Application of transcription factor BnaMYB52 in regulating drought resistance in rapeseed

Abstract

The present invention discloses the application of transcription factor BnaMYB52 in regulating rapeseed drought resistance, and relates to the field of biotechnology. The amino acid sequence of the transcription factor BnaMYB52 is shown in any one of SEQ ID NO.5‑8. The present invention reveals for the first time the negative regulatory role of transcription factor BnaMYB52 in rapeseed drought resistance, and provides a new molecular target and genetic improvement strategy for improving rapeseed drought resistance. Through in-depth research, it was found that knocking out the BnaMYB52 gene can significantly enhance the tolerance of rapeseed plants to drought environments. In addition, the new drought-resistant rapeseed germplasm obtained after genetic modification using this gene showed excellent adaptability under drought stress environments. This technological breakthrough not only helps to solve the practical problems faced by rapeseed cultivation in arid areas, but also lays a solid theoretical foundation and technical support for cultivating high-yield and high-quality rapeseed varieties that adapt to extreme climatic conditions.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of transcription factor BnaMYB52 in regulating the drought resistance of rapeseed. Background Art

[0002] Brassica napus (AACC, 2n=38, hereafter referred to as rapeseed) is an important oilseed crop, accounting for over 47% of total oilseed oil production and making it one of the most important sources of edible vegetable oil. Drought, as an abiotic stress factor, not only affects normal plant growth and development but also directly threatens crop yield and quality. Although recent research has focused on cloning and functional characterization of drought-resistance genes in rapeseed, the molecular mechanisms underlying drought resistance remain largely unknown, hindering the development of drought-resistant varieties. Therefore, identifying drought-resistance genes in rapeseed and elucidating their regulatory mechanisms are crucial for achieving high and stable yields.

[0003] To address these challenges, this study aims to identify drought-resistant genes in rapeseed and analyze their regulatory mechanisms under drought stress. This in-depth research will reveal the functions of these genes and their mechanisms of action in plant responses to drought. This will provide a theoretical basis and technical support for breeding rapeseed varieties with enhanced drought tolerance. This will also help improve rapeseed's adaptability to extreme environments, ensuring its production stability and economic benefits. Summary of the Invention

[0004] The present invention aims to provide a method for regulating drought resistance in rapeseed by using the transcription factor BnaMYB52, thereby overcoming the problems of the prior art. The present invention has found that the BnaMYB52 gene plays a negative regulatory role in drought resistance in rapeseed, and knocking out this gene can significantly enhance the tolerance of rapeseed plants to drought conditions.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of a biological material in constructing a highly drought-resistant rapeseed variety, wherein the biological material is used to knock out the BnaMYB52 gene;

[0007] The BnaMYB52 gene includes BnaA08.MYB52 gene, BnaA09.MYB52 gene, BnaC08.MYB52-1 gene and BnaC08.MYB52-2 gene whose nucleotide sequences are shown in SEQ ID NO.1-4 respectively;

[0008] The biological material is a CRISPR gene knockout vector. The gene knockout target of the CRISPR gene knockout vector is located in the second exon region of each gene, and its nucleotide sequence is shown in SEQ ID NO.11.

[0009] Furthermore, the method for constructing the CRISPR gene knockout vector comprises the following steps:

[0010] Using the pCBC-DT1T2 plasmid as a template, PCR amplification was performed using primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR to obtain PCR amplification products;

[0011] The PCR amplification product was digested and ligated with the pKSE401 plasmid to obtain the CRISPR gene knockout vector;

[0012] The nucleotide sequences of the primers DT1-BsF, DT1-F0, DT2-R0 and DT2-BsR are shown in SEQ ID NOs. 13-16, respectively.

[0013] Furthermore, the biological material is a recombinant microbial strain, and the recombinant microbial strain includes a CRISPR gene knockout vector for knocking out the BnaMYB52 gene.

[0014] Furthermore, the method for constructing the CRISPR gene knockout vector comprises the following steps:

[0015] Using the pCBC-DT1T2 plasmid as a template, PCR amplification was performed using primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR to obtain PCR amplification products;

[0016] The PCR amplification product was digested and ligated with the pKSE401 plasmid to obtain the CRISPR gene knockout vector;

[0017] The nucleotide sequences of the primers DT1-BsF, DT1-F0, DT2-R0 and DT2-BsR are shown in SEQ ID NOs. 13-16, respectively.

[0018] The present invention also provides a method for constructing a highly drought-resistant rapeseed variety, comprising the steps of knocking out the BnaMYB52 gene in the rapeseed plant to construct the highly drought-resistant rapeseed variety;

[0019] The BnaMYB52 gene includes BnaA08.MYB52 gene, BnaA09.MYB52 gene, BnaC08.MYB52-1 gene and BnaC08.MYB52-2 gene whose nucleotide sequences are shown as SEQ ID NO.1-4 respectively.

[0020] Furthermore, the BnaMYB52 gene is knocked out using a biological material; the biological material is a CRISPR gene knockout vector or a recombinant microbial strain, and the recombinant microbial strain includes the CRISPR gene knockout vector;

[0021] The gene knockout target of the CRISPR gene knockout vector is located in the second exon region of each gene, and its nucleotide sequence is shown in SEQ ID NO.11.

[0022] The present invention discloses the following technical effects:

[0023] The present invention reveals for the first time the negative regulatory role of the transcription factor BnaMYB52 in rapeseed drought resistance, providing a new molecular target and genetic improvement strategy for improving rapeseed drought resistance. Through in-depth research, it was found that knocking out the BnaMYB52 gene can significantly enhance the tolerance of rapeseed plants to drought environments. In addition, the new drought-resistant rapeseed germplasm obtained after genetic modification using this gene showed excellent adaptability under drought stress environments. This technological breakthrough not only helps to solve the practical problems faced by rapeseed cultivation in arid areas, but also lays a solid theoretical foundation and technical support for cultivating high-yield and high-quality rapeseed varieties that adapt to extreme climatic conditions. Therefore, the present invention has important application prospects in the selection and improvement of new drought-resistant rapeseed germplasm, can be widely used in modern agricultural production, and promote the sustainable development of the rapeseed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is the result of the detection of the relative expression level of the BnaMYB52 gene in the rapeseed overexpression transformation material;

[0026] Figure 2 This is an analysis diagram of the target site editing status of gene knockout mutant materials;

[0027] Figure 3 This is a phenotypic analysis diagram of the overexpression and gene knockout mutant materials in Example 5;

[0028] Figure 4 This is a statistical graph of the survival rates of the overexpression and gene knockout mutant materials in Example 5. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The primer information involved in the present invention is shown in Table 1:

[0035] Table 1 Primer information involved in the present invention

[0036]

[0037] Example 1 Cloning of the rapeseed BnaA09.MYB52 gene

[0038] The MYB52 gene, encoding the MYB DOMAIN protein, belongs to the MYB gene family. There are four copies of MYB52 in rapeseed: one copy each on chromosomes A08 and A09, and two copies on chromosome C08. These copies are BnaA08.MYB52 (BnaA08G0236300WE), BnaA09.MYB52 (BnaA09G0583300WE), BnaC08.MYB52-1 (BnaC08G0420000WE), and BnaC08.MYB52-2 (BnaC08G0199000WE). Among them, BnaA09.MYB52 and BnaC08.MYB52-1 have the highest nucleotide sequence similarity. The nucleotide sequences of the BnaA08.MYB52, BnaA09.MYB52, BnaC08.MYB52-1 and BnaC08.MYB52-2 genes are shown in SEQ ID NOs. 1-4, respectively, and the amino acid sequences of the proteins encoded therein are shown in SEQ ID NOs. 5-8, respectively.

[0039] SEQ ID NO.1:.

[0040] SEQ ID NO.2:ATGATGTGTAGTCGAGGACATTGGAGACCTGCAGAGGATGAGAAGCTTCGAGAACTCGTTGAACAGTTTGGTCCTCATAATTGGAACGCCATAGCTCAGAAGCTCTCTGGTCGATCTGGTAAAAGTTGTAGATTAAGATGGTTTAATCAGTTGAATCCTAGAATTAACCGAAACCCTTTCACGGAGGATGAAGAAGAAAGGCTTCTAGCTTCTCATCGGATCCATGGAAACAGATGGTCCGTGATCGCAAGATTTTTCCCCGGTCGAACCGATAACGCTGTTAAAAACCATTGGCACGTCATCATGGCTCGTCGTGGCCGAGAACTGTCCAAGCTACGTCCACGTGGTCTCGGCCATGATGGCACCACGGCTGCGACGATTGGTTATGACGGCTGCGATAAAAAGAGAAGATTGGCAACCGCAAGCACTATCAGTTATCCTCACCAGTTCTCTCATATTAGTCATTTTCAGCTCCTCAAAGAGTTCTTGACCGGAAAGATCGGGTTATGCAATAATACTACTCCAATCAACGAAGGAGCGATAAACCAAACCAAAAGACCGATTGAGTTCTACGATTTTCTCCAAGTCAAGTCGGATTCAAAGAAACCCGAAGTGATAGACAATTCAAGAAAATACGAAGAAGAAGATGGTGTTTCTGAACACAGCCACAATCACAACGAGAATTGTGTTCCCTTTATCGACTTTTTGTCTGTTGGAAACTCTGCCTCTCAGAGTTTATGTTAA.

[0041] SEQ ID NO.3:.

[0042] SEQ ID NO.4:.

[0043] SEQ ID NO.5:MMCSRGHWRPAEDEKLRELVQQFGPHNWNAIAQKLTGRSGKSCRLRWFNQLDPRINRTPFTEEEEKRLLASHRIHGNRWSVIARFFPGRTDNAVKNHWHVIMARRGRERSKMRPRSLGHDGTEAGPGMMGYKDCDKKRRLATTIDINYPYSFSHINHFQILKEFLTGKIGFCNNTTPINEGALDQTKRPMEFYNFLQVKTDSKPEVIHNSRKEEDEEEEDVDNCVPFIDFLSVGNSASKGLC.

[0044] SEQ ID NO.6:MMCSRGHWRPAEDEKLRELVEQFGPHNWNAIAQKLSGRSGKSCRLRWFNQLNPRINRNPFTEDEEERLLASHRIHGNRWSVIARFFPGRTDNAVKNHWHVIMARRGRELSKLRPRGLGHDGTTAATIGYDGCDKKRRLATASTISYPHQFSHISHFQLLKEFLTGKIGLCNNTTPINEGAINQTKRPIEFYDFLQVKSDSKKPEVIDNSRKYEEEDGVSEHSHNHNENCVPFIDFLSVGNSASQSLC.

[0045] SEQ ID NO.7:MMCSRGHWRPAEDEKLRELVEQFGPHNWNAIAQKLSGRSGKSCRLRWFNQLDPRINRNPFTEDEEERLLASHRIHGNRWSVIARFFPGRTDNAVKNHWHVIMARRGRELSKLRPRDLGHDGTKAATIGYDGCDKKRRLATATTISYPHQFSHISHFQLLKEFLTGKIGLCNNTTPINEGAINQSKRPMEFYDFLQVKTDSKKPEVLDNSRKYEEEDDVSEHNHNHNENCVPFIDFLSVGNSASQGLC.

[0046] SEQ ID NO.8: MMCSRGHWRPAEDEKLKELVQQFGPHNWNAIAQKLTGRSGKSCRLRWFNQLDPRINRNPFTEEEEERLLASHRIHGNRWSVIARFFPGRTDNAVKNHWHVIMARRGRERSKMRPRSLGH DGTAAGPGMMGYKDCDKKRKLATTTAINYPYSFSHINHFQILKEFLTGKIGFCNNTTPINDGAIDQTKRPMEFYNFLQVKTDSKKPEVIDHSRKDEEEEDVENCVPFFDFLSVGNSASHQGLC.

[0047] (1) RNA extraction

[0048] Total RNA was extracted using TansZol (catalog number ET101) from Quanshijin Company. Brassica napus seeds at 40 DAF were ground in liquid nitrogen. 100 mg of the ground sample was transferred to a 1.5 mL centrifuge tube and 1 mL of TransZol was added. The tube was vigorously inverted several times to mix thoroughly and allowed to stand at room temperature for 5 minutes. 0.2 mL of chloroform was added and the tube was vigorously shaken for 15 seconds and incubated at room temperature for 3 minutes. The tube was centrifuged at 10,000 × g and 4°C for 15 minutes. At this time, the sample was separated into three layers: a colorless aqueous phase (upper layer), a middle layer, and a pink organic phase (lower layer). The colorless aqueous phase was transferred to a new centrifuge tube, 0.5 mL of isopropanol was added, the tube was inverted to mix thoroughly, and the tube was incubated at room temperature for 10 minutes. The tube was centrifuged at 10,000 × g and 4°C for 10 minutes. The supernatant was removed and a colloidal precipitate formed on the side and bottom of the tube. 1 mL of Prepare 75% ethanol (prepared with diethylpyrocarbonate-treated water) by vigorous vortexing; centrifuge at 7500 × g at 4°C for 5 minutes; discard the supernatant and air-dry the pellet at room temperature; dissolve the pellet in 50-100 μL of RNA dissolution buffer; incubate at 55°C for 10 minutes. Measure the RNA concentration of 1 μL of the extracted total RNA using a Nanodrop microspectrophotometer. The concentration is determined based on an OD value of 1.8 < 0. 260 / OD 280 RNA purity was determined to be <2.0. 1 µL was also sampled for 1% agarose gel electrophoresis to check RNA integrity.

[0049] (2) cDNA synthesis

[0050] Reverse transcription was performed using the full-length Gold EasyScript ®One-Step gDNA Removal and cDNA Synthesis SuperMix (Catalog No. AE311). 1 μg of total RNA was used as a template, and 1 μL of Anchored Oligo (dT) 18 Primer, 10 μL of 2× ES Reaction Mix, and EasyScript ® 1 μL of RT / RI Enzyme Mix, 1 μL of gDNA Removal Agent, and enzyme-free sterile water were added to 20 μL. The above system was gently mixed and incubated at 42°C for 30 minutes. This step is to synthesize the first-strand cDNA and remove gDNA. Heat at 85°C for 5 seconds to inactivate EasyScript ® RT / RI and gDNA Remover. Add 180 μL of enzyme-free sterile water to dissolve the synthesized cDNA and set aside.

[0051] (3) Amplification of the BnaA09.MYB52 gene

[0052] The full-length CDS fragment of BnaA09.MYB52 (with the stop codon removed) was amplified using the above cDNA as a template using the forward primer BnaA09.MYB52-pCAMBIA2306-F and the reverse primer BnaA09.MYB52-pCAMBIA2306-R. TM PCR amplification was performed using 2× High-Fidelity Master Mix (Beijing Qingke Biotechnology Co., Ltd.).

[0053] PCR amplification system: 2×I-5 TM 2× High-Fidelity Master Mix 25 μL, forward primer (10 μmoL / L) 2.5 μL, reverse primer (10 μmoL / L) 2.5 μL, cDNA 3 μL, ddH2O 17 μL.

[0054] PCR amplification program: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 30 sec, 34 cycles; extension at 72°C for 5 min.

[0055] The amplified product was detected by agarose gel electrophoresis, and a 744 bp full-length CDS sequence of BnaA09.MYB52 was amplified and recovered using the Tiangen Agarose Gel Recovery Kit.

[0056] Example 2 Construction of BnaA09.MYB52 gene overexpression transformation vector

[0057] (1) Double-digest the vector 35S-pCAMBIA2306 with the fast restriction endonucleases Kpn I and Bam HI. The double-digestion system is: 10 μL of 5× fast digestion buffer, 1 μL of Kpn I, 1 μL of Bam HI, 20 μL of recovered product / plasmid, and 18 μL of ddH2O.

[0058] The enzyme digestion reaction was carried out in a 37°C water bath for 3 h, and the digestion products were recovered using the Tiangen DNA purification kit.

[0059] (2) Ligate the CDS fragment of the BnaA09.MYB52 gene with the fragment recovered after enzyme digestion of the vector obtained in step (1) to obtain a recombinant plasmid. The recombinant plasmid contains a constitutive expression promoter and an antibiotic marker. The ligation reaction system is: 6 μL of the CDS fragment of the BnaA09.MYB52 gene, 1 μL of the fragment recovered after enzyme digestion of the vector, 1 μL of Exnase II (Vazyme), and 2 μL of 5×CE II buffer.

[0060] Ligation reaction conditions: 37°C for 30 min.

[0061] (3) The recombinant plasmid was transformed into Escherichia coli DH5α. The transformation method is as follows:

[0062] 10 µL of the recombinant plasmid was added to 50 µL of DH5α competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.) and mixed by pipetting. The cells were placed on ice for 30 minutes. The cells were then incubated in a 42°C water bath for 1.5 minutes and then placed on ice for 3 minutes. The cells were then activated with 400 µL of antibiotic-free liquid LB medium and shaken at 37°C at 150 rpm for 45 minutes. 200 µL of the activated bacterial suspension was spread onto solid LB medium containing the corresponding resistance and incubated upside down at 37°C for 16 hours. Positive clones were screened and identified by enzyme digestion. Three positive clones were selected for sequencing. Analysis revealed that the CDS fragment of the BnaA09.MYB52 gene was successfully ligated into the vector, indicating the successful construction of the recombinant plasmid 35S-pCAMBIA2306-BnaA09.MYB52 for transforming plants.

[0063] (4) Introduce the correctly constructed recombinant plasmid into Agrobacterium GV3101 competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.), select positive single clones and store them in a -80℃ refrigerator. The introduction method is as follows:

[0064] a. Clean the cuvette: First, rinse with pure water, then with ultrapure water, discard, and then rinse with anhydrous ethanol (using a 1 mL pipette tip). Discard the anhydrous ethanol and place on a clean bench to dry.

[0065] b. Take 50 μL of competent Agrobacterium GV3101;

[0066] c. Take 1 μL of the correctly constructed recombinant plasmid and add it to 50 μL of competent medium. Mix thoroughly by gently pipetting to avoid creating bubbles.

[0067] d. Place the washed and dried electric rotating cup in ice to pre-cool, and then pour the above mixture against the wall of the cup;

[0068] e. Adjust the electroporator to 1800 V;

[0069] f. Remove the electroporation cup from the ice and wipe the outer wall of the electroporation cup with absorbent paper;

[0070] g. Place the electroporation cup into the instrument and start the electric shock;

[0071] h. After successful electroporation, add 400 μL of antibody-free LB to the cuvette, pipette to mix, and transfer to a sterile centrifuge tube.

[0072] i. Activate at 28°C for approximately 1 hour. Apply 100 μL of the solution to a plate containing the desired resistance. Seal with sealing film and incubate upside down at 28°C for 2 days. Perform spot detection.

[0073] (5) Agrobacterium colony detection

[0074] Select colonies and culture them in double-antibody LB medium at 28°C for 1 h. Take an appropriate amount of bacterial solution for PCR detection and save the positive Agrobacterium bacterial solution.

[0075] Example 3 Construction of BnaMYB52-CRISPR vector

[0076] The sgRNA-Cas9 system was used to create the rapeseed BnaMYB52 mutant. The experimental steps are as follows:

[0077] (1) Log in to the website http: / / crispr.hzau.edu.cn / CRISPR2 / and screen the target sites sgRNA1: ATAACGCTGTTAAAAACCAT (SEQ ID NO.11) and sgRNA2: GCTGCGACGATTGGTTATGA (SEQ ID NO.12), which are located in the second exon of genes BnaA09.MYB52 and BnaC08.MYB52-1, respectively. In BnaA08.MYB52 and BnaC08.MYB52-2, only the sgRNA1 target site is located in the second exon of the gene. Therefore, sgRNA1 can target all BnaMYB52 genes at the same time, while sgRNA2 can target BnaA09.MYB52 and BnaC08.MYB52-1.

[0078] (2) Primers DT1-BsF, DT1-F0, DT2-R0 and DT2-BsR were designed.

[0079] (3) PCR amplification: Four-primer PCR amplification was performed using a 100-fold diluted pCBC-DT1T2 plasmid (kindly provided by Professor Chen Qijun of China Agricultural University) as a template. DT1-BsF and DT2-BsR were at normal primer concentrations; DT1-F0 and DT2-R0 were diluted 20-fold.

[0080] Amplification system: 2×I-5 TM 2×High-Fidelity Master Mix 25 μL, DT1-BsF (10 μmol / L) 2 μL, DT2-BsR (10 μmol / L) 2 μL, DT1-F0 (0.5 μmol / L) 2 μL, DT2-R0 (0.5 μmol / L) 2 μL, pCBC-DT1T2 plasmid 3 μL, ddH2O 14 μL.

[0081] PCR amplification program: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 15 sec, annealing at 56°C for 30 sec, extension at 68°C for 30 sec, 34 circles; extension at 68°C for 5 min.

[0082] (4) Purify and recover the PCR product, and establish the following enzyme digestion-ligation system: 2 μL of PCR amplification product, 2 μL of pKSE401 plasmid (kindly donated by Professor Chen Qijun of China Agricultural University), 1.5 μL of 10×NEB T4 Buffer, 1.5 μL of 10× bovine serum albumin (BSA), 1 μL of BsaI (NEB), 1 μL of T4 Ligase (NEB), and 6 μL of ddH2O.

[0083] Reaction conditions: 37℃ for 5 h, 50℃ for 5 min, and 80℃ for 10 min.

[0084] (5) Transformation into E. coli DH5α: Take 5 μL of the ligation system to transform competent E. coli, screen on a Kan plate, identify positive clones by PCR, and sequence them. The vector that is correctly sequenced is the BnaMYB52-CRISPR gene knockout vector.

[0085] (6) Introduce the correctly constructed BnaMYB52-CRISPR gene knockout vector into Agrobacterium GV3101 competent cells, select positive single clones and store them in a -80℃ refrigerator. The introduction method is as follows:

[0086] a. Clean the cuvette: First, rinse with pure water, then with ultrapure water, discard, and then rinse with anhydrous ethanol (using a 1 mL pipette tip). Discard the anhydrous ethanol and place on a clean bench to dry.

[0087] b. Take 50 μL of competent Agrobacterium GV3101;

[0088] c. Take 1 μL of the correctly constructed recombinant plasmid and add it to 50 μL of competent medium. Mix thoroughly by gently pipetting to avoid creating bubbles.

[0089] d. Place the washed and dried electric rotating cup in ice to pre-cool, and then pour the above mixture against the wall of the cup;

[0090] e. Adjust the electroporator to 1800 V;

[0091] f. Remove the electroporation cup from the ice and wipe the outer wall of the electroporation cup with absorbent paper;

[0092] g. Place the electroporation cup into the instrument and start the electric shock;

[0093] h. After successful electroporation, add 400 μL of antibody-free LB to the cuvette, pipette a few times, and transfer to a sterile centrifuge tube.

[0094] i. Activate at 28°C for approximately 2 hours. Apply 100 μL of the solution to a plate containing the corresponding resistance. Seal with sealing film and incubate upside down at 28°C for 2 days. Perform spot detection.

[0095] (7) Agrobacterium colony detection

[0096] Select colonies and culture them in double-antibody LB medium at 28°C for 2 h. Take an appropriate amount of bacterial solution for PCR detection and save the positive Agrobacterium bacterial solution.

[0097] Example 4 Genetic transformation experiment

[0098] (1) Genetic transformation of overexpression materials and gene knockout mutant materials

[0099] Genetic transformation of rapeseed was performed using Agrobacterium tumefaciens containing the recombinant plasmid 35S-pCAMBIA2306-BnaA09.MYB52 constructed in Example 2 and Agrobacterium tumefaciens containing the BnaMYB52-CRISPR gene knockout vector constructed in Example 3. Westar, a Brassica napus plant, was used as the recipient for transformation. The detailed procedure is described in the reference: Dai C, Li Y, Li L, et al. An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus[J]. Molecular Breeding, 2020, 40: 1-13.

[0100] (2) Identification of overexpression transformed materials

[0101] Genomic DNA was extracted from the transformed rapeseed plants undergoing overexpression, and PCR was used to detect the insertion of the exogenous gene fragment. The overexpression backbone vector used in this invention is 35S-pCAMBIA2306. Primers pCAMBIA2306-R were designed on the vector backbone. PCR detection was performed using primers for the exogenous fragment (BnaA09.MYB52-pCAMBIA2306-F) and the vector backbone primers pCAMBIA2306-R. Transgenic seedlings were detected by PCR. The PCR reaction system consisted of: 5 µL of Taq polymerase mix; 0.5 µL of pCAMBIA2306-R (10 µmol / L); 0.5 µL of BnaA09.MYB52-pCAMBIA2306-F (10 µmol / L); 1 µL of gDNA; and 3 µL of ddH2O. PCR reaction conditions: pre-denaturation at 94°C for 5 min; 34 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 1 min; and extension at 72°C for 5 min.

[0102] qRT-PCR was performed on transgenic rapeseed seedlings identified by PCR to measure gene expression. RNA was extracted from leaves of individual transformed plants, and cDNA was synthesized (using the same method as in Example 1). Quantitative primers were designed using Primer 5 software, with product sizes ranging from 80 to 250 bp. BLAST comparisons were performed using reference sequences to ensure primer specificity. Finally, primers BnMYB52-RT-F and BnMYB52-RT-R were designed. BnaACTIN7-F / R were used as internal reference primers for qRT-PCR in rapeseed.

[0103] The qRT-PCR reaction system was as follows: 6.9 μL of cDNA diluted 10-fold, 0.3 μL of forward primer (10 μM), 0.3 μL of reverse primer 2 (10 μM), 2× TransStart ® Green qPCR SuperMix 7.5 μL.

[0104] The qRT-PCR reaction program was as follows: 94°C for 30 s, followed by 45 cycles of 94°C for 10 s, 60°C for 15 s, and 72°C for 30 s. Melting curves were generated. qRT-PCR was performed using the Bio-Rad CFX96 Real-Time Detection System.

[0105] Normalization was performed based on the internal reference primers, and the quantitative variation between replicates was determined using 2 -ΔΔCT Finally, the relative expression levels of rapeseed overexpression transformation materials OE-1 and OE-2 were obtained by analysis. Figure 1 .

[0106] (3) Identification of gene knockout mutant materials

[0107] The obtained rapeseed CRISPR-transformed plants were sequenced to screen for rapeseed mutants. The Cas9 protein was first identified using primers Cas9-570-F and Cas9-570-R. For Cas9 protein-positive plants, the target gene was specifically amplified and sequenced. The method for specific amplification of the target gene is as follows: primers MYB52(A8)-CRISPR-F and MYB52(A8)-CRISPR-R are used to specifically amplify BnaA08.MYB52; primers MYB52(A9)-CRISPR-F and MYB52(A9)-CRISPR-R are used to specifically amplify BnaA09.MYB52; primers MYB52(C8-1)-CRISPR-F and MYB52(C8-1)-CRISPR-R are used to specifically amplify BnaC08.MYB52-1; primers MYB52(C8-2)-CRISPR-F and MYB52(C8-2)-CRISPR-R are used to specifically amplify BnaC08.MYB52-2. The amplification method is as follows:

[0108] The PCR reaction system consisted of 20 μL of 2× Taq Master Mix, 2 μL of DNA template, 1.6 μL of forward primer, and 1.6 μL of reverse primer, supplemented with ddH₂O to 40 μL. The PCR reaction program was as follows: initial denaturation at 94°C for 5 min; 32 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 30 sec; and extension at 72°C for 5 min.

[0109] The amplified target fragments were sequenced, and the sequencing results were analyzed using the DSDecode online website (http: / / skl.scau.edu.cn / dsdecode / ) to analyze the editing status of the target sites. Sequencing results showed that two independent mutant lines (bnamyb52-1 and bnamyb52-2) were obtained in which all four BnaMYB52 homologous genes were knocked out; Figure 2 ).

[0110] Example 5 Phenotypic Analysis of Overexpression and Knockout Mutant Materials

[0111] Homozygous knockout mutant, overexpression, and wild-type seeds were harvested from the field and subjected to drought stress at the seedling stage in a greenhouse. The greenhouse temperature was maintained at 25°C with a 16 h / 8 h light / dark cycle. Control and treatment groups were established, with at least 30 seedlings planted for each line for sampling and survival analysis. Both seedlings were grown with normal watering until they were four weeks old. Watering was then discontinued for the treatment group and drought treatment began, while the control group maintained adequate water. After seven days of drought stress, phenotypic observations, photographs, and sampling were performed. Seedlings in the treatment groups were subjected to continued drought stress. After the majority of the lines that first exhibited drought-sensitive phenotypes had died, all drought-treated groups were rewatered (i.e., rewatered), and survival rates were calculated. Phenotypes and survival rates were verified in three replicates. Survival rate = number of surviving seedlings / total number of seedlings × 100%.

[0112] The results of the indoor drought test showed that there was no significant difference in the growth of 4-week-old seedlings in the control group under normal water supply conditions; after drought stress treatment, the two overexpression lines (OE-1 and OE-2) showed more severe wilting compared to the wild type (WT). In contrast, the two mutant lines (bnamyb52-1 and bnamyb52-2) showed stronger drought resistance. After a period of treatment, rewatering was performed, and the survival of the seedlings was counted after 3 days. After 3 days of rewatering, compared with the wild type, the mutant lines showed more single plant survival, while the overexpression lines showed more plant deaths ( Figure 3 The drought test was repeated three times, and the survival rate was calculated each time. The results showed that compared with the wild-type plants, the survival rate of the mutant plants was significantly increased, while the survival rate of the overexpression plants was significantly decreased ( Figure 3-Figure 4 ).

[0113] In summary, the experimental results show that gene BnaMYB52 plays an important role in regulating rapeseed drought resistance.

[0114] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An application of a biomaterial in improving the drought resistance of rapeseed, characterized in that: The biological material is used to knock out the BnaMYB52 gene; The BnaMYB52 gene includes BnaA08.MYB52 gene, BnaA09.MYB52 gene, BnaC08.MYB52-1 gene and BnaC08.MYB52-2 gene whose nucleotide sequences are shown in SEQ ID NO.1-4 respectively; The biological material is a CRISPR gene knockout vector. The gene knockout target of the CRISPR gene knockout vector is located in the second exon region of each gene, and its nucleotide sequence is shown in SEQ ID NO.

11.

2. The use according to claim 1, characterized in that The method for constructing the CRISPR gene knockout vector comprises the following steps: Using the pCBC-DT1T2 plasmid as a template, PCR amplification was performed using primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR to obtain PCR amplification products; The PCR amplification product was digested and ligated with the pKSE401 plasmid to obtain the CRISPR gene knockout vector; The nucleotide sequences of the primers DT1-BsF, DT1-F0, DT2-R0 and DT2-BsR are shown in SEQ ID NOs. 13-16, respectively.

3. The use according to claim 1, characterized in that The biological material is a recombinant microbial strain, and the recombinant microbial strain includes a CRISPR gene knockout vector for knocking out the BnaMYB52 gene.

4. The use according to claim 3, characterized in that The method for constructing the CRISPR gene knockout vector comprises the following steps: Using the pCBC-DT1T2 plasmid as a template, PCR amplification was performed using primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR to obtain PCR amplification products; The PCR amplification product was digested and ligated with the pKSE401 plasmid to obtain the CRISPR gene knockout vector; The nucleotide sequences of the primers DT1-BsF, DT1-F0, DT2-R0 and DT2-BsR are shown in SEQ ID NOs. 13-16, respectively.

5. A method for improving drought resistance of rapeseed, characterized in that: The method comprises the steps of knocking out the BnaMYB52 gene in rapeseed plants, and then amplifying and sequencing the rapeseed plants through PCR to construct a highly drought-resistant rapeseed variety. The BnaMYB52 gene includes BnaA08.MYB52 gene, BnaA09.MYB52 gene, BnaC08.MYB52-1 gene and BnaC08.MYB52-2 gene whose nucleotide sequences are shown in SEQ ID NO.1-4 respectively; The BnaMYB52 gene is knocked out using a biological material; the biological material is a CRISPR gene knockout vector or a recombinant microbial strain, and the recombinant microbial strain includes the CRISPR gene knockout vector; The gene knockout target of the CRISPR gene knockout vector is located in the second exon region of each gene, and its nucleotide sequence is shown in SEQ ID NO.11.

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