CN117467678A - Method for producing tanshinol - Google Patents

Method for producing tanshinol Download PDF

Info

Publication number
CN117467678A
CN117467678A CN202311194251.3A CN202311194251A CN117467678A CN 117467678 A CN117467678 A CN 117467678A CN 202311194251 A CN202311194251 A CN 202311194251A CN 117467678 A CN117467678 A CN 117467678A
Authority
CN
China
Prior art keywords
gene
cyp98a75
root
tanshinol
red sage
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202311194251.3A
Other languages
Chinese (zh)
Inventor
陈万生
冯婧娴
周正
霍俊成
段永豪
肖莹
邱实
季倩
杨颖博
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai University of Traditional Chinese Medicine
Original Assignee
Shanghai University of Traditional Chinese Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai University of Traditional Chinese Medicine filed Critical Shanghai University of Traditional Chinese Medicine
Priority to CN202311194251.3A priority Critical patent/CN117467678A/en
Publication of CN117467678A publication Critical patent/CN117467678A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • C12N9/0081—Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Nutrition Science (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the technical field of molecular biology and plant genetic engineering, in particular to a method for constructing a recombinant vector of CYP98A75 over-expression of salvia miltiorrhiza, and constructing recombinant agrobacterium on the basis of the recombinant vector; the hairy root of the red sage root which can over-express the CYP98A75 enzyme of the red sage root is obtained by taking the leaf disc of the red sage root as an explant through the mediation of agrobacterium, and the content of the salvianic acid A in the hairy root can be improved. The invention can obviously improve the content of the danshensu in hairy roots and has very important significance for the production of the danshensu.

Description

Method for producing tanshinol
Technical Field
The invention belongs to the technical fields of molecular biology and plant genetic engineering, and in particular relates to a method for producing tanshinol by establishing transgenic hairy roots of red sage roots through agrobacterium mediation.
Background
The molecular formula of tanshinol is C 9 H 10 O 5 The chemical name is D- (+) -beta (3, 4-dihydroxyphenyl) lactic acid, which is a phenolic aromatic acid compound containing catechol structure. Salvianic acid A has wide pharmacological effects including cardiovascular system effect, nervous system effect, antiinflammatory effect and antitumor effect. However, since salvianic acid is an intermediate product of the biosynthesis pathway of phenolic acid components, the accumulation level in the root of red-rooted salvia is low.
At present, the salvianic acid A is mainly extracted from the traditional Chinese medicinal material salvia miltiorrhiza, but the problems of large loss of phenolic components, unstable content and the like of the salvianic acid A exist in the traditional hydroalcoholic method, the enzyme extraction method and the microwave extraction method, and the traditional chemical total synthesis method of the salvianic acid A has the advantages of complex steps, poor selectivity, low yield and serious environmental pollution.
The biological method is adopted to synthesize the danshensu, which is a main direction, and has important significance for the production of the danshensu if the accumulation level of the danshensu in organisms can be improved.
Cytochrome P450 enzymes (CYP 450 s) are classified into P450 monooxidases (CYPs) and Cytochrome P450 reductases (cytochromes P450 Red, CPR). CYP98A is a cytochrome P450 family 98A subfamily enzyme, participates in the synthesis process of plant chlorogenic acid and rosmarinic acid, and CYP98A genes have been cloned from Arabidopsis thaliana, ruta graveolens, rabdosia rubescens and the like at present. It is thought that this gene may be involved in the synthesis of rosmarinic acid.
CN115216479 a discloses a recombinant genetically engineered bacterium, which uses coliform E.coliBL21 (DE 3) as host bacterium to construct a double-enzyme co-expression coliform system, and simultaneously expresses the transformed CYP98A75 gene and P450 reductase gene CPR, so that the substrate 4-hydroxy phenyllactic acid can be converted into salvianic acid.
The proposal needs to use specific substrate, can not improve the content of tanshinol in the plant body and can not realize the acquisition of the tanshinol from the red sage root.
Disclosure of Invention
The invention aims to provide application of the CYP98A75 gene of red sage root.
The invention also provides a method for generating tanshinol by utilizing hairy roots of the red sage root.
The technical scheme of the invention is as follows:
application of cytochrome P450 enzyme or radix Salviae Miltiorrhizae CYP98A75 gene in improving tanshinol content in hairy root of radix Salviae Miltiorrhizae is provided.
The cytochrome P450 enzyme is a protein shown in SEQ ID No.1 or a homologous protein thereof.
The gene of the salvia miltiorrhiza CYP98A75 codes a protein shown as SEQ ID No.1 or homologous protein thereof. Preferably, the CYP98A75 gene of the red sage root contains a nucleotide sequence shown in SEQ ID No. 2: (GenBank: KP 337735.1).
SEQ ID No.1:MAPLALLLLSLPVFFLLHNLYYRLRFRLPPGPRPWPIV GNLYDVKPLQFRCFADWAHSYGPIISVWFGSTLNVVVSSTELAKEVLKEKDQQLADRYRSRTATRLTKYGQDLIWADYGPHYVKVRKVCTVALFSAKSLESLRPIREDEVSAMVESIYNDCTAPGNSSKSLLLRKYLGAVSFNNITRIAFGKRYVDTEGRIHKQGEEMRSIADNRLRLGASRAIAEHIPWLRWMFPLNEEEFAKHAARRDRLTREIMEDHNIARQKSGGAKQHFCDALLTLKEKHDLTDDTIIGLLWDMIHAGMDTTAISVEWAMAELIRNPRVLQKVQEELDRVIGNERVMTELDFANLPYLRCVAKESLRLHPPTPLMLPHRASTNVKIGGYDIPKGSTVHVNVWAVARDPEVWKNPLEFRPERFLEDDVDIKGHDFRLLPFGAGRRICPGAQLGLDMVTSMLGRLLHHFKWAPPSGVSPEAINIAERPGVVTFMGTPLEAVATPRLPANLYERVAVDI
SEQ ID No.2:atggcgcctctcgctctcctcctcctctccctccctgtcttcttcctcctccacaacc tctactaccgcctccgcttccgcctccccccgggcccccgcccctggccgatcgtcggcaacctctacgacgtcaagccgctccagttccgctgcttcgccgattgggcccactcctacggccccatcatatccgtctggttcggctccaccctcaacgtcgtcgtttccagcaccgagctcgccaaggaggttctcaaggagaaggatcagcagctcgccgaccgctaccgcagccgcaccgccaccaggctcaccaagtacggtcaggacctgatttgggccgactatggccctcactacgtcaaggtgcggaaggtctgcaccgtcgcgctcttctccgccaagagcctcgaatcgttgaggccaattagagaggatgaggtctccgccatggtcgagtccatctacaatgactgcaccgcccctggcaactcgagtaagagcctgctgctaaggaaatatctaggagcagtgtcattcaacaacattacaagaatt gcattcgggaagagatatgtggatactgaagggagaatacacaagcaaggagaggagatgaggtccattgctgacaacaggttaaggctcggtgcctcccgtgccattgctgagcacattccatggctccgatggatgtttccactgaacgaagaggagtttgctaaacatgcagctcgcagggaccgtctcacacgagaaattatggaagaccacaacatcgcccgccagaagagtggaggagccaagcagcacttctgtgatgcattgctgacactaaaagagaaacatgatctaactgatgacaccatcattggccttctttgggacatgatccatgctggaatggacaccactgcgatatctgtggaatgggccatggcagagctgatcaggaacccaagggtgctacagaaggttcaagaggagctcgaccgtgtcattgggaacgaacgtgtgatgacagaacttgactttgcaaatcttccctacctacggtgtgtggccaaggaatcactcagattgcaccctccgaccccgctcatgcttcctcaccgtgccagtaccaatgtgaagatcggaggctacgacatccccaagggctcaaccgtgcatgtcaacgtgtgggcagttgcacgtgatcccgaggtgtggaaaaatcccctggagtttcgcccggagaggttccttgaggacgacgttgatataaaaggacatgattttaggcttctaccgttcggtgctggaagaagaatatgcccgggggcgcaactcggcctcgacatggtgacgtccatgcttggtcgccttctacaccacttcaaatgggctcctccaagcggggtgagcccagaagccatcaacattgcagagagacccggtgtcgtcaccttcatgggcactccattggaggcagttgctactcccagattaccggcaaacttgtacgagcgtgtcgccgtcgacatttga
The over-expression vector of the CYP98A75 gene of the red sage root can be used for improving the content of tanshinol in hairy roots of the red sage root. Preferably, the over-expression vector is PHB-Flag plasmid.
The agrobacterium containing the overexpression vector of the CYP98A75 gene of the salvia miltiorrhiza can be used for improving the content of tanshinol in hairy roots of the salvia miltiorrhiza. Preferably, the agrobacterium is agrobacterium C58C1.
The invention also provides a method for constructing hairy roots of red sage root for producing tanshinol, which comprises the following steps: the over-expression vector of the CYP98A75 gene of the red sage root is transformed into the agrobacterium, and the obtained recombinant agrobacterium is used for inducing the explant of the red sage root to obtain hairy root of the red sage root.
Preferably, the salvia miltiorrhiza explant is a leaf disc.
Preferably, the leaf discs are subjected to dip-dyeing in recombinant Agrobacterium solution at 20-30deg.C for 5-30min, preferably 8-15min.
The overexpression vector of the CYP98A75 gene of the red sage root is prepared by the following method:
(1) Amplifying the CYP98A75 gene of the red sage root, and carrying out double enzyme digestion on the over-expression vector and the amplified fragment by using BamH I and SpE I;
(2) And connecting the enzyme-cleaved over-expression vector with the enzyme-cleaved product of the amplification fragment of the CYP98A75 gene of the salvia miltiorrhiza to obtain the over-expression vector of the CYP98A75 gene of the salvia miltiorrhiza.
The over-expression vector is PHB-Flag plasmid, and the amplification primer sequence is as follows:
PHB-flag-SmCYP98A75-F：accagtctctctctcaagcttatggcgcctctcgctctc；
PHB-flag-SmCYP98A75-R：tttaaatcgataccggagctcaatgtcgacggcgaca。
transferring the connection product into competent engineering bacteria, culturing and expanding positive colony, and extracting over-expression vector of CYP98A75 gene of radix Salviae Miltiorrhizae. The engineering bacteria are preferably escherichia coli.
The method for transforming the agrobacterium comprises the following steps: the over-expression vector of the CYP98A75 gene of the red sage root is led into agrobacterium to obtain recombinant agrobacterium. Preferably, the over-expression vector of the CYP98A75 gene of the red sage root is introduced into the agrobacterium by a freeze thawing method. Recombinant agrobacteria were selected with rifampicin (Rif) and kanamycin (Kan).
Preferably, the agrobacterium is agrobacterium C58C1.
The hairy roots of the red sage root are screened by the following method:
culturing the impregnated leaf disc in a culture medium containing 0.5-0.8 mg/L Cef (cefotaxime sodium) until hairy roots appear; when the length of hairy root is 3-5cm, taking down the culture medium which is inoculated into and contains Cef and Hyg (hygromycin) for continuous culture. Preferably, the medium is 1/2MS.
In the growth process of hairy roots, gradient resistance reduction treatment is carried out on the used culture medium, and when the culture medium is replaced, the content of Cef is reduced to 0 from 0.5mg/L to 0.8 mg/L; meanwhile, the concentration of Hyg is increased in a gradient manner in the culture medium, and the content of Hyg is increased from 0 to 1-3 mug/L.
The hairy root of Salvia Miltiorrhiza after antibiotic adjustment is cultivated in a medium containing 1-3 μg/LHyg for 30-60 days, preferably 40-50 days.
A method for producing tanshinol comprises extracting tanshinol from hairy root of Saviae Miltiorrhizae radix obtained by the above method.
The invention has the beneficial effects that the salvia miltiorrhiza CYP98A75 gene is used for improving the content of danshensu in hairy roots of salvia miltiorrhiza, recombinant vectors of the overexpression of the salvia miltiorrhiza CYP98A75 are constructed, recombinant agrobacterium is constructed on the basis, based on the materials, the salvia miltiorrhiza leaf disc is used as an explant, and the hairy roots of the salvia miltiorrhiza, which can overexpress the enzyme of the salvia miltiorrhiza CYP98A75, are obtained through the mediation of the agrobacterium, so that the content of danshensu in the hairy roots of the salvia miltiorrhiza can be improved.
In the transformed hairy roots of the red sage root, the CYP98A75 gene expression level is 2-3.8 times of that of the wild hairy roots, the content of the tanshinol can reach 4.88 times of that of the wild hairy roots, the accumulation level of the tanshinol in the red sage root is obviously improved, and the method has very important significance for the production of the tanshinol.
The invention can obtain the hairy root of the red sage root with higher tanshinol yield, the hairy root grows rapidly and is easy to culture, the yield of the red sage root can be improved without adding exogenous substrate, and other components of the red sage root can be extracted and utilized, thus the invention has wide application prospect.
Drawings
FIG. 1 is a schematic diagram of construction of pHB-flag-CYP98A4 recombinant vector
FIG. 2 is a gel electrophoresis chart of the identification of different hairy root materials after PCR amplification, M is Marker, and PC is plasmid positive control
FIG. 3 shows the transcription level expression level (A) and the tanshinol content (B) of target genes of different hairy root lines
FIG. 4 is a schematic diagram of the construction of the pCB1300-Cas9-SmLAC75 vector of example 5
FIG. 5 is a gel electrophoresis pattern identified after PCR amplification of edited hairy root material in example 5, M is Marker, PC is plasmid positive control, WT is wild type, CYP98A75-I and CYP98A75-II are hairy roots subjected to gene editing.
FIG. 6 shows the gene transcript level expression level (A) and the tanshinol content (B) of each strain of the gene-edited hairy root.
Detailed Description
EXAMPLE 1 extraction of recombinant vector plasmid pHB-flag-SmCYP98A75 carrying target Gene
(1) Acquisition of ORF of the Gene SmCYP98A75
Extracting total RNA of the red sage root plant by using a plant RNA extraction kit, obtaining cDNA by using a reverse transcription kit, obtaining a target fragment by PCR amplification, and recovering an amplified product (the target fragment length is about 1500 bp) by using a 0.8% agarose gel electrophoresis and DNA gel recovery kit.
The PCR amplification primer of the target gene:
SmCYP98A75-F：5’-ATGGCGCCTCTCGCTCTCCTCCT-3’(SEQ ID NO.3)
SmCYP98A75-R：5’-GTCGACGGCGACACGCTCGTAC-3’(SEQ ID NO.4)
target gene PCR reaction system (30. Mu.L): 5 Xbuffer 6. Mu.L, cDNA sample 1. Mu.L, upstream and downstream primers 1. Mu.L each, dNTP 2.4. Mu.L, KD Plus 0.6. Mu.L, ddH 2 O makes up 30. Mu.L.
PCR reaction conditions: 94 ℃ for 5min;94℃30sec,55℃30sec,68℃60sec,35 cycles; 68 ℃ for 7min; constant temperature at 4 ℃.
(2) The recovered DNA product is connected to a quick connection carrier pEASY-Blunt Zero, the connection product is transferred into escherichia coli Top10 to be competent, bacterial liquid is coated on LB-Kan solid culture medium, and the culture is carried out for 12-16 hours in a 37 ℃ incubator.
Single colonies on the culture medium are picked, and the universal primer M13F/R (M13F/R is a necessary primer for biological sequencing) is used for identification, and the amplification conditions are the same as before.
Reaction system (10 μl): 5 Xbuffer 2. Mu.L, sample 1. Mu.L, upstream and downstream primers 0.5. Mu.L each, dNTP 0.8. Mu.L, KD Plus 0.3. Mu.L, ddH 2 O makes up 10. Mu.L.
The target fragment length was about 1500bp using 0.8% agarose gel electrophoresis, and the identified positive strain was kept at-80℃for further use.
EXAMPLE 2 construction of target Gene overexpression vector pHB-flag-SmCYP98A75 and host bacterium
(1) Designing target fragment amplification primers, amplifying under the same conditions as in the step (1) of the example 1, and recovering amplified products.
The amplification primer sequences were as follows:
PHB-flag-SmCYP98A75-F：accagtctctctctcaagcttATGGCGCCTCTCGCT CTC(SEQ ID NO.5)；
PHB-flag-SmCYP98A75-R：tttaaatcgataccggagctcAATGTCGACGGCGA CA (SEQ ID NO.6)。
(2) The pHB-flag vector plasmid was digested with BamH I and Spe I, the amplified product was digested with Bcl I and Spe I, the digested product was recovered by 0.8% agarose gel electrophoresis and DNA gel recovery kit, and the vector and amplified fragment were ligated with T4 ligase to give pHB-flag-CYP98A4 vector, the construction scheme of which is shown in FIG. 1.
(3) The procedure of example 1 was followed to transform E.coli competent, plate coating, incubate. Single colonies were picked and PCR identified.
Reaction system (10 μl): 5 Xbuffer 2. Mu.L, sample 1. Mu.L, upstream and downstream primers 0.5. Mu.L each, dNTP 0.8. Mu.L, KD Plus 0.3. Mu.L, ddH 2 O makes up 10. Mu.L.
The upstream primer is PHB-flag-SmCYP98A75-F;
downstream primer RBCSR: attaacttcggtcattagaggc (SEQ ID NO. 7).
Amplification conditions: 94 ℃ for 5min;94℃30s,55℃30s,68℃1min20s,30 cycles; 68 ℃ for 7min; constant temperature at 4 ℃.
The target fragment length was about 2000bp, amplified, and the amplified product was recovered using 0.8% agarose gel electrophoresis and a DNA gel recovery kit. The single colony strains with positive identification are sent to sequencing, and the strains with correct sequencing are preserved at-80 ℃ for standby.
(4) The recombinant expression vector plasmid pHB-flag-SmCYP98A75 was extracted from positive cells with a plasmid miniextraction kit and transferred into competent Agrobacterium C58C1.
10. Mu.L of pHB-flag-SmCYP98A75 plasmid is added into 100. Mu.L of competent cells, and the mixture is subjected to ice bath for 30min; quick-freezing with liquid nitrogen for 5min, and standing at room temperature for 3min; heat shock for 5min in 37 deg.c metal bath; adding 500 mu L of YEB liquid culture medium without antibiotics, and culturing for 4-6 h at a constant temperature of 28 ℃ and 120rpm in a shaking table in a dark place; the bacterial liquid is coated on a YEB-Kan-Rif solid screening culture medium and cultured in a constant temperature incubator at 28 ℃ for 48 hours.
Single colonies were picked and grown overnight in YEB-Kan-Rif liquid medium in a shaking table at constant temperature of 28 ℃. PCR identification of positive clones: the upstream primer of the identification primer is PHB-flag-SmCYP98A75-F, the downstream primer is RBCS R, the amplification and identification system is the same as the step (3), the target fragment length is about 2000bp, and the monoclonal bacterial colony strain with positive identification is preserved at-80 ℃ for standby.
EXAMPLE 3 Agrobacterium rhizogenes mediated genetic transformation
The recombinant agrobacterium C58C1 obtained in example 2 was used to induce hairy roots of red sage root, comprising the following steps:
(1) Activating the C58C1 strain with a plasmid vector, and performing expansion culture by using 20-40 mL of YEB-Kan-Rif liquid culture medium until the OD600 is approximately equal to 0.6; centrifugation was performed at 5000rpm at 4℃for 15min, the supernatant was discarded, and the cells were resuspended in an equal volume of 1/2MS liquid medium and incubated at 28℃for 30min with a shaking table at 80 rpm.
(2) Cutting 1cm from aseptic seedling of Saviae Miltiorrhizae radix 40d-60d after germination 2 Left and right leaf discs, inoculating on a 1/2MS solid culture medium, and standing overnight in a constant temperature incubator at 25 ℃ to serve as a receptor for genetic transformation;
the leaf disc is immersed in the recombinant agrobacterium culture solution activated in the step (1), and is immersed for 10min at a temperature of 28 ℃ by a shaking table and at a speed of 90 rpm.
(3) The leaf disc explant is clamped by forceps, redundant bacterial liquid on the leaf disc is sucked, the leaf disc is placed on a 1/2MS solid culture medium with the right side upwards, and the leaf disc is subjected to dark culture in a constant temperature incubator at 25 ℃ for 2 days; the leaf discs were transferred to 1/2MS-Cef (250 mg/L, 500. Mu.L/200 mL) solid medium for continued dark culture, and hairy roots were able to grow at the wound edges of the leaf discs around 2 weeks.
(4) When the hairy roots grow to about 3-5cm, they are cut off and inoculated onto a new 1/2MS-Cef-Hyg solid screening medium for culture.
In the growth process of hairy roots, gradient resistance reduction treatment is carried out on the used culture medium, the culture medium is replaced every 2 weeks, the consumption of Cef is reduced to 0 from 500 mu L/200mL, and the concentration of antibiotics is automatically regulated according to actual conditions; at the same time, the concentration of Hyg was increased in gradient in the medium, and the amount of Hyg (10 mg/L) was increased from 0 to 40. Mu.L/200 mL.
(5) Transferring the hairy root sample with the antibiotic adjustment to a 1/2MS-Hyg liquid culture medium for expansion culture, and replacing fresh culture medium every 2-4 weeks; the total incubation period of step (4) and step (5) was about 45 days.
Taking a proper amount of fresh hairy root material, extracting genome DNA by using a plant DNA extraction kit, wherein the extraction method refers to a specification, and the extracted DNA sample is stored at the temperature of minus 20 ℃.
And using the extracted DNA sample as a template, and identifying the DNA sample by using an identification primer. In addition, since all hairy root materials contain the rolB gene, DNA samples were used as templates to identify whether the target gene SmCYP98A75 was contained and whether the rolB gene unique to Agrobacterium C58C1 was contained.
Quickly freezing 100 mg/part of the hairy root with liquid nitrogen, and preserving at-80 ℃ for later use; and the other part is dried at 40 ℃ until the weight is not changed any more, and the material is ground by using a ball mill and stored in a shade and dry place for subsequent gene expression level detection and tanshinol content detection.
Identification primer of target gene CYP98A 75:
HPT-F：5’-CGATTTGTGTACGCCCGACAGTC-3’(SEQ ID NO.8)；
HPT-R：5’-CGATGTAGGAGGGCGTGGATATG-3’(SEQ ID NO.9)；
the roller B gene amplification primers are as follows:
rolB-F：5’-GCTCTTGCAGTGCTAGATTT-3’(SEQ ID NO.10)；
rolB-R：5’-GAAGGTGCAAGCTACCTCTC-3’(SEQ ID NO.11)；
the reaction system was (10 μl): mu.L of 5 Xbuffer, 1. Mu.L of sample, 0.5. Mu.L of each of the upstream and downstream primers, 0.8. Mu.L of dNTP, 0.8. Mu.L of KD Plus, and 10. Mu.L of ddH 2O.
The PCR reaction conditions were: 94℃for 5min,30 cycles (94℃for 30sec,55℃for 30sec,68℃for 60 sec) and 68℃for 7min.
After the PCR reaction, the amplified product was electrophoresed on an agarose plate. The agarose gel plates were placed under a gel imager and bands consistent in size with the rolB gene (423 bp) and the gene of interest (2000 bp) were observed, and the results are shown in FIG. 2, wherein PC was a plasmid positive control, indicating that the screening resulted in hairy roots containing the gene of interest induced by recombinant Agrobacterium C58C1.
Example 4
Taking a proper amount of fresh wild type red lead with a culture period of 45 days and a ginseng hairy root material containing a target gene induced by agrobacterium tumefaciens C58C1, drying at 40 ℃ until the weight is not changed any more, grinding the material by using a ball mill, and storing in a shade and dry place.
Detecting the expression level of target gene SmCYP98A75 in hairy roots of the red sage root and the content of tanshinol.
1. Gene expression level detection
Total RNA from the sample (quick frozen hairy roots stored at-80℃in example 3) was extracted using Beijing full gold Biotechnology Co., ltd TransZol Up Plus RNA Kit, the RNA content was determined, and the RNA was reverse transcribed into cDNA (-20℃for storage) and the procedure was as in example 1.
Real-time quantitative PCR (qPCR) was performed using cDNA as a template, and the expression of each target gene in different samples was detected using a SYBP qPCR super mix plus kit, qPCR system of 20. Mu.L.
The qPCR reaction conditions were: 95 ℃ for 10s;95 ℃ for 5 seconds, 60 ℃ for 30 seconds, 40 cycles; 95 ℃ for 15s; 30s at 60 ℃;95℃for 15s.
qPCR primers were as follows:
qPCR-F：CAGGCTCACCAAGTACGGT(SEQ ID NO.12)；
qPCR-R：ATTTCCTTAGCAGCAGGCT(SEQ ID NO.13)。
taking the salvia miltiorrhiza 18S as an internal reference, the salvia miltiorrhiza 18S internal reference primer is as follows:
18S-F：atgataactcgacggatcga；18S-R：cttggatgtggtagccgttt。
the expression level of the target gene of the hairy root of the wild control group was set to "1", and 2 was used -ΔΔCt The expression level of each target gene is calculated by the method, and the calculation method is as follows:
amount of target gene=2 -ΔΔCt
ΔΔct= (Ct target gene-Ct 18S) Experimental group - (Ct target gene-Ct 18S) Control group
2 -ΔΔCt Expression of the objective gene of the experimental group was expressed as a multiple of the expression of the objective gene of the control group, and the average value of three repeated experiments and the standard deviation thereof were taken.
The result of the transcriptional expression level of the target genes of different hairy root lines can be obtained through detection and calculation, and is shown in FIG. 3A.
The results showed that the transcription expression level of CYP98A75 gene in hairy roots was 2-3.8 times that of wild-type WT.
2. Detection of tanshinol
The salvianic acid content in the hairy root dry powder of the salvia miltiorrhiza of the example 3 is detected by mass spectrometry and chromatography.
The method for extracting the salvianic acid A comprises the following steps: the hairy root sample was dried in an oven at 40 ℃ and the dried sample was crushed using a ball mill. Precisely weighing hairy root sample powder 20mg, adding methanol 750 μl, ultrasonic extracting for 30min, centrifuging at 12000rpm at 4deg.C for 30min, and collecting supernatant; again, 750 μl of methanol was added and the two extracts were combined. Centrifuge at 12000rpm for 10min at 4deg.C, volatilizing 200 μL in a freeze concentrator (2000 rpm at 40deg.C), redissolving 1mL methanol, and adding 200 μL into sample injection vial.
Chromatographic conditions: waters xSELECT CSH TM C 18 The chromatographic column (2.1X105 mm,2.5 μm column temperature 35 ℃ mobile phase (0.1% formic acid+2 mmol/L ammonium acetate) water solution (A) and acetonitrile (B), elution gradient: 0-2.5min,7% B, 2.5-7min,7% -95% B. Flow rate 0.3mL/min, sample injection amount 10. Mu.L.
Mass spectrometry conditions: the target compounds were detected using a multiple reaction monitoring mode (Multiple reactions monitoring mode, MRM). And calculating the content measurement result of the target compound in the sample by using a standard curve method.
The results of the content of the danshensu in the different hairy root lines can be obtained by chromatographic mass spectrometry analysis, and are shown in figure 3B.
The result shows that the content of tanshinol in each gram of dry powder of the hairy root of the red sage root transferred into the over-expression vector is 2.66-4.88 times of that of the hairy root of the non-transferred root.
Example 5
The CRISPR/cas9 gene editing technology is used for editing target gene CYP98A75 and carrying out in-vivo functional research. The plasmid vectors used were psgR-Cas9 and pCAMBIA1300.
(1) Construction of the psgR-Cas9-CYP98A75 vector
Cleavage and recovery of the psgR-Cas9-At vector (20. Mu.L cleavage System): vector plasmid 1. Mu.g, bbs I0.5. Mu.L, fastAP (CIP) 1. Mu.L, 10 Xcutmart buffer 2. Mu.L, ddH 2 O makes up 20. Mu.L. And (3) recovering the enzyme-digested product by electrophoresis on 0.8% agarose gel at 37 ℃ for 30min to obtain a carrier recovery product.
Primer treatment (primer annealing to double strand, 10. Mu.L system): 1. Mu.L of the upstream primer, 1. Mu.L of the downstream primer, 1. Mu.L of 10 XT 4 buffer, and 0.4. Mu.L of T4 PNK; ddH 2 O makes up 10. Mu.L. 30min at 37 ℃; and (5) cooling to 25 ℃ in a gradient way at 95 ℃ for 5min to obtain a primer annealing product. The primer sequences were as follows:
Cas9-CYP98A75-F GATTGGTAGATGGACTCGACCATGGCGG；
Cas9-CYP98A75-R：AAACCCGCCATGGTCGAGTCCATCTAC；
ligation of primers to vector recovery product (20. Mu.L System): vector recovery product 50ng, primer annealing product 1. Mu.L, T4 ligase buffer 1. Mu.L, T4 ligase 0.5. Mu.L, ddH 2 O makes up 20. Mu.L, and the mixture is left at room temperature for 2h30min. The resulting ligation product was psgR-Cas9-CYP98A75.
(2) Construction of pCB1300-Cas9-SmLAC75 vector
The psgR-Cas9-CYP98A75 is directly used for transforming competent cells of the escherichia coli Top10, the transformation process is referred to the instruction, bacterial liquid is coated on LB-amp solid culture medium, and the bacterial liquid is cultured for about 16 hours in a constant temperature incubator at 37 ℃. Single colonies were picked up and cultured in 700. Mu.L LB-amp liquid medium for about 4-5 h in a constant temperature shaker at 37 ℃.
Bacterial liquid PCR identification of positive clones (10. Mu.L system): 2. Mu.L of 5 Xbuffer, 1. Mu.L of each of primer M13F and the downstream primer of the corresponding sample set, 1. Mu.L of bacterial solution, 0.8. Mu.L of dNTP, 0.3. Mu.L of KD Plus, ddH 2 O makes up 10. Mu.L. Amplification conditions: 94 ℃ for 5min;94℃for 30s,55℃for 30s,68℃for 30s,30 cycles; 68 ℃ for 7min; constant temperature of 4 DEG CTemperature. The PCR products were identified using 0.8% agarose gel electrophoresis and fragment sizes of about 400bp.
The bacterial liquid PCR identifies positive monoclonal and carries out sequencing, and a universal primer M13F is used as a sequencing primer.
And (3) inoculating the monoclonal sample bacterial liquid with the correct sequencing result into an LB-amp liquid culture medium for expansion culture, adding 700 mu L of the monoclonal sample bacterial liquid into 50% glycerol liquid nitrogen for quick freezing, and preserving strains at-80 ℃. The plasmid extraction kit is used for extracting the plasmid, and the extraction method refers to the product instruction book.
Cleavage, recovery and ligation of the pCAMBIA1300 vector and the psgR-Cas9-CYP98a75 vector: double digestion was performed using restriction enzymes EcoR I and Hind III, and the digested products were recovered using 0.8% agarose gel electrophoresis, and the recovered fragment size of the psgR-Cas9-CYP98A75 sample was about 5600bp. Ligation was performed using T4 ligase, and the ligation system was identical to the ligation of the "primer to vector recovery product" in step (1).
The ligation product was designated pCB1300-Cas9-CYP98A75, and its schematic construction is shown in FIG. 4.
The pCB1300-Cas9-CYP98A75 is directly transformed into the TOP competent cells of the escherichia coli, bacterial liquid is coated on LB-Kan solid culture medium, and the bacterial liquid is cultured for about 16 hours in a constant temperature incubator at 37 ℃. Single colonies were picked up and cultured in 700. Mu.L LB-Kan liquid medium in a shaking table at 37℃for about 4 to 5 hours.
Identifying positive clones by bacterial liquid PCR, and identifying a primer P2-5' -GCGATTAAGTTGGGTAACGC; the identification system of Ubi-At-R2-5' -TACATTCCGGTCAACCGGAA, PCR is the same as above. The PCR products were identified using 0.8% agarose gel electrophoresis and fragment sizes of about 400bp. The bacterial liquid PCR identifies positive monoclonal and carries out sequencing, and a primer P2 is used as a sequencing primer.
(3) Cultivation of hairy roots of red sage root
pCB1300-Cas9-CYP98a75, transformed into competent agrobacterium, and induced and cultured hairy roots of red sage root. Transformation, induction and cultivation are as described above.
Extracting DNA of hairy root for identification.
PCR identification of positive material: the identification primer uses P2/Ubi-At-R2, the identification system is the same as that of example 3, and the fragment size is about 700bp; the vector was identified using the vector universal identification primers rolB (423 bp) and rolC (622 bp).
Identification of CRISPR/Cas9 system edit hairy root DNA case:
amplification was performed using the extracted hairy root DNA sample as a template (30 μl amplification system): 5 X6. Mu.L of buffer, 1. Mu.L of each set of upstream and downstream primers, 2.4. Mu.L of dNTP, 1. Mu.L of each set of DNA, 0.6. Mu.L of KD Plus, and 30. Mu.L of ddH 2O. Amplification conditions: 94 ℃ for 5min;94℃for 30s,55℃for 30s,68℃for 30s,35 cycles; 68 ℃ for 7min; constant temperature at 4 ℃. The PCR products were identified using 0.8% agarose gel electrophoresis and fragment sizes were approximately 500bp-700bp. The results are shown in FIG. 5. WT is the unedited wild hairy root and CYP98A75-I and CYP98A75-II are the genetically edited hairy roots.
And sequencing samples positive to PCR identification, and calculating editing efficiency according to a sequencing result. The sequence of the identified primer is as follows:
JD-CYP98A75F：GGTTCTCAAGGAGAAGGATC；
JD-CYP98A75R：CAATGGACCTCATCTCCTC。
the sequencing results are shown in Table 1 below, and the gene editing conditions in each hairy root line are different, and the target sequence changes include deletion and mutation of bases.
TABLE 1 sequencing results of Gene-edited hairy root samples
(4) Gene expression and tanshinol content detection
And (3) detecting gene expression conditions: the procedure was as in example 4, and 100 mg/serving of the harvested gene-edited hairy root sample was snap frozen using liquid nitrogen, and kept at-80℃for further use. Total RNA of the samples was extracted using TransZol Up Plus RNA Kit (-80 ℃ C.) and stored for later use. And (3) determining the content of RNA, and reversely transcribing the RNA into cDNA (-20 ℃ for later use). Designing a synthetic qPCR primer, using an 18S internal reference primer in the red sage root, designing other primers by using laccase gene sequences of the red sage root, and enabling a target fragment to be 100bp-200bp.18SF
atgataactcgacggatcga；18SR cttggatgtggtagccgttt。qPCR-CYP98A75F CAGGCTCACCAAGTACGGT；
qPCR-CYP98A75R-ATTTCCTTAGCAGCAGGCT。
And carrying out real-time quantitative PCR (qPCR) by taking cDNA as a template, and detecting the expression condition of each target gene in different samples. Using SYBP qPCR super mix plus kit, 20. Mu.L of the system was reacted at 95℃for 10s;95 ℃ 5s 60 ℃ 30s 40 cycles; 95℃15s 60℃30s 95℃15s.
And (3) data processing: the expression level of each target gene was calculated by the 2- ΔΔct method with the expression level of the target gene of the wild control group hairy root set as "1", and the calculation method was as follows:
amount of target gene=2- ΔΔCt
ΔΔct= (Ct target gene-Ct 18S) Experimental group - (Ct target gene-Ct 18S) Control group
2- ΔΔCt Expression of the objective gene of the experimental group was expressed as a multiple of the expression of the objective gene of the control group, and the average value of three repeated experiments and the standard deviation thereof were taken.
The method for detecting the content of the compound is the same as in example 4.
The results are shown in FIG. 6. The expression of the target CYP98A gene in each gene-editing line was detected at the transcription level using qPCR (FIG. 6A), the hairy root sample lines were grouped on the abscissa, the expression of the gene of each target gene at the transcription level in the hairy root material was represented on the ordinate, and the single-factor analysis of variance was performed on each group of results. The expression of the target CYP98A gene varies from one gene editing strain to another, and the variation in expression levels may be caused by different degrees of gene editing. The expression level of the target gene is respectively reduced to different degrees (the reduction amplitude is 30% -80%) compared with the wild type WT group, and the result is statistically significant.
The accumulation of tanshinol content in each gene-edited group was compared with the wild-type control group (WT) (fig. 6B), and the statistical result was analyzed by single-factor analysis of variance. The content of tanshinol in the SmCYP98A75 monogenic editing strain group is reduced to 51% and 44% of that in the WT group compared with the WT group, and the results are statistically significant.
The above results show that decreasing the expression level of SmCYP98A75 gene in hairy roots of Salvia Miltiorrhiza can obviously decrease the accumulation level of tanshinol, which means that SmCYP98A75 is a key enzyme gene affecting the synthesis of tanshinol in Salvia Miltiorrhiza.

Claims (8)

1. Application of the CYP98A75 gene in improving the content of tanshinol in hairy roots of red sage root is provided.
2. The use according to claim 1, wherein the gene of the salvia miltiorrhiza CYP98a75 encodes a protein shown in SEQ ID No.1 or a homologous protein thereof.
3. The over-expression vector of the CYP98A75 gene of the red sage root is applied to the improvement of the content of tanshinol in hairy roots of the red sage root.
4. The application of agrobacterium containing over-expression vector of the CYP98A75 gene of the root of the red sage root in improving the content of tanshinol in hairy roots of the root of the red sage root.
5. A method for constructing hairy roots of salvia miltiorrhiza for producing tanshinol, which is characterized by comprising the following steps: and (3) transforming agrobacterium with an over-expression vector carrying the CYP98A75 gene of the salvia miltiorrhiza, and inducing the salvia miltiorrhiza with the agrobacterium to obtain hairy roots of the salvia miltiorrhiza.
6. The method of claim 5, wherein the over-expression vector is a PHB-Flag expression vector.
7. The method of claim 5, wherein the agrobacterium is agrobacterium C58C1.
8. A method for producing tanshinol, characterized in that tanshinol is extracted from hairy roots of salvia miltiorrhiza obtained by the method according to any one of claims 5-7.
CN202311194251.3A 2023-09-15 2023-09-15 Method for producing tanshinol Pending CN117467678A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311194251.3A CN117467678A (en) 2023-09-15 2023-09-15 Method for producing tanshinol

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311194251.3A CN117467678A (en) 2023-09-15 2023-09-15 Method for producing tanshinol

Publications (1)

Publication Number Publication Date
CN117467678A true CN117467678A (en) 2024-01-30

Family

ID=89622852

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311194251.3A Pending CN117467678A (en) 2023-09-15 2023-09-15 Method for producing tanshinol

Country Status (1)

Country Link
CN (1) CN117467678A (en)

Similar Documents

Publication Publication Date Title
CN105647943B (en) Saussurea involucrate cell squalene synthase gene SiSQS and coded product and application thereof
WO2021190632A1 (en) Hydroxylase gene and use thereof
CN109852644B (en) Method for preparing intermediate of brivaracetam
CN116640195B (en) Citrus CsPIF4 gene, protein coded by same and application thereof
CN115058374B (en) Recombinant zymomonas mobilis for synthesizing acetoin by utilizing pyruvic acid as well as construction method and application thereof
CN115074375B (en) Salvia 2-ketoglutarate-dependent dioxygenase gene and application thereof
CN117467678A (en) Method for producing tanshinol
CN113005130B (en) Tanshinone IIA synthase gene and application thereof
CN116790542A (en) Glycosyltransferase PpUGT3 for biosynthesis of rhizoma paridis saponin
CN112410353B (en) fkbS gene, genetic engineering bacterium containing fkbS gene, and preparation method and application of fkbS gene
CN104894150B (en) Saussurea involucrate cell phenylalanine ammonia lyase gene SiPAL and encoding product and application thereof
CN114317569A (en) Apple gene MdBGLU40 and application thereof in rot resistance of apple trees
CN117106836B (en) Application of phosphatidyl glycerol phosphatase coding gene in fermentation production of cytidine
CN115125180B (en) Recombinant zymomonas mobilis for producing acetoin by double ways and construction method and application thereof
CN114874961B (en) Recombinant zymomonas mobilis for synthesizing acetoin by using acetaldehyde, and construction method and application thereof
CN114480448B (en) Gene GbF3' H for promoting synthesis of ginkgo flavonol glycosides, and vector, protein and application thereof
CN117070537A (en) AmCAD1 gene for regulating and controlling Mongolian agropyron content, and encoding product and application thereof
CN118291461A (en) Grape promoter and application thereof in improving anthocyanin content of grape callus cells
Zhang Identification and Characterization of Apple Anthracnose Resistance Genes
CN115927406A (en) Nanguo pear PuAOX1a gene, overexpression vector and application
CN116606830A (en) Platycodin glycosyltransferase PgGT1 and encoding gene and application thereof
CN114437186A (en) PhlH protein mutant and application thereof in increasing mupirocin yield
CN115216479A (en) Engineering bacterium for converting 4-hydroxy phenyllactic acid to generate tanshinol and application thereof
CN117757813A (en) Penicillium digitatum transcription factor PDIDSM_85260 and application thereof
CN117165597A (en) Application of TdERF11 gene of larch in improving capacity of mountain novel Yang Kanglao

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication