Application of histone deacetylase smhda6 and its coding gene in regulating accumulation of salvianolic acids

Abstract

The application discloses histone deacetylase SmHDA6 and application of an encoding gene thereof in regulating accumulation of salvianolic acid. The application provides a histone deacetylase SmHDA6 for regulating biosynthesis of salvianolic acid in Danshen, wherein an amino acid sequence is shown as SEQ ID NO. 4, and a nucleotide sequence is shown as SEQ ID NO. 1. In the application, the SmHDA6 has a Histone deacetylase domain conservative functional domain for exerting histone deacetylation, is clustered in a Type I (RPD3-like superfamily) subfamily in the HDA family, expression amounts of the SmHDA6 gene in different parts of Danshen are obviously positively correlated with salvianolic acid content, and expression of the SmHDA6 gene is significantly up-regulated after being induced by ABA, MeJA and SA. Knocking out the SmHDA6 gene by a gene editing technology can significantly improve salvianolic acid content in Danshen, because knocking out the SmHDA6 gene activates expression of genes in a salvianolic acid metabolic pathway, and then increases biosynthesis of the salvianolic acid. Therefore, the SmHDA6 can regulate accumulation of the salvianolic acid in Danshen, can be applied to engineering bacteria and genetic engineering breeding of Danshen with high salvianolic acid content, and has great application value.

Description

Technical Field

[0001] This invention belongs to the field of medicinal plant genetic engineering technology, specifically relating to a histone deacetylase SmHDA6 and its encoding gene, and its application in promoting the accumulation of salvianolic acid. Background Technology

[0002] Salvia miltiorrhiza Bunge is a perennial, erect Chinese herbal medicine belonging to the genus Salvia in the family Lamiaceae. It is bitter and cold in nature, with a reddish-brown root bark and purplish-purple flesh (for sweating) and a shape resembling ginseng. It has the effects of removing blood stasis, relieving pain, promoting blood circulation, and regulating menstruation. It is a traditional Chinese medicine mainly used in the clinical treatment of cardiovascular and cerebrovascular diseases (Li Z, Xu S, Liu P. Salviamiltiorrhiza Burge (Danshen): a golden herbal medicine in cardiovasculartherapeutics. Acta Pharmacol Sin. 2018, 39(5): 802-824). At present, wild Salvia miltiorrhiza resources are decreasing day by day. Cultivated Salvia miltiorrhiza suffers from serious degradation in quality, long growth cycle, and low content of effective ingredients. As a result, the supply and demand contradiction between the limited high-quality Salvia miltiorrhiza resources and the huge market demand has become prominent, which restricts its practical application and has also become a bottleneck for the export of domestic Salvia miltiorrhiza and the quality control. Using genetic engineering techniques to introduce key enzyme genes (and / or regulatory transcription factors) in the biosynthesis pathway of active ingredients in Salvia miltiorrhiza into Salvia miltiorrhiza is one of the best ways to fundamentally improve the content of active ingredients and enhance the quality of the medicinal material (Wang Z, Peters RJ. Tanshinones: Leading the way into Lamiaceaelabdane-related diterpenoid biosynthesis. Curr Opin Plant Biol. 2022, 66:102189; Wu S, Zhu B, Qin L, Rahman K, Zhang L, Han T. Transcription factor: A powerful tool to regulate biosynthesis of active ingredients in Salvia miltiorrhiza. Front Plant Sci. 2021, 12: 622011).

[0003] The salvianolic acid components in Salvia miltiorrhiza mainly include salvianolic acid A, salvianolic acid B, rosmarinic acid, and caffeic acid. Their upstream biosynthesis is also composed of two pathways: the phenylalanine metabolic pathway and the tyrosine metabolic pathway. The key enzymes in the tyrosine metabolic pathway are known to be tyrosine aminotransferase (TAT) and p-hydroxyphenylpyruvate reductase (HPPR); the phenylalanine metabolic pathway is catalyzed by phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (C4H) and 4-coumarin coenzyme A ligase (4CL); the two branches generate rosmarinic acid through rosmarinic acid synthase (RAS) and P450 enzyme (CYP98A14) (Chen Q, LiJ, Ma Y, Yuan W, Zhang P, Wang G. Occurrence and biosynthesis of plantsesterterpenes (C25), a new addition to terpene diversity. Plant Commun. 2021, 2(5): 100184).

[0004] Based on the above synthetic pathways, from a molecular biology perspective, research has been widely conducted on increasing the expression levels of key synthetic enzyme genes or regulating the transcriptional levels of key transcription factors to influence the accumulation of salvianolic acid. Elucidating the underlying molecular mechanisms is not only a requirement of theoretical biology but also a necessary condition for the development of the *Salvia miltiorrhiza* industry (Zhao Y, Liu G, Yang F, Liang Y, Gao Q, Xiang C, Li X, Yang R, Zhang G, Jiang H, Yu L, Yang S. Multilayered regulation of secondary metabolism in medicinal plants. MolHortic. 2023, 3: 11). Unfortunately, most studies focus on transcription factor interaction mechanisms or key enzyme gene discovery, with relatively little research on the upstream epigenetics. In fact, epigenetics also has a powerful modifying effect on regulatory networks. For example, under the methylation inhibition of 5-azacytidine, multiple genes and transcription factor genes in Salvia miltiorrhiza exhibit demethylation, including the key tanshinone synthase gene KSL, thus increasing the tanshinone content in Salvia miltiorrhiza hairyroots by more than 1.5 times (Yang BC, Lee MS, Lin MK, Chang WT. 5-Azacytidine increases tanshinone production in Salvia miltiorrhiza hairyroots through epigenetic modulation. Sci Rep. 2022, 12(1): 9349). However, the role of histone acetylation modification in the synthesis of secondary metabolites in Salvia miltiorrhiza remains to be elucidated. Therefore, identifying the epigenetic regulators of tanshinone biosynthesis in Salvia miltiorrhiza will provide a scientific basis for increasing the content of medicinally active substances in Salvia miltiorrhiza using metabolic engineering methods. Summary of the Invention

[0005] The purpose of this invention is to provide a histone deacetylase SmHDA6 protein and its encoding gene, wherein histone deacetylase SmHDA6 can regulate the biosynthesis of salvianolic acid in tanshinone.

[0006] To achieve the above-mentioned objective, the present invention provides a histone deacetylase SmHDA6 that regulates the biosynthesis of salvianolic acid, wherein SmHDA6 has the nucleotide sequence shown in SEQ ID NO.1.

[0007] The present invention provides a protein encoded by the above-mentioned histone deacetylase SmHDA6 of tanshinone, the protein having an amino acid sequence as shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector containing the above-mentioned tanshinone deacetylase SmHDA6, wherein the recombinant vector is CRISPR-SmHDA6 (gene silencing).

[0009] The present invention also provides a recombinant microorganism containing the recombinant vector, wherein the microorganism is Agrobacterium rhizogenes C58C1 strain and the recombinant vector is CRISPR-SmHDA6.

[0010] Another object of the present invention is to provide a method for promoting the accumulation of salvianolic acid in tanshinone by the above-mentioned histone deacetylase SmHDA6, comprising the following steps:

[0011] (1) Using CRISPR / Cas9 gene editing technology, sgRNA was designed at the exon of the SmHDA6 gene, and the nucleotide sequence of the SmHDA6 gene is shown in SEQ ID NO.1;

[0012] (2) Insert the SmHDA6-sgRNA into pCAMBIA2300 to obtain the knockout vector CRISPR-SmHDA6;

[0013] (3) Transform the CRISPR-SmHDA6 knockout vector containing SmHDA6-sgRNA into Agrobacterium strain to obtain a strain with the CRISPR-SmHDA6; the host strain is preferably Agrobacterium rhizogenes C58C1 strain.

[0014] (4) The strain was genetically transformed into Salvia miltiorrhiza by Agrobacterium-mediated transformation, and resistant Salvia miltiorrhiza hairy roots were obtained by antibiotic screening. Transgenic Salvia miltiorrhiza hairy roots with the target gene SmHDA6 knocked out were obtained by qRT-PCR detection.

[0015] (5) The content of salvianolic acid in the hairy roots of Salvia miltiorrhiza with the SmHDA6 gene knocked out in (4) above was determined by HPLC, and the hairy roots of Salvia miltiorrhiza with increased salvianolic acid content were screened.

[0016] Another object of the present invention is to provide the application of the above-mentioned histone deacetylase SmHDA6 in the genetic improvement breeding of Salvia miltiorrhiza, wherein the application is preferably the application of SmHDA6 in the genetic engineering breeding of Salvia miltiorrhiza to regulate the accumulation of salvianolic acid.

[0017] This invention provides a histone deacetylase SmHDA6 and its encoding gene, belonging to the field of medicinal plant genetic engineering technology. The nucleotide sequence of the histone deacetylase SmHDA6 in *Salvia miltiorrhiza* is shown in SEQ ID NO.1. In this invention, SmHDA6 possesses a Histone deacetylase domain (PF00850) that performs histone deacetylation, clustering in the Type I (RPD3-like superfamily) subfamily of the HDA family. In different parts of *Salvia miltiorrhiza*, the expression level of the SmHDA6 gene is significantly positively correlated with the content of salvianolic acid, and its expression is significantly upregulated after induction treatment with ABA, MeJA, and SA. Knocking out the SmHDA6 gene using gene editing technology can significantly increase the content of salvianolic acid in *Salvia miltiorrhiza*. This is because knocking out the SmHDA6 gene can activate the expression of salvianolic acid metabolic pathway genes C4H, 4CL, HPPR, TAT, and RAS, thereby increasing the biosynthesis of salvianolic acid in *Salvia miltiorrhiza*. In summary, the SmHDA6 can regulate the accumulation of salvianolic acid in tanshinone, providing a stable and high-value gene resource for the large-scale production of salvianolic acid. Attached Figure Description

[0018] Figure 1 This is an electrophoresis image of the SmHDA6 histone deacetylase gene amplified in Danshen. The left side shows the molecular weight standards for nucleic acids, with the bands from top to bottom being 2000, 1500, 1000, 750, 500, 250, and 100 bp; the right side represents the RcWRKY51 gene.

[0019] Figure 2 This is the secondary structure of the histone deacetylase SmHDA6 protein from Tanshinone. Blue indicates α-helix, red indicates extended backbone, green indicates β-turn, and purple indicates random coil.

[0020] Figure 3 The image shows the tertiary structure of the histone deacetylase SmHDA6 protein from Danshen, with the red circle indicating the WRKY binding site.

[0021] Figure 4 Phylogenetic analysis was performed on SmHDA6 from *Salvia miltiorrhiza* and its members in the previously reported Arabidopsis thaliana histone deacetylase family. SmHDA6 clustered in the Type I (RPD3-like superfamily) subfamily and was most closely related to the Arabidopsis thaliana AtHDA19 protein.

[0022] Figure 5 The content of salvianolic acid and the expression level of the SmHDA6 gene in different parts of Salvia miltiorrhiza were measured. Different parts included the root ( Ro ot), stem ( St em), leaf ( Leaf) and flowers ( Fl (owr), bar represents the ± standard error (n ≥ 10) for each data set, and different lowercase letters indicate new negative range. Statistical comparison of different parts of Salvia miltiorrhiza showed significant differences at p < 0.01.

[0023] Figure 6 The expression level of the SmHDA6 gene in *Salvia miltiorrhiza* under ABA, MeJA, and SA induction treatments is given. The bar for each group represents the ± standard error (n ≥ 10). ** indicates that the Student's t-test showed a significant difference between the control group and the treatment group at p < 0.01.

[0024] Figure 7 This is a schematic diagram of the pCAMBIA2300-CRISPR-SmHDA6 knockout vector. The pCABIA2300 accession number is AF234315.

[0025] Figure 8 To investigate the effect of knocking out the SmHDA6 gene in *Salvia miltiorrhiza* on increasing salvianolic acid content. (AC) *Salvia miltiorrhiza* hairy roots; (D) HPLC determination of salvianolic acid content in *Salvia miltiorrhiza*; (E) SmHDA6 gene expression level; (F) salvianolic acid content. The bar represents ± standard error (n ≥ 10) for each data set. ** indicates a significant difference between the control group and the UV-treated group as shown in Student's t-test (p < 0.01).

[0026] Figure 9 This study focuses on the reported biosynthetic pathway of salvianolic acid and its key enzymes in Salvia miltiorrhiza.

[0027] Figure 10 This study evaluates the expression levels of key enzyme genes involved in the biosynthesis of salvianolic acid in *Salvia miltiorrhiza* plants with the SmHDA6 gene knocked out. Key enzyme genes include PAL, C4H, 4CL, TAT, HPPR, RAS, and CYP98A14. The bar represents ± standard error (n ≥ 10) for each data set. ** indicates a significant difference between the control group and the gene knockout group as shown by Student's t-test at p < 0.01. Detailed Implementation

[0028] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0029] Unless otherwise specified in the following examples, all experimental methods can be performed using conventional methods, such as those described in J. Sambrook et al.'s *Molecular Cloning: A Laboratory Manual*, F. Osborne et al.'s *A Concise Laboratory Manual of Molecular Biology*, or the manufacturer's instructions for use of the products employed.

[0030] The *Salvia miltiorrhiza* Bunge used in these examples was cultivated at the Medicinal Botanical Garden of Zhejiang University of Traditional Chinese Medicine (N30°5′, E119°53′; Hangzhou, China); the SteadyPure plant RNA extraction kit was purchased from Hunan Aikerui Biotechnology Co., Ltd. (catalog number: AG21019); the Evo M-MLV reverse transcription premix kit was purchased from Hunan Aikerui Biotechnology Co., Ltd. (catalog number: AG11728); *Escherichia coli* DH 5α was purchased from Shanghai Weidi Biotechnology Co., Ltd. (catalog number: DL1001); the GeneJET gel extraction kit was purchased from Thermo Scientific (catalog number: K0692); the Apex HFHS DNA polymerase premix was purchased from Hunan Aikerui Biotechnology Co., Ltd. (catalog number: AG12206); In-Fusion... ® The HD Cloning Kit was purchased from Takara (catalog number: 639648); Agrobacterium rhizogenes C58C1 was purchased from Shanghai Weidi Biotechnology Co., Ltd. (catalog number: AC1110); pMD18-T Vector was purchased from Takara (catalog number: D101A); the SYBR Green Pro Taq HS premixed qPCR kit was purchased from Hunan Aikerui Biotechnology Co., Ltd. (catalog number: AG11702); the Danshensu B standard was purchased from Sigma-Aldrich (catalog number: 49724); LB, YEB, MS, and SD media are commonly used media in this field, and their formulations are based on J. Sambrook et al.'s *Molecular Cloning: A Laboratory Manual*. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1 Cloning and Bioinformatics Analysis of the SmHDA6 Histone Deacetylase Gene from Danshen

[0032] (1) Extraction of total RNA from Salvia miltiorrhiza leaves and synthesis of the first strand of cDNA

[0033] 100 mg of freshly harvested Salvia miltiorrhiza leaves were ground into powder under liquid nitrogen. Total RNA was extracted from the Salvia miltiorrhiza leaves using the SteadyPure Plant RNA Extraction Kit (Hunan Aike Rui Biotechnology Co., Ltd., Catalog No.: AG21019) according to the instructions. The RNA was analyzed using a 1.0% agarose gel electrophoresis system (Biorad, California, USA) and NanoDrop. TMThe total RNA content and purity were determined using a 2000°C ultra-micro spectrophotometer (Thermo Scientific, Wisconsin, USA). 1000 ng of purified total RNA from *Salvia miltiorrhiza* leaves was used to synthesize the first strand of cDNA according to the instructions of the Evo M-MLV reverse transcription premixed kit (Hunan Aike Rui Biotechnology Co., Ltd., catalog number: AG11728). The reaction product was diluted to the required concentration and stored at -80°C for later use.

[0034] (2) Amplification and sequence analysis of the SmHDA6 gene of histone deacetylase in Danshen.

[0035] Using the cDNA from the *Salvia miltiorrhiza* leaves obtained above as a template, the corresponding upstream primer ATGGATACCGGAGGCAATTCT and downstream primer TTAGGCTAGGGGTTCAGAGCA for the *Salvia miltiorrhiza* histone deacetylase SmHDA6 gene were designed using Primer Premier 5.0 (Premier Biosoft, California, USA). The *Salvia miltiorrhiza* SmHDA6 gene sequence was obtained by PCR amplification using Apex HF HS DNA polymerase premix (Hunan Aike Rui Biotechnology Co., Ltd., catalog number: AG12206), as shown in SEQ ID NO.1. The PCR reaction system consisted of: 1.0 μL template, 25 μL 2× Apex HF FS PCR MasterMix, 1 μL upstream primer, 1 μL downstream primer, and sterile ddH2O to a total volume of 50 μL. PCR reaction procedure: 94°C pre-denaturation for 30 s, followed by 30 cycles (98°C 10 s, 55°C 10 s, 72°C 10 s). PCR products were detected using a 1.0% agarose gel electrophoresis system (Biorad, California, USA), and the target fragment was recovered using a GeneJET gel extraction kit (Thermo Scientific, catalog number: K0692) and ligated into a pMD18-T vector (Takara, catalog number: D101A). The ligation product was transformed into *E. coli* DH5α (Shanghai Weidi Biotechnology Co., Ltd., catalog number: DL1001) using the heat shock method and plated on a plate containing 100 μg / mL agarose gel. -1 The ampicillin antibiotic was incubated overnight at 37 °C on LB agar plates. Single colonies were picked as templates, and colony PCR was performed using the aforementioned upstream and downstream sequences as primers for verification. Positive clones were sent to Zhejiang Shangya Biotechnology Co., Ltd. for bacterial sequencing. The nucleotide sequence of the Tanshinone SmHDA6 gene was obtained as shown in SEQ ID NO.1.

[0036] The open reading frame (OPF) of the histone deacetylase SmHDA6 in Danshen is 1395 bp. Figure 1 The protein, encoding 464 amino acids (amino acid sequence shown in SEQ ID NO.2), has a predicted molecular weight of 52.44 kDa and a molecular formula of C2. 2321 H 3535 N 633 O 708 S 25 The theoretical isoelectric point (pI) is 5.15, the aliphatic index is 72.87, the instability index is 38.75, and the grand average of hydropathicity is -0.544. The histone deacetylase SmHDA6 protein of *Salvia miltiorrhiza* contains a conserved histone deacetylase domain (PF00850) at positions 36-324; E-value is 2.8 Ω·cm. -86 According to the pLoc-mPlant subcellular localization prediction software, the histone deacetylase SmHDA6 protein of *Salvia miltiorrhiza* was predicted to be located in the cell nucleus.

[0037] Analysis using the biological software SOPM showed that the secondary structure of the Tanshinone SmHDA6 protein contains 32.33% α-helix, 11.64% β-turn, 35.78% random coil, and 20.26% extended strand. Figure 2 The tertiary structure of the SmHDA6 protein in Tanshinone was deduced using SWISS-MODEL software as follows: Figure 3 As shown, its GMQE value is 0.85, and the sequence homology reaches 88.55%. Figure 3 ).

[0038] Further phylogenetic analysis showed that the histone deacetylase SmHDA6 protein belongs to the HDA family, clusters in the Type I (RPD3-like superfamily) subfamily, and is most closely related to the Arabidopsis thaliana AtHDA19 protein. Figure 4 ).

[0039] Example 2: Expression pattern analysis of the SmHDA6 gene, a histone deacetylase from Danshen.

[0040] Different tissues (roots, stems, leaves, and flowers) of *Salvia miltiorrhiza* were collected, and total RNA was extracted and reverse transcribed according to the method in Example 1-(1). Real-time quantitative PCR was performed using upstream primers (TGTCTGGAGATCGGTTAGGA) and downstream primers (CCACCACCTAACAACAGTAGAG) designed based on the SmHDA6 gene. A SYBR Green ProTaq HS premixed qPCR kit (Hunan Aike Rui Biotechnology Co., Ltd., catalog number: AG11702) was used for real-time quantitative PCR amplification. The reaction program was 95 °C denaturation for 30 s, followed by 40 cycles (95 °C 5 s, 60 °C 30 s). Data were obtained after running the reaction on an ABI 7500 quantitative PCR machine (Applied Biosystems, USA). −ΔΔCT The relative expression levels of each sample were calculated. The internal control was the tanshinone β-actin gene, with the upstream primer shown as AGCACCGAGCAGCATGAAGATT and the downstream primer shown as AGCAAAGCAGCGAACGAAGAGT.

[0041] The results showed that the SmHDA6 gene, a histone deacetylase from *Salvia miltiorrhiza*, was expressed in the roots, stems, leaves, and flowers of *Salvia miltiorrhiza*, with the highest expression in the roots, followed by the leaves and flowers. Figure 5 C). Furthermore, PlantCARE online software revealed numerous hormone-responsive elements (ABRE, CGTCA-motif, JERE, MYC, TCA-element, TGA-element, TGACG-motif) in the promoter region of the Tanshinone SmHDA6 gene. qRT-PCR analysis showed that the Tanshinone SmHDA6 gene exhibited significant upregulation after induction treatment with exogenous ABA, MeJA, and SA. Figure 6 The above results indicate that the SmHDA6 gene of histone deacetylase in Tanshinone is highly likely to regulate the accumulation of salvianolic acid in the plant through mediating hormone signaling pathways.

[0042] Example 3: The accumulation of salvianolic acid in different parts of Salvia miltiorrhiza was significantly positively correlated with the SmHDA6 gene.

[0043] The content of salvianolic acid in different parts of Salvia miltiorrhiza was determined according to the Salvia miltiorrhiza section of the Pharmacopoeia of the People's Republic of China. Approximately 0.15 g of powder from different parts of Salvia miltiorrhiza (passed through a No. 3 sieve) was accurately weighed and placed in a stoppered conical flask. 50 mL of methanol-water (8:2) mixed solution was accurately added, the flask was sealed, and the weight was measured. The flask was ultrasonically treated (power 140 W, frequency 42 kHz) for 30 min, cooled, and weighed again. The weight loss was made up with methanol-water (8:2) mixed solution, shaken well, filtered, and 5 mL of the subsequent filtrate was accurately measured and transferred to a 10 mL volumetric flask. Methanol-water (8:2) mixed solution was added to dilute to the mark, shaken well, filtered, and the subsequent filtrate was collected.

[0044] A standard C18 column (5 μm, 4.6 × 250 mm) was used; the mobile phase was acetonitrile-0.1% phosphoric acid (22:78); the detection wavelength was 286 nm; the column temperature was 20℃; and the flow rate was 1.2 mL / min. The theoretical plate number, calculated based on the salvianolic acid B peak, should be no less than 6000. 10 μL each of the salvianolic acid B standard solution (Sigma-Aldrich, catalog number: 49724) and the test solution from fruits at different developmental stages were accurately injected into the liquid chromatograph for determination.

[0045] The results showed that the theoretical plate number of the salvianolic acid B peak was 12080, and it achieved baseline separation from adjacent impurity peaks, meeting the testing requirements of the *Pharmacopoeia of the People's Republic of China*. The content of salvianolic acid B varied significantly in different parts of *Salvia miltiorrhiza* (root, stem, leaf, and flower), with the highest content in the root, followed by the leaves and flowers. Figure 5 B). This is significantly positively correlated with the expression level of the SmHDA6 gene, a histone deacetylase in Danshen (R). 2 = 0.91, p < 0.01). Therefore, the SmHDA6 gene is very likely to regulate the accumulation of salvianolic acid in different parts of Salvia miltiorrhiza.

[0046] Example 4: Knockout of the SmHDA6 gene in Danshen inhibits the biosynthesis of salvianolic acid.

[0047] (1) Construction of the SmHDA6 knockout vector for histone deacetylase in Danshen

[0048] Based on the nucleotide sequence of SmHDA6 from *Salvia miltiorrhiza* (SEQ ID NO.1), upstream primers (GATTGAGTAGAGACCATCAAATACAGG) and downstream primers (AAACCCTGTATTTGATGGTCTCTACTC) were designed at the EcoRI and Hind III restriction sites, respectively. The SmHDA6 sequence was obtained by high-fidelity amplification using *Salvia miltiorrhiza* leaf cDNA as a template. The sgRNA of SmHDA6 from *Salvia miltiorrhiza* was designed using the CRISPR / Cas9 gene editing system (Naito Y, Hino K, Bono H, Ui-Tei K. CRISPRdirect: software for designing CRISPR / Cas guide RNA with reduced off-target sites. Bioinformatics. 2015, 31(7): 1120-1123), and constructed into the plant expression vector pCAMBIA2300 (NCBI accession number: AF234315) containing the AtU6 and CaMV35S promoters. pCAMBIA2300 was digested with EcoRI and Hind III, and the CRISPR / Cas9-SmHDA6-sgRNA was digested with Bps I and inserted into the pMD-18T vector. In-Fusion sequencing was used. ® Following the instructions of the HD Cloning Kit (Takara, catalog number: 639648), the SmHDA6 knockout vector was constructed and named CRISPR-SmHDA6. Figure 8 ).

[0049] (2) The recombinant plasmid CRISPR-SmHDA6 was transformed into Agrobacterium rhizogenes C58C1

[0050] The knockout plasmid CRISPR-SmHDA6 was transformed into Agrobacterium rhizogenes C58C1 cells using a chemical transformation method. The specific steps were as follows: 100 μL of Agrobacterium rhizogenes C58C1 competent cells were mixed with 1 μg of the knockout plasmid CRISPR-SmHDA6. The mixtures were then placed on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, and immediately transferred to a 37 °C water bath for 5 min, followed by 5 min on ice. 700 μL of antibiotic-free LB medium was added, and the cells were cultured on a shaker at 28 °C and 100 rpm for 2 h. The culture was then plated onto 25 mL YEB plates (containing 50 mg / mL of the medium). -1 Kanamycin). Invert the plate and incubate it in a 28 °C incubator until colonies grow (about 2 days). Select positive clones for subsequent experiments.

[0051] (3) Agrobacterium-mediated genetic transformation of the SmHDA6 gene in the hairy roots of Salvia miltiorrhiza

[0052] Positive clones containing the knockout plasmid CRISPR-SmHDA6 were picked and placed in 50 mL of YEB liquid medium (containing 50 mg / mL). -1 Kanamycin, 50 mg / mL -1 Rifampicin), cultured at 28 °C with shaking at 200 rpm until OD. 600 = 0.6, add 0.2 mM acetylsyleugenone, and continue to incubate with shaking until OD. 600 = 0.8~1.0. Collect the bacterial culture by centrifugation at 5000 × g for 10 min at room temperature. Resuspend the precipitated Agrobacterium in 100 mL of osmotic buffer (0.2 mM acetylsylgenone, 10 mM MgCl2, 10 mM MES, pH 5.7), centrifuge at 5000 × g for 5 min at room temperature, discard the supernatant, and adjust the OD with osmotic buffer. 600 ≈ 0.6, cultured at 28 °C and 100 rpm for 30 min with shaking. Selected pre-cultured *Salvia miltiorrhiza* explants were infected with activated bacterial solution containing the knockout plasmid CRISPR-SmHDA6, and cultured in a shaker at 28 °C and 100 rpm for 10 min. The activated bacterial solution on the surface of the explants was blotted dry with sterile absorbent paper, and the explants were re-transferred onto 1 / 2 MS agar plates and incubated in the dark at 25 °C for 48 h. Multiple rounds of explant sterilization culture were performed, and positive *Salvia miltiorrhiza* hairy roots were screened using semi-quantitative PCR for subsequent experiments. *Salvia miltiorrhiza* hairy roots transformed with the empty plasmid were used as controls.

[0053] The results showed that, compared with the control (Mock), knocking out the SmHDA6 gene in *Salvia miltiorrhiza* using gene editing technology resulted in a stable *Salvia miltiorrhiza* hairy root line (pCAMBIA2300-CRISPR-SmHDA6) with the SmHDA6 gene knocked out. Figure 8 Further analysis revealed that the expression level of the SmHDA6 gene was significantly downregulated by 2.53-fold. Figure 8 E), the content of salvianolic acid increased by 6.84 times ( Figure 8 F). The above results indicate that the SmHDA6 gene of histone deacetylase in Tanshinone can regulate the accumulation of salvianolic acid.

[0054] Previous studies have shown that rosmarinic acid (RA) is a common precursor in the biosynthesis of salvianolic acids, originating from 4-coumaryl-CoA generated from the phenylalanine branch and 4-hydroxyphenyllactic acid generated from the tyrosine branch in the phenylpropane metabolic pathway. Therefore, the key enzymes in the biosynthetic pathway of salvianolic acids include phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), tyrosine aminotransferase (TAT), 4-hydroxyphenylpyruvate reductase (HPPR), rosmarinic acid synthase (RAS), and cytochrome P450 monooxygenase. CYP98A14), this pathway ( Figure 9 It has been conserved in plants such as white-flowered salvia, purple-flowered salvia, southern salvia, and sage (Zheng H, Fu X, Shao J, Tang Y, Yu M, Li L, Huang L, Tang K. Transcriptional regulatory network of high-value active ingredients inmedicinal plants. Trends Plant Sci. 2023, 28(7): 856).

[0055] Based on this, referring to the real-time fluorescence quantitative PCR detection method described in Example 2, using tanshinone β-actin as an internal reference gene, the expression patterns of key enzyme genes PAL, C4H, 4CL, TAT, HPPR, RAS, and CYP98A14 in the biosynthetic pathway of the above-mentioned salvianolic acid compounds were detected, and the upstream and downstream primers used were shown in SEQ ID NO.11 to SEQ ID NO.24 respectively.

[0056] Based on this, we found through gene expression analysis that in the hairy roots of *Salvia miltiorrhiza* with the SmHDA6 gene knocked out, the accumulation of tanshinone in *Salvia miltiorrhiza* was increased by upregulating the expression of genes C4H, 4CL, HPPR, TAT, and RAS in the tanshinone metabolism pathway. Figure 10 ).

[0057] The specific sequence list information is as follows:

[0058] >SmHDA6 nucleotide sequence (SEQ ID NO.1)

[0059]

[0060] >SmHDA6 amino acid sequence (SEQ ID NO.2)

[0061] MDTGGNSLASGADGVKRKVSYFYDPEVGNYYYGQGHPMKPHRIRMTHFLLAHYGLLQHMHVLKPNPARDKDLCRFHADDYVSFLRSITPETQQEQLRQLKRFNVGEDCPVFDGLYS FCQTYAGGSVGGAVKLNHGHCDIAVNWAGGLHHAKKCEASGFCYVNDIVLAILELLKVHERVLYVDIDIHHGDGVEEAFYTTDRVMTVSFHKFGDYFPGTGDVRDIGYGKGKYYSL NVPLDDGIDDESYQSLFKPIMGKVMEIFKPGAVVLQCGADSLSGDRLGCFNLSIKGHAECVKFMRSFNVPLLLLGGGGYTIRNVARCWCYETGVALGIELDDKMPQHEYYEYFGPD YTLHVAPSNMENKNSRHLLEEIRSKLLDNLSRLQHAPSVQFQERPPDSELPQMEEDHDGEDERYDPDSDMMDIDDERKPLPGRVKSEFPEPEPKDMDDAKEDEPNREVDLKCSEPLA

[0062] SmHDA6 nucleotide sequence amplification primers

[0063] upstream primer

[0064] ATGGATACCGGAGGCAATTCT

[0065] Downstream primer

[0066] TTAGGCTAGGGGTTCAGAGCA

[0067] The real-time quantitative PCR (RT-qPCR) primers used in SmHDA6

[0068] Upstream primer:

[0069] TGTCTGGAGATCGGTTAGGA

[0070] Downstream primer:

[0071] CCACCACCTAACAACAGTAGAG

[0072] Primers used for real-time quantitative PCR (RT-qPCR) of the internal reference gene β-actin

[0073] Upstream primer:

[0074] AGCACCGAGCAGCATGAAGATT

[0075] Downstream primer:

[0076] AGCAAAGCAGCGAACGAAGAGT

[0077] Primers used for constructing recombinant plasmid CRISPR-SmHDA6

[0078] Upstream primer:

[0079] GATTGAGTAGAGACCATCAAATACAGG

[0080] Downstream primer:

[0081] AAACCCTGTATTTGATGGTCTCTACTC

[0082] Primers for real-time quantitative PCR (RT-qPCR) of the TAT gene in the salvianolic acid metabolic pathway

[0083] Upstream primer:

[0084] CAACTGCTGGTCTTCCACAAAC

[0085] Downstream primer:

[0086] GCGAGCCAAAACGGACA

[0087] Primers for real-time quantitative PCR (RT-qPCR) of the PAL gene in the salvianolic acid metabolic pathway

[0088] Upstream primer:

[0089] GATAGCGGAGTGCAGGTCGTAC

[0090] Downstream primer:

[0091] CGAACTAGCAGATTGGCAGAGG

[0092] Primers for real-time quantitative PCR (RT-qPCR) of the C4H gene in the salvianolic acid metabolic pathway

[0093] Upstream primer:

[0094] CCAGGAGTCCAAATAACAGAGCCG

[0095] Downstream primer:

[0096] GCCACCAAGCGTTCACCAAGAT

[0097] Primers for real-time quantitative PCR (RT-qPCR) of the 4CL gene in the salvianolic acid metabolic pathway

[0098] Upstream primer:

[0099] ATTCGCATTCGCATTTCTCGG

[0100] Downstream primer:

[0101] GCGGCGTAGTGCTTCACCTTT

[0102] Primers for real-time quantitative PCR (RT-qPCR) of the HPPR gene in the salvianolic acid metabolic pathway

[0103] Upstream primer:

[0104] TGACTCCAGAAACAACCCACATT

[0105] Downstream primer:

[0106] CCCAGACGACCCTCCACAAG

[0107] Primers for real-time quantitative PCR (RT-qPCR) of the RAS gene in the salvianolic acid metabolic pathway

[0108] Upstream primer:

[0109] CGAGATCGCCTACTCCAAGTTCAAG

[0110] Downstream primer:

[0111] AGATGGCGTTACCGAAGTATCCCTG

[0112] Primers for real-time quantitative PCR (RT-qPCR) of the CYP98A14 gene in the salvianolic acid metabolic pathway

[0113] Upstream primer:

[0114] GGTCTGTACCGTCGTCCTCTTCTCC

[0115] Downstream primer:

[0116] ACAAGGCTGGTATTTGGGAAAAGGT

Claims

1. The application of the histone deacetylase SmHDA6 gene in promoting the accumulation of salvianolic acid in the hairy roots of Salvia miltiorrhiza, characterized in that, An sgRNA was designed at the exon of the SmHDA6 gene, and the CRISPR / Cas9-SmHDA6-sgRNA was inserted into pCAMBIA2300 to obtain a CRISPR-SmHDA6 knockout vector. The CRISPR-SmHDA6 knockout vector was transformed into Agrobacterium C58C1, and hairy roots of Salvia miltiorrhiza with the SmHDA6 gene knocked out were obtained by Agrobacterium-mediated transformation using Salvia miltiorrhiza explants. The nucleotide sequence of the SmHDA6 gene is shown in SEQ ID NO.1.

Images