Gene DG1C00225.1 for regulating and controlling flowering time of plant and application of gene DG1C00225.1

Abstract

The invention provides a gene DG1C00225.1 for regulating and controlling the flowering time of a plant and application of the gene DG1C00225.1, and belongs to the technical field of gene engineering, and the nucleotide sequence of the DG1C00225.1 is as shown in SEQ ID NO. 1. The blooming time of arabidopsis thaliana of the overexpressed gene DG1C00225.1 is obviously advanced, and the overexpressed gene DG1C00225.1 can be applied to cultivation of early flowering transgenic plants, shortens the breeding time, improves the breeding efficiency and promotes the development and utilization of gramineous forage grass.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a gene DG1C00225.1 for regulating the flowering time of plants and an application thereof. Background Art

[0002] Dactylis belongs to the genus Dactylis of the subfamily Festucoideae of the Poaceae family. It is a perennial cool-season clump forage grass widely cultivated worldwide. Dactylis has the advantages of high yield of leaves, shade tolerance, strong adaptability, good palatability and high nutritional value. It can be used for green feeding, hay preparation or silage. It is one of the four most widely distributed grasses in the world. About 14,000 tons of Dactylis seeds are produced globally each year, accounting for 3.3% of the world's temperate forage seeds. At present, Dactylis is cultivated in Qinghai, Gansu, Shaanxi, Shanxi, Henan, Jilin, Jiangsu, Hubei, Sichuan and Xinjiang provinces or regions. It is used for both mowing and grazing. Various poultry like to eat it. It has achieved good economic and ecological benefits and demonstrated broad prospects for utilization. The flowering period is an important agronomic trait of Dactylis, which is closely related to the quality and yield of Dactylis. The flowering period represents the transition of the plant from vegetative growth to reproductive growth, and the quality and yield of forage will also change accordingly. Duckgrass is suitable for mixed sowing with legume forage. 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. Summary of the invention

[0003] The purpose of the present invention is to provide a gene DG1C00225.1 for regulating the flowering time of plants and its application. The flowering time of Arabidopsis thaliana that overexpresses the gene DG1C00225.1 is significantly advanced, and it can be used to cultivate early-flowering transgenic plants, shorten the breeding time, improve the breeding efficiency, and thus promote the development and utilization of Poaceae forage.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention provides a gene DG1C00225.1 for regulating the flowering time of plants. The nucleotide sequence of the DG1C00225.1 is shown in SEQ ID NO.1.

[0006] The present invention also provides a protein encoded by the gene DG1C00225.1 for regulating the flowering time of plants. The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0007] The present invention also provides application of a gene DG1C00225.1 for regulating the flowering time of plants in Arabidopsis breeding.

[0008] Furthermore, the application includes promoting early flowering of Arabidopsis thaliana.

[0009] The present invention also provides an application of a protein encoded by the gene DG1C00225.1 for regulating the flowering time of plants in preparing an early-flowering Arabidopsis strain.

[0010] The present invention also provides an overexpression vector comprising the gene DG1C00225.1 for regulating the flowering time of plants.

[0011] The present invention also provides Agrobacterium comprising the gene DG1C00225.1 for regulating flowering time of plants.

[0012] The present invention also provides a method for cultivating early-flowering transgenic plants, which overexpresses the DG1C00225.1 gene in plants to obtain early-flowering transgenic plants;

[0013] Alternatively, the expression level of DG1C00225.1 protein in the plant is increased to obtain early-flowering transgenic plants;

[0014] Among them, the nucleotide sequence of the DG1C00225.1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the DG1C00225.1 protein is shown in SEQ ID NO.2.

[0015] Furthermore, the overexpression of the DG1C00225.1 gene in the plant to obtain the early-flowering transgenic plant specifically includes:

[0016] The DG1C00225.1 gene was homologously recombined with the vector pHG-35S to obtain the overexpression vector pHG-35S-DG1C00225.1;

[0017] The pHG-35S-DG1C00225.1 is transformed into plants, and early-flowering transgenic plants are obtained through screening and cultivation;

[0018] The plants include Arabidopsis thaliana.

[0019] Preferably, the step of transforming the pHG-35S-DG1C00225.1 into a plant specifically comprises:

[0020] The plant inflorescence was infected by the floral dip method to transform the pHG-35S-DG1C00225.1 into the plant.

[0021] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0022] The invention discloses a gene DG1C00225.1 for regulating the flowering time of a plant. Experiments have shown that overexpression of the DG1C00225.1 gene can promote early flowering of Arabidopsis thaliana. The DG1C00225.1 gene is applied to the genetic engineering modification of Arabidopsis thaliana to obtain an early flowering transgenic plant strain. The DG1C00225.1 gene is applied to the genetic engineering modification of Dactylis grass to obtain an early flowering Dactylis grass strain. The gene has the advantages of short breeding time and high breeding efficiency, has important utilization value for improving forage quality, and can effectively promote the development and utilization of the Gramineae forage Dactylis grass. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 This is the evolutionary tree analysis diagram of DG1C00225.1 protein and other homologous proteins.

[0025] Figure 2 This is the subcellular localization map of DG1C00225.1 protein.

[0026] Figure 3 is the relative expression level of DG1C00225.1 gene in different overexpression Arabidopsis strains.

[0027] Figure 4 This is a phenotype of flowering time for Arabidopsis wild type and DG1C00225.1 overexpressing strains.

[0028] Figure 5 The flowering time statistics of Arabidopsis wild type and DG1C00225.1 overexpressing strains.

[0029] Figure 6 Statistical chart of the number of rosette leaves during flowering in Arabidopsis wild type and DG1C00225.1 overexpressing strains.

[0030] Figure 7 Fluorescence quantification of flowering genes in Arabidopsis wild type and DG1C00225.1 overexpressing strains. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0032] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0033] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0034] The overall idea of ​​the present invention is as follows:

[0035] Many developmental processes in plants require the interaction between actin filaments and microtubules. Formins are large multidomain proteins that participate in a variety of actin-related processes, such as cell division and maintenance of cell polarity (Wasserman, 1998). In plant cells, formins promote actin nucleation as dynamic regulators of the cytoskeleton and can change plant growth phenotypes by interacting with microtubules and microfilaments. We performed genome-simplified sequencing on 249 Duckgrass accessions and found a key candidate gene DG1C00225.1 (DgFH18) that regulates Duckgrass flowering through genome-wide association analysis. We further studied its function and explored its regulatory role in Duckgrass flowering time.

[0036] It was found through experiments that overexpression of the DG1C00225.1 gene can promote the flowering time of Arabidopsis thaliana, and overexpression technology can also be used to increase the expression of DG1C00225.1 in Dactylis to promote early flowering of Dactylis. Dactylis is a cross-pollinated plant and its complex ploidy makes its genetic transformation difficult, and the verification of gene function is relatively lagging. Therefore, for the shortcomings of traditional forage improvement technology that is slow to take effect and has a long cycle, the present invention applies the DG1C00225.1 gene to cultivate early-flowering transgenic plants, which can shorten the breeding time, improve breeding efficiency, and promote the development and utilization of high-quality Gramineae forage Dactylis.

[0037] The following will describe in detail the gene DG1C00225.1 for regulating the flowering time of plants and its application in combination with the embodiments and experimental data.

[0038] Example 1

[0039] In this example, an evolutionary tree of DG1C00225.1 was constructed and the subcellular localization of DG1C00225.1 was performed.

[0040] 1. Evolutionary tree construction

[0041] The homologous sequences of DG1C00225.1 in 12 different species were searched and downloaded using the BLASTP tool on the NCBI website. The downloaded sequences were used to generate a phylogenetic tree using MEGA 7.0 software. The results showed that the DG1C00225.1 protein of Dactylis glomerata had the highest homology with the LrFH18 protein of Lolium rigidum ( Figure 1 )

[0042] 2. Subcellular Localization

[0043] 1. Amplification of target fragment

[0044] 1.1 Cloning of target fragment

[0045] The experimental material was the Duckgrass variety "2006-1", which was planted in the Wenjiang campus of Sichuan Agricultural University. The young leaves were used as the material to extract total RNA. The plant total RNA extraction kit of Tiangen (Beijing) Biochemical Technology Co., Ltd. was used for RNA extraction, and the operation was carried out according to the instructions attached. After RNA extraction, 1% agarose gel electrophoresis was used for integrity detection, and the RNA concentration and purity were determined using an ultra-micro spectrophotometer. The reverse transcription was performed using TaKaRa's PrimeScr ipt II 1st Strand cDNA Synthesis Kit, and the operation process was referred to the instructions attached.

[0046] Using RNA from "2006-1" as template, primers were designed by sequencing the full length of the

[0047] DG1C00225.1-F:5'-ATGAGGTGGCCGAGGAGCAGATT-3'

[0048] DG1C00225.1-R:5'-GTCGTCAGAATCAGATGAACTGG-3'.

[0049] The cDNA was used as a template for amplification. The 2×Phanta Max Master Mix (DyePlus) kit from Vazyme was used for amplification. The operation procedure was referred to the instruction manual attached.

[0050] 50uL reaction system:

[0051] 25uL 2×Phanta Max Master Mix(Dye Plus)

[0052] 2uL upstream primer (10μM)

[0053] 2uL downstream primer (10μM)

[0054] 4uL template DNA*

[0055] 17uL ddH2O.

[0056] Reaction procedure:

[0057] Pre-denaturation at 95°C for 3 min;

[0058] Denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 1 min, for 35 cycles;

[0059] Extension at 72°C for 5 min.

[0060] 1.2DNA electrophoresis and recovery:

[0061] 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, and take photos in a gel imaging system after the electrophoresis is completed.

[0062] Agarose gel DNA recovery (GK2042, Jierui Biotechnology):

[0063] 1) Carefully cut off the target DNA band and place it in a 1.5 mL EP tube.

[0064] 2) Add 400uL of banding B to the tube and place it in a 70℃ water bath until the gel is completely dissolved.

[0065] 3) Add 100uL of isopropanol to the tube, place at room temperature for 1 minute, and centrifuge at 5000rpm for 1 minute to pass through the column.

[0066] 4) Repeat step 3.

[0067] 5) Add 500uL wash buffer and wash twice at 12000rpm. Centrifuge at 10000rpm for 1 minute.

[0068] 6) Add 40uL of double distilled water to the column, incubate at 37°C for 2 minutes, and centrifuge at 12000 rpm for 1 minute to collect the product.

[0069] 2. Functional analysis of DG1C00225.1

[0070] Subcellular localization technology was used to determine the location of DG1C00225.1 protein. In order to determine the location of DG1C00225.1, the open reading frame (ORF) of DG1C00225.1 was inserted into the pAN580-35S-GFP vector, and the pAN580-35S-GFP empty vector was used as a control. The fusion vector and the control empty vector were transferred into tobacco leaves.

[0071] 2.1 Synthesis of DG1C00225.1 target gene amplification primers

[0072] pAN580-DG1C00225.1-F:AACACGGGGGACTTTGCAACatgaggtggccgaggagcag

[0073] pAN580-DG1C00225.1-R:CCTGAAGCGGCCGCTGTACAgtcgtcagaatcagatgaactggagc

[0074] 2.2 PCR system and procedure

[0075] PCR reaction was performed using cDNA as template.

[0076] Amplification system:

[0077] Ingredients volume Nuclease-freeWater 20uL BiorunPfuPCRMix 25uL Primer(+) 2uL Primer(-) 2uL Template 1uL Totalvolum 50uL

[0078] PCR procedure:

[0079]

[0080]

[0081] The PCR products were subjected to DNA electrophoresis and recovered as described above.

[0082] 2.3 Vector digestion

[0083] The vector used is pAN580-35S-GFP, and the restriction enzyme linking system is as follows:

[0084] Ingredients volume Nuclease-freeWater 12uL 10*buffer 2uL BnS 1uL Eco31I 1uL pBWA(V)HS-ccdb-GLosgfp 4uL Total volume 20uL

[0085] Reaction conditions:

[0086] temperature time 37℃ 1hours

[0087] The vector digestion product was purified using a PCR purification kit for the next recombination reaction.

[0088] 2.4 Recombination reaction

[0089] The ORF sequence of DG1 C00225.1 was recombined with the vector digestion product. The recombination reaction system was as follows:

[0090]

[0091] Reaction conditions:

[0092] temperature time 37℃ 30 hours

[0093] The ligation product was transformed into competent cells.

[0094] 2.5 Transformation of competent cells

[0095] 5-10uL of the ligation product was transformed into competent E. coli (see standard method for competent E. coli transformation) and transformed into Amp-resistant plates, cultured at 37°C for 12 hours, and then identified by colony PCR.

[0096] 2.6 Colony PCR Identification

[0097] Pick 10 colonies for 1.5mL EP tube inoculation and PCR identification, and design identification primers with pBWA(V)HS-ccdb-GLosgfp.

[0098] HS)35seq:tTCATTTGGAGAGAACACGGGggac(2861bp)

[0099] E7805(580C):ctttttcccctcggtcttgga(3503bp).

[0100] PCR system:

[0101]

[0102] PCR procedure:

[0103] step Number of cycles 94℃for5min 1 94℃for30sec 30 50℃for45sec 30 72℃for174sec 30 72℃for10min 1 16℃for30min 1

[0104] 2.7 Plasmid extraction from colonies

[0105] Inoculate the correct single colony into 3 mL of LB liquid culture medium containing Amp antibiotics, and culture overnight at 37°C and 200 rpm with shaking; take 1.5 mL of culture (3 mL for low-copy plasmid), centrifuge at 12,000 rpm for 30 seconds; aspirate the supernatant, 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 5 mol / L, pH 4.8), quickly mix up and down, let stand at room temperature for 5 minutes; centrifuge at 12,000rpm for 10 minutes; transfer the supernatant to another centrifuge tube, add 2 volumes of ethanol, mix; centrifuge at 12,000rpm for 10 minutes; remove the supernatant, wash the DNA precipitate with 70% ethanol, centrifuge at 12,000rpm for 1 minute, remove the supernatant; vacuum dry the precipitate; dissolve in 60μL double distilled water containing 10μg / mL RNase A.

[0106] 2.8 Tobacco leaf injection

[0107] (1) Tobacco cultivation: sow a number of tobacco seeds, culture them under 12-hour light conditions, and use them in experiments after one month of cultivation;

[0108] (2) Agrobacterium culture: The constructed vector plasmid was transformed into Agrobacterium (GV3101) by electroporation and cultured at 30°C for 2 days;

[0109] (3) Suspending Agrobacterium: Use an inoculation loop to scrape the Agrobacterium from the solid culture dish and inoculate it into 10 mL of YEB liquid culture medium with the corresponding resistance, and culture it at 170 rpm / min for 1 h;

[0110] (4) Collecting bacteria: centrifuge at 4000 rpm / min for 4 min and remove the supernatant;

[0111] (5) Resuspension: Resuspend the cells in 10 mM MgCl2 (containing 120 μM AS) suspension and adjust the OD600 to about 0.6;

[0112] (6) Injection: Select tobacco plants with good growth conditions and inject Agrobacterium solution into the lower epidermis of tobacco leaves using a 1 mL syringe without a pipette tip, and make a mark;

[0113] (7) Cultivation: Cultivate the injected tobacco plants under weak light for 2 days and then observe;

[0114] (8) Observation: Take the labeled tobacco leaves injected with Agrobacterium, make them into slides, observe them under a laser confocal microscope, and take pictures.

[0115] By injecting tobacco leaves, the fluorescence of DG1C00225.1 was observed under a laser confocal microscope and it was found that the protein may be localized in the nucleus and cytoplasm ( Figure 2 ).

[0116] Example 2

[0117] This example verifies the effect of DG1C00225.1 on the flowering time of Arabidopsis thaliana.

[0118] 1. Overexpression vector construction

[0119] 1.1 Connection of homology arms of target gene

[0120] According to the map of the vector pHG-35-GFP, BamHI / PstⅠ was designed as the insertion site and primers were synthesized. The primer sequences were:

[0121] pHG-35S-DG1C00225.1-F:CTCTCTCTCAAGCTTGGATCCatgaggtggccgaggagcagattg

[0122] pHG-35S-DG1C00225.1-R:ACGGGTCATGAGCTCCTGCAGgtcgtcagaatcagatgaactgg.

[0123] Using the target fragment DG1C00225.1 amplified in Part II, 1.1 of Example 1 as a template, a PCR reaction was performed to obtain a homology arm gene containing a pHB-35S restriction site. The correct PCR fragment was recovered using the same method as described above.

[0124] 1.2 pHG-35-GFP plasmid digestion:

[0125] 40μL enzyme digestion system:

[0126] Plasmid (30 μL)

[0127] 4 μL 10× digestion buffer

[0128] 4μL 10×BSA (with or without reference to the instruction manual)

[0129] 6U restriction enzyme (NEB)

[0130] Add water to make up to 40 μL

[0131] Incubate in a 37°C water bath for about 1 hour.

[0132] 1.3 Recombinant plasmid construction:

[0133] After the PCR fragment in step 1.1 was recovered by agarose gel, it was mixed with the empty vector recovered by restriction digestion in step 1.2 and added to the EasyGenoDNA recombination system (#VI201-02, Tiangen Biotechnology). 10 μL of the recombination system was as follows:

[0134] 5μL 2×EasyGeno Assembly Mix

[0135] 2.5 μL digested vector DNA

[0136] 2.5 μL fragment DNA

[0137] Add the reaction system into a 250μL EP tube, place it in a 50℃ water bath for 30 minutes, then transform the E. coli to plate, place it in a 37℃ incubator for 16 hours, pick the bacteria, send it for sequencing, and store the correctly sequenced plasmid at -20℃ for long-term storage.

[0138] 1.4 Plasmid extraction

[0139] Inoculate a single colony in 3 mL of LB liquid culture medium containing kanamycin, and culture overnight at 37°C and 200 rpm with shaking; take 1.5 mL of culture (3 mL for low-copy plasmid), centrifuge at 12,000 rpm for 30 seconds; aspirate the supernatant, 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 5 mol / L, pH 4.8), quickly mix up and down, let stand at room temperature for 5 minutes; centrifuge at 12,000rpm for 10 minutes; transfer the supernatant to another centrifuge tube, add 2 volumes of ethanol, mix; centrifuge at 12,000rpm for 10 minutes; remove the supernatant, wash the DNA precipitate with 70% ethanol, centrifuge at 12,000rpm for 1 minute, remove the supernatant; vacuum dry the precipitate; dissolve in 60μL double distilled water containing 10μg / mL RNase A.

[0140] 2. Arabidopsis Transformation

[0141] 2.1 Cultivation of Agrobacterium:

[0142] 1) Take the competent Agrobacterium stored at -80℃ and place it at room temperature or in the palm of your hand for a while until it partially melts. When it is in a state of ice-water mixing, insert it into ice.

[0143] 2) Add 0.1 μg (volume not more than 10 μL) plasmid DNA for every 100 μL competent medium, stir the bottom of the tube by hand to mix, 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.

[0144] 3) Add 700 μL of LB liquid culture medium without antibiotics and culture at 28°C and 200 rpm with shaking for 2 to 3 hours.

[0145] 4) Centrifuge at 6000rpm for one minute to collect the bacteria, take about 100μL of the supernatant, gently blow and resuspend the bacteria, spread it on the LB plate containing the corresponding antibiotics, and invert it in a 28℃ incubator for 2-3 days. Randomly select a single colony, perform colony PCR, identify the correct Agrobacterium single clone, and mark it for later use.

[0146] 5) Use a sterile pipette tip to pick up the marked Agrobacterium single clone and inoculate it into 1.5 mL LB liquid culture medium containing the corresponding antibiotics (use a 50 mL blue-capped centrifuge tube) and culture it at 30°C and 200 rpm for 24 hours.

[0147] 6) Inoculate the Agrobacterium culture in 100 mL of LB liquid medium containing antibiotics at a ratio of 1% and culture at 30°C with shaking until OD600 = about 1.0;

[0148] 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 about OD600 = 1.0.

[0149] The conversion buffer components are as follows:

[0150]

[0151] 2.2 Arabidopsis transformation:

[0152] 1) Water the Arabidopsis thaliana that is sprouting and flowering one day in advance;

[0153] 2) Invert the flower pot and place all the inflorescences into the Agrobacterium solution previously suspended in transformation buffer for about 30 seconds;

[0154] 3) After 7 days, repeat the transformation as described above. After 2-3 weeks, water as little nutrient solution as possible to accelerate aging. Collect the mature seeds in paper bags and place in a desiccator for 7 days.

[0155] 2.3 Screening of transgenic Arabidopsis

[0156] 1) Preparation of culture medium: Arabidopsis thaliana culture medium is 1 / 2MS (0.8% agar powder, without sucrose, Ph 5.8);

[0157] 2) Seed disinfection: 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 with sterile water 5 times;

[0158] 3) Resuspend the sterilized seeds with 100 μL sterile water, draw up with a 1 mL pipette tip, and spot on a 1 / 2 MS medium (selective antibiotics are added to the medium: 50 μg / mL KAN or 30 μg / mL HYG or 50 μM Glufosinate-ammonium) plate;

[0159] 4) Seal the plate, put it in a refrigerator at 4 degrees for 48 hours, and put it in an artificial climate chamber to start germination and growth. The plant growth environment is 60% relative humidity; constant temperature 20-22 degrees; photoperiod is 16 hours of light and 8 hours of darkness; light intensity is 80-200μmol / M 2 / S; 5) observe after 8-15 days, identify the positive ones and transplant them into planting soil;

[0160] 6) Preparation of planting soil: Mix peat soil and vermiculite at a ratio of 2:1 and set aside for later use;

[0161] 7) Soaking the soil: put the soil into the planting pot to about 1cm from the pot mouth, and use Huawuque compound fertilizer (N, P, K = 20%, 20%, 20%) to completely soak it;

[0162] 8) Transplantation: 20 days after germination, select the healthy and uniformly growing seedlings and transplant them into the culture soil that has been soaked in Huawuque beforehand. Cover it with plastic wrap and remove it after the seedlings grow strong.

[0163] 2.4 Verification of expression levels in homozygous Arabidopsis T3 lines

[0164] After the T3 transgenic Arabidopsis thaliana grew for 3 weeks, RNA was extracted to verify the expression level of DG1C00225.1 gene in the positive seedlings of 4 homozygous strains (overexpression strains are called OE) (e.g. Figure 3 ).

[0165] The quantitative primers are as follows: DG1C00225.1-qPCR-F: TCTTTGTCGCACGAAGTTGC

[0166] DG1C00225.1-qPCR-R: ATAAAGCGCTCCAGGTTGCT.

[0167] 3. Overexpression of DG1C00225.1 gene promotes early flowering in Arabidopsis

[0168] Phenotypic identification and statistics of flowering time and number of rosette leaves:

[0169] Flowering time determination: The flowering time of Arabidopsis thaliana was counted when the bolting height was 0.5 cm;

[0170] Determination of rosette leaf number: The number of rosette leaves was counted when Arabidopsis thaliana was flowering.

[0171] The results of the test are as follows Figures 4 to 6 As shown, through Figure 4 , Figure 5 and Figure 6 It can be seen that the flowering time of the DG1C00225.1 overexpressing lines OE6, OE7 and OE8 is earlier than that of the wild type WT, and the number of rosette leaves at flowering is also less than that of the wild type. Figure 5 , 6 In the table, different lowercase letters represent significant differences in flowering time / rosette leaves among different lines.

[0172] 4. Fluorescence Quantification of Genes Related to Flowering Time

[0173] Samples were taken from overexpressed and wild-type Arabidopsis plants after 3 weeks, and then frozen in liquid nitrogen, with 3 replicates for each sample. RNA was extracted and reverse transcribed from the obtained samples, and the experimental methods of RNA and reverse transcription were as above.

[0174] The results showed that overexpression of DG1C00225.1 promoted the expression of Arabidopsis flowering genes AtAP1, AtFUL and AtFT, while inhibiting the expression of flowering gene AtFLC. Therefore, DG1C00225.1 may be a gene that promotes flowering in Dactylis glomerata. Figure 7 ).

[0175] In the present invention, the full gene sequence of DG1C00225.1 is as follows:

[0176] atgaggtggccgaggagcagattgaaatggctcctcagcgcatgcctcatctctctcctgctcctcacgccgac

[0177] ggactgcaatgggctgcagcttgttgccgccatcaggaagaacttattttggccaccagcaccaccccatctgtctct

[0178] ttcgagccaaataggtgatacattggtggaacaattatggctcaactgtggtctagataggataatccttcaagaagt

[0179] taaaaatcaatcctattacacccctctattcaacatcatcagcaattcctaccgaacaaataaaaagatcgaggcagg

[0180] aatgttcacttctttgtcgcacgaagttgccaatactttccaggactgcttaagcaagcataatttcattttgtcgga

[0181] tggcttctctgggcatggccaggtagaagaggagaaggaggcaggaagcttattgtcaaataccaataaatttaaact

[0182] gccttttgcatcaaaaataagatatctactcgaaggaactccagcatcacacttatctttgtcaccgccagcaaaaga

[0183] agcaacctggagcgctttatcttccgaagcagagtctcgtccactggtcactagcataaaaaaaatcttccaagaccga

[0184] ggggaaaaagaagagcaaggacaaagactcagattcatccactgttgtcttgggtttggcagtgggctgcgtggcatt

[0185] gctggctcttattgtgtacttctgtgcttgtcgtggagatgattcagcgtcacdatagatctagggagggatgataa

[0186] accactcctaggtttgactgatctagcaggttcttcccgtaagtccagcgctacaccaatcgatgtcagtaggttggg

[0187] agcattgtcgcgcagttcatccgagatcccgcagtctgaatttgctgtaccaatcaagatgcatactacacagccatc

[0188] cgcgaagttgaagtcagtaggagcgatgtcaatgaaggcagaactaatggagcggcacagcaggctgagctcgcacga

[0189] gataacaaccgttgctggacgtccgtcggttgctaactccctagccgagaaggcttcaggttcttctgctactggtaa

[0190] tgcagttacacatgcaggtccacctccaccaccacctccagctcttcctggaaaggctcctccgcctcctccagttct

[0191] tcctggaaggactccgccacctcctcctcctgcacccggtgcacctgcaccaccaccactaccaggagcagcagcatc

[0192] agcaccaccaccacctccgaagccggccgggcctcctcctcctgggccaccaccacctcctgctccaagagctggagc

[0193] aggacctggacctccacctccaccacttaaaaaaggtggaccacctggagctggacctccgccaccagcaatgcccgg

[0194] tggtcctaaaaaaggtggaccaccaccgtttaagaagccaggagcggcagctcctgttgcagatacttcaaaaacaaa

[0195] attgaagcccttcttctgggacaaggttgctgcaagtccagatcaagcaatggtgtgggatcaaattaaagccggatc

[0196] cttccagtttaatgaggagatgatcgaaactctttttggttgcaacgctgttgacaagaaaagtaccgatggcaaaaa

[0197] ggagccagcaaaggaagcagcccaatttgttaggatcctcgaacctaaaaaggcacaaaatttggcaatttcactgaa

[0198] ggcactcagtgtttcagctgcagatgtacgtactgcagtgacagaagggtatgaactcccctctgatttgatacaaac

[0199] attgatacggtggatcccaactagcgacgaggagctacgacttcggctgtacactggagagatgagtcaacttggtca

[0200] ggccgagcaattcttgaagaccatcattgaaatcccatacattttccagcgcttggaggtgttacttttcatggccag

[0201] tttaccggaagaagccgcaggtgtgaagcagtcatttgaaaccctagaggtggcctgccaagagcttaggcacaaccg

[0202] tcttttcaagaagctgctggaggctgtacttaaaacaggcaaccgaatgaatgatggtaccttccgtgggggagcaca

[0203] agcgttcaaactggacaccctcctgaagctggctgatgtcaagggggtcgacggcaagacgacgctactgcatttcgt

[0204] cgtccaggagatcatccgctctgagggcgtccgcgcggtgcgggcagcgaaggagcagaacagcagcatttccagcgt

[0205] gagcagcaccgatgatctcaccgaggatgtcagcgacgacacggagcactataagcagctgggcctcgccgtggtgtc

[0206] caacctaggggaggacctccagaacgtccgcaaggcagccatcctggacgcggacgcgctgaccatcatggtggcgag

[0207] cctcgggcataggttggtgaaggcgaacgagttcttgaacacgagcatgaagagcttggaggaggagagcgggttcca

[0208] acgcaagttggtccagttcatagagcagtctcaggtgcaggtgacccacctgctagaggaggagaagaagcttcgctc

[0209] gcttgtgcgtaccactgtggattacttccacggcagcaccgggaaggacgaggggttgcgactgttcgtcatcgtccg

[0210] tgacttcctggcgatactggacagggtgtgcagggaggtgaaagaggcagccgccaaagcagccgctgctaacaagaa

[0211] agaggcggctgccgccaaagcagcagcggcaccgccagcaccacccacaaggggcaggcaaccgtcccagacgtctat

[0212] gtctttccgtgaccctcggcagcatctgaagcccgcgatccaaggacggagggggaaggcacacagcagctccagttc

[0213] atctgattctgacgactga.

[0214] The protein sequence of DG1 C00225.1 is as follows:

[0215] MRWPRSRLKWLLSACL I SLLLLTTPDCNGLQLVAAI RKNLFWPPAPPHLSLSSQIGDTLVEQLWLNCGLDRI IL

[0216] QEVKNQSYYTPLFN II SNSYRTNKKI EAGMFTSLSHEVANTFQDCLSKHNF ILSDGFSGHGQVEEEKEAGSLLSNTNK

[0217] FKLPFASKI RYLLEGTPASHLSLSPPAKEATWSALSSEAESRPLVTS IKKSSKTEGKKSKDKDSDSSTVVLGLAVGC

[0218] VALLAL IVYFCACRGDDSASPYDLGRDDKPLLGLTDLAGSSRKSSATPIDVSRLGALSRSSSEIPQSEFAVPI KMHTT

[0219] QPSAKLKSVGAMSMKAELMERHSRLSSHE I TTVAGRPSVANSLAEKASGSSSATGNAVTHAGPPPPPPPALPGKAPPPP

[0220] PVLPGRTPPPPPPAGAPAPPPLPGAAASAPPPPPPKPAGPPPPGPPPPPAPRAGGPPPPPPLKKGGPPGAGPPPPA

[0221] MPGGPKKGGPPPFKKPGAAAPVADTSKTKLKPFFWDKVAASPDQAMVWDQI KAGSFQFNEEM IETLFGCNAVDKKSTD

[0222] GKKEPAKEAAQFVRI LEPKKAQNLAI SLKALSVSAADVRTAVTEGYELPSDL IQTL IRWIPTSDEELRRLLYTGEMSQ

[0223] LGQAEQFLKT IIE IPYI FQRLEVLLFMASLPEEAAGVKQSFETLEVACQELRHNRLFKKLLEAVLKTGNRMNDGTFRG

[0224] GAQAFKLDTLLKLADVKGVDGKTTLLHFVVQE II RSEGVRAVRAAKEQNSS ISSVSSTDDLTEDVSDDTEHYKQLGLA

[0225] VVSNLGEDLQNVRKAAI LDADALTIMVASLGHRLVKANEFLNTSMKSLEEESGFQRKLVQFIEQSQVQVTHLLEEEKK

[0226] LRSLVRTTVDYFHGSTGKDEGLRLFVIVRDFLAI LDRVCREVKEAAAKAAAANKKEAAAAKAAAAPPAPPTRGRQPSQ

[0227] TSMSFRDPRQHLKPAIQGRRGKAHSSSSSSDSD.

[0228] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0229] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0230] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A gene DG1C00225.1 that regulates the flowering time of plants, characterized in that: The nucleotide sequence of DG1C00225.1 is shown in SEQ ID NO.

1.

2. The protein encoded by the gene DG1C00225.1 for regulating flowering time of plants according to claim 1, characterized in that: The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. Use of the gene DG1C00225.1 for regulating flowering time of plants as claimed in claim 1 in Arabidopsis breeding.

4. The use according to claim 3, characterized in that: The application includes promoting early flowering of Arabidopsis thaliana.

5. Use of the protein encoded by the gene DG1C00225.1 for regulating flowering time of plants as claimed in claim 2 in preparing early-flowering Arabidopsis strains.

6. An overexpression vector comprising the gene DG1C00225.1 for regulating flowering time of plants as claimed in claim 1.

7. Agrobacterium comprising the gene DG1C00225.1 for regulating flowering time of plants as claimed in claim 1.

8. A method for cultivating early-flowering transgenic plants, characterized in that: Overexpressing the DG1C00225.1 gene in plants to obtain early-flowering transgenic plants; Alternatively, the expression level of DG1C00225.1 protein in the plant is increased to obtain early-flowering transgenic plants; Among them, the nucleotide sequence of the DG1C00225.1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the DG1C00225.1 protein is shown in SEQ ID NO.

2.

9. The method for cultivating an early-flowering transgenic plant according to claim 8, characterized in that: The method of overexpressing the DG1C00225.1 gene in plants to obtain early-flowering transgenic plants specifically includes: The DG1C00225.1 gene was homologously recombined with the vector pHG-35S to obtain the overexpression vector pHG-35S-DG1C00225.1; The pHG-35S-DG1C00225.1 is transformed into plants, and early-flowering transgenic plants are obtained through screening and cultivation; The plants include Arabidopsis thaliana.

10. The method for cultivating an early-flowering transgenic plant according to claim 9, characterized in that: The step of transforming the pHG-35S-DG1C00225.1 into a plant specifically comprises: The plant inflorescence was infected by the floral dip method to transform the pHG-35S-DG1C00225.1 into the plant.

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