Chalcone synthase gene of Lonicera japonica and its application in increasing the content of flavonoids in plants

Abstract

The present invention provides a honeysuckle chalcone synthase gene and its application in increasing the content of plant flavonoids. The honeysuckle chalcone synthase gene has a sequence shown in SEQ ID NO: 1. The gene expresses the amino acid sequence shown in SEQ ID NO: 2. The gene can be used to increase the content of plant flavonoids.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a Lonicera japonica chalcone synthase gene (EVM0004700) and application thereof in increasing the content of plant flavonoids. Background Art

[0002] Lonicera maackii, also known as honeysuckle, is a deciduous shrub of the genus Lonicera in the family Caprifoliaceae. Its flowers are white or yellow, with yellow and white contrasting; its fruit is berry-like, usually red when ripe, and frost-resistant. It belongs to the same genus as honeysuckle (Lonicera ajaponica). Compared with the widely studied honeysuckle, the research on Lonicera maackii is still in its infancy. Preliminary studies have shown that Lonicera maackii has important medical potential in improving disease resistance, anti-inflammatory and anti-tumor activities, promoting blood circulation, removing blood stasis and detoxification. Its beneficial health factors are attributed to its biological natural active ingredients, such as flavonoids.

[0003] Flavonoids are indispensable chemicals in the secondary metabolism of plants, playing an important role in plant growth and development, and responses to biological and non-biological environmental factors. At the same time, they have a variety of biological activities such as antibacterial, anti-inflammatory, anti-tumor, and antioxidant properties. Studies have shown that chalcone synthase (CHS) is a key enzyme and rate-limiting enzyme in the flavonoid metabolic pathway in plants. It catalyzes the condensation reaction of three molecules of malonyl CoA and one molecule of β-coumaroyl CoA to produce chalcone, which is the basic carbon frame structure for the formation of flavonoid substances.

[0004] At present, chalcone synthase genes have been cloned from a variety of plants, such as Arabidopsis, tobacco, rice, Millettia reticulata, etc. However, there are no reports on the cloning of chalcone synthase genes in Lonicera japonica and its application in increasing the content of plant flavonoids. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. The present invention provides a nucleotide sequence of the Lonicera japonica EVM0004700 gene. The provided Lonicera japonica EVM0004700 gene can be used to regulate the biosynthesis of plant flavonoids.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect of the present invention, an isolated nucleic acid molecule is provided, wherein the nucleic acid molecule encodes Lonicera japonica chalcone synthase EVM0004700, and the nucleic acid molecule has a sequence shown in SEQ ID NO:1.

[0008] According to an embodiment of the present invention, the nucleic acid is cDNA.

[0009] In a second aspect of the present invention, a Lonicera japonica chalcone synthase EVM0004700 gene is provided, wherein the gene has a sequence shown in SEQ ID NO:1.

[0010] In a third aspect of the present invention, a Lonicera japonica chalcone synthase protein is provided, the protein having an amino acid sequence shown in SEQ ID NO: 2. The provided protein has Lonicera japonica chalcone synthase activity.

[0011] In the fourth aspect of the present invention, a construct is provided, comprising the nucleic acid molecule described in the first aspect or the gene described in the second aspect.

[0012] According to an embodiment of the present invention, the construct is at least one of a plasmid, a virus, and a bacteriophage.

[0013] In the fifth aspect of the present invention, a host cell is provided, comprising the nucleic acid molecule described in the first aspect, the gene described in the second aspect, or the construct described in the fourth aspect.

[0014] In the sixth aspect of the present invention, a transgenic plant is provided, comprising the nucleic acid molecule described in the first aspect, or the gene described in the second aspect, or the construct described in the fourth aspect, or the recombinant cell described in the fifth aspect.

[0015] According to an embodiment of the present invention, the transgenic plant includes at least one of Arabidopsis, tobacco, rice, wheat, soybean, corn, sorghum, sugarcane, peanut, potato, and tomato.

[0016] In a seventh aspect of the present invention, a method for increasing plant flavonoids is provided, comprising:

[0017] Introducing the nucleic acid molecule described in the first aspect, or the gene described in the second aspect, or the construct described in the fourth aspect, or the recombinant cell described in the fifth aspect into the plant;

[0018] Plants overexpressing Lonicera japonica chalcone synthase protein were screened and obtained.

[0019] Specifically, the drug can be introduced into the plant by Agrobacterium transformation.

[0020] In an eighth aspect, the present invention provides the use of the nucleic acid described in the first aspect or the protein described in the second aspect in increasing the flavonoid content of transgenic plants.

[0021] The beneficial effects achieved by the present invention are:

[0022] The invention provides a gene encoding a chalcone synthase of Lonicera japonica, which is obtained from Lonicera japonica for the first time, filling the gap of isolating and cloning chalcone synthase genes from Lonicera japonica. The content of flavonoids in a transgenic strain overexpressing the chalcone synthase EVM0004700 of Lonicera japonica is significantly higher than that in a wild-type plant, and the EVM0004700 gene can be used to increase the content of flavonoids in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the expression of the EVM0004700 gene in different tissues provided by the embodiments of the present invention.

[0024] Figure 2 It is a graph showing the results of subcellular localization analysis of the EVM0004700 protein provided according to an embodiment of the present invention.

[0025] Figure 3 The expression of EVM0004700 in wild-type and overexpressed Arabidopsis thaliana, as well as the flavonoid content determination results provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0026] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0027] The invention provides an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes Lonicera japonica chalcone synthase EVM0004700, and the nucleic acid molecule has a sequence shown in SEQ ID NO:1.

[0028] The sequence shown in SEQ ID NO: 1 is as follows:

[0029] ATGGTTACTGTCGAGGAGGTTCGTAATGCTCAACGCGCCCAAGGCCCTGCAACCGTC

[0030] ATGGCCATCGGCACTGCAACTCCTGCCAACTGCGTCGACCAAAGCGAGTACCCCGAT

[0031] TTCTACTTCCGCATCACCAACAGCGAACACAGGACTGAGCTCAAAGAAAAATTCAAG

[0032] CGCATGTGCGAGAAATCTATGATCAAGAAGCGTTATATGCACTTGACGGAGGAAATC

[0033] TTGAAAAGCAACCGGATATGTGCGCGTACATGGCACCTTCTCTAGATGCTAGGCAA

[0034] GATATTGTGGTGGTAGAGTACCGAAACTTGGTAAAGAAGCGGCAACAAAGGCGAT

[0035] CAAGGAATGGGGCCAGCCCAAGTCTAAAATTACCCACCTAGTCTTTTGCACCACTAG

[0036] CGGTGTCGACATGCCCGGTGCTGACTACCAGCTCACCAAGCTCCTCGGTCTACGCCC

[0037] TTCGGTCAAGCGCCTCATGATGTACCAACAGGGTTGCTTTGCTGGTGGCACGGTTCTT

[0038] CGTCTAGCCAAGGACCTAGCCGAGAAACAAAGGTGCTCGTGTACTTGTGGTTTGC

[0039] TCCGAGATCACCGCGGTCACGTTCCGTGGGCCTAGTGATACGCACCTTGACAGCCTT

[0040] GTGGGTCAGGCCTTGTTTGGTGATGGTGCAGCCGCTGTGATCATTGGTTCGGACCCA

[0041] GTGCCCGAGGTCGAGAAGCCATTATTTGAGCTAGTTTCAGCATCACAAACCATTCTA

[0042] CCAGATAGTGATGGGGCTATTGATGGACACCTACGTGAAGTTGGGCTAACGTTTCAC

[0043] CTTCTCAAGGACGTACCCGGGCTCATATCAAAGAACATAGAAAAGAGCCTTGTGGA

[0044] GGCATTCAAGCCTTTGGGTATATCGGATTGGAACTCGATCTTTTGGATTGCTCATCCG

[0045] GGTGGACCGGCTATATTGGACCAAGTTGGAAAAAAAGTTGGCACTTAAGCCTGAGAA

[0046] GATGGGAGCTACAAGGCATGTGCTTAGCGAGTATGGAAATATGTCTAGCGCTTGTGT

[0047] GCTGTTTATAATGGATGAGATGAGGAAGAAATCAGCCAACGAGGGGCTTAAGACCA

[0048] CCGGAGAGGGGCTCGAATGGGGTGTGCTCTTTGGGTTCGGACCAGGGCTCACCGTCG

[0049] AGACTGTTGTTCTCCATAGTGTGTCCACTTAA(SEQ ID NO:1)

[0050] According to a specific embodiment, the nucleic acid is cDNA.

[0051] The invention also provides a protein with Lonicera japonica chalcone synthase activity, wherein the protein has an amino acid sequence shown in SEQ ID NO:2.

[0052] The sequence shown in SEQ ID NO:2 is as follows:

[0053] MVTVEEVRNAQRAQGPATVMAIGTATPANCVDQSEYPDFYFRITNSEHRTELKEKFKR

[0054] MCEKSMIKKRYMHLTEEILKEQPDMCAYMAPSLDARQDIVVVEVPKLGKEAATKAIKE

[0055] WGQPKSKITHLVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLA

[0056] KDLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIIGSDPVPEVEK

[0057] PLFELVSASQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLVEAFKPLGISDWN

[0058] SIFWIAHPGGPAILDQVEKKLALKPEKMGATRHVLSEYGNMSSACVLFIMDEMRKKSAN

[0059] EGLKTTGEGLEWGVLFGFGPGLTVETVVLHSVST(SEQ ID NO:2)

[0060] The present invention provides a kind of construct again, including the nucleic acid molecule described above. The construct mentioned can be a cloning vector such as a T vector, a lambda phage vector, a P1 phage vector, a cosmid vector, a bacterial artificial chromosome, a yeast artificial chromosome, pGEM-T, pUC18, or an expression vector such as an adenovirus vector, a retrovirus vector or a plasmid vector. The plasmid vector can be a plant expression vector such as an expression vector selected from plant expression vectors pBIN19, pBI121, pBI221, pCAMBIA1300, pGreen, etc., and the type of the vector can be selected according to actual needs. According to an embodiment of the present invention, the construct is at least one of a plasmid, a virus, and a phage.

[0061] The present invention further provides a host cell, comprising the nucleic acid molecule described above or the construct described in the third aspect.

[0062] The host cell includes but is not limited to plant, bacteria, yeast or insect cells. According to a specific embodiment, the recombinant cell is a plant cell. For example, the host cell can be a honeysuckle cell or other plant cells, and other plant cells can be monocotyledonous plant cells and dicotyledonous plant cells. For example, monocotyledonous plants include but are not limited to corn, wheat, sugarcane, reed, sorghum, ginger, sand ginger, grass fruit, Amomum villosum, Curcuma, Curcuma zedoaria, Alpinia oxyphylla, Alpinia officinalis, ginger flower, cardamom, etc. Dicotyledonous plants include but are not limited to soybean, peanut, sunflower, potato, tomato, etc.

[0063] The present invention further provides a transgenic plant, comprising the nucleic acid molecule, or the construct, or the host cell.

[0064] The present invention further provides a method for increasing plant flavonoids, comprising:

[0065] Introducing the above-mentioned nucleic acid, or the construct, or the host cell into the plant;

[0066] Plants overexpressing Lonicera japonica chalcone synthase protein were screened and obtained.

[0067] The overexpression mentioned here means that the expression level of the Lonicera japonica chalcone synthase protein shows a significant difference compared to the unintroduced plant or wild plant. According to a specific embodiment, the significant difference mentioned here may be p<0.5.

[0068] For example, the isolated nucleic acid molecule or gene provided by the present invention as described above can be transformed into a recipient plant cell by Agrobacterium-mediated method or gene gun method, and differentiated into a complete plant to obtain a plant capable of highly expressing flavonoids.

[0069] Of course, gene editing technologies such as zinc finger nuclease technology (Zinc Finger Nuclease, ZFN), TALEN technology (Transcription activator-like effector nucleases), CRISPR / Cas (Clustered Regularly Interspaced Short palindromic Repeat Sequences) technology, etc., can be used to modify the genome sequence of the recipient plant genome so that it contains the isolated nucleic acid molecule or gene as described above, using the isolated nucleic acid molecule or the gene as described above as a template.

[0070] The nucleic acid molecules or genes or proteins described above can be used to increase the flavonoid content of transgenic plants. The nucleic acid molecules or genes provided by the present invention can increase the flavonoid content in transgenic plants by at least 1.2 to 1.5 times compared to wild-type plants without transfection.

[0071] The protection scheme of the present invention is described below by specific examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents mentioned in the examples can be obtained commercially.

[0072] Example 1

[0073] Example 1 The Lonicera japonica EVM0004700 gene was cloned by the following method.

[0074] (1) Extraction of RNA from Lonicera japonica

[0075] The roots, stems, leaves, flowers and fruits of Lonicera japonica were collected and ground in a liquid nitrogen environment for later use. RNA was then extracted according to the instructions of the plant total RNA extraction kit (RNeasy Plant mini Kit, manufacturer: Tiangen Biochemical Technology Beijing Co., Ltd.).

[0076] (2) cDNA synthesis

[0077] 1) Prepare the reverse transcription mixture according to the following system and place it in a 200 μL RNase free PCR tube. The system is shown in Table 1 below:

[0078] Table 1: Reverse transcription system

[0079] Components Dosage Total RNA / mRNA 50ng-5μg Anchored oligo(dT)18primer 1μL 2×TS Reaction Mix 10μL TransScript RT / RI Enzyme Mix 1μL gDNA Remover 1μL RNase-free Water to 20μL

[0080] 2) Mix thoroughly and then incubate at 42°C for 30 min;

[0081] 3) Heat at 85°C for 5 seconds to inactivate TransScript RT / RI and gDNA Remover;

[0082] 4) The obtained cDNA was stored at -20°C. All reagents used in the reaction were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0083] (3) Amplification of the target gene EVM0004700 and construction of the overexpression vector pCAMBIA1300-EVM0004700-GFP

[0084] 1) The coding region sequence of EVM0004700 was obtained by analyzing the results of high-throughput sequencing of the whole genome of Lonicera japonica, and homologous amplification primers with Kpn I and BamH I restriction sites were designed:

[0085] EVM0004700-F1:

[0086] GGGGGACGAGCTCGGTACC ATGGTTACTGTCGAGGAGGTT(SEQ ID NO:3)

[0087] EVM0004700-R1:

[0088] CCATGTCGACTCTAGAGGATCCAGTGGACACACTATGGAGA(SEQ ID NO:4)

[0089] 2) Using the cDNA sequence of Lonicera japonica as a template, the sequence of Lonicera japonica EVM0004700 was amplified according to the following PCR reaction system. The reagents and dosages used are shown in Table 2 below:

[0090] Table 2: PCR amplification system

[0091]

[0092] The PCR reaction procedure is as follows:

[0093] Pre-denaturation: 98℃2min;

[0094] (denaturation: 98°C for 30 s, annealing: 58°C for 30 s, extension: 72°C for 1.5 min) × 35 cycles;

[0095] Extension: 72°C for 5 min;

[0096] Keep warm at 4℃.

[0097] 3) The pCAMBIA1300 vector was digested with Kpn I and BamH I at 37°C for 4 h, and the amplified product and the digested product were purified using a recovery kit (manufacturer: Takara, China).

[0098] 4) Mix the recovered product of the EVM0004700 sequence amplified by PCR and the recovered product of the pCAMBIA1300 vector digested with KpnI and BamH I, react at 50°C for 15 minutes under the action of homologous recombinase, and transform the trans1-T1 yeast competent state (purchased from Beijing Quanshijin Biotechnology Co., Ltd., refer to the instructions for specific operation methods and dosages). Add 700μL LB culture solution, then place it in a constant temperature shaker, and resuscitate it at 37°C and 180rpm for 60min. Centrifuge the revived E. coli at 5,000rpm for 1min, draw 600μL of supernatant and discard it. The remaining 100μL is mixed with a pipette tip, evenly coated on the LB solid culture medium containing 50μg / mL Kan, and the culture medium is inverted in a constant temperature incubator and grown at 37°C for 12-16h. After the colonies grew out, single clones were picked and grown in 700 μL LB liquid culture medium containing 50 μg / mL Kan at 37°C for 6 h. 300 uL of bacterial solution was sent to the company for sequencing, positive clones were screened, and the vector was named pCAMBIA1300-EVM0004700-GFP.

[0099] The preparation method of LB liquid culture medium is as follows: weigh 5 g of tryptone, 5 g of NaCl, and 2.5 g of yeast extract, add ddH2O to a total volume of 500 mL, sterilize at 121°C with high pressure steam for 20 min, and cool to room temperature.

[0100] The preparation method of LB solid culture medium refers to LB liquid culture medium. Before making up the volume, add 5g agar powder, sterilize with high pressure steam at 121℃ for 20min, and cool to room temperature.

[0101] After sequencing, it was found that the nucleotide sequence of EVM0004700 was shown in SEQ ID NO. 1. The amino acid sequence encoding the Lonicera japonica chalcone synthase EVM0004700 was shown in SEQ ID NO: 2. 5) The vector pCAMBIA1300-EVM0004700-GFP was transformed into Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd., refer to the instruction manual for specific operation steps).

[0102] Example 2

[0103] Example 2 studied the expression differences of the EVM0004700 gene in different tissues.

[0104] (1) Referring to the method of Example 1, cDNAs of roots, stems, leaves, flowers and fruits of Lonicera japonica were used as templates.

[0105] (2) Primer design: 18S was selected as the endogenous reference gene, and specific primers for qRT-PCR were designed using Snapgene software:

[0106] EVM0004700-F:GGGTGGACCGGCTATATTGG(SEQ ID NO:5)

[0107] EVM0004700-R:CCCCTCGTTGGCTGATTTCT(SEQ ID NO:6)

[0108] 18S-F:CAACCATAAACGATGCCGA(SEQ ID NO:7)

[0109] 18S-R:AGCCTTGCGACCATACTCC(SEQ ID NO:8)

[0110] The qRT-PCR reaction was performed using ChamQ SYBR qPCR Master Mix of Nanjing Novozyme Biotechnology Co., Ltd., with 3 replicates for each reaction. The qRT-PCR reaction system is shown in Table 3 below:

[0111] Table 3 Reverse transcription system

[0112]

[0113]

[0114] The 2-ΔΔCT method was used to calculate the expression level of EVM0004700 gene in different tissues. The results showed that EVM0004700 gene was expressed in flowers, leaves and roots, with the highest expression level in flowers (e.g. Figure 1 shown).

[0115] Example 3

[0116] Example 3: Subcellular localization analysis of EVM0004700 protein.

[0117] The empty vector pCAMBIA1300-GFP was transferred into GV3101 as a control. The suspension of infiltration liquid was sucked with a sterile syringe and injected into tobacco leaves with good growth status. After the injection, the leaves were cultured in the dark for 1 day and then cultured in the light for 1 day. A tobacco leaf with a diameter of about 1 cm was placed on a slide and stained with DAPI for 15 minutes. The back was facing up and soaked with double distilled water. The leaves were fixed with a cover glass (to avoid bubbles) and observed under a laser confocal microscope.

[0118] The results showed that EVM0004700 protein was localized in the nucleus and cytoplasm (e.g. Figure 2 shown).

[0119] Example 4

[0120] Example 4 studied the function of the EVM0004700 gene.

[0121] (1) Transformation of Arabidopsis thaliana

[0122] 1) Preparation of infection solution:

[0123] The vector pCAMBIA1300-EVM0004700-GFP was transformed into Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd., for specific operation steps, refer to the instructions), and then a single colony was picked and inoculated into 700uL LB liquid culture medium, and cultured at 28°C overnight with shaking; 500μL of overnight culture solution was placed in 50mL LB culture medium, and cultured at 28°C overnight with shaking until OD 600 The bacterial solution was centrifuged at 5000 rpm for ten minutes, the supernatant was discarded, and then Arabidopsis transformation solution (50 g / L sucrose, 0.167‰ Silwet L-77, solvent is water) was added to resuspend the bacterial cells until OD 600 to 0.6-0.8 to obtain the Arabidopsis thaliana infection bacterial solution.

[0124] 2) Soak the Arabidopsis inflorescence in the above-mentioned Arabidopsis infection solution for 1 minute; cover with film to keep moisture, and culture in the dark for one day;

[0125] 3) taking out Arabidopsis thaliana, culturing them at a culture temperature of 22° C. with a light intensity of 16 hr / d and a dark intensity of 8 hr / d, and culturing the Arabidopsis thaliana until T0 generation seeds are harvested;

[0126] 4) The harvested T0 generation seeds were divided into 1.5 mL centrifuge tubes, and the seeds were sterilized with prepared disinfectant (13.6 mL anhydrous ethanol + 4 mL 30% hydrogen peroxide + water to 20 mL), and the seeds were evenly spotted on 1 / 2 MS solid screening medium (4.43 g MS / L, pH 5.8, 8 g agar / L, hygromycin 50 mg / L);

[0127] 5) Low temperature treatment at 4°C for 2 days, and then cultured in a plant incubator at 22°C;

[0128] 6) After the seeds germinate for about 10 days, select the positive seedlings;

[0129] 7) The positive plants preliminarily determined were transplanted into the soil. When the Arabidopsis to be screened grew to the fourth week, the leaf DNA was extracted for verification. The Arabidopsis expressing EVM0004700 was the transgenic Arabidopsis. The positive seedlings were cultured until the T1 generation of transgenic Arabidopsis seeds were harvested;

[0130] 8) Take T1 generation seeds and sow them on MS screening medium, and treat them before sowing in the same way as T0 generation positive seedlings (4)-(6); after growing for about ten days, select seeds that can grow normally on the screening medium and seeds that cannot grow normally, and transplant the seedlings of the strains that meet the 3:1 ratio after testing, and harvest the seeds to obtain T2 generation seeds;

[0131] 9) The treatment of T2 generation seeds before sowing is the same as that of T0 generation positive seedlings (4)-(6); after growing for about ten days, all those that can grow normally on the culture medium are selected as homozygous T3, and the seedlings are transplanted and T3 generation seeds are harvested.

[0132] (2) Expression analysis of EVM0004700 in wild-type and overexpressed Arabidopsis

[0133] According to the sequence of the target gene (shown in SEQ ID NO: 1), specific primers (SEQ ID NO: 5 and 6) and internal reference gene 18S primers (SEQ ID NO: 7 and 8) were designed, and the total RNA of the leaves of T3 homozygous Arabidopsis was extracted using RNA prep Pure polysaccharide polyphenol plant total RNA extraction kit (manufacturer: Tiangen Biochemical Technology (Beijing) Co., Ltd.), and then reversed into cDNA using a reverse transcription kit (manufacturer: Beijing Quanshijin Biotechnology Co., Ltd.), and the cDNA was diluted 10 times as a template for qRT-PCR. qRT-PCR reaction was performed using ChamQ SYBR qPCR Master Mix (manufacturer: Nanjing Novozyme Biotechnology Co., Ltd.), and each reaction was repeated 3 times. The specific system is as in Example 2.

[0134] The results showed that the expression level of EVM0004700 gene in overexpression strains OE-7 and OE-10 was significantly higher than that in wild type (WT) (e.g. Figure 3 (shown in A).

[0135] (3) Determination of flavonoid content in transgenic Arabidopsis

[0136] Weigh equal amounts of wild-type and transgenic Arabidopsis seedlings, place them in 2 mL centrifuge tubes, dry them in an oven, and then use a plant flavonoid content detection kit (manufacturer: Solebow) to determine the total flavonoid content. The specific steps are carried out according to the kit instructions.

[0137] The results are as follows Figure 3 As shown in B, compared with WT, the flavonoid content of strains OE-7 and OE-10 overexpressing the EVM0004700 gene was significantly increased.

[0138] In the description of this specification, reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention.

[0139] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes Lonicera japonica chalcone synthase EVM0004700, and the sequence of the nucleic acid molecule is shown in SEQ ID NO:

1.

2. The isolated nucleic acid molecule according to claim 1, characterized in that The nucleic acid molecule is cDNA.

3. A Lonicera japonica chalcone synthase protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

2.

4. The Lonicera japonica chalcone synthase protein according to claim 3, characterized in that The protein is encoded by the sequence shown in SEQ ID NO:

1.

5. A construct, characterized in that Comprising the nucleic acid molecule of claim 1 or 2.

6. The construct according to claim 5, characterized in that The construct is at least one of a plasmid, a virus, and a bacteriophage.

7. A host cell, characterized in that It comprises the nucleic acid molecule of claim 1 or 2 or the construct of claim 5 or 6.

8. A method for increasing plant flavonoids, characterized in that: include: Introducing the nucleic acid molecule according to claim 1 or 2, or the construct according to claim 5 or 6, or the host cell according to claim 7 into the plant; Screening and obtaining plants that overexpress the Lonicera japonica chalcone synthase protein; The plant is Arabidopsis thaliana.

9. The method according to claim 8, characterized in that The nucleic acid molecule, the construct, or the host cell is introduced into the plant by Agrobacterium transformation.

10. Use of the nucleic acid molecule according to claim 1 or 2 or the protein according to claim 3 or 4 in increasing the flavonoid content of a transgenic plant, wherein the transgenic plant is Arabidopsis thaliana.

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