A gene DG1C00225.1 for regulating plant flowering time and its application

Abstract

The present invention provides a gene DG1C00225.1 for regulating the flowering time of plants and its application, belonging to the technical field of genetic engineering. The nucleotide sequence of DG1C00225.1 is shown as SEQ ID NO.1. The flowering time of Arabidopsis thaliana overexpressing the gene DG1C00225.1 is significantly advanced. It can be applied to cultivate early-flowering transgenic plants, shorten the breeding time, improve the breeding efficiency, and promote the development and utilization of gramineous forage grasses.

Description

Technical Field

[0001] The present 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 its application. Background Art

[0002] Dactylis glomerata belongs to the genus Dactylis in the subfamily Festucoideae of the family Poaceae, and is a perennial cool-season tufted forage widely cultivated worldwide. Dactylis glomerata has the advantages of having many leaves and high yield, being shade-tolerant, having strong adaptability, good palatability and high nutritional value, and can be used for green forage, hay making or silage. It is one of the four widely distributed gramineous forages in the world, and about 14,000 t of Dactylis glomerata seeds are produced globally every year, accounting for 3.3% of the world's temperate forage seeds. At present, Dactylis glomerata is cultivated in provinces such as Qinghai, Gansu, Shaanxi, Shanxi, Henan, Jilin, Jiangsu, Hubei, Sichuan and Xinjiang. It is used for both mowing and grazing, and is liked by various poultry, and has achieved good economic and ecological benefits, showing broad application prospects. The flowering period is an important agronomic trait of Dactylis glomerata, which is closely related to the quality and yield of Dactylis glomerata. The flowering period represents the transition of plants from vegetative growth to reproductive growth, and the forage quality and yield will also change accordingly. Dactylis glomerata is suitable for mixed sowing with leguminous forages. By specifically cultivating Dactylis glomerata varieties with different flowering periods and cooperating with leguminous forage varieties with the same maturity period, the grazing time of the mixed sown grassland can be extended, and the comprehensive production performance and utilization efficiency can be improved. Summary of the Invention

[0003] The object 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 overexpressing the gene DG1C00225.1 is significantly advanced, and it can be applied to the cultivation of early-flowering transgenic plants, shorten the breeding time, improve the breeding efficiency, and thus promote the development and utilization of gramineous forages.

[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, and the nucleotide sequence of the DG1C00225.1 is shown as SEQ ID NO.1.

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

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

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

[0009] The present invention also provides the use of the encoded protein of gene DG1C00225.1 for regulating plant flowering time in the preparation of early-flowering Arabidopsis thaliana lines.

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

[0011] The present invention also provides Agrobacterium containing the gene DG1C00225.1 for regulating plant flowering time.

[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 the DG1C00225.1 protein in plants is increased to obtain early-flowering transgenic plants;

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

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

[0016] The DG1C00225.1 gene is 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 after screening and culturing, early-flowering transgenic plants are obtained;

[0018] The plants include Arabidopsis thaliana.

[0019] Preferably, the transformation of the pHG-35S-DG1C00225.1 into plants specifically includes:

[0020] The inflorescences of plants are infected by the floral dip method to transform the pHG-35S-DG1C00225.1 into plants.

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

[0022] The gene DG1C00225.1 for regulating plant flowering time in the present invention has been experimentally proven that overexpression of the DG1C00225.1 gene can promote early flowering of Arabidopsis thaliana. Applying the DG1C00225.1 gene to the genetic engineering transformation of Arabidopsis thaliana can obtain early-flowering transgenic plant lines. Applying it to the genetic engineering transformation of orchardgrass is expected to obtain early-flowering orchardgrass lines, and it has the advantages of short breeding time and high breeding efficiency, and has important utilization value for improving the quality of forage grass, and can effectively promote the development and utilization of the gramineous forage grass orchardgrass. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

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

[0025] Figure 2 It is a subcellular localization diagram of DG1C00225.1 protein.

[0026] Figure 3 It is the relative expression level of DG1C00225.1 gene in different overexpressed Arabidopsis thaliana lines.

[0027] Figure 4 It is a phenotypic diagram of the flowering time of wild-type Arabidopsis thaliana and overexpressed DG1C00225.1 lines

[0028] Figure 5 It is a statistical chart of the flowering time of wild-type Arabidopsis thaliana and overexpressed DG1C00225.1 lines.

[0029] Figure 6 It is a statistical chart of the number of rosette leaves at flowering of wild-type Arabidopsis thaliana and overexpressed DG1C00225.1 lines.

[0030] Figure 7 It is a fluorescence quantitative diagram of flowering genes of wild-type Arabidopsis thaliana and overexpressed DG1C00225.1 lines. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The following will specifically describe the present invention in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented therefrom. Those skilled in the art should understand that these specific embodiments 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 having the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of any contradiction, the present specification shall prevail.

[0033] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or can be 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 multi-domain proteins that participate in various actin-related processes, such as cell division and maintenance of cell polarity (Wasserman, 1998). In plant cells, formins act as regulators of the dynamic changes of the cytoskeleton to promote actin nucleation and can change the plant growth phenotype by interacting with microtubules and microfilaments. We performed genome-reduced sequencing on 249 Dactylis glomerata materials and found a key candidate gene DG1C00225.1 (DgFH18) that regulates the flowering of Dactylis glomerata through genome-wide association analysis. We further studied its function to explore its regulatory role in the flowering time of Dactylis glomerata.

[0036] It was found through experiments that overexpression of the DG1C00225.1 gene can promote the flowering time of Arabidopsis thaliana, and the expression level of DG1C00225.1 in Dactylis glomerata can also be increased by overexpression technology to promote early flowering of Dactylis glomerata. Dactylis glomerata is a cross-pollinated plant with complex ploidy, resulting in difficult genetic transformation and relatively lagged gene function verification. Therefore, for the disadvantages of slow effectiveness and long cycle of traditional forage improvement technologies, the present invention applies the DG1C00225.1 gene to the cultivation of early-flowering transgenic plants, which can shorten the breeding time, improve the breeding efficiency, and promote the development and utilization of the high-quality gramineous forage Dactylis glomerata.

[0037] The following will specifically describe a gene DG1C00225.1 for regulating plant flowering time and its application in the present application in combination with examples and experimental data.

[0038] Example 1

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

[0040] I. Construction of evolutionary tree

[0041] Search for and download the homologous sequences of DG1C00225.1 in 12 different species through the BLASTP tool on the NCBI website. The downloaded sequences were used to generate a phylogenetic tree with 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] II. Subcellular localization

[0043] 1. Amplification of the target fragment

[0044] 1.1 Cloning of the target fragment

[0045] The experimental material was the Dactylis glomerata variety "2006 - 1", which was planted in the Wenjiang Campus of Sichuan Agricultural University. Its young leaves were taken as materials to extract total RNA. The plant total RNA extraction kit from Tiangen (Beijing) Biochemical Technology Co., Ltd. was used for RNA extraction, and the operation was carried out according to the attached instructions. After RNA extraction, integrity detection was performed by 1% agarose gel electrophoresis, and a ultra-micro spectrophotometer was used to measure the RNA concentration and purity. For reverse transcription, the PrimeScript II 1st Strand cDNA Synthesis Kit from TaKaRa was selected, and the operation procedure was referred to the attached instructions.

[0046] Using the "2006 - 1" RNA as a template, primers were designed through the full-length sequence

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

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

[0049] Amplification was carried out using the cDNA as a template, and the 2×Phanta Max Master Mix (DyePlus) kit from vazyme was selected for amplification, and the operation procedure was referred to the attached instructions.

[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] 17 uL of ddH2O.

[0056] Reaction program:

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

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

[0059] Extension at 72°C for 5 min.

[0060] 1.2 DNA electrophoresis and recovery:

[0061] Electrophoresis: Add an appropriate amount of 10× loading buffer to each reaction tube and perform electrophoresis on a 1% - 3% (10 ul EB, 1 - 3 g agarose / 100 ml of 0.5× TBE buffer) agarose gel. Electrophoresis is carried out at 5 - 10 V / cm in 0.5× TBE buffer. After the electrophoresis is completed, take a photo in the gel imaging system.

[0062] Agarose gel DNA recovery (GK2042, Jie Rui Bio):

[0063] 1) Carefully cut the DNA target band and put it into a 1.5 mL EP tube.

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

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

[0066] 4) Repeat step 3.

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

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

[0069] 2. Functional analysis of DG1C00225.1

[0070] Use subcellular localization technology to determine the location of the DG1C00225.1 protein. To determine the location of DG1C00225.1, insert the open reading frame (ORF) of DG1C00225.1 into the pAN580 - 35S - GFP vector. Using the pAN580 - 35S - GFP empty vector as a control, transfer the fusion vector and the control empty vector into tobacco leaves.

[0071] 2.1 Synthesis of Primers for Amplifying the Target Gene DG1C00225.1

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

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

[0074] 2.2 PCR System and Program

[0075] Perform PCR reaction using cDNA as the template.

[0076] Amplification System:

[0077] Component Volume Nuclease-free Water 20uL Biorun Pfu PCR Mix 25uL Primer(+) 2uL Primer(-) 2uL Template 1uL Total volum 50uL

[0078] PCR Program:

[0079]

[0080]

[0081] Perform DNA electrophoresis and recovery on the PCR products using the method described above.

[0082] 2.3 Vector Digestion

[0083] The vector used is pAN580-35S-GFP. The digestion and ligation system is as follows:

[0084] Component Volume Nuclease-free Water 12uL 10*buffer 2uL BsaI 1uL Eco31I 1uL pBWA(V)HS-ccdb-GLosgfp 4uL Total volume 20uL

[0085] Reaction Conditions:

[0086] Temperature Time 37℃ 1hours

[0087] Purify the vector digestion products using a PCR purification kit for the next recombination reaction.

[0088] 2.4 Recombination Reaction

[0089] Recombine the ORF sequence of DG1C00225.1 with the vector digestion products. The recombination reaction system is as follows:

[0090]

[0091] Reaction Conditions:

[0092] Temperature Time 37℃ 30 hours

[0093] Transform the ligation product into competent cells.

[0094] 2.5 Transformation of competent cells

[0095] Transform 5 - 10 μL of the ligation product into Escherichia coli competent cells (see the standard method for transforming Escherichia coli competent cells), spread on an Amp-resistant plate, and culture at 37°C for 12 hours for colony PCR identification.

[0096] 2.6 Colony PCR identification

[0097] Pick 10 colonies and simultaneously inoculate them into 1.5 mL EP tubes and perform PCR identification. Design identification primers for pBWA(V)HS-ccdb-GLosgfp.

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

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

[0100] PCR system:

[0101] Component Volume Nuclease-free Water 9.5uL Biorun Magic PCR Mix 12.5uL HS)35seq 1uL E7805(580C) 1uL Template 1uL Total volum 25uL

[0102] PCR program:

[0103] Step Number of cycles 94℃ for 5min 1 94℃ for 30sec 30 50℃ for 45sec 30 72℃ for 174sec 30 72℃ for 10min 1 16℃ for 30min 1

[0104] 2.7 Plasmid extraction from colonies

[0105] Inoculate the correct single colony into 3 mL of LB liquid medium containing Amp antibiotic, culture at 37°C with shaking at 200 rpm overnight; take 1.5 mL of the culture (take 3 mL for low-copy plasmids), centrifuge at 12,000 rpm for 30 seconds; aspirate the supernatant completely, suspend the cells 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%), immediately mix gently up and down; add 150 μL of Solution III (KAc 5 mol / L, pH 4.8), mix quickly up and down, and let 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; discard the supernatant, wash the DNA pellet with 70% ethanol, centrifuge at 12,000 rpm for 1 minute, and remove the supernatant; dry the pellet in vacuo; dissolve it in 60 μL of double-distilled water containing 10 μg / 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 inoculating loop to scrape the Agrobacterium from the solid culture dish and inoculate it into 10 mL of YEB liquid 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 mark the leaves;

[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 pHG-35-GFP vector, 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 DG1C00225.1 target fragment amplified in the second part 1.1 of Example 1 as a template, perform a PCR reaction to obtain a homologous arm gene containing the pHB-35S restriction site. Recover the correct PCR fragment using the same method as described above.

[0124] 1.2 Digestion of vector pHG-35-GFP plasmid:

[0125] 40 μL digestion system:

[0126] Plasmid (30 μL)

[0127] 4 μL 10× digestion buffer

[0128] 4 μL 10× BSA (add or not according to the instruction manual)

[0129] 6 U restriction endonuclease (NEB)

[0130] Add water to make up to 40 μL

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

[0132] 1.3 Construction of recombinant plasmid:

[0133] After the PCR fragment in Step 1.1 is recovered by agarose gel electrophoresis, mix it with the empty vector recovered by digestion in Step 1.2, and add it to the EasyGeno DNA recombination system for ligation (#VI201-02, Tiangen Biotech). The 10 μL recombination system is as follows:

[0134] 5 μL 2× EasyGeno Assembly Mix

[0135] 2.5 μL digested vector DNA

[0136] 2.5 μL fragment DNA

[0137] Add the reaction system to a 250 μL EP tube, incubate in a 50°C water bath for 30 minutes, then transform Escherichia coli and spread on a plate. After incubating in a 37°C incubator for 16 hours, pick colonies, send them for sequencing, and store the plasmids with correct sequencing at -20°C for long-term preservation.

[0138] 1.4 Extraction of plasmid

[0139] Inoculate a single colony into 3 mL of LB liquid medium containing kanamycin antibiotic, and culture it overnight at 37°C with shaking at 200 rpm; take 1.5 mL of the culture (take 3 mL for low-copy plasmids), centrifuge at 12,000 rpm for 30 seconds; aspirate the supernatant completely, and suspend the bacterial cells 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), mix quickly 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 well; centrifuge at 12,000 rpm for 10 minutes; discard the supernatant, wash the DNA precipitate with 70% ethanol, centrifuge at 12,000 rpm for 1 minute, and remove the supernatant; dry the precipitate under vacuum; dissolve it in 60 μL of double-distilled water containing 10 μg / mL RNase A.

[0140] 2. Arabidopsis thaliana transformation

[0141] 2.1 Agrobacterium culture:

[0142] 1) Take the Agrobacterium competent cells stored at -80°C and let them stand at room temperature or in the palm of the hand for a moment until they are partially melted. When they are in an ice-water mixture state, insert them into ice.

[0143] 2) Add 0.1 μg (volume not exceeding 10 μL) of plasmid DNA to every 100 μL of competent cells, mix well by flicking the bottom of the tube, and let it stand 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 in sequence.

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

[0145] 4) Centrifuge at 6000 rpm for 1 minute to collect the bacteria, retain about 100 μL of the supernatant, gently resuspend the bacterial pellet by pipetting, and spread it on an LB plate containing the corresponding antibiotic. Incubate it upside down in a 28°C incubator for 2 - 3 days. Randomly select 1 single colony, perform colony PCR, identify the correct Agrobacterium monoclonal, and mark it for later use.

[0146] 5) Use a sterile pipette tip to pick the marked Agrobacterium monoclonal 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.

[0147] 6) Inoculate 100 mL of LB liquid medium containing antibiotics with a 1% inoculum of the Agrobacterium culture at a rate of 1% and incubate at 30°C with shaking until the OD600 reaches approximately 1.0.

[0148] 7) Centrifuge at 4,000 rpm for 15 min at 20°C to collect the cells; pipette the cells evenly with transformation buffer and resuspend them to approximately OD600 = 1.0.

[0149] The conversion buffer components are as follows:

[0150]

[0151] 2.2 Arabidopsis transformation:

[0152] 1) Water the Arabidopsis plants that are about to sprout and flower one day in advance;

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

[0154] 3) After 7 days, repeat the above transformation method. After 2-3 weeks, water the seeds with minimal nutrient solution to accelerate aging. Store the mature seeds in a paper bag 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 / 2 MS (0.8% agar powder, no sucrose, pH 5.8);

[0157] 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.

[0158] 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).

[0159] 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; 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 in a ratio of 2:1 and set aside;

[0161] 7) Soil soaking: Fill the planting pot with soil to about 1 cm from the pot mouth, and soak it completely with Flower Without Defect compound fertilizer (N, P, K = 20%, 20%, 20%).

[0162] 8) Transplanting: Select healthy and uniformly growing seedlings at 20 days after germination and transplant them into the culture soil previously soaked with Flower Without Defect. Cover it with plastic wrap and remove it after the seedlings survive.

[0163] 2.4 Verification of the expression level of the T3 homozygous lines of Arabidopsis thaliana

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

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

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

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

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

[0169] Measurement of flowering time: When the bolting height of Arabidopsis thaliana is 0.5 cm, its flowering time is counted;

[0170] Measurement of the number of rosette leaves: When Arabidopsis thaliana flowers, the number of rosette leaves is counted.

[0171] The measurement results are as Figures 4 - 6 shown. It can be seen from Figure 4 , Figure 5 and Figure 6 that the flowering time of the overexpression DG1C00225.1 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

[0172] 4. Fluorescent quantitative determination of genes related to flowering time

[0173] Samples of 3-week overexpressing and wild-type Arabidopsis thaliana were taken and then frozen in liquid nitrogen, with 3 replicates for each sample. RNA extraction and reverse transcription were performed on the obtained samples, and the experimental methods for RNA and reverse transcription were as described above.

[0174] The results showed that: the overexpressing DG1C00225.1 line promoted the expression levels of the genes AtAP1, AtFUL, and AtFT that promote Arabidopsis thaliana flowering, while inhibiting the expression level of the gene AtFLC that inhibits flowering. Therefore, DG1C00225.1 may be a gene that promotes the flowering of orchardgrass ( 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] agcaacctggagcgctttatcttccgaagcagagtctcgtccactggtcactagcataaaaaaatcttccaagaccga

[0184] ggggaaaaagaagagcaaggacaaagactcagattcatccactgttgtcttgggtttggcagtgggctgcgtggcatt

[0185] gctggctcttattgtgtacttctgtgcttgtcgtggagatgattcagcgtcaccatatgatctagggagggatgataa

[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] MRWPRSRLKWLLSACLISLLLLTPTDCNGLQLVAAIRKNLFWPPAPPHLSLSSQIGDTLVEQLWLNCGLDRI IL

[0216] QEVKNQSYYTPLFNI ISNSYRTNKKIEAGMFTSLSHEVANTFQDCLSKHNFILSDGFSGHGQVEEEKEAGSLLSNTNK

[0217] FKLPFASKIRYLLEGTPASHLSLSPPAKEATWSALSSEAESRPLVTSIKKSSKTEGKKKSKDKDSDSSTVVLGLAVGC

[0218] VALLALIVYFCACRGDDSASPYDLGRDDKPLLGLTDLAGSSRKSSATPIDVSRLGALSRSSSEIPQSEFAVPIKMHTT

[0219] QPSAKLKSVGAMSMKAELMERHSRLSSHEI TTVAGRPSVANSLAEKASGSSATGNAVTHAGPPPPPPPALPGKAPPPP

[0220] PVLPGRTPPPPPPAPGAPAPPPLPGAAASAPPPPPKPAGPPPPGPPPPPAPRAGAGPGPPPPPLKKGGPPGAGPPPPA

[0221] MPGGPKKGGPPPFKKPGAAAPVADTSKTKLKPFFWDKVAASPDQAMVWDQI KAGSFQFNEEMIETLFGCNAVDKKSTD

[0222] GKKEPAKEAAQFVRILEPKKAQNLAISLKALSVSAADVRTAVTEGYELPSDLIQTLIRWIPTSDEELRLRLYTGEMSQ

[0223] LGQAEQFLKTI IEIPYIFQRLEVLLFMASLPEEAAGVKQSFETLEVACQELRHNRLFKKLLEAVLKTGNRMNDGTFRG

[0224] GAQAFKLDTLLKLADVKGVDGKTTLLHFVVQEI IRSEGVRAVRAAKEQNSSISSVSSTDDLTEDVSDDTEHYKQLGLA

[0225] VVSNLGEDLQNVRKAAILDADALTIMVASLGHRLVKANEFLNTSMKSLEEESGFQRKLVQFIEQSQVQVTHLLEEEKK

[0226] LRSLVRTTVDYFHGSTGKDEGLRLFVIVRDFLAILDRVCREVKEAAAKAAAANKKEAAAAKAAAAPPAPPTRGRQPSQ

[0227] TSMSFRDPRQHLKPAIQGRRGKAHSSSSSSDSDD.

[0228] Finally, it should also be noted that the term "comprise", "include" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements 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 can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling 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 equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

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

1.

2. The coding protein of gene DG1C00225.1 for regulating plant flowering time 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 a gene DG1C00225.1 for regulating plant flowering time in Arabidopsis thaliana breeding, characterized in that, The application is to promote early flowering of Arabidopsis thaliana.

4. Use of the encoded protein of the gene DG1C00225.1 for regulating plant flowering time according to claim 2 in the preparation of an Arabidopsis thaliana line with early flowering.

5. An overexpression vector containing the gene DG1C00225.1 for regulating plant flowering time according to claim 1.

6. An Agrobacterium containing the gene DG1C00225.1 for regulating plant flowering time according to claim 1.

7. A method for cultivating an early-flowering transgenic plant, characterized in that, Overexpress the DG1C00225.1 gene in plants to obtain early-flowering transgenic plants; wherein, 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; The plant is Arabidopsis thaliana.

8. The cultivation method of an early-flowering transgenic plant according to claim 7, characterized in that, The overexpression of the DG1C00225.1 gene in plants to obtain early-flowering transgenic plants specifically includes: Perform homologous recombination of the DG1C00225.1 gene with the vector pHG-35S to obtain the overexpression vector pHG-35S-DG1C00225.1; Transform the pHG-35S-DG1C00225.1 into plants, and through screening and cultivation, obtain early-flowering transgenic plants.

9. The cultivation method of an early-flowering transgenic plant according to claim 8, characterized in that, The transformation of the pHG-35S-DG1C00225.1 into plants specifically includes: Infect the inflorescence of plants by the floral dipping method to transform the pHG-35S-DG1C00225.1 into plants.

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