CN115838757A - Method and application of gene editing technology to create dwarfing materials of Brassica napus - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering and biotechnology, and particularly relates to a method for creating a brassica napus dwarfing material by using a CRISPR/Cas9 gene editing technology and application thereof. The method comprises the steps of editing a cabbage type rape BnaA06G0083400WE gene by using a CRISPR/Cas9 gene editing technology to obtain a cabbage type rape dwarfing material; the nucleotide sequence of the coding gene mutant of the cabbage type rape dwarfing material is SEQ ID NO.1. The invention carries out gene editing on the Brassica napus BnaA06G0083400WE gene by a CRISPR/Cas9 gene editing technology to obtain dwarf Brassica napus, thereby providing precious gene resources and germplasm resources for Brassica napus breeding; the method has strong characteristics, is easy to obtain, is an effective way for realizing target character improvement and cultivating new materials, and can be applied to camellia oleifera breeding.

Description

Method and application of using gene editing technology to create dwarf materials of Brassica napus

technical field

The invention belongs to the field of plant genetic engineering and biotechnology, and relates to a method and application of site-directed mutation of Brassica napus BnaA06G0083400WE gene, and in particular to a method and application of using CRISPR/Cas9 gene editing technology to create dwarfing materials of Brassica napus.

Background technique

Plant height directly affects the lodging resistance and high-yield potential of crops, and has become one of the important indicators for modern crop breeding and ideal plant type breeding. Dwarf breeding is an important part of the Green Revolution. It first started with the wheat breeding revolution initiated by Norman Borlaug in 1950, and the discovery and application of the rice semi-dwarf gene sd1 and wheat Rht8 were milestones in this revolution. Since then, rice, The dwarf breeding of crops such as corn and cucumber has been carried out one after another.

At present, more than 90% of the rapeseed varieties in the main producing areas of my country are Brassica napus, 36% of which have a plant height higher than 180cm, and only 11% of which are lower than 160cm. Excessive plant height can easily cause production problems such as lodging, intolerance to fertilizers, and unsuitability for mechanized operations, which is one of the important factors restricting mechanized production of rapeseed. After rape lodging, the number of seeds per silique decreased by 17.5%, and the seed yield decreased by 16.2%. At the same time, the difficulty and machine damage rate of rapeseed mechanized harvesting increased, which in turn led to harvest reduction. At present, many researchers have obtained some rapeseed dwarf intermediate materials through mutagenesis, spontaneous mutation, etc. Dwarf rapeseed with excellent agronomic traits has not been used in production. Therefore, the cultivation of dwarf or semi-dwarf rapeseed varieties and their genetic analysis are of great significance for improving the mechanized harvesting of rapeseed.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a method and application of using gene editing technology to create dwarfing materials of Brassica napus, which can provide valuable genetic resources and germplasm resources for rape breeding, especially Brassica napus breeding.

Before carrying out the test of the present invention, the inventor has just carried out a large amount of research and summary.

The simulated lesion mutant is a kind of mutant that can spontaneously form programmed cell death similar to pathogen infection on the leaves when there is no obvious stress, injury or pathogen attack. Exist widely, such as: Arabidopsis, rice, corn, sorghum, wheat, barley and peanut have been reported. These mutants exhibit local and systemic resistance to many pathogens while undergoing programmed cell death. A typical phenotype of many mimic lesion mutants has been reported to be dwarfing. For example, the simulated lesion mutant ssi4 showed dwarfing and spontaneous cell death; the mutation of chs3-2D triggered immune activation, and the simulated lesion mutant chs3-2D caused extreme dwarfism and activation of defense responses; the simulated lesion mutant bir1 -1 exhibits an extreme dwarf phenotype, spontaneous cell death, and defense responses; a T-DNA insertion mutant of calmodulin-binding transcriptional activator (CAMTA) 3 exhibits enhanced disease resistance and dwarfism when grown at low temperature ; In addition, the simulated lesion mutants bon1 and snc4-1D, snc2-1D, mkk1 mkk2, bir1-1, ssi4, cpr22 and slh1 all showed different degrees of dwarf phenotype.

The tyrosine degradation pathway was first discovered in animals and bacteria. This metabolic pathway is degraded through five steps. The last step is the formation of acetoacetate and fumarate into the tricarboxylic acid under the action of fumarylacetoacetate hydrolase (FAH). The acid cycle breaks down completely. Genes and enzymes of the canonical tyrosine degradation pathway have been identified from Arabidopsis and demonstrated to have their respective catalytic activities in vitro. The inventor screened and identified a mutant sscd1 (short-day sensitive celldeath1) that forms simulated lesions in Arabidopsis under short-day conditions, and the mutant has no obvious difference from wild-type Arabidopsis under long-day conditions ; but under short-day conditions, without being infected by pathogens, some necrotic lesions appeared on the leaves. The SSCD1 gene has been isolated and identified by map-based cloning, and it is found that the gene encodes FAH.

The invention uses the CRISPR/Cas9 gene editing technology to edit the sixth exon of the Brassica napus FAH gene BnaA06G0083400WE to obtain dwarf Brassica napus. As an allotetraploid crop, Brassica napus has a complex genome with many homologous copies, and gene redundancy and gene additive effects often exist between different homologous copies. Mutation breeding has a large blindness, the direction and nature of mutagenesis cannot be controlled, there are few beneficial mutations, and the improved quantitative traits are poor. Therefore, using CRISPR/Cas9 gene editing technology with simple vector construction and high targeting specificity to edit genes is an effective way to improve target traits and cultivate new materials. The specific technical scheme is as follows:

In order to achieve the above object, the first aspect of the present invention provides a method of using gene editing technology to create a dwarf material of Brassica napus, by editing the BnaA06G0083400WE gene of Brassica napus using CRISPR/Cas9 gene editing technology, and obtaining the dwarf material of Brassica napus ( edited mutant material);

The nucleotide sequence of the coding gene mutant of the brassica napus dwarf material is SEQ ID NO.1.

Further, the method includes the following specific steps:

(1) Construction of Brassica napus BnaA06G0083400WE gene CRISPR/Cas9 expression vector:

1) Selection of sgRNA target sites:

The sgRNA target site is aimed at the structure and homology relationship of different copies of the BnaA06G0083400WE gene in Brassica napus westar, designed based on the CRISPRdirect website, and the nucleotide sequence is:

SEQ ID NO.2: 5'-agaggtcaaggccatccacaagg-3', named sgR-BnaA06G0083400WE;

2) Primer design and PCR amplification:

The primer sequences are as follows:

SEQ ID NO.3 (sgR-BnaA06G0083400WE-F): 5'-cagtGGTCTCagtcaagaggtcaaggccatccaca-3';

SEQ ID NO.4 (sgR-BnaA06G0083400WE-R): 5'-cagtGGTCTCaaaactgtggatggccttgacctct-3';

The PCR amplification process is as follows: According to the PCR reaction of 50 μ L system, the above and downstream primers are mixed, and the gRNA fragment (double-stranded DNA) is obtained by denaturation and annealing in a PCR instrument; in this technical solution, because the target sequence is relatively short, it can be obtained by denaturation and annealing. Get the target sequence;

3) Construction of CRISPR/Cas9 expression vector and Agrobacterium transformation:

Use T4 ligase to T4-ligate the gRNA fragment obtained in step 2) with the CRISPR/Cas9 plasmid digested with BsaI/Eco31I, then transform the ligated product into Escherichia coli DH5a and screen with antibiotics, then perform colony PCR identification and sequencing verification to obtain Expression vector;

Transform the vector containing sgRNA into Agrobacterium, and perform colony PCR verification to obtain the Agrobacterium strain containing the expression vector;

(2) Obtaining and identification of BnaA06G0083400WE gene CRISPR/Cas9 gene editing mutants in Brassica napus:

1) Place the pre-cultured hypocotyls in the Agrobacterium suspension containing the expression vector to induce callus, transfer the callus-producing hypocotyls to a medium for inducing embryogenic callus, and carry out The induction of embryogenic cells, the differentiation and screening of rapeseed genetically transformed seedlings are carried out at the same time, the corresponding antibiotics are added to the differentiation medium, divided into primary screening and secondary screening, and rooting culture of seedlings is carried out after positive seedlings are obtained;

2) Using PCR to detect the CRISPR/Cas9 expression vector in the positive Brassica napus seedlings, and detecting the editing status of the BnaA06G0083400WE gene in the CRISPR/Cas9 gene-edited plants by sequencing;

(3) To verify the differences in cell death and plant height between Brassica napus westar and BnaA06G0083400WE gene CRISPR/Cas9 gene editing mutants.

Further, the nucleotide sequence of the sgRNA target site is selected from the sixth exon region of the BnaA06G0083400WE gene in Brassica napus westar.

Further, the antibiotic is chloramphenicol.

The second aspect of the present invention provides an application of the above-mentioned method for creating dwarf materials of Brassica napus by using gene editing technology in rapeseed breeding.

Beneficial effects of the present invention:

The present invention uses the CRISPR/Cas9 gene editing technology to edit the sixth exon of the Brassica napus BnaA06G0083400WE gene to obtain dwarf Brassica napus, which can provide valuable genetic resources and germplasm resources for Brassica napus breeding.

The method of the present invention uses the CRISPR/Cas9 gene editing technology with simple vector construction and high targeting specificity to conduct directional editing of genes, has strong characteristics and is easy to obtain, and is an effective way to improve target traits and cultivate new materials.

Description of drawings

In Figure 1, A is the comparison chart of wesatr and sequenced CRISPR/Cas9-BnaA06G0083400WE sequence Blast results; B is the peak diagram of CRISPR/Cas9-BnaA06G0083400WE sequence sequencing; where wesatr is the wild type of Brassica napus, and CRISPR/Cas9-BnaA06G0083400WE is Edited mutant material;

Fig. 2 is a schematic diagram of the target site (shaded part) and missing base (shaded part white base) of Brassica napus BnaA06G0083400WE gene CRISPR/Cas9 gene editing of the present invention;

Fig. 3 is the BnaA06G0083400WE gene homozygous mutant material of the present invention and Brassica napus wild-type growth plant figure; Wherein, A is the mutant material (CRISPR/Cas9) of Brassica napus wild-type (westar) and editing for 30 days of long sunshine -BnaA06G0083400WE); B is Brassica napus wild type (westar) and edited mutant material (CRISPR/Cas9-BnaA06G0083400WE) with 40 days of long-day sunshine.

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Detailed ways

In order to better illustrate the technical solution of the present invention, the solution of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be performed according to the techniques described in documents or reference books in this field or according to the product instructions. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products.

The above-mentioned technical features of the present invention and the technical features specifically described in the following (such as examples of implementation) can be combined with each other to form a new or preferred technical solution.

Example 1: Construction of Brassica napus BnaA06G0083400WE Gene CRISPR/Cas9 Expression Vector

1. Cloning of the nucleic acid sequence of the full-length coding region of the BnaA06G0083400WE gene in Brassica napus westar:

Using the cDNA of Brassica napus westar as a template, using primers SEQ ID NO.5 (BnaA06FAH-ty-F): 5'-ACCATACATCATCCGTTT-3' and SEQ ID NO.6 (BnaA06FAH-ty-R): 5'-TCAAGGCAGTGAAGGTAAAA -3', clone the BnaA06G0083400WE gene in westar.

The cloned PCR product was subjected to 1% agarose gel electrophoresis, and the electrophoretic band consistent with the target fragment length was purified and recovered according to the TaKaRa gel recovery kit (MiniBEST Agarose Gel DNA Extraction Kit), and the recovered PCR product was purified Sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing to obtain the full-length coding region of the BnaA06G0083400WE gene in Brassica napus westar. The nucleic acid sequence is shown in the following SEQ ID NO.7:

ATG GCG TTG CTC AAG TCT TTC GTC GAT GTT GCT CCA CAC TCT CAC TTC CCTATC CAG AAC CTC CCT TAT GGCGTC TTC AAG CCC GAT TCC AAC TCT ACT CCC CGT CCCGCC GTC GCC ATC GGC GAT TCC GTC CTG GAC CTC TCAGCG ATC TCC GAA GCT GGG CTTTTC GAT GGT CCG ATC CTC AAC GGC TCC GAT TGC TTC CTT CAG CCT AAT CTGAAT AAGTTC TTA GCC ATG GGA CGA CCT GCT TGG AAG GAA GCG CGT TCT ACG CTT CAA AGA CTCTTG TCA TCTAGT GAG CCC ACT CTA CGA GAT AAC GAT GTT TTG AGG AGA AAG TCA TTTTAT GAG ATG AAT AAA GTG GAA ATGGTT GTT CCT ATG GTG ATT GGG GAC TAC ACA GACTTC TTT GCA TCC ATG CAT CAC GCC AAG AAC TGC GGT CTTATG TTT CGT GGG CCG CAGAAT GCT ATT AAC CCG AAT TGG TTT CGT CTT CCC ATT GCA TAT CAT GGA AGG GCATCATCT ATT GTC ATC TCT GGG ACT GAT ATT ATT CGA CCA AGA GGT CAA GGC CAT CCA CAAGGG GAC TCT GAACCG TAT TTT GGT CCT TCA AAG AAA CTT GAT TTT GAG CTT GAA ATGGCC GCT GTG GTT GGT CCA GGA AAT GAATTA GGA AAG CCT ATT GAC GTG AAC AAC GCAGCT GAC CAT ATA TTT GGC CTT GTA CTG ATG AAT GAC TGG AGTGCT AGG GAT ATT CAAGCG TGG GAG TAC GTA CCT CTT GGG CCT TTC CTA GGA AAG AGT TTC GGG ACT ACGGTATCT CCT TGG ATT GTT ACC TTA GAT GCG CTT GAA CCT TTC AGT TGT CAA GCT CCCAAG CAG GAT CCA CCT CCATTG CCA TAT CTA ACT GAG AAA GAA TCT GTC AAT TAC GATATC TCC TTG GAG GTT CAA CTC AAA CCT TCT GGCAAA GAT GAA TCT TCT GTA ATA ACAAAA AGC AAC TTC CAG AAC TTA TAC TGG ACC ATA ACG CAG CAG CTA GCGCAC CAT ACCGTT AAT GGT TGC AAC TTG AGA CCT GGT GAT CTC CTT GGA ACC GGA ACC ATA AGC GGACCC GAGCCA GAT TCA TAT GGG TGC CTA CTT GAG TTA ACT TGG AAT GGA CAG AAG CCTTTG TCA ATG AAC GGA ACA ACGCAG ACG TTT CTT CAA GAC GGA GAT CAA GTG ACC TTCTCA GGT GTA TGC AAG GGA GAT GGT TAC AAT GTC GGATTT GGA ACA TGC ACA GGG AAAATT TTA CCT TCA CTG CCT TGA

The amino acid sequence is shown in the following SEQ ID NO.8:

MALLKSFVDVAPHSHFPIQNLPYGVFKPDSNSTPRPAVAIGDSVLDLSAISEAGLFDGPILNGSDCFLQPNLNKFLAMGRPAWKEARSTLQRLLSSSEPTLRDNDVLRRKSFYEMNKVEMVVPMVIGDYTDFFASMHHAKNCGLMFRGPQNAINPNWFRLPIAYHGRASS IVISGTDIIRPRGQGHPQGDSEPYFGPSKKLDFELEMAAVVGPGNELGKPIDVNNAADHIFGLVLMNDWSARDIQAWEYVPLGPFLGKSFGTTVSPWIVT LDALEPFSCQ APKQDPPPLPYLTEKESVNYDISLEVQLKP SGKDESSVIT KSNFQNLYWT ITQQLAHHTV NGCNLRPGDL LGTGTISGPEPDSYGCLLELTWNGQKPLSMNGTTQTFLQDGDQVTFSGVCKGDGYNVGFGTCTGKILPSLP*。

2. Selection of sgRNA target sites and primer design

According to the target selection criteria, CRISPRdirect (http://crispr.dbcls.jp/) was used to design the target, located in the sixth exon of the BnaA06G0083400WE gene, the sequence is SEQ ID NO.2: 5'-agaggtcaaggccatccaca-3', named for sgR-BnaA06G0083400WE;

In order to facilitate the recombination of the target sequence and the expression vector, a BsaI/Eco31I restriction site GGTCTC is added to the front of the target sequence, so the primers are:

SEQ ID NO.3: (sgR-BnaA06G0083400WE-F): cagtGGTCTCagtcaagaggtcaaggccatccaca

SEQ ID NO.4: (sgR-BnaA06G0083400WE-R): cagtGGTCTCaaaactgtggatggccttgacctct

3. Obtaining double-stranded gDNA

The above pair of primers were sent to Shanghai Sangon Bioengineering Co., Ltd. for primer synthesis, and the synthesized primers were denatured and annealed by PCR to synthesize double-stranded gDNA for subsequent vector construction. The PCR reactions and procedures are shown in Table 1 and Table 2.

Table 1 PCR reaction

Table 2 PCR program

temperature time 95°C 10min 55°C 10min 14°C 5min

The PCR product obtained by denaturation and annealing was taken out from the PCR instrument and stored at 4°C to facilitate connection with the carrier.

4. Ligation of double-stranded gDNA to CRISPR/Cas9 vector:

The CRISPR/Cas9 plasmid was digested and ligated with BsaI/Eco31I restriction endonuclease in a constant temperature incubator at 37°C for 2 hours. The enzyme digestion and ligation reaction system is shown in Table 3.

Table 3 reaction system

Reagent volume T4 Buffer 1μL T4-ligase 0.5μL BsaI/Eco31I 0.5μL CRISPR/Cas9 vector 1.5μL dsgDNA 2μL Nuclease-free water 4.5μL 10μL (Total)

5. Recombinant plasmid transformation and identification

Take 5 μL of the ligation product and transform it into Escherichia coli DH5a competent, transform the recombinant vector into a plate coated with chloramphenicol resistance, incubate at 37°C for 12 hours, randomly select positive clones for colony PCR verification, and the primer is SEQ ID NO.9 (Forwardprimer): 5'-CCAGAAATTGAACGCCGAAG-3'; SEQ ID NO. 10 (Reverse primer): 5'-GTAAAACGACGGCCAGT-3'. The correct ones were verified and sent to Shanghai Sangong for sequencing, and the positive clones verified by the sequencing were shaken and plasmid extracted. Wherein, the colony PCR reaction system is shown in Table 4, and the PCR reaction conditions are shown in Table 5.

Table 4 Colony PCR reaction system

Reagent volume 5×buffer 4μL 2.5mM dNTPs 1.6μL Taq 0.2 μL bacterial suspension 5μL forward primer 1μL reverse primer 1μL Nuclease-free water 7.2 μL 20μL (Total)

Table 5 Colony PCR reaction system

Pre-denaturation at 94°C 2min 94°C 30sec 67°C 30sec 72°C 1min 15sec

6. Transformation of Agrobacterium

Transform the verified recombinant plasmid into Agrobacterium GV3101 by freezing and thawing with CaCl ₂ , select a single colony of Agrobacterium that has been verified by PCR and culture it, and culture it with shaking at 28°C and 220rpm for 18 to 24 hours. Centrifuge a large amount of activated bacterial liquid at 6000 g for 2 min, discard the supernatant, and resuspend the bacterial cells with Agrobacterium suspension (5% sucrose + 1/2MS + 0.02% Silwet-L77 + 0.01% 6-BA) until the _OD600 is between 0.6-1.0 between.

7. Genetic transformation of Brassica napus with CRISPR/Cas9 gene editing vector

(1) Prepare materials

Preparation of explants: soak rapeseed in 75% alcohol for 1 min, wash once with sterile water, then soak with 0.15% HgCl ₂ for 10-15 min, then wash with sterile water 4-5 times, 5 min each time. After sterilization, sow the seeds in 1/2 MS (or MS) medium and place them in a dark place for 4-5 days in dark (the germination of new seeds is different from that of old seeds). Hypocotyls are first pre-cultured in medium for 2-3 days. The tissue culture conditions are as follows: the photoperiod is 16h/d, and the temperature is 25±2°C.

(2) Genetic transformation of explants

The pre-cultured hypocotyls were placed in the Agrobacterium suspension, and after 8-10 minutes of infection, the Agrobacterium liquid on the surface of the hypocotyls was blotted dry with sterile filter paper.

(3) Co-cultivation of explants and Agrobacterium

Put the surface-dried hypocotyls in the co-cultivation medium at 25°C for 2-3 days in the dark. After infection, the hypocotyls were blotted dry with sterile filter paper.

(4) Induction of callus

After co-cultivation, the hypocotyls were transferred to the callus induction medium, the photoperiod was 16h/d, and the culture was 5-7d.

(5) Induction of embryogenic cells

The hypocotyls that produced the callus were transferred to the medium for inducing embryogenic callus, and the callus induced by the hypocotyls gradually turned green, and the induction time was 5-7 days.

(6) Differentiation and screening

The differentiation and screening of rapeseed genetically transformed seedlings are carried out at the same time. The corresponding antibiotics are added to the differentiation medium, which is divided into primary screening and secondary screening. Concentration, this stage is the second screening, which can screen out false positive seedlings before testing, increase the positive rate and reduce the workload in the later stage.

(7) Rooting of seedlings

The antibiotic chloramphenicol was added to the rooting medium, and the differentiated seedlings were selected again.

Example 2: Identification of Brassica napus BnaA06G0083400WE Gene CRISPR/Cas9 Gene Editing Mutants

Take 0.5 cm ² of the positive Brassica napus leaves obtained by screening, grind the samples, absorb the grinding liquid as a DNA template for PCR amplification, and determine the positive seedlings and positive rate by agarose gel electrophoresis. The amplified product was gel-cut and purified, its quality and concentration were tested, and it was sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing to detect the editing of the BnaA06G0083400WE gene. The sequencing results of the edited mutant material (CRISPR/Cas9-BnaA06G0083400WE) and the Blast comparison results with the BnaA06G0083400WE gene of wesatr are shown in Figure 1. From the comparison results in Figure 1, it can be seen that the screened Brassica napus It is a homozygous mutant for the deletion of base TCCA.

Example 3: Verification of the effect of BnaA06G0083400WE gene editing on plant height in Brassica napus

Through the sequencing results, a homozygous editing mutant of rapeseed that edited the sixth exon of the BnaA06G0083400WE gene was obtained. This homozygous mutant has a deletion of the base TCCA at the target site, and the amino acid sequence of its gene is shown in SEQ ID NO. 1. The comparison results with the normal BnaA06G0083400WE gene are shown in Figure 2.

The homozygous mutants were compared with wild-type westar plants, as shown in Figure 3A, 3B. It can be seen from Figure 3 that compared with the wild-type westar, the homozygous mutant has significantly shorter plant height and more branches. It shows that the present invention obtains the dwarf material (homozygous mutant material) of Brassica napus by using the CRISPR/Cas9 gene editing technology to edit the BnaA06G0083400WE gene of Brassica napus.

Finally, it should be emphasized that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the principles and principles shall be included within the protection scope of the present invention.

Claims (5)

1. A method of using gene editing technology to create dwarfing materials of Brassica napus, characterized in that, by utilizing the CRISPR/Cas9 gene editing technology to edit the BnaA06G0083400WE gene of Brassica napus, obtain the dwarfing materials of Brassica napus; The nucleotide sequence of the coding gene mutant of the brassica napus dwarf material is SEQ ID NO.1. 2. A kind of method utilizing gene editing technology to create Brassica napus dwarfing material according to claim 1, is characterized in that, comprises the following specific steps: (1) Construction of Brassica napus BnaA06G0083400WE gene CRISPR/Cas9 expression vector: 1) Selection of sgRNA target sites: The sgRNA target site is aimed at the structure and homology relationship of different copies of the BnaA06G0083400WE gene in Brassica napus westar, designed based on the CRISPRdirect website, and the nucleotide sequence is: SEQ ID NO.2: 5'-agaggtcaaggccatccacaagg-3'; 2) Primer design and PCR amplification: The primer sequences are as follows: SEQ ID NO.3: 5'-cagtGGTCTCagtca agaggtcaaggccatccaca-3'; SEQ ID NO.4: 5'-cagtGGTCTCaaaac tgtggatggccttgacctct-3'; The PCR amplification process is as follows: According to the PCR reaction of 50 μL system, the upper and lower primers are mixed, and denatured and annealed by a PCR instrument to obtain gRNA fragments; 3) Construction of CRISPR/Cas9 expression vector and Agrobacterium transformation: Use T4 ligase to T4-ligate the gRNA fragment obtained in step 2) with the CRISPR/Cas9 plasmid digested with BsaI/Eco31I, then transform the ligated product into Escherichia coli DH5a and screen with antibiotics, then perform colony PCR identification and sequencing verification to obtain Expression vector; Transform the vector containing sgRNA into Agrobacterium, and perform colony PCR verification to obtain the Agrobacterium strain containing the expression vector; (2) Obtaining and identification of BnaA06G0083400WE gene CRISPR/Cas9 gene editing mutants in Brassica napus: 1) Place the pre-cultured hypocotyls in the Agrobacterium suspension containing the expression vector to induce callus, transfer the callus-producing hypocotyls to a medium for inducing embryogenic callus, and carry out The induction of embryogenic cells, the differentiation and screening of rapeseed genetically transformed seedlings are carried out at the same time, the corresponding antibiotics are added to the differentiation medium, divided into primary screening and secondary screening, and rooting culture of seedlings is carried out after positive seedlings are obtained; 2) Using PCR to detect the CRISPR/Cas9 expression vector in the positive Brassica napus seedlings, and detecting the editing status of the BnaA06G0083400WE gene in the CRISPR/Cas9 gene-edited plants by sequencing; (3) To verify the difference in plant height between Brassica napus westar and BnaA06G0083400WE gene CRISPR/Cas9 gene editing mutants. 3. A method of using gene editing technology to create dwarfing materials in Brassica napus according to claim 2, wherein the nucleotide sequence of the sgRNA target site is selected from the BnaA06G0083400WE gene in Brassica napus westar The sixth exon region. 4. A method for creating dwarfing materials of Brassica napus by gene editing technology according to claim 2, characterized in that the antibiotic is chloramphenicol. 5. An application of the method for creating a dwarf material of Brassica napus by using gene editing technology according to any one of claims 1-4 in rapeseed breeding.

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