Application of salvia miltiorrhiza SmPYL4 gene in regulation and control of tanshinone content

Abstract

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

Description

Technical Field

[0001] The invention relates to application of the Salvia miltiorrhiza SmPYL4 gene in regulating tanshinone content, and belongs to the technical field of genetic engineering. Background Art

[0002] Salvia miltiorrhiza Bunge., first mentioned in Shennong's Herbal Classic, is a perennial plant of the genus Salvia in the family Lamiaceae. Its dried roots and rhizomes are used medicinally, boasting benefits such as promoting blood circulation and removing blood stasis, relieving pain through menstruation, clearing the heart and relieving restlessness, and cooling blood and eliminating carbuncles. Lipid-soluble tanshinones (including tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone) are the primary active components of danshen, exhibiting diverse pharmacological activities, including anti-tumor, anti-inflammatory, anti-hypertensive, and anti-fibrotic activities.

[0003] Current research focuses on increasing the content of tanshinone compounds in Danshen and cultivating superior varieties. With the continuous advancement of molecular biology and genetic engineering, the secondary metabolism of medicinal plants is becoming increasingly sophisticated. Overexpression or silencing of key enzyme genes and transcription factors involved in the biosynthesis of tanshinone components has become an effective means of increasing 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 various ABA-mediated abiotic stress responses. The model plant Arabidopsis thaliana contains 14 members of the PYR / PYL / RCAR protein family, including PYL7 / 8 / 9 / 10 in subfamily I, PYL4 / 5 / 6 / 11 / 12 / 13 in subfamily II, and PYR1 and PYL1 / 2 / 3 in subfamily III. Previous studies have shown that overexpressing VlPYL1 in grapes promotes anthocyanin accumulation in fruit, and overexpressing the NtPYL4 gene in tobacco reduces alkaloid content in tobacco roots. Similarly, overexpression of NtPYL4 in Arabidopsis also reduces alkaloid accumulation. So far, there are no reports on the role of PYL family genes in regulating root development and the synthesis and accumulation of tanshinone secondary metabolites in Salvia miltiorrhiza.

[0005] In previous studies, the inventors of the present invention screened and obtained a Salvia miltiorrhiza ABA receptor protein SmPYL4 based on the Salvia miltiorrhiza genome and transcriptomics. The protein has the highest expression level in Salvia miltiorrhiza roots, is weakly induced by ABA and strongly responds to exogenous PEG treatment. SmPYL4 can actively respond to drought stress. Summary of the Invention

[0006] In response to the above-mentioned prior art, the present invention provides a new use of the Salvia miltiorrhiza SmPYL4 gene - its use in regulating the content of tanshinones.

[0007] The present invention is achieved through the following technical solutions:

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

[0009] Furthermore, silencing or inhibiting the expression of the SmPYL4 gene in Salvia miltiorrhiza is used to increase the content of tanshinones. Still further, silencing or inhibiting the expression of the SmPYL4 gene in Salvia miltiorrhiza increases the content of tanshinones in the hairy roots of Salvia miltiorrhiza.

[0010] Furthermore, the specific method for silencing the SmPYL4 gene of Salvia miltiorrhiza is: constructing a silencing expression vector pK7GWIWG2R(Ⅱ)-SmPYL4, using Agrobacterium-mediated method to infect sterile leaves of Salvia miltiorrhiza, and culturing to obtain Salvia miltiorrhiza plants with silenced expression of the SmPYL4 gene of Salvia miltiorrhiza, the content of tanshinone in the hairy roots of the plants is higher than that of the wild-type plants.

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

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

[0013] Furthermore, the specific method is: constructing a silencing expression vector pK7GWIWG2R(Ⅱ)-SmPYL4, using Agrobacterium-mediated method to infect sterile leaves of Salvia miltiorrhiza, and culturing to obtain Salvia miltiorrhiza plants with silenced expression of the Salvia miltiorrhiza SmPYL4 gene, the content of tanshinone in the hairy roots of the plants is higher than that of the wild-type plants.

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

[0015] The present invention constructed silencing and overexpression vectors for the Salvia miltiorrhiza SmPYL4 gene. Using Agrobacterium-mediated infection of sterile Salvia miltiorrhiza leaves, the authors generated SmPYL4-silenced and overexpressing strains. Tanshinones were detected in hairy roots, as well as the expression levels of key enzyme genes involved in the synthesis pathway. The results showed that the SmPYL4 gene negatively regulates the synthesis and accumulation of tanshinones, and that silencing the gene effectively increases the content of tanshinones. This invention has important reference value and significance for the cultivation of superior Salvia miltiorrhiza varieties. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0017] Figure 2 : Relative expression levels of SmPYL4 gene in hairy roots of Salvia miltiorrhiza, where * represents P < 0.05, and ** represents P < 0.01.

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

[0019] Figure 4 : Determination results 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 biosynthesis pathway in hairy roots, where * represents P < 0.05 and ** represents P < 0.01. DETAILED DESCRIPTION

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

[0022] Unless otherwise specified, the instruments, reagents, and materials involved in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0023] Experiment 1: Effects of SmPYL4 gene expression on Salvia miltiorrhiza traits

[0024] The present invention respectively constructs a silencing expression system and an overexpression system of the Salvia miltiorrhiza SmPYL4 gene to study their effects on the development of the Salvia miltiorrhiza root system and the synthesis and accumulation of tanshinones secondary metabolites.

[0025] The nucleotide sequence of the SmPYL4 gene of Salvia miltiorrhiza is shown in SEQ ID NO. 1, as shown below (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] Sterile Salvia miltiorrhiza seedlings were subcultured in the tissue culture room of our laboratory under the following culture conditions: 25°C, 16 h light, 8 h dark.

[0032] 1.2 Vectors and strains

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

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

[0035] 2. Research Methods

[0036] 2.1 Construction of Salvia miltiorrhiza SmPYL4 gene overexpression vector

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

[0038] Table 1 Nucleotide sequences of specific primers involved in this study

[0039]

[0040] (2) PCR amplified the target gene containing homology arms and restriction sites, and purified the recovered product; enzyme digestion and purification were performed to obtain the pMDC202 linearized vector; using the homologous recombination kit, the target gene and vector were connected, and then transformed into DH5α competent cells, and a single clone was picked and expanded for culture. After PCR identification, it was sent to Boshang Biotechnology Co., Ltd. for sequencing. The positive plasmid pMDC202-SmPYL4 with correct sequencing was used for subsequent experiments.

[0041] 2.2 Construction of Salvia miltiorrhiza SmPYL4 gene silencing vector

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

[0043] (2) PCR amplification using specific primers containing attB linkers, and purification and recovery of target gene fragments.

[0044] (3) According to the instructions of the Gateway BP Clonase II Enzyme mix kit, the target fragment was ligated with the intermediate vector. The reaction system was as follows: 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 incubated in a PCR instrument at 37°C for 10 min to terminate the BP reaction.

[0045] (4) The ligation product was transformed into E. coli DH5α competent cells; a single clone was picked and expanded for culture, and the positive clone was identified by PCR in the bacterial solution, and then sent to Boshang Biotechnology Co., Ltd. for sequencing; the positive bacterial solution with correct sequencing was expanded for culture, and the pDONR221-SmPYL4 positive plasmid was extracted;

[0046] (5) According to the instructions of the Gateway LR Clonase II Enzyme mix kit, the pDONR221-SmPYL4 positive plasmid was ligated with 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°C for 5 h. After the reaction, 1 μL of Proteinase K was added to the centrifuge tube and incubated in a PCR instrument at 37°C for 10 min to terminate the LR reaction.

[0047] (6) The ligation products were transformed into competent E. coli cells; the positive bacterial solution 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 genetic transformation system of Salvia miltiorrhiza SmPYL4 gene

[0049] (1) The recombinant plasmids pMDC202-SmPYL4 and pK7GWIWG2R(II)-SmPYL4 were transformed into Agrobacterium rhizogenes Ar.Qual competent cells. Ar.Qual competent cells without any plasmid were used as a control. The specific operation was as follows:

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

[0051] b. Add 0.05 μg of recombinant plasmid to each 100 μL competent medium, quickly stir the bottom of the tube to mix, and place on ice, liquid nitrogen, 37°C water bath, and ice for 5 minutes each;

[0052] c. Add to 700 μL of LB liquid medium without antibiotics and culture in a constant temperature 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 the supernatant. Mix thoroughly and spread the mixture onto YEB solid medium plates containing 100 mg / L Spe antibiotic (silencing plasmid) and 50 mg / L Kan (overexpression plasmid). Incubate the plates upside down at 28°C in an incubator for 3 days.

[0054] e. Pick a single colony and inoculate it into 5 mL of YEB liquid medium containing the corresponding antibiotics. Culture overnight at 28°C and 200 rpm. Store the bacterial suspension that is positive by colony PCR at 4°C until use.

[0055] (2) 750 μL of the positive bacterial solution identified above was taken and inoculated into 75 mL of YEB liquid culture medium containing the corresponding antibiotics, and activated until the bacterial solution OD 600 = 0.6-1.0, centrifuged at 5000 rpm for 10 min, collected the bacterial pellet, and resuspended the bacterial cells in MS liquid culture medium. Then, 400 μmol / L acetosyringone was added and cultured in a shaking incubator at 28°C and 200 rpm for 20 min.

[0056] (3) Select the leaves of sterile Salvia miltiorrhiza seedlings that have been subcultured for 20 to 30 days and cut them into 0.5 cm 2 The size of the bacteria was pre-cultured on MS (pH=5.8) medium for 2 days; then it was completely immersed in the bacterial solution and infected for 10-15 minutes. After the surface bacterial solution was absorbed with sterile filter paper, it was spread flat on MS solid medium and cultured for 3 days.

[0057] (4) Rinse five times in sterile water, dry the excess water with sterile filter paper, and spread on MS solid medium containing a final concentration of 500 mg / L Cef for sterilization and culture. Change the medium every 7 days. The concentration of Cef in the medium is reduced from 500 mg / L to 400, 300, 200, 100, and 50 mg / L in sequence. 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, 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) Extract genomic DNA from transgenic hairy roots. The specific operation is as follows:

[0061] a. Grind 100 mg of liquid-cultured hairy roots thoroughly in liquid nitrogen. Immediately add 400 μL of Buffer A1 and 4 μL of RNase A to the ground sample. Vigorously flick the centrifuge tube to fully lyse the sample.

[0062] b. Bath in a 65°C water bath for 10 minutes, inverting 2-3 times;

[0063] c. Add 130 μL of Buffer A2, mix thoroughly, place on ice for 5 minutes, centrifuge at 14,000 rpm for 5 minutes, carefully aspirate the supernatant into a new 1.5 mL centrifuge tube, add 1.5 times the volume of the supernatant, and immediately mix by pipetting.

[0064] d. Transfer the mixture from step c to FastPure gDNA Columns IV placed in a collection tube, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0065] e. Add 600 μL of Buffer AW (with anhydrous ethanol added), centrifuge at 12,000 rpm for 30 seconds, discard the filtrate, and repeat this process.

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

[0067] g. Place the adsorption column in a new 1.5 mL centrifuge tube. Add 50 μL of Elution Buffer preheated to 65-75°C to the center of the membrane. Incubate at room temperature for 3-5 minutes. Centrifuge at 12,000 rpm for 1 minute. Measure the DNA concentration and quality using a microspectrophotometer. Store the obtained DNA in a refrigerator at -20°C.

[0068] (2) Design of identification primers. The specific operations were as follows: primers for identifying overexpression hairy root strains were designed using the hygromycin gene hpt on the pMDC202 vector and the pMDC202 vector, and the rolB gene on the Ri plasmid of Agrobacterium rhizogenes as templates; primers for identifying silent hairy root strains were designed using the 35S promoter in the pK7GWIWG2R(Ⅱ) vector and p35-F+SmPYL4-R. The primer sequences are shown in Table 1.

[0069] (3) Using the DNA extracted in 2.4(1) above as a template, perform PCR amplification and electrophoresis detection to identify positive strains.

[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 identified as positive, and qRT-PCR was used to detect changes in the expression levels of the SmPYL4 gene and related synthase genes in the tanshinone metabolic pathway. The primer sequences of the 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 the four reference substances, namely, tanshinone I, dihydrotanshinone I, tanshinone IIA, and cryptotanshinone, and place them in four 2 mL brown volumetric flasks. Use 100% chromatographic methanol to make the volume to 1 mg / mL. At the same time, prepare a mixed standard solution based on the four components and make the volume to 1 mg / mL. Dilute the solution to prepare standard solutions with concentrations of 0.5 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.01 mg / mL, and 0.005 mg / mL in sequence. Draw the standard curves of the four tanshinone components based on the concentration (X) and peak area (Y) of the standard solutions.

[0076] (2) The hairy root samples of wild type (WT), overexpression type (OE) and silenced type (RNAi) with the same growth cycle and freeze-dried for 48 hours 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, vortexed for 3 minutes, ultrasonically extracted at 100 W, 100 Hz, and 50°C for 50 minutes, centrifuged at 4000 rpm for 10 minutes, and the supernatant was aspirated and passed through a 0.22 μm organic filter to obtain the sample solution to be tested.

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

[0078] Table 3 Mobile phase gradient elution table

[0079]

[0080] 2.7 Data Processing and Analysis

[0081] SPSS 22.0 and other software were used to process and analyze the data, and GraphPad Prism 8.0 software was used to draw graphs.

[0082] 3. Results and Analysis

[0083] 3.1 Acquisition and identification of hairy roots

[0084] The leaves of Salvia miltiorrhiza were infected with Agrobacterium rhizogenes Ar.Qual containing the recombinant plasmids pMDC202-SmPYL4 and pK7GWIWG2R(Ⅱ)-SmPYL4, as well as those without any plasmids, to obtain overexpression (OE), silenced (RNAi) and wild-type (WT) hairy roots. Positive strains were screened by PCR amplification of genomic DNA, and 8 wild-type hairy roots (strain name WT, 10 strains tested), 6 overexpression hairy roots (strain name SmPYL4OE, 8 strains tested), and 5 silenced hairy roots (strain name SmPYL4RNAi, 8 strains tested) were obtained. The positive rates of hairy roots were 80%, 75%, and 62.5%, respectively. The results of electrophoresis detection of genomic DNA of hairy roots are shown in Figure 2. Figure 1 As shown, a single band was detected in the 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 Salvia miltiorrhiza genome.

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

[0086] The expression level of SmPYL4 gene in hairy roots was detected by qRT-PCR, and three strains with the most significant changes were selected for display. The relative expression level of SmPYL4 gene in hairy roots of Salvia miltiorrhiza is shown in Figure 2. Figure 2 As shown in the figure, gene expression levels in all overexpression lines were significantly higher than those in the wild-type line, while gene expression levels in the silenced lines were significantly lower than those in the wild-type line. Among them, the highest expression level of the SmPYL4 gene in line OE-6 was 23.99 times that of the wild-type line, while the lowest expression level in line RNAi-2 was reduced 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] In order to study the effect of SmPYL4 gene on the biosynthesis and accumulation of tanshinone components, the present invention expanded the culture of positive transgenic hairy root lines, and then ground and extracted the hairy roots to obtain the test sample solutions. The photos of the test sample solutions of the hairy roots of each line are shown in Figure 2. Figure 3As shown in the figure, compared with the wild-type strain, the color of the test sample solution corresponding to the silenced strain is redder, while the red color of the test sample solution corresponding to the overexpression strain is lighter. This preliminarily speculates 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 components in hairy roots was determined by HPLC. The results are as follows Figure 4 shown.

[0090] Regarding dihydrotanshinone I: In the wild-type strain, the content of dihydrotanshinone I was 0.5373 mg / g. In the overexpression strains OE-2, OE-3, and OE-6, the contents 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 content. In the silenced strains RNAi-1, RNAi-2, and RNAi-3, the contents were 0.9005 mg / g, 0.9307 mg / g, and 1.002 mg / g, respectively, representing 1.67-fold, 1.73-fold, and 1.86-fold of the wild-type content, respectively. The dihydrotanshinone I content in the transgenic strains was significantly different from that in the wild-type strain.

[0091] Regarding cryptotanshinone: In the wild-type strain, the cryptotanshinone content was 1.059 mg / g. In the overexpression strains 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 strain, respectively. In the silenced strains 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-fold, 3.99-fold, and 4.3-fold of the wild-type strain, respectively. The cryptotanshinone content in the transgenic strains was significantly different from that in the wild-type strain.

[0092] Regarding Tanshinone I: In the wild-type strain, the content of Tanshinone I was 0.8924 mg / g. In the overexpression strains OE-2, OE-3, and OE-6, the contents 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 content. In the silenced strains RNAi-1, RNAi-2, and RNAi-3, the contents were 1.126 mg / g, 1.168 mg / g, and 1.124 mg / g, respectively, representing 1.26-fold, 1.3-fold, and 1.25-fold of the wild-type content, respectively. Tanshinone I content in the transgenic strains was significantly different from that in the wild-type strain.

[0093] Regarding Tanshinone IIA: In the wild-type strain, the content of Tanshinone IIA was 0.7754 mg / g. In the overexpression strains OE-2, OE-3, and OE-6, the contents 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 levels. In the silenced strains RNAi-1, RNAi-2, and RNAi-3, the contents were 1.373 mg / g, 1.450 mg / g, and 1.456 mg / g, respectively, representing 1.77-fold, 1.87-fold, and 1.88-fold of the wild-type levels, respectively. Tanshinone IIA content in the transgenic strains was significantly different from that in the wild-type strain.

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

[0095] 3.4 Determination of gene expression levels of key enzymes in the tanshinone biosynthesis pathway in hairy roots

[0096] In order to further study the molecular mechanism of SmPYL4 gene regulating the biosynthesis and accumulation of tanshinone components, the expression of key enzyme genes SmCPS1, SmDXR, SmHDS, SmGGPPS, SmHMGR, SmMEK and SmDXS in hairy roots was detected by qRT-PCR. The relative expression levels of each gene were as follows: Figure 5As shown in the figure, the expression levels of the SmDXS and SmMEK genes in the overexpression strains OE-2, OE-3, and OE-6 were significantly reduced compared with the wild-type strain. In strain OE-6, the expression level of the SmDXS gene was only 12.68% of that in the wild-type strain, and the expression level of the SmMEK gene was only 25.15% of that in the wild-type strain. In strains OE-2 and OE-3, the expression levels of the SmDXS gene were 16.12% and 38.95% of that in the wild-type strain, respectively, and the expression levels of the SmMEK gene were 37.91% and 37.18% of that in the wild-type strain, respectively. In addition, the expression levels of the SmCPS1 gene were also reduced in strains OE-2, OE-3, and OE-6, with average increases of 37.72%, 22.23%, and 15.71% of that in the wild-type strain, respectively. In the silenced strains, however, the expression levels of these key enzyme genes underwent the opposite changes. Notably, the expression levels of the SmDXR and SmHDS genes varied relatively little across all strains, with expression levels in the silenced strain RNAi-2 being only 1.879- and 1.6-fold higher than those in the wild-type strain.

[0097] 4. Conclusion

[0098] The SmPYL4 gene is a negative regulatory factor in the synthesis and accumulation of tanshinones. Overexpression of the SmPYL4 gene will reduce the expression levels of tanshinone components (tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone), while silencing the SmPYL4 gene will increase the expression levels of tanshinone components (tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone).

[0099] The above examples are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. Application of the Salvia miltiorrhiza SmPYL4 gene in regulating tanshinone content, the nucleotide sequence of the Salvia miltiorrhiza SmPYL4 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Application of silencing or inhibiting the expression of Salvia miltiorrhiza SmPYL4 gene in increasing the content of tanshinones.

3. The use according to claim 2, characterized in that: Silencing or inhibiting the expression of the SmPYL4 gene in Salvia miltiorrhiza increases the content of tanshinones in the hairy roots of Salvia miltiorrhiza.

4. The use according to claim 2 or 3, characterized in that: The specific method for silencing the SmPYL4 gene of Salvia miltiorrhiza is as follows: constructing a silencing expression vector pK7GWIWG2R(Ⅱ)-SmPYL4, infecting sterile leaves of Salvia miltiorrhiza by Agrobacterium-mediated method, and culturing to obtain Salvia miltiorrhiza plants with silenced expression of the SmPYL4 gene of Salvia miltiorrhiza. The content of tanshinone in the hairy roots of the plants is higher than that of the wild-type plants.

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

6. The use according to claim 5, characterized in that: The tanshinones are tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone.

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

1.

8. The method for increasing the tanshinone content in hairy roots of Salvia miltiorrhiza according to claim 7, characterized in that: The silencing expression vector pK7GWIWG2R(Ⅱ)-SmPYL4 was constructed and infected into sterile leaves of Salvia miltiorrhiza mediated by Agrobacterium. After culture, Salvia miltiorrhiza plants expressing the SmPYL4 gene were obtained. The content of tanshinone in the hairy roots of the plants was higher than that of the wild-type plants.

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

10. The method for increasing the tanshinone content in hairy roots of Salvia miltiorrhiza according to claim 9, characterized in that: The tanshinones are tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone.

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