Method for improving synthesis of plant kaempferol-3-O-glucoside based on phoebe bournei PbUGT91BA1 gene and application

Abstract

The invention discloses a method for improving synthesis of kaempferol-3-O-glucoside of a plant based on a phoebe bournei PbUGT91BA1 gene and application of the method. The phoebe bournei PbUGT91BA1 gene is successfully transformed into arabidopsis thaliana through an agrobacterium tumefaciens mediated inflorescence infection method, overexpression of the gene is achieved, and molecular biology detection shows that the expression level of the PbUGT91BA1 gene in transgenic arabidopsis thaliana is remarkably higher than that of a wild plant. Furthermore, high performance liquid chromatography technology determination shows that the content of kaempferol-3-O-glucoside in the transgenic plant is obviously increased. According to the application, the phoebe bournei PbUGT91BA1 gene is overexpressed in arabidopsis thaliana for the first time, and a plant with the content of kaempferol-3-O-glucoside remarkably improved is successfully obtained.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology, and specifically relates to a method and application for improving the synthesis of kaempferol-3-O-glucoside in plants based on the PbUGT91BA1 gene of Phoebe bournei. Background Art

[0002] Plant secondary metabolites play important roles in plant growth and development, stress responses, and ecological adaptation. Among them, flavonoids have attracted much attention due to their antioxidant, antibacterial, and plant metabolism-regulating functions. Kaempferol, as an important flavonoid compound, its glycosylated derivatives (such as kaempferol-3-O-glucoside) have significant advantages in plant stress resistance, nutritional value, and medicinal potential. Glycosylation reaction is a key step in the modification of plant secondary metabolites, which is usually catalyzed by glycosyltransferase (GT). Among them, UDP-glycosyltransferase (UGT) is the most widespread class in the GT family, responsible for catalyzing the transfer of glycosyl from a donor molecule to a receptor molecule to form stable glycoside compounds.

[0003] In recent years, the role of UGT genes in the regulation of plant secondary metabolism has been intensively studied. For example, UGT genes related to the glycosylation of flavonoids have been identified in some plants, and these genes significantly affect the biological activity and accumulation of metabolites by catalyzing glycosylation reactions at specific sites. However, current studies on the functions of UGT genes mainly focus on model plants (such as Arabidopsis thaliana) or common economic crops, and little is known about the functions of UGT genes in rare tree species (such as Phoebe bournei). Phoebe bournei, as a unique subtropical evergreen tree in China, has important economic, ecological, and medicinal values. Its leaves, wood, and secondary metabolites are widely used in furniture manufacturing, landscaping, and traditional medicine. Nevertheless, the key genes and molecular mechanisms regulating the glycosylation of flavonoids in Phoebe bournei have not been fully elucidated, especially the research on the synthesis of kaempferol-3-O-glucoside is still blank.

[0004] In the prior art, although some UGT genes have been proven to increase the content of specific glycoside compounds in plants, most of these studies rely on heterologous expression systems in model plants and lack in-depth exploration and functional verification of genes specific to non-model plants (such as Phoebe bournei). In addition, the application of existing UGT genes mostly focuses on the regulation of single metabolic pathways, making it difficult to systematically increase the yield of target compounds, and there are technical bottlenecks in genetic transformation and metabolic engineering applications in rare tree species. Therefore, it is urgent to identify and utilize the key genes related to kaempferol-3-O-glucoside synthesis in Phoebe bournei and develop efficient metabolic regulation methods to increase the content of secondary metabolites in Phoebe bournei and its related plants, thereby expanding their application potential in the fields of medicine, ecology, and economy.

[0005] Based on the transcriptional regulation study of the key regulator PbMYB211 of kaempferol glycosides in Phoebe bournei, the present invention screened the gene PbUGT91BA1 related to kaempferol-3-O-glucoside synthesis. By heterologously transforming Arabidopsis thaliana and performing high-pressure liquid chromatography determination, it provides a theoretical basis for the study of glycosylation modification of secondary metabolites in Phoebe bournei. Summary of the Invention

[0006] To solve the above problems, the present invention provides a method and application for increasing the synthesis of kaempferol-3-O-glucoside in plants based on the PbUGT91BA1 gene of Phoebe bournei.

[0007] The present invention provides a PbUGT91BA1 gene of Phoebe bournei, and this gene is:

[0008] a) The nucleotide sequence shown in SEQ ID No:1; or

[0009] b) A nucleotide sequence that has one or more nucleotides substituted, deleted, or added in the nucleotide sequence shown in SEQ ID No.1 and has the function of catalyzing the synthesis of kaempferol-3-O-glucoside.

[0010] The present invention also provides the protein encoded by PbUGT91BA1 of Phoebe bournei, and this protein is composed of the amino acids shown in SEQ ID No.2.

[0011] The present invention also provides a method for increasing the content of kaempferol-3-O-glucoside in plants, and this method includes:

[0012] a) Constructing an expression vector containing the PbUGT91BA1 gene of Phoebe bournei;

[0013] b) Transforming the expression vector into plant cells;

[0014] c) Cultivate the plant cells to obtain transgenic plants, wherein the transgenic plants overexpress the Phoebe bournei PbUGT91BA1 gene, thereby increasing the content of kaempferol-3-O-glucoside.

[0015] In one embodiment, the above transformation is carried out by an Agrobacterium tumefaciens-mediated method.

[0016] In one embodiment, the above plant is Phoebe bournei or Arabidopsis thaliana.

[0017] The present invention provides the application of increasing the synthesis of kaempferol-3-O-glucoside in plants based on the Phoebe bournei PbUGT91BA1 gene. Prepare a modified plant containing the Phoebe bournei PbUGT91BA1 gene. Compared with the wild-type plant, in the modified plant, the expression of the Phoebe bournei PbUGT91BA1 gene is increased; wherein, the nucleotide sequence of the Phoebe bournei PbUGT91BA1 gene is as shown in SEQ ID NO: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process by which a gene undergoes transcription and translation to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression. Specifically, it can be an increase in the products obtained by transcription and translation.

[0018] In one embodiment, the above plant is Phoebe bournei or Arabidopsis thaliana.

[0019] In one embodiment, the above plant is Arabidopsis thaliana.

[0020] In one embodiment, the content of kaempferol-3-O-glucoside in the above transgenic plants is significantly higher than that in non-transgenic control plants.

[0021] The present invention also provides a transgenic plant, which contains the Phoebe bournei PbUGT91BA1 gene, and this gene is overexpressed in the transgenic plant, resulting in a higher content of kaempferol-3-O-glucoside than that in non-transgenic control plants.

[0022] The present invention successfully transformed the Phoebe bournei PbUGT91BA1 gene into Arabidopsis thaliana by the Agrobacterium tumefaciens-mediated floral dip method, realizing the overexpression of this gene. Molecular biology detection shows that the expression level of the PbUGT91BA1 gene in transgenic Arabidopsis thaliana is significantly higher than that in wild-type plants. Further determination using high-performance liquid chromatography (HPLC) technology shows that the content of kaempferol-3-O-glucoside in transgenic plants has increased significantly. This application overexpresses the Phoebe bournei PbUGT91BA1 gene in Arabidopsis thaliana for the first time and successfully obtains plants with a significantly increased content of kaempferol-3-O-glucoside, fully demonstrating the feasibility of artificially increasing the content of kaempferol-3-O-glucoside in plants by genetic engineering means. Description of the Drawings

[0023] Figure 1 Electrophoresis diagram of total RNA from the roots and leaves of Phoebe bournei in the present invention.

[0024] Figure 2 Electrophoresis diagram of PCR amplification of the PbUGT91BA1 gene of Phoebe bournei in the present invention.

[0025] Figure 3 Electrophoresis diagram of PCR detection of overexpressed plants of the PbUGT91BA1 gene of Phoebe bournei in the present invention.

[0026] Figure 4 Standard curve of kaempferol-3-O-glucoside in the present invention.

[0027] Figure 5 Histogram of kaempferol-3-O-glucoside content in Arabidopsis thaliana overexpressing the PbUGT91BA1 gene of Phoebe bournei in the present invention. Detailed implementation manners

[0028] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0030] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] 1 Materials

[0032] 1.1 Experimental materials

[0033] The Phoebe bournei materials selected are 1.5-year-old seedling container plants of the "Wuyuan No. 8" family.

[0034] Arabidopsis thaliana uses the Columbia (Columbia-0) wild type, which is cultured in the growth chamber of the Intelligent Experiment Building of Zhejiang A&F University, and the growth conditions are 25°C with 16 h / d of light.

[0035] 1.2 Experimental reagents and instruments

[0036] Experimental reagents: DL2000 DNA Marker, 10× Loading Buffer, and DNA gel extraction kit were purchased from Baori Biotechnology (Beijing) Co., Ltd.; M5 Plant RNeasy Complex Mini Kit RNA extraction kit and M5Hipure Next III Gelred nucleic acid dye were purchased from Beijing Polymer Beauty Biotechnology Co., Ltd.; 2× TransStartFastPfu PCR SuperMix, M5 Hiper ultra-fast mix, and pEASY-Blunt Zero Cloning Kit were purchased from Beijing TransGen Biotech Co., Ltd.; chemically competent cells such as DH5α and GV3101 were purchased from Shanghai Weidi Biotechnology Co., Ltd.; reverse transcription kit, quantitative PCR detection kit, and seamless cloning kit were purchased from Nanjing Novoprotein Scientific Inc.; 2× Easy Taq PCR SuperMix was purchased from Zhejiang Biosciences Co., Ltd.; restriction endonucleases were purchased from NEB (Beijing) Co., Ltd.

[0037] Experimental instruments: NanoDrop2000 ultra-micro spectrophotometer, micro centrifuge, basic electrophoresis apparatus, Bio-Rad Gel Doc XR gel imager, Bio-Rad S1000 PCR thermal cycler, CFX96 real-time fluorescence quantitative PCR system, micropipette (Eppendorf), laminar flow hood, water bath, ice maker, constant temperature shaking incubator and incubator, palm centrifuge, high-speed centrifuge, electronic balance, etc.

[0038] 1.3 Primer synthesis and sequencing

[0039] Both primer synthesis and sequencing were completed by Zhejiang Youkang Biotechnology Co., Ltd.

[0040] 2 Methods

[0041] 2.1 Total RNA extraction from Phoebe bournei

[0042] The M5 Plant RNeasy Complex Mini Kit was used to extract the total RNA from the roots and leaves of Phoebe bournei, and the steps were as follows:

[0043] (1) Take 1 mL of lysis buffer CLB into a centrifuge tube (if CLB has precipitation or sediment, it needs to be redissolved in a 65 °C water bath first), and add 5% β-mercaptoethanol to the lysis buffer CLB (50 μL of β-mercaptoethanol is added to 1 mL of CLB). After inverting and mixing evenly, preheat in a 65 °C water bath;

[0044] (2) Take about 0.1 g of the sample and put it into a mortar pre-cooled with liquid nitrogen, and grind it into a fine powder with liquid nitrogen;

[0045] (3) Transfer 100 - 200 mg of the fine powder to a centrifuge tube containing preheated lysis buffer CLB (already added with β-mercaptoethanol). Immediately vortex vigorously for 30 - 60 s or pipette up and down to mix until a satisfactory homogenate is obtained;

[0046] (4) Briefly place it back in a 65 °C water bath for 5 - 10 min, and occasionally invert it 1 - 2 times in the middle to assist in lysis;

[0047] (5) Centrifuge the lysate at 13,000 rpm for 10 min to precipitate the non-lysed fragments;

[0048] (6) Take the supernatant of the lysate (more supernatant can be taken if it does not exceed the capacity of the genomic DNA removal column, which can increase the yield) and transfer it to a new centrifuge tube. Add anhydrous ethanol (0.5 volume) equal to half of the volume of the supernatant. At this time, precipitation may occur, but it does not affect the extraction process. Immediately pipette up and down to mix, without centrifugation;

[0049] (7) Add the mixture (less than 720 μL each time, can be added in two portions at most) to a genomic DNA removal column, centrifuge at 13,000 rpm for 2 min, and discard the waste liquid;

[0050] (9) Place the genomic DNA removal column in a clean 2 mL centrifuge tube. Add 500 μL of lysis buffer RLT Plus to the genomic DNA removal column, centrifuge at 13,000 rpm for 30 s, collect the filtrate, accurately estimate the volume of the filtrate with a micropipette (usually about 450 - 500 μL, and the volume lost during filtration should be subtracted), add 0.5 volume of anhydrous ethanol, immediately pipette up and down to mix, without centrifugation;

[0051] (10) Immediately add the mixture (less than 720 μL each time, can be added in two portions at most) to an adsorption column RA (the adsorption column is placed in the collection tube), centrifuge at 13,000 rpm for 2 min, and discard the waste liquid;

[0052] (11) Add 700 μL of deproteinization solution RW1, let it stand at room temperature for 1 min, centrifuge at 13,000 rpm for 30 s, and discard the waste liquid;

[0053] (12) Add 500 μL of wash solution RW, centrifuge at 13,000 rpm for 30 s, and discard the waste liquid. Add 500 μL of wash solution RW and repeat once;

[0054] (13) Place the adsorption column RA back into the empty collection tube, centrifuge at 13,000 rpm for 2 min to remove as much wash solution as possible;

[0055] (14) Take out the adsorption column RA and place it in an RNase free centrifuge tube. Add 30 - 50 μL of RNase-Free H2O (heating in a 70 °C water bath in advance can increase the yield) to the middle part of the adsorption membrane according to the expected RNA yield. Let it stand at room temperature for 1 min and centrifuge at 12,000 rpm for 1 min;

[0056] (15) If the expected RNA yield > 30 μg, add 30 - 50 μL of RNase-Free H2O and repeat step 9. Combine the two washings, or use the first eluate and add it back to the adsorption column to repeat the step once.

[0057] 2.2 Synthesis of the first strand of reverse transcribed cDNA

[0058] The first strand of reverse transcribed cDNA of Phoebe bournei RNA (Total RNA) was synthesized with reference to the instructions of PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa).

[0059] (1) Prepare the following mixture for genomic DNA removal reaction:

[0060]

[0061] The reaction program is: 42 °C, 2 min; 4 °C, hold.

[0062] (2) Prepare the following mixture for reverse transcription reaction:

[0063]

[0064] The reaction program is: 37 °C, 15 min; 85 °C, 5 sec; 4 °C, hold.

[0065] 2.3 Cloning of the target gene

[0066] 2.3.1 Gene cloning

[0067] Design specific primers for PbUGT91BA1 (Table 1), and the amplification system and program are shown in Table 2.

[0068] Table 1 Cloning primers for PbUGT91BA1

[0069]

[0070] Table 2 Cloning system

[0071]

[0072] Reaction procedure: 98°C, 30 sec, 52°C, 5 sec, 72°C, 1 min, cycle 35 times; 72°C, 1 min; 16°C, hold.

[0073] 2.3.2 Recovery of target fragment

[0074] Prepare 1% agarose gel, detect the above PCR products by agarose gel electrophoresis. If the bands are correct, cut and recover the target fragment referring to the instructions of MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian).

[0075] (1) Prepare a sterilized 2 mL centrifuge tube and weigh the mass of the empty tube;

[0076] (2) Use a clean scalpel to cut out the gel block containing the target fragment under ultraviolet light, crush the gel block and put it into a 2 mL centrifuge tube, weigh and calculate the volume of the gel block (based on the standard of 1 mg = 1 μL);

[0077] (3) Add 3 times the gel volume of Buffer GM to the gel block, dissolve the gel block at room temperature, and mix intermittently by shaking;

[0078] (3) When the gel is completely dissolved, add isopropanol with a final concentration of 20%;

[0079] (4) Add the solution from the previous step to the Spin Column adsorption column, place it on the Collection Tube, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0080] (5) Add 700 μL of washing buffer Buffer WB to the adsorption column, centrifuge at 12000 rpm at room temperature for 1 min, and discard the waste liquid;

[0081] (6) Repeat step (5);

[0082] (6) Centrifuge empty at 12000 rpm for 1 min;

[0083] (7) Place the adsorption column in a sterilized 1.5 mL centrifuge tube, add 30 μL of sterilized water (preheated to 65°C) to the adsorption membrane, let it stand at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and store the collected liquid at -20°C.

[0084] 2.3.3 Ligation and transformation of target fragment

[0085] (1) Ligation: Use pEASY Blunt Zero vector (Transgene, -Blunt ZeroCloning Kit) and ligate it with the target gene. Mix the following solutions, gently mix well, and centrifuge briefly. PCR: 25 °C, 30 min;

[0086]

[0087] (2) Transformation: Take the competent cells of Escherichia coli Trans T1 (Transgene, Beijing). After thawing on ice, add 2 μL of the above ligation product, place it on ice for 30 min, heat shock in a 42 °C water bath for 30 s, then quickly and smoothly transfer the centrifuge tube to the ice bath for 2 min. Add 500 μL of LB medium without antibiotics, and culture it at 37 °C on a shaker at 200 rpm for 1 h. Centrifuge at 4000 rpm for 2 min, remove part of the supernatant, leave 100 μL of the bacterial solution, resuspend the cells by pipetting, and spread it on solid LB medium (containing 50 mg·mL -1 Kana), and incubate it upside down at 37 °C for 12 h;

[0088] (3) Bacterial detection: Pick white single colonies on the plate, add 500 μL of liquid LB medium (containing 50 mg·mL -1 Kana), and culture it at 37 °C on a shaker at 200 rpm for 3 - 5 h. Take 1 μL of the bacterial solution as a template for PCR detection. The primers are gene cloning primers, and the system and procedure are as follows:

[0089]

[0090] The reaction procedure is: 94 °C, 5 min; 94 °C, 30 sec, 52 °C, 30 sec, 72 °C, 1 min, cycle 35 times; 72 °C, 5 min; 16 °C, hold;

[0091] (4) Detect the PCR product of the bacterial solution by agarose gel electrophoresis, and select the positive clones to be sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing;

[0092] (5) After the sequencing results are correctly aligned by SnapGene, use the Transgene EasyPure Plasmid MiniPrep Kit to extract the plasmid, and use the obtained positive plasmid to construct the expression vector of the PbUGT91BA1 gene.

[0093] 2.3.4 Construction of the expression vector using homologous recombination method

[0094] According to II One Step Cloning Kit to construct the expression vector by homologous recombination method.

[0095] (1) First, add the linker sequences of pK2W7-eYGFPuv-3xFLAG and pYES2 to the specific primers of the target gene. Using the positive plasmid obtained in the previous step as a template, prepare the following PCR system (20 μL):

[0096]

[0097] Reaction program: 98 °C, 30 sec, 54 °C, 5 sec, 72 °C, 1 min, cycle 35 times; 72 °C, 5 min; 16 °C, hold;

[0098] (2) After ensuring that the product is a single band, refer to the MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian) instruction manual to cut and recover the target fragment from the gel;

[0099] (3) Double-digest the overexpression vector pK2W7-eYGFPuv-3xFLAG for linearization. Using the pK2W7-eYGFPuv-3xFLAG plasmid as a template, configure the following PCR system:

[0100]

[0101] Reaction program: 37 °C, 2 h; 16 °C, hold;

[0102] (4) Refer to the MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian) instruction manual to cut and recover the target fragment from the gel;

[0103] (5) The overexpression vector selected for genetic transformation is pK2W7-eYGFPuv-3xFLAG, and an overexpression vector of the PbUGT91BA1 gene is constructed. Prepare the following mixed solution and incubate at 25 °C overnight;

[0104]

[0105]

[0106] Optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol); Optimal amount of insert fragment used = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol);

[0107] Reaction program: 37 °C, 30 min; Cool to 4 °C or immediately place on ice for cooling;

[0108] (6) Take 2 μL for transforming Trans T1 (Transgene, Beijing) Escherichia coli competent cells, and then pick monoclonal colonies for bacterial liquid PCR detection. The primers are gene cloning primers, and the PCR system is as follows:

[0109]

[0110] The reaction program is: 94°C, 5 min; 94°C, 30 sec, 52°C, 30 sec, 72°C, 1 min, cycle 35 times; 72°C, 5 min; 16°C, hold;

[0111] (7) Detect the PCR products by agarose gel electrophoresis. After transforming into Trans T1 (Transgene, Beijing) Escherichia coli competent cells, select positive clones and send them to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. Extract the plasmids of the strains with correct sequencing results for later use.

[0112] 2.4 Transformation of Agrobacterium tumefaciens GV1301 by the liquid nitrogen method

[0113] (1) Take out the Agrobacterium tumefaciens competent cells stored at -80°C and thaw them on ice;

[0114] (2) Take 0.5 μg of the expression vector plasmid and add it to the Agrobacterium tumefaciens competent cells. After gently flicking to mix evenly, place it on ice, in liquid nitrogen, in a 37°C water bath, and in an ice-water mixture for 5 min each in turn;

[0115] (3) Add 700 μL of antibiotic-free YEP liquid medium to the centrifuge tube and incubate the bacterial liquid in a shaker (28°C, 200 rpm) for 4 - 6 h;

[0116] (4) Centrifuge at 6000 rpm for 2 min, pour off some of the supernatant, and leave about 70 - 100 μL of the remaining liquid, then pipette it to mix evenly;

[0117] (5) Spread the bacterial liquid evenly on YEP solid medium containing antibiotics (50 mg·mL -1 Spec and 50 mg·mL -1 Rif), and incubate it upside down in a 28°C biochemical incubator for 48 - 72 h;

[0118] (6) Pick monoclonal colonies on the plate for bacterial liquid PCR detection. The primers are gene cloning primers, and the PCR system is as follows:

[0119]

[0120]

[0121] The reaction procedure is as follows: 94°C, 5 min; 94°C, 30 sec, 46°C, 30 sec, 72°C, 20 sec, for 30 cycles; 72°C, 5 min; 16°C, hold;

[0122] (7) Add an equal volume of 50% glycerol to the monoclonal bacterial solution with a positive detection result, and store it in a -80°C refrigerator.

[0123] 2.5 Transformation of Arabidopsis thaliana by Agrobacterium-mediated floral dip method

[0124] Take out the Agrobacterium bacterial solution carrying the target gene vector from the -80°C refrigerator, streak it on a plate for activation, pick a monoclonal colony and pipette it into an EP tube containing 1 mL of YEP (containing 50 mg·mL -1 of Rif and 50 mg·mL -1 of Spec) liquid medium, and culture it on a shaker at 28°C for 16 h. Take 500 μL of the bacterial solution and transfer it to a 500 mL conical flask containing 200 mL of the same medium, and continue to culture it on a shaker at 28°C until the OD is between 0.6 and 0.8. Add 10 g of sucrose and 80 μL of silwet-L77 to 200 mL of the bacterial solution. Transfer the infection solution to a container with a large opening and low height, and use a syringe to aspirate and discharge until a large amount of foam is generated. Immerse the inflorescence of Arabidopsis thaliana with the fruit pods removed in the bacterial solution for 1 min, then take it out and absorb the excess bacterial solution on the surface with a tissue paper. Wrap it with plastic wrap to keep it moist, and place it flat in the growth chamber for dark culture for 2 d. Then carry out normal light culture, and carry out secondary infection after one week. Collect the mature seeds in batches, dry them at 37°C for 5 d, and store them in a 4°C refrigerator for later use.

[0125] 2.6 Identification of positive transgenic Arabidopsis thaliana

[0126] Use the M5 Hiper superluminal mix reagent to identify positive Arabidopsis thaliana plants, and the method is as follows:

[0127] (1) Take out the "Best Amplification Partner" and let it reach room temperature. Observe whether there is precipitation before use. Make sure that there is no precipitation in the lysis buffer, otherwise dissolve it at 37°C until it is clear before use;

[0128] (2) Cut a 2-square-millimeter leaf of transgenic Arabidopsis thaliana and put it into 20 μL of lysis buffer. Melt a yellow pipette tip with a flame to form a "grinding pestle" and crush the leaf tissue;

[0129] (3) Treat it at 98°C for 5 min in a PCR instrument;

[0130] (4) Centrifuge at 12,000 rpm for 2 min, take 2 μL as the PCR template, and the reaction system is as follows:

[0131]

[0132] The reaction procedure was as follows: 95°C for 3 min; 94°C for 25 s, 60°C for 25 s, 72°C for 1 min, with 34 cycles; 72°C for 5 min.

[0133] 2.7 Total flavonoid extraction and determination by high performance liquid chromatography (HPLC)

[0134] 2.7.1 HPLC parameter settings

[0135] The high performance liquid chromatography column used was a ZORBAX Eclipse XDB-C18 column (4.6 mm × 250 mm inner diameter 5 mm Agilent); the ultraviolet detector selected was a DVD detector; the flow rate was set at 1 mL / min; the detection wavelength was set at 276 nm; the column temperature was set at 35°C; the injection volume was set at 10 μL.

[0136] 2.7.2 Standard curve preparation

[0137] Take the kaempferol-3-O-glucoside standard (ChemFaces), dissolve it with methanol and dilute it to concentrations of 12.5, 25, 50, 100, and 200 μg / mL. Take 1 mL and pass it through a 0.22 μm microporous filter membrane into a liquid phase vial. According to the parameter settings in 2.7.1, use high performance liquid chromatography to obtain the peak area, then use the standard concentration as the abscissa and the peak area as the ordinate to plot the standard curve, obtain the linear regression equation, and judge its linear relationship according to the R of the linear regression equation. 2 Judge its linear relationship.

[0138] The mobile phase used was acetonitrile (A) and 0.2% acetic acid aqueous solution (B), and the linear gradient was set as follows: 0 - 10 min, 5% A to 24% A; 10 - 35 min, 24% A to 24% A.

[0139] 2.7.3 Total flavonoid extraction and detection

[0140] Freeze-dry the plant sample and grind it thoroughly. Weigh 0.1 g of the sample powder precisely into a 2 mL EP tube, add 2 mL of 80% methanol solution, extract it by ultrasonic wave at 55°C for 30 min, centrifuge it at 8000 rpm for 10 min, take the supernatant and pass it through a 0.22 μm microporous filter membrane into a liquid phase vial, then according to the parameter settings in 2.2.5.1, use high performance liquid chromatography to measure the peak area of the target product, and substitute it into the standard curve to calculate the concentration of the target product.

[0141] 3. Experimental results

[0142] 3.1 Total RNA extraction and analysis of the roots and leaves of Phoebe bournei

[0143] In this study, the M5 Plant RNeasy Complex Mini Kit was used to extract total RNA from the roots and leaves of Phoebe bournei, respectively. During the extraction process, the operation was carried out strictly according to the kit instructions. First, cells were rapidly lysed with lysis buffer, and an appropriate amount of β-mercaptoethanol was added to prevent RNA degradation. After centrifugation, column purification, and elution, the extracted RNA samples were quantitatively detected using a UV spectrophotometer. The OD260 / 280 ratios of all samples were maintained between 1.8 and 2.1, indicating that the content of proteins and other contaminants in the samples was low and the RNA purity was high. Meanwhile, agarose gel electrophoresis was used to detect RNA. The electrophoresis images showed that the two bands of 18S and 28S rRNA were clear and there was no obvious degradation phenomenon ( Figure 1 ), providing high-quality RNA samples that met the requirements for subsequent reverse transcription and gene cloning experiments.

[0144] 3.2 Cloning and vector construction of the PbUGT91BA1 gene sequence from Phoebe bournei

[0145] Using the total cDNA obtained by reverse transcription of the above-extracted total RNA as a template, specific primers were used to perform PCR amplification of the PbUGT91BA1 gene. The PCR amplification results showed a target band approximately 1404 bp long in the electrophoresis gel ( Figure 2 ), and the length of this band was consistent with the expectation, proving that the selected primers had good specificity. Subsequently, the PCR product with vector adaptors was ligated to the overexpression vector pK2W7-eYGFPuv-3xFLAG using homologous recombination technology. After the ligation reaction was completed, the recombinant product was transformed into Escherichia coli, and monoclonal screening was performed, followed by sending the samples to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the obtained cloned sequence was completely identical to the target PbUGT91BA1 gene, without mutations, deletions, or insertion errors. After extracting the correctly sequenced monoclonal plasmids, they were transformed into Agrobacterium tumefaciens GV3101, and the Agrobacterium was cultured to obtain a sufficient amount of bacterial liquid for subsequent Arabidopsis thaliana transformation.

[0146] 3.3 Identification of transgenic Arabidopsis thaliana positive plants with the PbUGT91BA1 gene sequence

[0147] Using the Agrobacterium tumefaciens-mediated floral dip method, Agrobacterium carrying the PbUGT91BA1 overexpression vector was transformed into Arabidopsis thaliana plants. After treatment, multiple transgenic positive plants were obtained by screening the collected Arabidopsis thaliana seeds, and the stable integration of the transgene was verified by PCR detection ( Figure 3 ). Molecular detection results showed that the expression level of the PbUGT91BA1 gene in transgenic plants was significantly higher than that in wild-type plants, indicating that the target gene was successfully introduced and highly expressed in Arabidopsis thaliana.

[0148] 3.4 Establishment of HPLC standard curve and content detection of kaempferol-3-O-glucoside

[0149] In order to quantitatively analyze the content of kaempferol-3-O-glucoside, the standard was first dissolved in methanol, and a series of standard solutions with a concentration gradient of 12.5, 25, 50, 100, and 200 μg / mL were prepared. The high performance liquid chromatography (HPLC) system was used for detection under the conditions of a ZORBAX Eclipse XDB-C18 column, a flow rate of 1 mL / min, a column temperature of 35°C, and a detection wavelength of 276 nm. The peak area of each concentration sample was recorded, and a standard curve was drawn with concentration as the horizontal axis and peak area as the vertical axis ( Figure 4 ). The experimental results show that the peak time of kaempferol-3-O-glucoside is about 14.6min, the regression equation of its standard curve is y=13.345x+21.756, and the correlation coefficient R 2 =0.9999, showing excellent linear relationship and quantitative accuracy.

[0150] Subsequently, the Arabidopsis samples that had been freeze-dried and ground and ultrasonically extracted with 80% methanol were pretreated and filtered through a 0.22μm filter membrane before HPLC detection. The data results showed that compared with the wild-type (WT) plants, the peak areas of kaempferol-3-O-glucoside (OE1, OE2, OE3) in transgenic Arabidopsis were significantly increased ( Figure 5 ), and the content was calculated by combining with the standard curve. This result proves that overexpression of PbUGT91BA1 gene in Phoebe thaliana can significantly promote the synthesis of kaempferol-3-O-glucoside in Arabidopsis, indicating the important regulatory role of this gene in the biosynthesis of 3-O-glycosylation.

[0151] In summary, this application systematically verifies the key role of the gene in regulating the synthesis of kaempferol-3-O-glucoside by cloning, constructing vectors, obtaining transgenic Arabidopsis thaliana and molecular identification of the PbUGT91BA1 gene of Phoebe fujianensis. The experimental results not only show that it is feasible to increase the content of kaempferol-3-O-glucoside by genetic engineering, but also provide a theoretical basis and practical foundation for further exploring the regulatory mechanism of plant secondary metabolism and developing transgenic plants with high economic benefits.

[0152] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Phoebe bournei PbUGT91BA1 gene, characterized in that, The gene is as follows: a) The nucleotide sequence shown in SEQ ID No:1; or b) A nucleotide sequence with one or more nucleotides substituted, deleted or added in the nucleotide sequence shown in SEQ ID No.1 and having the function of catalyzing the synthesis of kaempferol-3-O-glucoside.

2. The protein encoded by the gene according to claim 1, characterized in that, The protein consists of the amino acids shown in SEQ ID No.

2.

3. A method for increasing the content of kaempferol-3-O-glucoside in plants, characterized in that, The method includes: a) Constructing an expression vector containing the gene described in claim 1; b) Transforming the expression vector into a plant cell; c) Culturing the plant cell to obtain a transgenic plant, wherein the transgenic plant overexpresses the gene, thereby increasing the content of kaempferol-3-O-glucoside.

4. The method according to claim 3, characterized in that, The transformation is carried out by a method mediated by Agrobacterium tumefaciens.

5. The method according to claim 3 or 4, characterized in that, The plant is Phoebe bournei or Arabidopsis thaliana.

6. Application of improving the synthesis of kaempferol-3-O-glucoside in plants based on the Phoebe bournei PbUGT91BA1 gene, characterized in that, Preparing a modified plant containing the Phoebe bournei PbUGT91BA1 gene, in which the expression of the Phoebe bournei PbUGT91BA1 gene is increased as compared with the wild-type plant; wherein, the base sequence of the Phoebe bournei PbUGT91BA1 gene is as shown in SEQ ID NO:

1.

7. The application according to claim 6, wherein The plant is Phoebe bournei or Arabidopsis thaliana.

8. The application according to claim 7, wherein The plant is Arabidopsis thaliana.

9. The application according to claim 6, wherein The content of kaempferol-3-O-glucoside in the transgenic plant is significantly higher than that in the non-transgenic control plant.

10. A transgenic plant, characterized in that, The transgenic plant contains the gene described in claim 1, and the gene is overexpressed in the transgenic plant, resulting in a higher content of kaempferol-3-O-glucoside than that in the non-transgenic control plant.

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