WO2019219046A1 - 一种快速高效获得非转基因的定向基因突变植物的方法和应用 - Google Patents

一种快速高效获得非转基因的定向基因突变植物的方法和应用 Download PDF

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WO2019219046A1
WO2019219046A1 PCT/CN2019/087139 CN2019087139W WO2019219046A1 WO 2019219046 A1 WO2019219046 A1 WO 2019219046A1 CN 2019087139 W CN2019087139 W CN 2019087139W WO 2019219046 A1 WO2019219046 A1 WO 2019219046A1
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gene
transgenic
genetic
nucleic acid
plant
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赵云德
和玉兵
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华中农业大学
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • 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
    • A01H6/4636—Oryza sp. [rice]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • 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
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    • 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 invention belongs to the field of plant genetic engineering, and particularly relates to a method and an application for quickly and efficiently obtaining non-transgenic fixed-point gene mutant plants.
  • the method of the invention can rapidly obtain the site-directed mutant rice without the transgenic fragment, and can also be used for rapidly and efficiently constructing non-transgenic plants which are composed of multiple gene mutations. This method can help accelerate the functional study of rice genes, and at the same time facilitate complex multi-gene interaction studies, and in addition to accelerate the breeding and aggregation of good genes in rice.
  • SSNs sequence-specific nucleases
  • ZFN Zinc finger nucleases
  • TALEN Transcr iption activator-like effector nucleases
  • DSBs double-stranded breaks
  • NH EJ nonhomologous end joining
  • HDR homology-directed repair
  • the NH EJ repair method is mainly to directly connect the chromosomes at the break position, but this joint repair cannot guarantee very accurate repair, so that the deletion or insertion of nucleotides at the break position leads to mutation of the gene.
  • HDR repair mainly occurs in the presence of homologous sequences.
  • organisms can use the homologous sequence as a template to complete the repair of the fracture site. This method will produce accurate repair because of the presence of the template. If some artificial mutations are designed in the template, these mutations are accurately introduced into the genome of the organism.
  • CRISPR/CAS9 SSN-based gene site-directed mutagenesis techniques
  • CRISPR/CAS9 technology is simple to operate and low in cost, it can be achieved by changing a small RNA sequence to identify different target sites, so CRISPR/CAS9 technology is widely used.
  • Efficient targeting of editable DNA in any transformable organism provides an unprecedented tool for agricultural improvement.
  • CRISPR gene editing technology Since the first publication of the CRISPR/Cas9-meidated editing event in eukaryotes (Cong et al., 2013), CRISPR gene editing technology has also been widely used in plants. The timely removal of transgenic fragments from edited plants is a critical step in assessing the genetic and phenotypic stability of CRISPR-edited plants and is critical for crop improvement.
  • the sexual reproduction of plants needs to undergo generational alternation of sporozoites and gametophytes.
  • Sporozoites (2n) of sporophyte (2n) undergo meiosis to produce haploid gametophytes (n); male and female gametophytes combine to produce zygote by fertilization (2n) ), the zygote continues to grow and grow into sporozoites (2n).
  • the sporoblasts of the transgenic plants undergo meiosis to produce gametophytes carrying the transgenic construct and gametophytes without the transgenic construct, and the male and female embryoid bodies are freely combined to produce zygotes with fertilization.
  • Another reported strategy for isolating transgenic editing plants is to couple the CRISPR construct to an RNA interference element that targets the herbicide resistant P450 enzyme. This makes it possible to screen plants that do not contain transgenic fragments by means of specific herbicides (Lu et al., 2017). However, this strategy is still not rich in genetically modified plants, and it is necessary to plant and screen future generations, increasing the input of labor.
  • a genetically modified plant containing no transgene was obtained using the method of RNP (Cas9 protein-gRNA RiboNucleoProteins). RNPs typically utilize protoplast or immature embryo transformation methods (Liang et al., 2017) to obtain non-transgenic genetic editing materials.
  • protoplast transformation needs to undergo protoplast culture, transformation of protoplasts into callus, and differentiation of callus into seedlings.
  • the efficiency of each step is superimposed to limit the differentiation efficiency of monocotyledons.
  • the method of immature embryo transformation mainly bombards RNP into plant cells by a gene gun. Because neither method is screened with antibiotics, many plants that have not been mutated will be doped into the differentiated seedlings. Liang's research shows that only about 4% of plants have edited a single gene (Liang et al., 2017). Although offspring can be used to screen for mutant plants by PCR identification, this is also a labor-intensive task, especially when systematically studying multiple genes, the time and labor cost of obtaining available materials will become the limit of research and development. Speed factor.
  • BARNASE is a 12 kD extracellular small molecule ribonuclease produced by Bacillus amyloliquefaciens.
  • the BARNASE gene is highly toxic and can degrade RNA in cells, which will cause cell death. Previous studies have found that proteins expressed by this gene can kill plant cells (Lannenpaa et al., 2005).
  • the rice REG2 promoter can function very specifically during embryo development of seeds (Sun et al., 1996).
  • ORF79 or ORFH79 Two haplotypes of the MGL gene, ORF79 or ORFH79 (Hu et al., 2012), produce proteins that disrupt mitochondrial function and lead to male sterility during male gametophytic development. It has been reported that a haplotype ORF79 that drives the MGL using the CaMV35S constitutive promoter specifically kills pollen grains with the transgenic construct (Zou Yanzhen, 2006).
  • the present invention proposes a novel rapid and efficient non-transgenic fixed-point gene mutant plant for the mechanism of gene editing system.
  • the technical scheme combines a gene editing element with a gametophyte-specific lethal element or a seed-specific lethal element into a linked system, which is named TKE (Transgene Killer Editing).
  • the gene editing component in the TKE system can perform the function of gene editing, and the gametophyte-specific lethal component or the seed-specific lethal component can develop a seed-specific killing of the zygote or zygote that is combined with the gametophyte or gametophyte carrying the transgenic construct. dead.
  • the transgenic plants can autonomously eliminate their own transgenic offspring and independently screen out their own targeted mutant progeny without the transgenic construct.
  • the object of the present invention is to overcome the problem of the time-consuming and laborious screening in the process of obtaining non-transgenic mutants by site-directed mutagenesis technology, and to develop a method for rapidly obtaining non-transgenic targeted gene mutant plants, and the applicant named this method TKE. (Transgene Killer Editing: Elimination of Gene Editing of Transgenic Constructs) System.
  • a plant gene editing system such as the CRISPR/CAS9 system
  • the present invention will use a typical combination to demonstrate that the TKE system of the present invention can rapidly and efficiently obtain non-transgenic fixed-point gene mutant plants.
  • the basic steps are as follows: 1) The gene editing element uses the CRISPR/Cas9 gene editing element, including the Cas9 protein expression cassette. And sgRNA transcription cassettes. 2) Male gamete lethal element The 35S-MGL male gamete lethal element is used, and the MGL expression driven by the CaMV35S promoter will ensure that any male gametophyte containing MGL will be killed.
  • REG2-BARNASE embryo lethal element Female gametes or embryonic or endosperm lethal elements use the REG2-BARNASE embryo lethal element, the REG2 promoter functions very specifically during embryo development of the seed, and the BARNASE gene is placed under the control of the rice REG2 promoter, BARNASE Toxic proteins are not produced during callus or vegetative growth and are only produced during embryo development of the seed, so BARNASE driven by the REG2 promoter will ensure that any seed embryo containing BARNASE is killed.
  • the REG2-BARNASE and 35SMGL expression cassettes were introduced into the plasmid pCXUN-CAS9 carrying the CAS9 gene (He et al., 2017) (Fig. 1a), thereby completing vector construction of the TKE system.
  • the present invention is implemented by the following technical solutions:
  • Applicant provides a method for creating a non-transgenic plant (TKE system for short) that is genetically mutated, as follows:
  • the plant to be treated can be transgenic by introducing an exogenous nucleic acid molecule
  • step a) comprising introducing into the plant a construct comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule is a gene editing element (wherein the first nucleic acid molecule a genetic element capable of editing a nucleic acid; the second nucleic acid molecule is a lethal or stop developing element;
  • Plants which can be applied to the above steps a) and b) include plants of the family Gramineae, Leguminosae, Cruciferae, Compositae, Solanaceae, etc. which can obtain transgenic materials by Agrobacterium inoculation; preferably rice, corn, sorghum, barley, oats , wheat, millet, Brachypodium sylvestris, yarrow, sugar cane, soybean, canola, Arabidopsis, safflower, tomato, tobacco, alfalfa, potato, sweet potato, sunflower and cotton.
  • the genetic element capable of editing the nucleic acid described above may be selected from the group consisting of genetic elements of any one of the gene editing systems.
  • a specific embodiment includes a gene editing system preferably: for example, a ZFN gene editing system, a TALEN gene editing system, a CRISPR/CAS9 gene editing system, and a CRISPR/CPF1 gene editing system.
  • the gene elements of the above CRISPR/CAS9 gene editing system include the CAS9 gene (the nucleotide sequence of the CAS9 gene is the nucleotide sequence shown in SEQ ID NO: 1) and the sgRNA gene (the nucleotide sequence of the core skeleton of the sgRNA gene). As shown in SEQ ID NO: 2).
  • the second nucleic acid molecule is selected from the group consisting of gene element A, gene element B, and gene element C, wherein the gene element A is caused to cause death or stop development of the fertilized egg or embryo.
  • a genetic element, a genetic element B, is a genetic element that causes death or cessation of development of a fertilized polar nucleus or endosperm
  • a genetic element C is a combined genetic element, including a genetic element D and a genetic element A combination element, a genetic element D, and a genetic element B combination element
  • Gene element D and gene element E are combined elements, wherein gene element D is a genetic element that causes death or cessation of development of male gamete cells or pollen, and genetic element E is a genetic element that causes death or cessation of development of female gametophytic cells or polar nucleus cells.
  • the nucleotide sequence of the above gene element A is the nucleotide sequence shown in SEQ ID NO: 3.
  • the nucleotide sequence of the above gene element D is the nucleotide sequence shown in SEQ ID NO: 4.
  • the present invention actively and automatically eliminates any plant containing a transgenic construct, but still enables the plant to undergo targeted gene editing before the transgenic construct is removed.
  • the first nucleic acid molecule in the construct of the TKE system will be fixed to edit the target gene in the plant cell (see panel b in Figure 1).
  • the second nucleic acid molecule in the construct of the TKE system will kill or stop the development of the embryo or endosperm produced by the combination of the male gametophyte containing the TKE construct and the female gametophyte or the male and female embryonic bodies.
  • Any seed harvested from T0 plants transformed with the construct of the TKE system of the present invention is a non-transgenic seed containing no construct, and the target gene of these seeds contains the expected mutation (b diagram in Figure 1).
  • the present invention greatly reduces the time and labor required to isolate a gene-free plant without a transgenic fragment, greatly speeding up the progress of obtaining a transgenic mutant, and provides a very useful tool for crop improvement.
  • the present invention can prevent transgene drift caused by the scattering of pollen or seeds.
  • SEQ ID NO: 1 is the nucleotide sequence of the CAS9 gene. The sequence length is 4131 bp.
  • SEQ ID NO: 2 is the core skeletal nucleotide sequence of the sgRNA gene. The sequence length is 80 bp.
  • SEQ ID NO: 3 is the nucleotide sequence of gene element A. The sequence length is 2400 bp.
  • SEQ ID NO: 4 is the nucleotide sequence of gene element D. The sequence length is 1452 bp.
  • SEQ ID NO: 5 is the nucleotide sequence of the TKE plasmid. The sequence length is 18964 bp.
  • FIG. 1 Schematic diagram of self-elimination of the suicide transgene-mediated CRISPR/Cas9 construct after editing the target gene.
  • DESCRIPTION OF REFERENCE NUMERALS a) Schematic representation of three key components of the TKE (GMO Killer CRISPR) plasmid.
  • the cytoplasmic male sterility system gene MGL is under the control of the CaMV 35S promoter.
  • NOS refers to a terminator of the nopaline synthase gene from Agrobacterium tumefaciens.
  • the REG2 promoter is specific for early embryonic development and is used to drive the BARNASE gene, which encodes a toxic enzyme to plant cells.
  • the rbcs-E9 terminator was originally cloned from the pea rbcS-E9 gene. Codon-optimized Cas9 was placed under the control of the ubiquitin promoter UBQ of maize.
  • b) Flowchart of TKE-mediated isolation of rice plants without transgenic and CRISPR/Cas9 editing.
  • the TKE plasmid was transformed into rice callus by Agrobacterium-mediated transformation.
  • the BARNASE gene is not expressed during callus growth and vegetative growth, and the target gene may be edited by Cas9.
  • any male gametophyte containing Cas9 is killed by MGL and any embryo containing Cas9 is killed by BARNASE. Therefore, all seeds from TO plants are free of transgenes.
  • FIG. 2 Schematic representation of mutations and separation patterns in T1 plants produced by TKE-LAZY1. DESCRIPTION OF REFERENCE NUMERALS: a) The efficiency of transgene elimination and gene editing was tested using the TKE-LAZY1 plasmid. The target sequence (marked green) of the LAZY1 gene containing the PstI restriction site directly in front of the PAM site AGG was selected. Loss of function The lazy1 mutant showed a marked collateral phenotype. b) The PAM site "AGG" required for Cas9 cutting is marked in green. WT refers to wild type plant rice variety Zhonghua 11 (abbreviated as ZH11). The DNA sequence (genotype) of T1 plants from a single TO plant is shown.
  • a red “-” means deleting a base pair.
  • "a” in red and superscript refers to the insertion of "A”.
  • Progeny from T0 plant #34 produced only homozygous progeny.
  • the progeny from T0 plant #3 have three genotypes indicating the chimeric nature of the TO plant.
  • FIG. 3 A diagram of the TKE plasmid constructed in accordance with the present invention.
  • FIG. 4 Detection of transgenic fragments of T1 plants produced by TKE-LAZY1. DESCRIPTION OF REFERENCE NUMERALS: Five T0 generation plants with lazy1 phenotype (numbers #3, #30, #34, #40, #49, respectively) were randomly selected and germinated to obtain T1 generation seedlings for transgenic fragment detection. At the same time, it was tested whether the five T0 plants contained transgenic fragments. The results showed that the 5 plants with lazy1 phenotype were transgenic positive in the T0 generation and negative in the T1 generation.
  • the first nucleic acid molecule is exemplified by a genetic element of the CRISPR/CAS9 gene editing system; the second nucleic acid molecule is exemplified by a combination of the genetic element A and the genetic element D.
  • the feasibility verification gene of the technical scheme of the present invention is exemplified by the rice LAZY1 gene.
  • the transgenic method is exemplified by Agrobacterium-mediated rice stable transformation method, and is a conventional method (see the document published by the applicant's Huazhong Agricultural University related rice genetically modified patent authorization document or patent application).
  • the applicant's research has successfully constructed a plant gene editing vector pCXUN-CAS9 (He et al., 2017) using the genetic element of the CRISPR/CAS9 gene editing system (the nucleotide sequence of the CAS9 gene of the system is shown in SEQ. ID NO: 1). On this basis, the applicant added the gene element A and the gene element D to the pCXUN-CAS9 vector to verify the TKE system of the present invention.
  • the gene element A is exemplified by a BARNASE gene expression cassette (the nucleotide sequence of which is shown in SEQ ID NO: 3), and the gene element D is exemplified by an MGL gene expression cassette (nucleotide sequence is shown in SEQ ID NO: 4).
  • MGL and REG2 promoters were cloned ( Figure 1).
  • MGL gene was amplified using MGL-TAF (TGACAAATCTGCTCCGATG and MGL-TAR(CTTACTTAGGAAAGACTAC) as primers using genomic DNA of rice sterile line variety YTA (rice research institute from Guangdong Academy of Agricultural Sciences) as a template (Hu et al., 2012)
  • the DNA of the promoter of REG2 (Sun et al., 1996) was amplified using genomic DNA of rice cultivar 11 (ZH11) as a template and REG2P-TAF (GTCGACGAGCGAGTCATTAGCTAGTATAG) and REG2P-TAR (GGTGTTCGATCGATCCTAGCGGTG) as primers. Then, they were ligated into the T vector pEASY-T5 (TransGen Biotech) by TA cloning, respectively, to obtain two plasmids of MGL-TA and REG2P-TA
  • rE9T-F CTGCAGGAA TTCGATATCATTTAAATATTATGGCATTGGGAAAACTGTTT
  • rE9T-R GTAAAACGACGGCCAGTGCCAGTTTGGGATGTTTTACTCCTCATATTAAC
  • 35S-F GATTACGAATTCGAGCTCGGTACCCGGAGAGGCGGTTTGCGTATTGGCTA
  • 35S-R GATTACGAATTCGAGCTCGGTACCCGGAGAGGCGGTTTGCGTATTGGCTA
  • GAAGAGCCATCGGAGCAGATTTGTCATATCTCATTGCCCCCCGGATCTGCG primers
  • amplifying the 35S promoter DNA using MGL-TA plasmid as a template, MGL-TAF (TGACAAATCTGCTCCGATG) and MGL-R (AGCACATCCCCCTTTCGCCAGGGTTTAATTTTACTTAGGAAAGACTACACGAAT) are used to amplify MGL DNA; the above two PCR products are recovered by DNA digestion and mixed as a template to 35S-F (GATTACGAATTCGAGCTCGGTACCCGGAGAGGCGGTTTGCGTATTGGCTA) and MGL-R (AGCACATCCCCCTTTCGCCAGGGTTTAATTTTACTTAGGAAAGACTACAC
  • BAR-F CTGCAGGAATTCGATATCATTTAAATATGGCACAGGTTATCAACACG
  • BAR-R CAGTTTTCCCAATGCCATAATTTTAATTTTAAGAAAGTATGATGGTGATGTCGCAG
  • REG2P-TA plasmid was used as a template, REG2P-F (CTGCAGGAATTCGATATCATTTAAATGTCGACGAGCGAGTCATTAGCT) and REG2P-R (CGTGTTGATAACCTGTGCCATGGTGTTCGATCGATCCTAGCGGTG) were used as primers to amplify the promoter DNA of REG2, which was recovered by gelation and ligated into SwaI.
  • the 35S-MGL+BARNASE-pCXR9T vector gave the 35S-MGL+REG2-BARNASE-pCXR9T vector, the TKE plasmid (Fig. 3), and the complete nucleotide sequence of the plasmid is shown in SEQ ID NO: 5.
  • LAZY1 gene LAZY1 gene (LOC_Os11g29840) (Li et al., 2007) as a gene known to play an important role in a tropic response.
  • the loss of function lazy1 mutant showed a larger bifurcation angle (a map in Figure 2).
  • the visible phenotype of the lazy1 mutant allows qualitative evaluation of the editing efficiency of the constructs of the invention.
  • a specific sgRNA was designed using the LAZY1 gene in rice as a target gene, and the target sequence was GTCCGCGCCGGAGTACCTGC.
  • the final vector TKE (see Fig. 3) obtained in Example 1 was digested with Pme I into linear DNA, and sgRNA was introduced by overlapping PCR (conventional method) amplification.
  • the OsU6 promoter is used as a promoter of the sgRNA transcription unit, and the specific steps are as follows:
  • the TKE vector (Fig. 3) was ligated into the sgRNA element (the core skeletal nucleotide sequence of the sgRNA gene is shown in SEQ ID NO: 2).
  • the pCXUN-CAS9 vector He et al., 2017 with the OsU6P-sgRNA-OsU6T transcription cassette constructed in our laboratory was used as the template DNA, and the OsU6PF (GTCGTTTCCCGCCTTCAGTTTATGTACAGCATTACGTAGG) and LAZY1-U6R (GCAGGTACTCCGGGCGCGACAACCTGAGCCTCAGCGCAGC) primer pairs and OsU6TR were used respectively.
  • the sequenced positive plasmid TKE-LAZY1 was electrotransformed into Agrobacterium (EHA105) and infected with rice callus.
  • the transformed variety is “Zhonghua 11” (also known as ZH11, from the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences).
  • the specific transformation steps are as follows:
  • Agrobacterium strains were streaked with LA (LB+1.5% agar) and cultured at 28 ° C for 2 days; afterwards, all Agrobacterium were scraped into suspension medium; shaken at 28 ° C, 200 rpm Incubation for 0.5-1 h; measuring the concentration of the bacterial solution at a wavelength of 600 nm of a spectrophotometer, and adjusting to a value of 1.0 OD;
  • the resistant callus cultured in the screening medium is connected to the pre-differentiation medium, and the cultured callus is pre-differentiated for one week.
  • the resistant callus is transferred to the differentiation medium (50 ml/bottle; The flask or the flat-bottomed test tube was used as a culture flask; the culture was carried out at 25 ° C, 2000 Lux light, and the transgenic plants were obtained by regeneration.
  • CC-F TCCATATTTCATCTTCGGTGTCGT
  • CC-R AAGAGAGACCCATCATCATCAAGCTC;
  • the product size was 1105 bp.
  • the genomic DNA of flower wild type (ie non-transgenic) flower 11 (ZH11) was used as a negative control.
  • the loss-of-function lazy1 mutant showed a large bifurcation angle (a map in Fig. 2)
  • the increased phenotype of the lazy1 mutant's tiller angle can be used to qualitatively evaluate the editing efficiency of the construct of the present invention.
  • 29 had a distinct tillering angle, indicating that the CRISPR construct of the present invention is capable of producing a loss-of-function mutation on the target gene LAZY1.
  • Seeds were harvested from each individual TO plant and analyzed from progeny (T1 generation) from 5 independent positive TO plants, which have a visible lazy1 phenotype.
  • the first pair of primers is the first pair of primers:
  • MGL-429F TCTTCCATATTTCATCTTCGGTGT
  • MGL-429R GCATGACGTTATTTATGAGATGGG
  • the size of the amplified product was 429 bp.
  • BAR-377F AATTCAGACCGGATTCTTTACTCA
  • BAR-377R GTCGCTGATACTTCTGATTTGTTC;
  • the size of the amplified product was 377 bp.
  • the above genomic DNA was determined using ActinM-F (CTCAACCCCAAGGCTAACAG) and ActinM-R (ACCTCAGGGCATCGGAAC) as an internal control primer pair, and the quality of genomic DNA was determined by PCR amplification.
  • the genomic DNA of the T1 generation plants transformed with the common CRISPR vector pCXUN-CAS9 was subjected to PCR amplification using Hyg-280F and Hyg-280R primers to identify positive transgenic plants.
  • T1 T1 plants The results of T1 T1 plants are shown in Figure 4.
  • the positive results of transgenic positive are shown in Table 2.
  • LAZY1-GT1 CCTGCAACTGCATCACCGGGCTTG
  • LAZY1-GT2 TCCAAGGAAACCTCATGAAATAGTCAGCCA
  • TKE system of the present invention can automatically remove the transgenic T1 plant transgenic construct and ensure efficient site-directed mutagenesis of the progeny.
  • the invention can greatly reduce the time and manpower required for isolating rice without transgenic fixed-point DNA editing, and provides a very useful tool for crop genetic improvement. It can be easily applied to any other plant species that can be transgenicly manipulated by tissue culture.
  • CRISP RS an active interference element for a rapid and inexpensive selection of genome-edited, transgene-free rice plants. Plant Biotechnol J, 2017, 15(11): 1371-1373;

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Abstract

提供了一种获得非转基因的定向基因突变植株的方法和应用，通过创制定向基因突变的非转基因植株，消除转基因构建体的基因编辑，步骤为：a)将需要处理的植株通过引入外源核酸分子实施转基因；b)所述a)步骤的包括向植物引入构建体，其含有第一核酸分子和第二核酸分子，其中第一核酸分子为基因编辑元件；第二核酸分子为致死或停止发育元件。可用于植物基因编辑系统如CRISPR/CAS9中，能主动、自动消除植物转基因片段，留出足够时间使基因编辑元件在去除转基因片段之前进行靶向基因编辑，为基因编辑无转基因植株提供简单有效的方法。

Description

一种快速高效获得非转基因的定向基因突变植物的方法和应用 技术领域
本发明属于植物基因工程领域，具体涉及一种快速高效获得非转基因的定点基因突变植株的方法和应用。本发明的方法可以快速获得不含转基因片段的定点突变水稻，也可以用于快速高效构建集合多种基因突变的非转基因植株。该方法有助于加快水稻基因的功能研究，同时便于进行复杂的多基因互作研究，此外有助于加速改造和聚合水稻中优良基因的育种进度。
背景技术
近年来，随着生物技术的发展，基因组定点突变技术被逐步建立起来，其中主要依赖于一些序列特异核酸酶(Sequence-specific nucleases,SSNs)的功能解析及应用。其中主要包括三种SSN：锌指核酸酶(Zinc finger nucleases,ZFN)、转录激活样效应因子核酸酶(Transcr iption activator-like effector nucleases,TALEN)和成簇规律间隔短回文重复序列及其相关系统(Clustered regularly interspaced short palindromic repeats/CRISPR associated proteins,CRISPR/Cas system)。这几种SSN的共同特征是能够切割特定的DNA序列，诱导DNA双链断裂(Double-stranded breaks,DSBs)。随后生物体内的自我修复机制会自我启动，将断裂的DNA修复，根据修复方式的不同可以分为非同源重组连接(Nonhomologous end joining,NH EJ)和同源重组修复(homology-directed repair，HDR)(Symington and Gautier,2011)。NH EJ修复方式主要是通过直接将断裂位置的染色体连接起来，但是这个连接修复不能保证非常精确的修复，使得在断裂位置出现核苷酸的缺失或者插入从而导致基因发生突变。HDR修复主要发生在有同源序列存在的情况下，生物体在修复DSBs的时候可以以同源序列为模板来完成断裂位置的修复，这种方式因为有模板的存在，会产生精确的修复，如果在模板中设计一些人为的突变则会将这些突变精确的引入生物体的基因组中。
这几种基于SSN的的基因定点突变技术中，因为CRISPR/CAS9技术操作简单，成本低廉，仅仅通过改变一小段RNA序列即可达到识别不同靶位点，所以CRISPR/CAS9技术被广 泛应用于几乎任何可转化的生物体中高效地靶向编辑DNA，为农业改良提供前所未有的工具。自第一次在真核生物中报道CRISPR/Cas9-meidated编辑事件(Cong et al.,2013)以来，CRISPR基因编辑技术在植物中也得到了广泛的应用。从编辑的植物中及时清除转基因片段是评估CRISPR编辑的植物的遗传性和表型稳定性的关键步骤，对于作物改良至关重要。首先，在育种领域，如果在农作物品种中存在转基因片段，那么从政府管理机构获得商业种植的批准就非常困难，转基因的清除是CRISPR编辑作物获取监管批准进行商业应用的先决条件。其次，在研究领域，转基因片段上的基因编辑元件的存在大大增加了脱靶效应的风险，使得表型稳定性成为一个问题。此外，对于遗传学研究来说，转基因片段上的基因编辑元件的存在使得难以确定检测到的突变是从上一代遗传还是由当代新产生的。
植物的有性生殖需要经历孢子体和配子体的世代交替，孢子体(2n)的孢子母细胞(2n)经过减数分裂产生单倍体的配子体(n)；雌雄配子体通过受精作用结合产生合子(2n)，合子继续生长发育长成孢子体(2n)。转基因植物的孢子母细胞会经过减数分裂产生携带有转基因构建体的配子体和不带有转基因构建体的配子体，雌雄胚子体随着受精作用会自由组合产生合子。根据孟德尔的自由组合定律，当转基因构建体的拷贝数为1时，产生的合子中75％(理论值)的植株是带有转基因构建体，当转基因构建体的拷贝数大于1时，产生的合子中带有转基因构建体的数目会大于75％，并且随着拷贝数的增加这个比例还会继续增加。以上情况增加了获取不含有转基因构建体的难度。目前，从基因编辑体系中获得不带有转基因片段的方法主要有如下几种：
1)使用多代自交或者回交，通过遗传分离等传统方法鉴定无转基因，这种方法需要对植物进行多代种植或者杂交，非常费力和费时。2)使用在种子中特异性表达的mCherry荧光标记作为转基因片段存在的标记。这种方法目前主要在拟南芥中被使用，尽管荧光标记辅助选择无转基因的植物减少75％左右的筛选鉴定无转基因突变植株的工作量，但该策略仍然比较费时费力(Gao et al.,2016)。此外，荧光标记有助于鉴定不含转基因的植物，但它不会在T2代中富集或增加无转基因植物的比例。3)另一个报道的分离无转基因编辑植物的策略是将CRISPR构建体与靶向除草剂抗性P450酶的RNA干扰元件偶联。使得可以通过特定的除草剂筛选不含转基因片段的植物(Lu et al.,2017)。但是，这种策略仍然没有丰富无转基因 植物，同时需要对后代进行种植和筛选，增加了劳动力的投入。4)使用RNP(Cas9protein-gRNA RiboNucleoProteins)的方法获得不含转基因的基因编辑植株。RNP通常利用原生质体或幼胚转化的方法(Liang et al.,2017)来获得非转基因的基因编辑材料。但是原生质体转化需要经历原生质体培养、原生质体转变为愈伤组织、愈伤组织分化成幼苗，其中各步骤的效率叠加到一起就限制了单子叶植物的分化效率。幼胚转化的方法主要通过基因枪将RNP轰击进入植物细胞中。因为这两种方法均不加抗生素筛选，所以许多未发生突变的植株将掺杂到分化出的幼苗中。Liang的研究显示仅有4％左右的植株发生了单个基因的编辑(Liang et al.,2017)。虽说后代可以通过PCR的鉴定来筛选有突变的植株，但是这也是一项劳动强度比较大的工作，尤其是对于系统性研究多个基因时，获取可用材料的时间和人力成本将成为研发的限速因素。
BARNASE是解淀粉芽孢杆菌(Bacillus amyloliquefaciens)产生的一种12kD的胞外小分子核糖核酸酶。BARNASE基因具有强烈的毒性，能够降解细胞内的RNA，将造成细胞的死亡，前人的研究发现该基因表达的蛋白质可以杀死植物细胞(Lannenpaa et al.,2005)。
水稻REG2启动子可以非常特异地在种子的胚发育过程中发挥功能(Sun et al.,1996)。
MGL基因的两种单倍型ORF79或者ORFH79(Hu et al.,2012)产生的蛋白可以在雄性配子体发育时期破坏线粒体功能并导致雄性不育。有报道显示使用CaMV35S组成型启动子驱动MGL的一种单倍型ORF79可以特异的杀死带有转基因构建体的花粉粒(邹艳姣,2006)。
为了解决目前获取不含有转基因构建体的基因编辑植物需要耗费较多时间和人力成本的问题，本发明针对基因编辑系统的作用机理，提出了一种新型的快速高效获得非转基因的定点基因突变植株的技术方案。本技术方案将基因编辑元件与配子体特异性致死元件或种子特异性致死元件组合成一个连锁的系统，将其命名为TKE(Transgene Killer Editing：消除转基因构建体的基因编辑)系统。TKE系统中的基因编辑元件可以行使基因编辑的功能，配子体特异性致死元件或种子特异性致死元件可以将携带有转基因构建体的配子体或配子体结合成的合子或合子发育成的种子特异性的杀死。这样就能达到转基因植株自主消除自己的转基因后代，自主筛选出自己的不带有转基因构建体的定点突变后代的目的。
发明内容
本发明的目的在于克服现有通过定点突变技术获取非转基因突变体过程中费时费力的筛选难题，开发出一种快速获得非转基因的定向基因突变植株的方法，申请人将这种方法命名为TKE(Transgene Killer Editing：消除转基因构建体的基因编辑)系统。将本发明应用于植物基因编辑系统(如CRISPR/CAS9系统)中，能够主动且自动消除植物中转基因片段，但仍然留出足够的时间使基因编辑元件在去除转基因片段之前进行靶向基因编辑技术，为通过基因编辑培育无转基因植株提供了一种简单有效、省时省力的方法。
本发明将使用一个典型的组合来证明本发明的TKE系统可以快速高效的获取非转基因的定点基因突变植株，基本步骤如下：1)基因编辑元件使用CRISPR/Cas9基因编辑元件，包括Cas9蛋白表达盒和sgRNA转录盒。2)雄配子致死元件使用35S-MGL雄配子致死元件，利用CaMV35S启动子驱动的MGL表达将确保任何含有MGL的雄性配子体将被杀死。3)雌配子或胚胎或胚乳致死元件使用REG2-BARNASE胚胎致死元件，REG2启动子非常特异地在种子的胚发育过程中发挥功能，将BARNASE基因置于水稻REG2启动子的控制之下时，BARNASE毒蛋白不在愈伤组织或营养生长期间产生，只在种子的胚发育过程中产生，因此利用REG2启动子驱动的BARNASE将确保任何含有BARNASE的种子胚被杀死。将REG2-BARNASE和35SMGL表达盒导入带有CAS9基因的质粒pCXUN-CAS9中(He et al.,2017)(图1a)，从而完成TKE系统的载体构建。
具体地，本发明通过以下技术方案实现：
1.申请人提供了一种方法，该方法用于创制定向基因突变的非转基因植株(简称TKE系统)，步骤如下所述：
a)将需要处理的植株可以通过引入外源核酸分子实施转基因；
b)所述a)步骤的方法包括向植物引入构建体，该构建体含有第一核酸分子和第二核酸分子，其中所述第一核酸分子为基因编辑元件(其中所述的第一核酸分子为可以编辑核酸的基因元件)；第二核酸分子为致死或停止发育元件；
可应用于上述步骤a)和b)的植物包括禾本科、豆科、十字花科、菊科、茄科等可通过农杆菌浸染获取转基因材料的植物；优选水稻、玉米、高粱、大麦、燕麦、小麦、粟、二穗 短柄草、大刍草、甘蔗、大豆、油菜、拟南芥、红花、番茄、烟草、苜蓿、马铃薯、甘薯、向日葵和棉花。
上述可以编辑核酸的基因元件可选自任意一种基因编辑系统的基因元件构成的组。
一种具体的实施方案包括一种基因编辑系统优选：例如可以是ZFN基因编辑系统、TALEN基因编辑系统、CRISPR/CAS9基因编辑系统、CRISPR/CPF1基因编辑系统。
上述的CRISPR/CAS9基因编辑系统的基因元件包括CAS9基因(CAS9基因的核苷酸序列如SEQ ID NO：1所示的核苷酸序列)和sgRNA基因(sgRNA基因的核心骨架的核苷酸序列如SEQ ID NO：2所示)。
可以针对上述发明任务，采用下述之一的方法：第二核酸分子选自基因元件A、基因元件B、基因元件C构成的组，其中基因元件A为导致受精卵或胚致死或停止发育的基因元件，基因元件B为导致受精极核或胚乳致死或停止发育的基因元件，基因元件C为组合基因元件，包括基因元件D和基因元件A组合元件、基因元件D和基因元件B组合元件、基因元件D和基因元件E组合元件，其中基因元件D为导致雄性配子细胞或花粉致死或停止发育的基因元件，基因元件E为导致雌性配子细胞或极核细胞致死或停止发育的基因元件。
上述基因元件A的核苷酸序列如SEQ ID NO：3所示的核苷酸序列。
上述基因元件D的核苷酸序列如SEQ ID NO：4所示的核苷酸序列。
具体实施步骤可以详见实施例。
本发明的积极效果如下所述：
1.本发明可以主动和自动消除任何含有转基因构建体的植物，但仍能使植物体在转基因构建体被去除之前发生靶向基因编辑。当T0植物在愈伤生长和营养生长时期，TKE系统的构建体中的第一核酸分子将定点编辑植物细胞中的靶基因(见图1中的b图)。当T0植物进行生殖生长时，TKE系统的构建体中的第二核酸分子将使含有TKE构建体的雄性配子体和雌性配子体或雌雄胚子体结合产生的胚或胚乳致死或者停止发育。
2.本发明从TKE系统的构建体转化的T0植物上收获的任何种子是不含构建体的无转基因种子，并且这些种子的靶基因中含有预期的突变(图1中的b图)。
3.本发明极大地减少了分离无转基因片段的基因编辑植物所需的时间和劳力，极大地加 速了获取无转基因突变体的进度，为作物改良提供了非常有用的工具。
4.本发明可以防止由花粉或种子的飘散而引起的转基因漂移。
附图说明
序列表SEQ ID NO：1是CAS9基因的核苷酸序列。序列长度为4131bp。
序列表SEQ ID NO：2是sgRNA基因的核心骨架核苷酸序列。序列长度为80bp。
序列表SEQ ID NO：3是基因元件A的核苷酸序列。序列长度为2400bp。
序列表SEQ ID NO：4是基因元件D的核苷酸序列。序列长度为1452bp。
序列表SEQ ID NO：5是TKE质粒的核苷酸序列。序列长度为18964bp。
图1：编辑靶基因后，自杀转基因介导的CRISPR/Cas9构建体的自我消除示意图。附图标记说明：a)TKE(转基因杀手CRISPR)质粒的三个关键组成部分的示意图。细胞质雄性不育系统基因MGL处于CaMV 35S启动子控制之下。NOS是指来自根癌农杆菌的胭脂氨酸合酶基因的终止子。REG2启动子在早期胚胎发育中具有特异性，并被用于驱动BARNASE基因，该基因编码一种有毒的酶来植物细胞。rbcs-E9终止子最初是从豌豆rbcS-E9基因克隆的。密码子优化的Cas9被置于玉米的泛素启动子UBQ的控制之下。b)TKE介导的分离无转基因和CRISPR/Cas9编辑的水稻植物的流程图。通过农杆菌介导的转化将TKE质粒转化到水稻愈伤组织中。在愈伤生长和营养生长期间，BARNASE基因不被表达，并且可能由Cas9编辑靶基因。然而在繁殖阶段，任何含有Cas9的雄性配子体被MGL杀死，并且任何含有Cas9的胚胎被BARNASE杀死。因此，来自T0植物的所有种子都不含转基因。
图2：由TKE-LAZY1产生的T1植物中的突变和分离模式示意图。附图标记说明：a)使用TKE-LAZY1质粒测试转基因消除和基因编辑的效率。选择在PAM位点AGG正前方含有PstI限制位点的LAZY1基因的靶序列(标记为绿色)。功能丧失lazy1突变体显示明显的分蘖角表型。b)Cas9切割所需的PAM站点“AGG”以绿色标记。WT指野生型植物水稻品种中花11(简称ZH11)。显示了来自单个T0植物的T1植物的DNA序列(基因型)。红色的“-”是指删除一个碱基对。红色和上标中的“a”是指插入“A”。来自T0植物#34的后代仅产生纯合子代。来自T0植物#3的后代具有三种基因型，表明T0植物的嵌合体性质。
图3：本发明构建的TKE质粒图。
图4：TKE-LAZY1产生的T1植物的转基因片段检测。附图标记说明：随机选取5个T0代有lazy1表型的植株(编号分别为#3，#30，#34，#40，#49)收获种子后发芽获取T1代苗子后进行转基因片段检测。同时检测这5个T0代植株是否含有转基因片段。结果显示这5株有lazy1表型的植株在T0代均为转基因阳性，在T1代为转基因阴性。
具体实施方式
现以第一核酸分子以CRISPR/CAS9基因编辑系统的基因元件为例；第二核酸分子以基因元件A和基因元件D的组合元件为例。说明本发明技术方案可行性验证基因以水稻LAZY1基因为例。转基因方法以农杆菌介导的水稻稳定转化方法为例，为常规方法(见申请日前申请人华中农业大学相关水稻转基因专利授权文献或专利申请公开的文献)。
实施例1中间质粒载体、终载体的制备
本发明之前本申请人研究已经利用CRISPR/CAS9基因编辑系统的基因元件成功构建成了植物基因编辑载体pCXUN-CAS9(He et al.,2017)(该系统的CAS9基因的核苷酸序列见SEQ ID NO：1)。在此基础上申请人将基因元件A和基因元件D添加到pCXUN-CAS9载体上来验证本发明的TKE系统。其中基因元件A以BARNASE基因表达盒(其核苷酸序列见SEQ ID NO：3)为例，基因元件D以MGL基因表达盒(核苷酸序列见SEQ ID NO：4)为例。
具体构建步骤如下：
克隆MGL和REG2启动子(图1)。使用水稻不育系品种YTA(来自广东省农业科学院水稻研究所)的基因组DNA为模板以MGL-TAF(TGACAAATCTGCTCCGATG和MGL-TAR(CTTACTTAGGAAAGACTAC)为引物扩增出MGL基因(Hu et al.,2012)的DNA；利用水稻中花11(ZH11)的基因组DNA为模板，以REG2P-TAF(GTCGACGAGCGAGTCATTAGCTAGTATAG)和REG2P-TAR(GGTGTTCGATCGATCCTAGCGGTG)为引物扩增出REG2的启动子(Sun et al.,1996)的DNA，然后通过TA克隆分别将其连入T载体pEASY-T5(TransGen Biotech公司)得到MGL-TA和REG2P-TA两种质粒。
构建TKE载体。1)以pHEE401质粒(Wang et al.,2015)为模板，rE9T-F(CTGCAGGAA TTCGATATCATTTAAATATTATGGCATTGGGAAAACTGTTT)和rE9T-R(GTAAAACGACGGCCAGTGCCAGTTTGGGATGTTTTACTCCTCATATTAAC)为引物扩增出rbcsE9terminator DNA，然后将其切胶回收后连入被Hind III酶切过的pCXUN-CAS9载体，得到pCXR9T。2)以pCXUN-CAS9质粒(He et al.,2017)为模板，35S-F(GATTACGAATTCGAGCTCGGTACCCGGAGAGGCGGTTTGCGTATTGGCTA)和35S-R(GAAGAGCCATCGGAGCAGATTTGTCATATCTCATTGCCCCCCGGATCTGCG)为引物，扩增出35S启动子DNA；以MGL-TA质粒为模板，MGL-TAF(TGACAAATCTGCTCCGATG)和MGL-R(AGCACATCCCCCTTTCGCCAGGGTTTAATTTTACTTAGGAAAGACTACACGAAT)为引物扩增出MGL DNA；将以上两种PCR产物DNA切胶回收后混合作为模板，以35S-F(GATTACGAATTCGAGCTCGGTACCCGGAGAGGCGGTTTGCGTATTGGCTA)和MGL-R(AGCACATCCCCCTTTCGCCAGGGTTTAATTTTACTTAGGAAAGACTACACGAAT)为引物扩增出DNA，将该DNA切胶回收后连入被Kpn I酶切过的pCXR9T载体，得到35S-MGL-pCXR9T。3)以BpFULL1::BARNASE质粒(Lannenpaa et al.,2005)为模板，BAR-F(CTGCAGGAATTCGATATCATTTAAATATGGCACAGGTTATCAACACG)和BAR-R(CAGTTTTCCCAATGCCATAATTTTAATTTTAAGAAAGTATGATGGTGATGTCGCAG)为引物，扩增出BARNASE DNA；将该DNA切胶回收后连入被Swa I酶切过的35S-MGL-pCXR9T载体，得到35SMGL+BARNASE-pCXR9T载体。4)以REG2P-TA质粒为模板，REG2P-F(CTGCAGGAATTCGATATCATTTAAATGTCGACGAGCGAGTCATTAGCT)和REG2P-R(CGTGTTGATAACCTGTGCCATGGTGTTCGATCGATCCTAGCGGTG)为引物，扩增出REG2的启动子DNA，将其切胶回收后连入被Swa I酶切过的35S-MGL+BARNASE-pCXR9T载体，得到35S-MGL+REG2-BARNASE-pCXR9T载体即TKE质粒(图3)，该质粒的完整核苷酸序列见SEQ ID NO：5。
实施例2转化载体TKE-LAZY1的构建
为了检验本发明技术方案的有效性，申请人将LAZY1基因(LOC_Os11g29840)(Li et al.,2007)作为已知在向地性反应中发挥重要作用的基因。功能丧失lazy1突变体显示出较大的分蘖角(图2中的a图)。lazy1突变体的可见表型允许定性地评估本发明构建载体的编辑效率。
以水稻中的LAZY1基因为靶基因设计了特异性的sgRNA,靶序列为GTCGCGCCCGGAGTACCTGC。将实施例1中所得到的终载体TKE(见图3)，用Pme I消化成线性DNA，以重叠PCR(为常规方法)扩增的方法将sgRNA引入。本实施例采用OsU6启动子作为sgRNA转录单元的启动子，具体步骤如下：
将TKE载体(图3)连入sgRNA元件(sgRNA基因的核心骨架核苷酸序列见SEQ ID NO：2)。以本实验室已经构建的带有OsU6P-sgRNA-OsU6T转录盒的pCXUN-CAS9载体(He et al.,2017)为模板DNA，分别以OsU6PF(GTCGTTTCCCGCCTTCAGTTTATGTACAGCATTACGTAGG)和LAZY1-U6R(GCAGGTACTCCGGGCGCGACAACCTGAGCCTCAGCGCAGC)引物对和OsU6TR(CTGTCAAACACTGATAGTTTAAACGATGGTGCTTACTGTTTAG)和LAZY1-U6F(GTCGCGCCCGGAGTACCTGCGTTTTAGAGCTAGAAATAGCAAGTTA)引物对为模板扩增出两种DNA，将上述两种DNA切胶回收后混合作为模板，以OsU6PF和OsU6TR为引物扩增出完整的sgRNA转录单元DNA，然后将该DNA切胶回收后连入经过Pme I酶切过的TKE载体，得到TKE-LAZY1。
实施例3利用重组载体TKE-LAZY1转化农杆菌并对水稻宿主进行转化
将测序后的阳性质粒TKE-LAZY1电转化到农杆菌(EHA105)中，并侵染水稻愈伤组织。转化品种为水稻“中花11”(又称ZH11，来自中国农业科学院作物科学研究所)，具体转化步骤如下：
1)将水稻品种“中花11”的成熟胚去壳，先用70％乙醇浸泡1min，用0.15％升汞消毒20min，无菌水洗3～4次；将所得的外植体接种到诱导培养基上，于26℃暗培养诱导愈伤组织；
2)诱导培养35d后，取活力强、颗粒状的愈伤组织转入继代培养基进行继代培养；
3)取继代培养20d的愈伤组织的颗粒，接入预培养培养基上，于26℃下暗培养4d；
4)在预培养的第三天，用LA(LB+1.5％琼脂)划线接种农杆菌菌株，28℃静止培养2d；之后，将农杆菌全部刮入悬浮培养基；于28℃，200rpm振荡培养0.5-1h；在分光光度计600nm波长光测定菌液浓度，调至1.0OD值；
5)将预培养后的愈伤组织接入100ml锥形瓶(大约到40ml处)，加入调制好的农杆菌 菌液，浸泡30min，期间摇动数次。配制悬浮培养基:(500μl AS+5ml 50％葡萄糖)；
6)倒去菌液，将水稻愈伤组织置于灭菌滤纸上吸干表面菌液(一定要吸干菌液，使愈伤组织发白)，但不能直接在超净台上吹干)，接入共培养培养基(配方：250μl AS+5ml50％葡萄糖)，暗培养3d，再转入250ml共培养培养基上进行共培养；
7)将共培养后的愈伤组织用无菌水先快速摇动清洗两次；然后加入无菌水浸泡10min，使愈伤组织内部的菌体游离出来；倒去洗液，再加入含400mg/L的Cn无菌水浸泡15min；倒干洗液，将愈伤组织置于灭菌滤纸上吸干，接入筛选培养基；26℃暗培养。每3周继代一次，共继代两次。每转化一个质粒，需灭菌单蒸水1～2瓶，其中第一次筛选时，在300ml筛选培养基中加入500ul Cn和300ul Hn；第二次继代筛选时，在筛选培养基中加入400ul Cn和300ul Hn。
8)将筛选培养基培养的抗性愈伤组织接入预分化培养基，26℃暗培养一周)将预分化培养一周的抗性愈伤组织转入分化培养基(50ml/瓶；改用三角瓶或平底试管做培养瓶)；25℃，2000Lux光照培养，通过再生获得转基因植株。
10)待小植株3～5cm；转入生根培养基上促进生根。
11)将根系健壮的植株移入盆钵，在凉棚中过渡3～5d；然后移到自然条件下生长，直至成熟。
以上各种培养基配方见说明书文末。
实施例4转基因当代(T TKE-0)转基因片段检测和表型观察与统计
1)取成熟的转基因T0代水稻叶片，按常规CTAB法提取水稻基因组DNA；
2)设计检测转基因植物的阳性引物，序列如下：
CC-F:TCCATATTTCATCTTCGGTGTCGT，
CC-R:AAGAAGGACCTCATCATCAAGCTC；
PCR反应体系：
10×PCR Buffer 2μl
2.5mM dNTP 2μl
10μM CC-F 0.3μl
10μM CC-R 0.3μl
水稻基因组DNA 2μl
rTaq聚合酶 0.1μl
补充双蒸水加水补至 20μl
PCR扩增程序：
95℃ 5min
 
95℃ 30s
58℃ 30s
72℃ 1min
(跳至“95℃ 30s”,循环35次)
 
72℃ 7min
25℃ 1min
产物大小为1105bp。以水稻野生型(即非转基因)中花11(ZH11)的基因组DNA为阴性对照。
所有基因组DNA使用ActinM-F(CTCAACCCCAAGGCTAACAG)和ActinM-R(ACCTCAGGGCATCGGAAC)作为内对照引物对，通过PCR扩增确定基因组DNA的质量。
转基因阳性统计结果见表1。
表1 T0转基因阳性检测结果
被检测T0植株数目 含有构建体的植株比例
63 78％
由于功能丧失lazy1突变体显示出较大的分蘖角(图2中的a图)，所以lazy1突变体的分蘖角增大的表型可以被被用来定性评估本发明构建体的编辑效率。在本发明获得的63株T0植株中，29株具有明显的分蘖角型，表明本发明的CRISPR构建体能够在靶基因 LAZY1上产生功能缺失型突变。
实施例5T TKE-0后代(T TKE-1)转基因片段检测
从每个单独的T0植物收获种子，并分析来自5个独立的阳性T0植物的后代(T1代)，这5个独立的T0代植株具有可见的lazy1表型。
具体步骤如下：
1)取成熟的转基因T0代水稻叶片，按常规CTAB法提取水稻基因组DNA；
2)设计两对检测转基因植物的阳性引物：
第一对引物：
MGL-429F：TCTTCCATATTTCATCTTCGGTGT，
MGL-429R：GCATGACGTTATTTATGAGATGGG；
扩增产物大小为429bp。
第二对引物：
BAR-377F：AATTCAGACCGGATTCTTTACTCA，
BAR-377R：GTCGCTGATACTTCTGATTTGTTC；
扩增产物大小为377bp。
PCR反应体系：
10×PCR Buffer 2μl
2.5mM dNTP 2μl
10μM CC-F 0.3μl
10μM CC-R 0.3μl
水稻基因组DNA 2μl
rTaq聚合酶 0.1μl
补充双蒸水加水补至 20μl
PCR扩增程序：
95℃ 5min
 
95℃ 30s
58℃ 30s
72℃ 1min
(跳至“95℃30s”,循环35次)
 
72℃ 7min
25℃ 1min
上述基因组DNA使用ActinM-F(CTCAACCCCAAGGCTAACAG)和ActinM-R(ACCTCAGGGCATCGGAAC)作为内对照引物对，通过PCR扩增确定基因组DNA的质量。
使用Hyg-280F和Hyg-280R引物对普通CRISPR载体pCXUN-CAS9转化的T1代植株的基因组DNA进行PCR扩增来鉴定其阳性转基因植株。
TKE的T1代植株检测结果见图4。转基因阳性统计结果见表2。
表2 T1转基因阳性检测结果
Figure PCTCN2019087139-appb-000001
由表2，当使用常规的CRISPR/Cas9构建体时，至少75％的T1代转基因植物具有 CRISPR/Cas9构建体；当时用TKE质粒的技术方案时，来自5个独立T0代转基因植物中所有T1代转基因植物(总共59个)不含有CRISPR构建体，表明本发明的TKE质粒技术方案在消除转基因方面非常有效。
实施例6T TKE-1定向突变检测
使用LAZY1-GT1(CCTGCAACTGCATCACCGGGCTTG)和LAZY1-GT2(TCCAAGGAAACCTCATGAAATAGTCAGCCA)作为基因型检测引物，分别对6个独立的T1代家系中所有植株进行PCR扩增。然后，对PCR产物测序，通过Dsdecode网站(http://dsdecode.scgene.com/)对测序结果进行分析，鉴别出各个T1代植株的突变形式。
通过对5个独立的T0代转基因后代的59个T1代植物进行测序后发现：所有的转基因植物都在目标位点含有突变，突变效率为100％，具体突变形式和突变效率见图2。表明本发明的TKE系统既能自动清除转基因T1代植株转基因构建体又能保证后代发生高效的定点突变。本发明可大大减少分离无转基因定点DNA编辑的水稻所需的时间和人力，为作物遗传改良提供了非常有用的工具。可以很容易地被应用于其他任何能够通过组织培养进行转基因操作的植物物种。
附录：本发明涉及的各种培养基及其配方
诱导培养基
Figure PCTCN2019087139-appb-000002
PH值：5.9
补充蒸馏水至1000ml。
继代培养基
Figure PCTCN2019087139-appb-000003
PH值：5.9；
补充蒸馏水至1000ml。
预培养基
Figure PCTCN2019087139-appb-000004
PH值：5.6；
补充蒸馏水至250ml。
共培养培养基
Figure PCTCN2019087139-appb-000005
Figure PCTCN2019087139-appb-000006
PH值：5.6；
补充蒸馏水至250ml。
悬浮培养基
Figure PCTCN2019087139-appb-000007
PH值：5.4；
补充蒸馏水至100ml。
筛选培养基
Figure PCTCN2019087139-appb-000008
Figure PCTCN2019087139-appb-000009
PH值：6.0；
补充蒸馏水至250ml。
分化培养基
Figure PCTCN2019087139-appb-000010
PH值：6.0；
补充蒸馏水至1000ml。
生根培养基
Figure PCTCN2019087139-appb-000011
Figure PCTCN2019087139-appb-000012
pH值：5.8；
补充蒸馏水至1000ml。
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Claims (8)

  1. 一种用于创制定向基因突变的非转基因植株的方法，其特征包括：
    a)对定向基因突变的植物通过引入外源核酸分子实施转基因；
    b)实施转基因方法包括向定向基因突变的植物引入构建体，所述构建体含有第一核酸分子和第二核酸分子，其中第一核酸分子作为基因编辑元件，第二核酸分子作为致死或停止发育元件。
  2. 如权利要求1所述的方法，其中所述的第一核酸分子为可以编辑核酸的基因元件。
  3. 如权利要求1所述的方法，其中可以编辑核酸的基因元件选自任意一种基因编辑系统的基因元件构成的组。
  4. 如权利要求3所述的方法，所述的基因编辑系统，选自ZFN基因编辑系统，TALEN基因编辑系统，CRISPR/CAS9基因编辑系统或CRISPR/CPF1基因编辑系统。
  5. 如权利要求4所述的方法，其中所述的CRISPR/CAS9基因编辑系统的基因元件包括CAS9基因和sgRNA基因；其中：所述的CAS9基因的核苷酸序列如SEQ ID NO：1所示；所述的sgRNA基因的核心骨架的核苷酸序列如SEQ ID NO：2所示。
  6. 权利要求1-5之任一项所述的方法，其中第二核酸分子选自基因元件A，基因元件B，或基因元件C构成的组；其中基因元件A为导致受精卵或胚致死或停止发育的基因元件；基因元件B为导致受精极核或胚乳致死或停止发育的基因元件；基因元件C为组合基因元件，其包括基因元件D和基因元件A的组合元件；基因元件D和基因元件B的组合元件；基因元件D和基因元件E的组合元件；其中基因元件D为导致雄性配子细胞或花粉致死或停止发育的基因元件，基因元件E为导致雌性配子细胞或极核细胞致死或停止发育的基因元件。
  7. 如权利要求6所述的方法，其中基因元件A的核苷酸序列如SEQ ID NO：3所示。
  8. 如权利要求6所述的方法，其中基因元件D的核苷酸序列如SEQ ID NO：4所示。
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