Application of ghABF3 gene in regulating flowering time of plants

Abstract

The application provides a method for regulating flowering time of plants GhABF3 Application of a gene in regulating flowering time of plants, GhABF3 The nucleotide sequence of the gene is shown as SEQ ID NO. 5, and the amino acid sequence encoded by the gene is shown as SEQ ID NO. 6, GhABF3 Application of the gene in positively regulating flowering of cotton and Arabidopsis, through expression mode analysis, it is found that, GhABF3 The expression amount in early-maturing cotton varieties is significantly higher than that in late-maturing cotton varieties, which indicates that the gene can be specifically expressed at the flower bud transformation period, and is possibly related to the growth and development of cotton; the overexpression vector of the gene is constructed, and it is found that the flowering time of transgenic Arabidopsis with overexpression GhABF3 is obviously earlier than that of wild-type Arabidopsis. In addition, the VIGS technology is used for transient silencing of the gene, GhABF3 and it is found that the flowering time of the silenced plants is obviously later than that of the control plants; it is indicated that the gene has positive participation in regulating the flowering process of cotton.

Description

Technical Field

[0001] This invention belongs to the field of plant flowering technology, specifically relating to the application of the GhABF3 gene in regulating plant flowering period. Background Technology

[0002] Cotton is an important economic crop, and its fiber, as a vital natural fiber, is a crucial raw material for the textile industry. The low level of mechanization and long growth period of cotton cultivation significantly hinders the increase in cotton planting area and fiber yield. The short growth period and concentrated boll opening of early-maturing cotton make it suitable not only for direct sowing after wheat (oilseed) in cotton-growing areas of the Yellow River and Yangtze River basins, but also for increasing pre-frost flowering rate in the inland cotton-growing areas of Northwest China with short frost-free periods and insufficient accumulated temperature. The breeding of new early-maturing cotton varieties relies on the cloning and functional analysis of key genes regulating cotton precocity. The flowering integrator FLOWERINGLOCUS T (FT) combines with other transcription factors such as FD (Flowering locus D) containing the bZIP domain to form the flowering hormone FAC (Florigen activation complex), which participates in regulating the expression of the key flowering gene AP1 (APETALA1), thereby promoting the flowering regulation process (Tsuji et al. 2013). Furthermore, studies have shown that AP1 influences the expression of the flowering integrator SOC1 (Suppressor of overexpression of CONSTANS1), regulating the formation of floral meristems (Lee et al. 2010). SOC1, as a key integrator, is regulated by multiple flowering-related genes and also participates in regulating plant drought stress responses. In Arabidopsis, ABF3 (ABA-responsive element (ABRE)-binding factors 3), containing a bZIP domain, participates in responding to drought-induced ABA signaling, inducing SOC1 expression, and influencing and promoting Arabidopsis flowering (Hwang et al. 2019).

[0003] The bZIP family is one of the largest transcription factor families in plants, present in multiple species, and widely involved in regulating various processes such as plant hormone signaling, flower development, energy metabolism, senescence, and seed germination. In rice, overexpression of OsbZIP enhances tolerance to salt stress; compared to wild-type and mutant rice plants, overexpressing plants show a significant increase in the number of green leaves under salt stress (Xiang et al., 2008). In Chinese cabbage, BrABF3 promotes flowering by inducing CO transcription through direct binding to the promoter element of the CO (CONSTANS) gene (Zhang et al., 2022). In cotton, most studies on related bZIP genes focus on their effects on abiotic stress, with limited research on flowering. Summary of the Invention

[0004] The present invention addresses the shortcomings of the prior art by providing an application of the GhABF3 gene in regulating the flowering period of plants.

[0005] To achieve the above objectives, the solution of the present invention is:

[0006] Application of a GhABF3 gene in regulating plant flowering period.

[0007] Furthermore, the nucleotide sequence of the GhABF3 gene is shown in SEQ ID NO.5.

[0008] Furthermore, the amino acid sequence encoded by the GhABF3 gene is shown in SEQ ID NO.6.

[0009] Furthermore, the application of the GhABF3 gene in the positive regulation of plant flowering.

[0010] Furthermore, plants include cotton and Arabidopsis thaliana.

[0011] Furthermore, the expression level of the GhABF3 gene was higher in early-maturing cotton than in late-maturing cotton.

[0012] Furthermore, during PCR amplification of the GhABF3 gene, the sequence of the forward primer is shown in SEQ ID NO.1, and the sequence of the reverse primer is shown in SEQ ID NO.2.

[0013] Due to the adoption of the above solution, the beneficial effects of the present invention are:

[0014] This invention cloned the GhABF3 gene from upland cotton. Expression pattern analysis revealed significant differences in GhABF3 expression levels between the one-leaf and five-leaf stages in early-maturing and late-maturing varieties. Furthermore, the expression level in early-maturing cotton varieties was significantly higher than that in late-maturing varieties, indicating that this gene is specifically expressed during flower bud transition and may be related to cotton growth and development. An overexpression vector for this gene was constructed and transformed into Arabidopsis thaliana using the flower-dipping method. The results showed that transgenic Arabidopsis thaliana overexpressing GhABF3 flowered significantly earlier than wild-type Arabidopsis thaliana. In addition, using the upland cotton genetic standard line TM-1 as material, transient silencing of GhABF3 using the VIGS technique revealed that the flowering time of silenced plants was significantly later than that of control plants, demonstrating that this gene positively participates in regulating the cotton flowering process. Attached Figure Description

[0015] Figure 1 This is a diagram showing the expression pattern of GhABF3 in the one-leaf to five-leaf stages of Zhongmian Institute 50 (Z50) and Guoxin 11 (GX11) in Embodiment 1 of the present invention (Z50 is on the left and GX11 is on the right).

[0016] Figure 2 Phenotypic observations of wild-type Arabidopsis thaliana (WT) and three 35S::GhABF3 overexpression lines in Example 2 of this invention ((A) phenotypic observation, (B) quantitative fluorescence detection).

[0017] Figure 3 Phenotypic observations of pCLCrVA empty plants and pCLCrVA:GhABF3 silent plants in Example 3 of this invention ((A) cotton phenotypic observation, (B) quantitative fluorescence detection). Detailed Implementation

[0018] This invention provides an application of the GhABF3 gene in regulating the flowering period of plants.

[0019] Experimental materials

[0020] 1. Cotton material

[0021] The cotton materials selected for this experiment were upland cotton varieties TM-1, Zhongmian Institute 50, and Guoxin 11, which were planted in the plant light culture room (16 hours light / 8 hours darkness, 25℃) of the State Key Laboratory of Cotton Biology, Cotton Research Institute, Chinese Academy of Agricultural Sciences.

[0022] 2. Reagents and Consumables

[0023] 2.1 Enzymes and kits: GXL DNA Polymerase high-fidelity enzyme and gel extraction kit were purchased from Takara; RNA reverse transcription kit and KOD FX Neo enzyme (Code No. KFX-201) were purchased from Toyobo. The Ultra One Step Cloning Kit was purchased from Vazyme; the plasmid small-scale extraction kit was purchased from Magen; the restriction endonucleases (Xba I, Sac I, Spe I, and Asc I) were purchased from NEB; the DNA Marker and plant total RNA extraction kit were purchased from TIANGEN; and the TransStart Top Green qPCR SuperMix was purchased from TransGen.

[0024] 2.2 Other drugs: Agarose was a Spanish original product; peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, sucrose, Silwet L-77, phloroglucinol, etc. were domestically produced analytical grade; kanamycin, streptomycin sulfate, ampicillin, etc. were purchased from Takara Bio Engineering (Dalian) Co., Ltd.; Escherichia coli competent cells Trans5α were purchased from Beijing TransGen Biotech Co., Ltd.; Agrobacterium tumefaciens competent cells LBA4404 were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0025] 2.3 Culture Media: LB liquid medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride (NaCl) 10 g / L; LB solid medium: Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride (NaCl) 10 g / L, Agar powder 15 g / L, adjusted to 1 L; LB selective medium: Before plating LB, add the appropriate concentration of antibiotics after autoclaving and cooling to 55°C, shake well, and then plate. All reagent solutions mentioned but not listed in this invention were prepared according to the methods in the third edition of *Molecular Cloning: A Laboratory Manual*, and the biochemical reagents were analytical grade or higher.

[0026] 2.4 Main instruments: PCR amplification instrument (Eppendorf), high-speed centrifuge (Eppendorf 5427R), electrophoresis equipment (Beijing Liuyi), gel imaging system (BIO-RAD), real-time PCR instrument (ABI7500), fluorescence microscope (Olympus BX43), constant temperature incubator shaker (Shanghai Zhicheng), artificial climate test chamber (Saifu), etc.

[0027] Example 1:

[0028] 1. Expression pattern analysis of GhABF3 in early and late maturing varieties

[0029] 1.1 Experimental Materials: Upland cotton, early-maturing cotton variety Zhongmian 50, and late-maturing cotton variety Guoxin 11 were planted in the plant light culture room of the State Key Laboratory of Cotton Biology (16 hours light / 8 hours darkness, 25℃). The samples were true leaves of the early-maturing and late-maturing cotton varieties at the one-leaf and five-leaf stages. The collected materials were quickly frozen in liquid nitrogen and stored at -80℃ for later use.

[0030] 1.2 Leaf samples from the above-mentioned different materials were taken, and total RNA was extracted from the samples using a TIANGEN kit. The total RNA was reverse transcribed using a Toyobo FSQ-201 reverse transcription kit to obtain cDNA. The expression level of the GhABF3 gene in different materials was determined by fluorescence quantitative PCR.

[0031] (1) The steps for RNA extraction are as follows:

[0032] 1) Homogenization treatment: Take an appropriate amount of fiber sample and grind it into powder in liquid nitrogen. Add 700 μL of SL (add β-mercaptoethanol before use) and shake vigorously to mix the sample.

[0033] 2) Centrifuge at 12,000 rpm for 2 min;

[0034] 3) Transfer the supernatant to the CS filter column, centrifuge at 12,000 rpm for 2 min, and carefully aspirate the supernatant from the collection tube into a new RNase-free centrifuge tube, avoiding contact between the pipette tip and cell debris in the collection tube;

[0035] 4) Add 0.4 times the volume of supernatant in anhydrous ethanol, mix well, transfer the mixture into the CR3 adsorption column, centrifuge at 12,000 rpm for 15 s, discard the waste liquid in the collection tube, and put the CR3 adsorption column back into the collection tube.

[0036] 5) Add 350 μL of protein removal solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 15 s, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0037] 6) DNase I working solution: Take 10 μL of DNase I stock solution and 70 μL of RDD solution and mix gently;

[0038] 7) Add 80 μL of DNase I working solution to CR3 and let it stand at room temperature for 15 min;

[0039] 8) After standing, add 350 μL of protein removal solution RW1 to CR3, centrifuge at 12,000 rpm for 15 seconds, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0040] 9) Add 500 μL of washing buffer RW (add ethanol before use) to the adsorption column CR3, centrifuge at 12,000 rpm for 15 s, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube.

[0041] 10) Repeat step 9);

[0042] 11) Centrifuge at 12,000 rpm (~13,400 rpm) for 2 min. Place the adsorption column CR3 into a new RNase-Free centrifuge tube. Add 30-50 μL of RNase-Free ddH2O dropwise to the center of the adsorption membrane. Incubate at room temperature for 2 min, then centrifuge at 12,000 rpm (~13,400 rpm) for 1 min to obtain the RNA solution. Note: The elution buffer volume should not be less than 30 μL; a smaller volume will affect the recovery efficiency. Store the RNA sample at -70℃. If the expected RNA yield is greater than 30 μg, the RNA solution obtained in step 11) can be added back to the adsorption column CR3, incubated at room temperature for 2 min, then centrifuged at 12,000 rpm (~13,400 rpm) for 1 min to obtain the RNA solution.

[0043] (2) cDNA synthesis. 500 ng of RNA was reverse transcribed into cDNA using the Toyobo FSQ-201 reverse transcription kit. The reverse transcription system was as follows:

[0044] Prepare the RT reaction solution according to the following components (the reaction solution should be prepared on ice):

[0045] Table 1 Reverse Transcription System

[0046]

[0047] The reverse transcription reaction conditions are as follows:

[0048] 37℃ for 15 minutes (reverse transcription reaction),

[0049] 98℃ for 5 seconds (inactivation reaction of reverse transcriptase);

[0050] (3) Real-time quantitative PCR. The reverse transcription product cDNA solution was diluted 4 times as a template for qRT-PCR reaction.

[0051] Using GhActin as an internal reference gene, primers for real-time PCR were designed based on the reference CDS sequences of GhABF3 and GhActin on Cottongen, as follows:

[0052] Table 2 Primers for Quantitative Real-Time PCR

[0053]

[0054] Prepare the qRT-PCR reaction system on ice and perform the real-time PCR reaction.

[0055] The qRT-PCR reaction system is as follows:

[0056] Table 3. Real-time PCR reaction system

[0057]

[0058] qRT-PCR reaction procedure:

[0059] Table 4. Quantitative PCR reaction procedure

[0060]

[0061]

[0062] 1.3 The qRT-PCR results showed the expression pattern of GhABF3 from the one-leaf stage to the five-leaf stage of cotton, and the expression level of GhABF3 in the early-maturing variety Zhongmian Suo 50 (Z50) was significantly higher than that in the late-maturing cotton variety Guoxin 11 (GX11). Figure 1 This suggests that GhABF3 may be a gene related to cotton growth and development.

[0063] 2. Cloning of the cotton GhABF3 gene

[0064] 2.1 Gene cloning primers were designed based on the reference CDS sequence of GhABF3 on Cottongen and amplified from the upland cotton variety TM-1 by PCR. The open reading frame was 1281 bp, encoding 426 amino acids, with a relative molecular weight of 46.334 kDa and an isoelectric point of 10.205.

[0065] The gene CDS sequence (SEQ ID NO.5) is as follows:

[0066] >GhABF3

[0067]

[0068] The amino acid sequence (SEQ ID NO.6) is as follows:

[0069] >GhABF3

[0070] MGSHLNFKNFGDAPSMEGNESKPLGNFPLTRQSSIYSLTFDELQNTFSGIGKDFGSMNMDELLKNISTAEETQAFMTATVPGGEGSLSGGNLQRQGSLTLPRTLSQ KTVDEVWRNLMKENDGAKDGSSSGGGGGGGANLPQRQRTLGEMTLEEFLVKAGVVREDMQQFGVPNNTGFFGNNNSGVALGFQQINRNNGFLSNNNSVLSQPPRLPQN MTGTKSSQPQQQQQQQQQPPQQQQQPQARPLFPKQQTVAFAPSMHLMNTTQLASPGGRSSMVGIGDPSMNSNIVQSSGLQSGGMGIVGIGSPGSQISSDVISKNSVD TSSLSPVPYVFGRGRKCSAALEKVVERRQRRMIKNRESAARSRARKQAYTLELEAEVAKLKEINEELQKKQEEMMEMQKIQTLEAVNRAWGGGKRQCLRRTLTGPW.

[0071] 2.2 The specific process of cloning a gene is as follows:

[0072] (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.

[0073] (2) PCR amplification of the target gene

[0074] Dilute the above reverse transcription product cDNA solution 4-fold as a template for PCR. Prepare the following system on ice, according to TaKaRa... The GXL DNA Polymerase high-fidelity enzyme instruction manual specifies the PCR reaction system as follows:

[0075] Table 5. GXL High-Fidelity Enzyme PCR Amplification Reaction System

[0076]

[0077]

[0078] The PCR amplification procedure is as follows:

[0079] Table 6 High-fidelity enzyme PCR amplification program

[0080]

[0081] Primer sequences:

[0082] GhABF3-OE-F:5′-CACGGGGGACTCTAGAATGGGATCTCATCTGAATTTC-3′(SEQ ID NO.7)

[0083] GhABF3-OE-R:5′-GATCGGGGAAATTCGAGCTCCTACCAAGGGCCTGTGAGTGT-3′(SEQ IDNO.8)

[0084] After the reaction is complete, the sample is stored at 4°C and detected by 1% agarose gel electrophoresis. If the band size meets the expected design, the result is considered valid.

[0085] (3) The target fragment was cut and recycled using a gel recovery kit.

[0086] (4) Use the product from the above-mentioned recycled adhesive. The Ultra One Step Cloning Kit ligates the pBI121 vector, which has been double-digested with Xba I and Sac I, and transforms it into E. coli.

[0087] (5) Overnight culture at 37°C: After picking single clones from the resistant LB medium, culture them at 37°C by shaking.

[0088] (6) Bacterial PCR verification: Select positive clone samples and send them to Shangya Biotechnology Co., Ltd. for sequencing. Add a certain amount of glycerol to the bacterial solution with correct sequencing to make the final glycerol concentration about 20% and store at -80℃.

[0089] Example 2:

[0090] The GhABF3 gene was transformed into Arabidopsis thaliana using Agrobacterium-mediated transformation.

[0091] 1. Agrobacterium tumefaciens LBA4404 competent cells were transformed using the freeze-thaw method. The specific transformation process is as follows:

[0092] (1) Add 1 μg (2-10 μL) of the constructed pBI121 plasmid containing the target gene to 100 μL of Agrobacterium tumefaciens LBA4404 competent cells from Shanghai Weidi Biotechnology, mix well and incubate on ice for 30 min; then freeze in liquid nitrogen for 2-3 min and heat shock at 37℃ for 90 s.

[0093] (2) Incubate on ice for 5 minutes, then add 800 μL of LB liquid culture medium;

[0094] (3) After incubating at 190 rpm and 28 ℃ for 4 h, centrifuge at 4000 rpm for 5 min, remove the supernatant to the remaining 400-500 μL, repeatedly aspirate and mix, then take 200 μL of bacterial solution and spread it on the triple-antibody selection medium containing kanamycin, streptomycin sulfate and rifampin, incubate at 28 ℃ for about 36-48 h, and resistant colonies will be visible.

[0095] (4) Pick a single colony and incubate it in 1 mL of LB liquid medium containing three antibiotics for about 16 hours until it becomes turbid;

[0096] (5) Colony PCR and enzyme digestion identification were used to screen out positive Agrobacterium strains, and 20% glycerol bacterial solution was stored at -80℃.

[0097] 2. Transformation of Arabidopsis thaliana using inflorescence immersion method

[0098] (1) Inoculate 20 μL of Agrobacterium tumefaciens culture stored at -80℃ into 1 mL of LB liquid medium and culture overnight at 28℃ and 180 rpm with shaking. Take 200 μL of activated bacterial culture and add it to 20 mL of LB liquid medium and culture at 28℃ and 180 rpm with shaking.

[0099] (2) When the OD value of the bacterial solution is about 1.4, the bacterial solution is centrifuged at 3000 rpm to collect the bacterial cells;

[0100] (3) The conversion medium formula is: 5% sucrose, 0.03% silwet L-77 (Steven J, 1998);

[0101] (4) Suspend the bacterial cells in the above-mentioned transformation medium and adjust the OD. 600 =0.8 to begin staining;

[0102] (5) Place the Arabidopsis thaliana inflorescence in the transformation medium for 40s. After soaking, wrap the Arabidopsis thaliana with plastic wrap and culture in the dark for 24 hours. Then culture under normal conditions and harvest the seeds after maturity.

[0103] 3. Identification and Detection of Transgenic Arabidopsis Plants

[0104] 3.1 After harvesting the seeds, disinfect them with 0.1% HgCl solution, then purify them at 4℃ for 3-4 days, and then plant them on 1 / 2 MS containing kanamycin (agar concentration 0.6%). After about 10 days, the difference between positive and negative plants can be observed. Those that can grow normally are likely to be positive plants. Transplant the Arabidopsis thaliana that can grow normally to the culture room.

[0105] 3.2 The enzyme used for screening transgenic plants was the KOD FX Neo PCR enzyme. The most significant advantage of this enzyme is that it eliminates the need to extract Arabidopsis DNA; PCR can be performed directly on living leaves. For identification, Agrobacterium tumefaciens culture of GhABF3 was used as a positive control, and wild-type Arabidopsis was used as a negative control. The primers used for detection were:

[0106] Upstream primer F1 5'-GACGCACAATCCCACTATCC-3' (SEQ ID NO.9)

[0107] Downstream primer R1 5'-CTACCAAGGGCCTGTGAGTGT-3' (SEQ ID NO.10)

[0108] PCR reaction system:

[0109] Table 7 KOD FX Neo enzyme PCR amplification reaction system

[0110]

[0111]

[0112] PCR amplification procedure:

[0113] Table 8. KOD FX Neo enzyme PCR amplification program

[0114]

[0115] 3.3 Take appropriate amounts of the amplification products and perform electrophoresis on a 1% agarose gel. A band of about 1281 bp can be obtained, indicating that the GhABF3 gene has been integrated into the Arabidopsis thaliana genome.

[0116] 3.4 Based on the electrophoresis results, three T0 generation 35S::GhELF3 positive lines were selected, and T1 generation seeds were harvested.

[0117] 4. Identification, phenotypic observation, and quantitative detection of transgenic plants

[0118] 4.1 After disinfecting the harvested seeds, plant them on 1 / 2 MS containing kanamycin and vernalize them at 4°C for 3 days. Then transfer them to an artificial climate incubator. After about 10 days, the positive plants grow normally, while the cotyledons of the negative plants turn yellow and stop growing.

[0119] 4.2 Positive Arabidopsis thaliana plants were transplanted into small pots and allowed to grow for one month. DNA was then extracted and tested using PCR. Positive lines were tested for each generation of plants until the T3 generation was obtained, yielding homozygous transgenic Arabidopsis thaliana lines. Rosette leaves from the T3 generation transgenic plants and wild-type plants were sampled, and RNA was extracted for quantitative real-time detection.

[0120] Primer sequences for quantitative fluorescence:

[0121] AtActin-qF:5′-CTCCTTTGTTGCTGTTGACTAC-3′(SEQ ID NO.11)

[0122] AtActin-qR:5′-GCACAATGTTACCGTACAGATC-3′(SEQ ID NO.12)

[0123] GhABF3-qF:5′-ACGAGTCTAAGCCACTGGGGAA-3′(SEQ ID NO.13)

[0124] GhABF3-qR:5′-TCCACCTGGAACTGTAGCTGTCA-3′(SEQ ID NO.14)

[0125] 4.3 The T3 generation homozygous transgenic lines were used for plant phenotypic observation and quantitative analysis. The results are as follows: Figure 2 :

[0126] Phenotypic observations of T3 generation transgenic lines and wild-type Arabidopsis thaliana revealed that three 35S::GhABF3 overexpression lines flowered earlier than wild-type Arabidopsis thaliana. Figure 2 (A). Furthermore, quantitative real-time PCR results showed that the expression levels of GhABF3 in the three overexpression lines were significantly increased compared to wild-type Arabidopsis thaliana. Figure 2 (B). Therefore, it is speculated that GhABF3 has the effect of promoting flowering in Arabidopsis thaliana.

[0127] Example 3:

[0128] VIGS test for cotton

[0129] 1. Constructing a VIGS carrier

[0130] A silenced fragment of the GhABF3 gene CDS sequence was designed on the SGN VIGS website, and primers were designed using Oligo7. The silenced fragment was amplified from cDNA of *Cotton Upland TM-1*, and a VIGS vector was constructed. The primer sequences for amplifying the silenced fragment are as follows:

[0131] GhABF3-VA-F:5′-ATGCCTGCAGACTAGTATGGGATCTCATCTGAATTTC-3′(SEQ ID NO.15)

[0132] GhABF3-VA-R:5′-AGACCTAGGGGCGCGCCCAACGTTAATGATCCTTGCCTC-3′(SEQ IDNO.16)

[0133] The specific steps are as follows: The pCLCrVA plasmid was double-digested with SpeI and AscI, respectively. Using the CDS fragment of the gene as a template, primers containing the restriction sites were used for amplification. The target fragment and the large fragment product of the pCLCrVA vector were recovered by electrophoresis. The target gene fragment and the large fragment product of pCLCrVA were ligated overnight with T4 ligase. The ligation product was transformed into *E. coli* DH5α and cultured overnight at 37°C. Single clones were picked and cultured, and the sequence was sequenced to verify its correctness. The correctly sequenced plasmid was transformed into *Agrobacterium* for subsequent cotton infection experiments.

[0134] 2. Virus-induced silencing complex infects cotton.

[0135] 1) Inoculate 20 μL of Agrobacterium tumefaciens culture of pCLCrVA, pCLCrVA:GhABF3, pCLCrVA:PDS and pCLCrVB stored at -20℃ into 1 mL of LB liquid medium containing kanamycin and rifampin, and culture overnight at 28℃ with shaking at 180 rpm. Take 200 μL of activated bacterial culture and add it to 50 mL of LB liquid medium and culture at 28℃ with shaking at 180 rpm.

[0136] 2) When the OD600 value of the bacterial solution is about 1.5-2.0, centrifuge the bacterial solution at 3000 rpm to collect the bacterial cells and centrifuge for 10 min;

[0137] 3) The conversion medium formulation is as follows:

[0138] Table 9

[0139]

[0140] 4) Suspend the bacterial cells in the above transformation medium, adjust the OD to 1.5, and let them stand in the dark for three hours. Then, mix equal amounts of pCLCrVA, pCLCrVA:GhABF3, pCLCrVA:PDS and pCLCrVB.

[0141] 5) Once the cotyledons have fully expanded, gently make a small incision on the back of the cotyledons with a needle, and inject the bacterial solution into the cotton cotyledons using a 1mL syringe.

[0142] 3. Identification and quantitative detection of silent plants

[0143] Once the true leaves of the positive control pCLCrVA:PDS cotton plants showed whitening, it indicated that the cotton plants had been successfully infected by the virus. At this point, leaf samples were taken from the pCLCrVA and pCLCrVA:GhABF3 cotton plants, and DNA was extracted for PCR detection. RNA was extracted from the leaves, and the silencing efficiency of GhABF3 was detected by quantitative real-time PCR. Positive plants with bands were selected and transplanted into flowerpots for further cultivation in a cotton culture room, where the phenotypes of the pCLCrVA empty vector and the pCLCrVA:GhABF3 silenced plants were observed.

[0144] The primers used for cotton DNA testing are:

[0145] VA-F 5'-ATTTTGCGCCTGACTAGCCT-3'(SEQ ID NO.17)

[0146] VA-R 5'-CGAATTTTCAACGTTGCATACA-3'(SEQ ID NO.18)

[0147] VB-F 5'-ATGTACAGTTTAAAGAGTAGACG-3'(SEQ ID NO.19)

[0148] VB-R 5'-ATTATCCAATATAATCAAGGTCATAC-3'(SEQ ID NO.20)

[0149] The primers used for quantitative fluorescence detection are the same as those used for analyzing the expression patterns of early and late maturing cotton varieties.

[0150] 4. Phenotypic observation and data statistics

[0151] Phenotypic observation of unloaded and silenced plants revealed that the leaves of the positive control pCLCrVA:PDS plants exhibited whitishness, while cotton plants silenced with GhABF3 showed delayed flowering and reduced plant height compared to the pCLCrVA unloaded control. Figure 3 (A). Quantitative fluorescence results showed that the gene was effectively silenced. Figure 3 (B)

[0152] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

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Claims

1. A kind GhABF3 The application of genes in regulating plant flowering time; The GhABF3 The nucleotide sequence of the gene is shown in SEQ ID NO.5; The GhABF3 The amino acid sequence encoded by the gene is shown in SEQ ID NO. 6; The GhABF3 Application of genes in the positive regulation of plant flowering; The plant in question is cotton.

2. The application according to claim 1, characterized in that: The GhABF3 The expression level of the gene was higher in early-maturing cotton than in late-maturing cotton.

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