Polygonatum cyrtonema PcF3H gene and application thereof in promoting synthesis of flavanone compounds and catalyzing naringenin hydroxylation

Abstract

The invention discloses a Polygonatum cyrtonema PcF3H (Polygonatum cyrtonema PcF3H) gene. The invention also discloses a method for promoting the synthesis of the flavanone compound. The invention also discloses an application of the Polygonatum cyrtonema PcF3H gene in promoting the synthesis of flavanone compounds. The invention also discloses an application of the Polygonatum cyrtonema PcF3H gene in catalysis of naringenin hydroxylation. The polygonatum cyrtonema PcF3H gene is obtained through cloning, a plant expression vector is constructed, the polygonatum cyrtonema PcF3H gene is instantaneously transformed into tobacco, and compared with a control, the content of flavanone compounds in tobacco leaves over-expressed with the polygonatum cyrtonema PcF3H gene is remarkably increased. An applicant utilizes a prokaryotic expression system to induce and purify a protein product PcF3H protein expressing the polygonatum cyrtonema PcF3H gene, the PcF3H protein takes naringenin as a substrate, and a result shows that the product PcF3H protein of the polygonatum cyrtonema PcF3H gene has a function of catalyzing naringenin hydroxylation reaction.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and relates to a Polygonatum cyrtonema PcF3H gene and an application thereof in promoting the synthesis of dihydroflavonoid compounds and catalyzing the hydroxylation of naringenin. Background Art

[0002] Polygonatum cyrtonema Hua is a Chinese medicinal and edible plant that has the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. It is a dual-purpose plant for solving explicit and implicit hunger. The rhizomes of Polygonatum cyrtonema Hua are rich in a variety of flavonoid compounds, including homoisoflavonoids, dihydroflavonoids, chalcones, isoflavones, etc., accounting for 0.66% to 1.91% of the total dry weight. Modern medicine has proven that flavonoid compounds can help treat cardiovascular diseases, atherosclerosis, diabetes and other diseases. Among them, dihydroflavonoids account for a large proportion of flavonoid compounds. Recently, researchers have used high-performance liquid chromatography to identify dihydroflavonoid compounds with various structures including naringenin, hesperidin, hesperidin-5-O-glucoside, and pyrocatechol in Polygonatum cyrtonema Hua. At the same time, as an important intermediate in the biosynthesis of flavonoid compounds, dihydroflavonoids can be converted into downstream important flavonoid compounds through multi-step reactions, such as flavonoids, isoflavones, anthocyanins, flavonols, etc. At present, there are few studies on the synthesis pathway of dihydroflavonoid compounds in Polygonatum cyrtonema, and the molecular mechanism is still unclear.

[0003] The basic skeleton of flavonoid compounds is usually composed of two aromatic rings (A and B) and a pyran ring (C). Dihydroflavonoids, also known as dihydroflavone, have single bonds at the C2 and C3 positions of the C ring, which is also a key structural feature that distinguishes it from other flavonoids. Common dihydroflavonoid compounds in nature mainly include naringenin, hesperetin, eriodictyol, sakuranetin, homoeriodictyol, licorice, pinocembrin, etc., with about 350 species.

[0004] F3H belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) family and requires 2-ketoglutarate, oxygen, iron, and ascorbic acid for catalytic activity. The biological function of F3H is to catalyze the production of dihydrokaempferol or dihydroquercetin, important intermediates in the synthesis of flavonoids and isoflavones, using naringenin or eriodictyol as substrates. At the same time, it is also a key enzyme that directs carbon flow to the biosynthesis of 3-hydroxylated flavonoids, responsible for the biosynthesis of anthocyanidins and flavonols. In tomato (Solanum lycopersicum), overexpression of F3H in the F3H mutant restored the accumulation content of anthocyanidins and flavonoids to the same level as the wild type (WT). Downregulation of the F3H gene in strawberry resulted in a significant reduction in anthocyanidins and a moderate reduction in flavonols; an arginine to histidine mutation in flavanone-3-hydroxylase produced pink strawberry fruit. In carnation (Dianthus caryophyllus L.), silencing the DcF3H gene causes the flower color to become lighter or even colorless. Overexpression of Lycium barbarum LcF3H in tobacco increased the flavanol content and increased tolerance to drought stress. Overexpression of sorghum SbF3H redirected the biosynthesis of flavonoids, and the produced 3-deoxyanthocyanidins and flavonols acted as phytoalexins to reduce the sensitivity of sorghum to anthracnose. When Sichuan safflower was treated with methyl jasmonate (MeJA), CtF3H showed opposite expression patterns in different phenotypes of orange-yellow flowers and white flowers. The above facts indicate that the function of F3H in flavonoid biosynthesis is very complex, and its effect on the flavonoid spectrum may be species-specific.

[0005] So far, F3H genes have been cloned and characterized from a variety of plant species, such as petunia, barley, alfalfa, maize, Arabidopsis, and Tribulus. At the same time, there is a lot of evidence that F3H gene expression is related to a variety of environmental factors. For example, gibberellic acid and sucrose promote the expression of EsF3H and the accumulation of flavonoid-related bioactive compounds in Epimedium. Abscisic acid (ABA) and jasmonic acid (JA) regulate the expression of AtF3H related to flavonoid biosynthesis in Arabidopsis. However, the F3H gene in Polygonatum cyrtonema has not yet been cloned and characterized. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a PcF3H gene of Polygonatum cyrtonema and its application in promoting the synthesis of dihydroflavonoid compounds and catalyzing the hydroxylation of naringenin.

[0007] The present invention provides a Polygonatum cyrtonema PcF3H gene, which has a nucleotide sequence as shown in SEQ ID No: 1, or a sequence in which one or more nucleotides are deleted, added and / or substituted but the function remains unchanged. Preferably, the nucleotide sequence of the Polygonatum cyrtonema PcF3H gene is SEQ ID No: 1.

[0008] The invention also provides a protein encoded by the Polygonatum cyrtonema PcF3H gene, which is composed of the amino acids shown in SEQ ID No.2.

[0009] The present invention also provides a method for promoting the synthesis of dihydroflavonoid compounds, the method comprising preparing a modified plant containing the Polygonatum cyrtonema PcF3H gene, wherein the expression of the Polygonatum cyrtonema PcF3H gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcF3H gene is 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 is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the increase in the product obtained by transcription and the product obtained by translation can be an increase.

[0010] The present invention also provides the use of the Polygonatum cyrtonema PcF3H gene in promoting the synthesis of dihydroflavonoid compounds, and prepares a modified plant containing the Polygonatum cyrtonema PcF3H gene, wherein the expression of the Polygonatum cyrtonema PcF3H gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcF3H gene is 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 is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, specifically, an increase in the product obtained by transcription and the product obtained by translation. In one embodiment, the plant is Polygonatum cyrtonema or tobacco. Preferably, the plant is tobacco. In one embodiment, the Polygonatum cyrtonema PcF3H gene is connected to a vector, transformed into tobacco through Agrobacterium-mediated transformation, and screened, cultured, and transgenic strains are obtained.

[0011] The present invention also provides the use of the Polygonatum cyrtonema PcF3H gene in catalyzing the hydroxylation of naringenin, and prepares a modified plant containing the Polygonatum cyrtonema PcF3H gene, wherein the expression of the Polygonatum cyrtonema PcF3H gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcF3H gene is 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 is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the product obtained by transcription and the product obtained by translation can be increased. In one embodiment, the plant is Polygonatum cyrtonema or tobacco.

[0012] The present application obtains the PcF3H gene of Polygonatum cyrtonema by cloning, constructs a plant expression vector, and transiently transforms the PcF3H gene of Polygonatum cyrtonema into tobacco. Compared with the control, the content of dihydroflavonoid compounds in tobacco leaves overexpressing the PcF3H gene of Polygonatum cyrtonema significantly increases. At the same time, the applicant uses a prokaryotic expression system to induce and purify the protein product PcF3H protein expressing the PcF3H gene of Polygonatum cyrtonema. The PcF3H protein uses naringenin as a substrate, and uses ultra-high performance liquid chromatography mass spectrometry. The LC MS / MS results show that the PcF3H protein can catalyze naringenin to produce 3,5,7,3',4'-pentahydroxyflavanone. The results show that the product of the PcF3H gene of Polygonatum cyrtonema, the PcF3H protein, has the function of catalyzing the hydroxylation reaction of naringenin. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0014] Figure 1 The figure is the electrophoresis diagram of total RNA from the fibrous roots of Polygonatum cymbidium of the present invention.

[0015] Figure 2 The PCR amplification results of the PcF3H gene of the present invention, M in the figure: DL 2000 DNA Marker; 1-3: PcF3H gene amplification results.

[0016] Figure 3 The PCR bacterial test results of the PcF3H gene of the present invention, in which M: DL 5000 DNA Marker; 1-8: different single colonies of the PcF3H gene.

[0017] Figure 4 This is the electrophoresis diagram of the double enzyme digestion of the P1300-E35S-GFP vector of the present invention, where M: DL15000 DNA Marker; 1: P1300-E35S-GFP linear vector after enzyme digestion.

[0018] Figure 5 The PCR amplification result of the PcF3H gene of the present invention, M in the figure: DL5000 DNA Marker; 1: PCR amplification result of the PcF3H gene.

[0019] Figure 6 This is the bacterial test result after GV3101 was transformed with P1300-E35S-PcF3H-GFP of the present invention. In the figure, M: DL5000 DNA Marker; 1-6: single colonies with different PcF3H genes.

[0020] Figure 7This is a diagram of the construction of the P1300-E35S-PcF3H-GFP vector of the present invention.

[0021] Figure 8 The expression of the PcF3H gene of the present invention in the epidermal cells of tobacco leaves, Figure A: empty vector; Figure B: PcF3H.

[0022] Fig. 9 The figure is the secondary mass spectra of the five common dihydroflavonoid compounds of the present invention.

[0023] Fig.10 This is a heat map of the metabolite content of the dihydroflavonoid compound of the present invention, in which the normalized value range of the metabolite content is 0 to 1.00. The grayer the color, the higher the metabolite content, and the whiter the color, the lower the metabolite content.

[0024] Fig.11 The PCR amplification result of the PcF3H gene of the present invention and the electrophoresis diagram of the double enzyme digestion product of pGEX-4t-1, in which M: DL 5000 DNA Marker; 3-4: PcF3H; 5-8: pGEX-4t-1 linear vector fragment.

[0025] Fig.12 The bacterial test results after pGEX-4T-1-PcF3H of the present invention was transformed into dh5α, where M: DL 5000 DNA Marker; 1-4: PcF3H.

[0026] Fig.13 This is a construction diagram of the pGEX-4T-1-PcF3H vector of the present invention.

[0027] Fig.14 This is the SDS-PAGE analysis of the PcF3H protein of the present invention, where M is a two-color pre-stained protein marker; 1-6: PcF3H crude protein precipitation resuspension, supernatant resuspension.

[0028] Fig.15 For the present invention Figure 3 .15SDS-PAGE analysis of each component of PcF3H protein purification, in the figure, M is a two-color pre-stained protein marker; 1: pGEX-4t-1 empty supernatant resuspension; 2: PcF3H protein purification eluate; 3: column flow-through; 4: PcF3H crude protein supernatant resuspension; 5: PcF3H crude protein precipitate resuspension.

[0029] Fig.16 It is the linear regression equation of the standard protein solution concentration and absorbance of the present invention.

[0030] Fig.17 This is the UPLC analysis of the present invention using naringenin as a substrate.

[0031] Fig.18 The mass spectrum of the product 3,5,7,3',4'-pentahydroxyflavanone produced by the PcF3H catalysis of naringenin of the present invention is shown. DETAILED DESCRIPTION

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] 1. Experimental Materials and Reagents

[0036] 1.1 Plant materials

[0037] The Polygonatum multiflorum used in the experiment was obtained from the Forest Medicinal Materials Team of the State Key Laboratory of Zhejiang Agriculture and Forestry University (the culture conditions were natural conditions). The Nicotiana benthamiana used for subcellular localization and metabolite content determination was cultured in the plant growth room of this laboratory (culture temperature was 25°C, light time was 16h·d -1 ).

[0038] 1.2 Chemical reagents

[0039] The main reagents used in this experiment are shown in Table 1.1

[0040] Table 1.1 Primer sequences used for cloning PcF3H

[0041]

[0042]

[0043] Table 1.1 continued

[0044]

[0045] 1.3 Instruments and Equipment

[0046] Lanyan constant temperature metal bath; AB104-N electronic analytical balance; PCR instrument; Thermo nanodrop 2000 spectrophotometer; Sigma desktop high-speed refrigerated centrifuge; D-37520 high-speed centrifuge; DK-S24 electric constant temperature water bath; SpectraMax 190 absorbance microplate reader; DHG-9070HA precision forced convection drying oven; GENSYS10S UV-Vis ultraviolet spectrophotometer; ZEISS laser confocal microscope; ALPHA 1-2LD Plus freeze dryer, etc.

[0047] 1.4 Main solution formula

[0048] 1.4.1 LB medium

[0049]

[0050] Sterilize with high pressure steam at 121°C for 20 min, and store at 4°C after cooling. No agar is added to the liquid LB medium.

[0051] 1.4.2 Preparation of antibiotic stock solution

[0052] (1) Ampicillin (100 mg mL -1 )

[0053] Ampicillin (Apm) 1000mg

[0054] ddH2O (autoclaved) 10mL

[0055] After dissolution, the solution was sterilized by filtration through a 0.22 μm microporous filter membrane, dispensed into sterilized 2 mL centrifuge tubes, and stored at -20°C.

[0056] (2) Kanamycin (100 mg mL -1 )

[0057] Kanamycin (Kan) 1000mg

[0058] ddH2O (autoclaved) 10mL

[0059] After dissolution, the solution was sterilized by filtration through a 0.22 μm microporous filter membrane, dispensed into sterilized 2 mL centrifuge tubes, and stored at -20°C.

[0060] (3) Rifampicin (100 mg mL -1 )

[0061] Rifampicin (rif) 1000mg

[0062] Dimethyl sulfoxide (DMSO) 10mL

[0063] After dissolution, the solution was sterilized by filtration through a 0.22 μm microporous filter membrane, dispensed into sterilized 2 mL centrifuge tubes, and stored at -20°C.

[0064] 1.4.3 Preparation of injection buffer for transient expression in tobacco

[0065]

[0066] MES and Na3PO4 solutions are prone to bacterial infection, and the mother solution needs to be re-prepared after bacterial infection. Acetosyringone needs to be packaged in NN-dimethylformamide and dissolved before use, so as to avoid repeated freezing and thawing as much as possible. The injection buffer needs to be used now.

[0067] 2. Experimental Methods

[0068] 2.1 Extraction of total RNA from Polygonatum cymbidium

[0069] 2.1.1 Preparation of experimental supplies

[0070] (1) Wrap the mortar, pestle, tweezers and medicine spoon in tin foil and bake at 160℃ for 6 hours.

[0071] (2) Prepare RNA-free pipette tips and centrifuge tubes.

[0072] (3) Wear gloves, masks and lab coats throughout the process to avoid RNA degradation.

[0073] (4) Polygonatum cyrtonema T26_13 from Xiaoshankou Village, Miaoling Township, Xiushui City, Jiangxi Province was selected as the extraction material, and RNA was extracted from the fibrous roots.

[0074] 2.1.2 Extraction steps of total RNA from Polygonatum cyrtonema (kit)

[0075] The total RNA of Polygonatum cyrtonema was extracted using the MiniBEST Plant RNA Extraction Kit from TaKaRa (https: / / www.takarabiomed.com.cn / ).

[0076] 2.2 Detection of total RNA from Polygonatum cymbidium

[0077] Take an appropriate amount of Polygonatum cyrtonema total RNA sample and spot it for about 15 minutes at 110V electrophoresis to detect RNA integrity. The electrophoresis solution is replaced quickly to avoid RNA degradation to the greatest extent. RNA concentration and A230 / A260 and A260 / A280 are parameters for judging the quality of the extracted RNA, which are obtained by measuring 1μL of total RNA sample with a spectrophotometer.

[0078] 2.3 Acquisition of PcF3H gene

[0079] 2.3.1 Design of primers for the full length of PcF3H gene

[0080] The full-length primers of the CDS region of the PcF3H gene of Polygonatum cyrtonema were designed using the primer design software Primer5.0, and synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The primers are shown in Table 2.1 below:

[0081] Table 2.1 Primer sequences used for cloning PcF3H

[0082]

[0083] 2.3.2 Synthesis of the first strand of cDNA

[0084] RNA was collected from the fibrous roots of Polygonatum cyrtonema and analyzed according to PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription kit instructions to obtain cDNA. Remove genomic DNA reaction, the system is as shown in Table 2.2, gently mix, react at 42℃ for 2min, and place on ice after the reaction is completed.

[0085] Table 2.2 Reaction system for removing genomic DNA

[0086]

[0087] Then, reverse transcription reaction was performed, the system was as shown in Table 2.3, gently mixed, reacted at 37℃ for 15min, and then placed at 85℃ for 5sec. After the reaction, the mixture was stored at -20℃.

[0088] Table 2.3 Reverse transcription reaction system

[0089]

[0090] 2.2.3 Amplification of PcF3H gene fragment

[0091] The cDNA of Polygonatum cyrtonema obtained by the above reverse transcription was diluted 5 times and then PCR amplified. The 25 μL reaction system is as shown in Table 2.4.

[0092] Table 2.4 PCR amplification reaction system

[0093]

[0094] The PCR reaction program was as follows: initial denaturation: 98°C for 2 min; 33 cycles (denaturation: 98°C for 10 s; annealing at 58°C for 15 s; extension at 72°C for 1 min); final extension: 72°C for 3 min; storage: 4°C.

[0095] 2.2.4 Agarose gel electrophoresis

[0096] (1) Weigh 0.2 g agarose and 20 mL 1×TAE electrophoresis buffer, mix well, heat in a microwave oven until fully dissolved, and add 1 μL nucleic acid dye.

[0097] (2) Place the electrophoresis plate, insert the comb, slowly pour in the agarose solution prepared in (1), and let it stand at room temperature.

[0098] (3) After the gel solidifies, remove the comb and place the electrophoresis plate with the gel in the electrophoresis tank.

[0099] (4) Pour 1×TAE electrophoresis buffer into the electrophoresis tank so that the liquid level is 1-2 mm above the surface of the agarose gel.

[0100] (5) Spot the DNA maker and PCR products as controls into the wells.

[0101] (6) After running at 120 V for 15 min, remove the gel and place it in a gel imager for observation and photography.

[0102] 2.2.5 PCR product gel recovery

[0103] A DNA gel recovery kit (Jinsha Biotechnology) was used to recover the PCR product. The instructions for the kit can be found on the official website of Jinsha Biotechnology (https: / / genesand.com / ).

[0104] 2.2.6 Connection

[0105] The ligation system is shown in Table 2.5. Mix gently and react at room temperature for 5 minutes. After the reaction is completed, place on ice.

[0106] Table 2.5 Connection system

[0107]

[0108] 2.2.7 E. coli transformation

[0109] The E. coli used for transformation was TOP10 Chemically Competent Cell from Tsingke Biotechnology. For instructions, please refer to the official website of Tsingke Biotechnology (http: / / www.tsingke.net / ).

[0110] 2.2.8 Screening of positive clones

[0111] Pick a single colony from the long bacterial plate and put it into LB liquid containing the corresponding antibiotic, put it into a 37℃ shaker at 200r, and take out the turbid bacterial liquid after 5 hours for PCR verification. The system is as shown in Table 2.6. The PCR reaction program is: pre-denaturation: 98℃2min; 33 cycles (denaturation: 98℃10s; annealing 58℃15s; extension 72℃1min); final extension: 72℃3min; storage: 4℃.

[0112] Take 300 μL of the positive bacterial solution and send it to Youkang Biotechnology Co., Ltd. for sequencing verification.

[0113] Table 2.6 PCR verification reaction system

[0114]

[0115] 2.2.9 Extraction of positive clone plasmid

[0116] A high-purity plasmid DNA minipreparation kit (Jinsha Biotechnology) was used. The kit instructions can be found on the official website of Jinsha Biotechnology (https: / / genesand.com / ).

[0117] 2.3 Construction of P1300-E35S-PcF3H-GFP overexpression vector

[0118] The homologous recombination method was used to construct the P1300-E35S-PcF3H-GFP overexpression vector. The restriction sites on the P1300-E35S-GFP vector are SalI and BcuI. The primers required for constructing the P1300-E35S-PcF3H-GFP overexpression vector were designed using SnapGene and synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The primers are shown in Table 2.7.

[0119] Table 2. Primer sequences used for 7P1300-E35S-PcF3H-GFP overexpression vector

[0120]

[0121] 2.3.1 Double digestion of vector

[0122] In order to obtain the linearized vector, the plant expression vector P1300-E35S-GFP was digested with restriction endonucleases SalI and XholⅠ. The digestion reaction system is shown in Table 2.8, and the reaction was carried out at 37℃ for 1h and placed on ice.

[0123] Table 2.8 Enzyme digestion reaction system

[0124]

[0125] 2.3.2 Agarose gel electrophoresis

[0126] Same as 2.2.4.

[0127] 2.3.3 Rubber recycling

[0128] Same as 2.2.5.

[0129] 2.3.4 Connection

[0130] Use the full-form gold biological -Basic Seamless Cloning and Assembly Kit was used to connect the target gene with the linear vector after double enzyme digestion by homologous recombination. The system was as shown in Table 2.9, and the reaction was performed at 50℃ for 20min.

[0131] Table 2.9P1300-E35S-GFP vector connection system

[0132]

[0133] 2.3.5 E. coli transformation

[0134] Same as 2.2.7.

[0135] 2.3.6 Screening of positive clones

[0136] Same as 2.2.8.

[0137] 2.3.7 Extraction of positive clone plasmid

[0138] Same as 2.2.9.

[0139] 2.3.8 Agrobacterium transformation

[0140] The Agrobacterium used for transformation was GV3101 Chemically Competent Cell of Tsingke Biotechnology. The instructions can be found on the official website of Tsingke Biotechnology (http: / / www.tsingke.net / ).

[0141] 2.3.9 Screening and saving positive clones

[0142] (1) Pick a single colony from the plate and place it in LB liquid containing Rif (100 μg / mL) and Kan (100 μg / mL) and culture at 28°C with shaking for 2 days.

[0143] (2) PCR verification of Agrobacterium culture.

[0144] (3) Take 500 μL of positive bacterial solution, add 500 μL of 50% glycerol, and store in a -80°C ultra-low temperature refrigerator for later use.

[0145] 2.4 Tobacco transient transformation

[0146] (1) Take the bacterial solution stored at -80°C and spread it on Kan and Rif double-antibody plates and grow it at 28°C for two days. When Agrobacterium colonies grow, select a single colony and transfer it to 1 mL of double-antibody LB liquid medium and culture it at 28°C and 200 rpm. Continue to transfer the bacterial solution to 50 mL of double-antibody LB liquid medium and culture it overnight until the OD600 is about 0.6-0.8.

[0147] (2) Prepare an injection buffer for transient expression in tobacco.

[0148] (3) Centrifuge the bacterial solution at 3000 rpm for more than 10 min, discard the supernatant, add injection buffer to resuspend the precipitate, and continue centrifugation at 3000 rpm for 10 min. Discard the supernatant, and repeat twice.

[0149] (4) Resuspend the precipitate again with injection buffer, and the final OD600 of the Agrobacterium culture is between 0.3-0.5.

[0150] (5) Use a disposable 1 mL syringe to inject the Agrobacterium bacterial solution into the mesophyll from the back of the tobacco leaf and wipe off the residual bacterial solution on the leaf.

[0151] (6) The injected tobacco is cultured in the dark for 2-3 days.

[0152] 2.5 Confocal microscopy observation

[0153] Use a hole puncher to punch holes in tobacco leaves 3 days after injection of Agrobacterium solution, place the resulting small round leaves on a glass slide, add two drops of ddH2O and cover with a cover glass. Place the glass slide under a confocal microscope for observation.

[0154] Confocal microscopy conditions: 488 nm excitation light, 505-530 nm receiving light.

[0155] 2.6 Metabolome of transiently transformed tobacco

[0156] 2.6.1 Sample extraction

[0157] (1) Cut off tobacco leaves that show green fluorescence, wrap them in tin foil, and quickly freeze them in liquid nitrogen, and store them at -80°C.

[0158] (2) The leaves were placed in a freeze dryer and vacuum dried, and then ground into powder using a grinder.

[0159] (3) Dissolve 50 mg of sample powder in 1 mL of 70% methanol internal standard extract.

[0160] (4) Add 500 μL petroleum ether, vortex for 5 min, let stand to separate layers, and centrifuge at 12,000 rpm for 10 min at 4 °C.

[0161] (5) After centrifugation, all the supernatant was filtered through a 0.22 μm PTFE filter and stored at -20°C until LC-MS / MS analysis.

[0162] 2.6.2 Chromatographic mass spectrometry acquisition conditions

[0163] The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC) TM AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (Tandem massspectrometry, MS / MS) (Applied Biosystems 6500QTRAP, https: / / sciex.com.cn / ). Liquid phase conditions mainly include:

[0164] (1) Chromatographic column: Agilent SB-C18 1.8 μm, 2.1 mm × 100 mm;

[0165] (2) Mobile phase: Phase A is ultrapure water (with 0.1% formic acid added), phase B is acetonitrile (with 0.1% formic acid added);

[0166] (3) Elution gradient: 0.00 min B phase ratio is 5%, within 9.00 min B phase ratio increases linearly to 95% and maintains at 95% for 1 min, between 10.00 and 11.10 min B phase ratio decreases to 5% and equilibrates at 5% for 14 min;

[0167] 4) Flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0168] Mass spectrometry conditions mainly include:

[0169] The temperature of the electrospray ionization source (ESI) was 500°C; the ion spray voltage (IS) was 5500V (positive ion mode) / -4500V (negative ion mode); the ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and the collision-induced ionization parameters were set high. The QQQ scan used the MRM mode, and the collision gas (nitrogen) was set to moderate. Through further optimization of the declustering voltage (DP) and collision energy (CE), the DP and CE of each MRM ion pair were completed. According to the metabolites eluted in each period, a specific set of MRM ion pairs was monitored in each period.

[0170] 2.7 Verification of the in vitro function of Polygonatum cyrtonema PcF3H

[0171] 2.7.1 Construction of expression vector

[0172] The pGEX-4T-1-PcF3H overexpression vector was constructed by homologous recombination. The restriction sites on the pGEX-4T-1 vector are BamHⅠ and XholⅠ. The primers required for constructing the pGEX-4T-1-PcF3H overexpression vector were designed using SnapGene and synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The primers are shown in Table 2.10.

[0173] Table 2.10 Primer sequences used in pGEX-4T-1-PcF3H overexpression vector

[0174]

[0175] 2.7.1.1 Double digestion of vector

[0176] In order to obtain the linearized vector, the prokaryotic expression vector pGEX-4T-1 was digested with restriction endonucleases SpeⅠ and XholⅠ. The digestion reaction system is shown in Table 2.11, and the reaction was carried out at 37℃ for 1 hour and placed on ice.

[0177] Table 2.11 Enzyme digestion reaction system

[0178]

[0179] 2.7.1.2 Agarose gel electrophoresis

[0180] Same as 2.2.4.

[0181] 2.7.1.3 Rubber Recycling

[0182] Same as 2.2.5.

[0183] 2.7.1.4 Connection

[0184] Use the full-form gold biological -Basic Seamless Cloning and Assembly Kit was used to connect the target gene with the linear vector after double enzyme digestion by homologous recombination. The system was as shown in Table 2.12, and the reaction was performed at 50°C for 20 min.

[0185] Table 2.12 Vector ligation reaction system

[0186]

[0187] 2.7.1.5 E. coli transformation

[0188] Same as 2.2.7.

[0189] 2.7.1.6 Screening of positive clones

[0190] Same as 2.2.8.

[0191] 2.7.1.7 Extraction of positive clone plasmid

[0192] Same as 2.2.9.

[0193] 2.7.2 Prokaryotic expression and purification of pGEX-4T-1-PcF3H

[0194] 2.7.2.1 Transformation of target gene

[0195] Take the pGEX-4T-1-PcF3H plasmid and transform the Transetta (DE3) expression strain using the same method as above. After transformation, pick a single colony and add it to 1 mL LB liquid culture medium containing 100 mg / mL Amp and culture it at 37°C with shaking at 200 rpm until the bacterial solution becomes turbid. Add 50% glycerol to the strains that test positive and store them at -80°C.

[0196] 2.7.2.2 Induced expression of target protein

[0197] (1) Using empty Transetta (DE3) as a negative control, 500 μL of both the experimental group and the control group were added to 10 mL of LB liquid medium containing Amp and cultured at 37°C with shaking at 200 rpm until turbidity;

[0198] (2) Take 10 mL of activated bacterial solution, add 500 mL of LB liquid medium containing Amp, and culture at 16°C with shaking at 200 rpm until OD600 is between 0.5 and 0.6;

[0199] (3) Add IPTG inducer, and the final concentration of IPTG in the bacterial solution is 0.5 mM;

[0200] (4) Incubate at 16°C with shaking at 200 rpm for 12 h.

[0201] 2.7.2.3 Extraction of target protein

[0202] (1) Centrifuge the bacterial solution after IPTG induction at 5000 rpm at 4°C for 10 min, discard the supernatant, and collect the bacterial cells;

[0203] (2) Resuspend in 40 mL PBS solution, centrifuge at 5000 rpm for 10 min at 4°C, discard the supernatant and resuspend in 40 mL PBS solution;

[0204] (3) Use an ultrasonic cell crusher, No. 2 amplitude, 33% power, crush for 2 seconds, pause for 5 seconds, and continue for 30 minutes.

[0205] (4) Centrifuge the disrupted bacterial solution at 14,000 rpm at 4°C for 10 min, transfer the supernatant to a new centrifuge tube, add PBS solution to the precipitate and resuspend it, and keep both the supernatant and the resuspended solution for gel running.

[0206] (5) Take 1 mL of GST-tag and add 1 mL of PBS solution. Add it to the gravity column and discard the effluent. Repeat 4 times. For the last time, keep 1 mL of the mixture and add it to the supernatant. Rotate slowly on a shaker at 4°C for 1 h.

[0207] (6) The supernatant containing the GST-tag is passed through a gravity column, and the flowthrough is the effluent. The target protein binds to the GST-tag in the gravity column, and the protein is eluted with 5 mL of Elution Buffer to obtain the purified protein.

[0208] 2.7.2.4 SDS-PAGE electrophoresis analysis

[0209] (1) Using Omni-Easy TM One-step PAGE rapid preparation kit (10%) was used to prepare electrophoresis gel, and gel preparation was referred to the instructions of the Yazyme 10% SDS-PAGE rapid preparation kit.

[0210] (2) Take 40 μL of supernatant, precipitate resuspension, flowthrough and purified protein in a 1.5 mL centrifuge tube, add 10 μL of 5× protein loading buffer respectively, boil at 100°C for 10 min, and then centrifuge at 12000 rpm for 2 min.

[0211] (3) Take 20 μL of protein sample and load it on 10% PAGE gel at 120 V for 70 min.

[0212] (4) Remove the colloid, cut off the concentrated gel, place the separation gel in Coomassie Brilliant Blue rapid staining solution, stain for 30 min on a shaker at 20 rpm, and then decolorize with clean water overnight.

[0213] (5) The next day, observe the decolorization of the gel.

[0214] 2.7.2.5 Target protein concentration and desalination

[0215] (1) Add 10 mL PBS (pH = 8.0) to the upper layer of the ultrafiltration tube (50 kDa) and centrifuge at 4700 × g, 4°C, for 10 min;

[0216] (2) According to the electrophoresis results, the purified eluate containing PcF3H was added to the upper layer of the ultrafiltration tube, centrifuged at 4700 × g, 4°C, 20 min, and concentrated until the upper layer of the ultrafiltration tube was less than 1 mL, and the lower layer of effluent was discarded;

[0217] (3) Add 10 mL of PBS (pH = 8.0) to the upper layer of the ultrafiltration tube, centrifuge at 4700 × g, 4°C, 30 min, and concentrate until the upper layer of the ultrafiltration tube is less than 1 mL. Collect the upper layer of the ultrafiltration tube to obtain the concentrated PcF3H protein solution, and place it on ice.

[0218] 2.7.2.6 Protein concentration determination

[0219] The protein concentration was determined using the Easy Protein Quantitative Kit (Bradford). The instructions can be found in https: / / www.transgen.com / .

[0220] 2.7.3 In vitro enzyme function verification of PcF3H

[0221] (1) The in vitro enzymatic reaction system is shown in Table 2.13. The experimental group is a PBS solution after the pGEX-4T-1-PcF3H protein is induced and expressed and purified; the control group is a PBS solution with the same pH. The substrate is naringenin (concentration 1 mM). The reaction is carried out at 30°C and 300 rpm for 1 h.

[0222] Table 2.13 In vitro enzymatic reaction system

[0223]

[0224] (2) After the reaction is completed, liquid nitrogen is used to quench the reaction.

[0225] (3) At a temperature not higher than 30° C., use a evaporator at 1200 rpm to evaporate the reaction solution under reduced pressure for 3 h.

[0226] (4) Reconstitute with mass spectrometry grade methanol, sonicate in an ice-water bath for 30 min, and then vortex for 30 sec.

[0227] (5) Centrifuge at 12000 rpm for 30 min at 4°C. Take 100 μL of the supernatant and store it at -20°C.

[0228] 2.7.4 Detection of in vitro enzyme activity products

[0229] The samples were qualitatively determined by UHPLC-Q-TOF / MS. Chromatographic conditions: The samples were separated by Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC) C-18 column; column temperature 40°C; flow rate 0.4 mL / min; injection volume 2 μL; mobile phase composition A: water + 25 mM ammonium acetate + 0.5% formic acid, B: methanol; gradient elution program is as follows: 0---0.5 min, 5% B; 0.5---10 min, B linearly changes from 5% to 100%; 10.0---12.0 min, B is maintained at 100%; 12.0---12.1 min, B linearly changes from 100% to 5%; 12.1---16 min, B is maintained at 5%; the samples were placed in an automatic sampler at 4°C during the entire analysis process. In order to avoid the influence of fluctuations in instrument detection signals, the samples were analyzed continuously in a random order. Mass spectrometry conditions: AB Triple TOF 6600 mass spectrometer was used to collect the primary and secondary spectra of the samples. The ESI source conditions after chromatographic separation are as follows: Ion Source Gas1 (Gas1): 60, Ion Source Gas2 (Gas2): 60, Curtain gas (CUR): 30, source temperature: 600℃, Ion Sapary Voltage Floating (ISVF) ±5500V (positive and negative modes); TOF MS scan m / z range: 60-1000Da, production scan m / z range: 25-1000Da, TOF MS scan accumulation time 0.20s / spectra, product ion scan accumulation time 0.05s / spectra; The secondary mass spectrum was obtained by information dependent acquisition (IDA) in high sensitivity mode, Declustering potentia (l DP): ±60V (positive and negative modes), Collision Energy: 35±15eV, IDA settings are as follows: Exclude isotopes within 4Da, Candidate ions to monitor per cycle: 10. The mass spectrometry data were analyzed using MSDIAL (ver.4.9.221218 Windows x64).

[0230] 3 Experimental results

[0231] 3.1 Extraction of total RNA from Polygonatum cymbidium

[0232] The results of RNA concentration determination in the hairy roots of Polygonatum cyrtonema are shown in Table 3.1. The 260 / 280 values ​​of RNA in the hairy roots of Polygonatum cyrtonema are all in the range of 1.8-2.2. RNA agarose gel electrophoresis results Figure 1 As shown below, the 28S and 18S bands are bright and clear. Therefore, the RNA quality of the hairy roots is good and can be used in the subsequent reverse transcription cDNA experiment.

[0233] Table 3.1 RNA concentration determination in the root of Polygonatum cymbidium

[0234]

[0235] 3.2 Acquisition of PcF3H gene

[0236] Agarose gel electrophoresis was used to detect the target gene product amplified from cDNA. The results of agarose gel electrophoresis showed that the size of the target fragment of PcF3H gene was 1080 bp ( Figure 2 ). It can be seen from the electrophoresis results that the fragment size of the PCR amplification product is basically consistent with the theoretical gene length, so it is preliminarily determined that the product fragment obtained by PCR amplification is the PcF3H gene.

[0237] PcF3H was transformed and tested for bacteria. The test was positive ( Figure 3 ) was sent to the company for sequencing, and the sequencing results showed that the sequence was identical to that of the Polygonatum sibiricum genome, indicating that the PcF3H gene was successfully cloned.

[0238] 3.3 Construction of P1300-E35S-PcF3H-GFP overexpression vector

[0239] 3.3.1 Obtaining linear vector and target gene

[0240] like Figure 4 As shown, the circular P1300-E35S-GFP vector was double-digested with SalI and BcuI restriction endonucleases to obtain a linear P1300-E35S-GFP vector. After gel recovery, it was used to construct a vector. Figure 5 As shown, the target gene was amplified by PCR and the bands were clear.

[0241] 3.3.2 Vector construction and bacterial testing

[0242] After P1300-E35S-PcF3H-GFP was transformed into GV3101 Agrobacterium, a single clone plaque was picked from the plate. The bacterial test results were as follows: Figure 6 .

[0243] The P1300-E35S-PcF3H-GFP vector was successfully obtained and transformed into the Agrobacterium GV3101 strain. Figure 7shown.

[0244] 3.4 Subcellular localization

[0245] The successfully constructed P1300-E35S-PcF3H-GFP overexpression vector was transiently transformed into tobacco leaf cells, and tobacco leaves transformed with the P1300-E35S vector were used as negative controls. Three days after injection, tobacco leaves were observed under a laser confocal microscope. Figure 8 As shown, GFP fluorescence signals can be detected in the epidermal cells of tobacco leaves, indicating that the PcF3H gene is normally expressed in tobacco leaves. Observing the expression position of GFP fluorescence signals, it can be seen that the PcF3H protein is located in the cytoplasm.

[0246] 3.5 Metabolome analysis of PcF3H transgenic tobacco plants

[0247] Based on the UPLC-MS / MS detection platform and the Polygonatum cyrtonema broad-based targeted metabolome database, metabolomics analysis was performed on tobacco leaves overexpressing the PcF3H gene. Each sample group had three biological replicates. The metabolites of the samples were qualitatively and quantitatively analyzed by mass spectrometry. A total of 48 dihydroflavonoid compounds were detected, of which 5 compounds were shared by tobacco and Polygonatum cyrtonema ( Fig. 9 ).

[0248] Compared with the control, the content of dihydroflavonoids in tobacco leaves overexpressing the PcF3H gene increased significantly. Fig.10As shown in the figure, according to the heat map, compared with the control, after the overexpression of PcF3H gene, the contents of metabolites such as Eriodictyol-8-C-glucoside-4'-O-glucoside, Prunin, 5,6,7,4'-Tetramethoxyflavanone, Naringenin-7-O-Rutinoside-4'-O-glucoside, Didymin, and Poncirin decreased, while Chorin The contents of dihydroflavonoid metabolites such as erospondin, pinocembrin-7-O-(6”-O-malonyl) glucoside, naringenin-7-O-(6”-malonyl) glucoside, persicoside, aromadendrin, taxifolin-3’-O-glucoside, and taxifolin-2-O-glucoside were increased.

[0249] 3.6 Construction of pGEX-4T-1-PcF3H prokaryotic expression vector

[0250] 3.6.1 Obtaining linear vector and target gene

[0251] BamH I and Xho I were selected as double restriction sites of the empty pGEX-4t-1 plasmid, and the uncut vector plasmid and double restriction products were detected by agarose gel electrophoresis. Fig.11 As shown, after double enzyme digestion, the electrophoresis speed of the fragment slowed down, and the double enzyme digestion linear fragment of pGEX-4t-1 was obtained, which was used for the subsequent connection of the PcF3H gene fragment. Through PCR amplification, the PCR product was detected by agarose gel electrophoresis to obtain the PcF3H gene with homology arms, which was conducive to the next step of homologous recombination to obtain the prokaryotic expression vector.

[0252] 3.6.2 Vector construction and bacterial testing

[0253] After the gene fragment of PcF3H was recovered from gel, it was connected to the pGEX-4t-1 vector through homologous recombination and transformed into Escherichia coli. Four monoclonal colonies were randomly selected for PCR amplification. Fig.12As shown, all monoclonal colonies amplified specific bands with the same size as the target gene PcF3H, and these positive clones were sent to a sequencing company for sequencing.

[0254] The sequencing results showed that the pGEX-4T-1-PcF3H prokaryotic expression vector was successfully constructed, and then the pGEX-4T-1-PcF3H vector was transformed into the Transetta expression strain. Fig.13 shown.

[0255] 3.7PcF3H protein expression and purification

[0256] 3.7.1 pGEX-4T-1-PcF3H electrophoresis analysis

[0257] The supernatant, precipitate resuspension, flowthrough and purified pGEX-4T-1-PcF3H protein after prokaryotic expression and crushing and centrifugation were subjected to SDS-PAGE electrophoresis. After electrophoresis, the protein was stained with Coomassie Brilliant Blue Rapid Staining Solution and decolorized overnight. The results are shown in Fig.14 The Western blot results are shown in Fig.14 As shown in the figure, the size of PcF3H protein is 41.09 kDa, and the size of the GST tag on the pGEX-4T-1 vector is 26 kDa, so the size of the recombinant protein after the fusion of PcUGTs protein and GST tag is 67.09 kDa.

[0258] 3.7.2 Purification of target protein

[0259] Purification of the induced PcF3H crude protein by gravity column revealed a relatively pure PcF3H band in the eluate ( Fig.15 ), after collecting this component, subsequent protein concentration and desalting experiments were carried out.

[0260] 3.7.3 Determination of target protein concentration

[0261] Taking a single time as an example, the absorbance value of each well of the ELISA plate measured by the ELISA reader at a wavelength of 562nm is as shown in Table 3.2:

[0262] Table 3.2 Absorbance values ​​of each well at wavelength A562

[0263]

[0264] The linear regression equation was derived based on the absorbance ( Fig.16), and y=0.3099x+0.0856, R2=0.9991, where x is the concentration of the protein standard solution (mg / mL), and y is the absorbance of the measured protein standard solution at wavelength A562 (nm). The concentration of the PcF3H protein concentrate sample was calculated based on this equation for the subsequent catalytic characteristics study.

[0265] 3.8 Verification of PcF3H enzyme function in vitro

[0266] PcF3H in vitro enzyme activity assay Fig.17 As shown, compared with the blank control group, when naringenin was used as the substrate, the in vitro enzymatic activity of PcF3H obtained a product but not the theoretical product Aromadendrin.

[0267] In vitro catalytic experiments were carried out using the substrate naringenin. LC MS / MS results showed that PcF3H in vitro enzymatic activity produced the product 3,5,7,3',4'-pentahydroxyflavanone (3,5,7,3',4'-pentahydroxyflavanone) with an m / z of 303 [MH]-. Fig.18 )

[0268] 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 Polygonatum cyrtonema PcF3H gene, characterized in that: The Polygonatum cyrtonema PcF3H gene has the nucleotide sequence shown in SEQ ID No: 1, or a sequence in which one or several nucleotides are deleted, added and / or substituted but the function remains unchanged.

2. The gene according to claim 1, characterized in that The nucleotide sequence of the gene is SEQ ID No:

1.

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

2.

4. A method for promoting the synthesis of dihydroflavonoid compounds, the method comprising preparing a modified plant containing the Polygonatum cyrtonema PcF3H gene, characterized in that: Compared with the wild-type plant, in the modified plant, the expression of the Polygonatum cyrtonema PcF3H gene is increased; wherein the base sequence of the Polygonatum cyrtonema PcF3H gene is shown in SEQ ID NO:

1.

5. The application of the Polygonatum cyrtonema PcF3H gene in promoting the synthesis of dihydroflavonoid compounds, characterized in that: A modified plant containing the Polygonatum cyrtonema PcF3H gene is prepared, wherein the expression of the Polygonatum cyrtonema PcF3H gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcF3H gene is as shown in SEQ ID NO:

1.

6. The use according to claim 5, characterized in that: The plant is polygonatum or tobacco.

7. The use according to claim 6, characterized in that: The plant is tobacco.

8. The use according to claim 7, characterized in that: The PcF3H gene of Polygonatum cyrtonema is connected to a vector, transformed into tobacco through Agrobacterium-mediated transformation, and then screened, cultured and transgenic strains are obtained.

9. Use of the Polygonatum cyrtonema PcF3H gene in catalyzing the hydroxylation of naringenin, characterized in that: A modified plant containing the Polygonatum cyrtonema PcF3H gene is prepared, wherein the expression of the Polygonatum cyrtonema PcF3H gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcF3H gene is as shown in SEQ ID NO:

1.

10. The use according to claim 9, characterized in that: The plant is polygonatum or tobacco.

Images