Application of BdRFS protein or its related biomaterials in regulating flowering time, biomass or drought resistance of plants

Abstract

The application belongs to the technical field of genetic engineering, and discloses application of BdRFS protein or related biological materials in regulating flowering time, biomass or drought resistance of plants. The application proposes that the BdRFS protein can regulate flowering time, biomass and drought resistance of plants, and a recombinant expression vector containing a BdRFS gene fragment can be used to transform plant tissues, so that the obtained transgenic plants have the advantages of long flowering time, strong drought resistance and high biomass.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of BdRFS protein or related biological materials in regulating plant flowering period, biomass or drought resistance. Background Technology

[0002] Abiotic stresses in the environment, such as drought, salinity, cold damage, and heat damage, affect plant growth and development, leading to reduced crop yields and impacting the normal growth and quality of trees, fruit trees, flowers, and ornamental plants. Therefore, cultivating stress-resistant plant varieties is one of the main goals of the planting industry.

[0003] Flowering is an irreversible physiological developmental process in which plants transition from vegetative growth to reproductive growth. It is a crucial stage in the individual development and reproduction of plants and is induced by multiple factors, including photoperiod, temperature, hormones, and age. It occupies a central position in plant growth and species evolution, and the timing of vegetative and reproductive growth can be altered and adjusted through certain regulation.

[0004] *Brachys bipinnatifida* is an early-maturing Pooideae plant characterized by its small genome, short growth cycle, self-pollination, small size, ease of genetic transformation, and readily available mutants. Therefore, this invention aims to identify and clone new regulatory genes from *Brachys bipinnatifida* and apply them to molecular breeding of agricultural and forestry plants, with the goal of obtaining transgenic plant lines with high biomass, strong drought resistance, or long flowering periods. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes the application of BdRFS protein or related biological materials in regulating plant flowering period, biomass, or drought resistance. This invention proposes that BdRFS protein can regulate plant flowering period, biomass, and drought resistance, and that recombinant expression vectors containing BdRFS gene fragments can be used to transform plant tissues, resulting in transgenic plants with advantages such as long flowering period, strong drought resistance, and high biomass.

[0006] This invention provides the application of BdRFS protein in regulating plant flowering time, biomass, or drought resistance, and the amino acid sequence of the BdRFS protein is shown in SEQ ID NO: 4.

[0007] Preferably, the regulation involves delaying the flowering period, increasing plant biomass, or enhancing plant drought resistance. Increasing the expression of the BdRFS protein through regulation can delay the flowering period, increase plant biomass, and enhance plant drought resistance.

[0008] In this invention, the flowering period refers to the time required from the time a plant is sown until it flowers.

[0009] This invention also provides the application of biomaterials associated with BdRFS protein in regulating plant flowering time, biomass, or drought resistance, wherein the biomaterials contain at least one of the following (1)-(4):

[0010] (1) Nucleic acid molecules encoding the BdRFS protein;

[0011] (2) An expression cassette containing the nucleic acid molecule described in (1);

[0012] (3) A recombinant expression vector containing the nucleic acid molecule described in (1);

[0013] (4) Transformants containing the recombinant expression vector described in (3).

[0014] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the BdRFS protein is shown in SEQ ID NO: 3.

[0015] The present invention also provides a recombinant expression vector, which is constructed from a nucleic acid molecule encoding the BdRFS protein and a plant expression vector.

[0016] The plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors for monocotyledonous gene gun transformation; the binary Agrobacterium vectors include pBI121 and pCAMBIA series vectors; the plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., DNA fragments containing polyadenylate signals and any other effector mRNA processing or gene expression.

[0017] Preferably, the plant expression vector is pYLox.5.

[0018] When constructing the recombinant expression vectors described above, any strong or inducible promoter can be used before its transcription initiation nucleotide. These strong or inducible promoters include, but are not limited to, the ubiquitin promoter and the cauliflower mosaic virus (CaMV) 35S promoter, which can be used alone or in combination with other plant promoters. Furthermore, enhancers can also be used when constructing the recombinant expression vectors. These enhancer regions include, but are not limited to, the ATG start codon and its adjacent regions. The start codon must be identical to the reading frame of the coding sequence to ensure translation of the entire sequence. Translation control signals and start codons can come from various sources, including natural and synthetic ones. The translation initiation region can originate from the transcription initiation region or from structural genes.

[0019] Furthermore, the plant expression vectors used can be processed, including adding or replacing plant-selective markers, to facilitate the identification and screening of transgenic plants obtained using the aforementioned recombinant expression vectors. Usable selective markers include genes encoding herbicide-resistant enzymes or antibiotic resistance markers; the herbicides include glyphosate, glufosinate, etc., and the antibiotics include kanamycin, hygromycin, gentamicin, etc. From a safety perspective for transgenic plants, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0020] The present invention also provides a method for preparing the above-mentioned recombinant expression vector, comprising the following steps:

[0021] (1) Using cDNA of *B. bipinnatifida* as a template, the BdRFS gene fragment was amplified using primer pairs with nucleotide sequences as shown in SEQ ID NO: 1-2, and then ligated with the pGEM-T easy vector to construct the pGEM-BdRFS vector.

[0022] (2) Using the pGEM-BdRFS vector as a template, primer pairs with nucleotide sequences such as SEQ ID NO: 5-6 were used to introduce the BdRFS gene fragment into the Hind III and BamHⅠ restriction sites, and then ligated with the plant expression vector to construct the recombinant expression vector.

[0023] Preferably, the variety of Brachypodium distachyon (L.) Beauv. is Bd21-3.

[0024] The present invention also provides a method for cultivating drought-resistant, high-biomass, and late-maturing transgenic plants, wherein the above-mentioned recombinant expression vector is transformed into plant tissue, and the transformed plant tissue is cultured to obtain transgenic plants.

[0025] Preferably, the transformation methods include Agrobacterium-mediated transformation, gene gun transformation, electroporation, PEG vector transformation, and liposome transformation. Furthermore, those skilled in the art can also rationally select other transformation methods for the introduction of recombinant expression vectors according to actual needs.

[0026] Preferably, the plant tissue is a monocotyledonous plant.

[0027] More preferably, the plant tissue is a grass (Poaceae).

[0028] More preferably, the plant is a species of the genus *Brucea*.

[0029] More preferably, the plant tissue is *Bruguiera gymnorhiza*.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] This invention cloned the cDNA sequence BdRFS of an unknown functional gene from *Brachys bipinnatus*, and found that the expression of the BdRFS gene is affected by phosphorus, development, light, drought, cold, ABA, and MeJA. A method for cultivating transgenic plants using the BdRFS gene fragment is proposed. By ligating the BdRFS gene fragment to a plant expression vector to construct a recombinant expression vector and transforming it into plant tissues, drought-resistant, high-biomass, and late-maturing (i.e., long-flowering) plants can be obtained. Attached Figure Description

[0032] Figure 1 This is an agarose gel electrophoresis image of the PCR amplification of the open reading frame sequence of the BdRFS gene; where M is a standard DNA molecule; lane 1 is the amplified fragment of the BdRFS gene.

[0033] Figure 2 This is a schematic diagram of the expression frame of the recombinant expression vector pYLox.5-BdRFS.

[0034] Figure 3 This is a diagram showing the expression pattern of BdRFS in *Brachys bipinnatus*. (A) shows the effect of phosphorus deficiency on BdRFS in the aboveground and underground parts of *Brachys bipinnatus*. (B) shows the expression pattern of BdRFS during leaf development in *Brachys bipinnatus*: You (young leaf stage), Dev (developmental stage), Mat (maturity stage). (C) shows the expression pattern of BdRFS in *Brachys bipinnatus* under dark treatment: L (light environment), D (dark environment), RL (restored light environment). (D) shows the expression pattern of BdRFS in *Brachys bipinnatus* under dark treatment. (E) shows the expression pattern of BdRFS in *Brachys bipinnatus* under drought treatment; (F) shows the expression pattern of BdRFS in *Brachys bipinnatus* under cold treatment; and (F) shows the expression pattern of BdRFS in *Brachys bipinnatus* treated with 100 μM abscisic acid (ABA) and 100 μM methyl jasmonate (MeJA). The letters a, b, c, d, and e above the columns indicate significant differences between the different treatments (P ≤ 0.05). That is, the same letter above the data column indicates no significant difference, and different letters above the data column indicate significant differences.

[0035] Figure 4 This is an agarose gel electrophoresis image showing the PCR identification results of transgenic *Brucea diplostis* with the BdRFS gene introduced. In the image, W represents the wild type; P represents the pYLox.5-BdRFS vector, which is the positive control; H represents water; and numbers O1 to O21 represent different BdRFS transgenic *Brucea diplostis*.

[0036] Figure 5In the graphs (A), (B), and (C), we can see the real-time quantitative PCR results analysis, flowering time graph, and aboveground dry weight graph of transgenic *Brachys pubescens* with the BdRFS gene introduced, respectively. The letters a, b, and c above the columns indicate significant differences between different materials (P≤0.05).

[0037] Figure 6 The values ​​represent the relative water content of each plant lineage after drought treatment; the letters a, b, and c above the column indicate significant differences between different materials (P≤0.05).

[0038] Figure 7 The values ​​represent the relative electrical conductivity of each plant line after drought treatment; the letters a, b, and c above the column indicate significant differences between different materials (P≤0.05). Detailed Implementation

[0039] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments are merely preferred embodiments of this invention and do not constitute a limitation on the scope of protection claimed by this invention. Any modifications, substitutions, or combinations made without departing from the spirit and principle of this invention are included within the scope of protection of this invention.

[0040] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0041] The ecotype Bd21-3 of Brachypodium distachyon was kindly donated by Hunan Agricultural University.

[0042] Agrobacterium tumefaciens AGL1: purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0043] pYLox.5 overexpression vector: provided by the Genetic Engineering Laboratory of South China Agricultural University (Zhou H, Liu Q, Li J, et al. Photoperiod-and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA[J]. Cell Research, 2012, 22(4): 649-660.).

[0044] Escherichia coli DH5α: purchased from Shanghai Chaoyan Biotechnology Co., Ltd.

[0045] Example 1: Cloning of the BdRFS gene

[0046] (1) Preparation of cDNA template from *Brachys pubescens*

[0047] Seeds of *Brussels sprouts* (ecotype BD21-3) were surface-sterilized with a solution containing 30% (v / v) bleach and 0.05% (v / v) Tween 20, then treated in the dark at 4°C for 3 days, and germinated on 1 / 2 MS agar (1% w / v) plates. The plants were then grown in a growth chamber (light intensity 175 μmol·m²). -2 ·s -1 The plants were vertically cultured for 7 days at 22℃ / 18℃ with a 16-hour light-8-hour dark cycle, then transplanted into a culture medium (peat:perlite = 3:1) and grown in a greenhouse (24℃, 16-hour light-8-hour dark cycle) until maturity. Mature leaves of *Brachys bipinnatus* were collected, and total RNA was extracted using the Trizol method. cDNA templates were obtained by reverse transcription using the M-MLV reverse transcriptase kit (Promega) and stored at -20℃ for later use.

[0048] (2) Design primers for amplifying the BdRFS gene fragment

[0049] Based on the cDNA sequence of the BdRFS gene from the grass family *Brachypophthalmia ulmoides* in the GenBank database, primers were designed to amplify the open reading frame (ORF) of the BdRFS cDNA (synthesized by Shanghai Sangon Biotech). The upstream primer for amplifying BdRFS, BdRFS-f, has the nucleotide sequence: 5'-ATGGAGCTCCTGGACGTGG-3' (SEQ ID NO: 1); the downstream primer for amplifying BdRFS, BdRFS-r, has the sequence: 5'-GTACTGACAAGTGCAGTTCC-3' (SEQ ID NO: 2).

[0050] (3) Constructing a carrier

[0051] The cDNA template from step (1) was amplified by PCR using the upstream primer BdRFS-f and the downstream primer BdRFS-r from step (2):

[0052] The PCR reaction system (50 μL) is shown in Table 1.

[0053] Table 1 PCR reaction system

[0054]

[0055]

[0056] The KOD-Plus-DNA polymerase was purchased from TOYOBO.

[0057] PCR reaction program: 94℃ for 3 min; 94℃ for 0.5 min, 55℃ for 0.5 min, 72℃ for 0.5 min, 35 cycles; 72℃ for 10 min.

[0058] The amplification products were detected by 0.8% agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, the obtained BdRFS gene fragment is consistent with the expected fragment length (approximately 700 bp), indicating that the amplification was successful.

[0059] The obtained PCR product was recovered using a DNA gel extraction kit (Qiagen). The recovered PCR fragment was ligated with pGEM-T easy (TaKaRa) at a molar ratio of 3:1. The reaction system was as follows: 1 μL of pMD18-T Vector (0.03 pmol), 0.1 pmol to 0.3 pmol of the recovered target fragment, water was added to 5 μL, and then 5 μL of Solution I was added. The mixture was ligated at 16 °C for 2 h to obtain the ligation product.

[0060] Preparation of competent Escherichia coli cells: Using an inoculation loop, take E. coli DH5α bacterial suspension stored at -80℃ and streak it onto SOB solid medium. Incubate overnight at 37℃. Pick a single colony of E. coli DH5α and inoculate it into 1 mL of SOB liquid medium. Incubate at 37℃ with shaking at 200 rpm for about 6 hours. Inoculate it into 200 mL of SOB liquid medium and incubate at 37℃ with shaking at 200 rpm until OD550 = 0.6. Collect the bacterial cells by centrifugation at 2500 rpm for 10 min. Wash with pre-cooled 10% glycerol (glycerol:LB liquid medium) and centrifuge to collect the bacteria. Repeat the washing with pre-cooled 10% glycerol (glycerol:LB liquid medium) and centrifugation to collect the bacteria. Finally, aliquot the bacteria and store at -80℃ for later use.

[0061] Heat shock transformation of E. coli with the ligation product: The ligation product was added to 100 μL of DH5α competent cells and placed on ice for 30 min; after heat shock at 42℃ for 90 s, it was placed on ice for another 2 min; the heat-shocked cells were transferred to 1 mL of SOC liquid and cultured on a shaker at 37℃ with shaking at 200 rpm for 1 h. 100 μL of the bacterial culture was spread onto LB solid medium (containing 100 μg / mL Amp) containing IPTG and X-gal and incubated upside down overnight at 37℃.

[0062] (4) Screening, purification and sequencing of recombinant plasmid pGEM-BdRFS

[0063] Take the cultured plate. Colonies with blue spots do not contain the inserted fragment. Only pick colonies with white spots (positive, i.e., recombinant bacteria) for colony PCR detection.

[0064] Positive colonies verified by PCR were selected and inoculated into 2 mL of LB broth containing 100 μg / mL ampicillin. The culture was incubated overnight at 37°C with shaking at 200 rpm. 2 mL of the bacterial culture was then used to extract the plasmid using a plasmid DNA purification kit (Qiagen). The purified recombinant plasmid was named pGEM-BdRFS and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results included the following sequence:

[0065] 5'--3'(SEQID NO: 3).

[0066] Sequencing results were compared with the sequences in the library, confirming that the amplified PCR product was a fragment of the BdRFS gene.

[0067] The amino acid sequence of the BdRFS protein is shown in SEQ ID NO: 4:

[0068] MELLDVVPAEAIALRLYSLTAAANTVVSLCAWLVAALAAAAVGLWRVRAAGSSHKPGGAVVRSTLVDNKKIASESFDGPRPARSEPASPISEPSSPSKVRFTAYYGGTGSDG GDDGVVEGVKKCAERDEDDFNGESETAVLRRTASMRMRSTIKAPLMAAPDWEEKEMALRKRGDLGWYRHLDMAVLDGSVVRLWTGEVTAAVQASPRERRRAGLELHLSV(SEQ ID NO: 4).

[0069] Example 2: Construction of the recombinant expression vector pYLox.5-BdRFS

[0070] Based on the BdRFS open reading frame sequence obtained in Example 1, a primer pair with Hind III and BamHI restriction sites was designed for amplification:

[0071] An upstream primer cm188 for the Hind III restriction site was introduced, with the nucleotide sequence: 5'-TTTTTTAAGCTTATGGAGCTCCTGGACGTGG-3' (SEQ ID NO: 5);

[0072] A downstream primer cm189 with the BamHI restriction site was introduced, and its nucleotide sequence is: 5'-TTTTTTGGATCCGTACTGACAAGTGCAGTTCC-3' (SEQ ID NO: 6).

[0073] Using the cloning vector pGEM-BdRFS constructed in Example 1 as a template, and cm188 and cm189 as upstream and downstream primers, the BdRFS gene was amplified by PCR, and the amplified BdRFS gene fragment was purified and recovered.

[0074] The obtained BdRFS gene fragment and pYLox.5 overexpression vector were double-digested with restriction endonucleases HindIII (TaKaRa) and BamHI (TaKaRa), respectively. The target BdRFS gene fragment and the digested vector fragment were recovered. After ligation, the BdRFS gene was inserted between HindIII and BamHI at the multiple cloning site downstream of the promoter of the expression plasmid pYLox.5Ubi, to construct the recombinant expression vector, which was named pYLox.5-BdRFS. Figure 2 This is a schematic diagram of the expression frame of the recombinant expression vector pYLox.5-BdRFS.

[0075] Further sequencing of the insert fragment of the recombinant expression vector revealed that the sequence of the insert fragment was completely identical to the sequence of the BdRFS coding region, and the restriction enzyme sites at both ends of the insert fragment were also completely correct, thus proving that the recombinant expression vector pYLox.5-BdRFS was successfully constructed.

[0076] Example 3: Stress-induced expression of BdRFS

[0077] I. Obtaining a template of two-spike short-stalked grass under adverse conditions

[0078] Seeds of *Brussels sprouts* (ecotype BD21-3) were surface-sterilized with a solution containing 30% (v / v) bleach and 0.05% (v / v) Tween 20, then treated in the dark at 4°C for 3 days, and germinated on 1 / 2 MS agar (1% w / v) plates. The plants were then grown in a growth chamber (light intensity 175 μmol·m²). -2 ·s -1 Plants were vertically cultured for 7 days at 22℃ / 18℃ with a 16-hour light-8-hour dark cycle, then transplanted to a culture medium (peat:perlite = 3:1) and grown in a greenhouse (24℃, 16-hour light-8-hour dark cycle) until maturity. Leaf samples from mature plants were collected for phenotypic, biochemical, or molecular analysis.

[0079] Phosphorus starvation treatment involved placing sterilized seeds directly onto 1 / 2 MS agar containing 675 μM or 0 μM phosphate and growing them vertically in a growth chamber for 10 days.

[0080] To dehydrate the seedlings, immediately remove them from the pots, place them on Whatman 3MM paper, and let them dry at room temperature for 6 hours.

[0081] For freezing treatment, seedlings at the same developmental stage are transferred to a growth chamber at -4°C for 6 hours.

[0082] For plant hormone treatment, spray 100 μM abscisic acid (ABA) or methyl jasmonate (MeJA) onto the leaves of 3-week-old BD21-3 seedlings and cover with plastic film for 30 minutes.

[0083] Samples were taken from the target tissue at specified time points, and the samples were ground with liquid nitrogen. Total RNA was extracted from the leaves using TRE-Trizol reagent (TaKaRa). cDNA templates were prepared by reverse transcription using PrimeScript™ RT reagent Kit (TAKaRa).

[0084] II. Design of Specific Detection Primers

[0085] Based on the cDNA sequence of the BdRFS gene and the sequence of *Bradius breviscapus* UBC18 (Bradi4g00660), quantitative primers for detection were designed using Beacon Designer 7.0 software.

[0086] The upstream quantitative primer BdRFS-qf for the BdRFS gene has the following nucleotide sequence: 5'-CGATGGAGTAGTGGAAGGCG-3' (SEQ ID NO: 7);

[0087] The downstream quantitative primer BdRFS-qr for the BdRFS gene has the following nucleotide sequence: 5'-ATCAAGGGCGCCTTAATCGT-3' (SEQ ID NO: 8);

[0088] The upstream quantitative primer UBC18-qf for the UBC18 gene has the following nucleotide sequence: 5'-GGAGGCACCTCAGGTCATTT-3' (SEQ ID NO: 9);

[0089] The downstream quantitative primer UBC18-qr for the UBC18 gene has the following nucleotide sequence: 5'-ATAGCGGTCATTGTCTTGCG-3' (SEQ ID NO: 10).

[0090] III. Quantitative PCR detection of expression differences

[0091] The template cDNA prepared in step one was diluted 50 times and used as a template for quantitative PCR. The specific primers from step two were used for detection. The reaction system for quantitative detection is shown in Table 2.

[0092] Table 2 Reaction system for quantitative detection

[0093] Components Dosage SYBR Premix Ex Taq (2×) 5μL 10 μM upstream quantitative primers 0.5μL 10μM downstream quantitative primers 0.5μL cDNA 1μL <![CDATA[ddH2O]]> 3μL

[0094] The instrument used was a Bio-Rad Mini Option Real-Time PCR System. The PCR reaction conditions were set as follows: 95℃ for 10s; 94℃ for 5s, 59℃ for 25s, for 40 cycles. A negative control without cDNA template was used. Each sample was tested in triplicate. The housekeeping gene UBC18 was used as an internal reference gene. After the reaction, melting curve analysis was performed. The PCR amplification efficiency was above 95%. The relative expression level of the gene was automatically calculated using Bio-Rad CFX Manager (version 1.6) software.

[0095] according to Figure 3 The results of real-time quantitative PCR showed that: Figure 3 As shown in (A), BdRFS expression levels were significantly increased in both the aboveground and underground parts under phosphorus deficiency conditions; Figure 3 As shown in Figure (B), the expression level of BdRFS gradually increases during leaf development; Figure 3 As can be seen in (C), BdRFS expression is suppressed under dark conditions; Figure 3 As can be seen in (D), BdRFS expression is suppressed under drought conditions; Figure 3 As can be seen in Figure (E), BdRFS expression is strongly induced under frost damage conditions; Figure 3 As shown in Figure (F), BdRFS expression was inhibited by treatment with 100 μM ABA and 100 μM MeJA (F).

[0096] Example 4: Construction and Molecular Detection of Transgenic Two-Spike Short-Petaled Grass

[0097] I. The Production of Genetically Modified Two-Spike Short-Stalked Grass

[0098] 1. The recombinant expression vector pYLox.5-BdRFS was introduced into Agrobacterium tumefaciens AGL1.

[0099] The preparation of AGL1 competent cells was based on the method of J. Sambrook (Huang Peitang, Wang Jiaxi, Zhu Houchu, J. Sambrook, D.W. Russell, author [J]. Molecular Cloning: A Laboratory Manual, 2002: 27-30) with modifications: AGL1 culture was streaked on MYB plates and incubated at 28°C for 48 h. Single colonies were picked and inoculated into 50 mL of liquid SOC and incubated overnight at 28°C. 0.5 mL of the bacterial culture was then inoculated into 500 mL of liquid SOC and incubated at 28°C for 8 h until the OD600 reached 0.6. The culture was then cooled in an ice bath for 10 min and poured into sterilized 20 mL of liquid SOC. Centrifuge the cells in 0 mL centrifuge tubes at 4°C and 4000 rpm for 10 min after equilibration. Collect the cells, discard the SOC, invert the tubes onto sterile paper towels, drain the water, add 50 mL of pre-chilled 10% glycerol (glycerol: MYB liquid medium), shake on ice to suspend the cells, centrifuge at 4°C and 4000 rpm for 15 min, collect the cells, wash once more, add 2 mL of pre-chilled 10% glycerol (glycerol: MYB liquid medium), suspend the cells, aliquot into 25 μL / tube, flash freeze in liquid nitrogen, and store at -80°C. Thaw competent cells on ice. Pre-cool 0.2 cm inner diameter electroporation cuvettes on ice. In a clean bench, add 1.5 μL of pYLox.5-BdRFS plasmid (20 ng / μL) to 20 μL of thawed competent cells, gently tap the tube wall to mix, incubate on ice for 1 min, then transfer to an electroporation cuvette and place it between the electrodes of an electroporator (MicroPulser, Bio-RAD). Select program Agr and perform electroporation. After electroporation, quickly pour 1 mL of YEB liquid culture medium into the electroporation cuvette in a clean bench, then transfer it to a shaker tube using a pipette. Incubate at 28°C with gentle shaking for 2 h. Take 0.3 mL of bacterial culture and spread it on a YEB plate (containing 35 mg / L chloramphenicol and 50 mg / L kanamycin). Incubate upside down in a 28°C incubator for 48 h.

[0100] 2. Identification of Agrobacterium-positive colonies containing the recombinant expression vector pYLox.5-BdRFS

[0101] Single colonies were picked from the plate for colony PCR detection and labeled. PCR-positive colonies were then picked and cultured in 3 mL of YEB liquid medium (containing 35 mg / L chloramphenicol and 50 mg / L kanamycin) at 28°C with shaking for 40 h. 2 mL of the bacterial culture was used to extract plasmids using the alkaline lysis method. Restriction endonucleases kpn I (TaKaRa) and Spe I (TaKaRa) were used for digestion to confirm the presence of the plant expression vector pYLox.5-BdRFS in the positive clones. 0.8 mL of the confirmed Agrobacterium culture was added to 0.2 mL of 80% glycerol (glycerol: MYB liquid medium), and mixed thoroughly to obtain Agrobacterium AGL1 infection solution containing the recombinant expression vector pYLox.5-BdRFS. This solution was stored at -80°C for later use.

[0102] 3. Obtaining transgenic *Brachys pubescens* plants

[0103] (1) Induction and subculture of callus

[0104] Seeds of wild-type *Bruguiera divaricata* Bd21-3 were soaked at room temperature for 2 hours. After separating the top glumes (lemmas), the seeds were placed on sterile moist filter paper in a petri dish and vernalized in the dark at 4°C for 2 days, followed by germination at 25°C with 16 hours of light for 3-5 days. Seedlings were then transferred to a nutrient substrate, covered, and incubated at 22°C with 20 hours of light for 1-2 weeks. When sufficiently large, they were transferred to 11.4 cm pots (to begin tillering) and allowed to grow for 7-9 weeks, differentiating spikes with immature embryos. After harvesting the spikes at the correct stage (when the immature seeds are swollen but still green), the spikes were sprayed with 70% ethanol. Immature embryos (no longer than 0.3 mm) were isolated under a dissecting microscope and cultured in callus induction medium (CIM) at 24°C in the dark for 3 weeks. The induced embryogenic callus was subcultured and maintained in CIM at 24°C in the dark for another 2 weeks. Five weeks later, the callus tissue was separated again and transferred to a new CIM for one week.

[0105] (2) Infection, co-culture and the production of transgenic seedlings

[0106] Callus tissue was infected with Agrobacterium AGL1 containing the recombinant expression vector pYLox.5-BdRFS and 100 μM acetylsylgenin (AS). After vibratory vacuum infiltration for 10 minutes, the tissue was sonicated with cold water for 3-5 minutes, followed by vibratory vacuum infiltration for another 10 minutes. The callus tissue was then air-dried on several layers of sterile filter paper to remove excess Agrobacterium solution (placed in a laminar flow hood for 30 minutes). The callus tissue was placed on filter paper and co-cultured in a dark room at 24°C for 2-3 days (wrapped in plastic wrap and aluminum foil), then transferred to selective medium. The callus tissue on selective medium was passaged every 14 days until resistant callus tissue was formed. After 5-6 weeks of selection, healthy callus tissue (white and fluffy) was transferred to regeneration medium and cultured in a light room at 24°C, light / dark 16 / 8 hours. Shoots should appear within 2-4 weeks, then the tissue was transferred to rooting medium for rooting.

[0107] (3) PCR identification

[0108] When the plants were large enough, genomic DNA was extracted from young leaves using cetyltrimethylammonium bromide (CTAB). Positive transgenic lines were identified by PCR using primers designed from the selection marker gene hygromycin B phosphotransferase (forward primer: 5'-AAATCCGCGTGCACGAGGT-3' (SEQ ID NO: 11) and reverse primer: 5'-TCGTTATGTTTATCGGCACTTTGCA-3' (SEQ ID NO: 12)). Results are as follows: Figure 4 As shown, positive plants can amplify the corresponding band, which is the same size as the specific band amplified using the plant expression vector pYLox.5-BdRFS (positive control) as a template, while wild-type control plants cannot amplify this specific band, proving that the BdRFS gene has been introduced into the transgenic plants.

[0109] Example 5: Performance evaluation of transgenic two-spike short-stalked grass plants

[0110] I. Determination of Flowering Period

[0111] Seeds of wild-type and transgenic *Brachys edulis* (ecotype BD21-3) were surface-sterilized with a solution containing 30% (v / v) bleach and 0.05% (v / v) Tween 20, then treated in the dark at 4°C for 3 days and germinated on 1 / 2 MS agar (1% w / v) plates. The plants were then grown in a growth chamber (light intensity 175 μmol·m²). -2 ·s -1The plants were vertically cultured for 7 days in a 22℃ / 18℃, 16-hour light and 8-hour dark cycle, and then transplanted into a culture medium (peat: perlite = 3:1) and grown in a greenhouse (24℃, 16-hour light and 8-hour dark cycle) until flowering. The number of days from sowing to flowering was recorded.

[0112] II. Biomass Measurement

[0113] Seeds of wild-type and transgenic *Brachys edulis* (ecotype BD21-3) were surface-sterilized with a solution containing 30% (v / v) bleach and 0.05% (v / v) Tween 20, then treated in the dark at 4°C for 3 days and germinated on 1 / 2 MS agar (1% w / v) plates. The plants were then grown in a growth chamber (light intensity 175 μmol·m²). -2 ·s -1 The plants were vertically cultured for 7 days at 22℃ / 18℃ with a 16-hour light and 8-hour dark cycle, then transplanted to a culture medium (peat:perlite = 3:1) and grown in a greenhouse (24℃, 16-hour light and 8-hour dark cycle) until maturity (the maturity of *Bruguiera gymnorhiza* is indicated by seed maturity). The above-ground parts were collected, dried to constant weight, and weighed. Six individual plants from each line were measured, and the average value was calculated.

[0114] according to Figure 5 The results show that: Figure 5 As shown in (A), compared to the wild-type *Brachys bipinnatus*, the expression level of BdRFS in the transgenic *Brachys bipinnatus* was significantly increased; Figure 5 As shown in (B), compared to the wild-type *Brachys bipinnatus*, the transgenic *Brachys bipinnatus* has a longer flowering period; from Figure 5 As shown in (C), the aboveground biomass of the transgenic *Brachypodium distichum* was higher than that of the wild-type plant. These results indicate that the BdRFS gene can delay the flowering period and increase biomass in plants.

[0115] III. Drought Resistance Testing

[0116] Transgenic *Brachys bipinnatifida* and its wild-type seedlings were planted in the same substrate and culture pots. After growing in a greenhouse for 35 days, watering was stopped for 14 days. When the wild-type plants showed obvious wilting, mature leaves were collected to measure the relative water content (RWC) and ion leakage. The measurement methods are as follows:

[0117] Relative moisture content test: Fresh weight of leaves was measured immediately after cutting, then placed in beakers and left in the dark for 24 hours before being weighed at saturation. Afterwards, the leaves were dried in a forced-air drying oven at 80℃ for 48 hours, and the dry weight was measured. This was repeated three times, and the average value was taken as the relative moisture content of the sampled plant leaves. The calculation formula was: RWC(%) = (fresh weight - dry weight) / (saturation weight - dry weight) × 100%. Five individual plants from each line were measured, and the average value was calculated.

[0118] Conductivity test: Leaves were placed in Erlenmeyer flasks containing 20 mL of deionized water and incubated overnight at 4°C. The conductivity of the solution was measured using a conductivity meter (C1). The Erlenmeyer flasks were then placed in a boiling water bath for 20 min and cooled to room temperature. The conductivity of the solution was measured again using a conductivity meter (C2). The relative conductivity was calculated using the formula (C1 / C2) × 100. Five individual plants from each line were measured, and the average value was calculated.

[0119] from Figure 6 It can be seen that after 14 days of drought treatment, the relative water content of the transgenic *Brachys edulis* was higher than that of the wild type. From... Figure 7 It can be seen that after 14 days of drought treatment, the relative electrical conductivity of the transgenic *Brachys bipinnatifida* was lower than that of the wild type. This indicates that under drought stress, the transgenic *Brachys bipinnatifida* exhibited improved drought resistance compared to the wild type.

[0120] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application. Furthermore, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

Claims

1. The application of overexpression of BdRFS protein in delaying plant flowering, increasing biomass, or enhancing drought resistance, characterized in that, The amino acid sequence of the BdRFS protein is shown in SEQ ID NO: 4; The plant in question is *Hemiberlesia lataniae*.

2. The application of biomaterials associated with overexpression of BdRFS protein in delaying plant flowering, increasing biomass, or enhancing drought resistance, characterized in that... The biomaterial contains at least one of the following (1) to (4): (1) Nucleic acid molecules encoding the BdRFS protein; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant expression vector containing the nucleic acid molecule described in (1); (4) Transformants containing the recombinant expression vector described in (3); The amino acid sequence of the BdRFS protein is shown in SEQ ID NO: 4; The plant in question is *Hemiberlesia lataniae*.

3. The application according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the BdRFS protein is shown in SEQ ID NO:

3.

4. A method for cultivating drought-resistant, high-biomass, and late-maturing transgenic plants, characterized in that, The recombinant expression vector was transformed into plant tissue, and the transformed plant tissue was cultured to obtain transgenic plants; The plant in question is *Sophora bipinnata*. The recombinant expression vector was constructed from a nucleic acid molecule encoding the BdRFS protein and a plant expression vector; The amino acid sequence of the BdRFS protein is shown in SEQ ID NO:

4.

5. The method according to claim 4, characterized in that: The plant expression vector is pYLox.

5.

6. The method according to any one of claims 4 or 5, characterized in that, The transformation methods include Agrobacterium-mediated transformation, gene gun transformation, electroporation, PEG vector transformation, and liposome transformation.

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