Application of saposhnikovia divaricata c2' h gene and method for increasing coumarin content in saposhnikovia divaricata

Abstract

The application discloses application of a SdC2'H gene of Saposhnikovia divaricata and a method for increasing coumarin content in Saposhnikovia divaricata, and belongs to the technical field of bio-agriculture. The SdC2'H gene of Saposhnikovia divaricata and the biological material containing the SdC2'H gene are applied to the regulation of coumarin synthesis in Saposhnikovia divaricata. The method for increasing coumarin content in Saposhnikovia divaricata comprises the steps of increasing the expression amount and / or activity of the SdC2'H gene in Saposhnikovia divaricata. The SdC2'H gene is successfully cloned from Saposhnikovia divaricata for the first time, and a gene overexpression vector is constructed. The SdC2'H gene is positively regulated to control the synthesis of coumarin for the first time, and overexpression of the SdC2'H gene can significantly increase the product content. This not only deepens the understanding of the molecular mechanism of related biosynthesis, but also lays an important foundation for efficient production of high-value coumarin by using genetic engineering technology.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, specifically relating to the application of the SdC2'H gene in Saposhnikovia divaricata and a method for increasing the coumarin content in Saposhnikovia divaricata. Background Technology

[0002] Saposhnikovia divaricata (Turcz.) Schishk. is a perennial herb belonging to the Apiaceae family, and its dried, unbolted root is used medicinally. As an important traditional Chinese medicine, Saposhnikovia divaricata has a long history of medicinal use, having been applied in clinical practice for over two thousand years. Its main effects include relieving exterior syndromes and dispelling wind, eliminating dampness, and relieving pain and spasms. Modern pharmacological studies have revealed that Saposhnikovia divaricata has a complex and diverse chemical composition, including volatile oils, chromones, organic acids, coumarins, and polysaccharides. Among these, coumarins have been proven to be one of the main pharmacologically active components of Saposhnikovia divaricata, exhibiting significant anti-inflammatory, antiviral, antibacterial, cardioprotective, and cancer cell apoptosis-inducing pharmacological activities.

[0003] Coumarin compounds are synthesized via the phenylpropanoid metabolic pathway in plants. Based on the differences in the substituent groups and positions of their parent nucleus, they can be classified into simple coumarins, furanocoumarins, and pyranocoumarins. In this synthetic pathway, most related genes exist in the form of gene families. Notably, the enzyme gene C2'H, which catalyzes the key ortho-hydroxylation reaction, belongs to the 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily. This ortho-hydroxylation step is crucial for the formation of the coumarin backbone. C2'H can highly specifically catalyze the ortho-hydroxylation of substrates with coumaroyl-CoA and ferulicyl-CoA, thereby generating umbelliferone and scopolamine precursors, ultimately constructing the core structure of coumarin through spontaneous or enzymatic cyclization. Currently, the C2'H gene has been successfully cloned and reported in various plants such as Arabidopsis thaliana, sweet potato, Rut, and Angelica dahurica. However, the cloning of this gene in Saposhnikovia divaricata, a plant with important medicinal value, has not yet been reported.

[0004] In the field of medicinal plant research, the hairy root culture system, as an important biotechnology method, has attracted much attention due to its significant advantages such as good genetic stability, rapid proliferation rate, and ease of genetic manipulation. Hairy roots are a pathological manifestation of plants infected by *Agrobacterium rhizogenes*. They not only provide an ideal genetic manipulation platform for studying plant gene function but also possess the ability to synthesize and accumulate characteristic secondary metabolites of plants. In recent years, this technology has achieved significant results in the research of various medicinal plants such as *Salvia miltiorrhiza*, ginseng, and *Catharanthus roseus*, and has been widely used for the functional verification and metabolic regulation research of target genes. For example, overexpression of specific transcription factor genes in the hairy roots of *Salvia miltiorrhiza* can effectively increase the content of tanshinone; overexpression of cinnamic acid 4-hydroxylase (C4H) and 4-coumaryl-CoA ligase (4CL) genes in the hairy roots of *Scutellaria baicalensis* can significantly increase the accumulation of flavonoids; and overexpression of specific glycosyltransferase (UGT) genes in the hairy roots of *Polygonum multiflorum* can also significantly increase the content of target active ingredients. Nevertheless, there are currently no reports on using hairy root systems to verify gene function in Saposhnikovia divaricata, particularly on gene overexpression.

[0005] Therefore, how to overcome the technical bottleneck of cloning and functional verification of key enzyme genes of Saposhnikovia divaricata, and establish a gene function research platform suitable for Saposhnikovia divaricata, so as to provide an effective means for elucidating the synthesis pathway of its pharmacologically active substances and increasing the yield of active ingredients, has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] One of the objectives of this invention is to provide the application of the Saposhnikovia divaricata SdC2'H gene and biomaterials containing the Saposhnikovia divaricata SdC2'H gene in regulating the synthesis of Saposhnikovia divaricata coumarin.

[0007] The second objective of this invention is to provide a method for increasing the coumarin content in Saposhnikovia divaricata.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of this invention discloses the use of the Saposhnikovia divaricata SdC2'H gene and biomaterials containing the Saposhnikovia divaricata SdC2'H gene in regulating the synthesis of Saposhnikovia divaricata coumarin; the nucleotide sequence of the SdC2'H gene is:

[0010] In some embodiments of the present invention, the biological material containing the *Fangfeng* SdC2'H gene is an overexpression vector containing the *Fangfeng* SdC2'H gene.

[0011] In some embodiments of the present invention, the overexpression vector includes the *Fangfeng* SdC2'H gene and the pCAMBIA1304 vector, and the SdC2'H gene is inserted at two restriction enzyme sites, Bg1II and BstEII.

[0012] The second aspect of the present invention discloses a method for increasing the coumarin content in Saposhnikovia divaricata, comprising the step of increasing the expression level and / or activity of the SdC2'H gene in Saposhnikovia divaricata.

[0013] In some embodiments of the present invention, the step of increasing the expression level and / or activity of the SdC2'H gene in Saposhnikovia divaricata includes overexpressing the SdC2'H gene in Saposhnikovia divaricata.

[0014] In some embodiments of the present invention, the overexpression method includes introducing a recombinant vector containing the SdC2'H gene into Saposhnikovia divaricata.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention marks the first successful cloning of the SdC2'H gene from *Saposhnikovia divaricata*, construction of its gene overexpression vector, and transformation of *Saposhnikovia divaricata* cotyledons via *Agrobacterium rhizogenes*-mediated transformation, successfully obtaining hairy roots containing wild-type, empty vector control, and overexpressing SdC2'H gene. In the overexpression lines, SdC2'H gene expression was significantly upregulated. Functional studies showed that the overexpressed SdC2'H gene effectively catalyzes the ortho-hydroxylation of coumaroyl-CoA, generating a hydroxylation intermediate. This intermediate undergoes spontaneous lactonization to form umbelliferone, which is a precursor to various complex coumarins.

[0017] The key finding is that the coumarin content in hairy roots overexpressing the SdC2'H gene was significantly higher than that in wild-type and empty vector controls. This directly confirms that the SdC2'H gene positively regulates the biosynthetic pathway of this coumarin by enhancing catalytic efficiency.

[0018] In summary, this invention successfully constructed a hairy root system overexpressing the SdC2'H gene, and for the first time clearly revealed the key role of the SdC2'H gene in positively regulating coumarin synthesis, with its overexpression significantly increasing product content. This not only deepens our understanding of the relevant biosynthetic molecular mechanisms but also lays an important foundation for the efficient production of these high-value coumarins using genetic engineering technology. Attached Figure Description

[0019] Appendix Figure 1 The image shows the PCR amplification results of the SdC2'H gene, where 1-3 represent the SdC2'H gene; M represents the marker.

[0020] Appendix Figure 2 The hydrophilicity / hydrophobicity analysis diagram of SdC2'H;

[0021] Appendix Figure 3 The secondary structure prediction diagram for SdC2'H is shown, where Helix represents a spiral, Sheet represents a fold, Turn represents a turn, and Coil represents a coil.

[0022] Appendix Figure 4 The predicted tertiary structure of SdC2'H;

[0023] Appendix Figure 5 The image shows the predicted transmembrane domain of SdC2'H, where transmembrane represents the transmembrane region, inside represents the intracellular region, and outside represents the extracellular region.

[0024] Appendix Figure 6 Phylogenetic tree of the SdC2'H amino acid sequence;

[0025] Appendix Figure 7 Image of pCAMBIA1304 plasmid;

[0026] Appendix Figure 8 The images show the identification results of the hairy roots of *Saposhnikovia divaricata*. Image a shows the amplification of SdC2'H from the vector pCA-SdC2'H, with lanes 1-2 representing SdC2'H and M as the marker. Image b shows the amplification of hairy roots using rolB, with lanes 1-3 representing WT, NC, and SdC2'H respectively. + M is the Marker; c is the value from SdC2'H + Image of a 418bp fragment amplified from hairy roots, with lanes 1-2 being SdC2'H and M being the marker;

[0027] Appendix Figure 9 Images of the hairy roots of *Saposhnikovia divaricata* after 7 and 30 days of induction culture;

[0028] Appendix Figure 10 The relative expression level of SdC2'H in different hairy roots of Saposhnikovia divaricata (n=3); ****P<0.0001, vs WT;

[0029] Appendix Figure 11 The image shows the characteristics of hairy root samples after 21 days of culture in 1 / 2 liquid medium.

[0030] Appendix Figure 12 Figure showing the results of coumarin content analysis in the hairy roots of Saposhnikovia divaricata (n=3); where **P<0.01vs WT; △△△ P<0.001 vs NC;

[0031] Appendix Figure 9 ~Attached Figure 12 In the diagram, WT represents wild-type hairy roots; NC represents negative control hairy roots containing empty pCAMBIA1304; and SdC2'H...+ Hairy roots that overexpress SdC2'H. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0033] The reagents and instruments used in the embodiments of this invention are as follows:

[0034] Plant RNA extraction kit (ZP405), cDNA kit (AT311), rapid high-fidelity DNA polymerase, and homologous recombination seamless cloning kit (CU201) were purchased from Beijing TransGen Biotech Co., Ltd. anPrep column DNA gel extraction kit (B518131), SanPrep column plasmid DNA mini-extraction kit (B518191), plant DNA extraction kit (B518262), *E. coli* DH5α competent cells, restriction endonucleases Bg1II and BstEII were purchased from Shanghai Sangon Biotech Co., Ltd. NA markers (BM2000, BM15000) were purchased from TaKaRa; pMD-19T vector was purchased from TaKaRa; *Agrobacterium rhizogenes* K599 competent cells were purchased from Beijing Huayueyang Biotechnology Co., Ltd.; methanol (analytical grade) and acetonitrile (chromatographic grade) were purchased from Thermo Fisher Scientific, China.

[0035] PCR instrument ProFlex TM ThermoFish (USA), YAMATOIC412C constant temperature shaking incubator (USA), centrifuge (ThermoFish, USA), DYY-8C agarose gel electrophoresis apparatus (Beijing Liuyi), GIS-2010 gel imaging system (Shanghai Tanon).

[0036] In this embodiment of the invention, primer synthesis and sequencing were performed by Shanghai Sangon Biotech Co., Ltd. All primer information used is shown in Table 1, and all PCR procedures are shown in Table 2.

[0037] Table 1 Primer sequences

[0038]

[0039] Table 2 PCR Procedure

[0040]

[0041]

[0042] Example 1

[0043] This embodiment discloses the cloning and bioinformatics analysis of the *Saposhnikovia divaricata* SdC2'H gene. Details are as follows:

[0044] Total RNA was extracted from fresh Saposhnikovia divaricata leaf samples using a plant RNA extraction kit, and cDNA was obtained by reverse transcription using a cDNA synthesis kit (Sangon Biotech). Primers S1 (Table 1) were designed using VectorN TI, and the SdC2'H gene was amplified using PCR program 1 (Table 2). The 20 μL volume consisted of: 1.0 μL cDNA, 10.0 μL 2xTransTag HiFi PCR SuperMixⅡ, 1.0 μL SdC2'H-F1 (10 μmol·L⁻¹), and 1.0 μL SdC2'H-R1 (10 μmol·L⁻¹). -1 1.0 μL of ddH2O and 7.0 μL of ddH2O were added. The purified PCR product was recovered from the gel and ligated with the pMD-19T vector, and the reaction was carried out overnight at 16°C. The ligation product was transformed into E. coli DH5α competent cells and reacted with ddH2O at a concentration of 50 mg·L⁻¹. -1 Positive clones were screened on LB plates containing Amp antibiotics. Multiple positive clones were selected for PCR verification. The bacterial cultures with correct verification results were sent for sequencing.

[0045] Physicochemical properties of the *Saposhnikovia divaricata* SdC2'H sequence were analyzed using Expasy-ProtParam; the secondary structure of *Saposhnikovia divaricata* SdC2'H was analyzed using SOPMA; the tertiary structure of *Saposhnikovia divaricata* SdC2'H was modeled using Swiss-Model; the signal peptide of *Saposhnikovia divaricata* SdC2'H was predicted using Siqnal P6.0 Server; conserved domains in *Saposhnikovia divaricata* SdC2'H were predicted using NCBI's CD-search; 16 protein sequences from different species were selected from the GenBank database, and cluster analysis was performed on the above sequences and the *Saposhnikovia divaricata* SdC2'H cloned in this paper using MEGA 11 software. A phylogenetic tree was constructed using the nearest neighbor clustering (NJ) algorithm.

[0046] Depend on Figure 1The PCR amplification yielded a specific band approximately 813 bp in length. Sequencing results confirmed this band's actual length to be 813 bp, with the open reading frame (ORF) also measuring 813 bp. BLAST homology alignment showed a 93.33% similarity to the SdC2'H cDNA sequence of *Kitagawiaprearuptora*, and a 94.44% similarity in amino acid sequences, indicating that the cloned sequence is indeed the *Kitagawiaprearuptora* SdC2'H gene sequence. This sequence was registered in GenBank under the accession number PV169361. Physicochemical analysis of the *Kitagawiaprearuptora* SdC2'H protein sequence revealed its molecular formula to be C0.05. 1386 H 2191 N 357 O 401 S9, with a theoretical molecular weight of 30560.33 kDa, an isoelectric point of 6.61, and an instability index of 37.50 (<40), is an unstable protein; its hydrophilicity is -0.184 (<0), indicating that it is a hydrophilic protein (e.g., ...). Figure 2 (As shown).

[0047] Secondary structure prediction of the encoded protein revealed that the SdC2'H protein sequence contains 34.81% alpha helixes, 17.78% extended strands, and 47.41% random coils, indicating that the secondary structure of the SdC2'H protein is mainly composed of alpha helixes and coils (e.g., ...). Figure 3 As shown), the three-level structure model is as follows: Figure 4 As shown. The predicted transmembrane domains of encoded proteins are as follows: Figure 5 The results show that the probability of the SdC2'H protein being located in the transmembrane region or inside the membrane is very low; this protein is primarily located outside the membrane. Prediction results for the encoded protein signal peptide show that both the predicted values ​​for the signal peptide and the cleavage site are 0, indicating that its structure does not contain a signal peptide. Prediction results for the encoded protein conserved domains show that SdC2'H belongs to the PLN03178 superfamily. Cluster analysis results are as follows... Figure 6 As shown, the amino acid sequence of Saposhnikovia divaricata SdC2'H clusters with those of Angelica sinensis and Kitagawiapraeruptora, indicating a close phylogenetic relationship and suggesting that Saposhnikovia divaricata SdC2'H and C2'H of other Apiaceae plants have similar functions.

[0048] Example 2

[0049] like Figure 7 As shown in the figure, this embodiment discloses a method for constructing a plant binary expression vector pCA-SdC2'H that overexpresses the Saposhnikovia divaricata SdC2'H gene, and performs PCR verification.

[0050] The SdC2'H gene was inserted into the pCAMBIA1304 vector at two restriction sites, Bg1II and BstEII, as follows: Primers S2 were designed according to the seamless cloning kit (Sangon Biotech) (as shown in Table 1), and the target gene with the vector homologous arm was amplified using PCR program 2 (as shown in Table 2); the pCAMBIA1304 plasmid was double-digested with restriction endonucleases Bg1II and BstEII at 37℃ for 1 h; the amplified target gene and linearized vector were purified by gel extraction; the linearized vector and gene were ligated using the Basic Assembly reaction system (molar ratio of 2:3) at 50℃ for 15 min; the ligation product was transformed into E. coli Trans1-T1 competent cells and incubated at a concentration of 50 mg·L⁻¹. -1 Positive clones were screened on LB plates containing kan antibiotics; several positive clones were randomly selected for PCR and sequencing verification; the recombinant plasmid with correct verification results was named pCA-SdC2'H.

[0051] The results are as follows Figure 8 As shown in Figure a: A fragment of approximately 813 bp was amplified, which matches the length of the target sequence SdC2'H gene. Further sequencing results showed that the fragment had 100% identity with the above-mentioned registered sequence of the Fangfeng SdC2'H gene (PV169361), indicating that the binary expression vector carrying the Fangfeng SdC2'H gene was successfully constructed.

[0052] Example 3

[0053] This embodiment discloses the induction and culture of hairy roots of Saposhnikovia divaricata overexpressing the SdC2'H gene, as well as the detection of the expression level of the SdC2'H gene and the coumarin content in the hairy roots of Saposhnikovia divaricata obtained by induction and culture.

[0054] 1. Construction of recombinant Agrobacterium rhizogenes K599

[0055] The recombinant plasmid pCA-SdC2'H, prepared according to the method in Example 2, was introduced into Agrobacterium rhizogenes K599 competent cells using a freeze-thaw method. The plasmid was then cultured on YEB plates (containing 50 mg / L of Kan). -1 Str 50mg·L -1 Positive clones were screened using [a specific method / mechanism]. Primer S3 (as shown in Table 1) and PCR program 3 (as shown in Table 2) were used to verify and sequence positive colonies. The engineered bacteria with correct verification results were inoculated into YEB liquid medium and cultured with shaking at 28°C and 180 rpm until the logarithmic growth phase (OD2). 600 =0.6-1.0), collect the bacterial culture, centrifuge, and resuspend in an equal volume of 1 / 2 MS liquid medium.

[0056] 2. Induction, validation, and culture of hairy roots of Saposhnikovia divaricata overexpressing the SdC2'H gene

[0057] In a clean bench, leaves of aseptic seedlings of *Saposhnikovia divaricata* were cut and resuspended in an equal volume of 1 / 2 MS liquid culture medium in a prepared bacterial suspension. The mixture was incubated at 28°C with shaking for 10 minutes. Afterward, the explants were removed, dried on filter paper, and inoculated onto MS solid medium. The cultures were then incubated at 25°C for 3 days. After 3 days, the explants were rinsed with sterile water to remove excess *Agrobacterium*, dried, and transferred to a culture medium containing 50 mg / L... -1 Sterilization was performed in Cef solid MS medium. Simultaneously, wild-type Saposhnikovia root hairy roots (WT) were induced using Agrobacterium rhizogenes k599, while negative control (NC) and SdC2'H overexpressing Saposhnikovia root hairy roots were induced using Agrobacterium rhizogenes carrying the pCAMBIA1304 empty plasmid. + Subculture weekly, gradually reducing the concentration of Cef in the culture medium until hairy roots of *Saposhnikovia divaricata* are induced.

[0058] DNA was extracted from the hairy roots of each Saposhnikovia divaricata plant using a DNA extraction kit (Sangon Biotech). The rolB gene in the hairy root samples was verified by PCR using primer S4 (as shown in Table 1) and PCR program 4 (as shown in Table 2). The exogenous hrHyg gene in the Saposhnikovia divaricata hairy root samples overexpressing the SdC2'H gene was verified by PCR using primer S5 (as shown in Table 1) and PCR program 5 (as shown in Table 2).

[0059] Liquid culture was performed on the hairy roots of Saposhnikovia divaricata that had been verified above. The steps were as follows: Under aseptic conditions, 1.5 g of each hairy root sample with good growth status was weighed and transferred to 1 / 2 MS liquid medium. Each root system was replicated 3 times. The samples were cultured in a shaker at 25℃ and 130 rpm. The medium was changed every 7 days. After 21 days, the hairy root samples were collected, washed with distilled water, and used for subsequent determination of SdC2'H gene expression level and coumarin content.

[0060] Samples of Saposhnikovia divaricata hairy roots taken on days 7 and 30 post-infection are attached. Figure 9 As shown, the samples include: wild-type Saposhnikovia root hairy roots (WT), negative control hairy roots containing the pCAMBIA1304 empty plasmid (NC), and Saposhnikovia root hairy roots overexpressing the SdC2'H gene (SdC2'H). + All are growing well.

[0061] 3. Detection of SdC2'H gene expression level in hairy roots of Saposhnikovia divaricata

[0062] Total RNA was extracted from the above-mentioned hairy root systems using an RNA extraction kit (TransGold). cDNA was synthesized using a reverse transcription kit (Sangon Biotech). The reaction system was: 4.0 μL of Saposhnikovia root hairy RNA, Anchored Oligo(dT) 18 1 μL of 2xTSReaction Mix, 10 μL of TransScript RT / RI Enzyme Mix, 1 μL of gDNA Remover, and 1 μL of RNase-free Water were used. The reaction conditions were: incubation at 42°C for 30 min, followed by heating at 85°C for 5 s to inactivate TransScript RT / RI and gDNA Remover.

[0063] Using the *Saposhnikovia divaricata* EF1-α gene as an internal control, the expression level of the SdC2'H gene in *Saposhnikovia divaricata* hairy root samples was analyzed by RT-qPCR. Primer S6 (as shown in Table 1) and PCR program 6 (as shown in Table 2) were used to amplify EF1-α. The reaction system was: 1.0 μL of gene, upstream primer (10 μmol·L⁻¹). -1 1.0 μL of downstream primer (10 μmol·L⁻¹) -1 1.0 μL of SYBR Green Master Mix, 10.0 μL of ddH₂O, and 7.0 μL of ddH₂O were used. -△△CT The relative expression level of the SdC2'H gene was calculated using a method.

[0064] The results are as follows Figure 10 As shown, SdC2'H + The relative expression level of the SdC2'H gene in the root system was significantly higher than that in the WT root system.

[0065] 4. Determination of coumarin content in the hairy roots of Saposhnikovia divaricata

[0066] The hairy roots were rinsed with distilled water, dried with filter paper, and placed in a 60℃ oven. The dried hairy roots were ground into fine powder with a mortar and pestle, passed through a 30-mesh sieve, and 0.2g of sample was weighed. 6mL of methanol solution was added, and the sample was extracted with ultrasound at 60℃ for 1h. After cooling, the residue was removed by filtering with filter paper and then filtered through a 0.22μm filter membrane. The contents of four coumarins, namely psoralen, xanthotoxin, bergamot lactone, and imperatorin, were determined by HPLC. The determination method was the method described in the literature "Comprehensive Evaluation of Multi-Indicators of Saposhnikovia Root Material Quality" (Ma Bingru et al., Journal of Zhejiang A&F University, 2024, 41(4):715-723.DOI:10.11833 / j.issn.2095-0756.20230567.).

[0067] The growth of the hairy roots of each *Saposhnikovia divaricata* after 21 days of liquid culture is as follows: Figure 11As shown, all hairy root systems are growing well. The results of HPLC determination of the total content of four coumarins in the hairy roots of each *Saposhnikovia divaricata* are as follows: Figure 12 As shown, the results of the one-way ANOVA between samples indicate that: SdC2'H + The coumarin content in the samples was significantly higher than that in the WT and NC samples.

[0068] In summary, this invention successfully cloned the SdC2'H gene from *Saposhnikovia divaricata* for the first time. Its open reading frame (ORF) is 813 bp, encoding 270 amino acids. Bioinformatics analysis of the encoded protein (SdC2'H protein) revealed that the protein is hydrophilic and structurally unstable, lacking a typical signal peptide sequence. Its secondary structure is mainly composed of α-helices and random coils, preliminarily elucidating its unique folding conformation. Phylogenetic analysis further showed that the *Saposhnikovia divaricata* SdC2'H protein is most closely related to *Angelica sinensis* (Apiaceae family). Based on this, it is speculated that the SdC2'H gene may have a similar function to the known C2'H gene in *Angelica sinensis*. This discovery provides a crucial genetic resource for further elucidating the molecular basis of plant biosynthesis of active ingredients.

[0069] To verify the function of the SdC2'H gene, this invention, for the first time, constructed an overexpression vector for the SdC2'H gene and successfully induced hairy root systems in *Saposhnikovia divaricata* cotyledon explants using *Agrobacterium rhizogenes*-mediated genetic transformation technology. These hairy root systems included wild-type, empty plasmid negative control, and those overexpressing the SdC2'H gene. Experiments confirmed that the expression level of the SdC2'H gene in the hairy root system was significantly higher than that in the wild-type root system. Functional studies showed that the overexpressed SdC2'H gene in plant roots can efficiently catalyze the ortho-hydroxylation of coumaroyl-CoA, generating a key hydroxylation intermediate; this intermediate then undergoes spontaneous lactonization, ultimately leading to the formation of umbelliferones. Umbelliferones are important precursors to many complex plant active ingredients.

[0070] Crucially, the determination of the content of the target active ingredient in the hairy roots showed that the coumarin content in the hairy roots overexpressing the SdC2'H gene was significantly higher than that in the wild-type and empty plasmid negative control groups. This result directly confirms that the overexpression of the SdC2'H gene positively regulates the biosynthetic pathway of saposhnikovia coumarin by enhancing the catalytic efficiency of coumaroyl-CoA, thereby effectively increasing the accumulation level of the target product.

[0071] In summary, this invention, through the successful construction of a plant hairy root culture system overexpressing the SdC2'H gene, has for the first time clearly revealed the crucial role of the SdC2'H gene in regulating the biosynthesis of coumarin in saposhnikovia root. Overexpression of this gene significantly increases the coumarin content in hairy roots, providing a core basis for elucidating the molecular mechanisms of related biosynthesis and laying an important technical foundation for the efficient production of these valuable plant active ingredients using genetic engineering techniques.

[0072] The above-described experimental examples merely illustrate specific embodiments of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The use of overexpressing the *Saposhnikovia divaricata* SdC2'H gene, characterized in that, Application in improving the synthesis of saposhnikovia coumarin SdC2’H The nucleotide sequence of the gene is shown in Sequence 15.

2. Contains the windproof as described in claim 1 SdC2’H The use of gene-based biomaterials is characterized by, Applications in enhancing the synthesis of saposhnikovia coumarin, including saposhnikovia SdC2’H The biological material containing the gene is windproof SdC2’H Gene overexpression vectors.

3. The use according to claim 2, characterized in that, The overexpression vector includes *Saposhnikovia divaricata*. SdC2’H The gene and the pCAMBIA1304 vector were inserted using the Bg1II and BstEII restriction sites. SdC2’H Gene.

4. A method for increasing the coumarin content in Saposhnikovia divaricata, characterized in that, Including improvements as shown in Sequence 15 SdC2’H Steps for determining gene expression levels and / or activity.

5. The method according to claim 4, characterized in that, Improve wind resistance SdC2’H The steps of gene expression level and / or activity include overexpressing the gene in *Saposhnikovia divaricata*. SdC2’H Gene.

6. The method according to claim 5, characterized in that, Overexpression methods include containing SdC2’H The recombinant vector of the gene was introduced into Saposhnikovia divaricata.

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