Application of salvia miltiorrhiza mapk4 protein kinase and its coding gene in regulating content of tanshinone compounds in salvia miltiorrhiza

Abstract

The application discloses a tanshinone MAPK4 protein kinase and application of a coding gene thereof in regulating tanshinone content in Salvia miltiorrhiza. The application finds that the tanshinone MAPK4 protein kinase can improve gene transcription level and protein level of a key synthesis enzyme on a tanshinone synthesis path, and then improves tanshinone content (such as tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I) in Salvia miltiorrhiza. Therefore, the tanshinone MAPK4 protein kinase, the coding gene and an expression vector containing the coding gene can be used for regulating and improving tanshinone in Salvia miltiorrhiza.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biochemistry, and relates to plant gene regulation of secondary metabolites, in particular to application of MAPK4 protein kinase and a coding gene thereof in regulating content of tanshinone compounds in Salvia miltiorrhiza. BACKGROUND

[0002] Salvia miltiorrhiza is a perennial herb of the Labiatae family (Labiatae) Salvia. The medicinal active ingredients contained therein mainly exist 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 to remove blood stasis, cooling blood to resolve abscess, and anti-inflammatory and blood nourishing, and is widely used for treating cardiovascular and cerebrovascular diseases with significant curative effect. The liposoluble medicinal active ingredients in the roots of Salvia miltiorrhiza are mainly tanshinone compounds, including dihydrotanshinone I, cryptotanshinone, tanshinone I, IIA, IIB, V, VI, etc. In the Pharmacopoeia, the content of tanshinone IIA is used as an index of ketone compounds to judge the quality of Salvia miltiorrhiza medicinal materials. (Reference: Single Xiao, Hong Bangzhen, Liu Jie, et al. Research Progress and Prediction Analysis of Quality Markers of Salvia miltiorrhiza Chemical Components, Pharmacological Effects, and Clinical Applications. Chinese Journal of Chinese Medicine 2021, Vol. 46, No. 21, pp. 5496-5511.)

[0003] The medicinal active ingredients of Salvia miltiorrhiza mainly include liposoluble tanshinone compounds and water-soluble salvianolic acid compounds. Modern pharmacological studies have shown that tanshinone compounds are the main components of Salvia miltiorrhiza for exerting anti-tumor, anti-fibrosis, and clinical treatment of skin diseases. (Reference: Single Xiao, Hong Bangzhen, Liu Jie, et al. Research Progress and Prediction Analysis of Quality Markers of Salvia miltiorrhiza Chemical Components, Pharmacological Effects, and Clinical Applications. Chinese Journal of Chinese Medicine 2021, Vol. 46, No. 21, pp. 5496-5511.)

[0004] Therefore, it is of great significance to study the regulation mechanism of secondary substances tanshinone compounds in Salvia miltiorrhiza for production and academic research, which is conducive to improving the content of tanshinone compounds in Salvia miltiorrhiza. However, the existing technology is obviously insufficient. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art, and provides application of MAPK4 protein kinase and a coding gene thereof in regulating content of tanshinone compounds in Salvia miltiorrhiza, so as to improve the content of tanshinone compounds in Salvia miltiorrhiza.

[0006] The above-mentioned object of the present application is achieved by the following technical solutions:

[0007] The application of MAPK4 protein kinase in improving the content of tanshinone compounds in Salvia miltiorrhiza, wherein the sequence of the MAPK4 protein kinase is shown in Sequence NO. 1.

[0008] Further, the tanshinone compounds are tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

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

[0010] Further, the tanshinone compounds are tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

[0011] The application of an expression vector containing the coding gene of the above-mentioned Salvia MAPK4 protein kinase in improving the content of tanshinone compounds in Salvia.

[0012] Further, the tanshinone compounds are tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

[0013] Beneficial effects:

[0014] The present application finds that the Salvia MAPK4 protein kinase can improve the gene transcription level and protein level of key synthesis enzymes in the synthesis pathway of tanshinone compounds, and further improve the content of tanshinone compounds (such as tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I) in Salvia. Therefore, the Salvia MAPK4 protein kinase, its coding gene and the expression vector containing the coding gene can be used for regulating and improving the content of tanshinone compounds in Salvia. BRIEF DESCRIPTION OF DRAWINGS

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

[0016] Figure 2 pK7GWIWG2DII-MAPK4 vector plasmid for interference;

[0017] Figure 3 Total DNA agarose gel electrophoresis map in MAPK4 overexpressed hairy roots (MAPK4-OE);

[0018] Figure 4 Protein level map in MAPK4 overexpressed hairy roots (MAPK4-OE);

[0019] Figure 5 Transcription level of MAPK4 gene in MAPK4 overexpressed hairy roots (MAPK4-OE) and MAPK4-RNAi interference hairy roots;

[0020] Figure 6 Fluorescence detection map of MAPK4-RNAi interference hairy roots;

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

[0022] Figure 8 The transcription level of the synthetic enzyme gene of the tanshinone biosynthetic pathway;

[0023] Figure 9 The protein level of the synthetic enzyme of the tanshinone biosynthetic pathway. DETAILED DESCRIPTION

[0024] The substantial content of the present application will be specifically introduced below in combination with the drawings and examples, but the protection scope of the present application is not limited by this.

[0025] I. Experimental materials

[0026] anti-Flag antibody (Abbkine, California, USA), plant total RNA extraction kit (Norgen Biotek, Nanjing, China), standard tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I (Chengdu Efa, China).

[0027] II. Experimental methods

[0028] 1. Construction of MAPK4 plasmid vector

[0029] The sequence of the 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 then inserted into the pHB-3Flag plasmid to construct the overexpression pHB-MAPK4-3Flag vector plasmid. Figure 1 The MAPK4-RNAi vector was constructed by first amplifying the gene sequence of the MAPK4 fragment by PCR technology, then inserting it into the pENTR plasmid, and finally using gateway technology to combine this plasmid with the vector pK7GWIWG2DII to obtain the interfering pK7GWIWG2DII-MAPK4 vector plasmid. Figure 2 The two kinds of vector plasmids were respectively transformed into C58C1 Agrobacterium, and the Agrobacterium containing the pHB-MAPK4-3Flag vector plasmid and the empty vector plasmid was cultured on 50mg / L kanamycin and 50mg / L rifampicin plate medium, and the Agrobacterium containing the pK7GWIWG2DII-MAPK4 vector plasmid was cultured on 50mg / L rifampicin and 50mg / L spectinomycin plate medium.

[0033] Table 1 Primer list

[0034]

[0035]

[0036] 2. Agrobacterium rhizogenes C58C1-mediated genetic transformation of Salvia miltiorrhiza

[0037] 2.1 Preservation and activation of Agrobacterium rhizogenes strain

[0038] Single colonies were picked from the transformation plate and inoculated into 1mL LB medium containing the corresponding antibiotics (the Agrobacterium culture medium containing the pHB-MAPK4-3Flag vector plasmid and the empty vector plasmid contained 50mg / L kanamycin and 50mg / L rifampicin, and the Agrobacterium culture medium containing the pK7GWIWG2DII-MAPK4 vector plasmid contained 50mg / L rifampicin and 50mg / L spectinomycin), 200rpm, 28℃ shaking culture overnight. Add 200μL of the above bacterial solution to 10mL of LB medium containing the corresponding antibiotics, 200rpm, 28℃ shaking culture overnight, so that the bacterial solution concentration OD 600 4000rpm, 10min at room temperature, discard the supernatant, and dilute the bacterial body with 1 / 2MS (add acetyl-syringone AS, final concentration 100μM) liquid medium to OD 600 4000rpm, 10min at room temperature, discard the supernatant, and dilute the bacterial body with 1 / 2MS (add acetyl-syringone AS, final concentration 100μM) liquid medium to OD

[0039] 2.2 Pre-culture of explants

[0040] Sterile seedling leaves (0.5 cm x 0.5 cm) were cut and inoculated on pre-culture solid medium 1 / 2MS (with AS 100 μmol / L) and incubated in dark for 2 d at 25 °C.

[0041] 2.3 Co-culture of Agrobacterium and explants

[0042] Before infection, the pre-cultured leaves were taken out and immersed in the activated Agrobacterium 1 / 2MS liquid suspension for 10-20 min, and the explants were shaken gently to make full contact with the bacterial solution. The infected explants were taken out and the surface bacteria were absorbed with sterile water paper, and then transferred to co-culture medium (solid) 1 / 2MS (with AS 100 μmol / L) and co-cultured in dark for 2 d.

[0043] 2.4 Induction and culture of hairy roots

[0044] The co-cultured explants were transferred to the sterile medium 1 / 2MS + Cef 250 mg / L (solid) and cultured at 25 °C with 16 h / 8 h light. The hairy roots grew from the cut edges of the leaves in about two weeks. The hairy roots (about 2-3 cm) were cut and inoculated in the sterile medium 1 / 2MS + Cef 250 mg / L for two weeks. The fast-growing and well-branched hairy roots were selected and subcultured in the sterile medium 1 / 2MS + Cef 250 mg / L every two weeks. After 4-5 subcultures, the hairy roots were completely sterilized. The well-grown hairy roots of Salvia miltiorrhiza were transferred to the antibiotic-free 1 / 2MS medium and cultured for about 20 d to obtain the MAPK4 overexpressing and RNAi hairy roots of Salvia miltiorrhiza.

[0045] 2.5 Identification of positive hairy roots

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

[0047] The total DNA of about 100 mg of MAPK4 overexpressing hairy roots of Salvia miltiorrhiza was extracted by CTAB method (Stewart Jr, C. N., Via, L. E. (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 T-DNA of Agrobacterium. After agarose gel electrophoresis, the corresponding band indicated that the T-DNA of Agrobacterium was integrated into the root DNA.

[0048] 2.5.2 Western blot

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

[0050] 2.5.3 qRT-PCR method to identify positive root systems

[0051] Total RNA was extracted from hairy roots 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 was analyzed by qRT-PCR (Table 1) to identify the positive root systems (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.). The PCR running conditions were as follows: 95 °C, 10 min; then 95 °C, 10 s, 60 °C, 30 s, cycle 40 times, and the 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 Green fluorescence to identify positive root systems

[0053] Under the fluorescence microscope, the MAPK4-RNAi hairy roots emit green fluorescence, which indicates that this group of roots is positive.

[0054] 3. qRT-PCR analysis of gene expression

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

[0056] 4. Proteomic analysis of the enzyme protein level of the tanshinone synthesis pathway

[0057] Proteomic analysis of MAPK4-OE hairy roots and blank control hairy roots, ultrasonic treatment, protein extraction with phenol, add equal volume of Tris saturated phenol, centrifugation (5500 x g, 10 min, 4°C). The supernatant was precipitated with 0.1M ammonium acetate methanol solution, washed with pure methanol and pure acetone, and then redissolved with 8M urea. Gradually add 20% trichloroacetic acid, precipitate at 4°C for 2h, centrifuge (4500 x g, 5 min), discard the supernatant, and wash the precipitate with pre-cooled acetone 3 times. After drying, add 200mM tetraethylammonium bromide, ultrasonic dispersion. Add trypsin at a ratio of 1:50 for overnight enzymolysis. Add 5mM dithiothreitol, reduce at 56°C for 30min, then add 11mM iodoacetamide, incubate at room temperature for 15min in the dark. Use mixed ion mobility-quadrupole time-of-flight mass spectrometry (tims-QTOF-MS) for analysis. The sample is separated by C18 capture column and reversed-phase C18 analysis column, with mobile phase A (0.1% formic acid and 2% acetonitrile in water) and mobile phase B (0.1% formic acid in acetonitrile) establishing a 90min gradient elution, with a flow rate of 450nL·min-1. The mass spectrometer operates in parallel accumulation-serial fragmentation (PASEF) mode, with a capillary voltage of 2000V. MS and MS / MS spectra of m / z 100 to 1700 are collected, and the ion mobility scan range is 0.6 to 1.6V·s·cm-2. Each acquisition cycle includes one full MS scan and 10 PASEF MS / MS scans, with collision energy varying linearly according to mobility. Use DIA-NN software (V1.8.1) for library-free mode analysis of raw data from independent acquisition (DIA). Search the spectrum file in the local Danshen database (34,575 sequences) with the default settings for main parameters, including trypsin / P digestion, cysteine carbamoylation fixed modification, methionine oxidation, and protein N-terminal acetylation variable modification. Use MaxLFQ algorithm for protein intensity normalization, and quantitative analysis is based on the "PG.MaxLFQ" column in the DIA-NN output table.

[0058] 5. Determination of tanshinones content in positive transgenic hairy roots

[0059] Tanshinones were extracted from transgenic hairy roots by 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.) with tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I as standard to quantify the content of tanshinones.

[0060] III. Experimental results

[0061] 1. Identification of positive hairy roots

[0062] 1.1 Identification of MAPK4 overexpressing hairy roots

[0063] There are three methods to identify MAPK4 overexpressing hairy roots (MAPK4-OE). The first method is to extract total DNA from overexpressing hairy roots and then perform PCR amplification with primers specific to rolB and rolC genes in the rol family of Agrobacterium T-DNA, and the PCR products are detected by agarose gel electrophoresis, as shown in Figure 3 , the presence of corresponding rolB and rolC amplification bands indicates that the DNA of the hairy roots has integrated Agrobacterium T-DNA. The second method is to determine the protein level of MAPK4 in the MAPK4 overexpressing hairy roots, as shown in Figure 4 , the results of western blot experiment using anti-flag antibody show that MAPK4-flag protein is overexpressed in Salvia miltiorrhiza hairy roots. The third method is to detect the transcription level of MAPK4 in the overexpressing hairy roots, as shown in Figure 5 , the overexpression group of MAPK4 is 2-3 times that of the control group. The above results indicate that the MAPK4 overexpressing root system is a positive root system.

[0064] 1.2 Identification of MAPK4-RNAi interfering hairy roots

[0065] There are two methods to identify MAPK4-RNAi interfering hairy roots. The first method is to determine the transcription level of the gene MAPK4 in the interfering root system, as shown in Figure 5 , the interfering group is 5 times lower than the control group. The second method is to observe whether the RNA interfering root system emits green fluorescence under a fluorescence microscope, as shown in Figure 6It can be seen that the interference root emits green fluorescence under the inverted fluorescence microscope. It can be seen that the MAPK4-RNAi interference hairy root is a positive root.

[0066] 2. Determination of tanshinone content

[0067] The contents of representative substances tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I in tanshinone were determined (results are shown in Table 2 and Figure 7 It is found that the content of tanshinone I in the positive overexpression root is about 10.5 times that of the control group, and the content of tanshinone I in the interference group is about 3.2 times lower than that of the control group; the content of dihydrotanshinone I in the overexpression group is about 623.3 times higher than that of the control group, and the content of dihydrotanshinone I in the interference group is about 1.2 times lower than that of the control group; the content of tanshinone IIA in the overexpression group is about 5.9 times that of the control group, and the content of tanshinone IIA in the interference group is 2 times lower than that of the control group; the content of cryptotanshinone in the overexpression group is 176.1 times higher than that of the control group, and the content of cryptotanshinone in the interference group has no obvious change compared with the control group. It can be concluded that overexpression of MAPK4 protein kinase can increase the content of tanshinone in Salvia miltiorrhiza.

[0068] Table 2. Contents of representative substances in MAPK4 overexpression / hairy root (mean ± SD, n = 3)

[0069]

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

[0071] In order to explore whether MAPK4 protein kinase plays a regulatory role in the synthesis of tanshinone in Salvia miltiorrhiza, we studied the key enzymes in the synthesis pathway of tanshinone. First, we detected the transcription level of the gene (as shown in Table 2 and Figure 8 ), and the transcription level of the synthesis enzyme in the overexpression root increased significantly, and the transcription level of the related synthesis enzyme in the RNAi interference group decreased significantly. Secondly, we studied the protein level, as shown in Table 3 and Figure 9 , the protein level of the synthesis enzyme in the overexpression root increased significantly. It is speculated that MAPK4 protein kinase regulates the synthesis of tanshinone by regulating the transcription and expression of key enzyme genes in the synthesis pathway of tanshinone.

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

[0073] The above examples serve to specifically introduce the essential content of the present application, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific examples.

Claims

1. The application of Tanshinone MAPK4 protein kinase in increasing the content of tanshinone compounds in Tanshinone, wherein the sequence of the Tanshinone MAPK4 protein kinase is shown in SEQ ID NO.1, and the tanshinone compounds are tanshinone IIA, dihydrotanshinone I, cryptotanshinone and / or tanshinone I.

2. The application of the gene encoding the Tanshinone MAPK4 protein kinase as described in claim 1 in increasing the content of tanshinone compounds in Tanshinone, wherein the tanshinone compounds are tanshinone IIA, dihydrotanshinone I, cryptotanshinone, and / or tanshinone I.

3. The application of an expression vector containing the encoding gene of the Tanshinone MAPK4 protein kinase as described in claim 1 in increasing the content of tanshinone compounds in Tanshinone, wherein the tanshinone compounds are tanshinone IIA, dihydrotanshinone I, cryptotanshinone, and / or tanshinone I.

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