Application of the RcTPS1a gene in the rose 'Monthly Red' in regulating plant flowering time

Abstract

This invention discloses the application of the RcTPS1a gene in the rose 'Monthly Red' in regulating the flowering time of plants, belonging to the field of plant genetic engineering technology. The nucleotide sequence of the RcTPS1a gene is shown in SEQ ID NO.1. Overexpression of the RcTPS1a gene can promote the flowering time of Arabidopsis thaliana, while transient silencing of this gene in 'Monthly Red' delays flowering. Overexpression of this gene promotes flowering, demonstrating its function in promoting flowering.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically to the application of the 'Monthly Red' RcTPS1a gene in regulating the flowering time of plants. Background Technology

[0002] 'Rosa chinensis'Slater's Crismson China' is a plant belonging to the genus Rosa in the family Rosaceae. It possesses the trait of continuous flowering year-round, making it highly valuable for its ornamental and economic purposes. Therefore, understanding the functional genes controlling flowering is of significant theoretical and practical value for controlling the flowering period and achieving year-round ornamental value. Current research on rose flowering regulation mainly focuses on the formation of its continuous flowering trait and the photoperiodic and gibberellin pathways. The age-effect pathway and the molecular mechanisms by which the TPS gene regulates rose flowering remain unclear.

[0003] Trehalose-6-phosphate synthase (TPS) is a key gene for the synthesis of trehalose in plants. The TPS1 gene is a core member of the TPS gene family. It has been shown to have functions such as stress resistance and promoting flowering in model plants, but its function in other higher plants needs further research.

[0004] Therefore, providing the application of the 'Monthly Red' RcTPS1a gene in regulating the flowering time of plants is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides the application of the 'Monthly Red' RcTPS1a gene in regulating the flowering time of plants. Transiently silencing the 'Monthly Red' RcTPS1a gene can prolong the time from sowing to flowering, while overexpressing the 'Monthly Red' RcTPS1a gene can shorten the time from sowing to flowering. This demonstrates that the RcTPS1a gene has the function of regulating the flowering period of roses, thus solving the problem of inconsistent flowering time affecting variety breeding and agricultural production in practical applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The RcTPS1a gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence encoded by the RcTPS1a gene is shown in SEQ ID NO.2.

[0009] Furthermore, an overexpression vector pCambia1305-RcTPS1a contains the RcTPS1a gene.

[0010] Furthermore, a gene silencing vector pTRV2-RcTPS1a contains the RcTPS1a gene.

[0011] Furthermore, a recombinant engineered bacterium contains the overexpression vector pCambia1305-RcTPS1a or the gene silencing vector pTRV2-RcTPS1a.

[0012] Furthermore, the application of the RcTPS1a gene, the amino acid sequence encoded by the RcTPS1a gene, the overexpression vector pCambia1305-RcTPS1a, the gene silencing vector pTRV2-RcTPS1a, or the recombinant engineered bacteria in regulating plant flowering time.

[0013] Furthermore, the application of the RcTPS1a gene, the amino acid sequence encoded by the RcTPS1a gene, the overexpression vector pCambia1305-RcTPS1a, the gene silencing vector pTRV2-RcTPS1a, or the recombinant engineered bacteria in plant breeding.

[0014] Furthermore, the plants mentioned are Arabidopsis thaliana and the rose 'Monthly Red'.

[0015] The 'Monthly Red' flowering gene RcTPS1a provided by this invention can be used for the improvement and breeding of roses.

[0016] By transiently silencing the gene RcTPS1a, flowering can be delayed, thereby breeding late-flowering rose varieties to meet the market's demand for multi-season supply.

[0017] The 'Monthly Red' flowering gene RcTPS1a provided by this invention can also be used to regulate the flowering period of ornamental plants, providing a method to delay the flowering time of ornamental plants by transiently silencing this gene.

[0018] Overexpression of the gene RcTPS1a can advance flowering, thereby shortening the breeding cycle and improving breeding efficiency.

[0019] The 'Monthly Red' flowering gene RcTPS1a provided by this invention can also be used to regulate the flowering period of ornamental plants, and overexpression of this gene in ornamental plants can advance the flowering time of the plants.

[0020] As can be seen from the above technical solution, compared with the prior art, this invention discloses the application of the RcTPS1a gene in the rose variety 'Monthly Red' in regulating the flowering time of plants. It is the first time that the RcTPS1a gene in the rose variety 'Monthly Red', which has the function of regulating flowering time, has been disclosed, thus promoting the annotation of its gene function. Overexpression of the RcTPS1a gene can promote the flowering time of Arabidopsis thaliana, while transient silencing of the gene in 'Monthly Red' delays flowering. Overexpression of the gene promotes flowering, demonstrating that the gene has a function of promoting flowering. In traditional breeding, this can shorten breeding time and improve breeding efficiency. Furthermore, by transferring the RcTPS1a gene into ornamental plants for overexpression or transient silencing, the flowering period of the plant can be artificially regulated, which has social and economic value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The attached figure shows the subcellular localization of the protein encoded by the RcTPS1a gene of this invention.

[0023] Wherein: Bright: bright field of view; EGFP: GFP fluorescence; DsRed: cell membrane marker fluorescence; Merge: superimposed field of view;

[0024] Figure 2 The attached figure shows the flowering phenotype of the Arabidopsis T3 transgenic strain overexpressing the RcTPS1a gene according to this invention; photographed on the 40th day after sowing.

[0025] Figure 3 The attached figure shows the expression levels of flowering regulation-related genes in transgenic Arabidopsis thaliana overexpressing the RcTPS1a gene according to this invention;

[0026] Figure 4 The attached figure shows the expression levels of flowering regulation-related genes in 'Moonlight Red' cultivar with transient RcTPS1a gene silencing according to the present invention; where 1, 2, and 3 refer to 3 'Moonlight Red' plants with transient RcTPS1a silencing.

[0027] Figure 5 The attached figure shows the flowering phenotype of 'Monthly Red' variegated by transient silencing of the RcTPS1a gene according to this invention; photographed on day 58 post-infection.

[0028] Figure 6The attached figure shows the expression levels of flowering regulation-related genes in 'Monthly Red' that overexpress the RcTPS1a gene according to this invention; where A, E, and F refer to three 'Monthly Red' plants that overexpress the RcTPS1a gene.

[0029] Figure 7 The attached figure shows the flowering phenotype of 'Monthly Red' cultivar overexpressing the RcTPS1a gene according to this invention; photographed on day 49 post-infection. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The RcTPS1a CDS sequence (2934bp) is shown in SEQ ID NO.1.

[0032] GGAATTGTGAATGCTTGG AGGCCAACATTAATGCGCGATAGAATGTCTCTGCATGAAGGTTCTTCTGTGCTTGACCTCAAGGGTGACAACTACTTCTCTTGTGCTGTTGGGAGAAAGCGATCCAGTGCAAGATATCTACTTAAATCGTCAGACGATGTCGTTACTCTTTTAAAAGAGCTTGCTGAAGGTGGAGAACCAATGCCAGTCGTCTCGGAAAGTGAAGCAATGCCTCTCAACTCAGACGGTGTAGCAGTGCCTCTCGACTCAGAAGGCGTAGCACTGCCTCTCGACTCAGAAGGTGTAGTGCCTCTCAACTCAGAAGTTGGAGAACAGAGCACTGAGAATATCTAG; SEQ ID NO.1.

[0033] The amino acid sequence of RcTPS1a is shown in SEQ ID NO.2.

[0034] MPGNKYNCNPSTPRSRLERLLRERELRRSNLASQPNEGANRQAELSEYFLTDGENLALSGDEDFADGVAAARAFVDGCERQEGRPYKQRLLVVANRLPVSAVRKGEDSWQLEISVGGLVSALLGVKEFDARWIGWAGVNVPDSVGQKALTKALAEKRCIPVFLDEEIVHQYYNGYCNNILWPLFHYLGLPQEDRLATTRSFQSQFDAYKKANQMFADVVNEHYEDGDVVWCHDYHLMFLPKCLKEHNSKMKVGWFLHTPFPSSEIHRTLPSRSELLRSVLAADLVGFHTYDYARHFVSACTRILGLEGTPEGVEDQGKLTRVAAFPIGIDSDRFIRALELPQVQEHMK ELKERFAGRKVMLGVDRLDMIKGIPQKILAFEKFLEENLNWRDKVVLLQIAVPTRTDVPEYQKLTSQVHEIVGRINGRFGTLTAVPIHHLDRSLDFHALCALYAVTDVALVTSLRDGMNLVSYEFVACQASKKGVLILSEFAGAAQSLGAGAILVNPWNITEVAASIGYALNMPADEREKRHHHNFMHVTTHTSQEWAATFVSELNDTIVEAQLRTRQVPPLLPIKGSVDRYFQSSNRLLILGFNATLTEPKDTLGRRGGQIREMELKLHPDLKEPLKKLCNDSKTTIVVLSGSDRSVLDYNFGDYNMWLAAENGMFLRLTTGEWMTTMPENLNMDWVDSVKHVFEYFTERTPRSHFELRETSLVWNYKYADIEFGRLQARDLLQHLWTGPISNASVDVVQGARSVEVRAVGVTKGAAIDRILGEIVHNKGMKAPIDYVLCIGHFLPKDEDLYTFFEPELPCEVPVQTIPRPTSVPTPVNPSLPKISTGKSGSKGSRLKKQRSLSTLEKRANIGIVNAWRPTLMRDRMSLHEGSSVLDLKGDNYFSCAVGRKRSSARYLLKSSDDVVTLLKELAEGGEPMPVVSESEAMPLNSDGVAVPLDSEGVALPLDSEGVVPLNSEVGEQSTENI;SEQ ID NO.2。

[0035] Example 1

[0036] 1) Obtaining the target gene

[0037] The rose variety 'Monthly Red', grown at the Hunan Agricultural University Flower Base, was selected, and total RNA was extracted from fresh young leaves of its ground-grown seedlings. An RNA extraction kit (Hunan Aikerui Biotechnology Co., Ltd., AG21019) was used, and the procedure was performed according to the included instructions. After RNA extraction, the concentration was determined, and integrity was checked using 1% agarose gel electrophoresis. Using the extracted RNA as a template, cDNA synthesis was performed according to the instructions for the Aikerui Biotechnology cDNA synthesis kit (AG11615), following the included instructions.

[0038] 2) Cloning of the target gene

[0039] Primers for target gene amplification were designed using Primer 5.0 software. Using 'Yueyuehong' cDNA as a template, the target gene was amplified using P505 high-fidelity enzyme (Nanjing Novizan Biotechnology Co., Ltd., P505-d1). The specific amplification primer sequences are as follows:

[0040] KL-RcTPS1a-F: 5'-ATGCCAGGAAACAAGTATAACTGC-3'; SEQ ID NO.3;

[0041] KL-RcTPS1a-R:5'-TGCTAT CTAGATATTCTCAGTG -3';SEQ ID NO.4.

[0042] Amplification system: 2×PhantaMax Buffer 25μl, dNTP Mix 1μl, KL-RcTPS1a-F 2μl, KL-RcTPS1a-R 2μl, PhantaMax Super-Fidelity DNA Polymerase 1μl, cDNA 4μl, ddH2O 15μl. Reaction program: 95℃ for 3 min; 95℃ for 15 s, 56℃ for 15 s, 72℃ for 3 min, 35 cycles; 72℃ for 5 min; 4℃.

[0043] Prepare a 1% agarose gel and run electrophoresis to determine whether the RcTPS1a gene has been successfully amplified.

[0044] Add 10×DNALouding Buffer to the amplification product at a ratio of 10:1 (amplification product: 10×DNALouding Buffer), and mix thoroughly by pipetting. Prepare a 1% agarose gel, add all the sample to the wells, and incubate at 220V for 25 minutes. Purify the RcTPS1a gene amplification product according to the instructions of the Novozymes gel extraction kit (DC301), and store the purified product at -20℃.

[0045] Following the instructions of Tiangen Biotech's pLB cloning kit (VT205), the purified target gene product was ligated into the pLB cloning vector. The ligation product was then transformed into *E. coli* using the instructions of Weidi Biotech's DH5α competent cells (DL1001). After transformation, the bacterial culture was evenly spread on LB agar containing 50 mg / L Amp antibiotic and incubated upside down at 37°C for 14 h. Single colonies were picked and PCR amplified using 2× Rapid TaqMasterMix enzyme. The primer sequences are as follows:

[0046] pLB-F: 5'-CGACTCACTATAGGGAGAGCGGC-3'; SEQ ID NO.5;

[0047] pLB-R: 5'-AAGAACATCGATTTTCCATGGCAG-3'; SEQ ID NO. 6.

[0048] Amplification system: 12.5 μl of 2×Rapid TaqMasterMix, 1 μl of pLB-F, 1 μl of pLB-R, single colony, 10.5 μl of ddH2O. Reaction program: 95℃ for 3 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 45 s, 35 cycles; 72℃ for 5 min; 4℃.

[0049] Prepare a 1% agarose gel and run electrophoresis to determine whether the RcTPS1a gene has been successfully ligated to the pLB cloning vector.

[0050] In a clean bench, add 5 ml of LB liquid medium and 5 μl of 50 mg / mL Amp to the shaker tube. Then, use a pipette tip to pick up the identified positive single colonies and add them to the shaker. Incubate at 37°C and 200 rpm for 14 h. In a clean bench, transfer 300 μl of different positive single clone cultures to 1.5 ml centrifuge tubes, label them, add primers (pLB-F, pLB-R), and send them to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0051] In a clean bench, preserve the successfully sequenced positive monoclonal bacterial culture at a ratio of bacterial culture to 50% glycerol of 1:1.

[0052] Take the successfully sequenced bacterial culture and extract the pLB-RcTPS1a plasmid according to the instructions of Novizan Biosciences' plasmid extraction kit (DC201). After successful plasmid extraction, measure and record the concentration, and store at -20℃.

[0053] 3) Determine the expression location of the RcTPS1a gene using subcellular localization.

[0054] (1) Construction of the carrier:

[0055] Expression vectors were constructed using homologous recombination. The pBI121-EGFP vector, labeled with GFP (green fluorescent protein), was selected and digested with the restriction endonuclease BamHI. After digestion, the results were detected by gel electrophoresis, and the linearized vector was recovered from the gel. Primers with BamHI restriction sites and corresponding homologous arms were designed using Primer 5.0. The stop codon was removed from the reverse primer. The primer sequences are as follows:

[0056] pBI121-RcTPS1a-F: 5'-ACGGGGGACTCTAGAGGATCC ATGCCAGGAA ACAAGTATAACTGC -3';SEQ ID NO.7;

[0057] pBI121-RcTPS1a-R1:5'-ACTGACCACCCGGGGATCC GATATTCTCAGT GCTC -3';SEQ IDNO.8.

[0058] PCR amplification was performed using the designed primers and the previously constructed pLB-RcTPS1a vector as a template. The amplification system consisted of: 2×PhantaMax Buffer 25 μl, dNTP Mix 1 μl, pBI121-RcTPS1a-F 2 μl, pBI121-RcTPS1a-R1 2 μl, PhantaMax Super-Fidelity DNA Polymerase 1 μl, pLB-RcTPS1a 1 μl, and ddH2O 18 μl. The reaction program was: 95℃ for 3 min; 95℃ for 15 s, 70℃ for 15 s, 72℃ for 3 min, 35 cycles; 72℃ for 5 min; 4℃. The amplification results were detected by gel electrophoresis, and the target fragment was recovered from the gel. The target fragment was ligated into the linearized vector using SE homologous recombination ligase (ZC231) from Beijing Zhuangmeng International Biotechnology Co., Ltd. The ligation system and reaction conditions were specified in the kit instructions. The ligation product was transformed into DH5α Escherichia coli. After colony PCR, the bacteria were shaken and sequenced. After successful sequencing, the plasmid pBI121-EGFP-RcTPS1a-1 was extracted.

[0059] (2) Leaf injection method for tobacco infection:

[0060] After sowing tobacco in soil, place it in a refrigerator at 4℃ for 2 days, then place it in an incubator with 16 hours of light / 8 hours of darkness. After about 15 days, transplant it into seedling trays. When the tobacco has grown 6-8 true leaves, carry out infection.

[0061] Using a pipette tip, inoculate the preserved bacterial suspension containing pBI121-EGFP-RcTPS1a-1 or pBI121-EGFP (as a control) onto YEB + 50 mg / L Kan + 50 mg / L Rif plates and incubate at 28°C for 45 h. Pick a single colony and incubate in 5 ml of YEB liquid medium (containing 50 mg / L Kan and 50 mg / L Rif) at 28°C on a shaker for 24 h. Then, take 1 ml of the bacterial suspension and incubate in 50 ml of YEB liquid medium (containing 50 mg / L Kan and 50 mg / L Rif) at 28°C on a shaker for 12 h. Observe the OD of the bacterial suspension. 600 When the concentration of the bacterial culture reached 1.2, the culture was aliquoted into 50 ml centrifuge tubes, centrifuged at 6000 rpm for 8 min, the supernatant was discarded, and the culture was resuspended in resuspension buffer (10 mM MES + 10 mM MgCl2 + 0.2 mM AS, pH = 5.6) to OD. 600 =1.0, incubate in the dark for 2 hours. Make a wound on the underside of the tobacco leaf with a needle, and inject the bacterial solution into the wound using a syringe, ensuring the entire leaf is infected. Incubate in the dark at 22℃ for 48-52 hours, and observe the fluorescence under a fluorescence microscope. Results are shown below. Figure 1 .

[0062] Figure 1 The results showed that the green fluorescence emitted by pBI121-EGFP-RcTPS1a-1 partially overlapped with the red fluorescence of the cell membrane marker, indicating that RcTPS1a is located on the cell membrane and cytoplasm.

[0063] 4) Gene functional analysis

[0064] (1) Construction of overexpression vector

[0065] Using pLB-RcTPS1a plasmid as a template, primers were designed to amplify the full-length RcTPS1a gene. The primer sequences are as follows:

[0066] pBI121-RcTPS1a-F: Same as above;

[0067] pBI121-RcTPS1a-R2: 5'-GACCACCCGGGGATCCTTACTTATCGTCGTCATCCTTGTAATC GATATTCTCAGTGCTC -3';SEQ ID NO.9.

[0068] The amplification system and reaction procedure are the same as those in step 3) for the construction of the vector (1).

[0069] The pBI121-EGFP plasmid was digested with BamHI. PCR amplification of the RcTPS1a product showed a band position close to 3000 bp, consistent with the predicted band position. The single-enzyme digestion product of the pBI121-EGFP vector showed a significant difference in band position compared to the undigested plasmid control, confirming successful cleavage of the pBI121-EGFP vector and its suitability for subsequent experiments. Ligation of the target fragment and linearized vector with SE homologous recombinase, followed by transformation into DH5α competent cells and correct sequencing alignment, indicated successful construction of the overexpression vector pBI121-EGFP-RcTPS1a-2.

[0070] (2) Activation of bacterial culture

[0071] The constructed pBI121-EGFP-RcTPS1a-2 vector containing the 35S promoter was inoculated onto a YEB plate containing 50 mg / L Kan and 50 mg / L Rif, and incubated at 28°C for 45 h. Single colonies were picked and cultured in 5 ml of YEB liquid medium (containing 50 mg / L Kan and 50 mg / L Rif) at 28°C for 24 h using a shaker. Then, 1 ml of the bacterial culture was incubated in 50 ml of YEB liquid medium (containing 50 mg / L Kan and 50 mg / L Rif) at 28°C for 12 h using a shaker. The bacterial culture was then analyzed by measuring the OD value of the culture. 600 When the concentration of the bacterial culture reaches 1.2, aliquot the culture into 50ml centrifuge tubes, centrifuge at 6000rpm for 8min, discard the supernatant, and resuspend the culture in sterile 5% sucrose solution to OD200. 600 =1.0, left to stand in the dark for 2 hours.

[0072] (3) Inflorescence infection

[0073] Before infection, remove the Arabidopsis thaliana pods, retaining only the inflorescences that have not yet produced pods. Add Swilt-L-77 to the settled bacterial solution to a final concentration of 0.02%. Immerse the Arabidopsis thaliana inflorescences in the bacterial solution for 30 seconds. After infection, use cotton to absorb excess bacterial solution from the leaves and stems. Support the Arabidopsis thaliana with bamboo skewers, cover with a plastic bag, and place horizontally in the dark for 24 hours. Then transfer to an incubator with 16 hours of light / 8 hours of darkness for cultivation. Harvest the seeds after they mature and store them at 4℃.

[0074] (4) Screening of transgenic Arabidopsis thaliana

[0075] The collected T0 generation seeds were aliquoted into 2ml centrifuge tubes and sterilized in a laminar flow hood. First, the seeds were rinsed with sterile water, then sterilized with 1% NaClO for 10 minutes. During sterilization, one drop of Tween-80 was added to the NaClO to ensure thorough contact between the disinfectant and the seeds. The seeds were then rinsed 5-8 times with sterile water to completely remove the disinfectant. The seeds were inoculated onto MS medium supplemented with 50mg / L Kan and 200mg / L Cef (cephalosporin), vernalized at 4°C for 2 days, and then cultured in a 16h light / 8h dark incubator. Two weeks later, the plant phenotype was observed. Transgenic seedlings grew normally on the resistant medium, while non-transgenic seedlings turned yellow and died after germination.

[0076] (5) Identification of transgenic Arabidopsis thaliana

[0077] The selected transgenic seedlings were transplanted into small pots filled with substrate and placed in an incubator with 16 hours of light and 8 hours of darkness. After 10 days, leaves from wild-type and transgenic Arabidopsis thaliana were harvested, and DNA was extracted according to the instructions of the DNA extraction kit (DC104) from Novizan Biosciences. The DNA concentration was measured and stored at -80°C.

[0078] Using the extracted DNA as a template, PCR amplification was performed using 2×Rapid Taq MasterMix enzyme. The pBI121-EGFP-RcTPS1a-2 plasmid was used as a positive control, and sterile water and wild-type Arabidopsis DNA were used as negative controls. The primers designed for identification were as follows:

[0079] 35s-F: 5'-TCAACAAAGGGTAATATCCGG-3'; SEQ ID NO.10;

[0080] pBI121-RcTPS1a-R2: Same as above.

[0081] Amplification system: 12.5 μl of 2×Rapid Taq Master Mix, 1 μl of 35s-F, 1 μl of pBI121-RcTPS1a-R2, 1 μl of template, and 9.5 μl of ddH2O. Reaction program: 95℃ for 3 min; 95℃ for 15 s, 61℃ for 15 s, 72℃ for 45 s, 35 cycles; 72℃ for 5 min; 4℃.

[0082] (6) Screening of T3 generation transgenic plants

[0083] Seeds from the successfully identified T1 generation transgenic plants were collected and stored at 4℃. T2 generation transgenic plants were obtained by screening with MS medium supplemented with 50 mg / L Kan and 200 mg / L Cef. Seeds from the T2 generation transgenic plants were then screened again to obtain the T3 generation transgenic plants.

[0084] (7) Phenotypic observation and statistics of T3 generation transgenic plants

[0085] In screening T3 generation transgenic plants, wild-type Arabidopsis thaliana was sown in antibiotic-free MS medium. When the T3 generation transgenic plants reached four true leaves, they were transplanted into plug trays containing substrate and cultured in an incubator with 16 hours of light and 8 hours of darkness. The flowering time of wild-type and transgenic Arabidopsis thaliana was observed and statistically analyzed using GraphPad Prism 9.5.0 software, and differences were compared using an independent samples t-test. Results are shown below. Figure 2 .

[0086] Figure 2 The results showed that, according to statistical analysis, the flowering rate of T3 generation plants reached 67.5% on the 35th day after sowing, while the flowering rate of WT control plants was only 10% on the 35th day after sowing. The flowering time of T3 generation plants was on average 3.5 days earlier than that of WT, which was significantly earlier than that of WT, indicating that RcTPS1a can promote flowering in Arabidopsis thaliana.

[0087] (8) RT-qPCR detection of gene expression levels in T3 generation transgenic plants

[0088] When the T3 generation transgenic plants just showed the bolting phenotype, basal leaves of Arabidopsis thaliana were collected, and RNA was extracted (using the same RNA kit as in step 1). cDNA for quantitative PCR was synthesized using the Novizan cDNA synthesis kit (R233), following the kit's instructions.

[0089] Using wild-type and transgenic Arabidopsis cDNA as templates and the AtUBQ gene as an internal reference gene, the expression levels of the RcTPS1a gene and important flowering-regulating genes AtFT, AtSOC1, and AtSPL9 were measured. Data were analyzed using GraphPad Prism 9.5.0 software. The significance of the analytical methods was tested. Results are shown below. Figure 3 .

[0090] Figure 3 The results showed that, compared with WT plants, the expression levels of flowering-related genes AtFT, AtSPL9, and AtSOC1 in T3 generation transgenic Arabidopsis thaliana overexpressing RcTPS1a were higher than those in WT plants, indicating that the RcTPS1a gene can promote Arabidopsis flowering by affecting the expression of flowering-related genes AtFT, AtSPL9, and AtSOC1.

[0091] Example 2

[0092] 1) Carrier construction

[0093] Expression vectors were constructed using homologous recombination. The pTRV2 vector was double-digested with BamHI and EcoRI restriction endonucleases. After digestion, the results were detected by gel electrophoresis, and the linearized vector was recovered from the gel. A 348 bp fragment was selected from the CDS sequence of the target gene after removing the stop codon. Primers with restriction sites and homologous arms were designed using Primer 5.0. The reverse primer also had the stop codon removed. The primer sequences are as follows:

[0094] pTRV2-RcTPS1a-F:

[0095] 5'-CTGTGAGTAAGGTTACCGAATTC GGAATTGTGAATGCTTGG -3';SEQ ID NO.11;

[0096] pTRV2-RcTPS1a-R:

[0097] 5'-CGCGTGAGCTCGGTACCGGATCC GATATTCTCAGTGCTC TG-3'; SEQ ID NO.12.

[0098] PCR amplification was performed using the previously constructed pLB-RcTPS1a vector as a template. Amplification system: 2×PhantaMax Buffer 25 μl, dNTP Mix 1 μl, pTRV2-RcTPS1a-F 2 μl, pTRV2-RcTPS1a-R 2 μl, PhantaMax Super-Fidelity DNA Polymerase 1 μl, pLB-RcTPS1a 1 μl, ddH2O 18 μl. Reaction program: 95℃ for 3 min; 95℃ for 15 s, 70℃ for 15 s, 72℃ for 1 min, 35 cycles; 72℃ for 5 min; 4℃. Amplification results were detected by gel electrophoresis, and the target fragment was recovered from the gel.

[0099] The target fragment was ligated into a linearized vector using SE homologous recombinase. The ligation product was introduced into DH5α Escherichia coli, and after successful sequencing, the plasmid pTRV2-RcTPS1a was extracted. The pTRV1, pTRV2 empty vector, and pTRV2-RcTPS1a recombinant plasmid were transformed into Agrobacterium GV3101, respectively. After successful colony PCR identification, the bacterial culture was stored at -80℃.

[0100] 2) Momentary silence

[0101] After 30 days of rooting, the 'Monthly Red' cuttings were potted and allowed to continue growing for about 30 days. The plants that grew uniformly were then selected for subsequent infection.

[0102] (1) Preparation of bacterial culture

[0103] Bacterial suspensions containing pTRV1, pTRV2 empty vectors, and pTRV2-RcTPS1a recombinant plasmids were inoculated onto YEB plates containing 50 mg / L Kan and 50 mg / L Rif, and incubated at 28°C for 45 h. Single colonies were picked and incubated in 5 ml of YEB liquid medium (containing 50 mg / L Kan and 50 mg / L Rif) at 28°C for 24 h using a shaker. Then, 1 ml of bacterial suspension was incubated in 50 ml of YEB liquid medium (containing 50 mg / L Kan and 50 mg / L Rif) at 28°C for 12 h using a shaker. The bacterial suspension was then counted at OD0.05. 600 When the pH is 1.2-1.5, aliquot the bacterial culture into 50ml centrifuge tubes, centrifuge at 6000rpm for 8min, and discard the supernatant. Prepare a resuspension (10mM MES + 10mM MgCl2 + 0.2mM AS, pH = 5.6), and resuspend to OD. 600 =1.0, mix pTRV1 with pTRV2 and pTRV2-RcTPS1a in a 1:1 ratio, and let stand in the dark at 28°C for 3 hours.

[0104] (2) Vacuum infiltration method

[0105] The 'Monthly Red' cuttings were placed upside down in the bacterial solution, and aspiration was performed at 0.09 MPa for 5 minutes, repeated twice. After infection, excess bacterial solution was rinsed off with water, and the plants were placed in the dark at room temperature for 3 days. Then they were transplanted into the substrate and cultured in an incubator with 16 hours of light and 8 hours of darkness.

[0106] (3) Identification and phenotypic observation statistics

[0107] Fourteen days after infection, when new leaves emerged from the plants, uniformly sized young leaves were collected, and RNA was extracted to synthesize cDNA for quantitative real-time PCR. Using the cDNA from the infected plants as a template and the pTRV2 empty vector plasmid as a positive control, PCR amplification was performed using specific primers V2-F and V2-R to identify whether the silencing vector had entered the infected plants. The primer sequences are as follows:

[0108] V2-F: 5'-CGGTTACGACGAACCAAGGGAG-3'; SEQ ID NO.13;

[0109] V2-R: 5'-GTGCATTGCGAAACTCAAATGC-3'; SEQ ID NO. 14.

[0110] Amplification system: 12.5 μl of 2×Rapid TaqMasterMix, 1 μl of V2-F, 1 μl of V2-R, 1 μl of template, and 9.5 μl of ddH2O. Reaction program: 95℃ for 3 min; 95℃ for 15 s, 55℃ for 15 s, 72℃ for 15 s, 35 cycles; 72℃ for 5 min; 4℃.

[0111] Using cDNA from infected plants infected with the pTRV2 empty vector and pTRV2-RcTPS1a as templates, the expression levels of the RcTPS1a gene and the RcFT, RcSOC1, RcSPL1, and RcSPL14 genes were measured. One-way ANOVA was performed on the data using GraphPadPrism 9.5.0 software, and multiple comparison tests were conducted using Tukey analysis. Results are shown below. Figure 4 .

[0112] Figure 4 The results showed that the expression levels of the RcTPS1a gene and flowering regulation-related genes RcFT, RcSOC1, RcSPL1, and RcSPL14 in the three 'Yueyuehong' plants infected with pTRV2-RcTPS1a (1, 2, and 3) were significantly lower than those in the pTRV2 control plants. This indicates that silencing the RcTPS1a gene induces a decrease in the expression levels of flowering regulation-related genes RcFT, RcSOC1, RcSPL1, and RcSPL14, resulting in delayed flowering in 'Yueyuehong'.

[0113] The phenotypes of successfully identified silent plants were observed, and the results are shown in [the table below]. Figure 5 The flowering time of the plants compared with that of the control group infected with the pTRV2 empty vector was statistically analyzed using GraphPadPrism 9.5.0 software, and the differences were compared using an independent samples t-test. The average time from infection to flowering of 'Monthly Red' plants with silenced RcTPS1a was 82.7 days, significantly later than that of the control group (49 days), indicating that the RcTPS1a gene promotes flowering in 'Monthly Red'.

[0114] Example 3

[0115] 1) Carrier construction

[0116] Expression vectors were constructed using homologous recombination. The pCambia1305 vector was digested with BamHI restriction endonuclease. After digestion, the results were detected by gel electrophoresis, and the linearized vector was recovered from the gel. The full-length CDS sequence of the target gene was selected, and primers containing BamHI restriction sites and preceding and following homologous arms were designed using Primer 5.0. The stop codon was removed from the reverse primer. The primer sequences are as follows:

[0117] pCambia1305-RcTPS1a-F:

[0118] 5'-AGAGAACACGGGGGACGGATCC ATGCCAGGAAACAAGTATAAC TGC -3';SEQ ID NO.15;

[0119] pCambia1305-RcTPS1a-R:

[0120] 5'-ATGGTCTTTGTAGTCGGATCC GATATTCTCAGTGCTC -3';SEQ ID NO.16。

[0121] PCR amplification was performed using the previously constructed pLB-RcTPS1a vector as a template. The amplification system consisted of: 2×PhantaMax Buffer 25 μl, dNTP Mix 1 μl, pCambia1305-RcTPS1a-F 2 μl, pCambia1305-RcTPS1a-R 2 μl, PhantaMax Super-Fidelity DNA Polymerase 1 μl, pLB-RcTPS1a 1 μl, and ddH2O 18 μl. The reaction program was: 95℃ for 3 min; 95℃ for 15 s, 70℃ for 15 s, 72℃ for 3 min, 35 cycles; 72℃ for 5 min; 4℃. The amplification results were detected by gel electrophoresis, and the target fragment was recovered from the gel.

[0122] The target fragment was ligated into a linearized vector using SE homologous recombinase. The ligation product was introduced into DH5α Escherichia coli, and plasmids were extracted after successful sequencing. The pCambia1305 empty vector and pCambia1305-RcTPS1a recombinant plasmid were transformed into Agrobacterium GV3101, respectively. After successful colony PCR identification, the bacterial culture was stored at -80℃.

[0123] 2) Transient overexpression

[0124] After 30 days of rooting, the 'Monthly Red' cuttings were potted and allowed to continue growing for about 30 days. The plants that grew uniformly were then selected for subsequent infection.

[0125] (1) Preparation of bacterial culture

[0126] Prepare and preserve Agrobacterium bacterial cultures of pCambia1305 empty vector and pCambia1305-RcTPS1a.

[0127] (2) Vacuum infiltration method

[0128] The 'Monthly Red' cuttings were placed upside down in the bacterial solution, and aspiration was performed at 0.09 MPa for 5 minutes, repeated twice. After infection, excess bacterial solution was rinsed off with water, and the plants were placed in the dark at room temperature for 3 days. Then they were transplanted into the substrate and cultured in an incubator with 16 hours of light and 8 hours of darkness.

[0129] (3) Identification and phenotypic observation statistics

[0130] DNA was extracted from infected leaves 10 days after infection. PCR amplification was performed using the extracted DNA as a template to identify whether the recombinant plasmid had successfully integrated into the infected plant. Amplification primers: 35S-F;

[0131] pCambia1305-RcTPS1a-R. Amplification system: 2×Rapid TaqMasterMix 12.5 μl, 35s-F 1 μl, pCambia1305-RcTPS1a-R 1 μl, template 1 μl, ddH2O 9.5 μl. Reaction program: 95℃ 3 min; 95℃ 15 s, 67℃ 15 s, 72℃ 45 s, 35 cycles; 72℃ 5 min; 4℃.

[0132] RNA was extracted from infected leaves 10 days after infection. cDNA for quantitative real-time PCR was synthesized using a two-step method. Using cDNA from infected plants with the pCambia1305 empty vector and pCambia1305-RcTPS1a as templates, the expression levels of the RcTPS1a gene and the RcFT, RcSPL1, RcSPL14, and RcSOC1 genes were measured. One-way ANOVA was performed on the data using GraphPad Prism 9.5.0 software, and multiple comparison tests were conducted using Tukey analysis. Results are shown below. Figure 6 .

[0133] Figure 6 The results showed that the expression levels of the target gene RcTPS1a and the genes RcFT, RcSOC1, RcSPL1, and RcSPL14 in the three infected plants (A, E, and F) were significantly higher than those in the control plants infected with the pCambia1305 empty vector. This indicates that transient overexpression of the RcTPS1a gene in 'Yueyuehong' can increase the expression levels of RcFT, RcSOC1, RcSPL1, and RcSPL14 genes, promoting early flowering in 'Yueyuehong'.

[0134] The phenotypes of successfully infected plants were observed and identified, and the results are shown in [the table below]. Figure 7 (Three plants transiently overexpressing the pCambia1305-RcTPS1a vector were photographed). The flowering phenotypes of these plants and the control group infected with the pCambia1305 empty vector were compared. Data were analyzed using GraphPad Prism 9.5.0 software, and independent samples t-tests were used to compare differences. The average flowering time from infection to flowering for the five plants transiently overexpressing the pCambia1305-RcTPS1a vector was 48 days, while the average flowering time for the five control plants infected with the empty vector was 52.8 days. Overexpression of RcTPS1a advanced the flowering of 'Monthly Red' by 4.8 days. Overexpression of the RcTPS1a gene in 'Monthly Red' promotes flowering.

[0135] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. RcTPS1a Gene, RcTPS1a The amino acid sequence encoded by the gene, overexpression vector pCambia1305- RcTPS1a Gene silencing vector pTRV2- RcTPS1a Or the application of recombinant engineered bacteria in regulating plant flowering time; The RcTPS1a The nucleotide sequence of the gene is shown in SEQ ID NO.1; The RcTPS1a The amino acid sequence encoded by the gene is shown in SEQ ID NO.2; The overexpression vector pCambia1305- RcTPS1a The primer sequences are SEQ ID NO.15 and SEQ ID NO.16; The gene silencing vector pTRV2- RcTPS1a The primer sequences are SEQ ID NO.11 and SEQ ID NO.12; The recombinant engineered bacteria contains the overexpression vector pCambia1305- RcTPS1a or the gene silencing vector pTRV2- RcTPS1a ; The plant in question is a 'Monthly Red' rose.

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