CN111286508B

CN111286508B - Ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene and protein and application thereof

Info

Publication number: CN111286508B
Application number: CN201910806305.4A
Authority: CN
Inventors: 徐立安; 吴雅琼; 辛月; 胥猛; 黄淑婧
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2019-08-29
Filing date: 2019-08-29
Publication date: 2022-04-22
Anticipated expiration: 2039-08-29
Also published as: CN111286508A

Abstract

The invention discloses a ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene, and protein and application thereof, belonging to the technical field of genetic engineering. The base sequence of the flavonoid 3',5' -hydroxylase GbF3'5' H1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the flavonoid 3',5' -hydroxylase GbF3'5' H1 is shown as SEQ ID NO. 2. The gene sequence of the flavonoid 3',5' -hydroxylase GbF3'5' H1 is derived from ginkgo biloba. The invention clones the flavonoid 3',5' -hydroxylase GbF3'5' H1 gene from ginkgo, systematically identifies the function of the flavonoid 3',5' -hydroxylase gene, finds that the gene can improve the content of the related metabolites of plant flavonoids, and can participate in and promote the synthesis of the related metabolites of flavonoids in transgenic poplar.

Description

Ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene and protein and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene, and protein and application thereof.

Background

The CYP450 protein family is the most bulky family of plant genes, and plant CYP450 genes account for approximately 1% of the total genes in a plant genome. At present, CYP450 is widely spotlighted because of its basic function in plants, and is involved in the metabolism of exogenous chemical substances and endogenous biochemical substances. In particular, CYP450 participates in the secondary metabolism of plants, and mainly relates to the synthesis of structural macromolecules, pigments and defense compounds; and catabolism of various hormones or signaling molecules. CYP450 is a heme-dependent oxidase, and has a mixed function. Some of the important reactions catalyzed by CYP450 play an important role in the synthesis of plant phenylpropanes and flavonoids. For example, it may alter the content of anthocyanins downstream of the flavone pathway to affect the appearance of pigments.

Flavonoids are an important class of secondary metabolites in plants, have a variety of different physiological functions, and are considered to be important components of chemical defense mechanisms. The synthesis of the compound is started by phenylalanine (phenylalanine) and malonyl-coenzyme A (malonyl-coenzyme A) to gradually synthesize the aromatic compounds with various structures. Further modified into different subclasses of flavonoids by different classes of enzymes. At present, a plurality of genes involved in flavonoid synthesis are cloned, and the biosynthesis pathway of the flavonoid in arabidopsis thaliana is well researched. Compared with the single copy gene of Arabidopsis, the polygene family of Ginkgo biloba controls the steps of the biosynthetic pathway of flavones, forming a more complex network. Related studies have shown that flavonoid biosynthesis is regulated by key enzyme genes in the pathway. Although some of the key enzymes in the ginkgetin synthesis pathway (GbCHS, GbCHI, GbF3H and GbFLS) have catalytic activity in e.coli, it is not clear at present how these enzymes synthesize flavonoids in ginko in vivo due to the lack of related mutants.

Ginkgo biloba is an ancient writhing plant known as "activite", the only species existing in the Ginkgoaceae family. Its origin can be traced back to 2 hundred million years ago, and is a unique species representative of plant phylogeny, and has an important evolutionary position. Ginkgo has been grown around the world because of its good environmental and climatic adaptation. In addition, ginkgo biloba is an important medicinal tree species because its leaves are rich in flavonoids and terpenoids. Therefore, the ginkgo leaves are production type chemical plants of secondary metabolites with important pharmacological activity. Heretofore, the ginkgo biloba extract EGB761, one of the best known products of ginkgo biloba, has been widely used, including the prevention of senile dementia and cerebrovascular dysfunction. Although the supply of ginkgo leaves has many difficulties and chemical synthesis is far from being applied to industrial production, huge development potential and economic value exist. However, because a regeneration system of ginkgo biloba is not established, the ginkgo biloba genotype with high yield of flavonoids cannot be cultured by genetic transformation at present.

F3'5' H has extensive flavone substrate activity and is involved in the biosynthesis pathway of flavonoid-related metabolites. At present, the F3'5' H gene has been well studied in ornamental petunias. And F3'5' Hs have been isolated from various plants, such as grape, snapdragon, chamomile, tomato, etc., but have not been studied in Ginkgo biloba.

Disclosure of Invention

Aiming at the problems in the prior art, the invention solves the technical problem that F3'5' H gene participating in the synthesis of flavonoid-related metabolites is separated from ginkgo biloba, the invention separates and obtains ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene, provides the amino acid sequence of ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1, and provides the application of ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene in improving the content of the flavonoid-related metabolites in plants. The invention clones flavonoid 3',5' -hydroxylase GbF3'5' H1 gene from ginkgo, finds that the gene can improve the content of the related metabolites of plant flavonoid and can participate and promote the synthesis of the related metabolites of flavonoid in transgenic poplar.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

the base sequence of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene is shown as SEQ ID NO. 1.

The amino acid sequence of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 is shown as SEQ ID NO. 2.

An expression vector containing the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene.

The expression vector containing the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene clones ccdB gene located at the downstream of a CaMV 35S promoter onto a PBI121 vector by using Gateway technology to construct a gene vector containing Pro35S: : an expression vector of GbF3'5' H1.

The ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene is applied to improving the content of related metabolites of plant flavonoids. The method comprises the following steps: (1) constructing an expression vector containing the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene; (2) transforming the constructed expression vector into a plant or plant cell; (3) and culturing and screening to obtain transgenic plants.

The application of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene in improving the content of related metabolites of plant flavonoids is realized by cloning ccdB gene positioned at the downstream of CaMV 35S promoter onto a PBI121 vector by using Gateway technology to construct a gene containing Pro35S: : an expression vector of GbF3'5' H1.

The application of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene in improving the content of related metabolites of plant flavonoids utilizes agrobacterium strain EHA105 to transform a constructed expression vector into a plant.

The application of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene in improving the content of flavonoid-related metabolites of plants, wherein the plants are poplar.

The application of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene in improving the content of plant flavonoid-related metabolites, wherein the flavonoid-related metabolites comprise catechol, phenylalanine and 4', 5-dihydroxy-7-glucosyloxyflavanone.

Has the advantages that: compared with the prior art, the invention has the advantages that:

(1) the result of the invention not only provides information for the characteristics and functions of the gingko GbF3'5' H1, but also is helpful for better understanding the synthesis and potential molecular mechanism of the flavonoid compounds.

(2) The invention clones the gene of flavonoid 3',5' -hydroxylase GbF3'5' H1 from ginkgo, systematically identifies the function of the gene, finds that the gene can improve the content of the related metabolites of plant flavonoids, and can participate in and promote the synthesis of the related metabolites of the flavonoids in transgenic poplar.

Drawings

FIG. 1 is a qRT-PCR detection spectrum of gingko GbF3'5' H1 gene expression, wherein FIG. 1A is a diagram of expression rules of GbF3'5' H1 in various tissues, and FIG. 1B is a diagram of expression rules of gingko leaf GbF3'5' H1 gene at different periods;

FIG. 2 is a graph showing the results of transgenic plants and non-transgenic plants in GbF3'5' H1, wherein FIG. 2A is a graph of semi-quantitative PCR amplification of non-transgenic and transgenic poplar containing target fragments, FIG. 2B is a graph of relative expression levels of GbF3'5' H1 in 8 transgenic lines and non-transgenic poplar detected by qRT-PCR, and FIG. 2C is a graph of growth conditions of non-transgenic and transgenic poplar at 45 days;

FIG. 3 is a graph of the results of the measurement of differential metabolites between transgenic and non-transgenic plants, wherein FIG. 3A is an expression graph of differential metabolites between transgenic and non-transgenic seedlings, FIG. 3B is a graph of the results of the contents of 4', 5-dihydroxy-7-glucono flavanone in transgenic and non-transgenic seedlings, and FIG. 3C is a graph of the results of the contents of catechol, phenylalanine, piceatannol and resveratrol in transgenic and non-transgenic seedlings.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

Example 1 cloning and characterization of Ginkgo biloba flavonoid 3',5' -hydroxylase GbF3'5' H1 Gene

The extraction of DNA from ginkgo biloba is carried out by using Plant Genomic DNA Kit (cethyltetramethonium bromide (CTAB)) (Zoman, Beijing, China).

Total RNA was extracted from ginkgo leaves using the RNAprep Pure Plant kit (TIANGEN, Beijing, China), and specific primers for the GbF3'5' H1 gene were designed based on ginkgo transcriptome data (NCBI Short Reads Archive (SRA) database under access number SRP 137637). The full-length cDNA sequence of GbF3'5' H1 was cloned using Rapid Amplification CDNA Ends (RACE) technique. Nested primer design for amplification of full-length cDNA the SMATer RACE 5 '/3' Kit (Clontech, Japan) was used. All primers were designed using Oligo 6.0 software (as shown in table 1). PCR amplification, gel cutting, recovery and purification, and transferring into Escherichia coli competent cells via pMD19-T vector. Colonies were detected by PCR and positive colonies were selected for Sanger sequencing. The full length of GbF3'5' H1 was obtained by splicing the 5 'and 3' -RACE sequences and predicting the Open Reading Frame (ORF) using the NCBI ORF Finder. Thereafter, the GbF3'5' H1 ORF was PCR amplified in the following procedure: reacting at 95 ℃ for 3 min; at 35 cycles 95 ℃ for 30s, 55 ℃ for 40s, 72 ℃ for 90 s; finally, the extension is carried out for 10 minutes at 72 ℃. The PCR product was detected by 1% Agarose Gel electrophoresis, and the target fragment was recovered by Agarose Gel DNA purification kit, Agarose Gel DNA purification kit (TaKaRa, Dalian, China), and the target fragment was ligated to pMD19-T vector and transformed into E.coli TOP 10. And taking a single colony for culture. Screening positive clones were sequenced by Sanger.

TABLE 1 primer sequence information

In order to isolate the full-length cDNA of the GbF3'5' H gene, the predicted GbF3'5' H gene was subjected to 5 'RACE and 3' RACE detection, and then named GbF3'5' H1. The base sequence of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 is shown as SEQ ID NO. 2. The whole cDNA sequence of the gene is 1959bp, which contains 1527bp ORF, two sides are 42bp 5 '-UTR region and 387bp 3' -URT region, and the termination code is TGA.

Example 2 transcription and expression level of GbF3'5' H1 Gene

To detect the transcription and expression levels of the GbF3'5' H1 gene, a fluorescent quantitative PCR (qRT-PCR) analysis was performed. Total RNA was extracted using RNAprep plant kit (TIANGEN, Beijing, China), followed by PrimeScript^TMRT Master Mix (TAKAEA, Dalian, China) transcribes total RNA. The cDNA was diluted 5-fold as template.The GbF3'5' H1 primers and the internal reference gene primers designed for qRT-PCR amplification are shown in Table 1. qRT-PCR analysis was performed IN ABI ViiA 7Real-time PCR platform using FastStart Universal SYBR Green Master with ROX for RT-PCR Kit (Roche, Indianapolis, IN, USA). The 10 μ L reaction system and PCR reaction procedure were as follows: reacting at 95 ℃ for 2 min; 95 ℃ for 15s and 95 ℃ for 1min at 40 cycles. Three biological replicates were prepared per sample and the average relative expression was calculated. By using 2^-ΔΔCtThe method calculates relative expression levels. The duncan multiplex assay was performed using SPSS 22.0 software (SPSS inc., Chicago, IL, USA) and the difference was statistically significant for a p-value < 0.05.

The tissue expression research of the gene of ginkgo biloba is carried out by taking 1-year-old ginkgo biloba seedlings (leaves, stems, roots and petioles) and 25-year-old ginkgo biloba (kernels and buds) as materials. Taking 1-year-old ginkgo seedlings cultivated in a greenhouse of Nanjing forestry university as a test material, collecting leaves once every month from 4 months to 10 months at different leaf development stages, and researching the expression mode of the gene. After collection, the plant material was snap frozen in liquid nitrogen and placed in an ultra-low temperature freezer at-80 ℃.

The expression analysis results are shown in FIG. 3, where the expression level of the gene in the stem is set to 1, R in FIG. 1A means root, S means stem, L means leaf, and K means kernel; b refers to the bud, P refers to the petiole; in FIG. 1B, the expression level of GbF3'5' H1 gene at 6 months was set to 1, and the temporal expression pattern: 4. 5, 6, 7, 8, 9, 10 represent respectively month 4, month 5, month 6, month 7, month 8, month 9, month 10. The results indicate that GbF3'5' H1 accumulates in the leaf blades (significantly higher than elsewhere), followed by petioles, roots and nuclei without expression (as shown in fig. 1A). From month 4 to month 10, ginkgo biloba leaf develops through different stages (as shown in figure 1B). The expression result of GbF3'5' H1 gene shows that the expression level of mRNA of 4 months ginkgo leaves is the highest (more than 24 times of 6 months, and is obviously higher than other periods), and the expression level of 7 months ginkgo leaves is weak (the expression level is only 0.3).

Example 3 heterologous overexpression of GbF3'5' H1 in Populus

The ORF of GbF3'5' H1 cDNA was amplified by PCR and the ccdB gene located downstream of CaMV 35S promoter was cloned into PBI121 vector using Gateway technology (Invitrogen, CA, Carlsbad). The mixture containing Pro35S: : the vector of GbF3'5' H1 was introduced into Agrobacterium strain EHA105 for transformation. A stable and efficient genetic transformation system of Populus deltoids (Populus davidiana x Populus bolliana) is utilized to transfer GbF3'5' H1 gene of ginkgo into Populus deltoids. The tissue culture seedlings of the clone populus deltoids were grown under the conditions of 16h light and 8h dark photoperiod and under the conditions of 25 ℃ (day) and 18 ℃ (night).

To investigate the function of GbF3'5' H1 in plants, several 35S: : PCR validation was performed on the GbF3'5' H1 poplar transgenic line. 8 independent transgenic lines were randomly selected and the expression level of GbF3'5' H1 was detected by semi-quantitative PCR and qRT-PCR (primers see Table 1). The results show that GbF3'5' H1 is successfully expressed in 8 transgenic lines, and the results of the expression levels of the semi-quantitative PCR analysis and the qRT-PCR analysis are consistent (as shown in FIGS. 2A and 2B, WT in the figures is a non-transgenic poplar, and L1-L10 are transgenic poplar lines). The results of qRT-PCR analysis showed that GbF3'5' H1 has the highest expression level in transgenic line L4 (the relative expression level is 426 times that of non-transgenic seedlings), and then is transgenic line L1 and line L6. The expression quantity of 8 transgenic lines is higher than that of non-transgenic poplar. As seen in FIG. 2C, both transgenic and non-transgenic seedlings grew healthily.

Example 4 differential metabolites between transgenic and non-transgenic plants

To determine whether overexpression of GbF3'5' H1 affects flavonoid-related metabolite synthesis in transgenic plants, non-targeted metabolic assays and analyses of differential metabolites in transgenic poplar and non-transgenic shoot leaves in example 3 were used, and the relative concentrations of metabolite expression were determined. Non-targeted metabolome analysis uses the following method:

1. the sample treatment comprises the following steps:

1) each plant sample was precisely weighed to 60mg, placed into a 1.5mL centrifuge tube, and 40 μ L of internal standard (L-2-chloro-phenylalanine, 0.3mg/mL, methanol formulation) was added;

2) sequentially adding two small steel balls and 360 μ L of cold methanol, and standing in a refrigerator at-20 deg.C for 2 min;

3) grinding in a grinder (60Hz, 2 min);

4) performing ultrasonic extraction in ice water bath for 30 min;

5) adding 200 μ L chloroform, vortexing in a vortex machine (60Hz, 2min), adding 400 μ L water, and vortexing in a vortex machine (60Hz, 2 min);

6) performing ultrasonic extraction in ice water bath for 30 min;

7) standing at-20 deg.C for 30 min;

8) centrifuging at low temperature for 10min (13000rpm, 4 ℃), and filling 300. mu.L of supernatant into a glass derivatization bottle;

9) the quality control sample (QC) is prepared by mixing the extracting solutions of all samples in equal volume, and the volume of each QC is the same as that of the sample;

10) the samples were evaporated to dryness using a centrifugal concentration desiccator.

11) To a glass derivative vial was added 80. mu.L of methoxylamine hydrochloride pyridine solution (15mg/mL), vortexed and shaken for 2min, and then oximation reaction was performed in a shaking incubator at 37 ℃ for 90 min.

12) After the sample was taken out, 80. mu.L of BSTFA (containing 1% TMCS) derivative reagent and 20. mu.L of n-hexane were added, and 11 internal standards (C8/C9/C10/C12/C14/C16, 0.8 mg/mL; C18/C20/C22/C26/C28, 0.4mg/mL in chloroform) 10. mu.L, vortexed for 2min, and reacted at 70 ℃ for 60 min.

13) After the sample is taken out, the sample is placed at room temperature for 30min for GC-MS metabonomics analysis.

2. Gas chromatography-mass spectrometry conditions

Chromatographic conditions are as follows: DB-5MS capillary column (30m × 0.25mm × 0.25 μm, Agilent J & W Scientific, Folsom, CA, USA), carrier gas is high purity helium (purity not less than 99.999%), flow rate is 1.0mL/min, and injection port temperature is 260 deg.C. The sample introduction amount is 1 mu L, the split flow sample introduction ratio is 4: 1, and the solvent delay is 5 min.

Temperature programming: the initial temperature of the column incubator is 60 ℃, the temperature is increased to 125 ℃ by a program of 8 ℃/min, and the temperature is increased to 210 ℃ by 5 ℃/min; heating to 270 deg.C at 10 deg.C/min, heating to 305 deg.C at 20 deg.C/min, and maintaining for 5 min.

Mass spectrum conditions: and electron bombardment ion source (EI) with the ion source temperature of 230 ℃, the quadrupole rod temperature of 150 ℃ and the electron energy of 70 eV. The scanning mode is a full SCAN mode (SCAN), and the mass scanning range is as follows: m/z is 50-500.

3. Differential metabolite screening

And screening differential metabolites among groups by adopting a method combining multidimensional analysis and single-dimensional analysis. In the OPLS-DA analysis, variable weight values can be used for measuring the influence strength and the interpretability of the expression pattern of each metabolite on the classification and judgment of each group of samples, and differential metabolites with biological significance are mined, wherein the metabolites with VIP & gt 1 are generally regarded as differential metabolites. The significance of the differential metabolites between groups was further verified using the t-test (student's t test). The screening criteria were that the first principal component of the OPLS-DA model had a VIP value > 1 and a p value < 0.05. Wherein the fold change is the ratio of the average content of metabolites in the two groups.

Non-targeted GC-MS analysis showed 45 differential metabolites between the nontransformed genome and the transgenic group (as shown in fig. 3A). These 45 differential metabolites were mainly divided into 9 major groups, and these differential metabolites were probably caused by overexpression of the GbF3'5' H1 gene. The content of 17 different metabolites in transgenic poplars is higher than that of non-transgenic seedlings, the metabolites with up-regulated differences are probably caused by the over-expression of GbF3'5' H1 genes in poplars, so that the content of the 17 metabolites is obviously improved, and meanwhile, the improvement of the content of the metabolites has certain influence on the improvement of the content of related metabolites. Furthermore, 5 of the transgenic shoot metabolites (4 ', 5-dihydroxy-7-glucono flavanone, catechol, phenylalanine, piceatannol and resveratrol) and the flavone related metabolites showed differential expression, suggesting that GbF3'5' H1 may influence or interact with the content of different types of flavone compounds or other metabolites by influencing the content of these 5 metabolites. Of these, the content of 3 differential metabolites associated with flavones was significantly up-regulated. The flavonoids belong to phenols in plant secondary metabolites, 4', 5-dihydroxy-7-glucosyloxyflavanone (4 ', 5-dihydroxy-7-glucosyloxyflavanone) in transgenic seedlings is remarkably higher than the expression level in non-transgenic seedlings and is about 3 times of the content of the non-transgenic seedlings (figure 3B), which has a certain promotion effect on the improvement of the flavonoid content, and the overexpression of GbF3'5' H1 gene can generate a large amount of 4', 5-dihydroxy-7-glucosyloxyflavanone in plants. The transgenic plants had higher metabolite content of catechol (catechol) and phenylalanine (phenylalanine) than wild type plants (fig. 3C). Wherein, the increase of the phenylalanine content in the transgenic plant is beneficial to the biosynthesis of related metabolites such as downstream flavonoid and the like. This is because the biological synthesis of flavonoids starts from the phenylalanine synthesis pathway, which starts with phenylalanine and malonyl-coa to gradually synthesize aromatic compounds with diverse structures. Thus, an increase in phenylalanine content in transgenic plants favors the biosynthesis of downstream flavonoids and their associated metabolites.

Sequence listing

<110> Nanjing university of forestry

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Ala Val Leu Tyr Val Gly Phe Arg Tyr Val Leu Asn Ser Arg Lys Lys

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Arg Lys Leu Pro Pro Gly Pro Ser Gly Trp Pro Leu Val Gly Ser Leu

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Pro Leu Leu Gly Pro Met Pro His Val Thr Leu Tyr Asn Leu Ala Lys

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Lys His Gly Pro Ile Leu Tyr Leu Lys Leu Gly Thr Ser Ala Met Val

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Val Ala Ser Ser Pro Glu Thr Ala Lys Ala Phe Leu Lys Thr Leu Asp

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Leu Asn Phe Ser Asn Arg Pro Gly Asn Ala Gly Ala Thr Tyr Leu Ala

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Tyr Asp Ser Gln Asp Met Val Trp Ala Pro Tyr Gly Pro Arg Trp Lys

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Asp Asp Trp Gln Pro Val Arg Glu Ala Glu Met Gly His Met Leu Arg

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Leu Ile Leu Gln His Ser Ser Arg Arg Ser Asn Pro Val Val Asn Ile

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Arg Asn Met Lys Lys Leu His Lys Arg Phe Asp Ala Leu Leu Thr Arg

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Phe Leu Asp Ile Ile Met Ser His Arg Glu Asn Ala Asp Gly Asp Gly

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Gly Arg Leu Thr Asp Val His Ile Lys Ser Leu Leu Leu Asn Leu Phe

290 295 300

Thr Ala Gly Thr Asp Thr Ser Ser Ser Ile Ile Glu Trp Ala Val Ala

305 310 315 320

Glu Leu Ile His Asn Pro Glu Ile Ala Lys Arg Ala Gln Arg Glu Met

325 330 335

Asp Thr Val Ile Gly Arg Glu Arg Lys Leu Lys Glu Ser Asp Ile Ala

340 345 350

Asn Leu Pro Tyr Leu Val Ala Ile Cys Lys Glu Thr Phe Arg Lys His

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Pro Ser Thr Pro Leu Ser Leu Pro Arg Val Ala Asp Gln Asp Cys Leu

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Val Asp Gly Tyr Phe Ile Pro Lys Asp Thr Lys Leu Met Val Asn Val

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Trp Gly Ile Gly Arg Asp Pro Asp Leu Trp Glu Lys Pro Leu Glu Phe

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Asn Pro Asp Arg Phe Leu Thr Pro Lys Gly Ser Lys Ile Asp Pro Arg

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Gly Asn Asp Phe Glu Leu Ile Pro Phe Gly Ala Gly Arg Arg Ile Cys

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Ala Gly Thr Arg Met Gly Ile Lys Leu Val Glu Tyr Ile Leu Gly Ser

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Leu Ile His Ser Phe Asn Trp Asp Leu Pro Pro Asn Gln Lys Gln Leu

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Asn Met Asp Glu Ala Phe Gly Leu Ala Leu Gln Lys Ala Val Pro Phe

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Claims

1. The base sequence of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene is shown in SEQ ID NO. 1.

2. The amino acid sequence of the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 is shown in SEQ ID NO. 2.

3. An expression vector comprising the ginkgo flavonoid 3',5' -hydroxylase gene GbF3'5' H1 according to claim 1.

4. The expression vector containing the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene as claimed in claim 3, wherein the ccdB gene located downstream of CaMV 35S promoter is cloned onto PBI121 vector by using Gateway technology to construct an expression vector containing Pro35S:: GbF3'5' H1.

5. Use of the ginkgo flavonoid 3',5' -hydroxylase gene GbF3'5' H1 according to claim 1 for increasing the content of flavonoid-related metabolites in plants, wherein the plants are poplar, and the flavonoid-related metabolites are catechol, phenylalanine or 4', 5-dihydroxy-7-glucosyloxyflavanone.

6. The use of the ginkgo flavonoid 3',5' -hydroxylase gene GbF3'5' H1 according to claim 5 for increasing the content of plant flavonoid-related metabolites, comprising the steps of: (1) constructing an expression vector containing the ginkgo flavonoid 3',5' -hydroxylase GbF3'5' H1 gene; (2) transforming the constructed expression vector into a plant or plant cell; (3) and culturing and screening to obtain transgenic plants.

7. The use of the ginkgo biloba flavonoid 3',5' -hydroxylase gene GbF3'5' H1 gene for increasing the content of plant flavonoid-related metabolites according to claim 6, wherein the step (1) comprises cloning ccdB gene located downstream of CaMV 35S promoter into PBI121 vector by using Gateway technology to construct an expression vector containing Pro35S: GbF3'5' H1.

8. The use of ginkgo flavonoid 3',5' -hydroxylase gene GbF3'5' H1 gene according to claim 6 for increasing the content of plant flavonoid-related metabolites, wherein in step (2), the constructed expression vector is transformed into plants using Agrobacterium strain EHA 105.