Passion fruit PeBBX24 gene and application thereof in regulation and control of plant early blossoming

Abstract

The invention discloses a passion fruit PeBBX24 gene and application of the passion fruit PeBBX24 gene in regulation and control of plant early blossoming, and belongs to the technical field of gene engineering. The nucleotide sequence of a coding region of the passion fruit PeBBX24 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the protein coded by the passion fruit PeBBX24 gene is as shown in SEQ ID NO.2. Experiments prove that the heterologous overexpression passion fruit PeBBX24 gene in arabidopsis thaliana can obviously promote early flowering, the flowering time is advanced by 5-6 days, and the early flowering phenotype can be stably expressed under the conditions of medium and long sunshine and short sunshine, so that the gene has relatively strong adaptability to different light environments, and the application range of the gene under various cultivation conditions is widened. The gene can up-regulate the expression quantity of flowering promoting factors CO, FT, SOC1 and FUL and down-regulate the expression quantity of flowering inhibiting factors SVP and FLC, so that flowering transformation is effectively promoted, and early flowering regulation is realized. The gene can be used for cultivating passion fruit, arabidopsis thaliana and other early blossoming transgenic plants, and has important application value for shortening the breeding period and improving the breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a passion fruit PeBBX24 gene and its application in regulating early flowering in plants. Background Technology

[0002] Plant flowering time is a key agronomic and developmental trait that affects reproductive efficiency, cultivation cycle, and industrial economic benefits. Precisely controlling flowering time and cultivating early-flowering varieties using genetic engineering techniques is of great significance for shortening breeding cycles, optimizing planting patterns, and improving the production efficiency of crops and horticultural plants.

[0003] Passion fruit (Passiflora edulis) is a perennial vine belonging to the Passifloraceae family and the Passiflora genus. Its fruit has a unique flavor and is rich in nutrients, possessing both edible and economic value, and is widely cultivated in tropical and subtropical regions. However, the flowering time of passion fruit is easily affected by environmental factors. Stress such as high or low temperatures can lead to abnormal flower bud differentiation, yellowing and drop of flower buds, severely impacting flowering and fruiting. Furthermore, traditional breeding methods for early-flowering passion fruit varieties suffer from long cycles and low efficiency, making it difficult to meet the industry's urgent need for early-flowering varieties.

[0004] The BBX family consists of zinc finger transcription factors containing one or two B-box domains, widely involved in the regulation of various physiological processes in plants, including flowering time, photomorphogenesis, and stress response. Currently, the functions of several BBX genes have been identified in model plants such as Arabidopsis thaliana and rice. For example, AtBBX1 (CONSTANS) in Arabidopsis thaliana is a core gene regulating flowering in the photoperiod pathway, while AtBBX18 is involved in heat tolerance response; however, its function in regulating early flowering has not been reported. In passion fruit, the functions of BBX family genes, especially their role in flowering regulation, remain unclear, and related research is still lacking. Summary of the Invention

[0005] To address the above problems, this invention provides a passion fruit PeBBX24 gene and its application in regulating early flowering in plants. Overexpression of the passion fruit PeBBX24 gene in Arabidopsis thaliana significantly advances the flowering time, which can be applied to cultivate early-flowering transgenic plants, shorten the breeding time, improve breeding efficiency, and thus promote the development and utilization of horticultural plants.

[0006] This invention is achieved through the following technical solution: This invention provides a passion fruit PeBBX24 gene, the coding region of which has the nucleotide sequence shown in SEQ ID NO.1. The gene is 618 bp in length and encodes a protein containing 205 amino acids.

[0007] The present invention also provides a protein encoded by the passion fruit PeBBX24 gene described above, the amino acid sequence of which is shown in SEQ ID NO.2. This protein contains two conserved B-box domains, possesses transcriptional activation activity, and is located in the cell nucleus.

[0008] The present invention also provides an application of the passion fruit PeBBX24 gene as described above in regulating the flowering time of plants, the application being to promote early flowering in plants.

[0009] Furthermore, the plant includes Arabidopsis thaliana or passion fruit.

[0010] The present invention also provides a recombinant expression vector containing the passion fruit PeBBX24 gene described above, wherein the recombinant expression vector contains the complete coding sequence of the passion fruit PeBBX24 gene; the complete coding sequence of the passion fruit PeBBX24 gene is obtained by amplification using the primers shown in SEQ ID NO.3 and SEQ ID NO.4.

[0011] Furthermore, the recombinant expression vector uses the plant expression vector pGWB506 as the original vector; the transcription of the passion fruit PeBBX24 gene is driven by the p35S promoter.

[0012] The present invention also provides a recombinant engineered bacterium containing the passion fruit PeBBX24 gene described above, wherein the recombinant engineered bacterium comprises the recombinant expression vector described above.

[0013] The present invention also provides a primer set for quantitative PCR of the passion fruit PeBBX24 gene described above, the primer set comprising primers as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0014] A method for cultivating an early-flowering transgenic plant includes the following steps: (1) Using passion fruit leaf cDNA as a template, the complete coding sequence of the passion fruit PeBBX24 gene was amplified using primers shown in SEQ ID NO.3 and SEQ ID NO.4, and the coding sequence is shown in SEQ ID NO.1; (2) The coding sequence of the passion fruit PeBBX24 gene obtained in step (1) is ligated with the plant expression vector to construct a recombinant expression vector; (3) The recombinant expression vector obtained in step (2) is transformed into the target plant, and transgenic plants with overexpression of the PeBBX24 gene of passion fruit are obtained by screening.

[0015] Furthermore, in step (3), the transformation is performed using Agrobacterium, strain GV3101 mediated by the flower immersion method; The screening included: resistance screening on 1 / 2 MS solid medium containing 50 mg / mL hygromycin, and PCR molecular identification of transgenic plants using primers shown in SEQ ID NO.3 and SEQ ID NO.4.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention, through integrated transcriptome analysis and gene family identification, combined with functional verification, screened and identified PeBBX24, a key gene regulating early flowering in passion fruit. This gene is highly expressed in early-flowering varieties and is synergistically regulated by photoperiod and temperature. The discovery of its function not only fills a research gap in the field of flowering regulation within the passion fruit BBX gene family but also provides new gene resources and technical approaches for the precise improvement of plant flowering time. It has significant theoretical value and application prospects for promoting molecular breeding innovation in passion fruit and other horticultural plants and crops.

[0017] 2. The passion fruit PeBBX24 gene in this invention has a clear function of promoting early flowering, and it remains stable under different photoperiods. Experiments in this invention have demonstrated that heterologous overexpression of the passion fruit PeBBX24 gene in Arabidopsis thaliana can significantly promote early flowering, advancing the flowering time by 5-6 days. Furthermore, this early flowering phenotype is stably expressed under both long-day and short-day conditions, indicating that the gene has strong adaptability to different light environments and broadening its application range under various cultivation conditions.

[0018] 3. In this invention, the passion fruit PeBBX24 gene promotes early flowering by regulating the expression network of flowering-related genes, with a clearly defined molecular mechanism. This gene can upregulate the expression levels of flowering-promoting factors CO, FT, SOC1, and FUL, while downregulating the expression levels of flowering-inhibiting factors SVP and FLC, thereby effectively promoting the transition to flowering and achieving early flowering regulation. This clear molecular mechanism provides a reliable theoretical basis for subsequent gene function optimization and breeding applications.

[0019] 4. In this invention, the passion fruit PeBBX24 gene simultaneously participates in the temperature stress response, which helps enhance the plant's environmental adaptability to flowering. The expression of this gene is temperature-regulated; under high temperature conditions, its expression is suppressed, and the early flowering effect of the corresponding transgenic plants is also weakened. This characteristic indicates that this gene is synergistically regulated by photoperiod and temperature, enabling the plant to adjust its flowering process according to changes in ambient temperature, reducing the impact of adverse temperatures on flowering, and improving the plant's production stability under variable climate conditions.

[0020] 5. The PeBBX24 gene in this invention provides a new gene resource and technical approach for early flowering breeding of passion fruit and other plants. This gene is highly expressed in early-flowering passion fruit varieties, and overexpression in Arabidopsis thaliana significantly advances flowering, indicating that it also has an early-flowering function in different species. Using this gene to breed early-flowering transgenic plants can significantly shorten the traditional breeding cycle, improve breeding efficiency, and has important application value for improving the flowering period of passion fruit and other horticultural and agricultural crops. Attached Figure Description

[0021] Figure 1 WGCNA analysis of flowering development-related transcriptome data from two varieties with different flowering phenotypes revealed a network diagram showing the co-expression of multiple BBX members and the key flowering regulatory gene PeSVP.

[0022] Figure 2 The graph shows the expression analysis of the candidate gene PeBBX24; where A represents the expression level of PeBBX24 in different tissues, B represents the expression level in leaves of different varieties, and C represents the expression under different temperature treatments.

[0023] Figure 3 Phylogenetic relationship diagram of candidate gene PeBBX24 and members of the Arabidopsis AtBBX family.

[0024] Figure 4 The images show the transcriptional activity detection results and subcellular localization map of the PeBBX24 protein; where A is the transcriptional activity detection result image and B is the subcellular localization map.

[0025] Figure 5 A comparison of flowering phenotypes between Arabidopsis thaliana overexpressing PeBBX24 and wild-type Arabidopsis thaliana; where A is a comparison of root length, B is a comparison of root length growth rate at 3-6 days of age, and C is a comparison of flowering time.

[0026] Figure 6 This figure shows the expression levels of flowering-related genes in Arabidopsis thaliana overexpressing PeBBX24.

[0027] Figure 7 A comparison of flowering phenotypes between Arabidopsis thaliana overexpressing PeBBX24 and wild-type Arabidopsis thaliana under different day treatments.

[0028] Figure 8 This is a phenotypic comparison between Arabidopsis thaliana overexpressing PeBBX24 and wild-type Arabidopsis thaliana under high temperature stress; where A shows the phenotypic comparison after different treatment times under high temperature stress, and B shows the phenotypic inhibition after high temperature stress. Detailed Implementation

[0029] Members of the BBX gene family are widely involved in plant growth, development, and stress response processes, with some members confirmed to be related to flowering regulation. This invention, through transcriptome data analysis of different passion fruit varieties (early-flowering cultivar Guibai No. 1 and late-flowering cultivar Tainong No. 1), combined with weighted gene co-expression network analysis (WGCNA), screened for multiple transcription factors belonging to the BBX family that co-express with the known key flowering regulatory gene PeSVP. Furthermore, based on phylogenetic analysis and transcriptome- and qRT-PCR-based expression analyses, the candidate gene PeBBX24 was identified. Further experiments, including expression pattern analysis, subcellular localization, transcriptional activity detection, and Arabidopsis heterologous overexpression, validated its early flowering regulatory function and molecular mechanism, providing new gene resources and technical approaches for early flowering breeding in plants.

[0030] Experiments have shown that overexpression of the passion fruit PeBBX24 gene can promote flowering time in Arabidopsis thaliana, and overexpression technology can also be used to increase the expression level of PeBBX24 in passion fruit to promote early flowering. Addressing the shortcomings of traditional plant breeding techniques, which are slow to produce results and have long cycles, this invention applies the passion fruit PeBBX24 gene to the breeding of early-flowering transgenic plants, which can shorten breeding time, improve breeding efficiency, and promote the development and utilization of horticultural plants.

[0031] The present invention will be further described in detail below through embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of protection of the present invention.

[0032] Example 1: Screening, cloning, and sequence analysis of the PeBBX24 gene 1. Transcriptome analysis of flower development in varieties with different flowering phenotypes (1) Virus-free healthy seedlings (60 cm tall) of Guibai No. 1 and Tainong No. 1, planted in November, were selected as experimental materials. In early February of the following year, flower buds had formed on some branches of Guibai No. 1, while Tainong No. 1 was still in the vegetative growth stage. Samples of leaves, shoot tip meristems, and leaf axillary meristems at different nodes of the two passion fruit varieties were collected; as well as samples of different flower tissues at different stages of flower development of Tainong No. 1. Total RNA was extracted using the Novozymes RNA isolater Total RNA Extraction Reagent (Vazyme, R401, Nanjing, China). Approximately 1 μg of RNA from each sample was used for library construction, and transcriptome sequencing was performed on the Illumina NovaSeq 6000 platform.

[0033] (2) Sequencing data were used to obtain the FPKM expression matrix using the Fastp-HISAT2-FeatureCount pipeline. Further weighted gene co-expression network analysis (WGCNA) was performed using the R package WGCNA to screen for genes co-expressed in different tissues and during floral organ development in the two varieties. Figure 1 Light and temperature are key environmental factors influencing the differences in flowering phenotypes between Guibai No. 1 and Tainong No. 1. Transcription factors play important roles in regulating plant growth, development, and environmental signal responses. Previous studies have shown that many members of the BBX gene family are involved in the photoperiodic pathway regulating flowering, while genes such as SVP and FLM play crucial roles in regulating flowering time in response to changes in environmental temperature. Co-expression analysis revealed that multiple BBX gene family members appeared in the module containing the PeSVP gene, suggesting that BBX members may be involved in the regulation of flowering in response to light and temperature.

[0034] 2. Identification of Passion Fruit BBX Gene Family Members and Screening of Candidate Genes (1) Extract all Arabidopsis BBX family protein sequences from the Arabidopsis Information Resource (TAIR) database, and use these sequences as query sequences to search for passion fruit whole genome protein sequences by BLASTP alignment to screen homologous proteins in passion fruit. The E value threshold is set to <1e-5.

[0035] (2) Download the B-box domain model (Pfam00643) from the PFAM database and use HMMER 3.0 software to search for it in the whole genome protein sequence of passion fruit with an E value <1e-20 as the threshold.

[0036] (3) After removing redundancy from the protein sequences obtained by the two methods, the presence of B-box domains was further identified by online tools of NCBI-CDD and SMART databases. Finally, proteins containing B-box domains were retained as candidate BBX family members.

[0037] (4) Multiple sequence alignment of the amino acid sequences of Arabidopsis thaliana AtBBX and passion fruit BBX family was performed using MEGA 7.0 software, and a phylogenetic tree was constructed using the maximum likelihood (ML) method with 1000 Bootstrap repetitions.

[0038] (5) Based on the expression analysis and phylogenetic analysis results of all BBX members, a BBX transcription factor PeBBX24 that was significantly differentially expressed in the leaves of Guibai No. 1 and Tainong No. 1 was selected as the target gene.

[0039] (6) Use qRT-PCR technology to verify the expression pattern of PeBBX24 in different varieties and tissues, with the PeEF1a gene of passion fruit as the reference gene (upstream primer of PeEF1a gene qRT-PCR: TCCCTGACAAAGCAGAAGATG; downstream primer of PeEF1a gene qRT-PCR: GATGTAGCAGCCTACCTGAAAG).

[0040] The results showed that PeBBX24 was predominantly expressed in leaves during both the vegetative and reproductive stages of the adult Tainong No. 1 cultivar, with the highest expression abundance observed in leaves during the vegetative growth stage. Figure 2 A); Comparison results among different varieties showed that the expression of PeBBX24 in the leaves of Guibai No. 1 (GB) was significantly higher than that in the leaves of Tainong No. 1 (TN). Figure 2 B). Furthermore, in the leaves of Tainong No. 1, the expression of PeBBX24 was significantly downregulated after both high and low temperature treatments. Figure 2 C).

[0041] (7) Phylogenetic analysis showed that PeBBX24 is homologous to the Arabidopsis thaliana AtBBX18 gene. Figure 3 Previous studies have shown that AtBBX18 is negatively regulated by heat stress in Arabidopsis thaliana.

[0042] The results showed that PeBBX24 was predominantly expressed in leaves, with significantly higher expression in leaves of Guibai No. 1 (GB) than in leaves of Tainong No. 1 (TN). Furthermore, the expression of PeBBX24 was regulated by temperature, suggesting that this gene may be involved in the regulation of early flowering in Guibai No. 1, which is insensitive to low temperatures in early spring.

[0043] 3. Construction of PeBBX24-506 gene vector (1) Experimental materials: Passion fruit variety Tainong No. 1, planted in the artificial intelligence greenhouse of Fujian Agriculture and Forestry University, with temperature controlled at 25℃, photoperiod of 16h light / 8h dark, light intensity divided into three stages (6:30-7:30 30% i.e. 13000 lx, 7:30-20:30 50% i.e. 23000 lx, 20:30-22:30 30% i.e. 13000 lx), and humidity around 60%. Total RNA was extracted from young leaves.

[0044] (2) RNA extraction: Total RNA was extracted from passion fruit leaves using the Magen HiPure Plant RNA Mini Kit (R4151-03). Specific steps were followed according to the RNA extraction instructions. To prevent RNA contamination, masks and gloves were worn during the experiment. After RNA extraction, integrity was checked using 1.2% agarose gel electrophoresis, and RNA concentration and purity were determined using a micro spectrophotometer.

[0045] (3) Reverse transcription: cDNA was synthesized in vitro using the Novizan HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper) (R312). Gloves and masks were worn during the experiment, and the first step was performed on ice. The specific steps are as follows: 1) The extracted RNA was diluted to a concentration of 10 pg. The 10 pg RNA solution and RNase-free ddH2O were added to a sterile, enzyme-free centrifuge tube, with a total volume of 8 μL. The mixture was heated at 65°C for 5 min, then rapidly cooled on ice and incubated on ice for 2 min to denature the RNA template.

[0046] 2) Add 2 μL of 5×gDNA wiper Mix to the mixture from the previous step and gently mix with a pipette. Then, perform a PCR reaction at 42°C for 2 minutes to remove genomic DNA.

[0047] 3) According to the table below, put all reagents into the same EP tube, gently pipette and mix well to prepare the first-strand cDNA synthesis reaction solution.

[0048]

[0049] 4) The synthesis reaction solution was carried out at 25℃ for 5 min; 37℃ for 45 min; 85℃ for 5 sec to synthesize the first-strand cDNA. The synthesized cDNA was stored at -20℃ to avoid repeated freeze-thaw cycles.

[0050] (4) Gene cloning: Using cDNA as a template, primers pentr-PeBBX24-F (SEQ ID NO.3): 5'-ATGCGAACGCTATGCGAC-3' and pentr-PeBBX24-R (SEQ ID NO.4): 5'-CTTCTCAGGCTCCGCTTTAAAG-3' were designed using the full-length sequence. Amplification was performed using cDNA as a template with Hieff Canace® Plus PCR Master Mix (With Dye) (Yesen, 10154ES08, Shanghai, China). The procedure is described in the included instruction manual.

[0051] (5) 50 μL reaction system:

[0052] (6) Reaction procedure: Pre-denaturation 98℃, 3min; Denaturation: 98℃, 10sec; Extension: 68℃, 20sec; Cycle the denaturation to extension process 30-35 times; Final extension 72℃, 5min, then the reaction is terminated.

[0053] (7) DNA electrophoresis and recovery: After the reaction, all products were added to a 1% agarose gel for electrophoresis at 140V for 15min. The gel was then placed in a gel imaging system for imaging. A band of the same size as the target gene fragment was cut out and recovered using the Tiangen DNA Purification Gel Recovery Kit (Tangen, DP214, Beijing, China). For specific steps, please refer to the kit instructions.

[0054] (8) Sequence analysis: In-Fusion cloning technology was used, and OK Clon enzyme (Aikerui, Fuzhou, China) was used to cleave the target fragment with the entry vector (pENTR). TM Ligation and transformation were performed using pENTR™ / D-TOPO®. 1 μL of linearized pENTR™ / D-TOPO® vector, 1 μL of recovered target gene DNA fragment, 1 μL of OK Clonase, and 1 μL of ddH2O were added to a 200 μL PCR tube. The mixture was then pipetted to mix thoroughly, briefly centrifuged to allow the mixture to settle at the bottom of the PCR tube, and ligated in a 50°C metal bath for 1 hour. The mixture was then transformed into E. coli DH5α competent cells, and positive clones were selected for sequencing.

[0055] (9) Plasmid extraction: Use a pipette to aspirate the correctly sequenced bacterial culture into a 50 mL centrifuge tube containing 10 mL of Kan+ for propagation. After 12 h, collect the bacteria and extract the plasmid. Use the Megen HiPure Plasmid MicroKit (rapid type) rapid plasmid mini-prep kit to extract the plasmid (Megen, P1001, Guangdong, China). Refer to the kit instructions for specific steps.

[0056] (10) Construction of the final vector: The final expression vector selected was pGWB506 with a GFP (green fluorescent protein) tag, and Gateway® LRClonase was used. TM II. The EnzymeMix (Invitrogen, USA) kit was used for vector construction. The LR reaction system is shown in the table below. The recombinant intermediate vector, the final vector plasmid, and the LR enzyme were added to the PCR tube, mixed, and then incubated for 3 hours at 22°C in a metal bath. The tube was then transformed in E. coli using the heat shock method.

[0057]

[0058] The culture was spread on solid medium containing spectinomycin (Spec+) and incubated upside down at 37°C. The next day, single clones were picked for bacterial PCR identification. Bacterial cultures with the correct band size were selected for propagation, and the final recombinant vector plasmid was extracted.

[0059] (11) Freeze-thaw transformation of Agrobacterium: 1) Add the final vector containing the target gene into Agrobacterium GV3101, and sequentially place it on ice for 5 min, in liquid nitrogen for 5 min, in a water bath at 37℃ for 5 min, and on ice for 5 min. Then add 600 μL of blank LB liquid medium and revive in a shaker at 220 rpm and 28℃ for 2-3 h. 2) Centrifuge at 5000 rpm for 3 min to collect bacteria, spread them on solid culture medium containing spectinomycin (Spec+) and rifampin (Rif+) in a clean bench, and incubate upside down in an incubator at 28°C. 3) After 2-3 days, pick single colonies for bacterial PCR identification; add an equal amount of 50% glycerol to Agrobacterium containing the target fragment, mix well, and then freeze in liquid nitrogen and store at -80℃.

[0060] Sequencing results showed that the PeBBX24 gene coding region is 618 bp in length, with the nucleotide sequence shown in SEQ ID NO.1, encoding 205 amino acids, with the amino acid sequence shown in SEQ ID NO.2.

[0061] In this invention, the coding region nucleotide sequence (SEQ ID NO.1) of the PeBBX24 gene is as follows: ATGCGAACGCTATGCGACGTGTGCGAAAGCGCAGCCGCCATCCTTTTTTGCGCCGCCGATGAGGCTGCTCTTTGCCGTGCCTGTGACGAGAAGGTCCACTTGTGTAATAAGCTTGCTAGTCGACATGTACGTGTAGGGCTTGCTGATCCCAGTGATGTACCCCGTTGTGACATATGCGAAAGTGCACCAGATGGTAGTTCCCTCTGCCTGCAATGTGATACGATTGTGCATGTTGGCGGTAAAAGAACCCACGGTAGATATCTCCTATTGAGGCAGAGAATCGAGTTTCCAGGTGACAAACCTGGTCGTTCAGAGGAGCTAGGGCAGCAGCTGCTTGATCATAATGAAACAAAGAGGGACCAAACTCAATCACCTAAATTCGCATTGAGAGAAAATCAACATAACTACCAGGCATCTTATACTCCGATGTTGGAGAATAACACTGATGGTGATGGAAAAATGGACAATAAGTTGATAGATCTTAATACCAGGCCGCAGCGTGTATATGGACAAAATTCAACTAACCAGGAACATCGCCTAGATGTCATCAGTGGTGCCAATGGTGACTCTCCAGGAATGTTTCCTGTTGGACCCTTTAAAGCGGAGCCTGAGAAGTGA The protein sequence encoded by the PeBBX24 gene (SEQ ID NO.2) is as follows: MRTLCDVCESAAAILFCAADEAALCRACDEKVHLCNKLASRHVRVGLADPSDVPRCDICESAPDGSSLCLQCDTIVHVGGKRTHGRYLLLRQRIEFPGDKPGRSEELGQQLLDHNETKRDQTQSPKFALRENQHNYQASYTPMLENNTDGDGKMDNKLIDLNTRPQRVYGQNSTNQEHRLDVISGANGDSPGMFPVGPFKAEPEK Other PeBBX24-related sequences involved in this invention are as follows: PeBBX24 gene cloning upstream primer (SEQ ID NO.3): ATGCGAACGCTATGCGAC PeBBX24 gene cloning downstream primer (SEQ ID NO.4): CTTCTCAGGCTCCGCTTTAAAG PeBBX24 gene qRT-PCR upstream primer (SEQ ID NO.5): AGGCCGCAGCGTGTATATG PeBBX24 gene qRT-PCR downstream primer (SEQ ID NO.6): CTGGAGAGTCACCATTGGCA 4. Transcriptional activity analysis of the PeBBX24 gene To clarify the transcriptional activation function of the PeBBX24 gene-encoded protein, a recombinant bait vector, pGBKT7-PeBBX24, was constructed using a yeast two-hybrid system. After transformation into competent yeast cells, transcriptional activity was detected. The specific steps are as follows: (1) Construction of recombinant vector: The full-length CDS sequence of PeBBX24 was constructed into the yeast expression vector pGBKT7(BD), and primers pGBKT7-PeBBX24-F were designed: 5'- catatggccatggaggccgaattc ATGCGAACGCTATGCGAC-3' (the underlined portion is the homologous arm of the EcoRI restriction site in the pGBKT7 vector, and the last 18 bp is the upstream specific sequence of the PeBBX24 gene clone shown in SEQ ID NO.3, covering the ATG start codon) and pGBKT7-PeBBX24-R: 5'- agttatgcggc cgctgcaggtcga TCACTTCTCAGGCTCCGCTTTA-3' (the underlined part is the homologous arm of the Sal1 restriction site of the pGBKT7 vector, and the last 22 bp is the downstream specific sequence of the PeBBX24 gene clone shown in SEQ ID NO.4, covering the stop codon) is ligated into the restriction-digested pGBKT7 vector via in-fusion cloning.

[0062] (2) Yeast transformation: The constructed pGBKT7-PeBBX24 vector and the empty vector pGADT7 (AD) were co-transformed into competent yeast cells (Y2HGold). At the same time, the negative control plasmid combination pGADT7-T / pGBKT7-Lam and the positive control plasmid combination pGADT7-T / pGBKT7-53 were also transformed.

[0063] (3) Screening and identification: The transformed yeast strains were spread on a two-deficient medium (SD / -Leu-Trp) and incubated upside down in a constant temperature incubator at 30℃ for 3 days to observe the growth. The yeast strains with good growth were further transferred to a three-deficient medium (SD / -Leu-Trp-His) containing X-alpha-Gal to test whether they could activate the MEL1 reporter gene system in yeast cells. If the MEL1 reporter gene system in yeast cells could be activated, different concentrations of 3-AT (3-amino-1,2,4-triazole) were added to the three-deficient medium to screen for the lowest concentration that inhibited self-activation.

[0064] The results showed that yeast strains containing the BD-PeBBX24 protein vector grew well on the 2-deficient medium (SD / -Leu-Trp) and exhibited the same growth status as the positive control, indicating that the BD-PeBBX24 protein was non-toxic to the yeast strains. On the 3-deficient medium, yeast strains carrying the empty AD and BD-PeBBX24 plasmids grew normally, and on the 3-deficient medium containing X-alpha-Gal, yeast strains expressing the BD-PeBBX24 protein showed a blue mark, indicating that BD-PeBBX24 has the transcriptional activation ability to activate the reporter gene system in yeast. Figure 4 A).

[0065] 5. Subcellular localization analysis of PeBBX24 To determine the expression location of PeBBX24 protein in plant cells, the PeBBX24 gene fragment was constructed into the final vector pGWB506 to obtain the fusion expression vector p35S::PeBBX24-GFP. These fusion expression vectors and the empty vector control p35S::GFP were then transformed into Agrobacterium GV3101 using a freeze-thaw method. The Agrobacterium containing the target gene fragment was transferred to 10 mL of liquid medium containing Spec+ and Rif+, cultured overnight, and then collected. An infection solution was prepared to suspend the Agrobacterium at the bottom (10 mM morpholinoethanesulfonic acid, 100 µM acetylsalicylic acid, 10 mM MgCl2) and injected into the underside of leaves of three- to four-week-old Nicotiana benthamiana. The injected tobacco was then cultured in the dark in a greenhouse for 24 hours, followed by normal light cultivation. Appropriately sized sections of injected Nicotiana benthamiana were cut and stained with DAPI working solution. GFP fluorescence signals were detected using a laser scanning confocal microscope (Leica TCSSP8XDLS).

[0066] The results showed that, after injecting tobacco leaves, fluorescence observation under a laser confocal microscope revealed that the empty vector pGWB506 (p35S::GFP) was expressed in both the nucleus and cell membrane, while the fluorescence signal of p35S::PeBBX24-GFP was mainly distributed in the nucleus. Figure 4B), which conforms to the location characteristics of transcription factors.

[0067] Example 2: Effect of the PeBBX24 gene on flowering time in Arabidopsis thaliana To verify the regulatory role of the PeBBX24 gene on flowering time in plants, Arabidopsis thaliana (Col-0 ecotype) was used as experimental material. Transgenic plants overexpressing the PeBBX24 gene were constructed. By observing the differences in flowering phenotype between transgenic and wild-type plants, and combining the expression levels of flowering-related genes, the effect of the PeBBX24 gene on flowering time in Arabidopsis thaliana was clarified. The specific steps are as follows: (1) Infection of Arabidopsis thaliana: The p35S::PeBBX24-GFP vector was transformed into GV3101 competent cells via freeze-transformation. Single clones were picked for identification. Agrobacterium with the target gene fragment was transferred to 25 mL of LB liquid medium containing Spec+ and Rif+. After overnight culture, the bacteria were collected by centrifugation at 4000xg for 10 min. 50 mL of infection solution was prepared for each gene to suspend Agrobacterium at the bottom (MS powder: 0.111 g, sucrose: 2.5 g, ddH2O: 50 mL, Silweet-77: 20 µL). This was used to infect 4-week-old Arabidopsis thaliana with good growth. After the infection was completed, Arabidopsis thaliana was cultured in the dark for 24 h, then placed in a greenhouse for normal culture until the seeds matured. The seeds of the T0 generation were collected, dried at 37℃, and then stored at room temperature.

[0068] (2) Seed screening of transgenic Arabidopsis thaliana: A suitable amount of seeds were disinfected with 75% ethanol for 5 minutes, then washed with 95% ethanol for 15 seconds. The disinfected seeds were then dried on a blank plate. The dried seeds were then evenly sown on 1 / 2 MS solid medium containing hygromycin resistance and cultured at 4℃ in the dark for 3 days. Afterward, they were transferred to an artificial intelligence greenhouse for further cultivation. After about one week of cultivation, resistant seedlings with two cotyledons, roots, and a consistently green appearance were selected and transplanted into moist nutrient soil. Care was taken not to break the plant roots during transplanting. Tap water was sprayed onto the soil surface, and plastic wrap was used to maintain moisture. The film was removed every 2-5 days depending on the seedling growth. Regular watering was maintained to keep the soil moist. Arabidopsis DNA was extracted using the CTAB method. Positive plants were identified using PCR and continued to be cultured. Once the positive plants matured, T1 generation seeds were harvested from each plant. The Arabidopsis thaliana used in the experiment were all grown in an AI-controlled greenhouse, with the temperature controlled at 23 ℃, the photoperiod at 16 hours of light and 8 hours of darkness, and the humidity at approximately 70%.

[0069] (3) To determine whether PeBBX24 is involved in plant flowering, the recombinant plasmid 35S::PeBBX24 was transformed into wild-type Arabidopsis thaliana using the Arabidopsis flower dipping method. Colombian wild-type Arabidopsis thaliana (Col-0, WT) was used as a negative control. Based on the traits of the Arabidopsis transgenic lines, three T3 homozygous positive transgenic lines were selected from the PeBBX24 transgenic lines for further analysis.

[0070] The measurement results are as follows Figure 5 As shown, Figure 5 Image A shows an image taken on day 4 of normal growth of the PeBBX24 transgenic lines (OE-1, OE-2, and OE-3). The scale bar in the image is 0.5 cm. Compared with the control, the root length of the transgenic plants is significantly longer than that of the wild type. Figure 5 B represents data from 3-6 days of plant growth. Statistical analysis shows that the growth rates of the PeBBX24 transgenic lines (OE-1, OE-2, and OE-3) were 35.54%, 35.78%, and 44.02%, respectively, which were higher than those of the wild type (34.04%). Figure 5 The results showed that the PeBBX24 transgenic lines were not significantly different from the wild type at 15 days, and by 22 days the transgenic plants had begun to bolt and flower, while the control wild type did not bolt and produce flower buds until around 27 days. All transgenic lines flowered about 5-6 days earlier than the control plants. These data indicate that overexpression of PeBBX24 in Arabidopsis thaliana can promote early flowering.

[0071] (4) To better understand the molecular mechanism by which PeBBX24 participates in the regulation of flowering time, cDNA was extracted from wild-type and PeBBX24 transgenic Arabidopsis thaliana lines at different flowering days as templates, and qRT-PCR was used to analyze genes related to flowering regulation in Arabidopsis thaliana. Six genes involved in determining flowering time and flower development were searched from the Arabidopsis thaliana flowering database. Expression analysis of wild-type and overexpression seedlings at different flowering days revealed that the expression levels of flowering promoting factors CO, FT, SOC1, and FUL increased with flowering in Arabidopsis thaliana and were higher than those of WT. Conversely, the expression levels of flowering inhibiting factors SVP and FLC showed the opposite trend. Figure 6 These results further confirm that PeBBX24 promotes flower formation in Arabidopsis by regulating the expression of other flowering genes, suggesting that PeBBX24 may be an important gene promoting passion fruit flowering.

[0072] The primer sequences for quantitative analysis of key genes related to flowering in Arabidopsis thaliana are as follows: HK2 gene qRT-PCR upstream primer: GATTGGTGTCGCTGCTAGTCTC HK2 gene qRT-PCR downstream primer: AGAATTTGTGCCTCTTCGCTCTG AtCO gene qRT-PCR upstream primer: CGAGCTACGGGGGAGATAGA AtCO gene qRT-PCR downstream primer: GGATGAAATGTATGCGTTATGGT AtFT gene qRT-PCR upstream primer: AACGACGGTGATGATGCCT AtFT gene qRT-PCR downstream primer: TCCCACTGCCTGATTTTTGC AtSOC1 gene qRT-PCR upstream primer: TCGAGGAGCTGCAACAGATT AtSOC1 gene qRT-PCR downstream primer: GCTAGAGCTTTCTCCTTTTGCT AtFUL gene qRT-PCR upstream primer: ACGGGTCAGCAAGAAGGACAATTAG AtFUL gene qRT-PCR downstream primer: GCCGGAAGCAGAGAGTTTGGTTCCG AtSVP gene qRT-PCR upstream primer: ACCGGAAAACTGTTCGAGTTC AtSVP gene qRT-PCR downstream primer: TTAGTCGGTGGCTCTTGTCC AtFLC gene qRT-PCR upstream primer: GGCTAGCCAGATGGAGAATAATCA AtFLC gene qRT-PCR downstream primer: GTCACCGGAAGATTGTCGGA (5) Phenotypic observation of Arabidopsis thaliana under different day treatments: Arabidopsis thaliana plants that were 12 days old were selected and subjected to medium day (ND), short day (SD), and long day (LD) treatments. After 7 and 8 days of medium and long day treatment, the plants overexpressing PeBBX24 bolted and had flower buds. After 10 days of short day treatment, the plants overexpressing PeBBX24 bolted and had flower buds. Wild-type Arabidopsis thaliana plants in the same period did not bolt. Figure 7 After 20 days of treatment, it can be seen that the wild-type plants have bolted and flowered, while the PeBBX24-overexpressing plants in the same period of treatment have already produced siliques, indicating that the PeBBX24-overexpressing plants flower earlier than the wild-type Arabidopsis under medium-long-short day treatment.

[0073] (6) Figure 8A represents the expression level of the PeBBX24 gene in passion fruit plant leaves after treatment at 45℃ for 12h, 24h, and 48h, relative to the expression level before treatment. The relative expression levels of the PeBBX24 gene in passion fruit plants after 12h, 24h, and 48h were 0.04, 0.07, and 0.46, respectively. Figure 8 B represents the observation of Arabidopsis thaliana phenotypes under high-temperature stress: On the 15th day of Arabidopsis growth, WT and PeBBX24-overexpressing lines were subjected to 43℃ heat stress for 12h, 24h, and 48h, respectively, before being returned to the greenhouse to resume growth. Growth was observed and recorded on the 7th day of treatment (the 22nd day of Arabidopsis growth). Results showed that under normal conditions, the heading time of PeBBX24-overexpressing Arabidopsis plants was earlier than that of wild-type Arabidopsis. After heat treatment, the early flowering phenotype of PeBBX24-overexpressing Arabidopsis plants was gradually suppressed, and both wild-type and mutant plants died after 48h of heat treatment.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A passion fruit PeBBX24 gene, characterized in that, The nucleotide sequence of the coding region of the passion fruit PeBBX24 gene is shown in SEQ ID NO.

1.

2. A protein encoded by the passion fruit PeBBX24 gene as described in claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. An application of the passion fruit PeBBX24 gene as described in claim 1 in regulating plant flowering time, characterized in that, The application is to promote early flowering in plants.

4. The application of the passion fruit PeBBX24 gene in regulating plant flowering time according to claim 3, characterized in that, The plants mentioned include Arabidopsis thaliana or passion fruit.

5. A recombinant expression vector containing the passion fruit PeBBX24 gene as described in claim 1, characterized in that, The recombinant expression vector contains the complete coding sequence of the passion fruit PeBBX24 gene; the complete coding sequence of the passion fruit PeBBX24 gene was obtained by amplification using the primers shown in SEQ ID NO.3 and SEQ ID NO.

4.

6. The recombinant expression vector containing the passion fruit PeBBX24 gene according to claim 5, characterized in that, The recombinant expression vector uses the plant expression vector pGWB506 as the original vector; the transcription of the passion fruit PeBBX24 gene is driven by the p35S promoter.

7. A recombinant engineered bacterium containing the PeBBX24 gene of passion fruit as described in claim 1, characterized in that, The recombinant engineered bacteria comprises the recombinant expression vector as described in claim 5 or claim 6.

8. A primer set for quantitative PCR of the passion fruit PeBBX24 gene as described in claim 1, characterized in that, The primer set includes primers as shown in SEQ ID NO.5 and SEQ ID NO.

6.

9. A method for cultivating an early-flowering transgenic plant, characterized in that, Includes the following steps: (1) Using passion fruit leaf cDNA as a template, the complete coding sequence of the passion fruit PeBBX24 gene was amplified using primers shown in SEQ ID NO.3 and SEQ ID NO.4, and the coding sequence is shown in SEQ ID NO.1; (2) The coding sequence of the passion fruit PeBBX24 gene obtained in step (1) is ligated with the plant expression vector to construct a recombinant expression vector; (3) The recombinant expression vector obtained in step (2) is transformed into the target plant, and transgenic plants with overexpression of the PeBBX24 gene of passion fruit are obtained by screening.

10. The method for cultivating early-flowering transgenic plants according to claim 9, characterized in that, In step (3), the transformation is performed using Agrobacterium, strain GV3101 mediated by the flower immersion method; The screening included: resistance screening on 1 / 2 MS solid medium containing 50 mg / mL hygromycin, and PCR molecular identification of transgenic plants using primers shown in SEQ ID NO.3 and SEQ ID NO.4.

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