Application of transcription factor BnaMYB52 in regulation and control of drought resistance of rape

Abstract

The invention discloses application of a transcription factor BnaMYB52 in regulation and control of drought resistance of rape, and relates to the technical field of biology. The amino acid sequence of the transcription factor BnaMYB52 is as shown in any one of SEQ ID NO. 5 to 8. According to the invention, the negative regulation effect of the transcription factor BnaMYB52 in the drought resistance of the rape is disclosed for the first time, and a brand new molecular target and a genetic improvement strategy are provided for improving the drought resistance of the rape. Through in-depth study, it is found that knockout of the BnaMYB52 gene can significantly enhance the tolerance of rape plants to the drought environment. In addition, a new drought-resistant rape germplasm obtained after genetic modification of the gene shows excellent adaptive capacity in a drought stress environment. The technical breakthrough not only helps to solve practical problems faced by rape planting in arid regions, but also lays a solid theoretical foundation and technical support for cultivating high-yield and high-quality rape varieties adapting to extreme climate conditions.

Description

Technical Field

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

[0002] Brassica napus (AACC, 2n = 38, hereinafter referred to as rapeseed) is an important oil crop, and the rapeseed oil output accounts for more than 47% of the oil crops, being one of the most important sources of edible vegetable oil. As an abiotic stress factor, drought not only affects the normal growth and development of plants, but also directly threatens the yield and quality of crops. For rapeseed, although some studies have focused on the cloning and functional identification of its drought-related genes in recent years, the molecular mechanism of rapeseed drought resistance is still unclear, which limits the cultivation of drought-resistant varieties. Therefore, exploring rapeseed drought-resistant genes and analyzing their drought resistance regulation mechanisms are of great significance for high and stable yields of rapeseed.

[0003] To address the above challenges, the present invention is committed to exploring drought-resistant genes in rapeseed and analyzing their regulation mechanisms under drought stress conditions. Through in-depth research, revealing the functions of rapeseed drought-resistant genes and their action mechanisms in the process of plant response to drought will provide a theoretical basis and technical support for cultivating rapeseed varieties with higher drought resistance. At the same time, this will also help improve the adaptability of rapeseed in extreme environments and ensure its production stability and economic benefits. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of transcription factor BnaMYB52 in regulating the drought resistance of rapeseed to solve the problems existing in the above prior art. The present invention has found through research that the BnaMYB52 gene has a negative regulatory effect on the drought resistance of rapeseed, and knocking out this gene can significantly enhance the tolerance of rapeseed plants to drought environments.

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

[0006] The present invention provides the application of a biological material in constructing rapeseed varieties with high drought resistance, and the biological material is used to knock out the BnaMYB52 gene;

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

[0008] The biological material is a CRISPR gene knockout vector, and 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 as shown in SEQ ID NO.11.

[0009] Furthermore, the construction method of the CRISPR gene knockout vector includes the following steps:

[0010] Using the pCBC-DT1T2 plasmid as a template, PCR amplification is carried out using primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR to obtain a PCR amplification product;

[0011] The PCR amplification product is subjected to restriction enzyme ligation 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 respectively as shown in SEQ ID NO.13-16.

[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 construction method of the CRISPR gene knockout vector includes the following steps:

[0015] Using the pCBC-DT1T2 plasmid as a template, PCR amplification is carried out using primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR to obtain a PCR amplification product;

[0016] The PCR amplification product is subjected to restriction enzyme ligation 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 respectively as shown in SEQ ID NO.13-16.

[0018] The present invention also provides a method for constructing a rapeseed variety with high drought resistance, including the step of knocking out the BnaMYB52 gene in the rapeseed plant to construct the rapeseed variety with high drought resistance;

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

[0020] Further, the BnaMYB52 gene is knocked out using a biomaterial; the biomaterial 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 as shown in SEQ ID NO.11.

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

[0023] The present invention for the first time reveals the negative regulatory role of the transcription factor BnaMYB52 in the drought resistance of rapeseed, providing a brand-new molecular target and genetic improvement strategy for improving the drought resistance of rapeseed. Through in-depth research, it is 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 by genetic transformation using this gene shows excellent adaptability under drought stress conditions. This technological breakthrough not only helps to solve the practical problems faced by rapeseed cultivation in arid regions, but also lays a solid theoretical foundation and technical support for cultivating high-yield and high-quality rapeseed varieties adapted to extreme climate conditions. Therefore, the present invention has important application prospects in the breeding and improvement of new drought-resistant rapeseed germplasm, can be widely applied to modern agricultural production, and promotes the sustainable development of the rapeseed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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 use in the embodiments. Obviously, the drawings in the following description 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.

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

[0026] Figure 2 It is an analysis diagram of the target site editing of the gene knockout mutant material;

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

[0028] Figure 4 It is a statistical chart of the survival rates of the overexpression and gene knockout mutant materials in Example 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

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

[0030] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation 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 related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0034] The primer information related to the present invention is shown in Table 1 below:

[0035] Table 1 Primer Information Related to the Present Invention

[0036]

[0037] Example 1 Cloning of the Rapeseed BnaA09.MYB52 Gene

[0038] The MYB52 gene encodes a MYB DOMAIN protein and belongs to the MYB gene family. There are four copies of MYB52 in rapeseed, with one copy each on chromosomes A08 and A09, and two copies on chromosome C08, which are BnaA08.MYB52 (BnaA08G0236300WE), BnaA09.MYB52 (BnaA09G0583300WE), BnaC08.MYB52-1 (BnaC08G0420000WE), and BnaC08.MYB52-2 (BnaC08G0199000WE). Among them, the nucleotide sequence similarity between BnaA09.MYB52 and BnaC08.MYB52-1 is the highest. The nucleotide sequences of the BnaA08.MYB52, BnaA09.MYB52, BnaC08.MYB52-1, and BnaC08.MYB52-2 genes are shown in SEQ ID NO.1-4 respectively, and the amino acid sequences of the proteins they encode are shown in SEQ ID NO.5-8 respectively.

[0039] SEQ ID NO.1: ATGATGTGCAGCCGAGGCCATTGGAGACCTGCAGAAGATGAGAAGCTAAGAGAACTTGTCCAACAATTTGGTCCTCATAATTGGAACGCCATAGCTCAAAAGCTCACTGGTCGATCTGGTAAGAGTTGTAGATTGAGATGGTTTAATCAACTGGATCCTAGAATTAACCGAACCCCTTTTACGGAGGAAGAAGAAAAGAGGCTTTTAGCGTCTCATCGGATCCATGGGAACAGATGGTCCGTGATCGCAAGGTTTTTCCCCGGTCGAACTGATAACGCTGTTAAAAACCATTGGCACGTTATTATGGCTCGTCGTGGTCGAGAACGGTCCAAGATGCGTCCACGTAGCCTTGGCCATGATGGCACGGAGGCTGGGCCTGGGATGATGGGATATAAAGACTGTGACAAGAAGAGAAGATTGGCAACCACAATAGATATCAATTATCCTTATAGTTTCTCTCATATCAATCATTTTCAAATCCTCAAAGAGTTCTTGACTGGAAAGATCGGGTTTTGCAATAATACCACTCCAATCAATGAAGGAGCGCTAGACCAGACTAAAAGACCAATGGAGTTCTATAATTTTCTCCAAGTTAAGACGGATTCGAAACCTGAAGTAATACACAATTCAAGAAAAGAAGAAGACGAAGAAGAAGAAGATGTTGACAATTGTGTTCCCTTTATCGACTTTTTGTCTGTTGGAAACTCTGCCTCTAAGGGTTTATGTTAA.

[0040] SEQ ID NO.2: ATGATGTGTAGTCGAGGACATTGGAGACCTGCAGAGGATGAGAAGCTTCGAGAACTCGTTGAACAGTTTGGTCCTCATAATTGGAACGCCATAGCTCAGAAGCTCTCTGGTCGATCTGGTAAAAGTTGTAGATTAAGATGGTTTAATCAGTTGAATCCTAGAATTAACCGAAACCCTTTCACGGAGGATGAAGAAGAAAGGCTTCTAGCTTCTCATCGGATCCATGGAAACAGATGGTCCGTGATCGCAAGATTTTTCCCCGGTCGAACCGATAACGCTGTTAAAAACCATTGGCACGTCATCATGGCTCGTCGTGGCCGAGAACTGTCCAAGCTACGTCCACGTGGTCTCGGCCATGATGGCACCACGGCTGCGACGATTGGTTATGACGGCTGCGATAAAAAGAGAAGATTGGCAACCGCAAGCACTATCAGTTATCCTCACCAGTTCTCTCATATTAGTCATTTTCAGCTCCTCAAAGAGTTCTTGACCGGAAAGATCGGGTTATGCAATAATACTACTCCAATCAACGAAGGAGCGATAAACCAAACCAAAAGACCGATTGAGTTCTACGATTTTCTCCAAGTCAAGTCGGATTCAAAGAAACCCGAAGTGATAGACAATTCAAGAAAATACGAAGAAGAAGATGGTGTTTCTGAACACAGCCACAATCACAACGAGAATTGTGTTCCCTTTATCGACTTTTTGTCTGTTGGAAACTCTGCCTCTCAGAGTTTATGTTAA.

[0041] SEQ ID NO.3: ATGATGTGTAGTCGAGGACATTGGAGACCTGCAGAGGATGAGAAGCTTCGAGAACTCGTTGAACAGTTTGGTCCTCATAATTGGAACGCCATAGCTCAGAAGCTCTCTGGTCGATCTGGTAAAAGTTGTAGACTAAGATGGTTTAATCAGTTGGATCCTAGAATTAACCGAAACCCGTTCACGGAGGATGAAGAAGAAAGGCTTTTAGCTTCTCATCGGATCCACGGGAACAGATGGTCCGTGATCGCAAGATTTTTCCCCGGTCGAACCGATAACGCTGTTAAAAACCATTGGCACGTCATCATGGCTCGTCGTGGCCGAGAACTGTCCAAGCTACGTCCACGTGATCTTGGCCATGATGGCACGAAGGCTGCGACGATTGGTTATGACGGCTGCGATAAGAAGAGAAGATTGGCAACCGCAACCACTATCAGTTATCCTCACCAGTTCTCACATATTAGTCATTTTCAGCTCCTCAAAGAGTTCTTGACCGGAAAGATCGGGTTATGCAATAATACTACTCCAATCAACGAAGGAGCGATAAACCAAAGCAAAAGACCGATGGAGTTCTACGATTTTCTCCAAGTCAAGACGGATTCAAAGAAACCCGAAGTGTTAGACAATTCAAGAAAATACGAAGAAGAAGATGATGTTTCTGAACACAACCACAATCACAACGAGAATTGTGTTCCCTTTATCGACTTTTTGTCTGTTGGAAACTCTGCCTCTCAGGGTTTATGTTAA.

[0042] SEQ ID NO.4: ATGATGTGCAGCCGAGGCCATTGGAGACCTGCAGAAGATGAGAAGCTAAAAGAACTTGTCCAACAATTTGGTCCTCATAATTGGAACGCCATAGCTCAAAAGCTCACTGGTCGATCTGGTAAGAGTTGTAGATTGAGATGGTTTAATCAACTGGATCCTAGAATTAACCGAAACCCTTTTACGGAGGAAGAAGAAGAGAGGCTTTTAGCGTCTCATCGGATCCATGGGAACAGATGGTCTGTGATCGCAAGGTTTTTCCCCGGTCGAACTGATAACGCTGTTAAAAACCATTGGCACGTTATTATGGCTCGTCGTGGTCGAGAACGGTCCAAGATGCGTCCACGTAGCCTTGGCCATGATGGCACGGCGGCTGGGCCTGGGATGATGGGATATAAGGACTGTGACAAGAAGAGAAAATTGGCAACCACAACAGCTATCAATTATCCTTATAGTTTCTCTCATATCAATCATTTTCAAATCCTCAAAGAGTTCTTGACGGGAAAGATCGGGTTTTGCAATAATACCACTCCAATCAATGACGGAGCAATAGACCAGACTAAAAGACCAATGGAGTTCTACAATTTTCTCCAGGTTAAGACGGATTCGAAGAAACCTGAAGTGATAGACCATTCAAGAAAAGACGAAGAAGAAGAAGATGTTGAAAATTGTGTTCCATTTTTCGACTTTTTATCTGTTGGAAACTCTGCCTCTCATCAGGGTTTATGTTAA.

[0043] SEQ ID NO.5: MMCSRGHWRPAEDEKLRELVQQFGPHNWNAIAQKLTGRSGKSCRLRWFNQLDPRINRTPFTEEEEKRLLASHRIHGNRWSVIARFFPGRTDNAVKNHWHVIMARRGRERSKMRPRSLGHDGTEAGPGMMGYKDCDKKRRLATTIDINYPYSFSHINHFQILKEFLTGKIGFCNNTTPINEGALDQTKRPMEFYNFLQVKTDSKPEVIHNSRKEEDEEEEDVDNCVPFIDFLSVGNSASKGLC.

[0044] SEQ ID NO.6: MMCSRGHWRPAEDEKLRELVEQFGPHNWNAIAQKLSGRSGKSCRLRWFNQLNPRINRNPFTEDEEERLLASHRIHGNRWSVIARFFPGRTDNAVKNHWHVIMARRGRELSKLRPRGLGHDGTTAATIGYDGCDKKRRLATASTISYPHQFSHISHFQLLKEFLTGKIGLCNNTTPINEGAINQTKRPIEFYDFLQVKSDSKKPEVIDNSRKYEEEDGVSEHSHNHNENCVPFIDFLSVGNSASQSLC.

[0045] SEQ ID NO.7: MMCSRGHWRPAEDEKLRELVEQFGPHNWNAIAQKLSGRSGKSCRLRWFNQLDPRINRNPFTEDEEERLLASHRIHGNRWSVIARFFPGRTDNAVKNHWHVIMARRGRELSKLRPRDLGHDGTKAATIGYDGCDKKRRLATATTISYPHQFSHISHFQLLKEFLTGKIGLCNNTTPINEGAINQSKRPMEFYDFLQVKTDSKKPEVLDNSRKYEEEDDVSEHNHNHNENCVPFIDFLSVGNSASQGLC.

[0046] SEQ ID NO.8: MMCSRGHWRPAEDEKLKELVQQFGPHNWNAIAQKLTGRSGKSCRLRWFNQLDPRINRNPFTEEEEERLLASHRIHGNRWSVIARFFPGRTDNAVKNHWHVIMARRGRERSKMRPRSLGHDGTAAGPGMMGYKDCDKKRKLATTTAINYPYSFSHINHFQILKEFLTGKIGFCNNTTPINDGAIDQTKRPMEFYNFLQVKTDSKKPEVIDHSRKDEEEEDVENCVPFFDFLSVGNSASHQGLC.

[0047] (1) RNA extraction

[0048] Total RNA was extracted using TansZol (Catalog No. ET101) from TransGen Biotech. 40 DAF seeds of Brassica napus were ground into powder 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 mixture was vigorously vortexed several times to ensure thorough mixing and then allowed to stand at room temperature for 5 minutes. 0.2 mL of chloroform was added, and the mixture was vigorously shaken for 15 seconds and incubated at room temperature for 3 minutes. The sample was centrifuged at 10,000 ×g at 4°C for 15 minutes. At this time, the sample was divided into three layers: a colorless aqueous phase (upper layer), an intermediate 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, and the mixture was inverted to mix and incubated at room temperature for 10 minutes. The sample was centrifuged at 10,000 ×g at 4°C for 10 minutes, and the supernatant was removed, leaving a gelatinous precipitate on the side and bottom of the tube. 1 mL of 75% ethanol (prepared with diethyl pyrocarbonate-treated water) was added, and the mixture was vigorously vortexed. The sample was centrifuged at 7,500×g at 4°C for 5 minutes. The supernatant was discarded, and the precipitate was air-dried at room temperature. The precipitate was dissolved in 50 - 100 μL of RNA lysis buffer and incubated at 55°C for 10 minutes. 1 μL of the extracted total RNA was used to measure the RNA concentration with a Nanodrop micro-spectrophotometer. The RNA purity was determined based on 1.8 < OD 260 / OD 280 <2.0. At the same time, 1 μL was taken for 1% agarose gel electrophoresis to detect integrity.

[0049] (2) cDNA synthesis

[0050] Reverse transcription was performed using TransGen EasyScript ®One-Step gDNA Removal and cDNA Synthesis SuperMix (Catalog No. AE311). Using 1 μg of total RNA as a template, sequentially add 1 μL of Anchored Oligo(dT)18 Primer, 10 μL of 2×ES Reaction Mix, 1 μL of EasyScript ® RT / RI Enzyme Mix, 1 μL of gDNA Remover, and supplement with nuclease-free and sterile water to 20 μL. Gently mix the above system and place it at 42°C for 30 min. This step is to synthesize the first-strand cDNA and remove gDNA. Inactivate EasyScript ® RT / RI and gDNA Remover by heating at 85°C for 5 seconds. Add 180 μL of nuclease-free and sterile water to dissolve the synthesized cDNA for later use.

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

[0052] Using the above cDNA as a template, amplify the full-length CDS fragment of BnaA09.MYB52 (stop codon removed) with the forward primer BnaA09.MYB52-pCAMBIA2306-F and the reverse primer BnaA09.MYB52-pCAMBIA2306-R. Use I-5 TM 2×High-Fidelity Master Mix (Beijing Tsingke Biotechnology Co., Ltd.) for PCR amplification.

[0053] The PCR amplification system is: 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 is detected by agarose gel electrophoresis. The full-length CDS sequence of BnaA09.MYB52 with a length of 744 bp is obtained by amplification, and the product is recovered using the Tiangen agarose gel recovery kit.

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

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

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

[0059] (2) The CDS fragment of the BnaA09.MYB52 gene was ligated to the fragment recovered after digestion of the vector obtained in step (1) to obtain a recombinant plasmid. This recombinant plasmid contained a constitutive expression promoter and an antibiotic marker. The ligation reaction system was as follows: 6 μL of the CDS fragment of the BnaA09.MYB52 gene, 1 μL of the fragment recovered after 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 was as follows:

[0062] Pipette 10 µL of the recombinant plasmid into 50 µL of DH5α competent cells (purchased from Beijing Tsingke Biotechnology Co., Ltd.), mix well by pipetting, and place on ice for 30 min; incubate in a 42°C water bath for 1.5 min, and then place on ice for 3 min; add 400 µL of antibiotic-free liquid LB medium, and activate at 37°C in a shaker at 150 r / min for 45 min; pipette 200 µL of the activated bacterial solution and spread it on a solid LB medium with the corresponding resistance, and incubate upside down at 37°C for 16 h. After screening for positive clones, the plasmids were extracted and identified by digestion. Three positive clones were selected for sequencing. The analysis results showed that the CDS fragment of the BnaA09.MYB52 gene was successfully ligated to the vector, that is, the recombinant plasmid 35S-pCAMBIA2306-BnaA09.MYB52 for transgenic plants was successfully constructed.

[0063] (4) The correctly constructed recombinant plasmid was introduced into Agrobacterium tumefaciens GV3101 competent cells (purchased from Beijing Tsingke Biotechnology Co., Ltd.), and positive monoclonal clones were selected and stored in an -80°C refrigerator. The introduction method was as follows:

[0064] a. Wash the electroporation cuvette: First wash with pure water, then with ultrapure water, pour out, then wash with absolute ethanol (pipette with a 1 mL pipette tip), pour out the absolute ethanol, and place it in the laminar flow hood to dry.

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

[0066] c. Take 1 μL of the correctly constructed recombinant plasmid and add it to 50 μL of the competent cells. Gently pipette and mix well to avoid generating bubbles;

[0067] d. Place the washed and dried electroporation cuvette on ice for pre-cooling, and then pipette the above mixture along the wall of the cuvette;

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

[0069] f. Take the electroporation cuvette out of the ice and wipe the outer wall of the cuvette clean with absorbent paper;

[0070] g. Put the electroporation cuvette into the instrument and start the electroporation;

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

[0072] i. Activate at 28 °C for about 1 h, take 100 μL and spread it on a plate containing the corresponding antibiotic resistance. Seal it with a sealing film and incubate it upside down in a 28 °C incubator for 2 days, and pick colonies for detection.

[0073] (5) Detection of Agrobacterium tumefaciens colonies

[0074] Pick colonies into double-antibiotic LB, culture at 28 °C for 1 h, take an appropriate amount of the bacterial solution for PCR detection, and preserve the positive Agrobacterium tumefaciens bacterial solution.

[0075] Example 3 Construction of BnaMYB52-CRISPR vector

[0076] Use the sgRNA-Cas9 system to create Brassica napus BnaMYB52 mutants. The experimental procedures 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 regions of genes BnaA09.MYB52 and BnaC08.MYB52-1 respectively, while only the sgRNA1 target site of BnaA08.MYB52 and BnaC08.MYB52-2 is located in the second exon region of the gene. Therefore, sgRNA1 can target all BnaMYB52 genes simultaneously, while sgRNA2 can target BnaA09.MYB52 and BnaC08.MYB52-1.

[0078] (2) Design primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR.

[0079] (3) PCR amplification: Using the pCBC-DT1T2 plasmid (kindly provided by Professor Chen Qijun of China Agricultural University) diluted 100-fold as the template for four-primer PCR amplification. DT1-BsF and DT2-BsR are at normal primer concentrations; DT1-F0 and DT2-R0 are diluted 20-fold.

[0080] The amplification system is: 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 cycles; extension at 68°C for 5 min.

[0082] (4) Purify and recover the PCR product, and establish the following digestion-ligation system: 2 μL of the PCR amplification product, 2 μL of the pKSE401 plasmid (kindly provided 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), 6 μL of ddH2O.

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

[0084] (5) Transform Escherichia coli DH5α: Take 5 μL of the ligation system to transform Escherichia coli competent cells, screen on Kan plates, and identify and sequence the positive clones by PCR. The vector with correct sequencing is the BnaMYB52-CRISPR gene knockout vector.

[0085] (6) Introduce the correctly constructed BnaMYB52-CRISPR gene knockout vector into Agrobacterium tumefaciens GV3101 competent cells, and select positive monoclonal clones for storage in an -80°C refrigerator. The introduction method is as follows:

[0086] a. Wash the electroporation cuvette: First wash it with pure water, then with ultrapure water, pour out the water, and then wash it with anhydrous ethanol (pipette with a 1 mL pipette tip), pour out the anhydrous ethanol, and place it in the laminar flow hood to dry.

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

[0088] c. Take 1 μL of the correctly constructed recombinant plasmid and add it to 50 μL of the competent cells. Gently pipette to mix well, avoiding the generation of bubbles.

[0089] d. Place the washed and dried electroporation cuvette in ice for pre-cooling, and then pipette the above mixture along the wall of the cuvette.

[0090] e. Set the electroporator to 1800 V.

[0091] f. Take the electroporation cuvette out of the ice and wipe the outer wall of the cuvette clean with absorbent paper.

[0092] g. Put the electroporation cuvette into the instrument and start the electroporation.

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

[0094] i. Activate at 28°C for about 2 h, take 100 μL and spread it on a plate containing the corresponding antibiotic resistance. Seal it with parafilm and incubate it upside down in a 28°C incubator for 2 days, then pick colonies for detection.

[0095] (7)Agrobacterium colony detection

[0096] Pick colonies into double-antibiotic LB and culture at 28°C for 2 h. Take an appropriate amount of the bacterial solution for PCR detection and store the positive Agrobacterium bacterial solution.

[0097] Example 4 Genetic transformation experiment

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

[0099] The Agrobacterium containing the recombinant plasmid 35S-pCAMBIA2306-BnaA09.MYB52 constructed in Example 2 and the Agrobacterium containing the BnaMYB52-CRISPR gene knockout vector constructed in Example 3 were respectively used for genetic transformation of rapeseed. The Agrobacterium-mediated genetic transformation method was used. The receptor for rapeseed transformation in the present invention was Brassica napus Westar. For the specific operation process, please refer to 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 transformation materials

[0101] Genomic DNA of the obtained rapeseed overexpression transformation single plants was extracted, and the insertion of the exogenous gene fragment was detected by PCR. The overexpression backbone vector in the present invention was 35S-pCAMBIA2306. Primers pCAMBIA2306-R were designed on the vector backbone, and PCR detection was carried out by pairing with the exogenous fragment primer (BnaA09.MYB52-pCAMBIA2306-F). Transgenic positive seedlings were detected at the PCR level. The PCR reaction system was: Taq polymerase Mix 5 µL; pCAMBIA2306-R (10 μmol / L) 0.5 µL; BnaA09.MYB52-pCAMBIA2306-F (10 μmol / L) 0.5 µL; gDNA 1 µL; ddH2O 3 µL. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 1 min, for 34 cycles; extension at 72°C for 5 min.

[0102] The rapeseed transgenic positive seedlings obtained by PCR detection were subjected to qRT-PCR detection to detect the gene expression level. RNA of the transformed single plant leaves was extracted and cDNA was synthesized (the method was the same as in Example 1). The quantitative primers were designed using Primer 5 software, and the product size was between 80-250 bp. After design, BLAST alignment was performed with the reference sequence to ensure the specificity of the primers. Finally, the primers BnMYB52-RT-F and BnMYB52-RT-R were designed. BnaACTIN7-F / R was used as the internal reference primer for rapeseed qRT-PCR.

[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), and 7.5 μL of 2×TransStart ® Green qPCR SuperMix

[0104] The qRT-PCR reaction program was as follows: 30 s at 94°C; 10 s at 94°C, 15 s at 60°C, 30 s at 72°C, for 45 cycles; a melting curve was plotted. qRT-PCR was performed in a Bio-Rad CFX96 Real-Time detection system

[0105] Standardization was carried out according to the internal reference primer, and the quantitative variation between different replicates was calculated using the method of 2 -ΔΔCT . Finally, the relative expression levels of the rapeseed overexpression transformation materials OE-1 and OE-2 were analyzed and are shown in Figure 1 .

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

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

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

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

[0110] Example 5 Phenotypic Analysis of Overexpressing and Gene-Knockout Mutant Materials

[0111] Homozygous gene-knockout mutants, overexpressing and wild-type seeds were harvested in the field and subjected to a seedling drought experiment in the greenhouse. The greenhouse temperature was kept constant at 25 °C, and the light / dark time was 16 h / 8 h. The experiment had a control group and a treatment group, and more than 30 seedlings of each line were planted for sampling and survival rate statistics. The two groups of seedlings were watered normally and grown to 4 weeks old, then the watering of the treatment group was stopped to start drought treatment, while the control group maintained sufficient water supply. After 7 days of drought treatment, phenotypic observation, detection, photographing, and sampling were carried out. The seedlings in the treatment group were continuously subjected to drought stress. After most of the plants in the line that first showed drought-sensitive phenotypes died, re-watering treatment (i.e., re-watering) was carried out on all drought-treated groups, and the survival rate was statistically analyzed. The experiment was repeated three times to verify the phenotype and survival rate. Survival rate = number of surviving seedlings / total number of seedlings × 100%.

[0112] The results of the indoor drought experiment showed that there were no significant differences in the growth of 4-week-old seedlings in the control group under normal water supply conditions; after drought stress treatment, compared with the wild type (WT), both overexpressing lines (OE-1, OE-2) showed more severe wilting. On the contrary, the two mutant lines (bnamyb52-1 and bnamyb52-2) showed stronger drought resistance. After continuing the treatment for a period of time and then re-watering, the survival of the seedlings was statistically analyzed 3 days later. Three days after re-watering, compared with the wild type, the mutant lines showed more single-plant survival, while the overexpressing lines showed more plant deaths ( Figure 3 ). The drought experiment was repeated 3 times, and the survival rate was statistically analyzed each time. The results showed that compared with wild-type plants, the survival rate of mutant plants increased significantly, while the survival rate of overexpressing plants decreased significantly ( Figure 3 - Figure 4 ).

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

[0114] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a biological material in constructing a rapeseed variety with high drought resistance, characterized in that, The biological material is used for knocking out the BnaMYB52 gene; The BnaMYB52 gene includes the 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, and 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 application according to claim 1, wherein The construction method of the CRISPR gene knockout vector includes the following steps: Using the pCBC-DT1T2 plasmid as a template, performing PCR amplification with primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR to obtain a PCR amplification product; Performing enzymatic digestion and ligation of the PCR amplification product 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 NO.13-16 respectively.

3. The application according to claim 1, wherein 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 application according to claim 3, characterized in that, The construction method of the CRISPR gene knockout vector includes the following steps: Using the pCBC-DT1T2 plasmid as a template, performing PCR amplification with primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR to obtain a PCR amplification product; Performing enzymatic digestion and ligation of the PCR amplification product 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 NO.13-16 respectively.

5. A method for constructing a rape variety with high drought resistance, characterized in that, It includes the step of knocking out the BnaMYB52 gene in the rapeseed plant to construct the high drought-resistant rapeseed variety; The BnaMYB52 gene includes the 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.

6. The method according to claim 5, wherein Using the biological material to knock out the BnaMYB52 gene; 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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