CN113717988A - Method for directionally creating plant mutant - Google Patents
Method for directionally creating plant mutant Download PDFInfo
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- CN113717988A CN113717988A CN202110944666.2A CN202110944666A CN113717988A CN 113717988 A CN113717988 A CN 113717988A CN 202110944666 A CN202110944666 A CN 202110944666A CN 113717988 A CN113717988 A CN 113717988A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
Abstract
The invention belongs to the technical field of plant mutation, and particularly relates to a method for directionally creating plant mutants, which comprises the following steps of 1) impregnating plant calluses, introducing genes, and obtaining positive calluses of over-expressed genes; 2) preparing a nano material: comprising M2+‑N3+Hydrotalcite-like nanomaterials or nanotubes; 3) mixing the aqueous solution of the nano material in the step 2) with the specific gene gRNA fragment with the PAM structure; 4) and (3) impregnating the obtained mixed liquor with positive callus of the over-expressed gene, and culturing to obtain the plant mutant. The invention utilizes the combination of molecular technology and nanotechnology, through editing gene overexpression for DNA shearing to callus, then utilizes the hydrotalcite-like nanomaterial capable of loading and adsorbing nucleic acid molecules to mix the guide RNA for designing specific gene sequence, and introduces the mixed solution into gene overexpression plants through a direct spraying method, finally realizes the directional mutation of the plants, and can realize mass creation and simple operation.
Description
Technical Field
The invention belongs to the technical field of plant mutation, and particularly relates to a method for directionally creating a plant mutant.
Background
The creation of mutants is an important experimental material for researching plant gene functions, and is mainly realized by technical means such as chemical methods, physical mutagenesis or biological methods insertion or gene editing and the like.
The existing methods for creating mutants have defects to a certain extent, for example, the methods for creating mutants by chemical mutagenesis and T-DNA mutation have non-directionality, mutation positions need to be identified for obtaining mutants, and the workload is huge; the method of gene editing is used for creating mutants, the operation is relatively simple, but the method is not beneficial to simultaneously creating a plurality of mutants or creating a large number of mutants.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a method for directionally creating plant mutants, which combines gene editing technology with nanomaterials to achieve creation of a large number of directional mutants with simple operation.
The technical content of the invention is as follows:
the invention provides a method for directionally creating plant mutants, which comprises the following steps: 1) impregnating plant callus, introducing genes, and obtaining positive callus of over-expressed genes;
2) preparing a nano material: comprising M2+-N3+Hydrotalcite-like nanomaterials or nanotubes;
3) mixing the aqueous solution of the nano material in the step 2) with the specific gene gRNA fragment with the PAM structure;
4) and (3) dip-dyeing the mixed liquor obtained in the step 3) into the positive callus of the over-expressed gene in the step 1), culturing to obtain a seedling, namely a plant mutant, and extracting DNA of a mutant leaf for identification.
The plant variety in the step 1) comprises species such as arabidopsis, tobacco, poplar, camphor tree, apple, schima superba and the like;
the dip dyeing operation in the step 1) comprises the steps of dip dyeing plant callus by using an agrobacterium-mediated method: constructing an editing gene into a plant overexpression vector through enzyme digestion connection or a recombinant enzyme method, transforming a recombinant plasmid into an agrobacterium-induced state, infecting plant leaves pre-cultured on a differentiation culture medium with activated agrobacterium carrying a target vector, transferring the plant leaves to a resistance culture medium corresponding to resistance, performing differentiation and growth culture to generate callus, and screening out a positive callus of the overexpression gene;
the editing gene comprisesCas9 orCas12a gene;
the resistance culture medium is added with corresponding antibiotics of resistance genes carried by a plant overexpression vector on the basis of a differentiation culture medium, and the antibiotics comprise kanamycin Kana and hygromycin Hyg;
step 2) said M2+-N3+The hydrotalcite-like nano material adopts divalent metal ions M2+Trivalent metal ion N3+The prepared hydrotalcite nano material comprises Mg-Fe hydrotalcite-like nano material, and the preparation method comprises the following steps: and titrating Mg salt and Fe salt with the amount of methanol and other substances by using a NaOH solution to completely dissolve the Mg salt and the Fe salt, then filling nitrogen, fully mixing and stirring, performing high-temperature treatment, and then cooling to obtain the Mg-Fe hydrotalcite-like nanosheet layer with the thickness of about 2-100 nm.
The nanotubes include single-walled nanotubes, multi-walled nanotubes, and the like;
and 3) mixing the aqueous solution of the nano material and the gRNA fragments in a mixing ratio of (2-4): 1, mixing and standing overnight.
The invention has the following beneficial effects:
the invention relates to a method for directionally creating plant mutants, which utilizes the combination of molecular technology and nanotechnology and can be used for DNA shearingCas9 orCas12a gene is over-expressed in callus, then guide RNA for designing specific gene sequence is mixed in proportion by hydrotalcite-like nano material capable of loading and adsorbing nucleic acid molecule, and the mixed solution is introduced into callus via direct spraying methodCas9 orCas12a, performing over-expression on the plant, finally realizing directional mutation of the plant, extracting DNA after culturing for a period of time, and identifying the mutant by a PCR cloning and sequencing method, thereby greatly saving the time cost for creating the mutant; the invention combines gene editing technology with nanometer material, can realize the creation of a large number of directional mutants, and has simple operation.
Drawings
FIG. 1 is a diagram of gel electrophoresis of DNA adsorbed by the nanomaterial of example 1;
FIG. 2 is a graph showing the invasion of the stem differentiation of the Schima superba by the nanomaterial in example 1;
FIG. 3 shows the differentiation of seedlings from tobacco callus after transformation of the nanomaterial carrying recombinant plasmid in example 3.
Detailed Description
The present invention is described in further detail in the following description of specific embodiments and the accompanying drawings, it is to be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and modifications thereof by those skilled in the art after reading this disclosure that are equivalent to the above described embodiments.
All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.
Example 1
Method for directionally creating plant mutant
1) Impregnating plant callus: selecting stem of tissue culture seedling of schima superba, and culturingCas9 gene is constructed into a plant over-expression vector (pCAMBIA 1300) through enzyme digestion connection or a recombinant enzyme method to obtain a recombinant plasmid which is transformed into agrobacterium GV3101 competence, activated agrobacterium with a target vector is used for impregnating plant leaves which are pre-cultured for 12 h on a differential medium, the plant leaves are transferred to a resistance medium corresponding to resistance after being cultured for three days in darkness at room temperature, differential culture (30 d), selective culture (30 d) and rooting culture (30 d) are carried out to generate callus, and the over-expression is screenedCas9 gene positive callus;
the resistance culture media used for differentiation, selection and rooting culture are respectively as follows:
differentiation medium: WPM +30g/L sucrose + 100. mu. mol/L AS +1.0 mg/L NAA +2.0mg/L ZT +7.6g/L agar;
selecting a culture medium: WPM +30g/L sucrose +9mg/L Hyg +1.0 mg/L NAA +2.0mg/L ZT +7.6g/L agar;
rooting culture medium: WPM +30g/L sucrose +9mg/L Hyg +0.1mg/L NAA + 8.0 g/L agar;
2) preparation M2+-N3+Hydrotalcite-like nano material:
magnesium nitrate and ferric nitrate, which are equal in amount to those dissolved in methanol, were titrated by a 1M NaOH solution until they were completely dissolved;
then filling nitrogen, fully mixing and stirring for more than 30 min, treating at the high temperature of 100 ℃ for 60 h, cooling to 60 ℃, and finally preparing the Mg-Fe hydrotalcite-like nano-sheets with the thickness of 100 nm;
3) mixing the aqueous solution of the nano material obtained in the step 2) with the specific gene gRNA fragment with the PAM structure, wherein the mixing ratio is 3: 1, mixing, standing overnight;
4) soaking the positive callus of the over-expressed gene in the step 1) in the mixed solution obtained in the step 3), culturing to obtain a seedling, namely a plant mutant, and extracting DNA of a mutant leaf for identification.
As shown in the gel electrophoresis chart (schima superba) of fig. 1, it was revealed that mutant plants capable of implementing directed mutation were obtained;
as shown in fig. 2, it is a diagram of the differentiation state of the cell tissue after the nanomaterial invades the stem segment of the schima superba.
Example 2
Method for directionally creating plant mutant
1) Impregnating plant callus: selecting young leaf of tobacco, and mixingCasConstructing 12a gene into a plant over-expression vector (PBI 21) by enzyme digestion connection or a recombinant enzyme method to obtain a recombinant plasmid, transforming the recombinant plasmid into agrobacterium GV3101 competence, impregnating the activated agrobacterium with a target vector on a differentiation culture medium for pre-culturing plant leaves for 12 h, transferring the plant leaves to a resistance culture medium corresponding to resistance after three days of dark culture at room temperature, carrying out differentiation culture (30 d), selective culture (30 d) and rooting culture (30 d) to generate callus, and screening out the over-expression vectorCas12a gene positive callus;
the resistance culture media used for differentiation, selection and rooting culture are respectively as follows:
differentiation medium: MS +2.0mg/L NAA +0.5 mg/L6-BA;
selecting a culture medium: MS +2.0mg/L NAA +0.5 mg/L6-BA +250 mg/L Cb +50 mg/L Kana;
rooting culture medium: MS +50 mg/L Kana;
2) preparation M2+-N3+Hydrotalcite-like nano material:
magnesium nitrate and ferric nitrate, which are equal in amount to those dissolved in methanol, were titrated by a 1M NaOH solution until they were completely dissolved;
then filling nitrogen, fully mixing and stirring for more than 30 min, treating at the high temperature of 100 ℃ for 60 h, cooling to 60 ℃, and finally preparing the Mg-Fe hydrotalcite-like nano-sheets with the thickness of 50 nm;
3) mixing the aqueous solution of the nano material obtained in the step 2) with the specific gene gRNA fragment with the PAM structure, wherein the mixing ratio is 2: 1, mixing, standing overnight;
4) soaking the positive callus of the over-expressed gene in the step 1) in the mixed solution obtained in the step 3), culturing to obtain a seedling, namely a plant mutant, and extracting DNA of a mutant leaf for identification.
As shown in FIG. 3, it is a diagram of the state of the plant in which the tobacco callus transformed with the nanomaterial carrying recombinant plasmid has been differentiated into seedlings.
Example 3
Method for directionally creating plant mutant
1) Impregnating plant callus: selecting young leaf of tobacco, and mixingCasConstructing 12a gene into plant over-expression vector by enzyme digestion connection or recombinant enzyme method to obtain recombinant plasmid, transforming to Agrobacterium GV3101 competence, impregnating the plant leaves pre-cultured for 12 h on differentiation culture medium with activated Agrobacterium with target vector, transferring to resistance culture medium corresponding to resistance after dark culture at room temperature for three days, performing differentiation culture (30 d), selective culture (30 d) and rooting culture (30 d), generating callus, screening out over-expression vectorCas12a gene positive callus;
the resistance culture media used for differentiation, selection and rooting culture are respectively as follows:
differentiation medium: MS +2.0mg/L NAA +0.5 mg/L6-BA;
germination culture medium: MS +2.0mg/L NAA +0.5 mg/L6-BA +250 mg/L Cb +50 mg/L Kana;
rooting culture medium: MS +50 mg/L Kana;
resistance culture medium: MS +2.0mg/L NAA +0.5 mg/L6-BA + Hyg antibiotic of resistance gene carried by plant overexpression vector, and screening overexpression after culturingCas12a positive callus;
2) preparation M2+-N3+Hydrotalcite-like nano material:
magnesium nitrate and ferric nitrate, which are equal in amount to those dissolved in methanol, were titrated by a 1M NaOH solution until they were completely dissolved;
then filling nitrogen, fully mixing and stirring for more than 30 min, treating at the high temperature of 100 ℃ for 60 h, cooling to 60 ℃, and finally preparing the Mg-Fe hydrotalcite-like nano-sheets with the thickness of 2 nm;
3) mixing the aqueous solution of the nano material obtained in the step 2) with the specific gene gRNA fragment with the PAM structure, wherein the mixing ratio is 4: 1, mixing, standing overnight;
4) soaking the positive callus of the over-expressed gene in the step 1) in the mixed solution obtained in the step 3), culturing to obtain a seedling, namely a plant mutant, and extracting DNA of a mutant leaf for identification.
As shown in FIG. 3, it is a diagram of the state of the plant in which the tobacco callus transformed with the nanomaterial carrying recombinant plasmid has been differentiated into seedlings.
Example 4
Method for directionally creating plant mutant
1) Impregnating plant callus: selecting young leaf of poplar or camphor tree, and cuttingCas9 gene is constructed into plant over-expression vector by enzyme digestion connection or recombinant enzyme method, the obtained recombinant plasmid is transformed into agrobacterium GV3101 competence, activated agrobacterium with target vector is used to dip-dye plant leaves which are pre-cultured for 12 h on a differentiation culture medium, the plant leaves are transferred to a resistance culture medium corresponding to resistance after being cultured for three days in darkness at room temperature, differentiation culture (30 d), selective culture (30 d) and rooting culture (30 d) are carried out to generate callus, and over-expression is screened outCas9 gene positive callus;
the resistance culture media used for differentiation, selection and rooting culture are respectively as follows:
differentiation medium: MS +2.0mg/L NAA +1 mg/L6-BA;
germination culture medium: MS +2.0mg/L NAA +1 mg/L6-BA +300 mg/L Cb +50 mg/L Kana;
rooting culture medium: MS +50 mg/L Kana;
2) preparing single-walled nanotubes or multi-walled nanotubes;
3) mixing the aqueous solution of the nano material obtained in the step 2) with the specific gene gRNA fragment with the PAM structure, wherein the mixing ratio is 3: 1, mixing, standing overnight;
4) soaking the positive callus of the over-expressed gene in the step 1) in the mixed solution obtained in the step 3), culturing to obtain a seedling, namely a plant mutant, and extracting DNA of a mutant leaf for identification.
Claims (9)
1. A method for the targeted creation of plant mutants comprising the steps of:
1) impregnating plant callus, introducing genes, and obtaining positive callus of over-expressed genes;
2) preparing a nano material: comprising M2+-N3+Hydrotalcite-like nanomaterials or nanotubes;
3) mixing the aqueous solution of the nano material in the step 2) with the specific gene gRNA fragment with the PAM structure;
4) and (3) dip-dyeing the mixed liquor obtained in the step 3) into the positive callus of the over-expressed gene in the step 1), culturing to obtain a seedling, namely a plant mutant, and extracting DNA of a mutant leaf for identification.
2. The method for directionally creating plant mutants of claim 1, wherein the varieties of the plants of step 1) comprise arabidopsis, tobacco, poplar, camphor tree, apple and schima superba.
3. The method for the directed creation of plant mutants as claimed in claim 1, characterized in that the operation of the impregnation of step 1) comprises the impregnation of plant calli using an agrobacterium-mediated method: the editing gene is constructed into a plant overexpression vector through enzyme digestion connection or a recombinant enzyme method, then a recombinant plasmid is transformed to an agrobacterium-infected state, activated agrobacterium with a target vector is used for impregnating plant leaves pre-cultured on a differentiation culture medium, then the plant leaves are transferred to a resistance culture medium corresponding to resistance, differentiation and growth culture are carried out, callus is generated, and positive callus of the overexpression gene is screened out.
4. Method for the targeted creation of plant mutants as claimed in claim 3, characterized in that the editing genes compriseCas9 orCas12a gene.
5. The method for directionally creating plant mutants as claimed in claim 3, wherein the resistance medium is a differentiation medium supplemented with corresponding antibiotics of resistance genes carried by plant overexpression vectors, and the antibiotics comprise kanamycin Kana and hygromycin Hyg.
6. Method for the directed creation of plant mutants as claimed in claim 1, characterized in that step 2) said M2+-N3+The hydrotalcite-like nano material adopts divalent metal ions M2+Trivalent metal ion N3+The prepared hydrotalcite nano material.
7. Method for the directed creation of plant mutants as claimed in claim 1, characterized in that step 2) said M2+-N3+The hydrotalcite-like nano material comprises Mg-Fe hydrotalcite-like nano material, and the preparation method comprises the following steps: and titrating Mg salt and Fe salt with the amount of methanol and other substances by using a NaOH solution to completely dissolve the Mg salt and the Fe salt, then filling nitrogen, fully mixing and stirring, performing high-temperature treatment, and then cooling to obtain the Mg-Fe hydrotalcite-like nanosheet layer with the thickness of about 2-100 nm.
8. The method for directionally creating plant mutants as claimed in claim 1, wherein said nanotubes of step 2) comprise single-walled nanotubes and multi-walled nanotubes.
9. The method for directionally creating the plant mutant according to claim 1, wherein the mixing ratio of the aqueous solution of the nano material and the gRNA fragments in the step 3) is (2-4): 1, mixing and standing overnight.
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