Application of BnaALDH10A9 protein and its encoding gene in regulating aroma in Brassica crops

Abstract

This invention discloses the application of the BnaALDH10A9 protein and its encoding gene in regulating the aroma of Brassica crops. The BnaALDH10A9 protein includes BnaA06.ALDH10A9 protein with the amino acid sequence shown in SEQ ID NO:1 and / or BnaC03.ALDH10A9 protein with the amino acid sequence shown in SEQ ID NO:3. This invention confirms that knocking out the BnaALDH10A9 gene in rapeseed can obtain new Brassica napus germplasm that produces the aroma compound 2AP in different tissues. This invention designs target sites in the exon regions of two copies of the rapeseed BnaALDH10A9 gene (BnaA06.ALDH10A9 and BnaC03.ALDH10A9) and constructs a CRISPR / Cas9 gene editing vector, obtaining knockout mutants through genetic transformation. The aroma compound 2-acetyl-1-pyrrolline (2AP) was detected in the leaves, flowers, and seeds of the mutants (including single and double mutants), and the flowers and rapeseed oil emitted a distinct sweet aroma. This method is suitable for creating new aromatic rapeseed germplasm.

Description

Technical Field

[0001] This invention relates to the application of the BnaALDH10A9 protein and its encoding gene in regulating the aroma of Brassica crops, and belongs to the field of crop variety improvement. Background Technology

[0002] Rapeseed oil is rich in nutrients, containing abundant fatty acids and various vitamins. Low-erucic acid rapeseed oil has less than 7% saturated fatty acids, over 60% monounsaturated oleic acid, and approximately 9% linolenic acid, an essential fatty acid, making it one of the healthiest bulk vegetable oils. Furthermore, rapeseed has multiple uses, including as a vegetable, flower, feed, honey, and fertilizer. The multifunctional development and utilization of rapeseed can not only improve its planting efficiency but also drive the development of related industries such as local animal husbandry, beekeeping, and tourism (Zhang et al., 2018).

[0003] Aroma enhances the sensory experience of agricultural products, making them popular with consumers and thus giving them higher commercial value. For example, aroma is a key factor determining the quality and market price of rice. The aroma of rice mainly originates from 2-acetyl-1-pyrroline (2AP), which is produced due to a deletion mutation in the betaine aldehyde dehydrogenase 2 (BADH2) gene (Chen et al., 2008). The main function of BADH2 in plants is to convert γ-aminobutyraldehyde (GABald) into γ-aminobutyric acid (GABA). The absence or weakening of this enzyme's function leads to a large accumulation of GABA, which should have been converted to GABA, forming the intermediate 1-pyrroline (Δ1-pyrroline), which is ultimately converted into the aroma compound 2-AP. Previous reports have shown that mutations in the BADH2 gene can lead to the accumulation of the aroma compound 2AP in various cereal crops, including maize (Wang et al., 2021), sorghum (Zhang et al., 2022), and millet (Zhang et al., 2023). However, it remains unclear whether a gene with a similar function exists in the Brassica genus, and whether mutations in such a gene could impart aroma to rapeseed flowers, leaves, and seeds, thereby significantly enhancing the multifunctional development and utilization value of rapeseed. Summary of the Invention

[0004] Objectives of this invention: The first objective of this invention is to provide an application of the BnaALDH10A9 protein and its encoding gene in regulating the aroma of Brassica crops. The second objective of this invention is to provide a method for obtaining strongly aromatic Brassica crops.

[0005] Technical solution: The present invention provides an application of BnaALDH10A9 protein in regulating the aroma of Brassica crops, wherein the BnaALDH10A9 protein includes BnaA06.ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO:1 and / or BnaC03.ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO:3.

[0006] SEQ ID NO:1(MAITVPRRQLFIGGQWTEPLRRQTLPVVNPATEDIIGYIPAATSED VELAVEAARKALTRNQGKDWSKASGAVRARYLRAIAAKVTERKSELANLEAIDCGKPLDEAAWDMDDVAGCFEYYADLAQGLDSKQKAPLSLPLDTFKGYVLKEPIGVVGLITP WNYPLLMAVWKVAPALAAGCTAILKPSELASVTCLELADICREVGLPPGVLNILTGLGTEAGAPLASHPHVDKIVFTGSTATGSNIMTSAAKLVKPVSLELGGKSPIIVFDDVKI DKAVEWTMFGCFWTNGQICSATSRLLVHEKIADEFLDKLVKWTKNIKISDPFEEGCRLGPVVSKGQYERVVKFVSNARKEGATVLCGGARPGHLKKGYFVEPAIISNVTTSMEIW RDEVFGPVLCVKTFSTEDEAIQLANDSQYGLAGAVLSNDLERCDRVSKAFEAGIVWVNCSQPCFCQAPWGGTKRSGFGRELGEWGLENYLSVKQVTQYISNEPWGWYKPPSKL).

[0007] SEQ ID NO:3(MAITVPRRQLFIGGQWTEPLRRQTLPVVNPATEDIIGYIPAATSED VELAVEAARKALTRNKGNDWSKASGAVRARYLRAIAAKVTERKSELANLEAIDCGKPLDEAAWDMDDVAGCFEYYADLAEGLDAKQKAPLSLPLDTFKGYVLKEPIGVVGLITP WNYPLLMAVWKVAPALAAGCTAILKPSELASVTCLELADICREVGLPPGVLNILTGLGTEAGAPLASHPHVDKIVFTGSTATGSNIMTSAAKLVKPVSLELGGKSPIIVFDDVEI DKAVEWTMFGCFWTNGQICSATSRLLVHEKIADEFLDKLVKWTKNIKISDPFEEGCRLGPVVSKGQYERVVKFVSNARKEGATVLCGGARPGHFKKGYFVEPAIISNVTTSMEIW RDEVFGPVLCVKTFSTEDEAIQLANDSQYGLAGAVLSNDLERCDRVSKAFEAGIVWVNCSQPCFCQAPWGGTKRSGFGRELGEWGLGNYLSVKQVTQYISDEPWGWYKPPSKL).

[0008] The present invention also provides an application of the gene encoding the above-mentioned BnaALDH10A9 protein in regulating the aroma of Brassica crops. The gene sequence encoding BnaA06.ALDH10A9 protein is shown in SEQ ID NO:2, and the gene sequence encoding BnaC03.ALDH10A9 protein is shown in SEQ ID NO:4.

[0009]

[0010]

[0011] The present invention also provides a method for obtaining a strongly aromatic Brassica crop, the method comprising: downregulating or knocking out the expression of BnaALDH10A9 protein, wherein the BnaALDH10A9 protein comprises BnaA06.ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO:1 and / or BnaC03.ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO:3.

[0012] This invention also provides a method for obtaining aromatic Brassica crops based on CRISPR / Cas9 gene editing technology. The method involves designing target site sequences of BnaA06.ALDH10A9 and / or BnaC03.ALDH10A9, connecting the target site fragments with a linearized CRISPR / Cas9 vector, constructing a CRISPR / Cas9 gene editing vector, and then transferring it into Brassica crops.

[0013] Furthermore, the target site sequence is shown in SEQ ID NO:5.

[0014] Furthermore, the primer sequences used to synthesize the target sequence are shown in SEQ ID NO:6-7.

[0015] Furthermore, the CRISPR / Cas9 vector is psgR-Cas9(n).

[0016] Furthermore, the Brassica genus crops include Chinese cabbage, kale, and rapeseed.

[0017] Furthermore, the method also includes identifying the vector in the T0 generation of Brassica plants that have been transformed into the CRISPR / Cas9 gene editing vector, and then identifying the target sites of BnaA06.ALDH10A9 and / or BnaC03.ALDH10A9 in the positive seedlings. Plants with target site mutations are then self-pollinated.

[0018] Furthermore, the primer sequences used for vector identification are shown in SEQ ID NO:8-9, the primer sequences for identifying the BnaA06.ALDH10A9 target site are shown in SEQ ID NO:10-11, and the primer sequences for identifying the BnaC03.ALDH10A9 target site are shown in SEQ ID NO:12-13.

[0019] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention confirms that knocking out the BnaALDH10A9 gene in rapeseed can yield new rapeseed germplasm that produces the aroma compound 2AP in different tissues. This invention designs target sites in the exon regions of two copies of the BnaALDH10A9 gene (BnaA06.ALDH10A9 and BnaC03.ALDH10A9) and constructs a CRISPR / Cas9 gene editing vector, obtaining knockout mutants through genetic transformation. The aroma compound 2-acetyl-1-pyrrolline (2AP) can be detected in the leaves, flowers, and seeds of the mutants (including single and double mutants), and both the flowers and rapeseed oil emit a distinct sweet aroma. This method is suitable for creating new aromatic rapeseed germplasm. Attached Figure Description

[0020] Figure 1 Schematic diagram of the gene editing vector of the present invention: A: Gene structure and target site (red arrow) location and sequence of BnaA06.ALDH10A9 and BnaC03.ALDH10A9; CRISPR / Cas9 vector diagram.

[0021] Figure 2 Sanger sequencing T0 generation mutation results; Hetero: heterozygous mutation; Homo: homozygous mutation; Bi-allelic: biallelic mutation; Chimeric: chimera.

[0022] Figure 3 Determination of 2-acetyl-1-pyrrolidone (2AP) content in rapeseed by gas chromatography-mass spectrometry (GC-MS). (A) Total ion chromatograms of 2AP and 2,4,6-trimethylpyridine (TMP, as internal standard) in leaves of Bnaaldh10a9 mutant and wild type. (BD) Content of 2AP in leaves, flowers and seeds. AACC: wild type; aacc: bnaa06.aldh10a9bnac03.aldh10a9 double mutant; aaCC: bnaa06.aldh10a9 single mutant; AAcc: bnac03.aldh10a9 single mutant. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] Example 1: Construction of CRISPR / Cas9 gene editing vector

[0025] The sequence information of wild-type BnaA06.ALDH10A9 and BnaC03.ALDH10A9 genes was obtained from the BnIR database (https: / / yanglab.hzau.edu.cn / BnIR / ), with gene IDs of BnaA06g20780D and BnaC03g53290D, respectively. Target sites were designed using the CRISPR-P 2.0 online software (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Figure 1 The target site sequence is shown in SEQ ID NO: 5: GGGCTGTTCGTGCCAGATAC. The following primers with adapters, sgRNA-F (SEQ ID NO: 6: gtaaGTATCTGGCACGAACAGCCC) and sgRNA-R (SEQ ID NO: 7: aaacGGGCTGTTCGTGC CAGATAC), were synthesized by Qingke Biotechnology Co., Ltd., followed by primer annealing: 1 μL of sgRNA-F (10 μM), 1 μL of sgRNA-R (10 μM), and 8 μL of buffer (TE + 50 mM NaCl) were mixed, and then denatured at 95 °C for 10 min in a PCR instrument, followed by cooling to 20 °C at a rate of 0.1 °C / s.

[0026] The gene-editing vector psgR-Cas9(n) was provided by Professor Zhang Hui of Shanghai Normal University. This vector was modified based on the psgR-Cas9-At vector (Mao et al., 2013) by changing the restriction enzyme sites (from BbsI to BsaI). The psgR-Cas9(n) vector was digested with BsaI, and then the target site was ligated into the vector using the conventional fragment vector ligation method with T4 ligase. The ligation product was transformed into competent E. coli DH5a cells using conventional genetic transformation. After plasmid extraction, it was transformed into competent Agrobacterium tumefaciens GV3101 strain using the freeze-thaw method. Using the Agrobacterium-mediated hypocotyl genetic transformation method of Brassica napus (Dai et al., 2020), the gene-editing vector was transformed into the recipient material Brassica napus J9712.

[0027] Example 2: Positive identification and editing detection of tissue culture seedlings

[0028] Genomic DNA was extracted from transformed rapeseed J9712 tissue culture seedlings using the CTAB method. PCR detection was then performed using vector-specific primers U6F (SEQ ID NO:8: CCCAGGATTAGAATGATTAGGC) and sgRNA-R (SEQ ID NO:9: GGGCTGTTCGTGCCAGATAC). 46 positive seedlings were identified from 50 tissue culture seedlings. The PCR reaction system followed the instructions for 2×Taq Master Mix (Dye Plus) (Novizan, Nanjing, China), with a volume of 20 μL. The PCR reaction program was as follows: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1 min, for 33 cycles, followed by a final extension at 72℃ for 10 min. 1% agarose gel electrophoresis was performed after the PCR reaction.

[0029] Specific primers were used to amplify the target sites on positive seedlings. The specific primers for the BnaA06.ALDH10A9 target site were SEQ ID NO: 10-11 (F: CCTTCGCCGTCAAACACT; R: GTTACAAGCAACCAGGGTCC A), and the specific primers for the BnaC03.ALDH10A9 target site were SEQ ID NO: 12-13 (F: CCTTCGCCGTCAA ACACT; R: GCAAGGGTTACAAGCAACCAGGTAT). The PCR reaction system was the same as described above (Example 2).

[0030] The amplified products were then subjected to TA cloning and Sanger sequencing. Ultimately, we found that a total of 20 T0 generation plants had mutations at the target site. Figure 2 The majority of the detected mutations were single-base-pair insertions or deletions, with 62.5% being heterozygous mutations, 25% being homozygous or biallelic mutations, and 12.5% ​​being chimeric mutations. Figure 2 ).

[0031] Example 3: Determination of 2AP content in rapeseed tissue by GC-MS

[0032] We selected three Bnaaldh10a9 mutant strains for subsequent experiments. #17 was a single mutant of bnaa06.aldh10a9 (homozygous mutation); #2 was a single mutant of bnac03.aldh10a9 (heterozygous mutation); and #18 was a double mutant of bnaa06.aldh10a9 and bnac03.aldh10a9 (bnaa06.aldh10a9 was homozygous and bnac03.aldh10a9 was heterozygous). All three mutations were frameshift mutations caused by the deletion of a single cytosine nucleotide. Figure 2Through self-pollination, homozygous mutants were screened for each mutant line in the T1 generation. Subsequently, we extracted and determined the content of 2AP from the leaves, flowers, and seeds of the T2 generation homozygous mutant materials.

[0033] 2AP Extraction Method: Leaves (young leaves from 6-week-old plants), flowers, and dried mature seeds were collected from bnaaldh10a9 mutant and wild-type J9712 plants for 2AP extraction. Three biological replicates were set up for each tissue of each material. The specific procedures were as follows: For leaves, 20 g of each sample was weighed and cut into small pieces for subsequent processing; for flowers, 10 g of each sample was weighed and similarly cut into small pieces; and for dried mature seeds, 5 g of each sample was weighed and ground into a fine powder. Each sample was mixed with 150 mL of deionized water and placed in a round-bottom flask on one side of a double simultaneous distillation extractor. The heating temperature was set to 150 °C and heated to boiling. Simultaneously, 30 mL of dichloromethane and 0.05 mg / L of 2,4,6-trimethylpyridine (TMP) were added to the other flask of the double simultaneous distillation extractor as an internal standard, and the mixture was heated to 85 °C in a water bath. After 40 minutes of cyclic distillation, the dichloromethane extract was collected at the extraction port. The extract was then dried with anhydrous sodium sulfate, filtered through a 0.22 μm sterile filter membrane, and finally transferred to a sample vial for further analysis.

[0034] 2AP determination method: 2AP was quantified using gas chromatography-mass spectrometry (GGC-MS) (Trace ISQ, Thermo Fisher, USA). Chromatographic conditions were as follows: the gas chromatography column temperature program was 40℃ for 1 min, then increased to 65℃ at a rate of 2℃ / min and held for 4 min, followed by an increase to 250℃ at a rate of 10℃ / min. High-purity helium was used as the carrier gas at a flow rate of 1.0 mL / min. Mass spectrometry conditions were: ionization source temperature set to 250℃, scan mode selected ion monitoring (SIM), and mass-to-charge ratios of 43, 111 and 79, 121. The 2AP content was calculated using the formula: C2 = (A2 × C1 × V) / (A1 × W), where C1 is the TMP concentration, V is the injection volume, A1 and A2 are the peak areas of TMP and 2AP, respectively, and W is the sample mass.

[0035] A 2AP target peak at approximately 11.4 min was observed in all bnaaldh10a9 single mutant and double mutant tissues, but was not detected in the wild type. Figure 3 A). In all tissues examined, the 2AP content was similar in single mutants, but in double mutants, the 2AP content was 2.3 to 3.5 times higher than in single mutants. Figure 3BD). In the double mutant, the highest 2AP content was found in seeds (367.6 μg / kg), followed by flowers (54.4 μg / kg), and then leaves (31.0 μg / kg). Figure 3 (BD). From a sensory perspective, compared to wild-type materials, the double mutants exhibit a distinctly sweet aroma in their flowers and rapeseed oil. The rapeseed oil, in particular, emits a fragrance similar to "boiled corn." Sequence identification was performed on the three T2 generation homozygous mutant materials. The mutated bnaa06.aldh10a9 sequence is shown in SEQ ID NO:14 (ATGGCGATTACGGTGCCGCGACGGCAGCTCTTCATCGGCGGTCAATGGACA). GAGCCCCTTCGCCGTCAAACACTCCCTGTTGTCAATCCCGCCACGGAGGACATCATCGGTTACATCCCAGCTGCAACTTCTGAGGATGTGGAGCTCGCGGTGGAAGCTGCTAGGAAAGCACTTACAAGAAACCAAGGAAAGGATTGGTCTAAAGCATCCGGGGTGTTCGTGCCAGATAC TTACGTGCTATTGCAGCTAAGGTAACGGAGAGGAAGTCTGAGCTAGCTAATCTTGAGGCTATTGACTGCGGTAAACCTCTAGATGAAGCAGCATGGGACATGGATGATGTTGCTGGATGTTTTGAATATTATGCTGACCTAGCTCAAGGCTTAGATTCAAAGCAGAAGGCTCCTCTTTC TCTTCCCTTAGATACTTTTAAGGGGCTACGTTCTCAAGGAACCCATTGGTGTAGTTGGCCTGATTACTCCATGGAATTATCCGTTACTGATGGCTGTTTGGAAAGTCGCTCCTGCACTTGCCGCTGGGTGCACGGCAATACTGAAACCTTCTGAGTTGGCCTCCGTGACATGTTTGGAGC TCGCTGATATCTGCCGCGAGGTGGGTCTGCCACCTGGTGTTCTTAATATTCTGACTGGTTTAGGAACTGAAGCAGGTGCTCCATTGGCATCGCATCCACACGTTGACAAGATTGTTTTCACTGGAAGCACGGCAACTGGAAGCAACATTATGACTTCTGCTGCCAAATTGGTTAAACCTG

[0036] TTTCCTTGGAGCTTGGTGGGAAAAGCCCTATCATTGTCTTTGATGATGTCAAAATT

[0037] GACAAAGCTGTGGAATGGACTATGTTTGGTTGTTTCTGGACAAACGGTCAGATTT

[0038] GCAGTGCGACATCTCGACTTCTCGTGCATGAAAAGATTGCTGACGAATTTTTGGA

[0039] CAAGTTGGTAAAGTGGACAAAGAACATTAAGATTTCAGATCCTTTTGAAGAAGGC

[0040] TGTAGGCTTGGTCCTGTTGTCAGCAAAGGACAGTACGAGAGAGTAGTGAAGTTTG

[0041] TCTCAAACGCTAGGAAGGAAGGTGCAACTGTCCTCTGCGGAGGAGCTCGTCCTGG

[0042] GCATTTAAAAAAGGGTTATTTTGTTGAACCTGCTATAATTTCAAATGTGACTACTT

[0043] CAATGGAAATCTGGAGAGATGAAGTATTTGGTCCTGTTCTCTGTGTCAAAACATT

[0044] CTCCACTGAGGATGAGGCAATACAGCTGGCAAATGACTCCCAATATGGATTAGCA

[0045] GGCGCTGTATTATCAAATGATCTGGAGAGGTGTGATCGCGTTAGTAAGGCATTCG

[0046] AGGCGGGTATTGTGTGGGTCAACTGTTCTCAGCCATGTTTCTGTCAAGCTCCATGG

[0047] GGTGGAACCAAACGCAGTGGTTTTGGCCGTGAACTAGGAGAATGGGGACTTGAG

[0048] AACTACTTGAGTGTGAAGCAGGTGACGCAATATATATCTAATGAACCATGGGGAT

[0049] GGTACAAACCTCCTTCCAAGCTTTAA), the mutated bnac03.aldh10a9 sequence is as shown in SEQ ID

[0050] NO:15 (ATGGCGATTACGGTGCCGCGACGGCAGCTCTTCATTGGCGGTCAATG

[0051] GACAGAGCCCCTTCGCCGTCAAACACTCCCTGTTGTCAATCCCGCCACGGAGGAC

[0052] ATCATCGGTTACATTCCAGCTGCAACTTCTGAGGATGTGGAACTCGCGGTGGAAG

[0053] CTGCTAGGAAAGCACTTACAAGAAACAAAGGAAATGATTGGTCTAAAGCATCCG

[0054] GGGTGTTCGTGCCAGATACTTACGTGCTATTGCAGCTAAGGTAACGGAGAGGAAA

[0055] TCTGAACTAGCTAATCTTGAGGCTATTGATTGCGGTAAACCTCTAGATGAAGCAG

[0056] CATGGGACATGGATGATGTTGCTGGATGTTTTGAATATTATGCTGACCTAGCTGA

[0057] AGGCTTAGATGCAAAGCAGAAGGCTCCTCTTTCTCTTCCGTTAGATACTTTTAAGG

[0058] GCTACGTTCTCAAGGAACCCATTGGTGTAGTTGGGCTGATTACTCCATGGAATTA

[0059] TCCGTTACTGATGGCTGTTTGGAAAGTCGCTCCTGCACTTGCTGCTGGGTGCACGG

[0060] CAATACTGAAACCTTCTGAGTTGGCCTCCGTGACATGTTTGGAGCTCGCTGATATT

[0061] TGCCGCGAGGTGGGTCTGCCACCTGGTGTTCTTAATATTCTGACTGGTTTAGGAAC

[0062] TGAAGCAGGTGCTCCATTGGCATCGCATCCACACGTTGACAAGATTGTTTTCACT

[0063] GGAAGCACGGCAACTGGAAGCAACATTATGACTTCTGCTGCCAAATTGGTTAAAC

[0064] CTGTTTCCTTGGAGCTTGGTGGGAAAAGCCCTATCATTGTCTTTGATGATGTCGAA

[0065] ATTGACAAAGCTGTGGAATGGACTATGTTTGGTTGTTTCTGGACAAACGGTCAGA

[0066] TTTGCAGTGCGACATCTCGACTTCTCGTGCATGAAAAGATTGCTGACGAATTTTTG

[0067] GACAAGTTGGTAAAGTGGACAAAGAACATTAAGATTTCAGATCCTTTTGAAGAA

[0068] GGCTGTAGGCTTGGTCCTGTTGTCAGCAAAGGACAGTACGAGAGAGTAGTGAAG

[0069] TTTGTCTCAAACGCTAGGAAGGAAGGTGCAACTGTCCTCTGCGGAGGAGCTCGTC

[0070] CTGGGCATTTTAAAAAGGGTTATTTTGTTGAACCTGCTATAATTTCAAATGTGACT

[0071] ACTTCAATGGAAATCTGGAGAGATGAAGTATTTGGTCCTGTTCTCTGTGTCAAAA

[0072] CATTCTCCACTGAGGATGAGGCAATACAGCTGGCAAATGACTCCCAATATGGATT

[0073] AGCAGGCGCTGTATTATCAAATGATCTGGAGAGGTGTGATCGCGTTAGTAAGGCA

[0074] TTCGAGGCGGGTATTGTGTGGGTCAACTGTTCTCAGCCATGTTTCTGTCAAGTCCC

[0075] ATGGGGTGGAACCAAACGCAGTGGTTTTGGCCGTGAACTAGGAGAATGGGGACT

[0076] (TGGGAACTACTTGAGTGTGAAGCAGGTGACGCAGTATATATCTGATGAACCATGGGGATGGTACAAACCTCCTTCCAAGCTTTAA). Since Brassica rapa and Brassica napus are diploid ancestors of Brassica napus, they share high species homology. Therefore, this invention is also likely applicable to the genetic improvement of aroma in Brassica rapa and other Brassica rapa crops to enhance their flavor.

Claims

1. The application of BnaALDH10A9 protein knockout in enhancing the aroma of Brassica crops, characterized by, The BnaALDH10A9 protein is the BnaA06.ALDH10A9 protein with the amino acid sequence as shown in SEQ ID NO:1 and / or the BnaC03.ALDH10A9 protein with the amino acid sequence as shown in SEQ ID NO:3, and the Brassica genus crop is rapeseed.

2. The application of gene knockout of the BnaALDH10A9 protein as described in claim 1 in enhancing the aroma of Brassica crops, characterized in that, The gene for the BnaALDH10A9 protein is the gene sequence encoding BnaA06.ALDH10A9 protein as shown in SEQ ID NO:2 and the gene sequence encoding BnaC03.ALDH10A9 protein as shown in SEQ ID NO:4, and the Brassica genus crop is rapeseed.

3. A method for obtaining aromatic Brassica crops, characterized in that, The method includes: knocking out the expression of BnaALDH10A9 protein, wherein the BnaALDH10A9 protein is BnaA06.ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO:1 and / or BnaC03.ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO:3, and the Brassica genus crop is rapeseed.

4. A method for obtaining aromatic Brassica crops based on CRISPR / Cas9 gene editing technology, characterized in that, The method involves designing target site sequences for BnaA06.ALDH10A9 and / or BnaC03.ALDH10A9, ligating the target site fragments to a linearized CRISPR / Cas9 vector, constructing a CRISPR / Cas9 gene editing vector, and then transferring it into a Brassica oleracea crop, specifically rapeseed. BnaA06.ALDH10A9 The gene sequence is shown in SEQ ID NO:

2. BnaC03.ALDH10A9 The gene sequence is shown in SEQ ID NO:

4.

5. The method according to claim 4, characterized in that, The target site sequence is shown in SEQ ID NO:

5.

6. The method according to claim 5, characterized in that, The primer sequences used to synthesize the target site sequence are shown in SEQ ID NO:6~7.

7. The method according to claim 4, characterized in that, The CRISPR / Cas9 vector is psgR-Cas9(n).

8. The method according to claim 4, characterized in that, The method further includes identifying the vector in the T0 generation of Brassica plants transformed with the CRISPR / Cas9 gene editing vector, and then processing the positive seedlings. BnaA06.ALDH10A9 and / or BnaC03.ALDH10A9 Target site identification: Plants with identified target site mutations were then self-crossed.

9. The method according to claim 8, characterized in that, The primer sequences used for vector identification are shown in SEQ ID NO:8~9. BnaA06.ALDH10A9 The primer sequences for the target site are shown in SEQ ID NO:10~11, for identification. BnaC03.ALDH10A9 The primer sequences for the target site are shown in SEQ ID NO:12~13.

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