Use of a PpHCT5 gene in promoting biosynthesis of chlorogenic acid in plants

Abstract

The application clones a functional gene PpHCT5 from peach fruit and realizes the application of the gene in promoting the biosynthesis of chlorogenic acid in plants. PpHCT5 belongs to the BADH acyltransferase family, and the coding region is 1323 bp long and contains 440 amino acids. In vitro enzymatic test analysis shows that PpHCT5 protein is involved in the last step of the chlorogenic acid biosynthesis pathway and can catalyze the formation of chlorogenic acid from caffeoyl-CoA and quinic acid. Further in vivo functional verification shows that overexpression of the PpHCT5 gene promotes the accumulation of chlorogenic acid in transgenic peach fruit, tomato fruit and tobacco leaves. Based on this result, a new idea is provided for promoting the development and application of plant products rich in chlorogenic acid, and a foundation is laid for plant resistance breeding.

Description

Technical Field

[0001] This invention relates to the application of the PpHCT5 gene in promoting the biosynthesis of chlorogenic acid in plants, and belongs to the field of plant genetic engineering technology. Background Technology

[0002] Chlorogenic acid (CGA) is an important phenylpropanoid secondary metabolite found in plants, widely distributed in various plants, particularly in those of the Caprifoliaceae, Solanaceae, and Asteraceae families. It is found in very high concentrations in medicinal plants such as honeysuckle, eucommia, and coffee; it is also present in vegetables like potatoes, tomatoes, and carrots; furthermore, it is a major phenolic compound in many fruits, such as peaches, blueberries, pears, strawberries, and apples. With the resurgence of research into natural plant extracts like artemisinin, chlorogenic acid and other natural products have attracted widespread attention from scholars both domestically and internationally, and are now widely used in food, cosmetics, medicine, and chemical industries, earning it the internationally recognized title of "plant gold."

[0003] The peach [Prunus persica (L.) Batsch] originated in my country and is an important economic deciduous fruit tree. It is loved by consumers for its vibrant color, crisp and juicy flesh, captivating aroma, and rich nutritional value. Peach fruit is rich in nutrients, including protein, vitamins, soluble sugars, organic acids, and phenolic compounds. Chlorogenic acid is the most abundant phenolic acid in peach fruit, mainly existing in two forms: chlorogenic acid and its isomer, neochlorogenic acid, while also containing trace amounts of cryptochlorogenic acid. Chlorogenic acid plays a crucial role in enhancing the antioxidant capacity, disease resistance, and post-harvest preservation of peaches, making it an important entry point for improving the nutritional and resistant qualities of peach fruit. To date, research on chlorogenic acid in peach fruit has mainly focused on content determination and bioactivity; the biosynthetic pathway and key genes of peach chlorogenic acid remain undetermined.

[0004] Hydroxycinnamoyl-CoA:shikimate / quinate hydroxycinnamoyl transferase (HCT) is a key rate-limiting enzyme downstream of the chlorogenic acid biosynthesis pathway, and the HCT / HQT-mediated pathway is the main pathway for chlorogenic acid biosynthesis in most plants. In poplar, the expression abundance of the leaf PtHCT2 gene is significantly correlated with chlorogenic acid content and is regulated by WRKY transcription factors containing W-box elements (Zhang et al., 2018b). In mulberry, the expression level of the MaHCT4 gene is significantly positively correlated with chlorogenic acid content, and the MaHCT4 protein catalyzes the synthesis of p-coumaroylquinic acid and chlorogenic acid (Zhao et al., 2019). However, the role of the PpHCT gene in peach chlorogenic acid biosynthesis remains unclear. Summary of the Invention

[0005] The purpose of this invention is to provide a PpHCT5 gene that can be used to promote chlorogenic acid levels in plants. The technical solution adopted by this invention is as follows:

[0006] This invention provides an application of the PpHCT5 gene in promoting the biosynthesis of chlorogenic acid in plants, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Furthermore, the amino acid sequence of the protein encoded by the PpHCT5 gene is shown in SEQ ID NO.2.

[0008] Furthermore, PpHCT5 protein was induced to express in Escherichia coli.

[0009] Furthermore, the PpHCT5 protein catalyzes the conversion of caffeoyl-CoA and quinic acid into chlorogenic acid.

[0010] Furthermore, by upregulating the expression level of the PpHCT5 gene in plants, the chlorogenic acid content in plants can be increased.

[0011] Furthermore, the overexpression vector is the pCAMBIA1301-35SN vector, and the construction of the overexpression vector includes the following steps: selecting Sal I and Xba I restriction sites to insert the CDS of PpHCT5 into the pCAMBIA1301-35SN vector, and the constructed plasmid is named 35S-PpHCT5.

[0012] Furthermore, the DNA of the PpHCT5 gene was introduced into the recipient plant using the Agrobacterium-mediated transgenic method to obtain a transgenic plant, which had a higher chlorogenic acid content than the recipient plant.

[0013] Furthermore, the plants mentioned are peach, tomato, and tobacco.

[0014] The beneficial effects of this invention are:

[0015] This invention cloned the functional gene PpHCT5 from peach fruit and realized its application in promoting chlorogenic acid biosynthesis in plants. PpHCT5 belongs to the BADH acyltransferase family, with a coding region of 1323 bp containing 440 amino acids. In vitro enzymatic assays showed that the PpHCT5 protein participates in the final step of the chlorogenic acid biosynthesis pathway, catalyzing the conversion of caffeoyl-CoA and quinic acid into chlorogenic acid. Furthermore, in vivo functional verification showed that overexpression of the PpHCT5 gene promoted the accumulation of chlorogenic acid in transgenic peach fruit, tomato fruit, and tobacco leaves. Based on these results, new ideas can be provided for promoting the development and application of chlorogenic acid-rich plant products, and a foundation can be laid for plant resistance breeding. Attached Figure Description

[0016] Figure 1 Identification and purification of PpHCT5 protein expression. (A) SDS-PAGE results of PpHCT5 protein expression. Lane M: Protein Marker; Lane 1: Uninduced sample; Lanes 2-6: Induced samples. (B) SDS-PAGE results of PpHCT5 protein purification. Lane M: Protein Marker; Lane 1: Precipitate after disruption; Lane 2: Supernatant after disruption; Lane 3: Eluent; Lane 4: Washing sample; Lane 5: Elution sample.

[0017] Figure 2 High-performance liquid chromatography analysis of chlorogenic acid generated in vitro from PpHCT5 recombinant protein.

[0018] Figure 3 Construction of the PpHCT5 overexpression (OE) vector. (A) Schematic diagram of the PpHCT5-OE vector. (B) PCR amplification results of PpHCT5.

[0019] Figure 4 For the genetic transformation of tomatoes, (A) Agrobacterium infection, (B) co-culture, (C) selection culture, (D) callus culture, (E) rooting culture, (F) hardening off.

[0020] Figure 5 For the genetic transformation of tobacco, (A) Agrobacterium infection, (B) co-culture, (C) selection culture, (D) callus culture, (E) rooting culture, (F) hardening off.

[0021] Figure 6 GUS staining for the detection of transgenic plants. (A) GUS staining of PpHCT5-OE peach fruit. (B)

[0022] GUS staining of tomato fruit with PpHCT5-OE. (C) GUS staining of tobacco leaves with PpHCT5-OE. WT, wild type; EV, empty vector; OE, overexpression.

[0023] Figure 7 Functional analysis of the PpHCT5 gene in peach fruit. (A) Transient overexpression and transient silencing of PpHCT5 in 'Xiahui 5' peach fruit. (B) Chlorogenic acid content and gene expression pattern in peach fruit with transient PpHCT5 overexpression. (C) Chlorogenic acid and neochlorogenic acid content near the injection site in peach fruit. An empty vector (pCAMBIA1301-35SN) was used as a control. CGA, chlorogenic acid; NCGA, neochlorogenic acid. EV, empty vector; OE, overexpression. Error bars represent the standard deviation of three biological replicates. Statistical significance was determined by Student's t-test (*P<0.05, **P<0.01).

[0024] Figure 8 Functional analysis of the PpHCT5 gene in tomato fruits and tobacco leaves. (A) Stable overexpression of the PpHCT5 gene in tomato. Fruits from T1 generation transgenic plants were harvested 7 days after the color-breaking stage for analysis. (B) PpHCT5 gene expression and chlorogenic acid content in PpHCT5-OE transgenic tomato fruits. (C) Stable overexpression of the PpHCT5 gene in tobacco. Leaves from T2 generation transgenic seedlings were analyzed. (D) PpHCT5 gene expression and chlorogenic acid content in PpHCT5-OE transgenic tobacco leaves. CGA, chlorogenic acid; NCGA, neochlorogenic acid; CCGA, cryptochlorogenic acid. EV, empty vector; OE, overexpression. Error bars represent the standard deviation of three biological replicates. Statistical significance was determined by Student's t-test (*P<0.05, **P<0.01). ND, not detected. Detailed Implementation

[0025] The present invention is further illustrated below by way of examples, but is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or function as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following examples are commercially available.

[0026] Example 1: Isolation and Cloning of the PpHCT5 Gene

[0027] Weigh 200 mg of peach fruit samples from different developmental stages, and extract total RNA using the RNAprep Pure Plant Plus Kit (Tiangen, Beijing, China). Then, use a reverse transcription kit. cDNA was synthesized using the 1st Strand cDNA Synthesis Kit (gDNA digester plus). Based on the CDS sequence of the PpHCT gene, the primer sequences for PCR amplification were designed as follows:

[0028] Upstream primer PpHCT5-F: 5'-ATGGCGGTGGTCAACGTG-3' (SEQ ID No. 3)

[0029] Downstream primer PpHCT5-R: 5'-TTATATCTCATACAACAACTTGGAGAA-3' (SEQ ID No. 4)

[0030] Then, using the prepared cDNA as a template, PCR amplification was performed using the primer pairs described above. The PCR reaction system was 25 μL, including 2× PCR Master Mix 12.5 μL, forward and reverse primers 1 μL each, cDNA 1 μL, ddH2O 9.5 μL. The reaction program was as follows: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min.

[0031] The nucleotide sequence of the amplified PpHCT5 gene is shown in SEQ ID NO.1.

[0032] Further analysis of the above sequence using DNAMAN software showed that the PpHCT5 gene cloned in this invention contains two exons and one intron, with a coding region of 1323 bp and encoding 440 amino acids. The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0033] Example 2: Identification of PpHCT5 protein expression

[0034] The construction method of the prokaryotic expression vector is as follows: First, based on the cDNA sequence of the target gene PpHCT5 amplified in Example 1, BamHI and HindIII restriction sites were selected, and the pET-28a prokaryotic expression vector containing the His tag was constructed using homologous recombination. The recombinant plasmid was named His-PpHCT5, and the primer sequences for PCR amplification are as follows:

[0035] His-PpHCT5-F: 5'-CAAATGGGTCGCggatccATGGCGGTGGTCAACGTG-3' (SEQ ID

[0036] No. 5)

[0037] His-PpHCT5-R: 5'-GAGTGCGGCCGCaagcttTATCTCATACAACAACTTGGAGAA-3'

[0038] (SEQ ID No.6)

[0039] The method for inducing the expression of the target protein is as follows: The correctly sequenced recombinant plasmid His-PpHCT5 was transformed into BL21(DE3) competent cells. Positive single colonies were then picked and cultured in liquid medium containing kanamycin at 37°C until the bacterial OD600 reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mM, and the cells were cultured at 37°C for 4 hours. After centrifugation, the bacterial samples were collected and analyzed by SDS-PAGE. The results are shown below. Figure 1 A. Compared with the control group without IPTG (lane 1), 0.5 mM IPTG (lanes 2-6) can induce protein bands of 48.14 kD, indicating that the target protein is successfully expressed in vitro.

[0040] The method for purifying the target protein is as follows: Protein purification was performed using the His-tag Protein Purification Kit from Shanghai Beyotime Biotechnology Co., Ltd. After washing and elution, the recombinant protein was loaded for SDS-PAGE electrophoresis detection. The results are shown in the figure below. Figure 1 B.

[0041] Example 3: In vitro enzyme activity assay of PpHCT5

[0042] To verify the function of the candidate gene PpHCT5, the expressed PpHCT5 protein was used for in vitro enzyme activity assays. The 200 μL reaction system consisted of: 100 mM sodium phosphate buffer (pH = 7.4), 1.25 mM quinic acid, 0.625 mM caffeoyl coenzyme A, and 12.8 μg purified protein, with boiled inactivated protein as a control. The reaction mixture was incubated at 37 °C for 60 min, and the reaction was stopped by boiling for 10 min. The reaction product was extracted with 200 μL of methanol, filtered through a 0.22 μm organic filter, and then analyzed by HPLC. The results are shown in the figure. Figure 2 After adding purified PpHCT5 protein to the enzymatic reaction system, the content of caffeoyl-CoA decreased, and a new chromatographic peak appeared at 12.30 min. Figure 2(Middle). After comparing secondary chromatographic fragments and retention times, we deduced that this newly emerging chromatographic peak is chlorogenic acid (Middle). Figure 2 (Below). In contrast, no chlorogenic acid was detected after boiling PpHCT5 protein was added to the enzymatic reaction system. Figure 2 superior).

[0043] The HPLC chromatographic conditions were as follows: Determination was performed using an Agilent 1260 Infinity HPLC system with an Agilent ZORBAX SB-C18 column (4.6 mm × 250 mm, 5 μm) and a DAD detector. Mobile phase A was methanol (0.1% H3PO4), and mobile phase B was water (0.1% H3PO4). The injection volume was 5 μL, the flow rate was 1.0 mL / min, and the column temperature was 30 °C. Gradient elution was used, with the following elution conditions: 0–22 min, 5%–71% A; 22–30 min, 71%–5% A; 30–35 min, 5% A. The detection wavelength for chlorogenic acid was 320 nm.

[0044] Example 4: Construction of the PpHCT5 gene overexpression vector

[0045] The construction method of the overexpression vector is as follows: First, based on the cDNA sequence of the target gene PpHCT5 amplified in Example 1, Sal I and Xba I restriction sites were selected, and the CDS of PpHCT5 was inserted into the pCAMBIA1301-35SN vector using homologous recombination. Figure 3 The recombinant plasmid was named 35S-PpHCT5, and the primer sequences for PCR amplification are as follows:

[0046] 35S-PpHCT5-F: 5'-CTTATCGATACCgtcgacATGGCGGTGGTCAACGTG-3' (SEQ ID No. 7)

[0047] 35S-PpHCT5-R: 5'-GGATCCACTAGTtctagaTATCTCATACAACAACTTGGAGAA-3' (SEQ ID No. 8)

[0048] Example 5: Instantaneous transformation of peach fruit

[0049] The recombinant plasmid 35S-PpHCT5 extracted in Example 4 was transformed into GV3101 Agrobacterium competent cells. Single colonies of Agrobacterium were collected and cultured in liquid medium containing kanamycin and rifampicin resistance, with Agrobacterium containing the empty vector pCAMBIA1301-35SN as a control. The cells were cultured at 28°C and 220 rpm for 24-48 h. Then, each Agrobacterium was cultured in an Erlenmeyer flask containing 40 ml of liquid medium until the OD600 reached 0.8-1.0. The cells were centrifuged at 5000 rpm for 10 min, collected, and resuspended in infection solution (10 mM MgCl2, 10 mM MES, pH 5.7, 200 μM acetylsyleugenone) and incubated at 28°C in the dark for 2 h.

[0050] 'Xiahui 5' peaches at their second expansion stage (70 DAFB) were selected as experimental material. The above suspensions were injected into the peach fruits using a 1 ml syringe. Due to individual differences in the experimental fruits and variations in Agrobacterium injection efficiency, symmetrical portions of the same peach fruit were used as the experimental and control groups, with three biological replicates, each containing eight peaches. Five days later, the injected peaches were immediately frozen in liquid nitrogen and stored at -80°C.

[0051] Example 6 Agrobacterium-mediated tomato transgenic

[0052] Select two-week-old tomatoes, cut off two fully unfolded cotyledons, and place them face up on the pre-culture medium for pre-culture at 25°C in the dark. After 1 day, place the cotyledons into the OD medium. 600 Infect the leaves with a 0.4–0.6 g / L resuspension of bacteria (MS 4.74 g / L, sucrose 30 g / L, AS 100 mg / L, pH 5.8) for about 5 minutes. Blot the leaves dry with filter paper and place them face down on a co-culture medium. Co-culture is carried out at 25°C in the dark. After 2 days of co-culture, the leaves are transferred to selection medium A for further selection (25°C, 16 h light / 8 h dark). After 2–3 weeks, the leaves are transferred to selection medium B and cultured until resistant shoots appear. When the resistant shoots reach 1 cm in height, they are cut off and transferred to a rooting medium for rooting induction. After rooting, the tomato plants are hardened off in a tissue culture room for 3–4 days before being transplanted into pots filled with nutrient soil for further cultivation. Figure 4 Three transgenic lines were selected as three biological replicates, each containing 10 fruits. The fruits were harvested 7 days after the color-breaking stage, and the tomato fruits were frozen in liquid nitrogen and stored at -80°C.

[0053] The main culture medium formulations mentioned above are as follows:

[0054] Pre-culture medium: MS 4.74 g / L, sucrose 30 g / L, agar 9.0 g / L, zeatin 2 mg / L, pH 5.8.

[0055] Co-culture medium: MS 4.74 g / L, sucrose 30 g / L, agar 9.0 g / L, zeatin 2 mg / L, AS 100 mg / L, pH 5.8.

[0056] Screening medium A: MS 4.74 g / L, sucrose 30 g / L, agar 9.0 g / L, zeatin 2 mg / L, carbenicillin (Cab) 400 mg / L, hygromycin (Hyg) 10 mg / L, pH 5.8.

[0057] Screening medium B: MS 4.74 g / L, sucrose 30 g / L, agar 9.0 g / L, zeatin 0.2 mg / L, carbenicillin (Cab) 400 mg / L, hygromycin (Hyg) 10 mg / L, pH 5.8.

[0058] Rooting medium: MS 2.37 g / L, sucrose 30 g / L, agar 7.5 g / L, IBA 2 mg / L, carbenicillin (Cab) 400 mg / L, hygromycin (Hyg) 10 mg / L, pH 5.8.

[0059] Example 7 Agrobacterium-mediated leaf disc transformation of tobacco

[0060] The *Agrobacterium 35S-PpHCT5* from Example 5 was cultured in an Erlenmeyer flask containing 40 ml of liquid culture medium until the OD600 reached 0.4–0.6. The cells were collected by centrifugation, and an equal volume of MS liquid culture medium was added to resuspend the cells for 1 hour. One-month-old leaves of common tobacco were cut into small pieces in a clean bench and immersed in the bacterial solution for 10–15 minutes. The bacterial solution on the leaf surface was blotted dry with filter paper, and the leaves were placed face down on a co-culture medium and co-cultured at 25°C in the dark. After 2 days of co-culture, the leaves were transferred to a selection medium for selection culture (25°C, 16 hours light / 8 hours dark). When the resistant buds from the tobacco leaf callus reached 1 cm in height, the resistant buds were cut off and transferred to a rooting medium for rooting induction. After rooting, the tobacco plants were hardened off in a tissue culture room for 3–4 days before being transplanted into pots filled with nutrient soil for further cultivation. Figure 5 Three independent transgenic tobacco plants of generation T2 were selected as three biological replicates. The tobacco leaves were frozen in liquid nitrogen and stored at -80°C.

[0061] The main culture medium formulations mentioned above are as follows:

[0062] MS liquid medium: MS 4.74 g / L, sucrose 30 g / L, pH 5.8.

[0063] Co-culture medium: MS 4.74 g / L, sucrose 30 g / L, agar 8.5 g / L, 6-BA 3 mg / L, IBA 0.2 mg / L, pH 5.8.

[0064] Selective medium: MS 4.74 g / L, sucrose 30 g / L, agar 8.5 g / L, 6-BA 3 mg / L, IBA 0.2 mg / L, carbenicillin (Cab) 400 mg / L, hygromycin (Hyg) 20 mg / L, pH 5.8.

[0065] Rooting medium: MS 4.74 g / L, sucrose 30 g / L, agar 8.5 g / L, IBA 0.5 mg / L, carbenicillin 400 mg / L, hygromycin 20 mg / L, pH 5.8.

[0066] Example 8: Analysis of chlorogenic acid content and gene expression in transgenic plants

[0067] It should be noted that transgenic plants are detected through PCR amplification and GUS staining of plant genomic DNA. Figure 6 ).

[0068] 1. Detection of PpHCT5 gene expression level in transgenic plants:

[0069] qRT-PCR is used to detect the expression level of the PpHCT5 gene in transgenic plants, according to... qPCR The reaction was performed according to the instructions for the Green Master Mix kit, and the 20 μL reaction volume used was as follows: qPCR The reagents included 10 μL GreenMaster Mix, 1 μL cDNA, 0.8 μL each of upstream and downstream primers, and 7.4 μL ddH2O. The ABI 7500 real-time PCR instrument (Applied Biosystems, USA) was used with the following reaction program: 95°C denaturation for 5 min, 95°C denaturation for 10 s, 60°C annealing for 30 s, for a total of 40 cycles, followed by a 72°C extension for 30 s. Peach PpTEF2 was used as the internal control gene. The real-time primer sequences are as follows:

[0070] q-PpHCT5-F: 5'-GCAGATGGCCTTTCTGGTCT-3' (SEQ ID No. 9)

[0071] q-PpHCT5-R: 5'-TGTGATCGAATGCAGGCTGT-3' (SEQ ID No. 10)

[0072] 2. HPLC detection of chlorogenic acid compounds in transgenic plants

[0073] The extraction method of chlorogenic acid from transgenic plants is as follows: First, grind the plant sample with liquid nitrogen, accurately weigh 1g of sample, and add 7mL of methanol (0.1% H3PO4) extractant; then, extract the mixed solution with ultrasound in the dark for 30min, and centrifuge at 10000r / min for 10min at 4℃; finally, take the supernatant, filter it through a 0.22μm organic filter, and then enter the chromatographic column.

[0074] The determination of chlorogenic acid in transgenic plants was performed under the HPLC chromatographic conditions described in Example 3 above. The detection wavelength for chlorogenic acid compounds (chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid) was 320 nm. The content determination included three biological replicates, and the content was calculated based on the peak area.

[0075] Results of instantaneous injection of peach fruit Figure 7 As shown in Figure A. qRT-PCR results showed that the expression level of the PpHCT5 gene in peach fruits overexpressing the gene was significantly higher than that in peach fruits injected with the empty vector. Figure 7 B); meanwhile, HPLC analysis showed that, compared with peaches injected with empty vector, overexpression of the PpHCT5 gene significantly increased the content of chlorogenic acid and neochlorogenic acid near the injection site. Figure 7 C). Furthermore, the results of heterologous stable overexpression in tomato and tobacco are as follows: Figure 8 As shown. Compared with wild-type tomato fruits, overexpression of the PpHCT5 gene induced an increase in chlorogenic acid content in tomato fruits ( Figure 8 A and B). Similarly, similar results were observed in tobacco, where overexpression of the PpHCT5 gene not only promoted the biosynthesis of chlorogenic acid and neochlorogenic acid in tobacco leaves, but also increased the biosynthesis of cryptochlorogenic acid. Figure 8 (C and D). This result indicates that the PpHCT5 gene in this invention plays an important role in the biosynthesis of chlorogenic acid in plants.

Claims

1. The application of overexpression of the PpHCT5 gene in promoting chlorogenic acid biosynthesis in plants, characterized in that, The nucleotide sequence of the PpHCT5 gene is shown in SEQ ID NO. 1, and the plants are peach, tomato, and tobacco.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the PpHCT5 gene is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, PpHCT5 protein was induced to express in Escherichia coli.

4. The application according to claim 3, characterized in that, PpHCT5 protein catalyzes the conversion of caffeoyl-CoA and quinic acid into chlorogenic acid.

5. The application according to claim 1, characterized in that, By using gene overexpression technology, the expression level of the PpHCT5 gene was increased to promote the chlorogenic acid content in plants.

6. The application according to claim 5, characterized in that, The overexpression vector used was pCAMBIA1301-35SN.

7. According to claim 5, the DNA of the PpHCT5 gene is introduced into a recipient plant using the Agrobacterium-mediated transgenic method to obtain a transgenic plant, wherein the transgenic plant has a higher chlorogenic acid content than the recipient plant.

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