Gene DG1C01667.1 related to plant flowering time and application of gene DG1C01667.1

Abstract

The invention provides a gene DG1C01667.1 related to the flowering time of a plant and application of the gene DG1C01667.1, and belongs to the technical field of biology, and the nucleotide sequence of the DG1C01667.1 is as shown in SEQ ID NO. 1. The flowering time of arabidopsis thaliana overexpressed with the DG1C01667.1 gene is obviously advanced, and the DG1C01667.1 gene can be applied to genetic engineering modification of plants so as to regulate and control the flowering phase of the plants, shorten the breeding time, improve the breeding efficiency and promote the development and utilization of gramineous forage grass.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a gene DG1C01667.1 related to plant flowering time 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 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 feed, 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. It is used for both mowing and grazing. Various poultry like to eat it. It has achieved good economic and ecological benefits and has shown broad prospects for utilization.

[0003] Flowering period is an important agronomic trait of Dactylis turf, which is closely related to its quality and yield. The flowering period represents the transition of plants from vegetative growth to reproductive growth, and the quality and yield of forage will also change accordingly. Dactylis turf is suitable for mixed sowing with leguminous forages. Targeted cultivation of Dactylis turf varieties with different flowering periods and matching them with leguminous forage varieties with the same maturity period can extend the grazing time of mixed sowing grasslands and improve comprehensive production performance and utilization efficiency. Summary of the invention

[0004] The purpose of the present invention is to provide a gene DG1C01667.1 related to plant flowering time and its application. The flowering time of Arabidopsis thaliana overexpressing the DG1C01667.1 gene is significantly advanced, and it can be applied to plant genetic engineering modification to regulate plant flowering period, shorten breeding time, improve breeding efficiency, and promote the development and utilization of Poaceae forage.

[0005] The present invention is achieved through the following technical solutions:

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

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

[0008] The present invention also provides an application of a gene DG1C01667.1 related to plant flowering time in Arabidopsis breeding.

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

[0010] The present invention also provides an application of a protein encoded by a gene DG1C01667.1 related to the flowering time of a plant in preparing an early-flowering Arabidopsis strain.

[0011] The present invention also provides an overexpression vector comprising the gene DG1C01667.1 related to the flowering time of plants.

[0012] The present invention also provides Agrobacterium containing the gene DG1C01667.1 related to the flowering time of plants. Based on the same inventive concept, the present invention also provides a method for cultivating early-flowering transgenic plants, overexpressing the DG1C01667.1 gene in plants to obtain early-flowering transgenic plants;

[0013] Alternatively, the expression level of DG1C01667.1 protein in the plant is increased to obtain early-flowering transgenic plants;

[0014] Among them, 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 overexpression of the DG1C01667.1 gene in the plant to obtain the early-flowering transgenic plant specifically includes:

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

[0017] pHG-35S-DG1C01667.1;

[0018] The pHG-35S-DG1C01667.1 was transformed into plants, and early-flowering transgenic plants were obtained through screening.

[0019] Preferably, the plant comprises Arabidopsis thaliana.

[0020] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0021] The invention discloses a gene DG1C01667.1 related to the flowering time of a plant. 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 of Arabidopsis thaliana. The DG1C01667.1 gene is applied to the genetic engineering modification of Dactylis grass, which is expected to achieve the regulation of the flowering period of Dactylis grass, shorten the breeding time, and improve the breeding efficiency. The gene has important utilization value for improving forage quality, and promotes the development and utilization of the Gramineae forage Dactylis grass. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 This is the evolutionary tree analysis diagram of DG1C01667.1 protein and other homologous proteins.

[0024] Figure 2 This is the subcellular localization map of DG1C01667.1 protein.

[0025] Figure 3 The flowering time phenotypes of the wild-type and DG1C01667.1-overexpressing lines are shown.

[0026] Figure 4 The flowering time statistics of wild-type and DG1C01667.1-overexpressing lines are shown in Figure 2.

[0027] Figure 5 The number of rosette leaves at flowering time of wild type and DG1C01667.1 overexpressing lines. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] 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.

[0031] The overall idea of ​​the present invention is as follows:

[0032] MADS-box is an important type of transcriptional regulatory factor that exists almost throughout the plant kingdom and is involved in the development of plant floral tubes and the regulation of flowering time. We performed genome-simplified sequencing on 249 Duckgrass materials and found a key candidate gene DG1C01667.1 that regulates Duckgrass flowering through genome-wide association analysis. Tests have shown that overexpression of the DG1C01667.1 gene can promote early flowering in Arabidopsis and Duckgrass, which indicates that the Duckgrass DG1C01667.1 gene can participate in the regulation of plant flowering time and has important utilization value in improving forage quality. Using DG1C01667.1 to transfer into Duckgrass has important theoretical and application value for its research on the flowering time of Duckgrass.

[0033] Duckgrass is a cross-pollinated plant with complex ploidy, which makes its genetic transformation difficult and the verification of gene function is relatively lagging. So far, the research on the Duckgrass DG1C01667.1 gene is still blank. The present invention provides a gene related to the flowering time of plants-DG1C01667.1, and applies it to the genetic engineering modification of Duckgrass. For the shortcomings of traditional forage improvement technology that is slow to take effect and has a long cycle, this method can shorten the breeding time, improve the breeding efficiency, and promote the development and utilization of high-quality Gramineae forage Duckgrass.

[0034] The following will describe in detail a gene DG1C01667.1 related to plant flowering time and its application in combination with embodiments and experimental data.

[0035] Example 1

[0036] Phylogenetic tree analysis and subcellular localization of DG1C01667.1d.

[0037] 1. Evolutionary tree analysis

[0038] The homologous sequences of DG1C01667.1 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 DG1C01667.1 protein of Dactylis glomerata had the highest homology with the LrMADS18 protein of Lolium rigidum ( Figure 1 )

[0039] 2. Subcellular localization

[0040] 2.1 Target fragment amplification

[0041] 2.11 Cloning of target fragment

[0042] The experimental material was the Duckgrass variety "2006-1", which was planted in the Wenjiang campus of Sichuan Agricultural University. Its young leaves were used as materials 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 an ultra-micro spectrophotometer was used to determine RNA concentration and purity. TaKaRa's PrimeScript II 1st StrandcDNA Synthesis Kit was used for reverse transcription, and the operation process was referred to the instructions attached.

[0043] Using RNA from "2006-1" as template, primers were designed by sequencing the full length of the

[0044] DG1C01667.1-F:5'-ATGGGGCGGGCCGGTGCAGC-3',

[0045] DG1C01667.1-R:5'-TCTGTTACTGATGGTGCGGAGCATC-3'.

[0046] The cDNA was used as a template for amplification. The 2×Phanta Max Master Mix (DyePlus) kit of Vazyme was used for amplification. The operation procedure was referred to the instruction manual attached.

[0047] 50uL reaction system:

[0048] 25uL 2×Phanta Max Master Mix(Dye Plus)

[0049] 2uL upstream primer (10μM)

[0050] 2uL downstream primer (10μM)

[0051] 4uL template DNA*

[0052] 17uL ddH2O.

[0053] Reaction procedure:

[0054] Pre-denaturation at 95°C for 3 min;

[0055] 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;

[0056] Extension at 72°C for 5 min.

[0057] 2.12DNA electrophoresis and recovery:

[0058] 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). Perform electrophoresis at 5-10V / cm in 0.5×TBE buffer, and take photos in a gel imaging system after the electrophoresis is completed.

[0059] Agarose gel DNA recovery (GK2042, Jierui Biotechnology):

[0060] 1) Carefully cut off the target DNA band and place it in a 1.5 mL EP tube.

[0061] 2) Add 400uL of banding B to the tube and place it in a 70℃ water bath until the gel is completely dissolved.

[0062] 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.

[0063] 4) Repeat step 3.

[0064] 5) Add 500uL wash buffer and wash twice at 12000rpm. Centrifuge at 10000rpm for 1 minute.

[0065] 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.

[0066] 2.2 Functional analysis of DG1C01667.1

[0067] Subcellular localization technology was used to determine the location of DG1C01667.1 protein. In order 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 pAN580-35S-GFP empty vector was used as a control, and the fusion vector and the control empty vector were transformed into rice protoplasts.

[0068] 2.21 Synthesis of primers for amplification of the CDS of the target gene DG1C01667.1

[0069] pAN580-DG1C01667.1-F:AGTCCGGAGCTAGCTCTAGAatggggcgcgggccggtg

[0070] pAN580-DG1C01667.1-R:GCGGCCGCTGTACAGGATCCtctgttatactgatggtgcggagcatc

[0071] 2.22 PCR system and procedure

[0072] Perform PCR reaction using the cDNA prepared in step 2.1 as template.

[0073] Amplification system:

[0074] Ingredients volume Nuclease-free Water 20uL Biorun Pfu PCR Mix 25uL Primer(+) 2uL Primer(-) 2uL Template 1uL Total volume 50uL

[0075] PCR procedure:

[0076]

[0077]

[0078] The PCR products were subjected to DNA electrophoresis and recovered as described above.

[0079] 2.23 Vector digestion

[0080] The vector used is pAN580-35S-GFP, and the restriction enzyme linking system is as follows:

[0081]

[0082] Reaction conditions:

[0083] temperature time 37℃ 1hours

[0084] The vector digestion product was purified using a PCR purification kit for the next recombination reaction.

[0085] 2.24 Recombination reaction

[0086] The (ORF) sequence of DG1C01667.1 was recombined with the vector enzyme digestion product. The recombination reaction system is as follows:

[0087]

[0088]

[0089] Reaction conditions:

[0090] temperature time 37℃ 30 hours

[0091] The ligation product was transformed into competent cells.

[0092] 2.25 Transformation of competent cells

[0093] 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.

[0094] 2.26 Colony PCR Identification

[0095] Pick 10 colonies for 1.5mL EP tube inoculation and PCR identification, and design identification primers with pAN580-linker-esp3I.

[0096] Pan580-F:agttcatttcatttggagaggacag(2881bp)

[0097] D13519(633C):gatgaatatttgttgctccattcagct(3554bp).

[0098] PCR system:

[0099]

[0100]

[0101] PCR procedure:

[0102] step Number of cycles 94℃for 5min 1 94℃for 30sec 30 50℃for 45sec 30 72℃for 76sec 30 72℃for 10min 1 16℃for 30min 1

[0103] 2.27 Plasmid extraction from colonies

[0104] Inoculate the correct positive single colony into 3mL LB liquid culture medium containing Amp antibiotics, and culture overnight at 37℃ and 200rpm; take 1.5mL culture (3mL for low-copy plasmid), centrifuge at 12,000rpm for 30 seconds; aspirate the supernatant, suspend the bacteria in 100μL solution Ⅰ (Glucose 50mmol / L, EDTA 10mmol / L, Tris-HCl 25mmol / L, pH 8.0); add 200μL freshly prepared solution Ⅱ (NaOH 0.2mol / L, SDS1%), and immediately mix gently up and down; add 150μL solution Ⅲ (KAc 5mol / 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.

[0105] 2.28 Transformation of rice protoplasts

[0106] 1) After the rice seedlings were cultured in the dark at about 30℃ for 7-15 days, the stems and leaves were taken out, the surface dirt was washed with water, and the outermost leaf sheath was removed;

[0107] 2) Place the stems and leaves of the seedlings on a clean plastic board and cut them into small pieces with a sharp blade;

[0108] 3) Add 5-10 mL of enzymatic solution to completely immerse the tissue, at 28°C, and shake slowly for 4-5 hours;

[0109] 4) Filter the protoplasts with a 40 μm filter, then transfer to a 2 mL centrifuge tube and centrifuge at 600 rpm for 5 min. A turbid precipitate is visible.

[0110] 5) Directly aspirate the supernatant, wash twice with 10 mL of pre-cooled W5 solution, centrifuge at 600 rpm for 5 min, and centrifuge at 25°C;

[0111] 6) Add appropriate amount of MMG solution as needed to suspend the protoplasts to a concentration of 2*105 cells / mL. Microscopic examination: the protoplasts are round with few ruptures;

[0112] 7) Take 100uL protoplast suspension + 10uL DNA (plasmid solution prepared in step 2.27 or pAN580-35S-GFP empty vector), and the same volume of PEG 4000 solution, mix gently and evenly, and let stand at room temperature for 10-15min;

[0113] 8) Dilute the protoplasts with 1 mL of W5 and mix well to terminate the reaction;

[0114] 9) Centrifuge at 600 rpm for 5 min to collect the protoplasts and remove the supernatant;

[0115] 10) Add 1 mL of W5 solution for washing.

[0116] After transient expression in rice protoplasts, protoplast fluorescence was observed under a laser confocal microscope and it was found that the DG1C01667.1 protein may be localized in the nucleus and cytoplasm (such as Figure 2 ).

[0117] Example 2

[0118] This example verifies the effect of DG1C01667.1 on the flowering time of Arabidopsis thaliana.

[0119] 1. Overexpression vector construction

[0120] 1.1 Connection of homology arms of target gene

[0121] 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:

[0122] pHG-35S-DG1C01667.1-F:CTCTCTCTCAAGCTTGGATCCatggggcgcgggccggtg

[0123] pHG-35S-DG1C01667.1-R:ACGGGTCATGAGCTCCTGCAGtctgttatactgatggtgcggagcatc

[0124] Using the target fragment of DG1C01667.1 prepared in Section 2.1 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.

[0125] 1.2 pHG-35-GFP plasmid digestion:

[0126] 40μL enzyme digestion system:

[0127] Plasmid (30 μL)

[0128] 4 μL 10× digestion buffer

[0129] 4μL 10×BSA (with or without reference to the instruction manual)

[0130] 6U restriction enzyme (NEB)

[0131] Add water to make up to 40 μL

[0132] Incubate in a 37°C water bath for about 1 hour.

[0133] 1.3 Recombinant plasmid construction:

[0134] 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:

[0135] 5μL 2×EasyGeno Assembly Mix

[0136] 2.5 μL digested vector DNA

[0137] 2.5 μL fragment DNA

[0138] 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.

[0139] 1.4 Plasmid extraction

[0140] Inoculate a positive single colony into 3 mL of LB liquid culture medium containing kanamycin, and culture overnight at 37°C and 200 rpm; 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.

[0141] 2. Arabidopsis Transformation

[0142] 2.1 Cultivation of Agrobacterium:

[0143] 1) Take the competent Agrobacterium stored at -80℃ and place it at room temperature or in the palm of your hand for a while to partially melt it. When it is in a state of ice-water mixing, insert it into ice;

[0144] 2) Add 0.1 μg (volume not more than 10 μL) plasmid DNA for every 100 μL competent Agrobacterium, 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;

[0145] 3) Add 700 μL of LB liquid medium without antibiotics and culture at 28°C, 200 rpm with shaking for 2 to 3 hours;

[0146] 4) Centrifuge at 6000rpm for one minute to collect the bacteria, take about 100μL of the supernatant, gently blow and resuspend the bacteria, and 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;

[0147] 5) Use a sterile pipette tip to pick up the marked Agrobacterium monoclone and inoculate it into 1.5 mL LB liquid medium containing the corresponding antibiotics (use a 50 mL blue-capped centrifuge tube) and culture it at 30°C, 200 rpm and shaking for 24 hours;

[0148] 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;

[0149] 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.

[0150] The conversion buffer components are as follows:

[0151]

[0152] 2.2 Arabidopsis transformation:

[0153] 1) Water the Arabidopsis thaliana that is sprouting and flowering one day in advance;

[0154] 2) Invert the flower pot and place all the inflorescences into the Agrobacterium solution previously suspended in transformation buffer for about 30 seconds;

[0155] 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.

[0156] 2.3 Screening of transgenic Arabidopsis

[0157] 1) Preparation of culture medium: Arabidopsis thaliana culture medium is 1 / 2MS (0.8% agar powder, without sucrose, Ph 5.8);

[0158] 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;

[0159] 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;

[0160] 4) Seal the plate, put it in a refrigerator at 4 degrees for 48 hours, and place 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;

[0161] 6) Preparation of planting soil: Mix peat soil and vermiculite at a ratio of 2:1 and set aside for later use;

[0162] 7) Soil soaking: 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;

[0163] 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.

[0164] 3. Overexpression of DG1C01667.1 gene promotes early flowering in Arabidopsis

[0165] Phenotypic identification and statistics of flowering time and number of rosette leaves:

[0166] Flowering time determination: The flowering time of Arabidopsis thaliana was counted when the bolting height was 0.5 cm;

[0167] Determination of rosette leaf number: The number of rosette leaves was counted when Arabidopsis thaliana was flowering.

[0168] The results of the test are as follows Figures 3 to 5 As shown, through Figure 3 , Figure 4 and Figure 5 It can be seen that the flowering time of the DG1C01667.1 overexpressing lines OE4, OE5 and OE6 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 4 , 5 In the table, different lowercase letters represent significant differences in flowering time / rosette leaves among different lines.

[0169] Example 3

[0170] Identification of downstream target genes of DG1C01667.1.

[0171] To verify how DG1C01667.1 affects the flowering time of Dactylis glomerata, we performed DAP-seq sequencing of the DG1C01667.1 gene.

[0172] 1. DNA Library Preparation

[0173] (1) Kit extraction method

[0174] 1) Processing materials: Take 100 mg of fresh leaf tissue, add liquid nitrogen and grind thoroughly. Add 600 μL of lysis buffer PL preheated at 65°C, vortex for 1 min, add 5-6 μL RNaseA (20 mg / mL) at 37°C for 10 min to digest RNA before water bathing, and water bath at 65°C for 20-30 min. Invert and mix the sample 2-3 times during the water bathing process.

[0175] 2) Add 700 μL of chloroform or chlorine, mix thoroughly by inversion for a few minutes, and centrifuge at 12,000 rpm for 5 minutes.

[0176] 3) Pipette the supernatant into a new 1.5 mL centrifuge tube, being careful not to aspirate the interface material.

[0177] 4) Add 1.5 times the volume of binding solution PQ and immediately shake thoroughly for 15 seconds. Flocculent precipitation may appear at this time.

[0178] 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 place the adsorption column AC in a collection tube.

[0179] 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 place the adsorption column AC in a collection tube.

[0180] 7) Add 600 μL of washing solution WB, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and repeat this step once.

[0181] 8) Place the adsorption column AC back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column AC at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0182] 9) Transfer the adsorption column AC into a clean centrifuge tube, add 100 μL of elution buffer E to the middle part of the adsorption membrane, place at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 2 minutes, and collect the solution into the centrifuge tube.

[0183] (2) Genomic DNA fragmentation and purification

[0184] 1) Buffer 2.0, Take out Enzyme 2.0, thaw and mix thoroughly, centrifuge briefly to collect to the bottom of the tube, and place on ice for later use.

[0185] 2) Sample configuration and PCR reaction procedures are as follows:

[0186]

[0187]

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

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

[0190] The sample configuration and PCR reaction procedures are as follows:

[0191]

[0192] 5) Take out the magnetic bead solution from 2-8°C 30 minutes in advance and let it stand to allow its temperature to equilibrate to room temperature.

[0193] a) Invert or vortex to mix the magnetic beads thoroughly, and pipette 60 μL of Hieff Add DNA SelectionBeads to 100 μL Adapter Ligation product and vortex or pipette 10 times to mix thoroughly.

[0194] b) Incubate at room temperature for 5 minutes to allow the DNA to bind to the magnetic beads. Place the sample on a magnetic stand and wait for the solution to clear.

[0195] c) Keep the sample on the magnetic stand, 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.

[0196] d) Repeat the above steps once, rinsing twice in total.

[0197] e) Keep the sample on the magnetic rack and dry the magnetic beads with the lid open at room temperature for about 5 minutes.

[0198] f) Remove the sample from the magnetic rack, add an appropriate amount of nuclease-free water, vortex or use a pipette to mix thoroughly, and let it stand at room temperature for 2 minutes. Let it stand on the magnetic rack for 5 minutes until the solution is clear, carefully pipette the supernatant into a new nuclease-free centrifuge tube, and carefully remove the supernatant.

[0199] 2. Cell-free protein expression

[0200] (1) Expression vector construction: A PAS (PCR-based Accurate Synthesis) method was used to design full-length splicing primers. Protective base synthesis genes FSR were designed at both ends of the primers and connected between the seamless cloning-HindIII (AAGCTT) sites of the vector 30a-Halotag to obtain a recombinant plasmid.

[0201] (2) Cell-free protein expression experiment in wheat germ system Take out of the refrigerator SP6High-YieldWheat Germ Master Mix.

[0202] Add reactants according to the following ratio

[0203]

[0204] After adding all components to the reaction tube, mix gently. Incubate the reaction at 25°C for 2 h and perform WB verification.

[0205] 3. Binding of protein to DNA library

[0206] (1) Prepare protein samples, Halo-tag antibodies, and ProteinG magnetic beads. Each protein sample requires 15 μL of magnetic beads. Take out the corresponding 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. Operate on a magnetic stand. Finally, add PBS solution and evenly distribute it in the corresponding number of EP tubes. Aspirate the supernatant before use.

[0207] (2) Place the EP tube containing the PBS solution of magnetic beads on a magnetic rack for 2-5 minutes, discard the supernatant, add 200 μL of the above mixed solution (mixed solution: protein sample + Halo-tag antibody), and shake in a mixer at 4°C and 40 rpm for 2 hours.

[0208] (3) 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 adhered beads are washed off and settled to the bottom, discard the supernatant, immediately add 400 μL PBS, take out the tube and place it on a magnetic rack for 30 seconds and discard the supernatant.

[0209] (4) Repeat the above steps twice, washing 3 times in total

[0210] (5) Take 180 μL PBS and add it to a clean 1.5 mL EP tube. Then add 20 μL DNA library and mix by pipetting. Discard the PBS in the magnetic beads on the magnetic stand and add the mixed solution to the tube. Oscillate at 40 rpm at 4°C for 2 h.

[0211] (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, immediately add 400 μL PBS, take out and place on a magnetic rack for 30 seconds, and discard the supernatant.

[0212] (7) Repeat the above steps twice, a total of 3 times. In the last step, use a 10 μL pipette to remove the residual solution, add 30 μL 50 mM Tris-HCl, pH = 8.5, boil in a metal bath at 98°C for 10 min, place on a magnetic stand for 2 min, take the supernatant into a clean 1.5 mL EP tube, and store at -20°C. After the protein expression is detected, proceed to the next step of quantification.

[0213] (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 aspirate the supernatant into a clean centrifuge tube for later use. Take 20 μL of the supernatant and run SDS-PAGE.

[0214] 4. Library PCR plus index adapter and quantitative detection

[0215] (1) After thawing, invert and mix the Pro Amplification Mix and Primer Mix, centrifuge briefly to collect the contents at the bottom of the tube. Prepare the PCR reaction in a sterile PCR tube as follows:

[0216]

[0217]

[0218] (2) Use Hieff DNA Selection Beads are used to purify the reaction products:

[0219] 1) Take out the magnetic bead solution from 2-8°C 30 minutes in advance and let it stand to balance its temperature to room temperature.

[0220] 2) Invert or vortex to mix the magnetic beads thoroughly, and pipette 45 μL of Hieff Add DNA SelectionBeads to 50μL Library Amplification product and vortex or pipette 10 times to mix thoroughly.

[0221] 3) Incubate at room temperature for 5 minutes to allow the DNA to bind to the magnetic beads. Place the sample on a magnetic rack and carefully remove the supernatant after the solution has clarified.

[0222] 4) Keep the sample on the magnetic stand, 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.

[0223] 5) Repeat step 4 once, rinsing twice in total.

[0224] 6) Keep the sample on the magnetic rack and dry the magnetic beads at room temperature with the lid open for about 5 minutes.

[0225] 7) Remove the sample from the magnetic rack, add an appropriate amount of nuclease-free water, vortex or use a pipette to mix thoroughly, and let it stand at room temperature for 2 minutes. Let it stand on the magnetic rack for 5 minutes until the solution is clear, and carefully pipette the supernatant into a new nuclease-free centrifuge tube.

[0226] 5. High-throughput sequencing

[0227] The obtained DNA was sequenced using an Illumina HiSeq sequencer.

[0228] 6. DAP-seq Data Analysis

[0229] A total of 7490 potential sites binding to DG1C01667.1 were obtained through DAP-seq analysis. Among these sites, 88.09% were in intergenic regions, 3.42% were in promoter regions, and 3.38% were 2000bp downstream of the gene. In this experiment, we also detected many similar motifs. A flowering-related gene, ethylene response factor 1 (DG2C03697.1, DgERF1), was identified as a potential target gene of DG1C01667.1 that regulates flowering in Duckgrass.

[0230] In the present invention, the full gene sequence of DG1C01667.1 is as follows:

[0231] Atggggcgcgggccggtgcagctgcggcggatcgagaacaagataaaccggcaggtcaccttctccaagcgccggagcgggctgctcaagaaggcgcacgagatctccgtgctctgcgacgccgaggtcgcgctcatcgtcttctccaccaagggcaagctctacgagtactccagccaggacagtaacatggatgtcattcttgaacgttaccaacgttactcattcgaggaaagagctgtagtggaccaaaatattggaggccaggcaaattggggagatgaatatcgaagtttgaaaataaaactcgatgcactccagaagagtcaaaggcaactcttaggtgaacaattggacccactgaccacaaaagaacttcagcaattggaacagcagctagatagttctctgaagcacatcaggtcaagaaagaatcagcttctgtttgagtcaatatctgaacttcagaagaaggagaagtcacttaaagatcagaatggcgtcctgcagaagcacctcgtggagacacaaaaggagaaaaataacgttttatctaatattcatcaccgggaggagctgaatggagcagcaaatattcatcaccgggagcagctgaatggagcaacaaatattcatcaccgggagcagctgaatggagcagcaaatattcatcaccgggagcagctgaatggagcagcaaatattcatcaccgtgagcagctgaatggagcaacaaatattcacatccaggacgagcataatggagcaacaacaagctcaccgtcacctacaccagtgacggtcctagattccgtggcaactttaaatattgggtcatcgcattctagagaatcagcaggaggggagccagaatcacagccgtctccatcacaagcaaacagcggcaagctaccaccatggatgctccgcaccatcagtaacagatga.

[0232] The protein sequence of DG1C01667.1 is as follows:

[0233] MGRGPVQLRRIENKINRQVTFSKRRSGLLKKAHEISVLCDAEVALIVFSTKGKLYEYSSQDSNMDVILERYQRYSFEERAVVDQNIGGQANWGDEYRSLKIKLDALQKSQRQLLGEQLDPLTTKELQQLEQQLDSSLKHIRSRKNQLLFESISELQKK EKSLKDQNGVLQKHLVETQKEKNNVLSNIHHREELNGAANIHHREQLNGATNIHHREQLNGAANIHHREQLNGAANIHHREQLNGATNIHIQDEHNGATTSSPSPTPVTVLDSVATLNIGSSHSRESAGGEPESQPSPSQANSGKLPPWMLRTISNR.

[0234] 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.

[0235] 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.

[0236] 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 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 encoded protein is shown in SEQ ID NO.

2.

3. Use of the gene DG1C01667.1 related to plant flowering time as claimed in claim 1 in Arabidopsis breeding.

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

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

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

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

8. A method for cultivating early-flowering transgenic plants, characterized in that: Overexpressing the DG1C01667.1 gene in plants to obtain early-flowering transgenic plants; Alternatively, the expression level of DG1C01667.1 protein in the plant is increased to obtain early-flowering transgenic plants; Among them, 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.

9. The method for cultivating early-flowering transgenic plants according to claim 8, 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; The plants include Arabidopsis thaliana.

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