BoFBX gene of cauliflower, expression vector of BoFBX gene and application of BoFBX gene in regulating and controlling tightness degree of cauliflower flower ball

Abstract

The invention discloses a broccoli BoFBX gene, an expression vector of the broccoli BoFBX gene and application of the broccoli BoFBX gene to regulation and control of the tightness degree of broccoli flower balls. Through transcriptome differential genes and WGCNA analysis of compact cauliflower and loose cauliflower at different development stages, the BoFBX gene is found to be remarkably up-regulated in expression in the loose cauliflower ball-flower, and is not expressed in the compact cauliflower ball-flower; the nucleotide sequence of the BoFBX gene is as shown in SEQ ID No. 1; an agrobacterium tumefaciens-mediated genetic transformation method is adopted, an over-expression vector is introduced into a compact broccoli high-generation inbred line variety to obtain an over-expressed positive transgenic broccoli plant, and ball-flower phenotype field observation shows that the ball-flower of the positive transgenic broccoli plant with the over-expressed BoFBX gene is changed into a loose type from a compact type. The method has application value in the aspects of creating cauliflower germplasm with the ball-flower with ideal tightness or breeding loose type ball-flower cauliflower varieties and the like.

Description

Technical Field

[0001] This invention relates to genes isolated from vegetables and their applications, particularly to genes isolated from cauliflower. BoFBX Genes and their expression vectors, and their application in regulating the tightness of the flower head, belong to cauliflower. BoFBX Genes and their applications. Background Technology

[0002] Cauliflower (Brassica oleracea var. botrytis) is an important vegetable crop, widely favored by consumers for its high nutritional value, good taste, and wide adaptability. The tightness of the cauliflower head is a key trait determining product quality and market price, closely related to head appearance, taste, storage and transport resistance, and product consistency. Therefore, accurately controlling the tightness of the cauliflower head is a pressing technical challenge that needs to be overcome in current cauliflower breeding and production management.

[0003] Existing research indicates that head compactness is a typical quantitative trait, regulated by multiple genes, and closely related to plant hormones, developmental regulatory networks, and meristematic tissue development. However, knowledge of its key regulatory genes, molecular mechanisms, and their applications in variety improvement remains very limited, and there is a lack of clearly defined target genes for molecular breeding screening and precise improvement. This bottleneck severely restricts the pace of innovation in high-quality cauliflower varieties and also results in a lack of effective genetic control methods for head structural stability during industrial production.

[0004] The F-box protein family is a widely distributed and important component of the ubiquitin-proteasome pathway in plants. Initially discovered in the cyclin Cyclin F, it is a core component of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase complex (Glickman MH et al., 2002; Jain M., 2007). F-box proteins are named for their N-terminal F-box motif, which contains approximately 40-50 amino acids and a conserved Leu-Pro dipeptide and hydrophobic amino acids (Val, Ile, etc.). They bind to SKP1 to form the SCF complex and participate in various important biological processes, including plant growth and development, hormone signal transduction, photomorphogenesis, and stress regulation, by recognizing and degrading specific substrate proteins. Previous studies have shown that F-box proteins play a crucial role in plant development regulation, but their function in cauliflower head development, particularly in the formation of compactness, remains unclear. Summary of the Invention

[0005] One of the objectives of this invention is to provide a product isolated from cauliflower. BoFBX Genes and their encoded proteins.

[0006] A second objective of this invention is to provide a product containing the aforementioned extract from cauliflower. BoFBX Gene expression vectors and recombinant host cells containing said expression vectors.

[0007] The third objective of this invention is to separate the aforementioned substance from cauliflower. BoFBX Genes and their expression vectors are used to regulate the tightness of cauliflower heads. 。

[0008] To achieve the above objectives, the technical solution adopted by the present invention includes: One aspect of the present invention is to provide a method for isolating cauliflower. BoFBX The gene, wherein the nucleotide sequence of the cDNA of the gene is selected from any of the nucleotide sequences described in (a)-(e) below: (a) The polynucleotide sequence shown in SEQ ID No. 1; (b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2; (c) A polynucleotide sequence capable of hybridizing with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head; (d) A polynucleotide sequence having at least 75% or more identity with any of the polynucleotide sequences shown in (a)-(c), wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head; preferably, a polynucleotide sequence having at least 85% or more identity with any of the polynucleotide sequences shown in (a)-(c), wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head; more preferably, a polynucleotide sequence having at least 90% or more identity with any of the polynucleotide sequences shown in (a)-(c), wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head; most preferably, a polynucleotide sequence having at least 95% or more identity with any of the polynucleotide sequences shown in (a)-(c), wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head. (e) A polynucleotide sequence that is complementary to any of the polynucleotide sequences described in (a)-(d) and still has the function of regulating the tightness of the cauliflower head.

[0009] The percentage of sequence identity described in this invention can be obtained using well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0010] In addition, those skilled in the art can optimize the nucleotide sequence shown in SEQ ID No. 1 to enhance its expression efficiency in cauliflower.

[0011] The present invention described BoFBX Genes can be mutants obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID No. 1 or by performing missense mutations on one or more base pairs.

[0012] Those skilled in the art can readily employ known methods, such as directed evolution or point mutation, to modify the present invention. BoFBX The nucleotide sequence of the gene is mutated. Those that have been artificially modified and have the characteristics isolated from the present invention... BoFBX Nucleotides with 85% or higher nucleotide sequence identity to a gene, as long as they encode a protein with the amino acid sequence shown in SEQ ID No. 2, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0013] In addition, the nucleotides described in this invention can be DNA, such as cDNA, genomic DNA, or recombinant DNA; or they can be RNA, such as mRNA or hnRNA.

[0014] Another aspect of the present invention is to provide BoFBX The F-box, a gene-encoded protein, contains... BoFBX The amino acid sequence of the protein encoded by the gene is selected from any of the amino acid sequences shown in (a)-(d) below: (a) The amino acid sequence shown in SEQ ID No. 2; (b) A protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2, which still has the function or activity of regulating the tightness of cauliflower heads; (c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID No. 2, which still has the function or activity of regulating the tightness of cauliflower heads; (d) A protein with 85% or more identity to the amino acid sequence shown in SEQ ID No. 2, which still has the function or activity of regulating the tightness of cauliflower heads.

[0015] Another aspect of the present invention is to further provide a product containing the aforementioned... BoFBXThe gene expression cassette, recombinant expression vector, or recombinant host cell; preferably, the expression cassette, recombinant expression vector, or recombinant host cell is a recombinant eukaryotic expression cassette, recombinant eukaryotic expression vector, or recombinant plant cell; more preferably, the recombinant eukaryotic expression cassette or recombinant eukaryotic expression vector is a recombinant plant expression cassette or recombinant plant expression vector.

[0016] For reference, the present invention provides BoFBX Gene recombinant expression vectors for plants, including: BoFBX A recombinant plant expression vector is obtained by linking a gene with an expression regulatory element. This recombinant plant expression vector may consist of a 5′ non-coding region, the nucleotides shown in SEQ ID No. 1, and a 3′ non-coding region. The 5′ non-coding region may include a promoter sequence, an enhancer sequence, and / or a translational enhancement sequence. The promoter may be a constitutive promoter, an inducible promoter, or a tissue or organ-specific promoter, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the ubiquitin promoter of maize, which can be used alone or in combination with other plant promoters. The 3′ non-coding region may contain a terminator sequence, an mRNA cleavage sequence, etc. A suitable terminator sequence can be obtained from the Ti-plasmid of Agrobacterium tumefaciens, such as the terminator regions of octopine synthase and carmine synthase. The enhancer sequence may be an ATG start codon or an adjacent region start codon, but it must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translational enhancement sequence and start codon are widely available and can be natural or synthetic. Translation initiation regions can originate from transcription initiation regions or structural genes.

[0017] The recombinant plant expression vector may also contain selective marker genes for selecting transformed cells or tissues. These marker genes include genes encoding antibiotic resistance and genes conferring resistance to herbicides. Furthermore, the marker genes also include phenotypic markers, including but not limited to genes encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, no selective marker genes may be added, and transformed plants may be directly selected by stress screening.

[0018] Transformation protocols and the methods for introducing the polynucleotides or polypeptides into plants can vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the polynucleotides into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment. Transformed cells can be regenerated into stable transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0019] The present invention also provides a product containing the aforementioned BoFBX Recombinant host cells of gene expression cassettes or recombinant expression vectors.

[0020] Another aspect of the present invention is to... BoFBX Genes, their encoded proteins, containing the aforementioned BoFBX Recombinant plant expression vectors are used to regulate the tightness of cauliflower heads; wherein, the regulation of the tightness of cauliflower heads is preferably achieved by increasing the length of the head branches, increasing the angle between the head branches, or making the head looser.

[0021] In a preferred embodiment of the present invention, the method for controlling the tightness of the cauliflower head includes: BoFBX The gene was overexpressed in cauliflower, making BoFBX Enhanced expression of genes in cauliflower or BoFBX Enhanced function or activity of gene-encoded proteins can lead to increased branching in the flower head, a larger angle between branches, or a looser flower head.

[0022] Another aspect of the present invention provides a method for loosening cauliflower heads, comprising: taking BoFBX The gene was overexpressed in cauliflower, making BoFBX Enhanced expression of genes in cauliflower or BoFBX Enhanced function or activity of gene-encoded proteins can lead to increased branching in the flower head, a larger angle between branches, or a looser flower head.

[0023] Another aspect of the present invention provides a method for breeding pine cone cauliflower varieties, comprising: taking BoFBX The gene was overexpressed in cauliflower, making BoFBX Enhanced expression of genes in cauliflower or BoFBX Enhanced function or activity of gene-encoded proteins allows for the breeding of cauliflower varieties with increased flower head branching, larger branching angles, or looser flower heads.

[0024] This invention, through transcriptome differential gene and WGCNA analysis at different developmental stages of compact and loose cauliflower, discovered... BoFBXThe gene was significantly upregulated in loosely headed cauliflower, but not in compactly headed cauliflower, revealing... BoFBX Genes may play a key role in regulating the tightness of cauliflower heads; in order to study BoFBX The specific role of genes in regulating the tightness of cauliflower heads, and the construction of... BoFBX The gene overexpression vector, with the pCAMBIA3301 vector containing a 35S promoter as its backbone, was introduced into the compact cauliflower high-generation inbred line 'FQ-38' using Agrobacterium-mediated EHA105 genetic transformation. Transgenic plants were then screened, and finally, the desired results were obtained. BoFBX Field observations of positive transgenic cauliflower plants with overexpressed genes revealed changes in their flower head phenotype. BoFBX In transgenic cauliflower plants with overexpressed genes, the flower heads change from a compact to a loose shape. This invention discloses for the first time... BoFBX Gene regulation alters the tightness of cauliflower heads, which can be further applied to creating cauliflower germplasm with ideal head tightness, and has application value in breeding loose-headed cauliflower varieties.

[0025] Definitions of terms involved in this invention 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 invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, apparatus, and materials are now described.

[0026] The term "homology" refers to sequence similarity to a natural nucleic acid sequence. "Homology" includes nucleotide sequences that have preferably 85% or higher, more preferably 90% or higher, and most preferably 95% or higher identity with the nucleotide sequence of the regulatory fragment of the present invention. Homology can be evaluated visually or using computer software. Using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences.

[0027] The term "complementary" here refers to two nucleotide sequences comprising antiparallel nucleotide sequences that can pair with each other after forming hydrogen bonds between complementary base residues of the antiparallel nucleotide sequences. It is known in the art that the nucleotide sequences of two complementary strands are anticomplementary when viewed from the 5' to 3' direction. It is also known in the art that two sequences that can hybridize under a given set of conditions need not necessarily be 100% perfectly complementary.

[0028] The term "rigorous hybridization conditions" refers to conditions of low ionic strength and high temperature known in the field. Typically, under rigorous conditions, the detectability of a probe hybridizing with its target sequence is significantly higher than that with other sequences (e.g., at least twice the background level). Rigorous hybridization conditions are sequence-dependent and will vary under different environmental conditions; longer sequences hybridize specifically at higher temperatures. By controlling the rigor of hybridization or washing conditions, target sequences that are 100% complementary to the probe can be identified. For detailed guidance on nucleic acid hybridization, please refer to the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays. 1993."). More specifically, the stringent conditions are typically chosen to be approximately 5-10°C below the thermal melting point (Tm) of the specific sequence at a specified ionic strength and pH. Tm is the temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence in equilibrium (at a specified ionic strength, pH, and nucleic acid concentration) (because the target sequence is in excess, 50% of the probe is occupied in equilibrium at Tm). Stringent conditions may include a salt concentration below approximately 1.0 M sodium ion concentration at pH 7.0 to 8.3, typically approximately 0.01 to 1.0 M. The concentration of sodium ions (or other salts) must be M, and the temperature must be at least about 30°C for short probes (including, but not limited to, 10 to 50 nucleotides) and at least about 60°C for long probes (including, but not limited to, more than 50 nucleotides). Strict conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal can be at least twice the background hybridization, and, where appropriate, ten times the background hybridization. Exemplary strict hybridization conditions may be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubated at 65°C, washed in 0.2×SSC and washed in 0.1% SDS at 65°C. The washing can be performed for 5, 15, 30, 60, 120 minutes or longer.

[0029] The terms "host cell" or "recombinant host cell" refer to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotides may remain as, for example, non-integrating vectors of plasmids or may be integrated into the host genome.

[0030] The terms "polynucleotide" or "nucleotide" refer to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, either in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence implicitly encompasses variants of its conserved modifications (including, but not limited to, degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0031] The term "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region can be positioned relative to a nucleic acid sequence encoding a product of interest so that transcription of said nucleic acid sequence is directed by that promoter region. Therefore, the promoter region is "operably linked to that nucleic acid sequence".

[0032] The term "transformation" here refers to the process of introducing heterologous DNA into plant cells, plant tissues, or plants. Transformation of plant cells, plant tissues, or plants is understood to include not only the end products of the transformation process but also their progeny.

[0033] The terms “transformation,” “transgenic,” and “recombinant” refer here to a host cell or organism, such as a bacterial or plant cell (e.g., a plant), in which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome, or it can exist as an extrachromosomal molecule. Such an extrachromosomal molecule can self-replicate. Transformed cells, tissues, or plants are understood to include not only the end products of the transformation process but also their transgenic progeny. A “non-transformed,” “non-transgenic,” or “non-recombinant” host refers to a wild-type organism, such as bacteria or a plant, that does not contain a heterologous nucleic acid molecule.

[0034] The term "promoter" refers to any nucleic acid sequence (such as a DNA sequence) that is recognized and (directly or indirectly) bound by a DNA-dependent RNA polymerase during transcription initiation, resulting in the generation of an RNA molecule complementary to the transcribed DNA; this region may also be called the "5' regulatory region." A promoter is typically located upstream of the 5' untranslated region (UTR) preceding the coding sequence to be transcribed and has multiple regions that act as binding sites for RNA polymerase II and other proteins such as transcription factors to initiate the transcription of the operatively linked gene. The promoter itself may contain sub-elements (i.e., promoter motifs) that regulate the transcription of the operatively linked gene, such as cis-elements or enhancer domains. This promoter and the linked 5' UTR are also called the "promoter region." Attached Figure Description

[0035] Figure 1 The differences in floret phenotype between compact and loose cauliflower.

[0036] Figure 2 This is a WGCNA clustering tree.

[0037] Figure 3 This is the module in WGCNA analysis that is significantly associated with the head compactness trait.

[0038] Figure 4 These are genes that are differentially expressed in the module at different developmental stages of compact and loose cauliflower.

[0039] Figure 5 To express cauliflower BoFBX The test results of plants that tested positive for the gene.

[0040] Figure 6 Genetically modified cauliflower strain BoFBX -19 represents the phenotype of head tightness; a and b are overexpressing transgenic cauliflower lines. BoFBX -19 is the phenotype of the flower head 14 days after the flower head appears; c and d are the phenotypes of the flower head of the wild-type recipient material 'FQ-38' 14 days after the flower head appears. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments or test examples, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments or test examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0042] In the following specific implementations, unless otherwise specified, conventional experimental methods were followed and the recombinant techniques described (such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Sambrook J & Russell DW, 2001, etc.) were performed, or the conditions recommended in the manufacturer's instructions were followed.

[0043] Example 1: Transcriptome differential gene and WGCNA analysis at different developmental stages of compact and loose cauliflower Through compact cauliflower and loose cauliflower ( Figure 1 Transcriptome differential gene and WGCNA analysis at different developmental stages revealed... BoFBX The gene was significantly upregulated in loosely packed cauliflower heads, but not in compactly packed cauliflower heads. Figure 2 , Figure 3 , Figure 4 ),reveal BoFBX Genes may play a key role in the regulation of the tightness of cauliflower heads.

[0044] Example 2 Cauliflower BoFBX Cloning of genes 1. RNA extraction and cDNA cloning Approximately 1 g of cauliflower head tissue from the developing stage of loosely packed cauliflower 'SV105' was selected. After adding liquid nitrogen, the tissue and cells were lysed using a mortar and pestle, and then rapidly transferred to 1.5 mL centrifuge tubes. RNA was extracted from the cauliflower head using a plant total RNA extraction kit (RNAprep pureTissue Kit, TIANGEN). RNA concentration was detected using a spectrophotometer, and RNA integrity was assessed by agarose gel electrophoresis.

[0045] First-strand cDNA was synthesized using a reverse transcription kit (Prime Script Reverse Transcriptase kit, Takara). Further, PCR amplification was performed using loose cauliflower 'SV105' cDNA as a template. FBX-F: ATGCCGAAGACGAGGAGGAT (SEQ ID No. 3) and FBX-R: TCAGAATAGGTAATGGCAATTCTGAA (SEQ ID No. 4) were used as forward and reverse primers, respectively, and a 50µL high-fidelity enzyme, TransStart FastPfu Fly DNA Polymerase (Trans), was employed. PCR conditions were: 98 ℃ for 1 min; 30 cycles of denaturation at 98 ℃ for 10 s, 56 ℃ for 5 s, and 72 ℃ for 20 s; followed by 72 ℃ for 1 min. The PCR reaction system is shown in Table 1.

[0046] Table 1 PCR reaction system

[0047] The PCR products were recovered and ligated into the pMD18-T vector. Positive clones were screened and sequenced. Sequencing results. BoFBX The nucleotide sequence of the gene is shown in SEQ ID No. 1:

[0048] BoFBX The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 2: MPKTRRMSDLPQELVGEKILTKVPITSVKEVRSTCKKWEAITKSWVVLGKAAAAAAVPHEFLWFMTMNAKVYSLRFHLREEEEEDLVVDVSAKQVDLLNQLEVSKVYHC DGLVLCVATDHSKLVVWNPYLCQTRLIGPRENFNIRDNYAIGYSSSSSERDHKILRFVDDYSAPGPTRVFRCEIYGISSDSWRVLKPKPEWKIQINEHHRGVSVKGNTY FFAHERFVVIDDFGGRDCEVEDFLLCFDFTKERFGPRLPLPFHSYNEDCVTLSNVRDDQLAVLFGAFESDSFEIWVTLTLEPDHVSWSKFLLVEPGPALEFKLNDYFGG SFFVDEENKVAVVFEISDPHQHTAFVFGQAGYIQSVNLGQVTKIQYLCPYTTKHLEALLPPLVCSSSYRPSLVQVKRTPLHKRKRHLLRGKSKFIIMIQNCHYLF (SEQ ID No. 2).

[0049] Experimental Example 1 BoFBX Construction of gene overexpression vectors and Agrobacterium-mediated genetic transformation; phenotypic detection of flower head tightness in transgenic plants. 1. Experimental Methods 1.1 The PCR amplification product from Example 2 was recovered and purified. The F-Box gene was ligated into the vector pCAMBIA3301 plasmid (preserved in the inventor's laboratory, commercially available, purchase link: https: / / www.biofeng.com / zaiti / zhiwu / pCambia3301.html) using in-fusion technology. Positive clones were selected for sequencing after transformation.

[0050] 1.2 The correct plasmid was transformed into Agrobacterium EHA105 by liquid nitrogen freeze-thaw method. 50 mg / L Rif and 50 mg / L Kan were used as resistance screening. The obtained single clones were identified as positive by PCR.

[0051] 1.3 Add Agrobacterium to 50 mL LB medium containing 50 mg / L Rif and 50 mg / L Kan resistance, and incubate overnight at 28°C in a shaker. Measure the OD value; stop shaking when the OD value of the bacterial solution reaches 0.8–1.0. Centrifuge at 28°C, 4500 rpm for 10 min.

[0052] 1.4 Using Agrobacterium EHA105-mediated genetic transformation (Wang et al., 2022, Efficient generation of targeted point mutations in the Brassica oleracea var. botrytisgenome via a modified CRISPR / Cas9 system. Horticultural Plant Journal 8(4): 527-530), the above-mentioned... BoFBX Gene overexpression vectors were introduced into the compact cauliflower high-generation inbred line 'FQ-38' to obtain transgenic plants. Positive plants were detected using the universal primers Basta(277)-L and Basta(277)-R for glufosinate.

[0053] BoFBX Gene overexpression materials were isolated and observed in a dedicated plot. Each material was planted in a single row or in a section with a length of 5 meters, with a plant spacing of 45 cm and a row spacing of 50 cm. At the same time, the wild-type recipient material 'FQ-38' was planted in the same area as a control. The overall field performance and changes in flower heads during the heading period were observed.

[0054] 2. Experimental Results The results of detecting positive plants using the universal primers Basta(277)-L and Basta(277)-R for glufosinate are as follows: Figure 5 As shown.

[0055] overexpression BoFBX The phenotypic results of the tightness of the flower heads in transgenic cauliflower plants are as follows: Figure 6 As shown: Figure 6 a and Figure 6 b represents the overexpression transgenic cauliflower line. BoFBX -19 The phenotype of the flower head 14 days after the flower head appears; Figure 6 c and Figure 6 d represents the flower head phenotype of the wild-type receptor material 'FQ-38' 14 days after flower head formation. Compared with the wild-type receptor material, overexpression... BoFBX Genetically modified cauliflower strains BoFBX The -19 flower heads clearly showed characteristics such as increased branching, a larger angle between branching points, and a looser flower head, indicating that the cauliflower... BoFBXGenes play a key role in regulating the tightness of cauliflower heads.

Claims

1. Separated from cauliflower BoFBX Genes, characterized by, The aforementioned BoFBX The nucleotide sequence of the gene's cDNA is selected from any of the nucleotide sequences described in (a)-(e) below: (a) The polynucleotide sequence shown in SEQ ID No. 1; (b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 2; (c) A polynucleotide sequence capable of hybridizing with the polynucleotide sequence described in (a) or (b) under stringent hybridization conditions, wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head; (d) A polynucleotide sequence having at least 75% or more identity with any of the polynucleotide sequences shown in (a)-(c), wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head; preferably, a polynucleotide sequence having at least 85% or more identity with any of the polynucleotide sequences shown in (a)-(c), wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head; more preferably, a polynucleotide sequence having at least 90% or more identity with any of the polynucleotide sequences shown in (a)-(c), wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head; most preferably, a polynucleotide sequence having at least 95% or more identity with any of the polynucleotide sequences shown in (a)-(c), wherein the polynucleotide sequence still has the function of regulating the firmness of the cauliflower head. (e) A polynucleotide sequence that is complementary to any of the polynucleotide sequences described in (a)-(d) and still has the function of regulating the tightness of the cauliflower head.

2. The claim 1 BoFBX The gene encodes the protein F-box, characterized by, The amino acid sequence encoding the F-box protein is selected from any of the amino acid sequences shown in (a)-(d) below: (a) The amino acid sequence shown in SEQ ID No. 2; (b) A protein variant obtained by deleting or replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2, which still has the function or activity of regulating the tightness of cauliflower heads; (c) A protein variant obtained by inserting one or more amino acid residues into the amino acid sequence shown in SEQ ID No. 2, which still has the function or activity of regulating the tightness of cauliflower heads; (d) A protein with 85% or more identity to the amino acid sequence shown in SEQ ID No. 2, which still has the function or activity of regulating the tightness of cauliflower heads.

3. Containing the contents of claim 1 BoFBX Gene expression cassettes.

4. Containing the contents of claim 1 BoFBX Gene expression vector; preferably, the expression vector is an overexpression plant vector.

5. A recombinant host cell containing the recombinant expression vector of claim 4.

6. The claim 1 BoFBX The application of the gene, the protein F-box of claim 2, the expression cassette of claim 3, or the expression vector of claim 4 in regulating the tightness of cauliflower heads.

7. The application according to claim 6, characterized in that, include: Will BoFBX The gene was overexpressed in cauliflower, making BoFBX Increased expression of the gene in cauliflower may enhance the function or activity of the protein encoded by the F-box gene.

8. The application according to claim 6, characterized in that, The aforementioned regulation of the tightness of cauliflower heads involves increasing the length of the flower head branches, increasing the angle between the flower head branches, or making the flower head looser.

9. A method for cultivating a loose-headed cauliflower variety, characterized in that, Will BoFBX The gene was overexpressed in cauliflower, making BoFBX Increased expression of genes in cauliflower or enhanced function or activity of proteins encoded by F-box genes can lead to the selection of cauliflower varieties with increased flower head branching, larger flower head branching angles, or looser flower heads.

10. A method for loosening cauliflower heads, characterized in that, Will BoFBX The gene was overexpressed in cauliflower, making BoFBX Enhanced expression of genes in cauliflower or BoFBX Enhanced function or activity of the protein encoded by the gene causes cauliflower head to grow longer branches, larger branch angles, or looser heads.

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