Strawberry stolon regulation gene ZFP6 and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, and discloses a strawberry stolon regulation gene ZFP6 and application thereof, and the nucleotide sequence of the gene is as shown in SEQ ID NO.1. Researches prove that the ZFP6 gene is specifically expressed in axillary meristem of strawberry in the adult-plant stage, and the GA20ox4 gene can be directly or indirectly activated to start a biosynthesis pathway of GA and drive axillary buds to develop towards stolons. Through genetic interaction analysis of the ZFP6 gene, the GA20ox4 gene and the RGA1 gene, the function of the ZFP6 gene as an axillary bud fate decision upstream regulation factor is further verified, the blank of an endogenous regulation mechanism of the strawberry stolons is filled, and the ZFP6 gene is proved to be a molecular target for specifically regulating formation of the strawberry stolons. The gene sequence, the primer, the gene knockout expression vector and the matched technical system provided by the invention lay a foundation for strawberry molecular breeding and industrial application.

Description

Technical Field

[0001] This application belongs to the field of plant genetic engineering technology, specifically relating to the strawberry stolon regulatory gene ZFP6 and its application. Background Technology

[0002] strawberry( Fragaria × ananassa Stolon is one of the world's important horticultural economic crops. Its production mainly relies on asexual reproduction through stolons to achieve seedling propagation. The reproductive capacity of stolons not only determines the reproductive efficiency of the mother plant and the yield of seedlings, but also directly affects the balance of plant nutrient resources between stolon development and fruit development, ultimately affecting fruit yield.

[0003] Strawberry runners originate from axillary meristems, and their developmental fate is complexly regulated by various factors, including endogenous hormone levels, light quality signals, temperature, and developmental stage. Among these, GA (gibberellin) has been identified as a key hormone promoting runner formation. Exogenous application of GA can promote the formation of runners. The key gene GA20ox4 (Gibberellin 20-oxidase 4) in the GA biosynthesis pathway has five homologous genes in diploid forest strawberry, namely FvH4_2g35050, FvH4_5g19970, FvH4_7g12600, FvH4_7g12610 and FvH4_7g28670. Among them, only FvH4_2g35050 is expressed in axillary buds, and its expression level in axillary buds is significantly positively correlated with the transformation of axillary buds into runners. Although studies have reported the induction effects of environmental factors such as photoperiod and temperature on stolons, and have induced stolons by inducing FvH4_2g35050, the endogenous regulatory mechanisms, especially how upstream developmental signals precisely control the expression of GA20ox4 and its tissue-specific regulatory patterns, still lack clear genetic and molecular evidence.

[0004] Therefore, in strawberry breeding and propagation practices, there is still a lack of molecular targets that can specifically regulate stolon formation. Summary of the Invention

[0005] This application provides a strawberry runner regulatory gene ZFP6 and its application, aiming to solve the problems in the above-mentioned technologies.

[0006] To achieve the above-mentioned objectives, the technical solution of this application is as follows: This application provides a strawberry stolon regulatory gene ZFP6, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] This application also provides a protein encoded by the strawberry runner regulatory gene ZFP6, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] This application also provides a primer pair for amplifying the strawberry runner regulatory gene ZFP6, the nucleotide sequence of which is shown in SEQ ID NO.3-4.

[0009] This application also provides a method for amplifying the strawberry runner regulatory gene ZFP6, comprising: using strawberry genomic DNA as a template, performing PCR amplification with the primer pair to obtain the strawberry runner regulatory gene ZFP6; the PCR amplification program comprises: first, pre-denaturation at 94 °C for 3 min; then denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, for a total of 35 cycles; and finally extension at 72 °C for 5 min.

[0010] This application also provides the application of the strawberry runner regulatory gene ZFP6, the protein, or the strawberry runner regulatory gene ZFP6 obtained by the amplification method described above in regulating strawberry runners.

[0011] This application also provides a gene knockout expression vector for the strawberry runner regulatory gene ZFP6, wherein the gene knockout expression vector expresses sgRNA targeting the ZFP6 gene, wherein the sgRNA includes sgRNA1 and sgRNA2; the nucleotide sequence of sgRNA1 is shown in SEQ ID NO.5, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO.6.

[0012] Furthermore, the primer pairs used to synthesize the sgRNA double-stranded fragments, wherein the nucleotide sequences of the primer pairs corresponding to sgRNA1 are shown in SEQ ID NO.7-8, and the nucleotide sequences of the primer pairs corresponding to sgRNA2 are shown in SEQ ID NO.9-10.

[0013] This application also provides a method for constructing the gene knockout expression vector, comprising: synthesizing a double-stranded fragment of sgRNA1 and a double-stranded fragment of sgRNA2 based on the sgRNA1, the sgRNA2, and the corresponding primer pair; and ligating the double-stranded fragments of sgRNA1 and sgRNA2 with two first expression vectors respectively by enzyme digestion and ligation to obtain two sgRNA1... First expression vector and two sgRNAs First expression vector; based on the Golden Gate cloning method, the two sgRNAs1 The first expression vector is integrated into the second expression vector to obtain sgRNA1. sgRNA1-second expression vector; the two sgRNA2... The first expression vector is integrated into the second expression vector to obtain sgRNA2. sgRNA2-second expression vector; based on Gateway LR reaction, the sgRNA1 sgRNA1-second expression vector, sgRNA2 The sgRNA2 second expression vector, third expression vector, and fourth expression vector are assembled into the final gene knockout expression vector.

[0014] Furthermore, the restriction enzyme site is BsmBI; the first expression vector includes pYPQ131B and pYPQ132B vectors; the second expression vector includes pYPQ142 vector; the third expression vector includes pYPQ166 vector; and the fourth expression vector includes pJH23 vector.

[0015] This application also provides a method for reducing the number of strawberry runners, comprising: introducing the gene knockout expression vector, or the gene knockout expression vector constructed by the construction method, into strawberries.

[0016] This application provides a strawberry runner regulatory gene ZFP6, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO. 2. Examples confirm that, through a series of studies including gene cloning, gene knockout expression vector construction, genetic transformation, and functional verification, this application clarifies that the strawberry runner regulatory gene ZFP6 is specifically expressed in the axillary meristem of mature strawberry plants. It can directly or indirectly activate the expression of the GA20ox4 gene, initiate the GA biosynthesis pathway, and thus drive axillary buds to develop into runners. The ZFP6 loss-of-function mutant exhibits a significant "no runner" phenotype, with axillary buds instead developing into branched corollas. By analyzing the genetic interactions between the ZFP6 gene and other core factors in the GA synthesis and signaling pathways, such as the GA20ox4 and RGA1 genes, this study further validated the core function of the ZFP6 gene as an upstream regulator of axillary bud fate determination. This fills a gap in the endogenous regulatory mechanism of strawberry runners and confirms that the ZFP6 gene is a molecular target that specifically regulates strawberry runner formation. The gene sequence, primers, gene knockout expression vector, and supporting technology system provided in this application lay the foundation for strawberry molecular breeding and industrial applications. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart illustrating the gene knockout expression vector construction method provided in the embodiments of this application; Figure 2 This is a schematic diagram of the four-stage model of strawberry axillary bud development and key regulatory factors provided in the embodiments of this application; Figure 3 This application provides examples of differences in axillary bud fate and GA20ox4 gene expression at different developmental stages of strawberry. Figure 3 a: Development results of single leaf axils in the juvenile stage of diploid forest strawberry H4 seedlings at four weeks old; Figure 3 b: with Figure 3 a represents the developmental outcome of the leaf axils of the three-lobed leaves during the mature stage of the same plant; Figure 3 c: The morphology of the branched corolla of strawberry; Figure 3 d: Typical morphology of stolons formed in the leaf axils during the mature stage; Figure 3 e: Leaf type characteristics of a single leaf in the juvenile stage and a trifoliate leaf in the mature stage on the same strawberry plant; Figure 3 f: Schematic diagram of the development process of strawberry axillary buds from formation to fate determination stage; Figure 3 g: RT-qPCR results of GA20ox4 gene expression levels in axillary buds of juvenile and adult plants; Figure 4 These are the gene expression pattern analysis results of axillary buds at different developmental stages of strawberry provided in the embodiments of this application, wherein... Figure 4 a: Micrograph of axillary bud tissue of diploid forest strawberry H4 that will develop into a stolon; Figure 4 b: Micrograph of axillary bud tissue of diploid forest strawberry H4, which will develop into a branched corolla; Figure 4 c: Schematic diagram of tissue types and nomenclature of RNA-seq sequencing samples; Figure 4 d: Heatmap of expression of specific marker genes for different axillary bud types obtained based on RNA-seq analysis; Figure 5 This is an electrophoresis image of the PCR amplification products provided in the embodiments of this application; Figure 6 This is a schematic diagram of the ZFP6 gene structure provided in the embodiments of this application. The diagram shows the editing sites for gene knockout and also presents the specific situation of base insertion or deletion in each mutant. Figure 7This application provides a diploid forest strawberry H4, which is related to ZFP6. CR1 mutant strain, ZFP6 CR4 Comparison of overall phenotypic results of mutant strains under long-day conditions; Figure 8 The GA3 hormone treatment provided in this application embodiment affects ZFP6 CR1 Results of rescue experiments for mutant phenotypes; Figure 9 This is a comparison of the ZFP6 gene and RGA1 gene double mutant phenotypes and related control phenotypes provided in the embodiments of this application; Figure 10 The results of the correlation detection between the expression of the ZFP6 gene and the GA20ox4 gene provided in the embodiments of this application are as follows: Figure 10 a: RT-qPCR results of expression levels of ZFP6 and GA20ox4 genes in axillary buds of juvenile and adult plants; Figure 10 b: The GA20ox4 gene in diploid forest strawberry H4 and ZFP6 CR1 RT-qPCR results of expression levels in mutants; Figure 11 These are the multiple sequence alignment results of the ZFP6 protein in different species provided in the embodiments of this application. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] The flowchart shown in the attached diagram is for illustrative purposes only and does not necessarily include all content and operations / steps, nor does it necessarily have to be performed in the order described. For example, some operations / steps can be broken down, combined, or partially merged, so the actual execution order may change depending on the actual situation.

[0021] It should be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the application. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0022] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0023] The following detailed description of some embodiments of this application is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0024] In some embodiments, the strawberry stolon regulatory gene ZFP6 (Zinc FingerProtein 6) provided in this application has the nucleotide sequence shown in SEQ ID NO. 1; SEQ ID NO. 1: ATGAGTGCATCGGTTGCTGATCAGAACACCAAACCTTCATCATCAGCAATCAAGCTATTCGGGTTTTCGCTCATGGCCGATCCCTGTGAAGACGGAGTGGTGACAACGAAGAGCAAGAAGCAGTTTGTGTGTCACTACTGCGGTCGAGAGTTTGAGAATTCTCAAGCATTAGGTGGCCATCAAAATGCACACAAAAGAGAAAGGAAGCTAGCAAGAATGGCCATGTTGGAGCACGAG GAGGATCATCAGTATCAGGAATGCCAACACTTTCTTCCTCCTCAACGTAGTCAAGTACTGTTTCCAACCGGTGTCCGGTACCCCAGTATAAACCCTATGTTCGACACGAAATCTGGCGAGTCCGCAACCCAGAATTATATCGGCGGTGGTGCTGGTGCTGGTGCAGATCGGGTACTGCTCCCTAACGATTATGAGGGAAATGATCAGGTCGACATTGATCTACGTTTGAGACTTTAA.

[0025] In some embodiments, the protein encoded by the strawberry stolon regulatory gene ZFP6 provided in this application has the amino acid sequence shown in SEQ ID NO.2; SEQ ID NO.2: MSASVADQNTKPSSSAIKLFGFSLMADPCEDGVVTTTKSKKQFVCHYCGREFENSQALGGHQNAHKRERKLARMAMLEHEEDHQYQECQHFLPPQRSQVLFPTGVRYPSINPMFDTKSGESATQNYIGGGAGAGADRVLLPNDYEGNDQVDIDLRLRL*.

[0026] In some embodiments, the primer pair provided in this application for amplifying the above-mentioned strawberry stolon regulatory gene ZFP6 has the nucleotide sequence shown in SEQ ID NO.3-4; The primer pair includes an upstream primer and a downstream primer. The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.4. SEQ ID NO.3: ATGAGTGCATCGGTTGCTGATC; SEQ ID NO.4: TTAAAGTCTCAAACGTAGATC.

[0027] The method for amplifying the strawberry runner regulatory gene ZFP6 provided in this application includes the following steps: using strawberry genomic DNA as a template, PCR amplification is performed using the primer pair described above to obtain the strawberry runner regulatory gene ZFP6.

[0028] Since the ZFP6 gene has only one exon and no intron spacers, it can be directly amplified using strawberry genomic DNA as a template. By using the primer pairs mentioned above for PCR amplification, the strawberry stolon regulatory gene ZFP6 can be obtained.

[0029] The PCR amplification procedure includes: first, pre-denaturation at 94 °C for 3 min; then denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, for a total of 35 cycles; and finally extension at 72 °C for 5 min.

[0030] In some embodiments, the strawberry runner regulatory gene ZFP6, the protein, or the strawberry runner regulatory gene ZFP6 obtained by the amplification method described above are used in regulating strawberry runners.

[0031] Specifically, the application can be to influence the number and growth of strawberry runners by regulating the expression of the ZFP6 gene, for example, by using gene silencing (such as RNAi), gene knockout (such as CRISPR / Cas9), or gene overexpression technology (such as constructing a ZFP6 overexpression vector) to change the expression level of the ZFP6 gene in strawberry plants, thereby inhibiting or promoting runner growth; it can also be to target the ZFP6 protein, screen small molecule compounds that can specifically regulate its function, and develop strawberry runner-specific regulatory agents, for example, screening compounds that can inhibit the activity of ZFP6 protein, which can reduce runner occurrence after spraying and are suitable for fresh fruit cultivation; screening compounds that can activate the function of ZFP6 protein, which can promote runner growth after spraying and are suitable for seedling production; the specific application method is not limited here.

[0032] In some embodiments, the above-mentioned strawberry stolon regulatory gene ZFP6 provided in this application is used to knock out the expression vector. The gene knockout expression vector expresses sgRNA (Single Guide RNA) targeting the ZFP6 gene. The sgRNA includes sgRNA1 and sgRNA2. The nucleotide sequence of sgRNA1 is shown in SEQ ID NO.5, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO.6. SEQ ID NO.5: GGCCATGTTGGAGCACGAGG; SEQ ID NO.6: GAGAATTCTCAAGCATTAGG.

[0033] Furthermore, primer pairs for synthesizing sgRNA double-stranded fragments are provided, wherein the nucleotide sequences of the primer pairs corresponding to sgRNA1 are shown in SEQ ID NO.7-8, and the nucleotide sequences of the primer pairs corresponding to sgRNA2 are shown in SEQ ID NO.9-10.

[0034] The primer pair corresponding to sgRNA1 includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.8. The primer pair corresponding to sgRNA2 includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.10.

[0035] SEQ ID NO.7: 5'-ggtcAGGCCATGTTGGAGCACGAGG-3'; SEQ ID NO.8: 5'-aaacCCTCGTGCTCCAACATGGCCT-3'; SEQ ID NO.9: 5'-ggtcAGAGAATTCTCAAGCATTAGG -3'; SEQ ID NO.10: 5'-aaacCCTAATGCTTGAGAATTCTCT-3'.

[0036] like Figure 1 As shown, the method for constructing the gene knockout expression vector provided in this application includes steps S101 to S104.

[0037] Step S101: Based on sgRNA1, sgRNA2 and their corresponding primer pairs, synthesize the sgRNA1 double-stranded fragment and the sgRNA2 double-stranded fragment.

[0038] The primer pairs mentioned above all have preset restriction enzyme sites, and these restriction enzyme sites are consistent with the restriction enzyme sites of the first expression vector to be ligated later, providing a basis for the subsequent restriction enzyme ligation of the sgRNA double-stranded fragment to the first expression vector.

[0039] The preset restriction sites can be flexibly set according to the actual application and are not limited here. For example, it can be BsmBI, which is a Type IIs restriction endonuclease that can recognize non-palindromic sequences and cut outside the recognized sequence. It is often used for Golden Gate assembly.

[0040] Step S102: The sgRNA1 double-stranded fragment and the sgRNA2 double-stranded fragment are ligated to two different first expression vectors by enzyme digestion and ligation to obtain two sgRNA1... First expression vector and two sgRNAs The first carrier of expression.

[0041] The first expression vector is the basic backbone vector of sgRNA, which is used to provide the initial cloning site for the sgRNA double-stranded fragment. The choice of the first expression vector can be flexibly set according to the actual application and is not limited here. For example, the first expression vector is the pYPQ131B vector or the pYPQ132B vector.

[0042] For example, the two first expression vectors, pYPQ131B and pYPQ132B, are first digested with the preset restriction site BsmBI. Then, the obtained sgRNA1 double-stranded fragment and sgRNA2 double-stranded fragment are ligated to the digested pYPQ131B and pYPQ132B vectors, respectively, to finally obtain sgRNA1. pYPQ131B vector, sgRNA1 pYPQ132B vector, sgRNA2 pYPQ131B vector, sgRNA2 pYPQ132B vector.

[0043] Step S103: Based on the Golden Gate cloning method, the two sgRNAs1 The first expression vector is integrated into the second expression vector to obtain sgRNA1. sgRNA1-second expression vector; two sgRNA2... The first expression vector is integrated into the second expression vector to obtain sgRNA2. sgRNA2 - Second expression vector.

[0044] The second expression vector is an intermediate assembly vector for sgRNA. It can receive and accommodate two identical sgRNA expression units through the Golden Gate cloning method, thereby achieving centralized integration of sgRNA elements. The choice of the second expression vector can be flexibly set according to actual applications and is not limited here. For example, the second expression vector is the pYPQ142 vector.

[0045] For example, based on the Golden Gate cloning method, the obtained sgRNA1 pYPQ131B vector and sgRNA1 The pYPQ132B vector was integrated into the pYPQ142 vector to obtain sgRNA1. sgRNA1-pYPQ142 vector; the obtained sgRNA2 pYPQ131B vector and sgRNA2 The pYPQ132B vector was co-integrated into the pYPQ142 vector to obtain sgRNA2. sgRNA2-pYPQ142 vector.

[0046] Step S104: Based on the Gateway LR reaction, sgRNA1 sgRNA1 second expression vector, sgRNA2 The second, third, and fourth expression vectors of sgRNA2 are assembled into the final gene knockout expression vector.

[0047] The third expression vector contains the Cas9 protein-coding gene and provides the Cas9 nuclease required for gene knockout. The fourth expression vector is the final expression backbone vector containing elements such as the core promoter and terminator, which supports the transcription framework of the entire gene knockout expression vector. The selection of the third and fourth expression vectors can be flexibly set according to actual applications and is not limited here. For example, the third expression vector is the pYPQ166 vector and the fourth expression vector is the pJH23 vector.

[0048] For example, based on the Gateway LR reaction, the obtained sgRNA1 sgRNA1-pYPQ142 vector, sgRNA2 The sgRNA2-pYPQ142 vector, pYPQ166 vector, and pJH23 vector were assembled into the final gene knockout expression vector.

[0049] Using the above-described method for constructing gene knockout expression vectors, firstly, double-stranded sgRNA fragments are synthesized using primers with pre-defined restriction sites to ensure precise ligation with the first expression vector. Then, leveraging the efficient integration capabilities of the Golden Gate cloning method, the two sgRNAs—the first expression vector and the second expression vector—are integrated into the second expression vector, achieving orderly integration of the sgRNA elements. Finally, utilizing the site-specific recombination advantages of the Gateway LR reaction, the second expression vector containing the double sgRNAs is efficiently assembled with a third expression vector containing the Cas9 protein-coding gene and a fourth expression vector carrying core elements such as promoters and terminators, ultimately yielding a fully functional gene knockout expression vector.

[0050] In some embodiments, the method for reducing the number of strawberry runners provided in this application includes: introducing the gene knockout expression vector, or the gene knockout expression vector constructed by the above-described construction method, into strawberries.

[0051] The aforementioned gene knockout expression vectors are introduced into strawberry cells, such as strawberry leaf explants, using plant transformation technologies such as Agrobacterium-mediated transformation and gene gun transformation. After the vector enters the cell, the sgRNA in the vector can specifically recognize the target sequence of the ZFP6 gene, guiding the Cas9 protein to perform site-specific cleavage at the target site, resulting in DNA double-strand breaks. Subsequently, base insertion, deletion, or substitution mutations are randomly introduced at the cleavage site of the ZFP6 gene, leading to the loss of function of the ZFP6 gene. Ultimately, by knocking out the ZFP6 gene at a specific site, the number of strawberry runners is reduced and growth is inhibited.

[0052] Through a series of studies based on the embodiments of this application, the regulatory pathways for the development of strawberry axillary buds into stolons or branched corollas have been deduced, such as... Figure 2As shown, it can be roughly divided into the following four stages: Initiation: Regulated by LAM (Leafy Apical Meristem), the relevant developmental process is initiated; Maturation: Under the influence of red light, development is further promoted through PhyB (Phytochrome B) mediation; Fate Determination: The ZFP6 gene participates in regulation, acting on the GA20ox4 gene, which in turn affects the RGA1 (Repressor of ga1-3 1) gene, determining whether development is towards a branch crown or a stolon; Differentiation: MYB117a (Myeloblastosis-related transcription factor 117a) plays a role, through CK (Cytokinin... Cytokinin regulation ultimately leads to the differentiation and formation of branched corollas or stolons.

[0053] It should be noted that the GA20ox4 gene in the embodiments of this application specifically refers to its homolog FvH4_2g35050, and not other homologous genes in the GA20ox4 gene.

[0054] The following detailed description of the strawberry runner regulatory gene ZFP6 and its application provided in this application, through specific embodiments and experimental data, provides a detailed explanation.

[0055] Example 1 The observation of differences in axillary bud fate at different developmental stages of strawberry includes the following steps: (1) Experimental material cultivation and phenotypic observation diploid forest strawberry ( Fragaria vesca The Hawaii 4 (H4) strain was used as the wild-type experimental material. Strawberry seeds were first stratified in sterile water at 4 °C in the dark for at least 2 weeks, and then sown in soil. All strawberry plants were cultivated in a plant growth chamber under the following conditions: 16 h light / 8 h dark photoperiod, constant temperature of 22 °C, and relative humidity of 50%. White light at 110 μmol photons / m² was used. -2 s -1 Irradiate with light intensity.

[0056] The phenotypic characteristics of axillary bud fate in diploid forest strawberry H4 at different developmental stages provided in this embodiment are as follows: Figure 3As shown, it should be noted that the red and white arrows in the figure are shown to illustrate the phenotypic characteristics of the strawberry plant and are not actually displayed on the strawberry plant.

[0057] from Figure 3 As can be seen in a, axillary buds in the leaf axils of young single leaves tend to form branched corollas; from Figure 3 As seen in b, the axillary buds in the leaf axils of the trifoliate leaves in the mature plant stage mainly form stolons; from Figure 3 As can be seen in c, the typical branchcrown of strawberry corresponds to a single-leaved leaf; from Figure 3 As seen in d, the leaf type corresponding to the stolon is trifoliate; from Figure 3 As can be seen in e, the same plant can have both single leaves in the juvenile stage and three leaves in the mature stage at the same time, indicating that the fate of axillary buds is regulated by the plant development stage. Figure 3 The diagram in f clearly shows the entire process of an axillary bud from its inception to its formation as a branched corolla or stolon.

[0058] (2) Real-time quantitative PCR detection of genes (RT-qPCR) a) RNA extraction and cDNA synthesis Strawberry axillary bud meristems were collected, and total RNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. 1 μg of RNA was used for reverse transcription using Invitrogen's SuperScript IV VILO Master Mix to synthesize cDNA.

[0059] b) RT-qPCR detection RT-qPCR experiments were performed using a BioRad CFX96 real-time quantitative PCR system and an Applied Biosystems SYBR Green PCR MasterMix. The reaction mixture consisted of 1 μL cDNA template, 200 nM forward primer, 200 nM reverse primer, 5 μL 2×PowerUp SYBR Green Master Mix, and nuclease-free water to make up the remaining volume, for a total volume of 10 μL. The PCR reaction followed a three-step procedure: incubation at 50 °C for 2 min; pre-denaturation at 95 °C for 2 min; followed by denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, and extension at 72 °C for 30 s, for a total of 40 cycles. The forward and reverse primers used in the experiment are: Internal reference gene PP2a: PP2a-qPCR-F: 5'-ATGTCTGTGGTTGATGAACCG-3'; PP2a-qPCR-R: 5'-CCAATCCTACATAATGACTCCAC-3'; ZFP6 gene: ZFP6-qPCR-F: 5'-ATGAGTGCATCGGTTGCTGATC-3'; ZFP6-qPCR-R: 5'-TCTCAAACTCTCGACCGCAG-3'; GA20ox4 gene: GA20ox4-qPCR-F: 5'-CTGTGAAGCAATGACGAGTTTTGGCTC-3'; GA20ox4-qPCR-R: 5'-GCTCTTGTACAATCCATTCGATAGCGC-3'.

[0060] Relative gene expression levels were calculated using 2 -ΔΔCT The calculation method yields the following results: Figure 3 As shown in g.

[0061] from Figure 3 As can be seen, the expression level of the key enzyme gene GA20ox4 in adult axillary buds (Adult AXM) was significantly higher than that in juvenile axillary buds (Juvenile AXM), suggesting that this gene may participate in the determination of strawberry stolon development fate by regulating GA synthesis.

[0062] Example 2 The key gene ZFP6, which regulates differences in axillary bud fate at different developmental stages of strawberry, was identified, including the following steps: (1) Sample processing and total RNA extraction Axillary bud tissues of diploid forest strawberry H4 were dissected, with three biological replicates for each genotype to ensure the reliability of the results. Total RNA was extracted using the Thermo Fisher Scientific (USA) Arcturus PicoPure RNA Isolation Kit, and the procedure was strictly followed according to the kit's instructions.

[0063] (2) RNA sequencing library construction and sequencing 0.25 μg–2 μg of RNA was extracted from each sample and sent to Azenta US, Inc. for library construction, followed by sequencing using the Illumina NovaSeq platform. RNA sequencing libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina from New England Biolabs (Ipswich, Massachusetts, USA) and sequenced on the Illumina NovaSeq 6000 platform from Illumina (San Diego, California, USA). Each sample yielded approximately 20–30 million 150 bp paired-end reads.

[0064] (3) Sequencing data processing and differential expression analysis The raw sequencing data were processed using Trimmomatic software (v0.39) to remove adapter sequences and low-quality bases, resulting in clean reads. Subsequently, Salmon software (v0.11.2) was used to quasi-mapping the clean reads to the diploid forest strawberry H4 genome v4.0.a2, and transcript expression levels were quantified. The obtained transcript abundance data were imported into Tximport software (v1.10.1) and then used DESeq2 software (v1.22.2) for downstream differential expression analysis.

[0065] (4) Tissue-specific expression analysis First, the average TPM (transcriptions per million transcripts) of each tissue was calculated. Then, the TissueEnrich software (v1.2.1) was used to identify highly expressed specific genes in each tissue. The tissue-specific expression gene was defined as: a TPM value of not less than 1 and an expression level in that tissue that is at least 5 times that of any other tissue.

[0066] (5) Signature gene analysis The Seurat software (v5.0.1) was used to complete the following steps: The expression matrix was preprocessed and characteristic genes were identified. The expression values ​​of each sample were standardized by library size and log transformation to a standard distribution with a mean of 0 and a variance of 1. The characteristic genes of the predefined groups were identified by the FindMarkers function. The differential analysis method was set to "roc", and only genes with an AUC value of 1 were retained. When plotting the heatmap, the top 20 characteristic genes with the highest log2FC were selected for each phenotype group. Among them, only 11 characteristic genes were identified in the branched corolla group, so only 11 genes of this group are displayed.

[0067] (6) Gene Ontology (GO) enrichment analysis Using GO annotation information constructed in previous studies, enrichment analysis was performed using the topGO package (v2.54.0) in R. During the analysis, over-enriched GO items were identified using Fisher's exact test and the "classic" algorithm. To reduce interference from under-annotated GO items, the nodeSize parameter was set to 5 to prune the GO hierarchy. The analysis results were visualized using the ggplot2 package in R (see official website link: https: / / ggplot2.tidyverse.org).

[0068] The data results related to the discovery of ZFP6, a key gene regulating the differences in axillary bud fate at different developmental stages of strawberry, provided in this embodiment are as follows: Figure 4 As shown, it should be noted that the black circles in the figure are for illustrating the differentiation structure of the axillary bud tissue and are not actually shown on the axillary bud tissue.

[0069] Figure 4 The micrograph of a shows a stolon about to differentiate. Figure 4 The micrograph of b shows the branched corolla that is about to differentiate. The comparison between the two shows that the axillary buds of the two fate types have already shown initial differentiation in terms of tissue morphology. Figure 4 c clarifies the sampling tissue types and nomenclature for RNA-seq sequencing. Strawberry samples include the wild-type group (WT(H4)), the GA20ox4 mutant group (ga20ox4-1), and the phytochrome B mutant group (phyb). CR1 Among them, the axillary bud fate types during the developmental stages of the wild-type group include juvenile branched corolla (WT_J_BC) and mature stolon (WT_A_S); the axillary bud fate types during the developmental stages of the GA20ox4 mutant group include juvenile branched corolla (ga20ox4_J_BC) and mature branched corolla (ga20ox4_A_BC); and the axillary bud fate types during the developmental stages of the phytochrome B mutant group include juvenile immature (phyb_J_IM). Figure 4 The heatmap analysis revealed several marker genes specifically expressed in axillary buds. Among them, the GA20ox4 and ZFP6 genes were significantly highly expressed in axillary buds (Late Stolons) that were intended to form stolons in the mature stage, suggesting that the ZFP6 gene may be a key transcription factor that mediates the differentiation of axillary buds into stolons by regulating the expression of the GA20ox4 gene.

[0070] Example 3 The method for preparing the strawberry runner regulatory gene ZFP6 includes the following steps: (1) Extraction of strawberry genomic DNA Using diploid forest strawberry H4 as experimental material, a small piece of strawberry leaf was ground in liquid nitrogen with a blue pestle. 500 μL of cetyltrimethylammonium bromide (CTAB) DNA extraction buffer was added to a 1.5 mL centrifuge tube and incubated at 60 °C for 20 min. Then, 500 μL of chloroform was added, and the mixture was vortexed for 15 s. The tube was centrifuged at 13000 rpm for 5 min. Since the genomic DNA dissolved in the supernatant, 300 μL–400 μL of the supernatant was transferred to a new centrifuge tube. 1000 μL of ethanol (2–2.5 times the volume of the supernatant) was added, and the mixture was gently mixed and placed at -20 °C for 10 min. The tube was then centrifuged at 13000 rpm for 5 min. At this point, the DNA was present in the precipitate. The precipitate was washed with 1 mL of 75% ethanol, dried at room temperature, and resuspended in 20 μL of H2O. 1 μL of the resuspended precipitate was taken to obtain the genomic DNA.

[0071] The CTAB DNA extraction buffer formulation is as follows: 100 mM Tris-Cl (pH 8.0), 20 mM EDTA (pH 8.0), 1.4 M NaCl, 2% (w / v) CTAB, and 1% polyvinylpyrrolidone (PVP-4000).

[0072] (2) Cloning of the ZFP6 gene Using the obtained genomic DNA as a template, PCR amplification was performed using the upstream and downstream primers of the amplification primer pair. The nucleotide sequence of the upstream primer is as described in SEQ ID NO.3 above, and the nucleotide sequence of the downstream primer is as described in SEQ ID NO.4 above. The amplification system was as follows: 10 μL of Quantabio AccuStart II PCR ToughMix (2X), 0.2 μL of upstream primer, 0.2 μL of downstream primer, 8.6 μL of ddH2O, and 1 μL of DNA template, for a total volume of 20 μL. The amplification program was as follows: first, pre-denaturation at 94 °C for 3 min; then, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, for a total of 35 cycles; finally, extension at 72 °C for 5 min, yielding the desired result. Figure 5 As shown, the target fragment has a sequence length of 474 bp.

[0073] Example 4 Preparation and identification of transgenic plants of diploid forest strawberry H4 strain: (1) For the construction of the ZFP6 gene knockout expression vector, please refer to [link / reference]. Figure 1 This includes the following steps: a) Based on the pre-designed sgRNA1, sgRNA2 and corresponding primer pairs, synthesize double-stranded fragments of sgRNA1 and sgRNA2, each containing the pre-designed restriction enzyme site BsmBI. The nucleotide sequence of sgRNA1 is as described in SEQ ID NO.5 above, the nucleotide sequence of the upstream primer of its corresponding primer pair is as described in SEQ ID NO.7 above, and the nucleotide sequence of the downstream primer is as described in SEQ ID NO.8 above; the nucleotide sequence of sgRNA2 is as described in SEQ ID NO.6 above, the nucleotide sequence of the upstream primer of its corresponding primer pair is as described in SEQ ID NO.9 above, and the nucleotide sequence of the downstream primer is as described in SEQ ID NO.10 above. b) The two primary expression vectors, pYPQ131B and pYPQ132B, were digested with the pre-defined restriction site BsmBI. Subsequently, the obtained sgRNA1 and sgRNA2 double-stranded fragments were ligated into the digested pYPQ131B and pYPQ132B vectors, respectively, to obtain sgRNA1. pYPQ131B vector, sgRNA1 pYPQ132B vector, sgRNA2 pYPQ131B vector, sgRNA2 pYPQ132B vector; c) Based on the Golden Gate cloning method, the obtained sgRNA1 pYPQ131B vector and sgRNA1 The pYPQ132B vector was integrated into the pYPQ142 vector to obtain sgRNA1. sgRNA1-pYPQ142 vector; the obtained sgRNA2 pYPQ131B vector and sgRNA2 The pYPQ132B vector was co-integrated into the pYPQ142 vector to obtain sgRNA2. sgRNA2-pYPQ142 vector; d) Based on the Gateway LR reaction, the obtained sgRNA1 sgRNA1-pYPQ142 vector, sgRNA2 The sgRNA2-pYPQ142 vector, pYPQ166 vector, and pJH23 vector were assembled into the final ZFP6 gene knockout expression vector.

[0074] (2) Genotyping of infected explants and transgenic plants a) Take diploid forest strawberry H4 seedlings grown on MS medium, cut off their cotyledons or primary single leaves, and perform vacuum permeation treatment using a syringe sleeve and piston. Then, use Agrobacterium tumefaciens containing the ZFP6 gene knockout expression vector. Agrobacterium tumefaciens GV3101 strain infection; b) Infected explants were placed in 5++ medium (1×MS, 2% sucrose, 3.4 mg / L 6-benzylaminopurine (BA), 0.3 mg / L indolebutyric acid (IBA), 0.7% plant agar, pH 5.8) and incubated in the dark for 3 days. c) After rinsing the explants with sterile water, they were transferred to MS medium containing antibiotics (250 mg / L Timentin + 250 mg / L Carbenicillin) for further culture, followed by culture in the dark for 7 days to induce callus formation. d) The transformed callus tissue was transferred to 5++ medium containing 250 mg / L Timentin, 250 mg / L Carbenicillin and 4 mg / L Hygromycin for screening. The medium was replaced with fresh medium every 2 to 3 weeks until the buds formed and sprouted. e) After the buds grow, they are transferred to rooting medium (0.5×MS, 0.01 mg / L indolebutyric acid (IBA), 2% glucose, 0.7% plant agar, pH 5.8) and further screened under conditions containing 4 mg / L hygromycin. f) After approximately 1-2 months, the rooted plants were transplanted into soil, and genotyping was performed simultaneously. A total of 15 independent homozygous or biallelic mutants were obtained, from which 6 were selected for sequencing analysis. The results are as follows: Figure 6 As shown.

[0075] from Figure 6 As can be seen, under the targeting effect of sgRNA1 and sgRNA2, the gene sequences of these 6 mutants showed base insertion (such as "+1") or base deletion (such as "-5" or "-4") compared with the reference sequence (Ref). This indicates that the gene knockout expression vector was successfully applied to strawberry plants and achieved the targeted knockout effect of the ZFP6 gene.

[0076] (3) Phenotypic observation of transgenic strawberries a) Effects of ZFP6 gene knockout on strawberry runner formation Diploid forest strawberry H4 wild-type plants (WT(H4)) were used as the control group, and zfp6 were used as the control group. CR1 and zfp6 CR4 Gene knockout strawberry plants were designated as the experimental group. Both groups of plants were cultured under long-day conditions for 4 months, and the results were as follows: Figure 7 As shown.

[0077] from Figure 7 It is evident that the control group plants produced runners, while the experimental group showed no obvious runner formation. Furthermore, the axillary buds in the mature group developed into branched corollas rather than runners. Additionally, statistical results showed that all 31 mature axillary buds in the control group formed runners, whereas in the experimental group, zfp6... CR1 Of the 39 axillary buds, only 2 form stolons, zfp6 CR4 No stolons were formed at all. This indicates that the ZFP6 gene is a key regulator in stolon fate formation and a previously unidentified new regulator of stolon fate determination.

[0078] b) External GA3 treatment for ZFP6 CR1 Restorative effect of plant phenotype To clarify the role of the ZFP6 gene in the GA synthesis pathway, the zfp6 gene was analyzed. CR1 The axillary buds of the plants were treated. The experimental group was treated with exogenous 500 μmol / L GA3 every other day, while a control group was set up for control treatment. The results were observed after 16 days. Figure 8 As shown.

[0079] from Figure 8 It can be seen that only GA3 processes group zfp6 CR1 The formation of stolons was observed in the plants, while no significant changes were observed in the control group plants. This indicates that zfp6... CR1 The lack of stolons in the plants is due to the absence of GA, suggesting that the ZFP6 gene is located upstream of the GA synthesis pathway and affects stolon formation by regulating GA synthesis.

[0080] c) Double mutant experiments to verify the genetic relationship between the ZFP6 gene and the RGA1 gene. It should be noted that all genotype plants in this experiment were based on the diploid forest strawberry H4 genetic background. The white arrows in the figure are shown to illustrate the phenotypic characteristics of the strawberry plants and are not actually displayed on the strawberry plants.

[0081] To further verify the regulatory hierarchy of the ZFP6 gene in the GA synthesis pathway, rga1 was constructed. CR3 ;zfp6 CR1 The double mutant was observed, and its phenotype was determined. The results are as follows: Figure 9 As shown.

[0082] from Figure 9 As can be seen, rga1 CR3 ;zfp6 CR1 Double mutant phenotype and rga1 CR3The single mutants showed consistent phenotypes and were able to form a large number of runners normally. This result indicates that the RGA1 gene is located downstream of the ZFP6 gene, which is consistent with the regulatory model of "ZFP6 → GA20ox4 → GA → RGA1". This further confirms the molecular mechanism by which the ZFP6 gene regulates the fate of strawberry runners through the GA synthesis pathway.

[0083] d) Detection of expression levels of ZFP6 and GA20ox4 genes in axillary buds of strawberries at different developmental stages The expression levels of the ZFP6 and GA20ox4 genes in axillary buds of strawberry plants during the juvenile and mature stages were detected using the RT-qPCR method described above. The results are as follows: Figure 10 As shown.

[0084] from Figure 10 As can be seen, both the ZFP6 and GA20ox4 genes are highly expressed in adult axillary meristems, but lowly expressed in juvenile axillary meristems. This result is completely consistent with RNA-seq sequencing data, further confirming that the ZFP6 gene is a key regulatory factor in strawberry runner formation. Figure 10 b can be seen in zfp6 CR1 In the axillary buds of the mutant mature plants, the expression level of the GA20ox4 gene was only 0.02% of that of the wild type (WT(H4)), indicating that the expression of the GA20ox4 gene depends on the activity of the ZFP6 gene, which directly confirms the positive transcriptional regulation of the GA20ox4 gene by the ZFP6 gene.

[0085] Example 5 Conservation analysis of ZFP6 protein in different species To verify the sequence and functional conservation of ZFP6 protein in different species, the FVEZFP6 protein sequence of diploid forest strawberry H4 was used as a reference. First, highly conserved ZFP6 homologous protein sequences from different species were screened using the NCBI plant proteome database. Sequences from species with stolons were preferentially included in the analysis. Then, using BLASTP combined with domain annotation tools such as SMART, it was confirmed that all candidate sequences contained typical C2H2 zinc finger domains. Finally, multiple sequence alignment software, such as ClustalW, was used to analyze the conservation of the core domains of the candidate sequences. The results are as follows: Figure 11 As shown.

[0086] from Figure 11As can be seen from the figure, the conservation characteristics of the C2H2 zinc finger domain in homologous proteins from different species are displayed. The C2H2 zinc finger domain sequence in diploid forest strawberry H4 (FVEZFP6) is “FVCHYCGREFENSQALGGHQNAHKRE”. Further comparison of this sequence with C2H2 zinc finger domain sequences of homologous proteins from other species, including: alfalfa (TRIFOLIUM), banana (MACMA4_03_G01910.1), the invasive species Mikania micrantha, potato (SOLTU.DM.05G003320.1), and the invasive species Ipomoea pubescens, revealed that key core amino acid sites of the C2H2 zinc finger domain, such as cysteine ​​(Cys) and histidine (His), are highly conserved in homologous sequences across different species, indicating the stability of this domain during evolution.

Claims

1. A strawberry runner regulatory gene ZFP6, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A protein encoded by the strawberry runner regulatory gene ZFP6 as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A primer pair for amplifying the strawberry runner regulatory gene ZFP6 as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.3-4.

4. A method for amplifying the strawberry runner regulatory gene ZFP6 as described in claim 1, characterized in that, include: Using strawberry genomic DNA as a template, PCR amplification was performed using the primer pair described in claim 3 to obtain the strawberry stolon regulatory gene ZFP6; The PCR amplification procedure includes: first, pre-denaturation at 94 °C for 3 min; then denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, for a total of 35 cycles; and finally extension at 72 °C for 5 min.

5. The application of the strawberry runner regulatory gene ZFP6 as described in claim 1, the protein as described in claim 2, or the strawberry runner regulatory gene ZFP6 obtained by the amplification method described in claim 4 in regulating strawberry runners.

6. A gene knockout expression vector containing the strawberry runner regulatory gene ZFP6 as described in claim 1, characterized in that, The gene knockout expression vector expresses sgRNA targeting the ZFP6 gene, wherein the sgRNA includes sgRNA1 and sgRNA2; The nucleotide sequence of sgRNA1 is shown in SEQ ID NO.5, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO.

6.

7. The gene knockout expression vector for the strawberry runner regulatory gene ZFP6 according to claim 6, characterized in that, Primer pairs for synthesizing sgRNA double-stranded fragments, wherein the nucleotide sequences of the primer pairs corresponding to sgRNA1 are shown in SEQ ID NO.7-8, and the nucleotide sequences of the primer pairs corresponding to sgRNA2 are shown in SEQ ID NO.9-10.

8. A method for constructing a gene knockout expression vector as described in claim 6 or 7, characterized in that, include: Based on the sgRNA1, the sgRNA2 and the corresponding primer pair, synthesize the sgRNA1 double-stranded fragment and the sgRNA2 double-stranded fragment; The sgRNA1 double-stranded fragment and the sgRNA2 double-stranded fragment were ligated to two first expression vectors by enzyme digestion and ligation to obtain two sgRNA1... First expression vector and two sgRNAs The primary vehicle for expression; Based on the Golden Gate cloning method, the two sgRNA1 The first expression vector is integrated into the second expression vector to obtain sgRNA1. sgRNA1 - second expression vector; the two sgRNA2... The first expression vector is integrated into the second expression vector to obtain sgRNA2. sgRNA2 - Second expression vector; Based on the Gateway LR reaction, the sgRNA1 sgRNA1 second expression vector, sgRNA2 The sgRNA2 second expression vector, third expression vector, and fourth expression vector are assembled into the final gene knockout expression vector.

9. The method for constructing a gene knockout expression vector according to claim 8, characterized in that, The enzyme cleavage site is BsmBI; The first expression vector includes the pYPQ131B vector and the pYPQ132B vector; The second expression vector includes the pYPQ142 vector; The third expression vector includes the pYPQ166 vector; The fourth expression vector includes the pJH23 vector.

10. A method for reducing the number of strawberry runners, characterized in that, include: The gene knockout expression vector of any one of claims 6 to 7, or the gene knockout expression vector constructed by the construction method of any one of claims 8 to 9, is introduced into strawberries.

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