阅读说明：本技术 一种花椰菜单核苷酸突变的方法 (Method for single nucleotide mutation of cauliflower ) 是由 王桂香 宗梅 刘凡 田守卫 刘迪 武娅歌 韩硕 郭宁 段蒙蒙 于 2020-05-22 设计创作，主要内容包括：本发明公开了一种花椰菜单核苷酸突变的方法。本发明公开的花椰菜单核苷酸突变的方法包括：以下胚轴为外植体,利用BE3系统对花椰菜进行基因编辑,得到发生单核苷酸突变的花椰菜,花椰菜为花椰菜Korso,该方法采用PHSE901-Basta进行,PHSE901-Basta的序列为序列表中序列3,侵染外植体的菌液浓度满足OD<Sub>600</Sub>值为0.1。利用本发明的方法对花椰菜Korso的ALS基因和CENH3基因进行单核苷酸突变,ALS基因和CENH3基因的编辑效率分别为24.7％和80.0％。本发明在花椰菜乃至芸薹属蔬菜的种质改良、基于突变体库进行的基因分离和基因功能验证等方面都提供了一个有效的技术手段。(The invention discloses a method for single nucleotide mutation of cauliflower. The method for single nucleotide mutation of cauliflower disclosed by the invention comprises the following steps: using the hypocotyl as an explant, and utilizing a BE3 system to perform gene editing on cauliflower to obtain the cauliflower with single nucleotide mutation, wherein the cauliflower is cauliflower Korso, the method adopts PHSE901-Basta, the sequence of PHSE901-Basta is a sequence 3 in a sequence table, and the concentration of a bacterial liquid infecting the explant meets OD 600 The value was 0.1. By using the method of the inventionThe ALS gene and the CENH3 gene of cauliflower Korso are subjected to single nucleotide mutation, and the editing efficiency of the ALS gene and the CENH3 gene is 24.7 percent and 80.0 percent respectively. The invention provides an effective technical means in the aspects of germplasm improvement of cauliflower or brassica vegetables, gene separation based on a mutant library, gene function verification and the like.)

1. A method for single nucleotide mutation of a cauliflower gene, comprising: using the hypocotyl as an explant, and carrying out gene editing on the cauliflower by using BE3 to obtain the cauliflower with single nucleotide mutation.

2. The method of claim 1, wherein: the cauliflower is cauliflower Korso.

3. The method according to claim 1 or 2, characterized in that: the gene editing is carried out by adopting PHSE901-Basta, and the sequence of the PHSE901-Basta is a sequence 3 in a sequence table.

4. A method according to any one of claims 1-3, characterized in that: the concentration of the bacterial liquid infecting the explant meets OD₆₀₀The value was 0.1.

5. The method according to any one of claims 1-4, wherein: the method employs Basta for screening.

6. The method according to any one of claims 1-5, wherein: the co-culture medium used in the method is a culture medium obtained by adding 6-BA and NAA into an MS culture medium, and the concentrations of the 6-BA and the NAA in the co-culture medium are respectively 3mg/L and 0.1 mg/L;

and/or the screening culture medium used in the method is a culture medium obtained by adding Basta and carbenicillin to the co-culture medium, and the concentrations of the Basta and the carbenicillin in the screening culture medium are 3mg/L and 300mg/L respectively;

and/or the bud elongation culture medium used in the method is a culture medium obtained by adding Basta and carbenicillin to an MS culture medium, and the concentrations of the Basta and the carbenicillin in the bud elongation culture medium are 3mg/L and 200mg/L respectively.

7. The method according to any one of claims 1-6, wherein: the gene is ALS gene or CENH3 gene.

8. The method according to any one of claims 1-7, wherein: the gene is ALS gene, and the target sequence is sequence 1 in a sequence table;

the gene is a CENH3 gene, and the target sequence is a sequence 2 in a sequence table.

9. Use of the method of any one of claims 1 to 8 in cauliflower breeding.

10. Set of media for the editing of broccoli cuts, consisting of the PHSE901-Basta according to claim 3, the co-cultivation medium according to claim 6, the selection medium and the shoot elongation medium.

Technical Field

The invention relates to a method for single nucleotide mutation of cauliflower, belonging to the technical field of agricultural biology.

Background

Broccoli (Brassica oleracea var. botrytis) is a widely cultivated Brassica vegetable of the brassicaceae family. It has strong self-incompatibility and needs long-term vernalization and low-temperature induction for flowering. These properties significantly increase the difficulty of using physical or chemical mutagens to induce mutations and create new germplasm. Brassica vegetable crops are important vegetable groups, and along with the completion of whole genome sequencing and functional genome research of Chinese cabbages and cabbages, how to utilize functional gene editing mutation and directionally and genetically improve the existing germplasm becomes one of the most important subjects in the field of genetic breeding.

Gene editing technology can carry out efficient, accurate and specific modification on plant genomes. In particular to CRISPR/Cas9, and provides a revolutionary solution for creating targeted point mutation and germplasm innovation.

Mutations at several key sites in the ALS gene, i.e. the acetolactate synthase gene, at different sites of which single nucleotides are mutated, result in the alteration of the encoded amino acids, which may confer herbicide resistance to the plant, CENH3 (centromere-specific histone H3 variant) gene, which may affect the centromere localization function of its protein and may induce the material to be hybridized with it to be haploid. The single nucleotide mutation of the two genes can generate available excellent phenotypes, and the method has extremely high breeding application value.

Disclosure of Invention

The invention provides a high-efficiency, stable and simple technical method for creating site-specific mutation by gene editing aiming at the defects of the conventional cauliflower transgenosis and gene editing technology.

The invention provides a method for single nucleotide mutation of a cauliflower gene, which comprises the following steps: using the hypocotyl as an explant, and carrying out gene editing on the cauliflower by utilizing a BE3 system to obtain the cauliflower with single nucleotide mutation.

In the above method, the cauliflower may be Korso.

In the method, the gene editing can be carried out by adopting PHSE901-Basta, and the sequence of the PHSE901-Basta is a sequence 3 in a sequence table.

In the method, the concentration of the bacterial liquid infecting the explant can meet OD₆₀₀The value was 0.1.

The above method used Basta for screening.

The co-culture medium used in the above method may be a culture medium obtained by adding 6-BA and NAA to MS medium, the concentrations of 6-BA and NAA in the co-culture medium being 3mg/L and 0.1mg/L, respectively.

The screening medium used in the above method may be a medium obtained by adding Basta and carbenicillin to the co-culture medium, the concentrations of Basta and carbenicillin in the screening medium being 3mg/L and 300mg/L, respectively.

The bud elongation medium used in the above method may be a medium obtained by adding Basta and carbenicillin to an MS medium, the concentrations of Basta and carbenicillin in the bud elongation medium being 3mg/L and 200mg/L, respectively.

In the above method, the gene may be an ALS gene or a CENH3 gene.

In the above method, the gene is an ALS gene, and the target sequence may be sequence 1 in a sequence table;

the gene is CENH3 gene, and the target sequence can be sequence 2 in the sequence table.

Specifically, the method for mutating the single nucleotide of the cauliflower gene is a method for mutating the single nucleotide of an ALS gene in the cauliflower Korso or a method for mutating the single nucleotide of a CENH3 gene in the cauliflower Korso.

The method for mutating the single nucleotide of the ALS gene in the cauliflower Korso can comprise the following steps: will OD₆₀₀The EHA105/PHSE901-Basta-ALS bacterial liquid with the value of 0.1 infects hypocotyls of the cauliflower Korso to obtain an infected explant; co-culturing the infected explant in the co-culture medium to obtain a co-cultured explant; screening the explants subjected to co-culture in the screening culture medium to obtain resistant buds; culturing the resistant bud in the bud elongation culture medium to obtain a resistant seedling, and detecting the ALS gene of the resistant seedling to obtain cauliflower with the ALS gene having single nucleotide mutation;

the EHA105/PHSE901-Basta-ALS is a recombinant strain obtained by introducing PHSE901-Basta-ALS into an agrobacterium EHA105, and the PHSE901-Basta-ALS is a recombinant vector obtained by inserting double-stranded DNA shown in a sequence 1 in a sequence table into the PHSE901-Basta by BsaI.

The ALS gene may have a single nucleotide mutation of C₆C₇C₈-T₆T₇T₈、C₆C₇C₈-T₆T₇C₈、C₆C₇C₈-C₆T₇T₈、C₆C₇C₈-T₆C₇T₈、C₆C₇C₈-C₆T₇C₈Or C₆C₇C₈-T₆C₇C₈；

Said C is₆C₇C₈-T₆T₇T₈The 5 th-7 th cytosine deoxynucleotide of the sequence 1 in the ALS gene is mutated into thymine deoxynucleotide; said C is₆C₇C₈-T₆T₇C₈In the ALS gene, the 5 th and 6 th cytosine deoxynucleotides in the sequence 1 are mutated into thymine deoxynucleotides; said C is₆C₇C₈-C₆T₇T₈The genes are the 6 th and 7 th of the sequence 1 in the ALS geneThe cytosine deoxynucleotide of the position is mutated into thymine deoxynucleotide; said C is₆C₇C₈-T₆C₇T₈In the ALS gene, the 5 th and 7 th cytosine deoxynucleotides in the sequence 1 are mutated into thymine deoxynucleotides; said C is₆C₇C₈-C₆T₇C₈In order to mutate cytosine deoxynucleotide at the 6 th site of the sequence 1 in the ALS gene into thymine deoxynucleotide; said C is₆C₇C₈-T₆C₇C₈In order to mutate cytosine deoxynucleotide at the 5 th site of the sequence 1 in the ALS gene into thymine deoxynucleotide.

The method for single nucleotide mutation of the CENH3 gene in the cauliflower Korso can comprise the following steps: will OD₆₀₀The bacterial liquid EHA105/PHSE901-Basta-CENH3 with the value of 0.1 infects hypocotyls of the cauliflower Korso to obtain an infected explant; co-culturing the infected explant in the co-culture medium to obtain a co-cultured explant; screening the explants subjected to co-culture in the screening culture medium to obtain resistant buds; culturing the resistant bud in the bud elongation culture medium to obtain a resistant seedling, and detecting the ALS gene of the resistant seedling to obtain cauliflower with the ALS gene having single nucleotide mutation;

the EHA105/PHSE901-Basta-CENH3 is a recombinant strain obtained by introducing PHSE901-Basta-CENH3 into agrobacterium EHA105, and the PHSE901-Basta-CENH3 is a recombinant vector obtained by inserting double-stranded DNA shown in a sequence 2 in a sequence table into the PHSE901-Basta by BsaI.

The mononucleotide mutation of the CENH3 gene can be realized by mutating cytosine deoxynucleotide at the 4 th site of the sequence 2 in the CENH3 gene into thymine deoxynucleotide.

The application of the method for single nucleotide mutation of the cauliflower gene in the breeding of the cauliflower also belongs to the protection scope of the invention.

The invention also provides a kit for the editing of cauliflower menus, which consists of the PHSE901-Basta, the co-culture medium, the screening medium and the bud elongation medium.

In the present invention, the cauliflower may be cauliflower Korso.

The invention successfully obtains the cauliflower with the ALS gene and the CENH3 gene having single nucleotide mutation by taking the cauliflower hypocotyl as an explant and transforming by an agrobacterium-mediated method, wherein the editing efficiencies of the ALS gene and the CENH3 gene are respectively 24.7 percent and 80.0 percent. The invention provides an effective technical means in the aspects of germplasm improvement of cauliflower or brassica vegetables, gene separation based on a mutant library, gene function verification and the like.

Drawings

FIG. 1 shows regeneration of explant shoots after 2 weeks of TDZ medium culture.

FIG. 2 shows explants cultured in TDZ medium.

FIG. 3 shows the regeneration of explant shoots after 2 weeks of 6-BA medium culture.

FIG. 4 shows GUS staining of hypocotyls infected with body weight suspensions of various concentrations of bacteria.

FIG. 5 shows the acquisition of transformation resistant seedlings with cauliflower hypocotyls as explants. A. B is Basta screening culture after the hypocotyl explant agrobacterium infection; c is a transformant (namely a resistant regeneration plant) which is detected to be positive by a Basta test strip.

FIG. 6 shows PCR detection of T0 generation plants. The first and second rows are PCR products of gRNA-IDF and gRNA-IDR, and the third row is PCR products of nCas9-IDF and nCas 9-IDR. M: the leftmost side is the standard DNA molecular weight (Trans 2K plus DNAmarker); +: positive control (engineering bacteria), CK: negative control (untransformed cauliflower DNA); ntc: control without template; the other lanes are the detection results of the resistant regenerated plants, and the positive seedlings are the same as the positive control bands.

FIG. 7 shows the single base editing identification of cauliflower. a: korso (Ko) and Arabidopsis thaliana (At) ALS gene target sequence comparison, UGG is PAM area, three nucleotides in red font are target change sites, namely proline (P) 182 th coding site of ALS, cytosine deoxynucleotide in 4-9 th position in square frame can be mutated into thymine deoxynucleotide. b: korso (Ko) and Arabidopsis (At) CENH3 gene target sequences. c: related structures are edited for genes in the recombinant vector. d and e: PCR sequencing of two gene-editing strains respectively shows that C-T mutation occurs at the target site.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The broccoli Korso (Wang et al, Production and characterization of organic biomaterials beta, botanical and hair developers) in the examples described below was available to the public for the selection of advanced pre-branched materials, Plant Rep (2011)30: 1811-.

WX100, R8 and XLH65 in The following examples are described in The literature (Zhu et al, The genetic diversity and Relationships of Cauliflower (Brassica Oleracea var. Borrytis) organized Lines used by SSR Markers, PLoS One, December 6,2018; 13(12): e0208551.doi:10.1371/journal. hole.0208551), and The biological material is publicly available from The applicant, and is only used for repeating The relevant experiments of The present invention, and is not available for other uses.

The cauliflower in the following examples is also a product of Xiamen vegetable seed Co.

The broccoli Taisong in the following examples is a product of Zhejiang Shen Hainan Prolate Co.

The following examples of broccoli flourishing pine are products of cultivator and breeding industries, ltd.