Application of SmbHLH130 gene in increasing tanshinone content and salvianolic acid content in salvia miltiorrhiza

Abstract

The invention provides a SmbHLH130 gene and an application of the SmbHLH130 gene in increasing the content of tanshinone and salvianolic acid in salvia miltiorrhiza. According to the invention, a transcription factor SmbHLH130 is obtained through multi-omics data analysis in the early stage. A CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) technology is utilized, a SmbHLH130 gene editing carrier is constructed, mutant hairy roots of gene knockout SmbHLH130 are obtained through agrobacterium tumefaciens transformation mediated by agrobacterium rhizogenes ATCC15834, and whether knockout of the mutant hairy roots is successful or not is identified through sequencing. And determining the content of tanshinone and salvianolic acid in the hairy roots of the transgenic salvia miltiorrhiza by using a high performance liquid chromatography (HPLC). The tanshinone content and the salvianolic acid content of the obtained gene knockout hairy roots are remarkably improved. Real-time fluorescent quantitative PCR (RT-qPCR) analysis shows that the expression quantity of a key enzyme gene participating in the biosynthesis of the active component of the salvia miltiorrhiza is also remarkably increased. The invention provides a method for increasing tanshinone and salvianolic acid contents of hairy roots of salvia miltiorrhiza, which can provide a new thought and target gene for production of active components of salvia miltiorrhiza and molecular design and breeding of salvia miltiorrhiza, and has higher application value and research value.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and metabolic engineering technology, and specifically relates to a SmbHLH130 Gene knockout using CRISPR-Cas9 technology SmbHLH130 Gene, can simultaneously increase the content of tanshinone and salvianolic acid in Salvia miltiorrhiza. Background Art

[0002] Salvia miltiorrhiza is ( Salvia miltiorrhiza ) is a commonly used medicinal plant in my country. Its dried roots and rhizomes are the traditional Chinese medicine Danshen, which is widely used in the treatment of cardiovascular and cerebrovascular diseases, menstrual disorders and various inflammations. At the same time, Danshen is also an important source of raw materials for modern Chinese medicine preparations such as Danhong Injection, Danshen Polyphenolate Injection, and Danshen Pills.

[0003] Fat-soluble tanshinones and water-soluble phenolic acids are important material bases for the efficacy of salvia miltiorrhiza. Among them, fat-soluble tanshinones include cryptotanshinone, tanshinone I, tanshinone IIA, etc. Salvia miltiorrhiza phenolic acids include rosmarinic acid, tanshinone A, B, etc. The "Chinese Pharmacopoeia" stipulates that the total amount of tanshinone I, IIA, and cryptotanshinone shall not be less than 0.25%. The content of tanshinone B shall not be less than 3%. Due to the large market demand for salvia miltiorrhiza and its long growth cycle, the quality of cultivated salvia miltiorrhiza has gradually declined in recent years. How to increase the content of active ingredients in salvia miltiorrhiza has become an urgent need of the salvia miltiorrhiza industry.

[0004] Treating Salvia miltiorrhiza with inducers is an effective strategy to increase the active ingredients of Salvia miltiorrhiza. Studies have shown that treating Salvia miltiorrhiza with gibberellin (GA) can significantly increase the content of tanshinone and salvianolic acid. However, there are no reports on the simultaneous increase of tanshinone and salvianolic acid content using CRISPR-Cas9 gene editing technology. Summary of the invention

[0005] In order to solve the problem that the above-mentioned prior art fails to simultaneously increase the contents of tanshinone and salvianolic acid in salvia miltiorrhiza by genetic engineering technology, the present invention provides a SmbHLH130 Genes and their application in increasing the content of tanshinone and salvianolic acid in Salvia miltiorrhiza.

[0006] The present invention comprehensively applies transcriptome data analysis, gene cloning, vector construction, molecular detection, RT-qPCR analysis, target exploration, protein interaction, compound content determination and other means to screen out a gene encoding a transcription factor SmbHLH130 that interacts with the GA pathway signaling protein DELLA, namely, SmbHLH130 Gene, the full length of the gene is 2155bp, and the CDS sequence is 1185bp. SmbHLH130 The gene sequence is shown in SEQ ID NO.1, and the CDS sequence is shown in SEQ ID NO.2.

[0007] The present invention also provides SmbHLH130 Application of genes in increasing the content of tanshinone and salvianolic acid in Salvia miltiorrhiza by knocking out SmbHLH130 Genes to promote the production of tanshinone and salvianolic acid in Salvia miltiorrhiza hairy roots.

[0008] according to SmbHLH130 DNA sequence and CDS sequence of the gene, design of sgRNA sequence, construction SmbHLH130 Gene CRISPR-Cas9 expression vector; obtained by Agrobacterium rhizogenes-mediated genetic transformation SmbHLH130 The hairy roots were knocked out by gene knockout. RT-qPCR was used to analyze the expression of key enzyme genes in the biosynthesis pathway of tanshinone and tanshinone acid in the transgenic hairy roots. HPLC was used to determine the content of the active ingredients of danshen in the transgenic hairy roots. It was found that the maximum content of total tanshinone and tanshinone acid in the gene knockout strain could reach 2.34 mg / g DW, which was 5 times that of the control group. SmbHLH130 Its regulatory role in the biosynthesis of tanshinone provides important key regulatory genes for the metabolic engineering of tanshinone and tanshinone, and has important theoretical significance and application value in deeply understanding the laws of quality formation of salvia miltiorrhiza and providing targets for molecular design breeding of salvia miltiorrhiza.

[0009] Among them, the gene knockout method was used to knock out SmbHLH130 The steps of the gene are as follows: (1) Based on SmbHLH130 Gene, two sgRNA sequences were designed on the first exon as targets for gene knockout, the two target sequences were recombined into two primers respectively, and PCR cloning was performed using pCBC-DT1T2 as a template; (2) The PCR product cloned in step (1) was digested with Bsa I and recombined to obtain a gene knockout vector; (3) transferring the gene knockout vector obtained in step (2) into Agrobacterium rhizogenes; (4) The Agrobacterium rhizogenes transformed in step (3) was used to infect sterile Salvia miltiorrhiza seedling explants, and the hairy roots obtained were identified by PCR and sequencing to determine the successful knockout strain.

[0010] Furthermore, in step (1), two sgRNA sequences are designed according to the 19base-NGG principle, and the gene sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0011] Furthermore, in step (1), the two primers are primer pHEE401E-SmbHLH130-F and primer pHEE401E-SmbHLH130-R, and both have a Bsa I restriction site.

[0012] Furthermore, in step (2), the enzyme used for recombination is T4 ligase; and the gene knockout vector is pHEE401E-DsRED-SmbHLH130.

[0013] Furthermore, in step (3), the Agrobacterium rhizogenes is ATCC15834.

[0014] Furthermore, in step (4), the infection and PCR detection method of sterile Salvia miltiorrhiza seedling explants is as follows: (4.1) Pre-culture: Take the leaves of sterile Salvia miltiorrhiza seedlings as explants, inoculate them on 1 / 2MS solid culture medium and culture them in the dark for 2 days; Co-cultivation: Use a new sterile sharp surgical blade to dip into the bacterial solution of Agrobacterium rhizogenes transferred with the gene knockout vector obtained in step (3), cut through the leaves and veins of the pre-cultured sterile Salvia miltiorrhiza seedlings, and then place the leaves with cut veins on 1 / 2 MS solid culture medium and culture in the dark for 2-3 days; Sterilization: Place the co-cultured leaves on 1 / 2MS solid medium containing Timentin, and change the medium every two weeks. The concentration of Timentin is gradually reduced until there is no antibiotics; Isolation of monoclonal hairy roots: Cut different monoclonal hairy root systems separately and place them on separate 1 / 2MS solid medium for culture; Subculture: Cut off a part of the monoclonal hairy root system for subculture; (4.2) Identification of successful knockout strains: SmbHLH130 Specific primers were designed upstream and downstream of the sgRNA sequence of the gene, and the extracted monoclonal hairy root genomic DNA was used as a template for amplification. The amplified products were sequenced to identify the successful knockout strains.

[0015] Beneficial effects: The present invention can significantly increase the contents of tanshinone and salvianolic acid in salvia miltiorrhiza at the same time; in-depth analysis SmbHLH130 The regulatory mechanism of genes in the biosynthesis of tanshinone and salvianolic acid has important theoretical significance for in-depth understanding of the laws of quality formation of salvia miltiorrhiza, and also provides important gene resources for molecular design and breeding of salvia miltiorrhiza. The method of the present invention has reliable effects and low cost for obtaining tanshinone and salvianolic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 for SmbHLH130 Gene expression pattern analysis diagram; A is the gene expression pattern under GA treatment conditions SmbHLH130 Gene expression pattern analysis diagram; B is SmbHLH130 Analysis of gene tissue-specific expression; Figure 2 Map for CRISPR-Cas9 vector construction; Figure 3 Knockout SmbHLH130 Sequencing peaks of hairy roots; A is the target mutation site sequence of gene editing hairy roots; the wild-type (WT) DNA sequence is at the top, sgRNA is represented by blue font, PAM (NGG) site is represented by red font, '-' represents the number of bases reduced compared to the wild type, and '+' represents the number of bases increased compared to the wild type; the numbers represent the increase or decrease of the editing region compared to the wild type; B is the sequencing peaks of heterozygous mutation (bHLH130Cas-9), biallelic mutation (bHLH130Cas-13) and homozygous mutation (bHLH130Cas-36); Figure 4 Knockout SmbHLH130 Gene expression profile of key enzymes after genes; Figure 5 Knockout SmbHLH130 Figure 1. Determination of tanshinone and salvianolic acid contents in hairy roots of gene; Figure 6 This is the result of yeast single hybridization; Figure 7 Figure 1 is the result of EMSA; Figure 8 This is the Dul-LUC result diagram; Fig. 9 This is a diagram of the yeast two-hybrid experiment results; Fig.10 This is the result of the bimolecular fluorescence complementation experiment; Fig.11 This is a graph showing the results of the co-immunoprecipitation experiment. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with specific implementation examples. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the claims of the present application.

[0018] The experimental methods for which specific conditions are not specified in the following embodiments are usually carried out according to conventional conditions, such as the conditions described in molecular cloning, or according to the conditions recommended in the instructions attached to the reagents or kits provided by the manufacturer.

[0019] Example 1 Salvia miltiorrhiza SmbHLH130 Gene screening and cloning 1.1. Salvia miltiorrhiza SmbHLH130 Gene screening A GA-inhibited bHLH130 transcription factor was screened from the transcriptome database of Salvia miltiorrhiza treated with GA and was named SmbHLH130 Gene, SmbHLH130 The gene sequence is shown in SEQ ID No. 1.

[0020] 1.2 SmbHLH130 Gene cloning Weigh 0.1 g of Salvia miltiorrhiza hairy roots that are growing vigorously, freeze them quickly in liquid nitrogen, and grind them quickly in a mortar. Use the Quanshijin Plant Genomic DNA Extraction Kit to extract Salvia miltiorrhiza genomic DNA. Use the TIANGEN Polysaccharide Polyphenol RNA Extraction Kit to extract RNA. Reverse transcribe the RNA with good quality to obtain cDNA. SmbHLH130 The primers were designed based on the gene sequence. PCR amplification was performed using Danshen DNA and CDNA as templates and SmbHLH130-F / R as primers to obtain SmbHLH130 The full-length sequence and CDS sequence of the gene, the CDS sequence is shown in SEQ ID No. 2. The primer sequences are as follows:

[0021] Example 2 SmbHLH130 Gene subcellular localization 2.1 Vector construction To determine SmbHLH130 The location of the gene in the cell, the design of homologous recombination primers, and the construction of the primers into the GFP N-terminal fusion vector pEAQ by infusion homology method. The primer sequences are as follows:

[0022] 2.2 Expression of SmbHLH130-GFP fusion protein The constructed vector and empty vector were transformed into Agrobacterium GV3101, and the empty vector was used as a control. The cells were shaken overnight at 28°C to a concentration of OD 600 =0.4-0.6. Centrifuge and discard the supernatant, resuspend the bacteria in tobacco infection solution to make the bacterial solution concentration OD 600 =0.4-0.6, use a sterile syringe to absorb the bacteria, inject into about 1-month-old tobacco leaves, and observe with a laser confocal microscope 3 days later SmbHLH130 The location in the cell where a gene is expressed. Fig.10 As shown, SmbHLH130 The gene is localized in the cell nucleus, that is, SmbHLH130 is a nuclear-localized protein.

[0023] Example 3 Salvia miltiorrhiza SmbHLH130 Analysis of gene action mechanism 3.1 Yeast one-hybrid assay For research SmbHLH130The molecular mechanism of gene regulation of the active ingredients of Salvia miltiorrhiza was studied by using the PlantCARE website to analyze the promoters of key enzyme genes of the active ingredients of Salvia miltiorrhiza. The study found that the genes of the phenolic acid metabolic pathway SmC4H1, SmTAT1, SmHPPR1、SmCYP98A14 and tanshinone metabolic pathway genes SmDXS2, SmCPS1, SmKSL1, SmCYP76AH1 The promoter of the bHLH transcription factor has a cis-acting element E-box. Therefore, the SmbHLH130 transcription factor was constructed into the pGADT7 vector, and the synthetic key enzyme promoter element was constructed into the pAbAi vector and transformed into the yeast Y1H Gold. After self-activation verification, AbA concentration screening, spot plate and other experiments, such as Figure 6 As shown, SmbHLH130 Genes can be SmC4H1, SmTAT1, SmHPPR1, SmCYP98A14, SmDXS2, SmCPS1, SmKSL1, SmCYP76AH1 Target binding on gene promoters.

[0024] 3.2 Electrophoresis retardation assay (EMSA) To further study the binding of SmbHLH130 transcription factor to target gene promoters, SmbHLH130 protein was purified by prokaryotic expression system; at the same time, genes of tanshinone biosynthesis pathway were designed and synthesized. C4H1, TAT1, HPPR1, CYP98A14 and salvianolic acid biosynthetic pathway genes DXS2, CPS1, KSL1, CYP76AH1 EMSA experimental probe. The probe sequence is as follows:

[0025] After incubation, electrophoresis, color development and other steps, such as Figure 7 As shown, it was found that SmbHLH130 transcription factor can specifically C4H1, TAT1, HPPR1, CYP98A14 , DXS2, CPS1, KSL1, CYP76AH1 of probe binding.

[0026] 3.3 Dual-luciferase reporter assay (dual-LUC) For research SmbHLH130 Direct target of gene SmbHLH130 The gene sequence was transferred into the pGreen-62SK vector as an elicitor; the gene promoter (Promotor, Pro) of the key enzymes in the biosynthesis pathway of tanshinone and tanshinone was homologously recombined into the pGreen0800 vector to obtain the pGreen0800-Pro vector as a reporter gene. The correctly sequenced elicitor and reporter gene vectors were respectively transferred into Agrobacterium GV3101 (pSoup). After overnight culture, the bacteria were resuspended in the infection solution, the two bacterial solutions were mixed in equal volumes of 1:1, and injected into tobacco for dual-LUC experiments. Figure 8 As shown, the Dual-LUC experiment shows SmbHLH130 Genes can be repressed at the transcriptional level C4H1, TAT1, HPPR1, CYP98A14 , DXS2, CPS1, KSL1, CYP76AH1 of gene expression.

[0027]

[0028] Example 4 Interaction between SmbHLH130 and SmDELLA proteins in Salvia miltiorrhiza To verify whether SmbHLH130 interacts with the GA signal protein SmDELLA2, plasmids pGADT7-SmbHLH130 and pGBKT7-SmDELLA2 were first co-transformed into yeast strain AH109, ​​and the transformed yeast was placed in screening medium (SD / -Trp / -Leu / -His / -Ade) for screening, as shown in Figure 2 . Fig. 9 As shown, the yeast co-transformed with negative control pGADT7 and pGBKT7 did not grow, the yeast co-transformed with control pGADT7-SmbHLH130 and pGBKT7 did not grow, and the yeast co-transformed with control pGADT7 and pGBKT7-SmDELLA2 did not grow. Only the yeast co-transformed with plasmids pGADT7-SmbHLH130 and pGBKT7-SmDELLA2 could grow on the four-deficient medium. The results showed that SmbHLH130 and SmDELLA2 interacted. In addition, BiFC was used to verify the above results. N -SmbHLH130 and YFP C -SmDELLA2 vector was mixed with Agrobacterium to transform well-growing tobacco leaves. The mixed bacterial solution was injected into the tobacco. After 3 days, a 1 cm diameter tobacco leaf was taken out with a hole puncher and placed on a slide with the back facing up. It was soaked with double distilled water and fixed with a cover glass (to avoid bubbles). It was observed under a laser confocal microscope and only YFP was found. N -SmbHLH130 and YFP C -SmDELLA2, the yellow fluorescence signal can be detected. These results show that SmbHLH130 can interact with SmDELLA2 protein in vivo ( Fig.10 ). In order to further verify whether SmbHLH130 and SmDELLA2 interact with each other in Salvia miltiorrhiza, the SmbHLH130 protein was fused with the FLAG tag to form the FLAG-SmbHLH130 fusion protein as the bait protein; the SmDELLA2 protein was fused with the MYC tag to form the MYC-SmDELLA2 fusion protein, which was expressed using the tobacco transient transfection system. Fig.11As shown in: SmbHLH130 and SmDELLA2 were detected in the Input group, indicating that these two proteins exist in the sample; the band positions of SmbHLH130 and SmDELLA2 in the IP group are both shown, that is, these two proteins exist in the precipitation complex, proving that there is an interaction between SmbHLH130 and SmDELLA2, and their interaction may affect the regulation of bHLH130 transcription factors on target genes.

[0029] Example 5 SmbHLH130 Analysis of gene expression patterns 5.1 Analysis of tissue expression patterns RNA was extracted from roots, stems, leaves, flowers and other tissues of 2-year-old Salvia miltiorrhiza plants in full bloom in the medicinal botanical garden using the TIANGEN plant RNA extraction kit, and cDNA was synthesized using the TAKARA reverse transcription kit. RT-qPCR was performed SmbHLH130 Gene expression was detected using primers: RT-SmbHLH130-F and RT-SmbHLH130-R. Figure 1 As shown in B, the experimental results show SmbHLH130 The gene was mainly expressed in roots and leaves.

[0030] 5.2 GA-induced expression analysis GA with a final concentration of XµM was used to induce the hairy roots of Salvia miltiorrhiza 18 days after subculture. Samples were taken at 0, 1, 2, 4, 6, 8, 12, and 24 hours after treatment, and RNA was extracted and reverse transcribed into cDNA for gene expression determination. The primers were the same as those in Section 5.1. Figure 1 As shown in A, after GA treatment, SmbHLH130 The gene expression level gradually decreased, indicating SmbHLH130 Gene expression is repressed by GA.

[0031] Example 6 Salvia miltiorrhiza SmbHLH130 Obtaining gene knockout hairy roots 6.1 CRISPR / Cas vector construction According to the principle of 19base-NGG, SmbHLH130 Two sgRNA sequences were designed at the first exon position of the gene sequence as targets for gene knockout. The gene sequences of the two sgRNA sequences are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively. The designed sgRNA sequences (targets) were replaced into the following two template primers. The template primers are as follows:

[0032] Using pCBC-DT1T2 as a template, primers pHEE401E-SmbHLH130-F and pHEE401E-SmbHLH130-R were used for amplification (~750bp). The PCR product was purified and recovered, and the PCR product and pHEE401E-DsRED final vector were digested with BsaI, purified and recovered again, and recombined with T4 ligase. The correct sequence after sequencing was the pHEE401E-DsRED-SmbHLH130 gene knockout vector ( Figure 2 ).

[0033]

[0034] The pHEE401E-SmbHLH130 gene editing vector was successfully constructed. The vector structure is as follows Figure 2 As shown in the figure. The Arabidopsis AtU6 promoter was used to simultaneously initiate the dual-target expression of sgRNA1 and sgRNA2, and the promoter of the egg cell-specific EC1.2 gene was used to drive the expression of Cas9 in Salvia miltiorrhiza. Homozygous or biallelic mutants of the target gene can be effectively produced in the hairy roots of Salvia miltiorrhiza, greatly shortening the gene editing time and enhancing the gene editing effect.

[0035] 6.2 Obtaining gene knockout hairy roots by infecting Salvia miltiorrhiza leaves The pHEE401E-DsRED-SmbHLH130 gene knockout vector (plasmid) was transformed into Agrobacterium rhizogenes ATCC15834, and a single clone was picked for PCR verification. The bacterial solution was expanded and centrifuged, and resuspended in sterile liquid MS medium. The leaves of sterile Salvia miltiorrhiza seedlings that were subcultured for 30 days were taken as explants of sterile Salvia miltiorrhiza seedlings, and inoculated into 1 / 2MS solid medium for pre-culture at 25°C in the dark for 2 days. Use a new sterile sharp surgical blade to dip into the bacterial solution, scratch the leaves and veins of the pre-cultured sterile Salvia miltiorrhiza seedlings, so that Agrobacterium sticks to the wound of the leaves. Then place the leaves with the cut veins on 1 / 2MS solid medium for dark culture for 3 days as co-culture. The leaves after co-culture were transferred to 1 / 2MS solid medium containing 300 mg / L of timentin, and the medium was replaced every two weeks. The concentration of timentin was gradually reduced until there was no resistance, and hairy roots gradually grew in the process. When the root hairs are about 3-5 cm long, different monoclonal root hairs are cut and placed on separate 1 / 2MS solid culture medium for expansion culture and molecular identification; a part of the monoclonal root hairs is cut for subculture; and about 1 cm of root hairs are taken for positive identification.

[0036] 6.3 Molecular identification of gene knockout hairy roots The MagicPure 32 Plant Genomic DNA Kit was used to extract genomic DNA from Salvia miltiorrhiza hairy roots and perform PCR identification. The specific method is described in the instruction manual. The obtained hairy root genomic DNA was used as a template. rolB -F and rolB -R was used as a primer for PCR reaction to identify whether Agrobacterium successfully infects the leaf disc. At the same time, specific primers were designed at about 200 bp upstream and downstream of the sgRNA target. SmbHLH130 -CAS-Seq-F / R, sequence the PCR products. According to the sequencing results, hairy roots with homozygous mutations and biallelic mutations were selected for further study. Figure 3 It is the sequencing peak map and mutation site map of the knockout strain, which includes three different mutation types: heterozygous mutant (bHLH130Cas-9), biallelic mutant (bHLH130Cas-13), and homozygous mutant (bHLH130Cas-36).

[0037]

[0038] Example 7 SmbHLH130 Gene regulation of key enzyme gene expression 7.1 Hairy root culture Pick the positive hairy roots with good growth status, select the ones with more branches and cut 0.2g, transfer them into a 250mL conical flask containing 100mL 1 / 2MS culture medium, place it in a 25℃ shaking cabinet, and culture it in the dark at 110rpm for 30 days. Then remove the hairy roots, quickly dry the surface water with absorbent paper, and use part of it for RNA extraction, and part of it for the determination of tanshinone and salvianolic acid content.

[0039] 7.2 RNA extraction and reverse transcription RNA was extracted, and qualified RNA was used for reverse transcription into cDNA, and RT-PCR was used to measure the expression of related genes.

[0040] 7.3 Design and synthesis of RT-PCR primers Primers were designed based on the key enzyme genes in the biosynthesis pathway of tanshinone and salvianolic acid. The primers were synthesized by Shanghai Shenggong Bioengineering Co., Ltd. The primer sequences are as follows:

[0041] 7.4 RT-qPCR detection of gene expression in transgenic hairy roots The cDNA template obtained above was diluted 10 times and RT-qPCR amplified using the above primers. The experimental system used TB Green provided by TaKaRa ®Premix Ex Taq™ II FAST qPCR kit, β-actin gene was used as internal reference gene. The results showed that ( Figure 4 ), compared with the control, most of the key enzyme genes involved in the biosynthesis of tanshinone and salvianolic acid in the knockout strains of heterozygous mutation bHLH130Cas-9, biallelic mutation bHLH130Cas-13 and homozygous mutation bHLH130Cas-36 were upregulated to varying degrees.

[0042] 7.5 Determination of tanshinone and salvianolic acid content in hairy roots of Salvia miltiorrhiza Sample preparation: Dry the hairy roots with absorbent paper at 45℃ until constant weight, grind into fine powder. Accurately weigh 0.02g in 2ml 70% methanol, extract by ultrasonic for 45min (invert and mix every 15min), centrifuge at 8000rpm for 10min, and filter the supernatant through 0.22µm filter membrane for later use.

[0043] The content was determined by Waters e2695 binary high performance liquid chromatograph, the detector was Waters 2996 diode array detector, and the chromatographic column was Waters SunFire C18 (250mm×4.6mm, 5μm). The data were collected by Empower 3 data acquisition software. The chromatographic conditions were as follows: flow rate 1ml / min, column temperature 30℃, and sample volume 10μl. The detection wavelengths were 270nm (tanshinones) and 288nm (phenolic acids), respectively. The mobile phases were 0.026% phosphoric acid aqueous solution and acetonitrile, respectively, with gradient elution. The peak area of ​​each component was recorded and substituted into the linear regression equation to calculate the content of tanshinone and phenolic acid.

[0044] exist SmbHLH130 In the study of gene-knockout hairy roots, the contents of tanshinone and salvianolic acid components increased significantly compared to the control group. Specifically, the CA content in salvianolic acid increased sharply, reaching 10 times that of the control group; the RA content in the homozygous mutant bHLH130Cas-36 was 5 times that of the control group. Particularly significant was the SAB content, which increased to 20 times that of WT. As for tanshinone, the accumulation of CT increased most significantly, nearly 60 times higher than that of WT; the DT-Ⅰ content also increased nearly 5 times. The contents of TI and T-ⅡA in the knockout strains also increased significantly, 2 times and 17 times that of WT, respectively. The results showed SmbHLH130 Genes play a key role in regulating the synthesis of these two major types of medicinal ingredients. All differences were statistically significant (P<0.05) like Figure 5As shown, the contents of tanshinone (tanshinone Ⅰ (T-Ⅰ), tanshinone ⅡA (T-ⅡA), dihydrotanshinone I (DT-I) and cryptotanshinone (CT)) and salvianolic acid (caffeic acid (CA), rosmarinic acid (RA), salvianolic acid B (SAB)) produced by the CRISPR-Cas9 strain of the present invention were significantly increased compared with the control, and the total content could reach up to 2.34 mg / g DW (dry weight), which was 5 times that of the control group.

[0045] The present invention adopts gene knockout SmbHLH130 The genetic metabolic engineering strategy to obtain Salvia miltiorrhiza hairy roots with high yields of tanshinone and salvianolic acid provides the possibility for commercial mass production of tanshinone and salvianolic acid, and also provides a new strategy to meet the market demand for tanshinone and salvianolic acid.

[0046] The above-mentioned technologies not specifically mentioned are all referred to the prior art.

[0047] The description of the above embodiments is convenient for those skilled in the art to understand and use the present invention. It is obvious that those skilled in the art can easily make various modifications to the embodiments as needed, and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. One SmbHLH130 A gene characterized by Its gene sequence is shown in SEQ ID NO.

1.

2. according to claim 1 SmbHLH130 A gene characterized by The CDS sequence is shown in SEQ ID NO.

2.

3. A method according to claim 1 SmbHLH130 The application of genes in increasing the content of tanshinone and salvianolic acid in salvia miltiorrhiza is characterized in that: By knocking out SmbHLH130 Genes to promote the production of tanshinone and salvianolic acid in Salvia miltiorrhiza hairy roots.

4. The use according to claim 3, characterized in that: Knockout of gene in Salvia miltiorrhiza SmbHLH130 Gene, including the following steps: (1) Based on SmbHLH130 Gene, two sgRNA sequences were designed on the first exon as targets for gene knockout, the two target sequences were recombined into two primers respectively, and PCR cloning was performed using pCBC-DT1T2 as a template; (2) The PCR product cloned in step (1) was digested with Bsa I and recombined to obtain a gene knockout vector; (3) transferring the gene knockout vector obtained in step (2) into Agrobacterium rhizogenes; (4) The Agrobacterium rhizogenes transformed in step (3) was used to infect sterile Salvia miltiorrhiza seedling explants, and the hairy roots obtained were identified by PCR and sequencing to determine the successful knockout strain.

5. The use according to claim 4, characterized in that: In step (1), two sgRNA sequences are designed according to the 19base-NGG principle, and the gene sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

6. The use according to claim 4, characterized in that: In step (1), the two primers are primer pHEE401E-SmbHLH130-F and primer pHEE401E-SmbHLH130-R, and both have a Bsa I restriction site.

7. The use according to claim 4, characterized in that: In step (2), the enzyme used for recombination is T4 ligase; and the gene knockout vector is pHEE401E-DsRED-SmbHLH130.

8. The use according to claim 4, characterized in that: In step (3), the Agrobacterium rhizogenes is ATCC15834.

9. The use according to claim 4, characterized in that: In step (4), the infection and PCR detection methods of sterile Salvia miltiorrhiza seedling explants are as follows: (4.1) Pre-culture: Take the leaves of sterile Salvia miltiorrhiza seedlings as explants, inoculate them on 1 / 2MS solid culture medium and culture them in the dark for 2 days; Co-cultivation: Use a sterile sharp surgical blade to dip into the bacterial solution of Agrobacterium rhizogenes transferred with the gene knockout vector obtained in step (3), cut through the leaves and veins of the pre-cultured sterile Salvia miltiorrhiza seedlings, and then place the leaves with cut veins on 1 / 2 MS solid culture medium for dark culture for 2-3 days; Sterilization: Place the co-cultured leaves on 1 / 2MS solid medium containing Timentin, and change the medium every two weeks. The concentration of Timentin is gradually reduced until there is no antibiotics; Isolation of monoclonal hairy roots: Cut different monoclonal hairy root systems separately and place them on separate 1 / 2MS solid medium for culture; Subculture: Cut off a part of the monoclonal hairy root system for subculture; (4.2) Identification of successful knockout strains: SmbHLH130 Specific primers were designed upstream and downstream of the sgRNA sequence of the gene, and the extracted monoclonal hairy root genomic DNA was used as a template for amplification. The amplified products were sequenced to identify the successful knockout strains.

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