Application of Salvia miltiorrhiza SmPYL4 gene in regulating tanshinone content

Abstract

The application discloses application of a Salvia miltiorrhiza SmPYL4 gene in regulating tanshinone content, and a nucleotide sequence of the Salvia miltiorrhiza SmPYL4 gene is shown as SEQ ID NO. 1. Specifically, the expression of the Salvia miltiorrhiza SmPYL4 gene is silenced or inhibited, so that the content of tanshinone in the hairy roots of Salvia miltiorrhiza is increased. The tanshinone is any one or two or more of tanshinone I, dihydrotanshinone I, tanshinone II A and cryptotanshinone. The application further discloses a method for increasing the content of tanshinone in the hairy roots of Salvia miltiorrhiza. The application finds that the Salvia miltiorrhiza SmPYL4 gene has a negative regulation effect on the synthesis and accumulation of tanshinone components, and silencing the gene can effectively increase the content of tanshinone components. The application has important reference value and significance for the cultivation of excellent varieties of Salvia miltiorrhiza.

Description

Technical Field

[0001] This invention relates to the application of the SmPYL4 gene in regulating tanshinone content, and belongs to the field of genetic engineering technology. Background Technology

[0002] Salvia miltiorrhiza Bunge., first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), is a perennial plant belonging to the genus *Salvia* in the Lamiaceae family. Its dried roots and rhizomes are used medicinally, possessing properties such as promoting blood circulation and removing blood stasis, regulating menstruation and relieving pain, clearing the heart and relieving irritability, and cooling the blood and reducing swelling. Lipid-soluble tanshinone compounds (including tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone) are the main active components of Salvia miltiorrhiza, exhibiting various pharmacological activities such as antitumor, anti-inflammatory, antihypertensive, and anti-fibrotic effects.

[0003] Improving the content of tanshinone compounds in Salvia miltiorrhiza and cultivating superior varieties of Salvia miltiorrhiza are important research directions at present. With the continuous development of molecular biology and genetic engineering, the secondary metabolism engineering of medicinal plants is becoming increasingly sophisticated. Overexpression or silencing of key enzyme genes and transcription factors involved in the synthesis pathway of tanshinone components has become an effective means to increase the accumulation of tanshinone components.

[0004] PYR / PYL / RCAR proteins are functional abscisic acid (ABA) receptors in plants, belonging to the START (Star-related Lipid-transfer) superfamily of ligand-binding proteins. They play a crucial role in ABA-mediated responses to various abiotic stresses. The PYR / PYL / RCAR protein family in the model plant Arabidopsis thaliana comprises 14 members: PYL7 / 8 / 9 / 10 (subfamily 1), PYL4 / 5 / 6 / 11 / 12 / 13 (subfamily 2), and PYR1 and PYL1 / 2 / 3 (subfamily 3). Previous studies have shown that overexpression of VlPYL1 in grapes promotes anthocyanin accumulation in the fruit; overexpression of the NtPYL4 gene in tobacco reduces alkaloid content in tobacco roots; similarly, overexpression of the NtPYL4 gene in Arabidopsis thaliana also reduces alkaloid accumulation. There are currently no research reports on the role of PYL family genes in regulating the root development of Salvia miltiorrhiza and the synthesis and accumulation of tanshinone secondary metabolites.

[0005] In previous studies, the inventors of this invention screened a tanshinone ABA receptor protein, SmPYL4, based on the tanshinone genome and transcriptomics. SmPYL4 has the highest expression level in tanshinone roots and responds strongly to exogenous PEG treatment with weak ABA induction. SmPYL4 can make a positive response to drought stress. Summary of the Invention

[0006] In view of the above-mentioned prior art, the present invention provides a new use for the SmPYL4 gene of Tanshinone—its application in regulating the content of tanshinone.

[0007] This invention is achieved through the following technical solution:

[0008] The application of the SmPYL4 gene in regulating tanshinone content, the nucleotide sequence of the SmPYL4 gene is shown in SEQ ID NO.1.

[0009] Furthermore, the application of silencing or inhibiting the expression of the SmPYL4 gene in increasing tanshinone content. Even further, silencing or inhibiting the expression of the SmPYL4 gene in *Salvia miltiorrhiza* increases the tanshinone content in the hairy roots of *Salvia miltiorrhiza*.

[0010] Furthermore, the specific method for silencing the SmPYL4 gene in *Salvia miltiorrhiza* is as follows: construct the silencing expression vector pK7GWIWG2R(Ⅱ)-SmPYL4, infect sterile leaves of *Salvia miltiorrhiza* using Agrobacterium-mediated transformation, and culture them to obtain *Salvia miltiorrhiza* plants with silenced expression of the SmPYL4 gene. The content of tanshinone in the hairy roots of this plant is higher than that of the wild-type plant.

[0011] Furthermore, the tanshinone is any one or more of tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone.

[0012] One method to increase the tanshinone content in the hairy roots of Salvia miltiorrhiza is as follows: silencing or inhibiting the SmPYL4 gene in Salvia miltiorrhiza plants.

[0013] Further, the specific method is as follows: construct the silencing expression vector pK7GWIWG2R(Ⅱ)-SmPYL4, infect sterile leaves of Salvia miltiorrhiza using Agrobacterium-mediated transformation, culture, and obtain Salvia miltiorrhiza plants with silenced expression of the SmPYL4 gene. The content of tanshinone in the hairy roots of this plant is higher than that of the wild-type plant.

[0014] Furthermore, the tanshinone is any one or more of tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone.

[0015] This invention constructed a silencing expression vector and an overexpression vector for the SmPYL4 gene in *Salvia miltiorrhiza*. Using Agrobacterium-mediated transformation, sterile leaves of *Salvia miltiorrhiza* were infected to obtain silencing and overexpressing lines of the SmPYL4 gene. The content of tanshinone in hairy roots and the expression levels of key enzyme genes in the synthesis pathway were detected. The results showed that the SmPYL4 gene has a negative regulatory effect on the synthesis and accumulation of tanshinone components, and silencing this gene can effectively increase the content of tanshinone components. This invention has important reference value and significance for the breeding of superior varieties of *Salvia miltiorrhiza*. Attached Figure Description

[0016] Figure 1 Electrophoretic detection results of hairy root genomic DNA, where M: DNA marker (DL2000); N: negative control.

[0017] Figure 2 The relative expression level of the SmPYL4 gene in the hairy roots of *Salvia miltiorrhiza*, where * represents P < 0.05 and ** represents P < 0.01.

[0018] Figure 3 Photographs of the test sample solutions of the hairy roots of each strain.

[0019] Figure 4 Results of determination of tanshinone content in hairy roots, where * represents P < 0.05 and ** represents P < 0.01.

[0020] Figure 5 : Relative expression levels of key enzyme genes in the tanshinone synthesis pathway in hairy roots, where * represents P < 0.05 and ** represents P < 0.01. Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0022] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0023] Experiment 1: Study on the effect of SmPYL4 gene expression in Danshen on its traits

[0024] This invention constructs a silenced expression system and an overexpression system for the SmPYL4 gene in *Salvia miltiorrhiza*, respectively, and studies its effects on root development and the synthesis and accumulation of tanshinone secondary metabolites.

[0025] The nucleotide sequence of the SmPYL4 gene in *Salvia miltiorrhiza* is shown in SEQ ID NO.1, as follows (direction 5'-3'):

[0026] ATGCCCTCTTCTCTTCAGATTCAGAGGATCAACCCCATAAACCACATCGCGTGCATGCATCAGAAGGCGGCGAATCCCGCTTCCTGGGTATGCCCCGCCTCCGCCGCCGCCTCCCTGCCGGAGGAGATGCTCCACCACCACACCCACGCGGTGGCCCCGGGGCAGTGCTGCTCCGCGGCGGTGCAGCTGATCGACGCGTCGGTGGAGGCCGTGTGGTCCGTGGTGCGCCGCTTCGACAGGCCGCACGAGTACAAGAAGTTCCTCAAGAGCTGCCACGTCATCCTCGGCGACGGCGACGTCGGCACCCTCCGCGAGGTGCGTGTGGTGTCGGGGCTGCCGGCGGCGACCAGCACCGAGCGCCTCGAGATCCTCGACGATGAGAGGCACGTCATGAGCTTCAGCGTCGTCGGCGGCGACCACCGCCTGCGCAACTACAGCTCCGTCACCACCCTGCATGCGGCGGGCGGCGGTGGTGGCACGGTGGTGGTGGAGTCGTACGTGGTGGATGTGCCGCACGGGAACACCAAGGAGGAAACCTGCGCCTTTATCGATACCATCGTCAAATGTAATCTGCACTCGCTCGCCCAGATTGCTCAGAATTTGCCTAAAACATATTAG。

[0027] The amino acid sequence of the protein expressed by the Salvia miltiorrhiza SmPYL4 gene is shown in SEQ ID NO.2 and is as follows:

[0028] MPSSLQIQRINPINHIACMHQKAANPASWVCPASAAASLPEEMLHHHTHAVAPGQCCSAAVQLIDASVEAVWSVVR RFDRPHEYKKFLKSCHVILGDGDVGTLREVRVVSGLPAATSTERLEILDDERHVMSFSVVGGDHRLRNYSSVTTLHAAGG GGGTVVVESYVVDVPHGNTKEETCAFIDTIVKCNLHSLAQIAQNLPKTY。

[0029] 1. Experimental materials

[0030] 1.1 Plant materials

[0031] The sterile seedlings of Salvia miltiorrhiza were subcultured in the tissue culture room of our laboratory under the following conditions: 25℃, 16 hours of light, and 8 hours of darkness.

[0032] 1.2 Vectors and Strains

[0033] pMDC202 vector, pDONR221 vector, and pK7GWIWG2R(Ⅱ) vector are all commonly used plant expression vectors, which were purchased commercially and stored in the laboratory for internal use.

[0034] Escherichia coli DH5α competent cells and Agrobacterium rhizogenes Ar.Qual competent cells were both commercially purchased and stored in the laboratory for internal use.

[0035] 2. Research Methods

[0036] 2.1 Construction of the SmPYL4 gene overexpression vector in Danshen

[0037] (1) Based on the ORF sequence of the SmPYL4 gene, upstream and downstream primers containing XbaI & KpnI restriction sites were designed using Primer Premier 5.0. The primer sequences are shown in Table 1.

[0038] Table 1. Nucleotide sequences of the specific primers used in this study.

[0039]

[0040] (2) The target gene containing homologous arms and restriction enzyme sites was amplified by PCR, and the product was purified and recovered. The pMDC202 linearized vector was obtained by restriction enzyme digestion and purification. The target gene and the vector were ligated according to the homologous recombination kit, and then transformed into DH5α competent cells. Single clones were picked and cultured. After PCR identification, the cells were sent to Platinum Biotech Co., Ltd. for sequencing. The positive plasmid pMDC202-SmPYL4 with correct sequencing was used for subsequent experiments.

[0041] 2.2 Construction of the SmPYL4 gene silencing vector in Danshen

[0042] (1) Based on the principles of RNAi vector construction and Gateway principle, primers containing attB adapters were designed. The primer sequences are shown in Table 1.

[0043] (2) PCR amplification of specific primers containing attB adapters, and purification and recovery of the target gene fragment.

[0044] (3) Following the instructions of the Gateway BP Clonase II Enzyme Mix kit, the target fragment was ligated to the intermediate vector. The reaction mixture consisted of: target fragment, 1 μL; pDONR221 plasmid, 1 μL; BP enzyme, 1 μL; ddH2O, 3 μL. The reaction solution was incubated in a PCR instrument at 25°C for 5 h. After the reaction, 1 μL of Proteinase K was added to the centrifuge tube, and the mixture was incubated in a PCR instrument at 37°C for 10 min to terminate the BP reaction.

[0045] (4) The ligation product was transformed into Escherichia coli DH5α competent cells; single clones were picked and expanded for culture, and positive clones were identified by PCR in the bacterial culture. The culture was then sent to Platinum Biotech Co., Ltd. for sequencing; positive bacterial cultures with correct sequencing were expanded for culture, and pDONR221-SmPYL4 positive plasmid was extracted.

[0046] (5) Following the instructions of the Gateway LR Clonase II Enzyme Mix kit, the pDONR221-SmPYL4 positive plasmid was ligated to the pK7GWIWG2R(Ⅱ) vector to construct the plant silencing vector pK7GWIWG2R(Ⅱ)-SmPYL4. The reaction system was as follows: pDONR221-SmPYL4 positive plasmid, 1 μL; pK7GWIWG2R(Ⅱ) plasmid, 1 μL; LR enzyme, 1 μL; ddH2O, 2 μL. The reaction solution was incubated in a PCR instrument at 25℃ for 5 h. After the reaction, 1 μL of Proteinase K was added to the centrifuge tube, and the LR reaction was terminated by incubating in a PCR instrument at 37℃ for 10 min.

[0047] (6) The ligation product was transformed into competent E. coli cells; the positive bacterial culture with correct sequencing was expanded and cultured, and the pK7GWIWG2R(Ⅱ)-SmPYL4 positive plasmid was extracted for subsequent experiments.

[0048] 2.3 Construction of the SmPYL4 gene genetic transformation system in Danshen

[0049] (1) Recombinant plasmids pMDC202-SmPYL4 and pK7GWIWG2R(II)-SmPYL4 were transformed into Agrobacterium rhizogenes Ar.Qual competent cells, while Ar.Qual competent cells without any plasmids were used as controls. The specific operation is as follows:

[0050] a. Take out Ar.Qual competent cells, hold them in your palm for a moment, and when they are in an ice-water mixture, insert them into ice;

[0051] b. Add 0.05 μg of recombinant plasmid to each 100 μL of competent cells, quickly mix by tapping the bottom of the tube, and place the tube on ice, in liquid nitrogen, in a 37°C water bath, and on ice for 5 min each in sequence;

[0052] c. Add to 700 μL of antibiotic-free LB liquid medium and incubate in a shaker at 28°C for 3 h;

[0053] d. Centrifuge at 6000 rpm for 1 min, collect the bacterial pellet, and retain 100 μL of supernatant. Mix well and spread on YEB solid medium plates containing 100 mg / L Spe antibiotic (silencing plasmid) and 50 mg / L Kan (overexpression plasmid), respectively. Incubate upside down in an incubator at 28 ℃ for 3 days.

[0054] e. Pick a single colony and inoculate it into 5 mL of YEB liquid medium containing the corresponding antibiotic. Incubate overnight at 28°C and 200 rpm. Store the colony culture that is positive by colony PCR at 4°C for later use.

[0055] (2) Take 750 μL of the above-identified positive bacterial suspension and inoculate it into 75 mL of YEB liquid medium containing the corresponding antibiotic, and activate the bacterial suspension to OD. 600 =0.6~1.0, centrifuge at 5000rpm for 10min, collect the bacterial pellet, resuspend the bacterial cells in MS liquid medium, then add 400μmol / L acetylsuccinone, and incubate at 28℃ and 200rpm in a shaker for 20min.

[0056] (3) Select leaves from sterile Salvia miltiorrhiza seedlings that have been subcultured for 20-30 days and cut them into 0.5cm pieces. 2 The samples were pre-cultured on MS (pH=5.8) medium for 2 days; then completely immersed in the bacterial solution for 10-15 minutes. After blotting the surface bacterial solution with sterile filter paper, they were spread on MS solid medium and cultured for 3 days.

[0057] (4) Rinse 5 times in sterile water, absorb excess water with sterile filter paper, spread evenly on MS solid medium containing a final concentration of 500 mg / L Cef for sterilization culture, change the medium every 7 days, and reduce the concentration of Cef in the medium from 500 mg / L to 400, 300, 200, 100 and 50 mg / L in turn, and finally transfer to MS medium without antibiotics for culture.

[0058] (5) When the hairy roots grow to about 2 cm, separate the single root system. Culture on MS solid medium for about 30 days, then transfer to 50 mL of MS liquid medium and expand the culture in a shaker at 25 °C and 120 rpm.

[0059] 2.4 Molecular identification of transgenic hairy roots

[0060] (1) Genomic DNA was extracted from the transgenic hairy roots. The specific procedures are as follows:

[0061] a. Take 100 mg of hairy roots from liquid culture, add liquid nitrogen and grind thoroughly. Immediately add 400 μL Buffer A1 and 4 μL Rnase A to the ground sample, and vigorously shake the centrifuge tube to ensure complete lysis.

[0062] b. Incubate in a 65℃ water bath for 10 minutes, inverting the container 2-3 times during the process;

[0063] c. Add 130 μL of Buffer A2, mix thoroughly, place on ice for 5 min, centrifuge at 14000 rpm for 5 min, carefully aspirate the supernatant into a new 1.5 mL centrifuge tube, add 1.5 times the volume of Buffer A3 of the supernatant, and immediately pipette to mix.

[0064] d. Transfer the mixture from c to the FastPure gDNA Columns IV already placed in the collection tube, centrifuge at 12000 rpm for 30 s, and discard the filtrate;

[0065] e. Add 600 μL of Buffer AW (with anhydrous ethanol already added), centrifuge at 12000 rpm for 30 seconds, discard the filtrate; repeat this operation.

[0066] f. Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much of the washing solution as possible;

[0067] g. Place the adsorption column into a new 1.5 mL centrifuge tube, add 50 μL of preheated Elution Buffer (65–75 °C) to the center of the membrane, incubate at room temperature for 3–5 min, centrifuge at 12000 rpm for 1 min, and detect the concentration and quality of DNA using a micro spectrophotometer. Store the obtained DNA at -20 °C.

[0068] (2) Design of identification primers. The specific operation is as follows: use the hygromycin gene hpt on the pMDC202-F+SmPYL4-R and the rolB gene on the Agrobacterium rhizogenes Ri plasmid as templates to design primers for identifying overexpressing hairy root lines; and design primers for identifying silent hairy root lines using p35-F+SmPYL4-R based on the 35S promoter in the pK7GWIWG2R(Ⅱ) vector. The primer sequences are shown in Table 1.

[0069] (3) Using the DNA extracted in 2.4(1) above as a template, PCR amplification was performed, and positive strains were identified by electrophoresis.

[0070] 2.5 Determination of expression levels of key enzyme genes in the tanshinone synthesis pathway

[0071] RNA was extracted and reverse transcribed from the transgenic hairy root lines that were identified as positive. The changes in the expression levels of the SmPYL4 gene and related synthase genes in the tanshinone metabolic pathway were detected by qRT-PCR. The primer sequences of related synthase genes in the tanshinone metabolic pathway are shown in Table 2.

[0072] Table 2 Primer sequences of related synthase genes in the tanshinone metabolic pathway

[0073]

[0074] 2.6 Determination of Tanshinone Content

[0075] (1) Accurately weigh four reference standards: tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone, and place them in four 2mL brown volumetric flasks. Dilute each flask with 100% chromatographic methanol to 1mg / mL. At the same time, prepare a mixed standard solution based on the four components and dilute it to 1mg / mL. Dilute the solutions sequentially to prepare standard solutions with concentrations of 0.5mg / mL, 0.1mg / mL, 0.05mg / mL, 0.01mg / mL and 0.005mg / mL. Plot the standard curves of the four tanshinone components using the concentration (X) and peak area (Y) of the standard solutions.

[0076] (2) Wild-type (WT), overexpression (OE), and silent (RNAi) hairy root samples with consistent growth cycles and freeze-dried for 48 h were ground and passed through a 40-mesh sieve; 0.25 g of sample was accurately weighed into a 50 mL centrifuge tube, 25 mL of chromatographic methanol was added, the mixture was vortexed for 3 min, ultrasonically extracted for 50 min at 100 W, 100 Hz, and 50 °C, centrifuged at 4000 rpm for 10 min, the supernatant was collected and passed through a 0.22 μm organic filter to obtain the sample solution to be tested.

[0077] (3) The contents of four tanshinone components in the hairy root sample were determined by HPLC. The specific operation was as follows: 10 μL of sample was injected at a volume flow rate of 1.0 mL / min and a column temperature of 30±5℃ under the condition of detection wavelength of 270 nm. The mobile phase was 0.01% phosphoric acid as the aqueous phase (A) and acetonitrile as the organic phase (B). The elution gradient of the mobile phase is shown in Table 3.

[0078] Table 3 Gradient Elution of Mobile Phase

[0079]

[0080] 2.7 Data Processing and Analysis

[0081] The data was processed and analyzed using software such as SPSS 22.0, and graphs were created using GraphPad Prism 8.0 software.

[0082] 3. Results and Analysis

[0083] 3.1 Obtaining and Identifying Hairy Roots

[0084] Salvia miltiorrhiza leaves were infected with recombinant plasmids containing pMDC202-SmPYL4 and pK7GWIWG2R(Ⅱ)-SmPYL4, as well as Agrobacterium rhizogenes Ar.Qual without any plasmids, to obtain overexpressed (OE), silent (RNAi), and wild-type (WT) hairy roots. Positive lines were screened by PCR amplification of genomic DNA, yielding 8 wild-type hairy roots (strain name WT, 10 lines tested), 6 overexpressed hairy roots (strain name SmPYL4OE, 8 lines tested), and 5 silent hairy roots (strain name SmPYL4RNAi, 8 lines tested), with positive rates of 80%, 75%, and 62.5%, respectively. The electrophoretic detection results of the hairy root genomic DNA are shown below. Figure 1 As shown, a single band was detected in all positive transgenic hairy roots, and the band size was correct, indicating that the T-DNA region of the recombinant plasmid was successfully inserted into the Danshen genome.

[0085] 3.2 Determination of SmPYL4 gene expression in hairy roots

[0086] The expression level of the SmPYL4 gene in hairy roots was detected by qRT-PCR, and three lines with the most significant changes were selected for demonstration. The relative expression levels of the SmPYL4 gene in hairy roots of *Salvia miltiorrhiza* are shown below. Figure 2 As shown in the figure, gene expression levels in all overexpressing lines were significantly higher than those in wild-type lines, while gene expression levels in all silent lines were significantly lower than those in wild-type lines. Specifically, the highest expression level of SmPYL4 gene was observed in line OE-6, which was 23.99 times that of the wild-type line; the lowest expression level was observed in line RNAi-2, where SmPYL4 gene expression decreased to 23.47% of that in the wild-type line. These results further demonstrate that the obtained lines are positive lines and can be used for further research.

[0087] 3.3 Determination of Tanshinone Content in Hairy Roots

[0088] To investigate the effects of the SmPYL4 gene on the biosynthesis and accumulation of tanshinone compounds, this invention expanded the culture of positive transgenic hairy root lines, then ground and extracted the hairy roots to obtain test sample solutions. Photographs of the test sample solutions from the hairy roots of each line are shown below. Figure 3As shown, compared to the wild-type strain, the test sample solution of the silent strain is redder, while the test sample solution of the overexpression strain is lighter in color. This suggests that the tanshinone content may be higher in the SmPYL4 gene-silenced strain, while the tanshinone content may be lower in the overexpression strain.

[0089] To further verify this hypothesis, the content of tanshinone compounds in hairy roots was determined by HPLC, and the results are as follows: Figure 4 As shown.

[0090] Regarding dihydrotanshinone I: In wild-type lines, the content of dihydrotanshinone I was 0.5373 mg / g. In the overexpression lines OE-2, OE-3, and OE-6, the contents of dihydrotanshinone I were 0.1743 mg / g, 0.3056 mg / g, and 0.4604 mg / g, respectively, representing 32.43%, 56.87%, and 85.68% of the wild-type lines. In the silent lines RNAi-1, RNAi-2, and RNAi-3, the contents of dihydrotanshinone I were 0.9005 mg / g, 0.9307 mg / g, and 1.002 mg / g, respectively, representing 1.67 times, 1.73 times, and 1.86 times of the wild-type lines. The content of dihydrotanshinone I in the transgenic lines was significantly different from that in the wild-type lines.

[0091] Regarding cryptotanshinone: In wild-type lines, the cryptotanshinone content was 1.059 mg / g. In the overexpression lines OE-2, OE-3, and OE-6, the cryptotanshinone contents were 0.3855 mg / g, 0.3151 mg / g, and 0.6403 mg / g, respectively, representing 36.40%, 29.75%, and 60.46% of the wild-type contents. In the silent lines RNAi-1, RNAi-2, and RNAi-3, the cryptotanshinone contents were 1.245 mg / g, 4.235 mg / g, and 4.555 mg / g, respectively, representing 1.17 times, 3.99 times, and 4.3 times of the wild-type contents. The cryptotanshinone content in the transgenic lines differed significantly from that in the wild-type lines.

[0092] Regarding tanshinone I: In wild-type lines, the content of tanshinone I was 0.8924 mg / g. In the overexpression lines OE-2, OE-3, and OE-6, the contents of tanshinone I were 0.3235 mg / g, 0.4015 mg / g, and 0.5121 mg / g, respectively, representing 36.25%, 44.99%, and 57.38% of the wild-type lines. In the silent lines RNAi-1, RNAi-2, and RNAi-3, the contents of tanshinone I were 1.126 mg / g, 1.168 mg / g, and 1.124 mg / g, respectively, representing 1.26 times, 1.3 times, and 1.25 times of the wild-type lines. The content of tanshinone I in the transgenic lines was significantly different from that in the wild-type lines.

[0093] Regarding tanshinone IIA: In the wild-type lines, the content of tanshinone IIA was 0.7754 mg / g. In the overexpression lines OE-2, OE-3, and OE-6, the contents of tanshinone IIA were 0.1624 mg / g, 0.1020 mg / g, and 0.2001 mg / g, respectively, representing 20.94%, 13.15%, and 25.80% of the wild-type lines. In the silent lines RNAi-1, RNAi-2, and RNAi-3, the contents of tanshinone IIA were 1.373 mg / g, 1.450 mg / g, and 1.456 mg / g, respectively, representing 1.77 times, 1.87 times, and 1.88 times of the wild-type lines. The content of tanshinone IIA in the transgenic lines differed significantly from that in the wild-type lines.

[0094] In summary, the SmPYL4 gene has a negative regulatory effect on the synthesis and accumulation of tanshinone components, especially significantly increasing the content of cryptotanshinone and tanshinone IIA.

[0095] 3.4 Determination of expression levels of key enzyme genes in the tanshinone synthesis pathway in hairy roots

[0096] To further investigate the molecular mechanism by which the SmPYL4 gene regulates the biosynthesis and accumulation of tanshinone components, qRT-PCR was used to detect the expression of key enzyme genes in the tanshinone biosynthesis pathway, namely SmCPS1, SmDXR, SmHDS, SmGGPPS, SmHMGR, SmMEK, and SmDXS, in hairy roots. The relative expression levels of each gene are shown in the figure. Figure 5As shown in the figure, the expression levels of SmDXS and SmMEK genes in the overexpression lines OE-2, OE-3, and OE-6 were significantly lower than those in the wild-type lines. Specifically, in line OE-6, the expression level of SmDXS gene was only 12.68% of that in the wild-type line, and the expression level of SmMEK gene was only 25.15% of that in the wild-type line. In lines OE-2 and OE-3, the expression levels of SmDXS gene were 16.12% and 38.95% of that in the wild-type line, respectively, and the expression levels of SmMEK gene were 37.91% and 37.18% of that in the wild-type line, respectively. In addition, the expression level of SmCPS1 gene was also reduced in lines OE-2, OE-3, and OE-6, with average increases of 37.72%, 22.23%, and 15.71% compared to the wild-type line, respectively. In the silent strains, the expression levels of these key enzyme genes showed the opposite changes. Notably, the expression levels of the SmDXR and SmHDS genes varied relatively little across all strains, with the expression levels in the silent RNAi-2 strain being only 1.879 times and 1.6 times that of the wild-type strain, respectively.

[0097] 4. Conclusion

[0098] The SmPYL4 gene is a negative regulator in the synthesis and accumulation of tanshinone. Overexpression of the SmPYL4 gene reduces the expression levels of tanshinone components (tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone), while silencing the SmPYL4 gene increases the expression levels of tanshinone components (tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone).

[0099] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. The application of the SmPYL4 gene in regulating tanshinone content, characterized by: Silencing or inhibiting the expression of the SmPYL4 gene in *Salvia miltiorrhiza* increases the content of tanshinone in the hairy roots of *Salvia miltiorrhiza*; the nucleotide sequence of the SmPYL4 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The specific method for silencing the SmPYL4 gene in *Salvia miltiorrhiza* is as follows: construct the silencing expression vector pK7GWIWG2R(Ⅱ)-SmPYL4, infect sterile leaves of *Salvia miltiorrhiza* using Agrobacterium-mediated transformation, and culture them to obtain *Salvia miltiorrhiza* plants with silenced expression of the SmPYL4 gene. The content of tanshinone in the hairy roots of this plant is higher than that in the wild-type plant.

3. The application according to claim 1, characterized in that: The tanshinone is any one or more of tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone.

4. The application according to claim 3, characterized in that: The tanshinone is tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone.

5. A method for increasing the tanshinone content in the hairy roots of Salvia miltiorrhiza, characterized in that: Silencing or inhibiting the SmPYL4 gene in Salvia miltiorrhiza plants; the nucleotide sequence of the SmPYL4 gene is shown in SEQ ID NO.

1.

6. The method for increasing the tanshinone content in the hairy roots of Salvia miltiorrhiza according to claim 5, characterized in that: The silencing expression vector pK7GWIWG2R(Ⅱ)-SmPYL4 was constructed and infected with sterile leaves of Salvia miltiorrhiza using Agrobacterium-mediated transformation. After culturing, Salvia miltiorrhiza plants with silenced expression of the SmPYL4 gene were obtained. The content of tanshinone in the hairy roots of this plant was higher than that in the wild-type plant.

7. The method for increasing the tanshinone content in the hairy roots of Salvia miltiorrhiza according to claim 5, characterized in that: The tanshinone is any one or more of tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone.

8. The method for increasing the tanshinone content in the hairy roots of Salvia miltiorrhiza according to claim 7, characterized in that: The tanshinone is tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone.

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