Use of ghelf3 gene in regulating flowering time of plants

Abstract

The application discloses application of a GhELF3 gene in regulating flowering time of plants. The application provides application of any one of the following 1)-3) in regulating flowering time, bolting time and / or rosette leaf number of plants; 1) protein GhELF3; 2) a nucleic acid molecule coding protein GhELF3; and 3) a recombinant carrier, an expression cassette or a recombinant bacterium containing the nucleic acid molecule coding protein GhELF3. A cotton biological clock gene GhELF3 is cloned from Gossypium hirsutum, and the GhELF3 is an orthologous gene of Arabidopsis thaliana AtELF3. The GhELF3 shows an oscillation expression mode within one day, and an expression amount of the GhELF3 in a late-flowering cotton variety is significantly higher than that in an early-flowering cotton variety. An overexpression vector of the gene is constructed, and Arabidopsis thaliana is transformed by a dipping method. It is found that the flowering time of the transgenic Arabidopsis thaliana overexpressing the GhELF3 is obviously later than that of the wild-type Arabidopsis thaliana.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to application of GhELF3 gene in regulating flowering time of plants. BACKGROUND

[0002] Cotton is an important economic crop, and its fiber as an important natural fiber is an important raw material for the textile industry. The low degree of mechanization and long growth period of cotton greatly hinder the increase of cotton planting area and fiber yield. The short growth period and concentrated boll opening period of early-maturing cotton make it not only possible to be planted for early-maturing cotton and wheat (oil) after direct sowing in the cotton areas of the Yellow River Basin and the Yangtze River Basin, but also possible to improve the frost-before-flowering rate in the northwest inland cotton areas with short frost-free period and insufficient accumulated temperature. The cultivation of new early-maturing cotton varieties depends on the cloning and functional analysis of key genes for regulating the early-maturing property of cotton.

[0003] Early flower bud differentiation and early flowering are key factors determining the early-maturing of early-maturing cotton. Flower bud differentiation and flowering are complexly regulated by multiple pathways, including the photoperiod pathway, the vernalization pathway, the gibberellin pathway, the age pathway, and the autonomous pathway. Seasonal day length change (photoperiod) is the most conservative and important external environmental signal for regulating the flowering time of plants. Endogenous biological clock and external light signal jointly participate in the regulation of the flowering time of plants dependent on photoperiod. The key regulatory genes in the photoperiod pathway controlled by biological clock are expressed at a fixed time in a day, and the synchronization relationship between the expression phase of these genes and external light signal finally affects the expression of FLOWERING LOCUS T (FT) in the leaf phloem.

[0004] The input of external light signal to the core oscillator of biological clock synchronizes the internal physiological processes controlled by biological clock with the periodic changes of environment, ensuring that the plant starts the development stages such as reproductive growth at a suitable time. In cotton, the input mechanism of light signal to biological clock is unclear, and identifying the genes involved in the mechanism in cotton and studying their roles in regulating the flowering time of cotton are helpful for the cultivation of new early-maturing cotton varieties. SUMMARY

[0005] An object of the present application is to provide the use of any one of the following 1) to 3).

[0006] The present application provides the use of any one of the following 1) to 3) in regulating the flowering time, bolting time and / or rosette leaf number of plants;

[0007] 1) protein GhELF3;

[0008] 2) a nucleic acid molecule encoding protein GhELF3;

[0009] 3) a recombinant vector, expression cassette or recombinant bacteria containing a nucleic acid molecule encoding protein GhELF3;

[0010] The protein GhELF3 is as follows (1) or (2) or (3):

[0011] (1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;

[0012] (2) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing to which a tag sequence is added at the terminus;

[0013] (3) a protein derived from (1) or (2) by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 and having the same function.

[0014] In the above use, the nucleic acid molecule encoding the protein GhELF3 is a DNA molecule of any one of the following 1) to 3):

[0015] 1) a DNA molecule whose coding region is shown in SEQ ID NO: 1 in the sequence listing;

[0016] 2) a DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) and encodes a protein having the same function;

[0017] 3) a DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the DNA sequence defined in 1) and encodes a protein having the same function.

[0018] In the above use, the regulation of the flowering time of the plant is the prolongation of the flowering time of the plant.

[0019] In the above use, the regulation of the bolting time of the plant is the prolongation of the bolting time of the plant.

[0020] In the above use, the regulation of the number of rosette leaves of the plant is the increase in the number of rosette leaves of the plant.

[0021] The use of the above substance in the cultivation of a late-maturing plant is also within the scope of the present application; the late-maturing plant is specifically a plant that is late-flowering, late-bolting, and / or has a large number of rosette leaves.

[0022] Alternatively, the use of the above substance in the cultivation of a late-flowering plant, a late-bolting plant, or a plant with an increased number of rosette leaves is also within the scope of the present application.

[0023] The use of the above substance in the cultivation of a plant that is late-flowering, late-bolting, and / or has an increased number of rosette leaves is also within the scope of the present application.

[0024] Another object of the present invention is to provide a method for cultivating transgenic plants with extended flowering time, extended bolting time and / or increased number of rosette leaves.

[0025] The method provided by this invention is as follows: 1) or 2):

[0026] 1) The method includes the following steps: increasing the content and / or activity of protein GhELF3 in the target plant to obtain a transgenic plant;

[0027] 2) The method includes the following steps: increasing the expression of nucleic acid molecules encoding the protein GhELF3 in the target plant to obtain transgenic plants;

[0028] The flowering time of the transgenic plant is later than that of the target plant;

[0029] Alternatively, the bolting time of the transgenic plant is later than that of the target plant;

[0030] Alternatively, the number of rosette leaves in the transgenic plant is greater than that in the target plant;

[0031] The protein GhELF3 is as follows (1) or (2) or (3):

[0032] (1) A protein consisting of the amino acid sequence shown in Sequence 2 of the sequence listing;

[0033] (2) A protein consisting of an amino acid sequence shown in Sequence 2 of the sequence listing with a tag sequence added to the end;

[0034] (3) A protein derived from (1) or (2) with the same function by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in Sequence 2 of the sequence listing.

[0035] In the above methods, increasing the content and / or activity of protein GhELF3 in the target plant, or increasing the expression of nucleic acid molecules encoding protein GhELF3 in the target plant, both involve introducing the nucleic acid molecules encoding protein GhELF3 into the target plant.

[0036] The plants mentioned above are dicotyledonous or monocotyledonous plants.

[0037] This study cloned the cotton circadian rhythm gene GhELF3 from upland cotton. GhELF3 is a direct homolog of AtELF3 in Arabidopsis thaliana. GhELF3 exhibits an oscillating expression pattern within a day, and its expression level in late-flowering cotton varieties is significantly higher than that in early-flowering cotton varieties. An overexpression vector of this gene was constructed and transformed into Arabidopsis thaliana using the flower-dipping method. It was found that the flowering time of transgenic Arabidopsis thaliana overexpressing GhELF3 was significantly later than that of wild-type Arabidopsis thaliana. Attached Figure Description

[0038] Figure 1 Diurnal expression pattern of GhELF3 in CCRI 50 and GX11.

[0039] Figure 2 PCR amplification product of GhELF3.

[0040] Figure 3 PCR identification of 35S::GhELF3 positive plants.

[0041] Figure 4 Phenotype observation and data statistics of wild type Arabidopsis (WT) and 3 35S::GhELF3 overexpression lines. DETAILED DESCRIPTION

[0042] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0043] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0044] The cotton materials selected in this experiment are TM-1, CCRI 50 and GX11 (described in the following literature: Genomic analyses reveal the genetic basis of early maturity and identification of loci and candidate genes in upland cotton (Gossypium hirsutum L.), Plant Biotechnology Journal, 2020, 19(1), 109-123), which are planted in the plant illumination culture room of the State Key Laboratory of Cotton Biology, Cotton Research Institute, Chinese Academy of Agricultural Sciences (16 hours of light / 8 hours of darkness, 25℃).

[0045] The reagents and consumables used in the following examples are as follows:

[0046] 1. Enzymes and kits: GXL DNA Polymerase high-fidelity enzyme, gel recovery kit were purchased from Takara Company; RNA reverse transcription kit, KOD FX Neo enzyme (Code. No. KFX-201) were purchased from Toyobo Company; Ultra One Step Cloning Kit was purchased from Vazyme company; Plasmid mini-extraction kit was purchased from Magen company; Restriction endonuclease (Xba I, Sac I) was purchased from NEB company; DNA Marker, plant total RNA extraction kit were purchased from TIANGEN company; Fluorescent quantitative TransStart Top Green qPCR SuperMix was purchased from TransGen company.

[0047] 2、Other drugs: Agar was the original product of Spain, proteose peptone, yeast extract, chloroform, isopentanol, ethanol, isopropyl alcohol, sodium chloride, sucrose, silwet L-77, m-benzenetriol were domestic analytical pure, kanamycin, streptomycin sulfate, ampicillin were purchased from Baobiotechnology (Dalian) Co., Ltd., E. coli competent cells Trans5α were purchased from Beijing Quanshi Gold Biotechnology Co., Ltd., Agrobacterium competent cells LBA4404 were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0048] 3、Culture medium: LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L; LB solid medium: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar powder 15 g / L, constant volume to 1 L; LB selection medium: before plating, when the medium was cooled to 55℃ after high pressure sterilization, the corresponding concentration of antibiotic was added, and then the plate was shaken and plated. The reagent solution mentioned in this paper but not listed was prepared according to the method in the third edition of Molecular Cloning Experiment Guide. Biochemical reagents were analytical pure or above.

[0049] 4、Main instruments: PCR amplifier (Eppendorf), high-speed centrifuge (Eppendorf 5427R), electrophoresis equipment (Beijing Liyi), gel imaging system (BIO-RAD), fluorescent quantitative PCR instrument (ABI7500), fluorescent microscope (Olympus BX43), constant temperature culture oscillator (Shanghai Zhicheng), artificial climate test box (Saifu) and the like.

[0050] Example 1, discovery and cloning of GhELF3 gene

[0051] I. Analysis of diurnal expression pattern of GhELF3 in different flowering time upland cotton varieties

[0052] 1、Sample collection

[0053] The test material, early flowering variety of upland cotton CCRI50 and late flowering variety Guoxin 11, was planted in the plant illumination culture room of the National Key Laboratory of Cotton Biology (16 hours of illumination / 8 hours of darkness, 25°C). The sample was taken from the leaf blade at every 4 hours within 24 hours during the period of the 5th true leaf unfolding, and the material was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for standby.

[0054] 2. Fluorescent quantitative detection

[0055] The leaf blade sample of the above different materials was taken, total RNA of the sample was extracted by using the reagent kit of TIANGEN Company, cDNA was obtained by reverse transcription of the total RNA by using the reverse transcription reagent kit FSQ-201 of Toyobo, and the expression amount of GhELF3 gene in different materials was determined by fluorescent quantification.

[0056] (1) The extraction step of RNA is as follows:

[0057] 1) Homogenization treatment: an appropriate amount of fiber sample was quickly ground into powder in liquid nitrogen, 700 μl of SL (β-mercaptoethanol was added before use) was added, and the sample was immediately mixed uniformly by violent shaking;

[0058] 2) Centrifugation at 12,000 rpm for 2 min;

[0059] 3) The supernatant was transferred to the filter column CS, centrifugation was carried out at 12,000 rpm for 2 min, and the supernatant in the collection tube was carefully sucked and collected into a new RNase-Free centrifuge tube, and the suction head avoided contacting the cell debris in the collection tube;

[0060] 4) 0.4 times the supernatant volume of anhydrous ethanol was added, mixed, the mixture was transferred to the adsorption column CR3, centrifugation was carried out at 12,000 rpm for 15 sec, the waste liquid in the collection tube was poured off, and the adsorption column CR3 was placed back into the collection tube;

[0061] 5) 350 μl of deproteinization liquid RW1 was added to the adsorption column CR3, centrifugation was carried out at 12,000 rpm for 15 sec, the waste liquid in the collection tube was poured off, and the adsorption column CR3 was placed back into the collection tube;

[0062] 6) DNase I working solution: 10 μl of DNase I storage solution and 70 μl of RDD solution were gently mixed;

[0063] 7) 80 μl of DNase I working solution was added to CR3, and static treatment was carried out at room temperature for 15 min;

[0064] 8) After static treatment, 350 μl of deproteinization liquid RW1 was added to CR3, centrifugation was carried out at 12,000 rpm for 15 sec, the waste liquid in the collection tube was poured off, and the adsorption column CR3 was placed back into the collection tube;

[0065] 9) Add 500 μl of rinse solution RW (with ethanol added before use) to the adsorption column CR3, centrifuge at 12,000 rpm for 15 sec, discard the waste in the collection tube, and place the adsorption column CR3 back into the collection tube;

[0066] 10) Repeat step 9;

[0067] 11) Centrifuge at 12,000 rpm (~ 13,400 x g) for 2 min, place the adsorption column CR3 into a new RNase-Free collection tube, and add 30-50 μl of RNase-Free ddH20 to the middle of the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm (~ 13,400 x g) for 1 min, and obtain the RNA solution. Note: the volume of the elution buffer should not be less than 30 μl, and a small volume will affect the recovery efficiency. The RNA sample should be stored at -70 °C. If the expected RNA yield is greater than 30 μg, the RNA solution obtained by centrifugation in step 11 can be added to the adsorption column CR3 again, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm (~ 13,400 x g) for 1 min, and obtain the RNA solution.

[0068] (2) Synthesis of cDNA. 500 ng of RNA was reverse transcribed into cDNA using the reverse transcription kit FSQ-201 from Toyobo, and the reverse transcription system was as follows:

[0069] The RT reaction solution was prepared according to the following components (the reaction solution was prepared on ice):

[0070] Table 1 is the reverse transcription system

[0071]

[0072] The reverse transcription reaction conditions were as follows:

[0073] 37 °C for 15 min (reverse transcription reaction),

[0074] 98 °C for 5 s (inactivation reaction of reverse transcriptase);

[0075] (3) Real-time fluorescent quantitative PCR. The reverse transcription product cDNA solution was diluted 4-fold as the qRT-PCR reaction template.

[0076] GhActin was used as the internal reference gene, and the primers for fluorescent quantitative PCR were designed according to the reference CDS sequences of GhELF3 and GhActin on Cottongen as follows:

[0077] Table 2 is the fluorescent quantitative PCR primer

[0078]

[0079]

[0080] Prepare the qRT-PCR reaction system on ice and perform the real-time PCR reaction.

[0081] The qRT-PCR reaction system is as follows:

[0082] Table 3 shows the reaction system for quantitative real-time PCR.

[0083]

[0084] qRT-PCR reaction procedure:

[0085] Table 4 shows the reaction procedure for quantitative real-time PCR.

[0086]

[0087] The results of qRT-PCR are as follows Figure 1 As shown, GhELF3 exhibits an oscillating expression pattern throughout the day, and the expression level of GhELF3 in the late-flowering variety GX11 is significantly higher than that in the early-flowering variety Zhongmian50, suggesting that GhELF3 may be a circadian rhythm gene that inhibits cotton flowering.

[0088] II. Cloning of the cotton GhELF3 gene

[0089] Gene cloning primers were designed based on the reference CDS sequence of GhELF3 from Cottongen.

[0090] Primer sequences:

[0091] GhELF3-OE-F:5′-cacgggggactctagaATGAAGAGAGGAAAAGATGAT-3′

[0092] GhELF3-OE-R:5′-gatcggggaaattcgagctcTCATCTTAGTCCCGTTGCTC-3′

[0093] The specific process of cloning a gene is as follows:

[0094] (1) Experimental materials: Upland cotton variety TM-1 was grown in the plant light culture room of the State Key Laboratory of Cotton Biology (16 hours light / 8 hours darkness, 25℃). The samples were leaves at the 5th true leaf unfolding stage. The collected materials were quickly frozen in liquid nitrogen and stored at -80℃ for later use. Total RNA was extracted from the samples using a TIANGEN kit, and cDNA was obtained by reverse transcription of total RNA using a Toyobo reverse transcription kit FSQ-201.

[0095] (2) PCR amplification of the target gene

[0096] The reverse transcription product cDNA solution was diluted 4-fold as a PCR reaction template. The following system was prepared on ice, according to the TaKaRa GXL DNA Polymerase high-fidelity enzyme instructions, the PCR reaction system is as follows:

[0097] Table 5 is the GXL high-fidelity enzyme PCR amplification reaction system

[0098]

[0099] The PCR amplification procedure is as follows:

[0100] Table 6 is the high-fidelity enzyme PCR amplification procedure

[0101]

[0102] After the reaction, it was stored at 4°C, and detected by 1% agarose electrophoresis.

[0103] The results are shown in Figure 2 Lane M is MARKER, and lanes 1, 2 and 3 are the PCR target fragments of GhELF3. It can be seen that a band of 2160 bp is obtained, which is consistent with the expectation.

[0104] The PCR product was sent for sequencing, and the results showed that the PCR product had the GhELF3 gene shown in SEQ ID NO: 1 in the sequence table. The amino acid sequence of GhELF3 protein is SEQ ID NO: 2 in the sequence table. The open reading frame is 2121 bp, encoding 706 amino acids, and the relative molecular weight of the protein is 76.36 kDa, and the isoelectric point is 8.84.

[0105] Example 2, application of GhELF3 gene in promoting plant flowering

[0106] I. Preparation of recombinant vector

[0107] The PCR product of Example 1 was connected to the pBI121 vector (Wuhan Moli Biological Technology Co., Ltd. P0274) digested with XbaI and SacI using Ultra One Step Cloning Kit kit, to obtain the recombinant vector pBI121-GhELF3.

[0108] The recombinant vector pBI121-GhELF3 is a vector obtained by replacing the fragment between the XbaI and SacI enzyme digestion sites of the pBI121 vector with the GhELF3 gene shown in SEQ ID NO: 1 in the sequence table.

[0109] II. Preparation of recombinant bacteria

[0110] The competent cells of Agrobacterium tumefaciens LBA4404 from Shanghai Weidi Biology were transformed by freeze-thaw method, and the specific transformation process was as follows:

[0111] (1) 1 μg (2-10 μl) of the recombinant vector pBI121-GhELF3 prepared in the above step (1) was added to 100 μl of the competent cells of Agrobacterium tumefaciens LBA4404 from Shanghai Weidi Biology, and the mixture was uniformly mixed and then ice-bathed for 30 min; the mixture was quickly frozen in liquid nitrogen for 2-3 min and then heat-shocked at 37°C for 90 s;

[0112] (2) The mixture was ice-bathed for 5 min, and then 800 μl of LB liquid medium was added;

[0113] (3) After the mixture was cultured at 190 rpm and 28°C for 4 h, the mixture was centrifuged at 4000 rpm for 5 min, and the supernatant was removed to a remaining volume of 400-500 μl; after the mixture was repeatedly mixed by pipetting, 200 μl of the bacterial solution was taken and spread on a triple-antibiotic screening medium containing kanamycin, streptomycin sulfate and rifampicin, and the medium was cultured at 28°C for about 36-48 h, and the resistant colonies were visible;

[0114] (4) A single colony was picked and cultured in 1 ml of LB liquid medium containing triple antibiotics for about 16 h until turbidity;

[0115] (5) The colony was identified by PCR (using primers GhELF3-OE-F and GhELF3-OE-R to obtain a 2160 bp positive result) and enzyme digestion (using XbaI and SacI double enzyme digestion to obtain a 2130 bp positive result), and a positive Agrobacterium strain was screened, and the bacterial solution was stored at -80°C in 20% glycerol.

[0116] The positive Agrobacterium strain was named as recombinant bacteria LBA4404 / pBI121-GhELF3.

[0117] III. Transgenic Arabidopsis thaliana with GhELF3

[0118] 1. Floral dip method was used to transform Arabidopsis thaliana

[0119] (1) 20 μl of the recombinant bacteria LBA4404 / pBI121-GhELF3 solution stored at -80°C was inoculated into 1 ml of LB liquid medium, and the mixture was cultured at 28°C and 180 rpm overnight; 200 μl of the activated bacterial solution was added to 20 ml of LB liquid medium, and the mixture was cultured at 28°C and 180 rpm;

[0120] (2) When the OD value of the bacterial solution was about 1.2-1.6, the bacterial solution was centrifuged at 3000 rpm to collect the bacterial cells;

[0121] (3) Transformation medium formula: 5% sucrose, 0.03% silwet L-77 (Steven J, 1998);

[0122] (4) The bacterial body was suspended with the above transformation medium, and the OD was adjusted to 0.8 to start the infiltration; 600 = 0.8 to start the infiltration;

[0123] (5) The wild type Arabidopsis col-0 (NRR00220, Beijing Huayangyang Biotechnology Co., Ltd.) inflorescences were placed in the transformation medium for 30-50 s, and the Arabidopsis was wrapped with plastic wrap after infiltration. After 24 h of dark culture, it was placed under normal conditions for culture, and the seeds were harvested after maturation.

[0124] 2. Identification and detection of transgenic Arabidopsis plants

[0125] 1) Resistance screening

[0126] The harvested seeds in 1 above were disinfected with 0.1% HgCl solution, and then purified at 4°C for 4 days, and then planted on 1 / 2MS containing kanamycin (agar concentration 0.6%). After about 10 days, positive and negative plants could be distinguished, and plants that could grow normally were likely to be positive plants. The Arabidopsis that could grow normally was transplanted to the culture room, which was the resistant positive plant.

[0127] 2) Molecular identification

[0128] The enzyme used for screening of transgenic plants is KOD FX Neo PCR enzyme. The biggest feature of this enzyme is that it does not need to extract the DNA of the resistant positive plant, and can directly use the living leaf for PCR. When identifying, Agrobacterium LBA4404 / pBI121-GhELF3 of GhELF3 was used as a positive control, and wild type Arabidopsis was used as a negative control. The primers used for detection are:

[0129] Upstream primer F1 5'-GACGCACAATCCCACTATCC-3'

[0130] Downstream primer R1 5'-CTCATCTTAGTCCCGTTGCTC-3'

[0131] PCR reaction system:

[0132] Table 7 is the KOD FX Neo enzyme PCR amplification reaction system

[0133] Reagent name Reagent amount 2 x PCR Buffer for KOD FX Neo 25 μl 2 mM dNTPs 10 μl F1 (10 μM) 1 μl R1 (10 μM) 1 μl KOD FX Neo (1 U / μl) 1 μl 2 mm square leaf as needed sterilized distilled water up to 50 μl

[0134] PCR amplification program:

[0135] Table 8 is the KOD FX Neo enzyme PCR amplification program

[0136]

[0137] The leaf of the above-identified resistance positive strain was used as a template, and the upstream primer Fl and the downstream primer Rl were used for PCR amplification.

[0138] The appropriate amount of amplification product was taken and subjected to electrophoresis detection on a 1% agarose gel, respectively, and the results are shown in Figure 3 M lane is MARKER, 1-3 lanes are resistance positive strains, 4 and 5 lanes are positive control (pBI121-GhELF3 plasmid) and negative control (wild type Arabidopsis), it can be seen that the resistance positive strain can obtain a band of about 2270bp, indicating that the GhELF3 gene has been integrated into the Arabidopsis genome.

[0139] The strain with 2270bp band obtained by the above molecular identification is named T0 35S::GhELF3 positive strain, and a total of 3, T1 generation seeds are harvested.

[0140] 3. Identification of 35S::GhELF3 positive strains

[0141] 1) Phenotypic identification

[0142] The harvested T1 generation 35S::GhELF3 seeds were planted on 1 / 2MS containing kanamycin after disinfection, and after 3 days of 4°C vernalization, they were transferred to an artificial climate incubator, and after about 10 days, the positive plants grew normally, while the negative plants turned yellow and stopped growing.

[0143] 2) PCR identification

[0144] The positive Arabidopsis plants were transplanted into small flower pots for planting, and after growing for one month, the leaf DNA was extracted and detected by PCR.

[0145] The method is consistent with the above-mentioned molecular identification in 2), and the 2270bp band is the positive T1 generation 35S::GhELF3.

[0146] 3) Homozygous transgenic Arabidopsis strain

[0147] The positive T1 generation 35S::GhELF3 seeds were harvested, and the above steps were repeated until the T3 generation was propagated, and the plants of each generation were subjected to the above phenotypic identification and PCR identification, and the positive T3 generation 35S::GhELF3 was obtained, which was a homozygous transgenic Arabidopsis strain.

[0148] Four, phenotype observation of GhELF3 transgenic plants

[0149] The positive T3 generation 35S::GhELF3 seeds were sowed, and the seeds were planted on 1 / 2MS containing kanamycin after sterilization. The flowering time and bolting time were counted. The wild type Arabidopsis was used as a control. 24 seeds were used for each strain, and the average value was taken as the result.

[0150] The phenotypes of different strains were observed 28 days after sowing, and the results are shown in Figure 4 A. GhELF3-OE line1, GhELF3-OE line2 and GhELF3-OE line3 are different plants of the positive T3 generation 35S::GhELF3, and WT is the wild type Arabidopsis. It can be seen that 28 days after sowing, the three positive T3 generation 35S::GhELF3 strains are bolting, and the WT has already flowered.

[0151] The bolting time of different strains was counted, and the results are shown in Figure 4 B. line1, line2 and line3 are different plants of the positive T3 generation 35S::GhELF3, and WT is the wild type Arabidopsis. It can be seen that the 35S::GhELF3 overexpression strain has a later bolting time than the wild type Arabidopsis.

[0152] The flowering time of different strains was counted, and the results are shown in Figure 4 D. line1, line2 and line3 are different plants of the positive T3 generation 35S::GhELF3, and WT is the wild type Arabidopsis. It can be seen that the 35S::GhELF3 overexpression strain is 4-4.9 days later in flowering than the wild type Arabidopsis.

[0153] The number of rosette leaves of different strains was counted 32 days after sowing, and the results are shown in Figure 4 C. line1, line2 and line3 are different plants of the positive T3 generation 35S::GhELF3, and WT is the wild type Arabidopsis. It can be seen that the 35S::GhELF3 overexpression strain has more number of rosette leaves than the wild type Arabidopsis.

[0154] Therefore, the above results show that GhELF3 has the effect of delaying bolting and / or flowering of Arabidopsis. SEQUENCE LISTING <110>Chinese Academy of Agricultural Sciences, Institute of Cotton Research <120>Application of GhELF3 gene in regulating flowering time of plants <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 2121 <212> DNA <213> Artificial sequence <400> 1 atgaagagag gaaaagatga tgagaagaat atggagccta tgttccctag gcttcatgtt 60 aatgatacag agaaaggagg gccgagggct cctccaagga ataagatggc cctttatgaa 120 cagctaagta ttccctcaca gaggtttaac cctggcctct tgcctcataa cgcaagtaat 180 tcaggcagtt tggttcctcc tacttccaca agccagggaa gtagccttca aaatatgctt 240 tttccatctc atgtatcgca tttgacatct actaatcagg ctgagaagat tcatacaggc 300 cagcatggag gagcaagtgt aaatgctcct atggcacgcc ttgaaccgag aaagaaagtt 360 agagatgagg ttgatttctt ggttcctgtt tttgttaatt cagagacagg tctacaacat 420 agcaaaaata aggaatggtt tgatggggaa aaagcccgta atgttcatga taatgatcca 480 aatcggtgta gctcttctgg tgtcggttca agaaaggagg taagagatca gagtgaaggg 540 agcttgcaag catgctcaag tagggagcgt tcaataaaaa ctgctggaga ttcatcaact 600 agagaaaaga ttgatggatg tgccaaagag gccaatgtgt cccctgatca aggttgtgga 660 gagattccag catctagatt aagtggatcg catgaaaatg atgcttgctt ggtagaaaag 720 ttgagagctg gtagacaacc agttgataat ggatgtattg atgatgatat tgccttggtg 780 aaagtgattg gtgacggaat tctttctcgg aagagaagcc tgtctaattc agagaggaat 840 cacagtgttc ctgatgaaac tagtaatgac agtgaatgcc atgaagacag gacttgtggc 900 tcactgcagt gggcaaatgg agataaaagc gatgatgtct ctgagacttc tgtggtggat 960 actgtatcag gcttggaaat ctctcctgat gatgtggtgg gaataatagg tcagaagcgt 1020 ttctggaaag caagaagagc gattgctaat caacaaagag tatttgctgt acaagtgttt 1080 gagttacata ggctgattaa ggttcagaga tcaattgctg gattgccaca tctgttgctt 1140 gaggatactg catatttgag caaaccctct tacaaagact ctcctgggaa gaagcttcca 1200 ccagagttaa ttgtaaaacc agtgccccaa aataagcgca aagatgaagc tgaaaagcta 1260 agtcataaga gggaatgttc agcagaaaat gcggttggta ggacatccct ctcttctgtg 1320 aaaaatggta gtcagccttc aaataatggg cctttcctcg gaaataaacc accttcacca 1380 GCT ATG GTG AT AAA AAA AT GAACCCTTGG TGT TTC AAT C AAATGCTCGG GCACCAGTGG 1440 CTG GTCCCTG T CATGTCCCC CTCTGAAGG ACTGATATAC AAGCCCTATCC AGGACCTGGA 1500 TTCATGGGAT CTGCTTGTTG GAGGCTGTGG ACCTTTTGGAC AAAACCCAAT GACTGGA AAC 1560 TTCATGACAT CAGCTTACGG AGCTCAAGCT CCTCCTCATC AAGGACTTGG GGTTCACCA 1620 GGC ACTCCTT TAGTCGGTCA CTCCTACTTT CCTCCTCGTG GTATGCCAGT CATGAACCCA 1680 GCATTCTCAG GTTCTTCCAT GGAACAGATG AACCAATTTG CTGGAGCAGG TCCCATGCC 1740 CAAAGTGGCC AGTTATCTGG AAATGGA GCT AACTTTC AT ATTCAGCA ACAAAGTTCA TCT 1800 AATCTGCCGA GTGAGAAGAA TGTTCCTATT CCTCCTGTCG TGAAGTTTCA GGCATCTAAA 1860 GACACC GAGC AACGAAGATG TACAGCAAGC AGTCCAGGTG AGAGAGCAGA GAAAGACGGG 1920 ACCTGTAAT ACTGCTGAAGG AAA AAA AT ACACCTCTT ATCCTT ATAGCTCC AGCCTATCCA 1980 CAGGGAGCCT C GAAGCCT AACGAAACTG ACCAGAGGACA AGG GTGATACG AGTTGTGCCT 2040 CATAATCCTA GATCAGCCAC C GAATCAGC A GCACGTATTT TCCAATCTAT ACAAAAAGAG 2100 agagcaacgg gactaagatg a 2121 <210> 2 <211> 706 <212> PRT <213> Artificial sequence <400> 2 Met Lys Arg Gly Lys Asp Asp Glu Lys Asn Met Glu Pro Met Phe Pro 1 5 10 15 Arg Leu His Val Asn Asp Thr Glu Lys Gly Gly Pro Arg Ala Pro Pro 20 25 30 Arg Asn Lys Met Ala Leu Tyr Glu Gin Leu Ser Ile Pro Ser Gin Arg 35 40 45 Phe Asn Pro Gly Leu Leu Pro His Asn Ala Ser Asn Ser Gly Ser Leu 50 55 60 Val Pro Pro Thr Ser Thr Ser Gin Gly Ser Ser Leu Gin Asn Met Leu 65 70 75 80 Phe Pro Ser His Val Ser His Leu Thr Ser Thr Asn Gin Ala Glu Lys 85 90 95 Ile His Thr Gly Gin His Gly Gly Ala Ser Val Asn Ala Pro Met Ala 100 105 110 Arg Leu Glu Pro Arg Lys Lys Val Arg Asp Glu Val Asp Phe Leu Val 115 120 125 Pro Val Phe Val Asn Ser Glu Thr Gly Leu Gin His Ser Lys Asn Lys 130 135 140 Glu Trp Phe Asp Gly Glu Lys Ala Arg Asn Val His Asp Asn Asp Pro 145 150 155 160 Asn Arg Cys Ser Ser Ser Gly Val Gly Ser Arg Lys Glu Val Arg Asp 165 170 175 Gln Ser Glu Gly Ser Leu Gin Ala Cys Ser Ser Arg Glu Arg Ser Ile 180 185 190 Lys Thr Ala Gly Asp Ser Ser Thr Arg Glu Lys Ile Asp Gly Cys Ala 195 200 205 Lys Glu Ala Asn Val Ser Pro Asp Gin Gly Cys Gly Glu Ile Pro Ala 210 215 220 Ser Arg Leu Ser Gly Ser His Glu Asn Asp Ala Cys Leu Val Glu Lys 225 230 235 240 Leu Arg Ala Gly Arg Gin Pro Val Asp Asn Gly Cys Ile Asp Asp Asp 245 250 255 Ile Ala Leu Val Lys Val Ile Gly Asp Gly Ile Leu Ser Arg Lys Arg 260 265 270 Ser Leu Ser Asn Ser Glu Arg Asn His Ser Val Pro Asp Glu Thr Ser 275 280 285 Asn Asp Ser Glu Cys His Glu Asp Arg Thr Cys Gly Ser Leu Gin Trp 290 295 300 Ala Asn Gly Asp Lys Ser Asp Asp Val Ser Glu Thr Ser Val Val Asp 305 310 315 320 Thr Val Ser Gly Leu Glu Ile Ser Pro Asp Asp Val Val Gly Ile Ile 325 330 335 Gly Gln Lys Arg Phe Trp Lys Ala Arg Arg Ala Ile Ala Asn Gin Gin 340 345 350 Arg Val Phe Ala Val Gin Val Phe Glu Leu His Arg Leu Ile Lys Val 355 360 365 Gln Arg Ser Ile Ala Gly Leu Pro His Leu Leu Leu Glu Asp Thr Ala 370 375 380 Tyr Leu Ser Lys Pro Ser Tyr Lys Asp Ser Pro Gly Lys Lys Leu Pro 385 390 395 400 Pro Glu Leu Ile Val Lys Pro Val Pro Gin Asn Lys Arg Lys Asp Glu 405 410 415 Ala Glu Lys Leu Ser His Lys Arg Glu Cys Ser Ala Glu Asn Ala Val 420 425 430 Gly Arg Thr Ser Leu Ser Ser Val Lys Asn Gly Ser Gin Pro Ser Asn 435 440 445 Asn Gly Pro Phe Leu Gly Asn Lys Pro Pro Ser Pro Ala Asn Gly Asp 450 455 460 Lys Lys Met Asn Pro Trp Cys Phe Asn Gln Met Leu Gly His Gln Trp 465 470 475 480 Leu Val Pro Val Met Ser Pro Ser Glu Gly Leu Ile Tyr Lys Pro Tyr 485 490 495 Pro Gly Pro Gly Phe Met Gly Ser Ala Cys Gly Gly Cys Gly Pro Phe 500 505 510 Gly Gln Asn Pro Met Thr Gly Asn Phe Met Thr Ser Ala Tyr Gly Ala 515 520 525 Gln Ala Pro Pro His Gln Gly Leu Gly Val Leu Pro Gly Thr Pro Leu 530 535 540 Val Gly His Ser Tyr Phe Pro Pro Arg Gly Met Pro Val Met Asn Pro 545 550 555 560 Ala Phe Ser Gly Ser Ser Met Glu Gln Met Asn Gln Phe Ala Gly Ala 565 570 575 Gly Ser His Ala Gln Ser Gly Gln Leu Ser Gly Asn Gly Ala Asn Phe 580 585 590 Asn Ile Gln Gln Gln Ser Ser Ser Asn Leu Pro Ser Glu Lys Asn Val 595 600 605 Ala Ile Pro Pro Val Val Lys Phe Gin Ala Ser Lys Asp Thr Glu Gin 610 615 620 Arg Arg Cys Thr Ala Ser Ser Pro Gly Glu Arg Ala Glu Lys Asp Gly 625 630 635 640 Thr Cys Asn Thr Ala Glu Gly Lys Asn Thr Pro Leu Ile Leu Ile Ala 645 650 655 Pro Ala Tyr Pro Gin Gly Ala Ser Lys Pro Asn Glu Thr Asp Gin Arg 660 665 670 Thr Arg Val Ile Arg Val Val Pro His Asn Pro Arg Ser Ala Thr Glu 675 680 685 Ser Ala Ala Arg Ile Phe Gin Ser Ile Gin Lys Glu Arg Ala Thr Gly 690 695 700 Leu Arg 705

Claims

1. Use of any one of the following 1) - 3) in regulating the bolting time and / or rosette leaf number of a plant; 1) a protein GhELF3; 2) a nucleic acid molecule encoding the protein GhELF3; 3) a recombinant vector, expression cassette or recombinant bacteria containing the nucleic acid molecule encoding the protein GhELF3; the protein GhELF3 is as follows (1) or (2): (1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; (2) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing with a tag sequence added at the end; the regulation of the bolting time of a plant is to prolong the bolting time of a plant; the regulation of the rosette leaf number of a plant is to increase the rosette leaf number of a plant; the plant is Arabidopsis thaliana.

2. Use according to claim 1, characterized in that: the nucleic acid molecule encoding the protein GhELF3 is a DNA molecule whose coding region is shown in SEQ ID NO: 1 in the sequence listing.

3. Use of the substance as claimed in claim 1 or 2 in breeding a plant with late bolting or increased rosette leaf number; the plant is Arabidopsis thaliana.

4. Use of the substance as claimed in claim 1 or 2 in breeding a plant with prolonged bolting time and / or increased rosette leaf number; the plant is Arabidopsis thaliana.

5. A method for breeding a transgenic plant with prolonged bolting time and / or increased rosette leaf number, which is as follows 1) or 2): 1) the method comprises the following steps: increasing the content of the protein GhELF3 in a plant of interest to obtain a transgenic plant; 2) the method comprises the following steps: increasing the expression of the nucleic acid molecule encoding the protein GhELF3 in a plant of interest to obtain a transgenic plant; the bolting time of the transgenic plant is later than that of the plant of interest; the rosette leaf number of the transgenic plant is more than that of the plant of interest; the protein GhELF3 is as follows (1) or (2): (1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; (2) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing with a tag sequence added at the end; the plant is Arabidopsis thaliana.

6. The method according to claim 5, characterized in that: the content of the protein GhELF3 in the plant of interest is increased, or the expression of the nucleic acid molecule encoding the protein GhELF3 in the plant of interest is increased, by introducing the nucleic acid molecule encoding the protein GhELF3 into the plant of interest.

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