A breeding method of large kernel, high oil type brassica napus

Abstract

The application discloses a breeding method of large-grain and high-oil type Brassica napus, and the disclosed method comprises the following steps: knocking out BnaWIP2 genes in Brassica napus, and obtaining large-grain and high-oil type Brassica napus by mutating the BnaWIP2 genes. By simultaneously knocking out six copies of the BnaWIP2 genes, the application obtains CRISPR / Cas9 gene editing materials of Brassica napus with large grains and high oil, and the gene editing materials provide valuable gene resources and germplasm resources for the breeding of high-oil and high-product Brassica napus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering and biotechnology, and relates to Brassica napus BnaWIP2 Method for site-directed mutagenesis of genes and applications, in particular to a Brassica napus gene editing material based on CRISPR / Cas9. BACKGROUND

[0002] Edible oil is an important strategic material related to the national economy and people's livelihood, and is an important component of food security. Rapeseed is the first oil crop in China, with an annual planting area of about 120 million mu, and is the first source of domestic edible vegetable oil, accounting for more than 55% of the oil yield of domestic oil crops.

[0003] Rapeseed oil yield is mainly determined by seed oil content and yield. Studies have shown that a 1% increase in seed oil content is equivalent to a 2.5% increase in yield (Li Dianrong, Zheng Lei, Li Shaqin. 2014, Analysis of changes in yield per unit and oil content of new winter rapeseed varieties in China in the past thirteen years. Seed, 33(2): 96-100). Seed size and grain weight, as important factors of yield, have the advantages of strong environmental stability and high heritability. Therefore, synergistically improving seed size, grain weight and oil content is an important way to significantly increase rapeseed oil yield.

[0004] The zinc finger protein transcription factor family is one of the largest transcription factor families in plants and plays an important role in plant growth and development and nutrient metabolism (Han G., Li Y., Qian Z., et al., 2021, Advances in the regulation of epidermal cell development by C2H2 Zinc finger proteins in plants. Frontiers in Plant Science, 12: 754512). Arabidopsis AtWIP2 belongs to the C2H2 zinc finger protein transcription factor family. Studies have shown that, AtWIP2 is a gene necessary for the development of the transmitting tract of Arabidopsis carpels, AtWIP2 mutation will seriously affect the development of pollen tubes, the length of mature fruits is nearly 30% shorter than that of wild type, seeds are significantly larger, and the number of seeds is reduced by nearly 60% (Crawford B.C.W., Ditta G., Yanofsky M.F. 2007, The NTT gene is required for transmitting-tract development in carpels of Arabidopsis thaliana. Current Biology, 17(13): 1101-1108; Marsch-Martínez N., Zúñiga-Mayo V.M., Herrera-Ubaldo H., et al., 2014, The NTT transcription factor promotes replum development in Arabidopsis fruits. Plant Journal, 80: 69-81). AtWIP2 gain-of-function mutants and overexpression of AtWIP2 both inhibit the dehiscence of the silique (Chung K.S., Lee J. H., Lee J.S. et al., 2013, Fruit indehiscence caused by enhanced expression of NO TRANSMITTING TRACT in Arabidopsis thaliana . Molecular and Cells, 35: 519-525). AtWIP2 also interacts with the MADS-box transcription factor AtSTK (SEEDSTICK), co-targets KAWAK genes, and regulates the expression of cell wall polysaccharide and lipid deposition-related genes, thus determining the reproductive capacity of the plant (Herrera-Ubaldo H., Lozano-Sotomayor P., Ezquer I. et al., 2019, New roles of NO TRANSMITTING TRACT and SEEDSTICK during medial domain development in Arabidopsis fruits. Development, 146: dev.172395). In addition, it has been found that AtWIP2 with AtWIP4 , AtWIP5 redundantly mediate auxin signaling control of Arabidopsis root tip meristem initiation (Crawford B.C.W., Sewell J., Golembeski G. et al., 2015, Genetic control of distal stem cell fate within root and embryonic meristems. Science, 347: 655-659).

[0005] As an allopolyploid crop, Brassica napus has a complex genome with many homoeologous copies, and gene redundancy and gene additive effects often exist between different homoeologous copies (Schranz, M. E., Lysak, M. A., Mitchell-Olds, T. et al., 2006, The ABC's of comparative genomics in the Brassicaceae: building blocks of crucifer genomes. Trends in Plant Science, 11: 535-542). Traditional hybrid breeding is time-consuming and inefficient. Mutagenic breeding is highly random and uncontrollable, and has few beneficial mutations. SUMMARY

[0006] In view of the defects or deficiencies of the prior art, the present application provides a breeding method of large-grain and high-oil type Brassica napus.

[0007] Therefore, the breeding method of large-grain and high-oil type Brassica napus provided by the present application comprises knocking out BnaWIP2 genes in Brassica napus to obtain large-grain and high-oil type Brassica napus by gene mutation. BnaWIP2

[0008] The BnaWIP2 genes include BnaA04.WIP2 gene, BnaA07.WIP2 gene, BnaA09.WIP2 gene, BnaC04.WIP2 gene, BnaC06.WIP2 gene and BnaC08.WIP2 gene.

[0009] The BnaA04.WIP2 gene sequence is shown in SIQ ID NO. 1, BnaA07.WIP2 the gene sequence is shown in SIQ ID NO. 2, BnaA09.WIP2 the gene sequence is shown in SIQ ID NO. 3, BnaC04.WIP2 the gene sequence is shown in SIQ ID NO. 4, BnaC06.WIP2 the gene sequence is shown in SIQ ID NO. 5, BnaC08.WIP2 the gene sequence is shown in SIQ ID NO. 6.

[0010] Optionally, the CRISPR / Cas9 gene editing technology is used to knock out BnaWIP2 genes in Brassica napus. In a further aspect, the method comprises:

[0011] Step 1, constructing BnaWIP2 gene CRISPR / Cas9 expression vector and containing​BnaWIP2 Agrobacterium strain of CRISPR / Cas9 expression vector;

[0012] Step two, genetic transformation of Brassica napus varieties based on Agrobacterium-mediated method, and multiple gene editing mutant plant strains are obtained through hygromycin resistance screening;

[0013] Step three, screening of plants with large seeds and high seed oil content from multiple gene editing mutant plant strains.

[0014] Alternatively, in step one above, BnaWIP2 The sgRNA sequence of the gene sgR-BnaWIP2 is 5'-TACTCAGTTCTCTTGTCCTG-3'. The upstream primer sgR-BnaWIP2-F of sgR-BnaWIP2 in step one above is 5'-ATTGCAGGACAAGAGAACTGAGTA-3'; the downstream primer sgR-BnaWIP2-R of sgR-BnaWIP2 is 5'-AAACTACTCAGTTCTCTTGTCCTG-3'. In step two above, multiple gene editing mutant plant strains are obtained through hygromycin resistance screening based on Agrobacterium-mediated genetic transformation of Brassica napus varieties;

[0015] The CRISPR / Cas9-based Brassica napus gene editing material of the application utilizes CRISPR / Cas9 gene editing technology to simultaneously knockout BnaWIP2 six copies of the gene, and obtain Brassica napus CRISPR / Cas9 gene editing material with large seeds and high oil. These gene editing materials provide valuable gene resources and germplasm resources for the breeding of Brassica napus high-oil high-yield varieties. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 For BnaWIP2-Cas9 Agarose gel electrophoresis results of gene editing material identification. Const represents BnaWIP2-Cas9 plasmid, which is the positive control of the experiment; Westar represents wild type receptor, which is the negative control of the experiment.

[0017] Figure 2 For BnaWIP2 Related BnaWIP2 Sequencing results of homozygous mutants; wherein, Figure 2 A is BnaWIP2 CRISPR / Cas9 target site diagram of six copies of the gene; Figure 2 B is BnaWIP2 Homozygous mutant BnaWIP2- Cas9#4 Sequencing results of strains in the target site region; Figure 2 C isBnaWIP2 homozygous mutant BnaWIP2-Cas9#5 The sequencing results of the strain in the target site region.

[0018] Figure 3 The relevant BnaWIP2 The analysis results of mature seeds, seed quality, total seed fatty acid content and seed fatty acid component content of the mutant; wherein, Figure 3 A is the Brassica napus variety Westar (wild type) and BnaWIP2 mutant BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5 The photos of mature seeds of the strain; Figure 3 B is the Brassica napus variety Westar (wild type) and BnaWIP2 mutant BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5 The columnar statistical chart of seed quality of the strain; Figure 3 C is the Brassica napus variety Westar (wild type) and BnaWIP2 mutant BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5 The columnar statistical chart of total seed fatty acid content of the strain; Figure 3 D is the Brassica napus variety Westar (wild type) and BnaWIP2 mutant BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5 The columnar statistical chart of seed fatty acid component content of the strain. DETAILED DESCRIPTION

[0019] First of all, it needs to be pointed out that in the following examples, the reagents used are from commercial channels, and the test methods, detection methods, etc. involved, if not specially mentioned, are the conventional test methods, detection methods in the prior art.

[0020] The following gives specific embodiments of the present application, it should be noted that the present application is not limited to the following specific embodiments, any equivalent transformation made on the basis of the technical solutions of the present application falls within the protection scope of the present application.

[0021] In the following examples, Brassica napus uses the Brassica napus variety "Westar" known in the prior art. The reagents and materials used in the following examples are commercially available products.

[0022] In order to analyze the influence of Brassica napus BnaWIP2 gene on seed development and seed oil accumulation, this embodiment uses CRISPR / Cas9 gene editing technology to knockout six copies of Brassica napus BnaWIP2 BnaWIP2 gene BnaA04.WIP2 , BnaA07.WIP2 , BnaA09.WIP2 , BnaC04.WIP2 , BnaC06.WIP2 andBnaC08.WIP2 ), i.e. to obtain a Brassica napus gene editing material with larger seeds and higher fatty acid content.

[0023] CRISPR / Cas9 is a site-directed editing technology using RNA-guided Cas9 protein nuclease to cut target genes at specific sites, and the mutants created by CRISPR / Cas9 can be removed by self-crossing to remove the vector.

[0024] Example 1: Brassica napus BnaWIP2 Construction of CRISPR / Cas9 expression vector

[0025] 1.1 Selection of sgRNA target site and synthesis of upstream and downstream primers

[0026] In previous studies, the inventors used bioinformatics analysis methods to find multiple AtWIP2 homologous genes in Brassica napus variety Westar, which were named BnaA04.WIP2 , BnaA07.WIP2 , BnaA09.WIP2 , BnaC04.WIP2 , BnaC06.WIP2 and BnaC08.WIP2 according to different chromosomal locations. The full-length coding region nucleic acid sequences of the 6 copies of the BnaWIP2 gene in Brassica napus variety Westar are shown as SIQ ID NO. 1-6.

[0027] Based on the structure and homologous relationship of the 6 copies of the BnaWIP2 gene in Brassica napus variety Westar, an sgRNA sequence was designed on the first exon of the BnaWIP2 gene based on the CRISPRdirect website (http: / / crispr.dbcls.jp / ), and the selected sequence was 5'-TACTCAGTTCTCTTGTCCTG-3', as shown in Figure 1 .

[0028] This sgRNA sequence was synthesized into a pair of adapter primers for constructing a CRISPR / Cas9 expression vector.

[0029] According to the sgRNA sequence, adapter primers (named BnaWIP2-sgRNA-F and BnaWIP2-sgRNA-R) as shown in Table 1 were designed, and the first 4 bases of the adapter primers were the sticky ends of AarI restriction endonuclease.

[0030] Table 1 Primer sequence table

[0031]

[0032] 1.2 Obtaining double-stranded gDNA:

[0033] The above two pairs of adapter primers were sent to Shanghai Shengong Bioengineering Co., Ltd. for primer synthesis. The synthesized adapter primers were diluted to 10 µM, and 10 µL of each pair of primers were mixed and added to a PCR tube. The mixture was incubated at 95℃ for 5 min, and double-stranded gDNA was synthesized for subsequent vector construction.

[0034] 1.3 Double-stranded gDNA and CRISPR / Cas9 vector ligation:

[0035] The CRISPR / Cas9 plasmid was digested with AarI restriction endonuclease in a 37℃ constant temperature incubator for 10 hours. The enzyme digestion reaction system is shown in Table 2:

[0036] Table 2 Enzyme digestion reaction system

[0037]

[0038] After the enzyme digestion reaction was completed, the enzyme digestion products were purified according to the OMEGA gel recovery kit, and the product concentration and quality were detected using NanoDrop ND-1000. The linearized CRISPR / Cas9 plasmid after enzyme digestion and purification was added to the PCR tube with the above double-stranded gDNA, and T4 ligation was performed at 16℃ for 5-6 hours. The ligation reaction system is shown in Table 3:

[0039] Table 3 Ligation reaction system

[0040]

[0041] 1.4 Heat shock transformation of E. coli and identification of expression vector

[0042] The T4 ligation product was added to E. coli competent cells DH5α, mixed gently, placed on ice for 20 min, 42℃ water bath for 90 s, quickly placed on ice for 2 min, added with 700 µL of LB culture solution, and placed in a 37℃ shaking incubator for 30 min. The bacterial solution was spread on LB solid medium containing 100 ug / ml spectinomycin, and incubated at 37℃ constant temperature incubator overnight.

[0043] Single colony PCR was performed using the primers Cas9-F and BnaWIP2-sgRNA-R shown in Table 1. The single colony PCR reaction system is shown in Table 4, and the PCR reaction conditions are shown in Table 5:

[0044] Table 4 Single colony PCR reaction system

[0045]

[0046] Table 5 PCR reaction conditions

[0047]

[0048] PCR reaction is over, the product is verified by 1% agarose gel electrophoresis. The PCR detection band size is correct. Three colonies are selected and added to LB liquid medium containing 100 ug / ml spectinomycin, mixed and placed in a 37°C / 220 rpm shaker overnight culture, and single colony liquid is selected for sequencing in Shanghai Shenguo Bioengineering Co., Ltd. The sequencing results are correct. The single colony liquid is extracted according to the OMEGA plasmid extraction kit, and the CRISPR / Cas9 gene editing vector of BnaWIP2 is successfully constructed.

[0049] 1.6 Agrobacterium transformation

[0050] The above successfully constructed BnaWIP2 CRISPR / Cas9 gene editing vector is taken 5 μL and added to Agrobacterium competent cells GV3101, mixed and placed on ice for 30 min, then quickly frozen in liquid nitrogen for 5 min, immediately placed in a 37°C water bath for 5 min, then placed on ice for 3 min, added 700 μL LB culture solution (containing 25 ug / ml rifampicin), placed in a 28°C shaker for 2 hours, then the bacterial liquid was spread on LB solid medium containing 100 ug / ml spectinomycin and 25 ug / ml rifampicin, and placed in a 28°C constant temperature incubator overnight. Single colony PCR was performed using primers Cas9-F and BnaWIP2-sgRNA-R shown in Table 1, and the PCR reaction system is shown in Table 4 and the PCR reaction conditions are shown in Table 5.

[0051] Three positive colonies detected by PCR were selected and added to LB liquid medium containing 100 ug / ml spectinomycin and 25 ug / ml rifampicin, mixed and placed in a 28°C / 220 rpm shaker for shaking culture, and finally the bacterial liquid of the positive strain was stored in 33% glycerol at -80°C.

[0052] Example 2: Brassica napus BnaWIP2 Obtaining and identification of CRISPR / Cas9 gene editing mutants

[0053] The above constructed CRISPR / Cas9 gene editing vector was genetically transformed into Brassica napus based on plant tissue culture combined with Agrobacterium-mediated genetic transformation method, and two hygromycin-resistant CRISPR / Cas9 gene editing mutant Brassica napus lines were obtained.

[0054] The specific procedures for rapeseed genetic transformation and positive seedling identification are as follows: Westar seeds are selected and sterilized. The sterilized rapeseed seeds are inoculated onto MS medium and cultured for 7 days. Then, in a clean bench, sterile seedlings are removed with forceps, and cotyledon petioles and tips are cut off. Hypocotyls are cut into 1-2 cm segments as explants and placed on pre-culture medium for pre-culture. After 3 days, the activated seedlings containing... BnaWIP2 Agrobacterium, containing the CRISPR / Cas9 gene-editing vector, was inoculated into an Agrobacterium suspension, and the OD value was adjusted to approximately 0.2. Pre-cultured rapeseed explants were placed in the Agrobacterium suspension for 10 min, then air-dried on filter paper. The infected explants were then placed on a co-culture medium. Two days later, the explants were transferred to a screening medium and cultured for 7 days. Effective callus tissue was selected and transferred to a selection medium containing hygromycin for screening for approximately 15 days. This selection was repeated 2 to 3 times. Vigorously growing positive callus tissue was transferred to a differentiation medium to differentiate into seedlings. The differentiated seedlings were then transferred to a rooting culture medium for 7-10 days. The rooted seedlings were labeled, and five seedlings were selected on a hygromycin-resistant medium and named... BnaWIP2-Cas9#1~#5 A small amount of young rapeseed leaves were taken, and genomic DNA template was extracted for PCR amplification. Positive seedlings were identified by agarose gel electrophoresis. PCR verification was performed using primers Cas9-F and BnaWIP2-sgRNA-R as shown in Table 1. The PCR reaction system is shown in Table 4, and the PCR reaction conditions are shown in Table 5. The detection results are as follows: Figure 1 As shown, BnaWIP2-Cas9#1~#3 No stripes, but BnaWIP2-Cas9#4~#5 There are stripes, representing BnaWIP2-Cas9#4~#5 It is a positive strain.

[0055] by BnaWIP2-Cas9#4~#5 Using the genomic DNA of the strain as a template, and with the primers BnaX.WIP2-genotyping-F and BnaX.WIP2-genotyping-R (X represents A04, A07, A09, C04, C06, and C08) shown in Table 1, amplification was performed on... BnaWIP2 Six copies of the gene were amplified by PCR. The amplification products were excised and purified using a gel, and their quality and concentration were measured. The samples were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to detect the specific components of each CRISPR / Cas9 gene-editing mutant. BnaWIP2 Editing status of the copy.

[0056] BnaWIP2-Cas9#4 The sequencing results of the strain are as follows Figure 2 As shown in B, the editing sites for all six copies are located in the first exon, where: BnaA04.WIP2 The absence of 7 bases BnaA07.WIP2 Insert two bases, BnaA09.WIP2 20 bases missingBnaC04.WIP2 deletion of 7 bases, BnaC06.WIP2 deletion of 4 bases, BnaC08.WIP2 deletion of 7 bases.

[0057] BnaWIP2-Cas9#5 The sequencing results of the strains are shown in FIG. 6C, and the edited sites of the 6 copies are all on the first exon, wherein, Figure 2 deletion of 7 bases, BnaA04.WIP2 deletion of 7 bases, BnaA07.WIP2 insertion of 2 bases, BnaA09.WIP2 insertion of 1 base, BnaC04.WIP2 deletion of 5 bases, BnaC06.WIP2 deletion of 4 bases, BnaC08.WIP2 deletion of 5 bases.

[0058] Example 3: Verification of Brassica napus BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5 Effects of the mutants on seed development and seed oil accumulation

[0059] The detection method of total fatty acid content and each component fatty acid content in the seeds in this example is as follows: first, about 15 mg of rapeseed powder is weighed into a glass test tube, 4 mL of 2.5% sulfuric acid methanol extract solution (seventeen-methyl acid methyl ester (Cat No. CDAA-251017M, Anpel) with a final concentration of 25 pg / mL is added as an internal standard in advance) is added, the cap is tightly screwed, and the glass test tube is placed in a constant temperature water bath at 80°C for 2 h for esterification reaction. After the esterification reaction is completed, the glass test tube is taken out and cooled to room temperature, 2 mL of 0.9% NaCl (w / v) is added to terminate the esterification reaction, then 2 mL of n-hexane is added, vortexed for 40 s, and then centrifuged at 1500 rpm for 5 min, and then 800 pL of the upper organic phase is taken and transferred to a clean GC vial, and each fatty acid component is analyzed by a GC-2010 plus (Shimadzu, Japan) gas chromatograph. The chromatographic column (Supelcowax-10, Cat. No. 24079, Supelco) of the GC-2010 plus gas chromatograph is 30 m (length) x 0.25 mm (internal diameter) x 0.5 pm (liquid membrane thickness). The temperature program of the chromatographic analysis is set as follows: the initial temperature is 160°C and is maintained for 1 min, then is raised to 240°C at a rate of 4°C / min and is maintained for 16 min. After the chromatographic analysis is completed, each fatty acid component is determined according to the peak time, and each fatty acid component in the sample is uniformly processed by the internal standard.

[0060] The BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5After self-pollination to homozygosity, the Brassica napus variety Westar (wild type) and the BnaWIP2 mutant were compared. BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5 Seed development of the strain was observed. BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5 The mutant seeds are larger and heavier than the wild type. Figure 3 A, Figure 3 B). Further comparison of the Brassica napus variety Westar (wild type) with... BnaWIP2 mutant BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5 The total fatty acid content of the seeds of the strain was found to be... BnaWIP2-Cas9#4 and BnaWIP2-Cas9#5 The total fatty acid content in the seeds of the strain was significantly higher than that of the wild type. Figure 3 C). Analysis of the fatty acid content of each component revealed... BnaWIP2- Cas9#4 and BnaWIP2-Cas9#5 The content of C16:0, C18:0, C18:2, and C18:3 in the seeds of the strain was higher than that of the wild type, while the content of C18:1 was lower than that of the wild type. Figure 3 (D) This indicates that BnaWIP2 also has the function of regulating changes in fatty acid composition.

[0061] Based on the above embodiments, the present invention utilizes CRISPR / Cas9 gene editing technology to mutate... BnaWIP2 Large-particle, high-oil mutants obtained from the gene can be used as high-oil materials for Brassica napus after vector isolation.

[0062] Instruction manual nucleotide sequence

[0063] SIQ ID NO.1:

[0064] BnaA04.WIP2

[0065]

[0066] SIQ ID NO.2:

[0067] BnaA07.WIP2

[0068]

[0069] SIQ ID NO.3:

[0070] BnaA09.WIP2

[0071]

[0072] SIQ ID NO.4:

[0073] BnaC04.WIP2

[0074]

[0075] SIQ ID NO.5:

[0076] BnaC06.WIP2

[0077]

[0078] SIQ ID NO.6:

[0079] BnaC08.WIP2

[0080]

Claims

1. A breeding method of Brassica napus L. of large seed size and high total fatty acid content, characterized by, The method comprises obtaining a high-seed-size and high total fatty acid content Brassica napus by knocking out the gene in Brassica napus BnaWIP2 BnaWIP2 gene mutation​ The BnaWIP2 genes include BnaA04.WIP2 genes, BnaA07.WIP2 genes, BnaA09.WIP2 genes, BnaC04.WIP2 genes, BnaC06.WIP2 genes and BnaC08.WIP2 genes; The BnaA04.WIP2 the gene sequence is shown as SIQ ID NO. 1, BnaA07.WIP2 the gene sequence is shown as SIQ ID NO. 2, BnaA09.WIP2 the gene sequence is shown as SIQ ID NO. 3, BnaC04.WIP2 the gene sequence is shown as SIQ ID NO. 4, BnaC06.WIP2 the gene sequence is shown as SIQ ID NO. 5, BnaC08.WIP2 the gene sequence is shown as SIQ ID NO.

6.

2. The breeding method of Brassica napus of large seed grain, high total fatty acid content type according to claim 1, characterized by, Knocking out genes in Brassica napus using CRISPR / Cas9 gene editing technology BnaWIP2 genes.

3. The breeding method of the large-seeded, high total fatty acid content type Brassica napus according to claim 2, characterized by the method comprising: Step one, construction BnaWIP2 Gene CRISPR / Cas9 expression vector and Agrobacterium strain containing BnaWIP2 Gene CRISPR / Cas9 expression vector and Agrobacterium strain containing Step two, genetic transformation of Brassica napus varieties based on Agrobacterium-mediated method to obtain multiple gene editing mutant plants; Step three, screening of plants with large seeds and increased total seed fatty acid content from multiple gene editing mutant plants.

4. The breeding method of the large-seeded, high total fatty acid content type Brassica napus according to claim 3, characterized by, In step one BnaWIP2 The sgRNA sequence of the gene sgR-BnaWIP2 is 5'-TACTCAGTTCTCTTGTCCTG-3'.

5. The breeding method of the large-seeded, high total fatty acid content type Brassica napus according to claim 4, characterized by, The upstream primer sgR-BnaWIP2 of sgR-BnaWIP2 in step one is 5'-ATTGCAGGACAAGAGAACTGAGTA-3'; The downstream primer sgR-BnaWIP2 of sgR-BnaWIP2 is 5'-AAACTACTCAGTTCTCTTGTCCTG-3'.

6. The breeding method of the large-seeded, high total fatty acid content type Brassica napus according to claim 4, characterized by, Step two, genetic transformation of Brassica napus varieties based on Agrobacterium-mediated method to obtain multiple gene editing mutant plants.

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