Tea tree MYB transcription factor CsMYB330 gene and application thereof

Abstract

The invention discloses a tea tree MYB transcription factor CsMYB330 gene and application, the nucleotide sequence of the MYB transcription factor CsMYB330 gene is shown as SEQ ID NO: 1, the expression of the gene participates in regulating the flavonol content in plants, and the instantaneous silencing of the CsMYB330 gene in the tea tree can significantly reduce the flavonol content in the tea tree by 24.5% and reduce the bitter taste of the finished tea.

Description

(I) Technical Field

[0001] This invention belongs to the field of plant genetic engineering, and mainly relates to a tea plant MYB-type transcription factor CsMYB330 gene and its application. (II) Background Technology

[0002] tea tree( Camellia sinensis L. Tea, belonging to the genus *Camellia* of the family Theaceae, is a widely cultivated and important economic crop (HO Chi-tang et al., 2015). Tea possesses a unique taste and health benefits (Yarmolinsky, Jet al., 2015), and along with cocoa and coffee, it is one of the world's most popular non-alcoholic beverages (Zhu Yongxing & Jiang Aiqin, 2010). Studies have shown that flavonoids account for approximately 12%–24% of the dry weight of tea leaves, representing 70%–80% of the total polyphenolic compounds (Wan Xiaochun, 2003). Flavonols, an important branch of flavonoids, are widely found in the leaves of green plants, and their glycosidic forms are the main source of bitterness in tea. Flavonols are not only important factors affecting the quality and flavor of tea but also possess various pharmacological functions. Studies have shown that flavonols possess pharmacological functions such as anti-cancer (Xiong et al., 2018), anti-aging (Yuan et al., 2020), antioxidant (Lwxa et al., 2020), antiviral (Zhang et al., 2021), neuroprotective (Bernier., 2020), immune-regulating (Liu et al., 2019), and lipid-lowering and weight-loss (Yang et al., 2013). Therefore, in-depth research on the biosynthetic mechanism of flavonols in tea is of great significance for improving the quality and health benefits of tea.

[0003] Flavonols are the main coloring components of green tea infusion. Flavonol glycosides in tea leaves are primarily composed of... OFlavonoids in tea are primarily glycosides. Based on the degree of hydroxide oxidation and methyl oxidation at the C-3, C-4, and C-5 positions of the parent ring, they can be classified into kaempferol, quercetin, and myricetin (Wu et al., 2016). According to their aglycone type, flavonoids in tea can be further classified into myricetin glycosides (M-glycosides), quercetin glycosides (Q-glycosides), and kaempferol glycosides (K-glycosides); they can also be classified according to the number of glycosyl aglycones into monosaccharides, disaccharides, and trisaccharides (Fang et al., 2019). Quercetin is the most common and abundant flavonol compound in the diet, widely distributed in various foods. Studies have shown that quercetin content can reach 32.59 mg / 100 g in chili peppers (Capsicum annuum), 12.65~17.22 mg / 100 g in onions (Allium cepa), 4.56 mg / 100 g in tomatoes (Solanum lycopersicum), as high as 40.27 mg / 100 g in lettuce (Lactuca sativa), and only 2.47 mg / 100 g in apples (Malus domestica).

[0004] Flavonol synthesis is synergistically regulated by structural and regulatory genes (GAO GY et al., 2020). Key structural enzymes catalyzing this pathway include chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS), and dihydroflavonol-4-reductase (DFR). Transcription factors such as MYB, bHLH, WD40, and NAC directly bind to the promoters of flavonoid synthesis genes and exert their effects. MYB, bHLH, and WD40 can also interact to form the MBW (MYB-bHLH-WD40) complex, activating or inhibiting gene transcription, thereby regulating the biosynthesis of flavonoid compounds (Song et al., 2020; Tohge et al., 2005; Yan et al., 2021).

[0005] MYB (myeloblastosis) transcription factors are among the most important transcription factor (TF) families, widely distributed in eukaryotes (Dubos C et al., 2010), and play crucial roles in plant growth and development, responses to biotic and abiotic stresses, and the regulation of secondary metabolite synthesis (Niu YL, 2016). Researchers have found that CsMYB5 and CsMYB112 in tea plants positively regulate the expression of flavonol synthesis genes and negatively regulate the expression of anthocyanin synthesis genes. Previous studies have shown that MYB11, MYB12, and MYB111 are activators of flavonoid biosynthetic enzymes (REDDY VA et al., 2017). Different MYB genes play different regulatory roles in the synthesis of different types of flavonoids in tea plants, and further research on the functions of different MYB genes in the flavonoid synthesis pathway in tea plants is of great importance. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a tea plant MYB-type transcription factor. CsMYB330 Genes and their application in regulating the synthesis of flavonols in tea plants, through silencing transcription factors CsMYB330 The expression of this gene can significantly reduce the content of flavonols in tea leaves.

[0007] The technical solution adopted in this invention is:

[0008] In a first aspect, the present invention provides a tea plant MYB-type transcription factor CsMYB330 gene, wherein the largest open reading frame (coding region) of the transcription factor CsMYB330 gene is 879 bp, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0009] Furthermore, the amino acid sequence of the protein encoded by the transcription factor CsMYB330 gene is shown in SEQ ID NO: 2, containing 292 amino acids.

[0010] Secondly, this invention provides a silencing transcription factor CsMYB330 The antisense oligonucleotide sequence for gene expression, as shown in SEQ ID NO: 3 (TTGGAGTTGGCGGACTCACT).

[0011] Thirdly, the present invention provides the aforementioned transcription factor CsMYB330 Application of genes in regulating the synthesis of flavonols in tea plants.

[0012] Furthermore, the application is the silencing of transcription factors. CsMYB330 Gene expression in tea plants reduces the content of flavonols.

[0013] Furthermore, the application involves silencing transcription factors through the antisense oligonucleotide sequence. CsMYB330 Gene expression in tea plants.

[0014] Fourthly, the present invention provides a method for reducing the bitterness of tea, the method being the silencing of transcription factors. CsMYB330 Gene expression in tea plants is silenced by the aforementioned antisense oligonucleotide sequence, silencing transcription factors. CsMYB330 Gene expression in tea plants.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0016] This invention provides a novel tea plant MYB transcription factor. CsMYB330 This gene, whose expression is involved in regulating the flavonol content in plants, is transiently silenced in tea plants. CsMYB330 The gene can significantly reduce the content of flavonols in tea trees by 24.5%, thus reducing the bitterness of the finished tea. (iv) Description of the attached drawings

[0017] Figure 1 This is a comparison diagram of conserved domains of transcription factor CsMYB330.

[0018] Figure 2 This is an evolutionary diagram showing the relationship between transcription factor CsMYB330 and other MYB transcription factors in plants.

[0019] Figure 3 Micrographs showing the subcellular localization of transcription factor CsMYB330 and its control in tobacco leaves.

[0020] Figure 4 Silencing different antisense oligonucleotide sequences CsMYB330 In tea leaves after gene modification and control leaves CsMYB330 The expression level graph.

[0021] Figure 5 Silent for the tea tree CsMYB330 HPLC chromatogram of flavonols in tea leaves after gene expression; AsODN-1, AsODN-2, and AsODN-3 represent three replicates of A5 injection, and CK-1, CK-2, and CK-3 represent three replicates of ddH2O injection; standard represents a 2.5 mg / mL solution of isoquercitrin standard prepared in methanol.

[0022] Figure 6 Silent for the tea tree CsMYB330 A graph showing the flavonol content in tea leaves after gene expression. (V) Detailed Implementation Methods

[0023] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0024] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0025] Example 1: Isolation of transcription factors from tea plants CsMYB330 Genes and Cloning

[0026] 1. Transcription factors CsMYB330 Gene screening

[0027] Previously, the MYB family gene sequences of tea plants were obtained through transcriptome sequencing. CsMYB330 Amino acid sequence alignment of the gene-encoded amino acid sequence with MYB protein sequences from other species revealed that it possesses a conserved domain of the MYB protein. Figure 1 Evolutionary analysis revealed that CsMYB330 is closely related to GtMYBP5 and GtMYBP6, which have the function of regulating flavonoid synthesis. Therefore, it is speculated that CsMYB330 may have the function of regulating flavonoid synthesis in tea trees. Figure 2 ).

[0028] 2. Primer design:

[0029] Amplification was designed based on the sequence of SEQ ID NO: 1. CsMYB330 Full-length specific primers:

[0030] F: 5'-agctttcgcgagctcggtaccATGGGGAGATCACCTTGTTGTG -3';

[0031] R: 5'-cttgcatgcctgcaggtcgacTTATCTATGTAACCCATTTGTGCTGC -3'.

[0032] 3. RNA extraction:

[0033] Using FastPure from Nanjing Novizan Biotechnology Co., Ltd. ® Total RNA was extracted from the leaves of new shoots of 'Zijuan' tea tree using the Universal Plant Total RNA Isolation Kit (polysaccharide and polyphenol plant total RNA extraction kit). RNA integrity was analyzed using agarose gel electrophoresis, and RNA concentration (OD) was determined using an ultra-micro UV-Vis spectrophotometer from Hangzhou Mio Instruments Co., Ltd. 260 / OD 280RNA with a value between 1.8 and 2.1 is considered high-quality RNA. The remaining RNA should be stored at -80°C for later use.

[0034] 4. Reverse transcription to synthesize cDNA:

[0035] The RNA extracted in step 3 was reverse transcribed into cDNA using the HiScript II Q RTSuperMix for qPCR (+gDNA wiper) reagent from Nanjing Novizan Biotechnology Co., Ltd.

[0036] Add the following reagents (Table 1) sequentially to autoclaved RNase-Free centrifuge tubes, mix thoroughly with a pipette, and incubate at 42 °C for 2 min to remove gDNA from the RNA. Add the gDNA-free RNA to the cDNA synthesis reagent (5 × HiScriptⅡqRT SuperMixⅡ from Table 2). Incubate at 50 °C for 15 min, then at 85 °C for 5 sec to obtain the cDNA solution. Store at -20 °C for later use.

[0037] Table 1. Reaction system for gDNA removal

[0038]

[0039] Table 2. Reverse transcription system for cDNA synthesis

[0040]

[0041] 5. PCR amplification:

[0042] Using the cDNA from step 4 as a template, PCR amplification was performed using the primers from step 2. The amplification products were detected by agarose gel electrophoresis. The target gene fragment was purified and sequenced using the FastPure Gel DNA Extraction Mini Kit from Nanjing Novizan Biotechnology Co., Ltd. CsMYB330 The open reading frame is 879 bp, and the nucleotide sequence is shown in SEQ ID NO: 1, which encodes a total of 292 amino acids (amino acid sequence is shown in SEQ ID NO: 2).

[0043] PCR amplification system: cDNA 4 μL, 2×Phanta Max Master Mix (Dye Plus) 25 μL, primer F 2 μL, primer R 2 μL, ddH2O 17 μL.

[0044] PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 30 s, 34 cycles; 72℃ extension for 5 min.

[0045] SEQ ID NO: 1

[0046] ATGGGGAGATCACCTTGTTGTGAGAAAGCTCACACCAACAAAGGCGCCTGGACCAAAGAAGAAGACCAGCGCCTCATCAACTACATCCGCTCTCATGGCGAGGGCTGCTGGCGTTCCCTTCCCAAATCCGCCGGCTTGCTTCGCTGTGGCAAGAGTTGCAGACTCCGGTGGATTAACTATCTCCGTCCTGATCTCAAGAGAGGGAATTTCACTGAACAAGAAGACGAACTCATTATTAAACTTCACAGCATGCTCGGAAACAAATGGTCATTGATTGCTGGAAGATTACCAGGTAGAACCGACAACGAGATCAAGAACTACTGGAACACTCACATCAAGCGAAAACTCATCGTTCGGGGCCTCGATCCCCAAACTCACCGCCCTCTCAACGCCACCGCTTCAACCACCACCGCCAATTCCAAATTTGATTTCAGAAATGCCTCCCACTCCTATTCAACCCTAACTGACATCGATTTGACGATGAATCAAAACACATTCAAGCTTATTGCTATGGCGGACGATTCCTCATTCGACGACGGCAAGTGCAATAGCGGCACCACCGAGGAGGCTCCTCCTCAGCTCCTTCAACCACCACCACTTCTAGCTCATCATTCCGATCAAAACTGCCAATCCATCAATCTCGACCTCTCGATTGGACTCGTTCCGTTCCAATCCCTAACACGGAGTGAGTCCGCCAACTCCAACTCGGCGGAGATTGAGCTAAAGAAGGAGCAGGAGGAGGTGGTTCCGTATCAGTTTGTTGTGGCGGCGAGGCCGTCGGAGACGCCAGCGCCACAGGCGGTGTGTGTGTGTTGGCAGTTGTCGTTTCAGAGAGGTCAATTGTGTAGTAATTGCAGCACAAATGGGTTACATAGATAA

[0047] SEQ ID NO:2

[0048] MGRSPCCEKAHTNKGAWTKEEDQRLINYIRSHGEGCWRSLPKSAGLLRCGKSCRLRWINYLRPDLKRGNFTEQEDELIIKLHSMLGNKWSLIAGRLPGRTDNEIKNYWNTHIKRKLIVRGLDPQTHRPLNATASTTTANSKFDFRN ASHSYSTLTDIDLTMNQNTFKLIAMADDSSFDDGKCNSGTTEEAPPQLLQPPPLLAHHSDQNCQSINLDLSIGLVPFQSLTRSESANSNSAEIELKKEQEEVVPYQFVVAARPSETPAPQAVCVCWQLSFQRGQLCSNCSTNGLHR

[0049] Example 2: Construction of a device for detecting transcription factors CsMYB330 Recombinant plasmids for gene localization in tea plant subcellular structures and transformation

[0050] 1. Constructing recombinant plasmids

[0051] (1) Inserting a fragment

[0052] Using the 'Zijuan' tea cDNA obtained in Example 1 as a template, PCR amplification was performed using universal primers F / R and the amplification system and procedure of Example 1. The inserted fragment was recovered using the FastPure Gel DNA Extraction Mini Kit from Nanjing Novizan Biotechnology Co., Ltd.

[0053] Universal primer F:

[0054] cGagctcGgtaCCCGGggatccATGGGGAGATCACCTTGTTG

[0055] Universal primer R:

[0056] ccttgctcaccatGGTgtcgacTCTATGTAACCCATTTGTGC

[0057] (2) Linearized plasmids

[0058] Using the pCAMBIA2300-GFP plasmid (gifted by Professor Xu Yunmin of Zhejiang Agriculture and Forestry University) as an empty vector, after... BamH I and Kpn I. The plasmid was linearized by double enzyme digestion, and the linearized plasmid was recovered after analysis of the enzyme digestion results by agarose gel electrophoresis.

[0059] Enzyme digestion system: Kpn Ⅰ 2.5 μL BamH I 2.5 μL, 10×QuickCut Green Buffer 5 μL, plasmid n μL, (40-n) μL ddH2O, digested at 37 ℃ for 10 min.

[0060] (3) Recombinant plasmid

[0061] ClonExpress from Nanjing Novizan Biotechnology Co., Ltd. ® II. One Step Cloning Kit: Mix the linearized plasmid from step (2) and the insert fragment from step (1) according to the system in Table 3. React in a PCR instrument at 37 °C for 30 min. Immediately place the reaction tube on ice to cool. The ligation product is the recombinant plasmid.

[0062] Table 3 ClonExpress ® II One Step Cloning Kit Connection System

[0063]

[0064] 2. Transformation of recombinant plasmids into competent E. coli cells and identification of positive clones

[0065] (1) Thawing Escherichia coli DH5α competent cells (Trelief) from Beijing Qingke Biotechnology Co., Ltd. on ice ® 5α Chemically Competent Cell), add 100 μL of competent cells and 10 μL of the recombinant plasmid prepared in step 1 above to a centrifuge tube, gently aspirate and mix (do not shake violently), and let stand on ice for 30 min;

[0066] (2) After heat shock in a metal bath at 42℃ for 45 s, immediately transfer the sample to ice and place it for 2 min (do not shake the sample during this period to avoid reducing the conversion efficiency).

[0067] (3) Add 700 μL of LB liquid culture medium to the centrifuge tube and shake at 37℃ and 200 r / min for 1 h to revive the bacteria;

[0068] (4) Centrifuge the bacterial culture at 5000 r / min for 1 min at room temperature, discard 600 μL of supernatant, resuspend the bacterial cells with the remaining liquid culture medium in the tube, and spread them evenly on LB solid medium containing 50 μg / mL kanamycin; incubate in a 37℃ incubator for 12~16 h.

[0069] (5) Select multiple single clones and use the universal primers F and R from step 1 to perform PCR amplification using Green Taq Mix from Nanjing Novizan Biotechnology Co., Ltd. in accordance with the colony PCR reaction system (Table 4) to screen positive clones.

[0070] PCR amplification program: 95℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 30 s, 35 cycles; 72℃ extension for 5 min.

[0071] Single colonies of positive clones were amplified and sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing. After correct sequence alignment, plasmid was extracted and named pCAMBIA2300-GFP- CsMYB330 Store at -20℃ for later use.

[0072] Table 4 Colony PCR Reaction System

[0073]

[0074] 3. Transformation of Agrobacterium competent cells with recombinant vectors

[0075] (1) Take 100 μL of GV3101 ChemicallyCompetent Cell Agrobacterium tumefaciens cells purchased from Shanghai Angyu Technology Co., Ltd. from a -80℃ freezer and place them on ice to thaw;

[0076] (2) Add 1 μg of the plasmid correctly identified in step 2 (pCAMBIA2300-GFP- CsMYB330 After mixing, place on ice for 5 minutes, quick freeze in liquid nitrogen for 5 minutes, bath in water at 28°C for 5 minutes, and then bath in ice for 5 minutes.

[0077] (3) Add 700 μL of LB liquid medium and incubate at 28°C with shaking for 2-3 hours;

[0078] (4) Centrifuge at 6000 rpm for 1 min, and retain 100 μL of supernatant culture medium to resuspend the cells;

[0079] (5) Spread the bacterial culture evenly on LB solid medium plates containing 50 μg / mL Kana and 50 μg / mL Rif and incubate at 28℃ for 60-72 h;

[0080] (6) Pick the grown Agrobacterium colonies and test them again by PCR to screen for positive clones (PCR system, procedure, and primers are the same as in step 2 above), and label them as 35S:MYB330-GFP. Under the same conditions, pCAMBIA2300-GFP- CsMYB330Replace with pCAMBIA2300-GFP, and the resulting positive clone is denoted as 35S:GFP.

[0081] Example 3: Subcellular localization to determine the functional sites of CsMYB330 protein

[0082] (1) Pick the Agrobacterium positive single colony (35S:MYB330-GFP and 35S:GFP) from Example 2 above and put them into LB liquid medium containing 50 μg / mL Kana and 50 μg / mL Rif. Incubate at 28°C with shaking at 200 rpm for 16-20 h.

[0083] (2) Centrifuge at 5000 rpm for 10 min and discard the supernatant.

[0084] (3) Add the intrusion staining solution (10 mM MgCl2, 10 mM MES (2-morpholinoethanesulfonic acid) and 200 mM acetylsuccinone, solvent: water, pH 5.8) to adjust the OD of the bacterial culture. 600 After adjusting to 0.8, incubate the mixture in the dark at 28°C for 2-3 hours.

[0085] (4) The mixture after dark culture in step (3) was injected into transgenic tobacco expressing the fusion protein of nucleoprotein (H2B) and red fluorescent protein (RFP) using a syringe. Nicotiana benthamiana ) leaf (Song et al., 2024).

[0086] (5) After injection, the cells were cultured in a light incubator at 24°C in the dark for 36-48 hours. The expression location of the CsMYB330 gene was then observed using a confocal microscope. The results are shown in the figure. Figure 3 As shown, the results indicate that the CsMYB330 protein is located in the cell nucleus, indirectly suggesting that it is a transcription factor that may be involved in regulating gene expression.

[0087] Example 4, Tea Tree CsMYB330 Screening and detection of gene silencing antisense oligonucleotide sequences

[0088] 1. Design antisense oligonucleotide sequences:

[0089] Designed using the website https: / / sfold.wadsworth.org / cgi-bin / soligo.pl CsMYB330 The antisense oligonucleotide sequences (A1-A5) of the gene were synthesized at Zhejiang Shangya Biotechnology Co., Ltd., and diluted to 30 μM with ddH2O.

[0090] A1-CCATGAGAGCGGATGTAGTT

[0091] A2- GTCGGTTCTACCTGGTAATC

[0092] A3-TTCGCTTGATGTGAGTGTTC

[0093] A4-GGGATTGGAACGGAACGAGT

[0094] A5-TTGGAGTTGGCGGACTCACT (SEQ ID NO: 3)

[0095] 2. The method of silence:

[0096] Select the fifth leaf from a healthy 'Longjing 43' tea tree free from pests and diseases, and divide it into three groups as three biological replicates, with six leaves in each group. In each group, antisense oligonucleotides and control ddH2O were injected into the left and right halves of the same leaf, respectively. The specific operation method is as follows:

[0097] Draw 30 μM of antisense oligonucleotide solution from step 1 into a 1 mL syringe, and gently inject it onto the underside of the leaf, filling half of the leaf completely. In the same way, inject ddH2O into the other half of the leaf as a control.

[0098] After injection, the leaves were inserted into Hoagland nutrient solution (purchased from Beijing Cooler Technology Co., Ltd., product batch number NSP1020) and cultured at 24℃. After 6 h of culture, the leaves were quick-frozen in liquid nitrogen and stored at -80℃ for further detection and analysis.

[0099] 3. qRT-PCR analysis CsMYB330 Gene expression level

[0100] The tea leaves from step 2 were ground, RNA was extracted, and cDNA was obtained by reverse transcription. Quantitative fluorescence analysis showed that... CsMYB330 Gene expression was significantly suppressed by A5, specifically through the following steps:

[0101] according to CsMYB330 Designing specific primers and internal reference genes based on gene sequences CsPTBPrimers (see Table 5) were used to extract total RNA from tea leaf samples using the RNA prep Pμre polysaccharide and polyphenol plant total RNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The RNA was then reverse-transcribed into cDNA using the MonScript™ RTIII Super Mix with dsDNase reverse transcription kit from Mona Biotechnology Co., Ltd., and diluted 10-fold as a template for qRT-PCR. qRT-PCR reactions were performed using the ChamQ SYBR qPCR Master Mix from Nanjing Novizan Biotechnology Co., Ltd., with each reaction repeated in triplicate. The reaction products were analyzed using melting curves. Data were collected using a real-time quantitative PCR instrument from Jena Analytical Instruments (Beijing) Co., Ltd. CsMYB330 The relative expression level of a gene is represented by 2 times the CT value. -△△CT Method to calculate the number of blades in three treatments CsMYB330 Gene expression, results are shown in Figure 4 The results showed that tea leaves could be injected... CsMYB330 The antisense oligonucleotide A5 can significantly inhibit CsMYB330 The expression is used for subsequent functional analysis.

[0102] The qRT-PCR reaction system is as follows: 2 μL cDNA template, 10 μL 2×SYBR GreenMIX, 10 μmol / L... -1 Forward primer 0.4 μL, 10 μmol L -1 Add 0.4 μL of reverse primer to water until the total volume reaches 20 μL.

[0103] qRT-PCR program: 95℃, 10 min; 95℃, 10 s; 58℃, 10 s; 72℃, 15 s; 35 cycles; 72℃, 20 min.

[0104] Table 5. qRT-PCR detection of tea plants CsMYB330 Primer sequences for gene expression

[0105]

[0106] 4. HPLC analysis of flavonol content

[0107] Weigh the items determined in step 3. CsMYB330Three replicate tea leaf samples (0.2 g each) of gene-silenced (A5 injection) and control CK (ddH2O injection) were extracted in 1.5 mL of 1% HCl-methanol mixture (v:v) at 4℃ in the dark for 24 h, with periodic shaking to ensure complete extraction. After centrifugation at 12000 rpm for 10 min, the supernatant was filtered and placed in a 1.5 mL brown screw-top vial; analysis was performed using a Shimadzu LC-20AT high-performance liquid chromatography (HPLC) instrument. Chromatographic separation was performed using a C18 column (4.6 mm × 250 mm, 5 µm) with an injection volume of 10 µL. Solution A was prepared with Milli-Q water containing 10% (v:v) formic acid, and solution B was prepared with CH3CN containing 0.1% (v:v) formic acid. The linear elution gradient was as follows: 0–20 min, 10% B; 20–25 min, 30% B; 25–35 min, 10% B. Using isoquercitrin as a standard, a 2.5 mg / mL standard solution was prepared with methanol. The chromatographic peak of flavonols was detected at a wavelength of 354 nm. The results are shown in the figure. Figure 5 The flavonol content was calculated using a linear regression equation, and the results are shown below. Figure 6 The results showed that the injection CsMYB330 The flavonol content in tea leaf agarose (A5) containing antisense oligonucleotides was 0.20 mg / g, while the flavonol content in the control group (CK) was 0.27 mg / g. CsMYB330 After gene expression was suppressed, the flavonol content decreased significantly by 24.5%.

[0108] Using isoquercitrin as a standard, a standard solution was prepared with methanol. A standard curve was generated by HPLC. Linear regression analysis was performed using the standard concentration and the corresponding peak area to obtain the linear regression equation y = 18361586x - 506435 (R² = 0.9987), where x is the concentration value (mg / mL) and y is the peak area response value (mAU).

[0109] References: Song SS, Ran WX, Gao LH, Wang YC, Lv WY, Tao Y, Chen L, LiCF. A functional study reveals CsNAC086 regulated the biosynthesis offlavonols in Camellia sinensis. Planta. 259:147, 2024.

Claims

1. A tea plant MYB-type transcription factor CsMYB330 gene, characterized in that, The nucleotide sequence of the transcription factor CsMYB330 gene is shown in SEQ ID NO:

1.

2. The tea plant MYB class transcription factor CsMYB330 gene as described in claim 1, characterized in that, The amino acid sequence of the protein encoded by the transcription factor CsMYB330 gene is shown in SEQ ID NO:

2.

3. A method for silencing the transcription factor of claim 1 CsMYB330 Antisense oligonucleotide sequences for gene expression.

4. The antisense oligonucleotide sequence as described in claim 3, characterized in that, The antisense oligonucleotide sequence is shown in SEQ ID NO:

3.

5. A transcription factor according to claim 1 CsMYB330 Application of genes in regulating the synthesis of flavonols in tea plants.

6. The application as described in claim 5, characterized in that, The application is a silencing transcription factor. CsMYB330 Gene expression in tea plants reduces the content of flavonols.

7. The application as described in claim 6, characterized in that, The application involves silencing transcription factors using the antisense oligonucleotide sequence described in claim 4. CsMYB330 Gene expression in tea plants.

8. A method for reducing the bitterness of tea, characterized in that, The method is to silence transcription factors. CsMYB330 Gene expression in tea plants.

9. The method as described in claim 8, characterized in that, The method involves silencing transcription factors using the antisense oligonucleotide sequence described in claim 4. CsMYB330 Gene expression in tea plants.

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