A gene DG1C01667.1 related to plant flowering time and its application

Abstract

The present invention provides a gene, DG1C01667.1, associated with plant flowering time, and its application, belonging to the field of biotechnology. The nucleotide sequence of DG1C01667.1 is shown in SEQ ID NO. 1. Arabidopsis thaliana plants that overexpress the DG1C01667.1 gene significantly advance their flowering time. This gene can be applied to genetic engineering of plants to regulate their flowering period, shorten breeding time, improve breeding efficiency, and promote the development and utilization of grasses.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a gene DG1C01667.1 related to the flowering time of plants and its application. Background Technology

[0002] Orchardgrass, belonging to the genus *Dactylis* of the subfamily Festucoideae in the family Poaceae, is a widely cultivated perennial cool-season tufted forage grass worldwide. It boasts advantages such as abundant foliage, high yield, shade tolerance, strong adaptability, good palatability, and high nutritional value. It can be used for green fodder, haymaking, or silage, and is one of the four most widely distributed Poaceae forage grasses globally. Approximately 14,000 tons of orchardgrass seeds are produced annually worldwide, accounting for 3.3% of the world's temperate forage seeds. Currently, orchardgrass is cultivated in Qinghai, Gansu, Shaanxi, Shanxi, Henan, Jilin, Jiangsu, Hubei, Sichuan, and Xinjiang, serving both forage and grazing purposes. It is readily consumed by various poultry and has yielded significant economic and ecological benefits, demonstrating broad utilization prospects.

[0003] Flowering period is an important agronomical trait of orchardgrass, closely related to its quality and yield. The flowering period represents the transition from vegetative to reproductive growth, and the quality and yield of forage change accordingly. Orchardgrass is suitable for intercropping with leguminous forages. Targeted breeding of orchardgrass varieties with different flowering periods, combined with leguminous forage varieties that mature at the same time, can extend the grazing time of intercropped grasslands and improve overall production performance and utilization efficiency. Summary of the Invention

[0004] The purpose of this invention is to provide a gene DG1C01667.1 related to plant flowering time and its application. Overexpression of the DG1C01667.1 gene in Arabidopsis thaliana significantly advances the flowering time. It can be applied to the genetic engineering modification of plants to regulate the flowering period, shorten the breeding time, improve the breeding efficiency, and promote the development and utilization of gramineous forage grasses.

[0005] This invention is achieved through the following technical solution:

[0006] The present invention provides a gene DG1C01667.1 related to the flowering time of plants, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene-encoded protein of a gene DG1C01667.1 related to the flowering time of plants, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] This invention also provides the application of the gene DG1C01667.1, which is related to the flowering time of plants, in Arabidopsis thaliana breeding.

[0009] Furthermore, the application includes promoting early flowering in Arabidopsis thaliana.

[0010] The present invention also provides the application of the protein encoded by the gene DG1C01667.1, which is related to the flowering time of plants, in the preparation of early-flowering Arabidopsis thaliana lines.

[0011] The present invention also provides an overexpression vector containing the gene DG1C01667.1, which is associated with the flowering time of plants.

[0012] This invention also provides Agrobacterium containing the gene DG1C01667.1, which is associated with plant flowering time. Based on the same inventive concept, this invention also provides a method for cultivating early-flowering transgenic plants by overexpressing the DG1C01667.1 gene in plants to obtain early-flowering transgenic plants;

[0013] Alternatively, by increasing the expression level of the DG1C01667.1 protein in plants, early-flowering transgenic plants can be obtained;

[0014] The nucleotide sequence of the DG1C01667.1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the DG1C01667.1 protein is shown in SEQ ID NO.2.

[0015] Furthermore, the process of overexpressing the DG1C01667.1 gene in plants to obtain early-flowering transgenic plants specifically includes:

[0016] The DG1C01667.1 gene was homologously recombinated with the vector pHG-35S to obtain the overexpression vector pHG-35S-DG1C01667.1;

[0017] The pHG-35S-DG1C01667.1 was transformed into plants, and early-flowering transgenic plants were obtained through screening.

[0018] Preferably, the plant includes Arabidopsis thaliana.

[0019] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0020] This invention relates to a gene, DG1C01667.1, which is associated with the flowering time of plants. The DG1C01667.1 gene can affect the flowering time of Arabidopsis thaliana. Experiments have shown that overexpression of the DG1C01667.1 gene can promote early flowering in Arabidopsis thaliana. Applying the DG1C01667.1 gene to the genetic engineering modification of orchardgrass is expected to achieve regulation of orchardgrass flowering time, shorten breeding time, improve breeding efficiency, and has important utilization value for improving forage quality, thus promoting the development and utilization of orchardgrass, a grassy forage. Attached Figure Description

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

[0022] Figure 1 Phylogenetic tree diagram of DG1C01667.1 protein and other homologous proteins.

[0023] Figure 2 Subcellular localization map of DG1C01667.1 protein.

[0024] Figure 3 Flowering timetypes for wild-type and DG1C01667.1 overexpressing lines.

[0025] Figure 4 A statistical chart showing the flowering time of wild-type and DG1C01667.1 overexpressing lines.

[0026] Figure 5 The number of rosette leaves at flowering time for wild-type and DG1C01667.1 overexpressing lines. Detailed Implementation

[0027] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0028] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0030] The overall concept of this invention is as follows:

[0031] MADS-boxes are important transcriptional regulators found in almost the entire plant kingdom, participating in the development of floral organs and regulating flowering time. We performed simplified genome sequencing on 249 orchardgrass accessions and identified a key candidate gene, DG1C01667.1, regulating orchardgrass flowering through genome-wide association analysis. Testing showed that overexpression of DG1C01667.1 promoted early flowering in both Arabidopsis and orchardgrass, indicating that the DG1C01667.1 gene in orchardgrass can participate in the regulation of flowering time and has significant value for improving forage quality. Transforming DG1C01667.1 into orchardgrass for research on its effect on flowering time has important theoretical and practical value.

[0032] Orchardgrass is a cross-pollinated plant with complex ploidy, making genetic transformation difficult and gene function verification relatively slow. To date, research on the orchardgrass DG1C01667.1 gene is still lacking. This invention provides a gene related to plant flowering time—DG1C01667.1—and applies it to the genetic engineering modification of orchardgrass. Overcoming the shortcomings of traditional forage improvement techniques—slow results and long cycles—this method can shorten breeding time, improve breeding efficiency, and promote the development and utilization of high-quality gramineous forage grass, orchardgrass.

[0033] The following will provide a detailed description of the gene DG1C01667.1 related to plant flowering time and its application, in conjunction with embodiments and experimental data.

[0034] Example 1

[0035] Phylogenetic analysis and subcellular localization of DG1C01667.1d.

[0036] 1. Evolutionary Tree Analysis

[0037] Homologous sequences of DG1C01667.1 from 12 different species were searched and downloaded using the BLASTP tool on the NCBI website. Phylogenetic trees were generated from the downloaded sequences using MEGA 7.0 software. The results showed that the DG1C01667.1 protein from *Orchardgrass* had the highest homology with the LrMADS18 protein from *Lycium chinense*. Figure 1 )

[0038] 2. Subcellular localization

[0039] 2.1 Amplification of the target fragment

[0040] 2.11 Cloning of the target fragment

[0041] The experimental material was the orchardgrass variety "2006-1," planted at the Wenjiang Campus of Sichuan Agricultural University. Young leaves were used to extract total RNA. The plant total RNA extraction kit from Tiangen (Beijing) Biochemical Technology Co., Ltd. was used for RNA extraction, and the procedure was performed according to the included instructions. After extraction, RNA integrity was assessed using 1% agarose gel electrophoresis, and RNA concentration and purity were determined using a micro spectrophotometer. Reverse transcription was performed using the TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit, and the procedure was performed according to the included instructions.

[0042] Using the "2006-1" RNA as a template, primers were designed based on the full-length sequence.

[0043] DG1C01667.1-F:5'-ATGGGGCGGGCCGGTGCAGC-3',

[0044] DG1C01667.1-R:5'-TCTGTTACTGATGGTGCGGAGCATC-3'.

[0045] amplification was performed using cDNA as a template. The amplification was carried out using the 2×Phanta Max Master Mix (DyePlus) kit from Vazyme. The procedure was described in the included instruction manual.

[0046] 50uL reaction system:

[0047] 25uL 2×Phanta Max Master Mix(Dye Plus)

[0048] 2 μL upstream primer (10 μM)

[0049] 2 μL downstream primer (10 μM)

[0050] 4uL template DNA*

[0051] 17uL ddH2O.

[0052] Reaction procedure:

[0053] Pre-denaturation at 95℃ for 3 minutes;

[0054] 95℃ denaturation for 15s, 55℃ annealing for 15s, 72℃ extension for 1min, 35 cycles;

[0055] Extend at 72℃ for 5 minutes.

[0056] 2.12 DNA Electrophoresis and Recovery:

[0057] Electrophoresis: Add an appropriate amount of 10× loading buffer to each reaction tube and perform electrophoresis on a 1%-3% agarose gel (10ul EB, 1-3g agarose / 100ml 0.5×TBE buffer). Electrophoresis is performed at 5-10V / cm in 0.5×TBE buffer. After electrophoresis, take a picture using a gel imaging system.

[0058] Agarose gel DNA recovery (GK2042, Jereh Biotechnology):

[0059] 1) Carefully cut the target DNA band and place it into a 1.5 mL EP tube.

[0060] 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.

[0061] 3) Add 100 μL of isopropanol to the tube, let it stand at room temperature for 1 minute, centrifuge at 5000 rpm for 1 minute and pass it through the column.

[0062] 4) Repeat step 3.

[0063] 5) Add 500uL of wash buffer and wash twice at 12000rpm, then centrifuge at 10000rpm for 1 minute.

[0064] 6) Add 40 μL of double-distilled water to the column, place at 37°C for 2 minutes, and centrifuge at 12000 rpm for 1 minute to collect the product.

[0065] 2.2DG1C01667.1 Functional Analysis

[0066] The location of the DG1C01667.1 protein was determined using subcellular localization technology. To determine the location of DG1C01667.1, the open reading frame (ORF) of DG1C01667.1 was inserted into the pAN580-35S-GFP vector, and the fusion vector and the control empty vector were transformed into rice protoplasts using the pAN580-35S-GFP empty vector as a control.

[0067] 2.21 Synthesize amplification primers for the CDS of the target gene DG1C01667.1

[0068] pAN580-DG1C01667.1-F:AGTCCGGAGCTAGCTCTAGAatggggcgcgggccggtg

[0069] pAN580-DG1C01667.1-R:GCGGCCGCTGTACAGGATCCtctgttatactgatggtgcggagcatc

[0070] 2.22 PCR System and Procedure

[0071] The cDNA prepared in step 2.1 was used as a template for PCR reaction.

[0072] Amplification system:

[0073] Ingredients volume Nuclease-free Water 20uL Biorun Pfu PCR Mix 25uL Primer(+) 2uL Primer(-) 2uL Template 1uL Total volume 50uL

[0074] PCR procedure:

[0075]

[0076]

[0077] The PCR products were subjected to DNA electrophoresis and recovery, as described above.

[0078] 2.23 Vector digestion

[0079] The vector used is pAN580-35S-GFP, and the enzyme digestion and ligation system is as follows:

[0080]

[0081] Reaction conditions:

[0082] temperature time 37℃ 1 hour

[0083] The vector digestion products were purified using a PCR purification kit for use in the next recombinant reaction.

[0084] 2.24 Recombination reaction

[0085] The (ORF) sequence of DG1C01667.1 was recombined with the vector digestion product. The recombination reaction system is as follows:

[0086]

[0087]

[0088] Reaction conditions:

[0089] temperature time 37℃ 30 hours

[0090] The ligation product was transformed into competent cells.

[0091] 2.25 Transformed competent cells

[0092] Transform 5-10 μL of the ligation product into competent E. coli cells (see the standard method for transformation of competent E. coli cells), plate the transformed cells onto Amp resistant plates, incubate at 37°C for 12 hours, and perform colony PCR identification.

[0093] 2.26 Colony PCR Identification

[0094] Ten colonies were selected and simultaneously inoculated into 1.5 mL EP tubes for PCR identification. Primers for identification were designed using pAN580-l inker-esp3I.

[0095] Pan580-F:agttcatttcatttggagaggacag(2881bp)

[0096] D13519(633C):gatgaatatttgttgctccattcagct(3554bp).

[0097] PCR system:

[0098]

[0099]

[0100] PCR procedure:

[0101] step Cycle number 94℃ for 5 minutes 1 94℃ for 30 seconds 30 50℃ for 45 seconds 30 72℃ for 76 seconds 30 72℃ for 10 minutes 1 16℃ for 30 minutes 1

[0102] 2.27 Plasmid extraction from bacterial colonies

[0103] Inoculate a correctly identified positive single colony into 3 mL of LB broth containing Amp antibiotic and incubate overnight at 37°C with shaking at 200 rpm. Take 1.5 mL of the culture (3 mL of low-copy plasmid), centrifuge at 12,000 rpm for 30 seconds. Aspirate the supernatant and resuspend 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%) and immediately mix gently up and down. Add 150 μL of Solution III (KAc 5 mol / L, pH 8.0). 4.8) Quickly mix from top to bottom 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; remove the supernatant, wash the DNA precipitate with 70% ethanol, centrifuge at 12,000 rpm for 1 minute, remove the supernatant; vacuum dry the precipitate; dissolve in 60 μL of double-distilled water containing 10 μg / mL RNase A.

[0104] 2.28 Transformation of rice protoplasts

[0105] 1) Rice seedlings that have been cultured in the dark at around 30℃ for 7-15 days, take the stems and leaves, rinse the surface dirt with water, and remove the outermost leaf sheath;

[0106] 2) Place the seedling stems and leaves on a clean plastic board and cut them into small pieces using a sharp blade;

[0107] 3) Add 5-10 mL of enzymatic hydrolysate to completely soak the tissue, and gently shake at 28°C for 4-5 hours;

[0108] 4) Filter the protoplasts through a 40µm filter, then transfer them to a 2mL centrifuge tube and centrifuge at 600rpm for 5min. A turbid precipitate will be visible.

[0109] 5) Directly aspirate the supernatant, wash twice with 10 mL of pre-cooled W5 solution, centrifuge at 600 rpm for 5 min at a centrifugation temperature of 25℃;

[0110] 6) Add an appropriate amount of MMG solution to suspend the protoplasts as needed until the concentration is 2*105 protoplasts / mL. Microscopic examination: the protoplasts are round and have few ruptures.

[0111] 7) Take 100uL of protoplast suspension + 10uL of DNA (the plasmid solution prepared in step 2.27 or the pAN580-35S-GFP empty vector), and an equal volume of PEG 4000 solution, mix gently and evenly, and let stand at room temperature for 10-15 min.

[0112] 8) Dilute the protoplasts with 1 mL of W5, mix well, and terminate the reaction;

[0113] 9) Centrifuge at 600 rpm for 5 min to collect protoplasts and remove the supernatant;

[0114] 10) Add 1 mL of W5 solution to wash.

[0115] Transient expression of DG1C01667.1 protein in rice protoplasts was observed under a laser confocal microscope, revealing that the protein may be localized in the nucleus and cytoplasm (e.g., Figure 2 ).

[0116] Example 2

[0117] This embodiment verifies the effect of DG1C01667.1 on the flowering time of Arabidopsis thaliana.

[0118] 1. Construction of overexpression vectors

[0119] 1.1 Ligation of homologous arms of the target gene

[0120] Based on the pattern of the vector pHG-35-GFP, BamHI / PstⅠ was designed as the insertion site, and primers were synthesized. The primer sequences are as follows:

[0121] pHG-35S-DG1C01667.1-F:CTCTCTCTCAAGCTTGGATCCatggggcgcgggccggtg

[0122] pHG-35S-DG1C01667.1-R:ACGGGTCATGAGCTCCTGCAGtctgttatactgatggtgcggagcatc

[0123] Using the target fragment DG1C01667.1 prepared in section 2.1 of Example 1 as a template, a PCR reaction was performed to obtain the homologous arm gene containing the pHB-35S restriction site. The correct PCR fragment was recovered. The method was the same as described above.

[0124] 1.2 Enzyme digestion of the vector pHG-35-GFP plasmid:

[0125] 40μL enzyme digestion system:

[0126] Plasmid (30 μL)

[0127] 4 μL 10× enzyme digestion buffer

[0128] 4μL 10×BSA (refer to the instruction manual for optional use)

[0129] 6U restriction endonuclease (NEB)

[0130] Add water to make up to 40μL

[0131] Treat in a water bath at 37℃ for about 1 hour.

[0132] 1.3 Construction of recombinant plasmids:

[0133] After the PCR fragment in step 1.1 is recovered by agarose gel electrophoresis, it is mixed with the empty vector recovered by enzyme digestion in step 1.2, and ligated into the EasyGenoDNA recombination system (#VI201-02, Tiangen Biotech). The 10 μL recombination system is as follows:

[0134] 5μL 2×EasyGeno Assembly Mix

[0135] 2.5 μL of enzyme-digested vector DNA

[0136] 2.5 μL DNA fragment

[0137] Add the reaction system to a 250 μL EP tube, incubate at 50°C for 30 minutes, transform E. coli and plate the mixture, incubate at 37°C for 16 hours, pick bacteria, and send for sequencing. Extract plasmids with correct sequencing results and store them at -20°C for long-term storage.

[0138] 1.4 Plasmid Extraction

[0139] Inoculate a single positive colony into 3 mL of LB broth containing kanamycin and incubate overnight at 37°C with shaking at 200 rpm. Take 1.5 mL of the culture (3 mL of low-copy plasmid), centrifuge at 12,000 rpm for 30 seconds, aspirate the supernatant, and resuspend 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%) and immediately mix gently up and down. Add 150 μL of Solution III (KAc 5 mol / L, pH 8.0). 4.8) Quickly mix from top to bottom 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; remove the supernatant, wash the DNA precipitate with 70% ethanol, centrifuge at 12,000 rpm for 1 minute, remove the supernatant; vacuum dry the precipitate; dissolve in 60 μL of double-distilled water containing 10 μg / mL RNase A.

[0140] 2. Arabidopsis transformation

[0141] 2.1 Culture of Agrobacterium:

[0142] 1) Take the competent Agrobacterium cells stored at -80℃ and let them partially thaw at room temperature or in your palm for a moment. When they are in an ice-water mixture, insert them into ice.

[0143] 2) Add 0.1 μg (volume not exceeding 10 μL) of plasmid DNA to each 100 μL of competent Agrobacterium, mix well by hand by tapping the bottom of the tube, and incubate 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 antibiotic-free LB liquid medium and incubate at 28°C with shaking at 200 rpm for 2–3 hours;

[0145] 4) Centrifuge at 6000 rpm for one minute to collect the bacteria. Take approximately 100 μL of supernatant, gently pipette to resuspend the bacterial block, and spread it onto an LB agar plate containing the appropriate antibiotic. Invert the plate and incubate at 28°C for 2-3 days. Randomly select one single colony for colony PCR. Label the correctly identified Agrobacterium single clone for later use.

[0146] 5) Using a sterile pipette tip, pick up a labeled Agrobacterium monoclonal and inoculate it into 1.5 mL of LB liquid medium containing the corresponding antibiotic (in a 50 mL blue cap centrifuge tube). Incubate at 30°C and 200 rpm for 24 hours with shaking.

[0147] 6) Inoculate the Agrobacterium culture that has been shaken too much into 100 mL of LB liquid medium containing antibiotics at a ratio of 1%, and incubate at 30 °C with shaking until OD600 = 1.0;

[0148] 7) Centrifuge at 20℃, 4,000 rpm for 15 min and collect the bacterial cells; resuspend the bacterial cells in transformation buffer until OD600 = 1.0.

[0149] The components of the conversion buffer are as follows:

[0150]

[0151] 2.2 Arabidopsis transformation:

[0152] 1) Water the Arabidopsis thaliana plants that are currently bolting and flowering thoroughly one day in advance;

[0153] 2) Invert the flowerpot and place all the inflorescences upside down into the Agrobacterium bacillus solution that has been suspended in the conversion buffer for about 30 seconds;

[0154] 3) Repeat the transformation process once after 7 days. After 2-3 weeks, water with nutrient solution as little as possible to accelerate aging. Collect mature seeds in paper bags and dry them in a desiccator for 7 days.

[0155] 2.3 Screening of transgenic Arabidopsis thaliana

[0156] 1) Preparation of culture medium: Arabidopsis thaliana culture medium was prepared using 1 / 2 MS (0.8% agar powder, sucrose-free, 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, invert and mix for 5 minutes, and rinse 5 times with sterile water;

[0158] 3) Resuspend the disinfected seeds in 100 μL of sterile water, and use a 1 mL pipette tip to spot them onto a 1 / 2 MS medium (the medium contains screening antibiotics: 50 μg / mL KAN or 30 μg / mL HYG or 50 μM Glufosinate-ammonium) plate.

[0159] 4) Seal the petri dishes and vernalize them at 4 degrees Celsius for 48 hours. Then, place them in an artificial climate chamber to begin germination and growth. The plant growth environment is as follows: relative humidity 60%; constant temperature 20-22 degrees Celsius; photoperiod of 16 hours of light followed by 8 hours of darkness; light intensity 80-200 μmol / m². 2 / S;5) Observe after 8-15 days, and transplant positive ones into planting soil;

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

[0161] 7) Soaking the soil: Fill the planting pot with soil to about 1cm from the rim, and soak it completely with Flower Perfection compound fertilizer (N, P, K = 20%, 20%, 20%).

[0162] 8) Transplanting: 20 days after germination, select healthy seedlings with uniform growth and transplant them into potting soil that has been soaked in Flower Perfection solution beforehand. Cover them with plastic wrap and remove it after the seedlings have taken root.

[0163] 3. Overexpression of the DG1C01667.1 gene promotes early flowering in Arabidopsis thaliana.

[0164] Phenotypic identification and statistics on flowering time and number of rosette leaves:

[0165] Flowering time determination: The flowering time of Arabidopsis thaliana was recorded when the bolting height was 0.5 cm;

[0166] Rosette leaf number determination: The number of rosette leaves was counted when Arabidopsis thaliana was flowering.

[0167] The measurement results are as follows Figures 3-5 As shown, through Figure 3 , Figure 4 and Figure 5 It can be seen that the overexpression of DG1C01667.1 lines OE4, OE5 and OE6 flower earlier than the wild type WT, and the number of rosette leaves at flowering time is also less than that of the wild type. Figure 4 , 5 In the text, different lowercase letters represent significant differences in flowering time / roselet leaves between different strains.

[0168] Example 3

[0169] Identification of downstream target genes of DG1C01667.1.

[0170] To verify how DG1C01667.1 affects the flowering time of orchardgrass, we performed DAP-seq sequencing on the DG1C01667.1 gene.

[0171] 1. DNA library preparation

[0172] (1) Kit extraction method

[0173] 1) Material processing: Take 100 mg of fresh leaf tissue and grind it thoroughly with liquid nitrogen. Add 600 μL of lysis buffer PL preheated at 65℃, vortex for 1 min, add 5-6 μL of RNase A (20 mg / mL) before water bath, digest RNA at 37℃ for 10 min, and then incubate at 65℃ for 20-30 min, inverting the sample 2-3 times during the water bath process.

[0174] 2) Add 700 μL of chloroform or chlorine, invert and mix thoroughly for a few minutes, then centrifuge at 12,000 rpm for 5 minutes.

[0175] 3) Transfer the supernatant to a new 1.5mL centrifuge tube, being careful not to aspirate any interfacial material.

[0176] 4) Add 1.5 times the volume of binding solution PQ, and immediately shake thoroughly for 15 seconds. At this time, flocculent precipitate may appear.

[0177] 5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column AC, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column AC into a collection tube.

[0178] 6) Add 500 μL of inhibitor removal solution IR to the adsorption column AC, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column AC into the collection tube.

[0179] 7) Add 600 μL of rinsing buffer WB, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and repeat this step once.

[0180] 8) Place the adsorption column AC back into the collection tube, centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid. Place the adsorption column AC at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material.

[0181] 9) Transfer the adsorption column AC into a clean centrifuge tube, add 100 μL of elution buffer E to the middle of the adsorption membrane, incubate at room temperature for 2-5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0182] (2) Genomic DNA fragmentation and purification recovery

[0183] 1) Buffer 2.0 Remove the Enzyme 2.0, thaw it, mix it thoroughly, briefly centrifuge to collect the contents to the bottom of the tube, and place it on ice for later use.

[0184] 2) Sample preparation and PCR reaction procedure are as follows:

[0185]

[0186]

[0187] temperature time 37℃ 7min 72℃ 20min 4℃ Hold

[0188] 3) Dilute the Adapter to the appropriate concentration according to the amount of Input DNA.

[0189] 4) Take out Ligation Enhancer 2.0 and Rapid DNA Ligase 2.0, thaw them, mix them thoroughly, briefly centrifuge to collect them to the bottom of the tube, and place them on ice for later use.

[0190] The sample preparation and PCR reaction procedure are as follows:

[0191] Components volume Previous product 60μL Ligation Enhancer 2.0 30μL DNA Adapter 5μL Rapid DNA Ligase 2.0 5μL

[0192] temperature time 20℃ 15min 4℃ Hold

[0193] 5) Take the magnetic bead solution out of 2-8℃ 30 minutes in advance and let it stand to allow its temperature to equalize to room temperature.

[0194] a) Invert or vortex the liquid to thoroughly mix the magnetic beads, then take 60 μL of Hieff. Add DNA SelectionBeads to 100 μL of Adapter Ligation product and vortex or pipette 10 times to mix thoroughly.

[0195] b) Incubate at room temperature for 5 minutes to allow DNA to bind to the magnetic beads. Place the sample on a magnetic rack and wait for the solution to become clear.

[0196] c) Keep the sample on the magnetic rack at all times, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0197] d) Repeat the above steps once, for a total of two rinses.

[0198] e) Keep the sample on the magnetic rack at all times, and dry the magnetic beads at room temperature for about 5 minutes with the lid off.

[0199] f) Remove the sample from the magnetic rack, add an appropriate amount of nuclease-free water, vortex or pipette to mix thoroughly, and let stand at room temperature for 2 minutes. After the solution has clarified, let it stand on the magnetic rack for 5 minutes, then carefully aspirate the supernatant into a new nuclease-free centrifuge tube and carefully remove the supernatant.

[0200] 2. Cell-free protein expression

[0201] (1) Expression vector construction was carried out using the PAS (PCR-based Accurate Synthesis) method. Full-length splicing primers were designed, and protective base synthesis genes FSR were designed at both ends of the primers. They were then inserted between the seamless cloning-HindII (AAGCTT) sites of the vector 30a-Halotag; the resulting recombinant plasmid was obtained.

[0202] (2) Cell-free protein expression experiment of wheat germ system Take out of the refrigerator SP6High-Yield WheatGerm Master Mix.

[0203] Add reactants according to the following ratio.

[0204]

[0205] After adding all components to the reaction tube, mix gently. Incubate the reaction at 25°C for 2 hours, then perform Western blotting (WB) verification.

[0206] 3. Binding of proteins to DNA libraries

[0207] (1) Prepare protein samples, Halo-tag antibodies, and Protein G magnetic beads. Each protein sample requires 15 μL of magnetic beads. Take the appropriate volume of magnetic beads according to the number of samples and wash them 3-4 times with PBS at room temperature in a 1.5 mL EP tube. Perform the operation on a magnetic rack. Finally, add PBS solution and aliquot the corresponding number of EP tubes. Remove the supernatant before use.

[0208] (2) Place the EP tube containing magnetic beads and PBS solution on a magnetic rack for 2-5 min, discard the supernatant, add 200 μL of the above mixture (mixture: protein sample + Halo-tag antibody), and shake in a mixer at 40 rpm for 2 h at 4 °C.

[0209] (3) After incubation, place the tube on a magnetic rack at room temperature, use a pipette to aspirate the supernatant to wash the tube wall so that all the adhering beads are washed off and settled to the bottom. Discard the supernatant, immediately add 400 μL PBS, remove the tube and place it on a magnetic rack for 30 seconds, then discard the supernatant.

[0210] (4) Repeat the above steps twice, for a total of 3 washes.

[0211] (5) Add 180 μL of PBS to a clean 1.5 mL EP tube, then add 20 μL of DNA library, and mix well. At this time, discard the PBS in the magnetic beads on the magnetic rack, add the mixed solution, and oscillate at 40 rpm for 2 h at 4℃.

[0212] (6) After incubation, place the tube on a magnetic rack at room temperature. Use a pipette to aspirate the supernatant to rinse the tube wall so that all the beads sink to the bottom. Discard the supernatant and immediately add 400 μL of PBS. Remove the tube and place it on a magnetic rack for 30 seconds. Discard the supernatant.

[0213] (7) Repeat the above steps twice, for a total of 3 times. In the last step, use a 10 μL pipette to remove any remaining solution, add 30 μL of 50 mM Tris-HCl (pH = 8.5), boil in a metal bath at 98°C for 10 min, place on a magnetic rack for 2 min, and collect the supernatant into a clean 1.5 mL EP tube. Store at -20°C. After detecting the protein expression in the boiled sample, proceed with the next step of quantification.

[0214] (8) Add 30 μL of water and 30 μL of 2× loading buffer to the magnetic beads, boil at 99°C for 5-10 min, place on a magnetic rack for 30 sec, and transfer the supernatant to a clean centrifuge tube for later use. Take 20 μL of the supernatant for SDS-PAGE.

[0215] 4. Library PCR with index adapters and quantitative detection

[0216] (1) After thawing the Pro Amplification Mix and Primer Mix, invert and mix thoroughly, then briefly centrifuge to collect the residue at the bottom of the tube. Prepare the PCR reaction in the sterile PCR tube as follows:

[0217]

[0218]

[0219] (2) Using Hieff DNA Selection Beads are used to purify the reaction products:

[0220] 1) Take the magnetic bead solution out of 2-8℃ 30 minutes in advance and let it stand to allow its temperature to equalize to room temperature.

[0221] 2) Invert or vortex to thoroughly mix the magnetic bead solution, then take 45 μL of Hieff Add DNA SelectionBeads to 50 μL of Library Amplification product and vortex or pipette 10 times to mix thoroughly.

[0222] 3) Incubate at room temperature for 5 minutes to allow DNA to bind to the magnetic beads. Place the sample on a magnetic rack and carefully remove the supernatant after the solution has clarified.

[0223] 4) Keep the sample on the magnetic rack at all times, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0224] 5) Repeat step 4 once, for a total of two rinses.

[0225] 6) Keep the sample on the magnetic rack at all times, and open the lid to dry the magnetic beads for about 5 minutes at room temperature.

[0226] 7) Remove the sample from the magnetic rack, add an appropriate amount of nuclease-free water, vortex or pipette to mix thoroughly, and let stand at room temperature for 2 minutes. After the solution has clarified, let it stand on the magnetic rack for 5 minutes, then carefully aspirate the supernatant into a new nuclease-free centrifuge tube.

[0227] 5. High-throughput sequencing

[0228] The obtained DNA was sequenced using an Illumina HiSeq sequencer.

[0229] 6. DAP-seq data analysis

[0230] A total of 7490 potential binding sites for DG1C01667.1 were identified using DAP-seq analysis. Of these sites, 88.09% were located in intergenic regions, 3.42% in promoter regions, and 3.38% downstream of the gene. Many similar motifs were also detected in this experiment. A flowering-related gene, ethylene response factor 1 (DG2C03697.1, DgERF1), was identified as a potential target gene of DG1C01667.1 regulating orchardgrass flowering.

[0231] In this invention, the complete genome sequence of DG1C01667.1 is as follows:

[0232] Atggggcgcgggccggtgcagctgcggcggatcgagaacaagataaaccggcaggtcaccttctccaagcgccg

[0233] gagcgggctgctcaagaaggcgcacgagatctccgtgctctgcgacgccgaggtcgcgctcatcgtcttctccaccaa

[0234] gggcaagctctacgagtactccagccaggacagtaacatggatgtcattcttgaacgttaccaacgttactcattcga

[0235] ggaaagagctgtagtggaccaaaatattggaggccaggcaaattggggagatgaatatcgaagtttgaaaataaaact

[0236] cgatgcactccagaagagtcaaaggcaactcttaggtgaacaattggacccactgaccacaaaagaacttcagcaatt

[0237] ggaacagcagctagatagttctctgaagcacatcaggtcaagaaagaatcagcttctgtttgagtcaatatctgaact

[0238] tcagaagaaggagaagtcacttaaagatcagaatggcgtcctgcagaagcacctcgtggagacacaaaaggagaaaaa

[0239] taacgttttatctaatattcatcaccgggaggagctgaatggagcagcaaatattcatcaccgggagcagctgaatgg

[0240] agcaacaaatattcatcaccgggagcagctgaatggagcagcaaatattcatcaccgggagcagctgaatggagcagc

[0241] aaatattcatcaccgtgagcagctgaatggagcaacaaatattcacatccaggacgagcataatggagcaacaacaag

[0242] ctcaccgtcacctacaccagtgacggtcctagattccgtggcaactttaaatattgggtcatcgcattctagagaatc

[0243] agcaggaggggagccagaatcacagccgtctccatcacaagcaaacagcggcaagctaccaccatggatgctccgcac

[0244] catcagtaacagatga.

[0245] The protein sequence of DG1C01667.1 is as follows:

[0246] MGRGPVQLRRIENKINRQVTFSKRRSGLLKKAHEISVLCDAEVALIVFSTKGKLYEYSSQDSNMDVILERYQRY

[0247] SFEERAVVDQNIGGQANWGDEYRSLKIKLDALQKSQRQLLGEQLDPLTTKELQQLEQQLDSSLKHIRSRKNQLLFESI

[0248] SELQKKEKSLKDQNGVLQKHLVETQKEKNNVLSNIHHREELNGAANIHHREQLNGATNIHHREQLNGAANIHHREQLN

[0249] GAANIHHREQLNGATNIHIQDEHNGATTSSPSPTPVTVLDSVATLNIGSSHSRESAGGEPESQPSPSQANSGKLPPWM

[0250] LRTISNR.

[0251] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0252] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0253] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A gene DG1C01667.1 related to plant flowering time, characterized in that: The nucleotide sequence of DG1C01667.1 is shown in SEQ ID NO.

1.

2. The protein encoded by the gene DG1C01667.1 related to plant flowering time according to claim 1, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. The use of the gene DG1C01667.1 related to plant flowering time in Arabidopsis breeding according to claim 1, characterized in that: The application is to promote early flowering of Arabidopsis thaliana.

4. Use of the protein encoded by the gene DG1C01667.1 related to plant flowering time as claimed in claim 2 in preparing an early-flowering Arabidopsis strain.

5. An overexpression vector comprising the gene DG1C01667.1 related to plant flowering time as claimed in claim 1.

6. Agrobacterium comprising the gene DG1C01667.1 related to plant flowering time as claimed in claim 1.

7. A method for cultivating early-flowering transgenic plants, characterized in that: Overexpressing the DG1C01667.1 gene in plants to obtain early-flowering transgenic plants; Wherein, the nucleotide sequence of the DG1C01667.1 gene is shown in SEQ ID NO.1; The plant is Arabidopsis thaliana.

8. The method for cultivating early-flowering transgenic plants according to claim 7, characterized in that: The method of overexpressing the DG1C01667.1 gene in plants to obtain early-flowering transgenic plants specifically includes: The DG1C01667.1 gene was homologously recombined with the vector pHG-35S to obtain the overexpression vector pHG-35S-DG1C01667.1; The pHG-35S-DG1C01667.1 was transformed into plants, and early-flowering transgenic plants were obtained through screening.

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