Application of bodmr1 gene and bodmr6 gene in breeding disease-resistant broccoli varieties, expression vector and breeding method

Abstract

The application discloses application of BoDMR1 and BoDMR6 genes in cultivating disease-resistant broccoli varieties, an expression vector and a cultivating method, and relates to the technical field of plant molecular biology. A nucleotide sequence of a broccoli BoDMR1 gene is shown in SEQ ID No: 7, or a nucleotide after gene mutation of the nucleotide sequence shown in SEQ ID No: 7; and a nucleotide sequence of a broccoli BoDMR6 gene is shown in SEQ ID No: 8, or a nucleotide after gene mutation of the nucleotide sequence shown in SEQ ID No: 8. Through gene editing on endogenous BoDMR1 and / or BoDMR6 genes of plants, mutant plants showing resistance to diseases such as sclerotinia, black rot and alternaria blight can be obtained. Therefore, the application is beneficial to providing good resources for cultivating disease-resistant broccoli varieties.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and more specifically, to the application, expression vector, and cultivation method of the BoDMR1 and BoDMR6 genes in the breeding of disease-resistant broccoli varieties. Background Technology

[0002] Broccoli (Brassica oleracea L. var. italica), also known as Chinese broccoli, is a variety of Brassica oleracea belonging to the Brassicaceae family. Due to its superior average nutritional value and disease resistance compared to other vegetables, it is widely cultivated and has a large consumer market globally. It is one of my country's important major vegetable crops, with a current planting area of ​​approximately 1.5-1.8 million mu (100,000-120,000 hectares) and an annual output exceeding 4 million tons. All other provinces have achieved full coverage of broccoli cultivation, playing a vital role in my country's year-round vegetable supply. In recent years, with increasing consumer demand and expanding planting areas, crop rotation periods have become shorter, leading to a growing trend of increasing damage from pathogens such as black spot, black rot, and sclerotinia stem rot, posing a significant threat to broccoli cultivation.

[0003] Broccoli originated in the arid Mediterranean region of Italy. As a highly differentiated Brassica oleracea crop, its genetic resources are very limited, especially since commercial broccoli varieties are highly similar and generally lack strong disease resistance. Researching and utilizing plant immune genes and pathogen-pathogen interaction genes through biotechnology is of great scientific and practical significance for improving the disease resistance, quality, and economic benefits of broccoli, and for ensuring a balanced vegetable supply in my country.

[0004] Susceptibility genes are genes that promote pathogen transmission and play a negative role in plant defense. In contrast, resistance genes are essential for normal plant metabolism. Compared to resistance genes, susceptibility genes are more likely to provide durable resistance in the plant immune system. Therefore, inactivation of susceptibility genes is considered a prospective strategy for obtaining broad-spectrum and durable disease-resistant plants. The DMR1 (Downy mildew resistance gene 1) gene, found in Arabidopsis thaliana, encodes a homoserine kinase. Mutants dmr1-1 to dmr1-6 exhibit a one-base mutation (GA or CT) at 136bp, 352bp, 539bp, 604bp, 732bp, and 800bp, respectively, resulting in high homoserine content and thus resistance to downy mildew. The DMR6 (Downy mildew resistance gene 6) gene encodes a 2-oxoglutarate Fe(II)-dependent oxygenase of unknown function. A one-base mutation (GA) at 691 bp in the dmr6 gene, changing the codon from tryptophan to a stop codon, confers resistance to downy mildew in Arabidopsis thaliana dmr6 mutants. In tomato, the Sldmr6-1 mutant exhibits enhanced resistance to bacterial, oomycete, and fungal pathogens. This phenotype is associated with increased levels of the defensive hormone salicylic acid (SA) and enhanced transcriptional activation of the plant immune response. However, the defensive immune functions of the DMR1 and DMR6 genes in broccoli remain unclear, and the resistance of mutants to broccoli pathogens has not been tested.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the application, expression vector, and breeding method of the BoDMR1 and BoDMR6 genes in the breeding of disease-resistant broccoli varieties, thereby exploring the defensive effect of DMR1 and DMR6 gene knockout on broccoli pathogens and providing new material resources and strategies for disease-resistant broccoli breeding.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides the application of the broccoli BoDMR1 gene and / or BoDMR6 gene in the breeding of disease-resistant broccoli varieties or in improving the disease resistance of broccoli, characterized in that the nucleotide sequence of the broccoli BoDMR1 gene is as shown in SEQ ID No:7, or the nucleotide sequence shown in SEQ ID No:7 after gene mutation.

[0009] The nucleotide sequence of the broccoli BoDMR6 gene is shown in SEQ ID No:8, or is a nucleotide sequence of SEQ ID No:8 after gene mutation; the disease is selected from bacterial diseases and / or fungal diseases.

[0010] In a second aspect, the present invention provides a broccoli CRISPR / Cas9 gene editing system, characterized in that it comprises: Cas9 protein and sgRNA, wherein the nucleotide sequence of the sgRNA is shown as at least one of the sequences in SEQ ID NO:1-4.

[0011] Thirdly, the present invention provides an expression vector for a broccoli CRISPR / Cas9 gene editing system, wherein the expression vector is a plasmid comprising a nucleotide sequence of sgRNA and a gene sequence encoding Cas9 protein; the nucleotide sequence of sgRNA is shown in at least one of SEQ ID NO:1-4.

[0012] Fourthly, the present invention provides a method for breeding disease-resistant broccoli varieties or improving the disease resistance of broccoli, the method comprising: inhibiting the expression of the broccoli BoDMR1 gene and / or the broccoli BoDMR6 gene.

[0013] Fifthly, the present invention provides a kit or host cell for gene editing, the kit comprising: the above-described broccoli CRISPR / Cas9 gene editing system or the above-described expression vector; the host cell comprising: the above-described expression vector.

[0014] The present invention has the following beneficial effects:

[0015] This invention reveals the impact of loss of function of the BoDMR1 and / or BoDMR6 genes on disease resistance in broccoli. Through gene editing, the endogenous BoDMR1 and / or BoDMR6 genes in broccoli were rendered nonfunctional, resulting in mutant plants exhibiting resistance to diseases such as sclerotinia stem rot, black rot, and black spot. Therefore, this invention provides a valuable resource for breeding disease-resistant broccoli varieties.

[0016] This invention provides a CRISPR / Cas9 gene editing system for broccoli, which includes a specific sgRNA sequence that can target the BoDMR1 and / or BoDMR6 genes in the broccoli genome. The system has an editing efficiency of 25%-73.3% for the target site DNA, thus exhibiting high editing efficiency.

[0017] The gene editing system and expression vector described above can target the BoDMR1 and / or BoDMR6 genes in the broccoli genome, and the construction method of the expression vector is simple and rapid.

[0018] In addition, a kit for gene editing is provided, which has high editing activity and can be widely used in the field of broccoli gene editing. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A structural diagram of the gene editing expression vector for broccoli;

[0021] Figure 2 Image showing the transgenic detection results of resistant plants (M: DL2000 maker; +, +, +: pCas-sgBoDMR1, pCas-sgBoDMR6, pCas-sgBoDMR1+sgBoDMR6 vector plasmids, respectively; -: wild type; 1~28, 29~58, 59~78: resistant plant numbers).

[0022] Figure 3 Partial sequencing results of BoDMR1 mutation types in broccoli mediated by pCas9-sgBoDMR1;

[0023] Figure 4 Partial sequencing results of BoDMR6 mutation types in broccoli mediated by pCas9-sgBoDMR6;

[0024] Figure 5 Partial sequencing results of BoDMR1 and BoDMR6 mutation types in broccoli mediated by pCas9-sgBoDMR1+BoDMR6;

[0025] Figure 6 This is a graph showing the results of sclerotinia stem rot resistance identification.

[0026] Figure 7 Image showing the results of resistance identification for black rot disease;

[0027] Figure 8 This is a diagram showing the results of resistance identification for black spot disease. Detailed Implementation

[0028] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein, some methods and materials are described hereby. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0030] Unless otherwise specified, the practice of this invention will employ conventional techniques of plant physiology, plant molecular genetics, cell biology, molecular biology (including recombination techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Plant Physiology* (Cang Jing et al., 2017); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Current Protocols in Molecular Biology* (edited by F.M. Ausubel et al., 1987); *Plant Molecular Genetics* (by Monica A. Hughes et al.); and *PCR: The Polymerase Chain Reaction* (edited by Mullis et al., 1994). Each of these references is explicitly incorporated herein by reference.

[0031] In a first aspect, the present invention provides the application of the broccoli BoDMR1 gene and / or BoDMR6 gene in the breeding of disease-resistant broccoli varieties or in improving the disease resistance of broccoli. The nucleotide sequence of the broccoli BoDMR1 gene is shown in SEQ ID No:7, or the nucleotide sequence shown in SEQ ID No:7 after gene mutation.

[0032] The nucleotide sequence of the BoDMR6 gene in broccoli is shown in SEQ ID No:8, or the nucleotide sequence shown in SEQ ID No:8 after gene mutation; the diseases are selected from bacterial diseases and / or fungal diseases.

[0033] By using gene editing (such as gene knockout) to cause the loss of function of the endogenous BoDMR1 and / or BoDMR6 genes in broccoli; or by introducing nucleotides encoding the BoDMR1 and / or BoDMR6 genes through hybridization, the endogenous BoDMR1 and / or BoDMR6 genes in broccoli can also lose or partially lose their function. The resulting mutant plants exhibit resistance to diseases such as sclerotinia stem rot, black rot, and black spot. Therefore, this invention provides a valuable resource for breeding disease-resistant broccoli varieties.

[0034] In a preferred embodiment of the present invention, the fungal disease is selected from sclerotinia rot and / or black spot; the bacterial disease is black rot.

[0035] In a preferred embodiment of the present invention, gene mutation is caused by substitution, deletion or insertion; one or more nucleotides are substituted, deleted or inserted.

[0036] Deletion mutations can range in length from 1 nucleotide to over 100 nucleotides, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 4 The numbers 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 are listed. Such deletions can lead to almost no expression of the gene (RNA and / or protein), and the target protein will have no activity or reduced activity.

[0037] In a preferred embodiment of the present invention, the gene mutation in the nucleotide sequence after gene mutation of the nucleotide sequence shown in SEQ ID No:7 is selected from at least one of the following: an insertion of a T base or a G base between positions 191 and 192, a deletion of 7 bases between positions 183 and 191, or a deletion of 1 base at position 191.

[0038] In a preferred embodiment of the present invention, the gene mutation in the nucleotide sequence after gene mutation of the nucleotide sequence shown in SEQ ID No:8 is selected from at least one of the following: an insertion of a T base or a CC base between positions 144 and 145, a deletion of one base at position 145, or a deletion of four bases between positions 140 and 145.

[0039] In a preferred embodiment of the present invention, the application includes any of the following:

[0040] (1) When the nucleotide sequence of the BoDMR1 gene of broccoli is as shown in SEQ ID No:7, the endogenous BoDMR1 gene of broccoli is genetically modified so that it is not expressed, is not expressed at all, or the function of the BoDMR1 protein is partially or completely lost.

[0041] (2) When the nucleotide sequence of broccoli BoDMR1 is obtained by gene mutation from the nucleotide sequence shown in SEQ ID No:7, the obtained broccoli BoDMR1 mutant gene is introduced into broccoli cells.

[0042] (3) When the nucleotide sequence of the broccoli BoDMR6 gene is as shown in SEQ ID No:8, the endogenous BoDMR6 gene of broccoli is genetically modified so that it is not expressed, is not expressed at all, or the function of the BoDMR6 protein is partially or completely lost.

[0043] (4) When the nucleotide sequence of broccoli BoDMR6 is obtained by gene mutation from the nucleotide sequence shown in SEQ ID No:8, the obtained broccoli BoDMR6 mutant gene is introduced into broccoli cells.

[0044] (5) When the nucleotide sequence of the broccoli BoDMR1 gene is as shown in SEQ ID No:7 and the nucleotide sequence of the broccoli BoDMR6 gene is as shown in SEQ ID No:8, the endogenous BoDMR1 gene and BoDMR6 gene of broccoli are genetically modified to make them not expressed, basically not expressed, or express BoDMR1 protein and BoDMR6 protein with partial or complete loss of function.

[0045] (6) When the nucleotide sequence of the broccoli BoDMR1 gene is obtained by gene mutation from the nucleotide sequence shown in SEQ ID No:7, and the nucleotide sequence of the broccoli BoDMR6 gene is obtained by gene mutation from the nucleotide sequence shown in SEQ ID No:8, the obtained broccoli BoDMR1 mutant gene and broccoli BoDMR6 mutant gene are introduced into broccoli cells.

[0046] The above application method (5) is better than application methods (1) and (3) in improving the plant's disease resistance, that is, the double-target gene mutation of broccoli has a better disease resistance effect than the single-target gene mutation.

[0047] In one alternative implementation, the method introduced is selected from genetic transformation methods or genome editing methods.

[0048] The aforementioned genetic transformation methods include, but are not limited to, producing individuals with enhanced disease resistance through self-pollination or hybridization with other broccoli individuals using parental plants carrying the BoDMR1 mutant gene and / or the BoDMR6 mutant gene of broccoli. In other embodiments, the transformation methods include, but are not limited to, Agrobacterium-mediated gene transformation, gene gun transformation, and pollen tube pathway transformation.

[0049] Genome editing or gene mutation methods refer to methods readily conceived by those skilled in the art, such as conventional transgenic techniques and gene editing techniques (e.g., zinc-finger nucleases, transcription activator-like effector nucleases, or CRISPR / Cas9), to modify target plants to possess the BoDMR1 and / or BoDMR6 mutant genes of broccoli, thereby obtaining improved new varieties with enhanced disease resistance. Therefore, regardless of the technology used, as long as it utilizes the BoDMR1 and / or BoDMR6 mutant genes of broccoli provided by this invention to confer disease resistance in broccoli, it falls within the scope of protection of this invention.

[0050] In a preferred embodiment of the present invention, the genetic modification of the endogenous BoDMR1 gene and / or BoDMR6 gene in broccoli refers to gene modification using gene editing technology.

[0051] In a preferred embodiment of the present invention, the gene editing technology is zinc finger endonuclease technology, transcription activator effector nuclease technology, or CRISPR / Cas9 technology.

[0052] Secondly, the present invention provides a broccoli CRISPR / Cas9 gene editing system, comprising: Cas9 protein and sgRNA (single guide RNA, hereinafter referred to as sgRNA), wherein the nucleotide sequence of sgRNA is shown in at least one of SEQ ID NO:1-4.

[0053] SEQ ID NO:1-2 are used to target two sites of the BoDMR1 gene, respectively, and SEQ ID NO:3-4 are used to target two sites of the BoDMR6 gene, respectively.

[0054] BoDMR1-T1:5'-CGGTTAACGAGATCTTCGGT-3'(SEQ ID NO:1), BoDMR1-T2:5'-CCCGCCACCGTCGCTAATCT-3'(SEQ ID NO:2); BoDMR6-T1:5'-CCGGTTTCCGTCACTCTACT-3'(SEQID NO: 3), BoDMR6-T2: 5'-TATCTTCCACAGATCGATCT-3' (SEQ ID NO: 4).

[0055] This system uses the combined action of Cas9 protein and sgRNA to recognize, locate, cut, and edit target DNA.

[0056] The nucleotide sequence of the sgRNA can be one, two, three, or four different sequences. It can be any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; or two sequences shown in SEQ ID NO:1 and SEQ ID NO:2; or two sequences shown in SEQ ID NO:3 and SEQ ID NO:4; or two sequences shown in SEQ ID NO:1 and SEQ ID NO:3; or three sequences shown in SEQ ID NO:2 and SEQ ID NO:4; or three sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4; or four sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Those skilled in the art can choose the number of sgRNAs as needed.

[0057] In a preferred embodiment of the present invention, the nucleotide sequence of the sgRNA includes at least two sequences as shown in SEQ ID NO:1-4.

[0058] In a preferred embodiment of the present invention, the nucleotide sequence of the sgRNA includes: the nucleotide sequence shown in SEQ ID NO:1-2, and / or the nucleotide sequence shown in SEQ ID NO:3-4.

[0059] In a preferred embodiment of the present invention, the amino acid sequence of the Cas9 protein is shown in SEQ ID NO:5.

[0060] Thirdly, the present invention provides an expression vector for a broccoli CRISPR / Cas9 gene editing system, wherein the expression vector is a plasmid comprising a nucleotide sequence of sgRNA and a gene sequence encoding Cas9 protein; the nucleotide sequence of sgRNA is shown in at least one of SEQ ID NO:1-4.

[0061] The term "expression vector" refers to bacterial plasmids, yeast plasmids, or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within a host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0062] In a preferred embodiment of the present invention, the vector is a bacterial vector, such as an Agrobacterium expression vector.

[0063] Transforming host cells with a vector can be done using conventional techniques well known to those skilled in the art.

[0064] In one alternative embodiment, the method for introducing the expression vector into the host cell is selected from genetic transformation methods. In other embodiments, the transformation methods described above include, but are not limited to, Agrobacterium-mediated gene transformation, gene gun transformation, and pollen tube pathway transformation.

[0065] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0066] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline, ampicillin, and kanamycin resistance for Escherichia coli.

[0067] In one alternative embodiment, the expression vector further includes a signal peptide, such as a nuclear localization signal peptide.

[0068] In a preferred embodiment of the present invention, the vector includes an expression control element operatively linked to the sgRNA; the expression control element is a promoter, terminator, and / or enhancer.

[0069] In a preferred embodiment of the present invention, the nucleotide sequence of the sgRNA includes at least two sequences as shown in SEQ ID NO:1-4.

[0070] The nucleotide sequence of sgRNA includes: the nucleotide sequence shown in SEQ ID NO:1-2, and / or, the nucleotide sequence shown in SEQ ID NO:3-4.

[0071] In a preferred embodiment of this invention, the expression vector is obtained by ligating the nucleotide sequence of sgRNA to a base vector containing an expression cassette encoding the Cas9 protein. Specifically, the base vector encoding the Cas9 protein expression cassette is pCAMBIA2301 (laboratory-preserved), with the Cas9 protein expression cassette donated by Academician Liu Yaoguang of South China Agricultural University. Ligation methods include, but are not limited to, enzyme digestion ligation, seamless cloning, etc.

[0072] In a preferred embodiment of the present invention, the expression vector includes at least one of the following expression vectors:

[0073] (1) The gene sequence encoding the Cas9 protein expression cassette, the promoter for initiating sgRNA expression, the spacer sequence, the first sgRNA target sequence, the spacer sequence, the second sgRNA target sequence, and the terminator for terminating sgRNA expression; the first sgRNA target sequence is different from the second sgRNA target sequence, and the first sgRNA target sequence is selected from any one of sgRNA1 targeting the BoDMR1 gene of broccoli, sgRNA2 targeting the BoDMR1 gene of broccoli, sgRNA3 targeting the BoDMR6 gene of broccoli, and sgRNA4 targeting the BoDMR6 gene of broccoli, and its nucleotide sequence is shown in SEQ ID NO:1-4; the second sgRNA target sequence is selected from any one of sgRNA1 targeting the BoDMR1 gene of broccoli, sgRNA2 targeting the BoDMR1 gene of broccoli, sgRNA3 targeting the BoDMR6 gene of broccoli, and sgRNA4 targeting the BoDMR6 gene of broccoli, and its nucleotide sequence is shown in SEQ ID NO:1-4.

[0074] (2) The gene sequence encoding the Cas9 protein expression cassette, the promoter that initiates sgRNA expression, the spacer sequence, the first sgRNA target sequence, the spacer sequence, the second sgRNA target sequence, the terminator that terminates sgRNA expression, the promoter that initiates sgRNA expression, the spacer sequence, the third sgRNA target sequence, the spacer sequence, the fourth sgRNA target sequence, and the terminator that terminates sgRNA expression; and the first sgRNA target sequence, the second sgRNA target sequence, the third sgRNA target sequence, and the fourth sgRNA target sequence are different;

[0075] The first sgRNA target sequence is selected from any one of sgRNA1 targeting the BoDMR1 gene of broccoli, sgRNA2 targeting the BoDMR1 gene of broccoli, sgRNA3 targeting the BoDMR6 gene of broccoli, and sgRNA4 targeting the BoDMR6 gene of broccoli, and their nucleotide sequences are shown in SEQ ID NO:1-4 in sequence; the second sgRNA target sequence is selected from any one of sgRNA1 targeting the BoDMR1 gene of broccoli, sgRNA2 targeting the BoDMR1 gene of broccoli, sgRNA3 targeting the BoDMR6 gene of broccoli, and sgRNA4 targeting the BoDMR6 gene of broccoli, and their nucleotide sequences are shown in SEQ ID NO:1-4 in sequence; the third sgRNA target sequence is selected from any one of sgRNA1 targeting the BoDMR1 gene of broccoli, sgRNA2 targeting the BoDMR1 gene of broccoli, sgRNA3 targeting the BoDMR6 gene of broccoli, and sgRNA4 targeting the BoDMR6 gene of broccoli, and their nucleotide sequences are shown in SEQ ID NO:1-4 in sequence. As shown in NO:1-4; the fourth sgRNA target sequence is selected from any one of sgRNA1 targeting the BoDMR1 gene of broccoli, sgRNA2 targeting the BoDMR1 gene of broccoli, sgRNA3 targeting the BoDMR6 gene of broccoli and sgRNA4 targeting the BoDMR6 gene of broccoli, and their nucleotide sequences are shown in SEQ ID NO:1-4.

[0076] In a preferred embodiment of the present invention, the expression vector includes at least one of the following expression vectors:

[0077] (1) The gene sequence encoding the Cas9 protein expression cassette, the promoter that initiates sgRNA expression, the spacer sequence, sgRNA1 targeting the broccoli BoDMR1 gene, the spacer sequence, sgRNA2 targeting the broccoli BoDMR1 gene, and the terminator that terminates sgRNA expression; the nucleotide sequences of sgRNA1 and sgRNA2 are shown in SEQ ID NO:1-2 in sequence.

[0078] (2) The gene sequence encoding the Cas9 protein expression cassette, the promoter that initiates sgRNA expression, the spacer sequence, sgRNA3 targeting the broccoli BoDMR6 gene, the spacer sequence, sgRNA4 targeting the broccoli BoDMR6 gene, and the terminator that terminates sgRNA expression; the nucleotide sequences of sgRNA3 and sgRNA4 are shown in SEQ ID NO:3-4 in sequence.

[0079] (3) The gene sequence encoding the Cas9 protein expression cassette, the promoter that initiates sgRNA expression, the spacer sequence, sgRNA1 targeting the BoDMR1 gene of broccoli, the spacer sequence, sgRNA2 targeting the BoDMR1 gene of broccoli, the terminator that terminates sgRNA expression, the promoter that initiates sgRNA expression, the spacer sequence, sgRNA3 targeting the BoDMR6 gene of broccoli, the spacer sequence, sgRNA4 targeting the BoDMR6 gene of broccoli, and the terminator that terminates sgRNA expression; the nucleotide sequences of sgRNA1, sgRNA2, sgRNA3, and sgRNA4 are shown in SEQ ID NO:1-4.

[0080] The inventors' research shows that mutations in the BoDMR1 and BoDMR6 genes significantly improve resistance to diseases such as sclerotinia stem rot, black rot, and black spot in broccoli, while superimposed mutations of the two genes (BoDMR1 and BoDMR6) can further improve resistance to these diseases.

[0081] Gene editing causes mutations such as substitution, deletion, and insertion in the exon sequences of the BoDMR1 and BoDMR6 genes in broccoli, resulting in loss of gene function and enhancing the broccoli's resistance to sclerotinia rot, black rot, and black spot disease.

[0082] In a preferred embodiment of the present invention, the spacer sequence is tRNA, Csy4, or HH-HDV ribozyme; when the spacer sequence is tRNA, the nucleotide sequence of the spacer sequence is as shown in SEQ ID NO:6. The tRNA sequence plays a role in recognizing and improving the expression efficiency of target sites, and broadening the selection range of target sites.

[0083] In a preferred embodiment of the present invention, the promoter for initiating sgRNA expression is selected from any one of AtU6-1, AtU6-26, AtU3b, and AtU6-29. In other embodiments, the promoter for initiating sgRNA expression may also be the U6 promoter of broccoli.

[0084] In a preferred embodiment of the present invention, the terminator for terminating sgRNA expression is Tsp.

[0085] In a preferred embodiment of the present invention, the gene sequence encoding the Cas9 protein expression cassette includes a promoter for initiating Cas9 protein expression, a Cas9 protein coding sequence, and a terminator for terminating Cas9 protein expression.

[0086] In a preferred embodiment of the present invention, the promoter for initiating Cas9 protein expression is selected from any one of 35S, Ubi, UBQ, SPL, CmYLCV and tissue-specific promoters YAO, CDC45, rbcS, inducible promoter XEV, or combinations thereof.

[0087] In a preferred embodiment of the present invention, a first restriction enzyme site is present between the promoter that initiates sgRNA expression and the terminator that terminates Cas9 protein expression, and a second restriction enzyme site and / or a third restriction enzyme site are present at the 3' end of the terminator that terminates the expression of the second sgRNA.

[0088] There is a fourth restriction enzyme site between the promoter that initiates the expression of the third sgRNA and the terminator that terminates the expression of the second sgRNA.

[0089] The setting of restriction enzyme sites facilitates the low-cost and rapid construction of expression vectors. Those skilled in the art can set restriction enzyme sites according to the construction needs, and are not limited to the types of restriction enzyme sites in this invention.

[0090] In a preferred embodiment of the present invention, the first restriction enzyme site is NotI, the second restriction enzyme site is SbfI, the third restriction enzyme site is SacI, and the fourth restriction enzyme site is SbfI.

[0091] Fourthly, this invention provides a method for breeding disease-resistant broccoli varieties or improving the disease resistance of broccoli, the method comprising: inhibiting the expression of the broccoli BoDMR1 gene and / or the broccoli BoDMR6 gene. The diseases are selected from bacterial and / or fungal diseases.

[0092] In a preferred embodiment of the present invention, the fungal disease is selected from sclerotinia rot and / or black spot; the bacterial disease is black rot.

[0093] In a preferred embodiment of the present invention, the nucleotide sequence of the broccoli BoDMR1 gene is shown in SEQ ID No:7; and the nucleotide sequence of the broccoli BoDMR6 gene is shown in SEQ ID No:8.

[0094] In a preferred embodiment of the present invention, the method for inhibiting the expression of the broccoli BoDMR1 gene is to knock out, silence, or directionally mutate the broccoli BoDMR1 gene, or to introduce the nucleotide sequence shown in SEQ ID No:7 after gene mutation into broccoli cells through hybridization.

[0095] In a preferred embodiment of the present invention, the method for inhibiting the expression of the broccoli BoDMR6 gene is to knock out, silence, or directionally mutate the broccoli BoDMR6 gene, or to introduce nucleotides with gene mutation of the nucleotide sequence shown in SEQ ID No:8 into broccoli cells through hybridization.

[0096] In a preferred embodiment of the present invention, the knockout, silencing, or targeted mutation of the broccoli BoDMR1 gene and / or the broccoli BoDMR6 gene includes gene editing using the broccoli CRISPR / Cas9 gene editing system or the expression vector described above.

[0097] In a preferred embodiment of the present invention, the above-described broccoli CRISPR / Cas9 gene editing system or the above-described expression vector is used to transform and edit broccoli cells.

[0098] Fifthly, the present invention provides a method for gene editing using the above-mentioned broccoli CRISPR / Cas9 gene editing system or the above-mentioned expression vector, comprising the following steps: transforming and gene editing broccoli cells using the above-mentioned broccoli CRISPR / Cas9 gene editing system or the above-mentioned expression vector.

[0099] The specific steps include:

[0100] First, the broccoli CRISPR / Cas9 gene editing system is delivered to the target DNA environment to be cut, either intracellularly or in vitro. Then, the Cas9 protein in the CRISPR / Cas9 gene editing system recognizes the PAM sequence on the target DNA to be edited in the cell or in vitro. Next, the sgRNA in the CRISPR / Cas9 gene editing system forms a base complementary pair with the target DNA sequence to be edited in the cell or in vitro. Then, the Cas9 protein in the CRISPR / Cas9 gene editing system cuts the target site on the target DNA, causing double-strand breaks, thereby achieving targeted cutting of the target DNA in the in vitro environment. When in the cell, further repair is carried out through intracellular non-homologous end joining repair or homologous recombination repair pathways, thereby completing the gene editing of the target DNA in the cell.

[0101] In a sixth aspect, the present invention provides the application of the above-mentioned broccoli CRISPR / Cas9 gene editing system or the above-mentioned expression vector in broccoli cell gene editing.

[0102] In a preferred embodiment of the present invention, the broccoli CRISPR / Cas9 gene editing system or expression vector is introduced into broccoli competent cells, and then the gene-edited mutant strains are obtained through screening.

[0103] In one alternative embodiment, the method for introducing the expression vector into the host cell is selected from genetic transformation methods. In other embodiments, the transformation methods described above include, but are not limited to, Agrobacterium-mediated gene transformation, gene gun transformation, and pollen tube pathway transformation.

[0104] In a seventh aspect, the present invention provides a kit or host cell for gene editing, the kit comprising: the above-described broccoli CRISPR / Cas9 gene editing system or the above-described expression vector.

[0105] The host cell includes the expression vectors mentioned above.

[0106] In a preferred embodiment of the present invention, the host cell is selected from bacteria or fungi.

[0107] In a preferred embodiment of the present invention, the bacteria are Agrobacterium, Mycobacterium, Streptomyces, Escherichia coli, or Bacillus subtilis.

[0108] In a preferred embodiment of the present invention, the fungus is Trichoderma reesei or yeast.

[0109] The host cells mentioned above include transformants and transformed cells, which include primary transformed cells and their offspring, regardless of passage number. Offspring may not be entirely identical to parent cells in terms of nucleic acid content, but may contain mutations.

[0110] Eighthly, the present invention provides the application of the above-described broccoli CRISPR / Cas9 gene editing system, expression vector, kit, or host cell in the cultivation of disease-resistant broccoli. The diseases are selected from bacterial and / or fungal diseases.

[0111] Fungal diseases also include oomycete diseases. Anything that can reduce the severity of disease in broccoli, decrease the number of diseased leaves, or shrink diseased lesions falls within the scope of this invention.

[0112] In a preferred embodiment of the present invention, the fungal disease is selected from sclerotinia rot and / or black spot; the bacterial disease is black rot.

[0113] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0114] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0115] Example 1

[0116] This embodiment provides a BoDMR1 gene editing expression vector for broccoli, and the specific construction method is as follows:

[0117] (1) Cloning of the BoDMR1 gene sequence of broccoli:

[0118] Based on the Arabidopsis thaliana DMR1 genome sequence, homologous sequences of the Arabidopsis thaliana DMR1 genome were obtained from the broccoli genome resequencing library completed at our institution. Data analysis yielded the broccoli BoDMR1 gene and its information. Sequence alignment analysis was performed on the whole-genome resequencing data, and the BoDMR1 gene was located at the corresponding chromosomal position using visualization software. The associated splice sequence of the BoDMR1 gene was obtained, and specific primers were designed for amplification. The obtained PCR product was inserted into a pClone007 Simple Vector for single-clone sequencing to obtain the BoDMR1 gene sequence. The BoDMR1 gene sequence is shown in SEQ ID NO:7.

[0119] (2) Construction of BoDMR1 gene editing expression vector for broccoli.

[0120] Target sites for the BoDMR1 gene in broccoli were designed using an online website (http: / / skl.scau.edu.cn). Following sgRNA design principles, two target sites were selected: BoDMR1-T1: 5'-CGGTTAACGAGATCTTCGGT-3' and BoDMR1-T2: 5'-CCCGCCACCGTCGCTAATCT-3'. Homologous sequences of restriction enzymes were added to both ends of the target sites, and complementary primer pairs were synthesized: BoDMR1-T1-F: 5'-GATTCGGTTAACGAGATCTTCGGT-3', BoDMR1-T1-R: 5'-AAACACCGAAGATCTCGTTAACCG-3'; BoDMR1-T2-F: 5'-GATTCCCGCCACCGTCGCTAATCT-3', BoDMR1-T2-R: 5'-AAACAGATTAGCGACGGTGGCGGG-3'.

[0121] As shown in Table 1, the target site complementary primer pair was prepared into a reaction system with a total volume of 10 μL by adding ddH2O. The reaction was then denatured at 37℃ for 30 min and 95℃ for 5 min using a PCR amplification instrument. The temperature was then lowered to 25℃ at a rate of 0.2℃ / s and placed in a refrigerator at 4℃ for more than 1 h. The reaction product was diluted 200 times with ddH2O.

[0122] Table 1. Target site complementary primer reaction system

[0123]

[0124] The double-stranded Oligo targeting the above-obtained sites was digested with enzymes and ligated into the expression vector pCas9-sgRNA (pCAMBIA2301 (laboratory-preserved), with the Cas9 protein expression cassette donated by Academician Liu Yaoguang of South China Agricultural University). The ligation product was transformed into E. coli DH5α competent cells, plated on LB solid medium with 50 μg / mL kanamycin, and cultured at 37°C for 16 hours. Positive clones were screened to obtain the expression vector pCas9-sgBoDMR1 for broccoli gene editing. Figure 1 ).

[0125] Connection method:

[0126] As shown in Table 2, the pCas9-sgRNA plasmid was digested with the restriction enzyme BsaⅠ. The digestion product was subjected to agarose gel electrophoresis, and the linearized plasmid was recovered by gel excision. Then, as shown in Table 3, the sgBoDMR1-T1 complementary fragment and the digested linearized pCas9-sgRNA plasmid were ligated overnight at 4°C using T4 ligase. The ligation product was then transformed into E. coli DH5α competent cells, plated on LB solid medium with 50 μg / mL kanamycin, and cultured overnight at 37°C. Positive clones were screened, and plasmids were extracted.

[0127] The complementary fragment of sgBoDMR1-T2 was then ligated into the pCas9-sgRNA plasmid containing the sgBoDMR1-T1 fragment obtained in the previous step by digestion with the restriction enzyme BbsI.

[0128] Table 2 pCas9-sgRNA digestion reaction system

[0129]

[0130] Table 3 Target site assembly and ligation reaction system

[0131]

[0132]

[0133] The spacer sequence is tRNA; the nucleotide sequence of the spacer sequence is shown in SEQ ID NO:6. The amino acid sequence of the Cas9 protein is shown in SEQ ID NO:5. The BoDMR1 gene sequence is shown in SEQ ID NO:7.

[0134] Example 2

[0135] This embodiment provides a BoDMR6 gene editing expression vector for broccoli, and the specific construction method is as follows:

[0136] (1) Cloning of the BoDMR6 gene sequence of broccoli:

[0137] Based on the Arabidopsis thaliana DMR6 genome sequence, homologous sequences of the Arabidopsis thaliana DMR6 genome were obtained from the broccoli genome resequencing library completed at our institution. Data analysis yielded the broccoli BoDMR6 gene and its information. Sequence alignment analysis was performed on the whole-genome resequencing data, and the BoDMR6 gene was located at the corresponding chromosomal position using visualization software. The associated splice sequence of the BoDMR6 gene was obtained, and specific primers were designed for amplification. The obtained PCR product was inserted into a pClone007 Simple Vector for single-clone sequencing to obtain the BoDMR6 gene sequence. The BoDMR6 gene sequence is shown in SEQ ID NO:8.

[0138] (2) Construction of BoDMR6 gene editing expression vector in broccoli.

[0139] Target sites for the BoDMR6 gene in broccoli were designed using an online website (http: / / skl.scau.edu.cn). Following sgRNA design principles, two target sites were selected: BoDMR6-T1: 5'-CCGGTTTCCGTCACTCTACT-3' and BoDMR6-T2: 5'-TATCTTCCACAGATCGATCT-3'. Homologous sequences of restriction enzymes were added to both ends of the target sites, and complementary primer pairs were synthesized: BoDMR6-T1-F: 5'-GATTCCGGTTTCCGTCACTCTACT-3', BoDMR6-T1-R: 5'-AAACAGTAGAGTGACGGAAACCGG-3'; BoDMR6-T2-F: 5'-GATTTATCTTCCACAGATCGATCT-3', BoDMR6-T2-R: 5'-AAACAGATCGATCTGTGGAAGATA-3'.

[0140] As shown in Table 1 of Example 1, the target site complementary primer pair was prepared into a reaction system with a total volume of 10 μL by adding ddH2O. The reaction was then denatured at 37°C for 30 min and 95°C for 5 min using a PCR amplification instrument. The temperature was then lowered to 25°C at a rate of 0.2°C / s and placed in a refrigerator at 4°C for more than 1 h. The reaction product was diluted 200 times with ddH2O.

[0141] The double-stranded Oligo targeting the above-obtained sites was ligated with the expression vector pCas9-sgRNA (as in Example 1) via enzyme digestion. The ligation product was transformed into E. coli DH5α competent cells, plated on LB solid medium with 50 μg / mL kanamycin, and cultured at 37°C for 16 hours. Positive clones were screened to obtain the expression vector pCas9-sgBoDMR6 for broccoli gene editing. Figure 1 ).

[0142] Example 3

[0143] This embodiment provides gene editing expression vectors for broccoli BoDMR1 and BoDMR6, and the specific construction method is as follows:

[0144] (1) Cloning of the BoDMR1 and BoDMR6 gene sequences of broccoli:

[0145] The BoDMR1 and BoDMR6 gene sequences were obtained according to the methods of Example 1 and Example 2, respectively.

[0146] (2) Construction of expression vectors for editing BoDMR1 and BoDMR6 genes in broccoli.

[0147] Target sites for the BoDMR1 and BoDMR6 genes in broccoli were designed using an online website (http: / / skl.scau.edu.cn). Based on the sgRNA design principles, two target sites were selected: BoDMR1-T1: 5'-CGGTTAACGAGATCTTCGGT-3', BoDMR1-T2: 5'-CCCGCCACCGTCGCTAATCT-3'; BoDMR6-T1: 5'-CCGGTTTCCGTCACTCTACT-3', BoDMR6-T2: 5'-TATCTTCCACAGATCGATCT-3'. Homologous sequences of endonucleases were added to both ends of the target site, and complementary primer pairs for the target site were synthesized: BoDMR1-T1-F: 5'-GATTCGGTTAACGAGATCTTCGGT-3', BoDMR1-T1-R: 5'-AAACACCGAAGATCTCGTTAACCG-3'; BoDMR1-T2-F: 5'-GATTCCCGCCACCGTCGCTAATCT-3', BoDMR1-T2-R: 5'-AAACAGATTAGCG ACGGTGGCGGG-3'; BoDMR6-T1-F: 5'-GATTCCGGTTTCCGTCACTCTACT-3', BoDMR6-T1-R: 5'-AAACAGTAGAGTGACGGAAA CCGG-3'; BoDMR6-T2-F: 5'-GATTTATCTTCCACAGATCGATCT-3', BoDMR6-T2-R: 5'-AAACAGATCGATCTGTGGAAGATA-3'.

[0148] As shown in Table 1 of Example 1, the target site complementary primer pair was prepared into a reaction system with a total volume of 10 μL by adding ddH2O. The reaction was then denatured at 37°C for 30 min and 95°C for 5 min using a PCR amplification instrument. The temperature was then lowered to 25°C at a rate of 0.2°C / s and placed in a refrigerator at 4°C for more than 1 h. The reaction product was diluted 200 times with ddH2O.

[0149] The double-stranded Oligo targeting the above-obtained sites was ligated with the expression vector pCas9-sgRNA (as in Example 1) via enzyme digestion. The ligation product was transformed into E. coli DH5α competent cells, plated on LB solid medium with 50 μg / mL kanamycin, and cultured at 37°C for 16 hours. Positive clones were screened to obtain the expression vector pCas9-sgBoDMR1+sgBoDMR6 for broccoli gene editing. Figure 1 ).

[0150] Example 4

[0151] Genetic transformation of broccoli BoDMR1 and BoDMR6 gene editing vectors.

[0152] (1) The BoDMR1 and BoDMR6 gene editing expression vector plasmids constructed in Examples 1-3 were transformed into EHA105 Agrobacterium competent cells using the freeze-thaw method.

[0153] (2) Preparation of broccoli explants

[0154] Select plump and uniform broccoli seeds, disinfect them with 2% sodium hypochlorite for 2 hours, rinse once with sterile water, disinfect with 75% alcohol for 1 minute, rinse once with sterile water, disinfect with 10% sodium hypochlorite for 15 minutes, and then rinse with sterile water 5-6 times. During the process, shake the seeds continuously to ensure thorough disinfection. Sow the disinfected seeds evenly in 1 / 2 MS solid medium. Culture them at 25℃ under 16h light conditions for 7 days. Cut the hypocotyls of the sterile seedlings into small segments about 1 cm long and pre-culture them on broccoli differentiation medium (MS + 30 g / L sucrose + 10 g / L agar powder + 3.0 mg / L zeatin + 0.1 mg / L NAA pH 5.8) for 2 days.

[0155] (3) Agrobacterium infection of explants:

[0156] Single colonies of Agrobacterium were picked and placed in 10 ml of liquid medium containing Kan (50 mg / L) and Rif (50 mg / L), and cultured at 28°C for 2 days on a shaker at 225 rpm. On the day of infection, 600-700 μL of bacterial suspension was added to 50 mL of antibiotic-free YEB liquid and cultured at 28°C and 225 rpm for 4 hours until the OD600 value was approximately 0.6. The cells were centrifuged at 4°C and 4000 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in 20 ml of ice-cold MS liquid medium (MS + 20 g / L sucrose, pH 5.8). Hypocotyls that had been pre-cultured for 2 days were added to the suspension for 15 minutes of infection. The hypocotyls were then transferred to sterile filter paper to blot off excess bacterial suspension and then transferred to a differentiation culture medium for dark culture for 2 days.

[0157] (4) Explant selection and culture:

[0158] Hypocotyls cultured in the dark for 2 days were transferred to broccoli medium (MS + 3.0 mg / L zeatin + 0.1 mg / L NAA + 30 g / L sucrose + 10 g / L agar powder + 400 mg / L Cb) and cultured for 7 days. The hypocotyls were then transferred to selection medium (MS + 3.0 mg / L zeatin + 0.1 mg / L NAA + 30 g / L sucrose + 10 g / L agar powder + 400 mg / L Cb + 30 mg / L Kan) and cultured at 25°C under 16 h light conditions for 20 days. The selection medium was then changed every 14-20 days. Adventitious shoots that grew normally in the selection medium were cut off and inserted into medium (MS + 30 g / L sucrose + 7 g / L agar powder + 400 mg / L Cb + 30 mg / L Kan) and cultured for 14 days. The adventitious shoots were then cut off and transferred to rooting medium (MS + 1 mg / L NAA + 30 g / L sucrose + 7 g / L agar powder + 400 mg / L Cb + 30 mg / L Kan) and cultured until the roots grew to 1-2 cm. They were then transplanted into the soil.

[0159] Example 5

[0160] This embodiment demonstrates the detection of genetically modified broccoli and the screening of mutants.

[0161] (1) Genomic DNA was extracted from resistant plants using the SDS method. PCR amplification was performed using primers Cas9-F and Cas9-R (Cas9-F: 5'-CAAGTACGTGAACTTCCTCTACC-3', Cas9-R: 5'-GCTGGGAAAGGTCGATACGAGTC-3'). Agarose gel electrophoresis results showed that all 28 resistant plants with pCas9-sgBoDMR1 vector, 30 with pCas9-sgBoDMR6 vector, and 20 with pCas9-sgBoDMR1+sgBoDMR6 vector were transgenic plants. Figure 2).

[0162] (2) Screening of BoDMR1 and BoDMR6 mutants of broccoli.

[0163] To detect whether the BoDMR1 and BoDMR6 genes in the obtained transgenic plants had been edited, primer pairs BoDMR1-F1+BoDMR1-R1 and BoDMR6-F1+BoDMR6-R1 (BoDMR1-F1: 5'-ACCCTCTCCACCGTTATTCTCC-3', BoDMR1-R1: 5'-GCTCCTGCGATCATGGGAGGGTTT-3'; BoDMR6-F1: 5'-TGGCGGCAAAGCTTTTAT-3', BoDMR6-R1: 5'-TCGAAGCGCACAAAGCAAACCAGT-3') were designed upstream and downstream of the target site for PCR amplification. The PCR products were sequenced, and changes in the target site sequence or the appearance of duplicate peaks in the sequencing peak diagram were considered as gene mutations.

[0164] Sequencing results showed that 8 out of 28 transgenic plants had mutations at target site 2 of the BoDMR1 gene, with an editing efficiency of 28.6%. No mutations at target site 1 were detected. To further investigate the type of mutation, single-clone sequencing was performed on plants #1, #6, and #10. The sequencing results showed that in plant #1, a single-base insertion and a 7-base deletion occurred upstream of the PAM frame at target site 2 of the BoDMR1 gene; in plant #6, different single-base insertions occurred upstream of the PAM frame at target site 2 of the BoDMR1 gene; and in plant #10, a single-base insertion and a deletion occurred upstream of the PAM frame at target site 2 of the BoDMR1 gene. Figure 3 ).

[0165] Of the 30 transgenic lines obtained by transforming the pCas9-sgBoDMR6 vector, 22 showed overlapping peaks, with an editing efficiency of 73.3%. Further analysis revealed that no overlapping sequence occurred at the first target site of the BoDMR6 gene. Analysis of single-clone sequencing results from the mutant plants further confirmed that no editing occurred at the first target site, and the mutated sequences were all located at the second target site. Specifically, sequencing of 8 randomly selected single clones from plant #2 showed that 3 clones had small indels, and 5 clones did not mutate; sequencing of 7 randomly selected single clones from plant #4 showed that 5 clones had A (-A) deletions, and 2 clones did not mutate; sequencing of 10 randomly selected single clones from plant #20 showed that 5 clones had A (-A) deletions, and 5 clones did not mutate. Figure 4 ).

[0166] Twenty transgenic plants were obtained by transforming the pCas9-sgBoDMR1+sgBoDMR6 double knockout vector. PCR amplification of the BoDMR1 and BoDMR6 genes in the transgenic plants was performed using specific primers. The PCR products were directly sequenced to screen for mutant plants. Five plants showed overlapping peaks in both BoDMR1 and BoDMR6, indicating an editing efficiency of 25%. Single-clone sequencing of the BoDMR1 and BoDMR6 target sites in mutant plant #8 revealed that the mutations were all single-base insertions or deletions. Figure 5 The mutant plants were cultured and self-pollinated in an artificial climate chamber, and the offspring were screened for homogeneity and disease resistance.

[0167] Example 6

[0168] Identification of resistance to sclerotinia stem rot in broccoli plants with BoDMR1 and BoDMR6 gene mutations.

[0169] Select seedlings with four leaves and one bud for inoculation. Line a plastic box with two layers of absorbent paper and keep it moist to maintain a certain level of humidity. Place three true leaves in the plastic box, and then use a punch to create 6mm mycelial blocks at the edge of the colony (the sclerotinia rot strain was donated by Professor Song Hongyuan from the College of Horticulture and Landscape Architecture, Southwest University). Inoculate the mycelial blocks on both sides of the upper middle part of the leaves, away from the main vein. Then, place the sealed plastic box under 25℃ and 16 hours of light for 48 hours and observe the expansion of the lesions.

[0170] The susceptibility and resistance of mutant materials to sclerotinia stem rot were identified using an in vitro leaf agar block inoculation method, with the area of ​​lesion expansion as the indicator of resistance. The lesion expansion area was observed at 48 hours. The results showed that the lesion area of ​​both BoDMR1 and BoDMR6 mutant plants was significantly smaller than that of non-mutated plants. Furthermore, the lesion area of ​​the BoDMR1 and BoDMR6 double mutant plants was significantly smaller than that of the BoDMR1 and BoDMR6 single-gene mutant plants. Therefore, the results of the in vitro leaf inoculation identification demonstrate that BoDMR1 and BoDMR6 gene mutations play a significant role in improving the resistance of broccoli to sclerotinia stem rot, and the effect of double-gene superposition mutation is even more pronounced. Figure 6 ).

[0171] Example 7

[0172] Identification of resistance to black rot in broccoli plants with BoDMR1 and BoDMR6 gene mutations.

[0173] Select plump seeds for testing and disinfect them by soaking them in a 55℃ constant temperature water bath for 10 minutes. After drying, sow them into sterilized substrate in 72-well trays and place them in an artificial climate chamber for growth. When the two cotyledons of the seedlings are fully expanded, leave one strong seedling in each well. Inoculate the seedlings when they reach the four-leaf stage. Before inoculation, thoroughly water the seedlings and cover them with plastic film to maintain moisture for 24 hours (25℃ / 20℃). On the second day, spray 1×10⁻⁶ seeds with a small sprayer. 8 A CFU / mL infection solution (the black rot strain was donated by Professor Song Hongyuan of the College of Horticulture and Landscape Architecture, Southwest University) was evenly sprayed onto the plant leaves until no droplets fell from the leaves. After inoculation, the seedlings were kept moist with plastic film for 24 hours, and then placed under conditions of 14 hours of light, 28℃ / 24℃, and 90% humidity for growth management. The disease incidence was investigated on day 11 post-inoculation.

[0174] Blackening of leaf veins is the first visible symptom of broccoli infection by the pathogen. When the pathogen enters the midrib of the leaf through the hydathodes and smaller veins, the extracellular polysaccharides it produces cause vein blockage, leading to water stress and yellowing. Subsequently, a V-shaped necrotic area bounded by the chlorotic zone appears at the infected site. This is usually accompanied by a yellow halo. The lesions then gradually extend laterally and inward, causing the leaf tissue to yellow or die. Observation of the disease incidence in seedlings after spray inoculation with black rot revealed that both the BoDMR1 and BoDMR6 mutant plants showed significantly lower severity of black rot and a lower number of yellowed leaves compared to the non-mutant plants, indicating a certain degree of resistance to black rot. Figure 7 ).

[0175] Example 8

[0176] Identification of resistance to black spot disease in broccoli plants with BoDMR1 and BoDMR6 gene mutations.

[0177] Select plump seeds for testing and disinfect them by soaking them in a 55℃ constant temperature water bath for 10 minutes. After drying, sow them into sterilized substrate in 72-well trays and place them in an artificial climate chamber for growth. When the two cotyledons of the seedlings are fully expanded, leave one strong seedling in each well. When the seedlings reach the four-leaf stage, inoculate them with 5×10⁶ seeds. 4 A spore suspension of 1 spore / mL (the black spot disease strain was provided by Professor Gu Honghui of the Zhejiang Academy of Agricultural Sciences) was evenly sprayed onto the surface of the seedlings, preferably by dripping. After inoculation, the seedlings were kept moist with plastic film for 48 hours. Seven days after inoculation, the disease incidence on all leaves of each treatment was investigated.

[0178] Black spot disease primarily infects leaves, but in severe cases, it can also damage stems, petioles, flower heads, and seed pods. The disease typically progresses from older leaves to younger leaves. Initially, small chlorotic spots or black dots appear on the upper or lower surfaces of the leaves. Later, the center of the lesions turns brown and necrotic, gradually enlarging into circular or nearly circular lesions, often surrounded by a yellow halo. Observations of seedlings after spray inoculation with black spot disease revealed that both the BoDMR1 and BoDMR6 mutants had significantly fewer infected leaves, fewer infected lesions, and a lower disease severity compared to non-mutant plants, demonstrating a certain degree of resistance to black spot disease. Furthermore, the BoDMR6 mutant showed better resistance to black spot disease than the BoDMR1 mutant. Figure 8 ).

[0179] In summary, the BoDMR1 and BoDMR6 genes negatively regulate the disease resistance immunity of broccoli, and knocking out the BoDMR1 and BoDMR6 genes using technologies such as CRISPR / Cas9 can improve the disease resistance of broccoli.

[0180] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of knocking out the BoDMR1 and / or BoDMR6 genes in broccoli for breeding disease-resistant broccoli varieties or improving the disease resistance of broccoli, characterized in that, The nucleotide sequence of the broccoli BoDMR1 gene is shown in SEQ ID No:7; the application includes: knocking out the broccoli BoDMR1 gene and / or BoDMR6 gene; The nucleotide sequence of the BoDMR6 gene in broccoli is shown in SEQ ID No:8, and the disease is selected from any one of sclerotinia rot, black spot, and black rot.

2. The application according to claim 1, characterized in that, The application includes any of the following: (1) Gene modification of the endogenous BoDMR1 gene in broccoli to prevent it from being expressed or to completely lose the function of expressing BoDMR1 protein. (2) Gene modification of the endogenous BoDMR6 gene in broccoli to prevent it from being expressed or to completely lose the function of expressing BoDMR6 protein; (3) When the nucleotide sequence of the broccoli BoDMR1 gene is as shown in SEQ ID No:7 and the nucleotide sequence of the broccoli BoDMR6 gene is as shown in SEQ ID No:8, the endogenous BoDMR1 gene and BoDMR6 gene of broccoli are genetically modified so that they are not expressed or the function of expressing BoDMR1 protein and BoDMR6 protein is completely lost.

3. The application according to claim 2, characterized in that, The genetically modified endogenous BoDMR1 and / or BoDMR6 genes in broccoli refer to gene modification using gene editing technology. The gene editing technology mentioned is zinc finger endonuclease technology, transcription activator effector nuclease technology, or CRISPR / Cas9 technology.

4. A method for cultivating disease-resistant broccoli varieties or improving the disease resistance of broccoli, characterized in that, The method includes: inhibiting the expression of the broccoli BoDMR1 gene and / or the broccoli BoDMR6 gene; causing the BoDMR1 protein and / or the BoDMR6 protein to completely lose their function, wherein the disease is selected from any one of sclerotinia rot, black spot disease and black rot, the nucleotide sequence of the broccoli BoDMR1 gene is shown in SEQ ID No:7; the nucleotide sequence of the broccoli BoDMR6 gene is shown in SEQ ID No:

8.

5. The method for cultivating disease-resistant broccoli varieties or improving the disease resistance of broccoli according to claim 4, characterized in that, The method for inhibiting the expression of the BoDMR1 gene in broccoli is to knock out, silence, or perform targeted mutation of the BoDMR1 gene in broccoli. The method for inhibiting the expression of the broccoli BoDMR6 gene is to knock out, silence, or perform targeted mutation of the broccoli BoDMR6 gene.

6. The method for cultivating disease-resistant broccoli varieties or improving the disease resistance of broccoli according to claim 5, characterized in that, The knockout, silencing, or targeted mutation of the broccoli BoDMR1 and / or broccoli BoDMR6 genes includes gene editing using a CRISPR / Cas9 gene editing system or expression vector.

7. The method for cultivating disease-resistant broccoli varieties or improving the disease resistance of broccoli according to claim 6, characterized in that, CRISPR / Cas9 gene editing system or expression vector were used to transform and edit broccoli cells.

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