Application of GhLUX1 gene in regulating flowering time of plants

Abstract

The application discloses application of a GhLUX1 gene in regulating flowering time of plants. The application provides application of any one of the following 1) to 3) in regulating flowering time, bolting time and / or rosette leaf number of plants; 1) protein GhLUX1; 2) a nucleic acid molecule coding protein GhLUX1; and 3) a recombinant carrier, an expression cassette or a recombinant bacterium containing the nucleic acid molecule coding protein GhLUX1. A cotton biological clock gene GhLUX1 is cloned from Gossypium hirsutum, and the gene is an orthologous gene of Arabidopsis thaliana AtLUX. The GhLUX1 gene shows an oscillation expression mode within one day, and an expression amount of the GhLUX1 gene 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 GhLUX1 gene is obviously later than that of wild-type Arabidopsis thaliana.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to application of a GhLUX1 gene in regulation of flowering time of plants. BACKGROUND

[0002] Cotton is the most important natural fiber crop and also an important oil crop. It plays an important role in the national economy and people's life. Early-maturing cotton is an important cotton variety type, which has a shorter growth period and a concentrated boll opening period. In the cotton areas of the Yellow River Basin and the Yangtze River Basin, early-maturing cotton can be planted after wheat (oil) and then directly sowed, so as to realize double harvest of wheat (oil) and cotton in one year. Cloning and functional analysis of key genes in the molecular regulation pathway of early-maturing cotton help to accelerate the breeding of new early-maturing cotton varieties.

[0003] Early-maturing is closely related to flowering, and timely flowering determines the whole growth process of cotton. Plant flowering is complexly regulated by multiple pathways, including the photoperiod pathway, the vernalization pathway, the gibberellin pathway, the age pathway, the autonomous pathway and the like. The photoperiod pathway is the most conservative and important mechanism for regulating flowering time. In the flowering induction process of the photoperiod pathway, the change in day length induces the expression of the FLOWERING LOCUS (FT) gene in leaves, and the FT protein is transported to the apical meristem to induce plant flowering. The regulation of the biological clock on the flowering time of plants mainly reflects the expression rhythm and phase regulation of key genes such as CO / B-BOX1 (CONSTANS) in the photoperiod pathway, which sense the photoperiod signal of a specific day length and are regulated and modified by the biological clock system at multiple levels in transcription and post-transcription.

[0004] The biological clock core oscillator is composed of multiple interlocking transcription-translation feedback loops. Overexpression or mutation of the biological clock core oscillator gene in Arabidopsis and some major crops will cause changes in the flowering time of plants. However, the role of the biological clock in the regulation of the flowering time of cotton is still unclear. SUMMARY

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

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

[0007] 1) the protein GhLUX1;

[0008] 2) a nucleic acid molecule encoding the protein GhLUX1;

[0009] 3) a recombinant vector, expression cassette or recombinant bacteria containing the nucleic acid molecule encoding the protein GhLUX1;

[0010] The protein GhLUX1 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 GhLUX1 is a DNA molecule of any one of 1) to 3) below:

[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 delay of the flowering time of the plant.

[0019] In the above use, the regulation of the bolting time of the plant is the delay 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 breeding of late-maturing plants is also within the scope of the present application; the late-maturing plants are specifically plants that are late-flowering, late-bolting, and / or have an increased number of rosette leaves.

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

[0023] The use of the above substance in the breeding of plants that are late-flowering, late-bolting, and / or have 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 delayed flowering time, delayed 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 GhLUX1 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 GhLUX1 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 GhLUX1 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 GhLUX1 in the target plant, or increasing the expression of nucleic acid molecules encoding protein GhLUX1 in the target plant, both involve introducing the nucleic acid molecules encoding protein GhLUX1 into the target plant.

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

[0037] This study cloned the cotton circadian rhythm gene GhLUX1 from upland cotton. GhLUX1 is orthologous to AtLUX in Arabidopsis thaliana. GhLUX1 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 GhLUX1 was significantly later than that of wild-type Arabidopsis thaliana. Attached Figure Description

[0038] Figure 1 GhLUX1 diurnal expression pattern in CCRI 50 and GX11.

[0039] Figure 2 PCR amplification product of GhLUX1.

[0040] Figure 3 PCR identification of 35S::GhLUX1 positive plants.

[0041] Figure 4 Phenotype observation and data statistics of wild type Arabidopsis (WT) and 3 35S::GhLUX1 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 (recorded 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 GhLUX1 gene

[0051] I. Analysis of diurnal expression pattern of GhLUX1 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 GhLUX1 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, 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, the mixture was 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 ddH2O to the middle of the adsorption membrane, let 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 too 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 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 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 the fluorescent quantitative PCR were designed according to the reference CDS sequences of GhLUX1 and GhActin on Cottongen as follows:

[0077] Table 2 Fluorescent quantitative PCR primers

[0078]

[0079] qRT-PCR reaction system was prepared on ice and then fluorescence quantitative PCR reaction was performed.

[0080] qRT-PCR reaction system was prepared on ice and then fluorescence quantitative PCR reaction was performed.

[0081] Table 3 is the fluorescence quantitative PCR reaction system

[0082]

[0083] qRT-PCR reaction procedure:

[0084] Table 4 is the fluorescence quantitative PCR reaction procedure

[0085]

[0086] The results of qRT-PCR are shown in Figure 1 It can be seen that GhLUX1 shows oscillation expression pattern within one day and the expression amount of GhLUX1 in late flowering variety GX11 is significantly higher than that in early flowering variety Zhongmianso 50, which indicates that GhLUX1 is probably a biological clock gene with inhibiting cotton flowering effect.

[0087] II. Cloning of cotton GhLUX1 gene

[0088] Gene cloning primers were designed according to the reference CDS sequence of GhLUX1 on Cottongen,

[0089] Primer sequence:

[0090] GhLUX1-OE-F: 5'-cacgggggactctagaATGGGTCAAGAAGTGAAGATG-3'

[0091] GhLUX1-OE-R: 5'-gatcggggaaattcgagctcTCATTGGGAACCATATCTATT-3'

[0092] The process of cloning gene is as follows:

[0093] (1) Test material upland cotton variety TM-1 was planted in the plant illumination culture room of the State Key Laboratory of Cotton Biology (16 hours of illumination / 8 hours of darkness, 25°C). The sample was leaf blade at the 5th true leaf unfolding stage, which was quickly frozen in liquid nitrogen and stored in a refrigerator at -80°C for standby. Total RNA of the sample was extracted by using the kit of TIANGEN Company and cDNA was obtained by reverse transcription of total RNA by using the reverse transcription kit FSQ-201 of Toyobo.

[0094] (2) PCR amplification of target gene

[0095] The reverse transcription product cDNA solution was diluted 4 times as a PCR reaction template. The following system was prepared on ice, according to TaKaRa GXL DNA Polymerase high fidelity enzyme instruction, PCR reaction system as follows:

[0096] Table 5 is the GXL high fidelity enzyme PCR amplification reaction system

[0097]

[0098] The PCR amplification procedure is as follows:

[0099] Table 6 is the high fidelity enzyme PCR amplification procedure

[0100]

[0101] After the reaction, it was stored at 4°C, and detected by 1% agarose electrophoresis.

[0102] The results are shown in Figure 2 Lane M is MARKER, and lanes 1, 2 and 3 are the PCR target fragments of GhLUX1. It can be seen that a band with a size of 1050 bp is obtained, which is consistent with the expectation.

[0103] The PCR product was sent for sequencing, and the results showed that the PCR product has the GhLUX1 gene shown in Sequence 1 in the sequence table, and the amino acid sequence of the GhLUX1 protein is Sequence 2 in the sequence table. The open reading frame is 1014 bp, encoding 337 amino acids, and the relative molecular weight of the protein is 36.45 kDa, and the isoelectric point is 5.28.

[0104] Example 2, application of GhLUX1 gene in promoting plant flowering

[0105] I. Preparation of recombinant vector

[0106] 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-GhLUX1.

[0107] The recombinant vector pBI121-GhLUX1 is a vector obtained by replacing the fragment between the XbaI and SacI enzyme digestion sites of the pBI121 vector with the GhLUX1 gene shown in Sequence 1 in the sequence table.

[0108] II. Preparation of recombinant bacteria

[0109] The Agrobacterium tumefaciens LBA4404 competent cells are transformed by freeze-thaw method, and the specific transformation process is as follows:

[0110] (1) 100 μl of the Agrobacterium tumefaciens LBA4404 competent cells from Shanghai Weidi Biology are added with 11 μg (2-10 μl) of the prepared recombinant vector pBI121-GhLUX1, mixed, and then ice-bathed for 30 min; frozen in liquid nitrogen for 2-3 min, and then heat-shocked at 37 ℃ for 90 s;

[0111] (2) ice-bathed for 5 min, and then added with 800 μl of LB liquid medium;

[0112] (3) 190 rpm, 28 ℃, and cultured for 4 h, then centrifuged at 4000 rpm for 5 min, and the supernatant is sucked to remain 400-500 μl, mixed by repeatedly sucking and beating, and then 200 μL of the bacterial liquid is taken and coated on a three-antibiotic screening medium containing kanamycin, streptomycin sulfate and rifampicin, and cultured at 28 ℃ for about 36-48 h, and the resistant colonies can be observed;

[0113] (4) a single colony is picked and cultured in 1 ml of the LB liquid medium containing three antibiotics for about 16 h until turbid;

[0114] (5) colony PCR (the primers are GhLUX1-OE-F and GhLUX1-OE-R, and 1050 bp is positive) and enzyme digestion (doubly digested by XbaI and SacI, and 1020 bp is positive) are used for identification, and the positive Agrobacterium strain is screened, and the bacterial liquid of 20% glycerol is stored at -80 ℃.

[0115] The positive Agrobacterium strain is named as the recombinant bacteria LBA4404 / pBI121-GhLUX1.

[0116] III. Transgenic Arabidopsis thaliana with GhLUX1

[0117] 1. Floral dip method is used for transforming Arabidopsis thaliana

[0118] (1) 20 μl of the recombinant bacteria LBA4404 / pBI121-GhLUX1 liquid stored at -80 ℃ is inoculated into 1 ml of LB liquid medium, and cultured at 28 ℃ and 180 rpm overnight, and then 200 μl of the activated bacterial liquid is added into 20 ml of LB liquid medium and cultured at 28 ℃ and 180 rpm;

[0119] (2) when the OD value of the bacterial liquid is about 1.2-1.6, the bacterial liquid is centrifuged at 3000 rpm to collect the bacterial cells;

[0120] (3) the transformation medium formula is: 5% sucrose, 0.03% silwet L-77 (Steven J, 1998);

[0121] (4) The bacterial cells are suspended in the above transformation medium, and the OD is adjusted 600 = 0.8, and then immersed;

[0122] (5) The inflorescences of wild-type Arabidopsis col-0 (NRR00220 from Beijing Huayangyang Biotechnology Co., Ltd.) are immersed in the transformation medium for 30-50 s, and then the Arabidopsis is wrapped with plastic wrap after immersion. After 24 h of dark culture, the Arabidopsis is placed under normal conditions for culture, and the seeds are harvested after maturation.

[0123] 2. Identification and detection of transgenic Arabidopsis plants

[0124] 1) Resistance screening

[0125] The harvested seeds in 1) above are disinfected with 0.1% HgCl solution, and then purified at 4°C for 4 days. The seeds are then planted on 1 / 2MS containing kanamycin (agar concentration 0.6%). After about 10 days, positive and negative plants can be distinguished, and the plants that can grow normally are likely to be positive. The Arabidopsis plants that can grow normally are transplanted to the culture room.

[0126] 2) Molecular identification

[0127] The enzyme used for screening of transgenic plants is KOD FX Neo PCR enzyme. The most characteristic of this enzyme is that it does not need to extract Arabidopsis DNA, and can directly use the living leaves for PCR. During identification, the Agrobacterium liquid of GhLUX1 is used as a positive control, and wild-type Arabidopsis is used as a negative control. The primers used for detection are:

[0128] Upstream primer F1 5'-GACGCACAATCCCACTATCC-3'

[0129] Downstream primer R1 5'-TCATTGGGAACCATATCTATT-3'

[0130] PCR reaction system:

[0131] Table 7 is the KOD FX Neo enzyme PCR amplification reaction system

[0132]

[0133] PCR amplification program:

[0134] Table 8 is the KOD FX Neo enzyme PCR amplification program

[0135]

[0136]

[0137] The leaf of the positive strain identified in 1) above was used as a template, and PCR amplification was performed using the upstream primer Fl and the downstream primer Rl.

[0138] The amplification products were respectively taken in an appropriate amount and subjected to electrophoresis detection on a 1% agarose gel, and the results are shown in Figure 3 M lane is MARKER, 1-3 lanes are the positive strains, 4 and 5 lanes are the positive control (pBI121-GhLUX1 plasmid) and the negative control (wild-type Arabidopsis), and it can be seen that the positive strains obtain a band of about 1200bp, indicating that the GhLUX1 gene has been integrated into the Arabidopsis genome.

[0139] The strains with 1200bp bands obtained by the above molecular identification are named T0 35S::GhLUX1 positive strains, and a total of 3 are obtained, and T1 generation seeds are harvested.

[0140] 3. Identification of 35S::GhLUX1 positive strains

[0141] 1) Phenotypic identification

[0142] The harvested T1 generation 35S::GhLUX1 seeds were planted on 1 / 2MS containing kanamycin after disinfection, and after 3 days of vernalization at 4°C, 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 1200bp band is the positive T1 generation 35S::GhLUX1.

[0146] 3) Homozygous transgenic Arabidopsis strain

[0147] The positive T1 generation 35S::GhLUX1 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::GhLUX1 was obtained, which was a homozygous transgenic Arabidopsis strain.

[0148] IV. Phenotypic observation of GhLUX1 transgenic plants

[0149] The positive T3 generation 35S::GhLUX1 seeds were sown, and the seeds were planted on 1 / 2MS containing kanamycin after disinfection, and the flowering time and bolting time were counted. Wild-type Arabidopsis was used as a control. 24 seeds per strain, and the average value was taken.

[0150] The phenotypes of different lines were observed 28 days after sowing, and the results are as follows: Figure 4 As shown in Figure A, GhLUX1-OE line1, GhLUX1-OE line2, and GhLUX1-OE line3 are different plants of the positive T3 generation 35S::GhLUX1, while WT is a wild-type Arabidopsis thaliana. It can be seen that 28 days after sowing, when the three positive T3 generation 35S::GhLUX1 lines were bolting, WT had already flowered.

[0151] The bolting time of different strains was statistically analyzed, and the results are as follows: Figure 4 As shown in B, line 1, line 2, and line 3 represent different plants of the positive T3 generation 35S::GhLUX1, and WT represents wild-type Arabidopsis. It can be seen that the 35S::GhLUX1 overexpression line has a later bolting time than wild-type Arabidopsis.

[0152] The flowering time of different strains was statistically analyzed, and the results are as follows: Figure 4 As shown in D, line 1, line 2, and line 3 represent different plants of the positive T3 generation 35S::GhLUX1, and WT represents wild-type Arabidopsis. It can be seen that the 35S::GhLUX1 overexpression lines flowered on average 4-4.7 days later than wild-type Arabidopsis.

[0153] The number of rosette leaves for different plant lines was counted 32 days after sowing, and the results are as follows: Figure 4 As shown in C, line 1, line 2, and line 3 represent different plants of the positive T3 generation 35S::GhLUX1, and WT represents wild-type Arabidopsis thaliana. It can be seen that the 35S::GhLUX1 overexpression line has more rosette leaves than wild-type Arabidopsis thaliana.

[0154] Therefore, the above results indicate that GhLUX1 has the effect of delaying bolting and / or flowering in Arabidopsis thaliana. SEQUENCE LISTING <110> Cotton Research Institute, Chinese Academy of Agricultural Sciences <120> Application of GhLUX1 gene in regulating plant flowering time <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 1014 <212> DNA <213> Artificial sequence <400> 1 atgggtcaag aagtgaagat gagtgacttt gaagctaacg gcggagacga tgaagaagca 60 accgcaaacg ccattaccac catcgttgat gacgacgatg acgatgtcga agaaagagtt 120 atcgagtggg agatgggatt acctaactgc gatgatttaa ctccattatc ccaatcctta 180 atcccgccgg aacttgcctc cgctttcagc atctcgccgg agcctcgtcg gaccgtagtt 240 gatgtcaacc gcgcttctcg cagtactctc tcttctctcc gctctaccgg ggcccactcc 300 tcaaccacca ataacaataa cagtaatagc aatttccgtg atacgatagt cgttgaagcg 360 gaaggatatg ggtcggggtc gggctccgga tctgacccga agaagatgag gaagatggat 420 attgcagagg aagcggattc agcggttagg acgacggaga actcggacga tccgtctggg 480 aggacattga aacgaccgcg tttagtgtgg acgccgcagc tgcacaagag attcgttgat 540 gtggtggctc atctagggat aaaaagcgcg tttcccaaaa caatcatgca gttaatgaac 600 gtggaaggct tgacccgcga gaacgttgcc agtcatttgc aaaaatatcg gctttacttg 660 aagagaatgc aagggttaag caacgaaggc ccttcagctt ccgatcagct ttttgcatca 720 acaccggtgc cgcagagtct tcacgagact ggaagtggtg gcggtggtgg aggaggaagt 780 ggtggtggtg gtgccaatgg gaatgggaat gggcatttgg gcatggctat accgatgtct 840 tatggggcac caatgatgcc ggtcccgatg ccgatgtatg gacatgtggg gatgcatcaa 900 ggattgtatc atcaacaaag acaatatcat catcagaatg ggtacgaagc gaactcctat 960 gggatgatgc agcagagaga ctggtctgat gggaatagat atggttccca atga 1014 <210> 2 <211> 337 <212> PRT <213> Artificial sequence <400> 2 Met Gly Gln Glu Val Lys Met Ser Asp Phe Glu Ala Asn Gly Gly Asp 1 5 10 15 Asp Glu Glu Ala Thr Ala Asn Ala Ile Thr Thr Ile Val Asp Asp Asp 20 25 30 Asp Asp Asp Val Glu Glu Arg Val Ile Glu Trp Glu Met Gly Leu Pro 35 40 45 Asn Cys Asp Asp Leu Thr Pro Leu Ser Gln Ser Leu Ile Pro Pro Glu 50 55 60 Leu Ala Ser Ala Phe Ser Ile Ser Pro Glu Pro Arg Arg Thr Val Val 65 70 75 80 Asp Val Asn Arg Ala Ser Arg Ser Thr Leu Ser Ser Leu Arg Ser Thr 85 90 95 Gly Ala His Ser Ser Thr Thr Asn Asn Asn Asn Ser Asn Ser Asn Phe 100 105 110 Arg Asp Thr Ile Val Val Glu Ala Glu Gly Tyr Gly Ser Gly Ser Gly 115 120 125 Ser Gly Ser Asp Pro Lys Lys Met Arg Lys Met Asp Ile Ala Glu Glu 130 135 140 Ala Asp Ser Ala Val Arg Thr Thr Glu Asn Ser Asp Asp Pro Ser Gly 145 150 155 160 Arg Thr Leu Lys Arg Pro Arg Leu Val Trp Thr Pro Gln Leu His Lys 165 170 175 Arg Phe Val Asp Val Val Ala His Leu Gly Ile Lys Ser Ala Phe Pro 180 185 190 Lys Thr Ile Met Gln Leu Met Asn Val Glu Gly Leu Thr Arg Glu Asn 195 200 205 Val Ala Ser His Leu Gln Lys Tyr Arg Leu Tyr Leu Lys Arg Met Gln 210 215 220 Gly Leu Ser Asn Glu Gly Pro Ser Ala Ser Asp Gln Leu Phe Ala Ser 225 230 235 240 Thr Pro Val Pro Gin Ser Leu His Glu Thr Gly Ser Gly Gly Gly Gly 245 250 255 Gly Gly Gly Ser Gly Gly Gly Gly Ala Asn Gly Asn Gly Asn Gly His 260 265 270 Leu Gly Met Ala Ile Pro Met Ser Tyr Gly Ala Pro Met Met Pro Val 275 280 285 Pro Met Pro Met Tyr Gly His Val Gly Met His Gin Gly Leu Tyr His 290 295 300 Gln Gin Arg Gin Tyr His His Gin Asn Gly Tyr Glu Ala Asn Ser Tyr 305 310 315 320 Gly Met Met Gin Gin Arg Asp Trp Ser Asp Gly Asn Arg Tyr Gly Ser 325 330 335 Gln

Claims

1. Use of any of the following 1)-3) in regulating flowering time, bolting time and / or rosette leaf number of Arabidopsis thaliana; 1) protein GhLUX1; 2) nucleic acid molecule encoding protein GhLUX1; 3) recombinant vector, expression cassette or recombinant bacteria containing nucleic acid molecule encoding protein GhLUX1; said protein GhLUX1 is as follows (1) or (2): (1) protein consisting of amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; (2) protein consisting of amino acid sequence shown in SEQ ID NO: 2 in the sequence listing with a tag sequence added at the end; said regulating flowering time of Arabidopsis thaliana is delaying flowering time of Arabidopsis thaliana; said regulating bolting time of Arabidopsis thaliana is delaying bolting time of Arabidopsis thaliana; said regulating rosette leaf number of Arabidopsis thaliana is increasing rosette leaf number of Arabidopsis thaliana.

2. The use according to claim 1, wherein: said nucleic acid molecule encoding protein GhLUX1 is DNA molecule with coding region shown in SEQ ID NO: 1 in the sequence listing.

3. Use of any of the substances in claims 1-2 in breeding Arabidopsis thaliana with late flowering, late bolting or increased rosette leaf number.

4. Use of any of the substances in claims 1-2 in cultivating Arabidopsis thaliana with delayed flowering, delayed bolting and / or increased rosette leaf number.

5. A method for breeding transgenic Arabidopsis thaliana with delayed flowering, delayed bolting and / or increased rosette leaf number, which is as follows 1) or 2): 1) said method comprises the following steps: increasing the content of protein GhLUX1 in the target Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana; 2) said method comprises the following steps: increasing the expression of nucleic acid molecule encoding protein GhLUX1 in the target Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana; said transgenic Arabidopsis thaliana has later flowering time than the target Arabidopsis thaliana; or, said transgenic Arabidopsis thaliana has later bolting time than the target Arabidopsis thaliana; or, said transgenic Arabidopsis thaliana has more rosette leaf number than the target Arabidopsis thaliana; said protein GhLUX1 is as follows (1) or (2): (1) protein consisting of amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; (2) protein consisting of amino acid sequence shown in SEQ ID NO: 2 in the sequence listing with a tag sequence added at the end.

6. The method according to claim 5, wherein: said increasing the content of protein GhLUX1 in the target Arabidopsis thaliana, or said increasing the expression of nucleic acid molecule encoding protein GhLUX1 in the target Arabidopsis thaliana, is introducing the nucleic acid molecule encoding protein GhLUX1 into the target Arabidopsis thaliana.

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