阅读说明：本技术 一种藜科植物的遗传转化方法 (Genetic transformation method of Chenopodiaceae plant ) 是由 李峰 谢洪涛 于 2019-11-07 设计创作，主要内容包括：本发明提供了一种藜科植物的遗传转化方法,具体地,本发明提供了一种对藜科植物进行遗传转化的方法,包括步骤：(i)提供待遗传转化的藜科植物和外源DNA；(ii)将所述外源DNA转染所述待遗传转化的藜科植物的植物细胞,使得所述DNA与所述植物细胞中的染色体发生重组,从而获得经遗传转化的植物细胞；其中,所述植物细胞为生殖细胞；(iii)用步骤(ii)获得的植物细胞进行授精(授粉),并获得合子；(iv)获得由所述合子形成的种子；和(v)任选地,对所述种子发育形成的植株进行遗传转化情况的检测。本发明的方法可显著提高藜科植物的遗传转化效率。(The invention provides a genetic transformation method of a Chenopodiaceae plant, in particular to a genetic transformation method of a Chenopodiaceae plant, which comprises the following steps: (i) providing a Chenopodiaceae plant to be genetically transformed and exogenous DNA; (ii) transfecting the exogenous DNA into a plant cell of the chenopodiaceae plant to be genetically transformed, such that recombination of the DNA with a chromosome in the plant cell occurs, thereby obtaining a genetically transformed plant cell; wherein the plant cell is a germ cell; (iii) (iii) inseminating (pollinating) the plant cells obtained in step (ii) and obtaining zygotes; (iv) obtaining seeds formed from said zygotes; and (v) optionally, detecting the genetic transformation of the plant resulting from the development of said seed. The method of the invention can obviously improve the genetic transformation efficiency of Chenopodiaceae plants.)

1. A method for genetic transformation of a plant of the family chenopodiaceae comprising the steps of:

(i) providing a Chenopodiaceae plant to be genetically transformed and exogenous DNA;

(ii) transfecting the exogenous DNA into a plant cell of the chenopodiaceae plant to be genetically transformed, such that recombination of the DNA with a chromosome in the plant cell occurs, thereby obtaining a genetically transformed plant cell;

(iii) (iii) inseminating (pollinating) the plant cells obtained in step (ii) and obtaining zygotes;

(iv) obtaining seeds formed from said zygotes; and

(v) optionally, detecting the genetic transformation of the plant developed from the seed.

2. The method of claim 1, wherein the plant cell is a germ cell.

3. The method of claim 1, wherein said plant cell is from a reproductive organ, said reproductive organ comprising a flower.

4. The method of claim 1, wherein step (ii) is preceded by the step of (i'): subjecting the plant cell to a pretreatment.

5. The method of claim 4, wherein the pre-treatment comprises artificially removing hydrophobic materials from the surface of the plant.

6. The method of claim 1, wherein said Chenopodiaceae plant is selected from the group consisting of: quinoa, beet, spinach, chenopodium album, suaeda glauca, salsola collina, kochia scoparia, or combinations thereof.

7. The method of claim 1, wherein the transfection is by Agrobacterium infection or by gene gun bombardment.

8. The method of claim 1, wherein the transfection is performed at an initial flowering stage.

9. A method of preparing a genetically transformed plant cell comprising the steps of:

(i) providing a Chenopodiaceae plant to be genetically transformed and exogenous DNA;

(ii) transfecting the exogenous DNA into a plant cell of the chenopodiaceae plant to be genetically transformed, such that recombination of the DNA with a chromosome in the plant cell occurs, thereby obtaining a genetically transformed plant cell.

10. A method of making a genetically transformed plant comprising the steps of:

(a) preparing a genetically transformed plant cell by the method of claim 9; and

(b) regenerating said genetically transformed plant cell into a plant body, thereby obtaining said genetically transformed plant.

Technical Field

The invention relates to the technical field of biology, in particular to a genetic transformation method of Chenopodiaceae plants.

Background

Chenopodium quinoa Willd is a kind of whole grain with high nutritive value, is rich in protein, calcium, iron, zinc, vitamins and other micronutrients and all essential amino acids, and also has functional components for improving the nutrition level of people and preventing various diseases. Quinoa has the characteristics of high protein, high fiber, high vitamin, low fat, low sugar and the like, and is rich in total polyphenol, saponin, flavone, polysaccharide and the like, thereby playing an important role in the research and development of functional foods, cosmetics, medicines and biopesticides. Chenopodium quinoa is an excellent representative of 'functional food' aiming at reducing human chronic diseases, is positioned as space food by the American space agency, and mainly contains minerals, vitamins, fatty acids, phytohormones and antioxidants as functional components, and particularly has the effect of protecting brain neuron cell membranes. However, the planting of chenopodium quinoa in China is still restricted by climatic factors such as altitude, temperature and the like, so that the large-scale popularization and planting of chenopodium quinoa is quite difficult.

Chenopodium quinoa is listed as one of 10 global healthy nutritional foods by Food and Agricultural Organization (FAO) of the United nations, but because chenopodium quinoa transgenosis cannot be realized by means of tissue culture and the like, research on the chenopodium quinoa gene function by scientists and application of modern genetic engineering breeding technology to chenopodium quinoa breeding are seriously hindered.

Therefore, there is an urgent need in the art to develop a method capable of improving genetic transformation efficiency of quinoa.

Disclosure of Invention

The invention aims to provide a method capable of improving genetic transformation efficiency of quinoa.

In a first aspect, the present invention provides a method for genetic transformation of a Chenopodiaceae plant, comprising the steps of:

(i) providing a Chenopodiaceae plant to be genetically transformed and exogenous DNA;

(ii) transfecting the exogenous DNA into a plant cell of the chenopodiaceae plant to be genetically transformed, such that recombination of the DNA with a chromosome in the plant cell occurs, thereby obtaining a genetically transformed plant cell;

(iii) (iii) inseminating (pollinating) the plant cells obtained in step (ii) and obtaining zygotes;

(iv) obtaining seeds formed from said zygotes; and

(v) optionally, detecting the genetic transformation of the plant developed from the seed.

In another preferred embodiment, the plant cell is a germ cell.

In another preferred embodiment, said plant cell is from a reproductive organ, said reproductive organ comprising a flower.

In another preferred embodiment, said plant cell is from pistil and stamen.

In another preferred embodiment, the plant cell is from an ovary, an ovule and/or an anther.

In another preferred embodiment, the plant cell is a pollen cell and/or a male gametophyte and/or a female gametophyte.

In another preferred embodiment, the plant cell is a sperm cell and/or an egg cell and/or a fertilized egg.

In another preferred embodiment, the plant cell is from a bud, an ovary, or an ovule.

In another preferred embodiment, the pollen cells comprise pollen sperm cells and/or pollen vegetative cells.

In another preferred embodiment, step (iii) further comprises the step of subjecting said plant cells to dark culture at 20-35 ℃ (more preferably 24-26 ℃) for 1-5, preferably 1-3 days, more preferably 1-2 days, and then to light culture for 1-10 days, preferably 2-8 days, more preferably 3-5 days, and pollinating the plant.

In another preferred embodiment, before the step (ii), the method further comprises the step (i'): subjecting the plant cell to a pretreatment.

In another preferred example, the pre-treatment comprises artificially removing hydrophobic substances from the surface of the plant.

In another preferred embodiment, said chenopodiaceae plant is selected from the group consisting of: quinoa, beet, spinach, chenopodium album, suaeda glauca, salsola collina, kochia scoparia, or combinations thereof.

In another preferred embodiment, the transfection is carried out by Agrobacterium infection or gene gun bombardment.

In another preferred embodiment, in step (ii), the exogenous DNA is transferred into said plant cell by an Agrobacterium infection solution containing the exogenous DNA.

In another preferred example, the agrobacterium infection solution comprises an agrobacterium liquid and an infection solution.

In another preferred embodiment, the OD of the Agrobacterium liquid₆₀₀Is 0.4-2.5, preferably 0.6-2, more preferably 1-2.

In another preferred example, the staining solution comprises B5 dry powder, sucrose, 2-morpholine ethanesulfonic acid (MES), Acetosyringone (AS), gibberellin (GA3) and cytokinin (6-BA).

In another preferred embodiment, the concentration of the B5 dry powder in the staining solution is 0.05-5 g/L, preferably 0.1-1g/L, and more preferably 0.2-0.5 g/L.

In another preferred embodiment, the concentration of sucrose in the staining solution is 2-80g/L, preferably 10-60g/L, more preferably 20-40 g/L.

In another preferred embodiment, the MES concentration in the staining solution is 0.3-30mg/L, preferably 1-10mg/L, more preferably 2-6 mg/L.

In another preferred embodiment, the concentration of AS in the staining solution is 5-80mg/L, preferably 10-60mg/L, more preferably 20-50 mg/L.

In another preferred embodiment, the concentration of GA3 in the staining solution is 0.01-5mg/L, preferably 0.05-2mg/L, more preferably 0.1-1 mg/L.

In another preferred embodiment, the concentration of 6-BA in the staining solution is 0.1-10mg/L, preferably 0.5-6mg/L, more preferably 0.8-3 mg/L.

In another preferred example, the staining solution further comprises a surfactant.

In another preferred embodiment, the surfactant is selected from the group consisting of: silwet, MES (disodium fatty alcohol polyoxyethylene ether sulfosuccinate), DLS (disodium lauryl sulfosuccinate), or combinations thereof.

In another preferred embodiment, the concentration of the surfactant in the staining solution is 20-800 μ g/L, preferably 50-600 μ g/L, more preferably 100-500 μ g/L, more preferably 250-350 μ g/L.

In another preferred embodiment, the agrobacterium strain comprises agrobacterium tumefaciens and agrobacterium rhizogenes.

In another preferred embodiment, the agrobacterium strain is selected from the group consisting of: EHA105, GV3101, AgL1, LBA4404, or a combination thereof.

In another preferred embodiment, the transfection is performed at the initial flowering stage.

In another preferred embodiment, the transfection is performed at 2-10 days, preferably 3-5 days, of initial flowering.

In another preferred embodiment, the process is carried out under vacuum conditions.

In another preferred embodiment, the vacuum condition is: -0.3 MPa-0.01 MPa, preferably, -0.1-0.01 MPa, more preferably, -0.1-0.05 MPa.

In another preferred embodiment, the vacuum condition is maintained for a period of time of: 1-15min, preferably 2-10min, more preferably 3-10min, more preferably 4-6 min.

In another preferred example, in the step (v), the collected mature seeds are exposed to sunlight for 3-5 days (or dried in a drying oven for 2-6 days) to remove water, remove the shells, and soak the seeds (the soaking solution is water), so that the same germination efficiency is maintained.

In another preferred embodiment, in step (v), the genetic transformation is identified and screened 4-5 days after germination.

In another preferred embodiment, the genetic transformation is identified and screened by a method selected from the group consisting of: PCR, sequencing and marker gene screening.

In another preferred embodiment, the exogenous DNA is derived from a vector of interest.

In another preferred embodiment, the target vector is selected from the group consisting of: an overexpression plasmid, a gene editing plasmid, a gene silencing plasmid, or a combination thereof in a binary expression vector.

In another preferred embodiment, the target vector further comprises a selection marker gene.

In another preferred embodiment, the exogenous DNA is integrated into one or more plasmids selected from the group consisting of: plasmids PC3301, pBSE 401.

In another preferred embodiment, the selectable marker gene is selected from the group consisting of: herbicide resistance genes, antibiotic genes (e.g., hygromycin resistance genes), fluorescent protein genes, or combinations thereof.

In a second aspect, the present invention provides a method of preparing a genetically transformed plant cell comprising the steps of:

(i) providing a Chenopodiaceae plant to be genetically transformed and exogenous DNA;

(ii) transfecting the exogenous DNA into a plant cell of the chenopodiaceae plant to be genetically transformed, such that recombination of the DNA with a chromosome in the plant cell occurs, thereby obtaining a genetically transformed plant cell.

In another preferred embodiment, the plant cell is a germ cell.

In another preferred embodiment, said plant cell is from a reproductive organ, said reproductive organ comprising a flower.

In another preferred embodiment, said plant cell is from pistil and stamen.

In another preferred embodiment, the plant cell is from an ovary, an ovule and/or an anther.

In another preferred embodiment, the plant cell is a pollen cell and/or a male gametophyte and/or a female gametophyte.

In another preferred embodiment, the plant cell is a sperm cell and/or an egg cell and/or a fertilized egg.

In another preferred embodiment, the plant cell is from a bud, an ovary, or an ovule.

In another preferred embodiment, the pollen cells comprise pollen sperm cells and/or pollen vegetative cells.

In another preferred embodiment, before the step (ii), the method further comprises the step (i'): subjecting the plant cell to a pretreatment.

In another preferred example, the pre-treatment comprises artificially removing hydrophobic substances from the surface of the plant.

In another preferred embodiment, said chenopodiaceae plant is selected from the group consisting of: quinoa, beet, spinach, chenopodium album, suaeda glauca, salsola collina, kochia scoparia, or combinations thereof.

In another preferred embodiment, the transfection is carried out by Agrobacterium infection or gene gun bombardment.

In another preferred embodiment, in step (ii), the exogenous DNA is transferred into said plant cell by an Agrobacterium infection solution containing the exogenous DNA.

In another preferred example, the agrobacterium infection solution comprises an agrobacterium liquid and an infection solution.

In another preferred embodiment, the OD of the Agrobacterium liquid₆₀₀Is 0.4-2.5, preferably 0.6-2, more preferably 1-2.

In another preferred example, the staining solution comprises B5 dry powder, sucrose, 2-morpholine ethanesulfonic acid (MES), Acetosyringone (AS), gibberellin (GA3) and cytokinin (6-BA).

In another preferred embodiment, the concentration of the B5 dry powder in the staining solution is 0.05-5 g/L, preferably 0.1-1g/L, and more preferably 0.2-0.5 g/L.

In another preferred embodiment, the concentration of sucrose in the staining solution is 2-80g/L, preferably 10-60g/L, more preferably 20-40 g/L.

In another preferred embodiment, the MES concentration in the staining solution is 0.3-30mg/L, preferably 1-10mg/L, more preferably 2-6 mg/L.

In another preferred embodiment, the concentration of AS in the staining solution is 5-80mg/L, preferably 10-60mg/L, more preferably 20-50 mg/L.

In another preferred embodiment, the concentration of GA3 in the staining solution is 0.01-5mg/L, preferably 0.05-2mg/L, more preferably 0.1-1 mg/L.

In another preferred embodiment, the concentration of 6-BA in the staining solution is 0.1-10mg/L, preferably 0.5-6mg/L, more preferably 0.8-3 mg/L.

In another preferred example, the staining solution further comprises a surfactant.

In another preferred embodiment, the surfactant is selected from the group consisting of: silwet, MES (disodium fatty alcohol polyoxyethylene ether sulfosuccinate), DLS (disodium lauryl sulfosuccinate), or combinations thereof.

In another preferred embodiment, the concentration of the surfactant in the staining solution is 20-800 μ g/L, preferably 50-600 μ g/L, more preferably 100-500 μ g/L, more preferably 250-350 μ g/L.

In another preferred embodiment, the agrobacterium strain comprises agrobacterium tumefaciens and agrobacterium rhizogenes.

In another preferred embodiment, the agrobacterium strain is selected from the group consisting of: EHA105, GV3101, AgL1, LBA4404, or a combination thereof.

In another preferred embodiment, the transfection is performed at the initial flowering stage.

In another preferred embodiment, the transfection is performed at 2-10 days, preferably 3-5 days, of initial flowering.

In another preferred embodiment, the process is carried out under vacuum conditions.

In another preferred embodiment, the vacuum condition is: -0.3 MPa-0.01 MPa, preferably, -0.1-0.01 MPa, more preferably, -0.1-0.05 MPa.

In another preferred embodiment, the vacuum condition is maintained for a period of time of: 1-15min, preferably 2-10min, more preferably 3-10min, more preferably 4-6 min.

In another preferred embodiment, the exogenous DNA is derived from a vector of interest.

In another preferred embodiment, the target vector is selected from the group consisting of: an overexpression plasmid, a gene editing plasmid, a gene silencing plasmid, or a combination thereof in a binary expression vector.

In another preferred embodiment, the target vector further comprises a selection marker gene.

In another preferred embodiment, the exogenous DNA is integrated into one or more plasmids selected from the group consisting of: plasmids PC3301, pBSE 401.

In another preferred embodiment, the selectable marker gene is selected from the group consisting of: herbicide resistance genes, antibiotic genes (e.g., hygromycin resistance genes), fluorescent protein genes, or combinations thereof.

In a third aspect, the present invention provides a method of making a genetically transformed plant comprising the steps of:

(a) preparing a genetically transformed plant cell by the method of the second aspect of the invention; and

(b) regenerating said genetically transformed plant cell into a plant body, thereby obtaining said genetically transformed plant.

In a fourth aspect, the present invention provides a genetically transformed plant, which plant has been produced by a method according to the third aspect of the present invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventor of the invention has extensively and deeply studied, and unexpectedly found a method for improving the genetic transformation efficiency of quinoa for the first time, and specifically, the invention directly uses agrobacterium tumefaciens to dip-dye the inflorescence of quinoa, directly introduces exogenous genes into the germ cells of quinoa, and obtains quinoa transgenic positive plants after seeds are harvested and sowed and then identified and screened. The invention has great application value and can generate great economic benefit and social benefit. On this basis, the present inventors have completed the present invention.

Exogenous DNA

The term "exogenous DNA" as used herein refers to natural and synthetic deoxyribonucleic acid (DNA) sequences, which may optionally include synthetic nucleic acid analogs. The nucleic acids of the invention may optionally be optimized codons. Codon optimization means that the codon usage of the DNA is adapted to the cell or organism of interest in order to increase the transcription rate of the recombinant nucleic acid in the cell or organism of interest. It is well understood by the skilled person that nucleic acids can be modified at one position due to codon degeneracy, and that such modification still yields the same amino acid sequence at that position after translation, and thus efficient expression of nucleic acids can be achieved by codon optimization to take into account the species specificity of the target cell or organism. The DNA sequences described herein may be specifically codon optimized for use in the following non-limiting organisms: chenopodiaceae plant or any variety or subspecies of Chenopodiaceae plant (such as quinoa).

Object carrier

The term "vector of interest" as used herein refers to a transport means that incorporates exogenous DNA and delivers it to a target cell. The vector thus comprises a nucleic acid sequence, optionally including regulatory or localization sequences for direct or indirect delivery to a target cell of interest or a plant target structure in a desired cellular compartment of a plant. Vectors can also be used to introduce amino acid sequences into target cells or target structures. Generally, the vector used herein may be a plasmid vector. The term "transfection" or "transformation" as used herein refers to the introduction of a vector of interest containing exogenous DNA as described herein into a target cell or target structure of said plant and its integration into the genome of the target cell or target structure, wherein the material delivered by the vector will play its role during the transfection or transformation process. Specific methods may include Agrobacterium infection, gene gun bombardment, and other techniques known to those skilled in the art. The "agrobacterium infection" refers to the process that the target vector is introduced into agrobacterium cells in an active or passive mode under certain conditions, then the target plant cells are infected by agrobacterium, the target vector is introduced into the target plant cells or cell structures, and the carried exogenous DNA is recombined or integrated with plant genome, so that the plant genotype or phenotype is changed.

In the present invention, the target vector is selected from the group consisting of: an overexpression plasmid, a gene editing plasmid, a gene silencing plasmid, or a combination thereof in a binary expression vector.

In the present invention, a suitable vector may be a bacterial vector, for example an Agrobacterium, such as Agrobacterium tumefaciens (Ag. ro. bacterium tumefaciens).

Preprocessing

In the present invention, the pre-treatment comprises artificially removing hydrophobic substances on the surface of the plant and leaves around the infected part, for example, removing dust and granular secretion on the surface of the inflorescence, and cutting off the leaves around the inflorescence to make the infected part fully exposed and be convenient for being fully contacted with the infection.

Method of the invention

The invention provides a method for genetic transformation of Chenopodiaceae plants, which comprises the following steps:

(i) providing a Chenopodiaceae plant to be genetically transformed and exogenous DNA;

(ii) transfecting the exogenous DNA into a plant cell of the chenopodiaceae plant to be genetically transformed, such that recombination of the DNA with a chromosome in the plant cell occurs, thereby obtaining a genetically transformed plant cell;

wherein the plant cell is a germ cell;

(iii) (iii) inseminating (pollinating) the plant cells obtained in step (ii) and obtaining zygotes;

(iv) obtaining seeds formed from said zygotes; and

(v) optionally, detecting the genetic transformation of the plant developed from the seed.

Applications of

The invention can be used in the field of plant genetic engineering, for plant research and breeding, in particular for genetic improvement of crops, forestry crops or horticultural plants with economic value.

The main advantages of the invention include:

(1) the invention realizes genetic transformation of Chenopodiaceae plants (such as quinoa) by an agrobacterium inflorescence infection method for the first time, overcomes the technical bottleneck of Chenopodiaceae plant (such as quinoa) transgene, can greatly promote scientists to research the gene function of Chenopodiaceae plants (such as quinoa), accelerates the modern breeding process of Chenopodiaceae plants (such as quinoa), and is expected to realize popularization planting of Chenopodiaceae plants (such as quinoa) early.

(2) The method is simple and easy to operate, does not need fussy sterile operation and tissue culture process, and greatly saves labor cost and economic cost.

(3) The invention carries out genetic transformation on plant cells (such as germ cells) of Chenopodiaceae plants (such as quinoa) for the first time, and can improve the genetic transformation efficiency of the quinoa from 0 to 0.5 percent.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the present invention are commercially available without specific reference.