Flavonol synthase, coding gene, expression vector, host bacteria and application

Abstract

The application provides a flavonol synthase, a coding gene, an expression vector, a host bacterium and application, and belongs to the technical field of bioengineering. The application provides a flavonol synthase, and the amino acid sequence of the flavonol synthase is shown as SEQ ID NO. 1. The application can realize utilization of the Rhododendron simsii flavonol synthase and realize regulation and improvement of biosynthesis of plant flavonol and anthocyanin.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a flavonol synthase, its encoding gene, expression vector, host bacterium, and its applications. Background Technology

[0002] Flavonols, as the earliest and most widely distributed flavonoids in terrestrial plants, play important biological roles in plants. They compete with anthocyanin synthesis for substrates, thus affecting the color of plant tissues. In addition, they protect plant cells from UV damage, resist pathogens and herbivores, act as signaling molecules to promote interactions between plants and microorganisms, and influence pollen growth and development, as well as hormone transport within plants. Furthermore, numerous experiments have demonstrated a close relationship between flavonols and human health; they possess antioxidant, antiviral, and anti-cell proliferation activities, and are also important targets for molecular modification of economic crops, especially fruits and vegetables. Flavonol biosynthesis is an important upstream branch of the flavonoid synthesis pathway, using dihydroflavonol as a substrate and catalyzed by the flavonol synthase (FLS) gene to produce flavonols. Studies show that the FLS gene is a multi-gene family, and its coding region is relatively conserved, making it very important for evolutionary analysis. Mutations in the FLS gene or inactivation of this enzyme can affect and alter the color or anthocyanin content of target plants. For example, transferring the antisense FLS gene into petunias and tobacco reduced the flavonol content but increased the anthocyanin content, resulting in redder flowers. Meanwhile, Xu Feng et al. discovered that GbFLS in Ginkgo biloba can catalyze not only dihydroflavonol but also utilize naringenin as a substrate to generate calciferol, suggesting that GbFLS is a bifunctional enzyme in the flavonol synthesis pathway. This indicates that FLS is a crucial enzyme affecting the synthesis of flavonols and anthocyanins in plants, and also a target enzyme for improving plant color phenotypes and beneficial health-promoting components (flavonols).

[0003] FLS was first detected in 1981 in an extract from parsley cells. The FLS gene has since been cloned from plants such as Arabidopsis thaliana, Epimedium, tea tree, poplar, maize, citrus, and grape. With the continuous improvement of cDNA cloning technology, domestic researchers have also cloned the cDNA or genomic DNA sequences of the FLS gene from numerous plants. However, there are currently no reports on the cloning and functional studies of the FLS gene in Rhododendron simsii. Summary of the Invention

[0004] The purpose of this invention is to provide a flavonol synthase, which enables the utilization of flavonol synthase from Rhododendron simsii and the regulation and improvement of the biosynthesis of flavonols and anthocyanins in plants.

[0005] The present invention provides a flavonol synthase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The present invention also provides the encoding gene of the flavonol synthase described in the above technical solution, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides an expression vector containing the coding gene described in the above technical solution.

[0008] Preferably, the expression vector includes a prokaryotic expression vector or a binary expression vector.

[0009] The present invention also provides a host bacterium containing the expression vector described in the above technical solution.

[0010] The present invention also provides the application of the flavonol synthase described in the above-mentioned technical solutions, or the encoding gene described in the above-mentioned technical solutions, or the expression vector described in the above-mentioned technical solutions, or the host bacteria described in the above-mentioned technical solutions in controlling the synthesis of anthocyanins and / or flavonols in plants.

[0011] The present invention also provides the application of overexpressing the encoding gene of the flavonol synthase described in the above technical solution in reducing the content of anthocyanins and / or increasing the content of flavonols in plants.

[0012] The present invention also provides the application of the flavonol synthase, the encoding gene, the expression vector, or the host bacteria described in the above-mentioned technical solutions in improving plant flower color.

[0013] This invention also provides the application of overexpressing the gene encoding the flavonol synthase described in the above technical solution in reducing the color of plant petals.

[0014] This invention also provides the application of the flavonol synthase described in the above-mentioned technical solutions, or the encoding gene described in the above-mentioned technical solutions, or the expression vector described in the above-mentioned technical solutions, or the host bacteria described in the above-mentioned technical solutions, in any one or more of ① to ③:

[0015] ① Catalytic synthesis of quercetin from dihydroquercetin;

[0016] ② Catalytic synthesis of kaempferol from dihydrokaempferol;

[0017] ③ Catalytic synthesis of myricetin from dihydromyricetin.

[0018] This invention provides a flavonol synthase. Flavonol synthase is a key enzyme in the flavonol synthesis pathway and is essential for the biosynthesis and accumulation of flavonols in plants. This invention provides a flavonol synthase from *Rhododendron simsii*, which catalyzes the reaction of three dihydroflavonols (dihydroquercetin, dihydrokaempferol, and dihydromyricetin) to produce the corresponding flavonols. This controls flavonol accumulation and also affects anthocyanin accumulation, thereby improving flower color. Experimental results show that this invention is the first to clone a cDNA encoding a key enzyme gene (Flavonol synthase, FLS) related to flavonol metabolism in *Rhododendron simsii*. This invention utilizes the RdFLS gene to express recombinant RdFLS protein in *E. coli* cells. In in vitro enzyme activity experiments, this protein catalyzes the reaction of dihydroquercetin, dihydrokaempferol, and dihydromyricetin to produce quercetin, kaempferol, and myricetin, thus confirming that the recombinant protein expressed by the RdFLS gene in *E. coli* has the function of flavonol synthase. This invention involves transferring the RdFLS gene into Arabidopsis thaliana and tobacco. The resulting transgenic plants show a significant increase in flavonol accumulation and a decrease in anthocyanin accumulation. Simultaneously, the color of tobacco petals changes from pink to white or light pink, indicating that the above gene has potential application value in modifying the flower color of transgenic plants and improving the medicinal components of medicinal plants. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The diagram shows the results of multiple sequence alignment analysis of RdFLS1 provided by the present invention; in the box, the 2-ketoglutarate-Fe(II) oxygenation domain is represented, ● is an iron-binding residue, ☆ is a 2-ketoglutarate-binding residue, and Δ is a specific DHQ-binding residue.

[0021] Figure 2 The phylogenetic analysis results of RdFLS1 provided by this invention are shown in the figure.

[0022] Figure 3 The growth curve diagram provided for this invention;

[0023] Figure 4 The following diagrams show the detection results of RdFLS1 in vitro enzyme activity products provided by this invention; wherein, A: purification results of RdFLS recombinant protein; B: reaction results using dihydrokaempferol as a substrate; C: reaction results using dihydromyricetin as a substrate; D: reaction results using dihydroquercetin as a substrate.

[0024] Figure 5 The following figures illustrate the phenotypic and metabolite analysis results of RdFLS1 transgenic Arabidopsis thaliana plants provided by this invention; wherein, A: Phenotypic changes in RdFLS1 transgenic Arabidopsis thaliana; B: Quantitative analysis results of anthocyanins; C: Quantitative analysis results of flavonols.

[0025] Figure 6 The images provided by this invention show the phenotypic changes of RdFLS1 transgenic tobacco petals and the results of anthocyanin detection and analysis; wherein, A: phenotypic changes of RdFLS1 transgenic tobacco; B: petal extract image; C: anthocyanin quantitative analysis results; D: dihydroflavonol and flavonol quantitative analysis results. Detailed Implementation

[0026] This invention provides a flavonol synthase, the amino acid sequence of which is shown in SEQ ID NO.1: MEVERVRVQSLAHGGLHVLPAQFIRPASERPENSKALDGVTVPVISLSQPHDVVDEISRACSEWGFFLLTDHNVSPAAILRLKEVGEEFFNLPLKEKESYANDPSSGRFDGYGTKMTKNLDEKVEWVDYFFHVMYPPKKVNYDIWPKNPPSYRGATEEYSRELQQVTNKLLELLSEGLGLEGKALRSCLRDEEIEYEMKINMYPPCPQPELALGVEPHTDMSALTLLVPNDVPGLQVWKDSNWVAVNYLPNALFVHVGDQVEVLSNGKYKSVLHRSLVDKERTRMSWAVFVTPPHEAMIGPIPELINGENPSKYSTKTYAEYRHRKFNKIPQ.

[0027]

[0028] This invention also provides an expression vector containing the coding gene described in the above-described technical solution. In a specific embodiment, the expression vector includes a prokaryotic expression vector or a binary expression vector. In a specific embodiment, the prokaryotic expression vector can be constructed using pET32a(+) as the base vector, and the resulting prokaryotic expression vector can be named pET32-RdFLS; the binary expression vector can be constructed using pBI121 as the base vector, and the resulting binary expression vector can be named pBI121-RdFLS.

[0029] The present invention also provides a host bacterium containing the expression vector described in the above technical solution. In a specific embodiment, the host bacterium may be *Escherichia coli*.

[0030] The present invention also provides the application of the flavonol synthase described in the above-mentioned technical solutions, or the encoding gene described in the above-mentioned technical solutions, or the expression vector described in the above-mentioned technical solutions, or the host bacteria described in the above-mentioned technical solutions in controlling the synthesis of anthocyanins and / or flavonols in plants.

[0031] The present invention also provides the application of overexpressing the encoding gene of the flavonol synthase described in the above technical solution in reducing the content of anthocyanins and / or increasing the content of flavonols in plants.

[0032] The present invention also provides the application of the flavonol synthase, the encoding gene, the expression vector, or the host bacteria described in the above-mentioned technical solutions in improving plant flower color.

[0033] This invention also provides the application of overexpressing the gene encoding the flavonol synthase described in the above technical solution in reducing the color of plant petals.

[0034] This invention also provides the application of the flavonol synthase described in the above-mentioned technical solutions, or the encoding gene described in the above-mentioned technical solutions, or the expression vector described in the above-mentioned technical solutions, or the host bacteria described in the above-mentioned technical solutions, in any one or more of ① to ③:

[0035] ① Catalytic synthesis of quercetin from dihydroquercetin;

[0036] ② Catalytic synthesis of kaempferol from dihydrokaempferol;

[0037] ③ Catalytic synthesis of myricetin from dihydromyricetin.

[0038] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a flavonol synthase, its encoding gene, expression vector, host bacterium, and its applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Cloning of the RdFLS1 gene in Rhododendron simsii

[0041] Primers were designed based on the full-length transcriptome sequencing results of Rhododendron simsii (RdFLS-F1: GCCCATTCGCATCATACACTA, SEQ ID NO.3, RdFLS-R1: TGAGACGATTGCCTAGTAAA, SEQ ID NO.4) to amplify the cDNA of the FLS gene that controls flavonol synthesis in Rhododendron simsii. The amplified fragment with an open reading frame of 1002 bp (SEQ ID NO.2) was obtained by PCR amplification (94℃ for 8 min; 94℃ for 30 s, 54℃ for 1.5 min, 72℃ for 8 min, 30 cycles), encoding 333 amino acids (SEQ ID NO.1).

[0042] The ORF sequence of the RdFLS gene (SEQ ID NO.2):

[0043]

[0044] The protein sequence encoded by the nucleotide sequence of the RdFLS1 gene (SEQ ID NO.1):

[0045] MEVERVRVQSLAHGGLHVLPAQFIRPASERPENSKALDGVTVPVISLSQPHDVVVDEISRACSEWGFFLLTDHNVSPAAILRLKEVGEEFFNLPLKESYANDPSSGRFDGYGTKMTKNLDEKVEWVDYFFHVMYPPKKVNYDIWPKNPPSYRGATEEYSRELQQV TNKLLELLSEGLGLEGKALRSCLRDEEIEYEMKINMYPPCPQPELALGVEPHTDMSALTLLVPNDVPGLQVWKDSNWVAVNYLPNALFVHVGDQVEVLSNGKYKSVLHRSLVDKERTRMSWAVFVTPPHEAMIGPIPELINGENPSKYSTKTYAEYRHRKFNKIPQ.

[0046] Comparative analysis of RdFLS with known functional FLS from Arabidopsis thaliana, tobacco, Ginkgo biloba, and Camellia japonica showed that the FLS from Rhododendron simsii possesses an active site domain commonly found in flavonol synthases. Figure 1 Subsequently, a phylogenetic analysis was performed on the amino acid sequences of flavonol synthases from different plant sources and compared with RdFLS. The results showed that RdFLS belongs to the flavonol synthase family (…). Figure 2 Based on the above bioinformatics analysis results, it is speculated that the RdFLS gene of Rhododendron simsii may have the function of flavonol synthase.

[0047] Multiple sequence alignment method: DNAMAN was used to perform multiple sequence alignment of RdFLS with flavonol synthases from Arabidopsis thaliana, tobacco, ginkgo and camellia, and their conserved regions were marked according to the literature. Figure 1 The image shows the results of multiple sequence alignment analysis of RdFLS1; in the box, the 2-ketoglutarate-Fe(II) oxygenation domain is represented, ● represents ferrous binding residue, ☆ represents 2-ketoglutarate binding residue, and Δ represents a specific DHQ binding residue.

[0048] Evolutionary analysis method: The amino acid sequences of different plant flavonol synthases were downloaded from NCBI, multiple sequence alignment was performed using Clustalw, and then the phylogenetic tree was constructed using MEGA 6.0. Figure 2 The diagram shows the phylogenetic analysis results of RdFLS1, based on... Figure 2It can be seen that RdFLS1 aggregates with flavonol synthases from other species, and is most closely related to Camellia FLSa, indicating that RdFLS1 is likely to have the function of flavonol synthase.

[0049] Example 2

[0050] Functional study of RdFLS1 gene in Rhododendron simsii

[0051] 1. Enzyme activity detection of RdFLS1 in Rhododendron simsii

[0052] 1) Construction of prokaryotic expression vectors

[0053] To verify the enzymatic activity of the RdFLS protein, the cloned full-length RdFLS gene was used as a template. Primers containing EcoRI and NotI restriction sites (RdFLS-32F: GAATTCATGGAGGTGGAGAG, SEQ ID NO.5; RdFLS-32: GCGG CCGCTTATTGTGGAATCT, SEQ ID NO.6) were used for PCR amplification of the RdCHS4 open reading frame. PCR amplification yielded RdFLS products with EcoRI and NotI restriction sites at the 5' and 3' ends, respectively. These products were then recovered from the gel, ligated into T cells, transformed into JM109 competent cells, and identified by bacterial PCR and restriction digestion. The corresponding plasmids were then sequenced. Sequencing results showed that restriction sites were successfully introduced at both ends of the RdFLS gene. The correctly sequenced bacterial culture was inoculated, and plasmids were extracted in large quantities and digested with enzymes. After verifying that the size and brightness of the digestion products were correct, they were ligated into the pET32a(+) vector and transformed into JM109 competent cells. The next day, clones were randomly picked from the transformation plate and subjected to bacterial PCR and enzyme digestion verification. After the enzyme digestion results showed that the band size was as expected, the positive clones were sent for sequencing. The sequencing results were consistent with the original sequence, proving that the prokaryotic expression vector was successfully constructed. The successfully constructed recombinant plasmid was named pET32-RdFLS and introduced into E. coli BL21 cells for the large-scale preparation of soluble recombinant protein.

[0054] 2) Induction and purification of soluble recombinant proteins

[0055] The *E. coli* strains were streaked onto LB solid medium containing Amp (100 μg / ml) and incubated upside down at 37°C for 12–16 hours. Clones were picked and incubated overnight. The next day, 1% of the bacterial culture was inoculated into 5 ml tubes containing LB liquid medium and incubated at 37°C with shaking at 200 rpm. One tube was removed every half hour and temporarily stored at 4°C. One tube was left uninoculated as a blank control. 2 ml of the bacterial culture was collected at each time point, and the OD was measured. 600The values ​​were measured three times at each time point, and growth curves were plotted. The results showed that E. coli entered the logarithmic growth phase after 2 hours, and the growth rate was fastest and the bacterial state was optimal at 2.5 hours. Therefore, IPTG induction of RdFLS bacteria was chosen after 2.5 hours. Figure 3 The induction conditions were 37℃, 200rpm shaking culture for 2.5h, followed by the addition of IPTG to a final concentration of 1mM, and then continued induction culture in a constant temperature shaker. After exploring different IPTG concentrations and different induction times, the optimal induction conditions for RdFLS protein expression were finally determined to be 15℃, 1.0mM IPTG for 24h.

[0056] The protein was prepared in large quantities according to the above conditions, and the target protein was separated and purified by elution with nickel column and imidazole. The eluted protein was collected in large quantities and dialyzed. The target protein was concentrated using color-changing silica gel at low temperature. The obtained target protein was stored at -80℃ for enzyme activity detection. Figure 4 A in the middle.

[0057] The detailed steps for protein separation, purification, dialysis, and concentration are as follows:

[0058] ① Separation and purification:

[0059] a. Column packing: Slowly add the packing solution to the Ni-NTA pre-packed column, continuously compacting with 20% ethanol. When the packing volume reaches about 2 ml, further compact with 10 ml of 20% ethanol. Finally, equilibrate the column with 20 mM PBS buffer and store at 4°C for later use.

[0060] b. Large-scale preparation of RdFLS1 recombinant protein

[0061] (1) Under optimal induction conditions, Escherichia coli culture was prepared in large quantities after inoculation and shaking at a concentration of 1%.

[0062] (2) Aliquot the Escherichia coli culture into 50ml centrifuge tubes and centrifuge them in a high-speed centrifuge. Set the temperature to 4℃, the speed to 5000rpm, and centrifuge for 10min. Discard the supernatant and collect the bacterial cells.

[0063] (3) Add 5 ml of 20 mM PBS to each 50 ml of bacterial culture to suspend the bacterial cells, insert into ice, and incubate on ice for 30 min.

[0064] (4) Ultrasound: Set the ultrasound power to 45%, run for 5 seconds and stop for 5 seconds, set for 10 minutes, use ultrasound to break up cells, and repeat ultrasound once after the bacterial solution cools down.

[0065] (5) After the bacterial culture is broken, it is centrifuged in a high-speed centrifuge. The temperature is set to 4℃ and the speed is 6000rpm for 15min. The supernatant is the crude protein extract.

[0066] c. Sample loading: The collected supernatant solution was loaded onto the Ni column three times and equilibrated with three times the volume of PBS.

[0067] d. Imidazole elution: After equilibration, elute with elution buffers of imidazole concentrations of 10mM, 20mM, 50mM, 80mM, 200mM and 500mM in sequence. Collect 5 tubes of elution buffer for each concentration gradient, 2 ml / tube.

[0068] e. Column washing: Wash the column with 20mM PBS buffer, then soak it in 20% ethanol and store at 4°C.

[0069] f. SDS-PAGE electrophoresis verification: The eluent collected after column chromatography was subjected to SDS-PAGE electrophoresis to analyze the elution and purification of recombinant proteins.

[0070] ② Dialysis and Concentration:

[0071] a. Treatment of dialysis bags: Cut the dialysis bags to 15-20 cm, boil them in 2% (w / v) Na₂HCO₃ solution (containing 1 mM EDTANa₂, pH 8.0) for 10 minutes, rinse them thoroughly with deionized water, and then boil them in 1 mM EDTANa₂ (pH 8.0) for 10 minutes. After rinsing the dialysis bags thoroughly with deionized water, soak them in deionized water and store them at 4°C for later use. Used dialysis bags can be reused after being checked for leaks and boiled in distilled water for 30 minutes.

[0072] b. Based on the results of SDS-PAGE electrophoresis, collect the protein eluent with a single band and good concentration, and place it in a prepared dialysis bag. Dialyze the bag sequentially with 2L of dialysis buffer I, II, III, and IV in a 4°C cryo-chromatograph, for approximately 10 hours with each buffer. Dialysis buffer I: 20mM PBS, 500mM NaCl (pH 7.4); II: 20mM PBS, 300mM NaCl (pH 7.4); III: 20mM PBS, 100mM NaCl (pH 7.4); IV: 20mM PBS (pH 7.4).

[0073] c. Embed the protein packaged in the dialysis bag in pre-cooled color-changing silica gel and concentrate it in a 4°C freezer until it reaches about 1-2 ml. Take a small amount of sample for SDS-PAGE electrophoresis to verify the concentration. Store the remaining sample at -80°C for later use.

[0074] 3) Enzyme activity detection and analysis of RdFLS1

[0075] Using dihydroquercetin, dihydrokaempferol, and dihydromyricetin as substrates, the enzyme activity reaction systems were prepared according to Table 1. After mixing, the systems were placed in a 30°C water bath for 60 min. Subsequently, an equal volume of ethyl acetate (500 μL) was added to the reaction system for extraction (with vigorous shaking). The mixture was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant (ethyl acetate) was collected for HPLC analysis. Figure 4 ( Figure 4 The image shows the results of in vitro enzyme activity assays for RdFLS1. A: Purification of recombinant RdFLS protein; B: Reaction using dihydrokaempferol as a substrate; C: Reaction using dihydromyricetin as a substrate; D: Reaction using dihydroquercetin as a substrate. Results are as follows: Figure 4 As shown by the middle arrow, RdFLS can catalyze the reaction of dihydrokaempferol to produce kaempferol (B), the reaction of dihydromyricetin to produce myricetin (C), and the reaction of dihydroquercetin to produce quercetin (D), proving that RdFLS has the activity of flavonol synthase.

[0076] Table 1 Recombinant protease activity reaction system

[0077]

[0078] HPLC detection conditions:

[0079] Column temperature: 30℃; flow rate: 1mL / min; detection wavelength: 350nm; sample loading volume: 10μL (50μL); mobile phase: Aqueous phase A: 0.1% formic acid; Organic phase B: methanol; specific mobile phase conditions are shown in Table 2.

[0080] Table 2 Mobile phase conditions

[0081]

[0082] Effects of 2RdFLS1 on the synthesis of flavonols and anthocyanins in Arabidopsis and tobacco

[0083] 1) Construction of binary expression vectors

[0084] To verify the effect of the RdFLS1 gene on the synthesis of flavonols and anthocyanins in Arabidopsis thaliana and tobacco, the cloned full-length RdFLS1 gene was used as a template, and PCR amplification of the RdFLS1 open reading frame was performed using primers with BamHI and XbaI restriction sites (RdFLS-121F: GCTTAGAATGGAGGTGGAGAGGGTG, SEQ ID NO.7; RdFLS-121R: CGGGATCCTTATTGTGGAATCTTGTT, SEQ ID NO.8).

[0085] The target fragment was amplified extensively using the primers described above. After PCR product detection by 0.8% agarose gel electrophoresis, the target gene containing Xba I and BamHI restriction sites was recovered and verified. The validated gel-recovered product was ligated into the pMD18-T cloning vector and incubated overnight at 16°C. The next day, it was transformed into *E. coli* JM109 competent cells. After clones grew in the transformation plate, single clones were randomly selected for colony PCR. Positive clones were inoculated, plasmids were extracted, and verified by double digestion with Xba I and BamHI. The plasmid that tested positive was named recombinant plasmid T+RdFLS1(X+B) and sent for sequencing.

[0086] The recombinant plasmid T+RdFLS1(X+B) and the eukaryotic expression vector plasmid pBI121 were simultaneously digested with Xba I and BamHI restriction endonucleases. The digestion products were verified by 0.8% agarose gel electrophoresis. The correctly sized target fragment (RdFLS1) and linear vector (linearized pBI121) were recovered and verified. After successful verification, the linear vector and target fragment were mixed at a molar ratio of 1:10 and ligated overnight at 16°C.

[0087] All ligation products were transformed into E. coli JM109 competent cells and plated on Kansas. + On the surface of resistant LB solid medium; the next day, single clones were picked for bacterial PCR identification. Positive clones were inoculated, recombinant plasmids were extracted, and after successful identification by double digestion with XbaI and BamHI, they were sent for sequencing. The recombinant plasmid with correct sequencing results was named pBI121-RdFLS1.

[0088] 2) Phenotypic changes and analysis of flavonols and anthocyanins in transgenic plants

[0089] Using the constructed eukaryotic expression vector pBI121-RdFLS1, Arabidopsis thaliana was infected with Agrobacterium and overexpressing transgenic plants were obtained through Kan resistance screening. Individual transgenic Arabidopsis plants were cultured, and seeds were collected. The collected seeds were planted on 1 / 2 MS medium containing resistance and 3% sucrose, and phenotypic changes in T2 generation seedlings were observed. Compared with wild-type plants, the purple phenotype in the cotyledons and hypocotyls of transgenic plants was weakened. Anthocyanins and flavonols were extracted from both wild-type and transgenic seedlings and quantitatively analyzed using HPLC. Figure 5 The results showed that, compared with the wild-type control, the transgenic plants had reduced anthocyanin content and increased flavonol content.

[0090] The specific method is as follows:

[0091] Sow an appropriate amount of wild-type Arabidopsis seeds evenly in flowerpots filled with moist soil, cover with plastic wrap, poke holes in the plastic wrap with toothpicks for ventilation, and place in a constant temperature and light incubation room at 25℃. Water every 2-3 days. After germination, remove the film and thin out the seedlings to about 5-8 plants per pot. When the plants reach their peak flowering period, remove the pods and keep the flowers as material for Agrobacterium infection.

[0092] a. pBI121-RdFLS1 (GV3101) was inoculated into 5 ml of solution containing Rif (final concentration 50 mg / L) and Kan. + In LB liquid medium (final concentration 50 mg / L), culture at 30°C and 200 rpm for 48 h with shaking.

[0093] b. Inoculate all of the above bacterial suspension into 250 ml of solution containing Rif (final concentration 50 mg / L) and Kans. + Expand the culture in LB liquid medium (final concentration 50 mg / L) until the bacterial culture OD is reached. 600 The value is 1.0 or higher.

[0094] c. Transfer the cultured fresh bacterial solution into 50ml centrifuge tubes and centrifuge at 4℃ and 5000rpm for 15 minutes. Discard the waste liquid and retain the precipitate.

[0095] d. After resuspending the bacterial cells in a small amount of 5% sucrose solution, add the corresponding volume of 5% sucrose solution, and add 60 μl of silwet-77 (surfactant) for every 100 ml of suspension. Stir evenly on a magnetic stirrer.

[0096] e. After stopping stirring, take a pot of Arabidopsis and invert it into the bacterial solution, ensuring the flowers are completely submerged, and keep it submerged for 5 minutes. Stir the bacterial solution again thoroughly before infecting the next pot of Arabidopsis. After removing the Arabidopsis, place it horizontally in a tray, add a small amount of distilled water, cover, and incubate overnight at room temperature in a dark environment. The next day, transfer the Arabidopsis to a light-supported culture rack for normal cultivation, watering and spraying with nutrient solution regularly.

[0097] f. After the transgenic Arabidopsis thaliana reached maturity, the T1 generation seeds were harvested and named pBI121-RdFLS-T1.

[0098] g. Screening of transgenic T1 generation seeds:

[0099] (1) Take an appropriate amount of transgenic T1 seeds into a 1.5ml EP tube, add pasteurization solution at a ratio of 1:8 (pasteurization solution: water), and shake up and down for 15 minutes to wash the seeds.

[0100] (2) After washing, centrifuge at 12000 rpm for 1 min at room temperature.

[0101] (3) After rinsing the ultra-clean workbench three times with sterile water, the seeds were sown on 1 / 2 MS selection medium containing 1% sucrose (Kan final concentration 50 mg / L, Carbonicillin final concentration 100 mg / L).

[0102] (4) After vernalization at 4℃ for 2 days, place it on a light-incubated culture rack for culture.

[0103] (5) After the seeds germinate, the selected green resistant seedlings are transplanted into the soil.

[0104] h. At this point, the base of the petiole of some transgenic Arabidopsis plants has returned to purple. The plants with restored color are cultured to maturity, and T2 generation seeds are harvested from each plant.

[0105] i. Phenotypic observation: Take an appropriate amount of wild-type and T2 generation RdFLS transgenic seeds and sow them in a clean bench in 1 / 2 MS medium (anthocyanin induction medium) containing 3% sucrose. After vernalization, culture for about 5 days and observe the color difference of seedlings under a stereomicroscope.

[0106] j. Collect wild-type and phenotypically altered RdFLS transgenic Arabidopsis seedlings to extract total RNA, and use RT-PCR to verify whether the recombinant vector has been successfully transferred into Arabidopsis.

[0107] k. Collect wild-type and phenotypic RdFLS transgenic Arabidopsis seedlings to extract flavonols and anthocyanins, and perform qualitative and quantitative analysis.

[0108] Figure 5 Figure 1 shows the phenotypic and metabolite analysis results of RdFLS1 transgenic Arabidopsis thaliana plants; where A: phenotypic changes in RdFLS1 transgenic Arabidopsis thaliana; B: quantitative analysis of anthocyanins; C: quantitative analysis of flavonols.

[0109] Transgenic tobacco plants were obtained through injection and tissue culture. After successful rooting, they were transplanted into soil for further cultivation. Petal phenotypic changes were observed after flowering. Compared to wild-type plants, the tobacco petals were lighter in color to varying degrees, and the color changes in the petal extract were consistent with the petal color changes. Figure 6 ).

[0110] Figure 6 Figure 1 shows the phenotypic changes and anthocyanin detection results of petals from RdFLS1 transgenic tobacco; where A: phenotypic changes of RdFLS1 transgenic tobacco; B: petal extract; C: quantitative analysis of anthocyanins; D: quantitative analysis of dihydroflavonols and flavonols.

[0111] Enzyme activity assays revealed that RdFLS1 can react with dihydroquercetin, dihydrokaempferol, and dihydromyricetin to generate the corresponding flavonols, proving that the RdFLS cloned in this invention is indeed a flavonol synthase. When the RdFLS1 gene was transferred into wild-type Arabidopsis thaliana, it reduced anthocyanin synthesis and increased flavonol synthesis in the cotyledons and hypocotyls of the transgenic Arabidopsis, while simultaneously reducing the color of tobacco petals to varying degrees. In conclusion, the cloned RdFLS1 gene from Rhododendron simsii can control the synthesis of anthocyanins and flavonols, and can be applied to the improvement of anthocyanin and flavonol synthesis in other plants.

[0112] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A flavonol synthase, characterized in that, The amino acid sequence of the flavonol synthase is shown in SEQ ID NO.

1.

2. The gene encoding flavonol synthase according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. An expression vector containing the encoding gene of claim 2.

4. The expression vector according to claim 3, characterized in that, The expression vectors include prokaryotic expression vectors or binary expression vectors.

5. A host bacterium containing the expression vector of claim 3 or 4.

6. The use of overexpression of the gene encoding the flavonol synthase of claim 1 in reducing the anthocyanin content and / or increasing the flavonol content in plants; wherein the reduction of anthocyanin content is to decrease the anthocyanin content in Arabidopsis cotyledons and hypocotyls or to decrease the anthocyanin content in tobacco petals.

7. The use of overexpression of the gene encoding the flavonol synthase of claim 1 in lightening the color of tobacco petals.

8. The use of the flavonol synthase of claim 1, the encoding gene of claim 2, the expression vector of claim 3 or 4, or the host bacterium of claim 5 in any one or more of ① to ③: ① Catalytic synthesis of quercetin from dihydroquercetin; ② Catalytic synthesis of kaempferol from dihydrokaempferol; ③ Catalytic synthesis of myricetin from dihydromyricetin.

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