Application of salvia miltiorrhiza bHLH128 transcription factor and coding gene thereof in regulating and controlling content of tanshinone compounds in salvia miltiorrhiza

Abstract

The invention discloses application of a salvia miltiorrhiza bHLH128 transcription factor and a coding gene thereof in regulating and controlling the content of tanshinone compounds in salvia miltiorrhiza. It is found that the salvia miltiorrhiza bHLH128 transcription factor can be combined with a promoter of a gene CPS1, the level of key enzyme CPS1 on a tanshinone substance synthesis pathway is increased, 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 bHLH128 transcription factor, the coding gene of the salvia miltiorrhiza bHLH128 transcription factor 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 use of a Salvia miltiorrhiza bHLH128 transcription factor and a coding gene thereof for 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 a use of a Salvia miltiorrhiza bHLH128 transcription factor and a gene encoding the same for 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] The Salvia miltiorrhiza bHLH128 transcription factor is used for increasing the content of tanshinone compounds in Salvia miltiorrhiza. The sequence of the Salvia miltiorrhiza bHLH128 transcription factor is shown in Sequence NO.1.

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

[0009] The gene encoding the Salvia miltiorrhiza bHLH128 transcription factor is used to increase 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 expression vector containing the gene encoding the Salvia miltiorrhiza bHLH128 transcription factor is used to increase 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 found that the bHLH128 transcription factor of Danshen can be combined with the promoter of the gene CPS1 to increase the level of the key enzyme CPS1 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 bHLH128 transcription factor of Danshen, 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-bHLH128-3Flag;

[0016] Figure 2 To interfere with the pK7GWIWG2DII-bHLH128 vector plasmid;

[0017] Figure 3 Agarose gel electrophoresis of total DNA in bHLH128-overexpressing hairy roots (bHLH128-OE);

[0018] Figure 4 is a graph of protein levels in bHLH128-overexpressing hairy roots (bHLH128-OE);

[0019] Figure 5 The transcription level of bHLH128 gene in bHLH128 overexpressing hairy roots (bHLH128-OE) and interference hairy roots (bHLH128-RNAi);

[0020] Figure 6 This is a fluorescence detection diagram of interference with hairy roots (bHLH128-RNAi);

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

[0022] Figure 8 is the transcription level of SmCPS1 gene;

[0023] Fig. 9 This is a validation map of the transcription factor bHLH128 binding site. 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 bHLH128 plasmid vector

[0029] The sequence of the Salvia miltiorrhiza bHLH128 transcription factor is shown in Sequence NO.1.

[0030] Sequence No.1:

[0031]

[0032] The construction of bHLH128 overexpression vector was carried out by PCR technology, using specific primers bHLH128-OE (Table 1) to amplify the complete open reading frame sequence of bHLH128, and inserting it into pHB-3Flag plasmid to construct the overexpression pHB-bHLH128-3Flag vector plasmid ( Figure 1 The construction of bHLH128-RNAi vector is to first amplify the gene sequence of bHLH128 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-bHLH128 vector plasmid ( Figure 2 ). The two vector plasmids were respectively transferred into C58C1 Agrobacterium, and the Agrobacterium containing pHB-bHLH128-3Flag 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-bHLH128 vector plasmid was cultured on 50 mg / L rifampicin and 50 mg / L spectinomycin plate medium.

[0033] Table 1 Primer list

[0034]

[0035] 2. Genetic transformation mediated by Agrobacterium rhizogenes C58C1

[0036] 2.1 Storage and activation of Agrobacterium rhizogenes strains

[0037] 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-bHLH128-3Flag vector plasmid and the empty vector plasmid contains 50 mg / L kanamycin and 50 mg / L rifampicin, and the Agrobacterium culture medium of pK7GWIWG2DII-bHLH128 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.

[0038] 2.2 Explant pre-culture

[0039] 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.

[0040] 2.3 Co-cultivation of Agrobacterium and explants

[0041] 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.

[0042] 2.4 Induction and cultivation of hairy roots

[0043] The explants co-cultured for 2 days were transferred to the sterilized medium 1 / 2MS+Cef250 mg / 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), inoculate them in the sterilized medium 1 / 2MS+Cef250 mg / L and culture them for two weeks. Select the hairy roots with fast growth and good branching and transfer them to the sterilized medium 1 / 2MS+Cef250 mg / L for subculture screening. Subculture once every two weeks. After 4-5 subcultures, the hairy roots can be completely sterilized. Then transfer the well-growing Salvia miltiorrhiza hairy roots to the 1 / 2MS medium without antibiotics and continue to culture for about 20 days to obtain Salvia miltiorrhiza hairy roots with bHLH128 overexpression and RNA interference.

[0044] 2.5 Identification of positive hairy roots

[0045] 2.5.1 Extraction of total DNA from bHLH128-overexpressing Salvia miltiorrhiza hairy roots

[0046] About 100 mg of total DNA from bHLH128-overexpressing Salvia miltiorrhiza hairy roots 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 PCR amplification was performed using specific primers bHLH128-F and 3×flag-R. After agarose gel electrophoresis, the presence of corresponding bands indicated that the target gene was integrated into the DNA in the chromatin.

[0047] 2.5.2 Western blot

[0048] Western blot was used to verify whether the bHLH128-overexpressing hairy roots were positive roots, and the primary antibody was Anti-Flag antibody.

[0049] 2.5.3 qRT-PCR method to identify positive roots

[0050] 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 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.).

[0051] 2.5.4 Identification of positive roots by green fluorescence

[0052] Under a fluorescence microscope, if the bHLH128-RNAi interference hairy roots emitted green fluorescence, it indicated that the root group was positive.

[0053] 3. qRT-PCR Analysis of SmCPS1 Gene Expression

[0054] CPS1 enzyme is a key enzyme in the synthesis pathway of tanshinones in Salvia miltiorrhiza, so its transcription level was measured to confirm whether bHLH128 transcription factor 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.

[0055] 4. Exploring the binding site of transcription factor bHLH128 using the Y1H method

[0056] The upstream 2000-bp promoter of CPS1 was analyzed, and the E-boxes therein were screened and named E1-box, E2-box and E3-box. The 3×E-box sequences were synthesized and cloned into the pLacZi vector (Table 2), and bHLH128 was constructed into the pB42AD vector. The three pLacZi vectors were co-transformed into the yeast cell EGY48 with the pB42AD vector, and the transformed yeast was spread on the selective medium (SD / -Ura / -Trp), cultured at 30°C for 2-3 days, and the positive clones were screened. The positive clones were picked and inoculated into the liquid selective medium and cultured overnight at 30°C in a shaking incubator. The overnight cultured yeast solution was diluted to OD 600 ≈0.5, take 10 μL and spot it on solid color development medium (SD / -Ura / -Trp / +Raf / +X-Gal). Incubate at 30℃ for 1-2 days and observe whether the colony turns blue (lacZ activity positive).

[0057] Table 2 List of synthetic 3×E-box sequences

[0058]

[0059] 5. Determination of Tanshinone Content in Positive Transgenic Hairy Roots

[0060] 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.

[0061] 3. Experimental Results

[0062] 1. Identification of positive hairy roots

[0063] 1.1 Identification of hairy roots overexpressing bHLH128

[0064] There are three methods for identifying bHLH128-overexpressing hairy roots (bHLH128-OE). Method 1 is to extract the total DNA of the overexpressing hairy roots, amplify it with specific primers by PCR, and then detect it by agarose gel electrophoresis. Figure 3 As shown in the figure, the presence of corresponding bands indicates that the target gene bHLH128 is integrated into the DNA in the chromatin. Method 2 is to measure the protein level of bHLH128, such as Figure 4 Method three is to detect the transcription level of bHLH128 overexpressed in hairy roots, as shown in Figure 5 The results show that the bHLH128 overexpression group is more than 2 times that of the control group. The above results indicate that the bHLH128 overexpression root system is a positive root system.

[0065] 1.2 Identification of bHLH128-RNAi hairy roots

[0066] There are two methods for identifying bHLH128-RNAi interference with hairy roots. Method 1 is to measure the transcription level of the gene bHLH128 that interferes with the root system, such as Figure 5 The interference group was lower than the control group. Method 2 was to observe whether the RNAi root system emitted green fluorescence under a fluorescence microscope. Figure 6 It can be seen that the interference root system emits green fluorescence under fluorescence excitation, which shows that the bHLH128-RNAi hairy roots are positive roots.

[0067] 2. Determination of Tanshinone Content

[0068] The contents of representative substances in tanshinones, tanshinone IIA, dihydrotanshinone I, cryptotanshinone and tanshinone I, were determined (see Table 3, Figure 7 As shown in the figure, it was found that in the positive overexpression root system, the content of tanshinone I was about 1.9 times higher than that of the control group, and the content of tanshinone I in the interference group was about 6.4 times lower than that of the control group; the content of dihydrotanshinone I was about 1.5 times higher in the overexpression group than that of the control group, and about 3.3 times lower in the interference group than that of the control group; the content of tanshinone IIA was more than 4.3 times higher in the overexpression group than that of the control group, and the interference group did not change much compared with the control group; the content of cryptotanshinone was more than 1.9 times higher in the overexpression group than that of the control group, and the interference group was more than 1.2 times lower than that of the control group. It can be concluded that bHLH128 transcription factor can positively regulate the content of tanshinone substances in Salvia miltiorrhiza.

[0069] Table 3b Contents of representative tanshinone substances in hairy roots after HLH128 overexpression / interference (mean±SD, n=3)

[0070]

[0071]

[0073] 3. bHLH128 transcription factor regulates the expression of enzymes related to the synthesis of tanshinones

[0074] In order to explore whether the bHLH128 transcription factor plays a regulatory role in the synthesis of tanshinones in Salvia miltiorrhiza, we studied CPS1, a key enzyme in the synthesis pathway of tanshinones. First, we detected the transcription level of CPS1 gene ( Figure 8 ), the transcription level of CPS1 in the overexpressed root system increased significantly, and the transcription level of CPS1 in the RNAi interference group also decreased significantly. This indicates that bHLH128 regulates tanshinone biosynthesis by positively regulating the level of CPS1. It is speculated that the bHLH128 transcription factor may bind to the promoter of the CPS1 gene to regulate expression. To confirm this hypothesis, we used Y1H to verify ( Fig. 9 ),Depend on Fig. 9 It can be concluded that the bHLH128 transcription factor binds to the E2-box in the CPS1 promoter, thereby regulating gene expression and the synthesis of tanshinones.

[0075] Experiments have shown that bHLH128 affects the synthesis and production of tanshinones by regulating the gene expression of copalyl pyrophosphate synthase 1 (CPS1).

[0076] 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. Use of the bHLH128 transcription factor of Salvia miltiorrhiza for increasing the content of tanshinone compounds in Salvia miltiorrhiza, wherein the sequence of the bHLH128 transcription factor of Salvia miltiorrhiza 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 bHLH128 transcription factor according to claim 1 for 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 bHLH128 transcription factor according to claim 1 for 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.

Images