Method for creating dwarf material of brassica napus by gene editing technology and application
By using CRISPR/Cas9 gene editing technology to perform targeted editing of the sixth exon of the FAH gene in Brassica napus, the problems of blind breeding and poor improvement effect in existing technologies have been solved, and dwarf Brassica napus materials have been successfully bred, thus improving the mechanized production capacity of rapeseed.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YICHUN UNIVERSITY
- Filing Date
- 2022-09-09
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies make it difficult to effectively cultivate dwarf rapeseed varieties with excellent agronomic traits, which limits the mechanization of rapeseed production and leads to problems of blind breeding and poor improvement results.
The sixth exon of the FAH gene in Brassica napus was targeted and edited using CRISPR/Cas9 gene editing technology. A CRISPR/Cas9 gene editing vector was constructed, and dwarf Brassica napus material was obtained through gene editing methods with high targeting specificity.
This method effectively dwarfs rapeseed plant height, provides valuable genetic and germplasm resources, and enhances the mechanized production capacity and lodging resistance of rapeseed.
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Figure CN115838757B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of plant genetic engineering and biotechnology, and relates to the method and application of site-directed mutagenesis of the BnaA06G0083400WE gene in Brassica napus. In particular, it relates to a method and application of creating dwarfing materials of Brassica napus using CRISPR / Cas9 gene editing technology. Background Technology
[0002] Plant height directly affects a crop's lodging resistance and yield potential, making it one of the important indicators in modern crop breeding and the breeding of ideal plant types. Dwarfing breeding is an important part of the Green Revolution, which began with the wheat breeding revolution initiated by Norman Borlaug in 1950. The discovery and application of the rice semi-dwarf gene sd1 and wheat Rht8 were milestones in this revolution. Subsequently, dwarfing breeding work was carried out on crops such as rice, corn, and cucumber.
[0003] Currently, over 90% of rapeseed varieties in my country's main rapeseed producing areas are Brassica napus type, with over 36% having a plant height exceeding 180cm and only 11% below 160cm. Excessive plant height easily leads to lodging, insensitivity to fertilizer, and unsuitability for mechanized operations, making it a significant factor restricting mechanized rapeseed production. After lodging, the number of seeds per pod decreases by 17.5%, and seed yield drops by 16.2%. Simultaneously, the difficulty and damage rate of mechanized harvesting increase, resulting in reduced yield. Currently, several researchers have obtained some intermediate dwarf rapeseed materials through mutagenesis and spontaneous mutation, but due to factors such as excessively short plants, weak pre-winter growth, self-incompatibility of dwarf plants, and severe diseases, dwarf rapeseed with excellent agronomical traits has not yet been successfully applied in production. Therefore, breeding dwarf or semi-dwarf rapeseed varieties and conducting genetic analysis is of great significance for improving mechanized rapeseed harvesting. Summary of the Invention
[0004] This invention addresses the shortcomings of existing technologies by providing a method and application for creating dwarfing materials of Brassica napus using gene editing technology, which can provide valuable genetic and germplasm resources for rapeseed breeding, especially Brassica napus breeding.
[0005] Before conducting the experiments of this invention, the inventors carried out a great deal of research and summarization.
[0006] Lesion-mimicking mutants are a class of mutants that spontaneously develop programmed cell death on leaves, mimicking the effects of pathogen infection, even without significant adverse conditions, damage, or pathogen infestation. These mutants are widely found in plants such as Arabidopsis thaliana, rice, maize, sorghum, wheat, barley, and peanut. These mutants exhibit local and systemic resistance to many pathogens while undergoing programmed cell death. It has been reported that many lesion-mimicking mutants are typically dwarfed. For example, the lesion-mimicking mutant ssi4 exhibits dwarfism and spontaneous cell death; the mutation of chs3-2D triggers immune activation, and the lesion-mimicking mutant chs3-2D leads to extreme dwarfism and activation of the defense response; the lesion-mimicking mutant bir1-1 exhibits an extreme dwarfism phenotype, spontaneous cell death, and a defense response; the T-DNA insertion mutant of calmodulin-binding activator of transcription (CAMTA)3 exhibits enhanced disease resistance and dwarfism when grown at low temperatures; in addition, the lesion-mimicking mutants bon1 and snc4-1D, snc2-1D, mkk1 mkk2, bir1-1, ssi4, cpr22, and slh1 all exhibit varying degrees of dwarfism.
[0007] The tyrosine degradation pathway was first discovered in animals and bacteria. This metabolic pathway involves degradation through five steps, culminating in the final step where fumarate acetoacetate hydrolase (FAH) forms acetoacetic acid and fumaric acid, which then enter the tricarboxylic acid cycle for complete breakdown. Genes and enzymes involved in the typical tyrosine degradation pathway have been identified in Arabidopsis thaliana, and their respective catalytic activities in vitro have been demonstrated. The inventors screened and identified a mutant, sscd1 (short-day sensitive celldeath1), in Arabidopsis thaliana that forms lesion-like spots under short-day conditions. This mutant shows no significant difference from wild-type Arabidopsis thaliana under long-day conditions; however, under short-day conditions, without pathogen infection, it develops localized necrotic-like lesions on the leaves. The SSCD1 gene, encoding FAH, has been isolated and identified using map-based cloning.
[0008] This invention utilizes CRISPR / Cas9 gene editing technology to edit the sixth exon of the FAH gene BnaA06G0083400WE in Brassica napus, resulting in dwarfed Brassica napus. As an allotetraploid crop, Brassica napus has a complex genome with numerous homologous copies, often exhibiting gene redundancy and additive effects between different homologous copies. Mutagenesis breeding suffers from significant limitations; the direction and nature of mutations cannot be controlled, resulting in few beneficial mutations and poor improvement of quantitative traits. Therefore, using CRISPR / Cas9 gene editing technology, which features simple vector construction and high targeting specificity, for targeted gene editing is an effective way to achieve targeted trait improvement and cultivate new materials. The specific technical solution is as follows:
[0009] To achieve the above objectives, the first aspect of the present invention provides a method for creating dwarfing materials of Brassica napus using gene editing technology. The method involves editing the BnaA06G0083400WE gene of Brassica napus using CRISPR / Cas9 gene editing technology to obtain dwarfing materials of Brassica napus (edited mutant materials).
[0010] The nucleotide sequence of the coding gene mutant of the Brassica napus dwarfing material is SEQ ID NO.1.
[0011] Furthermore, the method includes the following specific steps:
[0012] (1) Construction of the CRISPR / Cas9 expression vector for the BnaA06G0083400WE gene in Brassica napus:
[0013] 1) Selection of sgRNA target sites:
[0014] The sgRNA target site was designed based on the structure and homology of different copies of the BnaA06G0083400WE gene in Brassica napus Westar, using the CRISPRdirect website. The nucleotide sequence is as follows:
[0015] SEQ ID NO.2: 5'-agaggtcaaggccatccacaagg-3', named sgR-BnaA06G0083400WE;
[0016] 2) Primer design and PCR amplification:
[0017] The primer sequences are as follows:
[0018] SEQ ID NO.3(sgR-BnaA06G0083400WE-F): 5'-cagtGGTCTCagtcaagaggtcaaggccatccaca-3';
[0019] SEQ ID NO.4(sgR-BnaA06G0083400WE-R): 5'-cagtGGTCTCaaaactgtggatggccttgacctct-3';
[0020] The PCR amplification process is as follows: PCR reaction is performed in a 50 μL system, with upstream and downstream primers mixed, and the mixture is denatured and annealed using a PCR instrument to obtain gRNA fragments (double-stranded DNA); in this technical solution, since the target sequence is relatively short, the target sequence can be obtained simply by denaturation and annealing.
[0021] 3) Construction of CRISPR / Cas9 expression vector and Agrobacterium-mediated transformation:
[0022] The gRNA fragment obtained in step 2) was ligated with the CRISPR / Cas9 plasmid digested with BsaI / Eco31I using T4 ligase. The ligation product was then transformed into E. coli DH5α and screened with antibiotics. Colony PCR identification and sequencing verification were performed to obtain the expression vector.
[0023] The vector containing sgRNA was transformed into Agrobacterium and colony PCR was performed to verify the Agrobacterium strain containing the expression vector.
[0024] (2) Obtaining and identifying the CRISPR / Cas9 gene editing mutant of the BnaA06G0083400WE gene in Brassica napus:
[0025] 1) The pre-cultured hypocotyls were placed in a suspension of Agrobacterium containing the expression vector to induce callus. The hypocotyls that produced callus were transferred to a medium for inducing embryogenic callus formation to induce embryogenic cells. The differentiation and screening of rapeseed genetically transformed seedlings were carried out simultaneously. The corresponding antibiotics were added to the differentiation medium. The seedlings were divided into primary screening and secondary screening. After obtaining positive seedlings, the seedlings were cultured for rooting.
[0026] 2) The CRISPR / Cas9 expression vector in positive Brassica napus seedlings was detected by PCR, and the editing status of the BnaA06G0083400WE gene in CRISPR / Cas9 gene-edited plants was detected by sequencing.
[0027] (3) Verify the differences in cell death and plant height between CRISPR / Cas9 gene-edited mutants of Brassica napus Westar and BnaA06G0083400WE genes.
[0028] Furthermore, the nucleotide sequence of the sgRNA target site is selected from the sixth exon region of the BnaA06G0083400WE gene in Brassica napus Westar.
[0029] Furthermore, the antibiotic is chloramphenicol.
[0030] In a second aspect, the present invention provides an application of the method for creating dwarfing materials of Brassica napus using gene editing technology in rapeseed breeding.
[0031] The beneficial effects of this invention are:
[0032] This invention utilizes CRISPR / Cas9 gene editing technology to edit the sixth exon of the BnaA06G0083400WE gene in Brassica napus, resulting in dwarf Brassica napus, which can provide valuable genetic and germplasm resources for Brassica napus breeding.
[0033] The method of this invention utilizes the CRISPR / Cas9 gene editing technology, which has simple vector construction and high targeting specificity, to perform targeted gene editing. It has strong characteristics and is easy to obtain, making it an effective way to improve target traits and cultivate new materials. Attached Figure Description
[0034] Figure 1 A in the figure is a Blast comparison of the wesatr sequence and the sequenced CRISPR / Cas9-BnaA06G0083400WE sequence; B is a peak diagram of the CRISPR / Cas9-BnaA06G0083400WE sequence; where wesatr is the wild type of Brassica napus and CRISPR / Cas9-BnaA06G0083400WE is the edited mutant material;
[0035] Figure 2 This is a schematic diagram of the target site (shaded area) and deleted bases (shaded white bases) of the CRISPR / Cas9 gene editing of the BnaA06G0083400WE gene in Brassica napus of this invention.
[0036] Figure 3 The images show the growth morphology of the homozygous mutant material of the BnaA06G0083400WE gene and the wild-type Brassica napus of this invention; wherein, A represents the wild-type Brassica napus (wester) and the edited mutant material (CRISPR / Cas9-BnaA06G0083400WE) under 30-day long-day conditions; B represents the wild-type Brassica napus (wester) and the edited mutant material (CRISPR / Cas9-BnaA06G0083400WE) under 40-day long-day conditions.
[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments. Detailed Implementation
[0038] To better illustrate the technical solution of the present invention, the following will explain the solution of the present invention in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques described in literature or reference books in the field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products.
[0039] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.
[0040] Example 1: Construction of CRISPR / Cas9 expression vector for BnaA06G0083400WE gene in Brassica napus
[0041] 1. Cloning of the full-length coding region nucleic acid sequence of the BnaA06G0083400WE gene in Brassica napus Westar:
[0042] Using cDNA from Brassica napus wester as a template, the BnaA06G0083400WE gene from wester was cloned using primers SEQ ID NO.5(BnaA06FAH-ty-F): 5'-ACCATACATCATCCGTTT-3' and SEQ ID NO.6(BnaA06FAH-ty-R): 5'-TCAAGGCAGTGAAGGTAAAA-3'.
[0043] The cloned PCR products were subjected to 1% agarose gel electrophoresis. The bands of the same length as the target fragment were purified and recovered using the TaKaRa gel extraction kit (MiniBEST Agarose Gel DNA Extraction Kit). The purified PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, yielding the full-length coding region of the BnaA06G0083400WE gene from Brassica napus (Wester). The nucleic acid sequence is shown in SEQ ID NO.7 below.
[0044] 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 CTCGAAC GTCGAC ATC 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 GGT TCT ACG CTT CAA AGA CTCTTG TCA TCTAGT GAG CCC ATTGA ATTACAT GAT TAG CGA GAT 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 CCCG CTT AAT GTT TGTTG GCATCATCT ATT GTC ATC TCT GGG ACT GAT 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 ATGGTG GCT GTG ATT GAT GAT A CCA CCA 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 CTAGGA 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
[0045] Attached to a specific SEQ ID NO.8 label:
[0046] MALLKSFVDVAPHSHPFPIQNLPYGVFKPDSNSTPRPAVAIGDSVLDLSAISEAGLFDGPILNGSDCFLQPNLNKFLAMGRPAWKEARSTLQRLLSSSEPTLRDNDVLRRKSFYEMNKVEMVVPMVIGDYTDFFASMHHAKNCGLMFRGPQNAINPNWFRLPIAYHGRASS IVISGTDIIRPRGQGHPQGDSEPYFGPSKKLDFELEMAAVVGPGNELGKPIDVNNAADHIFGLVLMNDWSARDIQAWEYVPLGPFLGKSFGTTVSPWIVT LDALEPFSCQ APKQDPPPLPYLTEKESVNYDISLEVQLKP SGKDESSVIT KSNFQNLYWT ITQQLAHHTV NGCNLRPGDL LGTGTISGPEPDSYGCLLELTWNGQKPLSMNGTTQTFLQDGDQVTFSGVCKGDGYNVGFGTCTGKILPSLP*.
[0047] 2. Selection of sgRNA target sites and primer design
[0048] Based on the target selection criteria, a target was designed using CRISPRdirect (http: / / crispr.dbcls.jp / ), located in the sixth exon of the BnaA06G0083400WE gene, with the sequence SEQ ID NO.2: 5'-agaggtcaaggccatccaca-3', named sgR-BnaA06G0083400WE;
[0049] To facilitate recombination of the target sequence with the expression vector, a BsaI / Eco31I restriction site, GGTCTC, is added to the front end of the target sequence. Therefore, the primers are:
[0050] SEQ ID NO.3: (sgR-BnaA06G0083400WE-F):cagtGGTCTCagtcaagaggtcaaggccatccaca
[0051] SEQ ID NO.4: (sgR-BnaA06G0083400WE-R):cagtGGTCTCaaaactgtggatggccttgacctct
[0052] 3. Obtaining double-stranded gDNA
[0053] The primer pair described above was sent to Shanghai Sangon Biotech Co., Ltd. for primer synthesis. The synthesized primers were then subjected to PCR reaction followed by denaturation and annealing to synthesize double-stranded gDNA for subsequent vector construction. The PCR reaction and procedure are shown in Tables 1 and 2.
[0054] Table 1 PCR reactions
[0055]
[0056]
[0057] Table 2 PCR Procedure
[0058] temperature time 95℃ 10min 55℃ 10min 14℃ 5min
[0059] Remove the PCR product obtained by denaturation and annealing from the PCR instrument and store it at 4°C to facilitate ligation with the vector.
[0060] 4. Ligation of double-stranded gDNA with CRISPR / Cas9 vector:
[0061] The CRISPR / Cas9 plasmid was digested and ligated using BsaI / Eco31I restriction endonuclease in a 37°C incubator for 2 hours. The digestion and ligation reaction system is shown in Table 3.
[0062] Table 3 Reaction System
[0063] reagents volume T4 Buffer 1μL T4-ligase 0.5μL BsaI / Eco31I 0.5μL CRISPR / Cas9 vector 1.5μL Double-stranded gDNA 2μL Nuclease-free water 4.5μL 10μL (Total)
[0064] 5. Transformation and identification of recombinant plasmids
[0065] 5 μL of the ligation product was transformed into *E. coli* DH5α competent cells, and the recombinant vector was transformed into chloramphenicol-resistant agar plates. The plates were incubated at 37°C for 12 hours. Positive clones were randomly selected for colony PCR verification. The primers were SEQ ID NO.9 (Forward primer): 5'-CCAGAAATTGAACGCCGAAG-3'; SEQ ID NO.10 (Reverse primer): 5'-GTAAAACGACGGCCAGT-3'. Verified clones were sent to Shanghai Sangon Biotech for sequencing. Positive clones with verified sequencing were then subjected to culture and plasmid extraction. The colony PCR reaction system is shown in Table 4, and the PCR reaction conditions are shown in Table 5.
[0066] Table 4 Colony PCR Reaction System
[0067] reagents volume 5×buffer 4μL 2.5 m M dNTPs 1.6μL Taq 0.2μL bacterial suspension 5μL forward primer 1μL Reverse primer 1μL Nuclease-free water 7.2μL 20μL (Total)
[0068] Table 5 Colony PCR Reaction System
[0069] 94℃ pre-denaturation 2min 94℃ 30 seconds 67℃ 30 seconds 72℃ 1 minute 15 seconds
[0070] 6. Transformation of Agrobacterium
[0071] The validated recombinant plasmid was transformed into Agrobacterium GV3101 using the CaCl2 freeze-thaw method. Single colonies of Agrobacterium that had been verified by colony PCR were selected and cultured at 28°C and 220 rpm for 18–24 h with shaking. The bacterial suspension was centrifuged at 6000g for 2 min to activate the bacteria, the supernatant was discarded, and the bacterial suspension was resuspended in Agrobacterium suspension (5% sucrose + 1 / 2 MS + 0.02% Silwet-L77 + 0.01% 6-BA) to OD200. 600 Between 0.6 and 1.0.
[0072] 7. Genetic transformation of Brassica napus using CRISPR / Cas9 gene editing vectors
[0073] (1) Prepare materials
[0074] Explant preparation: Rapeseed seeds were soaked in 75% ethanol for 1 min, rinsed once with sterile water, then soaked in 0.15% HgCl2 for 10-15 min, and rinsed 4-5 times with sterile water for 5 min each time. After sterilization, they were sown in 1 / 2 MS (or MS) medium and placed in the dark for 4-5 days (germination of new seeds differs from that of old seeds). Hypocotyls were pre-cultured in medium for 2-3 days. Tissue culture conditions were: photoperiod of 16 h / d and temperature of 25 ± 2℃.
[0075] (2) Genetic transformation of explants
[0076] Place the pre-cultured hypocotyl in an Agrobacterium suspension and infect for 8-10 minutes. Then, use sterile filter paper to absorb the Agrobacterium suspension from the surface of the hypocotyl.
[0077] (3) Co-culture of explants and Agrobacterium
[0078] Place the surface-dried hypocotyls in a co-culture medium and incubate in the dark at 25°C for 2-3 days. After infection, blot off excess bacterial solution with sterile filter paper.
[0079] (4) Callus induction
[0080] After co-culture, the hypocotyls were transferred to callus induction medium, with a photoperiod of 16 h / d, and cultured for 5-7 days.
[0081] (5) Induction of embryonic cells
[0082] The hypocotyls that produced callus were transferred to a medium that induces embryogenic callus formation. The callus induced by the hypocotyls slowly turned green, and the induction time was 5-7 days.
[0083] (6) Differentiation and selection
[0084] Differentiation and screening of rapeseed genetically transformed seedlings are carried out simultaneously. Appropriate antibiotics are added to the differentiation medium, and the process is divided into primary screening and secondary screening. The primary screening stage is in the early stage of differentiation, and the antibiotic concentration is low. After the differentiated seedlings elongate, the antibiotic concentration is increased, and this stage is the secondary screening. This can screen out false positive seedlings before testing, improve the positive rate, and reduce the workload in the later stages.
[0085] (7) Rooting of seedlings
[0086] The antibiotic chloramphenicol was added to the rooting medium, and the differentiated seedlings were screened again.
[0087] Example 2: Identification of CRISPR / Cas9 gene-editing mutants of the BnaA06G0083400WE gene in Brassica napus
[0088] Take 0.5cm 2 Positive leaves of Brassica napus obtained from screening were ground, and the ground sample solution was used as a DNA template for PCR amplification. Agarose gel electrophoresis was used to determine the number of positive seedlings and the positive rate. The amplified product was excised and purified, and its quality and concentration were measured. The product was then sent to Shanghai Sangon Biotech 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 Wesatr BnaA06G0083400WE gene are shown below. Figure 1 As shown, from Figure 1 The comparison results show that the selected rapeseed is a homozygous mutant with the missing base TCCA.
[0089] Example 3: Verification of the effect of gene editing of BnaA06G0083400WE on plant height in Brassica napus
[0090] Sequencing revealed a homozygous edited mutant of rapeseed with the sixth exon of the BnaA06G0083400WE gene edited. This homozygous mutant exhibited a deletion of the TCCA base at the target site. The amino acid sequence of this gene is shown in SEQ ID NO.1. A comparison with the normal BnaA06G0083400WE gene is shown below. Figure 2 As shown.
[0091] The homozygous mutant was compared with the wild-type Westar plant, such as Figure 3 As shown in A and 3B. From Figure 3It can be seen that, compared with the wild-type Westar, the homozygous mutant is significantly shorter and has more branches. This indicates that the present invention, by using CRISPR / Cas9 gene editing technology to edit the BnaA06G0083400WE gene of Brassica napus, obtained dwarfing materials (homozygous mutant materials) of Brassica napus.
[0092] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is 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 substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for creating dwarfing materials of Brassica napus using gene editing technology, characterized in that, Editing rapeseed using CRISPR / Cas9 gene editing technology BnaA06G0083400WE Genes were used to obtain dwarfing materials of Brassica napus; The nucleotide sequence of the coding gene mutant of the Brassica napus dwarfing material is SEQ ID NO.1; The method includes the following specific steps: (1) Brassica napus BnaA06G0083400WE Construction of CRISPR / Cas9 expression vectors for genes: 1) Selection of sgRNA target sites: The sgRNA target site is specific to the Westar argan oil of Brassica napus. BnaA06G0083400WE The structure and homology of different copies of the gene, designed based on the CRISPRdirect website, are as follows: SEQ ID NO.2: 5'-agaggtcaaggccatccacaagg-3'; The nucleotide sequence of the sgRNA target site is selected from the sixth exon region of the BnaA06G0083400WE gene in Brassica napus Westar. 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: PCR reaction is carried out in a 50 μL system, with upstream and downstream primers mixed, and the mixture is denatured and annealed using a PCR instrument to obtain gRNA fragments; 3) Construction of CRISPR / Cas9 expression vector and Agrobacterium-mediated transformation: The gRNA fragment obtained in step 2) was ligated with the CRISPR / Cas9 plasmid digested with BsaI / Eco31I using T4 ligase. The ligation product was then transformed into E. coli DH5α and screened with antibiotics. Colony PCR identification and sequencing verification were performed to obtain the expression vector. The vector containing sgRNA was transformed into Agrobacterium and colony PCR was performed to verify the Agrobacterium strain containing the expression vector. (2) Brassica napus BnaA06G0083400WE Obtaining and identifying CRISPR / Cas9 gene editing mutants: 1) The pre-cultured hypocotyls were placed in a suspension of Agrobacterium containing the expression vector to induce callus. The hypocotyls that produced callus were transferred to a medium for inducing embryogenic callus formation to induce embryogenic cells. The differentiation and screening of rapeseed genetically transformed seedlings were carried out simultaneously. The corresponding antibiotics were added to the differentiation medium. The seedlings were divided into primary screening and secondary screening. After obtaining positive seedlings, the seedlings were cultured for rooting. 2) PCR was used to detect the CRISPR / Cas9 expression vector in positive Brassica napus seedlings, and sequencing was used to detect the expression of CRISPR / Cas9 gene-edited plants. BnaA06G0083400WE The status of gene editing; (3) Verify the Westar variety of Brassica napus and BnaA06G0083400WE Differences in plant height among CRISPR / Cas9 gene-edited mutants.
2. The method for creating dwarfing materials of Brassica napus using gene editing technology according to claim 1, characterized in that, The antibiotic in question is chloramphenicol.
3. The application of a method for creating dwarfing materials of Brassica napus using gene editing technology as described in any one of claims 1-2 in rapeseed breeding.