进行筛选，理论上可以应用于所有基因序列的打靶。然而，要保证该技术应用的成功，需要有高效的农杆菌转化效率及大规模的转化量，更重要的是，还需要附加一套复杂的正负筛选系统以消除在农杆菌转化中产生的大量随机插入事件。尽管Terada等人连续成功地把该技术应用在水稻基因定向替换实验中(Terada et al.,2002；Terada et al.,2007)，但由于极低的整合效率一直无法得以提高，加上正负筛选系统构造复杂，该技术体系并没有得到推广应用。
近年来，随着基因定向编辑工具如锌指蛋白核糖核酸酶(Zinc finger nulease，ZFN)、转录激活样效应因子核酸酶(Transcription activator-like effectors nulease，TALEN)和规律成簇间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats，CRISPR)系统的推广应用，特别是后两者由于活性高，特异性好而且几乎没有序列的限制，近两年来发展非常迅速，并且在大量物种上得到成功应用(Li et al.,2012；Sun et al.,2012；Zhang et al.,2012；Dang et al.,2013；Feng et al.,2013；Li et al.,2013；Shan et al.,2013)。上述三种基因定向编辑工具的作用机理有相通之处，通过识别序列把核酸剪切酶引导至需要编辑的基因位点上，随后的剪切作用造成DNA序列的双链断裂(double strands break，DSB)，激活了宿主细胞的DNA修复途径，通过非同源末端连接(non-homologous end-joining，NHEJ)的方式，或者当同源模板在附近时，通过同源重组(homologous recombination，HR)的方式修复双链缺口。因此，利用基因定向编辑工具酶在靶位点附近制造一个DSB能在转化中极大地增加基因同源重组或定向插入的机率。
然而，相对于动物细胞和人类细胞，由于有细胞壁的阻碍，在植物细胞内进行基因重组或外源片段定点插入的一个巨大挑战就是如何把足够大量的供体分子输送到细胞中去。当然，对一些植物品种来说，例如蔬菜及一些观赏性植物，它们可以通过原生质体再生的方式重建植株(Davey et al.,2005)，这样，就可以利用原生质体作为受体，通过电转化或PEG介导的方式实现对大量细胞的转化，从而有足够的转化体对所期待的突变位点进行筛选(Zhang et al.,2012)。通过TALEN或CRISPR介导的基因打靶也已经在水稻、拟南芥、烟草等植物上得到成功(Zhang et al.,2012；Feng et al.,2013；Shan et al.,2013)，当然，这些基因打靶事件都是原生质体水平上实现的。但是对于水稻、拟南芥等植物品种， 通过原生质体重建植株在目前来说几乎是不可能的，只能开发其它技术去攻克这一难关。
前两年中国科学院遗传与发育研究所高彩霞课题组报道了利用TALEN系统与单链小片段DNA供体分子结合，通过基因枪共转化到小麦非成熟胚中，实现了对基因组中两个位点的定向插入，但效率较低，其中一个位点在69株再生植株中仅得到1株,而另一个位点在39株再生植株中亦仅有1株实现了定向插入(Wang et al.,2014)。最近该课题组用环状质粒作供体，以水稻EPSPS基因作靶标，通过基因枪与Cas9系统共转化的方式实现了目的基因的同源重组及定向插入，但试验成功率仍然不高，只有2％左右(Li et al.,2016)。当然，基因枪转化法需要配套的仪器耗材比较复杂，而且经常导致大量外源片段随机插入到基因组中。
对于大多数植物尤其是主要的大田作物来说，传统的遗传转化往往依赖于农杆菌或基因枪的介导，与动物细胞的转化相比通常难以产生足够大量的转化事件用作后期的筛选。目前在植物上，已经报道的基因打靶技术大多数基于靶标基因本身经靶向修饰后具备可筛选特性，这也是为什么在前述有关的植物基因定向打靶修饰的报道中，研究者们所选用的目标基因都与修饰后具备可选择特性的内源除草剂抗性或可见的表型变化有关。而实际上，生物体的大多数功能基因并不能提供这样的选择表型。
综上所述，本领域迫切需要开发这样的植物基因打靶技术：用农杆菌做一次常规转化即具有足够的打靶成功率，适用于大多数功能基因，利用广谱筛选标记如抗潮霉素基因或抗卡那霉素基因即可进行定向筛选。
发明内容
本发明的目的在于提供一种在植物细胞内表达外源基因的核酸构建物及其应用。
具体地，本发明提供了一种适用于运载外源基因在植物细胞内大量表达的工具，提供了一种在植物基因组中简单高效的实现同源重组或外源片段定点敲入的方法。利用WDV能大量复制外源片段的能力，同时利用高效的定点切割核酸酶例如ZFN、Talen、CRISPR/Cas9或者Cpf1等在靶标位点附近制造一个DSB，辅以定向的筛选方式，即可高效地实现植物细胞的基因同源重组或定向插入。
在本发明的第一方面，提供了一种核酸构建物，所述的构建物具有从5’至3’的式I结构：
A-B-C-D-E-F-G-H       (I)
式中，
A、B、C、D、E、F、G、H分别为用于构成所述构建物的元件；
各“-”独立地为键或核苷酸连接序列；
A为第一长基因间隔区序列；
B为任选的5’同源臂序列；
C为外源基因表达盒；
D为任选的3’同源臂序列；
E为任选的第二表达盒；
F为短基因间隔区序列；
G为玉米线条病毒属病毒的复制蛋白的编码序列；
H为第二长基因间隔区序列；
并且，所述第一长基因间隔区序列、短基因间隔区序列、和第二长基因间隔区序列来源于玉米线条病毒属(Mastrevirus)病毒。
在另一优选例中，所述玉米线条病毒属病毒包括小麦矮缩病毒(Wheat Dwarf Virus，WDV)、菜豆黄矮病毒(Bean Yellow Dwarf Virus,BeYDV)、玉米线条病毒(MSV)、烟草黄矮病毒(TYDV)。
在另一优选例中，所述的外源基因表达盒具有从5’至3’的式II结构：
C1-C2-C3       (II)
式中，
C1、C2、C3分别为用于构成所述外源基因表达盒的元件；
各“-”独立地为键或核苷酸连接序列；
C1为任选的第一启动子序列；
C2为外源蛋白的编码序列；
C3为终止子序列。
在另一优选例中，所述的外源基因表达盒具有式II结构，并且所述的构建物不含有元件B、元件D和元件E。
在另一优选例中，所述的外源基因表达盒具有式II结构，并且所述元件A的序列如SEQ ID NO.:1的第1-409位所示。
在另一优选例中，所述的第一启动子和终止子分别为植物启动子和植物终止子。
在另一优选例中，所述的第一启动子包括35S、Ubi、UBQ、SPL启动子或其组合。
在另一优选例中，所述的终止子包括NOS终止子、Poly A终止子。
在另一优选例中，所述的外源基因表达盒具有从5’至3’的式III结构：
Ci-Cii-Ciii-Civ        (III)
式中，
Ci、Cii、Ciii、Civ分别为用于构成所述外源基因表达盒的元件；
各“-”独立地为键或核苷酸连接序列；
Ci为第一外源蛋白的编码序列；
Cii为2A序列或IRES序列；
Ciii为第二外源蛋白(如筛选标记)的编码序列；
Civ为终止子序列。
在另一优选例中，所述元件Ci中的第一外源蛋白编码序列不包含起始密码子。
在另一优选例中，所述元件Ci中的第一外源蛋白编码序列不包含终止密码子。
在另一优选例中，所述元件Ci中的第一外源蛋白编码序列不包含起始密码子和终止密码子。
在另一优选例中，所述第一外源蛋白的N端还包含一柔性氨基酸接头序列。
在另一优选例中，所述的元件Cii具有自我剪切功能。
在另一优选例中，所述的2A序列如SEQ ID NO.:2的第733-798位所示。
在另一优选例中，所述元件Ciii中的第二外源蛋白(如筛选标记)的编码序列不包含起始密码子。
在另一优选例中，所述的筛选标记包括潮霉素抗性基因(Hyg)、卡那霉素抗性基因(NptII)、GFP基因、Amp抗性基因、草丁膦抗性基因(Bar)。
在另一优选例中，所述的外源基因表达盒具有式III结构，并且所述元件A为改造后的第一长基因间隔区序列，所述元件A不具有转录驱动能力。
在另一优选例中，所述元件A的序列如SEQ ID NO.:1的第1-329位所示。
在另一优选例中，所述的外源基因表达盒具有式III结构，并且所述的构建物含有的元件B、元件D和元件E不为无。
在另一优选例中，所述的外源基因表达盒具有式III结构，并且所述的构建物含有的元件B和元件D不为无。
在另一优选例中，所述的第二表达盒为定点切割核酸酶的靶向位点识别序列的表达盒。
在另一优选例中，所述的定点切割核酸酶包括锌指蛋白核糖核酸酶(Zinc finger nulease，ZFN)、转录激活样效应因子核酸酶(Transcription activator-like effectors nulease，TALEN)、规律成簇间隔短回文重复序列关联蛋白(CRISPR-associated proteins，Cas)和Cpf1(CRISPR from Prevotella and Francisella 1)。
在另一优选例中，所述的识别序列表达盒包括sgRNA表达盒。
在另一优选例中，所述的sgRNA表达盒从5’至3’依次包括以下元件：
(a)用于驱动snRNA(small nuclear RNA)的第二启动子序列；
(b)CRISPR靶向位点识别序列(small guid RNA,sgRNA)；
(c)RNA转录终止子。
在另一优选例中，所述的第二启动子包括：OsU3，OsU6a，OsU6b，OsU6c，AtU6-1，AtU3b，AtU3d，AtU6-1，AtU6-29。
在另一优选例中，所述的CRISPR靶向位点识别序列的长度为17-22nt。
在另一优选例中，所述的CRISPR靶向位点sg如SEQ ID NO.:3或SEQ ID NO.:4中第1-20nt所示。
在另一优选例中，所述的元件A和元件H之间可以形成环状复制子。
在另一优选例中，所述的元件B、元件C、元件D和元件E的总长度小于3000bp。
在另一优选例中，所述的元件B、元件C、元件D和元件E的总长度为0-10000bp，较佳地为0-3000bp。
在另一优选例中，所述的元件B和元件D的长度为0-1000bp，较佳地为400-800bp。
在另一优选例中，所述的元件B和元件D为所述定点切割核酸酶的靶向位点两侧的同源臂序列。
在另一优选例中，所述的元件G包括复制蛋白序列Rep和复制蛋白序列RepA。
在另一优选例中，所述的外源蛋白(第一外源蛋白、第二外源蛋白)包括：β-葡萄糖苷酸酶(Glucuronidase，GUS)、绿色荧光蛋白(Green Fluorescent Protein，GFP)、黄色荧光蛋白(Yellow Fluorescent Protein，YFP)、红色荧光蛋白(Red Fluorescent Protein，RFP)。
在另一优选例中，所述的元件F的序列如SEQ ID NO.:1的第422-608位所示。
在另一优选例中，所述的元件G的序列如SEQ ID NO.:1的第609-1750位所示。
在另一优选例中，所述的元件H的序列如SEQ ID NO.:1的第1751-2159位所示。
在另一优选例中，所述的核苷酸连接序列的长度为1-100bp，较佳地为1-50bp；更佳地为1-20bp。
在另一优选例中，所述的核苷酸连接序列包含限制性内切酶识别序列。
在另一优选例中，所述的构建物用于在植物细胞内表达外源基因。
在本发明的第二方面，提供了一种载体，所述的载体含有本发明第一方面所述的构建物。
在另一优选例中，所述的载体为植物表达载体。
在另一优选例中，所述的载体为pCambia载体。
在另一优选例中，所述的构建物整合到pCambia载体的T-DNA区。
在另一优选例中，所述的T-DNA区中还整合有Cas9表达盒(即同时整合有WDV复制区和Cas9表达盒)。较佳地，所述的Cas9表达盒含有Ubi启动子、Cas9完整的CDS和NOS终止子。
在另一优选例中，所述的载体还包含一筛选标记表达盒。
在另一优选例中，所述的载体还包含一定点切割核酸酶表达盒。
在另一优选例中，所述的载体还包含一定点切割核酸酶的靶向位点识别序列的表达盒。
在另一优选例中，所述的筛选标记表达盒、靶向位点识别序列的表达盒和定点切割核酸酶表达盒包含启动子序列和终止子序列。
在本发明的第三方面，提供了一种基因工程细胞，所述的细胞含有本发明第一方面所述的构建物，或其基因组整合有一个或多个本发明第一方面所述的构建物。
在另一优选例中，所述细胞的基因组整合有一个或多个所述构建物的元件C。
在另一优选例中，所述的细胞为植物细胞。
在另一优选例中，所述的植物选自下组：禾本科植物、豆科植物和十字花科植物。
在另一优选例中，所述的植物包括：拟南芥、小麦、大麦、燕麦、玉米、水稻、高粱、粟、大豆、花生、烟草和番茄。
在另一优选例中，所述的基因工程细胞是用选自下组的方法将本发明第一方面所述的构建物导入细胞的：农杆菌转化法、基因枪法、显微注射法、电击法、超声波法和聚乙二醇(PEG)介导法。
在本发明的第四方面，提供了一种制备转基因植物细胞的方法，包括步骤：
(i)将本发明第一方面所述的构建物、或本发明第二方面所述的载体转染植物细胞，使得所述构建物与所述植物细胞中的染色体发生定点重组，从而制得转基因植物细胞。
在另一优选例中，在步骤(i)中，还包括用包含定点切割核酸酶编码序列的构建物对所述植物细胞进行转染，从而对所述植物细胞的染色体进行定点切割。
在另一优选例中，在步骤(i)中，还包括用一定点切割核酸酶的靶向位点识别序列的表达盒对所述植物细胞进行转染，从而对所述植物细胞的染色体进行定点切割。
在另一优选例中，所述植物细胞的基因组中整合有定点切割核酸酶的编码序列。
在另一优选例中，所述定点切割的位点位于所述定点重组的位点附近。
在另一优选例中，所述的定点切割核酸酶为Cas蛋白。
在另一优选例中，所述的Cas蛋白包括Cas9蛋白。
在另一优选例中，所述的转染采用农杆菌转化法或基因枪轰击法。
在本发明的第五方面，提供了一种制备转基因植物细胞的方法，包括步骤：
(i)将本发明第一方面所述的构建物、或本发明第二方面所述的载体转染植物细胞，使得所述植物细胞含有所述构建物，从而制得转基因植物细胞。
在另一优选例中，所述的构建物以游离环化的形式存在于所述转基因植物细胞中。
在另一优选例中，所述构建物的拷贝数≥100，较佳地≥400。
在本发明的第六方面，提供了一种制备转基因植物的方法，包括步骤：
将本发明第四方面或本发明第五方面所述方法制备的转基因植物细胞再生为植物体，从而获得转基因植物。
在本发明的第七方面，提供了一种转基因植物，所述的植物是用本发明第六方面所述的方法制备的，或者所述植物的植物细胞中含有本发明第一方面所述的构建物或其基因组整合有一个或多个本发明第一方面所述的构建物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了小麦矮缩病毒(WDV)的基因组结构。具体包括长基因间隔区(long  intergenic region,LIR)，短基因间隔区(short intergenic region,SIR)，复合的两个复制蛋白(replicase proteins,Rep and RepA)，以及包衣蛋白(Coat Protein,CP)和迁移蛋白(Move Protein,MP)。改造的WDV可以去掉包衣蛋白和迁移蛋白的序列，用以装载外源基因片段(阴影部分)。
图2显示了pCambia-WDV-GUS转化子的结构。把一个完整的GUS表达基因(包含35S启动子，GUS基因的CDS和NOS终止子)克隆至改造后病毒的LIR及SIR区域之间，然后将整合后的病毒序列克隆到pCambia-1300的T-DNA区(LB与RB之间)。
图3显示了WDV-GUS基因复制子的结构。pCambia-WDV-GUS转化子导入植物细胞后，两个LIR之间的区域会形成环状复制子并大量复制。
图4显示了pCambia-WDV-GFP转化子的结构。把一个完整的GFP表达基因(包含35S启动子，GFP基因的CDS和NOS终止子)克隆至改造后病毒的LIR及SIR区域之间，然后将整合后的病毒序列克隆到pCambia-1300的T-DNA区(LB与RB之间)。箭头为检测环化复制子的引物结合位点。
图5显示了WDV-GFP基因复制子的结构。pCambia-WDV-GFP转化子导入植物细胞后，两个LIR之间的区域会形成环状复制子并大量复制。箭头为检测环化复制子的引物结合位点。
图6显示了用WDV或传统的pCambia表达GUS基因转化水稻愈伤组织后的GUS染色结果。Bar＝0.5cm。
图7显示了用WDV或传统的pCambia表达GFP基因转化水稻愈伤组织后在体视式荧光显微镜下观察GFP发光结果。Bar＝0.2cm。
图8显示了通过PCR检测WDV-GFP基因环化复制子的存在。环化片段PCR检测的引物结合位点见图4和图5。
图9A显示了检测WDV-GFP基因复制子的拷贝数。
图9B显示了检测WDV-GFP基因复制子的拷贝数。
图10显示了用WDV或传统的pCambia表达GUS基因转化水稻愈伤组织后不同时期的GUS染色结果。Bar＝1cm。
图11显示了用WDV或传统的pCambia表达GFP基因转化水稻愈伤组织后，用体视式荧光显微镜对同一愈伤组织持续追踪观察GFP发光结果。Bar＝0.2cm。
图12显示了pCambia-WDV-GFP(LIR)转化子的结构。把GFP基因盒的35S启动子去掉，直接由WDV的LIR驱动。
图13显示了不同GFP基因转化子转化水稻愈伤组织后在体视式荧光显微镜下观察GFP发光结果。Bar＝0.2cm。
图14显示了利用WDV复制的方式在水稻基因组中定点插入GFP基因的设计图。GFP-P2A-NptII基因盒位于左右同源臂之间。
图15显示了利用WDV复制的方式在水稻OsACT1位点3’端定点插入GFP基因的设计图。
图16显示了水稻OsACT1位点整合GFP基因的阳性愈伤组织，在荧光显微镜下观察发现有GFP发光的愈伤(箭头所示)。Bar＝1mm。
图17显示了GFP-P2A-NptII基因盒定点插入水稻OsACT1基因3’端(上)及野生型OsACT1基因(下)的示意图。ACT-F1/GFP-R1，NptII-F1/ACT-R1，ACT-F1/ACT-R1分别为5’融合边界，3’融合边界及内源OsGST基因的PCR检测引物。
图18显示了部分水稻T0代植株OsACT1位点整合外源基因的PCR检测结果。5’和3’融合边界及内源基因PCR检测引物的结合位点见图17。图中可见P1和P2为阳性植株，N1和N2为阴性植株。
图19显示了水稻OsACT1整合序列及重组边界的测序结果。蓝色为左同源臂的序列，浅绿色为右同源臂的序列，红色加粗的TAA为OsACT1翻译终止密码子，浅褐色为外源GFP-P2A-NptII基因盒的相关序列，12株阳性植株的测序结果用黑色标示。Cas9相关的sg序列、PAM序列及产生DSB的位点如图所示。
图20显示了利用WDV复制的方式在水稻OsGST基因3’端定点插入GFP基因的设计图。
图21显示了水稻OsGST基因3’端定点整合GFP-P2A-NptII基因盒(上)及野生型OsGST基因(下)的示意图。GST-F1/GFP-R1，NptII-F1/GST-R1，GST-F1/GST-R1分别为5’融合边界，3’融合边界及内源OsGST基因的PCR检测引物。
图22显示了部分水稻T0代植株OsGST位点整合外源基因的PCR检测结果。5’和3’融合边界PCR检测引物的结合位点见图22。图中可见6号为阳性植株，其它为阴性植株。
图23显示了水稻OsGST整合序列及重组边界的测序结果。浅红色为左同源臂的序列，浅蓝色为右同源臂的序列，红色加粗的TAA为OsGST翻译终止密码子，浅褐色为外源GFP-P2A-NptII基因盒的相关序列，3株阳性植株的测序结果用黑色标示。Cas9相关的sg序列、PAM序列及产生DSB的位点如图所示。
图24显示了WDV表达盒和Cas9表达盒在同一T-DNA区的核酸构建物示意图，该核酸构建物可用于野生型水稻定向敲入GFP-P2A-NptII基因盒。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现一种在植物细胞内表达外源基因的核酸构建物及其应用。本发明通过改造玉米线条病毒属一类的DNA病毒，具体地，以小麦矮缩病毒(WDV)为例，使之成为一种能在植物细胞内大量复制外源基因或供体片段的工具，并通过在供体片段后联结一个广谱的筛选标记，可以有效筛选同源重组或定点插入事件，从而实现外源基因片段在植物基因组的高效重组或定点插入，可以广泛应用于植物基因功能研究和作物遗传改良。
本发明提供了一种适用于运载外源基因在植物细胞内大量表达的工具，以及提高外源片段在植物基因组进行同源重组或定点敲入效率的方法。本发明还提供了包含该外源构建物的载体，植物细胞和植物。
术语
如本文所用，术语“同源臂”指打靶载体上待插入的外源序列两侧的与基因组序列完全一致的侧翼序列，用于识别并发生重组的区域。
如本文所用，“外源基因”指作用是阶段性作用的外源DNA分子。可用于本 申请的外源基因没有特别限制，包括转基因动物领域常用的各种外源基因。代表性例子包括(但并不限于)：β-葡萄糖苷酸酶基因、红色荧光蛋白基因、绿色荧光蛋白基因、溶菌酶基因、鲑鱼降钙素基因、乳铁蛋白、或血清白蛋白基因等。
如本文所用，“筛选标记基因”指转基因过程中用来筛选转基因细胞或转基因动物的基因，可用于本申请的筛选标记基因没有特别限制，包括转基因领域常用的各种筛选标记基因，代表性例子包括(但并不限于)：潮霉素抗性基因(Hyg)、卡那霉素抗性基因(NPTII)、新霉素基因、或嘌呤霉素抗性基因。
如本文所用，术语“表达盒”是指含有待表达基因以及表达所需元件的序列组件的一段多聚核苷酸序列。例如，在本发明中，术语“筛选标记表达盒”指含有编码筛选标记的序列以及表达所需元件的序列组件的多聚核苷酸序列。表达所需的组件包括启动子和聚腺苷酸化信号序列。此外，筛选标记表达盒还可以含有或不含有其他序列，包括(但并不限于)：增强子、分泌信号肽序列等。
在本发明中，适用于外源基因表达盒和筛选标记基因表达盒的启动子可以是任何一种常见的启动子，它可以是组成型启动子或诱导型启动子。较佳地，该启动子是组成型的强启动子，例如35S启动子等其它适用于真核表达的植物启动子。
如本文所用，术语“植物启动子”指能够在植物细胞中启动核酸转录的核酸序列。该植物启动子可以是来源于植物、微生物(如细菌、病毒)或动物等，或者是人工合成或改造过的启动子。
如本文所用，术语“植物终止子”指能够在植物细胞中可使转录停止的终止子。该植物转录终止子可以是来源于植物、微生物(如细菌、病毒)或动物等，或者是人工合成或改造过的终止子。代表性的例子包括(但并不限于)：Nos终止子。
如本文所用，术语“Cas蛋白”指一种核酸酶。一种优选的Cas蛋白是Cas9蛋白。典型的Cas9蛋白包括(但并不限于)：来源于酿脓链球菌SF370的Cas9。
如本文所用，术语“Cas蛋白的编码序列”指编码具有切割活性的Cas蛋白的核苷酸序列。在插入的多聚核苷酸序列被转录和翻译从而产生功能性Cas蛋白的情况下，技术人员会认识到，因为密码子的简并性，有大量多聚核苷酸序列可以编码相同的多肽。另外，技术人员也会认识到不同物种对于密码子具有一定的偏好性，可能会根据在不同物种中表达的需要，会对Cas蛋白的密码子进行优化，这些变异体都被术语“Cas蛋白的编码序列”所具体涵盖。此外，术语特定地包括了全长的、与Cas基因序列基本相同的序列，以及编码出保留Das蛋白功能的蛋白质的序列。
如本文所用，术语“植物”包括全植株、植物器官(如叶、茎、根等)、种子和植物细胞以及它们的子代。可用于本发明方法的植物的种类没有特别限制，一般包括任何可进行转化技术的高等植物类型，包括单子叶、双子叶植物和裸子植物。
如本文所用，术语“2A多肽编码序列”、“2A序列”指的是发现于病毒中的一段不依赖于蛋白酶的自剪切氨基酸序列，类似于IRES，利用2A可以实现单一启动子同时表达两个基因。它也广泛存在于各类真核细胞。与IRES不同的是，下游蛋白表达量不会减少。但剪切后2A多肽残基与上游蛋白连为一体，可在上游蛋白和2A多肽间加入一种Furin蛋白酶剪切点(4个碱性氨基酸残基，如 Arg-Lys-Arg-Arg)以从上游蛋白末端完全切除2A多肽残基。
本发明的构建物
本发明提供了一种在植物细胞内表达外源基因的核酸构建物，其是对小麦矮缩病毒(WDV)进行改造，利用其长基因间隔区(LIR)，短基因间隔区(SIR)和复制蛋白(Rep and RepA)，实现供体片段在植物细胞内的大量复制表达。
本发明的构建物如本发明第一方面所述。
本发明的构建物中所用的各种元件都是本领域中已知的，因此本领域技术人员可以用常规方法，如PCR方法、全人工化学合成法、酶切方法获得相应的元件，然后通过熟知的DNA连接技术连接在一起，就形成了本发明的构建物。
将本发明的构建物插入外源载体(尤其是适合转基因植物操作的载体)，就构成了本发明的载体。
将本发明的载体转化植物细胞从而介导本发明的载体对植物细胞染色体进行整合，制得转基因植物细胞。
将本发明的转基因植物细胞再生为植物体，从而获得转基因植物。
具体地，本发明构建物的构建流程和应用如下：
a)改造小麦矮缩病毒(WDV)以实现供体片段在植物细胞内的大量复制表达。野生型WDV基因组约3kb，包括长基因间隔区(long intergenic region,LIR)，短基因间隔区(short intergenic region,SIR)，复合的两个复制蛋白(replicase proteins,Rep and RepA)，以及包衣蛋白(Coat Protein,CP)和迁移蛋白(Move Protein,MP)(图1)。WDV侵染植物后通过Rep和RepA的作用能在植物细胞内大量复制，但其基因组一般不整合到植物基因组内，并且其复制子能在自身表达的包衣蛋白(CP)的作用下包埋成病毒颗粒，然后在迁移蛋白(MP)的作用下通过胞间连丝传递到旁边的细胞。为提高WDV对外源基因的装载能力，可以去除其包衣蛋白(CP)和迁移蛋白(MP)，仅保留长基因间隔区(LIR)，短基因间隔区(SIR)和复制蛋白(Rep and RepA)(图1，序列一)。改造后的病毒理论上没有了侵染及迁移能力，但保留了复制能力，两个LIR之间的区域会形成环状复制子，并在Rep及RepA蛋白的作用下大量复制。改造后的病毒可以利用基因枪的方式导入至植物细胞内，亦可将其克隆至pCambia的T-DNA区，通过农杆菌侵染的方式导入到植物细胞内。
b)为验证改造后的WDV复制模型，把一个完整的GUS表达基因盒和GFP表达基因盒(均包含35S启动子-CDS-NOS终止子)通过BamHI和SpeI限制性酶切位点分别克隆至改造后病毒的LIR及SIR区域之间，然后将整合后的病毒序列克隆到pCambia-1300的T-DNA区(图2，4)，通过农杆菌侵染的方式转化水稻日本晴的愈伤组织，进入水稻细胞后GUS基因表达盒和GFP基因表达盒会形成环状复制子并大量复制(图3，5)。转化后的愈伤通过GUS染色或在荧光显微镜下观察GFP发光，结果显示，通过WDV复制的GUS或GFP的表达强度明显高于普通的pCambia-GUS或pCambia-GFP(图6，7)。
c)为进一步确认改造后WDV的环化复制是否存在，分别提取了pCambia-GFP 和pCambia-WDV-GFP转化后愈伤组织的DNA，通过特异引物扩增检测到在pCambia-WDV-GFP转化中Rep片段及只有环化复制后才能产生的片段(图8)。
d)为进一步确认侵染水稻愈伤组织后WDV形成复制子的拷贝数，利用pCambia-GFP和pCambia-WDV-GFP转化后的愈伤组织分离原生质体，然后通过流式细胞仪分离并富集带有GFP信号的细胞，提取这些细胞的DNA，利用qPCR测定GFP片段的相对阈值，通过与标准样品的比较得出pCambia-WDV-GFP转化后平均每个带有GFP信号的细胞里GFP的绝对拷贝数约500个，是相应的pCambia-GFP转化的数百倍(图9)。
e)为进一步研究改造过的WDV侵染水稻愈伤组织后的持续表达时长，pCambia-WDV-GUS和pCambia-WDV-GFP农杆菌侵染过的愈伤组织从第三天起(一般农杆菌转化水稻愈伤后需要2-3天的共培养时间，所以从第三天开始观察)，每隔两天通过GUS染色或在体视式荧光显微镜下持续观察GFP发光，记录WDV所携带外源基因的持续表达情况。结果显示WDV所携带的GUS基因或GFP基因能持续高强度表达7天左右(从转化后第三天算起)，之后逐渐下降，但半个月后仍然能看到表达(图10，11)。
f)鉴于WDV具有强大的复制能力，而LIR区域本身也具有一定的双向驱动表达能力，猜测所携带的GUS基因或GFP基因即使删除其自身的35S启动子依然能大量表达。以去除35S启动子的WDV-GFP(LIR)做验证(图12)，转化水稻愈伤后GFP信号较原来的WDV-GFP(35S)有所减弱，但仍然明显高于pCambia-GFP对照(图13)。这一特性有助于缩短WDV所携带外源基因的长度，从而进一步增加复制子的拷贝数。
g)构建一个可以融合到植物功能基因3’端的并可定向筛选的GFP/Taq-P2A-NptII/Hyg基因盒。这样的一个基因盒子包括一个功能序列，如GFP可以用作靶标基因的亚细胞定位及动态示踪，也可以是一个标签如3XFlag，可以用作分离纯化靶标基因的蛋白。基因盒的后部分是一个广谱筛选标记，如抗潮霉素基因(Hyg)或抗卡那霉素基因(NptII)。中间是具有自剪切功能的2A多肽编码序列。下面以构建GFP-P2A-NptII基因盒为例(图14，序列二)：
其中的GFP仅包含CDS，而不包含转录启动子(如35S)及转录终止子(如NOS)，并且翻译起始密码子ATG及翻译终止密码子TAA也去掉。GFP编码序列的前端连接一段翻译柔性氨基酸的序列(连同BamHI酶切位点)，该序列是连接靶标基因和GFP基因的一段连桥，避免两个编码蛋白相互干扰，可根据不同类型的靶标基因灵活设计。紧随GFP后面的是具有自剪切功能的2A多肽编码序列，2A多肽编码序列后面通过限制性酶切位点KpnI连接NptII编码序列，同样的，NptII编码序列的翻译起始密码子ATG也去掉，但保留其翻译终止密码子TGA。当GFP-P2A-NptII基因盒融合到植物基因组内某功能基因的3’端，GFP及NptII编码序列可以跟随该基因的表达和翻译而起作用。而没有融合到特定序列的GFP-P2A-NptII基因盒则由于其自身没有转录启动子和翻译起始密码子ATG而不会产生作用。这样，可以通过NptII基因的作用而筛选富集定向插入事件从而避免大量随机插入事件的干扰，而GFP基因的作用既可在筛选水稻阳性愈伤克隆时选择有GFP信号的愈伤 从而提高选择的准确性，亦可在研究该靶标基因功能时发挥作用，例如观察其亚细胞定位及细胞分布流动情况。整个基因盒子的两端分别添加BamHI和PstI酶切位点，用以添加靶标基因的左同源臂和右同源臂。
h)改造LIR序列，使其失去转录驱动能力但保留复制能力。由于LIR区域本身也具有一定的双向驱动表达能力，为保证GFP/Taq-P2A-NptII/Hyg基因盒定向筛选特性，希望随机插入基因组的NptII/Hyg不会被转录，需要改造第一个LIR序列。经序列分析发现LIR序列后部分存在类似TATA盒子结构，故在第一个LIR把这部分序列掉，仅保留前面的序列用以连接GFP/Taq-P2A-NptII/Hyg基因盒(序列一)。
i)定点切割核酸酶DNA片段的制备。ZFN、Talen和CRISPR/Cas9技术都可以在植物基因组上制造定点切割产生双链DNA断裂。因此，表达这3种定点切割核酸酶的DNA元件都可以用于本发明。由于CRISPR/Cas9技术相对简单高效，本发明优选CRISPR/Cas9在植物基因组上制造定点切割。
j)遗传转化。将WDV复制区与表达定点切割核酸酶的DNA片段导入到植物受体中，上述两个片段可以整合到一个pCambia的T-DNA区内。导入方法包括但不局限于：基因枪法、显微注射法、电击法、超声波法和聚乙二醇(PEG)介导法等。受体植物包括但不限于水稻、大豆、番茄、玉米、烟草、小麦、高粱等。由于WDV的复制可使供体片段的拷贝数达到数百个，极大的提高了供体DNA片段定点插入到该基因组位点的效率，因此该发明不需要做大规模的农杆菌转化及后续的组织培养和筛选鉴定工作，通过常规的组织培养即可获得足够的定点敲入植株。
本发明的核苷酸序列
SEQ ID NO.:1显示了改造后WDV的结构序列LIR-SIR-Rep-LIR。其中，第1-409位为第一LIR序列，第422-608位为SIR序列，第609-1750位为Rep序列，第1751-2159位为第二LIR序列。该结构连接EGFP-P2A-NptII基因盒时，需要删除第一LIR序列的第330-409位。第一LIR序列SIR之间的BamHI(下划直线表示)及SpeI(下划波浪线标示)限制性酶切位点为插入外源基因的位置。
Figure PCTCN2017109119-appb-000001
Figure PCTCN2017109119-appb-000002
SEQ ID NO.:2显示了EGFP-P2A-NptII基因盒的序列。其中，第19-732位为EGFP的CDS序列，第733-798位为P2A的序列，第805-1599位为NptII的序列，第1-18位为一段翻译柔性氨基酸的序列。下划直线标示BamHI，下滑波浪线标示KpnI，下划虚线标示PstI，上述限制性酶切位点为方便克隆而设置。
Figure PCTCN2017109119-appb-000003
Figure PCTCN2017109119-appb-000004
SEQ ID NO.:5显示了水稻OsACT1定点插入EGFP-P2A-NptII基因盒的序列。大写字母表示OsACT1的相关序列，其序列参考水稻基因组注释(Rice Genome Annotation Project,http://rice.plantbiology.msu.edu/index.shtml),登录号：LOC_Os03g50885。小写字母表示外源插入的EGFP-P2A-NptII基因盒序列。下划波浪线为5’同源臂序列，下划直线为3’同源臂序列。方框内为sg(PAM)的序列。第2336-2338位加粗的TAA为OsACT1翻译终止密码子。第1-22位为引物ACT-F1的结合位点；第1207-1228位为引物GFP-R1的结合位点；第1765-1784位为引物NPTII-F1的结合位点；第2990-3009位为引物ACT-R1的结合位点。
Figure PCTCN2017109119-appb-000005
Figure PCTCN2017109119-appb-000006
SEQ ID NO.:6显示了水稻OsGST定点插入EGFP-P2A-NptII基因盒的序列。大写字母表示OsGST的相关序列，其序列参考水稻基因组注释(Rice Genome Annotation Project,http://rice.plantbiology.msu.edu/index.shtml),登录号：LOC_Os05g02530。小写字母表示外源插入的EGFP-P2A-NptII基因盒序列。下划波浪线为5’同源臂序列，下划直线为3’同源臂序列。方框内为sg(PAM)的序列。第2374-2376位加粗的TAA为OsGST翻译终止密码子。第1-22位为引物GST-F1的结合位点；第1236-1257位为引物GFP-R1的结合位点；第1794-1813位为引物NPTII-F1的结合位点；第3032-3053位为引物GST-R1的结合位点。
Figure PCTCN2017109119-appb-000007
Figure PCTCN2017109119-appb-000008
应用
本发明可应用于植物基因工程领域，用于改造各种不同的植物，尤其是具有经济价值的农作物和林业植物。
本发明的主要优点包括：
(a)通过改造玉米线条病毒属一类的DNA病毒，使之成为一种能在植物细胞内大量并持续复制外源供体片段的工具，供体的拷贝数可达数百甚至数千个，用作同源重组或定点插入的效率大幅提高，相对于传统的基于T-DNA插入的正负筛选系统，不需要做大规模的农杆菌转化及后续的组织培养和筛选鉴定工作，一次常规的农杆菌转化即可拿到足够的目标植株。
(b)病毒形成复制子并大量复制的过程为瞬时表达行为，其复制子不会随机整合到植物基因组内，相比基因枪轰击法，该发明避免了大量外源片段随机插入到 基因组的弊端。
(c)之前在植物上报道的基因重组或定向插入技术大多基于靶标基因本身经修饰后具备可筛选特性，而生物体内的大多数功能基因并不能提供这样的选择表型，该发明通过在供体片段后联结一个广谱的筛选标记，可以有效筛选定点重组或插入事件，适用于大多数功能基因。
(d)改造后的病毒在转化当代并不会对植株的生长发育造成严重的影响，其修饰的靶标基因可正常传递至下一代，并且整合到T-DNA区而插入到基因组内的病毒序列可通过自交分离。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
实施例1
在水稻内源OsACT1基因的CDS末端插入一个GFP-P2A-NptI I基因盒。
在水稻内源OsACT1基因的CDS末端设计并测试CRISPR-Cas9靶向位点(sg)，转化水稻品种日本晴的愈伤组织并拿到再生的植株群体后，抽样对靶标位点进行扩增测序发现高比例的各种敲除突变体(表1，图19)，说明该sg能在靶标序列高效地切割产生DSB。
表1 利用CRISPR-Cas9敲除OsACT1和OsGST的靶序列及T0代植株群体检测结果
Figure PCTCN2017109119-appb-000009
注：Ho纯合突变体；Bi等位双突变体；He杂合突变体；Chi嵌合突变体；WT野生型。下划线所示序列(AGG/TGG)为PAM序列。
以水稻品种日本晴的基因组DNA为模板，在靶标位点的左侧和右侧各扩增一段约500bp的同源臂(图19)，分别通过BamHI和PstI酶切位点连接至GFP-P2A-NptII基因盒的5’端和3’端。然后把CRISPR表达盒OsU6-ACT1sg-sgRNA通过SpeI酶切位点克隆至3’同源臂末端。这样，整个5’同源臂-GFP-P2A-NptII基因盒-3’同源臂-sgRNA表达盒整合至WDV的LIR和SIR复制区内。最后整个WDV表达盒克隆至pCambia-1300的T-DNA区域内(图15)。
通过农杆菌转化的方式把WDV表达盒运载到已经整合了Cas9基因的水稻品种日本晴的愈伤组织中，共培养3天后所转化的愈伤组织转移到含有G418筛选剂的筛选培养中。以15天为一个筛选周期，连续筛选两轮后得到阳性克隆的愈伤，在荧光显微镜下观察发现有GFP发光的愈伤(图16)。或者转化后的愈伤组织在筛选一个周期后，直接转移到含有G418筛选剂的分化培养基中再生植株，在T0代植 株群体中抽样提取DNA检测外源基因的整合情况。
利用特异性扩增5’和3’融合边界的引物对，通过PCR检测发现部分植株的5’和3’融合边界扩增均为阳性(图17，18)。
全部鉴定结果显示，利用WDV复制的方式实现定向基因插入的机率是传统的T-DNA提供供体方式的3倍，比基因枪大量轰击线性化供体片段的方式也提高了50％。然而，当不添加CRISPR表达盒OsU6-ACT1sg-sgRNA时，即使利用WDV复制的方式提供供体片段也没有检测出定向基因插入植株，说明在靶标位点引入DSB同样非常重要(表2)。
表2 不同转化方式及供体提供方式对水稻OsACT1靶向敲入外源基因的检测结果
Figure PCTCN2017109119-appb-000010
进一步对所有5’和3’融合边界扩增均为阳性的植株进行整合位点测序，发现全部植株的整合序列及其重组的边界序列均与理论参考序列一致(图19，SEQ ID NO.:5中的部分序列)，没有出现任何其它碱基的突变(缺失、插入或替换)。
实施例2
在水稻内源OsGST基因的CDS末端插入一个GFP-P2A-NptII基因盒(图20)
在水稻内源OsGST基因的CDS末端设计CRISPR-Cas9靶向位点，转化水稻品种日本晴的愈伤组织并拿到再生的植株群体后，抽样对靶标位点进行扩增测序发现高比例的各种敲除突变体，说明该sg能够高效地引导Cas9蛋白在靶标序列切割产生DSB(表1，图23)。以水稻品种日本晴的基因组DNA为模板，在靶标位点的左侧和右侧各扩增一段约500bp的同源臂，分别通过BamHI和PstI酶切位点连接至GFP-P2A-NptII基因盒的5’端和3’端。然后把CRISPR表达盒OsU6-GSTsg-sgRNA克隆至3’同源臂末端。这样，整个5’同源臂-GFP-P2A-NptII基因盒-3’同源臂-sgRNA表达盒整合至WDV的LIR和SIR复制区内。最后整个WDV复制区克隆至pCambia-1300的T-DNA区域内。
通过农杆菌转化的方式把WDV表达盒运载到已经整合了Cas9基因的水稻品种日本晴的愈伤组织中，共培养3天后所转化的愈伤组织转移到含有G418筛选剂的筛选培养中。愈伤组织在筛选一个周期后，直接转移到含有G418筛选剂的分化培养基中再生植株，在T0代植株群体中抽样提取DNA检测外源基因的整合情况。
利用特异性扩增5’和3’融合边界的引物对，通过PCR检测发现部分植株的5’和3’融合边界扩增均为阳性(图21，22)。
全部鉴定结果显示，利用传统的T-DNA提供供体片段没有实现定点整合，而利用WDV复制的方式实现定向基因插入的机率达到7.5％。同样，当不添加CRISPR表达盒OsU6-GSTsg时，即使利用WDV复制的方式提供供体片段也没有检测出定向 基因插入植株(表3)。
表3 不同转化方式及供体片段提供方式对水稻OsGST靶向敲入外源基因的检测结果
Figure PCTCN2017109119-appb-000011
进一步对所有5’和3’融合边界扩增均为阳性的植株进行整合位点测序，发现全部植株的整合序列及其重组的边界序列均与理论参考序列一致，没有出现任何其它碱基的突变(缺失、插入或替换)(图23，SEQ ID NO.:6中的部分序列)。
实施例3在水稻内源OsACT1和OsGST基因的CDS末端插入一个GFP-P2A-NptI I基因盒(转化野生型愈伤组织)
在该实施例中，进一步把Cas9表达盒(包含Ubi启动子、Cas9完整的CDS和NOS终止子)整合到实施例1和2中WDV所在的T-DNA区域。此外，位于WDV复制区之外，同时为了防止LIR对Cas9表达盒可能造成的干扰，在WDV复制区和Cas9表达盒之间放置了一个35S polyA转录终止子(图24)。
通过农杆菌转化的方式把该核酸构建物转化到野生型水稻品种日本晴的愈伤组织中，共培养3天后所转化的愈伤组织转移到含有G418筛选剂的筛选培养中。筛选15天后，直接转移到含有G418筛选剂的分化培养基中再生植株，在T0代植株群体中抽样提取DNA检测外源基因的整合情况。
鉴定结果显示，对OsACT1和OsGST两个位点的靶向敲入概率分别为8.5％和4.7％。这说明，WDV复制区和Cas9表达盒整合在一个T-DNA区域也能非常有效地进行外源基因定点敲入(表4)。
表4 WDV表达盒和Cas9表达盒同在的核酸构建物转化野生型水稻愈伤靶向敲入外源基因的检测结果。
Figure PCTCN2017109119-appb-000012
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种核酸构建物，其特征在于，所述的构建物具有从5’至3’的式I结构：
    A-B-C-D-E-F-G-H     (I)
    式中，
    A、B、C、D、E、F、G、H分别为用于构成所述构建物的元件；
    各“-”独立地为键或核苷酸连接序列；
    A为第一长基因间隔区序列；
    B为任选的5’同源臂序列；
    C为外源基因表达盒；
    D为任选的3’同源臂序列；
    E为任选的第二表达盒；
    F为短基因间隔区序列；
    G为玉米线条病毒属病毒的复制蛋白的编码序列；
    H为第二长基因间隔区序列；
    并且，所述第一长基因间隔区序列、短基因间隔区序列、和第二长基因间隔区序列来源于玉米线条病毒属(Mastrevirus)病毒。
  2. 如权利要求1所述的构建物，其特征在于，所述的玉米线条病毒属病毒包括小麦矮缩病毒(Wheat Dwarf Virus，WDV)、菜豆黄矮病毒(Bean Yellow Dwarf Virus,BeYDV)、玉米线条病毒(MSV)、烟草黄矮病毒(TYDV)。
  3. 如权利要求1所述的构建物，其特征在于，所述的外源基因表达盒具有从5’至3’的式II结构：
    C1-C2-C3     (II)
    式中，
    C1、C2、C3分别为用于构成所述外源基因表达盒的元件；
    各“-”独立地为键或核苷酸连接序列；
    C1为任选的第一启动子序列；
    C2为外源蛋白的编码序列；
    C3为终止子序列。
  4. 如权利要求1所述的构建物，其特征在于，所述的外源基因表达盒具有从5’ 至3’的式III结构：
    Ci-Cii-Ciii-Civ     (III)
    式中，
    Ci、Cii、Ciii、Civ分别为用于构成所述外源基因表达盒的元件；
    各“-”独立地为键或核苷酸连接序列；
    Ci为第一外源蛋白的编码序列；
    Cii为2A序列或IRES序列；
    Ciii为第二外源蛋白(如筛选标记)的编码序列；
    Civ为终止子序列。
  5. 如权利要求1所述的构建物，其特征在于，所述的第二表达盒为定点切割核酸酶的靶向位点识别序列的表达盒。
  6. 一种载体，其特征在于，所述的载体含有权利要求1所述的构建物。
  7. 一种基因工程细胞，其特征在于，所述的细胞含有权利要求1所述的构建物，或其基因组整合有一个或多个权利要求1所述的构建物。
  8. 一种制备转基因植物细胞的方法，其特征在于，包括步骤：
    (i)将权利要求1所述的构建物、或权利要求6所述的载体转染植物细胞，使得所述构建物与所述植物细胞中的染色体发生定点重组，从而制得转基因植物细胞。
  9. 一种制备转基因植物细胞的方法，其特征在于，包括步骤：
    (i)将权利要求1所述的构建物、或权利要求6所述的载体转染植物细胞，使得所述植物细胞含有所述构建物，从而制得转基因植物细胞。
  10. 一种制备转基因植物的方法，其特征在于，包括步骤：
    将权利要求8或权利要求9所述方法制备的转基因植物细胞再生为植物体，从而获得转基因植物。
PCT/CN2017/109119 2016-11-03 2017-11-02 在植物细胞内表达外源基因的核酸构建物及其应用 WO2018082611A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201610958988.1 2016-11-03
CN201610958988 2016-11-03
CN201710061566.9A CN108018306B (zh) 2016-11-03 2017-01-26 在植物细胞内表达外源基因的核酸构建物及其应用
CN201710061566.9 2017-01-26

Publications (1)

Publication Number Publication Date
WO2018082611A1 true WO2018082611A1 (zh) 2018-05-11

Family

ID=62075762

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/109119 WO2018082611A1 (zh) 2016-11-03 2017-11-02 在植物细胞内表达外源基因的核酸构建物及其应用

Country Status (1)

Country Link
WO (1) WO2018082611A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817766A (zh) * 2021-09-24 2021-12-21 沈阳农业大学 一种基因表达盒、重组表达载体及其制备方法与应用
CN114807198A (zh) * 2022-05-31 2022-07-29 南京农业大学 带有可视化蛋白融合抗生素筛选标记的CRISPR/Cas9载体及其构建方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012058762A1 (en) * 2010-11-04 2012-05-10 Medicago Inc. Plant expression system
CN104962580A (zh) * 2015-06-30 2015-10-07 浙江大学 一种重组植物弹状病毒载体及其构建方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012058762A1 (en) * 2010-11-04 2012-05-10 Medicago Inc. Plant expression system
CN104962580A (zh) * 2015-06-30 2015-10-07 浙江大学 一种重组植物弹状病毒载体及其构建方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LU XIONGBIN ET AL: "Plant Transgenic Methods and Progress", LIFE SCIENCES, vol. 10, no. 3, 15 June 1998 (1998-06-15), pages 125 - 131 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817766A (zh) * 2021-09-24 2021-12-21 沈阳农业大学 一种基因表达盒、重组表达载体及其制备方法与应用
CN114807198A (zh) * 2022-05-31 2022-07-29 南京农业大学 带有可视化蛋白融合抗生素筛选标记的CRISPR/Cas9载体及其构建方法和应用
CN114807198B (zh) * 2022-05-31 2024-03-22 南京农业大学 带有可视化蛋白融合抗生素筛选标记的CRISPR/Cas9载体及其构建方法和应用

Similar Documents

Publication Publication Date Title
US11384360B2 (en) Gene targeting in plants using DNA viruses
US11225668B2 (en) Modified FRT recombination site libraries and methods of use
WO2018103686A1 (zh) 叶绿体基因组编辑方法
WO2018099475A1 (zh) 基于Cpf1的植物基因组定点编辑方法
US20200190492A1 (en) Novel anti-crispr polynucleotides and polypeptides and methods of use
Schiermeyer et al. Targeted insertion of large DNA sequences by homology‐directed repair or non‐homologous end joining in engineered tobacco BY‐2 cells using designed zinc finger nucleases
WO2018082611A1 (zh) 在植物细胞内表达外源基因的核酸构建物及其应用
CN108018306B (zh) 在植物细胞内表达外源基因的核酸构建物及其应用
Hou et al. Retrotransposon vectors for gene delivery in plants
Meynard et al. Thirty years of genome engineering in rice: From gene addition to gene editing
WO2020171192A1 (ja) 植物細胞のゲノム編集用核酸及びその用途
AU2016214048A1 (en) Agrobacterium-mediated genome modification without T-DNA integration
US20080070276A1 (en) Transcriptional Termination of Transgene Expression Using Host Genomic Terminators
US20100257632A1 (en) Methods for generating marker-free transgenic plants
AU2008332063A1 (en) Methods for generating marker free transgenic plants

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17867986

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17867986

Country of ref document: EP

Kind code of ref document: A1