Method for creating non-transgenic mutant strain of chinese flowering cabbage

Abstract

The application discloses a method for creating non-transgenic mutant strains of Chinese cabbage. The method for creating non-transgenic mutant strains of Chinese cabbage provided by the application is realized by combining CRISPR / Cas9 technology and Chinese cabbage in situ transformation technology, and specifically comprises the following steps: introducing a CRIPSR / Cas9 gene editing vector carrying an sgRNA coding sequence of a target gene in a Chinese cabbage genome into a receptor Chinese cabbage by a vacuum infiltration in situ transformation method. Two PDS allele knockouts are obtained in 2032 seeds, and the non-transgenic editing mutant strains are obtained by the in situ transformation technology which is independent of tissue culture. The editing efficiency is greatly improved compared with the previously reported in situ transgenic efficiency, and the application provides a good research idea for transgenic safety and marker-free gene editing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for creating a non-transgenic mutant strain of rapeseed. Background Technology

[0002] The safety of genetically modified organisms (GMOs) has always been a major concern due to food safety and gene drift concerns. CRISPR / Cas9-mediated gene editing technology can replace traditional GMO techniques to obtain non-GMO improved materials. Safe non-GMO improved materials can typically be obtained by sexually reproducing GMO offspring and removing exogenous vectors containing Cas9 / sgRNA through marker selection, while retaining the edited mutations. CRISPR / Cas9 technology also offers the possibility of directly obtaining non-GMO mutant strains without the need for offspring segregation. These methods include Agrobacterium-mediated CRISPR / Cas9 transient expression (Chen et al., 2018; Iaffaldano et al., 2016) and Cas9 / gRNA complex (RNP) transfection (Woo et al., 2015; Murovec et al., 2018; Park et al., 2019), but all rely on tissue culture and in vitro regeneration.

[0003] As a major vegetable crop, Chinese cabbage has seen slow progress in transformation technology research, mainly due to the difficulty in explant germination and low transformation efficiency. Agrobacterium-mediated floral-dip transformation is a relatively simple transgenic method that does not rely on tissue culture. The advantages of this method are that it avoids tissue culture and in vitro regeneration, directly obtaining transgenic seeds; it is simple, time-saving, and labor-saving. In recent years, many scholars have used in-situ transformation methods to conduct transgenic research on Chinese cabbage, achieving some encouraging progress. Cao Mingqing (2000) successfully transformed non-heading Chinese cabbage using vacuum infiltration; Yan Jiyong (2003) transformed small Chinese cabbage using microinjection; in addition, Zhang Guanghui (1998), Yan Jiyong (2004), and Yu Zhandong (2007) successfully transformed large Chinese cabbage using vacuum infiltration, but the transformation efficiency was low, mostly around 0.01%. Chinese cabbage (Brassica campestris L.ssp.chinensis) is a conventionally cultivated variety of non-heading Chinese cabbage, with its young flower stalks as the main edible part. It takes about 40-60 days from sowing to harvest, making it a good material for studying in-situ transformation technology of Chinese cabbage.

[0004] While there are reports on genetic transformation of Chinese cabbage and the establishment of CRISPR / Cas9 gene editing systems, there are no reports on creating non-transgenic mutant strains of Chinese cabbage by combining in situ transformation with CRISPR / Cas9 technology. Summary of the Invention

[0005] The purpose of this invention is to provide a method for creating a non-transgenic mutant strain of rapeseed.

[0006] The method for creating non-GMO mutant strains of Chinese cabbage provided by this invention is achieved by combining CRISPR / Cas9 technology with in situ transformation technology of Chinese cabbage.

[0007] Further, the method may include the following steps: introducing a CRIPSR / Cas9 gene editing vector carrying an sgRNA coding sequence targeting a gene in the Chinese cabbage genome into the recipient Chinese cabbage through vacuum infiltration in situ transformation.

[0008] Furthermore, the method may include the following steps:

[0009] (A1) A CRIPSR / Cas9 gene editing vector carrying an sgRNA coding sequence targeting a gene in the Chinese cabbage genome was introduced into Agrobacterium to obtain recombinant Agrobacterium.

[0010] (A2) When the recipient Chinese cabbage enters the initial flowering stage, the flower buds are immersed in the resuspension of the recombinant Agrobacterium and vacuum treated for 10 minutes under a negative pressure of 100-105 Pa (e.g., 104 Pa).

[0011] The process of immersing the flower buds in the recombinant Agrobacterium suspension can be carried out as follows: use tweezers to open the flower buds that are about to open, and bend the branches while immersing the flowers so that the entire inflorescence is immersed in the recombinant Agrobacterium suspension.

[0012] (A3) The target gene knocked out non-transgenic mutant strain can be obtained from the seeds of the recipient Chinese cabbage that have undergone the (A2) flower soaking treatment.

[0013] Before subjecting the recipient cabbage to the flower soaking treatment, the procedure may include thoroughly watering the recipient cabbage the day before.

[0014] The process may further include removing open flowers and siliques before subjecting the recipient cabbage to the flower soaking treatment.

[0015] The OD600 of the resuspension of the recombinant Agrobacterium is 0.05. Specifically, the resuspension of the recombinant Agrobacterium can be prepared by suspending the recombinant Agrobacterium in 1 / 2 MS (containing 5% (i.e., 5 g / 100 mL) sucrose and 0.05% volume percentage Silwet L-77) until OD600 = 0.05.

[0016] In a specific embodiment of the present invention, the Agrobacterium is Agrobacterium tumefaciens, specifically Agrobacterium tumefaciens C58.

[0017] During the process of immersing the flower buds in the suspension of the recombinant Agrobacterium and vacuum treating them under a negative pressure of 100-105 Pa (e.g., 104 Pa) for 10 minutes, with a pause in between (a pause at 5 minutes to allow the pressure to slowly decrease to normal before repressurizing, which is more conducive to the plants being in a state that is receptive to bacterial invasion). Then, turn off the vacuum pump, slowly open the air valve, restore normal pressure, and remove the plants.

[0018] After subjecting the recipient cabbage to the flower-soaking treatment, the following steps may be included: using mature pollen from the untreated recipient cabbage to assist in pollination of the treated recipient cabbage.

[0019] After the assisted pollination is carried out, the following steps may also be included: cover the treated inflorescence with a perforated plastic bag, cover it with a silver-gray film, remove the plastic bag the next day, cover the inflorescence with a sodium sulfate paper bag, and remove the paper bag from the inflorescence at the end of the flowering period.

[0020] In a specific embodiment of the present invention, the CRIPSR / Cas9 gene editing vector is a pHSE401-Bar vector. The pHSE401-Bar vector is obtained by modifying the marker gene Hyg in the pHSE401 vector into the Bar gene.

[0021] In the method described above, the cabbage may be a non-heading cabbage.

[0022] Furthermore, the non-heading cabbage can be the flowering stalk.

[0023] In a specific embodiment of the present invention, the cabbage is specifically the flowering cabbage variety "Sijiu Cabbage Heart".

[0024] In a specific embodiment of the present invention, the target gene is the phytoene dehydrogenase gene.

[0025] Accordingly, the specific recognition sequence (corresponding to the spacer) in the sgRNA coding sequence of the target gene in the Chinese cabbage genome is shown in SEQ ID No. 1. The CRIPSR / Cas9 gene editing vector carrying the sgRNA coding sequence of the target gene in the Chinese cabbage genome is a recombinant vector obtained by replacing the small fragment between the two BsaI sites of the pHSE401-Bar vector with the DNA fragment shown in SEQ ID No. 1. The pHSE401-Bar vector is a vector obtained by modifying the marker gene Hyg in the pHSE401 vector into the Bar gene.

[0026] This invention builds upon and improves upon vacuum-mediated in situ transformation technology in Chinese cabbage. 2032 seeds of the Chinese cabbage 'Sijiu Caixin' variety were obtained, intended to have the phytoene desaturase gene (PDS) knocked out using a CRISPR / Cas9 system (the editing vector carrying the Bar gene). Starting with the sowing of the T0 generation seeds, phenotypic observation, Basta protein strip detection, and PDS gene sequencing were performed. Results showed that two plants exhibiting dwarfism and whitening (PDS gene knockout phenotype) tested negative for Basta protein strips, and PCR amplification did not detect the exogenous vector sequence. However, PDS gene sequencing confirmed a base deletion mutation in the target region, causing premature termination of the PDS gene knockout, and the wild-type PDS gene was not preserved. This successful example of gene editing knockout in Chinese cabbage through in situ transformation demonstrates that while the transformation vector did not integrate into the plant's genomic DNA, transient expression occurred in the host cells, achieving the desired gene editing result. Two PDS allele knockouts were obtained from 2032 seeds, marking the first time that a non-transgenic edited mutant strain was obtained through in situ transformation without tissue culture. The editing efficiency was significantly improved compared to previously reported in situ transgenic efficiency, and it also provides a promising research approach for marker-free gene editing. Attached Figure Description

[0027] Figure 1 Construction of the PDS gene editing knockout vector for *Cabbage Flowering Plant* and identification of in situ transformed plants. a: The first four exon sequences of the 'Sijiu Caixin' PDS gene. Different colored backgrounds represent different exons. The gRNA is located at the end of the first exon, underlined. The three bases in red text represent the PAM region. b: Schematic diagram of the left and right boundaries of the pHSE401-Bar-PDS transformation vector. The gRNA is transcribed by the U6-26 promoter and contains the Bar plant selection marker gene. c: Two of the 2032 seedlings were consistently weak and albino. d: PCR sequencing results of the target region of the PDS gene in the albino seedlings showed sequence diversity variations in the gRNA target region. e: Single-clone sequencing of the PCR products revealed different mutation types in the PDS target region of the two albino seedlings.

[0028] Figure 2 The results are from Basta protein strip tests. a: Basta protein strip sensitivity test: CK1 is a non-transformed normal plantlet, showing a negative reaction; CK2 is a single identified transformed plantlet, showing a positive reaction; 20, 40, and 80 are mixtures of one positive plantlet with 19, 39, and 79 non-transformed plantlets, respectively, all showing positive reactions. b: Partial test results from 2032 seedlings in situ transformed. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] The early-maturing rapeseed variety “Sijiu Caixin” mentioned in the following examples is a product of Hezhiyuan Seed Industry Co., Ltd.

[0032] The pHSE401 vector used in the following examples is an Addgene product, catalog number 62201.

[0033] Example 1: Creation of a non-transgenic mutant strain of Chinese cabbage

[0034] Knockout of the PDS gene (phytopene dehydrogenase gene) can cause plant albinism and is often used as a visual marker in gene editing research. This invention, based on the previous successful acquisition of in situ transformed seedlings of flowering cabbage, conducts research on in situ transformation gene editing technology for flowering cabbage.

[0035] I. Materials and Methods

[0036] 1. Materials

[0037] The recipient material for this invention is the early-maturing rapeseed variety "Sijiu Caixin," native to southern China, with a sowing-to-harvest period of approximately 40-50 days. Sowing is done in flowerpots in a greenhouse. Budding and flowering occur in about 30 days, with flowering plants reaching approximately 50 cm in height, allowing for convenient placement in vacuum-sealed containers.

[0038] Agrobacterium tumefaciens C58.

[0039] The CRIPSR / Cas9 gene editing vector pHSE401-Bar is a vector obtained by modifying the selection marker Hyg in the pHSE401 vector to the Bar gene, which is more suitable for Brassica vegetables. The original pHSE401 vector was digested with EcoRI and AsiSI to recover a large fragment of approximately 15 kb, which was then ligated to the synthesized vector SEQ ID No. 2, which contains EcoRI and AsiSI restriction sites at both ends. In SEQ ID No. 2, positions 126-803 are the 35S promoter, and positions 890-1442 are the Bar gene.

[0040] 2. Method

[0041] (1) Construction of CRIPSR / Cas9 gene editing recombinant vector

[0042] Based on the structure and sequence of the "Sijiu Caixin" PDS gene, a target site was selected on the first exon. The target sequence is as follows:

[0043] 5'-AACGAGAAGAAGCAAGCCT-3' (SEQ ID No. 1).

[0044] Design a pair of complementary nucleotide primers with adapters for the target sequence described above:

[0045] PDS-F: 5'-ATTGAACGAGAAGAAGCAAGCCT-3';

[0046] PDS-R: 5'-AAACAGGCTTGCTTCTTCTCGTT-3'.

[0047] Equal volumes of 100 μmol / L forward and reverse primers were mixed and incubated at 95 °C for 5 minutes, then slowly cooled to room temperature to obtain a double-stranded complementary DNA fragment with BsaI sticky ends. This DNA fragment was then digested and ligated with pHSE401-Bar using BsaI and T4 ligases, and the constructed vector pHSE401-Bar-PDS was sequenced for verification.

[0048] (2) Preparation of Agrobacterium resuspension

[0049] C58 strain carrying the pHSE401-Bar-PDS plasmid was streaked onto YEB medium containing Rif (25 mg / L) and Kana (50 mg / L) to cultivate single colonies. Single colonies were picked and cultured overnight in 20 ml of liquid YEB medium containing Rif (25 mg / L) and Kana (50 mg / L). After the culture became turbid, it was transferred to 200 ml of medium for expansion. When the OD600 reached approximately 1.0, the cells were collected by centrifugation at 4000 rpm for 15 min. The cells were resuspended in 1 / 2 MS (containing 5% (5 g / 100 mL) sucrose and 0.05% Silwet L-77) until the OD600 reached 0.05. The suspension was then poured into a beaker for later use.

[0050] (3) Vacuum infiltration in-situ transformation method

[0051] Once the plants enter the initial flowering stage, with most flower buds still unopened and ready for pollination, vacuum treatment and flower immersion transformation are performed. The day before immersion, the experimental materials are thoroughly watered. Before immersion, open flowers and siliques are removed, and the buds about to open are gently opened with tweezers. During immersion, the branches are bent so that the entire inflorescence is immersed in the Agrobacterium resuspension. Vacuum treatment is performed at 104 Pa negative pressure for 10 minutes, with a 5-minute pause to allow the pressure to slowly decrease to normal. The pressure is then increased again (this is to ensure the plants are in a more receptive state to bacterial invasion). The vacuum pump is then turned off, and the air valve is slowly opened to restore normal pressure before the plants are removed. After immersion, mature pollen from untreated experimental materials is used for pollination. After pollination, the treated inflorescence is covered with a perforated plastic bag and then covered with a silver-gray film. The plastic bag is carefully removed the next day. The inflorescence is then covered with a sodium sulfate paper bag, which is removed at the end of the flowering period. The plants are then properly managed. After the seeds mature, they will be collected for the next step of resistance screening.

[0052] (4) Basta protein test strip detection

[0053] The vector used for in situ transformation carries the Bar gene, which encodes resistance to the herbicide Basta. The BAR rapid test strip (Beijing Aochuang Jinbiao Biotechnology Co., Ltd., A07-13-413) can rapidly detect trace amounts of Basta protein, thus determining whether the Bar gene is expressed in the plant. The specific procedure is as follows: Take fresh, young leaves using a 0.5cm diameter punch, mix 30 leaves per sample, place them in a mortar, add approximately 20mL of clean ddH2O, and grind into a juice. Use the Basta protein test strip to test the liquid; a positive result shows two lines, while a negative result shows one line. If the mixed sample shows a positive reaction, the sample size can be reduced to find the target positive individual plant.

[0054] (5) PDS gene editing sequencing identification

[0055] Homozygous allelic knockout of the PDS gene produces an albino phenotype in plants. DNA was extracted from albino seedlings. The Cas9 region was amplified using primers nCas9-IDF and nCas9-IDR; the gRNA region was amplified using primers gRNA-IDF and gRNA-IDR. The PDS gene target region was amplified using primers PDS1 and PDS356. The PCR products were cloned and sequenced, with 15 clones from each sample sent for analysis.

[0056] nCas9-IDF: 5'-CATACCTCCCAGAACACAAATAAGC-3';

[0057] nCas9-IDR: 5'-ACTGAAGGGCAATAGTGAAGAATGT-3'.

[0058] gRNA-IDF: 5'-TGTCCCAGGATTAGAATGATTAGGC-3';

[0059] gRNA-IDR: 5'-CCCCAGAAATTGAACGCCGAAGAAC-3'.

[0060] PDS1: 5'-ATGCAACTGATCAATGCGGT-3';

[0061] PDS356: 5'-CGGGAATATCGCAGCTAAAG-3'.

[0062] II. Results and Analysis

[0063] 1. Gene editing target selection and vector construction

[0064] Phytochrome dehydrogenase (PDS), a key enzyme in the carotenoid biosynthesis pathway, is essential for chlorophyll synthesis. Therefore, knocking out the PDS gene produces an albino trait in plants, facilitating easy identification. This invention selected BrPDS (Gene ID: 103863556, NCBI database), the PDS gene of Chinese cabbage, as the target site for Cas9 gene editing knockout. A gRNA was selected from the first exon and ligated into the pHSE401-Bar vector for in situ transformation gene editing research in Chinese cabbage.

[0065] The first four exons of the PDS gene in "Sijiu Caixin" are as follows: Figure 1 As shown in Figure a. The recombinant vector, verified by sequencing, was named pHSE401-Bar-PDS. The structural description of this recombinant vector is as follows: the recombinant vector obtained by replacing the small fragment between the two BsaI sites of the pHSE401-Bar vector with the DNA fragment shown in SEQ ID No. 1 (structural diagram shown in Figure a). Figure 1 (As shown in b).

[0066] 2. Identification of PDS gene editing

[0067] In-situ transformation was performed in three batches, totaling 45 plants and yielding over 2000 seeds. After sowing, 2032 seedlings emerged. Phenotypic observation revealed that two seedlings remained albino and weak from emergence onwards (e.g., Figure 1 (As shown in c). DNA was extracted from two albino seedlings, and the target region contained in the PDS was amplified. Sequencing of the PCR products revealed disordered sequence changes in the target region. Figure 1 (d) The PCR products were cloned and subjected to single-clone sequencing analysis. Sequencing results showed that the PDS gene in the two albino seedlings underwent different gene editing and knockout, and no wild-type PDS alleles were preserved. Figure 1(e).

[0068] 3. Basta protein test strip results and analysis

[0069] Based on previous experience with mixed detection using Basta protein test strips, we mixed a confirmed positive transformed seedling (obtained through in vitro transformation) with 19, 39, and 79 non-transformed seedlings, respectively. The mixture was then placed in a mortar and the leaf extract was diluted with 30 mL of clean ddH2O. Test strips in all cases yielded positive results. Figure 2 (a) It is evident that the Basta protein test strips have very high sensitivity. For ease of operation and to ensure accurate results, this experiment used a method of sampling 30 plants at a time, diluting the leaf sap with 20 mL of clean ddH2O, and then testing the samples using Basta protein test strips. The results showed that all protein test strips were negative, and no positive plants were found at the protein level. Figure 2 (b) Simultaneously, amplification of the nCas9 and gRNA regions of the transformation vector using specific primers also failed to detect the target band.

[0070] This experiment, demonstrating the successful in-situ transformation of gene-edited knockout mutants in flowering cabbage, preliminarily indicates that while the transformation vector did not integrate into the plant's genomic DNA, transient expression occurred in the host cells, achieving the desired gene editing result. Two PDS allele knockouts were obtained from 2032 seeds, marking the first time non-transgenic edited mutants have been obtained through in-situ transformation without tissue culture. The editing efficiency is significantly higher than the previously reported efficiency of approximately one in ten thousand for in-situ transgenic methods, providing a valuable research approach for safe transgenic and marker-free gene editing.

[0071] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Beijing Academy of Agricultural and Forestry Sciences <120> A method for creating a non-transgenic mutant strain of rapeseed. <130> GNCLN201398 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial sequence <400> 1 aacgagaaga agcaagcct 19 <210> 2 <211> 1485 <212> DNA <213> Artificial sequence <400> 2 gggaattcgt cggtgtattc cacccaccga cgcgccattt ccgtgtgcat taagataatg 60 ctttccgccc catcggattg ctgttctgac atggtatatc attacgacag ccaatgtttg 120 gcggctcgta cgaatcatca agcggtcaac ctgtgctgtc aaccgtcacg atatgagcaa 180 gcaactcaaa cgctttccta ttcaccgcac gcgcgttgtt ggctgtttgg caggatatat 240 gccaacgtaa aaatgagggc aatcgattgt actgaatcgg attttcaagg gtctggccaa 300 aactattccg tgggcacctg gcacacgccc tggagtccgg cccgtttcca gttgagggtt 360 gtctacgctt agatgagaag gaaagttgtc caagacgaat cccagtgtcc tattaccaat 420 agccgacggt atcgataagc tagagatccg tcaacatggt ggagcacgac acgcttgtct 480 actccaaaaa tatcaaagat acagtctcag aagaccaaag ggcaattgag acttttcaac 540 aaagggtaat atccggaaac ctcctcggat tccattgccc agctatctgt cactttattg 600 tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca tcattgcgat aaaggaaagg 660 ccatcgttga agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga 720 gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata 780 tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta 840 tataaggaag ttcatttcat ttggagagga cagacctgca ggtcgatcca tgagcccaga 900 acgacgcccg gccgacatcc gccgtgccac cgaggcggac atgccggcgg tctgcaccat 960 cgtcaaccac tacatcgaga caagcacggt caacttccgt accgagccgc aggaaccgca 1020 ggagtggacg gacgacctcg tccgtctgcg ggagcgctat ccctggctcg tcgccgaggt 1080 ggacggcgag gtcgccggca tcgcctacgc gggcccctgg aaggcacgca acgcctacga 1140 ctggacggcc gagtcgaccg tgtacgtctc cccccgccac cagcggacgg gactgggctc 1200 cacgctctac acccacctgc tgaagtccct ggaggcacag ggcttcaaga gcgtggtcgc 1260 tgtcatcggg ctgcccaacg acccgagcgt gcgcatgcac gaggcgctcg gatatgcccc 1320 ccgcggcatg ctgcgggcgg ccggcttcaa gcacgggaac tggcatgacg tgggtttctg 1380 gcagctggac ttgagcctgc cggtaccgcc ccgtccggtc ctgcccgtca ccgagatctg 1440 atctcacgcg tctaggatcg acctgcagat cgttcgcgat ggcaa 1485

Claims

1. A method for creating non-transgenic mutant strains of Chinese cabbage, which is achieved by combining CRISPR / Cas9 technology with in situ transformation technology of Chinese cabbage; The method includes the following steps: (A1) A CRIPSR / Cas9 gene editing vector carrying an sgRNA coding sequence targeting a gene in the Chinese cabbage genome was introduced into Agrobacterium to obtain recombinant Agrobacterium; (A2) When the recipient Chinese cabbage enters the initial flowering stage, the flower buds are immersed in the resuspension of the recombinant Agrobacterium and vacuum treated for 10 minutes under a negative pressure of 104 Pa. During the process of immersing flower buds in the suspension of the recombinant Agrobacterium and vacuum treating them under a negative pressure of 104 Pa for 10 minutes, there was one pause in the middle; the pause was at 5 minutes, and the pressure was slowly reduced to normal pressure before being pressurized again. (A3) Obtain non-transgenic mutants with the target gene knocked out from the seeds finally produced by the recipient Chinese cabbage after the (A2) flower soaking treatment; The cabbage in question is a non-heading cabbage; The non-heading Chinese cabbage is the flowering stalk; The flowering stalks mentioned are of the variety "Sijiu Caixin"; The CRIPSR / Cas9 gene editing vector is used to insert the marker gene from the pHSE401 vector. Hyg Transformed into Bar Vectors obtained after gene sequencing; The target gene is the phytopene dehydrogenase gene; The specific recognition sequence in the sgRNA coding sequence of the target gene in the Chinese cabbage genome is shown in SEQ ID No.

1.

2. The method according to claim 1, characterized in that: Before subjecting the recipient cabbage to the flower soaking treatment, the procedure also includes thoroughly watering the recipient cabbage the day before.

3. The method according to claim 1, characterized in that: The procedure also includes removing open flowers and siliques before subjecting the recipient cabbage to the flower soaking treatment.

4. The method according to claim 1, characterized in that: After the recipient cabbage undergoes the flower-soaking treatment, the following steps are also included: taking mature pollen from the untreated recipient cabbage and using it to assist in pollination of the treated recipient cabbage.

5. The method according to claim 4, characterized in that: After the assisted pollination is carried out, the following steps are also included: cover the treated inflorescence with a perforated plastic bag, cover it with a silver-gray film, remove the plastic bag the next day, cover the inflorescence with a sodium sulfate paper bag, and remove the paper bag from the inflorescence at the end of the flowering period.

6. The method according to claim 1, characterized in that: The CRIPSR / Cas9 gene editing vector carrying the sgRNA coding sequence of the target gene in the Chinese cabbage genome is composed of two pHSE401-Bar vectors. Bsa The recombinant vector obtained by replacing the small fragment between sites I with the DNA fragment shown in SEQ ID No. 1, wherein the pHSE401-Bar vector is a marker gene from the pHSE401 vector. Hyg Transformed into Bar The vector obtained after gene generation.

Images