Application of bnCDS1 gene in disease prevention and / or growth of rapeseed and an sgRNA combination

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of BnCDS1 gene in plant disease prevention and control and / or growth and an sgRNA combination. The application edits the BnCDS1 gene of rapeseed, and the mutant rapeseed is not different from the wild type Westar in aspects of seedling stage plant phenotype, chlorophyll content, light energy conversion efficiency, nitrogen content and flowering time, but the plant height becomes shorter with the increase of the copy editing number, and the silique becomes smaller and shorter. It is indicated that BnCDS1 is involved in the regulation of rapeseed growth, seed setting and other traits. The edited BnCDS1 rapeseed mutant is inoculated with Sclerotinia sclerotiorum, Botrytis cinerea and Plasmodiophora brassicae, and it is found that the edited rapeseed has enhanced resistance to Sclerotinia sclerotiorum, Botrytis cinerea and Plasmodiophora brassicae, and the higher the editing copy number is, the stronger the resistance is. The BnCDS1 gene edited mutant shows a certain broad-spectrum disease resistance, and the BnCDS1 gene can be used as a new gene resource for green prevention and control of rapeseed diseases.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of the BnCDS1 gene in rapeseed disease control and / or growth and an sgRNA combination. Background Technology

[0002] Rapeseed is an important oilseed crop in my country and even the world. Its production has always been threatened by sclerotinia rot, gray mold, and clubroot, causing serious losses to rapeseed production every year. Among them, sclerotinia rot is caused by Sclerotinia sclerotiorum, which can infect 408 species of plants from 75 families; gray mold (Botrytis cinerea) is closely related to Sclerotinia sclerotiorum and is another important necrotic pathogen of rapeseed, causing gray mold; clubroot is caused by Plasmodiophora brassicae, which is an obligate parasite on the roots of cruciferous plants. Its dormant spores can survive in the soil for a long time, making it extremely difficult to control in production.

[0003] Currently, chemical agents are commonly used to control rapeseed diseases, such as sclerotinia oleifera, a dicarboximide fungicide. Developed abroad in the early 1970s, it is a broad-spectrum, contact-type, modern protective fungicide with some local therapeutic effect. However, with the overuse of chemical agents, rapeseed's sensitivity to these agents has decreased, leading to reduced control effectiveness. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art, and to provide the application of the BnCDS1 gene in the prevention and / or growth of rapeseed diseases and an sgRNA combination to enhance the ability of rapeseed to resist Sclerotinia sclerotiorum, Botrytis cinerea and Plasmodium falciparum.

[0005] This invention provides the application of knocking out or knocking down the BnCDS1 gene in the prevention and / or growth of rapeseed diseases; the BnCDS1 gene includes the BnaA09T0145500WE gene, the BnaC09T0175800WE gene, the BnaC04T0239200WE gene, and the BnaA03T0593500WE gene;

[0006] The amino acid sequence encoded by the BnaA09T0145500WE gene is shown in SEQ ID NO.1;

[0007] The amino acid sequence encoded by the BnaC09T0175800WE gene is shown in SEQ ID NO.2;

[0008] The amino acid sequence encoded by the BnaC04T0239200WE gene is shown in SEQ ID NO.3;

[0009] The amino acid sequence encoded by the BnaA03T0593500WE gene is shown in SEQ ID NO.4.

[0010] Preferably, the disease includes one or more of sclerotinia rot, gray mold, and clubroot.

[0011] Preferably, the rapeseed disease control includes knocking out or knocking down the BnCDS1 gene to improve rapeseed's resistance to pathogens.

[0012] Preferably, the pathogenic fungus includes one or more of Sclerotinias clerotiorum, Botrytiscinerea, and Plasmodiophora brassicae.

[0013] Preferably, the nucleotide sequence of the BnaA09T0145500WE gene is shown in SEQ ID NO.5;

[0014] The nucleotide sequence of the BnaC09T0175800WE gene is shown in SEQ ID NO.6;

[0015] The nucleotide sequence of the BnaC04T0239200WE gene is shown in SEQ ID NO.7;

[0016] The nucleotide sequence of the BnaA03T0593500WE gene is shown in SEQ ID NO.8.

[0017] The present invention also provides a combination of sgRNAs for knocking out the BnCDS1 gene, including sgRNA1, sgRNA2 and sgRNA3;

[0018] The nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.15;

[0019] The nucleotide sequence of the sgRNA2 is shown in SEQ ID NO.16;

[0020] The nucleotide sequence of the sgRNA3 is shown in SEQ ID NO.17.

[0021] The present invention also provides a gene editing vector for knocking out the BnCDS1 gene, comprising an editing backbone vector and a combination of sgRNA inserted into the editing backbone vector;

[0022] The sgRNA combination includes sgRNA1, sgRNA2, and sgRNA3;

[0023] The nucleotide sequence of the sgRNA1 is shown in SEQ ID NO.15;

[0024] The nucleotide sequence of the sgRNA2 is shown in SEQ ID NO.16;

[0025] The nucleotide sequence of the sgRNA3 is shown in SEQ ID NO.17.

[0026] Preferably, the editable skeleton carrier includes pRGEB35-Cas9.

[0027] This invention also provides the application of the sgRNA combination or gene editing vector described in the above technical solution in the prevention and / or growth of rapeseed diseases.

[0028] This invention also provides a method for improving the resistance of rapeseed to pathogens, which involves introducing the sgRNA combination or gene editing vector described in the above technical solution into rapeseed and then culturing it.

[0029] Beneficial effects:

[0030] This invention, through editing the BnCDS1 gene in rapeseed, revealed that the seedling phenotype, chlorophyll content, light conversion efficiency, nitrogen content, and flowering time of the BnCDS1 mutant rapeseed were indistinguishable from the wild-type Westar. However, plant height decreased with increasing copy number of editing, and siliques became smaller and shorter. These changes in agronomic traits indicate that BnCDS1 participates in the regulation of rapeseed growth and fruit setting. Inoculation of the edited BnCDS1 rapeseed mutant with *Sclerotinia sclerotiorum*, *Botrytis cinerea*, and *Plasmodiophora* showed that editing enhanced the rapeseed's resistance to these fungi, with higher copy numbers resulting in stronger resistance. The BnCDS1 gene-edited mutant exhibited broad-spectrum disease resistance, suggesting that the BnCDS1 gene could serve as a novel gene resource for the green control of rapeseed diseases. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0032] Figure 1 This diagram illustrates the structure of the BnCDS1 gene, the target site of the sgRNA, and the structure of the pRGEB35-Cas9-BnCDS1 editing vector. In A, red indicates the target site, green indicates the exon, yellow indicates the upstream / downstream bases, and horizontal lines indicate introns.

[0033] Figure 2 The results of identification of T0 generation rapeseed plants with the BnCDS1 gene are shown; among them, numbers 1 to 14 are 144 positive rapeseed plants with editing vectors.

[0034] Figure 3 This shows the editing status of the target site of the BnCDS1 gene in the T0 generation rapeseed mutant; where blue represents insertion mutations, green represents substitution mutations, "-" indicates deletion mutations, and red represents the PAM sequence; the numbers in parentheses indicate the number of inserted or deleted bases;

[0035] Figure 4 The results of identification and screening of BnCDS1 gene mutants in the T1 generation of rapeseed are shown. In the figure, the green part is the insertion mutation, "-" indicates the deletion mutation, and the red part is the PAM sequence. The number in parentheses is the number of inserted or deleted bases.

[0036] Figure 5 Phenotypes of the T2 generation rapeseed BnCDS1 mutant; from top to bottom, the phenotypes are rapeseed at the 5-leaf stage and rapeseed at the bolting and flowering stage, with a scale bar of 10 cm;

[0037] Figure 6 The results show the plant height, net photosynthetic rate, chlorophyll content, and nitrogen content of the T2 generation rapeseed mutant; where n≥8, and different lowercase letters indicate p<0.05.

[0038] Figure 7 The results of resistance analysis of T2 generation rapeseed mutants inoculated with Sclerotinia sclerotiorum and Botrytis cinerea were presented. Among them, A and D represent the lesions formed by Sclerotinia sclerotiorum and Botrytis cinerea on the leaves of BnCDS1 mutant; B and E represent the lesion area statistics; C and F represent the relative biomass expression statistics; different lowercase letters represent p < 0.05.

[0039] Figure 8 Analysis of resistance to clubroot bacteria in T2 generation rapeseed mutants; where A represents the disease phenotype of roots of BnCDS1 mutant 35 days after clubroot inoculation; B represents the statistical results of the number of diseased plants, n=27; C represents the statistical results of disease index; different lowercase letters represent p<0.05. Detailed Implementation

[0040] This invention provides the application of knocking out or knocking down the BnCDS1 gene in the prevention and / or growth of rapeseed diseases; the BnCDS1 gene includes the BnaA09T0145500WE gene, the BnaC09T0175800WE gene, the BnaC04T0239200WE gene, and the BnaA03T0593500WE gene;

[0041] The amino acid sequence encoded by the BnaA09T0145500WE gene is shown in SEQ ID NO.1;

[0042] The amino acid sequence encoded by the BnaC09T0175800WE gene is shown in SEQ ID NO.2;

[0043] The amino acid sequence encoded by the BnaC04T0239200WE gene is shown in SEQ ID NO.3;

[0044] The amino acid sequence encoded by the BnaA03T0593500WE gene is shown in SEQ ID NO.4.

[0045] In this invention, the disease preferably includes one or more of sclerotinia rot, gray mold, and clubroot, more preferably sclerotinia rot, gray mold, and clubroot. The rapeseed disease control of this invention preferably includes knocking out or downsampling the BnCDS1 gene to improve rapeseed resistance to pathogens; the pathogen preferably includes one or more of *Sclerotinia sclerotiorum*, *Botrytis cinerea*, and clubroot fungi, more preferably *Sclerotinia sclerotiorum*, *Botrytis cinerea*, and clubroot fungi.

[0046] In this invention, the preferred nucleotide sequence of the BnaA09T0145500WE gene is as shown in SEQ ID NO.5; the preferred nucleotide sequence of the BnaC09T0175800WE gene is as shown in SEQ ID NO.6; the preferred nucleotide sequence of the BnaC04T0239200WE gene is as shown in SEQ ID NO.7; and the preferred nucleotide sequence of the BnaA03T0593500WE gene is as shown in SEQ ID NO.8.

[0047] The sequence information of SEQ ID NO.1 to SEQ ID NO.8 of this invention is as follows:

[0048] SEQ ID NO.1:MEEESNVTSSSPSTPVQRLRHRKRSSTEVLDGDKVNASPLLVNDRNKYKSF MIRTYSTLWMIAGFVMVVYMGHLYITAMVLVIQIFMAKELFNLLRKAPEDKCLPGIKQLNWHFFFTAMLFVYGRILSQRLANTVTADQFLYRLVTGLIKYHMAICYFLYIIGFMWFILTLKKKMYKYQFGQYAWTHMILIVVFTQSSFTVANIFEGIFWFLLPASLIIINDIFAYIFGFFFGRTPLIKLSPKKTWEGFIGASVTTIISAFVLANVLGRFPWLTCPRQDLSTGWLQCDADPLFKPEPFTLPAWIPGWFPWKEMEVLPVQWHALCLGLFASIIAPFGGFFASGFKRAFKIKDFGDSIPGHGGITDRMDCQMVMAVFAYIYLQSFIVSQSVSVDKILDQILTNLSFEEQQALFSRLGQMIGNS*;

[0049] SEQ ID NO.2:MEEESNVTSSSPSTPVQRLRHRKRSSTEVLDGDKVNASPLLVNDRNKYKSFMVRTYSTLWMIAGFVMVVYMGHLYITAMVLVIQIFMAKELFNLLRKAPEDKCLPGIKQLNWHFFFTAMLFVYGRILSQRLANTVTADQFLYRLVSGLIKYHMAICYFLYIIGFMWFILTLKKKMYKYQFGQYAWTHMILIVVFTQSSFTVANIFEGIFWFLLPASLIIINDIFAYIFGFFFGRTPLIKLSPKKTWEGFIGASVTTIISAFVLANVLGRFPWLTCPRQDLSTGWLQCDADPLFKPEPFTLPAWIPGWFPWKEMEVLPVQWHALCLGLFASIIAPFGGFFASGFKRAFKIKDFGDSIPGHGGITDRMDCQMVMAVFAYIYLQSFIVSQSVSVDKILDQILTNLSFEEQQALLTRLGQMIGNS*;

[0050] SEQ ID NO.3:MEEESNVTSSPSTRLRHRKRSSTTEAVLDGNPSPLLVNDQNKYKSFMVRTYSTIWMIGGFVMVVYMGHLYITAMVLVIQIFMAKELFNLLRKAPEDKCLPGIKHLNWHFFFTAMLFVYGRILSQRLANTVTADQFLYRLVSGLIKYHMAICYFLYIIGFMWFILTLKKKMYKYQFGQYAWTHMILIVVFTQSSFTVANIFEGIFWFLLPASLIIINDIFAYIFGFFFGRTPLIKLSPKKTWEGFIGASVTTIISAFFLANIMGRFPWLTCPRQDLSTGWLQCDADPLFKPEPFTLPAWIPGWFPWKEMEVLPVQWHALCLGLFASIIAPFGGFFASGFKRAFKIKDFGDSIPGHGGITDRMDCQMVMAVFAYIYLQSFIVSQSVSVDKILDQILTNLTLEEQQALFTRLGQMIGYS*;

[0051] SEQ ID NO.4:MEEESNVSSSPSTRLRHRKQQRSSSAEAVVDGNPSPLLVNDQNKYKSFMVRTYSTIWMIGGFVMVVYMGHLYITAMVLVIQIFMAKELFNLLRKAPEDKCLPGIKHLNWHFFFTAMLFVYGRILSQRLANTVTADQFLYRLVSGLIKYHMAICYLLYIIGFMWFILTLKKKMYKYQFGQYAWTHMILIVVFTQSSFTVANIFEGIFWFLLPASLIIINDIFAYIFGFFFGRTPLIKLSPKKTWEGFIGASVTTIISAFFLANIMGRFPWLTCPRQDLSTGWLQCDADPLFKPEPFTLPAWIPGWFPWKEMEVLPVQWHSLCLGLFASIIAPFGGFFASGFKRAFKIKDFGDSIPGHGGITDRMDCQMVMAVFAYIYLQSFIVSQSVSVDKILDQILTNLTLEEQQALFTRLGQMIGYS*;

[0052]

[0053]

[0054]

[0055]

[0056] This invention, through editing the BnCDS1 gene in rapeseed, revealed that the seedling phenotype, chlorophyll content, light conversion efficiency, nitrogen content, and flowering time of the BnCDS1 mutant rapeseed were indistinguishable from the wild-type Westar. However, plant height decreased with increasing copy number of editing, and siliques became smaller and shorter. These changes in agronomic traits indicate that BnCDS1 participates in the regulation of rapeseed growth and fruit setting. Inoculation of the edited BnCDS1 rapeseed mutant with *Sclerotinia sclerotiorum*, *Botrytis cinerea*, and *Plasmodiophora* showed that editing enhanced the rapeseed's resistance to these fungi, with higher copy numbers resulting in stronger resistance. The BnCDS1 gene-edited mutant exhibited broad-spectrum disease resistance, suggesting that the BnCDS1 gene could serve as a novel gene resource for the green control of rapeseed diseases.

[0057] Based on the function of the BnCDS1 gene provided by this invention, this invention also provides a combination of sgRNAs for knocking out the BnCDS1 gene, including sgRNA1, sgRNA2 and sgRNA3; the nucleotide sequence of sgRNA1 is shown in SEQ ID NO.15; the nucleotide sequence of sgRNA2 is shown in SEQ ID NO.16; and the nucleotide sequence of sgRNA3 is shown in SEQ ID NO.17.

[0058] Based on the function of the BnCDS1 gene provided by this invention, this invention also provides a gene editing vector for knocking out the BnCDS1 gene, comprising an editing backbone vector and a combination of sgRNAs inserted into the editing backbone vector; the sgRNA combination is the sgRNA combination described in the above technical solution. In this invention, the editing backbone vector is preferably the pRGEB35-Cas9 vector, disclosed in Xie K, Minkenberg B, Yang Y. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci U SA. 2015 Mar 17; 112(11):3570-5. In this invention, sgRNA1, sgRNA2, and sgRNA3 are each linked to a tRNA and then tandemly connected, and this sequence is inserted into the editing backbone vector.

[0059] This invention also provides the application of the sgRNA combination or gene editing vector described in the above-mentioned technical solutions in the prevention and / or growth of rapeseed diseases. In this invention, the diseases preferably include one or more of sclerotinia rot, gray mold, and clubroot, more preferably sclerotinia rot, gray mold, and clubroot. The rapeseed disease prevention and control of this invention preferably includes knocking out or downsampling the BnCDS1 gene to improve rapeseed resistance to pathogens; the pathogens preferably include one or more of *Sclerotinia sclerotiorum*, *Botrytis cinerea*, and clubroot fungi, more preferably *Sclerotinia sclerotiorum*, *Botrytis cinerea*, and clubroot fungi.

[0060] This invention also provides a method for improving the resistance of rapeseed to pathogens, which involves introducing the sgRNA combination or gene editing vector described in the above-mentioned technical solution into rapeseed and then culturing it. This invention does not have strict requirements on the method of introduction; conventional methods in the art can be used.

[0061] To further illustrate the present invention, the application of the BnCDS1 gene in rapeseed disease control and / or growth and an sgRNA combination provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] 1. Test plants, strains, and culture conditions

[0064] The tested pathogenic microorganisms were: *Sclerotinia sclerotiorum* 1980, *Botrytis cinerea* B05.10 (published in: Yang G, Tang L, Gong Y, Xie J, Fu Y, Jiang D, Li G, Collinge DB, Chen W, Cheng JA cerato-plataninprotein SsCP1 targets plant PR1 and contributes to virulence of *Sclerotinias clerotiorum*. *New Phytol*. 2018 Jan; 217(2):739-755. doi:10.1111 / nph.14842. Epub 2017 Oct 27. PMID:29076546.) and *Plasmodiophora brassicae* (published in: Zhao Y, Li C, Chen X, Cheng J, Xie J, Lin Y, Fu Y, Jiang D, Chen T. Overexpression of chitinase PbChia1 from *Plasmodiophora brassicae*). improves broad-spectrum disease resistance of Arabidopsis. Virulence.2023Dec;14(1):2233147.doi:10.1080 / 21505594.2023.2233147.PMID:37431945;PMCID:PMC10337504.).

[0065] Test plant: Brassica napus Westar.

[0066] Culture conditions for pathogenic microorganisms: Sclerotinia sclerotiorum 1980 and Botrytis cinerea B05.10 were activated and cultured at 20°C on PDA medium (200g peeled potato filtrate, 20g glucose, 10g agar powder, distilled water added to 1L, autoclaved at 121°C for 20min).

[0067] Plant cultivation conditions: T0 generation BnCDS1 mutant rapeseed and some T1 and T2 generation plants produced by self-pollination were planted in nutrient-rich organic soil and cultured in a plant cultivation room. Cultivation conditions were 22℃, 16 hours of light, 8 hours of darkness, and 75% relative humidity. Some T1 and T2 plants were also cultivated at the Yezhihu Potted Plant Farm of Huazhong Agricultural University. Fertilization and watering were carried out according to the plants' needs.

[0068] 2. sgRNA design and CRISPR / Cas9 vector construction

[0069] (1) Determination of the target gene BnCDS1

[0070] The full-length amino acid sequence of AtCDS1 (corresponding gene number AT1G62430) was obtained from the Arabidopsis Information Resource website (https: / / www.arabidopsis.org / ). Homologous copy sequences from *Westar* rapeseed were compared using BLAST on the *Caulis Rapeseed Pangenomes* website (http: / / www.cbi.hzau.edu.cn / bnapus / ), yielding four homologous copy sequences: BnaA09T0145500WE, BnaC09T0175800WE, BnaC04T0239200WE, and BnaA03T0593500WE. The nucleotide sequences of the BnaA09T0145500WE, BnaC09T0175800WE, BnaC04T0239200WE, and BnaA03T0593500WE genes are shown in SEQ ID NO. 1–4, and their encoded amino acid sequences are shown in SEQ ID NO. 4. ID NO.5~8 as shown

[0071] (2) Based on the homology comparison results of the four genes obtained in step (1), the target sites for CRISPR / Cas9 gene editing were designed using the online design software CRISPR-P 2.0 (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRIS). The website contains the reference genome of Brassica napus Westar. The target copy gene name was selected directly on the webpage, or the gene sequence was submitted to obtain the locations and sequence information of all available target sites containing NGG (PAM structure) within the target region. Target sites sgRNA1 (S1), sgRNA2 (S2), and sgRNA3 (S3) were designed targeting the conserved regions of the four copies of the BnCDS1 gene. These three target sites are located in different exon regions (…). Figure 1 The three sgRNA segments (A) and their amplification primers are detailed in Table 1.

[0072] Table 1. sgRNA and its amplification primer sequence information

[0073]

[0074] In this sequence, lowercase letters represent adapter sequences amplified using pGTR as templates, which are used for tandem sequences; bolded parts represent PAM sequences.

[0075] After ligating sgRNA1, sgRNA2, and sgRNA3 each to a segment of tRNA, they were tandemly linked together, and this sequence was ligated to the AtUBI10P promoter to construct the CRISPR / Cas9 vector. Figure 1 (B), denoted as pRGEB35-Cas9-BnCDS1 (refer to existing technology: Xie K, Minkenberg B, Yang Y. Boosting CRISPR / Cas9 multiplexediting capability with the endogenous tRNA-processing system. ProcNatlAcad SciUSA. 2015, 112(11):3570-5).

[0076] Example 2

[0077] Agrobacterium-mediated genetic transformation of hypocotyls

[0078] We commissioned Wuhan Boyuan Biotechnology Co., Ltd. to transform the pRGEB35-Cas9-BnCDS1 editing vector constructed in Example 1 into rapeseed using the Agrobacterium-mediated Westar hypocotyl method, referring to existing technology (Zhou Y, Wang H, Gilmer S, Whitwill S, Keller W, Fowke LC. Control of petal and pollen development by the plantcyclin-dependent kinase inhibitor ICK1in transgenic Brassica plants. Planta, 2002, 215(2):248-257). Complete transgenic plants were obtained using tissue culture technology.

[0079] Example 3

[0080] 1. Positive detection and editing detection of single plants

[0081] (1) Take young leaves from the transgenic plants (T0 generation) of Example 2 and extract DNA. Based on the hygromycin (HygR) selection element and the Cas9 base sequence contained in the vector, design detection primers and perform PCR amplification with the extracted DNA. The target size band of the HygR gene was displayed at about 500bp. Transgenic plants in which the target size band of the Cas9 gene was detected in the range of 500-750bp were considered positive transgenic plants. Some detection results are shown below. Figure 2 As shown.

[0082] The primer sequences and PCR detection conditions for hygromycin detection are as follows:

[0083] Hyg-F: 5'-GTTGGCGACCTCGTATTGG-3' (SEQ ID NO. 18);

[0084] Hyg-R: 5'-TGCTTGACATTGGGGAGTTTA-3' (SEQ ID NO. 19);

[0085] The PCR amplification system consisted of: 1 μL DNA, 1 μL upstream primer F, 1 μL downstream primer R, 7.5 μL Mix, and 4.5 μL ddH2O. The PCR amplification program was: 95℃ for 5 min, 95℃ for 30 s, 57℃ for 30 s, 72℃ for 30 s, 30 cycles, 72℃ for 5 min, and 16℃ for 2 min.

[0086] The Cas9 detection primer sequences and PCR detection conditions are as follows:

[0087] Cas9-F: 5'-TGACCAGAAGCGACAAGAA-3' (SEQ ID NO. 20);

[0088] Cas9-R: 5'-CCACCACCAGCACAGAATA-3' (SEQ ID NO. 21);

[0089] The PCR amplification system consisted of: 1 μL DNA, 1 μL upstream primer F, 1 μL downstream primer R, 7.5 μL Mix, and 4.5 μL ddH2O. The PCR amplification program was: 95℃ for 5 min, 95℃ for 30 s, 57℃ for 30 s, 72℃ for 30 s, 30 cycles, 72℃ for 5 min, and 16℃ for 2 min.

[0090] (2) Specific primers were designed 100bp-150bp upstream and downstream of the target sites sgRNA1, sgRNA2, and sgRNA3 designed in Example 1. The sequence information is shown in Table 2. Genomic DNA was extracted from the 14 positive rapeseed plants obtained in step (1). The first round of PCR amplification was performed using the specific primers in Table 2 to obtain the first round of amplification products. The PCR amplification system was as follows: DNA 2μL (50ng), 10μmF 0.5μL, 10μm R 0.5μL, Mix: 3.5μL, ddH2O: 3.5μL. During PCR amplification, the amplification effect of the designed specific primers and the optimal annealing temperature were determined by gradient PCR. Amplification was performed for 28-32 cycles (minimizing the number of cycles while ensuring amplification).

[0091] 3 μL of the first-round amplification product was electrophoresed to determine its amplification specificity and amplification brightness. Based on this, 1 μL of the first-round amplification product was diluted 10–40 times and used as the DNA template for the second-round PCR amplification. The second-round PCR amplification added adapter sequences. The 10 μL reaction system consisted of: 0.2 μL of upstream universal primer F, 0.2 μL of downstream universal primer R, 3.7 μL of ddH2O, 0.1 μL of 2P-F, 0.1 μL of 2P-R, 3.7 μL of MIX, and 2 μL of diluted first-round PCR product. The upstream universal primers included 12 barcode primers F (F-1 to F-12), the downstream universal primers R included 8 barcode primers R (RA to RH), and 2P-R included 2P-1R to 2P-4R. Primer information is shown in Table 3.

[0092] Table 2 Primer sequences used in the first round of PCR amplification

[0093] Primer name Sequence (5'-3') A09-T2T1-F ggagtgagtacggtgtgcTACTCTACACTATGGATGATTG(SEQ ID NO.22) A09-T2T1-R gagttggatgctggatggTGGTATTTGATTAAGCCGGT(SEQ ID NO.23) C09-T2T1-F ggagtgagtacggtgtgcTACACTATGGATGATTTGC(SEQ ID NO.24) C09-T2T1-R gagttggatgctggatggTGATTAAGCCCGCTGACTA(SEQ ID NO.25) C04-T3T2-F ggagtgagtacggtgtgcCCTGATTGTGTAAGTTGTTGGA(SEQ ID NO.26) C04-T3T2-R gagttggatgctggatggGTTTGATACCAGGGAGACAT(SEQ ID NO.27) A03-T3T2-F ggagtgagtacggtgtgcCGGTCCAGTCAGCAAAAA(SEQ ID NO.28) A03-T3T2-R gagttggatgctggatggTGATTCCAGGGAGACATTTA(SEQ ID NO.29) A03-T1-F ggagtgagtacggtgtgcGCTGTATAATGTTCTTAAACTCGTC(SEQ ID NO.30) A03-T1-R gagttggatgctggatggAGCAGTGACAGTATTAGCCC(SEQ ID NO.31)

[0094] Table 3 Primer sequences used in the second round of PCR amplification

[0095]

[0096]

[0097] The amplification products from the second round of PCR amplification were identified by agarose gel electrophoresis and then purified using a gel extraction kit (OMEGAkit, D2500-02). The purified fragments were sequenced using Illumina (Annuo Genomics Co., Ltd.), and the results were analyzed on the Hi-TOM website (http: / / www.hi-tom.net / hi-tom / ). The results showed that the BnCDS1 gene was edited in 9 out of 14 positive rapeseed plants, with an editing efficiency of 64.29%. Editing types included single-base, double-base insertion, and single-base substitution, with single-base and multi-base deletions being the most common. The editing vector provided by this invention has a highly efficient editing capability in rapeseed, meeting expectations. Figure 3 ).

[0098] 2. Off-target rate detection

[0099] All potential off-target sites and their potential off-target efficiencies were predicted using the online software CRISPR-P.2.0 (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR), including mismatch sites with a nucleotide ratio of 4 or less. Target sites with high off-target efficiencies were selected, and nucleotide sequences of 100-200 bp before and after the sites were extracted based on their chromosomal physical location in the database for PCR primer design. Sequences containing potential off-target sites were extracted and submitted to the online software Primer 5.0 for batch primer design (Table 4). The primer pairs were set to amplify PCR lengths of 250bp-400bp. For each potential off-target site, DNA from edited T0 generation plants and untransformed Westar plants were used to perform PCR amplification. The PCR products were purified and ligated into the pMD19-T vector. Three clones from each sample were selected for Sanger sequencing analysis. The results showed that, except for the off-target efficiency of sgRNA1 off1-1, which was high at 47.1%, the off-target effects at other sites were very low, all below 20% (Table 5). From all off-target sites, three off-target sites (off-1, off-2, off-3) of sgRNA1, four off-target sites (off-1, off-2, off-3, off-4) of sgRNA2, and two off-target sites (off-1, off-2) of sgRNA3 were selected for Sanger sequencing. Analysis of 14 plants compared with wild-type Westar revealed no off-target effects, effectively avoiding the targeting of non-target genes. In summary, the editing vector provided by this invention exhibited high accuracy in the rapeseed genome, successfully preventing accidental editing.

[0100] Table 4 Primer sequences used for amplifying and predicting off-target sequences.

[0101]

[0102]

[0103] Table 5. Prediction and detection results of sgRNA off-target sites in T0 generation BnCDS1 mutant.

[0104]

[0105] Note: Y indicates no miss, N indicates miss.

[0106] Example 4

[0107] Screening of mutants with different mutation types and phenotypic determination

[0108] (1) T0 generation positive seedlings were cultured in a culture room and self-pollinated to obtain T1 generation BnCDS1 mutants with different editing types. In the culture room, some T1 generation mutants were cultured in medium-sized cups (27.5 cm in diameter, 31 cm in height) containing nutrient soil. The culture conditions were 22℃, 16 h light, 8 h dark, and 75% relative humidity. After about 4 weeks of growth, genomic DNA was extracted from individual mutants. Following the steps in Example 1, positive progeny were screened based on the Hygmycin (HygR) element and Cas9 base sequence, and their editing status was sequenced. Five different editing types of mutants were screened, including mutants with 4 copies (i.e., all 4 genes were mutated), 3 copies, 2 copies, 1 copy, and 0 copies. Figure 4 The mutants were named T2-5-2, T2-7-7, T2-3-8, T2-3-2, and T2-8-3, respectively. After 240 days of self-pollination and growth, the seeds of the T2 generation mutants were harvested individually at maturity and dried under natural conditions.

[0109] (2) T2 generation mutants with different copy number editing were cultured in medium-sized cups containing nutrient soil under indoor conditions: 22℃, 16h light, 8h dark, and 75% relative humidity. Phenotypic, chlorophyll content (plant nutrient analyzer), light conversion efficiency, and nitrogen content (photosynthesis analyzer: LI-CORLI-6800) were evaluated. Results showed that the mutant rapeseed at the 5-leaf stage did not differ significantly from the wild type. However, at the bolting and flowering stage, the 3-copy-edited T2-7-7 and the 4-copy-edited T2-5-2 were dwarfed compared to the wild-type Westar line. Figure 5 Simultaneously, at 45 days of rapeseed growth, the chlorophyll content of the mutants was measured using a plant nutrient analyzer, and the effects of the mutant BnCDS1 gene on the net photosynthetic rate and nitrogen content of rapeseed leaves were detected using a photosynthesis meter (LI-CORLI-6800). The results showed that the average nitrogen content of each BnCDS1 mutant was approximately 13.54 μmol / L, the chlorophyll content was approximately 41.49 μmol / L, and the net photosynthetic rate was approximately 9.14 μmol / L. -2 s -1 Around the same time, it is similar to the wild-type Westar, with no significant differences. Figure 6 The results showed that editing BnCDS1 in rapeseed affected its growth height to some extent, but did not affect seedling growth, chlorophyll content, net photosynthetic rate, or nitrogen content.

[0110] Example 5

[0111] Analysis of gene-edited rapeseed's resistance to Sclerotinia sclerotiorum and Botrytis cinerea.

[0112] (1) Activation of strains

[0113] Using an inoculation needle, take wild-type Sclerotinia sclerotiorum 1980 or wild-type Botrytis cinerea B05.10 mycelial blocks and inoculate them onto 90 mm diameter PDA medium with the mycelial side down. Incubate in a fungal culture room at 20 °C for three generations of continuous activation. Using a punch, take fresh mycelial blocks from the edge and place them onto 17.5 mL of 2×SY medium (medium: 10 g sucrose, 1 g yeast extract, 10 g agar, dissolved in distilled water and brought to a final volume of 1 L, sterilized by steam at 121 °C for 30 min) at a diameter of 90 mm. Incubate in a fungal culture room at 20 °C for 36 h.

[0114] (2) The T2 generation mutants T2-5-2, T2-7-7, T2-3-8, T2-3-2 and T2-8-3 obtained in Example 4 were planted in small cups containing nutrient soil and cultured in a plant culture room for about 45 days.

[0115] (3) Using a 3mm punch, mycelial blocks from 2×SY medium were taken and inoculated onto the right side of the leaves of both pure and Arabidopsis mutants near the veins, ensuring moisture retention. After inoculation with *Sclerotinia sclerotiorum* 1980 for 36 hours and *Botrytis cinerea* B05.10 for 48 hours, the diameter of the lesions was measured using the cross-sectional method, and the lesion area was calculated. The results showed that 36 hours after inoculation with *Sclerotinia sclerotiorum* 1980, the lesion area of ​​wild-type *Westar* was 270.46 mm². 2 The area of ​​the T2-3-2 lesion is approximately 250.18 mm². 2 The lesion area of ​​T2-3-8 was approximately 219.21 mm². 2 The area of ​​the T2-7-7 lesion is approximately 166.43 mm². 2 The area of ​​the T2-5-2 lesion is approximately 132.40 mm². 2 The area of ​​the T2-8-3 lesion was approximately 273.16 mm². 2 Analysis of lesion area differences revealed no significant differences between T2-3-2 and T2-8-3 and Wester, while the remaining mutants showed significant differences compared to Wester. However, T2-3-2 showed a 7.5% reduction in lesion area, while other mutants such as T2-3-8, T2-7-7, and T2-5-2 showed reductions of 18.95%, 38.46%, and 51.05%, respectively. Figure 7 (A and B); 48 hours after inoculation with Botrytis cinerea B05.10, the results showed that the lesion area of ​​wild-type Westar was 168.45 mm. 2 The area of ​​the T2-3-2 lesion is approximately 127.94 mm². 2 The lesion area of ​​T2-3-8 was approximately 103.31 mm². 2 The area of ​​the T2-7-7 lesion is approximately 83.05 mm². 2 The area of ​​the T2-5-2 lesion is approximately 53.14 mm².2 The lesion area of ​​T2-8-3 was approximately 167.52 mm². 2 Statistical analysis revealed that, compared to the wild-type Westar T2-3-2, the lesion area decreased by 24.05%, T2-3-8 by 38.67%, T2-7-7 by 50.69%, and T2-5-2 by 68.45%. This indicates that the edited mutant lesion area exhibits a similar disease resistance trend to that inoculated with *Sclerotinia sclerotiorum* 1980. Figure 7 (D and E). This indicates that the area of ​​the edited mutant lesions gradually decreases as the number of edited copies increases.

[0116] Using Ssβ-tubulin from Sclerotinia sclerotiorum as an internal reference gene, the relative biomass per unit area was detected by qPCR over 36 hours. The results showed that the relative biomass of Sclerotinia sclerotiorum gradually decreased with the increase of the number of edited copies. Figure 7 (C); Using qPCR to detect the relative biomass at 48 h, the results showed that as the number of edited copies increased, the relative biomass of *Botrytis cinerea* gradually decreased. Figure 7 (F). Editing BnCDS1 can significantly improve the resistance of rapeseed to Sclerotinia sclerotiorum and Botrytis cinerea.

[0117] Example 6

[0118] Inoculation and disease index statistics of clubroot bacteria

[0119] (1) Preparation of dormant spores of *Plasmodiophora stearothermiae*:

[0120] Rapeseed roots infected with clubroot were collected from the field. The roots were washed with tap water, treated with 70% ethanol for 2 minutes, and then with 10% hydrogen peroxide for 1 hour for disinfection. The roots were then rinsed thoroughly with sterile water 5 times. 10g of clubroot tissue was weighed, chopped, and mixed with 50mL of sterile water. The mixture was then ground using a juicer, filtered through multiple layers of gauze to remove root residue, and centrifuged at 3100 rpm for 10 minutes. The supernatant was discarded (this process was repeated 5 times). 5mL of 50% sucrose solution was added to the precipitate, mixed well, and centrifuged at 3100 rpm for 10 minutes. The supernatant was collected into a new sterile 50mL centrifuge tube. 30mL of sterile water was added, and the tube was centrifuged at 3100 rpm for 10 minutes. The supernatant was discarded (this process was repeated 5 times) to thoroughly wash away the sucrose from the dormant spores. The dormant spores were resuspended in 2% (w / v) chloramine-T solution and treated at room temperature for 20 minutes. The tubes were then centrifuged and washed 5 times with sterile water. The washed dormant spores were suspended in an antibiotic solution (1 mg / mL acetaminophen sulfate, 1 mg / mL vancomycin hydrochloride, 6 mg / mL cefotaxime sodium) and treated in the dark at 25°C for 24 h, followed by washing five times by centrifugation with sterile water. The concentration of the cleaned dormant spores was determined using a hemocytometer, and the prepared dormant spore solution was stored at 4°C for later use. The concentration of the *Plasmodiophora* dormant spore solution was adjusted to 1 × 10⁻⁶. 7spores / mL;

[0121] (2) The T2 generation mutants T2-5-2, T2-7-7, T2-3-8, T2-3-2, and T2-8-3 obtained in Example 4 were planted in small cups containing nutrient soil and cultured in a plant culture room. 1 mL of the mutants was inoculated into the roots of rapeseed after about one week of growth. Disease incidence was observed 3-4 weeks after inoculation, and the disease index was calculated (Grade 0: no disease; Grade 1: root swellings only attached to lateral roots, accounting for 1%-25% of the total root system; Grade 2: root swellings attached to the main root and root swellings on lateral roots). Grade 1: Root swellings account for 26%-49% of the total root system; Grade 2: Root swellings are attached to the main root, accounting for 50%-75% of the root system; Grade 3: Root swellings are attached to the main root, accounting for more than 75% of the root system. The results showed that wild-type Westar and all mutant rapeseed were affected. Statistics on the number of affected plants revealed that wild-type Westar and T2-8-3 had the highest number of affected plants, followed by T2-3-2 and T2-3-8, while T2-7-7 and T2-5-2 had the lowest number of affected plants. Figure 8 (A and B). Disease index statistics for inoculated *Plasmodiophora* fungi showed that the wild-type Westar was 44.44%, T2-3-2 was 33.33%, T2-3-8 was 31.48%, T2-7-7 was 22.22%, and T2-5-2 was 17.59%. Figure 8 (C). The results showed that editing BnCDS1 could significantly improve the resistance of rapeseed to clubroot fungus.

[0122] As can be seen from the above, the BnCDS1 gene can be used for plant disease control and to enhance the ability of plants, especially rapeseed, to resist Sclerotinia sclerotiorum, Botrytis cinerea, and clubroot fungi.

[0123] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Knockout BnCDS1 application of the gene in disease prevention and control of rapeseed; the BnCDS1 gene is BnaA09T0145500WE gene, BnaC09T0175800WE gene, BnaC04T0239200WE gene and BnaA03T0593500WE gene; The amino acid sequence coded by the BnaA09T0145500WE gene is shown as SEQ ID NO.

1. The amino acid sequence coded by the BnaC09T0175800WE gene is shown as SEQ ID NO.

2. The amino acid sequence coded by the BnaC04T0239200WE gene is shown as SEQ ID NO.

3. The amino acid sequence coded by the BnaA03T0593500WE gene is shown as SEQ ID NO.

4. The disease is one or more of sclerotinia, botrytis and clubroot.

2. Use according to claim 1, characterized in that, The rape disease prevention and control comprises improving the resistance of rape to pathogenic bacteria. The pathogenic fungi are one or more of Sclerotinia sclerotiorum Sclerotinia sclerotiorum , Botrytis cinerea Botrytis cinerea , and Plasmodiophora brassicae Plasmodiophora brassicae .

3. The use according to claim 1 or 2, characterized in that, The nucleotide sequence of the BnaA09T0145500WE gene is shown as SEQ ID NO.

5. The nucleotide sequence of the BnaC09T0175800WE gene is shown as SEQ ID NO.

6. The nucleotide sequence of the BnaC04T0239200WE gene is shown as SEQ ID NO.

7. The nucleotide sequence of the BnaA03T0593500WE gene is shown as SEQ ID NO.

8.

4. The sgRNA combination or gene editing vector of claim 1, wherein the sgRNA combination comprises sgRNA1, sgRNA2 and sgRNA3. BnCDS1 The sgRNA combination or gene editing vector of claim 1 in the application of disease prevention and control of Brassica napus; the sgRNA combination comprises sgRNA1, sgRNA2 and sgRNA3. The nucleotide sequence of the sgRNA1 is shown as SEQ ID NO.

15. The nucleotide sequence of the sgRNA2 is shown as SEQ ID NO.

16. The nucleotide sequence of the sgRNA3 is shown as SEQ ID NO.

17. The gene editing vector comprises an editing backbone vector and the sgRNA combination inserted into the editing backbone vector. The disease is one or more of sclerotinia, botrytis and clubroot.

5. Use according to claim 4, characterized in that, The editing backbone vector comprises pRGEB35-Cas9.

6. A method of increasing the resistance of Brassica oleracea to a pathogenic fungus, characterized in that, Knockout as described in claim 1 BnCDS1 Genes are introduced into rapeseed using sgRNA combinations or gene editing vectors and then cultured. The sgRNA combination comprises sgRNA1, sgRNA2 and sgRNA3. The nucleotide sequence of the sgRNA1 is shown as SEQ ID NO.

15. The nucleotide sequence of the sgRNA2 is shown as SEQ ID NO.

16. The nucleotide sequence of the sgRNA3 is shown as SEQ ID NO.

17. The gene editing vector comprises an editing backbone vector and the sgRNA combination inserted into the editing backbone vector. The pathogenic fungi are one or more of Sclerotinia sclerotiorum Sclerotinia sclerotiorum , Botrytis cinerea Botrytis cinerea , and Plasmodiophora brassicae Plasmodiophora brassicae .

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