Application of BnaALDH10A9 protein and coding gene thereof in regulating fragrance of brassica crops

Abstract

The invention discloses a BnaALDH10A9 protein and application of a coding gene of the BnaALDH10A9 protein in regulation and control of fragrance of brassica crops. The BnaALDH10A9 protein comprises a BnaA06. ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO: 1 and / or a BnaC03. ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO: 3. The invention proves that a new brassica napus germplasm capable of generating the aromatic substance 2AP in different tissues can be obtained by knocking out the brassica napus BnaALDH10A9 gene. According to the invention, target spots are designed for exon regions of two copy genes (BnaA06. ALDH10A9 and BnaC03. ALDH10A9) of the rape BnaALDH10A9, a CRISPR / Cas9 gene editing carrier is constructed, and a knockout mutant is obtained through genetic transformation. A flavor substance 2-acetyl-1-pyrroline (2AP) can be detected in leaves, flowers and seeds of mutants (including single mutants and double mutants), and obvious fresh and sweet flavor is emitted from the flowers and rapeseed oil. The method is suitable for creating new germplasm of fragrant rape.

Description

Technical Field

[0001] The invention relates to application of BnaALDH10A9 protein and a coding gene thereof in regulating the fragrance of Brassica crops, and belongs to the field of crop variety improvement. Background Art

[0002] Rapeseed is the largest oil crop in my country, producing more than 5 million tons of rapeseed oil each year, and is the largest source of domestic vegetable oil. Rapeseed oil is rich in nutrients, rich in fatty acids and multiple vitamins. The saturated fatty acid content of double-low rapeseed oil is less than 7%, the monounsaturated fatty acid oleic acid content is higher than 60%, and the essential fatty acid linolenic acid content of the human body is about 9%. It is one of the healthiest bulk vegetable oils. In addition, rapeseed has multiple functions such as vegetable, flower, feed, honey, and fertilizer. The multifunctional development and utilization of rapeseed can not only improve the planting effect of rapeseed, but also drive the development of local related industries such as breeding, beekeeping, and tourism (Zhang Zhe et al., 2018).

[0003] Aroma can enhance the sensory experience of agricultural products and is deeply loved by consumers, thus giving agricultural products a higher commodity value. For example, aroma is a key factor in determining the quality and market price of rice. The aroma of rice mainly comes from 2-acetyl-1-pyrroline (2AP), which is produced by the deletion mutation of 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 the enzyme function will lead to a large accumulation of GABAld, which should have been converted to GABA, thereby forming the intermediate product 1-pyrroline (Δ1-pyrroline), and finally converting into the synthetic aroma substance 2-AP. It has been reported that mutations in the BADH2 gene in a variety of cereal crops, such as corn (Wang et al., 2021), sorghum (Zhang et al., 2022), and millet (Zhang et al., 2023), can lead to the accumulation of the aroma substance 2AP in these crops. However, it is still unclear whether there are genes with similar functions in Brassica, and whether mutations in such genes can give rapeseed flowers, leaves, and seeds aroma, thereby greatly enhancing the multifunctional development and utilization value of rapeseed. Summary of the invention

[0004] Purpose of the invention: The first purpose of the present invention is to provide an application of BnaALDH10A9 protein and its encoding gene in regulating the aroma of Brassica crops. The second purpose of the present invention is to provide a method for obtaining a strong-flavored Brassica crop.

[0005] Technical solution: The present invention provides an application of a BnaALDH10A9 protein in regulating the aroma of Brassica crops, wherein the BnaALDH10A9 protein comprises a BnaA06.ALDH10A9 protein having an amino acid sequence as shown in SEQ ID NO:1 and / or a BnaC03.ALDH10A9 protein having 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 a gene encoding the BnaALDH10A9 protein in regulating the aroma of Brassica crops. The gene sequence encoding the BnaA06.ALDH10A9 protein is shown in SEQ ID NO:2, and the gene sequence encoding the BnaC03.ALDH10A9 protein is shown in SEQ ID NO:4.

[0009]

[0010]

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

[0012] The present invention also provides a method for obtaining strong-flavor Brassica crops based on CRISPR / Cas9 gene editing technology, the method comprising designing a target site sequence of BnaA06.ALDH10A9 and / or BnaC03.ALDH10A9, connecting the target site fragment and the linearized CRISPR / Cas9 vector, constructing a CRISPR / Cas9 gene editing vector and transferring the vector into the 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 NOs: 6-7.

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

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

[0017] Furthermore, the method also includes performing vector identification on T0 generation plants of Brassica crops transferred with CRISPR / Cas9 gene editing vectors, and then performing BnaA06.ALDH10A9 and / or BnaC03.ALDH10A9 target site identification on positive seedlings therein, and self-pollinating plants with target site mutations identified.

[0018] Furthermore, the primer sequences for vector identification are shown in SEQ ID NOs: 8-9, the primer sequences for identifying the BnaA06.ALDH10A9 target site are shown in SEQ ID NOs: 10-11, and the primer sequences for identifying the BnaC03.ALDH10A9 target site are shown in SEQ ID NOs: 12-13.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention confirms that knocking out the rapeseed BnaALDH10A9 gene can obtain a new rapeseed germplasm that can produce the aromatic substance 2AP in different tissues. The present invention designs targets for the exon region of the two copies (BnaA06.ALDH10A9 and BnaC03.ALDH10A9) of the rapeseed BnaALDH10A9 gene and constructs a CRISPR / Cas9 gene editing vector, and obtains a knockout mutant through genetic transformation. The aromatic substance 2-acetyl-1-pyrroline (2AP) can be detected in the leaves, flowers and seeds of the mutants (including single mutants and double mutants), and an obvious sweet fragrance is emitted from the flowers and rapeseed oil. This method is suitable for creating new aromatic rapeseed germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 . Schematic diagram of the gene editing vector of the present invention: A: BnaA06.ALDH10A9 and BnaC03.ALDH10A9 gene structure and target site (red arrow) position and sequence; 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 Results of GC-MS determination of 2-acetyl-1-pyrroline (2AP) content in rapeseed. (A) Total ion chromatograms of 2AP and 2,4,6-trimethylpyridine (TMP, as internal standard) in leaves of Bnaaldh10a9 mutant and wild type. (BD) 2AP content 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 DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with 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 / ), and the gene IDs were BnaA06g20780D and BnaC03g53290D, respectively. The target sites ( 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., and then primer annealing was performed: 1 μL sgRNA-F (10 μM), 1 μL sgRNA-R (10 μM) and 8 μL Buffer (TE+50 mM NaCl) were mixed, and then denatured at 95°C for 10 min in a PCR instrument, and then decreased 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. The vector was modified with restriction sites (from BbsI to BsaI) based on the psgR-Cas9-At vector (Mao et al., 2013). The psgR-Cas9(n) vector was digested with BsaI, and then the target site was connected to the vector using T4 ligase through conventional fragment-vector ligation. The ligation product was transferred into the competent Escherichia coli DH5a by conventional genetic transformation. After the plasmid was extracted, it was transformed into the competent Agrobacterium GV3101 strain by freeze-thaw method. The gene editing vector was transferred into the recipient material rapeseed J9712 using the Agrobacterium-mediated genetic transformation method of Brassica napus hypocotyls (Dai et al., 2020).

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

[0028] The genomic DNA of the transformed rapeseed J9712 tissue culture seedlings was extracted by the CTAB method, and then the vector-specific primers U6F (SEQ ID NO: 8: CCCAGGATTAGAATGATTAGGC) and sgRNA-R (SEQ ID NO: 9: GGGCTGTTCGTGCCAGATAC) were used for PCR positive detection. A total of 46 positive seedlings were identified in 50 tissue culture seedlings. The PCR reaction system was carried out according to the instructions of 2×Taq Master Mix (Dye Plus) (Novozyme, Nanjing, China), and the system was 20 μL. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, and full extension at 72°C for 10 min after 33 cycles of reaction. After the PCR reaction, 1% agarose gel electrophoresis was performed.

[0029] Specific primers were used to amplify the target site of the positive seedlings, wherein the specific primers for the BnaA06.ALDH10A9 target site were SEQ ID NOs: 10-11 (F: CCTTCGCCGTCAAACACT; R: GTTACAAGCAACCAGGGTCC A) and the specific primers for the BnaC03.ALDH10A9 target site were SEQ ID NOs: 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. Finally, we found that a total of 20 T0 plants had mutations at the target site ( Figure 2 The majority of mutations detected were 1-base-pair insertions or deletions, of which 62.5% were heterozygous mutations, 25% were homozygous or biallelic mutations, and 12.5% ​​were mosaic mutations ( Figure 2 ).

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

[0032] We selected three Bnaaldh10a9 mutants for subsequent experiments, among which #17 was a bnaa06.aldh10a9 single mutant (homozygous mutation); #2 was a bnac03.aldh10a9 single mutant (heterozygous mutation); and #18 was a bnaa06.aldh10a9bnac03.aldh10a9 double mutant (bnaa06.aldh10a9 was a homozygous mutation and bnac03.aldh10a9 was a heterozygous mutation). All three mutations were frameshift mutations caused by the loss of a single cytosine nucleotide ( Figure 2). Through self-pollination, each mutant line screened homozygous mutants in the T1 generation. Then we extracted and determined the content of 2AP in 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 of bnaaldh10a9 mutant and wild-type J9712 plants were collected for 2AP extraction. Three biological replicates were set for each tissue of each material. The specific operation was 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 cut into small pieces; and for dried mature seeds, 5 g of each sample was weighed and ground until a fine powder was formed. 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; at the same time, 30 mL of dichloromethane was added to the flask on the other side of the double simultaneous distillation extractor, and 2,4,6-trimethylpyridine (TMP) was added as an internal standard with a final concentration of 0.05 mg / L, and heated to 85 °C in a water bath. After 40 minutes of circulating distillation, the dichloromethane extract was collected at the extraction port. Subsequently, the extract was dried over anhydrous sodium sulfate, filtered through a 0.22 μm sterile filter membrane, and finally transferred to a sample bottle for subsequent analysis.

[0034] 2AP determination method: 2AP was quantified using gas chromatography-mass spectrometry (Trace ISQ, Thermo Fisher, USA). The chromatographic conditions were as follows: the column temperature of the gas chromatograph was maintained at 40°C for 1 min, then heated to 65°C at a rate of 2°C / min and maintained for 4 min, and then heated to 250°C at a rate of 10°C / min. High-purity helium was used as the carrier gas with a flow rate of 1.0 mL / min. The mass spectrometry conditions were as follows: the ionization source temperature was set at 250°C, the scanning mode was selected ion monitoring (SIM), and the mass-to-charge ratio was 43, 111 and 79, 121. The calculation formula for the 2AP content was: C2 = (A2×C1×V) / (A1×W), where C1 is the concentration of TMP, 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 and double mutant tissues, but was not detected in the wild type ( Figure 3 A) The 2AP levels in the single mutants were similar in all tissues examined, but in the double mutants the 2AP levels were 2.3 to 3.5 times higher than in the 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 the sensory experience, compared with the wild-type material, the double mutant's flowers and rapeseed oil exude a distinct sweet fragrance. Among them, the rapeseed oil exudes an aroma similar to "boiled corn". The above three T2 generation homozygous mutant materials were sequenced and the mutant 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 crops Chinese cabbage and cabbage are diploid ancestral species of Brassica napus, the species homology is relatively high. Therefore, the present invention is also likely to be applicable to the genetic improvement of the aroma of Brassica crops such as Chinese cabbage and cabbage to enhance the edible flavor.

Claims

1. A use of BnaALDH10A9 protein in regulating the aroma of Brassica crops, characterized in that: The BnaALDH10A9 protein includes a BnaA06.ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO:1 and / or a BnaC03.ALDH10A9 protein with an amino acid sequence as shown in SEQ ID NO:

3.

2. Use of the gene encoding the BnaALDH10A9 protein according to claim 1 in regulating the aroma of Brassica crops, characterized in that: The sequence of the gene encoding the BnaA06.ALDH10A9 protein is shown in SEQ ID NO:2, and the sequence of the gene encoding the BnaC03.ALDH10A9 protein is shown in SEQ ID NO:

4.

3. A method for obtaining a strong-flavored Brassica crop, characterized in that: The method comprises: down-regulating or knocking out the expression of BnaALDH10A9 protein, wherein the BnaALDH10A9 protein comprises a BnaA06.ALDH10A9 protein having an amino acid sequence as shown in SEQ ID NO:1 and / or a BnaC03.ALDH10A9 protein having an amino acid sequence as shown in SEQ ID NO:

3.

4. A method for obtaining strong-flavor Brassica crops based on CRISPR / Cas9 gene editing technology, characterized in that: The method comprises designing a target site sequence of BnaA06.ALDH10A9 and / or BnaC03.ALDH10A9, connecting the target site fragment and the linearized CRISPR / Cas9 vector, constructing a CRISPR / Cas9 gene editing vector and transferring the vector into Brassica crops.

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 sequence are shown in SEQ ID NOs: 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 Brassica crops include cabbage, kale and rapeseed.

9. The method according to claim 4, characterized in that The method further comprises performing vector identification on T0 generation plants of Brassica crops into which the CRISPR / Cas9 gene editing vector has been transferred, and then performing BnaA06.ALDH10A9 and / or BnaC03.ALDH10A9 target site identification on positive seedlings therein, and self-pollinating plants with target site mutations identified.

10. The method according to claim 9, characterized in that The primer sequences used for vector identification are shown in SEQ ID NOs: 8-9, the primer sequences for identifying the BnaA06.ALDH10A9 target site are shown in SEQ ID NOs: 10-11, and the primer sequences for identifying the BnaC03.ALDH10A9 target site are shown in SEQ ID NOs: 12-13.

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