A flowering-related gene DG1C02448.1 and its application

Abstract

The present invention discloses a flowering-related gene DG1C02448.1 and its application. The nucleotide sequence of the gene is shown in SEQ ID NO. 3, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 4. By transferring the gene into Arabidopsis thaliana for overexpression, it was found that the Arabidopsis overexpressing the DG1C02448.1 gene flowered significantly earlier, potentially suggesting that the gene is related to the flowering of Dactylis grass. The function of the gene has potential application value in the improvement of traditional forage grasses. At the same time, in order to address the shortcomings of traditional forage improvement technologies, such as slow results and long cycles, the gene and its potential function can shorten breeding time, improve breeding efficiency, and help promote the development and utilization of the high-quality grass forage Dactylis grass.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a flowering-related gene DG1C02448.1 and an application thereof. Background Art

[0002] Dactylis, a member of the genus Dactylis in the subfamily Festucoideae of the Poaceae family, is a widely cultivated perennial cool-season forage grass worldwide. It boasts abundant leaves, high yield, shade tolerance, strong adaptability, good palatability, and high nutritional value. It can be used for green fodder, hay, or silage, making it one of the four most widespread grasses in the world. Approximately 14,000 tons of Dactylis seeds are produced annually, accounting for 3.3% of the world's temperate forage seed supply. Currently, Dactylis is cultivated in Qinghai, Gansu, Shaanxi, Shanxi, Henan, Jilin, Jiangsu, Hubei, Sichuan, and Xinjiang provinces (autonomous regions). It is used for both mowing and grazing, and is favored by various poultry species. It has achieved excellent economic and ecological benefits and demonstrates broad potential for its utilization. Flowering period is a key agronomic trait of Dactylis, closely linked to its quality and yield. Flowering period marks the transition from vegetative to reproductive growth, with consequent changes in forage quality and yield. Duckgrass is suitable for mixed sowing with legume forages. Targeted cultivation of duckgrass varieties with different flowering periods and combination with legume forage varieties with the same maturity period can extend the grazing time of mixed grasslands and improve overall production performance and utilization efficiency.

[0003] Perennial grasses require a certain age to flower, and the molecular mechanisms of the plant aging pathway are a hot topic in the field of flowering. The aging pathway primarily regulates SPL family members through miR156, which in turn activates the expression of the florigen FT, ultimately leading to floral transformation. We have discovered a key candidate gene, DgSPL13, that regulates flowering in Dactylis grass. This gene is a homolog of TaSPL13 in wheat. Previous studies have shown that mutations in the microRNA156 recognition element lead to a roughly two-fold increase in TaSPL13 expression in wheat, resulting in an early-flowering phenotype in wheat. Therefore, the Dactylis grass DgSPL13 gene (DG1C02448.1) may be involved in regulating flowering time and has important potential applications in improving forage quality. Dactylis grass is a heterozygous plant with complex ploidy, making its genetic transformation difficult, and verification of gene function is relatively slow. So far, the research on Dactylis grass DG1C02448.1 gene is still blank. Using DgSPL13 to transfer it into Dactylis grass has important theoretical and application value for studying the flowering time of Dactylis grass. Summary of the Invention

[0004] The purpose of the present invention is to provide a flowering-related gene DG1C02448.1 and its application. Arabidopsis thaliana overexpressing the DG1C02448.1 gene blooms significantly earlier, potentially suggesting that the gene is related to the flowering of orchardgrass. The gene and its potential function can shorten breeding time, improve breeding efficiency, and help promote the development and utilization of high-quality Poaceae forage orchardgrass.

[0005] In order to achieve the above object, the present invention provides a flowering-related Dactylis grassi gene DG1C02448.1, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0006] The present invention also provides a protein encoded by the orchid grass gene DG1C02448.1, the amino acid sequence of which is shown in SEQ ID NO.4.

[0007] The orchardgrass gene DG1C02448.1 provided by the present invention can be used for the transformation of Arabidopsis thaliana, and can be particularly used for promoting early flowering of Arabidopsis thaliana and preparing early flowering Arabidopsis thaliana strains.

[0008] The present invention also provides an overexpression vector comprising the orchidgrass gene DG1C02448.1.

[0009] The present invention also provides an overexpression Agrobacterium comprising the orchidgrass gene DG1C02448.1.

[0010] The orchardgrass gene DG1C02448.1 provided by the present invention can be used for orchardgrass improvement.

[0011] Preferably, the above-mentioned Dactylis grassi improvement includes any one of the following: shortening the breeding time, improving the breeding efficiency and / or promoting the development of high-quality Dactylis grassi.

[0012] The orchardgrass gene DG1C02448.1 provided by the present invention can be used to prepare early-flowering orchardgrass.

[0013] The present invention has the following advantages:

[0014] This invention is the first to explore the function of the Dactylis grassi gene DG1C02448.1 and find that overexpression of this gene can significantly promote early flowering of Arabidopsis thaliana, suggesting that this gene is related to the flowering of Dactylis grassi. The function of this gene has potential application value in the improvement of traditional forage. At the same time, for the shortcomings of traditional forage improvement technology such as slow effect and long cycle, this gene and its potential function can shorten breeding time and improve breeding efficiency, which will help promote the development and utilization of high-quality Poaceae forage Dactylis grassi. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the expression results of the DG1C02448.1 gene at different stages in the present invention.

[0016] Figure 2 This is the protein subcellular localization result of the DG1C02448.1 gene in the present invention.

[0017] Figure 3 The expression results of DG1C02448.1 gene in the homozygous positive seedlings of the present invention are shown.

[0018] Figure 4 These are the phenotypic results of the wild-type and overexpressing DG1C02448.1 strains during flowering.

[0019] Figure 5 These are the statistical results of flowering time for the wild-type and DG1C02448.1-overexpressing strains in the present invention.

[0020] Figure 6 The figures are statistical results of the number of rosette leaves of the wild-type and overexpressing DG1C02448.1 lines during flowering.

[0021] Figure 7 These are the expression level determination results of genes (AP1, FLC, FUL) related to flowering time in the overexpressing DG1C02448.1 line of the present invention. DETAILED DESCRIPTION

[0022] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0023] Note: Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples, unless otherwise noted, are commercially available.

[0024] Experimental Example 1 Gene cloning

[0025] The experimental material was the Duckgrass cultivar 'Baoxing', grown at the Wenjiang campus of Sichuan Agricultural University. Total RNA was extracted from young leaves. RNA was extracted using the Tiangen (Beijing) Biochemical Technology Co., Ltd. Plant Total RNA Extraction Kit, following the included instructions. RNA integrity was assessed by 1% agarose gel electrophoresis, and RNA concentration and purity were determined using an ultra-micro spectrophotometer. Reverse transcription was performed using the TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit, following the included instructions.

[0026] Using Baoxing RNA as a template, primers were designed based on the full-length sequence, as follows:

[0027] Gene-F (SEQ ID NO. 1):

[0028] 5'-ATGGAGCGCAAGGACAAGTC-3',

[0029] Gene-R (SEQ ID NO. 2):

[0030] 5'-TATCTGATCTGGAAGTGGTTCAGC-3'.

[0031] The extracted RNA was converted into cDNA, and PCR amplification was performed using the cDNA as a template. The amplification was performed using the 2×Phanta Max MasterMix (Dye Plus) kit from Vazyme. The operation procedure was referred to the enclosed instruction manual.

[0032] The reaction system for PCR amplification was 50 μL: 25 μL 2×Phanta Max Master Mix (Dye Plus), 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), 4 μL template DNA, and 17 μL ddH2O.

[0033] The PCR amplification reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, 35 cycles; and extension at 72°C for 5 min.

[0034] The amplified product was recovered after electrophoresis as follows:

[0035] Electrophoresis: Add an appropriate amount of 10× loading buffer to each reaction tube and perform electrophoresis on a 1%-3% agarose gel (10 μL EB, 1-3 g agarose / 100 mL 0.5× TBE buffer). Perform electrophoresis at 5-10 V / cm in 0.5× TBE buffer. After electrophoresis, photograph the gel using a gel imaging system.

[0036] Agarose gel DNA recovery (GK2042, Jierui Biotechnology):

[0037] 1) Carefully cut off the target DNA band and place it in a 1.5 mL EP tube.

[0038] 2) Add 400 μL of banding B to the tube and place it in a 70°C water bath until the gel is completely dissolved.

[0039] 3) Add 100 μL of isopropanol to the tube, incubate at room temperature for 1 minute, and centrifuge at 5000 rpm for 1 minute to pass through the column.

[0040] 4) Repeat step 3.

[0041] 5) Add 500 μL wash buffer and wash twice at 12,000 rpm. Centrifuge at 10,000 rpm for 1 minute.

[0042] 6) Add 40 μL of double-distilled water to the column, incubate at 37°C for 2 minutes, and centrifuge at 12,000 rpm for 1 minute to collect the liquid.

[0043] The sequence of the gene recovered after amplification was sequenced, and the nucleotide sequence of the gene was shown as SEQ ID NO. 3. The specific sequence is as follows:

[0044] ATGGAGCGCAAGGACAAGTCCCGCAAGTCTTCCTCCGCGGCGGCC

[0045] ATGGCCGCGCTCGCCGCCGCAGCTGCAGTCGGAGCCGACGGCATCATGC

[0046] CTCCGTCCGGGGAGGAGGACAAGAAGCTGAACCTTGTGAACGTGCCCG

[0047] TGATCACCGTCGGCGCCTCGAGCTCCTCTTCCGCGGCTGTGGCGGTGAG

[0048] GAGGAACGGTGGTGCCGGTGGCGCCGTCGCGGCCGGGGCAGGCGGGC

[0049] CGATCTGCCAGGCTGAGAGGTGCGGCGTCGACCTCACCGACGCGAAGC

[0050] GTTACCACCGCAGGCACAAGGTGTGCGACGCGCACTCCAAGGCCGTCG

[0051] TCGCGATCGTCGGCGGCCTCCGCCAGCGCTTCTGCCAGCAATGCAGCCG

[0052] GTTCCACGAGCTTGTGGAGTTCGACGACACCAAGCGCAGCTGCCGCCG

[0053] GCGTCTAGCCGGGCACAACGAGCGGAGGAGGAAGAGCTCATCGGACGC

[0054] CAACGGCGGCGACGGATGCCGCCACGCCGATCAGGACGGCCGGGGCCA

[0055] TCCGGGAACCCTCCGCTGAACCACTTCCAGATCAGATAA.

[0056] The amino acid sequence of the protein expressed by this gene is shown in SEQ ID NO.4, and the specific sequence is as follows:

[0057] MERKDKSRKSSSAAAMAALAAAAAVGADGIMPPSGEEDKKLNLVNVPVITVGASSSSSAAVAVRRNGGAGGAVAAGAGGPICQAERCGVDLTDAKRYHRRHKVCDAHSKAVVAIVGGLRQRFCQQCSRFHELVEFDDTKRSCRRRLAGHNERRRKSSSDANGGDGCRHADQDGRGGHPGNPPLNHFQIR.

[0058] The gene was named DG1C02448.1.

[0059] Experimental Example 2: Functional Analysis of DG1C02448.1

[0060] 1. Expression of DG1C02448.1 gene at different stages

[0061] During the vernalization period, late spring flowering period, vegetative growth period, booting period, heading period and flowering period of Dactylis 'Baoxing', the whole genome was extracted from leaves and the transcriptome was analyzed. The expression levels of the DG1C02448.1 gene at different periods were as follows: Figure 1 As shown in the figure, the expression level of DG1C02448.1 gradually increased from the vernalization stage to the booting stage, and then gradually decreased during the heading and flowering stages after the booting stage. This suggests that this gene may be involved in the transition from the vegetative to the reproductive growth stage of Dactylis grass and is a potential candidate gene for regulating the heading and flowering of Dactylis grass.

[0062] 2. Localization of DG1C02448.1 protein

[0063] To determine the location of DG1C02448.1, the open reading frame (ORF) of DG1C02448.1 was inserted into the pAN580-35S-EGFP vector. The empty pAN580-35S-EGFP vector was used as a control. The fusion vector and the empty control vector were then transformed into rice protoplasts as follows:

[0064] 2.1 Synthesis of amplification primers for the DG1C02448.1 target gene

[0065] pAN580-DG1C02448.1-F (SEQ ID NO.5):

[0066] AAGTCCGGAGCTAGCTCTAGatggagcgcaaggacaagtccc

[0067] pAN580-DG1C02448.1-R(SEQ ID NO.6):

[0068] AGCGGCCGCTGTACAGGATCtctgatctggaagtggttcagcgg

[0069] 2.2 PCR system and amplification procedure

[0070] The system is 50 μL: Nuclease-free Water 20 μL, Biorun Pfu PCR Mix 25 μL, Primer-F2 μL, Primer-R 2 μL, and Template 1 μL.

[0071] Amplification program: 94°C for 5 min; 4°C for 30 sec, 50°C for 45 sec, 72°C for 76 sec, 30 cycles; 72°C for 10 min; 16°C for 30 min.

[0072] The PCR products were subjected to DNA electrophoresis and recovered as described above.

[0073] 2.3 Vector digestion

[0074] The enzyme digestion and ligation system is shown in Table 1. The reaction conditions are 37°C for 1 h. The vector digestion product is purified using a PCR purification kit for the next recombination reaction.

[0075] Table 1 Enzyme digestion reaction system

[0076]

[0077] 2.4 Recombination reaction

[0078] The recombination reaction system is shown in Table 2. The reaction conditions are 37°C for 30 minutes, and the ligation product is transformed into competent cells.

[0079] Table 2 Recombination reaction system

[0080]

[0081] 2.5 Transformation: 5-10 μL of the ligation product was transformed into competent E. coli (see standard method for competent E. coli transformation) onto an Amp-resistant plate, cultured at 37°C for 12 hours, and identified by colony PCR.

[0082] 2.6 Colony identification and plasmid extraction

[0083] After selecting the colonies for PCR identification, the correct single colony was obtained. The correct single colony was inoculated into 3 mL of LB liquid culture medium containing Amp antibiotics, and cultured at 37°C and 200 rpm overnight; 1.5 mL of culture (3 mL for low-copy plasmid) was taken and centrifuged at 12,000 rpm for 30 seconds; the supernatant was aspirated and the bacteria were suspended in 100 μL of solution I (Glucose 50 mmol / L, EDTA 10 mmol / L, Tris-HCl 25 mmol / L, pH 8.0); 200 μL of freshly prepared solution II (NaOH 0.2 mol / L, SDS 1%) was added and immediately mixed gently up and down; 150 μL of solution III (KAc 5 mol / L, pH 4.8), quickly mix up and down, and let it stand at room temperature for 5 minutes; centrifuge at 12,000 rpm for 10 minutes; transfer the supernatant to another centrifuge tube, add 2 volumes of ethanol, and mix; centrifuge at 12,000 rpm for 10 minutes; remove the supernatant, wash the DNA pellet with 70% ethanol, centrifuge at 12,000 rpm for 1 minute, and remove the supernatant; vacuum dry the pellet; and dissolve it in 60 μL of double-distilled water containing 10 μg / mL RNase A.

[0084] 2.7 Cultivation of Agrobacterium

[0085] 1) Take the competent Agrobacterium stored at -80℃ and place it in the palm of your hand for a while until it partially thaws. When it is in an ice-water mixture, insert it into ice.

[0086] 2) Add 0.1 μg (no more than 10 μL) of plasmid DNA per 100 μL of competent medium, mix by hand by flicking the bottom of the tube, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0087] 3) Add 700 μL of LB liquid medium without antibiotics and culture at 28°C, 200 rpm, and shake for 2 to 3 hours.

[0088] 4) Harvest the cells by centrifugation at 6000 rpm for one minute. Collect approximately 100 μL of the supernatant and gently pipette to resuspend the cells. Spread the suspension onto an LB plate containing the appropriate antibiotic and incubate inverted in a 28°C incubator for 2-3 days. Randomly select a single colony and perform colony PCR to identify the correct overexpressing Agrobacterium clone and label it for future use.

[0089] 5) Use a sterile pipette tip to pick up the marked Agrobacterium single colony and inoculate it into 1.5 mL of LB liquid medium containing the corresponding antibiotic (in a 50 mL blue-capped centrifuge tube) and culture it at 30°C with shaking at 200 rpm for 24 hours.

[0090] 6) Inoculate 100 mL of LB liquid medium containing antibiotics with the Agrobacterium culture at a ratio of 1% and culture at 30°C with shaking until the OD600 is approximately 1.0.

[0091] 7) Centrifuge at 20°C, 4,000 rpm for 15 min to collect the cells; pipette the cells evenly with transformation buffer and resuspend them to approximately OD600 = 1.0.

[0092] 2.8 Transformation of Rice Protoplasts

[0093] 1) After culturing rice seedlings in the dark at approximately 30°C for 7-15 days, remove stems and leaves, rinse surface dirt with water, and remove the outermost leaf sheaths.

[0094] 2) Place the stems and leaves of the seedlings on a clean plastic board and cut them into small pieces with a sharp blade;

[0095] 3) Add 5-10 mL of enzymatic solution to completely immerse the tissue, gently shake at 28°C for 4-5 hours;

[0096] 4) Filter the protoplasts through a 40 μm filter, transfer to a 2 mL centrifuge tube, and centrifuge at 600 rpm for 5 min until a turbid precipitate is visible.

[0097] 5) Directly aspirate the supernatant, wash twice with 10 mL of pre-cooled W5 solution, and centrifuge at 600 rpm for 5 minutes at 25°C.

[0098] 6) Add appropriate amount of MMG solution as needed to suspend the mixture to a concentration of 2*10 5 / mL, microscopic examination: protoplasts are round and rarely ruptured;

[0099] 7) Take 100 μL of protoplast suspension and 10 μL of DNA, add an equal volume of PEG4000 solution, mix gently and evenly, and let it stand at room temperature for 10-15 minutes;

[0100] 8) Dilute the protoplasts with 1 mL of W5 and mix well to terminate the reaction;

[0101] 9) Centrifuge at 600 rpm for 5 min to collect the protoplasts and remove the supernatant;

[0102] 10) Add 1 mL of W5 solution for washing.

[0103] After transient expression in rice protoplasts, the protein subcellular localization results of DG1C02448.1 gene were obtained. Figure 2 Observing the fluorescence of DG1C02448.1 under a laser confocal microscope revealed that the cell fluorescence signal of the cell nucleus was obvious, which confirmed that the protein was localized in the cell nucleus.

[0104] 3. Transform into Arabidopsis for functional verification

[0105] 3.1 Overexpression vector construction

[0106] 1) Connection of homology arms of target gene

[0107] According to the map of the vector pHB-35S, BamHI / PstⅠ was designed as the insertion site and primers were synthesized. The primer sequences were:

[0108] pHG-35S-DG1C02448.1-F(SEQ ID NO.7):

[0109] CTCTCTCTCAAGCTTGGATCCATGGAGCGCAAGGACAAGTC;

[0110] pHG-35S-DG1C02448.1-R(SEQ ID NO.8):

[0111] GATCAATTCGAGCTCCTGCAGTTATCTGATCTGGAAGTGGTTC.

[0112] Using the target fragment DG1C02448.1 as a template, perform a PCR reaction to obtain the homology arm gene containing the pHB-35S restriction site. Recover the correct PCR fragment using the method described above.

[0113] 2) Enzyme digestion of pHB-35S plasmid:

[0114] Enzyme digestion system 40 μL: 30 μL plasmid, 4 μL 10× enzyme digestion buffer, 4 μL 10× BSA (with or without reference to the instructions), 6 U restriction endonuclease (NEB), add water to 40 μL, and treat in a 37°C water bath for about 1 hour.

[0115] 3) Construction of recombinant plasmid:

[0116] After PCR fragments were recovered from an agarose gel, they were mixed with the empty vector recovered by enzyme digestion and ligated using the EasyGenoDNA Recombination System (#VI201-02, Tiangen Biotechnology). 10 μL of the recombination system consisted of 5 μL of 2× EasyGenoAssembly Mix, 2.5 μL of enzyme-digested vector DNA, and 2.5 μL of fragment DNA. The reaction mixture was added to a 250 μL EP tube and placed in a 50°C water bath for 30 minutes. E. coli was then transformed onto a plate and incubated at 37°C for 16 hours. The cells were then picked and sent for sequencing. Correctly sequenced plasmids were stored at -20°C for long-term storage.

[0117] 4) Plasmid extraction

[0118] Inoculate a single colony into 3 mL of LB liquid medium containing kanamycin and culture overnight at 37°C and 200 rpm; take 1.5 mL of culture (3 mL for low-copy plasmid) and centrifuge at 12,000 rpm for 30 seconds; aspirate the supernatant and suspend the bacteria in 100 μL of solution I (Glucose 50 mmol / L, EDTA 10 mmol / L, Tris-HCl 25 mmol / L, pH 8.0); add 200 μL of freshly prepared solution II (NaOH 0.2 mol / L, SDS 1%) and immediately mix gently up and down; add 150 μL of solution III (KAc 5mol / L, pH=4.8), quickly mix up and down, and let it stand at room temperature for 5 minutes; centrifuge at 12,000 rpm for 10 minutes; transfer the supernatant to another centrifuge tube, add 2 volumes of ethanol, mix well; centrifuge at 12,000 rpm for 10 minutes; remove the supernatant, wash the DNA precipitate with 70% ethanol, centrifuge at 12,000 rpm for 1 minute, and remove the supernatant; vacuum dry the precipitate; and dissolve it in 60 μL of double-distilled water containing 10 μg / mL RNase A.

[0119] 3.2 Arabidopsis transformation

[0120] To culture Agrobacterium using the above method, 1) water plants that are about to sprout and flower well one day in advance; 2) invert the small pot and place all inflorescences in a pre-suspended bacterial solution in transformation buffer for approximately 30 seconds; 3) repeat the transformation process 7 days later. After 2-3 weeks, minimize watering to accelerate aging. Mature seeds are stored in a paper bag and placed in a desiccator for 7 days. The transformation buffer for Arabidopsis transformation (per L) contains: 25 mL of MS macro (20×), 0.5 mL of MS or B5 micro (1000×), 5 mL of MS organic (200×), 2.5 mL of MS iron salt (200×), 50 g of sucrose, 10 μL of 6-BA (1 mg / mL), and 400 μL of SILWET-77. After saturation, adjust the pH to 5.8 with KOH.

[0121] (1) Screening of transgenic Arabidopsis

[0122] 1) Preparation of culture medium: Arabidopsis thaliana culture medium is 1 / 2 MS (0.8% agar powder, no sucrose, pH = 5.8);

[0123] 2) Seed disinfection: Incubate in 70% ethanol for 1 minute, add 1 mL of 7% sodium hypochlorite solution (containing 1 drop of Tween) for 10 minutes, mix by inversion for 5 minutes, and rinse 5 times with sterile water.

[0124] 3) Resuspend the sterilized seeds in 100 μL of sterile water, pipette with a 1 mL pipette tip, and spot onto a 1 / 2 MS medium plate (with selection antibiotics added: 50 μg / mL KAN, 30 μg / mL HYG, or 50 μM Glufosinate-ammonium).

[0125] 4) Seal the plate and place in a refrigerator at 4°C for 48 hours for vernalization. Place in an artificial climate chamber to begin germination and growth. The plant growth environment is 60% relative humidity, a constant temperature of 20-22°C, a photoperiod of 16 hours light and 8 hours dark, and a light intensity of 80-200 μmol / M 2 / S;

[0126] 5) Observe after 8-15 days and transplant the positive ones into the planting soil;

[0127] 6) Preparation of planting soil: Mix peat soil and vermiculite in a ratio of 2:1 and set aside;

[0128] 7) Soil soaking: Fill the soil into the planting pot to about 1 cm from the pot mouth, and use Huawuque compound fertilizer (N, P, K = 20%, 20%, 20%) to completely soak it;

[0129] 8) Transplanting: 20 days after germination, select healthy and uniformly growing seedlings and transplant them into potting soil that has been soaked in Huawuque beforehand. Cover with plastic wrap and remove once the seedlings have established themselves.

[0130] 9) Arabidopsis thaliana T1 generation resistance screening positive plants were sampled at the seedling stage and identified by PCR.

[0131] Samples were collected from 20-day-old seedlings and DNA was extracted using a plant genomic DNA extraction kit (DP305) according to the included instructions. PCR analysis was performed on the extracted DNA to identify transgenic Arabidopsis thaliana.

[0132] (2) Verification of expression levels in T3 homozygous strains

[0133] After the T3 transgenic plants grew for 3 weeks, RNA was extracted and the expression level of DG1C02448.1 gene in 6 homozygous positive seedlings was verified. Figure 3 .

[0134] 3.3 Statistics of Arabidopsis Flowering Time

[0135] (1) Determination of flowering time

[0136] The wild-type Arabidopsis WT and the homozygous lines OE2 and OE9 obtained above were selected, and their flowering time was counted when the bolting height of Arabidopsis thaliana was 0.5 cm. The phenotypic results of the wild-type and overexpressing DG1C02448.1 lines at flowering were obtained as follows: Figure 4 The flowering time statistics are shown in Figure 5 The statistical results of the number of rosette leaves at flowering are shown in Figure 6 As shown, through Figure 4 、 5 , 6 It can be seen that the flowering time of the DG1C02448.1 overexpressing line is earlier than that of the wild type, and the number of rosette leaves at flowering is also less than that of the wild type, indicating that the expression of the DG1C02448.1 gene can promote early flowering of Arabidopsis.

[0137] (2) Fluorescence quantitative determination of genes related to flowering time

[0138] Samples were collected from overexpression and wild-type Arabidopsis plants after 3 weeks and subsequently frozen in liquid nitrogen. Three replicates were obtained for each sample. RNA was extracted and reverse transcribed from the obtained samples. The experimental methods for RNA and reverse transcription were as described above.

[0139] The expression levels of genes related to flowering time (AP1, FLC, FUL) were measured by fluorescence quantitative analysis. Figure 7 The results are shown in the figure, where WT represents wild-type Arabidopsis and OE#SPL13 represents Arabidopsis overexpressing the DG1C02448.1 gene. The results showed that overexpression of the DG1C02448.1 gene promoted the expression of the flowering genes AP1 and FUL, while inhibiting the expression of the flowering gene FLC. Therefore, the DG1C02448.1 gene may be a gene that promotes flowering in Dactylis glomerata.

[0140] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A flowering-related orchardgrass gene DG1C02448.1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

2. A protein encoded by the orchardgrass gene DG1C02448.1 according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

4.

3. Use of the Dactylis grassi gene DG1C02448.1 as claimed in claim 1 in promoting early flowering of Arabidopsis thaliana.

4. Use of the Dactylis grassi gene DG1C02448.1 as claimed in claim 1 in preparing an early-flowering Arabidopsis thaliana strain.

5. An overexpression vector comprising the orchidgrass gene DG1C02448.1 as claimed in claim 1.

6. An overexpressing Agrobacterium comprising the Dactylis gracilis gene DG1C02448.1 as claimed in claim 1.

7. Use of the orchardgrass gene DG1C02448.1 according to claim 1 in orchardgrass improvement, wherein the orchardgrass improvement comprises any one of the following: Shorten breeding time; Improve breeding efficiency.

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