Application of GhbHLH093 gene in regulating flowering period of plants

Abstract

The present invention provides a <h2 style=";text-align:left;direction:ltr">GhbHLH093 The application of the gene in regulating the flowering period of plants, the nucleotide sequence of the gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2, <h2 style=";text-align:left;direction:ltr"> GhbHLH093 The expression level of the gene in early-maturing cotton is higher than that in late-maturing cotton. <h2 style=";text-align:left;direction:ltr"> GhbHLH093 The gene was most highly expressed in cotton roots and overexpressed <h2 style=";text-align:left;direction:ltr"> GhbHLH093 Genes promote flowering and reproductive growth and development in Arabidopsis thaliana; the present invention cloned a gene from upland cotton <h2 style=";text-align:left;direction:ltr"> GhbHLH093 The gene was heterologously expressed in Arabidopsis by constructing an overexpression vector, and the resulting overexpression transgenic strains flowered earlier than the wild type, indicating that <h2 style=";text-align:left;direction:ltr"> GhbHLH093 Genes play an important regulatory role in controlling the flowering period of cotton.

Description

Technical Field

[0001] The invention belongs to the technical field of plant flowering, and particularly relates to an application of a GhbHLH093 gene in regulating the flowering period of a plant. Background Art

[0002] Cotton is an important cash crop in my country and a strategically important material related to the national economy. my country is the world's largest cotton producer and consumer, and its textile and clothing exports are also among the highest in the world (Li Yuhua, 2021). The National Statistical Yearbook shows that although the planting area in Xinjiang, the largest cotton-producing area, has increased, other cotton-producing areas in my country have been directly affected by the competition between grain and cotton, reduced planting efficiency and natural disasters, resulting in a decline in the overall cotton production area and output. (Wang Xueying et al., 2022) Early-maturing upland cotton has a short growth cycle and fast growth and development. Taking advantage of its suitability for late spring sowing, early summer sowing and summer sowing, it can be used to achieve grain-cotton rotation with winter crops such as winter wheat and lettuce, and effectively improve the land multiple cropping index by optimizing the crop planting index. In addition, the breeding of early-maturing cotton can effectively increase the flowering rate of cotton before frost, and improve the quality of cotton in high-latitude areas such as Liaoning and Gansu where light and heat conditions are poor and temperature changes are large (Yu Shuxun et al., 1991). The early maturity of cotton is not only regulated by genes, but also affected by the environment. Studies have shown that the genetic correlation of seedling stage, bud stage, flowering stage, boll stage, boll opening stage, first fruit branch node, and pre-frost flowering rate is greater than the environmental correlation, which can be used as an indicator of cotton early maturity (Tian Jinghua, 1983; Yu Shuxun et al., 1990). Therefore, identifying cotton flowering-related genes, clarifying their regulatory mechanisms, and creating excellent early-maturing cotton germplasm resources are of great significance for the cultivation and industrialization of early-maturing cotton varieties.

[0003] Flowering is a sign that plants are shifting from vegetative growth to reproductive growth. It is a prerequisite for plants to complete fertilization and produce sexual reproductive organs. The flowering time of plants is affected by the combined effects of the natural environment and genetic mechanisms, and shows rich diversity between and within species. At present, there is a relatively complete framework for the regulatory pathways of plant flowering. Studies based on the model plant Arabidopsis thaliana have found that flowering time is regulated by a variety of environmental and endogenous pathways, with six main regulatory pathways, including light-dependent, autonomous, vernalization, gibberellin, temperature, and age pathways. (Fornara et al, 2010; Kinoshita & Richter, 2020) These pathways are both independent of each other and interact and coordinate with each other to form a complex and sophisticated network, which affects the flowering time of plants by regulating the expression of flowering-related genes.

[0004] When the plant begins to differentiate flower buds, flower organs are gradually formed, manifested as the opening of the corolla calyx, exposing the pistils and stamens, and having the ability to be fertilized and bear fruit. The flowering regulation mechanism of plants varies due to the differences in plant species morphology and environment, but the overall flowering process can be divided into three parts: flowering induction, flowering initiation, and flower development. Flower induction is the process in which plant reproductive growth replaces vegetative growth as the growth center. Flower initiation is the initiation of flower bud differentiation by the apical meristem of the plant through certain internal and external signal stimulation. Flower development is the process in which flower buds gradually differentiate into mature flower organs after the beginning of flower bud differentiation.

[0005] From the differentiation of flower buds to the cessation of plant growth, upland cotton has been in a stage of vegetative growth and reproductive growth. Compared with general dicotyledonous plants, cotton's floral organ development is very distinctive. In addition to sepals, petals, anthers and carpels, there is also a round of bracts on the outer layer of sepals (Zheng Shangyong et al., 2004). Due to the differences in floral development characteristics and structures of upland cotton, it is speculated that its molecular mechanism for regulating floral development is different from that of model plants such as Arabidopsis. However, there are few molecular biological studies related to cotton floral organ development, and its regulatory mechanism is still unclear. Summary of the invention

[0006] The present invention aims to provide an application of the GhbHLH093 gene in regulating the flowering period of plants in view of the deficiencies in the prior art.

[0007] To achieve the above object, the solution of the present invention is:

[0008] Application of GhbHLH093 gene in regulating flowering period of plants.

[0009] Preferably, the nucleotide sequence of the GhbHLH093 gene is shown in SEQ ID NO.1.

[0010] Preferably, the amino acid sequence encoded by the GhbHLH093 gene is shown in SEQ ID NO.2.

[0011] Preferably, the GhbHLH093 gene is used for positively regulating plant flowering.

[0012] Preferably, the plants include cotton and Arabidopsis thaliana.

[0013] Preferably, the expression level of the GhbHLH093 gene in early-maturing cotton is higher than that in late-maturing cotton.

[0014] Preferably, the GhbHLH093 gene is most highly expressed in cotton roots.

[0015] Preferably, overexpression of the GhbHLH093 gene promotes flowering and reproductive growth and development in Arabidopsis thaliana.

[0016] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0017] The present invention cloned the GhbHLH093 gene from upland cotton. The gene was heterologously expressed in Arabidopsis thaliana by constructing an overexpression vector, and the overexpression transgenic strain obtained flowered earlier than the wild type, indicating that the GhbHLH093 gene plays an important regulatory role in controlling the flowering period of cotton. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the expression level of the GhbHLH093 gene in the terminal buds of early- and late-maturing materials in Example 2 of the present invention.

[0019] Figure 2 This is a graph showing the expression levels of the GhbHLH093 gene in different cotton tissues in Example 2 of the present invention.

[0020] Figure 3 This is the subcellular localization map of the GhbHLH093 gene in Example 3 of the present invention.

[0021] Figure 4 This is a diagram showing that the GhbHLH093 gene promotes early flowering of overexpressed transgenic Arabidopsis thaliana in Example 3 of the present invention. DETAILED DESCRIPTION

[0022] The present invention provides an application of a GhbHLH093 gene in regulating the flowering period of a plant.

[0023] 1. Test materials

[0024] (1) Cotton material

[0025] The cotton materials selected in this experiment were the early-maturing upland cotton variety Zhongmian 50 and the late-maturing variety Guoxin 11. Among them, Zhongmian 50 and Guoxin 11 had extremely significant differences in flowering time and growth period (Table 1). They were planted in the experimental field of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences (Baibi Town, Anyang City, Henan Province), and the management measures were normal field management. The sampling method was to place the young shoots of the three cotton varieties from one leaf stage to five leaf stage in liquid nitrogen and store them at -80℃ before extracting the sample RNA.

[0026] Table 1 Significance test of traits of China Cotton Research Institute 50 and Guoxin Cotton 11

[0027]

[0028] (2) Reagents and consumables

[0029] Restriction endonucleases, modification enzymes, PCR reaction system-related enzymes, homologous recombination enzymes, gel recovery kits, cloning kits, and plasmid extraction kits were purchased from Novazon Biotechnology Co., Ltd., the fluorescence quantification kit was purchased from Kangwei Century Biotechnology Co., Ltd., and the RNA extraction kit was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0030] Other drugs: agarose is a Spanish original product, peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, sodium chloride, etc. are domestic analytical grade, kanamycin, etc. are from Solebold Biotechnology Co., Ltd., and Escherichia coli competent cells DH5α and Agrobacterium competent cells are purchased from Qingke Biotechnology Co., Ltd.

[0031] Culture medium: LB liquid culture medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L;

[0032] LB solid medium: Tryptone 10g / L, yeast extract 5g / L, sodium chloride (NaCl) 10g / L, agar powder 15g / L, fixed volume to 1L;

[0033] LB selection medium: before LB plating, add antibiotics of corresponding concentration when the medium is sterilized by high pressure and cooled to 55°C, shake well and then spread on the plate; 1 / 2MS solid medium: 1 / 2MS 22g / L, agar powder 8g / L, sucrose 30g / L.

[0034] Main instruments: PCR amplifier (BIO-RAD), high-speed centrifuge (Hettich MIKRO 200R), electrophoresis equipment (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR instrument (ABI7500), electric constant temperature incubator (Shanghai Senxin), constant temperature culture oscillator (Shanghai Zhicheng), artificial climate test chamber (Safe), artificial climate chamber.

[0035] Embodiment 1:

[0036] The CDS sequence and the amino acid sequence of the GhbHLH093 (Gh_D01G1087) gene were obtained from CottonFGD (http: / / www.cottonfgd.org / ). The open reading frame was 972 bp, encoding 323 amino acids. The gene was named GhbHLH093, and its function was studied.

[0037] The open reading frame sequence of GhbHLH093 (SEQ ID NO.1) is:

[0038] ATGGAGATCAATGAAGAAGGTTTGTTCGAAGAATTATTAGATGTGAGAGGAGAGAATTGGGACACAAATCCAACAGAAATGAGTGGGATTTTCTCTAATGGCACCTGGAACTTTGATGATCACAAACCTTCATCTACCTTTCTTCCATTGCCATTTCACCAAGATTATACTTACAACTTCAATCCAATCTACTGTCCCTTTGTTGATGAATTCTCTTCACAAAGCAACACATTTGATACACCCTCATTCCCACTCCAACAACAACATGATGACCAAGAATCCAGATTCCTTGTACATCAACTTCACAAGTTGGATGTTAAAGCTACTTGCAAAACCGAGCCTGTTCAATCACCTCACCCTGACAATCCAGCTAAAAAGTTGGAAAGGCAGCCTTCAAAGAATCTGATGGCTGAAAGAAGGAGACGAAAAAGGCTAAACGATCGCCTTTTGATGTTAAGATCCATTGTGCCTAAGATAAGCAAGGTAATTTACTGCAAAAATTCAGATTTCAAGCCTCTTATTACTGGTTTTATTTCCCATTTTTTGTCTCTTCAACAGATGGACCGTACATCCATACTTGGAGATACCATAGATTATACCAACGAACTCTTGGAGAGGATCAAAAGTTTGCAGCAAGAAGTTGAAGCAGGTTCAAACATGGATCATATTTTCAAGGGTGAAAAACCAAATGAAATGATAGTGAGAAATACACCCAAGTTTGAGGTTGAAAGAAGAAATGGGGATACAAGGATTGAGATTTGTTGCAGAGGGGATCCAGGATTGTTGTTATCAACCGTATCAACAATGGAAGCATCGGGGCTTGAGATTCAACAATGTGTCATTAGTTGTTTCAATGATTTTGCAATGCATGCTTCTTGCTCTGAGGATCTGGAACAGACAACATTAATGAGGTGTGAAGATATAAAGAAGGCATTATTTAGAAATGCTGGCTATGGTGGAAGATGTGTTTAG。

[0039] The amino acid sequence encoded by GhbHLH093 (SEQ ID NO.2) is:

[0040] MEINEEGLFEELLDVRGENWDTNPTEMSGIFSNGTWNFDDHKPSSTFLPLPFHQDYTYNFNPIYCPFVDEFSSQSNTFDTPSFPLQQQHDDQESRFLVHQLHKLDVKATCKTEPVQSPHPDNPAKKLERQPSKNLMAERRRRKRLNDRLLMLRSIVPKISKV IYCKNSDFKPLITGFISHFLSLQQMDRTSILGDTIDYTNELLERIKSLQQEVEAGSNMDHIFKGEKPNEMIVRNTPKFEVERRNGDTRIEICCRGDPGLLLSTVSTMEASGLEIQQCVISCFNDFAMHASCSEDLEQTTLMRCEDIKKALFRNAGYGGRCV.

[0041] Embodiment 2:

[0042] Studies have shown that cotton flower bud differentiation is closely related to early maturity traits and is a sign of cotton's transition from vegetative growth to reproductive growth. It directly affects flowering time. The early-maturing variety Zhongmiansuo 50 and the late-maturing variety Guoxinmian 11 were selected to extract flower bud RNA from the one-leaf stage to the five-leaf stage. The expression level of GhbHLH093 was detected by qRT-PCR technology. It was found that the expression level of this gene in the early-maturing variety Zhongmiansuo 50 from the one-leaf stage to the five-leaf stage was significantly higher than that in the late-maturing variety Guoxinmian 11.

[0043] To further explore the expression pattern of GhbHLH093 in different tissues, eight different tissues of cotton variety Zhongmian 50 were selected to analyze the gene expression level. The results showed that the GhbHLH093 gene was expressed most highly in the roots and also had a high expression in the flower organs. The specific steps for detecting the expression of GhbHLH093 are as follows:

[0044] <1> Sampling and grinding

[0045] The terminal buds of Zhongmiao 50 and Guoxin cotton 11 at the one-leaf to five-leaf stage were selected and placed in liquid nitrogen. They were ground into powder using a mortar and pestle, and approximately 1 g of the sample was taken into a 1.5 mL centrifuge tube.

[0046] <2> extract

[0047] RNA extraction was performed using the FastPure Universal Plant Total RNA Isolation Kit (Novizan, Nanjing, China), and the specific steps were as follows:

[0048] (1) The experiment was carried out at room temperature. 600 μL of Buffer PSL (polyphenol polysaccharide plant) was immediately added to the centrifuge tube containing the plant tissue. The tube was vortexed vigorously for 30 seconds to mix the sample and lysate thoroughly. The tube was centrifuged at 12,000 rpm (134,00 × g) for 5 minutes and the subsequent operation was immediately carried out.

[0049] (2) Take about 500 μL of the supernatant and transfer it to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube), centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, discard FastPure gDNA-Filter Columns III, and collect the filtrate;

[0050] (3) Add 0.5 times the volume of filtrate anhydrous ethanol (about 250 μL, adjusted according to the actual situation of the supernatant) to the collection tube and oscillate for 15 seconds. Transfer the above mixture to FastPure RNA Columns V, which have been placed in the collection tube, centrifuge at 12,000 rpm (13,400 × g) for 30 seconds, and discard the filtrate;

[0051] (4) Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm (13,400 × g) for 30 s, and discard the filtrate;

[0052] (5) Add 500 μL of Buffer RWB to FastPure RNA Columns V (please check whether 48 mL of anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (13,400 × g) for 30 s, and discard the filtrate;

[0053] (6) Repeat step (5);

[0054] (7) Place FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm (13,400 × g) for 2 min.

[0055] (8) Transfer FastPure RNA Columns V to new RNase-free Collection Tubes 1.5 mL centrifuge tubes, add 30-100 μL of RNase-free ddH2O to the center of the adsorption column membrane, and centrifuge at 12,000 rpm (13,400 × g) for 1 min;

[0056] ▲The elution volume is recommended to be no less than 30μL. A volume that is too small will affect the efficiency of nucleic acid recovery;

[0057] ▲The following steps can help increase the concentration of RNA products: preheat RNase-free ddH2O at 65℃; add RNase-free ddH2O and let stand at room temperature for 5 minutes; re-add the first eluent to the adsorption column for elution;

[0058] (9) The extracted RNA can be used directly in downstream experiments or stored at (-85)-(-65)℃.

[0059] <3> Synthesis of reverse transcribed cDNA

[0060] Reverse transcription cDNA synthesis using kit II Q RT SuperMix for qPCR (+gDNAwiper) (NoviZan, Nanjing, China), which can be divided into two parts: removal of genomic gDNA and reverse transcription of RNA. The reaction was carried out on ice. The reaction steps are as follows:

[0061] (1) Removal of genomic gDNA

[0062] Table 2 Reaction system preparation

[0063]

[0064]

[0065] Mix by gently pipetting. Incubate at 42°C for 2 minutes.

[0066] (2) Preparation of reverse transcription reaction system

[0067] Table 3

[0068]

[0069] Use a pipette to gently pipette and mix well. Place 20 μL of the mixed solution in a PCR instrument at 50°C for 15 min and 85°C for 5 s. The product can be used immediately for qPCR reaction or stored at -20°C and used within half a year.

[0070] <4> Fluorescence quantitative PCR

[0071] (1) Oligo 7 software was used to design specific primers for the GhbHLH093 gene, and the cotton His3 (AF024716) gene was used as an internal reference gene.

[0072] Table 4

[0073]

[0074] (2) Fluorescence quantitative PCR

[0075] The UltraSYBR Mixture (Low ROX) kit from Cwbio (China) and the Applied Biosystems 7500 instrument were used. The specific process is as follows:

[0076] 1) Dilute the above cDNA stock solution 5 times;

[0077] 2) Configuration of the reaction system (operation on ice):

[0078] Table 5

[0079]

[0080] The prepared system was mixed and centrifuged until there were no bubbles. Then, fluorescent quantitative PCR was performed using Applied Biosystems 7500: the PCR program was set according to the two-step method: pre-denaturation: 95°C, 2min; 95°C, 5s; 60°C, 34s ​​(this step collects fluorescence signals), and these two steps were set for 40 cycles; finally, melting curve analysis: 95°C, 15s; 60°C, 20s; 95°C, 15s. Microsoft Excel 2019 software was used to process the data, calculate the expression of genes, and Origin 2022 software was used for drawing.

[0081] <5> GhbHLH093 quantitative results analysis

[0082] The fluorescence quantitative results were calculated according to 2-ΔΔCt (Livak method) to obtain the relative expression level of GhbHLH093. Figure 1 It can be seen that the expression level of GhbHLH093 in the early-maturing varieties from the one-leaf stage to the five-leaf stage is higher than that in the late-maturing varieties, and the difference is significant; Figure 2 Tissue expression patterns showed that GhbHLH093 was highly expressed in both root and flower organs, indicating that this gene was related to cotton early maturity.

[0083] Embodiment 3:

[0084] Agrobacterium-mediated transient expression in tobacco and heterologous expression in Arabidopsis

[0085] Subcellular localization is closely related to the function of proteins. When studying proteins, determining their location in the cell is usually the first step. To verify the accuracy of the prediction results of the subcellular localization website (http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2 / ), a PBI-GFP-GhbHLH093 recombinant vector was constructed and the GhbHLH093 protein was transiently expressed in tobacco. It was found that the GhbHLH093 protein was localized in the nucleus. The full-length GhbHLH093 CDS sequence was connected to the PBI-121 vector to construct a 35S promoter vector. The 35S::GhbHLH093 recombinant vector was used to infect Arabidopsis thaliana by the Arabidopsis inflorescence infection method, and the T3 generation pure line plants were obtained by positive screening and purification of the offspring seeds. The phenotype and expression level of the offspring were analyzed, and it was found that overexpression of GhbHLH093 promoted early flowering of Arabidopsis thaliana.

[0086] <1> Gene primer design

[0087] According to the principle of homologous recombination primer design, specific primers were designed using Oligo 7 software to amplify the full length of the coding region of the GhbHLH093 gene. According to the CDS sequence of the gene, the corresponding restriction site sequences were added at the start codon ATG and the stop codon, so that the target gene fragment had the same terminal sequence as the linearized vector after restriction digestion. XbaI and SacI were selected as the restriction sites of the 35S promoter vector; the CDS sequence for subcellular localization was used to remove the stop codon to design specific primers, the restriction sites of the GFP vector were XbaI and SpeI, and the cDNA template used was upland cotton TM-1.

[0088] The 35S::GhbHLH093 specific primer sequence is as follows:

[0089]

[0090] The GhbHLH093-GFP specific primer sequences are as follows:

[0091]

[0092] <2> Gene cloning PCR system, procedure and product detection

[0093] (1) The reaction was carried out on ice. The reaction system was designed according to the kit Phanta Max Super-Fidelity DNA Polymerase (Novizan, Nanjing) as follows:

[0094] Table 6

[0095]

[0096] (2) PCR reaction procedure:

[0097]

[0098] (3) Detection of PCR products

[0099] Take 2 μL of PCR product, add 2 μL of 5× Loading Buffer, mix well, apply to 1% agarose gel, and check whether the band size is about 972 bp by electrophoresis.

[0100] (4) Gel recovery of PCR products

[0101] The Vazyme product purification kit was used as follows:

[0102] 1) After DNA electrophoresis, quickly cut the gel containing the target DNA fragment under UV light. It is recommended to use a paper towel to absorb the liquid on the surface of the gel and cut it into pieces, and try to remove excess gel. Weigh the gel (minus the weight of the empty tube), 100 mg of gel is equivalent to 100 μL volume, which is a gel volume;

[0103] 2) Add an equal volume of Buffer GDP. Incubate at 50-55℃ in a water bath for 7-10 minutes. Adjust the time appropriately according to the size of the gel to ensure that the gel blocks are completely dissolved. Invert and mix twice during the water bath to accelerate the dissolution;

[0104] 3) Briefly centrifuge to collect droplets on the tube wall. Place the FastPure DNA Mini Columns-G adsorption column in the Collection Tubes 2ml collection tube, transfer ≤700μL of sol to the adsorption column, and centrifuge at 12,000×g for 30-60s. If the sol volume is greater than 700μL, place the adsorption column in the collection tube, transfer the remaining sol to the adsorption column, and centrifuge at 12,000×g for 30-60s;

[0105] 4) Discard the filtrate and place the adsorption column in a collection tube. Add 300 μL of Buffer GDP to the adsorption column. Let stand for 1 min. Centrifuge at 12,000 × g for 30-60 s;

[0106] 5) Discard the filtrate and place the adsorption column in a collection tube. Add 700 μL of Buffer GW (with anhydrous ethanol) to the adsorption column. Centrifuge at 12,000 × g for 30-60 seconds;

[0107] 6) Repeat step 5);

[0108] 7) Discard the filtrate and place the adsorption column in a collection tube. Centrifuge at 12,000×g for 2 minutes;

[0109] 8) Place the adsorption column in a 1.5 mL sterile centrifuge tube, add 20-30 μL of sterile water to the center of the adsorption column, and place for 2 minutes. Centrifuge at 12,000×g for 1 minute. Discard the adsorption column and store the DNA at -20°C.

[0110] <3> Construction of plant expression vectors of GhbHLH093-GFP and 35S::GhbHLH093

[0111] (1) Double restriction digestion and gel recovery of PBI121 plasmid

[0112] The PBI121 plasmid was double-digested with XbaI and SacI, and the large fragment product of the PBI121 vector was recovered by electrophoresis. The enzyme digestion reaction system is as follows:

[0113] Table 7

[0114]

[0115] (2) Ligation of PCR gel-recovered products and restriction-digested PBI121 plasmid

[0116] The PCR product with the adapter and the double-digested PBI121 plasmid were treated with the Novogene Recombinase Reagent One Step Cloning Kit is used for ligation. The ligation reaction is as follows:

[0117] The system was set up on ice:

[0118] Table 8

[0119]

[0120] After the system is completed, mix the components by pipetting, react at 37°C for 30 min, immediately place in an ice-water bath for 5 min, and transform or store at -20°C.

[0121] (3) Transformation of ligation product into Escherichia coli

[0122] 1) Add 100 μL of E. coli DH5a competent cells to the ligation reaction system and incubate on ice for 30 min;

[0123] 2) Heat shock in a 42°C water bath for 45-90 seconds;

[0124] 3) Ice bath for 2 min; add 900 μL of LB liquid medium without resistance, incubate at 37°C, 190 rpm, and incubate for 1 h;

[0125] 4) Collect the bacteria by centrifugation at 4000 rpm for 3 min, discard the supernatant, keep about 100 μL, mix well and apply to LB plate containing kanamycin resistance;

[0126] 5) Incubate at 37°C overnight.

[0127] (4) Detection and sequencing of positive clones

[0128] 1) Pick up white colonies from the transformation plate, place them in liquid LB medium containing Kan, and culture them in a constant temperature shaker at 37°C for 8 hours;

[0129] 2) The positive clones were verified by colony PCR, and the verified single clones were sent to Shangya Biotechnology Co., Ltd. for sequencing, with 3 replicates for each sequence.

[0130] (5) Storage of positive bacterial solution

[0131] A certain amount of glycerol was added to the bacterial solution that was verified by PCR and sequenced correctly, so that the final concentration of glycerol was about 20%, and stored at -80°C. The plasmid with correct sequencing was returned for transformation of Agrobacterium.

[0132] (6) Transformation of Agrobacterium

[0133] The freeze-thaw method was used to transform Agrobacterium tumefaciens GV3101 competent cells. The specific transformation process is as follows:

[0134] 1) Melt Agrobacterium at -80℃ and insert into ice in the ice-water mixture state;

[0135] 2) Add 0.01-1 μg plasmid DNA to 100 μL competent medium, stir the bottom of the tube by hand to mix, and place on ice for 5 min, in liquid nitrogen for 5 min, at 37°C for 5 min, and in ice bath for 5 min;

[0136] 3) Add 700 μL of LB liquid medium without resistance and culture at 28°C with shaking for 2-3 hours;

[0137] 4) Place 100-150 μL of bacterial solution on an LB plate containing kanamycin, rifampicin, and streptomycin, and place it upside down in a 28°C incubator for 2-3 days;

[0138] 5) Select positive clones, culture them in LB liquid medium with resistance at 28°C for 48h, verify the correct bands by PCR, store the bacterial solution in glycerol at a final concentration of about 20%, and store at -80°C for future use.

[0139] <4> Agrobacterium-mediated transient expression in tobacco

[0140] The steps for tobacco transient expression are as follows:

[0141] (1) Tobacco cultivation: sow a number of tobacco seeds and cultivate tobacco under long-day conditions for one month for use in the experiment;

[0142] (2) Agrobacterium activation: Take 20 μL of GhbHLH093-GFP Agrobacterium culture stored at -80°C and inoculate into 1 mL of LB liquid medium (with corresponding antibiotics: kanamycin, rifampicin and streptomycin), and culture at 28°C, 180 rpm for 14-18 h;

[0143] Cultivate until the OD600 value of the bacterial solution is about 1.0-1.5 (about 18-20 hours), centrifuge at 5000 rpm for 8 minutes, discard the supernatant, and collect the bacteria;

[0144] (3) Resuspension: Resuspend the cells in 10 mM MgCl2, 10 mM MgCl2 (containing 120 uM AS, pH = 5.8) suspension and adjust the OD600 to about 0.8;

[0145] (4) Injection: Select tobacco plants with good growth conditions and use a 1 mL syringe without a gun tip to inject into the lower epidermis of the tobacco leaves and mark them;

[0146] (5) Cultivation: Cultivate the injected tobacco plants under weak light for 2 days and then observe;

[0147] (6) Observation: Take the labeled tobacco leaves injected with Agrobacterium, make them into slides, and observe and photograph them under a laser confocal microscope.

[0148] Note: During co-localization, transform the marker plasmid into Agrobacterium, suspend it together with the constructed vector plasmid Agrobacterium, mix them at a 1:1 ratio before injection, and then inject them into tobacco leaves.

[0149] <5> Subcellular localization results

[0150] Laser confocal microscopy revealed that the GFP signal of the GhbHLH093 fusion protein overlapped with the red nuclear anchor marker protein NLS-mCherry after transient expression in tobacco cells. Figure 3 , confirming the nuclear localization of GhbHLH093 protein.

[0151] <6> Agrobacterium-mediated infection of Arabidopsis inflorescence

[0152] (1) Arabidopsis thaliana cultivation

[0153] Colombian wild-type Arabidopsis thaliana cultured under long-day conditions (16 h of light and 8 h of darkness) were selected from robust strains of about 5 weeks old, the siliques were cut off, and the Arabidopsis thaliana was watered the day before infection to ensure the condition and humidity.

[0154] (2) Infection of Arabidopsis inflorescence

[0155] 1) Activation of bacterial culture: Take 20 μL of Agrobacterium culture containing the corresponding recombinant vector stored at -80°C, inoculate into 1 mL of LB liquid culture medium (add corresponding antibiotics: kanamycin, rifampicin and streptomycin), and culture at 28°C, 180 rpm for 14-18 h;

[0156] 2) Propagation: Take 200 μL of the activated bacterial solution and add it to 50 mL of LB liquid medium (add the corresponding antibiotics); culture at 28°C, 180 rpm until the OD600 value of the bacterial solution is about 1.2-1.6 (about 18-20 hours), centrifuge at 5000 rpm for 8 minutes, discard the supernatant, and collect the bacteria;

[0157] 3) Preparation of medium for infection and transformation: 1 / 2MS reduced by half, 6% sucrose, 0.02% Silwet L-77, and pH adjusted to 5.6-5.7 with NaOH;

[0158] 4) Suspend the above bacteria in transformation medium and adjust OD600 to 0.6-0.8;

[0159] 5) Infection: Place the Arabidopsis inflorescence (mainly unopened buds) in the transformation medium for 30-50 seconds. After infection, place the Arabidopsis flat in low light or dark conditions for 24 hours;

[0160] 6) Cultivate the treated Arabidopsis under normal conditions and spray the leaves of Arabidopsis with water every day within one week after infection; in order to improve the transformation efficiency, the infection can be repeated after about one week;

[0161] 7) After maturity, the Arabidopsis seeds are harvested, which are the transgenic T0 generation seeds.

[0162] <7> Phenotypic identification of transgenic Arabidopsis plants

[0163] (1) After the harvested seeds are disinfected, they are planted on 1 / 2MS containing kanamycin, and then vernalized at 4°C for 2 days. They are then transferred to an artificial climate test chamber. After about 10 days, the positive plants will grow normally, while the leaves of the negative plants will turn yellow and no longer grow.

[0164] (2) The positive Arabidopsis plants were transplanted into small flower pots and grown for one month before DNA was extracted for PCR testing.

[0165] The primers used in the detection are:

[0166] 35S-F GACGCACAATCCCACTATCC(SEQ ID NO.11)

[0167] GhbHLH093-RGACGGCCAGTGAATTCTTCAGCAGAAAGTTGTTTAGTATTG

[0168] (SEQ ID NO.12)

[0169] (3) Each generation of plants must be tested for positive lines until they are propagated to the T3 generation and homozygous transgenic Arabidopsis lines are obtained. The T3 generation lines are tested for qRT-PCR, and the process of fluorescence quantitative verification is as follows:

[0170] Extract RNA, reverse transcribe into cDNA, and use primers for fluorescence quantification of GhbHLH093

[0171] GhbHLH093-F ACGAAAAAGGCTAAACGATCGCC(SEQ

[0172] ID NO.13)

[0173] GhbHLH093-R ACCTGCTTCAACTTCTTGCTGC(SEQ ID

[0174] NO.14)

[0175] The qRT-PCR reaction system was prepared on ice and the fluorescence quantitative PCR reaction was performed. The fluorescence quantitative verification results confirmed that the transcription level of the GhbHLH093 gene in the transgenic plants was significantly higher than that in the non-transgenic Arabidopsis. Figure 4 .

[0176] (4) Transgenic T3 plants and non-transgenic plants were sterilized and cultured on 1 / 2MS medium. After two days of vernalization at 4°C, the Arabidopsis seedlings grew true leaves in about 10 days and were then moved to small flower pots for growth. Under the same conditions, phenotypic observations revealed that non-transgenic Arabidopsis flowered significantly later than overexpressing transgenic Arabidopsis ( Figure 4 ); It shows that overexpression of GhbHLH093 significantly promotes flowering and reproductive growth and development of Arabidopsis thaliana.

[0177] It will be apparent to those skilled in the art that the 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 the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. The nucleotide sequence is shown in SEQ ID NO.1 <h2 style=";text-align:left;direction:ltr">GhbHLH093 Application of genes in positive regulation of flowering time in Arabidopsis thaliana.

2. The use according to claim 1, characterized in that: Said <h2 style=";text-align:left;direction:ltr"> GhbHLH093 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: Overexpression <h2 style=";text-align:left;direction:ltr"> GhbHLH093 Genes promote flowering and reproductive growth and development in Arabidopsis thaliana.

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