Method for improving brassica napus drought resistance through BnJAM3 gene editing and application

Abstract

The invention relates to the technical field of plant genetic engineering application, in particular to a method for improving brassica napus drought resistance through BnJAM3 gene editing and application, all four copies of BnJAM3 in a brassica napus selfing line 'K407' are knocked out through CRISPR / Cas9 mediated gene editing, and a homozygous four-mutant J2-4 is obtained. Phenotypic analysis shows that compared with a wild type K407, the content of endogenous jasmonic acid in a J2-4 plant is remarkably increased. After drought stress and after-stress rehydration treatment, the survival rate of the J2-4 is obviously higher than that of the wild type K407. Meanwhile, the active oxygen scavenging capacity of the J2-4 mutant is also remarkably enhanced. In conclusion, the J2-4 four mutants of the BnJAM3 enhance the drought resistance of the plant by increasing the content of endogenous jasmonic acid. The rape BnJAM3 gene disclosed by the invention provides a new excellent gene resource for molecular design breeding, and has a good application prospect in the aspect of stress resistance 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 improving the drought resistance of Brassica napus through BnJAM3 gene editing. Background Art

[0002] Rapeseed 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 Brassicarapa L., AA, 2n=2x=20, Brassicanapus L., AACC, 2n=2x=38 and Brassicajuncea L., AABB, 2n=2x=36. Brassica rapeseed 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 China in the 1830s. Due to its advantages in seed yield and high oil content, it gradually replaced the production status of Brassica rapeseed and Brassica juncea and became the main cultivated 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] Rapeseed requires a lot of water during its growth and development. Drought at any stage during its growth period will lead to abnormal growth and development, short plants, and reduced yields. Shaanxi Province is located in the arid northwest region of my country. It is a major province for oilseed crop cultivation in my country and a major production area for rapeseed seed production in China. Therefore, screening or creating drought-resistant germplasm, exploring key genes for rapeseed to respond to drought stress, and cultivating new high-yield, high-quality, and drought-resistant rapeseed varieties are of great significance for promoting the innovative development of the oil crop industry in arid areas, ensuring the safety of edible oil in arid areas and even the whole country, and realizing agricultural modernization.

[0004] JAM3 is a basic helix-loop-helix transcription factor (bHLH transcription factor). Studies have found that bHLH transcription factor subfamily IIId members bHLH3, bHLH13, bHLH14 and bHLH17 play a negative regulatory role in the jasmonic acid signal transduction pathway as new targets of JAZ. Subfamily IIId members have redundant functions in negatively regulating JA responses. The quadruple mutants bhlh3bhlh13 bhlh14 bhlh17 show high sensitivity to JA-inhibited root growth and JA-induced anthocyanin accumulation, and show significantly enhanced JA-regulated plant defense against pathogen infection and insect pests. Subfamily IIId members also act downstream of the jasmonic acid signal receptor protein COI1, inhibiting different JA responses by antagonizing different transcriptional activators. Although researchers have isolated BnJAM3 genes from a variety of plants, the function of BnJAM3 in the plant stress response process in Brassica napus is still unclear. Therefore, exploring the function of BnJAM3 in Brassica napus in plant response to abiotic stress is of great significance for analyzing the molecular regulatory mechanism of plant response to drought stress and breeding new highly resistant rapeseed varieties.

[0005] To this end, the present application provides a method and application for improving the drought resistance of Brassica napus through BnJAM3 gene editing to solve the problems of the prior art. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application for improving the drought resistance of Brassica napus by BnJAM3 gene editing, so as to provide valuable gene resources and theoretical basis for rapeseed breeding.

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

[0008] On the one hand, the scheme of the present invention provides a method for improving the drought resistance of Brassica napus by editing the BnJAM3 gene. The method is based on the nucleic acid sequence characteristics of four copies of the BnJAM3 gene in the inbred line 'K407', designs a sgRNA sequence, clones it into a CRISPR / Cas9 vector, and introduces the vector into the inbred line 'K407' based on the Agrobacterium-mediated hypocotyl genetic transformation method, specifically comprising the following steps:

[0009] S1. Acquisition and vector construction of Brassica napus BnJAM3 gene:

[0010] S1-1. Acquisition of Brassica napus BnJAM3 gene:

[0011] Firstly, we searched the BnIR database using the search tool box and found that there were four copies of the BnJAM3 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.JAM3, BnaA03.JAM3, BnaC01.JAM3 and BnaC07.JAM3 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 the 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 BnJAM3 gene in the genome of the inbred line 'K407' also had four copies, namely: BnaA01.JAM3, BnaA03.JAM3, BnaC01.JAM3 and BnaC07.JAM3 encoded by the genome of the inbred line 'K407' had the same CDS sequences as these copies encoded by the genome of the 'Zhongshuang 11';

[0013] S1-2. Construction of CRISPR / Cas9 vector:

[0014] Based on the nucleic acid sequence characteristics of the four copies of the BnJAM3 gene in the inbred line ‘K407’, the sgRNA sequences were designed using the CRISPRdirect website;

[0015] For the primers targeting BnJAM3, 10 μL of forward primer and 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, T4 DNA Ligase was used to perform T4 ligation between the double-stranded gDNA product and the CRISPR / Cas9 vector linearized by AarI restriction endonuclease to construct the BnJAM3–Cas9 gene editing vector.

[0016] S2. Acquisition, screening and identification of gene-edited plants:

[0017] S2-1. Obtaining gene-edited plants:

[0018] After pre-culturing Agrobacterium containing the recombinant plasmid with the explants, the positive seedlings after spectinomycin selection, differentiation and rooting were defined as T1 transgenic plants;

[0019] S2-2. Screening and identification of transgenic plants:

[0020] The genomic DNA of young rapeseed leaves was extracted using a plant genomic DNA extraction kit;

[0021] First, the T1 transgenic plants were identified by PCR for the presence of the target gene and the transcription level of the target gene was detected;

[0022] Then, six T1-generation positive plants with higher transcription levels are selected and continued to be sown and planted 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.

[0023] Specific primers for each copy of the BnJAM3 gene were used to amplify each copy in the T1, T2, and T3 generation gene-edited mutant strains;

[0024] The PCR products were then sequenced to detect the editing status of each copy in the BnJAM3 gene editing mutant:

[0025] If the sequencing peak graph of the PCR amplification product is normal, without double peaks or overlapping peaks, it means that the gene in the amplification product is homozygous for editing;

[0026] If the sequencing peak graph of the PCR amplification product shows double peaks, 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 the cloned products are transformed into E. coli competent cells DH5α. Six monoclonal transformants are randomly selected for sequencing to determine the specific editing status of each copy of the BnJAM3 gene in each gene editing mutation.

[0027] By identifying the CRISPR / Cas9 vector in the T3 generation population, it was found that there were T3 generation homozygous individuals without CRISPR / Cas9 vectors in the T3 generation population. The copies of the BnJAM3 gene in these homozygous individuals were amplified and sequenced, and finally a homozygous quadruple mutant of the BnJAM3 gene was identified.

[0028] Furthermore, the primers and sequences used in step S1-2 are shown in the table:

[0029]

[0030] Furthermore, the BnJAM3-Cas9 identification primers and sequences in step S2-2 are shown in the following table:

[0031]

[0032] On the other hand, the present invention provides a method for improving the drought resistance of Brassica napus by BnJAM3 gene editing and its application in the stress resistance breeding of oil crops.

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

[0034] In the scheme of the present invention, all four copies of BnJAM3 in the Brassica napus inbred line 'K407' were knocked out by CRISPR / Cas9-mediated gene editing to obtain the homozygous quadruple mutant J2-4; phenotypic analysis showed that the endogenous jasmonic acid content of J2-4 plants was significantly increased compared with the wild type 'K407'; after drought stress and post-stress rehydration treatment, the survival rate of J2-4 was significantly higher than that of the wild type 'K407'; at the same time, the active oxygen scavenging ability of the J2-4 mutant was also significantly enhanced; in summary, it was shown that the J2-4 quadruple mutant of BnJAM3 enhanced the drought resistance of the plant by increasing the endogenous jasmonic acid content.

[0035] The rapeseed BnJAM3 gene provided by the scheme of the present invention provides a new excellent gene resource for molecular design breeding and has good application prospects in the stress resistance breeding of oil crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The BnJAM3 gene of Brassica napus is obtained. The Brassica napus genome encodes four BnJAM3 homologous genes (A is a schematic diagram of the chromosomal location of the BnJAM3 gene in the Brassica napus 'Zhongshuang11' genome, the chromosomal location data is obtained from the BnIR database, and the map is drawn using Mapchart software; B is a comparison of the amino acid sequence identity of JAM3 in Arabidopsis thaliana and the Brassica napus inbred line 'K407', and the sequence identity is expressed as a percentage);

[0037] Figure 2 Creation of gene editing materials (A is a schematic diagram of the gene structure, sequence-specific primers and CRISPR / Cas9 target sites of four copies of the BnJAM3 gene, the blue horizontal line indicates the protospacer sequence adjacent to the motif, the black horizontal line indicates the base sequence of the sgRNA, and the blue letters indicate the single nucleotide polymorphism of the target site; B is the nucleotide sequence alignment of the target site in the BnJAM3 T3 generation homozygous quadruple mutant and inbred line. The red letters are the inserted bases);

[0038] Figure 3 Determination of endogenous jasmonic acid content for the inbred line 'K407' and the homozygous gene-edited quadruple mutant J2-4;

[0039] Figure 4Evaluation of drought resistance of inbred line 'K407' and quadruple mutant J2-4 (A is leaf phenotypic analysis of inbred line 'K407' and quadruple mutant J2-4 under drought stress. Control, control group phenotype; Drought, drought treatment group phenotype; Re-water, treatment group phenotype after 3 days of rewatering, scale = 5 cm; B is survival rate after rewatering; C is DAB staining (upper) and NBT staining (lower) observation, scale = 1 cm; D is hydrogen peroxide content determination; E is superoxide dismutase activity determination; F is peroxidase activity determination, all index measurement values ​​are mean ± standard deviation (n = 3), one-way ANOVA multiple comparison was used, * indicates significant difference at p < 0.05 level). DETAILED DESCRIPTION

[0040] 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.

[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] Embodiment: The embodiment of the present invention provides a scheme for improving the drought resistance of Brassica napus based on the rape transcription factor BnJAM3. Based on the nucleic acid sequence characteristics of BnJAM3 in the inbred line 'K407', the present invention designs an sgRNA sequence, clones it into the CRISPR / Cas9 vector and introduces it into 'K407' through Agrobacterium-mediated genetic transformation, and knocks out all four copies of BnJAM3. T1 generation positive transgenic plants were obtained by hygromycin resistance screening. Further, sequence-specific primers were used to clone the target gene in the above-mentioned T1 generation transgenic plants, and the amplified products were Sanger sequenced to observe their editing. After three generations of screening and identification, a homozygous quadruple mutant J2-4 without a CRISPR / Cas9 vector was finally obtained in the T3 generation. Phenotypic analysis of J2-4 showed that the endogenous jasmonic acid content of J2-4 plants was significantly increased compared with the wild type 'K407'. After drought stress and post-stress rehydration, the survival rate of J2-4 was significantly higher than that of the wild type 'K407'. At the same time, the reactive oxygen scavenging ability of the J2-4 mutant was also significantly enhanced. Therefore, we found that by simultaneously targeting and editing the four copies of BnJAM3, we can obtain rapeseed materials with strong drought resistance.

[0043] The following is a specific implementation of the embodiment of the present invention:

[0044] 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 are all conventional test methods and detection methods in the prior art unless otherwise specified.

[0045] 1. Acquisition and vector construction of BnJAM3 gene of Brassica napus

[0046] 1.1. Acquisition of the BnJAM3 gene from Brassica napus

[0047] In order to identify the copy number of the BnJAM3 gene in the genome of the inbred line 'K407' of Brassica napus, the search tool box of the BnIR database was first used to search, and it was found that there were four copies of the BnJAM3 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.JAM3, BnaA03.JAM3, BnaC01.JAM3 and BnaC07.JAM3. Then, according to the nucleic acid sequences of these four copies in the genome of 'Zhongshuang 11', sequence-specific primers of 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 BnJAM3 gene in the genome of the inbred line 'K407'. The BnaA01.JAM3, BnaA03.JAM3, BnaC01.JAM3 and BnaC07.JAM3 encoded by the genome of the inbred line 'K407' have the same CDS sequences as these copies encoded by the 'Zhongshuang11' genome.

[0048] 1.2. Construction of CRISPR / Cas9 vector

[0049] In order to further study the biological functions of each copy of BnJAM3, based on the nucleic acid sequence characteristics of the four copies of the BnJAM3 gene in the inbred line 'K407', the sgRNA sequence was designed using the CRISPRdirect website. The primers used are shown in Table 1:

[0050] Table 1 Primer information for BnJAM3-Cas9 vector construction

[0051]

[0052] For the primers targeting the target BnJAM3, 10 μL of forward primer and 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, 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 BnJAM3-Cas9 gene editing vector.

[0053] 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.).

[0054] 2. Acquisition, screening and identification of gene-edited plants

[0055] 2.1. Obtaining gene-edited plants

[0056] The receptor materials used for rapeseed transformation were all Brassica napus inbred line 'K407', and the BnJAM3 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 spectinomycin selection, differentiation and rooting were defined as T1 transgenic plants.

[0057] 2.2 Screening and identification of transgenic plants

[0058] 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 of 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, 6 T1 positive plants 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 offspring single plant. At this time, the seeds harvested from the offspring of the single plant were T3 homozygous seeds, which can be used for subsequent experiments. At the same time, the specific primers for each copy of the BnJAM3 gene were used to amplify each copy in the T1, T2 and T3 gene editing mutant single plants. The PCR product was then sent directly to Shanghai Sangon Biotechnology Co., Ltd. (Xi'an, China) for sequencing to detect the editing of each copy in the BnJAM3 gene 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 6 monoclonal transformants are randomly selected and sent to Shanghai Sangon Biotechnology Co., Ltd. (Xi'an, China) for sequencing to determine the specific editing of each copy of the BnJAM3 gene 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 individuals without CRISPR / Cas9 vectors in the T3 generation population. The copies of the BnJAM3 gene in these homozygous individuals were amplified and sequenced, and finally a homozygous quadruple mutant of the BnJAM3 gene was identified.

[0059] Table 2BnJAM3-Cas9 identification primer information

[0060]

[0061] The planting method of all rapeseed materials in the present invention is as follows: first, about 30 rapeseed seeds of each genotype are taken and sown in plug trays with nutrient soil (nutrient soil: vermiculite = 4:1) as the matrix, and placed in an artificial climate culture room until two cotyledons are fully developed. The light cycle of the artificial climate culture room is set to 16h light / 8h dark, and the light intensity is set to 300-400μmol·m -2 ·s -1The 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.

[0062] 3. Effect of BnJAM3 on endogenous jasmonic acid content

[0063] The endogenous jasmonic acid content of the Brassica napus inbred line 'K407' and the homozygous quadruple mutant J2-4 was measured (Suzhou Mengxi Biotechnology Co., Ltd., high performance liquid chromatography). The results showed that compared with 'K407', the endogenous jasmonic acid content of the quadruple mutant J2-4 was significantly increased, indicating that BnJAM3 negatively regulates endogenous jasmonic acid synthesis.

[0064] 4. Effect of BnJAM3 on drought resistance of rapeseed

[0065] The drought resistance of the above-mentioned Brassica napus inbred line 'K407' and quadruple mutant J2-4 was comprehensively analyzed. The results showed that after 10 days of drought stress and 3 days of rewatering, the quadruple mutant J2-4 showed stronger drought tolerance. The survival rate of the inbred line 'K407' was 90.48%, while the survival rate of the quadruple mutant J2-4 reached 100%, which was significantly higher than that of K407. In order to explore the differences in antioxidant enzyme activity of materials under drought conditions, we took true leaves at 10 days of drought stress as samples to detect reactive oxygen and antioxidant enzyme activity in the leaves. The results showed that after drought stress, rapeseed leaves rapidly accumulated reactive oxygen, and the content of superoxide anion radicals and hydrogen peroxide in the leaves of the quadruple mutant J2-4 was lower than that of the inbred line 'K407'; while the peroxidase activity in the leaves of the quadruple mutant J2-4 was significantly higher than that of the inbred line 'K407'. These results indicate that under water-deficit conditions, the drought resistance of the quadruple mutant J2-4 is stronger than that of the inbred line 'K407'. The physiological mechanism may be that the quadruple mutant J2-4 rapidly produces more superoxide dismutase and peroxidase, which promotes the removal of intracellular reactive oxygen species and maintains the homeostasis of reactive oxygen species in the body.

[0066] 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 improving drought resistance of Brassica napus by BnJAM3 gene editing, characterized in that: The method is to design a sgRNA sequence based on the nucleic acid sequence characteristics of four copies of the BnJAM3 gene in the inbred line 'K407', clone it into a CRISPR / Cas9 vector, and introduce the vector into the inbred line 'K407' based on the Agrobacterium-mediated hypocotyl genetic transformation method, specifically comprising the following steps: S1. Acquisition and vector construction of Brassica napus BnJAM3 gene: S1-1. Acquisition of Brassica napus BnJAM3 gene: Firstly, we searched the BnIR database using the search tool box and found that there were four copies of the BnJAM3 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.JAM3, BnaA03.JAM3, BnaC01.JAM3 and BnaC07.JAM3 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 the 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 BnJAM3 gene in the genome of the inbred line 'K407' also had four copies, namely: BnaA01.JAM3, BnaA03.JAM3, BnaC01.JAM3 and BnaC07.JAM3 encoded by the genome of the inbred line 'K407' had the same CDS sequences as these copies encoded by the genome of the 'Zhongshuang 11'; S1-2. Construction of CRISPR / Cas9 vector: Based on the nucleic acid sequence characteristics of the four copies of the BnJAM3 gene in the inbred line ‘K407’, the sgRNA sequences were designed using the CRISPRdirect website; For the primers targeting BnJAM3, 10 μL of forward primer and 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, T4 DNA Ligase was used to perform T4 ligation between the double-stranded gDNA product and the CRISPR / Cas9 vector linearized by AarI restriction endonuclease to construct the BnJAM3–Cas9 gene editing vector. S2. Acquisition, screening and identification of gene-edited plants: S2-1. Obtaining gene-edited plants: After pre-culturing Agrobacterium containing the recombinant plasmid with the explants, the positive seedlings after spectinomycin 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 identified by PCR for the presence of the target gene and the transcription level of the target gene was detected; Then, six T1-generation positive plants with higher transcription levels are selected and continued to be sown and planted 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. Specific primers for each copy of the BnJAM3 gene were used to amplify each copy in the T1, T2, and T3 generation gene-edited mutant strains; The PCR products were then sequenced to detect the editing status of each copy in the BnJAM3 gene editing mutant: If the sequencing peak graph of the PCR amplification product is normal, without double peaks or overlapping peaks, it means that the gene in the amplification product is homozygous for editing; If the sequencing peak graph of the PCR amplification product shows double peaks, 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 the cloned products are transformed into E. coli competent cells DH5α. Six monoclonal transformants are randomly selected for sequencing to determine the specific editing status of each copy of the BnJAM3 gene in each gene editing mutation. By identifying the CRISPR / Cas9 vector in the T3 generation population, it was found that there were T3 generation homozygous individuals without CRISPR / Cas9 vectors in the T3 generation population. The copies of the BnJAM3 gene in these homozygous individuals were amplified and sequenced, and finally a homozygous quadruple mutant of the BnJAM3 gene was identified.

2. The method for improving drought resistance of Brassica napus by BnJAM3 gene editing according to claim 1, characterized in that: The primers and sequences used in step S1-2 are shown in the table:

3. The method for improving drought resistance of Brassica napus by BnJAM3 gene editing according to claim 1, characterized in that: The BnJAM3-Cas9 identification primers and sequences in step S2-2 are shown in the following table:

4. Application of the method for improving drought resistance of Brassica napus by BnJAM3 gene editing according to any one of claims 1 to 3 in stress resistance breeding of oilseed crops.

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