Camellia sinensis transcription factor and application thereof in linalool synthesis in tea leaves

Abstract

The application provides a tea tree transcription factor and application thereof in linalool synthesis of tea leaves. The tea tree transcription factor CsbHLH25 (CSS0046630) can directly combine with a promoter of a CsTPS13 gene and inhibit transcription activity of the CsTPS13 gene. Transient transformation tea leaf experiments show that CsbHLH25 transient overexpression can significantly inhibit CsTPS13 gene expression and reduce linalool production. Expression of the tea tree transcription factor CsbHLH25 is inhibited by yellow light. Yellow light treatment can inhibit expression of CsbHLH25, release inhibition of CsbHLH25 on the CsTPS13 gene, up-regulate CsTPS13 gene expression, and further improve linalool content. The application discloses a molecular mechanism of the transcription factor CsbHLH25 affecting linalool biosynthesis by negative regulation of the CsTPS13 gene, and provides a new technical means and application strategy for improving aroma quality of tea leaves.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a tea transcription factor and its application in the synthesis of linalool in tea. Background Technology

[0002] The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the world's most important economic crops. Aroma is a crucial identifying element of tea, largely determining its market acceptance and economic value. Research indicates that tea aroma consists of approximately 700 volatile compounds. Most aroma substances are primarily produced during tea processing, especially terpenoids. Among these, terpenols and jasmonic acid derivatives play a key role in enhancing the floral aroma quality of green tea. For example, withering or spreading techniques are employed in the processing of green tea, white tea, black tea, and oolong tea to improve their aroma characteristics. Among terpenoids, linalool is the most abundant monoterpene aroma compound, possessing floral characteristics and making a significant contribution to tea quality formation. Studies show that the content of linalool in fresh tea leaves is relatively low, but it increases significantly during withering or spreading. This phenomenon may be related to the activation of linalool synthesis pathway genes by various factors such as mechanical damage, water loss, and light exposure during this process. However, the biosynthetic mechanism of linalool under various stress conditions during tea processing still lacks in-depth research. Summary of the Invention

[0003] Technical Problem to be Solved: Addressing the lack of in-depth research on the regulatory mechanism of linalool biosynthesis during the withering of fresh tea leaves in existing technologies, the purpose of this invention is to provide a tea transcription factor and its application in linalool synthesis in tea. The transcription factor CsbHLH25 negatively regulates linalool synthesis by binding to the promoter of the CsTPS13 gene and inhibiting its transcriptional activity. This invention reveals the molecular mechanism by which the transcription factor CsbHLH25 affects linalool biosynthesis through negative regulation of the CsTPS13 gene during the yellow light withering of fresh tea leaves. This invention provides a new gene target and processing strategy for improving the aroma and quality of tea.

[0004] Technical solution: The present invention provides a tea tree transcription factor CsbHLH25, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0005] The amino acid sequence of the protein encoded by the tea tree transcription factor CsbHLH25 is shown in SEQ ID NO.2.

[0006] This invention also provides the application of the above-mentioned tea transcription factor CsbHLH25 in regulating the synthesis of linalool in tea.

[0007] This invention also provides a method for regulating the synthesis of linalool in tea leaves, specifically: spreading fresh tea leaves under light to inhibit the expression of the tea transcription factor CsbHLH25 and promote the expression of the CsTPS13 gene, thereby promoting the synthesis of linalool in tea leaves.

[0008] Preferably, the illumination is yellow light with a wavelength of 580-595 nm, the vertical distance between the light source and the leaf surface is 10-20 cm, the thickness of the fresh tea leaves is 0.5-2 cm, and the spreading time is 14-18 h.

[0009] The nucleotide sequence of the CsTPS13 gene is shown in SEQ ID NO.3.

[0010] The amino acid sequence of the protein encoded by the CsTPS13 gene is shown in SEQ ID NO.4.

[0011] Beneficial effects:

[0012] 1. This invention reveals the negative regulatory function and molecular mechanism of transcription factor CsbHLH25 in linalool synthesis. Transcription factor CsbHLH25 is a direct upstream regulator of the CsTPS13 gene. CsbHLH25 inhibits the transcriptional activity of the CsTPS13 gene by binding to its promoter. Overexpression of CsbHLH25 significantly inhibits CsTPS13 gene expression and reduces linalool yield. The key negative regulatory role of the CsbHLH25-CsTPS13 regulatory module in linalool biosynthesis has been clarified, providing new experimental evidence and theoretical support for the transcriptional regulation mechanism of linalool synthesis in tea.

[0013] 2. This invention elucidates the molecular mechanism by which yellow light spreading treatment increases linalool content through the CsbHLH25-CsTPS13 module. Experiments show that yellow light spreading treatment significantly inhibits the expression of the transcription factor CsbHLH25, thereby relieving its transcriptional repression of CsTPS13, upregulating CsTPS13 expression, and ultimately increasing linalool yield. This invention reveals the regulatory pathway of light conditions and aroma quality formation during tea processing at the molecular level, providing theoretical guidance for optimizing tea spreading technology.

[0014] 3. This invention provides new gene targets and processing strategies for improving the aroma and quality of tea. Based on the negative regulatory effect of transcription factor CsbHLH25 on linalool synthesis, the expression or activity of CsbHLH25 can be inhibited through gene editing, RNA interference, and other techniques to directionally increase the linalool content in tea. Simultaneously, yellow-light withering, as a physical processing method, does not require the introduction of exogenous genes or chemical reagents; it enhances aroma by regulating the expression of endogenous CsbHLH25, offering excellent ease of operation and application potential.

[0015] 4. This invention has significant industrial application prospects. The provided CsbHLH25 gene and its encoded protein, CsTPS13 gene and its encoded protein, and promoter sequence offer new gene targets for the efficient production of linalool. These targets can be applied to fields such as molecular breeding of tea trees, genetic engineering improvement, and optimization of tea processing techniques. This provides a new technical solution for improving the aroma and quality of tea and increasing product added value, and is of great significance for promoting the high-quality development of the tea industry. Attached Figure Description

[0016] Figure 1 The graph shows the correlation between the expression level and content of linalool biosynthesis genes and the qRT-PCR validation of candidate genes. In the graph: A shows the Pearson correlation analysis between differentially expressed genes in the linalool biosynthesis pathway and linalool content during the spreading process; B shows the change in expression level of CSS0012706 during the spreading process; C shows the change in expression level of CSS0032883 during the spreading process; D shows the change in expression level of CSS0012706 and CSS0032883 under yellow light induction.

[0017] Figure 2 This is a graph showing the differential expression analysis of candidate transcription factors based on transcriptome sequencing; in the graph: A shows the expression changes of transcription factors based on yellow light treatment of the transcriptome, CK represents the control, YL represents yellow light treatment; B shows the expression changes of 10 bHLH transcription factors during the spreading process, W1-W5 represent the spreading degree treatment;

[0018] Figure 3 Interaction diagram of CsbHLH25 and CsTPS13 promoters for yeast one-hybrid verification;

[0019] Figure 4 Subcellular localization map of CsTPS13 protein; In the figure: A is the subcellular localization of CsTPS13; B is the CsTPS13-His fusion protein separated by SDS-PAGE;

[0020] Figure 5 Subcellular localization map of CsbHLH25 protein;

[0021] Figure 6 This is a graph showing the in vitro enzyme activity analysis of CsTPS13 protein;

[0022] Figure 7 Figure 1 shows the effect of transient overexpression of CsTPS13 on linalool content in tea leaves; in the figure: A represents the expression analysis of CsTPS13; B represents the linalool content analysis.

[0023] Figure 8Figure 1 shows the results of the tobacco dual-luciferase report; Figure 2 shows the in vivo imaging analysis of CsbHLH25 dual-luciferase; Figure 3 shows the CsbHLH25 dual-luciferase activity assay, with values ​​representing LUC / REN values.

[0024] Figure 9 The figure shows the results of gel migration experiments to verify the interaction between CsbHLH25 and the CsTPS13 promoter; in the figure: A shows the EMSA experiment to verify the binding of CsbHLH25 and CACCTG; B shows the EMSA experiment to verify the binding of CsbHLH25 and CATTTG.

[0025] Figure 10 The figure shows the effect of transient overexpression of CsbHLH25 on the content of CsTPS13 in tea leaves; in the figure: A is the expression analysis of CsbHLH25; B is the expression analysis of CsTPS13.

[0026] Figure 11 Figure 1 shows the effect of transient overexpression of CsbHLH25 on the linalool content in tea leaves.

[0027] Figure 12 Figure 1 shows the expression of CsbHLH25 and the linalool content under different light quality spreading conditions. In the figure: A is the expression analysis of CsbHLH25; B is the expression analysis of CsTPS13; C is the linalool content analysis. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0029] The fresh tea leaves used in the experiment were purchased from Laoshantou Tea Co., Ltd., Mapu Town, Jinzhai County, Lu'an City, Anhui Province, China. The selected varieties were five locally cultivated green tea tree varieties: Shucha Zao (approximately 10-11 years old, altitude approximately 100-200 m), Wuniuzao (approximately 10-11 years old, altitude approximately 100-200 m), Longjing 43 (approximately 8-9 years old, altitude approximately 100-200 m), Qishan Quntizhong (approximately 20-40 years old, altitude approximately 800 m), and Dushan Quntizhong (approximately 20-40 years old, altitude approximately 400 m). The tenderness of the fresh leaves was measured from the second leaf.

[0030] Example 1

[0031] This embodiment describes the screening of key genes for linalool biosynthesis, and the specific method includes the following steps:

[0032] 1. Plant Materials and Treatment: Fresh tea leaves were evenly spread out under the following conditions: temperature 25℃, humidity 65%, and a leaf thickness of 2-3 cm. During the spreading process, the initial moisture content of the fresh leaves at 0 h was measured first. Subsequently, samples were taken whenever the moisture content decreased by 3% during spreading. Including the 0 h sample, samples were collected at five time points, named W1, W2, W3, W4, and W5. Details of the changes in fresh leaf moisture content are shown in Table 1. The fresh leaf samples were flash-frozen in liquid nitrogen and stored at -80℃ for subsequent transcriptomics and metabolic assays.

[0033] Table 1. Changes in moisture content of fresh leaves under different spreading conditions

[0034]

[0035] Sample preparation for yellow light withering: Yellow light treatment and a control group were set up, with specific parameters consistent with the conditions of the experiments on different withering degrees described above. Yellow light group: Fresh leaves were evenly spread in a small light-quality withering trough, which contained two yellow LED plant lamps (wavelength 580-595 nm), with the lamps approximately 15 cm away from the fresh tea leaves, and spread until the moisture content of the fresh leaves reached 69%. The control group did not have yellow LED lamps, but all other conditions remained the same. The fresh leaf samples were flash-frozen in liquid nitrogen and stored at -80℃, and then used for transcriptomics and metabolic assays.

[0036] 2. Transcriptome Sequencing: For experimental samples with different spread-out conditions, Beijing Novogene Bioinformatics Technology Co., Ltd. performed reference transcriptome sequencing using the Illumina Hiseq™ 2000 sequencing platform, with three biological replicates per sample. For samples with yellow spread-out conditions, Beijing Biomarker Biotechnology Co., Ltd. performed PE150 sequencing using the Illumina NovaSeq 6000 sequencing platform, with three biological replicates per sample. The latest genome of *Teaplanta spp.* published by Anhui Agricultural University (www.tpia.teaplants.cn) was used as a reference. The sequencing workflow mainly included sample RNA extraction, library construction, and sequencing. After raw data filtering, sequencing error rate checks, and GC content distribution checks, clean reads were obtained for subsequent analysis.

[0037] 3. Differentially expressed gene screening and candidate gene identification: Based on the integrated analysis of metabolomics and transcriptomics, key genes for linalool biosynthesis that simultaneously respond to water loss stress and yellow light induction during the fresh leaf spreading process were screened.

[0038] like Figure 1As shown in Figure A, a systematic analysis of the association between differentially expressed genes and metabolite accumulation revealed a significant positive correlation between changes in linalool content during the spreading process and four differentially expressed genes in the MEP pathway: 1-deoxy-d-xylulose 5-phosphate synthase gene (CSS0020368, r = 0.68, P < 0.01), geranyl pyrophosphate synthase gene (CSS0032883, r = 0.67, P < 0.01), terpene synthase gene (CSS0012706, r = 0.56, P < 0.05, and CSS0038720, r = 0.64, P < 0.05). Further comparison of the content changes of these key genes under yellow light induction showed that the expression levels of geranyl pyrophosphate synthase gene (CSS0032883) and terpene synthase gene (CSS0012706) were significantly increased.

[0039] like Figure 1 As shown in Figure BD, qRT-PCR verification revealed that the expression patterns of the two genes under the two spreading treatments (spreading degree and yellow light irradiation) were relatively consistent with the changes in the corresponding linalool content. When the water content of the spread leaves reached 68%, the expression level of the terpene synthase gene (CSS0012706) was significantly upregulated by 20.5 times compared with the fresh leaves at 0 h (P < 0.001), and it was 1.7 times higher in the yellow light treatment group than in the control group. The terpene synthase gene (CSS0012706) showed a stronger ability to respond to water loss stress, and its yellow light induction effect was the most significant. Therefore, the terpene synthase gene (CSS0012706) was identified as a key candidate gene and named CsTPS13, with its nucleotide sequence shown in SEQ ID NO.3.

[0040] like Figure 2 As shown in Figure A, based on transcriptome data, a total of 1183 differentially expressed transcription factors were identified in samples with different spreading levels, while 27 differentially expressed transcription factors were screened in samples treated with yellow light. Among the differentially expressed transcription factors induced by yellow light, 8 were upregulated and 19 were downregulated, with members of the bHLH (11) and ERF (8) families being the dominant ones.

[0041] Numerous published studies have shown that bHLH family transcription factors play a crucial role in regulating linalool biosynthesis in plants. Combined with expression pattern analysis during the spreading process, it was found that, except for CSS0046630, the remaining bHLH transcription factors exhibited significant differential expression. Figure 2 B).

[0042] Through promoter cis-acting element analysis, an E-box element specifically binding to the bHLH family was identified in the promoter region 1000 bp upstream of the ATG start codon in the CSTPS13 gene.

[0043] 4. Yeast one-hybrid assay to verify transcription factor binding to promoter:

[0044] (1) Construction of the bait vector: The promoter sequence of the CsTPS13 gene (SEQ ID NO.5) was cloned into the pAbAi vector to construct the bait vector pAbAi-Bait. The primer sequences are shown in Table 2.

[0045] Table 2 Primer Sequences

[0046]

[0047] (2) Y1H yeast competent cells were prepared using a classic yeast transformation kit. The bait vector pAbAi-Bait was linearized with BstbI enzyme and then transformed into yeast strain Y1H Gold. Positive clones were screened on SD / -Ura medium.

[0048] (3) Optimal concentration of abacitracin (AbA) for detecting bait yeast strains: Select positive bacteria and add them to 3 mL of SD / -Ura liquid medium, and incubate at 30℃ and 220 r / min for 12 h; resuspend the bacterial culture with 0.9% NaCl solution and adjust OD 600 To a concentration of 0.002; take 100 μL of bacterial culture and spread it on SD / -Ura plates containing different AbA concentrations (0, 100, 150, 200, 300 and 500 ng / mL), and incubate at 30℃ for 2-3 days; observe the growth of bait yeast on plates with different AbA concentrations to determine the optimal AbA concentration.

[0049] (4) Yeast one-hybrid screening library: The library plasmid was transformed into bait yeast strains using the large-scale yeast transformation method. The specific steps include: preparing the bait yeast strain into competent yeast cells; preparing a transformation premix: PEG Solution (1680 μL), 10×LiAc Solution (252 μL), Carrier DNA (40 μL), library plasmid (20 μL), and ddH2O (178 μL); adding 2170 μL of the premix to 600 μL of the competent bait cells and shaking to fully resuspend the cells; incubating in a 30℃ water bath for 50 min, mixing every 10 min; adding 160 μL of DMSO solution and heat-shocking in a 42℃ water bath for 30 min, mixing every 10 min; centrifuging at 4000 r / min for 5 min and discarding the supernatant; resuspending the precipitate with 3 mL of 2×YPDA solution, culturing at 30℃ and 220 r / min for 90 min, centrifuging at 4000 r / min for 5 min, and discarding the supernatant; adding 8 mL of DMSO solution... The bacterial cells were resuspended in 0.9% sodium chloride solution and divided into 50 equal portions. 160 μL of each portion was plated onto SD / -Leu / -Ura / AbA selection medium plates (50 plates) and incubated at 30℃ for 3-5 days. Single colonies of yeast with good growth were picked and subjected to yeast positivity detection using a 50 μL PCR system (2×E-Taq plus PCR Master Mix). 10 μL of the PCR stock solution was subjected to gel electrophoresis, and the remaining 40 μL of the PCR stock solution was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing using T7 primers. The obtained sequences were compared with the NCBI and tea tree genome databases to obtain their annotation information.

[0050] (5) Yeast one-hybrid point-to-point verification: The CDS sequences of the candidate interaction factors were amplified and ligated into the pGADT7 prey vector using homologous recombination. The primer sequences are shown in Table 3.

[0051] Table 3 Primer Sequences

[0052]

[0053] The pGADT7 prey vector containing candidate interaction factors and the empty vector (AD, negative control) were transformed into competent yeast cells used for screening the library, respectively; positive single colonies were picked and cultured, and the yeast culture was resuspended in 0.9% NaCl solution and diluted to OD. 600The concentrations were 0.2 and 0.02, respectively. 5 μL of bacterial suspension was spotted onto SD / -Ura / -Leu medium containing AbA concentrations of 0 and 175 ng / mL, respectively. The bacterial suspension transfected with the pGADT7-AD empty vector was used as a negative control, and p53-AD53 as a positive control. The cultures were incubated upside down at 30°C for 2-3 days, and the growth of blots was observed.

[0054] The results are as follows Figure 3 As shown, under the condition of normal growth of the positive control P53, only yeast cells transformed with CSS0046630 grew normally among the seven candidate transcription factors. The results indicate that CSS0046630 can specifically bind to the CsTPS13 gene promoter and is an upstream regulatory transcription factor of the CsTPS13 gene. CSS0046630 belongs to the bHLH-MYC subfamily, and based on Arabidopsis homology, it is named CsbHLH25.

[0055] Example 2

[0056] This example describes the cloning and sequence analysis of the transcription factors CsbHLH25 and CsTPS13 genes. The specific methods include the following steps:

[0057] 1. Young tea leaves were collected, and total RNA was extracted using a plant RNA extraction kit. First-strand cDNA was synthesized using the PrimerScript RT reagent kit. Specific primer sequences were designed based on gene sequences from the Tea Genome Database (TPIA), as shown in Table 4.

[0058] Table 4 Primer Sequences

[0059]

[0060] 2. Using cDNA as a template, PCR amplification was performed using Phanta® Max Super-Fidelity DNA Polymerase. After the amplification product was detected by gel electrophoresis (1%), the target fragment was excised and purified using an agarose gel purification kit. The purified product was ligated into a circular form with the pTOPO-Blunt Simple Vector intermediate vector and ligated at 25°C for 10 min. The ligation was then transformed into E. coli DH5α competent cells, plated on LB solid medium containing Amp antibiotic, and cultured overnight at 37°C. Positive single clones were picked for colony PCR verification and sequencing.

[0061] Sequencing results showed that the nucleotide sequence of transcription factor CsbHLH25 is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by transcription factor CsbHLH25 is shown in SEQ ID NO.2, encoding a total of 282 amino acids. The nucleotide sequence of the CsTPS13 gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the CsTPS13 gene is shown in SEQ ID NO.4, encoding a total of 545 amino acids; the promoter sequence of the CsTPS13 gene is shown in SEQ ID NO.5.

[0062] Example 3

[0063] This embodiment describes the subcellular localization of transcription factors CsbHLH25 and CsTPS13 genes. The specific method includes the following steps:

[0064] 1. The coding sequences of transcription factors CsbHLH25 and CsTPS13 were cloned into the pCAMBIA2300 vector, respectively. The vectors were constructed using homologous recombination, and double digestion with BamHI and XbaI was performed. Recombinant primers with added homologous arms were designed using appropriate restriction sites. The primer sequences are shown in Table 5.

[0065] Table 5 Primer Sequences

[0066]

[0067] Using a sequenced and accurate cloning vector plasmid as a template, amplification was performed using Phanta Max Super-Fidelity DNA Polymerase. The PCR product was detected by agarose gel electrophoresis, and the target band was recovered and purified. The vector plasmid was linearized with BamHI and XbaI, and the enzyme digestion system consisted of plasmid (500 ng / μL, 10 μL), FastDigest enzyme I / II (2.5 μL), 10×Buffer (5 μL), and ddH2O (30 μL). After incubation at 37℃ for 1 h, the intact plasmid was used as a control, and gel electrophoresis was performed to recover and purify the target fragment. The fragment was then transformed into *E. coli* DH5α competent cells using the ClonExpress II One Step Cloning Kit at 37℃ for 30 min, placed on ice, and cultured overnight at 37℃ on LB agar containing the appropriate antibiotics. Positive single clones were picked for colony PCR verification and then sent for sequencing.

[0068] 2. Using a chemical transformation method, the constructed recombinant plasmid was transformed into commercial Agrobacterium GV3101 competent cells and cultured at 28°C for 2-3 days on LB agar containing the appropriate antibiotic. Positive clones were picked and cultured until the bacterial culture reached OD.600 Once the OD reaches 0.6-0.8, centrifuge at 4000 r / min for 10 min, collect the bacterial cells, and resuspend them in a buffer solution (pH = 5.6) containing 10 mM MES, 10 mM MgCl2, and 150 μM MAS. Dilute the OD. 600 The final concentration was 0.2, and the mixture was left to stand at room temperature for 2 hours. Select healthy tobacco plants (do not water before transformation), remove the needle from a 1 mL syringe, draw up the bacterial solution, and gently inject it into the underside of the tobacco leaves, ensuring the solution completely saturates the entire leaf. Place the tobacco plants in a growth chamber and culture under normal light / dark cycles for 2-3 days. The optimal time for fluorescence observation is 36-48 hours after injection. Observe the tobacco epidermal cells using a fluorescence confocal microscope. The GFP excitation wavelength was 488 nm, the chloroplast fluorescence excitation wavelength was 560 nm, and the detection wavelength was 500-530 nm.

[0069] like Figure 4 As shown, the CsTPS13 protein acts on chloroplasts and has a protein size of 62.76 kDa.

[0070] like Figure 5 As shown, the CsbHLH25 protein is located in the cell nucleus and is a typical nuclear localized transcription factor.

[0071] Example 4

[0072] This embodiment describes the in vitro enzyme activity analysis of CsTPS13 protein. The specific method includes the following steps:

[0073] 1. The vectors used for prokaryotic expression were pET28a(+) (His tag) and pGEX-6P-1 (GST tag). The vector construction method was the same as in Example 3, and the primer sequences are shown in Table 6.

[0074] Table 6 Primer Sequences

[0075]

[0076] After all vectors were sequenced and confirmed to be correct, plasmids were extracted and transformed into Rosetta (DE3) competent cells. The specific steps for protein induction included: scaling up the prepared bacterial culture at a ratio of 2% (total 1 L), and incubating at 37°C and 220 r / min until OD reached. 600The value was 0.6-0.8, approximately 4 h; the bacterial culture was transferred to a shaker at 16 ℃ and 160 r / min and cultured for 30 min. After the bacterial culture cooled, IPTG was added to a final concentration of 0.5 mM, and low-temperature induction was continued for more than 16 h; the induced bacterial culture was enriched and centrifuged at 4 ℃ and 5000 r / min for 15 min; the supernatant was discarded, 15 mL of resuspension was added, and protease inhibitor was added at a ratio of 1:10000; the cells were disrupted using a JN-02C low-temperature high-pressure cell disruptor until the bacterial culture was clear, and then centrifuged at 4 ℃ and 14000 r / min for 1 h; after centrifugation, the supernatant was collected and placed entirely on ice for later use.

[0077] 2. His-tagged proteins were purified using Ni NTA Beads. The specific steps were as follows: Gravity column packing was performed, the column was fixed on an iron stand, and the lower and upper stoppers were removed sequentially, allowing the protective buffer to drain completely. Five column volumes of Lysis buffer were added to the column to equilibrate it. After draining the Lysis buffer, this process was repeated twice. The processed sample was added to the column, and the eluent was collected. Five volumes of Wash buffer were added to the column to wash away non-specifically adsorbed proteins. After draining the Wash buffer, this process was repeated three times, and the eluent was collected. The target protein was eluted using 10 mL of Elution buffer, with 0.5 mL collected per tube.

[0078] 3. GST-tagged proteins were purified using Glutathione Beads. The specific steps are as follows: Equilibrate the packed Glutathione Beads gravity column with 5 column volumes of equilibration buffer to ensure the packing material is in the same buffer system as the target protein. Repeat 2-3 times. Add the sample to the equilibrated gravity column and retain it for at least 2 minutes to ensure sufficient contact between the sample and the medium. Collect the eluent. Repeated loading can be used to increase binding efficiency. Wash with 10-15 column volumes of washing buffer to remove non-specifically adsorbed contaminating proteins and collect the washing buffer. Elute with 5-10 column volumes of elution buffer, collecting fractions, one 0.5 mL tube at a time.

[0079] 4. Detection of purified proteins using SDS-PAGE gels: The specific steps are as follows: Add loading buffer to the collected precipitate, eluent, impurity absorbent, and elution buffer, mix well, and denature the protein at 100℃ for 10 min; centrifuge at 12000 r / min for 5 min at 4℃, and spot the gel with 10 μL of supernatant; electrophoresis at 120 V for approximately 45 min; after gel running, add staining buffer, shake, and stain for 30 min; rinse the stained gel with distilled water, and then destain with staining buffer; identify the target bands by observing the gel, and quantify the protein using the BCA method; store the purified protein at -80℃ for later use, avoiding repeated freeze-thaw cycles.

[0080] 5. In vitro enzyme activity assays were performed using the purified target protein and the substrate geranyyl pyrophosphate (GPP) under suitable reaction conditions. The specific steps are as follows: 1 mL of reaction solution containing 5 mM DTT, 30 mM HEPES, 25 mM MgCl2, 60 μM substrate, and 10 μg of purified protein was prepared in a 25 mL headspace vial; the headspace vial was then sealed and incubated at 30°C for 1 h, followed by incubation at 45°C for 15 min; the extraction head (65 μm PDMS / DVB) was inserted into the reaction vial, and headspace solid-phase microextraction was performed for 30 min; after extraction, the extraction head was inserted into GC-MS for analysis.

[0081] The results are as follows Figure 6 As shown, in vitro enzyme activity experiments indicate that CsTPS13 protein can catalyze the formation of linalool from gerany pyrophosphate (GPP).

[0082] Example 5

[0083] This embodiment describes the effect of CsTPS13 overexpression on linalool in tea. The specific method includes the following steps:

[0084] 1. Using homologous recombination, a pMDC43 overexpression vector was constructed. The primer sequences are shown in Table 7.

[0085] Table 7 Primer Sequences

[0086]

[0087] After vector construction, positive clones were obtained through sequencing. Plasmids were extracted and transformed into Agrobacterium competent cells GV3101 containing the pSoup+p19 plasmid for subsequent transient transformation experiments on tea plants. The specific steps were as follows: The bacteria were propagated by shaking in 400 mL LB liquid medium; the bacterial resuspending was placed in a buffer solution (pH=5.6) containing 25 mM MES, 2 mM Na3PO4, 150 μM AS, and 0.5% D-glucose, and the OD was diluted... 600 To 1.0; the resuspended bacterial solution was activated by placing it in the dark at room temperature for 2-3 hours; for each treatment, 20 tea tree branches with normal and uniform growth (20 cm in diameter) and 3 leaves were selected; holes were made on the back of the leaves of the tea tree branches with a needle, and the branches were immersed in the activated bacterial solution for vacuum permeation treatment at a temperature of 25℃ and a light intensity of 300 μmol / m². 2 At 75% humidity and at a constant temperature, inoculated branches were hydroponically cultured in tea nutrient solution for 6 days before sampling. Positive seedlings were identified using real-time quantitative PCR.

[0088] 2. Extraction of volatiles: Take a positive sample, grind it thoroughly with liquid nitrogen, weigh 200 mg into a 5 mL centrifuge tube, add 1 mL of pre-cooled dichloromethane solution (containing 5 nmol of ethyl decanoate as an internal standard), extract at 4℃, 120 r / min, and in the dark for 12 h, then centrifuge at 4℃, 8000 r / min for 2 min, take the supernatant solution and filter it through a 0.22 μm filter membrane into a brown sample vial for testing.

[0089] 3. GC-MS Analysis: A TSQ8000 triple quadrupole gas chromatograph-mass spectrometer was used with a DB-5MS column (30 mm × 0.25 mm × 0.22 μm). High-purity helium (purity ≥ 99.99%) was used as the carrier gas at a flow rate of 1.0 mL / min. The temperature program was as follows: initial temperature 40℃ (hold for 2 min), increased to 230℃ at a rate of 10℃ / min (hold for 2 min), column oven 40℃, ion source EI, electron energy 70 eV, ion source temperature 230℃, and mass scan range m / z 35-400. The substances were identified using standards, RI values, NIST2014 database, and retention times. The quantification of volatiles was performed using the internal standard method.

[0090] like Figure 7 As shown in Figure A, compared with the empty vector, the expression level of the CsTPS13 gene in the positive tea tree branches was significantly increased, indicating that the positive samples were successfully constructed. Figure 7 As shown in Figure B, the linalool content in tea leaves was significantly increased after overexpression of CsTPS13 compared to the empty vector.

[0091] Example 6

[0092] This example demonstrates how Luciferase can validate the regulation of the CsTPS13 gene promoter by the transcription factor CsbHLH25. The specific method includes the following steps:

[0093] 1. Construction of dual-luciferase experimental vectors: The CsTPS13 gene promoter fragment (SEQ ID NO.5) was inserted into the pGreenII 0800-LUC reporter vector using homologous recombination, serving as the Reporter; the transcription factor was inserted into the pGreenII 62-SK vector, serving as the Effector. The primer sequences are shown in Table 8.

[0094] Table 8 Primer Sequences

[0095]

[0096] After all vectors were constructed, positive clones were obtained through sequencing. Plasmids were extracted, and the recombinant plasmid and the empty vector plasmid were transformed into Agrobacterium competent cells GV3101 containing the pSoup + p19 plasmid, respectively. Agrobacterium was transiently transformed into tobacco as follows: After activation, culture, and collection of Agrobacterium cells, 10 mL of permeate (containing 10 mM MES, 10 mM MgCl2, 150 μM AS, pH 5.6) was added to resuspend the cells. The cells were centrifuged at 4000 r / min for 5 min, the supernatant was discarded, and the cells were collected and resuspended in 5 mL of permeate. The OD of each Effector and Reporter was measured. 600 The OD value was adjusted to 0.8, and then the solution was mixed at an Effector:Reproter ratio of 5:1. The prepared inoculum was activated by incubating in the dark at room temperature for 2-3 hours. 1 mL of the inoculum was injected into the underside of tobacco leaves using a disposable syringe (watering was stopped 2-3 days before injection). Each combination was repeated three times. An empty vector (pGreenII 62-SK) + Effector mixture was used as a control. After injection, the leaves were labeled and returned to the light incubator for further cultivation. Fluorescence signals were detected using a live-cell fluorescence imaging system after 2-3 days. The activities of firefly luciferase (LUC) and reniform luciferase (REN) were detected using the Dual-Luciferase Reporter Assay System kit.

[0097] like Figure 8As shown, compared with the control group, the fluorescence signal of the treatment group co-expressing CsbHLH25 and CsTPS13 promoter fragments was significantly weakened; at the same time, its LUC / REN enzyme activity was significantly reduced. These results indicate that CsbHLH25 exerts an inhibitory effect through its interaction with the CsTPS13 promoter.

[0098] Example 7

[0099] This example demonstrates EMSA verification of the binding of transcription factor CsbHLH25 to the CsTPS13 gene promoter. The specific method includes the following steps:

[0100] 1. Prokaryotic expression and protein purification of transcription factor CsbHLH25 were performed using the same method as in Example 4. Primer sequences are shown in Table 9.

[0101] Table 9 Primer Sequences

[0102]

[0103] The promoter fragment containing the cis-element (SEQ ID NO.5) was synthesized, labeled with biotin at its 5' end, and the forward and reverse strands were annealed to form a double strand as an EMSA probe. Identical and mutated unlabeled oligonucleotides were used as cold competitive probes. The probe sequences are shown in Table 10.

[0104] Table 10 Primer Sequences

[0105]

[0106] EMSA experiments were performed using a chemiluminescence EMSA kit.

[0107] like Figure 9 As shown, the CsbHLH25 fusion protein specifically binds to both E-box probes, forming distinct migration bands; while the GST empty vector protein cannot bind to the labeled probes. Furthermore, the migration bands gradually become lighter with increasing concentrations of the unlabeled competitive probes; simultaneously, the binding signal between the probe and protein is significantly weakened after the addition of the mutant probe. These results indicate that the transcription factor CsbHLH25 can directly bind to the E-box element in the CsTPS13 promoter.

[0108] Example 8

[0109] This example illustrates the effect of CsbHLH25 overexpression on linalool in tea leaves. The construction method of the overexpression vector, Agrobacterium-mediated transient transformation of tea, volatile extraction, and GC-MS detection and analysis methods are the same as in Example 5. Primer sequences are shown in Table 11.

[0110] Table 11 Primer Sequences

[0111]

[0112] like Figure 10 As shown, the expression level of CsTPS13 in tea leaf slices overexpressing CsbHLH25 was significantly downregulated ( Figure 10 B), further confirming the negative regulatory effect of CsbHLH25 on CsTPS13. Further GC-MS analysis revealed that, compared to the empty control, the linalool content in the leaves of positive lines transiently overexpressing CsbHLH25 was significantly reduced (B). Figure 11 These results collectively indicate that CsbHLH25 inhibits linalool biosynthesis by downregulating the expression of CsTPS13.

[0113] Example 9

[0114] This example describes the expression of CsbHLH25 and the analysis of linalool content under yellow light. The specific method includes the following steps:

[0115] 1. Fresh tea leaves (one bud and two leaves) from Fuding Da Bai tea trees were obtained at the Tea Science Teaching Base of Huazhong Agricultural University (Wuhan, Hubei Province) and used as raw materials for the preparation of steamed green tea. After picking, the fresh leaves were spread out under a yellow LED light source, with the leaves not overlapping, until the leaf moisture content dropped to 62% (16 ± 2 h). All LED lamps used had a power of 18 W, a vertical distance of 15 cm between the lamp and the leaf surface, and a leaf thickness of 1 cm. The leaves were not turned during the spreading process to avoid mechanical damage that could lead to reddening. A dark spreading treatment (DDB) was also included as a control.

[0116] 2. The expression levels of CsTPS13 and CsbHLH25 were detected using CsTPS13-F / R (SEQ ID NO.10-11) and CsbHLH25-F / R (SEQ ID NO.14-15);

[0117] 3. The linalool content was determined, following the same steps as in Example 5.

[0118] To further illustrate the technical effects of the present invention, a comparative example is also provided, as follows:

[0119] Comparative Example 1

[0120] The difference between this comparative example and Example 9 is that red light spreading (RDB) is used in this comparative example.

[0121] Comparative Example 2

[0122] The difference between this comparative example and Example 9 is that this comparative example uses orange light spreading (ODB).

[0123] Comparative Example 3

[0124] The difference between this comparative example and Example 9 is that green light spreading (GDB) is used in this comparative example.

[0125] Comparative Example 4

[0126] The difference between this comparative example and Example 9 is that this comparative example uses blue light spreading (BDB).

[0127] Comparative Example 5

[0128] The difference between this comparative example and Example 9 is that this comparative example uses ultraviolet light spreading (PDB).

[0129] like Figure 12 As shown, under the same spreading conditions, compared with the control dark spreading (DDB) and other light-treated groups, the expression level of CsbHLH25 in the yellow light-treated group (YDB) was significantly downregulated. Figure 12 A), while the expression level of CsTPS13 and the content of linalool were significantly increased (A). Figure 12 B and Figure 12 C). Compared with other light treatment groups, the yellow light treatment group showed the greatest downregulation of CsbHLH25, and the highest expression levels of CsTPS13 and linalool content.

[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A tea plant transcription factor CsbHLH25, characterized in that: The nucleotide sequence of the tea tree transcription factor CsbHLH25 is shown in SEQ ID NO.

1.

2. The tea plant transcription factor CsbHLH25 according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the tea tree transcription factor CsbHLH25 is shown in SEQ ID NO.

2.

3. The application of the tea transcription factor CsbHLH25 according to claim 1 or 2 in regulating the synthesis of linalool in tea.

4. The application according to claim 3, characterized in that, The method for regulating linalool synthesis in tea involves spreading fresh tea leaves under light to inhibit the expression of the tea transcription factor CsbHLH25 and promote the expression of the CsTPS13 gene, thereby promoting the synthesis of linalool in tea. The light used is yellow light with a wavelength of 580-595 nm, the vertical distance between the light source and the leaf surface is 10-20 cm, the thickness of the spread tea leaves is 0.5-2 cm, and the spreading time is 14-18 h.

5. The application according to claim 4, characterized in that: The nucleotide sequence of the CsTPS13 gene is shown in SEQ ID NO.

3.

6. The application according to claim 4, characterized in that: The amino acid sequence of the protein encoded by the CsTPS13 gene is shown in SEQ ID NO.4.

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