Method for increasing oil content of rapeseeds based on rape transcription factor BnaA01.AP2 and application

Abstract

The invention relates to the technical field of plant genetic engineering application, in particular to a method for improving the oil content of rapeseeds based on a rape transcription factor BnaA01. AP2 and application. BnaA01. AP2 in a brassica napus inbred line 'K407' is knocked out through CRISPR / Cas9 mediated gene editing, and homozygous mutants L1 and L2 are obtained. Phenotypic analysis shows that compared with a wild type K407, the total seed yield and the oil yield of L1 and L2 mutant plants are remarkably improved, and meanwhile, various main agronomic characters of the mutant plants and the germination rate of the seeds under salt stress are not weakened compared with those of the wild type K407. The oilseed rape BnaA01. AP2 gene and mutants L1 and L2 disclosed by the invention provide new excellent gene resources and oilseed rape materials for molecular design breeding, and have good application prospects in the aspect of high-oil breeding of oil crops.

Description

Technical Field

[0001] The patent of this invention relates to the field of plant genetic engineering application technology, specifically, to a method and application for increasing rapeseed oil content based on rapeseed transcription factor BnaA01.AP2. Background Art

[0002] Brassica napus is a crop of the genus Brassica in the family Cruciferae. It can be divided into three categories based on chromosome composition and genetic background, namely Brassica napus L. (AA, 2n=2x=20), Brassica napus L. (AACC, 2n=2x=38) and Brassica juncea. Brassica napus accounts for about 80% of the total rapeseed planting area. It is named because its leaf shape and growth environment are similar to those of cabbage. It was introduced to my country in the 1830s. Due to its advantages in seed yield and high oil content, it gradually replaced Brassica napus and Brassica juncea in production and became the main variety in some parts of my country. As the largest oil crop in my country, rapeseed is the largest source of domestic edible vegetable oil. The high-quality edible oil it provides each year accounts for more than 50% of the domestic oil crop output, and it is the core of maintaining the security of the country's edible oil supply. At the same time, rapeseed provides about 8 million tons of high-protein rapeseed meal for my country's animal husbandry industry each year. In addition, rapeseed has multiple functions such as vegetable, nectar source, sightseeing, and fertilizer, and it occupies an important position in serving rural revitalization and meeting people's health needs in the new era.

[0003] The development of rapeseed seed industry in my country faces bottleneck problems such as lack of excellent germplasm resources and serious homogeneity of varieties. Improving rapeseed oil yield and improving its oil quality through biological breeding technology, creating excellent germplasm and cultivating breakthrough varieties are important measures to drive the transformation and upgrading of my country's rapeseed industry and ensure the security of my country's edible oil supply. Studies have shown that the effect of increasing oil content by manipulating a single structural gene is often not obvious, while by manipulating regulatory genes related to oil accumulation, the oil content of oil crop seeds can be significantly improved at the overall regulatory level. At present, my country's mining and functional analysis of original key regulatory genes that can be used for rapeseed biological breeding and significantly affect rapeseed seed oil accumulation are still behind those of foreign countries. Therefore, strengthening basic research, mining key regulatory genes that affect oil accumulation and quality improvement, and analyzing their molecular regulatory mechanisms have important application value and practical significance for creating excellent germplasm resources and cultivating high-yield and high-quality breakthrough rapeseed varieties.

[0004] AP2 belongs to the AP2 (APETALA 2) / ERF (Ethylene Responsive Factor) transcription factor family AP2 subfamily. In Brassica napus, AP2 has four homologous copies, namely BnaA01.AP2 (BnA01T0016800ZS), BnaA03.AP2 (BnA03T0561000ZS), BnaC01.AP2 (BnC01T0019400ZS), and BnaC07.AP2 (BnC07T0536500ZS). Studies have shown that silencing the expression of the four copies of BnAP2 using RNAi technology can lead to defects in the development of rapeseed flower organs and larger seeds, but reduced total yield. Using gene editing technology to simultaneously knock out the four copies of BnAP2, the mutant showed problems such as missing petals, reduced stamens, and reduced total yield. Mutations in AtAP2 (AT4G36920), the Arabidopsis homolog of the AP2 gene, can lead to larger seeds, significantly increased seed oil and storage protein content, and significantly reduced siliques per plant, silique length, and number of seeds per silique. Although knockout of BnAP2 and AtAP2 both lead to larger seeds and increased oil content, the total yield is significantly reduced. However, studies have shown that in allotetraploid Brassica napus, since most genes usually exist in multiple copies, it provides the possibility for functional differentiation of homologous genes. Therefore, analyzing the gene function of a single copy of BnAP2 and utilizing potential functional differentiation to fully utilize dominant traits have important application value and practical significance for improving rapeseed oil content and creating excellent germplasm resources.

[0005] To this end, the present application provides a method and application for increasing the oil content of rapeseed based on rapeseed transcription factor BnaA01.AP2 to solve the problems in the prior art. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application for increasing the oil content of rapeseed based on rapeseed transcription factor BnaA01.AP2, so as to solve the above-mentioned problems in the prior art and provide valuable gene resources and theoretical basis for rapeseed high-oil breeding.

[0007] The above-mentioned purpose of the present invention is achieved like this:

[0008] The scheme of the present invention provides a method for improving rapeseed oil content by editing the BnaA01.AP2 gene, the method comprising: designing a sgRNA sequence based on the nucleic acid sequence characteristics of the BnaA01.AP2 gene in the inbred line 'K407', and cloning the sgRNA sequence into a CRISPR / Cas9 vector; introducing the vector into 'K407' by Agrobacterium-mediated hypocotyl genetic transformation; obtaining T1 generation positive transgenic plants by hygromycin resistance screening; further, cloning the T1 generation transgenic plants using sequence-specific primers, and performing Sanger sequencing on the amplified products to observe their editing conditions; obtaining homozygous gene-edited mutants L1 and L2 without CRISPR / Cas9 vectors in the T3 generation population after three generations of screening and identification; specifically comprising the following steps:

[0009] S1. Acquisition and vector construction of rapeseed gene BnAP2:

[0010] S1-1. Acquisition and expression analysis of rapeseed gene BnAP2:

[0011] Using the search tool box of the BnIR database, we found that there were four copies of the BnAP2 gene in the genome of Brassica napus 'Zhongshuang11', located on chromosomes A01, A03, C01 and C07 respectively. According to the location of these four copies in the chromosomes, they were named BnaA01.AP2, BnaA03.AP2, BnaC01.AP2 and BnaC07.AP2 respectively;

[0012] Next, based on the nucleic acid sequences of the four copies in the 'Zhongshuang 11' genome, sequence-specific primers for each copy were designed upstream and downstream of its coding region, and PCR amplification was performed using the genomic DNA of the inbred line 'K407' as a template. By performing Sanger sequencing on all amplified products, it was found that the BnAP2 gene in the inbred line 'K407' genome also had four copies;

[0013] The BnaA01.AP2, BnaA03.AP2, BnaC01.AP2, and BnaC07.AP2 encoded by the ‘K407’ genome have identical CDS sequences to these copies encoded by the ‘Zhongshuang11’ genome;

[0014] S1-2, using NCBI to design specific primers for RT-qPCR based on the sequences obtained for the four copies of BnAP2 for gene expression analysis;

[0015] Four copies of CDS of BnAP2 were constructed into pCAMBIA1300-35S–GFP vector and transiently expressed in Nicotiana benthamiana leaves for subcellular localization analysis;

[0016] Four copies of CDS of BnAP2 were constructed into pGBKT7(BD) vector and analyzed for transcriptional activation activity;

[0017] S1-3, Construction of CRISPR / Cas9 vector, based on the nucleic acid sequence characteristics of BnaA01.AP2 gene in ‘K407’, sgRNA sequence was designed using CRISPRdirect website;

[0018] For the primers targeting the target BnaA01.AP2, 10 μL of the forward primer and the reverse primer were added to the PCR tube and mixed evenly. The mixture was incubated at 95°C for 5 min in a PCR instrument to obtain a double-stranded gDNA product. Then, the double-stranded gDNA product was ligated with the CRISPR / Cas9 vector linearized by the AarI restriction endonuclease using T4 DNA Ligase to construct the BnaA01.AP2–Cas9 gene editing vector.

[0019] Subsequently, each ligation product was transformed into E. coli competent cells DH5α by heat shock method, and transformants were selected on LB solid medium containing spectinomycin;

[0020] The monoclonal transformants were then verified by colony PCR using 2×Rapid Taq MasterMix, and the monoclonal transformants with the correct band size were added to LB liquid culture medium containing spectinomycin for expansion culture. Subsequently, the expanded culture culture fluid was sequenced, and the plasmid was extracted from the monoclonal culture fluid with the correct sequence according to the instructions of the plasmid mini-extraction kit.

[0021] S2. Acquisition, screening and identification of gene-edited plants:

[0022] S2-1. Obtaining gene-edited plants:

[0023] After pre-culturing the Agrobacterium containing the recombinant plasmid with the explants, the positive seedlings after hygromycin selection, differentiation and rooting were defined as T1 transgenic plants;

[0024] S2-2. Screening and identification of transgenic plants:

[0025] The genomic DNA of young rapeseed leaves was extracted using a plant genomic DNA extraction kit; first, the T1 transgenic plants were subjected to PCR to identify the presence of the target gene, and the transcription level of the target gene was detected;

[0026] Then, 10 T1 generation positive seedlings with higher transcription levels are selected for further sowing and planting until all the offspring seedlings of a certain T2 generation offspring are identified to contain the target gene fragment. At this time, the seeds harvested from the offspring of the single plant are T3 generation homozygous seeds.

[0027] Furthermore, the primers in step S1-3 are shown in the following table:

[0028]

[0029]

[0030] Furthermore, the primers in step S1-2 are shown in the following table:

[0031]

[0032] On the other hand, the scheme of the present invention provides a method for increasing rapeseed oil content based on rapeseed transcription factor BnaA01.AP2 for use in creating excellent germplasm resources and cultivating high-yield, high-quality breakthrough rapeseed varieties.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The scheme of the present invention knocks out BnaA01.AP2 in the Brassica napus inbred line 'K407' through CRISPR / Cas9-mediated gene editing, and obtains homozygous mutants L1 and L2. Phenotypic analysis shows that compared with the wild type 'K407', the total seed yield and oil yield of the L1 and L2 mutant plants are significantly improved, and the main agronomic traits of the mutant plants and the germination rate of the seeds under salt stress are not weakened compared with the wild type 'K407'. The rapeseed BnaA01.AP2 gene and mutants L1 and L2 provided by the scheme of the present invention provide new excellent gene resources and rapeseed materials for molecular design breeding, and have good application prospects in high-oil breeding of oil crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The expression patterns and transcriptional activation of four BnAP2 homologous genes (A is RT-qPCR analysis of the four copies of BnAP2 in the germinating seeds, roots, stems, expanding leaves and developing seeds of the wild type 'K407' of Brassica napus seeds), with BnGAPDH as the internal reference gene. The measured values ​​of each index are mean ± standard deviation (n = 3); B is the subcellular localization analysis of 4 copies of BnAP2 in tobacco (Nicotiana benthamiana) leaves. Bright, bright field; mCherry, nuclear localization protein fused with red fluorescent protein; GFP, green fluorescent protein; Merge, fusion of bright field, mCherry and GFP; C is the transcriptional activation activity analysis of 4 copies of BnAP2 in yeast, with the empty vector pGBKT7 (AD) as a negative control; transformed yeast cells were grown on SD / -Trp and SD / -Trp / -Ade / -His deficiency medium for 3 days and then photographed);

[0036] Figure 2 For the creation and phenotypic analysis of gene editing materials. (A is a schematic diagram of the gene structure and CRISPR / Cas9 target site of the BnaA01.AP2 gene. The red triangle indicates the location of the target sequence on BnaA01.AP2. The blue horizontal line and letters below indicate the PAM motif, and the black horizontal line and letters indicate the target sequence; B is BnaA01.AP2 Editing of T3 generation homozygous mutants L1 and L2, red letters indicate inserted bases; C is microscopic observation of silique development of wild type 'K407' and BnaA01.AP2 homozygous mutants (L1, L2) 28 days after pollination, scale bar = 1 cm; D is microscopic observation of mature seeds of 'K407' and BnaA01.AP2 homozygous mutants (L1, L2), scale bar = 500 μm; E, F are quantitative comparisons of total fatty acids (E) and main fatty acid components (F) in seeds of 'K407' and BnaA01.AP2 homozygous mutants (L1, L2), all index values ​​are mean ± standard deviation (n = 5), asterisk * indicates significant difference between mutant and wild type 'K407' phenotype, ** represents P < 0.01, *** represents P < 0.001);

[0037] Figure 3Seed germination phenotype analysis of wild type 'K407' and BnaA01.AP2 homozygous mutant L1 under salt stress (A is the seed germination phenotype of 'K407' and L1 mutant 96h after sowing under control and 200mM or 300mM NaCl treatment, the appearance of radicle was used as a morphological marker of seed germination, scale bar = 1cm; B is the statistical analysis of the germination rate of seeds of 'K407' and L1 mutant described in A, and the measured values ​​of each index are mean ± SD (n = 3));

[0038] Figure 4 Statistics of agronomic traits of wild type 'K407' and homozygous gene-edited materials L1 and L2 (the measured values ​​of each index are mean ± standard deviation (n = 3), multiple comparisons were performed using one-way ANOVA, * indicates significant difference at the p < 0.05 level). DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0040] First of all, it should be noted that in the following examples, the reagents used are all from commercial channels unless otherwise specified, and the test methods, detection methods, etc. involved, unless otherwise specified, are all conventional test methods and detection methods already available in the prior art.

[0041] The following combination Figure 1-Figure 4 The implementation of the present invention is described in detail with reference to the accompanying drawings and specific embodiments.

[0042] Example:

[0043] The scheme provided in this embodiment is a method and application for increasing the oil content of rapeseed based on the rapeseed transcription factor BnaA01.AP2. The present invention designs an sgRNA sequence based on the nucleic acid sequence characteristics of the BnaA01.AP2 gene in the inbred line 'K407' and clones it into the CRISPR / Cas9 vector. The vector is introduced into 'K407' by Agrobacterium-mediated hypocotyl genetic transformation. By screening for hygromycin resistance, T1 generation positive transgenic plants are obtained. Furthermore, sequence-specific primers are used to clone the target gene in the above-mentioned T1 generation transgenic plants, and the amplified products are subjected to Sanger sequencing to observe their editing. After three generations of screening and identification, homozygous gene-edited mutants L1 and L2 without CRISPR / Cas9 vectors were obtained in the T3 generation population.

[0044] The seed oil content and related agronomic traits of two homozygous gene-edited mutants of BnaA01.AP2, L1 and L2, were measured. The results showed that compared with the wild type 'K407', the mutant seed oil content was significantly increased, and the total seed yield and oil yield per plant were significantly increased. At the same time, plant height, number of branches, number of siliques per plant, silique length, number of seeds per silique, and seed germination rate under salt stress were not reduced compared with the wild type 'K407'. Therefore, we found that high-oil and high-yield rapeseed materials can be obtained by targeted editing of a single copy of BnaA01.AP2.

[0045] The following is the specific implementation of this implementation plan:

[0046] 1. Acquisition and vector construction of rapeseed gene BnAP2

[0047] 1.1. Acquisition and expression analysis of rapeseed gene BnAP2

[0048] Using the search tool box of the BnIR database, we found that there were four copies of the BnAP2 gene in the genome of Brassica napus 'Zhongshuang 11', located on chromosomes A01, A03, C01 and C07, respectively. According to the location of these four copies in the chromosomes, they were named BnaA01.AP2, BnaA03.AP2, BnaC01.AP2 and BnaC07.AP2. Then, based on the nucleic acid sequences of these four copies in the genome of 'Zhongshuang 11', sequence-specific primers for each copy were designed upstream and downstream of their coding regions, and PCR amplification was performed using the genomic DNA of the inbred line 'K407' as a template. By performing Sanger sequencing on all the amplified products, it was found that there were also four copies of the BnAP2 gene in the genome of the inbred line 'K407'. The BnaA01.AP2, BnaA03.AP2, BnaC01.AP2 and BnaC07.AP2 encoded by the 'K407' genome have the same CDS sequences as these copies encoded by the 'Zhongshuang11' genome.

[0049] NCBI was used to design specific primers for RT-qPCR based on the sequences obtained for the four copies of BnAP2 for gene expression analysis. The CDS of the four copies of BnAP2 were constructed into the pCAMBIA1300-35S-GFP vector and transiently expressed in Nicotiana benthamiana leaves for subcellular localization analysis. The CDS of the four copies of BnAP2 were constructed into the pGBKT7 (BD) vector for transcriptional activation activity analysis. The primers used are shown in Table 1:

[0050] Table 1 BnAP2 vector construction and RT-qPCR primer information

[0051]

[0052]

[0053] 1.2. Construction of CRISPR / Cas9 vector

[0054] In order to further study the biological function of the BnaA01.AP2 gene, the sgRNA sequence was designed using the CRISPRdirect website based on the nucleic acid sequence characteristics of the BnaA01.AP2 gene in 'K407'. The primers are shown in Table 2:

[0055] Table 2 Primer information for BnaA01.AP2–Cas9 vector construction

[0056]

[0057] For the primers targeting the target BnaA01.AP2, 10 μL of forward primer and reverse primer were added to a PCR tube and mixed evenly. The mixture was incubated at 95°C for 5 min in a PCR instrument to obtain a double-stranded gDNA product. Then, T4 DNA Ligase (Cat No. M0202S, New England Biolabs) was used to perform T4 ligation on the double-stranded gDNA product and the CRISPR / Cas9 vector linearized with AarI restriction endonuclease (Cat No. ER1581, Thermo Fisher Scientific) according to its instructions to construct the BnaA01.AP2-Cas9 gene editing vector.

[0058] Subsequently, each ligation product was transformed into E. coli competent cells DH5α (Cat No. DL1001, Weidi Biotechnology) by heat shock method, and the transformants were screened on LB solid medium containing spectinomycin. Then, the monoclonal transformants were verified by colony PCR according to the instructions of 2×RapidTaqMasterMix (Cat No. P222, Novozyme Biotechnology Co., Ltd.), and the monoclonal transformants with correct band size were added to LB liquid medium containing spectinomycin for expansion and culture. Subsequently, the expanded cultured bacterial solution was sent to Shanghai Sangon Biotechnology Co., Ltd. (Xi'an, China) for sequencing, and the monoclonal bacterial solution with correct sequencing was extracted with plasmid according to the instructions of plasmid mini-extraction kit (Cat No. DP103, Tiangen Biochemical Technology Co., Ltd.).

[0059] 2. Acquisition, screening and identification of gene-edited plants

[0060] 2.1. Obtaining gene-edited plants

[0061] The receptor materials used for rapeseed transformation were all Brassica napus inbred line 'K407', and the BnaA01.AP2 gene-edited mutant strain was obtained by the team of Professor Zhang Yanfeng from the Shaanxi Hybrid Rapeseed Research Center using Agrobacterium-mediated hypocotyl genetic transformation. After pre-culturing Agrobacterium containing the recombinant plasmid with explants, the positive seedlings formed by hygromycin selection, differentiation and rooting were defined as T1 transgenic plants.

[0062] 2.2 Screening and identification of transgenic plants

[0063] The genomic DNA of young rapeseed leaves was extracted using the SteadyPure Plant Genomic DNA Extraction Kit (Cat No. AG21011, Aikerui Biotechnology) according to the instructions provided by the kit. First, the T1 transgenic plants were identified for the presence of the target gene by PCR, and the transcription level of the target gene was detected. Then, 10 T1 positive seedlings with higher transcription levels were selected to continue sowing and planting until all the offspring seedlings of a certain single plant were identified to contain the target gene fragment in the T2 generation offspring single plant. At this time, the seeds harvested from the offspring of the single plant were T3 generation homozygous seeds, which can be used for subsequent experiments. At the same time, the target fragment was amplified in the T1, T2 and T3 generation gene editing mutant single plants using BnaA01.AP2 gene-specific primers. The PCR products were then sent directly to Shanghai Shenggong Biotechnology Co., Ltd. (Xi'an, China) for sequencing to detect the editing of the BnaA01.AP2 gene in each editing mutant. If the sequencing peak graph of the PCR amplification product is normal, there are no double peaks and overlapping peaks, indicating that the gene in the amplification product is homozygous editing. If the sequencing peak of the PCR amplification product shows a double peak, it indicates that the gene in the amplification product is heterozygous for editing. The amplification product needs to be further cloned into the pMD19-T vector, and then the cloned products are transformed into Escherichia coli competent cells DH5α (Cat No. DL1001, Weidi Biotechnology), and 8 monoclonal transformants are randomly selected and sent to Shanghai Sangon Biotechnology Co., Ltd. (Xi'an, China) for sequencing to determine the specific editing of BnaA01.AP2 in each gene editing mutation. By identifying the CRISPR / Cas9 vector of the T3 generation population, it was found that there were T3 generation homozygous single strains without CRISPR / Cas9 vectors in the T3 generation population. BnaA01.AP2 in these homozygous single strains was amplified and sequenced, and two homozygous mutants L1 and L2 of the BnaA01.AP2 gene were identified. The primers used are shown in Table 3:

[0064] Table 3 BnaA01.AP2-Cas9 identification primer information

[0065]

[0066] The planting method of all rapeseed materials in this study was as follows: First, about 30 rapeseed seeds of each genotype were taken and sown in plug trays with nutrient soil (nutrient soil: vermiculite = 4:1) as the matrix, and then placed in an artificial climate culture room until two cotyledons were fully developed. The light cycle of the artificial climate culture room was set to 16 h light / 8 h dark, and the light intensity was set to 300-400 μmol·m -2 ·s -1 The day and night temperature was set at 25 / 18°C. Then, the rapeseed seedlings with strong and consistent growth were selected and transplanted into pots with nutrient soil (nutrient soil: vermiculite = 4:1) as the matrix for further cultivation for 4 weeks. Subsequently, the seedlings were moved to the vernalization room for vernalization treatment for 5 weeks. The photoperiod in the vernalization room was set to 16h light / 8h dark, and the light intensity was set to 300-400μmol·m -2 ·s -1 , the temperature was set to 4°C. After vernalization, the plants were moved to the previous artificial climate culture room and continued to be cultured until the seeds matured.

[0067] 3. Statistics of fatty acid content and related agronomic traits of BnaA01.AP2 seeds

[0068] The mature seeds of the Brassica napus inbred line 'K407' and the homozygous mutant L1 were naturally dried to a constant weight, and the fatty acid content of the seeds was analyzed by gas chromatography (Shimadzu GC-2010plus) using the internal standard method (internal standard is methyl heptadecanoate C17:0). The results showed that compared with the inbred line 'K407', the total fatty acid content of the seeds of mutants L1 and L2 increased significantly, indicating that BnaA01.AP2 negatively regulates the accumulation of fatty acids in seeds.

[0069] Furthermore, the relevant agronomic traits of 'K407' and mutant L1 were statistically analyzed. The results showed that compared with 'K407', the plant height, silique width, seed size, 1000-grain weight, total seed yield per plant and oil yield of BnaA01.AP2 homozygous mutants L1 and L2 were significantly increased, while key agronomic traits such as branch number, silique number per plant and seed number per silique were not reduced. This shows that the homozygous mutants L1 and L2 of BnaA01.AP2 are two excellent rapeseed germplasms with high yield and high oil content.

[0070] 4. Effect of BnaA01.AP2 on salt tolerance of rapeseed seed germination

[0071] The germination of seeds of Brassica napus inbred line 'K407' and homozygous mutant L1 under NaCl stress was analyzed. The results showed that the germination rate of mutant L1 under salt stress was no different from that of 'K407', indicating that the mutation of BnaA01.AP2 did not lead to a decrease in the germination rate of BnaA01.AP2 seeds and its tolerance to salt stress.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for increasing the oil content of Brassica napus seeds by editing the BnaA01.AP2 gene, characterized in that: The method comprises the following steps: based on the nucleic acid sequence characteristics of the BnaA01.AP2 gene in the inbred line 'K407', an sgRNA sequence is designed and cloned into a CRISPR / Cas9 vector; the vector is introduced into 'K407' by Agrobacterium-mediated hypocotyl genetic transformation; T1 generation positive transgenic plants are obtained by hygromycin resistance screening; further, sequence-specific primers are used to clone the T1 generation transgenic plants, and Sanger sequencing is performed on the amplified products to observe their editing conditions; after three generations of screening and identification, homozygous gene editing mutants L1 and L2 without CRISPR / Cas9 vectors are obtained in the T3 generation population; the method comprises the following steps: S1. Acquisition and vector construction of rapeseed gene BnAP2: S1-1. Acquisition and expression analysis of rapeseed gene BnAP2: Using the search tool box of the BnIR database, we found that there were four copies of the BnAP2 gene in the genome of Brassica napus 'Zhongshuang11', located on chromosomes A01, A03, C01 and C07 respectively. According to the location of these four copies in the chromosomes, they were named BnaA01.AP2, BnaA03.AP2, BnaC01.AP2 and BnaC07.AP2 respectively; Next, based on the nucleic acid sequences of the four copies in the 'Zhongshuang 11' genome, sequence-specific primers for each copy were designed upstream and downstream of its coding region, and PCR amplification was performed using the genomic DNA of the inbred line 'K407' as a template. By performing Sanger sequencing on all amplified products, it was found that the BnAP2 gene in the inbred line 'K407' genome also had four copies; The BnaA01.AP2, BnaA03.AP2, BnaC01.AP2, and BnaC07.AP2 encoded by the ‘K407’ genome have identical CDS sequences to these copies encoded by the ‘Zhongshuang11’ genome; S1-2, using NCBI to design specific primers for RT-qPCR based on the sequences obtained for the four copies of BnAP2 for gene expression analysis; Four copies of CDS of BnAP2 were constructed into pCAMBIA1300-35S–GFP vector and transiently expressed in Nicotiana benthamiana leaves for subcellular localization analysis; Four copies of CDS of BnAP2 were constructed into pGBKT7(BD) vector and analyzed for transcriptional activation activity; S1-3, Construction of CRISPR / Cas9 vector, based on the nucleic acid sequence characteristics of BnaA01.AP2 gene in ‘K407’, sgRNA sequence was designed using CRISPRdirect website; For the primers targeting the target BnaA01.AP2, 10 μL of the forward primer and the reverse primer were added to the PCR tube and mixed evenly. The mixture was incubated at 95°C for 5 min in a PCR instrument to obtain a double-stranded gDNA product. Then, the double-stranded gDNA product was ligated with the CRISPR / Cas9 vector linearized by the AarI restriction endonuclease using T4 DNA Ligase to construct the BnaA01.AP2–Cas9 gene editing vector. Subsequently, each ligation product was transformed into E. coli competent cells DH5α by heat shock method, and transformants were selected on LB solid medium containing spectinomycin; The monoclonal transformants were then verified by colony PCR using 2×Rapid Taq MasterMix, and the monoclonal transformants with the correct band size were added to LB liquid culture medium containing spectinomycin for expansion culture. Subsequently, the expanded culture culture fluid was sequenced, and the plasmid was extracted from the monoclonal culture fluid with the correct sequence according to the instructions of the plasmid mini-extraction kit. S2. Acquisition, screening and identification of gene-edited plants: S2-1. Obtaining gene-edited plants: After pre-culturing the Agrobacterium containing the recombinant plasmid with the explants, the positive seedlings after hygromycin selection, differentiation and rooting were defined as T1 transgenic plants; S2-2. Screening and identification of transgenic plants: The genomic DNA of young rapeseed leaves was extracted using a plant genomic DNA extraction kit; first, the T1 transgenic plants were subjected to PCR to identify the presence of the target gene, and the transcription level of the target gene was detected; Then, 10 T1 generation positive seedlings with higher transcription levels are selected for further sowing and planting until all the offspring seedlings of a certain T2 generation offspring are identified to contain the target gene fragment. At this time, the seeds harvested from the offspring of the single plant are T3 generation homozygous seeds.

2. The method for increasing rapeseed oil content by BnaA01.AP2 gene editing according to claim 1, characterized in that: The primers in step S1-3 are shown in the following table:

3. The method for increasing rapeseed oil content by BnaA01.AP2 gene editing according to claim 1, characterized in that: The primers in step S1-2 are shown in the following table:

4. Application of the method for increasing rapeseed oil content by BnaA01.AP2 gene editing according to any one of claims 1 to 3 in creating excellent germplasm resources and cultivating high-yield, high-quality breakthrough rapeseed varieties.

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