Application of scutellaria baicalensis SbMYB8 gene in promotion of anthocyanin synthesis

Abstract

The invention discloses application of a scutellaria baicalensis SbMYB8 gene in promotion of anthocyanin synthesis, the application comprises the steps that the SbMYB8 gene is constructed to a plant expression vector and introduced into plant cells to promote synthesis of plant anthocyanin, and the nucleotide sequence of the SbMYB8 gene is shown as SEQ ID NO: 1, or the amino acid sequence coded by the SbMYB8 gene is shown as SEQ ID NO: 2. The MYB transcription factor SbMYB8 gene with the function of promoting anthocyanin synthesis is cloned and identified from the scutellaria baicalensis for the first time, and the blank of a transcription regulation mechanism for regulating anthocyanin metabolism in the scutellaria baicalensis is filled; the SbMYB8 gene can significantly promote accumulation of anthocyanin in various plants, shows good heterologous expression stability and adaptability, and has wide cross-species application potential.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology and relates to the application of the SbMYB8 gene in Scutellaria baicalensis in promoting anthocyanin synthesis. Background Technology

[0002] Anthocyanins are a class of water-soluble pigments widely found in plants, belonging to an important branch of flavonoids. They not only give plants vibrant colors in their flowers, fruits, and leaves, but also play important roles in plant stress resistance, antioxidant activity, UV protection, and pollinator attraction. Meanwhile, due to their significant antioxidant, free radical scavenging, and metabolic regulating activities, anthocyanins are widely used in food, medicine, and health products, possessing significant nutritional and economic value.

[0003] In plants, anthocyanin biosynthesis is primarily regulated by the synergistic action of various structural genes (such as CHS, CHI, F3H, DFR, and ANS) in the flavonoid synthesis pathway. The expression of these structural genes is finely regulated by transcription factors, particularly MYB family transcription factors. The MYB family is one of the largest transcription factor families in plants, with R2R3-MYB transcription factors playing a crucial role in regulating secondary metabolite synthesis. Previous studies have shown that in multiple plant species, including Arabidopsis thaliana, maize, and grape, specific MYB transcription factors can significantly promote anthocyanin accumulation by activating the expression of anthocyanin synthesis genes.

[0004] Scutellaria baicalensis, a plant belonging to the genus Scutellaria in the family Lamiaceae, is one of my country's traditional medicinal herbs, primarily known for its flavonoid components (such as baicalin and baicalein) found in its roots. However, recent studies have also discovered a certain amount of anthocyanins in Scutellaria baicalensis, but their synthetic regulatory mechanisms remain unclear, especially at the transcription factor level where systematic research is lacking.

[0005] Therefore, it is urgent to identify and clone key transcription factors that regulate anthocyanin synthesis from Scutellaria baicalensis, analyze their functions in plants, and explore their potential applications in genetic improvement, functional plant breeding, and efficient synthesis of natural pigments.

[0006] Based on the transcriptome and genome data of Scutellaria baicalensis, the inventors cloned an R2R3-MYB type transcription factor gene, SbMYB8, and verified through transient transformation and stable transgenic systems that it can significantly upregulate the expression of anthocyanin synthesis-related structural genes, promoting anthocyanin synthesis and accumulation. For the first time, the function of the SbMYB8 gene in regulating anthocyanin synthesis in Scutellaria baicalensis and other plants has been clarified, providing new genetic tools and theoretical basis for subsequent plant color improvement, functional enhancement and natural pigment development. Summary of the Invention

[0007] This invention aims to clone and functionally validate the R2R3-MYB transcription factor SbMYB8 gene derived from Scutellaria baicalensis, clarify its function in regulating anthocyanin synthesis in plants, and explore its application value in increasing anthocyanin content in plants. To achieve the above objectives, this invention provides the following technical solution:

[0008] On one hand, the present invention provides an application of the SbMYB8 gene of Scutellaria baicalensis in promoting anthocyanin synthesis. The application includes constructing the SbMYB8 gene into a plant expression vector and introducing it into plant cells to promote the synthesis of anthocyanins in plants. The nucleotide sequence of the SbMYB8 gene is shown in SEQ ID NO:1, or the amino acid sequence encoded by it is shown in SEQ ID NO:2.

[0009] According to the present invention, upregulation of the expression of the above-mentioned SbMYB8 gene can promote the synthesis of anthocyanins in the flavonoid biosynthesis pathway and increase the anthocyanin content of plants.

[0010] According to the present invention, the above-mentioned application includes introducing the SbMYB8 gene into Arabidopsis thaliana or tobacco, and expressing the exogenous gene through genetic transformation, thereby promoting the synthesis of anthocyanins in plants.

[0011] According to the present invention, the above-mentioned plant is Scutellaria baicalensis, Arabidopsis thaliana, or tobacco.

[0012] According to the present invention, the above-mentioned SbMYB8 gene is used to improve the flower color of ornamental plants or the anthocyanin content of functional plants.

[0013] On the other hand, the present invention also provides a recombinant expression vector containing the SbMYB8 gene and capable of being expressed in plant cells, preferably pMDC43.

[0014] On the other hand, the present invention also provides a transgenic plant containing the above-mentioned recombinant expression vector and capable of stably expressing the SbMYB8 gene, thereby significantly increasing its anthocyanin content.

[0015] According to the present invention, the above-mentioned plant is Scutellaria baicalensis, Arabidopsis thaliana, or tobacco.

[0016] On the other hand, the present invention also provides a method for constructing the above-mentioned transgenic plant, characterized in that the method includes the following steps:

[0017] (1) Cloning and synthesizing a DNA fragment containing the SbMYB8 coding sequence;

[0018] (2) The DNA fragment was inserted into the plant expression vector by restriction endonuclease digestion and ligation reaction;

[0019] (3) Transform the expression vector into Agrobacterium;

[0020] (4) Use Agrobacterium-mediated plant genetic transformation to obtain transgenic plants.

[0021] According to the present invention, the above-mentioned plant is Scutellaria baicalensis, Arabidopsis thaliana, or tobacco.

[0022] This invention is the first to clone and identify the MYB transcription factor SbMYB8 gene from Scutellaria baicalensis, which has the function of promoting anthocyanin synthesis, filling the gap in the transcriptional regulatory mechanism of anthocyanin metabolism in Scutellaria baicalensis. The SbMYB8 gene can significantly promote the accumulation of anthocyanins in various plants, showing good heterologous expression stability and adaptability, and has broad cross-species application potential. This gene has important application prospects in plant molecular breeding, industrial biosynthesis of natural pigments, and development of functional plant materials. Attached Figure Description

[0023] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0024] Figure 1 The images show the PCR electrophoresis diagram and nucleotide and amino acid sequences of the Scutellaria baicalensis SbMYB8 gene of this invention. A: PCR electrophoresis diagram; B: nucleotide and amino acid sequences.

[0025] Figure 2 For the bioinformatics analysis of the protein encoded by the SbMYB8 gene of Scutellaria baicalensis in this invention, A: signal peptide prediction; B: hydrophobicity prediction; C: tertiary structure prediction; D: transmembrane peptide prediction.

[0026] Figure 3 For the subcellular localization analysis of the SbMYB8 gene in Scutellaria baicalensis of this invention, A: Schematic diagram of the construction of the 35S::GFP recombinant vector; B: Subcellular localization of the 35S::SbMYB8::GFP fusion protein in tobacco leaves (from left to right: green fluorescence, bright field, superimposed field; scale bar = 50 μm).

[0027] Figure 4 This study analyzes the expression of the SbMYB8 gene in different flower colors of Scutellaria baicalensis, as described in this invention.

[0028] Figure 5To promote anthocyanin synthesis in tobacco through overexpression of the SbMYB8 gene of Scutellaria baicalensis in this invention, the following data are presented: A: Leaf and flower color of tobacco plants overexpressing the SbMYB8 gene; B: Expression level of the SbMYB8 gene in the leaves of wild-type WT and SbMYB8-OE seedlings cultured for 3 months; C: Anthocyanin content determination in wild-type WT and SbMYB8-OE seedlings.

[0029] Figure 6 For the identification of the response gene of Scutellaria baicalensis SbMYB8 gene overexpression in tobacco for the present invention, A: the number of differentially expressed genes (DEG) upregulated and downregulated between OE7 and WT; B: KEGG pathway analysis of differentially expressed genes.

[0030] Figure 7 This invention provides a qRT-PCR analysis of the transcriptional level of anthocyanin synthesis genes in transgenic tobacco. Detailed Implementation

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] 1. Materials

[0035] 1.1 Test Materials

[0036] The Medicinal Botanical Garden of Chengde Medical College in Chengde City, Hebei Province, was selected as the experimental area. Different colored Scutellaria baicalensis flowers were used as test materials, and samples were collected and stored at -80℃ for later use. Wild-type tobacco seeds were obtained using Nicotiana macrophylla, and the cultivation conditions were: temperature 28℃, 16 hours of light / 8 hours of darkness, and light intensity 2500 Lux.

[0037] 1.2 Main Reagents and Instruments

[0038] Gel extraction kit, plasmid extraction reagent, IPTG, X-Gal, peptone, yeast extract, sodium chloride, etc. were all purchased from Shanghai Sangon Biotech. rTaq enzyme (TAKARA), SYBR Premix Ex Taq™ II (Tli RNaseH Plus), Kpn I, Xba I restriction endonuclease, pMD19 (simple) vector and T4 DNA ligase, rifampin, ampicillin, kanamycin, hygromycin, etc. were all purchased from TAKARA. Agrobacterium tumefaciens GV3101 was a strain preserved in our laboratory.

[0039] PCR instrument, ultra-clean workbench (MCV-Bg1SCT), pipette, low-speed centrifuge, high-speed refrigerated centrifuge, electric thermostatic water bath (DK-526), ​​incubator, microwave oven, ice maker (sANYosxM-F-24), water purifier, drying oven, electronic balance, thermostatic heating magnetic stirrer, pH meter (pHS-3TC), ultraviolet analyzer, imaging system, thermostatic incubator shaker, etc.

[0040] 1.3 Primer synthesis and sequencing

[0041] Primer synthesis and sequencing were both completed by Qingke Biotechnology Co., Ltd.

[0042] 2. Methods

[0043] 2.1 Bioinformatics analysis of the SbMYB8 encoded protein from Scutellaria baicalensis

[0044] The physicochemical properties of the SbMYB8 protein, including amino acid number, molecular weight, molecular formula, theoretical isoelectric point, instability index, aliphatic index, hydrophilicity coefficient, and conservation, were predicted using the online ExPAsy tool (https: / / web.expasy.org / protparam). The signal peptide of the SbMYB8 protein was predicted using the SignalP 4.1 Server online website (https: / / services.healthtech.dtu.dk / services / SignalP-4.1 / ). The hydrophilicity / hydrophobicity of the SbMYB8 protein was predicted using the ProtScale online website (https: / / web.expasy.org / protscale / ). The transmembrane structure of the SbMYB8 protein was predicted using the TMHMMv.Server 2.0 online website (http: / / www.cbs.dtu.dk / services / TMHMM / ). The tertiary structure of the SbMYB8 protein was predicted using the online Swiss Model tool (https: / / swissmodel.expasy.org); the domains of the SbMYB8 protein were analyzed using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ).

[0045] 2.2 Construction of Overexpression Vectors

[0046] 2.2.1 Extraction of total RNA from Scutellaria baicalensis (referring to the Hi-Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Dual Column Type) (TSP0202)), the steps are as follows:

[0047] (1) Take 50 mg of plant sample, grind it into a fine powder with liquid nitrogen, and place it in a 1.5 mL pre-cooled centrifuge tube. Immediately add 750 μL of Buffer PR1 to the sample and immediately vortex to mix. Centrifuge at 12,000 rpm (~13,400×g) for 5 min;

[0048] (2) Place the gDNA filter column in the collection tube, transfer the supernatant to the filter column, centrifuge at 12,000 rpm (~13,400×g) for 1 min, discard the gDNA filter column, and aspirate the supernatant from the collection tube into a new centrifuge tube. Avoid contact between the pipette tip and the cell debris in the collection tube.

[0049] (3) Add 0.4 times the volume of supernatant of anhydrous ethanol (usually 300 μL) to the filtrate and pipette 3 to 5 times;

[0050] (4) Place the RNA adsorption column in a collection tube, transfer half of the mixture into the column, and centrifuge at 12,000 rpm (~13,400×g) for 1 min;

[0051] (5) Discard the waste liquid, put the column back into the collection tube, transfer the remaining mixture into the column, and centrifuge at 12,000 rpm (~13,400×g) for 1 min;

[0052] (6) Discard the waste liquid, put the column back into the collection tube, add 650 μL of Buffer PR2 into the column, and centrifuge at 12,000 rpm (~13,400×g) for 1 min;

[0053] (7) Discard the waste liquid, put the column back into the collection tube, add 650 μL of Buffer PR3 into the column, and centrifuge at 12,000 rpm (~13,400×g) for 1 min;

[0054] (8) Discard the waste liquid, put the column back into the collection tube, add 650 μL of anhydrous ethanol into the column, and centrifuge at 12,000 rpm (~13,400×g) for 1 min;

[0055] (9) Discard the waste liquid, reassemble the column into the collection tube, and centrifuge at 12,000 rpm (~13,400×g) for 2 min. Discard the waste liquid and place the column at room temperature for 10 minutes to thoroughly dry any remaining wash liquid;

[0056] (10) Transfer the column to a new centrifuge tube, add 50 μL of RNase-Free ddH2O dropwise to the center of the adsorption membrane, let stand at room temperature for 2 min, and centrifuge at 12,000 rpm (~13,400×g) for 1 min. Store the extracted RNA at -80℃.

[0057] (11) The A260 and A280 values ​​of total RNA were detected by NanoDrop2000, and the integrity of total RNA was detected by agarose gel electrophoresis.

[0058] 2.2.2 Prepare cDNA (10 μl reaction system) according to the following system and method.

[0059] Dissolve the total RNA in the sample and all components in the kit on ice before use.

[0060] Table 1 Reverse Transcription System 1

[0061]

[0062] Incubate at 42℃ for 2 min, then at 60℃ for 1 min, and immediately place on ice after the reaction.

[0063] Table 2 Reverse Transcription System 2

[0064]

[0065] Table 3 Reverse Transcription Procedure

[0066]

[0067] 2.2.3 Primer design for gene sequences

[0068] Based on the nucleotide sequence (864 bp) of the SbMYB8 gene in Scutellaria baicalensis (SEQ No. 1), homologous recombination primers were designed using the In-Fusion HD Cloning online website (https: / / www.takarabio.com / learning-centers / cloning / primer-design-and-other-tools) of Takara Bio Technology (Beijing) Co., Ltd. The primer sequences for SbMYB8 are as follows:

[0069] Table 4 SbMYB8 cloning primers

[0070]

[0071] 2.2.4 PCR amplification reaction

[0072] Using cDNA as a template and the primers designed above, PCR amplification was performed in the following reaction system:

[0073] (1) Prepare the following reaction solution in a 1.5 mL centrifuge tube:

[0074] Table 5 PCR reaction system

[0075]

[0076] (2) Reaction conditions:

[0077]

[0078]

[0079] 2.2.5 Agarose gel electrophoresis detection and gel recovery

[0080] The PCR products were subjected to 1.5% agarose gel electrophoresis using TAE (40 mM Tris-acetate, 1 mM EDTA) as the electrophoresis buffer and EB as the nucleic acid fuel. The electrophoresis results were detected under UV light, and the PCR products were cut and recovered. A DNA gel recovery kit was used for gel recovery, and the procedures are as follows:

[0081] 1) Column equilibration procedure: Add 500g of equilibration solution BL to the CA2 adsorption column (place the adsorption column in the collection tube), centrifuge at 12,000rpm (-13,400×g) for 1min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0082] 2) Cut a single target DNA band from the agarose gel (remove as much excess as possible) and place it in a clean centrifuge tube, then weigh it.

[0083] 3) Add an equal volume of PN solution to the gel block and place in a 50°C water bath, gently turning the centrifuge tube up and down continuously to ensure the gel block dissolves completely. If there is still undissolved gel block, continue to let it stand for a few more minutes or add more sol solution until the gel block is completely dissolved.

[0084] 4) Add the solution obtained in the previous step to an adsorption column CA2 (place the adsorption column in the collection tube), let it stand at room temperature for 2 min, centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, discard the waste liquid in the collection tube, and place the adsorption column CA2 into the collection tube.

[0085] 5) Add 600 PW of washing solution to the CA2 adsorption column (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400 x g) for 30-60 seconds, discard the waste liquid in the collection tube, and put the CA2 adsorption column into the collection tube.

[0086] 6) Repeat step 5.

[0087] 7) Place the CA2 adsorption column back into the collection tube and centrifuge at 12,000 rpm (~13,400 x g) for 2 min to remove as much wash solution as possible. Place the CA2 adsorption column at room temperature for several minutes to dry completely to prevent residual wash solution from affecting the next step of the experiment.

[0088] 8) Place the CA2 adsorption column into a clean centrifuge tube, add an appropriate amount of elution buffer EB dropwise to the center of the adsorption membrane, and incubate at room temperature for 2 min. Centrifuge at 12,000 rpm (~13,400 x g) for 2 min to collect the DNA solution, and store the recovered DNA solution at -20℃.

[0089] 2.2.6 Ligation of the recovered cDNA fragment with the pMDC43 vector

[0090] The pMDC43 vector was linearized by double digestion with Kpn I and Sac I.

[0091] Table 6 Double enzyme digestion system

[0092]

[0093] Mix gently, centrifuge briefly, digest at 37°C for 1 h, then at 80°C for 10 min.

[0094] According to the pMDC43 vector instructions, pMDC43 was ligated to the recovered DNA fragment in a sterile PCR reaction tube. The reaction system is as follows:

[0095] Table 7 Connection System

[0096]

[0097] Mix gently, centrifuge briefly, digest at 37°C for 30 min, and immediately place on ice to cool.

[0098] 2.2.7 Transformation, Screening and Identification

[0099] The recombinant product was transformed into competent E. coli cells, and the specific steps are as follows:

[0100] (1) Thaw DH5α Escherichia coli competent cells on ice.

[0101] (2) Add 10 μL of the recombinant product to 50 μL of competent cells, gently tap the tube wall to mix (do not shake to mix), and let stand on ice for 30 min.

[0102] (3) After being heat-shocked in a 42℃ metal bath for 45 seconds, it was immediately placed on ice to cool for 2 minutes.

[0103] (4) Add 500 μL of LB medium (without antibiotics) and shake at 37°C for 1 h (200 rpm).

[0104] (5) Centrifuge at 5,000 rpm for 5 min and discard 450 μL of supernatant. Resuspend the bacterial culture in the remaining culture medium and gently spread it evenly on an LB agar plate containing the corresponding resistance using a 200 μL pipette tip.

[0105] (6) Incubate upside down in a 37℃ incubator for 12-16 h.

[0106] (7) After E. coli colony PCR detection, positive clones were selected for sequencing verification. The sequencing results were analyzed using software such as BLAST, CLUSTAL, and MEGA4.0.

[0107] 2.2.8 Extraction of plant expression vector plasmids

[0108] Once the sequencing yields a completely correct sequence, plasmids are extracted. The plasmid extraction steps are as follows:

[0109] (1) Column equilibration step: Add 500 BL of equilibration solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (~13,400 x g) for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube (please use a column that has been processed on the same day).

[0110] (2) Take 1-5 ml of overnight cultured bacterial solution, add it to a centrifuge tube, and centrifuge at 12,000 rpm (~13,400 x g) for 1 min using a standard benchtop centrifuge. Try to remove the supernatant (if there is a large amount of bacterial solution, the bacterial precipitate can be collected into a centrifuge tube by multiple centrifugations).

[0111] (3) Add 250 μL of solution P1 to the centrifuge tube containing bacterial pellet (please check first whether RNase A has been added), and use a pipette or vortex mixer to thoroughly suspend the bacterial pellet.

[0112] (4) Add 250 μl of solution P2 to the centrifuge tube and gently invert it 6-8 times to fully lyse the bacteria.

[0113] (5) Add 350 μl of solution P3 to the centrifuge tube, and immediately gently invert it 6-8 times to mix thoroughly. A white flocculent precipitate will appear at this time. Centrifuge at 12,000 rpm (~13,400 x g) for 10 min.

[0114] (6) Transfer the supernatant collected in the previous step to the adsorption column CP3 using a pipette (place the adsorption column in the collection tube), being careful not to aspirate the precipitate. Centrifuge at 12,000 rpm (~13,400 x g) for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CP3 into the collection tube.

[0115] (7) Add 600 μl of washing solution PW to the adsorption column CP3 (please check if anhydrous ethanol has been added first). Centrifuge at 12,000 rpm (~13,400*g) for 30-60 seconds, discard the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

[0116] (8) Repeat step 7.

[0117] (9) Place the adsorption column CP3 into the collection tube and centrifuge at 12,000 rpm (~13,400 x g) for 2 min to remove the residual washing liquid in the adsorption column.

[0118] (10) Place the adsorption column CP3 in a clean centrifuge tube, add 50-100 μl of elution buffer EB to the middle of the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm (~13,400 x g) for 2 min and collect the plasmid solution into the centrifuge tube.

[0119] 2.2.9 Transformation of Agrobacterium

[0120] The plasmid was transformed into Agrobacterium competent cells. The procedure is as follows:

[0121] (1) Take GV3101 Agrobacterium competent cells stored at -80℃ and place them in your palm for a moment until they partially melt. When they are in an ice-water mixture, insert them into ice.

[0122] (2) Add 0.5 μg of plasmid DNA to each 50 μL competent cells, mix by hand by tapping the bottom of the tube, and incubate on ice for 5 min, liquid nitrogen for 5 min, metal bath at 37°C for 5 min, and then place on ice for 5 min.

[0123] (3) Add 500 μL of LB medium (without antibiotics) and shake at 28°C for 3 h (200 rpm).

[0124] (4) Centrifuge at 6,000 rpm for 5 min and discard 400 μl of supernatant. Resuspend the bacterial culture in the remaining culture medium and gently spread it evenly on an LB agar plate containing the corresponding resistance using a 200 μL pipette tip.

[0125] (5) Incubate upside down in a 28℃ incubator for 48-72 h.

[0126] (6) The method for detecting Agrobacterium colonies by PCR is the same as that for Escherichia coli colonies by PCR.

[0127] 2.2.10 Tobacco Genetic Transformation

[0128] 2.2.10.1 Obtaining and Propagating Sterile Seedlings

[0129] (1) Take an appropriate amount of seeds, put them into a sterile 1.5ml centrifuge tube, wash once with sterile water, centrifuge, discard the liquid, add 75% alcohol, and wash for 20 seconds.

[0130] (2) Centrifuge to remove alcohol, wash once with sterile water, add 0.1% HgCl2 and shake for 3 minutes. Centrifuge to remove HgCl2.

[0131] (3) Rinse three times with sterile water.

[0132] (4) Plant the seeds on Tob 1 medium and grow for about 5 weeks before genetic transformation.

[0133] 2.2.10.2 Agrobacterium preparation

[0134] Stir Agrobacterium bacteria stored at -80℃ onto a culture medium (YEB+Rif+Chl+Kan). Incubate at 28℃ upside down for 36–48 hours to activate the Agrobacterium. Pick a plump single colony and add it to a 50ml centrifuge tube containing 5ml of liquid antibiotic medium, and gently shake. Incubate at 28℃ and 200rpm for 16–24 hours with shaking. Take 1ml of the shaken bacterial suspension and inoculate it at a 1:50–100 ratio into an Erlenmeyer flask or culture flask containing antibiotic medium, and shake vigorously. Incubate at 28℃ and 200rpm for 8–14 hours or overnight until OD600 = 1.0–1.2. Transfer the bacterial suspension to a sterile 50ml centrifuge tube and centrifuge at 4000rpm for 9 minutes. Discard the supernatant, add M1 medium, and centrifuge at 4000rpm for 9 minutes. Discard the supernatant, add M2 medium to resuspend the bacterial suspension until the OD600 value is 0.8, and set aside for use.

[0135] 2.2.10.3 Agrobacterium infection

[0136] Take sterile young tobacco leaves, remove the main veins, cut into 3-5 pieces, and make 2-3 random cuts on each piece. Immerse the pieces in the bacterial solution for 20 minutes, shaking them every 2-3 minutes. Place the immersed tobacco leaves on sterile filter paper and blot dry. Lay them flat on Tob2 co-culture medium with the underside facing up, and incubate at 22℃ under low light for 3 days.

[0137] 2.2.10.4 Screening, regeneration, and rooting

[0138] Transfer the leaves to sterile culture flasks and wash them 2-3 times with sterile water + 500 mg / L Carb. Blot dry with sterile filter paper and transfer to selection medium Tob3. Culture conditions: 28℃, 16 hours light / 8 hours dark, light intensity 2500 Lux. Once green callus has formed on the leaf edges and wounds, cut off the callus and transfer to elongation medium Tob4, changing the medium every 3 weeks. Resistant shoots will emerge from the callus tissue. When the shoots elongate to 1-2 cm, transfer them to rooting medium Tob5. Culture conditions: 25℃, 16 hours light / 8 hours dark, light intensity 2500 lux.

[0139] (3) Microbial detection and sequencing:

[0140] Table 8 PCR Reaction System

[0141]

[0142] Single clones that conform to the expected fragment size were verified by bacterial culture PCR. 15-25 clones per sample were sent to Shanghai Sangon Biotech for sequencing.

[0143] 2.3 Transcriptome Sequencing Analysis of Transgenic Tobacco

[0144] 2.3.1 Experimental Materials

[0145] The tobacco was grown at 28℃ with 16 hours of light / 8 hours of darkness and a light intensity of 2500 Lux. Leaves from transgenic plants and wild-type plants with similar growth were selected as experimental samples. After collection, the samples were rapidly flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0146] 2.3.2 Database Construction and Data Analysis

[0147] 2.3.2.1 RNA extraction, purification, and RNA-seq

[0148] RNA extraction was performed using the same method as in 2.1.1. The extracted mRNA was enriched using mRNA Capture Beads; after purification with the magnetic beads, the mRNA was fragmented using high temperature; using the fragmented mRNA as a template, the first strand of cDNA was synthesized in a reverse transcriptase mixture; simultaneously with the synthesis of the second strand of cDNA, end repair and A-tailing were performed; then, adapters were ligated using Hieff. DNA Selection Beads were used to purify and select target fragments; then PCR library amplification was performed. After the constructed sequencing library passed quality control, paired-end sequencing was performed using an Illumina HiSeq 2500 at a sequencing depth of 100×.

[0149] 2.3.2.2 Raw Sequence Data Processing

[0150] Paired-end raw data obtained from high-throughput sequencers may contain adapters (introduced during library construction) and low-quality sequencing data (generated by the instrument itself). To ensure accurate and reliable analysis results, the raw data needs to be preprocessed to obtain valid data for subsequent information analysis. The sequencing data processing steps are as follows:

[0151] (1) Remove reads with adapters;

[0152] (2) Remove reads containing more than 10% N (N indicates that the base information cannot be determined);

[0153] (3) Remove reads that are all A bases;

[0154] (4) Remove low-quality reads (the number of bases with a quality value of Q≤20 accounts for more than 50% of the entire read).

[0155] 2.3.2.3 Reference Genome Alignment

[0156] Gene annotation was performed based on the following databases, and the annotation files were classified and statistically analyzed. In this study, TopHat2 was used to perform genome alignment with gene references on the preprocessed valid data, and the following statistics were compiled based on the gene location information specified by GTF: (1) Reads statistics of sequencing data and reference genome alignment; (2) Regional distribution summary of sequencing data and reference genome alignment, and a comprehensive evaluation was performed.

[0157] Table 9 Sequencing Data Analysis Database

[0158]

[0159] 2.3.2.4 Gene Expression Level Analysis

[0160] In this study, we used FPKM (Fragments Per Kilobase of exon model per Million mapped reads) to statistically analyze the expression abundance of known genes in different samples. FPKM represents the number of sequencing fragments per thousand transcriptome bases per million sequencing bases. After calculating gene expression levels, we used the cuffdiff command in the cufflinks software to perform differential expression analysis on newly constructed genes. Typically, the default threshold for differentially significant genes is: with biological replicates (q < 0.05 or P < 0.05), and without biological replicates (log2 fold change ≥ 1, P < 0.05). In this study, the threshold for differentially expressed genes was set to P < 0.05.

[0161] 2.3.2.5 Gene GO enrichment analysis and KEGG metabolic pathway analysis

[0162] Gene GO functional annotation and functional classification statistics were performed using Blast2GO (https: / / www.blast2go-.com / ) and WEGO (http: / / wego.genomics.org.cn / cgi-bin / wego / index.pl), respectively. GO classification was mainly divided into: biological processes, molecular functions, and cellular components. Both GO annotation and KEGG metabolic network analysis were tested using the Fisher exact test, requiring an error rate correction coefficient (FDR) ≤ 0.05. The MnBBP gene interaction network was analyzed using the online software String (https: / / string-db.org / ).

[0163] 2.4 Determination of total anthocyanins

[0164] 2.4.1 Sample Extraction

[0165] (1) Take a sufficient amount of sample and grind the sample using a grinder.

[0166] (2) Accurately weigh 0.1 g of sample, add 1 ml of 5% formic acid solution, vortex to mix, and extract by ultrasonication.

[0167] (3) Centrifuge at 12000 rpm for 4℃ for 10 min. Collect the supernatant into a new centrifuge tube. Add 1 ml of 5% formic acid solution to the precipitate and continue extraction.

[0168] (4) Repeat the above steps several times until the supernatant is colorless.

[0169] (5) Measure the absorbance at 530 nm, using 5% formic acid solution as a blank.

[0170] 2.4.2 Content Calculation

[0171] Plot the standard curve Y = 49.187x + 0.0426(R) 2 =0.9994), the anthocyanin content in the sample can be calculated according to the standard curve: anthocyanin content (μg / g) = C*V / M, where C is the result calculated according to the standard curve; M is the actual weighed mass; and V is the total volume of the extract.

[0172] 2.5 qRT-PCR analysis of SbMYB8 and anthocyanin synthesis-related genes

[0173] 2.5.1 Using SbMYB8 primers, quantitative real-time PCR analysis was performed on SbMYB8 and anthocyanin synthesis-related genes, with NTCP-23 as the mRNA internal reference gene.

[0174] 2.5.2 SbMYB8 real-time PCR, reaction system (20 μl) is as follows:

[0175] Table 10 qRT-PCR reaction system

[0176]

[0177] The quantitative PCR instrument used was an ABI 7500. Each reaction was performed in triplicate, with the following program: 95℃, 30 s; 40 cycles: 95℃, 10 s; 60℃, 20 s; 70℃, 10 s. After amplification, melting curves were analyzed to verify the single-product identity of the PCR products.

[0178] 2.5.3 Data Processing and Analysis of Quantitative Real-Time PCR

[0179] The preliminary processing of experimental data was performed using Excel 2007. The differential expression fold of each sample was calculated using the 2-Cq method, and the calculation formula is as follows: Ct=(Ct target-Ct control)Sample 2-(Ct target-Ct control)Sample 1. NTCP-23 was used as an internal reference gene to homogenize the expression level of the target gene.

[0180] 2.6 Data Statistics and Analysis

[0181] Data processing and analysis of qRT-PCR data were performed using SPSS 23.0, and Tukey's test was used to compare statistical significance (P < 0.05).

[0182] 3 Results and Analysis

[0183] 3.1 Cloning of the SbMYB8 gene CDS sequence from Scutellaria baicalensis

[0184] Based on differential expression analysis of the transcriptome of Scutellaria baicalensis with different flower colors, the SbMYB8 gene was screened. The gene sequence was obtained using Scutellaria baicalensis genome data and submitted to NCBI for sequence alignment; no similar sequences were identified in other organisms. The protein gene was cloned, and sequencing results showed that the open reading frame of the SbMYB8 gene is 864 bp. Figure 1 A), encoding 288 amino acids ( Figure 1 B).

[0185] 3.2 Bioinformatics analysis of the protein encoded by the SbMYB8 gene of Scutellaria baicalensis

[0186] Analysis of the protein encoded by the SbMYB8 gene of Scutellaria baicalensis revealed a molecular weight of 69454.99 Da and a theoretical isoelectric point of 5.13, classifying it as an acidic protein that carries a negative charge in a neutral environment. The average maximum signal peptide score among the protein's amino acid residues was 0.09545, indicating that the SbMYB8 gene of Scutellaria baicalensis lacks a signal peptide. Figure 2 A). The protein instability coefficient is 57.00, classifying it as an unstable protein; its aliphatic coefficient is 62.14; and its average hydrophilicity coefficient is -0.784, classifying it as a hydrophilic protein. Figure 2 B). Through tertiary structure analysis, it can be determined that ( Figure 2 C) The CrMYB4 protein contains two consecutive SANT domains (R), with a 180° fold between them. This spatial conformation allows it to utilize this folded domain region to bind to the promoter region of target genes, thus exercising its function as a transcription factor. Figure 2 D).

[0187] 3.3 Subcellular localization analysis of the SbMYB8 gene in Scutellaria baicalensis

[0188] The recombinant plasmid 35S::SbMYB8::GFP and the empty vector plasmid 35S::GFP (control) were injected into the lower epidermis of *Nicotiana benthamiana* leaves using Agrobacterium-mediated transformation. The subcellular localization of the SbMYB8 protein in the tobacco leaves was detected using confocal laser scanning microscopy. The results showed that the fluorescence signal of the SbMYB8-GFP fusion protein was localized in the cell nucleus, while the fluorescence signal of the empty vector was distributed in the cell nucleus, cytoplasm, and cell membrane. Figure 3 (A, B). This result indicates that the SbMYB8 gene transcription factor of Scutellaria baicalensis is specifically located in the cell nucleus and performs its biological function within the nucleus.

[0189] 3.4 Expression analysis of the SbMYB8 gene in Scutellaria baicalensis with different flower colors

[0190] To further investigate the expression characteristics of the SbMYB8 gene in Scutellaria baicalensis, its transcriptional level in different flower colors was analyzed by RT-qPCR. The results showed that the expression level of the SbMYB8 gene was highest in pink flowers and lowest in white flowers. Figure 4 The presence of this gene indicates that its expression has a certain tissue specificity, suggesting that the SbMYB8 gene in Scutellaria baicalensis may play a role in flower color formation.

[0191] 3.5 Overexpression of the SbMYB8 gene in Scutellaria baicalensis promotes anthocyanin synthesis in tobacco.

[0192] To determine whether the SbMYB8 gene in Scutellaria baicalensis is involved in anthocyanin synthesis, tobacco was transformed with a vector overexpressing (OE)SbMYB8 for phenotypic observation and identification. In the obtained independent SbMYB8-OE lines, plant phenotypes were observed, and it was found that overexpression of the SbMYB8 gene altered the color of tobacco leaves and resulted in deeper flower color. Figure 5 A). qRT-PCR was used to verify the expression level of the SbMYB8 gene in OE-3 and OE-7 tobaccos, and it was found that the expression level of the SbMYB8 gene in transgenic tobacco was higher than that in wild-type tobacco. Figure 5 B). After culturing wild-type WT and SbMYB8-OE lines in a greenhouse for 3 months, anthocyanin content was measured, and it was found that the SbMYB8 gene overexpression lines of Scutellaria baicalensis were significantly higher than those of wild-type WT plants. Figure 5 C).

[0193] 3.6 Transcriptome analysis of Scutellaria baicalensis SbMYB8 gene transgenic into tobacco

[0194] To explore the mechanism by which the SbMYB8 gene in Scutellaria baicalensis regulates anthocyanin synthesis in plants, we performed transcriptome analysis (RNA-seq) on leaves of WT and OE7 plants. Based on the p-value and the fold change threshold of >2 or <0.5 for FDR <0.05, 1967 upregulated and 768 downregulated differentially expressed genes were identified in the leaves of WT and OE7 plants. Figure 6 A). Differentially expressed genes were analyzed using KEGG, and among the 20 identified pathways, secondary metabolite biosynthesis, flavonoid biosynthesis, phenylalanine biosynthesis, etc., were identified. Figure 6 B).

[0195] 3.7 qRT-PCR analysis of the transcriptional level of anthocyanin synthesis genes in transgenic tobacco

[0196] To further understand the potential biological function of SbMYB8 in anthocyanin biosynthesis in Scutellaria baicalensis, RNA-seq data were used to map the expression patterns of anthocyanin synthesis-related genes in WT and OE7 samples, and qRT-PCR was used to quantitatively analyze the expression of these 13 heterogeneous genes. Quantitative data analysis revealed that the expression of these genes was consistent with the transcriptome data, showing significantly higher expression levels in transgenic tobacco than in wild-type tobacco. Figure 7 (A, B, C)

[0197] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An application of the SbMYB8 gene in promoting anthocyanin synthesis, characterized in that, The application includes constructing the SbMYB8 gene into a plant expression vector and introducing it into plant cells to promote the synthesis of anthocyanins in plants, wherein the nucleotide sequence of the SbMYB8 gene is shown in SEQ ID NO:1, or the amino acid sequence encoded by the SbMYB8 gene is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, Upregulation of the SbMYB8 gene can promote anthocyanin synthesis in the flavonoid biosynthesis pathway and increase the anthocyanin content of plants.

3. The application according to claim 1 or 2, characterized in that, The application includes introducing the SbMYB8 gene into Arabidopsis thaliana or tobacco, and expressing the exogenous gene through genetic transformation, thereby promoting the synthesis of anthocyanins in the plant.

4. The application according to claim 1 or 2, characterized in that, The plant in question is Scutellaria baicalensis, Arabidopsis thaliana, or tobacco.

5. The application according to claim 1, characterized in that, The SbMYB8 gene is used to improve the flower color of ornamental plants or the anthocyanin content of functional plants.

6. A recombinant expression vector, characterized in that, The vector contains the SbMYB8 gene as described in claim 1 and is expressed in plant cells, wherein the vector is preferably pMDC43.

7. A transgenic plant, characterized in that, The transgenic plant contains the recombinant expression vector as described in claim 6 and can stably express the SbMYB8 gene, thereby significantly increasing its anthocyanin content.

8. The transgenic plant according to claim 7, characterized in that, The plant in question is Scutellaria baicalensis, Arabidopsis thaliana, or tobacco.

9. A method for constructing the transgenic plant of claim 7, characterized in that, The method includes the following steps: (1) Cloning and synthesizing a DNA fragment containing the SbMYB8 coding sequence; (2) The DNA fragment is inserted into a plant expression vector by restriction endonuclease digestion and ligation reaction; (3) Transform the expression vector into Agrobacterium; (4) The genetic transformation of plants is mediated by the Agrobacterium to obtain the transgenic plants.

10. The method according to claim 9, characterized in that, The plant in question is Scutellaria baicalensis, Arabidopsis thaliana, or tobacco.

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