Application of Salvia miltiorrhiza MAPK4 protein kinase and coding gene thereof in regulating content of tanshinone compounds in Salvia miltiorrhiza

Abstract

The invention discloses application of Salvia miltiorrhiza MAPK4 protein kinase and a coding gene thereof in regulating the content of tanshinone compounds in Salvia miltiorrhiza. It is found that the salvia miltiorrhiza MAPK4 protein kinase can improve the gene transcription level and the protein level of key synthetase on a tanshinone substance synthesis pathway, and then the content of tanshinone substances (such as tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I) in salvia miltiorrhiza is increased. Therefore, the Salvia miltiorrhiza MAPK4 protein kinase, the coding gene thereof and the expression vector containing the coding gene can be used for regulating and improving tanshinone substances in Salvia miltiorrhiza.

Description

Technical Field

[0001] The invention belongs to the field of biochemistry, relates to plant gene regulation of secondary metabolites, and specifically relates to the application of salvia miltiorrhiza MAPK4 protein kinase and its encoding gene in regulating the content of tanshinone compounds in salvia miltiorrhiza. Background Art

[0002] Salvia miltiorrhiza is a perennial herb of the genus Salvia in the family Labiatae. The active pharmaceutical ingredients contained in it are mainly found in the roots, so the roots are often used as medicine. As a traditional Chinese medicine, Salvia miltiorrhiza has the effects of promoting blood circulation and removing blood stasis, cooling blood and eliminating carbuncle, and reducing inflammation and nourishing blood. It is widely used to treat cardiovascular and cerebrovascular diseases with significant therapeutic effects. The fat-soluble active pharmaceutical ingredients in the roots of Salvia miltiorrhiza are mainly tanshinone substances, including dihydrotanshinone I, cryptotanshinone, tanshinone I, IIA, IIB, V, VI, etc. In the pharmacopoeia, the content of tanshinone IIA is used as an indicator of ketone components to judge the quality of Salvia miltiorrhiza. (References: Shan Xiaoxiao, Hong Bangzhen, Liu Jie, et al. Research progress on the chemical composition, pharmacological effects, clinical application of Salvia miltiorrhiza and predictive analysis of quality markers. China Journal of Traditional Chinese Medicine, Vol. 46, No. 21, 2021, pp. 5496-5511.)

[0003] The active ingredients of Danshen are mainly divided into fat-soluble tanshinones and water-soluble salvianolic acids. Modern pharmacological studies have shown that tanshinones are the main components of Danshen for anti-tumor, anti-fibrosis and clinical treatment of skin diseases. (Reference: Shan Xiaoxiao, Hong Bangzhen, Liu Jie, et al. Research progress on chemical composition, pharmacological effects, clinical application and prediction analysis of quality markers of Danshen. China Journal of Traditional Chinese Medicine, Vol. 46, No. 21, 2021, pp. 5496-5511.)

[0004] Therefore, studying the regulatory mechanism of the accumulation of secondary biomass tanshinone compounds in Danshen is of great significance to production and academic research, and is conducive to increasing the content of tanshinone compounds in Danshen. However, the existing technology is obviously insufficient. Summary of the invention

[0005] The present invention aims to overcome the deficiencies of the prior art and provides the application of Salvia miltiorrhiza MAPK4 protein kinase and its encoding gene in regulating the content of tanshinone compounds in Salvia miltiorrhiza, so as to increase the content of tanshinone compounds in Salvia miltiorrhiza.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] Application of Salvia miltiorrhiza MAPK4 protein kinase in increasing the content of tanshinone compounds in Salvia miltiorrhiza. The sequence of the Salvia miltiorrhiza MAPK4 protein kinase is shown in Sequence NO.1.

[0008] Furthermore, the tanshinone compound is tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

[0009] The application of the gene encoding the above-mentioned Salvia miltiorrhiza MAPK4 protein kinase in increasing the content of tanshinone compounds in Salvia miltiorrhiza.

[0010] Furthermore, the tanshinone compound is tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

[0011] An application of an expression vector containing the gene encoding the above-mentioned Salvia miltiorrhiza MAPK4 protein kinase in increasing the content of tanshinone compounds in Salvia miltiorrhiza.

[0012] Furthermore, the tanshinone compound is tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

[0013] Beneficial effects:

[0014] The present invention finds that the Danshen MAPK4 protein kinase can increase the gene transcription level and protein level of the key synthase in the synthesis pathway of tanshinones, thereby increasing the content of tanshinones (such as tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I) in Danshen. Therefore, the Danshen MAPK4 protein kinase, its encoding gene and the expression vector containing the encoding gene can be used to regulate and increase the tanshinones in Danshen. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The vector plasmid for overexpressing pHB-MAPK4-3Flag;

[0016] Figure 2 To interfere with the pK7GWIWG2DII-MAPK4 vector plasmid;

[0017] Figure 3 Agarose gel electrophoresis of total DNA in MAPK4-overexpressing hairy roots (MAPK4-OE);

[0018] Figure 4 is the protein level diagram in MAPK4-overexpressing hairy roots (MAPK4-OE);

[0019] Figure 5 MAPK4-overexpression hairy roots (MAPK4-OE) and MAPK4-RNAi interfered with the transcription level of MAPK4 gene in hairy roots;

[0020] Figure 6 This is the fluorescence detection diagram of MAPK4-RNAi interference in hairy roots;

[0021] Figure 7is the content of tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I;

[0022] Figure 8 is the transcription level of the synthase genes in the tanshinone biosynthesis pathway;

[0023] Fig. 9 It is the protein level of the synthases in the tanshinone biosynthesis pathway. DETAILED DESCRIPTION

[0024] The essential contents of the present invention are described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0025] 1. Experimental Materials

[0026] anti-Flag antibody (Abbkine, California, USA), plant total RNA extraction kit (Novozyme RC411, Nanjing, China), and standards Tanshinone IIA, Dihydrotanshinone I, Cryptotanshinone, and Tanshinone I (Chengdu AiFa, China).

[0027] 2. Experimental Methods

[0028] 1. Construction of MAPK4 plasmid vector

[0029] The sequence of Salvia miltiorrhiza MAPK4 protein kinase is shown in Sequence NO.1.

[0030] Sequence No.1:

[0031]

[0032] The MAPK4 overexpression vector was constructed by PCR technology, using specific primers MAPK4-OE (Table 1) to amplify the complete open reading frame sequence of MAPK4, and insert it into the pHB-3Flag plasmid to construct the pHB-MAPK4-3Flag overexpression vector plasmid ( Figure 1 The construction of MAPK4-RNAi vector is to first amplify the gene sequence of MAPK4 fragment by PCR technology, then insert it into pENTR plasmid, and finally combine this plasmid with vector pK7GWIWG2DII by gateway technology to obtain the interference pK7GWIWG2DII-MAPK4 vector plasmid ( Figure 2 ). The two vector plasmids were respectively transformed into C58C1 Agrobacterium, and the Agrobacterium containing pHB-MAPK4-3Flag vector plasmid and empty vector plasmid were cultured on 50 mg / L kanamycin and 50 mg / L rifampicin plate medium, and the Agrobacterium containing pK7GWIWG2DII-MAPK4 vector plasmid was cultured on 50 mg / L rifampicin and 50 mg / L spectinomycin plate medium.

[0033] Table 1 Primer list

[0034]

[0035]

[0036] 2. Genetic transformation mediated by Agrobacterium rhizogenes C58C1

[0037] 2.1 Storage and activation of Agrobacterium rhizogenes strains

[0038] Pick a single colony from the transformation plate and inoculate it into 1 mL of LB medium containing the corresponding antibiotics (the Agrobacterium culture medium of pHB-MAPK4-3Flag vector plasmid and the empty vector plasmid contains 50 mg / L kanamycin and 50 mg / L rifampicin, and the Agrobacterium culture medium of pK7GWIWG2DII-MAPK4 vector plasmid contains 50 mg / L rifampicin and 50 mg / L spectinomycin), shake culture at 200 rpm and 28°C overnight. Add 200 μL of the above bacterial solution to 10 mL of LB medium containing the corresponding antibiotics, shake culture at 200 rpm and 28°C overnight, and make the bacterial solution concentration OD 600 The value was between 0.4 and 0.8. Centrifuge at 4000 rpm for 10 min at room temperature, discard the supernatant, and dilute the cells with 1 / 2MS (with acetosyringone AS added, final concentration 100 μM) liquid culture medium to OD 600 value of 0.4, and the suspension culture was continued for 2 h for inoculation of infected explants.

[0039] 2.2 Explant pre-culture

[0040] Sterile seedling leaves (0.5 cm × 0.5 cm) were cut and inoculated onto pre-cultured solid medium 1 / 2MS (added with AS 100 μmol / L), and cultured at 25°C in the dark for 2 days.

[0041] 2.3 Co-cultivation of Agrobacterium and explants

[0042] Before infection, take out the pre-cultured leaves and put them into the activated Agrobacterium 1 / 2MS liquid suspension for 10-20 minutes, and gently shake them to make the explants fully contact with the bacterial liquid. Take out the infected explants, use sterile absorbent paper to dry the remaining bacteria on the surface, and transfer them to the co-culture medium (solid) 1 / 2MS (added with AS100μmol / L), and co-culture them in the dark for 2 days.

[0043] 2.4 Induction and cultivation of hairy roots

[0044] The explants co-cultured for 2 days were transferred to the sterilized medium 1 / 2MS+Cef250mg / L (solid) and cultured at 25℃ with 16h / 8h light. Hairy roots grew out at the cut edge of the leaves in about two weeks. Cut the hairy roots (about 2-3cm), inoculated in the sterilized medium 1 / 2MS+Cef250mg / L and cultured for two weeks. The hairy roots with fast growth and good branching were selected and transferred to the sterilized medium 1 / 2MS+Cef250mg / L for subculture screening. The subculture was performed every two weeks, and the hairy roots were completely sterilized after 4-5 subcultures. The well-growing hairy roots of Salvia miltiorrhiza were then transferred to the 1 / 2MS medium without antibiotics and continued to be cultured for about 20 days to obtain Salvia miltiorrhiza hairy roots with MAPK4 overexpression and RNA interference.

[0045] 2.5 Identification of positive hairy roots

[0046] 2.5.1 Extraction of total DNA from hairy roots of MAPK4-overexpressing Salvia miltiorrhiza

[0047] About 100 mg of total DNA from hairy roots of MAPK4-overexpressing Salvia miltiorrhiza was extracted by CTAB method (Stewart Jr, CN, Via, LE (1993) A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques, 14, 748-749.), and amplified with specific primers of rolB and rolC genes in Agrobacterium T-DNA. After agarose gel electrophoresis, the presence of corresponding bands indicated that the Agrobacterium T-DNA was integrated into the hairy root DNA.

[0048] 2.5.2 Western blot

[0049] Western blot was used to verify whether the MAPK4-overexpressing hairy roots were positive roots, and the primary antibody was Anti-Flag antibody.

[0050] 2.5.3 qRT-PCR method to identify positive roots

[0051] Total RNA from hairy roots was extracted using a plant total RNA extraction kit and then reverse transcribed to generate cDNA (Yin, X., Nishimura, M., Hajika, M. and Komatsu, S. (2016) Quantitative proteomics reveals the flooding-tolerance mechanism in mutant and abscisic acid-treated soybean. J. Proteome Res., 15, 2008-2025.). The transcription level of the target gene can be analyzed by qRT-PCR (Table 1) for identification (Pei, T., Ma, P., Ding, K., Liu, S., Jia, Y., Ru, M., Dong, J. and Liang, Z. (2018) SmJAZ8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairyroots.J.Exp.Bot.,69,1663-1678.), the PCR operating conditions were as follows: 95°C, 10 min; then 95°C, 10 s, 60°C, 30 s, 40 cycles, β-actin (DQ243702.1) gene was used as the internal reference gene (Pei, T., Ma, P., Ding, K., Liu, S., Jia, Y., Ru, M., Dong, J. and Liang, Z. (2018) SmJAZ8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots. J. Exp. Bot., 69, 1663-1678.).

[0052] 2.5.4 Identification of positive roots by green fluorescence

[0053] Under a fluorescence microscope, if the MAPK4-RNAi interference hairy roots emitted green fluorescence, it indicated that this group of roots was positive.

[0054] 3. qRT-PCR Analysis of Gene Expression

[0055] The transcription level of enzymes in the synthesis pathway of tanshinones in Danshen can confirm whether MAPK4 protein kinase plays a regulatory role in the synthesis of tanshinones. The method is the same as step 2.5.3, and the primers are shown in Table 1.

[0056] 4. Proteomic analysis of enzyme protein levels in the tanshinone biosynthesis pathway

[0057] Proteomic analysis of MAPK4-OE hair roots and blank control hair roots was performed. Proteins were extracted with phenol by ultrasonic treatment, and an equal volume of Tris-saturated phenol was added and centrifuged (5500×g, 10min, 4℃). The supernatant was precipitated with 0.1M ammonium acetate methanol solution, washed with pure methanol and pure acetone, and then redissolved with 8M urea. 20% trichloroacetic acid was gradually added, precipitated at 4℃ for 2h, centrifuged (4500×g, 5min), the supernatant was discarded, and the precipitate was washed with pre-cooled acetone three times. After drying, 200mM tetraethylammonium bromide was added and ultrasonically dispersed. Trypsin was added at a ratio of 1:50 and enzymatic hydrolysis was performed overnight. 5mM dithiothreitol was added, reduced at 56℃ for 30min, and then 11mM iodoacetamide was added and incubated at room temperature in the dark for 15min. Analyzed using mixed ion mobility-quadrupole time-of-flight mass spectrometry (tims-QTOF-MS). The sample was separated by a C18 capture column and a reversed phase C18 analytical column, and a 90 min gradient elution was established with mobile phase A (0.1% formic acid and 2% acetonitrile in water) and mobile phase B (0.1% formic acid in acetonitrile) at a flow rate of 450 nL min-1. The mass spectrometer was operated in parallel accumulation-serial fragmentation (PASEF) mode with a capillary voltage of 2000 V. MS and MS / MS spectra were collected from m / z 100 to 1700, and the ion mobility scan range was 0.6 to 1.6 V·s·cm-2. Each acquisition cycle included a full MS scan and 10 PASEF MS / MS scans, and the collision energy varied linearly according to the mobility. The raw data of data independent acquisition (DIA) were analyzed in library-free mode using DIA-NN software (V1.8.1). The spectrum files were searched in the local Danshen database (34,575 sequences) with default settings for the main parameters, including trypsin / P digestion, fixed modification of cysteine ​​carbamidomethylation, variable modification of methionine oxidation and protein N-terminal acetylation, etc. Protein intensity was normalized using the MaxLFQ algorithm, and quantitative analysis was based on the “PG.MaxLFQ” column in the DIA-NN output table.

[0058] 5. Determination of Tanshinone Content in Positive Transgenic Hairy Roots

[0059] The tanshinones in the transgenic hairy roots were extracted with methanol and then determined by HPLC (Peng, L., Ru, M., Wang, K., Li, B., Wang, Y., Xia, P. and Liang, Z. (2014). Spaceflight environment-induced variation in root yield and active constituents of Salvia miltiorrhiza. Planta Med., 80, 1029-1035.). Standard substances tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I were used to quantify the content of tanshinones.

[0060] 3. Experimental Results

[0061] 1. Identification of positive hairy roots

[0062] 1.1 Identification of hairy roots overexpressing MAPK4

[0063] There are three methods for identifying MAPK4-overexpressing hairy roots (MAPK4-OE). Method 1 is to extract total DNA of overexpressing hairy roots, and use primers specific to rolB and rolC genes in the root cancer transposon gene family (rol) in Agrobacterium T-DNA for PCR amplification. The PCR products are detected by agarose gel electrophoresis. Figure 3 As shown in Figure 2, the presence of corresponding rolB and rolC amplification positive bands indicates that the T-DNA of Agrobacterium has been integrated into the DNA of the hairy roots. Method 2 is to determine the MAPK4 protein level in the hairy roots overexpressing MAPK4, such as Figure 4 The results of western blot experiments using anti-flag antibodies showed that MAPK4-flag protein was overexpressed in hairy roots of Salvia miltiorrhiza. Method three was to detect the transcription level of MAPK4 overexpressed in hairy roots, such as Figure 5 The MAPK4 overexpression group is 2-3 times higher than the control group. The above results indicate that the MAPK4 overexpression root system is a positive root system.

[0064] 1.2 Identification of hairy roots induced by MAPK4-RNAi

[0065] There are two methods for identifying MAPK4-RNAi interference in hairy roots. Method 1 is to measure the transcription level of the gene MAPK4 that interferes with the root system, such as Figure 5 The interference group shown is 5 times lower than the control group. Method 2 is to observe whether the RNA interference root system emits green fluorescence under a fluorescence microscope. Figure 6It can be seen that the interfered roots emit green fluorescence under an inverted fluorescence microscope, which shows that the MAPK4-RNAi interfered hairy roots are positive roots.

[0066] 2. Determination of Tanshinone Content

[0067] The contents of representative substances in tanshinones, tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I, were determined (the results are shown in Table 2 and Figure 7 As shown), it was found that in the positive overexpression root system, the content of tanshinone I was about 10.5 times higher than that of the control group, and the content of tanshinone I in the interference group was about 3.2 times lower than that of the control group; the content of dihydrotanshinone I in the overexpression group was about 623.3 times higher than that of the control group, and the content of dihydrotanshinone I in the interference group was about 1.2 times lower than that of the control group; the content of tanshinone IIA in the overexpression group was more than 5.9 times that of the control group, and the content of tanshinone IIA in the interference group was 2 times lower than that of the control group; the content of cryptotanshinone in the overexpression group was 176.1 times higher than that of the control group, and the content of cryptotanshinone in the interference group did not change significantly with that of the control group. It can be concluded that overexpression of MAPK4 protein kinase can increase the content of tanshinone substances in Salvia miltiorrhiza.

[0068] Table 2 Content of representative tanshinone substances in hairy roots of MAPK4 overexpression / interference (mean±SD, n=3)

[0069]

[0070] 3. MAPK4 protein kinase regulates the expression of enzymes related to the synthesis of tanshinone substances

[0071] In order to explore whether MAPK4 protein kinase plays a regulatory role in the synthesis of tanshinones in Salvia miltiorrhiza, we studied the key enzymes in the tanshinone synthesis pathway. First, we detected the transcription level of the gene ( Figure 8 ), the transcription level of the overexpressed root synthase increased significantly, and the transcription level of the related synthase in the RNAi interference group decreased significantly. Secondly, we studied the protein level, such as Fig. 9 As shown in the figure, the level of synthase protein in the overexpressed roots increased significantly. It is speculated that MAPK4 protein kinase regulates the synthesis of tanshinones by regulating the transcription and expression of key enzyme genes in the tanshinone synthesis pathway.

[0072] Experiments have shown that MAPK4 affects the synthesis and yield of tanshinones by regulating the transcription and expression of key enzyme genes in the tanshinone synthesis pathway.

[0073] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. Application of Salvia miltiorrhiza MAPK4 protein kinase in increasing the content of tanshinone compounds in Salvia miltiorrhiza. The sequence of the Salvia miltiorrhiza MAPK4 protein kinase is shown in Sequence NO.

1.

2. The use according to claim 1, characterized in that: The tanshinone compound is tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

3. Use of the gene encoding the Salvia miltiorrhiza MAPK4 protein kinase according to claim 1 in increasing the content of tanshinone compounds in Salvia miltiorrhiza.

4. The use according to claim 3, characterized in that: The tanshinone compound is tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

5. Use of an expression vector containing the gene encoding the Salvia miltiorrhiza MAPK4 protein kinase according to claim 1 in increasing the content of tanshinone compounds in Salvia miltiorrhiza.

6. The use according to claim 5, characterized in that: The tanshinone compound is tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

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