Kaempferol-3-O-rutinoside biosynthesis regulation and control method based on phoebe bournei PbUGT92AE1 gene and application of kaempferol-3-O-rutinoside biosynthesis regulation and control method

Abstract

The invention discloses a biosynthesis regulation and control method of kaempferol-3-O-rutinoside based on a phoebe bournei PbUGT92AE1 gene and application of the biosynthesis regulation and control method. According to the application, the phoebe bournei PbUGT92AE1 gene is successfully introduced into arabidopsis thaliana through an agrobacterium tumefaciens mediated inflorescence infection method, and a gene overexpression plant is obtained. Molecular detection results show that PbUGT92AE1 in a transgenic plant is remarkably and highly expressed, and the PbUGT92AE1 is remarkably different from a wild type. Furthermore, high performance liquid chromatography quantitative analysis is utilized to find that the content of kaempferol-3-O-rutinoside in the transgenic plant is obviously higher than that of the wild type. According to the application, the phoebe bournei PbUGT92AE1 gene is overexpressed in arabidopsis thaliana for the first time, so that the synthesis of kaempferol-3-O-rutinoside is effectively regulated and controlled, and the feasibility of improving the content of the secondary metabolite through a genetic engineering means is verified.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology and relates to a method for regulating the biosynthesis of kaempferol-3-O-rutinoside based on the PbUGT92AE1 gene of Phoebe bournei and its application. Background Art

[0002] Phoebe bournei belongs to the genus Phoebe in the Lauraceae family and is a subtropical evergreen tall tree. This species is not only a national second-class protected plant and precious timber tree species unique to China, but also an important original tree species of "golden nanmu". Phoebe bournei has a straight trunk, hard wood, and a unique fragrance. Its wood is commonly used in high-end furniture, handicrafts, and carving, with extremely high economic and ornamental values. In addition, the leaves of Phoebe bournei are evergreen throughout the year and the tree has a beautiful shape, making it an ideal tree species for roadside trees, landscape trees, and courtyard greening. At the same time, it has important ecological functions in fire prevention, soil fixation, and windbreak, and is suitable for constructing shelterbelts.

[0003] In plants, glycosylation, as a ubiquitous modification reaction, significantly improves the water solubility, biological activity, and stability of products by attaching specific sugar groups to natural product molecules, thereby regulating the growth and metabolic balance of plants. The glycosylation reaction is mainly catalyzed by a multi-membered family of glycosyltransferases (GTs), among which the UDP-glycosyltransferase (UGT) family is the most widely acting member. The highly conserved PSPG box (consisting of 44 amino acids) carried at the C-terminus of UGT is responsible for recognizing sugar donor molecules and catalyzing the glycosylation reaction. A large number of studies have shown that UGTs not only participate in the glycosylation modification of plant secondary metabolites (such as flavonoids, terpenoids, alkaloids, organic acids, etc.), but also play a crucial role in regulating physiological processes such as plant stress resistance, internal and external detoxification, and hormone balance. For example, ApUGT86C11 of Andrographis paniculata can catalyze the formation of a new glucoside at the C19-O site of diterpenoids; overexpression of UGT76E11 in Arabidopsis thaliana significantly increases the flavonoid content in transgenic plants, confirming its role in flavonoid metabolism regulation; in addition, UGT79B1 and UGT84A2 of Arabidopsis thaliana are involved in anthocyanin biosynthesis, and gene deletion of both leads to a significant reduction in anthocyanin content.

[0004] This application is based on the preliminary research on the biosynthesis regulation of kaempferol glycosides in Phoebe bournei. Through the screening of the regulatory network of the key transcription factor PbMYB211, the gene PbUGT92AE1 related to the synthesis of kaempferol-3-O-rutinoside was successfully identified. However, there is still a lack of systematic and in-depth research on the specific function, catalytic mechanism of this gene in the glycosylation modification of kaempferol-3-O-rutinoside, and its application technology for improving the accumulation of target products in transgenic plants. Therefore, it is urgent to develop a new method to make full use of the PbUGT92AE1 gene of Phoebe bournei and improve the biosynthesis efficiency of kaempferol-3-O-rutinoside in plants, providing a theoretical basis and technical support for the industrial development and economic application of this type of secondary metabolite. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for regulating the biosynthesis of kaempferol-3-O-rutinoside based on the PbUGT92AE1 gene of Phoebe bournei and its application.

[0006] The present invention provides a PbUGT92AE1 protein of Phoebe bournei, and the PbUGT92AE1 protein of Phoebe bournei is:

[0007] 1) A protein composed of the amino acids shown in SEQ ID No.2; or

[0008] 2) A protein derived from 1) with one or several amino acids substituted, deleted or added in the amino acid sequence shown in SEQ ID No.2 and having the same activity.

[0009] The present invention also provides a gene encoding the above PbUGT92AE1 protein of Phoebe bournei, which has the nucleotide sequence shown in SEQ ID No:1, or a sequence with one or several nucleotides deleted, added and / or substituted in the said sequence but with the same function.

[0010] The present invention also provides an expression vector, which contains a gene encoding the PbUGT92AE1 protein of Phoebe bournei; the expression vector also contains a promoter, a terminator capable of driving the expression of the target gene in plant cells, and at least one marker gene for screening or reporting.

[0011] The present invention also provides a method for regulating the biosynthesis of kaempferol-3-O-rutinoside based on the PbUGT92AE1 gene of Phoebe bournei, and the method includes the following steps:

[0012] a) Construct a plant expression vector containing the PbUGT92AE1 gene of Phoebe bournei;

[0013] b) Introduce the expression vector into plant cells or plants;

[0014] c) Screen out transgenic plants overexpressing the Phoebe bournei PbUGT92AE1 gene;

[0015] d) Collect the above transgenic plants and detect the accumulation of kaempferol-3-O-rutinoside by HPLC method.

[0016] In one embodiment, the above transgenic plants are Arabidopsis thaliana, and the expression level of the Phoebe bournei PbUGT92AE1 gene in the above transgenic plants is significantly higher than that of the untransformed wild-type plants.

[0017] The present invention also provides a transgenic plant that overexpresses the Phoebe bournei PbUGT92AE1 gene, thereby resulting in a significant increase in the content of kaempferol-3-O-rutinoside in the plant.

[0018] The present invention also provides the application of the Phoebe bournei PbUGT92AE1 gene in the regulation of kaempferol-3-O-rutinoside biosynthesis, to prepare a modified plant containing the Phoebe bournei PbUGT92AE1 gene. In the modified plant, compared with the wild-type plant, the expression of the Phoebe bournei PbUGT92AE1 gene is increased; wherein, the base sequence of the Phoebe bournei PbUGT92AE1 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 in 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.

[0019] In one embodiment, the above plant is Phoebe bournei or Arabidopsis thaliana. Preferably, the above plant is Arabidopsis thaliana.

[0020] In one embodiment, the Phoebe bournei PbUGT92AE1 gene is ligated to a vector and transformed into Arabidopsis thaliana through Agrobacterium-mediated transformation, screened, cultured, and transgenic lines are obtained.

[0021] In this application, the Phoebe bournei PbUGT92AE1 gene was successfully introduced into Arabidopsis thaliana by the floral dip method mediated by Agrobacterium tumefaciens, and gene overexpressing plants were obtained. Molecular detection results showed that PbUGT92AE1 was significantly highly expressed in the transgenic plants, with obvious differences compared with the wild type. Further quantitative analysis using high performance liquid chromatography (HPLC) found that the content of kaempferol-3-O-rutinoside in the transgenic plants was significantly higher than that of the wild type. This application overexpressed the Phoebe bournei PbUGT92AE1 gene in Arabidopsis thaliana for the first time, realized the effective regulation of kaempferol-3-O-rutinoside synthesis, verified the feasibility of improving the content of this secondary metabolite by genetic engineering means, and demonstrated its broad application prospects in agriculture and related industries. Description of the Drawings

[0022] Figure 1 Electrophoresis pattern of total RNA from Phoebe bournei roots and leaves of the present invention.

[0023] Figure 2 Electrophoresis pattern of PCR amplification of PbUGT92AE1 gene from Phoebe bournei of the present invention.

[0024] Figure 3 Electrophoresis pattern of PCR detection of overexpressing plants of PbUGT92AE1 gene from Phoebe bournei of the present invention.

[0025] Figure 4 Standard curve of kaempferol-3-O-rutinoside related to the present invention.

[0026] Figure 5 Histogram of kaempferol-3-O-rutinoside content in Arabidopsis thaliana overexpressing PbUGT91BA1 gene from Phoebe bournei of the present invention. Detailed implementation manners

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0028] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0029] 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.

[0030] 1 Materials

[0031] 1.1 Experimental materials

[0032] Phoebe bournei samples: The Phoebe bournei materials selected were 1.5-year-old container seedlings of the "Wuyuan No. 8" family.

[0033] Arabidopsis thaliana materials: The Columbia (Columbia-0) wild type was selected and cultivated in the growth chamber of the Intelligent Experiment Building of Zhejiang A&F University during the seedling stage. The growth conditions were 25°C and 16 hours of light per day.

[0034] 1.2 Experimental reagents and instruments

[0035] Experimental reagents: DL2000 DNA Marker, 10× Loading Buffer, and DNA gel extraction kit were purchased from Baoruiyi 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 Biotechnology Co., Ltd.; 2× Easy Taq PCR SuperMix was purchased from Zhejiang Yiside Biotechnology Co., Ltd.; restriction enzymes were purchased from NEB (Beijing) Co., Ltd.

[0036] 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.

[0037] 1.3 Primer synthesis and sequencing

[0038] Both primer synthesis and sequencing were completed by Zhejiang Youkang Biotechnology Co., Ltd.

[0039] 2 Methods

[0040] 2.1 Extraction of total RNA from Phoebe bournei

[0041] 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:

[0042] (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), add 5% β-mercaptoethanol to the lysis buffer CLB (50 μL of β-mercaptoethanol is added to 1 mL of CLB), invert and mix well, and preheat in a 65°C water bath;

[0043] (2) Take about 0.1 g of the sample and put it into a mortar pre-cooled with liquid nitrogen, and grind it to a fine powder in liquid nitrogen;

[0044] (3) Transfer 100 - 200 mg of the fine powder into a centrifuge tube containing preheated lysis buffer CLB (β-mercaptoethanol has been added). Immediately vortex vigorously for 30 - 60 s or pipette up and down to mix until a satisfactory homogenate is obtained.

[0045] (4) Briefly place it back in a 65 °C water bath for 5 - 10 min, and occasionally invert it 1 - 2 times during this period to assist in lysis.

[0046] (5) Centrifuge the lysate at 13,000 rpm for 10 min to precipitate the non-lysed fragments.

[0047] (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 supernatant volume. At this time, precipitation may occur, but it does not affect the extraction process. Immediately pipette up and down to mix, do not centrifuge.

[0048] (7) Add the mixture (less than 720 μL each time, can be added in two portions if necessary) to a genomic DNA removal column, centrifuge at 13,000 rpm for 2 min, and discard the waste liquid.

[0049] (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, the volume lost during filtration should be subtracted), add anhydrous ethanol at 0.5 volume, immediately pipette up and down to mix, do not centrifuge.

[0050] (10) Immediately add the mixture (less than 720 μL each time, can be added in two portions if necessary) 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.

[0051] (11) Add 700 μL of protein removal solution RW1, let it stand at room temperature for 1 min, centrifuge at 13,000 rpm for 30 s, and discard the waste liquid.

[0052] (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.

[0053] (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.

[0054] (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;

[0055] (15) If the expected RNA yield > 30 μg, add 30 - 50 μL of RNase-Free H2O and repeat step 9. Combine the two washing solutions, or add the first eluate back to the adsorption column and repeat the step once.

[0056] 2.2 Synthesis of the first strand of cDNA by reverse transcription

[0057] The first strand of cDNA of Phoebe bournei RNA (Total RNA) was synthesized by referring to the instruction manual of PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa).

[0058] (1) Prepare the following mixture for genomic DNA removal reaction:

[0059]

[0060] The reaction program is: 42 °C, 2 min; 4 °C, hold.

[0061] (2) Prepare the following mixture for reverse transcription reaction:

[0062]

[0063] The reaction program is: 37 °C, 15 min; 85 °C, 5 sec; 4 °C, hold.

[0064] 2.3 Cloning of the target gene

[0065] 2.3.1 Gene cloning

[0066] Design specific primers for PbUGT92AE1 (Table 1), and the amplification system and program are shown in Table 2.

[0067] Table 1 Cloning primers for PbUGT92AE1

[0068]

[0069] Table 2 Cloning system

[0070]

[0071] Reaction program: 98°C, 30 sec, 52°C, 5 sec, 72°C, 1 min, cycle 35 times; 72°C, 1 min; 16°C, hold.

[0072] 2.3.2 Recovery of target fragment

[0073] Prepare 1% agarose gel, and use agarose gel electrophoresis to detect the above PCR products. If the bands are correct, refer to the MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian) instruction manual to cut and recover the target fragment from the gel.

[0074] (1) Prepare a sterilized 2 mL centrifuge tube and weigh the mass of the empty tube.

[0075] (2) Use a clean scalpel to cut out the gel block containing the target fragment under ultraviolet light, chop the gel block into small pieces and put them into a 2 mL centrifuge tube, weigh and calculate the volume of the gel block (using the standard of 1 mg = 1 μL).

[0076] (3) Add 3 times the gel volume of Buffer GM to the gel block, dissolve the gel block at room temperature, and mix by intermittent shaking.

[0077] (3) When the gel is completely dissolved, add isopropanol with a final concentration of 20%.

[0078] (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.

[0079] (5) Add 700 μL of Wash Buffer WB to the adsorption column, centrifuge at 12000 rpm at room temperature for 1 min, and discard the waste liquid.

[0080] (6) Repeat step (5).

[0081] (6) Centrifuge empty at 12000 rpm for 1 min.

[0082] (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.

[0083] 2.3.3 Ligation and transformation of target fragment

[0084] (1) Ligation: Use the pEASY Blunt Zero vector (Transgene, -Blunt ZeroCloning Kit) and ligate it with the target gene. Mix the following solutions, gently mix well, and briefly centrifuge. PCR: 25°C, 30 min;

[0085]

[0086] (2) Transformation: Take Trans T1 (Transgene, Beijing) Escherichia coli competent cells, thaw them on ice bath, add 2 μL of the above ligation product, place it on ice bath for 30 min, heat shock in a 42°C water bath for 30 s, then quickly and steadily transfer the centrifuge tube to the ice bath for 2 min, add 500 μL of LB medium without antibiotics, and culture it in a shaker at 37°C and 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 culture it inverted at 37°C for 12 h;

[0087] (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 in a shaker at 37°C and 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.

[0088]

[0089] 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.

[0090] (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.

[0091] (5) After the sequencing results are correctly aligned by SnapGene, use the Transgene EasyPure Plasmid MiniPrep Kit to extract the plasmid, and construct the expression vector of the PbUGT92AE1 gene using the obtained positive plasmid.

[0092] 2.3.4 Construction of the expression vector using homologous recombination method

[0093] According to II One Step Cloning Kit to construct the expression vector by homologous recombination method.

[0094] (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):

[0095]

[0096] Reaction program: 98 °C, 30 sec, 54 °C, 5 sec, 72 °C, 1 min, cycle 35 times; 72 °C, 5 min; 16 °C, hold.

[0097] (2) After ensuring that the product is a single band, refer to the MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian) instruction manual to excise and recover the target fragment.

[0098] (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:

[0099]

[0100] Reaction program: 37 °C, 2 h; 16 °C, hold;

[0101] (4) Refer to the MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian) instruction manual to excise and recover the target fragment;

[0102] (5) The overexpression vector selected for genetic transformation is pK2W7-eYGFPuv-3xFLAG, and the overexpression vector of the PbUGT92AE1 gene is constructed. Prepare the following mixed solution, 25 °C, overnight;

[0103]

[0104] 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),

[0105] Reaction program: 37 °C, 30 min; cool to 4 °C or immediately place on ice for cooling;

[0106] (6) Take 2 μL for transformation of Trans T1 (Transgene, Beijing) Escherichia coli competent cells, and then pick single colonies for colony PCR detection. The primers are gene cloning primers, and the PCR system is as follows:

[0107]

[0108] The reaction procedure was as follows: 94°C, 5 min; 94°C, 30 sec, 52°C, 30 sec, 72°C, 1 min, for 35 cycles; 72°C, 5 min; 16°C, hold;

[0109] (7) The PCR products were detected by agarose gel electrophoresis. After transformation into the competent cells of Trans T1 (Transgene, Beijing) Escherichia coli, positive clones were selected and sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. The plasmids of the strains with correct sequencing results were extracted for later use.

[0110] 2.4 Transformation of Agrobacterium tumefaciens GV1301 by the liquid nitrogen method

[0111] (1) Take out the competent Agrobacterium tumefaciens stored at -80°C and thaw it on ice.

[0112] (2) Take 0.5 μg of the expression vector plasmid and add it to the competent Agrobacterium tumefaciens. After gently flicking to mix, place it on ice, in liquid nitrogen, in a 37°C water bath, and in an ice-water mixture for 5 min each in sequence.

[0113] (3) Add 700 μL of antibiotic-free YEP liquid medium to the centrifuge tube and incubate the bacterial solution in a shaker (28°C, 200 rpm) for 4 - 6 h.

[0114] (4) Centrifuge at 6000 rpm for 2 min, pour off some of the supernatant, leaving about 70 - 100 μL of liquid, and pipette to mix evenly.

[0115] (5) Spread the bacterial solution evenly on YEP solid medium containing antibiotics (50 mg·mL -1 Spec and 50 mg·mL -1 Rif), and incubate it inverted in a 28°C biochemical incubator for 48 - 72 h.

[0116] (6) Pick monoclonal colonies on the plate for bacterial solution PCR detection. The primers were the gene cloning primers. The PCR system was as follows:

[0117]

[0118]

[0119] The reaction procedure was 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;

[0120] (7) Add an equal volume of 50% glycerol to the positive monoclonal bacterial solution and store it in a -80°C refrigerator.

[0121] 2.5 Arabidopsis thaliana Transformation by Agrobacterium-mediated Inflorescence Infiltration

[0122] Take out the Agrobacterium liquid containing the vector with the target gene from the -80°C refrigerator, streak it on a plate for activation, pick a single colony and blow it into an EP tube containing 1 mL of YEP (containing 50 mg·mL -1 Rif and 50 mg·mL -1 Spec) liquid medium, and culture it in a shaker at 28°C for 16 h. Take 500 μL of the bacterial liquid and transfer it to a 500 mL conical flask containing 200 mL of the same medium, and continue to culture it in 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 liquid. Transfer the infiltration liquid to a container with a large opening and low height, and use a syringe to pump it until a large amount of foam appears. Immerse the inflorescence of Arabidopsis thaliana with the pods removed in the bacterial liquid for 1 min, then take it out and absorb the excess bacterial liquid 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 infiltration after one week. Collect the mature seeds in batches, dry them at 37°C for 5 d, and store them in a refrigerator at 4°C for later use.

[0123] 2.6 Identification of Positive Transgenic Arabidopsis thaliana

[0124] Use the M5 Hiper Supermix reagent to identify positive Arabidopsis thaliana plants as follows:

[0125] (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;

[0126] (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 over a fire to form a "grinding pestle" and crush the leaf tissue;

[0127] (3) Treat it in a PCR instrument at 98°C for 5 min;

[0128] (4) Centrifuge at 12,000 rpm for 2 min, take 2 μL as the PCR template, and the reaction system is as follows:

[0129]

[0130] The reaction program is: 95°C for 3 min; 94°C for 25 s, 60°C for 25 s, 72°C for 1 min, set 34 cycles; 72°C for 5 min.

[0131] 2.7 Total Flavonoid Extraction and HPLC Determination

[0132] 2.7.1 HPLC Parameter Settings

[0133] 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.

[0134] 2.7.2 Preparation of the standard curve

[0135] Take the kaempferol-3-O-rutinoside 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 filter it through a 0.22 μm microporous 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, with the standard concentration as the abscissa and the peak area as the ordinate, plot the standard curve, obtain the linear regression equation, and based on the R 2 of the linear regression equation, judge its linear relationship.

[0136] The mobile phase used was acetonitrile (A) and 0.2% acetic acid aqueous solution (B). The linear gradient was set as follows: 0 - 10 min, 5% A to 24% A; 10 - 35 min, 24% A to 24% A.

[0137] According to the above HPLC parameters, perform injection analysis and record the peak areas corresponding to each concentration. Plot the standard curve with the standard concentration as the abscissa and the peak area as the ordinate, and conduct linear regression analysis to obtain the regression equation and the correlation coefficient R 2 to evaluate the linear relationship and quantitative accuracy of the method.

[0138] 2.7.3 Extraction and content determination of total flavonoids

[0139] Freeze-dry the plant samples and grind them thoroughly into powder. Accurately weigh 0.1 g of the sample into a 2 mL EP tube, and add 2 mL of 80% methanol solution. After ultrasonic extraction of the sample at 55 °C for 30 minutes, centrifuge it at 8000 rpm for 10 minutes, and collect the supernatant. Filter the extract through a 0.22 μm microporous membrane, transfer it into a liquid-phase injection vial, and perform detection according to the above HPLC parameters. Record the peak area of the target substance in the sample, substitute it into the regression equation of the standard curve, and calculate the specific content of kaempferol-3-O-rutinoside.

[0140] 3. Experimental results

[0141] 3.1 Extraction of total RNA from the roots and leaves of Phoebe bournei

[0142] Extract total RNA from the root and leaf tissues of Phoebe bournei using the M5 Plant RNeasy Complex Mini Kit (Figure 1 ) and the purity was determined using an ultraviolet spectrophotometer. The results showed that the OD260 / 280 ratios of the RNA samples were all between 1.8 and 2.1, indicating that the extracted RNA had a high purity. Further, the integrity of the RNA was detected by 1% agarose gel electrophoresis. The results showed that the 18S and 28S rRNA bands were clear and sharp, and no obvious degradation was observed, indicating that the RNA quality was good and met the requirements of subsequent experiments.

[0143] 3.2 Cloning of Phoebe bournei PbUGT92AE1 Sequence and Vector Construction

[0144] Using the total cDNA obtained by reverse transcription of the total RNA extracted above as a template, PCR amplification was performed with PbUGT92AE1 specific primers, and a target band with a length of 1,586 bp was obtained ( Figure 2 ). Using the homologous recombination method, the specific primers with vector adapters after PCR amplification were ligated to the overexpression vector pK2W7-eYGFPuv-3xFLAG and transformed into Escherichia coli. After sequencing by Zhejiang Youkang Biotechnology Co., Ltd., the plasmid was extracted from the monoclonal with the correct sequence and then transformed into Agrobacterium tumefaciens GV3101, and the Arabidopsis thaliana was infected with the cultured Agrobacterium tumefaciens solution.

[0145] 3.3 Identification of Positive Transgenic Arabidopsis thaliana Plants with PbUGT92AE1

[0146] Agrobacterium tumefaciens 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. After PCR identification, the transgenic positive lines were obtained after the transformation of Phoebe bournei PbUGT92AE1 gene into Arabidopsis thaliana ( Figure 3 ). The molecular detection results showed that the expression level of the PbUGT91BA1 gene in the transgenic plants was significantly higher than that in the wild-type plants, indicating that the target gene was successfully introduced and highly expressed in Arabidopsis thaliana.

[0147] 3.4 Standard Curve and HPLC Analysis

[0148] The kaempferol-3-O-rutinoside standard was dissolved in methanol and diluted to concentrations of 12.5, 25, 50, 100, and 200 μg / mL. The peak areas were measured using an HPLC instrument, and a standard curve was plotted with the concentration as the abscissa and the peak area as the ordinate ( Figure 4 ). The results showed that the elution time of kaempferol-3-O-rutinoside was about 13.7 min, and the standard curve equation was y = 9.6429x + 13.91, with a correlation coefficient R 2 = 0.9999.

[0149] The HPLC results showed that, compared with WT, the PbUGT92AE1 transgenic Arabidopsis thaliana (OE1, OE2, OE3) contained more kaempferol-3-O-rutinoside ( Figure 5 ), indicating that PbUGT92AE1 is involved in the regulation of the biosynthesis of kaempferol-3-O-rutinoside in Phoebe bournei.

[0150] In summary, the successful overexpression of the PbUGT92AE1 gene in Arabidopsis thaliana significantly increased the accumulation of kaempferol-3-O-rutinoside, verifying the catalytic function of this gene in the glycosylation process of kaempferol. This application provides a theoretical basis and technical support for the subsequent research on the regulation mechanism of secondary metabolites in Phoebe bournei and metabolic engineering improvement, and has good application prospects.

[0151] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Phoebe bournei PbUGT92AE1 protein, characterized in that, The Phoebe bournei PbUGT92AE1 protein is: 1) a protein consisting of the amino acids shown in SEQ ID No. 2; or 2) a protein derived from 1) with one or several amino acids substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 2 and having the same activity.

2. The gene encoding the Phoebe bournei PbUGT92AE1 protein as claimed in claim 1, characterized in that, The gene has the nucleotide sequence shown in SEQ ID No: 1, or a sequence with one or several nucleotides deleted, added and / or substituted in the said sequence but with unchanged function.

3. An expression vector, characterized in that, The expression vector contains the gene described in claim 2; the expression vector also contains a promoter, a terminator capable of driving the expression of the target gene in plant cells, and at least one selection or reporter marker gene.

4. A method for regulating the biosynthesis of kaempferol-3-O-rutinoside based on the PbUGT92AE1 gene of Phoebe bournei, characterized in that, The method comprises the following steps: a) constructing a plant expression vector containing the gene described in claim 2; b) introducing the expression vector into plant cells or plants; c) screening transgenic plants with overexpression of the Phoebe bournei PbUGT92AE1 gene; d) collecting the transgenic plants and detecting the accumulation amount of kaempferol-3-O-rutinoside by HPLC method.

5. The method according to claim 4, characterized in that The transgenic plant is Arabidopsis thaliana, and the expression level of the Phoebe bournei PbUGT92AE1 gene in the transgenic plant is significantly higher than that of the untransformed wild-type plant.

6. A transgenic plant, characterized in that, The plant overexpresses the gene described in claim 2, thereby resulting in a significant increase in the content of kaempferol-3-O-rutinoside in the plant.

7. Application of Phoebe bournei PbUGT92AE1 gene in regulating biosynthesis of kaempferol-3-O-rutinoside, characterized in that, Preparing a modified plant containing the Phoebe bournei PbUGT92AE1 gene, in which the expression of the Phoebe bournei PbUGT92AE1 gene is increased relative to the wild-type plant; wherein, the base sequence of the Phoebe bournei PbUGT92AE1 gene is as shown in SEQ ID NO:

1.

8. The application according to claim 7, characterized in that, The plant is Phoebe bournei or Arabidopsis thaliana.

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

10. The application according to claim 7, wherein Connect the Phoebe bournei PbUGT92AE1 gene to a vector, transform it into Arabidopsis thaliana through Agrobacterium-mediated transformation, screen, culture and obtain transgenic lines.

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