Application of SmPYL8 gene in regulating tanshinone content in Salvia miltiorrhiza

Abstract

The application discloses application of a Salvia miltiorrhiza SmPYL8 gene in regulating tanshinone content, and a nucleotide sequence of the Salvia miltiorrhiza SmPYL8 gene is shown as SEQ ID NO. 1. Specifically, the expression of the Salvia miltiorrhiza SmPYL8 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. It is found by the application that the Salvia miltiorrhiza SmPYL8 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] The application relates to application of a Salvia miltiorrhiza Bunge. SmPYL8 gene in regulating tanshinone content and belongs to the technical field of genetic engineering. BACKGROUND

[0002] Salvia miltiorrhiza Bunge. is first recorded in Shennong Bencao Jing and belongs to a perennial plant of the Labiatae family, and the dried roots and rhizomes thereof are used as medicine and have the effects of activating blood and removing blood stasis, dredging channels and relieving pain, clearing heart and relieving heat, and cooling blood and resolving abscesses. Liposoluble tanshinone compounds (including tanshinone I, dihydrotanshinone I, tanshinone II A and cryptotanshinone) are main active ingredients in Salvia miltiorrhiza Bunge. and have multiple pharmacological activities such as anti-tumor, anti-inflammatory, anti-hypertensive and anti-fibrosis.

[0003] How to improve the content of tanshinone compounds in Salvia miltiorrhiza Bunge. and cultivate excellent varieties of Salvia miltiorrhiza Bunge. is an important research direction at present. With the continuous development of molecular biology and genetic engineering means, medicinal plant secondary metabolism engineering is becoming more and more perfect, and overexpression or silencing of key enzyme genes and transcription factors involved in the synthesis pathway of tanshinone compounds become effective means to improve the accumulation of tanshinone compounds.

[0004] PYR / PYL / RCAR proteins are functional abscisic acid (ABA) receptors in plants and belong to the START (Star-related lipid-transfer) superfamily of ligand-binding proteins and play an important role in ABA-mediated responses to various abiotic stresses. There are 14 members of the ABA receptor PYR / PYL / RCAR protein family in the model plant Arabidopsis thaliana, namely PYL7 / 8 / 9 / 10 of the first subfamily, PYL4 / 5 / 6 / 11 / 12 / 13 of the second subfamily, PYR1 and PYL1 / 2 / 3 of the third subfamily. It has been shown that overexpression of VlPYL1 in grape can promote the accumulation of anthocyanins in fruits, overexpression of NtPYL4 gene in tobacco can reduce the content of alkaloids in tobacco roots, and similarly, overexpression of NtPYL4 gene in Arabidopsis thaliana can also reduce the accumulation of alkaloids. At present, there is no research report on PYL family genes in regulating the development of Salvia miltiorrhiza Bunge. roots and the synthesis and accumulation of tanshinone secondary metabolites.

[0005] In the previous research, the inventors of the present application screened a Salvia miltiorrhiza Bunge. ABA receptor protein SmPYL8 based on the Salvia miltiorrhiza Bunge. genome and transcriptome, and the expression amount of SmPYL8 in stems is the highest, SmPYL8 strongly responds to exogenous PEG treatment with weak induction of ABA, and SmPYL8 can positively respond to drought stress. SUMMARY

[0006] In view of the prior art, the application provides a new use of a Salvia miltiorrhiza SmPYL8 gene, i.e., application in regulating tanshinone content.

[0007] The application is realized by the following technical scheme:

[0008] The application of the Salvia miltiorrhiza SmPYL8 gene in regulating tanshinone content, wherein the nucleotide sequence of the Salvia miltiorrhiza SmPYL8 gene is shown in SEQ ID NO. 1.

[0009] Further, the application of silencing or inhibiting expression of the Salvia miltiorrhiza SmPYL8 gene in increasing tanshinone content.

[0010] Further, the specific method for silencing the Salvia miltiorrhiza SmPYL8 gene is as follows: a silencing expression vector pK7GWIWG2R(II)-SmPYL8 is constructed, Agrobacterium-mediated infection is used to infect sterile leaves of Salvia miltiorrhiza, and cultivation is performed to obtain Salvia miltiorrhiza plants with silenced expression of the Salvia miltiorrhiza SmPYL8 gene, and the content of tanshinone in the hairy roots of the plants is higher than that in wild-type plants.

[0011] Further, the tanshinone is any one or two or more of tanshinone I, dihydrotanshinone I, tanshinone II A and cryptotanshinone.

[0012] A method for increasing tanshinone content in hairy roots of Salvia miltiorrhiza is as follows: silencing or inhibiting the Salvia miltiorrhiza SmPYL8 gene in Salvia miltiorrhiza plants.

[0013] Further, the specific method is as follows: a silencing expression vector pK7GWIWG2R(II)-SmPYL8 is constructed, Agrobacterium-mediated infection is used to infect sterile leaves of Salvia miltiorrhiza, and cultivation is performed to obtain Salvia miltiorrhiza plants with silenced expression of the Salvia miltiorrhiza SmPYL8 gene, and the content of tanshinone in the hairy roots of the plants is higher than that in wild-type plants.

[0014] Further, the tanshinone is any one or two or more of tanshinone I, dihydrotanshinone I, tanshinone II A and cryptotanshinone.

[0015] The application constructs a silencing expression vector and an overexpression vector of the Salvia miltiorrhiza SmPYL8 gene, uses Agrobacterium-mediated infection to infect sterile leaves of Salvia miltiorrhiza, and obtains a Salvia miltiorrhiza SmPYL8 gene silenced strain and an overexpression strain; the content of tanshinone in the hairy roots is detected, and the expression level of a key enzyme gene in a synthesis path is detected, and the results show that the Salvia miltiorrhiza SmPYL8 gene has a negative regulation on synthesis and accumulation of tanshinone components, and silencing the gene can effectively increase the content of tanshinone components. BRIEF DESCRIPTION OF DRAWINGS

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

[0017] Figure 2 Relative expression amount of Salvia miltiorrhiza SmPYL8 gene in hairy roots, wherein, * represents P<0.05, and ** represents P<0.01.

[0018] Figure 3 Photo of the sample solution to be tested of hairy roots of each strain.

[0019] Figure 4 Determination result of tanshinone content in hairy roots, wherein, * represents P<0.05, and ** represents P<0.01.

[0020] Figure 5 Relative expression amount of key enzyme gene of tanshinone synthesis path in hairy roots, wherein, * represents P<0.05, and ** represents P<0.01. DETAILED DESCRIPTION

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

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

[0023] Experiment 1: Study on the influence of expression of Salvia miltiorrhiza SmPYL8 gene on the properties of Salvia miltiorrhiza

[0024] The application constructs a silencing expression system and an overexpression system of Salvia miltiorrhiza SmPYL8 gene respectively, and studies the influence of the Salvia miltiorrhiza SmPYL8 gene on the root development of Salvia miltiorrhiza and the synthesis and accumulation of tanshinone secondary metabolites.

[0025] The nucleotide sequence of the Salvia miltiorrhiza SmPYL8 gene is shown in SEQ ID NO. 1, and is as follows (direction 5'-3'):

[0026] ATGATGAACATCAAAGGATTGAGCGCGGTGGAGAGTGAGTATACACTGAAGCATCACAGGCACCAGATTAAGGATAATCAATGCAGTTCGTTCCTCATCAAGCATATTAAAGCGCCCGTACATCTTGTTTGGTCTCTGGTCAGGAGGTTTGATCAACCACAGAAGTACAAACCTTTTGTTAGCCGTTGCATTGTGCAGGGAAATCTTGAAATTGGCAGTCTGAGGGAAGTTGATGTCAAGTCGGGTCTCCCTGCTACAACCAGCACCGAGAGATTGGAGCTTCTTGATGATAATGAACACATACTTAGCGTAAGGATCGTTGGTGGAGACCACAGACTCAGAAACTACTCTTCCATGGTGTCTGTTCATCCTGAGGTCATTGACGGGAGACCTGGGACAGTGGTGATTGAATCGTTTGTGGTGGATGTGCCAGAAGGGAACACAAAAGACGAAACATGCTACTTTGTCGAAGCACTGATCAGGTGTAATCTGAAATCACTGGCTGATGTTTCGGAGAGGCTGGCTGTGCAAGACAGGACTGAACCCATCAACCGTGCGTAA.

[0027] The amino acid sequence of the protein expressed by the Salvia miltiorrhiza SmPYL8 gene is shown as SEQ ID NO. 2, as follows:

[0028] MMNIKGLSAVESEYTLKHHRHQIKDNQCSSFLIKHIKAPVHLVWSLVRRFDQPQKYKPFVSRCIVQGNLEIGSLREV DVKSGLPATTSTERLELLDDNEHILSVRIVGGDHRLRNYSSMVSVHPEVIDGRPGTVVIESFVVDVPEGNTKDETCYFV EALIRCNLKSLADVSERLAVQDRTEPINRA.

[0029] 1. Experimental materials

[0030] 1.1 Plant material

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

[0032] 1.2 Vectors and strains

[0033] pMDC202 vector, pDONR221 vector, pK7GWIWG2R(II) vector, all are common plant expression vectors, purchased from commercial and self-stored in laboratory.

[0034] E. coli DH5α competent cells, Agrobacterium rhizogenes Ar. Qual competent cells, both purchased from commercial and self-stored in laboratory.

[0035] 2. Research methods

[0036] 2.1 Construction of Salvia miltiorrhiza SmPYL8 gene overexpression vector

[0037] (1) According to the ORF sequence of SmPYL8 gene, the upstream and downstream primers containing XbaI & KpnI restriction sites were designed by Primer Premier 5.0, and the primer sequences are shown in Table 1.

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

[0039]

[0040] (2) The target gene containing homologous arms and enzyme cutting sites was amplified by PCR, and the product was purified and recovered; the pMDC202 linearized vector was obtained by enzyme digestion and purification; according to the homologous recombination kit, the target gene and the vector were connected, then transformed into DH5α competent cells, and single colonies were picked and expanded. After PCR identification, it was sent to Platinum Biotechnology Co., Ltd. for sequencing. The positive plasmid pMDC202-SmPYL8 with correct sequencing was used for subsequent experiments.

[0041] 2.2 Construction of Salvia miltiorrhiza SmPYL8 gene silencing vector

[0042] (1) According to the principle of RNAi vector construction and Gateway principle, the primer containing attB adapter was designed, and the primer sequence is shown in Table 1.

[0043] (2) The specific primers containing attB adapter were amplified by PCR, and the target gene fragment was purified and recovered.

[0044] (3) According to the Gateway BP Clonase II Enzyme mix kit instructions, the target fragment is connected with the intermediate vector, and the reaction system is as follows: target fragment, 1 μL; pDONR221 plasmid, 1 μL; BP enzyme, 1 μL; ddH2O, 3 μL. The reaction solution is reacted in a PCR instrument at 25°C for 5h, after the reaction is completed, 1 μL of Proteinase K is added to the centrifuge tube, and incubated at 37°C for 10 min in a PCR instrument to terminate the BP reaction.

[0045] (4) The ligation product is transformed into E. coli DH5α competent cells; single colonies are picked and expanded, and positive clones are identified by PCR, and then sent to Plasmodium biotechnology Co., Ltd. for sequencing; the positive bacterial liquid of correct sequencing is expanded and cultured, and the pDONR221-SmPYL8 positive plasmid is extracted;

[0046] (5) According to the Gateway LR Clonase II Enzyme mix kit instructions, the pDONR221-SmPYL8 positive plasmid is connected with the pK7GWIWG2R(II) vector to construct the plant silencing vector pK7GWIWG2R(II)-SmPYL8, and the reaction system is as follows: pDONR221-SmPYL8 positive plasmid, 1 μL; pK7GWIWG2R(II) plasmid, 1 μL; LR enzyme, 1 μL; ddH2O, 2 μL. The reaction solution is reacted in a PCR instrument at 25°C for 5h, after the reaction is completed, 1 μL of Proteinase K is added to the centrifuge tube, and incubated at 37°C for 10 min in a PCR instrument to terminate the LR reaction.

[0047] (6) The ligation product is transformed into E. coli competent cells; the positive bacterial liquid of correct sequencing is expanded and cultured, and the pK7GWIWG2R(II)-SmPYL8 positive plasmid is extracted for subsequent experiments.

[0048] 2.3 Construction of Salvia miltiorrhiza SmPYL8 genetic transformation system

[0049] (1) The recombinant plasmids pMDC202-SmPYL8 and pK7GWIWG2R(II)-SmPYL8 are transformed into Agrobacterium tumefaciens Ar.Qual competent cells, and Ar.Qual competent cells without any plasmid are used as a control, and the specific operation is as follows:

[0050] a. Take the Ar.Qual competent cells, and insert them into ice when they are in an ice water mixed state;

[0051] b. Add 0.05 μg of recombinant plasmid to 100 μL of competent cells, mix quickly, and then place them on ice, liquid nitrogen, 37°C water bath, and ice for 5 min, respectively;

[0052] c. Add to 700 μL of LB liquid medium without antibiotics, and shake at 28°C for 3 h;

[0053] d. Centrifuge at 6000 rpm for 1 min, collect the bacterial precipitate, take 100 μL of supernatant, mix and then spread on YEB solid medium containing 100 mg / L Spe antibiotic (silent plasmid) and 50 mg / L Kan (overexpression plasmid), and invert culture at 28°C for 3 d;

[0054] e. Pick single colonies, inoculate in 5 mL of YEB liquid medium containing corresponding antibiotics, and culture at 28°C, 200 rpm overnight. The bacterial liquid identified as positive by colony PCR is stored at 4°C for standby.

[0055] (2) Take 750 μL of the above-mentioned bacterial liquid identified as positive, inoculate in 75 mL of YEB liquid medium containing corresponding antibiotics, activate to OD 600 = 0.6-1.0, centrifuge at 5000 rpm for 10 min, collect the bacterial precipitate, resuspend the bacteria with MS liquid medium, then add 400 μmol / L of acetyl-syringone, and shake at 28°C, 200 rpm for 20 min.

[0056] (3) Select the leaves of Salvia miltiorrhiza aseptic seedlings after 20-30 days of subculture, cut into 0.5 cm 2 size, and pre-culture on MS (pH = 5.8) medium for 2 d; then completely immerse in the bacterial liquid for 10-15 min, absorb the surface bacterial liquid with sterile filter paper, and then lay on MS solid medium for co-culture for 3 d.

[0057] (4) Wash 5 times with sterile water, absorb the excess water with sterile filter paper, and lay on MS solid medium containing Cef at a final concentration of 500 mg / L for sterilization culture. Replace the medium every 7 d, and the concentration of Cef in the medium is reduced from 500 mg / L to 400, 300, 200, 100, and 50 mg / L, respectively, 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 d, and then transfer to 50 mL of MS liquid medium for expansion culture at 25°C, 120 rpm.

[0059] 2.4 Molecular identification of transgenic hairy roots

[0060] (1) Extract the genomic DNA of transgenic hairy roots, and the specific operation is as follows:

[0061] a. Take 100 mg of hairy roots cultured in liquid medium, add liquid nitrogen to grind thoroughly, immediately add 400 μL Buffer Al and 4 μL Rnase A to the ground sample, shake the centrifuge tube vigorously to make thorough lysis;

[0062] b. 65°C water bath for 10 min, during which time invert 2-3 times;

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

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

[0065] e. Add 600 μL Buffer AW (to which anhydrous ethanol has been added), centrifuge at 12000 rpm for 30 s, discard the filtrate; repeat this operation;

[0066] f. Place the adsorption column back in the collection tube, centrifuge at 12000 rpm for 2 min, try to remove the rinse solution as much as possible;

[0067] g. Place the adsorption column in a new 1.5 mL centrifuge tube, add 50 μL Elution Buffer preheated to 65-75°C to the center of the membrane, place at room temperature for 3-5 min, centrifuge at 12000 rpm for 1 min, detect the concentration and quality of the DNA by microspectrophotometry, and store the obtained DNA in a refrigerator at -20°C.

[0068] (2) Design identification primers, the specific operation being as follows: design identification primers for overexpression-type hairy root strains using pMDC202-F+SmPYL8-R, the hpt gene on the pMDC202 vector, and the rolB gene on the Agrobacterium tumefaciens Ri plasmid as templates; design identification primers for silencing-type hairy root strains using p35-F+SmPYL8-R according to the 35S promoter in the pK7GWIWG2R(II) vector, and the primer sequences being shown in Table 1.

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

[0070] 2.5 Determination of the expression amount of a key enzyme gene in the tanshinone synthesis pathway

[0071] The transgenic hairy root strains identified as positive were subjected to RNA extraction and reverse transcription, and the expression levels of SmPYL8 gene and related synthetic enzyme genes in tanshinone metabolic pathway were detected by qRT-PCR, respectively. The primer sequences of the related synthetic enzyme genes in tanshinone metabolic pathway are shown in Table 2.

[0072] Table 2 Primer sequences of related synthetic enzyme genes in tanshinone metabolic pathway

[0073]

[0074] 2.6 Determination of tanshinone content

[0075] (1) Precisely weigh tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone four kinds of reference substances, and place them in four 2 mL brown volumetric flasks. Use 100% chromatographic methanol to make the concentration of each reference substance solution 1 mg / mL. Meanwhile, based on the four components, prepare a mixed standard solution with a concentration of 1 mg / mL. Dilute and 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 curve of the four tanshinone components with the concentration (X) and peak area (Y) of the standard solution.

[0076] (2) Grind the hairy root samples of wild type (WT), overexpression type (OE) and silencing type (RNAi) with consistent growth cycle and frozen for 48 h, and pass through a 40 mesh sieve. Precisely weigh 0.25 g of the sample in a 50 mL centrifuge tube, add 25 mL of chromatographic methanol, vortex for 3 min, and ultrasonically extract for 50 min under the conditions of 100 W, 100 Hz and 50°C. Centrifuge at 4000 rpm for 10 min, and then aspirate the supernatant, pass it through a 0.22 μm organic filter, and obtain the sample solution to be tested.

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

[0078] Table 3 Gradient elution table of mobile phase

[0079]

[0080] 2.7 Data processing and analysis

[0081] The data were processed and analyzed by using SPSS22.0 and other software, and the graphs were drawn by using GraphPad Prism 8.0 software.

[0082] 3. Results and analysis

[0083] 3.1 Obtaining and identification of hairy roots

[0084] The over-expression type (OE), the silencing type (RNAi) and the wild type (WT) hairy roots were obtained by infecting Salvia miltiorrhiza leaves with Agrobacterium rhizogenes Ar. Qual containing pMDC202-SmPYL8, pK7GWIWG2R(II)-SmPYL8 recombinant plasmid and no plasmid respectively. The positive strains were screened by PCR amplification of genomic DNA, and 8 wild type hairy roots (strain name WT, 10 strains were detected), 6 over-expression type hairy roots (strain name SmPYL8OE, 8 strains were detected) and 6 silencing type hairy roots (strain name SmPYL8RNAi, 8 strains were detected) were obtained. The positive rate of hairy roots was 80%, 75% and 75% respectively. The electrophoresis detection results of the genomic DNA of the hairy roots are shown in Figure 1 It can be seen that a single band is detected in the positive transgenic hairy roots, and the band size is correct, indicating that the T-DNA region of the recombinant plasmid is successfully inserted into the genome of Salvia miltiorrhiza.

[0085] 3.2 Determination of SmPYL8 gene expression amount in hairy roots

[0086] The expression amount of SmPYL8 gene in the hairy roots was detected by qRT-PCR, and the most significant 3 strains were selected for display. The relative expression amount of Salvia miltiorrhiza SmPYL8 gene in the hairy roots is shown in Figure 2 The gene expression amount in all over-expression type strains is significantly higher than that in the wild type strains, and the gene expression amount in the silencing type strains is significantly lower than that in the wild type strains. Among them, the expression amount of SmPYL8 in the highest strain OE-2 is 19.67 times that of the wild type strain, and the expression amount of SmPYL8 in the lowest strain RNAi-2 is reduced to 18.87% of the wild type strain. These results further indicate that these strains obtained are positive strains, which can be used for further research.

[0087] 3.3 Determination of tanshinone content in hairy roots

[0088] In order to study the influence of SmPYL8 gene on the biosynthesis and accumulation of tanshinone components, the positive transgenic hairy root strains were expanded, and then the hairy roots were ground and extracted to obtain the sample solution to be detected. The photos of the sample solution to be detected of the hairy roots of each strain are shown in Figure 3 It can be seen that compared with the wild type strain, the color of the sample solution to be detected corresponding to the silencing type strain is redder, and the red color of the sample solution to be detected corresponding to the over-expression type strain is lighter. It is preliminarily speculated that the tanshinone content in the silencing type strain of SmPYL8 gene may be higher, and the tanshinone content in the over-expression type strain may be less.

[0089] To further verify this presumption, the content of tanshinone components in hairy roots was determined by HPLC, and the results are shown in Table 1. Figure 4

[0090] For dihydrotanshinone I: in the wild type strain, the content of dihydrotanshinone I was 0.5373 mg / g. In the overexpression type strains OE-1, OE-2 and OE-4, the content of dihydrotanshinone I was 0.1830 mg / g, 0.4469 mg / g and 0.3075 mg / g, respectively, which was 34.05%, 83.17% and 57.23% of the wild type strain, respectively. In the silencing type strains RNAi-2, RNAi-4 and RNAi-6, the content of dihydrotanshinone I was 0.7538 mg / g, 0.6692 mg / g and 0.9673 mg / g, respectively, which was 1.4 times, 1.24 times and 1.8 times of the wild type strain, respectively. The content of dihydrotanshinone I in the transgenic strains was significantly different from that in the wild type strain.

[0091] For cryptotanshinone: in the wild type strain, the content of cryptotanshinone was 1.059 mg / g. In the overexpression type strains OE-1, OE-2 and OE-4, the content of cryptotanshinone was 0.4159 mg / g, 0.4837 mg / g and 0.5897 mg / g, respectively, which was 39.27%, 45.67% and 55.68% of the wild type strain, respectively. In the silencing type strains RNAi-2, RNAi-4 and RNAi-6, the content of cryptotanshinone was 1.6921 mg / g, 3.388 mg / g and 4.433 mg / g, respectively, which was 1.59 times, 3.19 times and 4.18 times of the wild type strain, respectively. The content of cryptotanshinone in the transgenic strains was significantly different from that in the wild type strain.

[0092] For tanshinone I: in the wild type strain, the content of tanshinone I was 0.8924 mg / g. In the overexpression type strains OE-1, OE-2 and OE-4, the content of tanshinone I was 0.3844 mg / g, 0.4619 mg / g and 0.5948 mg / g, respectively, which was 43.07%, 51.75% and 66.65% of the wild type strain, respectively. In the silencing type strains RNAi-2, RNAi-4 and RNAi-6, the content of tanshinone I was 1.452 mg / g, 1.278 mg / g and 1.178 mg / g, respectively, which was 1.62 times, 1.43 times and 1.32 times of the wild type strain, respectively. The content of tanshinone I in the transgenic strains was significantly different from that in the wild type strain.

[0093] ​For tanshinone ⅡA: in wild type strain, the content of tanshinone ⅡA was 0.7754 mg / g. In overexpression strains OE-1, OE-2 and OE-4, the content of tanshinone ⅡA was 0.2193 mg / g, 0.3978 mg / g and 0.5632 mg / g, respectively, which was 28.28%, 51.30% and 72.63% of the wild type strain, respectively. In silencing strains RNAi-2, RNAi-4 and RNAi-6, the content of tanshinone ⅡA was 2.731 mg / g, 1.915 mg / g and 1.412 mg / g, respectively, which was 3.52 times, 2.46 times and 1.82 times of the wild type strain, respectively. The content of tanshinone ⅡA in transgenic strains had significant difference compared with that in the wild type strain.

[0094] In summary, SmPYL8 gene has a negative regulatory effect on the synthesis and accumulation of tanshinone components, especially can significantly increase the content of cryptotanshinone and tanshinone ⅡA.

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

[0096] In order to further study the molecular mechanism of SmPYL8 gene regulating the biosynthesis and accumulation of tanshinone components, qRT-PCR was used to detect the expression of key enzyme genes SmCPS1, SmDXR, SmHDS, SmGGPPS, SmHMGR, SmMEK and SmDXS in tanshinone synthesis pathway in hairy roots, and the relative expression amount of each gene was as shown in Table 2. Figure 5 Compared with the wild type strain, the expression level of key enzyme genes in tanshinone synthesis pathway in the silencing strain was obviously increased, and the expression level of key enzyme genes in tanshinone synthesis pathway in the overexpression strain was obviously decreased. The expression amount of SmCPS1 gene in strain RNAi-4 was the most significantly increased, which was 2.856 times of the wild type strain, the expression amount of SmDXR gene was increased by 2.435 times, the expression amount of SmGGPPS gene was increased by 2.424 times, and the expression amount of SmHDS gene was increased by 2.655 times. The expression amount of SmHDS gene in strain OE-1 was the most significantly decreased, which was 36.58% of the wild type strain, and the expression amount of SmCPS1 gene was 40.32% of the wild type strain. The expression amount of SmHMGR gene in all strains had a smaller change range, and the expression amount in silencing strain RNAi-6 was 1.427 times of the wild type strain.

[0097] 4. CONCLUSION

[0098] The SmPYL8 gene is a negative regulatory factor in the synthesis and accumulation of tanshinones. Overexpression of the SmPYL8 gene reduces the expression of tanshinone components (tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone), and silencing of the SmPYL8 gene increases the expression of tanshinone components (tanshinone I, dihydrotanshinone I, tanshinone IIA and cryptotanshinone).

[0099] The foregoing examples are provided to give the person skilled in the art a complete disclosure and description of how the claimed embodiments are implemented and used, and are not intended to limit the scope of the disclosure disclosed herein. Modifications apparent to those skilled in the art will be within the scope of the appended claims.

Claims

1. Application of Salvia miltiorrhiza SmPYL8 gene in regulating tanshinone content, characterized in that: Silencing or inhibiting the expression of the SmPYL8 gene of Salvia miltiorrhiza, so that the content of tanshinone in the hairy roots of Salvia miltiorrhiza is increased; the nucleotide sequence of the SmPYL8 gene of Salvia miltiorrhiza is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The specific way of silencing the SmPYL8 gene of Salvia miltiorrhiza is as follows: a silencing expression vector pK7GWIWG2R(II)-SmPYL8 is constructed, Agrobacterium-mediated method is used to infect sterile leaves of Salvia miltiorrhiza, and culture is carried out, so that a Salvia miltiorrhiza plant with the SmPYL8 gene of Salvia miltiorrhiza being silenced is obtained, and the content of tanshinone in the hairy roots of the plant is higher than that of a wild type plant.

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

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

5. A method for increasing the content of tanshinone in the hairy roots of Salvia miltiorrhiza, characterized in that: The SmPYL8 gene of Salvia miltiorrhiza is silenced or inhibited in the plant of Salvia miltiorrhiza; the nucleotide sequence of the SmPYL8 gene of Salvia miltiorrhiza is shown as SEQ ID NO.

1.

6. The method for increasing the content of tanshinone in the hairy roots of Salvia miltiorrhiza according to claim 5, characterized in that: The specific way of silencing the SmPYL8 gene of Salvia miltiorrhiza is as follows: a silencing expression vector pK7GWIWG2R(II)-SmPYL8 is constructed, Agrobacterium-mediated method is used to infect sterile leaves of Salvia miltiorrhiza, and culture is carried out, so that a Salvia miltiorrhiza plant with the SmPYL8 gene of Salvia miltiorrhiza being silenced is obtained, and the content of tanshinone in the hairy roots of the plant is higher than that of a wild type plant.

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

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

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