Plant transcription factor and application thereof in improving flavonoids

Abstract

This invention provides a plant transcription factor and its application in enhancing flavonoid compounds, belonging to the fields of plant molecular biology and genetic engineering. It solves the technical problems of unclear flavonoid metabolism regulation mechanisms and difficulty in targeted enhancement of active ingredient accumulation levels in existing molecular breeding of Polygonatum cyrtonema. The amino acid sequence of the transcription factor PcMYB51 is shown in SEQ ID NO:2, and the preferred nucleotide sequence of its encoding gene is shown in SEQ ID NO:1. Overexpression of PcMYB51 in plants can significantly activate the flavonoid synthesis pathway. AtF3H , AtDFR , AtANS and AtBAN The expression of the structural gene increases the total flavonoid content in plants. This invention also provides the application of this gene in increasing the flavonoid and anthocyanin content of plants. It provides a core gene resource for the targeted improvement of the medicinal component content of medicinal plants such as Polygonatum multiflorum, and has extremely high application value.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biology and genetic engineering, specifically to a method derived from Polygonatum multiflorum (… Polygonatum cyrtonema The study focuses on the R2R3-MYB transcription factor PcMYB51 and its encoding gene, as well as the application of this gene in increasing the yield of flavonoids. Background Technology

[0002] Polygonatum multiflorum ( Polygonatum cyrtonema *Polygonatum multiflorum* (Hua) is a perennial herb belonging to the genus *Polygonatum* in the family Liliaceae, and is the original species of *Polygonatum* as defined in the Chinese Pharmacopoeia. *Polygonatum multiflorum* possesses both high medicinal and edible value, containing various active ingredients such as polysaccharides, saponins, flavonoids, and alkaloids, exhibiting pharmacological effects including antioxidant, antitumor, anti-aging, and immunomodulatory properties. Among these, flavonoids, especially the anthocyanin branch, are not only the main pigment components determining the color of plant organs (such as stems, fruits, and pedicels), but also important biochemical indicators for evaluating the quality and antioxidant capacity of the medicinal material.

[0003] Anthocyanins, as a class of natural water-soluble pigments, can endow plants with rich colors such as red, purple, and blue, and can effectively scavenge free radicals and protect cells from oxidative damage, resulting in a huge market demand in the health food and natural pigment industries. In plants, the biosynthesis of anthocyanins is finely regulated at the transcriptional level, with R2R3-MYB transcription factors playing a crucial "switch" role. Based on studies of model plants such as Arabidopsis thaliana, members belonging to Subgroup 6 (such as...) AtMYB75 / PAP1 , AtMYB90 / PAP2 It has been confirmed as a typical positive regulator of the anthocyanin synthesis pathway, activating the expression of downstream structural genes.

[0004] However, research on the molecular regulatory mechanisms of Polygonatum multiflorum, an important medicinal plant, is relatively lagging, especially on how to enhance the accumulation of total flavonoids and anthocyanins through endogenous gene regulation, which is still in its early stages. While some color-regulating genes have been identified in fruit trees such as tomatoes and apples, the regulatory efficiency and tissue specificity of these heterologous genes in Polygonatum multiflorum are difficult to predict due to evolutionary differences between species.

[0005] The main active components of Polygonatum multiflorum include flavonoids. Currently, research on the molecular mechanisms regulating flavonoid biosynthesis in Polygonatum multiflorum is insufficient, and regulatory elements capable of efficiently inducing anthocyanin accumulation in plants are lacking.

[0006] Therefore, cloning and identifying the sequence and biological function of PcMYB51, a key transcription factor in the endogenous S6 subgroup of Polygonatum odoratum, is of great theoretical significance and practical application prospects for achieving precise regulation of plant secondary metabolites through genetic engineering, and for directionally improving the content of medicinal components in Polygonatum odoratum and enhancing its industrial development value. Summary of the Invention

[0007] The technical objective of this invention is to solve the technical problems of unclear flavonoid metabolism regulation mechanism and difficulty in targeted improvement of active ingredient accumulation level in existing molecular breeding of Polygonatum cyrtonema. By cloning and identifying Polygonatum cyrtonema-specific R2R3-MYB transcription factor PcMYB51, and utilizing its significant transcriptional activation function, the total flavonoid and anthocyanin content of the plant can be increased exponentially, providing a new strategy that is both practical and convenient for the quality improvement of medicinal plants and the biomanufacturing of natural products.

[0008] To achieve the above-mentioned technical objectives of this invention, the following technical solution is adopted: In a first aspect, the present invention provides a plant transcription factor derived from Polygonatum polyantha (… Polygonatum cyrtonemaHua The nucleotide sequence of the encoding gene of the R2R3-MYB type transcription factor PcMYB51 is selected from any of the following: (a) The nucleotide sequence shown in SEQ ID NO:1; (b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2; or (c) A nucleotide sequence that is ≥95% homologous to the sequence defined in (a) or (b) and encodes a protein having positive regulatory activity in flavonoid synthesis. The transcription factor PcMYB51 is located in the cell nucleus.

[0009] In a second aspect, the present invention provides a recombinant expression vector comprising the PcMYB51 encoding gene and a promoter operatively linked to the gene.

[0010] A third aspect of the present invention provides a genetically engineered host material comprising the recombinant expression vector described above; the host material is selected from transformed Agrobacterium strains, plant cells, isolated plant tissues, callus tissues, or intact plants.

[0011] A fourth aspect of the present invention provides the application of PcMYB51 in enhancing the accumulation of plant active ingredients. This application involves overexpressing PcMYB51 in a recipient plant to increase the total flavonoid content of the plant, accompanied by a simultaneous increase in anthocyanin content. The recipient plant is Arabidopsis thaliana or Polygonatum sibiricum.

[0012] Preferably, the PcMYB51 exerts its transcriptional activation function by directly binding to the promoter region of a flavonoid pathway structural gene, wherein the structural gene includes PcFLS, PcDFR, PcLAR2, PcF3H1, PcF3H3 and PcCHS2 Any one or more of the following.

[0013] In a fifth aspect, the present invention provides a kit for elucidating the regulatory mechanism of flavonoid synthesis in plants, comprising the PcMYB51 encoding gene and reagents for verifying its interaction with a target gene, wherein the target gene is selected from the flavonoid synthesis pathway of Polygonatum odoratum. PcFLS, PcDFR, PcLAR2, PcF3H1, PcF3H3 and PcCHS2 .

[0014] In a sixth aspect, the present invention provides a method for preparing transgenic plants rich in flavonoids, comprising the step of transforming the recombinant expression vector into plant cells or tissues.

[0015] Compared with the prior art, the present invention achieves the following technical effects: Firstly, this invention clones the MYB transcription factor that regulates the biosynthesis of flavonoids in Polygonatum cyrtonema and conducts preliminary research on its regulatory mechanism. This provides gene targets and theoretical basis for improving the gene metabolic engineering of flavonoid bioactive substances in Polygonatum cyrtonema, and offers a new perspective on the comprehensive development and utilization of Polygonatum cyrtonema. It also provides important reference for elucidating the regulatory mechanisms of flavonoid compounds in other medicinal plants.

[0016] Secondly, this invention uses a homology method to screen structural genes involved in the biosynthetic pathway of flavonoids in Polygonatum cyrtonema, clones the full-length promoter, and utilizes yeast one-hybrid and dual-luciferase assays to investigate the target genes and regulatory patterns directly acted upon by PcMYB51. This reveals the mechanism by which PcMYB51 positively regulates the biosynthesis of flavonoids. Attached Figure Description

[0017] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0018] Figure 1 for PcMYB51 A graph showing the expression levels of genes in different tissues of Polygonatum cyrtonema.

[0019] Figure 2 Microscopic image showing the subcellular localization of PcMYB51 in tobacco leaf cells.

[0020] Figure 3 This is a graph showing the analysis of total flavonoids, anthocyanins, and proanthocyanidins in the leaves of transgenic Arabidopsis thaliana.

[0021] Figure 4 This is a graph showing the expression levels of genes related to flavonoid synthesis in transgenic Arabidopsis leaves.

[0022] Figure 5 This is a graph showing the results of yeast monohybridization analysis.

[0023] Figure 6 This is a graph showing the results of a dual-luciferase experiment. Detailed Implementation

[0024] The following are specific embodiments of the present invention, described in conjunction with the accompanying drawings, to further illustrate the technical solutions of the present invention. However, the present invention is not limited to these embodiments. Specific details, such as particular configurations, are provided in the following description merely to aid in a comprehensive understanding of the embodiments of the present invention. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0026] Example 1:

[0027] This embodiment describes the expression pattern and subcellular localization of the PcMYB51 gene in Polygonatum multiflorum, as detailed below: 1. Organizational Expression Analysis: Fresh samples of leaves, stems, and rhizomes of Polygonatum cyrtonema were collected at the same time point and flash-frozen in liquid nitrogen at -80 °C. Full-scale metallurgical analysis was performed. TransZol Total RNA was extracted using a Plant kit. Then, total RNA was extracted using a full-scale gold extraction method. TransScript ® The One-Step gDNARemoval and cDNA Synthesis SuperMix kit was used for reverse transcription. The reaction system was prepared according to the instructions, and the reverse transcription program was set to 42 ℃ for 30 min and 85 ℃ for 5 sec. The specific steps are as follows: Place the CA2 adsorption column into a collection tube, add 500 μL of equilibration buffer BL, centrifuge at 12000 rpm for 1 min, discard the waste liquid, then cut out the target band, place it in a centrifuge tube, add 300 μL of sol-gel buffer PN, incubate at 50 ℃ until the gel block dissolves, then transfer the liquid to the CA2 adsorption column, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, discard the waste liquid. Add 600 μL of wash buffer PW, centrifuge again and discard the waste liquid, repeat the wash once. Centrifuge the empty column for 2 min, open the cap and air dry at room temperature for 5 min. Transfer the adsorption column to a new centrifuge tube, add 30 μL of elution buffer EB, let it stand for 2 min, centrifuge for 2 min to collect the DNA solution, and store at -20 ℃. Based on sequence information from the Polygonatum polyphylla transcriptome data, qRT-PCR primers, qPcMYB51-F (5'-CGCCCTGATCTCAAGCGAGG-3') and qPcMYB51-R (5'-GCGCTTGATGTGCGTGTTCC-3'), and primers for the PcActin internal reference gene, qPcActin-F (5'-GGGCTCATAGACTGATCACAAG-3') and qPcActin-R (5'-TTGTGCTTACCGACCTAATGG-3'), were designed using the NCBI online tool Primer-BLSAT. The results were obtained using a Roche LightCycler 96 instrument. PcMYB51 Expression levels of Polygonatum multiflorum in different tissues. qRT-PCR technology was primarily used by Hieff UNICON. ® Real-time quantitative PCR amplification was performed using Universal Blue qPCRSYBR Master Mix. The PCR reaction program was set to 95 ℃ for 30 sec; 95 ℃ for 3 sec, 60 ℃ for 30 sec, for 40 cycles.

[0028] The results are as follows Figure 1 As shown, PcMYB51 The gene is expressed in leaves, stems, and rhizomes, but the expression levels differ significantly in different tissues. PcMYB51 The expression levels in leaves and stems were significantly higher than in roots, approximately twice the level in roots.

[0029] 2. Subcellular localization: Primers PcMYB51-XhoⅠ-F (5'-CATTTGGAGAGGACACGCATGGGCAGGTCTCCATGCT-3') and PcMYB51-linker-R (5'-GCAGCAGCCTCCTTTGCAGCAGCTTCTGCTACCCATCCTCTAATGGTCTGC-3') were designed using SnapGene software. The PcMYB51 gene was cloned using the synthesized cDNA as a template. The PCR reaction program was set as follows: 98℃ for 30 sec, 98℃ for 10 sec, 55℃ for 5 sec, 72℃ for 5 sec, 35 cycles, 72℃ for 1 min. The cloned PcMYB51 gene was ligated into the pEarleyGate 10-EGFP vector. Agrobacterium GV3101 competent cells were removed from a -80 ℃ freezer and thawed at room temperature to a mixture of ice and water. The cells were then placed in ice, and the recombinant plasmid was added to the freshly thawed competent cells. The mixture was then pipetted several times to mix thoroughly. The cells were then placed in ice for 5 min, flash-frozen in liquid nitrogen for 5 min, incubated in a 37 ℃ water bath for 5 min, and then incubated on ice for 5 min. 500 μL of LB liquid medium was added, and the mixture was incubated at 28 ℃ with shaking for 3 h. The cells were collected by centrifugation at 6000 rpm for 60 sec, and a portion of the supernatant was discarded, retaining approximately 100 μL of liquid. The cells were then resuspended and evenly spread on solid LB plates containing 50 mg / L kanamycin and 25 mg / L rifampicin. The plates were incubated upside down at 28 ℃ for 2-3 days. After colony growth, positive clones were screened and sequenced. Correct sequencing confirmed the successful construction of the recombinant plasmid vector pEarleyGate 101-PcMYB51-EGFP.

[0030] Agrobacterium GV3101 containing recombinant plasmids pEarleyGate 101-EGFP and pEarleyGate 101-PcMYB51-EGFP were injected into leaves of *Nicotiana benthamiana*. The method was as follows: 200 μL of Agrobacterium suspension containing the recombinant plasmid was inoculated into 20 mL of LB liquid medium containing 50 mg / L kan and 25 mg / L Rif, and cultured at 28 ℃ with shaking for 16 h. OD 600The bacterial cell concentration was approximately 1.2-1.5. The cells were collected by centrifugation at 6000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended once in 20 mL of tobacco resuspension (2 mL 0.5 MMES + 1 mL 1 M MgCl2 + 0.1 mL 100 mM As, adjusted to 100 mL with ddH2O), washed once, and centrifuged at 6000 rpm for 10 min to collect the cells, discarding the supernatant. The cells were resuspended in an appropriate amount of tobacco resuspension to achieve an OD600 of approximately 0.5-0.7, and incubated at room temperature in the dark for 2-4 h. The bacterial suspension obtained in step 4 was drawn up with a 1 mL syringe and injected into the lower epidermis of 5-week-old *Nicotiana benthamiana* leaves. Excess bacterial suspension was wiped off the leaf surface after injection. The cells were then incubated in the dark for one day, followed by one day of recovery incubation before fluorescence observation. The results are as follows: Figure 2 The GFP and DAPI signals of the PcMYB51 protein overlap with those in the cell nucleus, indicating that the PcMYB51 protein is located in the cell nucleus, exhibits a green fluorescent signal, and has the general characteristics of a transcription factor.

[0031] Example 2:

[0032] This embodiment verifies the stable overexpression function of the PcMYB51 gene from Polygonatum sibiricum in Arabidopsis thaliana, as detailed below: 1. Arabidopsis transformation Wild-type Arabidopsis thaliana was transformed with the recombinant plasmid pEarlyGate 101-PcMYB51-EGFP carrying the PcMYB51 gene using the inflorescence infection method. Resistant seedlings were obtained by screening on Basta-containing medium. Genomic DNA of transgenic Arabidopsis thaliana was extracted, and PCR identification was performed using Basta resistance gene-specific primers Blp-F (5'-TCAAATCTCGGTGACGGGC-3') and Blp-R (5'-ATGAGCCCAGAACGACGC-3'). A total of 5 PcMYB51 overexpressing transgenic lines were obtained.

[0033] 2. Determination of total flavonoid, anthocyanin and proanthocyanidin content 1) Determination of total flavonoid content Leaves from three-week-old Arabidopsis thaliana were collected, and the fresh samples were flash-frozen in liquid nitrogen at -80 °C for determination of total flavonoid content. Three biological replicates were set up for each line, and each replicate was measured three times. The specific method is as follows: First, freeze-dry the sample, grind it thoroughly through a 40-mesh sieve, accurately weigh 20 mg of powder and place it in a 5 mL centrifuge tube, add 2 mL of 60% ethanol, and extract by shaking at 60℃ for 2 h. Then, centrifuge at 12,000 rpm at room temperature for 10 min, and take the supernatant for testing. Next, accurately weigh 10 mg of rutin standard, dissolve it in 60% ethanol, and make up to 10 mL to obtain a 1 mg / mL rutin standard solution. Use this as the stock solution and dilute it with 60% ethanol to prepare 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg / mL rutin standard solutions for later use. Then, take 1 mL of each concentration gradient rutin standard solution and the supernatant to be tested and place it in a 5 mL volumetric flask, add 0.2 mL of 5% NaNO2, shake well and let stand for 6 min, then add 0.2 mL of 10% Al(NO3)3, shake well and let stand for 6 min, and then add 2 mL of... Add 4% NaOH, and finally dilute to the mark with 60% ethanol. Shake well and let stand for 15 min. After the reaction is complete, take 0.2 mL of liquid and add it to a 96-well microplate. Measure the absorbance of the sample at 510 nm in the microplate reader. Substitute the absorbance of each sample into the equation to calculate the total flavonoid content.

[0034] 2) Anthocyanin content determination Three-week-old Arabidopsis thaliana leaves were collected, and fresh samples were flash-frozen in liquid nitrogen at -80 °C for anthocyanin content determination. Three biological replicates were set up for each strain, and each replicate was measured three times. The specific method is as follows: First, the samples were freeze-dried, thoroughly ground, and passed through a 40-mesh sieve. 100 mg of powder was accurately weighed and placed in a 5 mL centrifuge tube. 1 mL of acidic methanol (80% methanol + 0.16% vitamin C + 0.16% TBHQ + 0.1% hydrochloric acid) was added, and the mixture was extracted at room temperature with shaking for 18 h. Then, the mixture was centrifuged at 12,000 rpm at 4 °C for 1 min. 400 μL of the supernatant was collected, and 600 μL of acidic methanol was added and mixed thoroughly before analysis. Finally, 0.2 mL of the liquid was added to a 96-well microplate, and the absorbance at 530 nm and 657 nm was measured using a microplate reader. The anthocyanin content was calculated using the formula (A...). 530 -0.25×A 657 ) / g.

[0035] 3) Determination of proanthocyanidin content Leaves from three-week-old Arabidopsis thaliana were collected, and fresh samples were flash-frozen in liquid nitrogen at -80 °C. The proanthocyanidin content was determined using the Solarbio Plant Proanthocyanidin (OPC) Content Detection Kit. Three biological replicates were set up for each line, and each replicate was measured three times. The specific method is as follows: First, freeze-dry the sample, grind it thoroughly through a 40-mesh sieve, accurately weigh 100 mg of powder into a 5 mL centrifuge tube, add 1 mL of extraction solution, sonicate at room temperature for 30 min, centrifuge at 12,000 rpm at room temperature for 10 min, and collect the supernatant for testing. Then, dilute the 10 mg / mL proanthocyanidin standard solution with the extraction solution to prepare standard solutions of 4, 2.5, 1.25, 0.625, 0.3125, and 0.15625 mg / mL for later use. Take 40 μL of each concentration gradient proanthocyanidin standard solution into a new centrifuge tube, add 160 μL of working solution as a standard tube, and add 40 μL of ddH2O and 160 μL of working solution to another new centrifuge tube as a blank tube. For each sample to be tested, take two 40 μL portions of the supernatant to be tested into two new centrifuge tubes, add 160 μL of working solution to one portion as the test tube, and add 160 μL of working solution to the other portion as the test tube. ddH2O was used as a control tube; the solution in the centrifuge tube was then vortexed and incubated at 30℃ for 30 min. After the reaction, 0.2 mL of the liquid was added to a 96-well microplate and detected at 500 nm. A standard curve was established with the concentration gradient of proanthocyanidin standard solution as the x-axis and the absorbance value ∆Astandard (∆Astandard = Astandard tube - Ablaze tube) as the y-axis to obtain the regression equation. Then, the absorbance value ∆A of each sample (∆Adetermined = Atest tube - Acontrol tube) was substituted into the equation to calculate the proanthocyanidin content.

[0036] The results are as follows Figure 3 The results showed that, compared with the wild type, overexpression of PcMYB51 significantly increased the content of total flavonoids, anthocyanins, and proanthocyanidins.

[0037] 3. Regulating the expression of structural genes Fresh samples of leaves from three-week-old Arabidopsis thaliana plants were collected and flash-frozen in liquid nitrogen at -80 °C. cDNA transcribed from total RNA in Arabidopsis leaves was used as the sample, and three biological replicates were established. qRT-PCR primers (as shown below) were designed using the NCBI online tool Primer-BLSAT (www.ncbi.nlm.nih.gov / tools / primer-blast / ). Atactin2 As an internal reference gene, based on The relative expression levels of AtCHS, AtCHI, AtF3H, AtFLS, AtDFR, AtANS, and AtBAN were determined using a qRT-PCR method. qRT-PCR was performed using a Roche LightCycler 96 instrument. The PCR reaction program was: 95℃ for 30 sec; 95℃ for 3 sec, 60℃ for 30 sec, for 40 cycles.

[0038] Primer sequences:

[0039] The results are as follows Figure 4 show, PcMYB51 Overexpression in plants AtF3H, AtDFR, AtANS, and AtBAN The expression levels of all of them were significantly increased. AtCHI and AtCHS The expression level did not change significantly. AtFLS The expression level of [a substance] showed an upregulation trend in lines 51-4 and 51-6, but this change did not reach a significant level in line 51-8.

[0040] Example 3:

[0041] This embodiment elucidates the regulatory mechanism of transcription factor PcMYB51 on structural genes involved in flavonoid biosynthesis, as detailed below: 1. Promoter cloning Genomic DNA was extracted from the leaves of *Polygonatum cyrtonema* seedlings grown in soil using a polysaccharide and polyphenol plant genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). The method is as follows: 1) Quickly transfer the fresh leaves of Polygonatum odoratum seedlings grown in soil to a mortar that has been pre-cooled with liquid nitrogen, and grind them thoroughly with a pestle, adding liquid nitrogen in small amounts several times during the process; 2) After grinding, weigh 100 mg of powder and transfer it to a 1.5 mL centrifuge tube. Then quickly add 400 μL GPSBuffer and 10 μL RNase A, vortex to mix, and incubate at 65 °C for 15 min, inverting and mixing several times during the incubation period. Then add 100 μL GPA Buffer and vortex for 1 min. 3) Centrifuge at 12,000 rpm at room temperature for 5 min, and carefully transfer 500 μL of the supernatant to the CS filter column; 4) Centrifuge at 12,000 rpm at room temperature for 1 min, transfer the filtrate to a new 1.5 mL centrifuge tube, add an equal volume of anhydrous ethanol, mix thoroughly, and then transfer the resulting solution and flocculent precipitate together to the CR2 filter column. 5) Centrifuge at 12,000 rpm at room temperature for 1 min, discard the waste liquid, and return the CR2 adsorption column to the collection tube; 6) Add 550 μL of protein removal solution RD to the adsorption column CR2, centrifuge at 12,000 rpm at room temperature for 1 min, discard the waste liquid, and put the adsorption column CR2 back into the collection tube. 7) Add 700 μL of washing solution PW to the adsorption column CR2, centrifuge at 12,000 rpm at room temperature for 1 min, discard the waste liquid, and put the adsorption column CR2 back into the collection tube. 8) Repeat step 7; 9) Centrifuge at 12,000 rpm at room temperature for 2 min, then open the lid and let stand at room temperature for 5 min to allow the rinsing solution to dry as much as possible; 10) Transfer the adsorption column CR2 to a new centrifuge tube, add 50 μL of elution buffer TB to the center of the membrane, let stand at room temperature for 2 min, centrifuge at 12,000 rpm at room temperature for 2 min to collect the DNA solution, and then store at -20 ℃ for later use.

[0042] Based on the genome sequence information of Polygonatum multiflorum, the promoter sequence of the structural gene for flavonoid biosynthesis in Polygonatum multiflorum was obtained. Specific adapter primers for amplifying the full-length promoter sequence were designed using SnapGene software (as shown below). PCR amplification was performed using Novozymes high-fidelity enzyme, with Polygonatum multiflorum genomic DNA as a template.

[0043]

[0044] PCR reaction program settings: 98 ℃ for 30 sec; 98 ℃ for 10 sec, 55 ℃ for 5 sec, 72 ℃ for 15 sec, 35 cycles; 72 ℃ for 1 min. The PCR product was cloned into the pHis2 vector.

[0045] 2. Yeast single-hybrid experiment: Based on the DNA sequence of the promoter of the flavonoid biosynthesis structural gene in Polygonatum cyrtonema, its cis-acting elements were predicted using the PlantCare website (https: / / www.plantcare.co.uk / ). Fragments rich in MYB-related binding sites were selected as bait sequences and ligated into the pHis2 vector. Through PCR amplification, the region rich in MYB-related binding sites was constructed into the pHis2 vector, yielding recombinant plasmids pHis2-CHS1, pHis2-CHS2, pHis2-F3H1, pHis2-F3H2, pHis2-F3H3, pHis2-F3'5'H, pHis2-IFS, pHis2-FLS, pHis2-FNS2, pHis2-DFR, pHis2-LAR2, pHis2-ANS, pHis2-UFGT1, and pHis2-ANR. PcMYB51The recombinant plasmids constructed using the pGADT7 and pHis2 vectors were co-transformed into the Y187 yeast strain using chemical transformation. The transformed yeast cells were first plated on a dual-deficient solid medium and incubated upside down at 30°C for 2-3 days to screen for positive clones successfully co-transformed with both plasmids. Single colonies were picked from the dual-deficient solid medium and inoculated into a dual-deficient liquid medium for overnight incubation. The bacterial culture was then transferred to fresh dual-deficient liquid medium and cultured until OD500. 600 The concentration was approximately 0.2, and it was subsequently diluted 10-fold, 100-fold, and 1000-fold with sterile water. Bacterial suspensions from each dilution were spotted onto triple-free solid medium containing 50 mM 3-AT and incubated upside down at 30°C for 2-3 days. Colony growth was observed. Results are as follows... Figure 5 As shown, PcMYB51 can communicate with PcFLS, PcDFR, PcLAR2, PcF3H1, PcF3H3 and PcCHS2 The promoter sequence binds.

[0046] 3. Dual-luciferase assay Using Polygonatum polyanthampton genomic DNA as a template, the following primers were designed using SnapGene software to clone promoter fragments of genes related to flavonoid biosynthesis (PcCHS1, PcDFR, PcF3H1, PcF3H3, PcANS, PcFLS, PcCHS2, PcLAR2).

[0047]

[0048] The region rich in MYB-related binding sites was constructed into the pGreenII 0800-LUC vector via PCR amplification, yielding recombinant plasmids LUC-ProCHS1, LUC-ProCHS2, LUC-ProF3H1, LUC-ProF3H3, LUC-ProFLS, LUC-ProDFR, LUC-ProLAR2, and LUC-ProANS. Simultaneously, the coding sequence of PcMYB51 was constructed into the pGreenII62-SK vector, yielding the effect plasmid SK-PcMYB51. The recombinant plasmids were transformed into Agrobacterium GV3101 (pSoup). An equal volume mixture of resuspension containing transcription factors and resuspension containing bait sequences was injected into tobacco as the experimental group, while an equal volume mixture of resuspension containing the empty pGreenII 62-SK vector and resuspension containing bait sequences was injected into tobacco as the control group. After injection, tobacco plants were cultured in the dark for 24 h, followed by recovery culture for 24-48 h. The leaves were then coated with D-luciferin potassium salt solution (150 μg / mL), and the LUC fluorescence signal was observed using a chemiluminescence detector.

[0049] Experimental results are as follows Figure 6As shown, PcMYB51 can bind to the promoters of PcDFR and PcF3H1 and activate their expression.

[0050] Those skilled in the art to which this application pertains may modify or supplement the specific embodiments described or use similar methods to replace them, but without departing from the inventive concept of this application or exceeding the scope defined by the appended claims.

Claims

1. A plant transcription factor, characterized in that, Derived from Polygonatum polyflorum ( Polygonatum cyrtonema Hua The nucleotide sequence of the encoding gene of the R2R3-MYB type transcription factor PcMYB51 is selected from any of the following: (a) The nucleotide sequence shown in SEQ ID NO:1; (b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2; or (c) A nucleotide sequence that is ≥95% homologous to the sequence defined in (a) or (b) and encodes a protein having positive regulatory activity in flavonoid synthesis.

2. A recombinant expression vector, characterized in that, It includes the PcMYB51 encoding gene as described in claim 1, and a promoter operatively linked to the gene.

3. A genetically engineered host material, characterized in that, It includes the recombinant expression vector of claim 2; the host material is selected from transformed Agrobacterium strains, plant cells, plant ex vivo tissues, callus tissues, or whole plants.

4. The application of PcMYB51 as described in claim 1 in enhancing the accumulation of plant active ingredients, characterized in that, The application involves overexpressing PcMYB51 in recipient plants to increase the total flavonoid content of the plants, accompanied by a simultaneous increase in anthocyanin content.

5. The application according to claim 4, characterized in that, The PcMYB51 exerts its transcriptional activation function by directly binding to the promoter regions of flavonoid pathway structural genes, which include... PcFLS, PcDFR, PcLAR2, PcF3H1, PcF3H3 and PcCHS2 Any one or more of the following.

6. A kit for elucidating the regulatory mechanism of plant flavonoid synthesis, characterized in that, The product comprises the PcMYB51 encoding gene as described in claim 1, and reagents for verifying its interaction with a target gene, wherein the target gene is selected from the flavonoid synthesis pathway of Polygonatum odoratum. PcFLS, PcDFR, PcLAR2, PcF3H1, PcF3H3 and PcCHS2 .

7. The transcription factor according to claim 1, characterized in that, The transcription factor PcMYB51 is located in the cell nucleus.

8. The application according to claim 4 or 5, characterized in that, The recipient plant is Arabidopsis thaliana or Polygonatum cyrtonema.

9. A method for preparing transgenic plants rich in flavonoids, characterized in that, It includes the step of transforming the recombinant expression vector of claim 2 into plant cells or tissues.

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