Brassica napus bnaa02.sabath1 gene and application thereof in prevention and control of sclerotinia stem rot of cruciferous crops

Abstract

This invention belongs to the fields of agricultural plant pathology and plant protection technology, and relates to the BnaA02.SABATH1 gene in Brassica napus and its application in controlling Sclerotinia sclerotinia disease in cruciferous crops. This invention constructs transgenic Brassica napus overexpressing BnaA02.SABATH1, and for the first time reveals the positive regulatory function of this gene on resistance to Sclerotinia sclerotinia disease. This invention provides a new approach and method for the control of Sclerotinia sclerotinia disease in Brassica napus and other cruciferous crops, and has broad agricultural application prospects and market value.

Description

Technical Field

[0001] This research relates to the fields of agricultural plant pathology and plant protection, with a particular focus on a Brassica napus BnaA02.SABATH1 gene and its application in controlling sclerotinia stem rot in cruciferous crops. Background Technology

[0002] Rapeseed (Brassica napus) is an important oilseed crop belonging to the genus Brassica in the family Brassicaceae. It is a major oilseed crop widely cultivated worldwide. Increasing rapeseed yield is mainly achieved through three methods: increasing rapeseed yield per unit area, increasing rapeseed oil content, and expanding the planting area. The primary goal of rapeseed breeders now is to strive to improve oil content, yield, and quality.

[0003] However, rapeseed production has long been threatened by sclerotinia rot. Sclerotinia rot is caused by infection with *Sclerotinia sclerotiorum*, a necrotrophic fungus and a major disease of oil crops and vegetables. This disease also reduces the oil content of rapeseed seeds and alters their fatty acid composition, thus affecting the quality of rapeseed oil. *Sclerotinia sclerotiorum* has a very wide host range, infecting more than 600 plant species from over 70 families, affecting important economic crops such as rapeseed, soybeans, and peanuts, causing significant economic losses annually. Currently, no rapeseed germplasm immune to sclerotinia rot has been found. It is generally believed that rapeseed resistance to sclerotinia rot is a complex quantitative trait controlled by multiple genes. Discovering and utilizing sclerotinia rot-resistant genes and breeding resistant varieties are the most economical and effective measures for controlling sclerotinia rot. However, the number and level of available resistance genes are limited. Therefore, identifying and cloning Sclerotinia stem rot resistance genes not only helps to elucidate the mechanism of Sclerotinia stem rot resistance in rapeseed, but also provides genetic resources for breeding rapeseed varieties with improved resistance by combining multiple resistance loci. Summary of the Invention

[0004] In view of this, the present invention provides a key target for the control of sclerotinia stem rot in cruciferous crops. By increasing the expression of the BnaA02.SABATH1 gene in rapeseed, the resistance of cruciferous crops to sclerotinia stem rot can be improved, providing a new technical means for the improvement of resistance to sclerotinia stem rot in cruciferous crops and for long-term and efficient control.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides an application of the Brassica napus BnaA02.SABATH1 gene in the control of sclerotinia stem rot in cruciferous crops, characterized in that the nucleotide sequence of the Brassica napus BnaA02.SABATH1 gene is shown in SEQ ID NO.1.

[0007]

[0008] On the other hand, the present invention provides an application of the protein encoded by the Brassica napus BnaA02.SABATH1 gene in the control of sclerotinia stem rot in cruciferous crops, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO.3.

[0009] Furthermore, the application of the Brassica napus BnaA02.SABATH1 gene in the control of sclerotinia stem rot in cruciferous crops is characterized by transforming cruciferous crops with biological materials used to improve the expression of the BnaA02.SABATH1 gene, thereby increasing the expression level of the BnaA02.SABATH1 gene.

[0010] Furthermore, the application of the Brassica napus BnaA02.SABATH1 gene in the control of sclerotinia stem rot in cruciferous crops is characterized in that the biological material includes at least one of a recombinant plasmid overexpressing the BnaA02.SABATH1 gene and a recombinant bacterium.

[0011] Furthermore, the application of the Brassica napus BnaA02.SABATH1 gene in the control of sclerotinia stem rot in cruciferous crops is characterized by the method for constructing a recombinant plasmid overexpressing the BnaA02.SABATH1 gene, comprising:

[0012] Total RNA was extracted from rapeseed, reverse transcribed into cDNA, and then the complete coding sequence of the BnaA02.SABATH1 gene was amplified using SEQ ID NO.4-5 as primers and cDNA as template. The cDNA was then recovered and purified to obtain the PCR purified product.

[0013] The purified PCR product was subjected to TA cloning to obtain the cloned product pEASY-BnaA02.SABATH1;

[0014] Using the cloned product pEASY-BnaA02.SABATH1 as a template, the target fragment was amplified using the primers shown in SEQ ID No. 6-7;

[0015] The target fragment and the overexpression vector PBI121S plasmid were digested with KpnI and XbaI to obtain the digestion product and the linearized PBI121S plasmid.

[0016] The digestion product and the linearized PBI121S plasmid were ligated using T4 DNA ligase to obtain a recombinant plasmid overexpressing the BnaA02.SABATH1 gene.

[0017] This invention does not impose any particular limitation on the overexpression method of the BnaA02.SABATH1 gene. Those skilled in the art can use conventional methods. In addition to constructing the BnaA02.SABATH1 gene into the PBI121S vector, other overexpression vectors can also be used. In addition to using the CaMV35S promoter, other constituent promoters or inducible promoters can also be used.

[0018] Furthermore, according to the application of the Brassica napus BnaA02.SABATH1 gene in the control of Sclerotinia sclerotinia in cruciferous crops, the characteristic is that rapeseed materials with increased resistance to Sclerotinia sclerotinia are obtained by creating transgenic rapeseed that overexpresses BnaA02.SABATH1.

[0019] Furthermore, the application is characterized by obtaining rapeseed materials with increased resistance to sclerotinia stem rot through the following steps:

[0020] The recombinant plasmid overexpressing the BnaA02.SABATH1 gene was transformed into Agrobacterium GV3101;

[0021] Recombinant plasmids were introduced into rapeseed using Agrobacterium-mediated transformation to obtain transgenic rapeseed overexpressing the BnaA02.SABATH1 gene;

[0022] Using BnaA02.SABATH1 gene expression as a detection index, the phenotypic segregation of transgenic plant offspring was detected, and transgenic rapeseed homozygous plants overexpressing BnaA02.SABATH1 that could stably inherit traits and whose offspring no longer segregated were obtained.

[0023] On the other hand, the present invention provides a method for controlling sclerotinia stem rot in cruciferous crops, characterized in that a biological material that enhances the expression of the BnaA02.SABATH1 gene is transformed into cruciferous crops, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0024] Besides the CRISPR / Cas9 gene editing system, the BnaA02.SABATH1 gene can be knocked out using methods such as EMS mutagenesis, homologous recombination, and T-DNA insertion. All of these methods can achieve the knockout of the BnaA02.SABATH1 gene and obtain BnaA02.SABATH1 gene knockout mutants. Furthermore, plant genetic transformation methods are not limited to Agrobacterium-mediated transformation; other genetic transformation methods such as pollen tube pathway, gene gun, and microinjection can also be used to obtain transgenic plants.

[0025] The advantages of this invention compared to the prior art are as follows:

[0026] This invention provides a method to enhance the resistance of cruciferous crops to sclerotinia stem rot. By utilizing host-induced gene overexpression technology, the expression of the key target gene BnaA02.SABATH1 in rapeseed is increased, which can enhance the resistance of cruciferous plants to sclerotinia stem rot. This provides a key technology for the prevention and control of sclerotinia stem rot in cruciferous crops and has broad prospects for agricultural application and market value. Attached Figure Description

[0027] Figure 1 The spectrum of the PBI121S vector;

[0028] Figure 2 A comparison of the relative expression levels of BnaA02.SABATH1 in plants overexpressing BnaA02.SABATH1;

[0029] Figure 3 Comparison of lesion lengths at different time points after inoculation with Sclerotinia sclerotiorum in plants overexpressing BnaA02.SABATH1;

[0030] Figure 4 Comparative images of lesions in plants overexpressing BnaA02.SABATH1 after inoculation with Sclerotinia sclerotiorum for 48 hours;

[0031] Figure 5 Design diagram of sgRNA for gene knockout of BnaA02.SABATH1;

[0032] Figure 6 Comparison of lesion length at different time points after inoculation with Sclerotinia sclerotiorum in mutant plants with the BnaA02.SABATH1 gene knocked out;

[0033] Figure 7 Comparative images of lesions in mutant plants with the BnaA02.SABATH1 gene knocked out 48 hours after inoculation with Sclerotinia sclerotiorum. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0035] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0036] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used herein to indicate amounts, percentages, or other quantities should, in all cases, be understood to be modified by the word "about." The term "about" has its usual meaning as indicating that a value includes the inherent variation in error of the equipment or method used to determine that value, or contains a value close to said value, for example, within 10% of said value (or a range of values). Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought.

[0037] Furthermore, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art. To make the above-mentioned objects, features, and advantages of this invention more apparent and understandable, specific embodiments of the invention will be described in detail below with reference to the accompanying drawings.

[0038] Example 1

[0039] This invention cloned a rapeseed gene, BnaA02.SABATH1, and for the first time elucidated the function of this gene in conferring resistance to Sclerotinia stem rot in rapeseed by constructing transgenic rapeseed with overexpression. Overexpression of the BnaA02.SABATH1 gene led to a significant increase in rapeseed resistance to Sclerotinia stem rot. Therefore, new rapeseed materials with enhanced resistance to Sclerotinia stem rot can be created and obtained by constructing homozygous lines of rapeseed with overexpression of the BnaA02.SABATH1 gene. These materials can be used for the creation and breeding of rapeseed varieties resistant to Sclerotinia stem rot, and for analyzing the function and mechanism of action of the BnaA02.SABATH1 gene. The main steps in creating and obtaining new rapeseed materials with improved resistance to Sclerotinia stem rot include:

[0040] 1.1 Cloning and preservation of the BnaA02.SABATH1 gene from rapeseed

[0041] Young leaf tissues of the rapeseed variety ZY821 were flash-frozen in liquid nitrogen and then total RNA was extracted using Trizol reagent (Invitrogen, catalog number 15596026). 1.0 μg of the extracted RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (Promega, catalog number M1701). Primers BnaA02.SABATH1-CDS-F (sequence shown in SEQ ID NO.4) and BnaA02.SABATH1-CDS-R (sequence shown in SEQ ID NO.5) were designed based on the BnaA02.SABATH1 sequence in the rapeseed genome database. Using the cDNA synthesized by reverse transcription as a template, the complete coding sequence of the BnaA02.SABATH1 gene was amplified using conventional PCR methods. The PCR amplification system consisted of 50 μl of dNTPs (10 mmol / L), 5 μl of 10× Taq buffer, 1 μl each of forward and reverse primers (10 μmmol / L), 0.5 μl of ExTaq (5 U / μl), 1 μl of cDNA template, and 37.5 μl of ddH2O. The PCR amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 45 s, 58℃ annealing for 45 s, and 72℃ extension for 1 min, repeated for 30 cycles; and a final extension at 72℃ for 10 min. The PCR products were identified by 1% (m / v) agarose gel electrophoresis. The purified PCR product (TIANGEN, catalog number DP209) was obtained by gel extraction and recovery.

[0042] The PCR-purified product was cloned using the pEASY-T1 Cloning Kit (TransGen Biotech, catalog number CT111). The reaction mixture consisted of 4 μL of PCR-purified product and 1 μL of pEASY-T1 Cloning Vector, incubated at 25°C for 5 min to obtain the TA clone product pEASY-BnaA02.SABATH1. The TA clone product pEASY-BnaA02.SABATH1 was transformed into *E. coli* DH5α competent cells using the heat shock method. After resuscitation with 250 μL of liquid LB medium, the cells were cultured at 37°C and 200 rpm for 30 min, followed by centrifugation at 6000 rpm for 1 min. 100 μL of the supernatant was resuspended and plated onto LB agar plates containing ampicillin. The cells were incubated overnight at 37°C. Transformants were validated using colony PCR, and positive transformants were sent to the company for gene sequencing to check for any mutations.

[0043] Sequencing analysis showed that the sequence contains a complete open reading frame, 1056 bases in length, as shown in SEQ ID No. 2.

[0044] E. coli carrying the pEASY-BnaA02.SABATH1 vector were transformed and stored at -80°C. They were then used for transgenic and other research.

[0045]

[0046] MAPTYTMAGGKGPNSYSQHSTYQRALLEVAKEKISEAISTKLEINSASNRFNIADFGCSTGPNTFLAVQNIIDAVGQKYRKETQKNPDDNIEFQVLFNDHSNNDFNTLFQTLPPTKGYFVAGVPGSFFGRVLPRDSLHVGHCSYSLHWLSQIPKGIADPNSPAWNKDIHCTGFSE KVAEAYLDQFKIDMGSFLKARGEELVSGGLLFLLGSCVPDGVKMSETMKGMLLDHLGNCLNDVAKEGLINQEELDSFNFPIYPAHVAEFKSVIEDNGCFTIEAFEKISHANEEFPLDPEFLATSNKVTFGGVIESRFGKEAMERTNELYEKKCPEILPELANAKSGMQFFIMLRRN (SEQ ID NO.3)

[0047] BnaA02.SABATH1-CDS-F:

[0048] ATGGCTCCGACGTACACG (SEQ ID NO.4)

[0049] BnaA02.SABATH1-CDS-R:

[0050] TTAATTTCTTCGAAGCATAATGAA (SEQ ID NO.5)

[0051] 1.2 Construction of BnaA02.SABATH1 gene overexpression plasmid and construction of homozygous transgenic lines

[0052] To verify the disease control potential of the BnaA02.SABATH1 gene, an overexpression vector for the BnaA02.SABATH1 gene was constructed in this embodiment. The original vector for the overexpression vector was the PBI121S vector stored in our laboratory, and its host bacterium was Escherichia coli. The vector map is shown below. Figure 1As shown. The construction process of the BnaA02.SABATH1 gene overexpression vector specifically includes: using the BnaA02.SABATH1 gene TA cloning product pEASY-BnaA02.SABATH1 obtained in Example 1.1 as a template, the target gene is obtained by PCR amplification using specific primers with restriction sites BnaA02.SABATH1-OE-F / R (as shown in SEQ ID No. 6 and SEQ ID No. 7). After purification of the amplification product, it is digested with KpnI and XbaI to obtain the digested product;

[0053] The overexpression vector PBI121S plasmid was double-digested with the same restriction enzymes KpnI and XbaI to obtain a linearized PBI121S plasmid. The digested product and the linearized PBI121S plasmid were then ligated with T4 DNA to obtain the overexpression vector. After transformation, screening, and sequencing, the PBI121S-BnaA02.SABATH1 overexpression vector was successfully constructed.

[0054] The sequence information mentioned above is as follows, where underlined sections indicate restriction enzyme sites:

[0055] BnaA02.SABATH1-OE-F:

[0056] CGG GGTAC CATGGCTCCGACGTACACG (SEQ ID NO.6)

[0057] BnaA02.SABATH1-OE-R:

[0058] CTA GTCTAGA TTAATTTCTTCGAAGCATAATGAA (SEQ ID No.7)

[0059] 1.3 Acquisition of Agrobacterium by transforming the BnaA02.SABATH1 gene overexpression vector PBI121S-BnaA02.SABATH1

[0060] The transformation of Agrobacterium GV3101 competent cells with the PBI121S-BnaA02.SABATH1 overexpression vector was carried out as follows: After freeze-thawing, 5 μL of the overexpression vector was added to the competent cells, mixed, and incubated on ice for 5 min; then frozen in liquid nitrogen for 5 min; and incubated in a 37℃ water bath for 5 min; fresh antibiotic-free liquid LB medium was added, mixed, and activated at 28℃ and 220 rpm / min for 2 h. The supernatant was centrifuged and plated onto LB agar plates containing kanamycin and rifampin, and incubated in the dark at 28℃ for 1 day. Positive single colonies were picked and amplified in kanamycin- and rifampin-resistant liquid LB medium. The resulting bacterial culture was then identified by KpnI and XbaI double digestion and PCR to obtain Agrobacterium carrying the BnaA02.SABATH1 gene overexpression vector PBI121S-BnaA02.SABATH1. This was used for genetic transformation of Brassica napus.

[0061] 1.4 Creation and Acquisition of Rapeseed Lines Transformed with BnaA02.SABATH1 Gene Overexpression Vector PBI121S-BnaA02.SABATH1

[0062] The PBI121S-BnaA02.SABATH1 overexpression vector was transformed into the Brassica napus recipient material ZS11 using Agrobacterium tumefaciens-mediated transformation to obtain transgenic T0 generation single plants. The operation steps are as follows: 1) Seed washing and germination: The seeds were disinfected with 75% ethanol for 1 min, treated with 0.15% mercuric chloride for 10 min, washed 6 to 8 times with sterile water, dried on filter paper, and then inoculated into germination culture flasks and cultured in the dark at 23℃ for 5-6 days; 2) Pre-culture: The hypocotyls of the germinated rapeseed seedlings were cut into 0.4-0 cm sections. 1) Cut explants into 0.6 cm segments, inoculate them into pre-medium, and culture at 23°C under light for 2-3 days; 2) Agrobacterium infection and co-culture: Select Agrobacterium single clones and culture them overnight in the culture medium to prepare Agrobacterium resuspension with OD600=0.2; Infect the explants in the Agrobacterium suspension for 10 min; Dry them on sterile filter paper, inoculate them into co-medium, and culture in the dark at 23°C for 48-72 h; 3) Destermination (delayed screening): Inoculate the co-cultured explants into destermination medium and culture at 23°C under light for 6 days; 4) Screening / differentiation: Inoculate the destermination-treated explants into screening / differentiation medium, 30 explants per plate, and culture at 23°C under light, changing the plate every 15 days; 5) Rooting culture: Inoculate the differentiated shoots into rooting medium and culture at 23°C under light. After rooting, transplant the seedlings into pots containing nutrient soil and vermiculite and culture in a greenhouse at 24°C to obtain T0 generation plants.

[0063] 1.5 Screening, identification, and acquisition of homozygous lines of rapeseed transgenic BnaA02.SABATH1 gene overexpression vector PBI121S-BnaA02.SABATH1

[0064] Genomic DNA was extracted from transgenic single plants using the CTAB method. Transgenic positive plants were screened by PCR using the BnaA02.SABATH1 gene-specific primer BnSAB1-JC-F (SEQ ID NO.21) and the vector-specific primer 121S-R (SEQ ID NO.23) (see Example 1.2 for the procedure). The positive plants were self-pollinated by bagging, and the homozygous lines obtained were used for subsequent sclerotinia disease resistance analysis.

[0065] BnSAB1-JC-F:

[0066] CGTGCGTGGAAAATGTGTGT (SEQ ID NO.21)

[0067] 121S-R:

[0068] GGCGAGAAAGGAAGGGAAGA (SEQ ID NO.23)

[0069] Total RNA was extracted from the second-to-last fully expanded leaf of the identified transgenic positive plants and reverse transcribed to synthesize cDNA. Using primers BnaA02.SABATH1-qF / R (sequences shown in SEQ ID No. 8 and SEQ ID No. 9), with the BnActin gene as an internal reference gene, quantitative real-time PCR was performed to detect the expression level of the transgenic plants. Primers BnActin-F and BnActin-R (sequences shown in SEQ ID No. 10 and SEQ ID No. 11) were used. The results are as follows: Figure 2 As shown (where (P < 0.01) The expression level of the BnaA02.SABATH1 gene in the transgenic positive plants OX-1, OX-2, and OX-4 was significantly higher than that in the recipient material ZS11. This proves that these plants are BnaA02.SABATH1 gene overexpression plants.

[0070] BnaA02.SABATH1-qF:

[0071] TCACTTGGGAAATTGCCTCAA (SEQ ID NO.8)

[0072] BnaA02.SABATH1-qR:

[0073] GCTTCTATCGTGAAACATCCGTTA (SEQ ID NO.9)

[0074] BnActin-F:

[0075] CTGGAATTGCTGACCGTATGAG (SEQ ID NO.10);

[0076] BnActin-R:

[0077] ATCTGTTGGAAAGTGCTGAGGG (SEQ ID NO. 11);

[0078] 1.6 Disease Resistance Detection and Analysis of Homozygous Rapeseed Lines Overexpressing BnaA02.SABATH1 Gene

[0079] Using the homozygous rapeseed line overexpressing the BnaA02.SABATH1 gene obtained in section 1.5 as material, we detected and analyzed its resistance to Sclerotinia sclerotiorum by inoculating it with Sclerotinia sclerotiorum, thereby clarifying the regulatory role of the BnaA02.SABATH1 gene in the resistance of rapeseed to Sclerotinia sclerotiorum, and laying the foundation for creating and obtaining rapeseed resistant to Sclerotinia sclerotiorum using this gene.

[0080] Activation culture of Sclerotinia sclerotiorum: Select plump and uncontaminated Sclerotinia sclerotiorum sclerotia, disinfect them with 75% ethanol, and cut the sclerotia in half with a sterile blade that has been flammed with an alcohol lamp. Place the cut side down on a PDA solid plate and incubate at 23°C in the dark for 3 days. Use a 4mm diameter punch to take a piece of hyphae 3-5mm inward from the edge of the colony. Inoculate the hyphae side down onto a new PDA solid plate and incubate at 23°C in the dark for about 36 hours before inoculation.

[0081] Inoculation of Sclerotinia sclerotiorum: Select rapeseed plants with uniform growth for inoculation. Use a 4mm diameter punch to take a mycelial block 3-5mm inward from the edge of the colony. Inoculate the mycelial block with the mycelial side down into the middle of a fully developed leaf, one mycelial block per leaf. Cover with a film to keep moist and place in a greenhouse at 23℃ for incubation. Take photos and record the results after an appropriate time (about 24 hours).

[0082] The inoculation experiment was repeated three times. Lesions were measured at 36 and 48 hours post-inoculation. Results showed that plants overexpressing BnaA02.SABATH1 were significantly more resistant to the disease than the transgenic negative control (ZS11). Lesion length analysis results are shown below. Figure 3 (in (P < 0.01) At 36 h post-inoculation, the lesion length of the ZS11 control plants was 21 mm, while the lesion lengths of the three overexpression lines were 18.1 mm, 17.3 mm, and 18.5 mm, respectively, representing only 85.8%, 82.1%, and 87.9% of the control. At 48 h post-inoculation, the lesion length of the ZS11 control plants was 27 mm, while the lesion lengths of the three overexpression lines were 23.8, 23.8, and 23.9 mm, respectively, approximately 88% of the control. Leaf lesion growth was photographed 48 h post-inoculation, and the results are shown below. Figure 4 The results showed that the lesion area of ​​plants overexpressing BnaA02.SABATH1 was smaller than that of control plants.

[0083] These results indicate that plants overexpressing BnaA02.SABATH1 showed significantly higher resistance to Sclerotinia stem rot than transgenic negative control plants. Overexpression of the BnaA02.SABATH1 gene led to a significant increase in rapeseed resistance to Sclerotinia stem rot; therefore, BnaA02.SABATH1 plays a positive regulatory role in rapeseed resistance to Sclerotinia stem rot. This invention successfully created a new rapeseed material with high resistance to Sclerotinia stem rot by constructing BnaA02.SABATH1-OX rapeseed.

[0084] Example 2 Construction of BnaA02.SABATH1 gene editing vector and acquisition of mutants

[0085] 2.1 Construction and acquisition of the CRISPR knockout structure of the BnaA02.SABATH1 gene

[0086] (1) Two target sites, gRNA1 and gRNA2, were designed based on the coding region (CDS region, sequence shown in SEQ ID NO.2) of the BnaA02.SABATH1 gene. Specifically, DNA fragments ending with the base NGG were selected as target sites in the exon region of Brassica napus BnaA02.SABATH1. One strand of each target site has a 5'-(N)XNGG-3' structure, where X in (N)X represents a sequence of X bases, N in (N)X represents any one of the bases A, G, C, or T, and X is 19 or 20. In this embodiment, the sequences of the two target sites, gRNA1 and gRNA2, are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively. Both gRNA1 and gRNA2 target the first exon of the BnaA02.SABATH1 gene. Figure 5 As shown;

[0087] gRNA1:

[0088] CGACGTACACGATGGCTGGAGG (SEQ ID NO.12)

[0089] gRNA2:

[0090] AGTCAACATTCAACGTATCAGG (SEQ ID NO.13)

[0091] (2) Based on the target site sequence, four single-stranded oligo DNA primer sequences were designed and synthesized, namely BnaA02.SABATH1-DT1-F0, BnaA02.SABATH1-DT1-BsF, BnaA02.SABATH1-DT2-R0, and BnaA02.SABATH1-DT2-BsR, and their sequences are shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 and SEQ ID NO.17, respectively;

[0092] BnaA02.SABATH1-DT1-F0:

[0093] TGCGACGTACACGATGGCTGGGTTTTAGAGCTAGAAATAGC (SEQ ID NO.14)

[0094] BnaA02.SABATH1-DT1-BsF:

[0095] ATATATGGTCTCGATTGCGACGTACACGATGGCTGGGTT (SEQ ID NO.15)

[0096] BnaA02.SABATH1-DT2-R0:

[0097] AACGATACGTTGAATGTTGACTCAATCTCTTAGTCGACTCTAC (SEQ ID NO.16)

[0098] BnaA02.SABATH1-DT2-BsR:

[0099] ATTATTGGTCTCGAAACGATACGTTGAATGTTGACTCAA (SEQ ID NO.17)

[0100] (3) Using pCBC-DT1T2 (provided by Professor Chen Qijun of China Agricultural University) diluted 100 times as a template, four-primer PCR amplification was performed to obtain the sgRNA expression cassette. BnaA02.SABATH1-DT1-BsF / -BsR was the normal primer concentration (10 μmol / L); BnaA02.SABATH1-DT1-F0 / -R0 was diluted 20 times (0.5 μmol / L). PCR system: pCBC-DT1T2 vector (diluted 100-fold) 1 μl, BnaA02.SABATH1-DT1-BsF 1 μl, BnaA02.SABATH1-DT2-BsR 1 μl, BnaA02.SABATH1-DT1-F0 1 μl, BnaA02.SABATH1-DT2-R0 1 μl, 2×PhantaMax MasterMix (Kangwei Century) 10 μl, ddH2O 5 μl; PCR reaction conditions: 95℃ for 3 min; 95℃ for 15 sec, 58℃ for 15 sec, 72℃ for 45 sec, 32 cycles; 72℃ for 7 min. PCR amplification yielded a 626 bp fragment, which was detected by 1% agarose gel electrophoresis. The target band was excised from the gel and recovered, dissolved in 30 μl ddH2O.

[0101] (4) The purified sgRNA expression cassette (626 bp) and pHSE401 backbone vector (provided by Professor Chen Qijun of China Agricultural University) were used to construct the gene editing vector CRISPR / Cas9 using a digestion-ligation method. The enzyme digestion-ligation system was as follows: 6 μl of purified sgRNA expression cassette, 2 μl of pHSE401 vector, 1.5 μl of 10×T4 ligase buffer (New England Biolabs, NEB), 1.5 μl of 10×BSA (NEB), 1 μl of BsaI (NEB), 1 μl of T4 ligase (NEB), and 2 μl of ddH2O, with a total reaction volume of 15 μl. The reaction conditions were: 37℃ for 5 h, 50℃ for 5 min, and 80℃ for 10 min. Five μl of the enzyme digestion-ligation product was transformed into competent E. coli DH5α cells. The cells were screened on Kans resistant plates and identified by colony PCR using primers U626-IDF (SEQ ID NO.18) and U629-IDR (SEQ ID NO.19) (726 bp). Positive clones were sequenced using primers U626-IDF (SEQ ID NO.18) and U629-IDF (SEQ ID NO.20). The correctly sequenced clones were identified as the constructed CRISPR / Cas9 vector pHSE401-BnaA02.SABATH1.

[0102] U626-IDF:

[0103] TGTCCCAGGATTAGAATGATTAGGC (SEQ ID NO.18)

[0104] U629-IDR:

[0105] AGCCCTCTTTCTTTCGATCCATCAAC (SEQ ID NO.19)

[0106] U629-IDF:

[0107] TTAATCCAAACTACTGCAGCCTGAC (SEQ ID NO.20)

[0108] 2.2 Acquisition of Agrobacterium by transforming the BnaA02.SABATH1 gene into the CRISPR / Cas9 vector

[0109] The recombinant plasmid pHSE401-BnaA02.SABATH1 was transformed into Agrobacterium GV1301 using the same method as in Example 1, and then genetically transformed into the rapeseed variety ZY821. The obtained transgenic material was identified by PCR using vector-specific primers. The nucleotide sequences of the upstream and downstream primers used for identification are shown in SEQ ID NO.18 and SEQ ID NO.19.

[0110] 2.3 Creation and Acquisition of CRISPR-knockout Brassica oleracea var. saccharigenes containing the BnaA02.SABATH1 gene

[0111] The CRISPR knockout structure pHSE401-BnaA02.SABATH1 of the BnaA02.SABATH1 gene was introduced into rapeseed using Agrobacterium-mediated transformation to obtain the T0 generation of rapeseed transgenic with pHSE401-BnaA02.SABATH1. The specific operation steps were the same as described in Example 1.4. PCR amplification was performed using upstream and downstream specific primers BnSAB1-JC-F (SEQ ID NO.21) and BnSAB1-JC-R (SEQ ID NO.22) for the target site. Plants with BnaA02.SABATH1 target site editing were screened from the transgenic progeny. The sequencing results after amplification with the identification primers were used to determine whether both alleles of the BnaA02.SABATH1 gene were successfully knocked out. Finally, mutant plants with successful BnaA02.SABATH1 gene knockout were identified.

[0112] BnSAB1-JC-F:

[0113] CGTGCGTGGAAAATGTGTGT (SEQ ID NO.21)

[0114] BnSAB1-JC-R:

[0115] AAAAATGGTTATCCCCGTGAAT(SEQ ID NO.22)

[0116] 2.4 Obtaining homozygous lines of rapeseed with BnaA02.SABATH1 gene CRISPR knockout structure

[0117] Homozygous rapeseed lines transfected with the BnaA02.SABATH1 gene CRISPR knockout structure (HSE401-BnaA02.SABATH1) were used as materials to test and analyze their resistance to sclerotinia stem rot. The inoculation method and disease resistance evaluation were the same as described in Example 1.6.

[0118] The BnaA02.SABATH1-CRISPR plants constructed in this invention were subjected to an inoculation experiment with Sclerotinia sclerotiorum. Lesions were measured at 36 and 48 hours post-inoculation, with transgenic negative samples used as controls. Results are shown below. Figure 6 (in (P < 0.01) The resistance of the gene-edited mutant to sclerotinia stem rot was significantly weakened. 36 h after inoculation, the lesion length of the ZY281 control plant was 20 mm, while the lesion length of the gene-edited mutant plants CR-5 was 24 mm, CR-11 was 23 mm, and CR-13 was 25 mm, representing increases of 20%, 15%, and 25% respectively compared to the control group. 48 h after inoculation, the lesion length of the ZY281 control plant was 26 mm, while the lesion length of the gene-edited mutant plants CR-5 was 31 mm, CR-11 was 30 mm, and CR-13 was 31 mm, representing increases of 19%, 15%, and 19% respectively compared to the control group. Leaf lesion photographs were taken 48 h after inoculation. Results are shown in the table below. Figure 7 The results showed that the lesion patches of BnaA02.SABATH1-CRISPR plants were larger than those of the control group.

[0119] In summary, this invention cloned the BnaA02.SABATH1 gene and demonstrated through gene overexpression and gene editing methods that BnaA02.SABATH1 positively regulates the resistance of Brassica napus to Sclerotinia sclerotinia, providing a theoretical basis and germplasm resources for the development of Sclerotinia sclerotinia resistance in Brassica napus.

Claims

1. Application of Brassica napus BnaA02.SABATH1 gene in prevention and control of Sclerotinia sclerotiorum, characterized in that, The nucleotide sequence of the Brassica napus BnaA02.SABATH1 gene is shown in SEQ ID NO.1; The application involves transforming rapeseed with biomaterials used to enhance the expression of the BnaA02.SABATH1 gene, thereby increasing the expression level of the BnaA02.SABATH1 gene.

2. Use of the protein encoded by the Brassica napus BnaA02.SABATH1 gene according to claim 1 for the control of Sclerotinia sclerotiorum in Brassica napus, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

3.

3. The use of Brassica napus BnaA02.SABATH1 gene in the prevention and control of Sclerotinia sclerotiorum according to claim 1, characterized in that, The biological material includes at least one of a recombinant plasmid overexpressing the BnaA02.SABATH1 gene and a recombinant bacterium.

4. The use of Brassica napus BnaA02.SABATH1 gene in the prevention and control of Sclerotinia sclerotiorum according to claim 3, characterized in that, The methods for constructing recombinant plasmids overexpressing the BnaA02.SABATH1 gene include: Total RNA was extracted from rapeseed, reverse transcribed into cDNA, and then the complete coding sequence of the BnaA02.SABATH1 gene (SEQ ID NO. 2) was amplified using SEQ ID NO. 4-5 as primers and cDNA as template. The cDNA was then recovered and purified to obtain the PCR purified product. The purified PCR product was subjected to TA cloning to obtain the cloned product pEASY-BnaA02.SABATH1; Using the cloned product pEASY-BnaA02.SABATH1 as a template, the target fragment was amplified using the primers shown in SEQ ID No. 6-7; The target fragment and the overexpression vector PBI121S plasmid were digested with KpnI and XbaI to obtain the digestion product and the linearized PBI121S plasmid. The digestion product and the linearized PBI121S plasmid were ligated using T4 DNA ligase to obtain a recombinant plasmid overexpressing the BnaA02.SABATH1 gene.

5. The use of Brassica napus BnaA02.SABATH1 gene according to claim 3 for the prevention and control of Sclerotinia sclerotiorum, characterized in that, We obtained rapeseed with increased resistance to sclerotinia stem rot by creating transgenic rapeseed that overexpresses BnaA02.SABATH1.

6. Use according to claim 4, characterized in that, The following steps are used to obtain rapeseed materials with increased resistance to sclerotinia stem rot: The recombinant plasmid overexpressing the BnaA02.SABATH1 gene was transformed into Agrobacterium GV3101; Recombinant plasmids were introduced into rapeseed using Agrobacterium-mediated transformation to obtain transgenic rapeseed overexpressing the BnaA02.SABATH1 gene; Using BnaA02.SABATH1 gene expression as a detection index, the phenotypic segregation of transgenic plant offspring was detected, and transgenic rapeseed homozygous plants overexpressing BnaA02.SABATH1 that could stably inherit traits and whose offspring no longer segregated were obtained.

7. A method for controlling sclerotinia stem rot in rapeseed, characterized in that, The biomaterials that enhance the expression of the BnaA02.SABATH1 gene were transformed into rapeseed, and the nucleotide sequence of the gene is shown in SEQ ID NO.1.

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