CN104830717A - Salvia miltiorrhiza endophyte with danshinolic acid B accumulation inducing effect, and use thereof - Google Patents
Salvia miltiorrhiza endophyte with danshinolic acid B accumulation inducing effect, and use thereof Download PDFInfo
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- salvia miltiorrhiza
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- 235000011135 Salvia miltiorrhiza Nutrition 0.000 title claims abstract description 24
- 238000009825 accumulation Methods 0.000 title claims abstract description 11
- 239000002253 acid Substances 0.000 title abstract description 14
- 241000304195 Salvia miltiorrhiza Species 0.000 title abstract 6
- 230000001939 inductive effect Effects 0.000 title description 5
- 241000675919 Pseudomonas psychrotolerans Species 0.000 claims abstract description 27
- 150000007965 phenolic acids Chemical class 0.000 claims abstract description 10
- 240000007164 Salvia officinalis Species 0.000 claims description 24
- 235000005412 red sage Nutrition 0.000 claims description 21
- 244000132619 red sage Species 0.000 claims description 19
- SNKFFCBZYFGCQN-UHFFFAOYSA-N 2-[3-[3-[1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyloxy]-3-(3,4-dihydroxyphenyl)propanoic acid Chemical compound C=1C=C(O)C=2OC(C=3C=C(O)C(O)=CC=3)C(C(=O)OC(CC=3C=C(O)C(O)=CC=3)C(O)=O)C=2C=1C=CC(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-UHFFFAOYSA-N 0.000 claims description 12
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- 238000005406 washing Methods 0.000 description 2
- QBLFZIBJXUQVRF-UHFFFAOYSA-N (4-bromophenyl)boronic acid Chemical compound OB(O)C1=CC=C(Br)C=C1 QBLFZIBJXUQVRF-UHFFFAOYSA-N 0.000 description 1
- IBGBGRVKPALMCQ-UHFFFAOYSA-N 3,4-dihydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1O IBGBGRVKPALMCQ-UHFFFAOYSA-N 0.000 description 1
- YMGFTDKNIWPMGF-AGYDPFETSA-N 3-(3,4-dihydroxyphenyl)-2-[(e)-3-[2-[(e)-2-(3,4-dihydroxyphenyl)ethenyl]-3,4-dihydroxyphenyl]prop-2-enoyl]oxypropanoic acid Chemical compound C=1C=C(O)C(O)=C(\C=C\C=2C=C(O)C(O)=CC=2)C=1/C=C/C(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 YMGFTDKNIWPMGF-AGYDPFETSA-N 0.000 description 1
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- VPLLTGLLUHLIHA-UHFFFAOYSA-N dicyclohexyl(phenyl)phosphane Chemical compound C1CCCCC1P(C=1C=CC=CC=1)C1CCCCC1 VPLLTGLLUHLIHA-UHFFFAOYSA-N 0.000 description 1
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- OJURWUUOVGOHJZ-UHFFFAOYSA-N methyl 2-[(2-acetyloxyphenyl)methyl-[2-[(2-acetyloxyphenyl)methyl-(2-methoxy-2-oxoethyl)amino]ethyl]amino]acetate Chemical compound C=1C=CC=C(OC(C)=O)C=1CN(CC(=O)OC)CCN(CC(=O)OC)CC1=CC=CC=C1OC(C)=O OJURWUUOVGOHJZ-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
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- XHALVRQBZGZHFE-UHFFFAOYSA-N rosmarinic acid methyl ester Natural products C=1C=C(O)C(O)=CC=1C=CC(=O)OC(C(=O)OC)CC1=CC=C(O)C(O)=C1 XHALVRQBZGZHFE-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The invention relates to a Salvia miltiorrhiza endophyte and its use in the improvement of danshinolic acid B accumulation in the Salvia miltiorrhiza hairy root culturing process. The Salvia miltiorrhiza endophyte is Pseudomonas psychrotolerans LG4 concretely, and has a preservation number of CCTCC NO:M2015085. The Salvia miltiorrhiza endophytec an promote the accumulation of a phenolic acid substance (danshinolic acid B) in Salvia miltiorrhiza hairy root.
Description
Technical field
The accumulation of salvianolic acid B can be improved when the present invention relates to a kind of red sage root endophyte and cultivate for Hairy Root Cultures of Salvia miltiorrhiza.
Background technology
The red sage root is Labiatae per nnial herb, have another name called Radix Campylotropis Hirtella (Herba Myrsines Africanae), red, red ginseng, Radix Salviae Miltiorrhizae, blood ginseng etc., be distributed in the areas such as China North China, East China, Central-South, northwest, southwest.Activeconstituents contained by its root and rhizome can be divided into following two classes by character: fat-soluble Diterpenoids from bulbus and water miscible phenolic acid components.Wherein water soluble component has: rancinamycin IV, coffic acid, isoferulic acid, rosmarinic acid, methyl rosmarinate, alkannic acid B, alkannic acid mono methyl ester, alkannic acid dimethyl ester, alkannic acid ethyl ester, Salvianic acid (Salvianic acidA) and salvianolic acid A, B, C, D, E, F, G, H, I, J etc.Pressure differential self all has very strong antioxygenation can remove superoxide anion and hydroxyl radical free radical, and anti-lipid peroxidation reacts.And wherein rosmarinic acid tool also have anti-inflammatory, antibacterial, antiviral, immunomodulatory, anti-thrombosis, anti-messangial cell propagation, antidepressant, etc. important biological activity, be widely used in food, makeup, medicine and other fields.
The market requirement of the red sage root increases just day by day, but wild Salvia miltiorrhiza resource-constrained, and different areas red rooted salvia difference in quality is very large, active component content poor stability.The growth cycle of planted rooted salvia is long and active constituent content is low in addition, and this brings certain difficulty all to Clinical practice and quality control.Recent domestic has carried out the research to red sage root tissue and cell cultures, shows to cultivate the primary bioactive components that directly can obtain the red sage root by the cell or tissue of the red sage root.Wherein, the Hairy Root Cultures of Salvia miltiorrhiza system formed by Agrobacterium rhizogenes infection plant, its speed of growth is very fast, genetic stability, becomes the good culture systems of producing red sage root active substance.But hairly root yield of active ingredients is low, affect enlarged culturing and even the suitability for industrialized production of hairly root.
For solving this contradiction, many investigators utilize inducible system to stimulate hairly root in recent years, by inducing the expression of relevant metabolic pathway enzyme system, promoting the accumulation of effective secondary metabolism thus improving output.The material of secondary metabolite can be produced be called elicitor (Elicitor) by inducing plant, be divided into abiotic elicitor and biotic induce.Abiotic elicitor comprises Ag
+deng metal ion, and biotic induce attached bag draws together yeast extract and some fungal fermented filtrate etc.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of red sage root endogenetic bacteria---Pseudomonas psychrotoleransLG4, and it can promote phenolic acid in Hairy Root Cultures of Salvia miltiorrhiza--the accumulation of content of danshinolic acid B.
In order to solve the problems of the technologies described above, the invention provides a kind of red sage root endophyte, be Pseudomonas psychrotoleransLG4, its preserving number is: CCTCC NO:M 2015085.
Present invention also offers the purposes of above-mentioned red sage root endophyte: the accumulation promoting phenolic acid content in Hairy Root Cultures of Salvia miltiorrhiza.Phenolic acid is salvianolic acid B.
This strain Pseudomonas psychrotolerans LG4 adopts plate dilution method to be separated to obtain a strain endogenetic bacteria from red sage root plant, through identifying that its fermented liquid has the effect promoting Hairy Root Cultures of Salvia miltiorrhiza salvianolic acid B Composition accumulation.
Separation method is:
1), by the root of the red sage root after surface sterilization process, add sterilized water and carry out grinding dilution, coat on nutrient agar medium, cultivate 1 ~ 3 day in 28 ~ 32 DEG C;
Nutrient agar medium is filled a prescription: peptone 10g, extractum carnis 3g, NaCl 5g, agar 18g and distilled water 1000mL, pH=7.2 ~ 7.4.
2), picking list bacterium colony in identical nutrient agar plate line purifying, in 28 ~ 32 DEG C cultivate 1 ~ 3 day;
3), repeating step 2), until obtain pure growth.
The preservation information of red sage root endophyte of the present invention is specific as follows: Pseudomonas psychrotolerans LG4, depositary institution: China typical culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M2015085, March 4 2015 preservation time.
Red sage root endophyte of the present invention--Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) bacterium colony after nutrient agar medium cultivates 24h is rounded, bacterium colony is less, neat in edge, surface drying, produce xanthein, cultivate 3-5 days rear surface projection folds.
Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) Gram-negative provided by the invention, aerobic, shaft-like, do not produce brood cell; Growth temperature be 20 ~ 40 DEG C, pH value is well-grown in the environment of 6.0 ~ 8.0; Energy metabolism glucose, pectinose, wood sugar etc. produce acid.
The 16S rDNA sequential analysis of this Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) shows, is 99% with the similarity of the strain Pseudomonas psychrotolerans recorded in Genbank international data center.
This Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) promotes the effect of salvianolic acid B accumulation in Hairy Root Cultures of Salvia miltiorrhiza culture system as elicitor.
Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) of the present invention can promote the raising of content of danshinolic acid B.Nontoxic to people and animals, environment is not polluted.Can be applied in Hairy Root Cultures of Salvia miltiorrhiza is cultivated, produce certain economic benefit.
When Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) of the present invention is for promoting that Hairy Root Cultures of Salvia miltiorrhiza salvianolic acid B accumulates, only need will utilize this red sage root endogenetic bacteria---elicitor prepared by Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) is connected in hairly root substratum, can promote Hairy Root Cultures of Salvia miltiorrhiza content of danshinolic acid B.
Beneficial effect of the present invention is mainly reflected in: utilize Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) fermented supernatant fluid elicitor provided by the invention, add the content that can improve salvianolic acid B in Hairy Root Cultures of Salvia miltiorrhiza culture system to, and then the content of phenolic acids in raising Hairy Root Cultures of Salvia miltiorrhiza, the Hairy Root Cultures of Salvia miltiorrhiza of inducing can be used for the extraction of salvianolic acid B list product.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
The isolation cultivation method of embodiment 1, Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085), carries out following steps successively:
1, gather wild Salvia miltiorrhiza plant in Sichuan Zhong Jiang, root cleans rear 25KHz ultrasonication 5 minutes;
2, aseptically, to step 1 gained for examination material sterilizing deionized water rinsing 3 times, and use alcohol and 3% (m/V) the chlorine bleach liquor surface sterilization of 75% (v/v) respectively;
3, in gnotobasis, by step 2 gained for examination material aseptic water washing 3 times, get the last aseptic water washing liquid coating of rinsing for 1 time of 100 μ l and be inoculated on nutrient agar medium, 30 DEG C cultivate 24h after, carry out aseptic checking.
As produced without bacterium colony, then continue follow-up step 4; Produce if any bacterium colony, then return step 2 and continue surface sterilization process.
4, in super clean bench, get the red sage root sample 1 ~ 2g handled well, fully grind in sterile mortar, and add 5mL sterilized water, mixing, leave standstill 15min, get 100 μ l supernatant dilution spreads in nutrient agar, be placed in 28 ~ 32 DEG C (better 30 DEG C) and cultivate 1 ~ 3 day;
5, the single bacterial classification produced in picking step 4 to be rule purifies and separates in same medium (nutrient agar), is placed in 28 ~ 32 DEG C (better 30 DEG C) and cultivates 1 ~ 3 day, continues picking list bacterium colony;
Repeat aforesaid operations till obtaining pure culture.
6, the single bacterium colony obtained in step 5 is carried out 16s rDNA sequencing, through sequence alignment, determine bacterial strain, and preservation.Obtain Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085).
The preparation method of embodiment 2, Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) elicitor, carries out following steps successively::
1, Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) is transferred in nutrient agar slopes, cultivate 2 days for 30 DEG C;
Nutrient agar slopes: peptone 10g, extractum carnis 3g, NaCl 5g, agar 18g and distilled water 1000mL, pH=7.2 ~ 7.4.
2, by the slant strains of transfering loop picking one ring step 1 gained, be inoculated in the 250ml triangular flask containing 50ml nutrient broth medium, cultivate 72h in 28 DEG C with 220rpm, obtain Pseudomonas psychrotolerans LG4CCTCC M 2015085 fermenation raw liquid;
Nutrient broth medium formula is: peptone 10g, extractum carnis 3g, NaCl 5g and distilled water 1000mL, pH=7.2 ~ 7.4.
Above-mentioned steps 1 and step 2 are all cultivated under natural light condition.
3, get step 2 gained fermenation raw liquid, in 4 DEG C, 12000rpm, centrifugal 2min, get supernatant and cross 0.22 μm of millipore filtration, and gained filtrate is Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) elicitor.
The promotion experiment that embodiment 3, Pseudomonas psychrotolerans LG4 (CCTCC NO:M 2015085) accumulate Hairy Root Cultures of Salvia miltiorrhiza salvianolic acid B:
1, the Hairy Root Cultures of Salvia miltiorrhiza (female root) of 0.3g is inoculated in the 250ml triangular flask containing 6, the 7-V substratum of 100ml, and 25 DEG C, 100rpm lucifuge cultivates 18 days, based on thing;
Experimental group: Pseudomonas psychrotolerans LG4 (CCTCC NO:M2015085) elicitor (embodiment 2 gained) adding 1.5ml in each basic thing, continue the same terms (25 DEG C, 100rpm, lucifuge) cultivate 6 days; As experimental group;
Negative control (blank): the sterile vegetative broth culture adding 1.5ml in each basic thing, continues the same terms (25 DEG C, 100rpm, lucifuge) and cultivates 6 days; Negative control;
Above-mentioned experimental group and blank group arrange 5 repetitions respectively;
The Hairy Root Cultures of Salvia miltiorrhiza of experimental group and blank gained is proceeded as follows respectively:
2, cultivate and terminate the hairly root that step 1 obtains by rear distilled water and clean three times, with in thieving paper suck dry moisture, claim fresh weight (fresh weight, FW); After will claim the hairly root of fresh weight to put into baking oven, 55 DEG C are dried to constant weight, measure dry weight (dry weight, DW).
3, the red sage root dried to constant weight in right amount is got, grind into powder.Get 70% (volume %) methyl alcohol that 0.5g adds 2.5mL, in 120W Ultrasonic Cleaners, (in ultrasonic procedure, water temperature can not be too high for ultrasonic extraction 60min, suitably can add ice cube, namely, control temperature≤20 DEG C), get supernatant after cooling, cross 0.45 μm of millipore filtration, carry out phenolic acid by the method for high performance liquid chromatography (HPLC)--the mensuration of content of danshinolic acid B.
Measurement result is as follows:
After adding Pseudomonas psychrotolerans LG4CCTCC M 2015085 elicitor in experimental group, hairly root dry weight remains unchanged (for blank), and content of danshinolic acid B is higher than blank group by 60%.
Comparative example, select following Pseudomonas psychrotolerans bacterial strain and this symbolic animal of the birth year closely to plant respectively bacterial strain as a comparison case, concrete bacterial strain is as follows:
The Code Number of A, German Culture Collection DSMZ preservation is the Pseudomonaspsychrotolerans of DSM 15758;
The Pseudomonas psychrotolerans of the Code Number CFCC 11317 of B, China Forest Microbiological Culture Collection administrative center CFCC preservation;
The Pseudomonas psychrotolerans of the Code Number CFCC 11072 of C, China Forest Microbiological Culture Collection administrative center CFCC preservation;
The Code Number of D, German Culture Collection DSMZ preservation is the Pseudomonasoleovorans of DSM 1045;
Carried out according to " the promotion experiment to the accumulation of Hairy Root Cultures of Salvia miltiorrhiza salvianolic acid B " described in " Elicitor preparation " described in above-described embodiment 2 and embodiment 3 by above-mentioned Pseudomonas bacterial strain, the result of final gained is as shown in table 1 below.
The impact that table 1.Pseudomonas psychrotolerans LG4CCTCC M 2015085 and comparative example accumulate Hairy Root Cultures of Salvia miltiorrhiza phenolic acid
| Numbering | Salvianolic acid B (mg/L) |
| A | 21.27±4.68 |
| B | 23.53±5.32 |
| C | 25.14±2.13 |
| D | 20.07±1.94 |
| Negative control group | 22.01±3.21 |
| P.psychrotolerans LG4 (the present invention) | 35.16±1.83 |
It is laboratory mean values ± standard deviation in table
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (3)
1. red sage root endophyte, is characterized in that: be Pseudomonas psychrotolerans LG4, its preserving number is: CCTCCNO:M 2015085.
2. the purposes of red sage root endophyte as claimed in claim 1, is characterized in that: the accumulation promoting phenolic acid content in Hairy Root Cultures of Salvia miltiorrhiza.
3. the purposes of red sage root endophyte according to claim 2, is characterized in that: phenolic acid is salvianolic acid B.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349431A (en) * | 2015-11-04 | 2016-02-24 | 中国人民解放军第二军医大学 | Endophytic fungus of radix salviae miltiorrhizae and application of endophytic fungus |
| CN117965393A (en) * | 2024-03-28 | 2024-05-03 | 南京农业大学三亚研究院 | Endophytic pseudomonas bacterium C145 and application thereof |
| CN117965393B (en) * | 2024-03-28 | 2024-06-21 | 南京农业大学三亚研究院 | Endophytic pseudomonas bacterium C145 and application thereof |
-
2015
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Non-Patent Citations (4)
| Title |
|---|
| 唐坤等: "一株促进丹参生长和提高丹酚酸含量的活性内生真菌", 《菌物学报》 * |
| 李文渊等: "诱导子对丹参有效成分次生代谢的诱导与调控", 《中国中药杂志》 * |
| 王萌等: "丹参内生真菌与其有效成分的相关性分析", 《中国实验方剂学杂志》 * |
| 阎岩: "微生物对丹参毛状根生长和次生代谢的影响极其机理", 《中国博士学位论文全文数据库农业科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349431A (en) * | 2015-11-04 | 2016-02-24 | 中国人民解放军第二军医大学 | Endophytic fungus of radix salviae miltiorrhizae and application of endophytic fungus |
| CN105349431B (en) * | 2015-11-04 | 2018-10-09 | 中国人民解放军第二军医大学 | A kind of Radix Salviae Miltiorrhizae endogenetic fungus and its application |
| CN117965393A (en) * | 2024-03-28 | 2024-05-03 | 南京农业大学三亚研究院 | Endophytic pseudomonas bacterium C145 and application thereof |
| CN117965393B (en) * | 2024-03-28 | 2024-06-21 | 南京农业大学三亚研究院 | Endophytic pseudomonas bacterium C145 and application thereof |
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