阅读说明：本技术 一种快速筛选非转基因定点突变植物的方法 (Method for rapidly screening non-transgenic site-directed mutant plants ) 是由 梁振 于 2020-05-11 设计创作，主要内容包括：本发明公开了一种快速筛选非转基因定点突变植物的方法,属于生物技术领域。该方法包括以下步骤：(1)将靶向特定基因靶位点的sgRNA导入含有正负筛选标记(coda::nptii或hpt::coda)的基因编辑载体中；(2)以基因编辑载体为基础构建基因敲除载体并将其通过农杆菌转化需要处理的植株,实施转基因,并通过nptii或hygromycin进行正向筛选获得第一代(拟南芥叫T1代,烟草、水稻等叫T0代)含有转基因元件的基因定点突变材料；(3)种植步骤(2)中所获得的第一代突变体,并收获种子。(4)利用负向筛选标记codA,对所收获的种子进行筛选,最终获得不含转基因成分的基因定点突变材料。(The invention discloses a method for rapidly screening non-transgenic site-directed mutant plants, belonging to the technical field of biology. The method comprises the following steps: (1) introducing sgRNA targeting a specific gene target site into a gene editing vector containing a positive and negative selection marker (cod:: nptii or hpt:: cod); (2) constructing a gene knockout vector based on a gene editing vector, transforming the gene knockout vector into a plant to be treated through agrobacterium, implementing transgenosis, and carrying out forward screening through nptii or hygromycin to obtain a first generation (arabidopsis called T1 generation, tobacco, rice and the like called T0 generation) gene site-directed mutant material containing a transgenic element; (3) planting the first generation mutant obtained in the step (2), and harvesting seeds. (4) And screening the harvested seeds by using a negative screening marker codA to finally obtain the gene site-directed mutation material without the transgenic component.)

1. A method for rapidly screening non-transgenic site-directed mutant plants is characterized by comprising the following steps: (1) introducing sgRNA targeting a specific gene target site into a gene editing vector containing a positive and negative screening marker coda, nptii or hpt, coda, wherein the gene editing vector comprises a gene editing element, a positive and negative screening marker and an agrobacterium vector skeleton fragment; (2) constructing a gene knockout vector on the basis of a gene editing vector, transforming the gene knockout vector into a plant to be treated through agrobacterium, implementing transgenosis, and carrying out forward screening through nptii or hygromycin to obtain a first generation of gene site-directed mutation material containing a transgenic element; (3) planting the first generation mutant obtained in the step (2), and harvesting seeds; (4) and screening the harvested seeds by using a negative screening marker codA to finally obtain the gene site-directed mutation material without the transgenic component.

2. The method for rapid screening of non-transgenic site-directed mutant plants according to claim 1, wherein the gene editing element is ZFNs, TALENs or CRISPR/Cas 9.

3. The method for rapid screening of non-transgenic site-directed mutant plants according to claim 1, wherein the positive and negative selection markers cod: nptii have the sequence shown in SEQ ID NO. 1.

4. The method for rapid screening of non-transgenic site-directed mutant plants according to claim 1, wherein the positive and negative selection markers hpt: coda have the sequence shown in SEQ ID NO. 2.

5. The method of claim 2, wherein the gene editing vector constructed in step (2) has a Cas9 gene driven by 35S promoter, and positive and negative selection markers are cod:: nptii or hpt:: coda, and is suitable for tobacco, soybean or rape.

6. The method of claim 2, wherein the gene editing vector constructed in step (2) has a Cas9 gene driven by the Ec1.1 promoter, and the positive and negative selection markers are hpt:: coda, and the gene editing vector is suitable for Arabidopsis thaliana.

7. The method of claim 2, wherein the Cas9 gene in the gene editing vector constructed in step (2) is driven by the Ubi-1 promoter of maize, and the positive and negative selection markers are cod:: nptii or hpt:: coda, and the gene editing vector is suitable for rice, wheat, maize or millet.

8. The method of claim 1, wherein the plant comprises tobacco, soybean, canola, arabidopsis, rice, wheat, maize or millet.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly screening non-transgenic site-directed mutant plants.

Background

Genome editing techniques, which can produce site-directed mutations in the genome, are emerging molecular biology techniques in the last decade, which mainly rely on Sequence-Specific Nucleases (SSNs) to cleave the genome directionally to generate DNA double-strand breaks (DSBs). Cells of organisms repair DSBs mainly through two pathways, non-homologous end joining (NHEJ) and Homologous Recombination (HR). The NHEJ repair pathway can generate insertion or deletion of a plurality of bases, so that frame shift mutation is caused, and gene knockout is caused; the HR repair pathway results in complete genome repair or, where a template is provided, base substitution or site-directed insertion. Currently, SSNs commonly used mainly include Zinc Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and Clustered regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeat/CRISPR, CRISPR/Cas) systems. The three sequence-specific nucleases can generate DSBs in a genome to induce gene site-directed mutagenesis, wherein the CRISPR/Cas9 technology has the characteristics of simplicity, convenience, high efficiency and the like, and is widely applied to various animals and cell lines thereof such as mice, rats, pigs, cows, dogs and the like and various plants including wheat, corns, rice, tobacco, arabidopsis thaliana, tomatoes and the like.

Conventional plant genome editing technology mainly utilizes agrobacterium or a gene gun to transfer SSNs such as CRISPR/Cas9 into plant cells in the form of DNA and randomly integrate into plant genomes so as to edit the genomes. Integration of SSNs, such as CRISPR/Cas9, into the plant genome can produce a variety of undesirable effects. First, in the basic research area: integration of DNA expression cassettes of SSNs, such as CRISPR/Cas9, into the genome presents the problem of increased potential off-target effects due to sustained expression of CRISPR/Cas 9. Secondly, SSNs belong to foreign DNA, integration into the plant genome involves transgenic problems and commercial plant approval from regulatory authorities is difficult. Thus, the elimination of transgenic components is an essential prerequisite for commercial application of genome editing crops.

At present, the following methods are mainly used for identifying and obtaining the genome editing crops without exogenous transgenic fragments: 1) through progeny selfing or backcross separation, extracting genome DNA and utilizing PCR aided Southern method to identify. In the method, the extraction of genome DNA is complicated, the identification workload is large, the false positive of PCR is high, and the Southern operation is very complicated. 2) And adding a specific expression mCherry fluorescent protein into the seeds as a transgenic marker element, and performing progeny separation and identification. This method is currently only applicable to arabidopsis, is not widely used in plants, and in addition requires a special fluorescence microscope, which is not available in all laboratories. 3) The CRISPR/Cas9 is transformed into plant protoplasts or immature embryos in the form of Ribonucleoprotein complexes (RNPs) to obtain site-directed mutagenesis material without foreign DNA. However, protoplast transformation requires protoplast culture, callus induction, differentiation and regeneration, and is difficult to achieve in monocots. Methods for transformation of young embryos are currently practiced only in wheat and maize. In addition, the two methods do not use antibiotic screening, and the subsequent identification consumes great manpower and material resources. 4) Coupling the CRISPR/Cas9 element with an RNAi element of a targeted herbicide-resistant P450 enzyme can also identify rice without transgene editing through herbicide screening. However, this method requires long-term planting and screening of the offspring, which increases the human input. Therefore, there remains a need in the art for a universal method for identifying site-directed mutants of non-transgenic offspring.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for rapidly screening non-transgenic site-directed mutant plants.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention is applicable to genome editing (such as CRISPR/Cas9) in both monocots and dicots. In the currently used genome editing method, SSNs vectors are transferred into cells by agrobacterium or gene gun, and the vectors usually include genome editing elements (such as CRISPR/Cas9) and transgenic forward selection markers (such as nptii and Hygromycin), and plants that are transformed and gene edited in the first generation enrichment are selected by using the forward selection markers. The bacterial cytosine deaminase gene (codA) can convert nontoxic 5-fluorocytosine into toxic 5-fluorouracil, so that the bacterial cytosine deaminase gene can be used for negative screening of transgenes. The invention fuses positive screening markers (such as nptii, Hygromycin and the like) and negative screening markers (codA) to generate new positive and negative screening markers (codA:: nptii or hpt:: codA), which can be used for positive screening of first generation transgenic site-specific mutant plants and rapid negative screening of progeny non-transgenic site-specific mutant plants.

The invention provides a method for rapidly screening non-transgenic site-directed mutant plants, which comprises the following steps: (1) introducing sgRNA targeting a specific gene target site into a gene editing vector containing a positive and negative selection marker (codon:: nptii or hpt:: codon), wherein the gene editing vector comprises a gene editing element, the positive and negative selection marker and an agrobacterium vector skeleton fragment; (2) constructing a gene knockout vector on the basis of a gene editing vector, transforming the gene knockout vector into plants (including tobacco, arabidopsis thaliana, rice and the like) needing to be processed by agrobacterium, implementing transgenosis, and carrying out forward screening by nptii or hygromycin to obtain a first generation (arabidopsis thaliana is called T1 generation, and tobacco, rice and the like are called T0 generation) gene site-directed mutant material containing a transgenic element; (3) planting the first generation mutant obtained in the step (2), and harvesting seeds. (4) And screening the harvested seeds by using a negative screening marker codA to finally obtain the gene site-directed mutation material without the transgenic component.

Further, the gene editing element is ZFNs, TALENs or CRISPR/Cas 9.

Furthermore, the sequence of the positive and negative screening marker coda is shown in SEQ ID NO. 1.

Further, the sequence of the positive and negative screening marker hpt < cod > is shown in SEQ ID NO. 2.

Furthermore, the Cas9 gene in the gene editing vector constructed in the step (2) is driven by a 35S promoter, and the positive and negative screening markers are coda:: nptii or hpt:: coda.

Furthermore, the Cas9 gene in the gene editing vector constructed in the step (2) is driven by an Ec1.1 promoter, and the positive and negative screening marks are hpt:: coda.

Further, the Cas9 gene in the gene editing vector constructed in the step (2) is driven by a corn Ubi-1 promoter, the positive and negative screening markers are coda:: nptii or hpt:: coda, and the gene editing vector is suitable for rice, wheat, corn or millet.

The plants suitable for the method provided by the invention comprise dicotyledonous plants such as tobacco, soybean, rape and arabidopsis thaliana and monocotyledonous plants such as rice, wheat, corn and millet.

Compared with the prior art, the invention has the following beneficial effects:

the invention greatly reduces a large amount of time and labor force required by the identification of the gene site-directed mutant material knockout transgenic component of the offspring, including PCR, Southern and the like, can effectively avoid false positive, and provides a practical and useful tool for mutant acquisition and offspring genetic improvement.

Drawings

FIG. 1 is a flow chart of rapid screening of non-transgenic site-directed mutant plants.

FIG. 2 is a schematic diagram of a series of gene editing vectors containing codA:. nptii or hpt:. codA positive and negative selection editing in examples 1-3.

FIG. 3 is a schematic diagram of genes NbXylt1 and NbXylt2 of tobacco in example 1.

FIG. 4 is a diagram showing the results of the PCR/RNP detection of pCNS-Xylt and pHCS-Xylt-induced gene site-directed mutagenesis and sequencing in example 1, in which the mutation band is shown by an arrow.

FIG. 5 is a graph of the results of screening tobacco T1 generation for transgenic-free xylt mutants using the CodA negative selection marker in example 1.

FIG. 6 shows the PCR identification of tobacco T1 generation mutant without transgene after CodA screening in example 1.

FIG. 7 is a schematic diagram of the Arabidopsis thaliana gene AtBRI1 in example 2.

FIG. 8 is a diagram of the site-directed mutation detection of the gene induced by pHCE-BRI1 in example 2 by Sanger sequencing.

FIG. 9 is a phenotype map of the Arabidopsis homozygous bri1 mutant in example 2.

FIG. 10 is a graph of the results of screening Arabidopsis thaliana T2 generation for the bri1 mutant without the transgene using the CodA negative selection marker in example 2.

FIG. 11 is a schematic view of the Osglosy 2 gene of rice in example 3.

FIG. 12 is a diagram showing the site-directed mutation of the genes induced by pCNU-gloss 2 and pHCU-gloss 2 detected by PCR/RE in example 3, wherein the arrows indicate the mutation bands.

Detailed Description