Application of brassica napus BnaA09.ADPG1 gene in improvement of brassica napus silique crack resistance and sgRNA

Abstract

The invention relates to the technical field of plant genetic engineering, and particularly discloses application of an oilseed rape BnaA09.ADPG1 gene in improving the cracking resistance of oilseed rape siliques and sgRNA. The nucleotide sequence of the BnaA09.ADPG1 gene is as shown in SEQ ID NO.1, the CDS sequence of the BnaA09.ADPG1 gene is as shown in SEQ ID NO.2, and the amino acid sequence of encoded protein is as shown in SEQ ID NO.3; the BnaA09.ADPG1 gene and the homologous gene thereof are enabled to lose functions through mutation, so that the cracking resistance of the rape silique is improved. The pod-shattering key gene BnaA09.ADPG1 provided by the invention is mutated by utilizing a CRISPR / Cas9 gene editing technology, so that the pod-shattering resistance of the rape can be improved, and other agronomic characters are not influenced, so that the pod-shattering key gene BnaA09.ADPG1 is used for creating new pod-shattering-resistant rape germplasm resources and rape pod-shattering-resistant breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to rapeseed BnaA09.ADPG1 Application of genes and sgRNA in improving the crack resistance of rapeseed siliques. Background Art

[0002] At present, a prominent problem in the mechanized harvesting of rapeseed is that most existing varieties have poor resistance to pod cracking, resulting in serious grain falling during mechanical harvesting, which can generally cause 8-15% yield loss, and even higher under adverse weather conditions. At present, due to concerns about grain falling losses, mechanical combined harvesting is generally carried out in the yellow maturity stage, but the moisture content of the harvested seeds is high at this time, and they need to be dried after harvesting, and the entrainment loss rate is high if they are not properly controlled. If mechanical harvesting is delayed until the dry maturity stage, pod cracking will lead to serious grain falling. Cultivating rapeseed varieties with strong resistance to pod cracking and suitable for mechanical harvesting is the fundamental way to solve this problem. Due to the narrow genetic background, very few pod cracking-resistant rapeseeds have been identified in rapeseed germplasm resources at home and abroad at this stage, making it difficult for breeders to improve the crack resistance of rapeseed pods through conventional breeding methods. Therefore, it is the key technical support to realize the mechanized production of rapeseed to discover key genes associated with the pod cracking trait of rapeseed from known databases, create pod cracking-resistant rapeseed germplasm resources, and then cultivate rapeseed varieties suitable for mechanized harvesting. Summary of the invention

[0003] In order to develop the function of existing genes in regulating the pod-breaking trait of rapeseed, the present invention provides a BnaA09.ADPG1 Application of genes in improving the crack resistance of rapeseed siliques and sgRNA. The key gene for cracking siliques provided by the present invention BnaA09.ADPG1 Mutation using CRISPR / Cas9 gene editing technology can improve rapeseed's resistance to pod shattering without affecting other agronomic traits, thereby being used to create new pod shatter-resistant rapeseed germplasm resources and rapeseed pod shatter-resistant breeding.

[0004] The present invention provides rapeseed BnaA09.ADPG1 Application of a gene in improving the crack resistance of rapeseed siliques, the BnaA09.ADPG1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3; BnaA09.ADPG1 The gene and its homologous genes lose their function, improving the crack resistance of rapeseed siliques.

[0005] The present invention uses a genetic engineering method to make rapeseed BnaA09.ADPG1 The gene mutation loses its function, thus improving the resistance of rapeseed to pod cracking. It can be used to create new rapeseed germplasm resources with resistance to pod cracking and to breed rapeseed with resistance to pod cracking.

[0006] Furthermore, CRISPR-Cas9 gene editing technology makesBnaA09.ADPG1 The gene undergoes mutation.

[0007] Furthermore, the CRISPR-Cas9 gene editing technology includes designing sgRNA such that BnaA09.ADPG1 the gene undergoes mutation.

[0008] Furthermore, the sgRNA contains target site 2 shown in SEQ ID NO.5 and target site 3 shown in SEQ ID NO.6.

[0009] The present invention also provides an sgRNA, which contains target site 2 shown in SEQ ID NO.5 and target site 3 shown in SEQ ID NO.6.

[0010] The present invention also provides a gene editing vector, which contains the above-mentioned sgRNA.

[0011] The present invention also provides a recombinant cell, which contains the above-mentioned gene editing vector.

[0012] The present invention also provides a method for improving the silique shattering resistance of rapeseed, which includes the following steps: Design an sgRNA target site for mutating the rapeseed BnaA09.ADPG1 gene, ligate the target site fragment with the gene editing vector and transform competent cells, and then infect, co-culture, screen, differentiate and root culture to obtain a rapeseed transgenic line with silique shattering resistance.

[0013] Furthermore, the steps of infecting, co-culturing, screening, differentiating and root culturing to obtain a rapeseed transgenic line with silique shattering resistance are as follows: Infect the hypocotyls of rapeseed with the Agrobacterium liquid containing the gene editing vector, screen and perform differentiation culture, cut the regenerated seedlings from the callus for root culture, transplant them after the roots grow out to obtain T0 generation transgenic seedlings, bag and self-cross the individual plants of T0 generation transgenic rapeseed, harvest the seeds and continue to plant to obtain T1 generation transgenic rapeseed seedlings with high silique shattering resistance.

[0014] The present invention also provides the application of the above-mentioned sgRNA, the above-mentioned gene editing vector or the above-mentioned recombinant cell in rapeseed breeding, and the above-mentioned sgRNA, gene editing vector or recombinant cell is used to improve the silique shattering resistance of rapeseed.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: By analyzing the publicly available transcriptome data of Zhongshuang 11 in rapeseed, the present invention quickly discovers a key gene for silique shattering BnaA09.ADPG1 . It is found through gene editing technology that mutating the BnaA09.ADPG1 gene significantly enhances the silique shattering resistance of transgenic rapeseed. In the present invention BnaA09.ADPG1Mutation of the gene can improve the pod shattering resistance of rapeseed without affecting other agronomic traits, and can be used to create new germplasm resources with pod shattering resistance and rapeseed breeding for pod shattering resistance.

[0016] The present invention also designs three sgRNA target sites. By ligating the three sgRNA target site fragments with a gene editing vector and transforming to obtain a recombinant editing vector, further transforming Agrobacterium and infecting rapeseed, the infected rapeseed is screened, differentiated and cultured to obtain transgenic rapeseed seedlings with improved pod shattering resistance. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 They are partial genes specifically expressed in the late stage of pod maturity found in the transcriptome of Zhongshuang 11.

[0019] Figure 2 It is ADPG1 Schematic diagram of gene structure.

[0020] Figure 3 It is BnaA09 . ADPG1 Schematic diagram of target site mutation of gene knockout mutant.

[0021] Figure 4 It is a schematic diagram of the K5-KRSN-ADPG1 gene editing vector (16373 bp).

[0022] Figure 5 It is BnaA09 . ADPG1 Identification results of pod shattering resistance index of gene knockout mutant and wild type control. Detailed Description of the Specific Embodiments

[0023] The following will describe the specific embodiments of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0024] Example 1: Mining of BnaA09.ADPG1 genes in rapeseed.

[0025] 1. Database screening Download the transcriptome sequencing data of Brassica napus cv. Zhongshuang 11 at the whole growth stage from the BnIR database (https: / / yanglab.hzau.edu.cn / ). Set the screening conditions: the FPKM value of gene expression level in silique pericarp is greater than 5, and the FPKM value in other tissues is less than 3. A total of 862 genes were screened out ( Figure 1 ). Further, two genes, BnaA09G0529500ZS and BnaA09G0529600ZS, which were specifically highly expressed in silique pericarp on the 60th day in the late stage of silique maturation, were found. These two genes are predicted to encode pectin polygalacturonase ADPG1 , which is involved in the degradation of cell wall pectin. Pectin is one of the three major components of the plant cell wall. The unique spatio-temporal expression patterns of these two ADPG1 homologous genes indicate that they are likely to be involved in the degradation of the abscission zone cell wall and silique shattering in the late stage of silique maturation, so they are regarded as key candidate genes.

[0026] 2. ADPG1 Analysis of homologous gene sequences The annotation of the ZS11_v0 reference genome shows that these two ADPG1 homologous genes are arranged in tandem repeats on the genome (such as Figure 2 ). Analyze the conserved domains of the predicted coding protein amino acid sequences. Conserved domain search analysis (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi) found that both of these two genes contain incomplete PLN02218 super family conserved domains, and the sequences are relatively short, only 144 aa and 175 aa respectively. These clues suggest that there may be annotation errors in these two ADPG1 genes. Extract the 4.6 kb genomic sequence including these two genes and use the FGENESH+ function of the Softberry website. The website address of the Softberry website is as follows:

[0027] http: / / www.softberry.com / berry.phtml?topic=fgenes_plus&group=programs&subgroup=gfs.

[0028] Using Arabidopsis thaliana ADPG1Using the genomic sequence of the homologous gene AT3G57510 as a reference sequence, a new open reading frame with a length of 1278 bp was predicted, and the predicted protein encoded has an amino acid sequence length of 425 aa. Analysis of conserved domain search indicated that this predicted protein contains a complete conserved domain of the PLN02218 super family. Sequence alignment analysis showed that the newly predicted ADPG1 open reading frame contains 8 exons, and this gene was named BnaA09.ADPG1 . The BnaA09.ADPG1 gene has a length of 2278 bp, and its genomic nucleotide sequence is shown in SEQ ID NO.1. The nucleotide sequence of the coding region is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

[0029] SEQ ID NO.1:

[0030] SEQ ID NO.2:

[0031] SEQ ID NO.3: MARCCVPLAIFLGVLLMLSWCEALSSNVDDGYGHEDGGYESDSLIKLNNDEVLTLKSSDKPTPESSTVSVSDFGAKGDGKTDDTQAFKKAWKKACSTKGVTSFLVPKGKTYLLKSTQFRGPCKSLRSFQILGTLSASTKRSDYNKDKNHWLILEDVNNLSVDGGSEGTVDGNGKIWWQNSCKIDRSKALTFYNLMNLNVKNLRVRNAQQIQISVEKCNNVNIKNVEITAPDDSPNTDGIHITNTQNIRVSNSHIGTGDDCISIEDGSQNVQINDLSCGPGHGISIGSLGDDNSKAYVLGVNVDGAKLTETDNGVRIKTYQGGSGTAKNIKFQNIRMENVKNPIIIDQNYCDKDKCEQQDSAVQVNNVVYRNISGTSATDVAITFDCSEKYPCKGIVLDNVNIKGGTASCKNANVKNQGNVSPQCS。

[0032] Example 2: Rapeseed BnaA09.ADPG1 Application of the gene in improving the silique shattering resistance of rapeseed.

[0033] I. BnaA09.ADPG1 Construction of gene editing vector 1. Design of sgRNA target sites: For the BnaA09.ADPG1 gene, 3 sgRNA target sites were designed using the website (http: / / cbi.hzau.edu.cn / ). The sgRNA target sites include target site 1 (T1) shown in SEQ ID NO.4, target site 2 (T2) shown in SEQ ID NO.5, and target site 3 (T3) shown in SEQ ID NO.6. As Figure 3 shown.

[0034] SEQ ID NO.4: ACGCCTAAGAAAATAGCAAG.

[0035] SEQ ID NO.5: CTAGCTTCTTGGTTCCTAAA.

[0036] SEQ ID NO.6: AACAATCTATCAGTCGACGG.

[0037] 2. Construction of gene editing vector: (1)Synthesize the T1-T3 template sequences (386 bp) containing 3 target sites as shown in SEQ ID NO.7, and clone them into the pUC18 cloning vector.

[0038] SEQ ID NO.7: ACGCCTAAGAAAATAGCAAGGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCACTAGCTTCTTGGTTCCTAAAGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAAACAATCTATCAGTCGACGG.

[0039] The underlined parts are the 3 target site sequences in turn.

[0040] (2)Amplification and recovery of the target site fragments Synthesize the following amplification primers shown in SEQ ID NO.8-SEQ ID NO.9 for PCR amplification. The PCR amplification reaction system is shown in Table 1.

[0041] ADPG1-F: CAGTGGTCTCATGCAACGCCTAAGAAAATAGCAAGGTTTCAGAGC (SEQ ID NO.8); ADPG1-R: CAGTGGTCTCAAAACCCGTCGACTGATAGATTGTTTGCACC (SEQ ID NO.9).

[0042] Table 1 PCR amplification reaction system PCR program: Pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 72°C for 25 sec, 30 cycles; finally, extension at 72°C for 5 min.

[0043] The PCR products (416 bp) were detected by 1.5% agarose gel electrophoresis at a voltage of 5 v / cm for 20 min. The electrophoretic bands containing the PCR products of T1 - T3 targets were cut out under ultraviolet light, recovered and purified using a common agarose DNA recovery kit. The recovery steps were referred to the kit instruction manual, and finally dissolved in 30 μL of ddH2O.

[0044] (3)Ligation and transformation of target fragments and gene editing vectors The CRISPR / Cas9 gene editing vector K5 - KRSN - ccdb used was provided by Wuhan Boyuan Biotechnology Co., Ltd. The vector map is shown in Figure 4 . The digestion and ligation system is shown in Table 2.

[0045] Table 2 Digestion and ligation system Digestion and ligation reaction conditions: 37℃ for 20 min; 37℃ for 10 min, 20℃ for 10 min, cycle 5 times; 37℃ for 20 min; 80℃ for 5 min.

[0046] 8 μL of the ligation product was transformed into Escherichia coli T1 competent cells, and spread on kanamycin (final concentration 50 mg / L) - resistant LB solid medium, and cultured at 37℃ for 12 hr for colony PCR identification.

[0047] (4)Colony PCR identification Take 6 sterile 1.5 mL centrifuge tubes, add 300 μL of kanamycin (final concentration 50 mg / L) - resistant liquid LB medium to each of them, randomly select 6 monoclonal colonies and add them to the centrifuge tubes, culture at 37℃ with shaking at 200 rpm for 5 hr, and take 1 μL of the monoclonal bacterial liquid as a template for colony PCR identification. The PCR identification detection primers are shown as SEQ ID NO.10 - SEQ ID NO.11, and the PCR system is shown in Table 3.

[0048] Table 3 PCR system M13 - F: gtaaaacgacggccagt (SEQ ID NO.10); M13 - R: ccagaaattgaacgccgaag (SEQ ID NO.11).

[0049] PCR program: Pre - denaturation at 94℃ for 3 min; Denaturation at 94℃ for 30 sec, annealing at 50℃ for 30 sec, extension at 72℃ for 30 sec, cycle 30 times; Finally, extension at 72℃ for 5 min.

[0050] The PCR products were detected by 1.5% agarose gel electrophoresis at a voltage of 5 v / cm for 20 min. The target band size was 1060 bp. Three positive clone bacterial solutions were selected, and plaque PCR was used to detect the forward or reverse primers for sequencing verification. One tube of positive clone bacterial solution with correct sequencing verification was taken for enlarged culture, and the recombinant editing vector was extracted and named K5-KRSN-ADPG1.

[0051] (5)Transformation of Agrobacterium tumefaciens with the recombinant editing vector The K5-KRSN-ADPG1 recombinant editing vector was transformed into Agrobacterium tumefaciens GV3101 competent cells by the liquid nitrogen freeze-thaw method. One tube of Agrobacterium tumefaciens GV3101 competent cells was thawed on ice. In a laminar flow hood, 5 μL of the K5-KRSN-ADPG1 recombinant plasmid was added to the freshly thawed GV3101 competent cells and left standing on ice for 20 min; the GV3101 competent cells added with the recombinant plasmid were successively placed in liquid nitrogen for 5 min, in a 37 °C water bath for 5 min, and finally left standing on ice for 5 min. 500 μL of blank liquid LB medium was added, and the cells were cultured with shaking at 28 °C and 200 rpm for 3 hr. 200 μL of the transformed bacterial solution was spread on an LB solid plate supplemented with kanamycin (final concentration 50 mg / L) and rifampicin (final concentration 25 mg / L), and incubated at 28 °C in an inverted position for 2 d. Six monoclonal colonies were randomly selected and subjected to positive detection by the plaque PCR detection method in the previous step. One tube of positive bacterial solution was selected, an equal volume of 50% glycerol was added, and it was stored at -80 °C in a refrigerator for later use.

[0052] II. Genetic transformation of rapeseed 1. Seed disinfection and germination Two hundred plump Zhongshuang 11 rapeseed seeds were selected, disinfected with 75% alcohol for 1 min, 1.5% mercuric chloride for 15 min, and rinsed 4 times with sterile distilled water. Then the seeds were placed on a sterile M0 medium (2.2 g / L MS powder, 7.5 g / L agar powder, pH = 6.5) and cultured in the dark at 24 °C for 6 days to obtain sterile seedlings. Two days before cutting the seedlings, the preserved Agrobacterium tumefaciens GV3101 strain was taken out, a small amount of bacterial solution was picked up with an inoculation loop, and streaked on an LB solid plate supplemented with kanamycin, gentamicin, and rifampicin at a final concentration of 50 mg / L each, and cultured at 28 °C for 2 days.

[0053] 2. Infection and co-culture A small amount of Agrobacterium tumefaciens cells were scraped with an inoculation loop and added to DM medium (4.4 g / L MS powder, 30 g / L sucrose, pH = 6.5, supplemented with 100 uM / L of acetosyringone before use), and cultured with shaking at 28 °C for 30 min until OD 600It is about 0.3; Cut the hypocotyls of rape after 6 days of dark culture into small segments about 1 cm long in a laminar flow hood, transfer the cut hypocotyl segments to DM medium, infect for 30 min, blot the excess bacterial liquid with filter paper, and then transfer the hypocotyl segments to M1 medium (4.4 g / L MS powder, 30 g / L sucrose, 18 g / L mannitol, 7.5 g / L agarose, 25 mg / L kanamycin, 0.3 mg / L kinetin, pH = 6.5, add 100 uM / L acetosyringone before use), and culture in the dark at 24°C for 2 days.

[0054] 3. Screening and differentiation Transfer the explants after 2 days of dark culture to M2 medium (4.4 g / L MS powder, 30 g / L sucrose, 18 g / L mannitol, 7.5 g / L agarose, 0.3 mg / L kinetin, pH = 6.5, add 50 mg / L kanamycin, 20 mg / L silver nitrate, 300 mg / L ticarcillin before pouring the plate), culture under light at 20°C for 3 weeks, and then transfer the explants to M3 medium (4.4 g / L MS powder, 10 g / L glucose, 0.25 g / L xylose, 0.6 g / L MES, 7.5 g / L agarose, 0.2 mg / L IAA, pH = 6.5, add 50 mg / L kanamycin, 300 mg / L ticarcillin, 2 mg / L trans-zeatin before use), culture under light at 20°C, and subculture every 2 weeks. Regenerated seedlings can be seen at this stage.

[0055] 4. Rooting Cut the regenerated seedlings from the callus and transfer them to M4 medium (3.21 g / L B5 powder, 20 g / L sucrose, 8.5 g / L agar powder, pH = 6.5) for rooting culture for 3 weeks. They can be transplanted after the roots grow out.

[0056] IV. Positive detection and target mutation detection of T0 and T1 transgenic rape After obtaining T0 transgenic seedlings, extract the genomic DNA of leaves using the conventional CTAB method, and then perform positive detection using the specific primers shown in SEQ ID NO.12 - SEQ ID NO.13 on the editing vector. The size of the amplification product is about 600 bp.

[0057] The sequences of the specific primers are as follows: F: ccacgagaagtaccctacga (SEQ ID NO.12); R: ggatggcgtccgacagattc (SEQ ID NO.13).

[0058] Further, target mutation detection primers were used to detect the mutation situation of the target sequence in the T0 generation positive transgenic materials. The target mutation amplification detection primers are as shown in SEQ ID NO.14 to SEQ ID NO.17.

[0059] The detection primers for target 1 are: ADPG1-T1-F: TACAATGATGAACTCAGACTC (SEQ ID NO.14); ADPG1-T1-R: CCTTAGCTCCGAAGTCTGATA (SEQ ID NO.15).

[0060] The detection primers for target 2 and target 3 are: ADPG1-T23-F: TATCAGACTTCGGAGCTAAGG (SEQ ID NO.16); ADPG1-T23-R: ATGTGTGACAGTGTGAGACA (SEQ ID NO.17).

[0061] The PCR products of target 1, target 2 and target 3 were sent to Wuhan Shengong Biological Engineering (Shanghai) Co., Ltd. for sequencing, and the reverse primers were used for sequencing respectively. It was found that heterozygous mutations occurred in target 2 and target 3, and no mutation occurred in target 1. The T0 generation transgenic rapeseed single plants with heterozygous mutations in the sequences of target 2 and target 3 were bagged and self-crossed, and the seeds were harvested and planted to continue the T1 generation. According to the above detection steps, the T1 generation materials were subjected to positive detection and target sequence mutation detection again. The homozygous mutants were screened and self-crossed for another generation to obtain 2 T2 generation homozygous mutant materials adpg1-1 and adpg1-2 .

[0062] V. Identification of silique shattering resistance index Identification of the silique shattering resistance index of the T2 generation gene-edited transgenic rapeseed: Before the siliques of the T2 generation transgenic rapeseed and wild-type seeds turned color and cracked, the whole plants were cut from the base of the stem. The single plants of each line were tied together with packaging tape and hung upside down in a well-ventilated warehouse to air dry naturally for 3 weeks; 30 single plants were taken from both the mutants and the wild-type controls, and 10 single plants were used as a biological replicate. 60 normally developed siliques were taken from each single plant and randomly divided into 3 parts as technical replicates for the identification of the silique shattering resistance index. The identification of the silique shattering resistance index was carried out according to the method described in the Technical Regulations for the Identification of Silique Shattering Resistance of Rapeseed NY / T3066-2016 for BnaA09.ADPG1 the siliques of the gene-edited T2 generation homozygous mutant transgenic rapeseed. The results are shown in Table 1.

[0063] Table 1 BnaA09.ADPG1 Identification results of the silique shattering resistance index of the transgenic rapeseed homozygous mutant lines Note: adpg1-1 and adpg1-2 both represent the homozygous mutant lines of transgenic rapeseed obtained after the mutations of target 2 and target 3.

[0064] As Figure 5 shown, compared with the wild-type Zhongshuang 11, BnaA09.ADPG1 the silique shattering resistance index of the gene-edited homozygous mutant materials is significantly improved, and the difference is extremely significant by one-way ANOVA test. It shows that BnaA09.ADPG1 mutating the gene can significantly improve the silique shattering resistance of rapeseed.

[0065] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept.

[0066] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and deformations.

Claims

1. Rapeseed BnaA09.ADPG1 The application of a gene in improving the crack resistance of rapeseed siliques is characterized in that: Said BnaA09.ADPG1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, the CDS sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3; BnaA09.ADPG1 The gene and its homologous genes lose their function, improving the crack resistance of rapeseed siliques.

2. The rapeseed according to claim 1 BnaA09.ADPG1 The application of a gene in improving the crack resistance of rapeseed siliques is characterized in that: Using CRISPR-Cas9 gene editing technology BnaA09.ADPG1 The gene mutates.

3. The rapeseed according to claim 2 BnaA09.ADPG1 The application of a gene in improving the crack resistance of rapeseed siliques is characterized in that: CRISPR-Cas9 gene editing technology involves designing sgRNA so that BnaA09.ADPG1 The gene mutates.

4. The rapeseed according to claim 3 BnaA09.ADPG1 The application of a gene in improving the crack resistance of rapeseed siliques is characterized in that: The sgRNA comprises target 2 shown in SEQ ID NO.5 and target 3 shown in SEQ ID NO.

6.

5. A sgRNA, characterized in that The sgRNA comprises target 2 shown in SEQ ID NO.5 and target 3 shown in SEQ ID NO.6 in claim 3.

6. A gene editing vector, characterized in that: The gene editing vector contains the sgRNA according to claim 5.

7. A recombinant cell, characterized in that The recombinant cell contains the gene editing vector according to claim 6.

8. A method for improving the crack resistance of rapeseed siliques, characterized in that: The steps include: Designing for mutant rapeseed BnaA09.ADPG1 The sgRNA target of the gene was identified, the target fragment was connected to the gene editing vector and transformed into competent cells, and then infection, co-cultivation, screening, differentiation and rooting culture were carried out to obtain the transgenic rapeseed strains with silique crack resistance.

9. Use of the sgRNA according to claim 5, the gene editing vector according to claim 6 or the recombinant cell according to claim 7 in rapeseed breeding, characterized in that: The sgRNA, gene editing vector or recombinant cell is used to improve the crack resistance of rapeseed siliques.

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